Formulation for administration of RNA

Abstract

To provide pharmaceutical products for the safe and efficient delivery of RNA encoding proteins with therapeutic value, such as vaccines, in patients and animals.SOLUTION: The present invention relates to compositions comprising polyplex formulations for delivery of RNA to a target organ or a target cell after parenteral administration, in particular after intramuscular administration. More precisely, the present invention relates to formulations for administration of RNA such as self-replicating RNA, in particular by intramuscular injection. In more detail, the formulations comprise polyplex particles from single stranded RNA and a polyalkyleneimine.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a polyplex formulation for delivering RNA to a target organ or cell after parenteral administration, particularly intramuscular administration. More specifically, the present invention relates to a formulation for administering RNA, such as a self-replicating RNA, particularly via intramuscular injection. More specifically, the polyplex particle comprises a single-stranded RNA, preferably a self-replicating or self-amplifying RNA, and a polyalkyleneimine. The RNA may encode a protein of interest, such as a pharmaceutically active protein. The RNA is taken up by cells, and the RNA is preferably translated into a peptide or protein capable of exhibiting its physiological activity. The composition of the present invention is applicable to inducing or enhancing an immune response. The composition of the present invention is also useful in the preventive and / or therapeutic treatment of diseases involving antigens such as proteins. Furthermore, the present invention relates to a method for producing a stable composition comprising an RNA-polyplex formulation, wherein the RNA-polyplex formulation comprises a single-stranded RNA and a polyalkyleneimine. The RNA-polyplex particle formulations described herein can be frozen and thawed, or dehydrated and rehydrated, without loss of product quality, particularly without substantial loss of RNA activity. In particular, the RNA-polyplex particle formulations described herein can be frozen or dehydrated by lyophilization, spray drying, or related methods, which allows for extended product shelf life with respect to liquid storage. Furthermore, the RNA-polyplex particle formulations described herein can comply with pharmaceutical product requirements, more specifically, GMP manufacturing requirements and pharmaceutical product quality requirements for parenteral application. The RNA-polyplex formulations described herein are particularly useful, for example, for vaccinating humans or animals against infectious diseases. [Background technology]

[0002] The introduction of exogenous nucleic acids encoding one or more polypeptides for prophylactic and therapeutic purposes has long been a goal of biomedical research. Prior art approaches share the commonality of delivering nucleic acid molecules to target cells or organisms, but differ in the type of nucleic acid molecule and / or delivery system. Safety concerns associated with the use of deoxyribonucleic acid (DNA) molecules have led to increased attention in recent years toward ribonucleic acid (RNA) molecules. Various approaches have been proposed, including the administration of single-stranded or double-stranded RNA in the form of naked RNA or in complexed or packaged forms in nonviral or viral delivery vehicles. In viruses and viral delivery vehicles, nucleic acids are typically encapsulated by proteins and / or lipids (virions). For example, genetically engineered RNA virus particles derived from RNA viruses have been proposed as delivery vehicles for plant therapy (WO 2000 / 053780(A2)) or mammalian vaccination (Tubulekas et al., 1997, Gene, vol. 190, pp. 191-195). Due to safety concerns, the medical and veterinary communities are reluctant to administer RNA virus particles to humans or animals. To develop therapeutics based on gene delivery, non-viral delivery vehicles applicable to RNA have been extensively investigated. However, for various reasons, the translation of non-viral gene delivery approaches into clinical therapy has not been very successful. Related reasons include insufficient levels of gene expression, technical and regulatory issues related to the pharmaceutical development of such complex products, and safety reasons. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2000 / 053780(A2) [Patent Document 2] International Publication No. 2014 / 071963(A1) [Patent Document 3] U.S. Patent Application No. 12 / 671,312 [Non-licensed literature]

[0004] [Non-licensed Document 1] Tubulekasら, 1997, Gene, Volume 190, Pages 191~195 [Non-licensed Document 2] "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by HGW Leuenberger, B. Nagel, and H. Koelbl, Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995) [Non-licensed Document 3] Molecular Cloning: A Laboratory Manual, 2nd edition, edited by J. Sambrook, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989 [Non-licensed Document 4] Koppel, D., J. Chem. Phys., Vol. 57, 1972, pp. 4814-4820 [Non-licensed Document 5] Joseら, Future Microbiol., 2009, Volume 4, Pages 837~856 [Non-licensed Document 6] Gould, 2010, Antiviral Res., Volume 87, Pages 111~124 [Non-licensed Document 7] Strauss & Strauss, Microbiol. Rev., 1994, Vol. 58, pp. 491-562 [Non-licensed Document 8] Rupp, 2015, J. Gen. Virology, Volume 96, pp. 2483-2500 [Non-licensed Document 9] Shirako & Strauss, 1994, J. Virol., Volume 68, pp. 1874-1885 [Non-licensed Document 10] Kim, 2004, Virology, Volume 323, pp. 153-163 [Non-licensed Document 11] Vasiljeva, 2003, J. Biol. Chem., Volume 278, Pages 41636~41645 [Non-licensed Document 12] Pettersson, 1980, Eur. J. Biochem., 105, pages 435~443 [Non-licensed Document 13] Rozanov, 1992, J. Gen. Virology, Vol. 73, pp. 2129-2134 [Non-licensed Document 14] Rubach, Virology, 2009, Volume 384, Pages 201~208 [Non-licensed Document 15] White, 1998, J. Virol., Volume 72, pp. 4320-4326 [Non-licensed Document 16] Frolov, 2001, RNA, Volume 7, Pages 1638~1651 [Non-licensed Document 17] McKinney, 1963, Am. J. Trop. Med. Hyg., 1963, Volume 12, Pages 597~603 [Non-licensed Document 18] Hardy & Rice, J. Virol., 2005, Vol. 79, pp. 4630-4639 [Non-licensed Document 19] Smith and Waterman, 1981, Ads App. Math., 2, 482 pages [Non-licensed Document 20] Neddleman and Wunsch, 1970, J. Mol. Biol., pp. 48, 443 [Non-licensed Document 21] Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85, 2444 pages. [Non-Patent Document 22] Current Protocols in Molecular Biology, edited by FM Ausubel et al., John Wiley & Sons, Inc., New York. [Non-Patent Document 23] Remington's Pharmaceutical Sciences, Mack Publishing Co. (ed. A. R Gennaro, 1985) [Non-Patent Document 24] Demoulins, Thomas et al., “Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines.” Nanomedicine: Nanotechnology, Biology and Medicine, (2015) Summary of the Invention [Problem to be solved by the invention]

[0005] Thus, there is a need for pharmaceutical products for the safe and efficient delivery of RNA encoding proteins of therapeutic value, such as vaccines, in patients and animals. Aspects and embodiments of the present invention as described herein address this need. [Means for solving the problem]

[0006] Immunotherapeutic strategies are promising options for the prevention and therapy of, for example, infectious and cancerous diseases. The increasing identification of pathogen- and tumor-associated antigens has led to a broad pool of targets suitable for immunotherapy. The present invention encompasses improved agents and methods suitable for the efficient expression of antigens suitable for immunotherapeutic treatment for the prevention and therapy of diseases.

[0007] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: (a) single-stranded RNA, and (b) Polyalkyleneimine A pharmaceutical composition comprising Regarding.

[0008] In a further aspect, the present invention provides a compound comprising: (a) single-stranded RNA, and (b) Polyalkyleneimine A composition comprising Regarding.

[0009] In one embodiment of all aspects of the present invention, the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the single-stranded RNA (N:P ratio) is 1.0 to 30, preferably 2.0 to 15.0, more preferably 6.0 to 12.0.

[0010] In a further aspect, the present invention provides a method for producing a composition comprising: (a) single-stranded RNA, and (b) Polyalkyleneimine A composition comprising: A composition in which the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the single-stranded RNA is 1.0 to 30.0, preferably 2.0 to 15.0, more preferably 6.0 to 12.0. Regarding.

[0011] In one embodiment of all aspects of the invention, the ionic strength of the composition is 50 mM or less, and preferably the concentration of positively charged monovalent ions is 25 mM or less and the concentration of positively charged divalent cationic free ions is 20 μM or less.

[0012] In a further aspect, the present invention provides a method for producing a composition comprising: (a) single-stranded RNA, and (b) Polyalkyleneimine A composition comprising: A composition having an ionic strength of 50 mM or less. Regarding.

[0013] In one embodiment, the concentration of positively charged monovalent ions is 25 mM or less and the concentration of positively charged divalent cationic ions is 20 μM or less.

[0014] In one embodiment of all aspects of the invention, the composition is for intramuscular administration, such as by intramuscular injection.

[0015] In one embodiment of all aspects of the invention, the single-stranded RNA and polyalkyleneimine are present in a polyplex particle.

[0016] In one embodiment of all aspects of the present invention, the polyalkyleneimine has the following general formula (I):

[0017] [ka]

[0018] (In the formula, R is H, an acyl group, or a compound of the following general formula (II):

[0019] [ka]

[0020] is a group comprising wherein R1 is H or a group represented by the following general formula (III):

[0021] [ka]

[0022] is a group comprising n, m, and l are independently selected from integers from 2 to 10; p, q, and r are integers, where the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5×10 2 From 10 7 Da, preferably 5000 to 10 5Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da). Includes.

[0023] In one embodiment, n, m, and l are independently selected from 2, 3, 4, and 5, preferably 2 and 3. In one embodiment, R is H. In one embodiment, R is H or an acyl group.

[0024] In one embodiment of all aspects of the invention, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine.

[0025] In one embodiment of all aspects of the invention, at least 92% of the N atoms in the polyalkyleneimine are protonatable.

[0026] In one embodiment of all aspects of the present invention, the composition of the present invention comprises one or more additives. In one embodiment, the one or more additives are selected from the group consisting of buffer substances, sugars, stabilizers, cryoprotectants, lyoprotectants, and chelating agents. In one embodiment of all aspects of the present invention, the composition of the present invention comprises one or more polymers. In one embodiment, the buffer substance comprises at least one selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), acetate buffer systems and analogs, phosphate buffer systems, or citrate buffer systems. In one embodiment of all aspects of the present invention, the composition of the present invention comprises a buffer for buffering to a pH range of between 4 and 8, preferably between 5 and 7.5. Examples of such buffer systems are acetate buffer, HEPES buffer, phosphate buffer, or acetate buffer. In one embodiment, the sugar is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides, preferably at least one selected from glucose, trehalose, sucrose, and dextran. In one embodiment, the additive is dextran having an average molar mass between 1 kDa and 100 kDa. In one embodiment, the cryoprotectant comprises at least one glycol, such as ethylene glycol, propylene glycol, and glycerol. In one embodiment, the chelating agent comprises EDTA. In one embodiment, the lipid comprises at least one lipid selected from the group consisting of cationic lipids, neutral lipids, and anionic lipids. In one embodiment, the composition of the present invention comprises one or more block copolymers comprising ethylene oxide building blocks and propylene oxide building blocks. In one embodiment, the composition of the present invention comprises a copolymer comprising ethylenediamine groups. In one embodiment, the composition of the present invention comprises an amphiphilic block copolymer, preferably comprising ethylene oxide building blocks and propylene oxide building blocks, and optionally also comprising ethylenediamine groups.

[0027] In one embodiment of all aspects of the invention, the composition comprises HEPES-buffered glucose (HBG or HBGx1), MES-buffered glucose (MBG or MBGx1), acetate-buffered glucose (ABG), or HEPES-buffered trehalose (HBT or HBTx1). In one embodiment of all aspects of the invention, the composition comprises glucose, trehalose, or sucrose in an acetate buffer at a concentration ranging from 0.1 mM to 10 mM. In one embodiment of all aspects of the invention, the composition comprises glucose, trehalose, or sucrose in a phosphate buffer at a concentration ranging from 0.1 mM to 10 mM.

[0028] In one embodiment of all aspects of the invention, the z-average size of the particles is less than 200 nm, preferably less than 150 nm, and more preferably less than 100 nm. In one embodiment, the z-average size of the particles is between 50 nm and 200 nm. In one embodiment of all aspects of the invention, the zeta potential of the particles is 20 mV or greater, preferably 25 to 40 mV. In one embodiment of all aspects of the invention, the electrophoretic mobility (μ) of the particles is between 1 and 1.6 μm*cm / V*S. In one embodiment of all aspects of the invention, the z-average size and / or zeta potential and / or electrophoretic mobility of the particles are determined in a suspension comprising polyplex particles and HEPES-buffered glucose (HBG) or HEPES-buffered trehalose (HBT). In one embodiment, the HBG comprises 5% glucose (w / v) and 10 mM HEPES, pH 7.1, or the HBT comprises 10% trehalose (w / v) and 10 mM HEPES, pH 7.1. In one embodiment, the z-average size of particles is determined by dynamic light scattering and data analysis using a cumulant algorithm. In one embodiment, the translational diffusion coefficient is measured by dynamic light scattering. The Stokes-Einstein equation is then used to calculate the z-average. In one embodiment, the electrophoretic mobility is measured by laser Doppler electrophoresis. The Henry equation or the Smoluchowski equation is then used to calculate the z-average potential.

[0029] In one embodiment of all aspects of the invention, the particles are neutral or positively charged, preferably at physiological pH or at a pH between 4.5 and 7.5.

[0030] In one embodiment of all aspects of the invention, the single-stranded RNA is a molecule of 6,000 to 15,000 bases, preferably 9,000 to 12,000 bases. In one embodiment of all aspects of the invention, the single-stranded RNA encodes at least one protein of interest. In one embodiment of all aspects of the invention, the single-stranded RNA is a replicon, preferably a self-replicating or self-amplifying RNA. In one embodiment, the replicon is capable of replication by a replicase derived from an alphavirus, and the replicon preferably comprises a 5' replication recognition sequence derived from an alphavirus or a variant thereof, and a 3' replication recognition sequence derived from an alphavirus or a variant thereof. In one embodiment of all aspects of the invention, the single-stranded RNA comprises an open reading frame encoding a peptide or protein of interest, such as a pharmaceutically active peptide or protein.

[0031] In one embodiment of all aspects of the invention, the compositions described herein are for use in therapy, hi one embodiment of all aspects of the invention, the compositions described herein are vaccine compositions.

[0032] In a further aspect, the present invention relates to the use of the compositions described herein for introducing RNA into a cell, in particular for expressing RNA in the cell. In one embodiment, the cell is a muscle cell.

[0033] In a further aspect, the present invention relates to the use of the compositions described herein for the intramuscular administration of RNA.

[0034] In a further aspect, the present invention relates to a method for intramuscular administration of RNA comprising the step of intramuscularly administering a composition described herein.

[0035] In a further aspect, the present invention provides a method for producing a composition comprising: (a) single-stranded RNA, and (b) Polyalkyleneimine 1. A frozen, freeze-dried, or spray-dried composition comprising: Compositions containing a cryoprotectant and / or a lyoprotectant, preferably a disaccharide such as trehalose or a polysaccharide such as dextran. Regarding.

[0036] In one embodiment, the composition further comprises a chelating agent, such as EDTA.

[0037] In one embodiment, the composition is prepared from an aqueous composition comprising 5-20% (w / v) disaccharide and, optionally, 20 μM to 10 mM, e.g., 80 μM to 5 mM, of a chelating agent. In one embodiment, the aqueous composition comprises trehalose, HEPES, and EDTA, e.g., 10% trehalose (w / v), 2.8 mM HEPES, 80 μM EDTA, pH 7.1.

[0038] In a further aspect, the present invention relates to an aqueous composition obtainable by thawing a frozen composition described herein or by reconstituting a freeze-dried or spray-dried composition described herein.

[0039] In a further aspect, the present invention provides a method for preparing a frozen, freeze-dried, or spray-dried composition, comprising the steps of: (i) preparing an aqueous composition comprising single-stranded RNA, a polyalkyleneimine, and a cryoprotectant and / or lyoprotectant, preferably a disaccharide such as trehalose or a polysaccharide such as dextran; (ii) freezing, lyophilizing, or spray-drying the composition; A method comprising: Regarding.

[0040] In one embodiment, the aqueous composition further comprises a chelating agent such as EDTA. In one embodiment, the aqueous composition comprises 5-20% (w / v) of a disaccharide, and optionally 20 μM to 10 mM, e.g., 80 μM to 5 mM, of a chelating agent. In one embodiment, the aqueous composition comprises trehalose, HEPES, and EDTA, e.g., 10% trehalose (w / v), 2.8 mM HEPES, 80 μM EDTA, pH 7.1.

[0041] In a further aspect, the present invention provides a method for producing a composition comprising: (a) single-stranded RNA, and (b) Polyalkyleneimine Use of a cryoprotectant and / or lyoprotectant, preferably a disaccharide such as trehalose or a polysaccharide such as dextran, for preparing a frozen, freeze-dried or spray-dried composition comprising Regarding.

[0042] In one embodiment, the disaccharide is used in combination with a chelating agent such as EDTA.

[0043] The frozen, lyophilized, or spray-dried composition, or an aqueous composition for preparing the frozen, lyophilized, or spray-dried composition, may comprise one or more of the following: (i) Non-aqueous solvents, such as ethylene glycol, glycerol, dimethyl sulfoxide, and dimethylformamide. (ii) Surfactants, such as Tween 80, Brij 35, Brij 30, Lubrol-px, Triton X-10; Pluronic F127 (polyoxyethylene-polyoxypropylene copolymer), also known as poloxamer, poloxamine, and sodium dodecyl sulfate. (iii) Disaccharides, such as trehalose, sucrose, lactose, and maltose. (iv) Polymers (which may have different MW), such as polyethylene glycol, dextran, poly(vinyl alcohol), hydroxypropyl methylcellulose, gelatin, polyvinylpyrrolidone, hydroxyethyl cellulose, ficoll, and albumin. (v) Amino acids, such as glycine, proline, 4-hydroxyproline, L-serine, glutamate, alanine, lysine, sarcosine, and gamma-aminobutyric acid.

[0044] In a further aspect, the present invention relates to a method for continuous flow production of RNA polyplex formulations by using a continuous flow pump and a mixing device, wherein two aqueous fluids are mixed through mm or μm sized channels. [Brief explanation of the drawings]

[0045] [Figure 1] A, Toxicity of free pure PEI in HEK-293 cells in vitro. IC50=77 μM nitrogen (free). B, Toxicity of PEI / replicon-RNA polyplexes in HEK-293 cells in vitro. IC50=542 μM nitrogen (polyplex formulation). [Figure 2] FIG. 1 shows the relative luminescence from C2C12 muscle cells after incubation with PEI / replicon-RNA polyplexes with an N / P of 11.6 under different storage conditions after one week of storage. [Figure 3] FIG. 1 shows the relative RNA integrity of PEI / replicon-RNA polyplexes with an N / P of 11.6 under different storage conditions after 2 weeks of storage. [Figure 4] FIG. 1 shows that poly(2-ethyl-2-oxazoline) can be obtained by ring-opening isomerization polymerization of 2-ethyl-2-oxazoline in the presence of an initiator. [Figure 5]Synthesis of fully deacylated linear PEI22, PEI87, and PEI217 by acid hydrolysis of PEOZ. Conditions: (i) 24% (wt / vol) HCl, 110°C, 96 h; n = 504 for 50 kDa PEOZ, 2,018 for 200 kDa PEOZ, and 5,044 for 500 kDa PEOZ. [Figure 6] Figure 1 shows the aggregation kinetics of IVT (A) and replicon (B) polyplexes at increasing salt concentrations. [Figure 7] Figure 1 shows physicochemical parameters of polyplexes before (initial) and after (final) filtration. A and B. Polyplex diameter and polydispersity were measured by DLS. C. RNA was released from polyplexes by heparin and measured by UV absorbance at 260 nm. D. PEI concentration was measured by CuSO assay. [Figure 8] Figure 1 shows a comparison of the chemical structures of highly pure PEI and conventionally pure PEI. For 25 kDa PEI, n = 58. The average number of -CH2CH2NH- monomers in 25 kDa PEI is 581, which is also the length of the contiguous stretch of potentially protonatable nitrogens. In conventional PEI 25, assuming a uniform distribution of N-propionyl moieties, the contiguous stretch of protonatable nitrogens is only 64. [Figure 9] FIG. 1 shows the in vitro transfection of C2C12 muscle cells with replicon-RNA polyplexes prepared at different N / P ratios and with different purity levels of PEI. [Figure 10] Figure 1 shows that replicon-RNA polyplexes with N / P ratios of 1 (-) and 11.6 (+) were prepared using highly pure PEI (jetPEI) and conventionally pure PEI (25 kDa). Free RNA was used as a control. The formulations were injected intramuscularly into the hind limbs of mice (n=3). Luminescence signals from the mouse muscles were recorded. [Figure 11]Figure 1 shows that replicon-RNA polyplexes with N / P ratios of 7.7 and 11.6 were prepared using highly pure PEI, jetPEI (Polyplus), PEI-Max 40000 (Polyscience), and Exgen 500 (Eurodamex). All formulations were prepared in HBGx1 buffer, except for the lyophilized formulation, which was prepared in HBTx1 buffer. The formulations were injected intramuscularly into the hind limbs of mice (n=3). Luminescence signals from the mouse muscles were recorded. [Figure 12] FIG. 1 shows lyophilized cakes of JetPEI / replicon-RNA polyplexes with an N / P of 11.6 prepared using different buffers. [Figure 13] Figure 1 shows C2C12 muscle cells were transfected in vitro with IVT-RNA encoding luciferase. The RNA was complexed with JetPEI at different N / P ratios in HBGx1 buffer. Luminescence signals were measured 24 hours after transfection. [Figure 14] IVT-RNA polyplexes with N / P ratios of 5.8 and 11.6 were prepared in HBGx1 buffer with pure PEI. Free IVT-RNA in HBGx1 buffer was used as a control. The formulations were injected intramuscularly into the hind limbs of mice (n=3) at RNA doses of 2-8 μg per injection. Luminescence signals from the mouse muscles were recorded 6 hours after injection. [Figure 15] Polyplexes of replicon-RNA and jetPEI with an N / P ratio of 11.6 were prepared in HBGx1 buffer at different RNA concentrations. For size measurement by DLS, the polyplexes were diluted to an RNA concentration of 10 mg / L. [Figure 16] Figure 17 shows that C2C12 muscle cells were transfected in vitro with the polyplexes of Figure 16. Luminescence signals were measured 24 hours after transfection. [Figure 17]Similar to Figure 16, this figure shows Rep-RNA polyplexes with an N / P ratio of 11.6 were prepared with pure PEI in HBGx1 buffer at different RNA concentrations. The formulations were injected intramuscularly into the hind limbs of mice (n=3) at RNA doses ranging from 2 to 8 μg per injection. Luminescence signals from the mouse muscles were recorded. [Figure 18] Figure 1 shows in vitro studies using PEI / replicon-RNA polyplexes in human dendritic cells (DCs) and mouse muscle cells (C2C12). A. Toxicity (expressed as % of surviving cells after treatment with polyplexes). B. Transfection (expressed as luminescence emission after treatment with polyplexes). Transfection results are shown only for C2C12 cells. [Figure 19] Rep-RNA polyplexes with an N / P ratio of 11.6 or 15.8 were prepared with PEI from Polyplus or Polytheragene in HBGx1 or Hepes 10 mM buffer. Prior to injection into mice, the polyplexes were diluted with HBGx1 or Opti-MEM buffer. The formulations were injected intramuscularly into the hind limbs of mice (n=3) at an RNA dose of 2 μg per injection. Luminescence signals from the mouse muscles were recorded. [Figure 20] A) 2 μg of unformulated (buffer solution) or formulated replicon-luciferase-encoding RNA was applied intramuscularly (i.m.) to both tibialis posterior muscles of Balb / c mice 4, 7, and 11 days after administration, and the animals were subjected to noninvasive in vivo bioluminescence imaging. Photons from the luciferase protein were collected over a 1-minute period and are shown overlaid with a photograph of the imaged mouse. B) Graphical representation of the measured photons / second (p / s) at the injection site. [Figure 21]A) Seven days after 2 μg of unformulated (buffer solution) or formulated replicon-luciferase-encoding RNA was applied intradermally (id) to two injection sites in the dorsal skin of Balb / c mice, the animals were subjected to noninvasive in vivo bioluminescence imaging. Photons from the luciferase protein were collected over a 1-minute period and are shown overlaid with a photograph of the imaged mouse. The black arrow indicates the injection site. B) Graphical representation of the measured photons / second (p / s) at the injection site. [Figure 22] FIG. 1 shows the beneficial effect of replicon-RNA formulations as vaccines. [Figure 23] FIG. 1 shows the beneficial effect of replicon-RNA formulations as vaccines. [Figure 24] FIG. 1 shows results from spray drying of replicon-RNA formulated with PEI in 10% (w:v) trehalose. [Figure 25] FIG. 1 shows normalized luminescence from C2C12 muscle cells after incubation with PEI / replicon-RNA polyplexes at different N / P ratios before (prepared) and after sterile filtration (post-filtered). [Figure 26] 26A and 26B show the effect of combining short- and long-chain PEI on in vitro transfection efficiency, according to Example 16. Figure 26A: Transfection efficiency of short-chain linear PEI and long-chain PEI polyplexes at 250 ng of RNA per well. Figure 26B: Transfection efficiency of short-chain branched PEI and long-chain PEI polyplexes at 250 ng of RNA per well. Compared to the benchmark, in vivo Jet PEI, and at the same overall NP ratio, higher expression levels were achieved by comparing short-chain PEI (Figure 26A: linear, Figure 26B: branched) with long-chain PEI (e.g., in vivo JetPEI) over different time frames. [Figure 27]Figure 17 shows the replicon-RNA transfection efficacy of long-chain Jet PEI + short-chain PEI polyplexes versus benchmark (i.e., in vivo JetPEI NP12): different combinations (NP4 + NP8 or NP1.15 + NP11) of short-chain PEI (branched, 1.8 kDA) and long-chain PEI when the overall NP is 12, according to Example 17. [Figure 28] Figure 28 shows the effect of salt variation (e.g., NaCl) on in vivo replicon (saRNA)-RNA transfection efficacy, according to Example 18. Bioluminescence signals were detected on days 3 (Figure 28A), 6 (Figure 28B), 9 (Figure 28C), and 13 (Figure 28D). Signal intensities are compared in Figure 28E. The strongest signal in the muscle region of mice could be detected 6 days after intramuscular injection in mice receiving PEI-replicon-RNA polyplexes (e.g., long-chain PEI N / P is 12) and the addition of low concentrations (5 to 10 mM) of salt. [Figure 29] This figure shows the effect of pH adjustment on the transfection efficiency of replicon (saRNA)-PEI formulations according to Example 18. Good results were obtained with saRNA-PEI polyplex formulations with pH values ​​between 6.5 and 7.1. The strongest signal was detected with the saRNA-long-chain PEI in the NP12 formulation, adjusted to pH 6.5. As a benchmark, saRNA-Jet PEI polyplexes in NP12, pH unadjusted (BM) or HBG (20 mM Hepes, pH 7.4, 5% glucose by weight), were used. [Figure 30] FIG. 10 shows the electrophoretic mobility of in vivo jetPEI / replicon-RNA polyplexes (N / P 4) adjusted to different pH values ​​according to Example 19. [Figure 31] FIG. 10 shows normalized luminescence from C2C12 muscle cells after incubation with different doses of in vivo jetPEI / replicon-RNA polyplexes at an N / P ratio of 4 and different pH values ​​(pH 6.5 to pH 8.5) according to Example 19. [Figure 32] FIG. 10 shows luciferase expression after transfection with different amounts of excess positive charges in PEI / PEI formulations according to Example 20. [Figure 33] FIG. 1 shows the optimization of polyplex transfection by using two-step conjugation according to Example 21. [Figure 34] FIG. 2 shows an immunization experiment according to Example 22, demonstrating the superior efficacy of saRNA-polyplexes formulated with MES-buffered glucose (MBG) compared to HEPES-buffered glucose (HBG). [Figure 35] Figure 1 shows that animals develop neutralizing antibody immune responses after intramuscular (i.m.) immunization with PEI-formulated self-amplifying RNA (saRNA) encoding the HA (H1N1 / Cf7-HA) of the A / California / 7 / 2009 (H1N1) virus. (A) BALB / c mice were immunized once on day 0 with buffer, a 1 / 25 dose of human vaccine, or 0.1 μg of PEI-formulated VEEV-saRNA or SFV-saRNA encoding H1N1 / Cf7-HA at a 12 / 1 N / P ratio. 28 and 48 days later, animals were bled, and serum was analyzed for antibodies against HA as measured by a virus neutralization assay (VNT; n=4). (B) Domestic piglets were immunized once on day 0 with buffer, one dose of human vaccine, or 90 μg of PEI-formulated VEEV-saRNA or SFV-saRNA encoding H1N1 / Cf7-HA at a 12 / 1 N / P ratio. On days 14, 21, 28, and 35 after immunization, pigs were bled and VNT (n=8; buffer group n=4) was performed to analyze neutralizing antibody immune responses to HA. The group of animals receiving the formulated VEEV-saRNA vaccine developed an immune response similar to that of animals injected with the positive control. SFV-saRNA also resulted in the development of neutralizing antibody immune responses, but at lower titers than after VEEV-saRNA immunization. The mean ± SEM is shown on the graph. [Figure 36]This figure shows that animals developed antibody immune responses after intramuscular (i.m.) immunization with PEI-formulated self-amplifying RNA (saRNA) encoding the porcine circovirus 2 (PCV2)-cap_EU protein. BALB / c mice were immunized twice on days 0 and 35 with buffer, 1 μg of PEI-formulated SFV-saRNA or VEEV-saRNA encoding PCV2-cap_EU at an N / P ratio of 12 / 1. On days 14, 34, and 56, animals were bled, and serum was analyzed for antibodies against PCV2-cap as determined by a commercially available ELISA assay (INgezim Circo IgG, Ingenasa; n = 4). Groups of animals receiving the formulated SFV-saRNA vaccine or the formulated VEEV-saRNA vaccine developed similar antibody responses against the PCV2-cap_EU protein. The antibody immune response after a single vaccination with SFV-saRNA was slightly higher than that with VEEV-saRNA. After two immunizations, antibody responses were nearly identical for both types of saRNA vaccines. Means ± SEM are shown on the graph. [Figure 37]This figure shows the complexation of replicon-RNA (saRNA) with N / P12 in different buffer systems and pH conditions. Both acetate and MES buffers contained a final concentration of 10 mM buffer and 5% w / v D-glucose. saRNA / PEI-polyplexes were stored at 4°C in each buffer for different periods (1, 2, 4, and 8 days after complexation). RNA integrity was measured directly (t=0) upon complexation of the different formulations. RNA integrity was measured by capillary electrophoresis. saRNA complexed in polyplexes can be released after 20 min of incubation at RT with a large excess of polyanions, which induces electrostatic interactions with the polymer, releasing the RNA encapsulated in the polyplexes. 200 μg of released RNA was used strictly following the protocol provided with the appropriate kit for capillary electrophoresis assays (Standard Sensitivity RNA Analysis Kit DF471). For each time point, a reference RNA was used to quantify saRNA integrity. Higher pH values ​​in the formulation buffer resulted in significantly increased degradation of saRNA. The least loss of saRNA integrity upon complexation was achieved at pH 4 using acetate buffer. [Figure 38]This figure shows that PEI-formulated saRNA-VEEV encoding A / California / 7 / 2009 HA (H1N1; Cf7 / HA) induces a stronger and longer-lasting antibody response compared to a commercial vaccine, but also induces a strong T cell response that protein-based vaccines lack. BALB / c mice were immunized intramuscularly twice on days 0 and 35 (indicated by arrows in the graph) with either buffer (black symbols), 20 μL of a licensed human vaccine against a seasonal influenza virus strain (Begripal 2016 / 2017; hLIC; gray symbols), or 0.5 μg of a PEI-formulated VEEV-saRNA-based vaccine encoding Cf7 / HA (dark gray symbols). At different time points, mice were sacrificed, and A) splenocytes were collected and single-cell suspensions were used to perform Cf7 / HA-specific ELISpot assays. For ELISpot analysis, different CF7 / HA-specific peptide pools were used to stimulate CD8+ T cell (left) or CD4+ T cell (right) responses as measured by IFNγ secretion. In addition, serum samples were collected and anti-Cf4 / HA-specific virus neutralization assays were performed on serum antibodies B) for their ability to inhibit viral cell infection. Note that for serological analysis, A / California / 4 / 2009 (H1N1; Cf4) virus was utilized, and data represent the mean ± SEM (n = 3 for buffer group, n = 4 for vaccine group). DETAILED DESCRIPTION OF THE INVENTION

[0046] Although the present invention is described in detail below, it should be understood that the present invention is not limited to the particular methodology, protocols, and reagents described herein, as the methodology, protocols, and reagents may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0047] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", edited by H.G.W. Leuenberger, B. Nagel, and H. Koelbl, Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0048] The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd ed., J. Sambrook et al., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

[0049] The elements of the present invention are described below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create further embodiments. The various described examples and preferred embodiments should not be construed as limiting the invention to only the explicitly described embodiments. The specification should be understood to disclose and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, unless otherwise indicated by context, any permutation and combination of all elements described in this application should be considered to be disclosed by the specification.

[0050] The term "about" means approximately or near, and in the context of numerical values ​​or ranges set forth herein, preferably means + / - 10% of the recited or claimed numerical value or range.

[0051] As used in the context of describing the present invention (particularly in the context of the claims), the terms "a," "an," and "the," and similar references, should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is intended merely to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples, or the use of exemplary language (e.g., "etc."), provided herein are intended merely to better illustrate the invention and do not pose a limitation on the scope of the invention as otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.

[0052] Unless expressly indicated otherwise, the term "comprises" is used in the context of this document to indicate that in addition to the members of the list preceding "comprises," further members may optionally be present. However, for certain embodiments of the present invention, it is intended that the term "comprises" encompasses the possibility that no further members are present; that is, in this embodiment, "comprises" should be understood to have the meaning of "consisting of."

[0053] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate such disclosure.

[0054] The following definitions apply to all aspects of the present invention.

[0055] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, preferably by 5% or more, 10% or more, 20% or more, more preferably by 50% or more, and most preferably by 75% or more. The term "inhibit" or similar expressions includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.

[0056] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%.

[0057] A "fragment" in reference to a nucleic acid sequence refers to a portion of the nucleic acid sequence, i.e., a sequence representing a nucleic acid sequence truncated at the 5' and / or 3' end. Preferably, a fragment of a nucleic acid sequence comprises at least 80%, preferably at least 90%, 95%, 96%, 97%, 98%, or 99% of the nucleotide residues from said nucleic acid sequence. In the present invention, fragments of RNA molecules that retain RNA stability and / or translation efficiency are preferred.

[0058] A "fragment" with respect to an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A fragment truncated at the C-terminus (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3' end of the open reading frame. A fragment truncated at the N-terminus (C-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence contains, for example, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues derived from the amino acid sequence.

[0059] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Ionic strength, I, is therefore mathematically represented by the following equation:

[0060]

number

[0061] where c is the molar concentration of a particular ionic species and z is the absolute value of its charge. The sum Σ is calculated over all the different types of ions (i) in the solution.

[0062] Preferably, the ionic strength of the compositions described herein is 50 mM or less, preferably 25 mM or less, preferably 20 mM or less, 19 mM or less, 18 mM or less, 17 mM or less, 16 mM or less, 15 mM or less, 10 mM or less, or 5 mM or less. Preferably, the ionic strength of the compositions described herein is sufficiently low to prevent aggregation of the polyplex particles.

[0063] According to the present invention, the term "ionic strength" preferably relates to the presence of monovalent ions. When referring to the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of a chelating agent is preferably sufficiently low to prevent RNA degradation. In a particularly preferred embodiment, the concentration or effective concentration of divalent ions is below the catalytic level for the hydrolysis of phosphodiester bonds between RNA nucleotides. In a particularly preferred embodiment, the concentration of free divalent ions is 20 μM or less, and preferably, no or essentially no free divalent ions are present.

[0064] The pH of the compositions described herein is preferably between 4 and 8, more preferably between 5.5 and 8, such as between 6 and 7.5, such as between 6.5 and 7.1, between 6.5 and 7, or between 6.5 and 6.9.

[0065] The term "disaccharide" refers to a carbohydrate composed of two monosaccharide residues linked by a glycosidic bond. Representative examples of disaccharides include trehalose, maltose, sucrose, lactose, lactulose, cellobiose, isomaltose, gentiobiose, laminarin disaccharide (laminarabiosu), chitobiose, xylobiose (xylobiose), inulin disaccharide, and mannobiose sugar. The preferred content of disaccharides in the compositions described herein is 5 to 20% (w / v), such as 5 to 15% (w / v), 7 to 15% (w / v), or 8 to 12% (w / v). According to the present invention, disaccharides with high glass transition temperatures are preferred.

[0066] The term "chelating agent" refers to a compound that forms a chelate with a metal ion, preferably a divalent or polyvalent metal ion. Chelating agents have multiple groups, such as -OH and -COOH, that can form a ring structure with a metal ion. Examples of chelating agents are ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), trans-1,2-diamino-cyclohexanetetraacetic acid monohydrate, N-hydroxyethylethylenediaminetriacetic acid (HEDTA), citric acid, and phosphate chelating agents (e.g., Dequest 2000). According to the present invention, ethylenediaminetetraacetic acid (EDTA) is preferred. The chelating agent is preferably present in the compositions described herein at a concentration of at least 20 μM, at least 40 μM, at least 60 μM, or at least 80 μM. Preferably, the chelating agent is present in the compositions described herein at a concentration of up to 10 mM, up to 5 mM, up to 2 mM, up to 1 mM, up to 0.5 mM, up to 0.2 mM, or up to 0.1 mM.

[0067] The term "freezing" refers to the solidification of a liquid, usually with the removal of heat.

[0068] The term "lyophilize" or "freeze-drying" refers to the lyophilization of a substance by freezing the substance and then reducing the surrounding pressure to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase.

[0069] The term "spray drying" refers to the spray drying of a substance by mixing a (heated) gas with an atomized (atomized) fluid in a vessel (spray dryer) to evaporate the solvent from the formed droplets, resulting in a dry powder.

[0070] The term "cryoprotectant" refers to a substance added to a formulation to protect the active ingredient during the freezing step.

[0071] The term "lyoprotectant" refers to a substance added to a formulation to protect the active ingredient during the drying step.

[0072] The term "reconstitute" refers to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as the product's original liquid state.

[0073] The term "autologous" is used to describe anything derived from the same subject. For example, "autologous cells" refer to cells derived from the same subject. Introduction of autologous cells into a subject is advantageous because they overcome immunological barriers that would otherwise result in rejection.

[0074] The term "allogeneic" is used to describe anything derived from different individuals of the same species. Two or more individuals are said to be allogeneic to each other if the genes at one or more loci are not identical.

[0075] The term "syngeneic" is used to describe individuals or tissues that have the same genotype, i.e., identical twins or inbred animals, or anything derived from their tissues or cells.

[0076] The term "xenogeneic" is used to describe something that is made up of multiple dissimilar elements. For example, introducing cells from one individual into a different individual constitutes a xenotransplant. A xenogene is a gene that originates from a source other than the subject.

[0077] According to the present invention, due to the instability of unprotected RNA, it is advantageous to provide the RNA molecule in the form of a complex. In particular, in some embodiments, the composition of the present invention comprises particles comprising RNA and a polyalkyleneimine.

[0078] When the system according to the present invention is formulated as a particulate formulation, each RNA species (e.g., replicon, replicase construct, and optional additional RNA species, such as RNA encoding a protein suitable for inhibiting IFN) can be formulated separately as an individual particulate formulation. In this case, each individual particulate formulation will contain one RNA species. The individual particulate formulations may be present as separate entities, for example in separate containers. Such a formulation can be obtained by providing each RNA species separately (typically each in the form of an RNA-containing solution) together with a particle-forming agent, thereby forming particles. Each particle exclusively contains the specific RNA species provided at the time the particle is formed (individual particulate formulation).

[0079] In one embodiment, the composition according to the present invention comprises two or more individual particle formulations. Each composition is referred to as a mixed particle formulation. The mixed particle formulation according to the present invention can be obtained by separately forming the individual particle formulations as described above and then mixing these individual particle formulations. The mixing step results in a formulation comprising a mixed population of RNA-containing particles (for illustration, for example, a first population of particles may contain a replicon, and a second particle formulation may contain a replicase construct). The individual particle populations may be combined in one container to comprise a mixed population of the individual particle formulations.

[0080] Alternatively, all RNA species of the composition (e.g., replicon, replicase construct, and optional additional species, such as RNA encoding a protein suitable for inhibiting IFN) can be formulated together as a complex particulate formulation. Such a formulation can be obtained by providing a complex formulation (typically a complex solution) of all RNA species together with a particle-forming agent, thereby forming particles. Unlike mixed particulate formulations, complex particulate formulations will typically contain particles containing two or more RNA species. In complex particulate compositions, different RNA species are typically present together within a single particle.

[0081] In one embodiment, the particulate formulation of the invention is a nanoparticulate formulation. In this embodiment, the composition according to the invention comprises RNA in the form of nanoparticles.

[0082] In a general definition, the term "nanoparticle" refers to any particle having a diameter between 1 nm and 1000 nanometers (nm).

[0083] In the context of the present invention, the term "particle" relates to a structured entity formed by molecules or molecular complexes. In one embodiment, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure.

[0084] "In vivo-jetPEITM", "in vivo jetPEITM", "in vivo jetPEI", "jetPEI", "jet PEI", and "JetPEI" all refer to the commercially available In vivo-jetPEITM reagent, catalog number 201-50G, manufactured by Polyplus-Transfection SA (Illkirch, France).

[0085] As used herein, the term "polyplex" refers to a complex of a polymer and a nucleic acid, such as RNA, formed through electrostatic interactions. When a polyplex contains RNA, it may also be called an "RNA complex" or an "RNA polyplex."

[0086] The present invention relates to polyplex particles formed from at least one type of single-stranded RNA and at least one type of polyalkyleneimine.

[0087] In one embodiment, the particles described herein have an average diameter of less than about 200 nm, preferably less than about 150 nm, and more preferably less than about 100 nm, hi one embodiment, the particles described herein have an average diameter of at least about 30 nm, at least about 40 nm, at least about 50 nm, at least about 60 nm, at least about 70 nm, at least about 80 nm, at least about 90 nm, or at least about 100 nm.

[0088] The term "mean diameter" refers to the average hydrodynamic diameter of the particles as measured by dynamic light scattering with data analysis using the so-called cumulant algorithm, which provides a so-called Z average , and the dimensionless polydispersity index (PI) results (Koppel, D., J. Chem. Phys., Vol. 57, 1972, pp. 4814-4820, ISO 13321). Herein, the "average diameter", "diameter", or "size" of particles refers to this Z average Used synonymously with the value of

[0089] The term "net charge" relates to the total amount of charges, such as positive and negative charges. For example, if a particle contains more negative charges than positive charges, the net charge of the particle is negative. If a particle contains more positive charges than negative charges, the net charge of the particle is positive. If a particle contains an equal number of positive and negative charges, the net charge of the particle is neutral, in particular electrically neutral. Thus, the net charge of the particles according to the present invention may be negative, positive, or neutral. In one embodiment, the net charge of the particles is positive. In one embodiment, the net charge of the particles is negative.

[0090] Terms such as "charged," "net charge," "negatively charged," or "positively charged" refer to the net electrical charge of a given compound or particle when dissolved or suspended in an aqueous buffer at a relevant pH (e.g., 7.1).

[0091] According to the present invention, "N / P ratio", "NP ratio", "N:P ratio", "N / P", and "NP" refer to the molar ratio of nitrogen atoms (N) in polyethyleneimine to phosphorus atoms (P) in RNA.

[0092] According to the present invention, the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the RNA (N / P ratio) is preferably 2.0 to 15.0, preferably 8.0 to 12.0, 6.0 to 14.0, or 6.0 to 12.0.

[0093] According to the present invention, the compositions described herein are preferably adjusted to a final N / P ratio in two or more steps, such as two, three, four, or more steps. For example, the composition may be adjusted in a first step to a first N / P ratio lower than the final N / P ratio, for example, using a long-chain polyalkyleneimine. The N / P ratio may be adjusted to the final N / P ratio by adding additional polyalkyleneimine, such as a short-chain polyalkyleneimine or a long-chain polyalkyleneimine, such as the long-chain polyalkyleneimine used in the first step. In one embodiment, the final N / P ratio is between 8 and 16, for example, between 9 and 14, for example, between 10 and 12. In one embodiment, the N / P ratio resulting from the first step is between 1 and 6, for example, between 2 and 5, such as 3 or 4.

[0094] Polyalkyleneimine As used herein, the polyalkyleneimine preferably has the following general formula (I):

[0095] [ka]

[0096] (In the formula, R is H, an acyl group, or a compound of the following general formula (II):

[0097] [ka]

[0098] is a group comprising wherein R1 is H or a group represented by the following general formula (III):

[0099] [ka]

[0100] is a group comprising n, m, and l are independently selected from integers from 2 to 10; p, q, and r are integers, where the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5×10 2 From 10 7 Da, preferably 5000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da). Includes.

[0101] In one embodiment, n, m, and l are independently selected from 2, 3, 4, and 5, preferably 2 and 3, more preferably 2. In one embodiment, R is H. In one embodiment, R is H or an acyl group.

[0102] In one embodiment, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 1.5×10 2 From 10 7 Da, preferably 5000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0103] According to the present invention, linear polyalkyleneimines, such as linear polyethyleneimine (PEI), are preferred. In one embodiment, linear PEI is obtained by ring-opening isomerization polymerization of 2-ethyl-2-oxazoline to obtain poly(2-ethyl-2-oxazoline) (PEOX, N-propionyl-PEI), which is then acid-hydrolyzed to cleave off the N-propionyl groups to produce PEI.

[0104] According to the present invention, linear PEI is preferably obtained by complete or essentially complete deacylation of PEOX. For example, linear PEI having a molecular weight of 22 kDa can be obtained from PEOX having a molecular weight of 50 kDa. It is preferred that at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or essentially 100% of the substituents on the nitrogen atoms in the polyalkyleneimine, such as polyethyleneimine, are hydrogen (i.e., R in the above formula is H). Therefore, it is preferred that at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or essentially 100% of the nitrogen atoms in the polyalkyleneimine, such as polyethyleneimine, are protonatable.

[0105] According to the present invention, a preferred polyalkyleneimine is polyethyleneimine (PEI), in particular linear polyethyleneimine. Such linear polyethyleneimine preferably has a molar mass between 15 kDa and 30 kDa and is preferably used in combination with a self-replicating or self-amplifying RNA, the N / P ratio preferably being between 6 and 15, and the linear polyethyleneimine and the self-replicating or self-amplifying RNA preferably being present in polyplex particles having a size of less than 200 nm, preferably less than 150 nm, even more preferably less than 100 nm.

[0106] In one embodiment of the present invention, the polyalkyleneimine is a combination of a short-chain polyalkyleneimine, such as a short-chain polyethyleneimine (linear and / or branched) between 0.6 and 11 kDa, preferably between 1 and 6 kDa or between 1 and 4 kDa, such as between 1 and 3 kDa, and a long-chain polyalkyleneimine, such as a long-chain polyethyleneimine (linear and / or branched) between 20 and 40 kDa, wherein the overall N / P ratio is preferably between 8 and 16, such as between 9 and 14, such as between 10 and 12. In one embodiment, the N / P ratio between the long-chain polyalkyleneimine and the RNA is between 1 and 6, such as between 2 and 5, such as 3 or 4.

[0107] Polyethyleneimine (PEI) is an organic polymer with a high cationic charge density that can compact nucleic acids into positively charged particles that can interact with anionic proteoglycans on the cell surface and facilitate particle entry by endocytosis.

[0108] There are several manufacturing methods for PEI. According to the present invention, linear polyethyleneimine is synthesized and prepared from a predetermined amount of monomer, 2-ethyl-2-oxazoline, preferably with a purity of more than 99%, by a method comprising the steps of sufficiently drying said amount of monomer and polymerizing said amount of monomer to obtain poly(2-ethyl-2-oxazoline) (PEOX) as follows:

[0109] - After a predetermined amount of acetonitrile has been sufficiently dried, said acetonitrile is used as a solvent for said amount of dried monomer, while adding a predetermined amount of a sufficiently dried initiator for the polymerization reaction and mixing them together; - purifying the PEOX obtained by evaporation to remove the solvent while carrying out at least three successive washing / precipitation steps with methanol and diethyl ether and corresponding filtrations; (The operations of drying, polymerization, and purification are carried out by: (i) 1Accurate identification of the PEOX polymer by performing an 1H NMR test, confirmation of the absence of monomers reaching levels below 1.0%, and confirmation of the absence of solvents reaching levels below 5.0%, and (ii) arranging so that the average molecular weight (Mw) of the PEOX exceeds 23,000 Da and the polydispersity is less than 1.5 by performing gel permeation chromatography) - hydrolyzing the PEOX with hydrochloric acid to 1 A step of sufficiently efficiently obtaining the PEI having a residual side chain or propionic acid in an amount less than 5% by performing an 1H-NMR test and identifying that this PEI is a single peak.

[0110] It should be understood that sufficiently drying a specific amount of monomer, acetonitrile, or initiator means obtaining a reduction in humidity lower than 10 ppm immediately before use, which can be obtained by drying over calcium hydride for 48 hours followed by distillation and collection of the monomer at a temperature higher than 129 °C.

[0111] According to the present invention, one or more of the following features are preferred: (i) The average molecular weight (Mw) of the PEOX is, for example, 40,000 Da < Mw < 60, Da, etc., (ii) The monomer / initiator ratio is about 500 (it should be understood that "about" means ±5%), (iii) The monomer / initiator ratio is 480, (iv) The monomer has a purity exceeding 99.95%, (v) The initiator is mixed with acetonitrile before being added to the monomer, (vi) The polymerization is carried out at a temperature exceeding 85 °C for a period exceeding 20 hours, (vii) The polymerization temperature is 105 °C or higher, (viii) After the initial filtration, the residue is washed thoroughly with a solvent such as MeOH, and after adding diethyl ether, poly(2-ethyl-2-oxazoline) naturally separates from the solution as an oil. The whole solvent is decanted, and after repeating the said washing and separation at least 4 times, it is dried in vacuo. (ix) The hydrolysis step 1 includes removing the discharged propionic acid obtained by azeotropic distillation from the reaction mixture periodically and for at least one day while monitoring the reaction process by 1H-NMR spectroscopy. (x) Dilute the residue obtained at the end of the reaction process with water, evaporate it at least 3 times to remove trace amounts of propionic acid, then dissolve the residue in water again and filter it before freeze-drying. (xi) The filtration is carried out by a sterile membrane having a mesh diameter between 0.20 μm and 0.25 μm, particularly a sterile cellulose acetate membrane.

[0112] Advantageously, the linear PEI for use according to the invention is characterized in that the intermediate PEOX has a molecular weight Mw, for example, 40,000 < Mw < 60,000 Da.

[0113] The degree of polymerization is controlled by the monomer / initiator ratio and the yield of the synthesis. The determination of the molecular weight can be carried out by gel permeation chromatography (GPC).

[0114] According to the present invention, the term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and locked nucleic acid (LNA). According to the present invention, nucleic acid includes genomic DNA, cDNA, mRNA, viral RNA, recombinantly prepared molecules, and chemically synthesized molecules. According to the present invention, nucleic acid may be in single-stranded or double-stranded form, and may be a linear molecule or a covalently closed circular molecule. Also according to the present invention, the term "nucleic acid" includes nucleic acids chemically derivatized at the nucleotide base, sugar, or phosphate, as well as nucleic acids containing non-natural nucleotides and nucleotide analogs. The nucleic acids described may be isolated and / or recombinant nucleic acids.

[0115] As used herein, the term "isolated" is intended to refer to a molecule that is substantially free of other molecules, such as other cellular material. According to the present invention, the term "isolated nucleic acid" means that the nucleic acid has been (i) amplified in vitro, for example, by polymerase chain reaction (PCR), (ii) recombinantly produced by cloning, (iii) purified, for example, by cleavage and gel electrophoretic fractionation, or (iv) synthesized, for example, by chemical synthesis. An isolated nucleic acid is a nucleic acid that is available for manipulation by recombinant techniques.

[0116] In the context of the present invention, the term "recombinant" means "produced by genetic engineering." Preferably, in the context of the present invention, a "recombinant" is not naturally occurring.

[0117] As used herein, the term "naturally occurring" refers to the fact that something can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a source in nature, and has not been intentionally modified by humans in the laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0118] According to the present invention, a "nucleic acid sequence" refers to the sequence of nucleotides in a nucleic acid, such as ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). The term may refer to an entire nucleic acid molecule (e.g., a single strand of an entire nucleic acid molecule, etc.) or to a portion thereof (e.g., a fragment).

[0119] According to the present invention, the "3' end of a nucleic acid" refers to the end having a free hydroxyl group. In a diagrammatic representation of a double-stranded nucleic acid, particularly DNA, the 3' end is always on the right side. According to the present invention, the "5' end of a nucleic acid" refers to the end having a free phosphate group. In a diagrammatic representation of a double-stranded nucleic acid, particularly DNA, the 5' end is always on the left side. 5' end 5'--P-NNNNNNN-OH-3' 3' end 3'-HO-NNNNNNN-P--5'

[0120] "Upstream" describes the relative location of a first element of a nucleic acid molecule with respect to a second element of the nucleic acid molecule, where both elements are contained within the same nucleic acid molecule and the first element is located closer to the 5' end of the nucleic acid molecule than the second element. The second element is then said to be "downstream" of the first element of the nucleic acid molecule. An element located "upstream" of a second element can be synonymously referred to as being located "5'" of the second element. With respect to double-stranded nucleic acid molecules, designations such as "upstream" and "downstream" are given with respect to the (+) strand.

[0121] According to the present invention, the term "gene" refers to a specific nucleic acid sequence that is responsible for the production of one or more cellular products and / or the performance of one or more inter- or intracellular functions. More specifically, the term relates to a nucleic acid section (DNA or RNA) that comprises a nucleic acid that codes for a specific protein or a functional or structural RNA molecule.

[0122] The term "vector" is used herein in its most general sense and includes, for example, any vehicle for a nucleic acid that allows said nucleic acid to be introduced into a prokaryotic and / or eukaryotic host cell and, where appropriate, integrated into the genome. Such vectors are preferably replicated and / or expressed intracellularly. Vectors include plasmids, phagemids, viral genomes, and fragments thereof.

[0123] In the context of the present invention, the term "RNA" refers to a molecule that contains ribonucleotide residues, preferably composed entirely or substantially of ribonucleotide residues, and includes all RNA types described herein. The term "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. The term "RNA" includes double-stranded RNA, single-stranded RNA, isolated RNA, e.g., partially or completely purified RNA, essentially pure RNA, synthetic RNA, and recombinantly produced RNA, such as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications can include the addition of non-nucleotide material, for example, to the ends of the RNA or internally, e.g., at one or more nucleotides of the RNA. Nucleotides within an RNA molecule can also include non-standard nucleotides, e.g., non-naturally occurring nucleotides, or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs are sometimes referred to as analogs, particularly analogs of naturally occurring RNA. The RNA used according to the present invention can have a known composition, or the composition of the RNA can be partially or completely unknown.

[0124] The term "stability" of RNA relates to the "half-life" of RNA. "Half-life" relates to the period required to eliminate half of the activity, amount, or number of a molecule. In the context of the present invention, the half-life of RNA indicates the stability of said RNA. The half-life of RNA may affect the "duration of expression" of RNA. It can be expected that RNA with a long half-life will be expressed for a long period of time.

[0125] The term "translation efficiency" relates to the amount of translation product produced by an RNA molecule within a specific period of time.

[0126] According to the present invention, "double-stranded RNA" or "dsRNA" means RNA having two partially or completely complementary strands.

[0127] According to the present invention, the RNA is preferably single-stranded RNA (ssRNA). The term "single-stranded RNA" generally refers to an RNA molecule that is not bound to a complementary nucleic acid molecule (typically, a complementary RNA molecule). Single-stranded RNA may contain self-complementary sequences, where a portion of the RNA may fold back to form secondary structural motifs such as, but not limited to, base pairs, stems, stem-loops, and bulges. Single-stranded RNA may exist as a minus strand [(-) strand] or a plus strand [(+) strand]. The (+) strand is the strand that contains or encodes genetic information. Genetic information may be, for example, a polynucleotide sequence encoding a protein. When the (+) strand RNA encodes a protein, the (+) strand can directly serve as a template for translation (protein synthesis). The (-) strand is the complement of the (+) strand. In the case of double-stranded RNA, the (+) strand and the (-) strand are two separate RNA molecules, and both of these RNA molecules bind to each other to form double-stranded RNA ("duplex RNA").

[0128] Particularly preferred single-stranded RNAs according to the present invention are mRNA and replicon RNA, such as self-replicating RNA. According to the present invention, the RNA may be coding RNA, i.e., RNA that encodes a peptide or protein. Preferably, the RNA is a pharmaceutically active RNA.

[0129] A "pharmaceutically active RNA" is an RNA that encodes a pharmaceutically active peptide or protein, such as an antigen, or an immunologically active compound (not encoding an antigen), or that is itself pharmaceutically active and has one or more pharmaceutical activities, such as those activities described for a pharmaceutically active protein.

[0130] According to the present invention, the term "RNA encoding a peptide or protein" means that the RNA, when present in the appropriate environment, preferably within a cell, directs a collection of amino acids to produce a peptide or protein during translation. Preferably, the coding RNA according to the present invention is capable of interacting with the cellular translation machinery to translate the coding RNA to produce the peptide or protein.

[0131] According to the present invention, the term "mRNA" means "messenger-RNA" and relates to a transcription product that is typically produced by using a DNA template and encodes a peptide or protein. Typically, mRNA comprises a 5'-UTR, a protein-coding region, a 3'-UTR, and a poly(A) sequence. mRNA is produced by in vitro transcription from a DNA template. In vitro transcription methods are known to those skilled in the art. For example, various in vitro transcription kits are commercially available. According to the present invention, mRNA can be modified by stabilizing modifications and capping.

[0132] The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) may be located 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR).

[0133] When present, a 3'-UTR is located at the 3' end of a gene, downstream of the stop codon of the protein-coding region; however, the term "3'-UTR" preferably does not include the poly(A) tail. Thus, a 3'-UTR is upstream of the poly(A) tail, e.g., directly adjacent to the poly(A) tail (if present). When present, a 5'-UTR is located at the 5' end of a gene, upstream of the start codon of the protein-coding region. A 5'-UTR is downstream of the 5'-cap, e.g., directly adjacent to the 5'-cap (if present). According to the present invention, 5'- and / or 3'-untranslated regions may be operably linked to an open reading frame such that these regions are associated with the open reading frame in such a way as to increase the stability and / or translation efficiency of an RNA comprising the open reading frame.

[0134] According to the present invention, the term "poly(A) sequence" or "poly(A) tail" refers to a continuous or discontinuous sequence of adenylate residues, typically located at the 3' end of an RNA molecule. A continuous sequence is characterized by consecutive adenylate residues. Continuous poly(A) sequences are typical in nature. Poly(A) sequences are not usually encoded by eukaryotic DNA, but are attached to the free 3' end of RNA by post-transcriptional template-independent RNA polymerase during eukaryotic transcription in the cell nucleus, and the present invention encompasses poly(A) sequences encoded by DNA.

[0135] Terms such as "5'-cap," "cap," "5'-cap structure," or "cap structure" are used interchangeably to refer to the dinucleotide found at the 5' end of some eukaryotic primary transcripts, such as messenger RNA precursors. A 5'-cap is a structure in which an (optionally modified) guanosine is attached to the first nucleotide of an mRNA molecule via a 5'-to-5' triphosphate linkage (or, in the case of certain cap analogs, a modified triphosphate linkage). These terms can also refer to a traditional cap or a cap analog.

[0136] RNA molecules according to the invention can be characterized by matching 5'-cap, 5'-UTR, 3'-UTR, poly(A) sequence, and / or codon usage.

[0137] RNA molecules for use according to the invention preferably have a size of more than 2000 bases, preferably more than 3000 bases, 4000 bases, 5000 bases, 6000 bases, 7000 bases, 8000 bases, 9000 bases, or more than 10000 bases. RNA molecules for use according to the invention preferably have a size of between 6000 and 20000 bases, preferably between 6000 and 15000 bases, preferably between 9000 and 12000 bases.

[0138] According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and / or protein. This term also includes partial expression of a nucleic acid. Furthermore, expression may be transient or stable. With respect to RNA, the terms "expression" or "translation" refer to the process in the ribosomes of a cell by which a chain of coding RNA (e.g., messenger RNA) directs the assembly of a sequence of amino acids to produce a peptide or protein.

[0139] The terms "transcription" and "transcribe" refer to the process in which a nucleic acid molecule ("nucleic acid template") having a specific nucleic acid sequence is read by an RNA polymerase, resulting in the production of a single-stranded RNA molecule by the RNA polymerase. During transcription, the genetic information in the nucleic acid template is transcribed. The nucleic acid template may be DNA, but, for example, in the case of transcription from an alphavirus nucleic acid template, the template is typically RNA. The transcribed RNA can then be translated into a protein. According to the present invention, the term "transcription" includes "in vitro transcription," which refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system. Preferably, a cloning vector is applied to generate the transcript. These cloning vectors are commonly called transcription vectors and, according to the present invention, are encompassed by the term "vector." The cloning vector is preferably a plasmid. According to the present invention, the RNA is preferably in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0140] The single-stranded nucleic acid molecule produced during transcription typically has a nucleic acid sequence that is the complementary sequence of the template.

[0141] According to the present invention, the term "template" or "nucleic acid template" or "template nucleic acid" generally refers to a nucleic acid sequence that can be replicated or transcribed.

[0142] The term "expression control sequence," according to the present invention, includes promoters, ribosomal binding sequences, and other control elements that control the transcription of a gene or the translation of the resulting RNA. In certain embodiments of the present invention, the expression control sequence can be regulated. The exact structure of an expression control sequence can vary depending on the species or cell type, but typically includes a 5'-nontranscribed sequence and 5'- and 3'-nontranslated sequences, which are involved in the initiation of transcription and translation, respectively. More specifically, a 5'-nontranscribed expression control sequence includes a promoter region encompassing a promoter sequence for transcriptional control of an operably linked gene. An expression control sequence may also include an enhancer sequence or upstream activator sequence. An expression control sequence of a DNA molecule typically includes a 5'-nontranscribed sequence and 5'- and 3'-nontranslated sequences, such as a TATA box, capping sequence, CAAT sequence, etc. An expression control sequence of an alphavirus RNA may include a subgenomic promoter and / or one or more conserved sequence elements. A specific expression control sequence according to the present invention is an alphavirus subgenomic promoter, as described herein.

[0143] The term "promoter" or "promoter region" refers to a nucleic acid sequence that controls the synthesis of a transcript, e.g., a transcript containing a coding sequence, by providing recognition and binding sites for RNA polymerase. A promoter region may contain additional recognition or binding sites for additional factors involved in regulating the transcription of the gene. A promoter can control the transcription of a prokaryotic or eukaryotic gene. A promoter may be "inducible," initiating transcription in response to an inducer, or "constitutive," in which transcription is not controlled by an inducer. Inducible promoters are expressed only to a very small extent or not at all in the absence of an inducer. In the presence of an inducer, the gene is "switched on," or the level of transcription increases, usually mediated by the binding of specific transcription factors. Particular promoters according to the present invention are alphavirus subgenomic promoters, as described herein. Other particular promoters are alphavirus genomic plus- or minus-strand promoters.

[0144] "Core promoter" refers to a nucleic acid sequence contained in a promoter. A core promoter is typically the minimal portion of a promoter required to properly initiate transcription. A core promoter typically contains a transcription initiation site and an RNA polymerase binding site.

[0145] The nucleic acid sequences specified herein, in particular transcribable coding nucleic acid sequences, can be combined with any expression control sequence, which may be homologous or heterologous to said nucleic acid sequence. The term "homologous" refers to the fact that the nucleic acid sequence is also naturally operably linked to an expression control sequence, and the term "heterologous" refers to the fact that the nucleic acid sequence is not naturally operably linked to an expression control sequence.

[0146] A nucleic acid sequence, particularly a nucleic acid sequence encoding a peptide or protein, and an expression control sequence are "operably" linked to each other when they are covalently linked to each other in such a way that transcription or expression of the transcribable and / or coding nucleic acid sequence is under the control or influence of the expression control sequence.

[0147] According to the present invention, "operably linked" or "operably linked" refers to a connection in a functional relationship. A nucleic acid is "operably linked" when it is functionally related to another nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operatively linked nucleic acids are typically contiguous to each other, but, where appropriate, are separated by further nucleic acid sequences.

[0148] In a particular embodiment, a nucleic acid is, according to the invention, operably linked to an expression control sequence, which may be homologous or heterologous to the nucleic acid.

[0149] "Polymerase" generally refers to a molecular entity capable of catalyzing the synthesis of a polymer molecule from monomer building blocks. "RNA polymerase" is a molecular entity capable of catalyzing the synthesis of an RNA molecule from ribonucleotide building blocks. "DNA polymerase" is a molecular entity capable of catalyzing the synthesis of a DNA molecule from deoxyribonucleotide building blocks. In the case of DNA and RNA polymerases, the molecular entity is typically a protein or an assembly or complex of multiple proteins. Typically, a DNA polymerase synthesizes a DNA molecule based on a template nucleic acid, which is typically a DNA molecule. Typically, an RNA polymerase synthesizes an RNA molecule based on a template nucleic acid that is either a DNA molecule (in which case the RNA polymerase is a DNA-dependent RNA polymerase, DdRP) or an RNA molecule (in which case the RNA polymerase is an RNA-dependent RNA polymerase, RdRP).

[0150] "RNA-dependent RNA polymerase" or "RdRP" is an enzyme that catalyzes the transcription of RNA from an RNA template. In the case of alphavirus RNA-dependent RNA polymerase, RNA replication results from the sequential synthesis of the complement of the (-) strand of the genomic RNA and the (+) strand of the genomic RNA. Therefore, alphavirus RNA-dependent RNA polymerase is synonymously referred to as "RNA replicase." In nature, RNA-dependent RNA polymerases are typically encoded by all RNA viruses except retroviruses. Typical representatives of viruses that encode RNA-dependent RNA polymerases are alphaviruses.

[0151] According to the present invention, "RNA replication" generally refers to an RNA molecule synthesized based on the nucleotide sequence of a given RNA molecule (template RNA molecule). The synthesized RNA molecule may, for example, be identical to or complementary to the template RNA molecule. Generally, RNA replication may occur via the synthesis of a DNA intermediate or directly by RNA-dependent RNA replication mediated by RNA-dependent RNA polymerase (RdRP). In the case of alphaviruses, RNA replication does not occur via a DNA intermediate but is mediated by RNA-dependent RNA polymerase (RdRP). A template RNA strand (first RNA strand) or a portion thereof serves as a template for the synthesis of a second RNA strand that is complementary to the first RNA strand or a portion thereof. The second RNA strand or a portion thereof may, in turn, optionally serve as a template for the synthesis of a third RNA strand that is complementary to the second RNA strand or a portion thereof. Thereby, the third RNA strand is identical to the first RNA strand or a portion thereof. Thus, an RNA-dependent RNA polymerase can directly synthesize a complementary RNA strand of a template, or indirectly synthesize an identical RNA strand (via a complementary intermediate strand).

[0152] According to the present invention, the term "template RNA" refers to an RNA that can be transcribed or replicated by an RNA-dependent RNA polymerase.

[0153] In a preferred embodiment of the invention, the RNA used in accordance with the present invention is a replicon RNA or simply "replicon", in particular a self-replicating RNA. In a particularly preferred embodiment, the replicon or self-replicating RNA is derived from or comprises elements derived from an ssRNA virus, in particular a positive-strand ssRNA virus, such as an alphavirus.

[0154] Generally, RNA viruses are a diverse group of infectious particles with RNA genome.RNA viruses can be classified into the subgroups of single-stranded RNA (ssRNA) viruses and double-stranded RNA (dsRNA) viruses, and ssRNA viruses can generally be further classified into positive-strand (+) viruses and / or negative-strand (-) viruses.At first glance, positive-strand RNA viruses are attractive as a delivery system for biopharmaceuticals because their RNA can directly serve as a template for translation in host cells.

[0155] Alphaviruses are typical representatives of positive-strand RNA viruses. Alphavirus hosts include a wide variety of organisms, including insects, fish, and mammals, such as domestic animals and humans. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, Vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5'-cap and a 3'-poly(A) tail. Alphavirus genomes encode nonstructural proteins (involved in viral RNA transcription, modification, and replication, as well as protein modification) and structural proteins (which form viral particles). Typically, two open reading frames (ORFs) are present in the genome. The four nonstructural proteins (nsP1 through nsP4) are typically co-encoded by a first ORF that begins near the 5' end of the genome, while the structural proteins of alphaviruses are co-encoded by a second ORF that is found downstream of the first ORF and extends to near the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with the ratio being approximately 2:1.

[0156] In cells infected with alphaviruses, only nucleic acid sequences encoding nonstructural proteins are translated from genomic RNA, while genetic information encoding structural proteins can be translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA) (Gould et al., 2010, Antiviral Res., 87:111-124). After infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts like a messenger RNA directly for the translation of the open reading frame encoding the nonstructural polyprotein (nsP1234). In some alphaviruses, an opal stop codon is present between the coding sequences for nsP3 and nsP4. Polyprotein P123, containing nsP1, nsP2, and nsP3, is generated upon translation termination at the opal stop codon, and polyprotein P1234, containing nsP4, is additionally generated upon readthrough of this opal codon (Strauss & Strauss, Microbiol. Rev., 1994, 58:491-562; Rupp et al., 2015, J. Gen. Virology, 96:2483-2500). nsP1234 is autoproteolytically cleaved into fragments nsP123 and nsP4. Polypeptides nsP123 and nsP4 combine to form a minus-strand replicase complex, which transcribes minus-strand RNA using the plus-strand genomic RNA as a template. Typically, at a later stage, the nsP123 fragment is completely cleaved into the individual proteins nsP1, nsP2, and nsP3 (Shirako & Strauss, 1994, J. Virol. 68:1874-1885). All four proteins form the (+)-strand replicase complex, which synthesizes new (+)-strand genomes using the complement of the (-)-strand of the genomic RNA as a template (Kim et al., 2004, Virology 323:153-163; Vasiljeva et al., 2003, J. Biol. Chem. 278:41636-41645).

[0157] In infected cells, subgenomic RNA and new genomic RNA are provided with a 5'-cap by nsP1 (Pettersson et al., 1980, Eur. J. Biochem. 105, pp. 435-443; Rozanov et al., 1992, J. Gen. Virology 73, pp. 2129-2134) and a poly-adenylate [poly(A)] tail by nsP4 (Rubach et al., Virology 2009, vol. 384, pp. 201-208). Thus, both subgenomic RNA and new genomic RNA resemble messenger RNA (mRNA).

[0158] Alphavirus structural proteins are typically encoded by a single open reading frame under the control of a subgenomic promoter (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562). The subgenomic promoter is recognized by cis-acting alphavirus nonstructural proteins. Specifically, the alphavirus replicase synthesizes a positive-strand subgenomic transcript using the complement of the negative-strand of the genomic RNA as a template. The positive-strand subgenomic transcript encodes the alphavirus structural proteins (Kim et al., 2004, Virology, vol. 323, pp. 153-163; Vasiljeva et al., 2003, J. Biol. Chem., vol. 278, pp. 41636-41645). The subgenomic RNA transcript serves as a template for translation of the open reading frame encoding the structural proteins into a single polyprotein, which is cleaved to produce the structural proteins. During later stages of alphavirus infection in host cells, a packaging signal located within the coding sequence of nsP2 ensures the selective packaging of genomic RNA into budding virions, which are packaged by structural proteins (White et al., 1998, J. Virol., 72:4320-4326).

[0159] In infected cells, negative-strand RNA synthesis is typically observed only during the first 3-4 hours postinfection and becomes undetectable at later stages, during which time only positive-strand RNA (both genomic and subgenomic) synthesis is observed. According to Frolov et al., 2001, RNA, 7, 1638-1651, a general model for the regulation of RNA synthesis suggests a dependency on processing of nonstructural polyproteins. Initial cleavage of the nonstructural polyprotein nsP1234 generates nsP123 and nsP4, which act as an RNA-dependent RNA polymerase (RdRp) that is active for negative-strand synthesis but inefficient for producing positive-strand RNA. Further processing of polyprotein nsP123, such as cleavage at the nsP2 / nsP3 junction, alters the template specificity of the replicase to increase positive-strand RNA synthesis and decrease or terminate negative-strand RNA synthesis.

[0160] Alphavirus RNA synthesis is also regulated by cis-acting RNA elements, including four conserved sequence elements (CSEs) (Strauss & Strauss, Microbiol. Rev., 1994, 58:491-562 and Frolov, 2001, RNA, 7:1638-1651).

[0161] In general, the 5' replication recognition sequences of alphavirus genomes are characterized by low overall homology among different alphaviruses, but have a conserved predicted secondary structure. The 5' replication recognition sequences of alphavirus genomes contain two conserved sequence elements, CSE1 and CSE2, that are involved not only in translation initiation but also in viral RNA synthesis. Secondary structure is thought to be more important than linear sequence for the function of CSE1 and CSE2 (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562).

[0162] In contrast, the 3' terminal sequence of alphavirus genomes, i.e., the sequence immediately upstream of the poly(A) sequence, is characterized by a conserved primary structure, in particular conserved sequence element 4 (CSE4), also called the "19-nt conserved sequence," which is important for the initiation of negative strand synthesis.

[0163] CSE3, also known as the "junction sequence," is a conserved sequence element on the (+) strand of alphavirus genomic RNA; its complement on the (-) strand acts as a promoter for subgenomic RNA transcription (Strauss & Strauss, Microbiol. Rev., 1994, 58:491-562; Frolov et al., 2001, RNA, 7:1638-1651). CSE3 typically overlaps with the region encoding the C-terminal fragment of nsP4.

[0164] In addition to alphavirus proteins, host cell factors, presumably proteins, may also bind to conserved sequence elements (Strauss & Strauss, supra).

[0165] Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the structural proteins of the alphavirus is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase (typically as polyprotein nsP1234), and the other nucleic acid molecule is capable of being replicated in trans by the replicase (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules capable of being replicated in trans by the replicase must contain certain alphavirus sequence elements to enable recognition by the alphavirus replicase and RNA synthesis.

[0166] According to the present invention, the term "alphavirus" will be understood broadly and include any viral particle having the characteristics of an alphavirus, including the presence of positive-strand RNA, which encodes suitable genetic information for replication in a host cell, including RNA polymerase activity. Further characteristics of many alphaviruses are described in Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562. The term "alphavirus" encompasses not only alphaviruses found in nature, but also any variants or derivatives thereof. In some embodiments, the variants or derivatives are not found in nature.

[0167] In one embodiment, the alphavirus is an alphavirus found in nature. Typically, alphaviruses found in nature are infectious to any one or more eukaryotic organisms, such as animals (including vertebrates such as humans and arthropods such as insects).

[0168] The alphavirus found in nature is preferably selected from the group consisting of: Barmah Forest virus complex (including Barmah Forest virus), Eastern equine encephalitis complex (including seven antigenic types of Eastern equine encephalitis virus), Middelburg virus complex (including Middelburg virus), Ndumu virus complex (including Ndumu virus), Semliki Forest virus complex (including Bebaru virus), Chikungunya virus, Mayaro virus and its subtype Una virus, O'Nyong virus and its subtype Igbo-Ora virus, Ross River virus and its subtype Bebaru virus, Getah virus, Sagiyama virus virus, including Semliki Forest virus and its subtype Me Tri virus), Venezuelan equine encephalitis complex (including Cabassou virus, Everglades virus, Mosso das Pedras virus, Mucambo virus, Paramana virus, Pixuna virus, Rio Negro virus, Trocara virus and its subtype Bijou Bridge virus, and Venezuelan equine encephalitis virus), Western equine encephalitis complex (including Aura virus, Babanki virus,virus, Kyzylagach virus, Sindbis virus, Ockelbo virus, Whataroa virus, Buggy Creek virus, Fort Morgan virus, Highlands J virus, including Western equine encephalitis virus, and some unclassified viruses including Salmon pancreatic disease virus, Sleeping Disease virus, Southern elephant seal virus, and Tonate virus. More preferably, the alphavirus is selected from the group consisting of the Semliki Forest virus complex (including virus types as indicated above, such as Semliki Forest virus), the Western equine encephalitis complex (including virus types as indicated above, such as Sindbis virus), the Eastern equine encephalitis virus (including virus types as indicated above), and the Venezuelan equine encephalitis complex (including virus types as indicated above, such as Venezuelan equine encephalitis virus).

[0169] In a further preferred embodiment, the alphavirus is Semliki Forest virus. In an alternative further preferred embodiment, the alphavirus is Sindbis virus. In an alternative further preferred embodiment, the alphavirus is Venezuelan equine encephalitis virus.

[0170] In some embodiments of the invention, the alphavirus is not an alphavirus found in nature. Typically, an alphavirus not found in nature is a variant or derivative of an alphavirus found in nature that is distinguished from an alphavirus found in nature by at least one mutation in the nucleotide sequence, i.e., genomic RNA. The mutation in the nucleotide sequence can be selected from an insertion, substitution, or deletion of one or more nucleotides compared to an alphavirus found in nature. The mutation in the nucleotide sequence may or may not be associated with a mutation in the polypeptide or protein encoded by the nucleotide sequence. For example, an alphavirus not found in nature can be an attenuated alphavirus. An attenuated alphavirus not found in nature is an alphavirus that typically has at least one mutation in its nucleotide sequence that distinguishes it from an alphavirus found in nature, such that it is not infectious at all, or is infectious but has a reduced ability to cause disease, or is completely incapable of causing disease. As an illustrative example, TC83 is an attenuated alphavirus distinct from the Venezuelan equine encephalitis virus (VEEV) found in nature (McKinney et al., 1963, Am. J. Trop. Med. Hyg., 1963, 12:597-603).

[0171] Members of the alphavirus genus can also be divided into those primarily associated with encephalitis and those primarily associated with fever, rash, and polyarthritis based on their relative clinical characteristics in humans.

[0172] The term "alphaviral" means found in an alphavirus, or originating from an alphavirus or derived from an alphavirus, for example by genetic engineering.

[0173] According to the present invention, "SFV" is an abbreviation for Semliki Forest virus. According to the present invention, "SIN" or "SINV" is an abbreviation for Sindbis virus. According to the present invention, "VEE" or "VEEV" is an abbreviation for Venezuelan equine encephalitis virus.

[0174] According to the present invention, the terms "alphaviral" or "derived from an alphavirus" refer to an entity that originates from an alphavirus. Illustratively, an alphavirus protein may refer to a protein found in and / or encoded by an alphavirus, and an alphavirus nucleic acid sequence may refer to a nucleic acid sequence found in and / or encoded by an alphavirus. Preferably, an "alphavirus" nucleic acid sequence refers to a nucleic acid sequence "of the alphavirus genome" and / or a nucleic acid sequence "of the alphavirus genomic RNA."

[0175] According to the present invention, the term "alphavirus RNA" refers to any one or more of the genomic RNA (i.e., (+) strand) of an alphavirus, the complement of the genomic RNA (i.e., (-) strand) of an alphavirus, and the subgenomic transcript (i.e., (+) strand), or any fragment thereof.

[0176] According to the present invention, "alphavirus genome" refers to the genomic (+) strand RNA of an alphavirus.

[0177] In accordance with the present invention, the term "native alphavirus sequence" and similar terms typically refer to a (e.g., nucleic acid) sequence of a naturally occurring alphavirus (an alphavirus found in nature). In some embodiments, the term "native alphavirus sequence" also encompasses sequences of attenuated alphaviruses.

[0178] According to the present invention, the term "5' replication recognition sequence" preferably refers to a contiguous nucleic acid sequence, preferably a ribonucleic acid sequence, that is identical to or homologous to the 5' fragment of an alphavirus genome. A "5' replication recognition sequence" is a nucleic acid sequence that can be recognized by an alphavirus replicase. The term 5' replication recognition sequence encompasses not only the native 5' replication recognition sequence, but also its functional equivalents, such as functional variants of the 5' replication recognition sequence of an alphavirus found in nature. The 5' replication recognition sequence is required for synthesis of the complement of the (-) strand of the alphavirus genomic RNA and is required for synthesis of the (+) strand viral genomic RNA from the (-) strand template. A native 5' replication recognition sequence typically encodes at least the N-terminal fragment of nsP1, but does not include the entire open reading frame encoding nsP1234. In view of the fact that a native 5' replication recognition sequence typically encodes at least the N-terminal fragment of nsP1, a native 5' replication recognition sequence typically includes at least one initiation codon, typically AUG. In one embodiment, the 5' replication recognition sequence comprises the alphavirus genome conserved sequence element 1 (CSE1) or a variant thereof, and the alphavirus genome conserved sequence element 2 (CSE2) or a variant thereof. The 5' replication recognition sequence can typically form four stem loops (SL), namely SL1, SL2, SL3, and SL4. The numbering of these stem loops begins at the 5' end of the 5' replication recognition sequence.

[0179] According to the present invention, the term "3' replication recognition sequence" preferably refers to a contiguous nucleic acid sequence, preferably a ribonucleic acid sequence, that is identical to or homologous to the 3' fragment of an alphavirus genome. A "3' replication recognition sequence" is a nucleic acid sequence that can be recognized by an alphavirus replicase. The term 3' replication recognition sequence encompasses not only the native 3' replication recognition sequence, but also its functional equivalents, such as functional variants of the 3' replication recognition sequence of an alphavirus found in nature. The 3' replication recognition sequence is required for synthesis of the complement of the (-) strand of the alphavirus genomic RNA. In one embodiment, the 3' replication recognition sequence comprises conserved sequence element 4 (CSE4) of the alphavirus genome or a variant thereof, and optionally the poly(A) tail of the alphavirus genome.

[0180] The term "conserved sequence element" or "CSE" refers to a nucleotide sequence found in alphavirus RNA. These sequence elements are called "conserved" because orthologs are present in the genomes of different alphaviruses, and orthologous CSEs of different alphaviruses preferably share a high percentage of sequence identity and / or similar secondary or tertiary structure. The term CSE encompasses CSE1, CSE2, CSE3, and CSE4.

[0181] According to the present invention, the terms "CSE1" or "44-nt CSE" refer synonymously to the nucleotide sequence required for (+)-strand synthesis from a (-)-strand template. The term "CSE1" refers to the sequence on the (+)-strand, and the complementary sequence of CSE1 (on the (-)-strand) functions as a promoter for (+)-strand synthesis. Preferably, the term CSE1 encompasses the 5'-most nucleotides of an alphavirus genome. CSE1 typically forms a conserved stem-loop structure. Without wishing to be bound by any particular theory, it is believed that in the case of CSE1, the secondary structure is more important than the primary structure, i.e., the linear sequence. In the genomic RNA of the model alphavirus Sindbis virus, CSE1 consists of a contiguous sequence of 44 nucleotides, which is formed by the 5'-most 44 nucleotides of the genomic RNA (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562).

[0182] According to the present invention, the terms "CSE2" or "51-nt CSE" refer synonymously to the nucleotide sequence required for negative-strand synthesis from a positive-strand template. The positive-strand template is typically an alphavirus genomic RNA or an RNA replicon (note that subgenomic RNA transcripts that do not contain CSE2 do not serve as templates for negative-strand synthesis). In alphavirus genomic RNA, CSE2 is typically located within the coding sequence of nsP1. In the genomic RNA of the model alphavirus Sindbis virus, the 51-nt CSE is located at nucleotide positions 155-205 of the genomic RNA (Frolov et al., 2001, RNA 7, 1638-1651). CSE2 typically forms two conserved stem-loop structures. These stem-loop structures are designated stem-loop 3 (SL3) and stem-loop 4 (SL4) because they are the third and fourth conserved stem-loops, respectively, in the alphavirus genomic RNA, counting from the 5' end of the alphavirus genomic RNA. Without wishing to be bound by any particular theory, it is believed that in the case of CSE2, the secondary structure is more important than the primary structure, i.e., the linear sequence.

[0183] According to the present invention, the terms "CSE3" or "junction sequence" refer synonymously to a nucleotide sequence derived from the alphavirus genomic RNA and comprising the initiation site of the subgenomic RNA. The complement of this sequence on the (-) strand acts to promote subgenomic RNA transcription. In the alphavirus genomic RNA, CSE3 typically overlaps the region encoding the C-terminal fragment of nsP4 and extends into a short non-coding region located upstream of the open reading frame encoding the structural proteins.

[0184] According to the present invention, the terms "CSE4," or "19-nt conserved sequence," or "19-nt CSE" refer synonymously to a nucleotide sequence derived from an alphavirus genomic RNA, located immediately upstream of the poly(A) sequence in the 3' untranslated region of the alphavirus genome. CSE4 typically consists of 19 consecutive nucleotides. Without wishing to be bound by any particular theory, CSE4 is understood to function as a core promoter for initiating negative-strand synthesis (Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856), and / or CSE4 and the poly(A) tail of the alphavirus genomic RNA are understood to function together for efficient negative-strand synthesis (Hardy & Rice, J. Virol., 2005, vol. 79, pp. 4630-4639).

[0185] According to the present invention, the term "subgenomic promoter" or "SGP" refers to a nucleic acid sequence upstream (5') of a nucleic acid sequence (e.g., a coding sequence) that controls transcription of the nucleic acid sequence by providing a recognition and binding site for an RNA polymerase, typically an RNA-dependent RNA polymerase, particularly a functional alphavirus nonstructural protein. SGPs may contain additional recognition or binding sites for additional factors. Subgenomic promoters are typically genetic elements of positive-strand RNA viruses, such as alphaviruses. Alphavirus subgenomic promoters are nucleic acid sequences contained in the viral genomic RNA. Subgenomic promoters are generally characterized in that they initiate transcription (RNA synthesis) in the presence of an RNA-dependent RNA polymerase, e.g., a functional alphavirus nonstructural protein. The RNA (-) strand, i.e., the complement of the alphavirus genomic RNA, serves as a template for the synthesis of a (+)-strand subgenomic transcript, which typically initiates at or near the subgenomic promoter. As used herein, the term "subgenomic promoter" is not limited to any particular location in a nucleic acid that comprises such a subgenomic promoter. In some embodiments, the SGP is identical to, overlaps with, or includes a CSE3.

[0186] The terms "subgenomic transcript" or "subgenomic RNA" refer synonymously to an RNA molecule obtainable as a result of transcription using an RNA molecule as a template ("template RNA"), where the template RNA includes a subgenomic promoter that controls transcription of the subgenomic transcript. Subgenomic transcripts can be obtained in the presence of an RNA-dependent RNA polymerase, particularly functional alphavirus nonstructural proteins. For example, the term "subgenomic transcript" can refer to an RNA transcript prepared in an alphavirus-infected cell using the complement of the negative strand of an alphavirus genomic RNA as a template. However, as used herein, the term "subgenomic transcript" is not limited thereto and also encompasses transcripts obtainable by using a heterologous RNA as a template. For example, a subgenomic transcript can also be obtained by using the complement of the negative strand of an SGP-containing replicon according to the present invention as a template. Thus, the term "subgenomic transcript" can refer not only to an RNA molecule obtainable by transcribing a fragment of an alphavirus genomic RNA, but also to an RNA molecule obtainable by transcribing a fragment of a replicon according to the present invention.

[0187] According to the present invention, a nucleic acid construct that can be replicated by a replicase, preferably an alphavirus replicase, is called a replicon. According to the present invention, the term "replicon" defines an RNA molecule that can be replicated by an RNA-dependent RNA polymerase to generate one or more identical or essentially identical copies of the RNA replicon without a DNA intermediate. "Without a DNA intermediate" means that no deoxyribonucleic acid (DNA) copy or complement of the replicon is formed in the process of forming the RNA replicon copy and / or no deoxyribonucleic acid (DNA) molecule is used in the process of forming the RNA replicon copy or its complement. The replicase function is typically provided by functional alphavirus nonstructural proteins.

[0188] According to the present invention, the terms "capable of replicating" and "replicable" generally describe that one or more identical or essentially identical copies of a nucleic acid can be prepared. When used together with the term "replicase," such as in "capable of replicating by a replicase," the terms "capable of replicating" and "replicable" describe functional characteristics of a nucleic acid molecule, e.g., an RNA replicon, with respect to the replicase. These functional characteristics include at least one of (i) the replicase's ability to recognize a replicon, and (ii) the replicase's ability to act as an RNA-dependent RNA polymerase (RdRP). Preferably, the replicase is capable of both (i) recognizing a replicon and (ii) acting as an RNA-dependent RNA polymerase.

[0189] The term "capable of recognizing" describes that the replicase is capable of physically associating with the replicon, and preferably that the replicase is capable of binding to the replicon non-covalently. The term "binding" may mean that the replicase has the ability to bind to any one or more of conserved sequence element 1 (CSE1) or its complementary sequence (if included by the replicon), conserved sequence element 2 (CSE2) or its complementary sequence (if included by the replicon), conserved sequence element 3 (CSE3) or its complementary sequence (if included by the replicon), or conserved sequence element 4 (CSE4) or its complementary sequence (if included by the replicon). Preferably, the replicase is capable of binding to CSE2 [i.e., the (+) strand] and / or CSE4 [i.e., the (+) strand], or is capable of binding to the complement of CSE1 [i.e., the (-) strand] and / or the complement of CSE3 [i.e., the (-) strand].

[0190] The phrase "capable of acting as an RdRP" means that the replicase is capable of catalyzing the synthesis of the complement of the (-) strand of an alphavirus genomic (+) strand RNA when the (+) strand RNA serves as a template and / or that the replicase is capable of catalyzing the synthesis of a (+) strand alphavirus genomic RNA when the (-) strand RNA serves as a template. In general, the phrase "capable of acting as an RdRP" can also encompass the ability of the replicase to catalyze the synthesis of a (+) strand subgenomic transcript when the (-) strand RNA serves as a template and synthesis of the (+) strand subgenomic transcript is typically initiated at an alphavirus subgenomic promoter.

[0191] The terms "capable of binding" and "capable of acting as an RdRP" refer to the ability under normal physiological conditions. In particular, these conditions refer to the conditions in cells expressing functional alphavirus nonstructural proteins or cells transfected with nucleic acids encoding functional alphavirus nonstructural proteins. The cells are preferably eukaryotic cells. The ability to bind and / or act as an RdRP can be tested experimentally, for example, in a cell-free in vitro system or in eukaryotic cells. Optionally, the eukaryotic cells are cells derived from a species infective with the particular alphavirus from which the replicase originates. For example, when an alphavirus replicase derived from a particular alphavirus infectious for humans is used, normal physiological conditions refer to the conditions in human cells. More preferably, the eukaryotic cells (in one example, human cells) are derived from the same tissue or organ as the tissue or organ in which the particular alphavirus from which the replicase originates is infectious.

[0192] According to the present invention, "when compared to a native alphavirus sequence" and similar terms refer to a sequence that is a variant of a native alphavirus sequence. A variant is typically not itself a native alphavirus sequence.

[0193] In one embodiment, the RNA replicon comprises replication recognition sequences, such as a 5' replication recognition sequence and a 3' replication recognition sequence. A replication recognition sequence is a nucleic acid sequence that can be recognized by a functional alphavirus nonstructural protein. In other words, the functional alphavirus nonstructural protein is capable of recognizing the replication recognition sequence. Preferably, the 5' replication recognition sequence is located at the 5' end of the replicon. In one embodiment, the 5' replication recognition sequence consists of or comprises CSE1 and CSE2. Preferably, the 3' replication recognition sequence is located at the 3' end of the replicon (if the replicon does not contain a poly(A) tail) or immediately upstream of the poly(A) tail (if the replicon contains a poly(A) tail). In one embodiment, the 3' replication recognition sequence consists of or comprises CSE4.

[0194] In one embodiment, the 5' and 3' replication recognition sequences are capable of directing replication of an RNA replicon in the presence of functional alphavirus nonstructural proteins. Thus, when present alone, or preferably together, these recognition sequences direct replication of an RNA replicon in the presence of functional alphavirus nonstructural proteins.

[0195] The functional alphavirus nonstructural proteins are preferably either cis (encoded as the protein of interest by an open reading frame on the replicon) or trans (encoded as the protein of interest by an open reading frame on a separate replicase construct that is capable of recognizing both the 5' and 3' replication recognition sequences of the replicon). In one embodiment, this is achieved when the 5' and 3' replication recognition sequences are native to the alphavirus from which the functional alphavirus nonstructural proteins are derived. By native, we mean that the natural origin of these sequences is the same alphavirus. In an alternative embodiment, the 5' and / or 3' replication recognition sequences are not native to the alphavirus from which the functional alphavirus nonstructural proteins are derived, provided that the functional alphavirus nonstructural proteins are capable of recognizing both the 5' and 3' replication recognition sequences of the replicon. In other words, the functional alphavirus nonstructural proteins are compatible with the 5' and 3' replication recognition sequences. Functional alphavirus nonstructural proteins are said to be compatible (cross-viral compatibility) if a non-native functional alphavirus nonstructural protein can recognize the respective sequences or sequence elements. Any combination of (3' / 5') replication recognition sequences and CSEs, respectively, with functional alphavirus nonstructural proteins is possible, as long as cross-viral compatibility exists. Cross-viral compatibility can be easily tested by one skilled in the art using this invention, for example, by incubating the functional alphavirus nonstructural protein to be tested with RNA (this RNA has the 3' and 5' replication recognition sequences to be tested) in a suitable host cell under conditions favorable for RNA replication. If replication occurs, the (3' / 5') replication recognition sequences and the functional alphavirus nonstructural protein are determined to be compatible.

[0196] In one embodiment of the present invention, the replicon is part of a trans-replication system, and thus the replicon is a trans-replicon. In this embodiment, the RNA replicon preferably does not contain an open reading frame encoding a functional alphavirus nonstructural protein. Thus, in this embodiment, the invention provides a system comprising two nucleic acid molecules: a first RNA construct for expressing functional alphavirus nonstructural proteins (i.e., encoding a functional alphavirus nonstructural protein), and a second RNA molecule, an RNA replicon. The RNA construct for expressing functional alphavirus nonstructural proteins is referred to herein interchangeably as an "RNA construct for expressing functional alphavirus nonstructural proteins" or a "replicase construct." Functional alphavirus nonstructural proteins are as defined above and are typically encoded by an open reading frame contained in the replicase construct. The functional alphavirus nonstructural protein encoded by the replicase construct may be any functional alphavirus nonstructural protein capable of replicating the replicon. According to the present invention, the replicase constructs may be present in the same composition, e.g., as a mixed or multi-particulate formulation, or may be present in separate compositions, e.g., as separate particulate formulations. When the system of the present invention is introduced into a cell, preferably a eukaryotic cell, the open reading frame encoding the functional alphavirus nonstructural proteins can be translated. After translation, the functional alphavirus nonstructural proteins are capable of replicating a separate RNA molecule (RNA replicon) in trans.

[0197] As used herein, trans (e.g., in the context of trans-acting, trans-regulatory) generally means "acting from different molecules" (i.e., between molecules). This is opposed to cis (e.g., in the context of cis-acting, cis-regulatory), which generally means "acting from the same molecule" (i.e., intramolecularly). In the context of RNA synthesis (including transcription and RNA replication), trans-acting elements include nucleic acid sequences containing genes encoding enzymes capable of RNA synthesis (RNA polymerases). RNA polymerases use a second nucleic acid molecule, i.e., a nucleic acid molecule other than the one in which they are encoded, as a template for synthesizing RNA. Both the RNA polymerase and the nucleic acid sequence containing the gene encoding the RNA polymerase are said to "act in trans" with respect to the second nucleic acid molecule. In the context of the present invention, the RNA polymerase encoded by the trans-acting RNA can be a functional alphavirus nonstructural protein. A functional alphavirus nonstructural protein is capable of using a second nucleic acid molecule, that is, an RNA replicon, as a template for the synthesis of RNA, including replication of the RNA replicon. An RNA replicon according to the present invention that can be replicated in trans by a replicase is herein synonymously referred to as a "trans-replicon."

[0198] According to the present invention, the role of the functional alphavirus nonstructural proteins is to amplify the replicon and, if a subgenomic promoter is present on the replicon, to prepare subgenomic transcripts. If the replicon encodes a gene of interest for expression, the level and / or duration of expression of the gene of interest can be regulated in trans by modifying the levels of functional alphavirus nonstructural proteins.

[0199] The trans-replication system of the present invention comprises at least two nucleic acid molecules. In preferred embodiments, the system consists of exactly two RNA molecules: a replicon and a replicase construct. In alternative preferred embodiments, the system comprises two or more replicons, each preferably encoding at least one protein of interest, and also comprises a replicase construct. In these embodiments, functional alphavirus nonstructural proteins encoded by the replicase construct can act on each replicon to promote replication and, optionally, production of subgenomic transcripts, respectively. For example, each replicon may encode a pharmaceutically active peptide or protein. This is advantageous, for example, when vaccination of a subject against several different antigens is desired.

[0200] Preferably, the replicase construct lacks at least one conserved sequence element (CSE) required for (+) strand template-based (-) strand synthesis and / or required for (+) strand template-based (-) strand synthesis. More preferably, the replicase construct does not contain any alphavirus conserved sequence elements (CSEs). In particular, among the four alphavirus CSEs (Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562; Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856), any one or more of the following CSEs: CSE1, CSE2, CSE3, and CSE4 are preferably absent from the replicase construct. In particular, in the absence of any one or more alphavirus CSEs, the replicase constructs of the present invention are much more similar to typical eukaryotic mRNAs than they are to alphavirus genomic RNAs.

[0201] The replicase constructs of the present invention preferably distinguish from alphavirus genomic RNA in that they are at least incapable of autonomous replication and / or do not contain an open reading frame under the control of a subgenomic promoter. If they are incapable of autonomous replication, they may also be referred to as "suicide constructs."

[0202] The replicase construct according to the present invention is preferably a single-stranded RNA molecule. The replicase construct according to the present invention is typically a (+)-strand RNA molecule. In one embodiment, the replicase construct according to the present invention is an isolated nucleic acid molecule.

[0203] In one embodiment, an RNA, such as a replicon, according to the present invention comprises at least one open reading frame encoding a peptide or protein of interest. In various embodiments, the peptide or protein of interest is encoded by a heterologous nucleic acid sequence. In accordance with the present invention, the term "heterologous" refers to the fact that the nucleic acid sequence is not naturally operatively or structurally linked to a nucleic acid sequence, such as an alphavirus nucleic acid sequence.

[0204] The RNA of the present invention may encode a single polypeptide or multiple polypeptides. The multiple polypeptides may be encoded as a single polypeptide (fusion polypeptide) or as separate polypeptides. In some embodiments, the RNA of the present invention may comprise two or more open reading frames, each of which may or may not be independently selected to be under the control of a subgenomic promoter in the case of a replicon. Alternatively, the polyprotein or fusion polypeptide may comprise individual polypeptides (e.g., foot-and-mouth disease virus 2A protein) or an intein, optionally separated by an autocatalytic protease cleavage site.

[0205] The protein of interest can be selected, for example, from the group consisting of a reporter protein, a pharmaceutically active peptide or protein, an inhibitor of intracellular interferon (IFN) signaling, and a functional alphavirus nonstructural protein.

[0206] According to the present invention, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably six or more, preferably twenty or more, up to a maximum of preferably fifty, preferably one hundred, or preferably one hundred and fifty consecutive amino acids linked together via peptide bonds. The term "protein" refers to large peptides, preferably peptides having at least 151 amino acids, although the terms "peptide" and "protein" are generally used synonymously herein.

[0207] The terms "peptide" and "protein" according to the present invention include substances that contain not only amino acid components but also non-amino acid components such as sugar and phosphate structures, and also substances that contain bonds such as ester, thioether, or disulfide bonds.

[0208] The term "variant," e.g., with respect to nucleic acid and amino acid sequences, includes, according to the present invention, any variant, particularly a mutant, viral strain variant, splice variant, conformation, isoform, allelic variant, species variant, and species homolog, especially one that occurs naturally. Allelic variants refer to alterations in the normal sequence of a gene, the significance of which is often unclear. Complete gene sequencing often identifies multiple allelic variants for a given gene. With respect to nucleic acid molecules, the term "variant" includes degenerate nucleic acid sequences, which according to the present invention are nucleic acids that differ from a reference nucleic acid in codon sequence due to the degeneracy of the genetic code (e.g., due to matching codon usage). Species homologs are nucleic acid sequences or amino acid sequences that have a different species origin from that of a given nucleic acid or amino acid sequence. Viral homologs are nucleic acid sequences or amino acid sequences that have a different viral origin from that of a given nucleic acid or amino acid sequence.

[0209] According to the present invention, nucleic acid variants include single or multiple nucleotide deletions, additions, mutations, substitutions, and / or insertions compared to a reference nucleic acid. Deletions include the removal of one or more nucleotides from the reference nucleic acid. Addition variants include 5'- and / or 3'-terminal fusions of one or more nucleotides, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more nucleotides. Substitutions involve the removal of at least one nucleotide in a sequence and the insertion of at least one other nucleotide in its place (e.g., transversions and transitions). Mutations include abasic sites, crosslinked sites, and chemically modified or modified bases. Insertions include the addition of at least one nucleotide to the reference nucleic acid.

[0210] According to the present invention, a "nucleotide change" may refer to a single or multiple nucleotide deletion, addition, mutation, substitution, and / or insertion when compared to a reference nucleic acid. In some embodiments, a "nucleotide change" is selected from the group consisting of a single nucleotide deletion, a single nucleotide addition, a single nucleotide mutation, a single nucleotide substitution, and / or a single nucleotide insertion when compared to the reference nucleic acid. According to the present invention, a nucleic acid variant may contain one or more nucleotide changes when compared to the reference nucleic acid.

[0211] A variant of a particular nucleic acid sequence preferably has at least one functional property of the particular sequence, and is preferably functionally equivalent to the particular sequence, e.g., a nucleic acid sequence exhibiting properties that are identical to or similar to the properties of the particular nucleic acid sequence.

[0212] Preferably, the degree of identity between a given nucleic acid sequence and a nucleic acid sequence that is a variant of the given nucleic acid sequence will be at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. The degree of identity is preferably provided over a region of at least about 30, at least about 50, at least about 70, at least about 90, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, or at least about 400 nucleotides. In a preferred embodiment, the degree of identity is provided over the entire length of the reference nucleic acid sequence.

[0213] "Sequence similarity" refers to the percentage of amino acids that are identical or that represent conservative amino acid substitutions. "Sequence identity" between two polypeptide or nucleic acid sequences refers to the percentage of amino acids or nucleotides that are identical between the sequences.

[0214] The term "% identity" is intended to refer in particular to the percentage of nucleotides that are identical in optimal alignment between the two sequences being compared, said percentage being purely statistical; in order to obtain optimal alignment between the two sequences, the differences between the two sequences may be randomly distributed over the entire length of the sequences, and the compared sequence may contain additions or deletions when compared to the reference sequence. The comparison of two sequences is usually carried out by comparing the sequences over a segment or "comparison window" in order to identify local regions of corresponding sequences after optimal alignment. Optimal alignment for comparison may be performed manually or using the local homology algorithm of Smith and Waterman, 1981, Ads App. Math., 2, 482, using the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol., 48, 443, and using the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85, 2444, or using computer programs that employ such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, Inc., 575 Science Drive, Madison, Wis.).

[0215] Percent identity is obtained by determining the number of identical positions in the compared sequences, dividing this number by the number of positions being compared, and multiplying this result by 100.

[0216] For example, the BLAST program "BLAST 2 sequences" available at the website http: / / www.ncbi.nlm.nih.gov / blast / bl2seq / wblast2.cgi may be used.

[0217] A nucleic acid is "hybridizable" or "hybridizes" to another nucleic acid if the two sequences are complementary to each other. A nucleic acid is "complementary" to another nucleic acid if the two sequences are capable of forming a stable duplex with each other. According to the present invention, hybridization is preferably carried out under conditions that allow specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in "Molecular Cloning: A Laboratory Manual," edited by J. Sambrook et al., 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or "Current Protocols in Molecular Biology," edited by F. M. Ausubel et al., John Wiley & Sons, Inc., New York, and refer to, for example, hybridization in a hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin, 2.5mM NaH2PO4 (pH 7), 0.5% SDS, 2mM EDTA) at 65°C. SSC is 0.15M sodium chloride / 0.15M sodium citrate, pH 7. After hybridization, the membrane onto which the DNA has been transferred is washed, for example, in 2xSSC at room temperature, and then in 0.1 to 0.5xSSC / 0.1xSDS at a temperature up to 68°C.

[0218] The percent complementarity indicates the percentage of contiguous residues in a nucleic acid molecule that can form hydrogen bonds (Watson-Crick base pairs) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Perfectly complementary" or "fully complementary" means that all contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. Preferably, the degree of complementarity according to the present invention is at least 70%, preferably at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, or most preferably at least 95%, 96%, 97%, 98%, or 99%. Most preferably, the degree of complementarity according to the present invention is 100%.

[0219] The term "derivative" includes any nucleic acid that has been chemically derivatized at the nucleotide base, sugar, or phosphate. The term "derivative" also includes nucleic acids containing nucleotides and non-naturally occurring nucleotide analogs. Preferably, derivatization of a nucleic acid increases its stability.

[0220] According to the present invention, a "nucleic acid sequence derived from a nucleic acid sequence" refers to a nucleic acid that may be a variant of the nucleic acid from which it is derived.

[0221] In one embodiment, the open reading frame encodes a reporter protein. In that embodiment, the open reading frame comprises a reporter gene. Certain genes may be chosen as reporters because the characteristics they confer on cells or organisms expressing them are easily identified and measurable, or because they are selectable markers. Reporter genes are often used as indicators of whether a particular gene has been incorporated into or expressed in a population of cells or organisms. Preferably, the expression product of a reporter gene is visually detectable. Common visually detectable reporter proteins typically possess fluorescent or luminescent proteins. Examples of specific reporter genes include genes encoding the jellyfish green fluorescent protein (GFP), which causes expressing cells to glow green under blue light, the enzyme luciferase, which catalyzes a reaction with luciferin to produce light, and red fluorescent protein (RFP). Variants of any of these specific reporter genes are also possible, so long as they retain the visually detectable property. For example, eGFP is a point mutant variant of GFP.

[0222] According to the present invention, in one embodiment, the RNA comprises or consists of a pharmaceutically active RNA. The "pharmaceutically active RNA" may be an RNA encoding a pharmaceutically active peptide or protein. Preferably, the RNA according to the present invention encodes a pharmaceutically active peptide or protein. Preferably, the open reading frame encodes a pharmaceutically active peptide or protein. Preferably, the RNA comprises an open reading frame encoding a pharmaceutically active peptide or protein, optionally in the case of an RNA replicon under the control of a subgenomic promoter.

[0223] A "pharmaceutically active peptide or protein" has a positive or beneficial effect on a condition or pathology of a subject when administered to the subject in a therapeutically effective amount. Preferably, a pharmaceutically active peptide or protein has curative or palliative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset, or lessen the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein may have preventative properties and can be used to delay the onset of a disease or lessen the severity of such a disease or pathological condition. The term "pharmaceutically active peptide or protein" encompasses whole proteins or polypeptides and can also refer to pharmaceutically active fragments thereof. The term may also encompass pharmaceutically active analogs of peptides or proteins. The term "pharmaceutically active peptide or protein" encompasses peptides and proteins that are antigens, i.e., the peptide or protein elicits an immune response in a subject that may be therapeutic, or partially or fully protective.

[0224] In one embodiment, the pharmaceutically active peptide or protein is or comprises an immunologically active compound, or antigen, or epitope.

[0225] According to the present invention, the term "immunologically active compound" relates to any compound that modifies the immune response, preferably by inducing and / or suppressing immune cell maturation, inducing and / or suppressing cytokine biosynthesis, and / or modulating humoral immunity by stimulating antibody production by B cells. In one embodiment, the immune response involves stimulation of a cellular response, such as an antibody response (usually comprising immunoglobulin G (IgG)) and / or a T cell response. Immunologically active compounds may possess potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and may also down-regulate other aspects of the immune response, for example by diverting the immune response away from a TH2 immune response, which is useful for the treatment of a wide range of TH2-mediated diseases.

[0226] According to the present invention, the term "antigen" or "immunogen" encompasses any substance that elicits an immune response. In particular, "antigen" relates to any substance that specifically reacts with antibodies or T lymphocytes (T cells). According to the present invention, the term "antigen" includes any molecule that contains at least one epitope. Preferably, an antigen in the context of the present invention is a molecule that, optionally after processing, induces an immune response that is preferably specific to that antigen. According to the present invention, any suitable antigen can be used that is a candidate for an immune response, which may be both a humoral and a cellular immune response. In the context of embodiments of the present invention, the antigen is preferably presented by cells, preferably antigen-presenting cells, in the context of MHC molecules, which results in an immune response against the antigen. The antigen preferably corresponds to or is a product derived from a naturally occurring antigen. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents and pathogens, or the antigen may be a tumor antigen. According to the present invention, an antigen may correspond to a naturally occurring product, for example a viral protein or part thereof. In a preferred embodiment, the antigen is a surface polypeptide, i.e., a polypeptide that is naturally present on the surface of a cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor. The antigen is capable of eliciting an immune response against the cell, pathogen, bacterium, virus, fungus, parasite, allergen, or tumor.

[0227] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, disease-associated antigens can be used for therapeutic purposes. Disease-associated antigens are preferably associated with infectious diseases caused by microorganisms, typically microbial antigens, or associated with cancer, typically tumors.

[0228] The term "pathogen" refers to a pathogenic biological agent capable of causing disease in an organism, preferably a vertebrate. Pathogens include microorganisms such as bacteria, unicellular eukaryotes (protists), fungi, and viruses.

[0229] The terms "epitope," "antigenic peptide," "antigenic epitope," "immunogenic peptide," and "MHC-binding peptide" are used interchangeably herein and refer to an antigenic determinant in a molecule such as an antigen, i.e., a portion or fragment thereof of an immunologically active compound that is recognized by the immune system, e.g., by T cells, particularly when presented in the context of an MHC molecule. An epitope of a protein preferably comprises a continuous or discontinuous portion of the protein and is preferably between 5 and 100, preferably between 5 and 50, more preferably between 8 and 30, and most preferably between 10 and 25 amino acids in length; for example, an epitope may be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. According to the present invention, an epitope may be an "MHC-binding peptide" or an "antigenic peptide" because it can bind to an MHC molecule, such as an MHC molecule on the surface of a cell. The term "major histocompatibility complex" and its abbreviation "MHC" encompass MHC class I and MHC class II molecules and refer to a complex of genes present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen-presenting or diseased cells in the immune response; they bind peptides and present them for recognition by T cell receptors. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments derived from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. Preferred immunogenic moieties bind to MHC class I or class II molecules. As used herein, an immunogenic moiety is said to "bind" to an MHC class I or class II molecule if such binding is detectable using any assay known in the art. The term "MHC-binding peptide" refers to a peptide that binds to an MHC class I and / or MHC class II molecule.For class I MHC / peptide complexes, binding peptides are typically 8-10 amino acids in length, although longer or shorter peptides may be effective. For class II MHC / peptide complexes, binding peptides are typically 10-25 amino acids in length, particularly 13-18 amino acids in length, although longer and shorter peptides may be effective.

[0230] In one embodiment, the protein of interest according to the present invention comprises an epitope suitable for vaccination of a target organism. Those skilled in the art will understand that one of the principles of immunobiology and vaccination is based on the fact that an immune protective response against a disease is induced by immunizing an organism with an antigen immunologically related to the disease being treated. According to the present invention, the antigen is selected from the group consisting of self-antigens and non-self-antigens. The non-self-antigen is preferably a bacterial antigen, a viral antigen, a fungal antigen, an allergen, or a parasitic antigen. Preferably, the antigen comprises an epitope capable of inducing an immune response in the target organism. For example, the epitope can induce an immune response against a bacterium, a virus, a fungus, a parasite, an allergen, or a tumor.

[0231] In some embodiments, the non-self antigen is a bacterial antigen. In some embodiments, the antigen elicits an immune response against a bacterium that infects animals, including mammals such as birds, fish, and farm animals. Preferably, the bacterium against which the immune response is elicited is a pathogenic bacterium.

[0232] In some embodiments, the non-self antigen is a viral antigen. The viral antigen may be, for example, a protein, polypeptide, or peptide derived from a viral surface protein. For example, it may be a membrane-bound glycoprotein, a capsid protein or polypeptide, or a spike protein or polypeptide. In some embodiments, the antigen induces an immune response against a virus that infects animals, including mammals such as birds, fish, and farm animals. Preferably, the virus against which the immune response is induced is a pathogenic virus.

[0233] In some embodiments, the non-self antigen is a polypeptide or protein derived from a fungus. In some embodiments, the antigen elicits an immune response against a fungus that infects animals, including mammals such as birds, fish, and farm animals. Preferably, the fungus against which the immune response is elicited is a pathogenic fungus.

[0234] In some embodiments, the non-self antigen is a polypeptide or protein derived from a unicellular eukaryotic parasite. In some embodiments, the antigen elicits an immune response against a unicellular eukaryotic parasite, preferably a pathogenic unicellular eukaryotic parasite. The pathogenic unicellular eukaryotic parasite may be, for example, from the genus Plasmodium, such as P. falciparum, P. vivax, P. malariae, or P. ovale; from the genus Leishmania; or from the genus Trypanosoma, such as T. cruzi or T. brucei.

[0235] In some embodiments, the non-self antigen is an allergenic polypeptide or protein, which is suitable for allergen immunotherapy, also known as hyposensitization therapy.

[0236] In some embodiments, the antigen is a self-antigen, particularly a tumor antigen. Tumor antigens and their determination are known to those skilled in the art.

[0237] In the context of the present invention, the term "tumor antigen" or "tumor-associated antigen" refers to a protein that is specifically expressed in a limited number of tissues and / or organs or at a particular developmental stage under normal conditions. For example, a tumor antigen may be specifically expressed in gastric tissue, preferably the gastric mucosa, in reproductive organs, such as the testis, in trophoblast tissue, such as the placenta, or in germline cells under normal conditions, and is expressed or aberrantly expressed in one or more tumor or cancer tissues. In this context, "a limited number" preferably means 3 or less, more preferably 2 or less. Tumor antigens in the context of the present invention include, for example, differentiation antigens, preferably cell-type-specific differentiation antigens, i.e., proteins that are specifically expressed in a particular cell type at a particular differentiation stage under normal conditions, cancer / testis antigens, i.e., proteins that are specifically expressed in the testis and sometimes the placenta under normal conditions, and germline-specific antigens. In the context of the present invention, tumor antigens are preferably associated with the cell surface of cancer cells and are preferably not or rarely expressed in normal tissues. Preferably, cancer cells are identified by tumor antigens or aberrant expression of tumor antigens. In the context of the present invention, the tumor antigen expressed by cancer cells in a subject, for example, a patient suffering from cancer disease, is preferably a self-protein in the subject. In a preferred embodiment, the tumor antigen in the context of the present invention is specifically expressed in non-essential tissues or organs under normal conditions, i.e., tissues or organs that do not lead to the death of the subject when damaged by the immune system, or in organs or body structures that are inaccessible or barely accessible to the immune system. Preferably, the amino acid sequence of the tumor antigen is identical between the tumor antigen expressed in normal tissues and the tumor antigen expressed in cancer tissues.

[0238] Examples of tumor antigens that may be useful in the present invention include p53, ART-4, BAGE, beta-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, such as CLAUDIN-6, CLAUDIN-18.2, and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MA GE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pm1 / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1, or RU2, SAGE, SART-1, or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, and WT. Particularly preferred tumor antigens include CLAUDIN-18.2 (CLDN18.2) and CLAUDIN-6 (CLDN6).

[0239] According to the present invention, it has been observed that an efficient immune response can be induced by immunization using PEI-formulated self-replicating (self-amplifying) RNA to deliver antigens. PEI-formulated self-replicating RNA derived from Venezuelan equine encephalitis virus (VEEV) was particularly effective in delivering antigens that are membrane proteins.

[0240] According to the present invention, the term "membrane protein" relates to proteins that are associated with or bound to the cell membrane. Membrane proteins include integral membrane proteins, which are permanently anchored or part of the membrane, and peripheral membrane proteins, which are only transiently attached to the lipid bilayer or other integral proteins. Integral membrane proteins are classified into transmembrane proteins, which span the membrane, and single-pass transmembrane proteins, which are attached to only one side of the membrane. For example, membrane proteins can be classified into various general types: 1) Type I membrane proteins: These proteins have a single transmembrane domain in the mature protein. The N-terminus is extracellular and the C-terminus is cytoplasmic. The N-terminus of the protein is characterized by a classical signal peptide sequence that directs the protein for import into the ER. Proteins are further classified into type Ia (containing a cleavable signal sequence) and type Ib (not containing a cleavable signal sequence). Examples of type I membrane proteins include, but are not limited to, influenza HA, insulin receptor, glycophorin, LDL receptor, and viral G proteins. 2) Type II membrane proteins: For these single membrane domain proteins, the C-terminus is extracellular and the N-terminus is cytoplasmic. The N-terminus may contain a signal anchor sequence. Examples of this protein type include, but are not limited to, influenza neuraminidase, Golgi galactosyltransferase, Golgi sialyltransferase, sucrase-isomaltase precursor, asialoglycoprotein receptor, and transferrin receptor. 3) Multi-pass transmembrane proteins: In type I and type II membrane proteins, the polypeptide crosses the lipid bilayer once, whereas in multi-pass membrane proteins, the polypeptide crosses the membrane multiple times. Multi-pass transmembrane proteins are also further classified as type IIIa and type IIIb. Type IIIa proteins have a cleavable signal sequence. Type IIIb proteins have their amino acid termini exposed to the outer surface of the membrane but do not have a cleavable signal sequence. Type IIIa proteins include, but are not limited to, the M and L peptides of the photoreaction center. Type IIIb proteins include, but are not limited to, cytochrome P450 and E. coli leader peptidase. Further examples of multi-pass transmembrane proteins are membrane transporters, such as sugar transporters (glucose, xylose), and ion transporters. 4) Lipid-chain-anchored membrane proteins: These proteins are associated with the membrane bilayer by one or more covalently attached fatty acid or other types of lipid chains, called prenyl groups. 5) GPI-anchored membrane proteins: These proteins are attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor. 6) Peripheral membrane proteins: These proteins are indirectly bound to the membrane by non-covalent interactions with other membrane proteins.

[0241] The term "membrane protein," as used herein, encompasses cellular membrane proteins of human or non-human cells and viral envelope proteins. An example of a membrane protein is influenza hemagglutinin (HA), a glycoprotein found on the surface of influenza viruses. Influenza HA is responsible for binding of the virus to cells with sialic acid on their membranes, such as cells in the upper respiratory tract or red blood cells. It is also responsible for the fusion of the viral envelope with the endosomal membrane after the pH is reduced. Other influenza virus membrane proteins are the M2 protein, which is abundantly expressed on the cell surface, and neuraminidase (NA).

[0242] Thus, in one aspect, the present invention provides a method for producing a composition comprising: (a) a single-stranded self-replicating RNA encoding a peptide or protein containing an antigen or epitope, and (b) Polyalkyleneimine A pharmaceutical composition comprising Regarding.

[0243] In a further aspect, the present invention provides a compound comprising: (a) a single-stranded self-replicating RNA encoding a peptide or protein containing an antigen or epitope, and (b) Polyalkyleneimine A composition comprising Regarding.

[0244] In one embodiment of all aspects of the present invention, the molar ratio of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the single-stranded RNA (N:P ratio) is 1.0 to 30, preferably 2.0 to 15.0, more preferably 6.0 to 12.0.

[0245] In a further aspect, the present invention provides a method for producing a composition comprising: (a) a single-stranded self-replicating RNA encoding a peptide or protein containing an antigen or epitope, and (b) Polyalkyleneimine A composition comprising: A composition in which the molar ratio (N:P ratio) of the number of nitrogen atoms (N) in the polyalkyleneimine to the number of phosphorus atoms (P) in the single-stranded RNA is 1.0 to 30.0, preferably 2.0 to 15.0, more preferably 6.0 to 12.0. Regarding.

[0246] In one embodiment of all aspects of the invention, the ionic strength of the composition is 50 mM or less, and preferably the concentration of positively charged monovalent ions is 25 mM or less and the concentration of positively charged divalent cationic free ions is 20 μM or less.

[0247] In a further aspect, the present invention provides a method for producing a composition comprising: (a) a single-stranded self-replicating RNA encoding a peptide or protein containing an antigen or epitope, and (b) Polyalkyleneimine A composition comprising: A composition having an ionic strength of 50 mM or less. Regarding.

[0248] In one embodiment, the concentration of positively charged monovalent ions is 25 mM or less and the concentration of positively charged divalent cationic ions is 20 μM or less.

[0249] In one embodiment of all aspects of the invention, the single-stranded self-replicating RNA is a cis replicon.

[0250] In one embodiment, the single-stranded self-replicating RNA is derived from Venezuelan equine encephalitis virus (VEEV). In one embodiment, the single-stranded self-replicating RNA corresponds to or essentially corresponds to the genomic RNA of VEEV or an attenuated form thereof, and the open reading frame encoding the structural proteins is replaced by an open reading frame encoding a peptide or protein comprising an antigen or epitope. In one embodiment, the antigen, or peptide or protein comprising an antigen or epitope, is a membrane protein, such as a viral envelope protein. In one embodiment, the antigen is influenza hemagglutinin.

[0251] In one embodiment, the single-stranded self-replicating RNA is derived from Semliki Forest virus (SFV). In one embodiment, the single-stranded self-replicating RNA corresponds to or essentially corresponds to the genomic RNA of SFV or an attenuated form thereof, and the open reading frame encoding the structural proteins is replaced by an open reading frame encoding a peptide or protein comprising an antigen or epitope. In one embodiment, the antigen, or peptide or protein comprising the antigen or epitope, is not a membrane protein. In one embodiment, the antigen is a viral capsid protein.

[0252] In one embodiment of all aspects of the invention, the composition is for intramuscular administration, such as by intramuscular injection.

[0253] In one embodiment of all aspects of the invention, the single-stranded RNA and polyalkyleneimine are present in a polyplex particle.

[0254] In one embodiment of all aspects of the present invention, the polyalkyleneimine has the following general formula (I):

[0255] [ka]

[0256] (In the formula, R is H, an acyl group, or a compound of the following general formula (II):

[0257] [ka]

[0258] is a group comprising wherein R1 is H or a group represented by the following general formula (III):

[0259] [ka]

[0260] is a group comprising n, m, and l are independently selected from integers from 2 to 10; p, q, and r are integers, where the sum of p, q, and r is such that the average molecular weight of the polymer is 1.5×10 2 From 10 7 Da, preferably 5000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da). Includes.

[0261] In one embodiment, n, m, and l are independently selected from 2, 3, 4, and 5, preferably 2 and 3. In one embodiment, R is H. In one embodiment, R is H or an acyl group.

[0262] In one embodiment of all aspects of the invention, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine.

[0263] In one embodiment of all aspects of the invention, at least 92% of the N atoms in the polyalkyleneimine are protonatable.

[0264] In one embodiment of all aspects of the present invention, the composition of the present invention comprises one or more additives. In one embodiment, the one or more additives are selected from the group consisting of buffer substances, sugars, stabilizers, cryoprotectants, lyoprotectants, and chelating agents. In one embodiment of all aspects of the present invention, the composition of the present invention comprises one or more polymers. In one embodiment, the buffer substance comprises at least one selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), acetate buffer systems and analogs, phosphate buffer systems, or citrate buffer systems. In one embodiment of all aspects of the present invention, the composition of the present invention comprises a buffer for buffering to a pH range between 4 and 6.5, between 3 and 5, or between 3.5 and 4.5. Examples of such buffer systems are acetate buffer, HEPES buffer, phosphate buffer, or acetate buffer. In one embodiment, the sugar is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides, preferably at least one selected from glucose, trehalose, sucrose, and dextran. In one embodiment, the additive is dextran having an average molar mass between 1 kDa and 100 kDa. In one embodiment, the cryoprotectant comprises at least one selected from the group consisting of glycols, such as ethylene glycol, propylene glycol, and glycerol. In one embodiment, the chelating agent comprises EDTA. In one embodiment, the composition of the present invention comprises one or more block copolymers comprising ethylene oxide building blocks and propylene oxide building blocks. In one embodiment, the composition of the present invention comprises a copolymer comprising ethylenediamine groups. In one embodiment, the composition of the present invention comprises an amphiphilic block copolymer, preferably comprising ethylene oxide building blocks and propylene oxide building blocks, and optionally also comprising ethylenediamine groups.

[0265] In one embodiment of all aspects of the invention, the composition comprises HEPES-buffered glucose (HBG or HBGx1), MES-buffered glucose (MBG or MBGx1), acetate-buffered glucose, or HEPES-buffered trehalose (HBT or HBTx1). In one embodiment of all aspects of the invention, the composition comprises glucose, trehalose, or sucrose in an acetate buffer at a concentration ranging from 0.1 mM to 10 mM. In one embodiment of all aspects of the invention, the composition comprises glucose, trehalose, or sucrose in a phosphate buffer at a concentration ranging from 0.1 mM to 10 mM.

[0266] In one embodiment of all aspects of the invention, the z-average size of the particles is less than 200 nm, preferably less than 150 nm, and more preferably less than 100 nm. In one embodiment, the z-average size of the particles is between 50 nm and 200 nm. In one embodiment of all aspects of the invention, the zeta potential of the particles is 20 mV or greater, preferably 25 to 40 mV. In one embodiment of all aspects of the invention, the electrophoretic mobility (μ) of the particles is between 1 and 1.6 μm*cm / V*S. In one embodiment of all aspects of the invention, the z-average size and / or zeta potential and / or electrophoretic mobility of the particles are determined in a suspension comprising polyplex particles and HEPES-buffered glucose (HBG) or HEPES-buffered trehalose (HBT). In one embodiment, the HBG comprises 5% glucose (w / v) and 10 mM HEPES, pH 7.1, or the HBT comprises 10% trehalose (w / v) and 10 mM HEPES, pH 7.1. In one embodiment, the z-average particle size is determined by dynamic light scattering and data analysis using a cumulant algorithm. In one embodiment, the translational diffusion coefficient is measured by dynamic light scattering. The Stokes-Einstein equation is then used to calculate the z-average. In one embodiment, the electrophoretic mobility is measured by laser Doppler electrophoresis. The Henry equation or the Smoluchowski equation is then used to calculate the z-potential.

[0267] In one embodiment, MBG contains 5% glucose (w / v) and 10 mM MES, hi one embodiment, acetate buffered glucose contains 5% glucose (w / v) and 10 mM acetic acid.

[0268] In one embodiment of all aspects of the invention, the particles are neutral or positively charged, preferably at physiological pH or at a pH between 4.5 and 7.5.

[0269] In one embodiment of all aspects of the invention, the single stranded RNA is a molecule of between 6,000 and 15,000 bases, preferably between 9,000 and 12,000 bases.

[0270] In one embodiment of all aspects of the invention, the compositions described herein are for use in therapy, hi one embodiment of all aspects of the invention, the compositions described herein are vaccine compositions.

[0271] In a further aspect, the invention relates to a composition as described herein for inducing an immune response. In one embodiment, the composition is administered by intramuscular administration. In one embodiment, the immune response is against an antigen or epitope.

[0272] In a further aspect, the invention relates to a method of inducing an immune response comprising administering a composition described herein. In one embodiment, the composition is administered by intramuscular administration. In one embodiment, the immune response is directed against an antigen or epitope.

[0273] In some embodiments, the pharmaceutically active peptide or protein need not be an antigen that elicits an immune response. Suitable pharmaceutically active proteins or peptides include cytokines and immune system proteins, such as immunologically active compounds (e.g., interleukins, colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, T-cell receptors, immunoglobulins), hormones (insulin, thyroid hormones, catecholamines, gonadotropins, trophic hormones, prolactin, oxytocin), and the like. , dopamine, bovine somatotropin, leptin, and the like), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, and the like), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, biosynthetic or degradative cholesterol, steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochromes, adenylate cyclase or guanylate cyclase cyclase, neuraminidase, and the like), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor binding proteins, and the like), transcription and translation factors, tumor growth suppressor proteins (e.g., proteins that inhibit angiogenesis), structural proteins (such as collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin), blood proteins (thrombin, serum albumin, Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (GCSF), or modified Factor VIII, anticoagulants, and the like).In one embodiment, the pharmaceutically active protein according to the invention is a cytokine involved in the regulation of lymphoid homeostasis, preferably a cytokine involved in, and preferably inducing or enhancing, the development, priming, expansion, differentiation, and / or survival of T cells. In one embodiment, the cytokine is an interleukin, such as IL-2, IL-7, IL-12, IL-15, or IL-21.

[0274] Another preferred protein of interest encoded by the open reading frame is an inhibitor of interferon (IFN) signaling. It has been reported that the viability of cells into which RNA has been introduced for expression may be reduced, particularly when the cells are transfected multiple times with the RNA, but IFN inhibitors have been found to improve the viability of cells in which the RNA is expressed (WO 2014 / 071963(A1)). Preferably, the inhibitor is an inhibitor of type I IFN signaling. By preventing extracellular IFN from engaging the IFN receptor and inhibiting intracellular IFN signaling in the cell, stable expression of the RNA in the cell is possible. Alternatively, or in addition, by preventing extracellular IFN from engaging the IFN receptor and inhibiting intracellular IFN signaling, cell survival is improved, particularly when the cells are repeatedly transfected with the RNA. Without wishing to be bound by theory, it is anticipated that intracellular IFN signaling may result in translation inhibition and / or RNA degradation. This can be addressed by inhibiting one or more IFN-induced antiviral effector proteins. The IFN-induced antiviral effector proteins can be selected from the group consisting of RNA-dependent protein kinase (PKR), 2',5'-oligoadenylate synthetase (OAS), and RNase L. Inhibiting intracellular IFN signaling can include inhibiting a PKR-dependent pathway and / or an OAS-dependent pathway. Suitable target proteins are proteins capable of inhibiting a PKR-dependent pathway and / or an OAS-dependent pathway. Inhibiting a PKR-dependent pathway can include inhibiting eIF2-alpha phosphorylation. Inhibiting PKR can include treating cells with at least one PKR inhibitor. The PKR inhibitor can be a viral inhibitor of PKR. A preferred viral inhibitor of PKR is vaccinia virus E3. When a peptide or protein (e.g., E3, K3) is intended to inhibit intracellular IFN signaling, intracellular expression of the peptide or protein is preferred.Vaccinia virus E3 is a 25 kDa dsRNA-binding protein (encoded by the gene E3L) that binds to and captures dsRNA, preventing the activation of PKR and OAS. E3 can directly bind to PKR and inhibit its activity, resulting in reduced phosphorylation of eIF2-alpha. Other suitable inhibitors of IFN signaling include Herpes simplex virus ICP34.5, Toscana virus NS, Bombyx mori nucleopolyhedrovirus PK2, and HCV NS34A.

[0275] In one embodiment, an inhibitor of intracellular or extracellular IFN signaling is encoded by a replicon. The replicon includes nucleic acid sequence elements that enable replication by the alphavirus replicase, typically CSE1, CSE2, and CSE4, and preferably also includes a nucleic acid sequence element that enables production of a subgenomic transcript, i.e., a subgenomic promoter, typically including CSE3. The replicon may further include one or more non-polypeptide sequence-modifying modifications, such as a cap, a poly(A) sequence, or codon usage adaptation, as described herein. When multiple open reading frames are present on the replicon, the inhibitor of intracellular IFN signaling may be encoded by any one of them, optionally under the control of a subgenomic promoter or not. In a preferred embodiment, the inhibitor of intracellular IFN signaling is encoded by the most upstream open reading frame of the RNA replicon. If the inhibitor of intracellular IFN signaling is encoded by the most upstream open reading frame of the RNA replicon, the genetic information encoding the inhibitor of intracellular IFN signaling will be translated early after introducing the RNA replicon into the host cell, and the resulting protein can then inhibit intracellular IFN signaling.

[0276] Additional preferred proteins of interest encoded by the open reading frames are functional alphavirus nonstructural proteins. The term "alphavirus nonstructural protein" encompasses any and all co- or post-translationally modified forms of alphavirus nonstructural proteins, including carbohydrate-modified (e.g., glycosylated) and lipid-modified forms.

[0277] In some embodiments, the term "alphavirus nonstructural proteins" refers to any one or more of the individual nonstructural proteins (nsP1, nsP2, nsP3, nsP4) of alphavirus origin, or a polyprotein comprising the polypeptide sequences of two or more nonstructural proteins of alphavirus origin. In some embodiments, "alphavirus nonstructural proteins" refers to nsP123 and / or nsP4. In other embodiments, "alphavirus nonstructural proteins" refers to nsP1234. In one embodiment, the proteins of interest encoded by the open reading frames consist of nsP1, nsP2, nsP3, and nsP4 all as a single, optionally cleavable polyprotein: nsP1234. In one embodiment, the proteins of interest encoded by the open reading frames consist of nsP1, nsP2, and nsP3 as a single, optionally cleavable polyprotein: nsP123. In that embodiment, nsP4 may be an additional protein of interest or may be encoded by an additional open reading frame.

[0278] In some embodiments, the alphavirus nonstructural proteins are capable of forming complexes or associations, e.g., within a host cell. In some embodiments, "alphavirus nonstructural proteins" refers to a complex or association of nsP123 (synonymously, P123) and nsP4. In some embodiments, "alphavirus nonstructural proteins" refers to a complex or association of nsP1, nsP2, and nsP3. In some embodiments, "alphavirus nonstructural proteins" refers to a complex or association of nsP1, nsP2, nsP3, and nsP4. In some embodiments, "alphavirus nonstructural proteins" refers to any one or more complexes or associations selected from the group consisting of nsP1, nsP2, nsP3, and nsP4. In some embodiments, the alphavirus nonstructural proteins include at least nsP4.

[0279] The term "complex" or "association" refers to two or more identical or different protein molecules in spatial proximity. The proteins of a complex are preferably in direct or indirect physical or physiochemical contact with each other. A complex or association may consist of multiple different proteins (heteromultimers) and / or multiple copies of one particular protein (homomultimers). In the context of alphavirus nonstructural proteins, the term "complex or association" describes a group of at least two protein molecules, at least one of which is an alphavirus nonstructural protein. A complex or association may consist of multiple copies of one particular protein (homomultimers) and / or multiple different proteins (heteromultimers). In the context of multimers, "multiple" means two or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more.

[0280] The term "functional alphavirus nonstructural protein" includes alphavirus nonstructural proteins that have replicase function. Thus, "functional alphavirus nonstructural protein" includes alphavirus replicases. "Replicase function" includes the function of an RNA-dependent RNA polymerase (RdRP), i.e., an enzyme capable of catalyzing the synthesis of negative-strand RNA from a positive-strand RNA template, and / or an enzyme capable of catalyzing the synthesis of positive-strand RNA from a negative-strand RNA template. Thus, the term "functional alphavirus nonstructural protein" can refer to a protein or complex that synthesizes negative-strand RNA using positive-strand (e.g., genomic) RNA as a template, a protein or complex that synthesizes new positive-strand RNA using the complement of the negative strand of genomic RNA as a template, and / or a protein or complex that synthesizes a subgenomic transcript using a fragment of the complement of the negative strand of genomic RNA as a template. Functional alphavirus nonstructural proteins may further possess one or more additional functions, such as, for example, a protease (for self-cleavage), a helicase, a terminal adenylyltransferase (for poly(A) tailing), a methyltransferase and a guanylyltransferase (to provide a 5'-cap to the nucleic acid), a nuclear localization site, a triphosphatase, etc. (Gould et al., 2010, Antiviral Res., 87:111-124; Rupp et al., 2015, J. Gen. Virol., 96:2483-500).

[0281] According to the present invention, the term "alphavirus replicase" refers to an RNA-dependent RNA polymerase of an alphavirus, including RNA-dependent RNA polymerases derived from naturally occurring alphaviruses (alphaviruses found in nature) and RNA-dependent RNA polymerases derived from variants or derivatives of alphaviruses, such as attenuated alphaviruses. In the context of the present invention, the terms "replicase" and "alphavirus replicase" are used interchangeably, unless the context dictates that any particular replicase is not an alphavirus replicase.

[0282] The term "replicase" includes all variants, particularly post-translationally modified variants, conformations, isoforms, and homologs, of alphavirus replicase that are expressed by alphavirus-infected cells or by cells transfected with nucleic acid encoding alphavirus replicase. The term "replicase" also includes all forms of replicase that are produced by, or can be produced by, recombinant methods. For example, replicase may be produced by recombinant methods that include a tag, e.g., a myc-tag, HA-tag, or oligohistidine tag (His-tag), that facilitates detection and / or purification of the replicase in the laboratory.

[0283] Optionally, the alphavirus replicase is additionally functionally defined by its ability to bind to any one or more of alphavirus conserved sequence element 1 (CSE1) or its complementary sequence, conserved sequence element 2 (CSE2) or its complementary sequence, conserved sequence element 3 (CSE3) or its complementary sequence, or conserved sequence element 4 (CSE4) or its complementary sequence. Preferably, the replicase is capable of binding to CSE2 [i.e., the (+) strand] and / or CSE4 [i.e., the (+) strand], or is capable of binding to the complement of CSE1 [i.e., the (-) strand] and / or the complement of CSE3 [i.e., the (-) strand].

[0284] The source of the replicase is not limited to any particular alphavirus. In a preferred embodiment, the alphavirus replicase comprises nonstructural proteins from Semliki Forest virus, including naturally occurring Semliki Forest virus and variants or derivatives of Semliki Forest virus, such as attenuated Semliki Forest virus. In an alternative preferred embodiment, the alphavirus replicase comprises nonstructural proteins from naturally occurring Sindbis virus and variants or derivatives of Sindbis virus, such as attenuated Sindbis virus. In an alternative preferred embodiment, the alphavirus replicase comprises nonstructural proteins from VEEV, including naturally occurring Venezuelan equine encephalitis virus (VEEV), and variants or derivatives of VEEV, such as attenuated VEEV. In an alternative preferred embodiment, the alphavirus replicase comprises nonstructural proteins from CHIKV, including naturally occurring Chikungunya virus (CHIKV), and variants or derivatives of CHIKV, such as attenuated CHIKV.

[0285] A replicase may also comprise nonstructural proteins from more than one alphavirus. Thus, heterologous complexes or assemblies comprising alphavirus nonstructural proteins and having replicase function are equally encompassed by the present invention. For illustrative purposes only, a replicase may comprise one or more nonstructural proteins (e.g., nsP1, nsP2) from a first alphavirus and one or more nonstructural proteins (nsP3, nsP4) from a second alphavirus. The nonstructural proteins from two or more different alphaviruses may be encoded by separate open reading frames or may be encoded by a single open reading frame as a polyprotein, e.g., nsP1234.

[0286] In some embodiments, the functional alphavirus nonstructural proteins are capable of forming membrane replication complexes and / or intracellular vacuoles in which the functional alphavirus nonstructural proteins are expressed.

[0287] When a functional alphavirus nonstructural protein, i.e., an alphavirus nonstructural protein with replicase function, is encoded by a nucleic acid molecule according to the present invention, the subgenomic promoter of the replicon, if present, is preferably compatible with said replicase. In this context, compatible means that the alphavirus replicase, if present, is capable of recognizing the subgenomic promoter. In one embodiment, this is achieved when the subgenomic promoter is native to the alphavirus from which the replicase is derived, i.e., when the natural origin of these sequences is the same alphavirus. In an alternative embodiment, the subgenomic promoter is not native to the alphavirus from which the alphavirus replicase is derived, provided that the alphavirus replicase is capable of recognizing the subgenomic promoter. In other words, the replicase is compatible with the subgenomic promoter (cross-viral compatibility). Examples of cross-viral compatibility involving subgenomic promoters and replicases originating from different alphaviruses are known in the art. Any combination of subgenomic promoters and replicases is possible, as long as cross-viral compatibility exists. Cross-viral compatibility can be easily tested by those skilled in the art by incubating the replicase to be tested with RNA (which RNA has the subgenomic promoter to be tested) under conditions suitable for RNA synthesis from the subgenomic promoter. If a subgenomic transcript is prepared, the subgenomic promoter and replicase are determined to be compatible. Various examples of cross-viral compatibility are known (reviewed by Strauss & Strauss, Microbiol. Rev., 1994, vol. 58, pp. 491-562).

[0288] In the present invention, open reading frames encoding functional alphavirus nonstructural proteins may be provided on an RNA replicon, or alternatively, may be provided as separate nucleic acid molecules, e.g., mRNA molecules. The separate mRNA molecules may optionally include, for example, a cap, a 5'-UTR, a 3'-UTR, a poly(A) sequence, and / or matching codon usage. The separate mRNA molecules may be provided in trans, as described herein.

[0289] When an open reading frame encoding functional alphavirus nonstructural proteins is provided on an RNA replicon, the replicon is preferably capable of replication via the functional alphavirus nonstructural proteins. In particular, an RNA replicon encoding functional alphavirus nonstructural proteins is capable of replication via the functional alphavirus nonstructural proteins encoded by the replicon. This embodiment is highly preferred when no nucleic acid molecules encoding functional alphavirus nonstructural proteins are provided in trans. In this embodiment, cis replication of the replicon is targeted. In a preferred embodiment, the RNA replicon contains not only an open reading frame encoding functional alphavirus nonstructural proteins, but also at least one additional open reading frame encoding a protein of interest, capable of replication via the functional alphavirus nonstructural proteins.

[0290] When multiple open reading frames are present on the replicon, the functional alphavirus nonstructural proteins may be encoded by any one of them, optionally under the control of a subgenomic promoter or not, but preferably not under the control of a subgenomic promoter. In a preferred embodiment, the functional alphavirus nonstructural proteins are encoded by the most upstream open reading frame of the RNA replicon. When the functional alphavirus nonstructural proteins are encoded by the most upstream open reading frame of the RNA replicon, the genetic information encoding the functional alphavirus nonstructural proteins will be translated early after introduction of the RNA replicon into the host cell, and the resulting proteins can then promote replication and, optionally, production of subgenomic transcripts in the host cell.

[0291] The presence of an open reading frame encoding a functional alphavirus nonstructural protein, whether contained in the replicon or in a separate nucleic acid molecule provided in trans, allows the replicon to replicate and, as a result, the gene of interest encoded by the replicon to be expressed at high levels under the control of a subgenomic promoter.

[0292] The RNA replicon is suitable for the expression of one or more genes encoding a peptide or protein of interest, optionally under the control of a subgenomic promoter. Various embodiments are possible. One or more open reading frames, each encoding a peptide or protein of interest, may be present on the RNA replicon. The most upstream open reading frame of the RNA replicon is referred to as the "first open reading frame." In some embodiments, the "first open reading frame" is the only open reading frame of the RNA replicon. Optionally, one or more additional open reading frames may be present downstream of the first open reading frame. One or more additional open reading frames downstream of the first open reading frame may be referred to as the "second open reading frame," "third open reading frame," etc., in the order in which they are present downstream of the first open reading frame (5' to 3'). Preferably, each open reading frame includes a start codon (base triplet), typically AUG (in the RNA molecule), which corresponds to ATG (in the respective DNA molecule).

[0293] When the replicon includes a 3' replication recognition sequence, it is preferred that all open reading frames are located upstream of the 3' replication recognition sequence.

[0294] When an RNA replicon containing one or more open reading frames is introduced into a host cell, the replicon can directly serve as a template for translation of the first open reading frame. Preferably, the replicon contains a 5'-cap, which facilitates direct expression of the gene encoded by the first open reading frame from the replicon.

[0295] In some embodiments, at least one open reading frame of the replicon is under the control of a subgenomic promoter, preferably an alphavirus subgenomic promoter. Alphavirus subgenomic promoters are highly efficient and therefore suitable for high-level heterologous gene expression. Preferably, the subgenomic promoter is a promoter for a subgenomic transcript in an alphavirus. This means that the subgenomic promoter is native to the alphavirus and, preferably, controls the transcription of an open reading frame encoding one or more structural proteins in the alphavirus. Alternatively, the subgenomic promoter is a variant of an alphavirus subgenomic promoter, with any variant that functions as a promoter for subgenomic RNA transcription in a host cell being suitable. When the replicon comprises a subgenomic promoter, the replicon preferably comprises conserved sequence element 3 (CSE3) or a variant thereof.

[0296] Preferably, at least one open reading frame under the control of a subgenomic promoter is located downstream of the subgenomic promoter. Preferably, the subgenomic promoter controls the production of a subgenomic RNA comprising a transcript of the open reading frame.

[0297] In some embodiments, the first open reading frame is under the control of a subgenomic promoter. When the first open reading frame is under the control of a subgenomic promoter, its localization is similar to that of open reading frames encoding structural proteins in the genome of an alphavirus. When the first open reading frame is under the control of a subgenomic promoter, the gene encoded by the first open reading frame can preferably be expressed from both the replicon and its subgenomic transcript (the latter in the presence of functional alphavirus nonstructural proteins). One or more additional open reading frames, each under the control of a subgenomic promoter, may be present downstream of the first open reading frame under the control of a subgenomic promoter. The gene encoded by one or more additional open reading frames, e.g., a second open reading frame, may be translated from one or more subgenomic transcripts, each under the control of a subgenomic promoter. For example, an RNA replicon may include a subgenomic promoter that controls the production of a transcript encoding a second protein of interest.

[0298] In other embodiments, the first open reading frame is not under the control of a subgenomic promoter. When the first open reading frame is not under the control of a subgenomic promoter, the gene encoded by the first open reading frame can be expressed from a replicon. One or more additional open reading frames, each under the control of a subgenomic promoter, can be present downstream of the first open reading frame. The gene encoded by the one or more additional open reading frames can be expressed from the subgenomic transcript.

[0299] In cells containing a replicon according to the present invention, the replicon may be amplified by functional alphavirus nonstructural proteins. Additionally, if the replicon contains one or more open reading frames under the control of a subgenomic promoter, one or more subgenomic transcripts are expected to be prepared by the functional alphavirus nonstructural proteins. The functional alphavirus nonstructural proteins may be provided in trans or may be encoded by the open reading frames of the replicon.

[0300] When a replicon contains two or more open reading frames encoding proteins of interest, each open reading frame preferably encodes a different protein, e.g., a different pharmaceutically active peptide or protein, e.g., the protein encoded by the second open reading frame is different from the protein encoded by the first open reading frame.

[0301] In some embodiments, the protein of interest encoded by the first and / or additional open reading frames, preferably the first open reading frame, is a functional alphavirus nonstructural protein or an inhibitor of IFN signaling, e.g., E3. In some embodiments, the protein of interest encoded by the first and / or additional open reading frames, e.g., the second open reading frame, is a pharmaceutically active peptide or protein, or a reporter protein.

[0302] In one embodiment, the protein of interest encoded by the first open reading frame is a functional alphavirus nonstructural protein. In that embodiment, the replicon preferably includes a 5'-cap. In particular, when the protein of interest encoded by the first open reading frame is a functional alphavirus nonstructural protein, preferably when the replicon includes a 5'-cap, the nucleic acid sequence encoding the functional alphavirus nonstructural protein can be efficiently translated from the replicon, and the resulting protein can then promote replication of the replicon and synthesis of subgenomic transcripts. This embodiment may be preferred when additional nucleic acid molecules encoding functional alphavirus nonstructural proteins are not used or are not present with the replicon. In this embodiment, cis replication of the replicon is targeted.

[0303] The compositions described herein can be administered to treat a disease, such as a disease described herein, e.g., a disease associated with the antigen encoded by the administered RNA.

[0304] The term "disease" refers to an abnormal condition affecting an individual's body. Disease is often understood as a medical condition associated with specific symptoms and signs. Diseases may be caused by factors originally from external sources, such as infectious diseases, or they may be caused by a malfunction within the body, such as autoimmune diseases. In humans, "disease" is often used in a broader sense to refer to any condition that causes pain, disability, suffering, social problems, or death to the affected individual, or that causes similar problems to those who come into contact with that individual. In this broader sense, "disease" may encompass injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical variations in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Living with many illnesses can alter one's outlook on life and personality, so illnesses typically affect individuals not only physically but also emotionally.

[0305] The term "antigen-associated disease" or "antigen-associated disease" refers to any disease that involves an antigen, e.g., a disease characterized by the presence of an antigen. The antigen-associated disease can be an infectious disease, an autoimmune disease, or a cancer disease or simply a cancer. As noted above, the antigen can also be a disease-associated antigen, e.g., a tumor-associated antigen, a viral antigen, or a bacterial antigen.

[0306] The term "infectious disease" refers to any disease (e.g., the common cold) that can be transmitted from individual to individual or organism to organism and is caused by a microbial agent. Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases caused by viruses, bacteria, and parasites, respectively. In this regard, an infectious disease may be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, influenza, animal diseases such as foot-and-mouth disease, petit ruminant disease, porcine reproductive and respiratory syndrome virus, or parasitic diseases such as Chagas, malaria, and others.

[0307] The term "autoimmune disease" refers to any disease in which the body mounts an immunogenic (i.e., immune system) response against some component of one's own tissues. In other words, the immune system loses its ability to recognize some tissue or system within the body as self and targets and attacks it as if it were foreign. Autoimmune diseases can be classified as those in which a single organ is primarily affected (e.g., hemolytic anemia and antiimmune thyroiditis) and those in which the autoimmune disease process spreads through many tissues (e.g., systemic lupus erythematosus). For example, multiple sclerosis is thought to be caused by T cells attacking the nerve sheaths surrounding nerve fibers in the brain and spinal cord. This results in loss of coordination, weakness, and blurred vision. Autoimmune diseases are known in the art and include, for example, Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis, and the like.

[0308] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, glioma, and leukemia. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, carcinoma of the genital and reproductive organs, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, kidney cancer, renal cell carcinoma, cancer of the renal pelvis, neoplasms of the central nervous system (CNS), neuroectodermal carcinoma, spinal axis tumor, glioma, meningioma, and pituitary adenoma. The term "cancer" according to the present invention also includes cancer metastasis.

[0309] The term "immune response" relates to the reaction of the immune system to an immunogenic organism, cell, or substance, such as a bacterium or virus. The term "immune response" encompasses innate and adaptive immune responses. Preferably, the immune response is associated with the activation of immune cells, induction of cytokine biosynthesis and / or antibody production.

[0310] The immune response induced by the composition of the present invention preferably comprises the steps of activating antigen-presenting cells, such as dendritic cells and / or macrophages, presenting an antigen or a fragment thereof by the antigen-presenting cells, and activating cytotoxic T cells resulting from this presentation.

[0311] The term "immune cell" refers to a cell of the immune system that is involved in the defense of an individual's body. The term "immune cell" encompasses specific types of immune cells and their precursors, including white blood cells, including macrophages; monocytes (progenitor cells of macrophages); granulocytes, such as neutrophils; eosinophils and basophils; dendritic cells; mast cells; and lymphocytes, such as B cells, T cells, and natural killer (NK) cells. Macrophages, monocytes (progenitor cells of macrophages), neutrophils, dendritic cells, and mast cells are phagocytic cells.

[0312] The term "immunotherapy" relates to the treatment of a disease or condition by inducing, enhancing, or suppressing an immune response. Immunotherapies designed to induce or amplify an immune response are classified as activating immunotherapies, while immunotherapies that reduce or suppress an immune response are classified as suppressing immunotherapies. The term "immunotherapy" encompasses antigen immunization or vaccination, or tumor immunization or vaccination. The term "immunotherapy" also relates to the manipulation of the immune response, such that an inappropriate immune response is modulated toward a more appropriate immune response, in the context of autoimmune diseases such as rheumatoid arthritis, allergies, diabetes, or multiple sclerosis.

[0313] The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the intent of inducing an immune response, for example, for therapeutic or prophylactic reasons.

[0314] The term "therapeutic treatment" or simply "treatment" refers to any treatment that improves the health status and / or prolongs (lengthens) the lifespan of an individual. The treatment may eliminate the disease in an individual, halt or slow the progression of the disease in an individual, inhibit or slow the progression of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or has previously had the disease.

[0315] The term "prophylactic treatment" or "preventative treatment" relates to any treatment intended to prevent a disease from appearing in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

[0316] The terms "protect," "prevent," "prophylactic," "preventive," or "protective" relate to the prevention and / or treatment of the appearance and / or spread of a disease, e.g., a tumor, in an individual. For example, prophylactic administration of immunotherapy, by administering a composition of the invention, can protect the recipient individual from tumor development. Therapeutic administration of immunotherapy, for example, by administering a composition of the invention, can halt disease development, e.g., result in inhibition of tumor progression / growth. This includes slowing tumor progression / growth, particularly preventing tumor progression, preferably resulting in tumor elimination. Therapeutic administration of immunotherapy can protect an individual, for example, from intrametastasis or metastasis of an existing tumor.

[0317] The term "individual" or "subject" relates to vertebrates, particularly mammals. For example, mammals in the context of the present invention are humans, non-human primates, domestic mammals such as dogs, cats, sheep, cows, goats, pigs, horses, etc., laboratory animals such as mice, rats, rabbits, guinea pigs, etc., and captive animals such as zoo animals. The term "subject" also relates to non-mammalian vertebrates such as birds (particularly domestic birds such as chickens, ducks, geese, turkeys, etc.), and fish (particularly farmed fish such as salmon or catfish). The term "animal" as used herein also includes humans.

[0318] Agents, such as the polyplex particles described herein, can be administered in the form of any suitable pharmaceutical composition. The term "pharmaceutical composition" relates to a formulation comprising a therapeutically active agent or a salt thereof, preferably together with pharmaceutical excipients such as buffers, preservatives, and osmolality adjusting agents. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administering the pharmaceutical composition to an individual. Pharmaceutical compositions are also known in the art as pharmaceutical formulations. Pharmaceutical compositions may be administered locally or systemically. In the context of the present invention, a pharmaceutical composition comprises the particles described herein.

[0319] The term "systemic administration" refers to the administration of a therapeutically active agent in a manner that distributes the agent widely throughout the body of an individual in significant amounts and exerts its biological effect. According to the present invention, administration is preferably by parenteral administration.

[0320] The term "parenteral administration" refers to the administration of a therapeutically active agent so that the agent does not pass through the intestine. The term "parenteral administration" includes, but is not limited to, intravenous, subcutaneous, intradermal, or intraarterial administration.

[0321] In a particularly preferred embodiment, the compositions according to the invention are administered to muscle cells, such as skeletal muscle, and therefore intramuscular administration, such as by intramuscular injection, is the preferred route of administration.

[0322] Administration can be achieved in a variety of ways. In one embodiment, the compositions according to the present invention are administered by injection. In a preferred embodiment, the injection is via a needle. Alternatively, needle-free injection may be used.

[0323] The pharmaceutical composition of the present invention may contain at least one adjuvant. The term "adjuvant" refers to a compound that, when administered to an individual in combination with an antigen or antigenic peptide, prolongs, enhances, or accelerates the immune response. Adjuvants are thought to exert their biological activity through one or more mechanisms, including increasing the surface area of ​​the antigen, prolonging antigen retention in the body, delaying antigen release, targeting antigens to macrophages, increasing antigen uptake, enhancing antigen processing, stimulating cytokine release, stimulating and activating immune cells such as B cells, macrophages, dendritic cells, and T cells, and non-specific activation of immune cells. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immunostimulating complexes. Examples of adjuvants include, but are not limited to, saponin, Freund's incomplete adjuvant, Freund's complete adjuvant, tocopherol, or alum.

[0324] Pharmaceutical compositions according to the present invention are generally applied as "pharmaceutically acceptable preparations" in "pharmaceutically effective amounts."

[0325] The term "pharmaceutically effective amount" refers to an amount that, alone or together with further doses, achieves the desired response or desired effect. When treating a specific disease, the desired response preferably relates to the inhibition of the disease process. This includes slowing the progression of the disease, and in particular, halting or reversing the progression of the disease. In the treatment of a disease, the desired response may also be delaying or preventing the onset of the disease or condition. The effective amount of the compositions described herein will depend on the condition to be treated, the severity of the disease, individual parameters of the patient, such as age, physiological state, size, and weight, the duration of treatment, the type of concomitant therapy (if any), the specific route of administration, and similar factors. Thus, the administered dose of the compositions described herein may depend on such various parameters. If the patient does not respond adequately to the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0326] The term "pharmaceutically acceptable" refers to the non-toxicity of a material that does not interact with the action of the active ingredients of the pharmaceutical composition.

[0327] Pharmaceutical compositions of the invention may contain salts, buffers, preservatives, carriers, and optionally other therapeutic agents. Preferably, pharmaceutical compositions of the invention include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0328] The term "excipient" is intended to refer to any substance in a pharmaceutical composition that is not an active ingredient, such as a binder, lubricant, thickener, surfactant, preservative, emulsifier, buffer, flavoring agent, or coloring agent.

[0329] The term "diluent" relates to an agent for diluting and / or thinning, and includes one or more of a fluid, liquid, or solid suspension, and / or mixing medium.

[0330] The term "carrier" refers to one or more compatible solid or liquid fillers or diluents suitable for administration to humans. The term "carrier" refers to a natural or synthetic organic or inorganic component with which the active ingredient is combined to facilitate application of the active ingredient. Preferably, the carrier component is a sterile liquid, such as water or oil, including mineral oil, animal or plant-derived oil, such as peanut oil, soybean oil, sesame oil, sunflower oil, etc. Salt solution and aqueous dextrose and glycerin solutions can also be used as aqueous carrier compounds.

[0331] Pharmaceutically acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, ed., 1985). Examples of suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, low-melting waxes, cocoa butter, and the like. Examples of suitable diluents include ethanol, glycerin, and water.

[0332] Pharmaceutical carriers, excipients, or diluents can be selected taking into account the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions of the present invention may comprise any suitable binder, lubricant, suspending agent, coating agent, and / or solubilizing agent as, or in addition to, the carrier, excipient, or diluent. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, and the like. Preservatives, stabilizers, dyes, and even flavoring agents can also be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

[0333] In one embodiment, the composition is an aqueous composition. The aqueous composition may optionally contain a solute, such as a salt. In one embodiment, the composition is in the form of a lyophilized composition. The lyophilized composition can be obtained by lyophilizing each aqueous composition.

[0334] The agents and compositions provided herein may be used alone or in combination with other therapeutic regimens, such as surgery, radiation, chemotherapy, and / or bone marrow transplantation (autologous, syngeneic, allogeneic, or unrelated).

[0335] The present invention will be described and illustrated in detail by means of figures and examples, which are used for illustrative purposes only and are not intended to be limiting. The description and examples will make available to those skilled in the art further embodiments that are also encompassed by the present invention. [Example]

[0336] Example 1 In vitro toxicity of polyplexes Materials and Methods In vivo-jetPEI™ reagent, catalog number 201-50G, was purchased from Polyplus-Transfection (Illkirch, France). In vivo-jetPEI™ was prepared at 150 mM (expressed as the concentration of nitrogen residue) in sterile, non-pyrogenic water. JetPEI was diluted to the desired concentration (expressed as the concentration of nitrogen residue) in HEPES 10 mM, pH 7.1, glucose 5% (HBGx1) buffer.

[0337] In vitro cytotoxicity assay HEK-293 cells were cultured at 2 x 10 cells per well. 4 Cells were seeded into 96-well flat-bottom plates at 100 μL / well at 37°C and 7.5% CO2. After 24 hours, the supernatant was discarded and replaced with 50 μL of DMEM medium (+10% FCS). PEI was diluted 1:5 in RPMI medium with 10% FCS and pre-incubated for approximately 15 minutes. 50 μL of PEI solution was then added to the cells to a final medium volume of 100 μL. After a further 18 hours, an XTT assay (XTT Cell Viability Kit #9095, New England Biolabs GmbH, Frankfurt, Germany) was performed according to the manufacturer's instructions. Cell death data as a function of PEI concentration were fitted using a sigmoidal curve equation.

[0338]

number

[0339] Figure 1A shows the toxicity of free pure PEI in HEK-293 cells in vitro. 50 = 77 μM nitrogen (free). Figure 1B shows the toxicity of PEI / replicon-RNA polyplexes in HEK-293 cells in vitro. IC 50= 542 μM nitrogen (polyplex formulation).

[0340] Results and Conclusions Free PEI leads to cell death at nitrogen concentrations higher than 18 µM (Figure 1A). To avoid toxicity issues, the final concentration of free PEI in the cell culture medium should be below the aforementioned limit. Although polyplexes cause less cell death than free PEI, their toxicity also increases as the concentration of PEI increases (Figure 1B). Cell death after addition of polyplexes can be calculated using the following equation: Cell death%=21.13+78.59 / (1+10^((-0.27-log(concentration))*3.13)) For polyplexes with an N / P of 11.6 (10 mg / l RNA concentration, PEI = 348 μM), cell death is 36.8%, while for polyplexes with an N / P of 15.8, cell death is 52.3%.

[0341] Example 2 Polyplex stability studies Materials and Methods The In vivo-jetPEI™ reagent was used from Example 1. Luciferase-encoding RNA, Construct D1-824 Replicon, ID R076 1, was prepared by the RNA Biochemistry unit (BioNTech RNA Pharmaceuticals GmbH, Mainz, Germany).

[0342] Preparation of polyplexes Prior to preparation, in-vivo jetPEI™ and sugar solution were equilibrated at room temperature. Preparation of in-vivo jetPEI™ / RNA complexes was carried out in a laminar flow hood using sterile sugar solution (Table 1). The final concentration of sugar in the formulation was 5-10% w / v. All formulations were prepared at an RNA concentration of 250 mg / l and an N / P ratio of 11.6. The preparation process was as follows: 1. Dilute the RNA using concentrated sugar buffer (HBG x 2, MBG x 2, or HBT x 2) to prepare a solution of 1 / 2 the final volume. Apply gentle vortexing. 2. The in-vivo jetPEI™ reagent was diluted with the same sugar buffer and sterile water to prepare a solution of 3 / 5 of the final volume. Gentle vortexing was applied. 3. Half the volume from the diluted in-vivo jetPEI™ was added quickly in one go to the diluted RNA and gentle vortexing was applied. 4. Polyplexes were incubated at room temperature for 15 to 20 minutes and then transferred to appropriate storage conditions.

[0343] [Table 1]

[0344] Lyophilization of polyplexes The formulation was placed in a benchtop manifold freeze dryer, Epsilon 2-4 LSCplus (Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). Samples were frozen to -40°C (approximately 2°C / min) at 1 atmosphere for 60-90 minutes. The pressure was reduced to 0.2 atmospheres over 10 minutes at -40°C. The samples were dried at 0.2 atmospheres and -40°C for 4 hours. The sample was heated (gradient) to −16° C. over 2 hours at 0.2 atmospheres. The sample was dried for an additional 4 hours at -16°C and 0.2 atmospheres. The pressure was reduced to 0.01 atmospheres over 10 minutes at -16°C. The sample was dried for an additional 4 hours at -16°C and 0.01 atmosphere. The sample was heated (gradient) to 20° C. over 2 hours at 0.01 atmosphere. The sample was further dried for 8 hours at 20° C. and 0.01 atmosphere.

[0345] RNA release from polyplexes Twelve test tubes containing 90 μl of sample were prepared according to Table 2.

[0346] [Table 2]

[0347] 10 μl of 50 g / l heparin in 500 mM NaCl was added to each tube, and the mixture was incubated at 30° C. for 20 minutes in a vortex machine with a shaking speed of 300 rpm.

[0348] RNA integrity Five microliters of released RNA was used for bioanalyzer measurements. The RNA was mixed with 5 μl of formamide. Quantification of RNA integrity was performed using an Agilent 2100 Bioanalyzer. The Agilent RNA 6000 Nano Kit was equilibrated at room temperature for 30 minutes. 400 μl of Nano Gel Matrix was centrifuged at 1,500 g for 10 minutes. 65 μl of the supernatant was mixed with 1 μl of well-vortexed Nano Dye Concentrate and centrifuged at 15,000 g for 10 minutes to obtain the Gel-Dye Mix. The prepared sample was denatured by heating at 70°C for 10 minutes, and the Nano Ladder was also heated at the same temperature for 2 minutes. The chip was primed using the Priming Station by adding 9 μL of Gel-Dye Mix to the G mark and applying pressure to the chip for 30 seconds. Next, 9 μL of "Gel-Dye-Mix" was added to the other two G positions. 7.5 μL of "Marker" was added to the ladder position, and 5 μL was added to each sample position. After adding 1.5 μL of denatured "Nano Ladder" to the ladder position, 1 μL of sample was added to all 12 sample wells (1 μL of HO was added to unused wells). The chip was placed in an IKA Vortex machine and vortexed at 2000 rpm for 1 minute. The chip was then measured by the machine, and the replicon RNA peak was detected between 47 and 57 seconds. RNA integrity was calculated using Expert 2100 software using smear analysis by selecting the replicon RNA region. The % relative RNA integrity was calculated using the following equation:

[0349]

number

[0350] In vitro transfection assay C2C12 cells, 2 x 10 cells per well 4The polyplexes were seeded into 96-well flat-bottom plates at a concentration of 100 μL each. Cells were maintained at 37°C and 7.5% CO2. After 24 hours, the supernatant was discarded and replaced with 50 μL of DMEM medium (+10% FCS). Polyplexes were diluted (1:5) in DMEM medium with 10% FCS and pre-incubated for approximately 15 minutes. 50 μL of polyplex solution was then added to the cells to a final medium volume of 100 μL. After an additional 48 hours, a Bright-Glo™ luciferase assay (catalog number E2610, Promega GmbH, Mannheim, Germany) was performed according to the manufacturer's instructions.

[0351] The relative luminescence % was calculated using the following equation:

[0352]

number

[0353] Figure 2 shows the relative luminescence from C2C12 muscle cells after incubation with PEI / replicon-RNA polyplexes with an N / P of 11.6 under different storage conditions after 1 week of storage.

[0354] Figure 3 shows the relative RNA integrity of PEI / replicon-RNA polyplexes with an N / P of 11.6 under different storage conditions after 2 weeks of storage.

[0355] Results and Conclusions The storage stability of polyplexes is poor in the liquid state (4 and 25°C). The stability of polyplexes is significantly better in the solid state (frozen or lyophilized) than in the liquid state. The storage stability of polyplexes in the solid state in HBT + EDTA buffer is significantly better than that in HBGx1 buffer. Long-term storage stability in the solid state is possible for polyplexes in HBT+EDTA, but long-term storage stability in the liquid state is highly unlikely for polyplexes in HBGx1. Polyplex formulations with replicon-RNA can be stabilized by the addition of trehalose 5–20% (w / v) and EDTA 80 μM–5 mM.

[0356] Example 3 Explanation of Mw calculation for linear PEI Linear PEI was synthesized from 2-ethyl-2-oxazoline in two steps. First, poly(2-ethyl-2-oxazoline) was obtained by ring-opening isomerization polymerization of 2-ethyl-2-oxazoline in the presence of an initiator (Figure 4). Next, PEOX(N-propionyl-PEI) was hydrolyzed with acid to remove the N-propionyl group and generate PEI (Figure 5).

[0357] Complete deacylation of PEOX (N-propionyl-PEI) with a molecular weight of 50 kDa yields linear PEI with a molecular weight of 22 kDa. The molecular weight of the intermediate product (PEOX) is determined by gel permeation chromatography using a refractive index detector or a multi-angle light scattering detector. Full technical details are described in U.S. Patent Application Serial No. 12 / 671,312, by Adib, Abdennaji, Fabrice Stock, and Patrick Erbacher, "Method for Manufacturing Linear Polyethylenimine (PEI) for Transfection Purposes and Linear PEI Obtained with Such Method."

[0358] According to the present invention, PEI synthesized from PEOX with a MW in the range of 40-60 kDa is a potent transfection reagent for replicon-RNA.

[0359] PEI for use according to the invention can be purchased commercially as in-vivo JetPEI from Polyplus-Transfection SA (Illkirch-Graffenstaden, France), PEI MAX 40000 from Polysciences Europe GmbH (Eppelheim, Germany), and Exgen 500 from Euromedex (Souffelweyersheim, France).

[0360] Example 4 Polyplex aggregation kinetics Materials and Methods Polyplexes were prepared at an RNA concentration of 200 mg / L in HBGx1 as previously described in Example 2. They were diluted with HBGx1 and phosphate-buffered saline, pH 7.4 (PBS), to an RNA concentration of 10 mg / L. PBS was used to increase the ionic strength of the solution. After rinsing the 96-well plate with filtered air, the diluted polyplexes were added to the plate. Size was measured using a DynaPro plate reader II instrument from WYATT technology GmbH (Dernbach, Germany). For size calculations of unimodal samples, cumulant fitting was used, while for multimodal samples, regularized fitting was used.

[0361] FIG. 6 shows the aggregation kinetics of polyplexes of IVT (A) and replicon (B) with JetPEI at increasing salt concentrations.

[0362] Results and Conclusions High ionic strength leads to an increase in polyplex size (Figure 6). The ionic strength of the polyplex formulation should be below 20 mM to prevent polyplex aggregation.

[0363] Example 5 Sterilization of polyplexes by filtration Materials and Methods Polyplexes were prepared at an RNA concentration of 100 mg / L and N / P ratios of 11.5, 13.5, and 15.5, as described above in the section "Polyplex Stability Studies."

[0364] RNA concentration and integrity RNA was released from polyplexes by incubation with heparin. 90 μl of free RNA (100 mg / L) or polyplexes was mixed with 10 μl of 20 g / L heparin in 10 mM Hepes, pH 7.4, and 1 mM EDTA. The mixture was incubated on a vortex machine for 20 minutes at 30°C. 5 μl of this mixture was mixed with 5 μl of formamide. RNA integrity was then measured using a bioanalyzer with a Picochip. RNA integrity was calculated as described in Example 2. RNA concentration was measured by the Ribogreen assay using the "Quant-iT RiboGreen RNA Reagent and Kit" (catalog number R11490, Thermo Fisher Scientific) according to the manufacturer's instructions for the highly sensitive method. Briefly, the polyplex and heparin mixture was incubated with Ribogreen fluorophore in a Tris 10 mM, pH 7.5, EDTA 1 mM buffer. Ribogreen fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0365] PEI concentration A solution of 23 mg of CuSO4 (anhydrous) in 100 ml of 0.1 M Na acetate, pH 5.4 (CSS reagent) was prepared. 600 μl of CSS reagent was mixed with the polyplexes and incubated at room temperature for 5 minutes. The absorbance of each solution was measured at 285 nm against a blank in a UV spectrophotometer using a 1 cm cuvette. A calibration curve with known PEI concentrations (0-1.55 mM) was prepared and used to calculate the PEI concentration of unknown samples. The background absorbance of free RNA was subtracted from all samples.

[0366] Measurement of electrophoretic mobility (μ) Polyplexes were diluted to an RNA concentration of 20 mg / L in 3.1 ml of HBG. They were then centrifuged at 600 g for 2 minutes. Three 1.05 ml samples were prepared for each formulation in plastic cuvettes. The electrophoretic mobility of polyplexes was measured by laser Doppler electrophoresis using a ζ-Wallis instrument (Corduan technologies, France). Medium-resolution measurements were performed for each sample in a sequence of 10 runs. Measurements with low signal-to-noise ratios or extreme μ values ​​(greater than 3 μm*cm / V*S or less than -3 μm*cm / V*S) were excluded from the final analysis. All formulations were measured in triplicate.

[0367] Filtration of polyplexes Three tubes containing 2.68 ml of polyplex were prepared at three different N / P ratios. Polyplexes (1.34 ml) were filtered through sterile Millex-GP Med Syringe Filter Units (catalog number SLMPL25SS, Merck Millipore) with 220 nm pores. Physiochemical properties were measured before and after filtration.

[0368] Figure 7 shows the physiochemical parameters of polyplexes before (prepared) and after (post-filtered) filtration. A and B. RNA was released from polyplexes by heparin. Replicon-RNA integrity (A) was then measured by capillary electrophoresis using a bioanalyzer instrument. Replicon-RNA concentration (B) was measured by Ribogreen fluorescence. C. PEI concentration was measured by CuSO4 assay. D. Electrophoretic mobility (μ) was measured by laser Doppler electrophoresis.

[0369] Results and Conclusions Syringe filter units are the preferred method for sterilizing polyplexes. Polyplexes must be small (<120 nm) to be sterilized by filtration. At an N / P ratio of 11.6, the physiochemical properties of the polyplexes are unchanged by filtration. And if the N / P ratio is increased above 11.6, loss of RNA occurs during filtration.

[0370] Example 6 Effect of PEI purity on transfection of replicon-RNA / PEI formulations Materials and Methods The following high-purity PEI was purchased: in-vivo JetPEI from Polyplus-Transfection SA (Illkirch-Graffenstaden, France) and PEI MAX 40000 from Polysciences Europe GmbH (Eppelheim, Germany). The following typical purity PEI was purchased: PEI 25000 from Polysciences Europe GmbH (Eppelheim, Germany). Polyplexes were prepared as previously described in Example 2 in HBGx1 at an RNA concentration of 100 mg / L.

[0371] In vivo transfection Mice were anesthetized with isoflurane, and the hair on the rear of the hind legs was shaved and sterilized with 70% EtOH solution. 20 μl of the test compound was injected into the tibialis posterior muscle using an insulin syringe equipped with a 30G cannula. The mice were observed for signs of pain, distress, and distress until they regained consciousness. On the day of measurement, mice were intraperitoneally injected with luciferin solution. Subsequently, mice were anesthetized with isoflurane and placed on a heat mat (37°C) in an IVIS® Spectrum (Perkin Elmer) imaging chamber while continuously receiving isoflurane / oxygen via an individual anesthesia mask. Five minutes after luciferin injection, a camera was used to detect bioluminescence light for one minute. The resulting images were analyzed using the software "LivingImage" (Perkin Elmer).

[0372] Figure 8 shows a comparison of the chemical structures of highly pure PEI and conventionally pure PEI. For 25 kDa PEI, n = 58. The average number of -CH2CH2NH- monomers in 25 kDa PEI is 581, which is also the length of the contiguous stretch of potentially protonatable nitrogens. In conventional PEI 25, assuming a uniform distribution of N-propionyl moieties, the contiguous stretch of protonatable nitrogens is only 64.

[0373] FIG. 9 shows the transfection of C2C12 muscle cells in vitro with replicon-RNA polyplexes prepared at different N / P ratios and with different purity levels of PEI.

[0374] According to Figure 10, replicon-RNA polyplexes with N / P ratios of 1 (-) and 11.6 (+) were prepared using highly pure PEI (jetPEI) and normal purity PEI (25 kDa). Free RNA was used as a control. The formulations were intramuscularly injected into the hind limbs of mice (n=3). Luminescence signals from the mouse muscles were recorded.

[0375] Results and Conclusions Polyplexes prepared using highly purified PEI transfect muscle cells in vitro better than PEI of normal purity. After intramuscular injection, polyplexes with an N / P ratio of 11.6 have the highest in vivo transfection efficacy.

[0376] Highly purified PEI-based cationic polyplexes with an N / P of 11.6 were able to transfect muscle cells in vivo significantly better (4-5 fold difference) than free replicon-RNA. Anionic polyplexes made with highly purified PEI and having an N / P ratio of 1, polyplexes made with normal purity PEI and having an N / P ratio of 1, and cationic polyplexes made with normal purity PEI and having an N / P ratio of 11.6 transfect muscle cells less efficiently than free replicon RNA.

[0377] Example 7 High transfection efficacy of replicon-RNA and lyophilized polyplexes with pure PEI from different suppliers Materials and Methods The following high-purity PEIs were purchased: in-vivo JetPEI from Polyplus-Transfection SA (Illkirch-Graffenstaden, France), Exgen 500 from Euromedex (Souffelweyersheim, France), and PEI MAX 40000 from Polysciences Europe GmbH (Eppelheim, Germany). Polyplexes were prepared in HBGx1 at an RNA concentration of 100 mg / L as previously described in Example 2. All formulations were prepared in HBGx1 buffer, except for the lyophilized formulations, which were prepared in HBTx1 buffer. Lyophilization of polyplexes in HBTx1 buffer was performed as previously described in Example 2. The formulations were injected intramuscularly into the hind limbs of mice (n=3), and luminescence signals from the mouse muscles were recorded as previously described in Example 6.

[0378] For the experiments in Figure 11, replicon-RNA polyplexes with N / P ratios of 7.7 and 11.6 were prepared using highly pure PEI, jetPEI (Polyplus), PEI-Max 40000 (Polyscience), and Exgen 500 (Eurodamex).

[0379] [Table 3]

[0380] FIG. 12 shows lyophilized cakes of JetPEI / replicon-RNA polyplexes with an N / P of 11.6 prepared using different buffers.

[0381] Results and Conclusions After intramuscular injection, replicon-RNA polyplexes efficiently transfected muscle tissue (Figure 11). High-purity PEI, jetPEI (Polyplus), PEI-Max 40000 (Polyscience), and Exgen 500 (Eurodamex) were used for polyplex preparation. Exgen-500 polyplexes transfected better when the N / P ratio was 7.7 rather than 11.6. Lyophilizates of polyplexes in trehalose have a cake-like morphology, whereas in glucose the lyophilizates collapse and are difficult to dissolve in water (Figure 12). For lyophilization of polyplexes, trehalose is preferred over glucose, and lyophilized polyplexes perform similarly in vivo to freshly prepared liquid polyplexes.

[0382] Example 8 Transfection of IVT-RNA into muscle cells using pure PEI polyplexes Materials and Methods In vivo-jetPEI™ reagent, catalog number 201-50G, was purchased from Polyplus-Transfection (Illkirch, France). In vitro transcribed (IVT) mRNA encoding luciferase, Construct pST1-475, was prepared by the RNA Biochemistry unit (Biontech RNA Pharmaceuticals, Mainz, Germany). Polyplexes of IVT-RNA and PEI were prepared as previously described in Example 2. Different N / P ratios were prepared by keeping the RNA concentration constant and increasing the PEI concentration.

[0383] In vitro transfection of muscle cells was performed as previously described in Example 2. In vivo transfection studies were performed as previously described in Example 6.

[0384] According to Figure 13, C2C12 muscle cells were transfected in vitro with IVT-RNA encoding luciferase. The RNA was complexed with JetPEI at different N / P ratios in HBGx1 buffer. Luminescence signals were measured 24 hours after transfection.

[0385] According to Figure 14, IVT-RNA polyplexes with N / P ratios of 5.8 and 11.6 were prepared in HBGx1 buffer with pure PEI. Free IVT-RNA in HBGx1 buffer was used as a control. The formulations were injected intramuscularly into the hind limbs of mice (n=3) at RNA doses of 2-8 μg per injection. Luminescence signals from the mouse muscles were recorded 6 hours after injection.

[0386] Results and Conclusions Polyplexes of IVT-RNA and highly purified PEI can efficiently transfect muscle cells in vitro. There is a positive correlation between transfection efficiency and the N / P ratio (Figure 13). Free IVT-RNA does not transfect cells in vitro. Polyplexes with highly purified PEI and N / P ratios of 5.8 and 11.6 transfected muscle tissue in vivo significantly worse than free IVT-RNA (Figure 14).

[0387] Example 9 Effects of particle size and preparation conditions on transfection by replicon-RNA polyplexes Materials and Methods Polyplexes of replicon-RNA and PEI were prepared as previously described in Example 2. Polyplexes were prepared with five different RNAs: 100, 250, 500, 750, and 1000 mg / L. The PEI concentration was increased accordingly to maintain an N / P ratio of 11.6 for all formulations. Polyplex sizes were measured as previously described in Example 4. In vitro transfection of muscle cells was performed as previously described in Example 2. In vivo transfection studies were performed as previously described in Example 6.

[0388] Polyplexes of replicon-RNA and jetPEI with an N / P ratio of 11.6 were prepared in HBGx1 buffer at different RNA concentrations, as shown in Figure 15. For size measurement by DLS, the polyplexes were diluted to an RNA concentration of 10 mg / L.

[0389] According to Figure 16, C2C12 muscle cells were transfected in vitro with the polyplexes of Figure 16. Luminescence signals were measured 24 hours after transfection.

[0390] As shown in Figure 17, Rep-RNA polyplexes with an N / P ratio of 11.6 were prepared with pure PEI in HBGx1 buffer at different RNA concentrations, similar to Figure 16. The formulations were injected intramuscularly into the hind limbs of mice (n=3) at RNA doses ranging from 2 to 8 μg per injection. Luminescence signals from the mouse muscles were recorded.

[0391] Results and Conclusions - Polyplexes are good transfection agents for replicon RNA. The biological activity of polyplexes with respect to the efficiency of RNA translation in muscle tissue is not affected by the RNA concentration (0.1-1 g / L) at the time of polyplex formation. - Polyplex size in the range of 60-200 nm has no effect on the transfection efficacy of polyplexes in vitro and in vivo (intramuscular injection).

[0392] Example 10 Different polyplexes behave differently after subcutaneous or intramuscular injection into mice Materials and Methods In vivo-jetPEI™ reagent, catalog number 201-50G, was purchased from Polyplus-Transfection SA (Illkirch, France). Linear PEI 22 kDa was provided by Professor Cheradamé (Polytheragene, EVRY cedex, France). Luciferase-encoding RNA, Construct D1-824 Replicon, ID 1600801, was prepared by the RNA Biochemistry unit (Biontech RNA Pharmaceuticals, Mainz, Germany).

[0393] Polyplexes with JetPEI were prepared in HBGx1 as previously described in Example 2 at an RNA concentration of 500 mg / L.

[0394] Polytheragene-PEI polyplexes were prepared similarly to the procedure for JetPEI, with the following two modifications. 1. For the preparation of polyplexes, HEPES 10 mM, pH 7.4 buffer was used instead of HBG × 1. An N / P ratio of 15.8 was used instead of 2.11.6.

[0395] Preparation of polytheragene-PEI polyplexes was performed according to the following publication: Demoulins, Thomas et al., "Polyethylenimine-based polyplex delivery of self-replicating RNA vaccines." Nanomedicine: Nanotechnology, Biology and Medicine, (2015).

[0396] Polyplexes were diluted in HBGx1 or Opti-MEM (catalog no. 31985062, Thermo Fisher Scientific, Schwerte, Germany) buffer to a final RNA concentration of 5 mg / l for in vitro studies and 100 mg / l for in vivo studies.

[0397] In vitro studies were carried out as described above in Examples 1 and 2. In vivo studies were performed as previously described in Example 6.

[0398] Results and Conclusions Figure 18 shows in vitro studies using PEI / replicon-RNA polyplexes in human dendritic cells (DCs) and mouse muscle cells (C2C12). A. Toxicity (expressed as % of surviving cells after treatment with polyplexes). B. Transfection (expressed as luminescence emission after treatment with polyplexes). Transfection results are shown only for C2C12 cells.

[0399] For the results shown in Figure 19, Rep-RNA polyplexes with an N / P ratio of 11.6 or 15.8 were prepared using PEI from Polyplus or Polytheragene in HBGx1 or Hepes 10 mM buffer. Prior to injection into mice, the polyplexes were diluted with HBGx1 or Opti-MEM buffer. The formulations were injected intramuscularly into the hind limbs of mice (n=3) at an RNA dose of 2 μg per injection. Luminescence signals from the mouse muscles were recorded.

[0400] Results and Conclusions

[0401] [Table 4]

[0402] Opti-MEM has an ionic strength higher than 20 mM. In Opti-MEM, polyplexes aggregate rapidly (Table 4), making this formulation unsuitable for drug development.

[0403] The polyplexes described herein (PEI, N / P 11.6, HBGx1, manufactured by Polyplus) have very different characteristics from the previously described polyplexes (PEI, N / P 15.8, manufactured by Polytheagene, Opti-MEM). In Opti-MEM, the polyplexes have diameters greater than 1000 nm and a multimodal size distribution. The polyplexes described herein have diameters of approximately 100 nm and a low polydispersity. In vitro, all polyplexes were more toxic to dendritic cells than to muscle cells, presumably because dendritic cells take up polyplexes after subcutaneous injection, whereas muscle cells are transfected with polyplexes after intramuscular injection. The in vitro transfection of muscle cells with the polyplexes described herein and the transfection of the polyplexes described previously is similar. The in vivo transfection of the polyplexes described herein and previously described polyplexes differs and depends on the route of administration. The polyplexes described herein transfect well after intramuscular injection but transfect poorly after subcutaneous injection. The previously described polyplexes do not transfect at all after intramuscular injection, but a weak signal was observed after subcutaneous injection.

[0404] Example 11 Administration of replicon RNA to mice by intramuscular versus intradermal injection Replicon RNA encoding the luciferase enzyme was dissolved in HBGx1 buffer or complexed with polyplexes as described in Example 2. These formulations were intramuscularly injected into the tibialis posterior muscle of Balb / c mice at a dose of 2 μg of RNA. Mice were anesthetized with isoflurane 4, 7, and 10 days after injection. Mice were then injected with luciferin substrate, and luminescence emission from the muscle was recorded with a CCD camera. Photons from the luciferase protein were collected over a 1-minute period and are overlaid on a photograph of the imaged mouse (Figure 20A). Figure 20B shows a graphical representation of the measured photons / second (p / s) at the injection site.

[0405] Seven days after intradermal (id) application of 2 μg of RNA encoding unformulated HBG (×1) or formulated replicon-luciferase to two injection sites in the dorsal skin of Balb / c mice, the animals were subjected to noninvasive in vivo bioluminescence imaging. Photons from the luciferase protein were collected over a 1-minute period and are shown overlaid with a photograph of the imaged mouse. The black arrow indicates the injection site (FIG. 21A). FIG. 21B shows a graphical representation of the measured photons / second (p / s) at the injection site.

[0406] Example 12 Beneficial effects of RNA formulations as vaccines Mice were immunized twice on days 0 and 21 of the study with either a PEI-formulated single-stranded replicon RNA composition encoding the hemagglutinin (HA) of the H1N1 influenza virus strain A / Puerto Rico / 8 / 1934 (H1N1 / PR8) at an N / P ratio of 11.6, or unformulated single-stranded RNA. All animals received a 1.25 μg dose of RNA. A third group received saline alone as a buffer control. As shown in Figure 22A, 19 days after the first immunization and shortly before the second immunization, all animals receiving the formulated RNA had developed an immune response to HA, as analyzed by a virus neutralization assay (VNT, detection limit 1280). In contrast, only five of the eight animals receiving unformulated RNA seroconverted to HA. As shown in Figure 22B, 35 days after the first immunization, all animals receiving RNA tested positive for HA-specific antibodies, and antibody titers to HA increased. Titers were significantly higher in animals in the formulated RNA group compared with the saline control group, but not in the unformulated RNA group. As shown in Figure 22C, 54 days after immunization, mice receiving 1.25 μg of formulated RNA encoding H1N1 / PR8-HA developed significantly higher antibody titers compared with animals receiving 1.25 μg of unformulated RNA encoding H1N1 / PR8-HA (significance calculated using one-way ANOVA; *p≦0.001).

[0407] Mice were immunized once on day 0 of the study with either a PEI-formulated single-stranded replicon RNA composition encoding the hemagglutinin (HA) of the H1N1 influenza virus strain A / Puerto Rico / 8 / 1934 (H1N1 / PR8) with a specific N / P ratio of 11.6, or unformulated single-stranded RNA. All animals received 0.25 μg of RNA. A third group received saline alone as a buffer control. As shown in Figure 23A, 54 days after immunization, all animals receiving RNA tested positive for HA-specific antibodies, and the titers of the formulated RNA group were significantly higher than those of the saline control group and animals receiving 0.25 μg of unformulated RNA encoding H1N1 / PR8-HA (significance calculated using one-way ANOVA; **p≦0.001, *p≦0.05). Fifty-five days after immunization, all mice were challenged with the median lethal dose (MLD) of H1N1 / PR8. 50 ) and survival was monitored. As shown in Figure 23B, all control animals died within 8 days. Four of the five animals that received unformulated RNA encoding H1N1 / PR8-HA survived the challenge infection. In contrast, all mice that received formulated RNA encoding H1N1 / PR8-HA survived the challenge infection, demonstrating the beneficial effect of the RNA / polyalkyleneimine composition.

[0408] Example 13 Spray drying of replicon RNA formulated with PEI The pipetting method given in Table 1 was followed twice and the resulting materials were combined to prepare a formulation with an N:P ratio of 12, resulting in 5.0 mL of replicon RNA polyplex with an RNA concentration of 0.1 mg / mL RNA.

[0409] [Table 5]

[0410] 3.5 mL of this formulation was spray-dried, and 533 mg of material was collected (yield: 76.1%). The particle size of the freshly prepared replicon RNA polyplexes was not determined because the material was discharged at the Buchi demo lab. The particle size (z-average) after reconstitution with water (mixture: 20 mg of spray-dried polyplexes and 200 μl of WFI, resulting in 10 mM trehalose and 0.1 mg / mL RNA concentration) was 289 nm, and the PDI was 0.238 (Nicomp, 15 min). Figure 24A shows an in vitro investigation of luciferase expression of saRNA polyplexes before and after spray drying. The luciferase activity of the saRNA was not lost due to the spray-drying process. The difference in absolute signal height is due to assay differences.

[0411] In further experiments, uncomplexed mRNA in 10% (w:v) trehalose was spray dried and the integrity of the mRNA after spray drying was investigated by capillary electrophoresis measurements.

[0412] 2.50 mL of messenger RNA (R36-05.2-DP, c(RNA) = 0.5 mg / mL, 10 mM Hepes, 0.1 mM EDTA, pH 7.0) was mixed with 2.50 mL of 20% (w:v) trehalose solution (1:1 volume ratio), resulting in the following composition: c(RNA) = 0.25 mg / mL, 10% (w / v) trehalose, 5 mM Hepes, 0.05 mM EDTA. The sample material was kept on ice during spray drying. A total of 2.5 mL of this solution was spray dried. During spray drying, the outlet temperature was increased from 33 to 37°C over a 10-minute period. To reduce the temperature increase, the gas flow was increased from 98 to 101 L / min. During the next 60 minutes of spray drying, the temperature was further increased to 41°C. After complete spraying, 165 mg of dry material was collected (yield: 66.0%). To analyze RNA integrity, this material was dissolved in wfi (mixture: 20 mg of spray-dried RNA and 200 μl of wfi, resulting in a trehalose content of 10% and an RNA concentration of 0.25 mg / mL). The dissolved RNA was analyzed using an Agilent 2100 Bioanalyzer. The results of these analyses are presented as an electropherogram of the spray-dried RNA in Figure 24B.

[0413] The integrity of the uncomplexed RNA was maintained, demonstrating that spray drying did not result in measurable RNA degradation.

[0414] The spray drying experiments were carried out as follows.

[0415] A Buchi Nanospray Dryer B-90 was used to spray dry the RNA-containing formulations. The following compounds were used to prepare these formulations: Messenger RNA (R36-05.2-DP, c(RNA) = 0.5 mg / mL, 10 mM Hepes, 0.1 mM EDTA, pH 7.0) Replicon RNA encoding luciferase (D2 RNA-A1310 29-01 pST1-SFV4-TRON-I2m2-A30L70, wfi, c(RNA) = 1 mg / mL) ·Water for injection (WFI) NaCl (1.5M in water) Trehalose 20% (w:v) in wfi (Pfanstiehl, Lot No. 35261A) 2X Hepes-buffered trehalose 20% (w:v) in wfi (Pfanstiehl, Lot No. 35261A, 20 mM Hepes) JetPEI (Polyplus, Lot No. 13081A1S, 150 mM nitrogen)

[0416] The following process parameters were chosen for spray drying: Nozzle: 4μm ·Inlet temperature: 80℃ ·Outlet temperature: 30℃ Gas flow: 98mL / min ·Applied current: 15.000V, 350μA

[0417] Before every experiment, the device was cleaned with RNAse Zapp and wiped with ethanol, and the cap was cleaned in an ultrasonic bath.

[0418] Example 14 Microfluidics for polyplex production A NanoAssemblr™ (Precision Nanosystems, Vancouver, BC, Canada) was used with the microfluidic chip (1029-036) provided by the manufacturer. For RNA, the company's own replicon RNA (batch number R071_1_2) was used. Polyethylenimine (Max PEI 40) was from Polysciences (Eppelheim, Germany). BD Plastipak 1 ml, 1508006, BD Biosciences (Heidelberg, Germany) was used as the syringe. The two components were mixed in a 1:1 ratio using a flow rate of 12 ml / min. Samples were prepared at two different concentrations: 50 mg / l and 250 mg / l. For particle size measurements using dynamic light scattering, a Nicomp 380 ZLS Submicron Particle / Zeta Potential Analyzer (PSS Nicomp, Santa Barbara, CA) was used.

[0419] For each condition, 1.5 ml was prepared. Samples were diluted with HBG buffer for size determination.

[0420] The pipetting method was as follows:

[0421] [Table 6]

[0422] Microfluidic mixing experiments were performed using a device equipped with a Y-type microfluidic mixer. Two components provided in standard syringes were mixed in this mixer. The two components were mixed in a 1:1 ratio using a constant flow rate of 12 ml / min. Samples were prepared at two different concentrations, namely, 50 mg / L and 250 mg / L, and the particle sizes of the resulting polyplexes were measured.

[0423] Polyplexes were produced using a microfluidic device, demonstrating the feasibility of continuous-flow and GMP manufacturing for upscaling. Particle formation was successful, with no signs of aggregate formation or blockage observed. Particle size was measured by dynamic light scattering. For polyplexes produced at 0.05 mg / L, the size was approximately 123 nm, and for particles produced at 0.25 mg / mL, the size was approximately 314 nm. Details of the results obtained are provided in the table below.

[0424] [Table 7]

[0425] The feasibility of microfluidic polyplex production was demonstrated. Production was performed using a simple Y-mixer, and no specific procedures, such as hydrodynamic focusing, were required to enable successful production. Under favorable conditions, particles with sizes well below 200 nm could be produced, allowing for final sterile filtration using established, GMP-compliant sterile filters. Since no signs of aggregation or blockage were observed, we conclude that upscaling to larger production batches would be possible without issue. Further options for upscaling include the parallelization of several identical devices. In summary, these results can be interpreted as an indication of the general feasibility of GMP-compliant microfluidic production of PEI / RNA polyplexes.

[0426] Example 15 Sterilization of polyplexes by filtration As previously described in the "Polyplex Stability Studies" section, polyplexes were prepared at a replicon-RNA concentration of 100 mg / L and N / P ratios of 11.5, 13.5, and 15.5. Three tubes containing 2.68 ml of polyplex were prepared at three different N / P ratios. Polyplexes (1.34 ml) were filtered through sterile Millex-GP Med Syringe Filter Units (catalog no. SLMPL25SS, Merck Millipore) with 220 nm pores. Polyplexes were diluted to an RNA concentration of 10 mg / L in HBG x 1. These polyplexes were then diluted to an RNA concentration of 5 mg / L in NaCl 0.9% 30 minutes before addition to cells. C2C12 cells, 2 x 10 cells per well 4The polyplexes were seeded into 96-well flat-bottom plates at a concentration of 100 μL each. Cells were maintained at 37°C and 7.5% CO2. After 24 hours, the supernatant was discarded and replaced with 50 μL of DMEM medium (+10% FCS). Polyplexes were diluted (1:5) in DMEM medium with 10% FCS and pre-incubated for approximately 15 minutes. 50 μL of polyplex solution was then added to the cells to a final medium volume of 100 μL. After an additional 48 hours, a Bright-Glo™ luciferase assay (catalog number E2610, Promega GmbH, Mannheim, Germany) was performed according to the manufacturer's instructions. In parallel with the transfection, cell number was measured using the Cell proliferation Kit II (XTT, Roche, #11465015001). In both assays, each polyplex sample was tested in biological triplicates. As a negative control, cells were seeded without treatment ("untreated"). For the XTT assay, cell-free medium equivalent to background (BG) was also seeded in triplicate. Finally, luminescence (Bright-Glo™) and absorbance (XTT) were measured using an "infinite 200pro" reader (Tecan). Normalized luminescence was calculated by dividing the luminescence signal by the absorbance (proportional to cell number).

[0427] Figure 25 shows the normalized luminescence from C2C12 muscle cells after incubation with PEI / replicon-RNA polyplexes at different N / P ratios before (prepared) and after (sterilized) sterile filtration.

[0428] It can be concluded that Syringe Filter Units are suitable for sterilization of polyplexes, and the transfection efficacy of polyplexes is not altered due to the sterilization process.

[0429] Example 16 Optimization of polyplex transfection by combining short- and long-chain PEI Luciferase-encoding replicon-RNA was complexed with short-chain PEI between 0.6 and 11 kDa (e.g., either linear short-chain PEI 2.5 kDa or branched short-chain PEI 1.8 kDa) and long-chain PEI between 20 and 40 kDa (e.g., 22 kDa in vivo / Jet PEI) in pure combinations with total NP of 10 or 12 in MBG buffer (final concentration: 5% w / v glucose, 10 mM MES, pH 6.1). In vivo / Jet PEI alone (NP of 12) was used as a benchmark. Complexation of short- and long-chain PEI polyplexes was performed in two steps. In the first step, replicon-RNA complexation was adjusted for the desired NP with in vivo / Jet PEI. In the second step, excess short-chain PEI was added to the formulation to reach the desired total NP ratio. That is, the first number defines the NP of long-chain PEI, and the second number defines the short-chain PEI (e.g., NP4+8=NP4 long-chain PEI and NP8 short-chain PEI). Luciferase assays were performed as previously described in Example 15. The results are shown in Figure 26. Figure 26A) shows the transfection efficiency of short-chain linear PEI and long-chain in vivo Jet PEI polyplexes at 250 ng of RNA per well. Figure 27B) shows the transfection efficiency of short-chain branched PEI and long-chain in vivo Jet PEI polyplexes at 250 ng of RNA per well. Conclusion: Compared to benchmark results involving only long-chain PEI (e.g., in vivo Jet PEI), transfection efficacy can be significantly improved by using a combination of long-chain PEI and short-chain PEI.

[0430] Interestingly, for the combination with linear short-chain PEI, the luciferase expression signal peaked in the first 24 h after transfection, whereas for the combination with branched short-chain PEI, the luciferase expression signal peaked in the first 48 h after transfection. Therefore, it is reasonable to carefully select the correct short-chain PEI / long-chain PEI combination in situations where expression within a specific time frame is desired.

[0431] Example 17 Optimization of polyplex transfection by reducing the amount of long-chain PEI Replicon-RNA encoding secreted nanoluciferase was complexed with short-chain PEI (e.g., branched 1.8 kDA) and long-chain PEI (e.g., in vivo / Jet PEI) in various combinations, including pure combinations with a total NP of 12, in MBG buffer (final concentration: 5% w / v glucose, 10 mM MES, pH 6.1). In vivo / Jet PEI (NP of 12) was used as a benchmark. Complexation of short-chain + long-chain PEI polyplexes was performed in two steps. In the first step, replicon-RNA complexation was adjusted with in vivo / Jet PEI to achieve the desired NP. In the second step, excess short-chain PEI was added to the formulation to reach the desired total NP ratio. That is, the first number defines NP, the long chain PEI, and the second number defines short chain PEI (e.g., NP4+8 = NP4 long chain PEI and NP8 short chain PEI). Secreted luciferase was measured in 125 ng of RNA per well according to the manufacturer's protocol (Nano-GLO, Promega, USA). Cell viability assays were performed as previously described in Example 15. The results are shown in Figure 27. Compared to the benchmark, and for the same overall NP ratio, higher expression levels were achieved with combinations such as NP4+8 and NP1.15+11. Thus, transfection efficacy can be significantly increased by reducing the concentration of long-chain PEI in the formulation and increasing the concentration of less toxic short-chain PEI (e.g., branched 1.8 kDa).

[0432] Example 18 Effect of salt variations and / or pH on transfection efficacy in vivo BALB / c mice were purchased from Janvier Laboratories and experiments were performed at 8 weeks of age. Before injection of the indicated test item, mice were anesthetized with isoflurane, and the hair on their hind legs was removed using an electric razor. Subsequently, replicon-RNA (saRNA)-PEI-polyplexes (e.g., saRNA-in vivo Jet PEI polyplex, NP12) were intramuscularly administered at a dose of 2 μg of RNA in a total volume of 20 μL to the tibialis posterior muscle of each of three mice per group. The formulations varied in terms of pH and / or salt concentration (e.g., NaCl). At the indicated time points, mice were intraperitoneally injected with D-luciferin solution (100 mg per kg body weight), and bioluminescence was noninvasively captured in isoflurane-anesthetized mice for 1 minute using an IVIS® Spectrum Device (Perkin Elmer). These photographs have a total of six values ​​per group, and the graphs display the photons / second [p / s] of bioluminescence signal in manually defined regions of interest (ROIs) for the mouse muscles (injection sites) as determined by Living Image® software (Perkin Elmer). The effect of varying salt (e.g., NaCl) on transfection efficacy is shown in Figure 28. Intramuscular injection of replicon-RNA-PEI polyplexes at different N / P ratios resulted in long-lasting bioluminescence signals in the muscle region of mice after measurements on days 3, 6, 9, and 13. The detected signal intensity increased from day 3 to day 6 (peak) after application, but was still detectable on day 13. The strongest signal in the muscle region of mice could be detected 6 days after intramuscular injection in mice that received replicon-RNA-PEI polyplexes (e.g., long-chain PEI N / P of 12) and the addition of low concentrations of salt (5 to 10 mM). The effect of pH variation on transfection efficacy is shown in Figure 29. Good results were obtained with replicon RNA (saRNA)-PEI polyplex formulations with pH values ​​between 6.5 and 7.1, preferably between 6.5 and 6.9. The strongest signal was detected with the replicon-RNA-long-chain PEI in the NP12 formulation, adjusted to pH 6.5. As a benchmark, replicon-RNA-Jet PEI polyplexes (NP12) in unadjusted pH (BM) or HBG (20 mM HEPES, pH 7.4, 5% glucose by weight) were used.

[0433] Example 19 pH-dependent effects on the in vitro electrophoretic mobility and transfection efficacy of PEI polyplexes Luciferase-encoding replicon-RNA was complexed with long-chain PEI (e.g., in vivo jet PEI) at an N / P ratio of 4 in HBG buffer (final concentration: 4.5% w / v glucose, 10 mM HEPES, pH 7.1) for 15 minutes at room temperature and divided into 11 samples. The pH of the samples was adjusted by adding HCl or NaOH depending on the final bulk pH to be tested. Electrophoretic mobility (μ) measurements were performed as described in Example 5. In vitro transfection of C2C12 mouse muscle cells, luciferase, and cell viability assays were performed as previously described in Example 15. The pH-dependent effect on the in vitro electrophoretic mobility and transfection efficacy of PEI polyplexes is shown in Figures 30 and 31. Figure 30 shows the electrophoretic mobility of in vivo jetPEI / replicon-RNA polyplexes adjusted to different pH values ​​(N / P 4). Figure 31 shows the normalized luminescence from C2C12 muscle cells after incubation with different doses of in vivo jetPEI / replicon-RNA polyplexes at an N / P ratio of 4 and different pH values ​​(pH 6.5 to pH 8.5).

[0434] The electrophoretic mobility of polyplexes was negatively correlated with pH. Neutral polyplexes were obtained at approximately pH 8.9. Increasing the positive charge density of PEI polyplexes by reducing the bulk pH of the polyplexes, preferably to a pH value between 6.5 and 7.1, resulted in higher transfection efficacy of C2C12 cells without affecting cell viability. PEI polymers contain primary and secondary amines, the protonation state of which depends on the bulk pH. Therefore, these results suggest an efficient method for controlling the charge of polyplexes and their transfection efficacy by adjusting and optimizing the bulk pH.

[0435] Example 20 Effect of excess positive charge in long-chain PEI blends Replicon-RNA encoding secreted nanoluciferase was complexed at different NP ratios between 2 and 12 in MBG buffer (final concentration: 5% w / v glucose, 10 mM MES, pH 6.1). Secreted luciferase was measured at 125 ng of RNA per well according to the manufacturer's protocol (Nano-GLO, Promega, USA). The excess positive charge was calculated based on the NP ratio of long-chain PEI (i.e., in vivo / Jet PEI) to replicon RNA and the exact NP ratio at which complete complexation of this replicon-RNA with in vivo Jet PEI occurs. The difference between the NP ratio used and the known NP ratio for complete complexation allowed the calculation of the excess positive charge of the formulation. As an example, results for in vivo Jet PEI polyplex transfection with 250 ng of RNA are shown in Figure 32. In general, increasing the concentration of positive charges in the formulation is exponentially proportional to the luciferase expression level. Excess positive charges, by increasing the amount of PEI, up to 30 nM, proved beneficial.

[0436] Example 21 Optimization of one-component PEI polyplex transfection by using two-step complexation. Even when only one PEI variant (e.g., long-chain PEI) is used, transfection efficiency can be improved by using a two-step complexation method. Replicon-RNA encoding secreted nanoluciferase was complexed in two steps to an overall NP of 12 in MBG buffer (final concentration: 5% w / v glucose, 10 mM MES, pH 6.1). One-step complexation with In vivo / Jet PEI (NP of 12) was used as a benchmark. For example, two-step complexation using In vivo Jet PEI was performed as follows: In the first step, the complexation of replicon-RNA was adjusted to the desired initial NP. In the second step, excess In vivo / Jet PEI was added to the formulation to reach the desired overall NP ratio. That is, the first number defines the initial PEI NP, and the second number defines the excess of a given in vivo Jet PEI in the second step (e.g., NP4+8: Jet PEI NP4 in the first step and Jet PEI NP8 in the second step). Secreted luciferase was measured in 125 ng of RNA per well according to the manufacturer's protocol (Nano-GLO, Promega, USA). Cell viability assays were performed as previously described in Example 15. The results of the two-step conjugation are shown in Figure 33. When compared to the benchmark one-step conjugation with vivo / Jet PEI (NP is 12), and for the same overall NP ratio, higher expression levels were achieved by the two-step conjugation.

[0437] Example 22 Effect of polyplex formulation buffer on immunization efficiency saRNA was formulated into polyplexes using in vivo-jetPEI. Polyplexes were produced using Hepes-buffered glucose (HBG) or MES-buffered glucose (MBG) (5% D-glucose, 10 mM MES, pH 6.1). Mice were immunized intramuscularly on day 0 in a single-dose experiment. Serum was collected 45 days after immunization, and the amount of Cf07-HA-specific antibodies in the serum was analyzed using an HA-specific ELISA. Endpoint titration of serum dilutions was performed to determine the area under the curve (AUC). The percentage increase in the area under the curve for polyplexes produced with MBG is expressed relative to polyplexes produced with HBG, which was set at 100%. The results are shown in Figure 34. Compared to polyplexes formulated with HBG, polyplexes prepared by using MES-buffered glucose produce much higher ELISA signals after a single vaccination of mice.

[0438] Example 23 Animals develop neutralizing antibody immune responses after intramuscular (im) immunization with PEI-formulated self-amplifying RNA (saRNA) encoding the HA (H1N1 / Cf7-HA) of the A / California / 7 / 2009 (H1N1) virus. BALB / c mice were immunized once on day 0 with buffer, a 1 / 25 dose of human vaccine, or 0.1 μg of PEI-formulated VEEV-saRNA or SFV-saRNA encoding H1N1 / Cf7-HA at a 12 / 1 N / P ratio. Animals were bled 28 and 48 days later, and serum was analyzed for antibodies against HA as measured by a virus neutralization assay (VNT; n=4). The results are shown in Figure 35(A).

[0439] The fed piglets were immunized once on day 0 with buffer, one dose of human vaccine, or 90 μg of PEI-formulated VEEV-saRNA or SFV-saRNA encoding H1N1 / Cf7-HA at an N / P ratio of 12 / 1. On days 14, 21, 28, and 35 after immunization, the pigs were bled and VNT (n=8; buffer group n=4) was performed to analyze the neutralizing antibody immune response to HA. The results are shown in Figure 35(B).

[0440] The group of animals that received the formulated VEEV-saRNA vaccine developed an immune response similar to that of animals injected with the positive control. SFV-saRNA also resulted in the development of a neutralizing antibody immune response, but at lower titers than after VEEV-saRNA immunization. The mean ± SEM is shown on the graph.

[0441] Example 24 Animals develop antibody immune responses after intramuscular (im) immunization with PEI-formulated self-amplifying RNA (saRNA) encoding the porcine circovirus 2 (PCV2)-cap_EU protein. BALB / c mice were immunized twice with buffer, 1 μg of PEI-formulated SFV-saRNA or VEEV-saRNA encoding PCV2-cap_EU at an N / P ratio of 12 / 1, on days 0 and 35. On days 14, 34, and 56, animals were bled and serum was analyzed for antibodies against PCV2-cap as determined by a commercial ELISA assay (INgezim Circo IgG, Ingenasa; n=4).

[0442] As shown in Figure 36, the groups of animals that received the formulated SFV-saRNA vaccine or the formulated VEEV-saRNA vaccine developed similar antibody responses to the PCV2-cap_EU protein. The antibody immune response after a single vaccination with SFV-saRNA was slightly higher than that of VEEV-saRNA. After two immunizations, the antibody responses were nearly identical for both types of saRNA vaccines. The mean ± SEM is shown on the graph.

[0443] Example 25 Effect of pH on the stability of self-amplifying RNA (saRNA) Replicon-RNA (saRNA) was complexed with N / P12 in different buffer systems and pH conditions. Both acetate and MES buffers contained a final concentration of 10 mM buffer and 5% w / v D-glucose. saRNA / PEI-polyplexes were stored at 4°C in each buffer for different periods (1, 2, 4, and 8 days after complexation). RNA integrity was measured directly (t=0) upon complexation of the different formulations. RNA integrity was measured by capillary electrophoresis. saRNA complexed in polyplexes could be released after 20 min of incubation at RT with a large excess of polyanions, which induces electrostatic interactions with the polymer, releasing the RNA encapsulated in the polyplexes. 200 μg of released RNA was used strictly following the protocol provided with the appropriate kit for capillary electrophoresis assays (Standard Sensitivity RNA Analysis Kit DF471). For each time point, a reference RNA was used for quantification of saRNA integrity.

[0444] Higher pH values ​​in the formulation buffer result in a significant increase in degradation of the saRNA, as shown in Figure 37. The least loss of integrity of the saRNA upon complexation was achieved at pH 4 using acetate buffer.

[0445] Example 26 PEI-formulated saRNA-VEEV encoding the A / California / 7 / 2009 HA (H1N1; Cf7 / HA) induces stronger and longer-lasting antibody responses compared to commercial vaccines, but also induces strong T cell responses that protein-based vaccines fail to induce. In Figure 38, BALB / c mice were immunized twice intramuscularly on days 0 and 35 (indicated by arrows in the graph) with either buffer (black symbols), 20 μL of a licensed human vaccine against seasonal influenza virus strains (Begripal 2016 / 2017; hLIC; gray symbols), or 0.5 μg of a vaccine based on PEI-formulated VEEV-saRNA encoding Cf7 / HA (dark gray symbols). At different time points, mice were sacrificed, and A) splenocytes were collected and Cf7 / HA-specific ELISpot assays were performed using single-cell suspensions. For ELISpot analysis, different Cf7 / HA-specific peptide pools were used to measure CD8+ / CD8+ responses as measured by IFNγ secretion. + T cells (left) or CD4 + T cell (right) responses were stimulated. Additionally, serum samples were collected and anti-Cf4 / HA-specific virus neutralization assays were performed on serum antibodies (B) for their ability to inhibit viral cell infection. Note that for serological analysis, A / California / 4 / 2009 (H1N1; Cf4) virus was utilized, and data represent the mean ± SEM (n = 3 for buffer group, n = 4 for vaccine group).

[0446] List of Abbreviations and Definitions ATM pressure C concentration CSS: Solution of 23 mg CuSO4 in 100 ml Na acetate 0.1 M, pH 5.4 DLS Dynamic Light Scattering EDTA Ethylenediaminetetraacetic acid FCS fetal calf serum h time HBG x 1 HEPES 10mM buffer (pH 7.1) glucose 5% HBG x 2 HEPES 20mM buffer (pH 7.1) glucose 10% HBT x 1 HEPES 10mM buffer (pH 7.1) Trehalose 10% HBT x 2 HEPES 20mM buffer (pH 7.1) Trehalose 20% HBT × 1 + EDTA HEPES 2.8 mM buffer (pH 7.1) Trehalose 10% and EDTA 80 μM HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid IVI Institute of Virology and immunology, Mittelhausern, Switzerland IVT in vitro transcribed mRNA kDa 1000 Daltons Lyo freeze drying min MBG x 1 5% D-glucose, 10mM MES, pH 6.1 MES 2-(N-morpholino)ethanesulfonic acid N / P The ratio between the number of amine groups in the PEI and the number of phosphate groups in the RNA. PEI Polyethyleneimine RNA ribonucleic acid UV ultraviolet light

Claims

1. For use as a medicine, (a) a single-stranded, self-replicating RNA encoding a peptide or protein comprising an antigen or epitope; and (b) Linear polyethyleneimine A composition comprising: A composition, wherein the molar ratio (N / P ratio) of the number of nitrogen atoms (N) in the linear polyethyleneimine to the number of phosphorus atoms (P) in the single-stranded RNA is 10.0 to 15.

0.

2. 2. A composition for use as a medicament according to claim 1, wherein the N / P ratio is greater than or equal to 10 and up to 12.

3. 3. A composition for use as a medicament according to claim 2, wherein the N / P ratio is between 10 and 12.

4. 4. A composition for use as a medicament according to any one of claims 1 to 3, wherein the composition has an ionic strength of 50 mM or less.

5. 5. The composition for use as a medicament according to claim 4, wherein the ionic strength is 40 mM or less, 30 mM or less, 25 mM or less, or 20 mM or less.

6. 6. A composition for use as a medicament according to any one of claims 1 to 5, wherein the concentration of monovalent cationic ions is 25 mM or less and the concentration of divalent cationic ions is 20 μM or less.

7. 7. A composition for use as a medicament according to any one of claims 1 to 6, comprising a combination of short-chain polyethyleneimine and long-chain polyethyleneimine.

8. 8. A composition for use as a medicament as claimed in claim 7, wherein the short-chain polyethyleneimine has a molecular weight between 0.6 and 11 kDa and the long-chain polyethyleneimine has a molecular weight between 20 and 40 kDa.

9. 9. A composition for use as a medicament according to any one of claims 1 to 8, wherein the single-stranded self-replicating RNA is a cis replicon.

10. A composition for use as a medicament according to any one of claims 1 to 9, wherein the single-stranded self-replicating RNA corresponds to the genomic RNA of VEEV or an attenuated form thereof, and the open reading frame encoding the structural proteins is replaced by an open reading frame encoding a peptide or protein comprising an antigen or epitope.

11. 11. A composition for use as a medicament according to claim 10, wherein the antigen, or a peptide or protein comprising an antigen or epitope, is a membrane protein.

12. The membrane protein comprises a type I membrane protein, a type II membrane protein, a multi-spanning membrane protein, a lipid chain-anchored membrane protein, a GPI-anchored membrane protein, a peripheral membrane protein, or a viral envelope protein; and / or 12. A composition for use as a medicament according to claim 11, wherein the antigen is influenza hemagglutinin.

13. A composition for use as a medicament according to any one of claims 1 to 9, wherein the single-stranded self-replicating RNA corresponds to the genomic RNA of SFV or an attenuated form thereof, and the open reading frame encoding the structural proteins is replaced by an open reading frame encoding a peptide or protein comprising an antigen or epitope.

14. 14. A composition for use as a medicament according to claim 13, wherein the antigen, or a peptide or protein comprising the antigen or epitope, is not a membrane protein.

15. 15. A composition for use as a medicament according to claim 13 or 14, wherein the antigen is a viral antigen.

16. 16. The composition for use as a medicament according to claim 15, wherein the viral antigen is a protein derived from a viral surface protein.

17. 17. The composition for use as a medicament according to claim 16, wherein the viral surface protein is a membrane-bound glycoprotein, a viral capsid protein, or a spike protein.

18. 18. A composition for use as a medicament according to any one of claims 1 to 17, wherein said use comprises intramuscular administration.

19. 19. A composition for use as a medicament according to any one of claims 1 to 18, wherein the single-stranded RNA and the linear polyethyleneimine are present in a polyplex particle.

20. The linear polyethyleneimine has the following general formula (I): 【Chemistry 1】 (In the formula, R is H, n is 2, p is an integer, The average molecular weight of the polymer is 1.5 x 10 2 From 10 7 Da, 5000 to 10 5 Da, 10,000 to 40,000 Da, 15,000 to 30,000 Da, or 20,000 to 25,000 Da).

20. A composition for use as a medicament according to any one of claims 1 to 19, comprising:

21. 21. The composition for use as a medicament according to any one of claims 1 to 20, wherein the composition comprises one or more additives selected from the group consisting of buffer substances, sugars, stabilizers, cryoprotectants, lyoprotectants and chelating agents.

22. 22. The composition for use as a medicament according to claim 21, wherein the buffering substance comprises at least one selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 2-(N-morpholino)ethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), acetic acid, acetate buffer, phosphoric acid and phosphate buffer, and citric acid and citrate buffer.

23. 23. A composition for use as a medicament according to claim 21 or 22, wherein the sugar is selected from the group consisting of monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides.

24. 24. The composition for use as a medicament according to claim 23, wherein the sugar comprises at least one selected from the group consisting of glucose, trehalose, and sucrose.

25. 25. A composition for use as a medicament according to any one of claims 21 to 24, wherein the cryoprotectant comprises at least one selected from the group consisting of glycols, including ethylene glycol and propylene glycol, and glycerol.

26. 26. A composition for use as a medicament according to any one of claims 21 to 25, wherein the chelating agent comprises EDTA.

27. 27. A composition for use as a medicament according to any one of claims 1 to 26, comprising HEPES buffered glucose (HBG), MES buffered glucose (MBG), acetate buffered glucose, or HEPES buffered trehalose (HBT).

28. 28. A composition for use as a medicament according to any one of claims 19 to 27, wherein the polyplex particles have a z-average determined by dynamic light scattering measurement of less than 200 nm, less than 150 nm, or less than 100 nm, and / or a polydispersity index determined by dynamic light scattering measurement of less than 0.5, less than 0.3, or less than 0.2, and / or a zeta potential of more than 20 mV, or between 25 and 40 mV.

29. 29. A composition for use as a medicament according to claim 27 or 28, wherein HBG comprises 5% glucose (w / v) and 10 mM HEPES, pH 7.1, HBT comprises 10% trehalose (w / v) and 10 mM HEPES, pH 7.1, and MBG comprises 5% glucose (w / v) and 10 mM MES, pH 6.

1.

30. 30. A composition for use as a medicament according to any one of claims 1 to 29, wherein the composition is for use in therapy and / or the composition is a vaccine composition and / or the composition is for inducing an immune response.

31. 31. A composition for use as a medicament according to claim 30, wherein the immune response is against an antigen or epitope.

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