RNA formulations suitable for therapy

Abstract

To provide formulations for efficient delivery of biologically active RNA to target cells or target tissue.SOLUTION: The present invention relates to compositions comprising RNA, preferably messenger RNA (mRNA), more preferably self-amplifying RNA (saRNA), and polymers, particularly cationic polymers, such as polyethylenimine (PEI), poly-L-lysin (PEL), polyvinylamine (PVA) or polyallylamine (PAA), where individual RNA molecules are present in solution. In the formulations, the RNA is preferentially present in the form of monomers, dimers, timers or oligomers, but not as aggregates comprising a large number of RNA molecules per aggregate, in particular large polyplex nanoparticles. The formulations display improved transfection efficacy. The formulations can be used for delivery of RNA to a subject. The formulations have an improved dose response relationship in comparison to formulations where large aggregates in the form of polyplex nanoparticles are present.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a composition comprising RNA, preferably messenger RNA (mRNA), more preferably self-amplifying RNA (saRNA), and a polymer, in particular a cationic polymer such as polyethyleneimine (PEI), poly-L-lysine (PLL), polyvinylamine (PVA), or polyallylamine (PAA), etc., wherein individual RNA molecules are present in solution. In the formulation, the RNA preferentially exists in the form of monomers, dimers, trimers, or oligomers, but does not exist as aggregates containing a large number of RNA molecules per aggregate, especially large polyplex nanoparticles. The formulation can be formed from RNA and a polymer that is highly in excess relative to the RNA. Moreover, the formulation can be formed at a very low concentration of RNA. The formulation exhibits improved transfection efficiency, the formulation can be used for the delivery of RNA to a subject, and the formulation has an improved dose-response correlation compared to formulations in which large aggregates in the form of polyplex nanoparticles are present. More precisely, the present invention relates to a formulation for the administration of RNA, preferably messenger RNA (mRNA), more preferably self-amplifying RNA (saRNA), which enables a reduced effective dosage with a significantly reduced risk of side effects. The RNA-polymer formulation described herein is particularly useful, for example, for human or animal vaccination against infectious diseases and the like.

Background Art

[0002] The use of RNA for the delivery of foreign genetic information into target cells presents an attractive alternative to DNA. Advantages of using RNA include transient expression and non-integrating properties. RNA does not need to enter the nucleus to be expressed and moreover has no potential to integrate into the host genome, thereby eliminating the risk of tumorigenesis. Messenger RNA (mRNA)-based therapeutic approaches are attracting increasing attention for applications in various fields, such as vaccination, cancer treatment, or protein replacement (Sahin et al. (2014) Nat. rev. 13(10): 759-780). Several mRNA-based drugs for cancer vaccination are being tested in clinical trials. To translate such novel therapeutic concepts into clinical practice, formulations suitable for administration to patients are required. mRNA needs to be protected from rapid degradation by RNase and enable cellular delivery to the target site and translation of the encoded protein. Conventional vehicles for nucleotide delivery are based on cationic lipids or cationic polymers and form nanoparticle formulations together with mRNA. Regarding polymeric delivery vehicles, polyethyleneimine (PEI) and its derivatives are among the most established carrier systems (Neuberg et al. (2014) Adv. in Gen. 88: 263-88) and have already been used in clinical trials for various applications.

[0003] Cationic polymers such as PEI are known to self-assemble with mRNA into nanoparticle complexes with controlled transfection properties, and in fact, PEI polyplex formulations are widely used for gene delivery. PEI is a cationic polymer composed of repeating units of one amino group and two carbon aliphatic spacers. PEI-based homopolymers can be classified according to their structure and size. A wide range of molecular weights (600 Da to 400 kDa) are available for both linear and branched forms. At the structural level, the main difference between linear and branched PEI depends on the type of amine, i.e., linear PEI has only secondary amines, while branched PEI has primary / secondary / tertiary amines in a ratio of 25 / 50 / 25. PEI is highly soluble in water at room temperature and is protonated in aqueous buffers, but is also soluble in organic solvents such as methanol, ethanol, or chloroform, etc.

[0004] The outstanding property of PEI as a non-viral delivery system relies precisely on the iminoethylene monomer that defines the high density of cationic charges. The protonated amines in the polymer can form electrostatic bonds with the anionic charges present in nucleic acids, mainly due to the phosphate backbone present in both DNA and RNA.

[0005] One important property of PEI polyplex formulations is the so-called N / P ratio, which gives the ratio of the number of nitrogen groups of PEI to the number of phosphate groups of RNA. Since nitrogen atoms (pH-dependent) are usually positively charged and phosphate groups are negatively charged, the N / P ratio correlates with the charge ratio. The N / P ratio at which charge balance exists is pH-dependent. At applicable pHs, charge balance is achieved by using an N / P ratio between 1 and 4. Thus, PEI formulations are often formed with N / P ratios greater than 4 and less than or equal to 12 because positively charged nanoparticles are considered favorable for transfection. In that case, the RNA is considered to be fully bound to the PEI nanoparticles and furthermore, there is considered to be excess free PEI relative to the nanoparticles. According to some publications, free PEI is potentially favorable for transfection by nanoparticles because PEI can be involved in the intracellular uptake mechanism and can be released from the endosomal compartment (Boeckle et al. (2004) J Gene Med. 6, 1102-1111; Cai et al. (2016) J Cont. Rel. 238, 71-79; Florea et al. (2002) AAPS 4, 1-11).

Prior Art Documents

Non-Patent Documents

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Problems to be Solved by the Invention

[0007] The development of formulations for the delivery of biologically active RNAs with improved safety and efficiency remains an unaddressed need. Thus, there is a need to provide a formulation for the efficient delivery of biologically active RNAs to target cells or target tissues, where the delivered RNA is translated into the peptide or protein it encodes.

Means for Solving the Problems

[0008] Formulations of RNAs with cationic polymers are described herein, where the RNAs mainly exist in the form of monomers, dimers, trimers, or oligomers, but not, or only to a limited extent, as aggregates containing multiple RNA molecules per aggregate, particularly large polyplex nanoparticles that are much larger than individual RNA molecules and contain more RNA copies. Surprisingly, it has been found that such phases can be formed by adding a very large excess of cationic polymer (with respect to the charge ratio) to the RNA and / or by forming the formulation at a very low concentration of RNA.

[0009] The conventional understanding of formulations containing RNA and cationic polymers is that so-called polyplex nanoparticles can be formed, which can exist in a size range from about 100 nm or less to several hundred nanometers. One common parameter in these nanoparticles is the so-called N / P ratio, which refers to the charge ratio between the positively charged nitrogen groups in the polymer and the negatively charged phosphate groups of the RNA. In the case of an excess of polymer, the RNA is considered to be fully bound to the nanoparticles, and the excess polymer is considered to be present in the free aqueous phase. In some publications, this excess free polymer is considered to be advantageous for the transfection efficiency of the polyplex nanoparticles.

[0010] Here, in addition to previously known polyplex nanoparticles, a new phase will be described. By adding increasing amounts of excess polymer to RNA at a concentration of about 0.1 mg / ml, a phase of individual RNA molecules in which the RNA molecules exist as monomers, dimers, trimers, or oligomers is formed, and the equilibrium between oligomers and monomers was found to shift towards the monomer side by adding increasing amounts of excess polymer. At RNA concentrations of 0.05 mg / ml or less, less excess PEI can form the new phase. For example, in the case of polyethyleneimine (PEI), RNA monomers are the dominant form at an N / P ratio of about 70 to 120 or more when the RNA concentration is 0.1 mg / ml. At an RNA concentration of only 0.05 mg / ml, preferably when the RNA:PEI mixing ratio is >1:1, excess PEI with an N / P ratio of 4, preferably 12, is sufficient. Without wishing to be bound by theory, individual RNA molecules are thought to associate with polyethyleneimine in a zwitterionic manner, giving rise to monomers, dimers, trimers, etc. of different molar masses. RNA in a composition containing PEI can be identified by different physicochemical techniques and physically separated from co-existing polyplex nanoparticles.

[0011] Surprisingly, it has been found that RNA in monomeric or oligomeric form in a PEI-containing composition has a much higher transfection efficiency than conventionally known polyplex nanoparticles. The highest transfection efficiency was found when the RNA was present entirely in monomeric form (when the RNA-PEI formulation was formed at a concentration of 0.1 mg / mL and an N / P ratio of about 70 - 120).

[0012] As demonstrated by the observation of reporter gene expression (luciferase, green fluorescent protein), the phases of individual RNA molecules demonstrated excellent transfection efficiency in vitro and in vivo. They are applicable for use as vaccine formulations, e.g., intramuscular administration, to obtain an improved antigen titer.

[0013] The novel phase of the RNA molecule in the polymer-containing composition can be easily formed by adding a suitable amount of the polymer to the RNA at a suitable concentration, a suitable mixing ratio, and suitable buffer conditions. The phases are advantageous for pharmaceutical use because they can be easily manufactured with controlled structural properties and there is no risk of large particle formation or aggregation.

[0014] The novel phase of the RNA in the polymer-containing composition can be formed by adding a solution of the polymer to a solution of the RNA or by dissolving the dehydrated (lyophilized) RNA using the polymer solution. Thus, pharmaceutical manufacturing and product control based on this are greatly facilitated.

[0015] It is possible to form an RNA-phase-based pharmaceutical product for intramuscular, intradermal, or subcutaneous administration.

[0016] One aspect of the present disclosure relates to a composition comprising RNA and a polymer in an aqueous phase, wherein a predominant fraction of the RNA molecules comprises individual molecules in solution. In one embodiment, the predominant fraction comprises more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% of the total amount of RNA in the composition.

[0017] In one embodiment of any aspect described herein and other aspects, the present disclosure relates to a composition comprising RNA and a polymer in an aqueous phase, wherein more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the total amount of RNA in the composition exists as a single molecular species. In one embodiment of any aspect described herein and other aspects, the present disclosure relates to a composition comprising RNA and a polymer in an aqueous phase, wherein more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the total amount of RNA in the composition is RNA that exists as an oligomer with 4 or fewer RNA molecules per RNA monomer or oligomer unit.

[0018] In one embodiment of any aspect described herein and other aspects, the present disclosure relates to a composition comprising RNA and a polymer in an aqueous phase, wherein more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the total amount of RNA in the composition is RNA that does not precipitate when centrifuged at 20,000 x g for 90 minutes.

[0019] In one embodiment of any aspect described herein and other aspects, the present disclosure relates to a composition comprising RNA and a polymer in an aqueous phase, wherein less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of the total amount of RNA in the composition exists as RNA polyplex nanoparticles with a size exceeding 50 nm or 60 nm and / or as RNA polyplex nanoparticles with 10 or more, or 20 or more, RNA copies per nanoparticle. In one embodiment, the composition essentially does not contain RNA polyplex nanoparticles with a size exceeding 50 nm or 60 nm and / or RNA polyplex nanoparticles with 10 or more, or 20 or more, RNA copies per nanoparticle.

[0020] In one embodiment of any aspect and other aspects described herein, the present disclosure relates to a composition comprising RNA and a polymer in an aqueous phase, wherein less than 20%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5% of the total amount of RNA in the composition is RNA present as aggregates containing multiple RNA molecules per aggregate.

[0021] The compositions described herein can be obtained by appropriately adjusting the amounts of RNA and the polymer, in particular by appropriately adjusting the N / P ratio.

[0022] In one embodiment of the compositions described herein, most of the RNA exists as a single molecular species. In various embodiments, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% of the total amount of RNA in the composition exists as a single molecular species.

[0023] In one embodiment of the compositions described herein, the polymer is a cationic polymer. In one embodiment of the compositions described herein, the polymer is a polycationic polymer. In one embodiment of the compositions described herein, the polymer comprises one or more selected from the group consisting of cationic or polycationic peptides or proteins including protamine, spermine or spermidine, polylysine, polyarginine, cationic polysaccharides including chitosan, poly(ethyleneimine), poly(propyleneimine), polybutrene, polyallylamine, and cationic polymers including polyvinylamine. In one embodiment, the polymer comprises a polyamidoamine (PAMAM) polymer.

[0024] In one embodiment of the composition described herein, the polymer comprises poly(ethyleneimine). In one embodiment of the composition described herein, the poly(ethyleneimine) is a linear polymer. In one embodiment of the composition described herein, the poly(ethyleneimine) is a branched polymer. In one embodiment of the composition described herein, the poly(ethyleneimine) has an average molar mass between 1000 Da and 150000 Da, between 5000 Da and 100000 Da, between 10000 Da and 50000 Da, between 15000 Da and 30000 Da, between 20000 Da and 25000 Da, or about 22500 Da. In one embodiment of the composition described herein, the poly(ethyleneimine) has an average molar mass between 22500 Da and 150000 Da.

[0025] In one embodiment of the composition described herein, the composition further comprises a buffering substance selected from the group consisting of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), 2-(N-morpholino)ethanesulfonic acid (MES), bis-tris buffer systems (e.g., bis-tris propane, bis-tris methane), acetate buffer systems, other carboxylic acid buffer systems, phosphate buffer systems, or citrate buffer systems, and the pH can be in the range of 4 to 8, more preferably 5 to 7.

[0026] In one embodiment of the composition described herein, the composition has an ionic strength of 50 mM or less, preferably, the concentration of positively charged monovalent ions is 25 mM or less, and the concentration of free positively charged divalent ions is 20 μM or less. In one embodiment of the composition described herein, the concentration of free positively charged divalent ions is 20 μM or less.

[0027] In one embodiment of the composition described herein, the RNA mainly consists of RNA molecules individually associated with the polymer. In various embodiments, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99% of the total amount of RNA in the composition exists as RNA molecules individually associated with the polymer.

[0028] In one embodiment of the composition described herein, the mass fraction of RNA present as an oligomer with 4 or fewer RNA copies per RNA monomer or oligomer unit is more than 60%, more than 70%, more than 80%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% of the total amount of RNA. In one embodiment of the composition described herein, the composition essentially does not contain RNA aggregates containing a large number of RNA molecules per aggregate.

[0029] In one embodiment of the composition described herein, the RNA is selected from the group consisting of mRNA, saRNA, siRNA, shRNA, miRNA, pre-miRNA, ribozyme, and antisense RNA.

[0030] In one embodiment of the composition described herein, the composition does not contain viral RNA particles.

[0031] In one embodiment of the composition described herein, the composition is formed by mixing RNA and an excess of polymer.

[0032] In one embodiment of the composition described herein, a polymer-containing solution and an RNA-containing solution are mixed, the final concentration of RNA is 0.1 mg / ml or less, and the ratio (N / P) of the number of nitrogen groups of the polymer to the phosphate groups of RNA is at least about 48. In one embodiment, the ratio (N / P) of the number of nitrogen groups of the polymer to the phosphate groups of RNA ranges from about 48 to 300, from about 60 to 200, or from about 80 to 150.

[0033] In one embodiment of the composition described herein, a polymer-containing solution and an RNA-containing solution are mixed, the final concentration of RNA is 0.05 mg / ml or less, and the ratio (N / P) of the number of nitrogen groups of the polymer to the number of phosphate groups of RNA is at least about 4. In one embodiment, the ratio (N / P) of the number of nitrogen groups of the polymer to the number of phosphate groups of RNA ranges from about 4 to 200, from about 12 to 150, or from about 24 to 120.

[0034] In one embodiment of the composition described herein, the RNA-containing solution is added to the polymer-containing solution, and the amount of the RNA-containing solution is equal to or in excess of the amount of the polymer-containing solution. Preferably, the volume ratio of the RNA-containing solution to the polymer-containing solution is between about 1:1 and 99:1. In one embodiment of the composition described herein, the RNA-containing solution is added to the polymer-containing solution, and the RNA-containing solution has an RNA concentration less than 2-fold, more preferably less than 1.5-fold, even more preferably less than 1.1-fold the concentration in the final composition. In one embodiment of the composition described herein, the RNA concentration in the mixture of the polymer-containing solution to which the RNA-containing solution is added is 0.5 mg / ml or less, 0.4 mg / ml or less, 0.3 mg / ml or less, 0.2 mg / ml or less, or 0.1 mg / ml or less. In one embodiment of the composition described herein, the RNA concentration in the RNA-containing solution added to the polymer-containing solution is 1 mg / ml or less, 0.9 mg / ml or less, 0.8 mg / ml or less, 0.7 mg / ml or less, 0.6 mg / ml or less, 0.5 mg / ml or less, 0.4 mg / ml or less, 0.3 mg / ml or less, 0.2 mg / ml or less, or 0.1 mg / ml or less.

[0035] In one embodiment of the composition described herein, the composition is formed by mixing a solution of RNA and a solution of a polymer.

[0036] In one embodiment of the composition described herein, the composition is formed by mixing an equal volume of a solution of RNA and a solution of a polymer.

[0037] In one embodiment of the composition described herein, the composition is formed by mixing a solution of RNA and a solution of a polymer at a volume mixing ratio of at least 4:1.

[0038] In one embodiment of the composition described herein, the composition is formed by dissolving dehydrated RNA in a solution of a polymer.

[0039] It is demonstrated herein that the RNA in the composition described herein exhibits a surprisingly dense packing and a very high packing density.

[0040] In one embodiment of any aspect and other aspects described herein, the present disclosure relates to a composition comprising RNA and a polymer in an aqueous phase, wherein the RNA is present in a very densely packed conformation, for example, the radius of gyration of the RNA obtainable by, for example, small-angle X-ray scattering measurement is smaller than the radius of gyration of the RNA in a 50 mM NaCl solution.

[0041] In one embodiment of any aspect and other aspects described herein, the present disclosure relates to a composition comprising RNA and a polymer in an aqueous phase, wherein the RNA is present in a very densely packed conformation, for example, the radius of gyration of the RNA obtainable by, for example, small-angle X-ray scattering measurement is 80% or less, preferably 60% or less, preferably 60% or less, preferably 50% or less, preferably 40% or less of the radius of gyration of the RNA in a 50 mM NaCl solution.

[0042] In one embodiment of any of the aspects and other aspects described herein, the present disclosure provides a composition comprising RNA and a polymer in an aqueous phase, wherein the RNA has a radius of gyration R of the RNA, which is obtainable, for example, by small-angle X-ray scattering measurements, etc. g (nm) and the cube root of the molar mass Mw (daltons) of the RNA, the ratio between them is given by the following formula:

[0043] [Number]

[0044] [wherein x is 0.17 nm·mol 1 / 3 *g -1 / 3 , preferably 0.15 nm·mol 1 / 3 *g -1 / 3 , more preferably 0.13 nm·mol 1 / 3 *g -1 / 3 , even more preferably 0.11 nm·mol 1 / 3 *g -1 / 3 , even more preferably 0.09 nm·mol 1 / 3 *g -1 / 3 , even more preferably 0.085 nm·mol 1 / 3 *g -1 / 3 , and relates to a composition present in a very densely packed conformation that can be represented by, for example, for an RNA having an R of about 12 nm g and a molar mass of 3×10 6 Da, the ratio would be about 0.083 nm·mol 1 / 3 *g -1 / 3 .

[0045] One aspect of the present disclosure relates to a composition comprising RNA and a polymer as described herein that is lyophilized. Such a lyophilized composition can be reconstituted by adding an aqueous solution to obtain a composition comprising the RNA and the polymer as described herein.

[0046] One aspect of the present disclosure relates to the compositions described herein for use in non-viral gene delivery. In one embodiment, the gene delivery should be performed into cells, which can be present, for example, in vitro, ex vivo, or in vivo in a subject.

[0047] One aspect of the present disclosure relates to a method of transfecting cells comprising the step of contacting the cells with the compositions described herein. In one embodiment, the cells can be present, for example, in vitro, ex vivo, or in vivo in a subject.

[0048] In one embodiment, the cells into which the gene is delivered or transfected are present in a subject, and the method comprises the step of administering the composition described herein to the subject. In one embodiment, the administration is by injection. In one embodiment, the composition is administered intramuscularly, intradermally, or subcutaneously.

Brief Description of the Drawings

[0049]

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DETAILED DESCRIPTION OF THE INVENTION

[0050] Although the present disclosure is described in detail below, it should be understood that since the specific methodologies, protocols, and reagents described herein may vary, the present disclosure is not limited thereto. It should also be understood that the technical terms used herein are for the purpose of describing only specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0051] Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H. G. W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0052] The practice of the present disclosure will, unless otherwise specified, use conventional methods in chemistry, biochemistry, cell physiology, immunology, recombinant DNA technology as described in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0053] In the following, elements of the present disclosure will be described. These elements are recited by specific embodiments; however, it should be understood that they can be combined in any way and in any number to create additional embodiments. The various examples and embodiments described should not be construed as limiting the present disclosure to only the explicitly described embodiments. This description should be understood to disclose and encompass embodiments that combine the explicitly described embodiments with any number of the disclosed elements. Further, any order and combination of all the described elements should be construed as being disclosed by this description unless indicated otherwise in the context.

[0054] The term "about" means approximately or nearly, and in relation to a numerical value or range described herein, in one embodiment, it means ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0055] As used in the context of describing the present disclosure, the terms "a", "an", "the" and similar indicators are to be construed as encompassing both the singular and the plural, unless otherwise specified herein or clearly negated by the context (especially in the context of the claims). The recitation of a range of values herein is merely intended to serve as a convenient way to individually refer to each separate value falling within that range. Unless otherwise specifically recited 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 specified herein or clearly precluded by the context. The use of any and all examples or exemplary language provided herein (e.g., "such as") is merely intended to better illustrate the present disclosure and is not intended to limit the claims. No language in this specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0056] Unless otherwise specifically defined, the term "comprising", as used in connection with this specification, is intended to indicate that there may optionally be further members in addition to the members of the list introduced by "comprising". However, as a particular embodiment of the present disclosure, it is also contemplated that the term "comprising" may encompass the possibility of the absence of further members, i.e., for the purposes of this embodiment, "comprising" should be understood to also have the meaning of "consisting of".

[0057] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instruction manuals, etc.) is hereby incorporated by reference in its entirety, regardless of the above and below. No description in this specification shall be construed as an admission that the present disclosure has no right to antedate such disclosure.

[0058] An aqueous phase consisting of a cationic polymer and RNA is described herein, where instead of RNA-polyplex nanoparticles, individual RNA molecules are present in the aqueous phase. Formulations of RNA solubilized in a polymer-containing solution have favorable transfection properties for the delivery of such RNA in vitro and in vivo. They are advantageous in pharmaceutical applications that require the delivery of RNA to target cells for various therapeutic approaches. A new phase has been discovered through a thorough investigation of the colloidal properties of the RNA / polymer system.

[0059] According to the classical understanding of polyplex formulations, an increase in the amount of cationic polymer approaches charge neutrality and beyond that, decreases the amount of free RNA until no free RNA remains. The RNA is thought to be fully inserted into the polyplex nanoparticles, which have different sizes and typically contain several copies of RNA and polymer molecules.

[0060] A thorough investigation of the PEI / RNA system revealed, surprisingly, that excess PEI allows for the detection of RNA existing as individually dissolved molecules. Moreover, even more surprisingly, the fraction of RNA present in the form of such individually dissolved molecules was found to increase with increasing amounts of the excess polymer. The RNA in the polyplex nanoparticles and the individually dissolved RNA could be separated from each other by centrifugation. Figure 2 shows the results of the centrifugation experiment, where the RNA content in the supernatant and pellet was quantified by UV measurement. The experiment was carried out using messenger RNA (mRNA) consisting of approximately 2000 nucleotides and self-amplifying RNA (saRNA) consisting of approximately 9000 nucleotides. For both types of RNA, mRNA and saRNA, their fractions did not precipitate under the applied centrifugation conditions. The figure demonstrates that the RNA fraction in the supernatant increased with increasing excess PEI. In Figure 3, the amounts of precipitated RNA and RNA in the supernatant were summed for each N / P ratio. Values close to 100% of the theoretical concentration were obtained, indicating that the findings clearly correlate with the true RNA concentration and are not due to any artifacts.

[0061] Particle size measurements from various fractions as a function of the N / P ratio were performed and are shown in Figure 4. The sizes in the unmodified system are shown after mixing the two components from the resuspended precipitate and supernatant and after centrifugation. Values close to those of the system before centrifugation were obtained from the resuspended pellet. In particular, measurements were also possible from the supernatant, in which case the size was between 25 nm and 50 nm. Although a certain error has to be taken into account for size measurements, the data indicate that in any case, some particle fraction was present in the supernatant.

[0062] The composition of the supernatant fraction was further investigated by agarose gel measurement. Figure 5 shows agarose gel traces for naked RNA in comparison with the supernatant at N / P 12, 36, and 72. The supernatant was measured either without treatment or after incubation with heparin to finally release the PEI-complexed RNA. In the case of the untreated supernatant, no RNA bands were seen, but after heparin treatment, they could be detected and the band intensity increased with the N / P ratio. This effectively confirms that RNA is present in the supernatant, but not in free form; rather, it is assumed to be present in a PEI-associated state and can be released from them by heparin treatment.

[0063] More systematic investigation of the supernatant fraction revealed quantitative insights into the shift of RNA from the nanoparticles to the supernatant phase. In Figure 6, the measurement of the supernatant fraction as shown in Figure 2 was extended to much higher N / P values. The supernatant fraction increased monotonically until all of the RNA shifted to the non-precipitating state at N / P values of approximately 130 to 150 (at N / P = 120, the supernatant contained 85% to 95% of the RNA depending on the RNA sample). Thus, at this N / P value, the RNA can be considered to have completely shifted to the supernatant state.

[0064] To elucidate insights into the molecular interference of RNA in the supernatant state, analytical ultracentrifugation (AUC) measurements were used. Figure 7A shows the results of AUC measurements at N / P 12. A large peak at a high sedimentation coefficient corresponding to the nanoparticles, and furthermore, a series of peaks at lower sedimentation coefficients similar to the fractionation of molecular moieties as monomers, dimers, trimers, etc. were identified. The peaks of RNA and PEI could be revealed independently or simultaneously. Thus, the RNA in the supernatant is considered to exist in the form of monomers, dimers, trimers, etc. of RNA, which is solvated by PEI stochiometrically. As observed in Figure 7C, further AUC analysis of PEI polyplexes containing iVT mRNA with increasing N / P showed that higher N / P ratios shifted the system towards individual monomeric RNA molecules. At N / P = 120, all iVT mRNA was found in a monomeric molecular arrangement.

[0065] As an example, the observations outlined here with PEI can be performed with other cationic polymers as demonstrated in Figure 8 or Figure 20B or Figure 22B where the RNA phase with several alternative cationic polymers is considered as a function of the N / P ratio. In all cases, an increase in the solubilized RNA fraction was found with increasing N / P. This demonstrates that the observations and conclusions with PEI as a model molecule are, in principle, equally applicable to other cationic polymers.

[0066] The novel PEI / RNA phase at high N / P ratios showed excellent transfection efficiency compared to classical polyplex nanoparticle systems. This is demonstrated in Figure 9, which shows the results of transfection experiments in vitro. RNA / PEI polyplex systems were assembled from RNA encoding luciferase at various N / P ratios, and luciferase expression was measured for the complete system or after separation of the pellet and supernatant by centrifugation. The activity of the supernatant phase increased much more with increasing N / P ratio than the overall system or the pelleted nanoparticles, which had very low activity. Thus, clearly, the supernatant phase provided an important contribution to the activity of the system, while the nanoparticles seemed to have very low or even harmful activity. The activity increased monotonically with the N / P ratio up to a value of about 120, above which it appeared to plateau. This coincides with the range in which RNA was quantitatively transfected into the monomeric form (Figures 10B, 10C). Thus, pure monomeric RNA present at N / P ratios above 120 seems to be most favorable for the best transfection efficiency.

[0067] The observations made in vitro could also be confirmed in vivo, as shown in Figures 11 - 13. Consistently, an increase in transfection efficiency with increasing N / P up to 120 was found. Higher luciferase expression was obtained, similar to the higher titers from the immune experiments. At the same time, the dose could be reduced, resulting in a reduction of the potential toxic effects of the vehicle. Moreover, the previous observations at high N / P ratios for excellent transfection efficiency, where the activity increased monotonically with the N / P ratio and plateaued in the range where RNA was quantitatively transfected in the monomeric form in the highest possible amount, could be observed for substantially different cationic polymers in Figure 20C or Figure 22C. This highlights the general applicability of the enhanced bioactivity in such RNA / PEI systems for other cationic polymers.

[0068] Definition The following definitions apply to all aspects of the present disclosure. The following terms, unless otherwise specified, have the following meanings. Any undefined terms have the meanings recognized in the relevant technical field.

[0069] Terms such as "reduce" or "inhibit", as used herein, mean, for example, the ability to cause an overall reduction of about 5% or more, about 10% or more, about 20% or more, about 50% or more, or about 75% or more at a certain level. The term "inhibit" or similar phrases includes complete or substantially complete inhibition, i.e., a reduction to zero or substantially zero.

[0070] Terms such as "increase" or "enhance", in one embodiment, relate to an increase or enhancement of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 80%, or at least about 100%.

[0071] As used herein, the term "aqueous phase" means a composition that contains water, either in whole or in part.

[0072] "Physiological pH", as used herein, means a pH of about 7.5.

[0073] As used in the present disclosure, "w / v%" means weight / volume percent, which is a unit of concentration meaning the amount of solute in grams (g) expressed as a percentage of the total volume of the solution in milliliters (ml).

[0074] As used in the present disclosure, "mol%" is defined as 100 times the ratio of the number of moles of one component to the total number of moles of all components.

[0075] In some cases, the ratio is given in weight percent, i.e., “wt%”. The term “wt%” means weight percent based on the total weight of the compound or composition.

[0076] As used herein, the term “mass fraction” means the mass ratio of a component to the total mass. Here, the mass fraction is typically expressed as a percentage (%), and is often referred to as weight percent and abbreviated as wt%. For example, the mass fraction of RNA present at a certain number of molecules greater than 60% means that more than 60% (by mass) of the total amount of RNA in the composition is present at this certain number of molecules.

[0077] The term “ionic strength” means the mathematical relationship between the number of different types of ionic species in a particular solution and their respective charges. Thus, the ionic strength I is given by the following equation:

[0078]

Equation

[0079] which is mathematically represented by the formula: where c is the molar concentration of a particular ionic species and z is the absolute value of its charge. The sum Σ is taken over all different ions (i) in the solution.

[0080] According to the present disclosure, in one embodiment, the term “ionic strength” is related to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, their concentration, or the effective concentration (presence of free ions) resulting from the presence of a chelating agent, is, in one embodiment, low enough to prevent the degradation of RNA. In one embodiment, the concentration or effective concentration of divalent ions is below the catalytic level for the hydrolysis of the phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent ions is 20 μM or less. In one embodiment, free divalent ions are absent or essentially absent.

[0081] "Osmolality" means the concentration of a solute expressed as the number of osmoles of solute per kilogram of solvent.

[0082] The term "freezing" usually relates to the solidification of a liquid by the removal of heat. The terms "lyophilizing" or "lyophilization" mean the freeze-drying of a substance by freezing the substance and then reducing the ambient pressure to directly sublime the frozen medium in the substance from the solid phase to the gas phase.

[0083] The term "spray drying" means spray drying a substance by mixing a (heated) gas with a fluid atomized (sprayed) in a vessel (spray dryer), and the solvent evaporates from the formed droplets, resulting in a dry powder.

[0084] The term "antifreeze agent" relates to a substance added to a formulation to protect the active ingredient during the freezing stage.

[0085] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying stage.

[0086] The term "reconstitute" relates to adding a solvent such as water to a dried product to return it to a liquid state, for example, its original liquid state and the like.

[0087] The term "recombinant", in the context of the present disclosure, means "produced by genetic engineering". In one embodiment, the "recombinant object" in the context of the present disclosure does not occur naturally.

[0088] As used herein, the term "naturally occurring" means the fact that the object 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 natural source, and has not been deliberately modified by humans in the laboratory is naturally occurring. The term "found in nature" means "naturally occurring" 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.

[0089] In the context of the present disclosure, the term "particle" relates to a structured entity formed by a molecule or molecular complex. In one embodiment, the term "particle" relates to a micro-sized or nano-sized structure, such as a micro-sized or nano-sized dense structure.

[0090] In the context of the present disclosure, the term "RNA particle" relates to a particle containing RNA. In one embodiment, the RNA particle is a nanoparticle.

[0091] As used in the present disclosure, "nanoparticle" preferably means a particle having an average diameter of at least about 50 nm.

[0092] The term "average diameter" provides a so-called Z 平均 value having a dimension of length and a dimensionless polydispersity (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814 - 4820, ISO 13321), and means the average hydrodynamic diameter of the particle as measured by dynamic light scattering (DLS) using data analysis using a so-called cumulant algorithm. Here, for a particle, "average diameter", "diameter", or "size" is used synonymously with this value of Z 平均 .

[0093] The "polydispersity index" is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis, as referred to in the definition of "average diameter". Under certain specific premises, it can be regarded as an indicator of the particle size distribution of a collection of nanoparticles.

[0094] As used herein, the terms "aggregate" or "complex" mean a larger entity such as a particle that forms from a plurality of (at least two) molecules. In this case, the individual molecules no longer exist independently. Aggregation is the process by which individual molecules associate non-covalently to form aggregates. Electrostatic interactions between positively charged molecules, such as polymers, etc., and negatively charged RNA can be involved in aggregate formation. This results in complexation and the spontaneous formation of RNA aggregates or RNA particles.

[0095] As used herein, the term "RNA aggregate" means an aggregate or unit containing a plurality of (at least two) RNA molecules. Thus, this term excludes a polyplex of one RNA molecule and one or more polymer molecules, and a polyplex is included in the terms "individual", "monomolecular", "unimolecular", or "monomer" when used herein with respect to RNA. The term "aggregate containing many RNA molecules", or simply "large RNA aggregate" or "large aggregate", as used herein, means an RNA aggregate containing 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 15 or more, 20 or more, 25 or more, or 50 or more RNA molecules, or even more RNA molecules.

[0096] As used herein, the term "polyplex" means an association formed by electrostatic interaction between a polymer and a nucleic acid, such as RNA. When the polyplex contains RNA, the term may also be referred to as an "RNA polyplex".

[0097] The terms "individual molecule", "single molecule", "monomolecule", or "monomer", as used herein, mean that a molecule does not exist as a plurality (at least two) of molecules of the same type and does not form a larger entity with other molecules of the same type by physical interaction. In particular, the terms "individual molecule", "single molecule", "monomolecule", or "monomer", as used herein, mean a unit that contains only one RNA molecule. The unit may contain molecules of any number of compounds other than RNA, such as polymers. Multimolecular RNA units (each of the latter containing two or more RNA molecules) can be distinguished by the different sizes of each unit. Typically, the terms "individual molecule", "single molecule", "monomolecule", or "monomer", as used herein, include RNA associated with a polymer, and the polymer and RNA do not form a unit with two or more RNA molecules per unit, but rather form a single-molecule RNA unit. In one embodiment, a single RNA molecule associates with a cationic polymer without intermolecular aggregation of the RNA molecules.

[0098] In this context, the term "fraction" relates to a part, such as a part of RNA, that can be separated from other fractions by a fractionation process, such as filtration, centrifugation, or chromatography. In one embodiment, a fraction can be associated with a unit (e.g., an RNA molecule-polymer complex) having a defined number of RNA molecules, such as one, two, three, four, five, etc. RNA molecules per unit. For example, different fractions can be related to single-molecule RNA fractions, bimolecular RNA fractions, trimolecular RNA fractions, tetramolecular RNA fractions, pentamolecular RNA fractions, etc., containing one, two, three, four, five, etc. RNA molecules per unit. Such various numbers of RNA molecules per unit are also designated herein as "molecularity".

[0099] As used herein, the term "dominant fraction" means a fraction of a certain number of RNA molecules that contains the most RNA (on a mass basis), such as a single-molecule RNA fraction, a two-molecule RNA fraction, a three-molecule RNA fraction, a four-molecule RNA fraction, a five-molecule RNA fraction, etc. For example, in a composition containing 30 ng of RNA, if 10 ng of RNA is present in the single-molecule RNA fraction, 8 ng of RNA is present in the two-molecule RNA fraction, 6 ng of RNA is present in the three-molecule RNA fraction, 4 ng of RNA is present in the four-molecule RNA fraction, and 2 ng of RNA is present in the five-molecule RNA fraction, the single-molecule RNA fraction is the dominant fraction, despite the fact that most of the RNA is present in fractions other than the single-molecule RNA fraction. If a composition contains only one type of RNA molecule, the dominant fraction is a fraction of a certain number of RNA molecules that contains the largest number of RNA molecules among all fractions.

[0100] One implementation commonly used to quantify RNA is the use of spectrophotometric analysis with a spectrophotometer. Spectrophotometric analysis is based on the principle that nucleic acids absorb ultraviolet light in a specific pattern. In the case of RNA, the sample is exposed to ultraviolet light at a wavelength of approximately 260 nanometers (nm), and a photodetector measures the light that has passed through the sample. A portion of the ultraviolet light will pass through, and a portion will be absorbed by the RNA. The higher the amount of light absorbed by the sample, the higher the RNA concentration in the sample.

[0101] According to the present invention, the terms "N / P ratio", "NP ratio", "N:P ratio", "N / P", and "NP" mean the molar ratio of the nitrogen atoms (N) of a polymer such as polyethyleneimine to the phosphorus atoms (P) of RNA.

[0102] RNA composition The present disclosure describes a composition comprising RNA and one or more polymers, wherein most of the RNA exists as individual molecules in solution. The polymer can associate with the RNA in various forms through non-covalent interactions with the RNA. The RNA described herein does not exist as a viral particle, particularly an infectious viral particle, i.e., it cannot infect cells with a virus. The RNA compositions described herein are typically formed from RNA and a cationic polymer, such as poly(ethyleneimine), etc. In some embodiments, the RNA composition comprises two or more types of RNA molecules, and the molecular parameters of the RNA molecules may be the same or different from each other with respect to molar mass or basic structural elements, such as molecular architecture, capping, coding region, or other features, etc.

[0103] Typically, a cationic polymer that can electrostatically bind to negatively charged RNA is used. These positively charged groups often consist of amines, which change their protonation state in the pH range between 5.5 and 7.5, and it is thought that this results in an ionic imbalance that causes endosome rupture. Natural polymers such as chitosan and synthetic polymers can be used herein.

[0104] "Polymer" as used herein is given its ordinary meaning, i.e., the molecular structure comprises one or more repeating units (monomers) connected by covalent bonds. All of the repeating units may be the same, or in some cases, two or more types of repeating units may be present in the polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties, may also be present in the polymer.

[0105] When two or more types of repeating units are present within a polymer, the polymer is said to be a "copolymer." In any embodiment using a polymer, it should be understood that the polymer used may, in some cases, be a copolymer. The repeating units forming the copolymer can be arranged in any manner. For example, the repeating units can be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., it can include one or more regions each containing a first repeating unit (e.g., a first block) and one or more regions each containing a second repeating unit (e.g., a second block), etc. A block copolymer can have two blocks (a diblock copolymer), three blocks (a triblock copolymer), or a greater number of different blocks.

[0106] In certain embodiments, the polymer is biocompatible. A biocompatible polymer is typically a polymer that does not cause much cell death at moderate concentrations as a result. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer can be chemically and / or biologically degraded in a physiological environment such as in the body. In the context of the present invention, the cationic or polycationic polymer is preferably selected from any cationic or polycationic polymer suitable for the formulation of RNA as described herein. Particularly preferred cationic or polycationic polymers are cationic or polycationic peptides or proteins, especially protamine, histone, spermine, spermidine, polyarginine, polylysine, for example, poly-L-lysine (PLL), etc.; cationic polysaccharides, for example, chitosan, diethylaminoethyl (DEAE) dextran, etc.; or cationic or polycationic polymers, for example, polyimines, for example, polyethyleneimine (PEI), poly(propyleneimine), poly(amidoamine) (PAA), polyaminoester (PAE), particularly, poly(β-aminoester), poly(allylamine), polyvinylamine, poly(dimethylaminoethyl methacrylate), hexadimethrine bromide (commercial brand name is Polybrene), and can be a nucleic acid-binding peptide or protein selected therefrom.

[0107] In certain embodiments, the polymer is a polyalkyleneimine, for example, polyethyleneimine (PEI), etc.

[0108] In one embodiment, the polymer comprises a polyamidoamine (PAMAM) polymer. Poly(amidoamine) or PAMAM is a class of dendrimers made of amide and amine functional repeating branched-chain subunits. PAMAM dendrimers have an overall spherical-like shape and are represented by an internal molecular architecture consisting of tree-like branches where each outer “layer” or generation has exponentially more branching points. This branched architecture distinguishes PAMAM from other dendrimers derived from conventional polymers, as they enable low polydispersity and a high level of structural control during synthesis, resulting in many surface sites compared to the total molecular volume. In one embodiment, PAMAM is functionalized with an L-lysine polymer.

[0109] Polyalkyleneimines Polyalkyleneimines for use herein include linear and branched polyalkyleneimines and mixtures thereof. The average molecular weight of the polyalkyleneimine is preferably between 1000 Da and 150000 Da, between 5000 Da and 100000 Da, between 10000 Da and 50000 Da, between 15000 Da and 30000 Da, between 20000 Da and 25000 Da, or about 22500 Da.

[0110] The polyalkyleneimine as used herein preferably has the following general formula (I):

[0111]

Chemical formula

[0112] [wherein, R is H, an acyl group or the following general formula (II):

[0113]

Chemical formula

[0114] is a group containing, and R1 is H or the following general formula (III):

[0115] [Chemical formula]

[0116] is a group containing, where n, m, and l are independently selected from integers from 2 to 10; p, q, and r are integers, and the sum of p, q, and r is preferably such that the average molecular weight of the polymer is between 1000 Da and 100000 Da, between 5000 Da and 75000 Da, between 10000 Da and 50000 Da, between 15000 Da and 30000 Da, between 20000 Da and 25000 Da, or about 20000 Da].

[0117] In one embodiment, n, m, and l are independently selected from 2, 3, 4, and 5, preferably from 2 and 3, and more preferably 2. In one embodiment, R1 is H. In one embodiment, R is H or an acyl group.

[0118] In one embodiment, the polyalkyleneimine contains polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine.

[0119] A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably between 1000 Da and 100000 Da, between 5000 Da and 75000 Da, between 10000 Da and 50000 Da, between 15000 Da and 30000 Da, between 20000 Da and 25000 Da, or about 20000 Da. Preferred according to the present invention is linear PEI. 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), and the poly(2-ethyl-2-oxazoline) is then acid-hydrolyzed to cleave the N-propionyl group to obtain PEI.

[0120] RNA concentration In certain embodiments of the present disclosure, the RNA in the compositions described herein is at a concentration of from about 0.0001 mg / mL to about 1 mg / mL, from about 0.0001 mg / mL to about 0.5 mg / mL, from about 0.00025 mg / mL to about 0.5 mg / mL, from about 0.0005 mg / mL to about 0.25 mg / mL, from about 0.0025 mg / mL to about 0.1 mg / mL, or from about 0.005 mg / mL to about 0.1 mg / mL. In certain embodiments, the RNA is at a concentration of from about 0.00025 mg / mL to about 0.1 mg / mL, from about 0.00025 mg / mL to about 0.09 mg / mL, from about 0.00025 mg / mL to about 0.08 mg / mL, from about 0.00025 mg / mL to about 0.07 mg / mL, from about 0.00025 mg / mL to about 0.06 mg / mL, or from about 0.00025 mg / mL to about 0.05 mg / mL.

[0121] RNA In the present disclosure, the term "RNA" relates to nucleic acid molecules containing ribonucleotide residues. In preferred embodiments, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" means a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA, such as substantially pure RNA like partially purified RNA, synthetic RNA, recombinantly produced RNA, and modified RNAs that differ from naturally occurring RNAs by addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations can mean addition of non-nucleotide material to internal RNA nucleotides or to the ends of the RNA. It is also contemplated herein that the nucleotides in the RNA can be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the context of the present disclosure, these modified RNAs are considered analogs of naturally occurring RNAs.

[0122] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As is established in the art, mRNA generally includes a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA means a nucleic acid containing deoxyribonucleotides.

[0123] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and 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. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0124] In certain embodiments of the present disclosure, the RNA is a replicon RNA or simply a "replicon", particularly self-replicating RNA (self-propagating RNA; saRNA). In one particularly preferred embodiment, the replicon or self-replicating RNA comprises elements derived from or related to an ssRNA virus, particularly a plus-strand ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of plus-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (see Jose et al., Future Microbiol., 2009, vol. 4, pages 837-856 for an overview of the alphavirus life cycle). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in the transcription, modification, and replication of viral RNA and protein modification) and structural proteins (forming viral particles). Typically, there are two open reading frames (ORFs) in the genome. Four non-structural proteins (nsP1-nsP4) are typically encoded together by the first ORF that begins near the 5' end of the genome, while the structural proteins of alphaviruses are found downstream of the first ORF and are encoded together by a second ORF that extends near the 3' end of the genome. Typically, the first ORF is longer than the second ORF, and the ratio is approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding non-structural proteins are translated from the genomic RNA, while the genomic 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., vol. 87, 111-124). After infection, i.e., at the initial stage of the virus life cycle, the (+)-strand genomic RNA behaves just like messenger RNA for the translation of the open reading frame encoding the non-structural polyprotein (nsP1234).For the delivery of foreign genetic information to target cells or target organisms, vectors derived from alphaviruses have been proposed. In a simple approach, the open reading frame encoding the structural protein of an alphavirus is replaced by an open reading frame encoding the protein of interest. The alphavirus-based trans-replication system relies on alphavirus nucleotide sequence elements on two separate nucleic acid molecules, i.e., one nucleic acid molecule encodes the viral replicase and the other nucleic acid molecule can be replicated by said replicase in trans (hence the designation of the trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. The nucleic acid molecule that can be replicated by the replicase in trans must contain certain alphavirus sequence elements in order to enable recognition and RNA synthesis by the alphavirus replicase.

[0125] In one embodiment, the RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine, pseudouridine (ψ), N1-methyl-pseudouridine (m 1 ψ), or 5-methyl-uridine (m 5 U).

[0126] In some embodiments, the RNA according to the present disclosure includes a 5'-cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA can be modified with a 5'-cap analog. The term "5'-cap" refers to the structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA by a 5'-5' triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position. Providing an RNA having a 5'-cap or a 5'-cap analog can be achieved by in vitro transcription, and the 5'-cap can be expressed co-transcriptionally onto the RNA strand or can be ligated to the RNA post-transcriptionally using a capping enzyme.

[0127] In some embodiments, the RNA according to the present disclosure includes a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region of a DNA molecule that is transcribed into an amino acid sequence but not translated, or the corresponding region in an RNA molecule, such as an mRNA molecule, etc. The untranslated region (UTR) can be present in the 5' (upstream) (5'-UTR) and / or 3' (downstream) (3'-UTR) of the open reading frame. The 5'-UTR, if present, is located at the 5'-end upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. The 3'-UTR, if present, is located at the 3'-end downstream of the stop codon of the protein-coding region, but the term "3'-UTR" preferably does not include the poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0128] In some embodiments, the RNA according to the present disclosure comprises a 3'-poly(A) sequence. The term "poly(A) sequence" typically relates to a sequence of adenyl (A) residues located at the 3' end of an RNA molecule. According to the present disclosure, in one embodiment, the poly(A) sequence comprises at least about 20, at least about 40, at least about 80, or at least about 100, and up to about 500, up to about 400, up to about 300, up to about 200, or up to about 150 A nucleotides, particularly about 120 A nucleotides. In one embodiment, the poly(A) sequence can be interrupted by one or more short sequences of between 5 and 20 nucleotides that include nucleotides other than A nucleotides, for example.

[0129] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code of a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.

[0130] With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell for instructing the assembly of a chain of mRNA into a peptide or protein of an amino acid sequence.

[0131] The RNA can be coding RNA, i.e., RNA that encodes a peptide or protein. The RNA can express the encoded peptide or protein. For example, the RNA can be RNA that encodes and expresses a pharmaceutically active peptide or protein. Alternatively, the RNA can be non-coding RNA, such as antisense RNA, microRNA (miRNA), or siRNA, etc.

[0132] The RNA used in this specification can be pharmaceutically active RNA. "Pharmaceutically active RNA" is RNA that encodes a pharmaceutically active peptide or protein or is itself pharmaceutically active RNA, for example, it has one or more pharmaceutical activities, such as those described for pharmaceutically active proteins, for example, immunostimulatory effects and the like. For example, the RNA can be one or more strands of RNA interference (RNAi). Such agents can include small interfering RNA (siRNA), or small hairpin RNA (shRNA), or precursors of siRNA or microRNA-like RNA that target a target transcript, for example, a transcript of an endogenous disease-related transcript of a subject.

[0133] Some aspects of the present disclosure involve targeted delivery of the RNA disclosed herein to a particular cell or tissue. In one embodiment, the present disclosure involves targeting the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen. The step of targeting the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen, is particularly preferred when the administered RNA is RNA encoding an antigen or epitope for inducing an immune response. In one embodiment, the target cells are spleen cells. In one embodiment, the target cells are antigen-presenting cells, such as specialized antigen-presenting cells in the spleen and the like. In one embodiment, the target cells are dendritic cells in the spleen. The "lymphatic system" is a part of the circulatory system and an important part of the immune system that includes a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphoid organs, a conducting network of lymphatic vessels, and circulating lymph fluid. Primary lymphoid organs or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary lymphoid organs or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphocytes and initiate an adaptive immune response.

[0134] In one embodiment, the target organ is the liver and the target tissue is liver tissue. In particular, delivery to such labeled tissue is preferred when the presence of RNA or the encoded peptide or protein in this organ or tissue is desirable, and / or when it is desired to express large amounts of the encoded peptide or protein, and / or when the systemic presence of the encoded peptide or protein in particularly substantial amounts is desired or necessary.

[0135] In one embodiment, after administration of the RNA composition described herein, at least a portion of the RNA is delivered to a target cell or target organ. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is an RNA encoding a peptide or protein, and the RNA is translated by the target cell to produce the peptide or protein. In one embodiment, the target cell is a liver cell. In one embodiment, the target cell is a muscle cell. In one embodiment, the target cell is an endothelial cell. In one embodiment, the target cell is a tumor cell or a cell in the tumor microenvironment. In one embodiment, the target cell is a blood cell. In one embodiment, the target cell is a lymph node cell. In one embodiment, the target cell is a lung cell. In one embodiment, the target cell is a skin cell. In one embodiment, the target cell is a splenocyte. In one embodiment, the target cell is an antigen-presenting cell, such as a specialized antigen-presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell of the spleen. In one embodiment, the target cell is a T cell. In one embodiment, the target cell is a B cell. In one embodiment, the target cell is a NK cell. In one embodiment, the target cell is a monocyte. Accordingly, the RNA composition described herein can be used to deliver RNA to such target cells. Accordingly, the present disclosure also relates to a method for delivering RNA to a target cell in a subject, the method comprising administering the RNA composition described herein to the subject. In one embodiment, the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is an RNA encoding a peptide or protein, and the RNA is translated by the target cell to produce the peptide or protein.

[0136] In certain embodiments, the RNA encodes a pharmaceutically active peptide or protein.

[0137] According to the present disclosure, the term "encoded by RNA" means that when the RNA is present in a suitable environment, such as within the cells of a target tissue, etc., it can direct the assembly of amino acids to produce the peptide or protein that it encodes during the process of translation. In one embodiment, the RNA can interact with a cellular translation mechanism that enables the translation of the peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or in the nucleus), secrete the encoded peptide or protein, or produce it on the surface.

[0138] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides and means a substance containing consecutive amino acids linked to each other by peptide bonds, about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, up to about 100, or up to about 150. The term "protein" means a large peptide, particularly a peptide having at least about 151 amino acids, although the terms "peptide" and "protein" are usually used synonymously herein.

[0139] A "pharmaceutically active peptide or protein" or a "therapeutic peptide or protein" has a favorable or beneficial effect on the condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In one embodiment, a pharmaceutically active peptide or protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "pharmaceutically active peptide or protein" includes the entire protein or polypeptide and can also mean pharmaceutically active fragments thereof. It can also include analogs of pharmaceutically active peptides or proteins.

[0140] Examples of pharmaceutically active proteins include, but are not limited to, cytokines and their derivatives, such as cytokine fusions (e.g., albumin-cytokine fusions), and immune system proteins, such as immunologically active compounds (e.g., interleukins, colony-stimulating factors (CSF), 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, chimeric antigen receptors (CAR), immunoglobulins, such as antibodies or bispecific antibodies, etc., e.g., for the production of immune-stimulating or neutralizing antibodies in the case of viral / bacterial infections, soluble major histocompatibility complex antigens, immunologically active antigens, such as bacterial, parasitic, or viral antigens, allergens, autoantigens, antibodies), hormones (insulin, thyroid hormones, catecholamines, gonadotropins, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, etc.), growth hormones (e.g., human growth hormone), growth factors (e.g., epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, cholesterol biosynthetic or degradative, steroidogenic enzymes, coenzymes, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochrome, adenylate or guanylate cyclase, neuraminidase, lysosomal enzymes, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (growth hormone or growth factor-binding proteins, etc.), transcription factors and translation factors, tumor growth inhibitory proteins (e.g., proteins that inhibit angiogenesis), structural proteins (e.g., collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin, etc.), 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,Examples include erythropoietin granulocyte colony-stimulating factor (GCSF), modified factor VIII, anticoagulants, etc.

[0141] The term "immunologically active compound" relates to any compound that modifies the immune response, for example, by inducing and / or suppressing the maturation of immune cells, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. Immunologically active compounds have potent immunostimulatory activities, such as, but not limited to, antiviral and antitumor activities, etc., and can also downregulate other aspects of the immune response, for example, by diverting the immune response from a TH2 immune response that is useful for the treatment of various TH2-mediated diseases. Immunologically active compounds can be useful as vaccine adjuvants.

[0142] In one embodiment, the pharmaceutically active peptide or protein includes cytokines. The term "cytokine" means a category of small proteins (about 5-20 kDa) that are important in cell signaling. The release of small proteins has an effect on the behavior of the cells around them. Cytokines are involved in autocrine signaling, paracrine signaling, and endocrine signaling as immunomodulatory agents. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones and growth factors (despite some overlap in the jargon). Cytokines are produced by a wide range of cells, such as immune cells like macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by two or more types of cells. Cytokines act via receptors and are particularly important in the immune system; cytokines regulate the balance between the humoral immune response and the cell-based immune response and regulate the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways. In one embodiment, the pharmaceutically active protein according to the present invention is a cytokine involved in the regulation of lymphatic homeostasis, preferably a cytokine involved in the growth, priming, proliferation, differentiation, and / or survival of T cells, preferably inducing or enhancing them. In one embodiment, the cytokine is an interleukin. In one embodiment, the pharmaceutically active protein according to the present invention is an interleukin selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

[0143] In one embodiment, the pharmaceutically active peptide or protein comprises a replacement protein. In this example, the invention provides a method for the treatment of a subject having a disorder that requires protein replacement (e.g., protein deficiency disorder), the method comprising administering to the subject an RNA as described herein that encodes a replacement protein. The term "protein replacement" means the introduction of a protein (including its functional variants) into a subject having a deficiency of such protein. The term also means the introduction of a protein into a subject in need of or benefiting from the provision of a protein, e.g., a subject suffering from protein deficiency. The term "disorder characterized by protein deficiency" means any disorder presented by a pathology caused by the absence or insufficient amount of a protein. The term encompasses protein folding diseases that result in a biologically inactive protein product, i.e., structural disorders. Protein deficiency may be involved in infectious diseases, immunosuppression, organ disorders, glandular disorders, radiation sickness, nutritional deficiencies, poisoning, or other environmental or external triggers. In one embodiment, the pharmaceutically active peptide or protein comprises one or more antigens or one or more epitopes, i.e., administration of the peptide or protein to a subject can induce an immune response against one or more antigens or one or more epitopes in a subject in which the administration can be therapeutic or partially or fully protective.

[0144] The term "antigen" relates to a causative agent containing an epitope capable of eliciting an immune response. The term "antigen" encompasses, in particular, proteins and peptides. In one embodiment, an antigen is presented by cells of the immune system, such as antigen-presenting cells like dendritic cells or macrophages. An antigen or its processing product, such as a T cell epitope, etc., is bound in one embodiment by a T cell receptor or a B cell receptor, or by an immunoglobulin molecule such as an antibody. Thus, an antigen or its processing product can specifically react with an antibody or a T lymphocyte (T cell). In one embodiment, an antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, etc., and an epitope is derived from such an antigen.

[0145] The term "disease-related antigen" is used in its broadest sense to mean any antigen associated with a disease. A disease-related antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cell antigen-specific immune response and / or a humoral antibody response against the disease. Thus, a disease-related antigen or its epitope can be used for therapeutic purposes. A disease-related antigen can be associated with an infection by a microorganism, typically a microbial antigen, or with cancer, typically a tumor.

[0146] The term "tumor antigen" means a component of a cancer cell that can be derived from the cytoplasm, cell surface, and cell nucleus. In particular, it means an antigen produced intracellularly or as a surface antigen on tumor cells.

[0147] The term "viral antigen" means any viral component having antigenic properties, i.e., capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0148] The term "bacterial antigen" means any bacterial component having antigenic properties, i.e., capable of eliciting an immune response in an individual. A bacterial antigen can be derived from the cell wall or the cytoplasmic membrane of a bacterium.

[0149] The term "epitope" means a part or fragment of a molecule such as an antigen that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or antibodies. The epitope of an antigen can include a continuous or discontinuous part of the antigen and can have an amino acid length between about 5 and about 100, for example, between about 5 and about 50, more preferably between about 8 and about 30, and most preferably between about 10 and about 25. For example, the epitope can preferably have an amino acid length of 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In one embodiment, the epitope has an amino acid length between about 10 and about 25. The term "epitope" encompasses T cell epitopes.

[0150] The term "T cell epitope" means a part or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" relate to a complex of genes that includes MHC class I and MHC class II molecules and are present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen-presenting cells or diseased cells in an immune response. MHC proteins or molecules bind to peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and show self-antigens (peptide fragments derived from the cell itself) and non-self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the bound peptide typically has an amino acid length between about 8 and about 10, although longer or shorter peptides can also be effective. In the case of class II MHC / peptide complexes, the bound peptide typically has an amino acid length between about 10 and about 25, particularly between about 13 and about 18, although longer and shorter peptides can also be effective.

[0151] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells), including cytolytic T cells. The term "antigen-specific T cell" or similar terms refer to a T cell that recognizes an antigen targeted by a T cell, particularly when presented on the surface of an antigen-presenting cell or a diseased cell such as a cancer cell in the context of an MHC molecule, and preferably exhibits an effector function of the T cell. A T cell is considered specific for an antigen when the cell kills a target cell expressing the antigen. T cell specificity can be evaluated using any of a variety of standard techniques, for example, within a chromium release assay or a proliferation assay. Alternatively, synthesis of a lymphokine (such as interferon-γ, etc.) can be measured.

[0152] In certain embodiments of the present disclosure, the RNA encodes at least one epitope. In certain embodiments, the epitope is derived from a tumor antigen. The tumor antigen can be a "standard" antigen that is generally known to be expressed in various cancers. The tumor antigen can also be a "neoantigen" that is specific to an individual tumor and has not been previously recognized by the immune system. Neoantigens or neoepitopes can result from one or more cancer-specific mutations in the genome of cancer cells that result in amino acid changes. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, such as, for example, CLAUDIN-6, CLAUDIN-18.2, and CLAUDIN-12, etc. 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, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / melanA, MC1R, myosin / m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml / 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, WT, and WT-1.

[0153] Cancer mutations vary from individual to individual. Thus, cancer mutations encoding novel epitopes (neoepitopes) represent attractive targets in the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy relies on the selection of cancer-specific antigens and epitopes capable of inducing a strong immune response within the host. RNA can be used to deliver patient-specific tumor epitopes to the patient. Dendritic cells (DCs), which are resident in the spleen, represent antigen-presenting cells of interest particularly for RNA expression of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to promote a therapeutic effect in tumor vaccine compositions. Rapid sequencing of the tumor mutanome can provide multiple epitopes, for example, for an individualized vaccine that can be encoded by the RNA described herein as a single polypeptide, which epitopes may optionally be separated by linkers. In certain embodiments of the present disclosure, the RNA encodes at least 1 epitope, at least 2 epitopes, at least 3 epitopes, at least 4 epitopes, at least 5 epitopes, at least 6 epitopes, at least 7 epitopes, at least 8 epitopes, at least 9 epitopes, or at least 10 epitopes. Exemplary embodiments include RNA encoding at least 5 epitopes (referred to as a "pentatope") and RNA encoding at least 10 epitopes (referred to as a "decatope").

[0154] A. Salts and ionic strength According to the present disclosure, the compositions described herein may include salts, such as organic or inorganic salts, examples of which include, but are not limited to, sodium chloride, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, potassium acetate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, sodium acetate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA) and amino acids.

[0155] Generally, the compositions described herein preferably contain salts at a concentration in the range of from 0 mM to about 100 mM, from about 5 mM to about 50 mM, or from about 5 mM to about 20 mM. In one embodiment, the composition contains an ionic strength corresponding to such salt concentration.

[0156] In one embodiment, the positively charged monovalent ions such as sodium chloride are at a concentration of from 0 mM to about 50 mM, from 0 mM to about 40 mM, or from about 10 mM to about 20 mM.

[0157] In one embodiment, the positively charged divalent ions are at a concentration (or effective concentration) of from 0 mM to about 20 μM, from 0 mM to about 10 μM, or from about 0 mM to about 5 μM.

[0158] B. Stabilizers The compositions described herein may also include stabilizers to avoid substantial degradation of the product quality, particularly substantial degradation of RNA activity during storage, freezing, lyophilization, and / or spray drying, for example, to reduce or prevent aggregation, RNA degradation, and / or other types of damage.

[0159] In certain embodiments, the stabilizer is a cryoprotectant or a lyoprotectant.

[0160] In certain embodiments, the stabilizer is a carbohydrate. As used herein, the term "carbohydrate" means and includes monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides. In certain embodiments, the stabilizer is sucrose and / or trehalose.

[0161] In certain embodiments, the stabilizer is an amino acid or a surfactant (e.g., poloxamer).

[0162] C. pH and Buffers According to the present disclosure, the compositions described herein have a pH suitable for the stabilization of the compositions, particularly the stabilization of RNA. In one embodiment, the compositions described herein have a pH of from about 4 to about 8, from about 5 to about 7, or from about 5.5 to about 6.5.

[0163] While not desiring to be bound by theory, the use of a buffer maintains the pH of the composition during the manufacture, storage, and use of the composition. In certain embodiments of the present disclosure, the buffer can be sodium bicarbonate, monosodium phosphate, disodium phosphate, monopotassium phosphate, dipotassium phosphate, [tris(hydroxymethyl)methylamino]propanesulfonic acid (TAPS), 2-(bis(2-hydroxyethyl)amino)acetic acid (bicine), 2-amino-2-(hydroxymethyl)propane-1,3-diol (tris), N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine), 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 1,4-piperazinediethanesulfonic acid (PIPES), dimethylarsinic acid, 2-morpholin-4-ylethanesulfonic acid (MES), 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), or phosphate buffered saline (PBS). Other suitable buffer systems can be acetic acid alone or in salt, citric acid alone or in salt, boric acid alone or in salt, and phosphoric acid alone or in salt, or amino acids and amino acid derivatives.

[0164] In certain embodiments, the buffer has a concentration of from about 2.5 mM to about 20 mM, or from about 2.5 mM to about 10 mM.

[0165] D. Chelating Agents Certain embodiments of the present disclosure contemplate the use of chelating agents in the compositions described herein. A chelating agent means a chemical compound that can form at least two coordination covalent bonds with a metal ion, thereby producing a stable and water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions that can induce the acceleration of RNA degradation in the present disclosure. Examples of suitable chelating agents include, but are not limited to, ethylenediaminetetraacetic acid (EDTA), salts of EDTA, desferrioxamine B, desferrioxamine, sodium dithiocarbamate, penicillamine, calcium pentetate, sodium salt of pentetate, succimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexane tetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), bis(aminoethyl) glycol ether-N,N,N',N'-tetraacetic acid, iminodiacetic acid, citric acid, tartaric acid, fumaric acid, or salts thereof. In certain embodiments, the chelating agent is EDTA or a salt of EDTA. In an exemplary embodiment, the chelating agent is disodium EDTA dihydrate.

[0166] In some embodiments, the EDTA is at a concentration of from about 0.05 mM to about 5 mM, from about 0.1 mM to about 2.5 mM, or from about 0.25 mM to about 1 mM.

[0167] E. Physical State of the Composition In embodiments, the compositions of the present disclosure are liquid or solid. Non-limiting examples of solids include frozen forms, or dehydrated forms, such as lyophilized or spray-dried forms, etc. In a preferred embodiment, the composition is liquid.

[0168] Pharmaceutical Composition The compositions described herein are useful as, or for the preparation of, a pharmaceutical composition or agent for therapeutic or prophylactic treatment.

[0169] The term "pharmaceutical composition" preferably relates to a formulation containing a therapeutically effective agent together with a pharmaceutically acceptable carrier, diluent, and / or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations. The pharmaceutical compositions of the present disclosure may include one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include heterogeneous groups of compounds such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum, etc.), bacterial products (e.g., Bordetella pertussis toxin, etc.), or immunostimulatory complexes, etc. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, chemokines, etc. Chemokines can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-γ, GM-CSF, LT-a. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide (registered trademark) ISA51, etc. Other adjuvants suitable for use in the present disclosure include lipopeptides such as Pam3Cys, etc. as well as lipophilic components such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid-A (MPL), monomycolyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).

[0170] The pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in a "pharmaceutically acceptable preparation".

[0171] The term "pharmaceutically acceptable" means the non-toxicity of a material that does not interact with the action of the active ingredient of the pharmaceutical composition.

[0172] The term "pharmaceutically effective amount" means an amount that achieves a desired reaction or desired effect, either alone or together with additional dosages. In the case of treating a particular disease, the desired reaction is preferably related to inhibiting the progression of the disease. This includes slowing down the progression of the disease and, in particular, interrupting or reversing the progression of the disease. The desired reaction in the treatment of a disease can also be a delay in the onset or prevention of the onset of the disease or condition. The effective amount of the compositions described herein will vary depending on the condition being treated, the severity of the disease, the 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 particular route of administration, and similar factors. Accordingly, the dosage administered of the compositions described herein can vary depending on such various parameters. If the patient's response is inadequate at the initial dosage, a higher dosage (or a higher dosage that can be effectively achieved by another, more local route of administration) can be used.

[0173] The pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0174] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0175] The term "excipient", as used herein, means a substance that can be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0176] The term "diluent" relates to an agent for dilution and / or viscosity reduction. Moreover, the term "diluent" includes any one or more of a fluid, a liquid, or a solid suspension and / or a mixed medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0177] The term "carrier" means a component that can be natural, synthetic, organic, or inorganic and is combined with an active ingredient to facilitate, enhance, or enable the administration of a pharmaceutical composition. When used herein, the carrier can be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, hydrogenated naphthalene, and in particular, biocompatible polylactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure includes isotonic saline.

[0178] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0179] Pharmaceutical carriers, excipients, or diluents can be selected in relation to the intended route of administration and standard pharmaceutical practice.

[0180] Route of administration of the pharmaceutical composition In one embodiment, the pharmaceutical composition described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration can include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" means administration by any method other than through the gastrointestinal tract, such as, for example, by intramuscular, intradermal, subcutaneous, or intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for intramuscular administration.

[0181] Use of the pharmaceutical composition The compositions described herein can be used in the therapeutic or prophylactic treatment of various diseases, particularly those in which the delivery of a peptide or protein to a subject results in a therapeutic or prophylactic effect. For example, the delivery of a virus-derived antigen or epitope can be useful in the treatment of viral diseases caused by that virus. The delivery of a tumor antigen or epitope can be useful in the treatment of cancer diseases in which cancer cells express the tumor antigen. The delivery of a functional protein or enzyme can be useful in the treatment of genetic disorders characterized by a dysfunctional protein, such as lysosomal storage diseases (e.g., mucopolysaccharidoses) or factor deficiencies. The delivery of a cytokine or cytokine fusion can be useful for modulating the tumor microenvironment.

[0182] The term "disease" (also referred to herein as "disorder") means an abnormal condition that affects an individual's body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. A disease can be caused by factors originating from external sources such as infections, or can be caused by internal dysfunctions such as autoimmune diseases. In humans, the term "disease" is often used more broadly to mean any condition that causes pain, dysfunction, suffering, social problems, or death to the affected individual, or similar problems to people in contact with the individual. In this broader sense, it often includes injuries, physical disabilities, disorders, syndromes, infections, single symptoms, deviant behaviors, and atypical variations of structure and function, while in other contexts and for other purposes, these may be regarded as distinguishable categories. A disease usually affects an individual not only physically but also emotionally, because in many diseases, living with it can change the person's view of life and personality.

[0183] In this context, the terms "treatment", "treating", or "treatment intervention" relate to the management and care of a subject for the purpose of combating a condition such as a disease or disorder. The terms are intended to encompass the full range of treatment for a given condition that a patient is suffering from, such as administering a therapeutically effective compound to reduce symptoms or complications, slow the progression of a disease, disorder, or condition, alleviate or sedate symptoms and complications, and / or cure or eliminate the disease, disorder, or condition, as well as to prevent the condition, where prevention should be understood as the management and care of an individual for the purpose of combating the disease, condition, or disorder and includes administering an active compound to prevent the onset of symptoms or complications.

[0184] The term "therapeutic treatment" relates to any treatment that improves the health condition of an individual and / or extends (increases) the lifespan. Such treatment can eliminate a disease in an individual, can prevent, inhibit, or slow the occurrence of a disease in an individual, can reduce the frequency or severity of symptoms in an individual, and / or can reduce the recurrence in an individual currently suffering from or having previously suffered from a disease.

[0185] The term "preventive treatment" or "prophylactic treatment" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "preventive treatment" and "prophylactic treatment" are used interchangeably herein.

[0186] The terms "individual" and "subject" are used interchangeably herein. They mean a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate), or any other non-mammal, e.g., bird (chicken), fish, etc., or any other animal species that can suffer from or be susceptible to a disease or disorder (e.g., cancer, infectious disease), whether or not having the disease or disorder, or can have a need for preventive intervention such as vaccination, or can have a need for intervention such as protein supplementation. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not specify a particular age and thus include adults, the elderly, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient".

[0187] The term "patient" means an individual or subject to treatment, particularly an individual or subject with a disease.

[0188] In one embodiment of the present disclosure, the objective is to provide protection against an infectious disease by vaccination.

[0189] In one embodiment of the present disclosure, the object is to provide secreted therapeutic proteins, such as antibodies, bispecific antibodies, cytokines, cytokine fusion proteins, enzymes, etc., to a subject, particularly a subject in need thereof.

[0190] In one embodiment of the present disclosure, the object is to provide protein replacement therapies, such as the production of erythropoietin, factor VII, von Willebrand factor, β-galactosidase, α-N-acetylglucosaminidase, to a subject, particularly a subject in need thereof.

[0191] In one embodiment of the present disclosure, the object is to regulate / reprogram immune cells in the blood.

[0192] In a subject in which the pharmaceutical composition described herein, comprising RNA encoding a peptide or protein comprising one or more antigens or one or more epitopes, can be therapeutic or partially or completely protective, it can be administered to the subject to elicit an immune response against the one or more antigens or one or more epitopes. Those skilled in the art will know that one of the principles of immunotherapy and vaccination is based on the fact that an immune protective response against a disease is generated by immunizing a subject with an antigen or epitope that is immunologically relevant to the disease being treated. Thus, the pharmaceutical compositions described herein are applicable for inducing or enhancing an immune response. Thus, the pharmaceutical compositions described herein are useful in the prophylactic treatment and / or therapeutic treatment of diseases involving an antigen or epitope.

[0193] The term "immunization" or "vaccination" describes the process of administering an antigen to an individual for the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.

[0194] The listing of documents and studies referenced in this specification is not intended as an admission that any of the foregoing is pertinent prior art. All statements regarding the content of these documents are based on information available to the applicant and do not constitute any admission as to the validity of the content of these documents.

[0195] The following description is presented to enable a person skilled in the art to make and use various embodiments. The description of specific devices, techniques, and applications is provided by way of example only. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described herein, but rather are to be accorded the scope consistent with the claims.

Examples

[0196] Materials and Methods Preparation of PEI Formulations Linear polyethyleneimine with a molecular weight of 20 kDa to 25 kDa is used (In vivo / Jet-PEI). For calculating the N / P ratio of polyplex formulations, the positive charge (N) of the nitrogen atoms of the amines in PEI and the negative charge (phosphate) (P) of the RNA are considered. The required RNA concentration is prepared in the final injection buffer. In separate tubes, the PEI for the formulation is diluted in injectable H2O. Complexation of the RNA will occur by mixing the RNA-PEI phases in the desired volume ratio. The formulation is incubated at room temperature for 15 minutes for stabilization. The buffer used for the formulation in all experiments is MGB (MES-buffered glucose (10 mM MES, pH 6.1, 5% D-glucose)).

[0197] Preparation of Formulations Using Viromer® (Aliphatic / Aromatic Substituted Polyalkyleneimine) Viromer® is a commercially available branched PEI that is highly modified with alkyl chains and aromatic motifs. For the calculation of the N / P ratio of the Viromer® formulation, the positive charge (N) of the nitrogen atoms of the amines in Viromer® and the negative charge (phosphate) of the RNA (P) are considered. The required RNA concentration is prepared in the final injection buffer. In separate tubes, Viromer® for the formulation is diluted in injectable H2O. Complexation of the RNA will occur by mixing the RNA-Viromer phases in the desired volume ratio. The formulation is incubated at room temperature for 15 minutes for stabilization. The buffer used for the formulation in all experiments is MGB (MES-buffered glucose (10 mM MES, pH 6.1, 5% D-glucose)).

[0198] Preparation of star poly-L-lysine PAMAM dendrimer (G5-PLL64) formulation A novel biodegradable cationic polymer of the poly(amidoamine) family, consisting of a G(5)-PAMAM dendrimer functionalized with 64 arms of penta-L-lysine polymer. For the calculation of the N / P ratio of the polyplex formulation, the positive charge (N) of the nitrogen atoms of the amines in G5-PLL64 and the negative charge (phosphate) of the RNA (P) are considered. The required RNA concentration is prepared in the final injection buffer. In separate tubes, G5-PLL64 for the formulation is diluted in injectable H2O. Complexation of the RNA will occur by mixing the RNA-G5-PLL64 phases in the desired volume ratio. The formulation is incubated at room temperature for 15 minutes for stabilization. The buffer used for the formulation in all experiments is MGB (MES-buffered glucose (10 mM MES, pH 6.1, 5% D-glucose)).

[0199] Luciferase and viability assays In order to reach the desired concentration in the culture medium, the formulated nanoparticles are appropriately diluted. Seed 5,000 C2C12 cells / well in a 96-well plate. 24 hours after seeding, aspirate the medium and apply 100 μl of culture medium with the diluted formulation. Maintain the plate in a cell incubator until the desired time point is reached. For luciferase measurement and viability measurement, strictly follow the manufacturer protocols of Nano-Glo and Cell Titer Glo 2.0. Perform luminescence measurements on a white bottom plate using a Tecan Infinite Pro200.

[0200] Size measurement Here, all size measurements are performed using dynamic light scattering (DLS) with a DynaPro PlateReader II to calculate the hydrodynamic size of the nanoparticles. The formulation is measured by diluting the sample to 0.01 mg / ml RNA with 120 μl of MGB5% in the wells of a 96-well plate. Record 10 data points per well, each over 10 seconds.

[0201] RNA quantification Quantify the RNA concentration using a Nanodrop. Use the microvolume option for RNA quantification, and more specifically, use 1.5 μl of the formulated RNA. Consider the 260 / 280 nm and 260 / 230 nm purity ratios for the calculation of the RNA concentration. Only data that meet the purity criteria are used in the RNA quantification. The purity criteria are defined as 1.8 - 2.2 for the 260 / 280 nm ratio and >1.8 for the 260 / 230 nm ratio. Use the formulation buffer as the blank solution for RNA quantification of each sample.

[0202] Free RNA quantification Free RNA in the formulation is measured by agarose gel electrophoresis under denaturing conditions. 100 ml of 1% agarose in TAE buffer is used. For RNA fluorescent labeling, 10 μl of GelRed is added before gel solidification. A formulation sample containing 1 μg of RNA is diluted to a final volume of 12 μl with gel loading buffer. This is placed on the agarose gel and the sample is run at 80 V, 50 mA for 40 minutes. UV fluorescence is measured after an exposure time of 0.1 second.

[0203] Centrifugation assay The sample is centrifuged at 20,000 G for 90 minutes at 4°C. After centrifugation, 90% of the supernatant is transferred to a new vial. Then, the pellet remaining after centrifugation is resuspended with the same volume as 90% of the supernatant transferred previously.

[0204] Ultracentrifugation assay The sample is measured by an analytical ultracentrifuge and light absorption is measured at two different wavelengths: 255 nm and 650 nm. MBG sample buffer is used as a blank sample for measurement. The sample is centrifuged at 80,000 G at room temperature.

[0205] PEI concentration assay The PEI concentration is determined by quantification of reduced copper(II) sulfate. The amount of reduced copper is proportional to the amount of secondary amine present in the solution. A 1.4 mM solution of copper(II) sulfate is prepared using 0.1 M sodium acetate at pH 5.4. A calibration curve of known PEI reference is prepared up to a maximum value of 1.55 mM. The formulated sample is diluted until the formulated RNA reaches 0.2 mg / ml and mixed 1:1 with the copper(II) sulfate solution to a final volume of 300 μl. The absorption of reduced copper is measured at 285 nm.

[0206] RNA release assay After complexation of RNA in the polyplex, release of the RNA can be triggered by using heparin, a strong polyanion. A solution of heparin at 100 mg / ml is prepared in 1 mM EDTA, 10 mM MES, pH 6.1. The heparin solution is diluted 1:10 in 100 μl of the formulated RNA sample. The heparin and the formulated RNA solution are incubated at 30 °C for 20 minutes. To avoid RNA degradation, the samples are stored immediately at 4 °C.

[0207] In vivo imaging BALB / C mice are used for in vivo testing of the formulation. Intramuscular injections of RNA and D-luciferin are performed in the posterior tibialis muscles of both hind legs of each mouse. When each desired time point is reached, luminescence is measured. The mice are anesthetized in a chamber ventilated with 2.5% isoflurane in oxygen and imaged 5 minutes after injection by an in vivo imaging system (IVIS, PerkinElmer, Waltham, MA). 200 μl of D-luciferin and 20 μL of the RNA / PEI polyplex formulation are injected intramuscularly. Luminescence is quantified using Living Image software (Perkin, Elmer).

[0208] ELISpot assay This assay is used to measure the frequency of CD4 + / CD8 + T cells secreting reactive IFNγ after in vivo vaccination. Nitrocellulose membrane 96-well plates (Multiscreen, Millipore) are coated with anti-mouse IFNγ monoclonal antibody (1-D1K; Mabtech). One day after coating with the IFNγ monoclonal antibody, the spleens of vaccinated mice are removed and homogenized for PBMC extraction. 1x10 6The PBMC solution of the cells was seeded into each well and stimulated with specific MHC1 & MHCII specific HA - peptides. The cells were incubated for 48 hours in RPMI1640 + GlutaMax (Gibco) + 10% FCS. IFNγ secretion was detected using capture and detection antibodies as indicated (Mabtech AB) and imaged using an ImmunoSpot Series Analyzer (Cellular Technology Ltd.).

[0209] ELISA assay Antibodies specific to the HA antigen of strain A / California / 07 / 2009 (H1N1) were measured in sera using a standardized ELISA. A Pierce streptavidin 96 - well plate (Nunc) was coated with 1 μg / mL of HA - biotinylated recombinant surface protein and incubated overnight at 4°C. The plate was blocked with 1% BSA in PBS. Bound IgG was detected using horseradish peroxidase (HRP) - conjugated goat anti - mouse IgG (AbD Serotec). A dilution series of recombinant mouse immunoglobulins was used as a standard for quantifying specific antibodies. After an 8 - minute incubation with 3,3',5,5' - tetramethylbenzidine (TMB), the reaction was stopped using sulfuric acid (25%). The optical density was read at 450 nm.

[0210] Virus neutralization assay (VNT) To determine the level of neutralizing antibodies against HA in the sera of animals, VNT was performed according to the Manual for the Laboratory Diagnosis and Virological Surveillance of Influenza (WHO Global Influenza Surveillance Network). Serial dilutions of serum samples starting at 1:10 were incubated for 2 hours with 100 TCID50 of infectious influenza virus. The final serum dilution of this assay was 1:1,280, which was the upper detection limit. Serum-virus mixtures were then applied to confluent Madin-Darby canine kidney (MDCK) monolayers in 96-well plates and incubated for an additional 3 days. Subsequently, 50 μL of the supernatant was incubated with 50 μL of 0.5% chicken erythrocytes (Lohmann Tierzucht, Cuxhaven, Germany), and hemagglutination was evaluated. The VNT titer was recorded as the reciprocal of the lowest dilution that inhibited agglutination (VNT / 50 μL).

[0211] Small-angle X-ray scattering (SAXS) SAXS measurements were performed at the EMBL P12 bioSAXS beamline at PETRA III, DESY (German Electron Synchrotron, Hamburg, Germany). The measurements were carried out at an X-ray energy of 10 keV and a flux of 5×10 12 ph / s; the beam size at the sample position was 0.2×0.3 mm 2 (v×h, full-width at half-maximum, FWHM), and a BioSAXS sample changer was used. A small volume (30 μL) of the sample was transferred to a vacuum-mounted quartz capillary tube, and images were collected using a PILATUS 6M detector. Images of the scattering signal were processed by the SASFLOW pipeline, i.e., all images were radially averaged, frames were compared for radiation damage, and frames not affected by radiation damage were averaged and imported into Origin 9.1. Background scattering was subtracted using a blank reference.

[0212] Automated High-Throughput Circular Dichroism (CD) Spectroscopy CD spectrum measurements were performed using a J-1500 CD spectrometer (JASCO, Inc.). 175 μL of the sample was transferred to a 96-PCR plate. Standard measurement parameters at a 2 mm cell path length: continuous scan mode with a wavelength range of 200 - 310 nm, a data acquisition interval of 0.1 nm, a response of 4 seconds, a scan speed of 50 nm / min, and a bandwidth of 1 nm. HT module parameters were set for the acquisition of 150 μl of sample volume and the injection of 575 μL of air to deliver the sample to the cell. Sample rinsing between samples was continuously performed with 2 × 200 μL of water, 2 × 200 μL of EtOh: water (1:1), and 2 × 200 μL of EtOH (96%). The drying of the tube was set to 200 seconds. A blank reference was used to subtract the background. Data analysis was performed using SpectraManager software (JASCO, Inc.).

[0213] Example 1: Characterization of Particle Fractionation To investigate the colloidal properties of PEI / RNA polyplex formulations, binding studies were performed by adding various amounts of RNA to PEI and quantifying the fraction of free RNA by agarose gel electrophoresis. Measurements were performed on two RNA species: messenger RNA (mRNA) and self-amplifying messenger RNA (saRNA). As shown in Figure 1A, in both cases, the fraction of residual free RNA decreased with an increase in the fraction of added PEI, allowing the binding of RNA to PEI to be monitored. Clearly, the binding characteristics also depended on individual nucleic acids that differed, for example, in sequence and length. Thus, 50% binding of saRNA required an N / P ratio of 0.8 (excess of RNA), while the compared mRNA required an N / P ratio of 1.4 (excess of PEI). At N / P ratios of 2 or more, free RNA was no longer measured, and the RNA was quantitatively bound to the polyplex nanoparticles.

[0214] At N / P ratios greater than 2, the RNA was fully bound, but there were some indications that the particle properties changed towards even higher N / P ratios. Figure 1B shows the particle size as determined by dynamic light scattering measurements. The particle size decreased up to an N / P value of approximately 6, beyond which the change in size was very small.

[0215] To elucidate the role of the excess polymer given in the formulation, a centrifugation assay was performed to separate the two main phases given in the formulation. The size of the RNA polyplexes is known to vary between 60 - 100 nm. The molecular weight of the free polymer is 22 kDa, while the free iVT mRNA is approximately 500 kDa and the saRNA is approximately 2900 kDa. From the knowledge of these specifications, the centrifugation time can be calculated for a given specific radius of rotation and centrifugal force (20,000 G).

[0216] The diagrams in Figures 2A / 2B show that at low N / P ratios, the RNA is abundant in the pellet, while with the increase in the overall PEI concentration, the RNA-rich fraction gradually shifts to the supernatant. As shown in Figure 3, after recovering the different fractions of RNA in the supernatant and pellet phases, approximately 100% of the initial RNA amount in the formulation can be recovered, which supports that the assay enables accurate quantitative analysis of the RNA / PEI polyplexes in different populations.

[0217] In further experiments (Figure 6), formulations with higher N / P ratios were tested. The RNA content in the supernatant fraction increased in correlation with the increment of N / P until all the RNA was found exclusively in the supernatant fraction. The N / P ratio at which no further RNA could be found in the pellet fraction is specific for each type of RNA.

[0218] Analysis of the sizes of the polyplexes detected in the phases of both fractionated RNA polyplexes (Figure 4) revealed that a significantly smaller-sized population of polyplexes was found in the supernatant, 30 - 40 nm smaller than the particles found in the pellet fraction. Especially at lower N / P (N / P < 72), due to the high polydispersity in this phase, the measured actual sizes for the supernatant population are not entirely accurate. The sizes of these particles suggest different molecular arrangements in the PEI-RNA formulations.

[0219] To confirm that the RNA in the supernatant is bound to PEI and does not exist in the form of free RNA, agarose gel electrophoresis is used. The use of an endogenous polyanion (heparin) can neutralize the cationic groups of PEI and, at the same time, release the RNA within the nanoparticles, allowing it to be detected as free RNA (Figure 5). The data confirm that free RNA cannot be detected in the supernatant phase after fractionation, but RNA bound to PEI can be detected, and the bound RNA can be released by heparin incubation.

[0220] Figure 7A shows the distinct co-sedimentation of PEI and RNA in different populations of RNA-PEI polyplexes in the formulation. When focusing on the polyplex population sedimenting at 50 - 400S, the molecular weight distribution strongly reassembles the complexed RNA monomers, dimers, trimers, tetramers, and pentamers. Analytical ultracentrifugation of the same but naked RNA in the formulation buffer showed clearly defined peaks, and it was found that 90% of the RNA sedimented at 23.56S. The amounts of RNA and PEI shown in Figure 7B for each population were quantified by integration of the said data shown in Figure 7A, i.e., by specifying the area under the curve of the sedimented PEI and RNA. Figure 7B shows that significant differences can be observed in the molecular arrangement of PEI particles in different populations. Finally, as shown in Figure 7C, by increasing the N / P ratio of the iVT mRNA PEI-polyplex, a distinct shift in the sedimentation coefficient profiles of different populations is achieved. This indicates that an increase in the N / P ratio shifts the complexed RNA towards a phase with a lower sedimentation coefficient, i.e., towards a lower molecular weight monomer population.

[0221] The structure of monomeric polymeric associated RNA was further investigated by small angle X-ray scattering (SAXS) measurements. RNA was measured in media containing purified monomeric RNA-PEI species, in buffer only for comparison, and in the presence of higher ionic strength by adding 50 mM of NaCl to the bulk phase. Figure 26A shows the direct scattering curves on a log-log scale. As already visually apparent, the curves are characterized by distinct different features. RNA in buffer only shows a curve with almost no structure, but in the presence of NaCl, a slight modulation by about 0.33 nm -1 is visually observed and the curve flattens towards low q. The curve of RNA in PEI shows even stronger structural features with at least two modulations at 0.32 nm -1 and 0.6 nm -1is already visually recognized. The flat region at low q extends longer than in the presence of salt. The modulation of the PEI curve points to the presence of dense, spherical particles with a very low polydispersity, which are thought to be individual PEI-dissolved RNA molecules. RNA in the presence of NaCl also shows some modulation, but it is less pronounced and occurs at higher q values. This indicates that here too, the RNA is somewhat condensed, but to a much lower extent and with a less dense packaging. These qualitative observations are confirmed by a more thorough analysis of the curves. In Figure 26B, Guinier plots of three data sets are given. In the Guinier approximation, the radius of gyration R g of the particles correlates with the intensity as follows:

[0222] [Number]

[0223] Therefore, a plot of the log intensity as a function of the square of the momentum transfer q results in a straight line, and R g can be derived from the slope -(R g ) 2 / 3. It should be noted that the Guinier approximation is only valid up to the maximum value of q. For example, in the case of a solid sphere, the range where the approximation is valid is usually considered to be q*Rg, < 1.3. For particles that are not rigid spheres with a smooth interface (anisotropic shape, rough interface, random coil conformation), this range is even lower, for example, q*Rg, < 1. In the Guinier plot, the RNA in PEI shows linear behavior over the entire range shown, from which a radius of gyration of 12 nm can be derived. In particular, this linear behavior is also observed towards the lower q end, down to near the lowest measured data point, which indicates that there are substantially no larger aggregates (dimers, oligomers, larger particles). If such larger particles were present, the intensity would show a curved shape and the intensity would increase more than the straight line in the Guinier plot.

[0224] Furthermore, the linear behavior is also maintained well up to q = 0.1 nm -1 (q 2 = 0.01 nm -2 ), i.e., up to the limit regarding scattering from the solid sphere. Therefore, it can be concluded that in reality, RNA exists in a very dense form with low anisotropy and low surface roughness.

[0225] Furthermore, the data in NaCl also show a linear range, but it is up to about 0.001 nm at most (10 times lower than in the case of the sample with PEI), and the slope is higher, which indicates a higher R -2 . From the analysis of this curve, a radius of gyration of about 29 nm is obtained. The data for RNA in buffer show linear behavior only in the lowest q range, where R g can be estimated to be 90 nm (where the approximation is valid only up to about 0.0001 nm at most, which is at the very limit or below the available q g -range in this measurement). -2 2 )

[0226] ​In Figure 26C, the data are given as a Kratky plot, where the scattering intensity multiplied by the square of the momentum transfer q is given as a function of q. In this Kratky plot, unfolded (very flexible) molecules should have a horizontal shape at high q, while dense globular macromolecules have a bell-shaped (Gaussian) peak. A distinct peak-like pattern indicating a dense globular organization is observed only for the PEI data. Figure 26D shows the calculated pair distribution function P(r), which gives information about the distances between the scattering parts in the particles. The peak gives the most abundant distance. Due to the extended conformation, the data cannot calculate the curve for RNA in buffer, so here only RNA in NaCl and PEI is shown. Here, the peak for RNA in PEI is at a much denser and significantly lower distance than that for RNA in NaCl, which supports the information already derived from the Guinier plot that the RNA particles in PEI are much denser. In summary, RNA dissolved in excess PEI shows a surprisingly dense organization, and there must be a very high packing density of RNA. Considering the simplest possible solid sphere model, the radius of gyration is (V = 4*π / 3r 3 , R g = r*(3 / 5) 1 / 2 ) 15600 nm 3 that would account for the volume of a single RNA molecule. This number can be compared to the volume of RNA with a molar mass of 3*10 6 Da complexed with PEI at an N / P ratio of 2, resulting in a total molar mass of approximately 4.15*10 6 Da (each nucleotide of 330 Da is accompanied by two PEI units of 43 Da each (thus the total mass is 330 / 456 times higher). Assuming a density of 1 along with Avogadro's number, the volume of the assembly for the following can be calculated:

[0227]

Number

[0228] The true density of RNA is somewhat higher than 1 g / mL, and this value is, surprisingly, 1.56×10 of the above calculated value 4 nm 3 close to. Counterions and water molecules are not included in the calculation. Moreover, in reality, the densified RNA is not a perfect sphere and must take into account a specific collapse with increasing radius and packing density, both of which result in an increase in the radius of gyration of a single molecule. Thus, these data indicate that the low radius of gyration measured here provides further confirmation that only a single RNA molecule can account for the scattering profile. The packing density is very high compared to other conditions where RNA is densified, such as high salt concentration, etc. For example, according to the R g data presented here, the volume occupied by RNA particles in PEI is approximately 15 times lower than in NaCl solution ((12 / 29) 3 ). Thus, the identification of a low radius of gyration could be a clear indicator for distinguishing PEI-densified RNA from other forms of PEI in solution.

[0229] Based on the previously described observations that can be derived from the investigation of monomeric RNA species by SAXS, the conformational structure of RNA in solutions containing PEI was investigated by circular dichroism. The RNA secondary-tertiary structure was investigated under increments of the bulk N / P ratio in the RNA / PEI formulation (0 - 24 - 48 - 72 - 120 - 240), as well as under increments of the NaCl concentration in the RNA / NaCl formulation. This last group was used to compare with another type of polyelectrolyte. The change in the peak position of the spectrum, as well as the variation of the ellipticity (mdeg) at this peak position, correlates with three major features of RNA: (1) the asymmetry of the sugar backbone and its specific rotation / orientation in the so-called A - B - Z forms, (2) the hydrogen bonding of nucleotide pairs in both cases of Watson - Crick base pairs or non-Watson - Crick base pairs, and (3) the π-cation-driven stacking interactions of the strands (Kypr, J. et al. (2012) Comprehensive Chiroptical Spectroscopy: Applications in Stereochemical Analysis of Synthetic Compounds, Natural Products, and Biomolecules, Volume 2: 575 - 586). In Figure 28A, there is a distinct shift of the peak position towards lower wavelengths and an increase in the measured ellipticity at this peak position. Both of these effects correlate with the increment of the NaCl concentration in the solution of RNA. The overall shift of the peak position towards lower wavelengths is accompanied by a proportional loss of the signal in the UV region, between 280 - 300 nm, where the stacking effect of the strands as mentioned previously can be detected. On the other hand, in contrast to the described effect of NaCl in the RNA solution, PEI caused the opposite effect on the conformational structure of RNA. In Figure 28B, there is a distinct shift of the peak position towards higher wavelengths and a decrease in the measured ellipticity. Both of these effects correlate with the monotonic increment of the N / P ratio in the RNA / PEI formulation. The overall shift of the peak position towards higher wavelengths is accompanied by a proportional gain of the signal between 280 - 300 nm.The nature of the interactions and effects in the RNA structure by PEI or NaCl can be continued in FIGS. 28C and 28D. By converting the PEI or NaCl concentration to the common X-axis of the positive charge concentration (mM), it summarizes the opposite effects of PEI and NaCl regarding the shift of the peak position and the effects on the ellipticity at the peak position. According to the effects by the SAXS analysis described previously, it can be seen that the condensation properties of PEI and NaCl are dramatically different, emphasizing that the monomeric RNA structure in the polycationic polymer is undoubtedly unique. The monomeric PEI-complexed RNA species is a very condensed structure with an inherent loss of the secondary structure of RNA, resulting from the π-cation-driven interaction between the cationic polymer and the nucleotides of RNA, which explains the loss of hydrogen bonds of nucleotides, a unique red shift in the spectrum, and the gain of signal at wavelengths of 280 - 300 nm. In contrast, NaCl can cause condensation physically restricted by the internal RNA folding (secondary structure), which is well characterized by the observable blue shift in the spectrum.

[0230] Example 2: The polymer-solvated RNA fraction can be detected in the supernatant after centrifugation of different cationic polymer / RNA polyplexes. To confirm this new concept expansion, apart from PEI-polyplexes, additional cationic polymers that are structurally related to or have similar cationic moieties to PEI are tested. Four different polymers other than PEI are tested, including DEAE-dextran, poly-L-lysine, polyvinylamine, and polyallylamine (Figure 8). There is a general trend observed in all the tested polymers that when the N / P ratio of the polyplex is increased, the RNA content in the supernatant after centrifugation increases proportionally. The increment of RNA in the supernatant population of the polyplexes varies among the tested polymers, being highest in DEAE-dextran and PEI-RNA-polyplexes, followed by PLL, PVA, and PAA. These results suggest a strong correlation between the structural arrangement of the cationic moieties in the polymer chain with respect to the binding properties to RNA and the formation of monomeric / oligomeric RNA fractions. Furthermore, two novel polymers with significantly different chemical / structural properties are tested against the previously observed effects of high N / P ratios in various different polymers that are chemically / structurally close to polyethyleneimine. For both G(5)-PLL(64) (i.e., PAMAM functionalized with G(5) by the (64) arms of penta-L-lysine) and Viromer® shown in Figures 20B and 22B respectively, it is shown that by increasing the N / P ratio of the formulation, a higher content of the monomeric RNA population is obtained. This clearly shows that the increment of N / P is not only closely related to polyethyleneimine but is a universal law applicable to a wide family of cationic polymers.

[0231] Example 3: The PEI-solvated RNA fraction observed in the supernatant after centrifugation is an important driving force in the overall biological activity of the PEI-polyplex formulation. After transfection of C2C12 cells with RNA encoding luciferase at various N / P ratios, the biological activity of different fractionated populations of PEI-polyplexes is investigated (Figure 9A, Figure 9B). The formulated nanoparticles and fractionated populations are combined to a total of 5 ng of formulated saRNA / iVT mRNA per well and diluted in culture medium according to the desired concentration (100 μL). The results clearly show that the supernatant population is the main driving force for biological activity. Indeed, these results suggest that the supernatant fraction of the formulation has even higher biological activity than the non-centrifuged parental formulation.

[0232] Example 4: High N / P ratios promise increased effects but are limited by toxicity at high doses in vitro. C2C12 cells transfected with RNA encoding luciferase at various N / P ratios are analyzed in a luciferase & viability assay. The formulated nanoparticles are diluted according to the desired concentration in the culture medium. At a dose of 50 ng of RNA per 5000 C2C12 cells / well (100 μL), luciferase activity is high at low PEI concentrations but decreases with increasing PEI concentration, along with viability (Figure 10A), and a completely different pattern is seen at a 10-fold lower RNA (and PEI) dose. In the lower-dose panel, no toxicity is observed, viability remains unchanged, and the measured luciferase activity increases with increasing PEI concentration (Figure 10B, Figure 10C). Thus, PEI has a toxic effect on transfected cells, but this is seemingly more than compensated for by the increased transfection effect at low doses, resulting in an overall equivalent effect. Moreover, in the case of both formulated RNAs (iVT mRNA or saRNA), an increment in the N / P ratio perfectly correlates with an increment in the amount of RNA found in the supernatant fraction and an increment in luminescence. As observed in both figures (Figure 10B, Figure 10C), the luminescence signal stops increasing as soon as all of the RNA is found in the supernatant fraction. Further increments in N / P do not lead to higher bioactivity; rather, they plateau. This indicates that the increment in luminescence is due only to an increment in the small-scale monomeric PEI-solvated RNA content in the formulation and not necessarily to the excess polymer itself. This observation is seen not only for the high N / P ratio bioactivity of PEI-solvated RNA but also for other cationic polymers such as Viromer® and G(5)-PLL(64), etc., and bioactivity is shown to increase monotonically with an increment in the amount of RNA found in the supernatant fraction (Figure 20B, Figure 22B) (Figure 20C, Figure 22C). For these two polymers, further increments in N / P beyond the point where the maximum amount of RNA in the supernatant fraction cannot increase lead to a plateau in bioactivity.

[0233] Example 5: Increased efficiency of bioluminescence expression in vitro after injection of a high N / P ratio at a reduced dose Fifteen female BALB / C mice are divided into five study groups. All groups are dosed intramuscularly in each leg with the same amount of saRNA (62.5 ng) encoding formulated luciferase. The first group is dosed with 62.5 ng of saRNA formulated at N / P 12, the second group with 62.5 ng of N / P 24, the third group with 62.5 ng of N / P 48, the fourth group with 62.5 ng of N / P 72, and the last group with 62.5 ng of N / P 96. The mice are observed by live imaging at five time points (24 hours, 72 hours, day 6, day 9, day 20). Figure 11A shows the bioluminescence signal detected 6 days after injection of the polyplex. The luminescence signal increases with increasing N / P ratio, reaching a maximum signal at N / P 72 (the N / P ratio of 96 appears to have reached the saturation limit). Total bioluminescence can be expressed as the area under the curve (Figure 11B), where again it is clearly shown that the increment in N / P leads to a higher effectiveness of the injected RNA dose and that the effectiveness can be increased by the plurality.

[0234] Example 6: The increased effectiveness at high N / P ratios can be used in vivo to reduce the RNA dose while benefiting from the same overall absolute biological performance. Nine female BALB / C mice are divided into three study groups. The first group is administered 500 ng of saRNA encoding luciferase formulated with PEI at an N / P ratio of 12. Group 2 is given 250 ng of RNA encoding luciferase formulated with PEI at an N / P ratio of 24, and group 3 is given 125 ng of RNA encoding luciferase formulated with PEI at an N / P ratio of 48. At five time points (24 hours, 72 hours, day 6, day 9, day 15), the mice are observed by live imaging. To reduce stress, only a randomly selected half of the mice in each group are examined at 24 hours and 72 hours. After day 6, half of all the mice (excluding the control group) are sacrificed and spleen samples are taken. The remaining mice are examined on day 9 and day 15 and then sacrificed to obtain blood and spleen samples.

[0235] After normalization of the luminescence signal with respect to the RNA concentration, it becomes visually apparent that the lowest dose (lowest concentration of RNA, basal concentration of PEI) with the highest N / P ratio shows the greatest effect (Figure 12A). Thus, by increasing the PEI ratio to RNA while maintaining a constant PEI concentration, it is possible to significantly reduce the RNA concentration without reducing the effect.

[0236] The spleen samples are used for the preparation of CD8-positive T cells. The T cell preparation is contacted with BALB / c MHC-I 1, 2, and 3 having antigen-presenting cells and pre-incubated with the firefly luciferase peptides GFQSMYTFV, VPFHHGFGM, and VALPHRTAC. Figure 12B shows that groups 1, 2, and 3 have similar strong CD8 responses, but after normalizing the CD8 response with respect to the RNA concentration applied to each group, a clear approximately five-fold increase in the effectiveness of the injected RNA is seen by increasing the N / P ratio of the formulation from 12 to 48. Thus, the relationship between dose and / or N / P ratio for the response applies not only to the transfection efficiency previously observed in vitro, but also to the bioluminescence and immunostimulatory capacity in vivo.

[0237] Example 7: A high N / P ratio results in higher anti-HA IgG and VNT even under a dose reduction scheme. Twenty-five female BALB / C mice are divided into five study groups. The first group is administered 500 ng of saRNA encoding California / 7 / 2009-HA formulated with PEI at an N / P ratio of 12. Group 2 is given 125 ng of RNA encoding luciferase formulated with PEI at an N / P ratio of 48, group 3 is given 83.3 ng of RNA encoding luciferase formulated with PEI at an N / P ratio of 72, group 4 is given 62.5 ng of RNA encoding luciferase formulated with PEI at an N / P ratio of 96, and group 5 is given 50 ng of RNA encoding luciferase formulated with PEI at an N / P ratio of 120. Serum samples are collected from each mouse at three time points (day 14, day 28, day 49). In Figure 13A, the absolute values of anti-HA IgG for the three time points clearly show that, even when reducing the dose of saRNA injected, there is a significant increment in the amount of specific IgG when the N / P ratio is increased proportionally, and the highest value is in group 3. After normalizing the anti-HA IgG levels in the serum against the amount of RNA injected per group, increasing the N / P ratio results in a significant increase in the efficacy of the injected RNA after 49 days, with an almost 12-fold increase at N / P 120 compared to N / P 12 (Figure 13B).

[0238] Example 8: The PEI-polymer length affects the formation of polymeric associated monomeric RNA species and overall bioactivity. Here, we investigate the effect of PEI polymer length in RNA formulations by systematically varying the polymer length, expressed as the number of repeating units (r.u.) of ethyleneimine. As a reference, we use PEI with a molecular weight of 22,500 Da, which is equivalent to approximately 500 r.u. of ethyleneimine. First, we evaluated the binding affinity of various PEI-polymer lengths to RNA (Figure 14A). There is a clear correlation between binding affinity and polymer length, such that longer polymers (>500 r.u.) bind very strongly to RNA even at very low N / P (<1.0), and no free RNA can be detected. In contrast, shorter polymers (<62 r.u.) require a higher N / P ratio (>1.3 - 1.5) to completely bind RNA. In parallel with the binding affinity, the amount of RNA found in the supernatant fraction strongly correlated with the polymer length used at a given N / P ratio (Figure 14B). The amount of RNA quantified in the supernatant after centrifugation assays at a given N / P ratio (any of 6, 12, 30, 60, or 120) increased monotonically with the PEI polymer length from 7 r.u. to 500 r.u. Above 500 r.u. of ethyleneimine in PEI, no increment in the fraction of monomeric RNA was detectable. This data suggests that not only the N / P ratio plays a role in the formation of such monomeric RNA-polymer association species, but also the polymer length plays a fundamental role. Finally, the in vitro activity, shown as luminescence measured from transfection of C2C12 cells, increased very strongly with the polymer length used for a given N / P ratio (Figure 14C). More specifically, by testing all polymer lengths in vitro at the same N / P (120), the increment in biological activity perfectly correlated with the increment in the amount of RNA in the supernatant (Figure 14B), suggesting that biological activity strongly depends on the amount of polymer-associated monomeric RNA and not on the excess positive charge that was kept constant in this experiment (Figure 14C).

[0239] Furthermore, 24 female BALB / C mice are divided into 8 study groups. All groups are administered with saRNA (125 ng) encoding luciferase in the same formulated amount with the same formulated N / P ratio (120) by intramuscular injection into each leg. The groups are administered with RNA formulated with different PEI-polymer lengths: 31 - 62 - 125 - 250 - 500 - 1000 - 2500 r.u. The last group is administered with only the formulated buffer. At 6 time points (day 1, day 3, day 6, day 9, day 13, day 20), the mice are observed by live imaging. Figure 15A shows the bioluminescence signals detected throughout the experiment after injection of PEI formulations of various polymer lengths. Figure 15B shows the bioluminescence observed at the peak point of expression (day 6) for all tested polymer lengths. The data clearly show that the bioluminescence, i.e., the biological activity, increases monotonically with the polymer length and is directly correlated with the amount of RNA in the supernatant (Figure 15B).

[0240] Example 9: Local RNA concentration plays an important role in the formation of polymer-associated monomeric RNA species. To understand the formation of monomeric RNA-polymer aggregates, in addition to the classical equal-volume mixing of the RNA-containing solution and the PEI-containing solution, additional volume mixing ratios were systematically investigated. By monitoring the hydrodynamic size of the formed complex, the content of polymeric associated monomeric RNA by centrifugation assay, and ultimately, by monitoring the biological activity in vitro, the effect of variations in the volume mixing ratio was evaluated. Due to the different starting concentrations of RNA and the cationic polymer, the final concentration of RNA, the final N / P ratio, and the final formulation volume were kept constant between test conditions, but the volume mixing ratio of the RNA-containing solution and the cationic polymer-containing solution was varied to perform the variable of the volume mixing ratio. The overall size of the complex decreases to 20 - 30 nm at the lowest RNA starting concentration (i.e., the highest initial RNA volume, the lowest initial PEI volume) for both types of RNA, as shown in Figure 16A. An increase in the starting RNA concentration results in a significant increment in the size of the complex up to 100 - 120 nm at the highest RNA starting concentration. This holds true not only for PEI-polymer but also for other cationic polymers, such as Viromer® or G(5)-PLL(64), as shown in Figure 21 or Figure 23A, and the same effect on the size of the complex can be observed by using the lowest RNA starting concentration (i.e., the highest initial RNA volume, the lowest PEI volume). The evaluation of the amount of polymeric associated monomeric RNA species in the supernatant after centrifugation of such formulations is shown in Figure 16B and Figure 23B. These figures show that the highest initial volume of RNA in excess of the initial polymer volume at a given fixed N / P ratio results in the highest amount of RNA found as polymeric associated monomeric RNA in the supernatant. A decrease in the mixing ratio of the initial RNA volume to the initial polymer volume results in a shortfall in the amount of RNA found in the supernatant (Figure 16B, Figure 23B). Furthermore, the biological activity at various variables of the volume mixing ratio was evaluated by transfection of C2C12 cells.Figure 16C shows that the biological activity decreases monotonically with an increase in the RNA starting concentration (with a decrease in the initial volume), which correlates with the decrease in RNA found in the supernatant in Figure 16B. The highest biological activity in Figure 16B is observed for the lowest RNA starting concentration in the formulation, i.e., for the highest volume mixing ratio of RNA initial volume to polymer initial volume. A further comparison of the observations in Figure 16B can be confirmed by the biological activity observed after transfection of C2C12 cells in Figure 17A. In this figure, an equal volume mixture at N / P 12 and a mixture with a 99:1 mixing ratio of initial RNA volume to initial PEI volume at N / P 12 are compared with an equal volume mixture at N / P 120. The data clearly show that when RNA is found exclusively as polymer-associated monomeric RNA species, an excess of positive charge (12 vs 120) plays little role. For the mixing ratios tested in Figure 16, different N / P ratios were tested in Figure 18, which showed that similar effects can be observed at N / P ratios lower than 12. Nevertheless, a stronger effect with the mixing ratios tested was observed at N / P 12 in Figure 18A, and further experiments focus on N / P 12.

[0241] Example 10: Polymer-associated monomeric RNA species are colloidal-stable in long-term liquid storage To evaluate the colloidal stability of monomeric RNA species found in the supernatant of the PEI-formulated RNA after centrifugation, two formulations were analyzed as polymer-associated monomeric RNA species, showing that they had the majority (>60%) of the RNA in the supernatant (Figure 19). The hydrodynamic size of the complex, the amount of complex counted in suspension, or the amount of RNA in the supernatant was monitored throughout the experiment. Overall, five different time points (0 h, 24 h, 48 h, 72 h, 96 h, 960 h) were monitored. Overall, there was no significant change in the hydrodynamic size of the complex over 960 h, either in the stored supernatant fraction, i.e., the polymer-associated monomeric RNA species, or in the bulk phase (Figure 19A). To confirm these findings, the concentration of particles can be evaluated by the amount of counts / second measured by DLS in parallel with the measurement of hydrodynamic size. As shown in Figure 19B, no significant change in counts / second was observed throughout the experiment, suggesting no shift between monomeric and non-monomeric RNA species in the bulk formulation or in the isolated RNA supernatant. This could be further confirmed in Figures 19C and 19D, where "aged" samples were compared to newly prepared samples under otherwise identical conditions. Again, both Figures 19C and 19D demonstrate the same size and counts / second, confirming what was previously shown in Figures 19A and 19B. Finally, by comparing the "aged" stored formulation to the newly prepared control, the amount of RNA in the supernatant was monitored throughout the process and shown in Figure 19E as the fold change in the quantified amount of RNA in the supernatant, detecting only minor variations within the error bars. The data shown in Figure 19E ultimately support that the amount of RNA found in the polymer-associated monomeric RNA species within the formulation does not change over 960 h.

[0242] Example 11: Lyophilization of RNA or RNA-Polymer Formulations Lyophilization of RNA or RNA-polymer formulations is an alternative to classical liquid / frozen storage of such formulations. Thus, the use of lyophilized RNA in a matrix suitable for direct reconstitution with an aqueous polymer-containing solution was investigated. In FIGS. 24 and 25, size, in vitro bioactivity, in vivo generation of anti-HA IgG, in vivo influenza neutralizing titers, and influenza HA-CD4 / CD8+ T cell responses were evaluated. In FIGS. 24C and 24D, the tested lyophilized RNA-PEI formulations were formulated at various N / P ratios and RNA:PEI volume mixing ratios. Fractions of this formulation were frozen or lyophilized. The lyophilized formulation was then reconstituted. The size of the complex did not change between the freshly prepared samples in FIG. 24C and the frozen or lyophilized samples. The bioactivity of such formulations was tested by transfection of C2C12. The bioactivity is shown in FIG. 24D, which shows that lyophilization of the RNA-PEI formulation maintains bioactivity. In FIGS. 24A and 24B, the lyophilization of the RNA was tested in various buffer solutions. Reconstitution of the lyophilized RNA was performed using an aqueous polymer-containing solution. The size after reconstitution of the lyophilized RNA, or the size of the new control formulation or the frozen control formulation, was evaluated in FIG. 24A, which shows equivalent sizes for all formulations, but does not show formulations made from RNA lyophilized in pure water. The bioactivity of the lyophilized RNA reconstituted with the polymer solution at a final N / P of 120 was evaluated in FIG. 24B. According to FIG. 24A, this experiment showed the highest bioactivity using RNA lyophilized in MBS and the lowest expression was obtained from RNA lyophilized in pure water. In FIG. 25, the biological performance of the lyophilized RNA-PEI formulation or the lyophilized RNA was further evaluated in vivo. Thirty-five female BALB / C mice were divided into seven study groups. The first group received only the formulation buffer. All six other groups received saRNA encoding California / 7 / 2009-HA formulated with PEI under various conditions.The second group was administered 500 ng of saRNA formulated with N / P 12, the third group was administered 125 ng of saRNA formulated with N / P 120, the fourth group was administered 125 ng of saRNA formulated with N / P 120 that had been previously lyophilized and reconstituted with water prior to injection into the mice of that group, the fifth group was administered 125 ng of saRNA that had been lyophilized in pure water and reconstituted with a polymer solution to have a final N / P 120, the sixth group was administered 125 ng of saRNA that had been lyophilized in MBG buffer and reconstituted with a polymer solution to have a final N / P 120, and the last group was administered 125 ng of saRNA that had been lyophilized in MBS buffer and reconstituted with a polymer solution to have a final N / P 120. Serum samples were collected from each mouse at three time points (day 14, day 28, day 56). In Figure 25A, the absolute values of anti-HA IgG obtained by ELISA are represented as the area under the curve for each group for the three time points. The anti-HA IgG levels were highest in the group injected with saRNA lyophilized in MBS, followed by the freshly prepared benchmark N / P 120 and the lyophilized N / P 120 RNA-PEI formulation. No clear IgG levels against HA could be detected in the group treated with RNA lyophilized in water (only 1 out of 5 mice was responsive). The previous findings were confirmed by the virus neutralization titers obtainable from the serological samples collected 56 days later (the final time point of the experiment) in Figure 25B. The data confirm that the highest VNT was observed in the group injected with saRNA lyophilized in MBS, followed by the freshly prepared benchmark N / P 120 and the lyophilized N / P 120 RNA-PEI formulation. Finally, in Figure 25C, the response of CD4 / CD8+ T cells to influenza peptides was evaluated in splenocytes of the spleen excised at the final time point measured by IFN-ELISPOT.The highest T cell responses (both CD4 and CD8) were obtained from the group treated with lyophilized RNA in MBS, which was two-fold higher than freshly prepared N / P120 or ten-fold (normalized to the dose) higher than freshly prepared N / P12. Overall, lyophilization not only provides longer-term stability for the use of such systems in vaccination approaches, but also has been shown to provide higher efficacy in such approaches at the same dose / conditions when lyophilization is performed in an appropriate matrix.

[0243] Example 12: Increased efficiency of in vivo bioluminescence expression after injection of high N / P ratios at reduced doses for other cationic polymers Twenty-seven female BALB / C mice were divided into nine study groups. All groups were subjected to intramuscular application of the same amount of saRNA (125 ng) encoding luciferase formulated in each leg. The first four groups were subjected to 125 ng of G(5)-PLL(64) / saRNA formulated at various N / P ratios of 12 - 24 - 48 - 96. The next four groups were subjected to 125 ng of Viromer® / saRNA formulated at various N / P ratios of 12 - 24 - 48 - 96. The last group was subjected to only the formulation buffer without saRNA or polymer. At four time points (day 1, day 3, day 6, day 8), the mice were observed by live imaging. Figures 27A and 27B show the bioluminescence signals detected throughout the experiment after injection of various polymer-polyplexes. Figure 27C shows the area under the curve for the expression of each group throughout the experiment as a function of the tested N / P ratio, classified into two different tested polymers. There is a clear increment in the luminescence signal due to the increase in the N / P ratio that reaches the maximum signal at N / P 48, while above this N / P ratio, there appears to be a deleterious effect along the in vitro observations for both of these cationic polymers, i.e., a local toxic effect that substantially precludes RNA expression, when the transfected cells die prior to RNA expression. The bioactivity of both polymers at higher N / P ratios in vivo supports previous study results (Figure 11) and shows the general applicability of increasing the N / P for other cationic polymers to enhance bioactivity by increasing the amount of RNA in the polymer-associated monomeric RNA species (Figures 8, 20B, 22B).

Claims

**Claim 1** A composition comprising RNA and a polymer in an aqueous phase, wherein a predominant fraction of the RNA molecules comprises individual molecules in solution.

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