RNA particles comprising polysarcosine

Abstract

To provide efficient methods and compositions for introducing RNA into cells that avoid disadvantages associated with the use of PEG.SOLUTION: The present disclosure relates to RNA particles and compositions comprising such RNA particles for delivering RNA to a target tissue after administration, in particular after parenteral administration, such as intravenous, intramuscular, subcutaneous or intratumoral administration. The RNA particle in one embodiment comprises single-stranded RNA such as mRNA encoding a peptide or protein of interest such as a pharmaceutically active peptide or protein. The RNA is taken up by the cells of the target tissue and the RNA may be translated into the encoded peptide or protein to exert its physiological activity.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to RNA particles for delivering RNA to target tissues after administration, particularly parenteral administration such as intravenous, intramuscular, subcutaneous or intratumoral administration, and compositions containing such RNA particles. In one embodiment, the RNA particles contain single-stranded RNA, such as mRNA, encoding a target peptide or protein, such as a pharmaceutically active peptide or protein. The RNA is taken up by cells in the target tissue, and the RNA can be translated into the encoded peptide or protein to exhibit its physiological activity. [Background technology]

[0002] The use of RNA to deliver foreign genetic information to target cells offers an attractive alternative to DNA. Advantages of using RNA include transient expression and non-transforming properties. RNA does not need to enter the nucleus to be expressed, and it cannot be integrated into the host genome, eliminating the risk of oncogenicity.

[0003] RNA can be delivered to subjects using a variety of delivery vehicles, primarily based on cationic polymers or lipids that combine with the RNA to form nanoparticles. Nanoparticles are designed to protect the RNA from degradation, enable delivery to the target site, and facilitate cellular uptake and processing by the target cells. In addition to molecular composition, parameters such as particle size, charge, or grafting with molecular moieties such as polyethylene glycol (PEG) or ligands play a role in delivery efficiency. PEG grafting is thought to reduce serum interactions, enhance serum stability, and increase circulation time, which may be useful for specific targeting approaches. Ligands that bind to receptors at the target site can help improve targeting efficacy. Furthermore, PEGylation can be used for particle manipulation. For example, when lipid nanoparticles (LNPs) are produced by mixing an aqueous phase of RNA with an organic lipid phase, a specific percentage of PEG-conjugated lipids in the lipid mixture is required; otherwise, the particles will aggregate during the mixing process. It has been shown that particle size can be adjusted by varying the molar fraction of PEG-lipids containing different molar masses of PEG. Similarly, particle size can be adjusted by changing the molar mass of the PEG moiety in PEGylated lipids. Typical accessible sizes range from 30 to 200 nm (Belliveau et al., 2012, Molecular Therapy - Nucleic Acids 1, e37). The particles thus formed have the added advantage of less interaction with serum components and a longer circulating half-life, which is desirable in many drug delivery approaches. In the absence of PEG-lipids, discrete particle sizes cannot be formed; particles form large aggregates and precipitate.

[0004] Therefore, in the case of LNPs formed from an ethanol phase and an aqueous phase, one of the primary roles of the PEG-lipid is to promote particle self-assembly by providing steric hindrance to the surface of nascent particles that form when nucleic acids are rapidly mixed in an ethanol solution containing RNA-binding lipids. The steric hindrance of the PEG prevents particle fusion and promotes the formation of a uniform population of LNPs with diameters less than 100 nm.

[0005] PEG is the gold-standard "stealth" polymer most widely used in drug delivery. PEG-lipids are typically incorporated into systems to prepare homogeneous, colloidally stable nanoparticle populations due to their hydrophilic steric hindrance properties (the PEG shell prevents electrostatic or van der Waals forces that lead to aggregation). PEGylation allows for the attraction of a water shell around the polymer, which shields RNA complexes from opsonization by serum proteins, prolongs serum half-life, and reduces rapid renal clearance, resulting in improved pharmacokinetic behavior. Varying the length of the lipid acyl chain (C18, C16, or C14) alters the stability of PEG-lipid incorporation into particles, resulting in altered pharmacokinetics. The use of PEG-lipids containing short (C14) acyl chains that dissociate from LNPs with half-lives of less than 30 minutes in vivo results in optimal hepatocyte gene silencing efficacy (Chen et al., 2014, J Control Release 196:106-12; Ambegia et al., 2005, Biochimica et Biophysica Acta 1669:155-163). Furthermore, tight control of particle size can be obtained by varying the PEG-lipid parameters; the higher the MW of PEG or the higher the PEG-lipid molar fraction within the particle, the smaller the particle.

[0006] Despite these advantages, PEGylation of nanoparticles can also lead to several adverse effects that adversely affect their intended use in drug delivery. PEGylation of liposomes and LNPs is known to reduce cellular uptake and endosomal escape, ultimately lowering overall transfection efficiency. Indeed, the PEG shell provides steric hindrance to efficient particle binding to cells and impedes endosomal escape by preventing membrane fusion between liposomes and endosomal membranes. Therefore, the type of PEG-lipid and the amount of PEG-lipid used must always be carefully adjusted to ensure sufficient stealth effect for in vivo and stabilization purposes without interfering with transfection on the one hand. This phenomenon is known as the "PEG dilemma."

[0007] In addition to reducing transfection efficiency, PEGylation is associated with accelerated blood clearance (ABC) and / or complement activation induced by anti-PEG antibodies, as well as storage diseases (Bendele A et al., 1998, Toxicological Sciences 42, 152-157; Young MA et al., 2007, Translational Research 149(6), 333-342; SM Moghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478). Ishida et al. and Laverman et al. reported that intravenous injection of PEG-grafted liposomes into rats can significantly alter the pharmacokinetic behavior of the second dose when administered after an interval of several days (Laverman P et al., 2001, J Pharmacol Exp Ther. 298(2), 607-12; Ishida et al., 2006, J Control Release 115(3), 251-8). The phenomenon of "accelerated blood clearance" (ABC) appears to be inversely proportional to the PEG content of the liposomes. The presence of anti-PEG antibodies in the plasma induces higher clearance of particles by the mononuclear phagocytic system (MPS), ultimately reducing the efficacy of the drug.

[0008] PEG is also thought to induce complement activation, which can lead to a hypersensitivity reaction also known as complement activation-associated pseudoallergy (CARPA). It is not yet clear from the literature whether complement activation is due to nanoparticles in general or to the presence of PEG in particular.

[0009] The presence of PEG in lipid nanoparticles may also induce specific immune responses. Semple et al. reported that liposomes containing PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotides or plasmid DNA elicited a strong immune response in mice, resulting in rapid blood clearance of the subsequent dose. The magnitude of this response was sufficient to induce significant morbidity and, in some cases, death. The rapid clearance of liposome-encapsulated ODN from the blood depended on the presence of PEG-lipids in the membrane, as the use of non-PEGylated liposomes or liposomes containing rapidly exchangeable PEG-lipids abolished the response. The generation of anti-PEG antibodies and presumed complement activation were likely explanations for the rapid clearance of vesicles from the blood (Semple et al., 2005, J Pharmacol Exp Ther. 312(3), 1020-6).

[0010] Because PEG can induce an immune response, it should be avoided in certain applications where multiple injections are required. An example is the use of mRNA, e.g., for protein replacement therapy. Here, the risk can be particularly high due to the potential inherent immunogenicity of RNA. [Prior art documents] [Non-patent literature]

[0011] [Non-Patent Document 1] Belliveau et al,2012,Molecular Therapy-Nucleic Acids 1,e37 [Non-patent document 2] Chen et al,2014,J Control Release 196:106-12 [Non-patent document 3] Ambegia et al., 2005, Biochimica et Biophysica Acta 1669:155-163 [Non-patent document 4] Bendele A et al.,1998,Toxicolocical Sciences 42,152-157 [Non-patent document 5] Young MA et al.,2007,Translational Research 149(6),333-342 [Non-patent document 6] SMMoghimi, J. Szebeni, 2003, Progress in Lipid Research 42:463-478 [Non-Patent Document 7] Laverman P et al.,2001,J Pharmacol Exp Ther.298(2),607-12 [Non-patent document 8] Ishida et al.,2006,J Control Release 115(3),251-8 [Non-Patent Document 9] Semple et al.,2005,J Pharmacol Exp Ther.312(3),1020-6 Summary of the Invention [Problem to be solved by the invention]

[0012] Thus, there remains a need in the art for efficient methods and compositions for introducing RNA into cells that avoid the disadvantages associated with the use of PEG. The present disclosure addresses these and other needs.

[0013] The inventors have surprisingly found that the RNA particle formulations described herein meet the above requirements. In particular, polysarcosine-lipid conjugates are demonstrated to be suitable components for the construction of RNA nanoparticles. Polysarcosine is composed of repeating units of the natural amino acid sarcosine (N-methylglycine) and is biodegradable. Polysarcosine-lipid conjugates enable the production of RNA nanoparticles by various techniques, resulting in defined surface properties and a controlled size range. Production can be carried out using a robust process that complies with pharmaceutical manufacturing requirements. The particles can be end-group functionalized with various moieties to adjust charge or introduce specific molecular moieties, such as ligands. [Means for solving the problem]

[0014] In one aspect, the invention provides a composition comprising a plurality of RNA particles, each particle comprising: (i) RNA; and (ii) one or more components that associate with RNA to form RNA particles Including, The present invention relates to a composition in which polysarcosine is conjugated to at least one of one or more components.

[0015] In one embodiment, the RNA particle is a non-viral RNA particle. In one embodiment, the one or more components that associate with the RNA to form the particle include one or more polymers. In one embodiment, the one or more polymers include a cationic polymer. In one embodiment, the cationic polymer is an amine-containing polymer. In one embodiment, the one or more polymers include one or more polymers selected from the group consisting of poly-L-lysine, polyamidoamine, polyethyleneimine, chitosan, and poly(β-amino ester).

[0016] In one embodiment, one or more components that associate with RNA to form particles include one or more lipids or lipid-like substances. In one embodiment, one or more lipids or lipid-like substances include cationic or cationically ionizable lipids or lipid-like substances. In one embodiment, the cationically ionizable lipids or lipid-like substances are positively charged only at acidic pH and do not remain cationic at physiological pH. In one embodiment, one or more lipids or lipid-like substances include one or more additional lipids or lipid-like substances. In one embodiment, polysarcosine is conjugated to at least one of the one or more additional lipids or lipid-like substances.

[0017] In a further aspect, the present invention provides a composition comprising a plurality of RNA-lipid particles, each particle comprising: (a) RNA; (b) Cationic or cationically ionizable lipids or lipid-like substances; and (c) Polysarcosine-lipid conjugates or conjugates of polysarcosine and lipid-like substances The present invention relates to a composition comprising:

[0018] In one embodiment, each particle comprises: (d) Non-cationic lipids or lipid-like substances Further includes:

[0019] In one embodiment, the cationic or cationically ionizable lipids or lipid-like materials comprise from about 20 mol % to about 80 mol % of the total amount of lipids and lipid-like materials present in the particle.

[0020] In one embodiment, the non-cationic lipids or lipid-like materials comprise from about 0 mol % to about 80 mol % of the total amount of lipids and lipid-like materials present in the particle.

[0021] In one embodiment, the polysarcosine-lipid conjugate or polysarcosine and lipid-like substance conjugate comprises from about 0.25 mol % to about 50 mol % of the total amount of lipid and lipid-like substances present in the particle.

[0022] In one embodiment, the non-cationic lipid or lipid-like substance comprises a phospholipid. In one embodiment, the non-cationic lipid or lipid-like substance comprises cholesterol or a cholesterol derivative. In one embodiment, the non-cationic lipid or lipid-like substance comprises a mixture of a phospholipid and cholesterol or a cholesterol derivative. In one embodiment, the phospholipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), or a mixture thereof. In one embodiment, the non-cationic lipid or lipid-like substance comprises a mixture of DSPC and cholesterol.

[0023] In one embodiment, the polysarcosine-lipid conjugate or the conjugate of polysarcosine with a lipid-like substance has the following general formula (I):

[0024] [ka]

[0025] Includes.

[0026] In one embodiment, the polysarcosine-lipid conjugate or the conjugate of polysarcosine with a lipid-like substance has the following general formula (II):

[0027] [ka]

[0028] wherein one of R1 and R2 comprises a hydrophobic group and the other is H, a hydrophilic group, or a functional group that may comprise a targeting moiety. Includes.

[0029] In one embodiment, the polysarcosine-lipid conjugate or the conjugate of polysarcosine with a lipid-like substance has the following general formula (III):

[0030] [ka]

[0031] where R is H, a hydrophilic group, or a functional group that may include a targeting moiety. Includes.

[0032] In one embodiment of all aspects of the invention, the particles do not comprise polyethylene glycol-lipid conjugates or conjugates of polyethylene glycol and lipid-like substances, and preferably do not comprise polyethylene glycol.

[0033] In one embodiment of all aspects of the invention, the RNA is mRNA.

[0034] In one embodiment of all aspects of the invention, the cationic or cationically ionizable lipid or lipid-like substance is N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP ... and mixtures thereof.

[0035] In one embodiment of all aspects of the invention, the polysarcosine comprises between 2 and 200 sarcosine units.

[0036] In one embodiment of all aspects of the invention, the polysarcosine-lipid conjugate or conjugate of polysarcosine with a lipid-like substance is a member selected from the group consisting of polysarcosine-diacylglycerol conjugates, polysarcosine-dialkyloxypropyl conjugates, polysarcosine-phospholipid conjugates, polysarcosine-ceramide conjugates, and mixtures thereof.

[0037] In one embodiment of all aspects of the invention, the particles are nanoparticles.

[0038] In one embodiment of all aspects of the invention, the particle comprises a nanostructured core.

[0039] In one embodiment of all aspects of the invention, the particles have a size of from about 30 nm to about 500 nm.

[0040] In one embodiment of all aspects of the invention, the polysarcosine conjugate inhibits particle aggregation.

[0041] In a further aspect, the invention relates to a method for delivering RNA to cells of a subject, the method comprising administering to the subject a composition described herein.

[0042] In a further aspect, the present invention relates to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject a composition described herein, wherein the RNA encodes the therapeutic peptide or protein.

[0043] In a further aspect, the present invention relates to a method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition described herein, wherein delivery of RNA to cells of the subject is beneficial in treating or preventing the disease or disorder.

[0044] In a further aspect, the present invention relates to a method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition described herein, wherein the RNA encodes a therapeutic peptide or protein, wherein delivery of the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.

[0045] In one embodiment, the subject is a mammal, hi one embodiment, the mammal is a human. [Brief explanation of the drawings]

[0046] [Figure 1] Relationship between particle size and molar fraction of polysarcosylated LNPs. Lipid nanoparticles were prepared using lipid mixtures containing increasing molar fractions of C14PSarc20. Under appropriate conditions, colloidally stable particles could be obtained. At very low PSarc fractions (0.5 and 1%), no measurable particles were formed, whereas at 2.5 mol% and above, particles with discrete sizes and low polydispersity indices were obtained. Particle size could be precisely tuned by varying the PSarc fraction. Particle size decreased monotonically from approximately 200–250 nm at 2.5 mol% PSarc to approximately 50 nm at 20 mol% PSarc. [Figure 2] The relationship between the polysarcosine length (polymerization units) of the PSarc lipid used in LNP formation and in vitro protein expression of luciferase-encoding mRNA LNPs in various cell lines was investigated. LNPs formulated with luciferase-encoding mRNA were tested in lung tumor cells (TC-1), muscle cells (C2C12), hepatocytes (Hep-G2), and macrophages (RAW 264.7). Bioluminescence signals were measured 24 hours after transfection. Regardless of cell line, increasing the number of polysarcosine polymerization units did not result in a decrease in protein expression levels, as is typically observed with PEG-lipids. [Figure 3]In vivo efficacy of LNPs containing a constant fraction (5%) of PSarc lipids, with polysarcosine lengths varied between 11 and 65 units. LNPs formulated with luciferase-encoding mRNA were intravenously injected into mice (10 μg of RNA, n=3). In vivo and ex vivo bioluminescence were measured. In all cases, the strongest signal was found in the liver. The figure shows data from ex vivo measurements of liver extracted 6 hours after injection. No significant effect of polysarcosine length on protein expression levels in the liver could be determined. This allows for engineering particles using a wide range of PSarc sizes without compromising transfection efficiency. [Figure 4] The effect of various polysarcosine end groups on particle size and zeta potential. PSarc, consisting of 20 repeating units with either amine, carboxylated, or acetylated end groups, was tested in a direct comparison. All other formulation parameters were held constant. LNPs were successfully formed with all tested end groups, and the correlation between PSarc fraction and particle properties (size and zeta potential) was similar. [Figure 5] In vitro characterization of LNPs containing polysarcosine lipids with different end groups was performed as described in Figure 4. PSarc lipids with a molar fraction of 5% and a length of 20 units were used. LNPs formulated with luciferase-encoding mRNA were tested in hepatocytes (Hep-G2), macrophages (RAW 264.7), muscle cells (C2C12), and embryonic kidney cells (HEK 293 T). 24 hours after transfection, bioluminescence signals were measured. Bioluminescence signals were obtained for all LNPs and cell lines. The dependence of signal intensity as a function of cell line was similar for all end groups. [Figure 6]In vivo efficacy of LNPs formulated with different end groups, as described in Figures 4 and 5. PSarc lipids were used at a molar fraction of 5% and 20 units in length. LNPs formulated with luciferase-encoding mRNA were injected intravenously (10 μg of RNA, n=3). In vivo and ex vivo bioluminescence was measured. In all cases, the strongest signal was found in the liver. The figures show data from ex vivo measurements from liver extracted 6 hours after injection. Similar signal intensities were determined for all end groups, indicating that all end groups are suitable for achieving similarly high transfection in vivo. [Figure 7] Effect of PEGylation and polysarcosylation on liposome size. Liposomes were prepared with DOTMA and DOPE (2:1 mol / mol) alone, or with a lipid mixture containing PEG-lipid or pSarcosylation at a 2% molar fraction. Both PEG and pSarcosylation resulted in a significant decrease in measured size, but the polydispersity index was higher (multimodal). [Figure 8] Lipoplex formation using liposomes containing PEG and PSarc, as described in Figure 7, resulted in the formation of lipoplexes with limited size and low polydispersity indices from all three types of liposomes (DOTMA and DOPE (2:1 mol / mol) alone, or containing PEG-lipids or pSarc at 2% molar fraction). The lipoplexes from PEGylated and polysarcosylated liposomes showed surprisingly low polydispersity indices (PDIs) compared with the liposome precursors, which had higher PDIs. This indicates that pSarc liposomes, with their high polydispersity index, may also be suitable for the formation of well-defined RNA lipoplexes with a fairly small size of 50 nm and a PDI of approximately 0.2. [Figure 9]In vitro characterization of lipoplexes composed of liposomes consisting of only DOTMA and DOPE (2:1 mol / mol) or the same lipid mixture containing PEG-lipid or pSarc at a 2% molar fraction. Lipoplexes formulated with luciferase-encoding mRNA were tested in hepatocytes (Hep-G2). 24 hours after transfection, bioluminescence signals were measured. PEGylation significantly reduced the signal, but this reduction was less pronounced in the presence of PSarc. PSarc appears to reduce transfection efficiency to a much lesser extent than PEG. [Figure 10] In vitro characterization of lipoplexes composed of liposomes consisting of only DOTMA and DOPE (2:1 mol / mol) or the same lipid mixture containing PEG-lipid or pSarc at a 2% molar fraction. Lipoplexes formulated with luciferase-encoding mRNA were tested in muscle cells (C2C12). 24 hours after transfection, bioluminescence signals were measured. PEGylation significantly reduced the signal, but this reduction was less pronounced in the presence of PSarc. PSarc appears to reduce transfection efficiency to a much lesser extent than PEG. [Figure 11] Relationship between particle size in formulation and polysarcosine chain length and molar ratio. [Figure 12] Scattering curves (SAXS) from polysarcosylated lipid nanoparticles. [Figure 13] RNA accessibility assessed by Quant-It Ribogreen assay. [Figure 14] Intravenous administration of various doses of EPO (erythropoietin)-encoding mRNA loaded into LNPs formulated with either PSarc or PEG-conjugated lipids. [Figure 15] Liver enzyme release as an early marker of hepatotoxicity after injection of LNPs formulated with increasing PSarc chain length. [Figure 16] C3a complex-mediated complement activation of PEGylated and polysarcosylated LNPs at theoretical human plasma concentrations. [Figure 17] Cryo-electron microscopy (TEM) image of LNPs formulated with DODMA:cholesterol:DSPC:PSarc 23 at 40:45:10:5 mole %. Scale bar = 200 nm. DETAILED DESCRIPTION OF THE INVENTION

[0047] Although the present disclosure will be described in detail below, it should be understood that this disclosure is not limited to the specific methodology, protocols and reagents described herein, and these may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0048] Preferably, the terms used herein are defined as set forth 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).

[0049] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0050] The elements of the present disclosure are described below. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and embodiments should not be construed as limiting the disclosure to only the embodiments explicitly described. This description should be understood to disclose and encompass embodiments combining the explicitly described embodiment with any number of the disclosed elements. Furthermore, any permutation and combination of all described elements should be considered disclosed by this description unless the context dictates otherwise.

[0051] The term "about" means approximately or approximately, and in the context of numerical values ​​or ranges described herein, means, in one embodiment, ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0052] As used in the context of describing the present disclosure (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better explain the disclosure and does not impose limitations on the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0053] Unless otherwise specified, the term "comprises" is used in the context of this document to indicate that additional members may optionally be present in addition to the members of the list introduced by "comprises." However, it is contemplated as a specific embodiment of the present disclosure that the term "comprises" encompasses the possibility that additional members are not present, i.e., for the purposes of this embodiment, "comprises" should be understood to have the meaning of "consisting of."

[0054] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure was not entitled to antedate such disclosure.

[0055] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated: Undefined terms have their art-accepted meanings.

[0056] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 20% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or essentially zero.

[0057] In one embodiment, terms such as "increase" or "enhancement" 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%.

[0058] As used herein, "physiological pH" refers to a pH of about 7.4.

[0059] As used in this disclosure, "% w / v" refers to weight-to-volume percent, which is a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of the solution in milliliters (mL).

[0060] As used in this disclosure, "mol %" is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

[0061] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Thus, ionic strength, I, is calculated by the formula:

[0062]

number

[0063] where c is the molar concentration of a particular ionic species and z is the absolute value of its charge. The sum Σ applies to all the different types of ions (i) in the solution.

[0064] According to the present disclosure, the term "ionic strength" in one embodiment relates to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, their concentration or effective concentration (presence of free ions) is, in one embodiment, sufficiently low to prevent RNA degradation due to the presence of a chelating agent. In one embodiment, the concentration or effective concentration of divalent ions is lower than the catalytic level for hydrolysis of 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.

[0065] "Osmolality" refers to the concentration of a solute expressed as osmoles of solute per kilogram of solvent.

[0066] The term "freezing" refers to a phase transition from a liquid to a solid state, which typically occurs when the temperature of a system is reduced below a critical temperature and is accompanied by a characteristic change in the enthalpy of the system.

[0067] The term "lyophilize" or "freeze-drying" refers to the lyophilization of a substance by freezing the substance and then reducing the surrounding pressure to cause the freezing medium in the substance to sublimate directly from the solid phase to the gas phase.

[0068] The term "spray drying" refers to spray drying a substance by mixing a (heated) gas with an atomized (atomized) fluid in a vessel (spray dryer), where the solvent from the formed droplets evaporates, resulting in a dry powder.

[0069] The term "cryoprotectant" relates to a substance added to a formulation to protect the active ingredient during the freezing step.

[0070] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying step.

[0071] The term "reconstitute" relates to adding a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

[0072] The term "recombinant" in the context of the present disclosure means "produced through genetic engineering." In one embodiment, a "recombinant entity" in the context of the present disclosure is not naturally occurring.

[0073] The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature", and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0074] In the context of the present disclosure, the term "particle" relates to a structured entity formed by molecules or molecular complexes. In one embodiment, the term "particle" relates to a micro- or nano-sized structure, such as a micro- or nano-sized dense structure dispersed in a medium.

[0075] In the context of the present disclosure, the term "RNA particle" refers to a particle containing RNA. The electrostatic interaction between positively charged molecules such as polymers and lipids and negatively charged RNA is involved in particle formation. This leads to complexation and spontaneous formation of RNA particles. In one embodiment, the RNA particle is a nanoparticle.

[0076] As used in this disclosure, "nanoparticles" refers to particles having an average diameter suitable for intravenous administration.

[0077] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, resulting in the so-called Z dimension, which has a length dimension. average , and the dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter", or "size" of a particle is this Z average Used synonymously with the value of

[0078] The "polydispersity index", as mentioned in the definition of "mean diameter", is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis. Under certain prerequisites, it can be interpreted as a measure of the size distribution of the nanoparticle ensemble.

[0079] Generally, the RNA-lipid particles described herein can be obtained by mixing RNA-containing phase with lipid-containing phase.This can be by mixing ethanol phase or other water-miscible solvents, including lipid, such as cationic lipids such as DODMA, and additional lipid with the aqueous phase that contains RNA.Another option is to mix the aqueous phase that contains lipid, for example, the lipid in the form of liposome or other types of lipid dispersion, with another aqueous phase that contains RNA.

[0080] RNA-containing particles Various types of RNA-containing particles have previously been described as suitable for delivering RNA in a microparticulate form (e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). For non-viral RNA delivery vehicles, nanoparticle encapsulation of RNA can physically protect the RNA from degradation and, depending on specific chemical properties, aid in cellular uptake and endosomal escape.

[0081] The present disclosure describes particles containing RNA and one or more components that associate with the RNA to form RNA particles, as well as compositions containing such particles. The RNA particles may contain RNA complexed to the particles in various forms through non-covalent interactions. The particles described herein are not viral particles, particularly infectious viral particles, i.e., they are unable to infect cells. The RNA-containing particles may be in the form of, for example, proteinaceous particles, polymer-containing particles, or lipid-containing particles. Suitable proteins, polymers, or lipids are included in the term "particle-forming component" or "particle-forming agent." The term "particle-forming component" or "particle-forming agent" refers to any component that associates with RNA to form an RNA particle. Such components include any component that can become part of an RNA particle.

[0082] Proteins, polymers, lipids, and other hydrophilic, hydrophobic, or amphiphilic compounds are typical components of RNA particle formulations.

[0083] Given their high degree of chemical flexibility, polymers are commonly used materials for nanoparticle-based delivery. Typically, cationic polymers are used to electrostatically condense negatively charged RNA into nanoparticles. These positively charged groups often consist of amines that change protonation state in the pH range of 5.5–7.5, leading to an ionic imbalance that triggers endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to RNA delivery. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-amino esters), in particular, are widely used in nucleic acid delivery due to their ease of synthesis and biodegradability.

[0084] As used herein, the term "polymer" is given its usual meaning, i.e., a molecular structure comprising one or more repeating units (monomers) connected by covalent bonds. The repeating units may all be identical, or in some cases, multiple types of repeating units may exist within a 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 as described herein, may also be present in the polymer.

[0085] When multiple 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 optionally be a copolymer. The repeating units forming the copolymer may be arranged in any manner. For example, the repeating units may be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., a copolymer comprising one or more regions each comprising a first repeating unit (e.g., a first block) and one or more regions each comprising a second repeating unit (e.g., a second block). A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more distinct blocks.

[0086] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are typically polymers that do not cause significant cell death at moderate concentrations. 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 within the body.

[0087] In certain embodiments, the particle-forming polymer can be protamine or a polyalkyleneimine, such as polyethyleneimine.

[0088] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) in place of somatic histones, particularly in association with DNA. In particular, the term "protamine" refers to a protein found in fish sperm that is strongly basic, soluble in water, does not coagulate with heat, and produces primarily arginine upon hydrolysis. In purified form, they are used to neutralize the anticoagulant effect of heparin in long-acting formulations of insulin.

[0089] In accordance with the present disclosure, the term "protamine" as used herein is intended to include any protamine amino acid sequence obtained or derived from natural or biological sources, and fragments thereof, and multimeric forms of said amino acid sequence or fragments thereof, as well as artificial, specifically designed for a particular purpose (synthetic) polypeptides that cannot be isolated from natural or biological sources.

[0090] In one embodiment, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75×10 2 ~10 7 Da, preferably 1000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0091] According to the present disclosure, linear polyalkyleneimines such as linear polyethyleneimine (PEI) are preferred.

[0092] lipid-containing particles In one embodiment, the RNA particles described herein comprise at least one lipid or lipid-like substance as particle-forming agent.The lipid carriers contemplated for use herein include any substance with which RNA can associate, for example, by forming a complex with RNA or by forming a vesicle in which RNA is enclosed or encapsulated.

[0093] The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are typically poorly soluble in water. In aqueous environments, their amphiphilic nature allows them to self-assemble into organized structures and various phases. One of these phases is composed of lipid bilayers, such as those found in vesicles, multilamellar / unilamellar liposomes, or membranes in aqueous environments. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups can contain polar and / or charged groups, including carbohydrates, phosphate, carboxylic acid, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other similar groups.

[0094] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Amphiphilic compounds often have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, and the non-polar portion is insoluble in water. Furthermore, the polar portion can have either a formal positive or a formal negative charge. Alternatively, the polar portion can have both a formal positive and a formal negative charge and can be a zwitterion or an internal salt. For purposes of this disclosure, an amphiphilic compound can be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0095] The terms "lipid-like substance," "lipid-like compound," or "lipid-like molecule" refer to substances that are structurally and / or functionally related to lipids but may not be considered lipids in the strict sense. For example, the term includes compounds that can form amphiphilic layers such as those found in vesicles, multilamellar / unilamellar liposomes, or membranes in aqueous environments, and includes surfactants or synthetic compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules containing hydrophilic and hydrophobic moieties with different structural organizations that may or may not resemble those of lipids. As used herein, the term "lipid" should be interpreted to encompass both lipids and lipid-like substances unless otherwise indicated herein or clearly contradicted by the context.

[0096] Specific examples of amphiphilic compounds that can be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0097] In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be classified into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, but fat is a subgroup of lipids called triglycerides. Lipids also include molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0098] Fatty acids, or fatty acid residues, are a diverse group of molecules made up of a hydrocarbon chain terminating in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically 4 to 24 carbons in length, can be saturated or unsaturated and can be bonded to functional groups including oxygen, halogens, nitrogen, and sulfur. When fatty acids contain double bonds, there is the possibility of cis or trans geometric isomerism, which significantly affects the configuration of the molecule. Cis double bonds result in bending of the fatty acid chain, an effect that can be compounded with more double bonds within the fatty acid chain. Other major lipid classes within the fatty acid category are fatty acid esters and fatty acid amides.

[0099] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the most well-known of which are fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is typically esterified with a different fatty acid. A further subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

[0100] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core attached by ester bonds to two fatty acid-derived "tails" and by a phosphate ester bond to a "head" group. Commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid), examples of glycerophospholipids are phosphatidylcholine (PC, also known as GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0101] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The predominant sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated and have chain lengths of 16–26 carbon atoms. The predominant sphingophospholipid in mammals is sphingomyelin (ceramide phosphocholine), while insects contain primarily ceramide phosphoethanolamine, and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues attached to a sphingoid base via glycosidic bonds. Examples of these are simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0102] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelins.

[0103] Glycolipids are compounds in which fatty acids are directly attached to a sugar backbone, forming structures compatible with membrane bilayers. In glycolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The best-known glycolipid is the acylated glucosamine precursor of the lipid A component of the lipopolysaccharide of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with as many as seven fatty acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli (E. coli) is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0104] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by classical enzymes as well as by iterative and multimodular enzymes that share mechanistic features with fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and possess great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0105] According to the present disclosure, lipids and lipid-like substances can be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH.

[0106] Preferably, the RNA particles described herein contain cationic or cationically ionizable lipids or lipid-like substances. Cationic or cationically ionizable lipids and lipid-like substances can be used to electrostatically bind to RNA. Cationic ionizable lipids and lipid-like substances are preferably substances that are positively charged only at acidic pH. This ionizable behavior is believed to enhance efficacy by aiding endosomal escape and reducing toxicity compared to particles that remain cationic at physiological pH. The particles may also contain non-cationic lipids or lipid-like substances. Collectively, anionic and neutral lipids or lipid-like substances are referred to herein as non-cationic lipids or lipid-like substances. Optimizing the formulation of RNA particles by adding other hydrophobic moieties, such as cholesterol and lipids, in addition to ionizable / cationic lipids or lipid-like substances can enhance particle stability and significantly increase the efficiency of RNA delivery.

[0107] In one embodiment, the cationic or cationically ionizable lipid or lipid-like substance comprises a head group that includes at least one nitrogen atom (N) that is positively charged or capable of being protonated.

[0108] Polysarcosine conjugates One or more of the particle-forming components described herein, such as polymers, lipids, or lipid-like substances used in the particles described herein, include polysarcosine (poly(N-methylglycine)).Polysarcosine can include acetylated (neutral end groups) or other functionalized end groups.In the case of RNA-lipid particles, in one embodiment, polysarcosine is conjugated, preferably covalently bound, to the non-cationic lipid or lipid-like substance contained in the particle.

[0109] In certain embodiments, the end groups of the polysarcosine can be functionalized with one or more molecular moieties that impart specific properties, such as a positive or negative charge, or a targeting agent that directs the particle to a particular cell type, population of cells, or tissue.

[0110] A variety of suitable targeting agents are known in the art. Non-limiting examples of targeting agents include peptides, proteins, enzymes, nucleic acids, fatty acids, hormones, antibodies, carbohydrates, mono-, oligo- or polysaccharides, peptidoglycans, glycopeptides, and the like. For example, any of a number of different substances can be used that bind to antigens on the surface of target cells. Antibodies against surface antigens of target cells generally exhibit the required target specificity. In addition to antibodies, Fab, Fab', F(ab')2 or scFv fragments or single domain antibodies (e.g., camelid V) can also be used. H Suitable immunoreactive fragments, such as antibody fragments (e.g., antibody fragments H fragments), can also be used. Many antibody fragments suitable for use in forming targeting mechanisms are already available in the art. Similarly, ligands for any receptors on the surface of target cells can be suitably used as targeting agents. These include any small molecule or biomolecule, natural or synthetic, that specifically binds to a cell surface receptor, protein, or glycoprotein found on the surface of the desired target cell.

[0111] In certain embodiments, the polysarcosine may be selected from the group consisting of 2 to 200, 2 to 190, 2 to 180, 2 to 170, 2 to 160, 2 to 150, 2 to 140, 2 to 130, 2 to 120, 2 to 110, 2 to 100, 2 to 90, 2 to 80, 2 to 70, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 150, 5 to 140, 5 to 1 and 30, 5-120, 5-110, 5-100, 5-90, 5-80, 5-70, 10-200, 10-190, 10-180, 10-170, 10-160, 10-150, 10-140, 10-130, 10-120, 10-110, 10-100, 10-90, 10-80, or 10-70 sarcosine units.

[0112] In certain embodiments, the polysarcosine has the following general formula (I):

[0113] [ka]

[0114] (where x refers to the number of sarcosine units) Through one of the bonds, the polysarcosine can be linked to a particle-forming component or a hydrophobic component. Through the other bond, the polysarcosine can be linked to a linker to a functional moiety such as H, a hydrophilic group, an ionizable group, or a targeting moiety.

[0115] cationic lipids In one embodiment, the RNA-lipid particles described herein comprise at least one cationic lipid. As used herein, "cationic lipid" refers to a lipid with a net positive charge. Cationic lipids bind negatively charged RNA to the lipid matrix through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety, such as a sterol, an acyl chain, a diacyl chain, or more acyl chains, and the head group of the lipid typically carries a positive charge. In certain embodiments, cationic lipids have a net positive charge only at a certain pH, particularly an acidic pH, but at a different, preferably higher pH, such as physiological pH, they preferably do not have a net positive charge, and are preferably uncharged, i.e., neutral. For the purposes of this disclosure, such "cationically ionizable" lipids are included in the term "cationic lipid" unless the context requires otherwise. Examples of cationic lipids include N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammoniumpropane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP); 1,2-dioleoyl-3-dimethylammoniumpropane (DODAP); 1,2-diacyloxy-3-dimethylammoniumpropane; 1,2-dialkyloxy-3-dimethylammoniumpropane; dioctadecyldimethylammonium ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanum trifluoroacetate (DOSPA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), Dioctadecylamidoglycylspermine (DOGS), 3-Dimethylamino-2-(cholest-5-ene-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy]-3-dimethyl-1-( cis,cis-9',12'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane dopane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N- (2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-Bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12 -dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC) ), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-aminium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA ), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N, 12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200). DODMA, DOTMA, DOTAP, DODAC, and DOSPA are preferred. In certain embodiments, at least one cationic lipid is DODMA.

[0116] In some embodiments, the cationic lipid may comprise from about 10 mol% to about 80 mol%, from about 20 mol% to about 60 mol%, from about 25 mol% to about 55 mol%, from about 30 mol% to about 50 mol%, from about 35 mol% to about 45 mol%, or about 40 mol% of the total lipid present in the particle.

[0117] More lipids In addition to cationic lipids, the RNA particles described herein may contain one or more additional lipids. Additional lipids may be incorporated that may or may not affect the overall charge of the RNA particle. In certain embodiments, the additional lipid is a non-cationic lipid. Non-cationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, "neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. In preferred embodiments, the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.

[0118] Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin, particularly diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBP), and the like. PC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, particularly diacylphosphatidylethanolamines such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with various hydrophobic chains.

[0119] In certain preferred embodiments, the additional lipid is DSPC, or DSPC and cholesterol.

[0120] In certain embodiments, the RNA particle comprises both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DODMA and the additional lipid is DSPC, or DSPC and cholesterol.

[0121] Without wishing to be bound by theory, the amount of at least one cationic lipid relative to the amount of at least one additional lipid can affect important RNA particle properties, such as charge, particle size, stability, tissue selectivity, and RNA biological activity. Thus, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

[0122] In some embodiments, non-cationic lipids, particularly neutral lipids (e.g., one or more phospholipids and / or cholesterol), may comprise from about 0 mol% to about 90 mol%, from about 20 mol% to about 80 mol%, from about 25 mol% to about 75 mol%, from about 30 mol% to about 70 mol%, from about 35 mol% to about 65 mol%, or from about 40 mol% to about 60 mol% of the total lipid present in the particle.

[0123] In certain preferred embodiments, the non-cationic lipid, particularly the neutral lipid, comprises a phospholipid such as DSPC that is about 5 mol% to about 50 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 40 mol%, about 5 mol% to about 35 mol%, about 5 mol% to about 30 mol%, about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol% of the total lipid present in the particle.

[0124] In certain preferred embodiments, the non-cationic lipid, particularly the neutral lipid, comprises from about 10 mol% to about 80 mol%, from about 10 mol% to about 70 mol%, from about 15 mol% to about 65 mol%, from about 20 mol% to about 60 mol%, from about 25 mol% to about 55 mol%, or from about 30 mol% to about 50 mol% of cholesterol or a derivative thereof based on the total lipid present in the particle.

[0125] In certain preferred embodiments, the non-cationic lipid, particularly the neutral lipid, comprises a mixture of (i) a phospholipid, such as DSPC, in an amount of about 5 mol% to about 50 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 40 mol%, about 5 mol% to about 35 mol%, about 5 mol% to about 30 mol%, about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol% of the total lipid present in the particle, and (ii) cholesterol or a derivative thereof, such as cholesterol, in an amount of about 10 mol% to about 80 mol%, about 10 mol% to about 70 mol%, about 15 mol% to about 65 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 55 mol%, or about 30 mol% to about 50 mol% of the total lipid present in the particle. As a non-limiting example, a lipid particle comprising a mixture of phospholipids and cholesterol may contain about 5 mol% to about 50 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 40 mol%, about 5 mol% to about 35 mol%, about 5 mol% to about 30 mol%, about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol% of the total lipid present in the particle, of DSPC, and about 10 mol% to about 80 mol%, about 10 mol% to about 70 mol%, about 15 mol% to about 65 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 55 mol%, or about 30 mol% to about 50 mol% of the total lipid present in the particle, of cholesterol.

[0126] Polysarcosine-lipid conjugates The RNA particles described herein, such as the above-mentioned RNA particles comprising cationic lipid and additional lipid, further comprise polysarcosine conjugates, such as polysarcosine-lipid conjugates.Polysarcosine can be conjugated with any particle-forming component, such as lipid or lipid-like substance, particularly covalently bonded or linked.Polysarcosine-lipid conjugates are molecules in which polysarcosine is conjugated with lipids described herein, such as cationic lipids or cationically ionizable lipids or additional lipids.Alternatively, polysarcosine is conjugated with lipids or lipid-like substances that are different from cationic or cationically ionizable lipids or additional lipids.

[0127] In certain embodiments, the polysarcosine-lipid conjugate or the conjugate of polysarcosine with a lipid-like substance has the following general formula (II):

[0128] [ka]

[0129] wherein one of R1 and R2 comprises a hydrophobic group and the other is a functional group that may comprise H, a hydrophilic group, an ionizable group, or a targeting moiety. In one embodiment, the hydrophobic group comprises a linear or branched alkyl group or an aryl group, preferably containing 10 to 50, 10 to 40, or 12 to 20 carbon atoms. In one embodiment, R1 or R2 comprising the hydrophobic group comprises a moiety such as a heteroatom, particularly N, linked to one or more linear or branched alkyl groups.

[0130] In certain embodiments, the polysarcosine-lipid conjugate or the conjugate of polysarcosine with a lipid-like substance has the following general formula (III):

[0131] [ka]

[0132] where R is a functional group that may contain H, a hydrophilic group, an ionizable group, or a targeting moiety. Includes.

[0133] The symbol "x" in the general formulae herein, such as general formulae (II) and (III), refers to the number of sarcosine units, which may be any number defined herein.

[0134] In certain embodiments, the polysarcosine-lipid conjugate or polysarcosine-lipid-like substance conjugate is a member selected from the group consisting of polysarcosine-diacylglycerol conjugates, polysarcosine-dialkyloxypropyl conjugates, polysarcosine-phospholipid conjugates, polysarcosine-ceramide conjugates, and mixtures thereof.

[0135] In particular examples, the polysarcosine-lipid conjugate is present in an amount of about 0.2 mol% to about 50 mol%, about 0.25 mol% to about 30 mol%, about 0.5 mol% to about 25 mol%, about 0.75 mol% to about 25 mol%, about 1 mol% to about 25 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 25 mol%, about 1 mol% to about 20 ... It may comprise about 1.5 mol% to about 5 mol%, about 1.5 mol% to about 25 mol%, about 1.5 mol% to about 20 mol%, about 1.5 mol% to about 15 mol%, about 1.5 mol% to about 10 mol%, about 1.5 mol% to about 5 mol%, about 2 mol% to about 25 mol%, about 2 mol% to about 20 mol%, about 2 mol% to about 15 mol%, about 2 mol% to about 10 mol%, or about 2 mol% to about 5 mol%.

[0136] Typically, the polysarcosine moiety has 2 to 200, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 150, 5 to 140, 5 to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, or 10 to 80 sarcosine units.

[0137] RNA-lipid particles "RNA-lipid particles" include lipid formulations that can be used to deliver RNA to a desired target site (e.g., a cell, tissue, organ, etc.). RNA-lipid particles are typically formed from a cationic lipid such as DODMA, one or more non-cationic lipids such as a phospholipid (e.g., DSPC), cholesterol or an analog thereof, and a polysarcosine-lipid conjugate.

[0138] Without intending to be bound by theory, it is believed that the cationic lipid and additional lipids bind to the RNA to form aggregates, binding the nucleic acid to the lipid matrix, and this spontaneous aggregation results in colloidally stable particles.

[0139] In some embodiments, the RNA-lipid particles contain multiple types of RNA molecules, where the molecular parameters of the RNA molecules may be similar or different from each other in terms of basic structural elements such as molar mass or molecular structure, capping, coding regions, or other features.

[0140] In some embodiments, the RNA-lipid particles contain, in addition to RNA, (i) cationic lipids, which may constitute from about 10 mol% to about 80 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 55 mol%, about 30 mol% to about 50 mol%, about 35 mol% to about 45 mol%, or about 40 mol% of the total lipids present in the particle; (ii) non-cationic lipids, particularly neutral lipids (e.g., one or more phospholipids and / or cholesterol), which may constitute from about 0 mol% to about 90 mol%, about 20 mol% to about 80 mol%, about 25 mol% to about 75 mol%, about 30 mol% to about 70 mol%, about 35 mol% to about 65 mol%, or about 40 mol% to about 60 mol% of the total lipids present in the particle; and (iii) non-cationic lipids, particularly neutral lipids (e.g., one or more phospholipids and / or cholesterol), which may constitute from about 0 mol% to about 90 mol%, about 20 mol% to about 80 mol%, about 25 mol% to about 75 mol%, about 30 mol% to about 70 mol%, about 35 mol% to about 65 mol%, or about 40 mol% to about 60 mol% of the total lipids present in the particle. The polysarcosine-lipid conjugate may constitute about 0.2 mol% to about 50 mol%, about 0.25 mol% to about 30 mol%, about 0.5 mol% to about 25 mol%, about 0.75 mol% to about 25 mol%, about 1 mol% to about 25 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, about 1 mol% to about 5 mol%, about 1.5 mol% to about 25 mol%, about 1.5 mol% to about 20 mol%, about 1.5 mol% to about 15 mol%, about 1.5 mol% to about 10 mol%, about 1.5 mol% to about 5 mol%, about 2 mol% to about 25 mol%, about 2 mol% to about 20 mol%, about 2 mol% to about 15 mol%, about 2 mol% to about 10 mol%, or about 2 mol% to about 5 mol% of the total lipids present in the solution.

[0141] In certain preferred embodiments, the non-cationic lipids, particularly neutral lipids, comprise from about 5 mol% to about 50 mol%, from about 5 mol% to about 45 mol%, from about 5 mol% to about 40 mol%, from about 5 mol% to about 35 mol%, from about 5 mol% to about 30 mol%, from about 5 mol% to about 25 mol%, or from about 5 mol% to about 20 mol% of the total lipids present in the particle.

[0142] In certain preferred embodiments, the non-cationic lipid, particularly the neutral lipid, comprises from about 10 mol% to about 80 mol%, from about 10 mol% to about 70 mol%, from about 15 mol% to about 65 mol%, from about 20 mol% to about 60 mol%, from about 25 mol% to about 55 mol%, or from about 30 mol% to about 50 mol% of cholesterol or a derivative thereof based on the total lipid present in the particle.

[0143] In certain preferred embodiments, the non-cationic lipid, particularly the neutral lipid, comprises a mixture of (i) a phospholipid, such as DSPC, in an amount of about 5 mol% to about 50 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 40 mol%, about 5 mol% to about 35 mol%, about 5 mol% to about 30 mol%, about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol% of the total lipid present in the particle, and (ii) cholesterol or a derivative thereof, such as cholesterol, in an amount of about 10 mol% to about 80 mol%, about 10 mol% to about 70 mol%, about 15 mol% to about 65 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 55 mol%, or about 30 mol% to about 50 mol% of the total lipid present in the particle. As a non-limiting example, a lipid particle comprising a mixture of phospholipids and cholesterol may contain about 5 mol% to about 50 mol%, about 5 mol% to about 45 mol%, about 5 mol% to about 40 mol%, about 5 mol% to about 35 mol%, about 5 mol% to about 30 mol%, about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol% of the total lipid present in the particle, of DSPC, and about 10 mol% to about 80 mol%, about 10 mol% to about 70 mol%, about 15 mol% to about 65 mol%, about 20 mol% to about 60 mol%, about 25 mol% to about 55 mol%, or about 30 mol% to about 50 mol% of the total lipid present in the particle, of cholesterol.

[0144] Typically, the polysarcosine moiety has 2 to 200, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 150, 5 to 140, 5 to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, or 10 to 80 sarcosine units.

[0145] In some embodiments, the RNA-lipid particles comprise, in addition to RNA, (i) DODMA, which may constitute from about 10 mol% to about 80 mol%, from about 20 mol% to about 60 mol%, from about 25 mol% to about 55 mol%, from about 30 mol% to about 50 mol%, from about 35 mol% to about 45 mol%, or about 40 mol% of the total lipid present in the particle; (ii) DODMA, which may constitute from about 5 mol% to about 50 mol%, from about 5 mol% to about 60 mol%, from about 25 mol% to about 55 mol%, from about 30 mol% to about 50 mol%, from about 35 mol% to about 45 mol%, or from about 40 mol% of the total lipid present in the particle; (iii) DSPC, which may constitute about 45 mol%, about 5 mol% to about 40 mol%, about 5 mol% to about 35 mol%, about 5 mol% to about 30 mol%, about 5 mol% to about 25 mol%, or about 5 mol% to about 20 mol% of the total lipid present in the particle; (iv) DSPC, which may constitute about 10 mol% to about 80 mol%, about 10 mol% to about 70 mol%, about 15 mol% to about 65 mol%, about 20 mol% to about 60 mol%, or about 25 mol% to about 55 mol% of the total lipid present in the particle; or about 30 mol% to about 50 mol%, and (iv) cholesterol, which may constitute about 0.2 mol% to about 50 mol%, about 0.25 mol% to about 30 mol%, about 0.5 mol% to about 25 mol%, about 0.75 mol% to about 25 mol%, about 1 mol% to about 25 mol%, about 1 mol% to about 20 mol%, about 1 mol% to about 15 mol%, about 1 mol% to about 10 mol%, about 1 mol% to about 5 mol%, about 1.5 mol% to about 25 mol%, about 1.5 mol% to about 20 mol%, about 1.5 mol% to about 15 mol%, about 1.5 mol% to about 10 mol%, about 1.5 mol% to about 5 mol%, about 2 mol% to about 25 mol%, about 2 mol% to about 20 mol%, about 2 mol% to about 15 mol%, about 2 mol% to about 10 mol%, or about 2 mol% to about 5 mol% of the polysarcosine-lipid conjugate.

[0146] In certain embodiments, the RNA particle comprises a polysarcosine-lipid conjugate according to general formula (II) or (III), DODMA, DSPC and cholesterol.

[0147] RNA particle size In one embodiment, the RNA particles described herein have a diameter of about 30 nm to about 1000 nm, about 30 nm to about 800 nm, about 30 nm to about 700 nm, about 30 nm to about 600 nm, about 30 nm to about 500 nm, about 30 nm to about 450 nm, about 30 nm to about 400 nm, about 30 nm to about 350 nm, about 30 nm to about 300 nm, about 30 nm to about 250 nm, about 30 nm to about 200 nm, about 30 nm to about 190 nm, about 30 nm to about 180 nm, about 30 nm to about 1 and having an average diameter in the range of 70 nm, about 30 nm to about 160 nm, about 30 nm to about 150 nm, about 50 nm to about 500 nm, about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 50 nm to about 190 nm, about 50 nm to about 180 nm, about 50 nm to about 170 nm, about 50 nm to about 160 nm, or about 50 nm to about 150 nm.

[0148] In certain embodiments, the RNA particles described herein have an average diameter in the range of about 40 nm to about 800 nm, about 50 nm to about 700 nm, about 60 nm to about 600 nm, about 70 nm to about 500 nm, about 80 nm to about 400 nm, about 150 nm to about 800 nm, about 150 nm to about 700 nm, about 150 nm to about 600 nm, about 200 nm to about 600 nm, about 200 nm to about 500 nm, or about 200 nm to about 400 nm.

[0149] For example, the RNA particles described herein, produced by the processes described herein, exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, or less than about 0.1. By way of example, the RNA particles may exhibit a polydispersity index ranging from about 0.1 to about 0.3.

[0150] RNA In the present disclosure, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends of the RNA (either or both). It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified RNAs are considered analogs of naturally occurring RNA. In certain embodiments, RNA according to the present invention comprises a population of different RNA molecules, e.g., a mixture of different RNA molecules that optionally encode different peptides and / or proteins. Thus, according to the present invention, the term "RNA" may include a mixture of RNA molecules.

[0151] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which is related to an RNA transcript encoding a peptide or protein. As established in the art, mRNA generally comprises 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 refers to a nucleic acid comprising deoxyribonucleotides.

[0152] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0153] In certain embodiments of the present disclosure, the RNA is a replicon RNA or simply "replicon," particularly a self-replicating RNA. In one particularly preferred embodiment, the replicon or self-replicating RNA is derived from or contains elements derived from a ssRNA virus, particularly a positive-strand ssRNA virus such as an alphavirus. Alphaviruses are a typical example of a positive-strand RNA virus. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication, as well as protein modification) and structural proteins (which form the virus particle). Typically, two open reading frames (ORFs) are present within the genome. The four nonstructural proteins (nsP1-nsP4) are typically encoded together by the first ORF, which begins near the 5' end of the genome, while the structural proteins of alphaviruses are encoded together by a second ORF found downstream of the first ORF and extending toward the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected with alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins is translatable from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87, pp. 111-124). After infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts like a messenger RNA to directly translate the open reading frame encoding the nonstructural polyprotein (nsP1-nsP4). Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or organisms.In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced with an open reading frame encoding the protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase, and the other nucleic acid molecule can be replicated in trans by said replicase (hence the term trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. Nucleic acid molecules that can be replicated in trans by the replicase must contain specific alphavirus sequence elements that allow recognition by the alphavirus replicase and RNA synthesis.

[0154] In certain embodiments of the present disclosure, the RNA in the RNA particles described herein has a concentration of about 0.002 mg / mL to about 5 mg / mL, about 0.002 mg / mL to about 2 mg / mL, about 0.005 mg / mL to about 2 mg / mL, about 0.01 mg / mL to about 1 mg / mL, about 0.05 mg / mL to about 0.5 mg / mL, or about 0.1 mg / mL to about 0.5 mg / mL. In certain embodiments, the RNA has a concentration of about 0.005 mg / mL to about 0.1 mg / mL, about 0.005 mg / mL to about 0.09 mg / mL, about 0.005 mg / mL to about 0.08 mg / mL, about 0.005 mg / mL to about 0.07 mg / mL, about 0.005 mg / mL to about 0.06 mg / mL, or about 0.005 mg / mL to about 0.05 mg / mL.

[0155] In one embodiment, the RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine, pseudouridine (ψ), N1-methylpseudouridine (m 1 ψ) or 5-methyluridine (m 5 U) is included.

[0156] In some embodiments, the RNA of 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 may be modified with a 5' cap analog. The term "5' cap" refers to the structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide linked to the mRNA by a 5'-5' triphosphate bond. In one embodiment, the guanosine is methylated at position 7. Providing an RNA with a 5' cap or 5' cap analog can be achieved by in vitro transcription, in which the 5' cap is co-transcriptionally expressed on the RNA strand, or can be attached to the RNA post-transcriptionally using a capping enzyme.

[0157] In some embodiments, an RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end, upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5' cap, e.g., directly adjacent to the 5' cap. If present, the 3'-UTR is located at the 3' end, downstream of the stop codon of the protein-coding region, although the term "3'-UTR" preferably does not include a poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) sequence, if present, e.g., directly adjacent to the poly(A) sequence.

[0158] In some embodiments, the RNA according to the present disclosure comprises a 3'-poly(A) sequence. The term "poly(A) sequence" refers to a sequence of adenyl (A) residues typically 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.

[0159] In the context of the present disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0160] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0161] RNA can be coding RNA, i.e., RNA that codes for peptide or protein. Said RNA can express the coded peptide or protein. For example, said RNA can be RNA that codes and expresses pharmacologically active peptide or protein. Alternatively, RNA can be non-coding RNA such as antisense RNA, microRNA (miRNA) or siRNA.

[0162] As used herein, RNA can be pharmaceutically active RNA. " Pharmacologically active RNA " is RNA that encodes pharmaceutically active peptide or protein, or is pharmacoactive in itself, for example, has one or more pharmacologic activities, such as immunostimulatory activity, as described for pharmacoactive protein.For example, RNA can be one or more strands of RNA interference (RNAi).Such agents include short interfering RNA (siRNA), or short hairpin RNA (shRNA), or the precursor of siRNA or microRNA-like RNA, that targets target transcript, for example, transcript that is related to the target endogenous disease.

[0163] Some aspects of the present disclosure include targeted delivery of the RNA disclosed herein to specific cells or tissues. In one embodiment, the present disclosure includes targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen. When the administered RNA encodes an antigen or epitope for inducing an immune response, targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen, is particularly preferred. In one embodiment, the target cells are spleen cells. In one embodiment, the target cells are antigen-presenting cells, such as professional antigen-presenting cells in the spleen. In one embodiment, the target cells are dendritic cells in the spleen. The "lymphatic system" is part of the circulatory system and is an important part of the immune system, including a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphocytes and initiate adaptive immune responses.

[0164] Lipid-based RNA delivery system has inherent selectivity to the liver.Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or lipid metabolism (liposome and lipid or cholesterol conjugate).In one embodiment, the target organ is the liver, and the target tissue is liver tissue.Delivery to such target tissue is preferred, particularly when it is desired to have RNA or encoded peptide or protein present in this organ or tissue, and / or when it is desired to express a large amount of encoded peptide or protein, and / or when it is desired or required to have the systemic presence of encoded peptide or protein, particularly when it is present in a significant amount.

[0165] In one embodiment, after administration of the RNA particles 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 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 cell in a lymph node. In one embodiment, the target cell is a lung cell. In one embodiment, the target cell is a blood cell. In one embodiment, the target cell is a skin cell. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in 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. Therefore, the RNA particles described herein can be used to deliver RNA to such target cells. Therefore, the present disclosure also relates to a method for delivering RNA to a target cell of a subject, comprising administering the RNA particles described herein to a subject. In one embodiment, the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is RNA that codes for a peptide or protein, and the RNA is translated by the target cell to produce the peptide or protein.

[0166] In one embodiment, the RNA encodes a pharmaceutically active peptide or protein.

[0167] According to the present disclosure, the term "RNA-encoded" means that the RNA, when present in the appropriate environment, such as within the cells of a target tissue, can direct the assembly of amino acids to produce the peptide or protein it encodes during the translation process. In one embodiment, the RNA can interact with the cellular translation machinery to enable translation of the peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm), secrete the encoded peptide or protein, or produce it on its surface.

[0168] According to this disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising 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, about 100, or about 150 consecutive amino acids joined together by peptide bonds. The term "protein" refers to large peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide" and "protein" are generally used synonymously herein.

[0169] A "pharmaceutically active peptide or protein" or "therapeutic peptide or protein" when provided to a subject in a therapeutically effective amount has a positive or beneficial effect on the subject's condition or pathology. In one embodiment, a pharmaceutically active peptide or protein has curative or palliative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A pharmaceutically active peptide or protein can have preventative properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathology. The term "pharmaceutically active peptide or protein" includes whole proteins or polypeptides and can also refer to pharmaceutically active fragments thereof. The term can also include pharmaceutically active analogs of peptides or proteins.

[0170] Examples of pharmaceutically active proteins include immune system proteins such as cytokines and their derivatives, such as cytokine fusions (such as albumin-cytokine fusions), as well as immunologically active compounds (e.g., interleukins, colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, ceretins, homing receptors, T cell receptors, chimeric antigen receptors (CARs), immunoglobulins, including antibodies or bispecific antibodies, for example for immune stimulation in case of viral / bacterial infections or for the production of neutralizing antibodies, immunologically active antigens, such as soluble major histocompatibility complex antigens, bacterial, parasitic, or viral antigens, allergens, autoantigens, antibodies), hormones (insulin, thyroid hormones, catecholamines, gonadotrophins, trophic hormones,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 enzymes, steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatase, cytochromes, adenylate or guanylate cyclase, neuraminidase, lysosomal enzymes, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (such as growth hormone or growth factor binding proteins), transcription and translation factors, tumor growth suppressor proteins (e.g., proteins that inhibit angiogenesis), structural proteins (such as collagen, fibroin, fibrinogen, elastin, tubulin, actin, myosin), blood proteins (such as thrombin, serum albumin, Factor VII, Factor VIII, insulin, Factor IX, Factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony-stimulating factor (GCSF) or modified Factor VIII, anticoagulants, and the like.

[0171] The term "immunologically active compound" refers to any compound that alters the immune response, for example, by inducing and / or suppressing immune cell maturation, inducing and / or suppressing cytokine biosynthesis, and / or altering humoral immunity by stimulating antibody production by B cells. Immunologically active compounds have potent immunostimulatory activity, including but not limited to antiviral and antitumor activity, and can also downregulate other aspects of the immune response, for example, shifting the immune response away from a TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunologically active compounds can be useful as vaccine adjuvants.

[0172] In one embodiment, the pharmaceutically active peptide or protein comprises a cytokine. The term "cytokine" refers to a category of small proteins (approximately 5-20 kDa) that are important in cell signaling. Their release affects the behavior of surrounding cells. Cytokines, as immunomodulators, participate in autocrine, paracrine, and endocrine signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (despite some overlap in terminology). Cytokines are produced by a wide range of cells, including immune cells such as 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 multiple cell types. Cytokines act through receptors and are particularly important in the immune system; they regulate the balance between humoral and cellular immune responses and regulate the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the actions of other cytokines in complex ways.

[0173] In one embodiment, the pharmaceutically active protein according to the present invention is a cytokine involved in regulating lymphoid homeostasis, preferably a cytokine involved in, preferably inducing or enhancing, the development, priming, proliferation, differentiation and / or survival of T cells. 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.

[0174] In one embodiment, the pharmaceutically active peptide or protein comprises a replacement protein. In this embodiment, the present invention provides a method for treating a subject with a disorder requiring protein replacement (e.g., a protein deficiency disorder), comprising administering to the subject RNA as described herein encoding the replacement protein. The term "protein replacement" refers to the introduction of a protein (including a functional variant thereof) into a subject with a deficiency of such a protein. The term also refers to the introduction of a protein into a subject who requires or would benefit from providing the protein, e.g., a subject suffering from a protein deficiency. The term "a disorder characterized by a protein deficiency" refers to any disorder exhibiting pathology caused by the absence or insufficient amount of protein. This term encompasses protein folding disorders, i.e., conformational disorders, that result in biologically inactive protein products. Protein deficiency may be involved in infection, immunosuppression, organ failure, glandular disorders, radiation sickness, nutritional deficiency, poisoning, or other environmental or external insults.

[0175] 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 elicits an immune response in the subject against the one or more antigens or one or more epitopes, which may be therapeutic or partially or fully protective.

[0176] The term "antigen" refers to an agent containing an epitope capable of generating an immune response. The term "antigen" includes, inter alia, proteins and peptides. In one embodiment, the antigen is presented by a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or a macrophage. The antigen or its processing product, such as a T cell epitope, is bound, in one embodiment, by a T cell or B cell receptor, or by an immunoglobulin molecule, such as an antibody. Thus, the antigen or its processing product can specifically react with an antibody or a T lymphocyte (T cell). In one embodiment, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, and the epitope is derived from such an antigen.

[0177] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, a disease-associated antigen or its epitope can be used for therapeutic purposes. A disease-associated antigen can be associated with infection by a microorganism, typically a microbial antigen, or can be associated with cancer, typically a tumor.

[0178] The term "tumor antigen" refers to a component of a cancer cell that can originate from the cytoplasm, cell surface, and cell nucleus. In particular, the term refers to an antigen that is produced intracellularly or as a surface antigen on a tumor cell.

[0179] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0180] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0181] The term "epitope" refers to a portion 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. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 10 to about 25 amino acids in length; for example, an epitope can be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0182] The term "T cell epitope" refers to a portion 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" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the 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 display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides can also be effective. For class II MHC / peptide complexes, the binding peptide is typically about 10 to about 25 amino acids long, particularly about 13 to about 18 amino acids long, although longer and shorter peptides can also be effective.

[0183] 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 refers to a T cell that recognizes the antigen it targets, particularly when presented on the surface of an antigen-presenting cell or diseased cell, such as a cancer cell, in association with an MHC molecule, and preferably exerts T cell effector function. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. T cell specificity can be assessed using any of a variety of standard techniques, such as a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured. In certain embodiments of the present disclosure, the RNA encodes at least one epitope.

[0184] In certain embodiments, the epitope is derived from a tumor antigen. The tumor antigen may be a "standard" antigen that is generally known to be expressed in various cancers. The tumor antigen may also be a "neoantigen" that is specific to an individual's tumor and has not previously been recognized by the immune system. The neoantigen or neoepitope may 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 claudin-6, claudin-18.2, and claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, 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, MUC These include 1, 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.

[0185] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding novel epitopes (neoepitopes) are attractive targets for the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a strong immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) present in the spleen are antigen-presenting cells that are particularly involved in RNA expression of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to enhance therapeutic efficacy in tumor vaccine compositions. Rapid sequencing of tumor mutagenesis can provide multiple epitopes for personalized vaccines that can be encoded by the RNA described herein, for example, as a single polypeptide in which the epitopes are optionally separated by a linker. 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 "pentatopes"), at least 10 epitopes (referred to as "decatopes"), and at least 20 epitopes (referred to as "eicosatopes").

[0186] Compositions Comprising RNA Particles The term "plurality of RNA particles" or "plurality of RNA-lipid particles" refers to a population of a specific number of particles. In certain embodiments, the term refers to a population of 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 1013 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 10 19 , 10 20 , 10 21 , 10 22 , or 10 23 It refers to a group of particles.

[0187] It will be apparent to one of ordinary skill in the art that the plurality of particles can include any portion of the aforementioned ranges or any range therein.

[0188] In embodiments, the compositions of the present disclosure are liquid or solid. Non-limiting examples of solids include frozen, freeze-dried, or spray-dried forms. In a preferred embodiment, the compositions are liquid.

[0189] In accordance with the present disclosure, the compositions described herein may include salts such as organic or inorganic salts, including, but 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.

[0190] The compositions described herein may also include stabilizers to avoid substantial loss of product quality, particularly substantial loss of RNA activity during storage, freezing, lyophilization, and / or spray drying, e.g., to reduce or prevent aggregation, particle collapse, RNA degradation, and / or other types of damage.

[0191] In one embodiment, the stabilizer is a cryoprotectant or lyoprotectant.

[0192] In one embodiment, the stabilizer is a carbohydrate. As used herein, the term "carbohydrate" refers to and includes monosaccharides, disaccharides, trisaccharides, oligosaccharides, and polysaccharides.

[0193] In one embodiment, the stabilizer is an amino acid or a surfactant (eg, a poloxamer).

[0194] According to the present disclosure, the RNA particle compositions described herein have a pH suitable for RNA particle stability, particularly RNA stability. In one embodiment, the RNA particle compositions described herein have a pH of about 4.0 to about 8.0, or about 5.0 to about 7.5. Without wishing to be bound by theory, the use of a buffer maintains the pH of the composition during its manufacture, storage, and use. In certain embodiments of the present disclosure, the buffer is selected from the group consisting of 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] The buffer system may be selected from the group consisting of 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 may be acetic acid, alone or in a salt thereof, citric acid, alone or in a salt thereof, boric acid, alone or in a salt thereof, and phosphoric acid, alone or in a salt thereof, or amino acids and amino acid derivatives.

[0195] Certain embodiments of the present disclosure contemplate the use of a chelating agent in the compositions described herein. A chelating agent refers to a compound capable of forming at least two coordinate covalent bonds with a metal ion, thereby forming a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions that may otherwise induce accelerated 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, deferoxamine, dithiocarbamate sodium, penicillamine, calcium pentetate, sodium pentetate, succinic acid, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic 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.

[0196] In some embodiments, EDTA is at a concentration of about 0.05 mM to about 5 mM, about 0.1 mM to about 2.5 mM, or about 0.25 mM to about 1 mM.

[0197] Pharmaceutical Composition Compositions comprising the RNA particles described herein are useful as, or for the preparation of, pharmaceutical compositions or medicaments for therapeutic or prophylactic treatment.

[0198] In one embodiment, the RNA particles described herein are present in a pharmaceutical composition. In another embodiment, the compositions described herein are pharmaceutical compositions.

[0199] The particles of the present disclosure may be administered in the form of any suitable pharmaceutical composition.

[0200] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably 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. A pharmaceutical composition is also known in the art as a pharmaceutical formulation. In the context of the present disclosure, a pharmaceutical composition comprises RNA particles as described herein.

[0201] The pharmaceutical compositions of the present disclosure may contain or be administered with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. 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, INFα, INF-γ, GM-CSF, or LT-α. Additional known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides such as Pam3Cys, as well as lipophilic components such as saponin, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid A (MPL), monomycoloylglycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).

[0202] Pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and a "pharmaceutically acceptable formulation."

[0203] The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the action of the active ingredients of the pharmaceutical composition.

[0204] The term "pharmaceutically effective amount" refers to an amount that, alone or together with further doses, achieves the desired response or desired effect. In the case of the treatment of a specific disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, particularly halting or reversing the progression of the disease. The desired response in the treatment of a disease can also be the delay or prevention of the onset of the disease or condition. The effective amount of the particles or compositions described herein depends on the condition being treated, the severity of the disease, individual patient parameters including age, physiological condition, size and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Thus, the dosage of the particles or compositions described herein can depend on such various parameters. If the patient's response is inadequate with the initial dose, a higher dose (or a substantially higher dose achieved by a different, more localized route of administration) can be used.

[0205] The pharmaceutical compositions of the present disclosure may include salts, buffering agents, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0206] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0207] The term "excipient" as used herein refers to a substance that may 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.

[0208] The term "diluent" refers to a diluting and / or thinning agent. Furthermore, the term "diluent" includes any one or more of a fluid, liquid or solid suspension and / or mixed medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0209] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which an active ingredient is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. As used herein, a carrier may 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 glycols, hydrogenated naphthalenes, and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0210] Pharmaceutically acceptable carriers, excipients or diluents for pharmaceutical use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0211] Pharmaceutical carriers, excipients, or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0212] Route of administration of pharmaceutical composition In one embodiment, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, transdermally, intramuscularly, or intratumorally. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, including absorption via the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration by any means other than via the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration.

[0213] Use of the Pharmaceutical Composition The RNA particles described herein can be used for the therapeutic or prophylactic treatment of various diseases, particularly diseases in which providing a peptide or protein to a subject provides a therapeutic or preventative effect. For example, providing an antigen or epitope derived from a virus can be useful in treating viral diseases caused by the virus. Providing a tumor antigen or epitope can be useful in treating cancer diseases in which cancer cells express the tumor antigen. Providing a functional protein or enzyme can be useful in treating genetic disorders characterized by dysfunctional proteins, such as lysosomal storage diseases (e.g., mucopolysaccharidoses) or factor deficiencies. Providing a cytokine or cytokine fusion can be useful in modulating the tumor microenvironment.

[0214] The term "disease" (also referred to herein as "disorder") refers to an abnormal condition affecting an individual's body. Disease is often interpreted as a medical condition associated with specific symptoms and signs. Disease can be caused by externally derived agents, such as infection, or by internal dysfunction, such as autoimmune disease. In humans, "disease" is often used more broadly to refer to a condition that causes pain, impairment, suffering, social problems, or death in the affected individual, or similar problems in those who come into contact with the individual. In this broader sense, disease sometimes includes impairment, incapacity, disability, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Because suffering from and living with many illnesses can alter one's outlook on life and personality, illnesses typically affect individuals not only physically but also emotionally.

[0215] In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject with the aim of combating a condition, such as a disease or disorder. This term is intended to include the full range of treatments for a given condition from which a subject is afflicted, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, slow the progression of a disease, disorder or condition, relieve or reduce symptoms and complications, and / or cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is to be understood as the management and care of an individual with the purpose of combating a disease, condition or disorder and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0216] The term "therapeutic treatment" relates to any treatment that improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, halt or delay the onset of the disease in an individual, inhibit or delay the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or has previously had the disease.

[0217] The term "prophylactic treatment" or "preventative treatment" relates to any treatment aimed at preventing a disease from occurring in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

[0218] The terms "individual" and "subject" are used interchangeably herein. These terms refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate), or any other non-mammal, including a bird (chicken), fish, or any other animal species, that may be suffering from or susceptible to a disease or disorder (e.g., cancer, infectious disease), but may or may not have the disease or disorder, or may require preventative intervention such as vaccination, or may be in need of intervention such as protein supplementation. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0219] The term "patient" refers to an individual or subject for treatment, particularly an afflicted individual or subject.

[0220] In one embodiment of the present disclosure, the objective is to provide protection against infectious diseases by vaccination.

[0221] In one embodiment of the present disclosure, the objective is to provide a secreted therapeutic protein, such as an antibody, bispecific antibody, cytokine, cytokine fusion protein, enzyme, etc., to a subject, particularly one in need thereof.

[0222] In one embodiment of the present disclosure, the objective is to provide protein replacement therapy, 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.

[0223] In one embodiment of the present disclosure, the goal is to regulate / reprogram immune cells in the blood.

[0224] Those skilled in the art will understand that one of the principles of immunotherapy and vaccination is based on the fact that immunizing a subject with the immunologically related antigen or epitope for the disease to be treated generates an immune protective response against the disease.Therefore, the pharmaceutical compositions described herein can be applied to induce or enhance immune response.Therefore, the pharmaceutical compositions described herein are useful in the preventive and / or therapeutic treatment of the disease that involves antigen or epitope.

[0225] The term "immunization" or "vaccination" refers to the process of administering an antigen to an individual with the intent of inducing an immune response, for example, for therapeutic or prophylactic reasons.

[0226] Citation of documents and tests referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements regarding the contents of these documents are based on information available to applicant and do not constitute an admission as to the accuracy of the contents of these documents.

[0227] The following description is presented to enable those skilled in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. 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 and shown herein, but are to be accorded the scope consistent with the claims. [Example]

[0228] Example 1 material and method material mRNA encoding luciferase or secreted NanoLuc® luciferase (secNLuc) was provided by the RNA Biochemistry Unit (BioNTech RNA Pharmaceuticals, Mainz, Germany) (mRNA concentrations are 2–5 mg / mL in water or 10 mM Hepes; 0.1 mM EDTA; pH 7.0).

[0229] The ionizable cationic lipid DODMA (1,2-dioleyloxy-N,N-dimethyl-3-aminopropane) and the helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) were purchased from Merck. The helper lipid DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) was obtained from Avanti Polar Lipids. Cholesterol was obtained from Sigma Aldrich. Sodium dodecyl sulfate (SDS) was obtained from Sigma Aldrich.

[0230] Prior to preparation, lipids are dissolved in absolute ethanol (Carl Roth) at a concentration of 5-100 mM and the ethanolic lipid solution is stored at -20 °C.

[0231] Prior to preparation, the ethanolic lipid solution, 100 mM pH 5.4 sterile citrate buffer and RNA were equilibrated at room temperature.

[0232] Protocol 1 for the preparation of lipid nanoparticles Lipid nanoparticles were prepared by mixing an ethanol phase containing lipids with an aqueous phase containing RNA using a microfluidic mixing device, the NanoAssemblr™ Benchtop Instrument (Precision NanoSystems, Vancouver, BC). One volume of ethanol containing a lipid mixture of 9 mM total lipid and three volumes of RNA at 0.15 mg / mL in 100 mM citrate buffer, pH 5.4, were mixed through a microfluidic cartridge at a total flow rate of 12 mL / min. The resulting mixture was then directly mixed with two volumes of 100 mM citrate buffer, pH 5.4. Unless otherwise noted, the particles were dialyzed against phosphate-buffered saline (PBS) for 2.5 hours in a 10K MWCO dialysis cassette (Slide-A-Lyser, ThermoFisher Scientific). The particles were then reconcentrated to a theoretical RNA concentration of approximately 0.2-0.5 mg / mL by ultrafiltration using Amicon® ultracentrifugal filters (30 kDa NMWL, Merck Millipore). Physicochemical characterization (size, polydispersity, zeta potential, RNA accessibility, and total RNA concentration) was performed on the day of preparation. After complete characterization, the formulations were stored at 4°C for no more than two days. Prior to in vitro testing or in vivo injection, the lipid nanoparticles were dissolved in PBS to the desired RNA concentration.

[0233] Protocol 2 for the preparation of lipid nanoparticles Lipid nanoparticles were prepared by mixing the lipid-containing aqueous phase with the RNA-containing aqueous phase. pSarc-liposomes were prepared by injecting 600 μl of 75 mM total lipids containing different molar fractions of cationic lipids and helper lipids with or without pSarc into a total volume of 14.4 ml of water with ethanol for 30 minutes while stirring. The liposomes were then added to the RNA in water at an N / P ratio of 4, followed by rapid vortexing to form pSarc-LPX with a final RNA concentration of 0.05 mg / ml. Physicochemical characterization (size, polydispersity, RNA accessibility, and total RNA concentration) was performed on the day of preparation. After complete characterization, the formulations were stored at 4°C for no more than two days. Prior to in vitro testing, the lipid nanoparticles were dissolved in water to the desired RNA concentration.

[0234] Particle size measurement The particle size and polydispersity index (PDI) of the lipid nanoparticles were measured by dynamic light scattering. The formulation was diluted with PBS to a final RNA concentration of 0.005 mg / mL. 120 μL of diluted sample was measured in triplicate in a 96-well plate. Size was measured using a DynaPro plate reader II instrument from WYATT technology GmbH (Dernbach, Germany).

[0235] Zeta potential (electrophoretic mobility) measurement RNA-lipid nanoparticles were diluted with 0.1x PBS in 1 ml to an RNA concentration of 0.01 mg / mL. Three 1.05 ml samples of each formulation were prepared in plastic cuvettes. The electrophoretic mobility of the particles was measured by laser Doppler electrophoresis using a ζ-Wallis instrument (Corduan technologies, France). For each sample, medium-resolution measurements were performed using one sequence of 10 analyses. Measurements with low signal-to-noise ratios or extreme mobilities (μ) (>3 or <-3 μm*cm / V*S) were excluded from the final analysis.

[0236] RiboGreen assay of RNA accessibility and total RNA concentration RNA-lipid nanoparticles were always concentrated to a final concentration of approximately 0.2–0.5 mg / mL RNA. The Quant-iT RiboGreen RNA assay (Thermo Fischer Scientific) was used to quantify RNA accessibility and total RNA concentration in the formulations. Briefly, encapsulation efficiency was determined using the RNA-binding dye RiboGreen by comparing fluorescence between samples in the presence and absence of 2% Triton X-100. In the absence of surfactant, fluorescence can only be measured from accessible free RNA, whereas in the presence of surfactant, fluorescence is measured from total RNA. The fluorescence of samples in the presence of surfactant Triton X-100 was also used to calculate total RNA concentration based on a calibration curve.

[0237] Lipid nanoparticle samples or PBS (negative control) were diluted with 1xTE buffer (Thermo Fisher Scientist) to an mRNA concentration of 2–5 μg / mL.

[0238] An aliquot of each diluted sample was further diluted 1:1 with 1xTE buffer (measurement of accessible mRNA) or 1:1 with 1xTE buffer containing 2% Triton-X100 (measurement of total mRNA, both accessible within particles and free mRNA). Samples were prepared in duplicate. To ensure sufficient lipid dissociation, samples were incubated at 37°C for 10 min. Quant-iT RiboGreen RNA reagent (1:100 dilution from a stock solution in TE buffer) was then added to each sample, and the dye's fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm (Tecan Infinite M200 Pro Multimode Plate Reader).

[0239] RNA accessibility was determined as follows:

[0240]

number

[0241] Total RNA concentrations were determined using an RNA standard curve in 1xTE buffer containing 2% Triton X-100.

[0242] Agarose gel electrophoresis Agarose gel electrophoresis was performed to assess free RNA. Gels were poured using 1 g of agarose dissolved in 100 mL of 1x TAE buffer (Tris-acetate-EDTA) (ThermoFisher), 1 mL of 5% sodium hypochlorite, and 10 μL of GelRed Nucleic Acid Gel Stain (Biotium). The gel was allowed to harden at room temperature for at least 25 minutes. The gel was then placed in a gel electrophoresis tank and run with 1x TAE running buffer (ThermoFisher). Prior to loading, samples were incubated at 40°C with or without 2% Triton X-100 for total RNA and free RNA, respectively. The gel was run at 80 V for 40 minutes. Images of the gel were captured using a Chemidoc XRS imaging system (Bio-Rad).

[0243] In vitro transfection assay and cell viability assay Cells were seeded into white 96-well flat-bottom plates at a concentration of 5,000 cells per well for C2C12 cells and 20,000 cells per well for HepG2 and TC1 cells. Cells were maintained at 37°C and 5% CO2, except for C2C12, at 7.5% CO2. After 18–24 h, the supernatant was discarded and replaced with 90 μL of the respective medium supplemented with 10% non-inactivated FCS. The formulations were diluted to a final concentration of 1–10 μg / mL in PBS. Next, 10 μl of lipid nanoparticle solution was added to the cells to obtain a final medium volume of 100 μl. The final amount of RNA in each well ranged from 33–100 ng. The plates were centrifuged at 500 g for 5 minutes at room temperature. After 24 hours of incubation with the cells, the Bright-Glo™ Luciferase Assay (Cat. No. E260, Promega GmbH, Mannheim, Germany) was performed according to the manual. The bioluminescence signal (RLU) was measured using a Tecan Infinite M200 Pro Multimode Plate Reader, and luciferase expression was calculated by subtracting the background from untransfected cells (PBS was used as a blank).

[0244] The same procedure was followed for measuring cell viability. After incubating the formulations with cells for 24 hours, the CellTiter-Glo™ assay (Cat. No. G9242, Promega GmbH, Mannheim, Germany) was performed according to the manual's instructions. Controls with PBS for 100% viability and DMSO for toxicity were included. Viability was calculated as follows:

[0245]

number

[0246] In vivo transfection in mice Mice were anesthetized with isoflurane and 200 μl of a test formulation of 0.05 mg / mL luciferase-encoding mRNA was injected intravenously into the retroorbital sinus using an insulin syringe pre-fitted with a 30 G cannula. Mice were observed for signs of pain, distress, and distress until they regained consciousness.

[0247] At the time of measurement (6 and 24 hours after administration), mice were intraperitoneally injected with 100 mg / kg body weight of D-luciferin solution. Subsequently, mice were anesthetized with isoflurane and placed on a heat mat (37°C) in an IVIS® Spectrum (Perkin Elmer) imaging chamber, with a constant supply of isoflurane / oxygen via an individual anesthesia mask. Five minutes after luciferin injection, bioluminescence was detected by a camera for 1 minute. Next, mice were sacrificed by neck extension, and organs, including the liver, lungs, spleen, heart, kidneys, brain, and lymph nodes, were collected and measured again ex vivo using the IVIS® Spectrum imaging device. The resulting images were analyzed using "LivingImage" software (Perkin Elmer). Regions of interest (ROIs) were drawn around the organs to quantify the total photon flux [p / s]. Blood was collected, and whole blood was centrifuged at 1000 × g for 3 minutes to obtain serum. Liver enzyme levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and LDH levels were determined by clinical chemistry analyzer-Indiko from Thermo scientific.

[0248] For EPO experiments, Balb / c mice (n=5) were intravenously administered a range of mRNA doses from 30 to 3 μg. After 3, 6, 24, and 48 hours, whole blood was collected and centrifuged at 13,000 rpm for 3 minutes to obtain plasma. EPO secretion was determined using the Mouse Erythropoietin DuoSet ELISA (R&D Systems).

[0249] Small angle X-ray scattering Small-angle X-ray scattering (SAXS) experiments were performed at the German Synchrotron-EMBL (DESY) Hamburg [P12]. The sample-to-detector distance was adjustable between 1.6 and 6 m to allow measurements from q = 0.6 Å to q = 3 Å. The concentrated LNP suspension was filled in situ into a quartz capillary tube using a syringe.

[0250] Complement activation In vitro C3a levels were determined using a Human C3a EIA kit (Quidel). Briefly, LNPs and controls (positive (Cremophore El) and negative (1x PBS and EDTA (18 mM))) were incubated with normal human serum complement (NHS, Quidel) at a ratio of 20:80 (specimen:NHS) for 1 hour at 37°C. LNPs were tested at 5x, 1x, and 0.02x the theoretical plasma concentration of a 1 mg / kg mRNA dose. The C3a EIA kit was performed according to the manufacturer's protocol.

[0251] Cryo-TEM Samples were stored in vitrified ice supported by a holey carbon film on 200-mesh copper grids (QuantiFoil® R2 / 1). Vitrification was performed in liquid ethane at -180 °C using a Leica EM GP. Grids were stored under liquid nitrogen until transferred to the electron microscope for imaging. Cryo-TEM imaging was performed on holey carbon-coated copper grids using a Zeiss Libra® 120 under liquid N2 cryo conditions. The microscope was operated at an accelerating voltage of 120 kV, and images were acquired with a Gatan UltraScan® CCD camera. Images of each grid were acquired at multiple scales to assess the overall distribution of the specimen.

[0252] <Example 2> Generation of RNA-lipid nanoparticles containing pSarc mRNA-lipid nanoparticles were prepared using amino acid-based polypeptoid lipids: polysarcosine-linked lipids.

[0253] LNPs were prepared by mixing an ethanol phase containing the lipids DODMA, cholesterol, DSPC, and C14pSarc20 in a molar ratio of 40:50-X:10:X with three volumes of RNA at 0.15 mg / mL in citrate buffer 100 mM pH 5.4.

[0254] [Table 1]

[0255] Figure 1 shows the relationship between particle size and molar fraction for polysarcosylated LNPs. Lipid nanoparticles were produced using lipid mixtures containing increasing molar fractions of C14PSarc20. Under appropriate conditions, colloidally stable particles could be obtained. At very low PSarc fractions (0.5 and 1%), no measurable particles were formed, whereas at 2.5 mol% and above, particles with discrete sizes and low polydispersity indexes were obtained. Particle size could be precisely tuned by varying the PSarc fraction. Particle size decreased monotonically from approximately 200–250 nm at 2.5 mol% PSarc to approximately 50 nm at 20 mol% PSarc.

[0256] Example 3 Particles containing pSarc-lipids with different lengths of sarcosine polymerization units LNPs were prepared by mixing one volume of an ethanol phase containing the lipids DODMA, cholesterol, DSPC, and C14pSarcX with different polymer lengths (X = 11, 20, 34, or 65) in a molar ratio of 40:45:10:5 with three volumes of RNA at 0.15 mg / mL in citrate buffer 100 mM pH 5.4.

[0257] [Table 2]

[0258] Figure 2 shows the relationship between the polysarcosine length (polymerization units) of the PSarc lipid used in LNP formation and in vitro protein expression of luciferase-encoding mRNA LNPs in various cell lines. LNPs formulated with luciferase-encoding mRNA were tested in lung tumor cells (TC-1), muscle cells (C2C12), hepatocytes (Hep-G2), and macrophages (RAW 264.7). Bioluminescence signals were measured 24 hours after transfection. Regardless of cell line, increasing the number of polysarcosine polymerization units did not result in a decrease in protein expression levels, as is typically observed with PEG-lipids.

[0259] Figure 3 shows the in vivo efficacy of LNPs containing a constant fraction (5%) of PSarc lipids, with polysarcosine lengths varied between 11 and 65 units. Mice were intravenously injected with LNPs formulated with luciferase-encoding mRNA (10 μg of RNA, n = 3). In vivo and ex vivo bioluminescence were measured. In all cases, the strongest signal was found in the liver. The figure shows data from ex vivo measurements of liver extracted 6 hours after injection. No significant effect of polysarcosine length on protein expression levels in the liver could be determined. This allows for the engineering of particles using a wide range of PSarc sizes without compromising transfection efficiency.

[0260] Example 4 Polysarcosine - Effect of lipid end groups LNPs were prepared by mixing one volume of an ethanol phase containing the lipids DODMA, cholesterol, DSPC, and C14pSarc20 with different terminal groups (NH2, COOH, and C2H3O) in a molar ratio of 40:50-x:10:x with three volumes of RNA at 0.15 mg / mL in citrate buffer 100 mM pH 5.4.

[0261] [Table 3]

[0262] Figure 4 shows the effect of various polysarcosine end groups on particle size and zeta potential. PSarc consisting of 20 repeating units with either amine, carboxylated, or acetylated end groups was tested in a direct comparison. All other formulation parameters were kept constant. LNP formation with all tested end groups was successful, and the correlation between PSarc fraction and particle properties (size and zeta potential) was similar.

[0263] Figure 5 shows the in vitro characterization of LNPs containing polysarcosine lipids with different end groups, as described in Figure 4. PSarc lipids with a molar fraction of 5% and a length of 20 units were used. LNPs formulated with luciferase-encoding mRNA were tested in hepatocytes (Hep-G2), macrophages (RAW 264.7), muscle cells (C2C12), and embryonic kidney cells (HEK 293 T). 24 hours after transfection, bioluminescence signals were measured. Bioluminescence signals were obtained for all LNPs and cell lines. The dependence of signal intensity as a function of cell line was similar for all end groups.

[0264] Figure 6 shows the in vivo efficacy of LNPs formulated with different end groups, as described in Figures 4 and 5. PSarc lipids were used at a molar fraction of 5% and 20 units in length. LNPs formulated with luciferase-encoding mRNA were injected intravenously (10 μg of RNA, n=3). In vivo and ex vivo bioluminescence was measured. In all cases, the strongest signal was found in the liver. The figure shows data from ex vivo measurements from liver extracted 6 hours after injection. Similar signal intensities were determined for all end groups, indicating that all end groups are suitable for achieving similarly high transfection in vivo.

[0265] <Example 5> Preparation of PSarc RNA-lipid nanoparticles using various cationic lipids The results of the following experiments demonstrate the versatility of polysarcosine to form RNA-lipid nanoparticles with various types of cationic moieties.

[0266] [Table 4]

[0267] Example 6 pSarc-liposomes and RNA-lipoplexes The results of the following experiments demonstrate that the inclusion of polysarcosine-conjugated lipids is suitable for the formation of liposomes and stealth RNA-lipoplexes. Under appropriate conditions, small particles with high transfection efficiency are formulated.

[0268] pSarc-liposomes were prepared by injecting 600 μl of a 75 mM total lipid ethanol solution containing cationic helper lipids and pSarc or PEG into a total volume of 14.4 ml of water with stirring for 30 min. The liposomes were then added to the RNA in water at N / P 4, followed by rapid vortexing to form pSarc-LPX.

[0269] Figure 7 shows the effect of PEGylation and polysarcosylation on liposome size. Liposomes were prepared with DOTMA and DOPE (2:1 mol / mol) alone, or with a lipid mixture containing PEG-lipid or pSarcosylation at a 2% molar fraction. Both PEG and pSarcosylation resulted in a significant decrease in measured size, but the polydispersity index was higher (multimodal).

[0270] Figure 8 shows lipoplex formation using liposomes containing PEG and PSarc, as described in Figure 7. All three types of liposomes (DOTMA and DOPE (2:1 mol / mol) alone, or containing PEG-lipids or pSarc at 2% molar fraction) formed lipoplexes with limited size and low polydispersity indexes. The lipoplexes from PEGylated and polysarcosylated liposomes showed surprisingly low polydispersity indexes (PDIs) compared with the liposome precursors, which had high PDIs. This indicates that pSarc liposomes, which have a high PDI, may also be suitable for the formation of well-defined RNA lipoplexes with a fairly small size of 50 nm and a PDI of approximately 0.2.

[0271] Figure 9 shows the in vitro characterization of lipoplexes composed of liposomes consisting of only DOTMA and DOPE (2:1 mol / mol) or the same lipid mixture containing PEG-lipid or pSarc at a 2% molar fraction. Lipoplexes formulated with luciferase-encoding mRNA were tested in hepatocytes (Hep-G2). 24 h after transfection, bioluminescence signals were measured. PEGylation significantly reduced the signal, but this reduction was less pronounced in the presence of PSarc. PSarc appears to reduce transfection efficiency to a much lesser extent than PEG.

[0272] Figure 10 shows the in vitro characterization of lipoplexes composed of liposomes consisting of only DOTMA and DOPE (2:1 mol / mol) or the same lipid mixture containing PEG-lipid or pSarc at a 2% molar fraction. Lipoplexes formulated with luciferase-encoding mRNA were tested in muscle cells (C2C12). 24 h after transfection, bioluminescence signals were measured. PEGylation significantly reduced the signal, but this reduction was less pronounced in the presence of PSarc. PSarc appears to reduce transfection efficiency to a much lesser extent than PEG.

[0273] Example 7 Further testing of pSarc particles Figure 11 shows the relationship between particle size in the formulation and polysarcosine chain length and molar ratio. With short polysarcosine chain lengths, particle formation is only possible at higher molar ratios, while with long polysarcosine chain lengths, particle formation is possible at a molar ratio of 1%. In general, particle size decreased with increasing polysarcosine chain length or molar ratio present in the formulation.

[0274] Figure 12 shows scattering curves (SAXS) from polysarcosylated lipid nanoparticles. LNPs were formulated using polysarcosine of varying chain lengths (11-34 units) and various molar ratios (2.5-10%). The scattering curves of the LNPs show that the LNPs are characterized by low internal organization, which decreases with increasing pSar chain length or molar ratio. The presence of two peaks indicates substantial contributions from individual lipid bilayer formation factors.

[0275] Figure 13 shows RNA accessibility assessed by Quant-It Ribogreen assay. PSarc-LNPs exhibit high RNA accessibility regardless of polysarcosine chain length and molar ratio.

[0276] Figure 14 shows the results of intravenous administration of various doses of EPO (erythropoietin)-encoding mRNA loaded into LNPs formulated with either PSarc or PEG-conjugated lipids. Plasma was collected 3, 6, 24, and 48 hours later, and EPO protein was quantified by ELISA. The results indicate that polysarcosine can directly replace other stealth moieties, such as PEG-conjugated lipids, without compromising efficacy. PSarc can even promote sustained protein secretion, which would be advantageous for protein replacement therapy.

[0277] Figure 15 shows the release of liver enzymes as an early marker of hepatotoxicity. Liver enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), and LDH, were measured in serum 6 and 24 hours after injection of LNPs formulated with increasing PSarc chain length. The data show that increasing the chain length of PSarc did not cause liver enzyme release (the horizontal line indicates the range of values ​​obtained in healthy mice), indicating that this biobased polymer is safe to use.

[0278] Figure 16 shows complement activation via the C3a complex of PEGylated and polysarcosylated LNPs at theoretical human plasma concentrations. Lipid formulations and controls (positive and negative) were incubated with human serum at a 20:80 ratio (sample:serum) for 1 hour at 37°C.

[0279] The data show reduced levels of C3a complexes with PSarc-LNP compared to PEG-LNP at high doses, i.e., five times the proposed human dose. At low doses, no C3a complex formation was observed compared to PBS (formulation buffer). These results suggest that PSarc may be less immunogenic than PEG-conjugated lipids.

[0280] Figure 17 shows cryo-TEM images of LNPs formulated with DODMA:cholesterol:DSPC:PSarc 23 at 40:45:10:5 mole %. The morphology of polysarcosinylated LNPs consists of small multilamellar vesicles in which mRNA may reside at the interface between closely apposed bilayers. Scale bar = 200 nm.

[0281] In summary, the results once again demonstrate that pSarc is a versatile platform to formulate small RNA nanoparticles, method-independent, for efficient RNA delivery.

Claims

1. 1. A composition comprising a plurality of RNA particles, each particle comprising: (i) RNA; and (ii) one or more components that associate with RNA to form RNA particles Including, A composition wherein polysarcosine is conjugated to at least one of said one or more components.

2. The composition of claim 1 , wherein the RNA particle is a non-viral RNA particle.

3. 3. The composition of claim 1 or 2, wherein the one or more components that associate with RNA to form particles comprise one or more polymers.

4. The composition of any one of claims 1 to 3, wherein the one or more polymers comprise a cationic polymer.

5. The composition of claim 4 wherein the cationic polymer is an amine-containing polymer.

6. 6. The composition of claim 3, wherein the one or more polymers comprise one or more polymers selected from the group consisting of poly-L-lysine, polyamidoamine, polyethyleneimine, chitosan, and poly(β-amino ester).

7. 3. The composition of claim 1, wherein the one or more components that associate with the RNA to form particles comprise one or more lipids or lipid-like substances.

8. 8. The composition of claim 7, wherein the one or more lipids or lipid-like substances comprise cationic or cationically ionizable lipids or lipid-like substances.

9. 9. The composition of claim 8, wherein the cationically ionizable lipid or lipid-like substance is positively charged only at acidic pH and does not remain cationic at physiological pH.

10. 10. The composition of claim 8 or 9, wherein the one or more lipids or lipid-like substances comprise one or more further lipids or lipid-like substances.

11. 11. The composition of claim 10, wherein the polysarcosine is conjugated to at least one of the one or more additional lipids or lipid-like substances.

12. 1. A composition comprising a plurality of RNA-lipid particles, each particle comprising: (a) RNA; (b) a cationic or cationically ionizable lipid or lipid-like substance; and (c) Polysarcosine-lipid conjugate or conjugate of polysarcosine and lipid-like substance A composition comprising:

13. Each particle is (d) non-cationic lipids or lipid-like substances The composition of claim 12 further comprising:

14. The composition according to any one of claims 1 to 13, wherein the particles do not comprise polyethylene glycol-lipid conjugates or conjugates of polyethylene glycol with lipid-like substances, preferably they do not comprise polyethylene glycol.

15. 15. The composition of any one of claims 12 to 14, wherein the cationic or cationically ionizable lipid or lipid-like material comprises from about 20 mol % to about 80 mol % of the total amount of lipid and lipid-like material present in the particle.

16. 16. The composition of any one of claims 13 to 15, wherein the non-cationic lipid or lipid-like material comprises from about 0 mol % to about 80 mol % of the total amount of lipids and lipid-like materials present in the particle.

17. 17. The composition of any one of claims 12 to 16, wherein the polysarcosine-lipid conjugate or polysarcosine and lipid-like substance conjugate comprises from about 0.25 mol % to about 50 mol % of the total amount of lipid and lipid-like substance present in the particle.

18. The composition of any one of claims 1 to 17, wherein the RNA is mRNA.

19. The cationic or cationically ionizable lipid or lipid-like substance is N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP ...

19. The composition of any one of claims 8 to 18, comprising ammonium chloride (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or a mixture thereof.

20. 20. The composition of any one of claims 13 to 19, wherein the non-cationic lipid or lipid-like substance comprises a phospholipid.

21. 21. The composition of any one of claims 13 to 20, wherein the non-cationic lipid or lipid-like substance comprises cholesterol or a cholesterol derivative.

22. 22. The composition of any one of claims 13 to 21, wherein the non-cationic lipid or lipid-like substance comprises a mixture of phospholipid and cholesterol or a cholesterol derivative.

23. 23. The composition of any one of claims 20 to 22, wherein the phospholipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), or a mixture thereof.

24. 24. The composition of any one of claims 13 to 23, wherein the non-cationic lipid or lipid-like substance comprises a mixture of DSPC and cholesterol.

25. 25. The composition of any one of claims 1 to 24, wherein the polysarcosine comprises from 2 to 200 sarcosine units.

26. The polysarcosine-lipid conjugate or the conjugate of polysarcosine and a lipid-like substance has the following general formula (I): 【Chemistry 1】 The composition of any one of claims 12 to 25, comprising:

27. The polysarcosine-lipid conjugate or the conjugate of polysarcosine and a lipid-like substance is represented by the following general formula (II): 【Chemistry 2】 (In the formula, R １ and R ２ one of which contains a hydrophobic group and the other is a functional group which may contain H, a hydrophilic group, or a targeting moiety. The composition of any one of claims 12 to 26, comprising:

28. The polysarcosine-lipid conjugate or the conjugate of polysarcosine and a lipid-like substance has the following general formula (III): 【Transformation 3】 where R is H, a hydrophilic group, or a functional group that may contain a targeting moiety. The composition of any one of claims 12 to 27, comprising:

29. 29. The composition of any one of claims 1 to 28, wherein the polysarcosine-lipid conjugate or a conjugate of polysarcosine with a lipid-like substance is a member selected from the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate, and mixtures thereof.

30. The composition of any one of claims 1 to 29, wherein the particles are nanoparticles.

31. The composition of any one of claims 1 to 30, wherein the particles comprise a nanostructured core.

32. The composition of any one of claims 1 to 31, wherein the particles have a size of about 30 nm to about 500 nm.

33. The composition of any one of claims 1 to 32, wherein the polysarcosine conjugate inhibits aggregation of the particles.

34. 34. A method for delivering RNA to cells of a subject, the method comprising administering to the subject a composition according to any one of claims 1 to 33.

35. 34. A method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject the composition of any one of claims 1 to 33, wherein the RNA encodes the therapeutic peptide or protein.

36. 34. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition of any one of claims 1 to 33, wherein delivery of said RNA to cells of said subject is beneficial in treating or preventing said disease or disorder.

37. 34. A method for treating or preventing a disease or disorder in a subject, comprising administering to the subject the composition of any one of claims 1 to 33, wherein the RNA encodes a therapeutic peptide or protein, wherein delivery of the therapeutic peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.

38. The method of any one of claims 34 to 37, wherein the subject is a mammal.

39. 39. The method of claim 38, wherein the mammal is a human.

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