Method for freezing and freeze-drying lipid nanoparticles (LNPs) and LNPs obtained thereby

Abstract

The present invention relates to a method for freezing or lyophilizing lipid nanoparticles (LNPs) comprising at least a nucleic acid and at least, as lipid components, a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or an ester thereof. The method comprises the steps of providing a liquid composition comprising said LNPs, spraying the composition in step a) under conditions suitable for obtaining droplets, and freezing the droplets obtained in step b) to obtain frozen LNPs. The method may also comprise the step of drying the obtained frozen LNPs to obtain lyophilized LNPs. The present invention also relates to freezing and lyophilizing LNPs.

Description

[Technical field]

[0001] The present invention relates to the field of pharmaceutical formulations and methods for their preparation. The present invention further relates to a method for freezing and lyophilizing lipid nanoparticles (LNPs). [Background technology]

[0002] Lipid nanoparticles (LNPs) have proven to be efficient in delivering various types of therapeutically active agents to cells (Thi et al., Vaccines, 2021, 9(4), 359). LNPs containing nucleic acids, such as LNP-mRNA, have attracted great interest and have recently demonstrated their efficacy and safety in the vaccine field, which may prove to be of dramatic importance in the management of the Covid-19 pandemic (Reichmuth et al., Therapeutic delivery, 2016, 7(5), 319-334; Khurana et al., Nano today, 2021, 38, 101142).

[0003] However, a significant drawback of the currently licensed mRNA-lipid nanoparticle (LNP) COVID-19 vaccines is that they must be stored at very low temperatures (Schoenmaker et al., International journal of pharmaceutics, 2021, 601, 120586). Indeed, the storage temperature requirements for both licensed vaccines are approximately -20°C for the Moderna vaccine and -80°C to -60°C for the BioNTech / Pfizer vaccine, respectively (Crommelin et al., J Pharm Sci. 2021;110(3):997-1001).

[0004] LNPs, being organized structures, can be adversely affected by the freezing process and such storage temperatures. Alterations in LNP structure can result in a reduction in the ability of LNPs to properly deliver their cargo to cells and achieve the intended therapeutic effect.

[0005] Moreover, such temperature conditions inevitably create multiple challenges in maintaining the cold chain throughout the manufacture, distribution, and packaging of the pharmaceutical composition. Any failure in cold chain management will inevitably result in product waste.

[0006] The pandemic situation requires rapid and global responses, but the temperature levels and control requirements needed to ensure LNP-based vaccine quality is maintained may slow the deployment of manufacturing facilities, global distribution organizations, and local supply and administration.

[0007] Freeze-drying (or lyophilization) is a common method for stabilizing unstable products in the pharmaceutical industry. Freeze-drying is a technique in which a product is solidified by freezing and the solvent containing the product, such as water, evaporates by sublimation when heated under low atmospheric pressure (or vacuum) or under a stream of cold, dry gas. Freeze-drying can be performed by conventional freeze-drying in vials or by spray-freeze-drying (SFD).

[0008] Methods and apparatus for spray freeze drying are disclosed in Adali et al., Processes. 2020;8(6), Wanning et al., Int J Pharm. 2015;488(1-2):136-153, WO 2009 / 109550 A1, WO 2013 / 050162 A1, WO 2013 / 050156 A1, or WO 2013 / 050159 A1.

[0009] Ali et al., (Int J Pharm. 2017;516(1-2):170-177) describe spray freeze drying of lipid nanoparticles.

[0010] Fukushige et al., (Int J Pharm. 2020;583:119338) describe spray freeze drying of liposomes containing protamine-siRNA complexes.

[0011] Zhao et al., Bioact Mater. 2020;5(2):358-363, reported a study on the stability of mRNA-containing lipid-like nanoparticles (LLNs) with different concentrations of cryoprotectants (sucrose, trehalose or mannitol) under the conditions of freezing or lyophilization processes.

[0012] However, freeze-drying of LNPs is also known to cause some stress leading to physical instability, such as aggregation, fusion, or content leakage of LNPs (Trenkenschuh et al., Eur J Pharm Biopharm. 2021 Aug; 165: 345-360). Ball et al. (Int J Nanomedicine, 2016, 12, 305-315) reported the effect of freeze-drying on LNP stability.

[0013] Therefore, there remains a need for methods of freezing or lyophilizing LNPs that have no or reduced adverse effects on LNP structure and / or stability.

[0014] There is a need for a method of freezing or lyophilizing LNPs that has no or reduced effect on LNP aggregation and size distribution of LNPs.

[0015] There is a need for a method of freezing or lyophilizing LNPs that has no or reduced effect on the encapsulation rate of the agent encapsulated in the LNP.

[0016] There is a need for LNP formulations that are suitable for being spray frozen or spray freeze dried with no or reduced adverse effects on LNP structure and / or stability.

[0017] There is a need for LNP formulations that are spray frozen or suitable to be spray frozen with no or reduced effect on LNP aggregation and size distribution of the LNPs.

[0018] There is a need for LNP formulations that can be spray frozen or are suitable to be spray frozen with no or reduced impact on the encapsulation rate of the agent encapsulated in the LNP.

[0019] There is a need for a method of lyophilization of LNPs and / or a formulation of LNPs suitable for lyophilization that allows for obtaining lyophilized LNPs that can be stored at 2-8°C with no or reduced effect on LNP aggregation and size distribution of the LNPs.

[0020] There is a need for a method of lyophilization of LNPs and / or LNP formulations suitable for lyophilization that allows for obtaining lyophilized LNPs that can be stored at 2-8° C. with no or reduced impact on the encapsulation rate of an agent to be encapsulated in the LNP, such as mRNA.

[0021] There is a need for a method for lyophilizing mRNA-containing LNPs and / or mRNA-containing LNP formulations suitable for providing lyophilized LNPs that can be stored at 2-8°C with no or reduced impact on the encapsulation rate of the encapsulated mRNA.

[0022] There is a need for methods of lyophilizing mRNA-containing LNPs and / or mRNA-containing LNP formulations suitable for being lyophilized that are suitable for providing lyophilized LNPs that can maintain or produce enhanced protein expression from the mRNA following administration.

[0023] The present invention aims to meet all or part of those needs. Summary of the Invention [Means for solving the problem]

[0024] According to one of its objects, the present invention provides a method for freezing lipid nanoparticles (LNPs), said LNPs comprising at least a cationic ionizable lipid, a neutral lipid and a steroid alcohol or an ester thereof as lipid components, said LNPs comprising at least one nucleic acid, said method comprising: a) providing a liquid composition comprising the LNPs; b) spraying the composition of step a) under conditions suitable to obtain droplets; c) freezing the droplets obtained in step b) to obtain frozen LNPs. The present invention relates to a method comprising the steps of:

[0025] As shown in the Examples section, the inventors have unexpectedly observed that freeze-drying of droplets containing LNPs, such as LNPs comprising at least cationic ionizable lipids, neutral lipids, and steroid alcohols or esters thereof, and optionally PEG-lipids, as lipid components, at a freezing temperature of, for example, -80°C, allowed for a reduction in aggregation of LNPs compared to freezing in vials. The size distribution of LNPs was maintained. Furthermore, for LNPs containing nucleic acids, such as mRNA, the nucleic acid encapsulation rate was observed to be higher for LNPs frozen in droplets compared to LNPs frozen in vials. The method as disclosed herein advantageously allows for maintaining the stability of frozen LNPs.

[0026] Reduction of LNP aggregation can be beneficial upon injection of formulations reconstituted from frozen LNPs, since aggregates that form large masses can cause adverse reactions such as pain. Furthermore, maintaining good nucleic acid encapsulation rates, such as mRNA, will improve corresponding protein expression and therefore therapeutic efficacy.

[0027] In some embodiments, the frozen LNPs may be obtained in frozen micropellets.

[0028] According to one of its objects, the present invention provides a method for freeze-drying lipid nanoparticles (LNPs), said method comprising the steps of: d) obtaining frozen LNPs according to the methods as disclosed herein; e) drying the frozen LNPs obtained in step d) under suitable conditions to obtain lyophilized LNPs. The present invention relates to a method comprising the steps of:

[0029] As shown in the Examples section, the inventors have unexpectedly observed that spray freeze-drying of LNPs, such as LNPs comprising at least a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or ester thereof, and optionally a PEG-lipid, as lipid components, makes it possible to prevent or reduce LNP aggregation. Furthermore, for LNPs containing nucleic acids, such as mRNA, the nucleic acid encapsulation rate was observed to be maintained over time compared to conventional freeze-drying processes (in vials). The methods disclosed herein advantageously make it possible to maintain the stability of freeze-dried LNPs.

[0030] Furthermore, as shown in the Examples section, the inventors unexpectedly observed that injection in mice of resuspended LNPs from spray freeze-dried LNPs containing nucleic acid, such as mRNA, encoding a protein, resulted in higher protein expression compared to resuspended LNPs from conventional freeze-dried LNPs.

[0031] Reduction of LNP aggregation can be beneficial upon injection of formulations reconstituted from lyophilized LNPs, since aggregates constituting large masses can cause adverse reactions such as pain. Furthermore, maintaining good nucleic acid encapsulation rates, such as mRNA, will improve corresponding protein expression and therefore therapeutic efficacy.

[0032] Furthermore, lyophilized LNPs obtained according to the spray freeze-drying method as disclosed herein dissolved more quickly in water for injection or in a buffer solution compared to lyophilized LNPs obtained according to conventional freeze-drying. Hence, the spray freeze-drying method as disclosed herein advantageously makes it possible to obtain lyophilized LNPs with reduced time for dissolution compared to conventional freeze-drying.

[0033] In some embodiments, the lyophilized LNPs may be obtained in lyophilized micropellets.

[0034] In some embodiments, the spraying in step b) may be performed with an electromagnetic droplet stream generator, a piezoelectric droplet stream generator, a hydraulic droplet aerosol generator, a compressed air nozzle, an ultrasonic atomizing nozzle, a thermal droplet stream generator, or an electrohydrodynamic droplet (EHD) generator. In some embodiments, the spraying may be performed with an electromagnetic droplet stream generator. In some embodiments, the spraying may be performed with a piezoelectric droplet stream generator.

[0035] The freezing of the droplets may be obtained by contacting the droplets with a freezing gas, a freezing liquid, or a freezing surface. In some embodiments, the freezing step c) may be performed by spraying the droplets into a cryogenic atmosphere, with pressurized carbon dioxide, into vapor above a cryogenic liquid, into a cryogenic liquid, or onto a cold solid surface. In the methods as disclosed herein, the freezing step may be performed by spraying the droplets into a cryogenic atmosphere.

[0036] The drying (or freeze-drying) in step e) can be carried out by rotary drum freeze-drying, air drying in a stream of cold air, vacuum chamber freeze-drying, or vacuum tunnel freeze-drying. In some embodiments, the drying in step e) can be carried out by vacuum chamber freeze-drying. In some embodiments, the drying in step e) can be carried out by rotary drum freeze-drying.

[0037] In some embodiments, the LNP comprises, in w / w % based on the total weight of the lipid component of the LNP: - about 20% to about 60%, or about 25% to about 60%, or about 30% to about 55%, or about 35% to about 55%, or about 35% to about 50%, or about 40% to about 50% of the ionizable cationic lipid, and / or - about 5% to about 50%, or about 5% to about 45%, about 9% to about 40%, or about 9% to about 30% of said neutral lipids, and / or - about 20% to about 55%, or about 20% to about 50%, or about 25% to about 45% of said steroid alcohol or ester thereof may include.

[0038] In some embodiments, - The ionizable cationic lipids are [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)yloxy)heptadecanedioate (L319); 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102);[(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315);[3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl] (Z)-Octadeca-9-enoate (DODAP);2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS);[(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC-Chol);Tetrakis(8-methylnonyl) 3,3',3'',3'''-(((methylazanediyl) Bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (306Oi10);Decyl (2-(dioctylammonio)ethyl)phosphate (9A1P9);Ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18);Bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate (BAME-O16B);1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200);3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);Hexa(octan-3-yl) 9,9',9'',9''',9'''',9''''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl)) tris(propane-3,1-diyl))tris(azanetriyl))hexanoate (FTT5);(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9''Z,9''Z,12Z,12'Z,12''Z,12''Z)-tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin);TT3;N; 1 ,N 3 ,N 5 -Tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide;N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5);Heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); [ka] and combinations thereof; and / or - the neutral lipid is selected from the group comprising: DSPC; DPPC; DMPC; POPC; DOPC; phosphatidylethanolamines, such as DOPE, DPPE, DMPE, DSPE, DLPE; sphingomyelin; ceramide, and combinations thereof; and / or - Sterols or their esters are cholesterol and its derivatives; ergosterol; desmosterol (3β-hydroxy-5,24-cholestadiene); stigmasterol (stigmasta-5,22-dien-3-ol); lanosterol (8,24-lanostadien-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); dihydrolanosterol (24,25-dihydrolanosterol); zymosterol (5α-cholesta-8,24-dien-3β-ol); la tosterol (5α-cholest-7-en-3β-ol); diosgenin ((3β,25R)-spirost-5-en-3-ol); sitosterol (22,23-dihydrostigmasterol); sitostanol; campesterol (campest-5-en-3β-ol); campestanol (5a-campestan-3b-ol); 24-methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol); cholesteryl margarate (cholest-5-en-3β-yl heptadecanoate); cholesteryl oleate; cholesteryl stearate; and combinations thereof.

[0039] The LNPs may further comprise at least one PEG-lipid as a lipid component.

[0040] LNPs may contain, by w / w %, about 0.5 to about 15%, or about 0.5% to about 10%, or about 0.8% to about 5%, or about 1% to about 3%, or about 1.5% to about 2% of the PEG-lipid, based on the total weight of the lipid component of the LNP.

[0041] The PEG-lipid may be selected from the group consisting of PEG-DAG; DMG-PEG-2000; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0042] In some embodiments, the LNP comprises, in w / w % based on the total weight of the lipid component of the LNP: - about 50% ionizable cationic lipid, about 10% neutral lipid, about 38.5% cholesterol, and about 1.5% PEG-lipid, or - about 46.3% ionizable cationic lipid, about 9.4% neutral lipid, about 42.7% cholesterol, and about 1.6% PEG-lipid, or - 47.4% ionizable cationic lipid, 10% neutral lipid, 40.9% cholesterol, and 1.7% PEG-lipid, or - about 40% ionizable cationic lipid, about 30% neutral lipid, about 28.5% cholesterol, and about 1.5% PEG-lipid, or - about 50% 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), about 10% DSPC, about 38.5% cholesterol, and about 1.5% DMG-PEG-2000, or - about 46.3% [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), about 9.4% DSPC, about 42.7% cholesterol, and about 1.6% 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), - about 47.4% [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), about 10% DSPC, about 40.9% cholesterol, and about 1.7% 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), or about 40% cKK-E10, about 30% DOPE, about 28.5% cholesterol, and about 1.5% DMG-PEG-2000, or - about 40% ML7 / OF-02, about 30% DOPE, about 28.5% cholesterol, and about 1.5% DMG-PEG-2000 may include.

[0043] In some embodiments, the nucleic acid contained in the LNP can be RNA. In some embodiments, the RNA can be mRNA.

[0044] The mRNA may comprise a 5'C cap structure, a 5'UTR sequence, an ORF sequence, a 3'UTR sequence, and a poly(A) tail.

[0045] The mRNA can be at least 30 nucleotides in length.

[0046] In some embodiments, the nucleic acid may be or encode a therapeutic agent, which may be a genome-editing polypeptide, a chemokine, a cytokine, a growth factor, an antibody, an enzyme, a structural protein, a blood protein, a hormone, a transcription factor, or an antigen.

[0047] In some embodiments, the nucleic acid may encode an antigen. The antigen may be selected from the group including bacterial antigens, viral antigens, and tumor antigens. The antigen may be an antigen from a strain of influenza A or influenza B virus, or from respiratory syncytial A or B virus, or from SARS-Cov2.

[0048] In some embodiments, the liquid composition comprising the LNPs further comprises at least one cryoprotectant. The cryoprotectant can be a polyol. The polyol can be selected from the group consisting of mannose, sucrose, lactose, trehalose, maltose, sorbitol, mannitol, glycerol, and inositol. The cryoprotectant can be trehalose.

[0049] In one of its aims, the invention relates to frozen LNPs obtained according to the methods as disclosed herein.

[0050] In one of its objects, the present invention relates to lyophilized LNPs obtained according to the methods as disclosed herein.

[0051] In one of its objects, the present invention relates to frozen LNPs comprising at least a nucleic acid and at least, as lipid components, a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or ester thereof, wherein the frozen LNPs are in a frozen micropellet. Such frozen LNPs may further comprise a PEG-lipid.

[0052] In one of its objects, the present invention relates to a lyophilized LNP comprising at least a nucleic acid and at least, as lipid components, a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or ester thereof, said lyophilized LNP being in a lyophilized micropellet. Such a lyophilized LNP may further comprise a PEG-lipid.

[0053] In one of its objects, the present invention relates to a method for the manufacture of a pharmaceutical agent, said method comprising at least the step of preparing frozen or lyophilized LNPs (wherein the LNPs comprise at least a nucleic acid) according to the method as disclosed herein. The method for the manufacture of the pharmaceutical agent further comprises the step of resuspending the lyophilized LNPs in a pharma- ceutically acceptable solvent or thawing the frozen LNPs.

[0054] In one of its aims, the present invention relates to lyophilized or frozen LNPs as disclosed herein and comprising at least a nucleic acid, for use as a medicament.

[0055] In one of its aims, the invention relates to frozen or lyophilized LNPs comprising at least one nucleic acid as disclosed herein and encoding an antigen from influenza A virus and / or influenza B virus, for use in preventing or treating influenza A and / or influenza B virus infection.

[0056] In one of its aims, the invention relates to frozen or lyophilized LNPs comprising at least one nucleic acid as disclosed herein and encoding an antigen from respiratory syncytial A virus and / or respiratory syncytial B virus, for use in preventing or treating respiratory syncytial A virus and / or respiratory syncytial B virus infection.

[0057] In one of its aims, the present invention relates to frozen or lyophilized LNPs comprising at least one nucleic acid encoding an antigen from influenza A virus and / or influenza B virus, as disclosed herein, for use as an immunogenic composition to combat influenza A and / or influenza B virus infection.

[0058] In one of its objects, the present invention relates to frozen or lyophilized LNPs as disclosed herein and comprising at least one nucleic acid encoding an antigen from respiratory syncytial A virus and / or respiratory syncytial B virus, for use as an immunogenic composition against respiratory syncytial A virus and / or respiratory syncytial B virus.

[0059] In one of its aims, the invention relates to the use of frozen or lyophilized LNPs as disclosed herein and comprising at least a nucleic acid, in the manufacture of a medicament.

[0060] In one of its objects, the present invention provides a method for the prevention and / or treatment of a disease in an individual in need thereof, the method comprising at least - resuspending lyophilized LNPs as disclosed herein in a pharma- ceutically acceptable solvent or thawing frozen LNPs to obtain thawed or resuspended LNPs, the frozen or lyophilized LNPs comprising at least a nucleic acid putatively active against said disease; - administering the thawed or resuspended LNPs to said individual. The present invention relates to a method comprising the steps of:

[0061] The invention is explained in more detail in the following description. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0062] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press can provide a general dictionary of many of the terms used in this disclosure to those skilled in the art. Although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, exemplary methods and materials are described below. In case of conflict, the present specification, including definitions, will control. In general, the nomenclature used in connection with and in the techniques of cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein is that well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0063] Units, prefixes, and symbols are shown in their Systeme International des Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise noted, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of this disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

[0064] Throughout this specification and the embodiments, the words "have" and "comprise", or variations such as "has", "having", "comprises", or "comprising", are understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of public documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0065] Whenever an embodiment is described herein with the language "comprising," it is understood that similar embodiments that are otherwise described with the terms "consisting of" and / or "consisting essentially of" are also provided.

[0066] The term "a" or "an" entity refers to one or more of that entity; for example, "a nucleotide sequence" is understood to refer to one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein.

[0067] Furthermore, "and / or", when used herein, should be considered as a specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended herein to include "A and B", "A or B", "A" (single), and "B" (single). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0068] The term "approximately" or "about" is used herein to mean approximately, roughly, around, or in the range of. When the term "about" is used in connection with a numerical range, it modifies that range by extending the boundaries above and below the indicated numerical values. In general, the term "about" can modify a numerical value above or below the stated value by a difference of, for example, 10 percent, above or below (higher or lower). In some embodiments, the term indicates a deviation from the stated numerical value by ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01%. In some embodiments, "about" indicates a deviation from the stated numerical value by ±10%. In some embodiments, "about" indicates a deviation from the stated numerical value by ±5%. In some embodiments, "about" indicates deviation from the stated numerical value by ±4%. In some embodiments, "about" indicates deviation from the stated numerical value by ±3%. In some embodiments, "about" indicates deviation from the stated numerical value by ±2%. In some embodiments, "about" indicates deviation from the stated numerical value by ±1%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.9%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.8%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.7%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.6%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.5%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.4%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.3%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.1%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.05%. In some embodiments, "about" indicates deviation from the stated numerical value by ±0.01%.

[0069] Depending on the context, the term "polynucleotide" or "nucleotide" encompasses a single nucleic acid as well as multiple nucleic acids. Within this disclosure, the terms "nucleic acid", "polynucleotide", and "oligonucleotide" are used interchangeably. They refer to a polymeric form of at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or their analogs. Nucleic acids can have any three-dimensional structure and can perform any function, known or unknown. In some embodiments, a polynucleotide is an isolated nucleic acid molecule or construct, such as messenger RNA (mRNA) or plasmid DNA (pDNA). In some embodiments, a polynucleotide comprises conventional phosphodiester bonds. In some embodiments, a polynucleotide comprises non-conventional bonds (e.g., amide bonds, such as those found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By "isolated" nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, that has been removed from its native environment. For example, a recombinant polynucleotide encoding a Factor VIII polypeptide contained in a vector is considered isolated for the purposes of this disclosure. Further examples of isolated polynucleotides include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of the polynucleotides of the present disclosure. Isolated polynucleotides or nucleic acids according to the present disclosure further include such molecules produced synthetically. In addition, the polynucleotide or nucleic acid can include regulatory elements such as a promoter, an enhancer, a ribosomal binding site, or a transcription termination signal.

[0070] "Nucleic acids", "polynucleotides", and "oligonucleotides" can be linear or circular. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, multiple loci (locuses) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, closed-end DNA (ceDNA), self-amplifying RNA (saRNA), stranded DNA (ssDNA), small interfering RNA (siRNA) and microRNA (miRNA), recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. Nucleic acids can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure can be imparted before or after assembly of the polymer. The sequence of the nucleic acid can be interrupted by non-nucleotide components. Nucleic acids can be further modified after polymerization, such as by conjugation with a labeling component. The term "complement of a nucleic acid" means a nucleic acid molecule that has a complementary base sequence and reverse orientation compared to a reference sequence such that it can hybridize with the reference sequence with complete fidelity. "Recombinant" as applied to nucleic acids means that the nucleic acid is the product of a variety of combinations of in vitro cloning, restriction and / or ligation steps, and other procedures that result in a construct that can potentially be expressed in a host cell.

[0071] As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as a plurality of "polypeptides" and refers to a molecule consisting of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids and does not refer to a specific length of the product. Thus, peptide, dipeptide, tripeptide, oligopeptide, "protein", "amino acid chain", or any other term used to refer to a chain or chains of two or more amino acids are included within the definition of "polypeptide", and the term "polypeptide" can be used in place of or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, proteolytic cleavage, or modification with non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced from recombinant technology, but is not necessarily translated from a specified nucleic acid sequence. It can be generated in any manner, including chemical synthesis.

[0072] An "isolated" polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural environment. No particular level of purification is required. For example, an isolated polypeptide can be easily removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in a host cell are considered isolated for the purposes of this disclosure, as are native or recombinant polypeptides that have been separated, fractionated, or partially or substantially purified by any suitable technique.

[0073] "Administer" or "administering", as used herein, refers to delivering a composition, e.g., a chimeric protein, described herein, to a subject. The composition, e.g., a chimeric protein, can be administered to a subject using methods known in the art. In particular, the composition can be administered intravenously, subcutaneously, intramuscularly, intradermally, or via any mucosal surface, e.g., orally, sublingually, lingually, nasally, rectally, vaginally, or via a pulmonary route. In some embodiments, administration is intravenous. In some embodiments, administration is subcutaneous. In some embodiments, administration is self-administration. In some embodiments, a parent administers the chimeric protein to a child. In some embodiments, the chimeric protein is administered to a subject by a medical professional, such as a physician, doctor, or nurse.

[0074] The term "antigen" includes any molecule, e.g., a peptide or protein, that elicits an immune response and / or contains at least one epitope against which an immune response is directed. For example, an antigen is a molecule that induces an immune response, e.g., specific for an antigen or a cell expressing the antigen, optionally after processing. After processing, the antigen can be expressed by an MHC molecule and reacts specifically with T lymphocytes (T cells). Thus, the antigen or a fragment thereof should be capable of being recognized by a T cell receptor and, in the presence of an appropriate co-stimulatory signal, be capable of inducing clonal expansion of T cells bearing T cell receptors that specifically recognize the antigen or fragment, resulting in an immune response against the antigen or a cell expressing the antigen.

[0075] According to the present disclosure, any suitable antigen that is a candidate for an immune response is contemplated. The antigen may correspond to or be derived from a naturally occurring antigen. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents, and the pathogen or antigen may also be a tumor antigen.

[0076] The expression "ionizable cationic lipid" refers to a lipid that contains one or more groups that can be protonated at physiological pH, but can be deprotonated at a pH above 8, 9, 10, 11, or 12. The ionizable cationic group can contain one or more protonatable amines that can form cationic groups at physiological pH. Cationic ionizable lipid compounds can also be further classified as C 6 ~C 24 They may contain one or more lipid components, such as two or more fatty acids with alkyl or alkenyl carbon groups. These compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0077] The expression "lipid component" refers to a group of organic compounds, including but not limited to esters of fatty acids, which are generally characterized by being poorly soluble in water but soluble in many organic solvents. Lipid is a general term that includes fats, fatty oils, essential oils, waxes, phospholipids, glycolipids, sulfolipids, aminolipids, chromolipids (lipochromes), and fatty acids. Within this disclosure, "lipid" includes neutral lipids, steroid alcohols or esters thereof, and PEGylated lipids.

[0078] The expression "lipid nanoparticle" (LNP) refers to a particle having at least one dimension on the order of nanometers (e.g., 10 to 800 nm, e.g., about 80 to about 200 nm, as measured by Nanoparticle Tracking Analysis (NTA)), which may be formulated with at least one lipid component as disclosed herein. In some embodiments, the LNP is included in a formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid, to a target site of interest (e.g., a cell, tissue, organ, tumor, etc.). Such lipid nanoparticles typically include a lipid component as disclosed herein.

[0079] The expression "frozen lipid nanoparticles" refers to a liquid composition of LNP that has been subjected to temperature conditions that cause its solvent components to solidify.

[0080] The expression "lyophilized lipid nanoparticles" refers to a liquid composition of LNPs that has been frozen and then subjected to drying conditions that cause the solvent components to evaporate.

[0081] The terms "micropellets" or "microbeads" are used interchangeably and are intended to refer to particles in the micrometer range that tend to be generally spherical / circular in shape. Frozen or lyophilized micropellets may have a mean value for diameter selected from the range of about 200 to about 1500 micrometers (μm), with a narrow size distribution of about ±50 μm around the various, preferably selected, values.

[0082] The expression "cationic ionizable lipid" refers to a lipid that contains one or more groups that can be protonated at physiological pH, but can be deprotonated at a pH above 8, 9, 10, 11, or 12. The ionizable cationic group can contain one or more protonatable amines that can form cationic groups at physiological pH. The cationic ionizable lipid compound can also further include C 6 ~C 24 They may contain one or more lipid components, such as two or more fatty acids with alkyl or alkenyl carbon groups. These compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0083] The expression "neutral lipid" refers to any lipid component that is either not ionizable or is a neutral zwitterionic compound at a selected pH, for example at physiological pH. Such lipids include, but are not limited to, neutral sphingolipids such as phosphatidylcholine, phosphatidylethanolamine, sphingomyelin (SM), or ceramide. Neutral lipids may be synthetic or naturally derived.

[0084] The phrases "PEG-lipid" or "PEGylated lipid" are used interchangeably and are intended to refer to a molecule that includes both a lipid moiety and a polyethylene glycol moiety. PEG-lipids are known in the art and include 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG), and the like.

[0085] Within this disclosure, the term "spherulization" refers to the process of solidifying small droplets of a liquid material falling into or against an upward flow of a cooling material, such as a cooling gas or refrigerant.

[0086] Within this disclosure, the term "significantly" as used in reference to a change is intended to mean that the change observed is notable and / or that it has statistical significance.

[0087] The expression "spray freeze drying" is intended to refer to a process in which a feed solution is broken down into droplets, the droplets are then frozen by contact with a low temperature medium, and the frozen droplets are then transferred to a freeze dryer to sublimate the water and obtain a dry powder, which may be dry micropellets.

[0088] Within this disclosure, the term "substantially" as used in connection with a feature of the disclosure is intended to specify a set of embodiments associated with this feature that largely, but not entirely, resemble this feature.

[0089] The expressions "steroid alcohol" or "sterol" are used interchangeably and are intended to refer to a group of lipids consisting of a sterane core with a hydroxyl moiety. Examples of steroid alcohols may include cholesterol, campesterol, sitosterol, stigmasterol, and ergosterol. An ester of a steroid alcohol or of a sterol refers to an ester of a carboxylic acid with a hydroxyl group of the steroid alcohol. Suitable carboxylic acids include, in addition to the carboxyl moiety, a saturated or unsaturated, linear or branched alkyl group. In some embodiments, the alkyl group is C 1 ~C 20It may be an alkyl group. In other embodiments, the carboxylic acid may be a fatty acid.

[0090] It is appreciated that certain features of the invention which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the invention which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention. All publications mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0092] The lists of sources, ingredients and components as set forth herein below are recited as including combinations and mixtures thereof that are contemplated and are within the scope of the present invention.

[0093] It is to be understood that every highest numerical limit given throughout this specification includes every lower numerical limit, as if such lower numerical limit were expressly written herein. Every lowest numerical limit given throughout this specification will include every upper numerical limit, as if such upper numerical limit were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical range were expressly written herein.

[0094] All lists of items, such as, for example, lists of ingredients, are intended to be and should be construed as Markush groups, and therefore all lists can be read and construed as a list of items "and combinations and mixtures thereof."

[0095] Trade names for components, including various raw materials, utilized in this disclosure may be mentioned herein. The inventors do not intend to be limited herein by materials under any particular trade name. Materials equivalent to those referenced by trade name (e.g., those obtained from different sources under different names or reference numbers) may be substituted and utilized in the description herein.

[0096] Spraying, freezing and drying The present invention relates to a method for spray freezing or spray freeze drying of lipid nanoparticles (LNPs). LNPs may contain at least a nucleic acid and at least, as lipid components, cationic ionizable lipids, neutral lipids, and steroid alcohols or esters thereof, wherein said method comprises the steps of:

[0097] The spray freezing method as disclosed herein comprises: a) providing a liquid composition comprising the LNPs; b) spraying the composition of step a) under conditions suitable to obtain droplets; c) freezing the droplets obtained in step b) to obtain frozen LNPs. may include.

[0098] Frozen LNPs may be obtained in frozen micropellets.

[0099] The spray freeze drying process as disclosed herein comprises: a) providing a liquid composition comprising the LNPs; b) spraying the composition of step a) under conditions suitable to obtain droplets; c) freezing the droplets obtained in step b) to obtain frozen LNPs; d) drying the frozen LNPs obtained in step c) under suitable conditions to obtain lyophilized LNPs. Includes.

[0100] The lyophilized LNPs may be obtained in the form of lyophilized micropellets, which may be obtained by pelleting and drying.

[0101] Freeze-drying, also known as lyophilization, is a process commonly used to dry labile products such as pharmaceuticals, biological materials, e.g., proteins, enzymes, microorganisms, and generally any heat- and / or hydrolysis-sensitive substances.

[0102] spray Spraying can be performed with an electromagnetic droplet stream generator, a piezoelectric droplet stream generator, a hydraulic droplet aerosol generator, a compressed air nozzle, an ultrasonic atomizing nozzle, a thermal droplet stream generator, or an electrohydrodynamic droplet (EHD) generator.

[0103] In some embodiments, the spraying may be performed with a piezoelectric droplet stream generator.

[0104] Electromagnetic or Piezoelectric Droplet Stream Generation Spheronization, also known as laminar jet breakup technique, allows the generation and solidification of calibrated monodisperse droplets of a liquid. Spheronization can be performed by electromagnetic or piezoelectric droplet stream generation and freezing of the droplets.

[0105] The main principle of electromagnetic or piezoelectric droplet stream generators is based on Rayleigh breakup of a liquid jet emerging from a capillary orifice using mechanical vibrations obtained by electromagnets or piezoelectric ceramic oscillators. Lord Rayleigh proposed a model for Newtonian fluids (Rayleigh L, Proc. London Math. Soc. 1978.10,4-13). For aqueous solutions emerging from small circular orifices at low pressure, the formation of small droplets down to a few micrometers in diameter is limited by surface tension and adhesion of the liquid to the nozzle walls. By piezoelectric excitation, the breakup length of the liquid jet can be shortened, and the signal type (e.g. sinusoidal, rectangular), frequency and amplitude affect both the average size and uniformity of the droplets.

[0106] The optimal wavelength for the fastest growing disturbance and jet breakup is

number

[0107] λ opt If is the optimal wavelength for jet collapse, then d j is the diameter of the jet, η is the viscosity of the fluid, ρ is the density of the fluid, and σ is the surface tension of the fluid.

[0108] The diameter of the droplets formed, d, is

number

[0109] The frequency f that must be applied to the fluid to achieve the desired result is determined by the jet velocity (and therefore the fluid flow rate) u. j and

number

[0110] Therefore, optimum conditions can be calculated knowing the process parameters and fluid properties. Depending on the nozzle diameter, the rheology and surface tension of the fluid, a range of frequencies and jet velocities exists to form uniform droplets (Meesters G., 1992. Mechanisms of droplet formation. Delft University Press, Delft, NL).

[0111] The suitable operating frequency can also be determined experimentally by visual assessment of the stability of the droplet formation. Standard spheronizing devices are equipped with a stroboscope light to observe the droplet formation: for a given product and given operating conditions, the frequency can be adjusted manually until a stable and motionless droplet chain is observed with this stroboscope light.

[0112] Spheronization makes it possible to generate monodisperse calibration droplets with diameters ranging, for example, from about 200 μm to about 1500 μm, or from about 300 μm to about 600 μm, with a narrow size distribution of ±25%, or ±10%.

[0113] The electromagnetic or piezoelectric droplet stream generator is a nozzle. Suitable nozzles and multi-headed nozzle systems have been developed for aseptic pelleting applications, such as those disclosed in Brandenberger et al., J. Biotechnol., 1998, 63, 73-80, or in WO 2016 / 012414 A1.

[0114] The nozzle may have an exit opening of about 250 μm to about 400 μm in diameter, and may be about 300 μm.

[0115] The prilling process can accommodate viscous liquids. The acceptable viscosity can be around 300 mPa.s.

[0116] The temperature of the supply vessel containing the liquid composition including the LNPs and of the nozzle must be controlled to avoid component or solvent crystallization prior to droplet formation. Those skilled in the formulation art know how to adjust the concentrations of different components in a stabilized formulation, taking into account possible interactions between excipients, to avoid uncontrolled crystallization and viscosity above a given limit.

[0117] Examples of nozzles for piezoelectric droplet stream generators are disclosed in Wanning at al. (Int J Pharm. 2015;488(1-2):136-153), the contents of which are incorporated by reference. Examples of nozzles for electromagnetic droplet stream generators are disclosed in WO 2016 / 012414 A1), the contents of which are incorporated by reference.

[0118] Other spraying methods Other methods of spraying, such as spraying with a hydraulic droplet aerosol generator, compressed air nozzle, ultrasonic atomizing nozzle, thermal droplet stream generator, or electrohydrodynamic droplet (EHD) generator, may be suitable for the methods disclosed herein. Such methods are disclosed in Adali et al., Processes. 2020; 8(6) and Wanning et al., Int J Pharm. 2015; 488(1-2): 136-153,), the contents of which are incorporated by reference.

[0119] In hydraulic nozzles, the spray is generated by forcing the fluid through an orifice. The required energy is provided by converting pressure into kinetic energy, and the droplet size varies as a function of the feed rate and viscosity, as well as the spray pressure.

[0120] In compressed air nozzles, the atomization energy is provided by a compressed gas stream (usually air) that interacts with the liquid and creates a shear field that results in a wide range of droplet sizes. These devices are also known as multi-fluid nozzles. For example, in a two-fluid nozzle, a liquid feed and a compressed gas are fed into the nozzle to create a shear field.

[0121] In ultrasonic nozzles, liquids are broken up into droplets when a high frequency electrical signal is converted into mechanical energy and transferred into the liquid. Typically, ultrasonic nozzles consist of two piezoelectric transducers that receive electrical input placed between two electrodes. This causes simultaneous mechanical expansion and contraction of the transducers, resulting in ultrasonic vibrations that are sent to the nozzle tip to atomize the feed. The droplet size depends on the operating frequency and the feed flow rate. The use of such devices allows for a high degree of control over particle size and provides a narrow droplet size distribution.

[0122] frozen Various techniques known in the art may be used to freeze the droplets, which is defined as the rendering of most or all of the solute phase to a hardened state by solidification of the solvent and removal of heat.

[0123] Freezing of the droplets can be obtained by contacting the droplets with a freezing gas, a freezing liquid, or a freezing plane. The freezing step can be carried out by spraying the droplets into a cryogenic atmosphere, with pressurized carbon dioxide, into vapor above a cryogenic liquid, into a cryogenic liquid, or onto a cold solid surface.

[0124] In some embodiments, the freezing step may be performed by spraying droplets into a cryogenic atmosphere.

[0125] Freezing in a cryogenic atmosphere In the methods disclosed herein, the freezing step may be carried out by spraying droplets into a cryogenic atmosphere.

[0126] In atmospheric freezing, the heat sink is gaseous, at ambient pressure with a nearly uniform temperature low enough to induce the formation of ice nuclei in the solution. Frictional stresses are generally low, and the size and roughly spherical shape of the droplets do not change as they solidify. Under these conditions, the cooling rate is limited by the rate of energy transfer across the droplet surface, which depends on the sliding velocity.

[0127] In some embodiments, freezing can be accomplished by allowing droplets to free fall into a cryogenic chamber where the temperature is maintained by a freezing medium in the range of about -100°C to about -160°C, e.g., about -110°C or -105°C. The freezing medium can be introduced into the freezing chamber by direct injection / spraying of freezing gas over all of the droplet passages. Alternatively, the freezing medium can be introduced as a flow of freezing gas countercurrent to the flow of droplets, or by maintaining a static freezing gas in the chamber at a pressure above atmospheric pressure (e.g., the overpressure can be 1.1 to 1.5 atmospheres). In some embodiments, the freezing medium is introduced into the freezing chamber by direct injection / spraying of freezing gas over all of the droplet passages.

[0128] In some embodiments, a spatial temperature profile can be configured in the cryogenic chamber, for example, the spatial temperature profile can be configured and maintained in the chamber such that a temperature range of -40°C to -60°C, e.g., -50°C to -60°C, is maintained in the top area of ​​the tower, and a temperature range of -150°C to -192°C, e.g., -150°C to -160°C, is maintained in the bottom area of ​​the tower.

[0129] The temperature in the cryogenic chamber can optionally be maintained or varied / cycled throughout from about -50°C to -190°C.

[0130] The droplets freeze during their free fall in the cryogenic chamber to form calibrated frozen particles. The minimum drop height for freezing the droplets into frozen droplets (i.e., ice crystal formation that solidifies the pellet) may depend on the size of the droplets to be frozen, the method used to freeze the droplets (i.e., direct injection / spray of freezing gas in the chamber, or countercurrent flow of freezing gas, or static freezing gas at pressure above atmospheric pressure).

[0131] The frozen droplets may have diameters ranging from about 200 μm to about 1500 μm, or from about 200 μm to about 800 μm, or from about 300 μm to about 600 μm, or at about 500 μm. The size of the particles may be measured with a particle size analyzer or an imaging particle size analyzer.

[0132] The frozen droplets obtained by such a method may be referred to as frozen micropellets.

[0133] To form freezing of droplets into circular micropellets in the size / diameter range of 100-800 μm, the approximate height of the cryogenic chamber can be 1-2 m (meters), whereas to form freezing of droplets into pellets in the size range down to 1500 μm (micrometers), the cryogenic chamber can be about 2-3 m, where the diameter of the cryogenic chamber can be about 50-150 cm for a height of 200-300 cm.

[0134] The freezing medium may have a temperature below -110°C.

[0135] The freezing medium is liquid or vapor nitrogen, liquid or vapor CO 2 , or liquid air and / or its vapor.

[0136] Other freezing methods Other methods of freezing may be suitable for the methods disclosed herein, such as by spraying droplets with compressed carbon dioxide into vapor above the cryogenic liquid, into the cryogenic liquid, or onto a cold solid surface.

[0137] Such methods are disclosed in Adali et al., Processes. 2020;8(6) and Wanning et al., Int J Pharm. 2015;488(1-2):136-153), the contents of which are incorporated by reference.

[0138] In spray drying with compressed carbon dioxide, the temperature of the aqueous spray liquor can also be reduced below the freezing point by Joule-Thompson cooling of the co-expanded carbon dioxide.

[0139] Freezing by spraying into vapor above a cryogenic liquid (SFV) can be performed by spraying droplets into a gaseous freezing medium above the freezing point of the liquid freezing medium and precipitating through the vapor layer onto the surface of the liquid freezing medium. Supercooling and freezing can occur in the floating gas and vapor or upon contact with the condensing refrigerant. The velocity of the small droplets drops rapidly due to atmospheric braking so that frictional stresses remain low and freezing conditions are similar to those during atmospheric freezing.

[0140] Spray freezing into liquid (SFL) can make it possible to achieve high freezing rates, since the solution to be frozen is injected directly into the cryogenic liquid at a high flow rate. Under these conditions, frictional stresses are high and the fluid dynamic conditions are not well defined. The particles formed are frequently small fragments. Alternatively, the solution can be dripped or sprayed at a lower rate from a nozzle into the liquid freezing medium. If the density of the solution to be frozen is lower than that of the cryogenic fluid, it can also be injected from the bottom of the freezing vessel, and the frozen particles are skimmed off the surface.

[0141] High cooling rates and uniform particulate matter can also be produced by spraying or dripping liquid onto a cold solid surface. Thus, freezing rates are accelerated compared to volatile cryogenic liquids because the Leidenfrost effect, in which a vapor layer limits the transfer of thermal energy to a heat sink, is avoided.

[0142] Similarly, in those methods, the freezing medium may have a temperature below -110° C. The freezing medium may be liquid nitrogen, liquid CO 2 Or it may be liquid air and / or its vapor.

[0143] After the freezing step, the frozen droplets are then collected and transferred to a spray dryer. Alternatively, they can be stored until they are freeze-dried. Such storage can be performed on pre-cooled trays in conditions that keep the frozen droplets below the glass transition temperature Tg' of their frozen concentrated phase to avoid any melting or agglomeration. For example, for Tg' values ​​in the range of -10°C to -45°C, the storage temperature should be at least -50°C or lower. The frozen LNPs disclosed herein can be stored at -70°C.

[0144] The frozen droplets are stored under suitable conditions to avoid any melting or agglomeration of the frozen droplets.

[0145] Drying (or freeze-drying) Drying (or freeze-drying) can be carried out by rotary drum vacuum freeze-drying, atmospheric drying in a current of cold air, vacuum chamber freeze-drying, or vacuum tunnel freeze-drying.

[0146] "Vacuum" is understood to mean low pressure or under pressure, below atmospheric pressure, as known to those skilled in the art. Vacuum conditions as used herein can mean pressures as low as 10 millibar, or 1 millibar, or 500 microbar, or 1 microbar. It should be pointed out that freeze-drying can generally be performed at different pressure regimes, for example, under atmospheric pressure.

[0147] In some embodiments, drying may be performed by rotary drum freeze drying, hi some embodiments, drying may be performed by freeze drying in a vacuum chamber.

[0148] The resulting frozen micropellets can be dried by subjecting them to sublimation conditions, which involve low heating temperatures and under vacuum to evaporate the solvent, i.e., from the frozen state to a gaseous state.

[0149] Drying by rotary drum vacuum freeze drying In some embodiments, the drying step can be carried out in a vacuum rotary drum dryer.Suitable vacuum rotary dryers are disclosed in WO 2013 / 050157 A1, WO 2013 / 050158 A1, WO 2013 / 050159 A1, Adali et al., Processes. 2020; 8(6), or Wanning et al., Int J Pharm. 2015; 488(1-2): 136-153), the contents of which are incorporated by reference.

[0150] A suitable rotary dryer may be placed in the vacuum chamber.

[0151] The drum of the tumble dryer may include a temperature controllable interior wall surface, for example with a double wall. Additionally or alternatively, other means for heating the micropellets during the freeze-drying process may be provided, for example microwave or infrared heating.

[0152] The temperature of the inner wall of the dryer can be controlled within the range of -60°C to +125°C.

[0153] During freeze-drying, the drum of the rotary dryer may be rotated to maximize the inner wall surface available for evaporation of the solvent. Typical rotation speeds during the freeze-drying process may include, but are not limited to, about 0.5 to 10 revolutions per minute (rpm), such as 1 to 8 rpm.

[0154] The lyophilized droplets may have a diameter in the range of about 200 μm to about 1500 μm, or about 200 μm to about 800 μm, or about 300 μm to about 600 μm, or about 500 μm.

[0155] The lyophilized droplets obtained by such a method may be referred to as lyophilized micropellets.

[0156] Other drying methods Other drying methods, such as air drying in a stream of cold air, vacuum chamber freeze drying, or vacuum tunnel freeze drying, may be suitable for the methods disclosed herein, which are suitable alternatives to drying in a rotating drum.

[0157] Such methods are disclosed in Adali et al., Processes. 2020;8(6) and Wanning et al., Int J Pharm. 2015;488(1-2):136-153), the contents of which are incorporated by reference.

[0158] In atmospheric freeze drying, cold and dry air or gas at atmospheric pressure passes over the frozen droplets and removes the solvent from their surface. With particulate drying material, the process gas can either rise through the bed of frozen droplets or, if the frozen droplets are on a permeable support, pass them in a downward flow. With a sufficiently fast upstream flow rate, a fluidized or entrained bed is formed, depending on the inertia of the frozen droplets, the geometry of the chamber and the gas dynamics. In downstream drying, the gas mainly seeps into the gaps between the frozen droplets.

[0159] In vacuum chamber freeze drying and vacuum tunnel freeze drying, the frozen droplets are placed in a sub-atmospheric pressure (vacuum) environment and low temperature. Application of a vacuum during the drying process allows for the removal of the solvent. The first drying removes the water from the formulation by sublimation of ice, then the second drying removes the non-frozen bound water.

[0160] In vacuum chamber freeze drying, frozen droplets are placed on trays and dried in layers, with the sublimation rate being determined by a bimodal particle size distribution, where the short-range diffusion of free solvent molecules is determined by the internal pores and their connectivity. The sublimation energy is provided by conduction from a heating plate below and / or by radiation from a radiant shelf. In some embodiments, drying is performed in a vacuum chamber freeze drying.

[0161] Vacuum tunnel freeze drying allows for shorter drying times and increased energy efficiency of freeze drying by reducing the thickness of the layer of frozen droplets and providing the sublimation energy by infrared or microwave radiation. The frozen droplets are deposited on trays which pass through an entrance lock into the vacuum tunnel and are unloaded through an exit lock in a quasi-continuous process.

[0162] For example, once a freeze dryer (vacuum chamber freeze drying and vacuum tunnel freeze drying) is loaded with trays, a vacuum is pulled in the chamber or tunnel to initiate conventional freeze drying (sublimation of ice) of the frozen droplets.

[0163] The following lyophilization parameters are examples of those used for formulations with Tg in the range of about -30°C to about -45°C: Initial drying: shelf temperature equal to -35°C, pressure equal to 50 μbar for 10 h. Second drying: shelf temperature equal to 20° C., pressure equal to 50 μbar for 3 h.

[0164] The lyophilization cycle must be designed to obtain a residual moisture content lower than 3%, preferentially. However, the moisture content can be optimized at higher values, on a case-by-case basis, if the stability of the material being lyophilized requires it.

[0165] The lyophilized droplets, or micropellets, can then be collected together. Storage conditions are suitable for dry, friable and hygroscopic particles. The bulk of the lyophilized droplets can then be filled into vials using dry powder filling techniques known in the art.

[0166] Lipid nanoparticles and manufacturing process Suitable lipid components for LNPs as disclosed herein can include as lipid components at least: one ionizable lipid, one neutral lipid, and one steroid alcohol or ester thereof.

[0167] Optionally, at least one PEG-lipid may also be implemented.

[0168] Ionizable Cationic Lipids LNPs as disclosed herein can include at least one ionizable cationic lipid.

[0169] The ionizable cationic lipids may contain one or more groups that can be protonated at physiological pH, but deprotonated at a pH above 8, 9, 10, 11, or 12. The ionizable cationic groups may contain one or more protonatable amines that can form cationic groups at physiological pH. Cationic ionizable lipids may also be further classified as C 6 ~C 24 They may contain one or more lipid components, such as two or more fatty acids with alkyl or alkenyl carbon groups. These compounds may be dendrimers, dendrons, polymers, or combinations thereof.

[0170] In some embodiments, the ionizable cationic lipid may include at least one protonizable amine moiety.

[0171] Suitable ionizable cationic lipids may be those from U.S. Pat. No. 9,512,073 or in U.S. Pat. No. 10,201,618, the contents of which are incorporated herein by reference.

[0172] Suitable ionizable cationic lipids are [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (Dlin-KC2-DMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (Dlin-DMA); di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102);[(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315);[3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl] (Z)-Octadeca-9-enoate (DODAP);2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS);[(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]30-henanthren-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC-Chol);Tetrakis(8-methylnonyl) 3,3',3'',3'''-(((methylazanediyl) Bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (306Oi10);Decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9);Ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(30-henanthrene-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18);Bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl) dipropionate (BAME-O16B);1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl)bis(dodecan-2-ol) (C12-200);3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12);Hexa(octan-3-yl)9,9',9'',9''',9'''',9''''''-(((benzene-1,3,5-tricarbonyl)iris(azanediyl)) Tris(propane-3,1-diyl))tris(azanetriyl))hexanoate (FTT5);(((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-tetrakis(octadeca-9,12-dienoate) (OF-Deg-Lin);TT3;N; 1 ,N 3 ,N 5 -Tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide;N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamide)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5);30-henanthrene-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); [ka] and combinations thereof.

[0173] The ionizable cationic lipid OF-02 is disclosed, inter alia, in PCT Application WO 2022 / 099003, the contents of which are incorporated by reference.

[0174] LNPs may contain, by w / w %, about 20% to about 60%, or about 25% to about 60%, or about 30% to about 55%, or about 40% to about 55%, or about 40% to about 50% ionizable cationic lipids based on the total weight of the lipid component of the LNP.

[0175] In one embodiment, a suitable ionizable cationic lipid can be (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, or Dlin-MC3-DMA (also known as MC3).

[0176] In one embodiment, a suitable ionizable cationic lipid may be 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), for example, present in an amount of about 50% w / w based on the total weight of the lipid components of the LNP.

[0177] In one embodiment, a suitable ionizable cationic lipid can be, for example, [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), present in an amount of about 46.3% or about 47.4% w / w based on the total weight of the lipid components of the LNP.

[0178] In one embodiment, a suitable ionizable cationic lipid may be cKK-E10, for example, present in an amount of about 40% w / w based on the total weight of the lipid component of the LNP.

[0179] In one embodiment, a suitable ionizable cationic lipid may be OF-02, for example, present in an amount of about 40% w / w based on the total weight of the lipid components of the LNP.

[0180] neutral lipid LNP as disclosed herein can comprise at least one neutral lipid.The presence of neutral lipid can improve the structural stability of lipid nanoparticles.Neutral lipid can be appropriately selected in consideration of the delivery efficiency of nucleic acid.

[0181] Neutral lipids differ from the ionizable cationic lipids disclosed herein in that neutral lipids are either not ionizable or are zwitterionic compounds that are neutral at a selected pH.

[0182] Suitable neutral lipids useful for LNPs may be selected from the group consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and ceramide.

[0183] Phosphatidylcholine and phosphatidylethanolamine are zwitterionic lipids. Sphingomyelin and ceramide are not ionizable lipids.

[0184] The phosphatidylcholine can be DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine).

[0185] The phosphatidylethanolamine can be DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-s / i-glycero-3-phosphoethanolamine), DLPE (1,2-dilauroyl-SM-glycero-3-phosphoethanolamine), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, or 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE).

[0186] The neutral lipid may be selected from the group consisting of phosphatidylcholines, such as DSPC, DPPC, DMPC, POPC, DOPC; phosphatidylethanolamines, such as DOPE, DPPE, DMPE, DSPE, DLPE; sphingomyelin; ceramide, and combinations thereof.

[0187] In one embodiment, the neutral lipid can be DSPC, DOPC, or DOPE, such as DSPC or DOPE.

[0188] In one embodiment, the neutral lipid can be DSPC.

[0189] LNPs may contain, in w / w %, about 5 to about 50%, or about 5 to about 45%, about 9 to about 40%, or about 9 to about 30% neutral lipids based on the total weight of the lipid component of the LNP.

[0190] In one embodiment, a suitable neutral lipid may be DSP, for example present in an amount of about 10% w / w based on the total weight of the lipid component of the LNP.

[0191] In one embodiment, a suitable neutral lipid may be DOPE, for example present in an amount of about 30% w / w based on the total weight of the lipid component of the LNP.

[0192] The neutral lipids may be present in the LNPs as disclosed herein in a molar ratio of ionizable cationic lipid:neutral lipid ranging from about 70:1 to about 1:2, such as from about 30:1 to about 1:1, such as from about 15:1 to about 2:1, such as from about 10:1 to about 4:1, more such as about 5:1.

[0193] Steroid alcohols or their esters LNPs as disclosed herein can include at least one steroid alcohol (i.e., sterol) or ester thereof. The presence of a sterol or an ester of a sterol can improve the structural stability of the lipid nanoparticle.

[0194] Sterols, i.e. steroid alcohols, are cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmasta-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5α-cholesta-8,24-dien-3β-ol), lathosterol (5α-cholesta-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, kaolin ... campestrol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol); BHEM-cholesterol (2-(((((3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]34-henanthrene-3-yl)oxy)carbonyl)amino)-N,N-bis(2-hydroxyethyl)-N-methylethane-1-aminium bromide); and combinations thereof.

[0195] An ester of a steroid alcohol, or sterol, refers to an ester of a carboxylic acid with a hydroxyl group of a steroid alcohol. Suitable carboxylic acids include, in addition to the carboxyl moiety, a saturated or unsaturated, linear or branched alkyl group. In some embodiments, the alkyl group is a C 1 ~C 20 Saturated or unsaturated, linear or branched alkyl groups, e.g. C 2 ~C 18 , for example C 4 ~C 16 , for example C 8 ~C 12The alkyl group may be saturated or unsaturated, linear or branched. In other embodiments, the carboxylic acid may be a fatty acid. For example, the fatty acid may be caprylic acid, caproic acid, lauric acid, stearic acid, margaric acid, oleic acid, linoleic acid, or arachidic acid.

[0196] In one embodiment, the ester of a sterol may be a cholesteryl ester.

[0197] The ester of a sterol or steroid alcohol may be selected from the group consisting of cholesteryl margarate (cholest-5-en-3β-yl heptadecanoate), cholesteryl oleate, cholesteryl stearate; and combinations thereof.

[0198] Sterols, i.e. steroid alcohols or their esters, are cholesterol or its derivatives, ergosterol, desmosterol (3β-hydroxy-5,24-cholestadiene), stigmasterol (stigmasta-5,22-dien-3-ol), lanosterol (8,24-lanostadien-3b-ol), 7-dehydrocholesterol (Δ5,7-cholesterol), dihydrolanosterol (24,25-dihydrolanosterol), zymosterol (5α-cholesta-8,24-dien-3β- ol), lathosterol (5α-cholest-7-en-3β-ol), diosgenin ((3β,25R)-spirost-5-en-3-ol), sitosterol (22,23-dihydrostigmasterol), sitostanol, campesterol (campest-5-en-3β-ol), campestanol (5a-campestan-3b-ol), 24-methylenecholesterol (5,24(28)-cholestadiene-24-methylene-3β-ol), cholesteryl margarate (cholest-5-en-3β-yl heptadecanoate), cholesteryl oleate, cholesteryl stearate, and combinations thereof.

[0199] Alternatively, the sterol may be a cholesterol derivative, such as oxidized cholesterol.

[0200] The oxidized cholesterol suitable for the present disclosure can be 25-hydroxycholesterol, 27-hydroxycholesterol, 20α-hydroxycholesterol, 6-keto-5α-hydroxycholesterol, 7-keto-cholesterol, 7β,25-hydroxycholesterol, 7β-hydroxycholesterol; and combinations thereof. For example, the oxidized cholesterol can be 25-hydroxycholesterol and 20α-hydroxycholesterol, for example, it can be 20α-hydroxycholesterol.

[0201] In one embodiment, the sterol or steroid alcohol, or ester thereof, can be cholesterol, a cholesteryl ester, or a cholesterol derivative, such as oxidized cholesterol. In one embodiment, the sterol or steroid alcohol can be cholesterol or a cholesteryl ester, such as cholesterol.

[0202] In one embodiment, the sterol or steroid alcohol can be cholesterol.

[0203] LNPs may contain, by w / w %, about 20% to about 55%, or about 20% to about 50%, or about 25% to about 45% of said steroid alcohol or ester thereof, based on the total weight of the lipid component of said LNP.

[0204] In one embodiment, the sterol or steroid alcohol can be cholesterol, and can be present, for example, in an amount of about 28.5%, or about 38.5%, or about 40.9%, or about 42.7% w / w based on the total weight of the lipid component of the LNP.

[0205] The sterol, i.e., steroid alcohol, or ester thereof, may be present in the LNP in a molar ratio of ionizable cationic lipid:steroid alcohol, or ester thereof, ranging from about 4:1 to about 1:2, such as from about 3.5:1 to about 1:1.8, such as from about 2:1 to about 1:1.5, such as from about 1.5:1 to about 1:1.2, e.g., from about 1.3:1 to about 1:1.3.

[0206] PEG-lipids The lipid nanoparticles may comprise PEG-lipids (or PEGylated lipids).

[0207] Possible PEG-modified lipids include C 6 ~C 20 The addition of PEG-modified lipids to LNP compositions can prevent complex aggregation and can also provide a means to enhance circulation lifetime and increase delivery of the composition or lipid nanoparticles to target cells.

[0208] Suitable PEGylated lipids include, for example, PEGylated diacylglycerols (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); PEGylated phosphatidylethanolamine (PEG-PE); PEG succinate diacylglycerols, such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-λ-0-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG). (PEG-S-DAG); PEGylated ceramide (PEG-cer); PEG dialkoxypropyl carbamates such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecyloxy)propyl)carbamate; 2,3-di(tetradecyloxy)propyl-N-(co-methoxy(polyethoxy)ethyl)carbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0209] In one embodiment, a suitable PEGylated lipid may be selected from the group consisting of EG-DAG; PEG-DMG; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

[0210] For example, the PEGylated lipid can be PEG-DMG PEG-PE, or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0211] In one embodiment, the PEG-lipid can be a PEG-PE, such as PEG-2000-PE.

[0212] In one embodiment, the PEG-lipid can be PEG-DMG, such as DMG-PEG-2000.

[0213] In one embodiment, the PEG-lipid can be 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0214] The LNP may contain PEG-lipid in a molar amount ranging from about 1 to about 15%, such as from about 1% to about 10%, such as from about 1% to about 5%, such as from about 1% to about 3.5%, based on the total molar amount of the lipid component of the LNP.

[0215] In one embodiment, the PEG-lipid can be DMG-PEG-2000, present in an amount of about 1.5%, for example w / w% based on the total weight of the lipid component of the LNP.

[0216] In one embodiment, the PEG-lipid can be 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), present in an amount of about 1.6% or about 1.7%, e.g., w / w % based on the total weight of the lipid component of the LNP.

[0217] The PEG-lipid and ionizable cationic lipid may be present in the LNP in a molar ratio of ionizable cationic lipid to PEG-lipid of about 70:1 to about 4:1, such as about 40:1 to about 10:1, such as about 35:1 to about 15:1, e.g., about 33:1 or about 14:1.

[0218] In one embodiment, an LNP may contain ionizable cationic lipids, neutral lipids, steroid alcohols or esters thereof, and PEG-lipids in molar amounts, w / w%, based on the total weight of the lipid components of the LNP, of about 20% to about 60% ionizable cationic lipids, about 5% to about 50% neutral lipids, 20% to about 55% steroid alcohols or esters thereof, and about 0.5% to about 15% PEG-lipids.

[0219] In one embodiment, an LNP may comprise ionizable cationic lipid, neutral lipid, steroid alcohol or ester thereof, and PEG-lipid in molar amounts, w / w%, based on the total weight of the lipid components of the LNP, of about 35% to about 55% ionizable cationic lipid, about 5% to about 35% neutral lipid, about 25% to about 45% steroid alcohol or ester thereof, and about 1.0% to about 2.5% PEG-lipid.

[0220] In one embodiment, an LNP may contain ionizable cationic lipids, neutral lipids, steroid alcohols or esters thereof, and PEG-lipids in molar amounts, w / w%, based on the total weight of the lipid components of the LNP, of about 40% to about 50% ionizable cationic lipids, about 9% to about 30% neutral lipids, about 28% to about 45% steroid alcohols or esters thereof, and about 1.5% to about 2.5% PEG-lipids.

[0221] In one embodiment, the molar ratio of the ionizable cationic lipid and the neutral lipid, steroid alcohol or ester thereof, and PEG-lipid can be about 35 / 16 / 46.5 / 1.5, about 50 / 10 / 38.5 / 1.5, about 57.2 / 7.1 / 34.3 / 1.4, about 40 / 15 / 40 / 5, about 50 / 10 / 35 / 4.5 / 0.5, about 50 / 10 / 35 / 5, about 40 / 10 / 40 / 10, about 35 / 15 / 40 / 10, or about 52 / 13 / 30 / 5.

[0222] In one embodiment, the molar ratio of the ionizable cationic lipid and the neutral lipid, the steroid alcohol or ester thereof, and the PEG-lipid can be about 35 / 16 / 46.5 / 1.5 or about 50 / 10 / 38.5 / 1.5.

[0223] In one embodiment, the LNP may comprise an ionizable cationic lipid, a neutral lipid, a steroid alcohol or ester thereof, and a PEG-lipid in molar amounts, w / w% based on the total weight of the lipid components of the LNP, of about 50% ionizable cationic lipid, about 10% neutral lipid, about 38.5% steroid alcohol or ester thereof, and about 1.5% PEG-lipid.

[0224] In one embodiment, the LNP may comprise an ionizable cationic lipid, a neutral lipid, a steroid alcohol or ester thereof, and a PEG-lipid in molar amounts, w / w% based on the total weight of the lipid components of the LNP, of about 46.3% ionizable cationic lipid, about 9.4% neutral lipid, about 42.7% steroid alcohol or ester thereof, and about 1.6% PEG-lipid.

[0225] In one embodiment, the LNP may comprise an ionizable cationic lipid, a neutral lipid, a steroid alcohol or ester thereof, and a PEG-lipid in molar amounts, w / w% based on the total weight of the lipid components of the LNP, of about 40% ionizable cationic lipid, about 30% neutral lipid, about 28.5% steroid alcohol or ester thereof, and about 1.5% PEG-lipid.

[0226] In one embodiment, the ionizable cationic lipid is Dlin-MC3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), or [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315); [ka] It could be.

[0227] In one embodiment, the neutral lipid can be DSPC or DOPE.

[0228] In one embodiment, the steroid alcohol can be cholesterol.

[0229] In one embodiment, the PEG-lipid can be PEG-PE (PEG-2000-PE) or PEG-DMG (PEG-2000-DMG).

[0230] In one embodiment, the ionizable cationic lipid can be Dlin-MC3-DMA, the neutral lipid can be DSPC, the steroid alcohol can be cholesterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0231] In one embodiment, the LNP may comprise, in w / w % based on the total weight of the lipid component of the LNP, 50% Dlin-MC3-DMA, 10% DSPC, 38.5% cholesterol, and 1.5% PEG-DMG (PEG-2000-DMG).

[0232] In one embodiment, the ionizable cationic lipid can be 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), the neutral lipid can be DSPC, the steroid alcohol can be cholesterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0233] In one embodiment, the LNP may comprise, in w / w % based on the total weight of the lipid component of the LNP, 50% 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG-2000.

[0234] In one embodiment, the ionizable cationic lipid can be [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), the neutral lipid can be DSPC, the steroid alcohol can be cholesterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0235] In one embodiment, the LNP may comprise, in w / w % based on the total weight of the lipid component of the LNP, 46.3% [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 9.4% DSPC, 42.7% cholesterol, and 1.6% 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0236] In one embodiment, the LNP may comprise, in w / w % based on the total weight of the lipid component of the LNP, 47.4% [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 10% DSPC, 40.9% cholesterol, and 1.7% 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159).

[0237] In one embodiment, the ionizable cationic lipid can be cKK-E10, the neutral lipid can be DOPE, the steroid alcohol can be cholesterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0238] In one embodiment, an LNP may comprise, in w / w % based on the total weight of the lipid component of the LNP, 40% cKK-E10, 30% DOPE, 28.5% cholesterol, and 1.5% DMG-PEG-2000.

[0239] In one embodiment, the ionizable cationic lipid can be ML7 / OF-02, the neutral lipid can be DOPE, the steroid alcohol can be cholesterol, and the PEG-lipid can be PEG-DMG (DMG-PEG-2000).

[0240] In one embodiment, an LNP may comprise, in w / w % based on the total weight of the lipid component of the LNP, 40% ML7 / OF-02, 30% DOPE, 28.5% cholesterol, and 1.5% DMG-PEG-2000.

[0241] Lipid Nanoparticles (LNPs) Lipid nanoparticles (LNPs) can be characterized by several parameters well known in the art, such as the mean diameter size, the mode diameter size, the polydispersity index (PI), which reflects the homogeneity of the size distribution of the LNPs, the pKa, and / or the zeta potential, which reflects the spherical surface charge of the LNPs.

[0242] The LNPs can be used to encapsulate at least one therapeutic agent. The encapsulation rate and the total content of such agents can also be used as parameters to characterize the LNPs.

[0243] Mode diameter size, mean diameter size and PI can be measured by a Malvern Nanoparticles Tracking Analysis (NTA) NS300 equipped with a 96 well plate autosampler or dynamic light scattering (DLS). pKa can be measured using the fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonic acid (TNS). Zeta potential can be measured using electrophoretic mobility or dynamic electrophoretic mobility measurements, for example on a Nicomp 380 ZLS system or a Malvern nanoZS.

[0244] The "mean diameter size" of an LNP may be measured by nanoparticle tracking analysis (NTA) and represents the average diameter of all particles analyzed in a sample. The "modal diameter size" represents the size of the most frequent particle population in a sample. In other words, it is the size of the most frequent particle. With respect to the size distribution profile of a sample, the modal diameter size represents the highest point of the peak seen in the distribution.

[0245] NTA utilizes the properties of both Brownian motion and light scattering to obtain the particle size distribution of a sample in liquid suspension. A laser beam is passed through a sample chamber and the particles in suspension in the beam path scattering light so that the particles can be viewed by a magnifying microscope to which a camera is attached. The particle motion is recorded frame by frame. The center of each observed particle is identified and tracked to obtain the average distance traveled in the x and y planes. This value helps to determine the particle diffusion coefficient (Dt), from which, knowing the sample temperature T and the solvent viscosity η, the spherical equivalent hydrodynamic diameter d of the particle can be calculated using the Stokes-Einstein equation:

number

[0246] LNPs can have a diameter that makes them suitable for systemic administration, e.g., parenteral administration, or for intramuscular, intradermal, or subcutaneous administration. Typically, lipid nanoparticles have an average diameter size of less than 600 nanometers (nm), e.g., less than 400 nm.

[0247] In one embodiment, the LNPs have a mean diameter size of less than 200 nm. Such a size is advantageously compatible with sterile filtration and is most suitable for transport through lymphatic vessels after intramuscular or subcutaneous administration. This size is also suitable for intravenous administration, since injection of larger particles can induce capillary thrombosis.

[0248] In some embodiments, the LNPs may have an average diameter size ranging from about 20 nm to about 300 nm, e.g., from about 25 nm to about 250 nm, e.g., from about 30 nm to about 200 nm, from about 40 nm to about 180 nm, from about 60 nm to about 170 nm, from about 70 to about 160 nm, and from about 80 to about 150 nm. In one embodiment, the LNPs may have an average diameter size ranging from about 85 to about 140 nm, as measured by NTA. In the liquid composition (step a) of the methods disclosed herein), the LNPs may have a mode diameter size of about 70 nm to about 250 nm, or about 80 nm to about 200 nm, or about 85 to about 140 nm, or about 90 to about 120 nm, as measured by NTA.

[0249] The NTA technique requires that the sample be liquid, therefore, for measurement of the mean diameter size of LNPs after a freezing or lyophilization step, the resulting frozen or lyophilized LNPs are thawed or resuspended in a solution such as an aqueous buffer or water for injection (WFI).

[0250] The freezing method and spray drying as disclosed herein may have zero or reduced impact on the mode size of LNPs that contain at least a cationic ionizable lipid, a neutral lipid, and a steroid alcohol, or an ester thereof, as lipid components. Also, the freezing method and spray drying as disclosed herein may have zero or reduced impact on the mode size of LNPs that contain at least a cationic ionizable lipid, a neutral lipid, a steroid alcohol, or an ester thereof, and a PEG-lipid, as lipid components. The stability of the LNPs is therefore unaffected or minimally affected by the methods as disclosed herein.

[0251] The stability of the LNP can be assessed by measuring the values ​​of several parameters that characterize the LNP before and after application of a method as disclosed herein, or after application of a method as disclosed herein and over a period of time.

[0252] Parameters of the LNP that can be measured to assess the stability of the LNP can be, for example, the mode diameter size as measured by NTA or the encapsulation rate of an agent, such as mRNA, that is optionally loaded into the LNP.

[0253] For example, the modal diameter size of LNPs can be measured for LNPs in a liquid composition prior to freezing, and for frozen LNPs. As noted above, frozen LNPs must be thawed or resuspended in solution, e.g., in an aqueous buffer or water for injection, before being subjected to measurement by NTA.

[0254] In some embodiments, the LNPs at the freezing step of the methods disclosed herein may have a mode diameter size as measured by NTA that is about 45% or less, or about 35% or less, or about 30% or less, or about 25% or less, or about 20% or less, or about 15% or less, or about 10% or less, or about 8% or less, or about 5% or less of the mode diameter size of the LNPs in the liquid composition (prior to freezing).

[0255] A change in the mode diameter size of the LNPs before and after the freezing step of less than about 45%, or less than about 35%, or less than about 30%, or less than about 25%, or less than about 20%, or less than about 15%, or less than about 10%, or less than about 8%, or less than about 5% may indicate a low or reduced aggregation effect during the freezing process.

[0256] The modal diameter size of the LNPs can also be measured on lyophilized LNPs that have been resuspended in solution, for example, in an aqueous buffer, or in water for injection, before being subjected to measurement by NTA.

[0257] In some embodiments, the LNPs in the lyophilization step of the methods disclosed herein may have an average diameter size as measured by NTA that is about 15% or less, or about 13% or less, or about 11% or less, or about 10% or less, or about 5% or less of the average diameter size of the LNPs in the liquid composition.

[0258] A change in the mode diameter size of the LNPs before and after the spray freeze drying step of less than about 15%, or less than about 13%, or less than about 11%, or less than about 10%, or less than about 5% may indicate low or reduced aggregation effects during the spray freeze drying process.

[0259] The effect of the spray freeze-drying method as disclosed herein can also be evaluated over time on freeze-dried LNP.In such a case, freeze-dried LNP can be stored at a constant temperature, for example, +5°C, and from time to time, for example, every 1 month, or 2 months, or 3 months, or 4 months, a sample of freeze-dried LNP can be resuspended in aqueous buffer or water for injection for mode size diameter measurement by NTA.The obtained measurement value can then be compared with a reference value, which can be the mode size diameter of LNP in the liquid composition before freeze-drying or immediately after freeze-drying, i.e., at T0.

[0260] The lipid nanoparticles may contain or encapsulate at least one therapeutic agent. Such agents may be encapsulated in and / or adsorbed onto the outer surface of the LNP. Such agents may have some positive or negative charge.

[0261] In the case of LNPs containing negatively charged therapeutic agents, such as nucleic acids, lipid nanoparticles can be formed, for example, during preparation, by adjusting the positive (+) to negative (-) charge ratio of the ionizable cationic lipids (cationic charge) to the negatively charged agent (e.g., anionic charge from phosphate in the case of nucleic acids). The charges of the ionizable cationic lipids and of the negatively charged agent are at a selected pH, such as physiological pH, which is about 6.5 to about 7.5.

[0262] The + / - charge ratio of ionizable cationic lipid to negatively charged agent in an LNP can be calculated by the following equation: (+ / - charge ratio) = [(amount of cationic lipid (mol)) * (total number of positive charges in cationic lipid)]: [(amount of negatively charged agent (mol)) * (total number of negative charges in negatively charged agent)].

[0263] The amount of negatively charged agent and the amount of ionizable cationic lipid can be easily determined by one skilled in the art taking into consideration the loading amount during preparation of LNP.

[0264] According to certain embodiments, the ratio of positive to negative charges in LNPs suitable for the present disclosure is such that they can have a global negative charge or a neutral or near-neutral global charge.

[0265] In one embodiment, the charge ratio of positive to negative charges in the LNP ranges from about 4:1 to about 15:1, such as from about 5:1 to about 12:1, such as from about 6:1 to about 9:1, such as from about 6:1 to about 8:1.

[0266] In one embodiment, the charge ratio of positive to negative charges in the LNP is about 6:1.

[0267] The present disclosure relates to frozen LNPs obtained according to the methods as disclosed herein.

[0268] The present disclosure relates to frozen LNPs comprising at least a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or ester thereof as lipid components, wherein the frozen LNPs are in a frozen micropellet. Such frozen LNPs may further comprise a PEG-lipid. Such frozen LNPs may further comprise a nucleic acid.

[0269] In one of its aims, the present invention relates to lyophilized LNPs obtained according to the methods as disclosed herein.

[0270] The present disclosure relates to lyophilized LNPs comprising at least a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or ester thereof as lipid components, said lyophilized LNPs being in a lyophilized micropellet. Such lyophilized LNPs may further comprise a PEG-lipid. Such lyophilized LNPs may further comprise a nucleic acid.

[0271] Lipid nanoparticle manufacturing process Methods for producing LNPs are known in the art.

[0272] In one embodiment, the LNPs containing a therapeutic agent comprise at least: i) solubilizing the lipid components of the LNPs in a water-miscible organic solvent; ii) mixing the organic solvent obtained in step a) with an aqueous solvent containing the nucleic acid; iii) obtaining said LNPs in an aqueous solvent; The method may be obtained by a method comprising the steps of:

[0273] In one embodiment, a method for producing LNPs includes at least: i) solubilizing in a water-miscible organic solvent at least one ionizable cationic lipid, at least a neutral lipid, at least one steroid alcohol or ester thereof, and at least one PEG-lipid; ii) mixing the organic solvent obtained in step a) with an aqueous solvent containing the nucleic acid; iii) obtaining lipid nanoparticles containing nucleic acids in an aqueous solvent; may include.

[0274] Useful water-miscible organic solvents can be any water-miscible organic solvents that can solubilize lipid compounds as disclosed herein and any other lipids added. Examples of suitable organic solvents can include ethanol or methanol, 1-propanol, isopropanol, t-butanol, THF, DMSO, acetone, acetonitrile, diglyme, DMF, 1,4-dioxane, ethylene glycol, glycerin, hexamethylphosphoramide, hexamethylphosphorous triamide. In one embodiment, the organic solvent can be ethanol and isopropanol.

[0275] Useful aqueous solvents in step ii) include aqueous buffers.

[0276] Examples of suitable aqueous buffers may include acidic buffers such as citrate buffer, sodium acetate buffer, succinate buffer, borate buffer or phosphate buffer. For example, the aqueous buffer solvent may be a citrate buffer or an acetate buffer.

[0277] The pH of the aqueous medium may range from about 3.5 to about 7.0, such as from about 4.0 to about 6.5, such as from about 4.5 to about 6.0, for example about 5.5. In one embodiment, the pH may be about 4.0.

[0278] In step ii), the organic solvent and the aqueous solvent may be mixed in a ratio organic solvent:aqueous solvent ranging from about 1:1 to about 1:6, in one embodiment, the ratio may range from about 1:2 to about 1:4, such as a ratio of about 1:3.

[0279] According to one embodiment, the organic solvent and the aqueous solvent may be mixed in step b) at a flow rate ranging from about 0.01 ml / min to about 12 ml / min. In some embodiments, the flow rate may range from about 0.02 ml / min to about 10 ml / min, from about 0.5 ml / min to about 8 ml / min, from about 1 ml / min to about 6 ml / min, or may be about 4 ml / min.

[0280] The mixing step can be carried out by any method known in the art. For example, both solvents can be mixed in a T-tube or Y-connector. Alternatively, mixing can be carried out by laminar mixing in a microfluidic micromixer as described by Belliveau et al. (Mol Ther Nucleic Acids. 2012;1(8):e37).

[0281] As indicated, the aqueous solvent in step b) comprises a nucleic acid. Suitable nucleic acids may be, for example, as detailed below.

[0282] The method may further include, if necessary, raising the pH from acidic to neutral.

[0283] In a further embodiment, the method may comprise step iv) of increasing the pH of the aqueous medium containing the LNPs obtained in step iii) to a pH in the range of about 5.5 to about 7.5, such as about 6.0 to about 7.5.

[0284] The step of increasing the pH can be performed by any method known in the art, for example, the change in pH can be performed by a dialysis or diafiltration step.

[0285] Furthermore, if necessary, the osmolality can be adjusted to reach a final osmolality close to 290 mOsmol / kg for infusing an isotonic solution into the body.

[0286] Additionally, the method for preparing LNPs may include any further steps suitable for harvesting, purifying, concentrating and / or sterilizing the lipid nanoparticles for further formulation as a pharmaceutical composition, e.g., as an immunogenic composition.

[0287] Freezing and lyophilization formulation of LNPs Prior to being frozen or lyophilized, a composition comprising LNPs can be mixed with an excipient, which can be a buffer, a bulking agent, a pH stabilizer, a pH adjusting agent, a heat stabilizer, a cryoprotectant, a lyoprotectant, or an antioxidant.

[0288] Compositions containing LNPs that are intended to be frozen or lyophilized can be isotonic (iso-osmotic).

[0289] Such excipients, such as cryoprotectants, can help stabilize the LNPs during the freezing or spray-freeze drying process.

[0290] Cryoprotectants and Lyoprotectants A liquid composition containing the LNPs may be supplemented with at least one cryoprotectant.

[0291] Cryoprotectants or lyoprotectants may be selected from disaccharides (such as lactose, trehalose, sucrose, maltose, and mannose), sorbitol, amino acids, peptides, polymers and proteins such as albumin (bovine serum albumin, human serum albumin) or gelatin.

[0292] In some embodiments, the cryoprotectant can be a carbohydrate. In one embodiment, the cryoprotectant is a carbohydrate selected from monosaccharides, disaccharides, trisaccharides, sugar alcohols, oligosaccharides or their corresponding sugar alcohols, and linear polyhydric alcohols. Exemplary disaccharide cryoprotectants include sucrose, trehalose, lactose, maltose, and the like.

[0293] In one embodiment, the cryoprotectant may be a polyol, hi some embodiments, the cryoprotectant may be selected from the group consisting of mannose, sucrose, lactose, trehalose, maltose, sorbitol, mannitol, glycerol, inositol, glucose, fructose, arginine, glycerin, dextran, and mixtures thereof.

[0294] In some embodiments, the cryoprotectant can be trehalose, hi some embodiments, the trehalose can be trehalose dihydrate.

[0295] In some embodiments, the cryoprotectant may be dextran.

[0296] In some embodiments, the cryoprotectant is a mixture of trehalose and dextran.

[0297] In one embodiment, trehalose is present in a concentration of about 5 to about 50 weight / volume percent (w / v%), or about 8 (w / v)% to about 40 (w / v)%, or about 10 (w / v)% to about 25 (w / v)%, or about 15 (w / v)% to about 20 (w / v)%, based on the total volume of the composition.

[0298] In one embodiment, trehalose is present at a concentration of about 16.25% (w / v).

[0299] Dextran having a molecular weight of 1,000 to 100,000 Da is preferred, and more preferably 1,000 to 10,000 Da may be used. Dextran may be used together with other cryoprotectants.

[0300] In one embodiment, dextran is present at a concentration of about 5 to about 25 weight / volume percent (w / v%), or about 8 (w / v)% to about 20 (w / v)%, or about 15 (w / v)% to about 18 (w / v)%.

[0301] In one embodiment, dextran is present at a concentration of about 16.25% (w / v).

[0302] In one embodiment, trehalose and dextran are present in equal amounts weight / volume percentages based on the total volume of the composition.

[0303] In one embodiment, the cryoprotectant is a mixture of trehalose at a concentration of about 16.25% (w / v) and dextran at a concentration of about 16.25% (w / v) based on the total volume of the composition.

[0304] In some embodiments, a composition containing an LNP according to the present disclosure may include a cryoprotectant consisting essentially of trehalose and dextran.

[0305] In some embodiments, the present disclosure relates to lyophilized micropellets comprising at least a nucleic acid and LNPs comprising, as lipid components, at least a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or ester thereof and a cryoprotectant as set forth above. In some embodiments, the cryoprotectant is a mixture of trehalose and dextran.

[0306] buffer solution The buffer may be selected from phosphate buffered saline, citrate buffer, Tris buffer, amino acid based buffer (such as histidine buffer, glycine buffer, etc.), sodium dihydrogen orthophosphate, disodium hydrogen orthophosphate, potassium dihydrogen orthophosphate, dipotassium hydrogen orthophosphate, TES, MOPS, PIPES, cocodylate, SSC, MES and HEPES.

[0307] In some embodiments, the buffer may be a Tris buffer.

[0308] In some embodiments, the buffer may be phosphate buffered saline.

[0309] In some embodiments, the formulation does not include a buffer.

[0310] In some embodiments, the formulation does not include Tris buffer.

[0311] Other excipients Compositions that contain LNPs and are intended to be frozen and lyophilized in the manner disclosed herein may further include additional excipients such as heat stabilizers, antioxidants, or bulking agents.

[0312] The heat stabilizer may be selected from mannitol, polymers (such as dextran, polyethylene glycol, polyvinylpyrrolidone, etc.) and proteins.

[0313] The antioxidant may be selected from vitamin A (retinol), vitamin C (ascorbic acid) and vitamin E (including tocotrienols and tocopherols).

[0314] Bulking agents may be selected from mannitol, polymers (such as dextran, polyethylene glycol, and polyvinylpyrrolidone), disaccharides (such as lactose, trehalose, sucrose, maltose, and mannose), sorbitol, and proteins such as albumin and gelatin.

[0315] nucleic acid Nucleic acids suitable for the present disclosure can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.

[0316] Nucleic acids may be single-stranded or double-stranded molecules, and may be covalently closed to form a line or a circle. Nucleic acids may be double-stranded RNA (dsRNA); single-stranded RNA (ssRNA); double-stranded DNA (dsDNA); single-stranded DNA (ssDNA); and combinations thereof.

[0317] Nucleic acid-containing LNPs can be used for the introduction of nucleic acids into cells, i.e., for transfection of cells, for example, for recombinant protein expression, for gene replacement, to reduce or increase expression of a host protein.

[0318] The nucleic acid may be of eukaryotic or prokaryotic origin, such as human, animal, plant, bacterial, yeast or viral origin. It may be obtained by any technique known to those skilled in the art, such as by screening libraries, by chemical synthesis or alternatively by mixed methods involving chemical or enzymatic modification of sequences obtained by screening libraries. It may be chemically modified.

[0319] The nucleic acid may be contained in a vector. Vectors are known to those skilled in the art and may include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC). Vectors include expression vectors as well as cloning vectors. Expression vectors include plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vivo expression system. Cloning vectors are generally used to manipulate and amplify certain desired DNA fragments and may lack functional sequences required for expression of the desired DNA fragment.

[0320] The nucleic acid can be messenger RNA (mRNA); microRNA (miRNA); short (or small) interfering RNA (siRNA); small hairpin RNA (shRNA); long non-coding RNA (lncRNA); asymmetric interfering RNA (aiRNA); self-amplifying RNA (saRNA); small nuclear RNA (snRNA); small nucleolar RNA (snoRNA); guide RNA (gRNA); antisense oligonucleotide (ASO); plasmid DNA (pDNA); closed loop DNA (ceDNA), and combinations thereof.

[0321] In some embodiments, the nucleic acid may be RNA.

[0322] In some embodiments, the nucleic acid can be messenger RNA (mRNA); microRNA (miRNA); short (or small) interfering RNA (siRNA); small hairpin RNA (shRNA); long non-coding RNA (lncRNA); asymmetric interfering RNA (aiRNA); self-amplifying RNA (saRNA); guide RNA (gRNA); and combinations thereof.

[0323] In some embodiments, the LNP may contain as a nucleic acid an mRNA encoding a CRISPR protein, such as CRISPR / Cas9, and a guide RNA (gRNA). The gRNA may be provided as a rRNA:tracrRNA duplex or as a single guide RNA (sgRNA). In some embodiments, the CRISPR protein may be provided directly as a polypeptide and not as an mRNA encoding the CRISPR protein.

[0324] In some embodiments, the RNA may be messenger RNA (mRNA).

[0325] In some embodiments, the nucleic acid may encode a genome editing polypeptide, a chemokine, a cytokine, a growth factor, an antibody, an enzyme, a structural protein, a blood protein, a hormone, a transcription factor, or an antigen, as described herein.

[0326] Messenger RNA (mRNA) mRNA is typically considered a type of RNA that conveys information from DNA to ribosomes. The existence of mRNA is typically very brief and involves processing and translation, followed by degradation. Typically, in eukaryotes, mRNA processing involves the addition of a "cap" on the N-terminal (5') end and a "tail" on the C-terminal (3') end.

[0327] A typical cap is a 7-methylguanosine cap, which is a guanosine that is attached to the first transcribed nucleotide by a 5'-5'-triphosphate bond. The presence of the cap is important to provide resistance to nucleases found in most eukaryotic cells. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; then, a guanosine triphosphate (GTP) is added to the terminal phosphate by a guanylyltransferase, creating a 5'5'5 triphosphate bond; then, the 7-nitrogen of the guanine is methylated by a methyltransferase.

[0328] A tail is typically a polyadenylation event whereby a polyadenylyl moiety is added to the 3' end of an mRNA molecule. The presence of this "tail" helps protect the mRNA from exonuclease degradation. Messenger RNA is translated by ribosomes into a series of amino acids that make up proteins.

[0329] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region (UTR). In some embodiments, the mRNA disclosed herein comprises a 5' UTR that includes one or more elements that affect the stability or translation of the mRNA. In some embodiments, the 5' UTR can be about 50-500 nucleotides in length. In some embodiments, the mRNA disclosed herein comprises a 3' UTR that includes one or more polyadenylation signals, binding sites for proteins that affect the stability of the mRNA's location within the cell, or one or more binding sites for miRNA. In some embodiments, the 3' UTR can be 50-500 nucleotides in length or more. In some embodiments, the mRNA disclosed herein comprises a 5' or 3' UTR that is derived from a gene different from that encoded by the mRNA transcript. In some embodiments, the mRNA disclosed herein comprises a 5' or 3' UTR that is unconventional.

[0330] The mRNA disclosed herein can be synthesized according to any of a variety of known methods. For example, the mRNA according to the present disclosure can be synthesized by in vitro transcription (IVT). In vitro transcription methods are known in the art. See, for example, Geall et al. (2013) Semin.Immunol.25(2):152-159; Brunelle et al. (2013) Methods Enzymol.530:101-14, the contents of which are incorporated by reference. Briefly, IVT is typically performed on a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RN polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor. The exact conditions will vary according to the specific application. The presence of these reagents is undesirable in the final mRNA product and is considered an impurity or contaminant, which must be purified to provide clean and homogenous mRNA suitable for therapeutic use. While mRNA provided from an in vitro transcription reaction may be desirable in some embodiments, other sources of mRNA can be used in accordance with the present disclosure, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals.

[0331] The mRNAs disclosed herein may be modified or unmodified. In some embodiments, the mRNAs disclosed herein contain one or more modifications that typically enhance RNNA stability. Exemplary modifications include backbone modifications, sugar modifications, or base modifications. In some embodiments, the disclosed mRNAs may contain purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), as well as modified nucleotide analogs or derivatives of purines and pyrimidines, such as 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-methylthio-N- ... -thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouracil (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-( uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-shodouracil, queosine, β-D-mannosyl-queosine, phosphoramidate, phosphorothioate, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine.In some embodiments, the disclosed mRNA comprises at least one chemical modification, including but not limited to, consisting of pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine. In some embodiments, the modified nucleotide comprises N1-methylpseudouridine. The preparation of such analogs is known to those skilled in the art from, for example, U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642, the contents of which are incorporated by reference.

[0332] The term "RNA" refers to a molecule that comprises, and for example consists entirely or substantially of, ribonucleotide residues. "Ribonucleotide" refers to a nucleotide that has a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. It includes isolated RNA, such as double-stranded RNA, single-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, or recombinantly produced RNA.

[0333] For clarity, mRNA encompasses any coding RNA molecule that can be translated into a protein by a eukaryotic host. A coding RNA molecule generally refers to an RNA molecule that includes a sequence that encodes a protein of interest and that can be translated by a eukaryotic host, said sequence starting with an initiation codon (ATG) and ending, for example, with a stop codon (i.e., TAA, TAG, TGA).

[0334] The RNA may be naturally occurring RNA or modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and / or alteration of at least one nucleotide. Such alterations may include the addition of non-nucleotide material at the end or within the RNA, for example, to at least one nucleotide of the RNA. The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These modified RNAs may be referred to as analogs or analogs of naturally occurring RNA.

[0335] mRNA can be produced by in vitro transcription using a DNA template. Alternatively, RNA can be obtained by chemical synthesis. Such methods are known to those skilled in the art. For example, there are various in vitro transcription kits available commercially.

[0336] RNA can be synthesized in vitro in a cell-free system using a suitable cell extract and a suitable DNA template. For example, a cloning vector is applied for the generation of the transcript. The promoter for controlling the transcription can be any promoter for any RNA polymerase. Some examples of RNA polymerases are T7, T3, and SP6 RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, for example, a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA. For example, a cloning vector is used to produce the transcript, which is generally a designated transcription vector.

[0337] The RNA can encode a protein or peptide, i.e., when present in the appropriate environment, e.g., within a cell, such as an antigen-presenting cell, e.g., a dendritic cell, the RNA can be expressed to produce the protein or peptide it encodes. The stability and translation efficiency of the RNA can be modified as needed.

[0338] In some embodiments, the mRNA may encode a genome editing polypeptide, a chemokine, a cytokine, a growth factor, an antibody, an enzyme, a structural protein, a blood protein, a hormone, a transcription factor, or an antigen, such as those described herein.

[0339] In some embodiments, the mRNA may encode an antigen.

[0340] The RNA molecules can be of variable length. Thus, they can be short RNA molecules, for example shorter than about 100 nucleotides, or long RNA molecules, for example longer than about 100 nucleotides, or even longer than about 300 nucleotides.

[0341] The mRNA may be at least 30 nucleotides in length.

[0342] An mRNA can include a 5' cap structure, a 5'-UTR sequence, an ORF sequence encoding a protein or peptide, a 3'-UTR sequence, and a poly(A) tail.

[0343] Typically, the mRNA may comprise or consist of the following general formula: [5'cap]w-[5'UTR]x-[gene of interest]-[3'UTR]y-[polyA]z wherein [5'cap] contains a methylguanine nucleotide attached to the mRNA by a 5'-5' linkage; In the formula, [5'UTR] and [3'UTR] are untranslated regions (UTRs), wherein the [5'UTR] contains a Kozak sequence, where [gene of interest] is any gene encoding a protein of interest, where [polyA] is the poly(A) tail, where w, x, y, and z are the same or different and equal to 0 or 1.

[0344] Kozak sequence refers to a sequence that is generally a consensus sequence occurring in eukaryotic mRNA and that plays a major role in the initiation of the translation process. Kozak sequences and Kozak consensus sequences are well known in the art.

[0345] The 3'UTR does not express any protein. The purpose of the 3'UTR is to increase the stability of the mRNA. According to one embodiment, the alpha-globin UTR is selected because it is known not to be unstable.

[0346] The sequence corresponding to the gene of interest may be codon optimized for satisfactory protein production in the host cell envisaged.

[0347] Poly(A) tails consist of multiple adenosine monophosphates, which are well known in the art. Poly(A) tails are generally produced during a step called polyadenylation, which is one of the post-translational modifications that generally occur during the production of mature messenger RNA. Such poly(A) tails contribute to the stability and half-life of mRNA and can be of variable length. For example, poly(A) tails can be 10A or more nucleotides, it can include 20A or more nucleotides, it can include 100A or more nucleotides, for example about 120A nucleotides.

[0348] RNA molecules are (i) a capped, unmodified RNA molecule; (ii) a capped modified RNA molecule; (iii) uncapped, unmodified RNA molecules; (iv) an uncapped modified RNA molecule may include.

[0349] Capped and uncapped RNA molecules A "capped RNA molecule" refers to a guanosine or modified guanosine, such as 7-methylguanosine (m-methylguanosine), linked to a 5'-5' triphosphate linkage or analog. 7 The term "5' cap" refers to an RNA molecule that has its 5' end attached to a 5' cap (G). This definition is equivalent to the most widely accepted definition of a 5' cap.

[0350] A "cap analog" is a 7-methylguanosine (m) bonded to a 5'-5' triphosphate linkage. 7 G) and therefore also include a cap that can be substituted without impairing protein expression of the corresponding messenger RNA in a eukaryotic host.

[0351] An example of a cap is m 7 GppN, m 7 GpppG, m 7 GppspG,m 7 GppspspG,m 7 GppspspG,m 7Gppppm7G,m 2 7’ , 3’ -OGpppG,m 2 7’ , 2’ -OGpppG,m 2 7’ , 2’ -OGppspsG, or m 2 7’ , 2’ -OGpppspsG may be mentioned.

[0352] Examples of cap analogs can be glyceryl, inverted deoxynucleotide abasic residues (moieties), 4',5' methylene nucleotides, 1-(beta-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, alpha-nucleotides, modified base nucleotides, threo-pentofuranosul nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5 dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties, 3'-3'-inverted nucleotide abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted nucleotide abasic moieties, 1,4-butanediol phosphate, 3'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 3' phosphorothioate, phosphorodithioate, or bridged or non-bridged methylphosphonate moieties.

[0353] Other examples of cap analogs include anti-reverse cap analogs (ARCA), N 1 -methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0354] Of note, among cap analogs, some are favorable for protein expression, whereas others may hinder protein expression. Such differences will be understood by those of skill in the art.

[0355] Providing a 5'-cap or 5'-cap analog to an RNA can be achieved by in vitro transcription of a DNA template in the presence of the 5'-cap or 5'-cap analog, where the 5'-cap can be incorporated into the generated RNA strand by co-transcription, or the RNA can be generated, for example, by in vitro transcription and the 5'-cap can be post-transcriptionally attached to the RNA using a capping enzyme, for example, cowpox virus capping enzyme.

[0356] An "uncapped RNA molecule" refers to any RNA molecule that does not fall within the definition of a "capped RNA molecule."

[0357] Thus, according to a general embodiment, "uncapped mRNA" may refer to an mRNA whose 5' end is not linked to 7-methylguanosine via a 5'-5' triphosphate linkage, or an analog as previously defined.

[0358] An uncapped RNA molecule, such as a messenger RNA, can be an uncapped RNA molecule having a (5')ρρρ(5'), (5')ρρ(5'), (5')ρ(5') or even a (5')OH end. Such RNA molecules can be abbreviated as 5'ρρRNA; 5'ρRNA; 5'ρRNA; 5'OHRNA, respectively.

[0359] Without limitation, the first base of the uncapped RNA molecule can be either adenosine, guanosine, cytosine, or uridine.

[0360] The RNA may be free of uncapped 5'-triphosphates. Removal of such uncapped 5'-triphosphates may be accomplished by treating the RNA with a phosphatase.

[0361] Modified and unmodified RNA molecules The RNA may include further modifications, such as an extension or truncation of the naturally occurring poly(A) tail, or alteration of the 5'- or 3'-untranslated region (UTR), such as the introduction of a UTR not related to the coding region of the RNA, e.g., replacement of an existing 3'-UTR with at least one, e.g., two copies, of a 3'-UTR derived from a globin gene, such as alpha2-globin, alpha1-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.

[0362] "Modified RNA molecule" refers to an RNA molecule that contains at least one modified nucleotide, nucleoside sugar, or base, such as a modified purine or modified pyrimidine. The modified nucleoside or base can be any nucleoside or base that is not A, U, C, or G (adenosine, uridine, cytidine, or guanosine, respectively, in reference to the nucleoside; and adenine, uracil, cytosine, or guanine when referring to only the sugar moiety).

[0363] "Unmodified RNA molecule" refers to any RNA molecule that does not meet the definition of a modified RNA molecule.

[0364] The terms "modified and unmodified" are considered differently from the terms "capped and uncapped" as the latter relates specifically to bases at the 5'-end of the RNA.

[0365] The presence of modified nucleotides can increase the stability and / or reduce the cytotoxicity of nucleic acids. The term RNA stability is related to the half-life of RNA, which is the period required to eliminate half of the activity, amount, or number of molecules. The half-life of RNA can indicate its stability. The half-life of RNA can affect the duration of expression of RNA. RNA with a long half-life can be expected to be expressed for an extended period of time.

[0366] Non-limiting examples of modified nucleotides, nucleosides and bases are disclosed in WO 2015 / 024667A1. Modified RNAs may contain modified nucleotides, nucleosides or bases, including backbone, sugar or base modifications. Modified bases and / or modified RNA molecules are known in the art and are taught, for example, in Warren et al. ("Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA"; Cell Stem Cell; 2010), the contents of which are incorporated by reference.

[0367] Sugar modifications include chemical modifications of the sugar of a nucleotide. Sugar modifications can consist in the substitution or modification of the 2' hydroxy (OH) group, which can be modified or replaced with a number of different "oxy" or "deoxy" substituents.

[0368] Examples of "oxy", -2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethylene glycol (PEG), -O(CH2CHO)nCH2CH2OR; "locked" nucleic acids (LNA), where the 2' hydroxyl is linked, e.g., by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups (-O-amino, where the amino group, e.g., NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, polyamino) or aminoalkoxy.

[0369] A "deoxy" modification includes hydrogen, amino (e.g., NH; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); or the amino group can be attached to the sugar via a linker, where the linker includes at least one of the atoms C, N, and O.

[0370] The sugar group can also contain at least one carbon that has the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, modified RNA can include nucleotides that contain, for example, arabinose as the sugar.

[0371] Backbone modifications include modifications in which the backbone phosphate of a nucleotide is chemically modified. The backbone phosphate group can be modified by replacing at least one of the oxygen atoms with a different substituent. Furthermore, modified nucleosides and nucleotides can include complete replacement of unmodified phosphates with modified phosphates as described herein.

[0372] Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenate, boranophosphate, boranophosphate ester, hydrogen phosphonate, phosphoramidate, alkyl or aryl phosphonate and phosphotriester.Phosphorodithioate has both non-bonding oxygens replaced by sulfur.Phosphate linker can also be modified by replacing the bonded oxygen with nitrogen (bridged phosphoramidate), with sulfur (bridged phosphorothioate) and with carbon (bridged methylene-phosphonate).

[0373] Base modifications include chemical modifications of the base portion of a nucleotide. In this context, the nucleotide analogs or modifications are selected from nucleotide analogs that are suitable for transcription and / or translation of RNA molecules in eukaryotic cells, for example. Modified nucleosides and nucleotides can be modified in the nucleobase portion. For example, the nucleosides and nucleotides can be chemically modified at the major groove surface. The major groove chemical modifications can include amino groups, thiol groups, alkyl groups, or halo groups. The modified base can be a modified purine base or a modified pyrimidine base. Examples of modified purine bases include modified adenosines and / or modified guanosines, such as hypoxanthine; xanthine; 7-methylguanine; inosine; xanthosine and 7-methylguanosine. Modified pyrimidine bases include modified cytidines and / or modified uridines, such as 5,6-dihydrouracil; pseudouridine; 5-methylcytidine; 5-hydroxymethylcytidine; dihydrouridine and 5-methylcytidine.

[0374] For example, nucleotide analogs / modifications include the following base modifications: 2-amino-6-chloropurine riboside-5'-triphosphate, 2-aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-amino-2'-deoxycytidine-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 2'-fluorothymidine-5'-triphosphate, 2'-O-methylinosine-5'-triphosphate. 4-thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine-5'-triphosphate, 5-bromo-2'-deoxycytidine-5'-triphosphate, 5-bromo-2'-deoxyuridine-5'-triphosphate, 5-iodocytidine- 5'-triphosphate, 5-iodo-2'-deoxycytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 5-propynyl-2'-deoxycytidine-5'-triphosphate, 5-propynyl-2'-deoxy Uridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurine riboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole- It may be selected from riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate, N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-triphosphate, and xanthosine-5'-triphosphate.

[0375] Modified nucleosides include pyridin-4-one ribonucleosides, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine. , 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine / 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.

[0376] Modified nucleosides and nucleotides include 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, and the like. pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.

[0377] Modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N 6 -(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyladenosine, 2-Methylthio-N6-threonylcarbamoyladenosine, N 6 ,N 6 -dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.

[0378] Modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N 2 ,N 2 -Dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N 2 -Methyl-6-thio-guanosine, and N 2 ,N 2 -dimethyl-6-thio-guanosine may be included.

[0379] RNA with an unmasked poly-A sequence can be translated more efficiently than RNA with a masked poly-A sequence. An "unmasked poly-A sequence" means that the poly-A sequence at the 3' end of the RNA molecule is terminated with an A in the poly-A sequence and that the poly-A sequence is not followed by any nucleotides other than the A located at the 3' end, i.e. downstream. Furthermore, a long poly-A sequence of about 120 base pairs provides optimal transcript stability and RNA translation efficiency.

[0380] Thus, to enhance RNA stability and / or expression, the poly-A sequence may be modified, for example, to have a length of 10-500, such as 30-300, for example 65-200, for example 100-150 adenosine residues. The poly-A sequence may have a length of approximately 120 adenosine residues. To further enhance RNA stability and / or expression, the poly-A sequence may be unmasked.

[0381] The incorporation of a 3'-untranslated region (UTR) into the 3'-untranslated region of an RNA molecule can result in improved translation efficiency. Synergistic effects can be achieved by incorporating two or more of such 3'-untranslated regions. The 3'-untranslated regions can be self-grafted or heterologous to the RNA into which they are introduced. The 3'-untranslated region can be derived from the human β-globin gene.

[0382] The combination of the above mentioned modifications, ie incorporation of a poly-A sequence, unmasking of the poly-A sequence and incorporation of at least one 3'-untranslated region, may have a synergistic effect on improving RNA stability and translation efficiency.

[0383] Expression of RNA can further be increased by modification of the peptide or protein encoding sequence, for example by increasing the GC content to increase RNA stability and / or by codon optimization to increase translation in the cell.

[0384] Nucleic Acids in LNPs The frozen or lyophilized LNPs obtained according to the methods disclosed herein contain a nucleic acid, such as an mRNA. The nucleic acid may encode a therapeutic agent. The nucleic acid, such as an mRNA, may be encapsulated in the LNP.

[0385] The encapsulation rate of nucleic acids in LNPs can be measured by any method known in the art, for example, fluorescent probes such as in the RiboGreen assay disclosed in the Examples section can be used.

[0386] The encapsulation rate can be used to assess the potential impact of the freezing or lyophilization method on the stability or maintenance of the tissue structure of the LNP.

[0387] The LNPs at the freezing step may contain a total amount of nucleic acid, such as mRNA, of about 5% or more, or about 4% or more, or about 3% or more, or about 2% or more, or about 1% or more of the total amount of nucleic acid, such as mRNA, in the LNPs in the liquid composition as measured by a RiboGreen assay.

[0388] The LNPs at the freezing step have a nucleic acid encapsulation rate of 25% or more, or 22% or more, such as 20% or more of mRNA, of the nucleic acid encapsulation rate of the LNPs in the liquid composition.

[0389] The LNPs at the drying step may have a nucleic acid encapsulation rate, such as mTNA, as measured by RiboGreen assay, at 3 months of 5% or more, or 2% or more, of the nucleic acid encapsulation rate of the LNPs at the drying step at TO when stored at +5° C. A change in nucleic acid encapsulation rate, such as mRNA, in the LNPs of less than 5% after 3 months of storage at +5° C. may indicate low or reduced structural changes of the LNPs during the storage period.

[0390] The LNPs at the drying step may contain a total amount of nucleic acid, such as mRNA, as measured by a RiboGreen assay, at 6 months that is about 5% or more, or about 2% or more, of the total amount of nucleic acid in the LNPs at the drying step at TO when stored at +5° C. A change in the total amount of nucleic acid in the LNPs after 6 months of storage at +5° C. of less than 5% may indicate low or reduced structural changes of the LNPs during the storage period.

[0391] The LNPs at the drying step may have a nucleic acid encapsulation rate, such as mTNA, as measured by RiboGreen assay, at 6 months of 10% or more, or 5% or more, of the nucleic acid encapsulation rate at the drying step at TO when stored at +5° C. A change in nucleic acid encapsulation rate in the LNPs after 6 months storage at +5° C. of less than 10% or more than 5% may indicate low or reduced structural changes of the LNPs during the storage period.

[0392] The LNPs at the drying step may contain a total amount of nucleic acid, such as mRNA, as measured by a RiboGreen assay of about 10% or more, or about 5% or more, of the total amount of nucleic acid in the LNPs at the drying step at TO, at 11 months when stored at +5° C. A change in the total amount of nucleic acid in the LNPs after 11 months of storage at +5° C. of less than 10% may indicate low or reduced structural changes of the LNPs during the storage period.

[0393] The LNPs at the drying step may have a nucleic acid encapsulation rate, such as mTNA, as measured by RiboGreen assay at 11 months of 10% or more, or 5% or more, of the nucleic acid encapsulation rate at the drying step at TO when stored at +5° C. A change in nucleic acid encapsulation rate in the LNPs after 3 months storage at +5° C. of less than 10% or more than 5% may indicate low or reduced structural changes of the LNPs during the storage period.

[0394] Treatment The nucleic acid may be a therapeutic agent or may encode a therapeutic agent, hi some embodiments, the nucleic acid may be an mRNA that encodes a therapeutic agent.

[0395] "Therapeutic agent" is intended to refer to an active ingredient proposed to prevent or reduce the risk of occurrence of a medical condition or a symptom of a medical condition, or to cure or reduce the intensity of a medical condition, or to cure or reduce at least one symptom of a medical condition, in an individual to whom it is administered. "Individual" is intended to refer to humans and animals.

[0396] The therapeutic agent can be a peptide, a protein, or a nucleic acid. In some embodiments, the therapeutic agent can be a nucleic acid. The nucleic acid can encode a variety of therapeutic peptides or proteins.

[0397] The therapeutic agent can be a genome-editing polypeptide, a chemokine, a cytokine, a growth factor, an antibody, an enzyme, a structural protein, a blood protein, a hormone, a transcription factor, or an antigen.

[0398] In one embodiment, the therapeutic agent can be a genome editing polypeptide. In some embodiments, the genome editing polypeptide is a CRISPR protein, such as CRISPR / Cas9, a restriction enzyme, a meganuclease, a transcription activator-like effector protein (TALE, such as TALE nuclease, TALEN), or a zinc finger protein (ZF, such as ZF nuclease, ZFN). See, e.g., WO 2020 / 139783.

[0399] The therapeutic agent may be a cytokine or chemokine suitable for stimulating or inhibiting an immune response, stimulating or preventing cell proliferation, or reducing inflammation. Examples of suitable cytokines or chemokines include insulin, insulin-like growth factors, human growth hormone (hGH), tissue plasminogen activator (tPA), interleukins (ILs), such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IL-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-69, IL-70, IL-71, IL-72, IL Cytokines such as IFN-33, interferon (IFN) alpha, IFN beta, IFN gamma, IFN omega or IFN tau, such as TNF alpha and TNF beta, TNF gamma, tumor necrosis factor (TNF), TNF-related apoptosis-inducing ligand (TRAIL); growth factors such as lymphotoxin-β (LT-β), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor (M-CSF), monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF). Production of erythropoietin or any other hormone growth factor is also included.

[0400] In some embodiments, the therapeutic agent may be an antibody. As used herein, the term "antibody" refers to a whole antibody, including two light chain polypeptides and two heavy chain polypeptides, or an antigen-binding fragment thereof. An antibody may be a monoclonal antibody (e.g., a full-length monoclonal antibody) that exhibits a single binding specificity and affinity for a particular epitope. The antigen-binding fragment may be a single chain antibody, a single chain Fv fragment (scFv), an Fd fragment, a Fab fragment, a Fab' fragment, or a F(ab')2 fragment. An antibody may recognize a tumor antigen or an infectious disease antigen, for example, an antigen expressed by a tumor cell, against which a protective or therapeutic immune response is desired. Examples of antibodies include, for example, adalimumab, infliximab, rituximab, ipilimumab, tocilizumab, canakinumab, itolizumab, or tralokinumab.

[0401] In some embodiments, the therapeutic peptide or protein may be an enzyme that has a desired use for regulating metabolism or growth in a subject. In some embodiments, the enzyme may be administered to replace an endogenous enzyme that is absent or dysfunctional. In some embodiments, the enzyme may be used to treat metabolic storage diseases. Metabolic storage diseases result from the systemic retention of metabolic products due to the absence or dysfunction of endogenous enzymes. Such metabolic products include lipids, glycoproteins, and mucopolycephaly. Examples of enzyme replacement therapy include lysosomal diseases such as Gaucher disease, Fabry disease, MPS I, MPS II (Hunter syndrome), MPS VI, and glycogen storage disease type II.

[0402] Structural proteins can be, for example, collagen, fibroin, fibrinogen, elastin, tubulin, actin, and myosin.

[0403] The blood protein can be, for example, 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, an anticoagulant, and the like.

[0404] The hormones can be, for example, insulin, thyroid hormones, gonadotropins, trophic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptin, and the like.

[0405] Transcription factors (TFs) form a complex system that recognizes specific DNA sequences to control chromatin and transcription, leading to the expression of the genome. There are several families of transcription factors, and members of each family may share structural characteristics. Examples of transcription factors may include helix-turn-helix (e.g., Oct-1), helix-loop-helix (e.g., E2A), zinc finger (e.g., glucocorticoid receptor, GATA proteins), basic protein-leucine zipper [cyclic AMP response element binding factor (CREB), activator protein-1 (AP-1)], or β-sheet motifs [e.g., nuclear factor-κB (NF-κB)].

[0406] The therapeutic agent may be an antigen suitable for triggering an immune response in the treatment of cancer or in the treatment of an infectious disease (eg, a viral, bacterial, fungal, protozoan or parasitic infection).

[0407] According to some embodiments, a composition containing an LNP as disclosed herein that comprises an antigen can therefore be an immunogenic composition or a vaccine composition.

[0408] Antigen-containing compositions can vary in terms of their valency. Valency refers to the number of antigenic components in a composition. Immunogenic or vaccine compositions can be monovalent or multivalent, i.e., bivalent, trivalent compositions, or more. Multivalent compositions can contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens or antigenic moieties (e.g., antigenic peptides, etc.). The antigenic components can be present on a single polynucleotide or on separate polynucleotides.

[0409] Compositions as disclosed herein can be used to prevent, treat, or cure infectious diseases resulting from contact with infectious agents, such as bacteria, viruses, fungi, protozoa, parasites, etc.

[0410] The compositions as disclosed herein may be used to prevent, treat, or cure cancer diseases.

[0411] According to some embodiments, the nucleic acid may encode at least one antigen selected from the group consisting of a bacterial antigen, a viral antigen, and a tumor antigen.

[0412] bacterial antigen The bacteria can be gram positive or gram negative. Bacterial antigens include Acinetobacter baumannii, Bacillus anthracis, Bacillus subtilis, Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, and others. botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Coagulase Negative Staphylococcus, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, enterotoxigenic Escherichia coli (ETEC), enteropathogenic E. coli, E. coli 0157:H7, Enterobacter sp.), Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Moraxella catarrhalis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis meningitides, Proteus mirabilis, Proteus sps.), Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Serratia marcesens, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans, Streptococcus pneumoniae pneumoniae, Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, and Yersinia pestis.

[0413] Viral antigens Viral antigens include adenovirus; herpes simplex, type 1; herpes simplex, type 2; encephalitis virus, papilloma virus, varicella zoster virus; Epstein-Barr virus; human cytomegalovirus; human herpes virus, type 8; human papilloma virus; BK virus; JC virus; smallpox; poliovirus, hepatitis B virus; human bocavirus; parvovirus B19; human astrovirus; Norwalk virus; coxsackievirus; hepatitis A virus; poliovirus; rhinitis virus; severe acute respiratory syndrome virus; hepatitis C virus; yellow fever virus; dengue fever virus; West Nile virus; rubella virus; hepatitis E virus; human immunodeficiency virus (HIV); influenza virus, type A or Type B; Guanarito virus; Junin virus; Lassa fever virus; Machupo virus; Sabia virus; Crimean-Congo hemorrhagic fever virus; Ebola virus; Marburg virus; Measles virus; Mumps virus; Parainfluenza virus; Respiratory syncytial virus (RSV); Human metapneumovirus; Hendra virus; Nipah virus; Rabies virus; Hepatitis D; Rotavirus; Orbivirus; Coltivirus; Hantavirus, Middle East respiratory coronavirus; SARS-Cov-2 virus; Chikungunya virus; Zika virus; Parainfluenza virus; Human enterovirus; Hantavirus; Japanese encephalitis virus; Swine vesicular rash virus; Eastern equine encephalitis source (Eastern equine encephalitisor); or Banna virus.

[0414] In one embodiment, the antigen is from an influenza A or influenza B virus strain, or a combination thereof. The influenza A or influenza B strain may be associated with birds, pigs, horses, dogs, humans, or non-human primates.

[0415] The nucleic acid may encode a hemagglutinin protein or a fragment thereof. The hemagglutinin protein may be H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, H18, or a fragment thereof. The hemagglutinin protein may or may not include a head domain (HA1). Alternatively, the hemagglutinin protein may or may not include a cytoplasmic domain.

[0416] In an embodiment, the hemagglutinin protein is a truncated hemagglutinin protein. The truncated hemagglutinin protein may or may not include a portion of the transmembrane domain.

[0417] In some embodiments, the virus may be selected from the group consisting of H1N1, H3N2, H7N9, H5N1, and H10N8 viruses or type B viruses.

[0418] In another embodiment, the antigen can be from a respiratory syncytial virus (RSV). Suitable RSV antigens can be from RSV A and / or RSV B. The RSV antigen can be, for example, the fusion glycoprotein F protein, or the attachment protein G protein.

[0419] In another embodiment, the antigen can be from a coronavirus, such as SARS-Cov-1 virus, SARS-Cov-2 virus, or MERS-Cov virus. In some embodiments, the antigen can be a SARS-Cov2 antigen, such as the spike protein from SARS-Cov2.

[0420] Tumor antigens The antigen may be a tumor antigen, i.e., a component of a cancer cell, such as a protein or peptide expressed in a cancer cell. The term "tumor antigen" refers to a protein that is specifically expressed under normal conditions in a limited number of tissues and / or organs or at a specific development stage, and is expressed or abnormally expressed in at least one tumor or cancer tissue. Tumor antigens include, for example, differentiation antigens, such as cell type-specific differentiation antigens, i.e., proteins that are specifically expressed under normal conditions in certain cell types at a certain differentiation stage, and germ lineage-specific antigens. For example, tumor antigens are presented by the cancer cells in which they are expressed.

[0421] For example, tumor antigens can include carcinoembryonic antigen, 1-fetoprotein, isoferritin, and fetal sulfoglycoprotein, cc2-H-ferroprotein, and gamma-fetoprotein.

[0422] Other examples of tumor antigens that may be useful in the present disclosure include p53, ART-4, BAGE, beta-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CD 4 / m, CEA, cell membrane 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, Gapl OO, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, e.g. MAGE- A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A1 1, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1 R, Myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl 90 minor BCR-abL, Pm l / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, Rul or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVrVIN, TEL / AMLl, TPI / m, TRP-1, TRP-2, TRP-2 / 1NT2, TPTE and WT, such as WT-1.

[0423] Pharmaceutical compositions and uses thereof The lyophilized or frozen LNPs obtained according to the methods disclosed herein can be used in pharmaceutical compositions, which can include the lyophilized or frozen LNPs by themselves or further combined with at least one pharma- ceutically acceptable excipient.

[0424] The present disclosure relates to lyophilized or frozen LNPs obtained according to the methods as disclosed herein and comprising at least one nucleic acid, for use as a medicament.

[0425] The present disclosure relates to lyophilized or frozen LNPs comprising at least a nucleic acid and, as lipid components, at least a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or ester thereof, optionally a PEG-lipid, for use as a medicament, wherein the lyophilized LNPs are in lyophilized micropellets, or the frozen LNPs are in frozen micropellets.

[0426] A method of manufacturing a medicament or pharmaceutical composition can include at least the step of preparing lyophilized LNPs according to the methods as disclosed herein, wherein the LNPs comprise at least a nucleic acid.

[0427] A method of manufacturing a medicament or pharmaceutical composition can include at least the step of preparing frozen LNPs according to the methods as disclosed herein, wherein the LNPs comprise at least a nucleic acid.

[0428] The method may further include packaging the lyophilized or frozen LNPs. The method may further include combining the lyophilized or frozen LNPs with at least one pharma- ceutically acceptable excipient. The method may further include resuspending the lyophilized LNPs in a pharma- ceutically acceptable solvent or thawing the frozen LNPs.

[0429] A "pharmaceutically acceptable solvent" can be any solvent suitable for resuspending or dissolving the lyophilized LNPs and that is pharmaceutically acceptable for enteral or parenteral administration to an individual in need thereof. The pharmaceutically acceptable solvent can be water for injection or a buffer such as saline, citrate buffer, histidine buffer, or phosphate buffer.

[0430] The pharmaceutical composition may be sterile.

[0431] General guidelines for the formulation and manufacture of pharmaceutical compositions and agents can be found, for example, in Remington's The Science and Practice of Pharmacy, 21 StEdition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Except for those excipients that may be incompatible with one or more components of the LNP, any pharma- ceutically acceptable excipient may be used in the pharmaceutical compositions.

[0432] Exemplary pharma- ceutically acceptable excipients that may be used may be selected from diluents such as water for injection or saline, such as amino acid buffers (histidine, arginine, glycine, proline, glycylglycine), saline buffers (inorganic salts NaCl, calcium chloride), phosphate buffers, acetate buffers, citrate buffers, succinate buffers, and the like; sugars or polyhydric alcohols such as dextrose, glycerol, ethanol, sucrose, trehalose, mannitol; surfactants such as polysorbate 80, polysorbate 20, poloxamer 188, and the like, and combinations thereof. In many cases, it will be preferable to include an isotonic agent in the composition, such as a sugar, polyhydric alcohol, or sodium chloride, and the formulation may also include antioxidants such as tryptamine and Tween 100. The composition may contain stabilizers such as ethanol, ethanolic acid, ethyl esters, etc., such as 20 or 80, other solvents such as monohydric alcohols, such as ethanol or isopropanol, and polyhydric alcohols, such as glycols, as well as edible oils, such as soybean oil, coconut oil, olive oil, safflower oil, cottonseed oil, and oily esters, such as ethyl oleate, isopropyl myristate, and the like; binders, adjuvants, solubilizers, thickeners, stabilizers, disintegrants, lubricants, buffers, emulsifiers, wetting agents, suspending agents, sweeteners, colorants, flavorings, preservatives, antioxidants, processing agents, drug delivery modifiers, and enhancers, such as calcium phosphate, magnesium stearate, talc, monosaccharides, disaccharides, starch, gelatin, cellulose, methylcellulose, sodium carboxymethylcellulose, dextrose, hydroxypropyl-β-cyclodextrin, polyvinylpyrrolidone, or polyethylene glycol. Pharmaceutically acceptable excipients may also include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, and the like that are physiologically compatible.

[0433] Frozen or lyophilized LNPs may be administered by any suitable route, depending on parameters known in the art, such as the form of the composition (solid or liquid), the individual being treated, and the nature of the therapeutic agent contained in the LNP.

[0434] For example, the resulting pharmaceutical compositions containing frozen or lyophilized LNPs can be administered systemically, orally, sublingually, intranasally, intradermally, or subcutaneously.

[0435] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent should first be rendered isotonic with sufficient saline or glucose. These aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be employed will be known to those skilled in the art.

[0436] In some embodiments, the resulting pharmaceutical composition containing frozen or lyophilized LNPs may be suitable for subcutaneous administration.

[0437] Pharmaceutical compositions containing frozen or lyophilized LNPs can be administered by a drug combination device, such as a multi-chamber syringe, in which at least one chamber contains the pharmaceutical composition in solid form and at least one chamber contains a pharma- ceutically acceptable solvent for suspending or dissolving the composition.

[0438] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs comprising at least a nucleic acid obtained according to the methods as disclosed herein and for use as a medicament.

[0439] In some embodiments, the present disclosure relates to lyophilized or frozen LNPs comprising at least a cationic ionizable lipid, a neutral lipid, a steroid alcohol, or an ester thereof, and optionally a PEG-lipid as lipid components, wherein the frozen LNPs are in frozen micropellets, or the lyophilized LNPs are in lyophilized micropellets, and the LNPs comprise at least a nucleic acid, for use as a pharmaceutical.

[0440] In one of its aims, the invention relates to frozen or lyophilized LNPs as disclosed herein and comprising at least one nucleic acid encoding an antigen from influenza A virus and / or influenza B virus for use in preventing or treating influenza A and / or influenza B virus infection.

[0441] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs comprising at least a cationic ionizable lipid, a neutral lipid, a steroid alcohol, or an ester thereof, and optionally a PEG-lipid as lipid components, wherein the frozen LNPs are in a frozen micropellet, or the lyophilized LNPs are in a lyophilized micropellet, and the LNPs comprise at least one nucleic acid encoding an antigen from influenza A virus and / or influenza B virus for use in preventing or treating influenza A and / or influenza B virus infection.

[0442] In one of its aims, the invention relates to frozen or lyophilized LNPs as disclosed herein and comprising at least one nucleic acid encoding an antigen from respiratory syncytial A virus and / or respiratory syncytial B virus for use in preventing or treating respiratory syncytial A virus and / or respiratory syncytial B virus infection.

[0443] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs comprising at least a cationic ionizable lipid, a neutral lipid, a steroid alcohol, or an ester thereof, and optionally a PEG-lipid as a lipid component, wherein the frozen LNPs are in a frozen micropellet, or the lyophilized LNPs are in a lyophilized micropellet, and the LNPs comprise at least one nucleic acid encoding an antigen from respiratory syncytial A virus and / or respiratory syncytial B virus for use in preventing or treating respiratory syncytial A virus and / or respiratory syncytial B virus infection.

[0444] In one of its aims, the invention relates to frozen or lyophilized LNPs as disclosed herein and comprising at least one nucleic acid encoding an antigen from influenza A virus and / or influenza B virus for use as an immunogenic composition against influenza A virus and / or influenza B virus.

[0445] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs comprising at least a cationic ionizable lipid, a neutral lipid, a steroid alcohol, or an ester thereof, and optionally a PEG-lipid as lipid components, wherein the frozen LNPs are in a frozen micropellet, or the lyophilized LNPs are in a lyophilized micropellet, and the LNPs comprise at least one nucleic acid encoding an antigen from influenza A virus and / or influenza B virus for use as an immunogenic composition against influenza A virus and / or influenza B virus.

[0446] In one of its aims, the invention relates to frozen or lyophilized LNPs as disclosed herein and comprising at least one nucleic acid encoding an antigen from respiratory syncytial A virus and / or respiratory syncytial B virus for use as an immunogenic composition against respiratory syncytial A virus and / or respiratory syncytial B virus.

[0447] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs comprising at least a cationic ionizable lipid, a neutral lipid, a steroid alcohol, or an ester thereof, and optionally a PEG-lipid as lipid components, wherein the frozen LNPs are in a frozen micropellet, or the lyophilized LNPs are in a lyophilized micropellet, and the LNPs comprise at least one nucleic acid encoding an antigen from respiratory syncytial A virus and / or respiratory syncytial B virus for use as an immunogenic composition against respiratory syncytial A virus and / or respiratory syncytial B virus.

[0448] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs obtained according to the methods as disclosed herein and comprising at least one nucleic acid encoding a SARS-Cov2 antigen for use in preventing or treating SARS-Cov-2 infection.

[0449] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs comprising at least a cationic ionizable lipid, a neutral lipid, a steroid alcohol, or an ester thereof, and optionally a PEG-lipid as lipid components, wherein the frozen LNPs are in a frozen micropellet, or the lyophilized LNPs are in a lyophilized micropellet, and the LNPs comprise at least one nucleic acid encoding a SARS-Cov2 antigen for use in preventing or treating SARS-Cov-2 infection.

[0450] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs obtained according to the methods as disclosed herein and comprising at least one nucleic acid encoding a SARS-Cov2 antigen for use as an immunogenic composition against SARS-Cov2.

[0451] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs comprising at least a cationic ionizable lipid, a neutral lipid, a steroid alcohol, or an ester thereof, and optionally a PEG-lipid as lipid components, wherein the frozen LNPs are in a frozen micropellet, or the lyophilized LNPs are in a lyophilized micropellet, and the LNPs comprise at least one nucleic acid encoding a SARS-Cov2 antigen for use as an immunogenic composition against SARS-Cov2.

[0452] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs comprising at least one nucleic acid obtained according to the methods as disclosed herein and in the manufacture of a medicament.

[0453] In some embodiments, the present disclosure relates to frozen or lyophilized LNPs comprising at least a cationic ionizable lipid, a neutral lipid, a steroid alcohol, or an ester thereof, and optionally a PEG-lipid as lipid components, wherein the frozen LNPs are in frozen micropellets or the lyophilized LNPs are in lyophilized micropellets, and the LNPs comprise at least one nucleic acid in the manufacture of a pharmaceutical agent.

[0454] In some embodiments, the present disclosure provides a method for preventing and / or treating a disease in an individual in need thereof, the method comprising at least: - resuspending the lyophilized LNPs obtained according to the methods disclosed herein in a pharma- ceutically acceptable solvent or thawing the frozen LNPs (frozen or lyophilized LNPs comprising at least one nucleic acid putatively active against said disease) to obtain resuspended or thawed LNPs; - administering the resuspended or thawed LNPs to the individual. The present invention relates to a method comprising the steps of:

[0455] In some embodiments, the present disclosure provides a method for preventing and / or treating a disease in an individual in need thereof, the method comprising: - resuspending the lyophilized LNPs in a pharma- ceutically acceptable solvent or thawing the frozen LNPs (wherein the LNPs comprise at least, as lipid components, a cationic ionizable lipid, a neutral lipid, a steroid alcohol or an ester thereof, and optionally a PEG-lipid, the frozen LNPs being in a frozen micropellet or the lyophilized LNPs being in a lyophilized micropellet, the frozen or lyophilized LNPs comprising at least one nucleic acid putatively active against said disease) to obtain resuspended or thawed LNPs; - administering the resuspended or thawed LNPs to the individual. The present invention relates to a method comprising the steps of:

[0456] In some embodiments, the nucleic acid may be RNA. In some embodiments, the RNA may be mRNA.

[0457] Also disclosed is a method of delivering LNPs to an individual, comprising (i) suspending or dissolving lyophilized LNPs in a pharma- ceutically acceptable solvent or thawing frozen LNPs to obtain a solution of LNPs, and (ii) administering the solution of LNPs to an individual in need thereof. Step (ii) is ideally performed within 24 hours of step (i), e.g., within 12 hours, 6 hours, 3 hours, or 1 hour after reconstitution of the LNPs with a liquid formulation.

[0458] The present disclosure should be understood to encompass all variations, combinations, and permutations of at least one limitation, element, clause, descriptive term, etc., from at least one of the enumerated claims being introduced into another claim (or any other claim, such as any other claim related thereto) that is dependent on the same base claim, unless otherwise stated or unless it is obvious to one skilled in the art that a contradiction or inconsistency would result. When elements are presented as a list, e.g., in a Markush group or similar format, it should be understood that each subgroup of elements is also disclosed and any element can be removed from the group. In general, when the present disclosure, or aspects of the present disclosure, are said to include certain elements, features, etc., they should also be understood to encompass embodiments that consist of or consist essentially of such elements, features, etc. For the sake of brevity, those embodiments have not been literally specifically set forth herein in every instance. It should also be understood that any embodiment or aspect of the present disclosure can be specifically excluded from the claims, regardless of whether a specific exclusion is enumerated in the specification. Publications and other reference materials mentioned herein to describe the background of this disclosure and to provide additional details regarding its practice are hereby incorporated by reference.

[0459] The following examples are offered for purposes of illustration and not limitation. EXAMPLES

[0460] Example 1: Materials and Methods Preparation of mRNA-containing lipid nanoparticles (LNPs) LNPs were prepared using DLin-MC3-DMA (i.e. (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate from SAI Life Science) as the ionizable cationic lipid; DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine-Avanti-Polar Lipids: ref. 850365) as the neutral lipid; cholesterol as the steroid alcohol-Avanti-Polar Lipids ref. 700000P; and DMG-PEG 2000 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] Avanti Polar Lipids ref. 880150P as the PEG-lipid.

[0461] The lipid components of the LNPs were dissolved in ethanol at a molar ratio of neutral lipid:steroid alcohol:PEG-lipid:ionizable cationic lipid of 10:38.5:1.5:50.

[0462] The encapsulated mRNA was non-replicating mRNA luminescence enzyme (mRNA-Luc; ref. L-7602 TriLink™ Biotechnologies. The mRNA was prepared in citrate buffer (pH 4.0) at a concentration of 900 μg / mL of mRNA.

[0463] The mRNA and lipids were mixed in a ratio of 3:1. The loading ratio used in all experiments was 6:1.

[0464] The LNPs were prepared according to the methods described herein below.

[0465] After purification and formulation, a stock solution of LNPs at 100 μg / mL mRNA and 3 mg / mL total lipids was obtained.

[0466] Preparation of the organic phase Preparation of 6 mL of organic phase for LNP formulation 46 mg of DSPC, 24 mg of DMG-PEG 2000, and 88 mg of cholesterol were dissolved in 3971 μL of ethanol, then 1872 μL of DLin-MC3-DMA stock solution (100 mg / mL in ethanol) was added to obtain a 20 mg / mL lipid phase solution.

[0467] Preparation of the aqueous phase Preparation of 1.8 mL aqueous phase for LNPs The mRNA concentration used in the aqueous phase was calculated to obtain a lipid nitrogen / mRNA phosphate loading ratio of 6 / 1 (N / P=6 / 1). This concentration was determined from the ionizable cationic lipid concentration assuming that 1 μg of mRNA corresponds to 0.003 μmol of phosphate. Since 1.5 mL of aqueous solution is required to produce 2 mL of LNPs when using a 3:1 aqueous to ethanol solution ratio in NanoAssemblR® (Nanoassemblr Benchtop from Precision Nanosystem; Belliveau et al., Molecular Therapy-Nucleic Acids (2012)), the required mRNA concentration was calculated to be 900 μg / mL.

[0468] The mRNA solution was prepared in 50 mM citrate buffer pH 4.0.

[0469] LNP preparation LNPs were prepared using the NanoAssemblR instrument according to the manufacturer's recommendations.

[0470] The aqueous and organic phases were loaded into the NanoAssemblR with suitable syringes, respectively, according to the manufacturer's recommendations. The flow rate was set up at a ratio of 3:1 and a total flow rate of 4 ml / min. The aqueous and lipid phases were then mixed to obtain LNPs.

[0471] LNP purification and harvesting The resulting LNPs were dialyzed against citrate buffer (50 mM, pH 4.0) to remove residual ethanol.

[0472] Preparation of LNP-containing formulations for freezing and lyophilization Prior to storage of the final product, the LNP formulations were prepared for freezing and lyophilization by adding excipients to the LNPs prepared as disclosed above.

[0473] First, a dialysis step was performed with citrate buffer to remove ethanol from the encapsulation process. A second dialysis was then performed with Tris buffer (50 mM), pH 7.5. Trehalose was then added (500 mM) as a cryoprotectant. The formulated LNPs were then sterile filtered (0.22 μm) before being filled into vials and frozen or lyophilized.

[0474] Freezing Process Formulated LNPs containing mRNA were subjected to two types of freezing processes: freezing in vials or freezing by spray freezing.

[0475] Freezing in vials was performed by filling 0.5 mL of formulated LNP (obtained as shown above) into 3 mL type I glass vials with lyostoppers (West ref. 7002-4333). Vials were frozen at minus 80° C. or minus -20° C. for liquid processes. Vials were frozen on lyophilizer shelves regulated at -45° C. under atmospheric pressure. Frozen vials were stored at minus 80° C. or minus 20° C., respectively, until use.

[0476] Spray freezing was performed by prilling a liquid jet of formulated LNPs (electromagnetic droplet flow nozzle - Meridion Technologies GmbH, Muellheim, Germany + prilling tower Gatt, Binzen, Germany) in a jacketed chamber (prilling tower) cooled by direct spraying / evaporation of liquid nitrogen (Adali et al., Processes 2020, 8, 709; Wanning et al., Int J Pharm. 2015; 488(1-2):136-153; WO 2013 / 050156 A1; WO 2013 / 050159 A1; or WO 2016 / 012414 A1). The atmosphere in the cooling chamber was cooled to below minus 105°C, the liquid flow rate was 20 mL / min, the frequency was 4000 Hz, and the nozzle diameter was 300 μm. The height of the column was 160 cm. The frozen micropellets (or microbeads) were poured onto pre-chilled trays at -50°C and freeze-dried at 50 μBar (freeze dryer SMH90, Elancourt, France). The frozen micropellets were harvested and stored at -80°C until use.

[0477] Freeze-drying (lyophilization) process The mRNA-containing LNPs were subjected to two types of freeze-drying processes: lyophilization in vials (or conventional spray freezing) and spray freeze drying (or prilling: freezing of droplets followed by drying).

[0478] Freeze-drying in vials (conventional freeze-drying) Freezing in vials was performed by filling 0.5 mL of formulated LNP (obtained as shown above) into 3 mL type I glass vials with lyostoppers (West ref. 7002-4333). The vials were then lyophilized in a Scientific Lyostar freeze dryer as follows.

[0479] [Table 1]

[0480] Lyophilized cakes of formulated LNPs were obtained and stored at +5° C. for 0, 1, 2, 3, 6 or 11 months prior to analysis.

[0481] Spray Freeze Drying Process Spray freezing was performed by prilling (electromagnetic droplet stream generator) as described above.

[0482] The frozen micropellets were harvested and then dried on the freeze-dryer shelves. The frozen micropellets were poured onto pre-chilled trays at -50°C and freeze-dried at 50 μBar (freeze-dryer USIFROID SMH90, Elancourt, France).

[0483] Lyophilized micropellets were harvested and filled into 5 mL Type I glass vials (100 mg / vial) with Lyo stoppers (West ref. 7002-4333) with a Powder Quantos Dosing System-Mettler Toledo, Columbus, Ohio, US) and stored at +5° C. until use. Lyophilized LNPs were stored for 0, 1, 2, 3, 6 or 11 months before analysis.

[0484] Resuspension of LNPs Frozen LNPs were thawed at room temperature.

[0485] Prior to analytical measurements or in vivo assays as described later herein, the lyophilized LNPs were resuspended in water for injection to obtain a resuspended LNP solution at 100 μg / mL RNA.

[0486] Analysis method The following analytical methods were used to characterize the LNPs after the freezing and lyophilization process.

[0487] LNP size and concentration The size and concentration of LNPs were measured by NTA (Nanoparticle Tracking Analysis).

[0488] NTA measurements were performed on NanoSight NS300 (Malvern) and Nano Sample Assistant (Malvern) instruments according to the manufacturer's recommendations. Frozen LNPs were thawed at room temperature and lyophilized LNPs were resuspended in water (0.5 mL) as indicated above. Resuspended or thawed LNPs were further diluted (1 / 2000) in Tris buffer (Tris 50 mM) and dispensed into 96-well plates with a Perkin Elmer dilution robot, Janus. The size (mean size and mode size) and concentration of LNPs were then measured (camera level 15 - detection threshold 5). SURF-CAL™ Particle Size Standard (Thermo Scientific PD-047B - size 47 ± 2 nm; concentration 1 × 10 10 particles / mL) was used as a control (camera level 15-detection threshold 3). NTA results presented here are the average results of samples in triplicate measurements.

[0489] LNP obtained after 0.22 μm filtration and before freezing or lyophilization was used as a control.

[0490] RNA encapsulation rate The percentage of encapsulated mRNA and the mRNA concentration in the LNT were measured using the Quant-iT Ribogreen RNA reagent kit according to the manufacturer's recommendations (Invitrogen Detection Technologies) and quantified with a fluorescence microplate reader. Standard curves using RiboGreen RNA standards (100 μg / ml) and an internal control (Clean Cap Fluo mRNA - ref. L7602-1 mg / mL) with or without Triton X100 (0.5%) were prepared in TE buffer (Tris 10 mM, EDTA 1 mM, pH 7.5) with 1-0.03125 μg / mL RNA for the standard curve and 100-3.125 μg / mL mRNA for the internal control, respectively.

[0491] Frozen LNPs were thawed at room temperature and lyophilized LNTs were resuspended in water (0.5 mL) as indicated above.

[0492] For quantification of unencapsulated RNA, LNPs were serially diluted in Tris / EDTA assay buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). For quantification of total RNA, LNPs were serially diluted in Tris / EDTA assay buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5) containing 0.5 (v / v)% Triton X100.

[0493] Controls and samples were distributed into 96-well plates.

[0494] Ribogreen dye (at 200x dilution) was added to the samples (50 / 50 mix; sample / Ribogreen reagent), mixed thoroughly and incubated for 5 min at room temperature in the dark. Fluorescence was measured on a SpectraMax plate reader (excitation and emission wavelengths: 485 and 528 nm).

[0495] The LNPs obtained after 0.22 μm filtration and before freezing or lyophilization were used as controls. After filtration, LNPs were sampled and used as LNP controls, which were not subjected to any process ("no freezing" and "no lyophilization").

[0496] mRNA integrity The integrity of mRNA in the LNPs was measured using capillary electrophoresis using a Bioanalyzer 2100 (AGILENT TECHNOLOGIES) according to the manufacturer's recommendations ("Quick Start Guide RNA 6000 Pico Kit G2938-90049" Rev. C Edition 08 / 2013).

[0497] Decapsulation of mRNA was performed by mixing samples with TE buffer-0.5% Triton X100 at a sample:buffer volume ratio of 1:9. Samples were further diluted (1:40 final) and thermally denatured (70°C) for 2 min.

[0498] The mRNA samples were then loaded onto the gel according to the manufacturer's recommendations ("Quick Start Guide RNA 6000 Pico Kit G2938-90049" Rev. C Edition 08 / 2013).

[0499] LNP obtained after 0.22 μm filtration and before freezing or lyophilization was used as a control.

[0500] In vivo bioluminescence LNPs containing mRNA-luc were prepared as described in Example 1. The LNPs were freeze-dried either by conventional freeze-drying in vials or by spray freeze-drying to obtain spray freeze-dried micropellets (see Example 1). The freeze-dried (or lyophilized) LNPs were stored at +5°C for 320 days, then resuspended in water for injection and injected into mice (5 animals per condition - SKH1 hairless female mice, 6 weeks old) by intramuscular route. Injections were performed in the right quadriceps. Each mouse received 3 μg of mRNA (35 μL injected). Measurements were taken at different time points after LNP mRNA-Luc injection: T0h, T6h, and T24h. Control mice received LNPs without mRNA.

[0501] After intramuscular injection, mice were imaged with an IVIS Spectrum CT machine to obtain background bioluminescence signals (T0). To generate bioluminescence, mice were administered 150 mg / kg D-luciferin by intraperitoneal route at T6h bioluminescence acquisition and at D1 for T24 acquisition, and at D3 at T72h. After 15 min, mice were anesthetized (isoflurane) and bioluminescence signal acquisition (luminescence enzyme) was performed. Acquisition was performed in the right quadriceps region.

[0502] To quantify the bioluminescence signal, a region of interest (ROI) was defined for each animal. The size and location of the ROI was adapted to the quadriceps muscle. The same size of ROI was used for each animal.

[0503] Quantification was performed with the ROI measurement tool from Living Image software. Results were expressed as total flux (photons / sec).

[0504] Example 2: Effect of the freezing process on LNP stability Experimental design LNPs containing mRNA-Luc were prepared as described in Example 1.

[0505] In the first set of experiments, LNPs were frozen in vials at -20°C and -80°C or spray frozen in micropellets at 80°C (see Example 1).

[0506] Frozen LNPs were thawed before measurements were taken.

[0507] The size and concentration of the LNPs were measured by NTA (see Example 1).

[0508] RNA encapsulation rates were measured by the Quant-iT™ RiboGreen® RNA Assay (see Example 1).

[0509] result The LNP mean and mode diameters and concentrations obtained by NTA are summarized in Table 1.

[0510] [Table 2]

[0511] The total RNA and RNA-encapsulated potassium rates are summarized in Tables 2 and 3.

[0512] [Table 3]

[0513] [Table 4]

[0514] The data show that spray-frozen LNPs at -80°C tend to have increased mRNA encapsulation rates compared to LNPs frozen in vials at -80°C or at -20°C.

[0515] This data indicates that spray freezing of LNPs improves the stability and mRNA encapsulation rate of frozen LNPs.

[0516] Taken together, the results indicate that spray freezing of LNPs improves the stability of frozen LNPs by reducing LNP aggregation and maintaining mRNA encapsulation rates.

[0517] Reduction of LNP aggregation can be beneficial during injection, since aggregates that form large masses can cause adverse reactions such as pain. Furthermore, maintaining a good mRNA encapsulation rate will improve the corresponding protein expression and therefore the efficacy of therapeutic exploration.

[0518] Example 3: Effect of the freeze-drying process on LNP stability Experimental design LNPs containing mRNA-luc were prepared as described in Example 1.

[0519] The LNPs were either conventionally lyophilized in vials (conventional) or spray-freeze dried by prilling (SFD) (see Example 1). The lyophilisates were stored at +5° C. for TO, 3, 6 or 11 months.

[0520] Before measurements were taken, the lyophilized LNPs were resuspended in water for injection.

[0521] The size and concentration of the resuspended LNPs were measured by NTA (see Example 1).

[0522] The RNA encapsulation rate of the resuspended LNPs was measured by the Quant-iT™ RiboGreen® RNA Assay (see Example 1).

[0523] result The mean and modal diameters of resuspended LNPs obtained by NTA, as well as concentrations, are summarized in Tables 4, 5 and 6.

[0524] [Table 5]

[0525] [Table 6]

[0526] [Table 7]

[0527] Taken together, the above data tend to indicate that spray-freeze-dried and conventionally freeze-dried LNPs are stable over time, especially when stored at +5°C.

[0528] The RNA encapsulation rates and total RNA of the resuspended LNPs are summarized in Tables 7 and 8.

[0529] [Table 8]

[0530] [Table 9]

[0531] The data show that when compared to the mRNA encapsulation rates in the liquid, prior to the lyophilization step, spray-freeze dried LNPs tend to have more stable mRNA encapsulation rates than conventionally lyophilized LNPs, especially after 11 months of storage.

[0532] This data indicates that spray freeze drying of LNPs improves the stability of mRNA encapsulation rate.

[0533] Taken together, the results indicate that spray freezing of LNPs improves LNP stability by preventing or reducing LNP aggregation and by maintaining mRNA encapsulation rates.

[0534] Reduction of LNP aggregation can be beneficial during injection, since aggregates that form large masses can cause adverse reactions such as pain. Furthermore, maintaining a good mRNA encapsulation rate will improve the corresponding protein expression and therefore the efficacy of therapeutic exploration.

[0535] Example 4: Effect of freezing and lyophilization processes on mRNA integrity Experimental design LNPs containing mRNA-luc were prepared as described in Example 1.

[0536] The LNPs were either conventionally freeze-dried in vials (conventional freeze-drying) or spray-freeze-dried by prilling (SFD) (see Example 1). The lyophilisates were stored at +5° C. for TO, 3 or 6 months.

[0537] The integrity of the mRNA was measured as described in Example 1.

[0538] result The predicted size of the luciferase mRNA (mRNA-Luc; reference number: L-7602 TriLink™ Biotechnologies) is 1941 bases.

[0539] Whatever the freeze-drying process, conventional freeze-drying versus spray freeze-drying, or duration of storage at +5° C., 0, 3 or 6 months, the integrity of the mRNA was maintained.

[0540] Example 5: Effect of the freeze-drying process on in vivo mRNA expression Experimental design LNPs containing mRNA-Luc were prepared as described in Example 1.

[0541] The LNPs were either conventionally freeze-dried in vials (conventional freeze-drying) or spray-freeze-dried (SFD) by prilling (see Example 1). The lyophilisates were stored at +5° C. for 320 days until further use.

[0542] Three groups of mice were treated: (1) LNPs with mRNA and conventionally freeze-dried, (3) LNPs with mRNA and spray-freeze-dried, and (3) LNPs without mRNA and not freeze-dried prior to use.

[0543] Bioluminescence was measured as described in Example 1.

[0544] result The bioluminescence results obtained are summarized in Table 9.

[0545] [Table 10]

[0546] As shown by the data above, at 6 and 24 hours post-injection, the levels of bioluminescence, and hence luciferase expression, were significantly greater in mice injected with spray-freeze-dried LNPs versus conventionally freeze-dried LNPs.

[0547] At 6 hours, the level of bioluminescence obtained with spray-freeze-dried LNPs was approximately 40-fold higher than that obtained with conventionally freeze-dried LNPs. Three days after injection, the level of bioluminescence obtained with spray-freeze-dried LNPs was still approximately 15-fold higher than that obtained with conventionally freeze-dried LNPs.

[0548] Enhanced expression of the protein encoded by the mRNA may find beneficial interest for therapeutic applications, such as, for example, in immunization and vaccine applications where enhanced expression of an antigen may serve to obtain an enhanced immune response.

[0549] [References] Adali MB,Barresi AA,Boccardo G,Pisano R.Spray Freeze-Drying as a Solution to Continuous Manufacturing of Pharmaceutical Products in Bulk.Processes.2020;8(6):709.https: / / doi.org / 10.3390 / pr8060709 Ali ME,Lamprecht A.Spray freeze drying as an alternative technique for lyophilization of polymeric and lipid-based nanoparticles.Int J Pharm.2017;.516(1-2):170-177.doi:10.1016 / j.ijpharm.2016.11.023 Ball, RL, Bajaj, P., & Whitehead, KA (2016). Achieving long-term stability of lipid nanoparticles: examining the effect of pH, temperature, and lyophilization. Int J Nanomedicine, 12, 305-315. https: / / doi.org / 10.2147 / IJN.S123062 Belliveau NM,Huft J,Lin PJ,et al.Microfluidic Synthesis of Highly Potent Limit-size Lipid Nanoparticles for In Vivo Delivery of siRNA.Mol Ther Nucleic Acids.2012;1(8):e37.Published 2012 Aug 14.doi:10.1038 / mtna.2012.28 Brandenberger H,Widmer F (1998) A new multinozzle encapsulation immobilisation system to produce uniform beads of alginate.J Biotechnol 63:73-80. Brunelle JL,Green R.In vitro transcription from plasmid or PCR-amplified DNA.Methods Enzymol.2013;530:101-114.doi:10.1016 / B978-0-12-420037-1.00005-1 Crommelin DJA,Anchordoquy TJ,Volkin DB,Jiskoot W,Mastrobattista E.Addressing the Cold Reality of mRNA Vaccine Stability.J Pharm Sci.2021;110(3):997-1001.doi:10.1016 / j.xphs.2020.12.006 Fukushige K,Tagami T,Naito M,et al.Developing spray-freeze-dried particles containing a hyaluronic acid-coated liposome-protamine-DNA complex for pulmonary inhalation.Int J Pharm.2020;583:119338.doi:10.1016 / j.ijpharm.2020.119338 Geall AJ,Mandl CW,Ulmer JB.RNA:the new revolution in nucleic acid vaccines.Semin Immunol.2013;25(2):152-159.doi:10.1016 / j.smim.2013.05.001; Khurana,A.,Allawadhi,P.,Khurana,I.,Allwadhi,S.,Weiskirchen,R.,Banothu,A.K.,Chhabra,D.,Joshi,K.,& Bharani,K.K.(2021).Role of nanotechnology behind the success of mRNA vaccines for COVID-19.Nano today,38,101142.https: / / doi.org / 10.1016 / j.nantod.2021.101142 Maier MA,Jayaraman M,Matsuda S,Liu J,Barros S,Querbes W,Tam YK,Ansell SM,Kumar V,Qin J,Zhang X,Wang Q,Panesar S,Hutabarat R,Carioto M,Hettinger J,Kandasamy P,Butler D,Rajeev KG,Pang B,Charisse K,Fitzgerald K,Mui BL,Du X,Cullis P,Madden TD,Hope MJ,Manoharan M,Akinc A.Biodegradable lipids enabling rapidly eliminated lipid nanoparticles for systemic delivery of RNAi therapeutics.Mol Ther.2013 Aug;21(8):1570-8.doi:10.1038 / mt.2013.124.Epub 2013 Jun 25.PMID:23799535;PMCID:PMC3734658. Meesters,G.M.H.(1992) Mechanisms of droplet formation.Thesis,Technische Universiteit Delft,Delft University Press. Rayleigh,L.(1878),On The Instability Of Jets.Proceedings of the London Mathematical Society,s1-10:4-13.https: / / doi.org / 10.1112 / plms / s1-10.1.4 Reichmuth,A.M.,Oberli,M.A.,Jaklenec,A.,Langer,R.,& Blankschtein,D.(2016).mRNA vaccine delivery using lipid nanoparticles.Therapeutic delivery,7(5),319-334.https: / / doi.org / 10.4155 / tde-2016-0006 Thi,T.,Suys,E.,Lee,J.S.,Nguyen,D.H.,Park,K.D.,& Truong,N.P.(2021).Lipid-Based Nanoparticles in the Clinic and Clinical Trials:From Cancer Nanomedicine to COVID-19 Vaccines.Vaccines,9(4),359.https: / / doi.org / 10.3390 / vaccines9040359 Trenkenschuh E,Friess W.Freeze-drying of nanoparticles:How to overcome colloidal instability by formulation and process optimization.Eur J Pharm Biopharm.2021 Aug;165:345-360.doi:10.1016 / j.ejpb.2021.05.024.Epub 2021 May 27.PMID:34052428. Schoenmaker,L.,Witzigmann,D.,Kulkarni,J.A.,Verbeke,R.,Kersten,G.,Jiskoot,W.,& Crommelin,D.(2021).mRNA-lipid nanoparticle COVID-19 vaccines:Structure and stability.Int J Pharm,601,120586.https: / / doi.org / 10.1016 / j.ijpharm.2021.120586 US 9,512,073-Amino acid-,peptide-and polypeptide-lipids,isomers,compositions,and uses thereof US 10,201,618-Alkenyl substituted 2,5-piperazinediones,compositions,and uses thereof U.S.Pat.No.4,373,071-Solid-phase synthesis of polynucleotides U.S.Pat.No.4,401,796-Solid-phase synthesis of polynucleotides U.S.Pat.No.4,415,732-Phosphoramidite compounds and processes U.S.Pat.No.4,458,066-Process for preparing polynucleotides U.S.Pat.No.4,500,707-Nucleosides useful in the preparation of polynucleotides U.S.Pat.No.4,668,777-Phosphoramidite nucleoside compounds U.S.Pat.No.4,973,679-Process for oligonucleotide synthesis using phosphoramidite intermediates U.S.Pat.No.5,047,524-Automated system for polynucleotide synthesis and purification U.S.Pat.No.5,132,418-Process for preparing polynucleotides U.S.Pat.No.5,153,319-Process for preparing polynucleotides U.S.Pat.No.5,262,530-Automated system for polynucleotide synthesis and purification U.S.Pat.No.5,700,642-Oligonucleotide sizing using immobilized cleavable primers Wanning S,Sueverkruep R,Lamprecht A.Pharmaceutical spray freeze drying.Int J Pharm.2015;488(1-2):136-153.doi:10.1016 / j.ijpharm.2015.04.053 WO 2009 / 109550 A1-Process for Stabilizing an Adjuvant Containing Vaccine Composition WO 2013 / 050156 A1-Process Line for The Production of Freeze -Dried Particles WO 2013 / 050158 A1-Heating Device for Rotary Drum Freeze-Dryer WO 2013 / 050159 A1-A Process for The Bulkware Production of Freeze-Dried Particles Under Closed Conditions WO 2013 / 050162 A1-Process Line for The Production of Freeze -Dried Particles WO 2015 / 024667A1-Method for Increasing Expression of Rna-Encoded Proteins WO 2016 / 012414 A1-Liquid Feeding Device for The Generation of Droplets WO 2020 / 139783 A2-Polypeptides Utiles Pour L’edition De Genes Et Procedes D’utilisation WO 2022 / 099003 A1-Lipid Nanoparticles for Delivering mRNA Vaccines Zhao P,Hou X,Yan J,et al.Long-term storage of lipid-like nanoparticles for mRNA delivery.Bioact Mater.2020;5(2):358-363.Published 2020 Mar 18.doi:10.1016 / j.bioactmat.2020.03.001

Claims

1. A method for freezing lipid nanoparticles (LNPs), the LNPs comprising at least a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or an ester thereof as lipid components, the LNPs comprising at least a nucleic acid, the method comprising: a) providing a liquid composition comprising said LNPs; b) atomizing the composition of step a) under conditions suitable to obtain droplets; c) freezing the droplets obtained in step b) to obtain frozen LNPs.

2. 10. The method of claim 1, wherein step b) of atomizing is performed with an electromagnetic droplet stream generator, a piezoelectric droplet stream generator, a hydraulic droplet aerosol generator, a compressed air nozzle, an ultrasonic atomizing nozzle, a thermal droplet stream generator, or an electrohydrodynamic droplet (EHD) generator.

3. 10. The method of claim 1, wherein the freezing step c) is carried out by spraying the droplets into a cryogenic atmosphere, with pressurized carbon dioxide, into vapor above a cryogenic liquid, into a cryogenic liquid, or onto a cold solid surface.

4. 1. A method for freeze-drying lipid nanoparticles (LNPs), said method comprising at least: d) obtaining frozen LNPs according to the method of claim 1; e) drying the frozen LNPs obtained in step d) under suitable conditions to obtain freeze-dried LNPs; A method comprising:

5. 5. The method of claim 4, wherein the drying step e) is carried out by rotary drum vacuum freeze drying, atmospheric drying in a stream of cold air, vacuum chamber freeze drying, or vacuum tunnel freeze drying.

6. The LNPs comprise, in w / w% based on the total weight of the lipid component of the LNP: 20 to 60%, or 25% to 60%, or 30% to 55%, or 35% to 55%, or 35% to 50%, or 40% to 50% of said ionizable cationic lipids, and / or - 5 to 50%, or 5% to 45%, or 9% to 40%, or 9% to 30% of said neutral lipids, and / or 20-55%, or 20%-50%, or 25%-45% of said steroid alcohol or ester thereof The method of claim 1 , comprising:

7. - the ionizable cationic lipids are [(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl] 4-(dimethylamino)butanoate (D-Lin-MC3-DMA); 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA); 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLin-DMA); di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102); [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315); [3-(dimethylamino)-2-[(Z)-octadec-9-enoyl]oxypropyl](Z)-oct-9-enoate (DODAP); 2,5-bis(3-aminopropylamino)-N-[2-[di(heptadecyl)amino]-2-oxoethyl]pentanamide (DOGS); [(3S,8S,9S, 10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-yl]N-[2-(dimethylamino)ethyl]carbamate (DC-Chol); tetrakis(8-methylnonyl) 3,3',3'',3'''-(((methylazanediyl)bis(propane-3,1-diyl))bis(azanetriyl))tetrapropionate (306Oi10); decyl(2-(dioctylammonio)ethyl)phosphate (9A1P9); ethyl 5,5-di((Z)-heptadec-8-en-1-yl)-1-(3-(pyrrolidin-1-yl)propyl)-2,5-dihydro-1H-imidazole-2-carboxylate (A2-Iso5-2DC18);Bis(2-(dodecyldisulfanyl)ethyl) 3,3'-((3-methyl-9-oxo-10-oxa-13,14-dithia-3,6-diazahexacosyl)azanediyl)dipropionate (BAME-O16B); 1,1'-((2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azane 3,6-bis(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2,5-dione (cKK-E12); hexa(octan-3-yl)9,9',9'',9''',9'''',9''''-((((benzene-1,3,5-tricarbonyl)iris(azanediyl)) Tris(propane-3,1-diyl))tris(azanetriyl))hexanonanoate (FTT5); (((3,6-dioxopiperazine-2,5-diyl)bis(butane-4,1-diyl))bis(azanetriyl))tetrakis(ethane-2,1-diyl) (9Z,9'Z,9''Z,9'''Z,12Z,12'Z,12''Z,12'''Z)-tetrakis(octadeca-9,12-di enoate) (OF-Deg-Lin); TT3; N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide; N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5); heptadecan-9-yl 8-((2-hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate (Lipid 5); 【Chemical 1】 and combinations thereof; and / or - said neutral lipid is selected from the group comprising DSPC; DPPC; DMPC; POPC; DOPC; phosphatidylethanolamines such as DOPE, DPPE, DMPE, DSPE, DLPE; sphingomyelin; ceramide, and combinations thereof; and / or The sterols or esters thereof include cholesterol and its derivatives; Lugosterol; Desmosterol (3β-hydroxy-5,24-cholestadiene); Stigmasterol (stigmasterol-5,22-dien-3-ol); Lanosterol (8,24-lanostadien-3b-ol); 7-dehydrocholesterol (Δ5,7-cholesterol); Dihydrolanosterol (24,25-dihydrolanosterol); Zymosterol (5α-cholesta-8,24-dien-3β-ol); Lathosterol (5α-cholesta-7- diosgenin ((3β,25R)-spirost-5-en-3-ol); sitosterol (22,23-dihydrostigmasterol); sitostanol; campesterol (campest-5-en-3β-ol); campestanol (5a-campestan-3b-ol); 24-methylenecholesterol (5,24(28)-cholestadien-24-methylene-3β-ol); cholesteryl margarate (cholest-5-en-3β-yl heptadecanoate); cholesteryl oleate; cholesteryl stearate; and combinations thereof, The method of claim 1.

8. The method of claim 1, wherein the LNP further comprises at least one PEG-lipid as a lipid component.

9. 9. The method of claim 8, wherein the LNP comprises 0.5-15%, or 0.5%-10%, or 0.8%-5%, or 1%-3%, or 1.5%-2% of the PEG-lipid, w / w% based on the total weight of the lipid component of the LNP.

10. 9. The method of claim 8, wherein the PEG-lipid is selected from the group consisting of PEG-DAG; DMG-PEG-2000; PEG-PE; PEG-S-DAG; PEG-S-DMG; PEG-cer; PEG-dialkoxypropylcarbamate; 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159); and combinations thereof.

11. The LNPs comprise, in w / w% based on the total weight of the lipid component of the LNP: 50% ionizable cationic lipid, 10% neutral lipid, 38.5% cholesterol, and 1.5% PEG-lipid, or 46.3% ionizable cationic lipids, 9.4% neutral lipids, 42.7% cholesterol, and 1.6% PEG-lipids, or 47.4% ionizable cationic lipid, 10% neutral lipid, 40.9% cholesterol, and 1.7% PEG-lipid, or 40% ionizable cationic lipid, 30% neutral lipid, 28.5% cholesterol, and 1.5% PEG-lipid, or 50% 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 10% DSPC, 38.5% cholesterol, and 1.5% DMG-PEG-2000, or 46.3% [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 9.4% DSPC, 42.7% cholesterol, and 1.6% 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), 47.4% [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl) bis(2-hexyldecanoate) (ALC-0315), 10% DSPC, 40.9% cholesterol, and 1.7% 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide (ALC-0159), or 40% cKK-E10, 30% DOPE, 28.5% cholesterol, and 1.5% DMG-PEG-2000, or 40% OF-02, 30% DOPE, 28.5% cholesterol, and 1.5% DMG-PEG-2000 The method of claim 8, comprising:

12. The method of any one of claims 1 to 11, wherein the nucleic acid is RNA.

13. 12. The method of any one of claims 1 to 11, wherein the nucleic acid is messenger RNA (mRNA); microRNA (miRNA); short (or small) interfering RNA (siRNA); small hairpin RNA (shRNA); long non-coding RNA (lncRNA); asymmetric interfering RNA (aiRNA); self-amplifying RNA (saRNA); guide RNA (gRNA); and combinations thereof.

14. The method of claims 1 to 11, wherein the nucleic acid encodes a therapeutic agent selected from among a genome-editing polypeptide, a chemokine, a cytokine, a growth factor, an antibody, an enzyme, a structural protein, a blood protein, a hormone, a transcription factor, or an antigen.

15. The method of any one of claims 1 to 11, wherein the liquid composition comprising the LNP comprises a cryoprotectant.

16. 16. The method of claim 15, wherein the cryoprotectant is a mixture of trehalose and dextran.

17. 17. The method of claim 16, wherein the trehalose and the dextran are present in equal amounts weight / volume percent relative to the total volume of the composition.

18. A freeze-dried LNP comprising at least a nucleic acid and at least, as lipid components, a cationic ionizable lipid, a neutral lipid, and a steroid alcohol or an ester thereof, wherein the freeze-dried LNP is in a freeze-dried micropellet.

19. The lyophilized LNP of claim 18, wherein the nucleic acid encodes a therapeutic agent selected from among a genome-editing polypeptide, a chemokine, a cytokine, a growth factor, an antibody, an enzyme, a structural protein, a blood protein, a hormone, a transcription factor, or an antigen.

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