A nucleic acid drug freeze-dried preparation, a preparation method and application thereof

Abstract

Provided are a nucleic acid-containing lipid nanoparticle lyophilized preparation and a preparation method and application thereof. A nucleic acid-containing lipid nanoparticle lyophilized preparation is provided, which comprises: i) a nucleic acid-containing lipid nanoparticle; ii) and a buffer reagent containing a lyoprotectant. The prepared nucleic acid-LNP lyophilized preparation has a uniform sample particle size, a small polydispersity coefficient, a high encapsulation rate, and a high RNA integrity after reconstitution. At the same time, nucleic acid leakage from the nanoparticles is avoided, and a lyophilized storage method is further provided, so that the nanoparticles can exist in a stable form under refrigerated conditions.

Description

A freeze-dried preparation of nucleic acid drug and its preparation method and application

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This disclosure claims priority to and the benefit of the following Chinese patent application filed with the State Intellectual Property Office of China: Chinese Patent Application No. 202311333065.3, filed on October 13, 2023, entitled “A Lyophilized Formulation of Nucleic Acid Drug, Preparation Method, and Use Thereof.” The entire text of the aforementioned patent application is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure belongs to the field of medical technology, and specifically relates to a freeze-dried preparation of a nucleic acid drug, a preparation method thereof, and an application thereof. Background Art

[0004] Spikevax and Comirnaty, the lipid nanoparticle (LNP)-encapsulated messenger RNA (mRNA) vaccines developed by Moderna and Pfizer BioNTech, respectively, have played a significant role in the global fight against SARS-CoV-2 due to their favorable safety profile and exceptionally high protective efficacy (>90%). The success of these two nucleic acid vaccines has broadened the therapeutic potential of nucleic acid drugs in the field of infectious diseases and other areas. This is primarily due to the LNP's unique core-shell structure, which encapsulates the nucleic acid drug in the center, protecting it from degradation by RNA endonucleases and exonucleases. Furthermore, the use of ionizable lipids in the LNP enhances its efficiency in intracellular delivery, thereby exerting both cellular and humoral immune responses. However, these nucleic acid drugs require relatively low storage temperatures (-80°C), and during storage, interactions between certain chemical groups within the encapsulated lipid nanoparticles, such as oxidation, hydrolysis, or transesterification, can lead to the shedding and degradation of mRNA and other nucleic acid drugs. This poses significant obstacles to the long-term storage and transportation of mRNA and other nucleic acid drugs.

[0005] Freeze-drying (lyophilization) is a common technique in the pharmaceutical industry. It removes moisture from products, creating a lyophilized powder, thereby improving product stability. It also facilitates the transportation of nucleic acid drugs, such as LNP-encapsulated mRNA (mRNA-LNP), to remote areas, eliminating the need for demanding cold chain transportation. This reduces transportation costs and expands the accessibility of mRNA nucleic acid drugs.

[0006] Summary of the Invention

[0007] The present disclosure aims to provide a lyophilized formulation containing nucleic acid lipid nanoparticles. By adding a stabilizer to the aqueous medium in which the lipid nanoparticles are prepared and simultaneously adding a suitable lyoprotectant to the resulting lipid nanoparticle suspension, the particle size uniformity of the nucleic acid-LNP (e.g., mRNA-LNP) nanoparticles, high nucleic acid encapsulation efficiency, mRNA integrity, and biological activity are ensured, while preventing mRNA leakage from the mRNA-LNP nanoparticles. Furthermore, a method for preserving the mRNA-LNP nanoparticles is provided, enabling the mRNA-LNP nanoparticles to be stored in a stable form under refrigerated conditions.

[0008] Another object of the present disclosure is to provide a freeze-drying method for a freeze-dried formulation containing nucleic acid lipid nanoparticles.

[0009] Another object of the present disclosure is to provide related uses of lyophilized formulations containing nucleic acid lipid nanoparticles.

[0010] The present disclosure provides a lyophilized formulation of lipid nanoparticles containing nucleic acids, the lyophilized formulation comprising:

[0011] i) lipid nanoparticles containing nucleic acids;

[0012] ii) and a buffer reagent containing a lyoprotectant.

[0013] In some embodiments, a lyophilized formulation of lipid nanoparticles containing nucleic acids, the lyophilized formulation comprising:

[0014] i) a lipid nanoparticle containing a nucleic acid, wherein the lipid nanoparticle includes a stabilizer;

[0015] ii) Buffer reagent containing lyoprotectant.

[0016] In some embodiments, a lyophilized formulation of lipid nanoparticles containing nucleic acids, the lyophilized formulation comprising:

[0017] i) lipid nanoparticles containing nucleic acids, wherein the lipid nanoparticles include a stabilizer, wherein the weight volume concentration (w / v%) of the stabilizer in the prepared buffer containing the nucleic acid lipid nanoparticles and the freeze-drying protectant is about 0.1-5%;

[0018] ii) Buffer reagent containing lyoprotectant.

[0019] In some embodiments, a lyophilized formulation of lipid nanoparticles containing nucleic acids, the lyophilized formulation comprising:

[0020] i) lipid nanoparticles containing nucleic acids, the lipid nanoparticles comprising nucleic acids, lipids, and a stabilizer; wherein the stabilizer has a mass volume concentration (w / v%) of about 0.1-5% in the prepared buffer containing the nucleic acid lipid nanoparticles and a freeze-drying protectant;

[0021] ii) Buffer reagent containing lyoprotectant.

[0022] In some embodiments, the lyophilized formulation containing nucleic acid lipid nanoparticles described in the present disclosure has an encapsulation rate of 85% or more, preferably 90% or more, and more preferably 92% or more after reconstitution.

[0023] In some embodiments, the lyophilized formulation containing nucleic acid lipid nanoparticles described in the present disclosure has a nucleic acid integrity of more than 90%, preferably more than 92%, and more preferably more than 93% after reconstitution of the lyophilized formulation.

[0024] In some embodiments, in the lyophilized formulation of nucleic acid-containing lipid nanoparticles disclosed herein, the absolute value of the change in nucleic acid integrity before and after lyophilization is less than 5%, preferably 2%, and more preferably 1%.

[0025] In some embodiments, the stabilizer is selected from any one of sucrose, trehalose, maltose, lactose, and mannitol.

[0026] In some embodiments, the stabilizer is selected from sucrose or trehalose.

[0027] In some embodiments, the weight volume concentration (w / v%) of the stabilizer in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.1% to about 4%.

[0028] In some embodiments, the weight volume concentration (w / v%) of the stabilizer in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.1% to about 3%.

[0029] In some embodiments, the weight volume concentration (w / v%) of the stabilizer in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.1% to about 2%.

[0030] In some embodiments, the weight volume concentration (w / v%) of the stabilizer in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.1% to about 1.5%.

[0031] In some embodiments, the weight volume concentration (w / v%) of the stabilizer in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.5% to about 1%.

[0032] In some embodiments, the stabilizer has a mass volume (w / v%) concentration of about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, or about 3.0% in the formulated buffer containing nucleic acid lipid nanoparticles and a freeze-drying protectant.

[0033] In some embodiments, the mass volume concentration (w / v %) of the stabilizer in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%.

[0034] In some embodiments, the buffering agent is selected from Tris, citrate, acetate, or phosphate buffers.

[0035] In some embodiments, the buffering reagent is selected from Tris buffer.

[0036] In some embodiments, the pH of the buffer reagent is 6.5-8.5.

[0037] In some embodiments, the pH of the buffer reagent is 6.9-7.9.

[0038] In some embodiments, the pH of the buffer reagent is 7.4.

[0039] In some embodiments, the buffering agent has a solubility of about 2 mM to about 30 mM.

[0040] In some embodiments, the buffering agent has a solubility of about 2 mM to about 20 mM.

[0041] In some embodiments, the buffering agent has a solubility of about 2 mM to about 15 mM.

[0042] In some embodiments, the buffering agent has a solubility of about 2 mM to about 9 mM.

[0043] In some embodiments, the buffering agent has a solubility of about 4 mM to about 9 mM.

[0044] In some embodiments, the buffering agent has a solubility of about 5 mM to about 9 mM.

[0045] In some embodiments, the lyoprotectant is selected from any one or more of maltose, lactose, sucrose, trehalose, isomaltose, nigerose, or kojibiose.

[0046] In some embodiments, the lyoprotectant is selected from sucrose or trehalose.

[0047] In some embodiments, the lyoprotectant is selected from sucrose.

[0048] In some embodiments, in the formulated buffer containing nucleic acid lipid nanoparticles and a lyoprotectant, the mass volume concentration (w / v%) of the lyoprotectant is about 4% to about 25%.

[0049] In some embodiments, in the formulated buffer containing nucleic acid lipid nanoparticles and a lyoprotectant, the mass volume concentration (w / v%) of the lyoprotectant is about 5% to about 25%.

[0050] In some embodiments, in the formulated buffer containing nucleic acid lipid nanoparticles and a lyoprotectant, the mass volume concentration (w / v%) of the lyoprotectant is about 5% to about 20%.

[0051] In some embodiments, in the formulated buffer containing nucleic acid lipid nanoparticles and a lyoprotectant, the mass volume concentration (w / v%) of the lyoprotectant is about 5% to about 15%.

[0052] In some embodiments, in the formulated buffer containing nucleic acid lipid nanoparticles and a freeze-drying protectant, the mass volume concentration (w / v%) of the freeze-drying protectant is about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, or about 15%.

[0053] In some embodiments, the weight volume (w / v%) concentration of the lyoprotectant in the formulated buffer containing nucleic acid lipid nanoparticles and the lyoprotectant is about 10%.

[0054] In some embodiments, the buffer reagent containing a lyoprotectant further comprises a surfactant.

[0055] In some embodiments, the surfactant is selected from poloxamers, such as any one or more of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, or poloxamer 407.

[0056] In some embodiments, the surfactant is selected from Poloxamer 188.

[0057] In some embodiments, the mass volume concentration (w / v%) of the surfactant in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.1% to about 3%.

[0058] In some embodiments, the mass volume concentration (w / v%) of the surfactant in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.1% to about 2%.

[0059] In some embodiments, the mass volume concentration (w / v%) of the surfactant in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.1% to about 1%.

[0060] In some embodiments, the mass volume concentration (w / v%) of the surfactant in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protectant is about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, 2.5%, or about 3.0%.

[0061] In some embodiments, the concentration of the surfactant by mass volume (w / v%) in the formulated buffer containing nucleic acid lipid nanoparticles and a freeze-drying protectant is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%.

[0062] In another aspect, the present disclosure provides a freeze-drying method for a freeze-dried formulation comprising nucleic acid lipid nanoparticles, the method comprising the following steps:

[0063] a) preparing an aqueous solution containing nucleic acid and an organic solution containing lipid, respectively, wherein the solution containing nucleic acid contains about 0.1-5% (w / v%) of a stabilizer; preferably, the organic solution is an ethanol solution;

[0064] b) preparing the lipid nanoparticles containing the nucleic acid, and then dissolving the lipid nanoparticles in a buffer reagent containing a lyophilization protectant to obtain a lipid nanoparticle suspension containing the nucleic acid;

[0065] c) freeze-drying the lipid nanoparticle suspension comprising the nucleic acid to form a lyophilized preparation.

[0066] In some embodiments, in a freeze-drying method comprising lipid nanoparticles, step a) further comprises the following steps:

[0067] a-1) preparing an aqueous solution containing nucleic acid: dissolving the nucleic acid in an aqueous medium, adding the stabilizer to the aqueous medium, wherein the stabilizer has a mass volume concentration (w / v%) of about 0.1% to 5%;

[0068] a-2) Preparation of ethanol solution containing lipid: The lipid compound is dissolved in ethanol.

[0069] In some embodiments, the aqueous medium is selected from citrate buffer or sodium acetate buffer, and the stabilizer is 0.1% to 1% trehalose or 0.1% to 2% sucrose, preferably 1% sucrose or 0.5% trehalose.

[0070] In some embodiments, the aqueous medium is selected from a citrate buffer or a sodium acetate buffer, and the pH of the buffer is 4 to 5. Preferably, the pH of the buffer is 4.

[0071] In some embodiments, the method for synthesizing the lipid nanoparticles containing nucleic acids in step b) adopts a classic microfluidic method. Specifically, an ethanol solution containing lipids and an aqueous solution containing nucleic acids are mixed by microfluidics to prepare lipid nanoparticles containing nucleic acids, which are then concentrated by ultrafiltration, and then the lipid nanoparticles containing nucleic acids are dissolved in a buffer reagent containing a lyoprotectant. Preferably, in the microfluidic method, the molar ratio of nitrogen atoms of the cationic lipid to phosphorus atoms of the mRNA (N / P ratio) is maintained at 5-7; most preferably, the N / P ratio is 6.

[0072] In some embodiments, in step c), the freeze drying in the present disclosure comprises the following steps: a pre-freezing stage, a sublimation drying stage, and an analytical drying stage, wherein:

[0073] Pre-freezing stage: temperature -50℃~-20℃, pre-freezing time is 1~8h; preferably temperature -50℃~-40℃, pre-freezing time is 1~5h;

[0074] Sublimation drying stage: temperature -50℃~-20℃, holding time 16~60h;

[0075] The drying stage is carried out at a temperature of 5 to 25°C for 6 to 24 hours. In some embodiments, in step b) of dissolving the nucleic acid-containing lipid nanoparticles in a buffer containing a lyoprotectant, the concentration of nucleic acid in the nucleic acid-containing lipid nanoparticles is 0.1 to 0.5 mg / ml, preferably 0.3 to 0.5 mg / ml.

[0076] The buffer reagent containing the freeze-drying protective agent is: 5mM TRIS buffer containing 5-15% sucrose by mass volume fraction (w / v%);

[0077] Preferably, the buffer reagent further contains 0.05-1% poloxamer 188 by mass volume fraction (w / v%);

[0078] More preferably, the buffer reagent contains 10% sucrose by mass volume fraction (w / v%) and 0.2% poloxamer 188 by mass volume fraction (w / v%), and has a pH of 7.4.

[0079] In some embodiments, the lyophilized formulation of nucleic acid-containing lipid nanoparticles described in the present disclosure is prepared using the aforementioned method of the present disclosure.

[0080] In some embodiments, the nucleic acid is selected from RNA.

[0081] In some embodiments, the RNA comprises one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, sgRNA, and tsRNA.

[0082] In some embodiments, the RNA is selected from mRNA.

[0083] In some embodiments, the nucleic acid is mRNA encoding coronavirus, influenza, herpes zoster virus, rabies, Zika virus, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human papillomavirus (HPV), human immunodeficiency virus (HIV), or Epstein-Barr virus (EBV).

[0084] In some embodiments, the nucleic acid-containing lipid nanoparticles include nucleic acids and lipids, wherein the lipids include one or more of cationic lipids, neutral helper lipids, cholesterol, and PEG-modified lipids, preferably two or more.

[0085] In some embodiments, the nucleic acid-containing lipid nanoparticles comprise a nucleic acid and a lipid, wherein the lipid comprises:

[0086] a) cationic lipids;

[0087] b) neutral helper lipids;

[0088] c) cholesterol; and

[0089] d) PEG-modified lipids.

[0090] In some embodiments, the nucleic acid-containing lipid nanoparticles have an N / P ratio of 5:1 to 10:1.

[0091] In some embodiments, the nucleic acid-containing lipid nanoparticles have an N / P ratio of 6.

[0092] In some embodiments, in the nucleic acid-containing lipid nanoparticles, based on the total molar amount of lipids as 100%, the molar ratio of each lipid component is:

[0093] a) Cationic lipid 45% to 50%;

[0094] b) neutral helper lipids 5% to 10%;

[0095] c) Cholesterol 38% to 48%;

[0096] and d) PEG-modified lipids 0-3%.

[0097] In some embodiments, the cationic lipid is selected from N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethanoloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315, or any one or more thereof.

[0098] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the cationic lipid is 45% to 48%.

[0099] In some embodiments, the neutral helper lipid is selected from any one or more of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) or phosphatidylethanolamine (DLPE).

[0100] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the neutral helper lipid is 6% to 10%.

[0101] In some embodiments, based on the total molar amount of lipid as 100%, the molar ratio of the neutral helper lipid is 8% to 10%.

[0102] In some embodiments, the PEG-modified lipid is selected from any one or more of methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), MG-PEG2000, DMG-PEG5000, and PEG2000.

[0103] In some embodiments, the molar ratio of the PEG-modified lipid is 1% to 3%, based on the total molar amount of the lipid being 100%.

[0104] In some embodiments, the molar ratio of the PEG-modified lipid is 1% to 2%, based on the total molar amount of the lipid being 100%.

[0105] In some embodiments, based on the total molar amount of lipids being 100%, the cholesterol accounts for 40% to 46%.

[0106] In some embodiments, based on the total molar amount of lipids being 100%, the cholesterol accounts for 42% to 45%.

[0107] In another aspect, the present disclosure also provides a method for preventing or treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the lyophilized formulation described herein reconstituted in a liquid medium.

[0108] In some embodiments, the reconstituted lyophilized formulation is administered intravenously.

[0109] In some embodiments, the reconstituted lyophilized formulation is administered intramuscularly.

[0110] In some embodiments, the reconstituted lyophilized formulation is administered via inhalation.

[0111] In some embodiments, the reconstituted lyophilized formulation is administered subcutaneously.

[0112] In some embodiments, the disease or condition comprises cancer or viral infection.

[0113] In some embodiments, the viral infection comprises coronavirus, influenza virus, herpes zoster virus, rabies virus, Zika virus, respiratory syncytial virus (RSV), cytomegalovirus (CMV), human papillomavirus (HPV), human immunodeficiency virus (HIV), and Epstein-Barr virus (EBV).

[0114] On the other hand, the present disclosure also provides a use of a lyophilized formulation containing nucleic acid lipid nanoparticles in the preparation of nucleic acid drugs.

[0115] In some embodiments, the nucleic acid drug can be used to treat cancer.

[0116] On the other hand, the present disclosure also provides a use of a freeze-dried preparation containing nucleic acid lipid nanoparticles in the preparation of a nucleic acid vaccine.

[0117] In some embodiments, the nucleic acid vaccine comprises a coronavirus vaccine, an influenza virus vaccine, a herpes zoster virus vaccine, a rabies virus vaccine, a Zika virus vaccine, a respiratory syncytial virus (RSV) vaccine, a cytomegalovirus (CMV) vaccine, a human papillomavirus (HPV) vaccine, a human immunodeficiency virus (HIV) vaccine, or an Epstein-Barr virus (EBV) vaccine.

[0118] Compared with the prior art, the present disclosure has the following beneficial effects:

[0119] The nucleic acid-LNP freeze-dried preparation prepared in this disclosure can be completely reconstituted within 10 seconds. The reconstituted sample has a uniform particle size, a low polydispersity coefficient, a high encapsulation efficiency, and high mRNA integrity. In addition, the in vitro transfection and in vivo immunogenicity after freeze-drying and reconstitution are not significantly different from those before freeze-drying. At the same time, stability results show that even after being placed at 2-8°C for two months, it still maintains a high level of in vivo biological activity, significantly reducing storage and transportation costs, improving transportation efficiency, and accessibility to remote areas.

[0120] Any embodiment of any aspect of the present disclosure may be combined with other embodiments, provided that no contradiction occurs. In addition, in any embodiment of any aspect of the present disclosure, any technical feature may be applicable to the technical feature in other embodiments, provided that no contradiction occurs.

[0121] Definitions and Explanations of Terms

[0122] Unless otherwise defined in the present disclosure, scientific and technical terms related to the present disclosure shall have the meanings understood by those of ordinary skill in the art. When a trade name appears in this document, it is intended to refer to its corresponding trade name or its active ingredient.

[0123] As used herein, the term "mRNA" refers to messenger ribonucleic acid. mRNA can be naturally occurring or non-naturally occurring or synthetic. For example, mRNA can include modified and / or non-naturally occurring components, such as one or more nucleobases, nucleosides, nucleotides, or linkers. mRNA can include a cap structure, a 5' transcription leader, a 5' untranslated region, a start codon, an open reading frame, a stop codon, a chain terminating nucleoside, a stem-loop, a hairpin, polyadenylic acid (polyA), a polyadenylation signal, and / or one or more cis-regulatory elements. mRNA can have a nucleotide sequence encoding a polypeptide. Translation of mRNA, such as in vivo translation of mRNA in mammalian cells, can produce a polypeptide. Traditionally, the basic components of a natural mRNA molecule include at least one coding region, a 5'-untranslated region (5'UTR), a 3'UTR, a 5' cap, and a polyA sequence.

[0124] As used herein, the term "untranslated region" or "UTR" refers to the portion of the mRNA upstream of the start codon and downstream of the stop codon that is not translated and is therefore referred to as the 5' untranslated region (5'UTR) and the 3' untranslated region (3'UTR), respectively. These regions are transcribed with the coding region and are therefore exonic when present in the mature mRNA.

[0125] As used herein, the term "5' untranslated region, 5'UTR" generally refers to a portion of an mRNA that is located 5' (i.e., "upstream") of the open reading frame and is not translated into protein. The 5'UTR is generally understood to be a specific segment of a messenger RNA (mRNA) that is located at the 5' end of the open reading frame of the mRNA. Typically, the 5'UTR starts at the transcription start site and terminates at one nucleotide before the start codon of the open reading frame. Preferably, the 5'UTR has a length of more than 20, 30, 40, or 50 nucleotides. The 5'UTR may contain elements for controlling gene expression, also referred to as regulatory elements. The regulatory elements may be, for example, ribosome binding sites. The 5'UTR may be modified post-transcriptionally, for example, by adding a 5'-cap. The 5'UTR of an mRNA is not translated into an amino acid sequence. The 5'UTR sequence is generally encoded by the gene that is transcribed into each mRNA during gene expression. The genomic sequence is first transcribed into pre-mRNA, which includes optional introns. The pre-mRNA is then further processed into mature mRNA during the maturation process. The maturation process comprises the following steps: 5' capping, splicing of the pre-mature mRNA to remove optional introns and 3' end modification (such as polyadenylation of the 3' end of the pre-mature mRNA and optional endonuclease / or exonuclease cleavage, etc.). Within the scope of the present disclosure, the 5'UTR corresponds to the mature mRNA sequence located between the start codon and, for example, the 5'-cap. Preferably, the 5'UTR corresponds to a sequence extending from the nucleotide located at the 3' side of the 5' cap, more preferably from the 3' side nucleotide immediately adjacent to the 5' cap, to the nucleotide located at the 5' side of the start codon of the protein coding region, preferably to the nucleotide immediately adjacent to the 5' side of the start codon of the protein coding region. The nucleotide immediately adjacent to the 3' side of the mature mRNA 5' cap typically corresponds to the transcription start site. The term "corresponding to" means that the 5'UTR sequence can be an RNA sequence in the mRNA sequence used to define the 5'UTR sequence, or a DNA sequence corresponding to this RNA sequence.

[0126] As used herein, the term "3' untranslated region, 3'UTR" generally refers to a part of mRNA that is located 3' (i.e., "downstream") of the open reading frame and is not translated into protein. Typically, 3'UTR is a part of the mRNA between the protein coding region (open reading frame (ORF) or coding sequence (CDS)) and the poly(A) sequence of the mRNA. In the context of the present disclosure, the term 3'UTR can also include elements that are not encoded in the template from which the RNA is transcribed, but are added after transcription during maturation, such as poly(A) sequences. The 3'UTR of mRNA is not translated into an amino acid sequence. The 3'UTR sequence is typically encoded by a gene that is transcribed into a respective mRNA during gene expression. The genomic sequence is first transcribed into a pre-mature mRNA comprising optional introns. The pre-mature mRNA is then further processed into a mature mRNA during maturation. The maturation process comprises the following steps: 5' capping, splicing of the pre-mature mRNA to remove optional introns and 3' end modification (such as polyadenylation of the 3' end of the pre-mature mRNA and optional endonuclease / or exonuclease cleavage, etc.). Within the scope of the present disclosure, the 3'-UTR corresponds to the stop codon of the protein coding region, preferably between the 3' end of the stop codon of the protein coding region and the poly (A) sequence of the mRNA. The term "corresponding to" means that the 3'-UTR sequence can be an RNA sequence in the mRNA sequence used to define the 3'-UTR sequence, or a DNA sequence corresponding to this RNA sequence. Preferably, the 3'UTR has a length of more than 20, 30, 40 or 50 nucleotides.

[0127] In some embodiments, the RNA comprises one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, sgRNA, and tsRNA. Preferably, the RNA is selected from mRNA.

[0128] As used herein, the terms "comprise" or "comprises" or "containing" and variations thereof such as comprises or comprising are to be understood in an open, non-exclusive sense, ie, "including but not limited to."

[0129] As used herein, the term "about" is used in this disclosure to mean approximately, around, roughly, or approximately. When the term "about" is used in conjunction with a numerical range, it modifies the range by extending the upper and lower limits of the numerical range stated. Unless otherwise specified, the term "about" is used herein to modify the upper and lower limits of the numerical value stated by a deviation of 10%.

[0130] As used herein, the term "internal aqueous phase" refers to the process of preparing lipid nanoparticles in which the nucleic acid is dissolved in an aqueous medium before the nucleic acid and lipid components are mixed to form nanoparticles. The aqueous medium is the internal aqueous phase.

[0131] As used herein, the term "external aqueous phase" refers to the process of preparing lipid nanoparticles, in which the nanoparticles are placed in an aqueous medium (such as a buffer reagent) after the nucleic acid and lipid components are mixed to form nanoparticles. The aqueous medium is the external aqueous phase.

[0132] As used herein, the term "therapeutically effective amount" means

[0133] An amount of a formulation of the present disclosure that (i) treats a specific disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a specific disease, condition, or disorder, or (iii) delays the onset of one or more symptoms of a specific disease, condition, or disorder described herein.

[0134] Typical routes of administration of the disclosed formulations or compositions include, but are not limited to, inhalation, intraperitoneal, mucosal, intramuscular, subcutaneous, and intravenous administration.

[0135] As used herein, the term "nitrogen to phosphorus ratio (N / P) of nucleic acid lipid nanoparticles" refers to the molar ratio of nitrogen atoms of the cationic lipid to phosphorus atoms of the mRNA in the nucleic acid lipid nanoparticles.

[0136] As used herein, the term "w / v%" represents mass volume concentration. For example, a mass volume concentration (w / v%) of about 4% to about 25% may represent about 4 to 25 g / 100 mL.

[0137] The term "cationic lipid" as used herein is ionizable, and the cationic ionizable lipid contains one or more groups that are protonated at physiological pH but can be deprotonated and are uncharged 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. Other examples of cationic lipids include, but are not limited to, N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium bromide. ammonium chloride (DOTAP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethanoloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315.

[0138] The structural compound of DLin-MC3-DMA has a CAS number of 1224606-06-7 and a structural formula as follows:

[0139] The structural compound of SM-102 has a CAS number of 2089251-47-6 and a structural formula as follows:

[0140] The structural compound of ALC-0315 has the CAS number 2036272-55-4 and the structural formula is as follows:

[0141] Other examples of the term "neutral lipid" as used herein include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), or phosphatidylethanolamine (DLPE).

[0142] Other examples of the term "PEG-modified lipids" as used herein include, but are not limited to, methoxypolyethylene glycol ditetradecylacetamide (ALC-0159), MG-PEG2000, DMG-PEG5000, PEG2000.

[0143] Other examples of the term "stabilizer" as used herein include, but are not limited to, sucrose, trehalose, maltose, lactose, and mannitol. Preferably, the stabilizer is selected from sucrose or trehalose.

[0144] In some embodiments, in the formulated buffer containing nucleic acid lipid nanoparticles and freeze-drying protective agent, the mass volume concentration (w / v%) of the stabilizer is about 0.1% to about 4% (i.e., 0.1-4 g / 100 mL). The mass volume concentration (w / v%) of the stabilizer is about 0.1% to about 3%. Or the mass volume concentration (w / v%) of the stabilizer is about 0.1% to about 2%. Or the mass volume concentration (w / v%) of the stabilizer is about 0.1% to about 1.5%. Or the mass volume concentration (w / v%) of the stabilizer is about 0.5% to about 1%. Or the mass volume concentration (w / v%) of the stabilizer is about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, about 2.5%, about 3.0%. Or the mass volume concentration (w / v) of the stabilizer is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%.

[0145] Other examples of the term "buffer" as used herein include, but are not limited to, Tris buffer, citrate buffer, acetate buffer, or phosphate buffer. The pH of the buffer is 6.5-8.5. Preferably, the pH of the buffer is 6.9-7.9. More preferably, the pH of the buffer is 7.4.

[0146] In some embodiments, the solubility of the buffering agent is from about 2 mM to about 30 mM. Alternatively, the solubility of the buffering agent is from about 2 mM to about 20 mM. Alternatively, the solubility of the buffering agent is from about 2 mM to about 15 mM. Alternatively, the solubility of the buffering agent is from about 2 mM to about 9 mM. Alternatively, the solubility of the buffering agent is from about 4 mM to about 9 mM. Alternatively, the solubility of the buffering agent is from about 5 mM to about 9 mM.

[0147] Other examples of the term "lyoprotectant" as used herein include, but are not limited to, maltose, lactose, sucrose, trehalose, isomaltose, nigerose, or kojibiose. Preferably, the lyoprotectant is selected from sucrose or trehalose. More preferably, the lyoprotectant is selected from sucrose.

[0148] In some embodiments, in the formulated buffer containing nucleic acid lipid nanoparticles and a freeze-drying protective agent, the mass volume concentration (w / v%) of the freeze-drying protective agent is about 4% to about 25% (i.e., 4 to 25 g / 100 mL). Or the mass volume concentration (w / v%) of the freeze-drying protective agent is about 5% to about 25%. Or the mass volume concentration (w / v%) of the freeze-drying protective agent is about 5% to about 15%. Or the mass volume concentration of the freeze-drying protective agent is about 5%, about 6%, about 7%, about 8%, about 9%, about 10%. Or the mass volume concentration (w / v%) of the freeze-drying protective agent is about 10%.

[0149] Other examples of the term "surfactant" as used herein include, but are not limited to, poloxamers, such as poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, or poloxamer 407. Preferably, the surfactant is selected from poloxamer 188, also known as Pluronic F68.

[0150] In some embodiments, in the formulated buffer containing nucleic acid lipid nanoparticles and a freeze-drying protective agent, the mass volume concentration (w / v%) of the surfactant is about 0.1% to about 3% (i.e., 0.1-3 g / 100 mL). The mass volume concentration (w / v%) of the surfactant is about 0.1% to about 2%. Alternatively, the mass volume concentration (w / v%) of the surfactant is about 0.1% to about 1%. Alternatively, the mass volume concentration (w / v%) of the surfactant is about 0.1%, about 0.5%, about 1.0%, about 1.5%, about 2.0%, 2.5%, or about 3.0%. Or the mass volume concentration (w / v%) of the surfactant is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about 1.9%, or about 2%. DETAILED DESCRIPTION

[0151] The present disclosure is further described below with reference to specific examples, and the advantages and features of the present disclosure will become more apparent as the description proceeds. Where specific conditions are not specified in the examples, conventional conditions or those recommended by the manufacturer were used. Reagents or instruments used, where the manufacturer is not specified, are commercially available conventional products.

[0152] Unless otherwise defined herein, scientific and technical terms related to the present disclosure shall have the meanings that are understood by those of ordinary skill in the art.

[0153] The embodiments of the present disclosure are merely exemplary and do not constitute any limitation on the scope of the present disclosure. It should be understood by those skilled in the art that the details and forms of the technical solutions of the present disclosure may be modified or replaced without departing from the spirit and scope of the present disclosure, but such modifications and replacements shall fall within the scope of protection of the present disclosure.

[0154] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0155] Materials and reagents

[0156] Other materials and reagents:

[0157] (1) The mRNA (XR00b-4) of the present invention was prepared by the present invention. The sequence is as follows:

[0158] For example, "Ψ" in the sequence listing herein represents a methyl-modified uracil.

[0159] The mRNA preparation process is as follows:

[0160] 1. Preparation of Linear Plasmid Template

[0161] According to conventional plasmid linearization and purification methods in the art, the mRNA construct plasmid is linearized using BsaI or BspQI restriction endonuclease.

[0162] 2. mRNA in vitro transcription and purification

[0163] An in vitro transcription system was prepared according to conventional systems in the art, and mRNA was transcribed and purified in vitro for subsequent preparation of mRNA-LNPs.

[0164] Example 1 Preparation of Lyophilized Samples of LNP-Encapsulated mRNA (mRNA-LNP)

[0165] Lyophilized preparations were prepared by adding lyophilized agents of different mass volume concentrations (w / v%) to the mRNA-LNP solution as shown in Table 1.

[0166] Table 1 Prescription of mRNA-LNP lyophilized preparation

[0167] Preparation process:

[0168] (1) Preparation of mRNA aqueous phase: Dilute 0.045 mg of purified mRNA to 0.1-0.4 mg / ml using 5-50 mM citrate buffer (pH 4.0).

[0169] (2) Preparation of lipid ethanol phase: ALC0315, DSPC, cholesterol and ALC0159 were dissolved in ethanol solution according to the molar ratio (molar ratio % = 46.3%:9.4%:42.7%:1.6%) to make the total lipid concentration 7.78 mg / ml to 30.58 mg / ml.

[0170] (3) Lipid nanoparticles were prepared using an MPE-L2 microfluidic device using a conventional microfluidic method in the art, maintaining a nitrogen-to-phosphorus ratio of 6 (the molar ratio of nitrogen atoms in cationic lipids to phosphorus atoms in mRNA, N / P ratio), and using pH 7.4 TRIS buffer as a diluent.

[0171] (4) Ultrafiltration was performed using an ultrafiltration tube for concentration. The lipid nanoparticle suspension was then dissolved in 5 mM TRIS buffer (pH 7.4) containing 5%, 10%, and 15% sucrose (w / v%) to prepare a lipid nanoparticle suspension. The volume was fixed to a specific mRNA concentration (0.1 mg / ml). Particle size, PDI, potential, and encapsulation efficiency were measured.

[0172] TRIS buffer: weigh 1 g of tromethamine (TRIS) and 6.67 g of tromethamine hydrochloride (TRIS HCl), add 10 kg of water for injection, and adjust the pH to 7.4 with 1 M HCl to obtain 5 mM pH 7.4 TRIS buffer.

[0173] (5) Freeze-drying process

[0174] Place the mRNA-LNP solution on a freeze dryer plate and prefreeze under vacuum at -50°C to -20°C for 1 to 5 hours. Sublime dry at -50°C to -20°C for 16 to 60 hours. Desorb and dry at 5°C to 25°C for 6 to 24 hours to obtain a solid freeze-dried mRNA-LNP powder.

[0175] Reconstitution of mRNA-LNP solid lyophilized powder: Remove the solid lyophilized powder from the lyophilizer and reconstitute the solid lyophilized powder with water for injection at 4°C to obtain a solution. The particle size, PDI, potential, and encapsulation efficiency of the reconstituted solution are then measured.

[0176] The particle size, PDI, and potential were determined by using a Zetasizer Ultra instrument to measure the LNP particle size and polydispersity index (PDI); and a potential analysis module to measure the potential value.

[0177] The total RNA concentration and encapsulation efficiency of the mRNA-LNP formulation were determined using the RiboGreen assay. The mRNA-LNP formulation solution was demulsified with Triton X100. 100 μL of the demulsified solution was added to a 96-well plate. Then, 100 μL of the RiboGreen dye solution was added. The plate was shaken on a plate shaker for 5 minutes at 600 rpm. Detection was performed using a SpectraMax iD3 multi-function microplate reader. Total RNA concentration was calculated using a standard curve.

[0178] Take 10 μL of the mRNA-LNP solution, add 990 μL of buffer, mix thoroughly, add 100 μL of RiboGreen dye solution, and place on a plate shaker for 5 minutes at 600 rpm. Detect using a SpectraMax iD3 multi-function microplate reader. Calculate the free RNA concentration using the standard curve. Calculate the encapsulation efficiency using the following formula.

[0179] Encapsulation efficiency (%) = 100% - (free mRNA concentration / total mRNA concentration) x 100%

[0180] The test results are shown in the following table.

[0181] Table 2 Sample test results

[0182] The results in Table 2 show that the encapsulation efficiency of lipid nanoparticles with sucrose added as a lyoprotectant is maintained at around 80% or higher after lyophilization. As the concentration of the lyoprotectant increases, the encapsulation efficiency decreases, and the overall lyotropic effect is good.

[0183] Example 2 Preparation of freeze-dried samples containing stabilizers in the aqueous phase of LNP

[0184] During the lipid nanoparticle preparation process, a stabilizer is added to the aqueous phase containing mRNA (the inner aqueous phase), which is then mixed with the lipid ethanol phase. The lipid nanoparticles are prepared using conventional microfluidics methods in the art, and the lipid nanoparticles are lyophilized to produce a lyophilized formulation. The specific formulation is shown in Table 3.

[0185] Table 3 Prescription of mRNA-LNP lyophilized preparation

[0186] Preparation process:

[0187] (1) Preparation of mRNA aqueous phase: 0.225 mg of purified mRNA was diluted to 0.1-0.4 mg / ml using 5-50 mM pH 4.0 citrate buffer, and 0.1%-1% trehalose or 0.1%-2% sucrose was added to the buffer at a mass volume fraction (w / v%).

[0188] (2) Preparation of lipid ethanol phase: ALC0315, DSPC, cholesterol and ALC0159 were dissolved in ethanol solution according to the molar ratio (molar ratio % = 46.3%:9.4%:42.7%:1.6%) to make the total lipid concentration 7.78 mg / ml to 30.58 mg / ml.

[0189] (3) Lipid nanoparticles were prepared using a conventional microfluidic method in the art, maintaining a nitrogen-phosphorus ratio of 6 (the molar ratio of nitrogen atoms in cationic lipids to phosphorus atoms in mRNA) using an MPE-L2 microfluidic device, and using pH 7.4 TRIS buffer as a diluent.

[0190] (4) Ultrafiltration was performed using an ultrafiltration tube for concentration, followed by dissolution in 5 mM TRIS buffer (pH 7.4) containing 10% sucrose (w / v%) to prepare a lipid nanoparticle suspension. The volume was fixed to a specific mRNA concentration (0.5 mg / ml). Particle size, PDI, potential, and encapsulation efficiency were measured.

[0191] (5) Freeze-drying process

[0192] Place the mRNA-LNP solution on a freeze dryer plate and prefreeze under vacuum at -50°C to -20°C for 1 to 5 hours. Sublime dry at -50°C to -20°C for 16 to 60 hours. Desorb and dry at 5°C to 25°C for 6 to 24 hours to obtain a solid freeze-dried mRNA-LNP powder.

[0193] Reconstitution of mRNA-LNP solid lyophilized powder: Remove the solid lyophilized powder from the lyophilizer and reconstitute the solid lyophilized powder with water for injection at 4°C to obtain a solution. The particle size, PDI, potential, and encapsulation efficiency of the reconstituted solution are then measured.

[0194] The determination and calculation methods of particle size, PDI, potential and encapsulation efficiency are the same as those in Example 1.

[0195] The test results are shown in the following table.

[0196] Table 4 Sample test results

[0197] The results in Table 4 show that the aqueous phases of Formulations 2-2 to 2-5 contained 0.1% to 1% trehalose, and the aqueous phases of Formulations 2-6 to 2-10 contained 0.1% to 2% sucrose. The particle size, PDI, zeta potential, and encapsulation efficiency of the mRNA-LNP samples of Formulations 2-2 to 2-10 were essentially identical before freeze-drying. After freeze-drying, under the same concentration conditions, the sucrose groups showed smaller changes in particle size and PDI than the trehalose group, and similar zeta potentials. However, the encapsulation efficiency of the groups containing sucrose in the aqueous phase was slightly higher than that of the group containing trehalose in the aqueous phase. Furthermore, the groups containing 1% sucrose and 0.5% trehalose in the aqueous phase showed the smallest changes in particle size, the smallest PDI, and the highest encapsulation efficiency.

[0198] Example 3 Stability Study of Lyophilized mRNA-LNP Samples with and without Stabilizer in the Aqueous Phase

[0199] The stability of the freeze-dried formulations of formulations 2-1 (without stabilizer in the internal aqueous phase), 2-4 (with 0.5% trehalose in the internal aqueous phase), and 2-9 (with 1% sucrose in the internal aqueous phase) was studied. The formulations were placed at -80°C, -20°C, 2-8°C, and 25°C, respectively. The particle size, PDI, zeta potential, and encapsulation efficiency were measured after reconstitution at 0 days, 15 days (15D), 2 months (2M), and 3 months (3M). The results are shown in Tables 5, 6, and 7.

[0200] Table 5 Stability results of prescription 2-1

[0201] Table 6 Stability results of formulations 2-4

[0202] Table 7 Stability results of formulations 2-9

[0203] The results in Tables 5, 6, and 7 show that when Prescriptions 2-1, 2-4, and 2-9 were stored at -80°C, -20°C, and 2-8°C for 2-3M, the particle size, PDI, Zeta potential, mRNA concentration, and encapsulation efficiency remained almost unchanged. When stored at 25°C for 3M, the particle size of Prescriptions 2-4 and 2-9 increased slightly, but the PDI, Zeta potential, mRNA concentration, and encapsulation efficiency did not change. This indicates that Prescriptions 2-4 and 2-9 can be stable for 3 months at -80°C, -20°C, 2-8°C, and 25°C. Compared to Prescription 2-1 (without a stabilizer in the internal aqueous phase), the encapsulation efficiencies of Prescriptions 2-4 (with 0.5% trehalose in the internal aqueous phase) and 2-9 (with 1% sucrose in the internal aqueous phase) were both 90%, which was higher than the group without a stabilizer in the internal aqueous phase (Prescription 2-1).

[0204] Example 4 Preparation of Lyophilized Samples of Low-Concentration mRNA Encapsulated in LNPs with Stabilizer in the Aqueous Phase (mRNA-LNP)

[0205] The mRNA-LNP lyophilized preparations differ from the formulations 2-4 and 2-9 of Example 2 in that a lyophilized preparation of low-concentration mRNA was prepared.

[0206] Table 8 Prescription of mRNA-LNP lyophilized preparation

[0207] Preparation process:

[0208] (1) Preparation of mRNA aqueous phase: 0.075 mg of purified mRNA was diluted to 0.1-0.4 mg / mL using 5-50 mM pH 4.0 citrate buffer, and 0.5% trehalose or 1% sucrose was added to the buffer at a mass volume fraction (w / v%).

[0209] (2) Preparation of lipid ethanol phase: ALC0315, DSPC, cholesterol and ALC0159 were dissolved in ethanol solution according to the molar ratio (molar ratio % = 46.3%:9.4%:42.7%:1.6%) to make the total lipid concentration 7.78 mg / ml to 30.58 mg / ml.

[0210] (3) Lipid nanoparticles were prepared using a conventional microfluidic method in the art, maintaining a nitrogen-phosphorus ratio of 6 (the molar ratio of nitrogen atoms in cationic lipids to phosphorus atoms in mRNA) using an MPE-L2 microfluidic device, and using pH 7.4 TRIS buffer as a diluent.

[0211] (4) Ultrafiltration was performed using an ultrafiltration tube for concentration, followed by dissolution in 5 mM TRIS buffer (pH 7.4) containing 10% sucrose (w / v%) to prepare a lipid nanoparticle suspension. The volume was fixed to a specific mRNA concentration (0.3 mg / ml). Particle size, PDI, potential, and encapsulation efficiency were measured.

[0212] (5) Freeze-drying process

[0213] Place the mRNA-LNP solution on a freeze dryer plate and prefreeze under vacuum at -50°C to -20°C for 1 to 5 hours. Sublime dry at -50°C to -20°C for 16 to 60 hours. Desorb and dry at 5°C to 25°C for 6 to 24 hours to obtain a solid freeze-dried mRNA-LNP powder.

[0214] Reconstitution of mRNA-LNP solid lyophilized powder: Remove the solid lyophilized powder from the lyophilizer and reconstitute the solid lyophilized powder with water for injection at 4°C to obtain a solution. The particle size, PDI, potential, and encapsulation efficiency of the reconstituted solution are then measured.

[0215] The determination and calculation methods of particle size, PDI, potential and encapsulation efficiency are the same as those in Example 1.

[0216] The test results are shown in the following table.

[0217] Table 9 Sample test results

[0218] The results in Table 9 show that the particle size, PDI, Zeta potential and encapsulation efficiency of the mRNA-LNP samples of Prescription 3-1 and Prescription 3-2 before freeze-drying are basically the same. After freeze-drying, the particle size, PDI, Zeta potential and encapsulation efficiency of the mRNA-LNP samples of Prescription 3-2 are basically the same as those of Prescription 3-1.

[0219] Example 5 Preparation of Lyophilized Samples of LNP-Encapsulated mRNA (mRNA-LNP)

[0220] Surfactants of different mass volume fractions (w / v%) were added to the mRNA-LNP solution as shown in the following table to prepare freeze-dried preparations.

[0221] Table 10 Prescription of mRNA-LNP lyophilized preparation

[0222] Preparation process:

[0223] (1) Preparation of mRNA aqueous phase: 0.075 mg of purified mRNA was diluted to 0.1-0.4 mg / mL using 5-50 mM pH 4.0 citrate buffer, and 1% sucrose was added to the buffer.

[0224] (2) Preparation of lipid ethanol phase: ALC0315, DSPC, cholesterol and ALC0159 were dissolved in ethanol solution according to the molar ratio (molar ratio % = 46.3%:9.4%:42.7%:1.6%) to make the total lipid concentration 7.78 mg / ml to 30.58 mg / ml.

[0225] (3) Lipid nanoparticles were prepared using a conventional microfluidic method in the art, maintaining a nitrogen-phosphorus ratio of 6 (the molar ratio of nitrogen atoms in cationic lipids to phosphorus atoms in mRNA) using an MPE-L2 microfluidic device, and using pH 7.4 TRIS buffer as a diluent.

[0226] (4) Ultrafiltration was performed using an ultrafiltration tube for concentration. The lipid nanoparticle suspension was then dissolved in 5 mM TRIS buffer (pH 7.4) containing 10% sucrose (w / v%) and 0%, 0.05%, 0.1%, 0.2%, and 0.3% poloxamer 188 (w / v%). The suspension was then fixed to a specific mRNA concentration (0.3 mg / ml). Particle size, PDI, potential, and encapsulation efficiency were measured.

[0227] (5) Freeze-drying process

[0228] Place the mRNA-LNP solution on a freeze dryer plate and prefreeze under vacuum at -50°C to -20°C for 1 to 5 hours. Sublime dry at -50°C to -20°C for 16 to 60 hours. Desorb and dry at 5°C to 25°C for 6 to 24 hours to obtain a solid freeze-dried mRNA-LNP powder.

[0229] Reconstitution of mRNA-LNP solid lyophilized powder: Remove the solid lyophilized powder from the lyophilizer and reconstitute the solid lyophilized powder with water for injection at 4°C to obtain a solution. The particle size, PDI, Zeta potential, encapsulation efficiency, and mRNA integrity of the reconstituted solution are measured.

[0230] The determination and calculation methods of particle size, PDI, potential and encapsulation efficiency are the same as those in Example 1.

[0231] mRNA integrity detection method:

[0232] Add 10% Triton X-100 to the mRNA-LNP sample to break the emulsion, obtaining a demulsified solution. Dilute the solution to a concentration of approximately 8 ng / μl using 1× TE buffer, mix thoroughly, denature at 70°C for 2 minutes, and quickly transfer to ice. The solution is then added to a 96-well plate, sealed with film, and centrifuged to remove air bubbles. mRNA integrity is assessed using an Agilent 5200 fragment analyzer.

[0233] Absolute value of change in mRNA integrity (%) = |mRNA integrity (%, before lyophilization) - mRNA integrity (%, after lyophilization) |

[0234] The test results are shown in the following table.

[0235] Table 11 Sample test results

[0236] As shown in Table 11, after the addition of poloxamer 188 surfactant, the reconstitution of freeze-dried mRNA-LNP samples from Formulations 4-2 and 4-5 revealed smaller particle sizes than those without poloxamer 188, with encapsulation efficiencies of 95%. Furthermore, mRNA integrity remained virtually unchanged before and after lyophilization. This suggests that the addition of poloxamer 188 can preserve the mRNA in the mRNA-LNP samples, resulting in minimal changes in encapsulation efficiency before and after lyophilization.

[0237] Example 6: Stability Study of Lyophilized Sample Preparation of LNP-Encapsulated mRNA (mRNA-LNP)

[0238] The lyophilized formulation of formula 4-4 was selected for stability study. The lyophilized formulation of formula 4-4 was placed at -80°C and 2-8°C, respectively. After reconstitution at 15 days (15D) and 1 month (1M), the particle size, PDI, Zeta potential, encapsulation efficiency and mRNA integrity were measured. The results are shown in the following table:

[0239] Table 12 Stability results of prescription 4-4

[0240] As shown in Table 12, when the freeze-dried sample of mRNA-LNP of Formulation 4-4 was stored at 1M at -80℃ and 2-8℃, the particle size, PDI, zeta potential, mRNA concentration, encapsulation efficiency and mRNA integrity remained almost unchanged, indicating that the addition of poloxamer 188 can stabilize the physicochemical properties of the freeze-dried mRNA-LNP sample.

[0241] Example 7 Preparation of Lyophilized Samples of LNP-Encapsulated mRNA (mRNA-LNP)

[0242] Formulas 7-1, 7-2, 7-3, 7-4, 7-5, 7-6, 7-7, 7-8, 7-9, and 7-10 were prepared with reference to the formula and preparation process of Example 2, except that in step (2), the lipid ethanol phase was prepared by dissolving ALC0315, DSPC, cholesterol, and ALC0159 in an ethanol solution at a molar ratio (molar ratio % = 47.5%:9.9%:40.8%:1.8%) to obtain a total lipid concentration of 7.78 mg / mL to 30.58 mg / mL.

[0243] The determination and calculation methods of particle size, PDI, potential, and encapsulation efficiency were the same as in Example 1. The results of the determination are similar to those in Table 4. Among them, the particle size, PDI, Zeta potential, and encapsulation efficiency of the mRNA-LNP freeze-dried samples containing stabilizers in the aqueous phase of prescriptions 7-2 to 7-10 were basically the same before and after freeze-drying. After freeze-drying, under the same concentration conditions, the sucrose group showed smaller changes in particle size and PDI than the trehalose group, and the Zeta potential was not much different. However, the encapsulation efficiency of the group containing sucrose in the aqueous phase was slightly higher than that of the group containing trehalose in the aqueous phase. In addition, the particle size change, PDI, and encapsulation efficiency of the groups containing 1% sucrose and 0.5% trehalose in the aqueous phase were the smallest, the smallest, and the highest.

[0244] Using the stability study method described in Example 3, we determined that formulations 7-2 to 7-10 exhibited minimal changes in particle size, PDI, zeta potential, mRNA concentration, and entrapment efficiency when stored at 3M at -80°C, -20°C, 2-8°C, and 25°C, demonstrating excellent stability. Compared to formulation 7-1 (without a stabilizer in the internal aqueous phase), formulations 7-2 to 7-10 exhibited higher entrapment efficiencies than the stabilizer-free formulation (formulation 7-1).

[0245] Example 8 Preparation of Lyophilized Samples of LNP-Encapsulated mRNA (mRNA-LNP)

[0246] Formulas 8-1, 8-2, 8-3, 8-4, and 8-5 were prepared with reference to the formula and preparation process of Example 5, except that in step (2), the lipid ethanol phase was prepared by dissolving ALC0315, DSPC, cholesterol, and ALC0159 in an ethanol solution at a molar ratio (molar ratio % = 47.5%:9.9%:40.8%:1.8%) to obtain a total lipid concentration of 7.78 mg / mL to 30.58 mg / mL.

[0247] The determination and calculation methods for particle size, PDI, potential, and encapsulation efficiency were the same as in Example 1. The results of the measurements are similar to those in Table 11. When the surfactant poloxamer 188 was added, the lyophilized samples of mRNA-LNPs from Formulations 8-2 to 8-5 were reconstituted and tested. The particle sizes were smaller than those in the groups without poloxamer 188, and the encapsulation efficiency was 95%. Furthermore, the mRNA integrity remained virtually unchanged before and after lyophilization. This suggests that the addition of poloxamer 188 can preserve the mRNA in the mRNA-LNP samples, and the encapsulation efficiency changes minimally before and after lyophilization.

[0248] Referring to the determination method of the stability study in Example 6, it was measured that when Formulations 8-2 to 8-5 were stored at -80°C and 2-8°C at 1M, the particle size, PDI, Zeta potential, mRNA concentration, encapsulation efficiency, and mRNA integrity remained almost unchanged, indicating that the addition of poloxamer 188 can stabilize the physicochemical properties of the mRNA-LNP freeze-dried samples.

Claims

1. A lyophilized preparation of lipid nanoparticles containing nucleic acid, wherein: The lyophilized preparation comprises: i) lipid nanoparticles containing nucleic acids; ii) and a buffer reagent containing a lyoprotectant.

2. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to claim 1, wherein The lyophilized formulation comprises: i) lipid nanoparticles containing nucleic acid, wherein the lipid nanoparticles comprise a stabilizer; ii) Buffer containing lyoprotectant.

3. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to claim 2, wherein The stabilizer is selected from any one of sucrose, trehalose, maltose, lactose and mannitol; preferably, the stabilizer is selected from sucrose or trehalose.

4. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to claim 2 or 3, wherein The lyophilized preparation comprises: i) a lipid nanoparticle containing a nucleic acid, wherein the lipid nanoparticle comprises a stabilizer, and in the prepared buffer containing the nucleic acid lipid nanoparticle and the freeze-drying protective agent, the mass volume concentration (w / v%) of the stabilizer is about 0.1-5%; ii) Buffer containing lyoprotectant.

5. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to claim 4, wherein In the prepared buffer containing nucleic acid lipid nanoparticles and freeze-drying protectants, the mass volume concentration (w / v%) of the stabilizer is about 0.1% to about 4%; preferably, the mass volume concentration (w / v%) of the stabilizer is about 0.1% to about 3%; more preferably, the mass volume concentration (w / v%) of the stabilizer is about 0.1% to about 2%.

6. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to any one of claims 1 to 5, wherein: The buffer reagent is selected from Tris, citrate, acetate or phosphate buffer.

7. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to any one of claims 1 to 6, wherein: The solubility of the buffer reagent is about 2 mM to about 30 mM; preferably, the solubility of the buffer reagent is about 2 mM to about 20 mM; more preferably, the solubility of the buffer reagent is about 2 mM to about 15 mM.

8. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to any one of claims 1 to 7, wherein The lyoprotectant is selected from any one or more of maltose, lactose, sucrose, trehalose, isomaltose, nigerose or kojibiose; preferably, the lyoprotectant is selected from sucrose or trehalose.

9. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to any one of claims 1 to 8, wherein In the prepared buffer containing nucleic acid lipid nanoparticles and a freeze-dried protective agent, the mass volume concentration (w / v%) of the freeze-dried protective agent is about 4% to about 25%; preferably, the mass volume concentration (w / v%) of the freeze-dried protective agent is about 5% to about 25%; more preferably, the mass volume concentration (w / v%) of the freeze-dried protective agent is about 5% to about 20%.

10. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to any one of claims 1 to 9, wherein: The buffer reagent containing the lyoprotectant further comprises a surfactant.

11. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to claim 10, wherein The surfactant is selected from poloxamers.

12. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to claim 10 or 11, wherein In the prepared buffer containing nucleic acid lipid nanoparticles and freeze-drying protectants, the mass volume concentration (w / v%) of the surfactant is about 0.1% to about 3%; preferably, the mass volume concentration (w / v%) of the surfactant is about 0.1% to about 2%; more preferably, the mass volume concentration (w / v%) of the surfactant is about 0.1% to about 1%.

13. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to any one of claims 1 to 12, wherein: In the prepared buffer containing nucleic acid lipid nanoparticles and freeze-drying protective agent, the concentration of nucleic acid in the lipid nanoparticle suspension containing nucleic acid is 0.1-0.5 mg / ml, preferably 0.3-0.5 mg / ml.

14. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to any one of claims 1 to 13, wherein: The nucleic acid is selected from RNA; the RNA includes one or more of antisense RNA, saRNA, mRNA, lncRNA, miRNA, siRNA, piRNA, sgRNA, and tsRNA.

15. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to any one of claims 1 to 14, wherein The RNA is selected from mRNA.

16. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to any one of claims 1 to 15, wherein: The nucleic acid-containing lipid nanoparticles include nucleic acid and lipids, and the lipids include: a) cationic lipids; b) neutral helper lipids; c) cholesterol; and d) PEG-modified lipids.

17. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to claim 16, wherein In the nucleic acid-containing lipid nanoparticles, the molar ratio of each lipid component is as follows, based on the total molar amount of lipids as 100%: a) cationic lipid 45% to 50%; b) neutral helper lipid 5% to 10%; c) Cholesterol 38% to 48%; and d) PEG-modified lipids 0-3%.

18. The lyophilized preparation of lipid nanoparticles containing nucleic acid according to claim 16 or 17, wherein The cationic lipid is selected from N,N-dimethyl-2,3-dioleoyloxypropylamine (DODMA), 1,2-dimyristyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DMRIE), N,N-dioleoyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOT AP), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 1,2-dimethoxy-N,N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 4-(N,N-dimethylamino)butyric acid (dilinoleyl) methyl ester (DLin-MC3-DMA), SM-102, ALC-0315, any one or more thereof; The neutral auxiliary lipid is selected from distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditri Any one or more of cosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), dioleoylphosphatidylethanolamine (DOPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE) or phosphatidylethanolamine (DLPE); The PEG-modified lipid is selected from any one or more of methoxypolyethylene glycol ditetradecyl acetamide (ALC-0159), MG-PEG2000, DMG-PEG5000, and PEG2000.

19. A method for preventing or treating a disease or condition in a subject, comprising administering to the subject a therapeutically effective amount of the lyophilized formulation of any one of claims 1 to 18 reconstituted in a liquid medium.

20. Use of a lyophilized preparation containing nucleic acid lipid nanoparticles according to any one of claims 1 to 18 in the preparation of nucleic acid drugs.

21. Use of a freeze-dried preparation containing nucleic acid lipid nanoparticles according to any one of claims 1 to 18 in the preparation of nucleic acid vaccines.

22. A method for preparing a lyophilized preparation of lipid nanoparticles containing nucleic acid, wherein: The method comprises the following steps: a) preparing an aqueous solution containing nucleic acid and an organic solution containing lipid respectively; preferably, the solution containing nucleic acid contains about 0.1-5% (w / v%) of a stabilizer; preferably, the organic solution is an ethanol solution; b) preparing the lipid nanoparticles containing nucleic acid, and then dissolving the lipid nanoparticles in a buffer reagent containing a lyophilization protectant to obtain a lipid nanoparticle suspension containing nucleic acid; c) freeze-drying the lipid nanoparticle suspension comprising the nucleic acid to form a lyophilized preparation.

23. The preparation method according to claim 22, wherein: Step a) further comprises the following steps: a-1) preparing an aqueous solution containing nucleic acid: dissolving the nucleic acid in an aqueous medium, adding the stabilizer to the aqueous medium, wherein the mass volume concentration (w / v%) of the stabilizer is about 0.1%-5%; a-2) Preparation of an ethanol solution containing lipids: the lipid compound is dissolved in ethanol; Preferably, the aqueous medium is selected from a citrate buffer or a sodium acetate buffer; the stabilizer is 0.1% to 1% trehalose or 0.1% to 2% sucrose; More preferably, the aqueous medium is selected from citrate buffer or sodium acetate buffer, and the pH of the buffer is 4-5; the stabilizer is 1% sucrose or 0.5% trehalose.

24. The preparation method according to claim 22 or 23, wherein: Step b) of the method for synthesizing the lipid nanoparticles containing nucleic acids comprises: using a classical microfluidic method, mixing an ethanol solution containing lipids and an aqueous solution containing nucleic acids by microfluidics to prepare lipid nanoparticles containing nucleic acids, concentrating by ultrafiltration, and then dissolving the lipid nanoparticles containing nucleic acids in a buffer reagent containing a lyophilization protectant; Preferably, in the microfluidic method, the molar ratio of nitrogen atoms of the cationic lipid to phosphorus atoms of the mRNA (N / P ratio) is maintained at 5-7; most preferably, the N / P ratio is 6.

25. The preparation method according to claim 24, wherein: In the step of dissolving the lipid nanoparticles containing nucleic acids in a buffer reagent containing a lyophilization protectant, the concentration of nucleic acids in the lipid nanoparticles containing nucleic acids is 0.1 to 0.5 mg / ml, preferably 0.3 to 0.5 mg / ml. The buffer reagent containing the freeze-dried protective agent is: 5mM TRIS buffer containing 5-15% sucrose by mass volume fraction (w / v%); Preferably, the buffer reagent further contains 0.05-1% poloxamer 188 by mass volume fraction (w / v%); More preferably, the buffer reagent contains 10% sucrose by mass volume fraction (w / v%) and 0.2% poloxamer 188 by mass volume fraction (w / v%), and the pH is pH 7.4.