Preparation for delivering nucleic acid in vivo

Abstract

The present invention relates to nasal, mucosal drug delivery systems, in particular to formulations for in vivo delivery of nucleic acids, which comprise nucleic acids for in vivo delivery can be used for mucosal administration of nucleic acids to treat or prevent diseases, such as infectious diseases.

Description

Technical Field

[0001] The present disclosure relates to the field of nucleic acid delivery, and in particular, to a preparation for in vivo delivery of nucleic acids. Background Art

[0002] The FDA approved ONPATTRO, the first RNA-based drug that uses lipid nanoparticles (lipid nanoparticles) to deliver siRNA to liver cells. This technology has generated widespread interest and has been used to deliver larger nucleic acids such as mRNA and genome editing components.

[0003] Lipid nanoparticles are usually composed of four lipids: (1) cationic lipids, (2) polyethylene glycol (PEG) lipids, (3) cholesterol, and (4) structural lipids. The first component, cationic lipids, are generally composed of a cationic head group and a lipid tail, and are located in the core of the lipid nanoparticle with nucleic acid. The second component, PEG lipids, have PEG chains anchored on the lipid head group. The third component is cholesterol, which is involved in the intracellular delivery of lipid nanoparticles. The fourth component, structural lipids, are mainly present on the surface of lipid nanoparticles and contribute to the packaging of nucleic acids and the stability of nanoparticles.

[0004] Studies have shown that when lipid nanoparticles of marketed products (see CN200980122413.4, CN201880017979.X, etc.) are delivered through different routes of administration (intramuscular, intradermal, nasal drops), the immunogenicity and expression of the same nucleic acid in the body are different, and the immune effect through nasal administration is significantly weaker than that through intramuscular and subcutaneous administration.

[0005] There is a need in the art for a preparation for in vivo delivery of nucleic acids that can also be efficiently expressed after nasal administration, stimulate immunity, and better protect the body's drug delivery system. Summary of the invention

[0006] In one aspect, the present disclosure provides a formulation for in vivo delivery of nucleic acid, comprising lipid nanoparticles, and nucleic acid encapsulated by the lipid nanoparticles.

[0007] In some embodiments, the lipid nanoparticles comprise 25.0-50.0 mol% of cationic lipids, 6.0-40.0 mol% of phospholipids, 20.0-47.0 mol% of structural lipids, and 1.0-3.0 mol% of polyethylene glycol-modified lipids, based on the total amount of all lipids in the lipid nanoparticles.

[0008] In some embodiments, the lipid nanoparticles comprise 25.0-50.0 mol% of cationic lipids, 6.0-30.0 mol% of phospholipids, 20.0-47.0 mol% of structural lipids, and 1.0-3.0 mol% of polyethylene glycol-modified lipids, based on the total amount of all lipids in the lipid nanoparticles.

[0009] In some embodiments, the lipid nanoparticle comprises 49.1 mol% cationic lipids, 27.9 mol% phospholipids, 20.1 mol% structural lipids, and 2.9 mol% polyethylene glycol-modified lipids, based on the total amount of all lipids in the lipid nanoparticle.

[0010] In some embodiments, the cationic lipid is selected from at least one compound of Formula 1, Formula 2, Formula 3, and Formula 4.

[0011]

[0012]

[0013] In some embodiments, the cationic lipid is a compound as shown in Formula 3.

[0014] In some embodiments, the structured lipid is selected from at least one of cholesterol, coproposterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatine, ursolic acid, and α-tocopherol.

[0015] In some embodiments, the structured lipid is cholesterol.

[0016] In some embodiments, the phospholipid is selected from at least one of DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, DUPC, POPC, DOPG, DPPG, DPPE, DMPE, DSPE, and DOTAP.

[0017] In some embodiments, the polyethylene glycol-modified lipid is selected from at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0018] In some embodiments, the PEG lipid is at least one of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000 or Chol-PEG2000, PEG2000.

[0019] In some embodiments, the PEG lipid is PEG2000.

[0020] In some embodiments, the nucleic acid comprises a DNA molecule and / or an RNA molecule.

[0021] In some embodiments, the DNA molecule comprises a linear DNA molecule and / or a circular DNA molecule.

[0022] In some embodiments, the RNA molecule comprises mRNA or circular RNA.

[0023] In some embodiments, the mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) is shown in one of Seq ID NO.4 or Seq ID NO.6.

[0024] In some embodiments, the mRNA molecule includes an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) is shown in Seq ID NO.4.

[0025] In some embodiments, the formulation for in vivo delivery of nucleic acid is used for administration to the nasal cavity or respiratory mucosa.

[0026] In some embodiments, the nasal administration comprises nasal instillation, nasal spray administration, or nasal inhalation.

[0027] In some embodiments, the average particle size of the lipid nanoparticles is 60nm to 300nm. In some embodiments, the average particle size of the lipid nanoparticles is 60nm to 100nm. In some embodiments, the average particle size of the lipid nanoparticles is 60nm to 90nm. In some embodiments, the average particle size of the lipid nanoparticles is 60nm to 80nm. In some embodiments, the average particle size of the lipid nanoparticles is 60nm.

[0028] In some embodiments, the lipid nanoparticles have a polydispersity index of 0 to 0.25. In some embodiments, the lipid nanoparticles have a polydispersity index of 0.10 to 0.20.

[0029] In some embodiments, the Zeta potential of the lipid nanoparticles is -20 mV to +20 mV. In some embodiments, the Zeta potential of the lipid nanoparticles is +10 mV to +20 mV.

[0030] In another aspect, the present disclosure also provides a pharmaceutical composition, which comprises the preparation for in vivo delivery of nucleic acid of the present disclosure, and optionally a pharmaceutically acceptable excipient.

[0031] In some embodiments, the preparation or pharmaceutical composition for in vivo delivery of nucleic acid disclosed herein is used for administration to the nasal cavity or respiratory mucosa.

[0032] The preparation according to claim 8, characterized in that the excipient is 1×PBS buffer, and the buffer has a pH of 7.4±0.05.

[0033] In some embodiments, the pharmaceutical composition is for nasal administration, and the concentration of nucleic acid in the pharmaceutical composition is 800-1000 μg / mL.

[0034] In some embodiments, the pharmaceutical composition is used for administration to the respiratory mucosa, and the concentration of nucleic acid in the pharmaceutical composition is 400-600 μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the specific embodiments of the present disclosure or the technical solutions in the prior art, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0036] Figure 1 The figures are test results of the cellular delivery efficiency of formulations F1 to F5 for in vivo delivery of nucleic acids.

[0037] Figure 2 The fluorescence detection results (mice) 3 hours after nasal instillation of formulations F1 to F5 for in vivo nucleic acid delivery.

[0038] Figure 3 The fluorescence detection results (mice) of formulations F1 to F5 for in vivo nucleic acid delivery 6 hours after nasal instillation.

[0039] Figure 4 The fluorescence detection results (mice) of formulations F1 to F5 for in vivo nucleic acid delivery 24 hours after nasal instillation.

[0040] Figure 5 The fluorescence detection results of the preparations F1 to F5 for in vivo delivery of nucleic acids 6 hours after nasal instillation.

[0041] Figure 6 The figures are test results of the cellular delivery efficiency of formulations F3-F4 and F6 for in vivo delivery of nucleic acids.

[0042] Figure 7 The fluorescence detection results (mice) 6 hours after nasal instillation of formulations F3-F4 and F6 for in vivo nucleic acid delivery.

[0043] Figure 8 The fluorescence detection results of the preparations F3-F4 and F6 for in vivo delivery of nucleic acid 6 hours after nasal instillation.

[0044] Fig. 9 The figures are test results of the cellular delivery efficiency of formulations F6 to F10 for in vivo delivery of nucleic acids.

[0045] Fig.10 The fluorescence detection results (mice) 3 hours after nasal instillation of preparations F6 to F10 for in vivo nucleic acid delivery.

[0046] Fig.11 The fluorescence detection results (mice) of formulations F6 to F10 for in vivo nucleic acid delivery 6 hours after nasal instillation.

[0047] Fig.12 The fluorescence detection results (mice) of formulations F6 to F10 for in vivo nucleic acid delivery 24 hours after nasal instillation.

[0048] Fig.13 The fluorescence detection results of the preparations F6 to F10 for in vivo delivery of nucleic acids 6 hours after nasal instillation.

[0049] Fig.14 The fluorescence detection results of mouse lungs 6 hours after nasal instillation of formulations F3, F4 and F6-F10 for in vivo nucleic acid delivery (mice).

[0050] Fig.15 The results are as follows: serum specific IgG titer results after nasal instillation (nasal administration) of formulations F11 to F13 for in vivo delivery of nucleic acids.

[0051] Fig.16 The results are shown for serum specific IgA titers after nasal instillation (nasal administration) of formulations F11 to F13 for in vivo delivery of nucleic acids.

[0052] Fig.17 The results of specific IgA titers in bronchopulmonary lavage fluid after nasal instillation (nasal administration) of preparations F11 to F13 for in vivo delivery of nucleic acids.

[0053] Fig.18 The results are as follows: specific IgA titer results of nasal lavage fluid after nasal instillation (nasal administration) of formulations F11 to F13 for in vivo delivery of nucleic acids.

[0054] Fig.19 These are the results of serum-specific IgG titers after aerosol inhalation of preparations F11 to F12 for in vivo delivery of nucleic acids.

[0055] Fig. 20These are the results of specific IgA titers in nasal lavage fluid after aerosol inhalation of preparations F11 to F12 for in vivo delivery of nucleic acids.

[0056] Fig.21 These are the test results of mouse lung lymphocytes after aerosol inhalation of preparations F11 to F12 for in vivo delivery of nucleic acids. DETAILED DESCRIPTION

[0057] Unless otherwise described, all technical and scientific terms used in the present disclosure have the same meanings as those generally understood by those of ordinary skill in the art in the present disclosure. For the purpose of explaining this specification, the following description in the present disclosure will be applied, and where appropriate, the present disclosure used in the singular will also include the plural form, and vice versa. All patents, applications, published applications and other publications are incorporated by reference in their entirety. If any description in the present disclosure stated conflicts with any document incorporated by reference in the present disclosure, the following description in the present disclosure stated shall prevail.

[0058] In the present disclosure, "lipid nanoparticles" refer to particles having at least one nanometer (nm) size (e.g., 1 to 1,000 nm) containing one or more types of lipid molecules. In some embodiments, the lipid nanoparticles include non-lipid molecules partially or completely encapsulated in the lipid shell. In some embodiments, the non-lipid molecules are nucleic acids (e.g., mRNA encoding viral proteins), and the lipid component of the lipid nanoparticles includes at least one cationic lipid. Without being bound by theory, during the formation of lipid nanoparticles, the interaction of cationic lipids with nucleic acids can promote the entry and / or encapsulation of nucleic acids into lipid nanoparticles.

[0059] In the present disclosure, "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or is capable of being positively charged in response to the pH value or hydrogen ion activity of its environment. In the present disclosure, "cation" encompasses "permanent cation" and "cationizable". In some embodiments, the positive charge in the cationic lipid is caused by the presence of a quaternary nitrogen atom.

[0060] In the present disclosure, "pharmaceutically acceptable carriers, diluents or excipients" include, but are not limited to, any adjuvants, carriers, excipients, glidants, sweeteners, diluents, preservatives, dyes / colorants, flavor enhancers, surfactants, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers approved by drug regulatory government agencies of various countries (such as the U.S. FDA, China CDE, etc.) for use in humans or livestock.

[0061] "Mol %" in the present disclosure refers to the molar percentage of a component relative to the total mol of all lipid components in the lipid nanoparticle (e.g., the total mol of cationic lipids, phospholipids, structural lipids, and polyethylene glycol-modified lipids, etc.).

[0062] In the present disclosure, "about" or "approximately" means an acceptable error of a particular value determined by a person of ordinary skill, which depends in part on the way of measuring or determining the value. In some embodiments, in the present disclosure, "about" or "approximately" means within 1, 2, 3 or 4 standard deviations. In some embodiments, in the present disclosure, "about" or "approximately" means within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, within 0.5%, within 0.05% or less of a given value or range. As used herein, when "about" is used in conjunction with a numerical range, "about" in the present disclosure is intended to apply to both ends of such modified ranges (e.g., "about 5 to 10" means "about 5 to about 10").

[0063] Provided herein is a preparation for in vivo delivery of nucleic acid, wherein the preparation includes a lipid nanoparticle in which nucleic acid is formulated, and lipid nanoparticles are delivered to a host cell, wherein the nucleic acid delivered is expressed in the host cell. In some embodiments, the host cell is separated and delivered by contacting the preparation for in vivo delivery of nucleic acid with the host cell under suitable conditions in vitro, wherein nucleic acid is expressed by the host cell. In some embodiments, the host cell is in its native environment in a subject, and delivery is performed by applying an appropriate amount of preparation for in vivo delivery of nucleic acid to the subject, wherein nucleic acid is expressed in the host cell of the subject.

[0064] In some embodiments, in vitro, for example, by contacting the host cell with a preparation for in vivo delivery of nucleic acids, or in vivo, for example, by administering the preparation for in vivo delivery of nucleic acids to a subject containing the host cell, the therapeutic agent and / or prophylactic agent encapsulated in the lipid nanoparticle can be delivered to the host cell. In some embodiments, after delivery, the therapeutic nucleic acid encapsulated in the lipid nanoparticle can be expressed by the host cell endogenous transcription and translation machinery.

[0065] Lipid nanoparticles can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of lipid nanoparticles. Dynamic light scattering or potentiometric methods (e.g., potentiometric titration) can be used to measure zeta potential. Dynamic light scattering can also be used to determine particle size. Instruments such as Zetasizer Nano ZS (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure multiple features of lipid nanoparticles, such as particle size, polydispersity index, and zeta potential.

[0066] In some embodiments, the average particle size of the lipid nanoparticles may be between tens of nanometers and hundreds of nanometers. For example, the average particle size may be about 60nm to about 300nm. In some embodiments, the average particle size of the lipid nanoparticles may be about 60nm to about 100nm, about 60nm to about 90nm, about 60nm to about 80nm. In some embodiments, the average particle size may be about 60nm.

[0067] Unless otherwise specified, when the preparation for in vivo delivery of nucleic acid herein is prepared as a nasal spray or an inhalation spray, or is used for nasal spray administration or spray inhalation administration, the average particle size of the lipid nanoparticles is the average particle size before administration. In some embodiments, the inhalation spray in the present disclosure comprises a nebulizer device and a preparation for in vivo delivery of nucleic acid contained in the nebulizer device, and the average particle size of the lipid nanoparticles is measured when contained in the nebulizer device, that is, the average particle size measured before administration.

[0068] The lipid nanoparticles can be relatively homogeneous. The polydispersity index (PDI) can be used to indicate the homogeneity of the lipid nanoparticles, such as the particle size distribution of the lipid nanoparticles. A smaller (e.g., less than 0.3) polydispersity index generally indicates a narrower particle size distribution. The polydispersity index of the lipid nanoparticles can be from about 0 to about 0.25. In some embodiments, the polydispersity index of the lipid nanoparticles can be from about 0.10 to about 0.20.

[0069] The zeta potential (Zeta potential) of lipid nanoparticles can be used to indicate the dynamic potential of the composition. For example, the Zeta potential can describe the surface charge of lipid nanoparticles. Lipid nanoparticles with positive or negative charges with lower absolute values ​​are preferred because substances with higher charges (higher absolute values) can interact undesirably with cells, tissues, and other components in the body. In some embodiments, the Zeta potential of lipid nanoparticles can be about -20mV to about +20mV, or about +10mV to about +20mV.

[0070] Encapsulation efficiency describes the amount of nucleic acid that is encapsulated by lipid nanoparticles or otherwise associated with lipid nanoparticles after preparation relative to the initial amount of nucleic acid provided. High encapsulation efficiency (e.g., close to 100%) is a preferred choice. In some embodiments, lipid nanoparticles include nucleic acids as therapeutic agents, and the encapsulation efficiency of the nucleic acids is at least about 80%. In some embodiments, the encapsulation efficiency of the nucleic acids is at least about 90%. In some embodiments, the encapsulation efficiency of the nucleic acids is at least about 97%.

[0071] Lipid nanoparticles are usually composed of four lipids: (1) cationic lipids, (2) polyethylene glycol (PEG) lipids, (3) cholesterol, and (4) structural lipids. The first component, cationic lipids, are generally composed of a cationic head group and a lipid tail, and are located in the core of the lipid nanoparticles with nucleic acids. The second component, PEG lipids, have PEG chains anchored on the lipid head group. The third component is cholesterol, which is involved in the intracellular delivery of lipid nanoparticles. The fourth component, structural lipids, is mainly present on the surface of lipid nanoparticles and helps the packaging of nucleic acids and the stability of nanoparticles. In addition, the second, third and fourth components are usually called auxiliary lipids in lipid nanoparticles, which can promote the structural transformation of lipid nanoparticles in endosomes and stimulate the cytoplasmic release of nucleic acids.

[0072] Phospholipids are structural lipids in lipid nanoparticles, mainly present on the surface of lipid nanoparticles, and contribute to the packaging of nucleic acids and the stability of nanoparticles. Without being bound by theory, it is expected that phospholipids can assemble into one or more lipid bilayer structures. In some embodiments, the phospholipids in the present disclosure are selected from at least one of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycerophosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phospho-racem-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), and 1,2-dioleoyl-3-co-acetylcholine (DOTAP).

[0073] In the present disclosure, "nucleic acid" and "polynucleotide" can be used interchangeably to refer to a polymer of deoxyribonucleotides (deoxyribonucleic acid, DNA) or a polymer of ribonucleotides (ribonucleic acid, RNA). "Nucleic acid sequence", "polynucleotide sequence" and "nucleotide sequence" can be used interchangeably to refer to the order of nucleotides in a polynucleotide. It should be understood by those skilled in the art that the DNA coding strand (sense strand) and the RNA it encodes can be regarded as having the same nucleotide sequence, and the deoxythymidylic acid in the DNA coding strand sequence corresponds to the uridine acid in the RNA sequence it encodes.

[0074] The open reading frame (ORF) described in the present disclosure is a continuous stretch of DNA or RNA that begins with a start codon (such as ATG or AUG) and ends with a stop codon (such as TAA, TAG or TGA, or UAA, UAG or UGA).

[0075] In some embodiments, based on the mRNA open reading frame sequence provided, a person of ordinary skill in the art will be able to obtain the corresponding circular RNA open reading frame sequence, and according to the contents of the open literature such as CN202180048567.4, a circular RNA complete sequence capable of encoding the same amino acid sequence is prepared. In some embodiments, based on the mRNA sequence provided, a person of ordinary skill in the art will be able to obtain the corresponding DNA sequence (e.g., uracil is converted to thymine). Similarly, based on the DNA sequence provided, a person of ordinary skill in the art will obtain the corresponding RNA sequence (e.g., thymine is converted to uracil). In some embodiments, based on the RNA or DNA sequence provided, a person of ordinary skill in the art will be able to obtain the corresponding amino acid sequence.

[0076] In some embodiments, the mRNA described in the present disclosure consists of a sequence including a 5' cap, a 5' UTR, an ORF, a 3' UTR and a 3' poly (A) tail in sequence from the 5' end to the 3' end.

[0077] The 5' untranslated region (UTR) described in the present disclosure refers to a sequence located just upstream (i.e., 5') of the start codon (i.e., the first codon of the mRNA transcript translated by the ribosome) of an mRNA that does not encode a polypeptide. When an RNA transcript is produced, the 5'UTR may include a promoter sequence. Such promoter sequences are known in the art. The RNA sequence of the 5'UTR is shown in Seq ID NO.16 and Seq ID NO.1, or a 5'UTR sequence disclosed before the filing date of the present disclosure may also be introduced into the present disclosure.

[0078] The 3' untranslated region (UTR) described in the present disclosure refers to a sequence located downstream of the stop codon of an mRNA that does not encode a polypeptide. The RNA sequence of the 3'UTR is shown in Seq ID NO.2, or a 3'UTR sequence disclosed before the filing date of the present disclosure may be introduced into the present disclosure.

[0079] The poly(A) tail described in the present disclosure is a sequence located downstream of the 3'UTR of mRNA containing multiple consecutive adenosine monophosphates. The poly(A) tail may contain 10 to 300 adenosine monophosphates. In cells and / or in vivo, the poly(A) tail is used to protect mRNA from enzymatic degradation and to help transcription termination, and / or mRNA export and translation from the nucleus.

[0080] In the present disclosure, the 5' cap can be co-transcriptionally added to the RNA during in vitro transcription (e.g., using the anti-reverse cap analog "ARCA"), or can be attached to the RNA after transcription using a capping enzyme. The 5' cap is selected from ARCA, mCAP, dmCAP, tmCAP, m7G(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, m7(3'OMeG)(5')ppp(5')(2'OMeG)pG, m7G(5')ppp(5')(2'OMeG)pG or dmCAP. In some embodiments, the 5' cap is m7Gppp(5')ppp(5')(2'OMeA)pG.

[0081] In some embodiments, one or more uridines in the mRNA are replaced with the modified nucleoside N1-methyl-pseudouridine (m1ψ).

[0082] On the other hand, the present disclosure also provides a pharmaceutical composition, which comprises the preparation for in vivo delivery of nucleic acid disclosed herein, and optionally a pharmaceutically acceptable excipient. In some embodiments, the preparation or pharmaceutical composition for in vivo delivery of nucleic acid disclosed herein is used for nasal or respiratory mucosal administration.

[0083] The nasal drug delivery system refers to a type of preparation that is administered through the nasal cavity to exert local or systemic therapeutic or preventive effects. It is particularly suitable for drugs that need to be highly expressed locally in the nasal cavity or drugs that avoid the first-pass effect of the liver. The advantages of nasal drug delivery are that the nasal mucosa has a large area and rich blood vessels, so the drug is absorbed quickly and takes effect quickly after administration. In addition, the first-pass effect of the liver is low, the systemic toxicity is low, and the bioavailability is high.

[0084] Common dosage forms for nasal administration include nasal drops, nasal sprays, powders, gel preparations and emulsions. Among them, the diffusion degree and dispersion area of ​​nasal spray drugs in the nasal mucosa are relatively wide. Currently, the commonly used atomization devices are quantitative pressure inhalers (MDI), dry powder inhalers (DPI) and nebulizers. After nasal spraying, the drug is deposited in the front part of the nasal cavity, and only a small part is slowly cleared into the throat, which prolongs the retention time of the drug in the nasal cavity, which is conducive to absorption and improves bioavailability.

[0085] Respiratory mucosal drug delivery refers to a method of drug delivery using a special device to exert systemic or local effects through the deep respiratory tract, cavity, mucosa, etc.

[0086] Common dosage forms for administration to the respiratory mucosa include inhalation aerosols, inhalation powder sprays, inhalation sprays, inhalation liquid preparations and preparations that can be converted into steam. Among them, inhalation spray is a traditional way of administration, which refers to a solution, suspension or emulsion that produces an aerosol for inhalation through a predetermined amount or a quantitative nebulizer. When used, the contents are released in the form of mist with the help of the pressure of a manual pump, ultrasonic vibration, high-pressure gas or other methods, so that a certain amount of atomized liquid can be inhaled in the form of an aerosol in one breath. It has the characteristics of atomization administration and avoids the use of propellants. It has low cost, rapid onset, easy use, and is safe and reliable. However, due to the large droplets sprayed, a large amount of liquid medicine is required.

[0087] In another aspect, the present disclosure also provides a method for treating or preventing a disease or condition, comprising administering a nucleic acid to a subject in a multiple dose regimen, wherein at least one dose is administered by a mucosal route using a formulation of the present disclosure or a pharmaceutical composition of the present disclosure.

[0088] In another aspect, the present disclosure further provides a nasal drop or nasal spray, comprising the preparation for in vivo delivery of nucleic acid of the present disclosure and a pharmaceutically acceptable excipient.

[0089] In another aspect, the present disclosure also provides an inhalation spray, which comprises the formulation for in vivo delivery of nucleic acid of the present disclosure and a pharmaceutically acceptable excipient.

[0090] The pharmaceutically acceptable excipients contained in the pharmaceutical composition, inhalation spray, nasal drops or nasal spray of the present disclosure may include propellants, such as trichloromonofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethane, 1,1,1,2-tetrafluoroethane, etc. The pharmaceutically acceptable excipients may also include one or more of water, sugar solution, electrolyte solution, and amino acid solution. For example, the pharmaceutically acceptable excipients include one or more of water, Ringer's solution, glucose solution, glucose sodium chloride solution, isotonic sodium chloride solution, fructose solution, dextran, amino acid solution, heparin solution, mannitol solution, sodium bicarbonate solution, and PBS buffer.

[0091] On the other hand, the present disclosure provides a method for preparing the aforementioned preparation for in vivo delivery of nucleic acid, characterized in that it comprises: mixing the mRNA and lipid nanoparticles to form a preparation for in vivo delivery of nucleic acid.

[0092] In some embodiments, in the method for preparing the preparation for in vivo delivery of nucleic acids, mRNA is dissolved in a buffer to obtain an aqueous phase, each lipid component of the liposome nanoparticle is measured and dissolved in an organic solvent to obtain an organic phase, and the aqueous phase and the organic phase are mixed and then the organic phase is removed to obtain the preparation for in vivo delivery of nucleic acids.

[0093] In some embodiments, the volume ratio of the aqueous phase to the organic phase is 1:2-4, preferably 1:3.

[0094] In some embodiments, the buffer comprises citrate buffer or sodium acetate, preferably citrate buffer.

[0095] In some embodiments, the pH of the buffer is 3-7, preferably 4.

[0096] In some embodiments, the concentration of mRNA in the aqueous phase is 0.05 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL.

[0097] In some embodiments, the organic solvent is selected from C1-C4 low-carbon alcohols, preferably anhydrous ethanol.

[0098] In some embodiments, the concentration of the lipid component in the organic phase is 5 mg / mL to 7 mg / mL, preferably 6 mg / mL.

[0099] In some embodiments, microfluidics is used to mix the aqueous and organic phases and tangential flow is used to filter the organic solvent;

[0100] Preferably, the microfluidic flow rate is >3 mL / min, more preferably 12 mL / min.

[0101] Example 1

[0102] Preparation of mRNA

[0103] 1. Synthesize RNA sequences using linearized plasmid DNA as template by in vitro transcription (IVT):

[0104] Add the following into the reactor: 1.0 μg template DNA, 2.0 μL 10× transcription buffer, 0.2 μL NTP (25 mM each), 20 U RNase inhibitor, 3000 U T7 RNA polymerase, 20.0 μL dH2O; incubate at 37°C for 3 to 5 hours. The transcription buffer composition is: 400 mM Tris-HCl pH 8.0, 190 mM MgCl2, 50 mM DTT, 10 mM spermidine.

[0105] 2. Enzymatic end-capping:

[0106] The RNA obtained by in vitro transcription in the above step was purified, and 60 μg of the obtained RNA was mixed with 72 μL dH 2 O, incubated at 65° C. for 5 minutes to denature the RNA, and the RNA denatured mixture was transferred to ice.

[0107] The RNA denaturation mixture, 10.0 μL 10× blocking buffer, 5.0 μL 20 mM GTP, 2.5 μL 20 mM S-adenosylmethionine, 100 U RNase inhibitor, 400 U 2'-O-methyltransferase, 40 U vaccinia blocking enzyme, and 28 μL dH2O were mixed and incubated at 37°C for 30 min.

[0108] The blocking buffer composition is 0.5M Tris-HCl (pH 8.0), 60mM KCl and 12.5mM MgCl2.

[0109] 3. Polyadenylation tailing reaction:

[0110] If the template DNA does not have a poly-T tail or in order to obtain a longer poly-A tail, a poly-A tailing reaction can be performed after enzymatic end-capping. Mix 100 μL RNA obtained after enzymatic end-capping, 20 U RNase inhibitor, 12.0 μL 10× tailing buffer, 6.0 μL 20 mM ATP, 20 U poly-A polymerase and 123 μL dH2O, and incubate at 37°C for 30 min.

[0111] The tailing buffer consisted of 0.5 M Tris-HCl (pH 8.0), 2.5 M NaCl and 100 mM MgCl2.

[0112] Example 2

[0113] Preparing formulations for in vivo delivery of nucleic acids

[0114] This embodiment provides a preparation for in vivo delivery of nucleic acid, comprising mRNA encoding fluorescein, wherein the mRNA includes a 5'UTR, an ORF region, a 3'UTR and a poly(A) tail from the 5' to the 3' end. The nucleotide sequence of the 5'UTR is shown in SEQ ID NO.1, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.2, and the nucleotide sequence of the poly(A) tail is shown in SEQ ID NO.3.

[0115] The lipids in the preparation for in vivo delivery of nucleic acid are respectively 49.1% of the cationic lipid of Formula 1, 6.9% of the structural lipid DSPC, 41.1% of cholesterol and 2.9% of polyethylene glycol 2000 by mole percentage.

[0116] The preparation method is as follows:

[0117] (a) RNA was dissolved in a pH 4 citrate buffer and the concentration was adjusted to 0.1 mg / mL to obtain an aqueous phase.

[0118] (b) Dissolving the cationic lipid, structural lipid, cholesterol and polyethylene glycol lipid in anhydrous ethanol according to the formulated amounts, adjusting the concentration of the lipid component in the organic phase to 6 mg / mL, and obtaining an organic phase.

[0119] (c) The aqueous phase of step (a) and the organic phase of step (b) were mixed in a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min, and the mixture was immediately diluted 100 times with the mixed solution, and the ethanol component in the solution was removed by tangential flow filtration (TFF), and concentrated to obtain formulation F1 for in vivo delivery of nucleic acids.

[0120] Example 3

[0121] Preparing formulations for in vivo delivery of nucleic acids

[0122] This embodiment provides a preparation for in vivo delivery of nucleic acid, comprising mRNA encoding fluorescein, wherein the mRNA includes a 5'UTR, an ORF region, a 3'UTR and a poly(A) tail from the 5' to the 3' end. The nucleotide sequence of the 5'UTR is shown in SEQ ID NO.1, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.2, and the nucleotide sequence of the poly(A) tail is shown in SEQ ID NO.3.

[0123] The lipids in the preparation for in vivo delivery of nucleic acid are respectively 49.1% of the cationic lipid of Formula 2, 6.9% of the structural lipid DOPE, 41.1% of cholesterol and 2.9% of polyethylene glycol 2000 according to molar percentage.

[0124] The preparation method is as follows:

[0125] (a) RNA was dissolved in a pH 4 citrate buffer and the concentration was adjusted to 0.1 mg / mL to obtain an aqueous phase.

[0126] (b) Dissolving the cationic lipid, structural lipid, cholesterol and polyethylene glycol lipid in anhydrous ethanol according to the formulated amounts, adjusting the concentration of the lipid component in the organic phase to 6 mg / mL, and obtaining an organic phase.

[0127] (c) The aqueous phase of step (a) and the organic phase of step (b) were mixed in a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min, and the mixture was immediately diluted 100 times with the mixed solution, and the ethanol component in the solution was removed by tangential flow filtration (TFF), and concentrated to obtain formulation F2 for in vivo delivery of nucleic acids.

[0128] Example 4

[0129] Preparing formulations for in vivo delivery of nucleic acids

[0130] This embodiment provides a preparation for in vivo delivery of nucleic acid, comprising mRNA encoding fluorescein, wherein the mRNA includes a 5'UTR, an ORF region, a 3'UTR and a poly(A) tail from the 5' to the 3' end. The nucleotide sequence of the 5'UTR is shown in SEQ ID NO.1, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.2, and the nucleotide sequence of the poly(A) tail is shown in SEQ ID NO.3.

[0131] The lipids in the preparation for in vivo delivery of nucleic acid are respectively 49.1% of the cationic lipid of formula 3, 6.9% of the structural lipid DOPE, 41.1% of cholesterol and 2.9% of polyethylene glycol 2000 by mole percentage.

[0132] The preparation method is as follows:

[0133] (a) RNA was dissolved in a pH 4 citrate buffer and the concentration was adjusted to 0.1 mg / mL to obtain an aqueous phase.

[0134] (b) Dissolving the cationic lipid, structural lipid, cholesterol and polyethylene glycol lipid in anhydrous ethanol according to the formulated amounts, adjusting the concentration of the lipid component in the organic phase to 6 mg / mL, and obtaining an organic phase.

[0135] (c) The aqueous phase of step (a) and the organic phase of step (b) were mixed in a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min, and the mixture was immediately diluted 100 times with the mixed solution, and the ethanol component in the solution was removed by tangential flow filtration (TFF), and concentrated to obtain formulation F3 for in vivo delivery of nucleic acids.

[0136] Example 5

[0137] Preparing formulations for in vivo delivery of nucleic acids

[0138] This embodiment provides a preparation for in vivo delivery of nucleic acid, comprising mRNA encoding fluorescein, wherein the mRNA includes a 5'UTR, an ORF region, a 3'UTR and a poly(A) tail from the 5' to the 3' end. The nucleotide sequence of the 5'UTR is shown in SEQ ID NO.1, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.2, and the nucleotide sequence of the poly(A) tail is shown in SEQ ID NO.3.

[0139] The lipids in the preparation for in vivo delivery of nucleic acid are respectively 49.1% of the cationic lipid of formula 3, 6.9% of the structural lipid DSPC, 41.1% of cholesterol and 2.9% of polyethylene glycol 2000 by mole percentage.

[0140] The preparation method is as follows:

[0141] (a) RNA was dissolved in a pH 4 citrate buffer and the concentration was adjusted to 0.1 mg / mL to obtain an aqueous phase.

[0142] (b) Dissolving the cationic lipid, structural lipid, cholesterol and polyethylene glycol lipid in anhydrous ethanol according to the formulated amounts, adjusting the concentration of the lipid component in the organic phase to 6 mg / mL, and obtaining an organic phase.

[0143] (c) The aqueous phase of step (a) and the organic phase of step (b) were mixed in a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min, and the mixture was immediately diluted 100 times with the mixed solution, and the ethanol component in the solution was removed by tangential flow filtration (TFF), and concentrated to obtain formulation F4 for in vivo delivery of nucleic acids.

[0144] Example 6

[0145] Preparing formulations for in vivo delivery of nucleic acids

[0146] This embodiment provides a preparation for in vivo delivery of nucleic acid, comprising mRNA encoding fluorescein, wherein the mRNA includes a 5'UTR, an ORF region, a 3'UTR and a poly(A) tail from the 5' to the 3' end. The nucleotide sequence of the 5'UTR is shown in SEQ ID NO.1, the nucleotide sequence of the 3'UTR is shown in SEQ ID NO.2, and the nucleotide sequence of the poly(A) tail is shown in SEQ ID NO.3.

[0147] The lipids in the preparation for in vivo delivery of nucleic acid are respectively 24.1% of cationic lipid of formula 4, 38.9% of structural lipid DOTAP, 34.1% of cholesterol and 2.9% of polyethylene glycol 2000 by mole percentage.

[0148] The preparation method is as follows:

[0149] (a) RNA was dissolved in a pH 4 citrate buffer and the concentration was adjusted to 0.1 mg / mL to obtain an aqueous phase.

[0150] (b) Dissolving the cationic lipid, structural lipid, cholesterol and polyethylene glycol lipid in anhydrous ethanol according to the formulated amounts, adjusting the concentration of the lipid component in the organic phase to 6 mg / mL, and obtaining an organic phase.

[0151] (c) The aqueous phase of step (a) and the organic phase of step (b) were mixed in a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min, and the mixture was immediately diluted 100 times with the mixed solution, and the ethanol component in the solution was removed by tangential flow filtration (TFF), and concentrated to obtain formulation F5 for in vivo delivery of nucleic acids.

[0152] Example 7

[0153] 1. Testing the delivery efficiency of preparations for in vivo delivery of nucleic acids in cells

[0154] Human embryonic kidney 293 (HEK293) cells were placed in a 5% CO2 incubator at 37°C. When the cells reached the logarithmic growth phase, they were digested with trypsin and collected. The cells were plated at a cell density of 5×10 3 Each well was inoculated into a 96-well plate and cultured overnight.

[0155] Luciferase mRNA positive control group: Add Opti-MEMTM Medium and transfection reagent to 1.5mL centrifuge tubes, mix well and let stand for 10 minutes, record as solution A. Add Opti-MEMTM Medium and mRNA to 1.5mL centrifuge tubes, mix well and let stand for 5 minutes, record as solution B. Then add solution A to solution B, mix well and let stand for 5 minutes, take 100μL of the mixture of A and B and add it dropwise to the cells in the 96-well plate.

[0156] Preparation group: The preparation was first diluted in Opti-MEMTM Medium, and 100 μL of the liquid was added dropwise to the cells in the 96-well plate.

[0157] PBS negative control group: add PBS to Opti-MEMTM Medium according to the same volume, and then take 100 μL of liquid and add it dropwise to the cells in the 96-well plate. Each group has 3 replicate wells and is placed in an incubator at 37°C and 5% CO2 for culture.

[0158] After 24 hours, according to the detection kit, add 100 μL of reagent to each well and let it stand for 10 minutes. After thorough mixing, detect it within 10 minutes using an enzyme-labeled instrument.

[0159] 2. Testing the delivery efficiency of preparations for in vivo delivery of nucleic acids in mice

[0160] Female Balb / c mice, 6 weeks old, weighing 20–25 g, were purchased from Zhuhai Baishitong Biotechnology Co., Ltd.

[0161] The mice were randomly divided into groups of 3, and PBS was used as a negative control group. The preparations F1 to F5 for in vivo nucleic acid delivery prepared in Examples 2 to 6 were taken, and the mRNA concentration in the preparations F1 to F5 was adjusted to 900 μg / mL with 1×PBS buffer (pH 7.4±0.05).

[0162] F1-F5 were administered by nasal drip (nasal administration), and D-luciferin potassium salt was injected intraperitoneally 3h, 6h, and 24h after administration, respectively, and then the fluorescence of the nasal cavity of mice was detected using a small animal imaging system.

[0163] The delivery efficiency of the preparations F1 to F5 for in vivo delivery of nucleic acid prepared in Examples 2 to 6 was tested in cells. The test results are shown in Figure 1 . Figure 1 The upper F0 is the experimental result of the Luciferase mRNA positive control group.

[0164] Compared with the negative control, the preparations for in vivo delivery of nucleic acids in groups F1 to F5 were successfully expressed in cells, and the delivery efficiency of the preparations for in vivo delivery of nucleic acids in groups F2 to F5 was higher than that of group F1. The preparations for in vivo delivery of nucleic acids prepared with cationic lipids of formula 3 had higher delivery efficiency in cells.

[0165] The fluorescence detection results of 3h, 6h and 24h after administration are shown in Figures 2 to 4 The results of fluorescence detection using a small animal imaging system were shown in the table below. Figure 5 .

[0166] from Figure 2As can be seen above, 6 hours after administration, the nasal cavity of most mice showed expression of luciferin protein. And the expression level of the preparations (F3 and F4) for in vivo delivery of nucleic acid prepared by cationic lipid of Formula 3 in the nasal cavity was higher than that of other groups at 6 hours.

[0167] Judging from the expression levels of the preparations for in vivo delivery of nucleic acids in cells and in vivo, the expression levels of the preparations for in vivo delivery of nucleic acids (F3 and F4) prepared with the cationic lipids of Formula 3 were higher than those of the cationic lipids of other groups.

[0168] Example 8

[0169] The preparations F1 to F5 for in vivo delivery of nucleic acid prepared in Examples 2 to 6 were taken and the particle size, PDI and Zeta potential of the preparations were measured using a particle size analyzer. The encapsulation efficiency of the preparations was then measured using a fluorescent dye. The test results are shown in Table 1.

[0170] Table 1

[0171] preparation Particle size (nm) PDI Zeta potential (mV) Encapsulation rate (%) F1 79 0.09 0.64 95.7 F2 79 0.18 3.03 98.9 F3 95 0.09 7.26 95.8 F4 113 0.09 2.93 94.5 F5 81 0.12 15.68 99.6

[0172] Example 9

[0173] Formulation F6 for in vivo delivery of nucleic acid was prepared according to the method described in Example 6, wherein the lipids were 49.1% of cationic lipid of formula 3, 6.9% of structural lipid DOTAP, 41.1% of cholesterol and 2.9% of polyethylene glycol 2000, respectively, based on molar percentage.

[0174] Preparations F3 to F4 for in vivo nucleic acid delivery prepared in Examples 4 to 5 and preparation F6 for in vivo nucleic acid delivery prepared in this example were taken, and the delivery efficiency in cells and the in vivo delivery efficiency in mice were detected according to the method described in Example 7; when detecting the in vivo delivery efficiency in mice, 1×PBS buffer (pH 7.4±0.05) was used to adjust the mRNA concentration in preparations F3 to F4 and F6 to 900 μg / mL.

[0175] The results of the cell delivery efficiency test are shown in Figure 6 Compared with the negative control, the preparations for in vivo delivery of nucleic acids in groups F3-F4 and F6 were successfully expressed in cells. The expression efficiency of the preparations for in vivo delivery of nucleic acids in group F6 was higher than that in groups F3-F4. D-luciferin potassium salt was injected intraperitoneally 6 hours after administration, and the results of fluorescence detection using a small animal imaging system are shown in Figure 7 and Figure 8 .

[0176] from Figure 7 and Figure 8As can be seen above, 6 hours after administration, the nasal cavity of mice in both the F3-F4 and F6 groups showed expression of luciferin protein. In addition, the expression efficiency of the preparation for in vivo delivery of nucleic acid in the nasal cavity of the F6 group was higher than that of the F3-F4 group at 6 hours.

[0177] Judging from the expression levels of the preparations for in vivo delivery of nucleic acids in cells and in vivo, the expression level of the preparations for in vivo delivery of nucleic acids using DOTAP as the structural lipid in the F6 group was higher than that of the preparations for in vivo delivery of nucleic acids using DOPE or DSPC as the structural lipids (F3 and F4 groups).

[0178] Example 10

[0179] The preparations F3-F4 and F6 for in vivo delivery of nucleic acid prepared in Examples 4-5 and Example 9 were taken and the particle size, PDI and Zeta potential of the preparations were measured using a particle size analyzer. The encapsulation efficiency of the preparations was then measured using a fluorescent dye. The test results are shown in Table 2.

[0180] Table 2

[0181] preparation Particle size (nm) PDI Zeta potential (mV) Encapsulation rate (%) F3 95 0.09 7.26 95.8 F4 113 0.09 2.93 94.5 F6 110 0.13 9.45 90.1

[0182] Embodiment 11

[0183] The preparation F7 for in vivo delivery of nucleic acid was prepared according to the method described in Example 6, wherein the lipids were 49.1% of the cationic lipid of Formula 3, 13.9% of the structural lipid DOTAP, 34.1% of cholesterol and 2.9% of polyethylene glycol 2000, respectively, based on molar percentage.

[0184] The formulation F8 for in vivo delivery of nucleic acid was prepared according to the method described in Example 6, wherein the lipids were 49.1% of the cationic lipid of Formula 3, 27.9% of the structural lipid DOTAP, 20.1% of cholesterol and 2.9% of polyethylene glycol 2000, respectively, based on molar percentage.

[0185] The preparation F9 for in vivo delivery of nucleic acid was prepared according to the method described in Example 6, wherein the lipids were 35.0% of the cationic lipid of Formula 3, 16.0% of the structural lipid DOPE, 46.5% of cholesterol and 2.5% of polyethylene glycol 2000, respectively, based on molar percentage.

[0186] The formulation F10 for in vivo delivery of nucleic acid was prepared according to the method described in Example 6, wherein the lipids were 30.0% of the cationic lipid of Formula 3, 39.0% of the structural lipid DOTAP, 30% of cholesterol and 1.0% of polyethylene glycol 2000, respectively, based on molar percentage.

[0187] Preparation F6 for in vivo nucleic acid delivery prepared in Example 9 and preparations F7 to 10 for in vivo nucleic acid delivery prepared in this example were taken, and the delivery efficiency in cells and in mice were detected according to the method described in Example 7; when detecting the in vivo delivery efficiency in mice, 1×PBS buffer (pH 7.4±0.05) was used to adjust the mRNA concentration in preparations F6 to F10 to 900 μg / mL.

[0188] The results of the cell delivery efficiency test are shown in Fig. 9 Compared with the negative control, the preparations for in vivo delivery of nucleic acids in groups F6 to F10 were successfully expressed in cells. Among them, the expression efficiency of the preparation for in vivo delivery of nucleic acids in group F8 was higher than that in other groups.

[0189] The fluorescence detection results of 3h, 6h and 24h after administration are shown in Figures 10-12 6 hours after administration, D-luciferin potassium salt was injected intraperitoneally, and the results of fluorescence detection using a small animal imaging system are shown in Fig.13 .

[0190] from Figures 10-12 As can be seen from the figure, the preparations for in vivo delivery of nucleic acids in groups F7 and F8 expressed luciferin protein in the nasal cavity for a longer period of time.

[0191] from Fig.13 As can be seen above, 6 hours after administration, the nasal cavity of mice in groups F6 to F10 showed expression of luciferin protein. And the expression efficiency of the preparation for in vivo delivery of nucleic acid in the nasal cavity of group F8 was higher than that of other groups at 6 hours.

[0192] Judging from the expression levels of the preparations for in vivo delivery of nucleic acids in cells and in vivo, the expression level of the preparations for in vivo delivery of nucleic acids in the F8 group was higher than that in the other groups.

[0193] Example 12

[0194] Female Balb / c mice, 6 weeks old, weighing 20-25 g, were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. They were randomly divided into groups, 3 mice in each group, and PBS was used as the negative control group.

[0195] Take the preparations F3 and F4 for in vivo delivery of nucleic acids prepared in Examples 3 to 4, the preparation F6 for in vivo delivery of nucleic acids prepared in Example 9, and the preparations F7 to 10 for in vivo delivery of nucleic acids prepared in Example 11, and adjust the mRNA concentration in the preparations F3, F4, and F6 to F10 to 900 μg / mL with 1×PBS buffer (pH 7.4±0.05). Administer by nasal drip (nasal administration), and inject D-luciferin potassium salt intraperitoneally 6 hours after administration, dissect the mice to obtain lung tissue, and then use the small animal imaging system to perform fluorescence detection on the lung tissue.

[0196] The results of fluorescence imaging of mouse lungs are shown in Fig.14 Only formulations F7 and F8, used for in vivo delivery of nucleic acids, showed expression of luciferin protein in the lung.

[0197] Embodiment 13

[0198] The preparations F6 to F10 for in vivo delivery of nucleic acid prepared in Example 9 and Example 10 were used to detect the particle size, PDI and Zeta potential of the preparations using a particle size analyzer. The encapsulation efficiency of the preparations was then detected using a fluorescent dye. The test results are shown in Table 3.

[0199] Table 3

[0200] preparation Particle size (nm) PDI Zeta potential (mV) Encapsulation rate (%) F6 110 0.13 9.45 90.1 F7 100 0.15 12.69 97 F8 69.1 0.14 19.39 99.2 F9 86 0.09 7.48 98.6 F10 85 0.13 10.18 99.3

[0201] Embodiment 14

[0202] This embodiment provides a preparation for in vivo delivery of nucleic acid, comprising mRNA encoding RSV pre-fusion F protein, the mRNA including 5'UTR, ORF region, 3'UTR and poly(A) tail from 5' to 3' end. The nucleotide sequence of 5'UTR is shown in SEQ ID NO.1, the nucleotide sequence of 3'UTR is shown in SEQ ID NO.2, and the nucleotide sequence of poly(A) tail is shown in SEQ ID NO.3.

[0203] The preparation method is as follows:

[0204] (a) RNA was dissolved in a pH 4 citrate buffer and the concentration was adjusted to 0.1 mg / mL to obtain an aqueous phase.

[0205] (b) Dissolving the cationic lipid, structural lipid, cholesterol and polyethylene glycol lipid in anhydrous ethanol according to the formulated amounts, adjusting the concentration of the lipid component in the organic phase to 6 mg / mL, and obtaining an organic phase.

[0206] (c) The aqueous phase of step (a) and the organic phase of step (b) are mixed in a volume ratio of 1:3 using a microfluidic device at a flow rate of 12 mL / min, the mixed solution is immediately diluted 100 times with the mixed solution, and the ethanol component in the solution is removed by tangential flow filtration (TFF), and concentrated to obtain a preparation for in vivo delivery of nucleic acids.

[0207] The lipids in the preparation for in vivo delivery of nucleic acids are, by molar percentage, 49.1% of the cationic lipid of formula 1, 6.9% of the structural lipid DSPC, 41.1% of cholesterol and 2.9% of polyethylene glycol 2000, wherein the amino acid sequence of the RSV pre-fusion F protein encoded by the mRNA is as shown in SEQ ID NO.5, and the nucleotide sequence of the ORF region is as shown in SEQ ID NO.4, thereby preparing preparation F11 for in vivo delivery of nucleic acids.

[0208] The lipids in the preparation for in vivo delivery of nucleic acids are, by molar percentage, 49.1% of the cationic lipid of formula 3, 27.9% of the structural lipid DOTAP, 20.1% of cholesterol and 2.9% of polyethylene glycol 2000, wherein the amino acid sequence of the RSV pre-fusion F protein encoded by the mRNA is as shown in SEQ ID NO.5, and the nucleotide sequence of the ORF region is as shown in SEQ ID NO.4, thereby preparing preparation F12 for in vivo delivery of nucleic acids.

[0209] The lipids in the preparation for in vivo delivery of nucleic acids are, by molar percentage, 49.1% of the cationic lipid of formula 3, 27.9% of the structural lipid DOTAP, 20.1% of cholesterol and 2.9% of polyethylene glycol 2000, wherein the amino acid sequence of the RSV pre-fusion F protein encoded by the mRNA is as shown in SEQ ID NO.7, and the nucleotide sequence of the ORF region is as shown in SEQ ID NO.6, thereby preparing preparation F13 for in vivo delivery of nucleic acids.

[0210] Embodiment 15

[0211] Female Balb / c mice, 6 weeks old and weighing 20-25 g, were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. They were randomly divided into groups, with 5 mice in each group.

[0212] Preparations F11 to F13 for in vivo nucleic acid delivery prepared in Example 14 were taken, and the mRNA concentration in the preparations F11 to F13 was adjusted to 900 μg / mL with 1× PBS buffer (pH 7.4±0.05).

[0213] Nasal drip (nasal administration) immune experiment: set up PBS negative control group, preparation F11 intramuscular injection group, preparation F11 nasal drip administration group, preparation F12 intramuscular injection group, preparation F12 nasal drip administration group, preparation F13 intramuscular injection group, preparation F13 nasal drip administration group.

[0214] A two-time immunization program was used, with the first administration time set as D0, the second administration on the 7th day after the first administration, blood was collected 7 days after the second immunization to separate serum, and bronchopulmonary lavage fluid and nasal lavage fluid were collected.

[0215] The specific IgG titer of serum was tested, and the specific IgA titer of serum, bronchopulmonary lavage fluid and nasal lavage fluid was tested.

[0216] The neutralizing activity detection method (specific IgG titer and specific IgA detection) of the serum (or bronchopulmonary lavage fluid, or nasal lavage fluid) of the preparation group for in vivo delivery of nucleic acid is as follows:

[0217] a) The antigen was diluted with PBS and added to the ELISA plate, incubated overnight, washed, added with 2% bovine serum albumin solution, and incubated in a constant temperature box.

[0218] b) Wash the ELISA plate with phosphate-Tween buffer and shake off the liquid in the well plate. Use phosphate-Tween buffer to dilute all samples in a gradient manner and add them to the ELISA plate, incubate and wash.

[0219] c) Add 100uL goat anti-mouse IgG or IgA to each well of the ELISA plate, and wash with phosphate-tween buffer after incubation.

[0220] d) Add 100uL TMB colorimetric solution to each well of the ELISA plate, add the prepared stop solution (2M H2SO4), and read the OD 450nm value on an ELISA reader.

[0221] The results of serum-specific IgG titers of preparations F11 to F13 for in vivo delivery of nucleic acids are shown in Fig.15 The results of serum-specific IgA titer test for preparations F11 to F13 for in vivo delivery of nucleic acids are shown in Fig.16 The results of the specific IgA titers of bronchopulmonary lavage fluid of preparations F11 to F13 for in vivo delivery of nucleic acids are shown in Fig.17 The results of the specific IgA titers of nasal lavage fluids of preparations F11 to F13 for in vivo delivery of nucleic acids are shown in Fig.18 .

[0222] from Figures 15 to 18 It can be seen from the above that the serum-specific IgG, specific IgA titers in bronchopulmonary lavage fluid and nasal lavage fluid, and serum-specific IgA titers of the preparation F12 for in vivo nucleic acid delivery are higher than those of the other groups, indicating that the delivery efficiency of the preparation F12 for in vivo nucleic acid delivery is higher than that of the preparation F11 for in vivo nucleic acid delivery, and that the mRNA encoding the RSV prefusion F protein (amino acid sequence SEQ ID NO.5) contained in the preparation F12 for in vivo nucleic acid delivery (the nucleotide sequence of the ORF region is shown in SEQ ID NO.4) can stimulate better immunogenicity.

[0223] Example 16

[0224] The preparations F11 to F13 for in vivo delivery of nucleic acid prepared in Example 15 were taken and the particle size, PDI and Zeta potential of the preparations were measured using a particle size analyzer. The encapsulation efficiency of the preparations was then measured using a fluorescent dye. The test results are shown in Table 4.

[0225] Table 4

[0226] Preparation name Particle size (nm) PDI Zeta potential (mV) Encapsulation rate (%) F11 69.38 0.168 0.31 97.23 F12 70.63 0.117 14.66 99.34 F13 66.66 0.089 12.83 98.74

[0227] Embodiment 17

[0228] Female Balb / c mice, 6 weeks old and weighing 20-25 g, were purchased from Zhuhai Baishitong Biotechnology Co., Ltd. They were randomly divided into groups, with 5 mice in each group.

[0229] The preparations F11 and F12 for in vivo delivery of nucleic acid prepared in Example 14 were taken, and the mRNA concentration in the preparations F11 and F12 was adjusted to 500 μg / mL with 1×PBS buffer (pH 7.4±0.05).

[0230] Aerosol inhalation immunity experiment: PBS negative control group, low / medium / high dose groups of preparation F11 spray inhalation, and low / medium / high dose groups of preparation F12 spray inhalation were set up. The dosage of preparation F11 and F12 low dose groups was 10μg mRNA, the dosage of preparation F11 and F12 medium dose groups was 50μg mRNA, and the dosage of preparation F11 and F12 high dose groups was 200μg mRNA.

[0231] The two-dose immunization program was used, with the first administration time set as D0, the second administration on the 7th day after the first administration, blood was collected 7 days after the second administration to separate serum, collect nasal lavage fluid, and collect all lung cells to verify the cellular immune response.

[0232] 1. Detect the serum-specific IgG titer and the serum and nasal lavage fluid-specific IgA titer.

[0233] The detection methods for specific IgG titer detection and specific IgA titer detection are shown in Example 15.

[0234] The results of serum-specific IgG titers of preparations F11-F12 for in vivo delivery of nucleic acids are shown in Fig.19 The results of the specific IgA titers of nasal lavage fluids of preparations F11-F12 for in vivo delivery of nucleic acids are shown in Fig. 20 .

[0235] from Figures 19-20It can be seen from the figure that the serum IgG and specific IgA titers in the nasal lavage fluid of the preparation F12 for in vivo delivery of nucleic acids were higher than those of the other groups, indicating that the delivery efficiency of the preparation F12 for in vivo delivery of nucleic acids was higher than that of the preparation F11 for in vivo delivery of nucleic acids.

[0236] 2. Detection of mouse lung lymphocytes

[0237] All lung cells of the immunized mice were collected 14 days after the first immunization (7 days after the second immunization), and CD3+CD4+ and CD3+CD8+ T lymphocytes were detected by flow cytometry. The specific operation steps are as follows:

[0238] a) The mouse lungs were removed, cut into pieces and digested. The lung cell suspension was passed through a cell sieve and the precipitate was collected after centrifugation.

[0239] b) Add red blood cell lysis buffer, let stand, then add RPMI-1640 basal medium to terminate the reaction. Centrifuge at room temperature and collect the precipitate.

[0240] c) Wash with RPMI 1640 basal medium and centrifuge.

[0241] d) Add Staining Buffer, then add 1 μg BD Fc block / 10^6 cells, incubate at 4℃ for 15 min. Centrifuge and keep the precipitate.

[0242] e) After washing the cells, stain the cell surface, resuspend them in Staining Buffer, and detect them by flow cytometry.

[0243] The results of the mouse lung lymphocyte detection are shown in Fig.21 .

[0244] from Fig.21 It can be seen from the above that the preparation F12 for in vivo delivery of nucleic acid is more capable of improving humoral immunity than the preparation F11 for in vivo delivery of nucleic acid.

[0245] Embodiment 18

[0246] The preparations F11-F12 for in vivo delivery of nucleic acid prepared in Example 17 were taken and the particle size, PDI and Zeta potential of the preparations were measured using a particle size analyzer. The encapsulation efficiency of the preparations was then measured using a fluorescent dye. The test results are shown in Table 5.

[0247] Table 5

[0248] Preparation name Particle size (nm) PDI Zeta potential (mV) Encapsulation rate (%) Collection method F11 75.74 0.144 3.694 96.61 Before atomization F11 274.7 0.265 -13.04 22.91 After atomization F12 99.93 0.221 17.29 98.6 Before atomization F12 269 0.391 10.53 98.17 After atomization

[0249] As can be seen from Table 5, the encapsulation rate of the preparation F11 for in vivo delivery of nucleic acids dropped sharply after atomization, and the Zeta potential also changed greatly; while the encapsulation rate and Zeta potential of the preparation F12 for in vivo delivery of nucleic acids were relatively stable before and after atomization. Therefore, the preparation F12 for in vivo delivery of nucleic acids is more suitable for spray inhalation administration.

Claims

1. A preparation for in vivo delivery of nucleic acid, characterized in that The preparation for in vivo delivery of nucleic acid comprises lipid nanoparticles and nucleic acid encapsulated by the lipid nanoparticles.

2. The preparation according to claim 1, characterized in that The lipid nanoparticle comprises 25.0-50.0 mol% of a cationic lipid, 6.0-40.0 mol% of a phospholipid, 20.0-47.0 mol% of a structural lipid, and 1.0-3.0 mol% of a polyethylene glycol-modified lipid, based on the total amount of all lipids in the lipid nanoparticle; Preferably, the lipid nanoparticle comprises 25.0-50.0 mol% of cationic lipids, 6.0-30.0 mol% of phospholipids, 20.0-47.0 mol% of structural lipids and 1.0-3.0 mol% of polyethylene glycol-modified lipids, based on the total amount of all lipids in the lipid nanoparticle; Preferably, the lipid nanoparticles contain 49.1 mol % of cationic lipids, 27.9 mol % of phospholipids, 20.1 mol % of structural lipids and 2.9 mol % of polyethylene glycol-modified lipids, based on the total amount of all lipids in the lipid nanoparticles.

3. The preparation according to claim 2, characterized in that The cationic lipid is selected from at least one compound of Formula 1, Formula 2, Formula 3, and Formula 4; Formula 1 Formula 2 Formula 3 Formula 4 Preferably, the cationic lipid is a compound as shown in Formula 3; Preferably, the structural lipid is selected from at least one of cholesterol, coprosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatine, tomatin, ursolic acid, and α-tocopherol; preferably, the structural lipid is cholesterol; Preferably, the phospholipid is selected from at least one of DSPC, DOPE, DLPC, DMPC, DOPC, DPPC, DUPC, POPC, DOPG, DPPG, DPPE, DMPE, DSPE, and DOTAP; preferably, the phospholipid is DOTAP; Preferably, the polyethylene glycol-modified lipid is selected from at least one of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol; preferably, the PEG lipid is at least one of PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000 or Chol-PEG2000, PEG2000; preferably, the PEG lipid is PEG2000.

4. The preparation according to claim 1, characterized in that The nucleic acid comprises a DNA molecule and / or an RNA molecule; Preferably, the DNA molecules include chain DNA molecules and / or circular DNA molecules; Preferably, the RNA molecule comprises mRNA or circular RNA.

5. The preparation according to claim 4, characterized in that The mRNA molecule comprises an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) is shown in one of Seq ID NO.4 or Seq ID NO.6; Preferably, the mRNA molecule comprises an open reading frame (ORF), and the nucleotide sequence of the open reading frame (ORF) is shown in Seq ID NO.

4.

6. The preparation according to claim 4, characterized in that The preparation for in vivo delivery of nucleic acid is used for administration to the nasal cavity and respiratory mucosa; Preferably, the nasal administration includes nasal drip, nasal spray administration or nasal inhalation.

7. The preparation according to claim 1, characterized in that The average particle size of the lipid nanoparticles is 60nm to 300nm; Preferably, the average particle size of the lipid nanoparticles is 60nm to 100nm; preferably, the average particle size of the lipid nanoparticles is 60nm to 90nm; preferably, the average particle size of the lipid nanoparticles is 60nm to 80nm; preferably, the average particle size of the lipid nanoparticles is 60nm; The polydispersity index of the lipid nanoparticles is 0 to 0.25; preferably, the polydispersity index of the lipid nanoparticles is 0.10 to 0.20; The Zeta potential of the lipid nanoparticles is -20mV to +20mV; preferably, the Zeta potential of the lipid nanoparticles is +10mV to +20mV.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the preparation for in vivo delivery of nucleic acid as claimed in claims 1 to 6, and optionally a pharmaceutically acceptable excipient; Preferably, the pharmaceutical composition is used for administration to the nasal cavity or respiratory mucosa.

9. The preparation according to claim 8, characterized in that The excipient is 1×PBS buffer, and the buffer has a pH of 7.4±0.

05.

10. The preparation according to claim 9, characterized in that The pharmaceutical composition is used for nasal administration, and the nucleic acid concentration in the pharmaceutical composition is 800-1000 μg / mL; The pharmaceutical composition is used for administration to the respiratory mucosa, and the nucleic acid concentration in the pharmaceutical composition is 400-600 μg / mL.

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