Polynucleotide encoding adrenocorticotropic hormone, and related composition and method thereof

Abstract

Provided is a nucleic acid molecule encoding adrenocorticotropic hormone, or a functional fragment or variant thereof. The present invention further relates to a composition containing the nucleic acid molecule, comprising a lipid nanoparticle, and a related treatment method and use for treating or preventing rheumatic diseases (including but not limited to gout inflammation), lung diseases, ophthalmologic diseases, kidney diseases or neurological diseases (including but not limited to infant spasms and multiple sclerosis) in a subject.

Description

Polynucleotides encoding adrenocorticotropic hormone and related compositions and methods

[0001] This application claims priority to Chinese patent application No. CN202310934800.X filed on July 27, 2023, which is hereby incorporated by reference in its entirety. Technical Field

[0002] The present disclosure relates to nucleic acid molecules encoding adrenocorticotropic hormone, its functional fragments or variants. The present disclosure also relates to compositions comprising the nucleic acid molecules, such as lipid nanoparticles (LNPs), and related treatment methods and uses for treating or preventing rheumatic diseases (including but not limited to gouty inflammation), lung diseases, ophthalmic diseases, kidney diseases or neurological diseases (including but not limited to infantile spasms, multiple sclerosis) in subjects. Background Art

[0003] ACTH (adrenocorticotropic hormone, ACTH) is a polypeptide hormone secreted by the adrenocorticotroph cells of the anterior pituitary gland. Its synthesis and secretion are stimulated by CRH (corticotropin-releasing hormone) secreted by the hypothalamus. The gene encoding ACTH is POMC (proopiomelanocortin), located on chromosome 2p23.3. POMC is initially transcribed and translated into a 241-amino acid polypeptide. This is cleaved by a series of proteases to produce a series of short peptide products, including ACTH. These products also include melanocyte-stimulating hormones (MSHs) and endorphins.

[0004] ACTH is 39 amino acid residues long, and its biological activity depends on amino acid residues 1-25. Fragments with less than 20 amino acids are completely inactive. Positions 25-39 play an important role in the stability of ACTH. ACTH levels under physiological conditions are affected by blood sugar, exercise, and circadian rhythms. After entering the circulation, ACTH binds to the MC2R receptor on adrenal cortical cells, promoting an increase in intracellular cAMP levels and activation of the PKA signaling pathway, thereby stimulating the adrenal cortex to synthesize and secrete steroid hormones such as cortisol. Cortisol plays an important role in sugar metabolism, protein metabolism, fat metabolism, water and salt balance, as well as anti-inflammatory and immune regulation. It can be seen that ACTH is very important for maintaining hormone balance and regulating various physiological functions. In addition, ACTH is also related to the development and functional regulation of the nervous system, as well as reproduction and growth.

[0005] Acthar Gel is an ACTH-producing drug manufactured by Mallinckrodt Pharmaceuticals and approved by the FDA in 1952. Acthar Gel has a variety of indications, some of which are commonly found:

[0006] Nephrotic Syndrome in Children: Acthar Gel can be used to treat idiopathic nephrotic syndrome in children, especially for patients with relapsed or refractory disease.

[0007] Acute exacerbations of Multiple Sclerosis: Acthar Gel can be used to treat acute exacerbations of Multiple Sclerosis to relieve symptoms during acute attacks.

[0008] Infantile Spasms: Acthar Gel can be used to treat infantile spinal muscular atrophy, helping to control epileptic seizures and improve patients' quality of life.

[0009] Rheumatoid Arthritis: Acthar Gel can be used to treat rheumatoid arthritis, especially in patients who have not responded to or cannot tolerate other treatment options.

[0010] In addition, Acthar Gel may also be used for other indications, such as systemic lupus erythematosus, idiopathic inflammatory myopathies, and acute attacks of gout.

[0011] Recent preclinical studies have shown that ACTH's treatment of acute gout attacks does not simply suppress inflammation by promoting the synthesis of steroid hormones such as cortisol in the adrenal cortex. In an acute gout model in adrenalectomized rats, ACTH can still alleviate acute gout attacks. Its mechanism of action may be to inhibit the recruitment of inflammatory immune cells.

[0012] Disadvantages of ACTH drugs:

[0013] High Cost: ACTH medications are expensive treatment options, making them unaffordable for some patients. As of 2021, Acthar Gel costs up to $35,000 per 5 ml vial. Some indications require multiple doses, making them costly.

[0014] Source limitations: Acthar Gel's active ingredient is natural ACTH extracted from the adrenal cortex of animals (usually pigs). After extraction, it undergoes a series of preparation and processing steps to obtain a final product that meets pharmaceutical quality and purity requirements. These steps include filtration, concentration, and viral inactivation to ensure product safety and quality. The animal extraction source and complex production process severely limit Acthar Gel's production capacity, and there are also risks such as large batch-to-batch variability and high immunogenicity.

[0015] Short half-life: Generally speaking, ACTH has a half-life of approximately 10 to 15 minutes. It is primarily metabolized and broken down in the body and cleared by the liver and kidneys. Furthermore, ACTH secretion is regulated by a negative feedback mechanism. When cortisol levels rise in the body, it inhibits ACTH secretion. This leads to frequent dosing of Acthar Gel, further affecting patient compliance.

[0016] In summary, ACTH is an important adrenal hormone that plays a crucial role in regulating hormone balance and multiple physiological functions. ACTH drugs play an important role in the treatment of gout, infantile spasms, and multiple sclerosis.

[0017] mRNA protein replacement therapy is a biotechnology based on mRNA technology. Its main function is to transmit the information encoding specific proteins to human cells, causing them to produce the corresponding proteins. It has the following advantages:

[0018] 1) Highly Customizable: The design and preparation of mRNA drugs can be tailored to specific therapeutic targets, making them highly personalized. By adjusting the mRNA sequence and structure, efficient expression and synthesis of specific proteins can be achieved, thereby achieving precise therapeutic effects.

[0019] 2) Sustained expression: Sequence-optimized mRNA drugs can be expressed in vivo for several days (or even longer), enabling lower dosing frequency and improving patient compliance.

[0020] 3) Broad Indications: mRNA drugs have the potential for a wide range of indications. They can be used to treat a variety of diseases, including infectious diseases, cancer, and genetic disorders. By adjusting the encoding information of mRNA, different therapeutic proteins can be produced to address a variety of different diseases.

[0021] 4) Relatively Short R&D Time: Compared to traditional drug development, the R&D time for mRNA drugs is likely to be relatively short. Once the coding information for the target protein is determined, the preparation and production of mRNA drugs can be carried out relatively quickly, helping to quickly respond to the challenges of emerging pathogens and diseases.

[0022] Currently, mRNA protein replacement therapy has been widely used in various fields, such as COVID-19 vaccines, cancer immunotherapy, and genetic disease treatment. The use of mRNA drugs to express adrenocorticotropic hormone may provide a promising approach for the treatment of gout and infantile spasms, with the potential to improve efficacy, safety, and convenience.

[0023] Summary of the Invention

[0024] The inventors of this application have solved the above-mentioned needs through creative work.

[0025] Specifically, the present disclosure relates to the following technical solutions:

[0026] In one embodiment, the present disclosure relates to a nucleic acid comprising an open reading frame (ORF) encoding adrenocorticotropic hormone, a functional fragment or variant thereof.

[0027] In further embodiments, the ORF encodes wild-type ACTH, mutant ACTH, truncated ACTH, or ACTH fusion protein that retains ACTH function.

[0028] In another embodiment, the amino acid sequence encoded by the ORF comprises a signal peptide and adrenocorticotropic hormone from the N-terminus to the C-terminus.

[0029] In yet another embodiment, the amino acid sequence encoded by the ORF comprises a signal peptide, adrenocorticotropic hormone, a linker, and a kappa light chain variable region (VLk) sequence from N-terminus to C-terminus.

[0030] In yet a further embodiment, the ACTH has the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO: 1.

[0031] In yet a further embodiment, the ORF comprises, in order from 5' to 3', a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding adrenocorticotropic hormone.

[0032] In yet a further embodiment, the ORF comprises, from 5' to 3', a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding adrenocorticotropic hormone, a nucleotide sequence encoding a linker, and a nucleotide sequence encoding a kappa light chain variable region (VLk) sequence.

[0033] In yet a further embodiment, the nucleic acid comprises, from 5' to 3', 5'-UTR, an ORF encoding an amino acid sequence comprising a signal peptide and adrenocorticotropic hormone or an amino acid sequence comprising a signal peptide, adrenocorticotropic hormone, a linker and a kappa light chain variable region (VLk) sequence, and 3'-UTR.

[0034] In yet further embodiments, the nucleic acid is DNA or RNA.

[0035] In another embodiment, the present disclosure relates to a vector or plasmid comprising the nucleic acid of any of the preceding embodiments.

[0036] In yet another embodiment, the present disclosure relates to a host cell comprising the nucleic acid or vector or plasmid of any one of the preceding embodiments.

[0037] In yet another embodiment, the present disclosure relates to a pharmaceutical composition comprising the nucleic acid of any of the preceding embodiments and a delivery agent.

[0038] In yet another embodiment, the present disclosure relates to a method for treating or preventing rheumatic diseases (including but not limited to gouty inflammation), lung diseases, ophthalmic diseases, kidney diseases or neurological diseases (including but not limited to infantile spasms, multiple sclerosis) in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a nucleic acid according to any one of the preceding embodiments or a pharmaceutical composition according to any one of the preceding embodiments.

[0039] In yet another embodiment, the present disclosure relates to a kit comprising a nucleic acid described herein and / or a pharmaceutical composition described herein, and instructions for use.

[0040] In yet another embodiment, the present disclosure relates to a method for enhancing expression of an open reading frame (ORF) encoding a corticotropin fusion protein in a nucleic acid, wherein the corticotropin fusion protein is a fusion protein of a signal peptide and corticotropin, and the signal peptide is fused to the N-terminus of the corticotropin, the method comprising linking the C-terminus of the corticotropin fusion protein in the ORF to a kappa light chain variable region (VLk) sequence via a linker.

[0041] The details of the present application are set forth in the accompanying description below. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, illustrative methods and materials are now described. Other features, objects and advantages of the present application will be apparent from the specification and claims. In the specification and the appended claims, the singular also includes the plural, unless the context clearly provides otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present application belongs. All patents and publications cited in this specification are incorporated herein by reference in their entirety.

[0042] The contents of all references cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are hereby expressly incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] FIG1 is a schematic diagram of the structure of the mRNA construct involved in the embodiments of the present invention.

[0044] 2a-b are the WB detection results and grayscale integration results of the cytoplasmic proteins after the ACTH mRNA construct was transfected into HEK293T cells in Example 2.

[0045] 3a-b are the WB detection results and grayscale integration results of cytoplasmic proteins after HEK293T cells were transfected with ACTH mRNA constructs with different UTRs in Example 2.

[0046] 4a-b are the WB detection results and grayscale integration results of cytoplasmic proteins after transfection of HEK293 cells with ACTH mRNA constructs containing different cap structures, modified bases and 3'-poly A tail sequences in Example 2.

[0047] 5a-b are the WB detection results and grayscale integration results of cytoplasmic proteins after HEK293T cells were transfected with ACTH mRNA constructs containing different secretion signal peptides in Example 2.

[0048] Figure 6 shows the ACTH expression levels in HEK293T cells transfected with different ACTH mRNA constructs in Example 3. The results show that among the mRNA construct sequences shown in SEQ ID NOs: 225-228, the mRNA construct shown in SEQ ID NO: 226 has the highest expression level in the cell supernatant.

[0049] FIG7 a shows the results of the ACTH blood levels in healthy mice at 0-24 hours after intravenous injection of the mRNA constructs represented by SEQ ID NOs: 229 and 230 in Example 5 in the form of LNPs. FIG.

[0050] FIG7 b shows the changes in ACTH blood levels in healthy mice from 0 to 396 hours after intravenous injection of the mRNA constructs represented by SEQ ID NOs: 231 and 232 in Example 5 in the form of LNPs.

[0051] FIG7 c shows the ACTH blood levels in healthy mice at 0-396 hours after intravenous injection of the mRNA construct represented by SEQ ID NO: 230 in Example 5 in the form of LNP.

[0052] 8a-k show the joint swelling results of rats with gout inflammation model induced by intramuscular and subcutaneous injection of the mRNA construct represented by SEQ ID NO: 230 in Example 6 in the form of LNPs, respectively.

[0053] Figures 9a-b show the changes in the levels of inflammatory factors IL-1beta and IL-6 in rats with gout inflammation model induced by intramuscular and subcutaneous injection of the mRNA construct represented by SEQ ID NO: 230 in Example 6 in the form of LNP.

[0054] FIG10 shows the blood ACTH levels in rats with gout inflammation model induced by intramuscular and subcutaneous injection of the mRNA construct represented by SEQ ID NO: 230 in Example 6 in the form of LNP.

[0055] FIG11a shows the expression level of ACTH in the cell supernatant of HEK293T cells transfected with the mRNA constructs shown in SEQ ID NOs: 233-241 and SEQ ID NO: 230 containing different secretion signal peptides in Example 7. FIG11a shows the expression level of ACTH in the cell supernatant of HEK293T cells transfected with the mRNA constructs shown in SEQ ID NOs: 233-241 and 230 containing different secretion signal peptides in Example 7.

[0056] Figure 11b shows the expression level of ACTH in the cell supernatant of HepG2 cells transfected with the mRNA constructs represented by SEQ ID NO: 234, SEQ ID NO: 235, SEQ ID NO: 237, and SEQ ID NO: 239 with different secretion signal peptides in Example 7.

[0057] FIG11c shows the expression level of ACTH in the cell supernatant of HEK293T cells transfected with the mRNA constructs represented by SEQ ID NOs: 253-266 and SEQ ID NO: 237 containing different secretion signal peptides in Example 7.

[0058] FIG12 shows the expression level of ACTH in the cell supernatant of HepG2 cells transfected with the mRNA construct represented by SEQ ID NO: 237 in Example 8 using different LNP formulations.

[0059] FIG13 shows the expression level of ACTH in the cell supernatant of HepG2 cells transfected with the mRNA constructs represented by SEQ ID NOs: 269-285 and SEQ ID NO: 237 in Example 10.

[0060] FIG14 shows the expression level of ACTH in the cell supernatant of HepG2 cells transfected with the mRNA constructs represented by SEQ ID NOs: 286-298 and SEQ ID NO: 237 in Example 11.

[0061] FIG15 shows the PK results of the mRNA constructs represented by SEQ ID NOs: 230 and 237 in Example 9 injected intravenously into rats in the form of LNP.

[0062] FIG16 is an anatomical diagram of the skin hardening and swelling after subcutaneous injection of the mRNA-LNP drug in Example 12.

[0063] FIG17 is a rat stability PK experiment of the highly secretory expression signal peptide construct in Example 13

[0064] Figures 18a-d show the joint swelling in the efficacy experiment of the construct SEQ ID NO: 289 in Example 14 in the MSU-induced gout inflammation model.

[0065] Figure 18e-f is a 24h significance analysis of joint swelling in the efficacy experiment of SEQ ID NO: 289 construct in the MSU-induced gout inflammation model in Example 14

[0066] Figure 18g shows the changes in plasma ACTH levels in the efficacy experiment of the construct SEQ ID NO: 289 in Example 14 in the MSU-induced gout inflammation model.

[0067] Figure 18h shows the changes in animal body weight in the efficacy experiment of the construct SEQ ID NO: 289 in Example 14 in the MSU-induced gout inflammation model.

[0068] Figure 18i-l shows the changes in cytokines, ALT, and AST in the efficacy experiment of the construct SEQ ID NO: 289 in Example 14 in the MSU-induced gout inflammation model.

[0069] Figures 19a-f show the ACTH expression levels of ACTH mRNA and ACTH-VLK mRNA in HepG2 cell supernatants in Example 15;

[0070] Figure 20 is a comparison of the in vivo half-life of ACTH short peptide and ACTH mRNA in Example 16;

[0071] FIG21a shows the clinical scores of each drug-dosing group in the EAU model experiment in Example 17;

[0072] Figure 21b is a significance analysis of each drug-dosing group at the time point of day 11 in the EAU model in Example 17;

[0073] FIG21c is a photograph of the animal eyeballs in each drug-dosing group in the EAU model experiment in Example 17;

[0074] FIG22a shows the clinical scores of each drug-dosing group in the AIA model experiment in Example 18;

[0075] Figures 22b-c show the joint swelling and paw swelling of each drug-dosing group in the AIA model experiment in Example 18;

[0076] FIG22d shows the body weight changes of each drug-dosing group in the AIA model in Example 18. DETAILED DESCRIPTION

[0077] Provided herein are nucleic acid molecules comprising an open reading frame (ORF) encoding adrenocorticotropic hormone, a functional fragment thereof, or a variant thereof. Also provided herein are compositions comprising the nucleic acid molecules, including lipid nanoparticles (LNPs), and related methods and uses for treating or preventing rheumatic diseases (including but not limited to gout inflammation), lung diseases, ophthalmic diseases, kidney diseases, or neurological diseases (including but not limited to infantile spasms, multiple sclerosis).

[0078] 1. General Technology

[0079] The techniques and procedures described or referenced herein include those generally well understood and / or commonly employed by those skilled in the art using conventional methods, such as, for example, the widely used methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed., 2001); Current Protocols in Molecular Biology (Ausubel et al., eds., 2003).

[0080] 2. Terminology

[0081] Unless otherwise described, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For the purpose of interpreting this specification, the following terminology will be applied, and where appropriate, terms 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 of a term set forth conflicts with any document incorporated herein by reference, the terminology set forth below shall prevail.

[0082] As used herein and unless otherwise indicated, the term "lipid" refers to a group of organic compounds that include, but are not limited to, fatty acid esters and are generally characterized by being poorly soluble in water but soluble in many non-polar organic solvents. Although lipids generally have poor water solubility, certain classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG2000) have limited water solubility and are soluble in water under certain conditions. Known lipid types include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be divided into at least three categories: (1) "simple lipids," including fats and oils, as well as waxes; (2) "compound lipids," including phospholipids and glycolipids (e.g., DMPE-PEG2000); and (3) "derivatized lipids," such as steroids. In addition, as used herein, lipids also include lipid-like compounds. The term "lipid-like compound," also referred to as "lipid-like," refers to lipid-like compounds (e.g., amphiphilic compounds with lipid-like physical properties).

[0083] The term "lipid nanoparticle" or "LNP" refers to a particle with at least one nanometer (nm) size (e.g., 1 to 1,000 nm) containing one or more types of lipid molecules. The LNP provided herein can further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule partially or completely encapsulated in a lipid shell. Specifically, in some embodiments, wherein the payload is a negatively charged molecule (e.g., mRNA encoding adrenocorticotropic hormone), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is expected that the cationic lipid can interact with the negatively charged payload molecule and promotes the payload to be incorporated into and / or be encapsulated in the LNP during LNP formation. Other lipids that can form a part for the LNP as provided herein include, but are not limited to, neutral lipids and charged lipids, such as steroids, polymer-conjugated lipids and various zwitterionic lipids.

[0084] The term "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or can be positively charged in response to the pH value or hydrogen ion activity of its environment (e.g., the environment of its intended use). Therefore, the term "cationic" encompasses both "permanent cations" and "cationizable". In certain embodiments, the positive charge in the cationic lipid is derived from the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that is positively charged in the environment of its intended use (e.g., at physiological pH).

[0085] The term "polymer-conjugated lipid" refers to a molecule that comprises both a lipid portion and a polymer portion. An example of a polymer-conjugated lipid is a pegylated lipid (PEG-lipid), wherein the polymer portion comprises polyethylene glycol.

[0086] The term "neutral lipid" encompasses any lipid molecule that exists in an uncharged form or in a neutral zwitterionic form at a selected pH value or within a selected pH range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment of the intended lipid use, such as a physiological pH value. As non-limiting examples, neutral lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); phosphatidylethanolamines, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(oleoyloxy)propyl))dimethylammonio)ethyl hydrogenphosphate (DOCP); sphingomyelin (SM); ceramides; steroids, such as sterols and their derivatives. The neutral lipids provided herein can be synthetic or derived from (isolated or modified from) natural sources or compounds.

[0087] The term "charged lipid" encompasses any lipid molecule that exists in a positively or negatively charged form at a selected pH value or within a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH conditions in the environment of the intended lipid use, such as physiological pH. As a non-limiting example, the neutral lipids that can be used in conjunction with the present disclosure include but are not limited to phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethanecarbamoylsterol (e.g., DC-Chol), 1,2-dioleoyl-sn-glycero-3-phospho-L-serine sodium salt (DOPS-Na), 1,2-dioleoyl-sn-glycero-3-phospho-(1′-racemic-glycerol) sodium salt (DOPG-Na) and 1,2-dioleoyl-sn-glycero-3-phospho-sodium salt (DOPA-Na). The charged lipids provided herein can be synthetic or derived from (isolated or modified from) natural sources or compounds.

[0088] As used herein, "lipoplex (LPX)" generally refers to a complex formed spontaneously by lipids (cationic lipids), for example, a complex formed by a cationic lipid and a negative nucleic acid (eg, mRNA).

[0089] As used herein, the terms "signal peptide" and "signal sequence" are used interchangeably and refer to sequences that can direct protein transport or extracellular export. The term encompasses signal sequence polypeptides and nucleic acid sequences encoding the signal sequence. Therefore, reference to a signal sequence in the context of nucleic acid actually refers to a nucleic acid sequence encoding a signal sequence polypeptide. In some embodiments, the nucleic acid comprises an ORF encoding a secretory signal peptide and adrenocorticotropic hormone. In other embodiments, the nucleic acid has an ORF encoding a secretory signal peptide, adrenocorticotropic hormone, a linker, and a VLk sequence. A secretory signal peptide comprising 15-60 amino acids at the N-terminus of a protein is typically required for transmembrane translocation on the secretory pathway, and therefore, generally controls most proteins in eukaryotes and prokaryotes to enter the secretory pathway. In eukaryotes, the signal peptide of the nascent precursor protein (preprotein) guides the ribosome to the rough endoplasmic reticulum (ER) membrane, and the growing peptide chain is transported through it for processing. ER processing produces mature protein, wherein signal peptide is usually cut from precursor protein by ER resident signal peptidase of host cell, or it remains uncut and acts as membrane anchor. Signal peptide can also promote protein targeting cell membrane. In some embodiments, the length of signal peptide can be 15-60 amino acids, and the length of corresponding signal sequence can be 45-180 nucleotides. Signal peptide from heterologous gene (its regulation expression of gene except adrenocorticotropic hormone) is known in the art, and its desired properties can be tested, then incorporated into nucleic acid of the present invention.

[0090] As used herein, the term "mRNA," also known as messenger RNA, generally refers to a type of single-stranded RNA transcribed from a DNA template, carrying genetic information and guiding protein synthesis. Once mRNA is transcribed from DNA according to the Watson-Crick principle of complementary base pairing, it contains base sequences corresponding to certain functional segments of the DNA molecule, serving as a direct template for protein biosynthesis. As used herein, mRNA comprises a nucleic acid molecule capable of encoding adrenocorticotropic hormone; that is, at least a portion of the mRNA is capable of encoding adrenocorticotropic hormone.

[0091] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye / colorant, flavor enhancer, surfactant, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier approved by the U.S. Food and Drug Administration as acceptable for use in humans or veterinary animals.

[0092] The term "composition" is intended to encompass a product containing specified ingredients (eg, mRNA molecules provided herein), optionally in specified amounts.

[0093] As used interchangeably herein, the terms "polynucleotide" or "nucleic acid" refer to nucleotide polymers of any length, and include, for example, DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may contain modified nucleotides, such as methylated nucleotides and their analogs. Nucleic acids can be in single-stranded or double-stranded form. As used herein and unless otherwise indicated, "nucleic acid" also includes nucleic acid mimics, such as locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholino nucleic acids. As used herein, "oligonucleotide" refers to a short synthetic polynucleotide, generally but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides is equally and fully applicable to oligonucleotides. Unless otherwise indicated, the left-hand end of any single-stranded polynucleotide sequence disclosed herein is the 5' end; the left-hand direction of a double-stranded polynucleotide sequence is referred to as the 5' direction. The direction of 5′ to 3′ addition of the nascent RNA transcript is called the transcription direction; the sequence region on the DNA chain that has the same sequence as the RNA transcript and is located at the 5′ end relative to the 5′ end of the RNA transcript is called the “upstream sequence”; the sequence region on the DNA chain that has the same sequence as the RNA transcript and is located at the 3′ end relative to the 3′ end of the RNA transcript is called the “downstream sequence”.

[0094] The term "isolated nucleic acid" refers to a nucleic acid, such as RNA, DNA, or mixed nucleic acid, that is substantially separated from other genomic DNA sequences and proteins or complexes (such as ribosomes and polymerases) that naturally accompany the native sequence. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules present in the natural source of the nucleic acid molecule. In addition, when prepared by recombinant technology, an "isolated" nucleic acid molecule, such as an mRNA molecule, can be substantially free of other cellular materials or culture medium, or when chemically synthesized, it can be substantially free of chemical precursors or other chemicals. In a specific embodiment, one or more nucleic acid molecules encoding adrenocorticotropic hormone as described herein are separated or purified. The term includes nucleic acid sequences removed from its naturally occurring environment, and includes recombinant or cloned DNA or RNA isolates and chemically synthesized analogs or analogs biosynthesized by heterologous systems. Substantially pure molecules can include isolated forms of molecules.

[0095] The term "coding nucleic acid" or its grammatical equivalents, when used to refer to a nucleic acid molecule, includes (a) a nucleic acid molecule that can be transcribed to produce mRNA and subsequently translated into peptides and / or polypeptides when in its natural state or manipulated by methods well known to those skilled in the art; and (b) the mRNA molecule itself. The antisense strand is the complementary sequence of such a nucleic acid molecule, and the coding sequence can be inferred therefrom. The term "coding region" refers to the portion of a coding nucleic acid sequence that is translated into a peptide or polypeptide. The term "untranslated region" or "UTR" refers to the portion of a coding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of the UTR relative to the coding region of the nucleic acid molecule, the UTR is referred to as a 5'-UTR if it is located at the 5' end of the coding region, and the UTR is referred to as a 3'-UTR if it is located at the 3' end of the coding region.

[0096] The term "corresponding DNA sequence" or its grammatical equivalents when used in relation to an RNA sequence means the DNA sequence from which the RNA is transcribed. For example, the corresponding DNA sequence of the RNA sequence GCUGGAGCCUCGGUGGC is GCTGGAGCCTCGGTGGC.

[0097] As used herein, an "open reading frame (ORF)" is a nucleotide sequence that begins with a start codon (e.g., methionine (ATG)) and ends with a stop codon (e.g., TAA, TGA or TAG), and encodes a polypeptide. It should be understood that an open reading frame (ORF) as used herein includes both DNA and RNA sequences.

[0098] As used herein, the term "codon optimization" refers to replacing one, at least one, or more than one codon in a parent polypeptide encoding nucleic acid with codons encoding the same amino acid residue with different relative usage frequencies in a cell. In some embodiments, the mRNA described herein comprises a codon-optimized nucleic acid sequence, wherein the amino acid sequence encoded by the open reading frame (ORF) with the codon-optimized nucleic acid sequence comprises a signal peptide and adrenocorticotropic hormone from N-terminus to C-terminus, or comprises a signal peptide, adrenocorticotropic hormone, a linker, and a kappa light chain variable region (VLk) sequence from N-terminus to C-terminus. Methods for codon optimization are known in the art. In some embodiments, codon optimization can be used to match the codon frequencies in the target and host organisms to ensure correct folding; bias towards GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcription and translation control regions; insert or remove protein trafficking sequences; remove / add post-translational modification sites in the encoded protein; add, remove or reorganize protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translation rate to ensure that the individual structures of the protein are properly folded; or reduce or eliminate problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art. In some embodiments, ORF sequences are optimized using optimization algorithms.

[0099] As used herein, the term "mRNA" refers to a messenger RNA molecule comprising one or more open reading frames (ORFs), which can be translated by a cell or organism having the mRNA to produce one or more peptide or protein products. The region containing one or more ORFs is referred to as the coding region of the mRNA molecule. In certain embodiments, the mRNA molecule further comprises one or more untranslated regions (UTRs).

[0100] The term "nucleobase" encompasses purines and pyrimidines, including the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural or synthetic analogs or derivatives thereof.

[0101] As used herein, the term "functional nucleotide analogue" refers to a modified form of a classical nucleotide A, G, C, U, or T that (a) retains the base pairing properties of the corresponding classical nucleotide and (b) contains at least one chemical modification of the (i) nucleobase, (ii) sugar group, (iii) phosphate group, or (iv) any combination of (i) to (iii) of the corresponding natural nucleotide. As used herein, base pairing encompasses not only the classical Watson-Crick adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between a classical nucleotide and a functional nucleotide analogue, or between a pair of functional nucleotide analogues, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonds to be formed between the modified nucleobase and the classical nucleobase, or between two complementary modified nucleobase structures. For example, a functional analogue of guanosine (G) retains the ability to base pair with cytosine (C) or a functional analogue of cytosine. An example of such non-classical base pairing is base pairing between a modified nucleotide inosine and adenine, cytosine, or uracil. As described herein, functional nucleotide analogs can be naturally occurring or non-naturally occurring. Thus, nucleic acid molecules containing functional nucleotide analogs can have at least one modified nucleobase, sugar group, and / or internucleoside bond. Provided herein are chemical modifications of the nucleobases, sugar groups, or internucleoside bonds of nucleic acid molecules.

[0102] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer having more than fifty (50) amino acid residues linked by covalent peptide bonds. That is, a description of a polypeptide equally applies to a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally occurring amino acid (e.g., an amino acid analog). As used herein, the terms encompass amino acid chains of any length, including full-length proteins (e.g., adrenocorticotropic hormone).

[0103] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules, or two or more nucleic acid molecules, as determined by sequence alignment." amino acid sequence identity percentage (%) " is defined as the percentage of the identical amino acid residues in the candidate sequence and the reference polypeptide sequence relative to the reference polypeptide sequence, after alignment and introduction of a gap factor. If necessary, maximum sequence identity percentage can be achieved and any conservative substitutions are not considered as part of sequence identity. The comparison for determining amino acid sequence identity percentage can be achieved in a variety of ways within the scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or MEGALIGN (DNAStar, Inc.) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequences.

[0104] As used herein, "modification" of an amino acid residue / position refers to an alteration of the primary amino acid sequence compared to the starting amino acid sequence, wherein the alteration is caused by a sequence change involving the amino acid residue / position. For example, typical modifications include replacing a residue with another amino acid (e.g., conservative or non-conservative substitution), inserting one or more (e.g., typically less than 5, 4, or 3) amino acids / positions near the residue, and / or deleting the residue / position.

[0105] The term "vector" refers to a material for carrying or including a nucleic acid sequence, including, for example, a nucleic acid sequence encoding an adrenocorticotropic hormone (ACTH) as described herein, so that the nucleic acid sequence is introduced into a host cell, or used as a transcription template to perform in vitro transcription in a cell-free system to produce mRNA. Suitable vectors include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include a selection sequence or marker for stable integration into the host cell chromosome. In addition, the vector may include one or more selective marker genes and appropriate transcription or translation control sequences. The selective marker gene included may, for example, provide resistance to antibiotics or toxins, supplement nutritional deficiencies, or provide key nutrients not present in the culture medium. Transcription or translation control sequences may include constitutive and inducible promoters, transcription enhancers, transcription terminators, and the like, as are well known in the art. When two or more nucleic acid molecules (for example, nucleic acid molecules encoding two or more different ACTHs) are co-transcribed or co-translated, the two nucleic acid molecules may be inserted into the same expression vector or in a separate expression vector. For single vector transcription and / or translation, the encoding nucleic acid can be operably linked to a common transcription or translation control sequence, or to different transcription or translation control sequences, such as an inducible promoter and a constitutive promoter. Methods well known in the art can be used to confirm that the nucleic acid molecule is introduced into the host cell. Such methods include: utilizing nucleic acid analysis such as Northern blot or polymerase chain reaction (PCR) amplification, immunoblotting for gene product expression or other suitable analytical methods to detect the introduced nucleic acid sequence or its corresponding expressed gene product. It will be understood by those skilled in the art that the nucleic acid molecule is expressed in an amount sufficient to produce the desired product (e.g., mRNA transcript of the nucleic acid as described herein), and it will also be understood that expression levels can be optimized by methods well known in the art to obtain sufficient expression products.

[0106] The term "administer" or "administration" refers to the operation of injecting or otherwise physically delivering a substance (e.g., a lipid nanoparticle composition described herein) present in vitro into a patient's body, such as by transmucosal, intradermal, intravenous, intramuscular delivery, and / or any other physical delivery method described herein or known in the art. When treating a disease, disorder, condition, or symptom thereof, the administration of the substance is typically performed after the onset of the disease, disorder, condition, or symptom thereof. When preventing a disease, disorder, condition, or symptom thereof, the administration of the substance is typically performed before the onset of the disease, disorder, condition, or symptom thereof.

[0107] "Long-term" administration, as opposed to acute mode, refers to administration of one or more doses in a continuous mode (e.g., over a period of time, such as days, weeks, months, or years), thereby maintaining the initial therapeutic effect (activity) over an extended period of time. "Intermittent" administration means that the treatment is not continued uninterrupted, but is cyclical in nature.

[0108] As used herein, the term "targeted delivery" or verb form "targeting" refers to a process in which the delivered agent (the therapeutic nucleic acid such as mRNA in the lipid nanoparticle composition as described herein) is promoted to reach a specific organ, tissue, cell and / or intracellular compartment (referred to as a target location) compared to delivery to any other organ, tissue, cell or intracellular compartment (referred to as a non-target location). Targeted delivery can be detected using methods known in the art, for example, by comparing the concentration of the delivered agent in the target cell population with the concentration of the delivered agent at the non-target cell population after systemic administration. In certain embodiments, targeted delivery makes the concentration at the target location at least 2 times higher than the concentration at the non-target location.

[0109] An "effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms; eliminate symptoms and / or potential causes; prevent the occurrence of symptoms and / or their potential causes; and / or improve or remedy damage caused by or associated with a disease, disorder, or condition. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactically effective amount.

[0110] As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., a therapeutic nucleic acid such as mRNA or pharmaceutical composition as described herein) sufficient to reduce and / or ameliorate the severity and / or duration of a given disease, disorder or condition, and / or its associated symptoms. A "therapeutically effective amount" of an agent of the present disclosure (e.g., a lipid nanoparticle composition as described herein) may vary according to a number of factors, such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. A therapeutically effective amount comprises an amount in which any toxic or deleterious effects of the agent are outweighed by the therapeutically beneficial effects of the agent. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a lipid nanoparticle composition as described herein or a therapeutic or prophylactic agent (e.g., therapeutic mRNA) contained therein that is effective to "treat" a disease, disorder, or condition in a subject or mammal.

[0111] A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended prophylactic effect, such as preventing a disease, disorder, condition, or related symptoms (e.g., rheumatic diseases (including but not limited to gout inflammation), lung diseases, ophthalmic diseases, kidney diseases, or neurological diseases (including but not limited to infantile spasms, multiple sclerosis), or symptoms caused therefrom), delaying its onset (or recurrence), or reducing the likelihood of its onset (or recurrence). Typically, but not necessarily, the prophylactic effective amount may be less than the therapeutically effective amount because the prophylactic dose is used for a subject before or at an early stage of a disease, disorder, or condition. A complete therapeutic or prophylactic effect does not necessarily occur by administering a single dose, but may only occur after administering a series of doses. Therefore, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

[0112] The terms "prevent," "preventing," or "prevention" refer to reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition, or associated symptoms, such as rheumatic diseases (including but not limited to gout inflammation), pulmonary diseases, ophthalmic diseases, renal diseases, or neurological diseases (including but not limited to infantile spasms, multiple sclerosis), or symptoms caused thereby.

[0113] The term "prophylactic agent" refers to any agent that can completely or partially inhibit the development, recurrence, onset, or spread of a disease and / or its associated symptoms in a subject.

[0114] The term "therapeutic agent" refers to any agent useful in treating, preventing, or alleviating a disease, disorder, or condition, including any agent useful in treating, preventing, or alleviating one or more symptoms of a disease, disorder, or condition and / or its associated symptoms.

[0115] The term "therapy" refers to any regimen, method, and / or agent that can be used to prevent, manage, treat, and / or improve a disease, disorder, or condition. In certain embodiments, the term "therapies" or "therapy" refers to biological therapies, supportive therapies, and / or other therapies known to those skilled in the art, such as medical personnel, that can be used to prevent, manage, treat, and / or improve a disease, disorder, or condition.

[0116] The terms "subject" and "patient" are used interchangeably. As used herein, in certain embodiments, the subject is a mammal, such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans). In a specific embodiment, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing a rheumatic disease (including but not limited to gouty inflammation), a lung disease, an ophthalmic disease, a kidney disease, or a neurological disease (including but not limited to infantile spasms, multiple sclerosis).

[0117] By "substantially all" is meant at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

[0118] As used herein and unless otherwise indicated, the term "about" or "approximately" means an acceptable error for a particular value determined by one of ordinary skill in the art, which depends in part on the manner in which the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms "about" and "approximately" mean 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.

[0119] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0120] All publications, patent applications, and other references cited in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The publications discussed herein provide their disclosures solely as of the filing date of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such publications by virtue of prior invention. Furthermore, the publication dates provided may differ from the actual publication dates, which may need to be independently confirmed.

[0121] A number of embodiments of the present invention have been described. However, it should be understood that various modifications can be made without departing from the spirit and scope of the present invention. Therefore, the descriptions in the experimental section and examples are intended to illustrate, but not to limit, the scope of the invention described in the claims.

[0122] 3. Therapeutic nucleic acids

[0123] 3.1 Open Reading Frame (ORF)

[0124] In one aspect, provided herein are therapeutic nucleic acid molecules for treating or preventing rheumatic diseases (including but not limited to gouty inflammation), lung diseases, ophthalmic diseases, kidney diseases, or neurological diseases (including but not limited to multiple sclerosis (MS) or infantile spasms (IS)). In some embodiments, the therapeutic nucleic acid comprises an open reading frame (ORF) encoding adrenocorticotropic hormone, its functional fragment or variant, which is expressed by cells in the subject to produce the encoded adrenocorticotropic hormone, its functional fragment or variant after being administered to a subject in need thereof. In some embodiments, the therapeutic nucleic acid molecule is a DNA molecule. In other embodiments, the therapeutic nucleic acid molecule is an RNA molecule. In a specific embodiment, the therapeutic nucleic acid molecule is an mRNA molecule.

[0125] In some embodiments, the ACTH, functional fragments thereof, or variants encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the ACTH, functional fragments thereof, or variants encoded by the mRNA payload can have a therapeutic effect when expressed in a cell.

[0126] In some embodiments, the nucleic acid molecules of the present disclosure include mRNA molecules. In specific embodiments, the nucleic acid molecules include at least one coding region (e.g., open reading frame (ORF)) encoding a protein of interest. In some embodiments, the nucleic acid molecules further include at least one untranslated region (UTR). In specific embodiments, the untranslated region (UTR) is located upstream (5' end) of the coding region and is referred to herein as a 5'-UTR. In specific embodiments, the untranslated region (UTR) is located downstream (3' end) of the coding region and is referred to herein as a 3'-UTR. In some embodiments, the nucleic acid molecules include both a 5'-UTR and a 3'-UTR. In some embodiments, the 5'-UTR includes a 5'-cap structure. In some embodiments, the nucleic acid molecules include a Kozak sequence (e.g., in a 5'-UTR). In some embodiments, the nucleic acid molecules include a poly-A region (e.g., in a 3'-UTR). In some embodiments, the nucleic acid molecules include a polyadenylation signal (e.g., in a 3'-UTR). In some embodiments, the nucleic acid molecules include a stabilizing region (e.g., in a 3'-UTR). In some embodiments, the nucleic acid molecules include a secondary structure. In some embodiments, the secondary structure is a stem-loop. In some embodiments, the nucleic acid molecule comprises a stem-loop sequence (e.g., in a 5′-UTR and / or a 3′-UTR). In some embodiments, the nucleic acid molecule comprises one or more intronic regions that can be excised during splicing. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from a 5′-UTR and a coding region. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from a coding region and a 3′-UTR. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from a 5′-UTR, a coding region, and a 3′-UTR.

[0127] In some embodiments, the ORF encodes a truncated ACTH, a mutant ACTH, or an ACTH fusion protein.

[0128] As used herein, adrenocorticotropic hormone (ACTH) is a polypeptide hormone secreted by the pituitary gland that stimulates adrenal cortex proliferation and promotes the synthesis and secretion of adrenocortical hormones.

[0129] In some embodiments, the ACTH can be selected from vertebrates of different species, preferably from humans.

[0130] In some embodiments, the ACTH can be wild-type ACTH.

[0131] In some embodiments, the ACTH can be any functional fragment of wild-type ACTH.

[0132] In some embodiments, the ACTH can be full-length or mutant, truncated wild-type ACTH, optionally in which one or more amino acids are replaced, or one or more amino acids are deleted; in some embodiments, the ACTH is humanized.

[0133] In some embodiments, the ACTH may be a fusion protein, optionally composed of functional fragments of ACTH from different species, or composed of functional fragments of ACTH and other peptide sequences or protein sequences.

[0134] In some embodiments, the ACTH comprises the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1.

[0135] In some embodiments, the ACTH fusion protein is a fusion protein of a secretion signal peptide and ACTH. The secretion signal peptide is fused to the N-terminus or C-terminus of the ACTH, preferably the N-terminus. In further embodiments, the C-terminus of the ACTH fusion protein is further linked to a kappa light chain variable region (VLk) sequence via a linker.

[0136] In some embodiments, the ACTH fusion protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 2-61, or an amino acid sequence that is at least 90%, 95%, 98%, or 99% identical to any one of SEQ ID NOs: 2-61.

[0137] In some embodiments, the amino acid sequence encoded by the ORF comprises a signal peptide and adrenocorticotropic hormone from the N-terminus to the C-terminus.

[0138] In some embodiments, the amino acid sequence encoded by the ORF comprises a signal peptide, adrenocorticotropic hormone, a linker, and a kappa light chain variable region (VLk) sequence from the N-terminus to the C-terminus.

[0139] In some embodiments, the linker comprises the amino acid sequence set forth in SEQ ID NO:55, or an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, or 99% identical to SEQ ID NO:55.

[0140] In some embodiments, the kappa light chain variable region (VLk) sequence comprises the amino acid sequence shown in SEQ ID NO:54, or an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO:54.

[0141] In some embodiments, the open reading frame (ORF) is codon optimized, for example, including but not limited to the optimization of G / C content, and preferably, the codon optimization does not change the amino acid sequence encoded by it. The guanosine / cytosine (G / C) content of the coding region of the mRNA involved in the present invention is optimized, but compared with the amino acid sequence encoded by the wild-type mRNA, the amino acid sequence encoded by the mRNA is unmodified. Wherein, the optimization of guanosine / cytosine (G / C) content can correspondingly improve the stability of the mRNA. Compared with the wild-type sequence, the modification (G / C content) of the RNA sequence can be based on the amino acid sequence encoded by it. For example, the form of the codon comprising A and / or U nucleotides is modified by replacing other codons (not comprising A and / or U or comprising lower levels of A and / or U nucleotides).

[0142] In some embodiments, the ORF comprises, from 5' to 3', a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding adrenocorticotropic hormone.

[0143] In some embodiments, the ORF comprises, from 5' to 3', a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding adrenocorticotropic hormone, a nucleotide sequence encoding a linker, and a nucleotide sequence encoding a kappa light chain variable region (VLk) sequence.

[0144] In some embodiments, the ORF encodes an amino acid sequence comprising any one of SEQ ID NOs: 2-61, or an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98%, or 99% identical to any one of SEQ ID NOs: 2-61.

[0145] In some embodiments, the ORF is codon-optimized, and preferably, the codon optimization does not change the amino acid sequence it encodes.

[0146] In some embodiments, the nucleotide sequence encoding the signal peptide comprises any one of SEQ ID NOs: 343-370 or its corresponding DNA sequence, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 343-370 or its corresponding DNA sequence.

[0147] In some embodiments, the adrenocorticotropic hormone encoding nucleotide sequence comprises any one of SEQ ID NOs: 108-167 or its corresponding DNA sequence, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 108-167 or its corresponding DNA sequence.

[0148] In some embodiments, the nucleotide sequence encoding the linker comprises SEQ ID NO: 169 or its corresponding DNA sequence, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 169 or its corresponding DNA sequence.

[0149] In some embodiments, the nucleotide sequence encoding the kappa light chain variable region (VLk) sequence comprises SEQ ID NO: 168 or its corresponding DNA sequence, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168 or its corresponding DNA sequence.

[0150] In certain embodiments, the therapeutic nucleic acid molecule is an mRNA molecule.

[0151] 3.2 5'-cap structure

[0152] Without being bound by theory, it is expected that the 5′-cap structure of the polynucleotide participates in nuclear export and increases polynucleotide stability, and binds to mRNA cap binding protein (CBP), which is responsible for polynucleotide stability in the cell and causes translational competence through the formation of mature circular mRNA species through the association of CBP with poly-A binding protein. The 5′-cap structure further facilitates the removal of 5′-proximal introns during mRNA splicing. Therefore, in some embodiments, the nucleic acid molecules of the present disclosure include a 5′-cap structure.

[0153] Nucleic acid molecules can be capped at the 5' end by the cell's endogenous transcriptional machinery, thereby generating a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue of the polynucleotide and the sense nucleotide transcribed at the 5' end. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides at the 5' end of the polynucleotide can also be optionally 2'-O-methylated. 5'-Decapping via hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0154] In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more alterations to the native 5'-cap structure produced by endogenous processes. Without being bound by theory, modifications to the 5'-cap can increase the stability of the polynucleotide, increase the half-life of the polynucleotide, and increase the translation efficiency of the polynucleotide.

[0155] Exemplary changes to the natural 5'-cap structure include creating a non-hydrolyzable cap structure to prevent decapping and thereby increase the half-life of the polynucleotide. In some embodiments, because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester bond, in some embodiments, modified nucleotides may be used during the capping reaction. For example, in some embodiments, a vaccinia capping enzyme from New England Biolabs (Ipswich, Mass.) may be used for α-thioguanosine nucleotides to create a phosphorothioate bond in the 5'-ppp-5' cap according to the manufacturer's instructions. Additional modified guanosine nucleotides, such as α-methylphosphonic acid and selenophosphate nucleotides, may be used.

[0156] Additional exemplary alterations to the native 5′-cap structure also include modifications at the 2′ and / or 3′ position of the capped guanosine triphosphate (GTP), replacement of the sugar ring oxygen (resulting in the oxygen of the carbocyclic ring) with a methylene moiety (CH2), modifications at the triphosphate bridge portion of the cap structure, or modifications at the nucleobase (G) portion.

[0157] Additional exemplary changes to the natural 5'-cap structure include, but are not limited to, 2'-O-methylation of the ribose sugar at the 5'-terminus of the polynucleotide and / or the 5'-terminal nucleotide on the sugar 2'-hydroxyl group (as described above). A plurality of different 5'-cap structures can be used to produce the 5'-cap of a polynucleotide (e.g., an mRNA molecule). Additional exemplary 5'-cap structures that can be used in conjunction with the present disclosure further include those described in International Patent Publications WO2008127688, WO 2008016473, and WO 2011015347, the entire contents of which are incorporated herein by reference.

[0158] In various embodiments, the 5'-terminal cap may comprise a cap analog. Cap analogs are also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, whose chemical structure differs from that of a natural (i.e., endogenous, wild-type, or physiological) 5'-cap while retaining cap function. Cap analogs can be synthesized and / or attached to a polynucleotide chemically (i.e., non-enzymatically) or enzymatically.

[0159] For example, the anti-reverse cap analog (ARCA) cap contains two guanosines linked via a 5′-5′-triphosphate group, wherein one of the guanosines contains an N7-methyl group as well as a 3′-O-methyl group (i.e., N7, 3′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, i.e., m7G-3′mppp-G, which can be equivalently referred to as 3′O-Me-m7G(5′)ppp(5′)G). The 3′-O atom of the other, unchanged guanosine is linked to the 5′-terminal nucleotide of the capped polynucleotide (e.g., mRNA). The N7- and 3′-O-methylated guanosines provide the terminal portion of the capped polynucleotide (e.g., mRNA). Another exemplary cap structure is mCAP, which is similar to ARCA but has a 2′-O-methyl group on the guanosine (i.e., N7, 2′-O-dimethyl-guanosine-5′-triphosphate-5′-guanosine, i.e., m7Gm-ppp-G).

[0160] In some embodiments, the cap analog can be a dinucleotide cap analog. As a non-limiting example, a dinucleotide cap analog can be modified with a boranophosphate or a phophoroselenoate at different phosphate positions, such as the dinucleotide cap analogs described in U.S. Patent No. 8,519,110, the entire contents of which are incorporated herein by reference in their entirety.

[0161] In some embodiments, the cap analog can be an N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analog known in the art and / or described herein. Non-limiting examples of N7-(4-chlorophenoxyethyl) substituted dinucleotide cap analogs include N7-(4-chlorophenoxyethyl)-G(5')ppp(5')G and N7-(4-chlorophenoxyethyl)-m3'-OG(5')ppp(5')G cap analogs (see, e.g., Kore et al., Bioorganic & Medicinal Chemistry 2013 21:4570-4574 for various cap analogs and methods for synthesizing cap analogs; the entire contents of which are incorporated herein by reference). In other embodiments, the cap analog that can be used in conjunction with the nucleic acid molecules of the present disclosure is a 4-chloro / bromophenoxyethyl analog.

[0162] In various embodiments, the cap analog may comprise a guanosine analog. Useful guanosine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0163] Without being bound by theory, it is expected that although cap analogs allow for simultaneous capping of polynucleotides in in vitro transcription reactions, up to 20% of transcripts remain uncapped. This, along with the structural differences between cap analogs and the natural 5′-cap structure of polynucleotides produced by the cell's endogenous transcription machinery, may lead to reduced translational capacity and cellular stability.

[0164] Therefore, in some embodiments, the nucleic acid molecules of the present disclosure can also be capped after transcription using enzymes to produce a more authentic 5′-cap structure. As used herein, the phrase “more authentic” refers to a feature that closely reflects or mimics an endogenous or wild-type feature in structure or function. That is, a “more authentic” feature better represents endogenous, wild-type, natural or physiological cellular function and / or structure than a synthetic feature or analog of the prior art, or it outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more aspects. Non-limiting examples of more authentic 5′-cap structures that can be used in conjunction with the nucleic acid molecules of the present disclosure are structures that have, in particular, enhanced binding to cap-binding proteins, increased half-life, reduced sensitivity to 5′-endonucleases and / or reduced 5′-decapping compared to synthetic 5′-cap structures known in the art (or compared to wild-type, natural or physiological 5′-cap structures). For example, in some embodiments, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can generate a classic 5'-5'-triphosphate bond between the 5'-terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, wherein the cap guanosine contains an N7-methylation and the 5'-terminal nucleotide of the polynucleotide contains a 2'-O-methyl group. This structure is referred to as a cap 1 structure. Compared to other 5' cap analog structures known in the art, for example, this cap results in higher translational capacity, cellular stability, and reduced activation of cellular proinflammatory cytokines. Other exemplary cap structures include 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), 7mG(5')-ppp(5')NlmpN2mp (cap 2), and m(7)Gpppm(3)(6,6,2')Apm(2')Apm(2')Cpm(2)(3,2')Up (cap 4).

[0165] In some embodiments, the 5'-cap structure used herein is selected from m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7GpppN, m7GpppNmpNp and m7GpppNmpNmp.

[0166] In a preferred embodiment, the 5'-cap structure used herein is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

[0167] Without being bound by theory, it is contemplated that the nucleic acid molecules of the present disclosure can be capped post-transcriptionally, and because this method is relatively efficient, nearly 100% of the nucleic acid molecules can be capped.

[0168] 3.3 Untranslated Region (UTR)

[0169] In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more untranslated regions (UTRs). In some embodiments, the UTR is located upstream of the coding region in the nucleic acid molecule and is referred to as a 5′-UTR. In some embodiments, the UTR is located downstream of the coding region in the nucleic acid molecule and is referred to as a 3′-UTR. The sequence of the UTR may be homologous or heterologous to the sequence of the coding region found in the nucleic acid molecule. Multiple UTRs may be included in the nucleic acid molecule and may have the same or different sequences and / or gene origins. According to the present disclosure, any portion of the UTR in the nucleic acid molecule (including without any portion) may be codon optimized, and any portion may independently contain one or more different structures or chemical modifications before and / or after codon optimization.

[0170] In some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise UTRs and coding regions that are homologous to each other. In other embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise UTRs and coding regions that are heterologous to each other. In some embodiments, to monitor the activity of UTR sequences, nucleic acid molecules comprising coding sequences of UTRs and detectable probes can be administered in vitro (e.g., in cell or tissue culture) or in vivo (e.g., to a subject), and the effects of the UTR sequences (e.g., regulating expression levels, cellular localization of the encoded product, or half-life of the encoded product) can be measured using methods known in the art.

[0171] In some embodiments, the nucleic acid molecules of the present disclosure comprise a 5′-UTR selected from SEQ ID NOs: 62-82 or their corresponding DNA sequences. In some embodiments, the nucleic acid molecules of the present disclosure comprise a 3′-UTR selected from SEQ ID NOs: 83-101 or their corresponding DNA sequences. In some embodiments, the nucleic acid molecules of the present disclosure comprise a 5′-UTR selected from SEQ ID NOs: 62-82 or their corresponding DNA sequences and a 3′-UTR selected from SEQ ID NOs: 83-101 or their corresponding DNA sequences. In specific embodiments, the nucleic acid molecules described herein can be in vitro transcribed RNA molecules.

[0172] 3.4 Polyadenylation (Poly-A) Region

[0173] During natural RNA processing, long chains of adenosine nucleotides (poly-A regions) are typically added to messenger RNA (mRNA) molecules to increase the molecule's stability. Immediately after transcription, the 3'-end of the transcript is cleaved to release the 3'-hydroxyl group. Subsequently, poly-A polymerase adds a string of adenosine nucleotides to the RNA. This process, called polyadenylation, adds a poly-A region between 100 and 250 residues in length. Without being bound by theory, it is contemplated that poly-A regions may confer a number of advantages to the nucleic acid molecules of the present disclosure.

[0174] Therefore, in some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise a polyadenylation signal. In some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise one or more polyadenylation (poly-A) regions. In some embodiments, the poly-A region is entirely composed of adenine nucleotides or functional analogs thereof. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 3' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5' end and at least one poly-A region at its 3' end.

[0175] According to the present disclosure, in different embodiments, the poly-A region may have different lengths. Specifically, in some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 30 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 35 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 40 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 45 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 50 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 55 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 60 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 65 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 70 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 75 nucleotides. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 80 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 85 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 90 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 95 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 100 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 110 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 120 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 130 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 140 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 150 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 160 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 170 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 180 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 190 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 200 nucleotides in length.In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 225 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 250 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 275 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 300 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 350 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 400 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 450 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 500 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 600 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 700 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 800 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 900 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is at least 1000 nucleotides in length. In some embodiments, the poly-A region of the nucleic acid molecules of the present disclosure is 1000 to 3000 nucleotides in length.

[0176] In some embodiments, the length of the poly-A region in a nucleic acid molecule can be selected based on the total length of the nucleic acid molecule or a portion thereof (e.g., the length of the coding region of the nucleic acid molecule or the length of the open reading frame, etc.). For example, in some embodiments, the poly-A region comprises about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the total length of the nucleic acid molecule containing the poly-A region.

[0177] Without being bound by theory, it is contemplated that certain RNA-binding proteins can bind to the poly-A region located at the 3′ end of an mRNA molecule. These poly-A binding proteins (PABPs) can regulate mRNA expression, for example, by interacting with the translation initiation machinery in the cell and / or protecting the 3′-poly-A tail from degradation. Thus, in some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise at least one binding site for a poly-A binding protein (PABP). In other embodiments, the nucleic acid molecule is bound to the PABP before being loaded into a delivery vehicle (e.g., a lipid nanoparticle). Typically, poly-A (e.g., a 3′-poly(A) sequence) is a continuous sequence of adenosine nucleotides. In some embodiments, the poly-A (e.g., a 3′-poly(A) sequence) comprises an adenosine polynucleotide and a linker sequence inserted therein to separate the adenine nucleotide sequence, i.e., the adenosine polynucleotide is an interrupting sequence.

[0178] In some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise poly-AG quadruplexes. A G quadruplex is a circular array of four hydrogen-bonded guanosine nucleotides that can be formed by G-rich sequences in DNA and RNA. In this embodiment, the G quadruplex is incorporated into one end of the poly-A region. The stability, protein yield, and other parameters of the resulting polynucleotide (e.g., mRNA) can be analyzed, including half-life at different time points. It has been found that the protein yield of the poly-AG quadruplex structure is at least 75% of the protein yield observed using only a poly-A region containing 120 nucleotides.

[0179] In some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure may include a poly-A region and may be stabilized by adding a 3′-stabilizing region. In some embodiments, the 3′-stabilizing region that can be used to stabilize nucleic acid molecules (e.g., mRNA) includes a poly-A or poly-AG quadruplex structure as described in International Patent Publication No. WO2013 / 103659, the contents of which are incorporated herein by reference in their entirety.

[0180] In other embodiments, 3′-stabilizing regions that can be used in conjunction with the nucleic acid molecules of the present disclosure include chain-terminating nucleosides such as, but not limited to, 3′-deoxyadenosine (cordycepin); 3′-deoxyuridine; 3′-deoxycytosine; 3′-deoxyguanosine; 3′-deoxythymidine; 2′,3′-dideoxynucleosides, such as 2′,3′-dideoxyadenosine, 2′,3′-dideoxyuridine, 2′,3′-dideoxycytosine, 2′,3′-dideoxyguanosine, 2′,3′-dideoxythymidine; 2′-deoxynucleosides; or O-methyl nucleosides: 3′-deoxynucleosides; 2′,3′-dideoxynucleosides; 3′-O-methyl nucleosides; 3′-O-ethyl nucleosides; 3′-arabinoside, as well as other alternative nucleosides known in the art and / or described herein.

[0181] In some embodiments, the nucleic acid molecules of the present disclosure comprise, from 5' to 3', the following: a 5'-UTR, an ORF encoding an amino acid sequence comprising a signal peptide and adrenocorticotropic hormone or an amino acid sequence comprising a signal peptide, adrenocorticotropic hormone, a linker and a kappa light chain variable region (VLk) sequence, a 3'-UTR, and a 3'-polyadenylation sequence.

[0182] In some embodiments, the 3'-poly (A) sequence comprises at least 10-400 adenosine nucleotides, preferably 50 to 400 adenosine nucleotides or 10 to 300 adenosine nucleotides; more preferably 50 to 250 adenosine nucleotides; most preferably 120 adenosine nucleotides;

[0183] Alternatively, the 3'-poly(A) sequence comprises an adenosine polynucleotide and a linker sequence inserted therein, wherein the linker sequence is used to separate the adenine nucleotide sequence.

[0184] In some embodiments, the nucleic acid comprises any one of SEQ ID NOs: 170-342, or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 170-342, or a DNA sequence corresponding thereto.

[0185] 3.5 Nucleobase Modification

[0186] In some embodiments, functional nucleotide analogs contain non-classical nucleobases. In some embodiments, the classical nucleobases in a nucleotide (e.g., adenine, guanine, uracil, thymine, and cytosine) can be modified or replaced to provide one or more functional analogs of the nucleotide. Exemplary modifications of nucleobases include, but are not limited to, one or more substitutions or modifications, including, but not limited to, alkyl, aryl, halo, oxo, hydroxyl, alkoxy, and / or thio substitutions; one or more fused rings or ring opening, oxidation, and / or reduction.

[0187] In some embodiments, the non-classical nucleobase is a modified uracil. Exemplary nucleobases and nucleosides with modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (s2U), 4-thio-uracil (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho5U), 5-aminoallyl-uracil, 5-halouracil (e.g., 5-iodouracil or 5-bromouracil), 3-methyluracil (m3U), 5-methoxyuracil (mo5U), uracil 5- Oxyacetic acid (cmo5U), uracil 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uracil (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm5U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm5U), 5-methoxycarbonylmethyl-uracil (mcm5U), 5-methoxycarbonylmethyl-2-thiouracil (mcm5s2U), 5-aminomethyl-2-thiouracil (nm5s2U), 5-methylaminomethyl-uracil (mnm5U), 5-methylaminomethyl-2- Thiouracil (mnm5s2U), 5-methylaminomethyl-2-selenouracil (mnm5se2U), 5-carbamoylmethyluracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm5s2U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurinemethyl-uracil (τm5U), 1-taurinemethyl-pseudouridine, 5-taurinemethyl-2-thiouracil (τm55s2U), 1-taurinemethyl-4- Thio-pseudouridine, 5-methyl-uracil (m5U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (Et1ψ), 5-methyl-2-thio-uracil (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5,6-dihydrouracil, 5-methyl-dihydrouracil (m5D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uracil (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uracil (m5U), 5-(isopentenylaminomethyl)-2-thio-uracil (m5s2U), 5,2'-O-dimethyl-uridine (m5Um), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonyl 5-(carbamoylmethyl)-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thiouracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thiouracil, 5-carboxymethyl-2-thiouracil, 5-cyanomethyl-uracil, 5-methoxy-2-thiouracil and 5-[3-(1-E-propenylamino)]uracil.

[0188] In specific embodiments, nucleic acid molecules of the present disclosure comprise modifications to uracil. In specific embodiments, nucleic acid molecules of the present disclosure comprise one or more pseudouracils (ψ). In specific embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the uracils in the nucleic acid molecules of the present disclosure are replaced by pseudouracils (ψ). In specific embodiments, all (100%) uracils in the nucleic acid molecules of the present disclosure are replaced by pseudouracils (ψ).

[0189] In specific embodiments, the nucleic acid molecules of the present disclosure comprise one or more 1-methylpseudouracils (m1ψ). In specific embodiments, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% of the uracils in the nucleic acid molecules of the present disclosure are substituted with 1-methyl-pseudouracils (m1ψ). In specific embodiments, all (100%) of the uracils in the nucleic acid molecules of the present disclosure are substituted with 1-methylpseudouracils (m1ψ).

[0190] Therapeutic nucleic acid molecules as described herein can be isolated or synthesized using methods known in the art. In some embodiments, the DNA or RNA molecules used in conjunction with the present disclosure are chemically synthesized. In other embodiments, the DNA or RNA molecules used in conjunction with the present disclosure are isolated from natural sources.

[0191] In some embodiments, the mRNA molecules used in conjunction with the present disclosure are biosynthesized using host cells. In specific embodiments, mRNA is produced by transcribing the corresponding DNA using host cells. In some embodiments, the DNA sequence encoding the mRNA sequence is integrated into an expression vector using methods known in the art, and the vector is then introduced into a host cell (e.g., E. coli). The host cell is then cultured under suitable conditions to produce mRNA transcripts. Other methods for producing mRNA molecules from DNA are known in the art. For example, in some embodiments, a cell-free (in vitro) transcription system comprising an enzyme of the host cell's transcriptional machinery can be used to produce mRNA transcripts.

[0192] 3.6 Methods for preparing mRNA

[0193] The present invention also provides a method for preparing mRNA containing an open reading frame (ORF) encoding adrenocorticotropic hormone, a functional fragment or variant thereof, which comprises the following steps: 1) synthesizing a DNA fragment for transcribing the mRNA, and cloning the DNA fragment into an expression vector to obtain a recombinant plasmid; 2) transferring the recombinant plasmid into a host cell, amplifying it, extracting the plasmid, and digesting the obtained plasmid with a restriction endonuclease to obtain a linearized DNA for in vitro transcription of the mRNA; and 3) transcribing the linearized DNA in vitro to obtain the target mRNA.

[0194] In some embodiments, a DNA fragment for transcribing an mRNA containing an open reading frame (ORF) encoding adrenocorticotropic hormone, its functional fragments, or variants is synthesized, and the DNA fragment is cloned into an expression plasmid to obtain a recombinant plasmid. In some embodiments, in addition to containing a DNA fragment corresponding to the mRNA encoding adrenocorticotropic hormone, the DNA fragment should also contain a T7 promoter sequence at the 5' end and a specific restriction endonuclease recognition sequence at the 3' end, and the restriction endonuclease can be selected from BspQI, BsaI, BsmI, NotI, etc. The present invention does not specifically limit the method for synthesizing the DNA fragment corresponding to the above-mentioned mRNA, and conventional DNA synthesis methods in the art can be used. In a preferred embodiment, the DNA fragment is synthesized by a commercial biotechnology company. In some embodiments, the expression plasmid does not contain a T7 promoter sequence and the recognition sequence of the specific restriction endonuclease (restriction endonuclease can be selected from BspQI, BsaI, BsmI, NotI, PvuI, etc.) used above. In some embodiments, the expression plasmid is preferably a pUC-GW plasmid. In a preferred embodiment, the DNA fragment is cloned into an expression plasmid by enzyme digestion. The present invention has no particular limitation on the specific operations of enzyme digestion and ligation, and conventional enzyme digestion and ligation operations in the art can be used.

[0195] After obtaining the recombinant plasmid, the recombinant plasmid is introduced into the host cell, amplified, the plasmid is extracted, and the obtained plasmid is digested with a restriction endonuclease to obtain a linearized DNA fragment for in vitro expression of mRNA. In a preferred embodiment, the host cell is an Escherichia coli cell or a competent form thereof. The present invention does not specifically limit the method of introduction, and conventional introduction methods in the art can be used. After obtaining the recombinant cells, positive recombinant cells are screened and colony sequencing is performed. In one embodiment, the screening of the positive recombinant cells is carried out on a solid culture medium containing kanamycin (kan), followed by colony PCR to select kan-resistant colonies. In some embodiments, the amplification procedure of the colony PCR is as follows: pre-denaturation at 98°C for 3 minutes; denaturation at 98°C for 10 seconds, annealing at 60°C for 5 seconds, extension at 72°C for 2 minutes, 34 cycles; and final extension at 72°C for 10 minutes. After the PCR amplification is completed, the colonies of the target bands are preferably determined by agarose gel electrophoresis, and then sequenced for verification. In some embodiments, the parameters of the agarose gel electrophoresis detection are as follows: 1% agarose, 5V / cm, 40min. In some embodiments, the plasmid of the recombinant cell with correct sequencing is extracted. The present invention does not specifically limit the extraction method of the plasmid, and preferably a plasmid extraction kit is used. In some embodiments, the specific restriction endonuclease mentioned above (the restriction endonuclease can be selected from BspQI, BsaI, BsmI, NotI, etc.) is used for enzyme digestion to obtain linearized DNA fragments of in vitro expressed mRNA. In some embodiments, the enzyme digestion system is designed with 50μl, preferably as follows:

[0196] In some embodiments, after the enzyme digestion reaction is completed, the amplified product is preferably subjected to agarose gel electrophoresis to determine whether the reaction is complete. The agarose gel electrophoresis detection parameters are preferably as follows: 1% agarose, 5V / cm, 40min. In some embodiments, the reaction is considered complete when only one band appears on agarose gel electrophoresis. After the enzyme digestion reaction is completed, the linearized DNA fragment is preferably purified. There is no special limitation on the purification method, and conventional purification methods in the art can be used. In the specific implementation process of the present invention, a DNA purification kit is preferably used. After the purification, NanoDrop is preferably used to detect the concentration of the purified linearized DNA template, as well as the ratios of OD260nm / OD280nm and OD260nm / OD230nm. When OD260nm / OD280nm is between 1.6-1.8, the linearized template is considered qualified.

[0197] After obtaining the linearized DNA template, the linearized DNA template is subjected to in vitro transcription to obtain the mRNA. The mRNA in vitro transcription system is designed as a 20 μl system and includes the following components:

[0198] In some embodiments, the Enzyme Mix includes T7 RNA polymerase, an RNase inhibitor, and an inorganic pyrophosphatase. In some embodiments, the in vitro RNA synthesis reaction is preferably performed in a 200 μl RNase-free tube at 37°C for 2 hours. The reaction reagents in the RNA in vitro transcription system are added in the following order: water, nucleotides, cap analog, transcription buffer, Enzyme Mix, and linearized DNA template.

[0199] After the in vitro transcription of the RNA is completed, it is preferably verified by agarose gel electrophoresis whether the in vitro synthesis of the mRNA is successful. The detection parameters of the agarose gel electrophoresis are as follows: 1% agarose, 5V / cm, 10min. In some embodiments, the appearance of the expected target band by agarose gel electrophoresis indicates that the reaction is successful. In some embodiments, the in vitro synthesized mRNA is subjected to steps such as removing the DNA template and recovering and purifying the mRNA. In some embodiments, the removal of the DNA template is preferably achieved by DNase I digestion. In some embodiments, the digestion is carried out as follows: 2μl of DNase I is mixed with the solution after the RNA in vitro transcription reaction and incubated at 37°C for 15min. After the digestion is completed, it is preferably detected for residual DNA fragments. In some embodiments, there is no limitation on the method for recovering and purifying the mRNA, and conventional purification methods in the art can be used. In the specific implementation process of the present invention, it is preferably carried out using an RNA purification kit. After the purified mRNA is recovered, the mRNA quality test is performed. The quality test includes the concentration of the mRNA, the OD260nm / OD280nm, and the OD260nm / OD230nm ratio of the mRNA. When the OD260nm / OD280nm range is 1.8-2.1 and the OD260nm / OD230nm range is greater than 2.0, the mRNA is considered qualified. After the purified mRNA is recovered, the purified mRNA is preferably packaged.

[0200] 3.7 Lipid Nanoparticles (LNP)

[0201] In some embodiments, the therapeutic composition may also include a delivery vehicle. The mRNA described herein may be formulated in nanoparticles or other delivery vehicles to prevent the mRNA from being degraded when delivered to a subject. In some embodiments, the mRNA may be encapsulated in nanoparticles. In a specific embodiment, nanoparticles are particles having at least one size (e.g., diameter) less than or equal to 1000nm, less than or equal to 500nm, or less than or equal to 200nm. In a specific embodiment, nanoparticles include lipids. Lipid nanoparticles may include, but are not limited to, liposomes and micelles. In a specific embodiment, the lipid nanoparticles may include cations and / or ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, pegylated lipids and / or structural lipids, or a combination thereof. In certain embodiments, lipid nanoparticles include one or more mRNA described herein, for example, mRNA encoding a protein of interest (e.g., adrenocorticotropic hormone).

[0202] The delivery vehicle in compositions as herein described can be nano lipid particle.Described nano lipid particle can comprise one or more cations and / or ionizable lipid." cationic lipid " generally refers to the net positive charge lipid that carries any number under certain pH (such as physiological pH).Described cationic lipid can include but is not limited to 3 (dilaurylamino) N1, N1, 4 tridodecyl 1 piperazine ethylamine (KL10), N1 [2 (docosylamino) ethyl] N1, N4, N4 tridodecyl 1, 4 piperazine diethylamine (KL22), 14, 25 tricosyl 15, 18, 21, 24 tetraaza octapore and alkane (KL25), DLin DMA, DLin K DMA, DLin KC2 DMA, Octyl CLinDMA, octyl CLinDMA (2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC Chol, DOSPA, DOGS, DODAP, DODMA and DMRIE. Additionally, a number of commercially available cationic and / or ionizable lipids can be used, e.g. (including DOTMA and DOPE) and (including DOSPA and DOPE). For example, the cationic lipid can be DLin MC3DMA. In some embodiments, the molar ratio of the cationic lipid in the lipid nanoparticle is about 40-70%. In a specific embodiment, the molar ratio of the cationic lipid in the lipid nanoparticle is about 50%.

[0203] In some embodiments, the nano lipid particles may comprise one or more non-cationic lipids. The non-cationic lipids may include anionic lipids. Anionic lipids suitable for use in the lipid nanoparticles of the present invention may include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylphosphatidylethanolamine, and other neutral lipids to which anionic groups are attached.

[0204] In some embodiments, the non-cationic lipids may include neutral lipids having a net charge of zero at physiological pH. Neutral lipids suitable for use in lipid nanoparticles of the present invention may include phospholipids, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylcholine (DP ... Oleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or a mixture thereof. In addition, lipids with a mixture of saturated and unsaturated fatty acid chains can be used. For example, the neutral lipids described herein can be selected from DOPE, DSPC, DPPC, POPC or any related phosphatidylcholine. In a specific embodiment, the neutral lipid is DSPC. In some embodiments, the molar ratio of the neutral lipid in the lipid nanoparticle is about 5-15%. In a specific embodiment, the molar ratio of the neutral lipid in the lipid nanoparticle is about 10%.

[0205] In some embodiments, the nano lipid particle can include lipid conjugates, and lipid conjugates include lipid moiety and polymer moiety, for example, the PEGylated lipid including lipid moiety and polyethylene glycol (PEG) moiety. Suitable lipid conjugates for use in the present invention include dimyristoylphosphatidylethanolamine-poly (ethylene glycol) 2000 (DMPE-PEG2000), DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-c-DOMG, PEG2000-c-DOPG etc. The molecular weight of operable poly (ethylene glycol) can be in the range of about 500 to about 10,000Da, or about 1,000 to about 5,000Da. The addition of these components can prevent lipid aggregation, and can also increase circulation duration, and is easy to lipid-nucleic acid composition delivery to target cells, or quickly releases nucleic acid. In a specific embodiment, the nano lipid particle can include PEG2000-DMG. In some embodiments, the molar ratio of the polyethylene glycol (PEG)-modified lipid molecules in the lipid nanoparticles is about 0.5-2%. In a specific embodiment, the molar ratio of the polyethylene glycol (PEG)-modified lipid molecules in the lipid nanoparticles is about 1.5%.

[0206] In some embodiments, the nanolipid particles may further comprise cholesterol. In some embodiments, the molar ratio of the cholesterol in the lipid nanoparticles is about 30-45%. In a specific embodiment, the molar ratio of the cholesterol in the lipid nanoparticles is about 38.5%.

[0207] In some embodiments, the nanolipid particles may include cationic lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipid molecules. In some embodiments, the molar ratio of the cationic lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipid molecules may be 45-55:35-45:5-15:0.5-2. In certain embodiments, the molar ratio of the cationic lipids, cholesterol, phospholipids, and polyethylene glycol-modified lipid molecules may be 50:38.5:10:1.5.

[0208] 3.8 Nanoparticle Composition

[0209] On the one hand, nucleic acid molecules as described herein are formulated for in vitro and in vivo delivery. In particular, in some embodiments, nucleic acid molecules are formulated into lipid-containing compositions. In some embodiments, lipid-containing compositions form lipid nanoparticles that enclose nucleic acid molecules in lipid shells. In some embodiments, lipid shells protect nucleic acid molecules from degradation. In some embodiments, lipid nanoparticles also help to transport enclosed nucleic acid molecules to intracellular compartments and / or mechanisms to provide expected preventive effects. In certain embodiments, when present in lipid nanoparticles, nucleic acids can resist degradation by nucleases in aqueous solutions. Lipid nanoparticles comprising nucleic acids and methods for their preparation are known in the art, such as those disclosed in U.S. Patent Publication No. 2004 / 0142025, U.S. Patent Publication No. 2007 / 0042031, PCT Publication No. WO 2017 / 004143, PCT Publication No. WO 2015 / 199952, PCT Publication No. WO 2013 / 016058, and PCT Publication No. WO 2013 / 086373, the entire disclosures of which are incorporated herein by reference in their entirety.

[0210] For example, multilamellar vesicles (MLVs) can be prepared by existing techniques, for example, by depositing a selected lipid on the inner wall of a suitable container or vessel; by dissolving the lipid in a suitable solvent and then evaporating the solvent to leave a thin film on the inside of the vessel, or by spray drying. An aqueous phase can be added to a rotating vessel, which results in the formation of MLVs. Unilamellar vesicles (ULVs) can then be formed by homogenization, sonication, or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[0211] Nanoparticle compositions as used herein include delivery vehicles (e.g., nanolipid particles), wherein mRNA can be associated with the surface of the lipid delivery vehicle and encapsulated therein. For example, when preparing nanoparticle compositions of the present disclosure, the cationic lipid delivery vehicle can be associated with mRNA through electrostatic interaction.

[0212] In the present invention, consider the size of target cell or tissue and the liposome application degree to be prepared to select the appropriate size of described lipid delivery vector (for example, lipid nano particle).In a specific embodiment, mRNA can be delivered to specific cell or tissue.For example, for targeting hepatocyte, can determine the size of lipid delivery vector (for example, lipid nano particle), make its size ratio the fenestration of endothelial lining liver sinusoidal space in liver less, make lipid delivery vector (for example, lipid nano particle) can easily permeate these endothelial fenestrations to arrive target hepatocyte.Lipid delivery vector (for example, lipid nano particle) can have enough large diameter to limit or obviously avoid being distributed in some cell or tissue.In some embodiments, the size (for example, diameter) of described lipid delivery vector (for example, lipid nano particle) can be in the range of about 25 to 250nm, for example, less than about 250nm, 175nm, 150nm, 125nm, 100nm, 75nm, 50nm, 25nm or 10nm. For example, the size (e.g., diameter) of the lipid delivery vehicle (e.g., lipid nanoparticle) can be in the range of about 25 to 250 nm, e.g., about 50 to 200 nm, about 75 to 175 nm, about 75 to 150 nm, or about 75 to 125 nm.

[0213] Nanoparticle compositions that can be used in conjunction with the present disclosure include, for example, lipid nanoparticles (LNPs), nanolipoprotein particles, liposomes, lipid vesicles, and lipid complexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and / or cross-linked to each other. The lipid bilayers can include one or more ligands, proteins, or channels.

[0214] 3.9 Preparation

[0215] According to the present disclosure, the nanoparticle compositions described herein can include at least one lipid component and one or more additional components, such as therapeutic and / or prophylactic agents. Nanoparticle compositions can be designed for one or more specific applications or goals. The components of a nanoparticle composition can be selected based on the specific application or goal, and / or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more components. Similarly, a specific formulation of a nanoparticle composition can be selected for a specific application or goal based on, for example, the efficacy and toxicity of a specific combination of each component.

[0216] The lipid component of the nanoparticle composition can include ionizable lipids, phospholipids (e.g., unsaturated lipids such as DOPE or DSPC), PEG lipids, and structural lipids. The components of the lipid component can be provided in a specific ratio.

[0217] In one embodiment, a nanoparticle composition is provided herein comprising a cationic or ionizable lipid compound, a therapeutic agent as provided herein, and one or more excipients. In one embodiment, the one or more excipients are selected from neutral lipids, phospholipids, steroids, and polymer-conjugated lipids. In one embodiment, the therapeutic agent is encapsulated within or associated with the lipid nanoparticles.

[0218] Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed for delivering therapeutic and / or prophylactic agents, such as RNA, to specific cells, tissues, organs, or systems or groups thereof in a mammal. The physicochemical properties of the nanoparticle compositions can be altered to increase selectivity for specific body targets. For example, the particle size can be adjusted based on the window size of different organs. The therapeutic and / or prophylactic agents contained in the nanoparticle compositions can also be selected based on one or more desired delivery targets. For example, therapeutic and / or prophylactic agents can be selected for specific indications, conditions, diseases, or disorders and / or for delivery to specific cells, tissues, organs, or systems or groups thereof (e.g., local or specific delivery). In certain embodiments, the nanoparticle compositions can include mRNA encoding a polypeptide of interest that can be translated intracellularly to produce a polypeptide of interest. Such compositions can be designed to be specifically delivered to a specific organ. In certain embodiments, the composition can be designed to be specifically delivered to the liver of a mammal.

[0219] The amount of the therapeutic and / or prophylactic agent in the nanoparticle composition can depend on the size, composition, desired target and / or application, or other characteristics of the nanoparticle composition, as well as the characteristics of the therapeutic and / or prophylactic agent. For example, the amount of RNA that can be used in the nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of the therapeutic and / or prophylactic agent and other components (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the wt / wt ratio of the lipid component to the therapeutic and / or prophylactic agent in the nanoparticle composition can be from about 5:1 to about 60:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 45:1, 50:1, and 60:1. For example, the wt / wt ratio of the lipid component to the therapeutic and / or prophylactic agent can be about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20: 1. The amount of the therapeutic and / or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., UV-visible spectroscopy).

[0220] In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and amounts thereof may be selected to provide a specific N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in the one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N:P ratio is selected. The one or more RNAs, lipids, and amounts thereof may be selected to provide an N:P ratio of about 2:1 to about 30:1, e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio may be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio can be about 5.0: 1, about 5.5: 1, about 5.67: 1, about 6.0: 1, about 6.5: 1, or about 7.0: 1. For example, the N:P ratio can be about 5.67:1.

[0221] The physical properties of a nanoparticle composition can depend on its components. For example, a nanoparticle composition comprising cholesterol as a structural lipid can have different characteristics than a nanoparticle composition comprising a different structural lipid. Similarly, the characteristics of a nanoparticle composition can depend on the absolute or relative amounts of its components. For example, a nanoparticle composition comprising a higher molar ratio of phospholipids can have different characteristics than a nanoparticle composition comprising a lower molar ratio of phospholipids. Characteristics can also vary depending on the method and conditions used to prepare the nanoparticle composition.

[0222] Nanoparticle compositions 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 the nanoparticle compositions. Zeta potential can be measured using dynamic light scattering or potentiometric methods (e.g., potentiometric titration). Dynamic light scattering can also be used to determine particle size. Instruments such as the Zetasizer Nano ZS (Malverm Instruments Ltd, Malvern, Worcestershire, UK) can also be used to measure multiple characteristics of the nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

[0223] In various embodiments, the average size of the nanoparticle composition can be between tens of nanometers and hundreds of nanometers. For example, the average size can be from about 40 nm to about 150 nm, such as about 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. In some embodiments, the average size of the nanoparticle composition can be from about 50 nm to about 100 nm, from about 50 nm to about 90 nm, from about 50 nm to about 80 nm, from about 50 nm to about 70 nm, from about 50 nm to about 60 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from about 60 nm to about 80 nm, from about 60 nm to about 70 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, from about 80 nm to about 100 nm, from about 80 nm to about 90 nm, or from about 90 nm to about 100 nm. In certain embodiments, the average size of the nanoparticle composition can be from about 70 nm to about 100 nm. In some embodiments, the average size can be about 80 nm. In other embodiments, the average size can be about 100 nm.

[0224] The nanoparticle composition can be relatively homogeneous. The polydispersity index can be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A smaller polydispersity index (e.g., less than 0.3) generally indicates a narrower particle size distribution. The polydispersity index of the nanoparticle composition can be about 0 to about 0.25, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.20, 0.21, 0.22, 0.23, 0.24, or 0.25. In some embodiments, the polydispersity index of the nanoparticle composition can be about 0.10 to about 0.20.

[0225] The zeta potential of a nanoparticle composition can be used to indicate the zeta potential of the composition. For example, the zeta potential can describe the surface charge of a nanoparticle composition. Nanoparticle compositions with relatively low positive or negative charges are generally desirable because highly charged species can interact undesirably with cells, tissues, and other components in the body. In some embodiments, the zeta potential of the nanoparticle composition can be about -10 mV to about +20 mV, about -10 mV to about +15 mV, about -10 mV to about +10 mV, about -10 mV to about +5 mV, about -10 mV to about 0 mV, about -10 mV to about -5 mV, about -5 mV to about +20 mV, about -5 mV to about +15 mV, about -5 mV to about +10 mV, about -5 mV to about +5 mV, about -5 mV to about 0 mV, about 0 mV to about +20 mV, about 0 mV to about +15 mV, about 0 mV to about +10 mV, about 0 mV to about +5 mV, about +5 mV to about +20 mV, about +5 mV to about +15 mV, or about +5 mV to about +10 mV.

[0226] The encapsulation efficiency of a therapeutic and / or prophylactic agent describes the amount of therapeutic and / or prophylactic agent encapsulated or otherwise associated with the nanoparticle composition after preparation relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., close to 100%). The encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and / or prophylactic agent in a solution containing the nanoparticle composition before and after disrupting the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and / or prophylactic agent (e.g., RNA) in the solution. For the nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and / or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency can be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

[0227] The nanoparticle compositions may optionally include one or more coatings. For example, the nanoparticle compositions may be formulated into capsules, films, or tablets having a coating. The capsules, films, or tablets containing the compositions described herein may have any useful size, tensile strength, hardness, or density.

[0228] 3.10 Pharmaceutical Compositions

[0229] According to the present disclosure, nanoparticle compositions can be formulated, in whole or in part, into pharmaceutical compositions. A pharmaceutical composition can comprise one or more nanoparticle compositions. For example, a pharmaceutical composition can comprise one or more nanoparticle compositions comprising one or more different therapeutic and / or prophylactic agents. The pharmaceutical composition can further comprise one or more pharmaceutically acceptable excipients or adjuvants, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents can be found, for example, in Remington's The Science and Practice of Pharmacy, 21st ed., A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and adjuvants can be used in any pharmaceutical composition, unless any conventional excipient or adjuvant is incompatible with one or more components of the nanoparticle composition. An excipient or adjuvant is incompatible with a component of the nanoparticle composition if the combination of the excipient or adjuvant with the component of the nanoparticle composition would result in any undesirable biological or other deleterious effect.

[0230] In some embodiments, the one or more excipients or adjuvants may constitute more than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, the one or more excipients or adjuvants may constitute 50%, 60%, 70%, 80%, 90% or more of the pharmaceutical customary percentage. In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and / or the International Pharmacopoeia.

[0231] The relative amounts of the one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated and further on the route of administration of the composition. For example, a pharmaceutical composition may contain between 0.1% and 100% (wt / wt) of the one or more nanoparticle compositions.

[0232] In some embodiments, nanoparticle compositions and / or pharmaceutical compositions of the disclosure are stored and / or shipped refrigerated or frozen (e.g., stored at 4°C or lower, for example, between about -150°C and about 0°C, or between about -80°C and about -20°C (e.g., about -5°C, -10°C, -15°C, -20°C, -25°C, -30°C, -40°C, -50°C, -60°C, -70°C, -80°C, -90°C, -130°C, or -150°C)). For example, the nanoparticle compositions and / or pharmaceutical compositions disclosed herein are stable for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months at a temperature of, for example, 4° C. or less (e.g., between about 4° C. and −20° C.). In one embodiment, the formulation is stable for at least 4 weeks at about 4° C. In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of: Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pH of the pharmaceutical compositions of the present disclosure is between about 7 and 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5 and 8, or between 7 and 7.8). For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein, Tris, saline, and sucrose, and has a pH of about 7.5-8, which is suitable for storage and / or transport at, for example, about -20°C. For example, a pharmaceutical composition of the present disclosure comprises a nanoparticle composition disclosed herein and PBS, and has a pH of about 7-7.8, which is suitable for storage and / or transport at, for example, about 4°C or lower. In the context of the present disclosure, "stability," "stabilized," and "stable" refer to the resistance of the nanoparticle compositions and / or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g., degradation, particle size changes, aggregation, changes in encapsulation, etc.) under given manufacturing, preparation, transportation, storage, and / or use conditions, for example, when stress is applied, such as shear force, freeze / thaw stress, etc.

[0233] Nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including patients or subjects who may benefit from the therapeutic effects provided by delivering a therapeutic and / or prophylactic agent to one or more specific cells, tissues, organs, or systems or groups thereof, such as the renal system. Although the descriptions of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein are primarily directed to compositions suitable for administration to humans, it will be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Modifications to compositions suitable for administration to humans in order to make the compositions suitable for administration to various animals are well known, and such modifications can be designed and / or performed by a veterinary pharmacologist of ordinary skill with only ordinary experimentation (if any). It is contemplated that subjects to whom the compositions may be administered include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0234] Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known or later developed in the art of pharmacology. Generally, such preparation methods comprise combining the active ingredient with an excipient and / or one or more other auxiliary ingredients, and then, if desired or necessary, dividing, shaping, and / or packaging the product into desired single-dose or multi-dose units.

[0235] Pharmaceutical compositions according to the present disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of the active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or a convenient fraction of such a dose, such as half or one-third of such a dose.

[0236] Pharmaceutical compositions can be prepared into various forms suitable for various routes and methods of administration. For example, pharmaceutical compositions can be prepared into liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders, and granules), dosage forms for topical and / or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants, and patches), suspensions, powders, and other forms.

[0237] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, the liquid dosage form may also contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may also contain additional therapeutic and / or prophylactic agents, additional agents, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or flavoring agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent, such as Cremophor™, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and / or combinations thereof.

[0238] Injectable preparations, such as sterile injectable aqueous or oily suspensions, can be prepared according to known techniques using suitable dispersants, wetting agents and / or suspending agents. Sterile injectable preparations can be sterile injectable solutions, suspensions and / or emulsions in non-toxic parenteral acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP and isotonic sodium chloride solution. Sterile fixed oils are typically used as solvents or suspending media. For this purpose, any mild fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used to prepare injections.

[0239] The injectable formulations can be sterilized, for example, by filtration through a bacteria-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

[0240] 3.11 Treatment Methods

[0241] The present disclosure features methods of delivering therapeutic and / or prophylactic agents to mammalian cells or organs, producing proteins of interest in mammalian cells, and treating a disease or condition in a mammal in need thereof, the methods comprising administering to the mammal a nanoparticle composition comprising the therapeutic and / or prophylactic agent and / or contacting mammalian cells with the nanoparticle composition.

[0242] In one aspect, the present disclosure provides a method for treating or preventing a rheumatic disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a therapeutic nucleic acid as described herein or a pharmaceutical composition comprising the therapeutic nucleic acid. In some embodiments, the subject is a human or non-human mammal.

[0243] In some embodiments, the nucleic acid or pharmaceutical composition is administered parenterally or enterally, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral, or rectal delivery.

[0244] In some embodiments, the nucleic acid-encapsulated lipid nanoparticles are endocytosed by cells in the subject.

[0245] In some embodiments, the nucleic acid is expressed by cells in the subject.

[0246] In a further embodiment, the administration is intravenous.

[0247] In further embodiments, the administration is about once a day, once every two days, twice a week, once a week, about once every two weeks, or about once a month.

[0248] In some embodiments, the rheumatic disease comprises gouty arthritis, rheumatoid arthritis, dermatomyositis / polymyositis, systemic lupus erythematosus, sarcoidosis, or psoriatic arthritis.

[0249] In another aspect, the present disclosure provides a method for treating or preventing a lung disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the therapeutic nucleic acid described herein or a pharmaceutical composition comprising the therapeutic nucleic acid.

[0250] In some embodiments, the subject is a human or non-human mammal.

[0251] In some embodiments, the nucleic acid or pharmaceutical composition is administered parenterally or enterally, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral, or rectal delivery.

[0252] In some embodiments, the nucleic acid-encapsulated lipid nanoparticles are endocytosed by cells in the subject.

[0253] In some embodiments, the nucleic acid is expressed by cells in the subject.

[0254] In a further embodiment, the administration is intravenous.

[0255] In further embodiments, the administration is about once a day, once every two days, twice a week, once a week, about once every two weeks, or about once a month.

[0256] In some embodiments, the lung disease comprises symptomatic pulmonary sarcoidosis.

[0257] In another aspect, the present disclosure provides a method for treating or preventing an ophthalmic disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the therapeutic nucleic acid described herein or a pharmaceutical composition comprising the therapeutic nucleic acid.

[0258] In some embodiments, the subject is a human or non-human mammal.

[0259] In some embodiments, the nucleic acid or pharmaceutical composition is administered parenterally or enterally, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral, or rectal delivery.

[0260] In some embodiments, the nucleic acid-encapsulated lipid nanoparticles are endocytosed by cells in the subject.

[0261] In some embodiments, the nucleic acid is expressed by cells in the subject.

[0262] In a further embodiment, the administration is intravenous.

[0263] In further embodiments, the administration is about once a day, once every two days, twice a week, once a week, about once every two weeks, or about once a month.

[0264] In some embodiments, the ophthalmic disease comprises keratitis, uveitis, or optic neuritis.

[0265] In yet another aspect, the present disclosure provides a method for treating or preventing a neurological disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of the therapeutic nucleic acid described herein or a pharmaceutical composition comprising the therapeutic nucleic acid.

[0266] In some embodiments, the subject is a human or non-human mammal.

[0267] In some embodiments, wherein the therapeutic nucleic acid or pharmaceutical composition is administered parenterally or enterally, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

[0268] In some embodiments, the therapeutic nucleic acid or pharmaceutical composition is administered about once a day, once every two days, twice a week, once a week, about once every two weeks, or about once a month.

[0269] In some embodiments, the lipid nanoparticles encapsulating the therapeutic nucleic acid are endocytosed by cells in the subject.

[0270] In some embodiments, wherein the therapeutic nucleic acid is expressed by cells in the subject.

[0271] In some embodiments, the neurological disease comprises multiple sclerosis, optic neuritis, or infantile spasms (IS).

[0272] 4. Technical Effects of the Present Disclosure

[0273] Through creative work, the inventors have discovered that by delivering the nucleic acid therapeutics described herein into the body, human cells can express ACTH, thereby treating rheumatic diseases (including but not limited to gout), lung diseases, ophthalmic diseases, kidney diseases, or neurological diseases (including but not limited to infantile spasms and multiple sclerosis). Compared to ACTH protein drugs, the therapeutic nucleic acids described herein have advantages such as a longer half-life, less frequent dosing, better therapeutic efficacy, and lower immunogenicity.

[0274] [Corrected 12.08.2024 in accordance with Rule 26] Sequence Listing

[0275] [Corrected on 12.08.2024 according to Rule 26] Note: In SEQ ID NO:2-61, the sequence indicated in bold on the N-terminal side is the signal peptide, and the sequence indicated in bold on the C-terminal side is the linker and VLk sequence; SP represents a signal peptide or a signal peptide coding sequence; Linker represents a linker or a linker coding sequence; VLk represents a VLk sequence or a VLk coding sequence; Flag represents a Flag tag or its coding sequence.

[0276] Example

[0277] The technical features involved in the different implementation plans described in this article can be implemented in combination with each other.

[0278] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0279] When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints may be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the understanding of the prior art by those skilled in the art and the description of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention may also be used to implement the present invention.

[0280] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields in the art. These techniques are well described in the literature, for example, by Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc. The reagents and raw materials used in the present invention are all commercially available.

[0281] Example 1: Preparation of mRNA constructs

[0282] In this example, mRNA encoding adrenocorticotropic hormone (ACTH) was designed and prepared by transcription reaction.

[0283] Design of mRNA constructs

[0284] Referring to the mRNA structure shown in Figure 1, a signal peptide (SP2-53) was introduced at the 5' end of the nucleotide sequence and the N-terminus of the amino acid sequence (as shown in SEQ ID NO: 1) of human adrenocorticotropic hormone (ACTH) to achieve extracellular secretion expression of the ACTH protein, or a Flag tag was added to the 3' end or C-terminus in parallel for protein expression detection, or a VLK sequence (linker: SEQ ID NO: 55; and VLK sequence: SEQ ID NO: 54) was introduced to the C-terminus through a linker to achieve a long half-life and high expression of the ACTH protein. Subsequently, a 5'-UTR was introduced at the 5' end of its ORF and a 3'-UTR and a Poly A tail structure were introduced at the 3' end to design an mRNA construct encoding adrenocorticotropic hormone (ACTH).

[0285] In this mRNA construct, the nucleotide sequence encoding adrenocorticotropic hormone (ACTH) was optimized for sequence, species, tissue specificity, and G / C content, and the optimized sequences are shown in SEQ ID NOs: 108-167. Furthermore, 5'-UTR (shown in SEQ ID NOs: 62-82), 3'-UTR (shown in SEQ ID NOs: 83-101), and Poly-A tail (shown in SEQ ID NOs: 103-107) functional elements were introduced, resulting in the design of 173 mRNA sequences (shown in SEQ ID NOs: 170-342).

[0286] Preparation of mRNA constructs

[0287] The DNA fragment encoding the mRNA construct of adrenocorticotropic hormone (ACTH) was synthesized and the mRNA was synthesized by in vitro transcription. Specifically:

[0288] Plasmid construction

[0289] 1) synthesizing a DNA fragment encoding the above-mentioned ACTH, wherein the DNA fragment comprises a sequence encoding adrenocorticotropic hormone and a T7 promoter sequence and 5'-UTR at the 5' end, and a 3'-UTR, a Poly-A sequence, and a specific restriction endonuclease recognition sequence (BspQI recognition sequence) at the 3' end;

[0290] 2) The synthesized DNA fragment and pUC-GW (purchased from GeneWeiZ) plasmid were then digested with PvuI (purchased from NEB, R3150), and the digested target DNA fragment and pUC-GW plasmid were ligated in vitro using T4 ligase (Thermo Scientific, EL0011) to obtain a recombinant plasmid.

[0291] 3) The recombinant plasmid obtained above was transformed into competent Escherichia coli and+ Culture on solid medium for 12 h;

[0292] 4) Then select the above kan + Perform colony PCR on single colonies on solid culture medium, and select colonies containing target bands in the colony PCR results for sequencing;

[0293] 5) Expand the colonies that have been sequenced correctly and use the omega D6915 Endo-free Plasmid Midi Kit for plasmid extraction.

[0294] 6) Use Nanodrop to measure the concentration of the extracted plasmid. Digest the correctly constructed plasmid with PvuI restriction endonuclease and verify by agarose gel electrophoresis. If the constructed plasmid is qualified, the electrophoresis results should show two distinct bands: one for the linearized plasmid and one for the desired product.

[0295] Preparation of linearized plasmid

[0296] The above plasmid was subjected to BspQI enzyme digestion reaction to linearize the plasmid. The reaction system was:

[0297] After 10 minutes of enzyme digestion reaction, the effect of plasmid digestion was detected by agarose gel electrophoresis. If the digestion was incomplete, the digestion reaction time could be extended appropriately.

[0298] Linearized plasmid purification

[0299] The linearized plasmid was digested with enzymes and purified using the Thermo Scientific GeneJET PCR Purification Kit, #K0702, as follows:

[0300] 1) Add Binding Buffer to the linearized enzyme digestion system at a 1:1 (v / v) ratio, mix thoroughly, observe the solution color change, and decide whether to add an appropriate amount of 3M sodium acetate;

[0301] 2) Pipette the solution from step 1) and transfer it to a GeneJET purification column. Centrifuge for 60 seconds and discard the filtrate.

[0302] 3) Add 700 μL Wash Buffer (+ethanol) to the GeneJET purification column, centrifuge for 60 seconds, and discard the filtrate;

[0303] 4) Centrifuge the empty GeneJET purification column for 1 min and discard the remaining wash buffer in the purification column;

[0304] 5) Place the GeneJET purification column in a new 1.5 mL centrifuge tube, add 50 μL of Elution Buffer, and centrifuge for 60 seconds.

[0305] 6) Discard the purification column, collect the purified DNA, and store at -20°C for later use;

[0306] 7) The purified linearized plasmid was verified and quality controlled by agarose gel electrophoresis.

[0307] Linearized plasmid in vitro transcription (IVT)

[0308] During the in vitro transcription (IVT) process of linearizing the plasmid to synthesize mRNA, modified nucleotides are added to the reaction system in a certain proportion. The modified nucleotides include N1-Methylpseudo-UTP, Pseudo-UTP, 5-Methoxy-UTP, and 5-Methyl-CTP. The mRNA is capped using anti-reversal Cap analogs, including Cap2 AG, Cap1 m6AG, Cap2 m6AG, and Cap1 AG.

[0309] Specifically:

[0310] 1) Perform in vitro transcription (IVT) of the linearized plasmid to obtain mRNA in the following reaction system. Add the following components at room temperature:

[0311] 2) Gently mix the components with a pipette, collect them by brief centrifugation, and incubate at 37°C for 2-3 hours;

[0312] 3) IVT products were verified and quality controlled by agarose gel electrophoresis;

[0313] 4) The IVT product was detected by agarose gel and was a single mRNA product band.

[0314] 5) Removal of DNA template from IVT reaction system:

[0315] An appropriate amount of DNase I enzyme was added to the reaction system, incubated at 37°C for 15 minutes to digest the transcribed DNA template, and verification and quality control were performed by agarose gel electrophoresis at the same time; by comparing the IVT products before and after DNase I digestion, no band of the same size as the linearized plasmid was detected in the IVT product after DNase I digestion, indicating that the DNA template in the IVT product has been completely removed.

[0316] Purification of in vitro transcription (IVT) products:

[0317] Purification was performed using NEB Monarch RNA Cleanup Kits, #T2050L method as follows:

[0318] 1) Add 100 μL RNA Cleanup Binding Buffer to the 50 μL DNase I digested IVT product.

[0319] 2) Take 150 μL of anhydrous ethanol and add it to the IVT product digested with DNase I. Mix thoroughly by gently pipetting.

[0320] 3) Place the purification column into the collection tube, add no more than 900 μL of sample at a time, centrifuge for 1 minute, and discard the filtrate until all the solutions in all steps are bound to the purification column;

[0321] 4) Add 500 μL RNA Cleanup Wash Buffer (+ethanol) to the purification column, centrifuge for 1 minute, and discard the filtrate;

[0322] 5) Centrifuge again for 1-2 minutes to completely remove the residual solution in the purification column;

[0323] 6) Transfer the purification column to a new RNase-free 1.5 mL centrifuge tube, add 50-100 μL of Elution Buffer or nuclease-free water to the purification column, centrifuge for 1 minute, collect the filtrate, and store at -70°C until use;

[0324] 7) The purified IVT product was verified and quality controlled by agarose gel electrophoresis.

[0325] Example 2: mRNA expression and analysis in cells

[0326] In order to determine the protein expression of the above mRNA constructs, the above mRNA encoding adrenocorticotropic hormone was transfected into HEK293T cells, and the cellular protein expression level was detected to obtain the preferred mRNA construct and possible optimization scheme.

[0327] Specifically:

[0328] mRNA transfection into cells

[0329] 1) Freshly revived HEK293T cells were cultured at 37°C, 5% CO2, and subcultured to P2 before use;

[0330] 2) Cells were plated in 24-well plates: 7x10 4Cells / well, complete culture medium (10% FBS, 1% double antibody) 500uL / well, 3 replicates for each condition, and continued to culture for 24 hours after plating;

[0331] 3) Prepare RNAiMAX-MIX transfection reagent and transfer 1 μg of mRNA into each well. The steps are as follows:

[0332] a. RNAiMAX: 5 μl + 50 μl opti-MEM (OMEM, no additives in the culture medium), mix well, and let stand for 5 minutes;

[0333] b. mRNA MIX: 1 μg mRNA (0.5 μg / μl) + 50 μl opti-MEM (OMEM, no additives in the culture medium) mixed evenly;

[0334] c. Add the above RNAiMAX to the mRNA MIX, mix thoroughly by pipetting, and let it stand for 15 minutes;

[0335] d. Prepare Lipo-mRNA MIX.

[0336] 4) Replace 400uL complete culture medium before cell transfection;

[0337] 5) Add 105 μl of the above-mentioned Lipo-mRNA MIX to each well for cell transfection;

[0338] 6) After transfection for a predetermined period of time, collect cytoplasm and supernatant samples.

[0339] 7) Cell supernatant: Centrifuge at 4000 rpm for 10 min at 4°C, transfer the supernatant and save the cytoplasm: Rinse once with pre-cooled PBS, add 100 μl of cell lysis buffer (containing protease inhibitors), and lyse on ice.

[0340] Protein expression Western blotting / WB detection

[0341] By detecting the expression of the Flag tag introduced at the C-terminus of the coding sequence, the expression level of ACTH in the mRNA was determined. Based on the expression level, the optimal mRNA construct was analyzed to screen the functional elements of the ACTH coding sequence and mRNA. The protein expression detection steps are as follows:

[0342] 1) Thaw and lyse the cytoplasmic samples, and quantify total protein using BCA.

[0343] 2) Add Loading Buffer to the lysate to a final concentration of 1x;

[0344] 3) Calculate the loading volume for 2 μg of total protein based on the BCA concentration;

[0345] 4) Load 2 μg of lysate and 10 μl of supernatant for SDS-PAGE protein electrophoresis;

[0346] 5) After electrophoresis, perform semi-dry transfer using an Iblot2 instrument for 7 minutes;

[0347] 6) Block with 5% skim milk for 1 hour and then wash once with PBST;

[0348] 7) Dilute the anti-flag antibody (HRP) at 1:3000, then soak the protein membrane in the antibody and incubate overnight at 4°C in the dark;

[0349] 8) Wash with PBST 5 times, 100 rpm, 10 min each time;

[0350] 9) Use ECL to expose and photograph in a protein imager.

[0351] analyze

[0352] This example evaluated the expression levels of ACTH in different mRNA constructs, mainly evaluating the effects of the coding sequence, UTR and PolyA functional elements, modified nucleotides and cap structure (5' Cap) of the mRNA.

[0353] 1. Screening of the coding sequence of adrenocorticotropic hormone

[0354] Twenty mRNA constructs (SEQ ID NO. 170-SEQ ID NO. 189) with different ACTH coding sequences were constructed based on conventional mRNA functional elements (structure: 5'UTR-1+SP12+different ACTH+Flag+3'UTR-1+PolyA-1). The more preferred constructs with ACTH coding sequences were identified based on Flag tag expression levels.

[0355] Referring to the WB results and grayscale integral results shown in Figures 2a-b, in the 60%-68% G / C content construct group, the constructs with the best and medium expression levels were SEQ ID NO.171 (the corresponding ACTH coding sequence was SEQ ID NO.109) and SEQ ID NO.177 (the corresponding ACTH coding sequence was SEQ ID NO.115), respectively. In the 53%-58% G / C content construct group, the constructs with the best and medium expression levels were SEQ ID NO.184 (the corresponding ACTH coding sequence was SEQ ID NO.122) and SEQ ID NO.189 (the corresponding ACTH coding sequence was SEQ ID NO.127). The WB results and grayscale integral calculation results shown in Figures 5a-b demonstrate the screening results of signal peptides.

[0356] 2. Screening of functional element sequences

[0357] Twenty mRNA constructs (SEQ ID NO. 190-SEQ ID NO. 209) using different functional elements were constructed based on the combination of the adrenocorticotropic hormone coding sequence 3 and the above-mentioned four 5'-UTRs and five 3'-UTRs. The more preferred functional element constructs were determined based on the Flag tag expression level detection.

[0358] Referring to the WB results and grayscale integral calculation results shown in Figures 3a-b, the expression level of the construct shown in SEQ ID NO: 190 is better, wherein the corresponding 5'-UTR is shown in SEQ ID NO: 62, and the 3-'UTR is shown in SEQ ID NO: 83.

[0359] 3. Cap structure, modified bases, and PolyA tail sequence

[0360] The mRNA construct containing 4 mRNA Cap caps (Cap2 AG, Cap1 m6AG, Cap2 m6AG, Cap1 AG) and 4 modified bases (N1-Methylpseudo-UTP, Pseudo-UTP, 5-Methoxy-UTP, 5-Methyl-CTP) was synthesized based on the mRNA construct shown in SEQ ID NO: 190, and the specific sequences containing 4 poly-A tail sequences are shown in SEQ ID NO.210-SEQ ID NO.213.

[0361] Referring to the WB results and grayscale integral calculation results shown in Figures 4a-b, the preferred Cap structure screened out was Cap1 AG, ​​the modified base type was N1-MethylpseudoUridine, and the Poly A tail sequence was SEQ ID NO.103 (the corresponding construct was SEQ ID NO.210).

[0362] 5. Conclusion

[0363] After screening the ACTH coding sequence, UTR sequence, capping type, modified bases, tailing type, signal peptide sequence, etc. in ACTH mRNA, the preferred functional elements are:

[0364] ACTH coding sequences: SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 122, SEQ ID NO: 127;

[0365] ACTH mRNA UTR combination: 5'-UTR (SEQ ID NO: 62) and 3'UTR (SEQ ID NO: 83);

[0366] ACTH mRNA capping type: Cap1 AG;

[0367] ACTH mRNA modified base: N1-Methylpseudo-UTP;

[0368] ACTH mRNA ending type: SEQ ID NO: 103;

[0369] Example 3. In vitro expression of constructs

[0370] Based on the ACTH mRNA coding sequence and functional elements screened out in the above experiments, four mRNA constructs (SEQ ID NO.225-SEQ ID NO.228) were constructed, which contained Flag tag proteins to verify the screened sequences and functional elements at the cellular level.

[0371] The mRNA constructs shown in SEQ ID NO. 225-SEQ ID NO. 228 were transfected into HEK293T cells, and the ACTH expression level in the cell supernatant was analyzed using an ACTH-ELISA kit or a Flag tag antibody. The specific experimental procedures are as follows:

[0372] 1) Coating: Add 100 μL of diluted Capture Antibody to a 96-well ELISA plate, seal the plate, and incubate at 4°C overnight.

[0373] 2) Washing: Wash the plate with 300 μL of Wash Buffer for a total of 5 times.

[0374] 3) Blocking: Add 250 μL Blocking Buffer to each well and block for 1 hour at room temperature;

[0375] 4) Wash the plate again: Wash the plate with 300 μL of Wash Buffer for a total of 5 times.

[0376] 5) Storage and Use: Seal the prepared ELISA plate with sealing film and store at 4°C until use.

[0377] 6) Experimental analysis

[0378] Sample loading: Add 100uL of sample / standard / QC sample to the coated ELISA plate, add 100uL of Assay Buffer to the blank well, and seal the ELISA plate with sealing film;

[0379] Incubation: Place the ELISA plate on a Microplate Shaker at 400 rpm, RT, and incubate for 2 h.

[0380] Wash the plate: Take 300uL Wash Buffer to wash the ELISA plate for a total of 5 times;

[0381] Primary antibody: Add 100uL of diluted detection antibody to each well of the ELISA plate and seal the plate with sealing film;

[0382] Incubation: Place the ELISA plate on a Microplate Shaker, 400 rpm, RT, incubate for 1 h;

[0383] Wash the plate: Take 300uL Wash Buffer to wash the ELISA plate for a total of 5 times;

[0384] Secondary antibody: Add 100 μL of diluted Peroxidase-conjugated Streptavidin to each well of the ELISA plate, seal the plate with sealing film, and incubate at room temperature for 1 hour;

[0385] Wash the plate: Take 300uL Wash Buffer to wash the ELISA plate for a total of 5 times;

[0386] Color development: Add 100 μL TMB to each well of the ELISA plate, seal the plate with sealing film, and incubate at room temperature for 0.5 h;

[0387] Termination: Add 100uL Stop Solution to each well of the ELISA plate to terminate the reaction;

[0388] Read the data: Use a microplate reader at a wavelength of 450 nm to read the plate, record the data and analyze.

[0389] Experimental result 1: First, the SEQ ID NO: 225 construct was selected, and its expression changes in cells were analyzed by transfecting different amounts of mRNA (0.5ug / 24-well cell, 1ug / 24-well cell, 2ug / 24-well cell). The results are shown in Figure 6 (left). With the increase of the transfected mRNA content, the expression level of ACTH in the cells also increased gradually. According to the experimental results, the final transfection amount of mRNA per 24-well cell was selected as 1ug.

[0390] Experimental result 2: The mRNA constructs represented by SEQ ID NO:225-SEQ ID NO:228 were transfected at a concentration of 1 μg per well for in vitro expression analysis. Quantification of ACTH in the cell supernatant ( Figure 6 , right) showed that the mRNA construct represented by SEQ ID NO:226 had the highest expression level in the cell supernatant.

[0391] Experimental conclusion: In the in vitro cell experiment, the amount of mRNA transfected per 24-well cell was 1 μg. Among the four candidate mRNA sequences (SEQ ID NO: 225-SEQ ID NO: 228), the mRNA construct represented by SEQ ID NO. 226 had the highest expression level in the cell supernatant.

[0392] Example 4. LNP formulated mRNA constructs

[0393] Based on the in vitro cell experiments results of the mRNA constructs shown in SEQ ID NO: 225-SEQ ID NO: 228, mRNA constructs without Flag tags (shown in SEQ ID NO: 229-232) were designed to study their in vivo expression in animals. The relevant mRNA was coated with LNP to form LNP particles containing the mRNA. The specific preparation process is as follows:

[0394] 1) Prepare the aqueous phase: Prepare the mRNA working solution in 100 mM sodium acetate (pH 4.0) at a concentration of 0.35 mg / mL.

[0395] 2) Preparation of the organic phase: 25 mM concentration, lipids dissolved in anhydrous ethanol, the composition percentages of the three formula components are as follows:

[0396] LNP Preparation 1:

[0397] DLin-MC3-DMA:DSPC:Cholesterol:DMPE-PEG2000=50:10:38.5:1.5

[0398] LNP Preparation 2:

[0399] SM102:DSPC:Cholesterol:DMPE-PEG2000=50:10:38.5:1.5

[0400] LNP Preparation 3:

[0401] ALC-0315:DSPC:Cholesterol:ALC-0159=50:10:38.5:1.5

[0402] 3) Mix by rapid pipetting using the LNP preparation system at a ratio of 3:1 aqueous phase to organic phase;

[0403] 4) LNP-mRNA is prepared by ultrafiltration, liquid exchange and concentration.

[0404] 5) Characterization of LNP encapsulation efficiency, particle size, PDI and Zeta potential:

[0405] The Qubit fluorescence method was used to determine the concentration of LNP-mRNA and the encapsulation efficiency was calculated. A particle size analyzer was used to measure the particle size, PDI, and Zeta potential of LNP-mRNA. The results are as follows:

[0406] Example 5. Animal experiments on mRNA construction

[0407] In this example, the mRNA constructs represented by SEQ ID NO: 229 to SEQ ID NO: 232 were intravenously injected into mice in the form of LNPs to study their expression in the animals.

[0408] Healthy mice aged 8-10 weeks were selected and raised under a standard feeding environment. One day before drug administration, all animals were weighed and grouped using StudyDirector™ (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). The "Matched distribution" randomization method was selected for grouping to ensure that the average weight of each group was as close as possible to the average weight of the other groups. The day of grouping was defined as Day 0. On the first day after grouping (Day 1), drug administration began according to the experimental design: the ACTH group was administered at a dose of 1 mpk (mpk in this article is an abbreviation for mg / kg), and the mRNA group (SEQ ID NO:229-SEQ ID NO:232) was administered at a dose of 1 mpk, and the administration method was intravenous administration. After group administration, routine testing was performed, including daily observation of the experimental animals' activity, food and water intake, eyes, fur, and other abnormalities outside the cage. Venous blood was also collected from the mice for subsequent detection of ACTH concentration in the blood.

[0409] Experimental results: The above-mentioned LNP-mRNA was injected intravenously into animals, and ACTH polypeptide was injected intravenously into animals as the positive drug group. As shown in Figures 7a-7c, the ACTH blood content was detected by ACTH ELISA test kit on samples of blood sampling points at different times. The results showed that the blood concentration of the ACTH positive drug group decreased rapidly after administration and dropped to the animal blood background level in about 12 hours. The mRNA construct shown in SEQ ID NO:230 was significantly detected to increase blood ACTH content around 3-4 hours after administration, and continued to have expression exceeding background levels until 12 hours. Other mRNA constructs (constructs shown in SEQ ID NO:229, SEQ ID NO:231, and SEQ ID NO:232) did not detect significant changes in ACTH blood content relative to animal background levels. By extending the animal feeding time, it was found that the SEQ ID NO:230 construct still had ACTH expression higher than animal blood background levels at 52 hours, demonstrating a more lasting blood drug concentration (shown in Figure 7c) of mRNA in animals.

[0410] Experimental conclusion: Consistent with the cell-level experiments, the mRNA construct represented by SEQ ID NO: 230 has a higher expression level than the other three candidate mRNAs both in the cell supernatant and in the animal blood.

[0411] Example 6. MSU-induced gout inflammation model experiment in rats

[0412] In order to verify the effect of the candidate mRNA screened in the above example in the treatment of acute gout, an MSU (monosodium urate)-induced gout inflammation model in rats was constructed, and the mRNA-LNP was administered by subcutaneous injection and intramuscular injection. Indicators such as joint swelling, blood inflammatory factor levels, and blood ACTH content were detected in the gout inflammation model rats to evaluate the effect of the mRNA construct shown in SEQ ID NO: 230 in the treatment of acute gout.

[0413] Establishment of MSU-induced gout inflammation model in rats

[0414] Healthy rats aged 8-10 weeks were selected and raised under a standard feeding environment. One day before the model was established, all animals were weighed and grouped using StudyDirector™ (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). The "Matched distribution" randomization method was selected to ensure that the average weight of each group was as close as possible to the average weight of the other groups. The day of grouping was defined as Day 0. On the first day after grouping (Day 1), 50uL of MSU-PBS solution was injected into the right ankle joint of the rat (the injection dose was 1.5mg MSU per rat), and 50uL of PBS solution was injected into the left ankle joint as a blank control. The swelling of the rat joints was observed and measured in real time. Routine tests were also performed, including daily observation of the experimental animals' activity, food and water intake, eyes, fur, and other abnormalities outside the cage. The quality of the acute gout inflammation model was assessed by measuring the swelling of the rat joints.

[0415] Efficacy of mRNA-LNP in MSU-induced gout inflammation model in rats

[0416] Dosage regimen for rat gout inflammation model:

[0417] In the gout inflammation model experiment, preventive drug administration to animals is involved, such as preventive administration of the positive drug Dexamethasone (10 mpk, intraperitoneal administration) 1 hour before the start of model construction, and preventive administration of ACTH short peptide and mRNA (SEQ ID NO: 230) (ACTH: 0.5 mpk; mRNA: 2 mpk) 0.5 hours before the start of model construction. The administration methods include subcutaneous administration and intramuscular injection. At the same time, a blank group (PBS injection) was set as a control. The swelling of the rat joints was observed and measured in real time, and the blood of the rats was collected for the detection of blood drug concentration and inflammatory factors. The time points for rat blood collection and swelling measurement were set as: 0h, 2h, 4h, 8h (swelling was measured at 8h, blood was collected at 7h), and 18h.

[0418] Swelling index

[0419] Experimental results: An animal gout inflammation model was constructed by injecting MSU into the joint cavity of rats, and the performance of the mRNA construct shown in SEQ ID NO: 230 in the gout inflammation model was analyzed. As shown in Figure 8a-k, at 18h, in the subcutaneous injection group, the mRNA administration group showed a significant relief of animal joint swelling compared to the modeling group (MSU Vehicle), and in some indicators, there was also a significant reduction in swelling. Compared with the subcutaneous injection group, no obvious mRNA swelling inhibition was observed in the intramuscular injection group, which may be caused by the different mechanisms of action of the two different administration methods.

[0420] Experimental conclusion: In the rat gout inflammation model, it can be clearly observed that the mRNA construct shown in SEQ ID NO: 230 can alleviate gout inflammation when administered subcutaneously, verifying the effectiveness of the SEQ ID NO: 230 construct mRNA in the treatment of gout inflammation.

[0421] Blood inflammatory factor indicators

[0422] Experimental results: An animal gout inflammation model was constructed by intra-articular injection of MSU in rats, and the performance of the mRNA construct shown in SEQ ID NO: 230 in the gout inflammation model was analyzed. Referring to the results shown in Figures 9a-b, compared with the modeling group (G3, PBS injection) and the ACTH short peptide group (G4, ACTH short peptide), the subcutaneous administration group (G5) of the mRNA construct shown in SEQ ID NO: 230 showed significant inhibitory effects on IL-1β and IL-6 inflammatory factors over time. At the same time, the inflammation inhibitory effect of the mRNA construct shown in SEQ ID NO: 230 was better than that of the ACTH short peptide group.

[0423] Experimental conclusion: In the gout inflammation model, the mRNA construct represented by SEQ ID NO: 230 exhibited a significant inflammation-suppressing effect, indicating that it has great potential in the treatment of acute gout.

[0424] Blood drug concentration (blood ACTH level) test

[0425] Experimental Results: A gout inflammation model was established in rats by intra-articular injection of MSU, and the performance of the mRNA construct represented by SEQ ID NO: 230 in this gout inflammation model was analyzed. Referring to the results shown in Figures 10a-b, compared to the modeling group (G3, injected with PBS), the groups receiving the mRNA construct represented by SEQ ID NO: 230 (G5 or G8) showed a significant increase in blood ACTH levels, with blood levels increasing by over 80%.

[0426] Experimental Conclusion: Administration of the mRNA construct represented by SEQ ID NO:230 in the form of mRNA-LNP to a gout inflammation model significantly increased the amount of ACTH expressed. This was consistent with the results of inflammatory factors and joint swelling in rats, indicating that the mRNA construct represented by SEQ ID NO:230 can reduce inflammation in this gout inflammation model.

[0427] Example 7 Screening of Highly Secretory Expression Signal Peptides

[0428] According to the results of the above examples, the mRNA construct shown in SEQ ID NO: 230 can show significant ACTH translation and expression in in vitro cell experiments and in vivo animal PK. At the same time, in a gout inflammation model, the great potential of mRNA-LNP in treating acute gout diseases was also verified. Improving the translation and expression efficiency of mRNA in vivo can not only reduce the cost of mRNA-LNP drugs, but also reduce the dosage for human administration, thereby reducing the side effects caused by the LNP liposome components. Therefore, a series of designs and screenings were made to improve the translation, secretion and expression of mRNA.

[0429] In this example, the inventors screened 21 signal peptides (such as the signal peptides in the sequences of SEQ ID NO: 14-SEQ ID NO: 34, wherein the encoding nucleotide sequences corresponding to some of the signal peptides are SEQ ID NO: 343-SEQ ID NO: 351) and randomly combined them with the structure of the mRNA construct shown in SEQ ID NO: 230, and further sequence optimization was performed to form mRNA constructs (SEQ ID NO: 233-SEQ ID NO: 241 and SEQ ID NO: 253-SEQ ID NO: 268). These constructs were transfected into HEK293T and HepG2 cells, and the cell supernatants were collected. The ACTH content in the cell supernatants was evaluated using an ACTH ELISA detection kit to verify their effectiveness in increasing ACTH secretion and expression.

[0430] Experimental results: As shown in Figures 11a and 11b, the mRNA constructs shown in SEQ ID NO: 233-SEQ ID NO: 241 were transfected into HEK293T cells, and the mRNA construct shown in SEQ ID NO: 230 was used as a positive control. The expression of each construct in the cell supernatant was analyzed. It was found that the expression of the mRNA construct shown in SEQ ID NO: 237 was significantly higher than that of the other constructs, and its expression level was increased by nearly 50 times compared with the mRNA shown in SEQ ID NO: 230. It was also found that the mRNA constructs shown in SEQ ID NO: 234 and SEQ ID NO: 235 had similar expression levels to the mRNA construct shown in SEQ ID NO: 230. In the validation experiment conducted on HepG2 human hepatocytes, the mRNA construct shown in SEQ ID NO: 237, which was highly secreted and expressed in HEK293T cells, still had relatively high cellular secretory expression in HepG2 cells, demonstrating the strong expression ability of the mRNA construct shown in SEQ ID NO: 237 in in vitro experiments. 11c , in another group of constructs screened for signal peptide optimization (SEQ ID NO.253-SEQ ID NO.266, the mRNA construct shown in SEQ ID NO:237 still showed a high level of ACTH secretion expression, which was more than an order of magnitude higher than that of the other constructs.

[0431] Experimental conclusion: By performing expression tests on constructs composed of these 17 signal peptides, a highly secretory expression construct SEQ ID NO: 237 was successfully screened, which had a secretory expression level nearly 50 times higher than that of the previous mRNA construct shown in SEQ ID NO: 230.

[0432] Example 8 Expression test of highly secretory expression construct in LNP liposomes

[0433] The highly secretory expression construct SEQ ID NO: 237 designed in Example 7 was prepared with a liposome formulation (cationic lipid DLin-MC3-DMA, SM102, ALC-0315, refer to the formulation shown in Example 4) for LNP formulation and transfection of human hepatocytes HepG2. A SEQ ID NO: 237 construct transfection reagent group (Thermo RNAiMAX kit) was also used as a blank control.

[0434] Experimental results: As shown in FIG12 , construct SEQ ID NO: 237 had similar expression levels in cationic lipid MC3 and SM102 liposome formulations, and was significantly higher than that in ALC-0315 liposome formulations.

[0435] Experimental conclusion: From the above experiments, it can be seen that MC3 and SM102 liposome preparations show good transfection efficiency. Therefore, subsequent animal experiments will use the MC3 cationic lipid formula to prepare mRNA-LNP liposomes.

[0436] Example 9 PK experiment of rats expressing highly secretory signal peptide construct

[0437] Rat PK experiment:

[0438] Based on the data at the in vitro experimental level, in order to verify the pharmacokinetics of the SEQ ID NO: 237 construct in animals, IVT mRNA was prepared based on the mRNA construct shown in SEQ ID NO: 237, and the mRNA was encapsulated using the MC3 cationic lipid formulation (refer to Example 4 for the formula). The rats were administered by intravenous injection, and the ACTH content in the animal blood was monitored in real time. At the same time, the mRNA construct shown in SEQ ID NO: 230 was introduced as a positive control, and the PBS group was used as a negative control. In this experiment, a different MC3 encapsulation process was used, with the total lipid concentration of the LNP encapsulation being 36 mg / mL and the mRNA concentration being 0.3 ug / uL. The detection results of the mRNA-LNP are shown in the following table:

[0439] Quantification of ACTH in rat blood ( FIG. 15 ) showed that, compared with the animal experiment with the mRNA construct represented by SEQ ID NO: 230 in Example 5, the blood concentration of the construct represented by SEQ ID NO: 230 was increased by an order of magnitude by changing the MC3 encapsulation process. Furthermore, the mRNA construct represented by SEQ ID NO: 230 maintained a relatively high blood level after 48 hours. The blood concentration of the mRNA construct represented by SEQ ID NO: 237 increased faster in animals than that of the construct represented by SEQ ID NO: 230, reaching Cmax at approximately 4 hours, and its blood concentration was still several times higher than that of the PBS group.

[0440] Experimental Conclusion: This example verifies that the mRNA construct shown in SEQ ID NO: 237 has a very rapid increase in blood drug concentration in rats, and its blood drug level is increased by nearly an order of magnitude compared with the PBS group; at the same time, under different MC3 encapsulation process conditions, the SEQ ID NO: 230 construct exhibits different rat blood drug concentrations. Compared with the previous rat blood drug level, in this example, its blood drug concentration is increased by nearly an order of magnitude, and a high blood drug level is still maintained after 48 hours.

[0441] Example 10 Further screening of highly secretory expression signal peptides

[0442] To further increase the secretory expression of the constructs, reduce the amount of LNP lipids used, and mitigate potential LNP-induced hepatotoxicity, the inventors designed a series of constructs (SEQ ID NO:269-SEQ ID NO:285 and SEQ ID NO:299-SEQ ID NO:315). These constructs were transfected into human HepG2 hepatocytes and their expression in the cell supernatant was assessed using an ACTH ELISA kit.

[0443] Experimental results: As shown in Figure 13, in the screening experiment of transfecting hepatocytes HepG2 with mRNA constructs represented by SEQ ID NO:269-SEQ ID NO:285, the mRNA constructs represented by SEQ ID NO:283 and SEQ ID NO:284 had very significant in vitro cell supernatant ACTH expression compared to the other constructs, and their protein secretion expression capabilities were close to that of the construct represented by SEQ ID NO:237.

[0444] Experimental conclusion: Through screening of the above constructs, the mRNA constructs shown in SEQ ID NO: 283 and SEQ ID NO: 284 were obtained, and their protein secretion expression capabilities were close to that of the construct shown in SEQ ID NO: 237.

[0445] Example 11 Design and screening of low immunogenic secretory signal peptide sequence constructs

[0446] Since the secretory signal peptide of the secretory expression construct SEQ ID NO: 237 is derived from rats, the inventors performed single point mutations and combined mutations on the amino acid residues in the secretory signal peptide (SP) sequence that were different from the human homolog. The corresponding signal peptide encoding nucleotide sequence was SEQ ID NO: 352-SEQ ID NO: 370. 19 mRNA constructs (SEQ ID NO: 286-SEQ ID NO: 298 and SEQ ID NO: 316-SEQ ID NO: 321) were obtained, and the constructs were transfected into human HEK293T or HepG2 cells. The ACTH content in the cell supernatant was detected by ELISA to verify the cellular expression of the humanized amino acid sequence.

[0447] Experimental results: As shown in Figure 14, based on the results of quantitative analysis of the ACTH content in the cell supernatant, the mRNA construct represented by SEQ ID NO:289 unexpectedly exhibited the highest extracellular secretion expression ability of ACTH, and its expression level was 5.7 times that of the mRNA construct represented by SEQ ID NO:237; at the same time, the mRNA construct represented by SEQ ID NO:298 also exhibited higher extracellular ACTH expression ability than the construct represented by SEQ ID NO:237, and its expression level was 2 times that of the mRNA construct represented by SEQ ID NO:237.

[0448] Experimental conclusion: Based on the construct SEQ ID NO: 237, by designing and optimizing the sequence of the secretion signal peptide and the amino acid sequence of ACTH encoding protein, a construct with higher extracellular expression ability was successfully screened, which also has the potential to reduce immunogenicity.

[0449] Example 12 Toxicity experiments in rats with different administration methods and different lipids

[0450] Early in vivo animal experiments were primarily based on MC3 cationic lipids for LNP preparation. During the experimental execution, individual animals occasionally experienced decreased activity, decreased food intake, and increased ocular secretions. We inferred that this might be caused by the cationic lipids, so we then compared the safety and mRNA expression levels of two cationic lipids, MC3 and SM102, in vivo. The results showed that based on the SEQ ID NO: 289 molecule, there was no significant difference in mRNA expression levels between MC3 and SM102, nor were there significant differences in physical and chemical properties such as LNP encapsulation efficiency, particle size, PDI, and zeta potential. However, under high-dose animal administration conditions, LNPs prepared with SM102 lipids had better safety than MC3 lipids. This result was also confirmed in our other projects, so for subsequent in vivo experiments, we selected SM102 cationic lipids as the lipid for LNP preparation.

[0451] To explore more administration routes, we also compared the effects of intravenous (iv), subcutaneous (sc), and intramuscular (im) administration of ACTH mRNA-LNP on animal safety and mRNA expression levels in rats. The results showed that expression levels showed a certain degree of dose-dependency at both the IV and IM dose regimens, while sc administration showed no significant dose-dependency. Furthermore, we found that subcutaneous injection of mRNA-LNP, regardless of low or high doses, resulted in hardening and swelling of the skin at the injection site. Dissection revealed the presence of oily, viscous tissue masses at the injection site (Figure 16; PBS injection on the left and 3 mg / kg ACTH mRNA (SEQ ID NO:289)-LNP injection on the right). This may be due to an inflammatory response after LNP injection or drug accumulation due to incomplete subcutaneous absorption. In summary, compared to subcutaneous administration of mRNA-LNP, intravenous and intramuscular injections are safer and more conducive to mRNA protein expression.

[0452] Example 13 PK experiment on the stability of the highly secretory expression signal peptide construct in rats

[0453] To investigate the inter-batch stability of short peptide mRNA protein replacement expression in animals, we selected the previously screened highly expressed molecule, SEQ ID NO: 289 (its CDS sequence amino acid sequence is SEQ ID NO: 38), for IVT mRNA preparation. The mRNA was encapsulated using an SM102 cationic lipid formulation (see Example 4 for the formulation). The mRNA was administered intravenously to rats, and the ACTH content in the animals' blood was monitored in real time. In this experiment, three independent batches were prepared using the same mRNA IVT and SM102 encapsulation process, and three independent animal experiments were conducted.

[0454] The experimental results are shown in Figure 17. The plasma ACTH content of each group of animals at the 0h time point was the SD rat's own blood ACTH background. The empty LNP group in group G1 did not change significantly over time. 4h after intravenous administration, the plasma ACTH content reached its highest level (Tmax = 4h). The average Cmax of the three batches of mRNA-LNPs 4h after administration was 828,998pg / mL, which was 337 times the 0h background. Subsequently, the plasma ACTH content gradually decreased over time. At the same time, we also found that the three batches of independently prepared mRNA-LNP drugs showed similar Cmax, Tmax and T-half in three batches of independent animal experiments (G2, G3, G4), indicating that the ACTH mRNA-LNP drug has good repeatability.

[0455] Experimental conclusion: The results of three independent batches of prepared mRNA-LNP drugs in three independent batches of animal experiments (G2, G3, and G4) show that the highly secreted expression signal peptide construct we currently screened has good repeatability and in vivo PK stability, which lays the foundation for subsequent in vivo animal PK and efficacy.

[0456] Example 14 Experimental Study on Highly Secreted Expression Signal Peptide Construct in MSU-Induced Rat Gout Inflammation Model

[0457] To evaluate the in vivo efficacy of the previously screened highly expressed molecule, SEQ ID NO:289 (its CDS amino acid sequence is SEQ ID NO:38), healthy rats aged 8-10 weeks were selected and housed under standard conditions. One day before model establishment, all animals were weighed and grouped using StudyDirector™ (version 3.1.399.19, StudyLog System, Inc., San Francisco, CA, USA). The "matched distribution" randomization method was used to ensure that the mean weight of each group was as close as possible to the mean weight of the other groups. The day of grouping was designated Day 0. On Day 1, the rats were injected with 50 μL of MSU-PBS solution (1.5 mg MSU per rat) into the right ankle joint. The left ankle joint was injected with 50 μL of PBS solution as a blank control. Joint swelling was observed and measured in real time. Routine monitoring was also performed, including daily observation outside the cage for activity, food and water intake, and observation of eye, coat, and other abnormalities. The quality of the established acute gout inflammation model was evaluated by measuring the swelling of the rat joints.

[0458] Efficacy of mRNA-LNP in MSU-induced gout inflammation model in rats

[0459] Dosage regimen for rat gout inflammation model:

[0460] In the gout inflammation model experiment, it is related to the preventive administration of animals, for example, the preventive administration of positive drug injection corticotropin (Shanghai First Biochemical, ACTH) is carried out 0.5h before the start of the model construction, and the administration of SEQ ID NO:289 construct LNP encapsulated drug is carried out simultaneously. The G6 group is the LNP empty bag group, which serves as the vehicle control. The administration method includes intravenous administration and intramuscular injection. At the same time, a blank non-modeling group (PBS injection) is set as a control. The swelling of the rat joints is observed and measured in real time, and the blood of the rats is collected for the detection of blood drug concentration and inflammatory factors. The time points of rat blood collection and swelling determination are set to: 0h, 4h, 8h, 18h, 24h.

[0461] Swelling index

[0462] Experimental results: An animal gout inflammation model was constructed by injecting MSU into the rat articular cavity, and the performance of the mRNA construct shown in SEQ ID NO: 289 in the gout inflammation model was analyzed. As shown in Figures 18a-d and 18e-f, compared with the non-modeling group in the G1 group, the modeling group G2 was treated with vehicle, and the animal joints showed obvious swelling, indicating that the experimental modeling was successful. The mRNA-LNP administration group (G8-G11), whether 1mpk (1mg / kg) or 0.3mpk (0.3mg / kg), showed smaller joint swelling than the control empty bag LNP group (G6) under both iv and im administration methods. At the same time, compared with the positive control drug group G4 group, the mRNA-LNP administration group (G8-G11) showed smaller joint swelling in all doses and administration methods. In addition, by comparing the mRNA-LNP administration doses of 1mpk and 0.3mpk, the high dose showed a better effect of alleviating joint swelling. At the 24h time point, all mRNA-LNP-treated groups (G8-G11) showed a significant decrease in animal joint swelling compared to the G2 control group.

[0463] Experimental Conclusion: In a rat gout inflammation model, the previously screened, highly secretory construct SEQ ID NO:289 successfully alleviated joint swelling in both IV and IM administration, with significant relief of joint swelling at the 24-hour time point at a 0.3 mpk dose. This effect was superior to the marketed injectable corticotropin. This experiment validated the efficacy of the construct SEQ ID NO:289 in a rat gout inflammation model, demonstrating a highly potent therapeutic effect against gout inflammation.

[0464] Blood drug concentration (blood ACTH level) test

[0465] Experimental results: While monitoring the efficacy of mRNA-LNP drugs in relieving joint swelling in rat gout inflammation models with different administration methods and dosages, the ACTH content in the plasma of the relevant animal groups was also monitored. Referring to the results shown in Figure 18g, groups G7-G9 were iv administration groups with dosages of 3mpk, 1mpk, and 0.3mpk, respectively, and groups G10-G11 were im administration groups with dosages of 1mpk and 0.3mpk, respectively. Both the iv and im administration groups showed obvious dose dependence at different doses. At the same time, at the same dosage, the iv exposure was dozens of times that of the im. The plasma ACTH levels of all administration groups reached Cmax at 4h and showed a gradually decreasing trend with a similar T-half over time.

[0466] Experimental Conclusions: Monitoring mRNA-LNP administration groups with different administration routes and dosages revealed that the SEQ ID NO:289 construct exhibited stable, controllable, and dose-dependent ACTH levels in animals. These data provided valuable insights into the selection of different administration routes and mRNA-LNP dosages. Furthermore, plasma ACTH levels in the different administration groups correlated closely with the drug's efficacy in relieving joint swelling in a gout inflammation model, demonstrating that precise treatment of varying degrees of gout inflammation can be achieved by controlling the dosage and route of administration.

[0467] Changes in animal body weight in the gout inflammation model

[0468] As shown in Figure 18h, changes in animal body weight, changes in animal plasma ACTH levels, and the mRNA-LNP dosage are correlated. At high doses of injectable corticotropin (3 mpk) and mRNA-LNP (3 mpk), animal body weight showed negative growth within 24 hours. As the dosage decreased, the effect on animal body weight gradually decreased. At a dose of 0.3 mpk mRNA-LNP, the mRNA drug had almost no effect on animal body weight, indicating that mRNA drugs can be used to control and treat gout inflammation at a safe dose.

[0469] Blood IL-6, IL-1β, AST, ALT indicators

[0470] Experimental Results: An animal gout inflammation model was established by intra-articular injection of MSU in rats, and the expression of inflammatory factors of the mRNA construct represented by SEQ ID NO: 289 in the gout inflammation model was analyzed. Referring to the results shown in Figures 18i-l, in terms of IL-1β, compared with the empty LNP control in the G6 group, the IL-1β levels in the G9 and G11 groups at a dose of 0.3 mpk were not higher than those in the G6 group. At the same time, there was no significant change in the IL-1β index level over time. In terms of IL-6, except for the mRNA-LNP-administered groups in the G9 and G11 groups, the other groups were similar to the control group, with no significant change in IL-6 levels. For the G9 group, which received 0.3 mpk mRNA-LNP IV administration, the value was close to the background level at 0 h, then increased at 4 h, reached a maximum at 8 h, and returned to normal background levels at 18 h. For the G11 group, which received 0.3 mpk mRNA-LNP im administration, the trend was similar to that of the G9 group, with IL-6 levels rapidly decreasing to background levels after 8 h. Throughout the entire process, the rise in IL-6 indices in the first 8 hours may be caused by the mRNA-LNP drug. After 8 hours, there was a rapid decline in the IL-6 index and even a cliff-like decline in G9. Referring to the previous PK of mRNA in the body, this may be due to the anti-inflammatory effect exerted by the expression of ACTH. Among the AST and ALT indicators, there was no obvious increase in ALT and AST indicators overall. The high data of G8 in individual groups at 8 hours and 24 hours were also due to the high dosage. This situation did not occur in the 0.3mpk dosage. Combined with the experimental data in the gout inflammation model, the 0.3mpk mRNA-LNP dose has shown a very obvious pharmacodynamics. Therefore, the SEQ ID NO: 289 construct can not affect the ALT and AST indicators at a dose that ensures pharmacodynamics.

[0471] Experimental Conclusion: In a gout inflammation model, the construct SEQ ID NO: 289 demonstrated superior efficacy to the marketed injectable corticotropin at a dose of 0.3 mpk. This dose did not cause changes in the cytokines IL-6 and IL-1β, or ALT and AST levels, demonstrating that SEQ ID NO: 289 has a very safe therapeutic window and has great potential for the treatment of acute gout.

[0472] Example 15 Design of long T-half high expression construct and in vitro screening experiment

[0473] The natural product of ACTH is a short peptide of 39 amino acids with a half-life of 15 minutes in human plasma. The dosing frequency of the marketed ACTH drug, injectable corticotropin, is twice a day, and the dosing frequency of the marketed drug, acthar gel, ranges from twice a day to once every 2-3 days, depending on the indication. Therefore, for a drug that is injected subcutaneously or intramuscularly, its short half-life is a key factor limiting its dosing frequency. The development of long T-half ACTH mRNA-LNP drugs is of great significance for the treatment of chronic diseases that are suitable for certain ACTH short peptides.

[0474] According to past experience and research, constructing ACTH fusion protein can effectively prolong the half-life of short half-life protein. In the present embodiment, by constructing signal peptide-ACTH-linker-VLK fusion construct (Fig. 1), the half-life of ACTH mRNA is attempted to be improved. The amino acid sequence of fusion construct linker is: SEQ ID NO:55, the mRNA sequence of linker is: SEQ ID NO:169, the amino acid sequence of VLK is SEQ ID NO:54, and the mRNA sequence of VLK is: SEQ ID NO:168. The complete mRNA sequence of the ACTH construct fused with VLK is: SEQ ID NO:335-340. The construct sequence plasmid of gene synthesis is subjected to plasmid linearization, IVT to produce mRNA stock solution, mRNA LNP encapsulation of SM102 prescription and physical and chemical property characterization of LNP drug, and the preparation of mRNA-LNP drug is completed.

[0475] The in vitro expression capacity and activity of the long T-half ACTH mRNA construct were verified in an in vitro cell-based assay. The mRNA stock solution was transfected into HepG2 cells using a transfection reagent. The cell supernatant containing the mRNA expression product was collected, and a blank control was prepared from the cell supernatant containing the mRNA. ACTH levels in the cell supernatant were quantified using the Abcam ACTH ELISA kit.

[0476] Experimental results 1: As shown in Figures 19a-f, a head-to-head comparison of the ACTH expression levels in the cell supernatants of the mRNA constructs fused with linker-VLK (SEQ ID NO: 335-340) and the ACTH mRNA constructs not fused with linker-VLK (SEQ ID NO: 237, SEQ ID NO: 289, SEQ ID NO: 298, SEQ ID NO: 316, SEQ ID NO: 318, SEQ ID NO: 319) showed that the ACTH expression levels of the cells of the ACTH mRNA constructs fused with linker-VLK were generally significantly higher than those of the ACTH mRNA constructs not fused with linker-VLK. By monitoring the ACTH expression trends in the cell supernatants of each construct 24h, 48h, and 84h after mRNA transfection, it was found that the ACTH expression levels of the cells of the ACTH mRNA constructs fused with linker-VLK have been showing an upward trend, while the ACTH expression levels of the ACTH mRNA constructs not fused with linker-VLK have been showing a downward trend over time. Combined with the previous expression advantage of SEQ ID NO: 289 at the in vitro cell level, the inventors finally selected SEQ ID NO: 336 (compared with SEQ ID NO: 289, the coding sequence of the linker and the VLK sequence were added) for subsequent in vivo verification.

[0477] Experimental Conclusion 1: By fusing the VLK sequence to ACTH mRNA via a linker, the expression level of ACTH mRNA can be significantly increased, and the short T-half during ACTH mRNA expression can also be reversed. The ACTH-VLK fusion construct significantly prolongs its expression T-half, which lays the foundation for obtaining a larger AUC and Cmax when administered to animals.

[0478] Example 16 In vivo expression of long T-half high expression construct

[0479] This example will examine the ability of two types of ACTH mRNA with and without linker-VLk sequences to express short ACTH peptides in vivo.

[0480] Experimental Results 1: As shown in Figure 20, the ACTH short peptide, SEQ ID NO:289, and SEQ ID NO:336 were administered to rats at doses of 1 mpk, 0.3 mpk, and 0.1 mpk, respectively. PK kinetic curves showed that the T-half of the ACTH short peptide, SEQ ID NO:289, and SEQ ID NO:336 was 20 minutes, 3.2 hours, and 3 days, respectively. This indicates that the T-half of the ACTH mRNA construct SEQ ID NO:289 without a VLK sequence fusion was significantly higher than that of the ACTH short peptide, while the T-half of the ACTH mRNA construct SEQ ID NO:336 with a VLK fusion was significantly higher than that of the SEQ ID NO:289 construct.

[0481] Experimental Conclusion 1: When administering ACTH in vivo via mRNA, its T-half is far superior to the short ACTH peptide. Furthermore, the T-half of the VLK-fused ACTH mRNA construct is far superior to the non-VLK construct SEQ ID NO: 289. Therefore, administering ACTH in vivo via mRNA can address the high dosing frequency caused by the short half-life of the ACTH peptide itself. Furthermore, mRNA constructs with different T-halfs can meet the administration requirements for a variety of acute and chronic indications, greatly expanding the scope of application of mRNA-LNP drugs.

[0482] Example 17 Experiment of Highly Secreted Expression Signal Peptide Construct in EAU Model

[0483] Experimental autoimmune uveitis (EAU) is an organ-specific, T cell-mediated autoimmune disease that targets the neural retina and related tissues; it is triggered by immunization with retinal antigens. The pathology of EAU closely resembles that of human uveitis and is hypothesized to be autoimmune, with patients displaying immune responses to retinal antigens. The EAU model has become a valuable tool for evaluating new immunotherapeutic and conventional therapeutic strategies. The EAU model can also be used to investigate the mechanisms underlying tolerance and autoimmunity to organ-specific antigens in immune-privileged sites. Therefore, EAU serves as a tool for both clinical and basic research into ocular and organ-specific autoimmunity. Peripheral immunity can be induced in susceptible animals using adjuvants containing several evolutionarily well-conserved uveal pathogens (purified protein antigens extracted from the retina or their peptides) or by adoptive transfer of lymphocytes specific for these antigens. The aim of this study was to test the efficacy of compounds against bovine binding protein-induced autoimmune uveitis in Lewis rats.

[0484] According to the animal weight, on day 0, 90 animals were randomly divided using BioBook software to ensure that the weight values ​​of each group of animals were similar to reduce deviations. Dosing began on Day 1, and the specific dosing method is shown in Table 1. Weigh and record twice a week. From the third day on, the eyes of the rats were examined with an ophthalmoscope every day, and the severity of the disease was assessed by the scoring system in Table 2. Among them, binocular photography was performed on Day 11 and Day 14. After the end of the in vivo observation (day 16), all rats were euthanized by excessive carbon dioxide.

[0485] Table 1 - Grouping and dosing regimen

[0486] Note: FTY720 is the positive control of fingolimod.

[0487] Table 2 - Clinical EAU scores of rats

[0488] a Each higher level includes the criteria of the previous level

[0489] Experimental results: As shown in Figure 21a-b, on day 11, the clinical score of the G3 0.4mpk ACTH short peptide treatment group decreased by 43% compared with the G2 vehicle control group, and the decrease was not significant; all ACTH mRNA groups (G5-G15) showed a very strong decrease in clinical scores. At the day 11 time point, the clinical scores of all ACTH mRNA groups were significantly different from those of the G2 vehicle group. In the experiment, the ACTH short peptide was administered once every two days, and the high expression construct SEQ ID NO: 289 (administered once every four days) and the long T-half high expression construct SEQ ID NO: 336 (single dose, administered once every two weeks) significantly reduced the frequency of administration while ensuring better efficacy. The above data were also verified in the intuitive visual photos of the animal eyeballs (Figure 21c).

[0490] Experimental Conclusions: In the EAU pharmacodynamic model, compared to the short ACTH peptide (ACTH, marketed as an injectable corticotropin, administered once every two days), the high-expression construct SEQ ID NO: 289 and the long T-half high-expression construct SEQ ID NO: 336 demonstrated significantly superior efficacy with significantly reduced dosing frequency. This demonstrates that the high-expression constructs SEQ ID NO: 289 and SEQ ID NO: 336 have a favorable therapeutic effect in EAU.

[0491] Example 18 Experiment of Highly Secreted Expression Signal Peptide Construct in AIA Model

[0492] Among the established RA animal models, the adjuvant-induced arthritis (AIA) model is one of the most commonly used standard arthritis models, reflecting many clinical features of human RA, including joint swelling and inflammation, synovial tissue proliferation, and destruction of cartilage and bone. It has been successfully used in preclinical studies for the evaluation of anti-rheumatic drugs and toxicity prediction. Indicators such as paw thickness, clinical scores, body, body weight, histopathology, pain assessment and X-ray photography have laid the foundation for the evaluation of these drugs. In this example, based on the results of Examples 20-22, a molecular validation experiment for therapeutic administration of the AIA model was designed. 0.1 mL of an inducer (Freund's complete adjuvant + 6 mg lipoidal amine) was administered intradermally in SD rats for modeling, and therapeutic administration was started when the clinical score was ≥3. Corticotropin (1.6 mpk) and prednisolone (5 mpk) were used as positive controls. The construct of SEQ ID NO:289 was administered at doses of 0.02 mpk and 0.1 mpk IV and 0.1 mpk im. The construct of SEQ ID NO:336 was administered at doses of 0.005 mpk IV and 0.005 mpk im. The animals were evaluated for AIA clinical scoring and joint swelling.

[0493] Experimental Results: As shown in Figure 22a, based on the clinical scores of the model data, the G2 vehicle group developed symptoms on day 12, and the clinical scores gradually improved over time. On day 13, with a clinical score ≥ 3, therapeutic administration of SEQ ID NO:289 and SEQ ID NO:336 was initiated. Following administration, significant decreases in clinical scores were observed in all treatment groups compared to the vehicle group and the LNP blank control group on day 19. Both SEQ ID NO:336-iv-0.005-single-dose and SEQ ID NO:389-iv-0.1-Q4D showed significant decreases in clinical scores compared to the positive control drug ACTH-im-1.6mpk-Q2D, which persisted until day 23. The prednisolone-treated group also showed a continued decrease in clinical scores after day 19 as the treatment continued. The same dose of im administration was inferior to the iv administration group in terms of efficacy. Similar results were also observed for the ankle diameter and paw volume indicators in Figures 22b-c. In Figure 22d, all treatment groups showed similar trends to those of the active drug and vehicle, especially the low-dose group, whose weight changes showed consistent effects with those of the vehicle and injectable corticotropin treatment groups.

[0494] Experimental Conclusions: Constructs of SEQ ID NO:289 and SEQ ID NO:336, administered at 0.1 mpk, 0.02 mpk, and 0.005 mpk, demonstrated comparable or superior efficacy in the AIA model compared to 1.6 mpk of corticotropin and 5 mpk of prednisolone, administered intravenously, without significant animal toxicity. These data demonstrate the potential of mRNA-LNP therapeutics for the treatment of AIA and even RA.

Claims

1. A nucleic acid comprising an open reading frame (ORF) encoding adrenocorticotropic hormone, a functional fragment or variant thereof.

2. The nucleic acid of claim 1, wherein the ORF encodes a wild-type ACTH, a mutant ACTH, a truncated ACTH, or an ACTH fusion protein that retains ACTH function.

3. The nucleic acid of claim 1 or 2, wherein the adrenocorticotropic hormone comprises the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:

1.

4. The nucleic acid according to any one of claims 1 to 3, wherein the ORF encodes a wild-type ACTH, a mutant ACTH, a truncated ACTH or an ACTH fusion protein that retains ACTH function and is linked to a secretion signal peptide.

5. The nucleic acid according to claim 4, wherein the secretion signal peptide sequence is contained in the amino acid sequence shown in any one of SEQ ID NOs: 2-61. 6 . The nucleic acid according to claim 2 , wherein the ACTH fusion protein is a fusion protein of a secretion signal peptide and ACTH.

7. The nucleic acid according to any one of claims 4 to 6, wherein the secretion signal peptide is fused to the N-terminus or C-terminus of the ACTH, preferably, the secretion signal peptide is fused to the N-terminus of the ACTH.

8. The nucleic acid according to any one of claims 2 to 7, wherein the C-terminus of the ACTH fusion protein is connected to a kappa light chain variable region (VLk) sequence via a linker.

9. The nucleic acid according to any one of claims 1 to 8, wherein the amino acid sequence encoded by the ORF comprises a signal peptide and adrenocorticotropic hormone from the N-terminus to the C-terminus.

10. The nucleic acid according to any one of claims 1 to 9, wherein the amino acid sequence encoded by the ORF comprises a signal peptide, adrenocorticotropic hormone, a linker and a kappa light chain variable region (VLk) sequence from the N-terminus to the C-terminus.

11. The nucleic acid of any one of claims 1-10, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:55 or an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO:

55.

12. The nucleic acid of any one of claims 1-11, wherein the kappa light chain variable region (VLk) sequence comprises the amino acid sequence shown in SEQ ID NO:54 or an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98% or 99% identical to SEQ ID NO:

54. 13 . The nucleic acid according to claim 1 , wherein the ORF comprises, in order from 5′ to 3′, a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding adrenocorticotropic hormone.

14. The nucleic acid according to any one of claims 1-13, wherein the ORF comprises, in order from 5' to 3', a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding adrenocorticotropic hormone, a nucleotide sequence encoding a linker, and a nucleotide sequence encoding a kappa light chain variable region (VLk) sequence.

15. The nucleic acid of any one of claims 1-14, wherein the ORF encodes an amino acid sequence comprising the amino acid sequence shown in any one of SEQ ID NOs: 2-61 or an amino acid sequence that is at least 70%, 80%, 90%, 95%, 98% or 99% identical to any one of SEQ ID NOs: 2-61.

16. The nucleic acid according to any one of claims 1 to 15, wherein the ORF is codon-optimized, and preferably, the codon optimization does not change the amino acid sequence encoded thereby.

17. The nucleic acid of any one of claims 13-16, wherein the nucleotide sequence encoding the signal peptide comprises any one of SEQ ID NOs: 343-370 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 343-370 or a DNA sequence corresponding thereto.

18. The nucleic acid of any one of claims 13-17, wherein the coding nucleotide sequence for adrenocorticotropic hormone comprises any one of SEQ ID NOs: 108-167 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 108-167 or a DNA sequence corresponding thereto.

19. The nucleic acid of any one of claims 13-18, wherein the encoding nucleotide sequence of the linker comprises SEQ ID NO: 169 or its corresponding DNA sequence or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 169 or its corresponding DNA sequence.

20. The nucleic acid of any one of claims 13-19, wherein the nucleotide sequence encoding the kappa light chain variable region (VLk) sequence comprises SEQ ID NO: 168 or its corresponding DNA sequence or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168 or its corresponding DNA sequence.

21. The nucleic acid of any one of claims 1-20, wherein the nucleic acid further comprises a 5'-untranslated region (5'-UTR).

22. The nucleic acid of claim 21, wherein the 5'-UTR comprises a DNA sequence of SEQ ID NO: 62-82 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a DNA sequence of SEQ ID NO: 62-82 or a DNA sequence corresponding thereto.

23. The nucleic acid according to any one of claims 1 to 22, wherein the nucleic acid further comprises a 3'-untranslated region (3'-UTR).

24. The nucleic acid of claim 23, wherein the 3'-UTR comprises a DNA sequence of SEQ ID NO: 83-101 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a DNA sequence of SEQ ID NO: 83-101 or a DNA sequence corresponding thereto.

25. The nucleic acid according to any one of claims 1-24, wherein the nucleic acid comprises, in order from 5' to 3': a 5'-UTR, an ORF encoding an amino acid sequence comprising a signal peptide and adrenocorticotropic hormone or an amino acid sequence comprising a signal peptide, adrenocorticotropic hormone, a linker and a kappa light chain variable region (VLk) sequence, and a 3'-UTR.

26. The nucleic acid according to claim 25, wherein the 5'-UTR is selected from any one of SEQ ID NOs: 62-82 or a DNA sequence corresponding thereto, and the 3'-UTR is selected from any one of SEQ ID NOs: SEQ ID NOs: 83-101 or a DNA sequence corresponding thereto.

27. The nucleic acid of any one of claims 1-26, wherein the nucleic acid further comprises a 3'-poly A sequence.

28. The nucleic acid according to claim 27, wherein the 3'-poly (A) sequence comprises at least 10-400 adenosine nucleotides, preferably 50 to 400 adenosine nucleotides or 10 to 300 adenosine nucleotides; more preferably 50 to 250 adenosine nucleotides; most preferably 120 adenosine nucleotides; Alternatively, the 3'-poly(A) sequence comprises an adenosine polynucleotide and a linker sequence inserted therein, wherein the linker sequence is used to separate the adenine nucleotide sequence.

29. A nucleic acid according to any one of claims 1-28, wherein the nucleic acid comprises any one of SEQ ID NOs: 170-342 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 170-342 or a DNA sequence corresponding thereto.

30. The nucleic acid of any one of claims 1-29, wherein the nucleic acid is DNA or RNA.

31. The nucleic acid of any one of claims 1-30, comprising at least one chemically modified nucleotide.

32. The nucleic acid of claim 31, wherein the chemical modification is selected from one or more of 5-methylcytidine, 5-methoxyuridine, pseudouridine, N6-methyladenosine, or N1-methylpseudouridine.

33. The nucleic acid of claim 31 or 32, wherein the chemical modification is N1-methylpseudouridine.

34. The nucleic acid of any one of claims 1-33, wherein at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or 100% of the uridine is chemically modified to N1-methylpseudouridine.

35. The nucleic acid according to any one of claims 1 to 34, which is an mRNA, and the mRNA comprises a 5'-cap structure.

36. The nucleic acid according to any one of claims 1 to 35, wherein the 5'-cap structure is selected from m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7GpppN, m7GpppNmpNp, m7GpppNmpNmp; preferably m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

37. The nucleic acid of any one of claims 1-36, wherein the nucleic acid is formulated in a nanoparticle.

38. The nucleic acid of claim 37, wherein the nanoparticle is a lipid nanoparticle.

39. A vector or plasmid comprising a nucleic acid according to any one of the preceding claims.

40. A host cell comprising the nucleic acid according to any one of claims 1-38 and / or the vector or plasmid according to claim 39.

41. A pharmaceutical composition comprising the nucleic acid of any one of claims 1-38 and a delivery agent.

42. The pharmaceutical composition of claim 41, wherein the delivery agent is selected from the group consisting of liposomes, lipid nanoparticles, lipidoids, polymers, lipoplexes, microvesicles, exosomes, peptides, proteins, cells transfected with polynucleotides, hyaluronidase, nanoparticle mimics, nanotubes, conjugates, and combinations thereof.

43. A pharmaceutical composition according to claim 41 or 42, wherein the delivery agent is a lipid nanoparticle.

44. A pharmaceutical composition according to any one of claims 41-43, wherein the pharmaceutical composition is formulated as a lipid nanoparticle encapsulating the nucleic acid in a lipid shell.

45. A pharmaceutical composition according to any one of claims 41-44, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient.

46. ​​A nucleic acid according to any one of claims 1 to 38 or a pharmaceutical composition according to any one of claims 41 to 45 for use as a medicament.

47. A nucleic acid according to any one of claims 1-38 or a pharmaceutical composition according to any one of claims 41-45 for use in treating or preventing a rheumatic disease, a pulmonary disease, an ophthalmic disease, a renal disease or a neurological disease in a subject.

48. A nucleic acid according to any one of claims 1-38 or a pharmaceutical composition according to any one of claims 41-45 for treating or preventing gouty arthritis, rheumatoid arthritis, dermatomyositis / polymyositis, systemic lupus erythematosus, sarcoidosis or psoriatic arthritis in a subject.

49. A nucleic acid according to any one of claims 1-38 or a pharmaceutical composition according to any one of claims 41-45 for use in treating or preventing symptomatic pulmonary sarcoidosis in a subject.

50. The nucleic acid according to any one of claims 1-38 or the pharmaceutical composition according to any one of claims 41-45 for use in treating or preventing keratitis, uveitis or optic neuritis.

51. The nucleic acid according to any one of claims 1-38 or the pharmaceutical composition according to any one of claims 41-45 for use in the treatment or prevention of focal interphase glomerulosclerosis or primary immunoglobulin A nephropathy.

52. The nucleic acid according to any one of claims 1-38 or the pharmaceutical composition according to any one of claims 41-45 for use in treating or preventing multiple sclerosis, optic neuritis or infantile spasms.

53. A method for treating or preventing a rheumatic disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a nucleic acid according to any one of claims 1-38 or a pharmaceutical composition according to any one of claims 41-45.

54. The method of claim 53, wherein the subject is a human or a non-human mammal.

55. The method of claim 53 or 54, wherein the administration is via parenteral or enteral administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

56. The method of any one of claims 53-55, wherein the administration is about once a day, once every two days, twice a week, once a week, about once every two weeks, or about once a month.

57. The method of any one of claims 53-56, wherein the nucleic acid is expressed by cells in the subject.

58. The method of any one of claims 53-57, wherein the rheumatic disease comprises gouty arthritis, rheumatoid arthritis, dermatomyositis / polymyositis, systemic lupus erythematosus, sarcoidosis, or psoriatic arthritis.

59. A method for treating or preventing a lung disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a nucleic acid according to any one of claims 1-38 or a pharmaceutical composition according to any one of claims 41-45.

60. The method of claim 59, wherein the subject is a human or a non-human mammal.

61. The method of claim 59 or 60, wherein the administration is via parenteral or enteral administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

62. The method of any one of claims 59-61, wherein the administration is about once a day, once every two days, twice a week, once a week, about once every two weeks, or about once a month.

63. The method of any one of claims 59-62, wherein the nucleic acid is expressed by cells in the subject.

64. The method of any one of claims 59-63, wherein the lung disease comprises symptomatic pulmonary sarcoidosis.

65. A method for treating or preventing an ophthalmic disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a nucleic acid according to any one of claims 1-38 or a pharmaceutical composition according to any one of claims 41-45.

66. The method of claim 65, wherein the subject is a human or a non-human mammal.

67. The method of claim 65 or 66, wherein the administration is via parenteral or enteral administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

68. The method of any one of claims 65-67, wherein the administration is about once a day, once every two days, twice a week, once a week, about once every two weeks, or about once a month.

69. The method of any one of claims 65-68, wherein the nucleic acid is expressed by cells in the subject.

70. The method of any one of claims 65-69, wherein the ophthalmic disease comprises keratitis, uveitis, or optic neuritis.

71. A method for treating or preventing a renal disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a nucleic acid according to any one of claims 1-38 or a pharmaceutical composition according to any one of claims 41-45.

72. The method of claim 71, wherein the subject is a human or a non-human mammal.

73. The method of claim 71 or 72, wherein the administration is via parenteral or enteral administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

74. The method of any one of claims 71-73, wherein the administration is about once a day, once every two days, twice a week, once a week, about once every two weeks, or about once a month.

75. The method of any one of claims 71-74, wherein the nucleic acid is expressed by cells in the subject.

76. The method of any one of claims 71-75, wherein the renal disease comprises focal segmental glomerulosclerosis or primary immunoglobulin A nephropathy.

77. A method for treating or preventing a neurological disease in a subject, comprising administering to the subject a therapeutically or prophylactically effective amount of a nucleic acid according to any one of claims 1-38 or a pharmaceutical composition according to any one of claims 41-45.

78. The method of claim 77, wherein the subject is a human or a non-human mammal.

79. The method of claim 77 or 78, wherein the administration is via parenteral or enteral administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

80. The method of any one of claims 77-79, wherein the administration is about once a day, once every two days, twice a week, once a week, about once every two weeks, or about once a month.

81. The method of any one of claims 77-80, wherein the nucleic acid is expressed by cells in the subject.

82. The method of any one of claims 77-81, wherein the neurological disease comprises multiple sclerosis, optic neuritis, or infantile spasms.

83. A kit comprising a nucleic acid according to any one of claims 1-38 and / or a pharmaceutical composition according to any one of claims 41-45, and instructions for use.

84. A method for enhancing the expression of an open reading frame (ORF) encoding an adrenocorticotropic hormone fusion protein in a nucleic acid, wherein the adrenocorticotropic hormone fusion protein is a fusion protein of a signal peptide and adrenocorticotropic hormone, and the signal peptide is fused to the N-terminus of the adrenocorticotropic hormone, the method comprising connecting the C-terminus of the adrenocorticotropic hormone fusion protein in the ORF to a kappa light chain variable region (VLk) sequence via a linker.

85. The method of claim 84, wherein the amino acid sequence encoded by the ORF comprises a signal peptide, adrenocorticotropic hormone, a linker, and a kappa light chain variable region (VLk) sequence from the N-terminus to the C-terminus.

86. The method according to claim 84 or 85, wherein the ORF comprises, from 5' to 3', a nucleotide sequence encoding a signal peptide, a nucleotide sequence encoding adrenocorticotropic hormone, a nucleotide sequence encoding a linker, and a nucleotide sequence encoding a kappa light chain variable region (VLk) sequence.

87. The method of any one of claims 84-86, wherein the nucleotide sequence encoding the signal peptide comprises any one of SEQ ID NOs: 343-370 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 343-370 or a DNA sequence corresponding thereto.

88. The method of any one of claims 84-87, wherein the adrenocorticotropic hormone encoding nucleotide sequence comprises any one of SEQ ID NOs: 108-167 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 108-167 or a DNA sequence corresponding thereto.

89. The method of any one of claims 84-88, wherein the encoding nucleotide sequence of the linker comprises SEQ ID NO: 169 or its corresponding DNA sequence or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 169 or its corresponding DNA sequence.

90. The method of any one of claims 84-89, wherein the nucleotide sequence encoding the kappa light chain variable region (VLk) sequence comprises SEQ ID NO: 168 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 168 or a DNA sequence corresponding thereto.

91. The method of any one of claims 84-90, wherein the nucleic acid further comprises a 5'-untranslated region (5'-UTR).

92. The method of claim 91, wherein the 5'-UTR comprises a DNA sequence of SEQ ID NO:62-82 or its corresponding sequence, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a DNA sequence of SEQ ID NO:62-82 or its corresponding sequence.

93. The method of any one of claims 84-92, wherein the nucleic acid further comprises a 3'-untranslated region (3'-UTR).

94. The method of claim 93, wherein the 3'-UTR comprises SEQ ID NO:83-101 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO:83-101 or a DNA sequence corresponding thereto.

95. The method of any one of claims 84-94, wherein the nucleic acid comprises, in order from 5' to 3': a 5'-UTR, an ORF encoding an amino acid sequence comprising a signal peptide and adrenocorticotropic hormone or an amino acid sequence comprising a signal peptide, adrenocorticotropic hormone, a linker and a kappa light chain variable region (VLk) sequence, and a 3'-UTR.

96. The method of claim 95, wherein the 5'-UTR is selected from any one of SEQ ID NOs: 62-82 or their corresponding DNA sequences, and the 3'-UTR is selected from any one of SEQ ID NOs: SEQ ID NOs: 83-101 or their corresponding DNA sequences.

97. The method of any one of claims 84-96, wherein the nucleic acid further comprises a 3'-poly(A) sequence.

98. The method according to claim 97, wherein the 3'-poly (A) sequence comprises at least 10-400 adenosine nucleotides, preferably 50 to 400 adenosine nucleotides or 10 to 300 adenosine nucleotides; more preferably 50 to 250 adenosine nucleotides; most preferably 120 adenosine nucleotides; Alternatively, the 3'-poly(A) sequence comprises an adenosine polynucleotide and a linker sequence inserted therein, wherein the linker sequence is used to separate the adenine nucleotide sequence.

99. The method of any one of claims 84-98, wherein the nucleic acid comprises any one of SEQ ID NOs: 170-342 or a DNA sequence corresponding thereto, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to any one of SEQ ID NOs: 170-342 or a DNA sequence corresponding thereto.

100. The method of any one of claims 84-99, wherein the nucleic acid is DNA or RNA.

101. The method of any one of claims 84-100, comprising at least one chemically modified nucleotide.

102. The method of claim 101, wherein the chemical modification is selected from one or more of 5-methylcytidine, 5-methoxyuridine, pseudouridine, N6-methyladenosine, or N1-methylpseudouridine.

103. The method of claim 101 or 102, wherein the chemical modification is N1-methylpseudouridine.

104. The method of any one of claims 84-103, wherein at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99% or 100% of the uridine is chemically modified to N1-methylpseudouridine.

105. The method according to any one of claims 84-104, which is mRNA, and the mRNA comprises a 5'-cap structure.

106. The method according to any one of claims 84-105, wherein the 5'-cap structure is selected from m7(3'OMeG)(5')ppp(5')(2'OMeA)pG, 3'-O-Me-m7G(5')ppp(5')G, m7G(5')ppp(5')(2'OMeA)pG, m7GpppN, m7GpppNmpNp, m7GpppNmpNmp; preferably m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.

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