WO2025021183A1 - Polynucleotide encoding adrenocorticotropic hormone, and related composition and method thereof - Google Patents

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

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

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 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) in subjects.

Background Art

ACTH (adrenocorticotropic hormone, ACTH) is a polypeptide hormone secreted by the adrenocorticotroph cells of the anterior pituitary gland. Its synthesis and secretion are promoted by CRH (corticotropin releasing hormone) secreted by the hypothalamus. The gene encoding ACTH is POMC (proopiomelanocortin), which is located on chromosome 2p23.3. After POMC is transcribed and translated, it initially generates a polypeptide of 241 amino acid residues. After a series of protease cleavage, a series of short peptide products including ACTH are finally generated. In addition to ACTH, products also include MSHs (melanocyte-stimulating hormones) and endorphin.

ACTH is 39 amino acid residues long, and its biological activity depends on the amino acid residues at positions 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 receptors on adrenal cortical cells, promoting the increase of intracellular cAMP levels and the 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.

Acthar Gel is an ACTH drug produced by Mallinckrodt Pharmaceuticals and approved by the FDA in 1952. Acthar Gel has a variety of indications, the following are some common indications:

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

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

Infantile Spinal Muscular Atrophy (Infantile Spinal Atrophy): Acthar Gel can be used to treat infantile Spinal Muscular Atrophy, helping to control epileptic seizures and improve the patient's quality of life.

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

In addition, Acthar Gel may also be used for other indications, such as systemic lupus erythematosus, idiopathic inflammatory myopathy, acute attack of gout, etc.

Recent preclinical studies have shown that ACTH in the treatment of acute gout attacks does not only inhibit inflammation by promoting the synthesis of steroid hormones such as cortisol in the adrenal cortex, because in the acute gout model of adrenalectomy in rats, ACTH can still alleviate the acute attack of gout. Its mechanism of action may be to inhibit the recruitment of inflammatory immune cells.

Disadvantages of ACTH drugs:

Cost: ACTH drugs are expensive treatment options, which makes them unaffordable for some patients. As of 2021, Acthar Gel costs up to $35,000 per vial (5 ml). Some indications require multiple doses, making them expensive.

Source restrictions: The active ingredient of Acthar Gel is natural ACTH extracted from the adrenal cortex of animals (usually pigs). After extraction, it needs to go through a series of preparation and processing steps to obtain a final product that meets the quality and purity requirements of the drug. These steps include filtration, concentration, virus inactivation, etc. to ensure the safety and quality of the product. The animal extraction source and complex production process severely limit the production capacity of Acthar Gel, and there are also risks such as large batch-to-batch differences and high immunogenicity.

Short half-life: Generally speaking, the half-life of ACTH is about 10 to 15 minutes. It is mainly metabolized and decomposed in the body and cleared by the liver and kidneys. In addition, the secretion of ACTH is also regulated by a negative feedback mechanism. When the level of cortisol in the body increases, it inhibits the secretion of ACTH. This also leads to the frequent administration frequency of Acthar Gel, which further affects the patient's compliance.

In short, ACTH is an important adrenal hormone that plays an important role in the regulation of hormone balance and multiple physiological functions. ACTH drugs play an important role in the treatment of gout, infantile spasms, and multiple sclerosis.

mRNA protein replacement therapy is a biotechnology based on mRNA technology. Its main function is to transmit the information encoded by a specific protein to human cells so that they can produce the corresponding protein. It has the following advantages:

1) Highly customized: The design and preparation of mRNA drugs can be customized according to specific therapeutic goals, so they are highly personalized. By adjusting the mRNA sequence and structure, efficient expression and synthesis of specific proteins can be achieved, thereby achieving precise therapeutic effects.

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

3) Broad indications: mRNA drugs have a wide range of potential indications. They can be used to treat a variety of diseases, including infectious diseases, cancer, genetic diseases, etc. By adjusting the coding information of mRNA, different therapeutic proteins can be produced to address a variety of different diseases.

4) Relatively short development time: Compared with traditional drug development, the development time of mRNA drugs may be relatively short. Once the coding information of the target protein is determined, the preparation and production of mRNA drugs can be a relatively rapid process, which helps to quickly respond to the challenges of emerging pathogens and diseases.

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

Summary of the invention

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

Specifically, the present disclosure relates to the following technical solutions:

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

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

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.

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 the N-terminus to the C-terminus.

In 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.

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.

In a further embodiment, 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.

In yet a further embodiment, the nucleic acid comprises, in order 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.

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

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

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

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.

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.

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.

In yet another embodiment, the present disclosure relates to a method for enhancing the 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 connecting the C-terminus of the corticotropin fusion protein in the ORF to a kappa light chain variable region (VLk) sequence via a linker.

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. According to the specification and claims, other features, objects and advantages of the present application will be apparent. In the specification and the appended claims, the singular form also includes the plural form, unless the context clearly provides otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as 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.

The contents of all references cited throughout this application (including literature references, granted 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 meanings commonly known to those of ordinary skill in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of the structure of the mRNA construct involved in the embodiments of the present invention.

Figure 2a-b shows 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.

Figure 3a-b shows the cytoplasmic protein WB detection results and grayscale integration results after HEK293T cells were transfected with ACTH mRNA constructs with different UTRs in Example 2.

Figure 4a-b shows the cytoplasmic protein WB detection results and grayscale integration results after HEK293 cells were transfected with ACTH mRNA constructs with different cap structures, modified bases and 3'-poly A tail sequences in Example 2.

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

Figure 6 shows the results of intracellular ACTH expression 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 NO: 225-228, the mRNA construct shown in SEQ ID NO: 226 has the highest amount in the cell supernatant.

Figure 7a shows the results of ACTH blood levels in healthy mice at 0-24 hours after intravenous injection of the mRNA constructs shown in SEQ ID NO: 229 and 230 in Example 5 in the form of LNP.

Figure 7b shows the changes in ACTH blood levels in healthy mice from 0 to 396 hours after intravenous injection of the mRNA constructs shown in SEQ ID NO: 231 and 232 in Example 5 in the form of LNP.

Figure 7c shows the results of ACTH blood levels in healthy mice at 0-396 hours after intravenous injection of the mRNA construct shown in SEQ ID NO:230 in Example 5 in the form of LNP.

Figures 8a-k respectively show the results of joint swelling in rats with gout inflammation model induced by intramuscular and subcutaneous injection of the mRNA construct shown in SEQ ID NO: 230 in Example 6 in the form of LNPs with MSU (monosodium urate).

Figure 9a-b shows the changes in the levels of inflammatory factors IL-1beta and IL-6 in rats with MSU-induced gout inflammation model injected intramuscularly and subcutaneously with the mRNA construct shown in SEQ ID NO:230 in Example 6 in the form of LNP.

Figure 10 shows the results of blood ACTH levels in rats with MSU-induced gout inflammation model injected intramuscularly and subcutaneously with the mRNA construct shown in SEQ ID NO: 230 in Example 6 in the form of LNP.

Figure 11a shows the expression level results of ACTH in the cell supernatant of HEK293T cells transfected with the mRNA constructs shown in SEQ ID NO:233-241 and the mRNA construct shown in SEQ ID NO:230 using different secretion signal peptides in Example 7.

Figure 11b shows the expression level of ACTH in the cell supernatant of HepG2 cells transfected with the mRNA constructs shown in 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.

Figure 11c shows the expression level results of ACTH in the cell supernatant of HEK293T cells transfected with the mRNA constructs shown in SEQ ID NO:253-266 and the mRNA construct shown in SEQ ID NO:237 using different secretion signal peptides in Example 7.

Figure 12 shows the expression level of ACTH in the cell supernatant of HepG2 cells transfected with the mRNA construct shown in SEQ ID NO:237 in Example 8 using different LNP preparations.

Figure 13 shows the expression level results of ACTH in the cell supernatant of HepG2 hepatocytes transfected with the mRNA constructs shown in SEQ ID NO: 269-285 and SEQ ID NO: 237 in Example 10.

Figure 14 shows the expression level results of ACTH in the cell supernatant of HepG2 hepatocytes transfected with the mRNA constructs shown in SEQ ID NO: 286-298 and the mRNA construct shown in SEQ ID NO: 237 in Example 11.

Figure 15 shows the PK results of the mRNA constructs shown in SEQ ID NO: 230 and 237 in Example 9 injected intravenously into rats in the form of LNP.

FIG. 16 is an anatomical diagram of the skin hardening and swelling after subcutaneous injection of the mRNA-LNP drug in Example 12.

FIG. 17 is a rat stability PK experiment of the highly secretory expression signal peptide construct in Example 13

Figure 18a-d shows the joint swelling in the efficacy experiment of the construct SEQ ID NO: 289 in Example 14 in the MSU-induced gout inflammation model

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

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

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

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

Figures 19a-f show the ACTH expression levels of ACTH mRNA and ACTH-VLK mRNA in the supernatant of HepG2 cells in Example 15;

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

FIG21a shows the clinical scores of each drug administration group in the EAU model experiment in Example 17;

Figure 21b is a significance analysis of each drug administration group at the time point of day 11 in the EAU model in Example 17;

FIG21c is a photograph of the eyeballs of animals in each drug-dosing group in the EAU model experiment in Example 17;

FIG22a shows the clinical scores of each drug administration group in the AIA model experiment in Example 18;

Figure 22b-c shows the joint swelling and paw swelling of each drug-dosing group in the AIA model experiment in Example 18;

FIG. 22d shows the body weight changes of each dosing group in the AIA model in Example 18.

DETAILED DESCRIPTION

Provided herein are nucleic acid molecules comprising an open reading frame (ORF) encoding adrenocorticotropic hormone, its functional fragment or variant. 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).

1. General Technology

The techniques and procedures described or referenced herein include those that are generally well understood and/or commonly employed by technicians in the field 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).

2. Terminology

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 term description will apply, 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 term description set forth below shall prevail.

As used herein and unless otherwise indicated, the term "lipid" refers to a group of organic compounds, including but not limited to fatty acid esters, and is generally characterized by being poorly soluble in water but soluble in many non-polar organic solvents. Although lipids are generally weakly water-soluble, 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" is also referred to as "lipid-like"; it refers to a lipid-like compound (e.g., an amphiphilic compound with lipid-like physical properties).

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 may 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 cationic lipids can interact with negatively charged payload molecules and promote the incorporation and/or encapsulation of payloads into the LNP during LNP formation. Other lipids that can form a part of 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.

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 "cation" encompasses "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).

The term "polymer-conjugated lipid" refers to a molecule comprising 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.

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 hydrogen phosphate (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.

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, 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.

As used herein, "lipoplex (LPX)" generally refers to a complex formed spontaneously by lipids (cationic lipids), such as a complex formed by a cationic lipid and a negative nucleic acid (such as mRNA).

As used herein, the terms "signal peptide" and "signal sequence" can be 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, the reference to a signal sequence in the context of a 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-terminal end of a protein is usually required for transmembrane translocation on a secretory pathway, and therefore, generally controls most proteins in eukaryotes and prokaryotes to enter the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (preprotein) guides the ribosome to the rough endoplasmic reticulum (ER) membrane, and the peptide chain growing vigorously 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 (it regulates the expression of gene except adrenocorticotropic hormone) is known in the art, and its desired property can be tested, and then incorporated into nucleic acid of the present invention.

As used herein, the term "mRNA", also known as messenger RNA, generally refers to a type of single-stranded ribonucleic acid that is transcribed from a DNA strand as a template, carries genetic information, and guides protein synthesis. Once DNA is used as a template, mRNA is transcribed according to the Watson-Crick base complementary pairing principle, and the mRNA contains a base sequence corresponding to certain functional fragments in the DNA molecule to serve as a direct template for protein biosynthesis. As used herein, mRNA contains a nucleic acid molecule that can encode adrenocorticotropic hormone, that is, at least a portion of the mRNA can encode adrenocorticotropic hormone.

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 for acceptable use in humans or livestock.

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

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 may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and/or analogs thereof, 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 analogs thereof. Nucleic acids may be in single-stranded or double-stranded form. As used herein and unless otherwise indicated, "nucleic acid" also includes nucleic acid mimetics, such as locked nucleic acids (LNA), peptide nucleic acids (PNA) and morpholino nucleic acids. As used herein, "oligonucleotide" refers to a short synthetic polynucleotide, which is 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”.

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. A "separated" 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, a "separated" nucleic acid molecule, such as an mRNA molecule, may be substantially free of other cell materials or culture medium, or when chemically synthesized, it may 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 that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA or RNA isolates and chemically synthesized analogs or analogs synthesized by heterologous systems. Substantially pure molecules may include isolated forms of molecules.

The term "coding nucleic acid" or its grammatical equivalents, when used to refer to nucleic acid molecules, includes (a) nucleic acid molecules that can be transcribed to produce mRNA and subsequently translated into peptides and/or polypeptides when in a natural state or manipulated by methods well known to those skilled in the art; and (b) mRNA molecules themselves. The antisense strand is the complementary sequence of such nucleic acid molecules, 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 called a 5'-UTR if it is located at the 5' end of the coding region, and the UTR is called a 3'-UTR if it is located at the 3' end of the coding region.

The term "corresponding DNA sequence" or its grammatical equivalent when used in reference 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.

As used herein, "open reading frame (ORF)" is a nucleotide sequence that starts 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 sequences and RNA sequences.

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 by using 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 an open reading frame (ORF) with a 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 frequency in the target and host organisms to ensure correct folding; bias GC content to increase mRNA stability or reduce secondary structure; minimize tandem repeat sequence codons or base runs that may impair gene construction or expression; customize transcription and translation control regions; insert or remove protein transport 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 the translation rate to allow the various structures of the protein to fold correctly; or reduce or eliminate problematic secondary structures within polynucleotides. Codon optimization tools, algorithms, and services are known in the art. In some embodiments, ORF sequences are optimized using optimization algorithms.

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 peptides 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).

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.

As used herein, the term "functional nucleotide analogue" refers to a modified form of a classical nucleotide A, G, C, U or T, which (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 not only encompasses the classical Watson-Crick adenine-thymine, adenine-uracil or guanine-cytosine base pairs, but also encompasses 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 a modified nucleobase and a classical nucleobase or between two complementary modified nucleobase structures. For example, a functional analogue of guanosine (G) retains the ability to base pair with a functional analogue of cytosine (C) or cytosine. An example of such non-classical base pairing is base pairing between modified nucleotides inosine and adenine, cytosine or uracil. As described herein, functional nucleotide analogs can be naturally occurring or non-naturally occurring. Therefore, nucleic acid molecules containing functional nucleotide analogs can have at least one modified nucleobase, sugar group and/or internucleoside bond. Chemical modifications to the nucleobase, sugar group or internucleoside bond of nucleic acid molecules are provided herein.

The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers having more than fifty (50) amino acid residues linked by covalent peptide bonds. That is, the description of polypeptides applies equally to the description of proteins, 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., amino acid analogs). As used herein, the terms encompass amino acid chains of any length, including full-length proteins (e.g., adrenocorticotropic hormone).

The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules determined by sequence alignment, or the sequences of two or more nucleic acid molecules." Amino acid sequence identity percentage (%)" is defined as the percentage of the amino acid residues identical in the candidate sequence and the reference polypeptide sequence and the reference sequence amino acid residues after sequence alignment and introduction of a gap factor relative to the reference polypeptide sequence. If necessary, the maximum sequence identity percentage can be achieved and any conservative substitution is not considered as part of the sequence identity. The comparison for determining the amino acid sequence identity percentage can be achieved in a variety of ways within the technical scope of the art, such as 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 sequence.

As used herein, "modification" of an amino acid residue/position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, wherein the change 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., usually less than 5, 4 or 3) amino acids/positions near the residue, and/or deleting the residue/position.

The term "vector" refers to a material for carrying or including a nucleic acid sequence, including, for example, a nucleic acid sequence encoding the ACTH described herein, so that the nucleic acid sequence is introduced into a host cell, or used as a transcription template to perform an in vitro transcription reaction 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 a host cell chromosome. In addition, the vector may include one or more selective marker genes and appropriate transcription or translation control sequences. The included selective marker genes may, for example, provide resistance to antibiotics or toxins, supplement nutrient deficiency nutrients, 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, etc., which 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: using 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 (such as the mRNA transcript of the nucleic acid described herein), and it will also be understood that the expression level can be optimized by methods well known in the art to obtain sufficient expression products.

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 mucosal, 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.

"Long-term" administration, as opposed to an 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 a longer period of time. "Intermittent" administration refers to treatment that is not continuous without interruption, but rather is cyclical in nature.

As used herein, the term "targeted delivery" or verb form "targeting" refers to a process in which the delivered agent (such as therapeutic nucleic acids such as mRNA in lipid nanoparticle compositions as described herein) reaches 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, such as 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.

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.

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 a pharmaceutical composition described herein) sufficient to reduce and/or improve the severity and/or duration of a given disease, disorder or condition, and/or its associated symptoms. The "therapeutically effective amount" of an agent of the present disclosure (e.g., a lipid nanoparticle composition described herein) may vary according to many 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 includes an amount in which the therapeutically beneficial effects of the agent outweigh any toxic or deleterious effects thereof. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a lipid nanoparticle composition as described herein or a therapeutic agent or prophylactic agent (e.g., therapeutic mRNA) contained therein that is effective to "treat" a disease, disorder, or condition of a subject or mammal.

A "prophylactically effective amount" is an amount of a pharmaceutical composition that, when administered to a subject, will have the intended preventive 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, since a preventive dose is used for a subject before a disease, disorder, or condition, or at its early stages, the preventive effective amount may be less than the therapeutically effective amount. A complete therapeutic or preventive effect may not necessarily occur by administering a single dose, but may occur only after a series of doses are administered. Therefore, a therapeutically or prophylactically effective amount may be administered in one or more administrations.

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 disease (including but not limited to gout inflammation), lung disease, ophthalmic disease, kidney disease, or neurological disease (including but not limited to infantile spasms, multiple sclerosis), or symptoms caused thereby.

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.

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.

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 "therapy" refers to biological therapy, supportive therapy and/or other therapy known to a person skilled in the art, such as a medical professional, that can be used to prevent, manage, treat and/or improve a disease, disorder or condition.

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 gout inflammation), a lung disease, an ophthalmic disease, a kidney disease, or a neurological disease (including but not limited to infantile spasms, multiple sclerosis).

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%.

As used herein and unless otherwise indicated, the term "about" or "approximately" means an acceptable error for a particular value determined by a person 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.

As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

All publications, patent applications and other references cited in this specification are incorporated herein by reference in their entirety, to the extent that each individual publication or patent application is specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided only as of the date of filing of the present application. Nothing herein should be construed as an admission that the present invention is not entitled to predate such publications by virtue of prior invention. In addition, the publication date provided may differ from the actual publication date, which may require independent confirmation.

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 rather than limit the scope of the invention described in the claims.

3. Therapeutic nucleic acids

3.1 Open Reading Frame (ORF)

In one aspect, provided herein are therapeutic nucleic acid molecules 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 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, and the therapeutic nucleic acid 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.

In some embodiments, the ACTH, its functional fragment or variant encoded by the mRNA can be of any size and can have any secondary structure or activity. In some embodiments, the ACTH, its functional fragment or variant encoded by the mRNA payload can have a therapeutic effect when expressed in a cell.

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 also 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 5'-UTR. In specific embodiments, the untranslated region (UTR) is located downstream (3' end) of the coding region and is referred to herein as 3'-UTR. In some embodiments, the nucleic acid molecules include both 5'-UTR and 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 the 5'-UTR). In some embodiments, the nucleic acid molecules include a poly-A region (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecules include a polyadenylation signal (e.g., in the 3'-UTR). In some embodiments, the nucleic acid molecules include a stabilizing region (e.g., in the 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.

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

As used herein, adrenocorticotropic hormone (ACTH) is a polypeptide hormone secreted by the pituitary gland, which has the function of stimulating adrenal cortex hyperplasia and promoting the synthesis and secretion of adrenal cortical hormones.

In some embodiments, the ACTH may be selected from vertebrates of different species, preferably from humans.

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

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

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

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

In some embodiments, the ACTH 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.

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

In some embodiments, the ACTH fusion protein comprises the amino acid sequence shown in any one of SEQ ID NO:2-61 or an amino acid sequence that is at least 90%, 95%, 98% or 99% identical to any one of SEQ ID NO:2-61.

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.

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.

In some embodiments, 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.

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.

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 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 replacing the codon containing A and/or U nucleotides by other codons (not containing A and/or U or containing A and/or U nucleotides with lower content) is modified.

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

In some embodiments, 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.

In some embodiments, the ORF encodes an amino acid sequence as shown in any one of SEQ ID NO: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 NO:2-61.

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

In some embodiments, the encoding nucleotide sequence of the signal peptide comprises any one of SEQ ID NO: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 NO:343-370 or its corresponding DNA sequence.

In some embodiments, the coding nucleotide sequence of adrenocorticotropic hormone comprises any one of SEQ ID NO: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 NO:108-167 or its corresponding DNA sequence.

In some embodiments, 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.

In some embodiments, the encoding nucleotide sequence of 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.

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

3.2 5'-cap structure

Without being bound by theory, it is expected that the 5'-cap structure of the polynucleotide is involved 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 via the association of CBP with poly-A binding protein to form mature circular mRNA species. 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.

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. Subsequently, this 5'-guanylate cap can 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.

In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more changes to a native 5'-cap structure produced by an endogenous process. Without being bound by theory, modifications to the 5'-cap may increase the stability of the polynucleotide, increase the half-life of the polynucleotide, and may increase the translation efficiency of the polynucleotide.

Exemplary changes to the native 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, 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 may be used, such as α-methylphosphonic acid and selenophosphate nucleotides.

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 (producing 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.

Additional exemplary changes to the native 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 nucleotide before the 5′-terminus (as described above). A plurality of different 5′-cap structures can be used to produce a 5′-cap for a polynucleotide (e.g., an mRNA molecule). Additional exemplary 5′-cap structures that may be used in conjunction with the present disclosure further include those 5′-cap structures described in International Patent Publications WO2008127688, WO 2008016473, and WO 2011015347, the entire contents of which are incorporated herein by reference.

In various embodiments, the 5'-terminal cap may include 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, which differ in chemical structure from a natural (i.e., endogenous, wild-type or physiological) 5'-cap while retaining cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or attached to a polynucleotide.

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).

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. Pat. No. 8,519,110, the entire contents of which are incorporated herein by reference in their entirety.

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.

In various embodiments, the cap analog may include 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.

Without being bound by theory, it is expected that although cap analogs allow 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 reduced cellular stability.

Therefore, in some embodiments, the nucleic acid molecules of the present disclosure may also be capped after transcription using an enzyme 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 cell 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 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 causes 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).

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.

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

Without being bound by theory, it is contemplated that the nucleic acid molecules of the present disclosure can be capped after transcription, and because this method is relatively efficient, nearly 100% of the nucleic acid molecules can be capped.

3.3 Untranslated Region (UTR)

In some embodiments, the nucleic acid molecules of the present disclosure include 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 the 5′-UTR. In some embodiments, the UTR is located downstream of the coding region in the nucleic acid molecule and is referred to as the 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 contain one or more different structural or chemical modifications independently before and/or after codon optimization.

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 may be administered in vitro (e.g., cells or tissue cultures) or in vivo (e.g., to a subject), and the effects of UTR sequences (e.g., regulating expression levels, cellular localization of the encoded product, or half-life of the encoded product) may be measured using methods known in the art.

In some embodiments, the nucleic acid molecules of the present disclosure comprise a 5′-UTR selected from SEQ ID NO:62-82 or a DNA sequence corresponding thereto. In some embodiments, the nucleic acid molecules of the present disclosure comprise a 3′-UTR selected from SEQ ID NO:83-101 or a DNA sequence corresponding thereto. In some embodiments, the nucleic acid molecules of the present disclosure comprise a 5′-UTR selected from SEQ ID NO:62-82 or a DNA sequence corresponding thereto and a 3′-UTR selected from SEQ ID NO:83-101 or a DNA sequence corresponding thereto. In specific embodiments, the nucleic acid molecules described herein may be RNA molecules transcribed in vitro.

3.4 Polyadenylation (Poly-A) region

During natural RNA processing, long chains of adenosine nucleotides (poly-A regions) are often added to messenger RNA (mRNA) molecules to increase the stability of the molecule. 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 is called polyadenylation, and a poly-A region of between 100 and 250 residues in length is added. Without being bound by theory, it is expected that the poly-A region can confer a number of advantages to the nucleic acid molecules of the present disclosure.

Therefore, in some embodiments, nucleic acid molecules (e.g., mRNA) of the present disclosure comprise polyadenylation signals. In some embodiments, 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.

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 length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 80 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 85 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 90 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 95 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 100 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 110 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 120 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 130 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 140 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 150 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 160 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 170 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 180 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 190 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 200 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 225 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 250 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 275 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 300 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 350 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 400 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 450 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 500 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 600 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 700 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 800 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 900 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is at least 1000 nucleotides. In some embodiments, the length of the poly-A region of the nucleic acid molecules of the present disclosure is 1000 to 3000 nucleotides.

In some embodiments, the length of the poly-A region in the 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 accounts for 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.

Without being bound by theory, it is expected that certain RNA binding proteins may bind to the poly-A region located at the 3' end of the mRNA molecule. These poly-A binding proteins (PABPs) may regulate mRNA expression, for example, interacting with the translation initiation machinery in the cell and/or protecting the 3'-poly-A tail from degradation. Therefore, 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, before the nucleic acid molecule is loaded into a delivery vehicle (e.g., lipid nanoparticle), it is formed into a conjugate or complex with PABP. Typically, poly-A (e.g., 3'-poly A sequence) is a continuous sequence of adenosine nucleotides. In some embodiments, poly-A (e.g., 3'-poly A sequence) comprises an adenosine polynucleotide and a linker sequence inserted therein, the linker sequence being used to separate the adenine nucleotide sequence, i.e., the adenosine polynucleotide is an interrupting sequence.

In some embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise poly-A-G quadruplexes. G quadruplexes are circular arrays 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 polynucleotides (e.g., mRNA) can be analyzed, including half-life at different time points. It has been found that the protein yield of the poly-A-G quadruplex structure is equal to at least 75% of the protein yield observed using only a poly-A region containing 120 nucleotides.

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-A-G quadruplex structure as described in International Patent Publication No. WO2013/103659, the contents of which are incorporated herein by reference in their entirety.

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.

In some embodiments, the nucleic acid molecules of the present disclosure comprise, from 5' to 3', in sequence: 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'-polyadenylic acid sequence.

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;

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.

In some embodiments, the nucleic acid comprises any one of SEQ ID NO:170-342 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 NO:170-342 or its corresponding DNA sequence.

3.5 Modification of Nucleobases

In some embodiments, the functional nucleotide analogs contain non-classical nucleobases. In some embodiments, the classical nucleobases in the 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 openings, oxidations, and/or reductions.

In some embodiments, the non-classical nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-ketoribonucleoside, 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-methylaminomethyluracil (mnm5U), 5-methylaminomethyl-2-thiouracil (mnm5s2U), 5-methylaminomethyl-2-selenouracil (mnm5se2U), 5-carbamoylmethyluracil (ncm5U), 5-carboxymethylaminomethyl-uracil (cmnm5U), 5-carboxymethylaminomethyl-2-thiouracil uracil (cmnm5s2U), 5-propynyl-uracil, 1-propynyl-pseudouridine, 5-taurine methyl-uracil (τm5U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thio-uracil (τm55s2U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uracil (m5U, i.e., with 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)uracil (m5U), methyl)-2-thio-uracil (m5s2U), 5,2'-O-dimethyl-uridine (m5Um), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m 3Um) and 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uracil, deoxythymidine, 5-(2-methoxycarbonylvinyl)-uracil, 5-(carbamoylhydroxymethyl)-uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil and 5-[3-(1-E-propenylamino)]uracil.

In a specific embodiment, the nucleic acid molecules of the present disclosure include modifications to uracil. In a specific embodiment, the nucleic acid molecules of the present disclosure include one or more pseudouracils (ψ). In a specific embodiment, 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 a specific embodiment, all (100%) uracils in the nucleic acid molecules of the present disclosure are replaced by pseudouracils (ψ).

In a specific embodiment, the nucleic acid molecules of the present disclosure comprise one or more 1-methyl pseudouracils (m1ψ). In a specific embodiment, 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 a specific embodiment, all (100%) uracils in the nucleic acid molecules of the present disclosure are substituted with 1-methyl pseudouracils (m1ψ).

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.

In some embodiments, the mRNA molecules used in conjunction with the present disclosure are biosynthesized using host cells. In a specific embodiment, mRNA is produced by using host cells to transcribe the corresponding DNA. In some embodiments, the DNA sequence encoding the mRNA sequence is integrated into an expression vector using methods known in the art, and then the vector is introduced into a host cell (e.g., E. coli). The host cell is then cultured under suitable conditions to produce mRNA transcripts. Other methods of 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 transcriptional machinery of a host cell can be used to produce mRNA transcripts.

3.6 Methods for preparing mRNA

The present invention also provides a method for preparing mRNA containing an open reading frame (ORF) encoding adrenocorticotropic hormone, its functional fragment or variant, 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, extracting the plasmid, and digesting the obtained plasmid with a restriction endonuclease to obtain a linearized DNA for in vitro transcription of mRNA; and 3) transcribing the linearized DNA in vitro to obtain the target mRNA.

In some embodiments, a DNA fragment for transcribing an mRNA containing an open reading frame (ORF) encoding adrenocorticotropic hormone, its functional fragment or variant is synthesized, and the DNA fragment is cloned into an expression plasmid to obtain a recombinant plasmid. In some embodiments, the DNA fragment, in addition to the DNA fragment corresponding to the mRNA encoding adrenocorticotropic hormone, 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 is not particularly limited to the method for synthesizing the DNA fragment corresponding to the above mRNA, and a conventional DNA synthesis method 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 above-mentioned specific restriction endonuclease recognition sequence (restriction endonuclease can be selected from BspQI, BsaI, BsmI, NotI, PvuI, etc.). 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.

After obtaining the recombinant plasmid, the recombinant plasmid is introduced into a host cell, amplified, and the plasmid is extracted. 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 the conventional introduction method in the art can be used. After obtaining the recombinant cell, positive recombinant cells are screened and colony sequencing is performed. In one embodiment, the screening of the positive recombinant cells is performed 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 3min; denaturation at 98°C for 10s, annealing at 60°C for 5s, extension at 72°C for 2min, 34 cycles; and finally extension at 72°C for 10min. After the PCR amplification is completed, the colony of the target band is 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 method for extracting the plasmid, and preferably a plasmid extraction kit is used. In some embodiments, the above-mentioned specific restriction endonuclease (restriction endonuclease can be selected from BspQI, BsaI, BsmI, NotI, etc.) is used for enzyme digestion to obtain a linearized DNA fragment of in vitro mRNA expression. In some embodiments, the enzyme digestion system is designed with 50μl, preferably as follows:

In some embodiments, after the end of the enzyme cleavage reaction, the amplified product is preferably subjected to agarose gel electrophoresis to determine whether the reaction is complete, and the agarose gel electrophoresis detection parameters are preferably as follows: 1% agarose, 5V/cm, 40min. In some embodiments, the reaction is considered to be complete when only one band appears in agarose gel electrophoresis. After the end of the enzyme cleavage reaction, the linearized DNA fragment is preferably purified. There is no special limitation on the purification method, and the conventional purification method in the art can be used. In the specific implementation process of the present invention, it is preferably carried out using a DNA purification kit. 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 to be qualified.

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:

In some embodiments, the Enzyme Mix includes T7 RNA polymerase, RNase inhibitor and inorganic pyrophosphatase. In some embodiments, the RNA in vitro synthesis is preferably reacted 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 order of water, nucleotides, cap analogs, transcription buffer, Enzyme Mix, and linearized DNA template.

After the in vitro transcription of the RNA is completed, it is preferred to verify whether the in vitro synthesis of the mRNA is successful by agarose gel electrophoresis, and 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 in 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 performed 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 preferred to perform a residual DNA fragment detection. In some embodiments, there is no limitation on the method for recovering and purifying the mRNA, and the conventional purification method in the art can be used. In the specific implementation of the present invention, it is preferred to use an RNA purification kit. After the purified mRNA is recovered, the mRNA quality test is performed, and the quality test includes the concentration of the mRNA, the ratio of OD260nm/OD280nm and OD260nm/OD230nm 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.

3.7 Lipid Nanoparticles (LNP)

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 avoid mRNA 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 cationic and/or ionizable lipids, anionic lipids, neutral lipids, amphipathic lipids, pegylated lipids and/or structural lipids, or a combination of the above. In certain embodiments, lipid nanoparticles include one or more mRNA described herein, for example, mRNA encoding a target protein (e.g., adrenocorticotropic hormone).

The delivery vehicle in the compositions described herein can be a nano lipid particle. The nano lipid particle can include one or more cations and/or ionizable lipids." Cationic lipids" generally refer to a net positive charge lipid carrying any number of lipids at a certain pH (such as physiological pH). The cationic lipids can include but are not limited to 3 (didodecylamino) N1, N1, 4 tridodecyl 1 piperazine ethylamine (KL10), N1 [2 (didodecylamino) ethyl] N1, N4, N4 tridodecyl 1, 4 piperazine diethylamine (KL22), 14, 25 tricosyl 15, 18, 21, 24 tetraaza octa-hole 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. In addition, many 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%.

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

In some embodiments, the non-cationic lipid may include a neutral lipid having a zero net charge at physiological pH. Neutral lipids suitable for 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 (DP ... Oleyl-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%.

In some embodiments, the nano lipid particle may include a lipid conjugate, and the lipid conjugate includes a lipid portion and a polymer portion, such as a PEGylated lipid comprising a lipid portion and a polyethylene glycol (PEG) portion. Lipid conjugates suitable for use in the present invention include dimyristoyl phosphatidylethanolamine-poly (ethylene glycol) 2000 (DMPE-PEG2000), DPPE-PEG2000, DMG-PEG2000, DPG-PEG2000, PEG2000-c-DOMG, PEG2000-c-DOPG, etc. The molecular weight of the poly (ethylene glycol) that can be used can range from about 500 to about 10,000Da, or from about 1,000 to about 5,000Da. The addition of these components can prevent lipid aggregation, and can also increase the circulation duration, and is easy to lipid-nucleic acid composition delivery to target cells, or rapidly release nucleic acid. In a specific embodiment, the nano lipid particle may include PEG2000-DMG. In some embodiments, the molar ratio of the lipid molecules modified by polyethylene glycol (PEG) in the lipid nanoparticles is about 0.5-2%. In a specific embodiment, the molar ratio of the lipid molecules modified by polyethylene glycol (PEG) in the lipid nanoparticles is about 1.5%.

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

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

3.8 Nanoparticle Composition

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 the enclosed nucleic acid molecules to intracellular compartments and/or mechanisms to exert expected preventive functions. In certain embodiments, when present in lipid nanoparticles, nucleic acid can resist the degradation of nucleases in aqueous solution. Lipid nanoparticles containing nucleic acids and methods for preparing them 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.

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

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.

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 the lipid delivery carrier (for example, lipid nano particle).In a specific embodiment, mRNA can be delivered to specific cells or tissues.For example, in order to target hepatocytes, the size of the lipid delivery carrier (for example, lipid nano particle) can be determined so that its size is smaller than the fenestration of the hepatic sinusoidal space of the endothelial lining in the liver, so that the lipid delivery carrier (for example, lipid nano particle) can easily penetrate these endothelial fenestrations to reach the target hepatocytes.Lipid delivery carrier (for example, lipid nano particle) can have a sufficiently large diameter to limit or obviously avoid being distributed in some cells or tissues.In some embodiments, the size (for example, diameter) of the lipid delivery carrier (for example, lipid nano particle) can be within 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.

Nanoparticle compositions that can be used in conjunction with the present disclosure include, for example, lipid nanoparticles (LNP), 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 aqueous compartments. The lipid bilayers can be functionalized and/or cross-linked to each other. The lipid bilayer can include one or more ligands, proteins, or channels.

3.9 Preparation

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

The lipid component of the nanoparticle composition may include ionizable lipids, phospholipids (eg, unsaturated lipids, such as DOPE or DSPC), PEG lipids, and structural lipids. The components of the lipid component may be provided in a specific ratio.

In one embodiment, a nanoparticle composition is provided herein, comprising a cationic or ionizable lipid compound, a therapeutic agent 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 in or associated with lipid nanoparticles.

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 mammals. The physicochemical properties of nanoparticle compositions can be changed 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, nanoparticle compositions can include mRNA encoding a polypeptide of interest, which 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.

The amount of the therapeutic and/or prophylactic agent in the nanoparticle composition may 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 may depend on the size, sequence and other characteristics of the RNA. The relative amounts of the therapeutic and/or prophylactic agent and other ingredients (e.g., lipids) in the nanoparticle composition may also vary. In some embodiments, the wt/wt ratio of the lipid component to the therapeutic and/or prophylactic agent in the nanoparticle composition may be 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).

In some embodiments, the nanoparticle composition comprises one or more RNAs, and 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 the number of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N: P ratio is selected. 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, such as 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 may 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 may be about 5.67:1.

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 of preparation of the nanoparticle composition.

Nanoparticle compositions can be characterized by a variety of methods. For example, the morphology and size distribution of nanoparticle compositions can be examined using microscopy (e.g., transmission electron microscopy or scanning electron microscopy). The 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 (Malvem Instruments Ltd, Malvem, Worcestershire, UK) can also be used to measure multiple characteristics of nanoparticle compositions, such as particle size, polydispersity index, and zeta potential.

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 about 40nm to about 150nm, such as about 40nm, 45nm, 50nm, 55nm, 60nm, 65nm, 70nm, 75nm, 80nm, 85nm, 90nm, 95nm, 100nm, 105nm, 110nm, 115nm, 120nm, 125nm, 130nm, 135nm, 140nm, 145nm or 150nm. In some embodiments, the average size of the nanoparticle composition can be about 50nm to about 100nm, about 50nm to about 90nm, about 50nm to about 80nm, about 50nm to about 70nm, about 50nm to about 60nm, about 60nm to about 100nm, about 60nm to about 90nm, about 60nm to about 80nm, about 60nm to about 70nm, about 70nm to about 100nm, about 70nm to about 90nm, about 70nm to about 80nm, about 80nm to about 100nm, about 80nm to about 90nm, or about 90nm to about 100nm. In certain embodiments, the average size of the nanoparticle composition can be about 70nm to about 100nm. In some embodiments, the average size can be about 80nm. In other embodiments, the average size can be about 100nm.

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 (e.g., less than 0.3) polydispersity index 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.

The zeta potential of a nanoparticle composition can be used to indicate the electrokinetic 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 materials with higher charges 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.

The encapsulation efficiency of therapeutic and/or prophylactic agents describes the amount of therapeutic and/or prophylactic agents encapsulated or otherwise associated with nanoparticle compositions after preparation relative to the initial amount provided. Encapsulation efficiency is expected to be high (e.g., close to 100%). Encapsulation efficiency can be measured, for example, by comparing the amount of therapeutic and/or prophylactic agents in a solution containing nanoparticle compositions before and after the nanoparticle compositions are destroyed with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic agents (e.g., RNA) in a solution. For nanoparticle compositions described herein, the encapsulation efficiency of therapeutic and/or prophylactic agents 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%.

The nanoparticle composition may optionally include one or more coatings. For example, the nanoparticle composition may be formulated into a capsule, film, or tablet having a coating. The capsule, film, or tablet containing the composition described herein may have any useful size, tensile strength, hardness, or density.

3.10 Pharmaceutical Compositions

According to the present disclosure, the nanoparticle composition can be formulated in whole or in part as a pharmaceutical composition. The pharmaceutical composition can include one or more nanoparticle compositions. For example, the pharmaceutical composition can include one or more nanoparticle compositions, and the one or more nanoparticle compositions include one or more different therapeutic agents and/or prophylactic agents. The pharmaceutical composition can further include one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents can be found in, for example, Remington’s The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional excipients and auxiliary ingredients can be used in any pharmaceutical composition, unless any conventional excipient or auxiliary ingredient is incompatible with one or more components of the nanoparticle composition. If the combination of the excipient or auxiliary ingredient with the components of the nanoparticle composition will result in any undesirable biological effect or other harmful effect, the excipient or auxiliary ingredient is incompatible with the components of the nanoparticle composition.

In some embodiments, the one or more excipients or auxiliary ingredients 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 auxiliary ingredients may constitute 50%, 60%, 70%, 80%, 90% or higher percentages of pharmaceutical practice. 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.

The relative amounts of 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 one or more nanoparticle compositions.

In some embodiments, nanoparticle compositions and/or pharmaceutical compositions of the present disclosure are stored and/or transported 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 the following: 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, the pharmaceutical compositions of the present disclosure comprise the nanoparticle compositions disclosed herein, Tris, saline, and sucrose, and have a pH of about 7.5-8, which is suitable for storage and/or transportation at, for example, about -20°C. For example, the pharmaceutical compositions of the present disclosure comprise the nanoparticle compositions disclosed herein and PBS, and have a pH of about 7-7.8, which is suitable for storage and/or transportation 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.

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 can benefit from the therapeutic effect provided by delivering therapeutic and/or prophylactic agents to one or more specific cells, tissues, organs or systems or groups thereof, such as the renal system. Although the description of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions provided herein is primarily directed to compositions suitable for administration to humans, it should be understood by those skilled in the art that such compositions are generally suitable for administration to any other mammal. Improvements to compositions suitable for administration to humans in order to make the compositions suitable for administration to various animals are well known, and veterinary pharmacologists with ordinary skills can design and/or perform such improvements only through ordinary experiments (if any). It is contemplated that subjects to whom the compositions are 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.

Pharmaceutical compositions comprising one or more nanoparticle compositions can be prepared by any method known or later developed in the art of pharmacology. In general, such preparation methods include 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 the desired single or multiple dosage units.

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 the subject and/or a convenient portion of such a dose, such as half or one-third of such a dose.

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.

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, isopropanol, 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.

Injectable preparations, such as sterile injectable aqueous or oily suspensions, may be prepared according to known techniques using suitable dispersants, wetting agents and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that may be used include water, Ringer’s solution, USP and isotonic sodium chloride solution. Sterile fixed oils are generally used as solvents or suspending media. For this purpose, any bland fixed oil may be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid may be used to prepare injections.

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 which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

3.11 Treatment Methods

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.

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 therapeutic or preventive 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.

In some embodiments, administration of the nucleic acid or pharmaceutical composition is via parenteral or enteral administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

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

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

In a further embodiment, the administration is intravenous.

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.

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

In yet 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.

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

In some embodiments, administration of the nucleic acid or pharmaceutical composition is via parenteral or enteral administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

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

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

In a further embodiment, the administration is intravenous.

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.

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

In yet 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.

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

In some embodiments, administration of the nucleic acid or pharmaceutical composition is via parenteral or enteral administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

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

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

In a further embodiment, the administration is intravenous.

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.

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

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.

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

In some embodiments, wherein the therapeutic nucleic acid or pharmaceutical composition is administered via parenteral administration or enteral administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery.

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.

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

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

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

4. Technical Effects of the Present Disclosure

Through creative work, the inventors have found that the nucleic acid therapeutic agent described herein can be delivered into the body to express ACTH in human cells, thereby treating gouty 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 and multiple sclerosis). Compared with ACTH protein drugs, the therapeutic nucleic acids described herein have the advantages of longer half-life, lower dosing frequency, better therapeutic effect, and lower immunogenicity.

[Corrected 12.08.2024 in accordance with Rule 26]
Sequence Listing

[Corrected 12.08.2024 in accordance with Rule 26]
Note: In SEQ ID NO:2-61, the sequence in bold on the N-terminal side is the signal peptide, and the sequence 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.

Example

The technical features involved in the different implementation schemes described in this article can be implemented in combination with each other.

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 embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the specification and claims of the present invention, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

When the embodiments give 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 can 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 grasp of the prior art by those skilled in the art and the record 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 can also be used to realize the present invention.

Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related conventional techniques in the field of the art. These techniques have been fully described in the existing literature, and specifically refer to 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, S an Diego；Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999；和METHODS IN MOLECULAR BIOLOGY, Vol.119, Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc. The reagents and raw materials purchased in the present invention are all commercially available.

Example 1: Preparation of mRNA constructs

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

Design of mRNA constructs

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

In this mRNA construct, the nucleotide sequence encoding adrenocorticotropic hormone (ACTH) was optimized in terms of sequence, species, tissue specificity and G/C content, and the optimized sequence is shown in SEQ ID NO: 108-167; and 5'-UTR (shown in SEQ ID NO: 62-82), 3'-UTR (shown in SEQ ID NO: 83-101) and Poly-A tail (shown in SEQ ID NO: 103-107) functional elements were introduced, and 173 mRNA sequences were designed (shown in SEQ ID NO: 170-342).

Preparation of mRNA constructs

The DNA fragment encoding the mRNA construct of adrenocorticotropic hormone (ACTH) is synthesized, and the mRNA is synthesized by in vitro transcription. Specifically:

Plasmid construction

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 a 5'-UTR at the 5' end, and comprises a 3'-UTR, a Poly-A sequence and a specific restriction endonuclease recognition sequence (a recognition sequence of BspQI) at the 3' end;

2) The synthesized DNA fragment and pUC-GW (purchased from Genewise) plasmid were then digested with PvuI (purchased from NEB, R3150) respectively. After the digestion, T4 ligase (Thermo Scientific, EL0011) was used to connect the digested target DNA fragment and pUC-GW plasmid in vitro to obtain the recombinant plasmid;

3) The recombinant plasmid obtained above was transferred into competent Escherichia coli and cultured on kan ⁺ solid medium for 12 h;

4) Then, a single colony on the kan ⁺ solid medium is selected for colony PCR, and a colony containing a target band in the colony PCR result is selected for sequencing;

5) Expand the colonies that have been sequenced correctly and use the omega D6915 Endo-free Plasmid Midi Kit to extract the plasmid.

6) Use Nanodrop to measure the concentration of the extracted plasmid, use PvuI restriction endonuclease to digest the correctly constructed plasmid, and verify it by agarose gel electrophoresis. If the qualified plasmid is constructed, the electrophoresis result should show two clear bands, one of which is the linearized plasmid after digestion and the other is the target product.

Preparation of linearized plasmid

The above plasmid was subjected to BspQI restriction enzyme digestion reaction to linearize the plasmid. The reaction system was:

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.

Linearized plasmid purification

The linearized plasmid was completely digested and purified using the Thermo Scientific, GeneJET PCR Purification Kit, #K0702 kit. The specific steps are as follows:

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

2) Pipette the solution in step 1), transfer it to the GeneJET purification column, centrifuge for 60 seconds, and discard the filtrate;

3) Add 700uL Wash Buffer (+ethanol) to the GeneJET purification column, centrifuge for 60s, and discard the filtrate;

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

5) Place the GeneJET purification column in a new 1.5mL centrifuge tube, add 50uL Elution Buffer, and centrifuge for 60s;

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

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

Linearized plasmid in vitro transcription (IVT)

During the in vitro transcription (IVT) linearized plasmid synthesis of mRNA, modified nucleotides are added to the reaction system in a certain proportion and randomly inserted into the mRNA sequence. The modified nucleotides include N1-Methylpseudo-UTP, Pseudo-UTP, 5-Methoxy-UTP, and 5-Methyl-CTP. Anti-reversal Cap analogs are used to cap the mRNA transcription. Cap analogs include Cap2 AG, Cap1 m6AG, Cap2 m6AG, and Cap1 AG.

Specifically:

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

2) Mix the components gently with a pipette, collect by brief centrifugation, and incubate at 37°C for 2-3 hours;

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

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

5) Removal of DNA template in IVT reaction system:

An appropriate amount of DNase I enzyme was added to the reaction system and 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, proving that the DNA template in the IVT product has been completely removed.

Purification of in vitro transcription (IVT) products:

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

1) Take 100uL RNA Cleanup Binding Buffer and add it to the above 50uL IVT product digested with DNase I;

2) Take 150uL of anhydrous ethanol and add it to the IVT product digested with DNase I, and mix it by gently pipetting;

3) Place the purification column in the collection tube, add no more than 900uL of sample each time, centrifuge for 1min, and discard the filtrate until all the solutions in all steps are bound to the purification column;

4) Add 500uL RNA Cleanup Wash Buffer (+ethanol) to the purification column, centrifuge for 1min, and discard the filtrate;

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

6) Transfer the purification column to a new RNase-free 1.5mL centrifuge tube, add 50-100uL Elution Buffer or nuclease-free water to the purification column, centrifuge for 1min, collect the filtrate, and store at -70℃ for later use;

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

Example 2: mRNA expression and analysis in cells

In order to determine the protein expression of the above mRNA construct, 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.

Specifically:

mRNA transfection cells

1) Culture the revived fresh HEK293T cells at 37°C and 5% _CO2 , and use them after subculturing to P2;

2) Use 24-well plates as cell plating: 7x10 ⁴ cells/well, complete medium (10% FBS, 1% double antibody) 500uL/well, 3 replicates for each condition, and continue culturing for 24 hours after plating;

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

a. RNAiMAX: 5ul + 50ul opti-MEM (OMEM, no additives in the culture medium), mix well and let stand for 5min;

b. mRNA MIX: 1μg mRNA (0.5μg/μl) + 50ul opti-MEM (OMEM, no additives in the culture medium) and mix well;

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

d. Prepare Lipo-mRNA MIX.

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

5) Add 105ul of the above Lipo-mRNA MIX to each well for cell transfection;

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

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

Protein expression Western Blotting/WB detection

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

1) After the above cytoplasmic samples were thawed and lysed, total protein was quantified using BCA;

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

3) Calculate the loading volume of 2ug total protein based on BCA concentration;

4) Load 2ug of lysate and 10ul of supernatant for SDS-PAGE protein electrophoresis;

5) After electrophoresis, use Iblot2 instrument to perform semi-dry transfer for 7 minutes;

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

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;

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

9) Use ECL to expose and take pictures in a protein imager.

analyze

This example evaluates the expression level 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 above mRNA.

1. Screening of the coding sequence of adrenocorticotropic hormone

Twenty mRNA constructs with different ACTH coding sequences (SEQ ID NO.170-SEQ ID NO.189) were constructed based on conventional mRNA functional elements (structure: 5'UTR-1+SP12+different ACTH+Flag+3'UTR-1+PolyA-1). The more preferred construct of ACTH coding sequence was determined based on the Flag tag expression level detection.

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), and 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 show the screening results of signal peptides.

2. Screening of functional element sequences

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

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, among which the corresponding 5'-UTR is shown in SEQ ID NO:62, and the 3-'UTR is shown in SEQ ID NO:83.

3. Cap structure, modified bases, PolyA tail sequence

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) is synthesized based on the mRNA construct shown in SEQ ID NO:190, and the specific sequence containing 4 poly-A tail sequences is shown in SEQ ID NO.210-SEQ ID NO.213.

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

5. Conclusion

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:

ACTH coding sequence: SEQ ID NO: 109, SEQ ID NO: 115, SEQ ID NO: 122, SEQ ID NO: 127;

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

ACTH mRNA capping type: Cap1 AG;

ACTH mRNA modified base: N1-Methylpseudo-UTP;

ACTH mRNA ending type: SEQ ID NO: 103;

Example 3. In vitro expression of constructs

Based on the above experiments, ACTH mRNA coding sequences and functional elements were screened, and 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.

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

1) Coating: Add 100uL of diluted Capture Antibody to a 96-well ELISA plate, seal the plate, and incubate at 4°C overnight;

2) Washing: Take 300uL Wash Buffer to wash the ELISA plate, wash 5 times in total;

3) Blocking: Add 250uL Blocking Buffer to each well and block for 1 hour at room temperature;

4) Wash the plate again: Take 300uL Wash Buffer to wash the ELISA plate, wash it 5 times in total;

5) Storage and use: Seal the prepared ELISA plate with sealing film and store at 4°C for future use;

6) Experimental analysis

Loading: Add 100uL sample/standard/QC sample to the coated ELISA plate, add 100uL Assay Buffer to the blank well, and seal the ELISA plate with sealing film;

Incubation: Place the ELISA plate on a Microplate Shaker, 400 rpm, RT, incubate for 2 h;

Wash the plate: Take 300uL Wash Buffer to wash the ELISA plate, wash 5 times in total;

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

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

Wash the plate: Take 300uL Wash Buffer to wash the ELISA plate, wash 5 times in total;

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

Wash the plate: Take 300uL Wash Buffer to wash the ELISA plate, wash 5 times in total;

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

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

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

Experimental result 1: First, we selected the construct SEQ ID NO: 225, and analyzed its expression changes in cells 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). As the content of transfected mRNA increased, the expression level of ACTH in cells also increased gradually. According to the experimental results, the final transfection amount of mRNA per 24-well cell was 1ug.

Experimental result 2: The mRNA constructs shown by SEQ ID NO:225-SEQ ID NO:228 were transfected with 1ug per well for in vitro expression analysis. The results of quantification of ACTH in the cell supernatant (Figure 6 right) showed that the mRNA construct shown by SEQ ID NO:226 had the highest expression level in the cell supernatant.

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

Example 4. LNP formulated mRNA constructs

Based on the in vitro cell experiment results of the mRNA constructs shown in SEQ ID NO: 225-SEQ ID NO: 228, mRNA constructs without Flag tags (as 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 mRNA. The specific preparation process is as follows:

1) Prepare the aqueous phase: prepare the mRNA working solution with 100 mM sodium acetate (pH 4.0) at a concentration of 0.35 mg/mL;

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

LNP Preparation 1:

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

LNP Preparation 2:

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

LNP Preparation 3:

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

3) Mix by rapid pipetting using the LNP preparation system at a ratio of 3:1 between aqueous phase and organic phase;

4) The LNP-mRNA is prepared by ultrafiltration, liquid exchange and concentration.

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

The concentration of LNP-mRNA was determined using the Qubit fluorescence method, and the encapsulation efficiency was calculated. The particle size, PDI and Zeta potential of LNP-mRNA were detected using a particle size analyzer. The results are as follows:

Example 5. Animal experiments on mRNA construction

In this example, the mRNA construct shown in SEQ ID NO:229-SEQ ID NO:232 was intravenously injected into mice in the form of LNP to study its expression in the animals.

Healthy mice aged 8-10 weeks were selected and raised under a standard feeding environment. One day before the start of drug administration, all animals were weighed and grouped using StudyDirectorTM (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). The "Matched distribution" random grouping method was selected for grouping to ensure that the average weight of each group was as close as possible to the average weight of 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 the abbreviation of 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 grouping and drug administration, routine tests were performed, including daily observation of the activity of experimental animals outside the cage, food and water intake, eyes, fur and other abnormalities. At the same time, venous blood was collected from mice for subsequent detection of ACTH concentration in the blood.

Experimental results: The above LNP-mRNA was injected intravenously into animals, and ACTH polypeptide was injected intravenously into animals as the positive drug group. The results are shown in Figures 7a-7c. The ACTH blood content of samples collected at different blood collection points was detected by ACTH ELISA detection kit. 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 can obviously detect an increase in blood ACTH content about 3-4 hours after administration, and it continues to be expressed beyond the background level until 12 hours. No obvious changes in ACTH blood content were detected for other mRNA constructs (constructs shown in SEQ ID NO: 229, SEQ ID NO: 231, and SEQ ID NO: 232) relative to the animal background level. By extending the animal feeding time, it was found that the SEQ ID NO: 230 construct still expressed ACTH above the animal blood background level at 52 hours, showing a more sustained blood concentration of mRNA in the animal (as shown in Figure 7c).

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

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

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

Establishment of MSU-induced gout inflammation model in rats

Healthy rats aged 8-10 weeks were selected and raised under a standard feeding environment. One day before the model was constructed, all animals were weighed and grouped using StudyDirectorTM (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). The "Matched distribution" random grouping method was selected for grouping to ensure that the average weight of each group was as close as possible to the average weight of other groups. The day of grouping was defined as day 0. On the first day after grouping (Day 1), 50uL MSU-PBS solution was injected into the right ankle joint of the rat (the injection dose was 1.5mg MSU per rat), and 50uL 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. At the same time, routine tests were performed, including daily observation of the activity of the experimental animals outside the cage, food and water intake, eyes, fur and other abnormalities. The quality of the establishment of the acute gout inflammation model was evaluated by measuring the swelling of the rat joints.

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

Dosage regimen for rat gout inflammation model:

In the gout inflammation model experiment, preventive administration of 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) 0.5 hours before the start of model construction (ACTH: 0.5 mpk; mRNA: 2 mpk), and 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 (8h for measuring swelling, 7h for collecting blood), and 18h.

Swelling index

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 obvious relief of animal joint swelling compared with 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.

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 through subcutaneous administration, which verifies the effect of SEQ ID NO: 230 construct mRNA on the treatment of gout inflammation.

Blood inflammatory factor indicators

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. 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.

Experimental conclusion: In the gout inflammation model, the mRNA construct shown in SEQ ID NO:230 exhibited significant inflammation inhibition effect, indicating that it has great potential in the treatment of acute gout.

Blood drug concentration (blood ACTH level) test

Experimental results: An animal gout inflammation model was constructed by injecting MSU into the rat joint cavity, 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 10a-b, compared with the modeling group (G3, PBS injection), the mRNA construct administration group shown in SEQ ID NO: 230 (G5 or G8) showed a significant increase in the animal blood ACTH content, and its blood content increased by more than 80%.

Experimental conclusion: The mRNA construct shown in SEQ ID NO: 230 was administered to the gout inflammation model in the form of mRNA-LNP, and the increase in the ACTH content expressed by its translation was clearly observed. This is consistent with the results of inflammatory factors and rat joint swelling. It shows that the mRNA construct shown in SEQ ID NO: 230 can play a role in reducing inflammation in the gout inflammation model.

Example 7 Screening of highly secretory expression signal peptides

According to the results of the above examples, the mRNA construct shown in SEQ ID NO: 230 can show obvious ACTH translation and expression in in vitro cell experiments and in vivo animal PK. At the same time, in the gout inflammation model, the great potential of mRNA-LNP in the treatment of acute gout disease 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 of human administration, thereby reducing the side effects of LNP liposome components. Therefore, a series of designs and screenings were made to improve the translation, secretion and expression of mRNA.

In this embodiment, the inventor screened 21 signal peptides (such as the signal peptides in the sequence of SEQ ID NO: 14-SEQ ID NO: 34, wherein the coding nucleotide sequences corresponding to some signal peptides are SEQ ID NO: 343-SEQ ID NO: 351) and randomly combined with the structure of the mRNA construct shown in SEQ ID NO: 230, and then optimized the sequence again 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 by means of an ACTH ELISA detection kit to verify their effects in increasing the secretion and expression of ACTH.

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 nearly 50 times higher than that of the mRNA shown in SEQ ID NO: 230. It was also found that the mRNA constructs shown in constructs 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 of HepG2 human hepatocytes, the mRNA construct shown in SEQ ID NO: 237, which was highly secreted and expressed in HEK293T cells, still had a relatively high cellular secretion expression in HepG2 cells, showing the strong expression ability of the mRNA construct shown in SEQ ID NO: 237 in in vitro experiments. As shown in Figure 11c, in another group of constructs screened by 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, and its expression level was more than one order of magnitude higher than that of other constructs.

Experimental conclusion: By performing expression tests on the constructs composed of these 17 signal peptides, we successfully screened out a high secretion expression construct SEQ ID NO: 237, which has a secretion expression level nearly 50 times higher than the previous mRNA construct shown in SEQ ID NO: 230.

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

The highly secretory expression construct SEQ ID NO: 237 designed in Example 7 was prepared with a liposome preparation (cationic lipids DLin-MC3-DMA, SM102, ALC-0315, refer to the formula shown in Example 4) for LNP formulation, and human hepatocytes HepG2 were transfected. At the same time, a SEQ ID NO: 237 construct transfection reagent group (Thermo RNAiMAX kit) was set up as a blank control.

Experimental results: As shown in Figure 12, construct SEQ ID NO:237 has similar expression levels in cationic lipid MC3 and SM102 liposome preparations, and is significantly higher than that in ALC-0315 liposome preparations.

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 MC3 cationic lipid formula to prepare mRNA-LNP liposomes.

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

Rat PK experiment:

Based on the data at the in vitro experimental level, in order to verify the pharmacokinetics of the construct SEQ ID NO: 237 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 preparation (refer to Example 4 for the formula), and the rats were administered by intravenous injection, and the ACTH content in the blood of the animals 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 a total lipid concentration of 36 mg/mL for LNP encapsulation and a mRNA concentration of 0.3 ug/uL, and the detection results of mRNA-LNP are shown in the following table:

By quantifying ACTH in rat blood (Figure 15), compared with the animal experiment of the mRNA construct shown by SEQ ID NO:230 in Example 5, the blood drug concentration of the construct shown by SEQ ID NO:230 was increased by an order of magnitude by changing the MC3 encapsulation process, and the mRNA construct shown by SEQ ID NO:230 still maintained a high blood drug level after 48 hours; the mRNA construct shown by SEQ ID NO:237 increased its blood drug concentration in animals faster than that of the SEQ ID NO:230 construct, reaching Cmax in about 4 hours, and its blood drug concentration was still several times higher than that of the PBS group.

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 nearly one order of magnitude higher than that of the PBS group; at the same time, under different MC3 encapsulation process conditions, the SEQ ID NO: 230 construct shows different rat blood drug concentrations. Compared with the previous rat blood drug level, in this example, its blood drug concentration is increased by nearly one order of magnitude, and a relatively high blood drug level is still maintained at 48 hours.

Example 10 Further screening of highly secretory expression signal peptides

In order to further increase the secretory expression of the construct, reduce the amount of LNP lipids, and reduce the potential hepatotoxicity caused by LNP, the inventors designed a series of constructs (SEQ ID NO: 269-SEQ ID NO: 285 and SEQ ID NO: 299-SEQ ID NO: 315), and transfected the relevant constructs into human hepatocytes HepG2, and evaluated the expression of the constructs in the cell supernatant using an ACTH ELISA detection kit.

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

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.

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

Since the secretory signal peptide of the secretory expression construct SEQ ID NO:237 is derived from rats, the inventors carried out single-point mutations and combined mutations in the amino acid residues in the secretory signal peptide (SP) sequence that are different from the human homologs. The corresponding signal peptide encoding nucleotide sequence is 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.

Experimental results: As shown in Figure 14, according to the quantitative analysis results of the ACTH content in the cell supernatant, the mRNA construct shown in SEQ ID NO: 289 unexpectedly showed the highest ACTH extracellular secretion expression ability, and its expression level was 5.7 times that of the mRNA construct shown in SEQ ID NO: 237; at the same time, the mRNA construct shown in SEQ ID NO: 298 also showed a higher extracellular ACTH expression ability than the construct shown in SEQ ID NO: 237, and its expression level was twice that of the mRNA construct shown in SEQ ID NO: 237.

Experimental conclusion: Based on the construct SEQ ID NO:237, through the sequence design and optimization of the secretion signal peptide and ACTH encoding protein amino acids, a construct with higher extracellular expression ability was successfully screened, which also has the potential to reduce immunogenicity.

Example 12 Toxicity test of different administration methods and different lipids in rats

The previous in vivo animal experiments were basically based on the preparation of LNPs using MC3 cationic lipids. During the experiment, some animals occasionally showed reduced activity, decreased food intake, and increased eye secretions. We inferred that this might be caused by cationic lipids, so we then compared the safety and mRNA expression levels of the 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 between MC3 and SM102 in terms of mRNA expression levels, and there was no significant difference 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 has also been verified in our other projects. Therefore, in subsequent in vivo experiments, we selected SM102 cationic lipids as the lipid for the preparation of LNPs.

In order to explore more ways of administration, we also compared the effects of three administration methods of ACTH mRNA-LNP on animal safety and mRNA expression levels in rats, namely intravenous injection (i.v.), subcutaneous injection (s.c.), and intramuscular injection (i.m.). The results showed that in the medium, high and low dosage regimens, the expression levels of i.v. and i.m. could show a certain degree of dose dependence, and s.c. administration did not have obvious dose dependence. At the same time, we also found that subcutaneous injection of mRNA-LNP, whether low or high dose, would cause the skin of the injected part to become hard and swollen. After dissection, it was found that there were oily and sticky tissue masses under the skin of the injection site (Figure 16, PBS was injected on the left side, and 3mg/kg ACTH mRNA (SEQ ID NO:289)-LNP was injected on the right side). This may be an inflammatory reaction caused by LNP injection, and it may also be that the drug cannot be completely absorbed subcutaneously, resulting in drug accumulation. In short, compared with subcutaneous administration of mRNA-LNP, intravenous and intramuscular injections are safer and more conducive to mRNA protein expression.

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

In order to explore the inter-batch stability of short peptide mRNA protein alternative expression in animals, the previously screened high expression molecule SEQ ID NO: 289 (its CDS sequence amino acid is SEQ ID NO: 38) was selected for the preparation of IVT mRNA, and the SM102 cationic lipid preparation (refer to Example 4 for the formula) was used for mRNA encapsulation. The rats were given the drug by intravenous injection, and the ACTH content in the animal blood was monitored in real time. In this experiment, three batches were prepared independently using the same mRNA IVT and SM102 encapsulation process, and three independent animal experiments were carried out.

The experimental results are shown in Figure 17. The plasma ACTH content of each group of animals at the 0h time point was the blood ACTH background of SD rats themselves, and the empty LNP group in group G1 did not change significantly over time. At 4h after intravenous injection, the plasma ACTH content reached the highest (Tmax=4h), and the average Cmax of the three batches of mRNA-LNP 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.

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

Example 14 Experiment of highly secretory expression signal peptide construct in MSU-induced rat gout inflammation model

In order to evaluate the in vivo efficacy of the previously screened highly expressed molecule SEQ ID NO: 289 (whose CDS sequence amino acid is SEQ ID NO: 38), healthy rats aged 8-10 weeks were selected and raised under a standard feeding environment. One day before the model was constructed, all animals were weighed and grouped using StudyDirectorTM (version 3.1.399.19, StudyLog System, Inc., S. San Francisco, CA, USA). The "Matched distribution" random grouping method was selected for grouping to ensure that the average weight of each group was as close as possible to the average weight of other groups. The day of grouping was defined as day 0. On the first day after grouping (Day 1), 50uL MSU-PBS solution was injected into the right ankle joint of the rat (the injection dose for each rat was 1.5mg MSU), and 50uL 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. At the same time, routine tests were performed, including daily observation of the animals' activity, food and water intake, eyes, fur, and other abnormalities outside the cage. The quality of the acute gout inflammation model was evaluated by measuring the swelling of the rat joints.

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

Dosage regimen for rat gout inflammation model:

In the gout inflammation model experiment, preventive drug administration to animals is involved, such as preventive administration of positive drug injection corticotropin (Shanghai First Biochemical, ACTH) 0.5h before the start of model construction, and administration of SEQ ID NO:289 construct LNP encapsulated drug at the same time. Group G6 is the LNP empty package group, which serves as the vehicle control. The administration methods include 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 for rat blood collection and swelling measurement are set as: 0h, 4h, 8h, 18h, and 24h.

Swelling index

Experimental results: An animal gout inflammation model was constructed by injecting MSU into the rat joint 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) showed smaller joint swelling than the control empty package LNP group (G6) in both i.v. and i.m. administration methods, whether 1mpk (1mg/kg) or 0.3mpk (0.3mg/kg). At the same time, compared with the positive control drug group G4, 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 administration dose showed a better effect in alleviating joint swelling. At the 24 h time point, all mRNA-LNP-treated groups (G8-G11) showed a significant decrease in animal joint swelling compared to the G2 control group.

Experimental conclusion: In the rat gout inflammation model, the high secretion expression construct SEQ ID NO: 289 screened in the early stage was verified to successfully relieve the swelling of the animal joints under both i.v. and i.m. administration methods, and at the 24h time point, the 0.3mpk administration dose had a significant effect on relieving the swelling of the animal joints. And its effect is better than the marketed drug injectable corticotropin. This experiment verified the efficacy of the SEQ ID NO: 289 construct in the rat gout inflammation model, showing a very powerful therapeutic effect on gout inflammation.

Blood drug concentration (blood ACTH level) test

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

Experimental conclusion: Monitoring the mRNA-LNP administration groups with different administration methods and dosages, the ACTH content expressed by the SEQ ID NO:289 construct in animals showed a stable and controllable dose dependence. The relevant data provided a very meaningful reference for us to choose different administration methods and mRNA-LNP dosages. At the same time, the plasma ACTH content of different administration groups corresponded to the efficacy of joint swelling in the gout inflammation model, indicating that precise treatment of gout inflammation of different degrees can be achieved by controlling the dosage and administration method.

Changes in animal body weight in the gout inflammation model

As shown in Figure 18h, the changes in animal weight and plasma ACTH content and the dosage of mRNA-LNP are correlated. At high injection corticotropin (3mpk) and mRNA-LNP (3mpk) dosages, the animal weight showed negative growth within 24 hours. As the dosage decreased, the effect on animal weight gradually decreased. At the dosage of 0.3mpk mRNA-LNP, the mRNA drug had almost no effect on the animal weight, indicating that we can achieve the control and treatment of gout inflammation with mRNA drugs at a safe dosage.

Blood IL-6, IL-1β, AST, ALT indicators

Experimental results: An animal gout inflammation model was constructed by injecting MSU into the rat joint cavity, and the inflammatory factor expression of the mRNA construct shown in SEQ ID NO:289 in the gout inflammation model was analyzed. Referring to the results shown in Figures 18i-l, in the IL-1β index, relative to the LNP empty bag control in the G6 group, the IL-1β in the 0.3mpk administration dose in the G9 and G11 groups was not higher than that in the G6 group. At the same time, there was no obvious change in the IL-1β index level over time. In the IL-6 index, except for the mRNA-LNP administration groups in the G9 and G11 groups, the other groups were similar to the control group, and there was no obvious change in the IL-6 level. For the G9 group with 0.3mpk mRNA-LNP i.v. administration, the value was close to the background level at 0h, and then increased at 4h, reached the maximum at 8h, and returned to the normal background level at 18h. For the G11 group with 0.3mpk mRNA-LNP i.m. administration, the trend was similar to that of the G9 group. After 8h, the IL-6 level quickly dropped to the background level. During the whole process, the increase in IL-6 index in the first 8 hours may be caused by the mRNA-LNP drug. After 8 hours, the IL-6 index dropped rapidly and even G9 dropped drastically. Referring to the previous PK of mRNA in the body, it may be due to the anti-inflammatory effect exerted by the expression of ACTH. In the AST and ALT indicators, there was no obvious increase in ALT and AST indicators as a whole. The high data of G8 in some groups at 8h and 24h were also caused by 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, so the SEQ ID NO:289 construct can ensure the efficacy without affecting the ALT and AST indicators.

Experimental conclusion: In the gout inflammation model, the SEQ ID NO: 289 construct showed better efficacy than the marketed drug injectable corticotropin at a dosage of 0.3 mpk. At the same time, this dose did not cause changes in the cytokines IL-6 and IL-1β, as well as ALT and AST indicators, indicating that SEQ ID NO: 289 has a very safe therapeutic window, indicating that it has great potential in the treatment of acute gout.

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

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 drug ACTH 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 shorter 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.

According to past experience and research, constructing ACTH fusion protein can effectively prolong the half-life of short half-life proteins. In this embodiment, the half-life of ACTH mRNA is improved by constructing a signal peptide-ACTH-linker-VLK fusion construct (Figure 1). The amino acid sequence of the fusion construct linker is: SEQ ID NO: 55, the mRNA sequence of the 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 gene-synthesized construct sequence plasmid is subjected to plasmid linearization, IVT to produce mRNA stock solution, mRNA LNP encapsulation of SM102 prescription, and characterization of the physical and chemical properties of LNP drugs to complete the preparation of mRNA-LNP drugs.

The in vitro expression ability and activity of the long T-half ACTH mRNA construct were verified in an in vitro cell experiment. The mRNA stock solution of the construct was transfected into HepG2 cells with a transfection reagent, and the cell supernatant with mRNA expression product was collected. At the same time, the cell supernatant with mRNA was prepared as a blank control. The ACTH level in the cell supernatant was quantified using the abcam ACTH ELISA kit.

Experimental result 1: As shown in Figure 19a-f, a head-to-head comparison of the ACTH expression levels in the supernatant of cells 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 cellular ACTH expression levels 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 trend of ACTH expression in the cell supernatant of each construct at 24h, 48h, and 84h after mRNA transfection, it was found that the ACTH expression level of the cells of the ACTH mRNA construct fused with linker-VLK has been on an upward trend, while the ACTH expression level of the ACTH mRNA construct without linker-VLK has been on 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 coding sequence and the VLK sequence were added) for subsequent in vivo verification.

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 after administration in animals.

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

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

Experimental results 1: As shown in Figure 20, 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. According to the PK kinetic curves, the T-half of ACTH short peptide, SEQ ID NO: 289 and SEQ ID NO: 336 were 20 min, 3.2 h and 3 days, respectively. This indicates that the T-half of the ACTH mRNA construct SEQ ID NO: 289 without VLK sequence fusion is significantly higher than that of the ACTH short peptide, while the T-half of the ACTH mRNA construct SEQ ID NO: 336 fused with VLK is significantly higher than that of the SEQ ID NO: 289 construct.

Experimental conclusion 1: The T-half of ACTH in vivo administered in the form of mRNA is far superior to the ACTH short peptide, and the T-half of the ACTH mRNA construct fused with VLK is far superior to the non-VLK construct SEQ ID NO: 289. Therefore, the administration of ACTH in vivo in the form of mRNA can solve the high administration frequency caused by the short half-life of ACTH short peptide itself. At the same time, mRNA constructs with different T-half can meet the administration status of various acute and chronic indications, greatly expanding the scope of application of mRNA-LNP drugs.

Example 17 Experiment of highly secretory expression signal peptide construct in EAU model

Experimental autoimmune uveitis (EAU) is an organ-specific, T-cell-mediated autoimmune disease that targets the neural retina and related tissues; it is caused by immunization with retinal antigens. The pathology of EAU closely resembles human uveitis and is assumed to be autoimmune in nature, 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 study the basic mechanisms of tolerance and autoimmunity to organ-specific antigens in immune-privileged sites. Therefore, EAU can serve as a tool for clinical and basic research on ocular and organ-specific autoimmunity. In susceptible animals, peripheral immunity can be induced by the use of some evolutionarily well-conserved uveal pathogens in adjuvants (purified protein antigens extracted from the retina or its peptides) or by adoptive transfer of lymphocytes specific for these antigens. The aim of this study was to test the therapeutic effects of compounds on Lewis rats with immune uveitis induced by bovine binding protein.

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 bias. 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, the eyes of the rats were examined with an ophthalmoscope every day, and the severity of the disease was evaluated using the scoring system in Table 2. Photos of both eyes were taken on Day 11 and Day 14. After the in vivo observation (day 16), all rats were euthanized by overdose of carbon dioxide.

Table 1 - Grouping and dosing regimen

Note: FTY720 is the positive control for fingolimod.

Table 2 - Clinical EAU scores of rats

a Each higher level includes the criteria of the previous level

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 solvent 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 solvent group. In the experiment, 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 and visual animal eye photos (Figure 21c).

Experimental conclusion: In the EAU pharmacodynamic model, compared with the short ACTH peptide (ACTH, once every two days, marketed injectable corticotropin), the high expression construct SEQ ID NO: 289 and the long T-half high expression construct SEQ ID NO: 336 showed better efficacy and significant differences when the dosing frequency was significantly reduced. It was verified that the high expression construct SEQ ID NO: 289 and the long T-half high expression construct SEQ ID NO: 336 have good therapeutic effects in EAU disease.

Example 18 Experiment of highly secretory expression signal peptide construct in AIA model

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 characteristics 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 lay 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 the 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. 1.6 mpk of corticotropin for injection and 5 mpk of prednisolone were used as positive references. The i.v. dose of SEQ ID NO:289 construct was 0.02 mpk and 0.1 mpk, and the i.m. dose was 0.1 mpk; the i.v. dose of SEQ ID NO:336 construct was 0.005 mpk and the i.m. dose was 0.005 mpk. The animals were evaluated for AIA disease clinical scoring, joint swelling, etc.

Experimental results: As shown in Figure 22a, according to the clinical score of the model data, the model G2 group vehicle group began to develop the disease on day 12, and the clinical score gradually increased with the passage of time. On day 13, clinical score ≥ 3, and therapeutic administration of SEQ ID NO: 289 and SEQ ID NO: 336 was started. After administration, it can be clearly observed that all the administration groups had a significant decrease in clinical score on day 19 compared with the vehicle group and the LNP blank control group. SEQ ID NO: 336-i.v.-0.005-single dose and SEQ ID NO: 389-i.v.-0.1-Q4D showed a significant decrease in clinical score compared with the positive control drug ACTH-i.m.-1.6mpk-Q2D, which lasted until day 23. The prednisolone administration group also showed a continuous decrease in clinical score after day 19 as the administration time continued. The i.m. group with the same dose was inferior to the i.v. group in terms of efficacy. The same results were also found in the ankle diameter and paw volume indicators in Figures 22b-c. In Figure 22d, all the groups had similar trends as the positive drug and vehicle, especially the low-dose group, whose weight change had a consistent weight effect with the vehicle and injectable corticotropin groups.

Experimental conclusion: The constructs of SEQ ID NO: 289 and SEQ ID NO: 336 showed the same or even better efficacy in the AIA model than 1.6 mpk of injectable corticotropin and 5 mpk of prednisolone at the dosage of 0.1 mpk, 0.02 mpk and 0.005 mpk by i.v., and no obvious animal toxicity was observed. The relevant data reflect the strong potential of mRNA-LNP drugs in the treatment of AIA and even RA.

Claims (106)

A nucleic acid comprising an open reading frame (ORF) encoding adrenocorticotropic hormone, a functional fragment or variant thereof. 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. The nucleic acid according to 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 has at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identity with the amino acid sequence shown in SEQ ID NO:1. 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. 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 NO:2-61. The nucleic acid according to any one of claims 2 to 5, wherein the ACTH fusion protein is a fusion protein of a secretion signal peptide and ACTH. 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. The nucleic acid according to any one of claims 2 to 7, wherein the C-terminus of the adrenocorticotropic hormone fusion protein is connected to the kappa light chain variable region (VLk) sequence via a linker. 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. 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. A nucleic acid according to 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. A nucleic acid according to 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. The nucleic acid according to any one of claims 1 to 12, wherein the ORF comprises, in order from 5' to 3', a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding adrenocorticotropic hormone. The nucleic acid according to any one of claims 1 to 13, wherein the ORF comprises, in order from 5' to 3', a signal peptide encoding nucleotide sequence, an adrenocorticotropic hormone encoding nucleotide sequence, a linker encoding nucleotide sequence and a kappa light chain variable region (VLk) sequence encoding nucleotide sequence. A nucleic acid according to any one of claims 1-14, wherein the ORF encodes an amino acid sequence as shown in any one of SEQ ID NO: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 NO:2-61. 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 therein. A nucleic acid according to any one of claims 13-16, wherein the signal peptide encoding nucleotide sequence comprises any one of SEQ ID NO: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 NO:343-370 or its corresponding DNA sequence. A nucleic acid according to any one of claims 13-17, wherein the coding nucleotide sequence of adrenocorticotropic hormone comprises any one of SEQ ID NO: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 NO:108-167 or its corresponding DNA sequence. A nucleic acid according to 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. A nucleic acid according to any one of claims 13-19, wherein the encoding nucleotide sequence of 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. The nucleic acid according to any one of claims 1 to 20, wherein the nucleic acid further comprises a 5'-untranslated region (5'-UTR). A nucleic acid according to claim 21, wherein the 5'-UTR comprises SEQ ID NO:62-82 or a DNA sequence corresponding thereto, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:62-82 or a DNA sequence corresponding thereto. The nucleic acid according to any one of claims 1 to 22, wherein the nucleic acid further comprises a 3'-untranslated region (3'-UTR). A nucleic acid according to claim 23, wherein the 3'-UTR comprises SEQ ID NO:83-101 or a DNA sequence corresponding thereto, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with SEQ ID NO:83-101 or a DNA sequence corresponding thereto. The nucleic acid according to any one of claims 1 to 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. The nucleic acid according to claim 25, wherein the 5’-UTR is selected from any one of SEQ ID NO: 62-82 or its corresponding DNA sequence, and the 3’-UTR is selected from any one of SEQ ID NO: SEQ ID NO: 83-101 or its corresponding DNA sequence. The nucleic acid according to any one of claims 1 to 26, wherein the nucleic acid further comprises a 3'-polyadenylic acid sequence. 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. A nucleic acid according to any one of claims 1-28, wherein the nucleic acid comprises any one of SEQ ID NO:170-342 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 NO:170-342 or its corresponding DNA sequence. The nucleic acid according to any one of claims 1 to 29, wherein the nucleic acid is DNA or RNA. The nucleic acid according to any one of claims 1 to 30, comprising at least one chemically modified nucleotide. 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. The nucleic acid according to claim 31 or 32, wherein the chemical modification is N1-methylpseudouridine. 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. The nucleic acid according to any one of claims 1 to 34, which is mRNA, and the mRNA comprises a 5'-cap structure. 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. The nucleic acid of any one of claims 1-36, wherein the nucleic acid is formulated in nanoparticles. The nucleic acid of claim 37, wherein the nanoparticle is a lipid nanoparticle. A vector or plasmid comprising the nucleic acid according to any one of the preceding claims. A host cell comprising the nucleic acid according to any one of claims 1 to 38 and/or the vector or plasmid according to claim 39. A pharmaceutical composition comprising the nucleic acid according to any one of claims 1-38 and a delivery agent. 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. The pharmaceutical composition of claim 41 or 42, wherein the delivery agent is a lipid nanoparticle. The pharmaceutical composition according to any one of claims 41-43, wherein the pharmaceutical composition is formulated as lipid nanoparticles encapsulating the nucleic acid in a lipid shell. A pharmaceutical composition according to any one of claims 41-44, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, diluent or excipient. 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. 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 treating or preventing a rheumatic disease, a pulmonary disease, an ophthalmic disease, a renal disease or a neurological disease in a subject. 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 treating or preventing gouty arthritis, rheumatoid arthritis, dermatomyositis/polymyositis, systemic lupus erythematosus, sarcoidosis or psoriatic arthritis in a subject. 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. 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 in treating or preventing keratitis, uveitis or optic neuritis. 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 in treating or preventing focal interphase glomerulosclerosis or primary immunoglobulin A nephropathy. 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 in treating or preventing multiple sclerosis, optic neuritis or infantile spasms. 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. The method of claim 53, wherein the subject is a human or non-human mammal. The method according to claim 53 or 54, wherein the administration is via parenteral administration or gastrointestinal administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery. 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. The method of any one of claims 53-56, wherein the nucleic acid is expressed by cells in the subject. 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. 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. The method of claim 59, wherein the subject is a human or non-human mammal. The method according to claim 59 or 60, wherein the administration is via parenteral administration or gastrointestinal administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery. 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. The method of any one of claims 59-62, wherein the nucleic acid is expressed by cells in the subject. The method of any one of claims 59-63, wherein the lung disease comprises symptomatic pulmonary sarcoidosis. 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 to 38 or a pharmaceutical composition according to any one of claims 41 to 45. The method of claim 65, wherein the subject is a human or non-human mammal. The method according to claim 65 or 66, wherein the administration is via parenteral administration or gastrointestinal administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery. 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. The method of any one of claims 65-68, wherein the nucleic acid is expressed by cells in the subject. The method of any one of claims 65-69, wherein the ophthalmic disease comprises keratitis, uveitis or optic neuritis. 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. The method of claim 71, wherein the subject is a human or non-human mammal. The method according to claim 71 or 72, wherein the administration is via parenteral administration or gastrointestinal administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery. 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. The method of any one of claims 71-74, wherein the nucleic acid is expressed by cells in the subject. The method of any one of claims 71-75, wherein the renal disease comprises focal segmental glomerulosclerosis or primary immunoglobulin A nephropathy. 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 to 38 or a pharmaceutical composition according to any one of claims 41 to 45. The method of claim 77, wherein the subject is a human or non-human mammal. The method according to claim 77 or 78, wherein the administration is via parenteral administration or gastrointestinal administration, preferably via intralesional, intramuscular, subcutaneous, intravenous, intraarterial, oral or rectal delivery. 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. The method of any one of claims 77-80, wherein the nucleic acid is expressed by cells in the subject. The method of any one of claims 77-81, wherein the neurological disease comprises multiple sclerosis, optic neuritis, or infantile spasms. A kit comprising the nucleic acid according to any one of claims 1 to 38 and/or the pharmaceutical composition according to any one of claims 41 to 45, and instructions for use. 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, and the method comprises 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. 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. The method according to claim 84 or 85, wherein the ORF comprises, from 5' to 3', in sequence: a signal peptide encoding nucleotide sequence, a corticotropin encoding nucleotide sequence, a linker encoding nucleotide sequence and a kappa light chain variable region (VLk) sequence encoding nucleotide sequence. The method according to any one of claims 84-86, wherein the encoding nucleotide sequence of the signal peptide comprises any one of SEQ ID NO: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 NO:343-370 or its corresponding DNA sequence. A method according to any one of claims 84-87, wherein the coding nucleotide sequence of adrenocorticotropic hormone comprises any one of SEQ ID NO: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 NO:108-167 or its corresponding DNA sequence. A method according to 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. 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 having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity thereto. The method according to any one of claims 84-90, wherein the nucleic acid further comprises a 5'-untranslated region (5'-UTR). A method according to claim 91, wherein the 5'-UTR comprises SEQ ID NO:62-82 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:62-82 or its corresponding DNA sequence. The method according to any one of claims 84-92, wherein the nucleic acid further comprises a 3'-untranslated region (3'-UTR). A method according to claim 93, wherein the 3'-UTR comprises SEQ ID NO:83-101 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:83-101 or its corresponding DNA sequence. The method according to 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. A method according to claim 95, wherein the 5’-UTR is selected from any one of SEQ ID NO: 62-82 or its corresponding DNA sequence, and the 3’-UTR is selected from any one of SEQ ID NO: SEQ ID NO: 83-101 or its corresponding DNA sequence. The method according to any one of claims 84-96, wherein the nucleic acid further comprises a 3'-poly A sequence. 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. A method according to any one of claims 84-98, wherein the nucleic acid comprises any one of SEQ ID NO:170-342 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 NO:170-342 or its corresponding DNA sequence. The method according to any one of claims 84-99, wherein the nucleic acid is DNA or RNA. The method according to any one of claims 84-100, comprising at least one chemically modified nucleotide. 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. The method according to claim 101 or 102, wherein the chemical modification is N1-methylpseudouridine. 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. The method according to any one of claims 84-104, which is mRNA, and the mRNA comprises a 5'-cap structure. 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.