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WO2025021183A1 - Polynucléotide codant pour l'hormone adrénocorticotropique, composition associée et méthode associée - Google Patents

Polynucléotide codant pour l'hormone adrénocorticotropique, composition associée et méthode associée Download PDF

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Publication number
WO2025021183A1
WO2025021183A1 PCT/CN2024/107743 CN2024107743W WO2025021183A1 WO 2025021183 A1 WO2025021183 A1 WO 2025021183A1 CN 2024107743 W CN2024107743 W CN 2024107743W WO 2025021183 A1 WO2025021183 A1 WO 2025021183A1
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nucleic acid
sequence
seq
mrna
acth
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Chinese (zh)
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单永强
刘锐
李振建
杨帆
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Shanghai Henlius Biologics Co Ltd
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Shanghai Henlius Biologics Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/67—General methods for enhancing the expression

Definitions

  • 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:
  • Acthar Gel can be used to treat idiopathic nephrotic syndrome in children, especially for patients with recurrent or refractory disease.
  • 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.
  • Acthar Gel may be used to treat rheumatoid arthritis, especially in patients who have not responded to or cannot tolerate other treatment options.
  • Acthar Gel may also be used for other indications, such as systemic lupus erythematosus, idiopathic inflammatory myopathy, acute attack of gout, etc.
  • 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.
  • ACTH 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.
  • 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.
  • Sequence-optimized mRNA drugs can be expressed in vivo for several days (or even longer), achieving lower dosing frequency and improving patient compliance
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • the present disclosure relates to a kit comprising a nucleic acid described herein and/or a pharmaceutical composition described herein, and instructions for use.
  • 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 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;
  • 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;
  • FIG. 22d shows the body weight changes of each dosing group in the AIA model in Example 18.
  • nucleic acid molecules comprising an open reading frame (ORF) encoding adrenocorticotropic hormone, its functional fragment or variant.
  • 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).
  • 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.
  • 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).
  • lipid nanoparticle 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).
  • the LNP comprises a non-lipid payload molecule partially or completely encapsulated in a lipid shell.
  • the payload is a negatively charged molecule (e.g., mRNA encoding adrenocorticotropic hormone)
  • the lipid component of the LNP comprises at least one cationic lipid.
  • 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.
  • 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”.
  • the positive charge in the cationic lipid is derived from the presence of a quaternary nitrogen atom.
  • the cationic lipid comprises a zwitterionic lipid that is positively charged in the environment of its intended use (e.g., at physiological pH).
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the selected pH value or range corresponds to the pH conditions in the environment of the intended lipid use, such as physiological pH.
  • 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
  • lipoplex 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).
  • 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.
  • the nucleic acid comprises an ORF encoding a secretory signal peptide and adrenocorticotropic hormone.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • mRNA contains a nucleic acid molecule that can encode adrenocorticotropic hormone, that is, at least a portion of the mRNA can encode adrenocorticotropic hormone.
  • 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.
  • composition is intended to encompass a product containing the specified ingredients (eg, mRNA molecules provided herein), optionally in the specified amounts.
  • nucleotide 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.
  • 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”.
  • 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.
  • 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.
  • 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.
  • coding nucleic acid 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.
  • coding region refers to the portion of a coding nucleic acid sequence that is translated into a peptide or polypeptide.
  • UTR untranslated region
  • 5'-UTR a 5'-UTR if it is located at the 5' end of the coding region
  • UTR a 3'-UTR if it is located at the 3' end of the coding region.
  • 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.
  • the corresponding DNA sequence of the RNA sequence GCUGGAGCCUCGGUGGC is GCTGGAGCCTCGGTGGC.
  • open reading frame 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.
  • start codon e.g., methionine (ATG)
  • stop codon e.g., TAA, TGA or TAG
  • 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.
  • 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.
  • ORF open reading frame
  • VLk light chain variable region
  • 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.
  • 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.
  • ORFs open reading frames
  • the region containing one or more ORFs is referred to as the coding region of the mRNA molecule.
  • the mRNA molecule further comprises one or more untranslated regions (UTRs).
  • nucleobase encompasses purines and pyrimidines, including the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural or synthetic analogs or derivatives thereof.
  • 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.
  • 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.
  • a functional analogue of guanosine (G) retains the ability to base pair with a functional analogue of cytosine (C) or cytosine.
  • 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.
  • 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).
  • the terms encompass amino acid chains of any length, including full-length proteins (e.g., adrenocorticotropic hormone).
  • 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.
  • modification 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.
  • 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.
  • 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.
  • 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.
  • the two nucleic acid molecules may be inserted into the same expression vector or in a separate expression vector.
  • 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.
  • 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.
  • administer 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.
  • a substance e.g., a lipid nanoparticle composition described herein
  • the administration of the substance is typically performed after the onset of the 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 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.
  • target delivery 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.
  • an effective amount is a therapeutically effective amount or a prophylactically effective amount.
  • 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
  • a therapeutically effective amount includes an amount in which the therapeutically beneficial effects of the agent outweigh any toxic or deleterious effects thereof.
  • 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).
  • a preventive dose 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.
  • prevent refers 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.
  • 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.
  • neurological disease including but not limited to infantile spasms, multiple sclerosis
  • 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.
  • 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.
  • 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 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).
  • a mammal such as a non-primate (e.g., cattle, pigs, horses, cats, dogs, rats, etc.) or a primate (e.g., monkeys and humans).
  • the subject is a human.
  • the subject is a mammal (e.g., a human) suffering from an infectious disease or a neoplastic disease.
  • 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).
  • a mammal e.g., a human
  • a rheumatic disease including but not limited to gout inflammation
  • a lung disease e.g., an ophthalmic disease
  • kidney disease e.g., a chronic fibrosis
  • a neurological disease including but not limited to infantile spasms, multiple sclerosis.
  • 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%.
  • 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.
  • 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.
  • the therapeutic nucleic acid molecule is a DNA molecule.
  • the therapeutic nucleic acid molecule is an RNA molecule.
  • the therapeutic nucleic acid molecule is an mRNA molecule.
  • 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.
  • the nucleic acid molecules of the present disclosure include mRNA molecules.
  • the nucleic acid molecules include at least one coding region (e.g., open reading frame (ORF)) encoding a protein of interest.
  • the nucleic acid molecules also include at least one untranslated region (UTR).
  • the untranslated region (UTR) is located upstream (5' end) of the coding region and is referred to herein as 5'-UTR.
  • the untranslated region (UTR) is located downstream (3' end) of the coding region and is referred to herein as 3'-UTR.
  • the nucleic acid molecules include both 5'-UTR and 3'-UTR.
  • 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.
  • the ORF encodes a truncated ACTH, a mutant ACTH, or an ACTH fusion protein.
  • adrenocorticotropic hormone 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.
  • the ACTH may be selected from vertebrates of different species, preferably from humans.
  • the ACTH can be wild-type ACTH.
  • the ACTH can be any functional fragment of wild-type ACTH.
  • 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.
  • 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.
  • 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.
  • the ACTH fusion protein is a fusion protein of a secretion signal peptide and ACTH.
  • the secretion signal peptide is fused to the N-terminus or C-terminus of the ACTH, preferably the N-terminus.
  • the C-terminus of the ACTH fusion protein is also connected to a kappa light chain variable region (VLk) sequence via a linker.
  • 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.
  • the amino acid sequence encoded by the ORF comprises a signal peptide and adrenocorticotropic hormone from the N-terminus to the C-terminus.
  • 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.
  • VLk light chain variable region
  • 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.
  • 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 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.
  • the optimization of guanosine/cytosine (G/C) content can correspondingly improve the stability of mRNA.
  • 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.
  • the ORF comprises, in order from 5' to 3', a nucleotide sequence encoding a signal peptide and a nucleotide sequence encoding adrenocorticotropic hormone.
  • 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.
  • VLk light chain variable region
  • 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 ORF is codon-optimized, and preferably, the codon optimization does not change the amino acid sequence it encodes.
  • 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.
  • 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.
  • 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 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 therapeutic nucleic acid molecule is an mRNA molecule.
  • 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.
  • CBP mRNA cap binding protein
  • the 5'-cap structure further facilitates the removal of 5'-proximal introns during mRNA splicing. Therefore, in some embodiments, the nucleic acid molecules of the present disclosure include a 5'-cap structure.
  • 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 nucleic acid molecules of the present disclosure comprise one or more changes to a native 5'-cap structure produced by an endogenous process.
  • 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.
  • modified nucleotides may be used during the capping reaction.
  • Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used for ⁇ -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 ⁇ -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.
  • GTP capped guanosine triphosphate
  • CH2 methylene moiety
  • 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.
  • 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.
  • 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).
  • 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).
  • the cap analog can be a dinucleotide cap analog.
  • 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.
  • 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).
  • the cap analog that can be used in conjunction with the nucleic acid molecules of the present disclosure is a 4-chloro/bromophenoxyethyl analog.
  • the cap analog may include a guanosine analog.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • the 5'-cap structure used herein is m7(3'OMeG)(5')ppp(5')(2'OMeA)pG.
  • 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.
  • the nucleic acid molecules of the present disclosure include one or more untranslated regions (UTRs).
  • the UTR is located upstream of the coding region in the nucleic acid molecule and is referred to as the 5′-UTR.
  • 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.
  • 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.
  • 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.
  • 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.
  • poly-A regions adenosine nucleotides
  • mRNA messenger RNA
  • 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.
  • nucleic acid molecules (e.g., mRNA) of the present disclosure comprise polyadenylation signals.
  • nucleic acid molecules (e.g., mRNA) of the present disclosure comprise one or more polyadenylation (poly-A) regions.
  • the poly-A region is entirely composed of adenine nucleotides or functional analogs thereof.
  • the nucleic acid molecule comprises at least one poly-A region at its 3' end.
  • the nucleic acid molecule comprises at least one poly-A region at its 5' end.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.).
  • 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.
  • the nucleic acid molecules may bind to the poly-A region located at the 3' end of the mRNA molecule.
  • These poly-A binding proteins 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.
  • the nucleic acid molecules e.g., mRNA
  • the nucleic acid molecules of the present disclosure comprise at least one binding site for a poly-A binding protein (PABP).
  • PABP poly-A binding protein
  • a delivery vehicle e.g., lipid nanoparticle
  • poly-A is a continuous sequence of adenosine nucleotides.
  • poly-A e.g., 3'-poly A sequence
  • poly-A 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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-ethy
  • 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.
  • VLk light chain variable region
  • 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;
  • 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.
  • 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 functional nucleotide analogs contain non-classical nucleobases.
  • the classical nucleobases in the nucleotide e.g., adenine, guanine, uracil, thymine, and cytosine
  • 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.
  • the non-classical nucleobase is a modified uracil.
  • exemplary nucleobases and nucleosides having a modified uracil include pseudouridine ( ⁇ ), 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 (c
  • the nucleic acid molecules of the present disclosure include modifications to uracil.
  • the nucleic acid molecules of the present disclosure include one or more pseudouracils ( ⁇ ).
  • 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 ( ⁇ ).
  • all (100%) uracils in the nucleic acid molecules of the present disclosure are replaced by pseudouracils ( ⁇ ).
  • the nucleic acid molecules of the present disclosure comprise one or more 1-methyl pseudouracils (m1 ⁇ ).
  • 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 (m1 ⁇ ).
  • all (100%) uracils in the nucleic acid molecules of the present disclosure are substituted with 1-methyl pseudouracils (m1 ⁇ ).
  • Therapeutic nucleic acid molecules as described herein can be isolated or synthesized using methods known in the art.
  • the DNA or RNA molecules used in conjunction with the present disclosure are chemically synthesized.
  • the DNA or RNA molecules used in conjunction with the present disclosure are isolated from natural sources.
  • the mRNA molecules used in conjunction with the present disclosure are biosynthesized using host cells.
  • mRNA is produced by using host cells to transcribe the corresponding DNA.
  • 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.
  • 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.
  • 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.
  • ORF open reading frame
  • 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.
  • 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.
  • the DNA fragment is synthesized by a commercial biotechnology company.
  • 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.).
  • the expression plasmid is preferably a pUC-GW plasmid.
  • 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.
  • 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.
  • 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.
  • positive recombinant cells are screened and colony sequencing is performed.
  • 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.
  • 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.
  • the colony of the target band is preferably determined by agarose gel electrophoresis, and then sequenced for verification.
  • the parameters of the agarose gel electrophoresis detection are as follows: 1% agarose, 5V/cm, 40min.
  • 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.
  • 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.
  • the enzyme digestion system is designed with 50 ⁇ l, preferably as follows:
  • 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.
  • the reaction is considered to be complete when only one band appears in agarose gel electrophoresis.
  • 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.
  • 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.
  • 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 ⁇ l system and includes the following components:
  • the Enzyme Mix includes T7 RNA polymerase, RNase inhibitor and inorganic pyrophosphatase.
  • the RNA in vitro synthesis is preferably reacted in a 200 ⁇ l 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.
  • the in vitro transcription of the RNA 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.
  • the digestion is performed as follows: 2 ⁇ l 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.
  • 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.
  • the mRNA is considered qualified. After the purified mRNA is recovered, the purified mRNA is preferably packaged.
  • 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.
  • the mRNA may be encapsulated in nanoparticles.
  • 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.
  • nanoparticles include lipids. Lipid nanoparticles may include, but are not limited to, liposomes and micelles.
  • 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.
  • 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.
  • cationic and/or ionizable lipids can be used, e.g. (including DOTMA and DOPE) and (including DOSPA and DOPE).
  • the cationic lipid can be DLin MC3DMA.
  • 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%.
  • 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.
  • 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), di
  • 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.
  • DOPE-mal dipalmitoylphosphatidylethanolamine
  • DMPE dimyristoylphosphoethanolamine
  • DSPE distearoyl-phosphatidyl-ethanolamine
  • 16-O-monomethyl PE 16-O-dimethyl PE
  • 18-1-trans PE 1-stearoyl-2-oleoyl-phosphatidylethanolamine
  • SOPE 1-stearoyl-2-oleoyl-
  • the neutral lipids described herein can be selected from DOPE, DSPC, DPPC, POPC or any related phosphatidylcholine.
  • the neutral lipid is DSPC.
  • 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%.
  • 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 nano lipid particle may include PEG2000-DMG.
  • 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%.
  • the nano lipid particles may further comprise cholesterol.
  • 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%.
  • the nanolipid particles may include cationic lipids, cholesterol, phospholipids, and lipid molecules modified with polyethylene glycol.
  • 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.
  • the molar ratio of the cationic lipids, cholesterol, phospholipids, and lipid molecules modified with polyethylene glycol may be 50:38.5:10:1.5.
  • nucleic acid molecules as described herein are formulated for in vitro and in vivo delivery.
  • nucleic acid molecules are formulated into lipid-containing compositions.
  • lipid-containing compositions form lipid nanoparticles that enclose nucleic acid molecules in lipid shells.
  • lipid shells protect nucleic acid molecules from degradation.
  • lipid nanoparticles also help to transport the enclosed nucleic acid molecules to intracellular compartments and/or mechanisms to exert expected preventive functions.
  • nucleic acid 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.
  • multilamellar vesicles 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.
  • 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.
  • delivery vehicles e.g., nanolipid particles
  • mRNA can be associated with the surface of the lipid delivery vehicle and encapsulated therein.
  • the cationic lipid delivery vehicle can be associated with mRNA through electrostatic interaction.
  • 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).
  • mRNA can be delivered to specific cells or tissues.
  • the size of the lipid delivery carrier for example, lipid nano particle
  • the size of the lipid delivery carrier 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
  • the size (for example, diameter) of the lipid delivery carrier can be within the range of about 25 to 250nm, for example, less than about 250nm,
  • the size (e.g., diameter) of the lipid delivery vehicle 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.
  • the nanoparticle composition is a vesicle comprising one or more lipid bilayers.
  • 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.
  • 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.
  • 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.
  • phospholipids eg, unsaturated lipids, such as DOPE or DSPC
  • PEG lipids e.g., PEG lipids
  • structural lipids e.g., structural lipids.
  • the components of the lipid component may be provided in a specific ratio.
  • a nanoparticle composition comprising a cationic or ionizable lipid compound, a therapeutic agent provided herein, and one or more excipients.
  • the one or more excipients are selected from neutral lipids, phospholipids, steroids, and polymer-conjugated lipids.
  • the therapeutic agent is encapsulated in or associated with lipid nanoparticles.
  • Nanoparticle compositions can be designed for one or more specific applications or targets.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the wt/wt ratio of the lipid component to the therapeutic and/or prophylactic agent can be about 10: 1 to about 40: 1.
  • 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).
  • 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.
  • 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.
  • the N: P ratio may be about 2: 1 to about 8: 1.
  • the N: P ratio is about 5: 1 to about 8: 1.
  • 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.
  • the N:P ratio may be about 5.67:1.
  • the physical properties of a nanoparticle composition can depend on its components.
  • a nanoparticle composition comprising cholesterol as a structural lipid can have different characteristics than a nanoparticle composition comprising a different structural lipid.
  • the characteristics of a nanoparticle composition can depend on the absolute or relative amounts of its components.
  • 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.
  • 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 Zet
  • the average size of the nanoparticle composition can be between tens of nanometers and hundreds of nanometers.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 0 mV to about +15 mV, about
  • 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.
  • free therapeutic and/or prophylactic agents e.g., RNA
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the one or more excipients or auxiliary ingredients may constitute 50%, 60%, 70%, 80%, 90% or higher percentages of pharmaceutical practice.
  • the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure.
  • the excipient is approved for human and veterinary use.
  • the excipient is approved by the U.S. Food and Drug Administration.
  • the excipient is pharmaceutical grade.
  • the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia and/or the International Pharmacopoeia.
  • a pharmaceutical composition may contain between 0.1% and 100% (wt/wt) of one or more nanoparticle compositions.
  • 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)).
  • 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,
  • 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).
  • the formulation is stable for at least 4 weeks at about 4°C.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • 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.
  • compositions can be prepared into various forms suitable for various routes and methods of administration.
  • 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 e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and elixirs
  • injectable forms e.g., solid dosage forms (e.g., capsules, tablets, pills, powders and granules)
  • dosage forms for topical and/or transdermal administration e.g.
  • 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.
  • 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.
  • inert diluents commonly used in the art, such as water or
  • 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.
  • additional agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents and/or flavoring agents.
  • the composition is mixed with a solubilizing agent, such as CremophorTM, 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 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.
  • 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.
  • 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.
  • the subject is a human or non-human mammal.
  • 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.
  • the nucleic acid-encapsulated lipid nanoparticles are endocytosed by cells in the subject.
  • the nucleic acid is expressed by cells in the subject.
  • the administration is intravenous.
  • 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 rheumatic disease comprises gouty arthritis, rheumatoid arthritis, dermatomyositis/polymyositis, systemic lupus erythematosus, sarcoidosis, or psoriatic arthritis.
  • 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.
  • the subject is a human or non-human mammal.
  • 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.
  • the nucleic acid-encapsulated lipid nanoparticles are endocytosed by cells in the subject.
  • the nucleic acid is expressed by cells in the subject.
  • the administration is intravenous.
  • 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 lung disease comprises symptomatic pulmonary sarcoidosis.
  • 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.
  • the subject is a human or non-human mammal.
  • 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.
  • the nucleic acid-encapsulated lipid nanoparticles are endocytosed by cells in the subject.
  • the nucleic acid is expressed by cells in the subject.
  • the administration is intravenous.
  • 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 ophthalmic disease comprises keratitis, uveitis, or optic neuritis.
  • 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.
  • the subject is a human or non-human mammal.
  • 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.
  • 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.
  • the lipid nanoparticles encapsulating the therapeutic nucleic acid are endocytosed by cells in the subject.
  • the therapeutic nucleic acid is expressed by cells in the subject.
  • the neurological disease comprises multiple sclerosis, optic neuritis, or infantile spasms (IS).
  • 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).
  • gouty rheumatic diseases including but not limited to gout inflammation
  • lung diseases including but not limited to gout inflammation
  • ophthalmic diseases including but not limited to gout inflammation
  • kidney diseases including but not limited to infantile spasms and multiple sclerosis
  • neurological diseases including but not limited to infantile spasms and multiple sclerosis.
  • the therapeutic nucleic acids described herein have the advantages of longer half-life, lower dosing frequency, better therapeutic effect, and lower immunogenicity.
  • 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; ⁇ 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.
  • mRNA encoding adrenocorticotropic hormone was designed and prepared by transcription reaction.
  • 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).
  • SEQ ID NO: 55 linker
  • VLK sequence SEQ ID NO: 54
  • 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).
  • ACTH adrenocorticotropic hormone
  • the DNA fragment encoding the mRNA construct of adrenocorticotropic hormone (ACTH) is synthesized, and the mRNA is synthesized by in vitro transcription. Specifically:
  • 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;
  • the above plasmid was subjected to BspQI restriction enzyme digestion reaction to linearize the plasmid.
  • the reaction system was:
  • the linearized plasmid was completely digested and purified using the Thermo Scientific, GeneJET PCR Purification Kit, #K0702 kit. The specific steps are as follows:
  • step 2) Pipette the solution in step 1), transfer it to the GeneJET purification column, centrifuge for 60 seconds, and discard the filtrate;
  • 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.
  • the IVT product was detected by agarose gel and was a single mRNA product band.
  • the purified IVT product was verified and quality controlled by agarose gel electrophoresis.
  • Example 2 mRNA expression and analysis in cells
  • 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.
  • RNAiMAX 5ul + 50ul opti-MEM (OMEM, no additives in the culture medium), mix well and let stand for 5min;
  • mRNA MIX 1 ⁇ g mRNA (0.5 ⁇ g/ ⁇ l) + 50ul opti-MEM (OMEM, no additives in the culture medium) and mix well;
  • 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.
  • 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:
  • HRP anti-flag antibody
  • 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.
  • the preferred functional elements are:
  • ACTH mRNA ending type SEQ ID NO: 103;
  • 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:
  • Blocking Add 250uL Blocking Buffer to each well and block for 1 hour at room temperature;
  • 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;
  • Wash the plate Take 300uL Wash Buffer to wash the ELISA plate, wash 5 times in total;
  • 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;
  • 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.
  • 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:
  • ALC-0315:DSPC:Cholesterol:ALC-0159 50:10:38.5:1.5
  • the LNP-mRNA is prepared by ultrafiltration, liquid exchange and concentration.
  • 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:
  • 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.
  • mice aged 8-10 weeks were selected and raised under a standard feeding environment.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • Dosage regimen for rat gout inflammation model Dosage regimen for rat gout inflammation model:
  • 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.
  • adjuvants purified protein antigens extracted from the retina or its peptides
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

L'invention concerne une molécule d'acide nucléique codant pour l'hormone adrénocorticotropique, ou un fragment fonctionnel ou un variant de celle-ci. La présente invention concerne en outre une composition contenant la molécule d'acide nucléique, comprenant une nanoparticule lipidique, et une méthode de traitement associée et une utilisation pour traiter ou prévenir des maladies rhumatismales (y compris, mais sans s'y limiter, une inflammation liée à la goutte), des maladies pulmonaires, des maladies ophtalmologiques, des maladies rénales ou des maladies neurologiques (y compris, mais sans s'y limiter, les spasmes du nourrisson et la sclérose en plaques) chez un sujet.
PCT/CN2024/107743 2023-07-27 2024-07-26 Polynucléotide codant pour l'hormone adrénocorticotropique, composition associée et méthode associée Pending WO2025021183A1 (fr)

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