Nucleic acid encoding thrombopoietin or precursor thereof, and nucleic acid composition

Abstract

Provided are a nucleic acid encoding thrombopoietin or a precursor thereof, and a composition and kit comprising same. Further provided is a use of the nucleic acid, the composition and the kit in treatment of thrombocytopenia.

Description

Nucleic acids and nucleic acid compositions encoding thrombopoietin or a precursor thereof

[0001] This application claims priority to Chinese Patent Application No. 202411380375.5, filed September 30, 2024, entitled “Nucleic acids and nucleic acid compositions encoding thrombopoietin or a precursor thereof,” the contents of which are incorporated herein in their entirety. TECHNICAL FIELD

[0002] The present application relates to the field of biological medicine, in particular to nucleic acids, compositions and kits encoding thrombopoietin or a precursor thereof and uses thereof. BACKGROUND

[0003] Platelets are one of the formed elements in mammalian blood. The main function of platelets is to promote hemostasis and accelerate blood clotting, while platelets also have the function of maintaining the integrity of capillary walls. Platelet count in blood circulation below the lower limit of normal is defined as thrombocytopenia, which increases the risk of bleeding. According to different causes, thrombocytopenia can be generally divided into primary thrombocytopenia (immune thrombocytopenia (ITP)) and secondary thrombocytopenia. ITP, also known as idiopathic thrombocytopenic purpura, is an acquired autoimmune hemorrhagic disease characterized by isolated peripheral blood platelet count reduction without clear cause. Foreign reports show that the annual incidence of adult ITP is (2-10) / 100,000, and the elderly over 60 years old are a high-risk group, and women of childbearing age are slightly higher than men of the same age group. The clinical manifestations of this disease vary greatly, and asymptomatic thrombocytopenia, skin and mucous membrane bleeding, severe internal organ bleeding, and fatal intracranial hemorrhage can all occur (Chinese Guidelines for the Diagnosis and Treatment of Adult Primary Immune Thrombocytopenia (2020 Edition)). In addition, more common secondary thrombocytopenia includes, for example, chemotherapy-induced thrombocytopenia (CIT) and chronic liver disease (CLD)-associated thrombocytopenia. CIT is a common adverse reaction of chemotherapy, and its incidence is related to tumor type, treatment regimen, and chemotherapy cycle, etc. CIT can lead to a reduction in the dose of chemotherapy drugs or a delay in the duration of chemotherapy, or even the need for platelet transfusion, thereby increasing treatment costs, reducing the effectiveness of chemotherapy and quality of life, and affecting prognosis (Chinese Society of Clinical Oncology (CSCO) 2022). In addition, thrombocytopenia is a common complication of liver disease and can adversely affect the treatment of cirrhosis.

[0004] Thrombopoietin (TPO) is an important factor in regulating platelet production. TPO, also known as megakaryocyte growth and development factor (MGDF), promotes the proliferation of hematopoietic stem cells, primitive progenitor cells, megakaryocytes, and platelets. After binding to TPO receptor (TPO-R, c-Mpl), TPO-R dimerizes, initiates downstream signaling, and promotes the proliferation and differentiation of hematopoietic stem cells into megakaryocytes, which in turn increases platelet production.

[0005] Thrombopoietin receptor agonists (TPO-RA), including recombinant human thrombopoietin (rhTPO), Eltrombopag, Hetrombopag, Avatrombopag, and Lusutrombopag, can be used to treat thrombocytopenia. Among them, rhTPO needs to be administered daily, and the convenience of administration is poor; Eltrombopag is limited in the field of CIT; and long-term use of small molecule TPO-RA causes liver damage. The population of patients with thrombocytopenia is huge, and the clinical benefit of existing therapies is limited. There is an unmet clinical need for drugs to treat thrombocytopenia. SUMMARY

[0006] In one aspect, the present application provides a nucleic acid comprising a polynucleotide encoding a thrombopoietin (TPO) precursor, wherein the TPO precursor comprises an N-terminal signal peptide and a TPO polypeptide, the signal peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 70, and 77.

[0007] In another aspect, the present application provides a thrombopoietin (TPO) precursor comprising an N-terminal signal peptide and a TPO polypeptide, wherein the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 70, and 77.

[0008] In some embodiments, the TPO polypeptide comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 16, 64, 65, and 66. In a preferred embodiment, the TPO polypeptide comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 16, and optionally: (1) comprises one or more valine substitutions at one or more positions corresponding to amino acids 46, 128, 131, and 141 of SEQ ID NO: 16, and / or (2) comprises one or more asparagine substitutions at one or more positions corresponding to amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16. In a preferred embodiment, the TPO polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 16, 64, 65, and 66.

[0009] In some embodiments, the polynucleotide encoding the TPO polypeptide comprises (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 17-20 and 95-98. In a preferred embodiment, the polynucleotide encoding the TPO polypeptide comprises the nucleotide sequence of any one of SEQ ID NOs: 17-20 and 95-98.

[0010] In some embodiments, the polynucleotide encoding the signal peptide comprises (1) (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 1, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2-5 and 92-94; (2) (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 70, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 82; or (3) (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 77, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 89. In a preferred embodiment, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of any one of SEQ ID NOs: 2-5, 92-94, 82, and 89.

[0011] In some embodiments, the TPO precursor comprises the amino acid sequence of any one of SEQ ID NOs: 29, 104, and 105.

[0012] In some embodiments, the polynucleotide encoding a TPO precursor comprises (1) (a) an amino acid sequence of SEQ ID NO: 29, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102; (2) (a) an amino acid sequence of SEQ ID NO: 104, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 106; or (3) (a) an amino acid sequence of SEQ ID NO: 105, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 107. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of any one of SEQ ID NOs: 30-33, 99-102, 106, and 107.

[0013] In some embodiments, the nucleic acid of the present application further comprises a 5'-UTR. In a preferred embodiment, the 5'-UTR comprises the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103.

[0014] In some embodiments, the nucleic acid of the present application further comprises a 3'-UTR. In a preferred embodiment, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 46 or 47.

[0015] In some embodiments, the nucleic acid of the present application comprises a 5'-UTR and a 3'-UTR, wherein the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 44 or 103, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 46; or the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 45, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 47.

[0016] In some embodiments, the nucleic acid of the present application further comprises a poly(A) sequence. In a preferred embodiment, the poly(A) sequence comprises at least 50 nucleotides.

[0017] In some embodiments, the nucleic acid of the present application comprises the nucleotide sequence of any one of SEQ ID NOs: 50-53, 108, and 109.

[0018] In some embodiments, the nucleic acid of the application is DNA or RNA. In an embodiment, the nucleic acid of the application is mRNA, circRNA, or saRNA. In an embodiment, the mRNA comprises 1-methylpseudouridine or pseudouridine modifications. In an embodiment, all of the uridine modifications in the mRNA are 1-methylpseudouridine or pseudouridine. In a preferred embodiment, the mRNA further comprises a 5’ cap.

[0019] In another aspect, the application also provides a composition comprising the nucleic acid of the application.

[0020] In some embodiments, the composition comprises a lipid encapsulating the nucleic acid of the application. In an embodiment, the composition comprises at least a first lipid. In a preferred embodiment, the first lipid comprises a cationic lipid. In a preferred embodiment, the first lipid comprises: (1) one or more of the compounds according to formula (01-I), (01-I-B), (01-I-B’), (01-I-B”), (01-I-C), (01-I-D), or (01-I-E), preferably the first lipid comprises one or more of the compounds listed in Table 1; (2) one or more of the compounds according to formula (02-I) or (02-II), preferably the first lipid comprises one or more of the compounds listed in Table 2; (3) one or more of the compounds according to formula (03-I), preferably the first lipid comprises one or more of the compounds listed in Table 3; or (4) one or more of the compounds according to formula (04-I) or (04-III), preferably the first lipid comprises one or more of the compounds listed in Table 4. In a preferred embodiment, the first lipid is Compound C1 or Compound C2. In some embodiments, the composition further comprises a polymer-bound lipid. In a preferred embodiment, the polymer-bound lipid is a PEGylated lipid.

[0021] In some embodiments, the composition comprises a first lipid, a polymer-bound lipid, and a steroid. In some embodiments, the composition comprises a first lipid, a polymer-bound lipid, a phospholipid, and a steroid. In a preferred embodiment, the first lipid comprises a cationic lipid. In a preferred embodiment, the polymer-bound lipid is a PEGylated lipid. In a preferred embodiment, the steroid is cholesterol. In an embodiment, the composition comprises Compound C1 or Compound C2, DMG-PEG 2000, DSPC, and cholesterol.

[0022] In some embodiments, the composition comprises 20-65 mol% cationic lipid, 5-40 mol% phospholipid, 0.5-5 mol% polymer conjugated lipid, and 20-50 mol% sterol. In a preferred embodiment, the composition comprises 30-55 mol% of compound CI or compound C2, 0.5-5 mol% of DMG-PEG 2000, 5-40 mol% of DSPC, and 20-50 mol% of cholesterol.

[0023] In some embodiments, the composition of the application is formulated as a lipid nanoparticle encapsulating the nucleic acid of the application in a lipid. In one embodiment, the composition of the application is a pharmaceutical composition.

[0024] In another aspect, the application also provides an expression vector comprising the nucleic acid of the application.

[0025] In yet another aspect, the application provides a host cell comprising the nucleic acid or the expression vector of the application.

[0026] The application also provides a method of producing thrombopoietin (TPO) comprising culturing the host cell of the application under suitable conditions and recovering the TPO from the host cell or its culture.

[0027] The application also provides the use of the nucleic acid or the composition of the application in the manufacture of a medicament for preventing and / or treating thrombocytopenia. In a preferred embodiment, the thrombocytopenia is selected from immune thrombocytopenia, chemotherapy-induced thrombocytopenia, liver disease-related thrombocytopenia, and aplastic anemia.

[0028] In some embodiments, the medicament further comprises another thrombopoietic agent.

[0029] The application also provides a kit comprising the nucleic acid or the composition of the application, and optionally further comprising another thrombopoietic agent.

[0030] In a preferred embodiment, the other thrombopoietic agent is selected from the group consisting of romiplostim, avatrombopag, eltrombopag, heptaprin, luspatercept, and combinations thereof. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 shows the ability of mRNA candidate molecules encoding human TPO to express TPO in vitro by ELISA analysis.

[0032] Figure 2 shows the ability of mRNA candidate molecules encoding human TPO to express TPO in vitro by ELISA analysis.

[0033] Figure 3 shows the concentration changes of TPO protein in the blood circulation of mice treated with mRNA-10-LNP and mRNA-3-LNP.

[0034] Figure 4 shows the ability of mRNA molecules encoding human TPO, constructed based on different signal peptides, to express TPO in vitro by ELISA analysis.

[0035] Figure 5 shows the concentration changes of TPO protein in the blood circulation of mice treated with mRNA-10-LNP, mRNA-17-LNP and mRNA-24-LNP.

[0036] Figure 6 shows the concentration changes of TPO protein in the blood circulation of mice treated with different mRNA-LNP formulations.

[0037] Figure 7 shows the ability of different drugs to activate the TPO signaling pathway using HEK-Blue TM TPO cell assay.

[0038] Figure 8 shows the platelet count in the blood circulation of mice treated with different mRNA-LNP.

[0039] Figure 9 shows the concentration changes of TPO protein in the blood circulation of mice treated with different doses of mRNA-10-LNP.

[0040] Figure 10 shows the platelet count in the blood circulation of CIT mice treated with PBS, control mRNA-LNP formulation or mRNA-10-LNP.

[0041] Figure 11 shows the platelet count in the blood circulation of long-term chemotherapy CIT mice treated with PBS, control mRNA-LNP formulation or mRNA-10-LNP.

[0042] Figure 12 shows the platelet count in the blood circulation of long-term chemotherapy ITP mice treated with PBS, control mRNA-LNP formulation or mRNA-10-LNP. DETAILED DESCRIPTION

[0043] General Definitions and Terminology

[0044] All patents, patent applications, scientific publications, manufacturer’s instructions and guidelines, etc., cited herein are incorporated by reference herein in their entirety. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0045] Unless otherwise indicated herein, the scientific and technical terms used in this document have the meanings that are commonly understood by one of ordinary skill in the art. Also, the nomenclature used in connection with, and the procedures and techniques of, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, are those well-known and commonly used in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2 nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). In addition, for purposes of the present invention, the following terms will be understood to have the following definitions.

[0046] As used herein, the terms "comprises," "comprising," "includes," "including," and "has," "having," and the like, are open-ended terms that are intended to denote the inclusion of elements, steps, or components that are listed, but do not exclude the presence of other elements, steps, or components. The terms "consisting of" and "consisting essentially of" do not include any element, step, or component not specified. The term "consisting essentially of" means that the recitation of elements, steps, or components is intended to be limited to those elements, steps, or components that do not materially affect the basic and novel characteristics of the claimed subject matter. It is understood that the terms "consisting essentially of" and "consisting of" are open-ended terms that are intended to encompass the inclusion of optional elements, steps, or components that do not materially affect the basic and novel characteristics of the claimed subject matter.

[0047] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. The terms "one or more" or "at least one" encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, or more.

[0048] As used herein, the conjunction "and / or" between elements of a list of more than two elements is understood to include the individual as well as the combined options. In other words, "and / or" includes "and" as well as "or." For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. More elements defined with "and / or" are understood in a similar manner and include any of the individual as well as any combination thereof.

[0049] The recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. Numerical values or ranges expressed herein are, unless expressly stated otherwise, modified by "about" to indicate that the value or range so qualified is understood to include the stated value or range ±20%, ±10%, ±5%, or ±3% as well.

[0050] As used herein, the term "percent identity" or "% identity" with respect to a sequence refers to the percentage of nucleotides or amino acids that are the same in the best alignment between the sequences to be compared. Differences between two sequences can be distributed at local regions (segments) or over the entire length of the sequences to be compared. Identity between two sequences is typically determined following optimal alignment of a segment or "comparison window" between the two sequences. Optimal alignment can be performed manually or with the aid of known algorithms, including but not limited to the local homology algorithm described by Smith and Waterman, 1981, Ads App. Math. 2, 482 and Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, the similarity search method described by Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 88, 2444, or using computer programs such as GAP, BESTFIT, FASTA, BLASTP, BLASTN, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis. For example, the percent identity of two sequences can be determined using the BLASTN or BLASTP algorithm publicly available on the National Center for Biotechnology Information (NCBI) website.

[0051] The % identity is obtained by determining the number of identical positions of the sequences to be compared, dividing this number by the number of positions compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100. In some embodiments, the degree of identity is given for at least about 50%, at least about 55%, 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%, or about 100% of the reference sequence. In some embodiments, the degree of identity is given for the full length of the reference sequence. Alignment for determining sequence identity can be performed with tools known in the art, preferably with optimal sequence alignment, for example, with Align, with standard settings, preferably EMBOSS::needle, Matrix: Blosum62, Gap Open 10.0, and Gap Extend 0.5.

[0052] In the present context, "nucleotides" include deoxyribonucleotides and ribonucleotides and derivatives thereof. As used herein, "ribonucleotides" are the constituent materials of ribonucleic acid (RNA), consisting of one molecule of base, one molecule of five-carbon sugar, and one molecule of phosphate, which refers to a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. Whereas "deoxyribonucleotides" are the constituent materials of deoxyribonucleic acid (DNA), also consisting of one molecule of base, one molecule of five-carbon sugar, and one molecule of phosphate, which refers to a nucleotide having a hydrogen group instead of a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. "Nucleotides" are generally referred to by a single letter representing the base therein: "A (a)" refers to a deoxyadenylate or adenylate containing adenine, "C (c)" refers to a deoxycytidylate or cytidylate containing cytosine, "G (g)" refers to a deoxyguanylate or guanylate containing guanine, "U (u)" refers to a uridylate containing uracil, and "T (t)" refers to a deoxythymidylate containing thymine.

[0053] As used herein, the terms "polynucleotide" and "nucleic acid" are used interchangeably to refer to a polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA). "Polynucleotide sequence," "nucleic acid sequence," and "nucleotide sequence" are used interchangeably to indicate the order of nucleotides in a polynucleotide. It will be understood by those skilled in the art that a DNA coding strand (sense strand) and its encoded RNA can be considered to have the same nucleotide sequence, with the deoxythymidylates in the DNA coding strand sequence corresponding to the uridylates in the encoded RNA sequence. Thus, although nucleotide sequences described herein can be represented as RNA sequences (including U), one skilled in the art will be able to determine the corresponding DNA sequence (i.e., replacing U with T), and vice versa. Thus, in the present context, when a DNA sequence comprising is represented as an RNA sequence, it will be understood that the DNA comprises the corresponding DNA sequence of the RNA sequence.

[0054] As used herein, the term "expression" includes transcription and / or translation of a nucleotide sequence. Thus, expression can involve production of a transcript and / or a polypeptide. The term "transcription" relates to the process of transcribing the genetic code in a DNA sequence into RNA (transcript). The term "in vitro transcription" refers to the synthesis of RNA, in particular mRNA, in vitro in a system free of cells, for example in an appropriate cell extract or reaction system (see, e.g., Pardi N., Muramatsu H., Weissman D., Kariko K. (2013). In: Rabinovich P. (eds) Synthetic Messenger RNA and Cell Metabolism Modulation. Methods in Molecular Biology (Methods and Protocols), vol 969. Humana Press, Totowa, NJ.).

[0055] As used herein, "encoding" refers to the inherent property of specific sequences of nucleotides in a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of the polymeric and large-molecular-weight molecules necessary for the functioning of the cell or other biological system. Thus, a gene codes for a protein if its mRNA, produced by transcription and translation in a cell or other biological system, results in the synthesis of the protein.

[0056] As used herein, the term "operably linked" refers to a functional spatial arrangement of two or more nucleotide sequences. For example, a nucleotide sequence encoding a signal peptide can be operably linked to a nucleotide sequence encoding a polypeptide of interest, such that the signal peptide facilitates secretory expression of the polypeptide of interest.

[0057] In the present context, a "vector" is a vehicle for introducing a foreign polynucleotide into a host cell, which allows the foreign polynucleotide to be amplified or expressed when the vector is transformed into an appropriate host cell. The vector is usually episomal, but can be designed to integrate the gene or part thereof into the chromosome of the genome. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, pox viral vectors, and baculoviral vectors, and the like.

[0058] As used herein, the term "expression vector" refers to a vector capable of expressing a polynucleotide of interest (including DNA and RNA). For example, in an expression vector, a polynucleotide (including DNA and RNA) encoding a polypeptide of interest can be operably linked to regulatory sequences (such as promoters and ribosome binding sites) capable of affecting expression of the polynucleotide. The regulatory sequences can include promoter and terminator sequences, and optionally can include origins of replication, selectable markers, enhancers, polyadenylation signals, and the like. The expression vector can be a plasmid, a bacteriophage vector, a recombinant virus, or other vector that, when introduced into an appropriate host cell, results in expression of the polynucleotide of interest. Suitable expression vectors are well known to those skilled in the art. An expression vector can be prepared as desired by one skilled in the art to be a vector that is replicable in a host cell, maintained episomally in a host cell, or integrated into the genome of a host cell.

[0059] As used herein, the term "host cell" refers to a cell used to receive, maintain, replicate, express a nucleic acid or vector. In some embodiments, a host cell can be a cell in which a polypeptide of the application is expressed.

[0060] The term "amino acid" is intended to encompass all molecules, whether natural or synthetic, which include both an amino functional group and an acid functional group and are capable of being included in a polymer of naturally occurring amino acids. Exemplary amino acids include naturally occurring amino acids; analogs, derivatives, and homologs; amino acid analogs with variant side chains; and all stereoisomers of any of the foregoing. As used herein, the term "amino acid" includes D- or L-optical isomers and peptidomimetics.

[0061] The terms "polypeptide," "peptide," and "protein" (if single chain) are used interchangeably herein to refer to polymers of amino acids of any length. The polymer can be straight or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. A polypeptide can be isolated from a natural source, produced by recombinant techniques from a eukaryotic or prokaryotic host, or it can be a product of synthetic procedures.

[0062] As used herein, the term "naturally occurring" means found in nature, unmodified by man, including naturally occurring mutant forms.

[0063] As used herein, the term "non-naturally occurring" means not found in nature, modified by man to differ from the naturally occurring counterpart. For example, a non-naturally occurring TPO can have one or more amino acid substitutions, deletions, or insertions compared to a naturally occurring TPO.

[0064] As used herein, the term "treatment" refers to therapeutic measures that cure, slow down, lessen the symptoms of, and / or halt or slow the progression of an existing diagnosed pathological condition or disorder. The term "prevention" or "preventive" refers to prophylactic measures that prevent the progression of an undiagnosed target pathological condition or disorder. Thus, a "subject in need thereof can include a subject already suffering from a disease; a subject predisposed to a disease; and a subject in need of prevention of a disease.

[0065] As used herein, "therapeutic effect" means an effect resulting from the treatment of an individual that alters, usually improves or ameliorates, the symptoms of a disease or disease condition, or cures the disease or disease condition.

[0066] The term "therapeutically effective amount" refers to the amount of an antibody, polypeptide, nucleic acid, small organic molecule, or other drug that is effective to treat a disease or condition in a "treatment" subject or mammal.

[0067] As used herein, the term "subject" describes an organism, such as a mammal, to which treatment with the nucleic acids, compositions, or pharmaceutical compositions of the application can be provided. Preferably, the subject is a rodent and a human.

[0068] As used herein and unless otherwise indicated, the term "lipid" refers to a group of organic compounds that includes, but is not limited to, fatty acid esters, and is generally characterized by poor solubility in water but solubility in many nonpolar organic solvents. Although lipids generally have weak water solubility, certain classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG 2000) have limited water solubility and can be soluble in water under certain conditions. Known lipid types include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids can be at least divided into three classes: (1) "simple lipids" including fats and oils, and waxes; (2) "compound lipids" including phospholipids and glycolipids (e.g., DMPE-PEG 2000); and (3) "derived lipids" such as sterol-like compounds. In addition, as used herein, lipids also include lipid-like compounds. The term "lipid-like compound" or simply "lipid-like" refers to a lipid-like compound (e.g., an amphiphilic compound having lipid-like physical properties).

[0069] The term "lipid nanoparticle" or "LNP" refers to a particle having at least one dimension in the nanometer (nm) scale (e.g., 1 to 1,000 nm) that contains one or more types of lipid molecules. The LNPs provided herein can further contain at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule that is partially or completely encapsulated within a lipid shell. In particular, in some embodiments, where the payload is a negatively charged molecule (e.g., an mRNA encoding a fusion protein), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is contemplated that the cationic lipid can interact with the negatively charged payload molecule and facilitate payload incorporation and / or encapsulation into the LNP during LNP formation. Other lipids that can form part of the LNP as provided herein include, but are not limited to, neutral lipids and charged lipids, such as sterols, and various zwitterionic lipids. In certain embodiments, the LNP according to the present disclosure comprises one or more lipids of Series 01, 02, 03, and 04, e.g., one or more lipids of formula (01-I) (and its subformulae (01-I-B), (01-I-B'), (01-I-B"), (01-I-C), (01-I-D), and (01-I-E)), (02-I), (02-II), (03-I), (04-I), and (04-III) as described herein.

[0070] The term "cationic lipid" refers to a lipid that is positively charged at any pH value or hydrogen ion activity of its environment, or is capable of becoming positively charged in response to the pH value or hydrogen ion activity of its environment, e.g., the environment of its intended use. Thus, the term "cationic" encompasses "permanent cationic" and "cationizable." In certain embodiments, the positive charge in the cationic lipid is caused by the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that is positively charged in the environment of its intended use, e.g., at physiological pH. In certain embodiments, the cationic lipid comprises one or more lipids of Series 01, 02, 03, and 04, e.g., one or more lipids of formula (01-I) (and its subformulae (01-I-B), (01-I-B'), (01-I-B"), (01-I-C), (01-I-D), and (01-I-E)), (02-I), (02-II), (03-I), (04-I), and (04-III) as described herein.

[0071] The term "polymer-conjugated lipid" refers to a molecule that comprises both a lipid moiety and a polymer moiety. An example of a polymer-conjugated lipid is a PEGylated lipid (PEG-lipid), wherein the polymer moiety comprises a polyethylene glycol.

[0072] The term“neutral lipid” encompasses any lipid molecule that exists in uncharged form or in a neutral zwitterionic form at a selected pH value or within a selected pH range. In some embodiments, the selected useful pH value or range corresponds to the pH conditions in the environment of a predetermined lipid use, such as a physiological pH value. As non-limiting examples, neutral lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); phosphatidylethanolamines such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 2-((2,3-bis(octadecyloxy)propyl)dimethylammonio)ethyl hydrogen phosphate (DOCP); sphingomyelin (SM); ceramides. The neutral lipids provided herein can be synthetic or derived from natural sources or compounds (isolated or modified therefrom).

[0073] The term“steroid” refers to a lipid with a cyclopentanoperhydrophenanthrene as a parent nucleus, including sterols and sterol derivatives. Steroid compounds do not contain bound fatty acids and are non-saponifiable lipids. In the present context, steroids can serve as structural lipids in the lipid component of the nanoparticle compositions. As non-limiting examples, steroids that can be used in conjunction with the present disclosure include, but are not limited to, cholesterol, coprostanol, sitosterol, ergosterol, campesterol, soysterol, and mixtures thereof.

[0074] The term“charged lipid” encompasses any lipid molecule that exists in a positively charged or negatively charged form at a selected pH value or within a selected pH range. In some embodiments, the selected pH value or range corresponds to the pH conditions in the environment of a predetermined lipid use, such as a physiological pH value. As non-limiting examples, charged lipids that can be used in conjunction with the present disclosure include, but are not limited to, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinates, dialkyldimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylphosphocholine, dimethylaminoethane carbamoyl sterol (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'-rac-glycerol) sodium salt (DOPG-Na), and 1,2-dioleoyl-sn-glycero-3-phospho sodium salt (DOPA-Na). The charged lipids provided herein can be synthetic or derived from natural sources or compounds (isolated or modified therefrom).

[0075] As used herein and unless otherwise indicated, the term "alkyl" refers to a saturated straight chain or branched chain hydrocarbon chain radical. In an embodiment, the alkyl group has, for example, 1 to 24 carbon atoms (Ci-C 24 alkyl), 4 to 20 carbon atoms (C4-C 20 alkyl), 6 to 16 carbon atoms (C6-C 16 alkyl), 6 to 9 carbon atoms (C6-C9 alkyl), 1 to 15 carbon atoms (Ci-C 15 alkyl), 1 to 12 carbon atoms (Ci-C 12 alkyl), 1 to 8 carbon atoms (Ci-C8 alkyl), or 1 to 6 carbon atoms (Ci-C6 alkyl), and is attached to the rest of the molecule by a single bond. Examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (iso-propyl), n-butyl, n-pentyl, 1,1-dimethylethyl (tert-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise specified, alkyl is optionally substituted.

[0076] As used herein and unless otherwise indicated, the term "alkenyl" refers to a straight chain or branched chain hydrocarbon chain radical that contains one or more carbon-carbon double bonds and is composed of carbon and hydrogen atoms only. As understood by one of ordinary skill in the art, the term "alkenyl" also encompasses radicals having "cis" and "trans" configurations or, alternatively, "E" and "Z" configurations. In an embodiment, the alkenyl group has, for example, 2 to 24 carbon atoms (C2-C 24 alkenyl), 4 to 20 carbon atoms (C4-C 20 alkenyl), 6 to 16 carbon atoms (C6-C 16 alkenyl), 6 to 9 carbon atoms (C6-C9 alkenyl), 2 to 15 carbon atoms (C2-C 15 alkenyl), 2 to 12 carbon atoms (C2-C 12 alkenyl), 2 to 8 carbon atoms (C2-C8 alkenyl), or 2 to 6 carbon atoms (C2-C6 alkenyl), and is attached to the rest of the molecule by a single bond. Examples of alkenyl include, but are not limited to, ethenyl, prop-1-enyl, but-1-enyl, pent-1-enyl, pent-1,4-dienyl, and the like. Unless otherwise specified, alkenyl is optionally substituted.

[0077] As used herein and unless otherwise indicated, the term "alkynyl" refers to a straight chain or branched chain hydrocarbon chain radical that contains one or more carbon-carbon triple bonds and is composed of carbon and hydrogen atoms only. In an embodiment, the alkynyl group has, for example, 2 to 24 carbon atoms (C2-C 24 alkynyl), 4 to 20 carbon atoms (C4-C 20 alkynyl), 6 to 16 carbon atoms (C6-C 16alkynyl), 6 to 9 carbon atoms (C6-C9alkynyl), 2 to 15 carbon atoms (C2-C 15 alkynyl), 2 to 12 carbon atoms (C2-C 12 alkynyl), 2 to 8 carbon atoms (C2-C8alkynyl), or 2 to 6 carbon atoms (C2-C6alkynyl), and is attached to the rest of the molecule by a single bond. Examples of alkynyl include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise indicated, alkynyl groups are optionally substituted.

[0078] As used herein and unless otherwise indicated, the term "alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a group, consisting solely of carbon and hydrogen atoms and being saturated. In one embodiment, the alkylene has, for example, 1 to 24 carbon atoms (C1-C 24 alkylene), 1 to 15 carbon atoms (C1-C 15 alkylene), 1 to 12 carbon atoms (C1-C 12 alkylene), 1 to 8 carbon atoms (C1-C8alkylene), 1 to 6 carbon atoms (C1-C6alkylene), 2 to 4 carbon atoms (C2-C4alkylene), 1 to 2 carbon atoms (C1-C2alkylene). Examples of alkylene include, but are not limited to, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is attached to the rest of the molecule through a single bond and to the group through a single bond. The points of attachment of the alkylene chain to the rest of the molecule and to the group can be through one carbon or any two carbons within the chain. Unless otherwise indicated, alkylene chains are optionally substituted.

[0079] As used herein and unless otherwise indicated, the term "alkenylene" refers to a straight or branched divalent hydrocarbon chain linking the rest of the molecule to a group, consisting solely of carbon and hydrogen atoms and containing one or more carbon-carbon double bonds. In one embodiment, the alkenylene has, for example, 2 to 24 carbon atoms (C2-C 24 alkenylene), 2 to 15 carbon atoms (C2-C 15 alkenylene), 2 to 12 carbon atoms (C2-C 12 alkenylene), 2 to 8 carbon atoms (C2-C8alkenylene), 2 to 6 carbon atoms (C2-C6alkenylene), or 2 to 4 carbon atoms (C2-C4alkenylene). Examples of alkenylene include, but are not limited to, ethynylene, propynylene, n-butenylene, and the like. The alkenylene is attached to the rest of the molecule through a single or double bond and to the group through a single or double bond. The points of attachment of the alkenylene to the rest of the molecule and to the group can be through one carbon or any two carbons within the chain. Unless otherwise indicated, alkenylene groups are optionally substituted.

[0080] As used herein and unless otherwise stated, the term "cycloalkyl" refers to a non-aromatic saturated monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms. Cycloalkyl groups may include fused or bridged ring systems. In one embodiment, the cycloalkyl group has, for example, 3 to 15 ring carbon atoms (C3-C4). 15 cycloalkyl groups, 3 to 10 cyclic carbon atoms (C3-C4) 10 Cycloalkyl groups (3 to 8 carbon atoms in a ring, C3-C8 cycloalkyl). The cycloalkyl group is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, adamantyl, norbornyl, decahydronaphthyl, 7,7-dimethyl-bicyclo[2.2.1]heptyl, etc. Unless otherwise stated, the cycloalkyl group is optionally substituted.

[0081] As used herein and unless otherwise stated, the term "cycloalkylene" refers to a divalent cycloalkyl group. Unless otherwise stated, cycloalkylene groups are optionally substituted.

[0082] As used herein and unless otherwise stated, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon group consisting only of carbon and hydrogen atoms and including one or more carbon-carbon double bonds. Cycloalkenyl groups may include fused or bridged ring systems. In one embodiment, the cycloalkenyl group has, for example, 3 to 15 cyclic carbon atoms (C3-C4). 15 Cycloalkenyl), 3 to 10 cyclic carbon atoms (C3-C 10 Cycloalkenyl groups (3 to 8 cyclic carbon atoms, C3-C8 cycloalkenyl groups) are cyclic. The cycloalkenyl group is attached to the rest of the molecule by a single bond. Examples of monocyclic cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, etc. Unless otherwise stated, the cycloalkenyl group is optionally substituted.

[0083] As used herein and unless otherwise stated, the term "cycloene imide" is a divalent cycloene group. Unless otherwise stated, cycloene imide groups are optionally substituted.

[0084] As used herein and unless otherwise indicated, the term "heterocyclyl" refers to a non-aromatic, monocyclic or polycyclic moiety containing one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorus, and sulfur. The heterocyclyl group can be attached to the main structure at any heteroatom or carbon atom. The heterocyclyl group can be monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic systems, where the polycyclic systems can be fused, bridged, or spiro systems. The heterocyclyl polycyclic system can contain one or more heteroatoms in one or more rings. The heterocyclyl group can be saturated or partially unsaturated. A saturated heterocyclyl group can be referred to as a "heterocycloalkyl group." A partially unsaturated heterocycloalkyl group can be referred to as a "heterocycloalkenyl group" when the heterocyclyl contains at least one double bond, or as a "heterocycloalkynyl group" when the heterocyclyl contains at least one triple bond. In an embodiment, the heterocyclyl group has, for example, 3 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 18 ring atoms (4- to 18-membered heterocyclyl), 5 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 8 ring atoms (4- to 8-membered heterocyclyl), or 5 to 8 ring atoms (5- to 8-membered heterocyclyl). When appearing herein, a numerical range, such as "3 to 18," refers to each integer within the given range; for example, "3 to 18 ring atoms" means that the heterocyclyl group can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, and so on, up to and including 18 ring atoms. Examples of heterocyclyl groups include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thiophenyl, pyridyl, piperidinyl, quinolinyl, and isoquinolinyl. Unless otherwise specified, a heterocyclyl group is optionally substituted.

[0085] As used herein and unless otherwise indicated, the term "heterocyclyl" refers to a non-aromatic, monocyclic or polycyclic moiety containing one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorus, and sulfur. The heterocyclyl group can be attached to the main structure at any heteroatom or carbon atom. The heterocyclyl group can be monocyclic, bicyclic, tricyclic, tetracyclic, or other polycyclic systems, where the polycyclic systems can be fused, bridged, or spiro systems. The heterocyclyl polycyclic system can contain one or more heteroatoms in one or more rings. The heterocyclyl group can be saturated or partially unsaturated. A saturated heterocyclyl group can be referred to as a "heterocycloalkyl group." A partially unsaturated heterocycloalkyl group can be referred to as a "heterocycloalkenyl group" when the heterocyclyl contains at least one double bond, or as a "heterocycloalkynyl group" when the heterocyclyl contains at least one triple bond. In an embodiment, the heterocyclyl group has, for example, 3 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 18 ring atoms (4- to 18-membered heterocyclyl), 5 to 18 ring atoms (3- to 18-membered heterocyclyl), 4 to 8 ring atoms (4- to 8-membered heterocyclyl), or 5 to 8 ring atoms (5- to 8-membered heterocyclyl). When appearing herein, a numerical range, such as "3 to 18," refers to each integer within the given range; for example, "3 to 18 ring atoms" means that the heterocyclyl group can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, and so on, up to and including 18 ring atoms. Examples of heterocyclyl groups include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isoxazolyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furanyl, tetrahydrofuranyl, thiophenyl, pyridyl, piperidinyl, quinolinyl, and isoquinolinyl. Unless otherwise specified, a heterocyclyl group is optionally substituted.

[0086] As used herein and unless otherwise indicated, the term "aryl" refers to a monocyclic aromatic group and / or a polycyclic monovalent aromatic group containing at least one aromatic hydrocarbon ring. In certain embodiments, the aryl group has 6 to 18 ring carbon atoms (C6-C 18 aryl), 6 to 14 ring carbon atoms (C6-C 14 aryl), or 6 to 10 ring carbon atoms (C6-C 10Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthracenyl, phenanthrenyl, pyrenyl, biphenyl, and terphenyl. The term "aryl" also refers to bicyclic, tricyclic, or other polycyclic hydrocarbon rings wherein at least one ring is aromatic and the other rings can be saturated, partially unsaturated, or aromatic, such as indanyl, indenyl, dihydroindenyl, or tetrahydronaphthyl / tetralinyl. Unless otherwise indicated, aryl groups are optionally substituted.

[0087] As used herein and unless otherwise indicated, the term "arylene" is a divalent aryl group. Unless otherwise indicated, arylene groups are optionally substituted.

[0088] As used herein and unless otherwise indicated, the term "heteroaryl" refers to a monocyclic aromatic group and / or a polycyclic aromatic group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more (e.g., one, one or two, one to three, or one to four) heteroatoms independently selected from O, S, and N. Heteroaryl groups can be attached to the main structure at any heteroatom or carbon atom. In certain embodiments, a heteroaryl group has 5 to 20, 5 to 15, or 5 to 10 ring atoms. The term "heteroaryl" also refers to bicyclic, tricyclic, or other polycyclic rings wherein at least one ring is aromatic and the other rings can be saturated, partially unsaturated, or aromatic, wherein at least one aromatic ring contains one or more heteroatoms independently selected from O, S, and N. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thiophenyl, oxadiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarinyl, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridyl, furopyridyl, thienopyridyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, and xanthenyl. Unless otherwise indicated, heteroaryl groups are optionally substituted.

[0089] As used herein and unless otherwise indicated, the term "heteroarylene" is a divalent heteroaryl group. Unless otherwise indicated, heteroarylene groups are optionally substituted.

[0090] When a group described herein is termed "substituted," it can be substituted with one or more of any suitable substituents. Illustrative examples of substituents include, but are not limited to, those found in the exemplary compounds and embodiments provided herein, as well as: a halogen atom, such as F, CI, Br, or I; a cyano group; an oxo group (=0); a hydroxyl group (-OH); an alkyl group; an alkenyl group; an alkynyl group; a cycloalkyl group; an aryl group; -(C=0)OR'; -0(C=0)R'; -C(=0)R'; -OR'; -S(0) x R'; -S-SR'; -C(=0)SR'; -SC(=0)R'; -NR'R'; -NR'C(=0)R'; -C(=0)NR'R'; -NR'C(=0)NR'R'; -OC(=0)NR'R'; -NR'C(=0)OR'; -NR'S(0) x R'; and -S(0) x R'; and -S(0) x NR'R', where: R' is, at each occurrence, independently H, C1-C 15 alkyl or cycloalkyl, and x is 0, 1, or 2. In some embodiments, the substituent is C1-C 12 alkyl. In other embodiments, the substituent is a cycloalkyl group. In other embodiments, the substituent is a halo group, such as a fluoro group. In other embodiments, the substituent is an oxo group. In other embodiments, the substituent is a hydroxyl group. In other embodiments, the substituent is an alkoxy group (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group (-NR'R').

[0091] As used herein and unless otherwise indicated, the term "optionally present" or "optionally" (e.g., optionally substituted) means that the event or circumstance subsequently described can or can not occur, and that the description includes situations where the event or circumstance occurs and where it does not. For example, "optionally substituted alkyl" means that the alkyl group can or can not be substituted, and that the description includes both substituted alkyl groups and alkyl groups without substitution.

[0092] As used herein and unless otherwise indicated, the term "prodrug" of a biologically active compound refers to a compound that can be converted to a biologically active compound under physiological conditions or by solvolysis. In one embodiment, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a biologically active compound. A prodrug can be inactive until converted to the biologically active compound when administered to a subject in need thereof. Prodrugs are often rapidly transformed in vivo to yield the parent biologically active compound, for example, by hydrolysis in blood. Prodrug compounds generally provide an advantage of solubility, tissue compatibility, or delayed release in a mammalian organism (see Bundgard, H., Design of Prodrugs (1985), pp. 7-9, 21-24 (Elsevier, Amsterdam)). A discussion of prodrugs is provided in Higuchi, T., et al., A.C.S. Symposium Series, Vol. 14, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

[0093] In one embodiment, the term "prodrug" is also intended to encompass any covalently bonded carriers, which release the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of a compound can be prepared by modifying functional groups present on the compound in such a way that their modifications can be cleaved in vivo to yield the parent compound. Prodrugs include compounds having a hydroxy, amino, or mercapto group bonded to any group which, when the prodrug of the compound is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or free mercapto group, respectively.

[0094] Examples of prodrugs include, but are not limited to, acetate, formate, and benzoate derivatives of alcohol functional groups or amide derivatives of amine functional groups in the compounds provided herein.

[0095] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable salt" includes both acid and base addition salts.

[0096] Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, salts of hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; and organic acids such as, but not limited to, acetic acid, 2,2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, camphor-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfic acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxo-glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-2-naphthoic acid, nicotinic acid, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid and the like.

[0097] Examples of pharmaceutically acceptable base addition salts include, but are not limited to, salts prepared by the addition of inorganic or organic bases to the free acid compounds. Salts derived from inorganic bases include, but are not limited to, sodium salts, potassium salts, lithium salts, ammonium salts, calcium salts, magnesium salts, iron salts, zinc salts, copper salts, manganese salts, aluminum salts, and the like. In one embodiment, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines; substituted amines including naturally occurring substituted amines; cyclic amines and basic ion-exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, deanol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, benethamine, benzathine, ethylenediamine, glucosamine, methylglucosamine, theobromine, triethanolamine, tromethamine, purines, piperizine, piperidine, N-ethylpiperidine, polyamine resins, and the like. In one embodiment, the organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

[0098] The compounds described herein may contain one or more asymmetric centers, thus yielding enantiomers, diastereomers, and other stereoisomers, which may be defined by absolute stereochemistry as (R)- or (S)-, or for amino acids as (D)- or (L)-. Unless otherwise stated, the compounds described herein are intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) vs. (-), (R)- vs. (S)-, or (D)- vs. (L)- isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques such as chromatography and fractional crystallization. Conventional techniques for the preparation / separation of individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of racemic mixtures (or racemic mixtures of salts or derivatives) using, for example, chiral high-performance liquid chromatography (HPLC). When the compounds described herein contain alkene double bonds or other geometrically asymmetric centers, unless otherwise stated, the compounds are intended to include E and Z geometric isomers. Similarly, all tautomers are also intended to be included.

[0099] As used herein and unless otherwise stated, the term "isomer" refers to different compounds having the same molecular formula. "Stereoisomers" are isomers that differ only in the spatial arrangement of their atoms. "Restricted rotational isomers" are stereoisomers obtained by restricted rotation around a single bond. "Enantiomers" are a pair of stereoisomers that are non-overlapping mirror images of each other. A mixture of any proportion of a pair of enantiomers may be called a "racemic" mixture. "Diadiaomers" are stereoisomers that have at least two asymmetric atoms but are not mirror images of each other.

[0100] "Stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In some embodiments, the compounds described herein are isolated as E or Z isomers. In other embodiments, the compounds described herein are mixtures of E and Z isomers.

[0101] "Tautomers" refer to the balanced isomers of a compound. The concentration of the isomers will depend on the environment in which the compound is present and can vary depending on, for example, whether the compound is a solid or in an organic solution or an aqueous solution.

[0102] It should also be noted that the compounds described herein may contain atomic isotopes in non-natural proportions at one or more atoms. For example, the compounds may contain radioactive isotopes, such as tritium ( 3 H), Iodine-125 ( 125 I), sulfur-35( 35 S) or carbon-14 ( 14 C) Radiolabeling, or isotope enrichment, such as deuterium ( 2 H), carbon-13 (13 C) or nitrogen-15 15 N). As used herein, an "isotopologue" is an isotopically enriched compound. The term "isotopically enriched" means that the isotopic composition of an atom differs from the natural isotopic composition of that atom. "Isotopically enriched" can also mean that the isotopic composition of at least one atom contained by a compound differs from the natural isotopic composition of that atom. The term "isotopic composition" refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, e.g., cancer therapeutic agents; research reagents, e.g., binding assay reagents; and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of the compounds described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds described herein are provided, e.g., isotopologues that are deuterium, carbon-13, and / or nitrogen-15 enriched. As used herein, "deuterated" means that at least one hydrogen (H) in a compound has been replaced with deuterium (indicated as D or 2 H). In some embodiments, the compound is enriched with deuterium at at least one position.

[0103] It should be noted that if there is a discrepancy between a depicted structure and the name of the structure, the depicted structure is intended to govern.

[0104] As used herein and unless otherwise indicated, the term "pharmaceutically acceptable carrier, diluent or excipient" includes any adjuvant, carrier, excipient, glidant, sweetening, diluting, preserving, dye / colorant, flavor-enhancing, surface-active, wetting or emulsifying agent that is approved by the United States Food and Drug Administration as being acceptable for use in humans or domestic animals.

[0105] The term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts optionally selected, e.g., an mRNA molecule provided herein.

[0106] Thrombopoietin precursor and nucleic acid encoding thrombopoietin precursor

[0107] In one aspect, the present application provides a nucleic acid comprising a polynucleotide encoding a thrombopoietin (TPO) precursor, wherein the TPO precursor comprises an N-terminal signal peptide and a TPO polypeptide, the signal peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 70, and 77.

[0108] In another aspect, the present application provides a thrombopoietin (TPO) precursor comprising an N-terminal signal peptide and a TPO polypeptide, and the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 70, and 77.

[0109] TPO and TPO precursor

[0110] “Thrombopoietin” or “TPO” in its general sense refers to a glycoprotein that is capable of binding to TPO receptor (TPO-R) and having thrombopoietic activity. Generally, TPO can comprise an EPO-like domain at the N-terminus and a glycosyl-rich domain at the C-terminus. It is generally believed that the EPO-like domain (e.g., amino acids 1-153 of SEQ ID NO: 16) is responsible for the specific binding to TPO-R; while the glycosyl-rich domain (e.g., amino acids 154-332 of SEQ ID NO: 16) can increase the stability of the TPO molecule and prolong its circulating half-life. It is believed that the naturally occurring TPO is synthesized as a precursor protein with an N-terminal signal peptide (e.g., a signal peptide with an amino acid sequence as set forth in SEQ ID NO: 6). After the N-terminal signal peptide is cleaved, the remaining amino acid sequence of the precursor protein is glycosylated into a glycoprotein. Unless specifically indicated or clearly contradicted by context, the definition of “thrombopoietin,” “TPO,” or “TPO polypeptide” as used herein encompasses any form of TPO polypeptide, including but not limited to naturally occurring TPO and modified and unmodified forms of non-naturally occurring TPO, the modifications including but not limited to glycosylation, phosphorylation, and disulfide bond formation.

[0111] As used herein, the term “thrombopoietin precursor” or “TPO precursor” refers to a polypeptide comprising an N-terminal signal peptide and a TPO polypeptide. The TPO precursor can be processed (e.g., in a host cell) into a TPO having thrombopoietic activity.

[0112] As used herein, “signal peptide” refers to a short peptide that directs the nascent protein to the secretory pathway, which can generally be about 5-30 amino acids in length. For secretory proteins, it is generally believed that the signal peptide is cleaved off before the nascent protein is secreted to the extracellular space. Exemplary amino acid sequences of signal peptides are set forth in Tables 5 and 8. The signal peptide can be fused directly or through a linker to the N-terminus of a TPO polypeptide. In one embodiment, the signal peptide is fused directly to the N-terminus of a TPO polypeptide.

[0113] The present inventors have surprisingly found that replacing the signal peptide (SEQ ID NO: 6) in a naturally occurring TPO precursor with a signal peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 70 and 77 can enhance the expression of TPO in vitro and in vivo. In particular, without wishing to be bound by any theory, the present inventors have surprisingly found that TPO produced from a TPO precursor comprising a signal peptide of the amino acid sequence of any one of SEQ ID NOs: 1, 70 and 77 has better pharmacokinetic characteristics. Thus, according to another specific aspect of the present application, a polynucleotide encoding a signal peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 70 and 77 operably linked to the 5' end of a polynucleotide encoding a TPO polypeptide is particularly advantageous in enhancing the expression of TPO and / or efficiently promoting thrombopoiesis. Thus, according to one specific aspect of the present application, the nucleic acid of the present application (in particular, a nucleic acid encoding a TPO precursor as set forth in any one of SEQ ID NOs: 29, 104 and 105, such as a nucleic acid comprising the nucleotide sequence of any one of SEQ ID NOs: 30-33, 99-102, 106 and 107) allows for high expression of TPO (e.g., in humans).

[0114] In preferred embodiments, the TPO polypeptide useful in the present application can be a mammalian TPO polypeptide, such as a human TPO polypeptide or a non-human primate TPO polypeptide. In one embodiment, the TPO polypeptide is a human TPO polypeptide. Examples of human TPO polypeptides can include naturally occurring human TPO polypeptides as described below.

[0115] The TPO polypeptide can be a naturally occurring TPO polypeptide or a non-naturally occurring TPO polypeptide.

[0116] The naturally occurring TPO polypeptide includes a TPO polypeptide produced from any naturally occurring TPO precursor. Exemplary amino acid sequences of naturally occurring human TPO polypeptides are set forth in SEQ ID NO: 16 (the amino acid sequence of its corresponding TPO precursor is set forth in, e.g., GenBank Accession No. NP_000451.1), SEQ ID NO: 64 (the amino acid sequence of its corresponding TPO precursor is set forth in, e.g., GenBank Accession No. NP_001171068.1), SEQ ID NO: 65 (the amino acid sequence of its corresponding TPO precursor is set forth in, e.g., GenBank Accession No. NP_001171069.1) and SEQ ID NO: 66 (the amino acid sequence of its corresponding TPO precursor is set forth in, e.g., GenBank Accession No. NP_001276926.1).

[0117] Non-naturally occurring TPO polypeptides can include variants of naturally occurring TPO polypeptides (see, e.g., WO2005010043A1 and WO0000612A1, the disclosures of which are incorporated herein in their entireties). For example, a non-naturally occurring TPO polypeptide can comprise one or more valine (Val) substitutions at one or more positions corresponding to amino acids 46, 128, 131, and 141 of SEQ ID NO: 16, as compared to a naturally occurring TPO polypeptide. A non-naturally occurring TPO can also comprise one or more additional N-linked glycosylation sites as compared to a naturally occurring TPO. For example, a non-naturally occurring TPO can comprise one or more asparagine (Asn) substitutions at one or more positions corresponding to amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16 (e.g., amino acids 108, 117, 153, 164, 193, 117, or 164, or amino acids 108 and 147, amino acids 108 and 164, amino acids 147 and 164, amino acids 108, 117, and 164, or amino acids 157 and 164), as compared to a naturally occurring TPO.

[0118] In some embodiments, the TPO polypeptide comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 16, 64, 65, and 66.

[0119] In a preferred embodiment, the TPO polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16. In an embodiment, the TPO polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16 and comprises one or more valine substitutions at one or more positions corresponding to amino acids 46, 128, 131, and 141 of SEQ ID NO: 16. In an embodiment, the TPO polypeptide comprises an amino acid sequence with a valine substitution at the position of amino acid 131 or 141 of SEQ ID NO: 16. In an embodiment, the TPO polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16 and comprises one or more asparagine substitutions at one or more positions corresponding to amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16 (e.g., amino acids 108, 117, 153, 164, 193, 117 or 164, or amino acids 108 and 147, amino acids 108 and 164, amino acids 147 and 164, amino acids 108, 117 and 164, or amino acids 157 and 164). In an embodiment, the TPO polypeptide comprises an amino acid sequence with one or more asparagine substitutions at one or more positions corresponding to amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16 (e.g., amino acids 108, 117, 153, 164, 193, 117 or 164, or amino acids 108 and 147, amino acids 108 and 164, amino acids 147 and 164, amino acids 108, 117 and 164, or amino acids 157 and 164).

[0120] In an embodiment, the TPO polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 16, 64, 65, and 66. In a preferred embodiment, the TPO polypeptide comprises the amino acid sequence of SEQ ID NO: 16.

[0121] In some preferred embodiments, the TPO precursor comprises an N-terminal signal peptide and a TPO polypeptide, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 and the TPO polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In a preferred embodiment, the TPO precursor comprises the amino acid sequence of SEQ ID NO: 29.

[0122] In some preferred embodiments, the TPO precursor comprises an N-terminal signal peptide and a TPO polypeptide, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 70 and the TPO polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In a preferred embodiment, the TPO precursor comprises the amino acid sequence of SEQ ID NO: 104.

[0123] In some preferred embodiments, the TPO precursor comprises an N-terminal signal peptide and a TPO polypeptide, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 77 and the TPO polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In a preferred embodiment, the TPO precursor comprises the amino acid sequence of SEQ ID NO: 105.

[0124] Nucleic acid

[0125] A nucleic acid of the present application can be single-stranded or double-stranded. Nucleic acids include, but are not limited to, DNA, cDNA, RNA (e.g., mRNA, circRNA, and saRNA), recombinantly produced, and chemically synthesized. A nucleic acid of the present application can be comprised in a vector. A nucleic acid of the present application can include naturally occurring, synthetic, and modified nucleotides. In some embodiments, a nucleic acid of the present application is used to express a TPO precursor described herein in a cell. A nucleic acid of the present application can be DNA or RNA.

[0126] A nucleic acid can comprise one or more segments (nucleotide fragments) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more segments). A nucleic acid can comprise a segment that encodes a polypeptide of interest (e.g., a TPO precursor). In particular embodiments, a nucleic acid can comprise a coding sequence for a polypeptide of interest (e.g., a TPO precursor) as well as regulatory sequences (including, but not limited to, transcriptional and translational regulatory sequences). In an embodiment, the regulatory sequences comprise one or more of the following: a promoter sequence, a 5’ untranslated region (5’-UTR) sequence, a 3’ untranslated region (3’-UTR) sequence, and a poly(A) sequence.

[0127] Coding sequence

[0128] As used herein, “coding sequence” refers to a nucleotide sequence in a nucleic acid that can be used as a template to synthesize a nucleotide sequence having a determined nucleotide sequence (e.g., tRNA and mRNA) or a determined amino acid sequence in a biological process. A coding sequence can be a DNA sequence or an RNA sequence. A DNA sequence or an mRNA sequence can be considered to encode a polypeptide if the mRNA corresponding to the DNA sequence (including the coding strand that is identical to the mRNA sequence and the template strand that is complementary to it) is translated into the polypeptide in a biological process.

[0129] As used herein, a "codon" refers to a sequence of three consecutive nucleotides in a nucleic acid (also referred to as a triplet code), which encodes a particular amino acid. The frequency of use of synonymous codons (codons that encode the same amino acid) varies among different species, and is referred to as "codon bias." It is generally accepted that, for a given species, coding sequences that use the preferred codons of that species can have higher translational efficiency and accuracy in that species' expression system. Thus, a nucleic acid can be "codon optimized," i.e., the codons in the nucleic acid are changed to reflect the codon bias of the host cell, preferably without changing the encoded amino acid sequence. Those skilled in the art will appreciate that, due to the degeneracy of the code, a nucleic acid of the application can comprise a coding sequence that is not identical to the coding sequences described herein (e.g., has about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the coding sequences described herein) but encodes the same amino acid sequence. In particular embodiments, a nucleic acid of the application comprises codons optimized for a host (e.g., a subject, particularly a human) cell, such that the encoded polypeptide is optimally expressed in the host (e.g., a subject, particularly a human).

[0130] In some embodiments, a nucleic acid of the application (e.g., an mRNA) comprises a coding sequence that encodes a TPO precursor as described herein. In one embodiment, the coding sequence that encodes a TPO precursor comprises a coding sequence that encodes a signal peptide and a coding sequence that encodes a TPO polypeptide.

[0131] In some embodiments, a nucleic acid of the application comprises a nucleotide sequence that is complementary to a coding sequence described herein. In some embodiments, the coding sequence comprises a start codon at its 5' end and a stop codon at its 3' end. In some embodiments, the coding sequence comprises an open reading frame (ORF) described herein.

[0132] In one embodiment, a nucleic acid of the application (e.g., an mRNA) comprises a polynucleotide that encodes a TPO polypeptide as described herein.

[0133] In an embodiment, the polynucleotide encoding a TPO polypeptide comprises a polynucleotide sequence encoding an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 16, 64, 65, and 66. In an embodiment, the polynucleotide encoding a TPO polypeptide comprises a polynucleotide sequence encoding an amino acid sequence that is (a) at least 95% identical to SEQ ID NO: 16, and optionally (b) (1) comprises one or more valine substitutions at one or more positions corresponding to amino acids 46, 128, 131, and 141 of SEQ ID NO: 16, and / or (2) comprises one or more asparagine substitutions at one or more positions corresponding to amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16. In an embodiment, the polynucleotide encoding a TPO polypeptide comprises a polynucleotide sequence encoding an amino acid sequence that has a valine substitution at the position of amino acid 131 or 141 of SEQ ID NO: 16. In an embodiment, the polynucleotide encoding a TPO polypeptide comprises a polynucleotide sequence encoding an amino acid sequence that has one or more asparagine substitutions at one or more positions of amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16 (e.g., amino acids 108, 117, 153, 164, 193, 117, or 164, or amino acids 108 and 147, amino acids 108 and 164, amino acids 147 and 164, amino acids 108, 117, and 164, or amino acids 157 and 164). In an embodiment, the polynucleotide encoding a TPO polypeptide comprises a polynucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 16, 64, 65, and 66.

[0134] In a preferred embodiment, the polynucleotide encoding a TPO polypeptide comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16. In an embodiment, the polynucleotide encoding a TPO polypeptide comprises (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 16, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 17-20 and 95-98. In an embodiment, the polynucleotide encoding a TPO polypeptide comprises the nucleotide sequence of any one of SEQ ID NOs: 17-20 and 95-98. In a preferred embodiment, the polynucleotide encoding a TPO polypeptide comprises the nucleotide sequence of SEQ ID NO: 17.

[0135] In an embodiment, the polynucleotide encoding the signal peptide comprises (a) an amino acid sequence of SEQ ID NO: 1, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2-5 and 92-94. In an embodiment, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of any one of SEQ ID NOs: 2-5 and 92-94. In a preferred embodiment, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 2.

[0136] In an embodiment, the polynucleotide encoding the signal peptide comprises (a) an amino acid sequence of SEQ ID NO: 70, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 82. In an embodiment, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 82.

[0137] In an embodiment, the polynucleotide encoding the signal peptide comprises (a) an amino acid sequence of SEQ ID NO: 77, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 89. In an embodiment, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 89.

[0138] In an embodiment, the nucleic acid of the application (e.g., mRNA) comprises a polynucleotide encoding a TPO precursor as described herein. In an embodiment, the polynucleotide encoding the TPO precursor comprises a polynucleotide encoding a signal peptide operably linked to the 5’ end of a polynucleotide encoding a TPO polypeptide.

[0139] In a preferred embodiment, the polynucleotide encoding the TPO precursor encodes the amino acid sequence of SEQ ID NO: 29. In an embodiment, the polynucleotide encoding the TPO precursor comprises (a) an amino acid sequence of SEQ ID NO: 29, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102. In an embodiment, the polynucleotide encoding the TPO precursor comprises the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102. In a preferred embodiment, the polynucleotide encoding the TPO precursor comprises the nucleotide sequence of SEQ ID NO: 30.

[0140] In a preferred embodiment, the polynucleotide encoding a TPO precursor encodes the amino acid sequence of SEQ ID NO: 104. In an embodiment, the polynucleotide encoding a TPO precursor comprises (a) an amino acid sequence encoding SEQ ID NO: 104, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 106. In an embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of SEQ ID NO: 106.

[0141] In a preferred embodiment, the polynucleotide encoding a TPO precursor encodes the amino acid sequence of SEQ ID NO: 105. In an embodiment, the polynucleotide encoding a TPO precursor comprises (a) an amino acid sequence encoding SEQ ID NO: 105, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 107. In an embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of SEQ ID NO: 107.

[0142] Regulatory sequences

[0143] As used herein, the term "untranslated region (UTR)" generally refers to a region in RNA (e.g., mRNA) that is not translated into an amino acid sequence (non-coding region), or the corresponding region in DNA. Typically, a UTR located 5' (upstream) of an open reading frame (start codon) can be referred to as a 5'-untranslated region (5'-UTR). A UTR located 3' (downstream) of an open reading frame (stop codon) can be referred to as a 3'-UTR. In the presence of a 5' cap, the 5'-UTR is located downstream of, e.g., directly adjacent to, the 5' cap. In particular embodiments, an optimized "Kozak sequence" can be included in the 5'-UTR, e.g., at a position proximal to the start codon, to improve translation efficiency. In the presence of a poly(A) sequence, the 3'-UTR is located upstream of, e.g., directly adjacent to, the poly(A) sequence.

[0144] As used herein, the term "poly(A) sequence" or "poly(A) tail" refers to a nucleotide sequence comprising consecutive or non-consecutive adenosine acids (or deoxyadenosine acids, for DNA). The poly(A) sequence is typically located at the 3' end of an RNA (or DNA encoding the RNA), e.g., the 3' end (downstream) of the 3'-UTR. In some embodiments, the poly(A) sequence does not comprise nucleotides other than adenosine acids at its 3' end. The poly(A) sequence can be produced during the preparation of the IVT-RNA, either by transcription from the coding sequence of the DNA template by a DNA-dependent RNA polymerase, or by ligation to the free 3' end of the IVT-RNA, e.g., the 3' end of the 3'-UTR, by a DNA-independent RNA polymerase (poly(A) polymerase).

[0145] In some embodiments, the nucleic acid of the application comprises a 5'-UTR. In a preferred embodiment, the 5'-UTR comprises the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103.

[0146] In some embodiments, the nucleic acid of the application comprises a 3'-UTR. In a preferred embodiment, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 46 or 47.

[0147] In some embodiments, the nucleic acid of the application comprises a 5'-UTR and a 3'-UTR. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 46 or 47. In a specific embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 44 or 103, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 46. In yet another specific embodiment, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 45, and the 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 47.

[0148] In some embodiments, the nucleic acid of the application comprises a poly(A) sequence. In an embodiment, the poly(A) sequence comprises consecutive adenosine acids (or deoxyadenosine acids, for DNA). In an embodiment, the poly(A) sequence can comprise at least 20, 30, 40, 50, 60, 70, 75, 80, 85, 95, or 100 and up to 120, 150, 180, 200, 300 adenosine acids (or deoxyadenosine acids, for DNA). In an embodiment, the sequence of consecutive adenosine acids (or deoxyadenosine acids, for DNA) in the poly(A) sequence is interrupted by a sequence comprising U (or T), C, or G nucleotides. In an embodiment, the poly(A) sequence comprises at least 50 nucleotides. In an embodiment, the poly(A) sequence comprises at least 80 nucleotides. In an embodiment, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 70, 80, 90, 100, 120, or 150 nucleotides. In a preferred embodiment, the poly(A) sequence comprises the nucleotide sequence A n wherein n is an integer from 75-175. In a preferred embodiment, n is an integer from 90-155, for example an integer from 120-150. In a preferred embodiment, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO: 48 or 49.

[0149] In an embodiment, the nucleic acid of the application comprises: (1) a 5'-UTR; (2) a polynucleotide encoding a TPO precursor, wherein the TPO precursor comprises an N-terminal signal peptide and a TPO polypeptide, the signal peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 70, and 77; (3) a 3'-UTR; and (4) optionally a poly(A) sequence. In a preferred embodiment, the TPO polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In a preferred embodiment, the TPO precursor comprises the amino acid sequence of any one of SEQ ID NOs: 29, 104, and 105.

[0150] In an embodiment, the polynucleotide encoding a TPO precursor comprises (1) (a) encodes the amino acid sequence of SEQ ID NO: 29, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102; (2) (a) encodes the amino acid sequence of SEQ ID NO: 104, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 106; or (3) (a) encodes the amino acid sequence of SEQ ID NO: 105, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 107. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102, 106, and 107. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of SEQ ID NO: 30.

[0151] In an embodiment, the nucleic acid of the application comprises: (1) a 5'-UTR comprising the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103; (2) a polynucleotide encoding a TPO precursor comprising (a) encodes the amino acid sequence of SEQ ID NO: 29, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102; (3) a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 46 or 47; and (4) an optional poly(A) sequence. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102. In an embodiment, the nucleic acid of the application comprises the nucleotide sequence of any one of SEQ ID NOs: 50-53.

[0152] In an embodiment, the nucleic acid of the application comprises: (1) a 5'-UTR comprising the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103; (2) a polynucleotide encoding a TPO precursor comprising (a) an amino acid sequence encoding SEQ ID NO: 104, and (b) a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of SEQ ID NO: 106; (3) a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 46 or 47; and (4) an optionally present poly(A) sequence. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of SEQ ID NO: 106.

[0153] In an embodiment, the nucleic acid of the application comprises: (1) a 5'-UTR comprising the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103; (2) a polynucleotide encoding a TPO precursor comprising (a) an amino acid sequence encoding SEQ ID NO: 105, and (b) a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of SEQ ID NO: 107; (3) a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 46 or 47; and (4) an optionally present poly(A) sequence. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of SEQ ID NO: 107.

[0154] In an embodiment, the nucleic acid of the application comprises: (1) a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 45; (2) a polynucleotide encoding a TPO precursor comprising the nucleotide sequence of any one of SEQ ID NOs: 30, 106, and 107; (3) a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 47; and (4) an optionally present poly(A) sequence. In a preferred embodiment, the nucleic acid of the application comprises the nucleotide sequence of any one of SEQ ID NOs: 50, 108, and 109.

[0155] RNA

[0156] In some embodiments, the nucleic acid of the application is an RNA. As used herein, the definition of "RNA" encompasses single-stranded, double-stranded, linear, and circular RNAs. The RNA of the application can be a non-amplified RNA or a self-amplifying RNA. The RNA of the application can be a chemically synthesized, recombinantly produced, and in vitro transcribed RNA. In some embodiments, the RNA of the application is used to express a TPO precursor described herein in a host cell.

[0157] In some embodiments, the RNA of the application is single-stranded RNA. In some embodiments, the RNA of the application is in vitro transcribed RNA (IVT-RNA). IVT-RNA can be obtained by in vitro transcription of a DNA template using an RNA polymerase (e.g., as described herein).

[0158] In some embodiments, the RNA of the application is messenger RNA (mRNA). Generally, mRNA can comprise a 5’-UTR, a coding sequence (e.g., a polynucleotide encoding a TPO precursor described herein), a 3’-UTR, and optionally a poly(A) sequence. mRNA can be produced, e.g., by in vitro transcription or chemical synthesis. In an embodiment, the mRNA of the application is obtained by in vitro transcription of a DNA template using an RNA polymerase (e.g., T7 RNA polymerase).

[0159] In some embodiments, the RNA of the application is circular RNA (circRNA). As used herein, the term “circRNA” or “circular RNA” refers to RNA that forms a circular structure through covalent bonds. Generally, circRNA is a single-stranded RNA that can comprise UTRs (5’-UTR and / or 3’-UTR) and an open reading frame, but does not comprise a 5’ cap and a poly(A) tail. CircRNA can comprise elements in the UTRs that promote cap-independent translation, such as an internal ribosome entry site (IRES), and optionally a polyadenosine (polyA) tract or a polyadenosine (polyAC) with interspersed cytosines. CircRNA can be produced using enzymatic or chemical methods. Generally, enzymatic methods can include, e.g., end joining of linear precursor RNA catalyzed by T4 ligase and formation of circRNA during IVT using ribozymes. Methods for making circRNA using ribozymes can include, e.g., methods based on type I introns, methods based on type II introns, and methods based on hairpin ribozymes (see, e.g., Wesselhoeft R.A. et al., Nature Communications, (2018) 9:2629; Petkovic, S. et al., Nucleic Acids Research, 2015, Vol. 43, No. 4, 2454-2465; Chen, XJ et al., Front. Bioeng. Biotechnol., 30 November 2021; and Lambowitz, A.M. et al., Cold Spring Harb Perspect Biol. 2011 Aug; 3(8): a003616).

[0160] In some embodiments, the RNA of the present application is a self-amplifying RNA (saRNA). As used herein, the terms “self-replicating RNA,” “self-amplifying RNA (saRNA),” and “replicon” can be used interchangeably to mean an RNA that can self-amplify and be translated. Generally, a saRNA can be derived from or comprise a self-replicating subgenomic of a viral (e.g., a positive-sense single-stranded RNA virus, such as an alphavirus, such as Venezuelan equine encephalitis virus (VEEV)) genome that includes viral genes encoding nonstructural proteins nsPl, nsP2, nsP3, and nsP4 that form a RNA-dependent RNA polymerase (RdRP), while viral genes encoding structural proteins are replaced by genes encoding a polypeptide of interest. A saRNA can comprise two open reading frames (ORFs) separated by a subgenomic promoter, i.e., a second ORF encoding a polypeptide of interest (e.g., a TPO precursor) followed by a subgenomic promoter drive at the 3’ end of a first ORF encoding nsPl-nsP4. A saRNA can also comprise a 5’ cap and a 5’-UTR at the 5’ end, and a 3’-UTR and a poly(A) sequence at the 3’ end. In addition, the untranslated regions of a saRNA can also comprise 5’ and 3’ conserved sequence elements (CSEs) required for self-amplification that are recognized by the RdRP. Like mRNA, a saRNA can also be produced by in vitro transcription or chemical synthesis. Detailed descriptions of saRNAs can be found, e.g., in WO2016135675A1 and WO2021183564A1.

[0161] In some embodiments, the RNA (e.g., mRNA, circRNA, and saRNA) of the present application comprises a polynucleotide encoding a TPO precursor as described herein.

[0162] In some embodiments, the RNA of the application further comprises structural elements that help to improve the stability and / or translational efficiency of the RNA, including but not limited to a 5’ cap, a 5’-UTR, a 3’-UTR, and a poly(A) sequence. In an embodiment, the RNA of the application is an mRNA comprising: (1) a 5’ cap; (2) a 5’-UTR; (3) a polynucleotide encoding a TPO precursor as described herein; (4) a 3’-UTR; and (5) a poly(A) sequence. In an embodiment, the RNA of the application is a circRNA comprising: (1) a 5’-UTR; (2) a polynucleotide encoding a TPO precursor as described herein; and (3) a 3’-UTR. In some embodiments, the circRNA of the application further comprises a spacer. In an embodiment, the RNA of the application is a saRNA comprising: (1) a 5’ cap; (2) a 5’-UTR; (3) a polynucleotide encoding an alphavirus nsPl, nsPl, nsP3, and nsP4; (4) a polynucleotide encoding a TPO precursor as described herein; (5) a 3’-UTR; and (6) a poly(A) sequence.

[0163] In an embodiment, the RNA of the application (e.g., mRNA, circRNA, and saRNA) comprises modified nucleotides.

[0164] In an embodiment, the RNA of the application (e.g., mRNA and saRNA) comprises a 5’ cap. As used herein, the term “5’ cap” generally relates to an N7-methylguanosine structure attached to the 5’ end of an mRNA via a 5’ to 5’ triphosphate linkage (also referred to as “m7G cap”, “m7Gppp-”). The 5’ cap can be co-transcriptionally added to the RNA in in vitro transcription (e.g., using anti-reverse cap analog “ARCA”), or can be attached to the RNA post-transcriptionally using a capping enzyme. In some embodiments, the RNA (e.g., IVT-RNA) is modified to a “Cap0 RNA” using a capping enzyme (e.g., vaccinia virus capping enzyme). In some embodiments, an additional methylation (e.g., by a 2’-O-methyltransferase) on the ribose 2’-O position of the nucleotide immediately adjacent to the m 7 G cap in the Cap0 RNA results in a “Cap1 RNA”. In some embodiments, a cap analog is used to generate the 5’ cap modified RNA. For a description of “cap analogs” see, e.g., Contreas, R. et al. (1982). Nucl. Acids Res. 10, 6353-6363 and US7074596B2. Examples of cap analogs include, but are not limited to, N7-methylguanosine-5’-triphosphate-5’ guanosine (m 7 G(5’)ppp(5’)G), N7-methylguanosine-5’-triphosphate-5’-adenosine (m 7G(5')ppp(5')A) and 3'-O-Me-m 7 G(5')ppp(5')G (ARCA).

[0165] The RNA (e.g., mRNA and saRNA) of the application can be an RNA comprising a Cap0 (m 7 G of the adjacent nucleotide), Cap1 (m 7 G of the adjacent nucleotide), or Cap2 (m 7 G downstream of the second nucleotide) structure. In a preferred embodiment, the mRNA of the application is a Cap1 mRNA. In a preferred embodiment, the mRNA of the application comprises 7Me GpppG 2’OMe .

[0166] In an embodiment, the RNA, particularly the mRNA, of the application comprises: (1) a 5'-UTR; (2) a polynucleotide encoding a TPO precursor, wherein the TPO precursor comprises an N-terminal signal peptide and a TPO polypeptide, the signal peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 70, and 77; (3) a 3'-UTR; and (4) an optional poly(A) sequence. In a preferred embodiment, the TPO polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In a preferred embodiment, the TPO precursor comprises the amino acid sequence of any one of SEQ ID NOs: 29, 104, and 105.

[0167] In an embodiment, the polynucleotide encoding a TPO precursor comprises (1) (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 29, and (b) is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102; (2) (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 104, and (b) is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 106; or (3) (a) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 105, and (b) is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 107. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102, 106, and 107. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of SEQ ID NO: 30.

[0168] In an embodiment, the RNA, particularly the mRNA, of the application comprises: (1) a 5'-UTR comprising the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103; (2) a polynucleotide encoding a TPO precursor comprising (a) an amino acid sequence encoding SEQ ID NO: 29, and (b) a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102; (3) a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 46 or 47; and (4) optionally a poly(A) sequence. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102. In an embodiment, the RNA, particularly the mRNA, of the application comprises the nucleotide sequence of any one of SEQ ID NOs: 50-53.

[0169] In an embodiment, the RNA, particularly the mRNA, of the application comprises: (1) a 5'-UTR comprising the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103; (2) a polynucleotide encoding a TPO precursor comprising (a) an amino acid sequence encoding SEQ ID NO: 104, and (b) a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of SEQ ID NO: 106; (3) a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 46 or 47; and (4) optionally a poly(A) sequence. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of SEQ ID NO: 106.

[0170] In an embodiment, the RNA, particularly the mRNA, of the application comprises: (1) a 5'-UTR comprising the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103; (2) a polynucleotide encoding a TPO precursor comprising (a) an amino acid sequence encoding SEQ ID NO: 105, and (b) a nucleotide sequence having at least 80%, 85%, 90%, 95%, or 99% identity to the nucleotide sequence of SEQ ID NO: 107; (3) a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 46 or 47; and (4) optionally a poly(A) sequence. In a preferred embodiment, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of SEQ ID NO: 107.

[0171] In an embodiment, the RNA, in particular the mRNA, of the present application comprises: (1) a 5'-UTR comprising the nucleotide sequence of SEQ ID NO: 45; (2) a polynucleotide encoding a TPO precursor comprising the nucleotide sequence of any one of SEQ ID NOs: 30, 106, and 107; (3) a 3'-UTR comprising the nucleotide sequence of SEQ ID NO: 47; and (4) an optionally present poly(A) sequence. In a preferred embodiment, the RNA, in particular the mRNA, of the present application comprises the nucleotide sequence of any one of SEQ ID NOs: 50, 108, and 109.

[0172] Modified nucleotides

[0173] In some embodiments, the nucleotides in the RNA (e.g., mRNA) of the present application can be naturally occurring nucleotides (e.g., naturally occurring ribonucleotides) and modified nucleotides. The modified nucleotides can be, for example, nucleotides that are not present in naturally occurring RNA, such as non-standard nucleotides or deoxy nucleotides. The modification of the nucleotides can occur on the nucleoside, such as on the ribose moiety and / or the nucleobase moiety. The modified nucleotides can be incorporated during transcription (e.g., in vitro transcription) or added during chemical synthesis of the RNA.

[0174] In some embodiments, the RNA (e.g., mRNA) of the present application is modified by comprising one or more modified nucleosides. In some embodiments, the modified nucleosides include modified cytidine, modified uridine, or a combination thereof.

[0175] Examples of modified uridines can include, but are not limited to: pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyluridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm5s2U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., with the nucleobase deoxythymine), 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (Et1ψ), 5-methyl-2-thio-uridine (m5s2U), 1-methyl-4-thio-pseudouridine (m1s4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (m5U), 5-(isopentenylaminomethyl)-2-thio-uridine (m5s2U), 5,2'-O-dimethyl-uridine (m5Um), 2-thio-2'-O-methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5- carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O- dimethyl-uridine (m3Um), and 5-(isopentenylaminomethyl)-2'-O-methyl- uridine (inm5Um), 1-thio-uridine, deoxythymidine, 5-(2- methoxycarbonylvinyl)-uridine, 5-(carbamoylhydroxymethyl)-uridine, 5- carbamoylmethyl-2-thio-uridine, 5-carboxymethyl-2-thio-uridine, 5- cyanomethyl-uridine, 5-methoxy-2-thio-uridine, and 5-[3-(1-E- propenylamino)]uridine.

[0176] Examples of modified cytidines can include, but are not limited to: 5-azacytidine, 6-azacytidine, pseudoisocytidine, 3-methylcytidine (m3C), N4- acetylcytidine (ac4C), 5-formylcytidine (f5C), N4-methyl-cytidine (m4C), 5- methyl-cytidine (m5C), 5-halo-cytidine (e.g. 5-iodo-cytidine), 5- hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolocytidine, pyrrolopseudoisocytidine, 2-thiocytidine (s2C), 2-thio-5-methylcytidine, 4- thiopseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1- deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2- methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4- methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), 5,2'-O-dimethyl-cytidine (m5Cm), N4-acetyl-2'-O-methyl-cytidine (ac4Cm), N4,2'-O-dimethyl-cytidine (m4Cm), 5-formyl-2'-O-methyl-cytidine (f5Cm), N4,N4,2'-O-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 5-hydroxy-cytidine, 5-(3-azidopropyl)-cytidine, and 5-(2-azidoethyl)-cytidine.

[0177] In some embodiments, one or more of the uridines in the RNA (e.g., mRNA) of the application are modified uridines. In some embodiments, the modified uridines include 1-methyl pseudouridine, pseudouridine, 5-methyl-uridine, or a combination thereof. In one embodiment, the modified uridines include pseudouridine. In one embodiment, the modified uridines include 5-methyl-uridine. In one embodiment, the modified uridines include 1-methyl-pseudouridine.

[0178] In one embodiment, the proportion of modified uridines in the RNA is 10-100%, e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%. In one embodiment, 20-100% of the uridines in the RNA are modified as 1-methyl pseudouridine. In one embodiment, 20-100% of the uridines in the RNA are modified as pseudouridine. In a preferred embodiment, all of the uridines in the RNA are modified as 1-methyl pseudouridine. In a preferred embodiment, all of the uridines in the RNA are modified as pseudouridine.

[0179] In one embodiment, the mRNA of the application comprises the nucleotide sequence of any one of SEQ ID NOs: 50-53, 108, and 109, and wherein one or more of the uridines are modified as 1-methyl pseudouridine or pseudouridine. In one embodiment, the mRNA of the application comprises the nucleotide sequence of any one of SEQ ID NOs: 50-53, 108, and 109, and wherein all of the uridines are modified as 1-methyl pseudouridine or pseudouridine. In a particular embodiment, the mRNA of the application comprises the nucleotide sequence of SEQ ID NO: 50, and wherein all of the uridines are modified as 1-methyl pseudouridine or pseudouridine.

[0180] DNA

[0181] In some embodiments, the nucleic acid of the application is DNA. Such DNA can be, for example, a DNA template for in vitro transcription of the RNA of the application or a DNA for expression of TPO in a host cell. The DNA can be double-stranded, single-stranded, linear, and circular DNA.

[0182] The DNA template can be provided in a suitable transcription vector. In general, the DNA template can be a double-stranded complex comprising a nucleotide sequence identical to the coding sequence described herein (coding strand) and a nucleotide sequence complementary to the coding sequence described herein (template strand). As known to one of skill in the art, the DNA template can comprise a promoter, a 5'-UTR, a coding sequence, a 3'-UTR, and an optionally present poly(A) sequence. The promoter can be a promoter usable by a suitable RNA polymerase, in particular a DNA-dependent RNA polymerase, known to one of skill in the art, including but not limited to promoters for SP6, T3, and T7 RNA polymerases. In some embodiments, the 5'-UTR, the coding sequence, the 3'-UTR, and the poly(A) sequence in the DNA template are the respective sequences comprised in or complementary to the RNA described herein. The DNA can be provided in a plasmid vector, e.g., a circular plasmid vector.

[0183] In some embodiments, the DNA of the application comprises a polynucleotide encoding a TPO precursor as described herein.

[0184] In some embodiments, the DNA of the application comprises a promoter sequence. In some embodiments, the DNA of the application comprises a 5'-UTR as described herein. In some embodiments, the DNA of the application comprises a 3'-UTR as described herein. In some embodiments, the DNA of the application comprises a 5'-UTR and a 3'-UTR as described herein. In some embodiments, the DNA of the application comprises a poly(A) sequence as described herein. In some embodiments, the DNA of the application comprises a sequence encoding a RNA of the application.

[0185] In one embodiment, the DNA of the application comprises (1) an optionally present promoter sequence; (2) a 5'-UTR; (3) a polynucleotide encoding a TPO precursor, wherein the TPO precursor comprises an N-terminal signal peptide and a TPO polypeptide, the signal peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 70, and 77; (4) a 3'-UTR; and (5) an optionally present poly(A) sequence.

[0186] Vectors and host cells

[0187] In yet another aspect, the application also provides expression vectors comprising the nucleic acids of the application. The expression vectors can further comprise additional nucleotide sequences, such as regulatory sequences and antibiotic resistance genes. The nucleic acids of the application can be present in one or more expression vectors.

[0188] The present application also provides a host cell comprising a nucleic acid or expression vector of the present application. The nucleic acid or expression vector of the present application can be introduced into a suitable host cell using various methods known in the art. Such methods include, but are not limited to, lipofection, electroporation, viral transduction, and calcium phosphate transfection, among others. In preferred embodiments, the host cell is used to express a polypeptide, antigen binding protein, or bispecific antibody of the present application. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, such as E. coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for expression include, but are not limited to, HeLa cells, human embryonic kidney cells (HEK cells, such as HEK293 cells, e.g., Expi293F cells derived from the 293 cell line), Chinese hamster ovary (CHO) cells, and other mammalian cells suitable for expression.

[0189] The present application also provides a method of making thrombopoietin (TPO), comprising culturing a host cell of the present application under suitable conditions and recovering the TPO from the host cell or culture thereof.

[0190] Compositions

[0191] In one aspect, the nucleic acids (particularly mRNA) described herein are formulated for delivery in vitro and in vivo. In one aspect, the present application provides a composition comprising a nucleic acid (particularly mRNA) of the present application. In particular, in some embodiments, the nucleic acid is formulated into a lipid-containing composition. In some embodiments, the composition comprises a lipid encapsulating the nucleic acid. In some embodiments, the lipid-containing composition forms a lipid nanoparticle that encloses the nucleic acid within a lipid shell. In some embodiments, the lipid shell protects the nucleic acid from degradation. In some embodiments, the lipid nanoparticle also facilitates transport of the encapsulated nucleic acid into intracellular compartments and / or mechanisms to exert the intended therapeutic function. In certain embodiments, the nucleic acid, when present in a lipid nanoparticle, is resistant to degradation by nucleases in aqueous solution. Lipid nanoparticles comprising nucleic acids and methods of making the same are known in the art, such as those disclosed in, e.g., US2004 / 0142025A1, US2007 / 0042031A1, WO 2017 / 004143A1, WO 2015 / 199952A1, WO 2013 / 016058A1, and WO 2013 / 086373A1, and WO 2023 / 098842A1, the entire disclosure of each of these publications is incorporated herein by reference in its entirety for all purposes.

[0192] Nanoparticle compositions that can be used in conjunction with the present disclosure include, for example, lipid nanoparticles (LNPs), nanolipoplex particles, liposomes, lipid vesicles, and lipoplexes. In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers can be functionalized and / or crosslinked to one another. The lipid bilayers can comprise one or more ligands, proteins, or channels.

[0193] In some embodiments, the composition comprises at least a first lipid (e.g., a cationic lipid), the first lipid comprising one or more of the compounds of the lipid series 01, 02, 03, and 04 as described herein, e.g., one or more of the compounds according to formulae (01-I) (and its subformulae (01-I-B), (01-I-B’), (01-I-B”), (01-I-C), (01-I-D), and (01-I-E)), (02-I), (02-II), (03-I), (04-I), and (04-III). In some aspects, the first lipid comprises one or more of the compounds listed in Tables 1, 2, 3, and 4. In some embodiments, the composition further comprises a polymer-bound lipid. In some aspects, the polymer-bound lipid is a PEGylated lipid.

[0194] In some embodiments, the composition comprises a first lipid, a polymer-bound lipid, and a sterol. In some embodiments, the composition comprises a first lipid, a polymer-bound lipid, a phospholipid, and a sterol. In some embodiments, the first lipid comprises a cationic lipid. In some embodiments, the polymer-bound lipid is a PEGylated lipid. In some embodiments, the sterol is cholesterol. In some embodiments, the composition comprises a cationic lipid, a PEGylated lipid, a phospholipid, and cholesterol.

[0195] In some embodiments, the composition is formulated as a lipid nanoparticle that encapsulates a nucleic acid in a lipid.

[0196] In some embodiments, the nanoparticle composition comprises a lipid component comprising at least one lipid, such as one or more of the compounds of Lipid Series 01, 02, 03, and 04 as described herein, e.g., one or more of the compounds according to Formulae (01-I) (and its subformulae (01-I-B), (01-I-B'), (01-I-B”), (01-I-C), (01-I-D), and (01-I-E)), (02-I), (02-II), (03-I), (04-I), and (04-III). For example, in some embodiments, the nanoparticle composition can comprise a lipid component comprising one of the compounds provided herein. The nanoparticle composition can further comprise one or more other lipid or non-lipid components, exemplary other lipid or non-lipid components can be found, e.g., in WO 2023 / 098842 A1.

[0197] Cationic Lipid

[0198] The cationic lipids include the following Lipid Series 01-04.

[0199] 1) Lipid Series 01

[0200] In an embodiment, the cationic lipid contained in the compositions, nanoparticle compositions, or nanoparticles described herein is a cationic lipid described in WO 2021204175 A1 (Lipid Series 01), which is incorporated herein by reference in its entirety.

[0201] In an embodiment, the cationic lipid is a compound of Formula (01-I):

[0202] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0203] G 1 and G 2 each independently is a bond, C2-C 12 alkylene, or C2-C 12 alkenylene, wherein one or more -CH2- in said alkylene or alkenylene is optionally replaced with -O-;

[0204] L 1 is -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1-SC(=O)R 1 -SC(=O)R a -SC(=O)R 1 -SC(=O)R b -SC(=O)R c -SC(=O)R a -SC(=O)R b -SC(=O)R c -SC(=O)R b -SC(=O)R c -SC(=O)R a -SC(=O)R 1 -SC(=O)R 1 -SC(=O)R 1 -SC(=O)R 1 -SC(=O)R 1 -SC(=O)R b -SC(=O)R c -SC(=O)R 10 -SC(=O)R 1 -SC(=O)R 1 -SC(=O)R 1 ;

[0205] -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R x -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R d -SC(=O)R 2 -SC(=O)R e -SC(=O)R f -SC(=O)R d -SC(=O)R e -SC(=O)R f -SC(=O)R e -SC(=O)R f -SC(=O)R d -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R 2 -SC(=O)R e -SC(=O)R f), -(C6-C 10 aryl)-R 2 , -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0206] R 1 and R 2 are each independently C6-C 32 alkyl or C6-C 32 alkenyl;

[0207] R a , R b , R d and R e are each independently H, C1-C 24 alkyl or C2-C 24 alkenyl;

[0208] R c and R f are each independently C1-C 32 alkyl or C2-C 32 alkenyl;

[0209] G 3 is C2-C 24 alkylene, C2-C 24 alkenylene, C3-C8cycloalkylene or C3-C8cycloalkenylene;

[0210] R 3 is -N(R 4 )R 5 ;

[0211] R 4 is C3-C8cycloalkyl, C3-C8cycloalkenyl, 4- to 8-membered heterocyclyl or C6-C 10 aryl; or R 4 , G 3 or G 3 together with the nitrogen to which they are attached form a cyclic moiety;

[0212] R 5 is C1-C 12 alkyl or C3-C8cycloalkyl; or R 4 , R 5 together with the nitrogen to which they are attached form a cyclic moiety;

[0213] x is 0, 1 or 2; and

[0214] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene and cyclic moiety is independently optionally substituted.

[0215] In an embodiment, the cationic lipid is a compound of Formula (01-I-B), (01-I-B'), (01-I-B"), (01-I-C), (01-I-D), or (01-I-E):

[0216] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0217] In an embodiment, G 1 and G 2 each independently is C3-C7alkylene. In an embodiment, G 1 and G 2 each independently is C5alkylene. In an embodiment, G 3 is C2-C4alkylene. In an embodiment, G 3 is C2alkylene. In an embodiment, G 3 is C4alkylene.

[0218] In an embodiment, R 3 has one of the following structures:

[0219] In an embodiment, R 1 , R 2 , R c , and R f each independently is branched C6-C 32 alkyl or branched C6-C 32 alkenyl. In an embodiment, R 1 , R 2 , R c , and R f each independently is branched C6-C 24 alkyl or branched C6-C 24 alkenyl. In an embodiment, R 1 , R 2 , R c , and R f each independently is -R 7 -CH(R 8 )(R 9 ), wherein R 7 is C0-C5alkylene, and R 8 and R 9 independently is C2-C 10 alkyl. In an embodiment, R 1 , R 2 , R c , and R feach independently -R 7 -CH(R 8 )(R 9 ), wherein R 7 is C0-C1alkylene, and R 8 and R 9 are independently C4-C8alkyl.

[0220] In an embodiment, the cationic lipid is a compound in Table 1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0221] Table 1.

[0222] 2) Lipid Series 02

[0223] In an embodiment, the cationic lipid contained in the compositions, nanoparticle compositions, or nanoparticles provided herein is a cationic lipid described in WO 2023 / 138611 Al (Lipid Series 02), which is incorporated herein by reference in its entirety. In an embodiment, the cationic lipid is a compound of Formula (02-I):

[0224] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0225] G 1 and G 2 are each independently C2-C 12 alkylene or C2-C 12 alkenylene, wherein one or more -CH2- in G 1 and G 2 is optionally replaced with -O-, -C(=O)O-, or -OC(=O)-;

[0226] each L 1 is independently -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR aC(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ), -NR a P(=O)(OR b )(OR c );

[0227] each L 2 is independently -OC(=O)R 2 , -C(=O)OR 2 , -OC(=O)OR 2 , -C(=O)R 2 , -OR 2 , -S(O) x R 2 , -S-SR 2 , -C(=O)SR 2 , -SC(=O)R 2 , -NR d C(=O)R 2 , -C(=O)NR e R f , -NR d C(=O)NR e R f , -OC(=O)NR e R f , -NR d C(=O)OR 2 , -SC(=S)R 2 , -C(=S)SR 2 , -C(=S)R 2 , -CH(OH)R 2 , -P(=O)(OR e )(OR f ), -NR d P(=O)(OR e )(OR f );

[0228] R 1 and R 2 are each independently C6-C 24alkyl or C2-C 24 alkenyl;

[0229] R a , R b , R d , and R e are each independently H, C1-C 24 alkyl or C2-C 24 alkenyl;

[0230] R c , and R f are each independently C1-C 24 alkyl or C2-C 24 alkenyl;

[0231] G 3 is C2-C 12 alkylene or C2-C 12 alkenylene, wherein a portion or all of the alkylene or alkenylene is optionally replaced with C3-C8cycloalkylene or C3-C8cycloalkenylene;

[0232] R 3 is -N(R 4 )R 5 , -OR 6 , or -SR 6 ;

[0233] R 4 is C1-C 12 alkyl, C2-C 12 alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0234] R 5 is H, C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0235] R 6 is hydrogen, C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, or C6-C 10 aryl;

[0236] x is 0, 1, or 2; and

[0237] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

[0238] In one embodiment, the cationic lipid is a compound of Formula (02-II):

[0239] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0240] G 1 and G 2 each independently is C2-C 12 alkylene or C2-C 12 alkenylene, wherein one or more -CH2- in G 1 and G 2 is optionally replaced by -O-, -C(=O)O-, or -OC(=O)-;

[0241] each L 1 independently is -OC(=O)R 1 , -C(=O)OR 1 , -OC(=O)OR 1 , -C(=O)R 1 , -OR 1 , -S(O) x R 1 , -S-SR 1 , -C(=O)SR 1 , -SC(=O)R 1 , -NR a C(=O)R 1 , -C(=O)NR b R c , -NR a C(=O)NR b R c , -OC(=O)NR b R c , -NR a C(=O)OR 1 , -SC(=S)R 1 , -C(=S)SR 1 , -C(=S)R 1 , -CH(OH)R 1 , -P(=O)(OR b )(OR c ), -NR a P(=O)(OR b )(OR c );

[0242] each L 2 independently is -OC(=O)R 2 , -C(=O)OR 2 , -OC(=O)OR 2 , -C(=O)R 2 , -OR2 -S(O) x R 2 -S-SR 2 -C(=O)SR 2 -SC(=O)R 2 -NR d C(=O)R 2 -C(=O)NR e R f -NR d C(=O)NR e R f -OC(=O)NR e R f -NR d C(=O)OR 2 -SC(=S)R 2 -C(=S)SR 2 -C(=S)R 2 -CH(OH)R 2 -P(=O)(OR) e (OR) f -NR d P(=O)(OR e (OR) f );

[0243] R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0244] R a R b R d and R e Each independently constitutes H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0245] R c and R f Each independently is C1-C 24 Alkyl or C2-C 24 alkenyl;

[0246] G 3 For C2-C 12 Alkylene or C2-C 12 The alkenyl group, wherein part or all of the alkenyl group is optionally replaced by a C3-C8 cycloalkyl or C3-C8 alkenyl group;

[0247] R 3 -N(R)4 )R 5 -OR 6 or -SR 6 ;

[0248] R 4 For C1-C 12 Alkyl, C2-C 12 alkenyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups;

[0249] R 5 For H, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl, C6-C 10 Aryl or 4- to 8-membered heterocyclic alkyl groups;

[0250] R 6 Hydrogen, C1-C 12 Alkyl, C3-C8 cycloalkyl, C3-C8 cycloalkenyl or C6-C 10 Aryl;

[0251] x is 0, 1, or 2; and

[0252] Each of the alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenyl groups is independently and optionally substituted.

[0253] In one embodiment, the cationic lipid is a compound listed in Table 2, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0254] Table 2.

[0255] 3) Lipid series 03

[0256] In one embodiment, the cationic lipid contained in the composition, nanoparticle composition or nanoparticles described herein is the cationic lipid (lipid series 03) described in WO2022152109A2, the entire contents of which are incorporated herein by reference.

[0257] In one embodiment, the cationic lipid is a compound of formula (03-I):

[0258] Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0259] G 1 and G 2 Each is an independent key, C2-C 12 Alkylene or C2-C12 alkenyl groups, in which G 1 and G 2 One or more of the -CH2- groups are optionally replaced by -O- groups;

[0260] Each L 1 Independently -OC(=O)R 1 -C(=O)OR 1 -OC(=O)OR 1 -C(=O)R 1 -OR 1 -S(O) x R 1 -S-SR 1 -C(=O)SR 1 -SC(=O)R 1 -NR a C(=O)R 1 -C(=O)NR b R c -NR a C(=O)NR b R c -OC(=O)NR b R c -NR a C(=O)OR 1 -SC(=S)R 1 -C(=S)SR 1 -C(=S)R 1 -CH(OH)R 1 -P(=O)(OR) b (OR) c -NR a P(=O)(OR b (OR) c -(C6-C) 10 (Aspartic)-R 1 -(6 to 10 yuan of heteroaryl)-R 1 -(4- to 8-membered heterocyclic group)-R 1 Or R 1 ;

[0261] Each L 2 Independently -OC(=O)R 2 -C(=O)OR 2 -OC(=O)OR 2 -C(=O)R 2 -OR 2 -S(O) x R 2 -S-SR2 -C(=O)SR 2 -SC(=O)R 2 -NR d C(=O)R 2 -C(=O)NR e R f -NR d C(=O)NR e R f -OC(=O)NR e R f -NR d C(=O)OR 2 -SC(=S)R 2 -C(=S)SR 2 -C(=S)R 2 -CH(OH)R 2 -P(=O)(OR) e (OR) f -NR d P(=O)(OR e (OR) f -(C6-C) 10 (Aspartic)-R 2 -(6 to 10 yuan of heteroaryl)-R 2 -(4- to 8-membered heterocyclic group)-R 2 Or R 2 ;

[0262] R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl;

[0263] R a R b R d and R e Each independently constitutes H, C1-C 24 Alkyl or C2-C 24 alkenyl;

[0264] R c and R f Each independently is C1-C 24 Alkyl or C2-C 24 alkenyl;

[0265] G 3 For C2-C 12 Alkylene or C2-C 12alkylene or part or all of the alkenylene group is optionally substituted by C3-C8cycloalkylene, C3-C8cycloalkenylene, C3-C8cycloalkynylene, 4- to 8-membered heterocyclyl, C6-C10aryl, or 5- to 10-membered heteroaryl; 10 arylene or 5- to 10-membered heteroarylene;

[0266] R 3 is hydrogen, C1-C 12 alkyl, C2-C 12 alkenyl, C2-C 12 alkynyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C3-C8cycloalkynyl, 4- to 8-membered heterocyclyl, C6-C 10 aryl, or 5- to 10-membered heteroaryl; or R 3 , G 1 or G 1 together with the nitrogen to which they are attached form a cyclic moiety; or R 3 , G 3 or G 3 together with the nitrogen to which they are attached form a cyclic moiety;

[0267] R 4 is C1-C 12 alkyl or C3-C8cycloalkyl;

[0268] x is 0, 1, or 2;

[0269] n is 1 or 2;

[0270] m is 1 or 2; and

[0271] wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

[0272] In one embodiment, the cationic lipid is a compound in Table 3, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

[0273] Table 3.

[0274] 4) Lipid Series 04

[0275] In one embodiment, the cationic lipid contained in the particle or composition provided herein is a cationic lipid described in WO 2022 / 247755 Al (Lipid Series 04), which is incorporated herein by reference in its entirety.

[0276] In one embodiment, the cationic lipid is a compound of Formula (04-I):

[0277] Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0278] G 1 and G 2 Each is an independent key, C2-C 12 Alkylene or C2-C 12 alkenyl;

[0279] L 1 -OC(=O)R 1 -C(=O)OR 1 -OC(=O)OR 1 -C(=O)R 1 -OR 1 -S(O) x R 1 -S-SR 1 -C(=O)SR 1 -SC(=O)R 1 -NR a C(=O)R 1 -C(=O)NR b R c -NR a C(=O)NR b R c -OC(=O)NR b R c -NR a C(=O)OR 1 -SC(=S)R 1 -C(=S)SR 1 -C(=S)R 1 -CH(OH)R 1 -P(=O)(OR) b (OR) c -(C6-C) 10 (Aspartic)-R 1 -(6 to 10 yuan of heteroaryl)-R 1 Or R 1 ;

[0280] L 2 -OC(=O)R 2 -C(=O)OR 2 -OC(=O)OR 2 -C(=O)R 2 -OR 2 -S(O) x R 2 -S-SR 2-C(=O)SR 2 -SC(=O)R 2 -NR d C(=O)R 2 -C(=O)NR e R f -NR d C(=O)NR e R f -OC(=O)NR e R f -NR d C(=O)OR 2 -SC(=S)R 2 -C(=S)SR 2 -C(=S)R 2 -CH(OH)R 2 -P(=O)(OR e )(OR f ), -(C6-C 10 arylene)-R 2 -(6- to 10-membered heteroarylene)-R 2 or R 2 ;

[0281] R 1 and R 2 are each independently C5-C 32 alkyl or C5-C 32 alkenyl;

[0282] R a , R b , R d and R e are each independently H, C1-C 24 alkyl or C2-C 24 alkenyl;

[0283] R c and R f are each independently C1-C 32 alkyl or C2-C 32 alkenyl;

[0284] R 0 is C1-C 12 alkyl, C2-C 12 alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl;

[0285] G 3 is C2-C 12 alkylene or C2-C 12 alkenylene;

[0286] R 4 is C1-C 12 alkyl, C2-C 12 alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0287] R 5 is C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0288] x is 0, 1, or 2;

[0289] s is 0 or 1; and

[0290] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene is independently optionally substituted.

[0291] In one embodiment, the cationic lipid is a compound of Formula (04-III):

[0292] or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

[0293] R 1 and R 2 each independently is C5-C 32 alkyl or C5-C 32 alkenyl;

[0294] R 0 is C1-C 12 alkyl, C2-C 12 alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl, or 4- to 8-membered heterocycloalkyl;

[0295] G 3 is C2-C 12 alkylene or C2-C 12 alkenylene;

[0296] G 4 is C2-C 12 alkylene or C2-C 12 alkenylene;

[0297] R 3 is -N(R 4 )R 5 or -OR 6 ;

[0298] R 4 is C1-C 12 alkyl, C2-C 12 alkenyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl;

[0299] R 5 is C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl, C6-C 10 aryl or 4- to 8-membered heterocycloalkyl; or R 4 , R 5 together with the nitrogen to which they are attached form a cyclic moiety;

[0300] R 6 is hydrogen, C1-C 12 alkyl, C3-C8cycloalkyl, C3-C8cycloalkenyl or C6-C 10 aryl; and

[0301] wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene and cyclic moiety is independently optionally substituted.

[0302] In one embodiment, the cationic lipid is a compound in Table 4, or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof.

[0303] Table 4.

[0304] In a preferred embodiment, the cationic lipid is compound C1. In a preferred embodiment, the cationic lipid is compound C2. In a preferred embodiment, the cationic lipid is compound 04-86. In a preferred embodiment, the cationic lipid is compound 04-92.

[0305] It should be understood that any embodiments of the compounds provided herein as set forth above, as well as any particular substituents and / or variables of the compounds provided herein as set forth above, can be independently combined with other embodiments and / or substituents and / or variables of the compounds to form embodiments not specifically set forth above. Moreover, where a list of substituents and / or variables of any particular group or variable is provided, it should be understood that each and every substituent and / or variable can be deleted from the particular embodiment and / or claim and the remaining list of substituents and / or variables is to be considered as being within the scope of the embodiments provided herein.

[0306] It should be understood that in the present specification the combinations of substituents and / or variables depicted by each formulae are permissible only if such contributions result in stable compounds.

[0307] Other ionizable lipids

[0308] As described herein, in some embodiments, the nanoparticle compositions provided herein comprise one or more charged or ionizable lipids in addition to the one or more ionizable lipids of Lipid Series 01, 02, 03, and 04, e.g., one or more lipids according to Formula (01-I) (and its subformulae (01-I-B), (01-I-B'), (01-I-B"), (01-I-C), (01-I-D), and (01-I-E)), (02-I), (02-II), (03-I), (04-I), and (04-III). Without being bound by theory, it is contemplated that certain charged or zwitterionic lipid compositions of the nanoparticle compositions improve cellular uptake of the nanoparticles, similar to the lipid composition in cell membranes. Exemplary additional charged lipids, ionizable lipids, or cationic lipids that can form part of the nanoparticle compositions of the present application can be found, e.g., in WO 2023 / 098842 Al, the contents of which are incorporated herein in their entirety.

[0309] Further, in some embodiments, the charged or ionizable lipids that can form part of the nanoparticle compositions of the present application are lipids comprising a cyclic amine group. Additional cationic lipids suitable for the formulations and methods disclosed herein include those described in WO2015199952 Al, WO2016176330 Al, and WO2015011633 Al, the entire contents of which are incorporated herein by reference in their entirety. Further, in some embodiments, the charged or ionizable lipids that can form part of the nanoparticle compositions of the present application are lipids comprising a cyclic amine group. Additional cationic lipids suitable for the formulations and methods disclosed herein include those described in WO2015199952 Al, WO2016176330 Al, and WO2015011633 Al, the entire contents of which are incorporated herein by reference in their entirety.

[0310] Polymer-bound lipids

[0311] In some embodiments, the lipid component of the nanoparticle composition can comprise one or more polymer-bound lipids, such as a pegylated lipid (PEG-lipid). Without being bound by theory, it is contemplated that the polymer-bound lipid component in the nanoparticle composition can improve colloidal stability and / or reduce protein adsorption of the nanoparticle. Exemplary polymer-bound lipids that can be used in conjunction with the present disclosure include, but are not limited to, PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the PEG-lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG2000, or Chol-PEG2000.

[0312] In an embodiment, the polymer-bound lipid is a pegylated lipid. For example, some embodiments include a pegylated diacylglycerol (PEG-DAG), such as 1-(monomethoxy-polyethylene glycol)-2,3-dimyristoylglycerol (PEG-DMG); a pegylated phosphatidylethanolamine (PEG-PE); a PEG succinate diacylglycerol (PEG-S-DAG), such as 4-O-(2',3'-ditetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG); a pegylated ceramide (PEG-cer); or a PEG dialkoxylpropyl carbamate, such as ω-methoxy(polyethoxy)ethyl-N-(2,3-ditetradecyloxypropyl)carbamate or 2,3-ditetradecyloxypropyl-N-(ω-methoxy)(polyethoxy)ethyl)carbamate.

[0313] In an embodiment, the pegylated lipid has the following formula:

[0314] or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0315] R 12 and R 13 each independently is a linear or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and

[0316] w has an average value ranging from 30 to 60.

[0317] In an embodiment, R 12 and R 13each independently is a linear saturated alkyl chain containing 12 to 16 carbon atoms. In other embodiments, w has an average value in the range of 42 to 55, for example, w has an average value of 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In some particular embodiments, w has an average value of about 49.

[0318] In one embodiment, the pegylated lipid has the following formula:

[0319] wherein w has an average value of about 49.

[0320] In some embodiments, the polymer-bound lipid (preferably, the pegylated lipid) is present at a concentration in the range of 0.5 mol% to 5 mol%. Preferably, the polymer-bound lipid is present at a concentration in the range of about 1.0 mol% to 2.5 mol%. More preferably, the polymer-bound lipid is present at a concentration in the range of about 1.2 mol% to 2 mol%. More preferably, the polymer-bound lipid is present at a concentration in the range of about 1.4 mol% to 1.8 mol%.

[0321] In some embodiments, the polymer-bound lipid is present at a concentration of 0.8 mol%, 0.9 mol%, 1.0 mol%, 1.1 mol%, 1.2 mol%, 1.3 mol%, 1.4 mol%, 1.5 mol%, 1.6 mol%, 1.7 mol%, 1.8 mol%, 1.9 mol%, 2.0 mol%, 2.1 mol%, 2.2 mol%, 2.3 mol%, 2.4 mol%, 2.5 mol%, 2.6 mol%, 2.7 mol%, 2.8 mol%, 2.9 mol%, or 3.0 mol%.

[0322] Structural Lipids

[0323] In some embodiments, the lipid component of the lipid nanoparticle comprises one or more structural lipids.

[0324] Exemplary structural lipids include, but are not limited to, cholesterol, coprostanol, sitostanol, ergostanol, elaidostanol, stigmasterol, brassicasterol, tomatidine, tomatin, ursolic acid, a-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid comprises cholesterol and a corticosteroid such as prednisolone, dexamethasone, prednisone, and hydrocortisone, or a combination thereof.

[0325] In some embodiments, the lipid nanoparticle provided herein comprises a sterol or a sterol analog. In some embodiments, the sterol or sterol analog is cholesterol.

[0326] In some embodiments, the structural lipid is present at a concentration ranging from 20 mol% to 50 mol%. In some embodiments, the structural lipid is present at a concentration ranging from 30 mol% to 50 mol%. Preferably, the structural lipid is present at a concentration ranging from 32 mol% to 46 mol%. More preferably, the structural lipid is present at a concentration ranging from 34 mol% to 44 mol%. Still more preferably, the structural lipid is present at a concentration ranging from 36 mol% to 42 mol%.

[0327] In some embodiments, the structural lipid is present at a concentration of about 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 38.5 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, or 50 mol%.

[0328] Phospholipids

[0329] In some embodiments, the lipid component of the lipid nanoparticle comprises one or more phospholipids, such as one or more (poly)unsaturated lipids.

[0330] Exemplary phospholipids include, but are not limited to, 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2- dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), and the like. Exemplary phospholipids can be found, for example, in WO 2023 / 098842 Al, the contents of which are incorporated herein in their entirety. In certain embodiments, the nanoparticle composition comprises DSPC. In certain embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

[0331] Still other exemplary phospholipids include, for example, dipalmitoyl phosphatidylglycerol (DPPG), palmitoyloleyl phosphatidyl ethanolamine (POPE), and dioleoyl phosphatidyl ethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidyl ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidyl ethanolamine (SOPE), and 1,2-diretoyl-sn-glycero-3-phosphoethanolamine (trans DOPE). In some embodiments, the phospholipid is l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In some embodiments, the phospholipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0332] In some embodiments, the phospholipid is phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), or phosphatidylglycerol (PG).

[0333] Further phospholipids that can form part of the lipid nanoparticles of the present application also include those described in WO 2017 / 112865 Al, the entirety of which is incorporated herein by reference.

[0334] In some embodiments, the phospholipid is present at a concentration ranging from 5 mol% to 40 mol%. In some embodiments, the phospholipid is present at a concentration ranging from 5 mol% to 15 mol%. Preferably, the phospholipid is present at a concentration ranging from 7 mol% to 13 mol%. More preferably, the phospholipid is present at a concentration ranging from 9 mol% to 11 mol%.

[0335] In some embodiments, the phospholipid is present at a concentration of about 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 9.5 mol%, 10 mol%, 10.5 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol%.

[0336] In some embodiments, the lipid component of the lipid nanoparticle comprises one or more of the lipids of series 01, 02, 03, and 04 described herein, for example one or more of the lipids according to formulae (01-I), (01-II), (02-I), (03-I), and (04-I) (and subformulae thereof), a phospholipid (such as an unsaturated lipid, preferably DOPE or DSPC), a polymer-bound lipid (preferably, a PEG-lipid), and a structural lipid (preferably, cholesterol).

[0337] In the above embodiments, preferably, the lipid nanoparticle comprises 20 mol% to 65 mol% cationic lipid. More preferably, the lipid nanoparticle comprises 25 mol% to 60 mol% cationic lipid. Still more preferably, the lipid nanoparticle comprises 30 mol% to 55 mol% cationic lipid. Still more preferably, the lipid nanoparticle comprises 35 mol% to 55 mol% cationic lipid. Still more preferably, the lipid nanoparticle comprises 40 mol% to 52 mol% cationic lipid.

[0338] In the above embodiments, preferably, the phospholipid is present at a concentration ranging from 5 mol% to 40 mol%. More preferably, the phospholipid is present at a concentration ranging from 5 mol% to 15 mol%. More preferably, the phospholipid is present at a concentration ranging from 7 mol% to 13 mol%. Still more preferably, the phospholipid is present at a concentration ranging from 9 mol% to 11 mol%.

[0339] In the above embodiments, preferably, the polymer-bound lipid (e.g., PEG-lipid) is present at a concentration ranging from 0.5 mol% to 5 mol%. More preferably, the polymer-bound lipid (e.g., PEG-lipid) is present at a concentration ranging from about 1.0 mol% to 2.5 mol%. Still more preferably, the polymer-bound lipid (e.g., PEG-lipid) is present at a concentration ranging from about 1.2 mol% to 2 mol%. Still more preferably, the polymer-bound lipid (e.g., PEG-lipid) is present at a concentration ranging from about 1.4 mol% to 1.8 mol%.

[0340] In the above embodiments, preferably, the structural lipid is present at a concentration ranging from 20 mol% to 50 mol%. More preferably, the structural lipid is present at a concentration ranging from 30 mol% to 50 mol%. Preferably, the structural lipid is present at a concentration ranging from 32 mol% to 46 mol%. More preferably, the structural lipid is present at a concentration ranging from 34 mol% to 44 mol%. Still more preferably, the structural lipid is present at a concentration ranging from 36 mol% to 42 mol%.

[0341] In the above embodiments, preferably, the molar ratio of cationic lipid to structural lipid ranges from 5: 1 to 1 : 1. More preferably, the molar ratio of cationic lipid to structural lipid ranges from 3: 1 to 1 : 1. Still more preferably, the molar ratio of cationic lipid to structural lipid ranges from 2: 1 to 1 : 1.

[0342] In the above embodiments, preferably, the molar ratio of cationic lipid to phospholipid is in the range of about 1 : 1 to about 10: 1. More preferably, the molar ratio of cationic lipid to phospholipid is in the range of about 2: 1 to about 8: 1. Still more preferably, the molar ratio of cationic lipid to phospholipid is in the range of about 3: 1 to 6: 1.

[0343] In the above embodiments, preferably, the molar ratio of cationic lipid to polymer- conjugated lipid is in the range of about 100: 1 to about 10: 1. More preferably, the molar ratio of cationic lipid to polymer-conjugated lipid is in the range of about 80: 1 to about 15: 1. Still more preferably, the molar ratio of cationic lipid to polymer-conjugated lipid is in the range of about 60: 1 to about 20: 1. Yet more preferably, the molar ratio of cationic lipid to polymer-conjugated lipid is in the range of about 40: 1 to about 25: 1. Yet more preferably, the molar ratio of cationic lipid to polymer-conjugated lipid is in the range of about 35: 1 to about 30: 1.

[0344] In some embodiments, the lipid nanoparticle comprises 20 mol% to 65 mol% cationic lipid, 5 mol% to 40 mol% phospholipid, 0.5 mol% to 5 mol% polymer-conjugated lipid, and 20 mol% to 50 mol% sterol.

[0345] In some embodiments, the molar ratio of cationic lipid, phospholipid (preferably, DSPC), structural lipid (preferably, cholesterol), and polymer-conjugated lipid (preferably, PEG-lipid) in the lipid nanoparticle is 30-55:5-40:20-50:0.5-5.

[0346] In some preferred embodiments, the cationic lipid comprises compound CI or C2.

[0347] In some preferred embodiments, the PEG-lipid comprises DMG-PEG, such as DMG-PEG2000.

[0348] In some most preferred embodiments, the lipid nanoparticle comprises 30-55 mol% compound CI or C2, 0.5-5 mol% DMG-PEG (such as DMG-PEG2000), 5-40 mol% DSPC, and 20-50 mol% cholesterol.

[0349] As used herein, "mol%" described in reference to lipids refers to the mole percentage of a component relative to the total moles of all lipid components in the LNP. Unless specifically indicated, the sum of the amounts (mol%) of all lipids in a lipid nanoparticle is 100 mol%.

[0350] In some embodiments, the lipid nanoparticle has an average diameter in the range of 50 nm to 180 nm. Preferably, the lipid nanoparticle has an average diameter in the range of 50 nm to 150 nm. More preferably, the lipid nanoparticle has an average diameter in the range of 50 nm to 120 nm. Still more preferably, the lipid nanoparticle has an average diameter in the range of 50 nm to 100 nm. Still more preferably, the lipid nanoparticle has an average diameter in the range of 60 nm to 85 nm.

[0351] In one embodiment, the nucleic acid in the composition is an mRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 50-53, 108, and 109. In a preferred embodiment, the mRNA comprises the nucleotide sequence of SEQ ID NO: 50.

[0352] In one embodiment, the composition comprises 30-55 mol% of Compound CI, 0.5-5 mol% of DMG-PEG 2000, 5-40 mol% of DSPC, 20-50 mol% of cholesterol, and an mRNA, wherein the mRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 50-53, 108, and 109. In a preferred embodiment, the mRNA comprises the nucleotide sequence of SEQ ID NO: 50.

[0353] In one embodiment, the composition comprises 30-55 mol% of Compound CI, 0.5-5 mol% of DMG-PEG 2000, 5-40 mol% of DSPC, 20-50 mol% of cholesterol, and an mRNA, wherein the mRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 50-53, 108, and 109. In a preferred embodiment, the mRNA comprises the nucleotide sequence of SEQ ID NO: 50.

[0354] In one embodiment, the composition comprises 30-55 mol% of Compound CI, 0.5-5 mol% of DMG-PEG 2000, 5-40 mol% of DSPC, 20-50 mol% of cholesterol, and an mRNA, wherein the mRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 50-53, 108, and 109. In a preferred embodiment, the mRNA comprises the nucleotide sequence of SEQ ID NO: 50.

[0355] In a specific embodiment, the composition comprises 30-55 mol% of Compound C2, 0.5-5 mol% of DMG-PEG 2000, 5-40 mol% of DSPC, 20-50 mol% of cholesterol, and mRNA, wherein the mRNA comprises the nucleotide sequence of any one of SEQ ID NOs: 50, 108, and 109, and wherein all uridines are modified as 1-methylpseudouridine or pseudouridine. In a preferred embodiment, the mRNA comprises the nucleotide sequence of SEQ ID NO: 50.

[0356] Nanoparticle compositions can be designed for one or more particular applications or targets. For example, a nanoparticle composition can be designed for delivery of a nucleic acid, such as an RNA, to a particular cell, tissue, organ, or system, or group thereof, in a mammal. The physicochemical properties of the nanoparticle composition can be altered to increase selectivity for a particular body target. For example, the particle size can be adjusted based on the fenestration size of different organs. The nucleic acid included in the nanoparticle composition can also be selected based on one or more desired delivery targets. For example, the nucleic acid can be selected for a particular indication, condition, disease, or disorder, and / or for delivery to a particular cell, tissue, organ, or system, or group thereof, e.g., local or specific delivery. In certain embodiments, the nanoparticle composition can include a nucleic acid, particularly mRNA, encoding a polypeptide of interest that is capable of being translated within a cell to produce the polypeptide of interest. Such compositions can be designed for specific delivery to a particular organ. In certain embodiments, the composition can be designed for specific delivery to the liver of a mammal.

[0357] The amount of nucleic acid in a nanoparticle composition can depend on the size, composition, desired target and / or application, or other properties of the nanoparticle composition, as well as the properties of the nucleic acid. For example, the amount of RNA that can be used in a nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of nucleic acid and other components, e.g., lipids, in a nanoparticle composition can also vary. In some embodiments, the wt / wt ratio of the lipid component to nucleic acid in a nanoparticle composition can be about 5: 1 to about 60: 1, such as about 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, 22: 1, 25: 1, 30: 1, 35: 1, 40: 1, 45: 1, 50: 1, and 60: 1. For example, the wt / wt ratio of the lipid component to nucleic acid can be about 10: 1 to about 40: 1. In certain embodiments, the wt / wt ratio is about 20: 1. The amount of nucleic acid in a nanoparticle composition can be measured, for example, using absorption spectroscopy, e.g., ultraviolet-visible spectroscopy.

[0358] In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and amounts thereof can be selected to provide a particular N:P ratio. The N:P ratio of a composition refers to the molar ratio of nitrogen atoms in the one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N:P ratio is selected. The one or more RNAs, lipids, and amounts thereof can be selected to provide an N:P ratio of about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N:P ratio can be about 2:1 to about 8:1. In other embodiments, the N:P ratio is about 5:1 to about 8:1. For example, the N:P ratio can be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. For example, the N:P ratio can be about 5.67:1.

[0359] Methods of characterizing nanoparticle compositions and various characteristics, such as particle size, polydispersity index, zeta potential, and encapsulation efficiency can be found, for example, in WO 2023 / 098842 Al, the contents of which are incorporated herein in their entirety.

[0360] The nanoparticle composition can optionally comprise one or more coatings. For example, the nanoparticle composition can be formulated as a capsule, film, or tablet having a coating. A capsule, film, or tablet comprising the compositions described herein can have any useful size, tensile strength, hardness, or density.

[0361] Pharmaceutical compositions / kit

[0362] According to the present disclosure, the nanoparticle compositions can be formulated, in whole or in part, into pharmaceutical compositions. The pharmaceutical compositions can comprise one or more nanoparticle compositions. For example, the pharmaceutical compositions can comprise one or more nanoparticle compositions comprising one or more different nucleic acids. The pharmaceutical compositions can also comprise one or more pharmaceutically acceptable carriers, diluents, excipients, or auxiliary ingredients, such as those described herein. General guidelines for the formulation and manufacture of pharmaceutical compositions and dosage forms can be found, for example, in Remington, The Science and Practice of Pharmacy, 21stEd., A.R. Gennaro; Lippincott, Williams & Wilkins, Baltimore, Md., 2006. Conventional carriers, diluents, excipients, and auxiliary ingredients can be used in any of the pharmaceutical compositions, unless the conventional carrier, diluent, excipient, or auxiliary ingredient is incompatible with one or more components of the nanoparticle composition. A carrier, diluent, excipient, or auxiliary ingredient is incompatible with a component of the nanoparticle composition if the combination of the carrier, diluent, excipient, or auxiliary ingredient and the component of the nanoparticle composition causes any undesirable biological effect or other adverse effect.

[0363] In some embodiments, the one or more carriers, diluents, excipients, or auxiliary ingredients can constitute greater than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, the one or more carriers, diluents, excipients, or auxiliary ingredients can constitute 50%, 60%, 70%, 80%, 90%, or a higher percentage of the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable carrier, diluent, excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the carrier, diluent, excipient is approved for human and veterinary use. In some embodiments, the carrier, diluent, excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the carrier, diluent, excipient is pharmaceutical grade. In some embodiments, the carrier, diluent, excipient meets the standards of the United States Pharmacopeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0364] The relative amounts of the one or more nanoparticle compositions, the one or more pharmaceutically acceptable carriers, diluents, excipients, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary, depending on the identity, size, and / or condition of the subject being treated and further depending on the route of administration of the composition. For example, the pharmaceutical composition can comprise between 0.1% and 100% (wt / wt) of the one or more nanoparticle compositions.

[0365] In certain embodiments, the nanoparticle compositions and / or pharmaceutical compositions of the present disclosure are stored and / or transported refrigerated or frozen. Refrigerated or frozen storage and / or transport conditions can be found, for example, in WO 2023 / 098842 Al, the contents of which are incorporated herein in their entirety.

[0366] In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier, diluent, excipient selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), physiological saline, PBS, and sucrose. In certain embodiments, the pH of the pharmaceutical compositions of the present disclosure is between about 7 and 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5 and 8 or between 7 and 7.8). For example, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein, Tris, physiological saline, and sucrose, and have a pH of about 7.5-8, which are suitable for storage and / or transport at, for example, about -20 °C. For example, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and PBS, and have a pH of about 7-7.8, which are suitable for storage and / or transport at, for example, about 4 °C or lower. In the context of the present disclosure, “stability,” “stabilization,” and “stable” refer to the nanoparticle compositions and / or pharmaceutical compositions disclosed herein being resistant to chemical or physical changes (e.g., degradation, changes in particle size, aggregation, changes in encapsulation, etc.) under given manufacturing, preparation, transport, storage, and / or use conditions, for example, when stress, such as shear, freeze / thaw stress, etc., is applied.

[0367] The nanoparticle compositions and / or pharmaceutical compositions comprising one or more nanoparticle compositions can be administered to any patient or subject, including one that can benefit from delivery of a nucleic acid to one or more particular cells, tissues, organs, or systems, or groups thereof. Although the description provided herein regarding the nanoparticle compositions and pharmaceutical compositions comprising the nanoparticle compositions is primarily directed to compositions suitable for administration to humans, those skilled in the art will appreciate 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 can be designed and / or performed by a veterinary pharmacologist of ordinary skill with only routine experimentation, if any. It is contemplated that subjects to which the compositions are administered include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals, such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats.

[0368] Pharmaceutical compositions comprising one or more nanoparticles can be prepared by any method known in or to be developed in the field of pharmacology. Generally, such preparation methods involve combining the active ingredient with a carrier, diluent, excipient and / or one or more other auxiliary components, and then, if desired or necessary, aliquoting, shaping and / or packaging the product into the desired single- or multi-dose units.

[0369] The pharmaceutical compositions according to this disclosure can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses. As used herein, a “unit dose” is a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of active ingredient is generally equal to the dose of the active ingredient to be administered to a subject and / or a convenient portion of that dose, such as half or one-third of that dose.

[0370] Pharmaceutical compositions can be formulated into various forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions can be formulated 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 application (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalers, and patches), suspensions, powders, and other forms.

[0371] Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may also contain inert diluents commonly used in this art, such as water or other solvents, solubilizers, and emulsifiers, such as ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, methyl benzoate, propylene glycol, 1,3-butanediol, dimethylformamide, oils (especially cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycol, and sorbitan fatty acid esters, and mixtures thereof. In addition to inert diluents, oral compositions may also contain additional therapeutic and / or preventative agents, additional agents such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, and / or aromatizers. In some embodiments for parenteral administration, the composition is mixed with a solubilizer, such as Cremophor. TM Alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers and / or combinations thereof.

[0372] Injectable formulations, such as sterile injectable aqueous or oily suspensions, can be formulated using suitable dispersants, wetting agents, and / or suspending agents according to known techniques. Sterile injectable formulations can be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenteral diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable media and solvents that can be used include water, Ringer's solution (USP), and isotonic sodium chloride solution. Sterile non-volatile oils are commonly used as solvents or suspension media. For this purpose, any mild non-volatile oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectable formulations.

[0373] Injectable formulations may be sterilized, for example by filtration via a bacterial trapping filter, and / or by incorporation of a sterilizing agent in the form of a sterile solid composition, which may be dissolved or dispersed in sterile water or other sterile injectable media prior to use.

[0374] The present invention provides methods for delivering nucleic acids to mammalian cells or organs, generating target peptides in mammalian cells, and treating diseases or conditions in mammals in need, the methods comprising administering a nanoparticle composition containing nucleic acids to the mammal and / or contacting mammalian cells with the nanoparticle composition.

[0375] The nucleic acids or compositions disclosed herein may also be provided in the form of a kit. Therefore, this disclosure also relates to a kit comprising the nucleic acids or compositions described herein. In some embodiments, the kit of the present invention further comprises other thrombopoietin-stimulating agents. In some embodiments, the other thrombopoietin-stimulating agents are selected from: roprostine, avatrombopag, eltrombopag, hetrombopag, rutrombopag, and combinations thereof. The kit may also include instructions for use or a suitable container as needed. In some embodiments, the kit also includes a device for administration. The kit generally includes a label indicating the intended use and / or method of use of the kit contents. The term "label" includes any written or recorded material provided on or with the kit or otherwise accompanied by the kit.

[0376] Therapeutic applications

[0377] In another general aspect, the present invention provides nucleic acids, compositions, pharmaceutical compositions or kits of the present invention for the prevention and / or treatment of thrombocytopenia, optionally in combination with other platelet-promoting drugs.

[0378] This invention provides the use of the nucleic acids, compositions, pharmaceutical compositions, or kits of the present invention in the preparation of medicaments for the prevention and / or treatment of thrombocytopenia. In some embodiments, the medicament further comprises other platelet-promoting drugs.

[0379] The present application also provides a method of preventing and / or treating thrombocytopenia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the nucleic acid, the composition, or the pharmaceutical composition of the present application. In some embodiments, the method further comprises administering to the subject a therapeutically effective amount of another thrombopoietic agent.

[0380] The other thrombopoietic agent can be any agent having thrombopoietic activity, such as a TPO-R agonist (TPO-RA), including but not limited to small molecule TPO- RAs (e.g., avatrombopag, eltrombopag, heptaprin, and lusfotrombopag) and TPO-R binding peptides (e.g., romiplostim). In some embodiments, the other thrombopoietic agent is selected from the group consisting of romiplostim, avatrombopag, eltrombopag, heptaprin, lusfotrombopag, and combinations thereof.

[0381] As used herein, the term “thrombocytopenia” refers to a condition in which the concentration of platelets in the blood is lower than the normal value or the level required for a healthy individual. In some embodiments, the thrombocytopenia is a platelet count of less than 150 x 10 9 , 140 x 10 9 , 130 x 10 9 , 120 x 10 9 , 110 x 10 9 , 100 x 10 9 , 95 x 10 9 , 90 x 10 9 , 85 x 10 9 , 80 x 10 9 , 75 x 10 9 , 70 x 10 9 , 65 x 10 9 , 60 x 10 9 , 55 x 10 9 , 50 x 10 9 , or 25 x 10 9 platelets per liter of blood.

[0382] As used herein, the term "thrombocytopenia" includes diseases of known etiology and primary thrombocytopenia. Thrombocytopenia is known to have many causes, including but not limited to: radiation therapy, chemotherapy, immunotherapy, immune thrombocytopenia (ITP, also known as primary immune thrombocytopenia), myelodysplastic syndrome (MDS), aplastic anemia, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), viral infection (including but not limited to human immunodeficiency virus (HIV), hepatitis C virus, and parvovirus infection), liver disease, severe myelosuppression, bone marrow transplant, stem cell transplant, peripheral blood stem cell transplant, TPO deficiency, and neutropenia.

[0383] The nucleic acids, compositions, pharmaceutical compositions, and kits of the present application can be used to prevent and / or treat thrombocytopenia, regardless of the single or multiple factors that cause the disease. In a particular embodiment, the thrombocytopenia is selected from immune thrombocytopenia, chemotherapy-induced thrombocytopenia, liver disease-associated thrombocytopenia, and aplastic anemia.

[0384] In an embodiment, preventing and / or treating thrombocytopenia comprises increasing the number of megakaryocytes, the number of platelets, reducing the risk of bleeding, and / or reducing platelet transfusions in a subject in need thereof.

[0385] Exemplary embodiments:

[0386] 1. A nucleic acid comprising a polynucleotide encoding thrombopoietin (TPO), wherein the polynucleotide encoding TPO comprises the nucleotide sequence of any one of SEQ ID NOs: 17-20, SEQ ID NOs: 25-28, and SEQ ID NOs: 21-23.

[0387] 2. The nucleic acid of embodiment 1, comprising a polynucleotide encoding a TPO precursor, wherein the polynucleotide encoding the TPO precursor comprises a polynucleotide encoding a signal peptide operably linked to the 5' end of the polynucleotide encoding TPO; preferably, the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 11, and 6.

[0388] 3. The nucleic acid of embodiment 2, wherein the polynucleotide encoding the signal peptide comprises (1) (a) an amino acid sequence encoding SEQ ID NO: 1, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2-5; or (2) (a) an amino acid sequence encoding SEQ ID NO: 11, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 12-15; or (3) (a) an amino acid sequence encoding SEQ ID NO: 6, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 7-10; preferably, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of any one of SEQ ID NOs: 2-5, SEQ ID NOs: 12-15, and 5, and SEQ ID NOs: 7-10; preferably, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of any one of SEQ ID NOs: 2-5 and SEQ ID NOs: 12-15; more preferably, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 2 or 12.

[0389] 4. A nucleic acid comprising a polynucleotide encoding a thrombopoietin (TPO) precursor, wherein the TPO precursor comprises an N-terminal signal peptide and a TPO, the signal peptide comprising the amino acid sequence of SEQ ID NO: 1 or 11.

[0390] 5. The nucleic acid of embodiment 4, wherein the TPO comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 16, 64, 65, and 66; preferably, the TPO comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16, and optionally: (1) comprises one or more valine substitutions at one or more positions corresponding to amino acids 46, 128, 131, and 141 of SEQ ID NO: 16, and / or (2) comprises one or more asparagine substitutions at one or more positions corresponding to amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16; more preferably, the TPO comprises the amino acid sequence of any one of SEQ ID NOs: 16, 64, 65, and 66.

[0391] 6. The nucleic acid of embodiment 4 or 5, wherein the polynucleotide encoding the TPO comprises (a) an amino acid sequence encoding SEQ ID NO: 16, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 17-20, SEQ ID NOs: 25-28, and SEQ ID NOs: 21-24; preferably, the polynucleotide encoding the TPO comprises the nucleotide sequence of any one of SEQ ID NOs: 17-20, SEQ ID NOs: 25-28, and SEQ ID NOs: 21-24; preferably, the polynucleotide encoding the TPO comprises the nucleotide sequence of any one of SEQ ID NOs: 17-20 and SEQ ID NOs: 25-28; more preferably, the polynucleotide encoding the TPO comprises the nucleotide sequence of SEQ ID NO: 17 or 25.

[0392] 7. The nucleic acid of any one of embodiments 4-6, wherein the polynucleotide encoding the signal peptide comprises (1) (a) an amino acid sequence encoding SEQ ID NO: 1, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2-5; or (2) (a) an amino acid sequence encoding SEQ ID NO: 11, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 12-15; preferably, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of any one of SEQ ID NOs: 2-5 and SED ID NOs: 12-15; more preferably, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO: 2 or 12.

[0393] 8. The nucleic acid of any one of embodiments 2-7, wherein the TPO precursor comprises the amino acid sequence of SEQ ID NO: 29 or 39.

[0394] 9. The nucleic acid of embodiment 8, wherein the polynucleotide encoding a TPO precursor comprises (1) (a) an amino acid sequence encoding SEQ ID NO: 29, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-33; or (2) (a) an amino acid sequence encoding SEQ ID NO: 39, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 40-43; preferably, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of any one of SEQ ID NOs: 30-33 and SED ID NOs: 40-43; more preferably, the polynucleotide encoding a TPO precursor comprises the nucleotide sequence of SEQ ID NO: 30 or 40.

[0395] 10. The nucleic acid of any one of embodiments 1-9, further comprising a 5’-UTR; preferably, the 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 44 or 45.

[0396] 11. The nucleic acid of any one of embodiments 1-10, further comprising a 3’-UTR; preferably, the 3’-UTR comprises the nucleotide sequence of SEQ ID NO: 46 or 47.

[0397] 12. The nucleic acid of any one of embodiments 1-11, comprising a 5’-UTR and a 3’-UTR, wherein the 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 44 and the 3’-UTR comprises the nucleotide sequence of SEQ ID NO: 46; or the 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 45 and the 3’-UTR comprises the nucleotide sequence of SEQ ID NO: 47.

[0398] 13. The nucleic acid of any one of embodiments 1-12, further comprising a poly(A) sequence; preferably, the poly(A) sequence comprises at least 50 nucleotides.

[0399] 14. The nucleic acid of any one of embodiments 1-13, comprising the nucleotide sequence of any one of SEQ ID NOs: 50-53 and SEQ ID NOs: 59-62; preferably, the nucleic acid comprises the nucleotide sequence of SEQ ID NO: 50 or 59.

[0400] 15. The nucleic acid of any one of embodiments 1-14, which is DNA or RNA.

[0401] 16. The nucleic acid of any one of embodiments 1-15, which is an mRNA, a circRNA, or a saRNA.

[0402] 17. The nucleic acid of embodiment 16, wherein all of the uridines in the mRNA are 1- methylpseudouridine or pseudouridine; preferably, the mRNA further comprises a 5’ cap.

[0403] 18. A polypeptide encoded by the nucleic acid of any one of embodiments 1-17.

[0404] 19. A composition comprising the nucleic acid of any one of embodiments 1-17.

[0405] 20. The composition of embodiment 18, comprising a lipid encapsulating the nucleic acid.

[0406] 21. The composition of embodiment 18 or 19, comprising at least a first lipid, wherein the first lipid comprises (1) one or more of the compounds according to formula (01-I), (01-I-B), (01-I-B’), (01-I-B”), (01-I-C), (01-I-D), or (01-I-E), preferably the first lipid comprises one or more of the compounds listed in Table 1; (2) one or more of the compounds according to formula (02-I) or (02-II), preferably the first lipid comprises one or more of the compounds listed in Table 2; (3) one or more of the compounds according to formula (03-I), preferably the first lipid comprises one or more of the compounds listed in Table 3; or (4) one or more of the compounds according to formula (04-I) or (04-III), preferably the first lipid comprises one or more of the compounds listed in Table 4; preferably, the first lipid is Compound C1 or Compound C2.

[0407] 22. The composition of embodiment 21, further comprising a second lipid, wherein the second lipid is a polymer-bound lipid; preferably, the polymer-bound lipid is a PEGylated lipid.

[0408] 23. The composition of any one of embodiments 19-22, comprising the first lipid, the second lipid, a phospholipid, and a steroid; preferably, the composition comprises Compound C1 or Compound C2, DMG-PEG 2000, DSPC, and cholesterol; more preferably, the composition comprises 30-55 mol% of Compound C1 or Compound C2, 0.5-5 mol% of DMG-PEG 2000, 5-40 mol% of DSPC, and 20-50 mol% of cholesterol.

[0409] 24. The composition of any one of embodiments 19-23, which is formulated as a lipid nanoparticle encapsulating the nucleic acid in a lipid.

[0410] 25. The composition of any one of embodiments 19-24, which is a pharmaceutical composition.

[0411] 26. An expression vector comprising the nucleic acid of any one of embodiments 1-17.

[0412] 27. A host cell comprising the nucleic acid of any one of embodiments 1-17 or the expression vector of embodiment 26.

[0413] 28. A method of making thrombopoietin (TPO), comprising culturing the host cell of embodiment 27 under suitable conditions and recovering the TPO from the host cell or its culture.

[0414] 29. Use of the nucleic acid of any one of embodiments 1-17 or the composition of any one of embodiments 19-25 in the manufacture of a medicament for preventing and / or treating thrombocytopenia; preferably, the thrombocytopenia is selected from immune thrombocytopenia, chemotherapy-induced thrombocytopenia, liver disease-related thrombocytopenia, and aplastic anemia.

[0415] 30. The use of embodiment 28, wherein the medicament further comprises another thrombopoietic agent; preferably, the another thrombopoietic agent is selected from the group consisting of: romiplostim, avatrombopag, eltrombopag, heptaprin, luspatercept, and combinations thereof.

[0416] 31. A kit comprising the nucleic acid of any one of embodiments 1-17 or the composition of any one of embodiments 19-25, and optionally further comprising another thrombopoietic agent; preferably, the another thrombopoietic agent is selected from the group consisting of: romiplostim, avatrombopag, eltrombopag, heptaprin, luspatercept, and combinations thereof.

[0417] 32. A thrombopoietin (TPO) precursor comprising an N-terminal signal peptide and a TPO polypeptide, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 1 or 11.

[0418] 33. The TPO precursor of embodiment 32, wherein the TPO comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs: 16, 64, 65, and 66; preferably, the TPO comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 16, and optionally: (1) comprises one or more valine substitutions at one or more positions corresponding to amino acids 46, 128, 131, and 141 of SEQ ID NO: 16, and / or (2) comprises one or more asparagine substitutions at one or more positions corresponding to amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16; more preferably, the TPO comprises the amino acid sequence of any one of SEQ ID NOs: 16, 64, 65, and 66.

[0419] 34. The TPO precursor of embodiment 32 or 33, comprising the amino acid sequence of SEQ ID NO: 29 or 39. Benefits

[0420] Benefits of the nucleic acids of the present application include at least the ability to express TPOs with high efficiency in vitro and / or in vivo. The nucleic acids of the present application can be delivered and expressed from nucleic acid compositions (e.g., mRNA-LNP formulations), further reducing cellular toxicity and immunogenicity.

[0421] Advantages of mRNA-LNP formulations include, but are not limited to: (1) support long-term concomitant chemotherapy administration treatment; (2) intravenous administration, which can achieve specific enrichment in the liver of animals, with significant advantages of in situ expression and enrichment; (3) extremely small doses can achieve sufficient and lasting therapeutic effects; (4) simple preparation process, without the need to use cell proliferation viruses or produce recombinant proteins; and / or (5) greatly reduced administration frequency, improving patient compliance.

[0422] Examples

[0423] The present application is further described by reference to the following examples. It should be understood that these examples are included merely to illustrate certain aspects of the present application, and should not be considered as limiting the scope of the application. The following materials and instruments are commercially available or prepared according to methods well known in the art. Unless otherwise indicated, the following experiments were performed according to the manufacturer’s instructions or according to methods and procedures well known in the art.

[0424] Example 1 Preparation of mRNA

[0425] 1.1. Design and synthesis of DNA templates

[0426] To screen for mRNA candidate molecules encoding human TPO precursor, different signal peptides and their coding sequences and TPO coding sequences were designed based on the naturally occurring human TPO precursor (GenBank Accession No. NP_000451.1, which comprises an N-terminal signal peptide (referred to herein as “SP2”; SEQ ID NO: 6) and a TPO polypeptide (SEQ ID NO: 16), the naturally encoded sequence of which is set forth in SEQ ID NO: 38 (GenBank Accession No.: CCDS3265.1)).

[0427] Plasmid DNA templates for in vitro transcribed mRNA were generated by cloning the 5’-UTR (selected from SEQ ID NOs: 44, 45 and 103), the open reading frame (ORF) sequence and the 3’-UTR sequence (selected from SEQ ID NOs: 46 and 47) into a plasmid (in-house developed, containing a kanamycin resistance gene, a T7 promoter sequence, a poly(A) sequence (A n , n is an integer from 75 to 175) and a unique type IIS restriction site downstream of the poly(A) sequence).

[0428] 1.2. In vitro transcription of mRNA from DNA templates

[0429] DNA linearization. The plasmid DNA prepared in Example 1.1 was linearized by digestion using the type IIS restriction enzymes Esp3I / BsmBI or Bsa I. Specifically, 10 pg of plasmid was mixed with 10 U of Esp3I / BsmBI or Bsa I and incubated at 37 °C for 4 hours to ensure complete linearization. The reaction was terminated by adding 1 / 10 volume of 3 M sodium acetate (pH 5.5) and 2.5 volumes of ethanol, mixed well and cooled at -20 °C for 1 hour. The linearized DNA was precipitated by centrifugation at 13800 g for 15 minutes at 4 °C, washed twice with 70% ethanol and resuspended in nuclease-free H2O.

[0430] In vitro transcription of mRNA. The 20 pL in vitro transcription reaction mixtures are shown in the table below, in which UTP was either not modified, or replaced by N1-methylpseudouridine-5’- triphosphate or pseudouridine-5’-triphosphate.

[0431] The reaction mixture was incubated at 37 °C for 6 hours, followed by the addition of 1 pL DNase I (RNase-free, 1 U / pL) to remove the DNA template and incubated at 37 °C for 30 minutes. The synthesized RNA was purified by LiCL precipitation and resuspended in nuclease-free H2O and stored at -20 °C.

[0432] mRNA capping. Per 10 pg of uncapped mRNA was heated at 65 °C for 10 min, placed on ice for 5 min, then mixed with 10 U of vaccinia virus capping enzyme, 50 U of mRNA Cap 2'-0-methyltransferase, 0.2 mM SAM, 0.5 mM GTP, and 1 U of RNAse inhibitor, and incubated at 37 °C for 60 min to generate Cap 1 (Capl) modified structure. The Capl mRNA thus obtained was purified by LiCl precipitation and resuspended in nuclease-free H20 and stored at -20 °C.

[0433] HPLC purification. RNA was purified by high-performance liquid chromatography (HPLC) using a C4 column (5 pm) (10 mm x 250 mm column) with buffer A containing 0.1 M triethylammonium acetate (TEAA) (pH = 7.0) and buffer B containing 0.1 M TEAA (pH = 7.0) and 25% acetonitrile.

[0434] Example 2 Preparation and characterization of mRNA-LNPs

[0435] Lipids of series 01 were synthesized following the methods described in WO2021204175A1; lipids of series 02 were synthesized following the methods described in WO2023138611A1; lipids of series 03 were synthesized following the methods described in WO2022152109A2; lipids of series 04 were synthesized following the methods described in WO2022247755A1.

[0436] mRNA was prepared as mRNA-LNP formulations. Briefly, cationic lipids (e.g., compound CI, compound C2, compound 04-86, or compound 04-92), DSPC, cholesterol, and PEG-lipid (e.g., DMG-PEG 2000) were dissolved in ethanol at a molar ratio of 30-55 / 5-40 / 20-50 / 0.5-5 (e.g., 50:10:38.5:1.5), and the prepared mRNA was diluted in 10-50 mM citrate buffer (pH = 4). LNP was prepared by mixing the ethanol lipid solution with the mRNA aqueous solution at a volume ratio of 1:2-4 using a microfluidic device with a total flow rate in the range of 9-30 mL / min, at a total lipid to mRNA weight ratio of about 10:1 to 30:1. Ethanol was removed using dialysis and replaced with DPBS. Finally, the lipid nanoparticles were filtered through a 0.2 pm sterile filter. In this context, the dose of mRNA-LNP formulations refers to the amount of mRNA contained therein.

[0437] Lipid nanoparticle size was determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern UK) using a 173° backscatter detection mode. Lipid nanoparticle encapsulation efficiency was determined using a Quant-it Ribogreen RNA quantification assay kit (Thermo Fisher Scientific, UK) according to the manufacturer’s instructions.

[0438] The apparent pKa of LNP formulations is related to the efficiency of LNP for nucleic acid delivery in vivo. The apparent pKa of each formulation was determined using an assay based on fluorescence of 2-(p-tolylamino)-6-naphthalenesulfonic acid (TNS). LNP formulations comprising cationic lipid / DSPC / cholesterol / DMG-PEG in PBS were prepared as described above. TNS was prepared as a 300 mM stock solution in distilled water. LNP formulations were diluted to 0.1 mg / mL total lipid in 3 mL of a buffer solution containing 50 mM sodium citrate, 50 mM sodium phosphate, 50 mM sodium borate and 30 mM sodium chloride with a pH in the range 3 to 9. An aliquot of TNS solution was added to give a final concentration of 0.1 mg / mL and, after vortex mixing, the fluorescence intensity was measured in a Molecular Devices Spectramax iD3 spectrometer using an excitation wavelength of 325 nm and an emission wavelength of 435 nm at room temperature. A sigmoidal curve best fit analysis was applied to the fluorescence data and the pKa value was measured as the pH value that gives half the maximum fluorescence intensity.

[0439] Example 3 mRNA construct and evaluation based on different signal peptides

[0440] 3.1 Design and preparation of mRNA

[0441] mRNA molecules encoding TPO precursors with different signal peptides were designed and prepared (Table 5 and Table 6).

[0442] Table 5

[0443] Table 6

[0444] Note: For mRNA-1 to mRNA-11, the poly(A) sequence is as set out in SEQ ID NO: 49. For mRNA-12 and mRNA-13, the poly(A) sequence is as set out in SEQ ID NO: 48.

[0445] 3.2 mRNA molecule evaluation based on in vitro expression

[0446] To detect the translation and expression efficiency of mRNA, Expi293F cells (Thermo Fisher Scientific, A14527) were chosen as host cells for mRNA transfection. Briefly, Expi293F cells passed more than 3 generations were transfected with Expi293 TM Expression medium (Gibco, A1435101) was used to resuspend the cells at a density of 1x10 6 cells / mL, and evenly spread in 24-well plates at 1 mL / well. According to the manufacturer's instructions, Lipofectamine TM 2000 transfection reagent (invitrogen, 11668019) and Opti- Ⅰ serum-free medium (Gibco, 31985070) were used to transfect 2 μg mRNA into cells, and the cells were cultured in a CO2 incubator at 37°C. After 24 h of transfection, the cell supernatant was collected by centrifugation at 300g for 5 min.

[0447] TPO protein in the cell supernatant was quantified by ELISA. The 96-well plate was coated overnight with 1 μg / mL human thrombopoietin receptor (hTPO-R) (Acro, THR-H52H7) diluted in PBS, 100 μL per well. The next day, the plate was washed 4 times with PBST, and then blocked with 3% BSA (sigma, B2064) for 1 h. Tebuconazole (Sanqunguojian, GMP No. S20050049) was diluted 50 times to serve as the starting concentration, and 2 times dilution was performed to obtain 12 concentrations for the standard curve. The cell supernatant was diluted 270 times. The diluted standard and sample were transferred to the 96-well plate and incubated at 37°C for 2 h. Then the plate was washed 4 times with PBST, and 100 μL / well of 1:1000 diluted Human Tpo Biotinylated antibody (RD, BAF288) was added to the plate and incubated for 2 h. Then 100 μL / well of 1:1000 diluted HRP Avidin was added to the plate and incubated for 1 h. Finally, the plate was washed 4 times with PBST, 100 μL of TMB (Sulebao, PR1200-500 mL) was added for color development, and after 3-5 min of incubation, 100 μL of ELISA stop solution (Sulebao, C1058) was added to stop the color development. The absorbance (OD 450 ) at 450 nm was detected using a microplate reader (SPARK). The results were analyzed using GraphPad Prism 9 software, and the TPO protein concentration in the cell supernatant was calculated by standard curve regression. The results are shown in Figures 1 and 2.

[0448] Surprisingly, in general, the efficiency of translation and expression of TPO protein from mRNA encoding TPO precursor containing signal peptide SP1 or SP3 is higher than that from mRNA encoding TPO precursor containing natural signal peptide SP2 among mRNAs with different coding sequences. In the mRNA encoding TPO precursor containing signal peptide SP3, the mRNA-10 containing TPO coding sequence shown in SEQ ID NO: 17 has the highest efficiency of translation and expression of TPO protein. In the mRNA encoding TPO precursor containing signal peptide SP1, the mRNA-3 containing TPO coding sequence shown in SEQ ID NO: 25 has the highest efficiency of translation and expression of TPO protein. Among all mRNA candidate molecules, the mRNA candidate molecules with the highest efficiency of translation and expression of TPO protein are mRNA-3 and mRNA-10. In particular, the translation efficiency of mRNA-3 and mRNA-10 is higher than that of mRNA-12 and mRNA-13 containing the natural coding sequence of human TPO precursor. Therefore, mRNA-3 and mRNA-10 are used for the development of mRNA-lipid nanoparticle (mRNA-LNP) formulations.

[0449] 3.3 Evaluation of mRNA molecules based on in vivo pharmacokinetics

[0450] mRNA-10 and mRNA-3 are prepared into mRNA-LNP formulations mRNA-10-LNP and mRNA-3-LNP (compound C1 as a cationic lipid) encapsulated by the same lipid nanoparticles, respectively. The pharmacokinetic changes after single tail vein administration of the two formulations are studied in BALB / C mice as test animals. Eight 6-8 week old female B-hFcRn mice are randomly divided into two groups according to body weight, 4 animals in each group, and single tail vein injection (i.v.) of mRNA-LNP formulations (2 μg / mouse). About 100 μL of whole blood is collected through the fundus sinus venosus at 0 h before administration, 6 h, 24 h, 48 h, 72 h and 120 h after administration. The collected whole blood is placed in a disposable coagulation separation gel vacuum blood collection tube, and after standing at room temperature for 30 min, the serum is separated by centrifugation at 4°C, 500g for 10 min, and all samples are stored at -80°C refrigerator for testing.

[0451] The concentration of TPO protein in mouse serum is detected by ELISA. For specific methods, see Example 3.2, except that the initial concentration of Thromboelastogram (Sanquin, China Pharmaceutical Code S20050049) is 1000 ng / mL, and different dilution multiples are taken for serum samples at different time points. The results are shown in Figure 3 and Table 7.

[0452] The results show that the mRNA-LNP preparation prepared from mRNA-10 has significantly higher maximum drug concentration (Cmax) and area under the curve (AUC) of TPO expression in vivo than the mRNA-LNP preparation prepared from mRNA-3, and has better pharmacokinetic characteristics.

[0453] Table 7

[0454] Example 4 mRNA construction and evaluation based on different signal peptides

[0455] 4.1 Design and preparation of mRNA

[0456] The results of Example 3 show that mRNA-10 constructed based on signal peptide SP3 efficiently expresses TPO in vitro and in vivo. In order to confirm the advantage of SP3 in TPO expression, referring to Example 1, more mRNA molecules were designed and prepared. These mRNA molecules (Table 8) have the same 5'-UTR, 3'-UTR and TPO coding sequence as mRNA-10, but encode different signal peptides.

[0457] Table 8

[0458] Note: For mRNA-14 to mRNA-25, the poly(A) sequence is shown in SEQ ID NO: 91.

[0459] 4.2 Evaluation of mRNA molecules based on in vitro expression

[0460] In order to detect the translation and expression efficiency of mRNA, Expi293F cells (Thermo Fisher Scientific, A14527) were selected as host cells for mRNA transfection. Expi293F cells passed more than 3 generations were collected, centrifuged at 300g for 5 min, and resuspended at a density of 1*10 TM Expression Medium (Gibco, A1435101) to a density of 1*10 6 cells / mL, and evenly plated in a 24-well plate at 1 mL / well, and placed in a 37°C incubator for use. 1 μg of mRNA was added to 30 μL of Opti- ⅠReduced Serum Medium (Gibco, 31985070) and mixed gently; 2 μL of Lipofectamine 1H 2000 Transfection Reagent (invitrogen, 11668019) was added to 30 μL of Opti- In the reduced serum medium, gently blow and mix. Mix the two, incubate for 10 min, form the liposome-mRNA complex, and then add to the cells. After transfection for 24 h, collect the cells by centrifugation at 300 g for 5 min.

[0461] The TPO protein in the cell supernatant was quantified by ELISA. Briefly, Human Thrombopoietin / Tpo Antibody (RD, MAB288) was diluted with PBS, plated in a 96-well plate at 1 pg / mL, 100 pL per well, and incubated overnight. The next day, after washing 4 times with PBST, 3% BSA (sigma, B2064) was used for blocking for 1 h. Human Thrombopoietin / TPO Protein (Acro, THN-H5216) was diluted with 1% BSA to 100 ng / mL as the starting concentration, and 2-fold dilution was performed to obtain 12 concentrations for standard curve drawing. The transfection supernatant was diluted with 1% BSA at an appropriate dilution, and the diluted standard and sample were transferred to the 96-well plate and incubated for 2 h. Then the plate was washed 4 times with PBST, and Human Tpo Biotinylated antibody (RD, BAF288) diluted 1:1000 with 1% BSA was added to the plate at 100 pL per well, and incubated for 2 h. HRP Avidin diluted 1:1000 with 1% BSA was added to the plate at 100 pL per well, and incubated for 1 h. Finally, the plate was washed 4 times with PBST, 100 pL of TMB (Solarbio, PR1200-500 mL) was added for color development, and after incubation for 3-5 min, 100 pL of ELISA stop solution (Solarbio, C1058) was added to stop color development. The absorbance (OD) at 450 nm was detected using a microplate reader (SPARK). 450 The results are shown in FIG. 4. The TPO protein concentration in the cell supernatant was calculated by standard curve.

[0462] As shown in FIG. 4, the TPO level expressed in vitro by mRNA-10, mRNA-17 and mRNA-24 was the highest. The results showed that among the numerous signal peptides, the mRNA based on signal peptides SP3, SP7 and SP14 had relatively high TPO expression efficiency in vitro.

[0463] 4.3 mRNA molecule evaluation based on in vivo pharmacokinetics

[0464] mRNA-10, mRNA-17 and mRNA-24 were prepared into mRNA-LNP preparations encapsulated by the same lipid nanoparticles.

[0465] Pharmacokinetic study was performed in mice. Briefly, 12 BALB / C mice (female, 6-8 weeks old; Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 3 groups according to body weight, 4 animals in each group, the specific grouping and dosing regimen are shown in Table 9. On the day of grouping (D0), the animals in each group were given corresponding drug treatment: single tail vein injection (i.v.) of different mRNA-LNP 10 μg / kg. And before administration (D0), 6h, 24h, 48h, 72h, 120h and 168h after administration. About 100 μL of whole blood was collected by retro-orbital sinus, and the collected whole blood was placed in a disposable coagulation separation gel vacuum blood collection tube. After standing at room temperature for 30 min, centrifugation at 4°C, 8000 rpm for 5 min to separate serum, all samples were stored at -80°C refrigerator.

[0466] The TPO protein in serum was quantified by ELISA. For specific method, please refer to Example 4.2. The results were analyzed using GraphPad Prism 9 software, and the TPO protein concentration in serum was calculated by standard curve. The results are shown in Figure 5.

[0467] The results show that mRNA-10 based on SP3 has the best expression efficiency in vivo.

[0468] Table 9

[0469] Example 5 In vivo pharmacokinetic study of TPO (SP3-TPO) expressed from mRNA-LNP

[0470] The results of Examples 3 and 4 show that mRNA-10 based on signal peptide SP3 can efficiently express TPO in vivo. In order to confirm the advantage of SP3 in expressing TPO in vivo, referring to Example 1, more mRNA molecules encoding SP3-TPO precursor protein (SEQ ID NO: 29) were designed and prepared (Table 10).

[0471] Table 10

[0472] These mRNA molecules were prepared into mRNA-LNP formulations encapsulated by the same lipid nanoparticles. Pharmacokinetic study was performed in mice. Briefly, 28 BALB / C mice (female, 6-8 weeks old; Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 7 groups according to body weight, 4 animals in each group for pharmacokinetic study, the specific grouping and dosing regimen are shown in Table 11. On the day of grouping (D0), the animals in each group were given corresponding drug treatment: a single intravenous (i.v.) injection of 10 pg / kg of different formulations. And before administration (D0), 6h, 24h, 48h, 72h, 120h and 168h after administration. About 100 pL of whole blood was collected by retro-orbital sinus, and the collected whole blood was placed in a disposable coagulation separation gel vacuum blood collection tube. After standing at room temperature for 30 min, centrifuge at 4°C, 8000 rpm for 5 min to separate serum, and all samples were stored at -80°C refrigerator.

[0473] The TPO protein in serum was quantified by ELISA. For specific method, please refer to Example 4.2. The results were analyzed using GraphPad Prism 9 software, and the TPO protein concentration in serum was calculated by standard curve. The results are shown in Figure 6.

[0474] The results show that the tested mRNAs with different sequence compositions encoding SP3-TPO exhibit comparable expression efficiency in vivo.

[0475] Table 11

[0476] Example 6 Detection of activation of HEK Blue TPO-R cell signaling pathway by TPO expressed from mRNA

[0477] To further determine the biological function of TPO expressed from mRNA (cell supernatant) and compare their functional activity with small molecule TPO-RA, TPO reporter cell line was used to detect their activation of downstream signaling pathways.

[0478] Preparation of HEK-Blue TM TPO cell (InvivoGen, Cat No: hkb-tpo) suspension: HEK-Blue TM TPO cells were prepared at 5 x 10 5Cells / mL were resuspended in fresh, pre-warmed experimental medium (90% DMEM + 10% FBS). Teboparin (Sanon, GMP No. S20050049), eltrombopag (Hengrui, GMP No. H20210021) and alitretinoin (Glaxo Operations UK Limited, GMP No. HJ20170388) were diluted to 1 pg / mL as the starting concentration. The supernatant of Expi293F cells transfected with mRNA-10 (TPO concentration was 474.7 ng / mL) was diluted 4 times as the starting concentration. Starting from the starting concentration, 3-fold dilution was performed, and a total of 10 concentrations were obtained. 100 pL / well of HEK-Blue TM TPO cell suspension (about 50,000 cells) and 100 pL of sample to be tested, then incubated overnight at 37°C, 5% CO2. The next day, prepare QUANTI-Blue solution (InvivoGen, item number: rep-qbs): add 1 mL of QB reagent and 1 mL of QB buffer to 98 mL of sterile water, vortex at room temperature for 10 minutes and stand by. Prepare a 96-well U-shaped plate (Corning, 3599), add the HEK-Blue TM TPO cell supernatant 20 pL and QUANTI-Blue solution 180 pL, incubate at 37°C for 30 minutes, and measure SEAP levels at 620 nm using a microplate reader (M3). The results are shown in Figure 7 and Table 12.

[0479] The results show that TPO expressed from mRNA (cell supernatant) activates downstream signaling pathways comparable to the control drug Teboparin, and better than small molecule TPO-RA.

[0480] Table 12

[0481] Top: Top signal value

[0482] Example 7 Affinity detection of TPO expressed from mRNA and various TPO-R

[0483] This example detects the affinity of TPO protein expressed from mRNA (cell supernatant) and different species of TPO-R (human, mouse, Cynomolgus and rat).

[0484] ​Octet R8 (Fortebio) was used to determine the affinity of TPO protein expressed from mRNA (supernatant of mRNA-10 transfected Expi293F cells) to hTPO-R (Acro, THR-H52H7), mouse TPO-R (Kisker, MPL-MM101), cynomolgus monkey TPO-R (Kisker, MPL-CM101), and rat TPO-R (Sino, 80346-R08B). The cell supernatant was diluted with 1x PBST (10x PBST: Shengao, C520004-0001, diluted 10 times with ultrapure water) for different species of TPO-R. Different species of TPO-R were diluted to 15 pg / mL for immobilization on HIS1K sensor (Fortebio, 18-5120).

[0485] The cell supernatant (TPO concentration of 1400 pg / mL) was started at a concentration of 100 nM-400 nM, 2-fold dilution, and a total of 4-5 concentrations were obtained. The diluted cell supernatant was then added to a 96-well black plate (Greiner bio-one, 655209) at 200 pL / well. The software parameters were set, the temperature was 30°C, and the frequency for collecting standard kinetic signals was 5.0 Hz; the HIS1K sensor was pre-wetted with 1x PBST for 10 minutes, and then detected on the machine. Each cycle contained the following steps: 1) non-specific binding investigation ① immersion in buffer for 60 s; ② whether the supernatant to be tested has non-specific binding to the sensor; ③ 10 mM glycine solution at pH 1.5 for regeneration for 30 s; and 2) affinity detection ① immersion in buffer for 80 s; ② TPO-R was immobilized on the sensor for 600 s (threshold 0.3-0.5 nm); ③ immersion in buffer for 100-120 s; ④ TPO binding to TPO-R for 60 s; ⑤ TPO dissociation from TPO-R for 80-100 s; and ⑥ sensor regeneration. Finally, using Fortébio Data Analysis 12.2 software, the TPO protein and TPO-R were fitted with a 1:1 binding model to determine the association rate (k on ) and dissociation rate (k off ), and to calculate the equilibrium dissociation constant (K D ).

[0486] The results are shown in Table 13. The affinity of TPO protein expressed from mRNA to different species of TPO-R was all in the order of 10 -8 , and the affinity was similar.

[0487] Table 13

[0488] Example 8 Optimization and pharmacodynamic study of mRNA-LNP formulation

[0489] mRNA was translated in vivo, and the delivery of mRNA was dependent on the lipid nanoparticles. To compare the delivery efficiency of mRNA encapsulated by lipid nanoparticles with compound C1 and compound C2 as cationic lipids, we prepared formulations mRNA-10-LNP (mRNA-LNP encapsulating mRNA-10, compound C1 as cationic lipid) and mRNA-10-LNP (mRNA-LNP encapsulating mRNA-10, compound C2 as cationic lipid), respectively, and then compared the pharmacokinetic profiles of the two formulations.

[0490] BALB / C mice (female, 6-8 weeks old; Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 2 groups according to body weight, 6 mice in each group, 3 animals in each group for pharmacodynamic study, and the specific grouping and administration scheme are shown in Table 14. On the day after grouping (D0), the animals in each group were given corresponding drug treatment: G1 animals were given a single intravenous injection (i.v.) of formulation mRNA-10-LNP (compound C1 as cationic lipid) 10 μg / kg, and G2 animals were given a single intravenous injection (i.v.) of formulation mRNA-10-LNP (compound C2 as cationic lipid) 10 μg / kg. Before administration (D0), 2 days (D2), 4 days (D4), 7 days (D7) and 10 days (D10) after administration, EDTA anticoagulated blood (about 30-50 μL) was collected through the ophthalmic venous sinus for platelet (PLT) count detection. The results are shown in Figure 8.

[0491] The results showed that the mRNA-LNP formulations prepared with compound C1 and compound C2 had comparable platelet increasing ability.

[0492] Table 14

[0493] Example 9 Pharmacokinetic study of TPO expressed from mRNA-LNP formulations

[0494] BALB / C mice (female, 6-8 weeks old; Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 3 groups according to body weight, 5 animals in each group, for pharmacokinetic study of mRNA-LNP formulations, and the specific grouping and administration scheme are shown in Table 15. On the day after grouping (D0), the animals in each group were given corresponding drug treatment: a single intravenous injection (i.v.) of formulation mRNA-10-LNP was given. And on D0, 6h, 24h, 32h, 48h and 72h after administration, about 100 μL of whole blood was collected through the ophthalmic venous sinus. The collected whole blood was placed in a disposable coagulation separation gel vacuum blood collection tube, and after standing at room temperature for 30 min, the serum was separated by centrifugation at 4°C, 500g for 10 min, and all samples were stored at 4°C refrigerator for testing.

[0495] The concentration of TPO protein in mouse serum was detected by ELISA. For details, see Example 3.2, except that the initial concentration of Thrombopoietin (Sanquin, GMP, Drug of China S20050049) was 1000 ng / mL, and different dilution multiples were taken for serum samples at different time points. The results are shown in Figure 9 and Table 16.

[0496] The results show that after single administration of mRNA-10-LNP, TPO expression can be induced, showing a dose-dependent relationship.

[0497] Table 15

[0498] Table 16

[0499] Example 10 In vivo pharmacodynamic study of mRNA-LNP formulation in mouse CIT model

[0500] According to the size of body weight, 16 BALB / C mice (female, 6-8 weeks old; Zhejiang Vantoll Life Experimental Animal Technology Co., Ltd.) were randomly divided into 4 groups, 4 animals in each group, and the specific grouping and administration scheme are shown in Table 17. On D0, D1 and D2, each animal was administered cyclophosphamide (Drug of China H32026196; Hengrui) 200.0 mpk by subcutaneous injection (s.c.) for three consecutive days to establish a CIT model. On D1, each group of animals was administered 10 μg / kg of control mRNA-LNP formulation (NST mRNA), 10 μg / kg of mRNA-10-LNP, or 10 μg / kg of Thrombopoietin by single tail vein injection (i.v.) according to the experimental scheme. On D0, D1, D4, D6, D8, D11, D13, EDTA anticoagulated blood (about 30-50 μL) was collected from the orbital venous sinus for platelet count detection. The results are shown in Figure 10.

[0501] The results show that mRNA-10-LNP has a good dose-dependent therapeutic effect on cyclophosphamide-induced thrombocytopenia in CIT mice. In terms of administration frequency, mRNA-10-LNP effectively restores platelets with single administration, while Thrombopoietin requires ten consecutive administrations (data not shown); in terms of platelet recovery speed, mRNA-10-LNP can restore platelets earlier than Thrombopoietin.

[0502] Table 17

[0503] Example 11 In vivo pharmacodynamic study of mRNA-LNP formulation in mouse long-term chemotherapy model (CIT)

[0504] Twenty BALB / C mice (female, 6-8 weeks old; Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 4 groups according to body weight, 5 animals in each group, and the specific grouping and administration scheme are shown in Table 18. CIT model was established by injecting cyclophosphamide into animals, and the specific method was as follows: on day 0 (D0), D1 and D2 of each modeling cycle, each animal was subcutaneously injected (s.c.) with cyclophosphamide (200.0 mpk (D0) + 150.0 mpk (D2, D3) for three consecutive days, and 3 weeks were a modeling cycle. In each cycle, on D1, each group of animals was respectively given a single tail vein injection (i.v.) of 10 pg / kg control mRNA preparation (NST mRNA) or 10 pg / kg mRNA-10-LNP, or subcutaneous injection (s.c.) of 10 pg / kg thymoglobulin for 10 consecutive days. In each cycle, on D0, D2, D5, D7, D9, D12, D14, D16 and D19, EDTA anticoagulated blood (about 30-50 pL) was collected from the orbital venous sinus for platelet count detection. The results are shown in Figure 11.

[0505] As shown in Figure 11, throughout the chemotherapy course, mRNA-10-LNP can continuously rebound platelets. While thymoglobulin gradually weakens the ability to rebound platelets from the second round of chemotherapy; until the third round of administration, it cannot rebound platelets (data not shown). The results show that mRNA-LNP delivery of human TPO has an advantage in terms of efficacy.

[0506] Table 18

[0507] Example 13 In vivo pharmacodynamics study of mRNA-LNP preparation in mouse ITP model

[0508] Twenty-five BALB / C mice (male, 6-8 weeks old; Zhejiang Vital River Laboratory Animal Technology Co., Ltd.) were randomly divided into 5 groups according to body weight, 5 animals in each group, and the specific grouping and administration scheme are shown in Table 19. On day 0 (D0), D3, D6, D9 and D12, each animal was administered R300 (Antibodies for Mouse Platelet Depletion (Emfret Analytics, Catalog No. R300)) by subcutaneous injection (0.2 mpk (D0, D3, D6) + 0.4 mpk (D9, D12)) to establish an ITP model. On D1, each group of animals was administered 10 μg / kg of control mRNA preparation (NST mRNA) and 10 μg / kg of mRNA-10-LNP by single tail vein injection (i.v.) according to the experimental scheme, and 10 μg / kg of Thrombocytulin was administered subcutaneously (s.c.) for 10 consecutive days or 10 μg / kg of Eltrombopag (Kyowa Kirin, Drug Standard Number SJ20220001) once a week. On D0, D2, D5, D7, D9 and D12, EDTA anticoagulated blood (about 30-50 μl) was collected from the orbital venous sinus for platelet count detection. The results are shown in Figure 12.

[0509] As shown in Figure 12, mRNA-10-LNP can rapidly restore platelets with once every two weeks administration. Thrombocytulin requires 10 consecutive administrations, and Eltrombopag requires once a week administration to restore platelets (data not shown). The results show that mRNA-10-LNP is significantly superior to Thrombocytulin and Eltrombopag in terms of platelet recovery speed, and can restore platelets in advance. Thrombocytulin and Eltrombopag have similar platelet recovery speeds.

[0510] Table 19

[0511] Sequence Listing

Claims

1. A nucleic acid comprising a polynucleotide encoding a thrombopoietin (TPO) precursor, wherein the TPO precursor comprises an N-terminal signal peptide and a TPO polypeptide, the signal peptide comprising the amino acid sequence of any one of SEQ ID NOs: 1, 70, and 77.

2. The nucleic acid of claim 1, wherein the TPO polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 16, 64, 65, and 66; preferably, the TPO polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16, and optionally: (1) comprises one or more valine substitutions at one or more positions corresponding to amino acids 46, 128, 131, and 141 of SEQ ID NO: 16, and / or (2) comprises one or more asparagine substitutions at one or more positions corresponding to amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16; more preferably, the TPO polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 16, 64, 65, and 66.

3. The nucleic acid of claim 1 or 2, wherein the TPO precursor comprises the amino acid sequence of any one of SEQ ID NOs: 29, 104, and 105.

4. The nucleic acid of any one of claims 1-3, wherein the polynucleotide encoding the TPO polypeptide comprises (a) an amino acid sequence encoding SEQ ID NO: 16, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 17-20 and 95-98; preferably, the polynucleotide encoding the TPO polypeptide comprises the nucleotide sequence of any one of SEQ ID NOs: 17-20 and 95-98; more preferably, the polynucleotide encoding the TPO polypeptide comprises the nucleotide sequence of SEQ ID NO:

17.

5. The nucleic acid of any one of claims 1-4, wherein the polynucleotide encoding the signal peptide comprises (1) (a) an amino acid sequence encoding SEQ ID NO: 1, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 2-5 and 92-94; (2) (a) an amino acid sequence encoding SEQ ID NO: 70, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 82; or (3) (a) an amino acid sequence encoding SEQ ID NO: 77, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 89; Preferably, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of any one of SEQ ID NOs: 2-5, 92-94, 82, and 89; More preferably, the polynucleotide encoding the signal peptide comprises the nucleotide sequence of SEQ ID NO:

2.

6. The nucleic acid of any one of claims 1-5, wherein the polynucleotide encoding the TPO precursor comprises (1) (a) an amino acid sequence of SEQ ID NO: 29, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 30-33 and 99-102; (2) (a) an amino acid sequence of SEQ ID NO: 104, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 106; or (3) (a) an amino acid sequence of SEQ ID NO: 105, and (b) a nucleotide sequence that is at least 80%, 85%, 90%, 95%, or 99% identical to the nucleotide sequence of SEQ ID NO: 107; Preferably, the polynucleotide encoding the TPO precursor comprises the nucleotide sequence of any one of SEQ ID NOs: 30-33, 99-102, 106, and 107; More preferably, the polynucleotide encoding the TPO precursor comprises the nucleotide sequence of SEQ ID NO:

30.

7. The nucleic acid of any one of claims 1-6, further comprising a 5’-UTR; preferably, the 5’-UTR comprises the nucleotide sequence of any one of SEQ ID NOs: 44, 45, and 103.

8. The nucleic acid of any one of claims 1-7, further comprising a 3’-UTR; preferably, the 3’-UTR comprises the nucleotide sequence of SEQ ID NO: 46 or 47.

9. The nucleic acid of any one of claims 1-8, comprising a 5’-UTR and a 3’-UTR, wherein the 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 44 or 103, and the 3’-UTR comprises the nucleotide sequence of SEQ ID NO: 46; or the 5’-UTR comprises the nucleotide sequence of SEQ ID NO: 45, and the 3’-UTR comprises the nucleotide sequence of SEQ ID NO:

47.

10. The nucleic acid of any one of claims 1-9, further comprising a poly(A) sequence; preferably, the poly(A) sequence comprises at least 50 nucleotides.

11. The nucleic acid of any one of claims 1-10, comprising the nucleotide sequence of any one of SEQ ID NOs: 50-53, 108, and 109; preferably, the nucleic acid comprises the nucleotide sequence of SEQ ID NO:

50.

12. The nucleic acid of any one of claims 1-11, which is DNA or RNA.

13. The nucleic acid of any one of claims 1-12, which is an mRNA, a circRNA, or a saRNA.

14. The nucleic acid of claim 13, wherein the mRNA comprises 1-methylpseudouridine or pseudouridine modifications; preferably, all of the uridine modifications in the mRNA are 1-methylpseudouridine or pseudouridine; preferably, the mRNA further comprises a 5’ cap.

15. A composition comprising the nucleic acid of any one of claims 1-14.

16. The composition of claim 15, comprising a lipid encapsulating the nucleic acid.

17. The composition of claim 15 or 16, comprising at least a first lipid; preferably, the first lipid is a cationic lipid; more preferably, the first lipid comprises (1) one or more of the compounds according to formula (01-I), (01-I-B), (01-I-B’), (01-I-B”), (01-I-C), (01-I-D), or (01-I-E), preferably the first lipid comprises one or more of the compounds listed in Table 1; (2) one or more of the compounds according to formula (02-I) or (02-II), preferably the first lipid comprises one or more of the compounds listed in Table 2; (3) one or more of the compounds according to formula (03-I), preferably the first lipid comprises one or more of the compounds listed in Table 3; or (4) one or more of the compounds according to formula (04-I) or (04-III), preferably the first lipid comprises one or more of the compounds listed in Table 4; preferably, the first lipid is Compound C1 or Compound C2.

18. The composition of claim 17, further comprising a polymer-bound lipid; preferably, the polymer-bound lipid is a PEGylated lipid.

19. The composition of claim 16, comprising a first lipid, a polymer-bound lipid, and a sterol; preferably, the first lipid comprises a cationic lipid; and / or the polymer-bound lipid is a PEGylated lipid; and / or the sterol is cholesterol.

20. The composition of claim 19, further comprising a phospholipid; preferably, the composition comprises Compound C1 or Compound C2, DMG-PEG 2000, DSPC, and cholesterol.

21. The composition of claim 20, comprising 20 mol% to 65 mol% of a cationic lipid, 5 mol% to 40 mol% of a phospholipid, 0.5 mol% to 5 mol% of a polymer-bound lipid, and 20 mol% to 50 mol% of a sterol; preferably, the composition comprises 30-55 mol% of Compound C1 or Compound C2, 0.5-5 mol% of DMG-PEG 2000, 5-40 mol% of DSPC, and 20-50 mol% of cholesterol.

22. The composition of any one of claims 15-21, which is formulated as a lipid nanoparticle encapsulating the nucleic acid in a lipid.

23. The composition of any one of claims 15-22, which is a pharmaceutical composition.

24. An expression vector comprising the nucleic acid of any one of claims 1-14.

25. A host cell comprising the nucleic acid of any one of claims 1-14 or the expression vector of claim 24.

26. A method of making thrombopoietin (TPO), comprising culturing the host cell of claim 25 under suitable conditions and recovering the TPO from the host cell or its culture.

27. Use of the nucleic acid of any one of claims 1-14 or the composition of any one of claims 15-23 in the manufacture of a medicament for preventing and / or treating thrombocytopenia; preferably, the thrombocytopenia is selected from the group consisting of immune thrombocytopenia, chemotherapy-induced thrombocytopenia, liver disease-related thrombocytopenia, and aplastic anemia.

28. The use of claim 27, wherein the medicament further comprises another thrombopoietic agent; preferably, the another thrombopoietic agent is selected from the group consisting of: romiplostim, avatrombopag, eltrombopag, heptaprin, luspatercept, and combinations thereof.

29. A kit comprising the nucleic acid of any one of claims 1-14 or the composition of any one of claims 15-23, and optionally further comprising another thrombopoietic agent; preferably, the another thrombopoietic agent is selected from the group consisting of: romiplostim, avatrombopag, eltrombopag, heptaprin, luspatercept, and combinations thereof.

30. A thrombopoietin (TPO) precursor comprising an N-terminal signal peptide and a TPO polypeptide, wherein the signal peptide comprises the amino acid sequence of any one of SEQ ID NOs: 1, 70, and 77.

31. The TPO precursor of claim 30, wherein the TPO polypeptide comprises an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs: 16, 64, 65, and 66; preferably, the TPO polypeptide comprises an amino acid sequence that is at least 95% identical to SEQ ID NO: 16, and optionally: (1) comprises one or more valine substitutions at one or more positions corresponding to amino acids 46, 128, 131, and 141 of SEQ ID NO: 16, and / or (2) comprises one or more asparagine substitutions at one or more positions corresponding to amino acids 108, 117, 153, 157, 164, and 193 of SEQ ID NO: 16; more preferably, the TPO polypeptide comprises the amino acid sequence of any one of SEQ ID NOs: 16, 64, 65, and 66.

32. The TPO precursor of claim 30 or 31, comprising the amino acid sequence of any one of SEQ ID NOs: 29, 104, and 105.

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