Recombinant Protein of Nerve Growth Factor Mutant and Its Use

Abstract

Disclosed is a recombinant protein comprising a variant nerve growth factor (NGF) protein and a heterologous signal peptide linked to said variant NGF protein. Also disclosed is a composition comprising an mRNA encoding said recombinant protein and a delivery vector, and the use of said composition for the treatment of toxic / genetic / metabolic peripheral neuropathies, nerve regeneration repair and / or central nervous system degenerative diseases.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically, to a recombinant protein of a nerve growth factor mutant containing mRNA encoding a recombinant protein, and its use in the treatment of nervous system diseases.

Background Art

[0002] Nerve growth factor (NGF) is a growth regulatory factor with neurotrophic function, and plays an important role in the development and maintenance of functional differentiation of basal forebrain cholinergic neurons (BFCNs) and peripheral nociceptive neurons, and is also important for maintaining the normal functions of mature central and peripheral neurons, self-protection and repair of nerve injury. As a powerful neurotrophic factor, NGF has attracted attention in the drug treatment of various central and peripheral nervous system diseases in the past 20 to 30 years.

[0003] During the process of clinical research, the use of NGF caused dose-dependent pain side effects. Alzheimer's disease (AD) is a progressive and fatal central nervous system degenerative disease characterized by cognitive impairment and memory impairment. In clinical trials, when high doses of NGF were used to treat AD patients, although both the cognitive ability and cerebral cortex metabolism of AD patients were significantly improved, it was found that the occurrence of side effects such as pain and weight loss offset the therapeutic effect, and ultimately the clinical trial was considered a failure. The same phenomenon was also observed when NGF was used to treat diabetic neuropathy and HIV peripheral neuropathy. This is because NGF is one of the important mediators that cause pain and hyperalgesia. Both the TrkA and p75NTR signaling pathways mediate the pain-inducing function of NGF, and it has been shown in many studies that the TrkA and p75NTR signaling pathways interact with each other. Therefore, the loss of either signaling pathway (TrkA or p75NTR) or the disruption of the balance between the TrkA and p75NTR signaling pathways affects the pain-inducing function of NGF.

[0004] The discovery of a "pain-free" nerve growth factor has brought great expectations for the clinical use of NGF. NGF R100W Subsequent hereditary sensory and autonomic neuropathy type V (HSAN V) was discovered in a family in northern Sweden. It is a mature NGF protein in which arginine at position 100 is replaced by tryptophan. Patients experience loss of pain and temperature sensation, but their intelligence is normal. This suggests that NGF R100W may selectively lose its pain-mediating function while retaining its neurotrophic function. The discovery of NGF R100W provides an important basis for studying the neurotrophic function and pain-mediating function of NGF. Research on its biological properties and related mechanisms of action may lead to new epoch-making discoveries of NGF in the treatment of toxic / hereditary / metabolic peripheral neuropathies, nerve regeneration and repair, and / or central nervous system degenerative diseases.

[0005] However, in the knock-in mouse model of HSAN V, mice expressing the NGF R100W mutant usually completely lose pain sensation at about 2 months after birth and in many cases cannot survive to adulthood. Furthermore, since the full-length NGF R100W mutant protein affects the cleavage of its mature form, mature NGF R100W cannot be normally secreted extracellularly and exert its neurotrophic function. Therefore, currently, there is no simple and efficient method to obtain the NGF R100W mutant protein, and the use of the NGF R100W mutant protein in the treatment of nerve diseases is greatly limited.

Summary of the Invention

[0006] The present application provides a recombinant protein comprising a nerve growth factor (NGF) protein mutant and a heterologous signal peptide linked to the NGF protein mutant, wherein the recombinant protein has at least one of the following characteristics: (1) compared with the wild-type NGF protein, the binding of the NGF protein mutant to the neurotrophic factor receptor p75 (p75NTR) is reduced; (2) the expression level and / or secretion level is increased by at least 50%; (3) it has a considerable expression level and / or secretion level, for example, it can reach at least 50% or more of the wild-type NGF protein; (4) pain is reduced; (5) the half-life is extended; and (6) it promotes the repair and regeneration of damaged nerve cells. The present application also provides a composition comprising the mRNA encoding the NGF protein or NGF protein mutant and a delivery vector. The recombinant protein and composition of the present application can be used to treat nervous system diseases such as peripheral neuropathy and neurodegenerative diseases and to promote nerve repair and regeneration. For example, the recombinant protein and composition of the present application can be used in the treatment of toxic / hereditary / metabolic peripheral neuropathy, nerve regeneration repair and / or central nervous system degenerative diseases (such as diabetic peripheral neuropathy, drug-related peripheral neuropathy, hereditary motor and sensory neuropathy, post-traumatic repair of peripheral nerves, traumatic optic neuritis and / or Alzheimer's disease).

[0007] In one aspect, the present application provides a recombinant protein comprising a nerve growth factor (NGF) protein variant and a heterologous signal peptide linked to the NGF protein variant, wherein the binding of the NGF protein variant to the neurotrophin receptor p75 (p75NTR) is reduced by at least about 50% compared to the wild-type NGF protein.

[0008] Compared to the signal peptide shown in SEQ ID NO: 29, the heterologous signal peptide increases the expression level and / or secretion level of the NGF protein variant by at least about 50%.

[0009] In some embodiments, the wild-type NGF protein comprises the amino acid sequence shown in SEQ ID NO: 32.

[0010] In some embodiments, the NGF protein variant comprises the amino acid sequence shown in SEQ ID NO: 33.

[0011] In some embodiments, the heterologous signal peptide is derived from brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and / or neurotrophin-4 (NT-4).

[0012] In some embodiments, the heterologous signal peptide is derived from Gp67, Gp64, honeybee melittin (HBM), albumin, IL-2, azurocidin preproprotein, mouse IgK, immunoglobulin heavy chain, and / or cystatin-S precursor.

[0013] In some embodiments, the recombinant protein further comprises a leader peptide.

[0014] In some embodiments, the recombinant protein comprises, in sequence from the N-terminus to the C-terminus, the heterologous signal peptide, the leader peptide, and the NGF protein variant.

[0015] In some embodiments, the recombinant protein further comprises the Fc region of an immunoglobulin.

[0016] In some embodiments, the recombinant protein, from the N-terminus to the C-terminus, sequentially comprises the heterologous signal peptide, the leader peptide, the NGF protein variant, and the Fc region.

[0017] In another aspect, the present application provides an isolated nucleic acid molecule, and the isolated nucleic acid molecule encodes the recombinant protein.

[0018] In some embodiments, the isolated nucleic acid molecule comprises DNA and / or RNA. In some embodiments, the isolated nucleic acid molecule is DNA. In some embodiments, the isolated nucleic acid molecule is RNA.

[0019] In some embodiments, the isolated nucleic acid molecule comprises modifications at one or more positions selected from a 5' cap, a 5' untranslated region, an open reading frame, a 3' untranslated region, and a poly A tail.

[0020] In some embodiments, the isolated nucleic acid molecule comprises at least one modified nucleotide.

[0021] In some embodiments, the modified nucleotides comprised in the isolated nucleic acid molecule are one or more nucleotides selected from N1-methylpseudo-UTP (N1-Methylpseudo-UTP), pseudo-UTP (pseudo-UTP), 5-methoxy-uridine-phosphate (5-Methoxy-U 5'-tri P), and 5-methyl-cytidine triphosphate (5-Methyl-CTP). T P) and 5-methyl-cytidine triphosphate (5-Methyl-CTP).

[0022] In some embodiments, the isolated nucleic acid molecule is codon-optimized.

[0023] In another aspect, the present application provides a composition comprising (a) an mRNA comprising a polynucleotide encoding a nerve growth factor (NGF) protein or a variant thereof, and (b) a delivery vector.

[0024] In some embodiments, the NGF protein comprises the amino acid sequence set forth in SEQ ID NO: 32.

[0025] In some embodiments, compared to the amino acid sequence set forth in SEQ ID NO: 32, the binding of the NGF protein variant to the neurotrophin receptor p75 (p75NTR) is reduced by at least about 50%.

[0026] In some embodiments, the NGF protein variant comprises the amino acid sequence set forth in SEQ ID NO: 33.

[0027] In some embodiments, the mRNA comprises a polynucleotide encoding a heterologous signal peptide, and compared to the signal peptide set forth in SEQ ID NO: 29, the heterologous signal peptide increases the expression level and / or secretion level of the NGF protein variant by at least about 50%.

[0028] In some embodiments, the heterologous signal peptide enables the expression level and / or secretion level of the NGF protein variant to reach 50% or more of the wild-type NGF protein.

[0029] In some embodiments, the heterologous signal peptide is derived from brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and / or neurotrophin-4 (NT-4).

[0030] In some embodiments, the heterologous signal peptide is derived from Gp67, Gp64, honeybee melittin (HBM), albumin, IL-2, azurocidin preproprotein, mouse IgK, immunoglobulin heavy chain, and / or cystatin-S precursor.

[0031] In some embodiments, the mRNA further comprises a polynucleotide encoding a leader peptide.

[0032] In some embodiments, the mRNA comprises, in sequence from the 5'-end to the 3'-end, a polynucleotide encoding the heterologous signal peptide, a polynucleotide encoding the leader peptide, and a polynucleotide encoding the NGF protein or a variant thereof.

[0033] In some embodiments, the mRNA comprises, in sequence from the 5'-end to the 3'-end, a polynucleotide encoding the IgK signal peptide, a polynucleotide encoding the leader peptide, and a polynucleotide encoding the NGF protein variant.

[0034] In some embodiments, the mRNA comprises, in sequence from the 5'-end to the 3'-end, a polynucleotide encoding the BDNF signal peptide, a polynucleotide encoding the leader peptide, and a polynucleotide encoding the NGF protein variant.

[0035] In some embodiments, the mRNA further comprises a polynucleotide encoding the Fc region of an immunoglobulin.

[0036] In some embodiments, the mRNA comprises, in sequence from the 5'-end to the 3'-end, a polynucleotide encoding the heterologous signal peptide, a polynucleotide encoding the leader peptide, a polynucleotide encoding the NGF protein variant, and a polynucleotide encoding the Fc region of an immunoglobulin.

[0037] In some embodiments, the mRNA comprises, in order from the 5'-end to the 3'-end, a polynucleotide encoding the IgK signal peptide, a polynucleotide encoding the leader peptide, a polynucleotide encoding the NGF protein variant, and a polynucleotide encoding the Fc region of the immunoglobulin.

[0038] In some embodiments, the mRNA encodes an amino acid sequence set forth in any one of SEQ ID NOs: 54-55.

[0039] In some embodiments, the mRNA comprises a modification at one or more positions selected from a 5' cap, a 5' untranslated region, an open reading frame, a 3' untranslated region, and a poly A tail.

[0040] In some embodiments, the mRNA comprises at least one modified nucleotide.

[0041] In some embodiments, the modified nucleotide comprised in the mRNA is N1-methylpseudouridine-5'-triphosphate (N1-Methylpseudo-UTP), pseudouridine-5'-triphosphate (pseudo-UTP), 5-methoxyuridine- 5'-tri phosphate (5-Methoxy-U T P), and 5-methylcytidine triphosphate (5-Methyl-CTP), and comprises one or more nucleotides selected therefrom.

[0042] In some embodiments, the mRNA is codon-optimized.

[0043] In some embodiments, the mRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 22-28 and 50-51. In some embodiments, the mRNA comprises a nucleotide sequence set forth in any one of SEQ ID NOs: 23 and 27.

[0044] In some embodiments, the delivery vector comprises liposomes.

[0045] In some embodiments, the delivery vector comprises lipid nanoparticles (LNP).

[0046] In some embodiments, the delivery vector comprises cationic lipids.

[0047] In some embodiments, the molar ratio of the cationic lipid is about 45% to about 55%.

[0048] In some embodiments, the cationic lipids are SM102 and DLin-MC3-DMA.

[0049] In some embodiments, the delivery vector comprises non-cationic lipids.

[0050] In some embodiments, the non-cationic lipids comprise phospholipids and / or lipid complexes.

[0051] In some embodiments, the molar ratio of the phospholipid is about 35% to about 40%.

[0052] In some embodiments, the phospholipid comprises distearoyl phosphatidylcholine (DSPC).

[0053] In some embodiments, the lipid complex comprises polyethylene glycol-modified lipid molecules.

[0054] In some embodiments, among the delivery vectors, the molar ratio of the lipid complex is about 1% to about 2%.

[0055] In some embodiments, the polyethylene glycol-modified lipid molecules comprise PEG2000-DMG.

[0056] In some embodiments, the delivery vector comprises cholesterol.

[0057] In some embodiments, among the delivery vectors, the molar ratio of the cholesterol is about 8% to about 12%.

[0058] In some embodiments, the delivery vector comprises a cationic lipid, cholesterol, a phospholipid and a lipid complex, and the mass ratio of the cationic lipid, cholesterol, phospholipid and lipid complex is 50:10:38.5:1.5.

[0059] In some embodiments, the delivery vector comprises DLin-MC3-DMA, cholesterol, DSPC and PEG2000-DMPE, and the mass ratio of DLin-MC3-DMA, cholesterol, DSPC and PEG2000-DMPE is 50:10:38.5:1.5.

[0060] In some embodiments, the diameter of the delivery vector is about 60 nm to about 500 nm.

[0061] In some embodiments, the diameter of the delivery vector is about 80 nm to about 200 nm.

[0062] In some embodiments, the mRNA is encapsulated in the delivery vector.

[0063] In another aspect, the present application provides a vector comprising the nucleic acid molecule.

[0064] In another aspect, the present application provides a cell comprising the nucleic acid molecule described in the present application and / or the vector, or expressing the recombinant protein described in the present application.

[0065] In another aspect, the present application provides a method for producing the recombinant protein, which comprises culturing the cell under conditions for expressing the recombinant protein.

[0066] In another aspect, the present application provides a pharmaceutical composition comprising the recombinant protein, the nucleic acid molecule, the composition, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0067] In another aspect, the present application provides a kit or a drug delivery device comprising the recombinant protein, the nucleic acid molecule, the composition, the vector, the cell, and / or the pharmaceutical composition.

[0068] In another aspect, the present application provides the use of the recombinant protein, the nucleic acid molecule, the composition, the vector, the cell, and / or the pharmaceutical composition in the manufacture of a medicament for the alleviation, prevention and / or treatment of central and / or peripheral nervous system diseases.

[0069] Those skilled in the art can easily gain insights into other aspects and advantages of the present application from the following detailed description. The following detailed description only shows and describes exemplary embodiments of the present application. As understood by those skilled in the art, the content of the present application enables those skilled in the art to make changes to the disclosed specific embodiments without departing from the spirit and scope of the invention related to the present application. Therefore, the descriptions in the drawings and the specification of the present application are merely exemplary and not restrictive.

[0070] Specific features of the invention related to the present application are described in the appended claims. The features and advantages of the invention related to the present application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as shown below.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0072] Hereinafter, embodiments of the present invention will be described by specific specific examples. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification.

[0073] [Definition of Terms] In the present application, the term "nerve growth factor (NGF)" generally refers to a secreted protein that functions in the growth and survival of various neurons. The term NGF as used herein is not limited to human NGF and includes all species orthologs of human NGF. The term "NGF" includes the pro-form of NGF (pro-form), proNGF (proNGF), full-length NGF, and any form of NGF produced by intracellular processes. The term also includes naturally occurring NGF variants such as splice variants, allelic variants, and isotypes.

[0074] NGF exists as a complex of approximately 130 kDa composed of three proteins, α-NGF, β-NGF, and γ-NGF, and this NGF is also referred to as proNGF (NGF precursor). ProNGF is also referred to as immature NGF. The γ subunit of the complex functions as a serine protease and can cleave the N-terminus of the β subunit, thereby activating the protein and generating functional NGF, namely β-NGF. NGF can bind to two receptors, the p75 neurotrophin receptor (p75NTR) and TrkA, a transmembrane tyrosine kinase. NGF is a fully validated target for pain and is known to mediate the sensitizing effect on nociceptors. The amino acid sequence of human full-length NGF can be referred to Genbank accession number NP_002497.2 or UniProtKB accession number P01138. Human full-length NGF generally contains 241 amino acids, among which positions 1-18 are the NGF signal peptide portion (amino acid sequence shown in SEQ ID NO: 29), positions 19-121 are the leader peptide portion (amino acid sequence shown in SEQ ID NO: 34), and positions 122-241 are mature NGF (mature NGF, amino acid sequence shown in SEQ ID NO: 32). Full-length NGF can become mature NGF through a splicing process.

[0075] In the present application, the term "variant" generally refers to an amino acid sequence having one or more amino acid changes (e.g., substitution, change, modification, replacement, deletion, or addition of one or more amino acids) or a nucleic acid sequence having one or more nucleotide changes (e.g., substitution, change, modification, replacement, deletion, or addition of one or more nucleotides) compared to a reference sequence. In the case of a polypeptide, variants include polypeptides having an amino acid sequence changed by one or more amino acid substitutions, deletions, or insertions. Variants can be naturally occurring or non-naturally occurring. Variants can retain one or more activities of the reference sequence, but the one or more activities of the variant compared to the reference sequence may be enhanced, decreased, or maintained without change.

[0076] In the present application, the term "NGF variant" generally refers to an amino acid sequence having one or more amino acid changes (e.g., substitution, change, modification, replacement, deletion or addition of one or more amino acids) as compared to the full-length NGF amino acid sequence (e.g., Genbank accession number NP_002497.2 or UniProtKB accession number P01138, or the amino acid sequence shown in SEQ ID NO: 13). For example, amino acids may change in the mature NGF portion. Compared to the wild-type NGF protein, the binding of the NGF protein variant to the neurotrophin receptor p75 (p75NTR) is reduced due to the amino acid change (e.g., mutation). For example, in the case of the full-length NGF sequence having the amino acid sequence shown in SEQ ID NO: 13, the amino acid mutation may be a mutation from arginine (R) at position 221 to tryptophan (W), and the mutated full-length NGF protein is NGF R221W (which may be referred to as the amino acid sequence shown in SEQ ID NO: 14). For example, in the case of the mature NGF sequence having the amino acid sequence shown in SEQ ID NO: 32, the amino acid mutation may be a mutation from arginine (R) at position 100 to tryptophan (W), and the mutated mature NGF protein is NGF R100W (which may be referred to as the amino acid sequence shown in SEQ ID NO: 33).

[0077] In the present application, the term "linked" is used interchangeably with "fused" or "fusion" and generally refers to linking two or more elements or components by any means including chemical conjugation or recombinant means. In the case of polypeptides, two polymers of amino acid residues are directly bonded to each other, or in the case of polynucleotides, two polymers of nucleotides are directly bonded to each other, or are separated by the insertion of amino acid residues or nucleotides within the same polypeptide or polynucleotide.

[0078] In the present application, the term "heterologous" generally refers to a polypeptide or nucleic acid comprising two or more sequences or subsequences that are not found in the same relationship to each other in nature. For example, recombination usually generates an expression cassette having two or more sequences from unrelated genes and placed in a new functional nucleic acid. The term "heterologous signal peptide" generally means that in the natural environment, the signal peptide and the amino acid sequence that binds to the signal peptide do not belong to the same origin, and may be derived from different species, different individuals, may be from the same species but different individuals, may be from the same individual but different proteins, or may be from the same species but different proteins. For example, a heterologous signal peptide and the amino acid sequence linked to the heterologous signal peptide are encoded by genes that are naturally unrelated and placed in a new polypeptide. For example, when a signal peptide is connected to an NGF protein or an NGF protein variant linked to the signal peptide, the signal peptide and the NGF protein or NGF protein variant are encoded by genes that are naturally unrelated, that is, when the gene encoding the signal peptide is unrelated to the gene encoding the NGF protein or the variant of the NGF protein, or when the protein naturally linked to the signal peptide is not an NGF protein or a variant of the NGF protein, the signal peptide can be said to be heterologous.

[0079] In the present application, the term "signal peptide" generally refers to a short peptide chain that guides the movement of a newly synthesized protein into the secretory pathway. The general length of a signal peptide is 5 to 30 amino acids. The signal peptide may be located at the N-terminus or C-terminus of the protein precursor, but in most cases, it exists as the N-terminal peptide of the protein precursor. The signal peptide may function to facilitate the translocation of the expressed polypeptide into the endoplasmic reticulum. The signal peptide is usually excised during this process. The signal peptide excised from the protein precursor may be heterologous or homologous to the organism used to produce the polypeptide. For example, the signal peptide of the present application may include functionally active fragments, cleavage products, and / or mutants of the wild-type signal peptide. For example, the signal peptide of the present application may have a function of inducing the binding of a substance containing the signal peptide to the cell membrane.

[0080] In the present application, the term "wild-type" generally refers to an amino acid or nucleic acid sequence that naturally exists within a certain species or population (e.g., human, mouse, rat, cell, etc.).

[0081] In the present application, the term "neurotrophin receptor p75" generally refers to the low-affinity nerve growth factor receptor (LNGFR), which binds to neurotrophins and is a member of the tumor necrosis factor receptor superfamily, and is also referred to as p75NTR. The amino acid sequence and other information of exemplary human p75NTR can be found under the accession number P08138 in the UniProtKB database. The p75NTR used herein is not limited to human p75NTR and includes all species orthologs of human p75NTR. The term "p75NTR" includes the precursor form of p75NTR, the p75NTR precursor, the full-length p75NTR, and any form of p75NTR generated by intracellular processes. The term also includes naturally occurring p75NTR variants such as splice variants, allelic variants, and isotypes.

[0082] In the present application, the term "neurotrophic factor" generally refers to proteins that support the survival, development, and normal function of neurons. It is a type of growth factor. Exemplary neurotrophic factors include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), neurotrophic factor-4 (NT-4), neurotrophic factor-6 (NT-6), neurotrophic factor-7 (NT-7), dehydroepiandrosterone (DHEA), DHEA sulfate (DHEA-S), GDNF ligand family, and ciliary neurotrophic factor (CNTF), and other biomolecules.

[0083] In the present application, the term "brain-derived neurotrophic factor (BDNF)" generally refers to one of the neurotrophic factors that is also commonly referred to as "abrineurin". BDNF is a member of the neurotrophic factor growth factor family and is related to classical nerve growth factors. BDNF is normally present in the brain and peripheries. BDNF as used herein is not limited to human BDNF and includes orthologs of all species of human BDNF. The term "BDNF" includes the precursor form of BDNF, pro-BDNF, full-length BDNF, and any form of BDNF produced by intracellular processes. The term also includes naturally occurring BDNF variants such as splice variants, allelic variants, and isotypes. The amino acid sequence of the human BDNF protein can be found under the accession number P23560 in the UniProtKB database. The full-length human BDNF protein usually has 247 amino acids and can include a signal peptide, a pro-region, a leader peptide, and mature BDNF. The BDNF signal peptide may be amino acids 1-18 of the full-length human BDNF protein. For example, the BDNF signal peptide can include the amino acid sequence shown in SEQ ID NO: 31.

[0084] In the present application, the term "neurotrophin-3 (NT-3)" generally refers to a neurotrophin of the NGF (nerve growth factor) family of neurotrophins. NT-3 is a protein growth factor that is active against specific neurons in the peripheral and central nervous systems. The amino acid sequence of exemplary human NT-3 can be found under accession number P20783 in the UniProtKB database.

[0085] In the present application, the term "neurotrophin-4 (NT-4)" generally refers to a neurotrophin of the NGF (nerve growth factor) family of neurotrophins. NT-4 mainly signals through the TrkB receptor tyrosine kinase. NT4 is also referred to as NT-5. The amino acid sequence of exemplary human NT-4 can be found under accession number P34130 in the UniProtKB database.

[0086] In the present application, the term "leader peptide" generally refers to a portion that is cleaved during the maturation or activation of a protein. Once cleaved, the leader peptide usually loses its independent biological function. The leader peptide can assist in the folding and proper expression of the protein. In some cases, the leader peptide can include a signal peptide or a part of the signal peptide. In some cases, the leader peptide does not include a signal peptide.

[0087] In the present application, the term "delivery vector" generally refers to a transport medium that can deliver a reagent (e.g., mRNA) to target cells. The delivery vector can deliver a reagent (e.g., mRNA) to a specific cell subtype. For example, the delivery vector targets a specific type of cell by virtue of the unique properties of the delivery vector or a moiety coupled to the vector, a moiety contained therein (or a moiety linked to the vector, a moiety that together with the delivery vector is sufficient to target the delivery vector). The delivery vector can also increase the in vivo half-life of the reagent to be delivered (e.g., mRNA) and / or the bioavailability of the reagent to be delivered. The delivery vector may include a viral vector, virus-like particle, polycationic vector, peptide vector, liposome, and / or hybrid vector. For example, when the target cell is a hepatocyte, the properties of the delivery vector (e.g., size, charge, and / or pH) can effectively deliver the delivery vector and / or a molecule encapsulated therein (e.g., mRNA) to the target cell, reduce immune clearance, and / or promote retention within the target cell.

[0088] In the present application, the term "lipid nanoparticle (LNP)" generally refers to a particle containing a plurality (i.e., two or more) of lipid molecules physically bonded to each other by intermolecular forces (e.g., covalent or non-covalent bonds). The lipid nanoparticle may be, for example, a microsphere (including monolayer and multilayer vesicles such as liposomes), a dispersed phase in an emulsion, a micelle, or an internal phase in a suspension. The lipid nanoparticle may include one or more lipids (e.g., cationic lipids, non-cationic lipids, and PEG-modified lipids).

[0089] In the present application, the term "liposome" generally refers to a vesicle having an internal space and isolated from the external medium through one or more bilayer membranes. For example, the bilayer may be formed from amphiphilic molecules such as synthetic lipids or natural lipids that include spatially isolated hydrophilic and hydrophobic domains, and also for example, the bilayer may be formed from amphiphilic polymers and surfactants.

[0090] In the present application, when the term "modified" is used with respect to a nucleic acid (e.g., RNA or DNA), it generally refers to the nucleic acid having different nucleotide molecules, different nucleotide sequences, being composed of different linkages, and / or incorporating non-natural moieties within its structure as compared to the corresponding wild type. For example, the modification may include modifications to nucleotides, e.g., the nucleotides may include modified bases, sugars, or phosphate groups. For example, the modification may include polypeptides or proteins having different nucleotide sequences but encoding the same amino acid sequence, or polypeptides or proteins having the same function. The modification may be a chemical modification and / or a biological modification. "Chemical modification" may include modifications that introduce chemical substances different from those found in wild-type or naturally occurring nucleic acids, e.g., covalent modifications, e.g., the introduction of modified nucleotides (e.g., nucleotide analogs, or the introduction of side groups not naturally found in these nucleic acid molecules). The term "modified nucleotide" generally refers to a unit in a nucleic acid polymer that includes a modified base, sugar, or phosphate group, or incorporates a non-natural moiety into its structure.

[0091] In the present application, when the term "codon optimization" is used with respect to a nucleic acid, it generally refers to a nucleic acid encoding a polypeptide that has been modified to have improved expression in a cell, such as a mammalian cell or a bacterial cell, by replacing one, at least one, or more than one codon of the nucleic acid encoding the parental polypeptide with a codon encoding the same amino acid residue but having a different relative frequency of use in the cell.

[0092] In the present application, the term "polynucleotide" generally includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA produced using nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleotide analogs), and hybrids thereof. The nucleic acid molecule may be single-stranded or double-stranded.

[0093] In this application, the term "isolated nucleic acid molecule" generally refers to a single-stranded or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' end to the 3' end, or an analog thereof, which is isolated from at least about 50% of the polypeptides, peptides, lipids, sugars, polynucleotides or other materials that are naturally found with the nucleic acid molecule when isolating the total nucleic acid from the source cell. For example, an isolated nucleic acid molecule is substantially free of other contaminating nucleic acid molecules or other molecules found in the natural environment of the nucleic acid that may interfere with its use or its therapeutic, diagnostic, prophylactic or research use.

[0094] In this application, the term "mRNA" generally refers to an RNA transcript that is processed to remove introns and can be translated into a polypeptide.

[0095] In this application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host and transports the inserted nucleic acid molecule within and / or between host cells. The vector can include vectors mainly used for inserting DNA or RNA into cells, vectors mainly used for replicating DNA or RNA, and expression vectors mainly used for transcription and / or translation of DNA or RNA. The vector can also include carriers having the above functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Usually, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product.

[0096] In this application, the term "cell" generally refers to an individual cell, cell line, or cell culture that can contain, or already contains, a plasmid or vector containing the nucleic acid molecules described in this application, or that can express the antibodies or antigen-binding fragments thereof described in this application. The cells can include the parental generation of a single host cell. Due to natural, accidental, or intentional mutations, the progeny cells are not necessarily identical to the original parental cells in morphology or genome, but it is sufficient if they can express the antibodies or antigen-binding fragments thereof described in this application. The cells can be obtained by in vitro transfection of cells using the vectors described in this application. The cells can be prokaryotic cells or eukaryotic cells.

[0097] In this application, the term "pharmaceutically acceptable carrier" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers, and the doses and concentrations at which they are employed are non-toxic to the cells or mammals to which they are exposed. Usually, a physiologically acceptable carrier is an aqueous pH-buffered solution. Examples of physiologically acceptable carriers can include buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides, and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium; and / or nonionic surfactants.

[0098] In this application, the term "pharmaceutical composition" generally refers to a formulation in a form that enables the biological activity of an active ingredient (e.g., the S protein variant, nucleic acid molecule of this application), and the formulation does not contain other components that have unacceptable toxicity to the subject to which the formulation is administered. These formulations can be sterile.

[0099] In the present application, the term "kit" generally refers to a packaged product containing components used for treating PD-1-mediated related diseases by administering the antigen-binding protein of the present application. The components of the kit may be contained in separate vials (i.e., a kit with separate parts), or may be provided in a single vial. The kit can include reagents such as buffers, protein stabilization reagents, signal generation systems (e.g., fluorescent signal generation systems), antibodies, control proteins, and test containers. The kit can also include instructions for carrying out the method.

[0100] In the present application, the term "drug delivery device" includes: (i) an injection module for administering a pharmaceutical composition containing an active ingredient to a subject; (ii) a pharmaceutical composition for injection, which contains an active ingredient selected from recombinant proteins, nucleic acid molecules, mRNA, vectors, cells, compositions, pharmaceutical compositions, or combinations thereof; and (iii) an optional efficacy monitoring module.

[0101] In the present application, the term "central and / or peripheral nervous system disease" generally refers to diseases associated with lesions of the central nervous system (brain or spinal cord) and the peripheral nervous system (nerves outside the brain or spinal cord). For example, central and / or peripheral nervous system diseases are caused by one or more of neuronal degeneration, inflammation or loss of oligodendrocytes, infarction, injury or tumor of the blood supply to the brain, and bacterial or viral infections. Central and / or peripheral nervous system diseases may include Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple sclerosis, encephalitis or meningitis, structural damage to the brain or spinal cord, and / or stroke.

[0102] In the present application, the term "neurodegenerative disease" generally refers to the progressive loss of neuronal function and structure that causes cognitive impairment. Neurodegenerative diseases can include Alzheimer's disease, Parkinson's disease, Huntington's chorea, early-onset Alzheimer's disease or early-onset Parkinson's disease, and / or amyotrophic lateral sclerosis.

[0103] In the present application, the term "peripheral neuropathy" generally refers to a disease or disorder caused by an abnormality of the peripheral nervous system. The peripheral nervous system includes all nerves other than the central nervous system (brain and spinal cord), such as the nerves connecting the head, face, eyes, nose, muscles, and ears to the brain (cranial nerves), the nerves connecting the spinal cord to the rest of the body, and the nerve cells distributed throughout the body.

[0104] In the present application, the term "subject" generally refers to a human or non-human animal (including mammals) in need of diagnosis, prognosis, improvement, prevention, and / or treatment of a disease, such as a human, non-human primate (apes, gibbons, gorillas, chimpanzees, orangutans, macaques), domestic animals (dogs and cats), farm animals (poultry such as chickens and ducks, horses, cows, goats, sheep, pigs), and laboratory animals (mice, rats, rabbits, guinea pigs). Human subjects include fetal, neonatal, infant, adolescent, and adult subjects. The subject can include an animal disease model.

[0105] In the present application, the terms "comprising", "consisting of", "having", "able to", "containing" and their variations are generally intended to be transitional phrases, terms or words without limitation that do not exclude the possibility of additional actions or structures. The term "consisting of" generally means that no other components (or similarly, features, integers, steps, etc.) can be present. Unless otherwise clearly indicated in the context, the singular forms in English such as "a", "an", "the", and in Japanese such as "one", "a kind", "said / the" generally include the plural forms of what is being referred to.

[0106] In this application, the term "about" generally means approximately, in the region of, roughly, or around. When the term "about" is used to refer to a range of values, the cutoff value or specific value is used to indicate that the recited value may vary by up to 10% from the recited value. Thus, the term "about" can be used to cover variations of ±10% or less, ±5% or less, ±1% or less, ±0.5% or less, or ±0.1% or less from a specific value.

[0107] Detailed Description of the Invention Recombinant Protein In one aspect, this application provides a recombinant protein comprising a nerve growth factor (NGF) protein variant, wherein the binding of the NGF protein variant to the neurotrophin receptor p75 (p75NTR) is reduced by at least about 50% (e.g., 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%, at least about 98% or more reduction) compared to the wild-type NGF protein. For example, the wild-type NGF protein may comprise the amino acid sequence shown in SEQ ID NO: 32.

[0108] In this application, compared to the amino acid sequence shown in SEQ ID NO: 32, the NGF protein variant may comprise one or more amino acid mutations. For example, compared to the amino acid sequence shown in SEQ ID NO: 32, the NGF protein variant may comprise an amino acid mutation at R100 (i.e., arginine at position 100). For example, compared to the amino acid sequence shown in SEQ ID NO: 32, the arginine at position 100 of the NGF protein variant mutates to tryptophan (i.e., R100W). For example, the NGF protein variant comprises the amino acid sequence shown in SEQ ID NO: 33.

[0109] As is known in the art, when a mature NGF protein undergoes an R100W mutation, its ability to be secreted extracellularly decreases. This application relates to NGF that exists naturally R100W NGF with increased expression level and / or secretion amount compared to a mutant protein R100W provides a recombinant protein of the mutant. The recombinant protein of this application is mature NGF secreted extracellularly R100W The amount of the mutant protein can be increased. For example, the recombinant protein of this application may include a heterologous signal peptide that may be located at the N-terminus of the NGF protein mutant. For example, compared to the signal peptide shown in SEQ ID NO: 29, the heterologous signal peptide increases the expression level and / or secretion amount of the NGF protein mutant by at least about 50% (for example, 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%, at least about 98% or more).

[0110] The length of the heterologous signal peptide described in this application may be 5 to 30 amino acids. For example, the length of the heterologous signal peptide described in this application may be 5 to 25 amino acids. For example, the length of the heterologous signal peptide described in this application may be 5 to 20 amino acids. For example, the length of the heterologous signal peptide described in this application may be 10 to 25 amino acids. For example, the length of the heterologous signal peptide described in this application may be 10 to 30 amino acids. For example, the length of the heterologous signal peptide described in this application may be 15 to 30 amino acids. For example, the length of the heterologous signal peptide described in this application may be 10 to 20 amino acids. For example, the length of the heterologous signal peptide described in this application may be 15 to 20 amino acids. For example, the length of the heterologous signal peptide described in this application may be 18 amino acids. For example, the length of the heterologous signal peptide described in this application may be 19 amino acids. For example, the length of the heterologous signal peptide described in this application may be 20 amino acids.

[0111] The heterologous signal peptides described in this application may be derived from prokaryotes such as bacteria (Escherichia coli, Staphylococcus aureus, Bacillus subtilis). The heterologous signal peptides described in this application may be derived from eukaryotes, such as yeast or mammalian cells (human, mouse, monkey, etc.).

[0112] The heterologous signal peptides described in this application may be derived from secreted proteins, periplasmic proteins and / or membrane-bound proteins, such as enzymes. For example, the signal peptide may be derived from Escherichia coli outer membrane protein A OmpA, OmpF, lambda phage receptor LamB, heat-stable enterotoxin ST, alkaline phosphatase PhoA, maltose-binding protein MBP, DsbA, Staphylococcus aureus protein A, Bacillus subtilis protease, neutral protease, Bacillus marginalis RNA enzymes, levanase. For example, the signal peptide may be derived from the natural signal peptide of exogenous proteins in the yeast expression system, α-factor signal peptide, sucrase gene SUC signal peptide sequence, acid phosphatase gene PHO1 and / or interstitial metalloproteinase, or may be derived from human serum albumin and bovine chymosin.

[0113] The heterologous signal peptides described in this application may be derived from Gaussia, Homo sapiens, Oikopleura dioica or Mus musculus. The heterologous signal peptides described in this application may include signal peptides derived from Gaussia luciferase, human trypsinogen-2, Oikosin1 variant, azurocidin preproprotein and / or Igκ. In some embodiments, the heterologous signal peptide is derived from Gp67, Gp64, honey bee melittin (HBM), albumin, IL-2, azurocidin preproprotein, mouse IgK, immunoglobulin heavy chain and / or cystatin-S precursor.

[0114] The heterologous signal peptides described in this application can include signal peptides derived from neurotrophic factors. For example, the heterologous signal peptide may be a signal peptide of brain-derived neurotrophic factor (BDNF), neurotrophic factor-3 (NT-3), and / or neurotrophic factor-4 (NT-4). For example, the heterologous signal peptide can include the amino acid sequence set forth in any one of SEQ ID NOs: 30, 31, and 35-43. For example, the heterologous signal peptide is an IgK signal peptide and / or a BDNF signal peptide. For example, the heterologous signal peptide includes the amino acid sequence set forth in any one of SEQ ID NOs: 30-31.

[0115] For example, the heterologous signal peptide is an IgK signal peptide, and for another example, the heterologous signal peptide includes the amino acid sequence shown in SEQ ID NO: 30.

[0116] For example, the heterologous signal peptide is a BDNF signal peptide, and for another example, the heterologous signal peptide includes the amino acid sequence shown in SEQ ID NO: 31.

[0117] For example, the heterologous signal peptide is an hIL-2 signal peptide, and for another example, the heterologous signal peptide includes the amino acid sequence shown in SEQ ID NO: 35.

[0118] For example, the heterologous signal peptide is an APP signal peptide, and for another example, the heterologous signal peptide includes the amino acid sequence shown in SEQ ID NO: 36.

[0119] For example, the heterologous signal peptide is an HC signal peptide, and for another example, the heterologous signal peptide includes the amino acid sequence shown in SEQ ID NO: 37.

[0120] For example, the heterologous signal peptide is a Cystatin signal peptide, and for another example, the heterologous signal peptide includes the amino acid sequence shown in SEQ ID NO: 38.

[0121] For example, the heterologous signal peptide is a Gp67 signal peptide, and for example, the heterologous signal peptide contains the amino acid sequence shown in SEQ ID NO: 39.

[0122] For example, the heterologous signal peptide is an HBM signal peptide, and for example, the heterologous signal peptide contains the amino acid sequence shown in SEQ ID NO: 40.

[0123] For example, the heterologous signal peptide is a Gp64 signal peptide, and for example, the heterologous signal peptide contains the amino acid sequence shown in SEQ ID NO: 41.

[0124] For example, the heterologous signal peptide is an Alb signal peptide, and for example, the heterologous signal peptide contains the amino acid sequence shown in SEQ ID NO: 42.

[0125] For example, the heterologous signal peptide is an SP signal peptide, and for example, the heterologous signal peptide contains the amino acid sequence shown in SEQ ID NO: 43.

[0126] In the present application, the recombinant protein further contains a leader peptide. The leader peptide may be a leader peptide of an NGF protein or a heterologous leader peptide derived from a non-NGF protein. For example, the leader peptide can contain the amino acid sequence shown in SEQ ID NO: 34.

[0127] In the present application, the recombinant protein further contains a leader peptide. In the present application, the recombinant protein can contain the heterologous signal peptide, the leader peptide, and the NGF protein variant in sequence from the N-terminus to the C-terminus.

[0128] In the present application, the recombinant protein further includes a leader peptide. In the present application, the recombinant protein can sequentially include the heterologous signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus, and the heterologous signal peptide includes an amino acid sequence shown in any one of SEQ ID NOs: 30-31 and 35-43, the leader peptide includes an amino acid sequence shown in SEQ ID NO: 34, and the NGF protein variant includes an amino acid sequence shown in SEQ ID NO: 33.

[0129] For example, the recombinant protein can sequentially include the IgK signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes an amino acid sequence shown in SEQ ID NO: 15.

[0130] For example, the recombinant protein can sequentially include the IgK signal peptide and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes an amino acid sequence shown in SEQ ID NO: 16.

[0131] For example, the recombinant protein can sequentially include the BDNF signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes an amino acid sequence shown in SEQ ID NO: 19.

[0132] For example, the recombinant protein can sequentially include the BDNF signal peptide and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes an amino acid sequence shown in SEQ ID NO: 20.

[0133] For example, the recombinant protein can sequentially include the SP signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes an amino acid sequence shown in SEQ ID NO: 58.

[0134] For example, the recombinant protein can sequentially include the hIL-2 signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes the amino acid sequence shown in SEQ ID NO: 59.

[0135] For example, the recombinant protein can sequentially include the Cystatin signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes the amino acid sequence shown in SEQ ID NO: 60.

[0136] For example, the recombinant protein can sequentially include the APP signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes the amino acid sequence shown in SEQ ID NO: 61.

[0137] For example, the recombinant protein can sequentially include the HC signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes the amino acid sequence shown in SEQ ID NO: 62.

[0138] For example, the recombinant protein can sequentially include the Gp67 signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes the amino acid sequence shown in SEQ ID NO: 63.

[0139] For example, the recombinant protein can sequentially include the HBM signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes the amino acid sequence shown in SEQ ID NO: 64.

[0140] For example, the recombinant protein can sequentially include the Gp64 signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes the amino acid sequence shown in SEQ ID NO: 65.

[0141] For example, the recombinant protein can sequentially include the Alb signal peptide, the leader peptide, and the NGF protein variant from the N-terminus to the C-terminus. For example, the recombinant protein includes the amino acid sequence shown in SEQ ID NO: 66.

[0142] In some embodiments, the recombinant protein further includes a tag peptide.

[0143] For example, the recombinant protein can sequentially include the heterologous signal peptide, the leader peptide, the NGF protein variant, and the tag peptide from the N-terminus to the C-terminus.

[0144] For example, the recombinant protein can sequentially include the heterologous signal peptide, the NGF protein variant, and the tag peptide from the N-terminus to the C-terminus.

[0145] For example, the tag peptide can include the amino acid sequence shown in (YPYDVPDYA)n, where n is an arbitrary integer from 1 to 5. Also for example, the tag peptide can include the amino acid sequence shown in YPYDVPDYA (SEQ ID NO: 68) or YPYDVPDYAYPYDVPDYA (SEQ ID NO: 69).

[0146] For example, the recombinant protein can sequentially include the IgK signal peptide, the leader peptide, the NGF protein variant, and the tag peptide from the N-terminus to the C-terminus. For example, the recombinant protein includes the amino acid sequence shown in SEQ ID NO: 70.

[0147] In some embodiments, the recombinant protein further comprises a transmembrane anchor region.

[0148] For example, the recombinant protein can sequentially comprise, from the N-terminus to the C-terminus, the heterologous signal peptide, the leader peptide, the NGF protein variant, and the transmembrane anchor region.

[0149] For example, the recombinant protein can sequentially comprise, from the N-terminus to the C-terminus, the heterologous signal peptide, the NGF protein variant, and the transmembrane anchor region.

[0150] For example, the transmembrane anchor region may be the C-terminal transmembrane anchoring domain of platelet-derived growth factor receptor (PDGFR). Also for example, the transmembrane anchor region can comprise the amino acid sequence shown in SEQ ID NO: 67.

[0151] For example, the recombinant protein can sequentially comprise, from the N-terminus to the C-terminus, the IgK signal peptide, the leader peptide, the NGF protein variant, and the transmembrane anchor region. For example, the recombinant protein comprises the amino acid sequence shown in SEQ ID NO: 17.

[0152] For example, the recombinant protein can sequentially comprise, from the N-terminus to the C-terminus, the IgK signal peptide, the NGF protein variant, and the transmembrane anchor region. For example, the recombinant protein comprises the amino acid sequence shown in SEQ ID NO: 18.

[0153] In some embodiments, the recombinant protein further comprises a half-life extension domain.

[0154] For example, the recombinant protein further comprises the Fc region of an immunoglobulin. The Fc region can extend the half-life of the recombinant protein. The Fc region may be located at the C-terminus of the recombinant protein. For example, compared with a recombinant protein that does not contain the Fc region, the half-life of the recombinant protein containing the Fc region can be extended by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more.

[0155] For example, the Fc region can be derived from human immunoglobulin and / or mouse immunoglobulin.

[0156] For example, the Fc region may be the Fc region of human IgG1.

[0157] For example, the Fc region may be the Fc region of mouse IgG.

[0158] For example, the Fc region can contain the amino acid sequence shown in any one of SEQ ID NOs: 56-57.

[0159] For example, the recombinant protein can sequentially contain the heterologous signal peptide, the leader peptide, the NGF protein variant, and the Fc region from the N-terminus to the C-terminus.

[0160] Also for example, the recombinant protein can sequentially contain the IgK signal peptide, the leader peptide, the NGF protein variant, and the Fc region from the N-terminus to the C-terminus.

[0161] For example, the Fc region can contain the amino acid sequence shown in any one of SEQ ID NOs: 54-55.

[0162] Nucleic acid molecule In another aspect, the present application provides an isolated nucleic acid molecule, and the isolated nucleic acid molecule encodes the recombinant protein. For example, the isolated nucleic acid molecule can encode a recombinant protein comprising the amino acid sequence set forth in any one of SEQ ID NOs: 15-20, 54-55, and 58-66.

[0163] For example, the isolated nucleic acid molecule can encode a recombinant protein comprising the amino acid sequence set forth in SEQ ID NO: 15. For example, the isolated nucleic acid molecule can encode a recombinant protein comprising the amino acid sequence set forth in SEQ ID NO: 19.

[0164] For example, the isolated nucleic acid molecule can comprise DNA and / or RNA. For example, the isolated nucleic acid molecule can be DNA and can encode a recombinant protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 19. For example, the isolated nucleic acid molecule can be RNA and can encode a recombinant protein comprising the amino acid sequence set forth in SEQ ID NO: 15 or 19.

[0165] For example, the isolated nucleic acid molecule can comprise modifications at one or more positions selected from a 5' cap, a 5' untranslated region, an open reading frame, a 3' untranslated region, and a poly A tail.

[0166] For example, the isolated nucleic acid molecule can comprise at least one modified nucleotide.

[0167] In the present application, the nucleic acid molecule can comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 2-8, 10-11, 23-28, 46-47, and 50-51. In the present application, the nucleic acid molecule can comprise a nucleotide sequence having at least 80% (e.g., 82%, 85%, 88%, 90%, 95%, 98%, 99% or more) sequence identity with the nucleotide sequence set forth in any one of SEQ ID NOs: 2-8, 10-11, 23-28, 46-47, and 50-51.

[0168] In the present application, the nucleic acid molecule can include a nucleotide sequence shown in any one of SEQ ID NOs: 11 to 12. In the present application, the nucleic acid molecule can include a nucleotide sequence having at least 80% (for example, 82%, 85%, 88%, 90%, 95%, 98%, 99% or more) sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 11 to 12.

[0169] In the present application, the nucleic acid molecule can include a nucleotide sequence shown in any one of SEQ ID NOs: 3, 11 and 23. In the present application, the nucleic acid molecule can include a nucleotide sequence having at least 80% (for example, 82%, 85%, 88%, 90%, 95%, 98%, 99% or more) sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 3, 11 and 23.

[0170] In the present application, the nucleic acid molecule can include a nucleotide sequence shown in any one of SEQ ID NOs: 7, 12 and 27. In the present application, the mRNA can include a nucleotide sequence having at least 80% (for example, 82%, 85%, 88%, 90%, 95%, 98%, 99% or more) sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 7, 12 and 27.

[0171] In one aspect, the present application provides an isolated RNA, particularly mRNA, wherein the pre-mRNA includes a nucleic acid molecule encoding a nerve growth factor (NGF) protein or a variant thereof, or at least a part of the mRNA can encode the NGF protein variant.

[0172] In some embodiments, the NGF protein includes the amino acid sequence shown in SEQ ID NO: 32.

[0173] In some embodiments, compared with the amino acid sequence shown in SEQ ID NO: 32, the binding of the NGF protein variant to the neurotrophin receptor p75 (p75NTR) is reduced by at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more).

[0174] In some embodiments, the NGF protein variant comprises the amino acid sequence shown in SEQ ID NO: 33.

[0175] In some embodiments, the mRNA comprises a polynucleotide encoding a heterologous signal peptide, and compared with the signal peptide shown in SEQ ID NO: 29, the heterologous signal peptide increases the expression level and / or secretion level of the NGF protein variant by at least 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more).

[0176] In some embodiments, the heterologous signal peptide can reach 50% or more of the expression level and / or secretion level of the wild-type NGF protein (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or more).

[0177] In some embodiments, the heterologous signal peptide is derived from a neurotrophic factor.

[0178] In some embodiments, the heterologous signal peptide is derived from brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and / or neurotrophin-4 (NT-4).

[0179] In some embodiments, the heterologous signal peptide is derived from Gp67, Gp64, honey bee melittin (HBM), albumin, IL-2, azurocidin preproprotein, mouse IgK, immunoglobulin heavy chain, and / or cystatin-S precursor.

[0180] In some embodiments, the heterologous signal peptide is an IgK signal peptide and / or a BDNF signal peptide.

[0181] In some embodiments, the heterologous signal peptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 30-31 and 35-43.

[0182] In some embodiments, the heterologous signal peptide comprises an amino acid sequence shown in any one of SEQ ID NOs: 30-31.

[0183] In some embodiments, the mRNA further comprises a polynucleotide encoding a leader peptide.

[0184] In some embodiments, the leader peptide comprises an amino acid sequence shown in SEQ ID NO: 34.

[0185] In some embodiments, the mRNA comprises, in sequence from the 5'-end to the 3'-end, a polynucleotide encoding the heterologous signal peptide, a polynucleotide encoding the leader peptide, and a polynucleotide encoding the NGF protein or a variant thereof.

[0186] In some embodiments, the mRNA comprises, in sequence from the 5'-end to the 3'-end, a polynucleotide encoding the IgK signal peptide, a polynucleotide encoding the leader peptide, and a polynucleotide encoding the NGF protein variant.

[0187] In some embodiments, the mRNA comprises, in order from the 5'-end to the 3'-end, a polynucleotide encoding the BDNF signal peptide, a polynucleotide encoding the leader peptide, and a polynucleotide encoding the NGF protein variant.

[0188] In some embodiments, the mRNA encodes an amino acid sequence shown in any one of SEQ ID NOs: 15 to 20. In some embodiments, the mRNA encodes an amino acid sequence shown in any one of SEQ ID NOs: 15 and 19.

[0189] In some embodiments, the recombinant protein further comprises a transmembrane anchor region.

[0190] For example, the recombinant protein can comprise, in order from the N-terminus to the C-terminus, the heterologous signal peptide, the leader peptide, the NGF protein variant, and the transmembrane anchor region.

[0191] For example, the recombinant protein can comprise, in order from the N-terminus to the C-terminus, the heterologous signal peptide, the NGF protein variant, and the transmembrane anchor region.

[0192] For example, the transmembrane anchor region may be a PDGFR transmembrane anchor region. Also for example, the transmembrane anchor region can comprise the amino acid sequence shown in SEQ ID NO: 67.

[0193] For example, the recombinant protein can comprise, in order from the N-terminus to the C-terminus, the IgK signal peptide, the leader peptide, the NGF protein variant, and the transmembrane anchor region. For example, the recombinant protein can comprise, in order from the N-terminus to the C-terminus, the IgK signal peptide, the NGF protein variant, and the transmembrane anchor region.

[0194] In some embodiments, the mRNA encodes an amino acid sequence set forth in any one of SEQ ID NOs: 17-18.

[0195] In some embodiments, the mRNA further comprises a polynucleotide encoding the Fc region of an immunoglobulin.

[0196] In some embodiments, the Fc region is derived from human immunoglobulin and / or mouse immunoglobulin.

[0197] In some embodiments, the Fc region is the Fc region of human IgG1.

[0198] In some embodiments, the Fc region is the Fc region of mouse IgG.

[0199] In some embodiments, the Fc region comprises an amino acid sequence set forth in any one of SEQ ID NOs: 56-57.

[0200] In some embodiments, the mRNA comprises, in order from the 5'-end to the 3'-end, a polynucleotide encoding the heterologous signal peptide, a polynucleotide encoding the leader peptide, a polynucleotide encoding the NGF protein variant, and a polynucleotide encoding the Fc region of the immunoglobulin.

[0201] In some embodiments, the mRNA comprises, in order from the 5'-end to the 3'-end, a polynucleotide encoding the IgK signal peptide, a polynucleotide encoding the leader peptide, a polynucleotide encoding the NGF protein variant, and a polynucleotide encoding the Fc region of the immunoglobulin.

[0202] In some embodiments, the mRNA encodes an amino acid sequence set forth in any one of SEQ ID NOs: 54-55.

[0203] In one aspect, the present application provides an isolated RNA, particularly mRNA, wherein the pre-mRNA contains a nucleic acid molecule encoding the recombinant protein, or at least a part of the mRNA can encode the recombinant protein. For example, the recombinant protein can contain the amino acid sequence shown in SEQ ID NO: 15.

[0204] In the present application, the RNA may be a naturally or non-naturally occurring RNA, for example, mRNA. The mRNA can contain one or more nucleobases, nucleosides or nucleotides. In the present application, the term "nucleoside" generally refers to a compound containing a sugar molecule (e.g., pentose or ribose) or its derivative and an organic base (e.g., purine or pyrimidine) or its derivative (also referred to as "nucleobase" herein). In the present application, "nucleotide" generally refers to a nucleoside containing a phosphate group.

[0205] In the present application, the mRNA can contain a 5'untranslated region (5'UTR), a 3'untranslated region (3'UTR) and / or a coding region (e.g., open reading frame).

[0206] In the present application, the mRNA may be modified. The modification may be a chemical modification or a biological modification. In the present application, the mRNA can contain one or more modified nucleobases, nucleosides or nucleotides. In this case, it may be referred to as "chemically modified mRNA", and may also be referred to as "modified mRNA" herein. Compared with an unmodified reference sequence (e.g., a naturally occurring or wild-type mRNA), the modified mRNA can have useful properties such as improved stability, increased intracellular retention, improved translation efficiency and / or reduced immunogenicity. Therefore, using modified mRNA can improve the efficiency of protein production, improve nucleic acid retention in cells, and reduce immunogenicity.

[0207] In some embodiments, the mRNA comprises modifications at one or more positions selected from a 5' cap, a 5' untranslated region, an open reading frame, a 3' untranslated region, and a poly A tail.

[0208] In the present application, the mRNA can comprise modified nucleobases, and the modified nucleobases may be modified uracil. In the present application, the mRNA can comprise modified nucleobases, and the modified nucleobases may be modified cytosine. In the present application, the mRNA can comprise modified nucleobases, and the modified nucleobases may be modified adenine. In the present application, the mRNA can comprise modified nucleobases, and the modified nucleobases may be modified guanine. In the present application, the mRNA can comprise a combination of one or more of the modified nucleobases (for example, a combination of 2, 3, or 4 of the modified nucleobases).

[0209] For example, the isolated nucleic acid molecule can be selected from one or more nucleotides of N1-methylpseudouridine-5'-triphosphate (N1-Methylpseudo-UTP), pseudouridine-5'-triphosphate (pseudo-UTP), 5-methoxyuridine- 5'-tri phosphate (5-Methoxy-U T P), and 5-methylcytidine triphosphate (5-Methyl-CTP). In some embodiments, the modified RNA of the present application can comprise a combination of one or more of the modified nucleobases (for example, a combination of 2, 3, or 4 of the modified nucleobases).

[0210] In the present application, the mRNA can be codon-optimized, and even after one or more nucleotides of the codon are changed, the codon still encodes the same amino acid.

[0211] In the present application, the mRNA can contain the nucleotide sequence shown in any one of SEQ ID NOs: 23 to 28 and 50 to 51. In the present application, the mRNA can contain a nucleotide sequence having at least 80% (e.g., 82%, 85%, 88%, 90%, 95%, 98%, 99% or more) sequence identity with the nucleotide sequence shown in any one of SEQ ID NOs: 23 to 28 and 50 to 51.

[0212] In the present application, the mRNA can contain the nucleotide sequence shown in SEQ ID NO: 23. In the present application, the mRNA can contain a nucleotide sequence having at least 80% (e.g., 82%, 85%, 88%, 90%, 95%, 98%, 99% or more) sequence identity with the nucleotide sequence shown in SEQ ID NO: 23.

[0213] In the present application, the mRNA can contain the nucleotide sequence shown in SEQ ID NO: 23. In the present application, the mRNA can contain a nucleotide sequence having at least 80% (e.g., 82%, 85%, 88%, 90%, 95%, 98%, 99% or more) sequence identity with the nucleotide sequence shown in SEQ ID NO: 23.

[0214] In the present application, the mRNA can contain the nucleotide sequence shown in SEQ ID NO: 27. In the present application, the mRNA can contain a nucleotide sequence having at least 80% (e.g., 82%, 85%, 88%, 90%, 95%, 98%, 99% or more) sequence identity with the nucleotide sequence shown in SEQ ID NO: 27.

[0215] The mRNA of the present application can be generated through any method feasible in the art, including but not limited to in vitro transcription (IVT) and synthetic methods. The mRNA can be manufactured using enzymatic, solid-phase, liquid-phase, combinatorial synthesis methods, small domain synthesis, and ligation methods. In one embodiment, the mRNA is manufactured using the IVT enzymatic synthesis method. Accordingly, the present application further relates to polynucleotides that can also be used for in vitro transcription of the mRNA in the present application, such as DNA, constructs, and vectors.

[0216] Composition In the present application, the composition can further include a delivery vector. The mRNA of the present application may be manufactured in nanoparticles or other delivery vectors, for example, to avoid degradation during delivery to a subject. In the present application, the mRNA may be encapsulated within nanoparticles. In certain embodiments, the nanoparticles have particles with at least one size (e.g., diameter) of about 1000 nM or less, about 500 nM or less, about 400 nM or less, about 300 nM or less, about 200 nM or less, or about 100 nM or less. In certain embodiments, the nanoparticles can include lipids. The lipid nanoparticles can include, but are not limited to, liposomes and micelles. In the present application, the lipid nanoparticles can include cationic and / or ionizable lipids, anionic lipids, non-cationic lipids, neutral lipids, amphiphilic lipids, polyethylene glycolylated lipids and / or structural lipids, or combinations of the above. In certain embodiments, the lipid nanoparticles include one or more mRNAs described in the present application, for example, mRNA, and also for example, mRNA encoding a target polypeptide (e.g., the recombinant protein or the NGF protein variant). For example, the lipid nanoparticles include one or more mRNAs encoding the recombinant protein or the NGF protein variant described in the present application.

[0217] The delivery vector in the composition described in this application may be a lipid nanoparticle. The lipid nanoparticle may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) cationic and / or ionizable lipids. A "cationic lipid" generally refers to a lipid having any number of net positive charges at a given pH (e.g., physiological pH). The cationic lipid may include, but is not limited to, 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethaneamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza-octatriacontane (KL25), DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, Octyl-CLinDMA, octyl-CLinDMA(2S), DODAC, DOTMA, DDAB, DOTAP, DOTAP.C1, DC-Choi, DOSPA, DOGS, DODAP, DODMA, and DMRIE. Furthermore, many commercially available cationic and / or ionizable lipids such as LIPOFECTIN® (including DOTMA and DOPE) and LIPOFECTAMINE® (including DOSPA and DOPE) can be used. For example, the cationic lipid may be DLin-MC3-DMA or DLin-KC2-DMA.

[0218] In some embodiments, the molar ratio of the cationic lipid in the lipid nanoparticle is about 40-70%, e.g., about 40-65%, about 40-60%, about 45-55%, or about 48-53%. In some embodiments, the molar ratio of the cationic lipid (e.g., DLin-MC3-DMA) in the lipid nanoparticle is about 50%.

[0219] In the present application, the lipid nanoparticles can comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) non-cationic lipids. The non-cationic lipids can include anionic lipids. Anionic lipids suitable for the lipid nanoparticles of the present application can include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, and other neutral lipids conjugated with an anionic group.

[0220] The non-cationic lipids can include neutral lipids. Neutral lipids suitable for the lipid nanoparticles of the present application can include phospholipids such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyl-oleoylphosphatidylcholine (POPC), palmitoyl oleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), or mixtures thereof. Further, lipids having a mixture of saturated and unsaturated fatty acid chains can be used. For example, the neutral lipids described in the present application can be selected from DOPE, DSPC, DPPC, POPC, or any related phosphatidylcholine.

[0221] In some embodiments, the molar ratio of the phospholipid in the lipid nanoparticles is about 30-45%, for example, about 33-42%, about 35-40% or about 38-39%. In some embodiments, the molar ratio of the phospholipid (e.g., DSPC) in the lipid nanoparticles is about 38.5%.

[0222] In the present application, the lipid nanoparticles can include lipid complexes such as polyethylene glycol (PEG)-modified lipids and derivative lipids. The PEG-modified lipid can include, but is not limited to, a polyethylene glycol chain up to 5 kDa covalently bonded to a lipid having an alkyl chain with a length of C6-C20. By adding these components, aggregation of lipids can be prevented, the circulation time can be extended, delivery of the lipid-nucleic acid composition to target cells can be promoted, or the nucleic acid can be rapidly released. For example, the polyethylene glycol (PEG)-modified lipid molecule may be a PEG ceramide having a shorter acyl chain (e.g., C14 or C18). For example, the lipid nanoparticles can include PEG2000-DMG.

[0223] In some embodiments, the molar ratio of the polyethylene glycol (PEG)-modified lipid molecule in the lipid nanoparticles is about 0.5-2%, for example, about 1-2%, about 1.2-1.8% or about 1.4-1.6%. In some embodiments, the molar ratio of the polyethylene glycol (PEG)-modified lipid molecule (e.g., PEG2000-DMG) in the lipid nanoparticles is about 1.5%.

[0224] In the present application, the lipid nanoparticles can further include cholesterol. In some embodiments, the molar ratio of the cholesterol in the lipid nanoparticles is about 5-15%, for example, about 6-14%, about 7-13%, about 8-12% or about 9-11%. In some embodiments, the molar ratio of the cholesterol in the lipid nanoparticles is about 10%.

[0225] In the present application, the lipid nanoparticles can include a cationic lipid, cholesterol, a phospholipid, and a polyethylene glycol-modified lipid molecule. In some embodiments, the molar ratio of the cationic lipid, cholesterol, phospholipid, and polyethylene glycol-modified lipid molecule may be 45-55:5-15:35-45:0.5-2. In some embodiments, the molar ratio of the cationic lipid, cholesterol, phospholipid, and polyethylene glycol-modified lipid molecule may be 50:10:38.5:1.5.

[0226] For example, the composition can include the mRNA and the lipid nanoparticles, wherein the mRNA can encode the amino acid sequence shown in SEQ ID NO: 15, the lipid nanoparticles can include a cationic lipid, cholesterol, a phospholipid, and a polyethylene glycol-modified lipid molecule, and the molar ratio of the cationic lipid, cholesterol, phospholipid, and polyethylene glycol-modified lipid molecule may be 50:10:38.5:1.5.

[0227] For example, the composition can include the mRNA and the lipid nanoparticles, wherein the mRNA can encode the amino acid sequence shown in SEQ ID NO: 15, the lipid nanoparticles can include DLin-MC3-DMA, cholesterol, DSPC, and PEG2000-DMG, and the molar ratio of DLin-MC3-DMA, cholesterol, DSPC, and PEG2000-DMG may be 50:10:38.5:1.5.

[0228] For example, the composition can include the mRNA and the lipid nanoparticles, wherein the mRNA can include the nucleotide sequence shown in SEQ ID NO: 23, the lipid nanoparticles can include a cationic lipid, cholesterol, a phospholipid, and a polyethylene glycol-modified lipid molecule, and the molar ratio of the cationic lipid, cholesterol, phospholipid, and polyethylene glycol-modified lipid molecule may be 50:10:38.5:1.5.

[0229] For example, the composition can include the mRNA and the lipid nanoparticles, where the mRNA can include the amino acid sequence shown in SEQ ID NO: 23, the lipid nanoparticles can include DLin-MC3-DMA, cholesterol, DSPC, and PEG2000-DMG, and the molar ratio of DLin-MC3-DMA, cholesterol, DSPC, and PEG2000-DMG may be 50:10:38.5:1.5.

[0230] Manufacturing method This application provides a method for manufacturing the delivery vector in the composition. For example, the selected lipid can be deposited on the inner wall of a suitable container or container, the lipid can be dissolved in a suitable solvent, and then the solvent can be evaporated to leave a thin film inside the container, or multilamellar vesicles (MLVs) can be manufactured through existing techniques such as performing spray drying. An aqueous phase is added to the rotating container to form MLVs. Next, unilamellar vesicles (ULVs) are formed by homogenization, sonication, or extrusion of the multilamellar vesicles. Furthermore, unilamellar vesicles can be formed by surfactant removal techniques.

[0231] In this application, the composition includes a delivery vector (e.g., lipid nanoparticles), in which the mRNA can associate with the surface of the lipid delivery vector and be encapsulated therein. For example, when manufacturing the composition of this application, the cationic lipid delivery vector may associate with the mRNA through electrostatic interaction.

[0232] In some embodiments, the composition includes a diagnostic radionuclide, a fluorescent substance, or other substances that can be detected in vivo and in vitro.

[0233] In the present application, an appropriate size of the lipid delivery vector (e.g., lipid nanoparticle) is selected in consideration of the size of the target cell or tissue and the degree of application of the liposome to be produced. In some embodiments, mRNA can be delivered to a specific cell or tissue. For example, to target hepatocytes, the size of the lipid delivery vector (e.g., lipid nanoparticle) is determined such that the size is smaller than the fenestrated gaps of the fenestrated sinusoids of the liver endothelium, so that the lipid delivery vector (e.g., lipid nanoparticle) can easily penetrate these endothelial fenestrated gaps and reach the target hepatocytes. The lipid delivery vector (e.g., lipid nanoparticle) can have a diameter large enough to limit or significantly avoid distribution within a specific cell or tissue. In the present application, the size (e.g., diameter) of the lipid delivery vector (e.g., lipid nanoparticle) may be in the range of about 25 to 250 nm, for example, about 250 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, 50 nm, 25 nm or less than 10 nm. For example, the size (e.g., diameter) of the lipid delivery vector (e.g., lipid nanoparticle) may be in the range of about 25 to 250 nm, for example, within about 50 to 200 nm, about 75 to 175 nm, about 75 to 150 nm or about 75 to 125 nm.

[0234] Vectors, Cells and Pharmaceutical Compositions In another aspect, the present application provides a vector comprising the nucleic acid molecule. For example, the vector may be a viral vector such as an adenovirus vector, an adeno-associated virus vector and / or a lentivirus vector.

[0235] In another aspect, the present application provides a cell comprising the nucleic acid molecule and / or the vector. In the present application, the cell may be a prokaryotic cell, for example, Escherichia coli. In the present application, the cell may be a eukaryotic cell such as a yeast cell, an insect cell, a plant cell and an animal cell. In the present application, the cell may be a mammalian cell such as a mouse cell and a human cell. In the present application, the cell may be a 293T cell or a P12 cell.

[0236] In another aspect, the present application provides a method for producing the recombinant protein, which includes culturing the cells under conditions for expressing the recombinant protein.

[0237] In another aspect, the present application provides a pharmaceutical composition including the recombinant protein, the nucleic acid molecule, the composition, the vector and / or the cell, and optionally a pharmaceutically acceptable carrier.

[0238] In another aspect, the present application provides a kit or drug delivery device including the recombinant protein, the nucleic acid molecule, the composition, the vector, the cell, and / or the pharmaceutical composition. It can include the recombinant protein, the nucleic acid molecule, the composition, the vector, the cell, and / or the pharmaceutical composition described in the present application in a single commonly used container, and can optionally be combined with one or more therapeutic agents and optionally be manufactured into a kit together.

[0239] Treatment method In another aspect, the present application provides a method for alleviating, preventing and / or treating central and / or peripheral nervous system diseases, which includes administering the recombinant protein, the nucleic acid molecule, the composition, the vector, the cell, and / or the pharmaceutical composition to a subject in need thereof. For example, the central and / or peripheral nervous system diseases can include neurodegenerative diseases. For example, the central and / or peripheral nervous system diseases can include peripheral neuropathy. For example, the peripheral neuropathy can include toxic peripheral neuropathy, hereditary peripheral neuropathy and / or metabolic peripheral neuropathy. For example, in some embodiments, the central and / or peripheral nervous system diseases can be included, and the peripheral neuropathy can include toxic peripheral neuropathy, hereditary peripheral neuropathy and / or metabolic peripheral neuropathy.

[0240] In some embodiments, the central and / or peripheral nervous system diseases include diabetic peripheral neuropathy, drug-related peripheral neuropathy, hereditary motor and sensory neuropathy, post-traumatic repair of peripheral nerves, traumatic optic neuritis, and / or Alzheimer's disease.

[0241] In another aspect, the present application provides the use of the recombinant protein, the nucleic acid molecule, the composition, the vector, the cell, and / or the pharmaceutical composition in the manufacture of a medicament for the alleviation, prevention, and / or treatment of central and / or peripheral nervous system diseases. For example, the central and / or peripheral nervous system diseases can include neurodegenerative diseases. For example, the central and / or peripheral nervous system diseases can include peripheral neuropathy. For example, the peripheral neuropathy can include toxic peripheral neuropathy, hereditary peripheral neuropathy, and / or metabolic peripheral neuropathy. For example, the central and / or peripheral nervous system diseases can be included in certain embodiments, and the peripheral neuropathy includes toxic peripheral neuropathy, hereditary peripheral neuropathy, and / or metabolic peripheral neuropathy.

[0242] In another aspect, a method for promoting the secretion and / or expression of nerve growth factor (NGF) protein is provided, which includes the recombinant protein, the nucleic acid molecule, the composition, the vector, the cell, and / or the pharmaceutical composition. For example, the method may be a method for non-therapeutic purposes. For example, the method may be an in vitro and / or ex vivo method.

[0243] In another aspect, a method for promoting the proliferation and / or differentiation of nerve cells is provided, which includes the recombinant protein, the nucleic acid molecule, the composition, the vector, the cell, and / or the pharmaceutical composition. For example, the method may be a method for non-therapeutic purposes. For example, the method may be an in vitro and / or ex vivo method.

[0244] In another aspect, there is provided a method for repairing damaged nerve cells, comprising the recombinant protein, the nucleic acid molecule, the composition, the vector, the cell, and / or the pharmaceutical composition. For example, the method may be a method for non-therapeutic purposes. For example, the method may be an in vitro and / or ex vivo method.

[0245] Without being bound by any theory, the following examples are only intended to illustrate various technical solutions of the present application and are not intended to limit the scope of the present application.

Examples

[0246] Example 1 NGF R100W Design optimization and synthesis of mRNA As shown in FIG. 1A or FIG. 1J, recombinant proteins containing NGF mutants were constructed by methods such as replacing the NGF endogenous signal peptide with an exogenous signal peptide, cleaving the leader peptide, and increasing the transmembrane anchor region. Here, NGFwt: from the N-terminus to the C-terminus, it is the full-length wild-type NGF protein which is sequentially the NGF signal peptide + leader peptide + mature NGF protein; NGFmut: from the N-terminus to the C-terminus, it is sequentially the NGF signal peptide + leader peptide + mature NGF R100W mutant protein, the full-length NGF R221W mutant protein; IGK19-NGFmut: from the N-terminus to the C-terminus, it is sequentially the IGK signal peptide + leader peptide + mature NGF R100W mutant protein; IGK122-NGFmut: from the N-terminus to the C-terminus, it is sequentially the IGK signal peptide + mature NGF R100W mutant protein; IGK19-NGF-PDGFRmut: from the N-terminus to the C-terminus, it is sequentially the IGK signal peptide + leader peptide + mature NGF R100W mutant protein + PDGFR transmembrane anchor region; IGK122-NGF-PDGFRmut: from the N-terminus to the C-terminus, it is sequentially the IGK signal peptide + mature NGF R100WMutant protein + PDGFR transmembrane anchor region; BDNF19NGFmut: From the N-terminus to the C-terminus, sequentially BDNF signal peptide + leader peptide + mature NGF R100W Mutant protein; BDNF122-NGFmut: From the N-terminus to the C-terminus, sequentially BDNF signal peptide + mature NGF R100W Mutant protein; The amino acid sequence and nucleotide sequence are as shown in Table 1 below.

[0247]

Table 1

[0248] Using T7 polymerase, the DNA sequence of the above coding region was obtained by in vitro transcription, an HA tag was added to the 3' end, cloned downstream of the T7 promoter of the pSG5L vector, a universal primer for the linear DNA template for in vitro transcription was obtained on the vector, and the DNA template for in vitro transcription was obtained by PCR. T7 RNA polymerase was used for in vitro transcription, DNA enzyme was used for digestion of the template, then E. coli poly(A) polymerase EPAP (E.coli Poly(A)Polymerase) was used for addition of the poly(A) tail, and finally, mRNA was obtained by purification and recovery.

[0249] The mRNA was expressed in the 293T tool cell line, and the expression level was detected using western blot. The results showed that protein expression was detected in the supernatant for both IGK19-NGFmut and BDNF19-NGFmut, as shown in Figure 1B. Also, Figure 1F shows that compared with the wild-type NGF protein (WT), NGF without a heterologous signal peptide R100WIt was shown that the secretion amount of the protein (Mut) into the supernatant was low, and most of it existed intracellularly in the form of proNGF. Furthermore, several other common signal peptides such as APP (Azurocidin preproprotein), HC (Immunoglobulin heavy chain), SP (Signal peptide), Cystatin (Cystatin-S precursor) HBM, Gp64, Alb (Albumin) (Figure 1J) were used, and as shown in Figures 1G - 1I, all the results indicated that the secretion of NGFmut could be promoted to a certain extent.

[0250] Example 2 Detection of the expression level of the mRNA-modified protein In this example, IGK19-NGFmut mRNA was modified, that is, nucleotides with different chemical modifications were added during transcription to obtain mRNA with chemical modifications, and the introduction of the chemically modified nucleotides was 100% substitution in all cases. The chemical modifications used were N1-methylpseudouridine-5'-triphosphate (N1-Methylpseudo-UTP, i.e., N1-UTP), pseudouridine-5'-triphosphate (pseudo-UTP), 5-methoxyuridine- 5'-tri phosphate (5-Methoxy-U T P, i.e., 5mO-UTP) and 5-methylcytidine triphosphate (5-Methyl-CTP, 5mCTP), respectively. As shown in Figure 1C, the protein expression levels obtained by the translation of IGK19-NGFmut mRNA containing different modifications were equivalent, and the protein expression level containing N1-UTP modification was the highest. The mRNA for subsequent biochemical and functional studies was modified with N1-UTP.

[0251] Example 3 Detection of the function of NGF R100W in promoting nerve cell differentiation The PC12 cell line is derived from transplantable mouse pheochromocytoma, and these cells showed a reversible neuronal phenotype response to NGF. When PC12 cells are exposed to the presence of NGF protein, the cells can respond within one week, mainly by arresting cell division and extending neurites. PC12 cells were cultured in RPMI1640 medium containing 5% fetal bovine serum and 5% horse serum. When the cells reached the logarithmic growth phase, they were transfected with NGFwt or IGK19-NGFmut mRNA. After 24 hours, the cells were stained with DAPI and HA, and cell morphology was observed using a fluorescence microscope and an electron microscope. As shown in Figures 1D and 1E, the results showed that IGK19NGFmut, similar to NGFwt, had its mRNA translated and expressed as a mutant protein, and could promote the differentiation of PC12 cells. R100W The mutant protein can be expressed and can promote the differentiation of PC12 cells.

[0252] Example 4: Obtaining lipid nanoparticles encapsulating NGF mRNA with lipids A mixture of NGF mRNA and lipids was encapsulated into a lipid nanoparticle (LNP) reaction system to deliver the mRNA, and the process is as shown in Figure 2. Four types of lipid molecules, namely cationic lipid (DLin-MC3-DMA), cholesterol, distearoyl phosphatidylcholine (DSPC), and polyethylene glycol-modified lipid molecule (PEG2000-DMG), were used. The four types of lipids were dissolved in ethanol at a molar ratio of 50:10:38.5:1.5. The lipids dissolved in ethanol and the mRNA transcribed in vitro were quickly mixed through a T-tube, and lipid nanoparticles with a diameter of about 100 μm were obtained by self-packaging. NGFwt and IGK19-NGFmut transcribed in vitro were mixed with lipids according to the ratio of mRNA:cationic lipid = 1:3 (molar ratio) and packaged into nanoparticle LNP. The particle size of the LNP was detected using a Malvern Zetasizer nanoparticle size and potential analyzer. As shown in Figure 3A, the sizes of NGFwt and IGK19-NGFmut were about 100 nm.

[0253] After collecting 2 μg of NGF LNP and directly dripping it onto 293T cells to perform cell transfection, as shown in Figure 3B, the expression of mature NGF protein secreted into the supernatant was detected in both the cells and the supernatant. By combining a fluorescence reporter system that oxidizes D-luciferin with luciferase to emit fluorescence and in vivo fluorescence imaging technology, the localization and expression dynamics of nanoparticle expression in the mouse body were determined. After injecting nanoparticles coupled with luciferase mRNA into the tail vein of mice, the luciferin substrate was intraperitoneally injected at different time points, and the intensity and localization of the bioluminescence generated by the luciferase oxidation of the substrate were detected using in vivo imaging of small animals. The results, as shown in Figure 3C, indicated that when administered via the circulatory pathway, the expression efficiency of the nanoparticles was higher and mainly concentrated in the liver.

[0254] Example 5 Verification of the Low Pain-Inducing Effect of IGK19-NGFmut LNP in Mice Eight 8-week-old male C57BL / 6 mice were divided into two groups of 4 mice each, and 2 μg of IGK19-NGFmut LNP and 2 μg of NGFwt LNP were injected into the plantar part of the hind paw of each mouse using plantar injection. Eight hours after the injection, a behavioral experiment of the mice was performed to detect the perception threshold of the thermal pain sensation on the plantar surface of the mice. Eight hours after the plantar injection, the mice were placed on a transparent frame, and infrared heat source stimuli of the same intensity were applied to the two groups of mice, and the latency until the mice lifted their feet was recorded.

[0255] Furthermore, ten 8-week-old male C57BL / 6 mice were divided into two groups of 5 mice each, and 2 μg of IGK19-NGFmut LNP and 2 μg of NGFwt LNP were injected in the same way, respectively. Eight hours after the plantar injection, the mice were placed on a grid frame, and mechanical stress of different magnitudes was applied to the plantar surface of the mice using von frey filaments, and the stress value that caused the mice to lift their feet was recorded.

[0256] As shown in Figure 4A, the results showed that the mice injected with IGK19-NGFmut LNP had a significantly higher sensitivity threshold to thermal pain compared to the control group of mice injected with NGFwt LNP. On the other hand, Figure 4B also shows that the expression of IGK19-NGFmut mRNA reduces the sensitivity of the mouse's mechanical pain response compared to the expression of NGFwt, indicating that the expression of IGK19-NGFmut LNP on the plantar surface of the mouse's foot has a lower pain-inducing effect on the mouse than NGFwt LNP.

[0257] Example 6 Verification of the Nerve Repair Effect of IGK19-NGFmut LNP in Mice with Peripheral Nerve Injury Modeling of peripheral nerve injury in mice was performed using 15 eight-week-old male C57BL / 6 mice by intraperitoneal injection of paclitaxel every other day. One week after the completion of modeling, the mice were divided into three groups, and 6 mice in each group were injected with IGK19-NGFmut LNP and NGFwt LNP, while only 3 mice were in the modeling group. Using plantar injection, 2 μg of IGK19-NGFmut LNP and 2 μg of NGFwt LNP were injected into the plantar part of the hind paw of each mouse, and the injection was performed once every two days. After 5 injections, the thermal pain perception threshold and mechanical pain threshold of the plantar surface of the mouse's foot were detected. The mouse was placed on a grid frame, and von frey filaments were used to apply mechanical stress of different sizes to the plantar surface of the mouse's foot, and the stress value that caused the mouse to lift its foot was recorded. After the mechanical pain threshold test was completed, a 2-hour buffer period was given, and then the two groups of mice were placed on a hot plate at 55 °C, and the latency of the mouse to lift its foot was recorded. The experimental process is as shown in Figure 5A.

[0258] The results of thermal and mechanical pain in Figure 5B showed that peripheral nerve injury in mice caused by 12 mg / kg paclitaxel injection may cause an increase in the pain threshold of the mice's feet. As shown in Figure 5C, as the number of injections increased, mice injected with IGK19-NGFmut LNP showed a significant increase in the continuity and density of peripheral nerves compared to the control group of mice injected with NGFwt LNP. On the other hand, Figure 5D also showed that the expression of IGK19-NGFmut mRNA made the mice more sensitive to mechanical and thermal pain compared to the expression of NGFwt, indicating that the expression of IGK19-NGFmut LNP on the soles of the mice's feet could repair the peripheral nerve injury caused by paclitaxel.

[0259] The above detailed description is provided in the form of explanations and examples and is not intended to limit the scope of the appended claims. At present, various changes in the embodiments listed in this application are obvious to those skilled in the art and are retained within the scope of the appended claims and their equivalent methods.

Claims

**Claim 1** A recombinant protein comprising a nerve growth factor (NGF) protein variant and a heterologous signal peptide linked to the NGF protein variant, wherein the amino acid sequence of the recombinant protein is shown in SEQ ID NO: 15, the recombinant protein. **Claim 2** An isolated nucleic acid molecule encoding the recombinant protein according to claim 1, wherein the nucleic acid molecule is RNA, the RNA contains modifications at one or more positions selected from a 5' cap, a 5' untranslated region, an open reading frame, a 3' untranslated region, and a poly A tail, the RNA contains at least one modified nucleotide, the nucleotide sequence of the RNA is shown in SEQ ID NO: 23, the nucleic acid molecule. **Claim 3** The nucleic acid molecule according to claim 2, wherein the modified nucleotide contained in the nucleic acid molecule comprises one or more nucleotides selected from N1-methylpseudouridine-5'-triphosphate (N1-Methylpseudo-UTP), pseudouridine-5'-triphosphate (pseudo-UTP), and 5-methoxyuridine-5'-triphosphate (5-Methoxy-UTP). **Claim 4** A composition comprising (a) an mRNA consisting of the nucleotide sequence shown in SEQ ID NO: 23, and (b) a delivery vector, wherein the mRNA contains modifications at one or more positions selected from a 5' cap, a 5' untranslated region, an open reading frame, a 3' untranslated region, and a poly A tail, the mRNA contains at least one modified nucleotide, the composition. **Claim 5** The composition according to claim 4, wherein the modified nucleotide contained in the mRNA comprises one or more nucleotides selected from N1-methylpseudouridine-5'-triphosphate (N1-Methylpseudo-UTP), pseudouridine-5'-triphosphate (pseudo-UTP), and 5-methoxyuridine-5'-triphosphate (5-Methoxy-UTP). **Claim 6** The composition according to claim 4 or claim 5, wherein the delivery vector comprises DLin-MC3-DMA, cholesterol, DSPC, and PEG2000-DMG, and the molar ratio of DLin-MC3-DMA, cholesterol, DSPC, and PEG2000-DMG is 50:10:38.5:1.

5. **Claim 7** ​ The composition according to claim 4 or claim 5, wherein the diameter of the delivery vector is about 80 nm to about 200 nm.

8. The composition according to claim 4 or claim 5, wherein the mRNA is encapsulated in the delivery vector.

9. A vector comprising the nucleic acid molecule according to claim 2.

10. A cell comprising the nucleic acid molecule according to claim 2 or claim 3, and / or the vector according to claim 9, or expressing the recombinant protein according to claim 1.

11. A method for producing the recombinant protein according to claim 1, comprising culturing the cell according to claim 10 under conditions for expressing the recombinant protein according to claim 1.

12. A pharmaceutical composition comprising the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or claim 3, the composition according to claim 4, the vector according to claim 9, and / or a cell, and optionally a pharmaceutically acceptable carrier, wherein the cell comprises the nucleic acid molecule according to claim 2 or claim 3 and / or the vector according to claim 9, or expresses the recombinant protein according to claim 1, said pharmaceutical composition.

13. A kit or drug delivery device comprising the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or claim 3, the composition according to claim 4, the vector according to claim 9, a cell, and / or the pharmaceutical composition, wherein the cell comprises the nucleic acid molecule according to claim 2 or claim 3 and / or the vector according to claim 9, or expresses the recombinant protein according to claim 1, wherein the pharmaceutical composition comprises the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or claim 3, the composition according to claim 4, the vector according to claim 9, and / or the cell, and optionally a pharmaceutically acceptable carrier, said kit or said drug delivery device.

14. Use of the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or claim 3, the composition according to claim 4, the vector according to claim 9, a cell, and / or the pharmaceutical composition in the manufacture of a medicament for the alleviation, prevention and / or treatment of central and / or peripheral nervous system diseases, wherein the cell comprises the nucleic acid molecule according to claim 2 or claim 3 and / or the vector according to claim 9, or expresses the recombinant protein according to claim 1, The pharmaceutical composition comprises the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or claim 3, the composition according to claim 4, the vector according to claim 9, and / or the cell, and an optional pharmaceutically acceptable carrier, The use. [

15. ] A method for expressing the recombinant protein according to claim 1, comprising using the nucleic acid molecule according to claim 2 or claim 3, the composition according to claim 4, the vector according to claim 9, the cell, and / or the pharmaceutical composition, wherein the cell comprises the nucleic acid molecule according to claim 2 or claim 3 and / or the vector according to claim 9, or expresses the recombinant protein according to claim 1, the pharmaceutical composition comprises the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or claim 3, the composition according to claim 4, the vector according to claim 9, and / or the cell, and an optional pharmaceutically acceptable carrier, the method is a method for non-therapeutic purposes, the method is an in vitro or ex vivo method, The method. [

16. ] A method for promoting the proliferation and / or differentiation of nerve cells, comprising using the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or claim 3, the composition according to claim 4, the vector according to claim 9, the cell, and / or the pharmaceutical composition, wherein the cell comprises the nucleic acid molecule according to claim 2 or claim 3 and / or the vector according to claim 9, or expresses the recombinant protein according to claim 1, the pharmaceutical composition comprises the recombinant protein according to claim 1, the nucleic acid molecule according to claim 2 or claim 3, the composition according to claim 4, the vector according to claim 9, and / or the cell, and an optional pharmaceutically acceptable carrier, the method is a method for non-therapeutic purposes, the method is an in vitro or ex vivo method, The method.

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