WO2020143515A1 - 人肝细胞生长因子突变体及其应用 - Google Patents

人肝细胞生长因子突变体及其应用 Download PDF

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WO2020143515A1
WO2020143515A1 PCT/CN2020/070010 CN2020070010W WO2020143515A1 WO 2020143515 A1 WO2020143515 A1 WO 2020143515A1 CN 2020070010 W CN2020070010 W CN 2020070010W WO 2020143515 A1 WO2020143515 A1 WO 2020143515A1
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hhgf
mutant
vector
nucleic acid
cells
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PCT/CN2020/070010
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English (en)
French (fr)
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聂李亚
许松山
马素永
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北京诺思兰德生物技术股份有限公司
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Priority to CN202080007253.5A priority Critical patent/CN113383012A/zh
Priority to US17/420,865 priority patent/US20220064242A1/en
Priority to KR1020217024747A priority patent/KR102667727B1/ko
Priority to EP20738308.4A priority patent/EP3909974A4/en
Priority to JP2021539421A priority patent/JP7246494B2/ja
Publication of WO2020143515A1 publication Critical patent/WO2020143515A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • C07K14/4753—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host

Definitions

  • the present application relates to mutants of human hepatocyte growth factor (hHGF).
  • the present application also relates to a nucleic acid molecule encoding the mutant, a vector containing the nucleic acid molecule, and a host cell containing the nucleic acid molecule or vector.
  • the present application also relates to a pharmaceutical composition containing the hHGF mutant or the nucleic acid molecule encoding the mutant, and the use of the hHGF mutant or the nucleic acid molecule encoding the mutant.
  • the hHGF mutant or the nucleic acid molecule encoding the mutant can be used, for example, to promote endothelial cell growth and/or migration, promote angiogenesis, or treat diseases that can benefit from the activity of natural hHGF (eg, treat lower limb arterial ischemia, myocardium Infarction and/or diabetic peripheral neuropathy), and therefore can be used to prepare drugs.
  • diseases that can benefit from the activity of natural hHGF eg, treat lower limb arterial ischemia, myocardium Infarction and/or diabetic peripheral neuropathy
  • Hepatocyte growth factor originally isolated from rat plasma and platelets, is a secreted heparin-affinity glycoprotein, also known as diffusion factor (SF). It is now known that HGF is produced by interstitial cells, binds to the receptor c-Met and activates the receptor's tyrosine kinase activity, and promotes hepatocytes, epithelial cells, endothelial cells, melanocytes, hematopoietic cells and other types of cells Growth, migration and morphogenesis. HGF plays an important role in the development of embryonic liver and placenta, participates in maintaining and renewing cells in organs such as liver, lung, and kidney, and promotes the regeneration and repair of these organs after injury. In addition, HGF can promote the invasion or growth inhibition of tumor cells from different sources. Therefore, HGF is a multifunctional cytokine with broad clinical application prospects.
  • SF diffusion factor
  • Mature HGF is a heterodimer composed of heavy chain ( ⁇ chain) and light chain ( ⁇ chain) connected by interchain disulfide bonds, where the ⁇ chain contains 463 amino acids, about 69kD; the ⁇ chain contains 234 amino acids , About 34kD.
  • the N-terminus of the ⁇ chain has a hairpin structure, and there are 4 plasmin-like Kringle structures (referred to as K1, K2, K3, K4 regions in turn) near its C-terminus, each Kringle structure consists of about 80 amino acids composition.
  • the hairpin structure and the K1 region are the key sites for binding HGF to the receptor c-Met; and the hairpin structure and the K2 region together constitute the necessary structure for the affinity of HGF with heparin and heparan sulfate; the ⁇ chain contains a serine protease Like folding region, but no serine protease activity.
  • the heavy chain and light chain each contain 2 N-glycosylation sites (hepatocyte growth factor) Molecular Biology Research. Journal of Bioengineering, 2002, 18:1-4).
  • C-Met is a specific cell membrane receptor for HGF, expressed in a variety of cells, such as cardiomyocytes, vascular endothelial cells, etc., mediating the biological role of HGF.
  • the HGF/C-Met system is widely expressed in a variety of tissues and participates in complex biological processes that regulate cell growth, movement, and tissue morphogenesis.
  • HGF is an endothelial growth factor that binds to its specific receptor C-Met, causing phosphorylation of the receptor's tyrosine residues, initiating the signaling process after the receptor; and also causing ERK phosphorylation, resulting in STAT3 (Ser727 ) Phosphorylation forms a dimer into the nucleus, promoting the expression of early growth response genes such as c-fos, thereby regulating cell growth at the transcriptional level.
  • HGF can activate MEK, P42/44MAPK and P90RSR, reduce cell death caused by hydrogen peroxide, and can activate BCL-2 gene expression and inhibit Bax protein translocation to the mitochondrial membrane surface, maintaining the mitochondrial membrane electrochemistry The gradient prevents the leakage of cytochrome C in mitochondria, inhibits the activity of Caspase-3 and Caspase-9, and produces anti-apoptotic effects.
  • HGF can also stimulate the expression of MMP-1, VEGF, HGF and C-Met in vascular endothelial cells and vascular smooth muscle cells, and significantly increase the mRNA expression and transcription activity of Ets-1, and play an important role in the process of neovascularization (Angiogenic properties) of hepatocyte growth factor is dependent on upregulation of essential transcription for factor for angiogenesis, ets-1.Circulation, 2003, 107:1411-1417).
  • the Ets pathway is also one of the molecular mechanisms by which HGF promotes angiogenesis (Therapeutic angiogenesis using hepatocyte growth factor. Current Gene. Therapy, 2004, 4:199-206).
  • the Ets family transcription factor has a DNA-binding domain and can bind to the core of the DNA sequence GGA. It plays a very important role in the expression of various genes involved in mitogenic signals, and may be involved in controlling the transcription of these genes. Regulation of angiogenesis.
  • the HGF gene contains many regulatory regions, such as B-cell and macrophage-specific transcription factor junction regions, interleukin 26 response element (IL26RE), transfer growth factor inhibitory element (TNFIE), and cAMP response element (CRE). Therefore, exogenous HGF can stimulate the expression of endogenous HGF by inducing ets activity, and endogenous HGF can promote the formation of small blood vessels through the automatic conduction function.
  • hHGF can be mutated to obtain hHGF mutants with enhanced biological activity.
  • natural hHGF and wild-type hHGF refer to biologically active, naturally occurring human hepatocyte growth factor (human hepatocyte growth factor, hHGF), both of which have the same meaning, And can be used interchangeably.
  • the amino acid sequences of natural hHGF or wild-type hHGF can be conveniently obtained from various public databases (for example, GenBank database). For example, the amino acid sequence of natural hHGF can be found in the GenBank database accession number: NP_000592.3.
  • natural hHGF when referring to the amino acid sequence of natural hHGF, it is described using the sequence shown in SEQ ID NO:1.
  • the expression "the 130th amino acid residue of natural hHGF” refers to the 130th amino acid residue of the protein shown in SEQ ID NO: 1.
  • natural hHGF can have multiple versions, they have substantially the same primary structure (ie, amino acid sequence) and higher structure (ie, spatial structure), and substantially the same biological function, However, there may still be slight differences in amino acid sequence between them. Therefore, in this application, natural hHGF is not limited to the protein shown in SEQ ID NO: 1, but is intended to cover all known natural hHGF.
  • natural hHGF should include various naturally occurring hHGFs with biological functions, including, for example, hHGF shown in SEQ ID NO: 1 and naturally occurring variants thereof. And, when describing the amino acid position of hHGF, it includes not only the specific amino acid position in SEQ ID NO: 1, but also the amino acid position corresponding to the specific amino acid position in its natural variant.
  • amino acid residue 130 of natural hHGF includes the amino acid residue 130 of SEQ ID NO: 1, and the corresponding amino acid position in its natural variant.
  • the expression “corresponding amino acid position” refers to an amino acid position at an equivalent position in the compared sequences when the sequences are optimally aligned, that is, when the sequences are aligned to obtain the highest percentage identity.
  • the expression “the position corresponding to the 130th position of SEQ ID NO: 1” means that when an optimal alignment is performed between a sequence and SEQ ID NO: 1, that is, when a sequence matches SEQ ID NO: 1.
  • native hHGF has the amino acid sequence shown in SEQ ID NO:1.
  • natural hHGF is a naturally occurring human hepatocyte growth factor with biological function, and its amino acid sequence has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • natural hHGF is a naturally-occurring biologically functional human hepatocyte growth factor, and its amino acid sequence has one or more (e.g., 1-10 compared to SEQ ID NO:1 1-5 or 1-3) amino acid differences (eg, conservative amino acid substitutions).
  • identity is used to refer to the sequence matching between two polypeptides or between two nucleic acids.
  • a position in two compared sequences is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or two A certain position in each of the polypeptides is occupied by lysine)
  • each molecule is the same at this position.
  • the "percent identity” between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions for comparison x 100. For example, if 6 of the 10 positions of the two sequences match, the two sequences have 60% identity.
  • the DNA sequences CTGACT and CAGGTT share 50% identity (3 of 6 positions match).
  • comparisons are made when two sequences are aligned to produce maximum identity.
  • Such an alignment can be achieved by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48:443-453 which is conveniently performed by a computer program such as the Align program (DNAstar, Inc.). You can also use the algorithms of E. Meyers and W. Miller (Comput. Appl.
  • the Needleman and Wunsch (JMoI Biol.48:444-453 (1970)) algorithms in the GAP program integrated into the GCG software package can be used, and the Blossum 62 matrix or PAM250 matrix and gap weights of 16, 14, 12, 10, 8, 6 or 4 and length weights of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences .
  • conservative substitution means an amino acid substitution that does not adversely affect or change the necessary characteristics of the protein/polypeptide containing the amino acid sequence.
  • conservative substitutions can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include substitutions that replace amino acid residues with amino acid residues that have similar side chains, such as those that are physically or functionally similar to the corresponding amino acid residues (eg, have similar size, shape, charge, chemical properties, including Substitution of residues such as the ability to form covalent bonds or hydrogen bonds.
  • a family of amino acid residues with similar side chains has been defined in the art.
  • These families include basic side chains (eg, lysine, arginine, and histidine), acidic side chains (eg, aspartic acid, glutamic acid), and uncharged polar side chains (eg, glycine) , Asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), non-polar side chains (such as alanine, valine, leucine, isole Amino acid, proline, phenylalanine, methionine), beta branched side chains (eg, threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, Phenylalanine, tryptophan, histidine).
  • basic side chains eg, lysine, arginine, and histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged polar side chains eg, glycine
  • polypeptide and “protein” have the same meaning and are used interchangeably.
  • amino acids are generally represented by one-letter and three-letter abbreviations known in the art.
  • alanine can be represented by A or Ala.
  • amino acid having a basic side chain has a meaning generally understood by those skilled in the art.
  • Amino acids usually have the following structure:
  • R is a side chain group.
  • the amino acid is an amino acid having a basic side chain.
  • the side chain of the amino acid can be dissociated to produce OH ⁇ , and it becomes basic.
  • the dissociated side chain of the amino acid will carry a positive charge. Therefore, amino acids with basic side chains are also referred to as basic amino acids or amino acids with positively charged side chain groups.
  • Typical examples of amino acids with basic side chains include, but are not limited to, lysine, arginine, and histidine.
  • the term “isolated” or “isolated” refers to obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a living animal naturally has a polynucleotide or polypeptide that has not been isolated, and the same high-purity polynucleotide or polypeptide isolated from this natural state is called an isolation. of.
  • the term “isolated” or “isolated” does not exclude the mixing of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.
  • the term "vector” refers to a nucleic acid vehicle into which a polynucleotide can be inserted.
  • the vector When the vector enables expression of the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell by transformation, transduction or transfection, so that the genetic material elements carried by it can be expressed in the host cell.
  • Vectors are well known to those skilled in the art and include, but are not limited to: plasmids (such as naked plasmids); phagemids; Kos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 origin Artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 bacteriophage; and viral vectors.
  • plasmids such as naked plasmids
  • phagemids such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1 origin Artificial chromosomes (PAC)
  • bacteriophages such as lambda phage or M13 bacteriophage
  • viral vectors include, but are not limited to: plasmids (such as naked plasmids); phagemids; Kos plasmids; artificial chromosomes, such as yeast
  • Viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses Tumor vacuole virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses poxviruses
  • baculoviruses papillomaviruses
  • Tumor vacuole virus such as SV40
  • a vector can contain multiple elements that control expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may contain an origin of replication.
  • the term "host cell” refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • prokaryotic cells such as E. coli or Bacillus subtilis
  • fungal cells such as yeast cells or Aspergillus
  • Insect cells such as S2 Drosophila cells or Sf9
  • animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK293 cells or human cells.
  • the term “pharmaceutically acceptable” means that it is recognized in the pharmaceutical arts that it can be used in animals, especially in humans.
  • pharmaceutically acceptable carrier and/or excipient refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, It is well known in the art (see eg Remington's Pharmaceuticals. Edited by Gennaro AR, 19th.
  • pH adjusting agents including but not limited to phosphate buffers
  • Surfactants including but not limited to cationic, anionic or nonionic surfactants, such as Tween-80
  • adjuvants including but not limited to sodium chloride
  • ionic strength enhancers including but not limited to sodium chloride
  • adjuvant refers to a non-specific immunopotentiator, which when delivered together with an antigen or in advance into the body, can enhance the body's immune response to the antigen or change the type of immune response.
  • adjuvants include, but are not limited to, aluminum adjuvant (such as aluminum hydroxide), Freund's adjuvant (such as complete Freund's adjuvant and incomplete Freund's adjuvant), Corynebacterium parvum, lipopolysaccharide, cytokines, etc. .
  • pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including oils derived from petroleum, animal, plant, or synthetic oils, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. .
  • water is the preferred carrier.
  • Saline solutions as well as aqueous dextrose and glycerol solutions can also be used as liquid carriers, especially for injectable solutions.
  • pharmaceutically acceptable excipients may include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, Sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, etc.
  • the pharmaceutical composition may also contain a wetting agent, or an emulsifying agent such as sodium hyaluronate, or a pH buffering agent.
  • the pharmaceutical composition can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like.
  • an effective amount refers to an amount sufficient to obtain or at least partially achieve the desired effect.
  • an effective amount for preventing a disease refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease
  • an effective amount for treating a disease refers to an amount sufficient to cure or at least partially prevent the disease and its complications of patients who already have the disease. It is well within the ability of those skilled in the art to determine such an effective amount.
  • the amount effective for therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and gender, the mode of drug administration, and other treatments administered simultaneously and many more.
  • the term "subject” refers to mammals, including but not limited to, humans, rodents (mice, rats, guinea pigs), dogs, horses, cattle, cats, pigs, monkeys, Chimpanzees, etc.
  • the subject is a human.
  • the term "disease that can benefit from the activity of natural hHGF” refers to a disease in which enhanced expression and/or activity of HGF can alleviate the symptoms of the disease, delay the progression of the disease, or cure or partially Cure the disease.
  • HGF has a variety of biological activities, including but not limited to one or more of the following activities: (1) promote the growth and/or migration of endothelial cells; (2) promote the development of blood vessels (such as microvessels) ; And/or, (3) Promote the repair of nerve damage (such as peripheral neuropathy, such as diabetic peripheral neuropathy).
  • HGF may have application prospects in many aspects, including but not limited to: (1) promoting endothelial cell growth and/or migration; (2) promoting the development of blood vessels (such as microvessels); (3) treating ischemic diseases, For example, coronary artery disease (CAD) or peripheral artery disease (PAD), such as lower limb arterial ischemia; (4) treatment of metabolic syndrome and diabetes and its complications (eg, diabetic peripheral neuropathy); (5) inhibition of restenosis; And (6) promote the repair of nerve damage (eg, neurodegenerative diseases, traumatic nerve damage, peripheral neuropathy).
  • CAD coronary artery disease
  • PDA peripheral artery disease
  • nerve damage eg, neurodegenerative diseases, traumatic nerve damage, peripheral neuropathy.
  • examples of the term "disease that can benefit from the activity of natural hHGF” include, but are not limited to, the above-mentioned diseases, for example, ischemic diseases, metabolic syndrome, diabetes and its complications, restenosis, nerve damage, and the like.
  • the inventors of the present application discovered after research that natural hHGF can be mutated to obtain hHGF mutants with enhanced biological activity. Specifically, the inventors of the present application found that by mutating the amino acid at position 130 of natural hHGF (referenced to SEQ ID NO: 1) to amino acids with basic side chains (eg, arginine, histidine, lysine) Amino acid), the resulting hHGF mutant has a stronger biological activity than natural hHGF.
  • the nucleic acid molecule encoding the hHGF mutant as a gene therapy drug exhibits a stronger therapeutic effect in the body of the subject than the nucleic acid molecule encoding natural hHGF.
  • this application provides a mutant of human hepatocyte growth factor (human hepatocyte growth factor, hHGF), which comprises the following mutations compared with natural hHGF: the natural hHGF corresponds to SEQ ID NO: The amino acid at the 130th position of 1 was mutated to an amino acid with a basic side chain.
  • human hepatocyte growth factor human hepatocyte growth factor, hHGF
  • the amino acid having a basic side chain is selected from arginine, histidine, and lysine. In certain preferred embodiments, the amino acid having a basic side chain is arginine. In certain preferred embodiments, the amino acid having a basic side chain is histidine. In certain preferred embodiments, the amino acid having a basic side chain is lysine.
  • the native hHGF has the amino acid sequence shown in SEQ ID NO:1.
  • the natural hHGF is a naturally occurring human hepatocyte growth factor with biological function, and its amino acid sequence has at least 85%, at least 90% compared with SEQ ID NO:1 , At least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity.
  • the natural hHGF is a naturally occurring human hepatocyte growth factor with biological function, and its amino acid sequence has one or more (e.g. 1 -10 or 1-5 or 1-3) amino acid differences (eg, conservative amino acid substitutions).
  • the mutant comprises the following mutation compared to the natural hHGF shown in SEQ ID NO:1: the amino acid at position 130 of SEQ ID NO:1 (ie, serine) is mutated to Arginine. In certain preferred embodiments, the mutant has the amino acid sequence shown in SEQ ID NO:2.
  • the mutant contains the following mutation compared to the natural hHGF shown in SEQ ID NO:1: the amino acid at position 130 of SEQ ID NO:1 (ie, serine) is mutated to Histidine. In certain preferred embodiments, the mutant has the amino acid sequence shown in SEQ ID NO:3.
  • the mutant comprises the following mutation compared to the natural hHGF shown in SEQ ID NO:1: the amino acid at position 130 of SEQ ID NO:1 (ie, serine) is mutated to Lysine. In certain preferred embodiments, the mutant has the amino acid sequence shown in SEQ ID NO:4.
  • the mutant has an amino acid sequence selected from SEQ ID NO: 2, 3, and 4.
  • the protein can be modified with polyethylene glycol (PEGylation modification) to improve the half-life of the protein in vivo. Therefore, in certain preferred embodiments, the mutant is modified. In certain preferred embodiments, the mutant is chemically modified. In certain preferred embodiments, the mutant is modified by PEGylation.
  • PEGylation modification polyethylene glycol
  • the hHGF mutant of the present application can be prepared by various known methods. In certain preferred embodiments, the hHGF mutant is prepared by recombinant expression. In certain preferred embodiments, the hHGF mutant is prepared by chemical synthesis. However, it is easy to understand that the hHGF mutant of the present application is not limited by its preparation method.
  • the hHGF mutant of the present application has stronger biological activity.
  • the inventors of the present application believe that the amino acid at position 130 (referred to as SEQ ID NO: 1) located in the first hairpin structure of the N-terminus of the ⁇ -chain of natural hHGF is mutated to have a basic side chain Amino acids (eg arginine, histidine, lysine) will change the conformation of the hairpin structure, enhance the binding of hHGF protein to the receptor c-Met, and thus enhance the biological activity of hHGF protein.
  • SEQ ID NO: 1 amino acid at position 130 located in the first hairpin structure of the N-terminus of the ⁇ -chain of natural hHGF is mutated to have a basic side chain Amino acids (eg arginine, histidine, lysine) will change the conformation of the hairpin structure, enhance the binding of hHGF protein to the receptor c-Met, and thus enhance the biological activity of hHGF protein.
  • the hHGF mutant of the present application may show stronger activity in one or more aspects selected from, for example: (1) promoting the growth and/or migration of endothelial cells; (2) promoting the development of blood vessels (such as microvessels) ; And/or, (3) Promote the repair of nerve damage (such as peripheral neuropathy, such as diabetic peripheral neuropathy).
  • the present application provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding a mutant of the present invention.
  • the nucleic acid molecule encodes a mutant having an amino acid sequence selected from SEQ ID NO: 2, 3, and 4.
  • the isolated nucleic acid molecule can be used to clone or express the mutant of the present invention.
  • the nucleotide sequence of the nucleic acid molecule may be codon-optimized according to cell preferences. Therefore, in certain preferred embodiments, the nucleotide sequence of the nucleic acid molecule is codon-optimized according to host cell preferences. In certain preferred embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized according to CHO cell preferences.
  • the nucleic acid molecule has a nucleotide sequence selected from SEQ ID NO: 6, 7 and 8.
  • the present application also provides a vector comprising the isolated nucleic acid molecule as described above.
  • the vector of the present invention may be a cloning vector or an expression vector.
  • the vector of the present invention may be, for example, a plasmid; a phagemid; a Coase plasmid; an artificial chromosome, such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), or an artificial chromosome derived from P1 (PAC); phages such as lambda phage or M13 phage; and, viral vectors, etc.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses Tumor vacuole virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses adeno-associated viruses
  • herpesviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papillomaviruses papillomaviruses
  • Tumor vacuole virus such as SV40
  • the vectors of the present invention are viral vectors, such as, but not limited to, retrovirus vectors (such as lentiviral vectors), adenovirus vectors, adeno-associated virus vectors, herpes virus vectors (such as Herpes simplex virus vectors), pox virus vectors, baculovirus vectors, papilloma virus vectors, papilloma polyoma vacuolar virus vectors.
  • the vectors of the present invention are selected from adenovirus vectors, adeno-associated virus vectors, and lentiviral vectors.
  • the vectors of the invention can be expressed or used to express mutants of the invention.
  • the vectors of the present invention can or are used to express the mutants of the present invention in a subject (eg, mammal, such as a human).
  • the vectors of the invention are used for gene therapy.
  • the vector of the present invention can be used as a gene therapy vector for expressing the mutant of the present invention and performing gene therapy in a subject (eg, mammal, such as a human).
  • the vectors of the present invention are plasmids containing isolated nucleic acid molecules as described above, such as naked plasmids.
  • the vector of the present invention is a pSN vector containing the isolated nucleic acid molecule as described above.
  • the pSN vector is disclosed in Chinese Patent CN 108611367B and has the nucleotide sequence shown in SEQ ID NO: 9.
  • the present application also provides a host cell comprising the isolated nucleic acid molecule or vector of the present invention.
  • host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.).
  • the cells of the invention may also be cell lines, such as CHO cells.
  • the present application also provides a method of preparing the mutant of the present invention, which comprises culturing the host cell of the present invention under suitable conditions, and recovering the mutation of the present invention from the cell culture of the host cell body.
  • the method includes the following steps:
  • step (3) the mutant of the present invention is isolated and recovered by anion exchange chromatography and heparin affinity chromatography.
  • the hHGF mutant of the present application Compared with natural hHGF, the hHGF mutant of the present application has stronger biological activity, and thus can be advantageously used as a medicine. Accordingly, compared to the nucleic acid molecule encoding natural hHGF, the nucleic acid molecule encoding the hHGF mutant of the present application exhibits a stronger therapeutic effect in the body of the subject, and can also be advantageously used as a medicine. Therefore, in another aspect, the present application also provides a pharmaceutical composition comprising the mutant or nucleic acid molecule or carrier of the present invention, and optionally, a pharmaceutically acceptable carrier and/or excipient .
  • the pharmaceutical composition contains a mutant according to the invention.
  • the mutant is unmodified.
  • the mutant is modified, such as PEGylated.
  • the pharmaceutical composition is used for gene therapy.
  • the pharmaceutical composition contains the nucleic acid molecule or carrier of the present invention.
  • the vector is a gene therapy vector capable of expressing the mutant of the present invention, such as a plasmid (eg, naked plasmid), adenovirus vector, adeno-associated virus vector, and lentiviral vector.
  • the pharmaceutically acceptable carrier and/or excipient is selected from pH adjusting agents (including but not limited to phosphate buffers), surfactants (including but not limited to cationic, anionic Or non-ionic surfactants, such as Tween-80), adjuvants, ionic strength enhancers (including but not limited to sodium chloride), diluents, excipients, media for containing or administering therapeutic agents, and their Any combination.
  • pH adjusting agents including but not limited to phosphate buffers
  • surfactants including but not limited to cationic, anionic Or non-ionic surfactants, such as Tween-80
  • adjuvants including but not limited to sodium chloride
  • ionic strength enhancers including but not limited to sodium chloride
  • the pharmaceutically acceptable carrier may be a sterile liquid, such as water and oil, including oil derived from petroleum, animal, plant, or synthetic oil, such as peanut oil, soybean oil, mineral oil , Sesame oil, etc.
  • the pharmaceutically acceptable carrier is selected from water, saline solution, aqueous dextrose, glycerin, and any combination thereof.
  • the pharmaceutically acceptable excipient may be selected from starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, monostearate Acid glycerin, talc, sodium chloride, milk powder, glycerin, propylene, ethylene glycol, water, ethanol, and any combination thereof.
  • the pharmaceutical composition may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders (eg, lyophilized powders), sustained-release formulations, and the like.
  • the pharmaceutical composition of the present invention can be administered in various suitable ways. Suitable modes of administration include, but are not limited to parenteral administration, such as intravenous, intradermal, subcutaneous, oral, intranasal (eg, inhalation), transdermal (eg, topical), transmucosal, and rectal administration.
  • parenteral administration such as intravenous, intradermal, subcutaneous, oral, intranasal (eg, inhalation), transdermal (eg, topical), transmucosal, and rectal administration.
  • the pharmaceutical composition is formulated according to conventional procedures into a pharmaceutical formulation suitable for intravenous, subcutaneous, intramuscular, oral, intranasal, or topical administration to humans.
  • compositions for injection are sterile and isotonic. If desired, such pharmaceutical compositions may also contain solubilizers and local anesthetics such as ergotamine to reduce pain at the injection site. In addition, the pharmaceutical composition for injection may also contain a preservative. In certain preferred embodiments, the pharmaceutical composition for injection may also be presented in unit dosage form (eg, stored in an ampoule or in a multi-dose container).
  • compositions for injection may take the form of suspensions, solutions, or emulsions in oily or aqueous media, and may contain formulations such as suspending agents, stabilizers, and/or dispersing agents. Alternatively, such pharmaceutical compositions may also be in powder form, which is dissolved with a suitable medium (eg, sterile, pyrogen-free water) before use.
  • a suitable medium eg, sterile, pyrogen-free water
  • the pharmaceutical composition is a lyophilized injection containing 0.01%-0.2% hHGF mutant, and 5% mannitol, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is a lyophilized injection containing 1-10 mg of the nucleic acid molecule or carrier according to the present invention, and a pharmaceutically acceptable carrier.
  • media suitable for parenteral dosage forms include, but are not limited to, water for injection; aqueous media, including but not limited to sodium chloride injection, Ringer's injection, glucose injection, glucose, and sodium chloride Injection, and lactated Ringer's injection; water miscible media, including but not limited to ethanol, polyethylene glycol, and polypropylene glycol; and, non-aqueous media including but not limited to corn oil, cottonseed oil, peanut oil, sesame oil, Ethyl oleate, isopropyl myristate, and benzyl benzoate.
  • HGF can stimulate the growth and migration of endothelial cells (Bussolino et al., J Cell Biol. 119:629 (1992); Nakamura et al., J J Hypertens 14:1067 (1996)); and, HGF can Used as a re-endothelialization stimulant (Yasuda et al., Circulation 101: 2546 (2000); Hayashi et al., Gene Ther 7: 1664 (2000)).
  • HGF can stimulate angiogenesis by regulating endothelial cell growth and vascular smooth muscle cell migration. Because of its angiogenic activity, HGF is considered a promising candidate for therapeutic angiogenesis. For example, previous studies have reported that HGF can be used to treat ischemic diseases such as coronary artery disease (CAD) or peripheral artery disease (PAD) (Miyagawa et al., Circulation 105: 2556 (2002); Azuma et al., Gene Ther. 13: 1206 (2006); Aoki et al., Gene Ther. 7:417 (2000); Funatsu et al., J. Thoracic Cardiovasc. Surg. 124: 1099 (2002))).
  • CAD coronary artery disease
  • PAD peripheral artery disease
  • HGF can improve vascular complications caused by diabetes (Peng et al., 2011) and is used to treat metabolic syndrome and diabetes and its complications (eg, diabetic peripheral neuropathy).
  • HGF can be used as an agent for inhibiting restenosis. Studies have shown that rapid endothelial surface reconstruction can inhibit smooth muscle cell proliferation and thus restenosis (Bauters et al., Prog Cardiovasc Dis. 40:107 (1997)).
  • VEGF vascular endothelial growth factor
  • HGF hepatocyte growth factor
  • HGF is a neurotrophic factor effective in multiple brain regions (Kato et al., 2009; Ebens et al., 1996) and can affect many types of neuronal cells, including motor neurons ( Elsen et al., 2009; Hayashi et al., 2006), hippocampal neurons (Lim et al., 2008), cerebellar granule cells (Ieraci et al., 2002) and sympathetic neurons (1999), and can simultaneously stimulate Neurogenesis and synapse generation (Shang et al., 2011; Wang et al., 2011).
  • HGF/c-Met signaling can promote neuronal injury healing (Trappal et al., 2008), especially after ischemic brain injury (Takeo et al., 2007). It has also been reported that the application of hepatocyte growth factor (HGF) in a murine or rat model with familial amyotrophic lateral sclerosis (ALS) disease can significantly slow down the degradation of motor neurons (Aoki et al. , 2009); reduce glial hyperplasia that contributes to the degenerative process (Kadoyama et al., 2007); delay the occurrence of paralysis (Kadayama et al., 2009); and increase lifespan (Sun et al., 2002). These findings indicate that HGF has therapeutic and neuroprotective effects in various neurological diseases, such as neurodegenerative diseases (e.g., ALS, Parkinson's disease, dementia), traumatic brain injury, and traumatic spinal cord injury.
  • neurodegenerative diseases e.g., ALS, Parkinson's disease, dementia
  • HGF has been shown to have application prospects in various aspects, including: (1) promoting endothelial cell growth and/or migration; (2) promoting the development of blood vessels (such as microvessels); (3) treating ischemic diseases, such as coronary Arterial disease (CAD) or peripheral arterial disease (PAD), such as lower limb arterial ischemia; (4) Treatment of metabolic syndrome and diabetes and its complications (eg, diabetic peripheral neuropathy); (5) Inhibition of restenosis; and ( 6) Promote the repair of nerve damage (eg, neurodegenerative diseases, traumatic nerve damage, peripheral neuropathy).
  • CAD coronary Arterial disease
  • PAD peripheral arterial disease
  • nerve damage eg, neurodegenerative diseases, traumatic nerve damage, peripheral neuropathy.
  • the hHGF mutant of the present application has stronger biological activity than natural hHGF, and thus can be advantageously used for the above-mentioned applications.
  • the present application provides a method of treating a disease in a subject that can benefit from the activity of natural hHGF, which comprises administering to a subject in need thereof a therapeutically effective amount of the invention Mutant or nucleic acid molecule or carrier or pharmaceutical composition.
  • the disease is selected from ischemic diseases, metabolic syndrome, diabetes and its complications, restenosis, and nerve damage.
  • the disease is an ischemic disease, such as coronary artery disease (CAD) or peripheral artery disease (PAD), such as myocardial infarction or lower limb arterial ischemia.
  • CAD coronary artery disease
  • PAD peripheral artery disease
  • the disease is diabetes or its complications, such as diabetic peripheral neuropathy.
  • the disease is restenosis, such as restenosis after surgery and restenosis after perfusion.
  • the disease is a nerve injury, such as a neurodegenerative disease (eg, amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia), traumatic nerve injury, peripheral neuropathy (Eg diabetic peripheral neuropathy).
  • a neurodegenerative disease eg, amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia
  • traumatic nerve injury eg, peripheral neuropathy (Eg diabetic peripheral neuropathy).
  • peripheral neuropathy Eg diabetic peripheral neuropathy
  • the disease is selected from lower limb arterial ischemia, myocardial infarction, and diabetic peripheral neuropathy.
  • the disease of the subject eg, lower limb arterial ischemia, myocardial infarction and /Or diabetic peripheral neuropathy.
  • the mutant is unmodified.
  • the mutant is modified, such as PEGylated.
  • the disease in the subject eg, lower limb arterial ischemia, myocardium Infarction and/or diabetic peripheral neuropathy.
  • the vector is a gene therapy vector capable of expressing the mutant of the present invention, such as a plasmid (eg, naked plasmid), adenovirus vector, adeno-associated virus vector, and lentiviral vector.
  • a suitable dose is an amount effective to treat the disease (eg, lower limb arterial ischemia, myocardial infarction, and/or diabetic peripheral neuropathy) when the above-mentioned pharmaceutical composition is administered.
  • the amount of active ingredient contained in a unit dosage form may be, for example, about 10 ⁇ g to 5 mg.
  • a suitable dosage will vary depending on the condition of the patient and the mode of administration, and may be, for example, about 1 ⁇ g to 100 ⁇ g/kg body weight.
  • the amount of active ingredient contained in a unit dosage form may be, for example, about 1-10 mg.
  • a suitable dosage will vary depending on the condition of the patient and the mode of administration, and may be, for example, about 10-200 ⁇ g/kg body weight.
  • the present application provides the use of the mutant or nucleic acid molecule or vector of the present invention in the preparation of a pharmaceutical composition for the treatment of a subject who can benefit from the activity of natural hHGF disease.
  • the disease is selected from ischemic diseases, metabolic syndrome, diabetes and its complications, restenosis, and nerve damage.
  • the disease is an ischemic disease, such as coronary artery disease (CAD) or peripheral artery disease (PAD), such as myocardial infarction or lower limb arterial ischemia.
  • CAD coronary artery disease
  • PAD peripheral artery disease
  • the disease is diabetes or its complications, such as diabetic peripheral neuropathy.
  • the disease is restenosis, such as restenosis after surgery and restenosis after perfusion.
  • the disease is a nerve injury, such as a neurodegenerative disease (eg, amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia), traumatic nerve injury, peripheral neuropathy (Eg diabetic peripheral neuropathy).
  • a neurodegenerative disease eg, amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia
  • traumatic nerve injury eg, peripheral neuropathy (Eg diabetic peripheral neuropathy).
  • peripheral neuropathy Eg diabetic peripheral neuropathy
  • the disease is selected from lower limb arterial ischemia, myocardial infarction, and diabetic peripheral neuropathy.
  • the pharmaceutical composition contains a mutant according to the invention.
  • the mutant is unmodified.
  • the mutant is modified, such as PEGylated.
  • the pharmaceutical composition is used for gene therapy.
  • the pharmaceutical composition contains the nucleic acid molecule or carrier of the present invention.
  • the vector is a gene therapy vector capable of expressing the mutant of the present invention, such as a plasmid (eg, naked plasmid), adenovirus vector, adeno-associated virus vector, and lentiviral vector.
  • mutant or nucleic acid molecule or carrier or pharmaceutical composition according to the present invention for use in the treatment of a disease in a subject that can benefit from the activity of natural hHGF.
  • the disease is selected from ischemic diseases, metabolic syndrome, diabetes and its complications, restenosis, and nerve damage.
  • the disease is an ischemic disease, such as coronary artery disease (CAD) or peripheral artery disease (PAD), such as myocardial infarction or lower limb arterial ischemia.
  • CAD coronary artery disease
  • PAD peripheral artery disease
  • the disease is diabetes or its complications, such as diabetic peripheral neuropathy.
  • the disease is restenosis, such as restenosis after surgery and restenosis after perfusion.
  • the disease is a nerve injury, such as a neurodegenerative disease (eg, amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia), traumatic nerve injury, peripheral neuropathy (Eg diabetic peripheral neuropathy).
  • a neurodegenerative disease eg, amyotrophic lateral sclerosis (ALS), Parkinson's disease, dementia
  • traumatic nerve injury eg, peripheral neuropathy (Eg diabetic peripheral neuropathy).
  • peripheral neuropathy Eg diabetic peripheral neuropathy
  • the disease is selected from lower limb arterial ischemia, myocardial infarction, and diabetic peripheral neuropathy.
  • the mutant is unmodified. In certain preferred embodiments, the mutant is modified, such as PEGylated. In certain preferred embodiments, the nucleic acid molecule or vector is used for gene therapy. In certain preferred embodiments, the vector is a gene therapy vector capable of expressing the mutant of the present invention, such as a plasmid (eg, naked plasmid), adenovirus vector, adeno-associated virus vector, and lentiviral vector.
  • a plasmid eg, naked plasmid
  • adenovirus vector eg., adeno-associated virus vector
  • lentiviral vector lentiviral vector.
  • the present application provides a method for promoting the growth and/or migration of endothelial cells, which comprises administering to the endothelial cells or subject in need thereof an effective amount of a mutant or nucleic acid molecule or carrier according to the present invention Or pharmaceutical composition.
  • the method is used in vivo.
  • the mutant or nucleic acid molecule or carrier or pharmaceutical composition of the present invention can be administered to a subject to promote the growth and/or migration of endothelial cells in the subject.
  • the method is used in vitro.
  • the mutant or nucleic acid molecule or carrier or pharmaceutical composition of the present invention can be administered to endothelial cells cultured in vitro to promote the growth and/or migration of endothelial cells in culture.
  • the endothelial cells are umbilical vein endothelial cells.
  • the present application provides a method of promoting angiogenesis, which comprises administering to a subject in need thereof an effective amount of the mutant or nucleic acid molecule or carrier or pharmaceutical composition of the present invention.
  • the angiogenesis is microangiogenesis.
  • the present application provides the use of the mutant or nucleic acid molecule or carrier of the present invention in the preparation of a pharmaceutical composition for promoting endothelial cell growth and/or migration or promoting angiogenesis.
  • the endothelial cells are umbilical vein endothelial cells.
  • the angiogenesis is microangiogenesis.
  • a mutant or nucleic acid molecule or carrier or pharmaceutical composition according to the present invention for use in promoting endothelial cell growth and/or migration or promoting angiogenesis.
  • the endothelial cells are umbilical vein endothelial cells.
  • the angiogenesis is microangiogenesis.
  • the hHGF mutant of the present application has stronger biological activity.
  • the inventors of the present application have found through research that the hHGF mutants of the present invention can exhibit stronger biological activity in the following aspects: (1) promoting endothelial cell growth and/or migration; (2) promoting blood vessels (eg Microvascularization); and/or, (3) promote the repair of nerve damage (eg, peripheral neuropathy, such as diabetic peripheral neuropathy).
  • nerve damage eg, peripheral neuropathy, such as diabetic peripheral neuropathy.
  • the hHGF mutants and nucleic acid molecules encoding hHGF mutants of the present invention can be more advantageously applied to one or more of the following aspects: (1) promote the growth and/or migration of endothelial cells; (2) promote blood vessels (such as microvessels) ) Occurs; (3) treatment of ischemic diseases, such as coronary artery disease (CAD) or peripheral artery disease (PAD), such as lower limb arterial ischemia; (4) treatment of metabolic syndrome and diabetes and its complications (eg, diabetes Peripheral neuropathy); (5) inhibit restenosis; and (6) promote nerve repair (eg, neurodegenerative disease, traumatic nerve injury, peripheral neuropathy) repair.
  • CAD coronary artery disease
  • PAD peripheral artery disease
  • nerve repair eg, neurodegenerative disease, traumatic nerve injury, peripheral neuropathy
  • Figure 1 shows the SDS-PAGE detection results of the four target proteins prepared in Example 1 (ie, natural hHGF, 130Arg-hHGF, 130His-hHGF, and 130Lys-hHGF), where lane 1: natural hHGF; lane 2: 130Arg-hHGF; lane 3: 130His-hHGF; lane 4: 130Lys-hHGF; lane 5: protein molecular weight markers.
  • SEQ ID NO: 1 amino acid sequence of natural hHGF
  • SEQ ID NO: 2 amino acid sequence of 130Arg-hHGF
  • SEQ ID NO: 3 amino acid sequence of 130His-hHGF
  • SEQ ID NO: 4 amino acid sequence of 130Lys-hHGF
  • SEQ ID NO: 5 (nucleotide sequence encoding natural hHGF)
  • SEQ ID NO: 6 nucleotide sequence encoding 130Arg-hHGF
  • SEQ ID NO: 7 (nucleotide sequence encoding 130His-hHGF)
  • SEQ ID NO: 8 (nucleotide sequence encoding 130Lys-hHGF)
  • SEQ ID NO: 9 nucleotide sequence of pSN vector
  • the amino acid sequence of natural hHGF (SEQ ID NO: 1) can be found in NCBI accession number NP_000592.3. Using SEQ ID NO: 1 as a template, the following three hHGF mutants were designed:
  • the nucleic acid molecules prepared as described above were cloned into expression vectors, respectively, and transformed into CHO host cells. Under conditions permitting the expression of foreign proteins, the transformed CHO host cells are cultured, and then the culture is collected and centrifuged to obtain a protein containing the target protein (native hHGF, 130Arg-hHGF, 130His-hHGF, or 130Lys-hHGF) Supernatant.
  • anion exchange chromatography medium (DEAE Sepharose Flow, GE Healthcare, 17-0709-10) to separate the target protein in the supernatant
  • heparin affinity chromatography medium Heparin Sepharose 6 Fast
  • the purified target protein obtained was detected by non-reduced polyacrylamide gel electrophoresis (non-reduced SDS-PAGE, Molecular Cloning Experiment Guide, Fourth Edition), and the results are shown in Figure 1.
  • the purity of the four purified target proteins are all greater than 98%, and can be used in subsequent studies.
  • pSN-hHGF which carries a polynucleotide (SEQ ID NO: 5) encoding natural hHGF (SEQ ID NO: 1);
  • pSN-130Lys-hHGF which carries a polynucleotide (SEQ ID NO: 8) encoding 130Lys-hHGF (SEQ ID NO: 4).
  • the content of the plasmid in each of the prepared stock solutions was determined.
  • the results show that, in the various raw liquid samples prepared, the plasmid content is in the range of 2.0-2.2 mg/mL.
  • the contents of the recombinant plasmids in the four stock solutions are: 2.12mg/mL (pSN-hHGF), 2.05mg/mL (pSN-130Arg-hHGF), 2.15mg/mL (pSN-130His-hHGF), And 2.10 mg/mL (pSN-130Lys-hHGF).
  • the chromatography column used was an anion exchange HPLC analysis column DNA-NPR, which was equilibrated with 20 mM Tris-HCl, 0.5 M NaCl, pH 8.8 buffer. After equilibration, load the sample for testing.
  • the sample load is 100 ⁇ l
  • the flow rate is 0.5 ml/min
  • the detection wavelength is 260 nm.
  • HPLC purity of the recombinant plasmids in the four stock solutions were: 97.5% (pSN-hHGF), 98.2% (pSN-130Arg-hHGF), 98.0% (pSN-130His-hHGF), 97.8% (pSN- 130Lys-hHGF).
  • the hHGF mutants (130Arg-hHGF, 130His-hHGF, and 130Lys-hHGF) prepared as above and natural hHGF are prepared with physiological saline to the desired concentration before use.
  • ECV304 cell line (umbilical vein endothelial cell), used to test the biological activity of HGF.
  • DMEM medium provided by Hyclone.
  • the preparation method is as follows: take 1 bag of DMEM medium powder (specification is 1L), add water to dissolve and dilute to 1000ml, and then add 2.1g of sodium bicarbonate. Then, the prepared medium was sterilized and filtered, and stored at 4°C.
  • Complete medium Take 100ml of fetal bovine serum and add DMEM medium to 1000ml.
  • Transwells Provided by Costar.
  • Carbon dioxide cell incubator Provided by Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory, model HH.CP.
  • Inverted microscope Provided by Chongqing Photoelectric Instrument Corporation, model XDS-1B.
  • Ultra-clean workbench Provided by Suzhou Purification Equipment Co., Ltd. Model SW-CJ-1F.
  • Optical microscope Provided by Chongqing Photoelectric Instrument Corporation, model BP104.
  • cell migration test was performed. Briefly, 600 ⁇ l of DMEM medium was added to each well of the tank under the migration plate, and Transwells were immersed in it. ECV304 cells digested with 0.1% trypsin were mixed with 1640 medium containing 10% fetal bovine serum into a cell suspension containing 1 ⁇ 10 6 cells per 1 ml. Add 200 ⁇ l of cell suspension to each well and incubate at 37°C for 1 h.
  • This test uses the migration cell count to evaluate the biological activity of the protein to be tested (native hHGF or hHGF mutant).
  • the method of quantitatively evaluating cell migration with an optical microscope is as follows: first select an area with uniform cell distribution under a low-power (4 times objective lens) optical microscope, and then use a medium-power microscope (20 times objective lens) with an additional grid in the eyepiece to randomly select continuously 5 fields of vision, counting the migrated cells. The measurement results were analyzed and evaluated by statistical t test method.
  • hHGF mutants and natural hHGF can induce/stimulate migration of endothelial cells, and that hHGF mutants have a stronger ability to induce endothelial cell migration than natural hHGF.
  • the three hHGF mutants (130Arg-hHGF, 130His-hHGF and 130Lys-hHGF) of the present application can better promote cell migration.
  • the rabbit lower limb arterial ischemia model was used to evaluate the effect of hHGF mutant and natural hHGF on the vascular and collateral circulation reformation of the rabbit lower limb ischemia model, so as to evaluate the therapeutic effect of hHGF mutant and natural hHGF.
  • the hHGF mutants (130Arg-hHGF, 130His-hHGF, and 130Lys-hHGF) prepared as above and natural hHGF are prepared with physiological saline to the desired concentration before use.
  • New Zealand male white rabbit 12-14 months old, weight 3.5-4.0kg, provided by Beijing Viton Lihua Company.
  • Therapeutics angiogenesis A single intraarterial bolus of vascular endothelial growth factor growth factors revascularization in laboratory abbit ischemic hindlimb model. J. Clin. Invest. 1994, 93: 662-670
  • the rabbit lower limb was established Blood model.
  • After intramuscular injection of 5 mg/kg dose of Xylazine the rabbit was anesthetized with 50 mg/kg dose of Ketamine. Sterilize the skin on the inside of the left thigh with alcohol and iodine.
  • an arterial cannula was inserted into the right carotid artery, a 3F catheter (Terumo, Japan) was inserted into the entrance of the left internal iliac artery, and 5 ml of contrast agent was infused at a rate of 1 ml per second for selective iliac Internal arteriography to confirm the establishment of sick animal models.
  • model control group (6 animals), hHGF test group (8 animals), 130Arg-hHGF test group (8 animals), 130His-hHGF test group (8 animals) and 130Lys- hHGF test group (8 animals).
  • the lower limb ischemic muscle tissues (adductor muscles and semimembranosus muscles) were removed and placed in O.C.T. compound (Miles Inc., Elkhart, USA) solution. After rapid freezing with liquid nitrogen, tissue cryosection was performed. According to the Indoxyl-tetrazolium method, capillary endothelial cells were stained with alkaline phosphatase. Under the microscope ( ⁇ 200), the number of capillary endothelial cells in the tissue is counted, and then converted into the number of capillaries per 1,000 muscle cells to quantitatively express the density of capillaries.
  • test results of the number of collateral vessels at the ischemic site and the number of collateral vessels at the ischemic site in each group of experimental animals before and after administration are shown in Table 2.
  • the treatment results of the three hHGF mutants were significantly different from those of the natural hHGF test group: rabbits receiving hHGF mutants had higher arteriolar density in the left hindlimb than rabbits receiving natural hHGF; of which, the 130Arg-hHGF test Group (p ⁇ 0.01), 130His-hHGF test group (p ⁇ 0.05) and 130Lys-hHGF test group (p ⁇ 0.01) were significantly better than natural hHGF test group in promoting the formation of collateral vessels.
  • the three hHGF mutants of the present application are unexpectedly superior to natural hHGF in the treatment of lower limb arterial ischemia.
  • Example 5 Evaluation of therapeutic effect of recombinant plasmid encoding hHGF or its mutants on rabbit lower limb arterial ischemia model
  • the rabbit lower limb arterial ischemia model was used to evaluate the effect of the recombinant plasmid encoding hHGF or its mutants on promoting the formation of collateral vessels, thereby evaluating the therapeutic effect of the recombinant plasmid encoding hHGF or its mutants.
  • the four recombinant plasmids (pSN-hHGF, pSN-130Arg-hHGF, pSN-130His-hHGF and pSN-130Lys-hHGF) prepared as above are prepared with physiological saline to the desired concentration before use.
  • New Zealand male white rabbit 12-14 months old, weight 3.5-4.0kg, provided by Beijing Viton Lihua Company.
  • Therapeutics angiogenesis A single intraarterial bolus of vascular endothelial growth factor growth factors revascularization in laboratory abbit ischemic hindlimb model. J. Clin. Invest. 1994, 93: 662-670
  • the rabbit lower limb was established Blood model.
  • After intramuscular injection of 5 mg/kg dose of Xylazine the rabbit was anesthetized with 50 mg/kg dose of Ketamine. Sterilize the skin on the inside of the left thigh with alcohol and iodine.
  • an arterial cannula was inserted into the right carotid artery, a 3F catheter (Terumo, Japan) was inserted into the entrance of the left internal iliac artery, and 5 ml of contrast agent was infused at a rate of 1 ml per second for selective iliac Internal arteriography to confirm the establishment of sick animal models.
  • the animals were randomly divided into a model control group (8 animals), a pSN-hHGF test group (8 animals), a pSN-130Arg-hHGF test group (8 animals), and a pSN-130His-hHGF test group ( 8) and pSN-130Lys-hHGF test group (8).
  • Table 3 show that the treatment results of the three recombinant plasmids encoding hHGF mutants are significantly different from the recombinant plasmid encoding natural hHGF: pSN-130Arg-hHGF test group (p ⁇ 0.01), pSN- The 130His-hHGF test group (p ⁇ 0.01) and the pSN-130Lys-hHGF test group (p ⁇ 0.01) were significantly better than the pSN-hHGF test group in promoting the formation of collateral vessels.
  • Example 6 Evaluation of the therapeutic effect of hHGF mutant and natural hHGF on rat diabetic peripheral neuropathy model
  • the hHGF mutants (130Arg-hHGF, 130His-hHGF, and 130Lys-hHGF) prepared as above and natural hHGF are prepared with physiological saline to the desired concentration before use.
  • Wistar rats (SPF grade, male, weighing 180-200 g, 2.5 to 3 months old) were provided by Beijing Viton Lihua Company. After the rats were repurchased, they were fed adaptively for 5 days and confirmed that the animals were in good condition. Ten animals were randomly selected as a normal control group, and the remaining 60 animals were modeled as follows. The rats were fasted for 12 hours without water, and then weighed, blood glucose was measured and numbered. In an ice bath environment, streptozotocin (STZ) was added to a previously prepared 0.1 mol/L citric acid/sodium citrate buffer (pH 4.4) to prepare a 2% STZ solution.
  • STZ streptozotocin
  • STZ was given to the model animals by left intraperitoneal injection at one time; animals in the normal control group received left intraperitoneal injection of equal dose of the same buffer.
  • the blood glucose of the rats was measured.
  • a total of 52 rats with blood glucose >16.7 mmol/L and urine glucose +++ to ++++ were selected as model animals.
  • a model rat with diabetic peripheral neuropathy was obtained.
  • model rats were randomly divided into model control group (10 rats), hHGF test group (10 rats), 130Arg-hHGF test group (10 rats), 130His-hHGF test group (11 rats), 130Lys-hHGF test group (11 animals).
  • Animals in the experimental group began to receive the drug 10 weeks after the successful modeling. 4 points were taken on the inside of the left thigh of each animal (1 point for the adductor muscle and 3 points for the semimembranosus muscle), and each point was intramuscularly administered with 250 ⁇ g/250 ⁇ l of the test drug (that is, each animal was given the test drug once A total of 1mg/1ml), administered once a day.
  • the control group of the model group was given an equal volume of normal saline for 20 days, with a total of 20 injections.
  • MNCV motor nerve conduction velocity
  • SNCV sensory nerve conduction velocity
  • MNCV measurement method is as follows: the recording electrode is vertically penetrated into the mid-abdominal part of the tibialis anterior muscle, and the stimulation electrode is used to stimulate the proximal end of the sciatic nerve with a stimulation current of 20mA.
  • the electromyograph displays and records the resulting action potential on the oscilloscope. The distance is calculated as MNCV.
  • the SNCV measurement method is as follows: the recording electrode is placed at the proximal end of the sciatic nerve, the stimulation electrode stimulates the proximal end of the sural nerve with a stimulation intensity of 30 mA, the electromyograph records the waveform obtained by the stimulation, and then the SNCV is calculated according to the distance between the two electrodes.
  • the model control group had the lowest MNCV and SNCV (significantly slower than the normal control group, p ⁇ 0.01); the hHGF test group followed (significantly slower than the normal control group, p ⁇ 0.05).
  • the MNCV and SNCV of the hHGF mutant group were slightly lower than the normal control group, but the difference was not statistically significant (p>0.05).
  • hHGF and hHGF mutants can promote the repair of diabetic peripheral neuropathy, restore the total cross-sectional area of the sural nerve myelinated nerve fibers and the average area of nerve fibers, and the therapeutic effect of hHGF mutants is superior to natural hHGF.
  • Example 7 Evaluation of the therapeutic effect of recombinant plasmid encoding hHGF or its mutants on rat diabetic peripheral neuropathy model
  • a rat diabetic peripheral neuropathy model was used to evaluate the effect of recombinant plasmid encoding hHGF or its mutants on diabetic peripheral neuropathy.
  • the four recombinant plasmids (pSN-hHGF, pSN-130Arg-hHGF, pSN-130His-hHGF and pSN-130Lys-hHGF) prepared as above are prepared with physiological saline to the desired concentration before use.
  • Wistar rats (SPF grade, male, weighing 180-200 g, 2.5 to 3 months old) were provided by Beijing Viton Lihua Company. After the rats were repurchased, they were fed adaptively for 5 days and confirmed that the animals were in good condition. Ten animals were randomly selected as a normal control group, and the remaining 60 animals were modeled as follows. The rats were fasted for 12 hours without water, and then weighed, blood glucose was measured and numbered. In an ice bath environment, streptozotocin (STZ) was added to a previously prepared 0.1 mol/L citric acid/sodium citrate buffer (pH 4.4) to prepare a 2% STZ solution.
  • STZ streptozotocin
  • STZ was given to the model animals by left intraperitoneal injection at one time; animals in the normal control group received left intraperitoneal injection of equal dose of the same buffer.
  • the blood glucose of the rats was measured.
  • a total of 52 rats with blood glucose >16.7 mmol/L and urine glucose +++ to ++++ were selected as model animals.
  • a model rat with diabetic peripheral neuropathy was obtained.
  • model rats were randomly divided into model control group (10 rats), pSN-hHGF test group (10 rats), pSN-130Arg-hHGF test group (10 rats), pSN-130His-hHGF test group (11 rats) , PSN-130Lys-hHGF test group (11 animals).
  • Animals in the experimental group began to receive the drug 10 weeks after the successful modeling.
  • Four points were taken on the inside of the left thigh of each animal (1 point of the adductor muscle and 3 points of the semimembranosus muscle), and each point was intramuscularly given 250 ⁇ g/250 ⁇ l of the test drug (that is, each animal was given the test drug once A total of 1mg/1ml), a total of 1 dose.
  • the model group was given an equal volume of saline.
  • MNCV motor nerve conduction velocity
  • SNCV sensory nerve conduction velocity
  • the measurement was performed 10 weeks after the administration. After anesthetizing the rat, the left sciatic nerve was surgically isolated, and Neuromatic-2000 electromyograph was used to determine the MNCV and SNCV of the rat.
  • the MNCV measurement method is as follows: the recording electrode is vertically penetrated into the mid-abdominal part of the tibialis anterior muscle, and the stimulation electrode is used to stimulate the proximal end of the sciatic nerve with a stimulation current of 20mA.
  • the electromyograph displays and records the resulting action potential on the oscilloscope. The distance is calculated as MNCV.
  • the SNCV measurement method is as follows: the recording electrode is placed at the proximal end of the sciatic nerve, the stimulation electrode stimulates the proximal end of the sural nerve with a stimulation intensity of 30 mA, the electromyograph records the waveform obtained by the stimulation, and then the SNCV is calculated according to the distance between the two electrodes.
  • the measurement was performed 10 weeks after the administration.
  • the distal end of the right sural nerve was fixed in 3% glutaraldehyde/0.1mol/L phosphate buffer and kept at 4°C overnight. Rinse with PBS buffer, fix with 1% osmium acid, rinse, dehydrate and embed with epoxy resin.
  • a 1 ⁇ m semi-thin cross-sectional slice was prepared, stained with 1% toluidine blue solution for 30 minutes, then washed with 85% alcohol, decolorized to a light blue background, and then sealed with gum.
  • the model control group had the lowest MNCV and SNCV (significantly slower than the normal control group, p ⁇ 0.01); the pSN-hHGF test group followed (significantly slower than the normal control group, p ⁇ 0.05).
  • the MNCV and SNCV of the recombinant plasmid test group encoding hHGF mutant were slightly lower than the normal control group, but the difference was not statistically significant (p>0.05).
  • the recombinant plasmid encoding natural hHGF and its mutants can promote the repair of diabetic peripheral neuropathy and restore damaged MNCV and SNCV, and the recombinant plasmid encoding the hHGF mutant (pSN-130Arg-hHGF, pSN-130His- The therapeutic effect of hHGF and pSN-130Lys-hHGF) is superior to the recombinant plasmid encoding natural hHGF (pSN-hHGF).
  • the total cross-sectional area of nerves and the average area of nerve fibers of animals in the recombinant plasmid test group encoding the hHGF mutant were significantly increased (p ⁇ 0.05).
  • the recombinant plasmid encoding natural hHGF and its mutants can promote the repair of diabetic peripheral neuropathy, restore the total cross-sectional area of the medullary nerve fibers of the sural nerve and the average area of nerve fibers, and three recombinant plasmids encoding hHGF mutants (pSN-130Arg-hHGF, pSN-130His-hHGF and pSN-130Lys-hHGF) have better therapeutic effects than the recombinant plasmid encoding natural hHGF (pSN-hHGF).
  • the treatment results of the three recombinant plasmids encoding hHGF mutants are significantly better than the recombinant plasmid encoding natural hHGF (pSN-hHGF).
  • Examples 3, 4 and 6 show that the hHGF mutant of the present application has significantly higher activity than natural hHGF in promoting the migration of umbilical vein endothelial cells, promoting the growth of lower extremity arterioles, and promoting the repair of diabetic peripheral neuropathy.
  • Examples 5 and 7 show that the recombinant plasmid encoding the hHGF mutant can be used as a gene therapy drug in the subject to promote the growth of lower extremity arterioles and promote the repair of diabetic peripheral neuropathy, and its therapeutic effect is significantly high Recombinant plasmid encoding natural hHGF.

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Abstract

提供了人肝细胞生长因子(hHGF)的突变体，还提供编码所述突变体的核酸分子，含有所述核酸分子的载体，含有所述核酸分子或载体的宿主细胞；同时提供含有所述hHGF突变体或编码所述突变体的核酸分子的药物组合物，以及所述hHGF突变体或编码所述突变体的核酸分子的用途。

Description

人肝细胞生长因子突变体及其应用 技术领域
本申请涉及人肝细胞生长因子(hHGF)的突变体。本申请还涉及编码所述突变体的核酸分子，含有所述核酸分子的载体，含有所述核酸分子或载体的宿主细胞。本申请还涉及含有所述hHGF突变体或编码所述突变体的核酸分子的药物组合物，以及所述hHGF突变体或编码所述突变体的核酸分子的用途。所述hHGF突变体或编码所述突变体的核酸分子例如可用于促进内皮细胞生长和/或迁移，促进血管发生，或治疗可受益于天然hHGF的活性的疾病(例如治疗下肢动脉缺血、心肌梗死和/或糖尿病周围神经病变)，并因此可用于制备药物。
背景技术
肝细胞生长因子(Hepatocyte growth factor,HGF)最初从大鼠血浆和血小板中分离得到,是一种分泌型肝素亲和糖蛋白,又称为扩散因子(Scatter factor,SF)。现已知HGF由间质细胞产生,与受体c-Met结合并激活该受体的酪氨酸激酶活性,促进肝细胞、上皮细胞、内皮细胞、黑色素细胞、造血细胞等多种类型细胞的生长、迁移和形态发生。HGF在胚肝和胎盘的发育中起重要作用,参与维持和更新肝、肺、肾等器官的细胞,并促进这些器官的再生和损伤后修复。此外,HGF对不同来源的肿瘤细胞具有促侵袭或者生长抑制作用。因此,HGF是一种多功能细胞因子,具有广阔的临床应用前景。
成熟HGF是由重链(α链)与轻链(β链)通过链间二硫键连接而成的异二聚体，其中，α链含463个氨基酸,约69kD；β链含234个氨基酸,约34kD。α链的N-末端具有一个发夹结构,且靠近其C-末端具有4个纤溶酶样Kringle结构(依次称为K1、K2、K3、K4区),每个Kringle结构由约80个氨基酸组成。现已知,发夹结构和K1区是HGF与受体c-Met结合的关键部位；而发夹结构和K2区一起构成HGF与肝素及硫酸乙酰肝素亲和的必需结构；β链含有丝氨酸蛋白酶样折叠区,但没有丝氨酸蛋白酶活性。整个HGF分子共有4个N-糖基化位点,分别位于Asn 294,Asn 402,Asn 566和Asn 653,重链和轻链各包含2个N-糖基化位点(肝细胞生长因子的分子生物学研究.生物工程学报，2002，18：1-4)。
C-Met是HGF的特异性细胞膜受体，表达于多种细胞，例如心肌细胞，血管内皮细胞等，介导HGF的生物学作用。HGF/C-Met系统广泛表达于多种组织，并参与调节细胞的生长，运动以及组织形态发生等复杂的生物学过程。HGF是一种内皮生长因子，与其特异性受体C-Met相结合，引起受体的酪氨酸残基磷酸化，启动受体后信号传导过程；并且还引起ERK磷酸化，导致STAT3(Ser727)磷酸化形成二聚体进入核内，促进早期生长反应基因如c-fos表达，从而在转录水平调节细胞生长。研究还发现，HGF可以激活MEK，P42/44MAPK以及P90RSR，减少过氧化氢引起的细胞死亡，并可以激活BCL-2基因表达并抑制Bax蛋白向线粒体膜表面移位，维持线粒体膜内外的电化学梯 度，阻止线粒体内的细胞色素C漏出，抑制Caspase-3和Caspase-9的活性，产生抗细胞凋亡作用。HGF还可以刺激MMP-1，VEGF，HGF以及C-Met在血管内皮细胞和血管平滑肌细胞表达，并显著增加Ets-1的mRNA表达和转录活性，在新生血管形成过程中发挥重要作用(Angiogenic property of hepatocyte growth factor is dependent on upregulation of essential transcription factor for angiogenesis,ets-1.Circulation,2003,107：1411-1417)。Ets途径也是HGF促进血管生成的分子学机制之一(Therapeutic angiogenesis using hepatocyte growth factor.Current Gene Therapy,2004,4:199-206)。Ets家族转录因子具有结合DNA的结构域，能结合到DNA序列GGA的核心上，对多种参与促有丝分裂信号的基因的表达起到非常重要的作用，并且可能通过控制这些基因的转录而参与了血管生成的调节。HGF基因包含有许多调节区，例如B细胞和巨噬细胞特异的转录因子连接区，白介素26反应元件(IL26RE)，转移生长因子抑制元件(TNFIE)和cAMP反应元件(CRE)等。因此，外源HGF可以通过诱导ets活性刺激内源性HGF表达，而内源性HGF又能通过自动传导功能，促进小血管生成。
本申请发明人在研究后发现，可对hHGF进行突变，获得生物学活性增强的hHGF突变体。
发明内容
在本申请中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本申请，下面提供相关术语的定义和解释。
如本文中所使用的，术语“天然hHGF”和“野生型hHGF”是指具有生物学活性的、天然存在的人肝细胞生长因子(human hepatocyte growth factor,hHGF)，二者具有相同的含义，且可互换使用。可方便地从各种公共数据库(例如，GenBank数据库)获得天然hHGF或野生型hHGF的氨基酸序列。例如，天然hHGF的氨基酸序列可见于GenBank数据库登录号：NP_000592.3。
如本文中所使用的，当提及天然hHGF的氨基酸序列时，其使用SEQ ID NO：1所示的序列来进行描述。例如，表述“天然hHGF的第130位氨基酸残基”是指，SEQ ID NO：1所示的蛋白的第130位氨基酸残基。然而，本领域技术人员理解，天然hHGF可具有多种版本，它们具有基本上相同的一级结构(即，氨基酸序列)和高级结构(即，空间结构)，以及基本上相同的生物学功能，但是它们彼此之间在氨基酸序列上仍然可以存在微小差异。因此，在本申请中，天然hHGF并不局限于SEQ ID NO：1所示的蛋白，而意欲涵盖所有已知的天然hHGF。因此，在本申请中，术语“天然hHGF”应包括各种天然存在的、具有生物学功能的hHGF，包括例如SEQ ID NO：1所示的hHGF以及其天然存在的变体。并且，当描述hHGF的氨基酸位置时，其不仅包括SEQ ID NO：1中的特定氨基酸位置，还包括其天然变体中与所述特定氨基酸位置对应的氨基酸位置。例 如，表述“天然hHGF的第130位氨基酸残基”包括，SEQ ID NO：1的第130位氨基酸残基，以及其天然变体中的相应氨基酸位置。根据本申请，表述“相应氨基酸位置”是指，当对序列进行最优比对时，即当序列进行比对以获得最高百分数同一性时，进行比较的序列中位于等同位置的氨基酸位置。类似地，表述“对应于SEQ ID NO:1的第130位的位置”是指，当对某一序列与SEQ ID NO:1进行最优比对时，即当某一序列与SEQ ID NO:1进行比对以获得最高百分数同一性时，进行比较的该序列中位于与SEQ ID NO:1的第130位等同位置的氨基酸位置。
在某些优选的实施方案中，天然hHGF具有如SEQ ID NO:1所示的氨基酸序列。在某些优选的实施方案中，天然hHGF是天然存在的、具有生物学功能的人肝细胞生长因子，并且其氨基酸序列与SEQ ID NO:1相比，具有至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，或至少99％的同一性。在某些优选的实施方案中，天然hHGF是天然存在的、具有生物学功能的人肝细胞生长因子，并且其氨基酸序列与SEQ ID NO:1相比，具有一个或多个(例如1-10个或1-5个或1-3个)氨基酸差异(例如，保守氨基酸置换)。
如本文中所使用的，术语“同一性”用于指两个多肽之间或两个核酸之间序列的匹配情况。当两个进行比较的序列中的某个位置都被相同的碱基或氨基酸单体亚单元占据时(例如，两个DNA分子的每一个中的某个位置都被腺嘌呤占据，或两个多肽的每一个中的某个位置都被赖氨酸占据)，那么各分子在该位置上是同一的。两个序列之间的“百分数同一性”是由这两个序列共有的匹配位置数目除以进行比较的位置数目×100的函数。例如，如果两个序列的10个位置中有6个匹配，那么这两个序列具有60％的同一性。例如，DNA序列CTGACT和CAGGTT共有50％的同一性(总共6个位置中有3个位置匹配)。通常，在将两个序列比对以产生最大同一性时进行比较。这样的比对可通过使用，例如，可通过计算机程序例如Align程序(DNAstar,Inc.)方便地进行的Needleman等人(1970)J.Mol.Biol.48：443-453的方法来实现。还可使用已整合入ALIGN程序(版本2.0)的E.Meyers和W.Miller(Comput.Appl Biosci.，4:11-17(1988))的算法，使用PAM120权重残基表(weight residue table)、12的缺口长度罚分和4的缺口罚分来测定两个氨基酸序列之间的百分数同一性。此外，可使用已整合入GCG软件包(可在www.gcg.com上获得)的GAP程序中的Needleman和Wunsch(J MoI Biol.48:444-453(1970))算法，使用Blossum 62矩阵或PAM250矩阵以及16、14、12、10、8、6或4的缺口权重(gap weight)和1、2、3、4、5或6的长度权重来测定两个氨基酸序列之间的百分数同一性。
如本文中所使用的，术语“保守置换”意指不会不利地影响或改变包含氨基酸序列的蛋白/多肽的必要特性的氨基酸置换。例如，可通过本领域内已知的标准技术例如定点诱变和PCR介导的诱变引入保守置换。保守氨基酸置换包括用具有相似侧链的氨基酸残基替代氨基酸残基的置换，例如用在物理学上或功能上与相应的氨基酸残基相似(例如具有相似大小、形状、电荷、化学性质，包括形成共价键或氢键的能力等)的残基进行的置换。已在本领域内定义了具有相似侧链的氨基酸残基的家族。这些家 族包括具有碱性侧链(例如，赖氨酸、精氨酸和组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)的氨基酸。因此，优选用来自相同侧链家族的另一个氨基酸残基替代相应的氨基酸残基。鉴定氨基酸保守置换的方法在本领域内是熟知的(参见，例如，Brummell等人，Biochem.32:1180-1187(1993)；Kobayashi等人Protein Eng.12(10):879-884(1999)；和Burks等人Proc.Natl Acad.Set USA 94:412-417(1997),其通过引用并入本文)。
如本文中所使用的，术语“多肽”和“蛋白质”具有相同的含义，可互换使用。并且在本发明中，氨基酸通常用本领域公知的单字母和三字母缩写来表示。例如，丙氨酸可用A或Ala表示。
如本文中所使用的，术语“具有碱性侧链的氨基酸”具有本领域技术人员通常理解的含义。氨基酸通常具有下述结构：
Figure PCTCN2020070010-appb-000001
其中，R为侧链基团。
因此，当侧链基团R呈碱性时，该氨基酸即为具有碱性侧链的氨基酸。在具有碱性侧链的氨基酸的溶液中，所述氨基酸的侧链能够解离产生OH -，而呈现碱性。相应地，解离后的所述氨基酸侧链将携带有正电荷。因此，具有碱性侧链的氨基酸也被称为碱性氨基酸或侧链基团带正电荷的氨基酸。具有碱性侧链的氨基酸的典型实例包括但不限于，赖氨酸、精氨酸和组氨酸。
如本文中所使用的，术语“分离的”或“被分离的”指的是，从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”或“被分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
如本文中所使用的，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒(例如裸质粒)；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体；以及，病毒载体等。可用作载体的病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、 疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可以含有多种控制表达的元件，包括但不限于，启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
如本文中所使用的，术语“宿主细胞”是指，可用于导入载体的细胞，其包括但不限于，如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如纤维原细胞，CHO细胞，COS细胞，NSO细胞，HeLa细胞，BHK细胞，HEK 293细胞或人细胞等的动物细胞。
如本文中所使用的，术语“药学上可接受的”意指，制药领域公认的可用于动物，特别是可用于人的。如本文中所使用的，术语“药学上可接受的载体和/或赋形剂”是指在药理学和/或生理学上与受试者和活性成分相容的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)，并且包括但不限于：pH调节剂(包括但不限于磷酸盐缓冲液)，表面活性剂(包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80)，佐剂，离子强度增强剂(包括但不限于氯化钠)，稀释剂，赋形剂，用于容纳或施用治疗剂的介质，以及其任何组合。
如本文中所使用的，术语“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂的典型实例包括但不限于，铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。
如本文中所使用的，药学上可接受的载体可以是无菌液体，诸如水和油，包括源自石油、动物、植物的或合成的油，诸如花生油、大豆油、矿物油、芝麻油等等。当静脉内施用药用组合物时，水是优选的载体。盐水溶液以及水性右旋糖和甘油溶液也可用作液态载体，特别是用于可注射溶液。
如本文中所使用的，药学上可接受的赋形剂可包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油、滑石、氯化钠、奶粉、甘油、丙烯、乙二醇、水、乙醇等等。如果需要，药物组合物还可以包含润湿剂，或乳化剂例如透明质酸钠，或pH缓冲剂。药物组合物可以采取溶液、悬浮液、乳状液、片剂、丸剂、胶囊、粉剂、缓释配方等形式。
如本文中所使用的，术语“有效量”是指足以获得或至少部分获得期望的效果的量。例如，预防疾病有效量是指，足以预防，阻止，或延迟疾病的发生的量；治疗疾病有效量是指，足以治愈或至少部分阻止已患有疾病的患者的疾病和其并发症的量。测定这样的有效量完全在本领域技术人员的能力范围之内。例如，对于治疗用途有效的量将取决于待治疗的疾病的严重度、患者自己的免疫系统的总体状态、患者的一般情况例如年龄，体重和性别，药物的施用方式，以及同时施用的其他治疗等等。
如本文中所使用的，术语“受试者”是指哺乳动物，包括但不限于，人，啮齿类动物(小鼠，大鼠，豚鼠)，狗，马，牛，猫，猪，猴，黑猩猩等。优选地，受试者是人。
如本文中所使用的，术语“可受益于天然hHGF的活性的疾病”是指这样的疾病，其 中HGF的增强的表达和/或活性能够缓解疾病的症状，迟滞疾病的进展，或治愈或部分治愈所述疾病。
如之前所报道的，HGF具有多种生物学活性，包括但不限于以下的一种或多种活性：(1)促进内皮细胞生长和/或迁移；(2)促进血管(例如微小血管)发生；和/或，(3)促进神经损伤(例如周围神经病变，例如糖尿病周围神经病变)修复。因此，HGF可在多个方面具有应用前景，包括但不限于：(1)促进内皮细胞生长和/或迁移；(2)促进血管(例如微小血管)发生；(3)治疗缺血性疾病，例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如下肢动脉缺血；(4)治疗代谢综合征和糖尿病及其并发症(例如，糖尿病周围神经病变)；(5)抑制再狭窄；和(6)促进神经损伤(例如，神经退行性疾病，创伤性神经损伤，周围神经病变)修复。
因此，术语“可受益于天然hHGF的活性的疾病”的实例包括但不限于上述疾病，例如，缺血性疾病，代谢综合征，糖尿病及其并发症，再狭窄，神经损伤等等。
本申请发明人在研究后发现，可对天然hHGF进行突变，获得生物学活性增强的hHGF突变体。具体而言，本申请发明人发现，通过将天然hHGF的第130位氨基酸(以SEQ ID NO:1为参照)突变为具有碱性侧链的氨基酸(例如，精氨酸、组氨酸、赖氨酸)，所产生的hHGF突变体具有比天然hHGF更强的生物学活性。相应地，编码所述hHGF突变体的核酸分子作为基因治疗药物，在受试者体内展现出比编码天然hHGF的核酸分子更强的治疗效果。
因此，在一个方面，本申请提供了一种人肝细胞生长因子(human hepatocyte growth factor,hHGF)的突变体，其与天然hHGF相比，包含下述突变：天然hHGF中对应于SEQ ID NO:1的第130位的位置上的氨基酸被突变为具有碱性侧链的氨基酸。
在某些优选的实施方案中，所述具有碱性侧链的氨基酸选自精氨酸、组氨酸和赖氨酸。在某些优选的实施方案中，所述具有碱性侧链的氨基酸为精氨酸。在某些优选的实施方案中，所述具有碱性侧链的氨基酸为组氨酸。在某些优选的实施方案中，所述具有碱性侧链的氨基酸为赖氨酸。
在某些优选的实施方案中，所述天然hHGF具有如SEQ ID NO:1所示的氨基酸序列。在某些优选的实施方案中，所述天然hHGF是天然存在的、具有生物学功能的人肝细胞生长因子，并且其氨基酸序列与SEQ ID NO:1相比，具有至少85％，至少90％，至少95％，至少96％，至少97％，至少98％，或至少99％的同一性。在某些优选的实施方案中，所述天然hHGF是天然存在的、具有生物学功能的人肝细胞生长因子，并且其氨基酸序列与SEQ ID NO:1相比，具有一个或多个(例如1-10个或1-5个或1-3个)氨基酸差异(例如，保守氨基酸置换)。
在某些优选的实施方案中，所述突变体与SEQ ID NO:1所示的天然hHGF相比，包含下述突变：SEQ ID NO:1的第130位氨基酸(即，丝氨酸)被突变为精氨酸。在某些优选的实施方案中，所述突变体具有如SEQ ID NO:2所示的氨基酸序列。
在某些优选的实施方案中，所述突变体与SEQ ID NO:1所示的天然hHGF相比， 包含下述突变：SEQ ID NO:1的第130位氨基酸(即，丝氨酸)被突变为组氨酸。在某些优选的实施方案中，所述突变体具有如SEQ ID NO:3所示的氨基酸序列。
在某些优选的实施方案中，所述突变体与SEQ ID NO:1所示的天然hHGF相比，包含下述突变：SEQ ID NO:1的第130位氨基酸(即，丝氨酸)被突变为赖氨酸。在某些优选的实施方案中，所述突变体具有如SEQ ID NO:4所示的氨基酸序列。
因此，在某些优选的实施方案中，所述突变体具有选自SEQ ID NO:2，3和4的氨基酸序列。
易于理解，可以对蛋白质进行各种修饰，以赋予蛋白期望的特性。例如，可以对蛋白质进行聚乙二醇修饰(PEG化修饰)，以改善蛋白质的体内半衰期。因此，在某些优选的实施方案中，所述突变体是经修饰的。在某些优选的实施方案中，所述突变体是经化学修饰的。在某些优选的实施方案中，所述突变体是经PEG化修饰的。
可通过各种已知的方法来制备本申请的hHGF突变体。在某些优选的实施方案中，所述hHGF突变体是通过重组表达的方法制备的。在某些优选的实施方案中，所述hHGF突变体是通过化学合成的方法制备的。然而，易于理解，本申请的hHGF突变体不受其制备方法所限制。
与天然hHGF相比，本申请的hHGF突变体具有更强的生物学活性。不拘于理论限制，本申请发明人认为，将位于天然hHGF的α链N-末端第一个发夹结构内的第130位氨基酸(以SEQ ID NO:1为参照)突变为具有碱性侧链的氨基酸(例如精氨酸、组氨酸、赖氨酸)，将会改变所述发卡结构的构象，增强hHGF蛋白与受体c-Met的结合，从而增强hHGF蛋白的生物学活性。因此，本申请的hHGF突变体可以在例如选自下述的一个或多个方面显示更强的活性：(1)促进内皮细胞生长和/或迁移；(2)促进血管(例如微小血管)发生；和/或，(3)促进神经损伤(例如周围神经病变，例如糖尿病周围神经病变)修复。
在另一个方面，本申请提供了一种分离的核酸分子，其包含编码本发明的突变体的核苷酸序列。在某些优选的实施方案中，所述核酸分子编码具有选自SEQ ID NO:2，3和4的氨基酸序列的突变体。
易于理解，所述分离的核酸分子可以用于克隆或表达本发明的突变体。在某些情况下，为了提高效率，可以根据细胞偏好，对核酸分子的核苷酸序列进行密码子优化。因此，在某些优选的实施方案中，所述核酸分子的核苷酸序列是根据宿主细胞偏好进行了密码子优化的。在某些优选的实施方案中，所述核酸分子的核苷酸序列是根据CHO细胞偏好进行了密码子优化的。
在某些优选的实施方案中，所述核酸分子具有选自SEQ ID NO:6，7和8的核苷酸序列。
在另一个方面，本申请还提供了一种载体，其包含如上所述的分离的核酸分子。
本发明的载体可以是克隆载体，也可以是表达载体。在某些优选的实施方案中，本发明的载体可以是例如，质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色 体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体；以及，病毒载体等。可用作载体的病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。因此，在某些优选的实施方案中，本发明的载体是病毒载体，例如但不限于，逆转录酶病毒载体(例如慢病毒载体)、腺病毒载体、腺相关病毒载体、疱疹病毒载体(如单纯疱疹病毒载体)、痘病毒载体、杆状病毒载体、乳头瘤病毒载体、乳头多瘤空泡病毒载体。在某些优选的实施方案中，本发明的载体选自腺病毒载体，腺相关病毒载体，和慢病毒载体。
在某些优选的实施方案中，本发明的载体能够表达或用于表达本发明的突变体。在某些优选的实施方案中，本发明的载体能够或用于在受试者(例如哺乳动物，例如人)体内表达本发明的突变体。在某些优选的实施方案中，本发明的载体用于基因治疗。在某些优选的实施方案中，本发明的载体能够用作基因治疗载体，用于在受试者(例如哺乳动物，例如人)体内表达本发明的突变体并进行基因治疗。
在某些优选的实施方案中，本发明的载体为包含如上所述的分离的核酸分子的质粒，例如裸质粒。在某些优选的实施方案中，本发明的载体为包含如上所述的分离的核酸分子的pSN载体。pSN载体公开于中国专利CN 108611367B中，具有如SEQ ID NO:9所示的核苷酸序列。
在另一个方面，本申请还提供了包含本发明的分离的核酸分子或载体的宿主细胞。此类宿主细胞包括但不限于，原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。本发明的细胞还可以是细胞系，例如CHO细胞。
在另一个方面，本申请还提供了制备本发明的突变体的方法，其包括，在合适的条件下培养本发明的宿主细胞，和从所述宿主细胞的细胞培养物中回收本发明的突变体。
在某些优选的实施方案中，所述方法包括以下步骤：
(1)构建表达载体，所述表达载体包含编码本发明的突变体(例如，具有如SEQ ID NO:2、SEQ ID NO:3或SEQ ID NO:4所示氨基酸序列的突变体)的核酸序列；
(2)将所述表达载体导入宿主细胞(例如CHO细胞)中，并在允许蛋白质表达的条件下，培养所述宿主细胞；和
(3)从所述宿主细胞的细胞培养物中分离和回收本发明的突变体。
在某些优选的实施方案中，在步骤(3)中，通过阴离子交换层析和肝素亲和层析来分离和回收本发明的突变体。
与天然hHGF相比，本申请的hHGF突变体具有更强的生物学活性，从而，可有利地用作药物。相应地，与编码天然hHGF的核酸分子相比，本申请的编码hHGF突变体的核酸分子在受试者体内展现出更强的治疗效果，也可有利地用作药物。因此，在另一个方面，本申请还提供了一种药物组合物，其含有本发明所述的突变体或核酸分子或载体， 以及任选地，药学上可接受的载体和/或赋形剂。
在某些优选的实施方案中，所述药物组合物含有本发明所述的突变体。在某些优选的实施方案中，所述突变体是未经修饰的。在某些优选的实施方案中，所述突变体是经修饰的，例如是PEG化修饰的。
在某些优选的实施方案中，所述药物组合物用于基因治疗。在某些优选的实施方案中，所述药物组合物含有本发明所述的核酸分子或载体。在某些优选的实施方案中，所述载体是能表达本发明突变体的基因治疗载体，例如质粒(例如裸质粒)，腺病毒载体，腺相关病毒载体，和慢病毒载体。
在某些优选的实施方案中，所述药学上可接受的载体和/或赋形剂选自pH调节剂(包括但不限于磷酸盐缓冲液)，表面活性剂(包括但不限于阳离子，阴离子或者非离子型表面活性剂，例如Tween-80)，佐剂，离子强度增强剂(包括但不限于氯化钠)，稀释剂，赋形剂，用于容纳或施用治疗剂的介质，以及其任何组合。
在某些优选的实施方案中，所述药学上可接受的载体可以是无菌液体，诸如水和油，包括源自石油、动物、植物的或合成的油，诸如花生油、大豆油、矿物油、芝麻油等等。在某些优选的实施方案中，药学上可接受的载体选自水，盐水溶液，水性右旋糖，甘油，以及其任何组合。
在某些优选的实施方案中，所述药学上可接受的赋形剂可选自淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、大米、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油、滑石、氯化钠、奶粉、甘油、丙烯、乙二醇、水、乙醇，以及其任何组合。
在某些优选的实施方案中，所述药物组合物可以采取溶液、悬浮液、乳状液、片剂、丸剂、胶囊、粉剂(例如冻干粉剂)、缓释配方等形式。
可通过各种合适的方式来施用本发明的药用组合物。合适的施用方式包括但不限于肠胃外施用，例如静脉内、皮内、皮下、口腔、鼻内(例如吸入)、经皮(例如局部)、经粘膜、和直肠施用。在某些优选的实施方案中，依照常规流程将药物组合物配制成适合于静脉内、皮下、肌肉内、口腔、鼻内、或局部施用于人的药物制剂。
通常，用于注射(例如静脉内施用，例如通过推注或连续输液)的药物组合物是无菌、等渗的。如果需要，此类药物组合物还可以包含增溶剂和局部麻醉剂诸如麦角胺，以减轻注射部位的疼痛。此外，用于注射的药物组合物还可以含有防腐剂。在某些优选的实施方案中，用于注射的药物组合物还可以以单位剂量形式存在(例如贮存在安瓿中或在多剂量容器中)。
用于注射的药物组合物可以采取诸如油性或水性介质中的悬浮液、溶液、或乳状液等形式，而且可以含有配制剂，诸如悬浮剂、稳定剂、和/或分散剂。或者，此类药物组合物还可以是粉末形式，其在使用前用合适介质(例如无菌、无热原水)溶解。在某些优选的实施方案中，所述药物组合物为冻干注射剂，其含有0.01％-0.2％的hHGF突变体，以及5％的甘露醇，以及药学上可接受的载体。在某些优选的实施方案中，所述药物组合物为冻干注射剂，其含有1-10mg的根据本发明所述的核酸分子或载体，以及药学上可接受的载体。
可用于提供肠胃外剂量形式的合适介质对于本领域技术人员而言是众所周知的。在某些优选实施方案中，适于肠胃外剂量形式的介质包括但不限于注射用水；水性介质，包括但不限于氯化钠注射液、Ringer氏注射液、葡萄糖注射液、葡萄糖和氯化钠注射液、以及乳酸化Ringer氏注射液；水易混介质，包括但不限于乙醇、聚乙二醇、和聚丙二醇；以及，非水性介质包括但不限于玉米油、棉籽油、花生油、芝麻油、油酸乙酯、肉豆蔻酸异丙酯、和苯甲酸苯甲酯。
之前的研究已经表明，HGF能够刺激内皮细胞的生长，并且刺激其迁移(Bussolino等，J Cell Biol.119：629(1992)；Nakamura等，J Hypertens 14：1067(1996))；并且，HGF可用作再内皮化刺激剂(Yasuda等，Circulation 101：2546(2000)；Hayashi等，Gene Ther 7：1664(2000))。
还已发现，HGF可通过调节内皮细胞生长和血管平滑肌细胞迁移来刺激血管生成。因为其血管发生活性，HGF被认为是治疗性血管发生的有前景候选物。例如，之前的研究报道，HGF可用于治疗缺血性疾病，比如冠状动脉疾病(CAD)或外周动脉疾病(PAD)(Miyagawa等，Circulation 105：2556(2002)；Azuma等，Gene Ther.13：1206(2006)；Aoki等，Gene Ther.7：417(2000)；Funatsu等，J.Thoracic Cardiovasc.Surg.124：1099(2002)))。
此外，还已报道，HGF能够改善糖尿病引起的血管并发症(Peng et al.，2011)，用于治疗代谢综合征和糖尿病及其并发症(例如，糖尿病周围神经病变)。
此外，还已报道，HGF可用作抑制再狭窄的药剂。研究表明，快速的内皮表面重建可抑制平滑肌细胞增殖，从而抑制再狭窄(Bauters等，Prog Cardiovasc Dis.40：107(1997))。向受损血管局部递送内皮生长因子(例如血管内皮生长因子(VEGF)或肝细胞生长因子(HGF))显示了抑制再狭窄的效果(Asahara等，Circulation 94：3291(1996)；Yasuda等，Circulation 101：2546(2000)；Hayashi等，Gene Ther 7：1664(2000)；Walter等，Circulation 110：36(2004))。
还已发现，HGF是一种在多个脑部区域有效的神经营养因子(Kato et al.，2009；Ebens et al.，1996)，能够影响多种类型的神经元细胞，包括运动神经元(Elsen et al.,2009；Hayashi et al.，2006)、海马神经元(Lim et al.，2008)、小脑颗粒细胞(Ieraci et al.，2002)和交感神经元(1999)，且能够同时刺激神经形成和突触发生(Shang et al.，2011；Wang et al，2011)。已报道，HGF/c-Met信号传导能够促进神经元损伤愈合(Trappal et al.，2008)，特别是在局部缺血性脑损伤之后(Takeo et al.，2007)。还已报道，在患有家族性肌萎缩性侧索硬化(ALS)疾病的鼠科动物或大鼠模型中应用肝细胞生长因子(HGF)能够显著减慢运动神经元的退化(Aoki et al.，2009)；减少助于退化过程的神经胶质增生(Kadoyama et al.，2007)；延迟瘫痪的发生(Kadayama et al.，2009)；和增加寿命(Sun et al.，2002)。这些研究结果表明，HGF在多种神经疾病，例如神经退行性疾病(例如ALS，帕金森氏病，痴呆病)，创伤性脑损伤，创伤性脊髓损伤中具有治疗作用和神经保护 作用。
因此，已显示HGF在多个方面具有应用前景，包括：(1)促进内皮细胞生长和/或迁移；(2)促进血管(例如微小血管)发生；(3)治疗缺血性疾病，例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如下肢动脉缺血；(4)治疗代谢综合征和糖尿病及其并发症(例如，糖尿病周围神经病变)；(5)抑制再狭窄；和(6)促进神经损伤(例如，神经退行性疾病，创伤性神经损伤，周围神经病变)修复。本申请的hHGF突变体具有比天然hHGF更强的生物学活性，从而可有利地用于上述应用。
因此，在另一个方面，本申请提供了在受试者中治疗可受益于天然hHGF的活性的疾病的方法，其包括，给有此需要的受试者施用治疗有效量的本发明所述的突变体或核酸分子或载体或药物组合物。
在某些优选的实施方案中，所述疾病选自缺血性疾病，代谢综合征，糖尿病及其并发症，再狭窄，以及神经损伤。在某些优选的实施方案中，所述疾病为缺血性疾病，例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如心肌梗死或下肢动脉缺血。在某些优选的实施方案中，所述疾病为糖尿病或其并发症，例如糖尿病周围神经病变。在某些优选的实施方案中，所述疾病为再狭窄，例如手术后再狭窄和灌注后再狭窄。在某些优选的实施方案中，所述疾病为神经损伤，例如神经退行性疾病(例如肌萎缩性侧索硬化(ALS)，帕金森氏病，痴呆病)，创伤性神经损伤，周围神经病变(例如糖尿病周围神经病变)。在某些优选的实施方案中，所述疾病选自下肢动脉缺血、心肌梗死和糖尿病周围神经病变。
在某些优选的实施方案中，通过给有此需要的受试者施用治疗有效量的本发明所述的突变体，从而治疗受试者的所述疾病(例如下肢动脉缺血、心肌梗死和/或糖尿病周围神经病变)。在某些优选的实施方案中，所述突变体是未经修饰的。在某些优选的实施方案中，所述突变体是经修饰的，例如是PEG化修饰的。
在某些优选的实施方案中，通过给有此需要的受试者施用治疗有效量的本发明所述的核酸分子或载体，从而治疗受试者的所述疾病(例如下肢动脉缺血、心肌梗死和/或糖尿病周围神经病变)。在某些优选的实施方案中，所述载体是能表达本发明突变体的基因治疗载体，例如质粒(例如裸质粒)，腺病毒载体，腺相关病毒载体，和慢病毒载体。
本领域普通技术人员知晓，施用的方式、频率和剂量将根据所治疗的病症、病况和个体的不同而异。一般来说，可以通过注射(例如皮内、肌内、静脉内或皮下)、局部施用(例如表皮施用)或滴加施用等方式施用。此外，还可以根据患者个体的不同，选择合理的施用途径和施用方案。合适的剂量为当施用上述的药物组合物后能够有效治疗所述疾病(例如下肢动脉缺血、心肌梗死和/或糖尿病周围神经病变)的量。
对于含有本发明所述的突变体的药物组合物而言，单位剂量形式包含的活性成分的量可以例如为大约10μg-5mg。合适的剂量将因患者病症以及给药方式而异，例如可以为大约1μg-100μg/kg体重。
对于含有本发明所述的核酸分子或载体的药物组合物而言，单位剂量形式包含的活 性成分的量可以例如为大约1-10mg。合适的剂量将因患者病症以及给药方式而异，例如可以为大约10-200μg/kg体重。
在另一个方面，本申请提供了本发明所述的突变体或核酸分子或载体在制备药物组合物中的用途，所述药物组合物用于治疗受试者中可受益于天然hHGF的活性的疾病。
在某些优选的实施方案中，所述疾病选自缺血性疾病，代谢综合征，糖尿病及其并发症，再狭窄，以及神经损伤。在某些优选的实施方案中，所述疾病为缺血性疾病，例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如心肌梗死或下肢动脉缺血。在某些优选的实施方案中，所述疾病为糖尿病或其并发症，例如糖尿病周围神经病变。在某些优选的实施方案中，所述疾病为再狭窄，例如手术后再狭窄和灌注后再狭窄。在某些优选的实施方案中，所述疾病为神经损伤，例如神经退行性疾病(例如肌萎缩性侧索硬化(ALS)，帕金森氏病，痴呆病)，创伤性神经损伤，周围神经病变(例如糖尿病周围神经病变)。在某些优选的实施方案中，所述疾病选自下肢动脉缺血、心肌梗死和糖尿病周围神经病变。
在某些优选的实施方案中，所述药物组合物含有本发明所述的突变体。在某些优选的实施方案中，所述突变体是未经修饰的。在某些优选的实施方案中，所述突变体是经修饰的，例如是PEG化修饰的。
在某些优选的实施方案中，所述药物组合物用于基因治疗。在某些优选的实施方案中，所述药物组合物含有本发明所述的核酸分子或载体。在某些优选的实施方案中，所述载体是能表达本发明突变体的基因治疗载体，例如质粒(例如裸质粒)，腺病毒载体，腺相关病毒载体，和慢病毒载体。
在另一个方面，提供了本发明所述的突变体或核酸分子或载体或药物组合物，其用于治疗受试者中可受益于天然hHGF的活性的疾病。
在某些优选的实施方案中，所述疾病选自缺血性疾病，代谢综合征，糖尿病及其并发症，再狭窄，以及神经损伤。在某些优选的实施方案中，所述疾病为缺血性疾病，例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如心肌梗死或下肢动脉缺血。在某些优选的实施方案中，所述疾病为糖尿病或其并发症，例如糖尿病周围神经病变。在某些优选的实施方案中，所述疾病为再狭窄，例如手术后再狭窄和灌注后再狭窄。在某些优选的实施方案中，所述疾病为神经损伤，例如神经退行性疾病(例如肌萎缩性侧索硬化(ALS)，帕金森氏病，痴呆病)，创伤性神经损伤，周围神经病变(例如糖尿病周围神经病变)。在某些优选的实施方案中，所述疾病选自下肢动脉缺血、心肌梗死和糖尿病周围神经病变。
在某些优选的实施方案中，所述突变体是未经修饰的。在某些优选的实施方案中，所述突变体是经修饰的，例如是PEG化修饰的。在某些优选的实施方案中，所述核酸分子或载体用于基因治疗。在某些优选的实施方案中，所述载体是能表达本发明突变体的基因治疗载体，例如质粒(例如裸质粒)，腺病毒载体，腺相关病毒载体，和慢病毒载体。
在另一个方面，本申请提供了促进内皮细胞生长和/或迁移的方法，其包括，给有此需要的内皮细胞或受试者施用有效量的本发明所述的突变体或核酸分子或载体或药物组合物。
在某些优选的实施方案中，所述方法用于体内。例如，可将本发明所述的突变体或核酸分子或载体或药物组合物施用给受试者，以促进受试者体内的内皮细胞生长和/或迁移。在某些优选的实施方案中，所述方法用于体外。例如，可将本发明所述的突变体或核酸分子或载体或药物组合物施用给体外培养的内皮细胞，以促进培养物中的内皮细胞生长和/或迁移。在某些优选的实施方案中，所述内皮细胞为脐静脉内皮细胞。
在另一个方面，本申请提供了促进血管发生的方法，其包括，给有此需要的受试者施用有效量的本发明所述的突变体或核酸分子或载体或药物组合物。在某些优选的实施方案中，所述血管发生为微小血管发生。
在另一个方面，本申请提供了本发明所述的突变体或核酸分子或载体在制备药物组合物中的用途，所述药物组合物用于促进内皮细胞生长和/或迁移或促进血管发生。在某些优选的实施方案中，所述内皮细胞为脐静脉内皮细胞。在某些优选的实施方案中，所述血管发生为微小血管发生。
在另一个方面，提供了本发明所述的突变体或核酸分子或载体或药物组合物，其用于促进内皮细胞生长和/或迁移或促进血管发生。在某些优选的实施方案中，所述内皮细胞为脐静脉内皮细胞。在某些优选的实施方案中，所述血管发生为微小血管发生。
发明的有益效果
与天然hHGF相比，本申请的hHGF突变体具有更强的生物学活性。特别地，本申请发明人通过研究发现，本发明的hHGF突变体可以在例如下述方面显示更强的生物学活性：(1)促进内皮细胞生长和/或迁移；(2)促进血管(例如微小血管)发生；和/或，(3)促进神经损伤(例如周围神经病变，例如糖尿病周围神经病变)修复。
因此，本发明的hHGF突变体和编码hHGF突变体的核酸分子可以更有利地应用于以下一个或多个方面：(1)促进内皮细胞生长和/或迁移；(2)促进血管(例如微小血管)发生；(3)治疗缺血性疾病，例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如下肢动脉缺血；(4)治疗代谢综合征和糖尿病及其并发症(例如，糖尿病周围神经病变)；(5)抑制再狭窄；和(6)促进神经损伤(例如，神经退行性疾病，创伤性神经损伤，周围神经病变)修复。
下面将结合附图和实施例对本申请的实施方案进行详细描述，但是本领域技术人员将理解，下列附图和实施例仅用于说明本申请，而不是对本申请的范围的限定。根据附图和优选实施方案的下列详细描述，本申请的各种目的和有利方面对于本领域技 术人员来说将变得显然。
附图说明
图1显示了，实施例1制备的4种目的蛋白(即，天然hHGF，130Arg-hHGF，130His-hHGF和130Lys-hHGF)的SDS-PAGE检测结果，其中，泳道1：天然hHGF；泳道2：130Arg-hHGF；泳道3：130His-hHGF；泳道4：130Lys-hHGF；泳道5：蛋白质分子量标记。
关于序列信息的说明
本发明涉及的序列的信息提供如下。
SEQ ID NO:1(天然hHGF的氨基酸序列)
Figure PCTCN2020070010-appb-000002
SEQ ID NO:2(130Arg-hHGF的氨基酸序列)
Figure PCTCN2020070010-appb-000003
SEQ ID NO:3(130His-hHGF的氨基酸序列)
Figure PCTCN2020070010-appb-000004
Figure PCTCN2020070010-appb-000005
SEQ ID NO:4(130Lys-hHGF的氨基酸序列)
Figure PCTCN2020070010-appb-000006
SEQ ID NO:5(编码天然hHGF的核苷酸序列)
Figure PCTCN2020070010-appb-000007
Figure PCTCN2020070010-appb-000008
SEQ ID NO:6(编码130Arg-hHGF的核苷酸序列)
Figure PCTCN2020070010-appb-000009
SEQ ID NO:7(编码130His-hHGF的核苷酸序列)
Figure PCTCN2020070010-appb-000010
SEQ ID NO:8(编码130Lys-hHGF的核苷酸序列)
Figure PCTCN2020070010-appb-000011
Figure PCTCN2020070010-appb-000012
SEQ ID NO:9(pSN载体的核苷酸序列)
Figure PCTCN2020070010-appb-000013
Figure PCTCN2020070010-appb-000014
具体实施方式
现参照下列意在举例说明本申请(而非限定本申请)的实施例来描述本申请。
除非特别指明，本申请中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，John Wiley&Sons,Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本申请，且不意欲限制本申请所要求保护的范围。
实施例1.hHGF以及其突变体的制备
天然hHGF的氨基酸序列(SEQ ID NO:1)可见于NCBI登录号NP_000592.3。以SEQ ID NO:1为模板，设计如下3个hHGF突变体：
(1)将SEQ ID NO:1第130位的Ser突变为Arg，得到hHGF突变体130Arg-hHGF，其氨基酸序列如SEQ ID NO:2所示；
(2)将SEQ ID NO:1第130位的Ser突变为His，得到hHGF突变体130His-hHGF，其氨基酸序列如SEQ ID NO:3所示；和
(3)将SEQ ID NO:1第130位的Ser突变为Lys，得到hHGF突变体130Lys-hHGF，其氨基酸序列如SEQ ID NO:4所示。
全基因合成分别编码天然hHGF(SEQ ID NO:1)与上述3个hHGF突变体(SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4)的多核苷酸，并在其5’端引入限制性酶切位点和起始密码子，在其3’端引入限制性酶切位点以及终止密码子，从而获得编码天然hHGF以及各种突变体的核酸分子。
将如上述制备的核酸分子分别克隆入表达载体，并转化到CHO宿主细胞中。在允许外源蛋白质表达的条件下，培养经转化的CHO宿主细胞，然后收集培养物，并进行离心，从而得到含有目的蛋白(天然hHGF，130Arg-hHGF，130His-hHGF，或130Lys-hHGF)的上清液。按照制造商的说明书，使用阴离子交换层析介质(DEAE Sepharose Fast Flow，GE healthcare，17-0709-10)分离上清液中的目的蛋白，并且，使用肝素亲和层析介质(Heparin Sepharose 6 Fast Flow,GE healthcare，17-0998-01)进一步纯化目的蛋白。
通过非还原聚丙烯酰胺凝胶电泳法(non-reduced SDS-PAGE，分子克隆实验指南，第四版)检测所获得的经纯化的目的蛋白，结果如图1所示。如图1所示，所获得的4种经纯化的目的蛋白(天然hHGF，130Arg-hHGF，130His-hHGF和130Lys-hHGF)的纯度均大于98％，可用于后续研究。
实施例2.编码hHGF以及其突变体的重组质粒的制备
全基因合成分别编码天然hHGF(SEQ ID NO:1)与上述3个hHGF突变体(SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4)的多核苷酸，其分别具有如SEQ ID NO:5-8所示的核苷酸序列。将如上制备的多核苷酸分子分别克隆入pSN载体(参见例如，中国专利CN 108611367B；其核苷酸序列如SEQ ID NO:9所示)，然后转化入大肠杆菌中。经筛选和测序验证后，获得含有目的重组质粒的工程菌株。发酵所构建的工程菌株，并提取质粒，从而获得含有目的重组质粒的原液，所述目的重组质粒具体如下：
(1)pSN-hHGF，其携带编码天然hHGF(SEQ ID NO:1)的多核苷酸(SEQ ID NO:5)；
(2)pSN-130Arg-hHGF，其携带编码130Arg-hHGF(SEQ ID NO:2)的多核苷酸(SEQ ID NO:6)；
(3)pSN-130His-hHGF，其携带编码130His-hHGF(SEQ ID NO:3)的多核苷酸(SEQ ID NO:7)；和
(4)pSN-130Lys-hHGF，其携带编码130Lys-hHGF(SEQ ID NO:4)的多核苷酸(SEQ ID NO:8)。
使用紫外分光光度计，测定所制备的各种原液中的质粒含量。结果显示，在所制备的各种原液样品中，质粒的含量均在2.0-2.2mg/mL范围内。具体而言，4种原液中的重组质粒的含量分别为：2.12mg/mL(pSN-hHGF)，2.05mg/mL(pSN-130Arg-hHGF)，2.15mg/mL(pSN-130His-hHGF)，和2.10mg/mL(pSN-130Lys-hHGF)。
取含有目的重组质粒的原液，用注射用水稀释至质粒浓度约为30μg/ml，然后用HPLC检测纯度，所使用的检测条件如下：
所使用的色谱柱为阴离子交换HPLC分析柱DNA-NPR，其用20mM Tris-HCl，0.5M NaCl，pH8.8缓冲液进行平衡。平衡后，加载样品，进行检测。上样量为100μl，流速为0.5ml/min，检测波长为260nm。上样后，用20mM Tris-HCl，0.5M NaCl，pH8.8缓冲液进行平衡(5min)，然后进行线性梯度洗脱，条件如下：(1)由100％A溶液(A溶液为20mM Tris-HCl，0.5M NaCl，pH8.8)线性过渡至100％B溶液(B溶液为20mM Tris-HCl，0.8M NaCl)，洗脱30min；(2)然后用20mM Tris-HCl，0.8M NaCl，pH8.8缓冲液洗脱5min。结果显示，在所制备的各种原液样品中，质粒的HPLC纯度均大于95.0％。具体而言，4种原液中的重组质粒的HPLC纯度分别为：97.5％(pSN-hHGF)，98.2％(pSN-130Arg-hHGF)，98.0％(pSN-130His-hHGF)，97.8％(pSN-130Lys-hHGF)。
实施例3.hHGF突变体与天然hHGF的体外生物学活性的评价
本实施例通过体外内皮细胞迁移实验来评价hHGF突变体与天然hHGF对内皮细胞迁移的影响，以评价hHGF突变体与天然hHGF的体外生物学活性。
1.材料与方法
1.1蛋白样品
如上制备的hHGF突变体(130Arg-hHGF，130His-hHGF和130Lys-hHGF)与天然hHGF，临用时用生理盐水配制成所需浓度。
1.2细胞株
ECV304细胞株(脐静脉内皮细胞)，用于测试HGF生物活性。
1.3试剂
DMEM培养基：Hyclone提供。配制方法如下：取DMEM培养基粉末1袋(规格为1L)，加水溶解并稀释至1000ml，再添加碳酸氢钠2.1g。然后，将配制的培养基除菌过滤，4℃保存。
完全培养基：取胎牛血清100ml，添加DMEM培养基至1000ml。
Transwells：Costar提供。
1.4仪器
二氧化碳细胞培养箱：上海博讯实业有限公司医疗设备厂提供，型号为HH.CP。
倒置显微镜：重庆光电仪器总公司提供，型号为XDS-1B。
超净工作台：苏州净化设备有限公司提供，型号为SW-CJ-1F。
台式细胞洗涤离心机：湖南星科科学仪器有限公司提供，型号为TDL-50B。
光学显微镜：重庆光电仪器总公司提供，型号为BP104。
1.5试验方法
根据“体外HGF活性检测试验”方法(The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes.J.Exp.Med.,1962,115:453–466)，进行细胞迁移试验。简言之，在迁移板下槽的每孔中加入600μl DMEM培养基，将Transwells浸没入其中。将经0.1％胰酶消化的ECV304细胞用含10％胎牛血清的1640培养基配成每1ml含1×10 6个细胞的细胞悬液。每孔加入200μl细胞悬液，并在37℃孵育1h。然后，用600μl含有2微克天然hHGF或hHGF突变体的培养基替代迁移板下槽中的原DMEM培养基，并继续培养2h。培养后，将Transwells转移至另一加有20％多聚甲醛的孔中，固定细胞10min。用棉签轻轻搽去膜上的未迁移细胞，随后用结晶紫染色5min。用手术刀片小心取下膜，放在载玻片上(有细胞的一面向上)，用光学显微镜观察。另设不使用天然hHGF或hHGF突变体的空白对照。
本试验使用迁移细胞计数来评价待测蛋白(天然hHGF或hHGF突变体)的生物学活性。用光学显微镜定量评价细胞迁移的方法如下：先在低倍(物镜4倍)光学显微镜下选取细胞分布均匀的区域，再用目镜中附加网格的中倍(物镜20倍)显微镜，随机连续挑选5个视野，计数迁移细胞。测定结果用统计学t检验方法进行分析和评价。
1.6统计分析
数据以均数±标准差
Figure PCTCN2020070010-appb-000015
表示。应用SPSS16.0统计软件，采用多因素析因设计资料的方差分析，进行统计学分析。
2.实验结果
如上所述，在体外试验中，通过用hHGF突变体与天然hHGF进行内皮细胞迁移试验，评价了hHGF突变体与天然hHGF对内皮细胞迁移的影响。实验结果如表1所示。
表1.hHGF突变体和天然hHGF对内皮细胞迁移的影响
Figure PCTCN2020070010-appb-000016
其中，“*”表示与空白对照组相比，p＜0.05；
“**”表示与空白对照组相比，p＜0.01；
“#”表示与天然hHGF试验组相比，p＜0.05；
“##”表示与天然hHGF试验组相比，p＜0.01。
如表1所示，在空白对照孔中，穿过迁移膜的内皮细胞很少；而在含有hHGF突变体或天然hHGF的测定孔中，迁移细胞数则明显增多，与空白对照孔相比，存在显著性差异(含有天然hHGF的测定孔：p＜0.05；含有hHGF突变体的测定孔：p＜0.01)。进一步，在含有hHGF突变体的测定孔中，迁移细胞数显著多于含有天然hHGF的测定孔(p＜0.01)。这些结果说明，hHGF突变体与天然hHGF都能够诱导/刺激内皮细胞迁移，并且，hHGF突变体诱导内皮细胞迁移的能力比天然hHGF更强。本申请的3种hHGF突变体(130Arg-hHGF，130His-hHGF和130Lys-hHGF)均能够更好地促进细胞迁移。
实施例4.hHGF突变体与天然hHGF对家兔下肢动脉缺血模型的治疗效果的评价
本实施例通过家兔下肢动脉缺血模型来评价hHGF突变体与天然hHGF对家兔下肢缺血模型的血管及侧支循环再形成的作用，以评价hHGF突变体与天然hHGF的治疗效果。
1.材料与方法
1.1蛋白样品
如上制备的hHGF突变体(130Arg-hHGF，130His-hHGF和130Lys-hHGF)与天然hHGF，临用时用生理盐水配制成所需浓度。
1.2动物模型
新西兰雄性白兔，12-14月龄，体重3.5-4.0kg，由北京维通利华公司提供。参照Takeshita等(Therapeutics angiogenesis.A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model.J.Clin.Invest.,1994，93：662-670)描述的方法，建立兔下肢动脉缺血模型。肌肉注射5mg/kg剂量的甲苯噻嗪(Xylazine)后，用50mg/kg剂量氯胺酮(Ketamine)对兔进行麻醉。用酒精和碘酒消毒左大腿内侧皮肤。在无菌状态下，切开左侧自腹股沟中点至膝关节的大腿皮肤，切开肌筋膜，分离肌肉，充分暴露股动脉，结扎其主干及分支，并切除从股动脉根部到腘动脉和大隐动脉分叉处的动脉血管。确保无出血后，缝合肌筋膜及皮肤。术后，连续肌肉注射庆大霉素(3mg/kg/d)3日以预防感染，同时肌肉注射吗啡(0.3mg/kg/d)10日进行镇痛。术后第10天，在右侧颈动脉插动脉套管，将3F导管(Terumo，Japan)插入至左侧髂内动脉入口处，以每秒1ml的速度灌入造影剂5ml，进行选择性髂内动脉造影术，以确认病态动物模型的建立。
1.3动物分组
建立动物模型后第十日，将动物随机分成模型对照组(6只)、hHGF试验组(8只)、130Arg-hHGF试验组(8只)、130His-hHGF试验组(8只)和130Lys-hHGF试验组(8只)。
1.4给药方法
在每只动物的左侧大腿内侧的缺血部位取4个点(内收肌1点，半膜肌3点)， 每点肌肉注射给予250μg/250μl受试药物(也即，每只动物一次给予受试药物共1mg/1ml)，每日给药1次。模型对照组给予等体积的生理盐水。持续给药15天，共计给药15次。
1.5用选择性髂内动脉造影术评价药物对血管生成的影响
由于下肢动脉缺血模型动物的侧支循环起源于髂内动脉分支，于术后第10日和第40日(首次给药后第30天)分别进行选择性髂内动脉造影术，以观察给药前后侧支循环形成情况。将3F导管(Terumo，Japan)插入右侧颈动脉，经腹主动脉，放置左侧髂内动脉入口，以1ml/秒的速度，共注入5ml造影剂，并进行Cine film摄影。在第4秒造影图上，在股骨上画三条垂直于股骨并将股骨等分为4个部分的直线，计数越过所述直线的血管数，重复计数3次，取平均值。
1.6毛细血管密度的组织学测定
于术后第40天，取下肢缺血肌肉组织(内收肌和半膜肌)，放入O.C.T.compound(Miles Inc.,Elkhart,USA)溶液中，用液氮速冻后，进行组织冷冻切片。按照Indoxyl-tetrazolium方法，用碱性磷酸酶染色毛细血管内皮细胞。在镜下(×200)，计数组织中毛细血管内皮细胞数，再换算成每1,000个肌细胞中的毛细血管数，以定量表示毛细血管的密度。
1.7统计分析
数据以均数±标准差
Figure PCTCN2020070010-appb-000017
表示。应用SPSS16.0统计软件，采用多因素析因设计资料的方差分析，进行统计学分析。
2.实验结果
各组实验动物给药前后缺血部位侧支血管数量，以及缺血部位新生侧支血管数量的测定结果，如表2所示。
表2.各组动物的侧支血管计数结果
Figure PCTCN2020070010-appb-000018
其中，“*”表示与模型对照组相比，p＜0.05；
“**”表示与模型对照组相比，p＜0.01；
“#”表示与hHGF试验组相比，p＜0.05；
“##”表示与hHGF试验组相比，p＜0.01。
如表2所示，在给药前，各组实验动物的侧支血管数(五个组之间)没有明显差异(p>0.05)。给药后，模型对照组的侧支血管数相比于给药前，无统计学差异 (p>0.05)；而四个试验组的侧支血管数均比给药前显著增加。在各组之间进行比较时，三个hHGF突变体试验组的血管增加数均显著大于hHGF试验组(p<0.05)。
表2的实验结果表明，3种hHGF突变体与天然hHGF对下肢动脉缺血都具有良好的治疗作用。与模型对照组比较，各试验组均能显著促进侧支血管形成，如血管造影术所显示的。治疗(从给药第一天计算)后第30日，试验组家兔的左后肢小动脉密度比模型对照组显著更高。进一步，3种hHGF突变体的治疗结果与天然hHGF试验组相比具有显著差异：接受hHGF突变体的家兔的左后肢小动脉密度比接受天然hHGF的家兔更高；其中，130Arg-hHGF试验组(p＜0.01)，130His-hHGF试验组(p＜0.05)和130Lys-hHGF试验组(p＜0.01)促进侧支血管形成的功效均显著优于天然hHGF试验组。这些结果表明，本申请的3种hHGF突变体在治疗下肢动脉缺血的效果方面，出乎意料地优于天然hHGF。
实施例5.编码hHGF或其突变体的重组质粒对家兔下肢动脉缺血模型的治疗效果的评价
本实施例通过家兔下肢动脉缺血模型来评价编码hHGF或其突变体的重组质粒促进侧支血管形成的作用，从而评价编码hHGF或其突变体的重组质粒的治疗效果。
1.材料与方法
1.1质粒样品
如上制备的4种重组质粒(pSN-hHGF，pSN-130Arg-hHGF，pSN-130His-hHGF和pSN-130Lys-hHGF)，临用时用生理盐水配制成所需浓度。
1.2动物模型
新西兰雄性白兔，12-14月龄，体重3.5-4.0kg，由北京维通利华公司提供。参照Takeshita等(Therapeutics angiogenesis.A single intraarterial bolus of vascular endothelial growth factor augments revascularization in a rabbit ischemic hind limb model.J.Clin.Invest.,1994，93：662-670)描述的方法，建立兔下肢动脉缺血模型。肌肉注射5mg/kg剂量的甲苯噻嗪(Xylazine)后，用50mg/kg剂量氯胺酮(Ketamine)对兔进行麻醉。用酒精和碘酒消毒左大腿内侧皮肤。在无菌状态下，切开左侧自腹股沟中点至膝关节的大腿皮肤，切开肌筋膜，分离肌肉，充分暴露股动脉，结扎其主干及分支，并切除从股动脉根部到腘动脉和大隐动脉分叉处的动脉血管。确保无出血后，缝合肌筋膜及皮肤。术后，连续肌肉注射庆大霉素(3mg/kg/d)3日以预防感染，同时肌肉注射吗啡(0.3mg/kg/d)10日进行镇痛。术后第10天，在右侧颈动脉插动脉套管，将3F导管(Terumo，Japan)插入至左侧髂内动脉入口处，以每秒1ml的速度灌入造影剂5ml，进行选择性髂内动脉造影术，以确认病态动物模型的建立。
1.3动物分组
建立动物模型后第十日，将动物随机分成模型对照组(8只)、pSN-hHGF试验组(8只)、pSN-130Arg-hHGF试验组(8只)、pSN-130His-hHGF试验组(8只)和 pSN-130Lys-hHGF试验组(8只)。
1.4给药方法
在每只动物的左侧大腿内侧的缺血部位取4个点(内收肌1点，半膜肌3点)，每点肌肉注射给予250μg/250μl受试药物(也即，每只动物一次给予受试药物共1mg/1ml)，共给药1次。模型对照组给予等体积的生理盐水。
1.5用选择性髂内动脉造影术评价药物对血管生成的影响
由于下肢动脉缺血模型动物的侧支循环起源于髂内动脉分支，于术后第10日和第40日(给药后第30天)分别进行选择性髂内动脉造影术，以观察给药前后侧支循环形成情况。将3F导管(Terumo，Japan)插入右侧颈动脉，经腹主动脉，放置左侧髂内动脉入口，以1ml/秒的速度，共注入5ml造影剂，并进行Cine film摄影。在第4秒造影图上，在股骨上画三条垂直于股骨并将股骨等分为4个部分的直线，计数越过所述直线的血管数，重复计数3次，取平均值。
1.6统计分析
数据以均数±标准差
Figure PCTCN2020070010-appb-000019
表示。应用SPSS16.0统计软件，采用多因素析因设计资料的方差分析，进行统计学分析。
2.实验结果
各组实验动物给药前后缺血部位侧支血管数量，以及缺血部位新生侧支血管数量的测定结果，如表3所示。
表3.各组动物的侧支血管计数结果
Figure PCTCN2020070010-appb-000020
其中，“*”表示与模型对照组相比，p＜0.05；
“**”表示与模型对照组相比，p＜0.01；
“#”表示与pSN-hHGF试验组相比，p＜0.05；
“##”表示与pSN-hHGF试验组相比，p＜0.01。
如表3所示，在给药前，各组实验动物的侧支血管数(五个组之间)没有明显差异(p>0.05)。给药后，模型对照组的侧支血管数相比于给药前，无统计学差异(p>0.05)；而四个试验组的侧支血管数均比给药前显著增加。在各组之间进行比较时，3个编码hHGF突变体的重组质粒试验组(pSN-130Arg-hHGF，pSN-130His-hHGF和pSN-130Lys-hHGF)的血管增加数均显著大于编码天然hHGF的重组质粒试验组(pSN-hHGF)(p<0.01)。
表3的实验结果表明，编码hHGF或其突变体的重组质粒对下肢动脉缺血都具有良好的治疗作用。与模型对照组比较，各试验组均能显著促进侧支血管形成。进一步，表3的实验结果还表明，3种编码hHGF突变体的重组质粒的治疗结果与编码天然hHGF的重组质粒相比具有显著差异：pSN-130Arg-hHGF试验组(p＜0.01)，pSN-130His-hHGF试验组(p＜0.01)和pSN-130Lys-hHGF试验组(p＜0.01)促进侧支血管形成的功效均显著优于pSN-hHGF试验组。这些结果表明，3种编码hHGF突变体的重组质粒(pSN-130Arg-hHGF，pSN-130His-hHGF和pSN-130Lys-hHGF)在治疗下肢动脉缺血的效果方面，出乎意料地优于编码天然hHGF的重组质粒(pSN-hHGF)。
实施例6.hHGF突变体与天然hHGF对大鼠糖尿病周围神经病变模型的治疗效果的评价
本实施例通过大鼠糖尿病周围神经病变模型来评价hHGF突变体与天然hHGF治疗糖尿病周围神经病变的效果。
1.材料与方法
1.1蛋白样品
如上制备的hHGF突变体(130Arg-hHGF，130His-hHGF和130Lys-hHGF)与天然hHGF，临用时用生理盐水配制成所需浓度。
1.2仪器
ONE-TOUCH稳择易型血糖仪，美国强生公司提供；
Neuromatic-2000型肌电图仪，丹迪公司提供；
EG1160型石蜡包埋机，德国Leica公司提供；
RM2255型切片机，德国Leica公司提供；
DM6000B光学显微镜，德国Leica公司提供。
1.3动物模型
Wistar大鼠(SPF级，雄性，体重180-200g，2.5到3月龄)由北京维通利华公司提供。大鼠购回后，适应性喂养5天，并确认动物状态良好。随机挑选10只作为正常对照组，其余60只如下进行造模。将大鼠禁食不禁水12h，然后称重，测血糖，并编号。在冰浴环境下将链脲佐菌素(STZ)加入事先配好的0.1mol/L柠檬酸/柠檬酸钠缓冲液(pH4.4)中，配制成2％的STZ溶液。按照65mg/kg剂量将STZ通过左侧腹腔注射一次性给予造模动物；正常对照组动物接受等剂量相同缓冲液的左侧腹腔注射。72小时后，测量大鼠血糖。选取血糖>16.7mmol/L、尿糖+++至++++的大鼠作为模型动物，共52只。喂养10周后，获得患糖尿病周围神经病变的模型大鼠。
1.4动物分组
将52只模型大鼠随机分为模型对照组(10只)、hHGF试验组(10只)、130Arg-hHGF试验组(10只)、130His-hHGF试验组(11只)、130Lys-hHGF试验组(11只)。
1.5给药方法
试验组动物于造模成功10周后开始接受给药。在每只动物的左侧大腿内侧取4 个点(内收肌1点，半膜肌3点)，每点肌肉注射给予250μg/250μl受试药物(也即，每只动物一次给予受试药物共1mg/1ml)，每日给药1次。模型组对照给予等体积的生理盐水，持续给药20天，共计注射20次。
1.6运动神经传导速度(MNCV)及感觉神经传导速度(SNCV)的测定
在首次给药后10周进行测定。在麻醉大鼠后，手术分离左侧坐骨神经，应用Neuromatic-2000型肌电图仪测定大鼠MNCV和SNCV。MNCV测定方法如下：将记录电极垂直刺入胫前肌肌腹中部，用刺激电极以20mA的刺激电流刺激坐骨神经近端，肌电图仪于示波器上显示并记录所得动作电位，然后根据两电极间距离计算出MNCV。SNCV测定方法如下：将记录电极置于坐骨神经近端，刺激电极以30mA的刺激强度刺激腓肠神经近端，肌电图仪记录刺激所得波形，然后根据两电极间距离计算出SNCV。
1.7腓肠神经有髓神经纤维的数量分析
在首次给药后10周进行测定。将右侧腓肠神经远端固定于3％戊二醛/0.1mol/L磷酸盐缓冲液中，保持4℃过夜。用PBS缓冲液冲洗，用1％锇酸后固定，然后冲洗、脱水，用环氧树脂包埋。制备1μm半薄横断面切片，用1％甲苯胺蓝溶液染色30分钟，然后用85％酒精清洗、脱色至背景为淡蓝色，然后树胶封片。以200倍放大率采集腓肠神经横切片图像，采用多功能真彩色病理图像分析系统进行有髓神经纤维计数，测定腓肠神经纤维总截面积、神经纤维密度及神经纤维平均截面积，以观察腓肠神经的病理改变。
1.8统计学处理
数据以均数±标准差
Figure PCTCN2020070010-appb-000021
表示。应用SPSS16.0统计软件，采用多因素析因设计资料的方差分析，进行统计学分析。
2.实验结果
2.1各组实验动物的MNCV及SNCV的测定
各组实验动物的MNCV及SNCV的测定结果如表4所示。
表4.首次给药后第10周各组动物的MNCV及SNCV
Figure PCTCN2020070010-appb-000022
其中，“*”表示与正常对照组相比，p＜0.05；
“**”表示与正常对照组相比，p＜0.01。
如表4所示，在首次给药后第10周，模型对照组的MNCV及SNCV最低(显著慢于正常对照组，p<0.01)；hHGF试验组次之(显著慢于正常对照组，p<0.05)。hHGF 突变体组的MNCV及SNCV略低于正常对照组，但差异无统计学意义(p>0.05)。这些结果表明，天然hHGF和hHGF突变体能够促进糖尿病周围神经病变修复，恢复受损的MNCV和SNCV，并且，hHGF突变体的治疗效果优于天然hHGF。
2.2腓肠神经有髓神经纤维的数量分析
各组实验动物的腓肠神经有髓神经纤维的数量分析结果如表5所示。
表5.首次给药后第10周各组动物的腓肠神经有髓神经纤维的数量分析
Figure PCTCN2020070010-appb-000023
其中，“*”表示与正常对照组相比，p＜0.05；
“**”表示与正常对照组相比，p＜0.01。
“#”表示与hHGF试验组相比，p＜0.05；
“##”表示与hHGF试验组相比，p＜0.01。
如表5所示，在首次给药后第10周，与正常对照组比较，模型对照组动物的神经总截面积和神经纤维平均面积均明显减小，且有显著性差异(p<0.01)；hHGF试验组动物的神经总截面积和神经纤维平均面积也出现明显减小(p<0.05)，但减小的程度有所下降；hHGF突变体试验组动物的神经总截面积和神经纤维平均面积减小不明显，没有显著性差异。进一步，与hHGF试验组相比，hHGF突变体试验组动物的神经总截面积和神经纤维平均面积均显著增加(p<0.05)。这些结果表明，天然hHGF和hHGF突变体能够促进糖尿病周围神经病变修复，恢复腓肠神经有髓神经纤维的截面总面积和神经纤维平均面积，并且hHGF突变体的治疗效果优于天然hHGF。
表4-5的结果表明，hHGF突变体与天然hHGF对糖尿病周围神经病变都有良好的治疗作用，能显著增加糖尿病大鼠的MNCV及SNCV，显著改善糖尿病大鼠腓肠神经纤维横截面总面积和神经纤维平均面积。并且，3种hHGF突变体的治疗结果均显著优于天然hHGF。本申请的3种hHGF突变体在治疗大鼠糖尿病周围神经病变的效果方面，出乎意料地优于天然hHGF。
实施例7.编码hHGF或其突变体的重组质粒对大鼠糖尿病周围神经病变模型的治疗效果的评价
本实施例通过大鼠糖尿病周围神经病变模型来评价编码hHGF或其突变体的重组质粒治疗糖尿病周围神经病变的效果。
1.材料与方法
1.1质粒样品
如上制备的4种重组质粒(pSN-hHGF，pSN-130Arg-hHGF，pSN-130His-hHGF和pSN-130Lys-hHGF)，临用时用生理盐水配制成所需浓度。
1.2仪器
ONE-TOUCH稳择易型血糖仪，美国强生公司提供；
Neuromatic-2000型肌电图仪，丹迪公司提供；
EG1160型石蜡包埋机，德国Leica公司提供；
RM2255型切片机，德国Leica公司提供；
DM6000B光学显微镜，德国Leica公司提供。
1.3动物模型
Wistar大鼠(SPF级，雄性，体重180-200g，2.5到3月龄)由北京维通利华公司提供。大鼠购回后，适应性喂养5天，并确认动物状态良好。随机挑选10只作为正常对照组，其余60只如下进行造模。将大鼠禁食不禁水12h，然后称重，测血糖，并编号。在冰浴环境下将链脲佐菌素(STZ)加入事先配好的0.1mol/L柠檬酸/柠檬酸钠缓冲液(pH4.4)中，配制成2％的STZ溶液。按照65mg/kg剂量将STZ通过左侧腹腔注射一次性给予造模动物；正常对照组动物接受等剂量相同缓冲液的左侧腹腔注射。72小时后，测量大鼠血糖。选取血糖>16.7mmol/L、尿糖+++至++++的大鼠作为模型动物，共52只。喂养10周后，获得患糖尿病周围神经病变的模型大鼠。
1.4动物分组
将52只模型大鼠随机分为模型对照组(10只)、pSN-hHGF试验组(10只)、pSN-130Arg-hHGF试验组(10只)、pSN-130His-hHGF试验组(11只)、pSN-130Lys-hHGF试验组(11只)。
1.5给药方法
试验组动物于造模成功10周后开始接受给药。在每只动物的左侧大腿内侧取4个点(内收肌1点，半膜肌3点)，每点肌肉注射给予250μg/250μl受试药物(也即，每只动物一次给予受试药物共1mg/1ml)，共给药1次。模型组对照给予等体积的生理盐水。
1.6运动神经传导速度(MNCV)及感觉神经传导速度(SNCV)的测定
在给药后10周进行测定。在麻醉大鼠后，手术分离左侧坐骨神经，应用Neuromatic-2000型肌电图仪测定大鼠MNCV和SNCV。MNCV测定方法如下：将记录电极垂直刺入胫前肌肌腹中部，用刺激电极以20mA的刺激电流刺激坐骨神经近端，肌电图仪于示波器上显示并记录所得动作电位，然后根据两电极间距离计算出MNCV。SNCV测定方法如下：将记录电极置于坐骨神经近端，刺激电极以30mA的刺激强度刺激腓肠神经近端，肌电图仪记录刺激所得波形，然后根据两电极间距离计算出SNCV。
1.7腓肠神经有髓神经纤维的数量分析
在给药后10周进行测定。将右侧腓肠神经远端固定于3％戊二醛/0.1mol/L磷酸盐缓冲液中，保持4℃过夜。用PBS缓冲液冲洗，用1％锇酸后固定，然后冲洗、脱水， 用环氧树脂包埋。制备1μm半薄横断面切片，用1％甲苯胺蓝溶液染色30分钟，然后用85％酒精清洗、脱色至背景为淡蓝色，然后树胶封片。以200倍放大率采集腓肠神经横切片图像，采用多功能真彩色病理图像分析系统进行有髓神经纤维计数，测定腓肠神经纤维总截面积、神经纤维密度及神经纤维平均截面积，以观察腓肠神经的病理改变。
1.8统计学处理
数据以均数±标准差
Figure PCTCN2020070010-appb-000024
表示。应用SPSS16.0统计软件，采用多因素析因设计资料的方差分析，进行统计学分析。
2.实验结果
2.1各组实验动物的MNCV及SNCV的测定
各组实验动物的MNCV及SNCV的测定结果如表6所示。
表6.给药后第10周各组动物的MNCV及SNCV
Figure PCTCN2020070010-appb-000025
其中，“*”表示与正常对照组相比，p＜0.05；
“**”表示与正常对照组相比，p＜0.01。
如表6所示，在给药后第10周，模型对照组的MNCV及SNCV最低(显著慢于正常对照组，p<0.01)；pSN-hHGF试验组次之(显著慢于正常对照组，p<0.05)。编码hHGF突变体的重组质粒试验组的MNCV及SNCV略低于正常对照组，但差异无统计学意义(p>0.05)。这些结果表明，编码天然hHGF及其突变体的重组质粒能够促进糖尿病周围神经病变修复，恢复受损的MNCV和SNCV，并且，编码hHGF突变体的重组质粒(pSN-130Arg-hHGF，pSN-130His-hHGF和pSN-130Lys-hHGF)的治疗效果优于编码天然hHGF的重组质粒(pSN-hHGF)。
(2)腓肠神经有髓神经纤维的数量分析
各组实验动物的腓肠神经有髓神经纤维的数量分析结果如表7所示。
表7.给药后第10周各组动物的腓肠神经有髓神经纤维的数量分析
Figure PCTCN2020070010-appb-000026
Figure PCTCN2020070010-appb-000027
其中，“*”表示与正常对照组相比，p＜0.05；
“**”表示与正常对照组相比，p＜0.01。
“#”表示与pSN-hHGF试验组相比，p＜0.05；
“##”表示与pSN-hHGF试验组相比，p＜0.01。
如表7所示，在给药后第10周，与正常对照组比较，模型对照组动物的神经总截面积和神经纤维平均面积均明显减小，且有显著性差异(p<0.01)；pSN-hHGF试验组动物的神经总截面积和神经纤维平均面积也出现明显减小(p<0.05)，但减小的程度有所下降；编码hHGF突变体的重组质粒试验组动物的神经总截面积和神经纤维平均面积减小不明显，没有显著性差异。进一步，与pSN-hHGF试验组相比，编码hHGF突变体的重组质粒试验组动物的神经总截面积和神经纤维平均面积均显著增加(p<0.05)。这些结果表明，编码天然hHGF及其突变体的重组质粒能够促进糖尿病周围神经病变修复，恢复腓肠神经有髓神经纤维的截面总面积和神经纤维平均面积，并且3种编码hHGF突变体的重组质粒(pSN-130Arg-hHGF，pSN-130His-hHGF和pSN-130Lys-hHGF)的治疗效果优于编码天然hHGF的重组质粒(pSN-hHGF)。
表6-7的结果表明，编码hHGF突变体的重组质粒与编码天然hHGF的重组质粒对糖尿病周围神经病变都有良好的治疗作用，能显著增加糖尿病大鼠的MNCV及SNCV，显著改善糖尿病大鼠腓肠神经纤维横截面总面积和神经纤维平均面积。并且，3种编码hHGF突变体的重组质粒(pSN-130Arg-hHGF，pSN-130His-hHGF和pSN-130Lys-hHGF)的治疗结果均显著优于编码天然hHGF的重组质粒(pSN-hHGF)。
3.结论
实施例3、4和6的结果表明，本申请的hHGF突变体在促进脐静脉内皮细胞迁移、促进下肢小动脉生长、促进糖尿病周围神经病变修复等方面具有显著高于天然hHGF的活性。
实施例5和7的结果表明，编码hHGF突变体的重组质粒能够作为基因治疗药物，在受试者体内用于促进下肢小动脉生长、促进糖尿病周围神经病变修复等方面，并且其治疗效果显著高于编码天然hHGF的重组质粒。
尽管本申请的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公开的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本申请的保护范围之内。本申请的全部范围由所附权利要求及其任何等同物给出。

Claims (12)

  1. 一种人肝细胞生长因子(human hepatocyte growth factor,hHGF)的突变体，其与天然hHGF相比，包含下述突变：天然hHGF中对应于SEQ ID NO:1的第130位的位置上的氨基酸被突变为具有碱性侧链的氨基酸。
  2. 权利要求1的突变体，其具有选自下列的一个或多个特征：
    (1)所述具有碱性侧链的氨基酸选自精氨酸、组氨酸和赖氨酸；
    (2)所述天然hHGF具有如SEQ ID NO:1所示的氨基酸序列；
    (3)所述突变体具有选自SEQ ID NO:2，3和4的氨基酸序列；
    (4)所述突变体是经修饰的，例如是经化学修饰的，例如是经PEG化修饰的；
    (5)所述hHGF突变体是通过重组表达的方法制备的，或者是通过化学合成的方法制备的；和
    (6)所述hHGF突变体与天然hHGF相比具有更强的生物学活性，例如，在选自下述的一个或多个方面显示更强的活性：(a)促进内皮细胞生长和/或迁移；(b)促进血管(例如微小血管)发生；和/或，(c)促进神经损伤(例如周围神经病变，例如糖尿病周围神经病变)修复。
  3. 一种分离的核酸分子，其包含编码权利要求1或2的突变体的核苷酸序列；
    优选地，所述核酸分子的核苷酸序列是根据宿主细胞(例如CHO细胞)偏好进行了密码子优化的；
    优选地，所述核酸分子具有选自SEQ ID NO:6，7和8的核苷酸序列。
  4. 一种载体，其包含权利要求3的分离的核酸分子；优选地，所述载体可以具有选自下列的一个或多个特征：
    (1)所述载体选自质粒(例如裸质粒)；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体；以及，病毒载体，例如逆转录酶病毒载体(例如慢病毒载体)、腺病毒载体、腺相关病毒载体、疱疹病毒载体(如单纯疱疹病毒载体)、痘病毒载体、杆状病毒载体、乳头瘤病毒载体、乳头多瘤空泡病毒载体；
    (2)所述载体用于表达(例如在受试者(例如哺乳动物，例如人)体内表达)所述突变体；和
    (3)所述载体是用于基因治疗的载体，例如质粒(例如裸质粒)，腺病毒载体，腺相关病毒载体，和慢病毒载体；优选地，所述载体为包含所述核酸分子的pSN载体。
  5. 一种宿主细胞，其包含权利要求3的分离的核酸分子或权利要求4的载体；优选地，所述宿主细胞选自原核细胞例如大肠杆菌细胞，以及真核细胞例如酵母细胞，昆虫细胞，植物细胞和动物细胞(如哺乳动物细胞，例如小鼠细胞、人细胞等)。
  6. 制备权利要求1或2的突变体的方法，其包括，在合适的条件下培养权利要求5的宿主细胞，和从所述宿主细胞的细胞培养物中回收所述突变体；
    优选地，所述方法包括以下步骤：
    (1)构建表达载体，所述表达载体包含编码所述突变体(例如，具有如SEQ ID NO:2、SEQ ID NO:3或SEQ ID NO:4所示氨基酸序列的突变体)的核酸序列；
    (2)将所述表达载体导入宿主细胞(例如CHO细胞)中，并在允许蛋白质表达的条件下，培养所述宿主细胞；和
    (3)从所述宿主细胞的细胞培养物中分离和回收本发明的突变体(例如通过阴离子交换层析和肝素亲和层析来分离和回收)。
  7. 一种药物组合物，其含有权利要求1或2的突变体或权利要求3的核酸分子或权利要求4的载体，以及任选地，药学上可接受的载体和/或赋形剂。
  8. 一种在受试者中治疗可受益于天然hHGF的活性的疾病的方法，其包括，给有此需要的受试者施用治疗有效量的权利要求1或2的突变体或权利要求3的核酸分子或权利要求4的载体或权利要求7的药物组合物；
    优选地，所述疾病选自缺血性疾病(例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如心肌梗死或下肢动脉缺血)，代谢综合征，糖尿病及其并发症(例如糖尿病周围神经病变)，再狭窄(例如手术后再狭窄和灌注后再狭窄)，以及神经损伤(例如神经退行性疾病(例如肌萎缩性侧索硬化(ALS)，帕金森氏病，痴呆病)，创伤性神经损伤，周围神经病变(例如糖尿病周围神经病变))；
    优选地，所述受试者为哺乳动物，例如人。
  9. 权利要求1或2的突变体或权利要求3的核酸分子或权利要求4的载体在制备药物组合物中的用途，所述药物组合物用于治疗受试者中可受益于天然hHGF的活性的疾病；
    优选地，所述疾病选自缺血性疾病(例如冠状动脉疾病(CAD)或外周动脉疾病(PAD)，例如心肌梗死或下肢动脉缺血)，代谢综合征，糖尿病及其并发症(例如糖尿病周围神经病变)，再狭窄(例如手术后再狭窄和灌注后再狭窄)，以及神经损伤(例如神经退行性疾病(例如肌萎缩性侧索硬化(ALS)，帕金森氏病，痴呆病)，创伤性神经损伤，周围神经病变(例如糖尿病周围神经病变))。
  10. 一种促进内皮细胞(例如脐静脉内皮细胞)生长和/或迁移的方法，其包括，给有此需要的内皮细胞或受试者施用有效量的权利要求1或2的突变体或权利要求3的核酸分子或权利要求4的载体或权利要求7的药物组合物。
  11. 一种促进血管发生(例如微小血管发生)的方法，其包括，给有此需要的受试者施用有效量的权利要求1或2的突变体或权利要求3的核酸分子或权利要求4的载体或权利要求7的药物组合物。
  12. 权利要求1或2的突变体或权利要求3的核酸分子或权利要求4的载体在制备药物组合物中的用途，所述药物组合物用于促进内皮细胞(例如脐静脉内皮细胞)生长和/或迁移或促进血管发生(例如微小血管发生)。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022226176A3 (en) * 2021-04-23 2022-12-01 Plantible Foods Inc. Ribulose-1,5-bisphosphate carboxylate-oxygenase (rubisco) protein isolate compositions for plant-based egg replacement products and plant-based milk replacement products

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CN109535243B (zh) 2019-09-24
EP3909974A4 (en) 2022-05-04
JP7246494B2 (ja) 2023-03-27
KR20210141453A (ko) 2021-11-23
CN109535243A (zh) 2019-03-29
JP2022522941A (ja) 2022-04-21
EP3909974A1 (en) 2021-11-17
US20220064242A1 (en) 2022-03-03
KR102667727B1 (ko) 2024-05-20

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