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WO2024119750A1 - Acide nucléique codant pour l'hgf humain et son utilisation - Google Patents

Acide nucléique codant pour l'hgf humain et son utilisation Download PDF

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Publication number
WO2024119750A1
WO2024119750A1 PCT/CN2023/099919 CN2023099919W WO2024119750A1 WO 2024119750 A1 WO2024119750 A1 WO 2024119750A1 CN 2023099919 W CN2023099919 W CN 2023099919W WO 2024119750 A1 WO2024119750 A1 WO 2024119750A1
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Prior art keywords
nucleic acid
hgf
sequence
mrna
seq
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Inventor
Jiayan Lang
Kai Wang
Andong Liu
Weijing Kong
Xue Wang
Zihang Pan
Xiaoyun Ma
Zhongqing LIN
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Beijing Jitai Pharmaceutical Technology Co Ltd
Peking University
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Beijing Jitai Pharmaceutical Technology Co Ltd
Peking University
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Publication of WO2024119750A1 publication Critical patent/WO2024119750A1/fr
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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
    • 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
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0658—Skeletal muscle cells, e.g. myocytes, myotubes, myoblasts
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • A61K48/0008—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—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 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric
    • 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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • the present invention relates to the field of biomedicines, in particular to a nucleic acid encoding human HGF and use thereof.
  • a phenomenon that the same amino acid has two or more codons is called the degeneracy of codons.
  • Synonymous codons are usually different at the third base.
  • the existence of the synonymous codons makes one protein or polypeptide sequence have many different nucleic acid coding sequences, while these different nucleic acid sequences are quite different in cell stability and protein expression efficiency. Searching for sequence design with better effect is one of the key research contents of nucleic acid medicine development.
  • Hepatocyte growth factor was originally isolated from rat plasma and platelets, and is a multifunctional cytokine. It is known that HGF is produced by interstitial cells, binds to a receptor c-Met and activates tyrosine kinase activity of the receptor, and promotes the growth, migration and morphogenesis of various types of cells such as hepatocytes, epithelial cells, endothelial cells, melanocytes and hematopoietic cells. HGF has a variety of biological activities, such as promoting the growth and/or migration of endothelial cells, promoting the occurrence of blood vessels (e.g. tiny blood vessels) , and promoting the repair of nerve damage (e.g. peripheral neuropathy, such as diabetic peripheral neuropathy) , and the like. Based on its biological activity, HGF is expected to be developed into a therapeutic medicine for HGF/c-Met related diseases.
  • HGF Hepatocyte growth factor
  • PAD Peripheral arterial disease
  • atherosclerosis which will lead to the stenosis or even occlusion of peripheral blood vessels, thus preventing blood flow to the distal of lower limbs.
  • PAD patients may ask for amputation of lower limbs, so as to relieve pain and improve their living environment.
  • angiography, stent or vascular surgery is often used to restore blood flow.
  • PAD patients are not necessarily suitable for vascular stent or vascular surgery. Therefore, the use of angiogenic factors for therapeutic angiogenesis to accelerate angiogenesis and microenvironment circulation, has been regarded as a promising PAD therapy in the last 20 years.
  • Angiogenesis refers to the growth of new capillary vessels originating from existing capillaries and postcapillary venules.
  • the growth and maturation of new vessels is a rather complicated and coordinated process.
  • the formation and development of vessels depend on the dynamic balance of angiogenesis promoting factors and angiogenesis inhibiting factors, which require the interaction between cells and cells, and between cells and matrixes, and a series of receptors are needed to be activated and regulated by a variety of angiogenesis promoting factors and angiogenesis inhibiting factors.
  • HGF hepatocyte growth factor
  • VEGF vascular endothelial growth Factor
  • BFGF basic fibroblast growth factor
  • PDGF platelet derived growth factor
  • angiogenesis promoting factors there are a plurality of medicinal forms of angiogenesis promoting factors, but all of them have obvious defects.
  • purified protein flows to an ischemic tissue through the blood system, but the immune system of an organism will generate immune responses to heterologous protein, and the injected heterologous protein will be quickly degraded by protease, which leads to poor therapeutic effect of the purified protein.
  • multiple injections of growth factors in ischemic area can lead to serious side effects.
  • the method of DNA therapy does not require surgery, but DNA is directly injected into a muscle tissue of the ischemic area, that is, in-situ therapy.
  • the application of viral vector transfection may insert the nucleic acid sequence of the virus vector into a host genome, having a certain risk in its use.
  • a first aspect of the present invention provides a nucleic acid encoding human hepatocyte growth factor (HGF) , wherein the nucleic acid comprises one or more open reading frames (ORF) , and the nucleic acid sequence of the ORF has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%sequence homology with the nucleic acid sequence selected from SEQ ID NOs: 21-31.
  • HGF human hepatocyte growth factor
  • the nucleic acid sequence of the ORF is selected from SEQ ID NOs: 21-31 or a transcribed RNA sequence thereof.
  • the nucleic acid further comprises a 5' cap.
  • the 5' cap is selected from m7G5'ppp5'Np, m7G5'ppp5'NmpNp, or m7G5'ppp5'NmpNmpNp.
  • the nucleic acid further comprises a 5'UTR.
  • the 5' UTR is selected from one of SEQ ID NOs: 1-8 or a combination thereof.
  • the nucleic acid further comprises a 3' UTR.
  • the 3' UTR is selected from one of SEQ ID NOs: 9-17 or a combination thereof.
  • the nucleic acid further comprises a poly-A sequence, and the poly-A sequence comprises 70-150 nucleotides.
  • the poly-A sequence comprises a sequence selected from SEQ ID NO: 18 or SEQ ID NO: 19.
  • the nucleic acid further comprises a sequence selected from SEQ ID NOs: 32, 33, 34, 35, 56, 57, 58, 60, 61, 63 and 65, or a transcribed RNA sequence thereof.
  • the nucleic acid further comprises at least one modified nucleoside selected from pseudouridine, N1-methyl-pseudouridine, or 5-methylcytidine.
  • the nucleic acid is DNA or mRNA. More preferably, the mRNA is an mRNA transcribed from a DNA encoding human HGF, the DNA comprises one or more open reading frames (ORF) , and a nucleic acid sequence of the ORF has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%sequence homology with a nucleic acid sequence selected from SEQ ID NOs: 21-31.
  • ORF open reading frames
  • the nucleic acid has a protein expression level which is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%higher than that of a natural sequence.
  • a second aspect of the present invention provides a nucleic acid construct, which comprises the nucleic acid of the present invention.
  • a third aspect of the present invention provides a vector, which comprises the nucleic acid or the nucleic acid construct of the present invention.
  • a fourth aspect of the present invention provides a cell, which comprises the nucleic acid, the nucleic acid construct, or the vector of the present invention.
  • a fifth aspect of the present invention provides a pharmaceutical composition, which comprises the nucleic acid, the nucleic acid construct, the vector, or the cell of the present invention.
  • a sixth aspect of the present invention provides use of the nucleic acid, the nucleic acid construct, the vector, the cell, or the pharmaceutical composition in the preparation of a medicine for preventing or treating a disease.
  • the disease is an ischemic disease or a disease with insufficient HGF expression.
  • a seventh aspect of the present invention provides a method for preventing or treating an ischemic disease or a disease with insufficient HGF expression, which comprises administering an effective amount of the nucleic acid, the nucleic acid construct, the vector, the cell or the pharmaceutical composition to a subject in need.
  • An eighth aspect of the present invention provides the nucleic acid, the nucleic acid construct, the vector, the cell or the pharmaceutical composition of the present invention for use as a medicament.
  • a ninth aspect of the present invention provides the nucleic acid, the nucleic acid construct, the vector, the cell, or the pharmaceutical composition of the present invention for use in a method for treating an ischemic disease or a method for treating a disease with insufficient HGF expression.
  • a tenth aspect of the present invention provides a method for expressing a polypeptide in a mammal, which comprises the step of contacting a mammalian cell with the nucleic acid, the nucleic acid construct, the vector, the cell, or the pharmaceutical composition of the present invention.
  • the nucleic acid, the nucleic acid construct, the vector, the cell, or the pharmaceutical composition comprises lipid nanoparticles for delivering mRNA.
  • the nucleic acid, the nucleic acid construct, the vector, the cell, or the pharmaceutical composition is an injection or topical preparation, such as an intramuscular injection preparation.
  • the ischemic disease is peripheral arterial disease.
  • the peripheral arterial disease is critical limb ischemia.
  • the peripheral arterial disease is lower limb ischemia related disease, such as lower limb ischemia caused by ulcer, intermittent claudication, lower limb pain and diabetes.
  • the present invention has the following beneficial effects.
  • the nucleic acid encoding human HGF provided by the present invention has lower predicted minimum folding energy (MFE) than that of the natural sequence, and the molecule is more stable;
  • nucleic acid provided by the present invention and the nucleic acid construct, the vector, the cell, or the pharmaceutical composition comprising the nucleic acid has a protein expression level superior to that of the natural sequence;
  • nucleic acid encoding human HGF provided by the present invention and the nucleic acid construct, the vector, the cell, or the pharmaceutical composition comprising the nucleic acid may activate HGF/c-met pathway and promote angiogenesis;
  • nucleic acid encoding human HGF provided by the present invention and the nucleic acid construct, the vector, the cell, or the pharmaceutical composition comprising the nucleic acid, are especially suitable for the treatment of the peripheral ischemic disease (such as critical limb ischemia) or the disease with insufficient HGF expression, and extremely safe and effective results can be obtained by directly administering the nucleic acid to ischemic sites or individuals with insufficient HGF expression.
  • peripheral ischemic disease such as critical limb ischemia
  • FIG. 1 is an agarose gel electrophoretogram of the in-vitro transcription product mRNA.
  • FIG. 2 is a graph showing the expression amount of luciferase mRNA in HSMC cells after co-transfection of the luciferase mRNA and HGF mRNA.
  • FIG. 3 is a graph showing the absolute amount of expression of the HGF mRNA in HSMC cells after co-transfection of the luciferase mRNA and the HGF mRNA.
  • FIG. 4 is a graph showing the relative amount of expression of the HGF mRNA in HSMC cells after co-transfection of the luciferase mRNA and the HGF mRNA.
  • FIG. 5 is a graph showing the expression amount of HGF over time in HSMC cells after HGF mRNA transfection.
  • FIG. 6 is a graph showing the cumulative amount of expression of the HGF over time in HSMC cells after the HGF mRNA transfection.
  • FIG. 7 shows the result of photographing A549 cells after 48 hours of wound closure assay.
  • FIG. 8 shows the wound closure degree of the A549 cells after 48 hours of wound closure assay.
  • FIG. 9 shows the flow cytometry detection results of FACS assay of HGF/c-met pathway in A549 cells.
  • FIG. 10 is a quantitative graph showing the activation degree of p-c-met in the FACS test of the HGF/c-met pathway in the A549 cells.
  • FIG. 11 shows the western blot results of the HGF/c-met pathway in A549 cells.
  • FIG. 12 shows the results of tube formation assay of HUVEC cells.
  • FIG. 13 shows the quantitative statistical results of the tube formation assay of the HUVEC cells.
  • FIG. 14 is an expression-time curve of HGF in gastrocnemius muscles of mice.
  • FIG. 15 is a graph showing expression amounts of HGF in gastrocnemius muscles of mice at different time points.
  • FIG. 16 shows the immunohistochemical staining picture of CD31 in rabbit muscle tissues.
  • a nucleic acid encoding human HGF comprises one or more open reading frames (ORF) , and the nucleic acid sequence of the ORF has at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%or 100%sequence homology with the nucleic acid sequence selected from SEQ ID NOs: 21-31.
  • the nucleic acid has a protein expression level superior to that of the natural sequence.
  • nucleic acid sequence of the ORF is selected from SEQ ID NOs: 21-31 or a transcribed RNA sequence thereof.
  • nucleic acid further comprises a 5' cap.
  • the 5' cap is selected from m7G5'ppp5'Np, m7G5'ppp5'NmpNp, or m7G5'ppp5'NmpNmpNp.
  • nucleic acid further comprises a 5' UTR.
  • the 5' UTR is selected from one of SEQ ID NOs: 1-8 or a combination thereof.
  • nucleic acid further comprises a 3' UTR.
  • the 3' UTR is selected from one of SEQ ID NOs: 9-17 or a combination thereof.
  • the nucleic acid further comprises a poly-A sequence
  • the poly-A sequence comprises 100-250 nucleotides, preferably 100-150 nucleotides, and more preferably 70-150 nucleotides.
  • poly-A sequence comprises a sequence selected from SEQ ID NO: 18 or SEQ ID NO: 19.
  • nucleic acid further comprises a sequence selected from SEQ ID NOs: 32, 33, 34, 35, 56, 57, 58, 60, 61, 63 and 65, or a transcribed RNA sequence thereof.
  • nucleic acid further comprises at least one modified nucleoside selected from pseudouridine, N1-methyl-pseudouridine, or 5-methylcytidine.
  • nucleic acid is selected from DNA or mRNA.
  • the nucleic acid has a protein expression level which is at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%higher than that of a natural sequence.
  • the present invention provides a nucleic acid construct comprising the nucleic acid.
  • the present invention provides a vector comprising the nucleic acid or the nucleic acid construct.
  • the present invention provides a cell comprising the nucleic acid, the nucleic acid construct, or the vector.
  • the present invention provides a pharmaceutical composition comprising the nucleic acid, the nucleic acid construct, the vector, or the cell.
  • the present invention provides a method for expressing a polypeptide in a mammal, which comprises the step of contacting a cell with the nucleic acid or the pharmaceutical composition in vivo or in vitro.
  • the present invention provides use of the nucleic acid, or the construct, the vector, the cell, or the pharmaceutical composition comprising the nucleic acid in treating a HGF/c-Met related disease and use in the preparation of a medicine for treating the HGF/c-Met related disease.
  • the HGF/c-Met related disease is an ischemic disease.
  • the ischemic disease is peripheral arterial disease.
  • the peripheral arterial disease is critical limb ischemia (CLI) .
  • CLI critical limb ischemia
  • polypeptide or"protein refers to a polymer in which amino acids are linked by peptide bonds.
  • Amino acids are selected from 20 natural amino acids or other unnatural amino acids.
  • the 20 natural amino acids refer to glycine, alanine, valine, leucine, isoleucine, methionine, proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.
  • Nucleotide is a compound composed of purine base or pyrimidine base, ribose or deoxyribose and phosphoric acid.
  • Nucleic acid refers to a polymer in which nucleotides are linked by 3', 5'-phosphodiester bonds.
  • the nucleic acid refers to single-stranded or double-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules and hybrid molecules thereof.
  • Examples of the nucleic acid molecules comprise, but are not limited to, messenger RNA (mRNA) , microRNA (miRNA) , small interfering RNA (siRNA) , self-amplified RNA (saRNA) and antisense oligonucleotide (ASO) .
  • mRNA messenger RNA
  • miRNA microRNA
  • siRNA small interfering RNA
  • saRNA self-amplified RNA
  • ASO antisense oligonucleotide
  • the nucleic acid is mRNA.
  • the nucleic acid may further be chemically modified.
  • the chemical modification of mRNA is selected from one or more of pseudouridine, N1-methyl-pseudouridine, 5-methoxyuridine, 5-methylcytosine, or the combinations thereof.
  • the open reading frame refers to a base sequence between an initiation codon and a terminator codon, which encodes a protein.
  • the mRNA molecule comprises ORF (s) , and optionally further comprises expression regulatory sequence (s) .
  • Typical expression regulatory sequences comprise, but are not limited to, 5' cap, 5' untranslated region (5' UTR) , 3' untranslated region (3' UTR) , polyadenylation sequence (polyA) and miRNA binding site.
  • the 5' cap of the mRNA is linked to the 5' end of the mRNA by a guanylic acid through a 5'-5' triphosphate bond.
  • the 5' guanylic acid may be further modified, such as methylation, to produce N7-methyl-guanylic acid residue.
  • the 1 st nucleotide or 2 nd nucleotide at the 5' end of the mRNA may be further modified, for example, the ribose part is subjected to 2'-O-methylation.
  • Examples of the 5' cap comprise, but are not limited to m7G5'ppp5'Np (O type) , m7G5'ppp5'NmpNp (I type) , and m7G5'ppp5'NmpNmpNp (II type) .
  • the 5'-end cap structure of the mRNA provides signals for ribosomes to recognize the mRNA and helps the ribosomes to bind to the mRNA.
  • the cap structure can increase stability of the mRNA and protect the mRNA from degradation by 5' ⁇ 3' exonuclease. In some embodiments, the cap is not present.
  • the untranslated region can be transcribed but not translated.
  • the 5' UTR comprises a sequence from the transcription initiation site to the initiation codon, but does not comprise the initiation codon.
  • the 3' UTR comprises a sequence from the terminator codon to the transcription termination signal, but does not comprise the terminator codon. Examples of the UTR comprise, but are not limited to the sequences shown in Table 1.
  • Polyadenylic acid protects the mRNA from degradation by 3' ⁇ 5' exonuclease, and increases the stability of the mRNA itself.
  • the length of the poly-A is 100-250 nucleotides, preferably 100-150 nucleotides, more preferably 70-150 nucleotides. Examples of the poly-Acomprise, but are not limited to the sequences shown in Table 2.
  • the miRNA can recognize and bind to miRNA binding sites of nucleic acid molecules, reduce stability of the nucleic acid molecules or inhibit the translation process, and further down-regulate the gene expression level. Removing the miRNA binding sites from a natural nucleic acid sequence can increase protein expression; or, one or more miRNA binding sites are added to the nucleic acid sequence to reduce protein expression.
  • the nucleic acid molecule of the present invention is added with a miR-122 binding site, thereby inhibiting the expression of the target gene in liver.
  • composition refers to any product containing specified amounts of each specified ingredient.
  • “Pharmaceutical composition” refers to a composition comprising active components, which may further comprise pharmaceutically acceptable adjuvants and other optional therapeutic components.
  • the pharmaceutical composition of the present invention comprises pharmaceutical compositions suitable for oral, rectal, topical and parenteral administration (comprising subcutaneous administration, intramuscular injection and intravenous administration) .
  • the pharmaceutical composition of the present invention can be conveniently prepared in a unit dosage form known in the art and by any preparation method known in the pharmaceutical field.
  • Treating HGF/c-Met related disease comprises but is not limited to (1) promoting growth and/or migration of endothelial cells; (2) promoting vasculogenesis (such as microvessel) ; (3) treating ischemic diseases, such as coronary vascular diseases or peripheral arterial diseases (such as critical limb ischemia) ; (4) treating metabolic syndrome and diabetes and complications thereof (for example, diabetic peripheral neuropathy) ; (5) inhibiting restenosis; and (6) promoting repair of nerve injury (for example, neurodegenerative diseases, traumatic nerve injury, and peripheral neuropathy) .
  • vasculogenesis such as microvessel
  • treating ischemic diseases such as coronary vascular diseases or peripheral arterial diseases (such as critical limb ischemia)
  • metabolic syndrome and diabetes and complications thereof for example, diabetic peripheral neuropathy
  • nerve injury for example, neurodegenerative diseases, traumatic nerve injury, and peripheral neuropathy
  • Lipid (s) refer to a group of organic compounds comprising but not limited to esters of fatty acids, and are characterized by being insoluble in water but soluble in many organic solvents.
  • the lipids are usually divided into at least three categories: (1) “simple lipid (s) " , which comprise fats and oils and waxes; (2) “compound lipid (s) “ , which comprise phospholipids and glycolipids; and (3) “derived lipid (s) " such as steroids.
  • Lipid nanoparticle means particle made of lipids (e.g., cationic lipids, ionizable lipids, conjugated lipids that prevent particle aggregation, neutral lipids, cholesterol, and the like) and nucleic acids, wherein the nucleic acids are encapsulated in the lipids.
  • the nucleic acid is resistant to degradation using nuclease in an aqueous solution.
  • ORF sequences SEQ ID NOs: 20-31 and SEQ ID NOs: 36-45 encoding the same human HGF natural protein were synthesized, wherein SEQ ID NO: 20 was a natural nucleic acid sequence (NM_000601.6) and the others were artificially designed nucleic acid sequences.
  • DNA plasmids comprising the ORF sequences and upstream and downstream regulatory sequences on both sides of the ORF sequences were constructed, amplified and extracted by a conventional molecular cloning method. Examples of 5' UTR, 3' UTR and polyA in the upstream and downstream regulatory sequences comprised but were not limited to the sequences shown in Table 1 and Table 2.
  • DNA plasmids containing SEQ ID NOs: 32-35 and SEQ ID NOs: 46-65 respectively encode mRNA products HGF-1 to HGF-24, and the corresponding relationships were shown in Table 3.
  • mRNAs were obtained by transcription in vitro.
  • the in-vitro transcription product mRNAs were detected by agarose gel electrophoresis, showing bright single bands without tailing and good integrity (FIG. 1) .
  • a typical preparation process was as follows.
  • the reagents were evenly mixed by vortex, instantaneous centrifuged by a centrifuge for 10 s, and reacted in a metal bath at 37°C for 3 h. If the amount of plasmid DNA added changed, the amounts of other additives were adjusted accordingly.
  • NanoDrop (Thermo Fisher) was used to detect the concentration of DNA, and then the DNA was stored in a refrigerator at-20°C.
  • a novoprotein in-vitro transcription kit was used for in-vitro transcription, wherein the item number was: E131-01A.
  • RNA Clean Beads were taken out from 2-8°C in advance for 30 minutes, equilibrate to room temperature, and then, magnetic beads were mixed thoroughly by inverting or vortex oscillation.
  • NanoDrop was used to detect the mRNA concentration and OD260/280 value, and then, the mRNA was marked and stored in a refrigerator at-80°C.
  • HSMC Human skeletal muscle cells with good growth status were selected, digested with trypsin at 37°C for 2 minutes, then added with HSMC complete medium (Gibco) to stop the digestion, centrifuged at 1,000 rpm for 5 minutes, and the cells were resuspended by using the HSMC complete medium, the cells were adjusted to a density of 1 ⁇ 10 5 cells/mL, and inoculated into a 48-well cell culture plate with 200 ⁇ L/well.
  • HSMC complete medium Gibco
  • RNA 100 ng of HGF mRNA and 100 ng of luciferase mRNA were taken from each well into a centrifuge tube, added into 20 ⁇ L Opti-METM (GIBCO) medium, and mixed sufficiently.
  • 0.6 ⁇ L of lipofectamine 2000 (Thermo Fisher) transfection reagent was taken from each well, and 20 ⁇ L of Opti-MEM medium was added, and mixed sufficiently.
  • the mRNA diluent was added into the Lip 2000 diluent, mixed sufficiently, and stood at room temperature for 10 minutes.
  • Opti-MEM medium 160 ⁇ L of Opti-MEM medium was added into the transfection complex and mixed sufficiently, and the medium in the original cell culture plate was discarded, and then 200 ⁇ L of the prepared transfection complex mixture was added into each well.
  • the supernatant in the cell culture plate was collected and the HGF expression amount was detected.
  • the luciferase expression amount was detected by a luciferase reporter gene detection system (Promega, item number E1501) .
  • the amount required for the test was calculated in advance, and a 5-fold cell lysate was diluted with deionized water into 1-fold application working solution.
  • a luciferase detection buffer was taken out from-20°C, and after being completely thawed, added into a luciferase detection substrate and dissolved completely to obtain the luciferase detection solution.
  • the detection results showed that there was no significant difference in the luciferase expression amount of the cells after the luciferase mRNA was transfected to the HSMC cells (FIG. 2) . Therefore, the luciferase could be used as an internal reference to calibrate the HGF expression amount.
  • HGF detection kit (Solarbio, SEKH-0201) was placed at room temperature for rewarming.
  • the volume of the diluted washing solution was calculated, and a 20 ⁇ concentrated washing solution was diluted into a 1 ⁇ application solution with deionized water.
  • SR2 detection diluent
  • step (3) The plate washing step in step (3) was repeated.
  • step (3) The plate washing step in step (3) was repeated, and the plate was washed for five times.
  • the detection results showed that after different HGF mRNAs were transfected into HSMC cells, the HGF expression amounts in the culture medium supernatants increased in different degrees (FIG. 3) , and the HGF concentration values were 200-1,040 ng/mL, wherein the HGF-23 mRNA sample had the highest HGF content, and the secretion amount of HGF in the cell culture supernatant was 1, 040 ng/mL.
  • the amount of mRNA added, the number and status of the cells, and the transfection efficiency will all affect the expression of HGF.
  • the expression of the luciferase mRNA was selected as an internal reference, and the RLU value of the HGF-10 sample was taken as one unit to calibrate the expression amount of HGF to obtain the relative amount of expression of HGF, and the calculation formula of each sample was as follows:
  • HGFn represented the expression amount of HGF (ng/ml) corresponding to the n th HGF sequence sample
  • RLUn represented the bioluminescence intensity corresponding to the n th HGF sequence sample, representing the expression amount of the luciferase.
  • HGF-7 the nucleic acid sequence of the obtained construct was SEQ ID NO: 32
  • HGF-10 the nucleic acid sequence of the obtained construct was SEQ ID NO: 33
  • HGF-15 the nucleic acid sequence of the obtained construct was SEQ ID NO: 34
  • HGF-23 the nucleic acid sequence of the obtained construct was SEQ ID NO: 35
  • nucleotide sequences of the four optimal nucleic acid constructs were as follows:
  • HSMC Human skeletal muscle cells
  • step (3) The transfection complex obtained in step (2) was evenly dripped into an plate.
  • the collected supernatants were determined by HGF ELISA, and the contents of HGF in CM were as shown in FIG. 5 and FIG. 6.
  • Cumulative amount of 12 h (amount of 4-6 h) + (amount of 6-12 h)
  • Cumulative amount of 24 h (amount of 4-6 h) + (amount of 6-12 h) + (amount of 12-24 h)
  • FIG. 5 showed that the secretion amount of HGF protein from the 24th to 48th hours (24-hour interval) was the highest, which was 55.5 ⁇ 1.1 ng/ml. From the 48th to 72th hours (24-hour interval) , the secretion amount decreased significantly, which was 26.5 ⁇ 2.4 ng/ml.
  • FIG. 6 showed that the cumulative amount of HGF protein reached 305.3 ⁇ 17 ng after 72 hours of transfection.
  • HGF mRNA was transfected into 293T cells with Lipofectamine 2000.
  • HGF began to express after 6 hours of transfection, at which time the medium was replaced with DMEM high-sugar culture medium and cultured for 24 hours, then the culture solution was pipetted and centrifuged, and the supernatant was collected.
  • A549 cells (adenocarcinoma human alveolar basal epithelial cells) were resuscitated and subcultured, and the medium used in the subculture was DMEM high-sugar medium+10%fetal bovine serum culture solution (hereinafter referred to as D10) .
  • D10 DMEM high-sugar medium+10%fetal bovine serum culture solution
  • A549 cells were inoculated into a 24-well plate with an inoculation density of 40,000 cells/well.
  • A549 cells were cultured at 37°Cand 5%CO 2 to adhere to the wall, and then the medium was replaced with DMEM high-sugar medium (serum-free medium, hereinafter referred to as D0) for 24 hours for later use.
  • the positive control D10 group, the negative control D0 group and the sample group (HGF-CM: 200 ng/mL HGF in CM, HGF Factor: 200 ng/mL HGF recombinant protein, Inhibitor: 20 nM Capmatinib) all had three repeats.
  • FIG. 7 and FIG. 8 showed that HGF-CM could significantly promote the migration of A549 cells.
  • A549 cells were inoculated in a 24-well plate with an inoculation density of 40,000 cells/well. After the inoculation, the A549 cells were cultured at 37°Cand 5%CO 2 to adhere to the wall, and then the medium was replaced with DMEM high-sugar medium for 24 hours for later use.
  • the negative control D0 group and the sample group (HGF-CM: 100 ng/mL HGF in CM, HGF Factor: 100 ng/mL HGF recombinant protein, Inhibitor: 20 nM Capmatinib) all had three repeats.
  • Target antibodies (c-met and p-c-met) were diluted at 1: 100, fully mixed with the cells, and incubated at 4°Cfor 15 minutes to allow the cells fully react with the antibodies.
  • Second antibody incubation an appropriate amount of fluorescent labeled second antibodies was added, fully mixed with the cells, and incubated at 4°Cfor 15 minutes to allow the cells fully react with the antibodies.
  • FIG. 9 Results of the flow cytometry detection were shown in FIG. 9, and the quantitative activation degree of p-c-met was shown in FIG. 10.
  • FIG. 9 and FIG. 10 showed that HGF-CM could significantly promote the activation of HGF/c-met in A549 cells.
  • the cell treatment was the same as the steps shown in 3.1. After a cell suspension was obtained, protein cleavage, protein denaturation, gel preparation, loading, electrophoresis, membrane transfer, rinsing, sealing, primary antibody incubation, secondary antibody incubation and exposure and developing were carried out. The results were shown in FIG. 11. The results showed that HGF-CM could significantly promote the activation of HGF/c-met pathway in A549 cells.
  • HUVEC cells were resuscitated and sub-cultured.
  • the medium used in the subculture was an Advanced DMEM/F12 medium containing 2%FBS, 1%P/S, 30 ⁇ g/mL vitamin C, 5 ng/mL EGF, 10 ng/mL FGF2, 2.5 ng/mL VEGFA and 25 ⁇ g/mL heparin (hereinafter referred to as EGM2 culture solution) .
  • EGM2 culture solution an Advanced DMEM/F12 medium containing 2%FBS, 1%P/S, 30 ⁇ g/mL vitamin C, 5 ng/mL EGF, 10 ng/mL FGF2, 2.5 ng/mL VEGFA and 25 ⁇ g/mL heparin.
  • EGM2 culture solution an Advanced DMEM/F12 medium containing 2%FBS, 1%P/S, 30 ⁇ g/mL vitamin C, 5 ng/mL EGF, 10 ng/mL FGF2, 2.5 ng/mL VEG
  • Matrigel was placed in an ice box and put in a refrigerator at 4°Covernight. Matrigel was added into a 96-well plate at a volume of 50 ⁇ L/well, and then placed in an incubator for 30 minutes until the Matrigel was solidified.
  • the cells were re-suspended in the above three media respectively and the cell densities were adjusted to 8 ⁇ 10 4 cells/mL.
  • the Matrigel was solidified, the 96-well plate was taken out, added with 100 ⁇ L of cell suspension into each well, the plate was marked, and put into a 5%CO 2 cell incubator at 37°Cfor 4-6 hours.
  • FIG. 12 showed the results of the tube formation assay of HUVEC cells.
  • FIG. 13 showed the quantitative statistical results of the tube formation assay of HUVEC cells.
  • FIG. 12 and FIG. 13 showed that the HGF mRNA of the present invention could express HGF protein in the transfected cells, and the HGF secreted into the supernatant could significantly promote the angiogenesis of the HUVEC cells.
  • the preparation methods of the lipid nanoparticles comprised microfluidic mixing, but were not limited to
  • N/P represented the ratio of the ionizable lipid (i.e. Lipid 5) to the active agent (i.e. HGF mRNA) , N represented cationic amine, and P represented anionic phosphoric acid.
  • mice Balb/c male mice, 8-10 weeks old, and three mice in each group.
  • the gastrocnemius was intramuscularly injected with a single dose of 0.5 mg/kg HGF mRNA LNP (mRNA equivalent) , and the total injection volume was 50 ⁇ L.
  • the control group was injected with Empty LNP.
  • the HGF protein expression level in the muscle tissue of the injected part was detected by ELISA. Specific steps were shown in Example 5.
  • mice After 6 hours of administration, the mice were sacrificed, the gastrocnemius muscles at the injected parts were separated, washed with pre-cooled PBS, and the PBS was blotted. The muscles were put in a 1.5 ⁇ L EP tube and frozen in a refrigerator at-80°C.
  • FIG. 14 was an expression-time curve of HGF in gastrocnemius muscles of mice.
  • FIG. 15 was a graph showing expression quantities of HGF in gastrocnemius muscles of mice at different time points. The results showed that HGF of the present invention could be well expressed in the mouse muscle, and the HGF protein expression could be detected at all time points.
  • the expression quantity of the HGF mRNA LNP group at 24 hours was 24.7 ng/mg.
  • the hHGF mRNA/LNP prepared as in Example 8 was prepared with DPBS to the required concentration immediately before use.
  • New Zealand male white rabbits were provided by Beijing Tonghe Litai Company.
  • W-B Pyun et al. naked DNA expressing two isoforms of Hepatocyte growth factor induces collateral artery augmentation in a rabbit model of limb ischemia. Gene Therapy (2010) 17, 1442–1452) , a rabbit model of lower extremity arterial ischemia was surgically established. After intramuscular injection of 5 mg/kg dose of Xylazine Hydrochloride, the rabbit was anesthetized with 50 mg/kg dose of Ketamine. The skin of the left inner thigh was disinfected with alcohol and iodine.
  • an arterial cannula was inserted into the right carotid artery, a2.7F catheter (Terumo, Japan) was inserted into the entrance of the left internal iliac artery, and 5 ml of contrast medium was injected at a rate of 1 ml per second, selective internal iliac arteriography was performed to confirm the establishment of the lower extremity ischemia model.
  • the animals were randomly divided into solvent control group (8 animals) , hHGF mRNA 5 ⁇ g/kg low-dose group (8 animals) , and hHGF mRNA 25 ⁇ g/kg high-dose group (8 animals) .
  • the lower limb ischemic muscle tissue (adductor muscle and semimembranosus) was removed, put into formalin solution, fixed for 24 hours, and then paraffin-embedded and sectioned. CD31 immunohistochemical detection was performed on each section. Observe the new blood vessels under the microscope ( ⁇ 40) .
  • Table 9 shows the assay results of the number of collateral vessels at the ischemic site and the number of new collateral vessels at the ischemic site before and after administration of the experimental animals in each group.

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Abstract

L'invention concerne un acide nucléique codant pour l'HGF humain et son utilisation. L'acide nucléique comprend un ou plusieurs cadres de lecture ouverts ORF, et la séquence d'acide nucléique de l'ORF a au moins 80 % d'homologie de séquence avec la séquence d'acide nucléique choisie parmi SEQ ID NO : 21-31. L'acide nucléique fourni est plus stable qu'une séquence naturelle et a un niveau d'expression de protéine supérieur à celui de la séquence naturelle.
PCT/CN2023/099919 2022-12-06 2023-06-13 Acide nucléique codant pour l'hgf humain et son utilisation Ceased WO2024119750A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007058776A2 (fr) * 2005-11-10 2007-05-24 Receptor Biologix, Inc. Proteines de fusion a intron du facteur de croissance des hepatocytes
WO2018104538A1 (fr) * 2016-12-08 2018-06-14 Curevac Ag Arn pour le traitement ou la prophylaxie d'une maladie du foie
WO2018104540A1 (fr) * 2016-12-08 2018-06-14 Curevac Ag Arn pour la cicatrisation des plaies
WO2020084161A1 (fr) * 2018-10-26 2020-04-30 Vrije Universiteit Brussel Nouveaux outils pour améliorer la thérapie génique, et utilisation correspondante

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007058776A2 (fr) * 2005-11-10 2007-05-24 Receptor Biologix, Inc. Proteines de fusion a intron du facteur de croissance des hepatocytes
WO2018104538A1 (fr) * 2016-12-08 2018-06-14 Curevac Ag Arn pour le traitement ou la prophylaxie d'une maladie du foie
WO2018104540A1 (fr) * 2016-12-08 2018-06-14 Curevac Ag Arn pour la cicatrisation des plaies
WO2020084161A1 (fr) * 2018-10-26 2020-04-30 Vrije Universiteit Brussel Nouveaux outils pour améliorer la thérapie génique, et utilisation correspondante

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE Nucleotide 9 October 2022 (2022-10-09), ANONYMOUS: "Homo sapiens hepatocyte growth factor (HGF), transcript variant 1, mRNA", XP093180209, retrieved from NCBI Database accession no. NM_000601.6 *
MAKAREVICH PAVEL, TSOKOLAEVA ZOYA, SHEVELEV ALEXANDER, RYBALKIN IGOR, SHEVCHENKO EVGENY, BELOGLAZOVA IRINA, VLASIK TATYANA, TKACHU: "Combined Transfer of Human VEGF165 and HGF Genes Renders Potent Angiogenic Effect in Ischemic Skeletal Muscle", PLOS ONE, vol. 7, no. 6, 13 June 2012 (2012-06-13), pages e38776, XP055826391, DOI: 10.1371/journal.pone.0038776 *

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