阅读说明：本技术 突变的肝细胞生长因子基因及其应用 (Mutant hepatocyte growth factor gene and application thereof ) 是由 李树民 于 2019-10-12 设计创作，主要内容包括：本申请涉及肝细胞生长因子(Hepatocyte growth factor,HGF)基因的突变的内含子4或其片段。本申请还涉及含有所述突变的内含子4或其片段的编码HGF蛋白的核酸分子,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核酸分子的药物组合物,以及所述药物组合物的用途。(The present application relates to a mutated intron 4 of the Hepatocyte Growth Factor (HGF) gene or a fragment thereof. The application also relates to a nucleic acid molecule containing the mutated intron 4 or the fragment thereof, which encodes the HGF protein, a vector containing the nucleic acid molecule, and a host cell containing the nucleic acid molecule or the vector. The application also relates to pharmaceutical compositions comprising said nucleic acid molecules, and to uses of said pharmaceutical compositions.)

1. a mutant Intron 4 of a Hepatocyte Growth Factor (HGF) gene, or a fragment thereof, wherein said mutant Intron 4 comprises a mutation at: corresponding to SEQ ID NO:1 at position 3815, 4774, and 4876; and, said fragment comprises the amino acid sequence of intron 4 of said mutation corresponding to SEQ ID NO:1 from position 1 to 246 and 3686 to 4926;

Preferably, said fragment further comprises a sequence corresponding to SEQ ID NO:1 from nucleotide 2686 to nucleotide 3685;

Preferably, the fragment further comprises, a linker sequence for linking the nucleotide fragments; preferably, the length of the linker sequence is 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, or 200-500 nucleotides; for example, the linker sequence is shown in SEQ ID NO 13.

2. The mutated intron 4 or fragment thereof of claim 1, wherein the fragment comprises or alternatively consists of:

(1) The mutant intron 4 has a sequence corresponding to SEQ ID NO:1, the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1 from 3686 to 4926 and optionally an adaptor sequence located between the two nucleotide fragments;

(2) The mutant intron 4 has a sequence corresponding to SEQ ID NO:1, and the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1 from 3686 to 4926, wherein the 3 'end of the first nucleotide fragment is directly linked to the 5' end of the second nucleotide fragment;

(3) the mutant intron 4 has a sequence corresponding to SEQ ID NO:1, and the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1 from 3686 to 4926, wherein the 3 'end of the first nucleotide fragment is linked to the 5' end of the second nucleotide fragment by an adaptor sequence;

(4) The mutant intron 4 has a sequence corresponding to SEQ ID NO:1, the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1 from position 2686 to 4926 and optionally a linker sequence between said two nucleotide fragments;

(5) The mutant intron 4 has a sequence corresponding to SEQ ID NO:1, and the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1 from position 2686 to 4926, wherein the 3 'end of the first nucleotide fragment is directly linked to the 5' end of the second nucleotide fragment; or

(6) The mutant intron 4 has a sequence corresponding to SEQ ID NO:1, and the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1, from position 2686 to 4926, wherein the 3 'end of the first nucleotide fragment is linked to the 5' end of the second nucleotide fragment by an adaptor sequence.

3. The mutated intron 4 or fragment thereof of claim 1 or 2, wherein the mutated intron 4 comprises a mutation selected from the group consisting of: in a nucleic acid sequence corresponding to SEQ ID NO:1 the nucleotide at position 3815 is mutated to an adenine nucleotide; in a nucleic acid sequence corresponding to SEQ ID NO:1 is mutated to a guanine nucleotide at the position 4774; in a nucleic acid corresponding to SEQ ID NO:1 at position 4876 is mutated to a guanine nucleotide; and, any combination thereof;

preferably, the mutated intron 4 comprises the following mutations: in a nucleic acid sequence corresponding to SEQ ID NO:1 the nucleotide at position 3815 is mutated to an adenine nucleotide; in a nucleic acid sequence corresponding to SEQ ID NO:1 is mutated to a guanine nucleotide at the position 4774; and, where the amino acid sequence corresponds to SEQ ID NO:1 at position 4876 is mutated to a guanine nucleotide.

4. The mutant intron 4 or fragment thereof of any of claims 1-3, wherein the hepatocyte growth factor is human hepatocyte growth factor;

Preferably, the human hepatocyte growth factor has the amino acid sequence as shown in SEQ ID NO: 12;

Preferably, the human hepatocyte growth factor gene has the following accession numbers as GenBank database: a nucleotide sequence set forth in NC _ 000007.14;

Preferably, the mutated intron 4 has the amino acid sequence as shown in SEQ ID NO: 9, or a fragment thereof having a nucleotide sequence selected from the group consisting of SEQ ID NOs: 10 and SEQ ID NO: 11.

5. A nucleic acid molecule encoding Hepatocyte Growth Factor (HGF) comprising exons 1-18 of an HGF gene, and mutated intron 4 of any one of claims 1-4, or a fragment thereof, located between exons 4 and 5;

Preferably, the hepatocyte growth factor is human hepatocyte growth factor;

preferably, the human hepatocyte growth factor has the amino acid sequence as shown in SEQ ID NO: 12;

preferably, exons 1-18 encode a polypeptide as set forth in SEQ ID NO: 12;

Preferably, the human hepatocyte growth factor gene has the following accession numbers as GenBank database: a nucleotide sequence set forth in NC _ 000007.14;

Preferably, the nucleic acid molecule has an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

6. A vector comprising a mutated intron 4 according to any one of claims 1 to 4 or a fragment thereof; preferably, the vector is used to clone the mutated intron 4 or a fragment thereof.

7. A vector comprising the nucleic acid molecule of claim 5;

Preferably, the vector is selected from the group consisting of plasmids; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophages such as lambda bacteriophage or M13 bacteriophage; and, viral vectors, such as retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors (e.g., herpes simplex viral vectors), poxvirus vectors, baculovirus vectors, papilloma viral vectors, papilloma polyomavirus vectors;

preferably, the vector is for expressing (e.g., expressing in a subject (e.g., a mammal, e.g., a human)) the HGF protein;

Preferably, the vector is a vector for gene therapy, such as a plasmid, an adenovirus vector, an adeno-associated virus vector, and a lentivirus vector;

Preferably, the vector has a nucleotide sequence selected from SEQ ID NO 6, SEQ ID NO 7 or SEQ ID NO 8.

8. A host cell comprising the nucleic acid molecule of claim 5 or the vector of claim 6 or 7;

preferably, the host cell is selected from prokaryotic cells, such as e.coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.);

preferably, the host cell is an E.coli cell, such as E.coli DH5 alpha cell; alternatively, the host cell is a 293T cell or a human cell.

9. A method of expressing or producing HGF protein comprising using a mutated intron 4 or fragment thereof according to any of claims 1-4;

preferably, the method comprises the use of a nucleic acid molecule according to claim 5 or a vector according to claim 7;

Preferably, the method comprises expressing the nucleic acid molecule according to claim 5 or the vector according to claim 7 in a host cell under conditions that allow the expression of the protein; and optionally, recovering the HGF protein expressed in the host cell.

10. Use of mutant intron 4 or a fragment thereof according to any of claims 1 to 4 for increasing the expression level of an HGF protein;

For example, said mutated intron 4 or fragment thereof is used to increase the expression level of HGF protein in vitro;

For example, the mutant intron 4 or fragment thereof is used to increase the expression level of HGF protein in a cell;

For example, the mutant intron 4 or fragment thereof is used to increase the expression level of HGF protein in cells in vitro;

for example, the mutant intron 4 or fragment thereof is used to increase the expression level of HGF protein in vivo;

for example, the mutant intron 4 or fragment thereof is used to increase the expression level of HGF protein in a patient (e.g., a mammal, such as a human).

11. use of the nucleic acid molecule of claim 5 or the vector of claim 7 for expressing or producing an HGF protein;

For example, the nucleic acid molecule or vector is used for in vitro expression or production of HGF protein;

For example, the nucleic acid molecule or vector is used for expression or production of HGF protein in a cell;

For example, the nucleic acid molecule or vector is used for in vitro, in cell expression or production of HGF protein;

for example, the nucleic acid molecule or vector is used for in vivo expression or production of HGF protein;

For example, the nucleic acid molecules or vectors are used to express or produce HGF protein in a patient (e.g., a mammal, such as a human).

12. a pharmaceutical composition comprising a nucleic acid molecule according to claim 5 or a vector according to claim 7, and optionally a pharmaceutically acceptable carrier and/or excipient;

preferably, the pharmaceutical composition is administered by injection;

preferably, the pharmaceutical composition is an injection or a lyophilized powder;

preferably, the nucleic acid molecule or vector is present in a therapeutically effective amount (e.g., an amount effective to treat an ischemic disease);

Preferably, the pharmaceutical composition is in unit dosage form.

13. use of the nucleic acid molecule of claim 5 or the vector of claim 7 in the preparation of a pharmaceutical composition for treating a disease in a subject that can benefit from native HGF activity;

Preferably, the disease is selected from ischemic diseases (e.g. Coronary Artery Disease (CAD) or Peripheral Artery Disease (PAD), such as myocardial infarction or lower limb arterial ischemia), metabolic syndrome, diabetes and its complications (e.g. diabetic peripheral neuropathy), restenosis (e.g. post-operative restenosis and post-perfusion restenosis), and nerve injury (e.g. neurodegenerative diseases (e.g. Amyotrophic Lateral Sclerosis (ALS), parkinson's disease, dementia disease), traumatic nerve injury, peripheral neuropathy (e.g. diabetic peripheral neuropathy));

Preferably, the subject is a mammal, e.g., a human;

Preferably, the pharmaceutical composition is administered by injection;

Preferably, the pharmaceutical composition is an injection or a lyophilized powder.

Technical Field

the present application relates to a mutated intron 4 of the Hepatocyte Growth Factor (HGF) gene or a fragment thereof. The application also relates to a nucleic acid molecule containing the mutated intron 4 or the fragment thereof, which encodes the HGF protein, a vector containing the nucleic acid molecule, and a host cell containing the nucleic acid molecule or the vector. The application also relates to pharmaceutical compositions comprising said nucleic acid molecules, and to uses of said pharmaceutical compositions.

Background

Hepatocyte Growth Factor (HGF), originally isolated from rat plasma and platelets, is a secreted heparin affinity glycoprotein, also known as a Spreading Factor (SF). HGF is produced by mesenchymal cells, can bind to a receptor c-Met and activate the tyrosine kinase activity of the receptor, and promotes the growth, migration and morphogenesis of various cells such as hepatocytes, epithelial cells, endothelial cells, melanocytes, hematopoietic cells and the like. HGF plays an important role in the development of embryonic liver and placenta, and is involved in maintaining and renewing the cells of organs such as liver, lung, kidney, etc., and promoting the regeneration and repair of these organs after injury. In addition, HGF has pro-invasive or growth-inhibitory effects on tumor cells of different origins.

the human HGF gene is located on the long arm of chromosome 7, is about 70Kb in length, and consists of 18 exons and 17 introns spaced apart. HGF gene can transcribe about 6kb transcript and synthesize one precursor polypeptide HGF728 comprising 728 amino acids. In addition, the HGF gene can also undergo another shearing, and synthesize a precursor polypeptide HGF723 consisting of 723 amino acids. After the inactive precursor polypeptide is cracked by protease and connected by disulfide bond, the mature HGF protein with biological activity is formed.

Since the half-life of HGF protein in vivo is very short (research progress of mechanism of anti-renal fibrosis by HGF, division of medical science and pathology in foreign countries, 2005, vol 25, No. 3), there are problems of repeated administration and excessive dosage when HGF protein is used to treat diseases. To avoid such problems, researchers have tried to apply HGF gene directly to the treatment of clinical diseases by gene therapy.

At present, there are cases of using the angiogenesis promoting factor gene to treat ischemic diseases in clinic, such as VEGF, FGF naked plasmid (the therapeutic base and the antigenic plasmid for the VIFCAD study-genetic for the recovery of biological activities in no-optionals using intracellular biological VEGF/FGF plasmid injection. post Kardiol Interw; 2006, 2: 116-. From the existing reports, the angiogenesis activity of the HGF gene therapy is stronger than that of VEGF and bFGF, and the safety is higher. Therefore, the HGF gene has better application prospect in treating the ischemic diseases of blood vessels.

It has been found that intron 4 located between exons 4 and 5 of HGF gene plays a role in controlling variable splicing in vivo, so that HGF gene can simultaneously express two natural HGF isoforms, HGF728 and HGF723 (hepatocytogenetic factor and its variant with a deletion of fine amino acids in the biological activity and biological structure. biochem Biophys Res Commun.1994 Apr 29; 200: 808-15). Chinese patent ZL03806534.7 reports that by inserting HGF genomic intron 4 or truncated sequence thereof between exons 4 and 5 of cDNA of natural HGF, hybrid gene capable of expressing both HGF728 and HGF723 protein can be generated, and the two proteins can exert synergistic effect and produce positive effect on disease treatment.

There remains a need in the art for further increasing the expression level of HGF gene. This is particularly advantageous at least for enhancing the gene therapy effect using the HGF gene.

disclosure of Invention

The inventors of the present application have unexpectedly found that intron 4 (e.g., SEQ ID NO: 1) or a fragment thereof of a natural hepatocyte growth factor gene (e.g., human HGF gene) may be mutated, and that the resulting mutated intron 4 or fragment thereof is capable of increasing the expression level of the HGF gene.

accordingly, in a first aspect of the present application, there is provided a mutant intron 4 of the Hepatocyte Growth Factor (HGF) gene, or a fragment thereof, wherein the mutant intron 4 comprises mutations at the following positions: corresponding to SEQ ID NO:1 at position 3815, 4774, and 4876; and, said fragment comprises the amino acid sequence of intron 4 of said mutation corresponding to seq id NO:1 from position 1 to 246 and 3686 to 4926. In certain preferred embodiments, the fragments may further comprise a linker sequence for linking the nucleotide fragments.

In certain preferred embodiments, the fragment further comprises a sequence corresponding to SEQ ID NO:1 from nucleotide 2686 to nucleotide 3685. In certain preferred embodiments, the fragment comprises the amino acid sequence of intron 4 of the mutation corresponding to SEQ ID NO:1 from position 1 to 246 and from position 2686 to 4926. In certain preferred embodiments, the fragments may further comprise a linker sequence for linking the nucleotide fragments.

in certain preferred embodiments, the fragment comprises or alternatively consists of: the mutant intron 4 has a sequence corresponding to SEQ ID NO:1, the first nucleotide fragment corresponding to positions 1 to 246 of SEQ id no:1 from position 3686 to 4926 and optionally an adaptor sequence located between the two nucleotide fragments. In certain preferred embodiments, the fragment comprises or alternatively consists of: the mutant intron 4 has a sequence corresponding to SEQ ID NO:1, and the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1 from 3686 to 4926, wherein the 3 'end of the first nucleotide fragment is directly linked to the 5' end of the second nucleotide fragment. In certain preferred embodiments, the fragment comprises or alternatively consists of: the mutant intron 4 has a sequence corresponding to SEQ ID NO:1, and the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1, from 3686 to 4926, wherein the 3 'end of the first nucleotide fragment is linked to the 5' end of the second nucleotide fragment by an adaptor sequence. Various linker sequences known in the art may be used. In certain preferred embodiments, the linker sequence is 1-500 nucleotides in length, e.g., 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-500 nucleotides in length. In certain preferred embodiments, the linker sequence is set forth in SEQ ID NO 13.

in certain preferred embodiments, the fragment comprises or alternatively consists of: the mutant intron 4 has a sequence corresponding to SEQ ID NO:1, the first nucleotide fragment corresponding to positions 1 to 246 of SEQ id no:1 from position 2686 to 4926 and optionally a linker sequence between said two nucleotide fragments. In certain preferred embodiments, the fragment comprises or alternatively consists of: the mutant intron 4 has a sequence corresponding to SEQ ID NO:1, and the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1 from 2686 to 4926, wherein the 3 'end of the first nucleotide fragment is directly linked to the 5' end of the second nucleotide fragment. In certain preferred embodiments, the fragment comprises or alternatively consists of: the mutant intron 4 has a sequence corresponding to SEQ ID NO:1, and the first nucleotide fragment corresponding to positions 1 to 246 of SEQ ID NO:1, from position 2686 to 4926, wherein the 3 'end of the first nucleotide fragment is linked to the 5' end of the second nucleotide fragment by an adaptor sequence. Various linker sequences known in the art may be used. In certain preferred embodiments, the linker sequence is 1-500 nucleotides in length, e.g., 1-5, 5-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-200, 200-500 nucleotides in length. In certain preferred embodiments, the linker sequence is set forth in SEQ ID NO 13.

In certain preferred embodiments, the mutated intron 4 comprises a mutation selected from the group consisting of: in a nucleic acid sequence corresponding to SEQ ID NO:1 the nucleotide at position 3815 is mutated to an adenine nucleotide; in a nucleic acid sequence corresponding to SEQ ID NO:1 is mutated to a guanine nucleotide at the position 4774; in a nucleic acid sequence corresponding to SEQ ID NO:1 at position 4876 is mutated to a guanine nucleotide; and, any combination thereof.

in certain preferred embodiments, the mutated intron 4 comprises the following mutations: in a nucleic acid sequence corresponding to SEQ ID NO:1 is mutated to an adenine nucleotide at position 3815. In certain preferred embodiments, the mutated intron 4 comprises the following mutations: in a nucleic acid sequence corresponding to SEQ ID NO:1 is mutated to a guanine nucleotide at the position 4774. In certain preferred embodiments, the mutated intron 4 comprises the following mutations: in a nucleic acid sequence corresponding to SEQ ID NO:1 at position 4876 is mutated to a guanine nucleotide.

In certain preferred embodiments, the mutated intron 4 comprises the following mutations: in a nucleic acid sequence corresponding to SEQ ID NO:1 the nucleotide at position 3815 is mutated to an adenine nucleotide; in a nucleic acid sequence corresponding to SEQ ID NO:1 is mutated to a guanine nucleotide at the position 4774; and, where the amino acid sequence corresponds to SEQ ID NO:1 at position 4876 is mutated to a guanine nucleotide.

In certain preferred embodiments, the hepatocyte growth factor is human hepatocyte growth factor. In certain preferred embodiments, the human hepatocyte growth factor has the amino acid sequence set forth in SEQ ID NO: 12. In certain preferred embodiments, the human hepatocyte growth factor gene has a sequence as set forth in GenBank database accession no: the nucleotide sequence shown in NC-000007.14.

in certain preferred embodiments, the mutated intron 4 has the amino acid sequence as set forth in SEQ ID NO: 9, or a nucleotide sequence shown in the specification. In certain preferred embodiments, the fragment has an amino acid sequence selected from SEQ ID NOs: 10 and SEQ ID NO: 11.

In another aspect of the present application, there is provided a nucleic acid molecule encoding Hepatocyte Growth Factor (HGF) comprising exons 1-18 of the HGF gene, and intron 4 or a fragment thereof, between exons 4 and 5, that is mutated as described herein.

In certain preferred embodiments, the hepatocyte growth factor is human hepatocyte growth factor. In certain preferred embodiments, the human hepatocyte growth factor has the amino acid sequence set forth in SEQ ID NO: 12. In certain preferred embodiments, exons 1-18 encode a polypeptide as set forth in SEQ ID NO: 12. In certain preferred embodiments, the human hepatocyte growth factor gene has a sequence as set forth in GenBank database accession no: the nucleotide sequence shown in NC-000007.14.

in certain preferred embodiments, the nucleic acid molecule has a sequence selected from SEQ ID NOs: 3, SEQ ID NO: 4 and SEQ ID NO: 5.

In yet another aspect of the present application, there is provided a vector comprising a mutated intron 4 or a fragment thereof according to the present application. In certain preferred embodiments, the vector is used to clone the mutated intron 4 or a fragment thereof.

In yet another aspect of the present application, there is provided a vector comprising a nucleic acid molecule according to the present application. In certain preferred embodiments, the vector is selected from the group consisting of a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophages such as lambda bacteriophage or M13 bacteriophage; and, viral vectors, such as retroviral vectors (e.g., lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors (e.g., herpes simplex viral vectors), poxvirus vectors, baculovirus vectors, papilloma viral vectors, papilloma polyomavirus vectors.

In certain preferred embodiments, the vector is for expressing (e.g., expressing in a subject (e.g., a mammal, such as a human)) the HGF protein. In certain preferred embodiments, the vector is a vector for gene therapy, such as a plasmid, an adenoviral vector, an adeno-associated viral vector, and a lentiviral vector.

In certain preferred embodiments, the vector has a nucleotide sequence selected from SEQ ID NO 6, SEQ ID NO 7 or SEQ ID NO 8.

In yet another aspect of the present application, there is provided a host cell comprising a nucleic acid molecule or vector according to the present application. In certain preferred embodiments, the host cell is selected from prokaryotic cells, such as e.coli cells, and eukaryotic cells, such as yeast cells, insect cells, plant cells, and animal cells (e.g., mammalian cells, such as mouse cells, human cells, etc.). In certain preferred embodiments, the host cell is an E.coli cell, such as E.coli DH5 a cell. In certain preferred embodiments, the host cell is a 293T cell or a human cell.

In yet another aspect of the present application, there is provided a method of expressing or producing HGF protein comprising using mutant intron 4 or fragment thereof according to the present application. In certain preferred embodiments, the method comprises the use of a nucleic acid molecule or vector according to the present application. In certain preferred embodiments, the method comprises expressing a nucleic acid molecule or vector according to the present application in a host cell under conditions that allow for expression of the protein; and optionally, recovering the HGF protein expressed in the host cell.

in yet another aspect of the present application, there is provided a use of mutant intron 4 or a fragment thereof according to the present application for increasing the expression level of an HGF protein. In certain preferred embodiments, said mutated intron 4 or fragment thereof is used to increase the expression level of HGF protein in vitro. In certain preferred embodiments, the mutated intron 4 or fragment thereof is used to increase the expression level of the HGF protein in a cell. In certain preferred embodiments, the mutated intron 4 or fragment thereof is used to increase the expression level of HGF protein in cells in vitro. In certain preferred embodiments, said mutated intron 4 or fragment thereof is used to increase the expression level of HGF protein in vivo. In certain preferred embodiments, the mutated intron 4 or fragment thereof is used to increase the expression level of HGF protein in a patient (e.g., a mammal, such as a human).

In yet another aspect of the application, there is provided a use of a nucleic acid molecule or vector according to the application for expressing or producing an HGF protein. In certain preferred embodiments, the nucleic acid molecule or vector is used for expression or production of an HGF protein in vitro. In certain preferred embodiments, the nucleic acid molecule or vector is used to express or produce an HGF protein in a cell. In certain preferred embodiments, the nucleic acid molecule or vector is used for expressing or producing an HGF protein in vitro, in a cell. In certain preferred embodiments, the nucleic acid molecule or vector is used to express or produce HGF protein in vivo. In certain preferred embodiments, the nucleic acid molecule or vector is used to express or produce HGF protein in a patient (e.g., a mammal, such as a human).

In a further aspect of the present application, there is provided a pharmaceutical composition comprising a nucleic acid molecule or vector according to the present application, and optionally, a pharmaceutically acceptable carrier and/or excipient.

The pharmaceutical compositions described herein may be administered by methods well known in the art, such as, but not limited to, administration by injection. In certain preferred embodiments, the pharmaceutical compositions described herein are injection solutions or lyophilized powders.

In certain preferred embodiments, the nucleic acid molecule or vector is present in a therapeutically effective amount (e.g., an amount effective to treat ischemic disease). In certain preferred embodiments, the pharmaceutical compositions described herein are presented in unit dosage form.

in yet another aspect of the application, there is provided the use of the nucleic acid molecule or vector in the preparation of a pharmaceutical composition for treating a disease in a subject that can benefit from natural HGF activity. In certain preferred embodiments, the disease is selected from ischemic diseases (e.g., Coronary Artery Disease (CAD) or Peripheral Artery Disease (PAD), such as myocardial infarction or lower limb arterial ischemia), metabolic syndrome, diabetes and its complications (e.g., diabetic peripheral neuropathy), restenosis (e.g., post-operative restenosis and post-perfusion restenosis), and nerve injury (e.g., neurodegenerative diseases (e.g., Amyotrophic Lateral Sclerosis (ALS), parkinson's disease, dementia disease), traumatic nerve injury, peripheral neuropathy (e.g., diabetic peripheral neuropathy)). In certain preferred embodiments, the subject is a mammal, e.g., a human. In certain preferred embodiments, the pharmaceutical composition is administered by injection. In certain preferred embodiments, the pharmaceutical composition is an injection or a lyophilized powder.

In yet another aspect of the application, there is provided a method of treating a disease that may benefit from natural HGF activity in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a nucleic acid molecule or vector or pharmaceutical composition according to the present application. In certain preferred embodiments, the disease is selected from ischemic diseases (e.g., Coronary Artery Disease (CAD) or Peripheral Artery Disease (PAD), such as myocardial infarction or lower limb arterial ischemia), metabolic syndrome, diabetes and its complications (e.g., diabetic peripheral neuropathy), restenosis (e.g., post-operative restenosis and post-perfusion restenosis), and nerve injury (e.g., neurodegenerative diseases (e.g., Amyotrophic Lateral Sclerosis (ALS), parkinson's disease, dementia disease), traumatic nerve injury, peripheral neuropathy (e.g., diabetic peripheral neuropathy)). In certain preferred embodiments, the subject is a mammal, e.g., a human. In certain preferred embodiments, the nucleic acid molecule or vector or pharmaceutical composition described herein is administered to the subject by injection.

in the present application, unless otherwise indicated, scientific and technical terms used herein have the meanings that are commonly understood by those of skill in the art. Also, cell culture, molecular genetics, nucleic acid chemistry, immunology laboratory procedures, as used herein, are conventional procedures that are widely used in the relevant art. Meanwhile, for better understanding of the present application, definitions and explanations of related terms are provided below.

as used herein, the term "hepatocyte growth factor" or "HGF protein" refers to a naturally occurring, biologically active Hepatocyte Growth Factor (HGF), which have the same meaning and are used interchangeably. As used herein, the term "human hepatocyte growth factor" or "hgf protein" refers to naturally occurring, biologically active human hepatocyte growth factors, which have the same meaning and are used interchangeably. The amino acid sequence of an HGF protein or an HGF protein can be conveniently obtained from various public databases (e.g., GenBank databases). For example, the amino acid sequence of a native hgf protein can be found in GenBank database accession numbers: NP _ 000592.3.

as used herein, the term "hepatocyte growth factor gene" or "HGF gene" refers to a gene encoding hepatocyte growth factor; the term "human hepatocyte growth factor gene" or "hHGF gene" refers to a gene encoding human hepatocyte growth factor. Generally, an HGF gene/HGF gene comprises 18 exons and 17 introns. As understood by those skilled in the art, in eukaryotic cells, DNA encoding a structural protein is typically separated by several non-coding intervening sequences (which are not translated into amino acid sequences); such non-coding intervening sequences are referred to as "introns," and each coding DNA sequence (which will be translated into an amino acid sequence) that is separated by an "intron" is referred to as an exon. Thus, the HGF gene/HGF gene comprises, in order, exon 1, intron 1, exon 2, intron 2, … …, exon 17, intron 17, and exon 18. In the examples of the present application, the nucleic acid sequence of the hHGF exon used is from GenBank (gi: 58533168). However, it is readily understood that degenerate sequences of said exon sequences may also be used, without affecting or changing the amino acid sequence of the expressed hgf protein. Thus, in the present application, exons 1-18 of an HGF gene/HGF gene are not limited to the particular nucleotide sequence used, and can be any nucleotide sequence capable of encoding an HGF protein/HGF protein (including the particular nucleotide sequence used, as well as degenerate sequences thereof). In some preferred embodiments, exons 1-18 of the hgf gene encode a polypeptide having the amino acid sequence set forth in SEQ ID NO: 12 in sequence. In this application, the term "intron 4" refers to the fourth intron which is located between exons 4 and 5. In certain preferred embodiments, the hgf gene has a sequence as set forth in GenBank database accession no: the nucleotide sequence shown in NC-000007.14. In this case, the nucleotide sequences of exons 1 to 18 and introns 1 to 17 in the hHGF gene can be easily determined by BLAST or by using the amino acid sequence of the hHGF protein. In some preferred embodiments, intron 4 of the hHGF gene has a nucleotide sequence as shown in SEQ ID NO. 1.

As used herein, when referring to hgf gene, reference is made to GenBank database accession No.: NC _ 000007.14; when referring to intron 4 of the hgf gene, reference is made to SEQ ID NO:1, is described. However, it is readily understood that the native hgf gene and intron 4 thereof may have multiple versions that have substantially the same nucleotide sequence and substantially the same biological function, but may still differ slightly in nucleotide sequence from each other. Thus, in the present application, hgf gene is not limited to GenBank database accession No.: NC _000007.14, and intron 4 thereof is not limited to the nucleotide sequence shown in SEQ ID NO: 1. The hgf gene of the present application is intended to encompass all naturally occurring hgf genes having biological function, including GenBank database accession numbers: the hgf gene represented by NC _000007.14 and naturally occurring variants thereof; and accordingly, intron 4 thereof is intended to encompass intron 4 encompassed by all such hgf genes, including SEQ ID NO:1, and naturally occurring variants thereof.

according to the present application, the expression "corresponding" refers to nucleotide positions or amino acid positions at equivalent positions in the sequences being compared when the sequences are optimally aligned, i.e., when the sequences are aligned for the highest percent identity. For example, the expression "position corresponding to position 3815 of SEQ ID NO: 1" refers to a nucleotide position in a sequence that is equivalent to position 3815 of SEQ ID NO:1, which is compared when the sequence is optimally aligned with SEQ ID NO:1, i.e., when the sequence is aligned with SEQ ID NO:1 to obtain the highest percent identity. Similarly, the expressions "position corresponding to position 4774 of SEQ ID NO: 1" and "position corresponding to position 4876 of SEQ ID NO: 1" have similar meanings.

As used herein, the term "nucleotide" is intended to include ribonucleotides and deoxyribonucleotides. For example, adenine nucleotides are intended to include adenine ribonucleotides and adenine deoxyribonucleotides, and may be selected according to actual needs. Similarly, guanine nucleotides are intended to include both guanine ribonucleotides and guanine deoxyribonucleotides, and can be selected according to actual needs. In certain preferred embodiments, the nucleotide is a deoxyribonucleotide. In certain preferred embodiments, the nucleotide is a ribonucleotide. In the present application, the nucleotide may be modified (e.g., chemically modified) or unmodified.

As used herein, the term "nucleic acid" is intended to include ribonucleic acids, deoxyribonucleic acids, and combinations thereof. Thus, in the present application, a nucleic acid molecule encoding Hepatocyte Growth Factor (HGF) may be RNA, DNA, or an RNA/DNA hybrid. In certain preferred embodiments, the nucleic acid molecule is RNA. In certain preferred embodiments, the nucleic acid molecule is DNA. In certain preferred embodiments, the nucleic acid molecule is an RNA/DNA hybrid. In the present application, the nucleic acid molecule may be modified (e.g., chemically modified) or unmodified.

as used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by an inserted polynucleotide, the vector is referred to as an expression vector. The vector may be introduced into a host cell by transformation, transduction, or transfection, and the genetic material elements carried thereby are expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: a plasmid; phagemid; a cosmid; artificial chromosomes such as Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs), or artificial chromosomes (PACs) derived from P1; bacteriophage such as lambda phage or M13 phage and viral vector. Viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, papilloma polyoma vacuolatum viruses (e.g., SV 40). A vector may contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may contain a replication initiation site.

As used herein, the term "host cell" refers to a cell that can be used to amplify or express a foreign gene (e.g., an HGF gene), which includes, but is not limited to, prokaryotic cells such as escherichia coli or bacillus subtilis, fungal cells such as yeast cells or aspergillus, insect cells such as S2 drosophila cells or Sf9, or animal cells such as fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, 293T cells, or human cells.

As used herein, the term "pharmaceutically acceptable" means that it is recognized in the pharmaceutical art as being useful for animals, particularly humans. As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to carriers and/or excipients that are pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, e.g., Remington's Pharmaceutical sciences. edited by geno AR,19the d. pennsylvania: machine Publishing Company,1995), and include, but are not limited to: 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 any combination thereof.

As used herein, a pharmaceutically acceptable carrier can be a sterile liquid, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. When the pharmaceutical composition is administered intravenously, physiological saline is a preferred carrier. Saline solutions as well as aqueous dextrose and glycerol solutions may also be employed as liquid carriers, particularly for injectable solutions.

As used herein, 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, glycerol, propylene, glycol, water, ethanol and the like. The pharmaceutical composition may also contain wetting agents, or emulsifiers such as sodium hyaluronate, or pH buffers, if desired. The pharmaceutical compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like.

As used herein, the term "subject" refers to a mammal, including, but not limited to, humans, rodents (mice, rats, guinea pigs), dogs, horses, cows, cats, pigs, monkeys, chimpanzees, and the like. Preferably, the subject is a human.

As used herein, the term "effective amount" refers to an amount sufficient to obtain, or at least partially obtain, a desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, or delay the onset of disease; a therapeutically effective amount for a disease is an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. It is well within the ability of those skilled in the art to determine such effective amounts. For example, an 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, e.g., age, weight and sex, the mode of administration of the drug, and other treatments administered concurrently, and the like.

Advantageous effects of the invention

As previously reported, HGF gene can be used for gene therapy. During gene therapy, HGF protein expressed in vivo with plasmids may have a variety of biological activities, including but not limited to one or more of the following: (1) promoting endothelial cell growth and/or migration; (2) promoting angiogenesis (e.g., microvascular); and/or, (3) promoting repair of nerve damage (e.g., peripheral neuropathy, e.g., diabetic peripheral neuropathy). Thus, HGF gene therapy may have application in a number of areas, including but not limited to: (1) promoting endothelial cell growth and/or migration; (2) promoting angiogenesis (e.g., microvascular); (3) treating 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 (e.g., diabetic peripheral neuropathy); (5) inhibiting restenosis; and (6) promoting repair of nerve injury (e.g., neurodegenerative disease, traumatic nerve injury, peripheral neuropathy).

The nucleic acid molecules and vectors of the present application are capable of expressing HGF protein at significantly higher levels within cells and are therefore particularly suitable for use in gene therapy, in the context of applications in the above-mentioned aspects.

Sequence information

Information on the sequences to which the present invention relates is provided in table 1 below.

Table 1: sequence information of SEQ ID NOS 1 to 13

Embodiments of the present application will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present application and do not limit the scope of the present application. Various objects and advantageous aspects of the present application will become apparent to those skilled in the art from the following detailed description of the preferred embodiments.

Detailed Description

The present application will now be described with reference to the following examples, which are intended to illustrate, but not limit, the present application.

Unless otherwise indicated, molecular biological experimental methods and immunoassays, as used herein, are essentially described with reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are described by way of example and are not intended to limit the scope of the claims to this application, as those skilled in the art will recognize.