WO2022107853A1 - 糖尿病に対する低用量の肝細胞増殖因子遺伝子治療 - Google Patents

糖尿病に対する低用量の肝細胞増殖因子遺伝子治療 Download PDF

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WO2022107853A1
WO2022107853A1 PCT/JP2021/042463 JP2021042463W WO2022107853A1 WO 2022107853 A1 WO2022107853 A1 WO 2022107853A1 JP 2021042463 W JP2021042463 W JP 2021042463W WO 2022107853 A1 WO2022107853 A1 WO 2022107853A1
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hgf
vector
dose
administration
promoter
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健一郎 小戝
恵理子 松田
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Kagoshima University NUC
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Kagoshima University NUC
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Priority to CN202180078077.9A priority Critical patent/CN116685340A/zh
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Priority to EP21894725.7A priority patent/EP4249003A4/en
Priority to US18/253,651 priority patent/US20230414718A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1833—Hepatocyte growth factor; Scatter factor; Tumor cytotoxic factor II
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/18—Drugs for disorders of the alimentary tract or the digestive system for pancreatic disorders, e.g. pancreatic enzymes
    • 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
    • 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
    • A61K48/0075—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 delivery route, e.g. oral, subcutaneous
    • 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
    • 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
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • C12N15/86—Viral vectors
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a gene therapy agent for diabetes that has both safety and therapeutic effect. More specifically, the invention contains a viral vector containing a nucleic acid encoding HGF downstream of a promoter capable of expressing a therapeutically effective amount of hepatocyte growth factor (HGF) and is administered at a low dose.
  • the present invention relates to a therapeutic agent for diabetes that does not substantially exhibit adverse events caused by viral vectors.
  • T2D type 2 diabetes
  • pancreatic ⁇ -cells suffer from insufficient insulin action due to compensatory insulin hypersecretion. It is a disease that tries to make up for it, but if the condition continues for a long time, it causes exhaustion of ⁇ cells, and it becomes impossible to secrete a sufficient amount of insulin, resulting in a hyperglycemic condition.
  • T1D type 1 diabetes
  • T1D type 1 diabetes
  • T1D is a disease in which insulin secretion is depleted due to the destruction of pancreatic ⁇ cells by an autoimmune mechanism, resulting in a hyperglycemic state.
  • T1D develops at an early age, but the details of its mechanism are still unknown.
  • Patients with T1D need to self-inject insulin for life to control their blood glucose.
  • T1D As a treatment method to cure T1D, expectations are increasing for regenerative medicine that reconstructs and reconstructs lost organ functions. One of them is ⁇ -cell replacement therapy by islet transplantation, and it has been reported that this treatment method is effective in improving glucose metabolism. However, the practical application of this technique is limited because T1D patients must use immunosuppressive agents after islet transplantation and there is a shortage of donors for islet transplantation.
  • HGF hepatocyte growth factor
  • HGF is expressed in endothelial cells and mesenchymal cells in the pancreas
  • c-Met which is a receptor for HGF, is also localized in pancreatic precursor cells, pancreatic islet cells, and pancreatic duct cells.
  • Non-Patent Document 1 discloses that a plasmid (20 ⁇ g) containing a nucleic acid encoding HGF was administered to mice before the onset of diabetes to maintain insulin secretion.
  • Non-Patent Document 2 discloses a high dose of an adeno-associated virus (AAV) vector containing a nucleic acid encoding HGF (3.0 ⁇ 10 11 vector genome (vg); about 1.5 ⁇ 10 13 per kg body weight. It is disclosed that vg) was administered to mice before the onset of diabetes through the pancreatic duct to suppress the increase in blood glucose.
  • AAV adeno-associated virus
  • Non-Patent Document 3 describes a high dose (1.0 ⁇ 10 11 virus particles (vp); about 5 ⁇ 10 12 per kg body weight) of an adenovirus (Ad) vector expressing HGF in mice after the onset of T1D. It has been reported that administration in the tail vein at vp) resulted in a partial response.
  • vp virus particles
  • Ad adenovirus
  • AAV vectors which were previously believed to be non-pathogenic and highly safe for a long period of time, have also been shown to be high in clinical trials of gene therapy for congenital myopathy. Three serious adverse events, especially deaths due to serious liver damage, were reported for patients receiving doses (3 x 10 14 vg / kg) of AAV vectors (Non-Patent Documents 5 and 6). ).
  • AAV vectors are currently the most widely used vector in clinical applications of in vivo gene therapy (systemic administration from blood vessels).
  • systemic vascular administration of high-dose vectors in vivo regardless of vector type, can cause serious adverse events, including dangerous liver damage. Therefore, the clinical application of high-dose vector in vivo gene therapy has been suspended and reviewed. Thus, AAV vector fatalities have become a global and historic event in the field of gene therapy.
  • Raper SE Chirmule N, Lee FS et al. Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol Genet Metab 2003; 80: 148-158. Audentes Therapeutics. Letter to the MTM disease community. Https://myotubulartrust.org/audentes-therapeutics-letter-23-june-2020/. Wilson JM, Flotte TR. Moving Forward After Two Deaths in a Gene Therapy Trial of Myotubular Myopathy. Hum Gene Ther 2020; 31: 695-696.
  • an object of the present invention is to provide a gene therapy means for diabetes such as T1D, which has both safety and therapeutic effect.
  • HGF cytomegalovirus
  • Non-Patent Documents 1-3 above cytomegalovirus
  • the present inventors need to increase the expression efficiency of the therapeutic gene in order to realize a low dose of the viral vector in order to ensure the safety of in vivo gene therapy and at the same time to obtain a desired therapeutic effect. I thought.
  • CA promoter CMV pre-early enhancer and modified chicken ⁇ -actin promoter
  • CA promoter CMV pre-early enhancer and modified chicken ⁇ -actin promoter
  • An Ad vector expressing HGF under the transcriptional control of a hybrid promoter (also referred to as "CAG promoter”, but referred to as "CA promoter” in the present specification) is produced and used as a model mouse after the onset of T1D.
  • the tail vein was administered at a dose 1 to 2 orders of magnitude lower than previously reported (corresponding to 3 ⁇ 10 8 pfu; 10 9 to 10 10 vp).
  • the increase in blood glucose was significantly suppressed for about 1 week after the administration as compared with the non-treated group, and the suppression tendency was maintained for a long period of time (at least 11 weeks after the administration).
  • Compensatory insulin hypersecretion from ⁇ -cells remaining in the acute phase of T1D was observed in the non-treated group, but normal insulin secretion was maintained in the HGF-treated group.
  • IPGTT the increase in blood glucose was significantly suppressed and the insulin secretion level was also significantly maintained on the 16th and 60th days after administration.
  • the present inventors by administering a viral vector that expresses HGF under the transcriptional control of a promoter capable of expressing a therapeutically effective amount of HGF at a lower dose than the existing dose. We have succeeded in treating T1D safely and effectively, and have completed the present invention.
  • HGF hepatocyte growth factor
  • the viral vector is an adenovirus (Ad) vector or an adeno-associated virus (AAV) vector.
  • the agent according to [8], wherein the intravenous administration is administration from a peripheral vein.
  • a method for protecting and regenerating pancreatic ⁇ cells in a mammal comprising administering a recombinant viral vector expressing HGF to a diabetic mammal, wherein the viral vector is 10 10 to 10 12 viral particles.
  • a method comprising a nucleic acid encoding HGF downstream of a promoter having transcriptional activity capable of conferring therapeutically effective blood HGF levels at a dose of (vp) / kg body weight.
  • FIGS. 1A and 1C show blood glucose between days -7 and 7 when an adenovirus vector was injected into the tail vein into mice that developed T1D due to streptozotocin (STZ) administration in two independent experiments. Shows daily changes in values. The day when the adenovirus vector was administered is the 0th day. 1B and 1D are taken from day -7 to day 77 (or day 70) in the T1D model mouse to which the adenovirus vector was administered (FIG. 1B corresponds to FIG. 1A and FIG. 1D corresponds to FIG. 1C, respectively). Shows weekly changes in blood glucose levels during the period.
  • STZ streptozotocin
  • Ad.CA-HGF is the viral vector of the present invention that expresses the HGF gene under the control of the CA promoter, and "Ad.CA-LacZ” is inserted with the ⁇ -galactosidase gene (LacZ) instead of HGF.
  • the control viral vector, "Intact” shows data from normal mice that have not undergone any treatment.
  • the horizontal axis of each figure shows the number of days (days) after administration of the viral vector, and the vertical axis shows the blood glucose level (mg / dl). *: P ⁇ 0.05
  • FIG. 2 shows the same mice as in FIG.
  • FIG. 1 (gray bar: T1D model mouse administered with Ad.CA-LacZ; black bar: T1D model mouse administered with Ad.CA-HGF; white bar: both STZ administration and viral vector administration. It is a graph which shows the result of having measured the plasma insulin concentration on the 7th day, the 14th day, and the 21st day in the normal mouse) which did not receive.
  • the vertical axis shows plasma insulin level (ng / ml). *: P ⁇ 0.05
  • FIG. 3 is a graph showing the results of measuring plasma AST levels on days 7, 14, and 21 in the same mice as in FIG. 1 (each bar is synonymous with FIG. 2).
  • the vertical axis shows plasma AST level (IU / L). The results of IPGTT in the same mice as in FIG.
  • FIG. 4A shows the blood glucose level (mg / dl) after glucose administration
  • FIG. 4B shows the time course of plasma insulin level (ng / ml) after glucose administration.
  • the results of IPGTT in the same mouse as FIG. 1 (60 days after administration of Ad vector) are shown.
  • FIG. 5A shows the blood glucose level (mg / dl) after glucose administration
  • FIG. 5B shows the time course of plasma insulin level (ng / ml) after glucose administration.
  • the present invention is a safe and effective protective / regenerating agent for pancreatic ⁇ cells in a diabetic mammal containing a recombinant viral vector expressing HGF (“hereinafter, ⁇ cell protecting / regenerating agent of the present invention”. Also called).
  • the ⁇ -cell protection / regeneration agent is administered at a dose of 10 10 to 10 12 viral particles (vp) / kg body weight, and the viral vector is a transcription that can provide therapeutically effective blood HGF levels at the dose. It is characterized by containing a nucleic acid encoding HGF downstream of the active promoter.
  • pancreatic ⁇ -cells As used herein, "protection and regeneration of pancreatic ⁇ -cells" is compared to controls that do not undergo therapeutic treatment while maintaining normal insulin secretion without causing compensatory insulin hypersecretion from ⁇ -cells. Insulin is significantly suppressed at least in the acute phase, and the tendency to suppress it is maintained for a long period of time (for example, 60 days or more, preferably 75 days or more, more preferably 90 days or more, still more preferably 120 days or more). It means that the function of the remaining ⁇ -cells is preserved and / or the ⁇ -cells proliferate to the extent that they are.
  • the " ⁇ cell protection / regeneration agent of the present invention” can also be a “hyperglycemia inhibitor”. ..
  • the " ⁇ -cell protection / regeneration agent of the present invention” is also a “diabetes treatment agent”. could be.
  • Non-Patent Document 3 hyperglycemia was suppressed by high-dose administration of an Ad vector expressing HGF under the control of the CMV promoter, but the blood insulin / glucose ratio was significantly increased.
  • the ⁇ -cell protection / regeneration agent of the present invention which can suppress hyperglycemia while maintaining normal insulin secretion without inducing compensatory insulin hypersecretion, causes ⁇ -cell exhaustion due to insulin hypersecretion, and thus ⁇ . It has beneficial effects such as reducing the risk of developing cell dysfunction.
  • the virus vector used for the HGF-expressing recombinant virus vector which is the active ingredient of the ⁇ -cell protection / regeneration agent of the present invention, is not particularly limited as long as it is generally used for gene therapy.
  • adenovirus (Ad). ) Vector, adeno-associated virus (AAV) vector, lentivirus vector, retrovirus vector, Sindbis virus vector, mad dog disease virus vector, Sendai virus vector, simple herpesvirus vector and the like can be used.
  • the Ad vector or AAV vector can be used from the viewpoints that chromosomal integration is infrequent and there is no risk of insertion mutagenesis, that it can be introduced into non-dividing cells, and that the transgene can be expressed in the medium to long term. preferable.
  • the gene expression period (usually 2-3 weeks) of the Ad vector is shorter than that of the AAV vector and the chromosome-integrated vector, but the pancreatic ⁇ -cell protective effect of HGF far exceeds the gene expression period (at least 60 days or more). Since it lasts for preferably 75 days or longer, more preferably 90 days or longer, and even more preferably 120 days or longer), it may be rather advantageous in that the risk of side effects such as carcinogenic risk due to long-term expression of HGF can be reduced or avoided. ..
  • the gene size that can be loaded on the AAV vector is as small as 4.7 kb, but the HGF coding sequence (CDS) is about 2.2 kb, and the entire expression cassette including the promoter and terminator is about 3 to 4 kb. , There is no problem in use.
  • Ad vector is known to accumulate in the liver, but even if HGF is introduced into cells of other organs and expressed, it can be secreted extracellularly and delivered to the pancreas by the bloodstream. Further, the tissue orientation of the AAV vector differs depending on the serotype, and examples of the serotype having the directivity toward the pancreas include types 6, 8 and 9. However, if a ubiquitous promoter is used, HGF introduced and expressed in cells of other organs can also be delivered to the pancreas by bloodstream, so that the serotype used is not particularly limited. Rather, when viral vectors accumulate in the pancreas, ⁇ -cells may be attacked by virus capsid antigen-specific killer T cells, so it may be preferable to express HGF in cells of other organs.
  • the HGF-expressing recombinant viral vector used in the present invention is downstream of a promoter having transcriptional activity capable of conferring therapeutically effective blood HGF levels when administered at a dose of 10 10-10 12 vp / kg body weight. It is characterized by containing a nucleic acid encoding HGF.
  • the "therapeutically effective blood HGF level” means a blood HGF level sufficient to exert the above-mentioned "protection / regeneration of pancreatic ⁇ cells” effect.
  • the blood HGF level is not particularly limited as long as it is within a concentration range that exerts a protective / regenerating effect on pancreatic ⁇ cells in diabetic mammals, but for example, the peak blood HGF level is preferably 2 ng / ml or more. It is 2 to 5 ng / ml, and the average blood HGF level for one week after administration can be 0.6 ng / ml or more, preferably 1 ng / ml or more.
  • the "promoter with transcriptional activity capable of conferring therapeutically effective blood HGF levels” is such that the above blood HGF levels can be achieved when administered at a dose of 10 10 to 10 12 bp / kg body weight. It is not particularly limited as long as it has transcriptional activity, but it is stronger than the CMV promoter used in existing HGF gene therapeutic studies for diabetes in various cell types and depending on the type of viral vector used. A promoter having a high transcriptional activity can be used.
  • CA promoters and promoters having equivalent transcriptional activity for example, polypeptide chain extension factor 1 ⁇ 1 (EF1A) promoter, polypeptide chain extension factor 1 ⁇ 1 short type (EFS) promoter, CBh promoter ( Hybrid promoter of pre-CMV early enhancer and modified chicken ⁇ -actin promoter different from CA promoter), splenic focal localization virus (SFFV) promoter, mouse stem cell virus (MSCV) promoter, Simian virus 40 (SV40) enhancer / early stage Ubiquitous promoters such as promoters, phosphoglycerate kinase (PGK) promoters, and ubiquitin C (UBC) promoters can be mentioned.
  • EF1A polypeptide chain extension factor 1 ⁇ 1
  • EFS polypeptide chain extension factor 1 ⁇ 1 short type
  • CBh promoter Hybrid promoter of pre-CMV early enhancer and modified chicken ⁇ -actin promoter different from CA promoter
  • SFFV splenic focal local
  • a promoter that is highly expressed specifically in the tissue or cell of the target organ for example, an albumin promoter in the liver, an ⁇ -fet protein promoter, or a tyrosin bond.
  • a promoter that is highly expressed specifically in the tissue or cell of the target organ for example, an albumin promoter in the liver, an ⁇ -fet protein promoter, or a tyrosin bond.
  • examples thereof include, but are not limited to, insulin promoters in pancreatic ⁇ cells, Pdx1 promoters, Ins2 promoters, myogenin promoters in muscles, skeletal muscle actin ⁇ 1 (ACTA1) promoters, MHCK7 promoters, SM22a promoters and the like, such as globulin promoters.
  • Tissue- or cell-specific promoters of any organ in which the secreted and expressed HGF can be delivered to the pancreas by bloodstream are also included).
  • the promoter to be used has a transcriptional activity capable of conferring therapeutically effective blood HGF levels, for example, a viral vector containing a nucleic acid encoding HGF downstream of the promoter, 10 10 to 10 12 vp / kg.
  • a viral vector in which a reporter gene such as GFP is inserted instead of HGF is infected with a panel of cultured human cells, and the transcription activity for the CMV promoter is measured using the reporter activity as an index. Can be done.
  • a CA promoter is mentioned as a promoter that drives the expression of HGF.
  • the CA promoter used in the present invention is a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1, or a nucleic acid capable of hybridizing with the complementary strand sequence of the nucleic acid under stringent conditions, and is represented by SEQ ID NO: 1.
  • SEQ ID NO: 1 includes nucleic acids having transcriptional activity equal to or higher than that of nucleic acids consisting of the nucleotide sequences to be sequenced.
  • nucleic acids include, for example, about 80% or more, preferably about 90% or more, more preferably about 95% or more, particularly preferably about 97% or more, and most preferably about 90% or more, with respect to the nucleotide sequence represented by SEQ ID NO: 1.
  • Hybridization can be performed according to a method known per se or a method similar thereto, for example, the method described in Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). In addition, when using a commercially available library, hybridization can be performed according to the method described in the attached instruction manual. Hybridization can preferably be performed according to high stringent conditions. Stringent conditions include (1) low ionic strength and high temperature for cleaning, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50 ° C, and (2) formamide.
  • nucleic acid encoding HGF used in the present invention, contains a nucleotide sequence represented by (corresponding to), or a nucleotide sequence that hybridizes with its complementary chain sequence under stringent conditions, and encodes a protein having activity equivalent to that of HGF (eg, pancreatic ⁇ -cell protection / regeneration activity). Nucleic acid to be mentioned.
  • the nucleic acid that hybridizes with the complementary strand sequence of the nucleotide sequence represented by SEQ ID NO: 2 under stringent conditions is, for example, about 60% or more, preferably about 70% or more with the nucleotide sequence represented by SEQ ID NO: 2. , More preferably about 80% or more, particularly preferably about 90% or more, most preferably about 95% or more of nucleic acids containing nucleotide sequences.
  • the "stringent condition" is synonymous with the case of the promoter.
  • the nucleic acid has an amino acid sequence having about 90% or more, preferably about 95% or more, more preferably about 97% or more, particularly preferably about 98% or more identity with the amino acid sequence represented by SEQ ID NO: 3. It encodes an amino acid sequence such that the protein containing the amino acid sequence has substantially the same activity (eg, pancreatic ⁇ -cell protection / regeneration activity) as the protein containing the amino acid sequence represented by SEQ ID NO: 3. It is a thing.
  • the nucleic acid encoding HGF may be an ortholog of a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 2 in a non-human mammal.
  • a nucleic acid encoding HGF derived from the mammal to be administered is not particularly limited as long as it has diabetes, and examples thereof include humans, mice, rats, rabbits, dogs, and monkeys, but humans are preferable.
  • the nucleic acid encoding HGF is a nucleic acid encoding human HGF (ie, a protein consisting of the amino acid sequence represented by SEQ ID NO: 3).
  • the nucleic acid encoding HGF is amplified by the PCR method using, for example, a synthetic DNA primer having a part of the nucleotide sequence of the CDS region of the HGF gene, or the DNA incorporated into an appropriate expression vector is incorporated into the CDS region of the HGF gene. It can be cloned by hybridization with a DNA fragment containing a nucleotide sequence or a labeled DNA. Hybridization is performed, for example, Molecular Cloning, 2nd ed. It can be performed according to the method described in (mentioned above). In addition, when a commercially available library is used, hybridization can be performed according to the method described in the instruction manual attached to the library.
  • the nucleotide sequence of DNA can be obtained by using a known kit, for example, MutanTM-superExpress Km (Takara Shuzo Co., Ltd.), MutanTM-K (Takara Shuzo Co., Ltd.), ODA-LA PCR method, Gappedduplex method, Kunkel method, etc. It can be converted according to a method known per se or a method similar thereto.
  • the cloned DNA can be used as it is for the purpose, digested with a restriction enzyme if desired, or after adding a linker.
  • the DNA may have ATG as a translation initiation codon on its 5'end and TAA, TGA or TAG as a translation termination codon on its 3'end. These translation initiation codons and translation termination codons can be added using a suitable synthetic DNA adapter.
  • An expression vector containing a nucleic acid encoding HGF is produced, for example, by cutting out a target fragment from the nucleic acid encoding the CDS region of the HGF gene and linking the fragment downstream of the promoter in the expression vector described above. can do.
  • the expression vector preferably contains a transcription termination signal, i.e., a terminator region downstream of the nucleic acid encoding HGF.
  • it further contains selectable marker genes for selection of transformed cells (genes that impart resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinosricin, genes that complement nutritional demand mutations, etc.). You can also do it.
  • the HGF-expressing recombinant viral vector of the present invention can be prepared by utilizing conventional genetic engineering technology, cell culture technology, and virus production technology [for example, Currant Protocols in Molecular Biology, F.I. Ausubel et al. eds. (1994) John Wiley & Sons, Inc. Molecular Cloning (A Laboratory Manual), 3rd ed. Volumes 1-3, Joseph Sambook & David W. Russeleds. , Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) (2001); Culture of Animal Cells; A Manual of Basic Techniq. Freshney eds. , 2nd ed. (1987), Wiley-Liss; Frank L. et al.
  • the ⁇ -cell protection / regeneration agent of the present invention can suppress hyperglycemia for a long period of time while maintaining normal insulin secretion even when administered at a low dose such that adverse events due to the viral vector do not substantially occur.
  • T1D and other diabetes mellitus that destroys pancreatic ⁇ -cells and requires insulin administration (eg, insulin resistance progresses and compensatory insulin hypersecretion causes ⁇ -cell exhaustion, resulting in impaired insulin secretion and thus ⁇ -cell death. It can be used for the treatment of T2D, etc.) and for suppressing the progression to complications.
  • the HGF-expressing recombinant virus vector of the present invention may be used as the drug substance as it is, but if necessary, it may be mixed with a pharmacologically acceptable carrier, such as an injection. It can also be used as a medicine after being made into various formulations of the above.
  • the pharmacologically acceptable carrier various conventional organic or inorganic carrier substances are used as the pharmaceutical material, and a solvent, a solubilizing agent, a suspending agent, an isotonic agent, and a buffering agent in a liquid pharmaceutical product are used. , As a pain-relieving agent, etc., pharmaceutical additives such as preservatives, antioxidants, and colorants can also be used.
  • the solvent include water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, cottonseed oil and the like.
  • solubilizers are polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, sodium acetate. And so on.
  • suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride and glycerin monostearate, such as polyvinyl alcohol and polyvinyl.
  • surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride and glycerin monostearate, such as polyvinyl alcohol and polyvinyl.
  • hydrophilic polymers such as pyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose and hydroxypropyl cellulose, polysorbates, and polyoxyethylene hydrogenated castor oil.
  • the tonicity agent include sodium chloride, glycerin, D-mannitol, D-sorbitol, glucose and the like.
  • buffer agent examples include buffer solutions such as phosphates, acetates, carbonates and citrates.
  • the pain-relieving agent include benzyl alcohol and the like.
  • preservatives include paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
  • antioxidants include sulfites, ascorbic acid salts and the like.
  • colorants include water-soluble food tar pigments (eg, food dyes such as Edible Red Nos. 2 and 3, Food Yellow Nos. 4 and 5, and Food Blue Nos. 1 and 2), water-insoluble lake pigments.
  • water-soluble food tar pigments eg, food dyes such as Edible Red Nos. 2 and 3, Food Yellow Nos. 4 and 5, and Food Blue Nos. 1 and 2
  • water-insoluble lake pigments e.g., aluminum salt of the water-soluble food coloring
  • natural dyes eg, ⁇ -carotene, chlorophyll, red iron oxide, etc.
  • Examples of the dosage form of the pharmaceutical composition include parenteral preparations such as injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.) and infusions.
  • parenteral preparations such as injections (eg, subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.) and infusions.
  • the ⁇ -cell protection / regeneration agent of the present invention can be produced by a method commonly used in the field of pharmaceutical technology, for example, the method described in the Japanese Pharmacopoeia.
  • the content of the viral vector as the active ingredient in the preparation varies depending on the dosage form, the dose of the active ingredient and the like, but is, for example, about 0.1 to 100% by weight.
  • the virus titer is, for example, 10 10 to 10 11 pfu / ml (the physical titer is several to 100 times higher than the biological titer, so when converted to virus particles, it is 2 ⁇ 10 10 to 2 ⁇ . It can be appropriately adjusted to be about 10 13 vp / ml), but is not limited to this range.
  • Suitable formulations for parenteral administration include aqueous and non-aqueous isotonic sterile injections. May contain an antioxidant, a buffer solution, an antibacterial agent, an isotonic agent, and the like. Examples thereof include aqueous and non-aqueous sterile suspensions, which may include suspending agents, solubilizers, thickeners, stabilizers, preservatives and the like.
  • the most suitable dosage form in the present invention is an injection solution.
  • the ⁇ -cell protective / regenerating agent of the present invention has a lower dose than the existing HGF gene therapy (5 ⁇ 10 12 to 1.5 ⁇ 10 13 vp / kg body weight) for diabetes, and is 10 10 to 10 12 vp / kg body weight. It is characterized by being administered to a human or other mammalian subject at a dose of. When administered at such a low dose, the ⁇ -cell protective / regenerating agent of the present invention can be safely used without causing adverse events (particularly liver dysfunction characterized by elevated AST or ALT levels). can do. Moreover, since the expression of HGF is driven by a promoter having strong transcriptional activity, it is possible to impart therapeutically effective blood HGF levels even at such a low dose, and sufficient protection of pancreatic ⁇ cells. -A reproduction effect can be obtained.
  • the dose of the preparation is determined by factors of the preparation itself such as vector type, promoter activity, infectivity of viral vector (physical titer: biological titer (vp: PFU) ratio, administration route, disease). It can be appropriately selected within the above range depending on the severity, the animal species to be administered, the drug acceptability of the administration target, the body weight, the age, and other external factors.
  • the Ad vector is a virus.
  • the vp: PFU ratio is an important parameter for viral dose determination, as the particles themselves can cause dose-dependent acute toxicity. For example, in the examples below, 3 ⁇ 108 PFU / mouse (body weight about).
  • the Ad vector of 20 g about 1.5 ⁇ 10 10 PFU per 1 kg in the tail vein has the effect of protecting and regenerating pancreatic ⁇ cells (suppression of hyperglycemia and normal insulin secretion).
  • the dose based on the physical titer is about 3 ⁇ 10 10 to about 3 ⁇ .
  • deaths due to acute liver injury have been reported due to hepatic artery administration of Ad vector with a body weight of 6 ⁇ 10 11 vp / kg (the above non-patent literature).
  • Ad vector When using an Ad vector, it is desirable to administer at a dose of 10 11 vp / kg body weight or less.
  • a higher dose of viral vector is accumulated in the liver. Therefore, it is speculated that other systemic administrations, especially when administering viral vectors from peripheral veins, can be safely used at doses of 10 12 bp / kg body weight, whereas AAV vectors have recently been used.
  • liver disease in the low-dose In a clinical study in which deaths due to severe liver damage were reported in the high-dose (3 ⁇ 10 14 vp / kg body weight) group, liver disease in the low-dose (1 ⁇ 10 14 vp / kg body weight) group.
  • Ad vectors produced for clinical application should be 1.5 ⁇ from peripheral veins.
  • FDA Food and Drug Administration
  • the ⁇ -cell protection / regeneration agent of the present invention is preferably administered parenterally (for example, intravenously, subcutaneously, intramuscularly, intraperitoneally, locally infused, etc.) by injection, catheter, balloon catheter, or the like.
  • Systemic administration eg, intravenous, intraarterial, intramuscular, intraperitoneal, etc.
  • Local administration into the pancreatic duct is considered to be advantageous because gene transfer is almost confined to the pancreas, and thus the risk of adverse events due to gene transfer into multiple organs can be avoided at first glance.
  • Systemic administration which allows gene transfer into other organs, is clinically advantageous because of the risk of ⁇ -cell destruction by cell attack.
  • the liver is particularly important as an adverse event due to the viral vector when used at a relatively high dose of 5 ⁇ 10 11 to 1 ⁇ 10 12 bp / kg body weight.
  • administration routes such as hepatic artery administration in which a high-dose virus vector is delivered to the liver, and to administer the ⁇ -cell protection / regeneration agent of the present invention from, for example, a peripheral vein. ..
  • the administration frequency of the ⁇ -cell protection / regeneration agent of the present invention is not particularly limited. As shown in the examples below, even when the Ad vector is used, a single administration can exert a certain degree of hyperglycemia-suppressing effect over a long period of at least about 2.5 months. In addition, the glucose-responsive insulin secretory capacity of pancreatic ⁇ cells can be maintained (improved glucose tolerance) for at least 60 days. The mice of the examples are further followed up, and it is fully expected that the above effects will last for a longer period of time. The same can be said when an AAV vector in which the transgene is not integrated into the chromosome is introduced into a dividing cell.
  • the expression of HGF can be sustained for a longer period of time, and thus the expression of HGF can be sustained for a longer period of time (for example, 6 months or longer, preferably 1 year or longer, more preferably 1 year or longer). (Several years or more), it is considered that it can exert an action of suppressing hyperglycemia and an action of improving glucose tolerance. Therefore, the ⁇ -cell protection / regeneration agent of the present invention is a non-chromophore-integrated type, and even when an Ad vector or AAV vector, which has been conventionally regarded as safer than a retrovirus or a lentiviral vector and has been frequently used, is used, for example, at least.
  • It can be administered at once every 60 days or longer, preferably 75 days or longer, more preferably 90 days or longer, and even more preferably 120 days or longer. Further, depending on the type of viral vector used, it is possible to administer it at an administration interval of once every 3 months to several years, and in still another embodiment, it may be a single administration.
  • mice injected with STZ were randomly divided into two groups after 3 days (day 0), and the 3 ⁇ 108 plaque forming unit (pfu) Ad.
  • AST plasma aspartate aminotransferase
  • IPGTT intraperitoneal glucose tolerance test
  • Intravenous administration of low-dose Ad vector does not cause hepatic dysfunction
  • intravenous administration of high-dose Ad vector in vivo causes gene transfer primarily into the liver and is therefore severe. It has been reported that it may cause liver damage (Mol Genet Metab 2003; 80: 148-158 .; Hum Gene Ther 2020; 31: 695-696). Therefore, in order to evaluate liver damage caused by intravenous administration of a low-dose Ad vector, the above 1.
  • Plasma AST and ALT levels were measured on days 7, 14, and 21 in the mice of each group.
  • Intravenous administration of low-dose Ad vector improves glucose tolerance in T1D model mice. IPGTT was performed 16 and 60 days after administration of Ad Vector to confirm suppression of postprandial hyperglycemia and glucose-responsive insulin secretory capacity. gone. In the HGF gene-administered group, suppression of blood glucose elevation after glucose administration was observed on both the 16th day (Fig. 4) and the 60th day (Fig. 5) of administration (FIGS. 4A and 5A), and glucose was also observed in the insulin secretion reaction. An increase in insulin secretion was confirmed in response to the stimulus (FIGS. 4B and 5B).
  • a recombinant virus vector that expresses HGF under the transcriptional control of a strong promoter such as a CA promoter shows a protective and regenerating effect on pancreatic ⁇ cells at a low dose, and has a hyperglycemic inhibitory effect while maintaining normal insulin secretion. Therefore, it is possible to avoid the risk of adverse events associated with administration of a high-dose virus vector, and it is assumed even when a non-chromosomally integrated virus vector, which has been considered to be relatively safe in the past, is used. The effect is maintained for a long period of time far beyond the gene expression period. Therefore, the ⁇ -cell protection / regeneration agent of the present invention can be a safe and effective clinically applicable gene therapy agent for diabetes.
  • pancreatic ⁇ -cell protection / regeneration agent of the present invention is an alternative and versatile regeneration for diabetes such as T1D. It is extremely useful as a medical means.

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WILSON JMFLOTTE TR: "Moving Forward After Two Deaths in a Gene Therapy Trial of Myotubular Myopathy", HUM GENE THER, vol. 31, 2020, pages 695 - 696, XP055831049, DOI: 10.1089/hum.2020.182

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