Low-dose hepatocyte growth factor gene therapy for diabetes

Abstract

The present invention provides an agent for protecting and regenerating pancreatic beta cells in a mammal having diabetes, the agent containing a recombinant viral vector that expresses hepatocyte growth factor (HGF), wherein the agent is characterized by being administered in a dose of 1010-1012 virus particles (vp) / kg body weight, and by the virus vector including a nucleic acid that encodes HGF downstream of a promoter having transcription activity capable of yielding a therapeutically effective HGF level in the blood at said dose.

Description

[Technical Field]

[0001] The present invention relates to a gene therapy agent for diabetes that achieves both safety and therapeutic efficacy. More specifically, the present invention relates to a diabetes treatment agent that contains a viral vector comprising a nucleic acid encoding hepatocyte growth factor (HGF) downstream of a promoter capable of expressing a therapeutically effective amount of HGF, and is administered at a low dose, thereby substantially not causing adverse events due to the viral vector. [Background technology]

[0002] According to the International Diabetes Federation (IDF), as of 2019, there were 463 million people with diabetes worldwide, and if this trend continues, it is projected to reach 700 million by 2045. Type 2 diabetes (T2D), which accounts for 90% of all diabetes cases, is a disease in which insulin sensitivity decreases due to genetic predisposition and environmental factors (lifestyle). When insulin sensitivity decreases, the beta cells of the pancreas try to compensate for the lack of insulin action by excessively secreting insulin as a compensatory mechanism. However, if this condition persists for a long time, it leads to exhaustion of the beta cells, making it impossible to secrete enough insulin, resulting in hyperglycemia.

[0003] On the other hand, type 1 diabetes (T1D) is a disease in which pancreatic β-cells are destroyed by an autoimmune mechanism, leading to a depletion of insulin secretion and a state of hyperglycemia. T1D develops at a young age, but the details of its mechanism are still unknown. Patients with T1D require lifelong self-injection of insulin to manage their blood sugar levels. However, even with strict insulin therapy, blood sugar control is difficult, and the recurring cycles of hyperglycemia and severe hypoglycemia make it challenging to prevent the progression of complications. Therefore, innovative treatment methods that can cure T1D are needed.

[0004] As a treatment method to cure T1D, there is growing expectation for regenerative medicine that regenerates and reconstructs lost organ function. One such method is β-cell replacement therapy using islet transplantation, and this treatment method has been reported to be effective in improving glucose metabolism. However, T1D patients must use immunosuppressants after islet transplantation, and furthermore, there is a shortage of islet donors, so the actual application of this technology is limited.

[0005] Therefore, as another regenerative medicine approach to T1D, attempts are being made to directly "protect (induce anti-cell death) and / or proliferate (regenerate) (induce regenerative healing)" the patient's own remaining β-cells in the body through in vivo gene therapy (hereinafter, this may be collectively referred to as "protection and regeneration"). For example, several studies using hepatocyte growth factor (HGF) as a therapeutic gene for T1D have been reported (Non-Patent Literature 1-3). HGF was initially identified as a potent hepatocyte mitogen, but it is now known to be a multifunctional cytokine, and in preclinical and clinical studies, it has exerted therapeutic effects such as cell protection, anti-fibrosis, and regeneration induction for numerous diseases. In the pancreas, HGF is expressed in endothelial cells and mesenchymal cells, and c-Met, the receptor for HGF, is also known to be localized in pancreatic progenitor cells, islet cells, and ductal cells.

[0006] Non-patent document 1 discloses that administering a plasmid (20 μg) containing nucleic acid encoding HGF to mice before the onset of diabetes maintained insulin secretion. Non-patent document 2 discloses that administering a high dose (3.0 × 10) of an adeno-associated virus (AAV) vector containing nucleic acid encoding HGF to mice was effective. 11 Vector genome (vg); when converted to per kg of body weight, it is approximately 1.5 × 10 13(vg) It has been disclosed that administering the HGF expression vector intraductally to mice before the onset of diabetes suppressed the rise in blood glucose. However, in both cases, the HGF expression vector was administered "before" the onset of T1D, so its usefulness for clinical application to T1D patients, in which many β cells have already been destroyed, has not been demonstrated. Furthermore, the latter intraductal administration method, although gene transfer is almost entirely limited to the pancreas, is highly invasive and not a practical method of administration in human clinical practice.

[0007] Non-patent document 3 describes the use of a high dose (1.0 × 10⁶) of an adenovirus (Ad) vector expressing HGF in mice after the onset of T1D. 11 Virus particles (VP); when converted to per kg of body weight, approximately 5 x 10 12 It has been reported that partial response was achieved when administered via tail vein (VP).

[0008] However, in 1999, in a clinical trial at the University of Pennsylvania for ornithine transcarbamylase deficiency, a congenital metabolic disorder, 6 × 10 11 vp / kg body weight (total weight 3.8 × 10) 13 A fatal accident occurred due to a systemic inflammatory response induced by hepatic artery administration of the Ad vector (vp) (Non-Patent Literature 4). Therefore, regardless of the target disease, high-dose Ad vectors have not been clinically applied in gene therapy involving systemic administration of Ad via blood vessels since then. Because this risk was thought to be limited to Ad, AAV vectors have since become commonly used in clinical applications of in vivo gene therapy (systemic administration via blood vessels) for congenital diseases and other conditions.

[0009] However, recently (June 23, 2020), it has been discovered that even AAV vectors, which had been believed to be non-pathogenic and highly safe for a long period of time, have shown adverse effects in clinical trials of gene therapy for congenital myopathy at high doses (3 × 10⁻¹⁰). 14For patients administered with AAV vectors at (dose unit: vg / kg), three cases of severe adverse events, particularly deaths due to severe liver damage, were reported (Non-Patent Document 5, Non-Patent Document 6). As described above, AAV vectors are currently the most widely used vectors in the clinical application of in vivo gene therapy (systemic administration from blood vessels). However, this report has just revealed that systemic administration of high-dose vectors from blood vessels in vivo may cause serious adverse events including dangerous liver damage regardless of the type of vector, and the clinical application of in vivo gene therapy with high-dose vectors has been interrupted and reviewed. Thus, the death incident caused by AAV vectors has become a world-historical major event in the field of gene therapy.

Prior Art Documents

Non-Patent Documents

[0010]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0011] Therefore, the object of the present invention is to provide a gene therapy method for diabetes, including T1D, that achieves both safety and therapeutic effect. [Means for solving the problem]

[0012] To solve the above problems, the inventors first focused on the HGF gene as a therapeutic gene. In the HGF gene therapy for T1D reported so far, HGF has been expressed under the transcriptional control of the cytomegalovirus (CMV) promoter (Non-Patent Documents 1-3 above). In the case of viral vectors, it has been administered at high doses of about 5×10 12 to about 1.5×10 13 vp / kg body weight. The inventors considered that in order to realize a low dose of the viral vector while ensuring the safety of in vivo gene therapy and at the same time obtain the desired therapeutic effect, it was necessary to increase the expression efficiency of the therapeutic gene. Therefore, a CA promoter (a hybrid promoter of the CMV immediate early enhancer and the modified chicken β-actin promoter; also called the "CAG promoter", but uniformly referred to as the "CA promoter" in this specification), which has been reported to exhibit stronger transcriptional activity than the CMV promoter and other commonly used ubiquitous promoters in various cell types, was used to produce an Ad vector that expresses HGF under its transcriptional control. A model mouse after the onset of T1D was intravenously injected into the tail vein at a dose (equivalent to 3×10 8 pfu; 10 9 to 10 10 vp), which is one to two orders of magnitude lower than the previously reported dose. As a result, in the HGF treatment group, the increase in blood glucose was significantly suppressed about 1 week after administration compared to the non-treatment group, and the suppression tendency was maintained for a long period (at least 11 weeks after administration). In addition, compensatory insulin hypersecretion from β cells remaining in the acute phase of T1D was observed in the non-treatment group, while normal insulin secretion was maintained in the HGF treatment group. In the 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. In the examples described below, although an Ad vector with transient expression (usually 2 to 3 weeks) was used for gene transfer, surprisingly, the hyperglycemic inhibitory effect and the glucose tolerance improvement effect were successfully sustained far beyond the predicted expression period. These results indicate that even with low-dose viral vector administration, HGF demonstrated protective and regenerative effects on β-cells, and that these effects persisted for a much longer period than expected. On the other hand, no liver damage was observed with viral vector administration, and there were no changes in appetite or exercise during the subsequent follow-up period; no adverse events were observed. Based on these findings, the inventors have succeeded in safely and effectively treating T1D by administering a viral vector expressing HGF under transcriptional control of a promoter capable of expressing a therapeutically effective amount of HGF at a lower dose than existing doses, thereby completing the present invention.

[0013] In other words, the present invention is as follows: [1] A protective and regenerative agent for pancreatic β-cells in mammals with diabetes, comprising a recombinant viral vector expressing hepatocyte growth factor (HGF), 10 10 ~10 12 A drug administered at a dose of viral particles (vp) / kg body weight, characterized in that the viral vector contains a nucleic acid encoding HGF downstream of a promoter having transcriptional activity capable of giving therapeutically effective blood HGF levels at that dose. [2] The agent according to [1], wherein the viral vector is an adenovirus (Ad) vector or an adeno-associated virus (AAV) vector. [3] The agent described in [2], administered as a single dose or multiple times with at least 60 days between doses. [4] The agent according to any one of [1] to [3], wherein the promoter is a CA promoter. [5] A drug described in any of [1] to [4], for diabetes mellitus of type 1. [6] The agent described in any of [1] to [5], wherein the mammal is a human. [7] The agent described in any of [1] to [6], which is administered by systemic administration. [8] The agent described in [7], for which systemic administration is intravenous administration. [9] The agent described in [8], in which intravenous administration is administered from a peripheral vein.

[10] A method for protecting and regenerating pancreatic β-cells in a mammal having diabetes, comprising administering a recombinant viral vector expressing HGF to the mammal, wherein the viral vector expresses HGF, 10 ~10 12 A method comprising administering a dose of viral particles (vp) / kg body weight, wherein the dose contains a nucleic acid encoding HGF downstream of a promoter having transcriptional activity capable of giving therapeutically effective blood HGF levels. [Effects of the Invention]

[0014] According to the present invention, even with low-dose administration, the desired protective and regenerative effects on pancreatic β-cells can be achieved over a long period of time. Therefore, while ensuring safety, it becomes possible to perform high-quality in vivo gene therapy using HGF for diabetes requiring insulin administration, including T1D, thereby increasing the feasibility of applying this therapy to human clinical practice. [Brief explanation of the drawing]

[0015] [Figure 1]Figures 1A and 1C show the daily changes in blood glucose levels from day -7 to day 7 in mice that developed T1D after streptozotocin (STZ) administration and were injected via tail vein with an adenovirus vector in two independent experiments. The day the adenovirus vector was administered is considered day 0. Figures 1B and 1D show the weekly changes in blood glucose levels from day -7 to day 77 (or day 70) in the T1D model mice administered with the adenovirus vector (Figure 1B corresponds to Figure 1A, and Figure 1D corresponds to Figure 1C). In the figures, "Ad.CA-HGF" is the viral vector of the present invention that expresses the HGF gene under the control of the CA promoter, "Ad.CA-LacZ" is a control viral vector in which the β-galactosidase gene (LacZ) is inserted instead of HGF, and "Intact" shows data from normal mice that have not received any treatment. In each figure, the horizontal axis shows the number of days after viral vector administration, and the vertical axis shows the blood glucose level (mg / dl). *:p<0.05 [Figure 2] Figure 2 is a graph showing the results of measuring plasma insulin concentrations on days 7, 14, and 21 in the same mice as in Figure 1 (gray bars: T1D model mice administered with Ad.CA-LacZ; black bars: T1D model mice administered with Ad.CA-HGF; white bars: normal mice that did not receive STZ or viral vector administration). The vertical axis shows plasma insulin levels (ng / ml). *: p<0.05 [Figure 3] Figure 3 is a graph showing the results of measuring plasma AST levels on days 7, 14, and 21 in the same mice as in Figure 1 (each bar is equivalent to that in Figure 2). The vertical axis represents plasma AST level (IU / L). [Figure 4] Figure 1 shows the results of IPGTT in the same mice (16 days after Ad vector administration). Blood samples were taken 30, 60, and 120 minutes after glucose (2 g / kg body weight) administration, and blood glucose and plasma insulin levels were measured. Figure 4A shows the time course of blood glucose (mg / dl) after glucose administration, and Figure 4B shows the time course of plasma insulin levels (ng / ml) after glucose administration. *: p<0.05 (compared to Ad.CA-LacZ); #: p<0.05 (compared to Intact); ns: no significant difference [Figure 5] Figure 1 shows the results of IPGTT in the same mice (60 days after Ad vector administration). After glucose (2 g / kg body weight) administration, blood samples were collected at 30, 60, and 120 minutes after administration, and blood glucose and plasma insulin levels were measured. Figure 5A shows the time course of blood glucose (mg / dl) after glucose administration, and Figure 5B shows the time course of plasma insulin levels (ng / ml) after glucose administration. *: p<0.05 (compared to Ad.CA-LacZ); #: p<0.05 (compared to Intact); ns: no significant difference [Modes for carrying out the invention]

[0016] The present invention provides a safe and effective recombinant viral vector expressing HGF, which is a protective and regenerative agent for pancreatic β-cells in mammals with diabetes (hereinafter also referred to as "the β-cell protective and regenerative agent of the present invention"). The β-cell protective and regenerative agent is 10 10 ~10 12 The viral vector is administered at a dose of viral particles (vp) / kg body weight, and is characterized in that the viral vector contains a nucleic acid encoding HGF downstream of a promoter having transcriptional activity capable of giving therapeutically effective blood HGF levels at that dose.

[0017] In this specification, "protection and regeneration of pancreatic β-cells" means that, without causing compensatory excessive insulin secretion from β-cells, normal insulin secretion is maintained, and at least acute hyperglycemia is significantly suppressed compared to a control without therapeutic treatment, and the function of remaining β-cells is preserved and / or β-cells proliferate to the extent that the suppressive trend is maintained over a long period thereafter (e.g., 60 days or more, preferably 75 days or more, more preferably 90 days or more, and even more preferably 120 days or more). In other words, since "protection and regeneration of pancreatic β-cells" in the present invention necessarily involves "suppression of hyperglycemia," the "β-cell protective and regenerative agent of the present invention" can also be a "hyperglycemia suppressant." Furthermore, since suppressing hyperglycemia and controlling blood glucose is of paramount importance in the treatment of diabetes and the prevention of progression to complications, the "β-cell protective and regenerative agent of the present invention" can also be a "diabetes treatment agent."

[0018] According to Non-Patent Document 3 mentioned above, high-dose administration of an Ad vector expressing HGF under the control of the CMV promoter suppressed hyperglycemia, but the blood insulin / glucose ratio was significantly increased, leading the authors to conclude that compensatory excessive insulin secretion occurred. Therefore, the β-cell protective and regenerative agent of the present invention, which can suppress hyperglycemia while maintaining normal insulin secretion without inducing compensatory excessive insulin secretion, offers advantageous effects such as reducing the risk of β-cell exhaustion and subsequent β-cell dysfunction due to excessive insulin secretion.

[0019] The viral vector used in the HGF-expressing recombinant viral vector, which is the active ingredient of the β-cell protection and regeneration agent of the present invention, is not particularly limited as long as it is a viral vector commonly used in gene therapy. For example, adenovirus (Ad) vectors, adeno-associated virus (AAV) vectors, lentiviral vectors, retroviral vectors, Sindbisvirus vectors, rabies virus vectors, Sendai virus vectors, herpes simplex virus vectors, etc., can be used. From the viewpoint of low frequency of chromosomal integration and no risk of insertional mutations, the ability to be introduced into non-dividing cells, and the ability to express the introduced gene over the medium to long term, it is preferable to use an Ad vector or an AAV vector.

[0020] Although the gene expression period of Ad vectors (usually 2-3 weeks) is shorter than that of AAV vectors and chromosome-integrated vectors, the pancreatic β-cell protective effect of HGF persists far beyond the gene expression period (at least 60 days, preferably 75 days, more preferably 90 days, and even more preferably 120 days or more). Therefore, it may actually be advantageous in that it can reduce or avoid the risk of side effects such as cancer risk due to long-term HGF expression. Furthermore, while AAV vectors have a small gene size of 4.7kb, the HGF coding sequence (CDS) is approximately 2.2kb, and the entire expression cassette including promoters and terminators is only about 3-4kb, so there are no problems with its use.

[0021] While Ad vectors are known to accumulate in the liver, HGF, even when introduced and expressed in cells of other organs, can be secreted extracellularly and delivered to the pancreas via the bloodstream. Furthermore, AAV vectors exhibit different tissue targeting depending on the serotype, with serotypes 6, 8, and 9 being examples of serotypes that target the pancreas. However, if a ubiquitous promoter is used, HGF introduced and expressed in cells of other organs can still be delivered to the pancreas via the bloodstream, so there are no particular restrictions on the serotype used. Rather, if viral vectors accumulate in the pancreas, there is a risk that β cells may be attacked by killer T cells specific to the viral capsid antigen, so it may be preferable to express HGF in cells of other organs.

[0022] The HGF-expressing recombinant viral vector used in the present invention is 10 10 ~10 12 The drug is characterized by containing a nucleic acid encoding HGF downstream of a promoter having transcriptional activity capable of giving therapeutically effective blood HGF levels when administered at a dose of vp / kg body weight. Here, "therapeutically effective blood HGF levels" means blood HGF levels sufficient to exert the aforementioned "protective and regenerative effect on pancreatic β-cells." The blood HGF level is not particularly limited as long as it is within the concentration range that exerts a protective and regenerative effect on pancreatic β-cells in mammals with diabetes, but for example, the peak blood HGF level may be 2 ng / ml or higher, preferably 2 to 5 ng / ml, and the average blood HGF level one week after administration may be 0.6 ng / ml or higher, preferably 1 ng / ml or higher.

[0023] "Promoters having transcriptional activity capable of giving therapeutically effective blood HGF levels" are defined as 10 10 ~10 12There are no particular restrictions as long as the promoter has transcriptional activity sufficient to achieve the above-mentioned blood HGF levels when administered at a dose of vp / kg body weight. However, depending on the cell type and the type of viral vector used, a promoter with stronger transcriptional activity than the CMV promoter used in existing HGF gene therapy research for diabetes can be used. Examples of such highly active promoters include the CA promoter and promoters with equivalent transcriptional activity, such as the polypeptide chain elongation factor 1α1 (EF1A) promoter, polypeptide chain elongation factor 1α1 short type (EFS) promoter, CBh promoter (a hybrid promoter of the CMV pre-initial enhancer and a modified chicken β-actin promoter different from the CA promoter), spleen-limited focal-forming virus (SFFV) promoter, mouse stem cell virus (MSCV) promoter, Simian virus 40 (SV40) enhancer / initial promoter, phosphoglycerate kinase (PGK) promoter, and ubiquitin C (UBC) promoter. Furthermore, when using highly tissue-targeting viral vectors or in the case of local administration, promoters that are specifically highly expressed in the tissue or cells of the target organ may also be used (for example, albumin promoter, α-fetoprotein promoter, thyroxine-binding globulin promoter in the liver, insulin promoter, Pdx1 promoter, Ins2 promoter in pancreatic β-cells, myogenin promoter, skeletal muscle actin α1 (ACTA1) promoter, MHCK7 promoter, SM22a promoter in muscle, etc., but not limited to these, and encompassing any tissue or cell-specific promoter of any organ from which HGF secreted and expressed can be delivered to the pancreas via the bloodstream). The promoter to be used must have transcriptional activity that can give therapeutically effective blood HGF levels, for example, by placing a viral vector containing the nucleic acid encoding HGF downstream of the promoter. 10 ~10 12The therapeutic effect can be confirmed by administering the drug to experimental animals such as mice at a dose equal to vp / kg body weight, and then measuring the HGF level in blood collected over time after administration using methods such as ELISA. More simply, this can also be done by, for example, infecting a panel of human cultured cells with a viral vector containing a reporter gene such as GFP instead of HGF, and then measuring the transcriptional activity toward the CMV promoter using reporter activity as an indicator.

[0024] In a particularly preferred embodiment, a CA promoter is used as a promoter that drives HGF expression. The CA promoter used in the present invention includes nucleic acids consisting of the nucleotide sequence represented by SEQ ID NO: 1, or nucleic acids that can hybridize with a complementary strand sequence of said nucleic acid under stringent conditions, and which have transcriptional activity equivalent to or greater than that of the nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 1. Examples of such nucleic acids include nucleic acids containing a nucleotide sequence that has about 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 1, preferably about 90% or more, more preferably about 95% or more, particularly preferably about 97% or more, and most preferably about 98% or more. The homology of nucleotide sequences in this specification can be calculated, for example, using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; gaps allowed; filtering = ON; match score = 1; mismatch score = -3).

[0025] Hybridization can be carried out according to known methods or similar methods, for example, the method described in Molecular Cloning, 2nd ed. (J. Sambrook et al., Cold Spring Harbor Lab. Press, 1989). When using commercially available libraries, hybridization can be carried out according to the method described in the accompanying instructions for use. Hybridization can preferably be carried out under high-stringent conditions. Examples of stringent conditions include (1) using low ionic strength and high temperature for washing, for example, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C, and (2) using a denaturing agent such as formamide, for example, 50% (v / v) formamide at 42°C with 50 mM sodium phosphate buffer (pH 6.5) containing 0.1% bovine serum albumin / 0.1% Ficol / 0.1% polyvinylpyrrolidone / 750 mM sodium chloride and 75 mM sodium citrate. Alternatively, stringent conditions may involve using 50% formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhart solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42°C, washing with 0.2x SSC and 50% formaldehyde at 55°C, followed by a highly stringent wash with 0.1x SSC containing EDTA at 55°C. Those skilled in the art can easily achieve the desired stringency by appropriately adjusting the temperature during the hybridization reaction and / or washing, the ionic strength of the buffer, etc., depending on factors such as probe length.

[0026] The "HGF-encoding nucleic acid" used in the present invention includes a nucleotide sequence represented by Sequence ID No. 2 (corresponding to the nucleotide sequence (CDS) from position 77 to 2263 of the human HGF mRNA sequence registered in GenBank as accession number: NM_000601), or a nucleotide sequence that hybridizes with its complementary strand sequence under stringent conditions, and which encodes a protein having activity equivalent to HGF (e.g., pancreatic β-cell protective and regenerative activity). Nucleic acids that hybridize under stringent conditions with the complementary chain sequence of the nucleotide sequence represented by SEQ ID NO: 2 include, for example, nucleic acids containing a nucleotide sequence that has approximately 60% or more identity with the nucleotide sequence represented by SEQ ID NO: 2, preferably approximately 70% or more, more preferably approximately 80% or more, particularly preferably approximately 90% or more, and most preferably approximately 95% or more. Here, "stringent conditions" is the same as in the case of the promoter described above. Furthermore, the nucleic acid is an amino acid sequence that has approximately 90% or more identity with the amino acid sequence represented by SEQ ID NO: 3, preferably approximately 95% or more, even more preferably approximately 97% or more, and particularly preferably approximately 98% or more, and encodes an amino acid sequence such that the protein containing the amino acid sequence has substantially equivalent activity (e.g., pancreatic β-cell protective and regenerative activity) to the protein containing the amino acid sequence represented by SEQ ID NO: 3.

[0027] The nucleic acid encoding HGF may be an ortholog in a non-human mammal of the nucleic acid consisting of the nucleotide sequence represented by Sequence ID No. 2. For example, it is desirable to use a nucleic acid encoding HGF derived from the mammal to which the agent is administered. The mammal to which the β-cell protective and regenerative agent of the present invention is administered is not particularly limited as long as it has diabetes, and examples include humans, mice, rats, rabbits, dogs, monkeys, etc., but humans are preferred. Therefore, in a preferred embodiment, the nucleic acid encoding HGF is a nucleic acid encoding human HGF (i.e., a protein consisting of the amino acid sequence represented by Sequence ID No. 3).

[0028] The nucleic acid encoding HGF can be cloned by, for example, amplifying it using PCR with a synthetic DNA primer having a portion of the nucleotide sequence of the CDS region of the HGF gene, or by hybridizing DNA incorporated into a suitable expression vector with a DNA fragment or synthetic DNA labeled with the nucleotide sequence of the CDS region of the HGF gene. Hybridization can be performed, for example, according to the method described in Molecular Cloning, 2nd ed. (mentioned above). Furthermore, when using a commercially available library, hybridization can be performed according to the method described in the instructions for use provided with the library.

[0029] The nucleotide sequence of DNA can be converted using known kits, such as Mutan™-super Express Km (Takara Shuzo Co., Ltd.) or Mutan™-K (Takara Shuzo Co., Ltd.), according to known methods such as ODA-LA PCR, Gappedduplex, Kunkel, or similar methods.

[0030] The cloned DNA can be used as is, or, depending on the purpose, after being digested with restriction enzymes or after linker addition as desired. The DNA may have an ATG translation start codon at its 5' end and a TAA, TGA, or TAG translation stop codon at its 3' end. These translation start and stop codons can be added using a suitable synthetic DNA adapter.

[0031] An expression vector containing a nucleic acid encoding HGF can be produced, for example, by excising a target fragment from the nucleic acid encoding the CDS region of the HGF gene and ligating the fragment downstream of the promoter in the expression vector described above. Preferably, the expression vector contains a transcription termination signal, i.e., a terminator region, downstream of the nucleic acid encoding HGF. Furthermore, it may also contain selection marker genes for transformed cell selection (such as genes that confer resistance to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin, or genes that complement nutrient requirement mutations).

[0032] The HGF-expressing recombinant viral vector of the present invention can be prepared using conventional gene engineering techniques, cell culture techniques, and virus production techniques. [For example, Current Protocols in Molecular Biology, F. Ausubel et al. eds. (1994) John Wiley & Sons, Inc.; Molecular Cloning (A Laboratory Manual), 3rd ed. Volumes 1-3, Joseph Sambrook & David W. Russelleds., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, New York) (2001); Culture of Animal Cells; A Manual of Basic Technique, R. Freshney eds., 2nd ed. (1987) Wiley-Liss; Frank L. Graham, Manipulation of adenovirus vector, Chapter 11, p109-p128; EJ Murray eds., Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression] Protocols (1991); Chen, SH. et al., Combination gene therapy for liver metastases of colon carcinoma in vivo., Proc. Natl. Acad. Sci. USA (1995) 92, 2477-2581, etc.].

[0033] The β-cell protective and regenerative agent of the present invention can suppress hyperglycemia over a long period of time while maintaining normal insulin secretion, even when administered at a low dose that substantially does not cause adverse events caused by the viral vector. Therefore, it can be used to treat T1D and other types of diabetes in which pancreatic β-cells are destroyed and insulin administration is required (for example, T2D in which insulin resistance progresses, leading to compensatory excessive insulin secretion that exhausts β-cells, resulting in insulin secretion disorders and ultimately β-cell death), and to suppress the progression to complications.

[0034] The β-cell protective and regenerating agent of the present invention may use the HGF-expressing recombinant viral vector of the present invention as is, but it can also be mixed with a pharmacologically acceptable carrier as needed to form various formulations such as injectable preparations, which can then be used as pharmaceuticals.

[0035] Here, various organic or inorganic carrier substances commonly used as pharmaceutical materials are used as pharmacologically acceptable carriers, and are incorporated as solvents, solubilizers, suspending agents, isotonic agents, buffers, and analgesics in liquid formulations. Pharmaceutical additives such as preservatives, antioxidants, and colorants may also be used as needed.

[0036] Suitable examples of solvents include sterile water for injection, physiological saline, Ringer's solution, alcohol, propylene glycol, polyethylene glycol, sesame oil, corn oil, olive oil, and cottonseed oil.

[0037] Suitable examples of solubilizers include polyethylene glycol, propylene glycol, D-mannitol, trehalose, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, sodium salicylate, and sodium acetate.

[0038] Suitable examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, and glyceryl monostearate; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethylcellulose, methylcellulose, hydroxymethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose; polysorbates; and polyoxyethylene hydrogenated castor oil.

[0039] Suitable examples of isotonic agents include sodium chloride, glycerin, D-mannitol, D-sorbitol, and glucose.

[0040] Suitable examples of buffering agents include buffer solutions such as phosphates, acetates, carbonates, and citrates.

[0041] Suitable examples of pain relievers include benzyl alcohol.

[0042] Suitable examples of preservatives include para-hydroxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, and sorbic acid.

[0043] Suitable examples of antioxidants include sulfites and ascorbic acid salts.

[0044] Suitable examples of colorants include water-soluble food tar dyes (e.g., food colorants such as Food Red No. 2 and 3, Food Yellow No. 4 and 5, Food Blue No. 1 and 2), water-insoluble lake dyes (e.g., aluminum salts of the aforementioned water-soluble food tar dyes), and natural pigments (e.g., β-carotene, chlorophyll, red iron oxide, etc.).

[0045] Examples of dosage forms for the pharmaceutical composition include injections (e.g., subcutaneous injections, intravenous injections, intramuscular injections, intraperitoneal injections, etc.) and parenteral preparations such as intravenous drips.

[0046] The β-cell protective and regenerative agent of the present invention can be manufactured by conventional methods in the pharmaceutical technology field, such as those described in the Japanese Pharmacopoeia. The content of the viral vector, which is the active ingredient in the formulation, varies depending on the dosage form, the amount of active ingredient administered, etc., but is, for example, about 0.1 to 100% by weight. The viral titer is, for example, 10 10 ~10 11 pfu / ml (The physical titer is several times to more than 100 times higher than the biological titer, so when converted to virus particles it is 2 × 10⁻¹⁰ 10 ~2×10 13 The concentration can be adjusted as needed to approximately vp / ml, but it is not limited to this range.

[0047] Suitable formulations for parenteral administration (e.g., intravenous injection, subcutaneous injection, intramuscular injection, local injection, intraperitoneal administration, etc.) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, isotonic agents, etc. Also, aqueous and non-aqueous sterile suspensions are examples, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. The most preferred dosage form in the present invention is an injection solution.

[0048] The β-cell protective and regenerative agent of the present invention is similar to existing HGF gene therapy (5 × 10) for diabetes. 12 or 1.5 × 10 13 10 (a lower dose than vp / kg body weight) 10 ~10 12 The present invention is characterized by administration to humans or other mammals at a dose of vp / kg body weight. By administering at such low doses, the β-cell protective and regenerative agent of the present invention can be used safely without substantially causing adverse events (particularly liver dysfunction characterized by elevated AST and ALT levels). Moreover, since HGF expression is driven by a promoter with potent transcriptional activity, even with such low doses, therapeutically effective blood HGF levels can be achieved, and sufficient protective and regenerative effects on pancreatic β-cells can be obtained.

[0049] The dosage of the formulation can be appropriately selected within the above range, depending on factors of the formulation itself, such as the type of vector, promoter activity, and infectivity of the viral vector (physical titer:biological titer (vp:PFU) ratio), as well as external factors such as the route of administration, the severity of the disease, the target animal species, the drug acceptability of the target, body weight, and age. In particular, for Ad vectors, the vp:PFU ratio is an important parameter for determining the viral dose, as the viral particles themselves can induce dose-dependent acute toxicity. For example, in the examples described later, 3 × 10⁻⁶ 8 PFU / mouse (approximately 20g of body weight; approximately 1.5 x 10 per kg) 10It has been confirmed that tail vein administration of an ad vector containing PFU (Plant-Voltage-Factor) produces protective and regenerative effects on pancreatic β-cells (suppression of hyperglycemia and normal insulin secretion). However, the vp:PFU ratio varies greatly, ranging from several times to more than 100 times, due to factors such as the viral extraction method. Therefore, the dose based on physical potency is approximately 3 × 10⁻⁶. 10 ~Approx. 3×10 12 It can be vp / kg body weight. However, as mentioned above, 6 × 10 11 Since there have been reports of deaths due to acute liver injury following hepatic artery administration of Ad vectors at a dose of vp / kg body weight (see Non-Patent Document 4 above), when using Ad vectors, 10 11 It is desirable to administer at a dose of 10 / kg body weight or less. However, in the case of hepatic artery administration as described in the literature, it is thought that a higher dose of the viral vector will accumulate in the liver, so in other systemic administrations, especially when administering the viral vector via peripheral veins, 10 12 It is presumed that it can be safely used even at doses of vp / kg body weight. On the other hand, in the case of AAV vectors, recently, high doses (3 × 10) have been observed. 14 In clinical trials where deaths due to severe liver damage were reported in the group receiving vp / kg body weight, low doses (1 × 10) were also observed. 14 In the group administered vp / kg body weight, no adverse liver events occurred despite the presence of liver disease, therefore, 10 12 It is presumed that it can be safely used even at doses of vp / kg body weight. Furthermore, the U.S. Food and Drug Administration (FDA) recommends a vp:PFU ratio of less than 30 for clinical-grade Ad vectors. Therefore, Ad vectors manufactured for clinical use should be administered via peripheral vein in a 1.5 × 10⁶ dose. 10 When administered at an infectious titer of PFU / kg body weight, 4.5 × 10 11 Since it will be administered at a dose less than vp / kg body weight, 6 × 10 11 This is considered to be a sufficiently low dose compared to hepatic arterial administration at a dose per kg of body weight.

[0050] The β-cell protective and regenerative agent of the present invention is preferably administered parenterally (e.g., intravenously, subcutaneously, intramuscularly, intraperitoneally, locally) by means of injection, catheter, balloon catheter, etc., and more preferably systemically (e.g., intravenously, intra-arterially, intramuscularly, intraperitoneally). Local administration into the pancreatic duct may seem advantageous because gene transfer is almost entirely confined to the pancreas, thus avoiding the risk of adverse events caused by gene transfer to multiple organs. However, there is a risk of β-cell destruction due to attack by killer T cells against the introduced gene or virus. Therefore, systemic administration, which enables gene transfer to other organs, is clinically advantageous. Among the dose ranges of the viral vector used in the present invention, a relatively high dose of 5 × 10⁻⁶ is preferred. 11 ~1 × 10 12 When using a dose of vp / kg body weight, in order to avoid liver damage, which is a particularly important adverse event caused by the viral vector, it is desirable to avoid administration routes that deliver high doses of the viral vector to the liver, such as hepatic artery administration, and instead administer the β-cell protective and regenerative agent of the present invention via, for example, a peripheral vein.

[0051] The frequency of administration of the β-cell protective and regenerative agent of the present invention is not particularly limited. As shown in the examples below, even when using an Ad vector, a single administration can produce a certain degree of hyperglycemia suppression for a long period of at least about 2.5 months. Furthermore, it can maintain the glucose-responsive insulin secretion ability of pancreatic β-cells (improve glucose tolerance) for at least 60 days. The mice in the examples are being observed further, and it is well expected that the above effects will persist for an even longer period. The same may be true when introducing an AAV vector, in which the transgene is not incorporated into the chromosome in principle, into dividing cells. Furthermore, when non-dividing cells are targeted using AAV vectors, HGF expression can be sustained for a longer period, and therefore, it is thought that the effects of suppressing hyperglycemia and improving glucose tolerance can be exerted for an even longer period (for example, 6 months or more, preferably 1 year or more, more preferably several years or more). Therefore, the β-cell protective and regenerative agent of the present invention is a non-chromosome-integrated type and can be administered at intervals of at least 60 days, preferably 75 days, more preferably 90 days, and even more preferably 120 days or more, even when using Ad vectors or AAV vectors, which have been conventionally considered safer and frequently used compared to retroviruses and lentiviral vectors. Furthermore, depending on the type of viral vector used, it is possible to administer it at intervals of once every 3 months to several years, and in yet another embodiment, it may even be administered as a single dose.

[0052] The present invention will be described in more detail below with reference to examples, but these are merely illustrative and do not limit the scope of the present invention in any way. [Examples]

[0053] Experimental method 1. Preparation of recombinant adenovirus vectors (Ad) Non-regenerative Ad (hereinafter referred to as "Ad.CA-HGF") expressing human HGF under transcriptional regulation by a hybrid promoter (CA promoter) of a cytomegalovirus immediate early enhancer and a modified chicken β-actin promoter, and non-regenerative Ad (hereinafter referred to as "Ad.CA-LacZ") expressing the LacZ gene under transcriptional regulation by the CA promoter, were prepared using the method described in HUMAN GENE THERAPY 10:2013-2017 (1999). The recombinant Ad thus prepared were amplified, purified, and titered using the conventional method described in Proc. Natl. Acad. Sci. USA 92: 2577-2581 (1995).

[0054] 2. Animal experiments Eight-week-old male c57 / BL / 6N mice (Kyudo Co., Ltd., Tosu) weighing 18g-20g were raised in a state where they could freely access food and water. Similar to a previously reported study (Diabetes 2010; 59: 1261-1265), streptozotocin (STZ; Sigma-Aldrich Japan, Tokyo), dissolved in 0.01M citrate buffer (pH 4.5), was administered intraperitoneally to 25 mice at a dose of 50 mg / kg once daily for 5 consecutive days (from day -7 to day -3). Three days later (day 0), the STZ-injected mice were randomly divided into two groups, and 3 × 10⁶ mice were divided into these groups. 8 Either Ad.CA-LacZ (n=13) or Ad.CA-HGF (n=12), both plaque-forming units (pfu), were injected once into the tail vein. Mice that had not received STZ injection or adenovirus gene therapy (n=8) were used as normal controls. Blood was collected from all mice, including the normal group. Blood collection was performed daily from day -7 to day 7, and weekly from day 14 to day 77. Blood glucose, plasma insulin, and plasma aspartate aminotransferase (AST) levels were tested at the time points shown in each figure. Intraperitoneal glucose tolerance tests (IPGTT) were performed on days 16 and 60 after Ad vector administration. After fasting mice for 14 hours, 2 g / kg body weight (equivalent to a 75 g oral glucose tolerance test (OGTT) in humans) of glucose was administered intraperitoneally. Blood was collected immediately before administration and 30, 60, and 120 minutes after administration, and blood glucose and plasma insulin levels were measured at each time point. Insulin levels were measured using a highly sensitive mouse insulin ELISA kit (Morinaga Institute of Life Science).

[0055] All animal experiments were conducted in accordance with the guidelines of the National Institutes of Health in the United States and with the approval of the Kagoshima University Animal Experiment Ethics Committee.

[0056] 3.Biochemical analysis Blood glucose levels were measured using Glucocard G Black (Arkray, Kyoto) and Medisafe Fit Pro II (Terumo, Tokyo), while plasma AST and ALT levels were measured using the SPOTCHEM SP-4430 clinical automated analyzer (Arkray). Plasma insulin levels were measured using an ELISA assay (Morinaga Ltd., Yokohama).

[0057] 4.Statistical analysis Data are presented as mean ± standard error (se). Multiple comparisons were tested using one-way ANOVA, and Student's t-test was used to determine whether there was a statistically significant difference between mice treated with Ad.CA-LacZ and mice treated with Ad.CA-HGF. A p-value of < 0.05 was defined as statistically significant.

[0058] Experimental results 1. Suppression of acute hyperglycemia in T1D model mice by low-dose Ad.CA-HGF gene therapy. In the untreated group (Ad.CA-LacZ administered group), blood glucose levels rapidly increased to 250 mg / dl on day 7 after vector administration (14 days after the first STZ injection) (Figure 1A), and continued to gradually increase thereafter, remaining at a high level of around 270 mg / dl (Figure 1B). 3×10 8 pfu (for clinical-grade Ad vectors, approximately 10 9 ~about 10 10 A single intravenous injection of Ad.CA-HGF (equivalent to vp) significantly suppressed the rise in blood glucose levels between days 3 and 6 (Figure 1A). During subsequent follow-up over approximately 3 months, blood glucose levels showed a tendency to be suppressed compared to the untreated group, demonstrating a long-term effect (Figure 1B). Repeated animal experiments using the same protocol yielded similar results, confirming the reproducibility of the hyperglycemia-suppressing effect of low-dose HGF administration (Figures 1C, 1D). In a previous report (Exp Mol Med 2003; 35: 494-500), an Ad vector expressing HGF under the control of the CMV promoter was described as 1 × 10⁶ 11 vp(approx. 5×10 12The dose administered is one to two orders of magnitude higher than the assumed value (vp / kg body weight) in this embodiment. Although a direct comparison is not possible due to the different administration routes, this dose is nearly 10 times higher than that used in cases of death caused by Ad vectors (Non-Patent Literature 4 above), which has hindered its application to human clinical practice. The present invention is extremely significant because, by using a CA promoter with more potent transcriptional activity, we have succeeded in achieving an equivalent or greater hyperglycemia suppression effect even at a dose 1 / 10 to 1 / 100 of that reported previously.

[0059] 2. Suppression of acute plasma insulin elevation in T1D model mice by low-dose Ad.CA-HGF gene therapy (protection and regeneration of residual β-cells). To evaluate the mechanism by which the therapeutic effect is obtained, plasma insulin concentrations were measured on days 7, 14, and 21 in the STZ-injected mice described in 1. above (Figure 2). On day 7, a significant increase in plasma insulin concentration was observed in the untreated group (Ad.CA-LacZ administered group). This phenomenon is consistent with previous findings (Environ Toxicol Pharmacol 2001; 9: 71-78) that STZ treatment destroys β-cells, and the remaining β-cells compensate by temporarily producing and secreting abnormally high levels of insulin, suggesting that β-cell death is progressing in the control mice. On the other hand, no increase in plasma insulin concentration was observed on day 7 in the Ad.CA-HGF administered group, and plasma insulin levels remained normal thereafter. These results suggest that systemic administration of low-dose Ad.CA-HGF allowed HGF secreted from gene-transfected cells to be delivered to the pancreas via the bloodstream, suppressing β-cell death (cytoprotective effect), resulting in a greater number of β-cells remaining, maintaining normal insulin secretion, and thus providing a therapeutic effect (hyperglycemia suppression). This offers further advantages over previous reports (Exp Mol Med 2003; 35: 494-500) which showed elevated insulin / glucose ratios suggesting compensatory excessive insulin secretion was induced by high-dose administration of HGF-expressing Ad vectors.

[0060] 3. Intravenous administration of low-dose Ad vectors does not cause hepatic impairment. In preclinical and clinical trials, it had been reported that high doses of Ad vector administered intravenously in vivo primarily resulted in gene transfer into the liver, potentially causing severe liver damage (Mol Genet Metab 2003; 80: 148-158; Hum Gene Ther 2020; 31: 695-696). Therefore, to evaluate liver damage caused by intravenous administration of low doses of Ad vector, plasma AST and ALT levels were measured on days 7, 14, and 21 in mice of each group described in 1. above. No significant increase in plasma AST levels was observed in either the Ad vector-administered mice (Ad.CA-LacZ group and Ad.CA-HGF group) until week 3 (Figure 3). Similarly, no significant increase in plasma ALT levels was observed until week 3. Throughout the subsequent follow-up period, all mice showed no changes in appetite or exercise, remained healthy, and survived until day 77, when the experiment concluded. These data suggest that a gene therapy strategy involving a single, low-dose intravenous injection of an Ad vector expressing the HGF gene under strong promoter transcriptional regulation is both effective and safe.

[0061] 4. Intravenous administration of low-dose Ad vectors improves glucose tolerance in T1D model mice. IPGTT was performed 16 and 60 days after administration of the Ad vector to confirm the suppression of postprandial hyperglycemia and glucose-responsive insulin secretion capacity. In the HGF gene administration group, suppression of the rise in blood glucose after glucose administration was observed on both day 16 (Figure 4) and day 60 (Figure 5) (Figures 4A, 5A), and an increase in insulin secretion in response to glucose stimulation was confirmed in the insulin secretion response (Figures 4B, 5B). [Industrial applicability]

[0062] Recombinant viral vectors expressing HGF under the transcriptional control of strong promoters such as the CA promoter exhibit protective and regenerative effects on pancreatic β-cells at low doses, suppressing hyperglycemia while maintaining normal insulin secretion, thus avoiding the risk of adverse events associated with high-dose viral vector administration. Furthermore, even when using non-chromosomal-integrated viral vectors, which have traditionally been considered relatively safe, these effects are maintained for a long period far exceeding the expected gene expression period. Therefore, the β-cell protective and regenerative agent of the present invention can be a safe and effective gene therapy agent for diabetes that can be clinically applied. The number of diabetes patients continues to increase worldwide, becoming a social problem, and the significance of the present invention is great. In particular, T1D develops at a young age, and existing islet transplantation therapy has limited use, so the pancreatic β-cell protective and regenerative agent of the present invention is extremely useful as an alternative and versatile means of regenerative medicine for diabetes, including T1D.

[0063] This application is based on Japanese Patent Application No. 2020-192844 (filed November 19, 2020) and Japanese Patent Application No. 2021-145795 (filed September 7, 2021), both of which are fully incorporated herein.

Claims

1. A therapeutic agent for type 1 or type 2 diabetes in mammals, comprising a recombinant viral vector expressing hepatocyte growth factor (HGF), 10 １０ A drug administered systemically at a dose of ~10¹¹ virus particles (vp) / kg body weight, characterized in that the viral vector, at that dose, contains a nucleic acid encoding HGF downstream of the CA promoter, wherein the viral vector is an adenovirus (Ad) vector or an adeno-associated virus (AAV) vector.

2. The agent according to claim 1, which is administered as a single dose or multiple times with an interval of at least 60 days between doses.

3. The agent according to claim 1 or 2, wherein the diabetes is type 1 diabetes.

4. The agent according to any one of claims 1 to 3, wherein the mammal is a human.

5. The agent according to any one of claims 1 to 4, wherein systemic administration is intravenous administration.

6. The agent according to claim 5, wherein intravenous administration is administered from a peripheral vein.

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