WO2013008881A9 - 虚血性疾患治療薬 - Google Patents
虚血性疾患治療薬 Download PDFInfo
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- WO2013008881A9 WO2013008881A9 PCT/JP2012/067797 JP2012067797W WO2013008881A9 WO 2013008881 A9 WO2013008881 A9 WO 2013008881A9 JP 2012067797 W JP2012067797 W JP 2012067797W WO 2013008881 A9 WO2013008881 A9 WO 2013008881A9
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- bifidobacterium
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- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/745—Bifidobacteria
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
Definitions
- the present invention relates to a gene transport carrier capable of specifically treating an ischemic disease site, a pharmaceutical composition, and a method for treating ischemic disease using them.
- Bifidobacteria which is a non-pathogenic intestinal bacterium that exists in the human intestine and forms flora and is known to be an extremely safe obligate anaerobic bacterium, has attracted attention.
- -Bifidobacteria transformed to express cytosine deaminase, an enzyme that converts the fluoroduracil prodrug 5-fluorocytosine to 5-FU have been developed. (See Patent Documents 4 and 5)
- angiogenic therapies have attracted attention as one of the options for patients who cannot revascularize due to arteriolar level impairment or who are unable to perform surgery due to invasive problems.
- many clinical trials have been conducted in recent years, particularly with respect to angiogenesis therapy by gene therapy.
- angiogenesis therapy using gene therapy is useful for treatment of ischemic diseases, but angiogenesis therapy using current gene therapy still has some problems.
- the currently used gene transport carriers have no lesion site specificity and are systemically disseminated when administered systemically, so intramuscular injection or intraarterial injection is mainly used for transgene administration.
- intramuscular injection or intraarterial injection is mainly used for transgene administration.
- these methods are used, it cannot be said that the administration is specific to the ischemic disease site and is uniformly administered to the disease site, and ischemic sites and non-ischemic sites are mixed. If this is the case, a large amount of transgene is delivered to the non-ischemic site, and angiogenesis is promoted at the non-ischemic site. There is concern that the phenomenon will occur.
- angiogenesis therapy is considered to have a significant advantage over other therapies in the elderly and diabetics because of its low invasiveness.
- malignant tumors in the elderly may have complications that can be exacerbated by angiogenesis, so current angiogenesis therapy with low disease site specificity is limited to its indication There is.
- Bifidobacteria are Bifidobacterium addressensetis, Bifidobacterium angratam, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium boum , Bifidobacterium breve, Bifidobacterium catenuratam, Bifidobacterium coelinum, Bifidobacterium coryneforme, Bifidobacterium kunicli, Bifidobacterium denticorens, Bifidobacterium Dentium, Bifidobacterium gallcum, Bifidobacterium gallinarum, Bifidobacterium globosum, Bifidobacterium indicum, Bifidobacterium infantis, Bif Bacterium Inopinatum, Bifidobacterium lactis, Bifidobacterium lactent
- Proteins useful for the treatment of ischemic disease are fibroblast growth factor (FGF), endothelial growth factor (ECGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) vascular growth factor (AGF), platelet-derived growth factor (PDGF), transforming growth factor ⁇ (TGF ⁇ ), angiopoietin, proteins having pro-angiogenic activity such as ephrin, factors related to vasodilation such as prostaglandins, granules Colony stimulating factors such as sphere colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophic factor
- the gene transport carrier according to (2) to (9), wherein the transformation plasmid is a non-shuttle plasmid.
- the gene transport carrier according to (2) to (10), wherein the transformation plasmid further comprises a gene sequence encoding a secretory signal peptide.
- the gene transport carrier according to (2) to (11), wherein the plasmid for transformation contains a pTB6 rep unit.
- the gene transport carrier according to (2) to (12), wherein the transformation plasmid has an expression cassette containing a gene encoding p37 promoter, HU terminator, and FGF2.
- the gene transport carrier according to (2) to (13), wherein the transformed plasmid comprises a DNA sequence encoding a polypeptide having the sequence set forth in SEQ ID NO: 40.
- the gene transport carrier according to (2) to (14), wherein the transformed plasmid is pFGF12a (SEQ ID NO: 38).
- a pharmaceutical composition for ischemic disease comprising the gene transport carrier of (1) to (15).
- An ischemic disease comprising administering an anaerobic bacterium capable of growing specifically in an ischemic disease site and expressing at least one protein useful for diagnosis or treatment of the ischemic disease. How to diagnose or treat. (19) The method according to (18), wherein the administration is systemic administration.
- the gene delivery carrier of the present invention is an anaerobic bacterium, it is engrafted and proliferated specifically to an ischemic disease site in an anaerobic environment, and a protein having therapeutic activity for ischemic disease is produced in the diseased tissue. It can be secreted, is extremely useful as a therapeutic agent for ischemic diseases, and can be expected as a high-quality gene transport carrier.
- it since it is engrafted specifically in the ischemic disease site, even if it is systemic administration such as intravenous injection, it is accumulated specifically in the ischemic disease site and exhibits an effect. Therefore, large doses and multiple doses are not required, and the burden on the administration subject can be reduced.
- ischemia continues, it grows for a long time because the anaerobic environment of the ischemic disease site is maintained, but once the ischemia is ameliorated, it is no longer an anaerobic environment, so it cannot grow and quickly Disappear.
- the gene delivery carrier of the present invention itself can express a protein useful for treatment, it is necessary to consider the efficiency of gene transfer into cells at or near ischemic sites as in the past. And can always exhibit high protein expression efficiency. Furthermore, since it is a carrier that is specifically delivered to an ischemic site, there is less risk of complications. Therefore, it is possible to perform angiogenesis treatment with higher safety and less invasiveness with fewer side effects and higher safety. Furthermore, by simultaneously expressing a marker on the gene delivery carrier of the present invention, it can also be used for ischemic site diagnosis and treatment monitoring. Furthermore, the gene carrier of the present invention can simultaneously deliver a plurality of genes, and it is considered that a more efficient and noninvasive treatment can be achieved by incorporating and administering a plurality of effective growth factors.
- FIG. 4 is a graph showing the number of bacteria in the affected limb and healthy limb at the time point of 168 hours after administration of the ischemic model local administration group.
- FIG. 5 is a graph showing changes in the number of B. longum bacteria in a healthy limb in the systemic administration group of ischemic model at a certain time point. In the systemic administration group, bacteria were hardly observed at 48 hours, and were not observed at 72 hours.
- FIG. 6 is a graph showing the change in blood flow ratio and the change in the number of bacteria with respect to elapsed time in the affected limb of the ischemic model model systemic administration group. It is observed that the number of bacteria increases with the passage of time after administration, and the number of bacteria decreases with the recovery of blood flow to the ischemic site.
- FIG. 7 is a Gram-stained image of muscle tissue at 4 days after administration in the ischemic model systemic administration group.
- FIG. 8 is a graph showing changes over time in the affected limb / health limb blood flow ratio of the lower limb ischemic model mouse (ischemic model) and the lower limb necrosis model mouse (necrotic model). It can be seen that the necrotic model has a longer ischemic duration and less recovery.
- FIG. 9 is a graph showing the change in blood flow ratio and the change in the number of bacteria with respect to the elapsed time in the affected limb of the necrotic model systemic administration group.
- the number of bacteria increased with the passage of time after administration, and B. longum bacteria were engrafted and proliferated while the ischemic state persisted, and it was observed that the number of bacteria decreased with the recovery of blood flow to the ischemic site Is done.
- FIG. 10 is a graph plotting the B. longum bacteria count against the affected limb / healthy limb blood flow ratio in the affected limb of the necrotic model systemic administration group. A state in which the number of bacteria decreases as the blood flow ratio increases is shown.
- FIG. 10 is a graph plotting the B. longum bacteria count against the affected limb / healthy limb blood flow ratio in the affected limb of the necrotic model systemic administration group. A state in which the number of bacteria decreases as the blood flow ratio increases is shown.
- FIG. 11 is a photograph showing the results of a test for detecting B. longum bacteria in affected limbs, lungs, kidneys, liver, spleen and heart in the systemic administration group of ischemic model.
- the upper left is the ischemic limb
- the upper middle is the lung
- the upper right is the kidney
- the lower left is the liver
- the lower middle is the spleen
- the lower left is the heart detection result.
- FIG. 12 is a graph showing the number of B. longum bacteria per elapsed time in the affected limb, blood, kidney, liver, spleen, and heart of the necrotic model systemic administration group. Immediately after administration, some bacteria are observed, but then disappear quickly. No bacteria were detected in the blood.
- FIG. 13 is a schematic diagram showing a ligation site and a bacterial administration site in a myocardial infarction model.
- FIG. 14 is a diagram showing a stained image of the heart of a myocardial infarction model minipig. a) is a TTC-stained image, indicating that the infarcted part (the part indicated by the arrow in the figure) is not stained and is an infarcted part. b) and c) respectively represent an MT stained image and an HE stained image at a normal myocardial site and an infarcted site. In both stained images, cardiomyocyte shedding and fibrosis progression were observed at the infarct site.
- FIG. 15 is a graph showing the number of B.
- FIG. 16 is a diagram showing a scheme for constructing pFGF12a, which is one embodiment of a transformation plasmid for transforming the gene delivery carrier of the present invention.
- FIG. 17 shows the results of Western blotting using the culture supernatant of Bifidobacterium longum 105A transformed with pFGF12a. Bifidobacterium longum105A transformed with pBEshuttle was used as a negative control, and hFGF2 was used as a positive control. A band is confirmed around 20 kDa, which is considered to be hFGF2.
- FIG. 18 shows the results of Western blotting using the culture supernatant of Bifidobacterium breve JCM1192 transformed with pFGF12a.
- Bifidobacterium breve JCM1192 transformed with pBEshuttle was used as a negative control, and hFGF2 was used as a positive control.
- a band is also confirmed around 20 kDa, which is considered to be hFGF2.
- FIG. 19 is a graph in which a pharmaceutical composition containing Bifidobacterium longum A105A / pFGF12a, which is a gene delivery carrier of the present invention, was intravenously injected into a mouse of a lower limb ischemia model and changes in blood flow at the lower limb ischemia site were observed. Although a slight difference in blood flow recovery has already occurred on the third day, the blood flow was significantly recovered in the group administered with the gene delivery carrier of the present invention after the sixth day.
- FIG. 19 is a graph in which a pharmaceutical composition containing Bifidobacterium longum A105A / pFGF12a, which is a gene delivery carrier of the present invention, was intravenously injected into a mouse of a lower limb ischemia model and changes in blood flow at the lower limb ischemia site were observed. Although a slight difference in blood flow recovery has already occurred on the third day, the blood flow was significantly recovered in the group administered with the gene delivery carrier of the present invention after the sixth day.
- the present invention relates to a gene transport carrier comprising anaerobic bacteria capable of growing specifically in the site of ischemic disease and expressing at least one protein useful for diagnosis or treatment of ischemic disease.
- ischemia means a state of oxygen / nutrient deficiency in the tissue due to a decrease in the amount of arterial blood supplied to the tissue due to stenosis or occlusion of the blood vessel. Cause degeneration, necrosis, etc.
- the gene delivery carrier of the present invention is mainly used in a treatment method for ameliorating an undesirable condition caused by ischemia, such as angiogenesis therapy and organ protection. Therefore, in the present specification, “ischemic disease” refers to a state in which tissue ischemia continues due to arterial stenosis or occlusion, regardless of the presence or absence of subjective symptoms, or an unfavorable state caused by such ischemia. Say.
- ischemic disease examples include, but are not limited to, ischemic heart disease such as angina pectoris and myocardial infarction, cerebral ischemia such as cerebral infarction, chronic cerebral ischemia such as moyamoya disease, spinal cord ischemia, Ischemic colitis, ischemic bowel diseases such as mesenteric artery occlusion, lower limb ischemia such as obstructive arteriosclerosis, retinal ischemia such as diabetic retinopathy, and the like.
- ischemic heart disease such as angina pectoris and myocardial infarction
- cerebral ischemia such as cerebral infarction
- chronic cerebral ischemia such as moyamoya disease
- spinal cord ischemia spinal cord ischemia
- Ischemic colitis ischemic bowel diseases such as mesenteric artery occlusion
- lower limb ischemia such as obstructive arteriosclerosis
- retinal ischemia such as diabetic retinopathy, and the like.
- ischemic site means a site where arterial blood volume, nutrition and oxygen are reduced due to ischemia, and is interchangeably used with “ischemic disease site” or “ischemic disease tissue”.
- ischemic disease tissue used.
- the “anaerobic bacterium” means a bacterium having an anaerobic property
- the “anaerobic property” means a property capable of growing under conditions with little or no oxygen.
- anaerobic bacteria can be classified into facultative anaerobes that can grow even in the presence of oxygen and obligate anaerobic bacteria that cannot grow in the presence of oxygen. Is preferred.
- the anaerobic property of the anaerobic bacterium of the present invention may be a property inherent to bacteria or may be obtained by transformation.
- the gene transport carrier of the present invention is composed of anaerobic bacteria, it can be specifically engrafted and proliferated at an ischemic disease site under anaerobic conditions. And since the gene transport carrier itself has gene transcription and translation functions, it is possible to express a protein useful for diagnosis or treatment at the time of engraftment in an ischemic disease tissue and supply it to the disease tissue. It is. Therefore, the gene transport carrier of the present invention contains a DNA sequence encoding a protein useful for diagnosis or treatment of ischemic disease.
- the gene transport carrier is transformed with a plasmid having a DNA sequence encoding a protein useful for diagnosis or treatment of ischemic disease.
- an ischemic disease includes an acute ischemic disease in which tissue is damaged by a rapid decrease in oxygen concentration due to ischemia, and a tissue is degenerated or necrotic due to a continuous decrease in oxygen concentration over a long period of time.
- Chronic ischemic disease that causes The gene delivery carrier of the present invention is preferably used for improving a chronic ischemic condition because it is selectively delivered to a disease site after administration, engrafts, and remains at the delivery site to develop an effect. It is done.
- chronic ischemic condition is used interchangeably with “chronic ischemic disease”, but is not limited thereto, for example, ischemic heart diseases such as angina pectoris, myocardial infarction, Examples include chronic cerebral ischemia such as moyamoya disease and lower limb ischemia such as obstructive arteriosclerosis. Examples of treatment for improving chronic ischemic disease include, but are not limited to, angiogenesis therapy.
- the gene transport carrier of the present invention Since the gene transport carrier of the present invention is premised on administration into the body, the anaerobic bacteria used need not be toxic or have low toxicity. Therefore, in one embodiment of the present invention, the gene transport carrier may be a pathogenic bacterium that has been mutated to low toxicity. However, a bacterium that has been mutated to low toxicity may be reversely mutated to return to the original pathogenic bacterium, and may exhibit harmful effects. Therefore, it is preferably a naturally non-pathogenic bacterium. Therefore, in a preferred embodiment of the present invention, the anaerobic bacteria use non-pathogenic enteric bacteria.
- Bifidobacterium Bifidobacteria
- Bifidobacteria Bifidobacterium addressentis, Bifidobacterium angratum, Bifidobacterium animalis, Bifidobacterium asteroides, Bifidobacterium bifidum, Bifidobacterium Um Boum, Bifidobacterium breve, Bifidobacterium catenuratam, Bifidobacterium coelinum, Bifidobacterium coryneforme, Bifidobacterium kunichli, Bifidobacterium denticorens, Bifido Bacterium Dentium, Bifidobacterium galcumum, Bifidobacterium galinarum, Bifidobacterium globosum, Bifidobacterium in
- the gene transport carrier of the present invention is engrafted specifically in the ischemic disease site, it is possible to diagnose the ischemic disease site by detecting the presence of the gene transport carrier.
- the detection of the gene transport carrier can be easily performed by labeling the gene transport carrier, for example. From the viewpoint of use in diagnosis of a disease, detection is preferably less invasive and preferably has less adverse effect on the delivery target due to the label. Therefore, in a preferred embodiment of the present invention, the gene transport carrier expresses a fluorescent protein as a protein useful for diagnosis. Examples of fluorescent proteins include various green fluorescent proteins (GFP) and red fluorescent proteins (RFP). As another preferred embodiment,
- the gene transport carrier of the present invention engrafts specifically on the ischemic disease site, the protein expressed at the engraftment site is necessarily delivered specifically on the ischemic disease site. Therefore, an ischemic disease can be effectively treated by expressing the protein used for the treatment of ischemic disease in the gene transport carrier of the present invention. Accordingly, in a preferred embodiment of the present invention, the gene transport carrier expresses a protein useful for the treatment of ischemic disease.
- proteins useful for the treatment of ischemic diseases include, but are not limited to, proteins having angiogenesis-promoting activity, proteins involved in vasodilation, and the like.
- proteins having angiogenesis-promoting activity include, but are not limited to, fibroblast growth factor (FGF), endothelial growth factor (ECGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), vascular growth factor (AGF), platelet-derived growth factor (PDGF), transforming growth factor ⁇ (TGF ⁇ ), angiopoietin, ephrin and the like.
- FGF fibroblast growth factor
- ECGF endothelial growth factor
- VEGF vascular endothelial growth factor
- HGF hepatocyte growth factor
- AGF vascular growth factor
- PDGF platelet-derived growth factor
- TGF ⁇ transforming growth factor ⁇
- angiopoietin ephrin
- Prostaglandins are involved in vas
- G-CSF granulocyte colony stimulating factor
- GM-CSF granulocyte-macrophage colony stimulating factor
- NGF nerve growth factor
- BDNF brain-derived neurotrophic Factors
- neurotrophins such as neurotrophin 3, insulin-like growth factor (IGF), and the like.
- shuttle plasmid means a plasmid that can replicate in two or more different hosts, and is used interchangeably with “shuttle vector plasmid”.
- non-shuttle plasmid means a plasmid that can replicate in only one host.
- the gene transport carrier of the present invention produces a protein encoded by a transformation plasmid in the microbial cell, but since such protein has a therapeutic effect only after being released outside the microbial cell, In order to exert a therapeutic effect using a protein that is not secreted, it is necessary to kill bacteria.
- the transformation plasmid further comprises a gene sequence encoding a secretory signal peptide.
- a non-shuttle plasmid can be prepared by removing the replication origin of bacteria other than transformed bacteria from this shuttle plasmid. Further, if desired, the promoter gene is replaced with a secretion signal that functions at least in bifidobacteria and its promoter gene, and the terminator gene is replaced with the terminator gene of the secretory signal peptide that functions in the bifidobacteria. A plasmid containing the gene sequence encoding the peptide can be generated.
- the present invention further relates to a pharmaceutical composition for ischemic disease comprising the gene transport carrier.
- the pharmaceutical composition of the present invention is not particularly limited as long as it contains the gene transport carrier of the present invention.
- the gene transport carrier of the present invention may be contained in at least one kind, and may be contained in two or more kinds.
- the pharmaceutical composition of the present invention can be used in combination with a pharmaceutical composition or a therapeutic agent for ischemic disease containing a compound having an effect of treating ischemic disease other than the gene transport carrier of the present invention.
- the pharmaceutical composition of the present invention may contain any component in addition to the gene transport carrier of the present invention as long as the effects of the present invention are not hindered.
- optional components include pharmacologically acceptable carriers, excipients, diluents and the like.
- the dosage form of the pharmaceutical composition of the present invention is not particularly limited, and examples thereof include a liquid or solid preparation containing the gene transport carrier of the present invention.
- the solution is prepared by purifying the anaerobic bacteria culture medium of the gene carrier of the present invention, and adding an appropriate physiological saline solution or a replacement solution or a pharmaceutical additive to the ampule or a vial as necessary. Can be manufactured.
- an appropriate protective agent is added to the liquid and filled into ampoules or vials, and then lyophilized or L-dried, or an appropriate protective agent is added to the liquid and lyophilized or L-dried. This can be manufactured by filling ampules or vials.
- the bifidobacteria of the present invention may be administered at 10 6 to 10 12 cfu per kg of body weight 1 to several times a day, one or more days as necessary, continuously or at appropriate intervals. And administer.
- “combined with X and Y” includes any of X and Y in different forms, and X and Y in the same form (for example, a form containing X and Y). Including cases. In the case where X and Y are in different forms, the case where both X and Y further contain other components is also included.
- One aspect of the present invention includes a method for diagnosing or treating an ischemic disease using a gene transport carrier comprising the above anaerobic bacteria.
- the gene transport carrier of the present invention is administered to a subject having an ischemic disease.
- an administration method both oral administration and parenteral administration are possible, but parenteral administration is preferable.
- intravenous injection, subcutaneous injection, local injection, intraventricular administration, etc. can be performed. Most preferred.
- the gene transport carrier of the present invention is administered systemically. Since the gene transport carrier of the present invention can deliver a gene in an ischemic disease site-specific manner, even if it is administered systemically such as intravenous injection without using a catheter or intramuscular injection, it is ischemic. Engraft and proliferate in the diseased area and exert its effect. Therefore, treatment that is very less invasive than conventional treatment methods is possible.
- B. longum bacteria transformed with the pBLES100 plasmid to become spectinomycin-resistant bacteria were subcultured twice in MRS medium (obtained from Kanto Chemical Co., Inc.). The culture solution that had been subcultured twice was subjected to centrifugal washing with physiological saline and suspended in physiological saline for use.
- MRS medium obtained from Kanto Chemical Co., Inc.
- the culture solution that had been subcultured twice was subjected to centrifugal washing with physiological saline and suspended in physiological saline for use.
- the B. Longum bacterium was administered to the ischemic limb and the healthy limb by intramuscular injection at 1 ⁇ 10 9 cfu / ml ⁇ 0.1 ml ⁇ 2 sites.
- the model experimental animal used was a male Göttingen minipig (15 kg) (obtained from Chugai Medical Research Institute). Ketalar (obtained from Sankyo Co., Ltd.) 15 mg / kg and atropine sulfate (obtained from Tanabe Seiyaku Co., Ltd.) (25 mg) were injected intramuscularly, followed by inhalation anesthesia Frocene (obtained from Takeda Pharmaceutical Company Limited) (2% -5 %) Maintained anesthesia. After thoracotomy, the left anterior descending coronary artery was ligated with 3-0 silk thread.
- Masson / Trichrome (MT) staining was performed by rinsing with running water for 10 minutes after fixing each section with Buan. After washing with water, it was immersed in a mixed solution of 10% potassium dichromate and 10% trichloracetic acid in an equal amount for 10 minutes, then immersed in iron hematoxylin staining solution for 5 minutes, and washed with running water for 5 minutes. After washing with water, it was immersed in a ponsoic acid fuchsin azofuroxin solution for 1 minute and then immersed in a 2.5% phosphotungstic acid solution for 10 minutes.
- the sections were immersed in 2% orange G solution for 5 minutes, washed with 1% aqueous acetic acid, stained with light green liquid for 3 minutes, and washed with 1% aqueous acetic acid. Dehydration, penetration, and encapsulation were performed in the same process as HE staining.
- the above vector and insert are connected using In-Fusion HD Cloning Kit and Cloning Enhancer (Takara Bio Inc.) (hereinafter referred to as in-fusion reaction), and a portion of the reaction solution is used to produce E. coli TOP10 chemically competent cell (Life Technologies Japan Co., Ltd.) was transformed. Details followed the product instructions.
- the transformed Escherichia coli was applied to LB (75 ⁇ g / mL spectinomycin-containing) agar medium and statically cultured at 37 ° C. overnight. The well-separated colonies on the plate were picked up and cultured overnight at 37 ° C. in LB (75 ⁇ g / mL spectinomycin-containing) liquid medium.
- FIG. 16 shows an outline of construction of human FGF2 expression (secretory) plasmid pFGF12a. PCR was performed using the above plasmid pFGF-P37 as a template and FGF35 primer (SEQ ID NO: 34) and FGF26 primer (SEQ ID NO: 35), and a DNA amplification product of about 4.2 kbp was used as a vector.
- P37 promoter base numbers 14 to 275 of SEQ ID NO: 38 -Structure of hFGF2 coding region: base numbers 276 to 902 of SEQ ID NO: 38
- Signal sequence base numbers 276 to 419 of SEQ ID NO: 38
- HFGF2 sequence base numbers 420 to 881 of SEQ ID NO: 38
- Histidine tag sequence base numbers 882 to 899 of SEQ ID NO: 38
- Stop codon base numbers 900 to 902 of SEQ ID NO: 38
- Hu terminator base numbers 903 to 1016 of SEQ ID NO: 38
- Bifidobacterium breve JCM1192 / pFGF12a and Bifidobacterium breve JCM1192 / pBEshuttle were also cultured.
- RCM medium was used instead of MRS medium.
- No vitamin C / cysteine solution was added.
- the recombinant bifidobacteria culture supernatant was trichloroacetic acid (TCA) precipitated according to a conventional method and redissolved in 1 ⁇ SDS sample buffer. This was heat-treated at 95 ° C. for 3 minutes and subjected to Western analysis.
- TCA trichloroacetic acid
- the sample was electrophoresed with 1 ⁇ SDS buffer using a mini-PROTEAN TGX gel (4-20%, Bio-Rad).
- Mini PROTEAN Tetra System Bio-Rad
- the gel after electrophoresis was transferred to iBlot Transfer Stacks using iBlot Gel Transfer Device. After the transfer, the membrane was washed 3 times with TTBS for about 5 minutes and then blocked with a 2% blocking solution (Block Ace).
- Anti-FGF-2-human rabbit poly H-131, Santa Cruz Biotechnology Inc. was added as a primary antibody and shaken overnight at 4 ° C.
- the common femoral artery, deep femoral artery, and superficial femoral artery were ligated with 8-0 polypropylene thread, and the superficial femoral artery was partially excised. After the wound was washed with physiological saline, the wound was sutured with 5-0 nylon thread.
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Abstract
Description
すなわち本発明は、以下に関する。
(1)虚血性疾患部位特異的に生育でき、かつ、少なくとも1種の、虚血性疾患の診断もしくは治療に有用なタンパク質を発現することができる嫌気性菌からなる遺伝子輸送担体。
(3)虚血性疾患が、慢性虚血性疾患である、(1)または(2)の遺伝子輸送担体。
(4)嫌気性菌が、非病原性の腸内細菌である、(1)~(3)の遺伝子輸送担体。
(5)嫌気性菌が、ビフィズス菌である、(1)~(4)の遺伝子輸送担体。
(8)虚血性疾患の診断に有用なタンパク質が、蛍光タンパク質である、(1)~(7)の遺伝子輸送担体。
(9)虚血性疾患の治療に有用なタンパク質が、線維芽細胞増殖因子(FGF)、内皮細胞増殖因子(ECGF)、血管内皮増殖因子(VEGF)、肝実質細胞増殖因子(HGF)血管増殖因子(AGF)、血小板由来増殖因子(PDGF)、トランスフォーミング増殖因子β(TGFβ)、アンギオポイエチン、エフリンなどの血管新生促進活性を有するタンパク質、プロスタグランジン類などの血管拡張に関与する因子、顆粒球コロニー刺激因子(G-CSF)、顆粒球-マクロファージコロニー刺激因子(GM-CSF)などのコロニー刺激因子、神経成長因子(NGF)、脳由来神経栄養因子(BDNF)、ニューロトロフィン3などのニューロトロフィン、インスリン様成長因子(IGF)からなる群から選ばれる一種である、(1)~(7)の遺伝子輸送担体。
(11)形質転換用プラスミドが、さらに分泌シグナルペプチドをコードする遺伝子配列を含む、(2)~(10)の遺伝子輸送担体。
(12)形質転換用プラスミドが、pTB6 repユニットを含む、(2)~(11)の遺伝子輸送担体。
(13)形質転換プラスミドが、p37プロモーター、HUターミネーター、およびFGF2をコードする遺伝子を含む発現カセットを有する、(2)~(12)の遺伝子輸送担体。
(14)形質転換プラスミドが、配列番号40に記載の配列を有するポリペプチドをコードするDNA配列を含む、(2)~(13)の遺伝子輸送担体。
(15)形質転換プラスミドが、pFGF12a(配列番号38)である、(2)~(14)の遺伝子輸送担体。
(16)(1)~(15)の遺伝子輸送担体を含む、虚血性疾患用医薬組成物。
(17)全身投与により投与される、(16)の医薬組成物。
(18)虚血性疾患部位特異的に生育でき、かつ、少なくとも1種の、虚血性疾患の診断または治療に有用なタンパク質を発現することができる嫌気性菌を投与することを含む、虚血性疾患を診断または処置する方法。
(19)投与が、全身投与である、(18)の方法。
本発明の遺伝子送達担体は、主に血管新生治療、臓器の保護など、虚血によって引き起こされる好ましくない状態を改善する処置法において用いられるものである。したがって本明細書において「虚血性疾患」とは、自覚症状の有無に拘わらず、動脈の狭窄や閉塞により、組織の虚血が持続している状態、またはかかる虚血により引き起こされる好ましくない状態をいう。虚血性疾患としては、これに限定するものではないが例えば、狭心症、心筋梗塞などの虚血性心疾患、脳梗塞などの脳虚血、もやもや病などの慢性脳虚血、脊髄虚血、虚血性大腸炎、腸間膜動脈閉塞症などの虚血性腸疾患、閉塞性動脈硬化症などの下肢虚血、糖尿病網膜症などの網膜虚血などが挙げられる。
本明細書において、「嫌気性菌」とは、嫌気的性質を有する細菌を意味し、「嫌気的性質」とは、酸素が少ないまたはない条件下で生育可能な性質を意味する。一般的に嫌気性菌は、酸素の存在下でも生育可能な通性嫌気性菌と、酸素の存在下では生育できない偏性嫌気性菌とに分類できるが、本発明においては偏性嫌気性菌が好ましい。本発明の嫌気性菌が有する嫌気的性質は、細菌が生来有する性質であっても、形質転換によって得られたものであってもよい。
上述の通り、本発明の遺伝子輸送担体である嫌気性菌が有する嫌気的性質は、形質転換によって得られたものであってもよく、したがって本発明の一態様において、嫌気性菌は、偏性嫌気性菌となるように形質転換されたものである。
また、ビフィドバクテリウム・ラクテンティスの株については、例えば、ビフィドバクテリウム・ラクテンティス標準株(JCM1220)を挙げることができる。
例えば、通常の手法に従って、形質転換菌と形質転換菌以外の菌、例えばビフィズス菌と大腸菌でそれぞれ機能する複製開始点を有するシャトルプラスミドに、少なくとも1種の、所望の虚血性疾患の診断または治療に有用な蛋白質をコードする遺伝子を挿入する事により、シャトルプラスミドを作製することができる。
さらに、所望に応じて、プロモーター遺伝子を、少なくともビフィズス菌で機能する分泌シグナルおよびそのプロモーター遺伝子に代え、ターミネーター遺伝子を、前記ビフィズス菌で機能する分泌シグナルペプチドのターミネーター遺伝子に代えることにより、さらに分泌シグナルペプチドをコードする遺伝子配列を含むプラスミドを作製することができる。
そして、形質転換する任意の嫌気性菌を、上記形質転換用プラスミドを用いて、遺伝子工学分野の公知の方法にしたがって形質転換する事により作製することができる。
本発明の医薬組成物は、本発明の遺伝子輸送担体を含有している限り特に制限はされない。本発明の遺伝子輸送担体は、少なくとも1種含まれていればよく、2種以上含まれていてもよい。さらに、本発明の医薬組成物は、本発明の遺伝子輸送担体以外の虚血性疾患治療効果を示す化合物を含有する医薬組成物や虚血性疾患治療剤と組み合わせて用いることができる。
本発明の医薬組成物の剤型は特に制限されないが、例えば、本発明の遺伝子輸送担体を含有する液剤あるいは固形製剤を挙げることができる。液剤は、本発明の遺伝子輸送担体の嫌気性菌の培養液を精製し、これに必要に応じて適当な生理食塩液もしくは補液または医薬添加物を加えてアンプルまたはバイアル瓶などに充填することにより製造することができる。また、固形製剤は、液剤に適当な保護剤を添加してアンプルまたはバイアル瓶などに充填した後凍結乾燥またはL乾燥するか、液剤に適当な保護剤を添加して凍結乾燥またはL乾燥した後これをアンプルまたはバイアル瓶などに充填することにより製造することができる。
本発明の医薬組成物の遺伝子輸送担体の投与量は、疾患部位において生育でき、且つ、有効治療量の活性タンパク質を発現するのに十分な量である限り特に制限はされないが、経済上の観点および副作用を可能な限り回避する観点から、必要な治療効果が得られる範囲においてできる限り少ない方が好ましい。
例えば、本発明の医薬組成物を用いる際には、用いる嫌気性菌の菌自体が示す疾患治療活性、用いる嫌気性菌が産生する疾患治療活性を有するタンパク質等の種類、および用いる嫌気性菌の当該活性タンパク質の産生量などによって、適宜設定する。
なお、本発明における「XとYと組み合わせてなる」には、XとYを別の形態としたもの、XとYを同一の形態(例えばXとYを含有する形態)としたもののいずれの場合も含む。また、XとYを別の形態としたものの場合、X、Yのいずれも他の成分をさらに含有している場合も含まれる。
本発明の方法において、本発明の遺伝子輸送担体を、虚血性疾患を有する対象に投与する。投与方法としては、経口投与、非経口投与共に可能であるが、非経口投与が好ましく、例えば静脈注射、皮下注射、局所注入、脳室内投与等を行うことができ、静脈注射、すなわち全身投与が最も好ましい。
本発明の遺伝子送達担体の、虚血性疾患部位への特異的送達性を検証するため、下肢虚血のマウスモデルを作製し、本発明の遺伝子送達担体を投与して検討した。
(1)モデル作製
実験動物は8-9週齢、オスのC57BL/6マウス(日本チャールズリバー社より入手)を使用し、以下の手順で下肢虚血モデル(ischemic model)マウスを作製した。麻酔は3.6%抱水クロラール(ナカライテスク株式会社より入手)を腹腔内に0.3ml注射した。腹部から下腿の除毛を行ったのち総大腿動脈から膝窩動脈を7-0ポリプロピレン糸にて結紮し、大腿動脈を切除した。創内を生理食塩水で洗浄後、創を5-0ナイロン糸にて縫合した。
マウスを麻酔後、レーザードップラー血流計(Moor Instruments社製)を用いて、(3)の下肢筋肉摘出前に血流測定を行った。下腿部から指先までの血流を両側について定量し、患側/健側比にて表した。血流測定はモデル作製直前、直後、1、2、3、4、5、7、10、14、21、28日後に行った。
pBLES100プラスミドで形質転換し、スペクチノマイシン耐性菌としたB.longum菌をMRS培地(関東化学株式会社より入手)で2回継代した。2回継代した培養液を、生理食塩水を用いて遠心洗浄を行い、生理食塩水で懸濁して使用した。局所投与モデルとして、下肢虚血モデル作製直後に虚血肢および健肢にそれぞれ上記B. Longum菌を1×109cfu/ml×0.1ml×2ヶ所筋肉内注射投与した。全身投与モデルとして、尾静脈にB.longum菌を1×109cfu/mlの濃度で0.2ml投与した。菌を投与してから1、2、3、4、5、7、10、14日後に、各投与モデルの、左右それぞれの下肢筋肉を摘出した(各n=5)。各組織をホモジナイズした後、BLプレートに塗布して37℃、嫌気的条件下で3日間培養した。培養後に各プレートのコロニー数を測定し、虚血肢および健肢の菌数を求めた。
(3)と同様にマウスへB.longum菌を投与した後、96時間後に下肢筋肉を摘出した。下肢筋肉を20%中性緩衝ホルマリンに48時間浸し組織を固定した。その後パラフィン包埋しパラフィンブロックとした。パラフィンブロックを3μmの厚さにカットし、スライドガラスにのせて24時間44℃で風乾し、パラフィン組織切片スライドとした。脱パラフィン後グラム染色を行った。グラム染色はグラム染色液neo-B&Mワコー(和光純薬工業株式会社)を用いた。染色後、光学顕微鏡で観察した。
(1)モデル作製
例1において、血流の回復とともにB.longum菌の生着数が減少していることが示唆されたが、このことを確認するため、より重度の虚血状態のモデルである下肢壊死モデル(necrotic model)マウスを作製した。実験動物は6-7週齢、オスのBALB/cマウス(日本チャールズリバー社より入手)を使用した。麻酔は3.6%抱水クロラール(ナカライテスク株式会社)を腹腔内に0.3ml注射した。腹部から下腿の除毛を行ったのち総大腿動脈から浅大腿動脈までを7-0ポリプロピレン糸にて結紮し、大腿動脈を切除した。創内を生理食塩水で洗浄後、創を5-0ナイロン糸にて縫合した。
例1と同様に、血流を測定した。結果を図8に示す。necrotic modelはischemic modelと比較して血流回復のタイミングが遅く、最終的な血流量も健肢と比較して約30%程度であり、約60%程度まで血流が回復するischemic modelと比較しても、回復の程度が半分程度と低いことがわかった。
例1と同様にB.longum菌を全身投与し、健肢および患肢筋肉組織を検査した。結果を図9および10に示す。necrotic modelでは、ischemic modelと比較して長期間にわたってB.longum菌が患肢に存在することがわかった。そしてischemic modelと同様、血流の回復とともに菌数が減少し、ある程度血流が回復した時点で菌が観察されなくなった。以上の結果から、B.longum菌は、どの程度の期間であっても、虚血状態が持続している間は患肢において生着、増殖しており、血流の回復(すなわち虚血状態からの回復)とともに菌数が減少を始めることがわかった。したがって、B.longum菌は虚血状態にある組織に特異的に送達され、導入した遺伝子を発現し、虚血状態の回復とともに消失する性質を有し、虚血性疾患の診断および治療において優れた効果を発揮することが期待できる。
上述の下肢虚血モデル(ischemic model)マウスおよび下肢壊死モデル(necrotic model)マウス両者について安全性試験を行った。菌を投与してから2,3,4,6,7日後に、各投与モデルの、腎臓、肝臓、脾臓、心臓を摘出した(各n=3)。またこれらの臓器に加え、ischemic modelでは肺を、necrotic modelでは血液をさらに摘出した。各組織をホモジナイズした後、BLプレートに塗布して37℃、嫌気的条件下で3日間培養した。培養後に各プレートのコロニー数を測定し、各組織の菌数を求めた。
別の代表的な虚血性疾患としては心筋梗塞などの虚血性心疾患が挙げられるが、近年虚血性心疾患に対しても血管新生治療、特に遺伝子導入による血管新生治療が試みられている。しかしながら他の虚血性疾患における問題点と同様の問題点が虚血性心疾患の治療においても挙げられる。特に虚血性心疾患においては、局所投与法としては冠動脈カテーテルを用いた動脈注射、冠動脈カテーテルを用いた局所注射、開胸下心筋内注射など、非常に高侵襲な方法が必要であり、血管新生療法は経皮的冠動脈形成術(PCI)、冠動脈バイパス術(CABG)などの他の治療法の適用ができない対象に対して特に恩恵があると考えられるにもかかわらず、現時点では他の治療法との併用が主となっている。
(1)モデル作製
実験動物は、オスのゲッチンゲン系ミニブタ(15kg)(中外医科学研究所より入手)を使用した。ケタラール(三共株式会社より入手)15mg/kgと硫酸アトロピン(田辺製薬株式会社より入手)(25mg)を筋肉注射したのち、吸入麻酔であるフローセン(武田薬品工業株式会社より入手)(2%~5%)により麻酔状態を維持した。開胸後、左冠状動脈前下行枝を3-0シルク糸で結紮した。術中に2%キシロカイン(アストラゼネカ株式会社より入手)(1ml~2ml)を投与した。閉胸前にラクテック(大塚製薬株式会社より入手)100mlで心嚢内を洗浄した。
心筋梗塞確認のために塩化トリフェニルテトラゾリウム(TTC)染色、ヘマトキシリン・エオジン(HE)染色およびマッソン・トリクローム(MT)染色を行った。TTC染色は心筋梗塞モデル作製7日後に摘出した心臓を5mmの厚さにスライスし、1%TTC液(SIGMAより入手)中、37℃で5分インキュベーションした。その後、肉眼的に観察した。
ヘマトキシリン・エオジン(HE)染色は脱パラフィン後、ヘマトキシリンで30秒から1分間染色し、流水にて10分間洗浄した後、0.5%エオジン溶液で30秒染色した。染色後、脱水、透徹および封入し、光学顕微鏡で観察した。
B.longum菌を例1と同様に調製し、心筋梗塞モデル作製直後にB.longum菌を1×109cfu/mlの濃度で、0.1mlを1cmおきに等間隔で健常部位と梗塞部位に心筋内投与した(図13参照)。菌を投与してから4日および7日後(各n=2)に心臓を摘出して梗塞部位と健常部位に分け、ホモジナイズした後、BLプレートに塗布して37℃、嫌気的条件下で3日間培養した。培養後に各プレートのコロニー数を測定し、梗塞部位および健常部位の菌数を求めた。
例5:hFGF2分泌Bifidobacteriumの構築
(1)発現プラスミドベクターp37の構築
プラスミドpCDshuttle(国際出願第2009/128272号)を鋳型として、CDvecF3プライマー(配列番号23)およびpUCoriR5プライマー(配列番号24)を用いたPCRを行い、PCR産物をベクターとした。Bifidobacterium 1ongum 105AのゲノムDNAを鋳型としてPins37Fプライマー(配列番号25)およびPins37Rプライマー(配列番号26)にて増幅したPCR産物をインサートとした。PCRは常法にて実施した。
上記プレート上のよく分離したコロニーを拾い、LB(75μg/mLスペクチノマイシン含有)液体培地にて37℃で一晩振とう培養後、これより、QIAprep Spin Miniprep Kit(株式会社キアゲン)を用いてプラスミドDNAを抽出した。このプラスミドDNAの全長についてシーケンス解析を行い、配列を確認した。完成したプラスミド名をp37(配列番号39)とした。
ヒトFGF2発現(非分泌型)プラスミドpFGF-P37の構築概要を図16に示した。ベクターを以下のように、調製した。即ち、プラスミドP37を鋳型として、FGF1プライマー(配列番号27)およびFGF7プライマー(配列番号28)を用いてPCRを行い、P37プロモーター(配列番号29)、Huターミネーター、pTB6レプリコン(ビフィズス菌中でのプラスミドレプリコン)、AAD9カセット(スペクチノマイシン耐性遺伝子発現ユニット)、pUCori部分(大腸菌中でのプラスミド複製起点)からなる約3.8kbpのDNAを増幅し、これをベクターとした。ベクターの末端15bpが、以下に示すインサート末端15bpと同配列になるようプライマーを設計した。DNAの増幅には、PrimeSTAR HS(Premix)(タカラバイオ株式会社)を使用し、PCR条件は同酵素の製品説明書に従った。
ヒトFGF2発現(分泌型)プラスミドpFGF12aの構築概要を図16に示した。上記プラスミドpFGF-P37を鋳型として、FGF35プライマー(配列番号34)およびFGF26プライマー(配列番号35)を用いてPCRを行い、約4.2kbpのDNA増幅産物をベクターとした。
別に、Bifidobacterium longum 105A株のゲノムDNAを鋳型として、FGF45プライマー(配列番号36)およびFGF17プライマー(配列番号37)にてPCRを行い、約0.2kbpのDNA増幅産物をインサートとした。インサートは、分泌シグナルを含んでおり、B. breve UCC2003のSec2(Shkoporov AN et. al., Biotechnol Lett (2008) 30: 1983-1988参照)のN末端配列がよく保存されている。ベクターおよびインサートの増幅には、PrimeSTAR HS(Premix)(タカラバイオ株式会社)を使用し、PCR条件は同酵素の製品説明書に従った。
上記ベクターおよびインサートを、上記、FGF発現プラスミド(非分泌型)の構築時と同様に、In-fusion反応にて連結し、大腸菌TOP10を形質転換、プラスミドのhFGF2発現ユニット部分のDNA配列を確認し、完成したプラスミド名をpFGF12a(配列番号38)とした。hFGF2発現ユニット部分の構成は以下の通りである。
・P37プロモーター:配列番号38の塩基番号14から275、
・hFGF2コード領域の構成:配列番号38の塩基番号276から902
・シグナル配列:配列番号38の塩基番号276から419
・hFGF2配列:配列番号38の塩基番号420から881
・ヒスチジンタグ配列:配列番号38の塩基番号882から899
・終始コドン:配列番号38の塩基番号900から902
・Huターミネーター:配列番号38の塩基番号903から1016
また、同ユニットがコードするアミノ酸配列を配列番号40に、更にその構成を以下に示す。
・hFGF2コード領域の構成:配列番号40のアミノ酸番号1から208
・シグナル配列:配列番号40のアミノ酸番号1から48(うち、アミノ酸番号1~37が推定シグナルペプチド領域であり、アミノ酸番号38~48が推定スペーサー領域であり、アミノ酸番号37と38との間が推定切断箇所)
・hFGF2配列:配列番号40のアミノ酸番号49から202
・ヒスチジンタグ配列:配列番号40のアミノ酸番号203から208
Bifidobacterium longum 105A株およびBifidobacterium breve JCM1192株のコンピテントセルを、Rossi et al., Letters in Applied Microbiology (1997) 24: 33-36に記載の方法に準じて作製した。即ち、IMR培地にて対数増殖期初期まで培養した上記ビフィズス菌をPBSバッファーにて洗浄後、KMRバッファーにて懸濁し、液体窒素にて急速凍結したものをコンピテントセルとした。コンピテントセルを氷上にて融解後、上記プラスミドpFGF12aあるいはpBEshuttle(国際公開第2011/093465号に記載のmockベクター、FGF発現なし)を混合し、Gene Pulser II(バイオ・ラッドラボラトリーズ株式会社)を用いてエレクトロポレーションを行った。エレクトロポレーションは、0.2cmギャップのキュベットを用いて、2kV、25μF、200Ωの条件で行った。形質転換後のビフィズス菌を、IMR(75μg/mLスペクチノマイシン含有)寒天培地に塗布し、37℃にて2日間嫌気培養を行なった。寒天培地上に形成したよく分離したコロニーを釣菌し、組換えビフィズス菌を取得した。
ビフィズス菌培養用培地として、MRS培地(oxide)10mLに、350mg/mLのアスコルビン酸および20mg/mLのL-システインを含むビタミンC/システイン溶液200μLおよび75mg/mLスペクチノマイシン溶液10μLを添加した。この調整培地に、上記組換えビフィズス菌B. longum 105A/pFGF12aおよびB. longum 105A/pBEshuttleを植菌し、37℃にて24時間嫌気培養した(活性化培養液)。活性化培養液を、上記調整培地にさらにリン酸ナトリウムバッファーを166mMになるよう添加した培地に植菌し、37℃にて18時間嫌気培養した。
上記組換えビフィズス菌培養液上清を常法に従いトリクロロ酢酸(TCA)沈殿し、1×SDSサンプルバッファーにて再溶解した。これを、95℃にて3分間加熱処理し、ウェスタン解析に供した。
培養上清を、TCA沈殿工程前まで上記と同様の方法にて調製し、human FGF2 ELISAキット(Quantikine(R) ELISAHuman FGF basic Immunoassay、R&D systems、カタログ番号:DFB50)にてhFGF2量を定量した。
培養上清中のhFGF2量は、B. longum 105A/pFGF12aにて33,058pg/mL、陰性コントロールのB. longum 105A/pBEshuttleでは0pg/mLであった。Bifidobacterium breve JCM1192/pFGF12aにて11,429pg/mL、陰性コントロールのBifidobacterium breve JCM1192/pBEshuttleでは0pg/mLであった。
本発明の遺伝子送達担体の、虚血性疾患部位での治療効果を検証するため、下肢虚血のマウスモデルを作製し、ヒトFGF2分泌ビフィズス菌を投与して検討した。
(1)モデル作製
実験動物は6-8週齢、メスのBalb/cマウス(日本チャールズリバー社より入手)を使用し、以下の手順で下肢虚血モデル(ischemic model)マウスを作製した。麻酔は3.6%抱水クロラール(ナカライテスク株式会社より入手)を腹腔内に0.3ml注射した。腹部から下腿の除毛を行ったのち総大腿動脈、深大腿動脈、浅大腿動脈を8-0ポリプロピレン糸にて結紮し、浅大腿動脈を部分切除した。創内を生理食塩水で洗浄後、創を5-0ナイロン糸にて縫合した。
マウスを麻酔後、レーザードップラー血流計(Moor Instruments社製)を用いて、血流測定を行った。下腿部から指先までの血流を両側について定量し、患側/健側比にて表した。血流測定はモデル作製直後、3~4、6および8日後に行った。
ヒトFGF2分泌ビフィズス菌およびコントロールとして非分泌ビフィズス菌をMRS培地(関東化学株式会社より入手)で2回継代した。2回継代した培養液を、生理食塩水を用いて遠心洗浄を行い、生理食塩水で懸濁して使用した。モデル作製翌日、眼窩静脈叢に菌を1×109cfu/mlの濃度で0.2ml投与し、上述(2)の如く下肢血流を測定した。
Claims (19)
- 虚血性疾患部位特異的に生育でき、かつ、少なくとも1種の、虚血性疾患の診断もしくは治療に有用なタンパク質を発現することができる嫌気性菌からなる遺伝子輸送担体。
- 嫌気性菌が、形質転換用プラスミドで形質転換されたものであり、該プラスミドが虚血性疾患の診断もしくは治療に有用なタンパク質をコードするDNA配列を含む、請求項1に記載の遺伝子輸送担体。
- 虚血性疾患が、慢性虚血性疾患である、請求項1または2に記載の遺伝子輸送担体。
- 嫌気性菌が、非病原性の腸内細菌である、請求項1~3のいずれか一項に記載の遺伝子輸送担体。
- 嫌気性菌が、ビフィズス菌である、請求項1~4のいずれか一項に記載の遺伝子輸送担体。
- ビフィズス菌が、ビフィドバクテリウム・アドレッセンティス、ビフィドバクテリウム・アングラタム、ビフィドバクテリウム・アニマリス、ビフィドバクテリウム・アステロイデス、ビフィドバクテリウム・ビフィダム、ビフィドバクテリウム・ボウム、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・カテヌラタム、ビフィドバクテリウム・コエリナム、ビフィドバクテリウム・コリネフォルメ、ビフィドバクテリウム・クニクリ、ビフィドバクテリウム・デンティコレンス、ビフィドバクテリウム・デンティウム、ビフィドバクテリウム・ガリクム、ビフィドバクテリウム・ガリナラム、ビフィドバクテリウム・グロボサム、ビフィドバクテリウム・インディクム、ビフィドバクテリウム・インファンティス、ビフィドバクテリウム・イノピナタム、ビフィドバクテリウム・ラクティス、ビフィドバクテリウム・ラクテンティス、ビフィドバクテリウム・リベロラム、ビフィドバクテリウム・ロンガム、ビフィドバクテリウム・マグナム、ビフィドバクテリウム・メリシクム、ビフィドバクテリウム・ミニマム、ビフィドバクテリウム・モンゴリエンス、ビフィドバクテリウム・パルブロラム、ビフィドバクテリウム・シュードカテヌラタム、ビフィドバクテリウム・シュードロンガム、ビフィドバクテリウム・サイクラエロフィラム、ビフィドバクテリウム・プロラム、ビフィドバクテリウム・ルミナレ、ビフィドバクテリウム・ルミナンチウム、ビフィドバクテリウム・サエクラレ、ビフィドバクテリウム・スカルドヴィ、ビフィドバクテリウム・サブタイル、ビフィドバクテリウム・スイス、ビフィドバクテリウム・テルマシドフィラム、およびビフィドバクテリウム・サーモフィラムからなる群より選ばれる1種である、請求項5に記載の遺伝子輸送担体。
- ビフィズス菌が、ビフィドバクテリウム・ロンガムである、請求項6に記載の遺伝子輸送担体。
- 虚血性疾患の診断に有用なタンパク質が、蛍光タンパク質である、請求項1~7のいずれか一項に記載の遺伝子輸送担体。
- 虚血性疾患の治療に有用なタンパク質が、線維芽細胞増殖因子(FGF)、内皮細胞増殖因子(ECGF)、血管内皮増殖因子(VEGF)、肝実質細胞増殖因子(HGF)血管増殖因子(AGF)、血小板由来増殖因子(PDGF)、トランスフォーミング増殖因子β(TGFβ)、アンギオポイエチン、エフリンなどの血管新生促進活性を有するタンパク質、プロスタグランジン類などの血管拡張に関与する因子、顆粒球コロニー刺激因子(G-CSF)、顆粒球-マクロファージコロニー刺激因子(GM-CSF)などのコロニー刺激因子、神経成長因子(NGF)、脳由来神経栄養因子(BDNF)、ニューロトロフィン3などのニューロトロフィン、インスリン様成長因子(IGF)からなる群から選ばれる一種である、請求項1~7のいずれか一項に記載の遺伝子輸送担体。
- 形質転換用プラスミドが、非シャトルプラスミドである、請求項2~9のいずれか一項に記載の遺伝子輸送担体。
- 形質転換用プラスミドが、さらに分泌シグナルペプチドをコードする遺伝子配列を含む、請求項2~10のいずれか一項に記載の遺伝子輸送担体。
- 形質転換用プラスミドが、pTB6 repユニットを含む、請求項2~11のいずれか一項に記載の遺伝子輸送担体。
- 形質転換プラスミドが、p37プロモーター、HUターミネーター、およびFGF2をコードする遺伝子を含む発現カセットを有する、請求項2~12のいずれか一項に記載の遺伝子輸送担体。
- 形質転換プラスミドが、配列番号40に記載の配列を有するポリペプチドをコードするDNA配列を含む、請求項2~13のいずれか一項に記載の遺伝子輸送担体。
- 形質転換プラスミドが、pFGF12a(配列番号38)である、請求項2~14のいずれか一項に記載の遺伝子輸送担体。
- 請求項1~15のいずれか一項に記載の遺伝子輸送担体を含む、虚血性疾患用医薬組成物。
- 全身投与により投与される、請求項16に記載の医薬組成物。
- 虚血性疾患部位特異的に生育でき、かつ、少なくとも1種の、虚血性疾患の診断または治療に有用なタンパク質を発現することができる嫌気性菌を投与することを含む、虚血性疾患を診断または処置する方法。
- 投与が、全身投与である、請求項18に記載の方法。
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US6984513B2 (en) | 1994-03-03 | 2006-01-10 | The Board Of Trustees Of The Leland Stanford Junior University | Anaerobe targeted enzyme-mediated prodrug therapy |
JP3642755B2 (ja) | 2000-09-21 | 2005-04-27 | 純 天野 | 嫌気性菌を用いた遺伝子治療用医薬 |
EP1227152A1 (en) * | 2001-01-30 | 2002-07-31 | Société des Produits Nestlé S.A. | Bacterial strain and genome of bifidobacterium |
CA2652689C (en) | 2006-05-24 | 2016-02-23 | Anaeropharma Science Inc. | Method of constructing gene transport support |
HUE033561T2 (hu) * | 2008-04-17 | 2017-12-28 | Anaeropharma Science Inc | Expresszió vektor |
JP5841432B2 (ja) | 2010-01-29 | 2016-01-13 | 株式会社アネロファーマ・サイエンス | 形質転換用プラスミド |
-
2012
- 2012-07-12 CA CA2841451A patent/CA2841451A1/en not_active Abandoned
- 2012-07-12 KR KR1020147003731A patent/KR20140046463A/ko not_active Application Discontinuation
- 2012-07-12 WO PCT/JP2012/067797 patent/WO2013008881A1/ja active Application Filing
- 2012-07-12 EP EP12811514.4A patent/EP2733207A4/en not_active Withdrawn
- 2012-07-12 CN CN201280034792.3A patent/CN103958680A/zh active Pending
- 2012-07-12 JP JP2013523979A patent/JPWO2013008881A1/ja active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10993972B2 (en) | 2015-01-19 | 2021-05-04 | Shinshu University | Therapeutic agent for ischemic diseases |
Also Published As
Publication number | Publication date |
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CA2841451A1 (en) | 2013-01-17 |
CN103958680A (zh) | 2014-07-30 |
EP2733207A4 (en) | 2014-12-10 |
WO2013008881A1 (ja) | 2013-01-17 |
EP2733207A1 (en) | 2014-05-21 |
JPWO2013008881A1 (ja) | 2015-02-23 |
KR20140046463A (ko) | 2014-04-18 |
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