WO2005089810A1

WO2005089810A1 - Method of inducing bone by transferring human osteogenetic factor gene with the use of electroporation method

Info

Publication number: WO2005089810A1
Application number: PCT/JP2004/014467
Authority: WO
Inventors: Kazuhisa Bessho; Mariko Kawai; Hiroki Maruyama
Original assignee: Kansai Technology Licensing Organization Co., Ltd.
Priority date: 2004-03-22
Filing date: 2004-09-24
Publication date: 2005-09-29
Also published as: JPWO2005089810A1

Abstract

It is intended to provide an osteogenesis or chondrogenesis agent containing a plasmid vector having human BMP-2 gene integrated thereinto; and an osteogenesis or chondrogenesis method with the use of the above-described osteogenesis or chondrogenesis agent whereby autologous bone regeneration can be less invasively, economically and conveniently carried out even by repeating electroporation for elevating gene transfer efficiency. Namely, an osteogenesis or chondrogenesis method which comprises integrating an osteogenetic factor BMP-2 into a plasmid vector and repeating electroporation to thereby enhance the BMP-2 transfer efficiency into target cells, thus inducing autologous bone regeneration in a bone defect site.

Description

Description Osteoinduction method by transduction of human bone morphogenetic factor using electro-boration method

The present invention relates to bone and cartilage formation using human BMP-2. Background art

BMP-2 (Bone Morphogenetic Protein-2) protein is known to have an osteoinductive effect (Wangeta 1 (1990), Proc. Natl. Accad. Sc i. USA 87, 2 220-2224). After the gene encoding human BMP-2 protein was isolated, the protein was purified from rhBMP-2 (recombinant human BMP-2), and ectopically purified in animal experiments. The results of bone formation and treatment of bone defects have been achieved (Yaskoeta 1 (1992), J. Bone Joint Design. Am. 74, 659-670; Fuji mu raeta 1., (1995), Biochems. Biophys. Res. Commun. 208, 316—322). Thus, various clinical applications of the protein in the dental, maxillofacial and orthopedic fields are expected.

However, BMP-2 protein is expensive and expensive to produce. Also, because of its solubility, it spreads immediately after transplantation, making it difficult to keep this protein at the target site. Thus, as an alternative to the method of inducing osteogenesis using the BMP-2 protein, gene therapy using a gene encoding the BMP-2 protein has been performed. Representative methods include a method using a viral vector and an electroporation method using a non-viral vector.

When a viral vector is used, there is a danger of causing an immune response in the host. On the other hand, in the elect opening method using a non-viral vector, a virus is used. Faster gene expression is possible and does not affect the host immune system. In addition, since the electoral-portation method can be applied to in ViVo, it is possible to repair bone defects in a living body. However, when the electro- volatilization method is used, a high voltage pulse is applied to the cells, so that damage to the cells is inevitable. For this reason, it has not been known that the efficiency of gene transfer is increased by a method that increases the invasion, such as repeating the electoral-portation.

The present invention provides an extremely low-invasive, inexpensive, and easy-to-use bone or cartilage-forming agent containing a plasmid vector into which the human BMP-2 gene has been incorporated, and an extremely low invasiveness even when electroporation is repeatedly performed to increase gene transfer efficiency. A main object of the present invention is to provide a bone or cartilage formation method using the bone or cartilage forming agent, which is capable of autogenous bone regeneration. Disclosure of the invention

The present inventors have been able to improve transfection efficiency by injecting a plasmid vector incorporating the human BMP-2 gene into a target site and repeatedly performing transcutaneous electrobolization, thereby improving bone formation. The present inventors have found that even if they are induced, and that repeated electoral poration is performed, the damage to the cells at the target site is extremely slight, and have completed the present invention.

The present invention provides the following bone or cartilage forming agent, and a method for forming bone or cartilage by introducing a bone morphogenetic gene using an electrolysis method.

Item 1. A bone or chondrogenic agent comprising a plasmid vector into which a human BM P-2 gene has been incorporated so that it can be expressed.

Item 2. A pharmaceutical composition comprising a plasmid vector into which a human BMP-2 gene has been incorporated so that it can be expressed, and a pharmaceutically acceptable carrier.

Item 3. The pharmaceutical composition according to Item 2 for bone or cartilage formation.

Item 4. The pharmaceutical composition according to Item 2, wherein the carrier is suitable for injection or transvascular administration.

Item 5. — Counter electrode, means for supplying electric pulses, Item 2. An apparatus for introducing the bone or cartilage forming agent according to Item 1 into cells at the site, comprising a means capable of fixing the pole.

Item 6. The pharmaceutical composition according to Item 2, further comprising two or more auxiliary components selected from the group consisting of anti-inflammatory agents and growth factors.

Item 7. A bone or cartilage forming agent comprising cells transformed by a plasmid vector into which a human BMP-2 gene has been expressed.

Item 8. A step of administering a plasmid vector incorporating a human BMP-2 gene to an invivo target site, and a step of introducing the gene into cells at the invivo target site by electroporation. Bone formation method including. Although it is not desired to limit the interpretation of the present invention, the present inventors consider the mechanism of action of the present invention as follows.

After injection of the plasmid vector containing the human BMP-2 gene into the target site, the human BMP-2 gene is introduced into cells at the target site by electroporation. Produces and secretes BMP-2 protein in cells. The secreted BMP-2 protein acts on the surrounding mesenchymal cells, induces osteoblasts, and forms bone tissue.

Injection of a plasmid vector incorporating the human BMP-2 (Bone Morphogenetic Protein-2) gene, which induces ectopic formation of bone composition, at the target site, followed by electoporation for human BMP-2 By introducing the gene into cells at the target site, bone formation is induced at the target site. At this time, efficient electrophoresis can be achieved by performing electroboration several times. In addition, since human BMP-2 induces ectopic bone formation, bone regeneration with autologous bone is possible even in patients who have lost bone due to surgery or the like.

In the present invention, "inducing ectopic bone formation" means that bone formation is possible even when there is no bone tissue nearby. That is, the use of the bone or cartilage-forming agent of the present invention enables repair, for example, when most of the mandible is lost. The term "target site" refers to a portion of a mammal intended for bone or cartilage formation in vivo, and the term "target site cells" refers to a bone or cartilage in mammals in vivo. Refers to any cell in the portion intended for formation. The BMP gene used in the present invention is a cytokin belonging to one of the TGFs (Transform Group Growth Factor) —) 3 supergene family, and has genes for osteoinductive factors such as osteogenic factors. Is widely included. Further, the BMP gene is preferably human BMP.

Hereinafter, the case where human BMP-2 is used will be described. However, changes in the case where another BMP gene is used can be easily conceived by those skilled in the art.

The plasmid vector incorporating the human BMP-2 of the present invention can be obtained by a conventional method such as screening and subcloning.

In preparing the human BMP-2_ plasmid vector of the present invention, it is preferable to perform screening using the primer sets of SEQ ID NOS: 1 and 2 as primers. In SEQ ID NO: 1, GAATTC at positions 9 to 14 from the 5 side represents an EcoRI recognition site, and ATG at positions 22 to 24 from the 5 'side represents an initiation codon. In SEQ ID NO: 2, GAATTC at positions 9 to 14 from the 5 'side represents an EcoRI recognition site, and CTA at positions 15 to 17 from the 5' side represents a stop codon.

Examples of the plasmid vector used for preparing the human BMP-2-plasmid vector of the present invention include pCAGGS, pUC BNP-2, pGL3Basic Vector, pDSRed 2-1 and the like, which are usually used for animal cell expression. A plasmid vector can be used as appropriate, but pCAGGS is preferred. The plasmid vector used in the present invention contains a promoter. The promoter is not particularly limited as long as it is commonly used by those skilled in the art, and those derived from viruses, for example, those derived from retrovirus, poliovirus, adenovirus, SV40, or those derived from non-virus (Eg, EF 1-α), those derived from bacteriophage λ, CAG, trp, lpp, lac, tac promoter, etc., and the CAG promoter is preferred. Although the human BMP-2-plasmid vector of the present invention is stable even at room temperature, it is preferably stored in a refrigerator at 4.

The human BMP-2-plasmid vector of the present invention can be used as a bone or cartilage forming agent, and the bone or cartilage forming agent contains a pharmaceutically acceptable carrier usually used by those skilled in the art. You can also. Carriers include sterile water, physiology Examples include saline and various buffer solutions. Similarly, it can also contain excipients and additives.

Excipients include sodium chloride, sodium citrate, and the like. Additives include thickeners, buffers, preservatives, and preservatives.

Examples of the thickener include methylcellulose (MC), carmellose sodium (CMC-Na), chondroitin sulfate, and the like.

Examples of the buffer include boric acid, sodium polyphosphate, trisodium phosphate, phosphoric acid phosphate, maleic acid, citric acid anhydride, dried sodium carbonate, acetate phosphate, sodium acetate, and the like.

Examples of preservatives or preservatives include ethyl parahydroxybenzoate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parabutyl benzoate, benzyl alcohol, chlorobutanol, benzalkonium chloride, benzethonium chloride, sodium acetate, and the like. .

Preferably, the bone or chondrogenic agent of the present invention is prepared as a formulation for topical administration.

The method for forming a bone using electroboration according to the present invention includes the following processes.

Process 1. Pre-treatment

In process 1, it is preferable to apply an appropriate amount of keratin cream or the like, for example, in order to remove the body hair at the target site if necessary and to improve the electrical conductivity in order to facilitate electroboration.

Process 2. Target site administration

The administration form of the bone or cartilage-forming agent containing the human BMP-2-plasmid vector of the present invention is not particularly limited as long as the plasmid vector can be administered to the target site. Although it can be used, injection is preferred.

The injection of the human BMP-2-plasmid vector is preferably carried out at a site to be energized between electrodes used for electroporation. In addition, for example, when forming bone of about 1 ml in volume, the dose of human BMP-2_ plasmid vector is: Approximately 1 ml, and the concentration of this plasmid vector is about 0.1 to 100.0 / ^ g / 1, preferably about 0.3 to 50.O ^ g / i1, more preferably about 0.5 to 8. OwgZ ^ l. The dose and concentration of the plasmid vector can be appropriately adjusted depending on the target patient and the target site.

In the case of performing the bone formation method of the present invention, it is possible to increase the bone formation rate by mixing an osteogenesis-promoting substance with an injection solution when injecting human BMP-2-plasmid vector. Examples of the bone formation promoting substance include PGE, (Prost ag l and i η Ε,), FGF (Fibr ob last Growth Fac tor), TG F-0, vitamin D ₃ , vitamin K _{2 and the} like. . The osteogenesis-promoting substance can also be administered after electrification.

Process 3. Elect mouth opening

The electrode used in the bone formation method according to the present invention is made of carbon, platinum, gold, titanium, silver, silver chloride, stainless steel, stainless steel, tungsten, copper, lead, copper chloride, or the like. It is preferably made of stainless steel. Further, it may be plated with a commonly used plating material such as platinum, gold, silver or copper. The shape of the electrode may be a needle electrode, but is preferably a plate electrode which can be passed through percutaneously from the viewpoint of invasiveness. Depending on the target site, the shape of the electrode can be appropriately changed to a tweezer type, a grip type, or the like. Means for fixing the electrodes include pinching with tweezers-type electrodes, insertion of needle-type electrodes, and the like. Prepare two electrodes and use them in pairs so as to sandwich the target site. Using the above-mentioned electrodes, inject human BMP-2-plasmid vector into the central part between the two electrodes, and perform electrofusion, that is, by applying an electric pulse to perform transfusion on cells at the target site. Can be. As the device for supplying the electric pulse, an electoral porosion system commonly used by those skilled in the art can be used.

As a method of the electric pulse, the voltage is about 25 to 500 V, preferably about 50 to 200 V, and the energizing time is 5 to: L about 000 milliseconds, preferably about 10 to 200 milliseconds, and the total is 1 to 100. Times, preferably 6 to 12 times, at an interval of about 0.25 to 30 seconds, preferably about 0.5 to 5 seconds. At this time, it is preferable that the energization in the second half (four times in the second half if the total is eight times) be performed with electric pulses of opposite polarity (reverse direction), and if possible, alternately energize electric pulses of different polarity Is more preferable. This series of injection of the human BM P-2—plasmid vector—and the series of electoral porations constitute one session. To induce bone formation by expressing the human BMP-2 gene in vivo, it is possible to perform the above treatment only for one session, but to induce bone formation reliably, it takes about 2 to 6 sessions. Preferably, about 3 to 5 sessions, and most preferably, 5 sessions are performed.

Since human BMP-2-plasmid vector is easily diffused in the body, electoral poration is performed immediately after injection, and the upper limit of the electoral polish is preferably six times per session. It is not preferable to carry out electroboration seven times or more, because the burden on the cells at the target site increases and the sensitivity of the cells decreases. When bone formation is continuously induced, it is preferable to allow about 24 hours for each session.

When energizing, it is preferable to check the accuracy of the current by measuring the resistance between the electrodes. At this time, it is preferable that the resistance between the electrodes is usually 800Ω or less. It is preferable that the distance between the electrodes is as short as possible.

For example, when the osteogenesis method of the present invention is applied to a patient having a partial humerus defect, the same volume as the bone portion lacking the human BMP-2 plasmid vector solution (8.0 Og / P-1) ( Assuming that the cross-sectional area of the humerus is 4 square cm, 40 cc) is injected, and 100 V is applied at a terminal or percutaneous election port placed at the stump of the bone. (4 times reverse the direction of current flow) at 1 second intervals. Do this 5 sessions every 24 hours. When applying to a bone defect at another site, those skilled in the art can easily determine the implementation conditions with reference to the above example of the humerus.

When forming a relatively large bone, make a terminal in the shape of the bone to be formed (or a cross-sectional shape) and energize it percutaneously, or insert a terminal made of an absorbent material etc. and energize it There is a method. At this time, a biodegradable and conductive plastic or the like can be used as the absorbent material. In addition, the administration of BM P-2 single plasmid vector and electoral poration were repeated many times, There is also a method of stopping when a suitable shape is obtained. In this case, a tweezers type electrode may be used.

When practicing the bone formation method according to the present invention, the patient may be anesthetized with an anesthetic commonly used by clinicians.

Process 4. Work-up

After electroporation, it is possible to confirm the presence or absence of bone formation by radiography. Further, after election port poisoning, it is preferable to fix the affected part by a method usually used by those skilled in the art (cast, bandage, etc.).

Since the bone formation method according to the present invention may cause mild but injured muscles and cause inflammation in the affected area, it is preferable to administer a commonly used anti-inflammatory agent after electoporation. Examples of the anti-inflammatory agent include aspirin, ferpinac, indomethacin, diclofenac sodium and the like. These anti-inflammatory agents can be administered together with or separately from the human BMP-2-plasmid vector prior to electroporation.

When the bone formation method according to the present invention is applied to a femoral bone defect or a deep bone defect, the electrode of the present invention can be used in the form of a needle or a terminal. In the case of deep bone loss, a method of surgically implanting a conductive film or the like into the body, performing electroporation using a needle electrode, and then removing the conductive film is applied. can do.

In another preferred embodiment of the present invention, bone or cartilage formation using transformed cells is demonstrated by a plasmid vector incorporating the human BMP-2 gene.

Transformation of cells with the human BMP-2-plasmid vector can be performed by a transformation method generally used by those skilled in the art.

Cells transformed by the human BM P-2-plasmid vector according to the present invention are not particularly limited as long as they are commonly used by those skilled in the art, and human-derived cells such as He1a cells. It is preferable that

When producing a transformed cell, it is preferable to incorporate a selection marker such as a drug resistance marker into the human BMP-2_plasmid vector. By injecting the transformed cells obtained as described above into, for example, a target site of bone of an osteoporosis patient, bone mass can be increased and symptoms can be improved. In addition, cartilage formation can be promoted by injecting into cartilage damage sites such as knee cartilage.

In addition, the target site is surgically implanted with a guide made of a resorbable material, such as a cylindrical or sheet-shaped guide, that is, a material that defines the final bone shape, and the transformed cells are injected there. It is also possible to induce bone and cartilage formation. At this time, it is preferable that the material of the absorption layer i is a material such as collagen, cellulose, chitin and chitosan that can supply blood from the host.

Bone and cartilage formation using the above-described transformed cells can be similarly carried out by the electroporation method of the present invention using human BMP-2-plasmid vector instead of the transformed cells.

The subject to which the bone formation method according to the present invention is applied includes, for example, patients who have had bone defects due to tumor surgery or accidents. In addition, even if no bone defect has occurred, diseases in the field of orthopedic surgery, such as crush fractures, osteoarthritis, congenital bone diseases (eg, osteogenesis imperfecta); cleft palate; chin formation and maxillary sinus The present invention can also be applied to diseases in the dental field, such as implant pretreatment such as raising the floor, and osteoporosis. Further, the bone formation method of the present invention is also applicable to veterinary surgery.

There are two main methods of pre-treatment of implants.One is to administer to a site with insufficient bone mass, and the other is to administer at the time of implant placement. There are ways to shorten the healing period by promoting it. In the latter case, it is possible to accelerate the healing rate even if only a very small amount of the human BMP-2-plasmid vector or a cell transformed with it is present on the surface of the implant.

Further, the bone or cartilage forming agent of the present invention can be used for regeneration of teeth, particularly for dentin formation. If tooth germ cells and stem cells are applied, cement and enamel can be regenerated.

Furthermore, the bone or chondrogenic agent of the present invention utilizes properties other than the bone forming ability of the BMP gene, for example, the renal cell differentiation activity of the BMP gene, and is used as a prophylactic and therapeutic agent for renal diseases. Can also be used. Brief Description of Drawings

Figure 1. Figure 1 shows X—ga 1 versus 8-galactosidase in muscle following transfection of the pCAGGS-1 ac Z gene with or without percutaneous electo-oral poration. The photograph of the staining is shown. Muscles were stained one day after one percutaneous electroporation (A) or five injections of 25 PCAGGS-1acZ without electroporation (B). As a control, untreated muscle (C) was stained in the same manner. All sections were stained with eosin. Scale bar: lmm

Figure 2. Figure 2 shows a photo of RT-PCR detection of human BMP-2 mRNA in muscle after transcutaneous electoporation of pCAGGS-BMP-2 or pCAGGS. 25, 100, and 400 g of PCAGGS-BMP-2 were electroporated, and 5 days (5 d) or 10 days (10 d), human BMP-2 mRNA was detected.

Figure 3. Figure 3 shows the transdermal electroporation of pCAGGS-BMP-2 (BMP-2 in the figure) or pCAG GS (C in the figure) in 1, 3, and 5 sessions (respectively, XI, X3, X5) The results of RT-PCT for the detection of human BMP-2 mRNA in muscle after the procedure are shown in the photograph.

Figure 4. Figure 4 shows BMP-2 production after a single electoporation of pCAGGS-BMP-2. After injection of 25 g, 100 fig, or 400 pCAG GS-BMP-2 and electoral poration, the results of Western blotting 5 days (5 d) or 10 days (10 d) were obtained. Shown in pictures. Figure 5. Figure 5 shows the BMP after 25 wg of PCAGGS—BMP—electrophoresis, 1, 3, or 5 sessions (XI, X3, and X5, respectively). 2 Production is shown in the photograph.

Figure 6. Figure 6 shows the pCAGGS-BMP-2 electo-portion, pathological changes in muscle after 1 (A, E), 3 (B, F), or 5 (C, G) sessions. The photographs show the localization of BMP-2 and BMP-2. A, B, C and D show hematoxylin and eosin (H & E) staining. Pathological changes were detected by hematoxylin and eosin (H & E) staining 7 days after electroporation. Identical sections were analyzed for BMP-2 localization by immunobiochemistry (E, F, G, H). Untreated muscle was stained in a similar manner (D, H). Scale bar: 500 m Figure 7. Figure 7 shows a soft x-ray of the target muscle 21 days after transfection of pCAGGS-BMP-2 or pCAGGS. After electroporation of pCAGGS-BMP-2 (pCAGGS-BMP-2 wi the 1 ectroporation), an opaque part was observed (arrow). Scale bar: 10 mm Figure 8. Figure 8 shows the results of histological examination 21 days after transfection of pCAGGS-BMP-2 or pCAGGS. After administration of pCAGGS-BMP-2, H & E staining (A, D) revealed osteoblast-like cells (red arrow: left arrow in the figure) and osteoclast-like cells (black arrow: right arrow in the figure) Indicated the presence of ectopic bone formation with Serial sections were stained for immunohistochemical detection of non-tissue specific phospholipase (TNALP; B, E). Tartrate-resistant acid phosphatase (TRAP) positive osteoclasts were detected by TRAP staining (C, F). Positive osteoclasts are stained red (C, F). No bone formation was observed in TNALP expression or TRAP positive osteoclasts 21 days after electoral poration of pCAGGS (G, H, Do scale bar: 100 m

Figure 9. Figure 9 shows a map of pCAGGS-BMP-2. BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to examples, but it is needless to say that the present invention is not limited to these examples.

Example 1. Plasmid vector

In order to obtain human BMP-2 cDNA, plasmid pUC BNP-2 (Oku boeta 1 (1999), Biochem. Biophys. Res. Commun. 262, 739-743), and SEQ ID NO: 1 And IE column number 2 PCR (Polymerase Chain Reaction) using one set of primers was performed. In SEQ ID NO: 1, GAATTC at positions 9 to 14 from the 5 'side represents an EcoRI recognition site, and ATG from position 22 counted from the 5, side represents an initiation codon. In SEQ ID NO: 2, GAATTC at positions 9 to 14 from the 5 'side represents an EcoRI recognition site, and CTAs at positions 15 to 17 from the 5' side represent a stop codon.

PCR was performed using 25 cycles of 15 seconds at 98, 2 seconds at 65, and 30 seconds at 74: using the K〇D DNA polymerase (Toyo bo, Osaka, Japan) ). This PCR product is digested using EcoRI, and to obtain pCAGGS-BMP-2, CAG (cyt om egalovirusi mm idiate—early encer / chiken β—actin hy brid) promoter Ligation was performed on the EcoR I-digestion cloning site of the pCAGGS expression vector, which contained the iwaeta 1., (1991), Gene 108, 193-199). The insert sequence of this 1197-bp was confirmed by DNA sequencing. The plasmid was then propagated in EscherichiacolicoDH5a. The plasmid vector was prepared using QiagenEndoFreepiamsidGigakite (Qiagen GmbH, Hi1den, Germany) (Maruyama et a 1., 2000). Immediately prior to injection, the DNA was diluted to 0.5, 2, or 8 / n1 in phosphate buffered saline (PBS).

Rat

Nine-week-old male Wistar rats were purchased from Kurea (Osaka, Japan) and housed under specific illuminant-free conditions.

Intramuscular DNA injection and transdermal electroporation

The rat was anesthetized by intraperitoneal administration of sodium pentovalpital (body weight: 100 g, 0.5 mg), and the hair in the target area of the foot was removed with a clipper. After covering the target site with keratin cream (Fukuda Denshi, Tokyo, Japan), a set of 5mm fixed length stainless steel plate electrodes Was attached. The accuracy of the current was confirmed by measuring the resistance between the electrodes (usually less than 800 Ω) surrounding the center of the gastrocnemius muscle without skin incision.

Next, 50/1 plasmid DNA (25 g, 100 g, or 400 wg) was injected in the middle between the electrodes using a 27 gauge needle. Immediately after the injection, electo-portionation was started by applying eight electrical pulses (100 V, 50 ms ec). After energizing four times, electric pulses of different polarities are performed four times, and these electric pulses are applied to the injection site at 1-second intervals using a quadrilateral electro-ballet (CUY21ED IT; Nepa Gene). Processed. In this example, electoral port por- tions were performed at intervals of 24 hours for 1 to 5 sessions. Statistical analysis

Mean The result is expressed as the standard error of the soil mean (SEM). Statistical analysis of differences between groups in ALP activity and Ca content was performed by analysis of variance (ANOVA), followed by Fisherscomparisonstest.

X—ga 1 staining

To clarify the in vivo gene transfer into skeletal muscle by percutaneous electoporation, it induces the cytoplasmic expression of E. coli i3-galactosidase (Niwa et al., (1991), Gene 108). Muscle tissue was stained histologically after donating 25 g of PCAGGS-1acZ. Rats are dissected one day after electoral poration, and target muscles are embedded in Tissue—Tek II. CT c oumpound (Sakura Finetechnical Co. Ltd., Tokyo, Japan). This tissue was frozen in a mixture of dry ice and acetone. Then, serial sections (5-m thickness) were prepared on a cryostat and placed on a slide glass coated with 3-amino-propyltriethoxysilane.

The sections were fixed in 1.5% dartaldehyde at room temperature for 10 minutes, washed three times with cold PBS (5 minutes per wash), and then placed in PBS (pH 7.4). , Containing lmg / ml Xg a 1, 2 mM MgC 12, 5 mM K ₄ Fe (CN) ₆ , 5 mM K ₃ Fe (CN) ₆ , and 0.5% Non i de t P-40, X— Incubate for 3 hours at 37 in gal staining solution I got it. The section was counterstained with eosin (Aihara and Miyazaki, (1998), Nat. Biotec hno l. 16, 16, 867-870).

After one 25 g electroporation with 25 g pCAGGS-lacZ

In Figure 1A, X-ga1 staining was much more extensive than after 5 injections of pCAGGS-1acZ (25 g) without electroporation (Figure IB). Muscle fibers were observed.

The results showed that transdermal electoporation increased the number of muscle fibers transfected by plasmid DNA. Example 2. RT-PCR

1, 5, or 10 days after gene transfer, rats injected with pCAGGS-BMP-2 (n = 1) or pCAGGS alone (n = l) were dissected under general anesthesia and targeted. The muscle (0.15 g) was excised. Total RNA, Isog en

(Nippon Gene, Tokyo, Japan) to isolate from muscle tissue. Using the primers shown in SEQ ID NOS: 3 to 6, human BMP-2 mRNA and dariseraldehyde-3-phosphate dehydrogenase (G3 PDH) mRNA were detected by RT-PCR.

The human BMP-2 forward primer, shown in SEQ ID NO: 4, was designed to hybridize to a sequence immediately downstream of the transcription initiation site of the CAG promoter to prevent contamination of the PCR product with plasmid or genomic DNA. . Incubate the RNA (1 fig) at 42 "C for 1 hour in a total volume of 201, then 94 for 3 minutes, followed by 94 for 1 minute, 55 ^ for 1 minute, and 72 The PCR product was analyzed by 2% agarose gel electrophoresis to detect 285-bp human BMP-2mRNA and 682-bp G3PDHmRNA.

5 or 10 days after one session of electroporation with pCAGGS—BMP—2 (25> 100 g or 400 g) or pCAGGS, a portion of the muscle is excised and RT—PCR is used to pCAGGS— The presence of human BMP-2 mRNA transcribed from BMP-2 was detected (Figure 2). In addition, pC A similar analysis was performed on muscles extracted one day after AGGS—81 \ 4? —2, 1, 3, or 5 sessions of electotomy (25 g each) (Figure 3).

Human BMP-2 mRNA was detected in all muscles 1, 5, and 10 days after transfection of pCAGGS-BMP-2 by percutaneous electoporation, but transfection of pCAGGS Was not observed in the lean muscle. Control G3PDH mRNA was observed in all groups (Figures 2 and 3). Therefore, RT-PCR results indicated that human BMP-2 was expressed in transfected cells in target muscle following electroporation of pCAGGS-BMP-2.

Example 3. Western blot analysis of BMP-2

After injection of the plasmid and electroporation, the gastrocnemius muscle was dissected. Tissue samples (0.2 g) were placed in 0.25 M sucrose in a Bio Mixer Polyethylene homogenizer (type ABM, Nissei Inc. Osaka, Jaan). Homogenized and centrifuged at 5000 g for 4 minutes at 4. This supernatant was mixed with heparin-Sepharose (Amersham, Arlint on Heigts, IL), and stirred with 喑 jar so that the protein was adsorbed on heparin sepharose. The heparin sepharose mixture was washed with PBS and centrifuged at 8000 g for 1 minute at 4. After removing the supernatant, the heparin sepharose mixture was mixed with an equal volume of sodium dodecyl sulfate gel electrophoresis loading buffer, heated at 70 for 10 minutes, and separated on a 12% acrylamide gel.

This protein was subjected to electroblotting on a nylon membrane (Mi11 ipore Co., Watermark, MA), and then the membrane was blotted onto a B1ock Ac e (D ainippon Se yaku, T ok yo). , Japan) at 4-blocking, and then room with anti-human BMP-2 monoclonal antibody (1: 200 dilution, Genetics Institute, Cambridge, MA) in blocking buffer. Incubated at room temperature for 2 hours. Then, wash the membrane and bring it to room temperature For 1 hour with horseradish peroxidase (HRP) -labeled anti-mouse antibody (1: 3000 dilution) and visualized using an electrochemiluminescence (ELC) detection system (Amersham). I dumb.

As shown by Western plotting, target muscle cells electroporated with pCAGGS-BMP-2 were shown to be capable of secreting BMP-2. Western blot analysis in target muscle treated with pCAGGS—BMP—2 (25 g> 100 ig or 400 g) showed BMP-2 at 5 or 10 days after electoporation in one session (Figure 4). In contrast, no BMP-2 production was observed in the group treated with pCAGGS. After one, three or five sessions of electroporation with pCAGGS-BMP-2 (25 g), production of BMP-2 was observed in all groups in rat muscle one day later. (Figure 5). These results indicate that BMP-2 gene transfer into skeletal muscle by percutaneous electoporation produces BMP-2.

Example 4. Evaluation by radiography

Rats were killed with an overdose of bentvalpital 21 days after plasmid injection with electoral poration or plasmid injection alone. Thereafter, the injected gastrocnemius muscle was excised, and soft X-ray photography was performed (SRO_M50, Sofron Inc., Tokyo, Japan).

The x-rays showed opaque areas with well-defined edges in the target muscles of all groups that received the pCAGGS-BMP-2 electoral portation (p CAGGS-BMP-2 2 w e t e l e c t r o p o r a t i o n). In contrast, pCAGGS electoporation of pCAGGS or a group receiving 5 injections of pCAGGSGS-BMP-2 without electoporation (pCAGGS_BMF—2 wi thou hou telectr opo) ration), soft X-rays did not detect opaque areas with sharp edges (Figure 7).

The opaque area with clear edges is the shape of six rats transfected with 258-800-81 ^? _ 2 in one session by electroporation. 2 (33%), 2 of 6 rats (33%) treated with 25 g of PCAGGS-BMP-2 in 3 sessions of electroporation, and 25 g of pCAGGS-BMP-2 Was observed in all six rats (100%) treated with 5 sessions of electroporation.

After transfection of 10 O ^ g or 40 Oxig of pCAGGS-BMP-2, opaque areas with clear edges on soft X-rays show similar trends as those detected in the 2 g treatment group. It was observed. That is, the probability of detecting an opaque portion increased with the repetition of the transcutaneous electroboration of pCAGGS-BMP-2.

Example 5. Histological and immunohistochemical analysis

To investigate the secretion and distribution of BMP-2 by electroporation with target 003-8 ^^?-2 in the target muscle, the muscle was immunohistochemically analyzed 7 days after electroporation. Samples were taken for analysis, fixed in 10% buffered formaldehyde, and embedded in paraffin. Thereafter, 4 / m thick sections of this sample were prepared. Monoclonal anti-human BMP-2 antibody was purchased from Genetics Institute (Cambridge, MA) and immunohistochemistry was performed using a labeled streptovidine-biotin staining kit (LSAB kit, DAKO, Carpinteria, CA). I did a dagger study. Performed deparaffinized sections were rehydrated, to inhibit the activity of endogenous Peruokishidaze were treated with 3% H _₂ 0 ₂ 15 min. The sections were then incubated overnight at 4 with anti-BMP-2 antibody (1: 100 dilution). The labeling reaction was visualized with diaminobenzidine (Wako Pure Chemical Industries, Ltd., Osaka, Japan), and counterstained with Mayer's matoxylin.

Eight days after the electoral poration of 1 session, 3 sessions, or 5 sessions of 81 ^? — 2 sessions, BMP-2 is highly expressed in the muscle surrounding blood vessels by immunological analysis. (Figures 6E, 6F and 6G). On the other hand, BMP-2 expression was not observed in the muscle that was not treated (FIG. 6H). These results were applied to the electoral port of pCAGGS-BMP-2. This indicates that gene transfer caused BMP-2 production and secretion in the target muscle.

To verify that the opaque area in the soft X-ray cavity was bone formation after electroporation of PCAGGS-BMP-2, a sample 21 days after electroporation was fixed with 10% buffered formaldehyde and paraffin was added. Embedded. Then, 4m thick sections were prepared and stained with H & E.

A new bone-like matrix was observed in the muscle fibers (FIG. 8A), and a bone marrow-like space containing blood cells was found in a portion of the new bone-like matrix. The new bone-like matrix was oval, hematoxylin-positive, and formed a line around the matrix. Many bones contained cells (Fig. 8D, red arrow: left arrow in the figure). There were also multinucleated osteoclast-like cells surrounding the bone-like matrix (Fig. 8D, black arrow: right arrow in the figure). On the other hand, no ectopic bone formation was observed in the group that received the electoporation of pCAGGS (Fig. 8G).

To stain serial sections with tartrate-resistant acid phosphatase (TRAP), a marker for osteoclasts, and to detect normal bone formation, tissues that are markers of calcification by osteoblast activity It was used for immunohistochemical detection of non-specific alkaline phosphatase (TNAL P). 0.1 mg mg ml NaF in 1 M acetate buffer (pH 5.0) ^^ ASML phosphate (Sigma, St. Lou is, M〇), 0.5% N, N-Dimethyl TRAP activity was detected by incubating with a mixture of formamide and 0. emg / m1 fastred AL salt (Sigma) at 37: for 30 minutes. Sections for TNALP immunohistochemistry, deparaffinized, rehydrated, and to inhibit endogenous Peruokishida Ichize were treated with 3% H ₂ 〇 ₂ 15 min. The sections were then incubated with anti-TNALP antibody (1: 200 dilution) at 4 (Odaetal, (1999), J. Biochem. 126, 694_699). Thereafter, the labeling reaction was visibly illuminated with diaminobenzidine, and the sections were comparatively stained with Mayoxy's matoxylin.

Ectopic bone-like matrix was tested for its ability to have osteoblast-contributing calcification activity. The expression of TNALP was detected in tissues surrounding the tissue and in the bone marrow-like space (FIGS. 8B and 8E).

Sections were stained with TRAP to determine whether these osteoclast-like cells were mature osteoclasts. TRAP-positive cells were scattered on the surface of the bone-like matrix. Many of these cells were consistent with osteoclast-like cells (Figure 8C and Example 6. Biochemical studies).

ALP activity (1 milligram of protein per international unit [IU / mg]) for quantitative analysis of bone formation after gene transfer with pCAGGS-BMP-2 using in vivo transdermal electroporation ) And tissue calcium (Ca) content (g / mg tissue) were measured. Samples (n = 5) were weighed and then homogenized in 0.25 M sucrose in a BioMixerPo1ytron homogenizer. The alkaline phosphatase (ALP) activity and total protein content of the resulting supernatant were determined by the 4_nitrophenylphosphatase method (Okuboeta 1., (1999), Biochem. Bi). ophy s. Res. Commun. 262, 739-743). The precipitate was desalted with 0.5N hydrochloric acid, and the calcium (Ca) content of the soluble fraction was determined by the ortho oc resol ph tha leinc omp lex method (Connery and Briggs, (1966), Am J. C li n. Pat ho l. 45, 2

90-296).

The ALP activity (3.6680 ± 0.055 I UZmg protein) and the Ca content (904600 ± 1.8452 gZmg tissue) were all obtained by adding pCAGG S-BMP-2 (25 g) every 24 hours. After the electoral port poration of the session, the significance (ρ <0 · 001) was higher than that of the same treatment using pCAGGS (0.000002 Sat. 0.001 IU). G protein mg, 0.0017 ± 0.0008 gZmg male).

From these data, it was found that the opaque areas observed on the soft radiographs were indicative of ectopic ossification. The ectopic bone contained osteoblasts, which differentiated and played a key role in bone matrix production, and mature osteoclasts. Example 7. Muscle damage by elect opening

Serial sections prepared in Example 5 were stained with hematoxylin and eosin (H & E) to analyze the damage caused by the electroporation of pCAGGS-BNP-2.

Muscle damage by transdermal electroporation of pCAGGS-BMP_2 was analyzed by microscopic and histological examination. Under the conditions of this example, no signs were seen in any of the rats during the three weeks of the test period. In addition, no minor muscle damage, such as burns and edema, was observed 1, 5, 7, or 10 days after electotomy. Seven days after five electroporations of pCAGGS-BMP-2, microscopic examination revealed significant infiltration of mononuclear cells into target muscle and necrosis, but none of them. It was not severe (Figure 6C).

On the other hand, muscles that had undergone a single electoporation showed only minimal infiltration of mononuclear cells and no necrosis was observed (Fig. 6A). Furthermore, it has been found that the degree of damage caused by electroporation does not depend on the amount of pCAGGS-BMP-2. The above-mentioned inflammatory response disappears by day 21 (Figure 8A), and damage from electroporation adversely affects bone formation, even after five percutaneous electoporation. Was not.

FIG. 9 shows PC AGGS-BMP-2 used in this example.

Industrial applicability

According to the present invention, a bone or cartilage-forming agent comprising a plasmid vector into which a human BMP-2 gene has been incorporated so that it can be expressed, and extremely low invasiveness even when repeatedly electoporation is performed to enhance gene transfer efficiency Thus, it is possible to provide a bone or cartilage formation method using the bone or cartilage forming agent, wherein the bone or cartilage formation agent can be easily and inexpensively regenerated.

Therefore, according to the present invention, it is possible to induce bone or cartilage formation in a minimally invasive, inexpensive, and simple manner.

Furthermore, since the bone or chondrogenic agent of the present invention contains a plasmid vector incorporating the BMP-2 gene, it can regenerate autologous bone in the vicinity of bone m ^ in vivo, and the bone defect site can be reduced. It can be applied to a wide range of cases.

The bone formation method of the present invention can be applied to a wide range of fields such as orthopedic surgery, dentistry, veterinary surgery, and the like.

Claims

The scope of the claims

1. A bone or chondrogenic agent consisting of a plasmid vector into which human BMP-2 gene has been incorporated so that it can be expressed.

2. A pharmaceutical composition comprising a plasmid vector into which human BMP-2 gene has been incorporated so that it can be expressed, and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition according to claim 2, for bone or cartilage formation.

4. The pharmaceutical composition according to claim 2, wherein the carrier is suitable for injection or transvascular administration.

5. A pair of electrodes, means for supplying an electric pulse, means capable of fixing the electrode to a site where bone or cartilage is to be formed, wherein the bone or cartilage-forming agent according to claim 1 is used for cells at the site. Equipment for introducing to the.

6. The pharmaceutical composition according to claim 2, further comprising two or more auxiliary components selected from the group consisting of an anti-inflammatory agent and a growth factor.

7. A bone or cartilage forming agent consisting of cells transformed by a plasmid vector into which the human BMP-2 gene has been incorporated so that it can be expressed.

8. A step of administering a plasmid vector incorporating the human BMP-2 gene to a target site of in vivo, and a step of introducing the gene into cells at the target site of invivo by electro-volume method in this order. Bone formation method.