WO2003083105A1 - Treatment and prevention of angiostenosis - Google Patents

Abstract

It is intended to provide means of treating and preventing angiostenosis. Namely, a replication vector showing vascular smooth muscle-specific expression and not acting on normal adult cells characterized by having the transcription initiation regulatory domain of a gene expressed specifically in vascular smooth muscle integrated into the upstream of a definite gene; medicinal compositions containing the same for treating and preventing angiostenosis; and method therefor.

Description

 Description Treatment and prevention of vascular stenosis

 The present invention relates to a vascular smooth muscle-specific expressing replicating viral vector that expresses a gene specifically in vascular smooth muscle and self-replicates and does not act on normal cells in adults, and a method for treating and preventing vascular stenosis of the vector. Regarding the use, specifically, a vascular smooth muscle-specific expression-replicating virus vector that does not act on adult normal cells, characterized by incorporating a transcription initiation control region of a gene expressed specifically in vascular smooth muscle upstream of a predetermined gene. The present invention relates to a pharmaceutical composition for treating and preventing vascular stenosis and a method for containing the same. Background art

 Atherosclerotic lesions, especially stenotic lesions of the main arteries such as the carotid and coronary arteries, are becoming a major problem in Japan. Treatment of these lesions with ballooii angioplasty is being established with the recent advances in endovascular surgery. However, even if stenosis is corrected once by this endovascular operation, restenosis occurs at a frequency of 30 to 50%, which is a major problem of this treatment method.

 In addition, it is expected that demand for various organ transplants, which has recently become a topic, will increase rapidly in the future. Although the survival of the organ itself has been improved by immunosuppressants, the stenosis of the vascular anastomosis between the transplanted organ and the patient remains a problem, which causes the incomplete function of the transplanted β and its loss. For example, angina and myocardial infarction after heart transplantation are problems that need to be overcome. The main pathological condition of these arteriosclerosis and vascular stenosis after organ transplantation is the proliferative change of the vascular smooth muscle, and developing therapeutic methods for this condition is of great interest.

Various approaches have been used to treat vascular restenosis, including various drugs and antibodies, but no effective treatment has yet been established. In the United States, clinical trials of cytostatic therapy using c-myc antisense and E2F decoy are being conducted. These approaches have no cell specificity. In addition, gene therapy using various gene transfer vectors has been attempted, but low in vivo gene transfer efficiency has been a problem, and good therapeutic effects have not been obtained.

 The present inventors have continued to study gene therapy for malignant tumors using a replicable recombinant herpes simplex virus (HSV) beta. Focusing on ICP4, which is expressed very early in HSV DNA replication and is essential for self-gene replication, connect the ICP4 gene downstream of a tissue-specific gene promoter and introduce it into ICP4-deficient mutant HSV Thus, a recombinant HSV specific to a specific cell is produced. Martuza and one of the present inventors, Taketake, et al., Obtained U.S. Pat. No. 5,728,379 issued in March 1998 ("replication of popular or cell-specific simple herpes virus").

 Clinical trials of malignant brain tumors using a replicable HSV vector that destroys cells in the proliferating phase have been conducted in the United States and the United Kingdom, but no attempt has been made to apply this vector to vascular disorders or ϋβ transplantation. Nare, This study is completely different from conventional gene therapy, which uses the virus as a mere carrier for gene transfer, and if the target tissue or cells are infected with even a small amount of the virus, the infection spreads to surrounding cells. It is completely original point that it breaks down. Viral vectors that violently rupture all cells in the proliferative phase have the potential to damage not only the proliferation of vascular smooth muscle, which is the main pathological condition of arteriosclerosis, but also the endothelium of the blood vessels. To do so, it is necessary to develop a vector that replicates and destroys only vascular smooth muscle during the growth phase. Disclosure of the invention

 Therefore, the present inventors have focused on a force luponin that is a protein specifically expressed in smooth muscle, and used a promoter of the force luponin, which has a smooth muscle phenotype and is an HSV vector that ruptures proliferating cells. And has confirmed and reported the efficacy and safety of gene therapy for leiomyosarcoma tumors (Cancer Res. 61: 3969-77, 2001; Japanese Patent Application No. 2001-143999).

One of the objects of the present invention is to treat an arterial sclerosing lesion using the HSV vector specific to the cell or tissue. On the other hand, stenotic lesions in transplanted blood vessels associated with organ transplantation are also a problem, and it has recently been revealed by Shimizu et al. That the pathology is the proliferation of host-derived vascular smooth muscle. In addition, the proliferation of a large number of viable luponin-positive vascular smooth muscle cells was observed (Nat Med. 7: 738-41, 2001). Therefore, it is considered that this virus can be applied to the treatment of this condition.

 That is, the present invention

 (1) A vascular smooth muscle-specific expression-replicating viral vector that does not act on adult normal cells, wherein a transcription initiation control region of a gene specifically expressed in vascular smooth muscle is incorporated upstream of a predetermined gene. In the above, the transcription initiation control region contains a region containing the nucleotide sequence shown in SEQ ID NO: 1, a region containing the human calponin gene promoter consisting of the nucleotide sequence shown in SEQ ID NO: 2, or a nucleotide sequence shown in SEQ ID NO: 3. The virus vector of the present invention, or the transcription initiation control region, has one or several bases deleted, substituted or added in the base sequence shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 A region of the present invention, which is a region comprising a base sequence that has been prepared and has a base sequence having an activity equivalent to the transcription initiation control activity of the gene. , Or;

 A viral vector of the present invention in which an enhancer, preferably a 4F2 enhancer, is incorporated upstream of the transcription initiation control region; or

 The viral vector of the present invention, wherein the expression-replicating virus vector is a simple virus virus or an adenovirus vector;

 The expression replication virus is ribonucleotide reductase

 A virus vector of the present invention lacking DNA encoding (Ribonucleotide reductase) and / or DNA encoding Thymidine kinase; or;

 The virus vector of the present invention, wherein the predetermined gene is a virus replication-related gene, specifically, a gene ICP4 encoding a transcription factor essential for initiation of virus replication; or

An apoptosis-related gene is linked further downstream of the predetermined gene, and is expressed under the control of the transcription initiation control region and the enhancer. A DNA encoding a protein having an anti-angiogenesis effect, which is linked further to the viral vector or the predetermined gene, and expressed under the control of the transcription initiation control region and the enhancer. A viral vector; or

(2) A pharmaceutical composition for treating or preventing vascular stenosis, comprising the viral vector of the present invention, preferably the pharmaceutical composition of the present invention, wherein the vascular stenosis is derived from an arteriosclerotic lesion or an organ transplant Or

 (3) A method for treating and preventing vascular stenosis, which comprises introducing an effective amount of the virus vector of the present invention into a living body and expressing and replicating the same. A method of the invention derived from a sexual lesion or organ transplant;

About. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing the results of transcriptional activity when a 5 ′ deletion mutant of the force-luponin gene promoter was transfected.

 Fig. 2 shows the effect of Enhansa-I on the transcription level of the human calponin expression control region in haploponin-positive tumor cells.

 Figure 3: d12 shows the structure of CALP and the results of an in vitro cytopathic assay.

 Figure 4: In vitro effect of dl 2. CALP on tumor cells.

 Figure 5: Selective injury activity of d12.CALP in vileluponin-positive cells in the mouth of invitro.

 Figure 6: Inhibition of dl 2. CALP on the formation of severe ulcers

 Fig. 7d 12. This is a view showing a nude mouse treated with CALP. FIG. 8 shows the replication of d12.CALP in vivo.

Figure 9 d 12. CALP spread and replicate at sites distant from the infected site in vivo _c BEST MODE FOR CARRYING OUT THE INVENTION

 (1) A vascular smooth muscle-specific expression-replicating virus vector that does not act on adult normal cells, wherein a transcription initiation control region of a gene specifically expressed in vascular smooth muscle is incorporated upstream of a predetermined gene.

 In the present invention, the term “gene specifically expressed in vascular smooth muscle” includes a haploid gene specifically expressed in smooth muscle, a human SM22α gene (a human SM22α gene, Sequences from 80 to 126; GenBank accession * D84342-D84344), Smemb-myosin expressed in smooth muscle during the growth phase, and the like.

 It has been found that human sarcoma tumor cells express the calpogene gene, a marker for differentiation of smooth muscle (Int. J. Cancer 79, 245-250, 1998, Sarcoma 3, 107-113, 1999, Intern. J. Cancer 82, 678-686, 1999). After that, in addition to bone soft tissue sarcoma, gastrointestinal stromal tumor (GIST), salivary gland sarcoma, fibrous sarcoma, malignant schwannomas and other nearly 20 types of human malignant tumors derived from mesenchymal cells, the lipoprotein gene was Abnormal expression has been reported one after another in Japan and overseas. The X-ray crystal structure and in vitro and in vivo functional analysis of the calponin (h1 or basic) revealed that it binds to the C-terminus of the actin molecule and suppresses the sliding movement of actin 'myosin. Puru (Biochem. Biophys. Res. Commun. 279, 150-157, 2000, J. Physiol. 529, 811-824, 2000). The force luponin gene is selectively expressed in smooth muscle cells in adults and is considered to be a marker for vascular differentiation (Physiol. Rev. 75, 487-517, 1995).

 Specific expression of the SM22α gene in smooth muscle is known, and its promoter region is also cloned (J Clin Invest. 100: 1006-1014, 1997, J

Biochem. 122: 157-167, 1997).

 Smemb-myosin expressed in proliferative smooth muscle is Circulation 94: 1118-1124,

1996.

In the present invention, the “transcription start control region” is a transcription start control region of the above gene, and means a region known as a promoter. This transcription initiation control region may include a part of the corresponding structural gene. As a specific example of this, in the case of the force luponin gene, a region containing the nucleotide sequence represented by SEQ ID NO: 1 from 126 to 119 of the human motor of the human calponin gene, preferably SEQ ID NO: 2 And the human calpond gene promoter consisting of the nucleotide sequence shown in SEQ ID NO: 3 and a region containing a part of the structural gene thereof. . In addition, the transcription initiation control region consists of the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3 in which one or several bases have been deleted, substituted or added. A base sequence having an activity equivalent to the transcription initiation control activity of a gene specifically expressed in a muscle is exemplified.

 Those skilled in the art can easily prepare a nucleotide sequence having such a mutation. In addition, an activity equivalent to the transcription initiation control activity of the gene is not required to have the same activity as the promoter of the gene as long as it is an activity capable of controlling the transcription initiation of the gene. For example, it can be measured by an ordinary method. Examples of a sequence having such a mutation include a region containing a region homologous to that of human, such as a mouse, rat, and puta-derived troponin promoter.

 In addition to the above, when targeting proliferating smooth muscle cells to attack, SM22Q; the open motor region of the gene, specifically _480 to 126 for the human SM22α gene GenBank accession * D84342-D84344, a region homologous to the SM22α gene promoter from mouse / rat or other mammals), or F1k-1 gene when targeting endothelial cells. A promoter region can be used. In these cases as well, a region containing a part of the 3tit gene can be used as a transcription start control region.

It is preferable to link an enhancer that activates transcription upstream of the transcription initiation control region. Examples of such enhancers include an adenovirus early gene, an enhancer of Moroni mouse leukemia virus terminal repeat, and histone H. 2 A gene enhancer, immunoglobulin enhancer, insulin gene enhancer, c-fos gene enhancer, T cell antigen receptor gene enhancer, muscle creatine kinase gene enhancer, human 4F An example is an array enhancer. When the transcription initiation control region is a region containing a sequence from 260 to +73 of the promoter of the luponin gene, only one transmembrane structure, which is considered to be an active factor for the amino acid transporter, is used. The 4F2 enhancer, such as the human 4F2 heavy chain transcription enhancer (SEQ ID NO: 4), which is the enhancer of the 4F2 heavy chain gene, which does not have the type 2 membrane glycoprotein, can significantly increase the transcription efficiency. preferable.

 The predetermined gene of the present invention, which is used for producing a vascular smooth muscle-specific expression replication resetter that does not act on normal cells in an adult, stops vascular smooth muscle proliferation that causes stenosis or restenosis of a blood vessel, Alternatively, it refers to a gene encoding a factor that damages or destroys cells or tissues derived from the proliferation. As such a factor gene, a gene necessary for initiation or maintenance of the virus to be used can be used. For example, an E1A gene of an adenovirus, an ICP6 (Ribonucleotide reductase) gene (Virology.1988 Sep; 166 (1): 41-51J Virol. 1988

Aug; 62 (8): 2970-7.). In particular, preferred is ICP4 (J Virol 56: 558-570, 1985), which is a gene encoding a transcription factor necessary for initiation of replication of a herpes virus. In addition, these genes may be those in which a part or all of the original structural gene located downstream of the transcription initiation control region and the above-mentioned predetermined gene are linked in frame. Specific examples include a DNA encoding a fusion protein of a part of the side and the ICP4 protein.

 In addition, as a gene for a factor that stops proliferation of vascular smooth muscle, which causes stenosis or restenosis of another blood vessel, or damages cells or tissues derived from the proliferation, or causes calculus, B c is an apoptosis-related gene. 1—Xs, Bok / Mtd, Bc1 Gs / Bra, Bel—GL, Bel—Rambo, Hrk / DP5, Bik / Nbk / Blk, Bad , B id, B i mL, S, EL / B o dL, M, S,

Proteins such as angiostatin, endostatin, FLK1, FLT1, FLT4, Tiel, and Tie2 as DNA that encodes apoptosis-promoting genes such as Noxa / APR and Puma, or proteins that have angiogenesis inhibitory proteins Specific examples of the DNA encoding It is not specified.

 The vascular smooth muscle-specific expression / replication virus vector of the present invention which does not act on adult normal cells, such as DNA encoding the apoptosis-related gene or the protein having an angiogenesis inhibitory action described above, further downstream of a predetermined gene. And a cell-specific expression-replicating virus vector that can be expressed under the control of the transcription initiation control region and the enhancer.

 In the adult of the present invention, the backbone of the vector used to prepare a cell-specific expression replicating virus vector that does not act on normal cells is preferably a vector that can infect smooth muscle cells, particularly vascular smooth muscle, or that can express by introducing a gene. . Examples of viral vectors include papovavirus such as SV40, vaccinia virus, adenovirus, adeno-associated virus vector, fowlpox virus, pseudorabies virus, retrovirus-derived vector, and simple virus vector (HSV vector). ), Among which HSV vectors and adenovirus vectors, particularly conditional replication-competent HSV vectors or conditional replication-competent adenovirus vectors, are highly efficient in gene expression and multiplication. It is preferable in terms of cell-specific cytotoxic activity and the like. As the conditional replicable HSV vector, for example, DNA that encodes liponucleotide reductase, DNA that encodes thymidine kinase, or a vector deficient in both of them can be used for the present invention. Thus, a vascular smooth muscle-specific expression / replication vector that does not act on adult normal cells can be suitably prepared.

 The term "adult" in "does not act on normal adult cells" refers to an individual except for childhood and generally refers to an adult.

(2) A pharmaceutical composition for treating and preventing vascular stenosis, comprising the viral vector of the present invention, and a vascular stenosis, which comprises introducing an effective amount of the vector into a living body and expressing and replicating the expression. For treating and preventing cancer

In the present invention, vascular stenosis means luminal stenosis of blood vessels caused by neointimal thickening, regardless of whether it is derived from arterial lesions or organ transplantation. In the present invention, vascular stenosis also includes restenosis after vascular endovascular surgery. If vascular stenosis is prevented by the pharmaceutical composition of the present invention, dysfunction of the transplanted organ and its dropout can be prevented, and angina pectoris after cardiac transplantation, myocardial infarction and the like can also be prevented.

 When the viral vector of the present invention is applied to treatment and prevention of vascular stenosis, the following methods can be applied.

 In the present invention, the vascular smooth muscle-specific expression / replication virus vector that does not act on normal cells in an adult is injected and replicated from a living cell tissue, preferably a blood vessel having vascular stenosis.

 The introduction and injection of the virus vector of the present invention to actually act as a medicament are performed by an appropriate administration route according to the disease, symptom and the like for the purpose of treatment. For example, it can be administered intravascularly (vein or artery). The dosage form may be, for example, in the form of a liquid preparation or the like. In general, the dosage form is an injection containing the viral vector of the present invention as an active ingredient, and if necessary, a carrier well-known in the art may be added.

The content of the viral vector of the present invention in the preparation can be appropriately adjusted depending on the disease to be treated, the age of the patient, body weight, and the like. Usually, the virus amount is 10 ⁹ -10 ¹⁰ pfu / ml (PFU: plaque forming It is expected that the dose will be about 0.1 ml at the concentration of (unit).

 By the administration of the virus vector of the present invention as described above, expression of a desired gene or infection or replication of the virus vector of the present invention occurs at a site where vascular stenosis has occurred or is about to occur, and the stenosis is reduced or eliminated. In this way, the treatment or prevention of vascular stenosis or vascular restenosis is achieved.

 Hereinafter, the present invention will be described specifically with reference to Preparation Examples and Examples, but the present invention is not limited to these Examples. Preparation example

 I. Cells used

The human leiomyosarcoma cell line SK-LMS-1 (HTB-88), the human osteosarcoma cell line HOS (CRL-1543), MNNG-HOS (CRL-1547), and Vero cells (CCL-81) American '' Type '' Riki Norechiichi '' Collection (American Type Culuture Collection).

 The human leiomyosarcoma cell line SKN (RCB 0513) and the human osteosarcoma cell line OST (RCB 0454) were purchased from RIKEN GENE BANK.

 Human synovial sarcoma and desmoid cell lines were established from tumor samples excised from each tumor patient. Synovial sarcoma was diagnosed by confirming the expression of the SYT-SSX fusion gene as described in the literature (Sarcoma 3, 107-113, 1999).

 Initially cultured human mesangial cells (HMC; provided by Dr. Yamabe of Hirosaki University School of Medicine; Nephrol. Dial. Transplant. 12, 438-442, 1997) were prepared from human fetal kidneys (16 and 18 weeks of gestation). (Dr, MR of University Hospital of Leiden

Established by Da), and those subcultured 4 to 6 times were used for the following Examples.

 The human umbilical vein endothelial cell line HUVEC (T200-05) was purchased from Toyobo Biochemical.

 ICP4 gene-introduced vegetative cells, E5 cells (Vero cells transfected with ICP4 gene)), and those provided by N. Deluca (University of Pittsburgh School of Medicine, Pittsburgh) Was. )

I I. Culture method

 SK-LMS-1 was cultured in Eagle MEM supplemented with 1 mM sodium pyruvate. HOS, MNNG—HOS, OST, Vero and E5 cells were cultured in DMEM. SKN cells were cultured in F12 medium. Synovial sarcoma cells and desmoid tumor cells were cultured in RPMI 1640 medium. Human mesangial cells were cultured in DMEM supplemented with lmg / ml D-glucose. All media contain 10%, 15% (for SKN) or 20% (for synovial sarcoma cells and desmoid) final concentrations of heat-inactivated fetal serum (Upstate).

Biotechnologies), 2 mM L-glutamine, 100 unitit / mL penicillin, and 100 / Z gZmL streptomycin. HUVEC were cultured in media according to the manufacturer's instructions. All the above cells were cultured at 37 ° C in C 0 ₂ under the conditions of 5 ° / _0, which is humidified.

I I I. Test method

 Chemiluminescence (ECL; Amers am Pharmacia Biotech) visualized the bound antibodies according to the manufacturer's protocol.

I V. Virus

 HSV ICP 4 deficient mutant d 120 (J. Virol. 56, 558-570, 1985) and HSV ICP 6 (ribonucleotide reductase) generated by infecting E5 cells or vegetative cells at low multiplicity, respectively. ) Defective mutant hr R3 was obtained from Dr. N. Deluca or Dr. S. Weller (University of Connecticut Health Center,

Farmington).

 In the examples, as a statistical analysis, statistical differences were confirmed using unpaired Student t'st-test. The difference was p <0.05, which was considered statistically significant. Preparation Example 1

Identification of expression control region of human calponin promoter I. Preparation of RNA and RT-PCR analysis

Total RNA was extracted from cells (OST, SK-LMS-1, Synovial cell sarcoma) cultured using the Isogene RNA extraction kit (Nippon Gene), and published in the literature (Int. J. Cancer). 79, 245-250, 1998). The semi-quantitative RT-PCR analysis described was performed. As conditions for PCR amplification, a cycle of denaturation at 94 ° C for 40 seconds, annealing at 60 ° C for 30 seconds, and extension reaction at 72 ° C for 90 seconds was repeated 30 times. As human calponin primers, 5′-gagtgtgcagacggaacttcagcc-3 ′ [forward primer 1 (FP 1); nt # 10-33 GenBank D17408; SEQ ID NO: 5] and 5′-gtctgtgcccagcttggggtc-3, [reverse primer 1 (RP 1 ); Nt # 660-680; SEQ ID NO: 6] as control GAPDH (glyceraldehyde 3-phosphate dehydrogenase) primers include 5'-cccatcaccatcttccagga-3 '[forward primer 2 (FP2); nt # 342-360; SEQ ID NO: 7] and 5'-ttgtcataccaggaaatgagc-3, [reverse Using primer-1 2 (RP 2); nt # 1052-1070; SEQ ID NO: 8], 671 bp and 731 bp of DNA were amplified, respectively.

I I. Isolation of the human canoleponin promoter

According to the method described in the literature (J. Biochem. 120, 18-21.1996), the human genome λΕΜBL3 phage library was screened using the amplified 671 bp and 731 bp DNA, and 5 ′ upstream of the human calponin gene. A genomic clone containing was isolated. By amplifying by PCR, 5, the side is a deleted fragment ρ- 1159 Luc, p-385 Lu c N ρ- 343 Lu c, p- 310 L uc, p-299Lu c N p- 288 Lu c was prepared p- 260Lu c, p- 239 L uc , p-219 Lu c, p- 201 Lu c, the _{p-176Lu c N p- 153 L} uc. Next, each fragment was transferred to the pGL2-Basic vector.

 (Promega) was subcloned to the multiple cloning site located on the 5th side of the luciferase gene.

 The numbers above indicate the 5 'end of the DNA fragment located upstream of the ATG translation initiation codon, hereafter denoted as +1. These deleted fragments have a common 3, terminus extending to position +73. Using the DQS-2000L DNA sequencer (manufactured by SfflMADZU) according to the manufacturer's protocol, the nucleotide sequence of the clone fragment was determined, and the sequence was determined according to the literature (J. Biochem. 120, 18-21, 1996).

(DDBJ / GenBank ™ EMBL database; accession No. D85611).

I I I. Identification of expression control region of human calpoyun promoter

To identify the minimal promoter region that regulates human calponin expression, plasmids containing the various leupoyun promoter luciferase constructs lacking the 5 'portion created in II above were transferred to a human osteosarcoma cell line. MNNG—HOS and Transfected into HOS and mesangial cell line HMC.

Twenty-four hours before transfection, the previously cultured cells were split and seeded on plates. According to the manufacturer's protocol, 1.2 μg of promoter plasmid, 0.3 xg of pCAGGS / iS-gal-related plasmid, and 3.75 μl of FuGENE ^™ 6 transfection reagent (Roche Made) and

The cells were injected into a 6 ゥ eldish and cells (5 × 10 ⁴ ) were transfected. Twenty-four hours after transfection, cells were collected in 100 1 wells of cell lysis buffer (PicaGene ™ luciferase analysis system, manufactured by Toyo Ink). After centrifugation at 12000 g x 5 minutes at 4 ° C, the supernatant (20 μl or 30/1) was used for luciferase assay (3-galactosidase assay), respectively. . Luciferase activity was measured using a BLR-201 luminescence reader (Aloka). -Galactosidase assay was performed using a one-galactosidase enzyme analysis system ( _promega ) according to the method described in the literature (J. Biochem. (Tokyo) 122, 157-167, 1997). All experiments were repeated a minimum of three times to confirm reproducibility. The transfection efficiency was determined by measuring the β-galactosidase activity of the cell extract, and the luciferase activity (optical unit) was detected according to the value. The transfection efficiency of various cell lines was evaluated by comparing the expression of the pSV2-Luc gene containing the SV40 enhancer and SV40 promoter. The data show the relative ratio of the normalized absorbance S. Ε. To 100% as 1 with respect to the PSV2-Luc value.

Mesangiumu cell line HMC are smooth muscle-like phenotype characteristic stably showed growth patterns (peaks and valleys), expressing the singular genes in smooth muscle such as a- smooth muscle Akuchin and SM22 _alpha . Of the three transfected cell lines, HMC had the highest expression of the haplo- luponin gene (Figure 1). As previously reported (Int. J. Cancer 79, 245-250, 1998), kyruponin shows moderate expression in the human osteosarcoma cell line HOS, but not at all in MNNG-HOS. (Figure 1).

p-288Luc fragment and p-260Luc fragment are transferred to pGL2-Basic vector As a result of a transient transfection assay on plasmids ρ-288Luc and p-260Luc produced by subcloning into (Promega) into HOS and HMC cells, the activities of both luciferases were: — Increased 4 fold in HOS cells and 6 fold in HMC cells than transfected with 1159 Luc. This indicates that there is an expression repressive region between 1-159 and -288 of the force-luponin promoter region. There was a significant correlation between the expression of the potent luponin mRNA and the transcriptional activity of the promoter region. From 260 to 219, both HOS cells and HMC cells showed a large decrease in promoter activity as bases were further removed. Furthermore, when a construct (p-201Luc, p-176Luc and!)-153Luc) in which a wide range of the 5th part of the force-luponin gene promoter region was removed, p-219Luc was transfected. When used, luciferase activity 1 "was comparable. These results indicate that the sequence from 260 to 119 is positive for transcription of the gene for luciferin in both HOS and HMC cells. It turns out that it is an expression control region.

One of the 260 to 219 regions of the promoter is composed of several sequence motifs similar to the consensus binding sequence of 258 Sox (AACAAT) and 250 GATA-1 (CACAATCAGC). Contains. Removing p-260Luc, a part of the L-239 promoter, from the L-239 gene promoter, reduces transcriptional activity by 50%. To determine whether the putative binding sites of Sox and GAT A-1 and the downstream region of 239 show expression control functions, plasmids!) 260 luc 255 /-254 (AA to GG), 246 / Three mutants with substitutions of —244 (A to G in -246, and 〇 to D in _244) and 232 / —231 (CC to TT) were prepared, and the plasmids were transfused. In the transfection experiments in HMC cells, the three mutants had p-260Luc activities of 73 ± 0.2%, 76 ± 0.2%, and 39 ± 0.1%, respectively. These results indicate that the transcriptional activity of the luluponin promoter requires an entire sequence of 1-260 to 1219. IV. Regulation at the transcriptional level by expression of the haplopoyun gene in human soft tissue and bone tumor cells

 Force luponin is expressed or not expressed in human soft tissue tumors and bone tumor cells to further investigate the correlation between force luponin expression and transcriptional activity of the force luponin promoter P-260Luc or human 4F double-stranded transcription enhancer (Mol. CellBiol. 9, 2588-2597, 1989) was introduced into various human cell lines (see Fig. 2) upstream of p-260Luc. Construction

 (p4F2-260Luc) was transfected. By RT-PCR analysis, the expression of kyruponin mRNA was determined in synovial sarcoma cells and leiomyosarcoma cells SK-LMS-1. In contrast, the expression of potent luponin mRNA in osteosarcoma cells OSΤ was negligible (Figure 2). As shown in FIG. 2, the transcriptional activity of p-260Luc and p4F2-260Luc was correlated with the expression level of the transcript of force luponin mRNA in all the measured cells. The results of these experiments indicate that the expression of the force / reponin gene in human soft tissue and bone tumor cells may be regulated at the transcriptional level by a sequence 260b upstream of the translation initiation site.

 As described above, when the 4F2 enhancer was inserted upstream of the lipoprotein promoter, the transcriptional activity of p-260Luc was detected in lipoprotein-positive synovial sarcoma cells and SK-LMS-1 cells. It increased from 3 times to 5 times. Therefore, in the experiments below, the 4F2 Enhansano 260-potency Lupoyun promoter sequence was used to regulate the expression of the HSV ICP4 gene in human soft tissue tumors and bone fl sever cells. Example 1

Preparation of Threaded HSV Vector

Based on the findings of the Preparation Example 1, to construct a HSV vector that replicates selectively in force Ruponin positive cells Contact Yopi proliferating cells, 4 F 2 Enhansa Z- 260 calponin promoter CP 4 _(P TK厶- The DNA fragment containing CALP-ICP4) was replaced with the IC SV IC Ρ 4 deletion mutant d 120. TK gene of Virol. 56, 558-570, 1985) And揷入the seat (U _L 2 3), to produce a d 1 2. CALP. The plasmid pTK-MAL-CALP-ICP4 contains two chimeric transgenes that express ICP4 protein containing E. coli-derived LacII and one galactosidase (FIG. 3A). d 1 2. The specific method of preparing CALP is as follows.

 A 4.1 kb blunt-ended S a1 I-MseI fragment from pGH108 (J. Virol. 56, 558-570, 1985) containing the coding region of ICP 4 (Johns Hopkins School of Medicine). Provided by Dr. Hayward) into the blunt-ended Hindill site downstream of the 33 33 b ρ human canoleponin promoter (260-h73) cloned into pAMP1 beta (pUC-type plasmid). Then, a human 4F double-stranded transcription enhancer (Mol. Cell Biol. 9, 2588-2597,

1989) (provided by Leiden of Harvard Medical School). The 444 bp NotI fragment was subcloned. The 4.7 kb fragment obtained by double digestion of the obtained pAMP1ZCALP—ICP4 vector with SalI and Hindill was converted into XbaI of pTKAL recombinant vector. Subcloned at the blunt end site. The ΤΚΔL recombinant vector has a 0.5 kb Bg1ΙΙ-ΚρnI region deleted TK coding sequence, Eschariciacoli-derived LacZ, upstream of the TK sequence (+53 of ΤΚ). (J. Virol. 71, 5124-5132, 1997), and the SV40-derived poly A signal (p Κ ΚΔL recombination vector

Prepared from pHSV106 released from GIBCX BRL with reference to JVirol. 71 (o. 7): 5124-5132, 1997). Iipofectamine ™ according to manufacturer's protocol

 (GIBCO / BRL), pΤΚΔ-CALP-ICP4 and d120 DNA obtained by linearizing the above plasmid at the Sa1I site were co-transfected into E5 cells. In the presence of ganciclovia (1 μ / ml), the recombinant viral vector identified as a single plaque, d 1 2. CALP, was converted to 5-promo 4-chloro-3-indolyl-J3-D-galactopira. Plaque purification was performed three times by staining blue with a nosid (X-ga 1) agarose overlay and performing isolation. After the DNA was purified, it was digested with restriction enzymes, and its recombination was confirmed by Southern plot and PCR analysis.

10 to 20 1 50 cm ² / tissue culture flasks (IWAKI CLASS) A virus-infected cell suspension was prepared by infecting the E5 cells of Example 1 and collecting the detached cells 48 hours later.

 Then, for virus recovery, infected E5 cells were collected by centrifugation at 400 X g for 5 minutes at 4 ° C and 10 ml of cold virus buffer (20 mM containing 150 mM NaCl). Tris—HC 1; pH 7.5). The cells were lysed by freezing and thawing three times in combination with sonication (six times per minute). After centrifugation at 1500 X g at 4 ° C for 5 minutes, the supernatant was further centrifuged at 15000 X g at 4 ° C for 45 minutes. The resulting pellet was suspended in a cold virus buffer, and the titer of d12.CALP purified by plaque assay in E5 cells was measured. Example 2

In vitro selective replication of recombinant HSV vectors in calpoen-positive cells. I. Analysis of cell destruction at the in vitro mouth.

Using a human cell line expressing force luponin or a human cell line not expressing force luponin, the selectivity of d12.CALP for viral replication was evaluated (FIG. 3B: RT-PCR results). The cell line prepared above (see FIG. 3C) was infected with a dl 2. CALP or ICP6-deficient mutant hrR3 having a multiplicity of infection of 0.001 for 48 hours. Virus replication was evaluated using plaque formation as an indicator (Fig. 3C). Infection with hr R3 is a positive control. In synovial sarcoma cells, SK-LMS-1 cells, and HOS cells positive for luluponin, dl 2. CALP showed the same cytopathic effect as hrR3. In contrast, no apparent cell lysis by dl 2. CALP was observed in SKN cells, OST cells, MNNG-HOS cells, and HUV EC cells, which were negative for luleponin. Desmoid cells showing the slowest growth rate express messenger luponin mRNA in the same level as SK-LM S-1 cells, but no apparent plaque formation by dl 2.C ALP was observed . The above results indicate that the cytopathic effect of dl 2.CALP is dependent on both the expression of kerupeuun and the rate of cell growth. As can be seen from FIGS. 4A and 4B (see Reference Photo 1), SK—LMS-1 cells and synovial sarcoma cells were exposed to d12.C ALP at a low multiplicity of infection (MOI: 0.001). ), Complete tumor destruction of cultures in 10 cm dishes occurs 96 hours after infection. It was also confirmed that the synovial sarcoma cell dysfunction expanded from cell to cell (Fig. 4A). Some infected SK-LMS-1 cells were multinucleated before lysis (Fig. 4B, arrow).

I I. In vitro virus replication analysis

 SK-LMS-1 cells or OST cells in 6-well ligneous tissue culture plate at a multiplicity of infection (MOI) of 0.01 to 0.000 Ipf uZcell in 1% heat-inactive FB SZP BS Were infected with the virus. Incubate such infected cells at 37 ° C for 1 hour, then add 1% FBS and 11.

The cells were cultured in the above-mentioned medium containing human IgG (m1) (manufactured by Jackson ImmunoResearch Lab.). 48 hours after infection, the number of plaque Zell was counted. For virus replication analysis, SK—LMS—1 cells or OS in 12-well tissue culture plates

Monolayer cultures of T cells (2 × 10 S_we 11) were infected with d12 CALP in 1% of 83 / -83 so that the multiplicity of infection (MOI) was 0.1. . The inoculated virus was removed one hour later, and the cells were incubated in the medium. At predetermined times (12 hours, 24 hours, 48 hours), infected cells were detached from the wells using 100 μl of virus buffer. Cell lysates (1 mu 1) ^10-3, was diluted to 10 ⁴ and 10_ ^5, and the titer of virus in the subsequent E 5 cells was evaluated by Shi ring Honoré step growth Atsu Si.

d 12. CALP replicated in cell-luponin-positive SK-LMS-1 cells, but d 12. CALP titers were 3-48 in cell-luponin-negative OST cells 48 hours post-infection. ^ 1 ^ 3-1 was reduced from 1 10 ⁶ to about lZl 0 ⁷ compared to the cells (Fig. 5

A)

Immunout analysis of ICP4 expression was performed in the same manner as described in the literature (Int. J. Cancer 79, 245-250, 1998). SK-LMS-l cells and OST cells so that the multiplicity of infection (MOI) becomes 0.01. Dl 2. CALP or virus 'Only one was infected, and cultured for 22 hours before harvesting. The same amount of protein was subjected to 9% SDS-PAGE gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was blocked with 5% skim milk (DIFCO Laboratories) at room temperature for 2 hours, and then a monoclonal antibody against HSV-1 or HSV-2 ICP4 protein (clone No. 1101,

Goodwin Institute for Cancer Research) (dilution ratio 1: 500) was used to incubate at 4 ° C.

 Immunoplot analysis of the cell extract 22 hours after infection revealed that the SK-LM S-1 cells expressed the ICP4 protein, but the OST cells did not express the ICP4 protein. This was consistent with the virus replication analysis (Figure 5B). In contrast, the d120 viral vector did not produce any progeny Winores in cultures of SK-LMS-1 and OST. Example 3

Treatment of human leiomyosarcoma xenografts with recombinant HSV vectors

To evaluate the effect of dl 2. CALP in treatment in vivo, SK-LMS-1 cells as leiomyosarcoma xenografts or OST cells as osteosarcoma grafts, and 6-week-old female athymic nude mice ( BALBZc S 1 c -nu / nu) (manufactured by Japan SLC) was injected subcutaneously into the body to fix the tumor. Tumors grew to approximately 6 to 7 mm in diameter in nude mice. 1 x 10 ⁷ pfu d12CA

50 1 (/ J) mass of viral suspension containing LP or the same volume of virus buffer (2 OmM Tris-HC1 containing 15 OmM NaC1; H7.

5) was injected into each tumor using a 30 gauge needle. Nine days later, the exact same treatment was repeated. The tumor is measured at a predetermined time after the injection, and the expression [0.53X length

X width squared] was used to calculate tumor volume. Prior to treatment, the tumor volume (138 ± 20 and 139 ± 28 mm ³ , respectively, n = 5) and the expression level of immunoreactive force luponin were not affected by dl 2.CALP No significant difference was found between the treated and control tumors. d 12. CALP infection was associated with growth inhibition of SK-LMS-1 tumors, but not with liponin-positive OST tumors (Figure 6A) . In contrast, treatment of SK-LMS-1 xenograft with viral buffer alone showed progressive tumor growth and death of all animals (n = 5) by day 89 after treatment. It was found that there was an association between the growth of advanced tumors and the death of animals (Figure 6B). Five weeks after the first dl 2. CALP infection, tumor regression was confirmed to be completely regressed in four out of five mice (FIG. 7; see reference picture 2). In one mouse, the tumor had regrown. Then, when the recurrent tumor of such a mouse was re-treated with d 12. CALP, the growth of the tumor was stably suppressed.

IX 10 ⁷ pf uZ tumor volume 10 for histochemical analysis with Xg a 1

After a single administration of 0 mm ³ d 12. CALP, mice with fl injuries were killed at a given B number. Removed subcutaneous tumors, 2% paraformaldehyde, with 0.5% of glutaminyl Rua aldehyde, in PBS containing 1 mM of M g C 1 _2, and fixed one晚at 4 ° C. Then, X—gal (lmg / ml),

_{1:? 3 6 (CN 6} ), the K ₄ F e (CN ₆₎ and 1 mM of Mg C 1 ₂ of 5mM substrate solution containing in PBS, soaked for 3 hours at 37 the tumor, then 3 The plate was washed with PBS containing% DMSO.

Histochemical staining using Xg a1 revealed that the expression of 3-galactosidase by introducing LacZ into the TK locus was based on dl 2. CALP-treated SK-LMS-1 tumor cells ( 8A and 8B; see Reference Photo 3), but could not be confirmed in control tumor cells. This identified the region where the d12 CALP virus spreads in vivo. Eighth, the death became prominent, and LacZ expression was deficient in this region (Fig. 8A, arrow). At higher magnification, blue-stained tumor cells showed polynuclear motility, as observed in in vitro cytopathic analysis (see FIG. 8C and reference photo 3, arrows). SK—LMS-1 cells lost their typical morphological appearance. As shown in FIG. 8D (see Reference Photo 3), in the virus-infected mice, LacZ expression in smooth muscle cells surrounding normal blood vessels was negative. This is a cell population in which vascular smooth muscle does not proliferate even when it expresses lupine. La c Z expression Is negative means that virus replication is not recognized, indicating that the safety of the present invention, which does not act on adult normal cells, was confirmed. To examine the distribution of the infected virus by PCR, DNA was prepared from infected or uninfected tumors and from tissues such as brain, lung, liver, kidney, heart, small intestine and uterus or testis. As conditions for PCR amplification, a cycle of denaturation at 94 ° C for 40 seconds, annealing at 60 ° C for 30 seconds, and extension reaction at 72 ° C for 90 seconds was repeated 30 times. ICP6 (ribonucleotide reductase) primers include 5'-gacagccatatcctgagc-3, [forward primer 3 (FP3); SEQ ID NO: 9] and 5'-actcacagatcgttgacgaccg-3 '[reverse primer-1 (RP3) SEQ ID NO: 10] as a primer for glycoprotein E:

5'-gagatgcgaatatacgaat-3 '[forward primer 4 (FP4); SEQ ID NO: 11] and 5'-gtgggtgggctcggccaaat-3' [reverse primer 4 (RP 4); SEQ ID NO: 12] As primers for Z, 5′-gcgttacccaacttaatcg-3 ′ [forward primer 5 (FP 5); SEQ ID NO: 13] and 5′-tgtgagcgagtaacaacc-3 ′ [reverse primer 5 (RP 5); SEQ ID NO: 14] As primers for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 5'-cccatcaccatcatttccagga-3 * [Forward Primer 6 (FP 6); SEQ ID NO: 15] and 5 Using '-ttgtcataccaggaaatggc-3' [Lipersprimer 6 (RP6); SEQ ID NO: 16], 221 bp, 320 bp, 320 bp, and 731 bp were used, respectively.

The DNA was amplified (J. Virol. 74, 3832-3841, 2000).

 d 12. In the DNA of brain, lung, liver, kidney, heart, small intestine or uterus prepared on day 8 after intra-tumor administration of C ALP, dl 2. Lac Z sequence specific for CALP Could not be confirmed. Histologically, Winores replication and LacZ expression did not occur in organs including the aorta and gastrointestinal smooth muscle. Again, the safety of this virus was confirmed. Example 4

Diffusion of recombinant HSV vector in tumor Duplicate d 12. Subcutaneous subcutaneous flanks on both sides of 6-week-old male nude mice to assess whether CALP can target tumor cells distant via blood vessels Then, SK-LMS-1 fl mass was engrafted, and the virus was injected into one of the tumors. That is, the SK-LMS-1 tumor mass in the right flank was inoculated intratumorally with d12 CALP, and the distribution of the virus in the SK-LMS-1 tumor mass in the left flank was examined. As shown in FIG. 9A (see Reference Photo 4), the expression of β-galactosidase was confirmed in the tumor cells on the opposite flank on the 20th day, similarly to the inoculated site. Histologically, extensive tumor deaths were observed in both the right flank tumor and the left flank tumor, but normal blood vessels were collected as shown in Figure 9Β (see Reference Photo 4). The effect of d12 CALP was not observed in the wound liponin-positive smooth muscle cells. The PCR method using primers for liponucleotide reductase (ICP6), glycoprotein E, or E. coli-derived Lac Z inserted at the TK locus yielded d 12. It was found that viral DNA derived from CALP spreads but does not spread to brain or testis tumor tissues (Fig. 9C; see reference photo 4). Industrial applicability

 The present invention uses a replicable simple herpesvirus (HSV) vector, which does not introduce a therapeutic gene but ruptures target cells one after another and spreads the infection. Yes, as shown in Stroke 30: 2431-2439, 1999, its therapeutic effect can be said to be epoch-making. The technique of limiting the cytotoxicity to only the target cell has been developed by the present inventors and is unique in the world. According to the present invention, in Japan, more than 100,000 patients with vascular restenosis are indicated annually, and it is possible to administer the virus intravascularly via a catheter, thus leading to a huge reduction in medical costs. It solves a major problem during organ transplantation and frees patients from fear of death.

Claims

 The scope of the claims

I. A vascular smooth muscle-specific expression-replicating viral vector that does not act on adult normal cells, wherein a transcription initiation control region of a gene specifically expressed in vascular smooth muscle is incorporated upstream of a predetermined gene.

 2. The virus vector according to claim 1, wherein the transcription initiation control region is a region containing the nucleotide sequence shown in SEQ ID NO: 1.

 3. The virus vector according to claim 1, wherein the transcription initiation control region is a region containing a human luponin gene promoter consisting of the nucleotide sequence shown in SEQ ID NO: 2.

 4. The viral vector according to claim 1, wherein the transcription initiation control region is a region containing the nucleotide sequence shown in SEQ ID NO: 3.

 5. The transcription initiation control region comprises a nucleotide sequence in which one or several bases are deleted, substituted or added in the nucleotide sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, 2. The viral vector according to claim 1, which is a region containing a nucleotide sequence having an activity equivalent to the transcription initiation control activity of the gene.

 6. The virus vector according to claim 1, which is a simple herpes virus vector or an adenovirus vector.

 7. The viral vector according to claim 1, wherein the DNA encoding ribonucleotide reductase and / or the DNA encoding thymidin kinase are deleted.

 8. The vector according to any one of claims 1 to 7, wherein the predetermined gene is a viral replication-related gene.

 9. The vector according to claim 8, wherein the viral replication-related gene is ICP4, a gene encoding a transcription factor essential for initiation of viral replication.

 10. A pharmaceutical composition for treating and preventing vascular stenosis, comprising the vector according to any one of claims 1 to 10.

 I I. The pharmaceutical composition according to claim 10, wherein the vascular stenosis is derived from an atherosclerotic lesion.

12. The pharmaceutical composition according to claim 10, wherein the vascular stenosis is derived from an organ transplant.

1 3. The transcription initiation control region of the gene specifically expressed in vascular smooth muscle A method for treating vascular stenosis, comprising introducing an effective amount of a vascular smooth muscle-specific expression-replicating virus vector, which does not act on adult normal cells, which is incorporated upstream of a gene, into an organism, and causes expression and replication. A way to prevent it.

 14. The method of claim 13, wherein the vascular stenosis is from an atherosclerotic lesion.

 15. The method of claim 13, wherein the vascular stenosis is from an organ transplant.