HK1063643A

HK1063643A - Paramyxovirus vector encoding angiogenesis gene and use thereof

Info

Publication number: HK1063643A
Application number: HK04106362.3A
Authority: HK
Inventors: 米满吉和; 居石克夫; 福村正之; 侯晓刚; 长谷川护
Original assignee: 株式会社载体研究所
Priority date: 2000-11-27
Filing date: 2001-11-27
Publication date: 2005-01-07

Description

Paramyxovirus vector encoding angiogenesis gene and use thereof

Technical Field

The present invention relates to paramyxovirus vectors encoding angiogenic genes and uses thereof.

Background

Studies on the treatment of ischemic diseases with growth factors inducing angiogenesis have been carried out in recent years. For example, the therapeutic effects of fibroblast growth factor 2(FGF2) (Baffour, R. et al, J.Vasc. Surg.16 (2): 181-91, 1992) and Endothelial Cell Growth Factor (ECGF)) (Pu, L.Q. et al, J.Surg, Res.54 (6): 575-83, 1993) on myocardial infarction and acute limb ischemia were investigated. Recent studies have shown that Vascular Endothelial Growth Factor (VEGF)/Vascular Permeability Factor (VPF) promotes angiogenesis in animal models of myocardial ischemia and limb ischemia (Takeshita, S. et al, Circulation 90(5 Pt 2): II228-34, 1994; Takeshita, S. et al, J.Clin, invest.93 (2): 662-70, 1994).

Recently, clinical trials using angiogenesis-inducing growth factors for human gene therapy have been initiated, and clinical applications of human gene therapy in angiogenesis therapy of ischemic limbs have also been studied. The endothelial cell-specific growth factor, vascular endothelial growth factor/vascular permeability factor (VEGF/VPF), is considered to be a potential therapeutic gene for achieving the above-mentioned object, and in fact, it has been reported that it has a superior therapeutic effect in gene therapy in which the gene is introduced into a human body by a plasmid vector (Baumgartner, I., et al, Circulation 97, 1114-. However, plasmid-mediated intramuscular gene transfer and its expression are not efficient and there are few reports on the adverse side effects or toxicity levels of intramuscular administration of VEGF genes. Recent reports have indicated that overexpression of VEGF mediated by a transgene (Thurston, G., et al, Science 286, 2511-2514(1999)) or an adenovirus (Thurston, G., et al, Nature Med.6, 460-463(2000)) causes abnormal angiogenesis in animals into which the gene has been introduced, and that plasmid-mediated VEGF gene introduction causes transient edema in ischemic limbs in humans (Baumgartner, I., et al, Circulation 97, 1114-1123 (1998); Isner, J.M., et al, J.Vasc.Surg.28, 964-973(1998)), but the detailed mechanism of these pathological phenomena is unclear. In addition, overexpression of VEGF disrupts the balance of angiogenic signals, and thus hemangioma-like fragile capillaries may be formed (Carmeliet, P., Naturemed.6, 1102-1103 (2000)). When the VEGF gene is introduced into the vessel wall in vivo, hemangioma-like endothelial growth makes the neointima significantly hypertrophic, possibly leading to extravasation of red blood cells (extravasation) extravascular (Yonmeitsu, Y., et al, Lab. invest.75, 313-doped 323 (1996)). The same pathological phenomena are observed when retroviral-mediated VEGF is persistently overexpressed in the myocardium (Lee, R.J., et al, Circulation 102, 898-901 (2000)). In addition, leakage of the locally expressed angiogenesis factors to the systemic circulatory level is an extremely important issue from the aspect of clinical applicability, and accidental angiogenesis complications caused by such leakage may lead to diabetic retinopathy or tumor growth.

On the other hand, acute critical limb ischemia due to acute aortic occlusion is mainly caused by thrombotic occlusion, and is an important target for therapeutic angiogenesis. The late curative effect of acute critical limb ischemia is not ideal, and amputation is mostly caused. Moreover, amputees have poor long-term prognosis, with an annual post-operative survival rate of only 50%. Plasmid-mediated gene expression levels are low and thus the efficacy for more severe acute arterial occlusion is not clear.

Disclosure of Invention

The object of the present invention is to provide a paramyxovirus vector encoding an angiogenic gene and use thereof. Specifically, the present invention provides paramyxovirus vectors encoding angiogenic genes, angiogenic compositions containing the vectors, and methods for promoting angiogenesis in ischemic tissues using the vectors.

In the preliminary studies of the present inventors, the introduction of the plasmid-based VEGF165 gene failed to rescue limbs of a mouse model of acute critical limb ischemia (data omitted). To confirm whether or not better results can be obtained by enhancing the expression of transgenes, the present inventors introduced therapeutic genes using a recombinant Sendai virus (SeV) -mediated gene introduction method that enables efficient gene introduction in various organs. In the examples, the present inventors focused on 2 recombinant SeV vectors as a therapeutic tool for limb ischemia, one of which expresses human VEGF165 and the other one expresses the murine fibroblast growth factor FGF2 (also referred to as bFGF) that shows angiogenic effects as protein administration (Baffour, R. et al, J.Vasc.Surg.16: 181-191 (1992)). Experiments using the above vectors were conducted to investigate (1) the expression level and kinetics of the introduced gene in SeV-mediated intramuscular gene transfer, (2) whether high expression of angiogenic factors prevents necrosis of acute ischemic limb or adversely affects, and (3) whether angiogenic proteins expressed at high levels in the muscle leak into the systemic circulation.

The following model was used as an ischemia model: BALB/C nu/nu mouse hindlimb amputation model (auto-aggregation model) which was completely excised from the iliac arteriovenous to the above-knee femoral arteriovenous (severe ischemia model) and C57BL/6 mouse limb salvage model (limbsalvage model) which was operated in the same manner as described above and in which hindlimb was not amputated due to physiological angiogenesis. Vectors expressing human VEGF165, mouse FGF2 or luciferase (SeV-hVEGF 165, SeV-mFGF2 or SeV-luciferase, respectively) were constructed, administered to thigh muscles and calf muscles two days before the ischemic surgery, and hindlimb status was observed to 10 days after the surgery.

When the luciferase gene was introduced into hindlimb skeletal muscle of mice, the SeV gene was expressed at a level 5 to 120 times higher than the expression level when 100. mu.g of plasmid (equivalent to 25 to 50 times the amount of 200mg/60kg of body weight used in clinical practice) was administered. In various cultured cells, the protein secretion levels of SeV-hVEGF165 and SeV-mFGF2 are high (50-500 ng/10)⁵One cell/24 hours). Compared with the non-administration group (baseline), intramuscular administration of SeV-mFGF2 increased FGF2 levels by 5-100 times, whereas intramuscular expression of FGF2 was limited (2 times of baseline at maximum) and endogenous VEGF expression was significantly increased when SeV-hVEGF165 was administered. The muscle tissue of the patient has large-area necrosis the next day after the SeV-hVEGF165 is given, the hindlimb drop is promoted, and the SeV-mFGF2 has obvious curative effect on limb rescue while the endogenous VEGF expression is increased. In both cases no significant leakage of protein from the carrier into the serum was found (< 5 pg/ml). In the limb salvage model, the non-administration group, the SeV-luciferase group and the SeV-mFGF2 group all salvaged successfully, and only 1/3 mice in the SeV-hVEGF group had hind limb detachment. In the hindlimb amputation model, only FGF2 group was effective in limb rescue, and almost all hindlimbs were amputated in the other groups.

The invention proves that the intramuscular administration of the recombinant Sendai virus vector can obviously enhance the expression of transgenes. The expression of the recombinant Sendai virus vector is 10-100 times that of the plasmid vector, but it is found that the administration of the recombinant Sendai virus vector expressing VEGF165 to mice with acute critical limb ischemia promotes limb shedding. To induce edema with SeV-hVEGF165 (example 4, FIG. 8), prevent blood flow restoration after ischemic surgery (example 5, FIGS. 11 and 12), and significantly increase the limb drop rate due to ischemia (example 5, FIGS. 9 and 10), the strong vascular permeability-enhancing effect of VEGF may be one of the causes of these pathological phenomena. In contrast, the Sendai virus vector expressing FGF2 was administered with high therapeutic effect. Recombinant proteins secreted into the systemic circulation were not detected in both models, thus indicating that SeV-mediated FGF2 treatment has little effect on other organs and a broad safety profile. The above results indicate that in human clinical applications, attention must be paid to the adverse effects of VEGF in specific limb states. Therefore, FGF2 gene therapy, which shows safety and efficacy in a wide range, can be a safe gene therapy system. In addition, the invention also proves the effectiveness of SeV vector which is a powerful tool for introducing therapeutic genes in vivo, so that the SeV vector can be used for clinical treatment of acute critical limb ischemia.

The present invention relates to paramyxovirus vectors encoding angiogenic factors and uses thereof, and in particular, the present invention relates to:

(1) a paramyxovirus vector encoding an angiogenesis gene capable of expression;

(2) the paramyxovirus vector of (1), wherein the angiogenic gene is fibroblast growth factor 2(FGF 2);

(3) the paramyxovirus vector according to item (1), wherein the paramyxovirus is Sendai virus;

(4) the paramyxovirus vector of item (1) in which the F gene is deleted;

(5) an angiogenic composition comprising the paramyxovirus vector according to (1) or a cell containing the vector and a pharmaceutically acceptable carrier;

(6) the composition according to the item (5) for treating ischemic tissue;

(7) the composition according to item (5) for intramuscular administration;

(8) a method for inducing angiogenesis, which comprises administering the angiogenesis composition according to any one of (5) to (7).

According to the invention, (1) limb ischemia-induced endogenous VEGF is not localized in muscle, but rather diffuses into the systemic circulation, but vector-mediated VEGF165 expression of the invention does not leak significantly into the systemic circulation; (2) even if the expression level is 5-100 times higher than that of endogenous FGF2, the exogenous FGF2 is not obviously diffused to the systemic circulation system; (3) the expression level of FGF2 induces the expression of endogenous VEGF, obviously increases limb blood flow and shows obvious limb rescue effect; (4) in contrast to FGF2, overexpression of VEGF165 induces limb injury. The results show that FGF2 is suitable for clinical application, and has wide safety range as a therapeutic angiogenesis factor for treating acute critical limb ischemia. In addition, the invention also discloses that the introduction of the VEGF165 gene may bring serious side effects to limb ischemia for the first time.

Interestingly, limb ischemia-induced endogenous VEGF is not concentrated in muscle but spreads to the systemic circulation. Although it is known that ischemic surgery induces the expression of endogenous VEGF in muscle and Endothelial Cells (EC) (Florkiewicz, R.Z. et al, J.cell. physiol.162, 388-399(1995)), the present invention also discloses for the first time experimental data on the possibility that endogenous VEGF induces a systemic rather than a local angiogenic response. Asahara et al have shown that systemic administration of VEGF increases the mobility of Endothelial Precursor Cells (EPCs) (Asahara, T. et al, EMBO J.18, 3964-3972(1999)), and that collateral vessel formation resulting from physiological responses to limb ischemia may depend to some extent on EPC-mediated "angiogenesis-like" neovascularization rather than proliferative EC-mediated local angiogenesis released from existing vessels (Isner J.M., J.Clin.Invest.106, 615-619 (2000)). It has been established that induction of VEGF introduced into ischemic limbs does not bring about sufficient perfusion of blood, leading to limb loss. In this case, VEGF may act primarily as an "vascular permeability factor" rather than an "angiogenic factor". The observation of muscle histology revealed that the VEGF 165-administered group had more severe intramuscular edema, which also supported the above-mentioned findings.

Secondly, FGF2 gene therapy alone is effective in treating ischemic limbs in accordance with the present invention, which is related to the function of endogenous VEGF in vivo. The total protein concentration of VEGF produced in muscle by the introduction of FGF2 gene was comparable to that when VEGF gene was introduced, but FGF2 gene therapy itself differs from VEGF gene therapy in that it showed sufficient therapeutic effect. These findings imply that not only VEGF, FGF2, is required during mature angiogenesis in order to perfuse the necessary blood without vascular leakage in the treatment of ischemic limbs. Angiopoietin-1, an angiogenic factor that has an inhibitory effect on VEGF-induced leakage of immature blood vessels, may contribute to this.

SeV-VEGF165 secretes the gene product at the same level in vitro as SeV-FGF2, but the expression level of SeV-VEGF165 injected into muscle in vivo is different from SeV-FGF2 or SeV-luciferase for completely unknown reasons. Like histological analysis, Laser Doppler Perfusion Image (LDPI) analysis also shows severe muscle tissue damage and low blood perfusion, and thus tissue damage such as VEGF 165-induced edema may impair the cellular mechanisms of SeV-mediated transcription such as tubulin (Moyer, s.a., et al, proc.natl.acad.sci.usa 83, 5405-5409(1986)) and phosphoglycerate kinase (Ogino, t.et al, j.biol.chem.274, 35999-36008 (1999)). Alternatively, it is possible that due to the lower level of expression of the endogenous VEGF165 gene (about 200pg/g muscle) there is a significant increase in critical ischemic muscle (1,400pg/g muscle), promoting limb drop-out. The above results strongly suggest that elevated concentrations of VEGF in muscle, for example even at low levels of about twice baseline, may lead to critical limb ischemia.

Angiogenesis is a well-coordinated process involving a number of factors, among which VEGF biological function is highly dose-dependent, and even deletion of only one allele can lead to fatal deletions (Carmeliet, p. et al, Nature 380, 435-. Throughout vascular perfection and maturation, VEGF must be expressed continuously, one-time overexpression of VEGF induces only a short angiogenic response (Pettersson, A. et al, Lab. invest.80, 99-115(2000)), and VEGF-induced capillary-like structures are not substantially associated with existing blood vessels (Springer, M.L., et al, mol. cell 2, 549-. Thus, the results of the present invention indicate that when insufficient FGF2 is present, the concentration of VEGF in muscle rises by a factor of two, and severe toxicity may occur. In view of the above considerations, while VEGF has great clinical potential in therapeutic angiogenesis, it should be noted more than ever before. In addition, the FGF2 gene introduced into muscle is proved to be safe and have obvious curative effect on limb rescue of acute critical limb ischemia.

In the present invention, the term "paramyxovirus vector" refers to a vector which is derived from paramyxovirus and can introduce a gene into a host cell. The paramyxovirus vector of the present invention may be Ribonucleoprotein (RNP) or a virus particle having infectivity. The term "infectivity" refers to the ability of a recombinant paramyxovirus vector to introduce a gene inside the vector into a cell to which the vector adheres, by maintaining its cell adhesion and membrane fusion ability. The paramyxovirus vector of the present invention may have replication ability or may be a defective vector having no replication ability. Herein, "replication ability" refers to the ability of a virus to replicate and produce infectious viral particles in a host cell infected with the viral vector. The replication ability can be investigated, for example, by using cell lines LLC-MK2 or CV-1 derived from monkey kidney.

In the present specification, the term "recombinant" paramyxovirus vector refers to a paramyxovirus vector constructed by genetic manipulation or an amplification product thereof. For example, a recombinant paramyxovirus vector can be prepared by reconstitution of a recombinant paramyxovirus cDNA.

In the present invention, "paramyxovirus" refers to a virus belonging to Paramyxoviridae or a derivative thereof. Paramyxoviruses suitable for use in the present invention include: sendai virus of Paramyxoviridae (Paramyxoviridae), Newcastle disease virus, mumps virus, measles virus, Respiratory Syncytial Virus (RSV), rinderpest virus, distemper virus, simian parainfluenza virus (SV5), I, II and type III of human parainfluenza virus, and the like. The virus of the present invention is preferably a virus of the genus paramyxovirus or a derivative thereof. Paramyxovirus viruses suitable for use in the present invention include, for example, human parainfluenza virus type I (HPIV-I), human parainfluenza virus type III (HPIV-III), bovine parainfluenza virus type III (BPIV-III), Sendai virus (also referred to as mouse parainfluenza virus type I), and simian parainfluenza virus type X (SPIV-X), and most other paramyxovirus viruses, with Sendai virus being the most preferred paramyxovirus of the present invention. These viruses may be natural strains, wild strains, mutant strains, laboratory-passaged strains, artificially constructed strains, and the like. Incomplete viruses such as DI particles (J.Virol., 1994, 68, 8413-8417), synthetic oligonucleotides, and the like can also be used as materials for preparing the viral vectors of the present invention.

Genes encoding paramyxovirus proteins include NP, P, M, F, HN, and L genes. "NP, P, M, F, HN, and L genes" refer to genes encoding nucleocapsid protein, phosphoprotein, matrix protein, fusion protein, hemagglutinin-neuraminidase protein, and macromolecular protein, respectively. The gene of each virus in the subfamily Paramyxovirinae is generally represented as follows, and the NP gene is also generally described as "N gene".

Paramyxovirus (Paramyxovirus) NP P/C/V M F HN-L

Rubella virus (Rubulavirus) NP P/V M F HN (SH) L

Measles virus (Morbillivirus) NP P/C/V M F H-L

For example, in the nucleotide sequence database, Sendai virus classified as Respirovirus of Paramyxoviridae has accession numbers of NP gene of M29343, M30202, M30203, M30204, M51331, M55565, M69046, and X17218; the P gene is M30202, M30203, M30204, M55565, M69046, X00583, X17007, and X17008; of the M genes are D11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584, and X53056; d00152, D11446, D17334, D17335, M30202, M30203, M30204, M69046, X00152, and X02131 for the F gene; of the HN gene are D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, and X56131; of the L genes are D00053, M30202, M30203, M30204, M69040, X00587, and X58886.

In the present invention, the term "gene" refers to genetic material, including nucleic acids such as RNA and DNA. The gene may or may not encode a protein, and may encode a functional RNA such as a ribozyme or antisense RNA. The gene may be a native sequence or an artificially designed sequence. In addition, in the present invention, the term "DNA" includes single-stranded DNA and double-stranded DNA.

The present invention provides paramyxovirus vectors encoding angiogenic factors and uses thereof. The present inventors demonstrated that high levels of transgene expression at the site of administration can be achieved by intramuscular administration of paramyxovirus vectors encoding angiogenic factors in vivo. The results of the mouse ischemic hind limb rescue experiments conducted by the present inventors showed that the administration of a recombinant paramyxovirus vector encoding an angiogenic factor (FGF2) prevented ischemic tissue necrosis and prevented hind limb detachment. The above vector can effectively induce angiogenesis and prevent necrosis in ischemic tissues, and thus, the vector of the present invention is suitable for gene therapy of ischemic diseases.

In addition, the present inventors found that the gene administered intramuscularly by the recombinant paramyxovirus vector was expressed continuously for 1 to 2 weeks. This result indicates that sustained therapeutic effects can be obtained when gene therapy is carried out using the recombinant paramyxovirus vector. Furthermore, the angiogenic factor expressed by the recombinant paramyxovirus vector administered intramuscularly is not detected in the systemic circulation system, and no adverse side effects are produced in tissues other than the target tissue. Therefore, according to the present invention, paramyxovirus vectors have various advantages in the introduction of angiogenic genes, which may bring great progress in gene therapy and the like targeting ischemic tissues.

From the viewpoint of safety, since the paramyxovirus vector is not pathogenic to humans, it means that it is preferably used in clinical trials for human gene therapy. First, in the expression of foreign genes mediated by plasmid DNA, the introduced DNA must be transported into the nucleus or its nuclear membrane must be eliminated, which is a major obstacle to increasing the success rate of gene expression. However, in the case of Sendai virus and the like, the expression of foreign genes is driven by the replication of the viral genome with cellular tubulin in the cytoplasm and RNA polymerase (L protein) which itself has. This indicates that Sendai virus does not interact with the host chromosome and is not considered to cause a safety problem such as canceration of cells due to chromosomal abnormality. Secondly, Sendai virus is known to be pathogenic to rodents, causing pneumonia, but it is not pathogenic to humans. One of the evidences is that intranasal administration of wild-type Sendai virus to non-human primates does not cause severe injury (Hurwitz J.L. et al, Vaccine, 1997, 15, 533-540). These characteristics suggest that Sendai virus vector can be applied to human therapy, and it is expected to become an option for gene therapy of angiogenic factors.

In the present invention, an angiogenic gene refers to a gene encoding a factor that directly or indirectly promotes angiogenesis and/or vasculogenesis. The factor may be a protein or a peptide, or may be a nucleic acid such as a functional RNA (ribozyme or antisense RNA). Angiogenic proteins include acidic fibroblast growth factor (aFGF), fibroblast growth factor 2(FGF2) (also known as basic fibroblast growth factor (bFGF)), Vascular Endothelial Growth Factor (VEGF), angiogenin (Ang) (including Ang-1 and Ang-2), Epidermal Growth Factor (EGF), transforming growth factor alpha (TGF-alpha), TGF-beta, platelet-derived endothelial growth factor (PD-ECGF), platelet-derived growth factor (PDGF), tumor necrosis factor alpha (TNF-alpha), Hepatocyte Growth Factor (HGF), insulin-like growth factor (IGF), Erythropoietin (EPO), Colony Stimulating Factor (CSF), macrophage-CSF (M-CSF), granulocyte/macrophage-CSF (GM-CSF), Interleukin (IL) -8, and Nitric Oxide Synthase (NOS), among others (Klagsbrun, m, and D' Amore, p, a.annu.rev.physiol.53: 217-39, 1991; folkman, j, and Shing, y., j.biol.chem.267 (16): 10931-4, 1992; symes, j.f. and Sniderman, a.d., curr. opin. lipidol.5 (4): 305-12, 1994).

Preferred examples of the angiogenic protein of the present invention include aFGF, FGF2, Ang-1, Ang-2, EGF, TGF- α, TGF- β, PD-ECGF, PDGF, TNF- α, HGF, IGF, EPO, CSF, M-CSF, GM-CSF, IL-8, and NOS, and vectors can be prepared using genes encoding proteins selected from the above-mentioned proteins.

Among the angiogenic proteins used in the present invention, a protein that allows a neovascular to be surrounded by parietal cells differentiated from neoendothelial cells connected to stromal cells is particularly preferred, rather than a protein that induces immature angiogenesis by VEGF. As the angiogenesis has three steps (vascularization, angiogenesis and vascular maturation), the results of experiments of removing various transcription factors show that various genes are related to the formation of mature vessels, specifically, the transcription factor SCL/tal-1 is mainly related to the vascularization, HIF-1, Id, ETS-1, HOXD3, COUP-TFII and MEF2C are related to the angiogenesis, and furthermore, after removing LKLF (lung ruppel-like factor) or dHAND gene, parietal cells cannot develop, thereby causing embryonic death.

Therefore, the angiogenic gene used in the present invention is more preferably a gene inducing transcription factors (including LKLF and dHAND) which are involved in the maturation of parietal cells in immature mesenchymal cells. It is thought that the stimulation with FGF2 is directly related to the induction of the above transcription factors, or promotes the proliferation and differentiation of mesenchymal cells by other growth factors, angiogenin, HGF, and the like.

The angiogenic protein preferably contains a secretion signal for secreting the protein. However, proteins such as FGF2 can be secreted extracellularly even without the typical secretion signal in nature (see examples), and these proteins do not necessarily have to contain a secretion signal. The gene encoding the above-mentioned angiogenic factor can be prepared by a known method, for example, PCR using primers designed based on the nucleotide sequence information. An especially preferred angiogenic factor for use in the present invention is FGF2, which exhibits stable therapeutic effect over a wide range of expression levels (Abraham, J.A. et al, 1986, EMBO J.5: 2523-2528; Moscatelli, D.A. et al, US 4994559; Baird, A. et al, US 5155214; Isner, J.M.US 6121246; WO 97/14307).

The angiogenic gene carried by the vector can exert its effect even if it is not homologous to the target individual to which the gene is to be introduced, but a gene homologous to the target individual is preferred. The angiogenesis gene carried by the vector is preferably mammalian angiogenesis gene, and in human, preferably human gene.

The paramyxovirus vector encoding an angiogenic gene of the present invention is particularly effective for treating ischemic tissues, i.e., the vector of the present invention can be used to introduce an angiogenic gene, thereby promoting angiogenesis and preventing necrosis due to ischemia. In the present invention, the ischemic tissue is not particularly limited as long as it is a tissue that has been or is being ischemic, and examples thereof include: muscle, brain, kidney and lung. The ischemic diseases which can be treated by the vector of the invention comprise cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, myocardial ischemia and the like. In the present invention, the treatment of ischemic tissue refers to the treatment of ischemic tissue or the prevention of ischemic injury, and specifically refers to the prevention of necrosis of ischemic tissue, the maintenance of survival of ischemic tissue, the promotion of angiogenesis and tissue regeneration in ischemic tissue, or the prevention or reduction of injury caused by ischemia.

The present invention provides a method for inducing angiogenesis, which comprises the step of administering a paramyxovirus vector encoding an angiogenesis gene. Further, the present invention provides a method for treating ischemic tissue, which comprises administering a paramyxovirus vector encoding an angiogenic gene. The subject to be administered is not particularly limited, and includes, for example, target mammals such as humans. In addition, as the administration target, non-human mammals, specifically, primates such as monkeys (apes such as prosperous apes and euapes and apes such as prosperous apes), rodents such as mice, rats and guinea pigs, and cows, dogs, cats, horses, sheep, and rabbits are specifically exemplified. The ischemia in the above-mentioned animals can be treated with the vector of the present invention, or the above-mentioned animals can be used as a model for the treatment of ischemia in humans (Morinaga, K. et al, 1987, J.Vasc. Surg.5: 719-730; Itoh, H. et al, 1994, Atherosclerosis 110: 259-270).

In the present invention, the method for inducing angiogenesis specifically comprises:

(1) a method for inducing angiogenesis, which comprises the step of administering a paramyxovirus vector encoding an angiogenesis gene or a cell containing the vector;

(2) the method according to item (1), wherein the angiogenic gene is fibroblast growth factor 2(FGF 2);

(3) the method of (1) or (2), wherein the administration is intramuscular administration;

(4) the method according to any one of (1) to (3), wherein the paramyxovirus is Sendai virus.

The method for treating ischemic tissues comprises the following steps:

(1) a method for treating ischemic tissue, which comprises the step of administering a paramyxovirus vector encoding an angiogenic gene or a cell containing the vector;

Administration can be in vivo or ex vivo (ex vivo). For in vivo administration, the paramyxovirus vector encoding the angiogenic gene can be injected by a method known to those skilled in the art, such as intramuscular injection, subcutaneous injection, and catheter administration. In ex vivo administration, the vector is introduced into cells in vitro, and the cells into which the vector has been introduced are injected into an individual by a method such as intramuscular injection, subcutaneous injection, or catheter administration. The vector-introduced cells used in ex vivo administration may be homologous or heterologous to the subject to whom they are administered, but are preferably homologous, more preferably cells taken from the subject to whom they are administered, and still more preferably cells taken from bone marrow or blood, including cells capable of forming or differentiating into vascular endothelial cells (i.e., vascular endothelial precursor cells). Angiogenesis of a tissue to be administered can be induced by administering a pharmaceutically effective amount of the vector of the present invention, and thus, treatment for preventing necrosis and exfoliation of the tissue can be performed in ischemia of the brain, heart, kidney, lung, limb, and the like.

In addition, the present invention also provides the use of paramyxovirus vectors encoding angiogenic genes for treating ischemic tissue and a method thereof, and specifically, the present invention provides:

(1) a paramyxovirus vector encoding an angiogenesis gene for treating ischemic tissue;

(2) the vector according to item (1), wherein the angiogenic gene is fibroblast growth factor 2(FGF 2);

(3) the vector according to (1) or (2) for intramuscular administration;

(4) the vector of any one of (1) to (3), wherein the paramyxovirus is Sendai virus.

The present invention also provides a composition for treating ischemic tissue, which comprises the above-mentioned paramyxovirus vector. In addition to the above-mentioned paramyxovirus vector, the composition may further contain a desired pharmaceutically acceptable carrier. For example, the vehicle of the present invention can be combined with a physiological solution to make an injection, or combined with a solid or semisolid substance (gel) to make an in vivo implant.

In the present invention, the paramyxovirus vector used for introduction of the angiogenic gene is not particularly limited, and suitable paramyxovirus vectors include, for example, vectors capable of replication and self-propagation. In general, the genome of a naturally occurring paramyxovirus contains a short 3 'leader followed by 6 genes encoding the N (nucleocapsid), P (phospho), M (matrix), F (fusion), HN (hemagglutinin-neuraminidase) and L (large) proteins, respectively, and a short 5' tail region at the other end. The vector of the present invention capable of autonomous replication can be obtained by designing a genome having the same structure as described above. In addition, a vector for expressing a foreign gene can be obtained by inserting the foreign gene into the genome of the above-mentioned vector. In the paramyxovirus vector of the present invention, the arrangement of viral genes may be different from that of wild-type viruses.

The paramyxovirus vector used in the present invention may be deleted for some of the genes contained in the wild-type paramyxovirus. For example, when a Sendai virus vector is reconstituted, it is generally considered that the proteins encoded by the NP, P/C, and L genes are necessary, but the virus vector of the present invention does not necessarily have to include the genes encoding the above proteins as its components. For example, an expression vector carrying a gene encoding the above protein may be co-transfected into a host cell with an expression vector encoding the vector genome, thereby reconstituting a viral vector. Alternatively, an expression vector encoding a vector genome is introduced into a host cell carrying a gene encoding the above protein, and the host cell supplies the above protein, thereby reconstituting a viral vector. The amino acid sequences of these proteins may differ from those from the starting virus, as long as they have an equivalent or stronger activity during nucleic acid introduction compared to the natural strain, and they may be mutated or substituted by a homologous gene of another virus.

It is considered that the proteins encoded by M, F and HN genes are essential for cell-cell propagation of paramyxovirus vectors. However, when the paramyxovirus vector is in the form of RNP, these proteins are not required. If the genome in RNP contains M, F and HN genes, when it is introduced into a host cell, the products of these genes are produced, and infectious virus particles are produced. Examples of RNP vectors that produce infectious viruses include RNPs containing viral genomic RNA and N, P and L proteins encoding N, P, M, F, HN and L genes. After the RNP is introduced into cells, the expression genome is expressed and replicated by the action of the N protein, P protein and L protein, and the infectious viral vector is amplified.

RNP can be introduced into cells as a complex with a transfection reagent such as lipofectamine (lipofectamine) and polycationic liposomes. Specifically, various transfection reagents can be used, for example, DOTMA (Boehringer), Superfect (QIAGEN #301305), DOTAP, DOPE, DOSPER (Boehringer #1811169), and the like. Chloroquine may be added to prevent degradation in endosomes (Calos, m.p., proc.natl.acad.sci.usa, 1983, 80, 3015). In the case of replication-competent viruses, the produced viruses can be amplified or passaged by reinfection with cultured cells, chicken eggs, or animals (e.g., mammals such as mice), etc.

A paramyxovirus vector lacking M, F and/or HN gene may also be used as the paramyxovirus vector of the present invention. These vectors can be reconstituted by, for example, exogenously supplying the deleted gene product. The viral vector thus prepared can still adhere to the host cell and induce cell fusion as with the wild-type virus, but the progeny viral particles do not have the same infectivity as the starting virus because the vector genome introduced into the cell lacks one of the above genes. Therefore, these vectors can be used as safe viral vectors capable of gene transfer only once. A gene deletion means that the function of the gene has been substantially lost, and when the gene encodes a protein, it means that a protein having the same function as the wild-type protein is not expressed. For example: the gene is not transcribed, the gene is a loss-of-function mutant, the gene is deleted, or the like. Preferably, a gene deletion means that the codon region of the gene is at least partially, preferably completely, deleted. For example, the vector for deleting the F gene is preferably a vector in which a part of, more preferably all, the codon region of the F protein is deleted. In the present invention, the F gene deletion vector more preferably has the F gene deleted in the minus strand, and further lacks the initiation signal sequence at the 3' -flanking region, thereby making it possible to suppress the expression of the polypeptide unnecessary at the deletion site. When an Open Reading Frame (ORF) encoding an unnecessary polypeptide is present at the deletion site, it is preferable to remove the ORF (described later) by site-directed mutagenesis or the like.

When an F gene-deleted vector is prepared, for example, a plasmid expressing the F gene-deleted recombinant paramyxovirus vector genome can be co-transfected into a host cell together with an expression vector for the F protein and expression vectors for the NP, P/C, and L proteins, thereby reconstituting a viral vector (WO00/70055 and WO 00/70070). Alternatively, it can be prepared, for example, by using a host cell in which the F gene is integrated into a chromosome. When these proteins are provided exogenously, their amino acid sequences may be different from those of the starting virus, so long as they have an equivalent or stronger activity during the nucleic acid introduction than that of the natural type, and they may be mutated or substituted with a homologous gene of another virus.

The following vectors can be prepared: the envelope protein includes a protein different from the viral envelope protein encoded by the vector genome. Such proteins are not limited and may include envelope proteins of other viruses, such as the G protein of Vesicular Stomatitis Virus (VSV) (VSV-G). The paramyxovirus vector of the present invention includes a pseudotype virus vector such as VSV-G, which carries an envelope protein derived from a virus other than a virus encoded by a genome.

The paramyxovirus vector of the present invention further comprises the following proteins on the surface of the viral envelope, for example: proteins such as adhesion factors, ligands and receptors that adhere to specific cells, or chimeric proteins comprising the above proteins on the outer surface of the virus and viral envelope-derived polypeptides on the inner side of the virus, thus producing vectors that target specific tissues. These proteins may be encoded by the viral genome itself, or may be provided by expression of genes other than the viral genome (e.g., genes of other expression vectors or host cell chromosomes) upon reconstitution of the viral vector.

The viral genes contained in the viral vector of the present invention may be altered, for example, to reduce antigenicity or to increase RNA transcription efficiency or replication efficiency. In particular, at least one of the replication factor NP, P/C, and L genes may be altered to enhance transcription or replication functions. One of the structural proteins, HN protein, has hemagglutinin activity and neuraminidase activity, and if the former activity is attenuated, it is possible to improve the stability of the virus in blood; altering the activity of the latter may then modulate infectivity. The fusion capacity of membrane fusion liposomes can be modulated by altering the F protein, which is involved in membrane fusion. For example, paramyxoviruses having reduced antigen presenting ability can be prepared by analyzing the antigen presenting epitope of F protein or HN protein, which may be cell surface antigen molecules.

The paramyxovirus vector of the present invention may further lack an auxiliary gene. For example: after deletion of one of the auxiliary genes of SeV, gene expression or replication in cultured cells is not affected, but the pathogenicity of SeV to hosts such as mice is significantly reduced (Kato, A.et al 1997 J.Virol.71: 7266-. Such an attenuated vector is particularly suitable as a viral vector for in vivo or ex vivo gene transfer.

The viral vectors of the invention may encode an angiogenic gene in genomic RNA. A recombinant paramyxovirus vector carrying a foreign gene can be prepared by inserting the foreign gene into the paramyxovirus vector genome. An angiogenic gene to be expressed in a target tissue (ischemic tissue or the like) can be used as the foreign gene. The foreign gene may be a gene encoding a native protein, or a gene encoding a protein obtained by modifying a native protein by deletion, substitution or insertion, as long as the modified protein has an equivalent function to the native protein. For example, for the purpose of gene therapy or the like, a therapeutic gene for a target disease may be inserted into DNA encoding the viral vector genome (viral vector DNA). In the case of introducing a foreign gene into Sendai virus vector DNA, it is preferable to insert a sequence comprising a multiple of 6 nucleotides between the transcription termination sequence (E) and the transcription initiation sequence (S) (Calain P. and Roux L., J.Virol., 1993, 67(8), 4822-4830). Foreign genes may be inserted upstream and/or downstream of each gene (NP, P, M, F, HN, and L genes) of the virus. In order not to interfere with the expression of the upstream and downstream genes, an E-I-S sequence (transcription termination sequence-intervening sequence-transcription initiation sequence) or a part thereof may be appropriately inserted upstream or downstream of the foreign gene, the E-I-S sequence may be placed between the respective genes, or the foreign gene may be inserted via IRES.

The expression level of the inserted foreign gene can be regulated by the type of transcription initiation sequence linked upstream of the gene (WO01/18223), and also by the position of the insertion point and the base sequences before and after the gene. For example, in Sendai virus, the closer the insertion site is to the 3' -end of the minus-strand RNA of the viral genome (the closer to the NP gene in the gene arrangement of the wild-type viral genome), the higher the expression level of the inserted gene. In order to obtain high-level expression of the foreign gene, it is preferable to insert it into an upstream region of the minus-strand genome, such as upstream of the NP gene (3' -flanking sequence on the minus strand), or between the NP and P genes. Conversely, the closer the insertion point is to the 5' -end of the negative strand RNA (the closer to the L gene in the gene arrangement of the wild-type viral genome), the lower the expression level of the inserted gene. In order to reduce the expression level of the foreign gene, it may be inserted into the 5 ' -most position on the minus strand, i.e., downstream of the L gene (5 ' -flanking region of the L gene on the minus strand) or upstream of the L gene (3 ' -flanking region of the L gene on the minus strand) of the wild-type viral genome. Thus, the insertion position of the foreign gene can be appropriately adjusted so as to obtain a desired gene expression level, or the combination of the insert and the gene encoding the viral protein in the periphery thereof can be optimized. For example, when a high-titer viral vector is administered to cause toxicity in the expression of an angiogenic factor at a high level, in addition to controlling the titer of the virus administered, the expression level of each viral vector can be lowered to obtain an appropriate therapeutic effect by setting the insertion point of the angiogenic gene at the 5' -end of the minus strand of the vector, or replacing the transcription initiation sequence with a sequence having a low efficiency, or the like.

In order to facilitate the insertion of a foreign gene, a cloning site may be designed at the insertion site. For example, the cloning site may be a recognition sequence for a restriction enzyme. In the vector DNA encoding the genome, a foreign gene may be inserted into the restriction site. The cloning site may be a multiple cloning site comprising a plurality of individual sequences recognized by restriction enzymes. The vector of the present invention may further comprise another foreign gene at a position other than the above-mentioned insertion position. These foreign genes are not limited, and may be other angiogenic genes or other genes.

The compounds can be prepared according to, for example, Hasan, m.k. et al, j.gen.virol, 1997, 78: 2813-2820, KatoA. et al, EMBO J., 1997, 16: 578-587 and Yu D, et al, Genes Cells, 1997, 2: the method described in 457- "466 was used to construct a recombinant Sendai virus vector carrying a foreign gene as follows.

First, a DNA sample containing the cDNA nucleotide sequence of the desired foreign gene is prepared. Preferably, the DNA sample can be confirmed to be a single plasmid by electrophoresis at a concentration of 25 ng/. mu.l or more. The following is an example of inserting a foreign gene into the NotI site of the viral genomic DNA. If the nucleotide sequence of the objective cDNA contains a NotI recognition site, it is preferable to previously modify the nucleotide sequence by site-directed mutagenesis or the like so as to remove the site, but not to modify the encoded amino acid sequence. Recovering the desired DNA fragment from the DNA sample by PCR amplification. In order to obtain an amplified fragment having NotI sites at both ends and to add a copy of a transcription termination sequence (E), an intervening sequence (I) and a transcription initiation sequence (S) (EIS sequence) of Sendai virus at one end, a forward DNA sequence and a reverse DNA sequence (antisense strand) were prepared as a primer pair comprising a NotI restriction enzyme recognition sequence, a transcription termination sequence (E), an intervening sequence (I), a transcription initiation sequence (S) and a partial sequence of a target gene.

For example, the forward synthetic DNA sequence contains any two or more nucleotides (preferably 4 nucleotides, excluding sequences from NotI recognition sites such as GCG and GCC, and more preferably ACTT) at the 5' -end to ensure digestion with NotI. A NotI recognition sequence GCGGCCGC was added to the 3' -end of the sequence. In addition, any 9 nucleotides or multiples of 9 plus 6 nucleotides are added as spacer sequences at the 3' -end. Further, a sequence of about 25 nucleotides of ORF, which starts from the start codon ATG of the desired cDNA and includes ATG, is also added at the 3' -end. Preferably, about 25 nucleotides from the desired cDNA are selected as the 3' -end of the forward synthetic oligo DNA, making the last nucleotide either a G or a C.

The reverse synthetic DNA sequence contains any two or more nucleotides (preferably 4 nucleotides, excluding sequences derived from NotI recognition sites such as GCG and GCC, and more preferably ACTT) at the 5' -end. A NotI recognition sequence GCGGCCGC was added to the 3' -end of the sequence. In addition, an insert oligo DNA was added to the 3' -end to adjust the length of the primer. The length of the oligo DNA was designed as follows: including the NotI recognition sequence GCGGCCGC, the complementary sequence of cDNA and the later-described EIS sequence derived from the Sendai virus genome, the total number of nucleotides is a multiple of 6 (so-called "rule of 6"; Kolakofski D. et al, J.Virol., 1998, 72, 891-4839; Calain P. and Roux L., J.Virol., 1993, 67, 4822-4830). Furthermore, the 3 ' -end of the insert is added with a sequence complementary to the S sequence of Sendai virus (preferably 5'-CTTTCACCCT-3'; SEQ ID NO: 1), an I sequence (preferably 5 ' -AAG-3 '), and a sequence complementary to the E sequence (preferably 5'-TTTTTCTTACTACGG-3'; SEQ ID NO: 2). Further, the 3 '-end was added with the complementary sequence of the desired cDNA so that the last nucleotide was G or C, and this last nucleotide was located about 25 nucleotides upstream of the termination codon of the cDNA, which was used as the 3' -end of the reverse synthetic oligo DNA.

PCR can be carried out by a conventional method using ExTaq polymerase (TaKaRa). The amplified target fragment is preferably digested with NotI using Vent polymerase (NEB), and then inserted into NotI site of the plasmid vector pBluescript. The nucleotide sequence of the resulting PCR product was detected by an automatic DNA sequencer, and a plasmid having the correct sequence was selected. The insert was excised from the plasmid by NotI digestion and cloned into the NotI site of the plasmid containing the paramyxovirus genomic cDNA. Alternatively, the recombinant Sendai virus cDNA can also be obtained by directly inserting the PCR product into NotI site without plasmid vector pBluescript.

For example, recombinant Sendai virus genomic cDNA can be constructed according to literature procedures (Kato, A. et al, EMBO J.16: 578-in 598, 1997; Hasan, M.K. et al, J.Gen.Virol., 78: 2813-in 2820, 1997; Yu, D. et al, Genes Cells, 1997, 2, 457-in 466; and Li, H.O. et al, J.Viroloy 74, 6564-in 6569, 2000). For example, an 18bp spacer sequence (5 ' - (G) -CGGCCGCAGATCTTCACG-3 '; SEQ ID NO: 3) with a NotI recognition site was first inserted into the cloned Sendai virus genomic cDNA (pSeV (+)) at a locus adjacent to the 5 ' -end of the leader sequence and the N-protein-encoding sequence to obtain a plasmid pSeV18+ b (+) (Hasan M.K., et al, J.General Virol., 1997, 78, 2813-2820) with a self-cleaving ribozyme site derived from the antigenomic strand of delta hepatitis virus. A recombinant Sendai virus cDNA into which a desired foreign gene has been inserted can be obtained by inserting a foreign gene fragment into the NotI site of pSeV18+ b (+).

The recombinant paramyxovirus vector DNA prepared as described above is transcribed in a test tube or a cell, and RNP is reconstructed in the presence of L, P of the virus and NP protein, thereby producing an expression vector containing the RNP. The present invention provides a method for producing a paramyxovirus vector encoding an angiogenic gene, which comprises the steps of transcribing a DNA encoding the genome of the paramyxovirus vector of the present invention in a cell in the presence of a protein that transcribes and replicates the genome, and recovering the resulting paramyxovirus vector. The proteins that transcribe and replicate the genome of the paramyxovirus vector of the invention include N, L, the P protein, and the like. The present invention also provides a DNA for producing the paramyxovirus vector of the present invention, which contains the above-mentioned DNA. The present invention also relates to the use of a DNA encoding the genome of the paramyxovirus vector of the invention for preparing the vector. Virus may be reconstituted from viral vector DNA according to well-known methods (WO 97/16539; WO 97/16538; Durbin A.P. et al, Virol., 1997, 235, 323-. Paramyxovirus vectors including parainfluenza virus, vesicular stomatitis virus, rabies virus, measles virus, rinderpest virus, sendai virus and the like, which are required for reconstitution from DNA by these methods. When F, HN and/or the M gene are deleted from the viral vector DNA, infectious viral particles cannot be formed, but it is possible to introduce these deleted genes and/or genes encoding other viral envelope proteins from another virus into a host cell and express them, thereby producing infectious viral particles.

The method for introducing the vector DNA into the cell comprises: (1) preparing a DNA pellet capable of incorporating into the desired cells, (2) preparing a complex comprising positively charged DNA which is suitable for incorporation into the desired cells and has low cytotoxicity, and (3) opening a transient pore of sufficient size to allow DNA to pass through on the plasma membrane of the desired cells using electric pulses.

Various transfection reagents can be used in method (2), for example, DOTMA (Boehringer), Superfect (QIAGEN #301305), DOTAP, DOPE and DOSPER (Boehringer #1811169), etc. In the method (1), transfection may be performed with calcium phosphate. In this method, DNA is taken up by the cells in the form of phagocytic vesicles, but sufficient amounts of DNA are known to be taken up in the nucleus of the Cell (Graham F.L. and VanDer Eb J., virology., 1973, 52, 456; Wigler M. and Silverstein S., Cell, 1977, 11, 223). Chen and Okayama studied the optimal conditions for the introduction of the technology, and they reported that: (1) the incubation conditions for the cells and the co-precipitate were: 2-4% CO2, 35 ℃, 15-24 hr, (2) circular DNA is more active than linear DNA, and (3) when the DNA concentration in the precipitation mixture solution is 20-30 μ g/ml, an optimal precipitate can be formed (Chen c. and Okayama h., mol. Method (2) is suitable for transient transfection. An earlier method was known to prepare a mixture of DEAE-dextran (Sigma # D-9885 M.W.5X 105) having a desired DNA concentration ratio for transfection. Chloroquine may be added to improve transfection efficiency as most complexes degrade in endosomes (Calos, m.p., proc.natl.acad.sci.usa, 1983, 80, 3015). Method (3) is called an electroporation method, and is more widely used than methods (1) and (2) because of the absence of cell selectivity. Transfection efficiency can be maximized by optimizing the duration of the pulse current, pulse form, electric field strength (inter-electrode gap, voltage), conductivity of the buffer, DNA concentration, and cell density.

Among the above three methods, the method (2) using a Transfection Reagent, preferably but not limited to Superfect transduction Reagent (QIAGEN, #301305) and DOSPER Liposomal transduction Reagent (Boehringer Mannheim #1811169), is simple to operate and can detect a large number of samples with a large number of cells, and thus is suitable for use in the present invention.

Specifically, the reconstitution from cDNA was performed as follows:

monkey kidney cells LLC-MK2 were cultured to 70-80% confluency in approximately 24-6 wells of plastic plates or 100mm Petri dishes in Minimal Essential Medium (MEM) containing 10% Fetal Calf Serum (FCS) and antibiotics (100U/ml penicillin G and 100. mu.g/ml streptomycin). Recombinant vaccinia virus vTF7-3 expressing T7 polymerase was inactivated in the presence of 1. mu.g/ml psoralen by UV exposure for 20 minutes and then infected with 2 PFU/cell (Fuerst T. R. et al, Proc. Natl. Acad. Sci. USA, 1986, 83, 8122-The duration of UV exposure. 1 hour after infection, 2-60. mu.g (more preferably 3-5. mu.g) of the above recombinant Sendai virus cDNA and a plasmid expressing a viral protein (24-0.5. mu.g pGEM-N, 12-0.25. mu.g pGEM-P and 24-0.5. mu.g pGEM-L, or more preferably 1. mu.g pGEM-N, 0.5. mu.g pGEM-P and 1. mu.g pGEM-L) (Kato. A. et al, GeneCells, 1996, 1, 569-579) which can be used for generating the full-length Sendai virus genome and is necessary were transfected by the lipofection method using Superfect (QIAGEN Co., Ltd.) or the like. Transfected Cells were cultured in MEM, which was serum-free and, if necessary, contained just 100. mu.g/ml rifampicin (Sigma) and preferably cytarabine (AraC) (Sigma) at a concentration of 40. mu.g/ml, in order to adjust the drug concentration to the optimum, thereby minimizing cytotoxicity of vaccinia virus and maximizing recovery of virus (Kato. A. et al, Genes Cells, 1996, 1, 569-. After transfection, cells were cultured for 48-72hr, then harvested and lysed by three freeze-thaw cycles. LLC-MK2 cells were transfected with cell lysates, cultured for 3-7 days, and the medium was collected. To reconstitute a viral vector that lacks the gene encoding the envelope protein and is incapable of replication, LLC-MK2 cells expressing the envelope protein can be transfected, or transfected with an expression plasmid for the envelope protein. Alternatively, the transfected cells are overlaid on top of LLC-MK2 cells expressing the envelope protein and cultured to propagate a defective viral vector (see WO00/70055 and WO 00/70070). The titer of the virus contained in the culture supernatant can be determined by measuring the Hemagglutinin Activity (HA). HA can be determined by "interior dilution" (Kato. A. et al, Genes Cells, 1996, 1, 569-. To remove any possible contaminating vaccinia virus vTF7-3, it may be diluted appropriately (e.g., 10%⁶Double) the allantoic fluid sample obtained, and performing re-amplification in chicken eggs, wherein the re-amplification can be repeated 3 times or more. The resulting virus can be stored at-80 ℃.

There is no particular limitation on the type of host cells used for reconstitution, so long as the viral vector can be reconstituted in these cells. The host cells include cultured cells such as LLC-MK2 cells derived from monkey kidney, CV-1 cells and BHK cells derived from hamster kidney, human-derived cells, and the like. In addition, in order to obtain a large amount of Sendai virus vector, the vector can be amplified by infecting a pregnant hen egg with the virus vector obtained from the above-mentioned host cell. A method for preparing a viral vector from chicken eggs has been developed (Advanced protocol in neuroscience study III, Molecular biology in neuroscience, edited by Nakanishi et al, Kouseisha, Osaka, 1993, 153-172). Specifically, for example, fertilized eggs are cultured in an incubator at 37 to 38 ℃ for 9 to 12 days to form embryos. The viral vector is inoculated into the allantoic cavity, and the fertilized egg is further cultured for several days to propagate the vector. The conditions such as culture time can be changed depending on the type of the recombinant Sendai virus used. Subsequently, allantoic fluid containing the virus is recovered. Sendai virus vectors can be isolated and purified from allantoic fluid samples by conventional methods (Tashiro M., Protocols in viruses experiments, edited by Nagai and Ishihama, MEDICAL VIEW, 1995, 68-73). In addition, in the case of mass production of the F gene-deleted Sendai virus described below, cells resistant to trypsin (for example, LLC-MK2 cells) are preferred.

An F gene-deleted Sendai virus vector can be constructed and prepared as follows (see WO00/70055 and WO 00/70070).

1. Construction of F Gene-deleted Sendai Virus genomic cDNA containing foreign Gene

The full-length cDNA of SeV, pSeV18, was digested with SphI/KpnI⁺b (+) (Hasan, M.K. et al, J.Gen.Virol.78, 2813-2820, 1997) ("pSeV 18)⁺b (+) "also called" pSeV18⁺"), the digested fragment (14673bp) was recovered. The digested fragments were cloned into pUC18 to obtain plasmid pUC18/KS, and then PCR-ligation was performed on this pUC18/KS in combination to construct F gene deletion site, F gene ORF (ATG-TGA. RTM. 1698bp) was removed, and the gap was filled with atgcatgccggcagatga (SEQ ID NO: 4), thereby constructing F gene-deleted SeV genomic cDNA (pSeV 18)⁺,/Δ F). In the PCR, the following primer pairs were used: upstream of the F gene: forward direction: 5' -gttgagatactgcaagagc (sequence number: 5), reverse: 5' -tttgccggcatgcatgtttcccaaggggagagaagtttttgcaacc (SEQ ID NO: 6); downstream of the F gene: forward direction: 5' -atgcatgccggcagatga (SEQ ID NO: 7), reverse: 5' -tggggtgaatgagaagaatcagc (SEQ ID NO: 8). The resulting PCR products were ligated with EcoT 22I. The plasmid obtained as described above was digested with SacI and SalI, and a fragment (4931bp) containing the F gene deletion site was recovered and cloned into pUC18 to obtain pUC 18/dFSS. The resulting pUC18/dFSS was digested with DraIII, and the fragments were recovered using pSeV18⁺The fragment containing DraIII in the F gene region was replaced, and the resulting plasmid pSeV18 was obtained⁺/ΔF。

The EIS sequence of the F gene (SeV-specific sequence, E, end; I, intergenic; S, start) remains in the above structure. Even if the downstream ORF of the F gene is removed, it is still possible for this structure to express the pentapeptide derived from the primer for the junction gap.

The foreign gene was inserted into the F gene deletion site using the NsiI and NgoMIV restriction enzyme sites of the F gene deletion site in pUC 18/dFSS. For this purpose, for example, a foreign gene fragment can be amplified using a primer ending with NsiI and a primer ending with NgoMIV.

For example, the EGFP gene was first amplified by PCR to construct cDNA containing the EGFP gene (pSeV 18)⁺/. DELTA.F-GFP). In order to make the number of nucleotides in the EGFP gene fragment 6-fold (Hausmann, S. et al, RNA 2, 1033-1045, 1996), PCR was carried out using a 5 '-primer (5' -atgcatatggtgatgcggttttggcagtac/SEQ ID NO: 9) having NsiI-terminated ends and a 3 '-primer (5' -tgccggctattattacttgtacagctcgtc/SEQ ID NO: 10) having NgoMIV-terminated ends. The PCR product was digested with NsiI and NgoMIV restriction enzymes, fragments recovered from the gel, ligated to the F gene deletion site in pUC18/dFSS using NsiI and NgoMIV restriction enzyme sites, and sequenced. Recovering DraIII fragment containing EGFP gene using pSeV18⁺The DraIII fragment containing the F gene region in the/. DELTA.F was substituted and ligated to obtain plasmid pSeV18⁺/ΔF-GFP。

Using pSeV18⁺/. DELTA.F or pSeV18⁺Insertion of foreign Gene into upstream of NP Gene at restriction enzyme NotI recognition site in/. DELTA.F-GFP. However, when pSeV18 was used⁺At/. DELTA.F, it is possible to express a pentapeptide derived from a primer linked to the deletion site of the F gene; when using pSeV18⁺GFP co-expression at/. DELTA.F-GFP. Therefore, if necessary, a gene construct not expressing the above-mentioned peptide and GFP was constructed by the following method:

digestion of pSeV18 with SalI and NheI⁺[ DELTA ] F-GFP, a fragment containing the F gene deletion site (6288bp) was recovered and cloned into Litmus38(New England Biolabs, Beverly, Mass.) to obtain Litmus SalINheifrg/. DELTA.F-GFP. EGFP containing EIS sequences upstream of the F-deleted gene was deleted by inverse PCR. That is, PCR was carried out using a reverse primer (5 '-gtttaccaggtggagagttttgcaaccaagcac/SEQ ID NO: 11) containing a recognition sequence for SexAI restriction enzymes at the upstream of the GEP gene and a forward primer (5' -ctttcacctggtacaagcacagatcatggatgg/SEQ ID NO: 12) containing a recognition sequence for SexAI restriction enzymes at the downstream of the GEP gene. A fragment of the desired size (10855bp) was excised and ligated to delete EGFP containing the EIS sequence upstream of the F-deleted gene.

However, due to the design of the primers, the resulting construct inserted an extra 15bp sequence between the two SexAI sites, and thus, E.coli SCS110 strain (dcm) was transformed^-/dam^-SCS110 strain, in which SexAI was methylated and therefore could not be digested), was prepared, and after digesting the plasmid with SexAI restriction enzyme, 1628bp and 9219bp gene fragments were recovered and ligated, and the extra 15bp sequence was removed, to construct litmus salinheifrg/Δ F (Δ 5aa), in which the EGFP gene including the EIS sequence upstream of the F gene was deleted, and the deleted region contained 6-fold nucleotides. Digesting the plasmid with SalI and NheI, recovering the fragment, and digesting the fragment with a plasmid containing pSeV18⁺Replacing and connecting SalI/DraIII fragment of middle F gene region to obtain plasmid pSeV18⁺/ΔF(Δ5aa)。

A foreign gene is inserted into the plasmid using, for example, a NotI restriction enzyme recognition sequence located upstream of the NP gene.

2. Construction of F Gene-deleted Sendai Virus genomic cDNA containing hFGF2 Gene

Various methods for obtaining human FGF2(hFGF2) cDNA are known, for example, by taking vascular smooth muscle cells from the great saphenous vein under conditions in which the patient's own consent was obtained, isolating cDNA therefrom by RT-PCR, and subcloning it into HindIII (5 ' -terminus) and EcoRI (3 ' -terminus) of pBluescriptSK + (Stratagene, La Jolla, Calif.), thereby preparing FGF2(hFGF2) cDNA. The sequence of the hFGF2 cDNA was determined in accordance with the sequence reported by Abraham et al (Abraham, J.A., et al, EMBO J.5(10), 2523-2528, 1986).

In order to insert the hFGF2 gene into the NotI restriction enzyme site upstream of the NP gene, a SeV specific sequence (EIS sequence) was added to the 3' -end of the hFGF2 gene, and a fragment having NotI recognition sequences at both ends was prepared. Specifically, the hFGF2 cDNA was used as a template, PCR was performed using an N-terminal primer (5 '-atccgcggccgccaaagttcacttatggcagccgggagcatcaccacgctgcccgccttgcccgaggatggcggcagcggcgcc/SEQ ID NO: 13) containing an initiation codon and a C-terminal primer (5' -atccgcggccgcgatgaactttcaccctaagtttttcttactacggtcagctcttagcagacattggaagaaaaagtatagc/SEQ ID NO: 14) containing a termination codon region and an EIS sequence, and the amplified fragment was digested with NotI and subcloned into the NotI site of pBluescriptSK + (Stratagene, La Jolla, Calif.), yielding pBS-hFGF 2. After determination of the nucleotide sequence, when there is a variation in the resulting gene, for example, QuickChange^TMThe Site-directed Mutagenesis Kit (Stratagene, La Jolla, Calif.) was corrected to the desired sequence by the method described in the Kit. After digestion of pBS-hFGF2 with NotI, a fragment containing hFGF2 cDNA was inserted into pSeV18⁺Construction of F-deleted Virus genomic cDNA pSeV18 comprising hFGF2 Gene at NotI site upstream of NP Gene in/. DELTA.F (5aa)⁺ hFGF2/ΔF(Δ5aa)。pSeV18⁺hFGF 2/. DELTA.F (. DELTA.5 aa) hereinafter also referred to as pSeV18⁺hFGF2/ΔF。

3. Construction of F expression plasmid

The Sendai virus F gene (SeV-F) can be expressed using a plasmid pCALNdLw (Cre/loxP inducible expression vector; Arai, T. et al, J.Virol.72(2), 1115 1121, 1998) designed so that the expression of the gene product can be induced by Cre DNA recombinase. pUC18/KS was digested with StyI and BstUI, and the fragment (1783bp) containing the SeV-F gene was excised, blunt-ended, and inserted into the specific SwaI site of pCALNdLw to construct an F-expressing plasmid pCALNdLw/F.

4. Preparation of helper cells for inducible expression of SeV-F protein

For example, LLC-MK2, a monkey kidney cell line commonly used for SeV amplification, can be used to establish a helper cell line expressing SeV-F protein and to recover infectious virions from the F gene-deleted genome. At 37 ℃ and 5% CO₂LLC-MK2 cells were cultured in MEM containing the following: heat treatment at 10% inactivated Fetal Bovine Serum (FBS), 50U/ml penicillin G sodium and 50. mu.g/ml streptomycin. The plasmid pCALNdLw/F described above, which was designed so that the expression of the F gene product could be induced by Cre DNA recombinase, was introduced into LLC-MK2 cells by the calcium phosphate method (Mammalian Transfection Kit; Stratagene, La Jolla, Calif.) according to a known protocol.

Specifically, for example, 10. mu.g of plasmid pCALNdLw/F was introduced into LLC-MK2 cells cultured in 10cm dishes to 40% confluency, followed by 10ml of 10% FBS-containing MEM medium at 37 ℃ and 5% CO₂Was cultured in an incubator for 24 hours. After 24 hours, the cells were detached, suspended in 10ml of a medium, and seeded into five 10ml culture dishes, for example, one 5ml dish, two 2ml dishes, and two 0.2ml dishes, and the stable gene-introduced strain was selected by culturing for 14 days in 10ml MEM medium containing G418(Gibco-BRL, Rockville, Md.) 1,200. mu.g/m and 10% FBS, and replacing the medium every two days. For example, 30 strains of G418-resistant cells propagated in the above medium are recovered using cloning rings. Each clone was expanded to confluency in a 10cm dish.

The method for selecting F gene stably introduced strain was as follows: expression of the F protein can be semi-quantitatively analyzed by western blotting. Cells were expanded to confluence in 6cm dishes and F protein expression was induced in each clone by infecting adenovirus AxCANCre with moi-3 multiplicity of infection as per Saito et al (Saito et al, Nucl. acids Res.23, 3816-3821, 1995; Arai, T. et al, J.Virol.72(2), 1115-1121, 1998). After 3 days of infection, the culture supernatant was removed, the cells were washed twice with PBS, and the cells were scraped off with a scraper toAfter centrifugation at 1500Xg for 5 minutes, the cells were recovered, stored at-80 ℃, thawed, suspended in 150. mu.l of PBS buffer, added with an equal amount of 2 XTTris-SDS-BME loading buffer (0.625M Tris (pH6.8), 5% SDS, 25% 2-ME, 50% glycerol and 0.025% BPB, obtained by Owl), heated at 98 ℃ for 3 minutes, and subjected to electrophoresis. The samples were run according to a known protocol (1X 10)⁵Cells/lane) were subjected to SDS-polyacrylamide gel electrophoresis and western blotting. SeV-F expression levels of individual clones were semi-quantitatively analyzed in Western blotting using an anti-SeV-F (F236) antibody diluted at a ratio of 1: 1000 as a primary antibody.

The establishment of LLC-MK2 cells that induce expression of the SeV-F gene product was confirmed by the above method. The cells before the SeV-F gene is induced to express are referred to as LLC-MK2/F, and the cells after the induction of the SeV-F gene are referred to as LLC-MK 2/F/Ad.

5. Reconstitution and amplification of F Gene-deleted SeV Virus

F gene-deleted Sendai virus genomic cDNA carrying an angiogenic gene is transfected into F gene-expressing helper cells and expressed, whereby the virus can be reconstituted. For example, when the above-mentioned Sendai virus genomic cDNA (pSeV 18) carrying the F gene deletion type of hFGF2 gene is used⁺hFGF 2/. DELTA.F), the cDNA was transfected into LLC-MK2 cells as follows. LLC-MK2 cells at 5X 10⁶The concentration of each cell/dish was seeded in a Petri dish with a diameter of 10cm, and after 24 hours of culture, recombinant vaccinia virus expressing T7 RNA polymerase (Fuerst, t.r. et al, proc. natl. acad. sci. usa 83, 8122-. UV irradiation of vaccinia virus is carried out, for example, with a UVStratalinker 2400 (catalog number 400676(100V), Stratagene, La Jolla, Calif., USA) equipped with 5 bulbs of 15 watts. After washing the cells twice, plasmid pSeV18 was added⁺hFGF2/Δ F, pGEM/NP, pGEM/P, pGEM/L (Kato, A., et al, Genes Cells1, 569-579, 1996) and pGEM/F-HN (WO00/70070) were suspended in OptiMEM (GIBCO) in amounts of 12 μ g, 4 μ g, 2 μ g, 4 μ g and 4 μ g per dish, respectively, and addedSuperFect transfection reagent (1. mu.g DNA/5. mu.l SuperFect, QIAGEN) was mixed, and after standing at room temperature for 15 minutes, 3ml OptiMEM containing 3% FBS was finally added. The resulting mixture was added to cells, cultured for 3 to 5 hours, and then the cells were washed twice with serum-free MEM, and then cultured for 24 hours in serum-free MEM containing 40. mu.g/ml cytosine-. beta. -D-arabinofuranoside (AraC, Sigma) and 7.5. mu.g/ml trypsin (GIBCO).

The culture supernatant was removed, and LLC-MK2/F/Ad cells, which were F gene-expressing helper cells cloned as described above, were layered on the cell top. Specifically, LLC-MK2/F/Ad dispersed in serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) was overlaid on the cells from which the culture supernatant was removed and culture was continued for 48 hours. The cells were recovered with a scraper and the particles were suspended in OptiMEM (10)⁷Individual cells/ml) and freeze-thaw repeated three times. Freeze-thaw lysates (200. mu.l/well) were added to the upper layer of LLC-MK2/F/Ad cells (in 12-well plates, 4X 10⁶One cell/well), 300. mu.l/well of serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) was added thereto, and the mixture was cultured for 15 to 24 hours. The culture supernatant was removed, the cells were washed with serum-free MEM, fresh serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) was added thereto, and the culture was carried out for 5 to 9 days, and the supernatant was collected. The resulting supernatant containing the reconstituted F gene-deficient SeV particles was collected, LLC-MK2/F/Ad cells were infected with the F gene-deficient SeV particles, and the F gene-deficient SeV cells were amplified by culturing in serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) in the same manner as described above (or repeating this procedure).

At this time, the culture supernatant containing F gene-deleted SeV particles was repeatedly filtered twice with a 0.22 μm filter, so that contamination with a recombinant vaccinia virus expressing T7 RNA polymerase during reconstitution could be substantially avoided. Specifically, the culture supernatant (sample after P2) amplified twice or more with AraC-containing serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) was filtered twice with a 0.22 μm filter and amplified once with AraC-containing serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) to obtain F gene-deleted SeV, which was used as SeV without recombinant vaccinia virus contamination.

When preparing defective viral vectors, for example, if two vectors defective in different envelope genes on their genomes are introduced into the same cell, each missing envelope protein is provided by expression of the other vector, and this complementation results in the production of infectious viral particles that can replicate and propagate. That is, if two or more envelope proteins complementary to the viral vector of the present invention are simultaneously inoculated in a mixed manner, a large amount of a mixture of various envelope gene-deficient viral vectors can be produced at a low cost. Since these viruses defective in envelope genes have a smaller genome, they allow insertion of a longer foreign gene. In addition, these viruses, which do not have the ability to infect themselves, are difficult to maintain in a co-infected state after extracellular dilution, and therefore they do not produce progeny and are less harmful to the environment.

If a viral vector is prepared using a gene for treating a disease as a foreign gene, the vector can be administered to carry out gene therapy. When the viral vector of the present invention is used for gene therapy, foreign genes having a desired therapeutic effect or endogenous genes whose supply in the body of a patient is insufficient can be expressed by direct administration or indirect (ex vivo) administration. The exogenous gene used herein is not particularly limited, and includes not only a nucleic acid encoding a protein but also a nucleic acid not encoding a protein, such as an antisense nucleic acid or ribozyme of a gene inhibiting angiogenesis.

The recovered paramyxovirus may be purified to be substantially pure. The purification method includes known purification and separation methods such as filtration, centrifugation, and column purification, or a combination thereof. "substantially pure" means that the virus is a main component of a sample in which the virus is present, and in general, a substantially pure virus vector can be identified when the proportion of proteins derived from the virus vector in the total proteins contained in the sample is 50% or more, preferably 70% or more, more preferably 80% or more, and still more preferably 90% or more. Specific purification methods of paramyxovirus include a method using cellulose sulfate ester or cross-linked polysaccharide sulfate ester (Japanese patent publication (Kokoku) Nos. 62-30752, 62-33879, and 62-30753) and a method of adsorbing fucose sulfuric acid-containing polysaccharide and/or its decomposition product (WO 97/32010).

The paramyxovirus vector of the present invention may constitute a composition together with a desired pharmaceutically acceptable carrier. Herein, "pharmaceutically acceptable carrier" refers to a substance that can be administered together with a carrier but does not inhibit gene transfer by the carrier. For example, the paramyxovirus vector of the present invention can be appropriately diluted with physiological saline, Phosphate Buffered Saline (PBS), or the like to prepare a composition. When the paramyxovirus vector of the invention is propagated in chicken eggs, the composition may contain allantoic fluid. The composition may further comprise a medium such as deionized water or a 5% aqueous glucose solution, and further, may further comprise a stabilizer, an antibiotic, and the like. The present invention also provides a method of preparing an angiogenic composition of the invention comprising the step of combining a carrier of the invention and a pharmaceutically acceptable carrier. In addition, the present invention relates to the use of a vector of the present invention for the preparation of an angiogenic composition of the present invention. The compositions of the present invention may also be used as pharmaceutical compositions. The present invention also relates to an ischemia therapeutic agent comprising the vector of the present invention and a pharmaceutically acceptable carrier, and the use of the vector of the present invention or the composition as a pharmaceutical.

The angiogenesis gene carried by paramyxovirus can be introduced by administering the paramyxovirus vector produced by the above-described method or a composition containing the vector. The present invention provides a method of inducing angiogenesis, which comprises administering the paramyxovirus vector of the present invention or the angiogenic composition of the present invention. The method is particularly effective for treating ischemic tissue. The site of administration is not particularly limited, and it is preferable to administer the agent locally to or around the ischemic tissue so that the transgene product is concentrated in the ischemic tissue during the ischemic treatment without leaking to the systemic circulatory system, or to express locally around the target tissue by a suitable gene delivery system. For example, gene delivery can be achieved by directly administering a composition containing the paramyxovirus vector of the present invention from the inside or outside of the ischemic tissue to express the foreign gene in the ischemic tissue. Alternatively, the administration may be indirect, for example, by injecting paramyxovirus vector-infected cells encoding an angiogenesis gene into ischemic tissue or into an artery that flows through the ischemic tissue.

Alternatively, local administration by catheter may be used. For example, the vector of the present invention may be administered by a double balloon catheter method in which a blood vessel is isolated by two balloons and the vector composition is injected into the isolated vascular space, or by a porous balloon administration method or the like (Jorgensen, B. et al, Lancet 1 (8647): 1106-8, 1989; Wolinsky, H. and Thung, S.N., J.Am Coll.Cardiol.15 (2): 475-81, 1990; WO 93/00051; WO 93/00052). In the above administration method, a hydrogel-coated balloon (Takeshita, S. et al, Lab. invest.75 (4): 487-501, 1996) can be used.

For example, in the treatment of myocardial infarction, angina pectoris, and other ischemic heart diseases, the carrier composition of the present invention can be directly injected into the myocardium via the intraventricular cavity using a catheter. In addition, angiogenesis and collateral circulation in the stenosed part of the coronary artery can be promoted by local injection of the vector of the present invention with a catheter.

However, the longer incubation time required to administer the vector via a catheter may cause vascular damage to the balloon, and it is often difficult to insert a catheter into a disseminated blood vessel of ischemic tissue. In the present invention, Intramuscular (IM) administration of the vector is particularly preferable in the treatment of ischemic tissue. Intramuscular injection is simpler than administration via a catheter and poses less risk of vascular injury. The vectors of the invention are administered, for example, to striated muscles, including skeletal muscles and cardiac muscles, in or adjacent to ischemic tissue. Bupivacaine may also be administered prior to administration of the viral vector, and is known to enhance expression of the transgene by inducing muscle regeneration. Further, Intradermal (ID) administration may also be selected. Methods for introducing the vector into muscle include percutaneous introduction, direct percutaneous introduction, and the like. When introducing the vector, measures must be taken to ensure that the extramuscular membrane is not damaged. Administration can be by needle and syringe, or by a biological syringe without a needle. Administration may be at one or more sites, and the number of administrations may be one or more.

The vectors of the present invention are also effective when administered in a matrix form. In the known method, the viral vector is dispersed in an arterial collagen (aterocollagen) matrix, the resulting mixture is solidified by freeze-drying, and then the matrix is slowly disintegrated. It has been reported that the above method can sustain the effect of transiently expressed adenovirus vectors or naked DNA (Ochida, T. et al, Nature Medicine 5, 707-710, 1999). The viral vector of the present invention may be formulated together with such an adjuvant, or may be lyophilized. In addition, lipid cations (lipid sites) may be added to enhance expression.

It is known that even a small amount of the matrix can slowly release growth factors and the like through an injection needle of about 18G over a long period of time. For example, in a protein preparation, the duration of growth hormone or the like in blood is longer, for example, 7 days or more, than when growth hormone or the like is given alone. It has been reported that the effect usually lasts for 10 days or more (JP 10-001440). Thereby reducing the number of administrations and the pain of the patient. The preparation can be used as solid injection (implant, etc.) or mucosa absorbent such as suppository, etc. for subcutaneous or intramuscular administration. Solid injections are usually in the form of a column or granules which can be administered through a syringe needle. Preferred shapes of the preparation include columnar shapes such as a square column and a cylinder, and granular shapes such as a sphere.

The non-oral formulation of the present invention may be sized according to the mode of administration, so long as it does not cause undue pain to the patient. When the injection is composed of a columnar matrix, the injection has a diameter of 3mm or less (e.g., 0.1 to 3mm) and a length of 30mm or less (e.g., 0.5 to 30mm), and preferably has a size such that it can be administered with a 14G or less or 14G injection needle having a diameter of 1.3mm or less (e.g., 0.1 to 1.2mm) and a length of 20mm or less (e.g., 0.5 to 20mm), more preferably has a diameter of 0.1 to 1mm and a length of 1 to 20mm, and is preferably columnar. The maximum diameter of the injection composed of the granular matrix is 1mm or less (e.g., 0.1 μm to 1mm), preferably 150 μm or less (e.g., 0.5 to 100 μm), and more preferably 1 to 100 μm. The weight of the base is selected according to the shape of the preparation, and the weight of the base for injection is usually 40mg or less than 40mg, preferably 1 to 25 mg.

The type of gene to be introduced by the paramyxovirus vector of the present invention is not particularly limited as long as it is a gene that promotes angiogenesis and/or vascularization. For example, genes encoding the above-mentioned aFGF, FGF2(bFGF), VEGF, Ang (including Ang-1 and Ang-2), EGF, TGF- α, TGF- β, PD-ECGF, PDGF, TNF- α, HGF, IGF, EPO, CSF, M-CSF, GM-CSF, IL-8 and NOS. These proteins include each member of the respective family and the isoform (isoform). A particularly suitable example of an angiogenic gene introduced using the paramyxovirus vector of the invention is the gene encoding FGF 2. FGF2 is expected to be useful in the treatment of acute ischemia, for example, it may have significant efficacy in the treatment of acute critical limb ischemia and the like. FGF2 is also effective in treating myocardial infarction (Yanagisawa-Miwa, A. et al., Science 257 (5075): 1401-3, 1992). The protein may be a secreted protein, a transmembrane protein, a cytoplasmic protein, a nucleoprotein, etc., preferably a secreted protein, or may be an artificially constructed protein including a fusion protein with other proteins, a dominant negative protein (including a soluble molecule of a receptor and a membrane-bound dominant negative receptor), a defective cell adhesion molecule, a cell surface molecule, etc. In addition, the protein may be a protein having a secretion signal, a membrane localization signal, a nuclear translocation signal, or the like. The introduced gene may be a gene whose expression is endogenously induced in ischemic tissues or a gene whose expression is not induced may be expressed at a different site. In addition, the function of an unwanted gene expressed in ischemic tissues can be inhibited by expressing an antisense RNA molecule or an RNA-cleaving ribozyme.

The vector of the present invention is expected to be applicable to gene therapy for various ischemic diseases and diseases treatable by angiogenesis. Such gene therapy includes treatment of ischemia caused by blood vessel cutting, infarction, blood flow interruption due to blood vessel separation, or the like. Ischemic diseases that can be treated with the vectors of the present invention include cerebrovascular ischemia, renal ischemia, pulmonary ischemia, limb ischemia, ischemic cardiomyopathy, myocardial ischemia, and the like. The tissue for gene therapy is not particularly limited and includes muscle, brain, kidney and lung. In addition, gene therapy can promote angiogenesis in transplantation, and is also useful for creating various disease models and for developing and evaluating therapeutic methods for disease models.

The promoting effect of angiogenesis by using a carrier can be determined by measuring the number and density of capillaries in a biopsy sample, analyzing an angiographic image, and the like, and also by blood flow analysis using doppler perfusion image analysis. The treatment effect of the ischemic tissue can be confirmed by visual observation or tissue section microscope observation of the necrosis or exfoliation of the tissue.

Administering a pharmaceutically effective amount of the paramyxovirus vector of the invention to a target tissue, thereby introducing the vector into cells of the target tissue. "pharmaceutically effective amount" refers to the amount of gene introduced into the cells of the target tissue at least partially to produce the desired therapeutic or prophylactic effect. By administering an effective amount of the paramyxovirus vector of the invention carrying a desired angiogenic gene, cells into which the vector is introduced produce an angiogenic factor. Preferably, significant levels of angiogenic factors are detected in the administered tissue by administering an effective amount of a paramyxovirus vector of the invention carrying the desired angiogenic gene. The "significant level" means that the expression level (the amount of a transcription product or a translation product) of a gene introduced by the vector of the present invention can be detected. For example, when a transgene's corresponding endogenous gene is present, the maximum expression level of the transgene is meant to be significantly elevated compared to the endogenous gene expression level. Preferably, the amount of expression of the angiogenic gene at the site of administration is about 1.2 times, preferably about 1.5 times, more preferably about 2 times, further preferably about 10 times or more than 10 times, and most preferably about 20 times or more than 20 times the amount of expression of the endogenous gene. However, the expression amount of the transgene should be determined in consideration of its effective expression amount and the level of intoxication.

The expression level of a transgene in a cell can be determined by methods known to those skilled in the art. For example, the transcription product of a gene can be detected by Northern hybridization, reverse transcription-polymerase chain reaction (RT-PCR), or RNA protection assay. Detection by Northern hybridization or RT-PCR can also be carried out in situ. The detection of the translation product can be performed as follows: western blotting, immunoprecipitation, RIA, enzyme-linked immunosorbent assay (ELISA), pull-down assay, etc. were performed using the antibody. To facilitate detection of the transgene expression product, the protein to be expressed may be tagged or a reporter gene may be inserted to ensure its expression. Reporter genes include, but are not limited to, genes encoding beta-galactosidase, Chloramphenicol Acetyltransferase (CAT), alkaline phosphatase, and Green Fluorescent Protein (GFP), among others.

The amount of the vector to be used may vary depending on the disease, the body weight, age, sex, symptoms of the patient, the purpose of administration, the kind of the gene to be introduced, etc., and may be appropriately determined by those skilled in the art. Preferably, the carrier comprises a pharmaceutically acceptable carrier and is administered in an amount of about 10⁵Cell Infection Unit (CIU)/ml-10¹¹CIU/ml, more preferably about 10⁷CIU/ml～10⁹CIU/ml, most preferably about 1X 10⁸CIU/ml～5×10⁸CIU/ml. Compositions comprising the viruses of the present invention can be administered to subjects including all mammals, including humans, monkeys, mice, rats, rabbits, sheep, cattle, and dogs, among others.

The dose for human administration is usually preferably 2X 10 per administration site⁸CIU～2×10¹⁰CIU, more preferably 2X 10⁹Quantity of about CIU, e.g. 5X 10⁸CIU～1×10¹⁰CIU, and the like. The number of administrations is one or more within the range of clinically acceptable side effects. The same applies to the number of administrations in one day. The number of administration sites can be increased or decreased or the amount of administration can be calculated for animals other than humans depending on the weight ratio or volume ratio of the administration site (ischemic tissue, etc.) to the body of the animal to be administered.

Brief Description of Drawings

Figure 1 shows the surgical procedure for moderate (left) and severe (right) acute hind limb ischemia in mice. The branch lines indicate the arteries and veins of the hind limb, and the thick lines indicate the sites of vascular resection.

FIG. 2 shows the expression of endogenous VEGF (filled) and FGF2 (open) in muscle (left panel) and serum (right panel) during hind limb ischemia. The ELISA was performed using a model of moderate and severe ischemia in C57BL/6 mice, and two days after surgery on the whole thigh muscle and gastrocnemius muscle (n-6) and serum (n-6). The result being muscular respectivelyThe total extracted protein or volume was normalized and expressed as mean ± s.d. The muscle data includes two sets of data for the thigh muscle and the gastrocnemius muscle (so each set is n-12). The average values are shown in the figure.^*P < 0.01, # P < 0.05 (one-way ANOVA).

FIG. 3 is a photograph of ischemia-induced VEGF expression detected by RT-PCR.

FIG. 4 shows the expression level (left panel) and the change with time (right panel) of the luciferase gene introduced into muscle by SeV. The untreated group, pCMV-luc (100. mu.g) group and SeV-luc (10. mu.g) were studied in a C57BL/6 mouse moderate ischemia model⁷pfu or 10⁸pfu) group (left panel). The right graph shows the change over time in the expression level of the luciferase gene introduced into the moderate ischemia model. Open circles to the group of C57BL/6 mice with pCMV-luc (100. mu.g), filled circles to SeV-luc (10)⁸pfu) C57BL/6 mice, shaded dots to SeV-luc (10)⁸pfu) with bold lines indicating cutoff values, above which significant transgene expression was indicated. The same log scale was used for gene expression levels in each figure.

FIG. 5 shows the results of measuring the in vitro secretion level of angiogenic proteins using Human Umbilical Vein Endothelial Cells (HUVEC), COS7 cells, bovine vascular smooth muscle cells (BSMC), and myocardial myoblasts (H9C 2). "basal release" means the amount of each factor produced in the absence of a carrier. By "cut-off value" is meant that the portion above which the transgene expression level is significant.

FIG. 6 shows the in vivo expression of FGF2(a) and VEGF (b) genes in muscle (left) and serum (right) exogenously introduced into moderate ischemic C57BL/6 mice. Immediately after the operation, 50. mu.l each of the carrier solutions was injected into the thigh muscle and the gastrocnemius muscle. Two days after the surgery, all thigh muscles and gastrocnemius muscles (n ═ 6) and serum (n ═ 6) were collected, and ELISA with mouse FGF2(a) and ELISA with mouse and human vegf (b), respectively. Results were normalized to total extracted protein or volume of muscle, respectively, expressed as mean ± s.d. The average values are shown in the figure. The scale is a log scale.

FIG. 7 is a graph showing the enhancement of the expression of endogenous murine VEGF mediated by means of gene transfer. I.e., enhancement of endogenous mouse VEGF expression mediated by gene transfer in limb muscles of C57BL/6 mice without surgery (left), moderate ischemia (middle), and severe ischemia (right). Immediately after the operation, 50. mu.l each of the carrier solutions was injected into the thigh muscle and the gastrocnemius muscle. Two days after the surgery, all thigh muscles and gastrocnemius muscles and serum (each group: n-6, all groups: n-12) were collected and subjected to ELISA for murine VEGF. Results were normalized to total extracted protein of muscle, expressed as mean ± s.d. The average values are shown in the figure.^*P < 0.01, # P < 0.05 (one-way ANOVA).

FIG. 8 is a photograph of a limb muscle tissue image of a gene-introduced mouse. The C57BL/6 mice were subjected to severe ischemic surgery and then treated as described in FIG. 7, and two days after surgery were histologically observed. Significant inflammatory infiltration and stromal cell edema were found in the thigh muscle (upper right) of mock-dosed (SeV-luciferase, mock) mice compared to untreated mice (upper left, non-ischemic). Severe muscle fiber injury, intracellular edema and inflammatory infiltration were observed in VEGF 165-treated mice (bottom left, VEGF165), and the introduction of FGF2 gene (bottom right, FGF2) inhibited the injury. The same results were obtained with 6 mice per group. Hematoxylin-eosin staining, magnification x 200.

FIG. 9 is a photograph showing the therapeutic or aggravating effect of the introduction of an exogenous angiogenic factor gene into severely ischemic muscles 10 days after left hind limb surgery in mice. Each figure shows the Limb Salvage Score (LSS). The upper half shows the typical exacerbation effect in the C57BL/6 mouse model of critical ischemia (limb salvage model), with complete detachment of the hind limb of the mice introduced with VEGF165 (upper middle panel), and limb salvage in the luciferase control group (upper left panel) and FGF2 treated group (upper right panel). The lower half shows typical efficacy in a BALB/c nu/nu mouse model of severe ischemia (auto-architecture model). Limbs were rescued in FGF 2-treated groups (lower right panel), and almost all hind limbs were detached in luciferase control groups (lower left panel) and VEGF 165-treated groups (lower right panel).

Fig. 10 is a graph showing the prognosis of hind limbs after administration of vehicle in the limb salvage model and the hind limb amputation model, showing the ratio of animal individuals in which hind limbs were retained after administration of vehicle (limb salvage rate). After introduction of the angiogenic gene into muscle, panel A shows the worsening effect of VEGF165 in the C57BL/6 mouse model of severe ischemia (salvage model), and panel B shows the therapeutic effect of FGF2 in the BALB/C nu/nu mouse model of severe ischemia (hind limb amputation model). Three independent experiments were performed for each group (n ═ 10). The curve was plotted by the Kaplen-Mayer method, data were analyzed by log-rank test,^*P＜0.0001。

FIG. 11 is a photograph showing the effect of angiogenesis in vivo measured by laser Doppler perfusion image analysis using a C57BL/6 mouse model of critical ischemia (limb salvage model). Mice were treated with 10⁷The amount of pfu was given to SeV-luciferase, SeV-VEGF165 and SeV-FGF2, and restoration of blood perfusion was observed. Each group showed the change over time of the same individual. The upper panel shows typical results of the change in blood flow with time in mice treated with SeV-luciferase (mock transfection). Blood reperfusion of the thigh muscles was observed 4 days after the operation, evident on day 7, but also no evident perfusion of the lower leg was detected on day 10, with consequent limb atrophy and signs of toe necrosis (right-most panel). The middle panel shows typical temporal changes in SeV-VEGF 165-treated mice. During the observation period, no significant reperfusion was found in the thigh and calf, with consequent limb loss (rightmost panel). The lower panel shows typical temporal changes in SeV-FGF 2-treated mice. Significant reperfusion of the thigh began on day 4 and significant blood flow was found throughout the limb beginning on day 10 with complete rescue of the limb (right-most panel).

FIG. 12 shows the recovery of blood perfusion by angiogenic gene therapy in the C57BL/6 mouse model of critical ischemia (limb salvage model). The average blood flow of the ischemic and non-ischemic limbs was calculated as the ratio of the left limb (ischemic limb) blood flow value/right limb (non-ischemic limb) blood flow value by performing the experiment in the same manner as described in fig. 11.^*P < 0.001 (compared with all other groups), # P < 0.05 (compared with all other groups), and # P < 0.05 (compared with the mock-administered group)]。

FIG. 13 shows the results of the chronological changes in the salvage rate in the mouse model of severe ischemia (limb drop model) in which a replicative SeV vector carrying hFGF2 gene or a F gene-deficient SeV vector carrying hFGF2 gene was administered. The number of test subjects (n) and the amount of vector administered are shown in the figure.

Best mode for carrying out the invention

The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples. In addition, the documents cited in the present specification are incorporated herein by reference in their entirety.

Recombinant SeV was prepared according to the methods described in the prior art (Yu, D.et al, Genes Cells 2 (7): 457-66, 1997; Yonemittsu, Y., et al, Nature Biotech.18, 970-.

Viral titers were determined by hemagglutination assay of chicken erythrocytes. High titer stock solution (10)⁹pfu/ml) was stored at-80 ℃ until use. Human VEGF165 cDNA was isolated by RT-PCR according to the methods described in the prior art (Yonemittsu, Y., et al, Lab. invest.75, 313-323 (1996)). Full-length FGF2 cDNA was prepared by PCR on the basis of the partial sequence of cDNA provided by Dr.village (National Institute of Bioscience and Human-Technology, Tsukuba, Japan) (Imamura, T., et al, Science 249, 1567-. Specifically, a fragment of mouse FGF2 cDNA (a fragment of nucleotides 7 to 435 in the nucleic acid having accession No. M30644) lacking the initiation codon region and the termination codon region was used as a template, and an N-terminal primer (5'-ACGTGCGGCCGCCAAAGTTCATCCACCATGGCTGCCAGCGGCATCACCTCGCTTCCC-3'/SEQ ID NO: 15) containing the initiation codon of mouse FGF2 cDNA and a C-terminal primer (5'-ACGTGCGGCCGCGATGAACTTTCACCCTAAGTTTTTCTTACTACGCGGATCAGCTCTTAGCAGACATTGGAAGAAACAGTATGGCCTTCTGTCCAGGTCCCGT-3'/SEQ ID NO: 16) containing the termination codon region and a SeV-specific sequence were used to amplify the full-length cDNA. In the determination of the methodAfter the nucleotide sequences of the obtained human VEGF165 cDNA and mouse FGF2 cDNA were correct, they were inserted into pSeV18⁺b (+) (Hasan, M.K., et al, 1997, J.general Virology 78: 2813-2820). Sendai virus vectors expressing human VEGF165 or mouse FGF2 were designated SeV-VEGF165 or SeV-FGF2, respectively. SeV-luciferase (Hasan, M.K., et al, J.Gen.Virol.78(Pt 11): 2813-2820, 1997; Yonemittsu, Y.et al, Nature Biotechnol.18: 970-973, 2000) and pCMV-luciferase (Yonemittsu, Y.et al, Nature Biotechnol.18: 970-973, 2000) were prepared as described above.

In statistical analysis, all data for the inventive examples are presented as mean ± s.d. Data from outside the salvage were analyzed by one-way anova with Scheffe correction. The salvage ratio was expressed as the salvage score (LSS) and the salvage data was analyzed by the Kaplen-Mayer method. Statistical significance of the salvage experiments was determined using the log-rank test, and in all statistical analyses, p < 0.05 was considered as a significant statistical difference.

[ example 1]Ischemia-induced endogenous VEGF expression does not cause local proteins in hindlimb muscle Accumulation of

To clarify the therapeutic or detrimental effects of angiogenic factors, the inventors constructed three limb ischemia models by two different procedures (fig. 1). Namely: (1) c57BL/6 mouse thigh-wide arteriovenous and saphenovenous excised moderate limb ischemia model (the moderate ischemia model shown in the left of FIG. 1) (Coffinhal, T., et al, am. J. Pathol.152, 1667-; (2) c57BL/6 mouse model of total excision of external iliac artery and vein, femoral artery and vein, external iliac artery and vein and branch vessel around femoral artery and vein (severe ischemia model shown on right of figure 1); (3) the model obtained by surgery using immunodeficient BALB/c nu/nu mice in the same manner as described in (2) above (i.e., BALB/c nu/nu mouse model of severe ischemia).

Mature male C57BL/6, BALB/C and BALB/C nu/nu mice (6-8 weeks old, Charles River Grade) were purchased from KBT Oriental Co.Ltd. (Tosu, Saga, Japan). Animal experiments were conducted using approved protocols in accordance with Laboratory animal feeding and Use methods recommended by the Kyushu university animal, the Committee for recombinant DNA and pathogenic infection experiments, and the laws (No. 105) and bulletins (No. 6) of the Japanese government, in addition to complying with Principles of Laboratory animal Care and guides for the Care and Use of Laboratory Animals (publication No. NIH 80-23, revised 1985) established by the national institutes of health.

Pentobarbital was injected intraperitoneally to anesthetize the mice thoroughly, and the skin was incised. In a moderate ischemia model, the entire superficial whole muscle artery and femoral vein and the saphenous artery and saphenous vein (from directly below the deep femoral artery to the popliteal artery) were ligated and excised (left in FIG. 1) (Couffinhal, T., et al, am.J.Pathol.152, 1667-. In severe ischemia models, external iliac arterio-venous and deep femoral arteries were also excised (fig. 1 right). The same operator (I.M.) performed 3-5 independent experiments to verify the reproducibility of the recovery state of the limbs of these models, using 10 or more mice per model in each operation. Each limb rescue experiment was repeated four times by the same operator under the same conditions.

The above model (1) never loses its limbs, only occasionally finding signs of toe necrosis; no limb necrosis (also referred to as "limb salvage model") occurred in model (2) (i.e., the C57BL/6 mouse model of severe ischemia); in model (3) (BALB/c nu/nu mouse model of severe ischemia) all mice had almost total limb amputation within 10 days after surgery (also referred to as hindlimb amputation model). Limb necrosis was observed in the immunocompetent BALB/c mice in the severe ischemia model to the same extent as in BALB/c nu/nu mice (data omitted). It has been reported that BALB/C mice are more sensitive to growth factors than C57BL/6 mice (Rohan, M.R., FASEB J.14, 871-.

The endogenous expression of VEGF and FGF2 in ischemic muscle and serum was studied using the above-described models of moderate and severe ischemia. Two days after the surgery, each limb muscle (thigh whole muscle and gastrocnemius muscle) and serum of the C57BL/6 mouse were collected, homogenized or dissolved, and subjected to enzyme-linked immunosorbent assay (ELISA). The amount of recombinant protein synthesized was determined using Quantikine immunoassay system (R & D Systems Inc., Minneapolis, MN) for human VEGF or mouse FGF2 according to the instructions thereof, and the total protein concentration was determined according to the Bradford method using protein assay system (Bio-Rad Laboratories, Hertfordshire, UK) and the results were normalized (Yonemittsu, Y., et al, Nature Biotech.18, 970-.

There was no significant difference in protein concentration between the whole thigh muscle and the gastrocnemius muscle, and therefore both were analyzed for each group. Interestingly, FGF2 protein concentration was significantly increased (P < 0.001) in both limb ischemia models of ischemia surgery (moderate model: 847.5 ± 187.7pg/g muscle, severe model: 895.4 ± 209.5pg/g muscle, n-12 per group) compared to the basal levels (489.7 ± 108.6pg/g muscle, n-12) in untreated mice (fig. 2). On the other hand, in the severe ischemic group, it was found that the expression of VEGF protein was enhanced by ischemia, but it was not clearly observed in muscle (untreated: 174.7. + -. 43.1; moderate: 119.2. + -. 53.4; severe: 242.5. + -. 244.3, n ═ 12). VEGF is known to be an endothelial mitogen that greatly enhances its expression through tissue ischemia (Shweiki, D. et al, Nature 359, 843-containing 845 (1992); Forsythe, J.A., et al, mol.cell.biol.16, 4604-containing 4613(1996)), but the above results seem to be inconsistent with this conclusion. Therefore, VEGF may leak into the systemic circulation, and based on this consideration, VEGF levels in serum are determined. The results are consistent with the hypothesis that serum VEGF protein levels were observed to increase in ischemia severity-dependent manner, but no FGF2 was detected in serum (fig. 2 right).

The present inventors hypothesized that limb ischemia can induce low molecular VEGF isoforms that are known to interact less with heparin sulfate than high or medium molecular weight VEGF (Cohen, T., et al, J.biol.chem.270, 11322-11326 (1995)). To confirm this hypothesis, the inventors analyzed the expression of VEGF isoforms in thigh muscle one day after surgery in male C57BL/6 mice (without surgery, moderate ischemia model and severe ischemia model) by RT-PCR using primer pairs that recognize the mouse VEGF splice isoforms containing VEGF188, 164, 144 and 120 (Burchardt, M., et al, biol. reproduct.60, 398-404 (1999)). The primer pairs were prepared using primers for rat VEGF exon 1 and exon 8 as described in the prior art (Burchardt, M., et al, biol. reproduction.60, 398-404 (1999)): a forward primer (5'-TGC ACC CAC GAC AGAAGG GGA-3'/SEQ ID NO: 17) and a reverse primer (5'-TCA CCG CCT TGG CTT GTC ACAT-3'/SEQ ID NO: 18) corresponding to the mouse VEGF isoform sequences. To detect the smallest VEGF isoform (VEGF115), the same forward primer and VEGF 115-specific reverse primer (5'-CTA CCA AAA GTT TCCCAG GCA G-3'/SEQ ID NO: 19) as described above were used (Sugihara, T. et al, J.biol.chem.273, 3033-3038 (1998)). RT-PCR conditions were the same as in the literature (Burchardt, M., et al, biol. reproduction.60, 398-404 (1999); Sugihara, T. et al, J.biol. chem.273, 3033-3038 (1998)).

As a result, endogenous VEGF expression associated with ischemia was observed only in the 164 isoform, while no significant expression of the other isoforms was detected (fig. 3). Also, expression of the known minimal isoform VEGF115(Sugihara, T. et al, J.biol.chem.273, 3033-3038(1998)) was not detected by RT-PCR analysis.

[ example 2]Kinetics of intramuscular introduction of genes into mouse hind limbs by recombinant Sendai Virus

The level of expression and changes over time of the transgene were first studied with firefly luciferase to analyze its kinetics. Luminometers (type LB 9507, EG) were used according to the literature method (Yonemittsu, Y., et al, Nature Biotech.18, 970-&G Berthold, germany) were used for luciferase assays and data were expressed in Relative Light Units (RLU)/mg protein. Luciferase measurements were normalized by measuring total protein concentration using a protein assay system (Bio-Rad laboratories, Hertfordshire, UK) according to the Bradford method. The limb muscles of the critical limb ischemia model are obviously seriously damagedThis means a reduction in transgene expression and was therefore analyzed in a moderate ischemia model (fig. 1 left). Genes were injected into the thigh muscle and the calf muscle at two sites during surgery, each site was injected in an amount of 25. mu.l, and the total of the two sites is indicated by the following dose. Mice (C57BL/6 mice) (6-8 weeks old, average body weight 30g) were given 100. mu.g of pCMV-luciferase (about 50 times the clinical amount) (Baumgartner, I., et al, Circulation 97, 1114) -1123 (1998); Isner, J.M., et al, J.Vasc.Surg.28, 964-973(1998)), which showed higher luciferase activity in the assay two days after gene transfer (average. + -. S.D.: 5.1. + -. 3.9X 10⁶RLU/mg protein, n ═ 6), but given with 10)⁷The group of SeV-luciferases that form plaque units (pfu) showed about 5-fold expression (2.4. + -. 3.8X 10)⁷RLU/mg protein, n-12), to 10⁸The group of pfu SeV showed about 120-fold expression (7.3. + -. 4.7X 10)⁸RLU/mg protein, n ═ 6) (fig. 4 left).

The temporal change in transgene expression was measured by administering a luciferase expression plasmid (pCMV-luc) or SeV-luc to a C57BL/6 mouse model with moderate ischemia in the same manner as described above. Intramuscular administration of 10⁸Expression was also found to decrease with time in the C57BL/6 mouse model of moderate ischemia with pfu SeV-luciferase (day 2: 7.3. + -. 4.3X 10)⁸RLU/mg protein, n-12; day 7: 3.4 +/-4.7 multiplied by 10⁷RLU/mg protein, n-12; day 14: 2.6. + -. 1.2X 10⁴RLU/mg protein, n-12) (fig. 4 right). Intramuscular administration of 10⁸The change with time in luciferase activity in a moderate ischemia model of pfu SeV-luciferase in BALB/C nu/nu mice up to day 7 was the same as that in C57BL/6 mice, but its expression was maintained later (day 2: 9.4. + -. 3.7X 10)⁸RLU/mg protein, n-12; day 7: 1.3. + -. 1.9X 10⁷RLU/mg protein, n-12; day 14: 0.9 +/-1.3X 10⁷RLU/mg protein, n ═ 12).

Then, the inventors measured the in vitro secretion of angiogenic proteins using a variety of cultured cells such as primary bovine vascular smooth muscle cells (BSMC), myocardial myoblasts (H9C2), primary human umbilical cord vascular endothelial cells (HUVEC), and COS7 cells. This implementationIn the examples, the FGF2 vector (SeV-FGF2) which does not contain the classical signal sequence required for protein secretion was used, since FGF2 which does not contain the secretion sequence can also be expressed extracellularly according to the previous studies of the present inventors and others (Piotrowicz, R.S. et al, J.biol.chem.272, 7042-7047 (1997); Qu, Z., et al, J.Histochem.Cytochem.46, 1119-1128 (1998); Florkiewicz, R.Z. et al, J.cell.Physiol.162, 388-399 (1995)). Consistent with expectations, dose-dependent efficient expression of FGF2 protein was detected in culture supernatants at levels comparable to VEGF165 (e.g., VEGF 165: FGF 2: 4,354 ± 2,794: 3,682 ± 1,063(HUVEC), 275 ± 58: 398 ± 154(BSMC), 16,987 ± 4,748: 5,976 ± 381(H9C2), 38,648 ± 4, 913: 1,547,237 ± 176,502(COS7) at MOI 100, with pg/10 in each group⁵Cells/24 hr, n ═ 3) (fig. 5).

[ example 3]Kinetics of intramuscular expression of SeV-mediated angiogenic factors in vivo

The present inventors determined the level of intramuscular expression of angiogenic factors following intramuscular administration of SeV-VEGF165 and SeV-FGF2 in vivo in a C57BL/6 mouse model of moderate ischemia (left side of FIG. 1). The administration site was in two places of thigh muscle and gastrocnemius muscle, each of which was 25. mu.l, and was administered only once at the time of surgery with a 26-gauge needle.

The results of in vivo expression of angiogenic factors are interesting compared to their results of in vitro expression and the results of in vivo expression of reporter genes. As shown in FIG. 6, SeV-FGF 2-mediated protein synthesis increased dose-dependently to achieve approximately 100-fold higher endogenous gene expression levels in the highest titer vector introductions (thigh muscle: baseline: 429. + -. 79, ischemia: 974. + -. 150, 10)⁶pfu：4,913±313，10⁷pfu：13,469±12,611，10⁸pfu: 46,703 + -12,092 pg/g muscle, n is 6 in each group; gastrocnemius muscle: baseline: 550 ± 104, ischemia: 720 +/-128, 10⁶pfu：1,376±158，10⁷pfu：8,252±8,190，10⁸pfu: 59,704 ± 35,297pg/g muscle, n ═ 6 in each group). In the group administered with SeV-FGF2, no significant serum FGF2 was detected even at the highest titers. In another aspect, the amount of VEGF165 isThe increase in dependence was much less than that of FGF2, at 10⁷pfu has not yet doubled at 10⁸pfu, on the other hand, hardly detected the expression of the human VEGF165 protein from SeV (thigh muscle: baseline: 176. + -. 44, ischemia: 143. + -. 64, 10)⁶pfu：159±67，10⁷pfu：224±216，10⁸pfu: < 5pg/g muscle, each group n ═ 6; gastrocnemius muscle: baseline: 173 ± 45, ischemia: 95 +/-28, 10⁶pfu：186±30，10⁷pfu：172±101，10⁸pfu: < 5pg/g muscle, each group n ═ 6). Serum levels of endogenous mouse VEGF were significantly increased in moderate limb ischemia (37.7 ± 15.4pg/ml, n ═ 6), but no human VEGF165 from the vector was significantly detected, indicating that the intramuscularly expressed VEGF165 did not spread to the systemic circulation.

[ example 4 ]]Introduction of angiogenic genes significantly enhanced ischemia-induced endogenous VEGF expression

The inventors considered whether the difference in the expression patterns of VEGF165 and FGF2 correlated with the expression of endogenous VEGF. Overexpression of endogenous VEGF exacerbates tissue ischemia by excessive penetration and may down-regulate SeV-mediated transcription. In addition, it has been reported that the angiogenic effect of FGF2 is caused in part by enhanced expression of endogenous VEGF in vivo and in vitro (Asahara, T., et al, Circulation 92, 365-.

Therefore, the inventors studied the change of endogenous mouse VEGF protein synthesis in the muscle by exogenously introduced angiogenic factor gene using mouse VEGF specific ELISA system. As shown in figure 7, introduction of the FGF2 gene instead of the VEGF165 gene significantly increased the intramuscular levels of endogenous murine VEGF in limb conditions of normal circulation (without surgery) and moderate ischemia. In critical limb ischemia, the expression of endogenous mouse VEGF is greatly enhanced by introducing two genes of angiogenesis factors, and particularly, the introduction of VEGF165 is about 7 times higher than that of ischemia per se (simulation).

To further investigate the above-mentioned problems, the present inventors examined the effect of introducing an angiogenic factor gene in a C57BL/6 mouse severe ischemia model histologically (FIG. 8). When simulated (mock) introduction was performed after ischemic surgery, scattered compact muscle fibers (diffuse cervical muscle fibers) developed inflammatory infiltrates with intracellular edema (intracellular edema) after two days. These results were significantly enhanced when VEGF165 gene was introduced, and significantly inhibited when FGF2 gene was introduced (FIG. 8)

[ example 5]Exogenously introduced VEGF165 is a limb injury in acute critical limb ischemia Factors other than limb salvage factors

Based on the above results, the present inventors investigated the efficacy of the introduction of the angiogenic factor gene in vivo using the above-mentioned models of moderate and severe ischemia. At the highest dose of 10⁸No production of the transgene product was found by pfu administration of VEGF165, so the inventors administered 10⁷pfu virus to observe in vivo efficacy. The degree of necrosis of the Limb was divided into 4-grade Salvage scores (Limb Salvage Score; LSS): LSS 4, complete limb salvage (complete limb salvage); LSS 3, necrosis below heel; LSS 2, necrosis below the knee; LSS 1, supraknee necrosis and LSS 0, total limb amputation around the inguinal ligament (total limb amputation around the exiting limb). From the above classification, the inventors studied the toxicity of SeV-mediated angiogenesis factor expression in a salvage model of C57BL/6 mouse critical limb ischemia. The method of introducing the angiogenic gene was the same as in example 2.

Two days prior to the ischemic surgery, the vehicle was injected and all groups of mice retained their limbs (data omitted). The vehicle was injected at the time of surgery, and mice in all groups including the FGF 2-administered group were completely salvaged (% LLS ═ 100%) except for the VEGF 165-administered group. As shown in fig. 9 and 10, individuals with limb loss were observed in mice given VEGF165 (5/10 limb loss,% LLS 52.5%, p < 0.0001 compared to the other groups) (fig. 10A). The results show that the VEGF165 gene introduced has limb injury effect. Then, the therapeutic effect of gene therapy of angiogenic factors was verified in BALB/c nu/nu mouse model of severe ischemia (amputation model). The result is: the mice were given significant inhibition of limb detachment with SeV-FGF2 (2/10 limb detachment,% LLS ═ 77.5%) (fig. 10B), while the mice given SeV-VEGF165(8/10 limb detachment,% LLS ═ 15.0%) did not improve hind limb prognosis as with luciferase-expressing SeV (5/6 limb detachment,% LLS ═ 16.7%) (fig. 10B). The above results clearly indicate that the FGF2 gene introduced has a limb-salvage effect.

The inventors then determined the effect of intramuscular gene introduction on the restoration of blood flow in the left leg subjected to severe ischemic surgery by laser Doppler perfusion image analysis (Coffinhal, T., et al, am. J. Pathol.152, 1667-1679 (1998); Murohara, T., et al, J. Clin. invest.105, 1527-1536 (2000)). Using the C57BL/6 mouse model of severe ischemia (limb salvage model), the conditions for gene transfer were exactly the same as those in FIG. 10A (limb salvage model) described above. The ratio of ischemic (left)/normal (right) limb blood flow was determined using a Laser Doppler Perfusion Image (LDPI) analyzer (Moorr instruments, Devon, UK) according to literature methods (Couffinhal, T., et al, am. J. Pathol.152, 1667-1679 (1998); Murohara, T., et al, J. Clin. invest.105, 1527-1536 (2000)). Specifically, to minimize data fluctuations due to body temperature, mice were maintained on a 37 ℃ thermostated plate prior to laser scanning. The target site (legs and feet) of each mouse was scanned twice in succession at predetermined times (2, 4,7 and 10 days before the experiment and after the operation) (fig. 11), and no substantial difference was found in the two scans. After laser scanning, the stored images were used to measure blood flow with a computer and to calculate the average blood flow for ischemic and non-ischemic feet. To reduce data fluctuations caused by ambient light or temperature effects, the LDPI index is expressed as a ratio of left limb (ischemic) blood flow value/right limb (non-ischemic) blood flow value.

Both the SeV-luciferase (mock) administered group and the SeV-FGF2 administered group detected significant blood flow around the upper thigh on day 4, particularly the FGF2 administered group found significant blood flow in the gastrocnemius muscle on days 4,7 and 10, while the contemporaneous luciferase administered group found limited blood flow in the thigh muscle (FIG. 11). In representative results, the limbs of the luciferase-administered group were slightly atrophied (millitrophic limb), whereas there was almost no limb damage in the FGF 2-administered group. In contrast, little blood flow was observed in the thigh muscle of the mice in the VEGF 165-administered group, with consequent hind limb loss (FIG. 11). All mice in the SeV-VEGF 165-administered group had limb loss at least at the level of the knee. The observation of the limb blood flow in each administration group is described in detail below.

1. Administering to an individual with SeV-luciferase

Most of the blood flow in the left hind limb disappeared immediately after surgery, and then gradually returned to approximately the center of the thigh by day 4, but not to the calf by day 10. The results are shown in the right most panel, with the hind limbs not rotted and slightly atrophied. 1/3, the same results were observed, and some individuals had better recovery degrees than the above.

2. Administering to a subject having SeV-VEGF165

As described above, most of the blood flow disappeared immediately after the operation, and little blood flow restoration was found in the femoral region in the subsequent observation. As a result, as shown in the right-most figure, the hind limb starts to fall off from the middle of the thigh. The results were identical for all 10 individuals.

3. Administering to an individual SeV-FGF2

As described above, most of the blood flow in the left hind limb disappeared immediately after the operation, and the extent of disappearance was substantially the same as that in the SeV-luciferase-administered group. Strong blood flow began in the thigh on day 4 (shown by the red dots), weak blood flow was found in the calf on day 7, and small but significant blood flow occurred in the entire left hind limb on day 10 (shown in blue). The results are shown in the right most figure, with the hind limb remaining and completely normal in appearance.

The inventors compared the thigh muscle blood flow of each group statistically by means of doppler images. As shown in FIG. 12, the blood flow values of the mice in the SeV-FGF 2-administered group were significantly higher than those of the mice in the SeV-luciferase-administered group during physiological restoration of limb blood flow. In contrast, the thigh muscle blood flow values were low in the mice of the SeV-VEGF 165-administered group, and most of the mice were detached from the middle of the lower limbs or from the upper part after 7 days of the operation.

[ example 6]Treatment of acute limb ischemia with replication incompetent Sendai virus vectors

1. Construction of F Gene-deleted Sendai Virus genomic cDNA carrying angiogenic Gene

First, the EGFP gene was amplified by PCR, and pSeV18 containing the full-length genomic cDNA of Sendai virus (SeV) was introduced⁺b (+) (Hasan, M.K., et al, J.Gen.Virol.78, 2813-2820, 1997) deletion of the F gene resulted in the plasmid pSeV18⁺/. DELTA.F (see WO00/70055 and WO00/70070), and then the EGFP gene was inserted into the F gene deletion site to obtain a plasmid (pSeV 18)⁺/. DELTA.F-GFP). In PCR, a 5 '-end primer (5' -atgcatatggtgatgcggttttggcagtac/SEQ ID NO: 9) ending with NisI and a 3 '-end primer (5' -tgccggctattattacttgtacagctcgtc/SEQ ID NO: 10) ending with NgoMIV were used so that the number of nucleotides of the EGFP gene fragment was a multiple of 6 (Hausmann, S. et al, RNA 2, 1033-1045, 1996). The PCR product was digested with NsiI and NgoMIV restriction enzymes and fragments recovered from the gel, ligated into pUC18/dFSS at the F gene deletion site between the NsiI and NgoMIV restriction enzyme sites, and verified by sequencing. The DraIII fragment containing the EGFP gene was recovered from the vector using pSeV18⁺The DraIII fragment containing the F gene region is replaced, and pSeV18 is obtained by connection⁺/. DELTA.F-GFP. However, even from pSeV18⁺DeltaF, the downstream ORF of the F gene was removed, and it was still possible to express the pentapeptide derived from the ligation fragment and the primer of the vector by the remaining EIS sequence of the F gene (SeV-specific sequence, E; end, I; intervening, S; start). In addition, the pSeV18⁺Since co-expression of GFP occurred in/. DELTA.F-GFP, a vector was constructed in which co-expression of the above pentapeptide and GFP was impossible. The recombination was carried out as follows.

Digestion of pSeV18 with SalI and NheI⁺Δ F-GFP, a fragment containing the deleted F gene region (6288bp) was recovered and cloned into Litmus38(New England Biolabs, Beverly, Mass.), to construct Litmus SalINheifrg/. DELTA.F-GFP. Deletion of F-containing Gene by inverse PCRThe EGFP gene of the EIS sequence upstream of the deletion region is deleted. Specifically, PCR was carried out using a reverse primer (5 '-gtttaccaggtggagagttttgcaaccaagcac/SEQ ID NO: 11) having a SexAI restriction enzyme recognition sequence at the upstream of the GFP gene and a forward primer (5' -ctttcacctggtacaagcacagatcatggatgg/SEQ ID NO: 12) having a SexAI restriction enzyme recognition sequence at the downstream of the GFP gene. A fragment (10855bp) of the desired size was excised and ligated to delete the EGFP gene containing the EIS sequence upstream of the deleted F gene region.

In this construct, an extra 15bp sequence was inserted between the SexAI sites due to the design of primers, and thus, E.coli SCS110 strain (in dcm) was transformed^-/dam^-SCS110 strain, in which SexAI is methylated and cannot be digested), plasmid was prepared, and after digesting the plasmid with SexAI restriction enzyme, 1628bp and 9219bp gene fragments were recovered and ligated, and the excess 15bp sequence was removed, to construct litmus salanheifrg/Δ F (Δ 5aa), in which the EGFP gene including the EIS sequence upstream of the F gene was deleted, and the deleted region contained multiple 6 nucleotides. Digesting the plasmid with SalI and NheI, recovering the fragment, and digesting the fragment with a plasmid containing pSeV18⁺Replacing SalI/NheI fragment of middle F gene region, and connecting to obtain plasmid pSeV18⁺And/. DELTA.F (. DELTA.5 aa). An angiogenic gene (e.g., human FGF2) is inserted into this plasmid using the NotI restriction enzyme recognition sequence located upstream of the NP gene as follows.

Vascular smooth muscle cells were harvested from the great saphenous vein under the consent of the patient himself, and cDNA of human FGF2(hFGF2) was isolated therefrom by RT-PCR and subcloned into the HindIII (5 '-end) and EcoRI (3' -end) sites of pBluescriptSK + (Stratagene, La Jolla, Calif.). The sequence of the hFGF2 cDNA was confirmed to be identical to the sequence reported by Abraham et al (Abraham, J.A., et al, EMBO J.5(10), 2523-2528, 1986).

To insert the hFGF2 gene into pSeV18⁺NotI restriction enzyme site upstream of NP Gene in/. DELTA.F (. DELTA.5 aa), first in hFGFSeV specific sequences (EIS sequences) were added to the 3' -end of the 2 gene to prepare fragments each having NotI recognition sequences at both ends. Specifically, the hFGF2 cDNA was used as a template, PCR was performed using an N-terminal primer (5 '-atccgcggccgccaaagttcacttatggcagccgggagcatcaccacgctgcccgccttgcccgaggatggcggcagcggcgcc/SEQ ID NO: 13) containing an initiation codon and a C-terminal primer (5' -atccgcggccgcgatgaactttcaccctaagtttttcttactacggtcagctcttagcagacattggaagaaaaagtatagc/SEQ ID NO: 14) containing a termination codon region and an EIS sequence, and the amplified fragment was digested with NotI, subcloned into the NotI site of pBluescriptSK + (Stratagene, La Jolla, Calif.) to obtain pBS-hFGF2, and sequenced. Digestion of pBS-hFGF2 with NotI gave a fragment containing hFGF2 cDNA, which was inserted into pSeV18⁺Construction of F-deleted Virus genomic cDNA pSeV18 comprising hFGF2 Gene at NotI site upstream of NP Gene in/. DELTA.F (. DELTA.5 aa)⁺hFGF 2/. DELTA.F (. DELTA.5 aa), also known as pSeV18⁺hFGF 2/. DELTA.F. In addition, pSeV18 encoding viral cDNA having replication ability was used⁺The NotI fragment containing hFGF2 cDNA was also inserted into the NotI site of/AF to construct pSeV18⁺hFGF 2. From pSeV18 using well known methods (Hasan, M.K. et al, J.Gen.Virol.78: 2813-2820, 1997; Kato, A. et al, 1997, EMBO J.16: 578-587; Yu, D. et al, 1997, Genes Cells 2: 457-466)⁺hFGF 2A replicative SeV vector expressing human FGF2 was prepared to construct SeV-hFGF 2.

3. Reconstitution and amplification of F Gene-deleted SeV Virus

F gene-deleted SeV vectors (cells before induction of expression of SeV-F gene are referred to as LLC-MK2/F, and cells after induction of expression are referred to as LLC-MK2/F/Ad) were reconstructed using helper cells (LLC-MK2/F, see WO00/70055 and WO00/70070) expressing F gene, which induced expression of Sendai virus F gene (SeV-F) by Cre DNA recombinase. LLC-MK2 cells at 5X 10⁶The concentration of each cell/dish was seeded into a 10cm diameter Petri dish, and after 24 hours of culture, recombinant vaccinia virus expressing T7 RNA polymerase (Fuerst, T.R. et al, Proc. Natl. Acad. Sci. USA 83, 8122-2). UV irradiation of vaccinia virus was performed using a UV Stratalinker2400 (Cat. No. 400676(100V), Stratagene, La Jolla, Calif., USA) equipped with 5 bulbs of 15 watts. After washing the cells twice, plasmid pSeV18 was added⁺hFGF 2/. DELTA. F, pGEM/NP, pGEM/P, pGEM/L (Kato, A., et al, Genes Cells1, 569-. The resulting mixture was added to cells, cultured for 3 to 5 hours, and then the cells were washed twice with serum-free MEM, and then cultured for 24 hours in serum-free MEM containing 40. mu.g/ml cytosine-. beta. -D-arabinofuranoside (AraC, Sigma) and 7.5. mu.g/ml trypsin (GIBCO).

The culture supernatant was removed, and LLC-MK2/F/Ad cells, which were F gene-expressing helper cells cloned as described above, were overlaid on the cell supernatant. Specifically, LLC-MK2/F/Ad dispersed in serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) was overlaid on the cells from which the culture supernatant was removed and culture was continued for 48 hours. The cells were recovered with a scraper and the particles were suspended in OptiMEM (10)⁷Individual cells/ml) and freeze-thaw repeated three times. Freeze-thaw lysates (200. mu.l/well) were added to the upper layer of LLC-MK2/F/Ad cells (in 12-well plates, 4X 10⁶One cell/well), 300. mu.l/well of serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) was added thereto, and the mixture was cultured for 15 to 24 hours. The culture supernatant was removed, the cells were washed with serum-free MEM, fresh serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) was added thereto, and the culture was carried out for 5 to 9 days, and the supernatant was collected. The recovered supernatant was infected with LLC-MK2/F/Ad cells and cultured in serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) in the same manner as described above, whereby F gene-deficient SeV was amplified.

After amplification, the culture supernatant containing F gene-deleted SeV particles was repeatedly filtered twice with a 0.22 μm filter, so that contamination with a recombinant vaccinia virus expressing T7 RNA polymerase during reconstitution could be substantially avoided. Specifically, the culture supernatant (sample after P2) amplified twice or more with AraC-containing serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin) was filtered twice with a 0.22 μm filter, amplified once with AraC-containing serum-free MEM (containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin), and the culture supernatant was recovered to obtain F gene-deleted SeV (SeV-hFGF 2/. DELTA.F) which was amplified and contained no recombinant vaccinia virus.

4. Gene therapy for limb ischemia by using SeV vector of replication-defective and replication-defective human FGF2 expression vector

The therapeutic effect of SeV vector, which is an expression vector for replication-and replication-defective human FGF2, was confirmed in a BALB/c nu/nu mouse model of severe ischemia (limb drop model) as described in example 1. The angiogenic gene was introduced in the same manner as in example 2. The carrier was injected during the operation, and the limb drop after the operation was observed, and the limb salvage rate (the ratio of individuals with limbs remaining) at each time point was calculated (fig. 13).

Mice in the control group given luciferase-expressing SeV (SeV-luciferase) showed a high limb shedding ratio to the same extent as those in the non-administered group, while administration of human FGF2 expression vectors (SeV-hFGF2 and SeV-hFGF 2/. DELTA.F) significantly inhibited limb shedding. This example shows that a paramyxovirus vector expressing human FGF2 has a high therapeutic effect as an angiogenic gene transfer vector for use in ischemic therapy, and that a replication ability-deficient paramyxovirus vector is effective for ischemic therapy.

Industrial applicability

The invention provides a basic technology of gene therapy of targeting ischemic tissues, which can effectively induce angiogenesis of the ischemic tissues and prevent necrosis.

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Claims

1. A paramyxovirus vector encoding an angiogenic gene capable of expression.

2. The paramyxovirus vector of claim 1, wherein the angiogenesis gene is fibroblast growth factor 2(FGF 2).

3. The paramyxovirus vector of claim 1, wherein the paramyxovirus is Sendai virus.

4. The paramyxovirus vector of claim 1, which lacks the F gene.

5. An angiogenic composition comprising the paramyxovirus vector of claim 1 or a cell comprising the vector and a pharmaceutically acceptable carrier.

6. The composition of claim 5, for treating ischemic tissue.

7. The composition of claim 5 for intramuscular administration.

8. A method for inducing angiogenesis, comprising the step of administering the angiogenic composition according to any one of claims 5 to 7.