HK1114104A

HK1114104A - Vectors with modified protease-dependent tropism

Info

Publication number: HK1114104A
Application number: HK08103866.7A
Authority: HK
Inventors: 喜纳宏昭; 井上诚; 上田泰次; 饭田章博; 长谷川护; 小林雅典
Original assignee: 株式会社载体研究所
Priority date: 2002-04-30
Filing date: 2006-02-18
Publication date: 2008-10-24

Description

Vectors with modified protease-dependent tropism

The application is a divisional application of Chinese application 03815574.5, the parent application is filed in 30/4/2003 with the international application number PCT/JP2003/005528, and enters the Chinese national stage in 30/12/2004, and the divisional application adopts the invention name consistent with the parent application.

Technical Field

The present invention relates to cell fusion vectors with modified protease-dependent tropism, and methods of making the same. The vectors of the present invention are useful as gene therapy vectors showing cancer-specific infection.

Background

The development of cancer gene therapy has progressed in recent years. At present, the present inventors have developed a gene therapy vector using Sendai virus (SeV). SeV is a virus of the Paramyxoviridae family, belonging to a group of viruses comprising as their genome a non-segmented negative-strand RNA virus. Paramyxovirus vectors can achieve high transfection efficiency and overexpression of heterologous genes, and are expected as gene therapy vectors for cancer. Some cancer treatments currently use paramyxoviruses. For example, mumps infected BHK21 cells were observed to show anti-tumor effects in tumor bearing nude mice (Minato, N. et al, J.Exp.Med.149, 1117-1133, 1979). Similarly, anti-tumor effects have been reported in other paramyxoviruses. Recently, the antitumor effect of the fusogenic protein (fusogenic) has attracted attention. Galanis et al, reported that cancer cells infected with adenovirus vectors carrying measles virus F and HN proteins form syncytia and thus produce an antitumor effect in vivo (Galanis, E. et al, hum. Gene ther.12, 811-821, 2001).

Obviously, cancers that do not undergo metastasis can be treated by surgical removal of the part, and metastatic cancers and malignant cancers are considered synonymous. Invasive metastatic cancers are known to overexpress Matrix Metalloproteinases (MMP) and/or plasminogen activator (uPA, tPA) (Cox, G., and O' Byrne, K.J., Anticancer Res.21, 4207-. This over-expression is believed to occur due to the fact that infiltration and metastasis are only possible after the surrounding extracellular matrix (ECM), which is a barrier to prevent cellular translocation during cancer cell metastasis and infiltration, is degraded by the cancer expressing enzymes (MMP, uPA, tPA) that degrade the ECM.

On the other hand, 3 problems have arisen with respect to gene therapy of cancer. First, genes cannot be transfected throughout cancer because of their low efficiency in gene transfer into cancer cells and the inability to easily achieve gene transfer into the core of solid cancers. Accordingly, the remaining cancer cells start to proliferate again, resulting in recurrence. Second, the gene is transfected not only into cancer cells but also into normal cells. Toxic genes damage normal cells, resulting in increased side effects. Third, as cancer becomes malignant, infiltration and metastasis occur, which becomes a problem in all types of cancer treatment. At present, no vector has been developed which can solve these problems.

Disclosure of Invention

The present invention provides a novel cell fusion vector having a modified protease-dependent tropism, which is infiltrated into surrounding cells only in the presence of a specific protease, and a method for preparing the same.

The paramyxovirus family of viruses (including Sendai virus) contains two proteins in their envelopes. The fusion (F) protein effects membrane fusion between the virus and its host cell, resulting in nucleocapsid release into the cytoplasm. The hemagglutinin-neuraminidase (HN) protein has hemagglutination activity and neuraminidase activity and plays a role in binding to host receptors. The F and HN proteins are also called spike proteins because of their presence on the surface of the viral envelope. The matrix (M) protein aligns along the envelope and imparts rigidity to the viral particle. The vectors of the present invention are characterized in that they transfer genes into a wide variety of cells and animal tissues with high efficiency and achieve high-level expression, as compared with existing vectors.

The F protein (F0) showed no cell fusion activity. Its fusion activity can only be shown by cleavage by host-derived proteases, which degrades F0 into F1 and F2. Thus, proliferation of viruses carrying the wild-type F protein is restricted to tissue types that express trypsin-like proteases that cleave the protein, such as respiratory mucus epithelium. There are several studies directed to modifying the tropism of infection or fusogenic power (fusogenicity) in paramyxoviruses by modifying the F protein. In the case of SeV, variants containing F cleaved only by alpha-chymotrypsin lose trypsin sensitivity and thus alter their tropism specific for the F cleavage sequence (Tashiro, M. et al, J.Gen.Virol.73(Pt 6), 1575-1579, 1992). Furthermore, in Newcastle disease virus and measles virus, it has been shown that syncytia forming ability is altered in a trypsin-dependent manner due to modification of the cleavage sequence for F (Li, Z. et al, J.Virol.72, 3789-3795, 1998; Maisner, A. et al, J.Gen.Virol.81, 441-449, 2000).

By modifying the cleavage sequence of the F protein as described above, the vector can infect and propagate in a specific tissue expressing a specific protease. However, one problem with paramyxovirus vectors is the secondary release of virus from cells, which occurs after the vector has been introduced into the target cell. In cells infected with replication-competent virus, virus particles are formed and progeny virus is released. Thus, viral particles also spread to sites other than the target tissue. Although the above-described viral particles comprising wild-type F protein do not show infectivity in the absence of trypsin-like enzyme, the viral particles themselves are released from the cells. For in vivo administration, it is of concern that virus that diffuses into the blood will diffuse throughout the body. Furthermore, the release of virus-like particles (VLPs) which lack replication ability was observed in F gene-deficient SeV transfected cells (Li, H.O. et al, J.Virol.74, 6564-6569, 2000; WO 00/70055; WO 00/70070). Infection of tissues other than the target tissue and induction of an immune response are both concerns about secondary release particles.

Thus, the present inventors have found that paramyxoviruses lacking the M gene in the viral envelope gene do not form particles, but they can undergo cell fusion-type infection by fusing infected cells with cells in contact with these infected cells to form syncytia (WO 00/09700). These M-deficient viruses replicate in transfected cells and are delivered to neighboring cells in the presence of trypsin. However, this phenomenon occurs only under conditions where F is cleaved and activated. In viruses containing wild-type F protein, transfer of the virus does not occur in the absence of trypsin-like protease. Therefore, the present inventors have presumed that a novel vector which does not produce particles for secondary release and can transmit infection only in a specific tissue can be produced by modifying the tropism of the F protein in such M-deficient virus. In particular, many invasive metastatic cancers are known to have enhanced protease (e.g., MMP, uPA, and tPA) activity, which can degrade the ECM. Therefore, the present inventors have utilized the protease-dependent cell fusion-type infection of this M-deficient SeV in combination with the phenomenon of overexpression of MMP, uPA, and tPA in cancer to prepare SeV vectors that specifically infect and spread to invasive metastatic cancers.

M-deficient viruses lack the M gene required for particle formation. Thus, viral particles are either not released or are extremely inhibited in the virus. When a conventional reconstitution method is used to prepare a replication-competent recombinant virus (Kato, A. et al, Genes Cells 1, 569-579, 1996), RNP of M-deficient virus can be prepared but infectious virus particles cannot be prepared (WO 00/09700). When an M-deficient vector is used as a cancer therapeutic agent, it is extremely useful to prepare an M-deficient virus as an infectious viral particle. Therefore, the present inventors developed a novel method for preparing an M-deficient virus into viral particles.

To achieve the goal of constructing a vector in which VLP release is inhibited, the present inventors contemplate the use of temperature sensitive mutations in the viral genes. Mutant virus strains that can grow at low temperatures but cannot grow at high temperatures have been reported. The present inventors have envisaged that muteins, in particular mutated M proteins, which inhibit virus particle formation at high temperatures, can be used to inhibit VLP formation, such that virus production can be carried out at low temperatures (e.g. 32 ℃), but practical applications of viruses such as gene therapy can be carried out at higher temperatures (e.g. 37 ℃). To this end, the present inventors constructed F gene-deficient recombinant Sendai virus vectors encoding mutated M and mutated HN proteins having all six temperature-sensitive mutations (three M proteins and three HN proteins) among the reported M and HN proteins. VLP release from the virus was detected and the level was determined to be about 1/10 or less for the wild-type virus. Furthermore, Sendai virus vector with suppressed VLP release was introduced into cells, and M protein subcellular localization in cells was analyzed by immunostaining using anti-M antibody. The results show that the introduction of a virus with suppressed VLP release can significantly reduce the aggregation of M protein on the cell surface compared to cells introduced with wild-type virus. Specifically, the concentration pattern (condensation pattern) of M protein is greatly reduced at high temperature (38 ℃). Subcellular localization of M and HN proteins in cells infected with SeV containing a temperature sensitive mutant M gene was precisely detected by confocal laser microscopy. The localization of the M protein on the cell surface was significantly reduced (even at low temperature (32 ℃)), and was observed to have a morphology similar to that of microtubules. At high temperatures (37 ℃), the M protein is localized on microtubules near the central body, i.e.close to the Golgi apparatus. Addition of a microtubule depolymerizing agent results in disruption of the M protein localization structure. This is seen in SeV containing the temperature-sensitive M gene and SeV containing the wild-type M gene. So that the M protein may actually act by being localized along microtubules. These findings confirm that the reduced level of secondary virus release in viruses with temperature sensitive mutations is due to insufficient intracellular localization of the M protein, a localization step that is believed to have a central role in particle formation. Thus, VLP formation can be effectively inhibited by preventing normal intracellular localization of the M protein. Furthermore, interaction with microtubules may be important for the function of M proteins. For example, secondary particle release may be reduced by disruption of subcellular localization of M protein, a step which may be achieved using genetic mutations or agents used to inhibit transport of M protein from the golgi apparatus along microtubules into the cells. Specifically, the present inventors have found that a recombinant viral vector having a reduced or eliminated particle-forming ability can be provided by preparing a viral vector containing a mutation that makes the M protein localization defective.

By eliminating the M gene from the virus, the present inventors constructed a virus in which aggregation of the M protein on the cell surface was completely inhibited in the virus-transfected cells. To this end, the inventors constructed helper cells that inducibly express wild-type M protein, which can be used to prepare M gene-deficient viruses. By using these cells, viral particles in which the RNP of the F-modified M gene deficient virus is contained in an envelope containing the wild-type M protein are recovered for the first time. The process of the invention makes it possible to produce concentrations of 1X 10⁸PFU/mL or higher, and thus provides for the first time a recombinant virus sufficient for practical use, particularly for clinical use. In addition, the virus production system of the present invention avoids the possibility of contamination with other viruses and can produce a highly safe, high-titer vector for gene therapy. By using the M-deficient SeV production system of the present invention, a practical F-modified M-deficient paramyxovirus is provided for the first time, which trans-provides the M protein using M-expressing cells.

The present inventors confirmed the actual antitumor effect in vivo using the infectious viral particles constructed as described above. M-deficient virus activated by Matrix Metalloprotease (MMP), the activity of which is enhanced in cancer, is administered to mice transplanted with cancer cells, and it is confirmed that the virus spreads throughout cancer tissues by cell fusion type infection. In cancers where wild-type virus is administered, the virus is restricted to the injection site even after several days. In contrast, the vector of the present invention shows high penetration force to cancer tissue, and the vector spreads throughout the cancer tissue. The inhibitory effect of the vectors of the present invention on cancer proliferation was evident compared to controls (no virus administration or administration of wild-type virus). Vectors targeting cells expressing MMPs have also been generated at present by retroviruses (Peng, K. -W. et al, Human Gene Therapy 8, 729-738, 1997; Peng, K. -W. et al, Gene Therapy 6, 1552-1557, 1999; Martin, F. et al, J.Virol.73, 6923-6929, 1999). However, they utilize a completely different design than the recognition sequences of the present invention. Furthermore, the goal of these reports is to specifically infect, i.e., target only, cancerous tissues. Thus, vectors for specific (intracellular) spread of infection in cancer tissues are provided for the first time by the present invention.

Furthermore, the present inventors succeeded in producing F-uncleaved virus of viral particles having uncleaved F protein on the surface of the virus by controlling the addition of protease during the formation of viral particles. As such, these viruses are not infectious; however, they show specific infectivity when treated with a protease that cleaves the F protein on the surface of a virus or when a virus is added to cells in the presence of such a protein. Such inducible infectious viral vectors allow the vector to specifically infect cancer cells that produce a particular protease.

In addition, the present inventors succeeded in using the wild-type F protein for the preparation of a vector comprising a modified F gene to develop a method for preparing virus particles using a protease that cleaves the wild-type F protein during the preparation of the vector. According to this method, virus amplification can be performed using helper cells expressing wild-type F protein and an enzyme that cleaves wild-type F protein, such as trypsin. The obtained viral particles have an envelope containing the wild-type F protein after cleavage and are infectious. However, due to the modified F gene (in which the cleavage site of the F protein encoded by the viral genome is modified), the infection spreads only in the presence of specific proteases. This method of preparing viruses using wild-type F proteins has advantages as it allows for independent integration of the modified F gene into the vector genome to produce viral particles.

As described above, the present invention provides a vector whose infection is spread only in the presence of a protease expressed in a specific tissue (e.g., cancer tissue). The vectors of the present invention do not produce significant amounts of viral particles, but the vectors can be transferred into surrounding cells by cell fusion. The vector of the present invention obtains infectivity by protease whose activity is particularly enhanced in cancer, and has a strong inhibitory effect on tumor growth. Therefore, gene therapy for cancer using these vectors is considered to be very effective.

Thus, the present invention relates to a cell fusion vector in which protease-dependent tropism is modified, and a method for preparing the vector. Specifically, the present invention relates to:

[1] a complex comprising paramyxovirus genomic RNA wherein (a) the nucleic acid encoding the M protein is mutated or deleted and (b) encodes a modified F protein having a cleavage site sequence replaced by a sequence cleavable by a protease that does not cleave the wild-type F protein, said complex further comprising the following properties:

(1) the ability to replicate the genomic RNA in a cell into which the complex has been introduced;

(2) a significant reduction or absence of viral particle production in the host's environment; and

(3) The ability to introduce the RNA into a cell contacted with a cell transfected with the complex in the presence of the protease.

[2] The complex of [1], wherein the complex is a viral particle.

[3] The complex of [2], further comprising a wild-type F protein.

[4] [1] to [3], wherein the paramyxovirus is Sendai virus.

[5] [1] to [4], wherein the protease is a protease whose activity is enhanced in cancer.

[6] [1] to [5], wherein the protease is a matrix metalloproteinase or a plasminogen activator.

[7] [1] to [6], wherein the sequence cleaved by the protease includes Pro-Leu-Gly, Pro-Gln-Gly or Val-Gly-Arg.

[8] [1] to [7], wherein the cytoplasmic domain of the wild-type F protein is partially deleted in the modified F protein.

[9] [1] to [8], wherein the modified F protein is fused to the HN protein.

[10] A method for producing a genomic RNA comprising a paramyxovirus in which (a) a nucleic acid encoding an M protein is mutated or deleted and (b) encodes a modified F protein whose cleavage site sequence is replaced with a sequence cleavable by a protease that does not cleave a wild-type F protein, said virus particle:

(1) Having the ability to replicate the genomic RNA in a cell into which the viral particle has been introduced;

(2) shows a significant reduction or absence of viral particle production in the host's internal environment; and

(3) the ability to introduce the genomic RNA into a cell that is contacted with a cell transfected with a viral particle comprising genomic RNA in the presence of the protease; the method comprises the following steps:

(i) amplifying RNPs comprising paramyxovirus N, P, and L proteins and genomic RNA in cells expressing paramyxovirus wild-type M protein; and

(ii) viral particles released into the cell culture supernatant were collected.

[11] A method for producing a virus particle comprising paramyxovirus genomic RNA in which (a) a conditionally mutated M protein is encoded, and (b) a modified F protein is encoded, the cleavage site sequence of which is replaced by a sequence cleavable by a protease that does not cleave the wild-type F protein, said virus particle:

i) Amplifying RNPs comprising paramyxovirus N, P, and L proteins and genomic RNA in a cell under permissive conditions for mutant M proteins; and

(ii) viral particles released into the cell culture supernatant were collected.

[12] The process of [10] or [11], wherein the step (i) is carried out at a temperature of 35 ℃ or less.

[13] The method of [10] or [11], further comprising the step of providing a protease that cleaves the modified F protein in at least one of steps (i) or (ii); or (iii) treating the virus particles collected in step (ii) with the protease.

[15] A therapeutic composition for cancer comprising the complex of [5] and a pharmaceutically acceptable carrier.

[16] A recombinant modified paramyxovirus F protein comprising Pro-Leu-Gly, Pro-Gln-Gly, or Val-Gly-Arg at the cleavage site and exhibiting cell-fusogenic properties in the presence of a matrix metalloproteinase or a plasminogen activator.

[17] A nucleic acid encoding the protein of [16 ].

[18] A viral particle comprising the protein of [16] or a nucleic acid encoding the protein.

[19] A fusion protein having a cell-fusogenic activity, comprising a transmembrane region of paramyxovirus F and HN proteins, wherein said F and HN proteins are cytoplasmic-bound to each other.

[20] [19] the fusion protein, wherein the cleavage site sequence of the protein is substituted by a sequence cleavable by a protease that does not cleave the wild type F protein.

[21] A nucleic acid encoding the protein of [19 ].

[22] A vector comprising the nucleic acid of [21 ].

[23] A viral particle comprising the protein of [19] or a nucleic acid encoding the protein.

The term "paramyxovirus" of the present invention refers to viruses belonging to the Paramyxoviridae family, and viruses derived therefrom. Paramyxoviruses are a group of viruses characterized by a non-segmented negative-strand RNA genome, including the subfamily Paramyxoviridae (including the genera Paramyxoviridae (also referred to as the genus Respirovirus, genus Rubulavirus) and Morbillivirus (Morblivirus)), and the subfamily Pneumoviridae (including the genera Pneumovirus and Metapneumovirus (Metapneumovirus)). specifically, paramyxoviruses that can be utilized herein include Sendai virus, Newcastle disease virus, mumps virus, measles virus, RS virus (respiratory syncytial virus), rinderpest virus, distemper virus, simian parainfluenza virus (SV5), and human parainfluenza viruses types 1, 2, and 3, etc. more specifically, for example, Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), Pestivirus (PDV), Canine Distemper Virus (CDV), dolphin Measles Virus (DMV), peste-des-petits-ruminants virus (PDPR), Measles Virus (MV), rinderpest virus (RPV), Hendra virus (Hendra), Ribovirus (Nipah), human parainfluenza virus-2 (HPIV-2), simian parainfluenza virus 5(SV5), human parainfluenza virus-4 a (HPIV-4a), human parainfluenza virus-4 b (HPIV-4b), Mumps virus (Mumps), and Newcastle Disease Virus (NDV). More preferred examples include viruses selected from the group consisting of: sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), seal distemper virus (PDV), Canine Distemper Virus (CDV), Dolphin Measles Virus (DMV), Peste des petits ruminants virus (PDPR), Measles Virus (MV), rinderpest virus (RPV), Hendra virus (Hendra), and Ribovirus (NipahVirus). The virus of the present invention preferably belongs to the subfamily Paramyxovirinae (including the genera Respirovirus, Rubulavirus, and morbillivirus), and more preferably the genus Respirovirus (also referred to as Paramyxovirus). The genus Respirovirus that can be used in the present invention includes human parainfluenza virus type 1(HPIV-1), human parainfluenza virus type 3(HPIV-3), bovine parainfluenza virus type 3 (BPIV-3), Sendai virus (also referred to as mouse parainfluenza virus type 1), simian parainfluenza virus type 10 (SPIV-10), and the like. The paramyxovirus of the present invention is most preferably Sendai virus. These viruses may be derived from natural strains, wild-type strains, mutant strains, laboratory-passaged strains, artificially constructed strains, and the like.

The terms "recombinant protein" and "recombinant virus" refer to proteins and viruses, respectively, produced by recombinant polynucleotides. The term "recombinant polynucleotide" refers to a polynucleotide that is unbound in its natural state. Specifically, the recombinant polynucleotide includes a polynucleotide whose polynucleotide strand has been artificially recombined, as well as a synthetic polynucleotide. The recombinant polynucleotide can be prepared by synthesizing the polynucleotide, treating the ribozyme, treating the ligase, and the like, using a conventional gene recombination method. Recombinant proteins can be prepared by expressing a combination of recombinant polynucleotides encoding the protein. Recombinant viruses can be prepared by the following steps: expressing a polynucleotide encoding a viral genome, said polynucleotide being constructed by genetic engineering; the virus is then reconstituted.

The term "gene" herein refers to genetic material, which includes nucleic acids such as RNA, DNA, and the like. In the present invention, a nucleic acid encoding a protein is referred to as a protein gene. However, the gene need not necessarily encode a protein, e.g., it can encode a functional RNA such as a ribozyme, antisense RNA, etc. The gene may be of natural origin or artificially designed sequence. The term "DNA" herein includes single-stranded DNA and double-stranded DNA. The term "encoding protein" refers to a polynucleotide comprising an antisense or sense ORF that encodes the amino acid sequence of the protein such that the polynucleotide is capable of being expressed under suitable conditions.

The present invention provides replication competent cell fusion vectors, which have been modified for protease-dependent tropism. In the host environment, such vectors do not release significant amounts of virus-like particles after transfection into cells, and only infiltrate into surrounding cells in the presence of specific proteases. Specifically, the vector of the present invention comprises:

a complex comprising paramyxovirus genomic RNA wherein (a) the nucleic acid encoding the M protein is mutated or deleted, and wherein (b) encodes a modified F protein having a cleavage site sequence replaced with a sequence cleavable by a protease that does not cleave the wild-type F protein, and which further comprises the following properties:

(3) the ability to introduce the RNA into a cell species that is in contact with a cell transfected with the complex in the presence of the protease. This complex is also referred to as a vector in the present invention because it has the function of replicating genomic RNA and transferring it to neighboring cells. The term "vector" refers to a vector for transferring a nucleic acid to a cell.

More specifically, the complex comprises genomic RNA of paramyxovirus and viral proteins bound to the RNA. The complex of the present invention may be composed of, for example, paramyxovirus genomic RNA and a viral protein, that is, Ribonucleoprotein (RNP). RNPs can be introduced into target cells, for example, in combination with desired transfection reagents. More specifically, this RNP is a complex comprising paramyxovirus genomic RNA, N protein, P protein and L protein. When RNPs are introduced into cells, the action of viral proteins results in transcription of a cistron encoding the viral protein from the genomic RNA; in addition, the genome itself can replicate and produce progeny RNPs. The replication of genomic RNA can be confirmed by detecting an increase in RNA copy number using RT-PCR, Northern hybridization, or the like.

More preferably, the complex is a paramyxovirus viral particle. The term "viral particle" refers to a small particle containing nucleic acid that is released from a cell by the action of viral proteins. The virus particles may be in various forms, such as spherical or rod-shaped, and differ according to the virus species. They are much smaller than cells, typically about 10nm to about 800 nm. The paramyxovirus particle has a structure such that the above RNP contains genomic RNA and viral proteins and is blocked by a lipid membrane (or envelope). The viral particles may or may not show infectivity (see below). For example, some viral particles do not inherently exhibit infectivity, but become infectivity after special treatment.

The term "paramyxovirus genomic RNA" refers to RNA that is capable of forming RNPs with paramyxovirus proteins and using these proteins to express genes from the genome to replicate nucleic acids and form progeny RNPs. The genome of paramyxovirus is a minus-strand single-stranded RNA, which is an RNA encoding a gene in an antisense mode. Typically, the paramyxovirus genome comprises a viral gene comprising an antisense series located between a 3 'leader region and a 5' trailer region. Between the Open Reading Frames (ORFs) of each gene, there are a series of sequences: a transcription termination sequence (E sequence), an intervening sequence (I sequence), and a transcription initiation sequence (S sequence). Thus, the RNA encoding the ORF of each gene is transcribed as an isolated cistron. The genomic RNA contained in the vectors of the present invention encodes (in antisense mode) nucleocapsid (N), phosphor (P), and large (L) protein. These proteins are required for the expression of the gene encoded by the RNA as well as for the autonomous expression of the RNA itself. In addition, the RNA encodes a fusion (F) protein in the antisense orientation that induces cell membrane fusion necessary for the RNA to diffuse to adjacent cells. Preferably, the genomic RNA further encodes the hemagglutinin-neuraminidase (HN or H) protein in the antisense orientation. However, in some cells, the HN protein is not essential for infection (Markwell, M.A. et al, Proc. Natl. Acad. Sci. USA 82(4), 978-. In addition, the vector can infect cells using a cell-binding protein other than HN as well as the F protein. Therefore, the vector of the present invention can be constructed using a genomic RNA that does not encode the HN gene.

Of viruses of the subfamily ParamyxovirinaeGenes are generally represented as follows: the N gene is also commonly referred to as "NP".

Respiratory virus genus mumps virus genus measles virus genus

NPNPNP

P/C/VP/VP/C/V

MMM

FFF

HNHNH

-(SH)-

LLL

For example, the database accession numbers of the nucleotide sequences of genes classified into Sendai virus of the genus Respirovirus of the family Paramyxoviridae are: the NP genes are M29343, M30202, M30203, M30204, M51331, M55565, M69046 and X17218; m30202, M30203, M30204, M55565, M69046, X00583, X17007, and X17008 for the P gene; d11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584, and X53056 for the M gene; d00152, D11446, D17334, D17335, M30202, M30203, M30204, M69046, X00152, and X02131 for the F gene; d26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, and X56131 for the HN gene; and the L gene is D00053, M30202, M30203, M30204, M69040, X00587, and X58886. Accession numbers for viral genes encoded by other viruses are as follows: the N genes are AF014953(CDV), X75961(DMV), D01070(HPIV-1), M55320(HPIV-2), D10025(HPIV-3), X85128(Mapuera), D86172 (mumps virus), K01711(MV), AF064091(NDV), X74443(PDPR), X75717(PDV), X68311(RPV), X00087(SeV), M81442(SV5), and AF079780 (Tupaia); the P genes are X51869(CDV), Z47758(DMV), M74081(HPIV-1), X04721(HPIV-3), M55975(HPIV-4a), M55976(HPIV-4b), D86173 (mumps virus), M89920(MV), M20302(NDV), X75960(PDV), X68311(RPV), M30202(SeV), AF052755(SV5), and AF079780 (Tupaia); the C genes are AF014953(CDV), Z47758(DMV), M74081(HPIV-1), D00047(HPIV-3), ABO16162(MV), X68311(RPV), AB005796(SeV), and AF079780 (Tupaia); m genes are M12669(CDV), Z30087(DMV), S38067(HPIV-1), M62734(HPIV-2), D00130(HPIV-3), D10241(HPIV-4a), D10242(HPIV-4b), D86171 (mumps virus), AB012948(MV), AF089819(NDV), Z47977(PDPR), X75717(PDV), M34018(RPV), U31956(SeV), and M32248(SV 5); genes F are M21849(CDV), AJ224704(DMV), M22347(HPN-1), M60182(HPIV-2), X05303(HPIV-3), D49821(HPIV-4a), D49822(HPIV-4b), D86169 (mumps virus), AB003178(MV), AF048763(NDV), Z37017(PDPR), AJ224706(PDV), M21514(RPV), D17334(SeV), and AB021962(SV 5); HN (H or G) is AF112189(CDV), AJ224705(DMV), U709498(HPIV-1), D000865(HPIV-2), AB012132(HPIV-3), M34033(HPIV-4A), AB006954(HPIV-4B), X99040 (mumps virus), K01711(MV), AF204872(NDV), Z81358(PDPR), Z36979(PDV), AF132934(RPV), U06433(SeV), and S76876 (SV-5). More than one strain is known per virus, and different strains may have genes comprising sequences other than those shown above.

The ORFs of these viral proteins are placed in the antisense orientation by the aforementioned E-I-S on the genomic RNA. On genomic RNA, the ORF closest to the 3 'end requires only the S sequence located between the 3' leader region and the ORF, and not the E and I sequences. On the other hand, the ORF closest to the end of genomic RNA5 'requires only the E sequence located between the 5' trailer region and the ORF, and not the I and S sequences. Both ORFs may be transcribed into the same cistron using, for example, IRES sequences. In this case, no E-I-S sequence is required between the two ORFs. In wild-type paramyxoviruses, the typical RNA genome has a 3 'leader sequence followed by sequences encoding the six ORFs for the N, P, M, F, HN, and L proteins in that order in antisense orientation, followed by a 5' trailer region at the other end. On the genomic RNA of the present invention, the structure of the viral gene is not limited thereto; however, it is preferred that the ORFs encoding the N, P, (M,) F, HN, and L proteins, in that order, be placed after the 3 'leader, followed by the 5' trailer, similar to the wild-type virus. In a particular type of paramyxovirus, the number of viral genes is not 6. However, even in this case, the position of each viral gene may be similar to that of the wild type described above, or may be appropriately changed. The ORF of the M protein will be described below. However, according to one embodiment of the vector of the invention, the ORF may be excluded or may encode a mutant M protein. Furthermore, in another embodiment of the vector of the present invention, the cleavage site of the genome-encoded F protein is modified to a sequence that can be cleaved by a protease that is not capable of cleaving the wild-type F protein (below). The genomic RNA of the invention may also encode one or more heterologous genes. Any gene of interest that is desired to be expressed in the target cell may also be used as a heterologous gene. The heterologous gene is preferably inserted into a site in a non-coding region of the genome. For example, it may be inserted between the 3 'leader sequence and the viral protein ORF closest to the 3' terminus, between each viral protein ORF, and/or between the viral protein ORF closest to the 5 'terminus and the 5' trailer region. In M gene-deficient genomes, a defective region may be inserted. When transferring a heterologous gene into a paramyxovirus, it is preferable that the polynucleotide of the insert placed in the genome has a chain length which is a multiple of 6 (Journal of virology 67(8), 4822-4830, 1993). The E-I-S sequence is located between the inserted heterologous gene and the viral ORF. Alternatively, the heterologous gene may be inserted by an IRES.

The expression level of a heterologous gene can be regulated by the type of transcription initiation sequence added upstream (3' side of the negative strand) of the gene (WO 01/18223). In addition, the expression level may be regulated depending on the position of insertion of the heterologous gene into the genome. The closer the heterologous gene is to the 3' end of the negative strand, the higher the expression level of the heterologous gene; similarly, the closer the heterologous gene is to the 5' end, the lower its expression level. Thus, the insertion site of the heterologous gene can be suitably adjusted to obtain the desired expression level of the heterologous gene and optimal combination with the upstream and downstream genes encoding the viral proteins. Generally, high expression levels are considered to be advantageous for heterologous genes. Therefore, the heterologous gene is preferably linked to a high efficiency transcription initiation sequence and inserted near the 3' end of the minus-strand genome. More specifically, it is preferably inserted between the 3 'leader and the viral protein ORF closest to the 3' end. Alternatively, a heterologous gene may be inserted between the ORF of the viral gene closest to D from the 3' end and the ORF of the next closest gene. Conversely, when high expression levels of the transgene are not preferred, the level of expression from the viral vector can be reduced by: for example, the insertion position of the gene in the vector is designed to be as close to the 5' side of the minus-strand genome as possible, or a transcription initiation sequence having low efficiency is used to produce an appropriate effect.

Any viral gene contained in the vector of the present invention may be modified from the wild-type gene, for example, to reduce the immunogenicity of the viral protein, or to enhance RNA transcription and replication efficiency. In particular, in paramyxovirus vectors, for example, the transcription or replication function can be enhanced by modifying at least one of the following replication factors: n, P, and L genes. The structural protein HN contains hemagglutinin and neuraminidase activities. If, for example, the hemagglutinin activity can be reduced, the stability of the virus in the blood can be increased. On the other hand, if, for example, the activity of neuraminidase can be modified, the infectivity can be adjusted. Furthermore, membrane fusion and/or particle formation ability can be modulated by modifying the F protein and its domains other than the cleavage site. For example, by analyzing the antigen-presenting epitopes of potential cell surface antigen molecules (e.g., F and HN proteins), viral vectors with reduced antigen-presenting ability for these proteins can be generated.

A vector having a defective auxiliary gene can be used as the vector of the present invention. For example, SeV pathogenicity in a host such as a mouse can be significantly reduced without impairing expression and replication of the gene in cultured cells by knocking out the V gene, the SeV helper gene (Kato, A. et al, J.Virol.71, 7266-. Such attenuated vectors are preferably used as viral vectors for in vivo and ex vivo (ex vivo) non-toxic gene transfer.

In a preferred embodiment, the complexes of the invention are substantially homogeneous. The term "substantially homogeneous" complex refers to a complex that is separated from a paramyxovirus RNP or viral particle that is not a complex of the invention, which is not a complex of the invention. That is, the substantially homogeneous complex of the present invention does not contain other paramyxovirus RNPs or virus particles having particle-forming ability. The term "particle-forming ability" herein refers to the ability of a vector to release infectious and/or non-infectious virus particles (referred to as virus-like particles) in cells infected with the viral vector, a process that is "secondary release". Furthermore, the complex of the present invention having a modified F protein cleavage site does not comprise a viral RNP comprising in its genome a gene encoding a wild-type F protein or an F protein having a fusion activity similar to the wild type, and does not comprise viral particles comprising this genome.

According to an embodiment of the present invention, the cleavage site sequence of the F protein encoded by the above genomic RNA is replaced with a sequence cleaved by another protease. Paramyxovirus F protein (F0) itself did not exhibit cell membrane fusion activity in its original form. However, when the extracellular region of the F0 fragment (or the ectodomain of a viral particle) is cleaved, it shows fusion activity. Two F protein fragments, the N-terminal side and C-terminal side fragments, F1 and F2, respectively, were generated by cleavage, which were linked by a disulfide bond. Cleavage of the F protein involves cleavage of the membrane F protein at a domain outside the cell membrane, resulting in the production of fragments with cell fusion forces. The term "cleavage site sequence" refers to an amino acid sequence or essential residues thereof required for cleavage by a protease. Cleavage sites for paramyxovirus F protein are known in the art and can be cleaved by trypsin-like intracellular proteases, such as furin (furin).

Furin is usually present in the golgi apparatus of most cells. The recognition motif for furin is Arg-X-Lys/Arg-Arg (RXK/RR) (two amino acids separated by "/" indicate either amino acid). The cleavage site of highly pathogenic human PIV3(RTKR), SV5(RRRR), mumps virus (RHKR), NDV (virulent strain), highly virulent strain (RQR/KR), measles virus (RHKR), RS virus (RKRR), etc., includes the sequence of these motifs. The F protein of a highly virulent strain is sensitive to proteases in all cells, and the virus of the strain is propagated in multiple steps in all organs by cleaving the F protein. Therefore, infection by these viruses is fatal. On the other hand, Sendai virus (PQSR), human PIV1(PQSR), and NDV (avirulent strain) weakly virulent strain (K/RQG/SR) having low toxicity do not contain this motif, but only Arg (which is a serine protease recognition sequence). The sequence of paramyxovirus F protein cleavage sites has been thoroughly analyzed and can be identified by those skilled in the art from the literature (see, e.g., "Uirusu-gaku (virology)", Hatanaka, M.ed., Tokyo, Asakura Shoten, 247-.

Furthermore, the cleavage site can be confirmed by identifying the cleavage site of F protein of a virus that grows in cells, tissues, individuals, etc. that can grow paramyxovirus or by identifying the cleavage site of F protein that is collected by expressing them in these cells, individuals, etc. Alternatively, the F protein may be cleaved or identified manually by treating cell surface expressed F protein with a protease, such as trypsin, that cleaves the cleavage site of the protein. According to an embodiment of the invention, the F protein comprises a modified F protein cleavage site that can be cleaved by another protease. To this end, the native cleavage sequence of the F protein may be modified by substitution, deletion, and/or insertion of one or more amino acids in order to reconstitute a sequence that can be cleaved by another protease. Modification of the amino acid sequence can be performed by conventional site-directed mutagenesis. Furthermore, the modified F protein may retain the property of being cleaved by a protease that cleaves the wild type F protein (see examples). Vectors encoding such modified F proteins have enhanced protease-dependent tropism as compared to wild-type F proteins.

Sequences cleaved by another protease may be those cleaved by the preferred protease. For example, a sequence that can be cleaved by a protease selectively expressed in a tissue or cell that is a preferred target for vector introduction can be used (WO 01/20989). By designing the vector using the F protein gene containing a sequence cleavable by a protease (active in the target tissue as described above), the vector can be subjected to good characteristics of proliferation and specific transfer to the surrounding cells under conditions having the protease activity. Vectors that specifically infiltrate only in specific tissues can be constructed, for example, by using cleavage sequences for proteases that are specifically expressed or activated in the specific tissues. In addition, vectors that specifically infiltrate under specific conditions (e.g., only in a specific disease lesion) can be constructed by utilizing cleavage sequences for proteases that are specifically expressed or activated under such conditions (e.g., disease). Both intracellular and extracellular proteases are available. For example, proteases secreted outside the cell, and proteases expressed on the membrane surface are preferable. Alternatively, the protease of choice may be any suitable protease present in the F protein transport pathway (from intracellular translation to secretion at the cell surface).

Many diseases are caused by abnormal expression of protease genes, including, for example, diseases belonging to all types of common diseases, such as metabolic diseases, circulatory diseases, inflammatory and immune diseases, infections, and malignant tumors. Specific examples include calpain in muscular dystrophy, disruption of the ubiquitin protein-protease system in autoimmune and neurological diseases, reduced expression of neprilysin in senile dementia, enhanced expression of MMPs in cancer infiltration and metastasis, pathogen-derived proteases from pathogenic microorganisms, serine proteases in hemostatic mechanisms, and aminopeptidases in placenta.

Calpains are calcium-dependent cysteine proteases which have been investigated as an enzyme involved in the hydrolysis of muscle proteins in muscle atrophy. Calpains undergo a specific activation mechanism, i.e., they are activated by binding to calcium, and calpains are thought to trigger muscle proteolysis by limited intracellular degradation of proteins, such as alpha-actin, troponin, and titin, which are important for skeletal muscle structure maintenance. For the cleavage sequence of calpain (Karlsson, J.O., et al, Cell biol. int.24, 235-243, 2000), Leu-Leu-Val-Tyr was used as the degradation substrate.

The ubiquitin protein-proteasome system is a selective and active intracellular proteolytic mechanism and is an important cell function regulatory system for signal transduction, cell cycle, and the like. The ubiquitin protein is composed of 76 amino acids, and is covalently bound to the protein by successive catalytic reactions by ubiquitin protein activating enzyme (E1), ubiquitin protein binding enzyme (E2), and ubiquitin protein ligase (E3). The ubiquitinylated proteins are degraded by the 26S proteasome. Several hundred E3 enzymes are known to exist and can be roughly classified into HECT type and RING finger type. A number of diseases suggest that these enzymes have aberrant activity. For example, Leu-Leu-Val-Tyr is used as a degradation substrate for the 26S proteasome (Reinheckel, T. et al, Arch. biochem. Biophys.377, 65-68, 2000).

Joint diseases, such as chronic rheumatoid arthritis, cause movement disorders by destroying articular cartilage tissue. The articular cartilage has a very low ability to regenerate and progressive joint destruction results from the destruction of cartilage architecture due to extracellular matrix degradation. In this chondrocyte extracellular matrix destruction, the relationship between the "disintegrin" and metalloprotease "(ADAM) molecules of MMPs and related gene families has recently been of interest. Specifically, ADAMTS (ADAM with thrombospondin motifs) molecules are considered to be essential enzymes for the degradation of cartilage proteoglycans (aggrecan) (Totorrella, M.D. et al, Science 284, 1664-1666, 1999). Sequences that lead to degradation of aggrecan by ADAMTS have been identified (Totorella, M.D., et al, J.biol.chem.275, 18566-18573, 2000).

Using the recognition sequences of these proteases, vectors specific for tissues expressing these proteases can be prepared.

Particularly preferred protease cleavage sequences of the invention include those proteases whose activity is enhanced in cancer. By constructing a vector using such a sequence, a vector that specifically infects cancer tissues can be constructed. Such a vector is very useful as a gene transfer vector for cancer therapy. A protease having "enhanced activity" in cancer is a protease that exhibits enhanced activity in a particular cancer tissue or cancer cell as compared to the activity in a corresponding normal tissue or normal cell. Wherein the term "enhanced activity" includes an increased level of expression of the protease and/or its own activity. Protease expression can be determined by the following method: such as Northern hybridization (using protease gene fragments as probes), RT-PCR (using primers that specifically amplify protease genes), or Western blotting, ELISA, and immunoblotting (using protease antibodies). The activity of the protease can be determined by degradation experiments using a protease substrate. The activity of many proteases in the body is known to be regulated by various known factors. The level of protease activity can be determined by measuring the expression level of these inhibitors.

For example, Extracellular matrix (ECM) degrading enzyme activity is especially enhanced in metastatic cancer (Nakajima, M.and Chop, A.M., Semin. cancer biol.2, 115. sup. 127, 1991; Duffy, M.J., Clin. exp. Metastasis 10, 145. sup. 155, 1992; Nakajima, M. "Extracellular matrix degrading enzyme (Japaese)", Seiki, M.ed., "Malignant transformation and metabolism of cancer", Chugai Igaku, 124. sup. 136, 1993). In animals, a matrix comprising proteins, such as collagen and proteoglycans, is formed in the intercellular spaces. Specific known components of the extracellular matrix include collagen, fibronectin, laminin, tenascin, elastin, proteoglycans, and the like. These ECMs have the function of regulating adhesion, development, metastasis, etc. of cells, as well as the function of regulating their distribution and activity by binding to soluble factors. Infiltration of ECM-degrading enzymes plays an important role in cancer metastasis, and many reports indicate that ECM-degrading enzyme inhibitors can inhibit metastasis and infiltration of basement membrane. Vectors for specific infectious infiltrating cancer tissues can be constructed by encoding modified F proteins with recognition sequences for ECM degrading enzymes to cleave at their cleavage sites.

ECM-degrading enzymes are classified into aspartic proteases, cysteine proteases, serine proteases, and metalloproteinases according to the kind of catalytic residues at their active centers. In particular, serine proteases and metalloproteases (which are neuroproteases) play an important role for ECM degradation in vivo. Serine proteases are widely distributed in microorganisms, animals, and plants. In higher animals, they are involved in a variety of biological reactions including, for example, digestion of food, blood coagulation, fibrinolysis, immune complement response, cell proliferation, ontogenesis, differentiation, aging, cancer metastasis and the like. Furthermore, the activity of serine proteases is often regulated by serine protease inhibitors (serpins), which are usually present in plasma and tissues, and quantitative or qualitative abnormalities of this inhibitor are known to cause inflammation.

Serine proteases that degrade the ECM include cathepsin G, elastin, plasmin, plasminogen activator, tumor trypsin, chymotrypsin-like neuroproteases, thrombin and the like. Plasmin is produced by limited degradation of plasminogen which exists in vivo in an inactive form. The limited degradation is regulated by Plasminogen Activator (PA) and its inhibitors, Plasminogen Activator Inhibitor (PAI). PA comprises tissue PA (tpa), which is involved in blood coagulation, and urokinase PA (upa), which is involved in ECM degradation (blast, f.and Verde, p., sen.cancer bio.1, 117-126, 1990). The function of these two PAs is inhibited by binding to PAI (Cajot, J.F. et al, Proc. Natl. Acad. Sci. USA 87, 6939-. uPA functions when bound to uPA receptors (uPAR) on the cell surface. Plasmin degrades fibronectin, tenascin, laminin, etc., but not collagen directly. However, it indirectly degrades collagen by activating a collagen-degrading enzyme by cleaving a part of a precursor of the enzyme. These enzymes generally show enhanced activity in Cancer cells and are well associated with metastatic capacity (Tanaka, N. et al, int.J. Cancer 48, 481-484, 1991; Boyd, D. et al, Cancer Res.48, 3112-3116, 1988; Hollas, W. et al, Cancer Res.51, 3690-3695, 1991; Correc, P. et al, int.J. Cancer 50, 767-771, 1992; Ohkoshi, M. et al, J.Natl. Cancer Inst.71, 1053-1057, 1983; Sakaki, Y. et al, New Horizon for Medicine (Japanese)17, 1815-1821, 1985).

Many studies have been made on the cleavage sequences of uPA and tPA (Rijken, D.C. et al, J.biol.chem.257, 2920. sup. 2925, 1982; Wallen, P. et al, Biochim.Biophys.acta 719, 318. sup. 328, 1982; Tate, K.M. et al, Biochemistry 26, 338. sup. 343, 1987) commonly used substrate sequences include VGR (Dooijewaard, G. and KLUFT, C., adv.Exp.Med.biol.156, 115. sup. klUFT, 1983) and the substrate S-2288 (lie-Pro-Arg) (Matsuo, O. et al, Jpn.J.Physiol.33, 1031. sup. 1037, 1983). Butenas et al used 54 fluorogenic substrates to identify sequences that were highly specific for tPA (Butenas, S. et al, Biochemistry 36, 2123-2131, 1997) and showed that tPA had high degradative activity for both sequences FPR and VPR. Therefore, these sequences are particularly preferred in the present invention.

Other ECM-degrading enzymes are classified as cysteine proteases or aspartic proteases. They are also involved in the metastasis and infiltration of cancer. Specific examples include: cathepsin B (Sloane, b.f., semin. cancer biol.1, 137-152, 1990) which utilizes laminin, proteoglycan, fibronectin, collagen, procollagen protease (activated by degradation) and the like as a substrate; cathepsin L (Kane, s.e. and Gottesman, m.m., semin. cancer biol.1, 127-; and cathepsin D (Rochefort, h., semin. cancer biol.1, 153-160, 1990) which utilizes laminin, fibronectin, proteoglycans, and cathepsins B and L (activated) as substrates. Cathepsins B and L are particularly highly expressed in breast cancer (Spyratos, F. et al, Lancet ii, 1115-. Disruption of their balance with their suppressors was suggested to be associated with malignant transformation of cancer (Sloane, B.F., Semin. cancer biol.1, 137-152, 1990; Kane, S.E.and Gottesman, M.M., Semin. cancer biol.1, 127-136, 1990).

Metalloproteases are metalloenzymes whose active center contains a metal element, such as Zn. Reported metalloproteases include caspases, aminopeptidases, angiotensin I converting enzyme, and collagenases. 16 or more Matrix Metalloproteinases (MMPs) have been reported for ECM-degrading metalloproteinases. Representative MMPs include collagenase-1, -2, and-3 (MMP-1, -8, and-13), gelatinases A and B (MMP-2 and-9), stromelysin (stromelysin)1, 2, and 3(MMP-3, -10, and-11), matrilysin (MMP-7), and membrane metalloprotease (MT1-MMP and MT 2-MMP). Typically, the active center of an MMP is Zn²⁺And which requires Ca for exhibiting enzymatic activity²⁺. In addition, MMPs are secreted as latent enzymes (called latent MMPs or prommps), which are activated extracellularly and degrade various ECMs present in vivo. Furthermore, the activity of MMPs is inhibited by common inhibitors, namely Tissue Inhibitors of Metalloproteinases (TIMPs). Other examples of ECM-degrading metalloproteinases include aminopeptidases that degrade ECM component proteins, such as aminopeptidase N/CD13 and aminopeptidase B. All of these proteases have been reported to be strongly associated with cancer based on experiments using inhibitors.

Among these proteases, collagenases (e.g., MMP-1, -8, and-13) cleave fibrillar collagens, i.e., collagen molecules type I, II, and III, at specific sites. Two gelatinases, gelatinase A (MMP-2) and gelatinase B (MMP-9), are known. Gelatinases are also known as type IV collagenases and, in addition to degrading type IV collagen (the major component of basement membrane), also degrade type V collagen and elastin. In addition, MMP-2 is also known to cleave type I collagen at the same location as MMP-1. MMP-9 does not degrade laminin and fibronectin; but MMP-2 degrades the proteins. Stromelysins (MMP-3 and-10) accept and degrade a wide range of substrates and degrade proteoglycans; type III, IV, and IX collagen; laminin; and fibronectin. Stromelysin (MMP-7) is a molecule lacking the hemopexin (hepexin) domain, which has the same substrate specificity as MMP-3, and has particularly high activity in degrading proteoglycans and elastin. Membrane-type metalloproteinases (MT-MMPs) (MT1-MMP, MT2-MMP, MT3-MMP, MT4-MMP, MT5-MMP, and MT6-MMP) comprise a transmembrane structure. MT-MMPs have an intervening sequence (approximately ten amino acids) located between the propeptide region and the active site. The insertion sequence comprises Arg-Xaa-Lys-Arg (Xaa is an amino acid) and is activated by cleavage by furin (an intracellular processing enzyme) during transport to the cell membrane. Known MT-MPPs include MT1-MMP (MMP-14), MT2-MMP (MMP-15), MT3-MMP (MMP-16), MT4-MMP (MMP-17), MT5-MMP (MMP-23), and MT-6-MMP (MMP-25). For example, MT1-MMP degrades type I, II, and III collagen, and MT3-MMP degrades type III collagen.

Overexpression of MMPs is known to be widely seen in cancer cells. They are classified as being caused by the cancer itself and by the cancer stromal cells. For example, interstitial collagen-degrading collagenase (MMP-1) is involved in the infiltration of cancer cells and its level of activity correlates with the metastatic potential of colon cancer (Wooley, D.E., cancer Metastasis Rev.3, 361-372, 1984; Tarin, D.et al, Br.J. cancer 46, 266-278, 1982). Furthermore, collagenase type IV (MMP-2 and MMP-9) activity is highly correlated with the metastatic potential of various epithelial cancers (Liotta, L.A. and Stetler-Stevenson, W.G., Semin. cancer biol.1, 99-106, 1990; Nakajima, M.Experimental Medicine 10, 246-. Furthermore, stromelysin (MMP-3) is also known to be associated with malignant alterations in cutaneous epitheliomas (dermal epithelial tumors) (Matrisian, L.M. and Bowden, G.T., semi.cancer biol.1, 107-. Stromerysin-3 (MMP-11) was observed to be highly expressed in breast and colon cancers (Basset, T. et al, Nature 348, 699-.

Many cleavage substrates for MMPs are known. Examples of substrate sequences which can be degraded by all MMPs include PLGLWAR (Bickett, D.M. et al, anal. biochem.212, 58-64, 1993), GPLGMRGL (Deng, S.J. et al, J.biol. chem.275, 31422-. Cleavage substrates for MMP-2 and-9 include PLGMWS (Netzel-Arnett, S. et al, anal. biochem.195, 86-92, 1991) and PLGLG (Weingarten, H. et al, Biochemistry 24, 6730-.

Recently, phage display peptide library scans demonstrated the degradation substrate sequences for MMP-9(Kridel, S.J. et al, J.biol.chem.276, 20572. 20578, 2001), MMP-2(Chen, E.I. et al, J.biol.chem.277, 4485. 4491, 2002), and MT1-MMP (Kridel, S.J. et al, J.biol.chem.in JBC Papersin Press, April 16, 2002, Manusccript M111574200). In these articles, the identified amino acid sequences are divided into four groups according to the presence or absence of the degrading ability by three MMPs. Group IV includes sequences specifically degradable by MT1-MMP, and for sequences lacking Arg, the VFSIPL and IKYHS sequences are considered substrates that are not degraded by MMP-9 and MMP-2, but are degraded by MT-MMP alone.

For example, the cleavage sequence for MMP-9 is Pro-X-X-Hy (wherein, X represents an arbitrary residue; Hy, a hydrophobic residue), and Pro-X-X-Hy- (Ser/Thr) is particularly preferred. Non-specific examples include Pro-Arg- (Ser/Thr) -Hy- (Ser/Thr) (cleavage occurs between X and Hy residues). Examples of Hy (hydrophobic residue) include Leu, Val, Tyr, Ile, Phe, Trp, and Met, but are not limited thereto. Other cleavage sequences have also been identified (see, e.g., groups I, II, IIIA, and IIIB in the following references; Kridel, S.J., et al, J.biol.chem.276, 20572-. The Pro-X-X-Hy described above can be used for MMP-2, and in addition, (Ile/Leu) -X-X-Hy, Hy-Ser-X-Leu, and His-X-X-Hy can also be used (see, e.g., groups I, II, III, and IV in the following documents: Chen, E.I., et al, J.biol.chem.277, 4485-. The cleavage sites for the MMP family, including MMP-7, MMP-1, MMP-2, MMP-9, MMP-3, and MT1-MMP (MMP-14), can be suitably selected by reference to the sequence of the native substrate proteins or by screening peptide libraries (Turk, B.E. et al, Nature Biotech.19, 661-. Examples of the 8 amino acid sequence of the cleavage site P4-P3-P2-P1-P1 ' -P2 ' -P3 ' -P4 ' (the cleavage occurs between P1 and P1 ') include VPMS-MRGG (corresponding to MMP-1), RPFS-MIMG (corresponding to MMP-3), VPLS-LTMG (corresponding to MMP-7), and IPES-LRAG (corresponding to MT1-MMP), but are not limited thereto. PLAYWAR (Nezel-Amett, S. et al, anal. biochem.195, 86, 1991) is an example of a cleavage sequence for MMP-8. Various synthetic substrates for MMPs are available and can be compared to each other (see, e.g., calbiohem  catalog, each MMP substrate in Merck).

Generally, MMP activity in tissues is regulated by the following process: latent enzyme production, latent enzyme activation, and inhibition of active enzyme by an inhibitor. MMP activity is involved in various physiological phenomena such as development and ovulation, fertilization, implantation into the endometrium, and wound healing. Dysregulation of MMP activity leads to a variety of pathologies, including, for example, cancer cell infiltration and metastasis, arthritis, gingivitis, arteriosclerosis, tumors, and fibrosis. For example, gelatinases (MMP-2 and-9) that degrade basement membrane components are known to be important for cancer metastasis. MMP-2 is activated by cleavage of pro-MMP-2 (pro MMP-2) by MT 1-MMP. On the other hand, there is another activation pathway for MMP-9, in which production of the first plasmin from plasminogen is via uPA activation of pro-MMP-3, and then activated MMP-3 activates pro-MMP-9. This pathway is involved in cancer metastasis. In order to develop the vector of the present invention into a vector targeting cancer, a sequence cleaved by the protease involved in metastasis of cancer may be introduced as a cleavage site of the F protein, in particular. Examples of such proteases include MMP-2, MMP-9, uPA, MMP-3, and MT1-MMP, more particularly MMP-2, MMP-9, and uPA.

When incorporating a protease cleavage sequence into the F protein, the protease cleavage sequence of interest is inserted into the cleavage site of the F protein, and preferably the pre-existing trypsin-like protease cleavage site is preferably degenerate. To achieve this, the amino acids around the cleavage site of the original trypsin-like protease may be replaced by the protease cleavage sequence of interest (recognition sequence). The modified F protein is cleaved by a protease of interest when expressed in a cell, and the cell membrane fusion activity of the F protein is maintained. The amino acid near the N-terminus of the F1 fragment (generated by cleavage of the F protein) is thought to play an important role in cell membrane fusion. Thus, unless cleavage is inhibited, the cleavage sequence is preferably designed such that the N-terminal sequence of the cleaved F1 fragment is identical to that of the wild-type F protein. Furthermore, in order to insert a linker into the cleavage site to induce an efficient cleavage reaction, it is preferable to add a minimum amount of amino acids to the N-terminus of the cleaved F1 fragment, as compared to wild-type F1. For example, less than five amino acids, preferably less than four amino acids, or more preferably less than three amino acids (e.g., one, two, or three amino acids) are added to the N-terminus after cleavage, as compared to wild-type F1. For example, the present invention demonstrates that addition of Met-Thr-Ser (SEQ ID NO: 1) to the N-terminus of the F1 fragment of the modified F protein after cleavage does not impair the MMP cleavage function or cell membrane fusion reaction. Therefore, the cleavage sequence is preferably designed such that Met-Thr-Ser, or a conservatively substituted sequence thereof or an amino acid comprising a partial sequence thereof is added to the N-terminus of F1 after cleavage. The term "conservative substitution" refers to a substitution between amino acids whose amino acid side chains have similar chemical properties. Specifically, Met may be substituted with Ile or Val, Thr may be substituted with Ser or Ala, and Ser may be substituted with Ala, Asn, or Thr. The substitution of the amino acid at each position can be performed independently.

More specific examples of preferred cleavage sequences for MMP-2 and MMP-9 include sequences comprising Pro-Leu/Gln-Gly (SEQ ID NO: 2). This sequence is a sequence commonly used as a substrate in synthetic substrates (Netzel-Arnett, S. et al, anal. biochem.195, 86-92, 1991), and the F protein is designed such that after cleavage of the modified F protein, this sequence is located at the C-terminus of the F2 fragment. To this end, the sequence containing the C-terminal amino acid of the F2 fragment was replaced by a sequence containing Pro-Leu/Gln-Gly after cleavage of the wild type F protein. The original sequence of one or more amino acids corresponding to the C-terminus of the F2 fragment of the F protein was appropriately deleted, and Pro-Leu/Gln-Gly was subsequently inserted (i.e., substituted). The number of amino acids deleted is equal to the number of amino acids inserted (e.g., three amino acids), or may be selected in the range of 0-10 amino acids. The F protein may be prepared so that the N-terminus of F1 is not impaired as long as the cleavage step by protease and membrane fusion are not impairedThe end is directly connected with the downstream of Pro-Leu/Gln-Gly. However, in the F protein of Sendai virus, the cleavage sequence and the F1 fragment are preferably linked by a suitable spacer. Examples of particularly preferred cleavage sequences comprising such spacers are those comprising Pro-Leu/Gln-Gly-Met-Thr-Ser (SEQ ID NO: 3) or Pro-Leu/Gln-Gly-Met-Thr (SEQ ID NO: 4). Met, Thr, and Ser may be conservatively substituted with other amino acids. More preferred examples of proteins include modified F proteins in which 1-10 residues (e.g., 1, 2, 3, 4, 5 or 6 residues) linked sequentially in the N-terminal direction from the C-terminal amino acid of cleaved F2 are substituted with a sequence comprising Pro-Leu/Gln-Gly-Met-Thr-Ser or Pro-Leu/Gln-Gly-Met-Thr. For example, as for Sendai virus F protein, the preferred protein may be one in which the sequence corresponding to the four C-terminal amino acids of the F2 fragment in the wild-type F protein (SEQ ID NO: 5) is different (although the sequences of different strains are different, usually, the sequence is different from strain to strain ¹¹³Pro-Gln-Ser-Arg¹¹⁶↓) is replaced by Pro-Leu/Gln-Gly-Met-Thr-Ser.

Any other desired sequence described herein can be used as a cleavage sequence for MMPs. Peptide libraries were used to analyze the substrate specificity of various MMPs (Turk, B.E., et al, Nature Biotech.19, 661-667, 2001). Detailed analyses were performed for MMP-2(Chen, E.I. et al, J.biol.chem.277(6), 4485-. In particular for MMP-9, the consensus sequence from P3 to P2 '(P3-P2-P1-P1' -P2 '; cleavage occurs between P1-P1') is proposed to be Pro-X-Hy- (Ser/Thr) (X ═ any residues; Hy ═ hydrophobic residues). This consensus sequence also matches one of the consensus sequences recommended for MMP-2 (Pro-X-X-Hy), and is therefore considered a good design to achieve specificity for MMP-2 and MMP-9. Therefore, in this respect, the above-mentioned sequence (Pro-Leu/Gln-Gly-Met-Thr-Ser or Pro-Leu/Gln-Gly-Met-Thr) is supported as a preferred embodiment. Specifically, the sequence of the F protein cleavage site preferably comprises Pro-X-X-Hy-Thr/Ser, and more preferably comprises Pro-X-X-Hy-Thr/Ser-Thr/Ser ("Thr/Ser" means Thr or Ser). For example, Pro-Leu-Gly-Leu-Trp-Ala and Pro-Gln-Gly-Leu-Tyr-Ala which do not pair with Pro-X-X-Hy-Thr/Ser are not preferred (FIG. 44). By inserting a peptide that pairs with the Pro-X-X-Hy-Thr/Ser sequence into the F protein cleavage site, a vector that exhibits high wettability in the presence of MMP can be constructed.

Another example of preferred cleavage sequences include those that are cleavable by plasminogen activator. Specific examples of cleavage sequences for uPA and tPA include sequences containing Val-Gly-Arg. The F protein was designed such that this sequence was located at the C-terminus of the F2 fragment of the modified F protein after cleavage. For this reason, after cleavage of the wild-type F protein, the sequence comprising the C-terminal amino acid of the F2 fragment may be substituted by a sequence comprising Val-Gly-Arg (SEQ ID NO: 6). More preferred examples of preferred proteins include modified F proteins in which 1 to 10 residues (e.g., 1, 2, 3, 4, 5 or 6 residues) sequentially linked in the N-terminal direction from the C-terminal amino acid of cleaved F2 are substituted with Val-Gly-Arg or a sequence comprising the sequence. For example, among Sendai virus F proteins, examples include an F protein in which the sequence corresponding to the three C-terminal amino acids of the F2 fragment in the wild-type F protein (SEQ ID NO: 5) is different (although the sequences of different strains are different, usually, the sequence is different¹¹⁴Gln-Ser-Arg¹¹⁶And ↓ (SEQ ID NO: 7)) is substituted by Val-Gly-Arg.

To efficiently identify modified F proteins that exhibit fusogenic properties in the presence of a particular protease, any experimental system using a plasmid vector can be used (example 31). Specifically, a plasmid vector expressing the modified F protein is transfected into cells, and the resulting cells are cultured in the presence of a protease to detect syncytia formation. The modified F protein encoded by this plasmid (which leads to syncytia formation) was cleaved with a protease to examine whether it showed fusion activity. For example, to determine MMP cleaved F protein, HT1080 cells expressing MMPs were used. Alternatively, MMPs can be added to the culture system. Modified F proteins with fusogenic properties can be readily obtained using the experimental systems developed in the present invention.

Vectors encoding modified F proteins can introduce the genomic RNA contained in the vector into cells that are relieved of cells transfected with the vector, a process that depends on the presence of a protease that cleaves the modified F protein. The action of the cleaved F protein results in cell fusion between the contacted cells and diffusion of RNPs to the fused cells. That is, the vector of the present invention does not form viral particles, however, it can transfer the vector to a localized area due to the infiltration of the vector into, for example, the contacted cells as described above. The protease may be expressed intracellularly or extracellularly, or may be added exogenously.

The modified F proteins provided by the invention exhibit specific protease dependent cellular fusogenic properties. By using this protein, viral vectors, drug-and gene-delivery vectors such as liposomes, which cause cell fusion or specific infection only in the presence of proteases, can be constructed. For example, by assembling a gene that modifies the F protein, which is cleaved by a protease specifically expressed in cancer cells, with the F gene of an adenovirus vector containing the F and HN genes (Galanis, E. et al, hum. Gene ther.12, 811-821, 2001), a vector that can cause cell fusion in the presence of a specific protease can be developed. Furthermore, for example, when retroviruses are pseudotyped (pseudotyping) with F and HN proteins (Spiegel, M. et al, J Virol.72(6), 5296-. As described above, the modified F proteins provided by the present invention and the nucleic acids encoding them can be used to develop various protease-dependent vectors in addition to the vectors of the present invention.

Furthermore, the present invention provides paramyxovirus vectors comprising a modified F protein that increases the cell fusogenic force by deleting the cytoplasmic domain. Part of the amino acids of the cytoplasmic region are deleted so that the amino acids from positions 0 to 28, preferably 1 to 27, more preferably 4 to 27 are present in the cytoplasmic region of the modified F protein. The term "cytoplasmic domain" refers to the cytoplasmic side of membrane proteins, and in the F protein, it corresponds to the C-terminal domain of The Membrane (TM) domain (see FIG. 42). For example, the cytoplasmic region of the F protein comprises amino acids 6-20, preferably 10-16, and more preferably 13-15, which show a significantly higher level of cellular fusogenicity compared to the wild-type F protein. Thus, paramyxovirus vectors comprising a modified F protein, the cytoplasmic region of which comprises about 14 amino acids, were prepared, thereby obtaining vectors with higher cell fusogenicity compared to vectors obtained with wild-type F protein. Preferably, the deleted F protein lacks 10 or more, more preferably 20 or more, more preferably 25 or more, and more preferably 28 or more of the C-terminal amino acids of the wild-type F protein. According to a most preferred aspect, the cytoplasmic domain deleted F protein lacks about 28 of the C-terminal amino acids of the wild-type F protein. Paramyxovirus vectors whose genome contains genes encoding these cytoplasmic domain-deleted F proteins have higher cell fusion power than conventional vectors and, therefore, are more strongly infiltrated into surrounding cells. Modification of the F protein cleavage site as described herein can result in a vector that exhibits high infiltration capacity only in the presence of a particular protease.

The invention also relates to a fusion protein consisting of two spike proteins carried by a paramyxovirus. Paramyxoviruses have a protein that functions in cell fusion (referred to as "F" protein) and a protein that functions in cell adhesion (referred to as "HN" or "H" protein). Herein, the former is generally referred to as F protein, and the latter is referred to as HN protein. These two proteins are expressed as fusion proteins, which show a very strong fusogenic force compared to when the proteins are expressed separately. In the fusion protein, the proteins are bound by a portion of their cytoplasmic domains. Specifically, the fusion protein comprises an F protein at the N-terminus, and an HN (or H) protein at the C-terminus thereof. When these proteins are fused, all the proteins are fused to each other, or alternatively, an F protein lacking part or all of the cytoplasmic domain may be fused to HN (or N). In the latter case, the number of amino acid residues from the downstream of the TM region of the F protein to the HN (or H) protein is 5 or more, preferably 10 or more, more preferably 14 or more, and still more preferably 20 or more. For example, when an F protein lacking the cytoplasmic domain is fused to an HN (or H) protein, the length is preferably adjusted by adding a linker peptide of an appropriate length to the C-terminus of the F protein portion. Specifically, it is preferable to use a cytoplasmic domain deleted F protein (containing 14 residues of the cytoplasmic domain) fused to the HN (or H) protein through any linker peptide. The amino acid sequence of the linker peptide is not particularly limited; however, it is preferred to use a polypeptide which does not have significant physiological activity, and a suitable example including this polypeptide is shown in FIG. 43(SEQ ID NO: 80).

The invention also relates to nucleic acids encoding these fusion proteins, and expression vectors comprising these nucleic acids. Cells transfected with these expression vectors exhibit strong fusogenicity and form syncytia by fusion with surrounding cells. The expression vector is not particularly limited, and includes, for example, plasmid vectors and viral vectors. For DNA vectors, it is preferred to use a strong promoter such as the CAG promoter (a chimeric promoter comprising the chicken β -actin promoter and the CMV enhancer) (Niwa, H., et al, Gene 108, 193-199, 1991). Viral vectors expressing the proteins of the invention produce strong fusions in cells after transfection. Examples of suitable viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, minus-strand RNA viral vectors, herpes simplex viral vectors, retroviral vectors, lentiviral vectors, Semliki forest (Semliki forest) viral vectors, Sindbis viral vectors, vaccinia viral vectors, fowlpox viral vectors, and other preferred viral vectors. Paramyxovirus vectors expressing the proteins of the present invention show high infiltration into various tissues. Specifically, using M gene-deleted paramyxovirus vectors encoding the fusion proteins of the present invention with modified F protein cleavage sites, vectors can be produced that induce strong cell fusion in specific tissues.

These recombinant viral vectors can be prepared according to methods known to those skilled in the art. For example, the adenoviral vectors most commonly used in gene therapy can be constructed by the Methods of Saito et al and other Methods (Miyake et al, Proc. Natl. Acad. Sci. USA, 93, 1320-24, 1996; Kanegae et al, actaPaediatotr. Jpn., 38, 182-188, 1996; Kanegae et al, "Baiomanyuaru shiriizu4-Idenshi-donyu to Hatsugen Kaisekiho (Biomanual Series 4: Methods for gene transfer, expression, and analysis)", Yodosha, 43-58, 1994; Kanegae et al, Cell Engineering, 13(8), 757-763, 1994). Furthermore, for example, retroviral vectors (Wakimoto et al, Protein Nucleic acid and Enzyme (Japanese)40, 2508-2513, 1995), adeno-associated viral vectors (Tamaki et al, Protein Nucleic acid and Enzyme (Japanese)40, 2532-2538, 1995) can be prepared by conventional methods. As specific methods for producing other viral vectors capable of transferring genes into mammals, methods for producing recombinant vaccinia viruses are known, which are described in International publication No. Hei 6-502069 of the Japanese translation edition published, examined published Japanese patent applications (JP-B) Hei 6-95937, and JP-B Hei 6-71429. Known methods for producing recombinant papillomaviruses include those described in JP-B Hei 6-34727, and published Japanese translation edition International publication Hei 6-505626. Further, known methods for producing recombinant adeno-associated viruses and recombinant adenoviruses are described in Japanese published patent application unexamined (JP-A) Hei 5-308975 and published Japanese translation edition International publication Hei 6-508039, respectively.

In the RNA genome, which is contained in the vector provided in one aspect of the present invention, the gene encoding the matrix (M) protein (i.e., the M gene) is mutated or deleted. According to the present invention, the cleavage site of the F protein is modified to a sequence cleavable by another protease, and M-priming is mutated or deleted to inhibit the particle-forming ability. Thus, vectors with entirely new properties (which do not release viral particles and only infiltrate a panel of cells expressing a particular protease) were successfully inverted. Mutations in the M gene abolish or significantly reduce granule-forming activity in the host's internal environment. Such mutations in cells expressing the M protein can be identified by detecting reduced aggregation of the protein at the cell surface (see examples).

According to the invention, the most efficient modification of the secondary release of the particles (i.e. the release of VLPs) is known, which is demonstrated by the absence of M protein. This fact is supported by the report on the role of the M protein in virion formation in Sendai virus (SeV) and other negative-strand RNA viruses. For example, strong expression of the M protein in Vesicular Stomatitis Virus (VSV) was found to cause VLP budding (Justice, p.a. et al, j.virol.69, 3156-; similarly, parainfluenza VLP formation has been reported to occur only when the M protein is overexpressed (Coronel, e.c. et al, j.virol.73, 7035-7038, 1999). Although this VLP formation by the M protein alone cannot be observed in all negative-strand RNA viruses, it is recognized that the M protein serves as a virion-forming core in negative-strand RNA viruses (Garoff, H. et al, Microbiol. mol. biol. Rev.62, 1171-1190, 1998).

The specific role of the M protein in virion formation is summarized below: viral particles are formed in so-called lipid rafts on cell membranes (Simons, K.and Ikonen, E., Nature 387, 569-572, 1997). These were originally identified as lipid fragments (Brown, D.A. and Rose, J.K., Cell 68, 533-544, 1992) that were insoluble in non-ionic detergents (e.g., Triton X-100). The formation of virions in lipid rafts has been shown for the following viruses: influenza virus (Ali, A. et al, J.Virol.74, 8709-8719, 2000), measles virus (MeV; Manie, S.N. et al, J.Virol.74, 305-. At these sites of lipid rafts, the M protein enhances virion formation, concentrating the envelope protein (also known as spike protein) and Ribonucleoprotein (RNP). In other words, the M protein may have an effect on viral assembly and shoot driver (Cathomen, T. et al, EMBO J.17, 3899-. Indeed, the M protein was shown to bind to the cytoplasmic tail of influenza spike proteins (Zhang, J. et al, J.Virol.74, 4634-one 4644, 2000), SeV (Sanderson, C.M. et al, J.Virol.67, 651-one 663, 1993). It also binds to RNP of influenza virus (Ruigrok, R.W. et al, Virology 173, 311-316, 1989), parainfluenza virus, SeV (Coronel, E.C. et al, J.Virol.75, 1117-1123, 2001), and the like. Furthermore, for SeV (Heggeness, M.H. et al, Proc. Natl. Acad. Sci. USA 79, 6232-. Thus, the M protein is thought to act as a driving force for viral assembly and budding due to its ability to function in binding to many viral components and lipids.

In addition, some reports suggest that modification of the envelope protein (spike protein) may also inhibit the release of VLPs. The following experimental examples are specifically reported where virion formation was indeed inhibited: deficiency of the G protein in Rabies Virus (RV) leads to a reduction in VLP formation 1/30 (Mebation, T. et al, Cell 84, 941-951, 1996). This level decreases to 1/500,000 or less when the M protein is deficient (Mebatson, T. et al, J.Virol.73, 242-390250, 1999), and in addition, cell-cell fusion is enhanced when the M protein is deficient for measles virus (MeV) (Catthomen, T. et al, EMBO J.17, 3899-3908, 1998). This is presumably due to the inhibition of virion formation (Li, Z et al, J.Virol.72, 3789-3795, 1998). Furthermore, similar fusion enhancements occur when mutations occur in the cytoplasmic tail (cytoplasmic-side tail) of the F or H proteins (Cathomen, T. et al, J.Virol.72, 1224-1234, 1998). Thus, introduction of a mutation resulting in deletion of the cytoplasmic tail of only the F and/or HN proteins can inhibit granule formation. However, since many VLPs were reported to exist in either F-deficient form (WO 00/70070) or HN-deficient form (Stricker, R.andRoux, L., J.Gen.Virol.72, 1703-1707, 1991), the effect of modifying these spike proteins was limited, especially in SeV. In addition, SeV is explained below: when SeV proteins F and HN are in the secretory pathway (specifically, when they are located in the Golgi apparatus, etc.), the cytoplasmic tail (of the F and HN proteins) binds to the M protein (Sanderson, C.M. et al, J.Virol.67, 651-663, 1993; Sanderson, C.M. et al, J.Virol.68, 69-76, 1994). Thus, this binding is believed to be important for efficient transfer of the M protein to the cell membrane lipid rafts in which the virions are formed. The M protein is thought to bind to F and HN proteins in the cytoplasm and to cause transfer to the cell membrane via the F and HN secretory pathways. As mentioned above, the M protein plays an important role in the formation of viral particles. The use of a modified M protein gene, which eliminates the aggregation of M protein on the cell surface, can result in a vector that does not have the ability to form virions.

Subcellular localization of the M protein can be determined by cell fractionation, or the localization of the M protein can be detected directly using immunostaining. In immunostaining, for example, M protein stained by a fluorescent-labeled antibody can be observed under a confocal laser microscope. Alternatively, after the cells are lysed, cell fractions can be prepared by using known cell fractionation methods, and can be subsequently localized by identifying M protein-containing fractions using, for example, immunoprecipitation or Western blotting, using M protein antibodies. Virions are formed in so-called membrane lipid rafts, i.e., lipid fragments that are insoluble in non-ionic detergents such as Triton X-100. Due to its ability to bind to the spike protein RNP, as well as to the M protein itself, and further to bind lipids, it is believed to be involved in the aggregation of viral components in lipid rafts. Thus, the putative M protein (detected by electrophoresis using lipid raft fragments) reflects aggregated M protein. That is, when the amount of detectable M protein is decreased, the aggregation of M protein on the cell surface is also decreased. The aggregation of the M protein on the cell surface can be directly observed using the immunocytological staining method used by the present inventors to detect subcellular localization. The method utilizes anti-M antibodies that can be used for immunocytological staining. When the M protein was aggregated, a very dense image was observed near the cell membrane when the study was performed using this method. When the M protein did not aggregate, there was neither a detectable concentration pattern nor a clear outline of the cell membrane. Thus, cell surface M protein aggregation is considered to be reduced when little or no concentration pattern is detected, cell membrane contours are unclear, and a light staining is observed through the cytoplasm.

Mutant M proteins with significantly reduced cell surface aggregation activity are considered to have significantly lower particle formation capacity than wild-type M proteins. The reduction in particle forming ability in the virus is statistically significant (e.g., at a significant level of 5% or less). Statistical tests are performed by using, for example, Student's t-test or Mann-Whitney U-test. The particle-forming ability of the viral vector comprising the mutant M gene in the host environment is reduced to a level of preferably 1/5 or less, more preferably 1/10 or less, more preferably 1/30 or less, more preferably 1/50 or less, more preferably 1/100 or less, more preferably 1/300 or less, and more preferably 1/500 or less. Most preferably, the vectors of the invention are substantially devoid of viral vector production capacity in the host environment. The term "substantial absence" means that no production of viral particles is detected in the host's internal environment. In this case, the virus particles are present at a concentration of 10³Less than ml, preferably 10²Less than ml, more preferably 10¹Below/ml.

The presence of virus particles can be directly confirmed by observation under an electron microscope or the like. Alternatively, viral nucleic acids or proteins can be used as indicators to detect or quantify them. For example, genomic nucleic acid in a viral particle can be detected and quantified by commonly used nucleic acid detection methods such as Polymerase Chain Reaction (PCR). Alternatively, viral particles comprising a heterologous gene can be quantified by infecting them into cells and detecting the expression of the gene. Non-infectious viral particles can be quantified by detecting gene expression after introducing the particles into cells using a transfection reagent. The viral particles of the invention comprise particles that are not infectious, such as VLPs.

In addition, the efficacy of a virus can be determined, for example, by measuring the Cell Infection Units (CIU) or the clotting activity (HA) (WO 00/70070; Kato, A. et al, Genes Cells 1, 569-579, 1996; Yonemittsu, Y. and Kaneda, Y., "hemagglutinating viruses of Japan-lipid-mediated gene delivery to Cells," Ed. by Baker, A.H., Molecular Biology of Molecular diseases, methods in Molecular medicine, Humana Press, 295-. For vectors labeled with a marker gene, such as the GFP gene, the viral titer can be quantified by directly counting infected cells using the marker as a marker (e.g., as GFP-CIU), as described in the examples. The titer thus determined can be considered to be equivalent to CIU (WO 00/70070). For example, when a cell is transfected with a sample containing viral particles, the loss of the production power of the viral particles can be demonstrated by the absence of a detectable infectivity titer. Virus particles (VLPs) without infectivity can be detected by transfection using lipofectin. Specifically, for example, DOSPER liposome transfection reagents (Roche, Basel, Switzerland; Cat. No.1811169) can be used. 100 ml of the solution with or without virus particles can be mixed with 12.5. mu.l DOSPER and kept at room temperature for 10 minutes. The mixture was shaken every 15 minutes and transfected into cells that were cultured to a full bottom in 6-well plates. VLPs can be detected the next day after transfection by the presence or absence of cells after transfection.

The term "environment within the host" refers to the environment within the hostWhere wild-type paramyxovirus (from which the vector is derived), typically proliferates naturally, or refers to an environment that allows for the propagation of an equivalent virus. The host environment may be, for example, optimal growth conditions for the virus. When the host of the paramyxovirus is a mammal, the host internal environment refers to the environment within the body of the mammal, or an equivalent thereof. I.e., a temperature of about 37 deg.C to 38 deg.C (e.g., 37 deg.C), corresponding to the temperature in a mammal. Examples of in vivo conditions include normal cell culture conditions, more specifically in a humid environment, in medium with or without serum (pH6.5 to 7.5), 37 ℃, 5% CO₂。

Important differences in the activity of modified M proteins due to environmental conditions include conditional mutations of the M protein, such as temperature sensitive mutations. The term "conditional mutation" refers to a post-mutation genotype that exhibits "loss of function" in the host environment, while exhibiting functional activity in another environment. For example, it may be preferable to use a gene encoding a temperature-sensitive mutant M protein, most or all of whose function is lost at 37 ℃ but restored at a lower temperature. The term "temperature-sensitive mutation" refers to a mutation whose activity is significantly reduced at high temperatures (e.g., 37 ℃) than at low temperatures (e.g., 32 ℃). The present inventors succeeded in producing viral particles whose particle-forming ability was greatly reduced at 37 ℃ (this temperature corresponds to the host internal environment) using a temperature-sensitive mutant of the M protein. The M protein mutant aggregates on the cell surface under low temperature conditions (e.g., 32 ℃); however, at the normal body temperature of the host (37 ℃), the protein loses its polymerization power and is unable to form viral particles. A vector comprising a nucleic acid encoding such a temperature-sensitive M protein mutant in the genome is preferable as the vector of the present invention. The M protein of such a viral vector encodes a conditionally mutated M protein that exerts its function under conditions of M protein function, i.e. permissive conditions, to form viral particles. When viral particles produced in this way are infected in the normal environment, the M protein cannot function and therefore no particles are formed.

The temperature-sensitive M gene mutation is not particularly limited, but includes, for example, at least one amino acid site selected from the group consisting of: g69, T116, and A118 of Sendai virus M protein preferably have two sites selected from them, and more preferably have all three sites. Other negative-strand RNA virus M proteins containing homologous mutations may also be suitably used. Herein, G69 refers to the 69 th amino acid in the M protein, i.e., glycine, T116 refers to the 116 th amino acid in the M protein, i.e., threonine, and a183 refers to the 183 th amino acid in the M protein, i.e., alanine.

The gene encoding the M protein (i.e., the M gene) is widely conserved in negative-strand RNA viruses and is known to interact with viral nucleocapsid and envelope proteins (Garoff, H. et al, Microbiol. mol. biol. Rev.62, 117-190, 1998). The amino acid sequence 104-119(104-KACTDLRITVRRTVRA-119/SEQ ID NO: 45) of the SeV M protein was predicted to form an alpha-helix and was identified as an important region for the formation of viral particles (Mottet, G. et al, J.Gen.Virol.80, 2977-2986, 1999). This region is widely conserved in negative-strand RNA viruses. The amino acid sequence of the M protein is similar in the minus-strand RNA virus. In particular, the term M protein known in viruses of the Paramyxovirinae sub-family is generally a protein having 330-380 amino acid residues. Their structures are similar over the entire region, but the C-terminal half has particularly high homology (Gould, A.R., R., Virus Res.43, 17-31, 1996; Harcourt, B.H., et al, Virology 271, 334-. Thus, for example, amino acids homologous to G69, T116 and A183 of SeV M protein can be easily identified.

The amino acid residues at the homologous positions of the other negative-strand RNA virus M proteins corresponding to G69, T116 and A183 of the SeV M protein can be identified by alignment with the SeV M protein by a person skilled in the art using amino acid sequence homology search programs such as BLAST or alignment-forming programs such as CLUSTAL W, which include alignment-forming functions. The M protein homology site corresponding to G69 in the M protein of SeV includes G68 in human parainfluenza virus-1 (HPIV-1); human parainfluenza virus-3 (HPIV-3); g70 for seal distemper virus (PDV) to neutralize Canine Distemper Virus (CDV); g71 in Dolphin Measles Virus (DMV); g70 in Peste des petits ruminants virus (PDPR), Measles Virus (MV) and rinderpest virus (RPV); g81 in Hendra virus (Hendra) and libaravirus (Nipah); g70 in human parainfluenza virus-2 (HPIV-2); e47 in human parainfluenza virus-4 a (HPIV-4a) and human parainfluenza virus-4 b (HPIV-4 b); and E72 in Mumps virus (Mumps). (the description in parentheses indicates abbreviation; letters and numbers indicate amino acids and positions). The M protein homology site corresponding to T116 in the M protein of SeV includes T116 in human parainfluenza virus-1 (HPIV-1); t120 in human parainfluenza virus-3 (HPIV-3); t104 in seal distemper virus (PDV) and Canine Distemper Virus (CDV); t105 in Dolphin Measles Virus (DMV); peste des petits ruminants virus (PDPR), Measles Virus (MV), T104 in rinderpest virus (RPV); t120 in Hendra virus (Hendra) and Nipah virus (Nipah); t117 in human parainfluenza virus-2 (HPIV-2) and simian parainfluenza virus 5(SV 5); t121 in human parainfluenza virus-4 a (HPIV-4a) and human parainfluenza virus-4 b (HPIV-4 b); t119 in Mumps virus (Mumps); and S120 in Newcastle Disease Virus (NDV). The M protein homology site corresponding to A183 in the M protein of SeV is A183 in human parainfluenza virus-1 (HPIV-1); f187 in human parainfluenza virus-3 (HPIV-3); y171 in seal distemper virus (PDV) and Canine Distemper Virus (CDV); y172 in Dolphin Measles Virus (DMV); y171 in peste des petits ruminants virus (PDPR), Measles Virus (MV) and rinderpest virus (RPV); y187 in Hendra virus (Hendra) and libavirus (Nipah); y184 in human parainfluenza virus-2 (HPIV-2); f184 in simian parainfluenza virus 5(SV 5); f188 in human parainfluenza virus-4 a (HPIV-4a) and human parainfluenza virus-4 b (HPIV-4 b); f186 in Mumps virus (Mumps); and Y187 in Newcastle Disease Virus (NDV). Among the above viruses, viruses suitable for use in the present invention include those comprising a genome encoding an M protein mutant in which an amino acid residue is substituted at one of the three positions, preferably at any two of the three positions, and more preferably at all three positions.

Amino acid mutations include mutations of any other desired amino acid. However, it is preferred to carry out the substitution with an amino acid whose side chain has different chemical properties. Amino acids can be grouped into basic amino acids (e.g., lysine, arginine, histidine); acidic amino acids (e.g., aspartic acid, glutamic acid); uncharged polar amino acids (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine); non-polar amino acids (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan); branched amino acids (e.g., threonine, valine, isoleucine); and aromatic amino acids (e.g., tyrosine, phenylalanine, tryptophan, histidine). An amino acid residue belonging to a particular amino acid group may be substituted with another amino acid belonging to a different group. Specific examples include, but are not limited to: substitution of an acidic or neutral amino acid with a basic amino acid; substitution of a non-polar amino acid with a polar amino acid; substituting an amino acid having a molecular weight less than the average value with an amino acid having a molecular weight greater than the average molecular weight of the naturally occurring amino acids in 20; and conversely, substituting an amino acid having a molecular weight greater than the average value with an amino acid having a molecular weight less than the average value. For example, sendai virus M protein comprising a mutation selected from G69E, T116A, and a183S or other paramyxovirus M protein comprising a mutation at a desired homologous position may be suitably used. Herein, G69E refers to a mutation in which the amino acid glycine of M protein at position 68 is substituted with glutamic acid, T116A refers to a mutation in which the amino acid threonine of M protein at position 116 is substituted with alanine, and A183S refers to a mutation in which the amino acid alanine of M protein at position 183 is substituted with serine. In other words, the G69, T116 and A183 in the M protein of Sendai virus and the homologous M protein sites in other viruses can be replaced by glutamic acid (E), alanine (A), and serine, respectively. These mutations are preferably used in combination, and particularly preferably comprise all three of the above mutations. M gene mutagenesis can be performed according to known mutagenesis methods. For example, as described in the examples, mutations can be introduced using oligonucleotides containing desired mutations.

For example, for measles virus, the M gene sequence of temperature sensitive strain P253-505, in which the epitope sequence of the anti-M protein monoclonal antibody has been altered, can be used (Morikawa, Y., et al, Kitasato Arch. exp. Med.64, 15-30, 1991). In addition, threonine at residue 104 of the measles virus M protein, or threonine at residue 119 of the mumps virus M protein (the above residues correspond to threonine at residue 116 of the SeV M protein), may be substituted with any amino acid (e.g., alanine).

According to a more preferred embodiment, the vector of the invention comprises an M gene defect. The term "M gene deficiency" refers to lack of M protein function, including the case where the vector has an M gene comprising a function-deficient mutation, and the case where the M gene is deleted from the vector. A functionally deficient M gene mutation can be generated, for example, by deletion of the M gene protein coding sequence, or insertion of another sequence. For example, a stop codon can be inserted into the M protein coding sequence (WO 00/09700). More preferably, the vectors of the invention are completely devoid of M protein coding sequences. Unlike vectors encoding the conditional mutant M protein, Open Reading Frame (ORF) vectors without the M protein are unable to produce viral particles under any conditions.

To prepare the vector of the present invention, a cDNA encoding paramyxovirus genomic RNA is transcribed in the presence of viral proteins required for RNP (which comprises paramyxovirus genomic RNA) reconstitution, i.e., N, P and L proteins. Viral RNPs can be reconstituted by forming either the negative strand genome (i.e., the same antisense strand as the viral genome) or the positive strand (the sense strand encoding the viral proteins). To improve the efficiency of reconstitution, the formation of the plus strand is preferred. The 3 'leader and 5' trailer sequences at the ends of the RNA preferably reflect the native viral genome as accurately as possible. To precisely control the 5' end of the transcription product, the T7 RNA polymerase recognition sequence can be used as a transcription initiation site to express RNA polymerase in cells. The 3' -end of the transcript can be controlled by: for example, an autonomously cleaving ribozyme was encoded at the 3' end to ensure its precise cleavage (Hasan, M.K. et al, J.Gen.Virol.78, 2813-2820, 1997; Kato, A. et al, EMBO J.16, 578-587, 1997; Yu, D. et al, Genes Cells 2, 457-466, 1997).

The cloning site for inserting the heterologous gene into the cDNA may be designed so as to facilitate insertion of the heterologous gene. The site may be inserted at any preferred position in the non-coding region of the protein of the genome. In particular, the site may be inserted between the 3 'leader and the viral protein ORF closest to the 3' terminus, between the viral protein ORFs, and/or between the viral protein ORFs closest to the 5 'terminus and the 5' trailer. In an M gene-deficient genome, the cloning site may be designed to be located at the deletion site of the M gene. The cloning site may be, for example, a recognition sequence for a restriction enzyme. The cloning site may be a so-called multiple cloning site comprising a number of restriction enzyme recognition sequences. The cloning site may be present at multiple sites in the genome, respectively, so that a large number of heterologous genes can be inserted at different locations in the genome.

Recombinant viral RNPs lacking particle-forming ability can be constructed as described, for example, in "Hasan, M.K. et al, J.Gen.Virol.78, 2813-2820, 1997", "Kato, A. et al, EMBO J.16, 578-587, 1997" and "Yu, D. et al, Genes Cells 2, 457-466, 1997". The method is described as follows:

to introduce a heterologous gene, a DNA sample containing the cDNA nucleotide sequence of the desired heterologous gene is first prepared. The DNA sample is preferably identified by electrophoresis as a single plasmid at a concentration of 25 ng/. mu.l or greater. The following example describes the use of a NotI site for inserting a heterologous gene into DNA encoding viral genomic RNA: if the target cDNA sequence contains a NotI recognition site, this site will be removed beforehand by techniques such as site-directed mutagenesis, to alter the nucleotide sequence, but not the amino acid sequence encoded thereby. The desired gene fragment is amplified and recovered from the DNA sample using PCR. Both ends of the amplified fragment become NotI sites by addition of NotI sites to the 5' regions of both primers. The E-I-S sequence or a portion thereof is included in the primers such that the E-I-S sequence is located between the ORFs on both sides of the viral gene and between the ORFs of the heterologous gene (after it is incorporated into the viral genome).

For example, to ensure NotI cleavage, the forward side (forward side) synthetic DNA sequence is as follows: two or more nucleotides (preferably four nucleotides, excluding sequences from the NotI recognition site such as GCG and GCC; more preferably ACTT) are arbitrarily selected on the 5 'side thereof, and the NotI recognition site "gcggccgc" is added to the 3' side thereof. In addition, a spacer (9 arbitrary nucleotides, or 9 ═ 6 multiple nucleotides), and ORF (a sequence of about 25 nucleotides and containing the start codon ATG of the ideal cDNA) were also added to the 3' side. Preferably, about 25 nucleotides are selected from the ideal cDNA such that G or C is the last nucleotide at the 3' end of the forward side synthetic oligo DNA.

The reverse-side synthetic DNA sequence is as follows: two or more arbitrary nucleotides (preferably four nucleotides, excluding sequences from the NotI recognition site such as GCG and GCC; more preferably ACTT) are selected from the 5 ' side, and the NotI recognition site "gcggccgc" is added to the 3 ' side thereof and further an oligo DNA insert is added to the 3 ' end to adjust the length. The length of the oligo DNA was designed such that the number of nucleotides of the NotI fragment product (containing the E-I-S sequence) amplified by the final PCR was a multiple of 6 (so-called "6-fold rule"; Kolakofski, D. et al, J.Virol.72, 891-899, 1998; Calain, P.andRoux, L., J.Virol.67, 4822-4830, 1993; Calain, P.and Roux, L., J.Virol.67, 4822-4830, 1993). A sequence that is different from the S sequence of Sendai virus, preferably 5'-CTTTCACCCT-3' (SEQ ID NO: 8), a sequence complementary to the I sequence, preferably 5 ' -AAG-3 ', and a sequence complementary to the E sequence, preferably 5'-TTTTTCTTACTACGG-3' (SEQ ID NO: 9), are further added to the 3 ' side of the inserted oligo DNA fragment. When these primers to which the E-I-S sequence is added are used, the 3' -end of the reverse-side synthetic DNA is formed by adding a complementary sequence corresponding to about 25 nucleotides (counting backwards from the stop codon of the ideal cDNA), and the length thereof is selected so that G or C becomes the last nucleotide.

PCR can be carried out by using Taq polymerase or the like according to a commonly used method. The ideal fragment thus amplified was digested with NotI and then inserted into the NotI site in the plasmid vector pBluescript. The nucleotide sequence of the PCR product thus obtained can be confirmed using a sequencer to select a plasmid containing the correct sequence. The inserted fragment was excised from the plasmid with NotI and cloned into the NotI site of the plasmid carrying the genomic cDNA. Alternatively, the recombinant sendai virus cDNA may be obtained by directly inserting the fragment into the NotI site without the mediation of a plasmid vector.

For example, recombinationSendai virus genomic cDNA can be constructed as described in the literature references (Yu, D.et al, Genes Cells 2, 457-2820, 1997; Hasan, M.K.et al, J.Gen.Virol.78, 2813-2820, 1997). For example, a spacer sequence comprising 18-bp of NotI restriction site (5 '- (G) -CGGCCGCAGATCTTCACG-3') (SEQ ID NO: 10) was inserted between the leader sequence of the cloned Sendai virus genomic cDNA (pSeV (+)) and the ORF of N protein, and thus a plasmid pSeV18 containing a self-cleaving ribozyme site from the hepatitis delta virus antigenomic strand was obtained⁺b (+) (Hasan, M.K., et al, J.general Virology 78, 2813-2820, 1997).

Furthermore, for example, in the case of deletion of the M gene, or in the case of introduction of a temperature-sensitive mutation, cDNA encoding genomic RNA is digested with a restriction enzyme, and a fragment containing the M gene is collected and cloned into an appropriate plasmid. M gene mutagenesis or the construction of M gene defect sites was performed using this plasmid. The mutation can be introduced by, for example, using QuikChange^TMSite-directed mutagenesis kit (Stratagene, La Jolla, Calif.) was performed according to the method described in the kit instructions. For example, M gene deficiency or deletion can be performed by using a combined PCR ligation method, whereby deletion of all or part of the M gene ORF, and ligation to an appropriate spacer sequence can be achieved. After obtaining the M gene mutated or defective sequence, a fragment containing the sequence is recovered, and the M gene region in the original full-length cDNA is replaced with the sequence. Thus, a viral genomic cDNA comprising the M gene can be produced. Using a similar method, mutations can be introduced into, for example, F and/or HN genes.

The vectors of the present invention can be reconstituted by intracellular transcription of DNA encoding genomic RNA in the presence of viral proteins. The present invention provides a DNA encoding the viral genomic RNA of the vector of the present invention, which can be used for preparing the vector of the present invention. In addition, the present invention relates to the use of DNA encoding the genomic RNA of the vector for the preparation of the vector of the present invention. Virus reconstitution from the (-) strand viral genomic cDNA can be carried out using known methods (WO 97/16539; WO 97/16538; Durbin, A.P. et al, Virology 235, 323-containing 332, 1997; Whelan, S.P. et al, Proc. Natl. Acad. Sci. USA92, 8388-containing 8392, 1995; Schnell. M.J. et al, EMBO J.13, 4195-containing 4203, 1994; Radecke, F. et al, EMBO J.14, 5773-containing 5784, 1995; Lawson, N.D. et al, Proc. Natl. Acad. Sci. USA92, 4477-containing Cel81, 1995; Garcin, D. et al, EMBO J.14, 6087-containing 6094, 1995; Kato, A. et al, Genes ls 1, 569, Sci. 9, Sci. 1996; Acad. D. et al, EMBO J.14, 6087-containing 6094, 1995; Kato, A. et al, Genes, St. 579, Sci. J.5, USA, 1997, Bart. J. 93, Klein, J. 93, J. 93, USA. 93). Using these methods, (-) strand RNA viruses or RNPs as viral components can be reconstituted from their DNA, including, for example, parainfluenza viruses, vesicular stomatitis viruses, rabies viruses, measles viruses, rinderpest viruses, Sendai viruses, and the like. The vectors of the invention may be reconstituted according to these methods.

Specifically, the vector of the present invention can be produced by the following steps: (a) transcribing a cDNA encoding paramyxovirus genomic RNA (negative strand RNA) or a complementary strand thereof (positive strand) in a cell expressing the N, P and L proteins; and (b) collecting the complex comprising genomic RNA from the cell or culture supernatant thereof. The transcribed genomic RNA replicates in the presence of N, L, and P proteins to form an RNP complex. When step (a) is carried out in the presence of the cleavage modified F protein encoded by the genome, the produced RNP is transferred to a cell in contact with the cell, infection spreads, and the vector is amplified. According to this method, the vector of the present invention can be produced in the form of RNP even in the absence of functional M protein.

The enzyme required to initiate transcription of genomic RNA from DNA, e.g., T7 RNA polymerase, can be provided by transfection of a plasmid or viral vector expressing the enzyme. Alternatively, the enzyme may be provided by incorporating its gene into the chromosome of the cell to induce expression in viral reconstitution. In addition, viral proteins required for reconstitution of genomic RNA and vectors are provided, for example, by introduction of plasmids expressing these proteins. To provide these viral proteins, helper viruses such as wild-type paramyxoviruses or specific mutant paramyxoviruses may be used. However, helper viruses are not preferred because they can lead to contamination by these viruses.

Methods for transferring DNA expressing genomic RNA into cells include, for example, the following methods: 1) a method for preparing a DNA precipitate that can be taken up by a target cell; 2) a method for preparing a complex containing positively charged DNA having cytotoxic activity and capable of being taken up by a target cell; and 3) a method of instantaneously opening a pore in a target cell membrane using electric pulses so that a DNA molecule can pass through.

In the above method 2), various transfection reagents can be used, and examples include DOTMA (Roche), Superfect (QIAGEN #301305), DOTAP, DOPE, DOSPER (Roche #1811169), and the like. Example of method 1) is a method of transfection using calcium phosphate, in which DNA entering the cell is incorporated into the phagosome, but also in a sufficient amount into the nucleus of the cell (Graham, f.l. and Van Der Eb, j., Virology 52, 456, 1973; wigler, m.and Silverstein, s., Cell 11, 223, 1977). Chen and Okayama have studied how to optimize this transfer technique and reported that optimal precipitation can be obtained under the following conditions: 1) in the presence of 2% -4% CO₂Incubating the cells and the co-precipitate together at 35 ℃ for 15-24 hours; 2) using a circular DNA having higher activity than the linear DNA; and 3) the concentration of DNA in the precipitation mixture is 20-30. mu.g/ml (Chen, C.and Okayama, H., mol.cell.biol.7, 2745, 1987). Method 2) is suitable for transient transfection. DEAE-dextran (Sigma # D-9885, M.W. 5X 10) was prepared in the past known method at the desired DNA concentration ratio ⁵) The mixture was mixed and transfected. Since many of the complexes are decomposed in the endosome, chloroquine is added to enhance results (Calos, m.p., proc.natl.acad.sci.usa 80, 3015, 1983). Method 3) refers to electroporation and is more varied than methods 1) and 2) because it does not involve cell sensitivity. Method 3) is said to be effective under the most ideal conditions of pulse current duration, pulse shape, electric field (gap between electrodes, volts) strength, buffer conductivity, DNA concentration and cell density.

Among the three, method 2) is easy to handle and simplifies the testing of many test samples using a large number of cells. Transfection agents are therefore suitable for the case of vector reconstitution for the introduction of DNA into cells. Preferably, a Superfect transfection reagent (QIAGEN, Cat. No.301305) or DOSPER liposome transfection reagent (Roche, Cat. No.1811169) is used, but the transfection reagent is not limited thereto.

Specifically, the reconstitution of a viral vector from cDNA can be performed, for example, as follows:

monkey kidney-derived LLC-MK2 cells were cultured to 100% confluency in 24-well to 6-well plastic plates or 100mm diameter plates using Minimal Essential Medium (MEM) containing 10% Fetal Calf Serum (FCS) and anti-biotin (100 units/ml penicillin G and 100. mu.g/ml streptomycin). These cells are then infected with recombinant vaccinia virus vTF7-3 expressing T7 polymerase, e.g., at 2 PFU/cell. The virus has been inactivated by treatment with UV radiation for 20 minutes in the presence of 1. mu.g/ml psoralen (psoralen) (Fuerst, T.R. et al, Proc. Natl. Acad. Sci. USA 83, 8122-. The amount of psoralen added and the time of UV irradiation can be appropriately adjusted. One hour after infection, cells can be transfected with 2. mu.g to 60. mu.g, more preferably 3. mu.g to 20. mu.g of the above DNA encoding the recombinant Sendai virus genomic RNA by lipofection or the like. This method uses Superfect (QIAGEN) and several plasmids expressing the transactivating viral proteins required for the production of viral RNP (0.5. mu.g-24. mu.g pGEM-N, 0.25. mu.g-12. mu.g pGEM-P and 0.5. mu.g-24. mu.g pGEM-L) (Kato, A. et al, Genes Cells 1, 569-. The ratio of expression vectors encoding N, P, and L is preferably 2: 1: 2. The amount of plasmid is suitably adjusted to, for example, 1. mu.g to 4. mu.g of pGEM-N, 0.5. mu.g to 2. mu.g of pGEM-P, and 1. mu.g to 4. mu.g of pGEM-L.

Transfected cells were cultured in serum-free MEM (containing 100. mu.g/ml rifampicin (Sigma) and cytarabine (AraC) as required, more preferably containing only 40. mu.g/ml cytarabine (AraC) (Sigma)). The reagent concentration can be optimized to minimize vaccinia virus-induced cytotoxic activity and maximize virus recovery (Kato, A. et al, Genes Cells 1, 569-. After transfection, cells were cultured for about 48 hours to about 72 hours, and then the freezing and thawing cycle was repeated three times to destroy the cells. LLC-MK2 was re-transfected with the disrupted cells and then cultured. RNPs can be introduced into cells as complexes with, for example, lipofectamine and polycationic liposomes. In particular, a variety of lipofection reagents can be utilized. Examples of such reagents are DOTMA (Roche), Superfect (QIAGEN #301305), DOTAP, DOPE, DOSPER (Roche #1811169), and the like. Chloroquine may be added to prevent decomposition in RNP endosomes (Calos, m.p., proc.natl.acad.sci.usa 80, 3015, 1983). In RNP-transfected cells, the step of expressing a viral gene from RNP and replicating RNP is followed by amplification of the vector. By diluting the cell lysate obtained and repeating the amplification, the vaccinia virus vTF7-3 could be completely removed. The re-amplification may be repeated, for example 3 times or more. The RNP obtained was stored at-80 ℃.

The host cell used for the reconstitution is not limited as long as the viral vector can be reconstituted. For example, in the case of reconstituting Sendai virus vectors, LLC-MK2 cells and CV-1 cells derived from monkey kidney, cultured cells such as BHK cells derived from hamster kidney, human-derived cells, and the like can be used. By expressing an appropriate envelope protein in these cells, infectious viral particles containing the protein in the envelope can be obtained.

When the M gene in the viral genome is defective or deleted, such virus-infected cells do not form viral particles. Therefore, although the vector of the present invention can be prepared into RNPs or RNP-containing cells by the above-described method, RNPs proliferating in the cells are transferred only to the contacted cells. Therefore, transfection spreads slowly, resulting in difficulty in producing a large amount of viral vector at a high degree. The present invention provides methods for preparing viral particles from the vectors of the invention. Viral particles in solution are more stable than RNPs. Furthermore, by rendering the viral particles infectious, the vector can be introduced into the target cells by simple contact without the need for transfection agents. Thus, viral particles are particularly useful in industrial applications. As a method for preparing the vector of the present invention into viral particles, the virus is reconstituted using a viral genome comprising an M gene having conditional mutations under permissive conditions. Specifically, the M protein is made to function to form particles by culturing cells transfected with the complex obtained in the above step (a) or steps (a) and (b) under permissive conditions. A method of producing a viral particle comprising genomic RNA encoding a mutant M protein having a conditional mutation, comprising the steps of: (i) amplifying RNPs comprising paramyxovirus N, P, and L proteins and genomic RNA under permissive conditions for mutating M protein in cells; and (ii) collecting the viral particles released into the cell culture supernatant. For example, the temperature-sensitive mutant M protein can be cultured at its permissive temperature.

Another method for preparing the vector of the present invention into viral particles uses helper cells expressing the M protein. By using M helper cells, the present inventors prepared a vector in which the cleavage site of the F protein is modified to a sequence cleaved by another protease and the M gene is mutated or deleted, as a viral particle. Since the method of the present invention does not require helper viruses such as wild-type paramyxovirus, M gene-containing viruses having particle-forming ability are not produced. Thus, the vectors of the present invention can be prepared in pure form. The present invention provides a viral particle comprising (i) paramyxovirus genomic RNA wherein (a) a nucleic acid encoding an M protein is mutated or deleted, and (b) encodes a modified F protein whose cleavage site sequence is replaced with a sequence cleaved by a protease that does not cleave a wild-type F protein, and which further (1) has the ability to replicate genomic RNA in a cell infected with the viral particle; (2) the production of viral particles in the host environment appears to be significantly reduced or eliminated; and (3) having the ability to introduce genomic RNA into cells contacted with cells transfected with viral particles containing the genomic RNA in the presence of the protease. According to a preferred embodiment, such viral particles will not produce viral particles.

A method for producing a viral particle of the invention in a cell expressing a functional M protein, comprising the steps of: (i) amplifying RNPs (which comprise paramyxovirus N, P and L proteins and genomic RNA) in cells expressing wild-type M protein of paramyxovirus or equivalent protein; (ii) viral particles released into the cell culture supernatant were collected. The wild-type M protein may be derived from a paramyxovirus of non-genomic RNA origin, as long as it has the ability to form viral particles. In addition, a marker peptide may be added to the protein, or alternatively, a linker peptide from a vector may be added to the protein when it is expressed by an appropriate expression vector. As described above, the protein to be used is not necessarily the wild-type M protein itself, but may be a protein having a virus particle-forming ability equivalent to the wild-type protein. The M protein expressed in cells expressing the M protein is contained in the envelope of viral particles produced from these cells; however, it does not contain the gene encoding the protein. Thus, wild-type M protein does not continue to be expressed in cells infected by the virus. Thus, viral particles cannot be formed.

The preparation of helper cells expressing the M protein can be performed as follows. For the preparation of vectors expressing the M protein in an inducible form, for example, inducible promoters can be used or a recombinant expression regulation system (e.g., Cre/loxP) can be used. The Cre/loxP inducible expression plasmid can be constructed, for example, using the plasmid pCALNdlw, which can be designed to inducibly express the gene product using Cre DNA recombinase (Arai, T. et al, J.virology 72, 1115 1121, 1998). As the cell capable of expressing the M protein, a helper cell line capable of continuously expressing the M protein is preferably established by inducing the M gene introduced into its chromosome. For example, a monkey kidney-derived cell line LLC-MK2 or the like can be used as such a cell. LLC-MK2 cells were cultured in MEM containing 10% heat-treated fixed Fetal Bovine Serum (FBS), 50 units/ml penicillin G sodium and 50. mu.g/ml streptomycin at 37 ℃ in 5% CO ₂The culture was performed in the atmosphere of (2). According to a known protocol, the above-described plasmid designed to inducibly express the M gene product using Cre DNA recombinase is introduced into LLC-MK2 cells using the calcium phosphate method (mammalian transfection kit (Stratagene)).

For example, a 10. mu. g M-expression plasmid can be introduced into LLC-MK2 cells grown to 40% confluency in a 10cm plate. These cells were then incubated at 37 ℃ in 10ml of MEM containing 10% FBS and 5% CO₂And (4) incubating. After 24 hours of incubation, cells were collected and suspended in 10ml of medium. The suspension was then added to 5 dishes 10cm in diameter: 5ml of suspension was added to one dish, 2 ml/dish to two dishes, and 0.2 ml/dish to two dishes. The cells in each dish were cultured in 10ml MEM containing 10% FBS and 1200. mu.g/ml G418(GIBCO-BRL) for 14 days(ii) a The culture medium was changed every two days. Thus, a cell line in which the gene is stably introduced is selected. The G418-resistant cells grown on the medium were collected using cloning rings. The collected cells of each clone were further cultured to a plate 10cm confluent.

High expression levels of the M protein in helper cells are important for recovery of high titer viruses. For this reason, for example, the selection of M-expressing cells as described above is preferably performed twice or more. For example, an M-expression plasmid containing a drug resistance marker gene is transfected, and cells containing the M gene are selected using a drug. Subsequently, an M-expression plasmid containing a marker gene that is resistant to a particular drug is transfected into the same cells, and the cells are selected using this second drug resistance gene. Selection of cells using the second marker is likely to express M protein at a higher, greater level than cells selected after the first transfection. Therefore, M helper cells constructed by two repeated transfections can be suitably used. Since M helper cells can express the F gene simultaneously, the preparation of infectious viral particles deficient in both F and M genes is possible (WO 03/025570). Therefore, it is also suggested to transfect the plasmid expressing the F gene more than twice to increase the level of F protein expression induction. The gene of the modified F protein described herein can be used as the F gene.

M protein inducible expression can be obtained by incubating the cells in a dish of 6cm and subsequently infecting these cells with MOI ═ 3, for example using adenovirus AxCANCre, according to the method of Saito et al (Saito et al, Nucleic Acids Res.23, 3816-.

The viral particles of the invention are prepared using cells expressing the wild-type M protein or its equivalent (M helper cells). The above-described RNPs of the present invention can be introduced into these cells and then cultured. RNP can be introduced into M helper cells by: for example, cell lysates containing RNP are transfected into M helper cells, or cell fusion is induced by co-culture of RNP producing cells and M helper cells. It can also be achieved by transcribing genomic RNA into M helper cells and resynthesizing RNP in the presence of N, P and L proteins.

The step (i) of the present invention (step of amplifying RNP using M helper cells) is preferably performed at low temperature. In preparing a vector using a temperature-sensitive mutant M protein, a method for preparing viral particles must be performed at a temperature below an allowable temperature. Surprisingly, however, the inventors have found that in the method of the invention efficient particle production is possible when the virus formation process is carried out at low temperatures, even when using wild-type M protein. In the present invention, the term "low temperature" means 35 ℃ or less, preferably 34 ℃ or less, more preferably 33 ℃ or less, and most preferably 32 ℃ or less.

According to the invention, the viral particles can be released into the extracellular fluid of virus-producing cells, for example, a virus titer of 1X 10⁵CIU/ml or more, preferably 1X 10⁶CIU/ml or more, more preferably 5X 10⁶CIU/ml or more, more preferably 1X 10⁷CIU/ml or more, more preferably 5X 10⁷CIU/ml or more, more preferably 1X 10⁸CIU/ml or more, and more preferably 5X 10⁸CIU/ml or above. The virus titre can be determined by methods described in the specification or elsewhere (Kiyotani, K. et al, Virology 177(1), 65-74, 1990; WO 00/70070).

A preferred embodiment of the method for reconstructing a recombinant viral vector from M-deficient viral genomic cDNA is as follows. Namely, the method comprises the steps of: (a) transcribing DNA encoding (negative-or positive-strand) genomic RNA in cells expressing viral proteins required for the formation of infectious viral particles (i.e., NP, P, L, M, F, and HN proteins); (b) co-culturing these cells with an M gene that is expressed integrated into the chromosome (i.e., M helper cells); (c) preparing a cell extract from the culture; (d) transfecting the extract to express the M gene integrated into the chromosome (i.e., M helper cells), and culturing the cells; and (e) recovering the viral particles from the culture supernatant. Step (d) is preferably carried out under the above-mentioned low temperature conditions. The obtained virus particles can be amplified by co-infection of helper cells (preferably at low temperatures). In particular, the virus may be reconstituted as described in the examples. The recovered virus particles can be diluted and then transfected again into the M helper cells to be expanded. The amplification step may be performed two or three or more times. The obtained virus strain can be stored at-80 ℃. Viral titers can be determined by measuring Hemagglutinin Activity (HA). HA can be determined by the "end point dilution method".

Specifically, LLC-MK2 cells may be, first, 5X 10⁶The density of cells/dish was plated on 100mm plates. When T7 RNA polymerase was used to induce transcription of genomic RNA, the cells were cultured for 24 hours and subsequently infected at room temperature with a recombinant T7 polymerase-expressing vaccinia virus (PLWUV-VacT7) that had been treated with psoralen and long wavelength ultraviolet light (365nm) for 20 minutes at MOI ═ about 2 for 1 hour at room temperature (Fuerst, t.r. et al, proc.natl.acad.sci.usa 83, 8122-. After washing the cells with serum-free MEM, the cells were transfected with appropriate lipofection reagents using plasmids expressing genomic RNA and plasmids expressing paramyxovirus N, P, L, F, and HN proteins, respectively. The ratio of the plasmids may be, for example, 6: 2: 1: 2, but is not limited thereto. After 5 hours of culture, the cells were washed twice with serum-free MEM and then cultured in MEM containing 40 μ g/ml cytosine- β -D-arabinofuranoside (AraC, Sigma, st. louis, MO) and 7.5 μ g/ml trypsin (GIBCO-BRL, Rockville, MD). After 24 hours of culture, the cells were cultured with cells that continuously expressed M protein (M helper cells) at approximately 8.5X 10 ⁶The cells/dish were overlaid and cultured for two more days at 37 ℃ in MEM containing 0. mu.g/ml AraC and 7.5. mu.g/ml trypsin (P0). Cultured cells were harvested and suspended in 2 ml/dish of ptiMEM. After three repeated freeze-thawing, lysates were directly transfected into M helper cells and cultured in serum-free MEM containing 40. mu.g/mL AraC and a protease cleaving the F protein (P1) at 32 ℃. After 3-14 days, a portion of the culture supernatant was collected and used to infect freshly produced M helper cells, which were then cultured in serum-free MEM containing 40. mu.g/mL AraC and protease (P2) at 32 ℃. 3-14 days later, M helper cells, which were just prepared, are reinfected and either in the presence (for preparing F-cleaved virus) or in the absence (for preparing F-uncleaved virus)Viral particles of (4) protease, and serum-free MEM (P3) was used for 3 to 7 days. By repeating the re-amplification 3 times or more, the initially used vaccinia virus can be completely eliminated. BSA was added to the collected culture supernatant at a final concentration of 1% and stored at-80 ℃.

The viral particles of the invention may be infectious particles in which the modified F protein is cleaved, or may be potentially infectious viral particles, i.e. particles which are not infectious in their original form but which are infectious after treatment with a protease which cleaves the modified F protein. The modified F protein encoded by the genome is present on the envelope of the viral particle, however, it lacks infectivity when uncut. Such viruses gain infectivity by treatment with a protease that cleaves a cleavage fragment of the modified F protein, or by contact with cells or tissues in the presence of a protease that cleaves the F protein. In order to obtain virus particles whose modified F protein is not cleaved by the production of the above-mentioned viruses using virus-producing cells, the final step of the virus amplification step may be carried out in the absence of a protease that cleaves the modified F protein. On the other hand, the preparation of the virus in the presence of the protease makes it possible to produce infectious viral particles having a cleaved F protein.

In addition, by expressing in a cell an envelope protein that is not encoded in the viral genome during production of the viral particle, a viral particle whose envelope comprises the protein can be produced. An example of such an envelope protein is the wild-type F protein. The genomic RNA of the viral particles produced in this way encodes the modified F protein and carries the wild-type F protein and this modified protein on its envelope. The viral particles are rendered infectious by cleavage of the wild type F protein on the viral particles by providing the wild type F protein in trans at the step of viral particle production and amplification in the presence of trypsin that cleaves the protein. According to this method, infectious virus particles can be produced at a high titer without using a protease that cleaves the modified F protein. Thus, the viral particle of the invention may be a viral particle comprising a paramyxovirus wild-type F protein. The wild-type F protein and the viral genome need not be from the same type of paramyxovirus, and the wild-type F protein may be an envelope protein from another paramyxovirus.

In addition, viral particles can be produced whose envelope comprises any desired viral envelope protein other than the wild-type F protein. In particular, in viral reconstitution, the desired envelope protein may be expressed in a cell to produce a viral vector comprising the envelope protein. These proteins are not particularly limited. Preferred examples include the G protein of Vesicular Stomatitis Virus (VSV) (VSV-G). The viral particles of the present invention include pseudotyped viral vectors containing envelope proteins such as VSV-G protein from viruses other than the virus from which the genomic RNA is derived. For the wild-type F protein, after infection of the viral particle into a cell, the protein is not expressed from the viral vector because the envelope protein is not encoded in the viral genomic RNA.

The viral particles of the invention may comprise a chimeric protein, e.g., a chimeric protein comprising an extracellular domain, one or more proteins capable of binding to a particular cell at the surface of the envelope, e.g., adhesion factors, ligands, receptors, and antibodies and fragments thereof, as well as polypeptides from the viral envelope in the intracellular domain. This can result in vectors that infect specific tissue targets. These vectors may be provided in trans by intracellular expression during viral vector reconstitution. Specific examples include fragments containing receptor binding regions for soluble factors such as cytokines, or antibody fragments for cell surface proteins (WO 01/20989).

In preparing a vector having a defective viral gene, for example, two or more types of vectors each having a defective viral gene in the viral genome may be introduced into the same cell. Each defective viral protein is then expressed and provided by another vector. This complementation results in the formation of infectious viral particles and the viral vector can be amplified in the replication cycle. That is, when two or more types of the vectors of the present invention are inoculated in combination with complementary viral proteins, a mixed viral gene defective viral vector can be produced on a large scale and at a low cost. Because these viruses lack viral genes, their genomes are smaller than the complete viral genome, and they may contain larger heterologous genes. Furthermore, these viruses are non-productive due to viral gene defects and are diluted extracellularly, so that co-infectivity is difficult to maintain in these viruses. These vectors are therefore non-reproductive, which is advantageous in terms of controlled environmental release.

A large number of viral vectors can be amplified by infecting them into embryonated chicken eggs by the above-described method. For example, M gene transgenic chickens can be produced and the vector can be inoculated into eggs for amplification. A basic method for preparing a viral vector using chicken eggs has been developed ("Shinkei-kagaku Kenkyu-noSaisentan Protocol III, Bunshi Shinkei Saibou Seirikaku (Leading edge technologies protocols III in neuroscience research, Molecular, cellular neurosurgery)", edited by Nakanishi, et al, KOSEISHA, Osaka, pp.153-172, 1993). Specifically, for example, fertilized eggs are transferred to an incubator and cultured at 37 ℃ to 38 ℃ for 9 to 12 days to grow embryos. The viral vector is then inoculated into the allantoic membrane cavity, and the fertilized egg is incubated for several days to propagate the viral vector. Subsequently, allantoic fluid contained in the virus was collected. The culture conditions, such as the duration of culture, vary depending on the recombinant virus amplified. Isolation and purification of viral vectors from allantoic fluid was performed according to conventional methods ("Protocols of Virology" by Mass Tashiro, edited by Nagai and dIshihama, Medical View, pp.68-73, 1995).

The recovered viral vector may be purified to substantial purity. Purification can be carried out by known purification and separation methods including filtration, centrifugation, column chromatography purification, and the like, or a combination thereof. The term "substantially pure" as used herein means that the viral vector as a component is the major portion of the sample in which it is present. In general, a substantially pure viral vector can be obtained by confirming that the ratio of the virus-derived protein to the total protein in the sample (excluding proteins added as a carrier or a stabilizer) is 10% or more, preferably 20% or more, more preferably 50% or more, more preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more. Specifically, paramyxovirus can be purified, for example, by a method using cellulose sulfate ester or cross-linked polysaccharide sulfate ester (JP-B No. Sho 62-30752; JP-B Sho 62-33879; JP-B Sho62-30753), a method using trehalose sulfate-containing polysaccharide and/or its decomposition product (WO97/32010), and the like.

The M gene defective vector having a modified cleavage site can be transferred into cells only by cell fusion type infection in the presence of a specific protease. Thus, the vectors of the invention may be used in gene therapy targeting tissues expressing specific proteases. The normal vector allows gene transfer to the surface layer of the target tissue; but they cannot penetrate into the tissue. In another aspect, the vectors of the invention have the ability to penetrate deep into target tissues with enhanced protease activity. For example, the vector of the present invention can infect cancer cells by infecting them into the surface layer, and can be transferred to the inside of cancer cells infiltrating into the deep part of normal tissues.

The vector of the present invention can be used for cancer, arteriosclerosis, joint diseases such as rheumatoid arthritis, and the like. For example, in joint diseases such as RA, destruction of the higher order structure of cartilage by extracellular matrix degradation occurs as described above, and the joint is destroyed. By removing cells whose ECM-degrading enzyme activity is enhanced due to the vector of the present invention, reduction in joint destruction is expected. In addition, macrophage-derived foam cells continue to aggregate during arteriosclerosis. These foam cells secrete large amounts of metalloproteases and disrupt fibroplasia leading to plaque breakdown. Treatment of such arteriosclerosis is achieved by killing macrophages expressing MMPs using the vectors of the invention. In addition, as described below, various proteases are activated in cancer. The vectors of the invention are useful as therapeutic vectors for specific infection and infiltration of cancer.

To prepare a composition comprising a carrier of the present invention, the carrier is combined, if necessary, with a desired pharmaceutically acceptable carrier or solvent. "pharmaceutically acceptable carrier or solvent" refers to a substance that can be co-administered with a vector without significantly inhibiting gene transfer by the vector. For example, the carrier may be formulated into a composition by appropriate dilution with physiological saline, phosphate-buffered physiological saline (PBS). When the vector is propagated in eggs, the composition may contain allantoic fluid. In addition, the composition comprising the carrier may contain a carrier or solvent, such as deionized water and a 5% dextran solution. In addition, the composition may also contain vegetable oils, suspending agents, detergents, biocides, and the like. In addition, preservatives and other additives may also be added to the composition. Compositions containing the vectors of the invention are useful as detergents and pharmaceuticals.

The vector dose depends on the type of disease, the patient's body weight, age, sex and symptoms, the purpose of administration, the form of the composition to be administered, the method of administration, the type of gene to be introduced, and the like. However, the correct dosage can be routinely determined by one skilled in the art. The preferred dosage of carrier administration is about 10⁵-10¹¹CIU/ml, more preferably about 10 ⁷-10⁹CIU/ml, more preferably about 1X 10⁸-5×10⁸CIU/ml. Preferably, the carrier is admixed with a pharmaceutically acceptable carrier. To administer cancer tissue, the vector may be administered to multiple target sites at multiple time points, such that the vector is evenly distributed. The preferred dose per administration to a human subject is 2X 10⁹-2×10¹⁰And (4) CIU. The administration may be performed once or more times within the limits of clinically acceptable side effects. Daily dosing frequency can be similarly determined. When the viral vector is administered to an animal other than a human, for example, the dose to be administered can be determined by converting the above-mentioned dose according to the body-to-weight ratio or volume ratio (e.g., average value) of the administration target site between the target animal and the human. Compositions comprising the vectors of the invention can be administered to all mammalian species, including humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, and the like.

The vectors of the invention are particularly useful for treating cancer. Cells infected with the vectors of the invention form syncytia by cell fusion in the presence of proteases. By taking advantage of this property, the vectors of the present invention can be used to treat cancers with enhanced activity of specific proteases. The present invention provides a therapeutic composition for cancer comprising a pharmaceutically acceptable carrier and a vector of the invention (which encodes an F protein cleaved by a protease that exhibits enhanced activity in cancer). Furthermore, the present invention relates to the use of a vector for the preparation of a cancer therapeutic composition. The invention also relates to methods of treating cancer comprising the step of administering such vectors to cancerous tissues. Since the activity of ECM-degrading enzymes is enhanced in invasive and metastatic malignant cancers, a vector comprising a gene that cleaves large F protein by ECM-degrading enzymes can be used to specifically infect malignant cancers, resulting in death of cancerous tissues.

The vectors of the invention also comprise a heterologous gene. The heterologous gene may be a marker gene that monitors infection of the cancer by the vector or therapeutic gene. Examples of therapeutic genes include apoptotic cell-induced genes; a gene encoding a cytotoxic protein; a cytokine; and hormones. Administration of the vectors of the invention to cancer may be directly (in vivo) administration to the cancer or indirectly (ex vivo) administration, wherein the vectors may be introduced into cells of patient origin or other cells, and the cells may be infused into the cancer.

The targeted cancer may be any cancer in which the activity of a specific protease is enhanced. Examples include the most invasive and metastatic malignancies (lung, stomach, colon, esophagus, breast, etc.). However, proteases such as MMPs, uPA, and tPA are expressed at lower levels in some malignant cancers. Thus, the ability to target cancer can be judged by the presence or absence of enhanced protease activity. The vectors of the invention are particularly useful for cancers that infiltrate into the submucosa of esophageal cancer, colon cancer that progresses to stage III and IV cancer in the internal sphincter, and invasive melanoma that infiltrates so deeply that it cannot be completely removed by surgery.

Brief Description of Drawings

FIG. 1 is a diagram showing the construction of F-deficient SeV genomic cDNA, in which a temperature-sensitive mutation has been introduced into the M gene.

FIG. 2 depicts the structure of viral genes constructed to suppress secondary particle release based on temperature-sensitive mutations introduced into the M gene, and viral genes constructed or used to detect and compare the effects of these introduced mutations.

FIG. 3 provides microscopic images representing GFP expression in cells that persistently express the F protein (LLC-MK2/F7/A), which were cultured for 6 days at 32 ℃ and 37 ℃ after infection with SeV18+/Δ F-GFP or SeV18+/MtsHNts Δ F-GFP, respectively.

FIG. 4 represents the results of a semi-quantitative determination of F protein expression levels over time using Western blotting in cells persistently expressing SeV-F protein (LLC-MK2/F7/A) cultured in trypsin-free, serum-free MEM at 32 ℃ or 37 ℃.

FIG. 5 provides microscopic images representing GFP expression in LLC-MK2 cells cultured at 32 deg.C, 37 deg.C or 38 deg.C for 3 days after infection with SeV18+ GFP, SeV18+/Δ F-GFP or SeV18+/MtsHNts Δ F-GFP at MOI ═ 3.

FIG. 6 depicts the Hemagglutination (HA) activity in the culture supernatant of LLC-MK2 cells sampled over time (with fresh medium added), cultured at 32 ℃, 37 ℃ or 38 ℃ after infection with SeV18+ GFP, SeV18+/Δ F-GFP or SeV18+/MtsHNts Δ F-GFP at MOI ═ 3.

FIG. 7 represents the ratio of M protein levels in cells to M protein levels in virus-like particles (VLPs). This ratio was determined by Western blotting using anti-M antibody. LLC-MK2 cells were infected with SeV18+ GFP, SeV18+/Δ F-GFP, or SeV18+/MtsHNts Δ F-GFP at MOI of 3, incubated at 37 ℃ for two days, and culture supernatants were recovered from the cell cultures. Each lane contains cultures equivalent to 1/10 from the cultures in each well of the 6-well plate.

FIG. 8 shows SEAP activity in culture supernatants of LLC-MK2 cells cultured at MOI of 3 after infection with SeV18+ SEAP/. DELTA.F-GFP or SeV18+ SEAP/MtsHNts. DELTA.F-GFP.

FIG. 9 shows HA activity in the culture supernatants of LLC-MK2 cells cultured 24, 50, or 120 hours after infection with SeV18+ SEAP/. DELTA.F-GFP or SeV18+ SEAP/. DELTA.F-GFP at MOI-3.

FIG. 10 shows the amount of VLPs determined by Western blotting using anti-M antibodies. LLC-MK2 cells were cultured for 5 days after infection with SeV18+ SEAP/. DELTA.F-GFP or SeV18+ SEAP/. DELTA.F-GFP at MOI. RTM.3. The culture supernatant was centrifuged to recover the virus. Each lane contains a culture equivalent to 1/10 of the contents of each well of a 6-well plate.

Figure 11 shows the cytotoxic activity estimated from the amount of LDH released into the cell culture medium. LLC-MK2, BEAS-2B or CV-1 cells were infected with SeV18+ GFP, SeV18+/Δ F-GFP or SeV18+/MtsHNts Δ F-GFP at an MOI of 0.01, 0.03, 0.1, 0.3, 1, 3, or 10. Cells were cultured in serum-free or 10% FBS-containing medium and cytotoxic activity experiments were performed 3 or 6 days post infection, respectively. The relative cytotoxic activity values of the cells are shown, with the cytotoxic activity of an equal number of cells, 100% of which were lysed by a cell denaturant (Triton), taken as 100%.

FIG. 12 shows the subcellular localization of M protein in LLC-MK2 cells, as observed by immunostaining with anti-M antibodies, where the cells were cultured for 2 days at 32 ℃, 37 ℃ or 38 ℃ after infection with SeV18+ GFP, SeV18+/Δ F-GFP or SeV18+/MtsHNts Δ F-GFP at MOI ═ 1.

Figure 13 provides a three-dimensional image of subcellular localization of M and HN proteins observed under confocal laser microscopy. A-10 cells were infected with SeV18+ SEAP/. DELTA.F-GFP or SeV18+ SEAP/MtsHNts. DELTA.F-GFP at MOI ═ 1 and then cultured in medium containing 10% serum at 32 ℃ or 37 ℃ for one day. These images were obtained by immunostaining with anti-M and anti-HN antibodies.

Figure 14 provides a three-dimensional image of subcellular localization of M and HN proteins observed under confocal laser microscopy. A-10 cells were infected with SeV18+ SEAP/. DELTA.F-GFP or SeV18+ SEAP/MtsHNts. DELTA.F-GFP at MOI ═ 1 and subsequently cultured in medium containing 10% serum at 32 ℃ or 37 ℃ for two days. These images were obtained by immunostaining with anti-M and anti-HN antibodies.

FIG. 15 shows the effect of microtubule disaggregating agents on the subcellular localization of M and HN proteins. A-10 cells were infected with SeV18+ SEAP/mtschntts Δ F-GFP at MOI ═ 1 and microtubule depolymerizing agents, colchicine or colchicine, were immediately added to these cells to give a final concentration of 1 μ M. These cells were cultured in a medium containing 10% serum at 32 ℃. After two days, the cells were immunostained using anti-M and anti-HN antibodies and then observed under a confocal laser microscope. These images show a stereoscopic three-dimensional image of subcellular localization of M and HN proteins.

FIG. 16 shows the effect of microtubule disaggregating agents on the subcellular localization of M and HN proteins. A-10 cells were infected with SeV18+/F-GFP or SeV18 +/mtschnts Δ F-GFP at MOI ═ 1, and the microtubule depolymerizing agent colchicine was immediately added to these cells to a final concentration of 1 μ M. These cells were cultured in a medium containing 10% serum at 32 ℃ or 37 ℃. After two days, the cells were immunostained using anti-M and anti-HN antibodies and then observed under a confocal laser microscope. These images show a stereoscopic three-dimensional image of subcellular localization of M and HN proteins.

FIG. 17 shows the construction of M-deficient SeV genomic cDNA comprising the EGFP gene.

FIG. 18 is a diagram showing the construction of F-deficient and M-deficient SeV genomic cDNAs.

FIG. 19 is a diagram showing the structure of F-deficient and/or M-deficient SeV genes constructed.

FIG. 20 shows the construction of M gene expression plasmid containing hygromycin resistance gene.

FIG. 21 shows that cloned cells inducibly expressing cloned M protein (and F protein) were infected with a recombinant adenovirus expressing Cre DNA recombinase (AcCANCre), and the expression levels of M and F proteins in the cells were semi-quantitatively compared by Western blotting.

FIG. 22 shows the viral reconstitution of M-deficient SeV (SeV18+/Δ M-GFP) with helper cell (LLC-MK2/F7/M) clones #18 and # 62.

FIG. 23 shows the viral productivity (CIU and HAU time course) of SeV18+/Δ M-GFP

FIG. 24 provides a picture and illustration of the results of RT-PCR used to confirm the structure of genes in SeV18+/Δ M-GFP virions.

FIG. 25 shows the results of a comparison of SeV18+/Δ M-GFP with SeV18+ GFP and SeV18+/Δ F-GFP, in which the viral structure of SeV18+/Δ M-GFP was confirmed from a protein point of view by Western blotting of viral proteins from LLC-MK2 cells and cell cultures after infection.

FIG. 26 shows a quantitative comparison of virus-derived proteins in supernatants of cultures of SeV18+/Δ M-GFP and SeV18+/Δ F-GFP (serial dilutions were made and detected using Western blot) infected LLC-MK2 cells, using anti-SeV antibodies.

FIG. 27 shows HA activity in LLC-MK2 cell culture supernatants (collected over time) infected with SeV18+/Δ M-GFP or SeV18+/Δ F-GFP at MOI ═ 3.

FIG. 28 provides fluorescence microscopy images obtained after 5 days from LLC-MK2 cells infected with SeV18+/Δ M-GFP or SeV18+/Δ F-GFP at MOI ═ 3.

FIG. 29 provides fluorescence micrographs of LLC-MK2 cells prepared as follows: LLC-MK2 cells were infected with SeV18+/Δ M-GFP or SeV18+/Δ F-GFP at MOI ═ 3, and culture supernatants were collected 5 days after infection and transfected into LLC-MK2 cells using cationic liposomes (Dosper). After two days, microscopic observation was performed.

FIG. 30 shows the design of the amino acid sequence of the F1/F2 cleavage site (F protein activation site). The recognition sequence of a protease (MMP or uPA) highly expressed in cancer cells is designed based on the recognition sequence of a synthetic substrate. Starting from the above, SEQ ID NO: 40-44.

FIG. 31 is a diagram showing the construction of M-deficient SeV vector cDNA in which the F activation site is modified.

FIG. 32 is a graph showing protease-dependent cell fusion-type infection of F-modified, M-deficient Sendai virus vectors. By using LLC-MK2, it was demonstrated that modification of F results in protease-dependent cell fusion-type infection. Cells were infected with each of M-deficient SeV (SeV/. DELTA.M-GFP (A, B, C, J, K, and L), SeV/F (MMP # 2). DELTA.M-GFP (D, E, F, M, N, and O), and SeV/F (uPA). DELTA.M-GFP (G, H, I, P, O, and R)), while adding 0.1. mu.g/ml collagenase (Clostridium) (B, E, and H), MMP-2(C, F, and I), MMP-9(J, M, and P), uPA (K, N, and Q), and 7.5. mu.g/ml trypsin (L, Q, and R)). After four days, the cells were observed under a fluorescent microscope. Only in LLC-MK2 with trypsin added, SeV/. DELTA.M-GFP containing unmodified F resulted in fusion of infected cells with surrounding cells, leading to fusogenic infection of the cells to form multinucleated cells, i.e.syncytia (L). In LLC-MK2 with collagenase, MMP-2, and MMP-9 added, SeV/F (MMP #2) Δ M-GFP, which contains the MMP degradation sequence for introduction of F, caused fusogenic infection of cells to form syncytia (E, F, and M). On the other hand, it was observed that SeV/(uPA) Δ M-GFP, which contains F-introduced urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA) degradation sequences, leads to fusogenic infection of cells in the presence of trypsin, and, after further modification, to the formation of syncytia in the presence of uPA (Q and R).

FIG. 33 shows protease-dependent cell fusion-type infection of cancer cells with F-modified, M-deficient Sendai virus vectors. Tests were performed to determine whether an infection with an endogenous protease-selective cell fusion type could be observed. The following cells were used: HT1080, MMP expressing cancer cell lines (a, D, and G); MKN28, cancer cell lines expressing tPA (B, E, and H); and SW620, cell lines that do not express any of these proteases (C, F, and I). In HT1080, only SeV/F (MMP #2) Δ M-GFP infection spread ten-fold or more (D). Only cell-confluent infections with SeV/F (uPA). DELTA.M-GFP were observed in tPA-expressing cell line MKN 28. In SW620 that does not express either of the above two enzymes, no spread of infection was observed.

FIG. 34 shows MMP induction by phorbol ester and induction of cell fusion type infection by F-modified, M-deficient Sendai virus vectors. The expression of MMP-2 and MMP-9 was confirmed by gelatin zymography (gelatinzymography), in which the portion where the gel hydrolysis (gelatinolytic) activity was present became clear (A). Lane C represents a control. Lane T is the result of the supernatant obtained after induction with 20nM PMA. Bands corresponding to MMP-9 were observed in HT1080 and Panc I, demonstrating induction of MMP-9. For MMP-2, latent MMP-2 with little or no activity was detected in PanCI prior to induction. As shown in FIG. 34B, SeV/F (MMP #2) Δ M-GFP showed a cell fusion type infection due to the induction of MMP-9.

FIG. 35 shows the in vivo cell fusion-type infection with the F-modified, M-deficient Sendai virus vector. Nude mice with HT1080 cancer were prepared. Wherein, animals with cancer diameter larger than 3mm 7-9 days after subcutaneous injection are used. 50 μ L of SeV was injected into the animals at one time. After two days, the cancer was observed under a fluorescence microscope. FIGS. A, D, G, and J are brightfield images; b, E, H, and K correspond to fluorescence images of GFP; and C, F, I, and L are magnified images. Fluorescence was observed only in the region around the site of injection of SeV-GFP and SeV/. DELTA.M-GFP, respectively (FIGS. E and H). In contrast, SeV/F (MMP #2) Δ M-GFP injection was observed to spread fluorescence throughout the cancer (panel K). In the enlarged view, the fluorescence in each cell was confirmed as SeV-GFP and SeV/. DELTA.M-GFP; however, the shape of the cells injected with SeV/F (MMP # 2). DELTA.M-GFP was unclear, suggesting that cell fusion occurred.

FIG. 36 shows in vivo cell fusion-type infection of F-modified, M-deficient Sendai virus vectors. In FIG. 35, the ratio of GFP to the whole cancer was measured by its area using NIH Image. Results SeV-GFP and SeV/. DELTA.M-GFP showed infection at 10% and 20%, respectively; whereas SeV/F (MMP #2) Δ M-GFP showed 90% infection, suggesting a clear spread of infection.

FIG. 37 shows the antitumor effect of the F-modified, M-deficient SeV vector in cancer-bearing nude mice. The volume of mouse cancer in fig. 35 was measured. Four groups of SeV were injected into cancers with a diameter of 3mm or more. Two days later, the injection was performed again, and the cancer volume was measured. The volume of the cancer injected with PBS, SeV-GFP, and SeV/. DELTA.M-GFP showed a rapid increase. Whereas the cancer volume injected with SeV/F (MMP #2) Δ M-GFP (spread throughout the cancer was shown in the experiment of FIG. 36) apparently did not proliferate and remained small. Compared with the other 3 groups, a significant antitumor effect was observed, P < 0.05 according to t-test.

FIG. 38 shows protease expression-selective infection of cancer cells with F-uncleaved, F-modified, M-deficient SeV vectors. The possibility of protease expression causing selective infection was examined in the following cell lines: HT1080 cell line expressing MMP, MKN28 cell line expressing tPA, and SW620 expressing little protease. Infection by SeV/F (MMP #2) Δ M-GFP was observed in the MMP-expressing HT1080 cell line but not in the tPA-expressing MKN28 cell line. Infection by SeV/F (uPA) Δ M-GFP was observed in the MKN28 cell line expressing tPA, but not in the HT1080 cell line expressing MMP.

FIG. 39 shows that the F-uncleaved, F-modified, M-deficient SeV vector acquired the ability to infect due to fibroblast induced MMP-3 and MMP-7. The infectivity change of the M-deficient SeV vector, which is F-modified by MMP induction by fibroblasts, was examined in vitro with SW480 and WiDr. Co-culture of human fibroblasts (hFB) with SW480 and WiDr resulted in infection with SeV/F (MMP #2) Δ M-GFP (B and D). This phenomenon was not observed in SW620 where no induction occurred (F).

FIG. 40 shows MMP-selective infection of human aortic smooth muscle cells with F-modified, M-deficient SeV vectors. Infection with SeV/. DELTA.M-GFP can only be continued by the addition of trypsin. Whereas infection with SeV/F (MMP #2) Δ M-GFP continues after the addition of collagenase, MMP-2, MMP-3, and MMP-9.

FIG. 41 shows cleavage of protease-dependent F protein in F-modified M-deficient SeV vectors. Western blotting confirmed that F0 of Sendai virus was cleaved protease-dependently to F1. M-deficient SeV vectors comprising unmodified F (shown in lanes 1, 4, 7, and 10), M-deficient SeV vectors having the MMP #2 sequence inserted in F (shown in lanes 2, 5, 8, and 11), and M-deficient SeV vectors having the uPA sequence inserted in F (shown in lanes 3, 6, 9, and 12) were treated with the above protease at 37 ℃ for 30 minutes (untreated (lanes 1, 2, and 3); 0.1ng/mL MMP-9 (lanes 4, 5, and 6); 0.1ng/mL uPA (lanes 7, 8, and 9); and 7.5. mu.g/mL trypsin (lanes 10, 11, and 12)). As a result, F1 cleavage occurs depending on the inserted protease substrate. That is, trypsin cleaves the F protein of the F-unmodified M-deficient SeV vector, MMP-9 cleaves the F protein of those M-deficient SeV vectors having the MMP #2 sequence inserted in the F protein, and uPA cleaves the F protein of those M-deficient SeV vectors having the uPA sequence inserted in the F protein.

FIG. 42 shows the generation of F inclusion region deletion mutants and comparison of their fusogenic potency by simultaneous expression with HN. FIG. 42A shows the construction of cytoplasmic domain deletion mutants of Sendai virus F protein. SEQ ID NO: 76-79. FIG. 42B shows the generation of cytoplasmic domain deletion mutants of the F protein and a comparison of the fusogenic forces obtained by simultaneous expression with HN. Cytoplasmic deletion mutant of Sendai virus F protein and HN were expressed simultaneously in LLC-MK2 cells supplemented with 7.5. mu.g/mL trypsin. Four days later, nuclear staining was performed with hematoxylin, and the number of nuclei forming syncytia was counted.

FIG. 43 shows a dramatic increase in the fusogenic force produced by the F/HN chimeric protein. FIG. 43A shows the structure of the F/HN chimeric protein. Linker sequences are set forth in SEQ ID NO: 80, respectively. FIG. 43B shows that the fusogenic force of the F/HN chimeric protein is increased by inserting a linker. Each of the F/HN chimeric proteins of Sendai virus and HN was simultaneously expressed in LLC-MK2 cells supplemented with 7.5. mu.g/mL trypsin.

FIG. 44 is a photograph summarizing the insertion of MMP substrate sequence into the F cleavage site of the F/HN chimeric protein. FIG. 44A is a diagram showing the structure of an F-modified F/HN chimeric protein into which an MMP substrate sequence is inserted. From above, SEQ ID NO: 81-89. FIG. 44B is a graph depicting syncytia formation due to expression of F-modified F/HN in HT1080 cells expressing MMPs.

Fig. 45 shows the modification of the F peptide (fusion peptide) and its concentration-dependent effect on syncytia formation. FIG. 45A is a fusion peptide modification construct. From above, SEQ ID NO: 90-93.

Figure 45B shows the fusogenic forces of MMP #2, MMP #6, and MMP #6G12A associated with the added collagenase (clostridial) concentrations.

FIG. 46 shows the genomic structure of modified F-modified M-deficient Sendai virus.

FIG. 47 is a graph depicting the spread of modified F-modified M-deficient Sendai virus in cancers that express MMPs at low levels. The figure shows the spread of cell fusion two days after infection with the modified F-modified M-deficient Sendai virus.

FIG. 48 is a graph showing the expression of MMP-2 and MMP-9 in cancer cell lines. The receptors for the gel signal recognition proteins of cancer cell line supernatants are shown.

FIG. 49 shows the spread of modified F-modified M-deficient Sendai virus in tumors expressing MMP at low levels. The figure indicates every 0.3cm two days after infection²The number of syncytia of (a). "Δ M" indicates SeV18+/Δ M-GFP, "# 2" indicates SeV18+/F (MMP #2) Δ M-GFP, "# 6" indicates SeV/F (MMP #6) Δ M-GFP, "# 6ct 14" indicates SeV (TDK)/Fct14(MMP #6) Δ M-GFP, and "F/HN chimera" indicates SeV (TDK)/Fct14(MMP # 6)/linker/HN Δ M-GFP.

Best mode for carrying out the invention

The following of the present invention specifically describes the embodiments; but should not be construed as limiting the invention. All references cited herein are incorporated by reference as part of the specification.

1. Construction of SeV vectors with reduced or defective particle formation

Example 1 construction of temperature-sensitive mutant SeV genomic cDNA:

SeV genomic cDNA was constructed in which a temperature-sensitive mutation was introduced into the M gene. FIG. 1 schematically shows the construction of the cDNA, which is described below. The F-deficient full-length Sendai virus genomic cDNA (pSeV18+/Δ F-GFP: Li, H. -O. et al, J.virology 74, 6564-. The fragment containing the M gene (4922bp) was separated by agarose gel electrophoresis. After the band of interest WAs excised, the DNA WAs recovered by QIAEXII gel extraction system (QIAGEN, Bothell, WA) and subcloned into pBluescript II (Stratagene, La Jolla, Calif.) at the EcoRV site (pBlueNaeIfrg-. DELTA. FGFP construction). Introduction of temperature sensitive mutation into M Gene of pBlueNaIFrg-. DELTA.FGFP by Using QuikChange^TMSite-directed mutagenesis kit (Stratagene, La Jolla, Calif.) was performed according to the method described in the kit instructions. Three types of mutations introduced into the M gene were G69E, T116A and A183S, according to the sequence of Cl.151 strain reported by Kondo et al (Kondo, T.et al, J.biol.chem.268, 21924-21930, 1993). The synthetic oligonucleotide sequences used to introduce the mutations were as follows:

G69E(5′-gaaacaaacaaccaatctagagagcgtatctgacttgac-3′/SEQ ID NO：11，5′-gtcaagtcagatacgctctctagattggttgtttgtttc-3′/SEQ ID NO：12)，

T116A (5'-attacggtgaggagggctgttcgagcaggag-3'/SEQ ID NO: 13, 5'-ctcctgctcgaacagccctcctcaccgtaat-3'/SEQ ID NO: 14) and

A183S(5′-ggggcaatcaccatatccaagatcccaaagacc-3′/SEQ ID NO：15，5′-ggtctttgggatcttggatatggtgattgcccc-3′/SEQ ID NO：16).

the M gene of plasmid pBlueNaeIfrg-. DELTA.FGFP contains the three mutations described above, which were digested with SalI and subsequently partially digested with ApaLI. The fragment containing all M genes was subsequently recovered (2644 bp). pSeV18+/Δ F-GFP was digested with ApaLI/NheI, and a HN gene-containing fragment (6287bp) was recovered. These two fragments were subcloned into Litmus38(New England Biolabs, Beverly, Mass.) SalI/NheI sites (Litmus SalI/NheIfrg-Mts. DELTA.FGFP constructs). By using QuikChange^TMSite-directed mutagenesis kit temperature-sensitive mutations were introduced into the Litmus SalI/NheIfrg-Mts. DELTA.FGFP HN gene in the same manner as the mutations were introduced into the M gene according to the method described in the kit instructions. The three mutations introduced into the HN gene were A262T, G264R and K461G, based on the sequence of ts271 strain reported by Thompson et al (Thompson, S.D., et al, Virology160, 1-8, 1987). The synthetic oligonucleotide sequences used to introduce the mutations were as follows:

A262T/G264R (5'-catgctctgtggtgacaacccggactaggggttatca-3'/SEQ ID NO: 17, 5'-tgataacccctagtccgggttgtcaccacagagcatg-3'/SEQ ID NO: 18), and

K461G(5′-cttgtctagaccaggaaatgaagagtgcaattggtacaata-3′/SEQ ID NO：19，5′-tattgtaccaattgcactcttcatttcctggtctagacaag-3′/SEQ ID NO：20).

although mutations can be introduced into the M and HN genes of different vectors, all mutations can also be introduced into both the M and HN genes by a plasmid (Litmus SalI/NheIfrg-. DELTA.FGFP) obtained by subcloning a fragment (8931bp) containing the M and HN genes at the SalI/NheI site of Litmus38, which is obtained by digesting pSev18 +/. DELTA.F-GFP with Sal I/Nhe I. The sequential introduction of mutations resulted in the introduction of a total of six temperature-sensitive mutations; three mutations were located in the M gene and three in the HN gene (LitmusSalI/NheIfrg-MtsHNts. DELTA. FGFP construct).

LitmusSalI/NheIfrg-MtsHNts. DELTA.FGFP was digested with SalI/NheI and an 8931bp fragment was obtained. Another fragment (8294bp) lacking the M and HN genes was obtained by digestion of pSeV18+/Δ F-GFP with SalI/NheI. These two fragments were ligated together to construct an F-deficient full-length Sendai virus genomic cDNA (pSeV18 +/MtsHNts. DELTA.F-GFP) containing 6 temperature-sensitive mutations in the M and HN genes, and the EGFP gene at the F deletion site (FIG. 2).

In addition, to quantify the expression level of the gene in the plasmid, a cDNA containing the secreted alkaline phosphatase (SEAP) gene was constructed. Specifically, a SEAP fragment (1638bp) containing the start signal of a terminator signal interference sequence located downstream of the SEAP gene was excised using NotI (WO 00/70070). The fragment was recovered and purified after electrophoresis. This fragment was subsequently inserted into the NotI site of each of pSeV18+/Δ F-GFP and pSeV18+/MtsHNts Δ F-GFP. The resulting plasmids were pSeV18+ SEAP/. DELTA.F-GFP and pSeV18+ SEAP/. DELTA.F-GFP, respectively (FIG. 2).

Example 2 reconstitution and amplification of viruses with temperature sensitive mutations introduced:

virus reconstitution was performed according to the method reported by Li et al (Li, H. -O. et al, J.virology 74, 6564-. F protein helper cells prepared using an inducible Cre/loxP expression system were used to reconstitute F-deficient viruses. This system utilizes the pCALNdLw plasmid designed to express Cre DNA recombinase-mediated inducible gene product expression (Arai, T, et al, J.Virol.72, 1115-1121, 1988). In this system, the inserted gene is expressed in the transformant carrying the plasmid using Saito et al (Saito, I. et al, Nucleic Acids Res.23, 3816-3821, 1995; Arai, T. et al, J.Virol.72, 1115-1121, 1998) method of infecting the transformant with a recombinant adenovirus (AxCANCre) expressing the creDNA recombinase. For SeV-F proteins, transformed cells containing the F gene are referred to herein as LLC-MK2/F7, and cells that continue to express F protein following AxCANCre induction are referred to herein as LLC-MK 2/F7/A.

Reconstitution of viruses containing temperature sensitive mutations was performed as follows: LLC-MK2 cells at 5X 10⁶Cells/dish were plated on 100-mm dishes and then cultured for 24 hours. These cells were infected (MOI 2) with recombinant vaccinia virus expressing T7 polymerase (which had been treated with psoralen and long wavelength ultraviolet light (365nm) for 20 minutes) (PLWUV-VacT 7: Fuerst, T.R. et al, Proc. Natl. Acad. Sci. USA 83, 8122-. Cells were washed with serum-free MEM. Plasmids, pSeV18+/MtsHNts Δ F-GFP, pGEM/NP, pGEM/P, pGEM/L and pGEM/F-HN (Kato, A. et al, Genes Cells 1, 569-579, 1996), were resuspended in Opti-MEM (Gibco-BRL, Rockville, Md.) in amounts of 12 μ g, 4 μ g, 2 μ g, 4 μ g and 4 μ g/dish, respectively. SuperFect transfection reagent (Qiagen, Bothell, WA) equivalent to 1. mu.g DNA/5. mu.L WAs added and mixed. The resulting mixture was left at room temperature for 15 minutes, and then 3ml of Opti-MEM containing 3% FBS was added. The mixture is added to the cells. After 5 hours of culture, the cells were washed twice with serum-free MEM and cultured in MEM containing 40. mu.g/ml cytosine-. beta. -D-arabinofuranoside (AraC: Sigma, St. Louis, Mo.) and 7.5. mu.g/ml trypsin (Gibco-BRL, Rockville, Md.). After 24 hours of culture, cells which continue to express the F protein (LLC-MK 2/F7/A: Li, H. -O. et al, J.virology 74, 6564- ⁶The amount of cells/dish was overlayed. These cells were then cultured in MEM containing 40. mu.g/mL AraC and 7.5. mu.g/mL trypsin at 37 ℃ for two more days (P0). Cells were collected and the pellet was washed with 2ml Opti-MEM/dish resuspension. Three freeze-thaw treatments and the lysates were transfected directly into LLC-MK 2/F7/A. Cells were cultured in serum-free MEM containing 40. mu.g/mL AraC and 7.5. mu.g/mL trypsin at 32 ℃ (P1). After 5-7 days, the freshly prepared LLC-MK2/F7/A was infected with a portion of the culture supernatant and cultured in the same serum-free MEM containing 40. mu.g/mL of LARAC and 7.5. mu.g/mL of trypsin at 32 ℃ (P2). After 3-5 days, the LLC-MK2/F7/A, which was prepared immediately before, was reinfected and the cells were cultured in serum-free MEM containing only 7.5. mu.g/mL trypsin for 3-5 days at 32 ℃ (P3). BSA was added to the recovered culture supernatant at a final concentration of 1% and the mixture was stored at-80 ℃. The stored virus solution was thawed and used in subsequent experiments.

The titers of the virus solutions prepared by this method were as follows: SeV18+/Δ F-GFP, 3X 10⁸；SeV18+/MtsHNtsΔF-GFP，7×10⁷；SeV18+SEAP/ΔF-GFP，1.8×10⁸；SeV18+SEAP/MtsHNtsΔF-GFP，8.9×10⁷GFP-CIU/mL (GFP-CIU is defined in WO 00/70070). On the other hand, for vectors containing GFP, CIU determined by direct detection of GFP is defined as GFP-CIU. It was confirmed that the GFP-CIU value was substantially equal to the corresponding CIU value (WO 00/70070). In the determination of the titre of SeV18+/Δ F-GFP and SeV18+/MtsHNts Δ F-GFP, post-infection spread of plaques of cells which persistently express the F protein (LLC-MK2/F7/A) was observed at 32 ℃ and 37 ℃. Figure 3 shows the pattern observed 6 days after infection. The SeV18+/MtsHNts Δ F-GFP plaques diffused to some extent at 32 ℃ but diffused to a much lesser extent at 37 ℃. This suggests that virion formation is reduced at 37 ℃.

Example 3 Effect of culture temperature (32 ℃) on Virus reconstitution:

in experimental reconstitution of temperature sensitive mutant-introduced viruses (example 2), P1 and all subsequent cultures were performed at 32 ℃. This temperature was used because the introduced control viruses used to evaluate temperature-sensitive mutations grew well at 32 ℃ (Kondo, T. et al, J.biol. chem.268, 21924-. Careful study of the test conditions revealed that for reconstitution of SeV (and for viruses other than those into which temperature-sensitive mutations have been introduced), the efficiency of reconstitution could be improved by performing P1 at 32 ℃ and subsequent culturing, and that there is a high possibility that viruses that were previously difficult to obtain could be recovered.

There are two reasons for the improved reconstitution efficiency at 32 ℃. First, it is considered that the cytotoxic activity caused by AraC (supplemented with it for the purpose of inhibiting vaccinia virus amplification) is inhibited when cultured at 32 ℃ unlike when cultured at 37 ℃. LLC-MK2/F7/A cells cultured in serum-free MEM containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin at 37 ℃ under virus reconstitution conditions resulted in cell destruction, including increased exfoliated cells, after 3-4 days. However, culture at 32 ℃ was effective for 7-10 days without damage to the cells. Success in reconstituting SeV with poor transcription and/or replication efficiency, or with poor formation of infectious virions, is considered to be a direct reflection of the culture duration. The second point is that the expression of F protein can be maintained in LLC-MK2/F7/A when the cells are cultured at 32 ℃. LLC-MK2/F7/A cells which continue to express F protein were cultured in 10% FBS-containing MEM at 37 ℃ until 6-well plates were confluent, then the medium was replaced with serum-free MEM containing 7.5. mu.g/ml trypsin, and the cells were further cultured at 32 ℃ or 37 ℃. Cells were recovered over time using a cell scraper and subjected to Western blotting with anti-F protein antibody (mouse monoclonal antibody) to semi-quantitatively analyze intracellular F protein. F protein expression was maintained at 37 ℃ for two days and subsequently decreased. However, expression lasted at 32 ℃ for at least 8 days (FIG. 4). These results confirm the effectiveness of virus reconstitution at 32 ℃ (after stage P1).

The Western blot was performed using the following method: cells recovered from one well of a 6-well plate were stored at-80 ℃ and then thawed in 100. mu.L of 1 Xdilution SDS-PAGE sample buffer (Red LoadingBuffer Pack; New England Biolabs, Beverly, Mass.). The sample was then heated at 98 ℃ for 10 minutes, centrifuged, and a 10- μ l aliquot of the supernatant was applied to an SDS-PAGE gel (multigel 10/20; Daiichi Pure Chemicals Co., Ltd., Tokyo, Japan). After 2.5 hours of electrophoresis at 15mA, proteins were transferred to PVDF membrane (Immobilon PVDF transfer membrane; Millipore, Bedford, Mass.) using a semidry method at 100mA for one hour. The transfer membrane was immersed in blocking solution (Block Ace; Snow Brand Milk Products Co., Ltd., Sapporo, Japan) at 4 ℃ for one hour or more, soaked in a primary antibody solution containing 10% Block Ace (supplemented with 1/1000 volumes of anti-F protein antibody), and then the membrane was allowed to stand overnight at 4 ℃. After washing 3 times with TBS (TBST) containing 0.05% Tween 20 and 3 times with TBS, the cells were soaked in a secondary antibody solution containing 10% Block Ace supplemented with 1/1000 volumes of HPR-conjugated anti-mouse IgG + IgM antibody (goat F (ab') 2 anti-mouse IgG + IgM, HRP; BioSource int., Camarillo, Calif.). The sample was then agitated at room temperature for 1 hour. The membrane was washed 3 times with TBST and 3 times with TBS. The membrane proteins were then detected by chemiluminescence (ECL western blot detection reagent; Amersham Pharmacia Biotech, Uppsala, Sweden).

Example 4 quantification of Secondary Release particles of Virus into which the temperature-sensitive mutation of the invention HAs been introduced (HA assay, Western blot):

the level of secondary release particles was compared with SeV18+/Δ F-GFP and SeV18+/MtsHNts Δ F-GFP using an autonomously replicating SeV containing all viral proteins and a GFP fragment (780bp) containing a stop signal-interfering sequence-start signal downstream of the GFP gene at the NotI site (SeV18+ GFP: FIG. 2).

LLC-MK2 cells were grown to confluency in 6-well plates. Will be 3X 10⁷Each virus solution of CIU/ml was added to these cells at 100 μ L per well (MOI ═ 3) and the cells were transfected for one hour. After washing the cells with MEM, serum-free MEM (1ml) was added to each well, and the cells were cultured at 32 ℃, 37 ℃ and 38 ℃ respectively. Samples were taken daily and 1ml of fresh serum-free MEM was added to the remaining cells immediately after sampling. Incubate and sample over time. Observation of GFP expression 3 days after infection under a fluorescence microscope revealed that the infection levels of the three types of viruses were almost equal under the three temperature conditions (32 ℃, 37 ℃ and 38 ℃) and that the GFP expression was similar (FIG. 5).

Secondary release particles hemagglutination Activity (HA Activity) assay was used according to Kato et al (Kato, A., et al, Genes Cell 1, 569-5) 79, 1996). Specifically, the virus solution was serially diluted with PBS using a round-bottom 96-well plate. Serial two-fold 50 μ L dilutions were performed in each well. mu.L of the preserved chicken blood (Cosmo Bio, Tokyo, Japan) was diluted to 1% with PBS, added to 50. mu.L of the virus solution, and the mixture was left at 4 ℃ for one hour. Erythrocyte agglutination was examined. The highest virus dilution in the agglutinated sample was determined as HA activity. In addition, one coagulation unit (HAU) was calculated to be 1X 10⁶Virus, and expressed as virus number (figure 6). The secondary release particle of SeV18+/MtsHNts Δ F-GFP was significantly reduced and was determined to be about 1/10 the level of the secondary release particle of SeV18+/Δ F-GFP at 37 ℃. SeV18+/MtsHNts Δ F-GFP virus particle formation is reduced at 32 ℃ and preparation can be carried out to some extent, although only some virus particles are produced.

Western blot was used to quantify the secondary release particles. LLC-MK2 cells were infected with the virus at MOI-3 in a similar manner to that described above, and culture supernatant and cells were recovered two days after infection. The culture supernatant was centrifuged at 48,000Xg for 45 minutes to recover virus particles. After SDS-PAGE, Western blotting was performed to detect these proteins using anti-M protein antibodies. The anti-M protein antibody is a newly prepared polyclonal antibody prepared from rabbit serum immunized with a mixture of three synthetic peptides: corresponding to amino acids 1-13(MADIYRFPKFSYE + Cys/SEQ ID NO: 21), 23-35(LRTGPDKKAIPH + Cys/SEQ ID NO: 22), and 336-348(Cys + NVVAKNIGRIRKL/SEQ ID NO: 23) of the SeV M protein. Western blotting was carried out according to the method described in example 3, wherein the primary antibody, anti-M protein antibody, was used at a dilution of 1/4000 and the secondary antibody, anti-rabbit IgG antibody conjugated with HRP (anti-rabbit IgG (goat) H + L conj.; ICN P., Aurola, OH) was used at a dilution of 1/5000. In the case of SeV18+/MtsHNts Δ F-GFP infected cells, M protein was widely expressed to a similar extent, but viral protein expression was reduced (FIG. 7). Western blot also confirmed the reduction of secondary released viral particles.

Example 5 expression level of Gene contained in Virus into which temperature-sensitive mutation was introduced (SEAP experiment):

the release of SeV18+/MtsHNts Δ F-GFP secondary particles was reduced. If the expression of the gene contained is simultaneously reduced, this modification is of no significance in the gene expression vector. Thus, gene expression levels were evaluated. LLC-MK2 cells were infected with SeV18+ SEAP/. DELTA.F-GFP or SeV18+ SEAP/MtsHNts. DELTA.F-GFP at MOI ═ 3 and culture supernatants were collected over time (12, 18, 24, 50 and 120 hours post infection). SEAP activity in the supernatant was determined using Reporter Assay Kit-SEAP (TOYOBO, Osaka, Japan) according to the Kit instructions. Both types of SEAP activity were comparable (fig. 8). The same samples were also assayed for hemagglutination activity (HA activity). The HA activity of SeV18+ SEAP/MtsHNts. DELTA.F-GFP decreased to one tenth (FIG. 9). Viral proteins were harvested from sample viruses by centrifugation at 48,000Xg for 45 minutes and then semi-quantitatively determined by Western blotting using anti-M antibodies. The level of viral proteins in the supernatant was also reduced (figure 10). These findings indicate that induction of the temperature sensitive mutation reduced the level of secondary particle release to about 1/10 without substantial reduction in the expression level of the contained gene.

Example 6 cytotoxic Activity of viruses having introduced temperature-sensitive mutations (LDH assay):

SeV infection is usually cytotoxic. The influence of the introduced mutation was examined in this respect. LLC-MK2, BEAS-2B and CV-1 cells were cultured at 2.5X 10 cells each⁴Cells/well were plated on 96-well plates (100. mu.L/well) and cultured. LLC-MK2 and CV-1 were cultured in MEM containing 10% FBS, and BEAS-2B was cultured in a 1: 1 mixed medium of D-MEM and RPMI (Gibco-BRL, Rockville, Md.) containing 10% FBS. After 24 hours of incubation, virus infection was performed by adding 5. mu.L/well of SeV18+/Δ F-GFP or SeV18+/MtsHNts Δ F-GFP solution (diluted in MEM containing 1% BSA). After 6 hours, the medium containing the virus solution was removed and replaced with the corresponding fresh medium (with or without 10% FBS). Culture supernatants were sampled 3 days post infection if FBS-free media was used, or 6 days post infection if FBS-containing media was used. Cytotoxic activity was analyzed by using the cytotoxic activity detection kit (Roche, Basel, Switzerland) according to the kit instructions. None of the viral vectors has cytotoxic activity in LLC-MK2And (4) sex. Furthermore, the cytotoxic activity of SeV18+/MtsHNts Δ F-GFP measured in CV-1 and BEAS-2B was comparable to or less than that of SeV18+/Δ F-GFP (FIG. 11). Therefore, inhibition of secondary particle release due to the introduction of temperature sensitive mutations does not induce cytotoxic activity.

Example 7 investigation of the mechanism of secondary particle release inhibition:

to illustrate part of the mechanism of secondary particle release inhibition associated with the introduction of temperature sensitive mutations, subcellular localization of the M protein was examined. LLC-MK2 cells were infected with SeV of each type (SeV18+ GFP, SeV18+/Δ F-GFP, SeV18+/MtsHNts Δ F-GFP) and cultured at 32 ℃ for two days, 37 ℃ or 38 ℃. The cells were immunostained with anti-M antibodies. Immunostaining was performed as follows: the cultured cells were washed once with PBS, methanol frozen to-20 ℃ was added, and the cells were fixed at 4 ℃ for 15 minutes. After washing the cells 3 times with PBS, they were blocked with PBS solution containing 2% goat serum and 0.1% Triton for one hour at room temperature. After washing three more times with PBS, the cells were incubated with a primary antibody solution (10. mu.g/mL anti-M antibody) containing 2% goat serum at 37 ℃ for 30 min. After washing 3 times with PBS, the cells were reacted with a secondary antibody solution containing 2% goat serum (10. mu.g/mL Alexa Fluor 488 goat anti-rabbit IgG (H + L) conjugate: Molecular Probes, Eugene, OR) for 15 min at 37 ℃. Finally, after washing three more times with PBS, the cells were observed under a fluorescence microscope. For self-replicating SeV18+ GFP containing F and HN proteins, concentrated M protein was detected on the cell surface at all detection temperatures (FIG. 12). This M protein concentration has been previously reported (Yoshida, T. et al, Virology 71, 143-. Specifically, for SeV18+ GFP, the M protein localization on the cell surface was shown to be normal at all temperatures, indicating that sufficient virions were formed. On the other hand, for SeV18+/Δ F-GFP, the concentration of M protein decreases dramatically at 38 ℃. It is believed that the M protein localizes to the cell surface and binds to the cytoplasmic tail of the F and HN proteins (Sanderson, C.M. et al, J.virology 68, 69-76, 1994; Ali, A. et al, Virology 276, 289-303, 2000). Since one of these two proteins, the F protein, was deleted in SeV18+/Δ F-GFP, the F protein deficiency was presumed to have an effect on M protein localization. The effect is expected to be stronger for SeV18+/MtsHNts Δ F-GFP, and it is also expected that even at 37 ℃, M protein localization will be disturbed and the number of particles released twice will be reduced.

Example 8 investigation of the inhibitory mechanism of secondary particle release (2):

to study the subcellular localization of SeV proteins in more detail, analysis was performed using a confocal laser microscope (MRC 1024; Bio-Rad Laboratories Inc., Hercules, Calif.). A-10 cells (rat myoblasts) were infected with SeV18+ SEAP/. DELTA.F-GFP and SeV18+ SEAP/. DELTA.F-GFP (MOI ═ 1), respectively, and then cultured in MEM containing 10% serum at 32 ℃ or 37 ℃. One or two days later, cells were immunostained using anti-M and anti-HN antibodies. Immunostaining was performed as follows: infected cell cultures were washed once with PBS. Methanol cooled to-20 ℃ was added to the cells and the cells were fixed at 4 ℃ for 15 minutes. The cells were washed 3 times with PBS and then blocked for one hour at room temperature with PBS solution containing 2% goat serum, 1% BSA and 0.1% Triton. The cells were reacted with a solution of anti-M first antibody (10. mu.g/mL anti-M antibody) containing 2% goat serum at 37 ℃ for 30 minutes. The cells were then reacted with an anti-HN primary antibody solution (1. mu.g/mL anti-HN antibody (IL4-1)) at 37 ℃ for 30 minutes. After three washes with PBS, the cells were reacted with a secondary antibody solution containing 2% goat serum (10. mu.g/mL Alexa Fluor 568 goat anti-rabbit IgG (H + L) conjugate and 10. mu.g/mL Alexa Fluor 488 goat anti-mouse IgG (H + L) conjugate: molecular probes, Eugene, OR) for 15 min at 37 ℃. Cells were washed three times with PBS and nuclei were stained with 4000-fold diluted TO PRO3(Molecular Probes, Eugene, OR). The cells were left at room temperature for 15 minutes. Finally, to prevent quenching (quenching), the liquid was replaced with Slow fade anti fade Kit solution (Molecular Probes, Eugene, OR), which observed the cells under a confocal microscope. Figure 13 shows the results one day after infection. Red represents M protein localization; green represents HN protein localization; and yellow is the co-location of the two. The deep red part (Far red) is color converted, so blue represents the nucleus. For SeV18+ SEAP/. DELTA.F-GFP, the localization pattern of each protein was not very different between 32 ℃ and 37 ℃ and localization of the M protein on the cell surface was observed. On the other hand, for SeV18+ SEAP/MtsHNts. DELTA.F-GFP, the localization of each protein at the above two temperatures was different from that in SeV18+ SEAP/. DELTA.F-GFP. In particular, there is hardly any M protein localization to the cell surface. Specifically, at 37 ℃, the M and HN proteins are almost completely separated, such that the M protein is localized at a site putatively proximal to a microtubule intermediate (e.g., proximal to the golgi apparatus). Similar results were obtained from cells cultured for two days after infection. In particular in SeV18+ SEAP/MtsHNts. DELTA.F-GFP-infected cells, subcellular M protein localization did not change between one and two days after infection (FIG. 14) and protein transport was shown to stop. The results also show that the reduced release of secondary particles by the temperature sensitive mutation introduced virus is due to a defect in the localization of the M protein, which plays an important role in particle formation.

When cultured at 32 ℃ after infection of cells with SeV18+ SEAP/MtsHNts. DELTA.F-GFP, the stained M protein had a morphology similar to that of microtubules (FIG. 13). To show the involvement of microtubules, agents that enhance microtubule depolymerization were added and the changes in localization of the M protein (and HN protein) were then studied. A-10 cells were infected with SeV18+ SEAP/MtsHNts. DELTA.F-GFP at MOI ═ 1 and the disaggregating reagents colchicine (Nakarai Tesque, Kyoto, Japan) or colchicine (Nakarai Tesque, Kyoto, Japan) were immediately added at a final concentration of 1 mM. The cells were then cultured at 32 ℃. Two days after infection, subcellular localization of M and HN proteins was observed by the same method as described above. In the absence of disaggregating agent, M protein distribution was morphologically similar to microtubules (fig. 13). However, the addition of a disaggregating agent resulted in the disruption of the structure and the protein was detected as a large fibrous structure (fig. 15). The structure may be a polymer of the M protein itself, or an M protein that binds to residues that depolymerize microtubules. In either case, as shown in FIG. 13, it appears to be plausible to conclude that the M protein is located on the microtubules of cells cultured at 32 ℃ after infection with SeV18+ SEAP/MtsHNts. DELTA.F-GFP.

To demonstrate that the localization of the above M protein in microtubules is a characteristic of temperature-sensitive viruses, the effect of changes in M protein (and HN protein) localization on microtubule depolymerizing agent (colchicine) pairs following infection with the viruses SeV18+/Δ F-GFP and SeV18+/MtsHNts Δ F-GFP was evaluated. A-10 cells were infected with SeV18+/Δ F-GFP or SeV18 +/mtschnts Δ F-GFP at MOI ═ 1, and the disaggregating agent colchicine was immediately added at a final concentration of 1 μ M. The cells were cultured at 32 ℃ or 37 ℃. Two days after infection, subcellular localization of the M protein (and HN protein) was observed by the same method as described above. The results are shown in fig. 16. Cells infected with both viruses showed similar characteristics. Specifically, when cultured at 32 ℃ after cell infection, the observed M protein was a large fibrous structure, similar to that in fig. 15. The coexistence of M protein and microtubules was only seen in the case of SeV18+/Δ F-GFP infection. Specifically, in cells infected with SeV18 +/MtsHNts. DELTA.F-GFP and cultured at 37 ℃, the M protein observed was localized to a region presumably near the Golgi apparatus.

From the above results, the following conclusions can be drawn: m protein is synthesized in the vicinity of the golgi apparatus; which is transported around the cell along microtubules (e.g., binding to motor proteins such as actin) and binds primarily to the cytoplasmic tail of the F and HN proteins (Sanderson, C.M. et al, J.virology 68, 69-76, 1994; Ali, A. et al, Virology 276, 289 303, 2000); and the M protein localizes to the cell surface and then forms particles. In viruses containing temperature sensitive mutations, all events up to intracellular transport along microtubules were normal at 32 ℃. However, translocation from the microtubules to the cell surface can be hindered, resulting in localization along the microtubules. At 37 ℃, it is presumed that intracellular transport even along microtubules is hindered, and localization near the golgi is thus observed. M protein synthesis is presumed to occur near the golgi. However, M protein aggregation may be observed at these sites, and regions that may synthesize themselves are elsewhere. However, tubulin, a component of microtubules, has been reported to activate and participate in SeV transcription and replication (Moyer, S.A. et al, Proc. Natl. Acad. Sci. U.S.A.83, 5405-Bus5409, 1986; Ogino, T.et al, J.biol. chem.274, 35999-Bus36008, 1999). However, since the golgi body is located near the central body where tubulin is expected to be abundantly present, the golgi body can be synthesized near the microtubule central body (i.e., near the golgi body). Furthermore, although the M gene of the SeV mutant F1-R contains a mutation, it modifies microtubules after infecting the cells, and the modification makes particle formation independent of the cell polarity of the F1-R strain (Tashiro, M. et al, J.Virol.67, 5902-5910, 1993). In other words, the results obtained in this example can be explained by presuming the intracellular transport of the M protein along tubulin. Among the putative mechanisms, introduction of temperature sensitive mutations into the M and HN genes can result in localization of defective subcellular M proteins, resulting in a reduction in secondary particle release.

Example 9 construction of genomic cDNA of M gene-deficient SeV comprising EGFP Gene:

the cDNA was constructed using the full-length genomic cDNA of M-deficient SeV, which is M gene-deficient (pSeV18+/Δ M: WO 00/09700). The construction scheme is shown in FIG. 17. A BstEII fragment (2098bp) containing the M defect site of pSeV18+/Δ M was subcloned into the BstEII site of pSE280(pSE-BstEIIfrg structure). The EcoRV recognition site of the pSE280 site was deleted by digestion with SalI/XhoI followed by ligation (Invitrogen, Groningen, Netherlands). pEGFP (TOYOBO, Osaka, Japan) containing the GFP gene was digested with Acc65I and EcoRI, and the 5' -end of the digest was blunt-ended by using a DNA truncation kit (Takara, Kyoto, Japan). The blunt-ended fragment was subsequently subcloned into pSE-BstEIIfrg, which had been digested with EcoRV and treated with BAP (TOYOBO, Osaka, Japan). The BstEII fragment containing the EGFP gene was returned to the original pSeV18+/Δ M to construct M gene-deficient SeV genomic cDNA (pSeV18+/Δ M-GFP) containing the EGFP gene at the M-deficient site.

Example 10 construction of cDNA of SeV deficient in genomic M Gene and in replication ability:

genomic cDNAs for M gene-deficient and F gene-deficient SeVs were constructed. The following construction scheme is shown in FIG. 18. The M gene was deleted with pBlueNaeFrg-. DELTA.FGFP by subcloning the NaeI fragment (4922bp) (pSeV18 +/DELTA.F-GFP: Li, H. -O. et al, J.virology 74, 6564 + 6569, 2000; WO 00/70070) of the full-length genomic cDNA of the F-deficient Sendai virus, which contained the EGFP gene at the site of the F gene defect, into pBluescript II (Stratagene, La Jolla, CA) EcoRV site. The deletion is designed such that the M gene can be directly cleaved using the ApaLI site immediately after the M gene. That is, after the ApaLI recognition site was inserted into the P gene so that the fragment to be excised became 6n, QuikChange was used for mutagenesis^TMSite-directed mutagenesis kit (Stratagene, LaJolla, Calif.) was performed according to the kit instructions. The synthetic oligonucleotide sequences used for mutagenesis were as follows:

5'-agagtcactgaccaactagatcgtgcacgaggcatcctaccatcctca-3'/SEQ ID NO: 24 and

5’-tgaggatggtaggatgcctcgtgcacgatctagttggtcagtgactct-3’/SEQ ID NO：25.

after mutagenesis, the resulting mutant cDNA WAs partially digested with ApaLI (at 37 ℃ C., 5 minutes), recovered using QIAquick PCR purification kit (QIAGEN, Bothell, WA), and then ligated. Again recovered with the QIAquick PCR purification kit, digested with BsmI and StuI, and used to transform H5 to prepare M gene-deficient (and F gene-deficient) DNA (pblueneaifrg- Δ M Δ FGFP).

The M gene-deficient pBlueNaIfrg-. DELTA.M.DELTA.FGFP (and F gene) was digested with SalI and ApaLI to recover a 1480bp fragment containing the M gene defect site. pSeV18+/Δ F-GFP was digested with ApaLI/NheI to recover a HN gene-containing fragment (6287bp), and these two fragments were cloned into the SalI/NheI site (Litmus SalI/NheIfrg-. DELTA.M Δ FGFP construct) of Litmus 38(New Engl and Biolabs, Beverly, Mass.). The 7767bp fragment recovered by SalI/NheI digestion of Litmus SalI/NheIfrg-. DELTA.M.DELTA.FGFP was ligated with another fragment (8294bp) obtained by SalI/NheI digestion of pSeV18 +/. DELTA.F-GFP, which does not contain genes such as the M and HN genes. Thus, M-and F-deficient Sendai virus full-length genomic cDNAs comprising the EGFP gene at the defect site (pSeV18+/Δ M Δ F-GFP) were constructed. The structure of the M-deficient (and M-and F-deficient) viruses thus constructed is shown in FIG. 19. The genomic cDNA can be used to construct M-deficient and F-deficient SeVs comprising the desired modified F protein.

EXAMPLE 11 preparation of helper cells expressing SeV-M protein

To prepare helper cells expressing M protein, Cre/loxP expression induction system was used. To construct this system, the plasmid pCALNdLw was used, which was designed to induce the expression of the gene product using Cre DNA recombinase (Arai, T, et al, J.Virol.72, 1115-1121, 1988). This system is also used for the preparation of helper cells for the F protein (LLC-MK2/F7 cells) (Li, H. -O. et al, J.virology 74, 6564-.

<1> Structure of plasmid expressing M Gene:

to prepare helper cells inducing the expression of F and M proteins, the M gene was transferred to these cells using the above-described system using LLC-MK2/F7 cells. Since pCALNdLw/F is used to transfer the neomycin resistance gene containing the F gene, it is necessary to insert a different drug resistance gene to enable the use of the same cell. Therefore, according to the scheme described in FIG. 20, the neomycin resistance gene of the plasmid containing the M gene (pCALNdLw/M: insertion of the M gene into the SwaI site of pCALNdLw) was replaced with the hygromycin resistance gene. That is, after pCALNdLw/M was digested with HincII and EcoT22I, the fragment containing the M gene (4737bp) was separated by electrophoresis on agarose, and the corresponding band was excised and recovered using the QIAEXII Gel extraction system. Meanwhile, pCALNdLw/M was digested with XhoI to recover a fragment (5941bp) not containing the neomycin resistance gene, and then further digested with HincII to recover a fragment of 1779 bp. The hygromycin resistance gene was prepared by PCR using pcdna3.1hygro (+) (Invitrogen, grongen, Netherlands) as a template and the following primer pairs:

hygro-5 ' (5'-tctcgagtcgctcggtacgatgaaaaagcctgaactcaccgcgacgtctgtcgag-3'/SEQ ID NO: 26) and

hygro-3’(5’-aatgcatgatcagtaaattacaatgaacatcgaaccccagagtcccgcctattcctttgccctcggacgagtgctggggcgtc-3’)/SEQ ID NO：27).

the PCR products were recovered using the QIAquick PCR purification kit and digested with XhoI and EcoT 22I. pCALNdLw-hygroM was constructed by ligating these three fragments.

<2> cloning of helper cells inducing expression of SeV-M and SeV-F proteins:

transfection was performed by product instructions using Superfect transfection reagent. Specifically, the following steps are performed: LLC-MK2/F7 cells at 5X 10⁵The cells/dish were plated in Petri dishes 60mm in diameter, and then cultured in D-MEM containing 10% FBS for 24 hours. pCALNdLw-hygroM (5. mu.g) was diluted in FBS-free and antibody-free D-MEM (150. mu.L total). The mixture was stirred and 30. mu.L of Superfect transfection reagent was added and the mixture was stirred again. After standing at room temperature for 10 minutes, D-MEM (1ml) containing 10% FBS was added. The transfection mixture thus prepared was stirred and LLC-MK2/F7 cells which had been washed once with PBS were added. In an incubator at 37 ℃ and 5% CO₂After 3 hours of incubation in the atmosphere, the transfection mixture was removed and the cells were washed three times with PBS. D-MEM containing 10% FBS was added to the cells that had been cultured for 24 hours. After culture, cells were trypsinized, plated in 96-well plates at a dilution of approximately 5 cells/well, and cultured in D-MEM (supplemented with 150. mu.g/ml hygromycin) (Gibco-BRL, Rockville, Md.) containing 10% FBS for approximately two weeks. Clones propagated from single cells were cultured and expanded in 6-well plates. A total of 130 clones were prepared and specifically analyzed as follows.

<3> analysis of helper cell clones inducing expression of one or more SeV-M (and SeV-F) proteins:

expression of M protein in 130 clones obtained as described above was semi-quantitatively analyzed by Western blotting. Each clone was plated in 6-well plates and, near confluent status, infected with recombinant adenovirus expressing Cre DNA recombinase (AxCANCre) (diluted in MEM containing 5% FBS) at MOI.5 according to the method of Saito et al (Saito, I. et al, Nucleic Acids Res.23, 3816-3821, 1995; Arai, T. et al, J.Virol.72, 1115-1121, 1998). After two days of incubation at 32 ℃, the culture supernatant was removed. Cells were washed once with PBS and isolated by using a cell scraper. SDS-PAGE was performed by loading 1/10 from the thus-recovered cells into each lane, and then Western blotting was performed according to the methods described in examples 3 and 4 using an anti-M protein antibody. Of the 130 clones, those with relatively high M protein expression levels were analyzed by Western blotting using anti-F protein antibody (F236: Segawa, H. et al, J.biochem.123, 1064-. Both results are shown in fig. 21.

Example 12 evaluation of helper cells inducing expression of SeV-M protein:

using the helper cells inducing the expression of SeV-M protein cloned in example 11, virus reconstitution of M-deficient SeV (SeV18+/Δ M-GFP) was carried out to evaluate the virus-producing ability of these cell clones. P0 lysate of SeV18+/Δ M-GFP was added to each clone and examined to see if GFP protein diffusion was observed (whether trans-supply of M protein was achieved). The P0 lysate was prepared as follows. LLC-MK2 cells at 5X 10 ⁶Cells/dish were plated on Petri dishes 100mm in diameter at 5X 10⁶Cells/dish, cultured for 24 hours, and then infected with pluuv-VacT 7 at room temperature for 1 hour at MOI ═ 2. Plasmids pSeV18+/Δ M-GFP, pGEM/NP, pGEM/P, pGEM/L, pGEM/F-HN and pGEM/M were suspended in Opti-MEM at weight ratios of 12 μ g, 4 μ g, 2 μ g, 4 μ g, 4 μ g and 4 μ g/dish, respectively. SuperFect transfection reagent equivalent to 1. mu.g DNA/5. mu.L was added to these suspensions and mixed. The mixture was left at room temperature for 15 minutes and finally added to 3ml of Opti-MEM containing 3% FBS. The mixture is added to the cells and the cells are subsequently cultured. After 5 hours of culture, the cells were washed twice with serum-free MEM and cultured in MEM containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin. After 24 hours of culture, LLC-MK2/F7/A cells were plated at 8.5X 10⁶The cells/dish were plated and further cultured in MEM containing 40. mu.g/ml AraC and 7.5. mu.g/ml trypsin at 37 ℃ for two days (P0). These cells were recovered and the pellet was resuspended in 2 ml/dish Opti-MEM, and P0 lysate was prepared by repeating three freeze and thaw cycles. At the same time, 10 different clones were plated in 24-well plates. When near full bottom, they were infected with AxCANCre at MOI ═ 5 and incubated at 32 ℃ for two days. These cells were transfected with P0 lysate of SeV18+/Δ M-GFP at 200 μ L/well and cultured at 32 ℃ using serum-free MEM containing 40 μ g/ml AraC and 7.5 μ g/ml trypsin. The spreading of the GFP protein by SeV18+/Δ M-GFP was observed in clones #18 and #62 (FIG. 36). Said diffusion being in grams Clone #62 was particularly fast and was used in subsequent experiments. These cells were hereafter referred to as LLC-MK2/F7/M62 prior to induction with AxCANCre. After incubation, the F and M proteins were expressed continuously and were designated LLC-MK 2/F7/M62/A. The preparation of SeV18+/Δ M-GFP cells was continued with LLC-MK2/F7/M62/A cells. Preparation of 9.5X 10 days 6 after P2 infection⁷GFP-CIU virus. Preparation of 3.7X 10 days 5 after P4 infection⁷GFP-CIU virus.

As shown in example 3, it is presumed that the culture at 32 ℃ after the P1 stage is particularly important for recovering SeV18+/Δ M-GFP virus. In SeV18+/Δ M-GFP, the trans-supply of the M protein from the expressing cells (LLC-MK2/F7/M62/A) is thought to be a cause; however, the spread of infection was very slow and was finally observed 7 days after P1 infection (fig. 22). Thus, in virus reconstitution experiments, "culturing at 32 ℃ after the P1 stage" was supported by its very effectiveness in reconstituting SeV that are poorly transcriptionally-replicating or that have a low capacity to form infectious viral particles.

Example 13 study of Virus production conditions Using helper cells inducing SeV-M protein expression:

the fertility of the above viruses was investigated. LLC-MK2/F7/M62/A cells were plated in 6-well plates and cultured at 37 ℃. When the cells were near full bottom, the temperature was changed to 32 ℃. One day later, the cells were infected with SeV18+/Δ M-GFP at MOI ═ 0.5. Culture supernatants were recovered over time and replaced with fresh medium. The supernatant thus recovered was used for determination of CIU and HAU. Most viruses were recovered 4-6 days post infection (FIG. 23). HAU remained for more than 6 days after infection, but cytotoxic activity was still strongly shown at this time point, suggesting that the reason was not the HA protein from the virion, but the activity of HA protein free or bound to cell debris. Therefore, for virus collection, the culture supernatant is preferably recovered on day 5 after infection.

Example 14 Structure confirmation of M Gene-deficient SeV:

the viral genes of SeV18+/Δ M-GFP' were confirmed by PCR, while the viral proteins were confirmed by Western blotting. In RT-PCR, P2 stage virus was used 6 days after infection. QIAamp viral RNA mini kit (QIAGEN, Bothell, WA) WAs used to recover RNA from the viral solution. The Thermoscript RT-PCR system (Gibco-BRL, Rockville, Md.) was used to prepare the cDNA. Both systems were used as described in the kit instructions. Random hexamers provided in the kit were used as primers for cDNA preparation. To confirm that the product was formed starting from RNA, RT-PCR was performed in the presence or absence of reverse transcriptase. PCR was performed using the cDNA prepared above as a template, using two pairs of primers: one composition of F3593 (5'-ccaatctaccatcagcatcagc-3'/SEQ ID NO: 28) on the P gene and R4993 (5'-ttcccttcatcgactatgacc-3'/SEQ ID NO: 29) on the F gene, and the other composition of F3208 (5'-agagaacaagactaaggctacc-3'/SEQ ID NO: 30) and R4993 on the P gene. Amplification of 1073bp and 1458bp DNA was observed from the former and latter compositions, respectively, as expected from the gene structure of SeV18+/Δ M-GFP (FIG. 24). When reverse transcriptase is absent (RT-), gene amplification does not occur. When the M gene was inserted instead of the GFP gene (pSeV18+ GFP), 1400bp and 1785bp of DNA were amplified, respectively. These DNAs are significantly different in size from those described above, supporting the fact that the virus is structurally defective in the M gene.

Protein confirmation was performed using Western blot. LLC-MK2 cells were infected with SeV18+/Δ M-GFP (shown as Δ M), SeV18+/Δ F-GFP (shown as F), and SeV18+ GFP (shown as 18+) at MOI ═ 3, respectively, and culture supernatants and cells were recovered 3 days post-infection. The culture supernatant was centrifuged at 48,000Xg for 45 minutes to recover the viral proteins. After SDS-PAGE, Western blotting was performed to detect proteins according to the methods in examples 3 and 4 using anti-M protein antibody, anti-F protein antibody, and DN-1 antibody (rabbit polyclonal), which mainly detects NP protein. In cells infected with SeV18+/Δ M-GFP, no M protein was detected and F and/or NP proteins were observed. Therefore, the virus also demonstrated a SeV18+/Δ M-GFP structure from a protein perspective (FIG. 25). No F protein was observed in cells infected with SeV18+/Δ F-GFP, but all viral proteins detected were detected in cells infected with SeV18+ GFP. In addition, very little NP protein was detected in the culture supernatant of cells infected with SeV18+/Δ M-GFP, indicating no or very little secondary release particles.

Example 15 quantitative analysis of Secondary Release particles of SeV deficient in the Presence or absence of M Gene:

As described in example 14, LLK-MK2 cells were infected with SeV18+/Δ M-GFP at MOI ═ 3, and culture supernatants were recovered three days after infection, filtered through a filter with a pore size of 0.45 μ M, and then centrifuged at 48,000xg for 45 minutes to recover viral proteins. The viral proteins in the culture supernatant were then detected semi-quantitatively using Western blotting. A sample prepared similarly from SeV18+/Δ F-GFP infected cells was used as a control. Serial dilutions of each sample were prepared and subjected to Western blotting to detect protein using DN-1 antibody (which recognizes primarily the NP protein). The viral protein level in the culture supernatant of SeV18+/Δ M-GFP infected cells was estimated to be about 1/100 for SeV18+/Δ F-GFP infected cells (FIG. 26). The HA activity of the SeV18+/Δ F-GFP sample was 64HAU, which was 2HAU lower than that of the SeV18+/Δ M-GFP sample.

The time course of the same experiment was examined. That is, LLC-MK2 cells were infected with SeV18+/Δ M-GFP at MOI ═ 3, and culture supernatants were recovered over time (daily) to determine HA activity (fig. 27). Over four days post infection, a small amount of HA activity was detected. However, measurement of LDH activity (cytotoxic activity indicator) showed significant cytotoxic activity in SeV18+/Δ M-GFP-infected cells at or above 4 days post-infection (fig. 28). This suggests that it is likely that the increased HA activity is not due to VLPs, but rather to the activity of HA proteins bound to or free from cell debris. In addition, culture supernatants obtained 5 days post infection were assayed using Dosper Lipofectation reagent (cationic liposomes) (Roche, Basel, Switzerland). The culture supernatant (100. mu.L) was mixed with Dosper (12.5. mu.L), left at room temperature for ten minutes, and then transfected into LLC-MK2 cells cultured in 6-well plates to a full bottom. Observations under a fluorescent microscope two days after transfection showed that many GFP-positive cells were observed in the supernatant of cells infected with SeV18+/Δ F-GFP (which contained the secondary release particles), while very few or almost no GFP-positive cells were observed in the supernatant of cells infected with SeV18+/Δ M-GFP (fig. 29). From the above results, it is inferred that the secondary release of the particles is almost completely inhibited by the M protein deficiency.

2. Construction of SeV vectors with reduced or defective particle formation due to modified protease-dependent tropism

SeV in which the F protein cleavage site was modified was constructed as follows using the M-deficient SeV reconstitution system constructed as described above.

Example 16 construction of M-deficient SeV genomic cDNA having a modified F protein activation site:

m-deficient SeV genomic cDNA inserted into the F protein F1/F2 cleavage site (activation site) was constructed, which has a recognition sequence for a protease highly expressed in cancer cells. Various sequences based on sequences that serve as synthetic substrates for MMP-2 and MMP-9 were constructed, as well as sequences based on the uPA substrate. FIG. 30 shows 4 sequences: two sequences designed based on the sequences used as synthetic substrates for MMP-2 and MMP-9 (Netzel-Arnett, S. et al, anal. biochem.195, 86-92, 1991) with or without modifications [ PLG ↓MTS (SEQ ID NO: 3) and PLG ↓LGL (SEQ ID NO: 31); hereinafter, the F proteins comprising these sequences are referred to as F (MMP #2) and F (MMP #3) ], respectively; another sequence was designed by inserting a sequence PLG of only 3 amino acids, which is common to MMP synthetic substrates (hereinafter, the F protein having this sequence is referred to as F (MMP # 4)); and a sequence designed based on a uPA substrate, VGR (SEQ ID NO: 6), (hereinafter, the F protein comprising this sequence is referred to as F (uPA)).

For the sequences currently designed to achieve selective effects on the MMPs of interest (MMP-2 and MMP-9), the sequences of commercially available synthetic substrates, and reports detailing the specificity of the substrates (Turk, B.E. et al, Nature Biotech.19(7), 661-667, 2001; Chen, E.I. et al, J.biol.chem.277(6), 4485-4491, 2002) are referenced. For MMP-9 in particular, the consensus sequence from P3-P2', Pro-X-Hy- (Ser/Thr) (X ═ any residue; Hy ═ hydrophobic residue) is recommended (Kridel, s.j. et al, j biol. chem.276(23), 20572-. Thus, F (MMP #2) was newly designed as the current design, PLG ↓ MTS, sequence from initial synthetic substrate, PLG ↓ MWS, so that it matched the consensus column.

The gene construction scheme is shown in FIG. 31. The full-length genomic cDNA (pSeV18+/Δ M-GFP) of the M-deficient site of M-deficient Sendai virus, in which the EGFP gene was inserted into the M-deficient site, was digested with SalI and NheI. The fragment containing the F gene (9634bp) WAs separated by agarose gel and electrophoresis, and then the corresponding band WAs excised and harvested using QIAEXII gel extraction system (QIAGEN, Bothell, WA). The resulting fragment was subcloned into the SalI/NheI site of LITMUS38(New Engl and Biolabs, Beverly, Mass.) (Structure of LitmusSalI/NheIfrg. DELTA.M-GFP). Mutagenesis of the F Gene on the Litmus SalI/NheIfrg. DELTA.M-GFP, QuickChange was used ^TMThe site-directed mutagenesis kit (Stratagene, La Jolla, Calif.) was performed according to the method described in the kit. The sequence of the synthetic oligonucleotides used for mutagenesis was as follows:

5’-CTGTCACCAATGATACGACACAAAATGCCccTctTggCatGaCGAGtTTCTTCGGTGCTGTGATTGGTACTATC-3’(SEQ ID NO：32)

and

5'-GATAGTACCAATCACAGCACCGAAGAAaCTCGtCatGccAagAggGGCATTTTGTGTCGTATCATTGGTGACAG-3' (SEQ ID NO: 33) for conversion to F (MMP # 2);

5’-CTGTCACCAATGATACGACACAAAATGCCccTctTggCCtGggGttATTCTTCGGTGCTGTGATTGGTACTATCG-3’(SEQ ID NO：34)

and

5'-CGATAGTACCAATCACAGCACCGAAGAATaaCccCaGGccAagAggGGCATTTTGTGTCGTATCATTGGTGACAG-3' (SEQ ID NO: 35) for conversion to F (MMP # 3);

5'-CAAAATGCCGGTGCTCCCCcGTtGgGATTCTTCGGTGCTGTGATT-3' (SEQ ID NO: 36) and

5'-AATCACAGCACCGAAGAATCcCaACgGGGGAGCACCGGCATTTTG-3' (SEQ ID NO: 37) for conversion to F (MMP # 4);

and

5’-GACACAAAATGCCGGTGCTCCCgtGggGAGATTCTTCGGTGCTGTGATTG-3’(SEQ ID NO：38)

and

5'-CAATCACAGCACCGAAGAATCTCccCacGGGAGCACCGGCATTTTGTGTC-3' (SEQ ID NO: 39) for conversion to F (uPA).

Lower case letters indicate mutated nucleotides.

The Litmus SalI/NheIfrg.DELTA.M-GFP containing the mutation of interest located on the F gene was digested with SalI/NheI to collect a fragment (9634bp) containing the F gene. F-deficient Sendai virus (pSeV18+/Δ F-GFP: Li, H. -O. et al, J.Virol.74, 6564-. The resulting plasmid was digested with SalI and NheI to collect a fragment (8294 bp). The collected fragments were ligated to each other to construct an M-deficient SeV cDNA (pSeV18+/F (MMP #2) Δ M-GFP, pSeV18+/F (MMP #3) Δ M-GFP, or pSeV18+/F (MMP #4) Δ M-GFP, which comprises an F (MMP #2), F (MMP #3) or F (MMP #4) gene (an F gene designed to be activated by MMP), and an M-deficient SeV cDNA (pSeV18+/F (uPA) Δ M-GFP), which comprises an F (uPA) gene (an F gene designed to be activated by uPA).

Example 17 reconstitution and amplification of M-deficient SeV vectors with modified F activation sites:

virus reconstitution was performed according to the method reported by Li et al (Li, H. -O. et al, J.Virol.74, 6564-. Since the virus is an M-deficient form, the helper cells described above that supply the M protein in trans are used (as in example 11). The Cre/loxP expression induction system is used for preparing helper cells. The system using the pCALNdLw plasmid was designed to induce gene product expression using Cre DNA recombinase (Arai, T. et al, J.Virol.72, 1115-1121, 1988). Thus, the transformants of this plasmid were infected with a recombinant adenovirus expressing Cre DNA recombinase (AxCANCre) to express the inserted gene using the method of Saito et al (Saito, I. et al, Nucleic Acids Res.23, 3816-3821, 1995; Arai, T. et al, J. Virol.72, 1115 1121, 1998) (see examples 11 and 12).

The reconstruction of M-deficient SeV in which the activation site of F is modified is performed as follows. LLC-MK2 cells at 5X 10%⁶Cells/dish were plated in 100-mm dishes and incubated for 24 hours. Recombinant vaccinia virus expressing T7 polymerase (PLWUV-VacT 7: Fuerst, T.R., et al, Proc. Natl. Acad. Sci. USA 83, 8122-. The cells were washed with serum-free MEM. pSeV18+/F (MMP #2) Δ M-GFP (optionally, pSeV18+/F (MMP #3) Δ M-GFP, pSeV18+/F (MMP #4) Δ M-GFP, or pSeV18+/F (uPA)) Δ M-GFP), pGEM/NP, pGEM/P, pGEM/L (Kato, A. et al, Genes Cells 1, 569-. SuperFect transfection reagent (Qiagen, Bothell, WA) corresponding to 5. mu.L/1. mu.g DNA WAs added to each solution, mixed and then left at room temperature for 15 minutes. Finally, the mixture was added to 3mL of Opti-MEM containing PBS at a final concentration of 3%, and then added to the cells for culture. After 5 hours of culture, the cells were washed twice with serum-free MEM and cultured in MEM containing 40. mu.g/mL cytidylic acid beta-D-arabinofuranoside (AraC: Sigma, St. Louis, MO) and 7.5. mu.g/mL trypsin (Gibco-BRL, Rockville, Md.). After 24 hours of culture, cells which continued to express the M protein (LLC-MK2/F7/M62/A) were incubated at 8.5X 10 ⁶The cells/dish were grown in density to layers and incubated in MEM containing 40. mu.g/mL AraC and 7.5. mu.g/mL trypsin for two more days at 37 ℃ (P0). These cells were collected and the pellet was suspended in 2 mL/dish of Opti-MEM. After three freeze-thaw repetitions, the lysates were directly transfected into LLC-MK2/F7/M62/A and cultured in serum-free MEM (ICN, Aurola, OH) (P1) containing 40. mu.g/mL AraC, 7.5. mu.g/mL trypsin, and 50U/mL collagenase type IV at 32 ℃. After 3-14 days, a portion of the culture supernatant was removed and transfected into freshly prepared LLC-MK2/F7/A and containing 40. mu.g/mL AraC, 7.5. mu.g/mL trypsin, and 50U/mL collagenase type IV (P/mL) at 32 ℃2) The culture in the serum-free MEM of (4). After 3-14 days, the cultures were re-transfected into the freshly prepared LLC-MK2/F7/M62/A and cultured in serum-free MEM containing 7.5. mu.g/mL trypsin and 50U/mL collagenase type IV (P3) at 32 ℃ for 2-7 days. BSA was added to the collected culture supernatant to make the final concentration 1%, and the culture was stored at-80 ℃. The virus strain solution was thawed for subsequent preparation and in vitro experiments.

Furthermore, helper cells capable of producing M-deficient SeV vectors at high titers (LLC-MK2/F7/M62- #33) were successfully obtained by introducing the SeV-M gene (and SeV-F gene) of the same system (pCALNdLw: Arai, T. et al, J.Virol.72, 1115-1121, 1988) into LLC-MK2/F7/M62 as helper cells (which supply the M protein in trans and continue the cloning of the cells). Using these cells, an M-deficient SeV vector (SeV18+/Δ M-GFP) in which the F gene is not mutated may be expressed at 1X 10 ⁸GFP-CIU/mL (GFP-CIU is defined in WO 00/70070) or higher. In addition, SeV18+/F (MMP # 2). DELTA.M-GFP and SeV18+/F (uPA). DELTA.M-GFP at 1X 10 were also achieved using these cells⁸Preparation of GFP-CIU/mL or higher titers.

When SeV18+/F (MMP #3) Δ M-GFP and SeV18+/F (MMP #4) Δ M-GFP were similarly reconstituted, viral particles could not be collected. To collect these viral particles, the reconstitution conditions must be further examined. Considering the fact that they cannot be collected under the same conditions, the F1/F2 cleavage site (activation site of F protein) in F (MMP #3) and F (MMP #4) may have problems, which result in, for example, low cleavage efficiency or weak activity of the cleaved F protein.

Example 18 preparation of in vivo samples of M-deficient SeV vectors with modified F activation sites:

various M-deficient SeV vectors for in vivo experiments were prepared by simple purification in which viral particles were precipitated by centrifugation. LLC-MK2/F7/M62- #33 grew to almost full bottom on 6-well plates, was infected with AxCANCre (MOI ═ 5), and was subsequently cultured at 32 ℃ for two days. These cells were infected with SeV18+/F (MMP #2) Δ M-GFP or SeV18+/Δ M-GFP at a MOI of 0.5. Subsequently, cells infected with SeV18+/F (MMP # 2). DELTA.M-GFP were placed in a medium containing 7.5. mu.g/mL of pancreas at 32 ℃ Culturing in serum-free MEM (1 mL/well) with protease and 50U/mL collagenase type IV for 3 days; and the SeV18+/Δ M-GFP infected cells were cultured in serum-free MEM (1 mL/well) containing only 7.5. mu.g/mL trypsin for 3 days. Supernatants were collected from six wells and mixed, then centrifuged at 2,190Xg for 15 minutes. The collected supernatant was passed through a filter (pore inner diameter 0.45. mu.M) and then further centrifuged at 40,000Xg for 30 minutes. The resulting pellet was suspended in 500. mu.L of PBS to prepare a purified virus solution. The titer of the M-deficient SeV vector prepared as described above was 1.3X 10⁹And 4.5X 10⁹GFP-CIU/mL (SeV 18+/F (MMP # 2). DELTA.M-GFP and SeV18 +/. DELTA.M-GFP, respectively). The F protein was cleaved in the viruses prepared in examples 17 and 18, and the viruses were infectious. Such SeV is called F-cleaved SeV or infectious SeV. Hereinafter, SeV18+/Δ M-GFP, SeV18+/F (MMP #2) Δ M-GFP, and SeV18+/F (uPA) Δ M-GFP are also simplified to SeV/Δ M-GFP, SeV/F (MMP #2) Δ M-GFP, and SeV/F (uPA) Δ M-GFP, respectively.

Example 19 evaluation of protease-dependent infection and cell fusion-type infection with F-modified M-deficient SeV vector:

<1> exogenous experiment:

the infection method in which extracellular proteases are added to cell lines is referred to as an exogenous experiment. The basic steps of the exogenous experiments performed in the following examples are as follows. The use of different conditions is described in the examples. LLC-MK2 was cultured in confluent 96-well plates (5X 10) ⁵Cells/well). After washing twice with MEM, the cells containing SeV (F-cleaved form: 1X 10)⁵CIU/mL, or F-uncleaved form: 1X 10⁷particles/mL (expressed as HA units; see example 25)]50 μ L of MEM was added and the cells were infected. At the same time, 50. mu.L of protease containing MEM was also added, and the cells were cultured at 37 ℃. After four days, the spread of infection was observed under a fluorescent microscope. Count 1mm²The number of GPF-expressing cells in the cells. The proteases used were purchased from ICN Biomedicals Inc. for collagenase (collagenase type IV), and MMP-2 (active MMP-2), MMP-3, MMP-7, MMP-9 (active MMP-9), and plasmin from COSMO BIO Co.

<2> endogenous experiments:

the method of infection by proteases expressed intracellularly without the addition of extracellular proteases is called endogenous experiment. The basic steps of the endogenous experiments performed in the following examples are described below. The use of different conditions is described in the examples. Various cancer cells were cultured in confluent 96-well plates (5X 10)⁵Cells/well). After washing twice with MEM, the cells containing SeV (F-cleaved form: 1X 10)⁵CIU/mL, or F-uncleaved form: 1X 10⁷particles/mL (expressed as HA units; see example 25)) of 50. mu.L MEM were added and infected cells. At the same time, 50. mu.L of protease containing MEM was also added, and the cells were cultured at 37 ℃. After four days, the spread of infection was observed under a fluorescent microscope. Count 1mm ²The number of GPF-expressing cells in the cells.

Example 20 protease-dependent cell fusion-type infection of M-deficient Sendai virus vectors modified by F (exogenous experiment):

the modification of F was confirmed using LLC-MK2 cells which hardly expressed protease, and assayed by the above exogenous experiment to determine whether it caused a protease-dependent cell fusion type infection (FIG. 32). Cells were infected with three classes of M-deficient SeV (as described in example 17), SeV/. DELTA.M-GFP, SeV/F (MMP # 2). DELTA.M-GFP, and SeV/F (uPA). DELTA.M-GFP. Meanwhile, various collagenase type IV (Clostridium histolyticum), active MMP-2, active MMP-9, or uPA, or trypsin of 7.5. mu.g/mL was added thereto at 0.1. mu.g/mL. After four days, the cells were observed under a fluorescent microscope. Only in LLC-MK2 with added trypsin, SeV/. DELTA.M-GFP with unmodified F resulted in fusion of infected cells with their surrounding cells, resulting in cell fusogenic infection and multinucleated cell (syncytial) formation (FIG. 32L). SeV/F (MMP #2) Δ M-GFP, which has MMP degradation sequence inserted into its F protein, shows cell fusion type infection of LLC-MK2 (with collagenase, active MMP-2, and active MMP-9 added), leading to syncytia formation (FIGS. 32E, 32F, and 32M). On the other hand, SeV/F (uPA) Δ M-GFP, in which urokinase-type plasminogen activator (uPA) and tissue-type PA (tPA) degradation sequences were inserted into the F protein, showed cell fusogenic infection in the presence of trypsin, and further modification of the F protein showed syncytia formation and formation of multinucleated cells in the presence of uPA (FIGS. 32Q and 32R). These results indicate that M-deficient SeV causes degradation substrate-dependent cell fusion-type infection due to incorporation of various protease degradation substrate sequences into the F protein, and spreads to contacted cells.

Example 21 MMP expression specific cell fusion type infection of cancer cell lines (endogenous experiment):

using SeV prepared in example 17, an endogenous experiment was conducted to determine the presence or absence of an endogenous protease-selective cell fusion type infection. Cancer cell lines expressing MMP, HT1080 (human fibroblast sarcoma) (Morodomi, T. et al, biochem. J.285(Pt 2), 603-611, 1992), cell lines expressing tPA, MKN28 (human gastric Cancer cell line) (Koshikawa, N. et al, Cancer Res.52, 5046-5053, 1992), and cell lines not expressing either protease, SW620 (human colon Cancer cell line) were used. MKN28 was obtained from the Riken Institute of Physical and Chemical Research (Cell No. RCB1000), while HT1080(ATCC No. CCL-121) and SW620(ATCC No. CCL-227), as well as SW480(ATCC No. CCL-228), WiDr (ATCC No. CCL-218), and Panc-1(ATCC No. CRL-1469) used in the following examples were obtained from the American Type Culture Collection (ATCC). The media used in the different studies of cells were provided for the experiments. In addition, FBS was added to all media at a final concentration of 1%. As shown in FIG. 33, in the MMP-expressing cell line, HT1080, only infection with SeV/F (MMP # 2). DELTA.M-GFP spread 10-fold or more. Furthermore, only cell-fused infections with SeV/F (uPA) Δ M-GFP spread in the tPA-expressing cell line, MKN 28. In SW620, which does not express either protease, no spread of infection was observed at all.

Example 22 cell confluent infection by phorbol ester-induced MMPs:

growth factors surrounding cancer cells have been reported to induce MMPs in cancer cells in vivo. This phenomenon can be used with phorbol ester, i.e. phorbol 12-myristate 13-acetic acid (PMA)) Replication in vitro. To investigate the infection that occurs under replicative conditions in which MMP expression is induced, PanCI, a pancreatic cancer cell line known to activate MMP-2 and induce MMP-9 by PMA, was used to test for the presence or absence of cell fusion type infection by F-modified M-deficient SeV vectors (Zervos, E.E., et al, J.Surg.Res.84, 162-167, 1999). PancI and other cancer cell lines were cultured in 96 wells to confluency (5X 10)⁵Cells/well). The endogenous experiments were performed with SeV prepared in example 7. After washing twice with MEM, 50. mu.L of the extract was mixed with 1X 10⁵MEM from CIU/mL SeV was added to infect (MOI ═ 0.01). The same amount (50. mu.L) of MEM (containing 40nM phorbol 12-myristate 13-acetic acid (Sigma)) was added, and at the same time, FBS was added to the medium to a final concentration of 1%.

The induced expression of MMP-2 and MMP-9 was confirmed by gel zymography, in which a partial clearing of the gel hydrolysis activity was present (Johansson, S., and Smedsrod, B., J.biol.chem.261, 4363-one 4366, 1986). Specifically, the supernatant of each culture was collected and dissolved in a sample buffer. The mixture was mixed with acrylamide to a final gel concentration of 1mg/mL to prepare an 8% acrylamide gel. After SDS polyacrylamide gel electrophoresis, the gel was washed with 10mM Tris (pH 8.0) and 2.5% Triton X-100 at 37 ℃ in gelatinase activation buffer (50mM Tris, 0.5mM CaCl) ₂，10^-6M ZnCl₂) Medium incubation was performed for one day and stained with 1% Coomassie blue R-250, 5% acetic acid, and 10% methanol (top half of FIG. 34). "C" represents control and "T" represents sample supernatant induced by 20nM PMA. The upper part of the figure shows that MMP-9 is induced in HT1080 and Panc I. Potential MMP-2 was detected prior to induction in Panc I. However, this latent form is known to have hardly any gel hydrolysis activity. Panc I infected with SeV/F (MMP #2) Δ M-GFP as in FIG. 34 (bottom half) indicated cell fusion type infection by MMP induction.

Example 23 spread of infection with SeV/F (MMP #2) Δ M-GFP in HT1080 cell lines in vivo:

nude mice with cancer HT1080 were prepared. 5X 10 of human fibroblastic tumor cell line HT1080⁶Individual cell (50. mu.L, 1X 10)⁸cells/mL), was injected subcutaneously into the right back skin of BALB/c nude mice (Charles River). After 7-9 days, animals with tumors greater than 3mm in diameter were used. The cancer volume (its shape is assumed to be elliptical) is 30-100mm³. 50 μ L of the following F-cleaved SeV were injected into the cancer at one time: MEM (control) (N ═ 5); SeV-GFP-containing MEM (1X 10)⁸CIU/mL) (N ═ 5); MEM (1X 10) containing SeV/. DELTA.M-GFP⁸CIU/mL) (N ═ 7); and MEM (1X 10) containing SeV/F (MMP #2) Δ M-GFP⁸CIU/mL) (N ═ 7). Two days later, the above cancers were observed under a fluorescence microscope (fig. 35). Fluorescence was observed only in the regions near the SeV-GFP and SeV/. DELTA.M-GFP injection sites (FIGS. 35E and 35H). In contrast, for SeV/F (MMP #2) Δ M-GFP, fluorescence was observed to spread throughout the cancer (FIG. 35K). The magnified images show the fluorescence from individual cells corresponding to SeV-GFP and SeV/. DELTA.M-GFP, whereas for SeV/F (MMP # 2). DELTA.M-GFP, the cell shape is unclear, suggesting cell fusion. In addition, the entire cancer region and GFP expression region were measured by NIH image in the above-mentioned images. The proportion of GFP-expressing regions throughout the cancer was 10% (corresponding to SeV-GFP) and 20% (corresponding to SeV/. DELTA.M-GFP), and conversely, the proportion of SeV/F (MMP # 2). DELTA.M-GFP was 90%, clearly indicating the spread of infection (FIG. 36). In tissues other than cancer tissues, fusogenic infection is hardly observed in fascia and subcutaneous connective tissues adjacent to cancer cells. Thus, under these conditions, it was determined that the infection did not spread to normal tissues other than the cancer tissues.

Example 24 antitumor Effect of F-modified M-deficient SeV vector on cancer-bearing nude mice:

HT1080 tumor bearing mice were prepared in the same manner as described in figure 35. After 8 or 9 days, animals with tumors greater than 3mm in diameter were selected and 50 μ L of the following four F-cleaved SeVs were injected into the cancer site: MEM (N ═ 5); containing SeV-GFP (1X 10)⁸CIU/mL) MEM (N ═ 5); containing MSeV/. DELTA.M-GFP (1X 10)⁸CIU/mL) MEM (N ═ 7); and SeV/F (MMP #2) containing Δ M-GFP (1X 10)⁸CIU/mL) in MEM (N ═ 7). Two days later, an equal amount of SeV was re-injected into the cancer site. The length of the major axis (a), minor axis (b) and thickness (c) of the cancer site were measured every other day. Presume the cancer is ellipseRound, the volume of cancer is calculated as V ═ pi/6 × abc. Cancers administered with PBS, SeV-GFP, and SeV/. DELTA.M-GFP, respectively, grew rapidly. In contrast, cancers administered SeV/F (MMP #2) Δ M-GFP, in which the vector spread throughout the cancer, as shown in FIG. 37, clearly showed no amplification and remained small. Analysis of significant differences by t-test showed significantly smaller volumes with P < 0.05 compared to the other three groups. This indicates that the vector has anticancer effects even without the therapeutic gene.

Example 25 preparation and Selective infection of F-uncleaved/F-modified M-deficient SeV vectors:

In the above-described SeV vector preparation procedure, culture was performed in a medium containing a high concentration of trypsin and 50U/mL collagenase to induce F cleavage, and the F-cleaved vector was collected (see examples 17 and 18). In this example, F-uncleaved SeV was produced by collecting SeV without adding protease during the preparation.

Specifically, LLC-MK2/F7/M62/A cells were cultured in 10cm dishes until the bottom was reached. Cells were infected with each F-modified M-deficient SeV prepared in example 17 (MOI ═ 5). After one hour, the supernatant was removed and washed twice with MEM medium. 4mL of MEM was added to the cells and cultured at 32 ℃. After 5 days, the supernatant was collected, and Bovine Serum Albumin (BSA) was added (final concentration of 1%). After determination of the HAU titer, the supernatant was stored at-70 ℃ until use. Collecting each F-modified M-deficient SeV to a concentration in the range of 2⁷-2¹⁰HAU/mL(1HAU＝1×10⁶Viral particles/mL, and thus corresponds to 1X 10⁸-1×10⁹particles/mL) and adjusted to 1 × 10 by dilution⁸。

The results of this exogenous experiment confirmed the production of vectors infecting LLC-MK2 in an MMP-dependent and uPA-or tPA-dependent manner by SeV/F (MMP #2) Δ M-GFP and SeV/F (uPA) Δ M-GFP, respectively (data for exogenous protease not shown). Furthermore, whether selective infection by protease expression was possible in HT1080 strain expressing MMP, MKN28 strain expressing tPA, and SW620 hardly expressing the protease was examined by endogenous experiments (fig. 38). SeV/F (MMP #2) Δ M-GFP infected the HT1080 strain expressing MMP, but not the MKN28 strain expressing tPA. SeV/F (uPA) Δ M-GFP infected MKN28 strain expressing tPA but not HT1080 strain expressing MMP. As described above, each SeV showed protease-dependent selective infection.

Example 26 infection with F-modified M-deficient SeV vectors due to the induction of MMP-3 and MMP-7 by human fibroblasts:

SW480 and WiDr induced MMP-3 and MMP-7, respectively, by co-culture with fibroblasts or in vivo culture (Kataoka, H. et al, Oncol. Res.9, 101-. These cells were used to investigate whether infection with the F-modified M-deficient SeV vector was altered in vivo. Each cancer cell line was cultured in 96-well plates to the full bottom (5X 10)⁴Cells/well). After washing twice with MEM, F-uncleaved SeV containing 1HAU/mL (1 HAU. multidot.1X 10) was added⁶Viral particles/mL and thus correspond to 1X 10⁶particles/mL) of 50 μ L MEM for infection. Normal human lung fibroblasts (TAKARA) at 5X 10⁴Cell/well concentration was added to the cells and incubated at 37 ℃ for four days (FIG. 39). SW480 and WiDr were infected with SeV/F (MMP # 2). DELTA.M-GFP by coculture with human fibroblasts. This phenomenon was not observed in SW620, which is not inducible.

Example 27 MMP-selective infection of human aortic smooth muscle cells with F-modified M-deficient SeV vector:

misexpression of MMPs (aberrant expression) has been reported in cancer, as well as in arteriosclerosis, rheumatoid arthritis, and wound healing (Galis, Z.S., and Khatri, J.J., circ. Res.90, 251-.

To demonstrate the applicability of the F-modified M-deleted SeV vectors to these diseases, MMP-selective infection of human aortic smooth muscle cells with the vectors was performed. Human smooth muscle cells (TAKARA) were cultured in 96-well plates to the full bottom (5X 1)0⁵Cells/well). After washing twice with MEM, 50. mu.L of a solution containing SeV (F-uncleaved form: 1HAU/mL (1X 10)⁶particles/mL)) was added to the cells for infection. An equal amount (50. mu.L) of the protease-containing MEM was added thereto, and the mixture was cultured at 37 ℃ for four days. Calculated per 1mm²The number of cells expressing GFP in the cells (FIG. 40). Infection with SeV/. DELTA.M-GFP was enhanced by the addition of trypsin alone, whereas infection with SeV/F (MMP # 2). DELTA.M-GFP was enhanced by the addition of collagenase, MMP-2, MMP-3 and MMP-9.

Example 28 protease-dependent cleavage of the F protein in F-modified M-deficient SeV vectors:

as shown in example 20, by incorporating each protease degradation sequence into the F protein, the F-modified M-deficient SeV vector showed cell fusion type infection depending on these degradation sequences. Furthermore, whether the cleavage of modified F0 occurred in a protease-dependent manner was confirmed by Western blotting. The virus was sampled by the following method. Three types of virions, SeV/Δ M, SeV/F (MMP #2) Δ M, and SeV/F (upa) Δ M, infected M protein-induced helper cells with MOI ═ 3. Two days after infection, the supernatant was collected and centrifuged at 18,500Xg for three hours, and the pellet was resuspended in PBS. For each viral suspension, protease was added to give a final concentration of 7.5. mu.g/mL trypsin, 0.1ng/mL MMP-9 and 0.1ng/mL uPA, and incubation was carried out at 37 ℃ for 30 minutes. Sample buffers were added to each mixture to prepare SDS-PAGE samples. SDS-PAGE and Western blotting were performed according to standard methods (Kido, H., et al, "Isolation and characterization of and tryptin-like protease found in and branched platelet derived cells. analytical activator of the virus fusion protein," J Biol Chem 267, 13573-. The rabbit anti-F1 antibody was obtained as an antiserum by immunization with a mixture of three synthetic peptides (FFGAVIGT + Cys: 117-124, EAREAKRDIALIK: 143-155, and CGTGRRPISQDRS: 401-413; SEQ ID NOS: 46, 47, and 48, respectively). HRP-labeled anti-rabbit IgG antibody (ICN, Aurola, OH) was used as a secondary antibody, and chemiluminescence (ECL Western blot detection reagent; Amersham Biosciences, Uppsala, Sweden) was used to detect the color developed. FIG. 41 shows the results of treating the following substances with the above protease at 37 ℃ for 30 minutes: m-deficient SeV vectors comprising unmodified F (1, 4, 7, and 10), M-deficient SeV vectors having an MMP #2 sequence with an inserted F (2, 5, 8, and 11), and M-deficient SeV vectors having a uPA sequence with an inserted F (3, 6, 9, and 12).

As shown in fig. 41, cleavage of F1 occurred, depending on each inserted protease substrate, in the following cases, respectively: the M-deficient SeV vector having an unmodified F is in the presence of trypsin, the M-deficient SeV vector having an MMP #2 sequence into which F is inserted is in the presence of MMP, and the M-deficient SeV vector having a uPA sequence into which F is inserted is in the presence of uPA. Although not shown, for the M-deficient SeV vector into which the uPA sequence was inserted, when the degradation time was extended to four hours, cleavage of F1 was observed in the presence of trypsin. This is in good agreement with the results of example 20 and indicates that syncytia formation occurs in an F-cut dependent manner.

Example 29 increase of fusion force by deletion of cytoplasmic region of F protein:

infiltration of the host by paramyxoviruses is achieved by fusing the viral membrane with the host cell membrane. In this infiltration mechanism, the HN protein of Sendai virus binds to sialic acid of the host, and the F protein causes cell membrane fusion. In this step, the conformational change of the F protein due to HN binding is suggested to be important (Russell, C.J., Jardetzky, T.S. and Lamb, R.A., "Membrane fusions of paramyxoviruses: capture of intermediates of fusion." EMBO J.20, 4024-34, 2001). Therefore, most of the F protein of paramyxoviruses does not show cell fusogenic ability when expressed alone on cells. Only cells simultaneously expressing HN protein had fusogenic properties. Deletion of F and HN protein cytoplasmic domains in paramyxoviruses is known to enhance their fusogenic ability (Cathogen, T., Naim, H.Y. and Cattaneo, R., "Measles virus with altered expression protein cytoplasmic tissue cell fusion" J.Virol.72, 1224-34, 1998). To determine which deletion mutant of cytoplasmic domain of F protein in Sendai virus resulted in the most increased fusogenic force, a deletion mutant was prepared and inserted into pCAGGS expression vector (Niwa, H., et al, Gene 108, 193-199, 1991). pCAGGS carrying HN was co-transfected and the resulting fusogenicity was confirmed by the number of syncytia formed.

PCR was performed for each mutant gene in which the cytoplasmic region of F had been deleted, and the resulting fragment was treated with XhoI and NotI using the following primers, and then ligated to pCAGGS vector. The primers used for PCR were as follows: fct27 primers (5'-CCGCTCGAGCATGACAGCATATATCCAGAGA-3'/SEQ ID NO: 49, and 5'-ATAGTTTAGCGGCCGCTCATCTGATCTTCGGCTCTAATGT-3'/SEQ ID NO: 50); fct14 primers (5'-CCGCTCGAGCATGACAGCATATATCCAGAGA-3'/SEQ ID NO: 51, and 5'-ATAGTTTAGCGGCCGCTCACCTTCTGAGTCTATAAAGCAC-3'/SEQ ID NO: 52); and

fct4 primers (5'-CCGCTCGAGCATGACAGCATATATCCAGAGA-3'/SEQ ID NO: 53, and 5'-ATAGTTTAGCGGCCGCTCACCTTCTGAGTCTATAAAGCAC-3'/SEQ ID NO: 54) (Kobayashi M. et al, J.Viol., 77, 2607, 2003).

To determine the fusogenic capacity of the cells, LLC-MK2 or HT1080 cells were plated in 24-well plates until confluent. mu.L Fugene 6 was mixed with 50. mu.L Opti-MEM. Mu.g of each pCAGGS expression plasmid was mixed with an equal amount of pCAGGS/EGFP and subsequently added to a mixture of Opti-MEM and Fugene 6. After standing at room temperature for 15 minutes, the mixture was added to a 24-well plate (the medium therein was replaced with 500. mu.L of MEM medium). At 37 ℃ 5% CO ₂After 3 hours of culture, the medium was replaced with 1% FBS-containing MEM (for HT1080) and with 7.5. mu.g/mL trypsin-containing MEM or a predetermined concentration of collagenase type IV (Clostridium) (for LLC-MK 2). After 48 hours of culture, the number of fused syncytia/100 visual field (0.3 cm)²) (inverted microscope). Alternatively, the cultured cells were fixed in 4% paraformaldehyde for 2 hours, transferred to 70% ethanol, and then transferred to distilled water, stained with hematoxylin for 5 minutes, and washed with water to count every 0.3cm²The number of nuclei in which syncytia are formed.

Three amino acids of the F protein whose cytoplasmic region is deleted are shown in fig. 42(a), and the fusion activity thereof is shown in fig. 42 (B). As shown in fig. 42(B), only the cells expressing the F protein were not fused, but cotransfection with HN induced fusion. In addition, the F protein (Fct14) having a sequence in which 28 amino acids are deleted was deleted so that the cytoplasmic region became 14 amino acids, which showed the highest fusogenic force.

[ example 30] F/HN chimeric proteins resulted in a greatly reduced fusogenic force:

paramyxovirus envelope proteins, i.e., the F and HN proteins, form trimers and tetramers, respectively, on the cell membrane and are known to interact through their extracellular domains as well as the M protein (Plemper, R.K., Hammond, A.L., and Cattaneo, R., "Measles enveloppe glycopyrrolate hepero-oligomer in the endoplastic reticulum." J.biol.chem.276, 44239-22346, 2001). As shown in fig. 42, the F protein alone did not show fusogenic force, and the HN protein was essential for its fusogenic force. Thus, a chimeric protein comprising F and HN proteins was produced to prepare a vector having enhanced fusogenic ability by expressing both F and HN proteins (as fusion proteins) on the cell membrane. The F protein is a type II membrane protein and HN is a type I membrane protein. Thus, as shown in FIG. 43(A), a chimeric protein (Fct14/HN) was prepared so as to form a U-shape on the cell membrane, and it contained two transmembrane domains. Fct14 showing high fusogenic force was used as the F protein. A linker sequence consisting of 50 amino acids was inserted between the two proteins (Fct 14/linker/HN). The linker sequence shows homology to any protein according to the currently searched database. (use of a nonsense sequence synthesized by reversing the N-terminus to C-terminus of the amino acid sequence of the cytoplasmic region of env of simian immunodeficiency virus (SIVagm)).

The method for preparing the expression plasmid for the F/HN chimeric protein gene is specifically described below. The F/HN chimeric protein gene is inserted into a pCAGGS vector. PCR was performed for the F gene and HN gene, respectively, and the obtained two fragments were ligated to pCAGGS. In this step, a 150-bp linker gene (50amino acids) was inserted into the F/HN gene or no gene was inserted thereinto. The sequences of the primers used were as follows: f gene primers (F-F: 5'-ATCCGAATTCAGTTCAATGACAGCATATATCCAGAG-3'/SEQ ID NO: 55 and

fct 14-R: 5'-ATCCGCGGCCGCCGGTCATCTGGATTACCCATTAGC-3'/SEQ ID NO: 56) (ii) a Joint/HN Gene primer (Joint-HN-F: 5'-ATCCGCGGCCGCAATCGAGGGAAGGTGGTCTGAGTTAAAAATCAGGAGCAACGACGGAGGTGAAGGACCAGAGGACGCCAACGACCCACGGGGAAAGGGGTGAACACATCCATATCCAGCCATCTCTACCTGTTTATGGACAGAGGGTTAGG-3'/SEQ ID NO: 57)

And HN-R: 5'-ATCCGCGGCCGCTTAAGACTCGGCCTTGCATAA-3'/SEQ ID NO: 58) (ii) a And HN gene primers (5'-ATCCGCGGCCGCAATGGATGGTGATAGGGGCA-3'/SEQ ID NO: 59 and 5'-ATCCGCGGCCGCTTAAGACTCGGCCTTGCA-3'/SEQ ID NO: 60).

As shown in fig. 43(B), although the chimeric protein without the linker sequence showed low fusogenic force, insertion of the linker significantly increased the fusion activity to about 5-fold of the sequence obtained by co-transfection of the F and HN proteins.

Example 31 function of fusion-inducing force and substrate specificity were maintained:

to obtain the fusogenicity, it is necessary not only to express the F protein simultaneously with the HN protein, but also to cleave the F protein into two subunits by proteases (F1 and F2). In FIGS. 42 and 43, the fusogenic force was determined in the presence of trypsin, and was shown to be completely absent in the absence of trypsin. The cleavage sequence of the F protein was modified in the Fct 14/linker/HN chimeric protein shown in fig. 43 so that it achieved fusogenic forces in an MMP dependent manner. Degradation substrate sequences for a number of MMPs are reported. Of these, 8 sequences were modified. The amino acid sequence of the cleavage site was modified as shown in FIG. 44(A), and QuickChange was used^TMSite-directed mutagenesis kit (Stratagene, La Jolla, Calif.). Protease cleavage of the sequence of the fusion peptide of monkeys is also contemplated in the modification. The N-terminus of the F1 region of paramyxovirus F protein, which is called fusion peptide, is reported to be important for its fusion activity, and the fusogenic force of the F protein is sometimes lost by mutation of amino acids in this region (Bagai, S. and Lamb, R.A., "A. glyc.) (The medicine "Virology 238, 283-90, 1997" in the analysis of the peptides by SV5 fusion peptides of the analysis of the fusion. Thus, the sequence of the N-terminal region of F1, the significance of which has been pointed out, is not changed. In the above case, the design of F1 after degradation by MMPs involves the addition of three residues to the N-terminus when inserting the common six-residue sequence of degradation substrates known as MMPs. This suggests that the addition may allow degradation by MMPs, but may affect the fusogenic capacity of the F protein. Therefore, in designing the F protein to be activated by MMP-dependent cleavage, the following two points must be considered: (1) substrate specificity of MMPs; and (2) retention of F protein fusogenicity after cleavage.

MMP #1 is the most well-known sequence as a substrate for MMP synthesis. The sequence is also useful for targeting other MMPs. MMP #3 and MMP #8 are also commercially available synthetic substrate sequences. The degradation substrate sequences of MMP-2 and MMP-9, PLGMWS, were modified to PLGMTS and PQGMTS (SEQ ID NOS: 61 and 62, respectively) as MMP #2 and MMP #6, respectively, following the consensus sequence revealed by phage display (Pro-X-X-Hy- (Ser/Thr) for MMP-9). MMP #5 was constructed as PQGLYA (SEQ ID NO: 63) as reported by Shneider et al (American society of Gene therapy, Annual meeting No.1163, 2002, Boston). In MMP #4, the sequence of the fusion peptide is not modified after degradation. The sequence of MMP7# was found by phage display of MMP-2.

Details of the preparation of expression plasmids having modified F activation sites located in the F/HN gene are shown below. After construction of the F/HN fusion gene, mutagenesis of the F protein activation site was performed on pBluescript F/HN. To introduce the mutations, QuikChange was used according to the kit instructions^TMSite-directed mutagenesis kit (Stratagene, La Jolla, Calif.). The sequences of the synthetic oligonucleotides used for mutagenesis were as follows:

f (MMP # 1): (5'-CTGTCACCAATGATACGACACAAAATGCCccTctTggCCtGggGttATTCTTCGGT GCTGTGATTGGTACTATCG-3'/SEQ ID NO: 64, and 5'-CGATAGTACCAATCACAGCACCGAAGAATaaCccCaGGccAagAggGGCATTTTGTGTCGTATCATTGGTGACAG-3'/SEQ ID NO: 65);

F (MMP # 2): (5'-CTGTCACCAATGATACGACACAAAATGCCccTctTggCatGaCGAGtTTCTTCGGTGCTGTGATTGGTACTATC-3'/SEQ ID NO: 32, and 5'-GATAGTACCAATCACAGCACCGAAGAAaCTCGtCatGccAagAggGGCATTTTGTGTCGTATCATTGGTGACAG-3'/SEQ ID NO: 33);

f (MMP # 3): (5'-CTGTCACCAATGATACGACACAAAATGCCccTctTggCCtGggGttATTCTTCGGTGCTGTGATTGGTACTATCG-3'/SEQ ID NO: 34, and 5'-CGATAGTACCAATCACAGCACCGAAGAATaaCccCaGGccAagAggGGCATTTTGTGTCGTATCATTGGTGACAG-3'/SEQ ID NO: 35);

f (MMP # 4): (5 '-CAAAATGCCGGTGCTCCCCcGTtGgGATTCTTCGGTGCTGTGATT-33'/SEQ ID NO: 36, and 5'-AATCACAGCACCGAAGAATCcCaACgGGGGAGCACCGGCATTTTG-3'/SEQ ID NO: 37);

f (MMP # 5): (5'-CTGTCACCAATGATACGACACAAAATGCCccTcagggCttGtatgctTTCTTCGGTGCTGTGATTGGTACTATC-3'/SEQ ID NO: 66, and 5'-GATAGTACCAATCACAGCACCGAAGAAagcataCaaGccctgAggGGCATTTTGTGTCGTATCATTGGTGACAG-3'/SEQ ID NO: 67);

f (MMP # 6): (5 '-CTGTCACCAATGATACGACACAAAATGCCccTcaaggCatGaCGAGtTTCTTCGGTGCTGTGATTGGTACTATC-33'/SEQ ID NO: 68, and 5'-GATAGTACCAATCACAGCACCGAAGAAaCTCGtCatGccttgAggGGCATTTTGTGTCGTATCATTGGTGACAG-3'/SEQ ID NO: 69);

F (MMP # 7): (5'-CTGTCACCAATGATACGACACAAAATGCCctTgcTtaCtataCGgctTTCTTCGGTGCTGTGATTGGTACTATC-3'/SEQ ID NO: 70, and 5'-GATAGTACCAATCACAGCACCGAAGAAagcCGtataGtaAgcAagGGCATTTTGTGTCGTATCATTGGTGACAG-3'/SEQ ID NO: 71); and

f (MMP # 8): (5'-CTGTCACCAATGATACGACACAAAATGCCccTctTggCttGgCGAGaTTCTTCGGTGCTGTGATTGGTACTATC-3'/SEQ ID NO: 72, and 5'-GATAGTACCAATCACAGCACCGAAGAAtCTCGcCaaGccAagAggGGCATTTTGTGTCGTATCATTGGTGACAG-3'/SEQ ID NO: 73).

Lower case letters indicate mutated nucleotides. After modification, the sequence was cleaved with EcoRI and ligated with pCAGGS.

Each vector containing each sequence, and a vector containing an EGFP gene (pCAGGS/EGFP) were mixed in equal amounts, and the mixture was transfected into HT1080 which highly expresses MMPs. As a result, only the genes of the sequences of MMP #2 and MMP #6 were introduced, and cell fusion occurred and syncytia were formed (fig. 44 (B)). These sequences have in common that after cleavage with a protease, a Hy-S/T-S/T sequence (MTS) is added to the N-terminus of the F1 protein. Therefore, it is considered that the addition of Hy-S/T-S/T sequences (particularly MTS sequences) is likely to satisfy the following requirements: (1) cleavage of the F protein by HT 1080-derived MMPs, and (2) retention of fusogenic forces of the F protein after cleavage. On the other hand, no cell fusion was observed at all for MMP #1, MMP #3, MMP #4, MMP #5, MMP #7, and MMP # 8. Since all sequences except for that of MMP #4 are derived from synthetic substrates for MMP and are expected to be cleaved by proteases, it was suggested that the addition of this three amino acid peptide to F1 could limit the activity of the cleaved F protein. For MMP #4, in this case, it is likely that cleavage itself does not occur. Although the data do not show, it is evident from the fact that syncytia formation was observed for MMP #4 due to the induction of MMPs in HT1080 by phorbol esters.

In addition to comparison of the fusogenic activities of the sequences of MMP #2 and MMP #6, the fusogenic activities of cells were also determined depending on the concentration of MMP in the sequence modified from G to A at residues 7 and 12 from the N-terminus of the sequence of the fusion peptide sequence of #6 (FIG. 45). The oligonucleotide sequences used for mutagenesis of this F/HN fusion gene are as follows:

5’-CTTCGGTGCTGTGATTGcTACTATCGCACTTGcAGTGGCGACATCAGCAC-3’(SEQ ID NO：74)

and

5'-GTGCTGATGTCGCCACTgCAAGTGCGATAGTAgCAATCACAGCACCGAAG-3' (SEQ ID NO: 75). Lower case letters indicate mutated nucleotides. Expression plasmids were prepared analogously to the above by, after mutagenesis, excision of the sequence with EcoRI and ligation to pCAGGS.

As a result, MMP #6 was found to have a fusion-inducing force 3-fold higher than MMP # 2. Importantly, MMP #6 induced cell fusion even at low protease concentrations. That is, F protein activation is achieved at low concentrations. However, when a mutation of G into A, which is reported to increase the fusogenic force of the F protein, was further introduced (PeisajoVich, S.G., Epand, R.F., Epand, R.M. and Shai, Y., "Sendai N-terminal fusion peptides of two similar peptides," of our J.biochem.269, 4342-50, 2002)) (#6G12A), the fusogenic force was reduced to 1/10 or less. These results explain that by simply inserting a protease cleavage sequence to modify the tropism by a protease, the activity of the F protein cannot be maintained and results in a loss of fusogenic capacity in many cases. When a virus is constructed by introducing a desired degradation sequence, the fusogenicity is confirmed by using this system. In addition, since pCAGGS carries Fct 14/linker/HN showing significant fusion activity, transfection of this plasmid is expected to have an anti-tumor effect. Furthermore, by introducing the chimeric protein into M-deficient Sendai virus, it is expected that the antitumor effect can be further increased.

Example 32 construction of an improved F-modified M-deficient SeV genomic cDNA having increased fusogenic force:

examples 29 and 30 show that the fusogenic capacity is increased by modifying the F protein carried by the pCAGGS vector. By making similar modifications to the M-deficient sendai virus vector, it is expected that improved F-modified Δ M SeV with increased fusogenic capacity can be prepared. The gene construction of the improved F-modified M-deficient SeV genomic cDNA was carried out by the following method. SeV/F (MMP # 6). DELTA.M-GFP was constructed in the same manner as in example 16. Mutation of the F Gene on LITMUSSHI/NheIfrg.DELTA.M-GFP the sequence of SEQ ID NO: 69, and QuikChange^TMSite-directed mutagenesis kit (Stratagene, La Jolla, Calif.) was performed according to the kit instructions. The cDNA of SeV/F (MMP # 6). DELTA.M-GFP was constructed by coupling SalI and NheI-digested fragments of the mutant LITMUSSlI/NheIfrg. DELTA.M-GFP and a fragment containing the NP gene (obtained by digesting the full-length genomic cDNA of an F-deficient Sendai virus carrying the EGFP gene located at the F deletion site with SalI and NheI (pSeV + 18/F-GFP; Li, H, et al, J.Viol.74, 6564-19 6569, 2000; WO00/70070)) (FIG. 46). Multiple purposeCloning site Sendai virus cDNA (named pSeV (TDK)) (JP-A2002-272465) was used to construct basic frameworks for M-deficient Sendai viruses in which 28 amino acids of the cytoplasmic domain of the F protein were deleted (SeV (TDK)/Fct14(MMP # 6). DELTA.M-GFP)) and M-deficient Sendai viruses carrying the F/HN chimeric protein (SeV (TDK)/Fct14(MMp # 6)/linker/HN. DELTA.M-GFP). M-deficient Sendai virus SeV (TDK)/Fct14(MMP # 6). DELTA.M-GFP in which the cytoplasmic region of the F protein was truncated was constructed as follows. Since TDK was used as a framework, pSeV (TDK)/Δ M-GFP was first constructed. GFP/EIS (GFP with addition of EIS sequences encoding transcription initiation and termination signals) was amplified by PCR using synthetic primers (Nhe-GFP-F: ATCCGCTAGCCCGTACGGCCATGGTGAGCAAG (SEQ ID NO: 94), and GFP-EIS-BssHII: ATCCGCGCGCCCGTACGATGAACTTTCACCCTAAGTTTTTCTTACTACGGAGCTTTACTTGTACAGCTCGTC (SEQ ID NO: 95)) using LITMUSSSalI/NheIfrg. DELTA.M-GFP as a template. NheI and BssHII treatments were performed on the multiple cloning site of Sendai virus cDNA and amplified GFP/EIS, and the resulting fragments were coupled to replace the M protein with GFP to prepare pSeV (TDK)/Δ M-GFP.

Fct14(MMP #6) was prepared by PCR using pCAGGS/Fct14(MMP # 6)/linker/HN prepared in example 13 as template, using synthetic primers, Mlv-F: ATCCACGCGTCATGACAGCATATATCCAGAG (SEQ ID NO: 96) and Fct 14-EIS-SalI: ATCCGTCGACACGATGAACTTTCACCCTAAGTTTTTCTTACTACTTTAACGGTCATCT GGATTACC (SEQ ID NO: 97). Fct14(MMP #6) was inserted into the position of F to replace the F gene, thereby constructing psev (tdk)/Fct14(MMP #6) Δ M-GFP (fig. 46). Subsequently, M-deficient Sendai virus carrying the F/HN chimeric protein (pSeV (TDK)/Fct14(MMP # 6)/linker/HN. DELTA.M-GFP) was constructed. GFP/EIS was amplified by PCR using GFP as a template and synthetic primers (Nhe-GFP-F: ATCCGCTAGCCCGTACGGCCATGGTGAGCAAG (SEQ ID NO: 98) and GFP-EIS-SalI: ATCCGCTAGCCCGTACGATGAACTTTCACCCTAAGTTTTTCTTACTACGGAGCTTTACTTGTACAGCTCGTC (SEQ ID NO: 99)). GFP/EIS and multiple cloning site Sendai virus cDNA were treated with NheI and SalI. The resulting fragments were coupled to delete the M and F genes and replaced with GFP to yield pSeV (TDK)/Δ M Δ F-GFP. Fct14(MMP # 6)/linker/HN amplification was performed by PCR using Fct14(MMP # 6)/linker/HN prepared in example 31 as a template and synthetic primers (F/HN5 'Nhe-F: ATCCGCTAGCAGTTCAATGACAGCATTATCCAGAG (SEQ ID NO: 100), and F/HN 3' Nhe-EIS-R: ATCCGCTAGCACGATGAACTTTCACCCTAAGTTTTTCTTACTACTTTTAAGACTCGGCCTTGCATAA (SEQ ID NO: 101)). pSeV (TDK)/Fct14(MMP # 6)/linker/HN. DELTA.M-GFP was constructed by coupling Fct14(MMP # 6)/linker/HN to the NheI site of pSeV (TDK)/Δ M. DELTA.F-GFP described above.

Example 33 reconstitution and amplification of modified F-modified M-deficient Sendai Virus:

the virus was reconstituted from the cDNA constructed in example 32 according to the method of Li et al (Li, H. -O. et al, J.virology 74, 6564-. However, since the cDNA is the M-deficient form in example 17, helper cells that supply the M protein in trans are used (example 11). The Cre/loxP expression induction system is used for preparing helper cells. This system uses the plasmid pCALNdLw, which is designed to inducibly express the gene product by Cre DNA recombinase (Arai, T, et al, J.Virol.72, 1115-1121, 1988). The inserted gene is expressed by infecting a recombinant adenovirus (AxCANCre), which expresses Cre DNA recombinase, into a transformant of this plasmid by the method of Saito et al (Saito, I. et al, Nucleic Acids Res.23, 3816-3821, 1995); arai, T, et al, J.Virol.72, 1115-1121, 1998). M-deficient SeV with the F protein activation site replaced was reconstituted as follows. LLC-MK2 cells at 5X 10%⁶Cells/dishes were plated on 100-mm dishes, cultured for 24 hours, and then infected with recombinant vaccinia virus expressing T7 polymerase (PLWUV-VacT 7: Fuerst, T.R. et al, Proc. Natl. Acad. Sci. USA 83, 8122-. Cells were washed with serum-free MEM. pSeV/F (MMP #6) Δ M-GFP (alternatively, pSeV (TDK)/Fct14(MMP #6) Δ M-GFP or pSeV (TDK)/Fct14(MMP # 6)/linker/HN Δ M-GFP), pGEM/NP, pGEM/P, pGEM/L (Kato, A. et al, Genes Cells 1, 569- bco-BRL, Rockville, Md.). SuperFect transfection reagent (Qiagen, Bothell, WA) equivalent to 5. mu.L/1. mu.g DNA WAs added to the mixture and mixed. After standing at room temperature for 15 minutes, the mixture was finally mixed with 3mL of Opti-MEM containing 3% FBS, added to the cells, and cultured. After 5 hours of culture, the cells were washed twice with serum-free MEM, and then cultured in MEM containing 40. mu.g/mL cytidylic acid beta-D-arabinofuranoside (AraC: Sigma, St. Louis, MO) and 7.5. mu.g/mL trypsin (Gibco-BRL, Rockville, Md.). After 24 hours of culture, cells which continued to express the F protein (LLC-MK 2/F7/M62/A: example 12) were incubated at 8.5X 10⁶The cells were layered on dish and incubated for two more days in MEM containing 40. mu.g/mL AraC and 7.5. mu.g/mL trypsin (P0) at 37 ℃. These cells were collected and the pellet was suspended in Opti-MEM at 2 mL/dish. After repeating the freeze-thaw cycle 3 times, lysates were directly transfected into LLC-MK2/F7/M62/A, and cells were cultured in serum-free MEM containing 40. mu.g/mLArac, 7.5. mu.g/mL trypsin, and 50U/mL collagenase type IV (ICN, Aurola, OH) (trypsin only for pSeV (TDK)/Fct14(MMP # 6)/linker/HN. DELTA.M-GFP) (P1). 3-14 days later, part of the culture supernatant was collected and transfected into the freshly prepared LLC-MK2/F7/M62/A, and the cells were cultured at 32 ℃ with 40. mu.g/mL AraC, 7.5. mu.g/mL trypsin, and 50U/mL collagenase type IV in serum-free MEM (trypsin only for pSeV (TDK)/Fct14(MMP # 6)/linker/HN. DELTA.M-GFP) (P2) 3-14 days later, infection of the freshly prepared LLC-MK2/F7/M62/A with a portion of the culture, and the cells were cultured for 307 days at 32 ℃ in serum-free MEM containing 7.5. mu.g/mL trypsin and 50U/mLIV type collagenase (trypsin only for pSeV (TDK)/Fct14(MMP # 6)/linker/HN. DELTA.M-GFP) (P3). Culture supernatants were collected and BSA (1% final concentration) was added and stored at-80 ℃. The preserved virus solution was thawed and used for later preparation and in vitro testing.

As described above, SeV/F (MMP #6) Δ M-GFP, in which the F protein cleavage site is changed from PLGMTS (SEQ ID NO: 61) to PQGMTS (SEQ ID NO: 62), SeV (TDK)/Fct14(MMP #6) Δ M-GFP, in which the cytoplasmic domain is deleted by 28 amino acids, and SeV (TDK)/Fct14(MMP # 6)/linker/HN Δ M-GFP, which carry the F/HN chimeric protein, were successfully prepared.

Example 34 increase of the fusogenic activity of the improved F-modified M-deficient Sendai virus vector:

to investigate the efficacy of the viruses prepared in example 33, various cancer cell lines with different expression levels of MMP-2 and MMP-9, and LLC-MK2 in which MMP expression was not detected were infected as follows, and the cellular fusogenic potency of the vector was determined (FIG. 47). Each cancer cell (HT1080, U87MG, A172, U251, SW480, and LLC-MK2) was plated onto 24-well plates (containing the supplier-specified media) until confluent. U87MG (ATCC No. HTB-14) and A172(ATCC No. CRL-1620) were purchased from ATCC. U251(IFO50288) was purchased from JCRB cell bank. After washing twice with MEM medium, each M-deficient sendai virus vector (SeV/AM-GFP) was infected at an MOI of 0.1. The cells were left at room temperature for 1 hour and washed with MEM medium, and then 0.5mL of MEM containing 1% FBS was added to the 24-well plate. After 48 hours of culture, the number of fused syncytia was counted per X100 visual field (0.3 cm) ²) (inverted microscope). Alternatively, cultured cells were fixed in 4% paraformaldehyde for two hours, transferred to 70% ethanol and then transferred to distilled water, stained with hematoxylin for 5 minutes, and washed with water to count every 0.3cm²The number of nuclei forming syncytia. The results are shown in FIG. 49.

The expression of MMP-2 and MMP-9 was confirmed by the receptor of the gel signal recognition proteosome performed in example 22 (FIG. 48). As a result, the expression of MMP2 in HT1080, U87MG, and a172 was confirmed. In addition, low levels of MMP-9 expression were demonstrated in U251 and SW 480. The apparent expression of MMP-2 in LLC-MK2 is due to MMP-2 activity in 1% of the serum contained in the medium. GFP diffusion was observed two days after infection of each cancer cell line. As a result, a fusogenic activity was observed in U251 and SW480, which did not show spread of infection with the conventional SeV/F (MMP #2) Δ M-GFP (infected with the modified F-modified M-deficient Sendai virus vector). Specifically, those cells infected with M-deficient Sendai virus vector (SeV (TDK)/Fct14(MMP # 6)/linker/HN. DELTA.M-GFP) carrying the F/HN chimeric protein showed fusogenic activity. Although data not shown, mouse Lewis lung carcinoma and mouse colon-26 carcinoma also showed fusogenic activity due to infection with the modified M-deficient Sendai virus vector. The improvement of the carrier is expected to further enhance the effect and show an effect on cancer having a low concentration of MMP.

Industrial applicability

The present invention provides vectors for the specific spread of infection in the presence of a protease of interest. The vectors of the invention do not show significant production of virus-like particles and are transferred into surrounding cells only by cell fusion. Thus, the vectors of the present invention can be used to infect vectors located in a restricted area of the tissue of interest. In particular, the invention provides vectors that specifically spread their infection to cancer. These vectors have a strong inhibitory effect on tumor proliferation. Gene therapy of cancer using the vector of the present invention is likely to be a new cancer therapy with little side effect.

Sequence listing

<110> Kyoco research institute of vector (DNAVEC RESEARCH INC.)

<120> vector having modified protease-dependent tropism

<130>D3-A0202P

<150>JP 2002-129351

<151>2002-04-30

<160>101

<170>PatentIn version 3.1

<210>1

<211>3

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence for proteolytic cleavage

<400>1

Met Thr Ser

1

<210>2

<211>3

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence for proteolytic cleavage

<220>

<221>misc_feature

<222>(2)..(2)

<223> Xaa' at position 2 represents Leu or Gln.

<400>2

Pro Xaa Gly

1

<210>3

<211>6

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence for proteolytic cleavage

<220>

<221>misc_feature

<222>(2)..(2)

<223> Xaa' at position 2 represents Leu or Gln.

<400>3

Pro Xaa Gly Met Thr Ser

1 5

<210>4

<211>5

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence for proteolytic cleavage

<220>

<221>misc_feature

<222>(2)..(2)

<223> Xaa' at position 2 represents Leu or Gln.

<400>4

Pro Xaa Gly Met Thr

1 5

<210>5

<211>4

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence derived from Sendai Virus

<400>5

Pro Gln Ser Arg

1

<210>6

<211>3

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence for proteolytic cleavage

<400>6

Val Gly Arg

1

<210>7

<211>3

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence derived from Sendai Virus

<400>7

Gln Ser Arg

1

<210>8

<211>10

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized sequence derived from Sendai Virus

<400>8

ctttcaccct 10

<210>9

<211>15

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized sequence derived from Sendai Virus

<400>9

tttttcttac tacgg 15

<210>10

<211>18

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized sequence containing Not I site

<400>10

cggccgcaga tcttcacg 18

<210>11

<211>39

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized sequence for site-directed mutagenesis of Sendai virus

<400>11

gaaacaaaca accaatctag agagcgtatc tgacttgac 39

<210>12

<211>39

<212>DNA

<213> Artificial

<220>

<400>12

gtcaagtcag atacgctctc tagattggtt gtttgtttc 39

<210>13

<211>31

<212>DNA

<213> Artificial

<220>

<400>13

attacggtga ggagggctgt tcgagcagga g 31

<210>14

<211>31

<212>DNA

<213> Artificial

<220>

<400>14

ctcctgctcg aacagccctc ctcaccgtaa t 31

<210>15

<211>33

<212>DNA

<213> Artificial

<220>

<400>15

ggggcaatca ccatatccaa gatcccaaag acc 33

<210>16

<211>33

<212>DNA

<213> Artificial

<220>

<400>16

ggtctttggg atcttggata tggtgattgc ccc 33

<210>17

<211>37

<212>DNA

<213> Artificial

<220>

<400>17

catgctctgt ggtgacaacc cggactaggg gttatca 37

<210>18

<211>37

<212>DNA

<213> Artificial

<220>

<400>18

tgataacccc tagtccgggt tgtcaccaca gagcatg 37

<210>19

<211>41

<212>DNA

<213> Artificial

<220>

<400>19

cttgtctaga ccaggaaatg aagagtgcaa ttggtacaat a 41

<210>20

<211>41

<212>DNA

<213> Artificial

<220>

<400>20

tattgtacca attgcactct tcatttcctg gtctagacaa g 41

<210>21

<211>14

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence for immunization

<400>21

Met Ala Asp Ile Tyr Arg Phe Pro Lys Phe Ser Tyr Glu Cys

1 5 10

<210>22

<211>13

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence for immunization

<400>22

Leu Arg Thr Gly Pro Asp Lys Lys Ala Ile Pro His Cys

1 5 10

<210>23

<211>14

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence for immunization

<400>23

Cys Asn Val Val Ala Lys Asn Ile Gly Arg Ile Arg Lys Leu

1 5 10

<210>24

<211>48

<212>DNA

<213> Artificial

<220>

<400>24

agagtcactg accaactaga tcgtgcacga ggcatcctac catcctca 48

<210>25

<211>48

<212>DNA

<213> Artificial

<220>

<400>25

tgaggatggt aggatgcctc gtgcacgatc tagttggtca gtgactct 48

<210>26

<211>55

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized sequence for amplifying hygromycin resistance gene

<400>26

tctcgagtcg ctcggtacga tgaaaaagcc tgaactcacc gcgacgtctg tcgag 55

<210>27

<211>83

<212>DNA

<213> Artificial

<220>

<400>27

aatgcatgat cagtaaatta caatgaacat cgaaccccag agtcccgcct attcctttgc 60

cctcggacga gtgctggggc gtc 83

<210>28

<211>22

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized sequence derived from Sendai Virus

<400>28

ccaatctacc atcagcatca gc 22

<210>29

<211>21

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized sequence derived from Sendai Virus

<400>29

ttcccttcat cgactatgac c 21

<210>30

<211>22

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized sequence derived from Sendai Virus

<400>30

agagaacaag actaaggcta cc 22

<210>31

<211>6

<212>PRT

<213> Artificial

<220>

<223> Artificial Synthesis sequence for quantitative hydrolytic cleavage

<400>31

Pro Leu Gly Leu Gly Leu

1 5

<210>32

<211>74

<212>DNA

<213> Artificial

<220>

<400>32

ctgtcaccaa tgatacgaca caaaatgccc ctcttggcat gacgagtttc ttcggtgctg 60

tgattggtac tatc 74

<210>33

<211>74

<212>DNA

<213> Artificial

<220>

<400>33

gatagtacca atcacagcac cgaagaaact cgtcatgcca agaggggcat tttgtgtcgt 60

atcattggtg acag 74

<210>34

<211>75

<212>DNA

<213> Artificial

<220>

<400>34

ctgtcaccaa tgatacgaca caaaatgccc ctcttggcct ggggttattc ttcggtgctg 60

tgattggtac tatcg 75

<210>35

<211>75

<212>DNA

<213> Artificial

<220>

<400>35

cgatagtacc aatcacagca ccgaagaata accccaggcc aagaggggca ttttgtgtcg 60

tatcattggt gacag 75

<210>36

<211>45

<212>DNA

<213> Artificial

<220>

<400>36

caaaatgccg gtgctccccc gttgggattc ttcggtgctg tgatt 45

<210>37

<211>45

<212>DNA

<213> Artificial

<220>

<400>37

aatcacagca ccgaagaatc ccaacggggg agcaccggca ttttg 45

<210>38

<211>50

<212>DNA

<213> Artificial

<220>

<400>38

gacacaaaat gccggtgctc ccgtggggag attcttcggt gctgtgattg 50

<210>39

<211>50

<212>DNA

<213> Artificial

<220>

<400>39

caatcacagc accgaagaat ctccccacgg gagcaccggc attttgtgtc 50

<210>40

<211>11

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence derived from Sendai Virus F protein

<400>40

Gly Val Pro Gln Ser Arg Phe Phe Gly Ala Val

1 5 10

<210>41

<211>13

<212>PRT

<213> Artificial

<220>

<223> artificially synthesized sequence of F protein derived from Sendai Virus mutagenesis

<400>41

Gly Val Pro Leu Gly Met Thr Ser Phe Phe Gly Ala Val

1 5 10

<210>42

<211>13

<212>PRT

<213> Artificial

<220>

<400>42

Gly Val Pro Leu Gly Leu Gly Leu Phe Phe Gly Ala Val

1 5 10

<210>43

<211>10

<212>PRT

<213> Artificial

<220>

<400>43

Gly Val Pro Leu Gly Phe Phe Gly Ala Val

1 5 10

<210>44

<211>11

<212>PRT

<213> Artificial

<220>

<400>44

Gly Val Pro Val Gly Arg Phe Phe Gly Ala Val

1 5 10

<210>45

<211>16

<212>PRT

<213> Artificial

<220>

<223> amphipathic α -helical domain of Sendai virus

<400>45

Lys Ala Cys Thr Asp Leu Arg Ile Thr Val Arg Arg Thr Val Arg Ala

1 5 10 15

<210>46

<211>8

<212>PRT

<213> Artificial

<220>

<223> Synthesis of polypeptide

<400>46

Phe Phe Gly Ala Val Ile Gly Thr

1 5

<210>47

<211>13

<212>PRT

<213> Artificial

<220>

<223> Synthesis of polypeptide

<400>47

Glu Ala Arg Glu Ala Lys Arg Asp Ile Ala Leu Ile Lys

1 5 10

<210>48

<211>13

<212>PRT

<213> Artificial

<220>

<223> Synthesis of polypeptide

<400>48

Cys Gly Thr Gly Arg Arg Pro Ile Ser Gln Asp Arg Ser

1 5 10

<210>49

<211>31

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primers

<400>49

ccgctcgagc atgacagcat atatccagag a 31

<210>50

<211>40

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primers

<400>50

atagtttagc ggccgctcat ctgatcttcg gctctaatgt 40

<210>51

<211>31

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primers

<400>51

ccgctcgagc atgacagcat atatccagag a 31

<210>52

<211>40

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primers

<400>52

atagtttagc ggccgctcac cttctgagtc tataaagcac 40

<210>53

<211>31

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primers

<400>53

ccgctcgagc atgacagcat atatccagag a 31

<210>54

<211>40

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primers

<400>54

atagtttagc ggccgctcac cttctgagtc tataaagcac 40

<210>55

<211>36

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primer, F-F

<400>55

atccgaattc agttcaatga cagcatatat ccagag 36

<210>56

<211>36

<212>DNA

<213> Artificial

<220>

<223> Fct 14-Synthesis of primer, Fct14-R

<400>56

atccgcggcc gccggtcatc tggattaccc attagc 36

<210>57

<211>152

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primer, linker-HN-F

<400>57

atccgcggcc gcaatcgagg gaaggtggtc tgagttaaaa atcaggagca acgacggagg 60

tgaaggacca gaggacgcca acgacccacg gggaaagggg tgaacacatc catatccagc 120

catctctacc tgtttatgga cagagggtta gg 152

<210>58

<211>33

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primer, HN-R

<400>58

atccgcggcc gcttaagact cggccttgca taa 33

<210>59

<211>32

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primers

<400>59

atccgcggcc gcaatggatg gtgatagggg ca 32

<210>60

<211>30

<212>DNA

<213> Artificial

<220>

<223> Synthesis of primers

<400>60

atccgcggcc gcttaagact cggccttgca 30

<210>61

<211>6

<212>PRT

<213> Artificial

<220>

<223> MMP cleavage sequence

<400>61

Pro Leu Gly Met Thr Ser

1 5

<210>62

<211>6

<212>PRT

<213> Artificial

<220>

<223> MMP cleavage sequence

<400>62

Pro Gln Gly Met Thr Ser

1 5

<210>63

<211>6

<212>PRT

<213> Artificial

<220>

<223> MMP cleavage sequence

<400>63

Pro Gln Gly Leu Tyr Ala

1 5

<210>64

<211>75

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>64

ctgtcaccaa tgatacgaca caaaatgccc ctcttggcct ggggttattc ttcggtgctg 60

tgattggtac tatcg 75

<210>65

<211>75

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>65

cgatagtacc aatcacagca ccgaagaata accccaggcc aagaggggca ttttgtgtcg 60

tatcattggt gacag 75

<210>66

<211>74

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>66

ctgtcaccaa tgatacgaca caaaatgccc ctcagggctt gtatgctttc ttcggtgctg 60

tgattggtac tatc 74

<210>67

<211>74

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>67

gatagtacca atcacagcac cgaagaaagc atacaagccc tgaggggcat tttgtgtcgt 60

atcattggtg acag 74

<210>68

<211>74

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>68

ctgtcaccaa tgatacgaca caaaatgccc ctcaaggcat gacgagtttc ttcggtgctg 60

tgattggtac tatc 74

<210>69

<211>74

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>69

gatagtacca atcacagcac cgaagaaact cgtcatgcct tgaggggcat tttgtgtcgt 60

atcattggtg acag 74

<210>70

<211>74

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>70

ctgtcaccaa tgatacgaca caaaatgccc ttgcttacta tacggctttc ttcggtgctg 60

tgattggtac tatc 74

<210>71

<211>74

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>71

gatagtacca atcacagcac cgaagaaagc cgtatagtaa gcaagggcat tttgtgtcgt 60

atcattggtg acag 74

<210>72

<211>74

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>72

ctgtcaccaa tgatacgaca caaaatgccc ctcttggctt ggcgagattc ttcggtgctg 60

tgattggtac tatc 74

<210>73

<211>74

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>73

gatagtacca atcacagcac cgaagaatct cgccaagcca agaggggcat tttgtgtcgt 60

atcattggtg acag 74

<210>74

<211>50

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>74

cttcggtgct gtgattgcta ctatcgcact tgcagtggcg acatcagcac 50

<210>75

<211>50

<212>DNA

<213> Artificial

<220>

<223> synthetic oligonucleotide for mutagenesis

<400>75

gtgctgatgt cgccactgca agtgcgatag tagcaatcac agcaccgaag 50

<210>76

<211>49

<212>PRT

<213> Artificial

<220>

<223> partial sequence of Sendai virus F protein

<400>76

Val Ile Val Ile Val Leu Tyr Arg Leu Lys Arg Ser Met Leu Met Gly

1 5 10 15

Asn Pro Asp Asp Arg Ile Pro Arg Asp Thr Tyr Thr Leu Glu Pro Lys

20 25 30

Ile Arg His Met Tyr Thr Lys Gly Gly Phe Asp Ala Met Ala Glu Lys

35 40 45

Arg

<210>77

<211>34

<212>PRT

<213> Artificial

<220>

<223> partial sequence of Sendai virus F protein

<400>77

Val Ile Val Ile Val Leu Tyr Arg Leu Lys Arg Ser Met Leu Met Gly

1 5 10 15

Asn Pro Asp Asp Arg Ile Pro Arg Asp Thr Tyr Thr Leu Glu Pro Lys

20 25 30

Ile Arg

<210>78

<211>21

<212>PRT

<213> Artificial

<220>

<223> partial sequence of Sendai virus F protein

<400>78

Val Ile Val Ile Val Leu Tyr Arg Leu Lys Arg Ser Met Leu Met Gly

1 5 10 15

Asn Pro Asp Asp Arg

20

<210>79

<211>11

<212>PRT

<213> Artificial

<220>

<223> partial sequence of Sendai virus F protein

<400>79

Val Ile Val Ile Val Leu Tyr Arg Leu Lys Arg

1 5 10

<210>80

<211>50

<212>PRT

<213> Artificial

<220>

<223> linker sequence

<400>80

Ala Ala Ala Ile Glu Gly Arg Trp Ser Glu Leu Lys Ile Arg Ser Asn

1 5 10 15

Asp Gly Gly Glu Gly Pro Glu Asp Ala Asn Asp Pro Arg Gly Lys Gly

20 25 30

Val Gln His Ile His Ile Gln Pro Ser Leu Pro Val Tyr Gly G1n Arg

35 40 45

Val Arg

50

<210>81

<211>12

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>81

Ala Gly Val Pro Gln Ser ArgPhe Phe Gly Ala Val

1 5 10

<210>82

<211>12

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>82

Ala Pro Leu Gly Leu Trp Ala Phe Phe Gly Ala Val

1 5 10

<210>83

<211>12

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>83

Ala Pro Leu Gly Met Thr Ser Phe Phe Gly Ala Val

1 5 10

<210>84

<211>12

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>84

Ala Pro Leu Gly Leu Gly Leu Phe Phe Gly Ala Val

1 5 10

<210>85

<211>12

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>85

Ala Gly Val Pro Pro Leu Gly Phe Phe Gly Ala Val

1 5 10

<210>86

<211>12

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>86

Ala Pro Gln Gly Leu Tyr Ala Phe Phe Gly Ala Val

1 5 10

<210>87

<211>12

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>87

Ala Pro Gln Gly Met Thr Ser Phe Phe Gly Ala Val

1 5 10

<210>88

<211>12

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>88

Ala Leu Ala Tyr Tyr Thr Arg Phe Phe Gly Ala Val

1 5 10

<210>89

<211>12

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>89

Ala Pro Leu Gly Leu Ala Arg Phe Phe Gly Ala Val

1 5 10

<210>90

<211>23

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>90

Gln Ser Arg Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly Val

1 5 10 15

Ala Thr Ser Ala Gln Ile Thr

20

<210>91

<211>26

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>91

Pro Leu Gly Met Thr Ser Phe Phe Gly Ala Val Ile Gly Thr Ile Ala

1 5 10 15

Leu Gly Val Ala Thr Ser Ala Gln Ile Thr

20 25

<210>92

<211>26

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>92

Pro Gln Gly Met Thr Ser Phe Phe Gly Ala Val Ile Gly Thr Ile Ala

1 5 10 15

Leu Gly Val Ala Thr Ser Ala Gln Ile Thr

20 25

<210>93

<211>26

<212>PRT

<213> Artificial

<220>

<223> F protein cleavage site

<400>93

Pro Gln Gly Met Thr Ser Phe Phe Gly Ala Val Ile Ala Thr Ile Ala

1 5 10 15

Leu Ala Val Ala Thr Ser Ala Gln Ile Thr

20 25

<210>94

<211>32

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized oligonucleotide

<400>94

atccgctagc ccgtacggcc atggtgagca ag 32

<210>95

<211>72

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized oligonucleotide

<400>95

atccgcgcgc ccgtacgatg aactttcacc ctaagttttt cttactacgg agctttactt 60

gtacagctcg tc 72

<210>96

<211>31

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized oligonucleotide

<400>96

atccacgcgt catgacagca tatatccaga g 31

<210>97

<211>66

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized oligonucleotide

<400>97

atccgtcgac acgatgaact ttcaccctaa gtttttctta ctactttaac ggtcatctgg 60

attacc 66

<210>98

<211>32

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized oligonucleotide

<400>98

atccgctagc ccgtacggcc atggtgagca ag 32

<210>99

<211>72

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized oligonucleotide

<400>99

atccgctagc ccgtacgatg aactttcacc ctaagttttt cttactacgg agctttactt 60

gtacagctcg tc 72

<210>100

<211>36

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized oligonucleotide

<400>100

atccgctagc agttcaatga cagcatatat ccagag 36

<210>101

<211>67

<212>DNA

<213> Artificial

<220>

<223> artificially synthesized oligonucleotide

<400>101

atccgctagc acgatgaact ttcaccctaa gtttttctta ctacttttaa gactcggcct 60

tgcataa 67

Claims

1. A fusion protein having a cell-fusogenic activity, comprising the transmembrane regions of paramyxovirus F and HN proteins, wherein said F and HN proteins are cytoplasmic-bound to each other.

2. The fusion protein of claim 1, wherein the sequence of the protein cleavage site is substituted with a sequence cleavable by a protease that does not cleave wild-type F protein.

3. A nucleic acid encoding the protein of claim 1.

4. A vector comprising the nucleic acid of claim 3.

5. A viral particle comprising the protein of claim 1 or a nucleic acid encoding the protein.