Temperature-sensitive negative-strand RNA virus or viral vector and RNA genomes thereof

Abstract

To provide a temperature-sensitive negative-strand RNA virus or viral vector, and to provide RNA genomes thereof.SOLUTION: Provided is a negative-strand RNA virus or viral vector having a negative-strand RNA genome, in which a phosphoprotein (P) on the RNA genome has an amino acid mutation corresponding to a substitution at an amino acid in the phosphoprotein (P) corresponding to any one or all of D433, R434, and K437, and may have an amino acid mutation at L511, corresponding to a further amino acid substitution.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a temperature-sensitive negative-strand RNA virus or viral vector and its RNA genome.

Background Art

[0002] Negative-strand RNA viruses or viral vectors such as Sendai virus are known as cytoplasmic RNA viral vectors and are considered advantageous in regenerative medicine and in vivo use because they are less likely to damage the nucleus. In addition, negative-strand RNA viruses or viral vectors such as Sendai virus may exhibit high gene transfer efficiency and / or gene expression efficiency both in vivo and in vitro, and are considered particularly beneficial for the purpose of gene transfer and / or gene expression, and development is actively underway.

[0003] In order to enhance the safety of the use of viruses or viral vectors, techniques for removing (for example, reducing the amount or disappearing) viruses or viral vectors that have infected cells from inside the cells are required. Temperature-sensitive mutants have been produced as techniques for removing viruses or viral vectors. Specifically, those containing the L511F mutation in the P protein or those having the D433A / R434A / K437A mutation have been proposed (Patent Documents 1 to 4). In addition, a method of introducing (N1197S / L1558I / K1796E) into the L protein to enhance temperature sensitivity has been proposed. (Patent Documents 3 and 4).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Document

[0005]

Non-Patent Document 1

Summary of the Invention

[0006] According to the present invention, a temperature-sensitive negative-strand RNA virus or virus vector and its RNA genome are provided. According to the present invention, virus or virus vector-infected cells can be cultured while maintaining the infection according to the temperature conditions of the culture, and then the virus or virus vector can be removed (reduced).

[0007] According to the present invention, the following inventions are provided. [1] An RNA genome of a negative-strand RNA virus or virus vector, wherein the P protein encoded by the viral genome has substitution mutations in the amino acids of the P protein corresponding to any one or all of D433, R434, and K437, and the substitution mutations include mutations to any of G, T, and S, and may further have substitution mutations in the amino acids of the P protein corresponding to L511. The substitution mutation of L511 is a mutation to Y or F. An RNA genome of a negative-strand RNA virus or virus vector. [2] The negative-strand RNA virus or virus vector according to [1] above, wherein the P protein encoded by the viral genome has substitution mutations in the amino acids of the P protein corresponding to all of D433, R434, and K437, and the substitution mutations include mutations to any of G, T, and S. An RNA genome of a negative-strand RNA virus or virus vector. [3] The negative-strand RNA virus or virus vector according to [2] above, wherein the substitution mutation is a mutation to T or S. An RNA genome of a negative-strand RNA virus or virus vector. [4] The RNA genome of the negative-strand RNA virus or viral vector according to [3] above, wherein the substitution mutation is a mutation to T. [5] The RNA genome of the negative-strand RNA virus or viral vector according to [3] above, wherein the substitution mutation is a mutation to S. [6] The negative-strand RNA virus or viral vector according to any one of [1] to [3] above, wherein the P protein encoded by the viral genome (1S) has amino acid mutations corresponding to D433S, R434A, K437A, and L511F, (2S) has amino acid mutations corresponding to D433S, R434A, K437A, and L511Y, (3S) has amino acid mutations corresponding to D433A, R434S, K437A, and L511F, (4S) has amino acid mutations corresponding to D433A, R434S, K437A, and L511Y, (5S) has amino acid mutations corresponding to D433A, R434A, K437S, and L511F, (6S) has amino acid mutations corresponding to D433A, R434A, K437S, and L511Y, (7S) has amino acid mutations corresponding to D433S, R434S, K437A, and L511F, (8S) has amino acid mutations corresponding to D433S, R434S, K437A, and L511Y, (9S) has amino acid mutations corresponding to D433S, R434A, K437S, and L511F, (10S) has amino acid mutations corresponding to D433S, R434A, K437S, and L511Y, (11S) has amino acid mutations corresponding to D433A, R434S, K437S, and L511F, (12S) has amino acid mutations corresponding to D433A, R434S, K437S, and L511Y, Having amino acid mutations corresponding to (13S) D433S, R434S, K437S, and L511F Having amino acid mutations corresponding to (14S) D433S, R434S, K437S, and L511Y Having amino acid mutations corresponding to (1T) D433T, R434A, K437A, and L511F Having amino acid mutations corresponding to (2T) D433T, R434A, K437A, and L511Y Having amino acid mutations corresponding to (3T) D433A, R434T, K437A, and L511F Having amino acid mutations corresponding to (4T) D433A, R434T, K437A, and L511Y Having amino acid mutations corresponding to (5T) D433A, R434A, K437T, and L511F Having amino acid mutations corresponding to (6T) D433A, R434A, K437T, and L511Y Having amino acid mutations corresponding to (7T) D433T, R434T, K437A, and L511F Having amino acid mutations corresponding to (8T) D433T, R434T, K437A, and L511Y Having amino acid mutations corresponding to (9T) D433T, R434A, K437T, and L511F Having amino acid mutations corresponding to (10T) D433T, R434A, K437T, and L511Y Having amino acid mutations corresponding to (11T) D433A, R434T, K437T, and L511F Having amino acid mutations corresponding to (12T) D433A, R434T, K437T, and L511Y Having amino acid mutations corresponding to (13T) D433T, R434T, K437T, and L511F Having amino acid mutations corresponding to (14T) D433T, R434T, K437T, and L511Y (1ST) Having amino acid mutations corresponding to D433S, R434T, K437S, and L511F, or (2ST) Having amino acid mutations corresponding to D433S, R434T, K437S, and L511Y, The genomic RNA of a negative-strand RNA virus or a viral vector. [7] A negative-strand RNA virus or viral vector according to any one of [1] to [6] above, wherein the large protein (L protein) has an amino acid mutation corresponding to either or both of L1361C and L1558I, the genomic RNA of a negative-strand RNA virus or viral vector. [8] The genomic RNA of a negative-strand RNA virus or viral vector according to any one of [1] to [7] above, carrying at least one exogenous target gene. [9] The genomic RNA of a negative-strand RNA virus or viral vector according to any one of [1] to [8] above, which is Sendai virus or a Sendai virus vector.

[10] A nucleic acid encoding the genomic RNA of a negative-strand RNA virus or viral vector according to any one of [1] to [9] above {preferably DNA, more preferably an expression vector for the genomic RNA (particularly, a plasmid, etc.)}.

[11] A negative-strand RNA virus or viral vector containing the genomic RNA of a negative-strand RNA virus or viral vector according to any one of [1] to [9] above.

[12] A negative-strand RNA virus or viral vector according to

[11] above, containing a P protein that is less temperature-sensitive than the P protein encoded on the genome or a P protein that is not temperature-sensitive.

[13] When the L protein encoded on the viral genome is temperature-sensitive, containing an L protein that is less temperature-sensitive than the L protein encoded on the genome or an L protein that is not temperature-sensitive, A negative-strand RNA virus or viral vector according to

[11] or

[12] above.

[14] A composition comprising the negative-strand RNA virus or viral vector according to any one of

[11] to

[13] above.

[15] A method for expressing a target gene in a cell, a method for producing a cell that has expressed the target gene, or a method for culturing a cell, comprising: Infecting the cell with the negative-strand RNA virus or viral vector according to any one of

[11] to

[13] above, and then culturing the cell under a first temperature condition to express the target gene in the cell; Subsequently culturing the cell under a second temperature condition to remove some or all of the negative-strand RNA virus or viral vector from the cell. A method.

[0008]

[16] A method for expressing a target gene in a cell, comprising: Infecting the cell with the negative-strand RNA virus or viral vector according to any one of

[11] to

[13] above, and then culturing the cell under a first temperature condition to express the target gene in the cell; Subsequently culturing the cell under a second temperature condition to remove some or all of the negative-strand RNA virus or viral vector from the cell. A method.

[17] A method for producing a cell that has expressed a target gene, comprising: Infecting the cell with the negative-strand RNA virus or viral vector according to any one of

[11] to

[13] above, and then culturing the cell under a first temperature condition to express the target gene in the cell; Subsequently culturing the cell under a second temperature condition to remove some or all of the negative-strand RNA virus or viral vector from the cell. A method.

[18] A method for culturing a cell, comprising: Infecting the cell with the negative-strand RNA virus or viral vector described in any one of

[11] to

[13] above, and then culturing under a first temperature condition to express the target gene in the cell; Subsequently, culturing further under a second temperature condition to thereby remove some or all of the negative-strand RNA virus or viral vector from the cell; Method.

[0009]

[19] A method for expressing a target gene in a cell, Infecting the cell with the negative-strand RNA virus or viral vector described in any one of

[11] to

[13] above, and then culturing under a first temperature condition to express the target gene in the cell; Method.

[20] A method for producing a cell expressing a target gene, Infecting the cell with the negative-strand RNA virus or viral vector described in any one of

[11] to

[13] above, and then culturing under a first temperature condition to express the target gene in the cell; Method.

[21] A method for culturing a cell, Infecting the cell with the negative-strand RNA virus or viral vector described in any one of

[11] to

[13] above, and then culturing under a first temperature condition to express the target gene in the cell; Method.

[0010]

[22] A method for expressing a target gene in a cell, Infecting the cell with the negative-strand RNA virus or viral vector described in any one of

[11] to

[13] above, and then culturing under a second temperature condition to express the target gene in the cell (and preferably removing the virus or viral vector from the cell); Method.

[23] A method for producing a cell expressing a target gene, Infecting the cell with the negative-strand RNA virus or virus vector described in any one of

[11] to

[13] above, and then culturing the cell under a second temperature condition to express the target gene in the cell (and preferably removing the virus or virus vector from the cell), comprising: Method.

[24] A method for culturing a cell, Infecting the cell with the negative-strand RNA virus or virus vector described in any one of

[11] to

[13] above, and then culturing the cell under a second temperature condition to express the target gene in the cell (and preferably removing the virus or virus vector from the cell), Method.

[0011]

[25] The invention according to any one of the above, wherein the negative-strand RNA virus or virus vector is Sendai virus.

[26] The invention according to any one of the above, wherein the negative-strand RNA virus or virus vector has an RNA genome and all of the N protein, P protein, M protein, F protein, HN protein, and L protein {wherein the RNA genome preferably does not have a gene encoding a functional F protein}.

[0012]

[0101] An amino acid sequence having an amino acid mutation corresponding to one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or all amino acid mutations selected from the group consisting of 4I, 18K, 36L, 50I, 57K, 59F, 63A, 68L, 90V, 98I, and 105R with respect to the amino acid sequence set forth in SEQ ID NO: 18, preferably a degradation-inducible protein derived from FKBP12.

[0102] An amino acid sequence having one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all amino acid mutations selected from the group consisting of 4I, 18K, 50I, 57K, 59F, 63A, 68L, 90V, 98I, and 105R with respect to the amino acid sequence set forth in SEQ ID NO: 18, and an amino acid mutation corresponding to 36L (and preferably 90V and / or 106P), a degradation-inducible protein, preferably a degradation-inducible protein derived from FKBP12.

[0103] The degradation-inducible protein according to

[0101] or

[0102] above, having amino acid mutations corresponding to all of 4I, 18K, 36L, 50I, 57K, 59F, 63A, 68L, 90V, 98I, and 105R with respect to the amino acid sequence set forth in SEQ ID NO: 18.

[0104] A degradation-inducible fusion protein comprising a target protein and the degradation-inducible protein according to any one of

[0101] to

[0103] above.

[0105] A nucleic acid (e.g., DNA or RNA) encoding the protein according to any one of

[0101] to

[0104] above.

[0106] An antisense oligo against the nucleic acid encoding the protein according to any one of

[0101] to

[0103] above {preferably, comprising an antisense portion against the nucleic acid encoding one or more of the above amino acid mutations}.

[0107] An antibody that binds to the protein according to any one of

[0101] to

[0103] above.

[0108] An antibody that binds to the protein according to any one of

[0101] to

[0103] above with a stronger binding affinity than to the protein having the amino acid sequence set forth in SEQ ID NO: 18.

[0109] An antibody {preferably a monoclonal antibody or a polyclonal antibody, which may be isolated} that binds to the protein according to any one of the above

[0101] to

[0103] with a binding affinity that is more than 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 100-fold, 1,000-fold, or 10,000-fold stronger than that to the protein having the amino acid sequence set forth in SEQ ID NO: 18.

[0110] An animal cell containing any of the above proteins.

[0111] An animal cell containing any of the above nucleic acids.

[0112] A negative-strand RNA virus or vector containing any of the above proteins.

[0113] A negative-strand RNA virus or vector containing any of the above nucleic acids.

[0114] The negative-strand RNA genome of the virus or vector according to the above

[0113] containing any of the above nucleic acids.

[0115] A nucleic acid encoding the genome according to the above

[0114] , or a vector containing the nucleic acid.

[0013]

[0131] An M protein having the amino acid sequence set forth in SEQ ID NO: 20.

[0132] A nucleic acid encoding the M protein according to the above

[0131] .

[0133] An HN protein having the amino acid sequence set forth in SEQ ID NO: 21.

[0134] A nucleic acid encoding the HN protein according to the above

[0133] .

[0135] An animal cell containing the M protein according to the above

[0131] and / or the HN protein according to the above

[0133] .

[0136] An animal cell containing the M protein according to the above

[0131] and the HN protein according to the above

[0133] .

[0137] A negative-strand RNA virus or vector comprising the M protein described in the above

[0131] and / or the HN protein described in the above

[0133] .

[0138] A nucleic acid encoding the negative-strand RNA virus or vector described in the above

[0137] , or a vector containing the nucleic acid.

[0139] An animal cell containing the nucleic acid described in the above

[0132] and / or

[0134] .

[0140] The genome (negative-strand RNA genome) of a negative-strand RNA virus or vector containing the nucleic acid described in the above

[0132] and / or

[0134] .

[0141] A negative-strand RNA virus or vector containing the genome of the above

[0140] .

Brief Description of Drawings

[0014]

Figure 1A

Figure 1B

Figure 2

Figure 3

Figure 4

Figure 5A

Figure 5B

Figure 6

Figure 7A

Figure 7B

Figure 8

Figure 9A

Figure 9B

Figure 10

Figure 11A

Figure 11B

Figure 12

[0015] As used herein, the term "negative-strand RNA virus vector" refers to a recombinant virus obtained by modifying a virus having a negative-strand RNA as its genome (i.e., a negative-strand RNA virus) for introducing a target gene. Examples of negative-strand RNA viruses include viruses of the Orthomyxoviridae family (Orthomyxoviruses such as influenza virus), Paramyxoviridae family (Paramyxoviruses such as Morbillivirus), Rhabdoviridae family (Rhabdoviruses such as rabies virus), Filoviridae family (Filoviruses such as Ebola and Marburg virus), and Bunyaviridae family (Bunyaviruses such as hantavirus). Examples of Paramyxoviruses include viruses of the Orthoparamyxovirinae subfamily. Examples of viruses of the Orthoparamyxovirinae subfamily include viruses of the genus Respirovirus, such as Sendai virus. In all embodiments, the negative-strand RNA virus and the negative-strand RNA virus vector are preferably Sendai virus (also referred to as murine parainfluenza type 1 virus) or a Sendai virus vector.

[0016] As used herein, a "packaging cell" is a cell that produces a viral vector. Generally, from the perspective of improving safety, the viral genome of the viral vector has factors responsible for one or more functions selected from the group consisting of its growth, replication, and spread (including infection of other cells) disrupted, and is engineered so that it cannot grow, replicate, or spread after cell infection. However, when producing a viral vector, in order to enable its growth, replication, and spread, a viral vector is produced while complementing the disrupted factors in the packaging cell. For this reason, the packaging cell expresses a part of the disrupted viral factors so as to complement the disrupted viral factors and restore the viral production amount in order to produce a viral vector. The packaging cell may stably hold such factors in the genome or may hold them temporarily. In any case, during virus production, the complemented viral factors are supplied into the cells of the packaging cell. As an example, for instance, a Sendai virus vector is classically obtained by producing a Sendai virus having a genome lacking the F gene using a packaging cell that supplies the F gene. F genes that are activated in the presence of trypsin but also types of F genes (F5R) that are activated by furin, which is ubiquitously present in cells, have been developed, enhancing the convenience of virus production (see, for example, WO2005 / 071085A). In recent years, techniques for producing Sendai virus from cDNA have been developed. For example, vectors have been prepared that are based on the attenuated Z strain and have been further engineered to enhance safety for medical applications in humans. For example, techniques have been developed to delete any one or more of the M, F, and HN genes from the viral genome to abolish the transmissibility of the virus. For example, an F gene-deleted viral genome is preferably used. The viral genome is operably linked to a control sequence (e.g., the T7 promoter), and the production of the viral genome can be driven by the above control sequence. Thereby, a Sendai virus genome can be produced in a packaging cell from cDNA.When using the T7 promoter as the first control array, the T7 RNA polymerase can be supplied, for example, by a helper virus such as vaccinia virus. In packaging cells, N, P, F, and L are expressed operably linked to a control array that drives transcription by an RNA polymerase (e.g., polII), thereby supplying virus particle components and enabling the formation of virus particles in the packaging cells. As the packaging cells, for example, LLC-MK2 cells derived from monkey kidneys are used. Thereby, virus particles that can infect cells once but cannot then spread to other cells are obtained. The virus particles can be used after concentration and / or purification as needed. When incorporating a foreign gene into the virus, a virus-specific control array is introduced as needed to enable transcription by the RNA-dependent RNA polymerase.

[0017] According to the present invention, there is provided a method for producing a negative-strand RNA virus or a negative-strand RNA virus vector. Examples of the negative-strand RNA virus include Orthomyxoviridae (Orthomyxoviruses such as influenza virus), Paramyxoviridae (Paramyxoviruses such as Morbillivirus), Rhabdoviridae (Rhabdoviruses such as rabies virus), Filoviridae (Filoviruses such as Ebola and Marburg virus), and Bunyaviridae (Bunyaviruses such as Hantavirus). The negative-strand RNA virus is preferably a virus of the family Paramyxoviridae, more preferably a paramyxovirus, and even more preferably Sendai virus.

[0018] Taking Sendai virus as an example, the composition of a negative-strand RNA virus or a negative-strand RNA virus vector will be described. Sendai virus includes a negative-strand RNA genome and nucleoprotein (N protein), phosphoprotein (P protein), matrix protein (M protein), fusion protein (F protein), hemagglutinin-neuraminidase (HN protein), and large protein (L protein). The N protein, P protein, and L protein can form an RNP complex with the RNA genome, and the M protein, F protein, and HN protein can constitute the outer shell of the virus particle. The genome is considered to be encapsulated in the outer shell. Sendai virus can infect cells using the F protein. By constructing the outer shell to have the F protein and modifying (such as deleting) the genome so that it cannot produce the F protein, the Sendai virus that increases from the genome after infection lacks the F protein and thus cannot spread further. Note that the nucleoprotein (N protein), phosphoprotein (P protein), and large protein (L protein) are considered necessary for the autonomous replication of the viral genome or vector genome in the introduced cells. Examples of the phosphoprotein (P protein) include P proteins having amino acid sequences registered under GenBank accession numbers BAN84668.1, AAB06197.1, P04859.1, P14252.1, AAB06291.1, AAX07439.1, BAM62828.1, BAM62834.1, P04860.1, BAM62840.1, BAD74220.1, P14251.1, BAM62844.1, BAM62842.1, BAF73480.1, BAD74226.1, BAF73486.1, Q9DUE2.1, BAC79134.1, NP_056873.1, and ABB00297.1. In a preferred embodiment, examples of the phosphoprotein (P protein) include, for example, the amino acid sequence of the P protein described in GenBank accession number BAN84668.1 (SEQ ID NO: 1), or a P protein having a sequence corresponding to the said sequence.Examples of the large protein (L protein) include an L protein having the amino acid sequence described in GenBank accession number BAN84672.1 (SEQ ID NO: 5) or a sequence corresponding to the said sequence. The P protein of Sendai virus may have an amino acid sequence corresponding to any of the amino acid sequences of the said P proteins. The amino acid numbers of the P protein referred to in this specification are the amino acid numbers in the amino acid sequence of SEQ ID NO: 1. A person skilled in the art will be able to identify the amino acid corresponding to the amino acid of the said amino acid number by aligning the amino acid sequences, etc., even in the case of a P protein having other amino acid sequences. The L protein of Sendai virus has an amino acid sequence corresponding to the amino acid sequence of the said L protein. The amino acid numbers of the L protein referred to in this specification are the amino acid numbers in the amino acid sequence of SEQ ID NO: 5. A person skilled in the art will be able to identify the amino acid corresponding to the amino acid of the said amino acid number by aligning the amino acid sequences, etc., even in the case of an L protein having other amino acid sequences.

[0019] In this specification, a "control sequence" refers to a sequence that drives a gene operably linked thereto and has the activity of transcribing RNA from the gene. The control sequence is, for example, a promoter. Examples of the promoter include a class I promoter (which can be used for the transcription of rRNA precursors), a class II promoter (which is composed of a core promoter and an upstream promoter element and can be used for the transcription of mRNA), and a class III promoter (which is further classified into type I, type II, and type III).

[0020] In certain embodiments, a negative-strand RNA virus vector has, on its genome, factors related to the growth or infection of negative-strand RNA viruses in cells disrupted (e.g., deleted), and has reduced growth or infectivity in cells other than packaging cells, or has substantially no infectivity. As described above, such a vector is provided with an initial infectivity to cells by being produced under conditions where the packaging cells supply the factors related to growth or infection that have been disrupted. Thereby, the vector obtained from the packaging cells has infectivity, but thereafter, the vector that has infected cells other than the packaging cells cannot produce further infectious particles, thereby improving the safety of the vector.

[0021] As used herein, "exogenous" means derived from an organism other than the host. Typically, a control sequence can be exogenous. Also, the gene to be expressed can be exogenous. Thus, a gene operably linked to a control sequence can be such that both the control sequence and the gene can be exogenous. "Endogenous" means that which is possessed by the host itself.

[0022] As used herein, "corresponding to a certain amino acid of Protein A" means being present at the position corresponding to the certain amino acid of Protein A when aligned in a homolog or ortholog of Protein A.

[0023] <The virus or virus vector of the present invention and its RNA genome> According to the present invention, there are provided a negative-strand RNA virus or virus vector and its RNA genome. According to the present invention, the negative-strand RNA virus can preferably be a mononegavirus, more preferably a virus of the family Paramyxoviridae, still more preferably a virus of the genus Respirovirus of the family Paramyxoviridae, and most preferably Sendai virus. And in the present invention, virus vectors prepared from these viruses can be provided. The above virus or viral vector may include a negative-strand RNA genome (hereinafter sometimes simply referred to as "viral genome"), a nucleoprotein (N protein), a phosphoprotein (P protein), a matrix protein (M protein), a fusion protein (F protein), a hemagglutinin-neuraminidase (HN protein), and a large protein (L protein). The above virus or viral vector may have an RNP complex containing the N protein, the P protein, and the L protein, as well as the viral genome or the viral vector genome. The above virus or viral vector may also have an outer shell of a viral particle containing the M protein, the F protein, and the HN protein. In certain embodiments of the present invention, the above virus or viral vector has at least the P protein. In this embodiment, the P protein is preferably included in the RNP complex. Also, in this embodiment, the above virus or viral vector preferably has an outer shell of a viral particle containing the M protein, the F protein, and the HN protein.

[0024] Previous studies have revealed that Sendai virus or a Sendai virus vector prepared by replacing the P protein with a P protein mutant exhibits temperature sensitivity. Specifically, a P protein mutant (SEQ ID NO: 2) containing all of the L511F mutation and the D433A / R434A / K437A mutations is a temperature-sensitive mutation, and it has been shown that the virus or viral vector having the P protein mutant is removed from cells by increasing the temperature (WO2010 / 008054, 2017 / 082174).

[0025] Hereinafter, temperature-sensitive mutants of the P protein and temperature-sensitive mutants of the L protein will be described. These mutants relate to the P protein and L protein encoded by the viral genome. In contrast, the presence or absence of temperature sensitivity of the P protein and L protein as constituent proteins of a virus or viral vector having the viral genome is not questioned. That is, after infecting target cells, the virus or viral vector amplifies in the cells using the proteins translated from the viral genome. As long as the P protein and preferably the L protein of the virus and viral vector that amplify in the cells have temperature sensitivity, the intended effect of the present invention will be achieved.

[0026] Therefore, in one aspect, the P protein and L protein of a virus or viral vector having the viral genome may have a lower temperature sensitivity than the temperature-sensitive mutants of the P protein and L protein encoded by the viral genome, and preferably, they do not have temperature sensitivity (for example, have the same temperature sensitivity as the wild type). By configuring in this way, it becomes difficult for the production amount of the virus or viral vector at the culture temperature of the packaging cells to decrease, and it can be expected to increase the production efficiency of the virus or viral vector. Also, in one aspect, the P protein and L protein as protein components of a virus or viral vector having the viral genome may have the temperature-sensitive mutant of the P protein described below, and preferably the temperature-sensitive mutant of the L protein. The presence or absence and strength of temperature sensitivity at a specific temperature can be determined based on the amount of the virus or viral vector after culture at the specific temperature.

[0027] The temperature-sensitive mutants of the P protein and the temperature-sensitive mutants of the L protein will be described. In the present invention, the P protein encoded by the viral genome has a temperature-sensitive mutation and can confer temperature sensitivity to a virus or a viral vector. In the present invention, the temperature sensitivity of a virus or a viral vector means that its growth is significantly reduced, its amount is significantly decreased, or its amount reaches below the detection limit in a specific temperature range as compared with a virus or a viral vector that does not have a corresponding temperature-sensitive mutation on the viral genome. The specific temperature range is not limited as long as it is a temperature at which cell growth is not inhibited. For example, it is about 35°C to about 39°C, preferably 36°C to 38.5°C. In one aspect of the present invention, the P protein has a substitution mutation in the amino acid of the P protein corresponding to any one or more or all of D433, R434, and K437, and the substitution mutation includes a mutation to any one of G, T, and S. That is, in one aspect of the invention, the P protein has the amino acid sequence of SEQ ID NO: 4 {except for SEQ ID NOs: 1 and 2}. By doing so, the P protein mutant can confer temperature sensitivity to Sendai virus or a Sendai virus vector. In a preferred aspect of the present invention, the P protein mutant can exhibit higher temperature sensitivity than the P protein mutant containing the D433A / R434A / K437A mutation.

[0028] In one aspect of the present invention, the P protein has a substitution mutation in the amino acid of the P protein corresponding to all of D433, R434, and K437, and the substitution mutation includes a mutation to any one of G, T, and S. In one aspect of the present invention, the P protein has a substitution mutation in the amino acid of the P protein corresponding to all of D433, R434, and K437, and the substitution mutation is a mutation to any one of G, T, and S. In one aspect of the present invention, the P protein has a substitution mutation in the amino acid of the P protein corresponding to all of D433, R434, and K437, and the substitution mutation is a mutation to T. In one aspect of the present invention, the P protein has a substitution mutation in the amino acid of the P protein corresponding to all of D433, R434, and K437, and the substitution mutation is a mutation to S.

[0029] In certain embodiments of the present invention, the P protein may have substitution mutations in the amino acid sequence corresponding to L511. In certain embodiments of the present invention, it is preferred that the amino acid sequence of the L protein corresponding to L511 is mutated to F or Y. For example, the P protein may be a P protein variant having an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 3. Also, for example, the P protein may be a P protein variant having an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 4.

[0030] In certain embodiments of the present invention, the P protein has a mutation to an amino acid selected from the group consisting of S, A, and T in the amino acid corresponding to D433, a mutation to an amino acid selected from the group consisting of S, A, and T in the amino acid corresponding to R434, a mutation to an amino acid selected from the group consisting of S, A, and T in the amino acid corresponding to K437, and may have a mutation to F or Y in the amino acid corresponding to L511. Also, in certain embodiments of the present invention, the P protein (1S) has mutations of the amino acids corresponding to D433S, R434A, K437A, and L511F, (2S) has mutations of the amino acids corresponding to D433S, R434A, K437A, and L511Y, (3S) has mutations of the amino acids corresponding to D433A, R434S, K437A, and L511F, (4S) has mutations of the amino acids corresponding to D433A, R434S, K437A, and L511Y, (5S) has mutations of the amino acids corresponding to D433A, R434A, K437S, and L511F, (6S) has mutations of the amino acids corresponding to D433A, R434A, K437S, and L511Y, (7S) has mutations of the amino acids corresponding to D433S, R434S, K437A, and L511F, (8S) has mutations of the amino acids corresponding to D433S, R434S, K437A, and L511Y, (9S) Having amino acid mutations corresponding to D433S, R434A, K437S, and L511F (10S) Having amino acid mutations corresponding to D433S, R434A, K437S, and L511Y (11S) Having amino acid mutations corresponding to D433A, R434S, K437S, and L511F (12S) Having amino acid mutations corresponding to D433A, R434S, K437S, and L511Y (13S) Having amino acid mutations corresponding to D433S, R434S, K437S, and L511F (14S) Having amino acid mutations corresponding to D433S, R434S, K437S, and L511Y (1T) Having amino acid mutations corresponding to D433T, R434A, K437A, and L511F (2T) Having amino acid mutations corresponding to D433T, R434A, K437A, and L511Y (3T) Having amino acid mutations corresponding to D433A, R434T, K437A, and L511F (4T) Having amino acid mutations corresponding to D433A, R434T, K437A, and L511Y (5T) Having amino acid mutations corresponding to D433A, R434A, K437T, and L511F (6T) Having amino acid mutations corresponding to D433A, R434A, K437T, and L511Y (7T) Having amino acid mutations corresponding to D433T, R434T, K437A, and L511F (8T) Having amino acid mutations corresponding to D433T, R434T, K437A, and L511Y (9T) Having amino acid mutations corresponding to D433T, R434A, K437T, and L511F (10T) Having amino acid mutations corresponding to D433T, R434A, K437T, and L511Y (11T) Having amino acid mutations corresponding to D433A, R434T, K437T, and L511F (12T) Having amino acid mutations corresponding to D433A, R434T, K437T, and L511Y (13T) Having amino acid mutations corresponding to D433T, R434T, K437T, and L511F (14T) Having amino acid mutations corresponding to D433T, R434T, K437T, and L511Y (1ST) Having amino acid mutations corresponding to D433S, R434T, K437S, and L511F, or (2ST) Preferably having amino acid mutations corresponding to D433S, R434T, K437S, and L511Y.

[0031] In certain embodiments of the present invention, the virus or viral vector has genes encoding at least the P protein and the L protein. The P protein encoded by the RNA genome has any of the above mutations. Further, the L protein encoded by the RNA genome has a temperature-sensitive mutation and may be able to confer temperature sensitivity to the virus or viral vector. In the present invention, the temperature sensitivity of the virus or viral vector means that its growth is significantly reduced, its quantity is significantly decreased, or its quantity reaches below the detection limit in a specific temperature range as compared to a virus or viral vector that does not have the corresponding temperature-sensitive mutation. In certain embodiments, it preferably has substitution mutations in one or more or all of the amino acids corresponding to N1197, L1361, L1558, and K1796. The mutation of the amino acid corresponding to N1197 is preferably, for example, a mutation to S. The mutation of the amino acid corresponding to L1361 is preferably, for example, a mutation to C. Further, the mutation of the amino acid corresponding to L1558 is preferably, for example, a mutation to I. Also, the amino acid mutation corresponding to K1796 is preferably, for example, a mutation to E. Thus, in certain embodiments, the L protein preferably has any one or more or all (preferably all) of N1197, L1361C, L1558I, and K1796. In certain embodiments, it preferably has any one, or preferably all, of the mutations of N1197S, L1558I, and K1796, and more preferably further has L1361C. That is, in certain embodiments of the invention, the L protein has the amino acid sequence of SEQ ID NO: 6 {excluding SEQ ID NO: 5}.

[0032] Thus, in certain embodiments of the present invention, the virus or viral vector has genes encoding at least the P protein and the L protein, the P protein encoded by the RNA genome has any of the above mutations, and the L protein encoded by the RNA genome preferably has any of the above mutations. That is, in certain embodiments, the P protein has the amino acid sequence of SEQ ID NO: 4 {excluding SEQ ID NOs: 1 and 2}, and the L protein preferably has the amino acid sequence of SEQ ID NO: 6 {excluding SEQ ID NO: 5}. In certain embodiments, the P protein has substitution mutations in the amino acids of the P protein corresponding to all of D433, R434, and K437, the substitution mutations include mutations to any of G, T, and S, and the L protein preferably has either or both of L1361C and L1558I (preferably both). Also, in certain embodiments, the P protein has substitution mutations in the amino acids of the P protein corresponding to all of D433, R434, and K437, the substitution mutations include mutations to any of G, T, and S, the P protein has substitution mutations in the amino acid sequence corresponding to L511 or L511, the substitution mutations are mutations to F or Y, and the L protein preferably has either or both of L1361C and L1558I (preferably both). In certain embodiments, the P protein has any of the mutations of (1S)-(14S) and (1T)-(14T) and (1ST)-(2ST) above, and the L protein preferably has either or both of L1361C and L1558I (preferably both). The mutations of the P protein can be used in combination with mutations of other proteins. Also, the mutations of the L protein can be used in combination with mutations of other proteins.

[0033] In certain embodiments of the present invention, the virus or viral vector may have an M protein or a variant thereof. The variant of the M protein may have substitution mutations in the amino acids of the M protein corresponding to 1 to 3 amino acids selected from the group consisting of G69, T116, and A183. The variant of the M protein may have a mutation in the amino acid corresponding to G69, for example, the mutation may be a mutation corresponding to G69E. The variant of the M protein may have a mutation in the amino acid corresponding to G116, for example, the mutation may be a mutation corresponding to T116A. The variant of the M protein may have a mutation in the amino acid corresponding to A183, for example, the mutation may be a mutation corresponding to A183T or A183S, preferably a mutation corresponding to A183S. In certain embodiments, the variant of the M protein may have amino acid mutations corresponding to G69E, T116A, and A183T, or preferably, amino acid mutations corresponding to G69E, T116A, and A183S. The mutation of the M protein may reduce the cytotoxicity of SeV. The mutation of the M protein can be used in combination with mutations of other proteins.

[0034] In certain embodiments of the present invention, the virus or viral vector may have an HN protein or a variant thereof. The variant of the HN protein may have substitution mutations in the amino acids of the HN protein corresponding to 1 to 3 amino acids selected from the group consisting of A262, G264, and K461. The HN protein may have a mutation in the amino acid corresponding to, for example, A262, and for example, the mutation may be a mutation corresponding to A262T. The HN protein may have a mutation in the amino acid corresponding to, for example, G264, and for example, the mutation may have a mutation corresponding to G264R or G264K, preferably a mutation corresponding to G264K. The HN protein may have a mutation in the amino acid corresponding to, for example, K461, and for example, the mutation may be a mutation corresponding to K461E. In certain embodiments, the variant of the HN protein may have amino acid mutations corresponding to A262T, G264R, and K461E, or preferably, may have amino acid mutations corresponding to A262T, G264K, and K461E. The mutation to the HN protein may reduce the cytotoxicity of SeV. The mutation of the HN protein can be used in combination with mutations of other proteins.

[0035] In certain embodiments of the present invention, the P protein, the L protein, the M protein, and the HN protein can each have the mutations described above.

[0036] The amino acid substitutions defined above may be conservative amino acid substitutions with respect to the amino acids after the mutation. For example, a mutation to a non-charged polar amino acid may be a mutation to another non-charged polar amino acid, and for example, a mutation to a non-polar amino acid may be a mutation to another non-polar amino acid. A conservative substitution means a group of acidic amino acids (aspartic acid (D) and glutamic acid (E)), a group of basic amino acids (arginine (R), lysine (K), and histidine (H)), a group of non-charged polar amino acids (asparagine (N), glutamine (Q), serine (S), threonine (T), and tyrosine (Y)), and Group of non-polar amino acids (alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W), and cysteine (C)) Amino acid substitutions within each of the groups, where non-conservative substitutions mean amino acid substitutions that are not conservative substitutions.

[0037] In certain embodiments, the virus or viral vector may further comprise a gene of interest (GOI). The loading position of the GOI on the RNA genome is before the N gene (hereinafter referred to as “+”), between the P gene and the M gene (hereinafter referred to as “PM”), between the M gene and the HN gene (hereinafter referred to as “MHN”), and between the HN gene and the L gene (hereinafter referred to as “HNL”), and it is known that the gene expression level decreases in this order of loading positions.

[0038] In one aspect, the virus or viral vector may include a genome encoding a nucleoprotein (N protein), a phosphoprotein (P protein), a matrix protein (M protein), a fusion protein (F protein), a hemagglutinin-neuraminidase (HN protein), and a large protein (L protein). In one aspect, it may include a genome that does not include a gene encoding a functional fusion protein (F protein) (preferably having a deletion of the gene), and includes genes encoding a nucleoprotein (N protein), a phosphoprotein (P protein), a matrix protein (M protein), a hemagglutinin-neuraminidase (HN protein), and a large protein (L protein). In all aspects of the present invention, the gene encoding the P protein on the genome and, optionally, the gene encoding the L protein preferably each have the temperature-sensitive mutation. By doing so, the virus or viral vector that has proliferated in cells after infection can exhibit the effect of temperature sensitivity. In all aspects of the present invention, on the virus or viral vector particles, the P protein and the L protein may or may not have the temperature-sensitive mutation. The virus or viral vector may be pseudotyped. A pseudotyped virus or viral vector refers to one in which the envelope protein has been altered, for example, the envelope protein may be altered to one derived from another virus. This makes it possible to change the cell tropism of the virus. In a preferred aspect, the genome of the virus or viral vector has the P protein and / or L protein having the temperature-sensitive mutation, and the virus particle itself of the virus or viral vector may include the wild-type P protein and / or the wild-type L protein, and further may not include the temperature-sensitive P protein and the temperature-sensitive L protein.

[0039] In one aspect, the virus or viral vector has an RNP complex containing a genome and nucleoprotein (N protein), phosphoprotein (P protein), and large protein (L protein), and a virus particle containing matrix protein (M protein), fusion protein (F protein), and hemagglutinin-neuraminidase (HN protein), and the virus particle contains the complex.

[0040] In one aspect, the virus or viral vector may contain a gene of interest operably linked to a control sequence. The virus or viral vector can express the gene of interest operably linked to the control sequence in infected cells. The gene of interest is transcribed into RNA, and the RNA may exhibit biological activity, or is translated into a protein via the RNA, and the protein may exhibit biological activity. "Control sequence" refers to a sequence that drives a gene operably linked thereto and has the activity of transcribing RNA from the gene. The gene of interest can be exogenous to the virus or viral vector. The gene of interest can also be exogenous to the cells to be infected. Here, "exogenous" means not originally possessed by that species. The control sequence is, for example, a promoter. Promoters include, for example, class I promoters (which can be used for transcription of rRNA precursors), class II promoters (composed of a core promoter and an upstream promoter element and can be used for transcription of mRNA), and class III promoters (further classified into type I, type II, and type III).

[0041] The virus or viral vector can infect animal cells (especially mammalian cells, preferably human cells). The virus or viral vector can preferably amplify its genome in animal cells (especially mammalian cells, preferably human cells). The virus or viral vector can preferably exhibit temperature sensitivity at 37°C or higher, 37.5°C or higher, 38°C or higher, or 38.5°C or higher. Due to this temperature sensitivity, the virus or viral vector can be removed from the cells.

[0042] The above virus or viral vector can be produced by a conventional method for producing a virus or viral vector. In the production, a gene encoding any of the above P protein mutants can be used as the gene encoding the P protein. Also, in the production, a gene encoding any of the above L protein mutants can be used as the gene encoding the L protein. Further, the RNA genome of the above virus or viral vector can be obtained by transcription from a nucleic acid (preferably DNA, more preferably an expression plasmid containing the nucleic acid) encoding the RNA genome of the above virus or viral vector. The nucleic acid having a region encoding the RNA genome of the above virus or viral vector preferably further has a control sequence, and the above region is operably linked to the control region. The nucleic acid having a region encoding the RNA genome of the above virus or viral vector is preferably carried on an expression vector of the nucleic acid. The expression vector is not particularly limited, and examples thereof include plasmid vectors, viral vectors, cosmid vectors, artificial chromosomes, and the like. The expression vector may further contain an origin of self-replication (Ori), a positive selection marker gene (for example, a drug resistance gene, a gene encoding a fluorescent protein, etc.) in addition to the expression cassette of the RNA genome operably linked to the control sequence. It is known that the RNA genome of a virus or viral vector is produced in a packaging cell that supplies viral protein components and the like. The packaging cell supplies the components contained in the produced virus and viral vector particles. In the packaging cell, it is preferable that a large amount of virus and viral vector particles are produced. From this viewpoint, the protein supplied to the packaging cell preferably does not have the temperature sensitivity described in this specification, and for example, it is preferably a wild-type protein. In this way, the titer of the produced virus can be measured based on a conventional method. The titer is 1×10 5 CIU / mL or more, 2×10 5Above CIU / mL, 3×10 5 Above CIU / mL, 4×10 5 Above CIU / mL, 5×10 5 Above CIU / mL, 6×10 5 Above CIU / mL, 7×10 5 Above CIU / mL, 8×10 5 Above CIU / mL, 9×10 5 Above CIU / mL, 1×10 6 Above CIU / mL, 2×10 6 Above CIU / mL, 3×10 6 Above CIU / mL, 4×10 6 Above CIU / mL, 5×10 6 Above CIU / mL, 6×10 6 Above CIU / mL, 7×10 6 Above CIU / mL, 8×10 6 Above CIU / mL, 9×10 6 Above CIU / mL, 1×10 7 Above CIU / mL, 2×10 7 Above CIU / mL, 3×10 7 Above CIU / mL, 4×10 7 Above CIU / mL, or 5×10 7 It may be above CIU / mL.

[0043] <Method for culturing cells infected with the above virus or viral vector> According to the present invention, there are provided a method for infecting cells with a virus or viral vector, and a method for culturing cells infected with a virus or viral vector.

[0044] According to the present invention, the method for infecting cells with a virus or viral vector includes contacting the cells with the virus or viral vector of the present invention described above in an environment suitable for infection. By loading a selection marker gene (for example, a visualization marker gene such as a fluorescent protein and a drug resistance gene) onto the virus or viral vector, cells infected with the virus or viral vector can be selected using the expression of the selection marker gene as an index.

[0045] According to the present invention, there is provided a method for culturing cells infected with the virus or viral vector of the present invention. The method for culturing cells infected with the virus or viral vector of the present invention includes culturing the cells infected with the virus or viral vector of the present invention in an environment suitable for the maintenance or proliferation of the cells. Those skilled in the art can appropriately infect cells with the virus or viral vector of the present invention by conventional methods. According to the present invention, cells infected with the virus or viral vector of the present invention can also be provided.

[0046] In one aspect, the method for culturing cells infected with a virus or viral vector includes culturing the cells under a first temperature condition. In one aspect, the method for culturing cells infected with a virus or viral vector may include culturing the cells under a first temperature condition and then culturing the cells under a second temperature condition.

[0047] The first temperature condition is a condition suitable for the maintenance or proliferation of the virus or viral vector that has infected the cells.

[0048] Also, the second temperature condition is a condition suitable for the decrease or disappearance of the amount of the virus or viral vector.

[0049] Also, the first temperature is lower than the second temperature.

[0050] When maintaining the virus or viral vector of the present invention in cells, for example, cells infected with the virus or viral vector of the present invention can be cultured under first temperature conditions. Also, when reducing or eliminating the virus or viral vector of the present invention, for example, cells infected with the virus or viral vector of the present invention can be cultured under second temperature conditions. The virus or viral vector of the present invention can be maintained or propagated in infected cells, but by raising the culture temperature, maintenance or propagation can be inhibited, and the virus or viral vector can be removed from the infected cells. Those skilled in the art will be able to appropriately determine the first temperature and the second temperature.

[0051] The method of culturing cells infected with a virus or viral vector may include culturing the cells under conditions suitable for the maintenance or propagation of the virus or viral vector. The conditions suitable for maintenance or propagation preferably include temperature conditions under which the virus or viral vector is not affected by temperature sensitivity due to mutations in the P protein or L protein. For example, when it is necessary to maintain or propagate the virus or viral vector of the present invention in cells (for example, when highly expressing a target gene carried by the virus or viral vector), the infected cells can be cultured under conditions suitable for the maintenance or propagation of the virus or viral vector of the present invention. Temperature conditions under which the virus or viral vector is not affected by temperature sensitivity due to mutations in the P protein or L protein can be appropriately determined by those skilled in the art. For example, if it is not affected by temperature sensitivity at 37°C, the infected cells can be cultured at 37°C. For example, when affected by temperature sensitivity at 37°C, the infected cells can be cultured under lower temperature conditions, for example, temperatures of 35°C or higher and lower than 37°C. However, the conditions suitable for maintaining or increasing the virus or viral vector of the present invention are those that do not significantly adversely affect the maintenance and growth of the cells.

[0052] A method of culturing cells infected with a virus or viral vector may include culturing the cells under conditions suitable for reducing the amount of the virus or viral vector. In one aspect, a method of culturing cells infected with a virus or viral vector may include culturing the cells under conditions suitable for maintaining or growing the virus or viral vector, and then culturing the cells under conditions suitable for reducing the amount of the virus or viral vector. Conditions suitable for reducing the amount of the virus or viral vector preferably include temperature conditions under which the virus or viral vector is affected by temperature sensitivity due to a mutation in the P protein or L protein. For example, when it is necessary to reduce the virus or viral vector of the present invention in cells (for example, when removing the virus or viral vector from the cells after expressing the target gene carried by the virus or viral vector), the infected cells can be cultured under conditions suitable for reducing the virus or viral vector of the present invention. The temperature conditions under which the virus or viral vector is affected by temperature sensitivity due to a mutation in the P protein or L protein can be appropriately determined by those skilled in the art. For example, if it is not affected by temperature sensitivity at 37°C, the infected cells can be cultured at a temperature above 37°C (for example, a temperature above 37°C and not exceeding 39°C, for example, 37.5°C to 38.5°C). For example, when it is affected by temperature sensitivity at 37°C, the infected cells can be cultured at a temperature of 37°C or higher. In this way, when culturing the infected cells at a temperature (the first temperature) that is not affected by temperature sensitivity or is less likely to be affected, and it is desired to remove the virus or viral vector from the infected cells, the culture temperature can be raised to the second temperature. However, the conditions suitable for reducing the virus or viral vector of the present invention do not cause significant adverse effects (especially irreversible disorders or damages, etc.) on the maintenance and growth of the cells. By doing so, the virus or viral vector can be removed from the cells infected with the virus or viral vector. Therefore, in one aspect, a method of obtaining virus-free or viral vector-free cells from cells infected with the virus or viral vector of the present invention is provided.Cells that do not have a virus or virus vector can be tested, for example, by detecting no viral or virus vector genome within the cells. Detection of the genome can be appropriately determined by those skilled in the art using well-known techniques such as polymerase chain reaction (PCR). According to the method for culturing the above cells, transient gene introduction and gene expression can be achieved.

[0053] According to the present invention, culturing of cells containing or infected with the virus or virus vector of the present invention may be carried out at a second temperature from the beginning. That is, it takes time until the virus or virus vector of the present invention is removed from the infected cells. Therefore, until it is removed, genes and the like carried by the virus or virus vector within the cells are expressed, and the intended purpose can also be achieved. Therefore, it may be possible to culture at the second temperature without culturing at the first temperature.

[0054] The first temperature and the second temperature only need to be included in a certain temperature range, but preferably can be temperatures at a constant temperature (± error range). The certain temperature range is, for the first temperature, a temperature range (higher temperature region) suitable for maintaining or growing the virus or virus vector, and for the second temperature, a temperature range (lower temperature region) suitable for removing (decreasing) the virus or virus vector. For the purpose of further maintaining or culturing the cells after removal of the virus or virus vector, neither temperature range should cause significant harm to the survival of the cells nor cause significant damage to the cells. Preferably, neither temperature range should have a substantial impact on the survival of the cells.

[0055] According to the present invention, a method for inducing cell differentiation, infecting the cells with the virus or virus vector of the present invention, then culturing the infected cells to induce cell differentiation, After induction of differentiation, raise the culture temperature to remove the virus or viral vector of the present invention from the differentiated cells, There is provided a method comprising the virus or viral vector, which is capable of expressing a differentiation-inducing factor. Induction of differentiation means differentiating cells into other stable cell states. Therefore, the method of inducing differentiation of cells is synonymous with the method of producing differentiated cells. As a method of differentiating a certain cell into another stable cell state, various known methods can be used. The virus or viral vector of the present invention can carry a differentiation-inducing factor that promotes differentiation induction. Cells can be induced to differentiate into somatic cells such as, for example, nerve cells, cardiomyocytes, chondrocytes, osteocytes, blood cell lineage cells (such as white blood cells), hepatocytes, and pancreatic β cells. Induction of differentiation into these somatic cells can be carried out by methods known to those skilled in the art.

[0056] According to the present invention, there is provided a method of reprogramming (nuclear reprogramming) cells, infecting the cells with the virus or viral vector of the present invention, then culturing the infected cells to reprogram the cells, after induction of differentiation, raising the culture temperature to remove the virus or viral vector of the present invention from the reprogrammed cells, There is provided a method comprising the virus or viral vector, which is capable of expressing a reprogramming factor. The reprogramming factor is not particularly limited, and examples thereof include Yamanaka factors (OCT4, SOX2, KLF4, and optionally c-MYC), Thomson factors (OCT4, SOX2, NANOG, LIN-28), and other reprogramming factors. The reprogramming factor can also be, for example, KLF4, OCT4, SOX2, and MYCL.

[0057] Without being limited to the above, the present invention can be used in various applications where it is desired to transiently express proteins or RNAs in cells and then remove the vector. The protein components and, if necessary, the RNA components for genome editing systems (e.g., zinc finger nucleases, TALENs, and CRISPR / Cas systems (e.g., CRISPR / Cas9 system)) can be expressed intracellularly, and if necessary, RNA-protein complexes (RNP complexes) can be formed to edit nucleic acids (e.g., DNA) such as the genome of cells and then remove the vector after editing.

[0058] <Removal of Viruses or Viral Vectors from Infected Cells Using Degrons> In the present invention, degrons can also be used to remove viruses or viral vectors that have infected cells from the infected cells. The degrons may be used in combination with the removal of viruses or viral vectors that utilize the temperature-sensitive mutants and temperature changes described above. As degrons, for example, various degrons such as degrons derived from FK506-binding protein (FKBP12) and degrons derived from dihydrofolate reductase (DHFR) can be used (see US2009 / 0215169A). The destabilizing domains (DDs) of these proteins are degradation-inducing and can be used to induce the degradation of the target protein by fusing with other proteins (target proteins). The DD tag and its variants having degradation-inducing properties can be added, for example, to the N-terminus or C-terminus of the protein.

[0059] In the present invention, the FKBP12-derived DD may have an amino acid mutation corresponding to any amino acid mutation selected from the group consisting of F15S, V24A, H25R, E60G, L106P, D100G, M66T, R71G, D100N, E102G, and K105I. The FKBP12-derived DD preferably has a mutation in the amino acid corresponding to F36, particularly F36V. Further, the FKBP12-derived DD preferably has L106P in particular. Moreover, the FKBP12-derived DD preferably has F36V and L106P. In the present invention, preferably, it has one or more mutations selected from the group consisting of 4I, 18K, 36L, 50I, 57K, 59F, 63A, 68L, 90V, 98I, and 105R, and more preferably has all the mutations.

[0060] In the present invention, there are provided an N protein, a P protein, an M protein, an F protein, an HN protein, and an L protein fused with the above DD, and a gene encoding any of them. In the present invention, there are provided a cell or a Sendai virus or a Sendai virus vector or a Sendai virus genome containing one or more selected from the group consisting of an N protein, a P protein, an M protein, an F protein, an HN protein, and an L protein fused with the above DD, and / or one or more of the genes encoding them. These fusion proteins may contain variants of each protein described above. The variants are as described in this specification.

[0061] In the present invention, in particular, there are provided a P protein fused with the above DD and a gene encoding the P protein. In the present invention, there are provided a cell or a Sendai virus or a Sendai virus vector or a Sendai virus genome containing a P protein fused with the above DD and / or a gene encoding the P protein. Here, instead of the P protein, a variant of the P protein described in this specification may be used.

[0062] When the P protein fused with the above DD is essential for the intracellular growth of a negative-strand RNA virus or vector, the negative-strand RNA virus or vector can be removed from the cell in the presence of a degradation-promoting molecule such as dTAG-13 or its derivative. When it is desired to maintain the negative-strand RNA virus or vector in the cell, the cell can be cultured in the presence of a stabilizing molecule such as Shield1 or its derivative. Thus, in the present invention, the removal (and preferably maintenance) of a virus or viral vector in a cell can be controlled using a degradation-promoting molecule and preferably further using a stabilizing molecule. dTAG-13 is a PROTAC (abbreviation for Proteolysis Targeting Chimera) having a site that binds to the above DD or its mutant and a site that binds to a ubiquitin E3 ligase, and induces the degradation of the fusion protein of the above DD or its mutant.

[0063]

Chemical formula

[0064]

Chemical formula

[0065] <Packaging cells and method for producing the same> In the present invention, a negative-strand RNA virus or vector such as SeV can be produced using an existing packaging cell. However, in the present invention, a new packaging cell can be produced, and a virus or vector may be produced using the obtained packaging cell.

[0066] In one aspect, the packaging cell is a cell line, preferably a stably established cell line. The packaging cell may be any type of cell that can be infected by a negative-strand RNA virus or vector, and is not particularly limited, and can be, for example, LLC-MK2 cells and Vero cells. The packaging cell typically expresses one or more components of a virus, such as the F protein. The packaging cell may additionally carry a PKR inhibitory factor (such as VAI, etc.).

[0067] The packaging cell has a gene encoding one or more components of a virus such as the F protein, and preferably exhibits expression at a certain level or higher for the gene. The gene is operably linked to a control sequence, and its expression is driven by the control sequence. The expression level is determined by various factors such as the control sequence, the position on the genome where the gene is integrated, and the copy number of the gene inserted into the genome. In order to ensure a high expression level, a marker gene (for example, a drug resistance gene and a visualization marker gene (for example, a fluorescent protein)) linked under the same promoter can be utilized during gene introduction. That is, first, for obtaining a high-expression strain, a control sequence has a marker gene operably linked via a first site-specific recombination sequence, and further, via a second site-specific recombination sequence, a gene encoding one or more components of a virus such as the F protein is linked. In this configuration, the marker gene will be present between the first site-specific recombination sequence and the second site-specific recombination sequence (referred to as the intervening sequence). The first site-specific recombination sequence and the second site-specific recombination sequence undergo recombination in the presence of a recombinase, and the sequence (intervening sequence) between them can be removed. After removing the intervening sequence by the recombinase, it is designed such that a gene encoding one or more components of a virus such as the F protein is driven by the above control sequence. Furthermore, a marker gene is present in the intervening sequence, and based on the expression intensity of the marker gene, cells in which the marker gene is strongly expressed can be selected. When the marker gene is a drug resistance gene, by selecting cells with a certain concentration of the drug, cells showing expression of the drug resistance gene at a certain level or higher are selected. When the marker gene is a visualization marker, by measuring the expression intensity of the visualization marker (for example, measuring the fluorescence intensity of GFP if it is GFP), cells showing expression of the visualization marker at a certain level or higher are selected. In this way, cells with a high expression intensity of the marker gene are selected. In the selected cells, it is suggested that the above gene is highly expressed due to various factors such as the position on the genome where the gene is integrated and the copy number of the gene inserted into the genome.Clone the selected cells and allow a site-specific recombinase that recognizes the site-specific recombination sequence to act on the resulting clones to induce recombination. Then, the intervening sequence is removed, and a gene encoding one or more components of a virus, such as the F protein, is operably linked to the control sequence. In this way, packaging cells can be obtained. The site-specific recombinase can be introduced into cells, for example, by a removable negative-strand RNA virus vector, such as a Sendai virus vector (e.g., temperature-sensitive SeV). After introducing the site-specific recombinase, the vector can be removed to below the detection limit so that the cells can be preferably used as packaging cells. After removal of the intervening sequence, one site-specific recombination sequence may remain between the control sequence and the introduced gene.

[0068] Site-specific recombinases and their recognition sequences are not particularly limited. For example, a system of Cre recombinase (hereinafter sometimes simply referred to as "Cre"), which is a site-specific recombinase, and loxP, its recognition sequence; a system of Flp recombinase (hereinafter sometimes simply referred to as "Flp"), which is a site-specific recombinase, and FRT, its recognition sequence; a system of Dre recombinase and rox, its recognition sequence (Anastassiadis, K. et al., Dis. Model. Mech. (2009) 2: 508-515); and a site-specific recombination reaction system such as a system of φC31 recombinase and attP / attB, its recognition sequence (Belteki G et al., Nat. Biotechnol. (2003) 21: 321-324) can be used. Modified systems such as the SCre-SloxP system and the VCre-VloxP system (E. Suzuki and M. Nakayama, Nucleic Acid Res. (2011) 39 (8): e49) that improve the Cre-loxP system can also be used. In addition, mutant FRT sequences such as the FRT sequence described in International Publication No. 2001 / 023545 (for example, FRT(f2161) and FRT(f2262)), and sequences of mutant loxP sequences such as the loxP sequence described in Japanese Patent Application Laid-Open No. 11-196880 can be used. Such recognition sequences of site-specific recombinases can be freely selected and used by those skilled in the art.

[0069] In the present invention, a genome of a negative-strand RNA virus or a vector containing a nucleic acid encoding any of the above proteins is provided. In the present invention, a nucleic acid encoding the genome and a vector containing the nucleic acid are provided. The vector can be a cloning vector and an expression vector for the genome. In a preferred embodiment, the expression vector can transcribe the genome in packaging cells.

Examples

[0070] Example 1: Preparation of Minus-Strand RNA Virus Vector A minus-strand RNA virus vector was expressed in virus packaging cells. In particular, the temperature sensitivity of introducing mutations into the P protein during expression was evaluated.

[0071] As the minus-strand RNA virus vector, Sendai virus (SeV) was used. Sendai virus was obtained by introducing a plasmid mixture into F gene-expressing cells and collecting it from the culture supernatant after 3 days. The plasmid mixture contained pSeV-EmGFP carrying a Sendai virus genome operably linked to a T7 promoter. The Sendai virus genome carried a gene encoding EmGFP so that the growth of the genome could be detected by fluorescence. The Sendai virus genome had a deletion in the F gene.

[0072] (1) F gene-expressing cells were prepared as follows. The F gene of SeV (with Kozak sequence added and optimized for human codons) was inserted into pCAGGS-neo to construct pCAGGS-F-neo. The resulting plasmid was transfected into Vero cells or LLC-MK2 cells using ViaFect (Promega) and selected using a 1-2 mg / mL G418 disulfate solution (Nacalai Tesque) to obtain F gene-expressing cells. The obtained cells were designated as Vero-F and LLC-MK2-F.

[0073] (2) F gene-deleted SeV was prepared as follows. Based on the sequence information of the SeV-Z strain of ACCESSION: AB855655, pSeV / dF in which the F gene-deleted SeV genome is transcribed by the T7 promoter was constructed. In order to minimize the cytotoxicity of the SeV-Z strain, the following amino acid mutations were added to pSeV / dF: M protein: 69E, 116A, and 183S, HN protein: 262T, 264R, and 461E, and L protein: 1197S and 1796E (see, for example, WO2003 / 025570). To evaluate the temperature sensitivity and removal of SeV, EmGFP, a mutant of GFP, was incorporated into the genome as the gene of interest (GOI). The gene expression levels decreased in the order of the positions where the GOI was incorporated: in front of the N gene (hereinafter referred to as "+"), between the P gene and the M gene (hereinafter referred to as "PM"), between the M gene and the HN gene (hereinafter referred to as "MHN"), and between the HN gene and the L gene (hereinafter referred to as "HNL"). The position where the EmGFP gene was incorporated mainly used the + position in front of the N gene of SeV. In this example, on the SeV genome, mutations by amino acid substitution were introduced into any one or more of D433, R434, K437, and L511 of the P protein having the amino acid sequence of SEQ ID NO: 1. In the packaging cells, either a temperature-sensitive P protein or a non-temperature-sensitive P protein (for example, wild type) can be used, but the production efficiency of the virus was higher when a non-temperature-sensitive P protein (for example, wild type) was used. Therefore, in this example, the genome carried a gene encoding a temperature-sensitive mutant P protein, but the packaging cells expressed the wild-type P protein and the wild-type P protein was introduced into the virus particles.

[0074] (3) The plasmid for SeV reconstruction was prepared as follows. With reference to WO2005 / 071092, Kozak sequence addition and optimization for human codons were performed to construct pCAGGS-NPco, pCAGGS-P4C(-)co, pEF1-Lco, and pCAGGS-F5Rco. For T7, mutations 430P, 849I, and 880Y were introduced with reference to P2001-54387A, and mutations 644Y and 667Y were introduced with reference to P2003-61683A. pCAGGS-T7mco carrying the obtained T7m sequence was constructed. P4C(-) is a mutation that abolishes the expression of the C protein from the P locus.

[0075] The types of mutations to the P protein and the mutant names are as follows. P2: 433A, 434A, 437A, 511F (SEQ ID NO: 2) P2Y: 433A, 434A, 437A, 511Y (SEQ ID NO: 8) P2m3SY: 433S, 434S, 437S, 511Y (SEQ ID NO: 9) P2m3TY: 433T, 434T, 437T, 511Y (SEQ ID NO: 10) P2m3GY: 433G, 434G, 437G, 511Y (SEQ ID NO: 11) P2m3SF: 433S, 434S, 437S, 511F (SEQ ID NO: 12) P2m433S: 433S, 434A, 437A, 511F (SEQ ID NO: 13) P2m433SY: 433S, 434A, 437A, 511Y (SEQ ID NO: 14) P2m434SY: 433A, 434S, 437A, 511Y (SEQ ID NO: 15) P2m437SY: 433A, 434A, 437S, 511Y (SEQ ID NO: 16)

[0076] The ratio of the plasmid for SeV reconstruction and the transfection reagent followed WO2005 / 071092. Specifically, the following weights of plasmid and transfection reagent (TransIT-LT1 Reagent or ViaFect) were mixed to obtain a plasmid mix.

[0077] NP, P4C(-), F5R, T7: 0.5 μg each L: 2 μg pSeV: 5 μg Total plasmid: 9 μg TransIT-LT1 Reagent or ViaFect: 15 μL

[0078] Hereinafter, unless otherwise specified, the volume of the transfection reagent and the weight of the plasmid were mixed so that the ratio was 9:15.

[0079] The plasmid and the transfection reagent were mixed in 225 μL of OptiMEM and transfected into F gene-expressing cells in a 12-well plate cultured in 500 μL of medium (10% FBS / E-MEM), and then cultured at 37°C. From the day after transfection, the cells were cultured at 32°C. The medium was changed daily with serum-free medium (ITS-X / NEAA / E-MEM) supplemented with 2.5 μg / mL trypsin. The culture supernatant on the third day after transfection was collected and used to infect F gene-expressing cells seeded in a culture flask to amplify SeV. The culture supernatant from the third to fifth days in the flask was collected, diluted 10-fold to 100,000-fold, and used to infect Vero cells seeded in a 96-well plate. The number of GFP-positive cells 3 days after infection was measured using a fluorescence microscope system ECLIPSE Ti2-E (Nikon), and the infectious titer was calculated.

[0080] The obtained SeV mutant was used to infect Vero cells (MOI = 20). The infected cells were cultured for 3 days under temperature conditions of 35°C or 38.5°C. Since the SeV vector expresses EmGFP intracellularly and the infected cells emit fluorescence according to the amount of SeV intracellularly, the amount of SeV in the infected cells was estimated by measuring the fluorescence from the cells. The results were as shown in Figures 1A and 1B. As shown in Figure 1A, the P2m3SY mutant showed strong fluorescence at 35°C, but the fluorescence decreased strongly at 38.5°C. Figure 1B shows the numerical values of the fluorescence amount. For the P2m3SY mutant, the fluorescence amount decreased to about 0.013% at 38.5°C compared to 35°C. For the P2 mutant, the fluorescence amount decreased to about 5.7% at 38.5°C compared to 35°C. From this, it was shown that the P2m3SY mutant has a temperature sensitivity more than 400 times higher than that of the P2 mutant.

[0081] (4) Introduction of Further Temperature-Sensitive Mutations Next, the obtained SeV mutant was used to infect Vero cells (MOI = 20). The infected cells were cultured for 3 days under temperature conditions of 35°C or 38.5°C, and the amount of SeV in the infected cells was estimated by measuring the fluorescence from the cells in the same manner as above. Taking the fluorescence amount from the culture under the 35°C condition of P2 as 1, the relative values were used to obtain each fluorescence amount. The results were as shown in Figure 2 and Table 1. As shown in Figure 2, each mutant showed a little temperature sensitivity at 37°C, but when cultured under the temperature condition of 38.5°C, the fluorescence amount decreased significantly. Comparing the relative fluorescence amounts in Table 1, it became clear that all mutants had improved temperature sensitivity compared to P2. Among the mutants, P2m433S had the highest temperature sensitivity.

[0082]

Table 1

[0083] The obtained SeV mutants were infected into Vero cells (MOI = 10). Then, they were cultured under temperature conditions of 32°C, 35°C, 37°C, or 38.5°C. The SeV vector expresses EmGFP intracellularly, and the infected cells emit fluorescence according to the amount of intracellular SeV. Each mutant was cultured under each temperature condition, and the amount of fluorescence from the cells was measured 3 days later. The results were as shown in FIG. 3 and Table 2. It was expressed by the relative fluorescence intensity when P2Y was set to 100%. As shown in FIG. 3, each mutant showed temperature sensitivity even at 37°C, but when cultured under the temperature condition of 38.5°C, the amount of fluorescence decreased significantly. When comparing the relative amounts of fluorescence in Table 2, it became clear that the temperature sensitivity of each mutant was improved compared to P2.

[0084]

Table 2

[0085] For the above mutants, mutations to the L protein having the amino acid sequence set forth in SEQ ID NO: 5 were further added on the viral genome. The mutations were introduced into N1197, L1361, L1558, and K1796 of the L protein. The specific mutations are as follows. LmCI :N1197S, L1361C, L1558I, K1796E (SEQ ID NO: 17)

[0086] Sendai virus (SeV) obtained from packaging cells was used to infect Vero cells (MOI = 20). Subsequently, the cells were cultured under temperature conditions of 35°C, 37°C, or 38.5°C. The SeV vector expresses EmGFP intracellularly, and infected cells emit fluorescence corresponding to the amount of intracellular SeV. Each mutant was cultured under each temperature condition, and the amount of fluorescence from the cells was measured 3 days later. The results were as shown in Figure 4 and Table 3. The results were expressed as the relative fluorescence intensity when P2+LmCI was set to 100%. As a result, for all of P2m3SY+LmCI, P2m3TY+LmCI, and P2m3GY+LmCI, the amount of fluorescence decreased after culturing at 37°C, and no fluorescence was detected after culturing at 38.5°C. P2m3TY+LmCI and P2m3GY+LmCI also showed strong temperature sensitivity even when cultured under temperature conditions of 35°C and 37°C. In addition, when a mutant having the amino acid sequence set forth in SEQ ID NO: 7 was used as the L protein, temperature sensitivity was shown, but the mutant having the amino acid sequence set forth in SEQ ID NO: 17 had higher temperature sensitivity. From this, by introducing either or both of the mutation at L1361 and the mutation at L1558, the L protein can confer temperature sensitivity to SeV.

[0087]

Table 3

[0088] Similarly, various mutants having LmCI were obtained from packaging cells and used to infect Vero cells at 35°C (MOI = 20). The cells were cultured for 3 days under the temperature condition of 35°C, passaged, and then further cultured for 7 or 14 days under temperature conditions of 35°C, 37°C, or 38.5°C. The fluorescence intensity from the cells was measured. The results were as shown in Figure 5 and Table 4. As shown in Figure 5A and Table 4, P2m3SY+LmCI showed a certain degree of temperature sensitivity even under the temperature condition of 37°C, but showed strong temperature sensitivity under the temperature condition of 38.5°C.

[0089]

Table 4

[0090] In addition, various mutants having Lm1558I were obtained from packaging cells and used to infect Vero cells at 35°C (MOI = 20). The cells were cultured for 3 days under the temperature condition of 35°C, passaged, and then further cultured for 7 or 14 days under the temperature conditions of 35°C, 37°C, or 38.5°C. The fluorescence intensity from the cells was measured. The results were as shown in FIG. 5B and Table 4-1. As shown in FIG. 5B and Table 4-1, P2m3SY+Lm1558I showed a certain degree of temperature sensitivity even under the temperature condition of 37°C, but showed strong temperature sensitivity under the temperature condition of 38.5°C.

[0091]

Table 4-1

[0092] (5) Introduction of the target gene into cells by SeV Human fibroblasts were infected with SeV carrying a growth factor, and the cell number was counted. As SeV, P2m3SY was used, and cyclin D1 and CDK4 (R24C) were carried as growth factors in the genome. A growth factor-carrying SeV vector as a test vector and a SeV vector carrying EmGFP as a negative control were used to infect human fibroblasts (MOI = 20), passaged every 7 days, and the cell number was counted. The results were as shown in FIG. 6. In the growth factor-carrying SeV vector, the cell number increased to 156 times the initial number. In contrast, in the negative control, the cell number remained at 9 times the initial number. From this, it was found that the temperature-sensitive vector of the present invention is suitable for introducing a foreign gene into infected cells and expressing it in the infected cells. In addition, after culturing, by raising the temperature, the SeV vector can be removed from the cells, and the amount of intracellular vector becomes below the detection limit. As a result, it was clarified that a removable SeV vector system could be constructed after gene introduction.

[0093] Furthermore, in this example, a combination of CyclinD1, CDK4 (R24C), and TERT (hereinafter referred to as "CCT") was introduced into human umbilical cord matrix-derived mesenchymal stem cells (MSCs) and human neonatal foreskin-derived fibroblasts (NHDF cells) using the above temperature-sensitive vector (SeV / TSdF-P2m434SY) to confirm its effect on cell proliferation. The Sendai virus genome carried CCT at the PM site and mEmerald at the MHN site. The cells were cultured in DMEM / F12 medium containing 10% FBS on a collagen plate. They were passaged once every 7 days, and the cell count was measured each time they were passaged. When passaging, a certain number of cells were collected and seeded into a certain amount of fresh medium. The cell count was measured 42 days later, and the growth rate was calculated. The results were as shown in Table 5. As shown in Table 5, while the control MSCs grew 137-fold, the MSCs into which CCT was introduced using the SeV of the present invention grew up to 13.2 million-fold. Also, while the control NHDF grew 12.1-fold in 42 days, the NHDF into which CCT was introduced using the SeV of the present invention grew up to 1.47 million-fold.

[0094]

Table 5

[0095] Furthermore, in this example, human fibroblasts were infected with a Sendai virus vector carrying reprogramming factors (MOI = 20), and the cells after infection were observed by immunocytochemical staining using an anti-NANOG antibody. As the reprogramming factors, KLF4, OCT4, SOX2, and MYCL were used. The vectors were those having P2m3SY and LmCI mutations, and a vector co-loaded with KLF4, OCT4, and SOX2 (KOS) and a vector loaded with MYCL were prepared. Human fibroblasts were infected with the two types of vectors, passaged 5 days later, and the medium was changed to StemFit medium 6 days later. The expression of NANOG in the cells was observed using an anti-NANOG antibody 11 days later. Then, as shown in Fig. 7A, the human fibroblasts treated with the Sendai virus vector were reprogrammed and the expression of NANOG was induced. After the reprogramming of the cells, when the temperature of the medium was raised to 38.5 °C, the Sendai virus vector was removed from the cells, and the amount of intracellular vector became below the detection limit. However, when the fibroblasts into which the reprogramming factors were introduced were cultured in StemFit AK92N medium on a plate coated with iMatrix-511, the SeV vector was gradually removed from the cells even under the temperature condition of 37 °C, and the fluorescence of EmGFP expressed from SeV became below the detection limit on the 28th day (see Fig. 7B).

[0096] (6) Promotion of further removal of SeV by degron By adding a proteolytic tag (degron) to the P protein, the removal of SeV can be promoted. The DD tag is a type of proteolytic tag (see, for example, US2009 / 0215169A). The DD tag (see SEQ ID NO: 18) is a 107 - amino - acid - long protein obtained by introducing F36V and L106P into FKBP12. When the small - molecule compound Shield1 is added, it is stabilized, and when dTAG - 13 is added, the degradation of the protein fused to the tag is also promoted. In this example, a DD mutant (DDm; see SEQ ID NO: 19) having 11 mutations (4I, 18K, 36L, 50I, 57K, 59F, 63A, 68L, 90V, 98I, and 105R) with respect to the DD tag was prepared. In this example, in order to examine the protein - degradation - promoting effect of DDm, the DD tag and its mutants (including DDm) were added to the C - terminus of EmGFP and mounted at the + position of the SeV vector (having the mutations of the above - mentioned WO2003 / 025570). The obtained SeV was infected into HeLa cells (MOI = 10), and on the day after infection, 1 μM of Shield1 or 5 μM of dTAG - 13 was added, and the fluorescence from the cells was observed 3 days after infection. The results were as shown in Tables 6 and 7. In addition, when a DDrs tag having the amino - acid sequence described in SEQ ID NO: 28 was prepared and added to the C - terminus of EmGFP, the stabilizing effect of EmGFP by Sheild1 was not obtained, and it showed only about 20% of the fluorescence intensity of DD - tagged EmGFP (Figure 8).

[0097]

Table 6

[0098]

Table 7

[0099] As shown in Tables 6 and 7, DDm had improved stability against Shield1 and an improved degradation - promoting effect by dTAG - 13. As a result, the Shield1 / dTag - 13 ratio was significantly improved.

[0100] Next, a DDm tag was added to the C-terminus of various temperature-sensitive mutants of the P protein. EmGFP was mounted at the + position of the SeV genome. HeLa cells were infected with SeV expressing various temperature-sensitive mutants to which the DDm tag was added (MOI = 10), and the fluorescence intensity derived from EmGFP was confirmed 3 days later. The results were as shown in Fig. 9 and Table 8.

[0101]

Table 8

[0102] As shown in Fig. 9A and Table 8, P2m3TYddm and P2m2S434TYddm to which DDm was added improved the removal efficiency of SeV and promoted the removal of SeV from cells even under the temperature condition of 37.5°C. This result was obtained in the absence of dTAG-13, suggesting that the addition of the DDm tag destabilizes the P protein even in the absence of dTAG-13. As shown in Fig. 9B and Table 9, the destabilization of the P protein to which the DDm tag was added was further promoted in the presence of dTAG-13.

[0103]

Table 9

[0104] (7) Reduction of the cytotoxicity of SeV SeV induces cytotoxicity. For the purpose of reducing this cytotoxicity, amino acid mutations can be introduced into the M protein and the HN protein. As described above, the SeV of this example has basically already introduced such mutations (WO2003 / 025570). In this example, the amino acid mutations of each of the M protein and the HN protein were further investigated. Specifically, in addition to G69E and T116A, A183T was introduced into the M protein (see SEQ ID NO: 20). Also, in addition to A262T and K461E, G264K was introduced into the HN protein (see SEQ ID NO: 21). L511Y was introduced into the P protein, and N1197S and K1796E were introduced into the L protein. Then, SeV having EmGFP at the + position was constructed, and Vero cells were infected, and the infected cells were cultured at 32°C for 20 days. The fluorescence intensity derived from EmGFP in Vero cells was measured.

[0105] The results were as shown in FIG. 10. As shown in FIG. 10, the M protein having A183T increased the fluorescence intensity 2.6-fold compared to the control, and the HN protein having G264K increased the fluorescence intensity 3.9-fold. The detachment from the dish of EmGFP-positive cells observed in the control was reduced in the SeV-infected cells having the above mutants. Therefore, it was suggested that the above mutations reduce the cytotoxicity of SeV. When an initialization factor was loaded onto the SeV vector having the mutant of the M protein and the fibroblasts were infected, reprogramming of the fibroblasts could be induced.

[0106] (8) Preparation of packaging cells Packaging cells for negative-strand RNA viruses or viral vectors were established from Vero cells and LLC-MK2 cells. Specifically, a gene encoding F and a gene encoding a PKR inhibitor were incorporated into a vector and introduced into packaging cells to obtain a packaging cell expressing the gene encoding F and the gene encoding the PKR inhibitor as a stable strain. VAI was used as the PKR inhibitor. The insertion position of VAI is not particularly limited, but in this example, a sequence encoding VAI was introduced downstream of the gene encoding F (see Fig. 11A). These incorporated genes can be driven by any promoter, but in this example, they were driven by the CAG promoter (see Fig. 11A). However, between the CAG promoter and the gene encoding F, a region flanked by two site-specific recombination sequences (here loxP) was interposed, and the vector was constructed such that the gene encoding F could be strongly driven by the CAG promoter by removing the region flanked by loxP with Cre recombinase or the like. Between loxP, a gene encoding a fusion protein formed by linking iCaspase9 and NeoR (neomycin resistance gene) with a 2A sequence was incorporated. This made it possible to select clones with a high expression level of NeoR (including clones with a large number of introduced copies) to the extent that they could survive in the presence of a drug such as a certain concentration of G418. Based on such a design concept, the pCALiNdLv-F vector was constructed from the pCALNdL vector (JOURNAL OF VIROLOGY, Feb. 1998, p. 1115-1121) (Fig. 11A). SeV-Cre carried a gene encoding Cre recombinase at the + position and a fluorescent protein mEmerald at the MHN position. Thereby, the growth of the virus can be confirmed by the tendency of mEmerald. SeV-Cre can also have a genome that exhibits temperature sensitivity and is removed from cells in a temperature-dependent manner.

[0107] After introducing the pCALiNdLv-F vector into LLC-MK2 cells and Vero cells, drug selection with G418 was performed at 37°C. SeV-Cre was introduced into the obtained clones, and the cells were cultured at 32°C. As a result, as shown in Fig. 11B (1st), both LLC-MK2 cells and Vero cells were infected well. The supernatant from the 1st cells was brought into contact with the 2nd cells. If the supernatant contains a virus with infectivity, it should infect the 2nd cells and produce fluorescence derived from mEmerald. As a result of the infection experiment on the 2nd cells, the supernatant infected both LLC-MK2 cells and Vero cells, causing fluorescence derived from mEmerald in the cells (see 2nd in Fig. 11B). When the supernatant does not contain a virus with infectivity, as shown in the lower panel of 2nd for LLC-MK2 cells and the middle panel of 2nd for Vero cells, the 2nd cells are not infected with the virus, and fluorescence derived from mEmerald cannot be produced in the cells. In this example, since viruses with infectivity can be produced reproducibly in both LLC-MK2 cells and Vero cells, those skilled in the art can understand that viruses with infectivity can be produced as appropriate. More specifically, the above results indicate that high-expression clones of F and PKR inhibitors could be selected by drug selection with G418 in LLC-MK2 cells and Vero cells (1.3 - 2.5 mg / mL of G418 was used for LLC-MK2 cells and 0.9 - 1.5 mg / mL of G418 was used for Vero cells), and that the sequence between loxP was removed by the action of Cre recombinase carried by SeV, resulting in high expression of F. As a result, SeV production was observed in the 1st cells and the 2nd cells were also infected, indicating that a virus with infectivity could be produced in the 1st cells. Thus, it was suggested that the above plasmid is a useful tool for generating packaging cells from 1st cells (normal LLC-MK2 cells and Vero cells). In this system, it is considered that SeV-Cre remains in the 1st cells. Therefore, by removing SeV-Cre in the 1st cells thereafter, the obtained cells can be utilized as packaging cells for SeV reconstitution.The removal of SeV-Cre can be achieved by simply subculturing cells. However, various methods such as the use of temperature-sensitive strains and the utilization of microRNAs are known to enhance the removal efficiency, and it can be removed as appropriate. Subsequently, when the cells were recloned under temperature conditions of 37°C, F-expressing cells from which SeV had been removed were obtained. Due to the expression of F, LLC-MK2 cells and Vero cells could be used as packaging cells, and it can be understood that F may be supplied from the cell side, supplied by a plasmid, or supplied from the Sendai virus genome.

[0108] Sequence Listing: SEQ ID NO: 1: An example of the amino acid sequence of the wild-type P protein of Sendai virus MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITDRGGKTDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL LPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 Sequence number 2: Amino acid sequence of the Sendai virus P protein variant (P2) MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITAAGGATDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL FPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO:3: Amino acid sequence of Sendai virus P protein mutant (L511F) MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITDRGGKTDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL FPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO:4: Amino acid sequence of temperature-sensitive P protein mutant of Sendai virus MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITXXGGXTDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL XPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO:5: An example of the amino acid sequence of the wild-type L protein of Sendai virus MDGQESSQNP SDILYPECHL NSPIVRGKIA QLHVLLDVNQ PYRLKDDSII NITKHKIRNG 60 GLSPRQIKIR SLGKALQRTI KDLDRYTFEP YPTYSQELLR LDIPEICDKI RSVFAVSDRL 120 TRELSSGFQD LWLNIFKQLG NIEGREGYDP LQDIGTIPEI TDKYSRNRWY RPFLTWFSIK 180 YDMRWMQKTR PGGPLDTSNS HNLLECKSYT LVTYGDLVMI LNKLTLTGYI LTPELVLMYC 240 DVVEGRWNMS AAGHLDKKSI GITSKGEELW ELVDSLFSSL GEEIYNVIAL LEPLSLALIQ 300 LNDPVIPLRG AFMRHVLTEL QTVLTSRDVY TDAEADTIVE SLLAIFHGTS IDEKAEIFSF 360 FRTFGHPSLE AVTAADKVRA HMYAQKAIKL KTLYECHAVF CTIIINGYRE RHGGQWPPCD 420 FPDHVCLELR NAQGSNTAIS YECAVDNYTS FIGFKFRKFI EPQLDEDLTI YMKDKALSPR 480 KEAWDSVYPD SNLYYKAPES EETRRLIEVF INDENFNPEE IINYVESGDW LKDEEFNISY 540 SLKEKEIKQE GRLFAKMTYK MRAVQVLAET LLAKGIGELF SENGMVKGEI DLLKRLTTLS 600 VSGVPRTDSV YNNSKSSEKR NEGMENKNSG GYWDEKKRSR HEFKATDSST DGYETLSCFL 660 TTDLKKYCLN WRFESTALFG QRCNEIFGFK TFFNWMHPVL ERCTIYVGDP YCPVADRMHR 720 QLQDHADSGI FIHNPRGGIE GYCQKLWTLI SISAIHLAAV RVGVRVSAMV QGDNQAIAVT 780 SRVPVAQTYK QKKNHVYEEI TKYFGALRHV MFDVGHELKL NETIISSKMF VYSKRIYYDG 840 KILPQCLKAL TKCVFWSETL VDENRSACSN ISTSIAKAIE NGYSPILGYC IALYKTCQQV 900 CISLGMTINP TISPTVRDQY FKGKNWLRCA VLIPANVGGF NYMSTSRCFV RNIGDPAVAA 960 LADLKRFIRA DLLDKQVLYR VMNQEPGDSS FLDWASDPYS CNLPHSQSIT TIIKNITARS 1020 VLQESPNPLL SGLFTETSGE EDLNLASFLM DRKVILPRVA HEILGNSLTG VREAIAGMLD 1080 TTKSLVRASV RKGGLSYGIL RRLVNYDLLQ YETLTRTLRK PVKDNIEYEY MCSVELAVGL 1140 RQKMWIHLTY GRPIHGLETP DPLELLRGIF IEGSEVCKLC RSEGADPIYT WFYLPDNIDL 1200 DTLTNGCPAI RIPYFGSATD ERSEAQLGYV RNLSKPAKAA IRIAMVYTWA YGTDEISWME 1260 AALIAQTRAN LSLENLKLLT PVSTSTNLSH RLKDTATQMK FSSATLVRAS RFITISNDNM 1320 ALKEAGESKD TNLVYQQIML TGLSLFEFNM RYKKGSLGKP LILHLHLNNG CCIMESPQEA 1380 NIPPRSTLDL EITQENNKLI YDPDPLKDVD LELFSKVRDV VHTVDMTYWS DDEVIRATSI 1440 CTAMTIADTM SQLDRDNLKE MIALVNDDDV NSLITEFMVI DVPLFCSTFG GILVNQFAYS 1500 LYGLNIRGRE EIWGHVVRIL KDTSHAVLKV LSNALSHPKI FKRFWNAGVV EPVYGPNLSN 1560 QDKILLALSV CEYSVDLFMH DWQGGVPLEI FICDNDPDVA DMRRSSFLAR HLAYLCSLAE 1620 ISRDGPRLES MNSLERLESL KSYLELTFLD DPVLRYSQLT GLVIKVFPST LTYIRKSSIK 1680 VLRTRGIGVP EVLEDWDPEA DNALLDGIAA EIQQNIPLGH QTRAPFWGLR VSKSQVLRLR 1740 GYKEITRGEI GRSGVGLTLP FDGRYLSHQL RLFGINSTSC LKALELTYLL SPLVDKDKDR 1800 LYLGEGAGAM LSCYDATLGP CINYYNSGVY SCDVNGQREL NIYPAEVALV GKKLNNVTSL 1860 GQRVKVLFNG NPGSTWIGND ECEALIWNEL QNSSIGLVHC DMEGGDHKDD QVVLHEHYSV 1920 IRIAYLVGDR DVVLISKIAP RLGTDWTRQL SLYLRYWDEV NLIVLKTSNP ASTEMYLLSR 1980 HPKSDIIEDS KTVLASLLPL SKEDSIKIEK WILIEKAKAH EWVTRELREG SSSSGMLRPY 2040 HQALQTFGFE PNLYKLSRDF LSTMNIADTH NCMIAFNRVL KDTIFEWARI TESDKRLKLT 2100 GKYDLYPVRD SGKLKTISRR LVLSWISLSM STRLVTGSFP DQKFEARLQL GIVSLSSREI 2160 RNLRVITKTL LDRFEDIIHS ITYRFLTKEI KILMKILGAV KMFGARQNEY TTVIDDGSLG 2220 DIEPYDSS 2228 Sequence No. 6: Amino acid sequence of the temperature-sensitive L protein mutant of Sendai virus MDGQESSQNP SDILYPECHL NSPIVRGKIA QLHVLLDVNQ PYRLKDDSII NITKHKIRNG 60 GLSPRQIKIR SLGKALQRTI KDLDRYTFEP YPTYSQELLR LDIPEICDKI RSVFAVSDRL 120 TRELSSGFQD LWLNIFKQLG NIEGREGYDP LQDIGTIPEI TDKYSRNRWY RPFLTWFSIK 180 YDMRWMQKTR PGGPLDTSNS HNLLECKSYT LVTYGDLVMI LNKLTLTGYI LTPELVLMYC 240 DVVEGRWNMS AAGHLDKKSI GITSKGEELW ELVDSLFSSL GEEIYNVIAL LEPLSLALIQ 300 LNDPVIPLRG AFMRHVLTEL QTVLTSRDVY TDAEADTIVE SLLAIFHGTS IDEKAEIFSF 360 FRTFGHPSLE AVTAADKVRA HMYAQKAIKL KTLYECHAVF CTIIINGYRE RHGGQWPPCD 420 FPDHVCLELR NAQGSNTAIS YECAVDNYTS FIGFKFRKFI EPQLDEDLTI YMKDKALSPR 480 KEAWDSVYPD SNLYYKAPES EETRRLIEVF INDENFNPEE IINYVESGDW LKDEEFNISY 540 SLKEKEIKQE GRLFAKMTYK MRAVQVLAET LLAKGIGELF SENGMVKGEI DLLKRLTTLS 600 VSGVPRTDSV YNNSKSSEKR NEGMENKNSG GYWDEKKRSR HEFKATDSST DGYETLSCFL 660 TTDLKKYCLN WRFESTALFG QRCNEIFGFK TFFNWMHPVL ERCTIYVGDP YCPVADRMHR 720 QLQDHADSGI FIHNPRGGIE GYCQKLWTLI SISAIHLAAV RVGVRVSAMV QGDNQAIAVT 780 SRVPVAQTYK QKKNHVYEEI TKYFGALRHV MFDVGHELKL NETIISSKMF VYSKRIYYDG 840 KILPQCLKAL TKCVFWSETL VDENRSACSN ISTSIAKAIE NGYSPILGYC IALYKTCQQV 900 CISLGMTINP TISPTVRDQY FKGKNWLRCA VLIPANVGGF NYMSTSRCFV RNIGDPAVAA 960 LADLKRFIRA DLLDKQVLYR VMNQEPGDSS FLDWASDPYS CNLPHSQSIT TIIKNITARS 1020 VLQESPNPLL SGLFTETSGE EDLNLASFLM DRKVILPRVA HEILGNSLTG VREAIAGMLD 1080 TTKSLVRASV RKGGLSYGIL RRLVNYDLLQ YETLTRTLRK PVKDNIEYEY MCSVELAVGL 1140 RQKMWIHLTY GRPIHGLETP DPLELLRGIF IEGSEVCKLC RSEGADPIYT WFYLPDXIDL 1200 DTLTNGCPAI RIPYFGSATD ERSEAQLGYV RNLSKPAKAA IRIAMVYTWA YGTDEISWME 1260 AALIAQTRAN LSLENLKLLT PVSTSTNLSH RLKDTATQMK FSSATLVRAS RFITISNDNM 1320 ALKEAGESKD TNLVYQQIML TGLSLFEFNM RYKKGSLGKP XILHLHLNNG CCIMESPQEA 1380 NIPPRSTLDL EITQENNKLI YDPDPLKDVD LELFSKVRDV VHTVDMTYWS DDEVIRATSI 1440 CTAMTIADTM SQLDRDNLKE MIALVNDDDV NSLITEFMVI DVPLFCSTFG GILVNQFAYS 1500 LYGLNIRGRE EIWGHVVRIL KDTSHAVLKV LSNALSHPKI FKRFWNAGVV EPVYGPNXSN 1560 QDKILLALSV CEYSVDLFMH DWQGGVPLEI FICDNDPDVA DMRRSSFLAR HLAYLCSLAE 1620 ISRDGPRLES MNSLERLESL KSYLELTFLD DPVLRYSQLT GLVIKVFPST LTYIRKSSIK 1680 VLRTRGIGVP EVLEDWDPEA DNALLDGIAA EIQQNIPLGH QTRAPFWGLR VSKSQVLRLR 1740 GYKEITRGEI GRSGVGLTLP FDGRYLSHQL RLFGINSTSC LKALELTYLL SPLVDXDKDR 1800 LYLGEGAGAM LSCYDATLGP CINYYNSGVY SCDVNGQREL NIYPAEVALV GKKLNNVTSL 1860 GQRVKVLFNG NPGSTWIGND ECEALIWNEL QNSSIGLVHC DMEGGDHKDD QVVLHEHYSV 1920 IRIAYLVGDR DVVLISKIAP RLGTDWTRQL SLYLRYWDEV NLIVLKTSNP ASTEMYLLSR 1980 HPKSDIIEDS KTVLASLLPL SKEDSIKIEK WILIEKAKAH EWVTRELREG SSSSGMLRPY 2040 HQALQTFGFE PNLYKLSRDF LSTMNIADTH NCMIAFNRVL KDTIFEWARI TESDKRLKLT 2100 GKYDLYPVRD SGKLKTISRR LVLSWISLSM STRLVTGSFP DQKFEARLQL GIVSLSSREI 2160 RNLRVITKTL LDRFEDIIHS ITYRFLTKEI KILMKILGAV KMFGARQNEY TTVIDDGSLG 2220 DIEPYDSS 2228 Accession No. 7: Amino acid sequence of a mutant (N1197S / L1558I / K1796E) of Sendai virus L protein MDGQESSQNP SDILYPECHL NSPIVRGKIA QLHVLLDVNQ PYRLKDDSII NITKHKIRNG 60 GLSPRQIKIR SLGKALQRTI KDLDRYTFEP YPTYSQELLR LDIPEICDKI RSVFAVSDRL 120 TRELSSGFQD LWLNIFKQLG NIEGREGYDP LQDIGTIPEI TDKYSRNRWY RPFLTWFSIK 180 YDMRWMQKTR PGGPLDTSNS HNLLECKSYT LVTYGDLVMI LNKLTLTGYI LTPELVLMYC 240 DVVEGRWNMS AAGHLDKKSI GITSKGEELW ELVDSLFSSL GEEIYNVIAL LEPLSLALIQ 300 LNDPVIPLRG AFMRHVLTEL QTVLTSRDVY TDAEADTIVE SLLAIFHGTS IDEKAEIFSF 360 FRTFGHPSLE AVTAADKVRA HMYAQKAIKL KTLYECHAVF CTIIINGYRE RHGGQWPPCD 420 FPDHVCLELR NAQGSNTAIS YECAVDNYTS FIGFKFRKFI EPQLDEDLTI YMKDKALSPR 480 KEAWDSVYPD SNLYYKAPES EETRRLIEVF INDENFNPEE IINYVESGDW LKDEEFNISY 540 SLKEKEIKQE GRLFAKMTYK MRAVQVLAET LLAKGIGELF SENGMVKGEI DLLKRLTTLS 600 VSGVPRTDSV YNNSKSSEKR NEGMENKNSG GYWDEKKRSR HEFKATDSST DGYETLSCFL 660 TTDLKKYCLN WRFESTALFG QRCNEIFGFK TFFNWMHPVL ERCTIYVGDP YCPVADRMHR 720 QLQDHADSGI FIHNPRGGIE GYCQKLWTLI SISAIHLAAV RVGVRVSAMV QGDNQAIAVT 780 SRVPVAQTYK QKKNHVYEEI TKYFGALRHV MFDVGHELKL NETIISSKMF VYSKRIYYDG 840 KILPQCLKAL TKCVFWSETL VDENRSACSN ISTSIAKAIE NGYSPILGYC IALYKTCQQV 900 CISLGMTINP TISPTVRDQY FKGKNWLRCA VLIPANVGGF NYMSTSRCFV RNIGDPAVAA 960 LADLKRFIRA DLLDKQVLYR VMNQEPGDSS FLDWASDPYS CNLPHSQSIT TIIKNITARS 1020 VLQESPNPLL SGLFTETSGE EDLNLASFLM DRKVILPRVA HEILGNSLTG VREAIAGMLD 1080 TTKSLVRASV RKGGLSYGIL RRLVNYDLLQ YETLTRTLRK PVKDNIEYEY MCSVELAVGL 1140 RQKMWIHLTY GRPIHGLETP DPLELLRGIF IEGSEVCKLC RSEGADPIYT WFYLPDSIDL 1200 DTLTNGCPAI RIPYFGSATD ERSEAQLGYV RNLSKPAKAA IRIAMVYTWA YGTDEISWME 1260 AALIAQTRAN LSLENLKLLT PVSTSTNLSH RLKDTATQMK FSSATLVRAS RFITISNDNM 1320 ALKEAGESKD TNLVYQQIML TGLSLFEFNM RYKKGSLGKP LILHLHLNNG CCIMESPQEA 1380 NIPPRSTLDL EITQENNKLI YDPDPLKDVD LELFSKVRDV VHTVDMTYWS DDEVIRATSI 1440 CTAMTIADTM SQLDRDNLKE MIALVNDDDV NSLITEFMVI DVPLFCSTFG GILVNQFAYS 1500 LYGLNIRGRE EIWGHVVRIL KDTSHAVLKV LSNALSHPKI FKRFWNAGVV EPVYGPNISN 1560 QDKILLALSV CEYSVDLFMH DWQGGVPLEI FICDNDPDVA DMRRSSFLAR HLAYLCSLAE 1620 ISRDGPRLES MNSLERLESL KSYLELTFLD DPVLRYSQLT GLVIKVFPST LTYIRKSSIK 1680 VLRTRGIGVP EVLEDWDPEA DNALLDGIAA EIQQNIPLGH QTRAPFWGLR VSKSQVLRLR 1740 GYKEITRGEI GRSGVGLTLP FDGRYLSHQL RLFGINSTSC LKALELTYLL SPLVDEDKDR 1800 LYLGEGAGAM LSCYDATLGP CINYYNSGVY SCDVNGQREL NIYPAEVALV GKKLNNVTSL 1860 GQRVKVLFNG NPGSTWIGND ECEALIWNEL QNSSIGLVHC DMEGGDHKDD QVVLHEHYSV 1920 IRIAYLVGDR DVVLISKIAP RLGTDWTRQL SLYLRYWDEV NLIVLKTSNP ASTEMYLLSR 1980 HPKSDIIEDS KTVLASLLPL SKEDSIKIEK WILIEKAKAH EWVTRELREG SSSSGMLRPY 2040 HQALQTFGFE PNLYKLSRDF LSTMNIADTH NCMIAFNRVL KDTIFEWARI TESDKRLKLT 2100 GKYDLYPVRD SGKLKTISRR LVLSWISLSM STRLVTGSFP DQKFEARLQL GIVSLSSREI 2160 RNLRVITKTL LDRFEDIIHS ITYRFLTKEI KILMKILGAV KMFGARQNEY TTVIDDGSLG 2220 DIEPYDSS 2228 Sequence number 8: Example of Sendai virus P protein variant MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITAAGGATDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL YPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 Sequence number 9: Example of a P protein mutant of Sendai virus MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITSSGGSTDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL YPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 Sequence number 10: Example of a P protein mutant of Sendai virus MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITTTGGTTDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL YPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 Sequence number 11: Example of Sendai virus P protein variant MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITGGGGGTDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL YPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO: 12: Example of Sendai virus P protein variant MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITSSGGSTDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL FPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO: 13: Example of Sendai virus P protein variant MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITSAGGATDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL FPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO:14: Example of Sendai virus P protein variant MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITSAGGATDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL YPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO:15: Example of Sendai virus P protein variant MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITASGGATDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL YPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO:16: Example of Sendai virus P protein variant MDQDAFILKE DSEVEREAPG GRESLSDVIG FLDAVLSSEP TDIGGDRSWL HNTINTPQGP 60 GSAHRAKSEG EGEVSTPSTQ DNRSGEESRV SGRTSKPEAE AHAGNLDKQN IHRAFGGRTG 120 TNSVSQDLGD GGDSGILENP PNERGYPRSG IEDENREMAA HPDKRGEDQA EGLPEEVRGS 180 TSLPDEGEGG ASNNGRSMEP GSSHSARVTG VLVIPSPELE EAVLRRNKRR PTNSGSKPLT 240 PATVPGTRSP PLNRYNSTGS PPGKPPSTQD EHINSGDTPA VRVKDRKPPI GTRSVSDCPA 300 NGRPIHPGLE TDSTKKGIGE NTSSMKEMAT LLTSLGVIQS AQEFESSRDA SYVFARRALK 360 SANYAEMTFN VCGLILSAEK SSARKVDENK QLLKQIQESV ESFRDIYKRF SEYQKEQNSL 420 LMSNLSTLHI ITAAGGSTDN TDSLTRSPSV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL YPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO: 17: Example of Sendai virus L protein variant MDGQESSQNP SDILYPECHL NSPIVRGKIA QLHVLLDVNQ PYRLKDDSII NITKHKIRNG 60 GLSPRQIKIR SLGKALQRTI KDLDRYTFEP YPTYSQELLR LDIPEICDKI RSVFAVSDRL 120 TRELSSGFQD LWLNIFKQLG NIEGREGYDP LQDIGTIPEI TDKYSRNRWY RPFLTWFSIK 180 YDMRWMQKTR PGGPLDTSNS HNLLECKSYT LVTYGDLVMI LNKLTLTGYI LTPELVLMYC 240 DVVEGRWNMS AAGHLDKKSI GITSKGEELW ELVDSLFSSL GEEIYNVIAL LEPLSLALIQ 300 LNDPVIPLRG AFMRHVLTEL QTVLTSRDVY TDAEADTIVE SLLAIFHGTS IDEKAEIFSF 360 FRTFGHPSLE AVTAADKVRA HMYAQKAIKL KTLYECHAVF CTIIINGYRE RHGGQWPPCD 420 FPDHVCLELR NAQGSNTAIS YECAVDNYTS FIGFKFRKFI EPQLDEDLTI YMKDKALSPR 480 KEAWDSVYPD SNLYYKAPES EETRRLIEVF INDENFNPEE IINYVESGDW LKDEEFNISY 540 SLKEKEIKQE GRLFAKMTYK MRAVQVLAET LLAKGIGELF SENGMVKGEI DLLKRLTTLS 600 VSGVPRTDSV YNNSKSSEKR NEGMENKNSG GYWDEKKRSR HEFKATDSST DGYETLSCFL 660 TTDLKKYCLN WRFESTALFG QRCNEIFGFK TFFNWMHPVL ERCTIYVGDP YCPVADRMHR 720 QLQDHADSGI FIHNPRGGIE GYCQKLWTLI SISAIHLAAV RVGVRVSAMV QGDNQAIAVT 780 SRVPVAQTYK QKKNHVYEEI TKYFGALRHV MFDVGHELKL NETIISSKMF VYSKRIYYDG 840 KILPQCLKAL TKCVFWSETL VDENRSACSN ISTSIAKAIE NGYSPILGYC IALYKTCQQV 900 CISLGMTINP TISPTVRDQY FKGKNWLRCA VLIPANVGGF NYMSTSRCFV RNIGDPAVAA 960 LADLKRFIRA DLLDKQVLYR VMNQEPGDSS FLDWASDPYS CNLPHSQSIT TIIKNITARS 1020 VLQESPNPLL SGLFTETSGE EDLNLASFLM DRKVILPRVA HEILGNSLTG VREAIAGMLD 1080 TTKSLVRASV RKGGLSYGIL RRLVNYDLLQ YETLTRTLRK PVKDNIEYEY MCSVELAVGL 1140 RQKMWIHLTY GRPIHGLETP DPLELLRGIF IEGSEVCKLC RSEGADPIYT WFYLPDSIDL 1200 DTLTNGCPAI RIPYFGSATD ERSEAQLGYV RNLSKPAKAA IRIAMVYTWA YGTDEISWME 1260 AALIAQTRAN LSLENLKLLT PVSTSTNLSH RLKDTATQMK FSSATLVRAS RFITISNDNM 1320 ALKEAGESKD TNLVYQQIML TGLSLFEFNM RYKKGSLGKP CILHLHLNNG CCIMESPQEA 1380 NIPPRSTLDL EITQENNKLI YDPDPLKDVD LELFSKVRDV VHTVDMTYWS DDEVIRATSI 1440 CTAMTIADTM SQLDRDNLKE MIALVNDDDV NSLITEFMVI DVPLFCSTFG GILVNQFAYS 1500 LYGLNIRGRE EIWGHVVRIL KDTSHAVLKV LSNALSHPKI FKRFWNAGVV EPVYGPNISN 1560 QDKILLALSV CEYSVDLFMH DWQGGVPLEI FICDNDPDVA DMRRSSFLAR HLAYLCSLAE 1620 ISRDGPRLES MNSLERLESL KSYLELTFLD DPVLRYSQLT GLVIKVFPST LTYIRKSSIK 1680 VLRTRGIGVP EVLEDWDPEA DNALLDGIAA EIQQNIPLGH QTRAPFWGLR VSKSQVLRLR 1740 GYKEITRGEI GRSGVGLTLP FDGRYLSHQL RLFGINSTSC LKALELTYLL SPLVDEDKDR 1800 LYLGEGAGAM LSCYDATLGP CINYYNSGVY SCDVNGQREL NIYPAEVALV GKKLNNVTSL 1860 GQRVKVLFNG NPGSTWIGND ECEALIWNEL QNSSIGLVHC DMEGGDHKDD QVVLHEHYSV 1920 IRIAYLVGDR DVVLISKIAP RLGTDWTRQL SLYLRYWDEV NLIVLKTSNP ASTEMYLLSR 1980 HPKSDIIEDS KTVLASLLPL SKEDSIKIEK WILIEKAKAH EWVTRELREG SSSSGMLRPY 2040 HQALQTFGFE PNLYKLSRDF LSTMNIADTH NCMIAFNRVL KDTIFEWARI TESDKRLKLT 2100 GKYDLYPVRD SGKLKTISRR LVLSWISLSM STRLVTGSFP DQKFEARLQL GIVSLSSREI 2160 RNLRVITKTL LDRFEDIIHS ITYRFLTKEI KILMKILGAV KMFGARQNEY TTVIDDGSLG 2220 DIEPYDSS 2228 Sequence number 18: An example of a DD tag GVQVETISPG DGRTFPKRGQ TCVVHYTGML EDGKKVDSSR DRNKPFKFML GKQEVIRGWE 60 EGVAQMSVGQ RAKLTISPDY AYGATGHPGI IPPHATLVFD VELLKP 106 SEQ ID NO:19: Example of DDm tag GVQIETISPG DGRTFPKKGQ TCVVHYTGML EDGKKLDSSR DRNKPFKFMI GKQEVIKGFE 60 EGAAQMSLGQ RAKLTISPDY AYGATGHPGV IPPHATLIFD VELLRPE 107 SEQ ID NO:20: Variant of M protein MADIYRFPKF SYEDNGTVEP LPLRTGPDKK AIPHIRIVKV GDPPKHGVRY LDLLLLGFFE 60 TPKQTTNLES VSDLTEPTSY SICGSGSLPI GVAKYYGTDQ ELLKACTDLR ITVRRAVRAG 120 EMIVYMVDSI GAPLLPWSGR LRQGMIFNAN KVALAPQCLP VDKDIRLRVV FVNGTSLGAI 180 TITKIPKTLA DLALPNSISV NLLVTLKTGI STEQKGVLPV LDDQGEKKLN FMVHLGLIRR 240 KVGKIYSVEY CKSKIERMRL IFSLGLIGGI SFHVQVNGTL SKTFMSQLAW KRAVCFPLMD 300 VNPHMNMVIW AASVEITGVD AVFQPAIPRD FRYYPNVVAK NIGRIRKL 348 SEQ ID NO:21: Variant of HN protein MDGDRGKRDS YWSTSPSGST TKPASGWERS SKADTWLLIL SFTQWALSIA TVIICIIISA 60 RQGYSMKEYS MTVEALNMSS REVKESLTSL IRQEVIARAV NIQSSVQTGI PVLLNKNSRD 120 VIQMIDKSCS RQELTQHCES TIAVHHADGI APLEPHSFWR CPVGEPYLSS DPEISLLPGP 180 SLLSGSTTIS GCVRLPSLSI GEAIYAYSSN LITQGCADIG KSYQVLQLGY ISLNSDMFPD 240 LNPVVSHTYD INDNRKSCSV VTTKTRGYQL CSMPTVDERT DYSSDGIEDL VLDVLDLKGR 300 TKSHRYRNSE VDLDHPFSAL YPSVGNGIAT EGSLIFLGYG GLTTPLQGDT KCRTQGCQQV 360 SQDTCNEALK ITWLGGKQVV SVIIQVNDYL SERPKIRVTT IPITQNYLGA EGRLLKLGDR 420 VYIYTRSSGW HSQLQIGVLD VSHPLTINWT PHEALSRPGN EECNWYNKCP KECISGVYTD 480 AYPLSPDAAN VATVTLYANT SRVNPTIMYS NTTNIINMLR IKDVQLEAAY TTTSCITHFG 540 KGYCFHIIEI NQKSLNTLQP MLFKTSIPKL CKAES 575 Sequence number 22: An example of the domain of bovine FKBP12 corresponding to the DD domain of human FKBP12 MGVQVETISP GDGRTFPKRG QTCVVHYTGM LEDGKKFDSS RDRNKPFKFV LGKQEVIRGW 60 EEGVAQMSVG QRAKLTISPD YAYGATGHPG IIPPNATLIF DVELLKLE 108 Sequence number 23: An example of the domain of rhesus monkey FKBP12 corresponding to the DD domain of human FKBP12 MGVEIETISP GDGRTFPKKG QTCVVHYTGM LQNGKKFDSS RDRNKPFKFR IGKQEVIKGF 60 EEGAAQMSLG QRAKLTCTPD VAYGATGHPG VIPPNATLIF DVELLNLE 108 SEQ ID NO: 24: An example of human FKBP12 MGVQVETISP GDGRTFPKRG QTCVVHYTGM LEDGKKFDSS RDRNKPFKFM LGKQEVIRGW 60 EEGVAQMSVG QRAKLTISPD YAYGATGHPG IIPPHATLVF DVELLKLE 108 SEQ ID NO: 25: An example of the domain of mouse FKBP12 corresponding to the DD domain of human FKBP12 MGVEIETISP GDGRTFPKKG QICVVHYTGM LQNGKKFDSS RDRNKPFKFR IGKQEVIKGF 60 EEGTAQMSLG QRAKLTCTPD VAYGATGHPG VIPPNATLIF DVELLSLE 108 SEQ ID NO: 26: An example of the domain of avian FKBP12 corresponding to the DD domain of human FKBP12 MGVQVETISP GDGRTFPKRG QTCVVHYTGM LEDGKKFDSS RDRNKPFKFM LGKQEVIRGW 60 EEGVAQMSVG QRAKLTISPD YAYGATGHPG IIPPHATLVF DVELLKLE 108 SEQ ID NO: 27: An example of the domain of rat FKBP12 corresponding to the DD domain of human FKBP12 MGVEIETISP GDGRTFPKKG QICVVHYTGM LQNGKKFDSS RDRNKPFKFR IGKQEVIKGF 60 EEGAAQMSLG QRAKLTCTPD VAYGATGHPG VIPPNATLIF DVELLNLE 108 SEQ ID NO: 28: An example of the DDrs tag GVQVETISPG DGRTFPRRGQ TCVVHYTGML EDGRRVDSSR DRNRPFRFML GRQEVIRGWE 60 EGVSQMSVGQ RSRLTISPDY SYGSTGHPGI IPPHSTLVFD VELLRPE 107

Claims

1. The RNA genome of a negative-strand RNA virus or viral vector, The RNA genome of a negative-strand RNA virus or viral vector, wherein the P protein encoded by the viral genome has substitution mutations in amino acids of the P protein corresponding to any one or more or all of D433, R434, and K437, the substitution mutations including any of G, T, and S, and may further have a substitution mutation in an amino acid of the P protein corresponding to L511, the substitution mutation of L511 being a mutation to Y or F.

2. 2. The RNA genome of a negative-strand RNA virus or viral vector according to claim 1, wherein the P protein encoded by the viral genome has substitution mutations in amino acids of the P protein corresponding to all of D433, R434, and K437, and the substitution mutations include mutations to any of G, T, and S.

3. The RNA genome of a negative-strand RNA virus or viral vector according to claim 2, wherein the substitution mutation is a mutation to T or S.

4. The RNA genome of a negative-strand RNA virus or viral vector according to claim 3, wherein the substitution mutation is a mutation to T.

5. The RNA genome of a negative-strand RNA virus or viral vector according to claim 3, wherein the substitution mutation is a mutation to S.

6. The negative-strand RNA virus or viral vector according to any one of claims 1 to 3, wherein the P protein encoded by the viral genome is (1S) having amino acid mutations corresponding to D433S, R434A, K437A, and L511F; (2S) having amino acid mutations corresponding to D433S, R434A, K437A, and L511Y; (3S) having amino acid mutations corresponding to D433A, R434S, K437A, and L511F; (4S) having amino acid mutations corresponding to D433A, R434S, K437A, and L511Y; (5S) having amino acid mutations corresponding to D433A, R434A, K437S, and L511F; (6S) having amino acid mutations corresponding to D433A, R434A, K437S, and L511Y; (7S) having amino acid mutations corresponding to D433S, R434S, K437A, and L511F; (8S) having amino acid mutations corresponding to D433S, R434S, K437A, and L511Y; (9S) having amino acid mutations corresponding to D433S, R434A, K437S, and L511F; (10S) having amino acid mutations corresponding to D433S, R434A, K437S, and L511Y; (11S) Amino acids corresponding to D433A, R434S, K437S, and L511F having a mutation of (12S) having amino acid mutations corresponding to D433A, R434S, K437S, and L511Y; (13S) having amino acid mutations corresponding to D433S, R434S, K437S, and L511F; (14S) having amino acid mutations corresponding to D433S, R434S, K437S, and L511Y; (1T) having amino acid mutations corresponding to D433T, R434A, K437A, and L511F; (2T) having amino acid mutations corresponding to D433T, R434A, K437A, and L511Y; (3T) having amino acid mutations corresponding to D433A, R434T, K437A, and L511F; (4T) having amino acid mutations corresponding to D433A, R434T, K437A, and L511Y; (5T) having amino acid mutations corresponding to D433A, R434A, K437T, and L511F; (6T) having amino acid mutations corresponding to D433A, R434A, K437T, and L511Y; (7T) having amino acid mutations corresponding to D433T, R434T, K437A, and L511F; (8T) having amino acid mutations corresponding to D433T, R434T, K437A, and L511Y; (9T) having amino acid mutations corresponding to D433T, R434A, K437T, and L511F; (10T) having amino acid mutations corresponding to D433T, R434A, K437T, and L511Y; (11T) having amino acid mutations corresponding to D433A, R434T, K437T, and L511F; (12T) having amino acid mutations corresponding to D433A, R434T, K437T, and L511Y; (13T) having amino acid mutations corresponding to D433T, R434T, K437T, and L511F; (14T) having amino acid mutations corresponding to D433T, R434T, K437T, and L511Y; (1ST) having amino acid mutations corresponding to D433S, R434T, K437S, and L511F, or (2ST) having amino acid mutations corresponding to D433S, R434T, K437S, and L511Y; The RNA genome of a negative-stranded RNA virus or viral vector.

7. The RNA genome of a negative-strand RNA virus or viral vector according to any one of claims 1 to 6, wherein a large protein (L protein) encoded on the viral genome has an amino acid mutation corresponding to either or both of amino acids L1361C and L1558I.

8. The RNA genome of the minus-strand RNA virus or viral vector according to any one of claims 1 to 7, which carries at least one exogenous gene of interest.

9. The RNA genome of a minus-strand RNA virus or viral vector according to any one of claims 1 to 8, which is a Sendai virus or a Sendai virus vector.

10. A nucleic acid encoding the RNA genome of the negative-strand RNA virus or viral vector according to any one of claims 1 to 9.

11. A minus-strand RNA virus or viral vector comprising the RNA genome of the minus-strand RNA virus or viral vector according to any one of claims 1 to 9.

12. The negative-strand RNA virus or viral vector according to claim 11 , comprising a P protein that is less temperature sensitive than the P protein encoded on the genome, or a P protein that has no temperature sensitivity.

13. A negative-strand RNA virus or viral vector described in claim 11 or 12, which contains an L protein that is less temperature sensitive than the L protein encoded on the genome, or which contains an L protein that is not temperature sensitive, when the L protein encoded on the viral genome is temperature sensitive.

14. A composition comprising the negative-strand RNA virus or viral vector according to any one of claims 11 to 13.

15. A method for expressing a target gene in a cell, a method for producing a cell expressing a target gene, or a method for culturing a cell, comprising the steps of: Infecting the cell with the minus-strand RNA virus or the viral vector according to any one of claims 11 to 13, and then culturing the cell under a first temperature condition to express the gene of interest in the cell; and then further culturing the cells under a second temperature condition, thereby removing a part or all of the negative-strand RNA virus or viral vector from the cells. method.

16. A method for expressing a target gene in a cell, a method for producing a cell expressing a target gene, or a method for culturing a cell, comprising the steps of: Infecting the cell with the minus-strand RNA virus or the viral vector according to any one of claims 11 to 13, and then culturing the cell under a first temperature condition to express the gene of interest in the cell. method.

17. A method for expressing a target gene in a cell, a method for producing a cell expressing a target gene, or a method for culturing a cell, comprising the steps of: Infecting the cells with the minus-strand RNA virus or the viral vector according to any one of claims 11 to 13, and then culturing the cells under a second temperature condition to express the target gene in the cells and remove the virus or the viral vector from the cells. method.

18. A degradation-inducing protein having an amino acid sequence having two or more or all amino acid mutations selected from the group consisting of 4I, 18K, 50I, 57K, 59F, 63A, 68L, 90V, 98I, and 105R, and an amino acid mutation corresponding to 36L, relative to the amino acid sequence set forth in SEQ ID NO:

18.

19. The degradation-inducing protein of claim 18, having amino acid mutations corresponding to all of 4I, 18K, 36L, 50I, 57K, 59F, 63A, 68L, 90V, 98I, and 105R relative to the amino acid sequence set forth in SEQ ID NO:

18.

20. A degradation-inducing fusion protein comprising a target protein and the degradation-inducing protein described in claim 18 or 19.

21. A nucleic acid encoding the protein according to any one of claims 18 to 20.

22. An animal cell comprising the protein according to any one of claims 18 to 20 and / or the nucleic acid according to claim 21.

23. A minus-strand RNA virus or vector comprising a protein according to any one of claims 18 to 20 and / or a nucleic acid according to claim 21.

24. A negative-strand RNA virus or vector comprising a negative-strand RNA genome comprising the nucleic acid of claim 21.

25. M protein having the amino acid sequence set forth in SEQ ID NO:

19.

26. 26. A nucleic acid encoding the M protein of claim 25.

27. HN protein having the amino acid sequence set forth in SEQ ID NO:

20.

28. A nucleic acid encoding the HN protein of claim 27.

29. 28. An animal cell comprising the M protein of claim 25 and / or the HN protein of claim 27.

30. A negative-strand RNA virus or vector comprising the M protein of claim 25 and / or the HN protein of claim 27.

31. 28. An animal cell comprising a nucleic acid according to claim 26 and / or 27.

32. 28. A negative-strand RNA virus or vector comprising a negative-strand RNA genome comprising a nucleic acid according to claim 26 and / or 27.

33. A nucleic acid encoding the genome of a minus-strand RNA virus or vector comprising the nucleic acid according to claim 26 and / or 27, or a vector comprising said nucleic acid.

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