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

Abstract

To provide a temperature-sensitive negative-strand RNA virus or a viral vector, and an RNA genome thereof.SOLUTION: A negative-strand RNA virus or viral vector is provided, in which at least one viral protein encoded in the genome (preferably the P protein or a temperature-sensitive P protein) is in the form of a fusion protein with a BromoTag.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 technology]

[0002] Negative-strand RNA viruses or viral vectors such as Sendai virus are known as cytoplasmic RNA viral vectors, and are thought to be advantageous for regenerative medicine and in vivo use because they are less likely to damage the nucleus. Furthermore, negative-strand RNA viruses or viral vectors such as Sendai virus can sometimes exhibit high gene transfer and / or gene expression efficiency both in vivo and in vitro, and are thought to be particularly useful for the purposes of gene transfer and / or gene expression, and their development is actively progressing.

[0003] To improve the safety of using viruses or viral vectors, there is a need for a technology to remove (e.g., reduce or eliminate) viruses or viral vectors that have infected cells from within the cells. Temperature-sensitive mutants have been created as a technology for removing viruses or viral vectors. Specifically, mutants containing the L511F mutation or the D433A / R434A / K437A mutations in the P protein have been proposed (Patent Documents 1 to 4). Another method has been proposed in which N1197S / L1558I / K1796E are introduced into the L protein to enhance temperature sensitivity (Patent Documents 3 and 4). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2012 / 029770 [Patent Document 2] WO2010 / 008054 [Patent Document 3] WO2017 / 082174 [Patent Document 4] WO2003 / 025570 [Non-patent literature]

[0005] [Non-Patent Document 1] PNAS,108(34):14234-14239,2011 Summary of the Invention

[0006] The present invention provides a temperature-sensitive minus-strand RNA virus or viral vector and its RNA genome. According to the present invention, cells infected with the virus or viral vector can be cultured while maintaining the infection by adjusting the culture temperature conditions, and then the virus or viral vector can be removed (reduced).

[0007] According to the present invention, the following inventions are provided. [1] The RNA genome of a minus-strand RNA virus or viral vector, wherein at least one of the viral proteins encoded by the genome (preferably the P protein or the temperature-sensitive P protein) is in the form of a fusion protein with a bromotag. [2] The minus-strand RNA virus or viral vector according to [1] above, The temperature-sensitive P protein encoded by the viral genome has substitution mutations at amino acids of the P protein corresponding to any one or more or all of D433, R434, and K437, the substitution mutations including a mutation to any of G, T, and S, and may further have a substitution mutation at an amino acid of the P protein corresponding to L511, the substitution mutation at L511 being a mutation to Y or F, or The RNA genome of a negative-strand RNA virus or viral vector, wherein the temperature-sensitive 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 the minus-strand RNA virus or viral vector according to [2] above, wherein the substitution mutation is a mutation to T or S. [4] The RNA genome of the minus-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 minus-strand RNA virus or viral vector according to [3] above, wherein the substitution mutation is a mutation to S. [6] The minus-strand RNA virus or viral vector according to any one of [1] to [3] above, wherein the temperature-sensitive P protein encoded by the viral genome is (1S) having amino acid mutations corresponding to D433S, R434A, K437A, and L511F; (2S) with amino acid mutations corresponding to D433S, R434A, K437A, and L511Y; (3S) with amino acid mutations corresponding to D433A, R434S, K437A, and L511F; (4S) with amino acid mutations corresponding to D433A, R434S, K437A, and L511Y; (5S) with amino acid mutations corresponding to D433A, R434A, K437S, and L511F; (6S) with amino acid mutations corresponding to D433A, R434A, K437S, and L511Y; (7S) with amino acid mutations corresponding to D433S, R434S, K437A, and L511F; (8S) with amino acid mutations corresponding to D433S, R434S, K437A, and L511Y; (9S) with amino acid mutations corresponding to D433S, R434A, K437S, and L511F; (10S) with amino acid mutations corresponding to D433S, R434A, K437S, and L511Y; (11S) with amino acid mutations corresponding to D433A, R434S, K437S, and L511F; (12S) with amino acid mutations corresponding to D433A, R434S, K437S, and L511Y; (13S) with amino acid mutations corresponding to D433S, R434S, K437S, and L511F; (14S) with amino acid mutations corresponding to D433S, R434S, K437S, and L511Y; (1T) having amino acid mutations corresponding to D433T, R434A, K437A, and L511F; (2T) with amino acid mutations corresponding to D433T, R434A, K437A, and L511Y; (3T) having amino acid mutations corresponding to D433A, R434T, K437A, and L511F; (4T) with amino acid mutations corresponding to D433A, R434T, K437A, and L511Y; (5T) with amino acid mutations corresponding to D433A, R434A, K437T, and L511F; (6T) with amino acid mutations corresponding to D433A, R434A, K437T, and L511Y; (7T) with amino acid mutations corresponding to D433T, R434T, K437A, and L511F; (8T) with amino acid mutations corresponding to D433T, R434T, K437A, and L511Y; (9T) with amino acid mutations corresponding to D433T, R434A, K437T, and L511F; (10T) with amino acid mutations corresponding to D433T, R434A, K437T, and L511Y; (11T) with amino acid mutations corresponding to D433A, R434T, K437T, and L511F; (12T) with amino acid mutations corresponding to D433A, R434T, K437T, and L511Y; (13T) with amino acid mutations corresponding to D433T, R434T, K437T, and L511F; (14T) with amino acid mutations corresponding to D433T, R434T, K437T, and L511Y; (1ST) has amino acid mutations corresponding to D433S, R434T, K437S, and L511F, or (2ST) with amino acid mutations corresponding to D433S, R434T, K437S, and L511Y; The genomic RNA of a negative-strand RNA virus or viral vector. [7] The genomic RNA of a minus-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 the amino acids L1361C and L1558I. [8] The genomic RNA of the minus-strand RNA virus or viral vector according to any one of [1] to [7] above, which carries at least one exogenous gene of interest. [9] The genomic RNA of the minus-strand RNA virus or viral vector according to any one of [1] to [8] above, which is a Sendai virus or a Sendai virus vector.

[10] A nucleic acid encoding the genomic RNA of the minus-strand RNA virus or viral vector described in any one of [1] to [9] above {preferably, DNA, more preferably, an expression vector for the genomic RNA (particularly, a plasmid, etc.)}. [11A] A minus-strand RNA virus or viral vector comprising the genomic RNA of a minus-strand RNA virus or viral vector described in any one of [1] to [9] above, wherein preferably at least one of the viral proteins encoded by the genome (preferably the P protein or temperature-sensitive P protein) is in the form of a fusion protein with a bromo tag, and / or at least one of the viral proteins constituting the virus or viral vector (preferably the P protein or temperature-sensitive P protein) is in the form of a fusion protein with a bromo tag. [11B] A minus-strand RNA virus or viral vector comprising the genomic RNA of a minus-strand RNA virus or viral vector described in any one of [1] to [9] above, wherein preferably at least one of the viral proteins encoded by the genome (preferably the P protein or the temperature-sensitive P protein) is in the form of a fusion protein with a bromotag. [11C] A minus-strand RNA virus or viral vector comprising the genomic RNA of a minus-strand RNA virus or viral vector described in any one of [1] to [9] above, wherein preferably at least one of the viral proteins constituting the virus or viral vector (preferably the P protein or the temperature-sensitive P protein) is in the form of a fusion protein with a bromotag. [11D] A minus-strand RNA virus or viral vector comprising the genomic RNA of a minus-strand RNA virus or viral vector described in any one of [1] to [9] above, wherein preferably at least one of the viral proteins encoded by the genome (preferably the P protein or temperature-sensitive P protein) is in the form of a fusion protein with a bromo tag, and at least one of the viral proteins constituting the virus or viral vector (preferably the P protein or temperature-sensitive P protein) is in the form of a fusion protein with a bromo tag. [11E] A minus-strand RNA virus or viral vector in which at least one of the viral proteins encoded in the genome (preferably the P protein or temperature-sensitive P protein) is in the form of a fusion protein with a bromo tag, and / or at least one of the viral proteins constituting the virus or viral vector (preferably the P protein or temperature-sensitive P protein) is in the form of a fusion protein with a bromo tag. In the following, [11A] to [11E] will be collectively referred to as

[11] .

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

[11] above, which comprises a P protein that is less temperature-sensitive than the temperature-sensitive 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, the L protein may be less temperature sensitive than the L protein encoded on the genome, or may be temperature insensitive. The minus-strand RNA virus or viral vector according to

[11] or

[12] above.

[14] A composition comprising the minus-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 expressing a target gene, or a method for culturing a cell, comprising: Infecting the cells with the minus-strand RNA virus or viral vector according to any one of

[11] to

[13] above, and then culturing the cells under a first temperature condition to allow the target gene to be expressed in the cells; and then further culturing the cells under a second temperature condition, thereby removing a part or all of the minus-strand RNA virus or viral vector from the cells. method.

[0008]

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

[11] to

[13] above, and then culturing the cells under a first temperature condition to allow the target gene to be expressed in the cells; and then further culturing the cells under a second temperature condition, thereby removing a part or all of the minus-strand RNA virus or viral vector from the cells. method.

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

[11] to

[13] above, and then culturing the cells under a first temperature condition to allow the target gene to be expressed in the cells; and then further culturing the cells under a second temperature condition, thereby removing a part or all of the minus-strand RNA virus or viral vector from the cells. method.

[18] A method for culturing cells, comprising: Infecting the cells with the minus-strand RNA virus or viral vector according to any one of

[11] to

[13] above, and then culturing the cells under a first temperature condition to allow the target gene to be expressed in the cells; and then further culturing the cells under a second temperature condition, thereby removing a part or all of the minus-strand RNA virus or viral vector from the cells. method.

[0009]

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

[11] to

[13] above, and then culturing the cells under a first temperature condition to allow the target gene to be expressed in the cells. method.

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

[11] to

[13] above, and then culturing the cells under a first temperature condition to allow the target gene to be expressed in the cells. method.

[21] A method for culturing cells, comprising: Infecting the cells with the minus-strand RNA virus or the viral vector according to any one of

[11] to

[13] above, and then culturing the cells under a first temperature condition to allow the target gene to be expressed in the cells. method.

[0010]

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

[11] to

[13] above, and then culturing the cells under a second temperature condition to express the gene of interest in the cells (and preferably removing the virus or viral vector from the cells). method.

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

[11] to

[13] above, and then culturing the cells under a second temperature condition to express the gene of interest in the cells (and preferably removing the virus or viral vector from the cells). method.

[24] A method for culturing cells, comprising: Infecting the cells with the minus-strand RNA virus or viral vector according to any one of

[11] to

[13] above, and then culturing the cells under a second temperature condition to express the gene of interest in the cells (and preferably removing the virus or viral vector from the cells). method.

[0011]

[25] Any of the above-mentioned inventions, wherein the minus-strand RNA virus or viral vector is Sendai virus.

[26] A negative-strand RNA virus or viral vector contains an RNA genome and an N protein, a P protein or a temperature-sensitive P protein, an M protein, an F protein, and an H protein. Any of the above-described inventions, which has all of the N protein and the L protein {wherein the RNA genome preferably does not have a gene encoding a functional F protein}.

[0012]

[0101] A degradation-inducing protein, preferably a degradation-inducing protein derived from FKBP12, having an amino acid sequence having amino acid mutations corresponding to 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or all of the amino acid mutations selected from the group consisting 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.

[0102] A degradation-inducing protein, preferably a degradation-inducing protein derived from FKBP12, having 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 relative 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).

[0103] A degradation-inducing protein described in

[0101] or

[0102] above, which has 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 described in SEQ ID NO: 18.

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

[0101] to

[0103] above.

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

[0101] to

[0104] above.

[0106] An antisense oligonucleotide against a nucleic acid encoding a protein according to any one of

[0101] to

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

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

[0101] to

[0103] above.

[0108] An antibody that binds to a protein described in any one of

[0101] to

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

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

[0101] to

[0103] above with a binding affinity that is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 100 times, at least 1,000 times, or at least 10,000 times stronger than that binding to a protein having the amino acid sequence described 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 minus-strand RNA virus or vector comprising any of the above proteins.

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

[0114] A minus-strand RNA genome of the virus or vector described in

[0113] above, which contains any of the above nucleic acids.

[0115] A nucleic acid encoding the genome described in

[0114] above, or a vector containing said nucleic acid.

[0013]

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

[0132] A nucleic acid encoding the M protein described in

[0131] above.

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

[0134] A nucleic acid encoding the HN protein described in

[0133] above.

[0135] An animal cell comprising the M protein described in

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

[0133] above.

[0136] An animal cell comprising the M protein described in

[0131] above and the HN protein described in

[0133] above.

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

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

[0133] above.

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

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

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

[0132] and / or

[0134] above.

[0140] A genome (minus-strand RNA genome) of a minus-strand RNA virus or vector, comprising the nucleic acid described in

[0132] and / or

[0134] above.

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

[0140] above.

[0014]

[0201] A negative-strand RNA virus or vector comprising one or more proteins selected from the group consisting of an N protein, a P protein or a temperature-sensitive P protein, an M protein, an F protein, an HN protein, and an L protein to which a peptide having the amino acid sequence set forth in SEQ ID NO: 29 has been added.

[0202] A negative-strand RNA virus or vector comprising a nucleic acid encoding one or more proteins selected from the group consisting of an N protein, a P protein or a temperature-sensitive P protein, an M protein, an F protein, an HN protein, and an L protein to which a peptide having the amino acid sequence set forth in SEQ ID NO: 29 has been added.

[0203] A minus-strand RNA virus or vector described in

[0201] or

[0202] above, further comprising a nucleic acid encoding a target protein to which a peptide having the amino acid sequence set forth in SEQ ID NO: 29 has been added.

[0204] A genome (minus-strand RNA genome) of a minus-strand RNA virus or vector described in any one of

[0201] to

[0203] above.

[0205] A nucleic acid encoding the genome described in

[0204] above.

[0206] A gene expression vector comprising the nucleic acid described in

[0205] above.

[0207] A cell (e.g., an animal cell, e.g., a host cell or a packaging cell) comprising a minus-strand RNA virus or vector described in any one of

[0201] to

[0203] above, a genome described in

[0204] above, a nucleic acid described in

[0205] above, or a gene expression vector described in

[0206] above.

[0015]

[0251] A method for producing a minus-strand RNA virus or vector, comprising: A method comprising introducing a nucleic acid encoding a minus-strand RNA virus or vector described in any one of

[0201] to

[0203] above into a packaging cell to obtain a minus-strand RNA virus or vector.

[0252] A method for treating cells infected with a minus-strand RNA virus or vector described in any one of

[0201] to

[0203] above, comprising contacting the cells with an effective amount of a binding molecule that binds to the peptide and ubiquitin E3 ligase, thereby removing part or all of the minus-strand RNA virus or vector from the cells.

[0253] The method described in

[0252] above, wherein the binding molecule is (S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl (R)-2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)butanoate.

[0254] The method described in

[0252] or

[0253] above, which is carried out under temperature conditions of 37°C to 38.5°C.

[0255] A method according to any one of

[0252] to

[0254] above, which is carried out in vitro.

[0256] A method according to any one of

[0252] to

[0254] above, which is carried out in vivo.

[0257] A binding molecule that binds to the peptide and ubiquitin E3 ligase, or a composition containing the binding molecule, for use in the method described in

[0256] above.

[0258] A binding molecule that binds to the peptide and ubiquitin E3 ligase, or a composition containing the binding molecule, for use in removing a minus-strand RNA virus or vector from the body of a subject having the minus-strand RNA virus or vector described in any of

[0201] to

[0203] above.

[0259] The binding molecule or a composition comprising the binding molecule according to

[0257] or

[0258] above, wherein the binding molecule is (S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl (R)-2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)butanoate. [Brief explanation of the drawings]

[0016] [Figure 1A] FIG. 1A is a fluorescence micrograph of cells infected with various temperature-sensitive mutants of the P protein, showing the temperature sensitivity of the mutants. [Figure 1B] FIG. 1B shows the relative fluorescence intensity from cells infected with various temperature-sensitive mutants of the P protein, indicating the temperature sensitivity of the mutants. [Figure 2] FIG. 2 shows the relative fluorescence intensity from cells infected with various temperature-sensitive mutants of the P protein, indicating the temperature sensitivity of the mutants. [Figure 3] FIG. 3 shows the relative fluorescence intensity from cells infected with various temperature-sensitive mutants of the P protein, indicating the temperature sensitivity of the mutants. [Figure 4] FIG. 4 shows the relative fluorescence intensity from cells infected with various temperature-sensitive mutants having mutations in the P protein and L protein, indicating the temperature sensitivity of the mutants. [Figure 5A] FIG. 5A shows the relative fluorescence intensity from cells infected with various temperature-sensitive mutants having mutations in the P protein and L protein, indicating the temperature sensitivity of the mutants. [Figure 5B] FIG. 5B shows the relative fluorescence intensity from cells infected with various temperature-sensitive mutants having mutations in the P protein and L protein, obtained from cells packaging various mutants having Lm1558I, indicating the temperature sensitivity of the mutants. [Figure 6] FIG. 6 is a graph showing that the growth of cells into which a cell growth factor has been introduced using a temperature-sensitive vector is promoted. [Figure 7A] FIG. 7A is a fluorescence micrograph showing the expression of NANOG, a pluripotency marker, in cells into which cell reprogramming factors were introduced using a temperature-sensitive vector. [Figure 7B]Figure 7B shows microscopic images of fibroblasts transfected with reprogramming factors 21 and 28 days after transfection. Cells were cultured at 37°C. GFP (mEmerald) is encoded by the SeV genome, and GFP intensity indicates the amount of SeV genome present in the cells. [Figure 8] FIG. 8 shows the effect of stabilizing EmGFP by the DDrs tag. [Figure 9A] FIG. 9A shows the ability of DDm, a mutant of the degradation-inducing protein DD, to remove a temperature-sensitive vector from cells in the absence of a degradation-promoting factor (dTAG-13). [Figure 9B] FIG. 9B shows the ability of DDm, a mutant of the degradation-inducing protein DD, to remove a temperature-sensitive vector from cells in the presence of a degradation-promoting factor (dTAG-13). [Figure 10] Figure 10 shows the effect of introducing mutations into the M protein and HN protein on cytotoxicity due to viral vector infection. The vertical axis shows the fluorescence intensity of EmGFP. EmGFP is encoded in the SeV genome. In this experiment, the control showed cell detachment and a decrease in fluorescent-positive cells, and the EmGFP intensity reflected the number of viable cells. [Figure 11A] FIG. 11A shows the structure of an F protein transfer vector for producing packaging cells. [Figure 11B] Figure 11B shows the production of SeV vectors (i.e., successful functional introduction of the F protein) in cells (LLC-MK2 cells and Vero cells) transfected with the vector of Figure 11A and SeV expressing a nucleic acid encoding Cre recombinase, and the temperature-sensitive elimination of the produced SeV vectors from the cells. [Figure 12] FIG. 12 shows an alignment of the FKBP12 domains of various animal species corresponding to the destabilization domain (DD) derived from human FKBP12. [Figure 13] Figure 13 shows the ability of a temperature-sensitive vector carrying a bromo-tagged P protein to be removed in the presence of a PROTAC. [Figure 14]Figure 14 shows the ability of a temperature-sensitive vector carrying a bromo-tagged P protein to be removed in the presence of a PROTAC. Specific Description of the Invention

[0017] As used herein, the term "negative-strand RNA viral vector" refers to a recombinant virus obtained by modifying a virus having a negative-strand RNA genome (i.e., a negative-strand RNA virus) for the purpose of introducing a target gene. Examples of negative-strand RNA viruses 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 viruses), and Bunyaviridae (bunyaviruses such as hantavirus). Examples of paramyxoviruses include viruses of the Orthoparamyxovirinae subfamily. Examples of viruses of the Orthoparamyxovirinae subfamily include viruses of the Respirovirus genus, such as Sendai virus. In all embodiments, negative-strand RNA viruses and negative-strand RNA viral vectors are preferably Sendai virus (also known as murine parainfluenza type 1 virus) or Sendai virus vectors.

[0018] As used herein, "packaging cells" refer to cells that produce viral vectors. To improve safety, the viral genome of a viral vector is generally engineered to have one or more factors responsible for its growth, replication, and spread (including infection of other cells) disrupted, preventing growth, replication, or spread after cell infection. However, when producing a viral vector, the disrupted factors are complemented in the packaging cells to enable growth, replication, and spread. Therefore, to produce a viral vector, packaging cells express some of the disrupted viral factors to complement the disrupted viral factors and restore virus production. Packaging cells may stably or transiently harbor such factors in their genomes. In either case, the complemented viral factors are supplied intracellularly to the packaging cells during virus production. For example, Sendai virus vectors are classically produced by using a Sendai virus having a genome lacking the F gene in packaging cells that supply the F gene. The supplied F gene is activated in the presence of trypsin, but other types of F genes (F5R) that are activated by furin, which is ubiquitously present in cells, have also been developed, making virus production more convenient (see, for example, WO 2005 / 071085A). In recent years, technologies for producing Sendai virus from cDNA have been developed. For example, vectors have been created using the attenuated Z strain as a base, with modifications to further enhance safety for human medical applications. For example, technologies have been developed to delete one or more of the M, F, and HN genes from the viral genome, thereby eliminating viral transmissibility. For example, F gene-deleted viral genomes are preferably used. The viral genome is operably linked to a regulatory sequence (e.g., a T7 promoter), and the production of the viral genome can be driven by this regulatory sequence. This allows the Sendai virus genome to be produced from cDNA in packaging cells.When a T7 promoter is used as the first control sequence, T7 RNA polymerase can be supplied by a helper virus such as vaccinia virus. N, P, F, and L, operably linked to a control sequence driving transcription by RNA polymerase (e.g., pol II), are expressed in the packaging cells, thereby supplying viral particle components for viral particle formation within the packaging cells. For example, LLC-MK2 cells derived from monkey kidney are used as packaging cells. This produces viral particles that can infect cells once but cannot subsequently spread to other cells. The viral particles can be concentrated and / or purified as needed before use. When incorporating a foreign gene into the virus, a viral-specific control sequence is optionally introduced to enable transcription by RNA-dependent RNA polymerase.

[0019] According to the present invention, a method for producing a minus-strand RNA virus or a minus-strand RNA virus vector is provided.Minus-strand RNA viruses 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).Minus-strand RNA viruses are preferably viruses of the Paramyxoviridae family, preferably paramyxoviruses, and preferably Sendai virus.

[0020] The construction of a negative-strand RNA virus or negative-strand RNA viral vector will be explained using Sendai virus as an example. Sendai virus contains 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 coat of the virus particle. The genome is thought to be encapsulated within the outer coat. Sendai virus can infect cells using the F protein. The outer coat is constructed to contain the F protein, and the genome is modified (deleted, etc.) so that the F protein cannot be produced. This prevents further propagation of the Sendai virus that replicates from the genome after infection due to the lack of F protein. The nucleoprotein (N protein), phosphoprotein (P protein), and large protein (L protein) are believed to be necessary for the autonomous replication of the viral genome or vector genome in the transfected cells. Examples of phosphoproteins (P proteins) 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, the phosphoprotein (P protein) includes, for example, a P protein having the amino acid sequence of the P protein set forth in GenBank accession number BAN84668.1 (SEQ ID NO: 1), or a sequence corresponding to said sequence.Examples of large proteins (L proteins) include L proteins having the amino acid sequence set forth in GenBank accession number BAN84672.1 (SEQ ID NO: 5) or a sequence corresponding to said sequence. The Sendai virus P protein may have an amino acid sequence corresponding to any of the amino acid sequences of the above-mentioned P proteins. The amino acid numbers of the P protein referred to herein are those in the amino acid sequence of SEQ ID NO: 1. Those skilled in the art would be able to identify amino acids corresponding to the amino acid numbers of P proteins having other amino acid sequences, for example, by aligning the amino acid sequences. The Sendai virus L protein has an amino acid sequence corresponding to the amino acid sequence of the above-mentioned L protein. The amino acid numbers of the L protein referred to herein are those in the amino acid sequence of SEQ ID NO: 5. Those skilled in the art would be able to identify amino acids corresponding to the amino acid numbers of L proteins having other amino acid sequences, for example, by aligning the amino acid sequences.

[0021] As used herein, the term "regulatory sequence" refers to a sequence that drives a gene operably linked thereto and has the activity of transcribing RNA from the gene. An example of a regulatory sequence is a promoter. Examples of promoters include class I promoters (which can be used to transcribe rRNA precursors), class II promoters (which are composed of a core promoter and upstream promoter elements and can be used to transcribe mRNA), and class III promoters (which are further broadly divided into types I, II, and III).

[0022] In one embodiment, a minus-strand RNA viral vector has disrupted (e.g., deleted) factors in its genome that are involved in the proliferation or infection of minus-strand RNA viruses in cells, and has reduced or substantially no ability to proliferate or infect cells other than packaging cells. As described above, such a vector is conferred with initial cell infectivity by producing it under conditions in which the disrupted proliferation or infection factors are supplied to the packaging cells. As a result, the vector obtained from the packaging cells is infectious, but the vector that subsequently infects cells other than the packaging cells is unable to produce any more infectious particles, thereby improving the safety of the vector.

[0023] As used herein, "exogenous" means derived from an organism other than the host. Typically, the regulatory sequence may be exogenous. The gene to be expressed may also be exogenous. Thus, in the case of a gene operably linked to a regulatory sequence, both the regulatory sequence and the gene may be exogenous. "Endogenous" means that it is contained in the host itself.

[0024] As used herein, "corresponding to a certain amino acid in protein A" means that in a homolog or ortholog of protein A, the amino acid is present at a position corresponding to the certain amino acid in protein A when aligned.

[0025] <Virus or viral vector of the present invention and its RNA genome> The present invention provides a minus-strand RNA virus or viral vector and its RNA genome. The minus-strand RNA virus may be preferably a mononegavirus, more preferably a virus of the Paramyxoviridae family, even more preferably a virus of the Respirovirus genus of the Paramyxoviridae family, and most preferably a Sendai virus. The present invention also provides viral vectors constructed from these viruses. The virus or viral vector may contain a negative-strand RNA genome (hereinafter sometimes simply referred to as the "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 virus or viral vector may have an RNP complex containing the N protein, P protein, and L protein, as well as the viral genome or viral vector genome. The virus or viral vector may also have a viral particle coat containing the M protein, F protein, and HN protein. In one embodiment of the present invention, the virus or viral vector has at least the P protein. In this embodiment, the P protein is preferably contained in an RNP complex. Also in this embodiment, the virus or viral vector preferably has a viral particle coat comprising an M protein, an F protein, and an HN protein. The P protein may include a functional variant thereof. A functional variant may be, for example, a protein that has 90% or more sequence identity with the amino acid sequence of the P protein set forth in SEQ ID NO: 1 and that functions as a P protein. Functional variants include temperature-sensitive mutants of the P protein. A functional variant may also be, for example, a fragment of the P protein. The fragment may be the C-terminal domain of the P protein, a region containing the N-binding site (in the case of Sendai virus, the region of amino acids 479 to 568 of SEQ ID NO: 1) and the L-binding site (in the case of Sendai virus, the region of amino acids 411 to 445 of SEQ ID NO: 1), or a region further containing an oligomerization site in addition to the above regions (in the case of Sendai virus, the region of amino acids 320 to 446 of SEQ ID NO: 1), or the C-terminal region (in the case of Sendai virus, the region of amino acids 320 to 568 of SEQ ID NO: 1) (see, for example, Blanchard L. et al., Virology. (2004) 319, 201-211). Alternatively, the fragment may be a protein that shares 90% or more sequence identity with the amino acid sequence of the region and has the function of a P protein (e.g., the ability to form a tetramer and / or the function as a polymerase subunit). In one embodiment, the fragment is a C-terminal domain of the P protein, a region containing the N-binding site (in the case of Sendai virus, the region of amino acids 479 to 568 of SEQ ID NO: 1) and the L-binding site (in the case of Sendai virus, the region of amino acids 411 to 445 of SEQ ID NO: 1), or a region that further contains an oligomerization site in addition to the above regions (in the case of Sendai virus, the region of amino acids 320 to 446 of SEQ ID NO: 1), or a protein that has 90% or more sequence identity with the amino acid sequence of the region and has the function of the P protein (e.g., the ability to form a tetramer).In one embodiment, the fragment is the C-terminal domain of the P protein, and may be the C-terminal region (in the case of Sendai virus, the region of amino acids 320 to 568 of SEQ ID NO: 1), or a protein that has 90% or more sequence identity with the amino acid sequence of that region and has the function of the P protein (e.g., function as a polymerase subunit).

[0026] Previous studies have shown that Sendai viruses or Sendai virus vectors prepared by replacing the P protein with a P protein mutant exhibit temperature sensitivity. Specifically, it has been shown that the P protein mutant (SEQ ID NO: 2) containing both the L511F mutation and the D433A / R434A / K437A mutations is a temperature-sensitive mutation, and that viruses or viral vectors containing this P protein mutant are removed from cells by increasing the temperature (WO2010 / 008054, 2017 / 082174).

[0027] Below, temperature-sensitive mutants of the P protein and temperature-sensitive mutants of the L protein are described, but these mutants are described with respect to the P protein and L protein encoded by the viral genome. In contrast, the temperature sensitivity of the P protein and L protein as constituent proteins of a virus or viral vector carrying the viral genome is not an issue. That is, after infecting a target cell, the virus or viral vector amplifies within the cell using proteins translated from the viral genome. As long as the P protein and preferably the L protein of the virus and viral vector amplifying within the cell are temperature-sensitive, the desired effects of the present invention will be achieved.

[0028] Thus, in one embodiment, the P protein and L protein of a virus or viral vector having the viral genome may have a temperature sensitivity that is weaker than that of a temperature-sensitive mutant of the P protein and L protein encoded by the viral genome, and preferably have no temperature sensitivity (e.g., have temperature sensitivity equivalent to that of the wild type). This configuration is expected to reduce the decrease in virus or viral vector production at the packaging cell culture temperature, thereby improving the efficiency of virus or viral vector production. Furthermore, in one embodiment, the P protein and L protein as protein components of a virus or viral vector having the viral genome may have a temperature-sensitive mutant of the P protein, and preferably a temperature-sensitive mutant of the L protein, as described below. The presence or absence and strength of temperature sensitivity at a specific temperature can be determined based on the amount of virus or viral vector after culture at that specific temperature.

[0029] Temperature-sensitive mutants of the P protein and the L protein are described below. In the present invention, the P protein encoded by the viral genome has a temperature-sensitive mutation, which can confer temperature sensitivity to a virus or viral vector. In the present invention, the temperature sensitivity of a virus or viral vector is such that its proliferation is significantly reduced, its amount is significantly reduced, or its amount reaches or falls below the detection limit within a specific temperature range, compared to a virus or viral vector that does not have the corresponding temperature-sensitive mutation in the viral genome. The specific temperature range is not limited as long as it is a temperature at which cell proliferation is not inhibited, but is, for example, about 35°C to about 39°C, preferably 36°C to 38.5°C. In one embodiment of the present invention, the P protein has substitution mutations in amino acids of the P protein corresponding to any one or all of D433, R434, and K437, including G, T, and S. That is, in one embodiment of the invention, the P protein has the amino acid sequence of SEQ ID NO: 4 (excluding SEQ ID NOs: 1 and 2). In this way, the P protein mutant can confer temperature sensitivity to the Sendai virus or Sendai virus vector. In a preferred embodiment of the present invention, the P protein mutant can exhibit higher temperature sensitivity than a P protein mutant containing the D433A / R434A / K437A mutations.

[0030] In one embodiment of the present invention, the P protein has substitution mutations at amino acids in the P protein corresponding to all of D433, R434, and K437, and the substitution mutations include mutations to any of G, T, and S. In one embodiment of the present invention, the P protein has substitution mutations at amino acids in the P protein corresponding to all of D433, R434, and K437, and the substitution mutations are mutations to any of G, T, and S. In one embodiment of the present invention, the P protein has substitution mutations at amino acids in the P protein corresponding to all of D433, R434, and K437, and the substitution mutations are mutations to T. In one embodiment of the present invention, the P protein has substitution mutations at amino acids in the P protein corresponding to all of D433, R434, and K437, and the substitution mutations are mutations to S.

[0031] In one embodiment of the present invention, the P protein may have a substitution mutation in the amino acid sequence corresponding to L511. In one embodiment of the present invention, the amino acid sequence of the L protein corresponding to L511 is preferably mutated to F or Y. For example, the P protein may be a P protein mutant having an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 3. Alternatively, for example, the P protein may be a P protein mutant having an amino acid sequence corresponding to the amino acid sequence set forth in SEQ ID NO: 4.

[0032] In one embodiment of the present invention, the P protein may have a mutation at the amino acid corresponding to D433 to an amino acid selected from the group consisting of S, A, and T, a mutation at the amino acid corresponding to R434 to an amino acid selected from the group consisting of S, A, and T, a mutation at the amino acid corresponding to K437 to an amino acid selected from the group consisting of S, A, and T, and a mutation at the amino acid corresponding to L511 to F or Y. Also, in one embodiment of the present invention, the P protein may have (1S) having amino acid mutations corresponding to D433S, R434A, K437A, and L511F; (2S) with amino acid mutations corresponding to D433S, R434A, K437A, and L511Y; (3S) with amino acid mutations corresponding to D433A, R434S, K437A, and L511F; (4S) with amino acid mutations corresponding to D433A, R434S, K437A, and L511Y; (5S) with amino acid mutations corresponding to D433A, R434A, K437S, and L511F; (6S) with amino acid mutations corresponding to D433A, R434A, K437S, and L511Y; (7S) with amino acid mutations corresponding to D433S, R434S, K437A, and L511F; (8S) with amino acid mutations corresponding to D433S, R434S, K437A, and L511Y; (9S) with amino acid mutations corresponding to D433S, R434A, K437S, and L511F; (10S) with amino acid mutations corresponding to D433S, R434A, K437S, and L511Y; (11S) with amino acid mutations corresponding to D433A, R434S, K437S, and L511F; (12S) with amino acid mutations corresponding to D433A, R434S, K437S, and L511Y; (13S) with amino acid mutations corresponding to D433S, R434S, K437S, and L511F; (14S) with amino acid mutations corresponding to D433S, R434S, K437S, and L511Y; (1T) having amino acid mutations corresponding to D433T, R434A, K437A, and L511F; (2T) with amino acid mutations corresponding to D433T, R434A, K437A, and L511Y; (3T) having amino acid mutations corresponding to D433A, R434T, K437A, and L511F; (4T) with amino acid mutations corresponding to D433A, R434T, K437A, and L511Y; (5T) with amino acid mutations corresponding to D433A, R434A, K437T, and L511F; (6T) with amino acid mutations corresponding to D433A, R434A, K437T, and L511Y; (7T) with amino acid mutations corresponding to D433T, R434T, K437A, and L511F; (8T) with amino acid mutations corresponding to D433T, R434T, K437A, and L511Y; (9T) with amino acid mutations corresponding to D433T, R434A, K437T, and L511F; (10T) with amino acid mutations corresponding to D433T, R434A, K437T, and L511Y; (11T) with amino acid mutations corresponding to D433A, R434T, K437T, and L511F; (12T) with amino acid mutations corresponding to D433A, R434T, K437T, and L511Y; (13T) with amino acid mutations corresponding to D433T, R434T, K437T, and L511F; (14T) with amino acid mutations corresponding to D433T, R434T, K437T, and L511Y; (1ST) has amino acid mutations corresponding to D433S, R434T, K437S, and L511F, or (2ST) It is preferable to have amino acid mutations corresponding to D433S, R434T, K437S, and L511Y.

[0033] In one embodiment of the present invention, the virus or viral vector has genes encoding at least a P protein and an L protein. The P protein encoded by the RNA genome has any of the mutations described above. The L protein encoded by the RNA genome may also have a temperature-sensitive mutation, conferring temperature sensitivity to the virus or viral vector. In the present invention, the temperature sensitivity of a virus or viral vector is such that its proliferation is significantly reduced, its amount is significantly reduced, or its amount reaches or falls below the detection limit in a specific temperature range, compared to a virus or viral vector lacking the corresponding temperature-sensitive mutation. In one embodiment, the virus or viral vector preferably has substitution mutations in one or all of the amino acids corresponding to N1197, L1361, L1558, and K1796. The amino acid mutation corresponding to N1197 is preferably, for example, a mutation to S. The amino acid mutation corresponding to L1361 is preferably, for example, a mutation to C. The amino acid mutation corresponding to L1558 is preferably, for example, a mutation to I. The amino acid mutation corresponding to K1796 is preferably, for example, a mutation to E. Thus, in one embodiment, the L protein preferably has one or more or all (preferably all) of the N1197, L1361C, L1558I, and K1796 mutations. In one embodiment, it is preferred to have any or preferably all of the N1197S, L1558I, and K1796E mutations, and more preferably to further have L1361C. That is, in one embodiment of the invention, the L protein has the amino acid sequence of SEQ ID NO: 6 (excluding SEQ ID NO: 5).

[0034] Thus, in one embodiment of the present invention, the virus or viral vector has genes encoding at least a P protein and an L protein, and the P protein encoded by the RNA genome preferably has any of the mutations described above, and the L protein encoded by the RNA genome preferably has any of the mutations described above. That is, in one embodiment, the P protein preferably 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 one embodiment, the P protein preferably 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 G, T, or S, and the L protein preferably has either or both (preferably both) of L1361C and L1558I. In one embodiment, the P protein has substitution mutations at amino acids in the P protein corresponding to all of D433, R434, and K437, and the substitution mutations include a mutation to any of G, T, and S; the P protein has substitution mutations in L511 or the amino acid sequence corresponding to L511, and the substitution mutations are a mutation to F or Y; and the L protein preferably has either or both (preferably both) of L1361C and L1558I. In one embodiment, the P protein has substitution mutations at amino acids in the P protein corresponding to all of D433, R434, and K437, and the substitution mutations are a mutation to G, T, and S; the P protein has substitution mutations in L511 or the amino acid sequence corresponding to L511, and the substitution mutations are a mutation to F or Y; and the L protein preferably has either or both (preferably both) of L1361C and L1558I. In one embodiment, the P protein preferably has any of the above-mentioned (1S)-(14S), (1T)-(14T), and (1ST)-(2ST) mutations, and the L protein preferably has either or both of L1361C and L1558I (preferably both). Mutations in the P protein can be used in combination with mutations in other proteins. Furthermore, mutations in the L protein can be used in combination with mutations in other proteins.

[0035] In certain embodiments of the present invention, the virus or viral vector may have an M protein or a mutant thereof. The M protein mutant may have substitution mutations at amino acids in the M protein corresponding to one to three amino acids selected from the group consisting of G69, T116, and A183. The M protein mutant may, for example, have a mutation at an amino acid corresponding to G69, e.g., a mutation corresponding to G69E. The M protein mutant may, for example, have a mutation at an amino acid corresponding to G116, e.g., a mutation corresponding to T116A. The M protein mutant may, for example, have a mutation at an amino acid corresponding to A183, e.g., a mutation corresponding to A183T or A183S, preferably a mutation corresponding to A183S. In certain embodiments, the M protein mutant may have amino acid mutations corresponding to G69E, T116A, and A183T, or preferably, amino acid mutations corresponding to G69E, T116A, and A183S. Mutations in the M protein can reduce the cytotoxicity of SeV, and can be used in combination with mutations in other proteins.

[0036] In certain embodiments of the present invention, the virus or viral vector may have an HN protein or a mutant thereof. The mutant HN protein may have a substitution mutation at an amino acid in the HN protein corresponding to one to three amino acids selected from the group consisting of A262, G264, and K461. The HN protein may have a mutation at an amino acid corresponding to A262, for example, a mutation corresponding to A262T. The HN protein may have a mutation at an amino acid corresponding to G264, for example, a mutation corresponding to G264R or G264K, preferably a mutation corresponding to G264K. The HN protein may have a mutation at an amino acid corresponding to K461, for example, a mutation corresponding to K461E. In certain embodiments, the mutant HN protein may have amino acid mutations corresponding to A262T, G264R, and K461E, or preferably, amino acid mutations corresponding to A262T, G264K, and K461E. Mutations in the HN protein can reduce the cytotoxicity of SeV. HN protein mutations can be used in combination with mutations in other proteins.

[0037] In certain embodiments of the present invention, the P protein, L protein, M protein, and HN protein can each have the mutations described above.

[0038] The amino acid substitutions defined above may be conservative substitutions for the mutated amino acid, e.g., a mutation to an uncharged polar amino acid may be mutated to another uncharged polar amino acid, and vice versa. Conservative substitutions are: 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)); the group of uncharged polar amino acids (asparagine (N), glutamine (Q), serine (S), threonine (T), and tyrosine (Y)), and A group of nonpolar amino acids (alanine (A), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), methionine (M), tryptophan (W), and cysteine ​​(C)) The term "non-conservative substitution" refers to an amino acid substitution that is not a conservative substitution.

[0039] In some embodiments, the virus or viral vector may further carry a gene of interest (GOI). The GOI is located in front of the N gene (hereafter referred to as "+"), between the P and M genes (hereafter referred to as "PM"), between the M and HN genes (hereafter referred to as "MHN"), or between the HN and L genes (hereafter referred to as "HNL") on the RNA genome, and it is known that gene expression levels decrease in this order of location.

[0040] In one embodiment, the virus or viral vector may comprise a genome including genes encoding the nucleoprotein (N protein), phosphoprotein (P protein), matrix protein (M protein), fusion protein (F protein), hemagglutinin-neuraminidase (HN protein), and large protein (L protein). In another embodiment, the virus or viral vector may comprise a genome that does not include a gene encoding a functional fusion protein (F protein) (preferably, the gene is deleted), but includes genes encoding the nucleoprotein (N protein), phosphoprotein (P protein), matrix protein (M protein), hemagglutinin-neuraminidase (HN protein), and large protein (L protein). In all embodiments of the present invention, the gene encoding the P protein on the genome, and optionally the gene encoding the L protein, each preferably contain the temperature-sensitive mutation described above. This allows the virus or viral vector propagated in infected cells to exhibit temperature-sensitive effects. In all embodiments of the present invention, the P protein and L protein on the virus or viral vector particle may or may not contain temperature-sensitive mutations. 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 have been altered to that derived from another virus. This allows the cell tropism of the virus to be altered. In a preferred embodiment, the genome of the virus or viral vector has a P protein and / or an L protein with the above-mentioned temperature-sensitive mutation, and the virus particle of the virus or viral vector itself may contain a wild-type P protein and / or a wild-type L protein, or may not contain a temperature-sensitive P protein or a temperature-sensitive L protein.

[0041] In one aspect, the virus or viral vector has a viral particle comprising an RNP complex including a genome, a nucleoprotein (N protein), a phosphoprotein (P protein), and a large protein (L protein), and a matrix protein (M protein), a fusion protein (F protein), and a hemagglutinin-neuraminidase (HN protein), and the viral particle comprises the complex.

[0042] In certain embodiments, the virus or viral vector may contain a gene of interest operably linked to a regulatory sequence. The virus or viral vector can express the gene of interest operably linked to the regulatory sequence in infected cells. The gene of interest may be transcribed into RNA, which exhibits physiological activity, or translated via RNA into a protein, which exhibits physiological activity. A "regulatory sequence" refers to a sequence that drives a gene operably linked to it and has the activity of transcribing RNA from the gene. The gene of interest may be exogenous to the virus or viral vector. The gene of interest may also be exogenous to the infected cell. Here, "exogenous" means that the gene is not naturally present in the infected cell. An example of a regulatory sequence is a promoter. Examples of promoters include class I promoters (which can be used to transcribe pre-rRNA), class II promoters (which are composed of a core promoter and upstream promoter elements and can be used to transcribe mRNA), and class III promoters (further classified into types I, II, and III).

[0043] The virus or viral vector can infect animal cells (particularly mammalian cells, preferably human cells). The virus or viral vector can preferably amplify its genome in animal cells (particularly 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. This temperature sensitivity allows the virus or viral vector to be removed from the cell.

[0044] The virus or viral vector can be produced by a conventional virus or viral vector production method. During production, a gene encoding any of the P protein variants can be used as the gene encoding the P protein. Furthermore, during production, a gene encoding any of the L protein variants can be used as the gene encoding the L protein. Furthermore, the RNA genome of the 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 virus or viral vector. The nucleic acid having a region encoding the RNA genome of the virus or viral vector preferably further comprises a regulatory sequence, and the region is operably linked to the regulatory sequence. The nucleic acid having a region encoding the RNA genome of the virus or viral vector is preferably carried in an expression vector for the nucleic acid. Expression vectors include, but are not limited to, plasmid vectors, viral vectors, cosmid vectors, artificial chromosomes, and the like. In addition to the expression cassette for the RNA genome operably linked to the regulatory sequence, the expression vector may further comprise an origin of autonomous replication (Ori) and a positive selection marker gene (e.g., a drug resistance gene, a gene encoding a fluorescent protein, etc.). It is known that the RNA genome of a virus or viral vector is produced in packaging cells that supply viral protein components and the like. The packaging cells supply the components contained in the produced virus and viral vector particles. It is preferable that the virus and viral vector particles are produced in large quantities in the packaging cells. In this respect, it is preferable that the protein supplied to the packaging cells does not have the temperature sensitivity described herein, and for example, it is preferable that it is a wild-type protein. The virus produced in this way can be titered by conventional methods. The titer is 1 x 10 5 CIU / mL or more, 2×10 5CIU / mL or more, 3×10 5 CIU / mL or more, 4×10 5 CIU / mL or more, 5×10 5 CIU / mL or more, 6×10 5 CIU / mL or more, 7×10 5 CIU / mL or more, 8×10 5 CIU / mL or more, 9×10 5 CIU / mL or more, 1×10 6 CIU / mL or more, 2×10 6 CIU / mL or more, 3×10 6 CIU / mL or more, 4×10 6 CIU / mL or more, 5×10 6 CIU / mL or more, 6×10 6 CIU / mL or more, 7×10 6 CIU / mL or more, 8×10 6 CIU / mL or more, 9×10 6 CIU / mL or more, 1×10 7 CIU / mL or more, 2×10 7 CIU / mL or more, 3×10 7 CIU / mL or more, 4×10 7 CIU / mL or greater, or 5 × 10 7 It can be CIU / mL or higher.

[0045] <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 a viral vector, and a method for culturing cells infected with a virus or a viral vector.

[0046] According to the present invention, a method for infecting cells with a virus or viral vector comprises contacting cells with the virus or viral vector of the present invention described above under an environment suitable for infection. By incorporating a selectable marker gene (e.g., a visualization marker gene such as a fluorescent protein, and a drug resistance gene) into the virus or viral vector, cells infected with the virus or viral vector can be selected using the expression of the selectable marker gene as an indicator.

[0047] The present invention provides 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 comprises culturing 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 using conventional methods. The present invention also provides cells infected with the virus or viral vector of the present invention.

[0048] In one aspect, a method of culturing cells infected with a virus or viral vector comprises culturing the cells under a first temperature condition. In one aspect, a method of culturing cells infected with a virus or viral vector can comprise culturing the cells under a first temperature condition and then culturing the cells under a second temperature condition.

[0049] The first temperature condition is a condition suitable for the maintenance or proliferation of the virus or viral vector that has infected the cells.

[0050] The second temperature conditions are suitable for reducing or eliminating the amount of virus or viral vector.

[0051] The first temperature is lower than the second temperature.

[0052] When maintaining the virus or viral vector of the present invention in cells, cells infected with the virus or viral vector of the present invention can be cultured, for example, under a first temperature condition. When reducing or eliminating the virus or viral vector of the present invention, cells infected with the virus or viral vector of the present invention can be cultured, for example, under a second temperature condition. 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 proliferation 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 determine the appropriate first and second temperatures.

[0053] A method for culturing cells infected with a virus or viral vector can 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 the 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 (e.g., when overexpressing a gene of interest carried by the virus or viral vector), 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 the 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 the virus or viral vector is not affected by temperature sensitivity at 37°C, infected cells can be cultured at 37°C. For example, if the virus or viral vector is affected by temperature sensitivity at 37°C, infected cells can be cultured at a lower temperature, for example, between 35°C and 37°C. However, conditions suitable for the maintenance or propagation of the virus or viral vector of the present invention are those that do not significantly adversely affect the maintenance or propagation of cells.

[0054] A method for culturing cells infected with a virus or viral vector can include culturing the cells under conditions suitable for reducing the amount of the virus or viral vector. In one embodiment, a method for culturing cells infected with a virus or viral vector can include culturing the cells under conditions suitable for maintaining or propagating 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 amount of the virus or viral vector of the present invention in cells (e.g., when expressing a gene of interest carried by the virus or viral vector and then removing the virus or viral vector from the cells), infected cells can be cultured under conditions suitable for reducing the amount of 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 determined appropriately by those skilled in the art. For example, if the virus or viral vector is not affected by temperature sensitivity at 37°C, infected cells can be cultured at temperatures above 37°C (e.g., temperatures above 37°C and below 39°C, e.g., 37.5°C to 38.5°C). For example, if 37°C is affected by temperature sensitivity, infected cells can be cultured at a temperature equal to or higher than 37°C. In this way, infected cells can be cultured at a temperature (first temperature) that is not or is less affected by temperature sensitivity, and when it is desired to remove the virus or viral vector from the infected cells, the culture temperature can be raised to a second temperature. However, conditions suitable for reducing the virus or viral vector of the present invention are those that do not significantly adversely affect the maintenance or proliferation of the cells (particularly, irreparable damage or injury, etc.). In this way, the virus or viral vector can be removed from cells infected with the virus or viral vector. Thus, in one embodiment, a method is provided for obtaining cells free of the virus or viral vector from cells infected with the virus or viral vector of the present invention.Cells that do not contain a virus or a viral vector can be tested by, for example, detecting the absence of the virus or viral vector genome within the cells. Genome detection can be appropriately determined by those skilled in the art using well-known techniques such as polymerase chain reaction (PCR). The above-described cell culture method allows for transient gene transfer and gene expression.

[0055] According to the present invention, cells containing or infected with the virus or viral vector of the present invention may be cultured at the second temperature from the beginning. That is, it takes time for the virus or viral vector of the present invention to be removed from the infected cells. Therefore, by the time of removal, genes and the like carried by the virus or viral vector can be expressed in the cells, achieving the intended purpose. Therefore, it may be acceptable to culture the cells at the second temperature rather than at the first temperature.

[0056] The first temperature and the second temperature may each be within a certain temperature range, but preferably may be a certain temperature (± error range). The certain temperature range, for the first temperature, is a temperature range (higher temperature range) suitable for maintaining or propagating a virus or viral vector, and for the second temperature, is a temperature range (lower temperature range) suitable for removing (reducing) the virus or viral vector. For the purpose of further maintaining or culturing cells after removing the virus or viral vector, neither temperature range significantly harms cell viability or causes significant damage to the cells. Preferably, neither temperature range substantially affects cell viability.

[0057] According to the present invention, there is provided a method for inducing differentiation of cells, comprising the steps of: infecting the cells with the virus or viral vector of the present invention; Then, culturing the infected cells to induce differentiation of the cells; After differentiation induction, increasing the culture temperature to remove the virus or viral vector of the present invention from the differentiation-induced cells; The present invention provides a method comprising the steps of: (a) inducing differentiation into a stable cell state; and (b) expressing a differentiation-inducing factor in the virus or viral vector. Differentiation induction means differentiating a cell into another stable cell state. Therefore, a method for inducing differentiation of a cell is synonymous with a method for producing a differentiation-induced cell. Various known methods can be used to differentiate a cell into another stable cell state. The virus or viral vector of the present invention can be equipped with a differentiation-inducing factor that promotes differentiation induction. The cells can be induced to differentiate into somatic cells such as neurons, cardiomyocytes, chondrocytes, bone cells, blood cells (e.g., leukocytes), hepatocytes, and pancreatic beta cells. Differentiation into these somatic cells can be achieved by methods known to those skilled in the art.

[0058] According to the present invention, there is provided a method for reprogramming a cell (nuclear reprogramming), comprising: infecting the cells with the virus or viral vector of the present invention; Then, culturing the infected cells to reprogram the cells; After differentiation induction, increasing the culture temperature to remove the virus or viral vector of the present invention from the reprogrammed cells; The method includes carrying a reprogramming factor in the virus or viral vector in an expressible manner. Examples of the reprogramming factor include, but are not limited to, Yamanaka factors (OCT4, SOX2, KLF4, and optionally c-MYC), Thomson factors (OCT4, SOX2, NANOG, LIN-28), and other reprogramming factors. The reprogramming factor may also be, for example, KLF4, OCT4, SOX2, and MYCL.

[0059] The present invention can be used in a variety of applications, including but not limited to those described above, in which it is desired to transiently express proteins or RNA in cells and then remove the vector. Protein components for genome editing systems (e.g., zinc finger nucleases, TALENs, and CRISPR / Cas systems (e.g., CRISPR / Cas9 systems)) and, if necessary, RNA components are expressed in cells, and if necessary, an RNA-protein complex (RNP complex) is formed to edit nucleic acids (e.g., DNA) such as the genome of the cell, and the vector is then removed after editing.

[0060] <Removal of virus or viral vector from infected cells using degrons> In the present invention, degrons can also be used to remove viruses or viral vectors that have infected cells. Degrons may be used in combination with the above-mentioned temperature-sensitive mutants and temperature-shift-based virus or viral vector removal. Various degrons can be used, such as those derived from FK506-binding protein (FKBP12) and dihydrofolate reductase (DHFR) (see US 2009 / 0215169A). The destabilizing domains (DD) of these proteins are degradation-inducible and can be used to induce degradation of other proteins (target proteins) by fusing them with the target proteins. The DD tag and its degradation-inducible variants can be added, for example, to the N- or C-terminus of proteins.

[0061] In the present invention, the FKBP12-derived DD may have an amino acid mutation corresponding to any one of the amino acid mutations 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 at the amino acid corresponding to F36, particularly F36V. Furthermore, the FKBP12-derived DD preferably has L106P. Furthermore, the FKBP12-derived DD preferably has F36V and L106P. In the present invention, the FKBP12-derived DD preferably 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 of the mutations.

[0062] The present invention provides the N protein, P protein, M protein, F protein, HN protein, and L protein fused with the above-mentioned DD, as well as genes encoding any of these. The present invention also provides a cell, Sendai virus, Sendai virus vector, or Sendai virus genome containing one or more proteins selected from the group consisting of the N protein, P protein, M protein, F protein, HN protein, and L protein fused with the above-mentioned DD, and / or one or more genes encoding these proteins. These fusion proteins may contain mutants of the above-described proteins in place of each of the proteins. The mutants are as described herein.

[0063] The present invention particularly provides a P protein fused with the above-mentioned DD and a gene encoding the P protein. The present invention also provides a cell, Sendai virus, Sendai virus vector, or Sendai virus genome containing the P protein fused with the above-mentioned DD and / or a gene encoding the P protein. Here, a mutant of the P protein described herein may be used in place of the P protein.

[0064] If the P protein fused to the DD is essential for the intracellular proliferation of a minus-strand RNA virus or vector, the minus-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. To maintain the minus-strand RNA virus or vector within the cell, cells can be cultured in the presence of a stabilizing molecule such as Shield1 or its derivative. Thus, the present invention allows for the control of the removal (and preferably maintenance) of a virus or viral vector within the cell using a degradation-promoting molecule, and preferably also using a stabilizing molecule. dTAG-13 is a PROTAC (Proteolysis Targeting Chimera) that has a site that binds to the DD or its mutant and a site that binds to ubiquitin E3 ligase, and induces the degradation of the fusion protein of the DD or its mutant. [ka] [ka]

[0065] Bromotags can also be used as degrons (Bond et al., J. Med. Chem., 2021, 64, 20, 15477-15502). Examples of bromotags include Brd4, a peptide having the amino acid sequence set forth in SEQ ID NO: 29. BD2(L387A)and a portion of the Brd4 protein having the corresponding amino acid sequence. Bromotags have proteolysis-inducing properties and can be used to induce degradation of other proteins (target proteins) by fusing them with the target proteins. Bromotags have 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 29 and are capable of inducing degradation of the target protein fused to the tag in the presence of a bromotag ligand. Bromotags can be added to the N-terminus or C-terminus of a protein. In this way, a fusion protein of the target protein and bromotag can be obtained.

[0066] The present invention provides the N protein, P protein, M protein, F protein, HN protein, and L protein fused with the above-mentioned bromotag, as well as genes encoding any of these. The present invention also provides a cell, Sendai virus, Sendai virus vector, or Sendai virus genome containing one or more proteins selected from the group consisting of the N protein, P protein, M protein, F protein, HN protein, and L protein fused with the above-mentioned bromotag, and / or one or more genes encoding these proteins. These fusion proteins may contain mutants of the above-described proteins instead of each of the proteins. The mutants are as described herein.

[0067] The present invention particularly provides the P protein fused with the bromo tag and a gene encoding the P protein. The present invention also provides a cell, a Sendai virus, a Sendai virus vector, or a Sendai virus genome comprising the P protein fused with the bromo tag and / or a gene encoding the P protein. Here, a P protein mutant described herein may be used instead of the P protein. In one embodiment, the bromo tag is fused to the C-terminus of the P protein. In one embodiment, the bromo tag is fused to a temperature-sensitive variant of the P protein. In one embodiment, the temperature-sensitive variant of the P protein can be any of those described above. In one embodiment, the bromo tag is fused to the C-terminus of a temperature-sensitive variant of the P protein. Temperature-sensitive variants can be, for example, P2m3SY, P2m2S434TY, and P2m3T. The temperature-sensitive mutant fused with the bromo tag has, for example, the amino acid sequence of any of SEQ ID NOs: 30 to 32. In one embodiment, the bromo tag is fused to a temperature-sensitive variant of the L protein. In one embodiment, the bromotag is fused to the C-terminus of a temperature-sensitive variant of the L protein. In one embodiment, the temperature-sensitive variant of the L protein can be any of those described above.

[0068] When the P protein fused with the bromotag is essential for the intracellular proliferation of a minus-strand RNA virus or vector, the minus-strand RNA virus or vector can be removed from the cell in the presence of a degradation-promoting molecule such as a bromotag ligand (e.g., AGB1) or its derivative. Thus, the present invention allows the removal (and preferably maintenance) of a virus or viral vector in the cell to be controlled using a degradation-promoting molecule. The bromotag ligand (e.g., AGB1) is a PROTAC (Proteolysis Targeting Chimera) that has a site that binds to the bromotag or its mutant and a site that binds to ubiquitin E3 ligase, and induces the degradation of the fusion protein of the bromotag or its mutant. AGB1 is (S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl (R)-2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)butanoate.

[0069] <Packaging cells and their manufacturing method> In the present invention, minus-strand RNA viruses or vectors such as SeV can be produced using existing packaging cells. However, in the present invention, new packaging cells can be produced, and the resulting packaging cells can be used to produce viruses or vectors.

[0070] In one embodiment, the packaging cells are cell lines, preferably stably established cell lines. Packaging cells may be any type of cell that can be infected with a minus-strand RNA virus or vector, including, but not limited to, LLC-MK2 cells and Vero cells. Packaging cells typically express one or more viral components, such as the F protein. Packaging cells may also be loaded with a PKR inhibitor (e.g., VAI).

[0071] The packaging cells contain genes encoding one or more viral components, such as the F protein, and preferably express the genes at a certain level or higher. The genes are operably linked to regulatory sequences, and their expression is driven by the regulatory sequences. The expression level is determined by various factors, such as the regulatory sequences, the genomic location where the genes are integrated, and the copy number of the genes inserted into the genome. To ensure high expression levels, marker genes (e.g., drug resistance genes and visualization marker genes (e.g., fluorescent proteins)) linked under the same promoter can be used during gene transfer. Specifically, to obtain a high-expression strain, a marker gene is first operably linked to a regulatory sequence via a first site-specific recombination sequence, and then a gene encoding one or more viral components, such as the F protein, is linked via a second site-specific recombination sequence. In this configuration, the marker gene is located between the first and second site-specific recombination sequences (referred to as an intervening sequence). The first and second site-specific recombination sequences recombine in the presence of a recombinase, removing the intervening sequence. After the intervening sequence is removed by a recombinase, the control sequence is designed to drive genes encoding one or more viral components, such as the F protein. Furthermore, a marker gene is present in the intervening sequence, and cells in which the marker gene is strongly expressed can be selected based on the expression intensity of the marker gene. If the marker gene is a drug resistance gene, cells that express the drug resistance gene at a certain level or higher are selected by selecting cells with a certain concentration of the drug. If the marker gene is a visible marker, cells that express the visible marker at a certain level or higher are selected by measuring the expression intensity of the visible marker (e.g., measuring its fluorescence intensity in the case of GFP). In this way, cells with a high expression intensity of the marker gene are selected. It is suggested that the gene is highly expressed in the selected cells due to various factors, such as the location on the genome where the gene is integrated and the copy number of the gene inserted into the genome.The selected cells are cloned, and the resulting clones are subjected to the action of a site-specific recombinase that recognizes the site-specific recombination sequence and induces recombination. This removes the intervening sequence, and a gene encoding one or more viral components, such as an F protein, is operably linked to the control sequence. In this manner, packaging cells can be obtained. The site-specific recombinase can be introduced into cells, for example, using a removable minus-strand RNA viral vector, such as a Sendai virus vector (e.g., temperature-sensitive SeV). After the site-specific recombinase is introduced, the vector can be removed to reduce it to below the detection limit, allowing the cells to be suitably used as packaging cells. After the intervening sequence is removed, one site-specific recombination sequence may remain between the control sequence and the introduced gene.

[0072] The site-specific recombinase and its recognition sequence are not particularly limited, and examples of site-specific recombination reaction systems that can be used include the system of the site-specific recombinase Cre recombinase (hereinafter sometimes simply referred to as "Cre") and its recognition sequence loxP, the system of the site-specific recombinase Flp recombinase (hereinafter sometimes simply referred to as "Flp") and its recognition sequence FRT, the system of Dre recombinase and its recognition sequence rox (Anastassiadis, K. et al., Dis. Model. Mech. (2009) 2: 508-515), and the system of φC31 recombinase and its recognition sequence attP / attB (Belteki G et al., Nat. Biotechnol. (2003) 21: 321-324). The SCre-SloxP system and the VCre-VloxP system (E. Suzuki and M. Nakayama, Nucleic Acid Res. (2011) 39(8): e49), which are improvements on the Cre-loxP system, can also be used. Mutant FRT sequences (e.g., FRT(f2161) and FRT(f2262)) such as the FRT sequence described in International Publication No. 2001 / 023545, and mutant loxP sequences such as the loxP sequence described in Japanese Patent Application Laid-Open No. 11-196880, may also be used. Those skilled in the art can freely select and use the recognition sequences for such site-specific recombinases.

[0073] The present invention provides a genome of a minus-strand RNA virus or vector, comprising a nucleic acid encoding any of the above proteins. The present invention also provides a nucleic acid encoding the genome, and a vector comprising the nucleic acid. The vector may be a cloning vector or an expression vector for the genome. In a preferred embodiment, the expression vector is capable of transcribing the genome in a packaging cell. [Example]

[0074] Example 1: Preparation of minus-strand RNA viral vector A negative-strand RNA viral vector was expressed in viral packaging cells, and the temperature sensitivity of the P protein mutation during expression was evaluated.

[0075] Sendai virus (SeV) was used as the negative-strand RNA viral vector. The Sendai virus was harvested from the culture supernatant after 3 days after transfection of a plasmid mixture into F gene-expressing cells. The plasmid mixture contained pSeV-EmGFP, which carries the Sendai virus genome operably linked to a T7 promoter. The Sendai virus genome carried a gene encoding EmGFP, allowing detection of genome replication by fluorescence. The Sendai virus genome contained a deletion of the F gene.

[0076] (1) F gene-expressing cells were prepared as follows. The SeV F gene (with a Kozak sequence and optimized for human codons) was inserted into pCAGGS-neo to construct pCAGGS-F-neo. The resulting plasmid was transfected into Vero or LLC-MK2 cells using ViaFect (Promega), and selection was performed using 1–2 mg / mL G418 disulfate solution (Nacalai Tesque) to obtain F gene-expressing cells. The resulting cells are designated Vero-F and LLC-MK2-F.

[0077] (2) F gene-deficient SeV was constructed as follows. Based on the sequence information of the SeV-Z strain (Accession No. AB855655), we constructed pSeV / dF, which allows transcription of an F gene-deleted SeV genome driven by the T7 promoter. To minimize the cytotoxicity of SeV-Z, 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, e.g., WO 2003 / 025570). To evaluate the temperature sensitivity and deletion of SeV, we incorporated a GFP mutant, EmGFP, into the genome as a gene of interest (GOI). The gene expression level decreases in the following order: before the N gene (hereafter referred to as "+"), between the P gene and the M gene (hereafter referred to as "PM"), between the M gene and the HN gene (hereafter referred to as "MHN"), and between the HN gene and the L gene (hereafter referred to as "HNL"). The EmGFP gene was mainly placed at the "+" position before the N gene of SeV. In this example, amino acid substitution mutations were introduced into 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 SeV genome. While both temperature-sensitive and temperature-insensitive P proteins (e.g., wild-type) can be used in packaging cells, the use of temperature-insensitive P proteins (e.g., wild-type) resulted in higher virus production efficiency. Therefore, in this example, a gene encoding a P protein with a temperature-sensitive mutation was carried in the genome, but the wild-type P protein was expressed in packaging cells and introduced into virus particles.

[0078] (3) The plasmid for SeV reconstruction was constructed as follows. Based on WO2005 / 071092, we added a Kozak sequence and optimized the codons to human codons to construct pCAGGS-NPco, pCAGGS-P4C(-)co, pEF1-Lco, and pCAGGS-F5Rco. We introduced 430P, 849I, and 880Y mutations into T7 based on P2001-54387A, and 644Y and 667Y mutations based on P2003-61683A. pCAGGS-T7mco, carrying the resulting T7m sequence, was constructed. P4C(-) is a mutation that eliminates expression of the C protein from the P locus.

[0079] The types of mutations in the P protein and the names of the mutant strains 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)

[0080] The ratio of SeV reconstitution plasmid to transfection reagent was determined in accordance with WO 2005 / 071092. Specifically, the following weight amounts of plasmid and transfection reagent (TransIT-LT1 Reagent or ViaFect) were mixed to obtain a plasmid mix.

[0081] NP, P4C(-), F5R, T7: 0.5μg each L: 2 μg pSeV: 5 μg Total plasmid: 9 μg TransIT-LT1 Reagent or ViaFect: 15 μL

[0082] Unless otherwise specified below, the volume of the transfection reagent and the weight of the plasmid were mixed at a ratio of 9:15.

[0083] The plasmid and transfection reagent were mixed in 225 μL of OptiMEM and transfected into F gene-expressing cells in 500 μL of medium (10% FBS / E-MEM) in a 12-well plate. The cells were then cultured at 37°C. From the day after transfection, the cells were cultured at 32°C. The medium was replaced daily with serum-free medium (ITS-X / NEAA / E-MEM) supplemented with 2.5 μg / mL trypsin. The culture supernatant was collected on day 3 after transfection and used to infect F gene-expressing cells seeded in a culture flask for SeV amplification. The culture supernatant was collected from the flask on days 3 to 5, diluted 10- to 100,000-fold, and used to infect Vero cells seeded in a 96-well plate. The number of GFP-positive cells was counted 3 days after infection using an ECLIPSE Ti2-E fluorescence microscope (Nikon), and the infectious titer was calculated.

[0084] Vero cells were infected with the resulting SeV mutants (MOI = 20). Infected cells were cultured at 35°C or 38.5°C for 3 days. Because the SeV vector expresses EmGFP intracellularly, infected cells emit fluorescence proportional to the amount of SeV in the cells. The amount of SeV in infected cells was estimated by measuring the fluorescence from the cells. The results are shown in Figures 1A and 1B. As shown in Figure 1A, the P2m3SY mutant exhibited strong fluorescence at 35°C but significantly decreased at 38.5°C. Figure 1B shows the fluorescence intensity values. At 38.5°C, the fluorescence intensity of the P2m3SY mutant decreased to approximately 0.013% compared to 35°C. At 38.5°C, the fluorescence intensity of the P2 mutant decreased to approximately 5.7% compared to 35°C. These results indicate that the P2m3SY mutant is more than 400-fold more temperature-sensitive than the P2 mutant.

[0085] (4) Introduction of additional temperature-sensitive mutations Next, Vero cells were infected with the resulting SeV mutants (MOI = 20). The infected cells were cultured at 35°C or 38.5°C for 3 days, and the amount of SeV in the infected cells was estimated by measuring the fluorescence from the cells as described above. The fluorescence intensity from the P2 culture at 35°C was set at 1, and the relative fluorescence intensity was calculated. The results are shown in Figure 2 and Table 1. As shown in Figure 2, each mutant showed slight temperature sensitivity at 37°C, but the fluorescence intensity significantly decreased when cultured at 38.5°C. Comparison of the relative fluorescence in Table 1 revealed that all mutants had improved temperature sensitivity compared to P2. Among the mutants, P2m433S exhibited the greatest temperature sensitivity.

[0086] [Table 1]

[0087] Vero cells were infected with the resulting SeV mutants (MOI = 10) and then cultured at 32°C, 35°C, 37°C, or 38.5°C. The SeV vector expresses EmGFP intracellularly, and infected cells emit fluorescence corresponding to the amount of SeV in the cells. Each mutant was cultured at each temperature, and the amount of fluorescence from the cells was measured after 3 days. The results are shown in Figure 3 and Table 2. The fluorescence intensity is expressed relative to P2Y, which is defined as 100%. As shown in Figure 3, each mutant exhibited temperature sensitivity even at 37°C, but the fluorescence intensity significantly decreased when cultured at 38.5°C. Comparison of the relative fluorescence intensities in Table 2 revealed that all mutants exhibited improved temperature sensitivity compared to P2.

[0088] [Table 2]

[0089] For the above mutants, additional mutations were introduced into the L protein of the viral genome, which has the amino acid sequence set forth in SEQ ID NO: 5. 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)

[0090] Vero cells were infected with SeV from the packaging cells (MOI = 20). Then, the cells were cultured at 35°C, 37°C, or 38.5°C. The SeV vector expresses EmGFP intracellularly, and infected cells emit fluorescence corresponding to the amount of SeV in the cells. Each mutant was cultured at each temperature, and the amount of fluorescence from the cells was measured after 3 days. The results are shown in Figure 4 and Table 3. The fluorescence intensity is expressed relative to that of P2+LmCI, which is set at 100%. The fluorescence intensity of P2m3SY+LmCI, P2m3TY+LmCI, and P2m3GY+LmCI all decreased after culture at 37°C, and was not detected after culture at 38.5°C. P2m3TY+LmCI and P2m3GY+LmCI exhibited strong temperature sensitivity, even when cultured at 35°C and 37°C. Although temperature sensitivity was also observed when a mutant having the amino acid sequence of SEQ ID NO: 7 was used as the L protein, the mutant having the amino acid sequence of SEQ ID NO: 17 was more temperature sensitive. Thus, by introducing either or both of a mutation at L1361 and a mutation at L1558, the L protein can confer temperature sensitivity to SeV.

[0091] [Table 3]

[0092] Similarly, various mutants carrying LmCI were obtained from packaging cells and infected into Vero cells at 35°C (MOI = 20). The cells were cultured at 35°C for 3 days, subcultured, and then cultured at 35°C, 37°C, or 38.5°C for an additional 7 or 14 days. The fluorescence intensity from the cells was measured. The results are shown in Figure 5 and Table 4. As shown in Figure 5A and Table 4, P2m3SY+LmCI showed some thermosensitivity even at 37°C, but showed strong thermosensitivity at 38.5°C.

[0093] [Table 4]

[0094] Furthermore, various mutants carrying Lm1558I were obtained from packaging cells and infected into Vero cells at 35°C (MOI = 20). The cells were cultured at 35°C for 3 days, subcultured, and then cultured at 35°C, 37°C, or 38.5°C for an additional 7 or 14 days. Fluorescence intensity from the cells was measured. The results are shown in Figure 5B and Table 4-1. As shown in Figure 5B and Table 4-1, P2m3SY+Lm1558I exhibited some temperature sensitivity even at 37°C, but exhibited strong temperature sensitivity at 38.5°C.

[0095] [Table 4-1]

[0096] (5) Introduction of target genes into cells by SeV Human fibroblasts were infected with SeV carrying growth factors and counted. P2m3SY SeV was used, carrying cyclin D1 and CDK4(R24C) growth factors in its genome. Human fibroblasts were infected with the growth factor-carrying SeV vector as the test vector and the EmGFP-carrying SeV vector as the negative control (MOI = 20), passaged every 7 days, and counted. The results are shown in Figure 6. The growth factor-carrying SeV vector increased the cell number to 156-fold over the initial number. In contrast, the negative control only achieved a 9-fold increase over the initial number. These results demonstrate that the temperature-sensitive vector of the present invention is suitable for introducing foreign genes into infected cells and expressing them in the infected cells. Furthermore, after incubation, the SeV vector can be removed from the cells by increasing the temperature, reducing the intracellular vector amount below the detection limit. This demonstrates the construction of a SeV vector system that can be removed after gene transfer.

[0097] Furthermore, in this example, a combination of Cyclin D1, 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 temperature-sensitive vector (SeV / TSdF-P2m434SY) to examine the effect on cell proliferation. The Sendai virus genome contained CCT at the PM position and mEmerald at the MHN position. Cells were cultured on collagen plates in DMEM / F12 medium containing 10% FBS. They were passaged every 7 days, and cell numbers were counted after each passage. At each passage, a fixed number of cells was collected and seeded in a fixed volume of fresh medium. After 42 days, cell numbers were counted and the proliferation rate was calculated. The results are shown in Table 5. As shown in Table 5, control MSCs proliferated 137-fold, whereas MSCs into which CCT was introduced using the SeV of the present invention proliferated 13.2 million-fold. Moreover, control NHDFs proliferated 12.1-fold over 42 days, whereas NHDFs into which CCT was introduced using the SeV of the present invention proliferated 1.47 million-fold.

[0098] [Table 5]

[0099] Furthermore, in this example, human fibroblasts were infected with Sendai virus vectors carrying reprogramming factors (MOI = 20) and then examined by immunocytochemical staining using an anti-NANOG antibody. The reprogramming factors used were KLF4, OCT4, SOX2, and MYCL. Vectors carrying P2m3SY and LmCI mutations were prepared, one carrying KLF4, OCT4, and SOX2 (KOS) and one carrying MYCL. Human fibroblasts were infected with the two vectors and passaged after 5 days. The medium was replaced with StemFit medium after 6 days. After 11 days, NANOG expression in the cells was examined using an anti-NANOG antibody. As shown in Figure 7A, human fibroblasts treated with Sendai virus vectors underwent reprogramming and induced NANOG expression. After cell reprogramming, raising the medium temperature to 38.5°C eliminated the Sendai virus vector from the cells, and the intracellular vector level became undetectable. However, when fibroblasts transfected with reprogramming factors were cultured in StemFit AK92N medium on iMatrix-511-coated plates, the SeV vector was gradually eliminated from the cells even at 37°C, and the fluorescence of EmGFP expressed by SeV fell below the detection limit by day 28 (see Figure 7B).

[0100] (6) Degron promotes further SeV elimination Addition of a proteolytic tag (degron) to the P protein can facilitate SeV removal. 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 protein obtained by introducing F36V and L106P into FKBP12. Addition of the low-molecular-weight compound Shield1 stabilizes the tag, and addition of dTAG-13 promotes degradation of the tag-fused protein. In this example, a DD mutant (DDm; see SEQ ID NO: 19) was constructed with 11 mutations in the DD tag (4I, 18K, 36L, 50I, 57K, 59F, 63A, 68L, 90V, 98I, and 105R). In this example, to examine the effect of DDm on promoting protein degradation, a DD tag and its mutants (including DDm) were added to the C-terminus of EmGFP and then incorporated into the + position of an SeV vector (containing the mutations described in WO2003 / 025570). The resulting SeV was infected into HeLa cells (MOI = 10). On the day after infection, 1 μM Shield1 or 5 μM dTAG-13 was added, and fluorescence from the cells was observed 3 days after infection. The results are shown in Tables 6 and 7. Furthermore, when a DDrs tag having the amino acid sequence set forth in SEQ ID NO: 28 was prepared and added to the C-terminus of EmGFP, the stabilization effect of Shield1 was not achieved, and the fluorescence intensity was only approximately 20% of that of DD-tagged EmGFP (Figure 8).

[0101] [Table 6]

[0102] [Table 7]

[0103] As shown in Tables 6 and 7, DDm improved the stability of Shield1 and enhanced the degradation-promoting effect of dTAG-13, resulting in a significant improvement in the Shield1 / dTag-13 ratio.

[0104] Next, a DDm tag was added to the C-terminus of various temperature-sensitive mutants of the P protein. EmGFP was loaded at the + site of the SeV genome. HeLa cells were infected with SeV expressing various DDm-tagged temperature-sensitive mutants (MOI = 10), and the fluorescence intensity derived from EmGFP was measured 3 days later. The results are shown in Figure 9 and Table 8.

[0105] [Table 8]

[0106] As shown in Figure 9A and Table 8, the DDm-tagged P2m3TYddm and P2m2S434TYddm proteins improved SeV removal efficiency and promoted SeV removal from cells even at 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 Figure 9B and Table 9, the destabilization of the DDm-tagged P protein was further enhanced in the presence of dTAG-13.

[0107] [Table 9]

[0108] (7) Reduction of SeV cytotoxicity SeV induces cytotoxicity. To reduce this cytotoxicity, amino acid mutations can be introduced into the M protein and HN protein. As described above, the SeV used in this example essentially had such mutations already introduced (WO 2003 / 025570). In this example, amino acid mutations in the M protein and HN protein were further investigated. Specifically, A183T was introduced into the M protein in addition to G69E and T116A (see SEQ ID NO: 20). G264K was introduced into the HN protein in addition to A262T and K461E (see SEQ ID NO: 21). L511Y was introduced into the P protein, and N1197S and K1796E were introduced into the L protein. Furthermore, SeV carrying EmGFP at the + site was constructed, infected into Vero cells, and the infected cells were cultured at 32°C for 20 days. The intensity of EmGFP-derived fluorescence in Vero cells was measured.

[0109] The results are shown in Figure 10. As shown in Figure 10, the M protein with A183T increased the fluorescence intensity 2.6-fold compared to the control, and the HN protein with G264K increased the fluorescence intensity 3.9-fold. The detachment of EmGFP-positive cells from the dish observed in the control was reduced in the SeV-infected cells with the above mutants. This suggests that the above mutations reduce the cytotoxicity caused by SeV. Fibroblasts were infected with SeV vectors carrying the above mutants of the M protein, loaded with reprogramming factors. When this was done, it was possible to induce reprogramming of fibroblasts.

[0110] (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 vector carrying a gene encoding F and a gene encoding a PKR inhibitor was introduced into packaging cells to obtain stable lines of packaging cells expressing the genes encoding F and the PKR inhibitor. VAI was used as the PKR inhibitor. While the insertion site of VAI is not particularly limited, in this example, a sequence encoding VAI was inserted downstream of the gene encoding F (see Figure 11A). These genes can be driven by any promoter, but in this example, they were driven by the CAG promoter (see Figure 11A). However, a region flanked by two site-specific recombination sequences (here, loxP) was inserted between the CAG promoter and the F-encoding gene. The vector was constructed so that the F-encoding gene was strongly driven by the CAG promoter by removing the loxP region using Cre recombinase or similar. Between the loxP regions, a gene encoding a fusion protein consisting of iCaspase9 and NeoR (a neomycin resistance gene) linked by a 2A sequence was inserted. This enabled the selection of clones with high NeoR expression levels (including clones with high copy numbers) that could survive in the presence of a certain concentration of drugs such as G418. Based on this design concept, the pCALiNdLv-F vector was constructed from the pCALNdL vector (JOURNAL OF VIROLOGY, February 1998, pp. 1115-1121) (Figure 11A). SeV-Cre contains a gene encoding Cre recombinase at the + site and the fluorescent protein mEmerald at the M-H site. This allows viral growth to be monitored by monitoring the mEmerald activity. SeV-Cre can also be engineered to have a temperature-sensitive genome that is excised from cells in a temperature-dependent manner.

[0111] After transfection of LLC-MK2 and Vero cells with the pCALiNdLv-F vector, drug selection with G418 was performed at 37°C. The resulting clones were transfected with SeV-Cre and cultured at 32°C. As shown in Figure 11B (1st), both LLC-MK2 and Vero cells were successfully infected. The supernatant from the 1st cells was then contacted with the 2nd cells. If the supernatant contained infectious virus, it would infect the 2nd cells and produce mEmerald-derived fluorescence. Infection experiments with the 2nd cells showed that the supernatant infected both LLC-MK2 and Vero cells, producing mEmerald-derived fluorescence (see Figure 11B, 2nd). If the supernatant did not contain infectious virus, the virus would not infect the 2nd cells, as shown in the bottom panel of 2nd for LLC-MK2 cells and the middle panel of 2nd for Vero cells, and the cells would not produce mEmerald-derived fluorescence. In this example, infectious viruses were reproducibly produced in both LLC-MK2 and Vero cells, and those skilled in the art will understand that they can appropriately produce infectious viruses. More specifically, the results suggest that clones highly expressing F and PKR inhibitors were selected by drug selection with G418 in LLC-MK2 and Vero cells (1.3 to 2.5 mg / mL of G418 was used for LLC-MK2 cells, and 0.9 to 1.5 mg / mL of G418 was used for Vero cells), and that the Cre recombinase carried by SeV removed the sequence between the loxP sites, resulting in high F expression. As a result, SeV was produced in the first-stage cells, and it also infected the second-stage cells, demonstrating that infectious viruses were successfully produced in the first-stage cells. These results suggest that the above-mentioned plasmids are useful tools for generating packaging cells from first-stage cells (typical LLC-MK2 and Vero cells). In this system, SeV-Cre is thought to remain in the primary cells, and therefore, by subsequently removing SeV-Cre from the primary cells, the resulting cells can be used as packaging cells for SeV reconstitution.Although SeV-Cre can be removed by simple cell passage, various methods are known to enhance removal efficiency, such as the use of temperature-sensitive strains and microRNAs, and removal can be performed appropriately. Further recloning of the cells at 37°C yielded F-expressing cells from which SeV had been removed. While LLC-MK2 and Vero cells could be used as packaging cells due to the expression of F, it is understood that F can be supplied by the cells themselves, via a plasmid, or from the Sendai virus genome.

[0112] (9) Virus removal by bromotag HeLa cells were cultured with P protein variants (P2m3SY, P2m2S434TY, and P2m3TY) containing Brd4 at the C-terminus. BD2(L387A) The cells were infected with SeV (MOI = 20) carrying proteins with P2m3SYb, P2m2S434TYb, and P2m3Tb (respectively) at 32-38.5°C. As a control, HeLa cells were infected with SeV (MOI = 20) carrying a protein (P2m3SY) with ddm added to a P protein variant (P2m3SYddm) under the same conditions as above. On the day after SeV infection, 5 μM dTAG-13 or 1 μM BromoTAG™ AGB1 was added as a degrader to induce degradation of the P protein variants, and EmGFP fluorescence was evaluated on day 2 after SeV infection. AGB1 binds von Hippel-Lindau (VHL) E3 ligase to Brd4. BD2(L387A) Recruit to Brd4 BD2(L387A)AGB1 promotes the degradation of attached proteins. AGB1 is (S)-13-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbonyl)-14,14-dimethyl-11-oxo-3,6,9-trioxa-12-azapentadecyl (R)-2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)butanoate. As shown in Figure 13, the combination of Brd4BD2(L387A) and BromoTAG promoted SeV removal more effectively than the combination of ddm and dTAG-13.

[0113] Similar experiments were performed using various P protein variants. The results are shown in Figure 14 and Tables 10 and 11. Tables 10 and 11 summarize the results of Figure 14.

[0114] [Table 10]

[0115] [Table 11]

[0116] As shown in Figure 14 and Table 10, in the absence of a ligand (degrader) for the tag, SeV containing all P protein variants was detected at 37°C, whereas in the presence of the ligand, SeV containing the bromo-tagged P protein was below the detection limit. Furthermore, it was revealed that the SeV removal effect was higher than that of dmm.

[0117] Next, bromotags were used to control the expression of the harboring gene. HeLa cells were infected at 37°C with SeV (MOI = 10) carrying EmGFP (EmGFPb), a Brd4BD2(L387A)-tagged version of EmGFP. HeLa cells were also infected with SeV+EmGFPb / TSdF at an MOI = 10. One micromolar of BromoTAG™ AGB1 was added the day after SeV infection, and EmGFPb fluorescence was assessed two days after SeV infection. The results are shown in Table 12. As shown in Table 12, the addition of AGB1 attenuated EmGFPb fluorescence to 2.3% of that observed without AGB1.

[0118] [Table 12]

[0119] Based on the above, this disclosure demonstrates that it is possible to remove SeV and control the expression (degradation) of specific proteins using bromotags and their corresponding PROTACs.

[0120] 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 SEQ ID NO: 2: Amino acid sequence of the P protein variant (P2) 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 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 the P protein mutant (L511F) 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 FPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTN 568 SEQ ID NO: 4: Amino acid sequence of a 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 SEQ ID NO: 6: Amino acid sequence of a 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 SEQ ID NO: 7: Amino acid sequence of the mutant (N1197S / L1558I / K1796E) 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 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 SEQ ID NO: 8: Example of a Sendai virus P protein mutant 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 SEQ ID NO: 9: Example of a Sendai virus P protein mutant 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 SEQ ID NO: 10: Example of a Sendai virus P protein mutant 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 SEQ ID NO: 11: Example of a Sendai virus P protein mutant 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 a Sendai virus P protein mutant 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 a Sendai virus P protein mutant 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 a Sendai virus P protein mutant 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 a Sendai virus P protein mutant 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 a Sendai virus P protein mutant 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 a Sendai virus L protein mutant 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 SEQ ID NO: 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: M protein mutant 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: Mutant 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 SEQ ID NO: 22: An example of a domain of bovine FKBP12 that corresponds to the DD domain of human FKBP12 MGVQVETISP GDGRTFPKRG QTCVVHYTGM LEDGKKFDSS RDRNKPFKFV LGKQEVIRGW 60 EEGVAQMSVG QRAKLTISPD YAYGATGHPG IIPPNATLIF DVELLKLE 108 SEQ ID NO: 23: An example of a domain in rhesus monkey FKBP12 that corresponds 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 a domain of mouse FKBP12 that corresponds 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 a domain of rabbit 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 a domain of rat FKBP12 that corresponds to the DD domain of human FKBP12 MGVEIETISP GDGRTFPKKG QICVVHYTGM LQNGKKFDSS RDRNKPFKFR IGKQEVIKGF 60 EEGAAQMSLG QRAKLTCTPD VAYGATGHPG VIPPNATLIF DVELLNLE 108 SEQ ID NO: 28: Example of a DDrs tag GVQVETISPG DGRTFPRRGQ TCVVHYTGML EDGRRVDSSR DRNRPFRFML GRQEVIRGWE 60 EGVSQMSVGQ RSRLTISPDY SYGSTGHPGI IPPHSTLVFD VELLRPE 107 SEQ ID NO: 29: Brd4BD2 (L387A) (bromotag) VKDVPDSQQH PAPEKSSKVS EQLKCCSGIL KEMFAKKHAA YAWPFYKPVD VEALGAHDYC 60 DIIKHPMDMS TIKSKLEARE YRDAQEFGAD VRLMFSNCYK YNPPDHEVVA MARKLQDVFE 120 MRFAKMPDE SEQ ID NO: 30: P2m3SYb 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 TDSLTRSPV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL YPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTNKL GSGSVKDVPD SQQHPAPEKS SKVSEQLKCC 600 SGILKEMFAK KHAAYAWPFY KPVDVEALGA HDYCDIIKHP MDMSTIKSKL EAREYRDAQE 660 FGADVRLMFS NCYKYNPPDH EVVAMARKLQ DVFEMRFAKM PDE sequence number 31:P2m2S434TYb 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 ITSTGGSTDN TDSLTRSPV FAKSKENKTK ATRFDPSMET LEDMKYKPDL 480 IREDEFRDEI RNPVYQERDT EPRASNASRL YPSKEKPTMH SLRLVIESSP LSRAEKVAYV 540 KSLSKCKTDQ EVKAVMELVE EDIESLTNKL GSGSVKDVPD SQQHPAPEKS SKVSEQLKCC 600 SGILKEMFAK KHAAYAWPFY KPVDVEALGA HDYCDIIKHP MDMSTIKSKL EAREYRDAQE 660 FGADVRLMFS NCYKYNPPDH EVVAMARKLQ DVFEMRFAKM PDE sequence number 32:P2m3TYb 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 EDIESLTNKL GSGSVKDVPD SQQHPAPEKS SKVSEQLKCC 600 SGILKEMFAK KHAAYAWPFY KPVDVEALGA HDYCDIIKHP MDMSTIKSKL EAREYRDAQE 660 FGADVRLMFS NCYKYNPPDH EVVAMARKLQ DVFEMRFAKM PDE Sequence number 33: EmGFPb MVSKGEELFT GVVPILVELD GDVNGHKFSV SGEGEGDATY GKLTLKFICT TGKLPVPWPT 60 LVTTLTYGVQ CFARYPDHMK QHDFFKSAMP EGYVQERTIF FKDDGNYKTR AEVKFEGDTL 120 VNRIELKGID FKEDGNILGH KLEYNYNSHK VYITADKQKN GIKVNFKTRH NIEDGSVQLA 180 DHYQQNTPIG DGPVLLPDNH YLSTQSALSK DPNEKRDHMV LLEFVTAAGI TLGMDELYKL 240 GSGSVKDVPD SQQHPAPEKS SKVSEQLKCC SGILKEMFAK KHAAYAWPFY KPVDVEALGA 300 HDYCDIIKHP MDMSTIKSKL EAREYRDAQE FGADVRLMFS NCYKYNPPDH EVVAMARKLQ 360 DVFEMRFAKM PDE

Claims

1. An RNA genome of a negative-strand RNA virus or viral vector, The RNA genome of a minus-strand RNA virus or viral vector, wherein at least one of the viral proteins encoded by the genome (preferably the P protein or the temperature-sensitive P protein) is in the form of a fusion protein with a bromotag.

2. The minus-strand RNA virus or viral vector according to claim 1, The temperature-sensitive P protein encoded by the viral genome has substitution mutations at amino acids of the P protein corresponding to any one or more or all of D433, R434, and K437, the substitution mutations including a mutation to any of G, T, and S, and may further have a substitution mutation at an amino acid of the P protein corresponding to L511, the substitution mutation at L511 being a mutation to Y or F, or The RNA genome of a negative-strand RNA virus or viral vector, wherein the temperature-sensitive 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 minus-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 minus-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 minus-strand RNA virus or viral vector according to claim 3, wherein the substitution mutation is a mutation to S.

6. The minus-strand RNA virus or viral vector according to any one of claims 1 to 3, wherein the temperature-sensitive 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) 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) having amino acid mutations corresponding to D433S, R434T, K437S, and L511Y; The RNA genome of a negative-strand RNA virus or viral vector.

7. The RNA genome of a minus-strand RNA virus or viral vector according to any one of claims 1 to 6, wherein the 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 minus-strand RNA virus or viral vector according to any one of claims 1 to 9.

11. A minus-strand RNA virus or viral vector in which at least one of the viral proteins encoded in the genome (preferably the P protein or the temperature-sensitive P protein) is in the form of a fusion protein with a bromo tag, and / or at least one of the viral proteins constituting the virus or viral vector (preferably the P protein or the temperature-sensitive P protein) is in the form of a fusion protein with a bromo tag, or 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 minus-strand RNA virus or viral vector according to claim 11, comprising a P protein that is less temperature-sensitive than the temperature-sensitive P protein encoded on the genome, or a P protein that is not temperature-sensitive.

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

14. A composition comprising the minus-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: Infecting the cells with the minus-strand RNA virus or viral vector according to any one of claims 11 to 13, and then culturing the cells under a first temperature condition to express the gene of interest; and then further culturing the cells under a second temperature condition, thereby removing a part or all of the minus-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: Infecting the cells with the minus-strand RNA virus or viral vector according to any one of claims 11 to 13, and then culturing the cells under a first temperature condition to express the gene of interest. 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 cells are infected with the minus-strand RNA virus or viral vector according to any one of claims 11 to 13, and then cultured under a second temperature condition to express the target gene in the cells and remove the virus or viral vector from the cells. including 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 according to 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 the protein according to any one of claims 18 to 20 and / or the nucleic acid according to claim 21.

24. A minus-strand RNA virus or vector comprising a minus-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. A nucleic acid encoding the M protein of claim 25.

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

30. A minus-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 minus-strand RNA virus or vector comprising a minus-strand RNA genome comprising the nucleic acid of claim 26 and / or 27.

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

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