JP2008105946A - Method of reconstructing blood cell from gene-transfer hematopoietic cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of producing a hematopoietic cell transplantation composition containing gene-transfer hematopoietic cells by using chromosome non-integration virus vector, and a method of reconstructing blood cells from the gene-transfer hematopoietic cells by using the chromosome non-integration virus vector. <P>SOLUTION: The method of producing the hematopoietic transplantation composition enables to reconstruct blood cells expressing transferred gene by transferring a minus chain RNA virus vector into hematopoietic cells and hematopoietic cell-transplanting these cells. The gene-transfer hematopoietic cells can be differentiated into leucocytes, erythrocytes and thrombocytes in the body to which the hematopoietic cells are transplanted. Furthermore, a safe gene-transferring system causing no danger of damaging host chromosomes in gene therapy in hematopoietic cell transplantation. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a composition for transplanting hematopoietic cells, including transgenic hematopoietic cells. The present invention also relates to a method for reconstructing blood cells from transgenic hematopoietic cells. The method of the present invention can be applied to cell chromosomal undamaged gene therapy in hematopoietic cell transplantation.

Gene therapy is expected as a new treatment for refractory diseases caused by single gene abnormalities such as X-linked SCID. In 2000, it was reported in France that a common gamma chain (causal gene) was introduced into CD34 positive hematopoietic stem cells using a retroviral vector and was effective in 9 out of 11 cases. (Cavazzana-Calvo M. et al., Science 2000; 288: 669-672). However, T-cell leukemia occurred in 2 patients and warnings for similar vectors and treatment systems were made (Kaiser J., Science 2003; 299: 495; Hacein-Bey-Abina S. et al. Science 2003; 302: 415-419). Subsequent analysis has reported that these two cases show constitutive activation of LMO2, a gene that is required for T cell development and should be lost in the thymus (Hacein-Bey-Abina S. et al. al., Science 2003; 302: 415-419; Fischer A. et al., N Eng Journal Med 2004; 350: 2526-2527). At present, it is thought that this constitutive activation of LMO2 may be specific to this clinical research protocol, but the incorporation of the retroviral vector into the host chromosome of the proviral genome itself is not effective for the tumor suppressor gene. The possibility of activation and activation of oncogenes cannot be denied, and is considered a fundamental problem. This potential risk is also envisioned for adeno-associated virus vectors (AAV) and lentiviral vectors that incorporate genes into chromosomes as well. In some cases of congenital immunodeficiency disease, bone marrow transplantation is not effective, so these children are the only option left with gene therapy. Therefore, it is desired to construct a new and safe vector system based on a concept different from conventional chromosomally integrated vectors.
Cavazzana-Calvo M. et al., Science 2000; 288: 669-672 Kaiser J., Science 2003; 299: 495 Hacein-Bey-Abina S. et al., Science 2003; 302: 415-419 Hacein-Bey-Abina S. et al., Science 2003; 302: 415-419 Fischer A. et al., N Eng Journal Med 2004; 350: 2526-2527

  The present invention provides a method for producing a hematopoietic cell transplant composition containing transgenic hematopoietic cells using a non-chromosomally integrated viral vector. The present invention also provides a method for reconstructing blood cells from hematopoietic cells transfected with a non-chromosomal viral vector.

  The present inventors have developed a method using a non-chromosomal integrated virus in order to enable safe gene transfer without damaging the chromosomes of hematopoietic cells in hematopoietic cell transplantation. For this purpose, a gene was introduced into a hematopoietic cell using a minus-strand RNA virus, which is one of the non-chromosomal viruses, and the blood cell system was reconstructed from the hematopoietic cell. First, bone marrow cells were prepared by removing erythrocytes and T cells from the mouse femur, and infected with a minus-strand RNA virus incorporating a foreign gene to produce transgenic bone marrow cells. Then, an experiment was performed in which the transgenic bone marrow cells were injected into mice subjected to myeloablation by irradiation. After studying the engraftment dynamics and immunological reaction of the transferred bone marrow cells in vivo, the bone marrow cells into which the minus-strand RNA viral vector was introduced engraft in the recipient, and the blood cell lineage cells including T cells Repopulation was confirmed. The present invention is the first to disclose blood cell reconstruction by gene-introduced hematopoietic cells using a non-chromosomal cytoplasmic vector. By using a minus-strand RNA viral vector, a gene can be obtained without recombination of the cell chromosome. This demonstrates that repopulation of the blood cell lineage is possible from the introduced transplanted hematopoietic cells. In particular, it has been found that if a minus-strand RNA viral vector having a mutation or deletion in a gene encoding an envelope-constituting protein is used, blood cells can be stably reconstructed for a long period of time. According to the present invention, it is possible to safely carry out gene therapy in hematopoietic cell transplantation without worrying about chromosome damage.

That is, the present invention relates to a gene-transduced hematopoietic cell composition suitable for hematopoietic cell transplantation and a method for producing the same, and a method for reconstructing blood cells from hematopoietic cells that have been gene-transduced using a minus-strand RNA viral vector. The present invention relates to the invention described in each claim. In addition, the invention which consists of one or several combination of the invention as described in the claim which cites the same claim is already intended by the expression as described in those claims. That is, the present invention
[1] A method for producing a hematopoietic cell transplant composition, comprising introducing a minus-strand RNA viral vector into a hematopoietic cell, and preparing a composition comprising the hematopoietic cell and a pharmaceutically acceptable medium,
[2] The method according to [1], wherein the minus-strand RNA viral vector is mutated or deleted in one or more genes encoding envelope-constituting proteins,
[3] The method according to [2], wherein all genes encoding envelope-constituting proteins are mutated or deleted.
[4] The method according to [2] or [3], wherein at least one gene encoding an envelope constituent protein is deleted,
[5] The method according to any one of [1] to [4], wherein the minus-strand RNA virus is a Paramyxoviridae virus,
[6] The method according to [5], wherein the Paramyxoviridae virus is Sendai virus,
[7] A hematopoietic cell transplant composition obtained by the method according to any one of [1] to [6],
[8] A method for generating gene-introduced blood cells from hematopoietic cells,
A method comprising: (a) contacting a minus-strand RNA viral vector with a hematopoietic cell; and (b) injecting the hematopoietic cell of step (a) into an animal.
[9] The method according to [8], further comprising a step of immunosuppressing the animal before the injection in step (b).
[10] The method according to [8] or [9], wherein in the minus-strand RNA viral vector, one or more genes encoding envelope-constituting proteins are mutated or deleted,
[11] The method according to [10], wherein all genes encoding envelope-constituting proteins are mutated or deleted.
[12] The method according to [10] or [11], wherein at least one gene encoding an envelope constituent protein is deleted,
[13] The method according to any one of [8] to [12], wherein the minus-strand RNA virus is a Paramyxoviridae virus,
[14] The method according to [13], wherein the Paramyxoviridae virus is Sendai virus.

  In the present invention, it was demonstrated for the first time that hematopoietic cells introduced with a non-chromosomally integrated cytoplasmic vector can be repopulated in vivo. In recipients, T cells derived from donors expressing foreign genes were detected, supporting that repopulation occurred from hematopoietic stem cells into which a minus-strand RNA viral vector was introduced. Negative-strand RNA viruses transcribe and replicate in the cytoplasm and do not interact with chromosomal genes in the cell nucleus. The present invention enables host chromosome non-injury gene therapy for hematopoietic diseases such as congenital immunodeficiency.

  The present invention provides a hematopoietic cell transplant composition containing a hematopoietic cell into which a minus-strand RNA viral vector has been introduced and a pharmaceutically acceptable medium, and a method for producing the same. In the present invention, the minus-strand RNA virus is a virus that contains, as a genome, RNA of a minus strand (an antisense strand complementary to a strand that senses a viral protein). Negative strand RNA is also called negative strand RNA.

  A minus-strand RNA virus vector refers to a minus-strand RNA virus as a carrier (vector) for introducing a gene into a cell. The minus-strand RNA viral vector may or may not have a foreign gene.

  A recombinant virus refers to a virus produced via a recombinant polynucleotide, or an amplification product of the virus. A recombinant polynucleotide refers to a polynucleotide in which both ends or one end are not joined as in the natural state. Specifically, a recombinant polynucleotide is a polynucleotide in which the binding of polynucleotide strands has been artificially altered (cut and / or joined). A recombinant polynucleotide can be produced by a known genetic recombination method by combining polynucleotide synthesis, nuclease treatment, ligase treatment and the like. A recombinant virus can be produced by expressing a polynucleotide encoding a viral genome constructed by genetic engineering and reconstructing the virus. For example, a method of reconstructing a virus from cDNA encoding a viral genome is known (Y. Nagai, A. Kato. Microbiol. Immunol. 1999: 43; 613-624).

  In the present invention, gene refers to genetic material and refers to a nucleic acid encoding a transcription unit. The gene may be RNA or DNA. In the present invention, a nucleic acid encoding a protein is called a gene of the protein. In general, a gene may not encode a protein. For example, a gene may encode a functional RNA such as a ribozyme or an antisense RNA. In general, a gene can be a naturally derived or artificially designed sequence. In the present invention, “DNA” includes single-stranded DNA and double-stranded DNA. In addition, encoding a protein means that an ORF encoding the amino acid sequence of the protein is included in sense or antisense so that the polynucleotide can express the protein under appropriate conditions.

  Examples of minus-strand RNA viruses that are preferably used in the present invention include the Paramyxoviridae virus Sendai virus, Newcastle disease virus, Mumps virus, measles Virus (Measles virus), RS virus (Respiratory syncytial virus), rinderpest virus, distemper virus, simian parainfluenza virus (SV5), human parainfluenza virus type 1,2,3, orthomyxovirus Influenza virus of Orthomyxoviridae, Vesicular stomatitis virus of Rhabdoviridae, Rabies virus, etc., specifically Sendai virus (SeV) , Human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), phocine diste mper virus (PDV), canine distemper virus (CDV), dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV), rinderpest virus (RPV), Hendra virus (Hendra), Nipah virus (Nipah), human parainfluenza virus-2 (HPIV-2), simian parainfluenza virus 5 (SV5), human parainfluenza virus-4a (HPIV-4a), human parainfluenza virus-4b (HPIV-4b), mumps virus (Mumps ), And Newcastle disease virus (NDV). More preferably, Sendai virus (SeV), human parainfluenza virus-1 (HPIV-1), human parainfluenza virus-3 (HPIV-3), phocine distemper virus (PDV), canine distemper virus (CDV), dolphin molbillivirus (DMV ), Peste-des-petits-ruminants virus (PDPR), measles virus (MV), rinderpest virus (RPV), Hendra virus (Hendra), Nipah virus (Nipah), Respiratory syncytial virus (RSV) Viruses and the like.

  The minus-strand RNA virus used in the present invention is particularly preferably a single-strand minus-strand RNA virus (also referred to as a non-segmented minus-strand RNA virus). A “single-stranded negative strand RNA virus” refers to a virus having a single-stranded negative strand [ie, minus strand] RNA in the genome. Such viruses include Paramyxovirus (including Paramyxoviridae; Paramyxovirus, Morbillivirus, Rubulavirus, and Pneumovirus genera), Rhabdoviridae (including Vesiculovirus, Lyssavirus, and Ephemerovirus genera), Filovirus (Filoviridae), Family such as Orthomyxovirus (including Orthomyxoviridae; Infuluenza virus A, B, C, and Thogoto-like viruses), Bunyavirus (including Bunyaviridae; Bunyavirus, Hantavirus, Nairovirus, and Phlebovirus genera), Arenavirus (Arenaviridae) Viruses belonging to are included.

  The minus-strand RNA virus used in the present invention is more preferably a virus belonging to the Paramyxovirus subfamily (including the Respirovirus genus, Rubravirus genus, and Morbillivirus genus), or more preferably a derivative thereof. A virus belonging to the genus Respirovirus (also referred to as Paramyxovirus) or a derivative thereof. Derivatives include viruses in which viral genes have been modified, chemically modified viruses, and the like so as not to impair the ability to introduce genes by viruses. Examples of respirovirus viruses to which the present invention can be applied include human parainfluenza virus type 1 (HPIV-1), human parainfluenza virus type 3 (HPIV-3), and bovine parainfluenza virus type 3 (BPIV-3). , Sendai virus (also called mouse parainfluenza virus type 1), and monkey parainfluenza virus type 10 (SPIV-10). In the present invention, the paramyxovirus is most preferably Sendai virus. These viruses may be derived from natural strains, wild strains, mutant strains, laboratory passage strains, artificially constructed strains, and the like.

  The minus-strand RNA viral vector antisenseally encodes the gene carried on the viral genomic RNA. Viral genomic RNA has the function of forming a ribonucleoprotein (RNP) together with the viral protein of the minus-strand RNA virus, the gene in the genome is expressed by the protein, and this RNA is replicated to form daughter RNP RNA. Genomic RNA may be one in which a viral gene has mutation and / or deletion, or has been modified by incorporating a foreign gene. In general, the genome of a minus-strand RNA virus has a structure in which a viral gene is arranged as an antisense sequence between a 3 ′ leader region and a 5 ′ trailer region. Between each gene's ORF, there is a transcription termination sequence (E sequence)-an intervening sequence (I sequence)-a transcription initiation sequence (S sequence), so that the RNA encoding each gene's ORF is a separate cistron. Transcribed. The genomic RNA contained in the virus of the present invention encodes a minus-strand RNA viral protein that constitutes RNP with the RNA. However, since the genomic RNA of a minus-strand RNA virus is a minus strand, these proteins are encoded as antisense. Genomic RNA and the negative-strand RNA virus protein that composes RNP form a complex with the genomic RNA of the negative-strand RNA virus, replication of the genomic RNA, expression of the genes encoded in the genome, and the RNA itself A group of viral proteins required for replication. These proteins form the core excluding the viral envelope, and are typically N (nucleocapsid), P (phospho), and L (large) proteins. Depending on the virus species, the notation may differ, but the corresponding protein is obvious to those skilled in the art (Anjeanette Robert et al., Virology. 1998: 247; 1-6). For example, N may be written as NP. The RNA may encode an M (matrix) protein necessary for the formation of virus particles. Further, the RNA may encode an envelope protein necessary for virus particle infection. Examples of envelope proteins of minus-strand RNA viruses include F (fusion) protein, which is a protein that causes cell membrane fusion, and HN (hemagglutinin-neuraminidase) protein (or H (hemagglutinin)) necessary for adhesion to cells. In addition, viral envelope proteins other than F protein and / or HN (or H) protein may be encoded. However, as described later, the minus-strand RNA virus used in the present invention preferably has a mutation and / or deletion of a gene encoding an envelope-constituting protein.

For example, each gene in each virus belonging to the Paramyxovirus subfamily is generally expressed as follows. In general, the NP gene is also denoted as “N”. HN is expressed as H (hemagglutinin) when it has no neuraminidase activity.
Respirovirus NP P / C / VMF HN-L
Rubravirus NP P / VMF HN (SH) L
Mobilivirus NP P / C / VMFH-L

  For example, the accession numbers in the base sequence database for each gene of Sendai virus are M29343, M30202, M30203, M30204, M51331, M55565, M69046, X17218 for the NP gene, and M30202, M30203, M30204, M55565, M69046 for the P gene. , X00583, X17007, X17008, M gene is D11446, K02742, M30202, M30203, M30204, M69046, U31956, X00584, X53056, F gene is D00152, D11446, D17334, D17335, M30202, M30203, M30204, M69046, X00152 , X02131, and the HN gene, see D26475, M12397, M30202, M30203, M30204, M69046, X00586, X02808, X56131, and the L gene, D00053, M30202, M30203, M30204, M69040, X00587, and X58886. Examples of viral genes encoded by other viruses include the N gene, CDV, AF014953; DMV, X75961; HPIV-1, D01070; HPIV-2, M55320; HPIV-3, D10025; Mapuera, X85128; Mumps , D86172; MV, K01711; NDV, AF064091; PDPR, X74443; PDV, X75717; RPV, X68311; SeV, X00087; SV5, M81442; and Tupaia, AF079780, for the P gene, CDV, X51869; DMV, Z47758; HPIV -l, M74081; HPIV-3, X04721; HPIV-4a, M55975; HPIV-4b, M55976; Mumps, D86173; MV, M89920; NDV, M20302; PDV, X75960; RPV, X68311; SeV, M30202; SV5, AF052755 ; And Tupaia, AF079780 for the C gene, CDV, AF014953; DMV, Z47758; HPIV-1. M74081; HPIV-3, D00047; MV, ABO16162; RPV, X68311; SeV, AB005796; and Tupaia, AF079780, for the M gene Is CDV, M12669; DMV Z30087; HPIV-1, S38067; HPIV-2, M62734; HPIV-3, D00130; HPIV-4a, D10241; HPIV-4b, D10242; Mumps, D86171; MV, AB012948; NDV, AF089819; PDPR, Z47977; PDV, X75717; RPV, M34018; SeV, U31956; and SV5, M32248 CDV, M21849; DMV, AJ224704; HPN-1.M22347; HPIV-2, M60182; HPIV-3.X05303, HPIV-4a, D49821; HPIV-4b, D49822; Mumps, D86169; MV, AB003178; NDV, AF048763; PDPR, Z37017; PDV, AJ224706; RPV, M21514; SeV, D17334; and SV5, AB021962, CDV, AF112189 for the HN (H or G) gene; DMV, AJ224705; HPIV-1, U709498; HPIV- 2.D000865; HPIV-3, AB012132; HPIV-4A, M34033; HPIV-4B, AB006954; Mumps, X99040; MV, K01711; NDV, AF204872; PDPR, Z81358; PDV, Z36979; RPV, AF132934; SeV, U06433; And SV-5, S76876. However, a plurality of strains are known for each virus, and there are genes having sequences other than those exemplified above depending on the strain.

  The ORFs encoding these viral proteins and the ORFs of foreign genes are arranged in an antisense manner in the genomic RNA via the E-I-S sequence described above. The closest 3 'ORF in genomic RNA requires only the S sequence between the 3' leader region and the ORF, and not the E and I sequences. The ORF closest to the 5 ′ in the genomic RNA requires only the E sequence between the 5 ′ trailer region and the ORF, and does not require the I and S sequences. Two ORFs can also be transcribed as the same cistron using a sequence such as IRES. In such a case, no E-I-S sequence is required between these two ORFs. For example, in the case of wild-type paramyxoviruses, a typical RNA genome follows the 3 ′ leader region, followed by six ORFs that antisensely encode N, P, M, F, HN (H), and L proteins. In order, followed by a 5 ′ trailer region at the other end. In the present invention, the genomic RNA is not limited to the arrangement of the viral gene, but preferably, like the wild-type virus, the 3 'leader region is followed by N, P, M, F, HN (or H), and the ORF encoding the L protein are preferably arranged in order, followed by the 5 ′ trailer region. In some types of viruses, the viral genes are different, but even in such a case, it is preferable to arrange each viral gene in the same manner as in the wild type as described above. In general, a vector carrying N, P and L genes autonomously expresses a gene from an RNA genome in a cell, and the genomic RNA is replicated. Furthermore, infectious virus particles are formed and released extracellularly by the action of genes encoding envelope spike proteins such as the F and HN (or H) genes and the M gene. Therefore, such a vector becomes a viral vector having transmission ability. When a foreign gene is loaded on a vector, it may be inserted into a protein non-coding region in this genome as described later.

  The minus-strand RNA viral vector used in the present invention preferably has a mutation and / or deletion in one or more envelope protein genes. It has been found that the use of such a mutant negative-strand RNA virus significantly increases the efficiency of blood cell repopulation. Here, the mutation is a mutation that significantly reduces the infectious virus-producing ability of the virus substituted with the mutant gene as compared with the virus having the wild-type gene. The term “deficiency” means that the function of the wild-type gene is substantially lost, which includes modification so as not to express a functional protein, and deletion of the gene. The fact that the function of the wild type gene is substantially lost means that the virus production at 37 ° C. is reduced to 1/10 or less, preferably 1/20 or less, more preferably 1/50 or less of the wild type due to the gene deficiency. It is to be. The envelope constituent protein refers to a viral protein that is a component of the viral envelope, and includes a spike protein that is exposed on the surface of the envelope and functions to adhere to or infect cells, and a backing protein that functions to form an envelope. Typically, genes of envelope constituent proteins include F, HN, and M. Depending on the virus species, genes such as H, M1, and G are included. Viruses in which one or more of these envelope protein genes have been mutated and / or deleted are highly safe because the ability to form infectious virus particles in infected cells is reduced. In addition, cytotoxicity is significantly reduced. Examples of the gene to be mutated and / or deleted in the genome include F gene, HN (or H) gene, M gene, or any combination thereof. Particularly preferably, at least the F gene is mutated or deleted, more preferably deleted. More preferably, the F gene and the HN (or H) gene are mutated or deleted, more preferably deleted. More preferably, the F gene, HN (or H) gene and M gene are mutated or deleted.

  In particular, it is preferable to use a vector in which at least the F gene is deleted and the HN (or H) gene is mutated or deleted. Once such a viral vector has entered the cell, the F protein is not expressed, and the HN (or H) protein is expressed at a very low level or not. When many HN proteins are expressed in vector-infected cells, the present inventors have confirmed that cell adsorption and aggregation may occur, and in fact, the present inventors have confirmed that such a phenomenon can occur in vitro. Therefore, better results can be obtained in hematopoietic cell transplantation by using a vector having a temperature-sensitive mutation in the HN (or H) gene, more preferably a vector lacking the HN (or H) gene. Furthermore, better engraftment dynamics can be obtained by mutating or deleting the M gene.

  Numerous temperature sensitive mutations are known in envelope constituent proteins. Viruses having these temperature-sensitive mutant protein genes can be preferably used in the present invention. A temperature-sensitive mutation is a mutation whose activity is significantly reduced at a normal temperature (eg, 37 ° C.) of a virus host as compared to a low temperature (eg, 32 ° C.). Such a protein having a temperature-sensitive mutation is useful because a virus can be produced under permissive conditions (low temperature) without complementation with a wild-type protein or the like.

  For example, the temperature-sensitive mutation of the M gene is not particularly limited, but is at least one selected from the group consisting of G69, T116, and A183 in the Sendai virus M protein, preferably arbitrarily selected. Those in which two, more preferably all three amino acid sites, or sites that are homologous to those of other minus-strand RNA virus M proteins are mutated can be suitably used. Here, G69 is the 69th amino acid Gly of the M protein, T116 is the 116th amino acid Thr of the M protein, and A183 is the 183rd amino acid Ala of the M protein.

  The gene encoding the M protein (M gene) is widely conserved in minus-strand RNA viruses and is known to have a function of interacting with both the viral nucleocapsid and the envelope (Garoff, H. et al., Microbiol. Mol. Biol. Rev. 1998: 62; 117-190). In addition, 104-119 (104-KACTDLRITVRRTVRA-119 / SEQ ID NO: 1), which is expected to be amphiphilic α-helix in the SeV M protein, has been identified as an important region for particle formation (Genevieve Mottet et al., J Gen. Virol. 1999: 80; 2977-2986), but this region is well conserved among minus-strand RNA viruses. The amino acid sequence of the M protein is similar in minus-strand RNA viruses. In particular, in the Paramyxovirus subfamily, the known M protein is a basic protein consisting of approximately 330 to 380 amino acids in length, and is similar over the entire region. However, the similarity in the C-terminal half is particularly high (Gould, AR Virus Res. 1996: 43; 17-31, Harcourt, BH et al., Virology. 2000: 271; 334-349). Therefore, for example, amino acids homologous to G69, T116, and A183 of SeV M protein can be easily identified.

  For those skilled in the art, the amino acid of the homologous site of the other minus-strand RNA virus M protein corresponding to SeV M protein G69, T116, and A183 can be obtained by using a homology search program for amino acid sequences (such as BLAST). Can be identified by aligning with amino acids of SeVM protein using an alignment creation program such as CLUSTAL W. For example, the homologous site of each M protein corresponding to G69 of SeV M protein may be G69, human parainfluenza virus-3 (HPIV-3) if human parainfluenza virus-1 (HPIV-1) (parentheses are abbreviated). G73 for phocine distemper virus (PDV) and canine distemper virus (CDV), G71 for dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV), and G70 for rinderpest virus (RPV), G81 for Hendra virus (Hendra) and Nipah virus (Nipah), G70 for human parainfluenza virus-2 (HPIV-2), human parainfluenza virus-4a (HPIV-4a) ) And human parainfluenza virus-4b (HPIV-4b) and E72 for mumps virus (Mumps) (letters and numbers represent amino acids and their positions). In addition, homologous sites of each M protein corresponding to T116 of SeV M protein include T116 for human parainfluenza virus-1 (HPIV-1), T120 for human parainfluenza virus-3 (HPIV-3), phocine T104 for distemper virus (PDV) and canine distemper virus (CDV), T105 for dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV) and rinderpest virus (RPV) T104, Hendra virus (Hendra) and Nipah virus (Nipah) T120, human parainfluenza virus-2 (HPIV-2) and siman parainfluenza virus 5 (SV5) T117, human parainfluenza virus-4a ( HPIV-4a) and human parainfluenza virus-4b (HPIV-4b) include T121, mumps virus (Mumps) is T119, and Newcastle disease virus (NDV) is S120. The homologous sites of each M protein corresponding to A183 of SeV M protein include human parainfluenza virus-1 (HPIV-1) A183, human parainfluenza virus-3 (HPIV-3) F187, phocine distemper virus Y171 for PDV and canine distemper virus (CDV), Y172 for dolphin molbillivirus (DMV), peste-des-petits-ruminants virus (PDPR), measles virus (MV) and rinderpest virus (RPV). Y171, Y187 for Hendra virus (Hendra) and Nipah virus (Nipah), Y184 for human parainfluenza virus-2 (HPIV-2), F184 for human parainfluenza virus 5 (SV5), human parainfluenza virus- F188 for 4a (HPIV-4a) and human parainfluenza virus-4b (HPIV-4b), F186 for mumps virus (Mumps), and Y187 for Newcastle disease virus (NDV). In the viruses listed here, each M protein is a mutant M protein in which one of the above three sites, preferably a combination of any two sites, and more preferably all three sites of amino acids are replaced with other amino acids. A virus having a genome encoding is preferably used in the present invention.

  The amino acid mutation may be a substitution with another desired amino acid, but is preferably a substitution with an amino acid having a different side chain chemistry. For example, amino acids are basic amino acids (eg lysine, arginine, histidine), acidic amino acids (eg aspartic acid, glutamic acid), uncharged polar amino acids (eg glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar amino acids (Eg alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched amino acids (eg threonine, valine, isoleucine), and aromatic amino acids (eg tyrosine, phenylalanine, tryptophan, histidine) etc. For example, a certain amino acid may be substituted with an amino acid other than the amino acid of the group to which the amino acid belongs. Specifically, basic amino acids are substituted with acidic or neutral amino acids, polar amino acids are substituted with nonpolar amino acids, and amino acids with molecular weights greater than the average molecular weight of 20 natural amino acids. For example, substitution with an amino acid smaller than the average molecular weight, and conversely, substitution with an amino acid larger than the average molecular weight is not limited thereto.

  Specifically, a mutation selected from the group consisting of G69E, T116A, and A183S in Sendai virus M protein, or another paramyxovirus M protein containing a mutation at a position homologous thereto can be used. Here, G69E is a mutation in which the 69th amino acid Gly of the M protein is replaced with Glu, T116A is a mutation in which the 116th amino acid Thr of the M protein is replaced with Ala, and A183S is the 183 of the M protein. A mutation in which the first amino acid Ala is replaced with Ser. That is, the homologous sites of Sendai virus M protein G69, T116, and A183 or other viral M proteins can be replaced with Glu (E), Ala (A), and Ser (S), respectively. It is preferable to have these mutations in combination, and it is more preferable to retain all of the above three mutations. Mutation can be introduced into the M gene according to a known mutation introduction method. For example, as described in Examples, it is possible to introduce using an oligonucleotide having a target mutation.

  Further, for example, in measles virus, P253-505 (Morikawa, Y. et al., Kitasato Arch. Exp. Med. 1991: 64; 15-30), a temperature-sensitive strain in which the epitope for the monoclonal antibody of the M protein is changed. ) M gene sequence may be used. Alternatively, the 104th Thr of the measles virus M protein corresponding to the 116th Thr of the SeV M protein or the 119th Thr of the M protein of the mumps virus may be substituted with another amino acid (for example, Ala).

  In a further preferred embodiment, the vector used in the present invention is deficient in the M gene. M gene deficiency means that the function of the wild-type M gene is substantially lost, and includes cases of having an M gene having a loss-of-function mutation and deletion of the M gene. A loss-of-function mutation of the M gene can be prepared, for example, by deleting the protein coding sequence of the M gene or inserting another sequence. For example, a stop codon can be designed in the middle of the M protein coding sequence (WO00 / 09700). Most preferably, the vector of the present invention is completely deleted of the coding sequence of the M protein. A vector lacking the ORF of the M protein has lost the ability to form virus particles under any conditions, unlike a vector encoding a temperature-sensitive mutant M protein.

  The temperature-sensitive mutation of the HN gene is not particularly limited. For example, at least one selected from the group consisting of A262, G264, and K461 of Sendai virus HN protein, preferably arbitrarily selected 2 More preferably, those containing mutations at all three amino acid sites or at sites homologous to those of other minus-strand RNA virus M proteins can be preferably used. Here, A262 refers to the 262th amino acid Ala of the HN protein, G264 refers to the 264th amino acid Gly of the HN protein, and K461 refers to the 461st amino acid Lys of the HN protein. The amino acid mutation may be a substitution with another desired amino acid, but is preferably a substitution with an amino acid having a different side chain chemistry like the mutation of the M protein described above. For example, as described above, substitution with a different group of amino acids may be mentioned. Specifically, for example, a Sendai virus HN protein containing a mutation selected from the group consisting of A262T, G264R, and K461G or a mutation homologous thereto can be preferably used. Here, A262T is a mutation in which the 262nd amino acid Ala of the HN protein is replaced with Thr, G264R is a mutation in which the 264th amino acid Gly of the HN protein is replaced with Arg, and K461G is the 461 of the HN protein. A mutation in which the second amino acid Lys is replaced with Gly. That is, the homologous sites of Sendai virus HN protein A262, G264, and K461 or other viral HN proteins can be replaced with Thr (T), Arg (R), and Gly (G), respectively. It is preferable to have these mutations in combination, and it is more preferable to retain all of these three mutations.

  In addition, for example, in mumps virus, referring to the Urabe AM9 strain (Wright, KE et al., Virus Res. 2000: 67; 49-57), which shows a temperature-sensitive property and is used as a vaccine, It is preferable in the present invention to introduce a mutation into amino acids 464 and 468. Amino acid mutations at homologous positions can be applied to other minus-strand RNA viruses.

  The minus-strand RNA virus may have a mutation in the P gene or L gene. Specifically, such mutations include the 86th Glu (E86) mutation of the SeV P protein, the substitution of the 511st Leu (L511) of the SeV P protein with other amino acids, or other negative strands. Examples include substitution of homologous sites in RNA virus P protein. The amino acid mutation may be a substitution with another desired amino acid, but is preferably a substitution with an amino acid having a different side chain chemistry as described above. For example, as described above, substitution with a different group of amino acids may be mentioned. Specific examples include substitution of E86 with Lys (E86K) and substitution of L511 with Phe (L511F). In the L protein, substitution of the 1197th Asn (N1197) and / or the 1795th Lys (K1795) with other amino acids, or substitution of homologous sites of other minus-strand RNA virus L proteins can be mentioned. L protein genes having both of these two mutations of L protein are particularly preferred. The amino acid mutation may also be a substitution with another desired amino acid, but is preferably a substitution with an amino acid having a different side chain chemistry as described above. For example, as described above, substitution with a different group of amino acids may be mentioned. Specific examples include substitution of N1197 with Ser (N1197S) and substitution of K1795 with Glu (K1795E). Having both P gene and L gene mutations can significantly enhance the effects of persistent infectivity, suppression of secondary particle release, or suppression of cytotoxicity. Furthermore, by combining mutations and / or deletions in the envelope protein gene, these effects can be dramatically increased.

  The minus-strand RNA viral vector may be one lacking an accessory gene. For example, knocking out the V gene, one of the SeV accessory genes, significantly reduces the virulence of SeV against hosts such as mice without impairing gene expression and replication in cultured cells (Kato, A et al. J. Virol. 1997: 71; 7266-7272, Kato, A. et al. EMBO J. 1997: 16; 578-587, Curran, J. et al., WO01 / 04272, EP1067179). Such an attenuated vector is particularly useful as a viral vector for gene transfer with lower toxicity.

  Minus-strand RNA viruses transcribe and replicate only in the cytoplasm of the host cell, and do not have integration into the chromosome (Lamb, RA and Kolakofsky, D., Paramyxoviridae: The viruses and their replication) In: Fields BN, Knipe DM, Howley PM, (eds). Fields of virology. Lippincott-Raven Publishers: Philadelphia, 1996: 2; 1177-1204). For this reason, safety problems such as canceration and immortalization due to chromosomal abnormalities do not occur. This feature of the minus-strand RNA virus is thought to contribute greatly to the safety of vectorization. As a result of heterologous gene expression, for example, even when Sendai virus (SeV) is passed through multiple successive passages, almost no base mutation is observed, the stability of the genome is high, and the inserted heterologous gene is expressed stably over a long period of time. (Yu, D. et al., Genes Cells. 1997: 2: 457-466). There are also merits in properties such as the size of the transgene or packaging flexibility due to the absence of capsid structural proteins. SeV vectors can introduce foreign genes up to at least 5 kb, and two or more genes can be expressed simultaneously by adding a transcription unit.

  In particular, Sendai virus is known to be pathogenic to rodents and cause pneumonia, but is not pathogenic to humans. This is also supported by previous reports that nasal administration of wild-type Sendai virus does not show serious adverse effects in non-human primates (Hurwitz, JL et al., Vaccine. 1997: 15; 533-540, Bitzer, M. et al., J. Gene Med. 2003: 5; 543-553, Slobod, KS et al., Vaccine. 2004: 22; 3182-3186). In addition, it shows extremely high gene transfer and expression efficiency that can not be obtained with existing vectors in various cells and organs (Yonemitsu Y. et al., Nature Biotechnol 2000; 18: 970-973; Masaki I. et al., FASEB J 2001; 15: 1294-1296; Yamashita A. et al., J Immunol 2002; 168: 450-457; Masaki I. et al., Circ Res 2002; 90: 966-973; Onimaru M. et al. Circ Res 2002; 91: 723-730; Shoji F. et al., Gene Ther 2003; 10: 213-218; Okano S. et al., Gene Ther 2003; 10: 1381-1391). Even in vitro introduction into human hematopoietic stem cells, high-efficiency gene introduction is possible while maintaining the differentiation ability into three strains (Jin CH. Et al., Gene Ther. 2003; 10: 272-277). These features of Sendai virus support the use of Sendai virus vectors as gene therapy vectors in hematopoietic cell transplantation into humans.

  A minus-strand RNA viral vector can encode a desired foreign gene in genomic RNA. A recombinant viral vector containing a foreign gene can be obtained by inserting the foreign gene into the genome of the above viral vector. As the insertion position of the foreign gene, for example, a desired site in the protein non-coding region of the viral genome can be selected. For example, between each 3 'leader region of genomic RNA and the viral protein ORF closest to the 3' end, It can be inserted between the protein ORF and / or between the viral protein ORF closest to the 5 ′ end and the 5 ′ trailer region. Further, in a genome in which an envelope protein gene such as M, F or HN gene is deleted, a nucleic acid encoding a foreign gene can be inserted into the deleted region. When introducing a foreign gene into a paramyxovirus, it is desirable to insert it so that the polynucleotide chain length of the inserted fragment into the genome is a multiple of 6 (Kolakofski, D. et al., J. Virol. 1998). : 72; 891-899; Calain, P. and Roux, LJ Virol. 1993: 67; 4822-4830). An E-I-S sequence is constructed between the inserted foreign gene and the viral ORF. Two or more foreign genes can be inserted in tandem via the E-I-S sequence.

  The foreign gene introduced by the minus-strand RNA virus is not particularly limited, and examples of natural proteins include cytokines, other humoral factors, growth factors, receptors, intracellular signal molecules, enzymes, peptides, and the like. It is done. The protein can be a secreted protein, a membrane protein, a cytoplasmic protein, a nucleoprotein, and the like. Artificial proteins include, for example, fusion proteins such as chimeric toxins, dominant negative proteins (including soluble receptors or membrane-bound dominant negative receptors), deletion-type cell adhesion molecules and cell surface molecules. Can be mentioned. Moreover, the protein which added the secretion signal, the membrane localization signal, or the nuclear translocation signal etc. may be sufficient. Antisense RNA molecules or RNA-cleaved ribozymes can be expressed as transgenes to suppress the function of specific genes. If a viral vector is prepared using a disease therapeutic gene as a foreign gene, this vector can be administered to perform gene therapy. As an application of the viral vector of the present invention to gene therapy, a foreign gene that can be expected to have a therapeutic effect can be supplied in a patient's body by either gene expression by direct administration or gene expression by indirect (ex vivo) administration. It is possible to express deficient endogenous genes and the like from dendritic cells. The method of the present invention can also be used as a gene therapy vector in regenerative medicine.

Examples of loaded genes are given below.
CD45: SCID cause 1 gene, located on chromosome 1q31-32. This is a phosphatase that regulates Src kinase with a common lymphocyte CD antigen, and this deficiency results in T and NK deficient SCID (Hermiston. Et al., Annu. Rev. Immunol. 2003: 21; 107-137; Buckley RH., Annu. Rev. Immunol. 2004: 22; 625-655)
IL-7 receptor alpha: SCID cause 1 gene, located on chromosome 5p13. A receptor subunit of IL-7 that is important for differentiation of lymphocytes, especially T cells, and homeostasis. This deficiency results in T-deficient SCID (Peschon JJ., J. Exp. Med. 1994: 180; 1955-1960 ; Buckley RH., Annu. Rev. Immunol. 2004: 22; 625-655)
RAG1 / 2: SCID cause 1 gene, located on chromosome 11p13. An important recombinase in the production of T cell receptors and antibodies. This deficiency results in T and B deficient SCID (schwarz. Et al., Science. 1996: 274; 97-99; Buckley RH., Annu. Rev. Immunol. 2004: 22; 625-655)
Adenosine Deaminase (ADA): SCID cause 1 gene, located on chromosome 20q13.11. It is an enzyme in the recycling pathway of purine metabolism, and this gene deficiency causes lymphocytes to become apoptotic due to accumulation of purine metabolites, resulting in T and NK deficient SCID (Noguchi M., Cell, 1999: 73; 147-157; Puck JM., Hum. Mol. Gemet., 1993: 1099-1104; Buckley RH., Annu. Rev. Immunol. 2004: 22; 625-655)
common gamma chain (γc): SCID cause 1 gene, located on chromosome Xq13.1. It is a subunit common to IL-2, IL-4, IL7, IL-7, IL-15, and IL-21 receptors, and becomes a T, B, NK deficient SCID (so-called X-linked SCID) (Buckley RH. Et al., J. Pediatr. 1997: 130; 378-387; Buckley RH. Et al., N, Engl. J. Med. 1999: 340; 508-516, Buckley RH., Annu. Rev. Immunol 2004: 22; 625-655)
DAF and CD59: Glycosil phosphatidylinositol linked protein that suppresses complement activity. Both deficiencies cause hemolysis due to erythrocyte self-complement activity and cause severe nocturnal hemoglobinuria (Johnson RJ. Et al ., J. Clinic. Pathol., 2002: 55; 145-152)
copper transporting P-type ATPase (ATP7B, Wilson disease protein): A causative gene of Wilson disease. This protein is located on chromosome 13 and is important for copper metabolism in the bile duct. This defect causes copper deposition in the liver, cornea, and brain (Ferenci P., Clin liver Dis. 1998: 2; 31-49)
GFP (Owan jellyfish fluorescent chromophore), luciferase: These can be used as a marker of transgenic cells for analysis of in vivo kinetics and determination of gene introduction efficiency.
FGF-2: (WO2002 / 042481)
Others, Btk (deficient in companion agammaglobulinemia), CD40 / CD40 ligand (deficient in high IgM syndrome), (Cooper MD. Et al., Hematology, 2003: 313-330), WASP (Wiskott-Aldrich syndrome) Snapper SB. Et al., Annu. Rev. immunol. 1999: 17; 905-929), and the like.

  The expression level of a foreign gene mounted on a vector can be regulated by the type of transcription initiation sequence added upstream (3 ′ side of the negative strand (negative strand)) of the gene (WO01 / 18223). Moreover, it can control by the insertion position of the foreign gene on a genome, and an expression level becomes so high that it inserts near 3 'of minus strand, and an expression level becomes low, so that it inserts near 5'. Thus, the insertion position of the foreign gene can be appropriately adjusted so as to obtain a desired expression level of the gene and so that the combination with the genes encoding the preceding and subsequent viral proteins is optimal. In general, since it is considered advantageous to obtain high expression of a foreign gene, the foreign gene is preferably linked to a transcription start sequence having high efficiency and inserted near the 3 ′ end of the minus-strand genome. Specifically, it is inserted between the 3 ′ leader region and the viral protein ORF closest to 3 ′. Alternatively, it may be inserted between the viral protein gene closest to 3 ′ and the ORF of the second viral protein gene, or between the second and third viral protein genes from 3 ′. In wild-type paramyxoviruses, the viral protein gene closest to 3 'of the genome is the N gene, the second gene is the P gene, and the third gene is the M gene. On the other hand, if high expression of the transgene is not desired, for example, set the insertion position of the foreign gene as 5 'side of the minus-strand genome as much as possible, or make the transcription start sequence less efficient, etc. It is also possible to obtain an appropriate effect by suppressing the expression level from the above.

  As an S sequence to be added when inserting a nucleic acid encoding a foreign gene into the genome, for example, a desired S sequence of a minus-strand RNA virus can be used, but if it is Sendai virus, 3'-UCCCWVUUWC-5 ' The sequence of (W = A or C; V = A, C, or G) (SEQ ID NO: 2) can be preferably used. Particularly preferred are 3′-UCCCAGUUUC-5 ′ (SEQ ID NO: 3), 3′-UCCCACUUAC-5 ′ (SEQ ID NO: 4), and 3′-UCCCACUUUC-5 ′ (SEQ ID NO: 5). These sequences are expressed as 5′-AGGGTCAAAG-3 ′ (SEQ ID NO: 6), 5′-AGGGTGAATG-3 ′ (SEQ ID NO: 7), and 5′-AGGGTGAAAG- 3 '(SEQ ID NO: 8). As the E sequence of the Sendai virus vector, for example, 3′-AUUCUUUUU-5 ′ (SEQ ID NO: 9) (5′-TAAGAAAAA-3 ′ (SEQ ID NO: 10) for DNA encoding a plus strand) is preferable. The I sequence may be, for example, any 3 bases, and specifically, 3′-GAA-5 ′ (5′-CTT-3 ′ in plus strand DNA) may be used.

  In order to produce a minus-strand RNA viral vector, (a) in a mammalian cell, the genomic RNA of the minus-strand RNA virus is encoded in the presence of a viral protein necessary for reconstitution of the RNP containing the genomic RNA of the minus-strand RNA virus. And (b) a step of recovering the produced minus-strand RNA virus. The viral proteins necessary for the reconstitution of the above RNP typically refer to N, P, and L proteins. Transcription can generate a negative-strand genome (ie, the same antisense strand as the viral genome), or a positive strand (antigenome, complementary strand of genomic RNA) can reconstruct a viral RNP. it can. In order to increase the efficiency of vector reconstitution, a plus strand is preferably generated. It is preferable that the RNA ends reflect the ends of the 3 ′ leader sequence and the 5 ′ trailer sequence as accurately as possible in the same manner as the natural viral genome. This can be achieved, for example, by adding a self-cleaving ribozyme to the 5 ′ end of the transcript and accurately cutting the end of the minus-strand RNA viral genome with the ribozyme. Alternatively, in order to accurately control the 5 ′ end of the transcript, the RNA polymerase recognition sequence of bacteriophage is used as a transcription initiation site, and the RNA polymerase is expressed in the cell. In order to control the 3 ′ end of the transcript, for example, the 3 ′ end of the transcript can be encoded with a self-cleaving ribozyme, and the 3 ′ end can be accurately cut out by this ribozyme (Hasan, MK et al., J. Gen. Virol. 1997: 78; 2813-2820, Kato, A. et al., EMBO J. 1997 ,: 16; 578-587 and Yu, D. et al., Genes Cells. 1997: 2; 457-466). As the ribozyme, a self-cleaving ribozyme derived from the antigenomic strand of hepatitis delta virus can be used.

  For more specific recombination of recombinant viruses, reference can be made, for example, to the following documents (WO97 / 16539; WO97 / 16538; WO00 / 70055; WO00 / 70070; WO01 / 18223; WO03 / 025570; Durbin, AP et al. Virology, 1997: 235; 323-332; Whelan, SP et al. Proc. Natl. Acad. Sci. USA, 1995: 92; 8388-8392; Schnell. MJ et al., EMBO J. 1994: 13; Radecke, F. et al., EMBO J. 1995: 14; 5773-5784; Lawson, ND et al., Proc. Natl. Acad. Sci. USA 92: 4477-4481; Garcin, D. et al. EMBO J., 1995: 14; 6087-6094; Kato, A. et al., Genes Cells. 1996: 1; 569-579; Baron, MD and Barrett, T., J. Virol. 1997: 71; 1265-1271; Bridgen, A. and Elliott, RM, Proc. Natl. Acad. Sci. USA. 1996: 93; 15400-15404; Hasan, MK et al., J. Gen. Virol., 1997: 78; 2813 -2820, Kato, A. et al., EMBO J. 1997: 16; 578-587, Yu, D. et al., Genes Cells, 1997: 2; 457-466, Tokusumi, T. et al. Virus Res 2002: 86; 33-38, Li, H.-O. et al., J. Virol. 2000: 74; 6564-6569). By these methods, minus-strand RNA viruses including parainfluenza, vesicular stomatitis virus, rabies virus, measles virus, Linder pest virus, Sendai virus and the like can be reconstituted from DNA.

When transfecting plasmid vectors to express viral proteins and viral genomic RNA in virus-producing cells, for example, the calcium phosphate method (Graham, FL and Van Der Eb, J., Virology. 1973: 52; 456; Wigler, M. and Silverstein, S., Cell. 1977: 11; 223; Chen, C. and Okayama, H. Mol. Cell. Biol., 1987: 7; 2745), methods using various transfection reagents, or electroporation The law etc. can be used. For transfection reagents, DEAE-dextran (Sigma # D-9885 MW 5 × 10 ⁵ ), DOTMA (Roche), Superfect (QIAGEN # 301305), DOTAP, DOPE, DOSPER (Roche # 1811169), TransIT-LT1 (Mirus , Product No. MIR 2300). Chloroquine can be added to prevent the transfection reagent-DNA complex from being degraded in the endosome (Calos, MP, Proc. Natl. Acad. Sci. USA 1983: 80; 3015).

  When using a protein having a temperature-sensitive mutation, production of the recombinant virus is performed at an allowable temperature (for example, 35 ° C. or lower, more preferably 34 ° C. or lower, more preferably 33 ° C. or lower, most preferably 32 ° C. or lower). Even when a wild-type protein is used, efficient particle formation can be achieved by performing virus production at a low temperature. In the case of producing a recombinant virus deficient in an envelope constituent protein gene, in a virus-producing cell, by introducing and expressing such a deficient gene and / or a gene encoding an envelope protein of another virus into the cell, Infectious virus particles can be formed (Hirata, T. et al., J. Virol. Methods, 2002: 104; 125-133; Inoue, M. et al., J. Virol. 2003: 77; 6419-6429; Inoue M. et al., J Gene Med. 2004; 6: 1069-1081).

  For example, in virus production, use envelope proteins derived from viruses that infect human cells, such as the V protein (VSV-G) of Vesicular stomatitis virus (VSV) and the amphotropic envelope protein of retroviruses. Can do. The VSV-G protein may be derived from any VSV strain. For example, VSV-G protein derived from Indiana serotype strain (J. Virology, 1981: 39; 519-528) can be used, but is not limited thereto. Thus, the minus-strand RNA viral vector may be produced by arbitrarily combining envelope proteins derived from other viruses. As an amphotropic envelope protein, for example, an envelope protein derived from murine leukemia virus (MuLV) 4070A strain can be used. An envelope protein derived from MuMLV 10A1 can also be used (for example, pCL-10A1 (Imgenex) (Naviaux, RK et al., J. Virol. 1996: 70; 5701-5705). Examples include herpes simplex virus gB, gD, gH, gp85 protein, EB virus gp350, gp220 protein, etc. Examples of hepadnaviridae proteins include hepatitis B virus S protein. These proteins may be used as a fusion protein in which the extracellular domain is bound to the intracellular domain of F protein or HN protein. Thus, the viral vector used in the present invention includes VSV-G protein and the like. Pseudotype virus vectors containing envelope proteins derived from viruses other than the virus from which the genome is derived are included. By designing the Beropu protein so as not to be encoded in the genomes, after the virus particles infects a cell, does not this protein from the viral vector is expressed.

  In addition, proteins such as adhesion factors, ligands and receptors that can adhere to specific cells on the envelope surface, antibodies or fragments thereof, or these proteins in the extracellular region, derived from the envelope protein of minus-strand RNA viruses It is also possible to produce a virus containing a chimeric protein having the above polypeptide in the intracellular region. This can control the specificity of the viral vector infection. These may be encoded in the viral genome or supplied by expression from a gene other than the viral genome (for example, a gene on another expression vector or a host chromosome) at the time of virus reconstitution.

Helper cells that express envelope-constituting proteins can be produced, for example, by transfecting a gene encoding a deficient envelope-constituting protein or another envelope protein (WO00 / 70055 and WO00 / 70070; Hasan, MK et al. al., J. General Virology. 1997: 78; 2813-2820). In order to enable inducible expression, for example, envelope in a vector having a recombinase target sequence such as Cre / loxP inducible expression plasmid pCALNdlw (Arai, T. et al., J. Virology, 1998: 72; 1115-1121). It is preferable to incorporate a protein gene. As the cells, for example, monkey kidney-derived cell line LLC-MK ₂ cells (ATCC CCL-7) can be used. LLC-MK ₂ cells were cultured in MEM supplemented with 10% heat-treated non-immobilized fetal bovine serum (FBS), penicillin G sodium 50 units / ml, and streptomycin 50 micro-g / ml at 37 ° C., 5% CO _2. Incubate at Since the SeV-F gene product is cytotoxic, the above plasmid pCALNdLw / F designed to induce and express the envelope protein gene product by Cre DNA recombinase is obtained by the calcium phosphate method (mammalian transfection kit (Stratagene)). Gene transfer is performed on LLC-MK ₂ cells according to well-known protocols. After cloning the cells by limiting dilution, the cells are expanded and the cell lines that highly express the transgene are selected. For this purpose, for example, adenovirus AxCANCre is prepared by the method of Saito et al. (Saito et al., Nucl. Acids Res. 1995: 23; 3816-3821; Arai, T. et al., J. Virol. 1998: 72; 1115 -1121), for example, at moi = 3-5, and select cells by Western blotting or virus production.

  The titer of the recovered virus can be determined by, for example, CIU (Cell Infecting Unit) measurement or hemagglutination activity (HA) measurement (WO00 / 70070; Kato, A. et al., Genes Cells. 1996: 1; 569-579; Yonemitsu, Y. & Kaneda, Y., Hemaggulutinating virus of Japan-liposome-mediated gene delivery to vascular cells. Ed. By Baker AH. Molecular Biology of Vascular Diseases. Method in Molecular Medicine: Humana Press. 1999: 295-306; Kiyotani, K. et al., Virology. 1990: 177; 65-74). In addition, for a vector carrying a marker gene such as GFP (green fluorescent protein), the titer can be quantified by directly counting infected cells using the marker as an index (for example, as GFP-CIU). The titer measured in this way can be handled equivalently to CIU (WO00 / 70070).

As long as the virus reconstitutes, the host cell used for reconstitution is not particularly limited. For example, cultured cells such as monkey kidney-derived LLC-MK ₂ cells and CV-1 cells (eg, ATCC CCL-70), hamster kidney-derived BHK cells (eg, ATCC CCL-10), human-derived cells, etc. it can. By expressing an appropriate envelope protein in these cells, infectious virus particles having the protein in the envelope can also be obtained. In addition, in order to obtain a large amount of viral vector, it is possible to amplify the vector by infecting a chicken egg with a viral vector obtained from the above host. A method for producing viral vectors using eggs has already been developed (Nakanishi et al., (1993), "Advanced Protocol III for Neuroscience Research, Molecular Neuronal Physiology", Koseisha, Osaka, pp.153-172. ). Specifically, for example, fertilized eggs are placed in an incubator and cultured at 37-38 ° C. for 9-12 days to grow embryos. Viral vectors are inoculated into the allantoic cavity and eggs are cultured for several days (eg, 3 days) to propagate the viral vectors. Conditions such as the culture period may vary depending on the recombinant Sendai virus used. Thereafter, the urine fluid containing the virus is collected. Separation and purification of Sendai virus vector from urine can be performed according to a conventional method (Yasuhito Tashiro, “Virus Experiment Protocol”, supervised by Nagai and Ishihama, Medical View, pp.68-73, (1995)).

  The recovered viral vector can be purified to be substantially pure. The purification method can be carried out by a known purification / separation method including filtration (filtration), centrifugation, adsorption, column purification and the like, or any combination thereof. “Substantially pure” means that the virus component occupies a major proportion in the solution containing the virus vector. For example, a substantially pure virus vector composition has a 10% (by weight) protein content as a viral vector component of the total protein contained in the solution (except for proteins added as carriers and stabilizers). / Weight) or more, preferably 20% or more, more preferably 50% or more, preferably 70% or more, more preferably 80% or more, still more preferably 90% or more. For example, in the case of a paramyxovirus vector, as a specific purification method, a method using a cellulose sulfate ester or a crosslinked polysaccharide sulfate ester (Japanese Examined Patent Publication Nos. 62-30752, 62-33879, and 62). -30753), and a method of adsorbing to a sulfated-fucose-containing polysaccharide and / or a degradation product thereof (WO97 / 32010), and the like, but are not limited thereto.

  A hematopoietic cell transplant composition can be produced by infecting hematopoietic cells with the above-described minus-strand RNA viral vector. The present invention relates to a method for producing a hematopoietic cell transplant composition using a minus-strand RNA viral vector, and the use of a minus-strand RNA viral vector in the production of a hematopoietic cell transplant composition.

  Hematopoietic cells refer to cells that can differentiate into blood cells of at least one of white blood cells, red blood cells, and platelets. Hematopoietic cells include bone marrow cells, hematopoietic progenitor cells, hematopoietic stem cells (HSC) and the like. The hematopoietic cell used in the present invention is preferably a cell having the ability to differentiate into at least three lineages of white blood cells, red blood cells, and platelets. A hematopoietic progenitor cell is a cell that can differentiate into at least one of the blood cell lineages. Cells that have differentiated into each lineage of blood cells and lost their multipotency (lymphocyte system, granulocyte system, erythroid system, Cells that differentiate into the platelet system). A hematopoietic stem cell is a cell having self-replicating ability and pluripotency, and is capable of differentiating into at least three types of leukocytes, erythrocytes, and platelets. Hematopoietic cells may be those that can differentiate into somatic cells (nervous system, liver, heart, etc.) (Weissman IL. Et al., Science, 2000: 287; 1442-1446; Lagasse E. et. al., Immunity, 2001: 14; 425-436; Anderson DJ. et al. Nat Med, 2001: 7; 393-395).

  Each blood cell can be identified from its morphology and staining specificity. As staining methods, Giemsa staining, Wright staining, May-Giemsa staining, May-Grünwald-Giemsa staining, peroxidase staining, elastase staining, PAS staining, etc. can be used (Neochemistry Laboratory, Blood (above), 1987. , See pages 8-15).

Hematopoietic stem cells are present not only in the bone marrow but also in peripheral blood and umbilical cord blood. In the present invention, hematopoietic cell transplantation refers to transplantation of a cell composition containing hematopoietic cells, and includes bone marrow transplantation (BMT), peripheral blood stem cell transplantation (PBSCT), umbilical cord blood stem cell transplantation (CBSCT), and the like. That is, the present invention can be applied not only to bone marrow cell transplantation but also to peripheral blood hematopoietic stem cell transplantation and umbilical cord blood transplantation. Identification of HSC can be carried out by calculating granulocyte and monocyte progenitor cells by a colony assay method, or measuring CD34 (antigen) positive cells, which are considered to be characteristic of HSC, by a flow cytometer. Specifically, as a hematopoietic cell, c-kit ⁺ , Thy1.1 ^low , lin ⁻ , Sca-1 ⁺ (Osawa M, Hanada K, Hamada H, Nakauchi H. Science. 1996; 273: .. 242-245), CD34 ⁺ in ^{humans, Thy +, Lin -, c} -kit low (Bhatia M, Wang JC, Kapp U, Bonnet D, Dick JE Proc Natl Acad Sci US A. 1997; 94: 5320- And cells having a phenotype of 5325). lin ^- phenotype (lineage negative), which can be identified and selected using the appropriate commercially available kits such as, for example GPA, CD3, CD2, CD56, CD24, CD19, CD14, CD16, and CD99b are all negative belongs to.

  For the method of isolating human hematopoietic stem cells, the following literature can also be referred to (Leary AG, Blood 69: 953, 1987; Sutherland HJ, Blood 74: 1563, 1989; Andrews RG, J Exp Med 169: 1721. 1989; Terstappen LWMM, Blood 77: 1218, 1991; Lansdorp PM, J Exp Med 175: 1501, 1992; Briddell RA, Blood 79: 3159, 1992; Gunji Y, Blood 80: 429, 1992; Craig W, J Exp Med 177: 1331, 1993; Gunji Y, Blood 82: 3283, 1993; Traycoff CN, Exp Hematol 22: 215, 1994; Huang S, Blood 83: 1515, 1994; DiGinsto D, Blood 84: 421, 1994; Murray L, Blood 85 : 368, 1995; Hao QL, Blood 86: 3745, 1995; Laver JH, Exp Hematol 23: 1515, 1995; Berardi AC, Science 267: 104, 1995; Kawashima I, Blood 87: 4136, 1996; Leemhuis T, Exp Hematol 24: 1215, 1996; Civin CI, Blood 88: 4102, 1996; Larochelle A, Nature Med 2: 1329, 1996; Tajima S, J Exp Med 184: 1357, 1996; Sakabe H, Stem Cells 15: 73, 1997 Sakabe H, Leukemia 12: 728, 1998; Harada Mine et al., New hematopoietic stem cell transplantation, Nanedo, 1998, pages 9-23).

For example, mouse bone marrow cells are collected from the femur and tibia by flushing, and after removing red blood cells with Lysing buffer (0.38% NH ₄ Cl in Tris-HCl, pH7.65), anti-Thy1.2 antibody And after removal of mature T cells by antibody complement reaction using rabbit complement (Tomita Y, Mayumi H, Eto M, Nomoto K. J Immunol. 1990; 144: 463-473.). Or, the collected bone marrow cells are anti-B220 (B cell) antibody, anti-CD3 (T cell) antibody, anti-Gr-1 (granulocyte) antibody, anti-Mac-1 (macrophage) antibody, anti-DX-5 (NK cell) antibody. It can be obtained by removing the lineage with an antibody such as beads or by centrifugation (Sato T, Laver JH, Ogawa M. Blood. 1999; 94: 2548-2554.).

When collecting human hematopoietic cells from bone marrow, collect several milliliters of bone marrow at a time from 3 to 5 places on the left and right sides of the iliac bones in the prone position under general anesthesia (to avoid contamination with peripheral blood) ). In the case of allogeneic transplantation, the number of cells is 3 (2-5) x 10 ⁸ / kg (per patient weight), and in the case of autologous transplantation, bone marrow is collected with the goal of 1-3 x 10 ⁸ / kg. Because there is contamination of bone tissue, etc., it is packed in a bag after passing through the mesh. Transplant with a coarse filter when transplanting. In the case of allogeneic transplantation, it is preferable to select a human lymphocyte antigen (HLA) compatible donor. However, transplantation is possible even for donors who do not match HLA due to the establishment of GVHD prevention methods, the development of new immunosuppressants, and the purification technology of CD34 positive cells. As donors, it is preferable that 4 types or more, more preferably 5 types or more, and more preferably all of 3 groups 6 antigens of HLA-A, -B, and -DR coincide with the recipient. In the case of ABOmajor incompatible transplantation, one obtained by removing red blood cells by centrifugation is used. If ABOminor is incompatible, use a product from which plasma has been removed by centrifugation. (See Morishita et al., “Hematopoietic Stem Cell Transplant Manual” 2nd edition, supervised by Kodera and Saito, Nippon Medical Museum, Tokyo, (1999) p260-277).

When collecting peripheral blood stem cells from peripheral blood, G-CSF 400 μg / m ² (or 10 μg / kg) daily is divided into 1 or 2 doses and subcutaneously injected every day for 5 days or until the end of collection. Obtained by collecting hematopoietic stem cells from peripheral blood (venous) using a blood cell separator 1 to 3 times during the period from the 4th to 6th day after the start of administration (See the guideline for stem cell transplantation (3rd edition revised on April 21, 2003)). For mobilization of peripheral blood stem cells (PBSC), see Harada M et al., J. Hematother 5: 63-71, 1996, Waller CF et al., Bone Marrow Transplant 18: 279-283, 1996, Anderlini P et al. , Blood 90: 903-908, 1997, Harada Mine et al., New hematopoietic stem cell transplantation, Nanedo, 1998, 67-72 pages, Nagoya BMT Group, Hematopoietic cell transplantation manual revised third edition, 2004, pages 237-240 That. As G-CSF used for mobilization, a derivative having a modified N-terminus (such as nartograstin) or a modified protein (such as lenograstin) having a sugar chain added can be used in addition to a wild-type protein. G-CSF and other hematopoietic factors may be used in combination. For example, GM-CSF, IL-3, or SCF can be used in combination (Huhn RD et al., Exp Hematol 24: 839-847, 1996; Begley CG et al., Blood 90: 3378-3389, 1997 Lane TA et al., Blood 85: 275-282, 1995). During G-CSF administration, aspirin can be administered to alleviate systemic symptoms such as low back pain, bone pain, and fever, and prevent increased platelet aggregation.

In order to obtain human peripheral blood hematopoietic stem cells (CD34 ⁺ cells), CD34-positive cells were enriched with CD34 antibody magnetic beads from the peripheral blood stem cells obtained above, Isolex system (such as Baxter), CliniMACS (AmCell), Concentrate through an avidin column (CEPRATE, Cellpro), immobilize CD34 antibody in the flask, adsorb the reacting cells to the flask, and wash away the rest (CELLECTOR, AIS). (See Morishita et al., “Hematopoietic Stem Cell Transplantation Manual” 2nd edition, supervised by Kodera, Saito, Nippon Medical Museum, Tokyo, (1999) p260-277).

  In human umbilical cord blood cell transplantation, erythrocyte sedimentation agent blood cells are added to the collected umbilical cord blood and divided into each blood fraction by weight, and the necessary hematopoietic stem cells are obtained by removing the plasma fraction and erythrocyte fraction which are unnecessary during transplantation. In addition, CD34 positive cells were isolated by Isolex system (Baxter), CliniMACS (AmCell), concentration method through avidin column (CEPRATE, Cellpro), CD34 antibody was immobilized on the flask, and the cells that were reacted were adsorbed to the flask. Umbilical cord blood CD34-positive hematopoietic stem cells can be obtained by purification by washing away (CELLECTOR, AIS), etc. (Morishita et al., “Hematopoietic stem cell transplantation manual” 2nd edition, supervised by Kodera, Saito, Nippon Medical Center, Tokyo, (1999), p661-680).

The isolated hematopoietic cells can be cultured by adding, for example, SCF, IL-3, GM-CSF, G-CSF, and Epo by the methyl sesulose method (Sonoda Y. et al., Blood 84: 4099- 4106, 1994; Kimura T. et al., Blood 90: 4767-4778, 1997). Hematopoietic cells are added at about 1 × 10 ² to 1 × 10 ⁴ / ml and cultured, for example, at 37 ° C., 5% CO ₂ , 5% O ₂ . However, the culture conditions may be adjusted as appropriate.

In autologous transplantation, peripheral blood stem cell transplantation is often selected from the viewpoint of convenience, but autologous bone marrow transplantation is also applied. In that case, usually, in the recovery period of hematopoiesis without the influence of chemotherapy, after confirming normal hematopoiesis of the bone marrow, it is collected from the iliac and sternum under general anesthesia. The target cell number is about 3 × 10 ⁸ to 5 × 10 ⁸ / kg of bone marrow nucleated cells. Since the cell suspension obtained by the continuous blood component separation apparatus contains very little granulocytes or erythrocytes, the mononuclear cell separation operation can be omitted. When many granulocytes and erythrocytes are contained, mononuclear cells can be separated by Ficoll specific gravity centrifugation. In the case of bone marrow fluid, mononuclear cells can be separated by Ficoll treatment or a blood separation device in order to remove and concentrate granulocytes and erythrocytes.

Since infusion of contaminated platelets may cause embolization, it is preferable to remove them after collection. For example, it can be collected in a double bag and weakly centrifuged (200 g, 15 minutes) or dispensed into a centrifuge tube and centrifuged at 1600 rpm for 10 minutes to remove platelets and replaced with RPMI1640 medium. When the collected cells are cryopreserved, they are suspended in RPMI1640 medium containing 10% autoserum and 10% DMSO, frozen, frozen with a programmed freezer, and stored in liquid nitrogen. The cell concentration can be 2 × 10 ⁷ to 6 × 10 ⁷ / ml. For relatively short-term cell preservation, ice suspension should be used in cell suspension (concentration of 1 × 10 ⁸ / ml or less) to a final concentration of 6% Hydrodyethyl Starch (HES), 5% DMSO, 4% albumin. Equal amounts of chilled stock solutions can be mixed and stored frozen in a -80 ° C deep freezer (Knudsen LM et al., J. Hematother. 5: 399-406, 1996).

The number of CD34 positive cells required for safe autotransplantation is approximately 2 × 10 ⁶ / kg (Schots R et al., Bone Marrow Transplant. 17: 509-515, 1996; Zimmerman ™ et al., Bone Marrow Transplant 15: 439-444, 1995). The number of CD34 positive cells can be measured by ordinary two color flowcytometry. Specifically, after removing red blood cells from bone marrow cells and peripheral blood apheresis cells by hemolysis, the cells are developed with forward scatter and anti-CD45 antibody, gates are determined, and red blood cells and platelets are removed. Next, this gate is developed with side scatter and anti-CD34 antibody, and the number of cells in the fraction excluding nonspecific reaction parts such as neutrophils is calculated as a ratio to the total number of cells. From this value and the number of blood cells measured in advance with a blood cell counter, the total number of CD34 positive cells can be calculated.
The number of viable stem cells contained in the cryopreserved cells can be determined by counting CFU-GM by the colony formation method. The standard of CFU-GM required for transplantation is generally 1 × 10 ⁵ to 2 × 10 ⁵ / kg.

  The minus-strand RNA viral vector can be introduced into the hematopoietic cell by bringing the hematopoietic cell prepared as described above into contact with the minus-strand RNA viral vector. Minus-strand RNA viral vectors can be contacted with hematopoietic cells in vivo (in vivo) or in vitro (in vitro or ex vivo). For example, culture solution, physiological saline, blood, plasma, serum, body fluid, etc. What is necessary is just to implement in the physiological aqueous solution of this.

  In the contact between the vector and the hematopoietic cell, the MOI (multiplicity of infection; the number of infectious viruses per cell) is preferably between 1 and 500, more preferably 2 to 300, still more preferably 3 to 200. More preferably, it is 5-100, More preferably, it is 7-70. The contact between the vector and the hematopoietic cells is sufficient even for a short time, for example, 1 minute or longer, preferably 3 minutes or longer, 5 minutes or longer, 10 minutes or longer, or 20 minutes or longer, for example, about 1 to 60 minutes. More specifically, it may be about 5 to 30 minutes. Of course, it may be contacted for a longer time, for example, for several days or longer.

  The hematopoietic cells into which the vector has been introduced can be combined with a desired pharmacologically acceptable carrier or vehicle to form a hematopoietic cell transplant composition. The “pharmaceutically acceptable carrier or vehicle” is not limited as long as it is a solution capable of suspending living cells. Examples include phosphate buffered saline (PBS), sodium chloride solution, Ringer's solution, and culture solution.

  The hematopoietic cell transplant composition may contain hematopoietic cells into which no vector has been introduced. The engraftment efficiency of hematopoietic cells can be increased by mixing hematopoietic cells into which the vector has been introduced and hematopoietic cells into which the vector has not been introduced. The mixing ratio is not limited thereto, but for example, vector-introduced hematopoietic cells: non-introduced hematopoietic cells may be appropriately adjusted between 1:10 and 10: 1.

  The present invention also relates to a method for reconstructing blood cells from hematopoietic cells into which a minus-strand RNA viral vector has been introduced. This method includes the steps of (a) contacting a minus-strand RNA viral vector with hematopoietic cells, and (b) injecting the hematopoietic cells of step (a) into an animal. A minus-strand RNA viral vector is a cytoplasmic vector and does not integrate into the chromosomes of hematopoietic cells, so there is no risk of chromosomal damage like retroviruses. Moreover, in the present invention, the minus-strand RNA virus replicates in the cytoplasm of the hematopoietic cell, and even if the hematopoietic cell divides, the vector is distributed to the daughter cell, and the expression of the transgene can be sustained in the reconstructed blood cell. Proven.

  A blood cell refers to a cell component in blood, and specifically includes white blood cells, red blood cells, and platelets. Producing blood cells means producing any of these blood cells, preferably all of white blood cells, red blood cells, and platelets. Leukocytes include granulocytes, monocytes, and lymphocytes. In the present invention, leukocytes mean any of those cells. By the method of the present invention, hematopoietic cells into which a minus-strand RNA viral vector has been introduced can be differentiated into three lines. In other words, leukocytes, erythrocytes, and platelets are generated from hematopoietic cells into which the minus-strand RNA viral vector has been introduced by hematopoietic cell transplantation.

Transplantation of hematopoietic cells can be performed according to well-known transplantation protocols as appropriate. There are no particular restrictions on the animal to be subjected to hematopoietic cell transplantation, but examples include desired mammals, such as non-human mammals such as mice, rats, dogs, pigs, cats, cows, rabbits, sheep, goats, monkeys, etc. And human. For example, in mice, the number of cells administered is per mouse, 5.0 x 10 ⁶ from 2.0 x 10 ^7, preferably about 1.0x10 ^7, preferably about 2.0x10 ⁷ the tail vein, penile veins or the retinal vein from one shot veins, It can be administered by injection (Tomita Y, Mayumi H, Eto M, Nomoto K. J Immunol. 1990; 144: 463-473.). In human bone marrow cell transplantation, 2 x 10 ⁸ / kg to 3 x 10 ⁸ / kg, preferably about 3 x 10 ⁸ / kg are intravenously administered per individual. For human peripheral blood hematopoietic stem cell transplantation, 2 x 10 ⁶ / kg or more for autologous hematopoietic stem cell transplantation per individual, 5 x 10 ⁶ / kg or more for allogeneic transplantation, preferably about 5 x 10 ⁶ / kg for autologous hematopoietic stem cell transplantation In the case of allogeneic transplantation, about 10 x 10 ⁶ / kg is administered intravenously. In human umbilical cord blood hematopoietic stem cell transplantation, the necessary number of transplants per individual is infused intravenously at 2 × 10 ⁷ / kg or more, preferably about 4 × 10 ⁷ / kg. (See Morishita et al., “Hematopoietic Stem Cell Transplant Manual” 2nd edition, supervised by Kodera and Saito, Nippon Medical Museum, Tokyo, (1999) p260-277, p661-680).

  Pre-administration of antihistamines (such as hydroxyzine 25 mg or chlorphenirmine 5 mg) or corticosteroids (hydrocortisone 100 mg) can be performed to prevent blood clots and emboli due to platelet contamination in the infusion. When frozen cells are used for transplantation, tubule injury can be prevented by infusion of 4,000 units of a haptoglobin preparation when hemolysis of erythrocytes in the transplanted cell solution becomes a problem. In the case of peripheral blood stem cell transplantation, it is also preferable to infuse in two divided days.

  In order to promote the engraftment of donor hematopoietic cells, it is preferable to immunosuppress the recipient prior to the injection of hematopoietic cells. For example, in bone marrow transplantation in leukemia, total body irradiation (TBI) can be performed before hematopoietic cell transplantation in order to kill leukemia cells and inactivate host immunocompetent cells. Such treatment is also commonly referred to as preparative regimen or conditioning. TBI is targeted for both acute and chronic leukemia. At this time, it can be used in combination with anticancer agents such as melphalan and cyclophosphamide (CPA). However, TBI can also cause various disorders, and pretreatment with only anticancer drugs, such as busulfan and CPA BUCY, can be selected to avoid these disorders (Inoue, T. et al., Strahlenther Onkol 169: 250 -255, 1993; Blaise, D. et al., Blood. 1992: 79; 2578-2582; Blume, KG. Et al., Blood. 1993: 81; 2187-2193; Hartman, A. et al., Bone Marrow Transplant. 1998: 22; 439-443). In addition, it is possible to reduce the pretreatment dose by performing Donor Lymphocyte Infusion (DLI) simultaneously with bone marrow transplantation.

  When performing radiation irradiation, irradiation methods such as long SAD (Source axis distance) (or SSD (Source skin distance) method, sweep beam method, beam movement method, treatment bed movement method, etc. can be applied. The long SAD (SSD) method is simple in that it does not require a special device, and is often used.The irradiation may be single irradiation or divided irradiation, but divided irradiation is more effective for normal cells. For example, in the case of a human, 10Gy (6cGy / min) per dose, 12Gy (6cGy / min) 6 times / 6 days, 2 times 1.8Gy / time Examples include ˜3 times / day (total dose of about 15 Gy) or 2 to 3 Gy / times of 1-2 times / day (total dose of about 12 Gy), but the dose may be adjusted as appropriate.

  Moreover, it has been proved by animal experiments that hematopoietic cells can be engrafted in the bone marrow if a large number of donor hematopoietic cells are injected without pretreatment. This indicates that donor hematopoietic cell engraftment does not necessarily require myeloablative pretreatment. Based on this, hematopoietic stem cell transplantation using a non-myeloablative pretreatment that performs only host immunosuppression, so-called mini-transplant (Mini-transplant), has been developed, and the present invention is applied to this. It is also possible. Mini-transplants, unlike transplants with myeloablative pretreatment, have fewer complications and extend the indication to older people and patients with organ damage. In the present invention, immunosuppression includes not only myeloablative treatment such as TBI but also non-myeloablative treatment with an immunosuppressant or the like. As an immunosuppressant used for pretreatment, anticancer agents such as fludarabine and cladribine and antithymocyte globulin are used. By performing hematopoietic stem cell transplantation after such pretreatment, it is possible to create a mixed chimera of bone marrow in which the hematopoietic cells of the patient and the donor are mixed, or a complete chimera in which the bone marrow is replaced with all of the donor hematopoietic cells. For non-malignant diseases such as aplastic anemia, a mixed chimera with non-myeloablative pretreatment can be applied, but in tumors such as leukemia, a large amount of lymphocytes are injected to completely chimerize. Examples of diseases for which hematopoietic cell transplantation without bone marrow destruction is considered effective include renal cancer, malignant melanoma, low-grade lymphoma, and aplastic anemia. Bone marrow destruction is preferred for tumors with a high risk of recurrence. When irradiation is performed as a pretreatment for mini transplantation, for example, a small dose TBI of about 1 to 2 Gy / time is applied. Reduced conditioning therapy has also been developed, which is an intermediate pretreatment between conventional pretreatment and mini-transplantation. These pretreatments can also be used in the present invention.

  When a large dose of anticancer drug or whole body irradiation is performed in transplantation, whole body hematopoiesis is suppressed, and resistance to infection is significantly reduced. Therefore, it is preferable to spend in a sterile room during the transplantation period to prevent infection from the outside world. However, since allogeneic peripheral blood stem cell transplantation (allo-PBSCT) using peripheral blood as the source of HSC can be recovered at an early stage, hematopoietic ability can be recovered, so transplantation can be performed without managing a completely sterile room. Is possible. If transplantation is successful, the white blood cell count increases in about 20 days. In general, peripheral blood stem cell transplantation recovers faster than bone marrow transplantation. If the increase in blood cells is small, hematopoietic stem cell transplantation can be further performed.

  In the case of autologous transplantation, there is no problem in engraftment if the number of transplanted hematopoietic stem cells is sufficient, but in the case of allogeneic transplantation, unless the donor and recipient are genetically identical, the recipe for the antigens of the graft T cells of the ent react and cause engraftment failure. Therefore, pretreatment before transplantation requires an immunosuppressive effect for the engraftment of donor HSCs. Conversely, when HSCs engraft and donor hematopoietic capacity is restored, donor-derived T cells attack recipient cells and develop graft-versus-host disease (GVHD). Because GVHD is an important factor in determining the prognosis after allogeneic transplantation, immunosuppressive therapy is performed to prevent the onset. Acute GVHD usually develops within 2 to 6 weeks after allogeneic transplantation. In addition, infectious diseases such as cytomegalovirus and microangiopathy due to immunosuppressive agents may occur. For diagnosis, biopsy from the skin, stomach, duodenum, large intestine, rectum, etc. is useful. Treat patients with corticosteroids if they are moderately ill or above. If they do not respond to steroids, use immunosuppressants such as antithymocyte globulin and tacrolimus. Cyclosporine and methotrexate are often used as prophylactics for relative transplants, and tacrolimus and methotrexate are often used for unrelated relatives. Chronic GVHD develops at a constant rate in long-term survivors who have received allogeneic transplants, typically around 100 days after transplantation. The clinical findings are similar to autoimmune diseases such as scleroderma, lichen planus, Sjogren syndrome, and primary biliary cirrhosis, and can be diagnosed by biopsy of the lips and skin, liver biopsy, etc. Treatment is mainly cyclosporine and corticosteroids, and phototherapy such as PUVA (Psoralens with Ultraviolet A) can be performed for skin lesions.

Hematopoietic cell transplantation in the present invention includes, for example, aplastic anemia, leukemia (acute leukemia such as acute myeloid leukemia and acute lymphoblastic leukemia, and chronic myelogenous leukemia), myelodysplastic syndrome, lymphoma (Hodgkin's disease, non-Hodgkin) (Including lymphomas), blood diseases such as myeloma, malignant tumors (including breast cancer, germ cell tumors, etc.) for which chemotherapy is effective, and congenital diseases. Applicable diseases can also be applied to diseases caused by single gene deficiency or abnormal expression system (promoter deficiency or abnormal control), or diseases caused by two or more gene deficiencies or abnormal expression systems. In addition, it is applicable not only to abnormalities in blood cells but also to diseases caused by abnormalities in somatic cells such as neural cells and hepatocytes that can be differentiated from bone marrow stem cells. Moreover, it can be applied not only to a gene abnormality disease but also to a disease that can be expected to have a therapeutic effect when used as a supply cell for a secreted protein or the like. For example, single-gene deficiencies include X-linked SCID, ADA deficiency, hemophilia, ammunoglobulinemia, hyper-IgMemia, chronic granulomatosis, etc. Hemoglobinuria and the like can be mentioned, and therapeutic effects can be expected by transplanting each causative gene into hematopoietic stem cells and then transplanting the cells. Examples of diseases and genes for which gene therapy in hematopoietic cell transplantation is considered to be effective include, for example, Hurler disease, Scheie disease, and Hurler-Scheie disease (α-L-iduronidase or iduronate sulfatase), Sanfilippo disease (N-sulfatase, N-acetyl-α-D-glucosaminidase, α-D-flucosaminidase-N-acetyltransferase), Morquio disease (galactosamine-β-sulfate sulfatase or β-galactosidase), Maroteaux-Lamy syndrome (N-acetylgalactosamine-4-sulphatase), Sly disease (β-glucuronidase), Gaucher disease (β-glucocerebrosidase), Farber disease (acid ceramidase), Niemann-Pick disease (acid sphingomyekinase), Krabbe disease (galactocerebroside β-galactosidase), Metachromatic leucodystrophy (arylsufryase A), (Α-galactosidase A), SCID (adenosine deaminase), X-linked SCID (gamma-c receptor), X-linked agammaglobulinemia (XLA) (Bruton's tyrosine kinase), JAK-3 deficiency (Jak-3), Aspartyglycoamin uria (aspartyglycosaminidase), Fucosidoses (α-L-fucosidase), Guibaud-Vainsel syndrome (carbonic anhydrase II), Thallassemias (α-, β-globin), G6PD deficiency (glucose-6-phosphate dehydrogenase), PK deficiency ( pyruvate kinase L type), Erythropoietic porphyia (uroporphyrinogen III synthase), and the like. Purine nucleoside phosphorylase (PNP) is the causative gene for purine nucleoside phosphorylase deficiency, IL-2 receptor γ chain (γC) for X-linked severe combined immunodeficiency, and X-linked chronic granulomatous disease Can be exemplified by gp91 ^phox, which is one of the subunits constituting NADPH oxidase. In addition, when transplanting hematopoietic stem cells of solid malignant tumors or hematologic malignant tumors, it is considered possible to induce enhancement of the antitumor effect by introducing a chimeric toxin gene that targets tumor-specific molecules. It is also possible to introduce the multi-drug resistance gene MDR-1 to reduce the side effects of cancer chemotherapy. Furthermore, gene therapy of anti-HIV genes (eg RRE decoy, env antisense) is possible for the treatment of acquired immune deficiency syndrome (AIDS).

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, this invention is not restrict | limited to these Examples. All documents cited in this specification are incorporated as a part of this specification.

Animals used 7-week-old female C57BL / 6J mice and Balb / c mice (KBT Oriental), GFP transgenic mice C57BL / 6-TgN (act-EGFP) OsbC14-Y01-FM131 (Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. FEBS Lett. 1997; 407: 313-319) were used as donors, and C57BL / 6 and Balb / c nu / nu were used as recipients.

Construction of vectors (SeV ¹⁸⁺ / dF-GFP, SeV ¹⁸⁺ / dFM ^ts HN ^ts PLmut-GFP)
The plasmid pSeV ¹⁸⁺ / dF-GFP, which is the vector template for SeV ¹⁸⁺ / dF-GFP (hereinafter SeV / dF-GFP), was inserted between the leader sequence and the NP gene by inserting the full-length cDNA of Sendai virus Z strain. It was prepared by inserting GFP cDNA, a donor nucleic acid, into the NotI cleavage site artificially constructed in the spacer sequence and then deleting the F gene region. For SeV ¹⁸⁺ / dFM ^ts HN ^ts PLmut-GFP (hereinafter tsSeV / dF-GFP), M gene (G69E, T116A, A183S), HN gene (A262T, G264R, K461G), and P, Mutations were inserted into the L gene (P gene: L511F, L gene: N1197S, K1795E) (Inoue M, et al. J Virol 2003; 77: 3238-3246; Inoue M, et al. Mol. Ther. 2003; 7 (5): S37).
For the construction of both vectors, a reverse genetics method for producing a minus-strand RNA virus from cDNA was used (Inoue M, et. Al. J Virol 2003; 77: 3238-3246). Specifically, pSeV / dF-GFP or pSeV ¹⁸⁺ / dFM ^ts HN ^ts PLmut-GFP and a plasmid expressing N, P, L, and F proteins are simultaneously transfected into monkey kidney-derived LLC-MK ₂ cells. Created by. Since the F gene product is necessary for vector reconstitution, it was amplified by feeding it into trans using LLC-MK ₂ cells that continuously express the F protein.

Gene transfer into hematopoietic cells and transplantation into mice All of the following experiments were performed aseptically.
Bone marrow cells collected from the femur of donor mice were flushed with a syringe with a 23 gauge needle in 10% FBS + RPMI1640 (Gibco BRL). Thereafter, erythrocytes were removed with Lysing buffer (0.38% NH ₄ Cl in Tris-HCl, pH7.65), and anti-Thy1.2 antibody (mouse ascites IgM monoclonal antibody, Meiji Dairies) was used to completely remove mature T cells. Health Science Institute, Tokyo) and rabbit complement (CL3051, CEDARLANE, Ontario CANADA) were reacted to bone marrow cells.
T cell-depleted bone marrow cells were adjusted to 1 × 10 ⁷ / ml, then infected with the vector at a multiplicity of infections (MOI) = 10, and incubated in a 37 ° C. incubator for about 1 hour. Thereafter, a part of the cells was plated and then GFP gene expression was confirmed 48 hours later by flow cytometry (FACSCaliber, Becton Dickinson, CA). The collected bone marrow cells were washed three times, centrifuged, and diluted with serum free RPMI. Recipient mice each: C57BL / 6 were injected with ^{2 x 10 7 / 200μl, nu} / nu-BALB / C is 4 x 10 ⁷ / 200μl each 30-gauge syringe from the tail vein. These recipient mice were irradiated with gamma rays 4-6 hours before transplantation, with C57BL / 6 giving a lethal dose of 10 Gry and nu / nu-BALB / C giving a sublethal dose of 7 Gry. In the FK506 administration group for cellular immunosuppression, FK506 (tacrolimus, Fujisawa Pharmaceutical Co., Ltd., Osaka) was intraperitoneally administered daily at 5 mg / kg every two weeks after bone marrow transplantation. In addition, no-spleen mice were laparotomized 2 days before transplantation to remove the spleen.

Flow cytometry Peripheral blood was collected from the tail vein 1, 4, 8, and 12 weeks after bone marrow transplantation, and GFP positive cells were analyzed by FACS. First, erythrocytes were removed from peripheral blood with lysing buffer, washed twice with HANKS solution, and adjusted to about 1 × 10 ^{5 to} 1 × 10 ⁶ / ml. Prior to analysis, PI (propidium iodide) staining was performed to exclude dead cells. In addition, to confirm the fraction of GFP-positive cells in the spleen, bone marrow, and thymus of each mouse, and to determine dendritic cells (DC) and monocytes / macrophages (MФ) after Fc block, anti- CD11bPE (1: 4, Biosciences Pharmingen CA) and anti-CD11cAPC (1: 5, Biosciences Pharmingen CA), anti-B220APC (1:13, Becton Dickinson CA) and anti-IgMPE for B-cell detection (1:10, Becton Dickinson CA), helper T-cell (hT-cell) and cytotoxic T-cell (cT-cell) for determination of anti-CD3APC (1: 5, Biosciences Pharmingen CA), anti-CD8PE ( 1: 10, Becton Dickinson CA) or anti-CD4PE (1:10, Becton Dickinson CA) and natural killer (NK) cells, NKT cells, and T-cells for anti-CD3APC and anti-CD3 -DX5PE (1: 5, Biosciences Pharmingen CA) antibody was used for staining for 30 minutes at 4 ° C and analyzed by FACS Caliber (Becton Dickinson CA). Data were evaluated using FlowJo (TreeStar Inc., San Carlos, CA) or CellQuest ^™ software (Becton Dickinson CA).

Measurement of serum anti-SeV antibody titer 1, 4, 8, 12 weeks after bone marrow transplantation, peripheral blood is collected from the tail vein of mice with a blood collection tube, left at room temperature for 30 minutes, and then centrifuged at 10,000 rpm for 5 minutes did. The supernatant was stocked and stored at -80 ° C. At the time of examination, the sample was thawed at room temperature, and the serum anti-SeV antibody titer was measured using MONILISA (R) HVJ Enzyme-linked Immunosorbent Assay kit (Wakamoto Pharmaceutical Co. Ltd., Tokyo).

Blood test 1, 4, 8, 12 weeks after bone marrow transplantation, peripheral blood is collected from the tail vein of the mouse using a blood collection tube, diluted with 50 μl of a hemocytometer dilution solution to 20 μl of peripheral blood, The examination was performed using a fully automatic hemocytometer (CelltacαMEK-6158, Nihon Kohden, Tokyo).

Measurement of CTL activity Grind recipient mouse spleen with glass slide, lysing, dilute 5 x 10 ⁶ / ml in RPMI + penicillin + DM containing medium with 10% FCS, mix with SeV peptide 1 mM, 24 It was seeded in a well plate and cultured for 5 days. On the third day of the culture, 3 μl (30 U / ml or more) of IL-2 was added per 1 ml of the medium along with the medium exchange, and co-cultured with IL-2 for 48 hours.
Target cells (EL4: American Type Culture Collections) was adjusted to 1 x 10 ⁶ / 200μl, a SeV peptides were added 50 [mu] M. Further, 0.1 mCi of Na ₂ ⁵¹ CrO ₃ was added to the cells, and after shaking well, the cells were incubated for 1.5 hours with shaking every 15 minutes under 10% CO ₂ condition.
Cells cultured for 5 days were collected and seeded in 3 wells at 1 × 10 ⁶ , 5 × 10 ⁵ , 2.5 × 10 ⁵ , 1.25 × 10 ⁵ in a U-bottom 96-well plate. 100 μl each of the target cells was added thereto, incubated for 4 hours, washed twice and then subjected to a γ counter, and SeV-specific Na ₂ ⁵¹ CrO ₃ release was calculated according to the following formula.
% Specific release = ((experimental cpm-spontaneous cpm) / (maximum cpm-spontaneous cpm)) x 100
Maximum release was obtained by cell lysis using 1% triton X-100 (Wako Pure Chemical Industries Ltd., Osaka, Japan). The natural release amount was obtained by culturing the cells only with 0.2 ml of the culture solution. Natural release ranged from 15% to 20% of maximum release. Three specimens were prepared for each individual.
As the SeV peptide, an amino acid sequence NH ₂ -FAPGNYPAL-COOH (SEQ ID NO: 11) of an NP protein that has been shown to be a target sequence of CTL already reported and presented to class I antigens is synthesized. Used. The tsSeV / dF-GFP NP protein has no mutation at the same site.

Statistical analysis All statistical analysis values were expressed as mean ± standard error. Data was analyzed using the Mann Whitney U-test, and P <0.05 was considered statistically significant.

[Example 1] SeV gene transfer efficiency and engraftment efficiency of mouse hematopoietic cells First, gene transfer using SeV / dF-GFP and tsSeV / dF-GFP to C57BL6 mouse bone marrow cells (after removal of T cells) Efficiency was examined (Figure 1a). Each vector solution was added to the bone marrow cell culture solution at MOI = 10, washed 1 hour later with fresh culture solution, and analyzed for GFP positive rate 48 hours later by FACS. A typical analysis diagram is shown in FIG. The GFP positive rate by SeV / dF-GFP is 80.7 ± 2.9% (n = 3) and that by tsSeV / dF-GFP is 88.8 ± 1.9% (n = 18). Both have high gene transfer efficiency of 80% or more. Indicated.
Next, the engraftment efficiency of bone marrow cells into which GFP gene was introduced with SeV in irradiated allogeneic mice was examined using GFP positive rate in peripheral blood cells as an index (FIG. 1b). When a positive control GFP transgenic mouse (GFP-TG) was used as a donor, the engraftment rate was always 98% or more during the course, and this efficiency was maintained from 4 to 8 weeks after transplantation ( n = 3). When bone marrow cells treated with SeV / dF-GFP were transplanted, the GFP positive rate in peripheral blood was lower than that of the positive control (0.1-2.3%, n = 9) from the early stage of transplantation (1 week). Cells disappeared. On the other hand, when bone marrow cells treated with tsSeV / dF-GFP were transplanted, a relatively high GFP positive rate was shown early in the transplantation (45-85%, n = 10). Thereafter, the GFP positive rate gradually decreased, and at 4 weeks after transplantation, although there were individual differences (2.0-26%), on average, 10% or more of peripheral blood cells were still GFP positive.

[Example 2] In vivo distribution of hematopoietic cells transfected with tsSeV / dF-GFP and GFP positive rate in each cell fraction In Fig. 1, six mice with relatively good engraftment efficiency were used. Then, the distribution of GFP-expressing cells in the hematopoietic system was examined (FIG. 2). The GFP positive rate at 5 weeks in these 6 peripheral bloods was 55.9 ± 5.9%, relatively high in the spleen and thymus, but 17.1 ± 8.8% in the lower limb femur bone marrow.
Next, the GFP positive cell rate in each cell fraction was analyzed by FACS in the spleen and bone marrow (FIG. 3). The identification of the cell ^{fraction, NK cells = CD3 - / DX5} +, NKT cells = CD3 + / DX5 +, whole T-cells = CD3 + / DX5 -, helper T-cells = CD3 + / CD4 +, cytotoxic ^{T-cells = CD3 + / CD8} +, dendritic cells (DCs) = CD11b + / CD11c +, monocytes / macrophages (MΦ) = CD11b + / CD11c -, with ^{B-cells = B220 + / IgM} +. NK, DC, MΦ, and B were relatively positive in both spleen and bone marrow. Although the positive rate for NKT and T is relatively low, the GFP-positive NKT / T cells detected in the spleen and bone marrow of the recipient were transferred because the T cell fraction of the transplanted cells was almost completely removed. It was thought to be derived from hematopoietic stem cells rather than differentiated NKT / T cells.

[Example 3] Engraftment dynamics of hematopoietic cells transfected with tsSeV / dF-GFP in recipients after 5 weeks From the above results, bone marrow cells transfected with tsSeV / dF-GFP are allogenic. It was shown to be repopulation in the recipient. However, it became clear that the GFP positive rate gradually decreased. In order to examine this decrease in the number of GFP positive cells in more detail, longer-term engraftment dynamics were examined.
When bone marrow cells treated with tsSeV / dF-GFP were transplanted in a similar manner to a total of 53 C57BL6 mice, about 40% of the mice died 5-7 weeks after transplantation (FIG. 4a). For these mice, peripheral blood was collected at 1 and 4 weeks after transplantation to confirm GFP positive cells. However, in the blood collection at 4 weeks of dead mice, all cases had a relatively good GFP positive rate ( In addition to more than 10%), dilution of blood was observed. Therefore, the number of blood cells in mice with a good survival rate at 4 weeks after transplantation was examined.
As shown in FIG. 4b, bone marrow cells treated with tsSeV / dF-GFP as compared to untreated mice (n = 6) and recipient mice transplanted with GFP-TG mouse bone marrow (n = 6). Mice (10% or more of peripheral blood GFP-positive cells at 4 weeks out of all 6 mice: n = 3) transplanted with a white blood cell count (WBC), red blood cell count (RBC), platelets at 4 weeks after transplantation (PLT) All were significantly reduced.

[Example 4] Examination of immunological reaction in recipients against transplanted hematopoietic cells transfected with tsSeV / dF-GFP
In order to investigate the cause of pancytopenia in recipient mice transplanted with tsSeV / dF-GFP transgenic bone marrow, we investigated the immune response to SeV in the recipient mice.
4-1. Cellular immune response
Examination of peripheral blood GFP-positive cell rate at 4 weeks in recipient mice transplanted with tsSeV / dF-GFP transgenic bone marrow and specific cytotoxic T cell activity (CTL activity) against Sendai virus-infected cells in the same individual did.
As shown in FIG. 5a, individuals with a high peripheral blood GFP positive cell rate (64.9%) showed significant CTL activity, but the level remained low.
In order to examine whether the slight increase in CTL activity obtained in Fig. 5a is significantly related to the decrease in the number of GFP positive cells derived from tsSeV / dF-GFP, daily administration of FK506 mainly suppressing cellular immunity (From 2 weeks after transplantation, 5 mg / kg / day) and cell engraftment dynamics in nude mice were examined. As shown in FIG. 5b, since no increase in cell engraftment was observed in both cases, the involvement of CTL in the elimination of transgenic cells in recipient mice transplanted with tsSeV / dF-GFP transgenic bone marrow was It was considered relatively low.

4-2. Next, regarding the humoral immune response in recipient mice (n = 34) transplanted with tsSeV / dF-GFP transgenic bone marrow, the relationship between the peripheral blood GFP positive cell rate at 4 weeks and anti-SeV antibody Was examined.
As shown in FIG. 6a, 11 of 34 died at 5 weeks and 23 survived for a long time. From these, the number of peripheral blood GFP-positive cells collected 4 weeks after transplantation and the anti-SeV antibody titer at the same time were examined. Individuals with a high GFP positive rate had a low anti-SeV antibody titer, and individuals with a low GFP positive rate. A tendency of high anti-SeV antibody titers was observed (FIGS. 6a and 6b). Since the ELISA system for detection of anti-SeV antibodies used in the present invention recognizes SeV membrane proteins, recipients can detect antibodies against the few membrane proteins expressed in bone marrow cells infected with tsSeV / dF-GFP. It was thought to have been produced.

[Example 5] Study on post-transplantation kinetics of gene-transduced and non-transfected mixed hematopoietic cells Finally, at least a portion of tsSeV / dF-GFP transgenic bone marrow cells in recipient bone marrow or spleen blood cells Since there are donor-derived GFP-positive T cells that should have been removed, it is considered that these GFP-positive T cells may be derived from hematopoietic stem cells in the donor cells gener- ated by tsSeV / dF-GFP. On the other hand, the recipients of tsSeV / dF-GFP gene-introduced bone marrow cells include a group that dies with relatively high repopulation and a group that is rapidly eliminated after 4 weeks. It was hypothesized that the gene transfer efficiency was low in the latter.
To examine this, equal amounts of transgenic bone marrow cells and non-introduced bone marrow cells by tsSeV / dF-GFP were mixed and transplanted into recipient mice (FIG. 7). In this experiment, all the recipient mice died by the 8th week in the group transplanted with only the transgenic cells, but the GFP-positive peripheral blood cell rate in all 5 cases rapidly in the mixed transplant group after 4 weeks. Went down. This result was considered to be consistent with the above hypothesis.

  According to the present invention, hematopoietic cell transplantation using transgenic hematopoietic cells using a non-chromosomal cytoplasmic vector has become possible. Since the method of the present invention does not involve integration into the host chromosome, the gene can be introduced without risk of inactivation of the tumor suppressor gene or activation of the oncogene. It is possible to build. In some cases of congenital immunodeficiency diseases, hematopoietic cell transplantation is not effective, and these treatments are the only options that remain with gene therapy. It is expected that the method of the present invention is applied to new gene therapy for these intractable diseases.

a. Gene transfer efficiency analysis by SeV / dF-GFP and tsSeV / dF-GFP into T cell-removed donor (C57BL6 mouse) bone marrow cells by flow cytometry. It is a figure which shows a typical profile. The gene introduction efficiency of SeV / dF-GFP was 80.7 ± 2.9% (n = 3), and that of tsSeV / dF-GFP was 88.8 ± 1.9% (n = 18). b. Peripheral blood cells when bone marrow collected from C57BL6 strain GFP transgenic mice (GFP-TG) or C57BL6 mouse bone marrow transfected with SeV / dF-GFP or tsSeV / dF-GFP is transplanted to allogeneic recipients It is a figure which shows a time-dependent change of the GFP positive rate in a cell. The vertical axis is a logarithmic display. It is a figure which shows the GFP positive rate (5 weeks after a transplant, a total of 6 heads) in various organs when the C57BL6 mouse | mouth bone marrow introduce | transduced by tsSeV / dF-GFP by the flow cytometry is transplanted to an allogeneic recipient. Among the individuals analyzed in FIG. 1, those with good survival rate were selected and analyzed from peripheral blood at 4 weeks. A representative profile is shown. Numbers in parentheses indicate mean ± standard error% (n = 6). GFP positive rate in each blood cell fraction in spleen and bone marrow when C57BL6 mouse bone marrow transfected with tsSeV / dF-GFP is transplanted to allogeneic recipients by flow cytometry (5 weeks after transplantation, total of 6) FIG. It is a figure which shows the time-dependent change (a total of 53 heads) of a survival rate when the C57BL6 mouse | mouth bone marrow introduce | transduced with tsSeV / dF-GFP is transplanted to an allogeneic recipient. b. Peripheral blood leukocyte count (WBC), erythrocyte count (RBC), platelet count when untreated C57BL6 mice, tsSeV / dF-GFP transgenic or bone marrow transplanted to allogeneic recipients (PLT) (4 weeks after transplantation). A marked pancytopenia is observed only in tsSeV / dF-GFP. a. SeV-specific CTL activity (4 weeks after transplantation) when C57BL6 mouse bone marrow transfected with tsSeV / dF-GFP is transplanted into allogeneic recipients. The numbers in parentheses indicate the percentage of peripheral blood GFP positive cells in each individual at the same time. For each individual, three culture wells are seeded with target cells, each data is an average of three wells, and error bars indicate standard errors. b. When C57BL6 mouse bone marrow transfected with tsSeV / dF-GFP is transplanted into allogeneic recipients (n = 10), and FK506 (5 mg / kg / day) is injected into the peritoneal cavity from the second week after transplantation into similar mice. It is a figure which shows the time-dependent change of the peripheral blood GFP positive cell at the time of transplanting the bald / c mouse | mouth bone marrow which introduce | transduced the internal biday (n = 7) and tsSeV / dF-GFP into the allogeneic nude mouse. None of the operations failed to prevent the decrease in the GFP positive rate. a. When transplanted with C57BL6 mouse bone marrow transfected with tsSeV / dF-GFP to allogeneic recipients (total population n = 34), death group (n = 11, white triangle) within 5 weeks and long-term survival group ( It is a figure which shows the correlation (4 weeks after a transplant) of the peripheral blood GFP positive cell rate (vertical axis) and serum anti-SeV antibody titer (horizontal axis) in n = 23 and a black square. Note that in the dead population, peripheral blood GFP positive rate is high and anti-SeV antibody titer is low. b. A bar graph in which the data shown in a. above is divided into two groups (dead group, surviving group) and the GFP positive rate (left) and anti-SeV antibody titer (right) are compared. Error bars indicate standard error. When C57BL6 mouse bone marrow transfected with tsSeV / dF-GFP is transplanted to allogeneic recipients alone (ts-BMT, n = 3) and when the same bone marrow is mixed with the same amount of untreated bone marrow ( It is a figure which shows the time-dependent change of the peripheral blood GFP positive cell rate in ts-Mix-BMT, n = 5). In the ts-BMT group, all cases died within 8 weeks although the GFP positive rate was relatively maintained. On the other hand, in the mixed cell transplantation group, the rate of GFP positive cells rapidly decreased, and all cases survived for a long time. The vertical axis is a logarithmic display.

Claims (14)

  A method for producing a hematopoietic cell transplant composition, comprising introducing a minus-strand RNA viral vector into a hematopoietic cell and preparing a composition comprising the hematopoietic cell and a pharmaceutically acceptable medium.   The method according to claim 1, wherein the minus-strand RNA viral vector is mutated or deleted in one or more genes encoding envelope-constituting proteins.   The method according to claim 2, wherein all genes encoding envelope-constituting proteins are mutated or deleted.   The method according to claim 2, wherein at least one gene encoding an envelope-constituting protein is defective.   The method according to claim 1, wherein the minus-strand RNA virus is a Paramyxoviridae virus.   The method according to claim 5, wherein the Paramyxoviridae virus is Sendai virus.   A hematopoietic cell transplant composition obtained by the method according to any one of claims 1 to 6. A method of generating transgenic blood cells from hematopoietic cells,
(A) a step of bringing a minus-strand RNA viral vector into contact with a hematopoietic cell, and (b) a step of injecting the hematopoietic cell of step (a) into an animal.   9. The method of claim 8, further comprising the step of immunosuppressing the animal prior to the injection of step (b).   The method according to claim 8, wherein in the minus-strand RNA viral vector, one or more genes encoding envelope-constituting proteins are mutated or deleted.   The method according to claim 10, wherein all genes encoding envelope-constituting proteins are mutated or deleted.   The method according to claim 10, wherein at least one gene encoding an envelope-constituting protein is missing.   The method according to claim 8, wherein the minus-strand RNA virus is a Paramyxoviridae virus.   The method according to claim 13, wherein the Paramyxoviridae virus is Sendai virus.