JP2007068473A - Expression vector containing transcription factor-binding sequence of mhc gene - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To increase the expression amount of an introduced gene in a nucleic acid medicine using an expression vector, and to increase the expressed amount of the introduced gene in the nucleic acid medicine, especially a gene vaccine and to enhance antigen-specific immune reaction against a protein synthesized from the introduced gene. <P>SOLUTION: The expression vector contains a modified promoter containing a transcription factor-binding sequence having a binding ability with a major histocompatibility gene complex (MHC) enhanceosome in the cell of a vertebrate. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an expression vector for expressing a target gene and a gene expression method using the vector. In addition, the present invention relates to a nucleic acid drug such as a gene vaccine containing the vector, and further to a disease prevention or treatment method using the nucleic acid drug.

  In recent years, technological development of nucleic acid medicine, which is a medicine using nucleic acid as a main component, has been actively developed. In particular, research that belongs to the category of gene therapy or gene vaccine that treats or prevents human or animal diseases by introducing a target gene into a living body using a vector has entered the stage of practical application. Some have already gone into clinical research.

  In general, gene therapy artificially strengthens or weakens the function of a gene by introducing the gene using an expression vector or the like for a disease caused by an abnormal function of a specific gene in an animal. The treatment is performed by. As a general problem of this gene therapy, a common main problem is that sufficient expression efficiency cannot be obtained for the introduced gene, and therefore a sufficient therapeutic effect cannot be obtained. On the other hand, gene vaccines use an expression vector to introduce a foreign gene into a living body, and artificially induce a specific immune response against the protein expressed from the introduced gene, thereby infectious diseases. Or to prevent or treat malignant tumors. Therefore, in gene vaccines, it is necessary to develop a vector specialized for that purpose. That is, the development of a vector that can induce a specific immune reaction more effectively.

  For this reason, many attempts have been made to develop and improve expression vector promoters in order to increase gene expression efficiency with expression vectors. However, among promoters that can actually be used for gene therapy and gene vaccines, expression efficiency is good. Currently, CMV promoters, CAG promoters, and the like are widely used. Although attempts have been made to use tissue-specific promoters, their transcriptional activity is very weak compared to CMV and CAG promoters.

  In this context, in addition to the conventional combination of the target gene and promoter, a promoter-enhancing transcription factor binding sequence such as an enhancer, and a gene encoding a transcription factor that binds to this transcription factor binding sequence to enhance transcription activity In combination with an expression vector, the idea of a self-promoting expression system composed of a facilitating interaction between the transcription factor expressed by the vector and the transcription factor binding sequence thereto was proposed. (See Patent Document 1). However, the above-mentioned Patent Document 1 is a so-called paper example in which the experimental method is described but the experimental result is not described.

  The inventors of the present application have studied the self-promoting expression system independently of the above-mentioned Patent Document 1 so far, and can implement the self-promoting expression system through actual experiments. We have demonstrated and reported on this (for example, see Non-Patent Document 1). That is, the inventors of the present application, in order to actually realize a self-promoting expression system, the types of promoters and transcription factors to be modified, the range of transcription factor binding sequences to be incorporated, the promoters to be incorporated, It has been clarified that elements such as the positional relationship with the transcription factor binding sequence are very important, and the combination of these elements greatly changes the level of expression efficiency.

  On the other hand, as described above, when the expression vector is used as a gene vaccine, in addition to merely increasing the expression level of the transgene, the gene expression from the transgene is finally converted into an antigen in vivo. Must lead to enhanced specific immune response. Antigens such as proteins and peptides recognized as foreign substances in vivo are presented extracellularly as antigens by major histocompatibility complex (MHC) molecules. Therefore, activating the antigen-presenting function by MHC is essential for inducing an antigen-specific immune response. MHC has type I and type II, and transcription of both MHCI type and type II genes is activated by a class II transactivator (CIITA), which is a transcription factor (Non-Patent Document). 2-6). Specifically, CIITA binds to transcription factors binding sequences of MHCI type and MHCII type genes through formation of a protein complex called MHC enhanceosome with several other proteins. Then, the transcription of each MHC gene is activated. For this reason, attempts have been reported to activate the immune response using CIITA as a target (see, for example, Non-Patent Document 7).

Japanese Patent Laid-Open No. 11-176 Abstracts of the 16th Annual Meeting of the Japanese Society for Biological Defense Elsen, PJ. Et al., Current Opinion in Immunology, 16, 67-75, 2004 Masternak, K. et al., Genes Dev, 14, 1156-1166, 2000 Zhu, XS. Et al., Mol Cell Biol, 20, 6051-6061, 2000 Jabrane-Ferrat, N., et al., Int immunol, 15, 467-475, 2003 Rousseau, P. et al., Immunology, 111, 53-65, 2004 Martin, BK. Et al., J Immunol, 162, 6663-70, 1999

  As described above, in conventional nucleic acid medicine using an expression vector, high level expression of a transgene is obtained. In addition, in nucleic acid medicine, in gene vaccine, in addition to high level expression of a transgene, It was difficult to induce a high level of antigen-specific immune response to the expressed protein.

  Therefore, the present invention is intended to improve such problems of the prior art, and aims to further increase the expression level of a transgene in a nucleic acid drug using an expression vector. The purpose is to further increase the expression level of the transgene and to further enhance the antigen-specific immune response to the protein synthesized from the transgene.

  In order to achieve the above object, the inventors of the present application have conceived to combine the self-promoting expression system with the antigen-presenting activation action by CIITA. That is, in the self-promoting expression system, the transcription factor binding sequence of the MHC gene that plays an essential role for antigen presentation (region that binds to MHC enhanceosome) is used as the transcription factor binding sequence, and MHC is used as the transcription factor. An expression vector system using CIITA, a gene transcription factor, was constructed. However, the target of the transcription factor binding sequence is not a simple transcription factor, but a protein complex called MHC enhanceosome (at least three transcription factors in this complex bind directly to the transcription factor binding sequence). Thus, the basic configuration of the present invention is different from the prior art. And, as a result of examining the expression vector system constructed in this way and the expression of the target (antigen) gene and the antigen-specific immune reaction against the expressed protein, the expression vector system was compared with the conventional expression vector, It has been found that not only a high level of gene expression efficiency can be obtained, but also a high level of antigen-specific immune response against the expressed protein can be induced.

  That is, in the first aspect, the expression vector of the present invention has a modified promoter containing a transcription factor binding sequence capable of binding to a major histocompatibility complex (MHC) enhanceosome in a vertebrate cell. It is characterized by. In a preferred embodiment, the expression vector includes a target gene operably linked to the modified promoter, and the transcription factor binding sequence promotes transcription of the target gene. The present invention also provides an expression vector mixture comprising the expression vector and a CIITA expression vector. In these expression vectors and / or expression vector mixtures, the transcription factor binding sequence is preferably a transcription factor binding sequence of an MHC class I, MHC class II and / or β2 microglobulin gene.

  In a different aspect of the present invention, there is provided a gene expression composition comprising the above expression vector or expression vector mixture, or a nucleic acid drug for vertebrates. These nucleic acid drugs can be used as vaccines for the prevention or treatment of malignant tumors, multiple sclerosis, Alzheimer's disease, or other diseases.

  The expression vector of the present invention can efficiently express a target gene. In addition, the gene vaccine containing the expression vector can induce a high level of antigen-specific immune response against the protein expressed from the transgene in addition to the high level expression of the transgene. Furthermore, the nucleic acid medicament of the present invention can be used not only for the prevention or treatment of infectious diseases, but also for the prevention or treatment of malignant tumors, multiple sclerosis, Alzheimer's disease, and other diseases.

(MHC enhanceosome and its transcription factor binding sequence)
The MHCI type II gene encodes a cell surface glycoprotein, the antigen peptide recognized as a foreign substance is presented on the cell surface by MHC, and the MHC antigen peptide complex is expressed on the T cell receptor (TCR surface of the T lymphocyte). ) Causes T cell activation. The MHCI type protein presents endogenous peptides to CD8 positive T cells, while the MHC II type protein presents exogenous peptides to CD4 positive T cells. These MHC-peptide-TCR interactions play an important role in antigen-specific immune responses. MHCI type subtype genes (such as HLA-A, HLA-B, HLA-BE, and HLA-C), MHCII type subtype genes (such as HLA-DRA), and β2 microglobulin gene are on the promoter sequence. Has a unique transcription factor binding sequence called SXY module, and a transcription factor complex called MHC enhanceosome (RFX (regulatory factor X) with CIITA as a core, CREB (cAMP response element binding protein), NFY ( Transcription of each gene is activated by interacting with nuclear factor Y), transcription factor complex composed of CBP (CREB-binding protein), BRG1 and the like. Here, it is RFX, CREB, and NFY that actually combine with the SXY module, but CIITA integrates these. The SXY module is a transcription factor binding sequence module having four transcription factor binding sequences of S or W box, X1 box, X2 box, and Y box as constituent elements. The transcription factor that binds to the S or W box is unknown, but RFX binds to the X1 box, CREB binds to the X2 box, and NFY binds to the Y box.

(Basic configuration of the expression vector or expression vector mixture of the present invention)
The basic element in one embodiment of the present invention includes a promoter and the transcription factor binding sequence (a sequence that interacts with the MHC enhanceosome and activates transcription), and is further functionally linked to the promoter. An expression vector containing a gene. The basic element in another embodiment is an expression vector further comprising a gene encoding CIITA in the basic element, wherein the CIITA gene is expressed by a promoter different from the target gene. In still another embodiment, the basic element is an expression vector mixture comprising an expression vector incorporating a gene encoding CIITA separately from the expression vector containing the basic element. What is important in the latter two aspects is that CIITA expressed in cells interacts with the transcription factor binding sequence to enhance transcriptional activity and increase expression of the target gene. Although all of the elements may be contained therein or CIITA may be expressed by different expression vectors, it is preferable that the promoter controlling the expression of CIITA does not contain the transcription factor binding sequence. This is because the expression level of the target gene is decreased when CIITA is expressed in a self-promoting manner. In the present invention, the term “promoter” includes a core promoter element such as a TATA box to which an RNA polymerase complex binds during transcription as an essential element, and in addition, a gene including various transcription factor binding sequences upstream thereof. This refers to the DNA region necessary for transcription. The facilitating “transcription factor binding sequence” according to the present invention may be generally referred to as “enhancer”, but is a base sequence that promotes transcription by binding of the corresponding transcription factor, and is 5 ′ upstream or It exists 3 'downstream.

(Promoter serving as the base of the expression vector of the present invention)
The promoter used as a base for preparing the modified promoter incorporating the transcription factor binding sequence may be any promoter as long as it is a promoter that functions in vertebrate cells. However, since the object of the present invention is to increase the gene expression efficiency, it is desirable to use a promoter having a strong promoter activity as a base promoter. Therefore, for example, a CMV promoter or a CAG promoter is preferable as a base promoter.

(Transcription factor binding sequence of the expression vector of the present invention)
The transcription factor binding sequence according to the present invention binds to an MHC enhanceosome in a vertebrate cell and has promoter activity when the transcription factor binding sequence is present at an appropriate position in the positional relationship with the promoter. It is a sequence that enhances. At least one SXY module must be included, but other transcription factor binding sequences such as an NF-κB binding sequence (enhancer A) and an interferon-stimulated response as long as they have an action of enhancing transcription activity as a whole An element (ISRE) or the like may be included. The SXY module may be incorporated in the positive direction or in the negative direction. Further, a positive SXY module and a negative SXY module may be connected and incorporated. Specific sequences of the SXY module are shown in SEQ ID NOs: 1 to 5. SEQ ID NO: 1 is the SXY module of MHCI type HLA-A, SEQ ID NO: 2 is the SXY module of MHCI type HLA-B, and SEQ ID NO: 3 is the SXY proximal to the transcription start point of MHCII type HLA-DLA SEQ ID NO: 4 is an SXY module (hereinafter abbreviated as S'X'Y 'module) that is located distal to the transcription start point of MHCII type HLA-DLA and whose sequence direction is opposite to that of the SXY module. SEQ ID NO: 5 is the SXY module of β2 microglobulin. The SXY module is not limited to these sequences. As long as it retains the ability to bind to the MHC enhanceosome and enhance transcription activity, it hybridizes with a DNA sequence complementary to these sequences under stringent conditions. Also includes sequences that soy. Here, as shown in FIGS. 4, 5, and 6, the SXY module has a linker between the S box and the X1X2 box, and between the X1X2 box and the Y box. It seems that the degree of freedom of mutation is relatively high.

  The term “hybridizes under stringent conditions” is a well-known hybridization experimental condition for those skilled in the art. Specifically, it means that two nucleic acid fragments hybridize to each other under the hybridization conditions described in Sambrook, J., Molecular Cloning: A Laboratory Manual, 3rd edition. More specifically, “stringent conditions” refer to hybridization at 6.0 × SSC at about 45 ° C. and then washing at 2.0 ° SSC at 50 ° C. For the selection of stringency, the salt concentration in the washing step is selected, for example from about 2.0 × SSC, 50 ° C. as low stringency to about 0.2 × SSC, 50 ° C. as high stringency, Can do. Further, the temperature of the washing step can be increased from a low stringency condition of room temperature, about 22 ° C., to a high stringency condition of about 65 ° C. A person skilled in the art can realize hybridization conditions of stringency similar to the above conditions by appropriately selecting various conditions such as SSC dilution, formamide concentration, and temperature.

(Location of transcription factor binding sequence in expression vector of the present invention)
The position where the transcription factor binding sequence that interacts with the MHC enhanceosome exists may be inside the base promoter sequence or in the upstream region outside the base promoter sequence. In general, it is preferable to insert the transcription factor binding sequence at a position where it usually exists. Therefore, when viewed in relation to the base promoter sequence, the transcription factor binding sequence is preferably present in the base promoter sequence rather than in the upstream region of the base promoter sequence. Expressed numerically, it is preferably present approximately 30 to 1000 base pairs upstream of a core promoter element such as a TATA box. Further, more preferably within about 600 base pairs upstream of the core promoter element. Also most preferably within about 300 base pairs upstream of the core promoter element. In addition, there is no problem even if the number of incorporation is one or plural. An array including the SXY module in the positive direction and an array including the SXY module in the negative direction may be incorporated.

(CIITA gene in the expression vector of the present invention)
The CIITA gene to be incorporated into the expression vector can be used as long as it is a vertebrate-derived CIITA gene, but a human-derived CIITA gene is particularly preferable. The sequence is not limited to a natural type (for example, SEQ ID NO: 6), and a mutation may be introduced as long as it can maintain the characteristics of forming an MHC enhanceosome as CIITA and stimulating transcriptional activity. . That is, for example, in the base sequence described in SEQ ID NO: 6, it may be a base sequence in which one or more bases are substituted, deleted, added, and / or inserted.

(Target gene of the expression vector of the present invention)
A “target gene” is any foreign gene that is desired to be expressed in a host cell. In a genetic vaccine, it refers to all antigen genes derived from pathogenic microorganisms, malignant neoplasms, etc., as well as all genes introduced together with antigen genes for the purpose of optimizing immune responses. All genes introduced for the purpose of gene therapy are also included. Specifically, gene therapy for congenital / acquired deficiency includes all or part of the responsible gene, and gene therapy for malignant neoplasms in addition to the slaughter gene as well as genes introduced to increase drug / radiation sensitivity.・ Gene therapy for circulatory disorders such as lower limb arterial occlusion introduces genes introduced for the purpose of revascularization, and transplantation medicine covers genes introduced for suppression of rejection and control of graft-versus-host disease. . In vitro, genes introduced for protein production using cultured cells are targeted. Specifically, various immunoregulatory molecules such as cytokines, interferons, costimulatory factors, transcription factors, antibodies or fragments thereof, various growth factors, hormones, hematopoietic factors, blood coagulation factors, neurotransmitters, neuropeptides, vaccines Examples include molecules used as antigens, molecules used as adjuvants, various enzymes, and genes such as glycoproteins.

  In a preferred embodiment of the present invention, the expression vector further comprises a nucleic acid sequence encoding a transcription factor other than CIITA. That is, it is a nucleic acid sequence that encodes a transcription factor that binds to a facilitating transcription factor binding sequence present in each of the modified promoters and enhances transcription activity. These transcription factors may be transcribed by a promoter different from that of the desired gene, or may be the same promoter. Alternatively, the desired gene and transcription factor may be operably linked to an internal ribosome binding site (IRES) to form two or more cistrons placed under the transcriptional control of a single promoter. In this case, it is important to place the “desired gene” in the vicinity of the promoter 3 ′ and the DNA encoding the transcription factor in the vicinity of the IRES 3 ′. If two genes are arranged in the reverse order, the gene expression enhancing effect cannot be expected.

  In another embodiment of the present invention, an expression vector comprising a plurality of one or more desired genes operably linked to one or more promoters, wherein the plurality of genes are each There are provided a multiple promoter expression vector having a promoter, and a multiple cistron expression vector in which at least one of the plurality of genes is linked to another gene via an internal ribosome binding site to form a multiple cistron. The multiple promoter expression system or the multiple cistron expression system of the present embodiment can simultaneously express the respective antigen genes of hepatitis virus, influenza virus, and herpes virus, for example, and hepatitis, influenza, and herpes infections. It can be used as a multivalent vaccine against infectious diseases. In gene therapy, a plurality of deficient genes can be supplemented at the same time in the same manner. Furthermore, it can be used in various application methods such as simultaneously expressing an antigen and an adjuvant, or simultaneously expressing each subunit of a multimeric protein.

(Sequence that can be added to the expression vector of the present invention)
In addition to its basic elements, the expression vector of the present invention can be added with any sequence that has the effect of enhancing expression efficiency and any sequence that has the effect of enhancing immunogenicity. Increase the immunogenicity of antigens such as mRNA stabilizing sequences such as WPRE (woodchuck hepatitis virus post-transcriptional response element) (Garg, S et al. Journal of Immunology 173: 550-558, 2004) and CpG motifs Examples include addition of a sequence (Klinman DM. Et al. Journal of Immunology. 158: 3635-3639, 1997).

(Types of vectors that can be used in the expression vector of the present invention)
The expression vector of the present invention may be a viral vector such as an adenovirus vector used in the field of gene therapy, or a naked DNA such as a plasmid or a PCR amplification product. There may be. In recent years, physical methods such as electroporation and gene guns, and chemical methods such as liposomes and peptide-mediated methods have been improved, and simple and safe in vivo gene transfer using non-viral vectors has been carried out (Niidome , T. and Huang, L. Gene Therapy (2002) vol. 9, pp. 1647-1652), the specific method will be described later.

(Gene transfer into host cells)
The gene transfer of the present invention is performed by introducing the expression vector into a host cell. The desired gene to be expressed and the nucleic acid sequence encoding the transcription factor may be constructed on the same DNA, or may be constructed as separate DNA and cotransfected (cotransformed) into the same host cell. Good. In the case of co-transfection, the desired gene expression level can be optimized by changing the amount ratio of each vector DNA. The amount ratio of the vector DNA is appropriately adjusted so as to be optimal depending on the type of host cell to be used.

  Multiple promoter expression system or multiple cistron expression by incorporating one or more desired genes operably linked to one or more promoters into one vector When constructing a multiple cistoronic expression system, the following points should be noted in particular. A gene under the transcriptional control of a modified promoter introduced with a transcription factor binding motif increases the expression level in the presence of the corresponding transcription factor, but if two or more modified promoters are incorporated into one vector, the gene generated by the modified promoter The effect of enhancing expression is reduced. When creating a vector that co-expresses a desired gene and a transcription factor using a multiple promoter expression system, the desired gene is placed downstream of the modified promoter, and the gene encoding the transcription factor is placed downstream of the normal promoter. This is very important. This is the same when a double cistron expression system using IRES is combined to construct a quadruple cistron expression system, and only one modified promoter should be used.

(Transformation of cultured cells)
The expression vector of the present invention can transform a host cell, and by culturing the obtained transformed cell, the expression vector itself of the present invention and a desired gene product expressed thereby can be produced. . Here, the host is not particularly limited as long as it can replicate and maintain the expression vector of the present invention. For example, bacteria such as Escherichia coli and Bacillus subtilis, and Saccharomyces Yeast such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, animal cells such as COS cells and CHO cells, or insect cells such as Sf9 and Sf21 can be used.

  E. coli is preferable as a host cell for preparing the expression vector of the present invention. After culturing the transformed cell and autonomously replicating a plurality of copies of the expression vector of the present invention, the transformed cell is disrupted to produce the present invention. The expression vector can be recovered and purified. Examples of the method for introducing the expression vector of the present invention into bacteria or yeast include a method using calcium ions, an electroporation method, a spheroplast method, and a lithium acetate method.

  As a host cell for performing the gene expression of the present invention, a vertebrate cell is preferable, and a mammalian cell is particularly preferable. For example, monkey cell COS-7, Vero, Chinese hamster ovary cell (CHO cell), mouse 3T3 cell. And L cells, rat GH3, human 293, and HeLa cells are used, but not limited thereto. Examples of the method for introducing the expression vector of the present invention into animal cells include an electroporation method, a calcium phosphate method, and a lipofection method.

As a medium for culturing transformed cells obtained using animal cells as a host, generally used RPMI1640 medium, DMEM medium, or a medium obtained by adding fetal calf serum or the like to these mediums is used. The culture is usually performed at 37 ° C. for 1 to 30 days in the presence of 5% CO ₂ . During culture, antibiotics such as kanamycin and penicillin may be added to the medium as necessary. If serum coexistence is not desirable, a serum-free medium suitable for the purpose may be used.

  After culturing, when a desired gene product is produced in the cell, the gene product is extracted by disrupting the cell. When the desired gene product is produced outside the cell, the culture solution is used as it is, or the cell is removed by centrifugation or the like. Then, by using a general biochemical method used for isolation and purification of the gene product, for example, ammonium sulfate precipitation, gel chromatography, ion exchange chromatography, affinity chromatography, etc. alone or in appropriate combination, The gene product can be isolated and purified from the culture.

(Use for gene vaccine and gene therapy as nucleic acid medicine of the present invention)
Using the method of the present invention, an antigen gene can be introduced into a living body with the intention of inducing an immune response against the gene product. In addition, a therapeutic gene can be introduced into a living body in the hope that the gene product itself will have some therapeutic effect. It does not matter whether the transgene is a plasmid DNA (naked plasmid DNA) or a recombinant viral vector. The administration method when using plasmid DNA is not particularly limited. A method in which DNA is dissolved in physiological saline and injected as it is, a method using a carrier such as a liposome, various polymers, and a gene gun that sprays DNA coated on fine gold particles at a high pressure to reach the skin, Various introduction methods such as a powder jet and similar high-pressure injection gene introduction device, electroporation, and sonoporation can be applied. Administration site is muscle, subcutaneous, intradermal, intravascular, respiratory and genitourinary mucosa, brain, liver, kidney, lung, etc., luminal organ walls such as digestive tract, abdominal cavity, thoracic cavity, joint cavity, brain All possible parts such as body cavities such as the spinal cavity, visual instruments, and hearing instruments are targeted. For details on vectors and gene transfer devices used for administration, refer to the following documents (Methods, Nucleic Acid Vaccines (ed., JW. Morrow and NA. Sheikh), 31 (3), pp181-262. , 2003. Elseviers Science; The Journal of Gene Medicine, Supplement: Gene Therapy Vectors Vol. 6, Issue S1, 2004. John Wiley & Sons, Ltd; Somiari S et al. Theory and in vivo application of electroporative gene delivery; Molecular Therapy : The Journal of the American Society of Gene Therapy. 2 (3): 178-87, 2000).

(Dose for administration to individuals)
The dosage of the pharmaceutical composition of the present invention varies depending on the age of the administration subject, the administration route, and the frequency of administration, and cannot be generally defined. However, the dosage is appropriate and appropriate for the physiologically active substance produced by the expression vector of the present invention. The effective amount to be administered as a combination with various diluents and pharmacologically usable carriers can be selected from a dose in the range of 0.01 mg to 1000 mg per kg body weight per dose, preferably 1 day per day. It is divided into several times and administered for one day or more.

(Target animal of the pharmaceutical composition of the present invention)
All vertebrates with MHC are subject to the pharmaceutical composition of the present invention. That is, basically, all vertebrates except for jawless species (evolutionally lower type of fish) that do not have MHC molecules are targeted. Therefore, the pharmaceutical composition of the present invention prevents humans, non-human mammals, birds, amphibians, reptiles and fish that need to be treated or prevented for various diseases that are subject to general gene vaccines or gene therapy. Or it is applicable to treatment. In addition, a method for treating or preventing non-human vertebrates using the pharmaceutical composition of the present invention is also a subject of the present invention.

(Basic techniques such as molecular biology)
In practicing the present invention, for general methods such as molecular biology, microbiology, cell biology, and recombinant DNA technology, and the prior art, the practitioner will use standard reference books in the field unless otherwise indicated. You can refer to it. These include, for example, Molecular Cloning: A Laboratory Manual 3rd edition (Sambrook & Russell, Cold Spring Harbor Laboratory Press, 2001); DNA vaccines Methods and Protocols (Lowrie & Whalen, edited by Humana Press, 1999); Successful gene transfer and expression analysis protocol (Nakajima and Kitamura, edited by Yodosha, 2003); Methods, Nucleic Acid Vaccines (ed., JW. Morrow and NA. Sheikh) 31 (3), pp181-262, 2003. Elseviers Science .; The Journal of Gene Medicine, Supplement: Gene Therapy Vectors Vol. 6, Issue S1, 2004. John Wiley & Sons, Ltd .; Somiari S et al. Theory and in vivo application of electroporative gene delivery .; Molecular Therapy: See The Journal of the American Society of Gene Therapy. 2 (3): 178-87, 2000. Reagents and kits for cell culture and cell biology experiments referred to in this specification are Sigma, Aldrich, Invitrogen / GIBCO, Clontech, Stratagene, Qiagen, Promega, Roche Diagnostics Available from commercial vendors such as Becton-Dickinson and Takara Bio.

  Specific examples relating to the present invention will be described below, but these are not intended to limit the present invention, but to illustrate the present invention.

(Construction of an expression vector containing a modified CMV promoter)
The expression vector of the present invention was constructed based on a pGA vector (see Ross, TM et al. Nature Immunology vol. 1 p127-131, 2000) containing a human cytomegalovirus IE promoter / enhancer (CMV promoter). The pGA vector was provided by Professor HLRobinson of the University of Emory University School of Medicine Vaccine Research Center. The pGA vector is used for transcription in eukaryotic cells, the cytomegalovirus immediate early promoter (CMV-IE), intron A, and bovine growth hormone polyadenylation signal (BGH poly A), as well as prokaryotic growth. The Col E1 Origin is composed of a kanamycin resistance gene for selection with antibiotics and a λT0 terminator for enhancing the stability of the target gene. First, in order to generally increase the gene expression efficiency of the expression vector, an mRNA stabilizing sequence WPRE (woodchuck hepatitis virus post-transcriptional response element) motif was incorporated into the XhoI-AVRII restriction enzyme site of the pGA vector. The construct obtained by adding WPRE to this pGA vector was named “pGA-IA (donor) / MCS-WPRE vector” and used as the basis of an expression vector containing a modified CMV promoter (see FIG. 2). The WPRE motif was provided by Dr. S. Garg of the US Centers for Disease Control. A luciferase gene was functionally incorporated between intron A and WPRE. Next, by site-directed mutagenesis in the upstream region of the TATA box (located at positions 571 to 577 in the base sequence of FIG. 3) in the CMV promoter of pGA-IA (donor) / MCS-WPRE incorporating luciferase. A plurality of restriction enzyme sites were installed (FIG. 3). That is, six restriction enzyme sites of XmaI, MluI, MfeI, XbaI, AgeI and AscI sites were placed in order from the upstream. The transcription factor-binding sequence of the present invention is substituted with the sequence of the region sandwiched between the restriction enzyme sites of each set, with the three sets of XmaI-MluI, MfeI-XbaI and AgeI-AscI set as above as a unit. I was able to incorporate it. FIG. 4 is a diagram schematically showing the promoter region of MHCI type II and type II and β2 microglobulin gene, and an SXY module is commonly present in the vicinity of the transcription start site. In type II, an array similar to the SXY module exists in the opposite direction to the SXY module approximately 2 kb upstream of the SXY module (FIGS. 5 and 6). Here, a module existing in the upstream area of the type II SXY module is represented as an S′X′Y ′ module. In addition, the SXY module (SEQ ID NO: 3) existing in the MHC II type promoter region (HLA-DRA among type II) is abbreviated as P, and the S′X′Y ′ module ( Sequence number 4) is abbreviated as D. The P, D and P + D (hereinafter abbreviated as PD) were incorporated into the above three sets of restriction enzyme sites in various combinations. The case where the transcription factor binding sequence was not incorporated into the restriction enzyme site was abbreviated as X. FIG. 7 schematically shows how the transcription factor binding sequence was actually incorporated. In addition, the region (SEQ ID NO: 2) containing the SXY module of the type I promoter (using HLA-B of type I) and the two κBE and ISRE located upstream thereof, not MHCII type, one each at three locations When incorporated, it was abbreviated as “111”. The MHCI type (HLA-B) and type II (HLA-DRA) transcription factor binding sequences used above were both donated by Professor HLLobinson of the Emory University School of Medicine Vaccine Research Center.

(Construction of human CIITA expression vector)
A human CIITA expression vector used for co-expression with the modified CMV promoter-containing expression vector containing the SXY module was constructed as follows. Based on the human CIITA expression vector provided by Dr. Koichi Suzuki of National Institute of Infectious Diseases (see Montani, V et al. Endocrinology, 139: 280-289, 1998 for the construction and structure of the vector). A fragment (SEQ ID NO: 6) corresponding to CIITA cDNA was excised, and the fragment was functionally inserted between pGA vector intron A and BGH poly A to construct a human CIITA expression vector in the present invention.

(Many expression vector preparation)
For the large-scale preparation of each expression vector for transfection into animal cultured cells, first, Escherichia coli transformed with each expression vector is mass-cultured in an LB medium containing an antibiotic, and then Qiagen Maxiprep kit (Qiagen , Hilden, Germany) and the plasmid was extracted and purified according to the method described in the manufacturer's protocol. The vaccine vectors for animal immunization of Examples 4 and 5 described later were also prepared in large quantities by the same method.

(Examination of transgene expression efficiency in cultured cells-Luciferase assay)
The expression efficiency of the transgene in the vector of the present invention was examined by a luciferase reporter assay. That is, examination of the transgene expression efficiency at the cellular level was based on the luminescence reaction caused by the activity of the translation product of the firefly luciferase gene incorporated in the expression vector as a reporter gene. NIH-3T3, Vero, HeLa, and L929 cells used in the luciferase assay were cultured at 37 ° C. in the presence of 5% CO ₂ in RPMI1640 medium or DMEM medium or medium supplemented with fetal calf serum or the like. . FuGene6 (Roche Applied Science, Mannheim, Germany or PolyFect (Qiagen, Hilden, Germany) was used for transfection of the luciferase expression vector. The Luciferase assay was performed 48 hours after transfection with the Bright-Glo kit (Promega). The cells were lysed to cause a luminescence reaction, and the luciferase activity was calculated by measuring the fluorescence intensity using Fluostar Galaxy (BMG Labtechnologies, Duren, Germany) to determine the expression level of the luciferase gene.

(Combination of SXY modules and influence on expression efficiency by CIITA co-expression)
The gene expression levels of expression vectors containing various modified CMV promoters schematically illustrated in FIG. 50 ng of 10 ng of a luciferase gene expression vector containing a modified CMV promoter or an unmodified CMV promoter (negative control) in which SXY module and S′X′Y ′ module are incorporated in various combinations at any three restriction enzyme sites The NIH-3T3 cells in 96 wells were transfected with the CIITA expression vector or mock plasmid (Kara vector not containing the CIITA gene). Cells were harvested after 48 hours and luciferase activity was measured.

  The result is shown in FIG. Each bar graph represents the average value of luciferase activity of three samples. When mock plasmids (irrelevant plasmids) are co-transfected, gene expression levels are increased in most modified promoters compared to unmodified promoters (naive). On the other hand, when the CIITA expression vector is administered simultaneously, the gene expression level is markedly increased in all modified promoters compared to the unmodified promoter. Furthermore, in the comparison between the same promoters, the expression level of the target gene was increased by co-expressing CIITA with the CIITA expression vector in all the modified promoters as compared with the case of the modified promoter expression vector alone. . Not only when three type II SXY modules are incorporated (PPP; abbreviated as 222 in Example 2) but also when three type I SXY modules are incorporated (111), the gene expression level is significantly increased. It was observed. From the above experiments, it is possible to obtain some increase in gene expression level by simply incorporating the SXY module into the promoter without co-expression of CIITA, and further increase the degree of increase by co-expression of CIITA. Proven. And in the increase of these expression levels, the difference between the SXY module and the S'X'Y 'module, the difference depending on the combination and the number of incorporation, and the difference depending on the installation location are not clear, but in either case the difference in degree is However, it has been proved that a sufficient increase in the expression level can be obtained as compared with the unmodified promoter.

(Examination of gene expression efficiency in multiple cell lines)
Next, whether or not the effect of increasing the gene expression level by the vector of the present invention found in Example 1 is also observed in cell lines other than the NIH-3T3 cells used in Example 1, We investigated using. In this example, a luciferase expression vector containing a modified promoter (222; abbreviated as PPP in Example 1) incorporating three type II (HLA-DRA) SXY modules, which had a remarkable effect in Example 1. Experiments were conducted on four types of cell lines. That is, using four types of cell lines of Vero, HeLa, L929, and NIH-3T3 under the same conditions as in Example 1, a control non-modified promoter or a modified promoter incorporating a transcription factor binding sequence (222) In contrast, CIITA expression vectors or mock plasmids were co-transfected, and the gene expression level was examined based on the luciferase activity of the reporter gene.

  The result is shown in FIG. Each cell line shows luciferase activity as a relative value when the value of luciferase activity when co-transfected with a mock plasmid using a non-modified promoter (naive + mock) is 1. Each treatment group has 3 samples, and the bar graph represents the mean and standard error of relative luciferase activity. When CIITA is not co-expressed, the gene expression level of the modified promoter (222 + mock) incorporating the SXY module is significantly increased in all cell lines examined compared to the non-modified promoter (naive + mock). Was. Furthermore, when CIITA was co-expressed by co-transfection of a CIITA expression vector (222 + CIITA), the gene was further increased in all cells examined than when only the modified promoter vector was transfected (222 + mock). The expression level was increased. From the above results, in human cultured cells, it is possible to increase the gene expression level by introducing a gene using a modified promoter incorporating an SXY module, and in addition, CIITA is co-expressed with an expression vector. As a result, it was found that the expression level of the transgene can be further increased.

(Examination of gene expression efficiency in the presence of interferon)
Usually, there are various endogenous interferons in the living body of animals, and it has been reported that the expression efficiency of the expression vector introduced into the living body is attenuated by these interferons. Therefore, in an experimental system for investigating the expression efficiency of a vector using a cell line, it was examined whether the expression vector of the present invention was effective even under conditions where interferon was present. Among the expression vectors constructed in Example 1, “111” or “222”, which is a luciferase expression vector in which MHCI type II or type II SXY modules are respectively incorporated into all three sets of restriction enzyme sites of the CMV promoter, Transcriptional activity was compared with the modified CMV promoter luciferase vector “naive” in the presence or absence of interferon β or γ by luciferase assay. The cell lines used for the assay are HeLa and NIH-3T3. The interferon concentration was 500 mU / ml.

  This result is shown in FIG. Each treatment group has 3 samples, and the bar graph represents the mean value and standard error of luciferase activity. In HeLa cells, in the absence of interferon, 111 and 222 modified CMV promoter-containing expression vectors incorporating the SXY module showed a marked increase in transcriptional activity compared to the unmodified naive CMV promoter expression vector. On the other hand, in the presence of interferon β, the transcriptional activity of the unmodified naive promoter is reduced to a little less than 30% in the absence of interferon β. However, the 111 or 222 modified promoter is not modified in the presence of interferon β. A 2-3-fold increase in activity is observed for the naive promoter. The remarkable increase by these modified promoters was similar in the presence of interferon γ. Further, in experiments with NIH-3T3 cells, although there was a difference in the degree of increase, a significant increase effect by the modified promoter in the presence of interferon was observed as in the case of HeLa cells. That is, the above experiment proved that the transcription activity of the MHC gene transcription factor binding sequence of the present invention was enhanced by the SXY module even in the presence of interferon β or γ. Therefore, these modified promoters have a slight decrease in transcriptional activity in the presence of interferon compared to the non-modified CMV promoter, and therefore, transcriptional activity is reduced in gene transfer using a viral vector that induces induction of interferon. Expected to be effective in maintaining.

(Construction of vector for gene vaccine)
An expression vector for a gene vaccine comprising an antigen gene (LacZ) under the control of a promoter containing an MHC gene transcription factor binding sequence and a CIITA gene under the control of a separate promoter in a single vector Was constructed as follows. First, the antigen gene LacZ was ligated downstream of the promoter of the pGA vector. Then, downstream of LacZ, using a XhoI-AvrII site, WPRE (woodchuck hepatitis virus post-transcriptional response element) having an mRNA stabilizing action was incorporated in order to increase the expression efficiency. Then, in the CMV promoter of this LacZ expression pGA vector, three regions (SEQ ID NO: 2 or 3) each containing an MHCI type or type II SXY module were prepared in the same manner as the expression vector construction method in Example 1. Incorporated one by one. And the NotI site located in the 3 'side vicinity of the PolyA site previously introduced by the site-directed mutagenesis method is digested with the restriction enzyme. In parallel with this, a CIITA expression vector based on the pGA vector is constructed, and only the non-modified CMV promoter, CIITA gene and PolyA region are excised from the CIITA expression vector with NotI. Then, the excised fragment was ligated with a LacZ expression plasmid cleaved with NotI to construct the gene vaccine vector of the present invention. FIG. 1 schematically shows the vaccine vector thus completed (including three type I SXY modules). This is named pDX CMV-111-LacZ-WPRE / CIITA vector and is abbreviated as “111-LacZ-WPRE / CIITA”.

(Investigation of antigen-specific immune response in individual animals)
In order to investigate the effect of the expression vector for gene vaccine “111-LacZ-WPRE / CIITA” (FIG. 1) prepared above, mice were used using the following 6 types of constructs as shown in FIG. An immunization experiment was conducted. Those using SXY of type II instead of type I described in the previous paragraph are abbreviated as “222-LacZ-WPRE / CIITA”, and the control does not include CIITA expression cassette and WPRE motif, Each of the promoters was prepared as a non-modified CMV, 111 modified CMV, or 222 modified CMV, and abbreviated as “naive-LacZ”, “111-LacZ”, and “222-LacZ”, respectively. A LacZ expression vector (abbreviated as “naive-LacZ-WPRE”) in which only the WPRE motif was incorporated in an unmodified CMV promoter was also prepared.

  Antigen-specific cellular immune responses induced by each LacZ expression vector constructed above were examined using 6-8 week old BALB / C female mice as test animals. Each group was tested with 5 to 8 animals. First, for the first immunization of mice, 50 micrograms of LacZ expression vector dissolved in physiological saline was injected into the exposed quadriceps muscle, and then a tweezers type platinum electrode (diameter 10 mm, distance between electrodes about 5 mm, CUY650- 10, NepaGene Chiba, Japan) directly on the quadriceps and directly energized six times with a rectangular wave electroporator (CUY21EDIT, NepaGene Chiba, Japan) (voltage 30 V, energization time 50 msec, energization interval 100 msec) It went by. Four weeks later, booster immunization was performed under the same conditions. Two weeks after the last immunization, the spleen was removed from the mice, lymphocytes were isolated, and LacZ-specific cytotoxic lymphocyte (CTL) activity was measured. A CTL assay for measuring CTL activity is an assay kit CytoTox 96 Non-radioactive using the method described by Decker et al. (Decker, T. et al. J. Immunol Meth. Vol 115, p61-69, 1988). A cytotoxicity assay (Promega) was used.

  FIG. 12 shows the experimental results of the above CTL assay. The CTL assay is an experiment that examines how much damage activated cells (target cells) isolated from a vaccinated animal can have on antigen-bearing cells (target cells). In contrast to the black circle graph pulsed with the antigen peptide, the white circle graph shows a negative control not pulsed with the antigen peptide. The vertical axis (% Specific Lysis) indicates what percentage of target cells are damaged and lysed, and the horizontal axis (E / T Ratio) indicates the ratio of effector cells / target cells. If high-efficiency cell lysis is observed despite the small effect cell / target cell ratio, this indicates that the cytotoxicity is very strong. First, in the comparison of “naive-LacZ”, “111-LacZ” and “222-LacZ” not containing the CIITA expression cassette, no significant difference in cell lysis rate was observed. “Naive-LacZ-WPRE” containing WPRE which is an mRNA stabilizing sequence has an increased cell lysis rate compared to “naive-LacZ” not containing WPRE. This WPRE-containing non-modified promoter “naive-LacZ-WPRE” is replaced with “111-LacZ-WPRE / CIITA” or “222-LacZ-WPRE / CIITA” containing a CIITA expression cassette and a modified 111 or 222 CMV promoter. When "111-LacZ-WPRE / CIITA" shows a significant increase in cell lysis rate, "222-LacZ-WPRE / CIITA" also shows a slight increase. Therefore, from these experiments, the antigen gene linked to the promoter containing the SXY module is co-expressed with the CIITA gene under a separate promoter, thereby inducing a high level of antigen-specific cellular immunity. It was shown that it can be done.

A single vector comprising an antigen gene (LacZ) under the control of a CMV promoter containing a transcription factor binding sequence of an MHCI type gene and a class II transactivator (CIITA) gene under the control of a separate CMV promoter It is the cleavage map of the plasmid construct of the expression vector for gene vaccines contained in it. The WHO (woodchuck hepatitis virus post-transcriptional response element) motif, which is an mRNA stabilizing sequence, is added to the XhoI-AVRII restriction enzyme site of the pGA vector based on the cytomegalovirus early promoter (CMV-IE) and intron A. It is a cleavage map of the integrated plasmid construct (pGA-IA (donor) / MCS-WPRE vector). It is the figure which showed the arrangement | sequence of the promoter (naive CMV promoter) used as the basis of the modified promoter which introduce | transduced six restriction enzyme sites into the CMV-IE promoter by site-directed mutagenesis. It shows the transcription factor binding sites such as TATA box and CREB, which are core promoter elements existing inside. It is the figure which showed typically the promoter area | region of MHCI type | mold and II type | formula, and (beta) 2 microglobulin gene, and represents the conservation nature of the SXY module in each gene. It is the figure which showed the positional relationship of the SXY module (Distal promoter) and the S'X'Y 'module (Proximal promoter) which exists in the reverse direction upstream in the promoter area | region of MHCII type | mold (HLA-DRA). It is the figure which showed the similarity of the arrangement | sequence of a SXY module (Distal promoter) and a S'X'Y 'module (Proximal promoter) about the promoter area | region of MHCII type | mold (HLA-DRA). Within the three sets of restriction enzyme sites in the CMV-IE promoter sequence shown in FIG. 3, the MHCII type HLA-DRA promoter region SXY module (abbreviated as P), S'X'Y 'module (abbreviated as D), And P + D (abbreviated as PD) are schematically shown in what combinations were incorporated to create a modified promoter (however, abbreviated as X when nothing was incorporated). Transcription of the modified CMV promoter-containing luciferase expression vector incorporating the SXY module of the MHC gene promoter region shown in FIG. 7 in various combinations with NIH3T3 cells together with the mock plasmid or CIITA expression vector. It is the graph which showed activity. The modified CMV promoter-containing luciferase expression vector (222) or the unmodified CMV promoter-containing luciferase expression vector (naive) incorporating three SXY modules of the promoter region of the MHCII type gene shown in FIG. Is a graph showing the transcriptional activity when co-administered with a mock plasmid or CIITA expression vector. A modified CMV promoter-containing luciferase expression vector (222) incorporating three SXY modules of the promoter region of the MHCII type gene shown in FIG. 7, and a modified CMV incorporating three SXY modules of the promoter region of the MHCI type gene FIG. 5 is a graph comparing the transcriptional activity of a promoter-containing luciferase expression vector (111) when transfected into two types of cells in the presence of interferon with the transcriptional activity of an unmodified CMV promoter-containing luciferase expression vector (naive). . In mice, the effect of a gene vaccine vector containing a transcription factor binding sequence of an MHCI type gene (a cleavage map is shown in FIG. 1) and a gene vaccine vector containing a transcription factor binding sequence of an MHCII type gene will be examined. It is the schematic diagram of each plasmid construct used for the immunity experiment for. It is the graph which showed the antigen specific CTL activity in the mouse | mouth by each vector for gene vaccines shown in FIG.

Claims (20)

  An expression vector comprising a modified promoter comprising a transcription factor binding sequence capable of binding to a major histocompatibility complex (MHC) enhanceosome in a vertebrate cell.   The expression vector according to claim 1, comprising a target gene operably linked to the modified promoter, wherein the transcription factor binding sequence promotes transcription of the target gene.   The expression vector according to claim 1 or 2, wherein the modified promoter is constructed by adding the transcription factor binding sequence to a CMV promoter or a CAG promoter.   The expression vector according to claim 3, wherein the transcription factor binding sequence is located approximately 30 to 1000 base pairs upstream of the TATA box of the CMV promoter or CAG promoter.   The expression vector according to any one of claims 1 to 4, further comprising a nucleic acid sequence encoding an MHC class II transactivator (CIITA), wherein the CIITA is expressed separately from a target gene. .   6. The nucleic acid sequence encoding CIITA is operably linked to an internal ribosome binding site (IRES) or is operably linked to a promoter separate from the modified promoter. Expression vector.   The expression vector according to claim 6, wherein the separate promoter is a CMV promoter or a CAG promoter.   An expression vector mixture comprising the expression vector according to any one of claims 1 to 4 and a CIITA expression vector.   The expression vector mixture according to claim 8, wherein the promoter of the CIITA expression vector is a CMV promoter or a CAG promoter.   The expression vector or expression according to any one of claims 1 to 9, wherein the transcription factor binding sequence is a transcription factor binding sequence of MHC class I, MHC class II and / or β2 microglobulin gene. Vector mixture.   The transcription factor binding sequence hybridizes under stringent conditions with DNA comprising the base sequence described in any one of SEQ ID NOs: 1 to 5, or DNA comprising a base sequence complementary to the DNA; The expression vector or expression vector mixture according to claim 10, wherein the expression vector or the mixture of expression vectors is DNA having an ability to bind to an MHC enhanceosome.   The expression vector or the expression vector mixture according to any one of claims 1 to 9, wherein the transcription factor binding sequence includes an SXY module.   A composition for gene expression comprising the expression vector or expression vector mixture according to any one of claims 1 to 12.   A gene expression method using the expression vector or the expression vector mixture according to any one of claims 1 to 12.   A nucleic acid medicine for vertebrates comprising the expression vector or the expression vector mixture according to any one of claims 1 to 12.   The nucleic acid medicine according to claim 15, wherein the vertebrate needs to be vaccinated for prevention or treatment of infectious diseases.   The nucleic acid medicine according to claim 15, wherein the vertebrate needs to be vaccinated for the prevention or treatment of malignant tumor, multiple sclerosis, Alzheimer's disease, or other diseases.   The nucleic acid medicine according to claim 15, wherein the vertebrate needs to receive gene therapy.   A method for preventing or treating an infectious disease for a non-human vertebrate having MHC, comprising using the nucleic acid drug according to claim 16.   A method for preventing or treating a malignant tumor, multiple sclerosis, Alzheimer's disease, or other disease targeting a non-human vertebrate having MHC, wherein the nucleic acid drug according to claim 17 or 18 is used.

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