JPWO2009051078A1 - Therapeutic gene expression system - Google Patents

Abstract

The present application includes (1) an expression unit comprising a transcription regulatory sequence in which transcription is induced by a trans-acting factor, a therapeutic gene arranged in a form in which expression can be controlled by the sequence, and (2) (1) Trans-acting factor precursor that does not have the activity to induce transcription from the transcriptional regulatory sequence and is converted to an active trans-acting factor by the action of a specific factor, or a gene encoding the precursor is expressed A gene expression system comprising: an expression unit arranged in a possible form is provided. By designing appropriate trans-acting factor precursors, disease-specific therapeutic gene expression is achieved.

Description

  The present invention relates to a nucleic acid construct useful for treating or preventing a disease using a therapeutic gene as an active ingredient.

  Gene therapy is a new disease that has not been originally possessed by a cell by correcting the function of the cell by adding the correct genetic information for a disease (genetic disease, cancer, etc.) caused by the “incorrect genetic information” of the cell. It has been developed as a treatment method for treating or preventing a disease by adding a “protective gene”.

  At present, unlike the above-described embodiment, the gene encoding a product that exhibits cytotoxicity is also used energetically for the purpose of selectively eliminating cells harmful to the living body (such as cancer cells or cells infected with pathogenic microorganisms). Has been studied. For example, when a hermetic virus thymidine kinase gene is expressed in a target cell and then a harmless precursor drug (ganciclovir) is administered to a living body, the precursor drug is activated specifically for the target cell, resulting in cytotoxicity of the drug. Target cells are destroyed.

  Toxins encoded by prokaryotic toxin-antitoxin genes are known as polypeptides that exhibit cytotoxicity. MazF (non-patent document 1), which is a mazE-mazF-based toxin, and PemK (non-patent document 2), which is a pemI-pemK-based toxin, both recognize single-stranded RNA-specific endoribonuclease activity. It is known that these toxins are used for the treatment of proliferative diseases (Patent Document 1).

  Human immunodeficiency virus (HIV) infects and destroys cells expressing CD4 molecules, such as T cells. For this reason, a decrease in CD4 positive T cells (helper T cells) and a decrease in cellular immunity occur in the human body that has been infected with HIV. Eventually, the patient becomes severely immunocompromised and develops an opportunistic infection like Carini pneumonia. This condition is referred to as acquired immunodeficiency syndrome (AIDS).

  Antiviral agents (reverse transcriptase inhibitors, protease inhibitors, etc.) and vaccines that block the HIV life cycle are being developed as treatment methods for HIV infection, and some antiviral agents have already been put into practical use. ing. However, in the case of HIV having a high mutation frequency, a mutant that does not have the effect of the above-mentioned drug may appear in the infected individual, and the situation has not necessarily been completed as a specific medicine. As another approach, attempts to develop gene therapy drugs that inhibit the growth of HIV using nucleic acids such as RNA decoys and ribozymes, proteins such as transdominant mutant proteins and intracellular antibodies as active ingredients have yet to be completed. Not.

  In addition, methods for causing cell death specifically in HIV-infected cells have been devised (for example, Patent Document 2, Non-Patent Documents 3, 4, and 5). In the above method, a gene encoding a product showing cytotoxicity is connected downstream of the LTR promoter derived from HIV, but no clinically applied examples have been known so far.

  Patent Document 3 describes the use of a single-stranded RNA-specific endoribonuclease as the above-mentioned cytotoxic product. In this method, the expression of single-stranded RNA-specific endoribonuclease is induced in a manner dependent on the Tat protein expressed with HIV infection, and the single-stranded RNA in the cell containing the HIV genome is degraded. As a result, HIV replication and budding are prevented in the cells. When the RNA derived from HIV is degraded to stop the expression of Tat protein, and the expressed Tat protein disappears from the cell, the expression of endoribonuclease also stops. During this time, ribosomes and tRNA in the cells are not destroyed, and normal protein synthesis is resumed as the expression of the endoribonuclease is stopped. Therefore, cells that are not destroyed at this point resume proliferation.

International Publication No. 2004/113498 Pamphlet US Pat. No. 5,554,528 International Publication No. 2007/020873 Pamphlet Molecular Cell, Vol. 12, p913-923 (2003) Journal of Biological Chemistry (J. Biol. Chem.), 279, p20678-20684 (2004) Human Gene Therapy, Volume 2, p53-60 (1991) Proceedings of National Academy of Sciences of USA (Proc. Natl. Acad. Sci. USA), Vol. 91, p. 365-369 (1994) Human Gene Therapy, Vol. 10, p103-112 (1999)

  The technique described in Patent Document 3 has been shown to be able to express an effective amount of endoribonuclease triggered by HIV infection, and is an excellent therapeutic gene expression system, but is difficult to use for other than HIV infection. It is. The present invention has been made in view of the above prior art, and an object of the present invention is to provide a therapeutic gene expression system applicable to a wide range of diseases.

  The present inventors examined an expression system capable of expressing a therapeutic gene for various diseases. As a result, it was found that by expressing a trans-acting factor that turns on / off a transcriptional regulatory sequence of a therapeutic gene as a precursor, it is possible to express a disease-specific therapeutic gene, and the present invention was completed. .

That is, the present invention
[1] The following components:
(1) a transcriptional regulatory sequence whose transcription is induced by a trans-acting factor, an expression unit comprising a therapeutic gene arranged in a form in which expression can be controlled by the sequence, and (2) the trans-acting described in (1) An expression unit in which the precursor of the factor or the gene encoding the precursor of the trans-acting factor described in (1) is arranged to be expressed, wherein the precursor is derived from the transcriptional regulatory sequence described in (1) A gene expression system comprising: an active trans-acting factor that has no activity of inducing transcription of and is converted into an active trans-acting factor by the action of a specific factor;
[2] The gene expression system according to [1], wherein the specific factor is a polypeptide expressed in cells by canceration or viral infection,
[3] The gene expression system according to [1] or [2], wherein the therapeutic gene is a gene encoding a polypeptide, a ribozyme, an antisense RNA, or an RNA that exhibits RNA interference.
[4] The gene expression system according to [3], wherein the therapeutic gene is a gene encoding a polypeptide selected from a polypeptide having protease activity, a polypeptide having nuclease activity, and a cytokine.
[5] The gene expression system according to [4], wherein the therapeutic gene is a gene encoding a polypeptide having endoribonuclease activity specific to single-stranded RNA.
[6] The gene expression system according to any one of [1] to [5], wherein the transcriptional regulatory sequence is a promoter derived from an LTR of an immunodeficiency virus.
[7] The gene expression system according to [6], wherein the trans-acting factor is a polypeptide derived from Tat protein of immunodeficiency virus,
[8] A trans-acting factor precursor is a polypeptide that is converted into an active trans-acting factor by a modification selected from phosphorylation, dephosphorylation, glycosylation, glycosylation, and limited degradation [1] [7] the gene expression system according to any one of
[9] The gene expression system according to [8], which is a polypeptide in which a precursor of a trans-acting factor is converted into an active trans-acting factor by the action of a virus-derived protease.
[10] The gene expression system according to [8], wherein the trans-acting factor precursor is a polypeptide that is converted into an active trans-acting factor by the action of a protease derived from hepatitis C virus.
[11] The gene according to [9], wherein the precursor of the trans-acting factor is a fusion polypeptide in which another polypeptide is fused to the N-terminal side of the trans-acting factor via a peptide chain that can be limitedly decomposed by a virus-derived protease. Expression system.
[12] The gene expression system according to any one of [1] to [11], wherein the expression unit of (1) and the expression unit of (2) are inserted on the same vector,
[13] The method according to any one of [1] to [11], comprising a first vector in which the expression unit of (1) is inserted and a second vector in which the expression unit of (2) is inserted. Gene expression system,
[14] A precursor of a trans-acting factor, having no activity to induce transcription from a transcriptional regulatory sequence whose transcription is induced by the trans-acting factor, and an active trans-acting factor by the action of a specific factor A trans-acting factor precursor that is converted to
[15] Transcriptional regulation in which a trans-acting factor and another polypeptide are artificially fused via a peptide chain that can be limitedly decomposed by a protease, and the trans-acting factor has an intrinsic activity of inducing transcription [14] a trans-acting factor precursor characterized in that it has lost the activity of inducing transcription to a sequence
[16] The trans-acting factor precursor of [15], wherein the trans-acting factor is a polypeptide derived from the Tat protein of immunodeficiency virus,
[17] The trans-acting factor precursor of [15] or [16] fused via a peptide chain that can be limitedly degraded by the action of a virus-derived protease,
[18] The trans-acting factor precursor of [17] fused via a peptide chain that can be limitedly decomposed by the action of a protease derived from a virus derived from hepatitis C virus,
[19] The trans-acting agent precursor according to claim 15, wherein another polypeptide is artificially fused to the N-terminal side of the trans-acting factor via a peptide chain that can be limitedly decomposed by a protease.
[20] A step of introducing a nucleic acid encoding the trans-acting factor precursor according to any one of [14] to [19] and the gene expression system according to any one of [21] [1] to [13] into a living body or a cell. A method of treating or preventing a disease comprising
About.

  According to the present invention, a therapeutic gene expression system capable of selectively expressing a therapeutic gene for various diseases is provided. This expression system can design an appropriate system according to various diseases, and can be widely applied to gene therapy. In addition, the retroviral vector described above is used for cancer and viral infections. Useful for treatment and prevention.

It is a figure which shows the structure of pMT-HLTR-MazF-PL. It is a figure which shows the structure of pMTD3-U3TAR-MazF-PL. It is a figure which shows the structure of pMTD3H-ZsGreen / SN. It is a figure which shows the measurement result of the fluorescent protein ZsGreen expression cell by a flow cytometer, and is a figure which shows the difference in Tat inactivation by the difference in the position of Tat and HSA. It is a figure which shows the measurement result of the fluorescent protein ZsGreen expression cell by a flow cytometer, and is a figure which shows that Tat is activated when NS3 protease acts on the fusion protein of HSA and Tat.

  The therapeutic gene used in the present invention is not particularly limited as long as it exhibits a preferable action in a disease for which treatment or prevention is desired. Polypeptide, ribozyme, antisense RNA, RNA exhibiting RNA interference action Etc. are exemplified. Since the expression system of the present invention brings about the expression of therapeutic genes in response to the onset of diseases such as carcinogenesis or viral infection, it exhibits genes useful for eliminating cancer cells and virus-infected cells, such as cytotoxicity. Possible genes can be used.

  The therapeutic gene encoding a polypeptide used in the present invention includes a gene encoding a polypeptide having protease activity, a gene encoding a polypeptide having nuclease activity, a gene encoding a cytokine, and nucleic acid metabolism. Examples include genes.

    A preferred embodiment of the present invention is exemplified by a gene encoding a polypeptide having a single-stranded RNA-specific endoribonuclease activity. Among the prokaryotic toxin-antitoxin gene toxins, there is a polypeptide having sequence specificity and having an activity of degrading single-stranded RNA in a ribosome-independent manner, and is particularly suitable for the present invention. For example, as described in Molecular Cell, Vol. 12, p913-923 (2003), Journal of Biological Chemistry (J. Biol. Chem.), Vol. 279, p20678-20684 (2004), Genes encoding MazF and PemK, which are endoribonucleases constituting toxin-antitoxin toxins, can be used in the present invention, but are not limited thereto. These endoribonucleases are suitable for use in the present invention because they are not inhibited by cytoplasmic ribonuclease inhibitors, such as human placenta-derived ribonuclease inhibitors (WO 2004/113498). When the endoribonuclease is continuously expressed, the cell does not synthesize a new protein, so that the cell is inhibited from growing and further cell death is induced. In addition, since the virus that infects the cells inhibits the synthesis of the proteins that constitute the virus, virus replication and budding do not occur.

  The expression of the therapeutic gene is controlled by a transcriptional regulatory sequence whose transcription is induced by a trans-acting factor. The “transcriptional regulatory sequence whose transcription is induced by a trans-acting factor” used in the present invention is not particularly limited as long as it is a sequence on / off of transcription by the binding / dissociation of a specific trans-acting factor. Under the control of the transcription regulatory sequence, for example, a therapeutic gene expression unit can be prepared by placing a therapeutic gene downstream thereof. The trans-acting factor is preferably a polypeptide whose amino acid sequence has been clarified, and is converted into a precursor using the amino acid information.

  The precursor of a trans-acting factor used in the present invention does not have an activity of inducing transcription from a transcription regulatory sequence, but is converted into an active trans-acting factor that retains the activity by a specific factor. Here, the conversion from the precursor to the active form is not particularly limited, but can be achieved by, for example, ligand binding or polypeptide modification. Examples of the modification of the polypeptide include phosphorylation, dephosphorylation, glycosylation, glycosylation, limited degradation, etc., utilizing the action of enzymes such as kinase, phosphatase, glycosyltransferase, glycosidase and protease, respectively. Conversion to the active form can be performed. The precursor can be prepared by genetic engineering by selecting means such as introduction or deletion of a phosphorylation site or sugar chain binding site, or addition of an appropriate polypeptide. Preferably, an appropriate means is selected in consideration of the characteristics of the target cell and target disease.

  For example, a precursor that can be converted into an active form by the action of a polypeptide that is expressed or released in the cell due to canceration of the cell or viral infection of the cell can be designed. For example, some viruses are known to require the action of their own proteases in their replication (eg, immunodeficiency virus, hepatitis C virus, etc.) and are targeted for therapeutic drug discovery. By designing the precursor of the trans-acting factor so that conversion to the active form occurs as a result of limited degradation by such protease, it is possible to construct a system in which the expression of a therapeutic gene is induced by viral infection . In addition, when multiple types of polypeptides that are expressed or released intracellularly in a disease-specific manner are known, it is possible to design precursors that can be converted into active forms by the action of any polypeptide. Is possible. For example, when two types of proteases with different substrate specificities are expressed by viral infection, a precursor that can be converted into an active form by the action of either protease can be designed. Furthermore, it is possible to design a plurality of precursors that cause the conversion to the active form by the action of any of the plurality of polypeptides. For example, when two types of proteases with different substrate specificities are expressed or released by viral infection, a precursor can be designed for each protease and used together for the treatment of viral infection.

  A precursor of a trans-acting factor that can be used in the present invention can be prepared, for example, by the following method. First, a nucleic acid encoding a fusion polypeptide in which an amino acid sequence that is recognized and cleaved by the protease described above is added to a natural trans-acting factor, and an arbitrary polypeptide is added across the amino acid sequence is prepared. Next, a vector in which the nucleic acid is arranged downstream of an appropriate promoter is prepared. A cell into which a nucleic acid having an appropriate reporter gene connected downstream of a transcription regulatory sequence whose transcription is induced by the trans-acting factor is prepared, and the expression of the reporter gene is expressed when the vector is introduced into this cell. If not found, this vector encodes a precursor of a trans-acting factor that has lost its ability to induce transcription. Here, the reporter gene may be any gene as long as the presence or absence of expression of the gene can be confirmed by any method, and has no physiological activity as long as an appropriate detection means, for example, an antibody that can be used for detection exists. It may be a polypeptide. Furthermore, by selecting a vector containing a gene encoding a fusion polypeptide that is converted into an active transactivator by the action of a protease from the vectors thus selected, the trans action usable in the present invention is selected. A precursor of the factor and a gene encoding the precursor can be obtained. In addition, a fusion polypeptide obtained by adding an arbitrary polypeptide containing an amino acid sequence recognized and cleaved by the protease to a natural trans-acting factor can be used.

  Amino acid sequences that are recognized and cleaved by HIV-derived protease and hepatitis C virus-derived protease have been studied [for example, Journal of Biological Chemistry, Vol. 266, p14554-14561 (1991), Journal of Virology (J. Virol. ), 67, p2832-2843 (1993), Journal of Virology, 71, p6208-6213 (1997)], based on this information, the fusion polypeptide gene can be designed. In addition, any polypeptide fused to a trans-acting factor is not particularly limited as long as it can suppress the activity of the factor when fused with a trans-acting factor. For example, a library of fusion polypeptide genes to be used for screening may be constructed using a library of nucleic acids encoding random polypeptides, a library of cDNA derived from a certain organism or cell, or a fragment thereof.

  A precursor of a trans-acting factor prepared and selected by the above operation, or a gene encoding the precursor, is introduced into a cell together with or before or after the therapeutic gene expression unit. It is used to control a therapeutic gene transcription unit. Preferably, an expression unit of said precursor is used, in which the gene encoding the precursor of the trans-acting factor is placed under the control of a suitable promoter, for example downstream thereof. Since it is preferable that the precursor of the trans-acting factor is constantly expressed in the cell, a promoter having a constant transcription activity is usually used for its expression. For example, promoters derived from housekeeping genes, virus-derived promoters (CMV promoter, SV40 promoter) and derivatives thereof are exemplified. When introducing a precursor of a trans-acting factor in the form of a polypeptide, a known means for introducing a protein into a cell may be used.

  As a particularly preferred embodiment of the present invention, an immunodeficiency virus, for example, a transcriptional regulatory sequence derived from HIV, ie, a promoter derived from LTR, is used as a transcriptional regulatory sequence for expression of a therapeutic gene, and its transcriptional activity is expressed by Tat of immunodeficiency virus. Examples include therapeutic gene expression systems that are controlled by precursors derived from proteins. Tat protein derived from HIV binds to the TAR sequence of the LTR promoter and remarkably enhances its transcriptional activity. For this reason, a significant amount of a therapeutic gene is transcribed and expressed in accordance with the conversion of the Tat protein precursor to the active Tat, and the therapeutic effect is exhibited. The Tat protein precursor does not have TAR binding activity, or binding to TAR does not enhance transcription from the promoter.

  The transcriptional regulatory sequence is not limited to the LTR promoter of the natural base sequence, but is modified appropriately for the sequence, for example, deletion of a binding site with a transcription factor derived from a host cell present in the promoter or Tat protein specific It may have been subjected to deletion of a region unnecessary for typical transcription. Examples of the latter deletion of the region unnecessary for Tat protein-specific transcription include deletion of the U5 region of the LTR and the region downstream of the TAR sequence (part of the R region and U5 region).

The expression unit comprising the above (1) a transcription regulatory sequence in which transcription is induced by a trans-acting factor, and a therapeutic gene arranged in a form in which expression can be controlled by the sequence, and (2) (1) The therapeutic gene expression system of the present invention is constructed by the expression unit in which the precursor of the trans-acting factor or the gene encoding the precursor of the trans-acting factor described in (1) can be expressed. . The therapeutic gene expression system may further include other components, for example, a vector used for introduction of the system into cells, an introduction reagent, and the like.

  The gene expression system of the present invention can be used by being mounted on an appropriate vector for introduction into cells. As a mode thereof, (A) a system in which a therapeutic gene expression unit and a trans-acting factor precursor expression unit are mounted on the same vector, and (B) a therapeutic gene expression unit and a trans-acting factor precursor expression unit are different. There are two types of systems installed in Vector. Which system is to be selected may be appropriately selected according to the cell or disease to be applied, difficulty in handling it, and the like. In the above-described embodiment in which the precursor of a trans-acting factor is introduced in the form of a polypeptide, a vector carrying only a therapeutic gene expression unit is introduced into the cell.

  The vector used in the system of the present invention is not particularly limited as long as it can be used for gene transfer and gene expression into a desired host, for example, a mammalian cell. A non-viral vector is exemplified by a plasmid vector. The plasmid vector can be introduced into a target cell using, for example, a method using a carrier such as liposome or ligand-polylysine, a calcium phosphate method, an electroporation method, a particle gun method, or the like.

  The viral vector that can be used in the present invention is not particularly limited, and known viral vectors used in gene transfer methods such as retroviral vectors (including lentiviral vectors and pseudotype vectors), adenoviral vectors, and adeno-associated vectors. Viral vectors, simian virus vectors, vaccinia virus vectors, Sendai virus vectors, and the like can be used. As the above-mentioned viral vector, those lacking replication ability are preferable so that they cannot self-replicate in infected cells. Particularly preferably, retroviral vectors, adenoviral vectors or lentiviral vectors are used.

  In cells into which the therapeutic gene of the present invention has been introduced, an active trans-acting factor is first generated by the action of a factor that is expressed or released in the cell by canceration or viral infection. A therapeutic gene is expressed. It is a feature of the present invention that an appropriate precursor of a trans-acting factor can be designed according to the type of disease, and can be applied to many diseases. In addition, since the therapeutic effect is exhibited depending on the onset and progression of the disease, the use of the system of the present invention makes it possible to treat and prevent the disease using the function of the therapeutic gene to the maximum. . A method for treating or preventing a disease using the therapeutic gene expression system of the present invention is also included in the present invention.

  When the therapeutic gene expression system of the present invention is used for treatment or prevention of a disease, the system is introduced into a living body. As the means, an in vivo method in which the therapeutic gene expression system of the present invention (or 1 or 2 vector on which the system is mounted) is directly introduced into a living body, Two examples are ex vivo methods in which a therapeutic gene expression system (or one or two vectors carrying the system) is introduced and then the cells are transplanted into a living body. Which method should be selected may be determined in consideration of the type of disease, the action mechanism and properties of the therapeutic gene used, the type of vector, and the like.

  The present invention is a precursor of a trans-acting factor, has no activity of inducing transcription from a transcriptional regulatory sequence whose transcription is induced by the trans-acting factor, and an active trans-acting by the action of a specific factor A precursor of a trans-acting factor that is converted to a factor is provided.

  As a preferred embodiment, an artificially constructed precursor of a trans-acting factor that is converted into an active trans-acting factor retaining transcription-inducing activity from a transcriptional regulatory sequence by limited degradation with a disease-specific protease is used. provide.

  The precursor of the trans-acting factor of the present invention is a fusion polypeptide of a trans-acting factor and another polypeptide, and induces transcription to a transcriptional regulatory sequence that exhibits an activity of inducing transcription by nature. Loss of activity. Other polypeptides fused to the trans-acting factor may be fused to the N-terminal side or the C-terminal side as long as the transcription-inducing activity of the trans-acting factor can be lost.

  The precursor of the trans-acting factor is an amino acid that is recognized and cleaved by a disease-specific protease between a region derived from a trans-acting factor and a region derived from another polypeptide and / or within a region derived from another polypeptide. Has an array. When the amino acid sequence is cleaved, it is converted into a trans-acting factor that retains transcription-inducing activity. Further, it may be a fusion polypeptide in which an arbitrary polypeptide containing an amino acid sequence that is recognized and cleaved by the protease is added to a trans-acting factor. When a plurality of disease-specific proteases are present, the precursor of the trans-acting factor includes two or more protease recognition sequences corresponding to the recognition sequences of these proteases, the region derived from the trans-acting factor, and the like. Between the regions derived from one polypeptide and / or within a region derived from another polypeptide.

  The precursor of the trans-acting factor of the present invention may be prepared based on any trans-acting factor. One embodiment is exemplified by a precursor of a trans-acting factor constructed based on the Tat protein of immunodeficiency virus. Tat protein is particularly suitable for the purposes of the present invention because of its low molecular weight and ability to exhibit transcription-inducing activity alone. When Tat protein is used as a trans-acting factor, a preferred embodiment of the trans-acting precursor of the present invention is not particularly limited. However, a trans-acting factor in which another polypeptide is fused to the N-terminal side of the Tat protein. Examples are precursors.

  The precursor of the trans-acting factor of the present invention can be obtained by designing the precursor by the above-mentioned method and selecting one having a desired function. In the construction, a protease capable of activating it can be arbitrarily selected, and can be freely designed by introducing an amino acid sequence recognized and cleaved by the protease. As a particularly preferred embodiment, a trans-acting factor precursor having a recognition and cleavage sequence of a protease derived from immunodeficiency virus or hepatitis C virus is exemplified. The precursor is used for treatment and prevention of immunodeficiency virus infection and hepatitis C virus infection using the therapeutic gene expression system of the present invention.

  In the trans-acting factor precursor of the present invention, the polypeptide fused to the trans-acting factor is not particularly limited as long as it can suppress the activity when fused with the trans-acting factor. For example, a polypeptide having a molecular weight of about 10 to 100 kDa and capable of existing as a monomer is used. Although not specifically limited to the present invention, human serum albumin (HSA, NCBI Acc. No. AAH34023.1), brain abundant, membrane attached signal protein 1 (BASP1, RefSeq ID: NP_0) described in Examples below. And laminin α1 (RefSeq ID: NP — 005550) or a partial sequence thereof, and homologues of these polypeptides are exemplified as polypeptides fused to trans-acting factors. Although not particularly limited, the homologue of a polypeptide in the present specification means at least 70%, preferably at least 80% or 90% or more, more preferably 95% or more identity on the amino acid sequence. It refers to the polypeptide that it has. In the trans-acting factor precursor of the present invention, two or more of the same or different types of polypeptides fused in series may be fused to the trans-acting factor.

  In the precursor of the trans-acting factor of the present invention, the amino acid sequence recognized and cleaved by a disease-specific protease is not particularly limited. However, the recognition sequence of an immunodeficiency virus-derived protease or hepatitis C virus-derived NS3 Examples include protease recognition sequences. The consensus sequence recognized by NS3 protease is (Asp / Glu) -Xaa-Xaa-Xaa-Xaa- (Cys / Thr) ↓ (Ser / Ala) [Xaa represents all amino acid residues, and ↓ represents NS3 It indicates the position of the peptide bond cleaved by the protease] [Journal of Virology (Vol. 67), p2832-2843 (1993)]. Suitable examples of such NS3 protease recognition sequences include NS5a / 5b site, NS4b / 5a site, and NS4a / 4b site [J. Virol., Vol. 68, p8147-8157. (1994)]. Examples of amino acid sequences of NS5a / 5b site, NS4b / 5a site, and NS4a / 4b site are Glu-Asp-Val-Val-Cys-Cys-Ser, Asp-Cys-Ser-Thr-Pro-, respectively. Examples include amino acid sequences including Cys-Ser and Asp-Glu-Met-Glu-Glu-Cys-Ala. The sequence that is recognized and cleaved by a disease-specific protease in the precursor of the trans-acting factor of the present invention may have a plurality of these protease recognition sequences.

  The gene encoding the precursor of the trans-acting factor of the present invention is also one aspect of the present invention.

  The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to the scope of the examples.

Example 1 Construction of MazF Expression Plasmid (1) Construction of MazF Expression Retroviral Vector Plasmid pMT-HLTR-MazF-PL Encodes gag, sgGFP and RRE inserted in plasmid pQBI-LTRgagGFP (manufactured by Quantum Biotechnologies Inc.) The region was removed by double digestion with restriction enzymes SalI and XbaI, and a chemically synthesized DNA having a base sequence shown in SEQ ID NO: 2 in the sequence listing and encoding MazF was inserted therein. The thus constructed plasmid was designated as pQBI-LTRMazFcv1. Using pQBI-LTRMazFcv1 as a template and primers of SEQ ID NO: 3 and SEQ ID NO: 4, the region encoding MazF from HIV-LTR was amplified by PCR, and the resulting amplified fragment was digested with restriction enzymes NotI and Eco52I. Digested. This DNA fragment was named HIV-LTR-MazF cassette.

  Using a pZsGreen1-N1 plasmid (manufactured by Clontech) as a template and using the primers shown in SEQ ID NO: 5 and SEQ ID NO: 6, a DNA fragment containing a poly A signal derived from herpes simplex virus type 1 was obtained by PCR. The amplified fragment was digested with restriction enzymes MluI and NotI. This DNA fragment was named pA fragment.

  Using pQBI-pgk (manufactured by Quantum Biotechnologies Inc.) as a template, using the primers described in SEQ ID NO: 7 and SEQ ID NO: 8, a fragment containing the mouse PGK promoter was amplified by PCR, and the resulting amplified fragment was used as a restriction enzyme. After digestion with MluI, the ends were blunted and further digested with BamHI. This DNA fragment was named pPGK fragment.

  Human low affinity by PCR using the genome of retrovirus-producing cell SFCMM-3 [Human Gene Therapy, Vol. 9, p2243-251 (1998)] as a template and the primers of SEQ ID NO: 9 and SEQ ID NO: 10. The extracellular domain of Nerve Growth Factor Receptor was amplified, and the resulting amplified fragment was digested with restriction enzymes EcoRI and XhoI. This DNA fragment was named LNGFR fragment.

  Recombinant retrovirus shown in FIG. 1 by sequentially inserting the above gene fragments into the MluI and BamHI sites of the pMT vector [pM vector described in Gene Therapy, Vol. 7, pages 797 to 804 (2000)]. The vector plasmid pMT-HLTR-MazF-PL was obtained. This vector is characterized in that the HIV-LTR-MazF cassette is inserted in the opposite direction to the transcription of the retroviral vector genome.

(2) Construction of MazF-expressing retroviral vector plasmid pMTD3-U3TAR-MazF-PL For the purpose of comparison with pMT-HLTR-MazF-PL, the HIV-LTR-MazF cassette is in the same direction as the transcription of the retroviral vector genome. A self-inactivating retroviral vector pMTD3-U3TAR-MazF-PL was constructed (FIG. 2). For this preparation, the plasmid vector pMTD3 in which the region spanning the 43rd to 309th bases of the 3′LTR U3 region of the pMT vector was deleted, and the DNA fragment prepared in (1) at the MluI and BamHI sites. Was constructed as shown in FIG.

Example 2 Establishment of Retrovirus-Producing Cells and Preparation of Retrovirus Vector Transient virus production using pMT-HLTR-MazF-PL and Retrovirus Packaging Kit Eco (manufactured by Takara Bio Inc.) was performed, and ecotropic MT-HLTR -Obtained MazF-PL virus. The ecotropic MT-HLTR-MazF-PL virus was infected with GaLV retrovirus packaging cell PG13 (ATCC CRL-10686) in the presence of 8 μg / mL of polybrene (Sigma Aldrich) to obtain a transgenic cell. The cells were seeded at a limiting dilution in a 96-well plate, the culture supernatant of the clones that had proliferated was collected, and the virus titer was measured by the real-time PCR method described in Example 3. The clone 1 strain in which high titer virus production was observed was selected to establish a retrovirus vector-producing cell line PG13 / MT-HLTR-MazF-PL.

PG13 / MT-HLTR-MazF-PL was cultured in Dulbecco's modified Eagle (DMEM) medium (manufactured by Sigma) containing 10% fetal bovine serum (manufactured by Invitrogen) (containing 50 U / mL penicillin and 50 μg / mL streptomycin). When grown semiconfluent, the medium was replaced with fresh 10% fetal calf serum containing 5 mM sodium butyrate, 50 U / mL penicillin, 50 μg / mL streptomycin-containing DMEM medium (0.1 mL / cm ² ), and the supernatant was replaced after 24 hours. A GaLV / MT-HLTR-MazF-PL recombinant retrovirus solution was obtained by filtration through a 0.45 μm filter (Millipore). The obtained virus solution was divided and dispensed, stored in a −80 ° C. freezer, and subjected to the following gene transfer experiment.

Example 3 Measurement of virus titer Real-time PCR reaction was performed using 1 μL of virus solution as a template using Retro-X qRT-PCR Titration Kit (manufactured by Clontech), and the RNA copy number in the virus solution was calculated from a calibration curve. The titer of the GaLV / MT-HLTR-MazF-PL virus solution obtained in Example 2 was 9.78 × 10 ⁹ copies / mL.

Example 4 Production of Cell Line Retaining MazF Gene A human cell line is infected with the GaLV / MT-HLTR-MazF-PL recombinant retrovirus produced in Example 2. A polyclonal cell line transformed with the virus is prepared by selecting the transfected cells with CD271 microbead kit (Miltenyi Biotech).

Example 5 Production and Selection of Tat Precursor Gene A gene encoding Tat protein is synthesized or isolated from a commercially available plasmid (for example, plasmid pQBI-tatGFP manufactured by Quantum Biotechnologies Inc.), and an expression vector for eukaryotic cells is prepared. A vector inserted downstream of the promoter is prepared. A structural gene in which a synthetic DNA encoding an amino acid sequence that is recognized and cleaved by NS3 protease of hepatitis C virus is inserted into the C-terminal part of the Tat protein coding region of this vector, and further randomly amplified from a human cDNA library downstream thereof A vector in which the fragments are connected is prepared. This vector is a library of genes for fusion polypeptides containing the Tat protein.

  The polyclonal cell line prepared in Example 4 is transformed with this vector. A transformed cell that retains the growth ability is a cell that does not express MazF, and it can be expected that the transformed cell has been transformed with a vector encoding a fusion polypeptide that has lost its ability to induce transcription of the Tat protein.

  NS3 protease gene is introduced into the grown cells to express the gene. As a result, cells that have undergone cell death are cells in which active Tat protein is generated by NS3 protease and MazF is expressed. A vector is recovered from the cells, and a Tat protein fusion polypeptide to which transcription-inducing activity is imparted by NS3 protease digestion, that is, a gene encoding the trans-acting factor precursor of the present invention is obtained.

  Alternatively, the vector is collected from the grown cells and the amino acid sequence of the fusion polypeptide encoded therein is confirmed. Further, this fusion polypeptide is prepared and tested for cleavage by NS3 protease, and a gene encoding a Tat protein fusion polypeptide to which transcription-inducing activity is imparted by NS3 protease digestion is obtained.

Example 6: Preparation of TAR-ZsGreen-introduced cells (1) Construction of pMTD3H-ZsGreen / SN plasmid First, in order to prepare a vector in which the U3 region of the 3 ′ LTR of Moloney Murine Leukemia Virus (MMLV) was deleted, the pMT vector PCR was carried out using the SCV3LB primer described in SEQ ID NO: 11 and the SCV3LRI primer described in SEQ ID NO: 12 using as a template, and the resulting PCR fragment was cloned into a pGEM-T Easy vector (manufactured by Promega). The thus constructed plasmid was designated as pGEM-T Easy-3′LTR plasmid.

  Next, PCR was performed using the 3′LTR-2 primer represented by SEQ ID NO: 13 and the SCV3LRI primer represented by SEQ ID NO: 12 using the pMT vector as a template, and the resulting PCR fragment was cleaved with restriction enzymes NheI and SalI. And cloned into the NheI-SalI site of the pGEM-T Easy-3 ′ LTR plasmid. The thus constructed plasmid was designated as preMTD3 plasmid.

  Subsequently, the preMTD3 plasmid was cleaved with restriction enzymes BamHI and SalI, and the resulting fragment was cloned into the BamHI-SalI site of the pMT vector. The thus constructed pMTD3 plasmid was named. The pMTD3 plasmid is a plasmid lacking U3 of 3′LTR.

  Next, PCR was performed using the pQBI-LTR gagGFP vector (manufactured by Quantum Biotechnologies Inc.) as a template and the 5′HIV1-U3 primer described in SEQ ID NO: 14 and the 3′HIV-TAR primer described in SEQ ID NO: 15. The PCR fragment was cleaved with restriction enzymes MluI and ApaI and cloned into the MluI-ApaI site of the pMTD3 plasmid. The thus constructed plasmid was designated as pMTD3H plasmid.

  Next, PCR was performed using the pZsGreen-N1 vector (manufactured by Clontech) as a template using the ZsGreen5 primer described in SEQ ID NO: 16 and the ZsGreen3 primer described in SEQ ID NO: 17, and the resulting PCR fragment was digested with restriction enzymes BamHI and XhoI. After cutting, it was cloned into the BamHI-XhoI site of the pMTD3H plasmid. The thus constructed plasmid was designated as pMTD3H-ZsGreen plasmid.

  Finally, the pDON-AI vector (Takara Bio Inc.) was cleaved with restriction enzymes SalI and XhoI, and the resulting fragment was cloned into the XhoI site of the pMTD3H-ZsGreen plasmid. The thus constructed plasmid was designated as pMTD3H-ZsGreen / SN plasmid. FIG. 3 shows an outline of the pMTD3H-ZsGreen / SN plasmid.

(2) Preparation of MTD3H-ZsGreen / SN retroviral vector The plasmid pGPD, pE contained in Retrovirus Packaging Kit Ampho (manufactured by Takara Bio Inc.) was prepared by using the pMTD3H-ZsGreen / SN plasmid prepared in Example 6- (1). -Transformed HEK293T cells with ampho. After culturing the obtained transformed cells for 2 days, a cell supernatant was obtained, filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore), and retrovirus vector (MTD3H-ZsGreen / SN / Ampho retroviral vector).

(3) Preparation of transformed cells The human fibrosarcoma cell HT1080 was infected with the MTD3H-ZsGreen / SN / Ampho retroviral vector prepared in Example 6- (2) using polyprene having a final concentration of 8 μg / mL. Thereafter, the transfected cells are cultured in DMEM medium containing 500 μg / mL geneticin (containing 10% fetal bovine serum, 50 U / mL penicillin, 50 μg / mL streptomycin), and the transfected cells are selected and controlled by the U1 TAR promoter of HIV1. HT1080 cells (HT1080; named as ZsG) into which a ZsGreen gene and a neomycin resistance gene controlled by the SV40 promoter were introduced were obtained.

  The MTD3H-ZsGreen / SN / Ampho retroviral vector lacks the 3′LTR U3, and when introduced into the genome after being infected with HT1080 ;; the 5′LTR replaces the 3′LTR sequence, It does not have U3 promoter activity on the genome. Therefore, ZsGreen is controlled only by the U1 TAR promoter of HIV1.

Example 7: Preparation of NS3 protease gene-introduced cell (1) Construction of HCV NS3 protease expression plasmid DNA sequence corresponding to HCV NS3-NS4a (NCBI Acc. No. 1027-1711 of AAF 65964.1) (NCBI Acc. No. A restriction enzyme BglII site and an initiation codon (ATG) are added upstream of AF20774.1 3408-5462), a stop codon and a restriction enzyme NotI site are added downstream, and further, the base amino acid is not changed in four positions. The sequence-substituted sequence (SEQ ID NO: 18) was chemically synthesized and cloned into a pMD19-T vector (Takara Bio Inc.). The thus constructed plasmid was designated as SYN1619-17 plasmid.

  Next, an approximately 2.1 kb DNA fragment obtained by cleaving the pSYN1619-17 plasmid with restriction enzymes BglII and NotI was cloned into the BglII-NotI site of the pLPCX vector (Clontech). The thus constructed plasmid was designated as pLPCX NS3-4a plasmid.

(2) Construction of pDON5SHyg-NS3 plasmid The pTriEx-3 Hygro vector (Novagen) was cleaved with restriction enzymes HindIII and BstXI, and the resulting fragment was blunt-ended using DNA Blunting kit (Takara Bio). . The obtained DNA fragment was cloned into a site obtained by blunting a pMEI-5 Neo vector (manufactured by Takara Bio Inc.) with restriction enzymes FbaI and XhoI. The thus constructed plasmid was designated as pMEI-5 Hygro plasmid.

  Next, the pDON-AI-2 vector (manufactured by Takara Bio Inc.) was cleaved with restriction enzymes SspI and MluI, and the resulting fragment was cloned into the SspI-MluI site of the pMEI-5 Hygro plasmid. The thus constructed plasmid was designated as pDON5-Hygro vector.

  Subsequently, the pLPCX NS3-4a plasmid prepared in Example 7- (1) was cleaved with the restriction enzyme BglII, then blunt-ended using DNA Blunting Kit (Takara Bio Inc.), further cleaved with NotI, and pDON5-Hygro The vector was cloned into the PmaCI-NotI site of the vector. The thus constructed plasmid was designated as pDON5SHyg-NS3 plasmid.

(3) Preparation of DON5SHyg-NS3 retroviral vector The pDON5SHyg-NS3 plasmid prepared in Example 7- (2) was transformed into HEK293T cells together with plasmids pGP and pE-ampho contained in Retrovirus Packaging Kit Ampho. . After culturing the transformed cells for 2 days, a cell supernatant was obtained, filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore), and retrovirus vector (DON5SHyg-NS3 / Ampho retrovirus vector) Naming).

(4) Preparation of transformed cells HT1080 produced in Example 6- (3) was prepared using the DON5SHyg-NS3 / Ampho retrovirus vector produced in Example 7- (3) with a final concentration of 8 μg / mL of polyprene. Infecting ZsG, culturing the infected cells in DMEM medium containing 200 μg / mL hygromycin (containing 10% fetal calf serum, 50 U / mL penicillin, 50 μg / mL streptomycin), and selecting transgenic cells; HT1080 cells (HT1080 ;; named ZsG + NS3) into which the ZsGreen gene controlled by the U3TAR promoter, the neomycin resistance gene controlled by the SV40 promoter, and the NS3 gene controlled by the MMLV LTR promoter were obtained.

Example 8: Preparation of Tat fusion protein-expressing retroviral vector (1) Preparation of pDON5-Neo vector pDON-AI-2 vector (Takara Bio Inc.) was cleaved with restriction enzymes SspI and MluI, and the resulting fragment was pMEI -5 It was cloned into the SspI-MluI site of Neo vector (Takara Bio). The vector thus constructed was designated as pDON-5-Neo vector.

(2) Construction of Tat protein expression plasmid PCR was performed using the plasmid pQBI-tatGFP (manufactured by Quantum Biotechnologies Inc.) as a template and the Tat Sac2 F primer described in SEQ ID NO: 24 and the Tat EcoR1 R primer described in SEQ ID NO: 20. The obtained PCR fragment was cleaved with restriction enzymes SacII and EcoRI, blunt-ended using DNA Blunting kit (Takara Bio), and cloned into the PmeI site of pDON-AI vector (Takara Bio). The plasmid thus obtained was designated as pDON-Tat plasmid.

(3) Cloning of HSA ORF cDNA PCR was performed using the Human Mammary Gland cDNA library (manufactured by Takara Bio Inc.) as a template and the hALBF1 primer described in SEQ ID NO: 21 and the hALBR1 primer described in SEQ ID NO: 22. Using the obtained PCR fragment as a template, PCR was performed using the phosphorylated hALBF2 primer described in SEQ ID NO: 23 and the phosphorylated hALBR1 primer described in SEQ ID NO: 22, and cDNA corresponding to the coding region of the material Human Serum Albumin (HSA) (NCBI Acc. No. BC034023.1 ORF 73-1830) was amplified. The obtained PCR fragment was cloned into the pUC18 vector that had been dephosphorylated after digestion with the restriction enzyme HincII, and the constructed plasmid was named pUhA2 plasmid.

(4) Cloning of HIV Tat ORF Using the pDON-Tat plasmid prepared in Example 8- (2) as a template, the phosphorylated Tat Sac2 F primer described in SEQ ID NO: 24 and the phosphorylated TatBmR1 primer described in SEQ ID NO: 25 were used. PCR was performed. The obtained PCR fragment was cloned into the pUC18 vector that had been dephosphorylated after digestion with the restriction enzyme HincII to obtain the pUTatBm plasmid.

(5) Construction of Tat-NS3 protease site plasmid After annealing the synthetic DNA stop jctU described in SEQ ID NO: 26 and stop jctL described in SEQ ID NO: 27, this was transformed into the pDON5-Neo vector prepared in Example 8- (1). A plasmid cloned into the BamHI-HpaI site was prepared. At the SacII-Aor51HI site of this plasmid, a fragment of about 0.27 kb obtained by cleaving the pUTatBm plasmid prepared in Example 8- (4) with SacII and BamHI, and the synthetic DNA siteA1U described in SEQ ID NO: 28 and SEQ ID NO: 29 The DNA fragment obtained by annealing with the described synthetic DNA site A1L was cloned. The thus constructed plasmid was designated as pD5NTatA1 plasmid. Further, instead of using a DNA fragment obtained by annealing a synthetic DNA siteA1U and a synthetic DNA siteA1L, a DNA fragment obtained by annealing a synthetic DNA siteB1U described in SEQ ID NO: 30 and a synthetic DNA siteB1L described in SEQ ID NO: 31 was used to obtain pD5NTatA1 A plasmid was constructed in the same manner as the construction of the plasmid, and this was designated as pD5NTatB1 plasmid.

(6) Construction of pD5NTA1H plasmid The pUhA2 plasmid prepared in Example 8- (3) was cleaved with restriction enzymes EcoRI and HindIII to obtain a DNA fragment of about 1.8 kb containing the HSA gene. This DNA fragment was blunt-ended using DNA Blunting Kit (manufactured by Takara Bio Inc.) and then inserted into the Aor51HI site of the pD5NTatA1 plasmid prepared in Example 8- (5), and NS3 protease was added to the C-terminal side of the Tat protein. A plasmid (named pD5NTA1H plasmid) having a sequence encoding a fusion protein linked with HSA across the recognition sequence NS3 / 4a site was obtained.

(7) Construction of pD5NHA1T plasmid PCR was performed using the NSatF1 primer described in SEQ ID NO: 32 and the TatRHpa primer described in SEQ ID NO: 33 using the pDON-Tat plasmid prepared in Example 8- (2) as a template. Using the obtained PCR product as a template, PCR was performed using the NSatF2 primer described in SEQ ID NO: 34 and the TatRHpa primer described in SEQ ID NO: 33, and a DNA fragment in which a part of the NS3 protease recognition sequence NS3 / 4A site was added to the Tat gene was obtained. Amplified. The obtained PCR fragment was cloned into the HincII site of the pUC118 vector and then cleaved with restriction enzymes SacII and HpaI to obtain a DNA fragment of about 0.3 kb. This DNA fragment was cloned into the SacII-HpaI site of the pDON5-Neo vector prepared in Example 8- (1), and the constructed plasmid was designated as pD5NT plasmid.

  Next, PCR was performed using the HSAKSR primer described in SEQ ID NO: 35 and the HSANSR1 primer described in SEQ ID NO: 36 using the pUhA2 plasmid prepared in Example 8- (3) as a template. Using the obtained PCR product as a template, PCR was performed using the HSAKF primer represented by SEQ ID NO: 35 and the HSANSR2 primer represented by SEQ ID NO: 37, and a DNA fragment in which a part of the NS3 protease recognition sequence NS3 / 4A site was added to the HSA gene was obtained. Amplified. The obtained PCR fragment was cloned into the HincII site of the pUC118 vector, and then cleaved with restriction enzymes SacII and SalI. The obtained DNA fragment of about 1.8 kb was cloned into the SacII-SalI site of the pD5NT plasmid and encoded a fusion protein in which the Tat protein was linked to the C3 terminal side of the HSA protein across the NS3 / 4a site. A plasmid (designated pD5NHA1T plasmid) having the sequence

(8) Construction of pD5NHB1T plasmid The pD5NHA1T plasmid prepared in Example 8- (7) was cleaved with restriction enzymes SacII and BamHI to obtain a DNA fragment of about 1.8 kb containing the HSA gene. This DNA fragment was inserted into the SacII-BamHI site of the pD5NTatB1 plasmid prepared in Example 8- (5), replacing the Tat gene. The thus constructed plasmid was designated as pD5NHB1 plasmid.

  Next, using the pDON-Tat plasmid prepared in Example 8- (2) as a template, PCR was performed using the TatFAor primer described in SEQ ID NO: 38 and the TatRHpa primer described in SEQ ID NO: 33, and a DNA fragment containing the Tat gene was amplified. . The obtained PCR fragment was cloned into the HincII site of the pUC118 vector and then cleaved with restriction enzymes Aor51HI and HpaI to obtain a DNA fragment of about 0.3 kb. This DNA fragment is inserted into the Aor51HI-HpaI site of the pD5NHB1 plasmid, and a plasmid (pD5NHB1T plasmid having a sequence encoding a fusion protein in which Tat is linked across the NS3 protease recognition sequence NS5a / 5b site on the C-terminal side of the HSA protein. Named).

(9) Preparation of Tat-expressing retroviral vector HEK293T cells were transformed with the pDON-Tat plasmid prepared in Example 8- (2) together with the plasmids pGP and pE-ampho contained in Retrovirus Packaging Kit Ampho. After culturing the transformed cells for 2 days, a cell supernatant was obtained, filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore), and a retroviral vector expressing Dat (DON-Tat / Ampho retroviral vector).

(10) Preparation of Tat and HSA Fusion Protein Expression Retroviral Vector The pD5NTA1H plasmid prepared in Example 8- (6) was transformed into HEK293T cells together with plasmids pGP and pE-ampho contained in Retrovirus Packaging Kit Ampho. did. After culturing the transformed cells for 2 days, a cell supernatant was obtained, filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore), and N-terminal via the NS3 recognition sequence NS3 / 4A site. A retroviral vector (named D5NTA1H / Ampho retroviral vector) expressing a fusion protein having Tat on the side and HSA on the C-terminal side was obtained.

  In addition, the pD5NHA1T plasmid prepared in Example 8- (7) was transformed into HEK293T cells together with plasmids pGP and pE-ampho contained in Retrovirus Packaging Kit Ampho. After culturing the transformed cells for 2 days, a cell supernatant was obtained, filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore), and N-terminal via the NS3 recognition sequence NS3 / 4A site. A retroviral vector (named D5NHA1T / Ampho retroviral vector) expressing a fusion protein having HSA on the side and Tat on the C-terminal side was obtained.

  Further, the pD5NHB1T plasmid prepared in Example 8- (8) was transformed into HEK293T cells together with plasmids pGP and pE-ampho contained in Retrovirus Packaging Kit Ampho. After culturing the transformed cells for 2 days, a cell supernatant was obtained, filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore), and N-terminal via the NS3 recognition sequence NS5A / 5B site. A retroviral vector (named D5NHB1T / Ampho retroviral vector) expressing a fusion protein having HSA on the side and Tat on the C-terminal side was obtained.

Example 9: Measurement of Tat activity (1) Measurement of difference in Tat inactivation due to difference in position of Tat and HSA HT1080 prepared in Example 6- (3) ;; ZsG in Example 8- (10) The prepared D5NTA1H / Ampho retroviral vector, D5NHA1T / Ampho retroviral vector, and the DON-Tat / Ampho retroviral vector prepared in Example 8- (9) were each infected, cultured for 3 days, and then flow cytometer ( Beckman Coulter, Inc.). FIG. 4 shows the measurement results of fluorescent protein ZsGreen expressing cells using a flow cytometer.

  HT1080 infected with the positive control DON-Tat / Ampho retroviral vector; 62.21% of ZsG cells were positive, and the majority of cells expressed the ZsGreen gene from the U3TAR promoter of HIV1 by Tat. [FIG. 4B]. HT1080 infected with a D5NTA1H / Ampho retroviral vector expressing a fusion protein having Tat at the N-terminal side and HSA at the C-terminal side through the NS3 / 4A site, NS3 / 4A sites; 35.71% of ZsG cells Was positive, and inactivation of Tat by HSA was not complete [FIG. 4 (c)].

  On the other hand, HT1080 infected with a D5NHA1T / Ampho retrovirus vector expressing a fusion protein having HSA on the N-terminal side and Tat on the C-terminal side through the NS3 / 4A site of NS3 ;; ZsG is 3.09% Cells were positive, and Tat was almost completely inactivated by HSA [FIG. 4 (d)]. From this, in order to inactivate the trans-acting factor, it was found that addition of the inactivating protein to the N-terminal side of Tat was effective.

(2) Measurement of Tat activation by NS3 protease HT1080 prepared in Example 6- (3); ZsG and HT1080 prepared in Example 7- (4); The D5NHB1T / Ampho retroviral vector prepared in (1) and the DON-Tat / Ampho retroviral vector prepared in Example 8- (9) were each infected, cultured for 3 days, and then analyzed with a flow cytometer. FIG. 5 shows the measurement results of cells expressing fluorescent protein ZsGreen using a flow cytometer.

  Cells infected with the positive control DON-Tat / Ampho retroviral vector were HT1080 ;; 91.7% cells positive with ZsG; HT1080 ;; 93.4% cells positive with ZsG + NS3 and NS3 protease In most cells regardless of the presence or absence of ZsGreen gene, the Tsat expressed the ZsGreen gene from the U3TAR promoter of HIV1 [FIG. 5 (b)]. On the other hand, cells infected with the D5NHB1T / Ampho retroviral vector were HT1080 ;; 0.79% of the cells were positive in ZsG, and Tat was almost completely inactivated in the absence of NS3 protease, but HT1080; ; 15.2% of the cells were positive with ZsG + NS3, and the recognition sequence was cleaved by NS3 protease to activate Tat. It was found that the activated Tat expressed a gene regulated by the U3 TAR promoter of HIV1. [FIG. 5 (c)].

Example 10 Cloning of DNA Fragment Encoding Polypeptide Used as Tat Fusion Partner (1) Cloning of DNA Encoding Human Laminin α1 Partial Sequence Using Human Placental Total RNA (Clontech) as a template, PrimeScript High Fidelity RT-PCR RT-PCR was performed using Kit (manufactured by Takara Bio Inc.), A1F5482 (AgeI) primer described in SEQ ID NO: 47 and A1R8608 (NotI) primer described in SEQ ID NO: 48. A library in which the obtained PCR fragment is inserted into the pMD20-T vector attached to the kit using the Mighty TA-cloning Kit for PrimeSTAR (manufactured by Takara Bio Inc.) is encoded, and a partial sequence of human laminin α1 chain is encoded. A clone carrying a gene fragment (cDNA corresponding to positions 5482-8608 of GenBank accession number NM_005559) was selected. The plasmid of this clone was named pMD20-A1 (5482-8608) 35.

  Using the above-mentioned pMD20-A1 (5482-8608) 35 as a template, PCR using the phosphorylated LMF1 primer described in SEQ ID NO: 49 and the phosphorylated LMR1 primer described in SEQ ID NO: 50, and phosphorylation described in SEQ ID NO: 51 PCR was performed using the LMF2 primer and the phosphorylated LMR2 primer described in SEQ ID NO: 52. The PCR fragment obtained by each reaction was cloned into the HincII site of pUC18, and the constructed plasmids were named pULM1 plasmid and pULM2 plasmid, respectively.

(2) Cloning of BASP1 gene Homo sapiens brain abundant, membrane attached signal protein 1 (BASP1) cDNA clone (manufactured by OriGene Technologies, Inc.) with primer number S described in SEQ ID NO: 57 as a primer, and SEQ ID NO: 57 as a primer with the sequence number 57 as a template. PCR was performed, and the obtained PCR fragment was cloned into the HincII site of the pUC118 vector (Takara Bio Inc.). The thus constructed plasmid was designated as pUC-BASPKFNSR1 plasmid.

  Further, a plasmid was constructed in the same manner as the pUC-BASPKFNSR1 plasmid except that the BASPFH2 primer described in SEQ ID NO: 63 and the BASPRH2 primer described in SEQ ID NO: 64 were used as PCR primers, and the constructed plasmid was named pUC-BASPFH2RH2 plasmid. did.

Example 11 Production of Retroviral Vector Plasmid for Expression of Fusion Protein of Tat and Other Polypeptide (1) Construction of Retroviral Vector Plasmid for Expression of Fusion Protein with Fusion Partner Fused to Nat Side of Tat-1
Example 10-SacII-BamHI site of the pD5NHB1T plasmid prepared in Example 8- (8) was obtained by digesting the pULM1 plasmid obtained in (1) with restriction enzymes SacII and BamHI. The DNA fragment was replaced with the DNA fragment encoding HSA. The resulting plasmid was named pD5NLB1T plasmid.

  The pUC-BASPKFNSR1 plasmid obtained in Example 10- (2) was digested with restriction enzymes PmaCI and BamHI to obtain a DNA fragment of about 700 bp containing the BASP1 gene. On the other hand, the pD5NHB1T plasmid prepared in Example 8- (8) was cleaved with ApaI, then blunted with DNA Blunting Kit (manufactured by Takara Bio Inc.), and further cleaved with BamHI to obtain a DNA fragment encoding HSA. Removed. A DNA fragment containing the above BASP1 gene was inserted to replace the removed DNA fragment. The thus constructed plasmid was designated as pD5NBB1T plasmid.

(2) Construction of a retroviral vector plasmid for expression of a fusion protein in which a fusion partner is fused to the C-terminal side of Tat. Obtained by digesting the pULM2 plasmid obtained in Example 10- (1) with restriction enzymes Aor51HI and HpaI. The 0.99 kbp DNA fragment was inserted into the Aor51HI-HpaI site of the plasmid pD5NTatB1 prepared in Example 8- (5). The resulting plasmid was named pD5NTB1L plasmid.

  The pUC-BASPFH2RH2 plasmid obtained in Example 10- (2) was cleaved with the restriction enzyme HincII to obtain a DNA fragment of about 700 bp containing the BASP1 gene. This DNA fragment was inserted into the Aor51HI site of the pD5NTatB1 plasmid prepared in Example 8- (5). The thus constructed plasmid was designated as pD5NTB1B plasmid.

(3) Characteristics of the fusion protein encoded by the DNA fragment inserted in the viral vector plasmid constructed in Examples 11- (1) and 11- (2) In Examples 11- (1) and 11- (2) Table 1 shows the characteristics of the fusion protein encoded by the DNA fragment inserted into the constructed viral vector plasmid. Each fusion protein has an NS3 protease recognition sequence NS5a / 5b between Tat and another polypeptide.

Example 12 Production of Various Retroviral Vectors The viral vector plasmid, pD5NLB1T plasmid, pD5NBB1T plasmid, pD5NTB1L plasmid, and pD5NTB1B plasmid constructed in Examples 11- (1) and 11- (2) are included in Retrovirus Packaging Kit Ampho, respectively. The HEK293T cells were transformed with the plasmids pGP and pE-ampho and cultured for 2 days, and then the cell supernatant was obtained and filtered through a 0.45 μm filter (Milex HV, manufactured by Millipore) D5NLB1T / Ampho retroviral vector, D5NBB1T / Ampho retroviral vector, D5NTB1L / Ampho retroviral vector, D5NTB1B / Amp O The retrovirus vector was obtained, respectively.

Example 13 Reconstitution of transformed cells (1) HT1080 prepared in Example 6- (3) ;; ZsG was limited in DMEM medium (containing 10% fetal bovine serum, 50 U / mL penicillin, 50 μg / mL streptomycin) A single clone (HT1080 ;; named ZsGSC) was obtained by the dilution method.

(2) HT1080 prepared in Example 7- (4) ;; ZsG + NS3 is obtained by limiting dilution with DMEM medium (containing 10% fetal bovine serum, 50 U / mL penicillin, 50 μg / mL streptomycin). did. The resulting single crane was infected with the DON5SHyg-NS3 / Ampho retrovirus vector prepared in Example 7- (3) using a final concentration of 8 μg / mL of polyprene, and the infected cells were infected with 200 μg / mL of hygromycin. Cultivated in a DMEM-containing medium (containing 10% fetal bovine serum, 50 U / mL penicillin, 50 μg / mL streptomycin) to select transgenic cells, and a neomycin controlled by the HIV3 U3TAR promoter and the SV40 promoter HT1080 cells (HT1080; named ZsG + NS3W) into which a resistance gene and an NS3 gene controlled by the MMLV LTR promoter were additionally introduced were obtained.

Example 14 Inactivation of Tat activity and measurement of Tat activation by NS3 protease HT1080 prepared in Example 13- (1); ZsGSC and HT1080 prepared in Example 13- (2); ZsG + NS3W Each of the retroviral vectors prepared in Example 12 and the DON-Tat / Ampho retroviral vector prepared in Example 8- (9) were each infected, cultured for 3 days, and then flow cytometer (manufactured by Beckman Coulter, Inc.) ). Table 2 shows the measurement results of the fluorescent protein ZsGreen expressing cells by flow cytometer.

  Cells infected with the positive control DON-Tat / Ampho retroviral vector were HT1080 ;; 96.4% cells positive with ZsGSC, HT1080; 96.9% cells positive with ZsG + NS3W, NS3 protease The ZsGreen gene was expressed from the U3TAR promoter of HIV1 by Tat in most cells regardless of the presence or absence.

  HT1080 infected with D5NTB1L / Ampho or D5NTB1B / Ampho expressing a fusion protein having a fusion partner having a Tat at the N-terminal side and a fusion partner at the C-terminal side through the NS3 / 4A site of NS3; ZsGSC is 90% or more The cells were positive and Tat inactivation by the fusion partner was not observed.

  On the other hand, HT1080 infected with D5NLB1T / Ampho or D5NTBB1T / Ampho expressing a fusion protein having a fusion partner having Tat at the C-terminal side and a fusion partner at the N-terminal side via the NS3 / 4A site of NS3; .3% and 1.3% of the cells were positive and Tat was almost completely inactivated by the fusion partner. From this, in order to inactivate the trans-acting factor, it was confirmed that addition of an inactivating protein to the N-terminal side of Tat was effective.

  In addition, HT1080 infected with D5NLB1T / Ampho ;; HT1080 infected with ZsG + NS3W and D5NTBB1T / Ampho ;; 22.7% and 49.5% of cells were positive with ZsG + NS3W, respectively. Therefore, when a fusion protein having a Tat at the C-terminal side and a fusion partner at the N-terminal side via the NS3 / 4A site is used, the recognition sequence is cleaved by the NS3 protease to activate Tat. It was confirmed that the gene regulated by the U3TAR promoter of HIV1 was expressed by the converted Tat.

  The present invention provides a therapeutic gene expression system capable of inducing gene expression in a disease-specific manner. The system can be designed to produce a desired effect based on the characteristics of various diseases. The therapeutic gene expression system of the present invention is useful for the treatment and prevention of various diseases, particularly cancer and viral infections.

SEQ ID NO: 2; Synthetic DNA encoding MazF.
SEQ ID NO: 3; Primer to amplify HIV-LTR-MazF cassette.
SEQ ID NO: 4; Primer to amplify HIV-LTR-MazF cassette.
SEQ ID NO: 5; Primer to amplify pA fragment.
SEQ ID NO: 6; Primer to amplify pA fragment.
SEQ ID NO: 7; Primer to amplify pPGK fragment.
SEQ ID NO: 8; Primer to amplify pPGK fragment.
SEQ ID NO: 9; Primer to amplify LNGFR fragment.
SEQ ID NO: 10; Primer to amplify LNGFR fragment.
SEQ ID NO: 11-17; PCR primer.
SEQ ID NO: 18; Synthetic DNA encoding HCV NS3 protease.
SEQ ID NO: 19-25; PCR primer.
SEQ ID NO: 26-27; Synthetic DNA to remove a stop codon.
SEQ ID NO: 28-29; Synthetic DNA to introduce the NS3 protease NS3 / 4a cleavage site.
SEQ ID NO: 30-31; Synthetic DNA to introduce the NS3 protease NS5a / 5b cleavage site.
SEQ ID NO: 32-68; PCR primer.

Claims (21)

A gene expression system that includes the following components:
(1) a transcriptional regulatory sequence whose transcription is induced by a trans-acting factor, an expression unit comprising a therapeutic gene arranged in a form in which expression can be controlled by the sequence, and (2) the transcriptional regulatory according to (1) A trans-acting factor precursor that has no activity to induce transcription from a sequence and is converted to an active trans-acting factor by the action of a specific factor, or a form capable of expressing a gene encoding the precursor An expression unit located in   The gene expression system according to claim 1, wherein the specific factor is a polypeptide expressed in cells by canceration or viral infection.   The gene expression system according to claim 1 or 2, wherein the therapeutic gene is a gene encoding a gene selected from a polypeptide, a ribozyme, an antisense RNA, and an RNA that exhibits RNA interference.   The gene expression system according to claim 3, wherein the therapeutic gene is a gene encoding a polypeptide selected from a polypeptide having protease activity, a polypeptide having nuclease activity, and a cytokine.   The gene expression system according to claim 4, wherein the therapeutic gene is a gene encoding a polypeptide having a single-stranded RNA-specific endoribonuclease activity.   The gene expression system according to any one of claims 1 to 5, wherein the transcriptional regulatory sequence is a promoter derived from an LTR of an immunodeficiency virus.   The gene expression system according to claim 6, wherein the trans-acting factor is a polypeptide derived from Tat protein of immunodeficiency virus.   The precursor of a trans-acting factor is a polypeptide that is converted into an active trans-acting factor by a modification selected from phosphorylation, dephosphorylation, glycosylation, glycosylation, and limited degradation. The gene expression system according to any one of claims.   9. The gene expression system according to claim 8, wherein the precursor of the trans-acting factor is a polypeptide that is converted into an active trans-acting factor by the action of a virus-derived protease.   The gene expression system according to claim 9, wherein the precursor of the trans-acting factor is a polypeptide that is converted into an active trans-acting factor by the action of a protease derived from hepatitis C virus.   The gene expression according to claim 9, wherein the precursor of the trans-acting factor is a fusion polypeptide in which another polypeptide is fused to the N-terminal side of the trans-acting factor via a peptide chain that can be limitedly degraded by a protease derived from a virus. system.   The gene expression system according to any one of claims 1 to 11, wherein the expression unit of (1) and the expression unit of (2) are inserted on the same vector.   The gene expression according to any one of claims 1 to 11, comprising a first vector in which the expression unit of (1) is inserted and a second vector in which the expression unit of (2) is inserted. system.   A precursor of a trans-acting factor that does not have the activity of inducing transcription from a transcriptional regulatory sequence whose transcription is induced by the trans-acting factor, and is converted to an active trans-acting factor by the action of a specific factor. Precursor of trans-acting factor.   Transcription to a transcriptional regulatory sequence in which a trans-acting factor and other polypeptide are artificially fused via a peptide chain that can be limitedly decomposed by a protease, and the trans-acting factor has an intrinsic activity of inducing transcription The precursor of a trans-acting factor according to claim 14, characterized in that it has lost the activity of inducing.   The precursor of a trans-acting factor according to claim 15, wherein the trans-acting factor is a polypeptide derived from the Tat protein of immunodeficiency virus.   The trans-acting factor precursor according to claim 15 or 16, which is fused via a peptide chain that can be limitedly degraded by the action of a virus-derived protease.   The precursor of a trans-acting factor according to claim 17, which is fused via a peptide chain that can be limitedly degraded by the action of a protease derived from a virus derived from hepatitis C virus.   The precursor of a trans-acting factor according to claim 15, wherein another polypeptide is artificially fused to the N-terminal side of the trans-acting factor via a peptide chain that can be limitedly decomposed by a protease.   A nucleic acid encoding the precursor of the trans-acting factor according to any one of claims 14 to 19.   A method for treating or preventing a disease comprising a step of introducing the gene expression system according to any one of claims 1 to 13 into a living body or a cell.

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