KR20030032443A - Tetracyclin inducible hiv-1 packaging cell line and a method for producing hiv-1 defective interfering particle using same - Google Patents

Abstract

PURPOSE: A tetracycline inducible HIV-1 packaging cell line and a method for producing an HIV-1 defective interfering(DI) particle using the same are provided. The HIV-1 packaging cell line can regulate expression of subsidiary proteins requisite for the expression of an HIV-1 DI particle to inhibit its cell toxicity, and is thus useful for the production of the HIV-1 DI particle. CONSTITUTION: A gene construct functioning as a helper virus is prepared by removing the packaging signal, Env, Nef and 3'-LTR from the full-length HIV-1 genome. A tetracycline reactive packaging construct is prepared by inserting the gene construct into a vector expressing tetracycline(Tet) response unit, wherein the vector is selected from the group consisting of pTRE, pTet-ON, pTet-OFF, pRetro-ON and pRetro-OFF. The tetracycline inducible HIV-1 packaging cell line is prepared by transformation with the tetracycline reactive packaging construct.

Description

TETRACYCLIN INDUCIBLE HIV-1 PACKAGING CELL LINE AND A METHOD FOR PRODUCING HIV-1 DEFECTIVE INTERFERING PARTICLE USING SAME}

The present invention relates to a tetracycline inducible packaging cell line, specifically a gene construct in which a portion of the packaging signal, Env, Nef and 3 'LTR in the HIV-1 genome is removed to act as a helper virus; A tetracycline reactive packaging construct comprising the gene construct; A packaging cell line transfected with the packaging construct to express a gene of interest only in the presence of tetracycline to package HIV-1 DI (defective interfering) particles; And to a method of producing HIV-1 DI particles using the same.

Retroviral vectors and packaging cells are used as the most important tool for transferring heterologous genes into eukaryotic cells. Replication defective retroviral vectors contain cis-acting sequences for efficient viral replication, while packaging cell lines provide the viral protein required on trans for viral replication. The introduction of a retroviral vector into a suitable packaging cell line allows the propagation of the vector virus in the absence of a replication-competent virus (RCV). To date, many packaging cell lines have been established in a variety of gene delivery methods and used for delivery of heterologous genes.

Human immunodeficiency virus type-1 (HIV-1) is a very complex retrovirus. The genome of HIV-1 encodes gag, env and pol as well as accessory proteins such as vif, vpr, vpu, tat, rev and nef. HIV-1 has a chronic and slow pathogenicity when infected, and its value is increasing as a carrier of gene therapy for treating diseases. HIV-1-based vectors can be used for the introduction of genes of interest for the treatment of diseases, and expression of these genes is induced only after HIV-1 infection. HIV-1-base vectors also have the potential to introduce genes into non-dividing cells. The development of the HIV-1-basic packaging cell system has enabled the widespread application of HIV-1 vectors and the specific delivery of heterologous genes into CD4 + cells. Moreover, a single cycle of HIV-1 replication can be used in HIV-1 vectors and packaging cell systems to facilitate the study of HIV-1 infection rates, HIV-1 latency and the role of accessory proteins during HIV-1 replication. Used to build. In addition, the production of vector viruses in the absence of replicable HIV-1 provides a safer system for HIV-1 replication studies.

HIV-1 is known to actively reproduce its replication even in asymptomatic periods of 8 to 10 years without symptoms after infection. A new concept of DI particles has been devised for HIV-1 infection, with all the elements that can act as an antiviral agent against these viruses, and they can be useful as a tool to deliver genes for therapeutic purposes to the destination. In particular, the application of the central nervous system (CNS) to nondividing cells is expected.

In order for these DI particles to act as antiviral agents, the following three conditions must be met. First, since HIV-infected cells in the patient's body actively produce viruses, they must be able to act simultaneously as target cells and helper cells for HIV-1 DI particles, and second, naturally occurring DI. Thirdly, it is necessary to produce a gene product different from the particle, and to suppress the production of infected wild type HIV-1 without drastically inhibiting the production of wild-type virus particles. You have to have all the signals for packaging.

HIV-1-basic packaging cells have been reported several times before, but the problem is that the titer of the vector virus produced by these packaging cells is very low. There are at least two ways to build a packaging cell line using HIV-1 compared to other packaging cell lines based on other oncoretroviruses such as murine leukemia virus and spleen necrosis virus. Additional technical difficulties exist. One is that additional regulatory proteins required for replication expressed by HIV should be expressed only at specific stages to suit their purpose, and the other is the constitutive expression of several HIV proteins, including Env, protease, Vpr, etc. constitutive expression) is cytotoxic. Therefore, although it is possible to construct a packaging cell line that substantially expresses HIV proteins, the use of relatively specific cell lines with stronger resistance to the constitutive expression of HIV-1 proteins than in normal cell lines is required, which is why this is used. Limiting the possible cell types. In other words, in constructing a packaging cell line, it is important to remove cytotoxicity in order to stably express these proteins.

The existing lenti-virus is used as a viral vector by removing a certain part of the entire gene and inserting the target gene into the removed part. However, the recovery of viral particles through this constructed DNA construct cannot be expressed alone in a cell. Therefore, cotransfection of the DNA capable of expressing the removed portion (cotransfection) to obtain the desired virus.

In order to overcome the technical limitations in constructing a packaging cell line using an existing viral vector, the present inventors have used a tetracycline expression induction system to stably express an accessory protein in a cell and exhibit no cytotoxicity. The present invention has been completed by constructing an HIV-1 packaging cell line that regulates their expression.

It is an object of the present invention to provide a packaging cell line which does not exhibit cytotoxicity while stably expressing an accessory protein in a cell.

Figure 1 shows a schematic diagram of the Tetracycline inducible expression system devised by Gossen and Bujard,

FIG. 2 is a schematic diagram of the gene construct pHyPC of the present invention in which a portion of the packaging signal, Env, Nef and 3 'LTR, in full-length HIV-1 may be removed to act as a helper virus. Shown,

FIG. 3 shows a tetracycline inducible packaging construct pTRE-Pg in which pTRE vector having a Tet response unit is inserted except for the 3 'LTR of the helper virus construct pHyPC,

Figure 4 shows the packaging system of the invention to produce the desired DI particles using the packaging cell line transfected with the tetracycline inducible packaging construct pTRE-Pg of Figure 3,

5 is a result confirmed by ELISA that the Tet-induced HIV-1 packaging cell line of the present invention can regulate the expression of the gene of interest depending on the presence of tetracycline.

In order to achieve the above object, the present invention provides an HIV-1 packaging cell line that stably expresses an accessory protein without causing cytotoxicity using a tetracycline expression induction system.

The present invention also provides a method for producing HIV-1 DI particles (defective interfering) using the HIV-1 packaging cell line.

Hereinafter, the present invention will be described in detail.

The present invention provides a gene construct capable of removing a portion of the full-length HIV-1 genome and inserting a gene of interest to act as a helper virus; A tetracycline reactive packaging construct comprising the gene construct; The packaging construct is transfected to provide a packaging cell line that expresses a gene of interest only in the presence of tetracycline to package HIV-1 DI particles.

The inventors have established a packaging cell line that can efficiently package HIV-1 DI particles to develop a new concept of targeted DI particles for gene therapy for Acquired Immune Deficiency Syndrome (AIDS). .

HIV-1 packaging cell line of the invention

1) constructing a helper virus construct by precision gene fusion;

2) constructing an HIV-1 DI packaging construct with the helper virus construct inserted therein;

3) preparing a cell line expressing a tetracycline regulatory element;

4) packaging the HIV-1 DI particles by transfecting the cell line with the HIV-1 DI packaging construct of step 2); And

5) prepared by a packaging system comprising measuring the packaging effect of HIV-1 DI particles on HIV-1 infected cells.

As mentioned above, HIV-1 is a highly complex retrovirus whose genomes include not only retrovirus-specific genes such as gag, pol, and env, but also accessory proteins such as vif, vpr, vpu, tat, rev, and nef. accessory proteins are also coded. All of these proteins act as toxic substances in the cells, causing a major obstacle to cell growth. In order to solve these problems and to establish a stable cell line capable of packaging them, the inventors devised such a tetratracyclin inducible expression system. The expression induction system of the present invention uses a switch ON / OFF system that regulates the expression of a specific gene in the presence or absence of tetracycline (Tet).

The tetracycline expression induction system was devised by Gossen and Bujard in 1992 (see Fig. 1) (Gossen M and Bujard H, Proc. Natl. Acad. Sci. USA 89 (12): 5547 ~ 5551, 1992), to date it has been used in the study for the regulation of various genes. The system is based on regulatory elements of E. coli's Tn10-specific tetracycline-resistant operon in which transcription of resistance-mediated genes is negatively regulated by tetracycline inhibitor (tetR). In the presence of tetracycline, tetracycline binds to tetR, causing a conformational change of tetR, which prevents tetR from binding to an effector (tetO) located within the promoter region of the operon, thereby preventing transcription. Is done. By combining tetR with the C-terminal domain of HSV-derived VP16, which is essential for the transcription of early viral genes immediately, it forms a hybrid transactivator (tTA) that stimulates the minimum promoter fused with the tetracycline effector sequence. . The promoter is substantially inactive when the concentration of tetracycline is low, which means that tetR binds to tetO and the tetracycline-regulatory transactivator binds to the tetO sequence since tetR is not sufficiently inactivated by tetracycline. Because it is suppressed.

The packaging cell line of the present invention, when a Tet is present in the tetracycline expression induction system, the regulatory unit expressed by the Tet stimulates the response unit, and the promoter is activated by the activity of the stimulated response unit. It uses a Tet-ON system that works to induce the expression of the desired gene.

In order to construct the helper virus construct in step 1), 749 to 787 in the full-length HIV-1 genome of GenBank #; AF324493 described by SEQ ID NO: 1 by precision gene fusion. Gene construct pHyPC that can act as a helper virus was removed by removing some of the packaging signals, Env from 6307 to 7755, Env from 8651 to 9709, Nef and 3 'LTR. (See Figure 2). The gene construct pHyPC is inserted into a Tet reactive plasmid to complete an expression inducible packaging cell line that induces expression of the desired gene only in the presence of Tet in the cell.

In step 2), a packaging construct is prepared by inserting the helper virus construct pHyPC constructed above into a vector having a Tet response unit (see FIG. 3). As a vector capable of expressing a Tet reaction unit, pTRE, pTet-ON, pTet-OFF, pRetro-ON, or pRetro-OFF may be used, and in the present invention, in a pHyPC capable of expressing a helper virus as a preferred embodiment, PTRE-Pg is prepared by inserting the remaining portion excluding the 3 'LTR into the pTRE vector containing the Tet reaction unit. The expression of the gene of interest inserted in the packaging construct is regulated by an inducible promoter consisting of a minimal cytomegalovirus (CMV) promoter that is linked to a tetracycline operator sequence.

In step 3), as the Tet regulatory element, regulatory elements of E. coli's Tn10-specific tetracycline resistant operon are used. The regulatory element expresses the tTA transactivator, a fusion protein composed of the tetracycline repressor and the active domain of the herpes simplex virus VP16 protein, thereby inhibiting the expression of the target gene from the inducible promotor. Can be transactivated.

If tetracycline is present when transfecting the HIV-1 packaging cell line to the cell line expressing the Tet regulatory element in step 4), tetracycline binds to the tTA transactivator protein expressed from the cell line expressing the Tet regulatory element, resulting in pHyPC. Stimulates the reaction unit of the inserted packaging construct pTRE-Pg and induces the expression of Gag, Pol and Env proteins from pHyPC. On the other hand, if tetracycline is absent, tetracycline does not bind to the tTA transactivator protein expressed from the Tet regulatory element and thus does not stimulate the reaction unit of the packaging construct pTRE-Pg with pHyPC inserted, thereby preventing Gag, Pol and Env. Expression of the protein is not induced (see FIG. 4).

As such, the Tet-induced HIV-1 packaging cell line capable of expressing the gene of interest only in the presence of tetracycline and packaging HIV-1 DI particles was named pHyPC-HD, and the Korea Biotechnology Research Institute on August 24, 2001 Deposited to the Gene Bank (Accession Number: KCTC 10054BP).

When tetracycline is treated in the HIV-1 packaging cell line constructed above, the Tet control unit stimulates the reaction unit, whereby the activated reaction unit expresses pHyPC, a helper virus gene, to produce HIV-1 DI particles. do. That is, the present invention has an advantage of enabling packaging by constructing a packaging system using a tetracycline expression induction system to remove cytotoxicity that interferes with the expression of accessory proteins essential for the expression of HIV-1 DI particles. Therefore, the method for producing HIV-1 DI particles of the present invention induces the expression of DI particles through a stable cell line expressing pHyPC, and thus can be used as an important tool for the production of targeted DI particles.

The information obtained through the production of HIV-1 DI particles using the HIV-1 packaging cell line of the present invention can be usefully used for the treatment of diseases caused by other cellular dysfunction as well as various disorders caused by HIV. .

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

Example 1 Fabrication of Packaging Construct

First, in order to construct a gene construct capable of acting as a helper virus, packaging signals 749 to 787 from the full-length HIV-1 genome sequence described in SEQ ID NO: 1, 6307 to 7755 Gene construct pHyPC was constructed by removing Env from No. 8, Env from No. 8651 to 9709, and Nef and 3 'LTR. Specifically, HIV-1 pNL4-3 (US DEPARTMENT OF HEALTH AND HUMAN SERVICES, USA) is treated with the restriction enzyme Sna B I / Sal I to process part of the packaging signal, restriction enzyme Bam H I / Sac I to treat part of the Env, Restriction enzymes Sac I / Pvu I were treated to remove portions of Env, Nef and 3 ′ LTR (FIG. 2). The helper virus construct pHyPC (7,399 bp) thus constructed includes the HIV gag, pol, tat, and vif gene open reading frames (ORFs), and the packaging signals, vpr, vpu, rev, env, nef, and 3'LTR. Mutation, deletion or destruction.

In order to prepare a Tet reactive vector by inserting the helper virus construct pHyPC constructed as described above into a plasmid containing a Tet reaction unit, pHyPC was treated with restriction enzyme Bss HII ( Clenow enzyme treatment) / Nhe I and restriction enzyme. PTRE plasmid digested with Eco RI / Xba I (3.1 kb, Clontech) was inserted. The pTRE plasmid and the Eco R I / Bss HII portion of pHyPC are filled in by ligation and the Xba I / Nhe I portion is connected through one cohesive and blunt end junction. The packaging construct pTRE-Pg was constructed (FIG. 3). At this time, a 6,688 bp sized pHyPC fragment was inserted into the multiple cloning site of the pTRE plasmid to be under the control of the tetracycline-responsive inducible promoter of the pTRE plasmid.

Example 2 Preparation of HIV-1 Packaging Cell Line

To prepare an HIV-1 packaging cell line, gene transfer by lipofectin (Lipofectin, Bodytech) was first carried out using a pUHD10-3 plasmid (Clontech) capable of expressing a tetracycline regulatory element. HeLa cell line (ATCC # CCL-2) was transfected through the method. At the time of transfection, the ratio of lipofectin and pUHD10-3 plasmid DNA was 10: 1 and the reaction was performed at 37 ° C. for 2 hours. After transfection, the cell lines were cultured in DMEM medium containing 10% fetal bovine serum, 400 μg / ml G418 to select only cell lines transfected with pUHD10-3 plasmid.

The plasmid pTK-Hygro expressing the packaging construct pTRE-Pg and the hygromycin resistance gene prepared in Example 1 to the cell line containing the selected tetracycline regulatory element in the same manner as described above Cotransfection. At this time, the plasmid pTK-Hygro was used as the backbone of pCR3 (Invitrogen), and the high fusion of TK (Thymidine Kinase) gene of Herpes simplex virus to Nhe I / Eco RI restriction enzyme site It was prepared by inserting the gromycin resistance gene. Transfected cells were selected by culturing in DMEM medium containing 200 μg / ml of hygromycin.

In order to confirm that each of the clones selected from the Tet-induced HIV-1 packaging cell line was screened for the expression of HIV-1 envelope protein by the following fusion assay (fusion assay). Specifically, the selected cells were mixed with HeLa T4 cells at a ratio of 1: 1, and then added to a 24 well plate at a concentration of 2 × 10 ⁴ total cells / well to incubate for 4 hours with or without tetracycline. It was. After that, the cells were observed under a microscope to determine the formation of synctia, and the supernatants obtained therefrom were used for reverse transcriptase activity assay.

As a result, cell clones forming multinucleated bodies with HeLa T4 cells and positive for RT were selected as Tet-induced HIV-1 packaging cell line and named pHyPC-HD. Deposited to the Engineering Research Institute Gene Bank (Accession Number: KCTC 10054BP).

Example 3 Expression of Tet Inducible HIV Protein from HIV-1 Packaging Cell Line

In order to confirm whether the Tet-induced HIV-1 packaging cell line selected above can express Gag and Env proteins required for virus replication, the following experiment was performed.

Specifically, the packaging cell line was cultured in the presence of tetracycline and then cells were treated with 1 × Lammmli buffer (125 mM Tris, 2% sodium dodecyl sulfate [SDS], 5% 2-mercaptoethanol, 10% glycerol, 0.001% Hemolysis was performed by adding bromophenol blue, pH 6.8). At this time, a packaging cell line cultured in a culture solution without tetracycline was used as a control. P24 protein isolated from each cell incubated for 24 to 48 hours with or without tetracycline was quantified using the p24 ELISA kit (Cellular Products Inc.). Specifically, 50 minutes of protein-A-Sepharose (Protein-A-Sepharose, pH 7.4, Sigma) dissolved in 100 mM / ml of 10 mM Tris-HCl was added to each isolated p24 protein for 30 minutes. The reaction was carried out at room temperature for a while and then the procedure was performed according to the manufacturer's instructions.

As a result, little p24 protein was expressed from the packaging cells cultured in the tetracycline-free culture, whereas the packaging cells cultured in the tetracycline-added culture expressed very high levels of p24 protein. From this, it was confirmed that the Tet-inducible HIV-1 packaging cell line of the present invention can regulate the expression of a gene of interest depending on the presence or absence of tetracycline.

As discussed above, the Tet-inducible HIV-1 packaging cell line of the present invention can stably express adjunct proteins in cells without producing detectable replication-capable viruses, using a tetracycline expression induction system. By regulating their expression, accessory proteins do not exhibit cytotoxicity in the cell, and thus can be usefully used for the packaging of HIV-1 DI particles. The Tet inducible HIV-1 packaging cell line of the present invention can also be applied to the development of HIV-1 vectors as a tool for delivering genes to specific cells of interest.

Claims (8)

Gene constructs capable of acting as a helper virus by removing some of the packaging signals, Env, Nef, and 3 'LTR from the full-length HIV-1 genome. The method of claim 1, In the full length HIV-1 genome described in SEQ ID NO: 1, the packaging signal portions 749 through 787, the Env portions 6307 to 7755, and the Env, Nef and 3 'LTR portions 8651 to 9709 Gene construct characterized in that the pHyPc gene construct is removed. A tetracycline reactive packaging construct in which the gene construct of claim 1 is inserted into a vector expressing a tetracycline response unit. The method of claim 3, wherein A packaging construct, wherein the vector expressing a tetracycline reaction unit is selected from the group consisting of pTRE, pTet-ON, pTet-OFF, pRetro-ON, and pRetro-OFF. The method of claim 3, wherein A packaging construct characterized in that the remaining portion of the pHyPC gene construct that can act as a helper virus, except for the 3 'LTR, is a pTRE-Pg packaging construct inserted into a pTRE vector containing a Tet response unit. The method of claim 5, gene expression of the pTRE-Pg packaging construct is regulated by an inducible promotor consisting of a minimal cytomegalovirus (CMV) promoter linked to a tetracycline operator sequence Packaging Construct. A cell line wherein the tetracycline reactive packaging construct of claim 3 is transfected to regulate expression of HIV-1 accessory protein genes by a tetracycline expression induction system. The method of claim 7, wherein HIV-1 packaging cell line pHyPC / HD, characterized in that pTRE-Pg packaging constructs are transfected to express HIV-1 DI (defective interfering) particles by expressing an accessory protein gene only in the presence of tetracycline (Accession No .: KCTC 10054BP).