JP7462863B2 - Method for propagating recombinant measles virus and host cells - Google Patents

Description

The present invention relates to a method for propagating a recombinant measles virus and a host cell for propagating the recombinant measles virus.

Measles virus (MeV) is a highly pathogenic virus that belongs to the genus Morbilivirus and family Paramyxoviridae. It infects humans as the natural host and causes immunosuppression and respiratory symptoms.

The measles virus is one of the most researched viruses, and a wide range of knowledge has been accumulated regarding its virological properties and pathological expression mechanisms. Currently, it is expected that the characteristics of the measles virus can be utilized for applications in fields such as genetic engineering and medicine. In fact, clinical research is already being conducted on the measles virus as an oncolytic virus. Furthermore, because it is possible to express a large number of foreign genes and target expression only in specific cells, it is also used as a gene transfer vector.

For example, since it has been shown that the measles virus infects tumor cells and induces tumor regression (Non-Patent Document 1), the measles virus has attracted attention as a tool for viral cancer therapy. To date, clinical research on viral therapy using measles virus based on vaccine strains has been conducted on ovarian cancer and myeloma (Non-Patent Document 2).

On the other hand, measles viruses generally have a low ability to produce infectious particles, making it difficult to obtain high titers of the virus. Furthermore, various modifications are made to the viral genome depending on the application, so there are concerns that the virus's proliferation ability will be reduced compared to wild-type viruses. In particular, attenuated viruses are used for medical applications, so improving the production cells to obtain high titers of the virus is important.

Grote et al., Blood 97:3746-3754 2001. Galanis et al., Cancer research 70:875-882 2010.

The present invention has been made in consideration of these problems, and aims to provide a method for propagating a recombinant measles virus that can efficiently produce a high-titer recombinant measles virus, and a host cell for propagating the recombinant measles virus.

The method for propagating a recombinant measles virus according to the present invention is characterized by using a host cell in which RPAP3 has been knocked down or knocked out.

The method for propagating a recombinant measles virus according to the present invention is characterized by comprising a receptor molecule expression step of causing a host cell to express human SLAM, a receptor molecule necessary for measles virus infection, a modification step of knocking down or knocking out RPAP3 in a host cell expressing human SLAM, and a culture step of culturing the recombinant measles virus using a host cell in which RPAP3 has been knocked down.

The host cells for propagating the recombinant measles virus of the present invention are host cells in which RPAP3 has been knocked down or knocked out.

The host cell of the present invention is characterized in that it expresses human SLAM, a receptor molecule necessary for measles virus infection, and that it is used to propagate a recombinant measles virus in which RPAP3 has been knocked down or knocked out.

According to the present invention, high-titer recombinant measles virus can be efficiently produced.

FIG. 1 is a diagram illustrating the proteins that constitute the R2TP complex. FIG. 1 shows that increased intracellular viral RNA is observed in RPAP3 knockdown cells compared to control cells. FIG. 1 shows that measles virus was detected at titers approximately 10-fold higher in RPAP3 knockdown cells than in control cells.

The following describes in detail an embodiment of the present invention with reference to the attached drawings. However, this embodiment is intended to facilitate understanding of the principles of the present invention, and the scope of the present invention is not limited to the embodiment described below. Other embodiments in which a person skilled in the art appropriately replaces the configuration of the embodiment described below are also included in the scope of the present invention.

The method for propagating a recombinant measles virus according to the present invention uses host cells in which RPAP3 has been knocked down or knocked out.

The method for propagating a recombinant measles virus according to the present invention comprises a receptor molecule expression step of causing a host cell to express human SLAM, a receptor molecule necessary for measles virus infection, a modification step of knocking down or knocking out RPAP3 in the host cell expressing this human SLAM, and a culture step of culturing the recombinant measles virus using the host cell in which RPAP3 has been knocked down.

The host cell of the present invention is a host cell for propagating a recombinant measles virus in which RPAP3 has been knocked down or knocked out.

The host cells of the present invention are host cells used to grow recombinant measles viruses, in which human SLAM, a receptor molecule necessary for measles virus infection, is expressed and RPAP3 is knocked down or knocked out.

As shown in Figure 1, the R2TP complex is a protein complex formed by four different proteins, RuvBL1-RuvBL2-RPAP3-PIH1D1. The inventors have newly discovered that knocking down or knocking out RPAP3 (RNA polymerase-associated protein 3), which is a component of the host factor R2TP complex, increases the amount of measles virus RNA and produces a virus with a titer approximately 10 times higher. Based on this fact, the present invention has been completed.

Host cells are not particularly limited as long as they are capable of propagating the recombinant measles virus, and examples include bacterial cells (e.g., Streptococcus, Staphylococcus, Escherichia coli, Streptomyces, Bacillus subtilis), insect cells (e.g., Drosophila S2, Spodoptera SF9), animal cells (e.g., Vero, CEF, A549, CHO, COS, HeLa, C127, 3T3, BHK, HEK293, Bowes melanoma cells), and plant cells.

The recombinant measles virus to be propagated in the present invention is not particularly limited, and examples thereof include IC323-EGFP, MV-dF-OSKL-EGFP, MV3F, MV4FN, 2 seg-MV (Takeda M et al, J. Virol., 80: 4242-8, 2006), SI-AcGFP (Seki F et al, J. Virol., 85: 11871-82, 2011), and AIK-C (Nakayama T et al, J. Gen. Virol., 82: 2143-50, 2001).

The method for knocking down or knocking out RPAP3 is not particularly limited, but examples include a method using siRNA or shRNA targeting RPAP3. Expression suppression using siRNA suppresses gene expression by introducing artificially synthesized siRNA into cells, but the effect is temporary, for example, about one week. On the other hand, a method for expressing siRNA in cells in the form of shRNA using a vector takes time to establish due to drug selection, etc., but results in cells with RPAP3 permanently knocked down. For knockout, genome editing using the CRISPR-Cas9 system is also possible. In the CRISPR-Cas9 system, a guide RNA (gRNA) is designed for the target gene, and a gRNA expression vector and a Cas9 expression vector are introduced simultaneously into the cells, and knockout cells are established by drug selection.

The present inventors have demonstrated that the proliferation of mumps virus is suppressed in host cells in which RPAP3 is knocked down, and that the R2TP complex is a co-chaperone of Hsp90, which is important for the proliferation of mumps virus (The Role of the R2TP Complex in Mumps Virus Infection, 7th Negative Strand Virus-Japan, Kato, Hiroshi et al.). On the other hand, in the present invention, the proliferation of recombinant measles virus is improved in host cells in which RPAP3 is knocked down. This is presumably due to the following reasons. That is, RPAP3 is a factor that negatively controls the RNA synthesis of paramyxoviruses (including measles virus and mumps virus), and when RPAP3 is knocked down, an increase in the amount of viral RNA is observed in both viral infections. However, in mumps virus infection, the excessive increase in viral RNA induces the host's natural immune response, and as a result, viral proliferation is suppressed. On the other hand, in measles virus infection, the proliferation inhibition seen in mumps virus infection is not observed. Measles virus has an accessory protein called C protein that suppresses innate immunity, which is not found in mumps virus, and this is thought to be due to differences in resistance to innate immunity between paramyxovirus species. This method of using host cells with RPAP3 knockdown to grow recombinant measles virus is completely different from conventional techniques.

(1) Introduction of siRNA
A549/hSLAM cells (Takeda M et al., J. Virol. 74: 6643-7, 2005) were used, which were A549 cells (derived from human alveolar basal epithelial adenocarcinoma) expressing human SLAM (signaling lymphocyte-activation molecule), a receptor molecule required for measles virus infection. RPAP3-targeting siRNA (5'-uugaaggauaguucugucgaa-3' (SEQ ID NO: 1), Yoshida M et al., Biochem. Biophys. Res. Commun. 430: 320-4, 2013) was transfected into A549/hSLAM cells using Lipofectamine RNAiMax (Thermo Fisher Scientific). Cells transfected with a non-targeting siRNA pool (Dharmacon) were used as negative controls.

(2) Cell culture
48 hours after siRNA introduction, the cells were washed once with serum-free medium and inoculated with a recombinant measles virus (IC323-EGFP) solution at an MOI of 0.05. After incubation at 37°C for 1 hour, the cells were washed three times with serum-free medium and cultured in 10% serum-containing medium.

(3) Measurement of viral RNA quantity and viral titer
Total RNA was extracted from recombinant measles virus-infected cells 0, 24, 48, and 72 hours later using the RNeasy Mini Kit (Qiagen). Reverse transcription was performed at 37°C for 15 minutes using oligo(dT) primer and PrimeScript RTase (TaKaRa bio). Quantitative PCR (qPCR) was performed using the LightCycler 480 system (Roche). The N gene of measles virus was detected using Universal ProbeLibrary Probe #85 (Roche) and primers (5'-tcacatgatgatccaagcagt-3' (sequence number 2) and 5'-tttccttgttctcgaaccatc-3' (sequence number 3), unpublished). In addition, the hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene was detected as an internal standard gene using Universal ProbeLibrary Probe #62 (Roche) and primers (5'-gggaggccatcacattgtag-3' (sequence number 4) and 5'-cactatttctattcagtgtttga-3' (sequence number 5) (Katoh H et al., J. Virol. 91: e02220-16, 2017). The amount of measles virus RNA was calculated by relative quantification based on the expression level of the internal standard gene. As a result, an increase in intracellular viral RNA was observed in RPAP3 knockdown cells compared to control cells (Figure 2).

After 0, 24, 48, 72, and 96 hours, the culture supernatant of recombinant measles virus-infected cells was collected, and the infectious virus titer in the culture supernatant was calculated by the focus method using hSLAM-expressing Vero cells (Vero/hSLAM cells, Ono N et al, J. Virol. 75: 4399-401, 2001). Vero/hSLAM cells were washed once with serum-free medium, and the Vero/hSLAM cells were infected with 10-fold serially diluted culture supernatant. After 1 hour of incubation at 37°C, the cells were cultured in 0.5% agarose-containing 5% serum-containing medium. After 72 hours of culture, the number of foci was measured using the EGFP signal as an indicator, and the virus titer was calculated. As a result, when RPAP3 was knocked down, measles virus was detected in the culture supernatant 72 and 96 hours after infection at a titer approximately 10 times higher than that of control cells (Figure 3).

It can be used in regenerative medicine and gene therapy.

SEQ ID NO: 1: siRNA
SEQ ID NOs: 2 to 5: Primers

Claims (2)

A method for propagating a recombinant measles virus, comprising using a host cell in which RPAP3 has been knocked down or knocked out. a receptor molecule expression step of expressing a receptor molecule, human SLAM, necessary for measles virus infection in a host cell;
A modification step of knocking down or knocking out RPAP3 in a host cell in which human SLAM is expressed;
a culturing step of culturing a recombinant measles virus using the host cell in which RPAP3 has been knocked down;
A method for propagating a recombinant measles virus, comprising the steps of :