JP7075653B2 - Methods and Pharmaceutical Compositions for Treating α-Herpesvirus Infections - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Law Abstracts of the 160th Annual Meeting of the Japanese Society of Veterinary Medicine, p. 377

The present invention relates to methods and pharmaceutical compositions for treating α-herpesvirus infections.

In general, antiviral treatment targets for viral infections are targeted at viral proteins with the aim of blocking the viral replication cycle. On the other hand, there is still no effective treatment to eradicate the latently infected virus from the host cell. In latent infection with a latent virus, the host's immune system is evaded and the virus is reactivated at any time, which poses a lasting risk for the host's life. An example of a latent virus is the herpes virus, which is one of the most prevalent pathogens.

Current vaccines against α-herpes, such as porcine herpesvirus type 1 which is the pathogen of Oeski's disease, which is a neurological disease in pigs, and chickenpox-zoster virus, which is the pathogen of chickenpox, are all aimed at preventing the onset. It cannot prevent latent infection of the virus and subsequent reactivation, and does not lead to eradication of the disease. For example, Patent Document 1 describes compositions and methods for selectively treating viral infections using a guided nuclease system.

Special Table 2017-518575 Publication No.

However, with the composition and method described in Patent Document 1, it is difficult to completely prevent the latent infection of the herpes virus, and once the latent infection is established, it is difficult to suppress the reactivation of the virus. One aspect of the present invention is to provide a method capable of suppressing the reactivation of α-herpesvirus latently infected in a host.

The first aspect is to introduce a first gene containing a base sequence encoding a guide RNA having a sequence specific to the UL41 gene of α-herpes virus and a base sequence encoding Cas9 nuclease into the cells of the virus host. And a method of treating an α-herpesvirus infection comprising cleaving the UL41 gene in the cell with the Cas9 nuclease in the presence of the guide RNA.

The method may be a method of suppressing the growth of α-herpesvirus in a virus host, or a method of suppressing reactivation of latent α-herpesvirus in a virus host in which α-herpesvirus is latently infected. good. Further, the first gene may be integrated into a vector or may be integrated into a modified virus. Further, the sequence specific to the UL41 gene may contain a base sequence complementary to any of the base sequences of SEQ ID NOs: 2 to 11.

The second aspect is a guide RNA having a base sequence complementary to any of the base sequences of SEQ ID NOs: 13 to 22.

The third aspect is a nucleic acid molecule containing any of the nucleotide sequences of SEQ ID NOs: 13 to 22.

The fourth aspect is a nucleic acid molecule having a base sequence encoding Cas9 nuclease and a base sequence encoding a guide RNA having a sequence specific to the UL41 gene of α-herpes virus. The base sequence encoding the guide RNA may contain any of the base sequences of SEQ ID NOs: 2 to 11.

A fifth aspect is a pharmaceutical composition containing the nucleic acid molecule. The pharmaceutical composition may be used in the treatment of α-herpesvirus infection.

According to one aspect of the present invention, it is possible to provide a method capable of suppressing the reactivation of α-herpesvirus latently infected in a host.

It is a schematic diagram which shows the structural example of a plasmid.

Treatment method for α-herpes virus infection The treatment method for α-herpes virus infection is a first gene containing a base sequence encoding a guide RNA having a sequence specific to the UL41 gene of α-herpes virus and a base sequence encoding Cas9 nuclease. Is introduced into the cell of the viral host and the UL41 gene in the cell is cleaved with the Cas9 nuclease in the presence of the guide RNA. By expressing the guide RNA and Cas9 nuclease in the cells of the viral host by the first gene introduced, for example, the UL41 gene expressed in the early stage of activation of α-herpesvirus can be selectively destroyed. This makes it possible to suppress the growth of α-herpesvirus in the virus host. In addition, it is possible to suppress the reactivation of the latent α-herpes virus in the virus host in which the α-herpes virus is latently infected.

The treatment method for α-herpesvirus infection may be any treatment applied to the infection, for example, prevention of onset, treatment of infectious disease, improvement and suppression of progression (prevention of deterioration), reactivation in latent infection. Examples include suppression of infection.

The α-herpesviruses to be treated include simple herpesviruses (Human herpesvirus 1, Herpes simplex virus 1; HSV-1, Human herpesvirus 2, Herpes simplex virus 2, herpesviruses virus 2; HSV-2). -Zoster herpes (VZV), porcine herpesvirus type 1 (Suid herpesvirus 1: Pseudorabies virus; PRV), hihiherpesvirus type 2 (Papine herpesvirus 2), onagasaru herpesvirus type 16 (Cerpes) 1, Cercopisecine herpesvirus 1), horse herpesvirus type 1 (Equid herpesvirus 1), bovine herpesvirus type 1 (Bovine herpesvirus 1), bovine herpesvirus type 2 (Bovine herpes 1), etc. can be mentioned.

The UL41 gene in α-herpesvirus is described in, for example, J. Vet. Med. Sci. 72 (9): As described in 1179-1187, 2010, etc., it is well conserved among various α-herpesvirus strains, and its gene product is known to inhibit protein synthesis in host cells. .. As a result of analyzing the reactivation mechanism in latent infection of PRV, which is a kind of α-herpesvirus, the present inventors have found that the UL41 gene is expressed in the early stage of reactivation. Based on this new finding, we have succeeded in developing a method for suppressing the reactivation of α-herpesvirus latently infected in the host by selectively suppressing the expression of the UL41 gene in the host cell. Since the UL41 gene is highly conserved among various α-herpesvirus strains, a highly versatile treatment method for α-herpesvirus infection can be realized by targeting a sequence having particularly high homology.

Specific examples of the base sequence of the UL41 gene include the base sequence of the UL41 gene (SEQ ID NO: 1) derived from the PRV wild strain YS-81.

The homology of the UL41 gene of another strain to the UL41 gene derived from the PRV wild strain YS-81 is, for example, 93% or more in porcine herpesvirus, 74% or more in salherpesvirus, and 74% or more in horse herpesvirus. It shows 70% or more, 76% or more in bovine herpesvirus, and 70% or more in human herpesvirus. The gene homology is calculated using the BLAST program.

In the treatment method of the present embodiment, a first gene having a base sequence encoding a guide RNA having a sequence specific to the UL41 gene of α-herpes virus and a base sequence encoding Cas9 nuclease is placed in the cells of the virus host. Introduce. A method of cleaving a target gene using a guide RNA and Cas9 nuclease is known as the CRISPR-Cas9 system. The CRISPR-Cas9 system utilizes a short guide RNA (gRNA) and a Cas9 nuclease complexed with it to cleave DNA sequence-specifically upstream of the protospacer flanking motif (PAM) at any genomic position. do. For more information on the CRISPR-Cas9 system, see, eg, Cell. Res. 23: 465-472, 2013; Nat. Biotechnol. 31: 227-229, 2013; Nucl. Acids Res. 1-11, 2013, Japanese Patent Application Laid-Open No. 2016-093196 and the like can be referred to.

The wild-type Cas9 nuclease causes double-strand breaks at at least two sites in the target gene. As a result, for example, a small insertion defect can be introduced into the target gene by non-homologous end binding (NHEJ), and the expression of the UL41 gene, which is the target gene, can be suppressed. The Cas9 nuclease may be a wild type or a mutant type.

The guide RNA has a base sequence specific to the UL41 gene of α-herpes virus, and a base sequence necessary for recruiting Cas9 nuclease, for example, a base sequence having a scaffold function. Since the guide RNA has a base sequence specific to the UL41 gene, Cas9 nuclease can be recruited to a specific site of the UL41 gene. The recruited Cas9 nuclease disrupts the UL41 gene in a sequence-specific manner and suppresses the expression of the UL41 gene.

The sequence specific to the UL41 gene is a base sequence complementary to the corresponding base sequence of the UL41 gene. Specifically, a base sequence complementary to the base sequence of, for example, 20 bases from the protospacer adjacent motif (PAM) sequence to the 5'end side in the base sequence of the UL41 gene can be mentioned. That is, the base sequence of 20 bases on the 3'end side of the guide RNA specifically recognizes the base sequence at the target position of the UL41 gene, which is the target gene, and is located immediately before the protospacer adjacent motif (PAM). The Cas9 / guide RNA complex is recruited to the DNA target site via RNA-DNA sequence formation.

The sequence specific to the UL41 gene can be determined based on the selected PAM sequence by searching and selecting the PAM sequence from the base sequence of the UL41 gene. Specifically, the sequence specific to the UL41 gene can be a base sequence complementary to the base sequence of, for example, 20 bases from the selected PAM sequence to the 5'end side. As the PAM sequence to be selected, any PAM sequence that can suppress the expression of the UL41 gene may be selected, and among them, the PAM located on the upstream side of the UL41 gene (for example, around 300 bases from the 5'side). It is preferable to select the sequence. Further, the PAM sequence may be selected from, for example, the base sequence of SEQ ID NO: 1 or may be selected from the base sequence complementary to the base sequence of SEQ ID NO: 1.

Specific examples of the base sequence of the UL41 gene to be recognized for the UL41 gene-specific sequence in the guide RNA include PRVvhs (SEQ ID NO: 2), PRVvhs11 (SEQ ID NO: 3), and PRVvhs26 (SEQ ID NO: 4) shown in the table below. , PRVvhs34 (SEQ ID NO: 5), PRVvhs41 (SEQ ID NO: 6), PRVvhs51 (SEQ ID NO: 7), PRVvhs61 (SEQ ID NO: 8), PRVvhs81 (SEQ ID NO: 9), PRVvhs93 (SEQ ID NO: 10), PRVvhs103 (SEQ ID NO: 11). And so on. Among them, at least one selected from the group consisting of PRVvhs, PRVvhs34, PRVvhs41, PRVvhs61, PRVvhs81, PRVvhs93, and PRVvhs103 is preferable, and at least one selected from the group consisting of PRVvhs, PRVvhs34, PRVvhs61, PRVvhs61, PRVvhs81, and PRVvhs93. ..

The guide RNA preferably contains a scaffold sequence in addition to the base sequence specific to the UL41 gene. Examples of the base sequence encoding the scaffold sequence include the following base sequences.

Therefore, specific examples of the base sequence encoding the guide RNA include the base sequences shown below.

As the base sequence encoding the guide RNA contained in the first gene, a base sequence complementary to the base sequence of the guide RNA can be used. As the base sequence encoding Cas9 nuclease, a base sequence contained in a commercially available vector or the like in which the Cas9 nuclease gene is incorporated (for example, pGuide-it-ZsGreen1 Vector (Linear): manufactured by Takara Bio Inc., etc.) can be used. can. The first gene can be easily constructed, for example, by incorporating a gene having a base sequence encoding a guide RNA into a vector in which a base sequence encoding Cas9 nuclease is incorporated. This gives a first gene having a base sequence encoding a guide RNA and a base sequence encoding a Cas9 nuclease on the same nucleic acid molecule. Further, the base sequence encoding the guide RNA and the base sequence encoding the Cas9 nuclease may be present on different nucleic acid molecules.

The virus host into which the first gene is introduced includes animals that can be infected with α-herpes virus, animals that are infected with α-herpes virus, and animals that are latently infected with α-herpes virus. In addition, animals include mammals, and mammals include humans.

The introduction of the first gene into the cells of a viral host can be performed by a variety of methods known in the art, including non-viral and viral vectors. Examples of the non-viral vector method include lipofection, nucleofection, microinjection, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, artificial virions and the like, all of which may be appropriately selected from known methods.

As the virus vector, α-herpes virus can be used in addition to commonly used virus vectors such as retrovirus, lentivirus, adenovirus, and adeno-associated virus. The method using a viral vector can be carried out by constructing a modified virus in which the first gene is expressively incorporated into the virus. For example, when α-herpes virus is used, a modified virus can be constructed by recombining the first gene in the region of the UL41 gene. Specifically, for example, known genetic manipulations described by Sambrook et al., 1989, Molecular cloning: a laboratory manual. 2nd edition, Cold Spring Harbor, New York: Cold Spring Harbor Press. It can be done by technology as well as cell engineering technology.

Guide RNA
Another aspect of the invention is a guide RNA used in a method of treating an alpha virus infection. The guide RNA is composed of a base sequence specific to the UL41 gene of α-herpes virus and a base sequence that enables recruitment of Cas9 nuclease to a target site. Specific examples of the guide RNA include a nucleic acid molecule (for example, RNA) encoded by any of the base sequences of SEQ ID NOs: 13 to 22.

Nucleic Acid Molecular Another embodiment of the present invention is a nucleic acid molecule containing a base sequence encoding the guide RNA. The nucleic acid molecule may contain a base sequence encoding Cas9 nuclease in addition to the base sequence encoding the guide RNA, and if necessary, a promoter or the like necessary for efficiently expressing these genes. It may contain a group of genes.

The method for treating α-herpesvirus infection according to the present invention also suppresses the growth of the virus by suppressing the expression of the UL41 gene of the α-herpesvirus, and thereby suppresses the onset of the virus in the initial infection and reactivates the latent virus. It has the advantage of suppressing. The non-viral vector or viral vector used in this method can suppress the onset of α-herpesvirus infection by primary administration. Furthermore, the reactivation of the latently infected α-herpes virus can be suppressed by the secondary administration.

By the way, the method for treating α-herpes virus infection can be carried out by applying a nuclease system other than the CRISPR-Cas9 system as long as it is a method for suppressing the expression of the UL41 gene of α-herpes virus. For example, zinc finger nucleases, transcriptional activator-like effector nucleases (TALENs), meganucleases, and the like can also be applied.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to these Examples.

Cloning and nucleotide sequence determination of YS-81 UL41 gene Using the PRV field strain YS-81 genome as a template and the primers shown below (SEQ ID NOs: 23 and 24), 94 ° C. × 30 seconds, 55 ° C. × 30 seconds, 72 ° C. The UL41 gene was amplified by 45 cycles of PCR for 1 minute and directly cloned into the pTA2 cloning vector. For the obtained vector, we requested a sequence from Hokkaido System Science Co., Ltd. and determined the entire base sequence. The determined base sequence was the base sequence shown as SEQ ID NO: 1.

Construction of pGuide-it-ZsGreen1 vector The PAM sequence is searched based on the information of the base sequence of UL41, and the UL41 gene corresponding to the UL41 gene-specific sequence of the guide RNA (sgRNA) (hereinafter, also referred to as the guide sequence). The following PRVvhs sequence (SEQ ID NO: 2) was selected as the base sequence. An oligo DNA having a selected base sequence was commissioned to Sigma-Aldrich to synthesize it, and the obtained oligo DNA was cloned into a pGuide-it-ZsGreen1 plasmid vector (manufactured by Takara Bio) to obtain a guide RNA and Cas9 nuclease. A plasmid having the encoding base sequence (pGuide-it CRISPR / Cas9 YSvhs; hereinafter, also simply referred to as "YSvhs") was prepared. The block diagram of the obtained plasmid is shown in FIG.

Similarly, plasmids (YSvhs11 to YSvhs103) having the base sequences (SEQ ID NOs: 3 to 11) shown in the table below as the base sequence of the UL41 gene corresponding to the guide sequence were prepared.

Plaque suppression test 1 μg / hole of the plasmid obtained above was mixed with 4 μl of jetPRIME and 100 μl of a dedicated buffer, and the plasmid was introduced into a 6-well plate inoculated into pig kidney cells PK-15 seeded with 1 × 10 ⁶ cells. .. After culturing at 37 ° C. under 5% CO ₂ for 24 hours, YS-81 100PFU / hole was inoculated, and 5 days after culturing, the number of plaques appearing by staining with a 10% formalin solution containing crystal violet was counted. As a control, a pGuide-it-ZsGreen1 plasmid in which the guide sequence was not incorporated was used. The results are shown in the table below. It should be noted that the smaller the number of plaques, the greater the inhibitory effect on YS-81.

From Table 8, it can be seen that the proliferation of YS-81 is suppressed by introducing a plasmid having a base sequence encoding the guide RNA and Cas9 nuclease into the pig kidney cell PK-15.

Preparation of Latent Infection Model Mice A 4-week-old Balb / c mouse was intraperitoneally administered with 0.25 ml of porcine antiviral serum diluted 1: 128 using PBS (-). Thirty minutes later, 0.5 ml of the field virus YS ^- 81 diluted with PBS (-) equivalent to 102 LD50 was intraperitoneally administered to attack. Mice that survived the attack for more than 2 months were used as latent infection model mice.

Intraventricular administration of latent PRV reactivation suppression test In the brain of latent infection model mice, 1 μg of plasmid (pGuide-itCRISPR / Cas9 YSvhs) was mixed with 100 μl of TransIT-Neural and inoculated. Twenty-four hours later, the latent virus was reactivated by intraperitoneal inoculation with acetylcholine chloride. As follows, nasal swabs were collected day after day and viral DNA was detected by PCR.

For the nasal swab, 100 μl of MEM culture solution is flowed from the right nostril through the right nostril under triple anesthesia (medetomidin 0.3 mg / kg, midazolam 4 mg / kg, butorphanol 5 mg / kg), and the washing solution is collected with a cotton swab. Then, it was mixed with 300 μl of MEM culture solution, and this was used as a nasal swab. The obtained nasal swab sample was mixed with a double amount of ISOGEN (LS) (manufactured by Wako Pure Chemical Industries, Ltd.), and DNA was extracted according to the protocol of the kit.

To detect viral DNA, a PCR reaction was performed on the gG gene using the extracted DNA as a template. Using the primers shown in the table below, the program performed 30 cycles of 94 ° C x 2 minutes, 94 ° C x 1 minute, 56 ° C x 1 minute, and 72 ° C x 2 minutes, and extended at 72 ° C x 7 minutes. .. The presence or absence of a specific band of 531 bp was confirmed for the amplified product by electrophoresis using 1.0% agarose. The results are shown in Table 10. In addition, Mock in the table means a Balb / c mouse which is not latently infected.

From Table 10, it can be seen that by inoculating the plasmid encoding the guide RNA and Cas9 nuclease into the brain, the virus is not excreted even if the latent infection model mouse is given a reactivation stimulus. That is, it can be seen that plasmid inoculation suppresses virus reactivation in latent infection model mice. The result of the χ ² test was p = 0.005613.

Claims (12)

Introducing a first gene containing a base sequence encoding a guide RNA having a sequence specific to the UL41 gene of α-herpes virus and a base sequence encoding Cas9 nuclease into cells of a viral host other than humans .
By cleaving the UL41 gene in the cell with the Cas9 nuclease in the presence of the guide RNA,
Including
A method for treating an α-herpesvirus infection, wherein the sequence specific to the UL41 gene comprises a base sequence complementary to the base sequence of any of SEQ ID NOs: 2, 5, 6 and 8 to 11 . The method according to claim 1, wherein the growth of α-herpesvirus in the virus host is suppressed. The method according to claim 1 or 2, wherein the virus host is an animal that is latently infected with α-herpes virus and suppresses reactivation of the latent α-herpes virus. The method according to any one of claims 1 to 3, wherein the first gene is integrated into a vector. The method according to any one of claims 1 to 4, wherein the first gene is incorporated into a modified virus. The method according to any one of claims 1 to 5, wherein the sequence specific to the UL41 gene comprises a base sequence complementary to any of the base sequences of SEQ ID NOs: 2, 5 and 8 to 11 . A guide RNA having a base sequence complementary to any of the base sequences of SEQ ID NOs: 13, 16, 17 and 19 to 22 . The guide RNA according to claim 7, which has a base sequence complementary to any of the base sequences of SEQ ID NOs: 13, 16 and 19 to 22 . It has a base sequence encoding Cas9 nuclease and a base sequence encoding a guide RNA having a sequence specific to the UL41 gene of α-herpes virus.
A nucleic acid molecule in which the base sequence encoding the guide RNA has a base sequence complementary to any of the base sequences of SEQ ID NOs: 2, 5, 6 and 8 to 11 . The nucleic acid molecule according to claim 9, wherein the base sequence encoding the guide RNA includes any of the base sequences of SEQ ID NOs: 2, 5 and 8 to 11 . A pharmaceutical composition comprising the nucleic acid molecule of claim 9 or 10. The pharmaceutical composition according to claim 11, which is used for treating an α-herpesvirus infection.

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