JP2019135930A - METHODS AND PHARMACEUTICAL COMPOSITIONS FOR TREATING α HERPES VIRUS INFECTION - Google Patents

Abstract

To provide methods for inhibiting reactivation of latently infecting α herpes viruses in hosts.SOLUTION: Disclosed herein is a method for treating α herpes virus infection comprising introducing a first gene into a host cell of the virus, where the first gene comprises a base sequence encoding a guide RNA having a sequence specificity to UL41 gene of α herpes virus and a base sequence encoding Cas9 nuclease; and cleaving the UL41 gene in the host cell with the Cas9 nuclease in the presence of the guide RNA.SELECTED DRAWING: Figure 1

Description

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

  In general, antiviral treatment targets against viral infections are aimed at blocking the viral replication cycle and targeted by viral proteins. On the other hand, there is still no effective treatment for eradicating latently infected viruses from host cells. In a latent infection with a latent virus, the host's immunity is avoided and the virus is reactivated at any point of time, creating a persistent risk for the life of the host. An example of a latent virus is a herpes virus, which is one of the most prevalent pathogens.

  Current vaccines against alpha herpes such as porcine herpesvirus type 1 which is a pathogen of Aujeszky's disease, which is a neurological disease of swine, and chickenpox-zoster virus which is a pathogen of chicken pox are for the purpose of preventing the onset, Virus latent infection and subsequent reactivation cannot be prevented and will not lead to eradication of the disease. For example, U.S. Patent No. 6,057,049 describes compositions and methods for selectively treating viral infections using a guided nuclease system.

Special table 2017-518075 gazette

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

  In the first aspect, a first gene comprising a nucleotide sequence encoding a guide RNA having a sequence specific to the UL41 gene of α-herpesvirus and a nucleotide sequence encoding a Cas9 nuclease is introduced into a cell of a viral host. And 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 viral host, or a method of suppressing the reactivation of latent α-herpesvirus in a virus host that is latently infected with α-herpesvirus. Good. The first gene may be incorporated into a vector or a modified virus. Further, the sequence specific to the UL41 gene may include a base sequence complementary to any one of SEQ ID NOs: 2 to 11.

  The second embodiment is a guide RNA having a base sequence complementary to any one of SEQ ID NOS: 13 to 22.

  The third aspect is a nucleic acid molecule comprising any one 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 herpesvirus. The base sequence encoding the guide RNA may include any one of SEQ ID NOs: 2 to 11.

  The fifth aspect is a pharmaceutical composition comprising the nucleic acid molecule. The pharmaceutical composition may be used for the treatment of alpha herpesvirus infection.

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

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

Method for treating α herpesvirus infection The method for treating α herpesvirus infection is a first gene comprising a base sequence encoding a guide RNA having a sequence specific to the UL41 gene of α herpesvirus and a base sequence encoding a Cas9 nuclease. In a cell of a viral host, and cleaving the UL41 gene in the cell with the Cas9 nuclease in the presence of the guide RNA. By expressing the guide RNA and Cas9 nuclease in the virus host cell by the introduced first gene, for example, the UL41 gene expressed at the early stage of activation of α-herpesvirus can be selectively destroyed. Thereby, the growth of α herpesvirus in the virus host can be suppressed. In addition, reactivation of the latent α-herpesvirus in a virus host in which the α-herpesvirus is latently infected can be suppressed.

  The treatment method for α herpesvirus infection may be any treatment given to the infection, such as prevention of onset, treatment of infection, improvement and suppression of progression (prevention of deterioration), reactivation in latent infection And the like.

  Α herpesviruses to be treated are herpes simplex viruses (Human herpesvirus 1, Herpes simplex virus 1; HSV-1, Human herpesvirus 2, Herpes simplex virus 2; -Zoster virus (VZV), swine herpesvirus type 1 (Suid herpesvirus 1: Pseudorabies virus; PRV), baboon herpesvirus type 2 (Papiine herpesvirus 2), rhesus herpes virus 16 type (Cercopetic virus B) s 1, Cercopithecine herpesvirus 1), equine herpesvirus type 1 (Equid herpesvirus 1), bovine herpesvirus type 1 (Bovine herpesvirus 1), mention may be made of bovine herpes virus type 2 (Bovine Herpesvirus 2) or the like.

  The UL41 gene in α-herpesvirus is described in, for example, J. Org. Vet. Med. Sci. 72 (9): 1179-1187, 2010, etc., are well conserved among various α herpesvirus strains, and their gene products are known to inhibit host cell protein synthesis. . 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 at the early stage of reactivation. Based on this new knowledge, the inventors succeeded in developing a method for suppressing reactivation of α-herpesvirus latently infected in the host by selectively suppressing the expression of UL41 gene in the host cell. Since the UL41 gene is highly conserved among various α herpes virus strains, a highly versatile treatment method for α herpes virus infection can be realized by targeting a particularly highly homologous sequence.

  Specific examples of the base sequence of the UL41 gene include, for example, 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 other strains with respect to the UL41 gene derived from the PRV wild strain YS-81 is, for example, 93% or more in swine herpes virus, 74% or more in simian herpes virus, and in horse herpes virus 70% or more, 76% or more for bovine herpesvirus, and 70% or more for human herpesvirus. The gene homology is calculated using the BLAST program.

  In the treatment method of this embodiment, a first gene having a base sequence encoding a guide RNA having a sequence specific to the UL41 gene of α-herpesvirus and a base sequence encoding a Cas9 nuclease is introduced into a cell of a virus host. Introduce. A method of cleaving a target gene using guide RNA and Cas9 nuclease is known as a CRISPER-Cas9 system. The CRISPER-Cas9 system utilizes a short guide RNA (gRNA) and a Cas9 nuclease complexed with it to cleave DNA in a sequence-specific manner upstream of a protospacer adjacent motif (PAM) at any genomic location To do. For details of the CRISPER-Cas9 system, see, for example, Cell. Res. 23: 465-472, 2013; Nat. Biotechnol. 31: 227-229, 2013; Nucl. Acids Res. 1-11, 2013, JP-A-2006-093196, and the like can be referred to.

  Wild-type Cas9 nuclease causes double-strand breaks in at least two locations of the target gene. Thereby, for example, a small insertion defect is introduced into the target gene by non-homologous end joining (NHEJ), and the expression of the target gene UL41 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-herpesvirus 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, a Cas9 nuclease can be recruited to a specific site of the UL41 gene. The UL41 gene is cleaved in a sequence-specific manner by the recruited Cas9 nuclease, thereby suppressing 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, for example, a base sequence complementary to a base sequence of 20 bases from the protospacer adjacent motif (PAM) sequence to the 5 'end side in the base sequence of the UL41 gene can be exemplified. That is, the base sequence of 20 bases on the 3 ′ end side of the guide RNA specifically recognizes the base sequence that is the target position of the UL41 gene that is the target gene, and is located immediately before the protospacer adjacent motif (PAM). Recruit the Cas9 / guide RNA complex to the DNA target site via RNA-DNA base pairing.

  A sequence specific to the UL41 gene can be determined by searching a PAM sequence from the nucleotide sequence of the UL41 gene and selecting it, based on the selected PAM sequence. Specifically, the sequence specific to the UL41 gene can be a base sequence complementary to, for example, a base sequence of 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 in particular, the PAM located upstream of the UL41 gene (for example, about 300 bases from the 5 ′ side). It is preferred to select a sequence. Further, the PAM sequence may be selected from, for example, the base sequence of SEQ ID NO: 1 or may be selected from a base sequence complementary to the base sequence of SEQ ID NO: 1.

  Specific examples of the base sequence of the UL41 gene that is the recognition target of the sequence specific to the UL41 gene in the guide RNA include, for example, 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) Etc. 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, PRVvhs81, PRVvhs93, and PRVvhs93. .

  The guide RNA preferably includes a scaffold sequence in addition to a 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 included in the first gene, a base sequence complementary to the base sequence of the guide RNA can be used. In addition, as a base sequence encoding Cas9 nuclease, a base sequence contained in a commercially available vector or the like in which a Cas9 nuclease gene is incorporated (for example, pGuide-it-ZsGreen1 Vector (Linear): manufactured by Takara Bio Inc.) may be used. it can. The first gene can be easily constructed by, for example, incorporating a gene having a base sequence encoding a guide RNA into a vector in which a base sequence encoding Cas9 nuclease is integrated. Thereby, the 1st gene which has the base sequence which codes guide RNA and the base sequence which codes Cas9 nuclease on the same nucleic acid molecule is obtained. Further, the base sequence encoding guide RNA and the base sequence encoding Cas9 nuclease may be present on different nucleic acid molecules.

  Viral hosts into which the first gene is introduced include animals that can be infected with α herpes virus, animals that are infected with α herpes virus, animals that are latently infected with α herpes virus, and the like. In addition, mammals include mammals, and mammals include humans.

  Introduction of the first gene into the cells of the viral host can be performed by various methods known in the art including non-viral vectors and viral vectors. Non-viral vector methods include lipofection, nucleofection, microinjection, liposomes, immunoliposomes, polycations or lipid: nucleic acid conjugates, artificial virions, etc., all of which may be appropriately selected from known methods.

  As a viral vector, α herpes virus can be used in addition to a commonly used viral vector such as a retrovirus, a lentivirus, an adenovirus, and an adeno-associated virus. The method using a viral vector can be performed by constructing a modified virus in which a first gene is incorporated into a virus so that the first gene can be expressed. For example, when α-herpesvirus is used, a modified virus can be constructed by recombining the first gene in the UL41 gene region. Specifically, for example, known genetic manipulation described by Sambrook et al. (J. Sambrook et al., 1989, Molecular cloning: a laboratory manual. 2nd edition, Cold Spring Harbor, New York: Cold Spring Harbor Press). This can be done by techniques as well as by cell engineering techniques.

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

Nucleic acid molecule Another aspect of the present invention is a nucleic acid molecule comprising 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 guide RNA, and further, if necessary, such as a promoter necessary for efficiently expressing these genes. It may contain a gene group.

  The method for treating α-herpesvirus infection according to the present invention suppresses the expression of the UL41 gene of α-herpesvirus, thereby suppressing the growth of the virus, thereby preventing the onset of the initial infection and reactivating 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, reactivation of latently infected α herpesvirus can be suppressed by secondary administration.

  By the way, the method for treating α-herpesvirus infection can be carried out by applying a nuclease system other than the Crisper-Cas9 system as long as it suppresses the expression of the UL41 gene of α-herpesvirus. For example, zinc finger nuclease, transcription activator-like effector nuclease (TALEN), meganuclease and the like can be applied.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.

Cloning and nucleotide sequencing of YS-81 UL41 gene Using PRV field strain YS-81 genome as a template, 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 PCR for 45 cycles of x 1 minute and directly cloned into the pTA2 cloning vector. About the obtained vector, the Hokkaido system science company was requested to sequence and the whole base sequence was determined. The determined base sequence was the base sequence shown as SEQ ID NO: 1.

Construction of pGuide-it-ZsGreen1 vector A PAM sequence is searched based on information on the base sequence of UL41, and a UL41 gene corresponding to a sequence specific to the UL41 gene of guide RNA (sgRNA) (hereinafter also referred to as guide sequence) is detected. The following PRVvhs sequence (SEQ ID NO: 2) was selected as the base sequence. Oligo DNA having the selected base sequence was synthesized by requesting Sigma-Aldrich, and the obtained oligo DNA was cloned into pGuide-it-ZsGreen1 plasmid vector (manufactured by Takara Bio Inc.) to obtain guide RNA and Cas9 nuclease. A plasmid (pGuide-itCRISPR / Cas9 YSvhs; hereinafter simply referred to as “YSvhs”) having a base sequence to be encoded was prepared. A structural diagram of the resulting plasmid is shown in FIG.

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

Plaque suppression test 1 μg / well of the plasmid obtained above was mixed with 4 μl of jetPRIME and 100 μl of dedicated buffer, and inoculated into porcine kidney cells PK-15 seeded at 1 × 10 ⁶ cells in a ^6-well plate to introduce the plasmid. . After culturing at 37 ° C. under 5% CO ₂ for 24 hours, YS-81 100PFU / well was inoculated, and the number of plaques that appeared after staining with 10% formalin solution with crystal violet added after 5 days of culture was counted. As a control, pGuide-it-ZsGreen1 plasmid not incorporating a guide sequence was used. The results are shown in the table below. In addition, it means that the suppression effect with respect to YS-81 is so large that the number of plaques is small.

  From Table 8, it can be seen that introduction of a plasmid having a base sequence encoding guide RNA and Cas9 nuclease into porcine kidney cell PK-15 suppresses the growth of YS-81.

Preparation of latent infection model mouse A 4-week-old Balb / c mouse was intraperitoneally administered with 0.25 ml of porcine antiviral serum diluted with PBS (-) equivalent to 1: 128. Thirty minutes later, 0.5 ml of the field virus YS-81 diluted to the equivalent of 10 ² LD50 with PBS (−) was administered intraperitoneally and attacked. Mice that survived more than 2 months from the challenge were used as latent infection model mice.

Inhibition test of latent PRV reactivation by intraventricular administration 1 μg of plasmid (pGuide-itCRISPR / Cas9 YSvhs) was mixed with TransIT-Neural 100 μl and inoculated into the brain of latent infection model mice. After 24 hours, latent virus was reactivated by intraperitoneal inoculation with acetylcholine chloride. As described below, nasal swabs were collected daily and viral DNA was detected by PCR.

  For nasal swabs, 100 μl of MEM culture solution was allowed to flow from the right nostril using a micropipette under three-kind anesthesia (medetomidine 0.3 mg / kg, midazolam 4 mg / kg, butorphanol 5 mg / kg), and the washing solution was collected with a cotton swab. Then, it was mixed with 300 μl of MEM culture solution to obtain a nasal swab. The sample of the obtained nasal swab was mixed with twice the amount of ISOGEN (LS) (manufactured by Wako Pure Chemical Industries, Ltd.), and DNA was extracted according to the protocol of the kit.

  For detection of viral DNA, 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 was carried out for 30 cycles of 94 ° C. × 2 minutes, 94 ° C. × 1 minute, 56 ° C. × 1 minute, and 72 ° C. × 2 minutes, and extended at 72 ° C. × 7 minutes. . The presence or absence of a 531 bp specific band was confirmed by electrophoresis using 1.0% agarose. The results are shown in Table 10. In the table, Mock means a Balb / c mouse that is not latently infected.

From Table 10, it can be seen that by inoculating a plasmid encoding guide RNA and Cas9 nuclease into the brain, the virus is not excreted even when a reactivation stimulus is given to a latently infected model mouse. That is, it can be seen that plasmid inoculation suppresses virus reactivation in a latently infected model mouse. The result of the χ ² test was p = 0.000513.

Claims (12)

introducing a first gene comprising a base sequence encoding a guide RNA having a sequence specific to the UL41 gene of α-herpesvirus and a base sequence encoding a Cas9 nuclease into a cell of a viral host;
Cleaving the intracellular UL41 gene with the Cas9 nuclease in the presence of the guide RNA;
A method of treating alpha herpesvirus infection.   The method according to claim 1, wherein the method inhibits the growth of α-herpesvirus in the viral host.   The method according to claim 1 or 2, wherein the viral host is an animal latently infected with α-herpesvirus and suppresses reactivation of the latent α-herpesvirus.   The method according to any one of claims 1 to 3, wherein the first gene is incorporated 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 nucleotide sequence complementary to any nucleotide sequence of SEQ ID NOs: 2 to 11.   A guide RNA having a base sequence complementary to any base sequence of SEQ ID NOs: 13 to 22.   A nucleic acid molecule having any one of the nucleotide sequences of SEQ ID NOS: 13 to 22.   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-herpesvirus.   The nucleic acid molecule according to claim 9, wherein the base sequence encoding the guide RNA comprises any one of SEQ ID NOs: 2 to 11.   A pharmaceutical composition comprising the nucleic acid molecule according to claim 9 or 10.   The pharmaceutical composition according to claim 11, which is used for treatment of alpha herpesvirus infection.