WO2024011980A1 - 一种用于单纯疱疹病毒感染性疾病的基因治疗药物 - Google Patents

一种用于单纯疱疹病毒感染性疾病的基因治疗药物 Download PDF

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WO2024011980A1
WO2024011980A1 PCT/CN2023/088908 CN2023088908W WO2024011980A1 WO 2024011980 A1 WO2024011980 A1 WO 2024011980A1 CN 2023088908 W CN2023088908 W CN 2023088908W WO 2024011980 A1 WO2024011980 A1 WO 2024011980A1
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grna
crispr
hsv
vlp
plasmid
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凌思凯
汪啸渊
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上海本导基因技术有限公司
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the invention belongs to the field of genetic medicine and relates to a gene therapy medicine for herpes simplex virus infectious diseases.
  • Herpes simplex virus belongs to the ⁇ subfamily of the human herpesviridae family and is an enveloped spherical virus. Divided into herpes simplex virus type I (Herpes simplex virus I, HSV-1) and herpes simplex virus type II (Herpes simplex virus II, HSV-2). They are all pathogenic viruses that can cause herpetic encephalitis, herpetic keratitis, genital herpes or neonatal encephalitis. There is currently no cure and no vaccine to prevent infection.
  • Herpes simplex keratitis is an infectious corneal disease caused by HSV-1, which can cause blindness in severe cases.
  • HSV-1 contains approximately 152-kb double-stranded linear DNA and has multiple subtypes.
  • HSV-1 infection can cause a variety of diseases. Among them, infection of the cornea causes viral keratitis, which is the main cause of blindness caused by infectious corneal diseases in the world. Humans are the only natural host of this virus. Human infection with HSV-1 is very common in nature. According to statistics, 50%-80% of people in the world are carriers of HSV-1, and 1.5 million new people are infected every year. Among patients with keratitis caused by HSV virus, 40,000 patients have visual impairment or blindness.
  • HSV-1 Humans are the only natural host of the HSV-1 virus, and infection with HSV-1 is very common. Most people are carriers. Once infected with HSV-1, the virus will lie dormant in neurons for life. When the body's immunity is low, it may be attacked by HSV-1 and cause inflammation. Among them, HSV-1 infects the cornea of the eye, causing viral corneal disease. inflammatory symptoms. HSK caused by HSV-1 is the world's leading cause of blindness from corneal scarring and corneal opacification.
  • Acyclovir Acyclovir
  • Ganciclovir Ganciclovir
  • other similar broad-spectrum antiviral drugs Mild patients often use antiviral drugs very effectively for the first time, but they can only inhibit the replication of HSV-1 in the affected area for a short time and cannot clear the virus. They also have no countermeasures against the virus lurking in the nerves, leading to recurrence of the disease. . The same is true for other drugs in the small molecule inhibitor class that are being studied.
  • patients who use broad-spectrum antiviral drugs for a long time are prone to side effects of drug resistance, which affects the effectiveness of subsequent disease treatment. Severe HSK patients need corneal transplant surgery to restore vision, but corneal donor resources are scarce and cannot meet the transplant needs of all patients. In addition, there is a possibility of disease recurrence after corneal transplantation.
  • HSV-2 is a common human pathogen that affects approximately 1.5 billion people worldwide. If left untreated, HSV-2 can cause The virus has a mortality rate of up to 70%. HSV-2 infection usually causes characteristic lesions of the mucous membranes and skin surrounding the genitals, ultimately leading to genital herpes. After primary infection, HSV-2 replicates in the central nervous system after entering peripheral sensory nerves and ascending to the dorsal root ganglia, subsequently establishing a latent infection in which periodic reactivation and viral efflux can occur, causing infection of the tissue. disease. In addition, women are more likely to be infected with HSV-2 than men, and transmission of HSV-2 during delivery can lead to neonatal complications, brain damage, or death. Meanwhile, HSV-2 infection increases the risk of HIV infection by about three times. ACV can be used to prevent viral replication. The ensuing problems of drug resistance and recurrence are the key to the cure of the disease.
  • gene therapy has taken shape. Gene therapy is divided into gene compensation gene therapy and gene editing therapy. At that time, gene compensation was the fastest to enter clinical application, while gene editing therapy made little progress. It was not until the emergence of the efficient gene editing tool CRISPR in 2012 that the development of gene therapy technology was rapidly advanced.
  • CRISPR-based in vivo Gene editing is a major breakthrough in the history of gene therapy. It can be applied not only to genetic mutation diseases, but also to acquired diseases and viral infectious diseases.
  • in vivo gene editing therapy can be used to eliminate the infectious virus HSV to achieve curative effect.
  • Genes in the HSV genome that are closely related to viral replication can no longer replicate after being knocked out by CRISPR.
  • the technology of VLP delivery of mRNA is used to transiently deliver CRISPR to the patient's corneal stromal cells, targeting the infected HSV Genes related to -1 replication are deleted so that HSV-1 cannot continue to replicate and is eliminated. It also has a good antiviral effect on HSV-2.
  • the object of the present invention is to provide a gene therapy drug for herpes simplex virus infectious diseases in view of the shortcomings of the above-mentioned prior art.
  • the present invention uses the technology of VLP to deliver mRNA to transiently deliver CRISPR into cells infected with HSV virus, targeting genes related to HSV replication and performing gene knockout, so that HSV cannot continue to replicate and is eliminated. It has a good removal effect on both HSV-1 and HSV-2.
  • the invention provides a lentiviral vector for targeting HSV virus.
  • Its packaging plasmid includes a plasmid pMD.2G that expresses membrane proteins, a plasmid that expresses lentiviral GagPol long-chain protein containing RNA binding protein, and a plasmid that expresses wild-type lentivirus GagPol.
  • a plasmid expressing Cas9 mRNA and a plasmid expressing gRNA in the plasmid expressing gRNA,
  • the backbone gene of gRNA is placed in the 3’-LTR region of the lentiviral vector, and expression is initiated by the promoter.
  • the corresponding lentiviral vector is designated as VLP-CRISPR;
  • VLP-CRISPR-2gRNA place the backbone gene of one gRNA in the 3'-LTR region of the lentiviral vector, and place the backbone gene of another gRNA between the 5'-LTR and 3'-LTR regions, each driven by two identical or different promoters. Start expression, and the corresponding lentiviral vector is designated as VLP-CRISPR-2gRNA.
  • helper plasmid pRSV-REV is also included.
  • the backbone gene of the gRNA includes a universal gRNA with a sequence shown in SEQ ID NO. 9 or an optimized Osp.gRNA with a sequence shown in SEQ ID NO. 10.
  • the plasmid expressing the lentiviral GagPol long-chain protein containing RNA-binding protein is to place n copies of the RNA-binding protein at the N-terminal or C-terminal of the lentiviral GagPol long-chain protein, or At the same time, the N-terminus and the C-terminus; the amino acid sequence of the RNA-binding protein is shown in SEQ ID NO. 2; n is any copy number that the RNA-binding protein can achieve.
  • the plasmid expressing Cas9 mRNA places the gene of the stem-loop structure recognized by the RNA-binding protein at the 5'-end or/and 3'-end of the Cas9 gene; the number of stem-loop structures is Any copy number that can be achieved (can be 1 to 12 copies); 1 to several copies of nuclear localization signals can be added to the 5'-end or/and 3'-end of the Cas9 gene; the stem-loop structural sequence As shown in SEQ ID NO.3.
  • the plasmid expressing Cas9 mRNA contains 2 copies of NLS and 6 copies of the neck loop structure, and is designated as pCMV-2 ⁇ NLS-Cas9-6 ⁇ MS2.
  • Cas9 includes Sp.Cas9, Sa.Cas9, xCas9, SpG, SpRY, SpCas9-HF1 and variants with similar functions to these Cas9s.
  • the present invention also provides a method for preparing a lentiviral vector for targeting HSV virus.
  • the plasmid pMD.2G expressing membrane protein, the plasmid expressing the lentiviral GagPol long-chain protein containing RNA binding protein, and the plasmid expressing wild-type lentivirus
  • the GagPol long-chain protein plasmid, helper plasmid pRSV-REV, Cas9 mRNA-expressing plasmid and gRNA-expressing plasmid were co-transfected into virus production cells; the supernatant was collected, concentrated, and purified.
  • the plasmid expressing Cas9 mRNA is pCMV-2 ⁇ NLS-Cas9-6 ⁇ MS2; the plasmid expressing gRNA is plasmid pLV-egfp-U3-Osp.gRNA.
  • the construction of plasmid pLV-egfp-U3-Osp.gRNA includes the following steps:
  • step S2 Use plasmid pCCL-PGK-egfp as a template, use primer 5'-gcatctagctagaattaatt-3' and primer 5'-ttgtcttcgttgggagtgaa-3' to amplify the DNA fragment; use the gene synthesized in step S1 as a template, use primer 5'-ttcactcccaacgaagacaagagggcctatttcccatgat -3' and primer 5'-aattaattctagctagatgctgctagagattttccacact-3' amplify the DNA fragment;
  • the present invention also provides a gene therapy drug for herpes simplex virus infectious diseases, which uses the aforementioned lentiviral vector to deliver gRNA targeting HSV replication-related genes.
  • Genes related to HSV replication include UL5, UL8, UL9, UL29, UL30, UL42, and UL52.
  • guide sequences targeting any one or two genes related to HSV replication are placed at the front end of the gRNA backbone in VLP-CRISPR or VLP-CRISPR-2 gRNA respectively.
  • the targeted HSV replication-related genes are UL29 and UL8, and the lentiviral vector VLP-CRISPR-UL29/UL8 is obtained;
  • the guide sequence can be any on the selected gene that conforms to the CRISPR guide sequence rules.
  • Region that is, the region containing the PAM sequence (NGG) at the 3'-end of its sequence.
  • the gRNA sequence of UL29 is 5’-gcgagcgtacacgtatcccc-3’
  • the gRNA sequence of UL8 is 5’-ggggcagccataccgcgtaa-3’.
  • the present invention uses CRISPR/Cas9 to simultaneously target two genes related to HSV-1 replication, UL8 and UL29, so that these two genes are knocked out or the HSV-1 genome is broken, and the HSV-1 genome is finally eliminated.
  • the targeted HSV replication-related genes are UL52 and UL8, and the lentiviral vector VLP-CRISPR-UL52/UL8 is obtained.
  • the guide sequence can be any region on the selected gene that conforms to the CRISPR guide sequence rules, that is, the region containing the PAM sequence (NGG) at the 3'-end of the sequence.
  • the gRNA sequence of UL52 is 5'-acggagcagccctcgcccct-3'
  • the gRNA sequence of UL8 is 5'-ggggcagccataccgcgtaa-3'.
  • the universal VLP-CRISPR-UL52/UL8 can effectively eliminate HSV-1 and HSV-2 and can be used for gene editing treatment of diseases caused by HSV-1 or HSV-2 virus infection.
  • the present invention also provides a method for preparing a gene therapy drug with the function of clearing HSV-1.
  • the plasmid pMD.2G expressing membrane protein, the plasmid expressing the long-chain lentivirus GagPol protein containing RNA binding protein, and the plasmid expressing wild-type lentivirus
  • the plasmid of GagPol long-chain protein, the helper plasmid pRSV-REV, the plasmid expressing Cas9 mRNA (pCMV-2 ⁇ NLS-Cas9-6 ⁇ MS2) and the plasmid expressing gRNA (pLV-U6-UL29-U3-UL8) were co-transfected into 293T cells, the supernatant is collected, concentrated, purified, and dissolved with excipients.
  • the present invention also provides a method for preparing a universal gene therapy drug with the function of clearing HSV-1 and HSV-2.
  • the plasmid pMD.2G expressing membrane proteins and the plasmid expressing lentiviral GagPol long chain protein containing RNA binding protein are used.
  • plasmid expressing wild-type lentiviral GagPol long-chain protein helper plasmid pRSV-REV, plasmid expressing Cas9 mRNA (pCMV-2 ⁇ NLS-Cas9-6 ⁇ MS2) and plasmid expressing gRNA (pLV-U6-UL52-U3- UL8) was co-transfected into 293T cells, and the supernatant was collected, concentrated, purified, and dissolved with excipients.
  • the present invention has the following beneficial effects:
  • VLP-CRISPR-UL29/UL8 can directly clear HSV-1, rather than just temporarily inhibiting virus replication
  • Universal VLP-CRISPR-UL52/UL8 can effectively eliminate HSV-1 and HSV-2.
  • Figure 1 is a schematic diagram comparing the gene editing efficiency of different nuclear localization signals and stem-loop structure copy numbers
  • FIG. 2 is a schematic diagram of VLP-CRISPR-gRNA
  • Figure 3 is a schematic diagram of the proportion of HSV-1 (expressing GFP) positive cells
  • Figure 4 is a schematic diagram of the titer of HSV-1-GFP in cell supernatant
  • Figure 5 is a schematic diagram of HSV-1 and HSV-2 titers in cell supernatants.
  • the technical modifications involved in the gene therapy drug for herpes simplex virus infectious diseases of the present invention include:
  • VLP vector so that it can deliver Cas9 mRNA and gRNA simultaneously to obtain VLP-CRISPR;
  • VLP-CRISPR To produce VLP-CRISPR, you need to combine a plasmid expressing membrane protein (pMD.2G), a plasmid expressing lentiviral GagPol long-chain protein containing RNA-binding protein, and a plasmid expressing wild-type lentiviral GagPol long-chain protein (pMDlg/PRRE-D64V ), helper plasmid pRSV-REV, plasmid expressing Cas9mRNA and plasmid expressing gRNA Co-transfected into virus production cells; collect the supernatant, concentrate and prepare.
  • pMD.2G plasmid expressing membrane protein
  • pMDlg/PRRE-D64V a plasmid expressing wild-type lentiviral GagPol long-chain protein
  • helper plasmid pRSV-REV helper plasmid pRSV-REV
  • Cas9mRNA plasmid expressing Cas9m
  • RNA-binding protein 1 or 2 copies of the RNA-binding protein (gene sequence SEQ ID NO.1, amino acid sequence SEQ ID NO.2) can be placed in the lentivirus respectively.
  • GagPol long chain protein N-terminus or C-terminus or both N-terminus and C-terminus.
  • the RNA-binding protein can also be present in any copy number achievable.
  • the gene of the stem-loop structure recognized by the RNA-binding protein is placed at the 5’-end or/and 3’-end of the Cas9 gene.
  • the number of its stem-loop structure can be from 1 (SEQ ID NO.3) to 12 copies (SEQ ID NO.4), etc., or it can be any copy number that can be achieved.
  • the 5'-end or/and 3'-end of the Cas9 gene can be added with 1 to several copies of nuclear localization signal (NLS, Cas9 protein N-terminal NLS sequence: SEQ ID NO.5; Cas9 protein C-terminal NLS sequence : SEQ ID NO.6) to increase the efficiency of protein entering the nucleus;
  • Cas9 can be Sp.Cas9, Sa.Cas9, xCas9, SpG, SpRY and SpCas9-HF1 and other Cas9 or variant forms with DNA cutting function.
  • gRNA backbone gene For the plasmid expressing gRNA, place the gRNA backbone gene in the 3’-LTR region of the lentiviral vector; expression is initiated by the U6 promoter (SEQ ID NO.7) or H1 (SEQ ID NO.8) promoter.
  • the backbone of gRNA can be a universal version (SEQ ID NO.9) and an optimized version (Osp.gRNA, SEQ ID NO.10).
  • VLP-CRISPR-2 gRNA that can target two genes so that it can deliver Cas9 mRNA and gRNA that targets two genes at the same time;
  • VLP-CRISPR-2 gRNA In order to produce VLP-CRISPR-2 gRNA, it is necessary to continue to transform the plasmid expressing gRNA.
  • This plasmid needs to contain 2 gRNA series, one of which is as described in 1.c, and the other gRNA is placed in the 5'-LTR and 3 of the plasmid. '-LTR.
  • the gRNA can also be Osp.gRNA with an optimized backbone sequence.
  • VLP-CRISPR Modify the VLP-CRISPR that eliminates HSV-1 virus or/and HSV-2 virus so that it can simultaneously deliver Cas9 mRNA and gRNA targeting HSV-1 and HSV-2 replication-related genes.
  • the guide sequence of the gene (separate or common for HSV-1 and HSV-2 viruses) is placed at the front end of the gRNA backbone in VLP-CRISPR or VLP-CRISPR-2gRNA; the guide sequence can be any guide sequence on the selected gene that conforms to the CRISPR guide sequence rules.
  • the region that is, the region containing the PAM sequence (NGG) at the 3'-end of its sequence.
  • the gRNA backbone used is universal gRNA or optimized Osp.gRNA.
  • guide sequences in two important genes related to HSV-1 replication, UL8 and UL52 are selected to prepare a universal gene therapy drug for treating herpes simplex virus infectious diseases.
  • VLP vector simultaneously delivers Cas9mRNA and Osp.gRNA
  • VLP vector The realization of simultaneous delivery of Cas9mRNA and Osp.gRNA by VLP vector first requires the transformation of the production plasmid.
  • the constructed VLP-CRISPR is a plasmid expressing membrane protein (pMD.2G) and a plasmid expressing lentiviral GagPol long-chain protein containing RNA binding protein (pMS2M-PH-Gag–Pol-D64V, sequence As shown in SEQ ID NO.12), plasmid expressing wild-type lentiviral GagPol long-chain protein (pMDlg/PRRE-D64, sequence as shown in SEQ ID NO.13), helper plasmid (pRSV-REV), expressing Cas9mRNA Plasmid (containing 2 copies of NLS and 6 copies of the neck loop structure, pCMV-2 ⁇ NLS-Cas9-6 ⁇ MS2, the sequence is shown in SEQ ID NO.14) and plasmid expressing gRNA (pLV-egfp-U3 -Osp.gRNA, the sequence is shown in SEQ ID NO.15) was co-transfected into 293T cells, and the supern
  • the Osp.gRNA expression box of plasmid pLV-egfp-U3-Osp.gRNA is in the 3’LTR region.
  • the steps to construct the plasmid are:
  • plasmid pCCL-PGK-egfp (the third generation lentiviral packaging plasmid, used to carry the delivered foreign gene, the gene sequence is shown in SEQ ID NO.11) as a template, and use the primer 5'-gcatctagctagaattaatt-3' and primer 5'-ttgtcttcgttggggagtgaa-3' to amplify the DNA fragment; use the gene synthesized in step 1 as a template, use primer 5'-ttcactcccaacgaagacaagagggcctatttcccatgat-3' and primer 5'-aattaattctagctagatgctgctagagattttccacact-3' to amplify the DNA fragment;
  • This example also verified the gene editing efficiency of the VLP vector packaged by the expression Cas9mRNA plasmid containing 1 or 2 copies of NLS and 6 or 12 copies of the neck loop structure, with the AAVS1 site (expressed when packaging VLP
  • the plasmid of AAVS1-gRNA is pLV-egfp-U3-Osp.gRNA-AAVS1, which consists of the annealed oligonucleotide chains "5'-CACCGGGGCCACTAGGGACAGGAT-3'" and "5'-AAACATCCTGTCCCTAGTGGCCCC-3'" inserted into AarI digestion Compare the editing efficiency results of the plasmid pLV-egfp-U3-Osp.gRNA).
  • a plasmid expressing membrane protein pMD.2G
  • a plasmid expressing lentiviral GagPol long-chain protein containing RNA binding protein pMS2M-PH-Gag– Pol-D64V
  • plasmid expressing wild-type lentiviral GagPol long-chain protein pMDlg/PRRE-D64V
  • helper plasmid pRSV-REV
  • plasmid expressing Cas9 mRNA containing 2 copies of NLS and 6 copies of the neck loop
  • the structure, pCMV-2 ⁇ NLS-Cas9-6 ⁇ MS22) and the plasmid expressing gRNA pLV-U6-UL29-U3-UL8, SEQ ID NO.16 were co-transfected into 293T cells, and the supernatant was collected, concentrated and purified. , obtained by dissolving it with excipients.
  • the sixth plasmid (plasmid expressing gRNA) is changed to pLV-U6-UL29-U3-UL8, the UL8-Osp.gRNA expression box is in the 3'LTR region, and the expression of UL29-Osp.gRNA box replaces the GFP expression box. Plasmids are composed of genetically synthesized DNA sequences and then circularized.
  • the schematic diagram of VLP-CRISPR-Osp.gRNA is shown in Figure 2.
  • the gRNA expression cassette of VLP-CRISPR-UL29/UL8 targets the UL29 and UL8 genes respectively.
  • the guide sequence can be any region on the selected gene that conforms to the CRISPR guide sequence rules, that is, the region containing the PAM sequence (NGG) at the 3’-end of the sequence.
  • the gRNA sequence of UL29 was selected to be 5'-gcgagcgtacacgtatccc-3', and the gRNA sequence of UL8 was 5'-ggggcagccataccgcgtaa-3' to conduct the HSV-1 genome cleavage experiment.
  • 2VLP-CRISPR-UL29/UL8 infection Inoculate 293T cells in a 96-well plate, 2 x 10 4 cells per well (total volume 100 ⁇ L), and culture in a 37°C, 5% CO 2 cell culture incubator (Note: used for 293T The culture medium is DMEM+6%FBS+1%PS). After 24 hours, p24 200ng VLP-CRISPR-UL29/UL8 was added to each well. VLP-CRISPR (p24 200ng) without gene targeting was added to the Scramble Control group. No addition was added to the MOCK group and Blank group. Each group had 3 duplicate wells. Continue. nourish. After 14h-18h, take out the 96-well plate and change the medium (200 ⁇ L/well).
  • HSV-1-GFP Extracorporation of the Green Fluorescent Ptein into the Herpes Simplex Virus Type 1 Capsid.P Desai and S Person.1998Sep;72(9):7563–7568.
  • PFU HSV-1-GFP Extracorporation of the Green Fluorescent Ptein into the Herpes Simplex Virus Type 1 Capsid.P Desai and S Person.1998Sep;72(9):7563–7568.
  • HSV-1-GFP for 40-48h to observe the expression of GFP. When there is more (>50%) GFP expression in the Scramble Control group and MOCK group, collect the cell supernatant and cell pellet.
  • the HSV-1-GFP used in this example expresses GFP protein, and GFP expression represents HSV-1.
  • Flow cytometry can be used to detect the proportion of GFP-positive cells (representing HSV-1) and measure the elimination effect of HSV-1.
  • Centrifuge (700g, 5min) to remove the supernatant, add 150 ⁇ L PBS to resuspend the cells, and analyze the proportion of GFP-positive cells by flow cytometry. The results are shown in Figure 3;
  • Vero cells were seeded in a 12-well plate, 3 x 10 5 cells per well (total volume 1 mL), and placed at 37°C and 5% CO 2 cells Culture in an incubator (Note: the culture medium used by Vero is DMEM+6% FBS+1% PS). After 24 hours, add the dilution of the cell supernatant to be tested. In this example, the dilution was 100 times. Add 100 ⁇ L of diluted virus solution to each well to adsorb cells. Gently shake the culture plate to distribute the virus evenly, and place it in a 37°C, 5% CO2 cell culture incubator for 2 hours.
  • Infection titer N lesions ⁇ dilution factor/infection volume. The data are shown in Figure 4. There are too many plaques in the Scramble Control group to count, so the Scramble Control group is not counted and only the other groups are counted. StudentT-test was used for statistical analysis, ** P ⁇ 0.01.
  • a plasmid expressing membrane protein (pMD.2G) and a plasmid expressing lentiviral GagPol long-chain protein containing RNA binding protein (pMD.2G) are required.
  • pMS2M-PH-Gag–Pol-D64V plasmid expressing wild-type lentiviral GagPol long-chain protein
  • pRSV-REV helper plasmid
  • Cas9 mRNA containing 2 copies of NLS and 6 copies of the neck loop structure
  • pCMV-2 ⁇ NLS-Cas9-6 ⁇ MS2 plasmid expressing Cas9 mRNA
  • pLV-U6-UL52-U3-UL8, SEQ ID NO.17 were co-transfected into 293T cells, and collected The supernatant is concentrated, purified, and dissolved with excipients.
  • the sixth plasmid (plasmid expressing gRNA) is pLV-U6-UL52-U3-UL8, the UL8-Osp.gRNA expression cassette is in the 3'LTR region, and the UL52-Osp.gRNA expression cassette is Replace the GFP expression box. Plasmids are composed of genetically synthesized NDA sequences and then circularized.
  • the schematic diagram of VLP-CRISPR-Osp.gRNA is shown in Figure 2.
  • the gRNA expression cassette of VLP-CRISPR-UL52/UL8 targets the UL52 and UL8 genes respectively.
  • the guide sequence can be any region on the selected gene that conforms to the CRISPR guide sequence rules, that is, the region containing the PAM sequence (NGG) at the 3’-end of the sequence.
  • VLP-CRISPR-UL52/UL8 eliminates HSV-1 and HSV-2 in vitro
  • the gRNA sequence of UL52 is 5'-acggagcagccctcgcccct-3'
  • the gRNA sequence of UL8 is 5'-ggggcagccataccgcgtaa-3' for HSV genome cleavage gRNA.
  • Example 3 200ng p24VLP-CRISPR-UL52/UL8 per experimental well was used to eliminate HSV-1 and HSV-2 respectively, and the secretion amount of HSV-1/HSV-2 in the cell supernatant was detected by plaque assay. The results showed that both were effective in clearing HSV-1. Judging from the test results of the HSV-2 elimination effect, VLP-CRISPR-UL52/UL8 has a higher effect than VLP-CRISPR-UL29/UL8. The data are shown in Figure 5. StudentT-test is used for statistical analysis, ** P ⁇ 0.01, *** P ⁇ 0.001, ns means not significant.

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Abstract

本发明公开了一种用于单纯疱疹病毒感染性疾病的基因治疗药物；本发明利用慢病毒载体VLP-CRISPR或VLP-CRISPR-2gRNA递送靶向HSV复制相关基因的gRNA；具体是将靶向HSV复制相关的基因中任意一个或者两个基因的向导序列分别放到VLP-CRISPR或者VLP-CRISPR-2gRNA中gRNA骨架前端；得到VLP-CRISPR-gRNA1或VLP-CRISPR-gRNA1/gRNA2。具体的，得到VLP-CRISPR-UL29/UL8或VLP-CRISPR-UL52/UL8；其中VLP-CRISPR-UL29/UL8可直接清除HSV-1；通用型VLP-CRISPR-UL52/UL8可有效直接清除HSV-1和HSV-2，而不仅仅是暂时抑制病毒的复制通路。

Description

一种用于单纯疱疹病毒感染性疾病的基因治疗药物 技术领域
本发明属于基因药物领域，涉及一种用于单纯疱疹病毒感染性疾病的基因治疗药物。
背景技术
单纯疱疹病毒(Herpes simplex virus，HSV)属于人类疱疹病毒科α亚科，是一种有包膜的球形病毒。分单纯疱疹病毒Ⅰ型(Herpes simplex virus Ⅰ，HSV-1)和单纯疱疹病毒Ⅱ型(Herpes simplex virus Ⅱ，HSV-2)。都是致病性病毒，可引起疱疹性脑炎、疱疹性角膜炎、生殖器疱疹或新生儿脑炎，至今无药可根治，也没有疫苗预防感染。
单纯疱疹病毒性角膜炎(Herpes simplex keratitis，HSK)是由HSV-1引起的感染型眼角膜疾病，严重时致盲。HSV-1含有约152-kb双链线性DNA，有多种亚型。HSV-1感染可引起多种疾病，其中，感染眼角膜引起病毒性角膜炎，这是世界上感染性角膜病致盲的主要原因。人类是这种病毒的唯一天然宿主，自然状态下人类感染HSV-1的情况非常普遍，据统计，世界上50％-80％人是HSV-1的携带者，每年新增150万由感染了HSV病毒引起的角膜炎患者，其中4万患者视力损坏或失明。
人类是HSV-1病毒的唯一天然宿主，且被HSV-1感染的情况非常普遍。大多数人为携带者，一旦感染HSV-1，病毒将就终身潜伏在神经元中，在机体免疫力低下时可能受到HSV-1的攻击引发炎症，其中，HSV-1感染眼角膜引起病毒性角膜炎症状。HSV-1引起的HSK是世界上角膜瘢痕形成和角膜混浊致盲的主要原因。
现如今，临床上治疗HSK的药物首选是阿昔洛韦(Acyclovir，ACV)、更昔洛韦(Ganciclovir)等类似的广谱性抗病毒药物。轻症患者初次使用抗病毒药物时时抗病毒效果很好，但只能短时抑制患病部位的HSV-1的复制，无法清除病毒，而且对于潜伏在神经中的病毒毫无对策，从而疾病复发。其它正在研究的小分子抑制剂类的药物也是如此。另外，患者长期使用广谱性抗病毒药物易产生耐药性的副作用，所以影响后续的疾病治疗效果。HSK重症患者需要通过角膜移植手术恢复视力，但是角膜供体资源少，不能解决所有患者的移植需求，另外，角膜移植后也有疾病复发的可能。
HSV-2是一种常见的人类病原体，全世界约有15亿人感染了HSV-2，如果不治疗， 这种病毒的死亡率高达70％。HSV-2感染通常会引起生殖器周围粘膜和皮肤的特征性病变，终导致生殖器疱疹。在原发感染后，HSV-2在进入周围感觉神经并上升至背根神经节后，在中枢神经系统中复制，随后建立一种潜在感染，可发生周期性重激活和病毒流出，感染组织引起疾病。此外，女性的HSV-2感染率高于男性，分娩期间HSV-2的传播可导致新生儿并发症，致脑损伤或死亡。同时，HSV-2感染使感染HIV的风险增加了大约三倍。ACV可用来阻止病毒复制，随之而来的耐药性和反复发作的问题是困扰疾病根治的关键。
近三十年来，基因治疗的疗法初见雏形。基因治疗分为基因补偿型基因疗法和基因编辑疗法。彼时更快进入临床应用的是基因补偿型，而基因编辑疗法的进展甚微。直到2012年高效的基因编辑工具CRISPR的出现，才快速推进了基因治疗技术的发展。如今已经有很多应用CRISPR的基因编辑治疗进入临床试验阶段，而绝大多数基因编辑疗法都是离体基因编辑，而正在做的在体基因编辑疗法的临床试验只有2例，基于CRISPR的在体基因编辑是基因治疗发展史中的一大突破，不仅可以应用于基因突变性疾病，还可以用于获得性疾病和病毒感染性疾病。对于无根治方法、易复发的单纯疱疹性疾病，可以用在体基因编辑治疗的方法清除感染的病毒HSV以达到疗效。HSV的基因组中与病毒复制密切相关的基因被CRISPR敲除后亦不能再进行复制，本发明中，应用VLP递送mRNA的技术将CRISPR瞬时递送到患者角膜基质层细胞中，靶向已感染的HSV-1复制相关的基因，进行基因敲除，使HSV-1无法继续复制从而被清除。对于HSV-2也有很好的抗病毒效果。现如今还没有类似的已上市基因治疗药物。
发明内容
本发明的目的在于针对上述现有技术存在的不足，提供一种用于单纯疱疹病毒感染性疾病的基因治疗药物。本发明应用VLP递送mRNA的技术将CRISPR瞬时递送到感染了HSV病毒的细胞中，靶向HSV复制相关的基因，进行基因敲除，使HSV无法继续复制从而被清除。对于HSV-1和HSV-2都有很好的清除效果。
本发明的目的是通过以下技术方案来实现的：
<第一方面>
本发明提供一种用于靶向HSV病毒的慢病毒载体，其包装质粒包括表达膜蛋白的质粒pMD.2G、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、表达野生型慢病毒GagPol长链蛋白的质粒、表达Cas9mRNA的质粒和表达gRNA的质粒上；所述表达gRNA的质粒中，
将gRNA的骨架基因置于慢病毒载体的3’-LTR的区域，由启动子启动表达，对应的慢病毒载体记为VLP-CRISPR；
或将一个gRNA的骨架基因置于慢病毒载体的3’-LTR的区域，另一个gRNA的骨架基因置于5’-LTR和3’-LTR区域之间，分别由两个相同或者不同启动子启动表达，对应的慢病毒载体记为VLP-CRISPR-2gRNA。
在一些实施例中，还包括辅助质粒pRSV-REV。
作为本发明的一个实施方案，所述gRNA的骨架基因包括序列如SEQ ID NO.9所示的通用型gRNA或序列如SEQ ID NO.10所示的优化型Osp.gRNA。
通用型gRNA骨架序列SEQ ID NO.9：
优化型Osp.gRNA骨架序列SEQ ID NO.10：
作为本发明的一个实施方案，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒，是将RNA结合蛋白n个拷贝分别置于慢病毒GagPol长链蛋白N-端或C-端或者同时N-端和C-端；所述RNA结合蛋白的氨基酸序列如SEQ ID NO.2所示；n为RNA结合蛋白所能达到的任意拷贝数。
作为本发明的一个实施方案，所述表达Cas9mRNA的质粒，是将RNA结合蛋白所识别的茎环结构的基因置于Cas9基因的5’-端或/和3’-端；茎环结构数量为所能达到的任意拷贝数(可以为1～12个拷贝)；Cas9基因的5’-端或/和3’-端均可以加入1到几个拷贝的核定位信号；所述茎环结构序列如SEQ ID NO.3所示。
进一步的，所述表达Cas9mRNA的质粒含有2个拷贝的NLS和6个拷贝的颈环结构，记为pCMV-2×NLS-Cas9-6×MS2。
进一步的，Cas9包括Sp.Cas9、Sa.Cas9、xCas9、SpG、SpRY、SpCas9-HF1以及与这些Cas9功能相似的变体形式。
<第二方面>
本发明还提供一种用于靶向HSV病毒的慢病毒载体的制备方法，将表达膜蛋白的质粒pMD.2G、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、表达野生型慢病毒 GagPol长链蛋白的质粒、辅助质粒pRSV-REV、表达Cas9mRNA的质粒和表达gRNA的质粒共转染入病毒生产细胞；收集上清，浓缩，纯化而得。
作为本发明的一个实施方案，所述表达Cas9mRNA的质粒为pCMV-2×NLS-Cas9-6×MS2；所述表达gRNA的质粒为质粒pLV-egfp-U3-Osp.gRNA。
作为本发明的一个实施方案，质粒pLV-egfp-U3-Osp.gRNA的构建包括如下步骤：
S1、通过基因合成的方法合成U6或H1启动子和Osp.gRNA的DNA序列；
S2、以质粒pCCL-PGK-egfp为模板，用引物5’-gcatctagctagaattaatt-3’和引物5’-ttgtcttcgttgggagtgaa-3’扩增DNA片段；以步骤S1合成的基因为模板，用引物5’-ttcactcccaacgaagacaagagggcctatttcccatgat-3’和引物5’-aattaattctagctagatgctgctagagattttccacact-3’扩增DNA片段；
S3、纯化、连接两个DNA片段；将连接体系转化到细菌感受态中，验证质粒序列正确后，大量扩增单克隆，提取质粒。
<第三方面>
本发明还提供一种用于单纯疱疹病毒感染性疾病的基因治疗药物，利用前述的慢病毒载体递送靶向HSV复制相关基因的gRNA。HSV复制相关的基因包括UL5、UL8、UL9、UL29、UL30、UL42、UL52。
作为本发明的一个实施方案，将靶向HSV复制相关的基因中任意一个或者两个基因的向导序列分别放到VLP-CRISPR或者VLP-CRISPR-2gRNA中gRNA骨架前端。
作为本发明的一个实施方案，所述靶向HSV复制相关基因为UL29、UL8，得到慢病毒载体VLP-CRISPR-UL29/UL8；其向导序列可以是所选基因上的任意符合CRISPR向导序列规则的区域，即其序列3’-端含有PAM序列(NGG)的区域。如：UL29的gRNA序列是5’-gcgagcgtacacgtatccc-3’，UL8的gRNA序列是5’-ggggcagccataccgcgtaa-3’。本发明利用CRISPR/Cas9同时靶向HSV-1复制相关的两个基因，UL8和UL29，使这两个基因被敲除或者HSV-1基因组断裂，最终清除HSV-1基因组。
作为本发明的一个实施方案，所述靶向HSV复制相关基因为UL52、UL8，得到慢病毒载体VLP-CRISPR-UL52/UL8。其向导序列可以是所选基因上的任意符合CRISPR向导序列规则的区域，即其序列3’-端含有PAM序列(NGG)的区域。UL52的gRNA序列是5’-acggagcagccctcgcccct-3’，UL8的gRNA序列是5’-ggggcagccataccgcgtaa-3’。该通用型VLP-CRISPR-UL52/UL8可有效清除HSV-1和HSV-2，可用于HSV-1或HSV-2病毒感染导致疾病的基因编辑治疗。
<第四方面>
本发明还提供一种具有清除HSV-1功能的基因治疗药物的制备方法，将表达膜蛋白的质粒pMD.2G、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、表达野生型慢病毒GagPol长链蛋白的质粒、辅助质粒pRSV-REV，表达Cas9mRNA的质粒(pCMV-2×NLS-Cas9-6×MS2)和表达gRNA的质粒(pLV-U6-UL29-U3-UL8)共转染进293T细胞，收取上清液浓缩、纯化、用赋形剂溶解所得。
<第五方面>
本发明还提供一种具有清除HSV-1和HSV-2功能的通用型基因治疗药物的制备方法，将表达膜蛋白的质粒pMD.2G、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、表达野生型慢病毒GagPol长链蛋白的质粒、辅助质粒pRSV-REV，表达Cas9mRNA的质粒(pCMV-2×NLS-Cas9-6×MS2)和表达gRNA的质粒(pLV-U6-UL52-U3-UL8)共转染进293T细胞，收取上清液浓缩、纯化、用赋形剂溶解所得。
与现有技术相比，本发明具有如下有益效果：
1)可单次给药治疗疾病；
2)VLP-CRISPR-UL29/UL8可直接清除HSV-1，而不仅仅是暂时抑制病毒的复制；
3)通用型VLP-CRISPR-UL52/UL8可有效清除HSV-1和HSV-2。
附图说明
通过阅读参照以下附图对非限制性实施例所作的详细描述，本发明的其它特征、目的和优点将会变得更明显：
图1为比较不同核定位信号和茎环结构拷贝数基因编辑效率示意图；
图2为VLP-CRISPR-gRNA示意图；
图3为HSV-1(表达GFP)阳性细胞数比例示意图；
图4为细胞上清中HSV-1-GFP的滴度示意图；
图5为细胞上清中HSV-1、HSV-2滴度示意图。
具体实施方式
本发明的用于单纯疱疹病毒感染性疾病的基因治疗药物涉及的技术改造包括：
1、改造VLP载体，使其能同时递送Cas9mRNA和gRNA，得到VLP-CRISPR；
生产VLP-CRISPR，需要将表达膜蛋白的质粒(pMD.2G)、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、表达野生型慢病毒GagPol长链蛋白的质粒(pMDlg/PRRE-D64V)、辅助质粒pRSV-REV，表达Cas9mRNA的质粒和表达gRNA的质粒 共转染入病毒生产细胞；收集上清，浓缩，制备而得。
首先改造用于生产VLP载体的质粒体系。
a.表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒，可将RNA结合蛋白(基因序列SEQ ID NO.1，氨基酸序列SEQ ID NO.2)的1或2个拷贝分别置于慢病毒GagPol长链蛋白N-端或C-端或者同时N-端和C-端。RNA结合蛋白也可以是所能达到的任意拷贝数。
b.表达Cas9mRNA的质粒，将RNA结合蛋白所识别的茎环结构的基因置于Cas9基因的5’-端或/和3’-端。其茎环结构(SEQ ID NO.3)数量可以是1(SEQ ID NO.3)到12个拷贝(SEQ ID NO.4)等，也可以是所能达到的任意拷贝数。Cas9基因的5’-端或/和3’-端都可以加入1到几个拷贝的核定位信号(NLS，Cas9蛋白N-端NLS序列：SEQ ID NO.5；Cas9蛋白C-端NLS序列：SEQ ID NO.6)来增加蛋白进入细胞核的效率；Cas9可以是Sp.Cas9、Sa.Cas9、xCas9、SpG、SpRY和SpCas9-HF1等其他具有DNA切割功能的Cas9或变体形式。
c.表达gRNA的质粒，将gRNA的骨架基因置于慢病毒载体的3’-LTR的区域；由U6启动子(SEQ ID NO.7)或H1(SEQ ID NO.8)启动子启动表达。gRNA的骨架可以是通用型版本(SEQ ID NO.9)和优化版本(Osp.gRNA，SEQ ID NO.10)。
2、改造能靶向2个基因的VLP-CRISPR-2gRNA，使其能同时递送Cas9mRNA和靶向2个基因的gRNA；
为了能生产出VLP-CRISPR-2gRNA，需要继续改造表达gRNA的质粒，此质粒需要包含2个gRNA系列，其中1个如1.c所述,另一个gRNA放到质粒的5’-LTR和3’-LTR之间。gRNA也可以是优化骨架序列的Osp.gRNA。
3、改造清除HSV-1病毒或/和HSV-2病毒的VLP-CRISPR，使其能同时递送Cas9mRNA和靶向HSV-1和HSV-2复制相关基因的gRNA.
为了生产出靶向HSV-1或HSV-2或HSV-1与HSV-2病毒的通用型VLP-CRISPR，需要继续改造表达gRNA的质粒，将靶向HSV复制相关的基因中任意一个或者两个基因的向导序列(HSV-1和HSV-2病毒单独或通用)分别放到VLP-CRISPR或者VLP-CRISPR-2gRNA中gRNA骨架前端；其向导序列可以是所选基因上的任意符合CRISPR向导序列规则的区域，即其序列3’-端含有PAM序列(NGG)的区域。所用到的gRNA骨架为通用型的gRNA或优化的Osp.gRNA。本发明中，选择UL8和UL52两个HSV-1复制相关的重要基因中的向导序列制成通用型治疗单纯疱疹病毒感染性疾病的基因治疗药物。
下面结合实施例对本发明进行详细说明。以下实施例将有助于本领域的技术人员进一步理解本发明，但不以任何形式限制本发明。应当指出的是，对本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干调整和改进。这些都属于本发明的保护范围。
实施例1、VLP载体同时递送Cas9mRNA和Osp.gRNA
VLP载体同时递送Cas9mRNA和Osp.gRNA的实现首先需要改造生产质粒。
在本实施例中，构建的VLP-CRISPR是将表达膜蛋白的质粒(pMD.2G)、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒(pMS2M-PH-Gag–Pol-D64V，序列如SEQ ID NO.12所示)、表达野生型慢病毒GagPol长链蛋白的质粒(pMDlg/PRRE-D64，序列如SEQ ID NO.13所示)、辅助质粒(pRSV-REV)，表达Cas9mRNA的质粒(含有2个拷贝的NLS和6个拷贝的颈环结构，pCMV-2×NLS-Cas9-6×MS2，序列如SEQ ID NO.14所示)和表达gRNA的质粒(pLV-egfp-U3-Osp.gRNA，序列如SEQ ID NO.15所示)共转染进293T细胞，收取上清液浓缩、纯化所得。其中本实施例中除了质粒pLV-egfp-U3-Osp.gRNA，都是通过基因合成DNA序列后再环化成质粒。
质粒pLV-egfp-U3-Osp.gRNA的Osp.gRNA表达框在3’LTR区域，构建质粒的步骤是：
①通过基因合成的方法合成U6启动子和Osp.gRNA的DNA序列；
②以质粒pCCL-PGK-egfp(第三代慢病毒包装质粒，用于携带所递送的外源基因，基因序列如SEQ ID NO.11所示)为模板，用引物5’-gcatctagctagaattaatt-3’和引物5’-ttgtcttcgttgggagtgaa-3’扩增DNA片段；以步骤①合成的基因为模板，用引物5’-ttcactcccaacgaagacaagagggcctatttcccatgat-3’和引物5’-aattaattctagctagatgctgctagagattttccacact-3’扩增DNA片段；
③用DNA纯化试剂盒纯化两个DNA片段；
④采用HiFi DNA Assembly试剂连接两个DNA片段；
⑤将连接体系转化到细菌感受态中，挑取单克隆，扩增培养，质粒小提；
⑥用引物5’-gagggcctatttcccatgat-3’进行Sanger测序验证质粒是否与预期序列相符；
⑦验证质粒序列正确后，大量扩增单克隆，提取质粒。
本实施例中还验证了含有1个或2个拷贝的NLS和6个或12个拷贝的颈环结构的表达Cas9mRNA质粒包装的VLP载体的基因编辑效率，以AAVS1位点(包装VLP时表达 AAVS1-gRNA的质粒为pLV-egfp-U3-Osp.gRNA-AAVS1，由退火后的寡核苷酸链“5’-CACCGGGGCCACTAGGGACAGGAT-3’”和“5’-AAACATCCTGTCCCTAGTGGCCCC-3’”插入AarI酶切的质粒pLV-egfp-U3-Osp.gRNA得来)的编辑效率结果做对比。实验时，将293T细胞接种到96孔板里，1×104/孔。24h后分别加入待验证的VLP，150ng p24每孔。感染72h后收集细胞，提取基因组，用引物5’-ttcgggtcacctctcactcc-3’和5’-ggctccatcgtaagcaaacc-3’扩增AAVS1位点周围的DNA片段，用引物5’-ttcgggtcacctctcactcc-3’Sanger测序，将测序文件输入TIDE网站分析工具计算Indel(碱基插入/缺失)结果，感染结果如图1所示。带有2NLS的VLP基因编辑效果更好。6个拷贝和12个拷贝的MS2递送Cas9mRNA包装而成的VLP都能达到较高的基因编辑效率。StudentT-test做统计分析，**P<0.01,n.s.代表不显著。
实施例2、具有清除HSV-1功能的VLP-CRISPR-UL29/UL8
生产具有清除HSV-1功能的VLP-CRISPR-UL29/UL8，需要将表达膜蛋白的质粒(pMD.2G)、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒(pMS2M-PH-Gag–Pol-D64V)、表达野生型慢病毒GagPol长链蛋白的质粒(pMDlg/PRRE-D64V)、辅助质粒(pRSV-REV)，表达Cas9mRNA的质粒(含有2个拷贝的NLS和6个拷贝的颈环结构，pCMV-2×NLS-Cas9-6×MS2)和表达gRNA的质粒(pLV-U6-UL29-U3-UL8，SEQ ID NO.16)共转染进293T细胞，收取上清液浓缩、纯化、用赋形剂溶解所得。
与实施例1不同的是，第6个质粒(表达gRNA的质粒)改为pLV-U6-UL29-U3-UL8，UL8-Osp.gRNA表达框在3’LTR区域，UL29-Osp.gRNA的表达框替换GFP表达框。质粒由基因合成DNA序列后再环化构成。VLP-CRISPR-Osp.gRNA示意图见图2,其VLP-CRISPR-UL29/UL8的gRNA表达盒分别是靶向UL29和UL8基因的。其向导序列可以是所选基因上的任意符合CRISPR向导序列规则的区域，即其序列3’-端含有PAM序列(NGG)的区域。
实施例3、VLP-CRISPR-UL29/UL8体外清除HSV-1
按照实施例2的方法生产VLP-CRISPR-UL29/UL8，质粒转染时，按照比例pMD.2G:pRSV-REV:pMDlg/PRRE-D64V:pMS2M-PH-Gag–Pol-D64V:pCMV-2×NLS-Cas9-6×MS2:pLV-U6-UL29-U3-UL8＝3.75：3：6.5：6.5：13：13，质粒总量和细胞数之间的关系为0.76μg/cm2，所用赋形剂为磷酸缓冲液。本实施例中选择UL29的gRNA序列是5’-gcgagcgtacacgtatccc-3’，UL8的gRNA序列是5’-ggggcagccataccgcgtaa-3’来进行HSV-1基因组切割实验。
①用p24ELISA检测试剂盒检测VLP-CRISPR-UL29/UL8的物理滴度；
②VLP-CRISPR-UL29/UL8感染：将293T细胞接种于96孔板，每孔2 x 104cells(总体积100μL)，放入37℃、5％CO2细胞培养箱中培养(注：293T所用培养基为DMEM+6％FBS+1％PS)。24h后每个孔加入p24 200ng VLP-CRISPR-UL29/UL8，Scramble Control组加入无基因靶向性的VLP-CRISPR(p24 200ng)，MOCK组和Blank组不加，每组3个复孔，继续培养。14h-18h后拿出96孔板，换液(200μL/孔)。加入2×104PFU HSV-1-GFP(Incorporation of the Green Fluorescent Ptein into the Herpes Simplex Virus Type 1 Capsid.P Desai and S Person.1998Sep；72(9):7563–7568.)，Blank组不加。轻摇培养板使病毒均匀分布，放回37℃、5％CO2细胞培养箱中继续培养。加HSV-1-GFP40-48h观察GFP表达情况，当Scramble Control组和MOCK组有较多(>50％)GFP表达时收集细胞上清和细胞沉淀。
③流式分析GFP阳性细胞数比例：本实施例中用的HSV-1-GFP是表达GFP蛋白的，GFP表达代表HSV-1。通过流式分析的方法可以检测GFP阳性细胞(代表含有HSV-1)的比例，衡量HSV-1的清除效果。每样品加入400μL 4％PFA固定细胞15min。离心(700g，5min)去上清，加150μL PBS重悬细胞，流式分析GFP阳性细胞数比例，结果见图3；
StudentT-test做统计分析，***P<0.001。
④空斑试验检测细胞上清中HSV-1-GFP的分泌量：将Vero细胞接种于12孔板，每孔3 x 105cells(总体积1mL)，放入37℃、5％CO2细胞培养箱中培养(注：Vero所用培养基为DMEM+6％FBS+1％PS)。24h后加入待测细胞上清液的稀释液，本实施例中，稀释100倍。每孔加入100μL稀释后的病毒液吸附细胞。轻摇培养板使病毒均匀分布，放入37℃、5％CO2细胞培养箱中培养2h。尽可能吸出所有液体，每孔加入1mL固体培养基(固体培养基温度约为37℃)。将12孔板放到4℃冰箱中约5min，待固体培养基凝固后，放回37℃、5％CO2细胞培养箱中继续培养。72h后每个孔加入400μL 4％PFA，于室温固定10min。弃去PFA，每个孔加入500μL 1％结晶紫溶液，室温染色1.5-2h。弃去染色液，将固体取出。用ddH2O小心润洗三次12孔板，晾干后肉眼计数病斑数。感染滴度(PFU·mL-1)＝N病斑×稀释倍数/感染体积，数据见图4，其中Scramble Control组空斑过多难以计数，遂不统计Scramble Control组只统计其它组。StudentT-test做统计分析，**P<0.01。
结果显示，VLP-CRISPR-UL29/UL8组的HSV-1数量明显少于Scramble Control组(不靶向HSV-1的VLP-CRISPR)和Mock组(未添加VLP-CRISPR)，说明VLP-CRISPR-UL29/UL8 具有清除HSV-1的作用。
实施例4、具有清除HSV-1和HSV-2功能的通用型VLP-CRISPR-UL52/UL8
生产具有清除HSV-1和HSV-2功能的通用型VLP-CRISPR-UL52/UL8，需要将表达膜蛋白的质粒(pMD.2G)、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒(pMS2M-PH-Gag–Pol-D64V)、表达野生型慢病毒GagPol长链蛋白的质粒(pMDlg/PRRE-D64V)、辅助质粒(pRSV-REV)，表达Cas9mRNA的质粒(含有2个拷贝的NLS和6个拷贝的颈环结构，pCMV-2×NLS-Cas9-6×MS2)和表达gRNA的质粒(pLV-U6-UL52-U3-UL8，SEQ ID NO.17)共转染进293T细胞，收取上清液浓缩、纯化、用赋形剂溶解所得。
与实施例2不同的是，第6个质粒(表达gRNA的质粒)为pLV-U6-UL52-U3-UL8，UL8-Osp.gRNA表达框在3’LTR区域，UL52-Osp.gRNA的表达框替换GFP表达框。质粒由基因合成NDA序列后再环化构成。VLP-CRISPR-Osp.gRNA示意图见图2,其VLP-CRISPR-UL52/UL8的gRNA表达盒分别是靶向UL52和UL8基因的。其向导序列可以是所选基因上的任意符合CRISPR向导序列规则的区域，即其序列3’-端含有PAM序列(NGG)的区域。
实施例5、VLP-CRISPR-UL52/UL8体外清除HSV-1和HSV-2
按照实施例4的方法生产VLP-CRISPR-UL52/UL8，质粒转染时，按照比例pMD.2G:pRSV-REV:pMDlg/PRRE-D64V:pMS2M-PH-Gag–Pol-D64V:pCMV-2×NLS-Cas9-6×MS2:pLV-U6-UL52-U3-UL8＝3.75：3：6.5：6.5：13：13，质粒总量和细胞数之间的关系为0.76μg/cm2，所用赋形剂为磷酸缓冲液。本实施例中以UL52的gRNA序列是5’-acggagcagccctcgcccct-3’，UL8的gRNA序列是5’-ggggcagccataccgcgtaa-3’做HSV基因组切割的gRNA。
按照实施例3中的方法，用每实验孔200ng p24VLP-CRISPR-UL52/UL8分别清除HSV-1和HSV-2，空斑试验检测细胞上清中HSV-1/HSV-2的分泌量。结果显示两者都能有效清除HSV-1。从清除HSV-2的效果检测结果看，VLP-CRISPR-UL52/UL8的效果高于VLP-CRISPR-UL29/UL8。数据见图5。StudentT-test做统计分析，**P<0.01,***P<0.001,n.s.代表不显著。
以上对本发明的具体实施例进行了描述。需要理解的是，本发明并不局限于上述特定实施方式，本领域技术人员可以在权利要求的范围内做出各种变形或修改，这并不影响本发明的实质内容。

Claims (10)

  1. 一种用于靶向HSV病毒的慢病毒载体，其包装质粒包括表达膜蛋白的质粒pMD.2G、表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒、表达野生型慢病毒GagPol长链蛋白的质粒、表达Cas9 mRNA的质粒和表达gRNA的质粒；所述表达gRNA的质粒中，
    将gRNA的骨架基因置于慢病毒载体的3’-LTR的区域，由启动子启动表达，对应的慢病毒载体记为VLP-CRISPR；
    或将一个gRNA的骨架基因置于慢病毒载体的3’-LTR的区域，另一个gRNA的骨架基因置于5’-LTR和3’-LTR区域之间，由启动非编码短序列RNA的启动子启动表达，对应的慢病毒载体记为VLP-CRISPR-2gRNA。
  2. 根据权利要求1所述的用于靶向HSV病毒的慢病毒载体，其特征在于，所述gRNA的骨架基因包括序列如SEQ ID NO.9所示的通用型gRNA或序列如SEQ ID NO.10所示的优化型Osp.gRNA。
  3. 根据权利要求1所述的用于靶向HSV病毒的慢病毒载体，其特征在于，所述表达含RNA结合蛋白的慢病毒GagPol长链蛋白的质粒，是将RNA结合蛋白n个拷贝分别置于慢病毒GagPol长链蛋白N-端或C-端或者同时N-端和C-端；n为RNA结合蛋白所能达到的任意拷贝数。
  4. 根据权利要求3所述的用于靶向HSV病毒的慢病毒载体，其特征在于，所述RNA结合蛋白的氨基酸序列如SEQ ID NO.2所示。
  5. 根据权利要求1所述的用于靶向HSV病毒的慢病毒载体，其特征在于，所述表达Cas9 mRNA的质粒，是将RNA结合蛋白所识别的茎环结构的基因置于Cas9基因的5’-端或/和3’-端；茎环结构数量为所能达到的任意拷贝数；Cas9基因的5’-端或/和3’-端均可以加入1到几个拷贝的核定位信号；所述茎环结构序列如SEQ ID NO.3所示。
  6. 根据权利要求5所述的用于靶向HSV病毒的慢病毒载体，其特征在于，Cas9包括Sp.Cas9、Sa.Cas9、xCas9、SpG、SpRY、SpCas9-HF1中的任意一种或任意一种的变体。
  7. 一种用于单纯疱疹病毒感染性疾病的基因治疗药物，其特征在于，利用如权利要求1-6中任一项所述的慢病毒载体递送靶向HSV复制相关基因的gRNA，HSV复制相关的基因包括UL5、UL8、UL9、UL29、UL30、UL42、UL52。
  8. 根据权利要求7所述的基因治疗药物，其特征在于，将靶向HSV复制相关的基 因中任意一个或者两个基因的向导序列分别放到VLP-CRISPR或者VLP-CRISPR-2gRNA中gRNA骨架前端。
  9. 根据权利要求7所述的基因治疗药物，其特征在于，所述靶向HSV复制相关基因为UL29、UL8，得到慢病毒载体VLP-CRISPR-UL29/UL8。
  10. 根据权利要求7所述的基因治疗药物，其特征在于，所述靶向HSV复制相关基因为UL52、UL8，得到慢病毒载体VLP-CRISPR-UL52/UL8。
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