WO2024113302A1 - Grna targeting hsv essential genes, crispr/cas gene editing system, delivery system, and use - Google Patents

Abstract

Disclosed are gRNA targeting HSV essential genes, a CRISPR/Cas gene editing system, a delivery system, and use. Aiming at an HSV clinical strain genome sequence, the present invention designs and screens a gRNA series that directly targets the HSV essential genes VP16, ICP27, ICP4, or gD, and uses a viral vector or a non-viral vector to deliver nuclease Cas9 and gRNA targeting a viral gene. The HSV key gene-targeted gene editing can effectively inhibit HSV replication and spread of infection, and the gRNA combination cocktail strategy shows an effect superior to that of single-target editing. The method of the present invention and the gene therapy mediated by the viral vector can effectively eliminate HSV viruses with latent infections or recurrent infections, thus providing a potentially important means for the treatment of refractory HSV-1-related diseases such as viral encephalitis, herpes simplex keratitis, and infectious blindness.

Description

gRNA targeting HSV essential genes, CRISPR/Cas gene editing system, delivery system and application Technical Field

The patent of this invention relates to a method of using CRISPR/Cas gene editing for gene therapy, and in particular to gRNA targeting HSV essential genes, a CRISPR/Cas gene editing system, a delivery system and applications.

Background technique

Herpes simplex virus 1 (HSV-1) is the prototype member of the human herpesviridae family. Epidemiological surveys have shown that herpes simplex virus type 1 (HSV-1) is very common and prevalent worldwide, with a global prevalence of about 70%, and most infections occur in childhood. HSV-1 is a highly contagious virus that is mainly transmitted through oral-oral contact. Autopsy studies have shown that the HSV-1 viral genome is present in the trigeminal ganglion of 85-90% of individuals, and is the most common cause of cold sores, viral encephalitis, and infectious blindness. HSV-1 usually causes mild or even asymptomatic infections in healthy or immunocompetent individuals, but can cause severe corneal infections and neuroencephalitis caused by severe central nervous system (CNS) infections in newborns or immunocompromised patients.

Clinical symptoms caused by HSV infection include latent infection without clinical symptoms, oral ulcers and corneal inflammation, and severe central nervous system infection causing neurological encephalitis. In North America, HSV-1 is the most common cause of encephalitis in children and adults. HSV-1 is a common neurotropic pathogen that can reach the central nervous system without obvious clinical symptoms. HSV-1 can infect the terminals of olfactory neurons and enter the CNS through retrograde axonal transport of neurons, eventually reaching the olfactory bulb in the brain. HSV-1 can also be reactivated from neurons in the trigeminal ganglion and reach the skin or CNS through anterograde transport; after entering the CNS, the virus can either remain dormant in this tissue or be activated to cause severe acute necrotizing encephalitis. Herpes simplex encephalitis (HSE) is one of the most serious manifestations of HSV-1 infection, with a mortality rate of up to 97%. Studies have shown that although the use of the antiviral drug acyclovir (ACV) can reduce the mortality rate of HSE patients, most patients have permanent neurological sequelae after recovery, including symptoms of cognitive, memory and behavioral disorders, and an increased incidence of epilepsy.

HSV-1 infection can cause a chronic immune inflammatory disease in the cornea, herpes simplex keratitis (HSK), which can cause irreversible damage and blindness in infected people. During ocular infection, the virus enters the corneal epithelium from the trigeminal ganglion latently and causes an inflammatory response through reactivation. Patients are characterized by corneal opacity and new blood vessel formation on the surface of the eye. Recurrent inflammation caused by periodic reactivation of the virus may even lead to blindness. Studies have shown that HVEM is an important host cell protein receptor in the immune pathogenesis of HSV-1 infection in the eye.

There is currently no vaccine for the prevention of HSV infection. Both primary viral infection and reactivation after latent infection may cause chronic inflammatory processes of varying degrees. Commonly used clinical therapeutic drugs are nucleoside analogs represented by oral acyclovir. Since the 1980s, ACV and its derivatives have become the first choice for the prevention and treatment of herpes virus infections. The continuous emergence of drug-resistant strains has also put forward higher requirements for antiviral treatment. Due to the large prevalence of HSV-1 and the serious problem of latent infection, people have to pay attention to the problem of drug resistance, and it also suggests that the development of new drugs is imminent.

Herpes simplex virus type 1 (HSV-1) is a major cause of neuroencephalitis and infectious blindness. However, current drugs for the treatment of HSV cannot eliminate latent HSV virus or reactivated virus in the trigeminal ganglion. Therefore, the development of new therapeutic strategies against latent HSV has become a priority. CRISPR-based genome editing technology represents a promising approach to combat viral infection and replication by modifying or destroying the viral genetic material.

Summary of the invention

In order to address the deficiencies in the prior art, the present invention aims to provide gRNA, CRISPR/Cas gene editing system, delivery system and application targeting HSV essential genes.

The present invention designs and screens a series of gRNAs that directly target HSV essential genes VP16, ICP27, ICP4 or gD, VP16 (2gRNA), ICP27 (2gRNA), ICP4 (2gRNA) or gD (4gRNA), and specifically provides new technologies, methods and delivery strategies based on CRISPR-Cas gene editing to resist HSV infection.

The technical solution of the present invention is as follows:

The first aspect of the present invention provides a gRNA targeting an essential gene of the HSV virus, wherein the gRNA is selected from one of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.10.

Furthermore, the gRNA is selected from one of the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.7.

A second aspect of the present invention provides a gRNA composition targeting essential genes of the HSV virus, wherein the gRNA composition is selected from two or more of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.10.

Furthermore, the gRNA composition is a composition of the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.7.

The third aspect of the present invention provides a gRNA expression vector targeting essential genes of the HSV virus, which comprises the gRNA, or the gRNA composition.

Furthermore, the expression vector is a viral vector or a non-viral vector;

The viral vector is a lentiviral vector, a retroviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an adenoviral vector or a virus-like particle (VLP);

The non-viral vector is a plasmid or mRNA.

A fourth aspect of the present invention provides a CRISPR/Cas gene editing system, which comprises the gRNA, or the gRNA composition, and a Cas protein.

Further, the Cas protein is one or more of SpCas9 protein derived from Streptococcus pyogenes, Cas9 from Neisseria meningitidis, Cas9 from Streptococcus thermophilus, Cas9 from Staphylococcus aureus, Cpf1 from Acidaminococcus, Cpf1 from Corynebacterium glutamicum, micro-SaCas9 from Staphylococcus aureus, and C2c2 from Oral Cibotium;

Preferably, the Cas protein is SpCas9 protein derived from Streptococcus pyogenes.

A fifth aspect of the present invention provides a delivery system for the CRISPR/Cas gene editing system, the delivery system comprising:

1) the gRNA expression vector;

2) Cas protein expression vector.

Furthermore, the Cas protein expression vector is a viral vector or a non-viral vector;

The viral vector is a lentiviral vector, a retroviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an adenoviral vector or a virus-like particle (VLP);

The non-viral vector is a plasmid or mRNA.

A sixth aspect of the present invention provides a kit comprising:

1) the gRNA, or the gRNA composition, or the gRNA expression vector;

2) Cas protein expression vector.

Further, the Cas protein is one or more of SpCas9 protein derived from Streptococcus pyogenes, Cas9 from Neisseria meningitidis, Cas9 from Streptococcus thermophilus, Cas9 from Staphylococcus aureus, Cpf1 from Acidaminococcus, Cpf1 from Corynebacterium glutamicum, mini-SaCas9 from Staphylococcus aureus, and C2c2 from Oral Cibotium;

Preferably, the Cas protein is SpCas9 protein derived from Streptococcus pyogenes.

Furthermore, the Cas protein expression vector is a viral vector or a non-viral vector;

The viral vector is a lentiviral vector, a retroviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an adenoviral vector or a virus-like particle (VLP);

The non-viral vector is a plasmid or mRNA.

The seventh aspect of the present invention provides the use of the gRNA, or the gRNA composition, or the gRNA expression vector, or the CRISPR/Cas gene editing system, or the delivery system, or the kit in the preparation of drugs for preventing and/or treating HSV infection-related diseases.

Furthermore, the HSV infection-related disease is an HSV-1 or HSV-2-related disease;

Preferably, the HSV infection-related diseases include viral encephalitis, herpes simplex virus keratitis and infectious blindness.

The eighth aspect of the present invention provides the use of the gRNA, or the gRNA composition, or the gRNA expression vector, or the CRISPR/Cas gene editing system, or the delivery system, or the kit in the preparation of drugs for preventing and/or treating anti-HSV.

Furthermore, the HSV is HSV-1 or HSV-2.

The ninth aspect of the present invention provides a pharmaceutical composition comprising a delivery system for the CRISPR/Cas gene editing system.

The tenth aspect of the present invention provides a method for inhibiting HSV replication and/or expression of HSV essential genes, the method comprising: before or after HSV infection, transfecting the target cells with the delivery system of the CRISPR/Cas gene editing system, and the ribonucleoprotein complex formed by the Cas-gRNA expressed by the cells targetedly and precisely shears and destroys the HSV genome, thereby blocking viral replication and transmission.

The beneficial effects of the present invention are:

The present invention provides a new technology for resisting herpes simplex virus infection by using CRISPR-Cas genome editing technology. A series of gRNAs directly targeting HSV essential genes VP16, ICP27, ICP4 or gD are designed and screened for the genome sequence of HSV clinical strains, and a gene therapy viral vector or a non-viral vector is used to deliver nuclease Cas9 and gRNA targeting viral genes. Gene editing targeted to HSV key genes can effectively inhibit HSV replication and spread infection, and each gRNA can effectively inhibit HSV viral replication when mediated alone. Among them, the gRNAs of aVP16-2, aICP27-2, aICP4-2 or agD-1 can inhibit HSV viral replication better than the same group, and the gene editing of gRNAs in pairs can more effectively inhibit HSV viral replication. The mixed gene editing (named Cocktail) of four gRNAs of aVP16-2, aICP27-2, aICP4-2 and agD-1 inhibits HSV viral replication better than the gene editing mediated by a single gRNA, and the cocktail strategy of gRNA combination shows an effect better than single target editing. In animal experiments, in vivo gene editing using CRISPR-Cas9/gRNA delivered by viral vectors can effectively block HSV replication and transmission.

The present invention uses the method of gRNA mixed gene editing (Cocktail), whether it is a prevention strategy or a treatment strategy Cocktail, which can effectively inhibit HSV virus replication and inter-tissue transmission. The earlier the gRNA mixed gene editing (Cocktail) treatment is after HSV infection, the more obvious the inhibitory effect. The current drugs for treating HSV cannot eliminate the HSV virus lurking in the trigeminal ganglion or the virus relapses. The gene therapy mediated by the method of the present invention and viral vectors can effectively eliminate latently infected or recurrently infected HSV viruses, providing a potential important means for the treatment of refractory HSV-1 related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a diagram showing the screening of HSV target genes for CRISPR gene editing and the design of gRNA;

Figure 2 is an identification diagram of CRISPR Cas9/gRNA plasmid;

Figure 3 is a diagram showing the effect of Cas9/gRNA single plasmid transfection and anti-HSV replication;

Figure 4 is the determination of virus titer in the cell supernatant after Cas9/gRNA single plasmid transfection editing;

Figure 5 is a diagram showing the design of the Cas9/gRNA plasmid optimization dual-plasmid combination strategy and the anti-HSV replication effect;

Figure 6 is a diagram showing the cocktail strategy of four types of gRNAs of YouScreen and the anti-HSV replication effect at the cellular level;

FIG7 is a determination of virus titer in the cell supernatant after editing using the cocktail combination strategy;

Figure 8 is a comparison of the preventive and therapeutic effects of Cocktail multi-target editing;

FIG9 is a diagram showing the packaging preparation of gene delivery Cas9/gRNA lentivirus and its anti-HSV effect;

Figure 10 shows that the LV-Cas9/gRNA control virus cannot inhibit HSV replication;

Figure 11 is a diagram showing the effect of LV-Cas9/gRNA single virus on anti-HSV replication in vivo;

Figure 12 shows the LV-Cas9/gRNA virus cocktail strategy against HSV replication;

FIG13 shows the dose effect of LV-Cas9/gRNA virus combination (Cocktail);

Figure 14 is a diagram showing the effect of LV-Cas9/gRNA virus combination (Cocktail) in treating HSV infection.

Detailed ways

The present invention is described in detail below in conjunction with the accompanying drawings and embodiments. Unless otherwise specified, all are conventional techniques in the art. The reagents or materials, unless otherwise specified, are derived from commercial channels.

The present invention designs and screens a series of gRNAs that directly target HSV essential genes VP16, ICP27, ICP4 or gD, VP16 (2 gRNAs), ICP27 (2 gRNAs), ICP4 (2 gRNAs) or gD (4 gRNAs). Based on CRISPR/Cas9 gene editing technology, the present invention uses vectors to deliver Cas9/gRNA to target cells to achieve editing of targeted genes. The vectors include commonly used viral vectors and non-viral vectors, wherein viral vectors include lentiviral vectors, retroviral vectors, adeno-associated viral vectors, herpes simplex virus vectors, adenoviral vectors, virus-like particles (VLPs), etc.; non-viral vectors include plasmids, mRNA, etc.

In a specific embodiment, the present invention constructs 10 lentiviral vector core plasmids carrying SpCas9 and corresponding gRNA, and then packages and prepares lentiviral vectors of gene delivery CRISPR editing system. First, the inhibitory effects of 10 SpCas9/gRNAs were screened in cell experiments. Two days after infection with H129-GFP virus, the virus in the control group (NC and Vec) rapidly proliferated, and almost all cells expressed green fluorescent protein, while the fluorescence ratios of the 10 gRNA groups targeting the cutting of 4 genes decreased to varying degrees; by detecting the HSV virus titer of the cell supernatant, the virus titer of the experimental group was significantly lower than that of the control group, and the inhibitory effects of the four gRNA groups of agD-1, aICP4-2, aVP16-2, and aICP27-2 were screened out as the most obvious, thus serving as an alternative for packaging lentiviral vectors.

In addition, the combined gRNA strategy ("cocktail" strategy) clearly showed superior effects over single gRNA gene editing. The present invention tests whether the combination of Cas9/gRNA for targeted editing of 4 genes can achieve better HSV inhibition effects. Cells were transfected with gRNA plasmids of different concentrations (0.5μg, 1μg, 2μg) in combination (Cocktail), and then the HSV-GFP virus was infected with the transfected cells at MOI=0.01 and MOI=0.001, respectively. It was found that the Cocktail group showed a significant inhibitory effect on HSV replication at low doses, and there was an obvious dose effect. As the amount of Cas9/gRNA plasmids increased, the stronger the effect of inhibiting viral proliferation. These results show that the combined Cas9/gRNA strategy for targeted editing of 4 genes can effectively inhibit HSV viral replication and proliferation, and show a significant dose effect.

In response to the question of the therapeutic effect of the CRISPR editing system, the present invention conducted an experiment of first infecting the HSV virus and then treating it with the CRISPR editing system. The HSV-GFP virus was infected 12 hours, 6 hours and 1 hour in advance, respectively, and then the Cas9/gRNA-cocktail plasmid was transfected. It was found that during the high-speed proliferation stage of acute HSV virus infection (12 hours of viral proliferation), Cas9/gRNA intervention had a certain inhibitory effect but it was not obvious. If intervention was given earlier after HSV infection - CRISPR treatment in the 6-hour viral proliferation group and the 1-hour viral proliferation group could significantly reduce the proliferation of HSV virus, and the earlier the intervention, the better.

The present invention uses packaged lentivirus to carry out animal experiments in vivo. In the in vivo experiment, the Cas9/gRNA combination (Cocktail) strategy can effectively inhibit virus proliferation and spread. In the control group (NC) without any intervention, and the lentivirus empty control group (Vec), the virus has been efficiently proliferated 5 days after H129-GFP was injected into the mouse V1 brain region, and spread to brain regions such as LGd along the neural circuit. The infected neurons are highlighted by fluorescent protein, and the lentivirus empty administration has no therapeutic effect. When the lentivirus carrying the expression of Cas9/gRNA is used for treatment alone, the effects of inhibiting viral replication (few fluorescent cells at the injection site) and viral spread (no mark in the LGd brain region) are shown, and the effects of the two groups of LV-aICP4 and LV-aICP27 are better than LV-gD and LV-aVP16. The Cas9/gRNA combination (Cocktail) treatment effect is relatively the best, and no or only a small number of cells are observed to be infected and marked at the injection site V1, and it also shows a dose effect. Another important thing about the present invention is that it also verifies the "infection first, then treatment" experiment, where the mouse V1 brain region is first infected with H129-GFP, and then lentivirus cocktail treatment is administered one day later. It is found that the replication and spread of HSV can be effectively inhibited, and the effect is better than single lentivirus administration.

CRISPR gene editing targeting the four key genes of HSV, VP16, ICP27, ICP4, and gD, can effectively inhibit the replication and proliferation of HSV in the lytic replication cycle, and the Cas9/gRNA combination (Cocktail) strategy is more efficient. It can be imagined that the use of CRISPR genome editing technology to eliminate latent HSV viruses in a latent infection state with much lower viral titers may have a more significant inhibitory effect.

In summary, the Cas9/gRNA genome editing of the present invention can effectively inhibit the infection and spread of HSV at the prevention level and the treatment level, especially in the in vivo animal experiment. Compared with the control group, whether Cas9/gRNA is administered alone or in combination with Cocktail, the proliferation of HSV is significantly inhibited, and the number of labeled nerve cells decreases exponentially compared with the control group. In addition, no related toxic side effects were observed in the Cas9/gRNA combination (Cocktail) treatment, indicating the safety of this strategy. This method of eliminating HSV infection using CRISPR genome editing provides a new means for the potential treatment of refractory HSV-1 related diseases, especially when traditional treatments are ineffective.

The present invention is described in detail below with reference to the embodiments.

Example 1: Screening of HSV target genes for CRISPR gene editing and design and synthesis of gRNA

HSV essential genes VP16, ICP27, ICP4 and gD were selected as potential targets for CRISPR/Cas9 strategy editing. In this example, four essential viral genes were designed and targeted, and guide RNAs combined with Cas9 were developed to test their ability to inhibit HSV-1 replication.

sgRNA was designed based on the target site sequence of the selected gene and screened by CRISPR DESIGN system (http://crispr.mit.edu/). The sgRNA oligonucleotide sequences are shown in Table 1. A total of 10 gRNAs were designed: VP16 targeting (2 gRNAs, sequences see SEQ ID NO.1 and 2), ICP27 targeting (2 gRNAs, sequences see SEQ ID NO.3 and 4), ICP4 targeting (2 gRNAs, sequences see SEQ ID NO.5 and 6) and gD targeting (4 gRNAs, sequences see SEQ ID NO.7-10) (Figure 1, Table 1). The corresponding 10 core plasmids of lentiviral vectors carrying SpCas9 and gRNA were constructed and verified (Figure 1) and the corresponding lentivirus was packaged.

Table 1 CRISPR/Cas9-edited HSV1 gene and designed sgRNA sequences

Example 2: Construction of CRISPR Cas9/gRNA lentiviral core plasmid

sgRNA was designed according to the target sequence of the selected gene and screened by the CRISPR design system (http://crispr.mit.edu/). The sgRNA oligonucleotide sequences are shown in Table 1. The empty vector lentiCRISPR v2 vector (addgene, #52961, sequence see SEQ ID NO.11) was digested with BsmBI, and the annealed oligonucleotides were cloned into the single guide RNA scaffold to obtain the CRISPR Cas9/gRNA lentiviral core plasmid. The packaging plasmid was then used to produce lentiCRISPR-sgRNA viruses in HEK293T cells as previously described (Cold Spring Harb Protoc. 2018 Apr 2; 2018 (4)). In brief, the above lentivirus constructs were co-transfected into 293T cells with three lentiviral packaging vectors (pGAG/POL, pREV, and pMD2.G), and the supernatant was collected 2 to 3 days later to obtain the targeted virus. The titers of the virus stocks of the five lentiviruses produced (LV-vec, LV-agD-1, LV-aVP16-2, LV-aICP4-2, and LV-aICP27-2) were all 1×10 ⁸ PFU/ml.

Example 3: Identification of CRISPR Cas9/gRNA plasmid

After the plasmid was constructed, it was sent to a sequencing company for sequencing, and the sequencing result was successful. At the same time, the plasmid was used as a template to amplify the target fragment. The reaction system and PCR program were as follows:

PCR procedure:

After the PCR program was completed, 1% agarose gel was prepared and run in TAE electrophoresis buffer at 180V for 23 minutes. The results are shown in Figure 2. The target fragments of the ten plasmids were consistent in size and were identified correctly.

Example 4: Cas9/gRNA single plasmid transfection and anti-HSV replication effect

BHK cells in the logarithmic growth phase were collected and inoculated into six-well plates at a density of 1×10 ⁶ cells/mL. After the cells adhered to the wall, the cell culture medium was replaced with 400μL Opti-MEM serum-free medium. Prepare a 1.5mL sterile EP tube, add 2μg Cas9/gRNA single plasmid, 50μL Opti-MEM medium, gently shake and mix, and incubate at room temperature for 5min. Then add 4μL Lipofectamine 2000 reagent and 50μL Opti-MEM medium to the sterile EP tube, shake and mix gently, and let stand at room temperature for 15min. Pipette 100μL of the mixture of plasmid and Lipofectamine 2000 into the plate wells, mix well, and culture in a cell culture incubator at 37℃, 5% CO _2. After transfection for 24h, discard the cell supernatant. Each well was infected with 100 μL HSV 129-EGFP at MOI = 0.1, and the virus solution was discarded after adsorption for 1 hour, and 2 mL of DMEM medium containing 2% serum was added, and cultured in a cell culture incubator at 35°C, 3% CO ₂ for 48 hours. The cells were observed under a fluorescence microscope and photographed.

The virus supernatant was collected and the virus titer was measured by plaque assay. BHK cells in the logarithmic growth phase were taken and inoculated into 12-well plates at a density of 2×10 ⁵ /mL, and the cells were allowed to adhere to the wall. The virus supernatant was diluted 10 times 6 times, and 100 μL of the diluted virus supernatant was successively drawn into the well plate. After adsorption for 1 hour, the virus liquid was discarded, and an equal volume of 4% DMEM culture medium and 1% agarose solution were added. After standing, the agarose concentration was placed in a 35°C, 3% CO ₂ cell culture incubator for 24 hours, and the spots were observed and counted under a fluorescence microscope. The results are shown in Figure 3. Compared with the control group, the expression of green fluorescent protein was reduced after Cas9/gRNA single plasmid transfection.

In addition, the titer of HSV is another measure of the effectiveness of Cas9/gRNA compared with the control group, as shown in Figure 4, which shows a significant reduction in the HSV titer in the Cas9/gRNA transfection group. Among them, agD-1, aICP4-2, aICP27-2, and aVP16-2 had the best inhibitory effect on the proliferation of HSV, so these Cas9/gRNA plasmids were selected for subsequent experiments and lentiviral packaging.

Example 5: Cas9/gRNA plasmid optimization dual plasmid combination strategy design and anti-HSV replication effect

The four Cas9/gRNA plasmids agD-1, aICP4-2, aICP27-2, and aVP16-g2 with the highest inhibition efficiency on HSV replication in Case 4 were selected and combined in pairs (agD-g1+aICP4-g2; agD-g1+aICP27-g2; agD-g1+aVP16-g2; aICP27-g2+aICP4-g2; aVP16-g2+aICP4-g2; aICP27-g2+aVP16-g2;) and transfected into BHK cells together. After 24 hours, the cells were infected at an MOI of 0.1. After adsorption for 1 hour, the virus solution was discarded, 2 mL of DMEM medium containing 2% serum was added, and the cells were cultured in a cell culture incubator at 35°C and 3% _CO2 for 48 hours. The cells were observed and imaged using a fluorescence microscope. As shown in Figure 5, the expression of green fluorescent protein was reduced after the optimized double-plasmid group was combined and transfected compared with the control group.

Example 6: Cocktail strategy of four types of gRNAs of YouScreen and anti-HSV replication effect at the cellular level

The four plasmids were mixed and transfected into BHK cells according to a concentration gradient. Each plasmid was transfected with 0.5μg, 1μg and 2μg. After 24 hours, HSV H129-EGFP was infected at MOI=0.001 and MOI=0.01, respectively. Cell fluorescence expression was imaged after 48 hours, and the supernatant virus titer was measured. The results are shown in Figures 6 and 7. From the perspective of HSV titer or the expression abundance of the reporter gene green fluorescent protein, the cocktail strategy can effectively inhibit the proliferation of HSV. HSV infection MOI=0.01 or 0.001, a clear trend can be seen at 2d.p.i. In addition, no significant statistical difference in virus titer was detected between different dose groups, indicating that the cocktail strategy had no dose effect in this set of experiments.

Example 7: Comparison of preventive and therapeutic effects of cocktail multi-target editing

To mimic the treatment of HSV-infected patients, cells were first infected with HSV H129-GFP (MOI=0.01), and then co-transfected with four gRNA plasmids at 1H, 6H, and 12H after HSV infection. Compared with 6H.P.I and 12H.P.I., the best inhibitory effect was shown by the plasmid expression of Cas9/gRNA in 1H.P.I. The Cocktail multi-target editing prevention group expressed Cas9/gRNA 24 hours before viral infection and also showed good inhibitory effect. As shown in Figure 8.

Example 8: Packaging and preparation of gene delivery Cas9/gRNA lentivirus

1) 24 hours before transfection, digest the 293T cells in the logarithmic growth phase with trypsin, adjust the cell density to 1.2 x ¹⁰⁷ cells/20ml with a medium containing 10% serum, re-inoculate in a 15cm cell culture dish, and culture in a 37℃, 5% _CO2 incubator. When the cell density reaches 70% to 80% after 24 hours, it can be used for transfection. The cell state is crucial for virus packaging, so it is necessary to ensure a good cell state and a low number of passages.

2) 2 h before transfection, the cell culture medium was replaced with serum-free medium.

3) Add to a sterile centrifuge tube:

Each prepared DNA solution (20 μg of pGC-LV vector, 15 μg of pHelper 1.0 vector, and 10 μg of pHelper 2.0 vector) was mixed evenly with the corresponding volume of Opti-MEM, and the total volume was adjusted to 2.5 ml. The solution was incubated at room temperature for 5 minutes.

4) Gently shake the Lipofectamine 2000 reagent, take 100 μl of Lipofectamine 2000 reagent and mix it with 2.4 ml Opti-MEM in another tube, and incubate at room temperature for 5 minutes.

5) Mix the diluted DNA with the diluted Lipofectamine 2000 and gently invert to mix without shaking. Mixing must be done within 5 minutes.

6) After mixing, incubate at room temperature for 15 to 20 minutes to form a transfection complex of DNA and Lipofectamine 2000 dilution solution.

7) Transfer the mixture of DNA and Lipofectamine 2000 into the culture medium of 293T cells, mix well, and culture in a cell culture incubator at 37°C, 5% CO2.

8) After culturing for 4 to 6 hours, the culture medium containing the transfection mixture was discarded, 10 ml of cell culture medium containing 10% serum was added to each culture dish cell, and the cells were cultured in a 37°C, 5% CO ₂ incubator for 48 hours. The supernatant was then collected to obtain the packaged lentivirus.

293T cells (human kidney epithelial cell line) were then used to further verify the inhibitory effect of the lentivirus expressing Cas9/gRNA designed to target HSV replication. The results were consistent with the plasmid effect on BHK cells. The results not only showed that Cas9/gRNA editing can effectively inhibit HSV replication in different cell lines, but also that the packaged lentivirus has a good effect. See Figure 9.

Example 9: LV-Cas9/gRNA control virus cannot inhibit HSV replication

On day 0, 5 × 10 ⁴ TU of empty lentivirus and PBS were injected into adult male C57BL/6 mice (n=3). Animals were anesthetized by intraperitoneal injection (80 mg/kg) of sodium pentobarbital and placed in a stereotaxic apparatus. All animals were kept anesthetized with isoflurane (1-1.5%) during surgery and virus injection. The skull above the target area coordinates: V1 (AP, -2.80 mm; ML, -2.40 mm; and DV, -0.90 mm) was thinned with a dental drill, and skull fragments were carefully removed. A microinjector was connected to a glass micropipette with a tip diameter of 10-15 mm, and injections were performed at a rate of 30 nl per minute using a syringe pump. After the injection was completed, the mouse was left for an additional 10 minutes, and then the needle was slowly withdrawn. After surgery, lincomycin hydrochloride and lidocaine hydrochloride gel should be applied to the sutured wound to prevent inflammation and relieve pain in the mice. On day 5, 500 H129-EGFP viral particles were injected into the same brain region.

The results of the experiment in which PBS and LV-VEC were injected into the primary visual cortex V1 as controls and then H129-EGFP was injected 5 days later showed that a large amount of EGFP expression could be observed in the H129-EGFP injection site and the downstream output areas of V1, such as LGD and contralateral V1, indicating that the infection and replication of the disease were not inhibited (Figure 10).

Example 10: Effect of LV-Cas9/gRNA single virus on HSV replication in vivo

On day 0, 5×10 ⁴ TU of experimental groups of lentivirus expressing agD-1, aICP4-2, aICP27-2, aVP16-g2, respectively, were injected into adult male C57BL/6 mice (n=3). Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg) and placed in a stereotaxic apparatus. All animals were kept anesthetized with isoflurane (1-1.5%) during surgery and virus injection. The skull above the target region coordinates: V1 (AP, -2.80 mm; ML, -2.40 mm; and DV, -0.90 mm) was thinned with a dental drill, and skull fragments were carefully removed. A microinjector was connected to a glass micropipette with a tip diameter of 10-15 mm, and injections were performed at a rate of 30 nl per minute using a syringe pump. After the injection was completed, the mouse was left for an additional 10 minutes, and then the needle was slowly withdrawn. After surgery, lincomycin hydrochloride and lidocaine hydrochloride gel was applied to the sutured wound to prevent inflammation and relieve pain in mice. On day 5, 500 H129-EGFP virus particles were injected into the same brain area.

Lentivirus expressing agD-1, aICP4-2, aICP27-2, and aVP16-g2 were injected into the primary visual cortex V1 in advance, and then H129-EGFP was injected 5 days later. It was observed that there was no EGFP expression in the downstream output area LGD of the H129-EGFP injection site V1 and the contralateral V1, indicating that the infection and replication of the virus were significantly inhibited (see Figure 11).

Example 11: LV-Cas9/gRNA virus cocktail strategy to prevent HSV replication

On day 0, 5×10 ⁴ TU of a homogenous mixed preparation of lentivirus expressing agD-1, aICP4-2, aICP27-2, and aVP16-g2 were injected into adult male C57BL/6 mice (n=3). Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg) and placed in a stereotaxic apparatus. All animals were kept anesthetized with isoflurane (1-1.5%) during surgery and virus injection. The skull above the target area coordinates: V1 (AP, -2.80 mm; ML, -2.40 mm; and DV, -0.90 mm) was thinned with a dental drill, and skull fragments were carefully removed. A microinjector was connected to a glass micropipette with a tip diameter of 10-15 mm, and injections were performed at a rate of 30 nl per minute using a syringe pump. After the injection was completed, the mouse was left for an additional 10 minutes, and then the needle was slowly withdrawn. After surgery, lincomycin hydrochloride and lidocaine hydrochloride gel were applied to the sutured wound to prevent inflammation and relieve pain in mice. On day 5, 500 H129-EGFP virus particles were injected into the same brain area.

It can be observed that there is no EGFP expression in the downstream output region LGD of the H129-EGFP injection site V1 and the contralateral V1, indicating that the infection and replication of the virus are significantly inhibited (see Figure 12).

Example 12: LV-Cas9/gRNA virus combination (Cocktail) dose effect

On day 0, 1×10 ⁵ TU of a homogenous mixed preparation of lentivirus expressing agD-1, aICP4-2, aICP27-2, and aVP16-g2 were injected into adult male C57BL/6 mice (n=3). Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg) and placed in a stereotaxic apparatus. All animals were kept anesthetized with isoflurane (1-1.5%) during surgery and virus injection. The skull above the target area coordinates: V1 (AP, -2.80 mm; ML, -2.40 mm; and DV, -0.90 mm) was thinned with a dental drill, and skull fragments were carefully removed. A microinjector was connected to a glass micropipette with a tip diameter of 10-15 mm, and injections were performed at a rate of 30 nl per minute using a syringe pump. After the injection was completed, the mouse was left for an additional 10 minutes, and then the needle was slowly withdrawn. After surgery, lincomycin hydrochloride and lidocaine hydrochloride gel were applied to the sutured wound to prevent inflammation and relieve pain in mice. On day 5, 500 H129-EGFP virus particles were injected into the same brain area.

It can be observed that there is no EGFP expression in the downstream output area LGD and the contralateral V1 of the H129-EGFP injection site V1, indicating that the infection and replication of the virus are significantly inhibited. And compared with the experimental group injected with only 5×10 ⁴ TU, no neurons were observed to be infected by the virus at the injection site, indicating that the LV-Cas9/gRNA virus combination (Cocktail) has a dose effect. As shown in Figure 13.

Example 13: Effect of LV-Cas9/gRNA virus cocktail in treating HSV infection

On day 0, 500 H129-EGFP viral particles were injected into the same brain region into adult male C57BL/6 mice (n=3). Animals were anesthetized by intraperitoneal injection of sodium pentobarbital (80 mg/kg) and placed in a stereotaxic apparatus. All animals were kept anesthetized with isoflurane (1-1.5%) during surgery and virus injection. The skull above the target area coordinates: V1 (AP, -2.80 mm; ML, -2.40 mm; and DV, -0.90 mm) was thinned with a dental drill, and skull fragments were carefully removed. A microinjector was connected to a glass micropipette with a tip diameter of 10-15 mm, and virus injections were performed at a rate of 30 nl per minute using a syringe pump. After the injection was completed, the mouse was left for an additional 10 minutes, and then the needle was slowly withdrawn. After surgery, lincomycin hydrochloride and lidocaine hydrochloride gel should be applied to the sutured wound to prevent inflammation and relieve pain in the mice. On day 5, 5×10 ⁴ TU of lentivirus mixtures expressing agD-1, aICP4-2, aICP27-2, and aVP16-g2 were injected into the same brain region.

Compared with the two control groups, very little EGFP expression can be observed in the H129-EGFP injection site V1 and the contralateral V1, and no EGFP expression in the downstream output area LGD, indicating that the infection and replication of the virus are strongly inhibited (see Figure 14).

Claims (19)

1. A gRNA targeting an essential gene of the HSV virus, characterized in that the gRNA is selected from one of the nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.10.
2. The gRNA according to claim 1 is characterized in that the gRNA is selected from one of the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.7.
3. A gRNA composition targeting essential genes of HSV virus, characterized in that the gRNA composition is selected from two or more nucleotide sequences shown in SEQ ID NO.1-SEQ ID NO.10.
4. The gRNA composition according to claim 3 is characterized in that the gRNA composition is a composition of the nucleotide sequences shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 and SEQ ID NO.7.
5. A gRNA expression vector targeting an essential gene of the HSV virus, characterized in that it comprises the gRNA described in claim 1 or 2, or the gRNA composition described in claim 3 or 4.
6. The gRNA expression vector according to claim 5, characterized in that the expression vector is a viral vector or a non-viral vector;

   The viral vector is a lentiviral vector, a retroviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an adenoviral vector or a virus-like particle;

   The non-viral vector is a plasmid or mRNA.
7. A CRISPR/Cas gene editing system, characterized in that it comprises the gRNA described in claim 1 or 2, or the gRNA composition described in claim 3 or 4, and a Cas protein.
8. The CRISPR/Cas gene editing system according to claim 7, characterized in that the Cas protein is one or more of SpCas9 protein derived from Streptococcus pyogenes, encephalococcus Cas9, Streptococcus thermophilus Cas9, Staphylococcus aureus Cas9, Acidaminococcus Cpf1, Corynebacterium glutamicum Cpf1, Staphylococcus aureus micro-SaCas9, and oral filamentous fungi C2c2;

   Preferably, the Cas protein is SpCas9 protein derived from Streptococcus pyogenes.
9. The delivery system of the CRISPR/Cas gene editing system according to claim 7 or 8, characterized in that the delivery system comprises:

   1) The gRNA expression vector according to claim 5 or 6;

   2) Cas protein expression vector.
10. The delivery system according to claim 9, characterized in that the Cas protein expression vector is a viral vector or a non-viral vector;

    The viral vector is a lentiviral vector, a retroviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an adenoviral vector or a virus-like particle;

    The non-viral vector is a plasmid or mRNA.
11. A kit, characterized in that it comprises:

    1) The gRNA according to claim 1 or 2, or the gRNA composition according to claim 3 or 4, or the gRNA expression vector according to claim 5 or 6;

    2) Cas protein expression vector.
12. The kit according to claim 11, characterized in that the Cas protein is one or more of SpCas9 protein derived from Streptococcus pyogenes, encephalococcus Cas9, Streptococcus thermophilus Cas9, Staphylococcus aureus Cas9, Acidococcus ammoniae Cpf1, Corynebacterium glutamicum Cpf1, Staphylococcus aureus micro-SaCas9, and oral filamentous fungi C2c2;

    Preferably, the Cas protein is SpCas9 protein derived from Streptococcus pyogenes.
13. The kit according to claim 11, characterized in that the Cas protein expression vector is a viral vector or a non-viral vector;

    The viral vector is a lentiviral vector, a retroviral vector, an adeno-associated viral vector, a herpes simplex viral vector, an adenoviral vector or a virus-like particle;

    The non-viral vector is a plasmid or mRNA.
14. Use of the gRNA described in claim 1 or 2, or the gRNA composition described in claim 3 or 4, or the gRNA expression vector described in claim 5 or 6, or the CRISPR/Cas gene editing system described in claim 7 or 8, or the delivery system described in claim 9 or 10, or the kit described in any one of claims 11-13 in the preparation of drugs for preventing and/or treating diseases related to HSV infection.
15. The use according to claim 14, characterized in that the HSV infection-related disease is an HSV-1 or HSV-2-related disease;

    Preferably, the HSV infection-related diseases include viral encephalitis, herpes simplex virus keratitis and infectious blindness.
16. Use of the gRNA of claim 1 or 2, or the gRNA composition of claim 3 or 4, or the gRNA expression vector of claim 5 or 6, or the CRISPR/Cas gene editing system of claim 7 or 8, or the delivery system of claim 9 or 10, or the kit of any one of claims 11 to 13 in the preparation of drugs for the prevention and/or treatment of anti-HSV.
17. The use according to claim 16, characterized in that the HSV is HSV-1 or HSV-2.
18. A pharmaceutical composition, characterized in that it comprises the delivery system of the CRISPR/Cas gene editing system according to claim 9 or 10.
19. A method for inhibiting HSV replication and/or HSV virus essential gene expression, characterized in that the method comprises: before or after HSV infection, transfecting the target cells with the delivery system of the CRISPR/Cas gene editing system according to claim 9 or 10, and the ribonucleoprotein complex formed by the Cas-gRNA expressed by the cells targets and precisely shears and destroys the HSV genome, thereby blocking viral replication and transmission.

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