Low-virulence, long-acting and high-capacity herpes simplex virus vector and use thereof

Abstract

Provided are a low-virulence, long-acting and high-capacity herpes simplex virus vector and the use thereof. Both copies of the neurovirulence factor V34.5 gene in the HSV-1 viral genome are completely knocked out, and an exogenous gene expression cassette is inserted at the original neurovirulence factor V34.5 gene locus. The exogenous gene expression cassette is selected from expression cassette I or expression cassette II, wherein the expression cassette I comprises a promoter, a fluorescent protein gene, a 2A peptide encoding gene, a Cre encoding gene, a WPRE transcription enhancement element and PA, which are connected in sequence, and the expression cassette II comprises a promoter, a SpCas9 or other nuclease encoding genes, a 2A peptide encoding gene, a fluorescent protein gene, a promoter, a sgRNA for targeted editing of the target gene and PA, which are connected in sequence. The vector and the use thereof expand the application scope of attenuated ItHSV vectors in neural circuit labeling and gene delivery transduction, providing a new vector for neural circuit tracing labeling and long-term gene delivery and expression of exogenous genes.

Description

A low-toxic, long-acting, high-load herpes simplex virus vector and its application Technical Field

[0001] The present invention belongs to the field of biotechnology, relates to neurobiology and molecular virology, and specifically relates to a low-toxic, long-acting, high-load herpes simplex virus vector and its application. Background Art

[0002] In recent years, viral transsynaptic tracing has been increasingly used in neural circuit analysis. Traditional neural network tracing methods, such as dyes, compound tracers, and protein peptides, can be transported along axons but cannot cross synapses, effectively marking only localized neuronal morphology. Compared to traditional neural network tracing methods, neurotropic viruses offer distinct advantages as tracing tools: 1) efficient infection of neurons; 2) ability to spread across synapses; 3) controllable transsynaptic direction, allowing for specific reverse or forward transmission; 4) viral self-replication after crossing synapses, without signal attenuation; and 5) the ability to carry complex regulatory elements and diverse markers.

[0003] Currently, the commonly used neurotropic viruses include pseudorabies virus (PRV) and herpes simplex virus (HSV) from the alphaherpesviridae family, and rabies virus (RV) from the rhabdoviridae family. Others include vesicular stomatitis virus (VSV). Among them, PRV Bartha strain and RV are retrograde transsynaptic infections, and VSV can be transmitted in both directions. The HSV-1 H129 strain can specifically transmit anterograde transsynaptically, which is consistent with the direction of nerve impulse transmission and is very suitable for labeling output neural networks.

[0004] Herpes simplex virus type 1 (HSV1) is a ubiquitous, conditionally pathogenic pathogen. It is a large, enveloped virus with a diameter of approximately 200 nm. Its core is a double-stranded DNA genome of approximately 153 kb, consisting of covalently linked long segments (UL) and short segments (US). Each segment contains an inverted repeat sequence at the end, so it can form four isomers.

[0005] The neurotropic virus strain HSV-1 H129, due to its primary anterograde transsynaptic transmission within neural circuits and its large capacity for exogenous genes, has become one of the most promising tools for tracing anterograde neural circuits. After years of development as a neural tracing viral vector, HSV-1 H129 has been genetically engineered to produce a diverse array of anterograde neural circuit tracing tools, forming a relatively comprehensive suite of anterograde tracing tools.

[0006] Summary of the Invention

[0007] In order to address the deficiencies in the prior art, the present invention aims to provide a low-toxic, long-acting, high-load herpes simplex virus vector and its application.

[0008] The specific technical solutions of the present invention are as follows:

[0009] On the one hand, the present invention provides a low-toxicity, long-acting, high-load herpes simplex virus, which is prepared by completely knocking out two copies of the neurovirulence factor γ34.5 gene in the HSV-1 viral genome and inserting an exogenous gene expression cassette into the original gene site of the neurovirulence factor γ34.5. The exogenous gene expression cassette is selected from expression cassette I or expression cassette II, and the expression cassette I includes a promoter, a fluorescent protein gene, a 2A peptide encoding gene, a Cre encoding gene, a WPRE transcription enhancer element, and a PA connected in sequence. The expression cassette II includes a promoter, a SpCas9 or other nuclease encoding gene, a 2A peptide encoding gene, a fluorescent protein gene, a promoter, an sgRNA for targeting the target gene for editing, and a PA connected in sequence. Preferably, the HSV-1 virus is strain HSV-1 H129.

[0010] Furthermore, the promoter is selected from one of the group consisting of hUbC promoter, CMV promoter, CAG promoter, EF1α promoter, pTH promoter, and pChAT promoter;

[0011] The fluorescent protein gene is tdTomato or EGFP;

[0012] The 2A peptide is selected from one of P2A, T2A, E2A, and F2A.

[0013] Furthermore, the expression cassette I includes a promoter hUbC, an EGFP encoding gene, a P2A peptide encoding gene, a Cre encoding gene, a WPRE transcription enhancing element, and PA connected in sequence;

[0014] The expression cassette II includes a promoter CAG, a SpCas9 or other nuclease encoding gene, a P2A peptide encoding gene, an EGFP encoding gene, a promoter U6, an sgRNA targeting the target gene for editing, and PA connected in sequence.

[0015] The present invention also provides a low-toxic, long-acting, high-load herpes simplex virus, wherein the exogenous gene expression cassette is selected from expression cassette I.

[0016] The present invention also provides a low-toxic, long-acting, high-load herpes simplex virus, wherein the exogenous gene expression cassette is selected from expression cassette II.

[0017] The present invention also provides a method for preparing the low-toxic, long-acting, high-load herpes simplex virus, comprising the following steps:

[0018] (1) Knock out both copies of the neurovirulence factor γ34.5 gene in the HSV-1 viral genome to obtain a recombinant virus;

[0019] (2) constructing a targeting vector, wherein the targeting vector is a vector for inserting an exogenous gene expression cassette between the upstream and downstream homology arms of the neurovirulence factor γ34.5 gene of the vector pH129ΔRL1, the nucleotide sequence of the vector pH129ΔRL1 is shown in SEQ ID NO.1, the nucleotide sequence of the upstream homology arm of the neurovirulence factor γ34.5 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the downstream homology arm of the neurovirulence factor γ34.5 gene is shown in SEQ ID NO.3, and the exogenous gene expression cassette is selected from the above-mentioned expression cassette I or expression cassette II;

[0020] (3) The targeting vector obtained in step (2) and the recombinant virus prepared in step (1) are co-transfected into cells and then recombined. After spot picking and purification, the low-toxicity, long-acting, high-load herpes simplex virus is obtained.

[0021] The present invention also provides the use of a low-toxic, long-acting, high-load herpes simplex virus prepared by a targeting vector in which an exogenous gene expression cassette is selected from expression cassette I in neural circuit labeling.

[0022] Furthermore, the neural circuit marker is a reverse neural circuit marker.

[0023] Furthermore, the low-toxicity, long-acting, high-load herpes simplex virus prepared by the targeting vector containing the exogenous gene expression cassette selected from expression cassette I is injected into an animal expressing the Cre-dependent fluorescent protein gene, or the low-toxicity, long-acting, high-load herpes simplex virus prepared by the targeting vector containing the exogenous gene expression cassette selected from expression cassette I is co-injected with a virus expressing the Cre-dependent fluorescent protein gene.

[0024] The present invention also provides the use of a low-toxic, long-acting, high-load herpes simplex virus prepared by a targeting vector in which an exogenous gene expression cassette is selected from expression cassette II in target gene editing.

[0025] The present invention also provides a gene editing vector, which is a low-toxic, long-acting, high-load herpes simplex virus prepared by a targeting vector in which an exogenous gene expression cassette is selected from expression cassette II, and carries gene editing expression cassette elements, including a promoter, SpCas9 or other nuclease encoding gene, 2A peptide encoding gene, fluorescent protein gene, promoter, sgRNA for targeted editing of the target gene, and PA connected in sequence.

[0026] The beneficial effects of the present invention are:

[0027] The present invention is based on the fact that the ltHSV-hUbC-tdTomato-WPRE recombinant virus after attenuation of the HSV-1 virus has low toxicity, the ability to absorb reverse markers through axon terminals, and exhibits very stable and efficient exogenous gene expression capabilities. It is further transformed into a viral vector for delivering Cre recombinase to expand the application range of ltHSV in neural circuit labeling, named ltHSV-hUbC-EGFP-2A-Cre-WPRE, or transformed into a viral vector for targeted editing of target genes, for editing target genes, named ltHSV-SpCas9-2A-EGFP-sgtdT4, providing a new vector system for neural circuit tracing labeling and long-term gene delivery and expression of exogenous genes.

[0028] Using ltHSV carrying Cre recombinase in Ai14 mice, we tracked brain regions upstream of the ventral tegmental area of ​​the midbrain, confirming the ltHSV's ability to stably and efficiently reverse-directedly label and deliver genes. In vitro and in vivo experiments confirmed the ltHSV's ability to efficiently deliver and edit genes, demonstrating the CRISPR / Cas9-carrying ltHSV's ability to efficiently deliver and edit genes. This invention provides a new vector system for tracing neural circuits and for long-term gene delivery and expression of large or multiple exogenous genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 is a schematic diagram of the ltHSV-hUbC-EGFP-2A-Cre viral genome and its recombinant vector construction; Figure 1A is a schematic diagram of the ltHSV-hUbC-EGFP-2A-Cre viral genome; Figure 1B is the agarose gel electrophoresis result of the gene fragment P2A-Cre; Figure 1C is the agarose gel electrophoresis result of pH129ΔRL1-hUbC-EGFP-WPRE-PA after BsrGI digestion; Figure 1D is the agarose gel electrophoresis result of single colony PCR products;

[0030] Figure 2 shows the recombination, purification and molecular identification of the ltHSV-hUbC-EGFP-2A-Cre virus; Figure 2A shows the molecular identification results of the ltHSV-hUbC-EGFP-2A-Cre virus; Figure 2B shows a fluorescence image of BHK-21 cells infected with the ltHSV-hUbC-EGFP-2A-Cre recombinant virus;

[0031] Figure 3 is a comparison of the growth curves of ltHSV-hUbC-EGFP-2A-Cre, H129-WT, and the parent virus ltHSV-hUbC-tdTomato-WPRE;

[0032] Figure 4 is a cell-level verification of the expression efficiency of Cre recombinase delivered by the ltHSV-hUbC-EGFP-2A-Cre vector;

[0033] FIG5 is an in vivo characterization of the expression efficiency of Cre recombinase delivered by the ltHSV-hUbC-EGFP-2A-Cre vector;

[0034] Figure 6 is a schematic diagram of the genome structure of the ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing virus and its recombinant vector molecular construction;

[0035] Figure 7 shows the reconstruction, purification and molecular identification of the ltHSV-SpCas9-2A-EGFP-sgtdT4 viral vector;

[0036] Figure 8 shows that the ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing vector has high and precise editing efficiency at the in vitro cell level;

[0037] Figure 9 shows an in vivo experiment in Ai14 mice verifying that the ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing virus has high delivery and editing efficiency. DETAILED DESCRIPTION

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0039] Example 1: Construction of pH129ΔRL1-hUbC-EGFP-2A-Cre-WPRE-PA

[0040] The ltHSV-hUbC-tdTomato-WPRE virus is obtained by completely knocking out both copies of the neurovirulence factor γ34.5 gene in the HSV-1 H129-WT viral genome. The virus is prepared based on a targeting vector, which is a vector in which a complete fluorescent protein tdTomato expression cassette is inserted between the upstream and downstream homology arms of the neurovirulence factor γ34.5 gene of the vector pH129ΔRL1. The virus is named pH129ΔRL1-hUbC-tdTomato-WPRE-PA. The nucleotide sequence of the vector pH129ΔRL1 is shown in SEQ ID NO.1, the nucleotide sequence of the upstream homology arm of the neurovirulence factor γ34.5 gene is shown in SEQ ID NO.2, the nucleotide sequence of the downstream homology arm of the neurovirulence factor γ34.5 gene is shown in SEQ ID NO.3, and the nucleotide sequence of pH129ΔRL1-hUbC-tdTomato-WPRE-PA is shown in SEQ ID NO.4. The preparation method of the targeting vector and the ltHSV-hUbC-tdTomato-WPRE virus refers to Chinese patent CN110117577A. In previous studies, the inventors found that the ltHSV-hUbC-tdTomato-WPRE virus has low toxicity, the ability to absorb reverse labeling through axon terminals, and exhibits very stable and efficient reporter gene expression capabilities. This example further transforms it into a virus for gene delivery, constructs a virus that delivers Cre recombinase, and is named ltHSV-hUbC-EGFP-2A-Cre to expand the application range of ltHSV in neural circuit labeling. The schematic diagram of the ltHSV-hUbC-EGFP-2A-Cre viral genome structure is shown in Figure 1A. The ltHSV-hUbC-EGFP-2A-Cre virus is prepared based on the targeting vector pH129ΔRL1-hUbC-EGFP-2A-Cre-WPRE-PA.

[0041] The nucleotide sequence of the targeting vector pH129ΔRL1-hUbC-EGFP-2A-Cre-WPRE-PA is shown in SEQ ID NO. 5. The construction method is as follows: tdTomato in pH129ΔRL1-hUbC-tdTomato-WPRE-PA was replaced with EGFP, and the resulting vector was named pH129ΔRL1-hUbC-EGFP-WPRE-PA; 579-P2ACre-F (5'-CTCGGCATGGACGAGCTGTACAAGGGAAGCGGAGCTACTAACTTCAGCCTGC-3' SEQ ID NO. 7) and 579-P2ACre-R (5'-GAGGTTGATTTCTAGATTAATCGCCATCTTCCAGCAGGCGCACC-3' SEQ ID NO. 8) primer pair, the gene fragment P2A-Cre was amplified, and the fragment size was 1138 bp. The agarose gel electrophoresis results after the PCR reaction are shown in Figure 1B. After successfully obtaining the P2A-Cre expression cassette, it was inserted into the EGFP gene sequence of pH129ΔRL1-hUbC-EGFP-WPRE-PA after digestion with BsrGI. The agarose gel electrophoresis results after BsrGI digestion of the pH129ΔRL1-hUbC-EGFP-WPRE-PA plasmid showed a fragment size of 9226 bp (Figure 1C). The ligation product was transformed into Escherichia coli competent cells. After culturing in a 37°C incubator for 16 hours, single colonies were picked for PCR identification and sequencing. The agarose gel electrophoresis results of the single colony PCR products are shown in Figure 1D. Positive monoclonal colonies No. 2 and No. 7 were selected for amplification culture. Plasmids were extracted after 12 to 16 hours. The obtained pH129ΔRL1-hUbC-EGFP-2A-Cre-WPRE-PA targeting vector was double-digested with EcoRI and KpnI. DNA electrophoresis verified the double bands of 9150 bp + 1214 bp. The agarose gel electrophoresis results after double enzyme digestion are shown in Figure 1E, indicating that the enzyme digestion results of the constructed plasmid were correct.

[0042] Example 2: ltHSV-hUbC-EGFP-2A-Cre virus recombination, purification, molecular identification and preparation

[0043] After obtaining the pH129ΔRL1-hUbC-EGFP-2A-Cre-WPRE-PA targeting vector and verifying its correctness through sequencing, a pure ltHSV-hUbC-EGFP-2A-Cre monoclonal strain was obtained through homologous recombination with the parent virus ltHSV-hUbC-tdTomato-WPRE and plaque purification.

[0044] To verify that the P2A-Cre expression cassette in the obtained monoclonal strain was correctly inserted into the position of the RL1 gene, the virus ltHSV-hUbC-EGFP-2A-Cre was inactivated at 100°C for 10 minutes, and the P2A-Cre gene fragment (1138 bp) was amplified by PCR using the primer pair 579-P2ACre-F and 579-P2ACre-R. The results are shown in Figure 2A. The negative control showed no band, and the positive control was the constructed pH129ΔRL1-hUbC-EGFP-2A-Cre-WPRE-PA targeting vector, which amplified a target band of approximately 1138 bp by PCR. No target band was amplified from the supernatant of the inactivated virus of the parent virus ltHSV-hUbC-tdT-WPRE, while a target band of approximately 1138 bp was amplified using the ltHSV-hUbC-EGFP-2A-Cre as a template. These results indicate that the P2A-Cre expression cassette was correctly inserted into the position of the RL1 gene.

[0045] After successful molecular identification, the purified ltHSV-hUbC-EGFP-2A-Cre recombinant virus was mass-produced and concentrated and purified by infecting BHK-21 cells grown on 10 cm culture dishes. Two days after virus infection, the results, as shown in Figure 2B, showed that all BHK-21 cells showed obvious EGFP fluorescence expression, and all diseased cells expressed fluorescence, indicating that the recombinant virus had indeed been purified. After all cells with obvious CPE were collected, the supernatant containing the recombinant virus was collected into a 50 mL centrifuge tube, and cell debris was removed by centrifugation (6400 rpm, 10 minutes). The supernatant was filtered using a 0.22 μm filter membrane and finally concentrated using a Beckman high-speed centrifuge (30000 rpm, 3 hours). The concentrated recombinant virus precipitate was resuspended in a small amount of PBS (pH = 7.4) and kept at 4 ° C overnight with continuous shaking. On the second day, the virus solution was mixed, and then the resuspended recombinant virus solution was added to the upper layer of 20% sucrose solution for ultracentrifugation (30000 rpm, 3 hours), concentrated and purified. Finally, the dissolved virus was aliquoted and frozen in a -80 ° C refrigerator, and a titer of 2×10 9 The concentrated purified virus was purified at a concentration of PFU / mL. This concentrated virus will be used to verify the in vitro Cre recombinase expression efficiency of ltHSV-hUbC-EGFP-2A-Cre and the in vivo reverse transcription gene delivery efficiency of ltHSV.

[0046] Example 3: In vitro replication characteristics of the ltHSV-hUbC-EGFP-2A-Cre delivery vector

[0047] After confirming the correctness of the genetic information of the obtained ltHSV-hUbC-EGFP-2A-Cre virus strain, in order to evaluate whether the insertion of the exogenous gene P2A-Cre expression frame has an effect on the growth kinetics of HSV, this example determines the growth curve of the virus. Specifically, the virus titers of H129-WT, the parent virus ltHSV-hUbC-tdT-WPRE and ltHSV-hUbC-EGFP-2A-Cre viruses were determined at 3h, 6h, 12h, 24h, 36h, 48h, 60h, and 72h after infection with MOI=0.1. The titer of the virus supernatant collected at each time point was repeated 3 times, and the average value was finally taken. The virus growth curve drawn based on the collected data is shown in Figure 3. The results showed that there was no significant difference in the growth curves of the three viruses. The titers of the three viruses reached the highest 24-36 hours after infection, and the virus titer could reach 10 7 PFU / mL, and then entered the plateau phase. This indicates that the in vitro replication characteristics of the attenuated ltHSV-hUbC-tdT-WPRE and ltHSV-hUbC-EGFP-2A-Cre viruses are not significantly different from those of the wild-type strain, indicating that the newly recombinant viruses have comparable replication abilities in vitro to the wild-type strain.

[0048] Example 4: In vitro characterization of the expression efficiency of Cre recombinase delivered by the ltHSV-hUbC-EGFP-2A-Cre vector

[0049] To characterize the in vitro expression efficiency of Cre recombinase delivered by the ltHSV-hUbC-EGFP-2A-Cre vector, in this example, BHK-21 cells were infected with ltHSV-hUbC-EGFP-2A-Cre (as a "control") and H129-CAG-DIO-tdTomato (as a "reporter") alone or in equal proportions. Cells infected with the single-virus "control" ltHSV-hUbC-EGFP-2A-Cre expressed the green fluorescent protein EGFP (Figures 4A, B). However, cells infected with the single-virus "reporter" H129-CAG-DIO-tdTomato did not express the red fluorescent protein tdTomato due to the lack of Cre recombinase (Figures 4C, D). When the "control" ltHSV-hUbC-EGFP-2A-Cre and the "reporter" H129-CAG-DIO-tdTomato viruses were co-infected in BHK-21 cells, Cre recombinase expression allowed the cells to simultaneously express the green fluorescent protein EGFP and the red fluorescent protein tdTomato (Figure 4E, F). This demonstrates that ltHSV-hUbC-EGFP-2A-Cre can efficiently express Cre recombinase in vitro as a gene delivery vector.

[0050] Example 5: Efficient labeling of the VTA input circuit using the ltHSV-hUbC-EGFP-2A-Cre vector delivering Cre recombinase

[0051] After confirming that the ltHSV-hUbC-EGFP-2A-Cre gene delivery vector efficiently expressed Cre recombinase in vitro, we further characterized the in vivo expression efficiency of Cre recombinase delivered by the ltHSV-hUbC-EGFP-2A-Cre vector and used the Cre recombinase-delivered ltHSV-hUbC-EGFP-2A-Cre vector to efficiently label the VTA input circuit. We injected the ltHSV-hUbC-EGFP-2A-Cre vector into the VTA of Ai14 mice (expressing a Cre-dependent tdTomato reporter gene driven by a CAG promoter) (Figure 5A). Because Cre recombinase acts as a biocatalyst, even a small amount of Cre recombinase can initiate fluorescent expression of the Cre-dependent tdTomato reporter gene. Therefore, using Ai14 mice allows for more sensitive monitoring of the spread of ltHSV-hUbC-EGFP-2A-Cre. After perfusion sampling on the 7th day after injection, slice imaging observation showed that in addition to the large amount of red and green fluorescent protein expression detected in the VTA brain region at the injection site (Figure 5B), consistent with the ltHSV-hUbC-tdT-WPRE labeling results, a large amount of fluorescent signals were also observed in the brain region directly projecting to the VTA (Figure 5C). It is worth noting that the experimental results showed that the number of green neurons directly labeled by ltHSV-hUbC-EGFP-2A-Cre upstream of the VTA was less than the number of tdTomato red fluorescent neurons expressed after Cre recombinase-mediated recombination, indicating that this strategy HSV can further label more corresponding upstream projection neurons.

[0052] Example 6: Construction and molecular identification of ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing recombinant plasmid vector

[0053] Because HSV has a broad host range and can infect a variety of cell types, this property makes it a potential treatment for a wide range of diseases. In particular, HSV's neurotropism, enabling efficient neuronal infection, offers potential for treating neurological diseases and makes it an attractive gene delivery vector. CRISPR / Cas9 has developed into a mature gene editing technology, enabling precise editing and modification of target genes. Combining these two technologies, the inventors hope to enable more efficient gene therapy and research. After successfully constructing and verifying the efficiency of gene delivery of the ltHSV-hUbC-EGFP-2A-Cre vector, the present invention further constructs a gene editing vector ltHSV-SpCas9-2A-EGFP-sgtdT4 based on low toxicity, reverse labeling and large capacity, which is prepared based on the pH129ΔRL1-CAG-SpCas9-2A-EGFP-U6-sgtdT-PA targeting vector. The gene expression frame hUbC-tdTomato-WPRE-PA inserted between the upstream and downstream homologous arms of the ICP34.5 gene in the ltHSV-hUbC-tdT-WPRE virus is replaced with CAG-SpCas9-2A-EGFP-U6-sgtdT-PA to construct a targeting vector of the ltHSV gene editing vector.

[0054] To construct the gene expression cassette CAG-SpCas9-2A-EGFP-U6-sgtdT-PA, gene fragments sgtdT1#–4# were designed based on the target sequence of the selected tdTomato gene and screened using the CRISPR DESIGN system (http: / / crispr.mit.edu / ). All designed sgRNA sequence oligonucleotide fragments are listed in Table 1. First, the empty vector pSpCas9(BB)-2A-GFP(PX458) (Addgene, #48138) was digested with BbsI enzyme. The digestion product was then excised from a gel and a 9229 bp fragment was recovered (Figure 6B). Simultaneously, the designed and synthesized sgRNA sequence oligonucleotide fragments were annealed to obtain sgtdT1#–4# fragments with sticky ends. The sgtdT1#–4# fragments were then ligated into the guide RNA scaffold of the empty PX458 vector. The resulting plasmids were named sequentially in the format PX-sgtdT1#–4#. PX-sgtdT1#~4# was double-digested with SbfⅠ and FspⅠ and dephosphorylated, and a 7.8k fragment was recovered to obtain the CAG-SpCas9-2A-EGFP-U6-sgtdT1#~4#-PA gene expression cassette (Figure 6C). The successfully obtained gene expression cassette was enzymatically ligated with a 6.5kb fragment recovered and dephosphorylated after double-digestion of the pH129-ΔRL1 plasmid with SbfⅠ and HpaⅠ restriction endonucleases. In this way, the exogenous gene expression cassette CAG-SpCas9-2A-EGFP-U6-sgtdT1#~4#-PA was inserted between the upstream and downstream homology arms of the γ34.5 gene in the pH129-ΔRL1 plasmid. The four ligation products were transformed into competent E. coli cells and cultured in a 37°C incubator for 16 hours. Single colonies were then picked for PCR identification and sequencing (Figures 6D-G). Appropriate positive single clones were selected for amplification and culture, and plasmids were extracted 12-16 hours later. The resulting plasmids were the targeting vectors of the ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing vector, named pH129ΔRL1-CAG-SpCas9-2A-EGFP-U6-sgtdT1#-4#-PA. The resulting targeting vectors of the ltHSV gene editing vector were double-digested with Hind III and EcoR V and verified by sequencing.

[0055] Table 1 Sequences of oligo fragments required for synthesizing the tdTomato gene

[0056] Example 7: Construction and analysis of gene editing ltHSV-SpCas9-2A-EGFP-sgtdT4 recombinant virus

[0057] After obtaining the pH129ΔRL1-CAG-SpCas9-2A-EGFP-U6-sgtdT4#-PA targeting plasmid and sequencing verification, its nucleotide sequence is shown in SEQ ID NO. 6. Through homologous recombination with the parent virus ltHSV-hUbC-tdT-WPRE and plaque picking purification, a pure progeny ltHSV-SpCas9-2A-EGFP-sgtdT4 monoclonal strain was obtained. To verify that the obtained monoclonal strain had correctly inserted the CAG-SpCas9-2A-EGFP-U6-sgtdT4#-PA gene expression cassette into the RL1 gene, PCR amplification of a 1529 bp fragment containing the sgtdT4# sequence was performed. The results are shown in Figure 7A. The negative control showed no band, while the positive control, the constructed pH129ΔRL1-CAG-SpCas9-2A-EGFP-U6-sgtdT4#-PA targeting plasmid, amplified a 1529 bp target band. The parent virus ltHSV-hUbC-tdT-WPRE inactivated virus supernatant sample did not amplify the target band, while the recombinant virus ltHSV-SpCas9-2A-EGFP-sgtdT4 was used as a template to amplify the correct target band of approximately 1529 bp. These results indicate that the CAG-SpCas9-2A-EGFP-U6-sgtdT4#-PA expression cassette was correctly inserted into the RL1 gene. After successful molecular identification, the purified ltHSV-SpCas9-2A-EGFP-sgtdT4 recombinant virus was mass-produced and concentrated by infecting BHK-21 cells grown on 10 cm culture dishes. Two days after virus infection, the results are shown in Figure 7B. BHK-21 cells showed obvious EGFP fluorescence expression, and all diseased cells expressed fluorescence, indicating that the recombinant virus has been purified. The culture supernatant was collected and concentrated by centrifugation, and a titer of 1×10 8 The concentrated purified virus with a concentration of PFU / mL will be used for the one-step growth curve determination of ltHSV-SpCas9-2A-EGFP-sgtdT4 and the in vivo validation experiment of the ltHSV gene editing vector.

[0058] Example 8: In vitro characterization of the efficiency of the ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing vector

[0059] In order to characterize the gene editing efficiency of the ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing vector in vitro, ltHSV-EGFP and H129ΔTK-tdT (the two recombinant viral vectors were constructed and preserved by the laboratory in the early stage, and ltHSV-EGFP was used as a control virus without CRISPR gene editing elements) were first mixed in equal proportions to infect BHK-21 cells. The experimental principle is shown in Figure 8A. Because the two viruses express green and red fluorescent proteins, respectively, and do not inhibit the expression of each other's proteins, a large number of red and green co-labeled cells can be observed (Figure 8B). Then, the ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing vector targeting the tdTomato gene was mixed with H129ΔTK-tdT in equal proportions and then infected with BHK-21 cells. The experimental principle is shown in Figure 8C. Because the virus expressing green fluorescent protein will cut the tdTomato gene and interfere with the expression of red protein, the number of red and green co-labeled cells can be observed to be significantly reduced (Figure 8D). The experimental results show that almost all cells expressing green no longer express red fluorescent protein, and the gene editing effect is close to 100%. This shows that ltHSV-SpCas9-2A-EGFP-sgtdT4 has a very high gene editing efficiency in vitro as a gene editing vector.

[0060] Example 9: In vivo characterization of the efficiency of the ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing vector

[0061] In order to characterize the gene editing efficiency of ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing vector in vivo, a titer of 1×10 8 PFU / mL of ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing vector and AAV1-hSyn-EGFP-P2A-Cre (2×10 11 VG) were mixed in a volume ratio of 3:1 and injected into the VTA brain region of Ai14 mice (Figure 9A). Since a small amount of AAV1 expressing Cre recombinase can initiate the fluorescent expression of the Cre-dependent tdTomato reporter gene, and the ltHSV titer is low, this strategy is very challenging for the gene editing efficiency of the ltHSV gene editing vector in vivo. After perfusion sampling on the 14th day after injection, slice imaging observation (Figure 9B) showed that red fluorescent protein expression could be detected in the VTA brain region at the injection site, but red fluorescent protein was not expressed in neurons expressing green fluorescent protein, and red and green were not co-labeled. This shows that the ltHSV-SpCas9-2A-EGFP-sgtdT4 gene editing vector targeting the tdTomato gene can effectively exert its gene editing ability in vivo.

[0062] In summary, the present invention constructs an attenuated HSV-1 (ltHSV) vector system by knocking out the double-copy gene of the neurovirulence factor ICP34.5 of herpes simplex virus type 1 (HSV1), and the study found that it has the characteristics of efficient gene delivery and low toxicity. A series of ltHSV recombinant virus tools were further constructed, including ltHSV viral vectors that deliver Cre recombinase and CRISPR gene editing systems for large genes, respectively. In the method validation experiment, the upstream brain area of ​​the ventral tegmental area of ​​the midbrain was tracked using ltHSV carrying Cre recombinase on Ai14 mice, confirming that ltHSV has stable and efficient reverse gene delivery and expression capabilities. Further utilizing its low toxicity characteristics and the advantages of large vector capacity, it was verified in in vitro and in vivo experiments that the ltHSV viral vector carrying CRISPR / Cas9 elements has the ability of efficient gene delivery and gene editing. The present invention provides a new vector system for neural circuit tracing and long-term gene delivery to express large or multiple exogenous genes.

[0063] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A low-toxicity, long-acting, high-loading herpes simplex virus, characterized in that, The low-toxicity, long-acting, high-loading herpes simplex virus is obtained by completely knocking out two copies of the neurovirulence factor γ34.5 in the HSV-1 virus genome and inserting an exogenous gene expression cassette at the original gene locus of the neurovirulence factor γ34.

5. The exogenous gene expression cassette is selected from Expression Cassette I or Expression Cassette II. Expression Cassette I includes a promoter, a fluorescent protein gene, a 2A peptide coding gene, a Cre coding gene, a WPRE transcriptional enhancer element, and PA connected in sequence. Expression Cassette II includes a promoter, a SpCas9 or other nuclease coding gene, a 2A peptide coding gene, a fluorescent protein gene, a promoter, an sgRNA targeting the gene to be edited, and PA connected in sequence.

2. The low-toxicity, long-acting, high-loading herpes simplex virus according to claim 1, wherein The HSV-1 virus is selected from the strain HSV-1 H129.

3. The low-toxicity, long-acting, high-loading herpes simplex virus according to claim 1, characterized in that, The promoter is selected from one of the hUbC promoter, CMV promoter, CAG promoter, EF1α promoter, pTH promoter, and pChAT promoter; The fluorescent protein gene is tdTomato or EGFP; The 2A peptide is selected from one of P2A, T2A, E2A, and F2A.

4. The low-toxicity, long-acting, high-loading herpes simplex virus according to claim 1, characterized in that, Expression Cassette I includes a promoter hUbC, an EGFP coding gene, a P2A peptide coding gene, a Cre coding gene, a WPRE transcriptional enhancer element, and PA connected in sequence; Expression Cassette II includes a promoter CAG, a SpCas9 or other nuclease coding gene, a P2A peptide coding gene, an EGFP coding gene, a promoter U6, an sgRNA targeting the gene to be edited, and PA connected in sequence.

5. A low-toxicity, long-acting, high-loading herpes simplex virus, characterized in that, The exogenous gene expression cassette is selected from Expression Cassette I.

6. A low-toxicity, long-acting, high-loading herpes simplex virus, characterized in that, The exogenous gene expression cassette is selected from Expression Cassette II.

7. The preparation method of the low-toxicity, long-acting, high-loading herpes simplex virus according to any one of claims 1-6, characterized in that, It includes the following steps: (1) Knock out two copies of the neurovirulence factor γ34.5 in the HSV-1 virus genome to obtain a recombinant virus; (2) Construct a targeting vector, which is a vector with an exogenous gene expression cassette inserted between the upstream and downstream homologous arms of the neurovirulence factor γ34.5 gene in the vector pH129ΔRL1. The nucleotide sequence of the vector pH129ΔRL1 is shown in SEQ ID NO.

1. The nucleotide sequence of the upstream homologous arm of the neurovirulence factor γ34.5 gene is shown in SEQ ID NO.

2. The nucleotide sequence of the downstream homologous arm of the neurovirulence factor γ34.5 gene is shown in SEQ ID NO.

3. The exogenous gene expression cassette is selected from Expression Cassette I or Expression Cassette II as claimed in claim 1; (3) Co-transfect the targeting vector obtained in step (2) and the recombinant virus prepared in step (1) into cells for recombination. After plaque purification, the low-toxicity, long-acting, high-loading herpes simplex virus as claimed in any one of claims 1-6 is obtained.

8. Use of the low-toxicity, long-acting, high-loading herpes simplex virus as claimed in claim 5 in neural circuit labeling.

9. The application according to claim 8, characterized in that The neural circuit labeling is retrograde neural circuit labeling.

10. The application according to claim 8, characterized in that The low-toxicity, long-acting, high-loading herpes simplex virus described in claim 5 is injected into an animal with Cre-dependent fluorescent protein gene expression, or the low-toxicity, long-acting, high-loading herpes simplex virus described in claim 5 is co-injected with a virus containing Cre-dependent fluorescent protein gene expression.

11. Use of the low-toxicity, long-acting, high-loading herpes simplex virus described in claim 5 in target gene editing.

12. A gene editing vector, characterized in that, It is the low-toxicity, long-acting, high-loading herpes simplex virus described in claim 6, carrying a gene editing expression cassette element, including a promoter, a SpCas9 or other nuclease-encoding gene, a 2A peptide-encoding gene, a fluorescent protein gene, a promoter, an sgRNA targeting the gene to be edited, and a PA, which are connected in sequence.

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