Lentivirus-like particle for treating Huntington disease

Abstract

The invention discloses a lentivirus-like particle for treating a Huntington disease. The lentivirus-like particle VLP-HD is composed of an RNP complex composed of Cas9 protein wrapped by a VLP vector and gRNA of a targeted HTT gene. After cells are infected, VLP-HD releases Cas9: gRNA RNP, the mutant HTT gene can be efficiently knocked out in a targeted mode, generation of mutant HTT protein with PolyQ is prevented from the source, and therefore the effect of treating HD is achieved. The VLP-HD can be a single-target VLP, a mutant gene can be knocked out in a mode that CRISPR / Cas9 is delivered to enable the mHTT gene to be subjected to frame shift mutation, and the VLP-HD can also be a double-target VLP, two CRISPR / Cas9 are delivered to delete CAG nucleotide repeated fragments, inducing disease occurrence, in the mHTT gene, and generation of PolyQ is eradicated.

Description

Technical Field

[0001] The present invention belongs to the field of gene therapy, and relates to a lentivirus-like particle for treating Huntington's disease, and particularly to a modified lentivirus-like particle VLP suitable for treating Huntington's chorea. Background Art

[0002] Huntington's disease (HD) is a hereditary neurological disease characterized by psychiatric disorders, cognitive disorders, and choreiform involuntary movements. Huntington's disease is an autosomal dominant genetic disease induced by an increase in the CAG nucleotide repeat on exon 1 of the HTT gene on chromosome 4 of patients. The HTT gene product, the HTT protein, is widely expressed in human neurons and has multiple functions. In healthy individuals, the HTT gene has an average of 17-20 CAG repeats. When the number of CAG repeats reaches more than 40 times, polyglutamine (PolyQ) is formed, leading to the aggregation of the huntingtin protein. The aggregated protein enters the nucleus and causes gene transcriptional dysregulation. The aggregated protein disrupts protein homeostasis in the cytoplasm, including synaptic dysfunction, mitochondrial toxicity, and reduced axonal transport rate, resulting in the occurrence of Huntington's disease. HD patients generally develop cognitive and motor impairments in middle age and increase with age.

[0003] The worldwide prevalence of HD is approximately 2.7 / 100,000. In Western countries, the prevalence of HD is 10.6-13.7 per 100,000 people. Currently, the clinical treatment method is symptomatic treatment, and there is no causative treatment method. With the development of gene therapy technology, it is possible to treat genetic diseases caused by gene mutations. Recently, a therapy AMT-130 that uses an AAV vector to deliver microRNA (rAAV5-miHTT) to knockdown the mutant HTT (mHTT) gene has been proposed for the treatment of Huntington's disease. A phase II clinical trial (NCT05243017) is underway to investigate the safety and efficacy of AMT-130 in early Huntington's adult patients in Europe. The treatment strategy of this treatment method is to use an AAV vector to long-term express microRNA targeting the mHTT gene in neurons, and knockdown the mutant protein by inhibiting the translation of mHTT mRNA. However, this method requires the long-term colonization of foreign genes in cells, the stability effect of foreign genes is unknown, and there is a risk of foreign gene integration into the host cell genome. Summary of the Invention

[0004] The purpose of the present invention is to provide a solution to the safety and efficacy problems in the treatment of Huntington's chorea in the prior art, and to provide a lentivirus-like particle for treating Huntington's disease. Specifically, this treatment method is a gene editing treatment method; it is achieved by delivering CRISPR RNP by VLP to target and knockout the HTT gene.

[0005] Specifically, the object of the present invention is achieved by the following technical solutions:

[0006] <First aspect>

[0007] The present invention relates to a lentivirus-like particle, which is composed of an RNP complex formed by a VLP vector, a Cas9 protein, and a gRNA targeting the HTT gene. The lentivirus-like particle VLP is composed of: a modified lentiviral envelope that encapsulates an RNP complex composed of a Cas9 protein and a targeting gRNA. The modified lentiviral envelope is a lentiviral envelope formed by fusing an RNA-binding protein with a lentiviral GagPol long-chain protein.

[0008] As an embodiment of the present invention, the gene sequences expressing the VLP vector components are respectively located on ① a plasmid expressing a membrane protein, ② a plasmid expressing a lentiviral GagPol long-chain protein containing an RNA-binding protein, ③ a plasmid expressing a lentiviral GagPol long-chain protein, ④ an auxiliary plasmid pRSV-Rev, ⑤ a plasmid expressing the Cas9 protein required for forming the RNP, and ⑥ a plasmid expressing a gRNA containing an RNA stem-loop structure recognized by the RNA-binding protein.

[0009] As an embodiment of the present invention, the plasmid expressing the Cas9 protein required for forming the RNP and the plasmid expressing the gRNA containing an RNA stem-loop structure recognized by the RNA-binding protein can be the same plasmid. That is, the gene sequences expressing the VLP vector components are respectively located on ① a plasmid expressing a membrane protein, ② a plasmid expressing a lentiviral GagPol long-chain protein containing an RNA-binding protein, ③ a plasmid expressing a lentiviral GagPol long-chain protein, ④ an auxiliary plasmid pRSV-Rev, and ⑤ a plasmid expressing the Cas9 protein required for forming the RNP and the gRNA containing an RNA stem-loop structure recognized by the RNA-binding protein.

[0010] As an embodiment of the present invention, the membrane protein in the VLP vector components includes a protein expressing VSV-G, a CD4 recognition protein, a CD8 recognition protein, RD114, an endogenous baboon retrovirus membrane protein, or a modified membrane protein with cell infection specificity.

[0011] As an embodiment of the present invention, the amino acid sequence of VSV-G is SEQ ID NO.7.

[0012] As an embodiment of the present invention, the modified membrane protein with cell infection specificity, namely the NVR-G protein, has neuronal infection specificity, and the amino acid sequence of NVR-G is SEQ ID NO.8.

[0013] As an embodiment of the present invention, the gene sequence of NVR-G is SEQ ID NO.29.

[0014] As an embodiment of the present invention, the amino acid sequence of the lentiviral GagPol long-chain protein containing the RNA-binding protein is SEQ ID NO.9.

[0015] As an embodiment of the present invention, the gRNA backbone sequence containing the RNA stem-loop structure recognized by the RNA-binding protein is SEQ ID NO.10.

[0016] As an embodiment of the present invention, the targeting site of the guide sequence carried on the gRNA targeting the HTT gene can be any site on the HTT gene. The VLP carrying 1 kind of Cas9:gRNA complex targets the mutant HTT (mHTT) gene to generate Indel (insertion / deletion), resulting in a frameshift mutation and knocking out the mutant HTT (mHTT) gene; the VLP carrying 2 kinds of Cas9:gRNA complexes targets the HTT gene at both ends of the CAG repeat sequence, cutting off the CAG repeat sequence to prevent the production of proteins that cause aggregation.

[0017] As an embodiment of the present invention, the gRNA sequences targeting the HTT gene are:

[0018] gRNA1: 5'-GGCCTTCATCAGCTTTTCCA-3',

[0019] gRNA4: 5'-GAAGGACTTGAGGGACTCGA-3',

[0020] gRNA7: 5'-GACCCTGGAAAAGCTGATGA-3',

[0021] gRNA8: 5'-GGAGACCGCCATGGCGACCC-3',

[0022] gRNA9: 5'-CAGCTTTTCCAGGGTCGCCA-3',

[0023] gRNA10: 5'-CTTTTCCAGGGTCGCCATGG-3

[0024] or gRNA11: 5'-AGGCCTTCATCAGCTTTTCC-3'.

[0025] As an embodiment of the present invention, at least two of the following sequences are respectively selected as the gRNA sequences targeting the HTT gene:

[0026] gRNA1: 5’-GGCCTTCATCAGCTTTTCCA-3’、

[0027] gRNA2: 5’-TGAGGAAGCTGAGGAGGCGG-3’、

[0028] gRNA3: 5’-GGCGGCGGCTGAGGAAGCTG-3’、

[0029] gRNA4: 5’-GAAGGACTTGAGGGACTCGA-3’、

[0030] gRNA5: 5’-TTCATTGCCCCGGTGCTGAG-3’、

[0031] gRNA6: 5’-GGCTGAGGAAGCTGAGGAGG-3’、

[0032] gRNA7: 5’-GACCCTGGAAAAGCTGATGA-3’、

[0033] gRNA8: 5’-GGAGACCGCCATGGCGACCC-3’、

[0034] gRNA9: 5’-CAGCTTTTCCAGGGTCGCCA-3’、

[0035] gRNA10: 5’-CTTTTCCAGGGTCGCCATGG-3’、

[0036] gRNA11: 5’-AGGCCTTCATCAGCTTTTCC-3’、

[0037] gRNA12: 5’-GAGTCGGCCCGAGGCCTCCG-3’、

[0038] gRNA13: 5’-GGCGGCTGAGGAAGCTGAGG-3’、

[0039] gRNA14: 5’-AGCGGGCCCAAACTCACGGT-3’、

[0040] gRNA15: 5’-AGCAGCGGCTGTGCCTGCGG-3’、

[0041] gRNA16: 5’-GGAAGCTGAGGAGGCGGCGG-3’、

[0042] gRNA17: 5’-GCCGGGACAGGGAGCTGCAG-3’、

[0043] gRNA18: 5'-TGAGGAGGCGGCGGCGGCGG-3'.

[0044] As an embodiment of the present invention, the gRNA plasmid targeting the HTT gene is a plasmid combination of at least one of gRNA1, gRNA4, gRNA5, gRNA7-12 and at least one of gRNA2, gRNA3, gRNA6, gRNA13-18.

[0045] <Second aspect>

[0046] The present invention relates to a method for preparing the aforementioned lentivirus-like particles, the method comprising the following steps: transfecting a plasmid containing the lentiviral vector genome sequence into virus-producing cells, and preparing by (collecting the supernatant, concentrating and purifying); the lentiviral vector genome sequences are respectively located on ① a plasmid expressing a membrane protein, ② a plasmid expressing a lentiviral GagPol long-chain protein containing an RNA-binding protein, ③ a plasmid expressing a lentiviral GagPol long-chain protein, ④ an auxiliary plasmid pRSV-Rev, and ⑤ a plasmid expressing the Cas9 protein required for forming RNP and ⑥ a plasmid expressing an HTT gRNA containing an RNA stem-loop structure recognized by an RNA-binding protein, or on ⑤ a plasmid expressing the Cas9 protein required for forming RNP and an HTT gRNA plasmid containing an RNA stem-loop structure recognized by an RNA-binding protein.

[0047] The backbone part of the plasmid (the part other than the core sequence expressed) is not limited.

[0048] <Third aspect>

[0049] The present invention relates to the use of a lentivirus-like particle in the preparation of a drug for treating Huntington's disease.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1) The VLP-HD described in the present invention is a lentivirus-like particle encapsulating the RNP formed by the Cas9 protein and gRNA. After infecting cells, VLP-HD can efficiently target and knockout the mutant HTT gene, prevent the production of mutant HTT protein with PolyQ from the source, remove the cause of HD disease at the gene level, and thus achieve the effect of treating HD causally.

[0052] 2) The VLP-HD described in the present invention can be a single-target VLP, delivering one CRISPR / Cas9 to knockout the gene by causing a frameshift mutation in the mHTT gene, or a double-target VLP, delivering two CRISPR / Cas9 to cut off the CAG nucleotide repeat fragment that induces disease occurrence in the mHTT gene, and prevent the production of PolyQ.

[0053] 3) Since VLP-HD delivers the RNP structure formed by Cas9 protein and gRNA, and CRISPR / Cas9 is degraded by intracellular proteases and nucleases after performing the gene cleavage function in cells, the gene editing of VLP-HD is transient. The transiently present CRISPR / Cas9 will greatly reduce the risk of off-target effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Other features, objects, and advantages of the present invention will become more apparent by reading the following detailed description of non-limiting embodiments with reference to the accompanying drawings:

[0055] Figure 1 Plasmid map for expressing single gRNA;

[0056] Figure 2 Gene editing efficiency of VLP-HD;

[0057] Figure 3 Gene editing effect of dual-target VLP-HD;

[0058] Figure 4 Schematic diagram of dual-target VLP-HD knocking out HTT protein;

[0059] Figure 5 Schematic diagram of the structure of VLP-HD;

[0060] Figure 6 Efficiency of neuron-specific envelope protein in lentivirus infecting different sources of cells;

[0061] Figure 7 Gene editing effect of neuron-specific VLP-HD;

[0062] Figure 8 Data of VLP-HD-NVR-G improving the motor coordination and balance ability of Huntington mice. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0063] The present invention will be described in detail below with reference to the embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those of ordinary skill in the art can make several adjustments and improvements without departing from the concept of the present invention. These all fall within the protection scope of the present invention.

[0064] Example 1: Construction of VLP-CRISPR RNP (single-target and dual-target)

[0065] In this example, the method for constructing VLPs containing CRISPR RNP is as follows: Co-transfect the plasmid expressing membrane protein (pMD.2G), the plasmid expressing the lentiviral GagPol long-chain protein containing an RNA-binding protein (pMS2M-PH-GagPol-D64V, the plasmid sequence is shown in SEQ ID NO.1), the plasmid expressing the wild-type lentiviral GagPol long-chain protein (pMDlg / PRRE-D64V, the plasmid sequence is shown in SEQ ID NO.2), the helper plasmid (pRSV-REV), the plasmid expressing Cas9 protein (pCMV-2NLS-Cas9, the plasmid sequence is shown in SEQ ID NO.3), and the plasmid expressing gRNA containing MS2 stemloop (pU6-Osp.gRNAMS2in, the plasmid sequence is shown in SEQ ID NO.4) into 293T cells, and collect the supernatant for concentration and purification. The concentration of VLP p24 was measured using a lentivirus titer ELISA kit for quantification.

[0066] Construct VLP-CRISPR RNP targeting two sites. On the basis of the above production plasmids, add a pU6-Osp.gRNAMS2in targeting the second site, that is, a total of 2 plasmids expressing gRNA; or place the expression cassettes of the 2 gRNAs on the same plasmid, that is, insert the expression cassettes (U6-Osp.gRNAMS2in) of the 2 gRNA sequences in a cis or trans tandem manner into the plasmid pU6-Osp.gRNAMS2in to replace the original single-copy expression cassette.

[0067] Example 2: Design gRNA sequences and predict gene editing efficiency

[0068] Input the HTT gene into the CRISPRon (v1.0) web tool to design gRNAs and predict gene editing efficiency, and screen the gRNAs from the obtained results. Screen the gRNA sequences in the 5'-direction of the CAG repeat region on exon 1 of the HTT gene, such as gRNA1, gRNA4, gRNA 7-11. These gRNAs can be used to construct single-target VLP-HD, or can be combined with the gRNAs in the 3'-direction of the CAG repeat region (such as gRNA2, gRNA3, gRNA6, and gRNA13-18) screened out to form double-target VLP-HD. Screen the gRNA sequences in the 5'-UTR region (such as gRNA5 and gRNA12), and combine them with gRNA2, gRNA3, gRNA6, or gRNA13-18 to form double-target VLP-HD. The predicted gene editing efficiency of each gRNA is shown in the following table:

[0069]

[0070] The gene editing efficiencies at different gRNA sites are different. Among the screened gRNA sequences, except for gRNA9-11, the software-predicted efficiency is >50%.

[0071] Example 3: HTT gene editing efficiency of single-target VLP-HD and verification of the efficiency of HTT gRNA

[0072] 1. Produce VLP-HD. VLP-HD carries Cas9:gRNA RNP targeting the HTT gene. Co-transfect plasmids pMD.2G, pMS2M-PH-GagPol-D64V, pMD1g / PRRE-D64V, pRSV-REV, pCMV-2NLS-Cas9, and pU6-Osp.gRNAMS2in-HTT into 293T cells, collect the supernatant, concentrate and purify to obtain VLP particles. Determine the VLP p24 concentration using a lentivirus titer ELISA kit.

[0073] Construct plasmid pU6-Osp.gRNAMS2in-HTT. Select several gRNA sequences targeting the HTT gene, namely gRNA1: 5'-GGCCTTCATCAGCTTTTCCA-3' (PAM: GGG); gRNA2: 5'-TGAGGAAGCTGAGGAGGCGG-3' (PAM: CGG); gRNA3: 5'-GGCGGCGGCTGAGGAAGCTG-3' (PAM: AGG); gRNA4: 5'-GAAGGACTTGAGGGACTCGA-3' (PAM: AGG); gRNA5: 5'-TTCATTGCCCCGGTGCTGAG-3' (PAM: CGG) gRNA12: 5'-GAGTCGGCCCGAGGCCTCCG-3' (PAM: GGG); gRNA15: 5'-AGCAGCGGCTGTGCCTGCGG-3' (PAM: CGG).

[0074] Add the above gRNA sequences between the promoter and the gRNA backbone of plasmid pU6-Osp.gRNAMS2in to construct plasmids pU6-Osp.gRNAMS2in-HTT1, pU6-Osp.gRNAMS2in-HTT2, pU6-Osp.gRNAMS2in-HTT3, pU6-Osp.gRNAMS2in-HTT4, pU6-Osp.gRNAMS2in-HTT5, pU6-Osp.gRNAMS2in-HTT12, pU6-Osp.gRNAMS2in-HTT15. The schematic diagram of the plasmid map carrying gRNA is shown in Figure 1(The gRNA positions shown in the figure can be replaced with different gRNAs), and the plasmids can be obtained by gene synthesis according to the nucleotide sequences. The VLPs produced using these plasmids are VLP-HD1, VLP-HD2, VLP-HD3, VLP-HD4, VLP-HD5, VLP-HD12, and VLP-HD15, respectively. Among them, VLP-HD1, VLP-HD4, and VLP-HD5 are single-target VLPs-HD with the function of knocking out polyQ in the mutant HTT gene; VLP-HD2, VLP-HD3, and VLP-HD15 need to be used in combination with any other VLP-HD to have the function of knocking out the polyQ gene.

[0075] 2. Verify the gene editing efficiency of VLP-HD.

[0076] Seed 293T cells into 96-well plates at a density of 2×104 cells / well and culture for 24 h. Then infect with various VLPs-HD at a concentration of 50 ng / well. Harvest the cells 72 h after infection and extract genomic DNA. Use primers (F: 5’-agggctgtcaatcatgctgg-3’ and R: 5’-ggttgctgggtcactctgtc-3’) to perform PCR amplification of the DNA fragment around the target site, followed by Sanger sequencing and TIDE (https: / / tide.nki.nl / ) detection of Indel. As Figure 2 shown, the highest gene editing efficiency is close to 90%. Compared with the gene editing efficiency of the predicted gRNA sequences: the gene editing efficiencies of VLP-HD 1-4 are different from but not significantly different from those of the predicted gRNAs 1-4; the gene editing efficiency of VLP-HD5 (89.3%) is much higher than that of the predicted gRNA15 (68.594%); the gene editing efficiencies of VLP-HD12 and VLP-HD12 (Indel (insertion or deletion) efficiencies are 5.8% and 8.5% respectively) are much lower than those of the predicted gRNAs 12 and gRNA15 (60.424% and 62.264% respectively, see Example 2).

[0077] Example 4: Knockout of the HTT gene by dual-target VLP-HD

[0078] 1. Produce dual-target VLP-HD. VLP-HD carries CRISPR RNP targeting two HTT gene loci. Co-transfect plasmid pMD.2G, pMS2M-PH-GagPol-D64V, pMDlg / PRRE-D64V, pRSV-REV, pCMV-2NLS-Cas9 and two pU6-Osp.gRNAMS2in-HTT plasmids expressing HTT gRNA into 293T cells, collect the supernatant, concentrate and purify to obtain VLP particles. Determine the VLP p24 concentration using a lentivirus titer ELISA kit.

[0079] The two plasmids expressing HTT gRNA are combined as gRNA1+gRNA2, gRNA1+gRNA3, gRNA1+gRNA6, gRNA5+gRNA2, gRNA5+gRNA3, gRNA5+gRNA6. pU6-Osp.gRNAMS2in-HTT5 expresses gRNA5: 5’-TTCATTGCCCCGGTGCTGAG-3’ (PAM: CGG); pU6-Osp.gRNAMS2in-HTT6 expresses gRNA6: 5’-GGCTGAGGAAGCTGAGGAGG-3’ (PAM: CGG). The plasmids can be obtained by gene synthesis according to the nucleotide sequence.

[0080] 2. Verify the gene cleavage of dual-target VLP-HD.

[0081] Seed HeLa cells into 96-well plates, 2×10 4 / well, culture for 24 h and infect with VLP-HD, 100 ng / well. Harvest the cells and extract the genome 72 h after infection. Use primers (F: 5’-GGACGGGTCCAAGATGGACG-3’ and R: 5’-caaactcacGGTCGGTGCAG-3’) to perform PCR amplification of the DNA fragment around the target site and perform agarose gel nucleic acid electrophoresis. Since it is dual-target cleavage, a fragment containing CAG nucleotide repeats will be removed, and it is expected to produce two DNA fragments, one is the original fragment and the other is the cleaved fragment. gRNA1+gRNA2 is expected to produce 410 bp + 282 bp bands; gRNA1+gRNA3 is expected to produce 410 bp + 273 bp bands, gRNA1+gRNA6 is expected to produce 410 bp + 279 bp bands, gRNA5+gRNA2 is expected to produce 410 bp + 208 bp bands, gRNA5+gRNA3 is expected to produce 410 bp + 199 bp bands, gRNA5+gRNA6 is expected to produce 410 bp + 205 bp bands. As Figure 3, after VLP-HD infected cells, the DNA cleavage bands were consistent with the expectation, and the fragment with CAG nucleotide repeats was cleaved off, demonstrating the gene editing ability of VLP-HD. When VLP-HD was applied to mHTT gene editing, all CAG nucleotide fragments could also be cleaved off, thus preventing the production of PolyQ and causing protein aggregation.

[0082] Example 5: Dual-target VLP-HD knockout of HTT protein

[0083] Produce VLP-HD(gRNA1+gRNA2) and VLP-HD(gRNA1+gRNA3) according to the method in Example 3, infect HeLa cells, collect cell pellets after 5 days of infection, and lyse them with RIPA (containing 1% PMSF). Add 10% loading buffer and treat in a metal bath at 98 °C for 10 min to prepare samples, and then perform Western Blot. Incubate with the primary antibodies anti-Huntingtin antibody (3E10) and anti-β-actin overnight, incubate with the secondary antibody anti-MouseIgG at room temperature, and then add the developing solution for development. Figure 4 It shows that after VLP-HD cleaved the HTT gene, the HTT protein was knocked out.

[0084] The structural schematic diagram of VLP-HD is as Figure 5 shown. It can be seen from Figure 5 that VLP-HD is a lentivirus-like particle (VLP) packaging the structure of Cas9:gRNARNP, where the RNA is not limited to one type, and there is no vector RNA gene of traditional lentivirus in VLP.

[0085] Example 6: NVR-G envelope protein has neurocyte infection specificity

[0086] 1. Construct the plasmid pNVR-G expressing the fusion protein NVR-G, replace the gene of VSV-G in the plasmid pMD.2G with the gene expressing NVR-G (the sequence is SEQ ID NO.29), and construct the plasmid pNVR-G. The plasmid sequence is as shown in SEQ ID NO.5.

[0087] 2. Production of lentivirus expressing GFP with NVR-G protein on the envelope. The method is as follows: Co-transfect the plasmid expressing the membrane protein (pNVR-G), the plasmid expressing the wild-type lentiviral GagPol long-chain protein (pMDlg / PRRE-D64V, the plasmid sequence is shown in SEQ ID NO.2), the helper plasmid (pRSV-REV), and the plasmid expressing GFP protein (pCCL-PGK-eGFP, the plasmid sequence is shown in SEQ ID NO.6) into 293T cells, collect the supernatant, and concentrate and purify the obtained product. Measure the VLP p24 concentration with a lentivirus titer ELISA kit for quantification.

[0088] 3. Production of lentivirus expressing GFP with VSV-G protein on the envelope. The method is as follows: Co-transfect the plasmid expressing the membrane protein (pMD.2G), the plasmid expressing the wild-type lentiviral GagPol long-chain protein (pMDlg / PRRE-D64V), the helper plasmid (pRSV-REV), and the plasmid expressing GFP protein (pCCL-PGK-eGFP) into 293T cells, collect the supernatant, and concentrate and purify the obtained product. Measure the VLP p24 concentration with a lentivirus titer ELISA kit for quantification.

[0089] 4. Seed THP-1 (human monocytic leukemia cells), SH-SY5Y (human neuroblastoma cells), HEB (human brain astrocytes), and HeLa cell lines into 48-well plates at 4×10 4 cells / well. After 24 hours, each type of cell is infected with 20 ng of lentivirus with NVR-G envelope protein and lentivirus with VSV-G envelope protein respectively. After 48 hours, use a flow analyzer (BD&LSR Fortessa) to detect the proportion of GFP-expressing cells in each group.

[0090] The experimental results are as Figure 6 shown. The lentivirus with VSV-G envelope protein has broad infectivity, can infect a variety of cells and generally has a high infection efficiency. The lentivirus with NVR-G envelope protein only has a high infection efficiency when infecting the neuronal cell line SH-SY5Y. VLP-HD needs to edit the mHTT gene of neuronal cells to play a therapeutic role. The lentiviral particles coated with NVR-G envelope protein can specifically infect nerve cells. Therefore, using NVR-G envelope protein to produce the lentiviral particles VLP-HD-NVR-G for treating Huntington's disease may have a certain targeting ability to infect neuronal cells.

[0091] Example 7: Editing of the HTT gene by VLP-HD-NVR-G

[0092] Using the method in Example 3, plasmid pNVR-G was used to replace plasmid pMD.2G to produce VLP-HD-NVR-G (gRNA1 + gRNA2). 293T cells were used to verify the gene editing efficiency.

[0093] Seed 293T cells into 96-well plates at 2×10 4 cells / well. After 24 h, infect with 100 ng of VLP-HD-NVR-G (gRNA1 + gRNA2). Harvest the cells 72 h after infection, extract the genomic DNA, and perform PCR amplification of the DNA fragment around the target site using primers (F: 5’-GGACGGGTCCAAGATGGACG-3’ and R: 5’-caaactcacGGTCGGTGCAG-3’) for agarose gel electrophoresis of nucleic acids. Since it is a dual-target cleavage, a fragment containing CAG nucleotide repeats will be removed, and two DNA fragments are expected to be produced, one is the original fragment and the other is the cleaved fragment. gRNA1 + gRNA2 is expected to produce bands of 410 bp + 282 bp. As Figure 7 shown, the PCR fragments of the gene-edited samples ( Figure 7 lanes 1 + 2 in

[0094] Example 8, VLP-HD-NVR-G Treatment of HD Mice, Efficacy Test

[0095] To study the potential of VLP-HD to alleviate the abnormal phenotypes of Huntington's disease, in vivo efficacy and safety studies were conducted using an HD disease-specific mouse model in which exon 1 of endogenous mouse Htt was replaced with exon 1 of human mutant Htt, with approximately 190 CAG repeats. One of the advantages of the mouse model is that it has a high level of expression of mutant huntingtin protein, which enables the rapid onset and progression of HD symptoms. This makes it a useful model for studying the early stages of the disease and for testing. The motor and balance abilities of Huntington mice are dysregulated, and the motor coordination and balance abilities can be evaluated using the beam walking test. The mouse walks 1 m on a grid beam. During walking, it is possible for the mouse's paws to fall or slip between the wires, and if this occurs, it is recorded as an error. The total number of steps and the total time are recorded. Inject VLP-HD-NVR-G into 8-week-old Huntington mice by intracranial injection, and inject PBS into the control group. Analyze the results 4 weeks later. The data show that in the beam walking test, untreated HD mice (HD Model) showed significantly more foot slips than wild-type (WT) mice, with a significant difference (**P < 0.01). After treatment with VLP-HD-NVR-G, the performance of the mice was not significantly different from that of WT mice (n.s.) ( Figure 8 ). This indicates that VLP-HD can improve the motor coordination and balance abilities of HD mice.

[0096] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art can make various modifications or alterations within the scope of the claims, which do not affect the essence of the present invention.

Claims

1. A lentivirus-like particle, characterized in that, The lentivirus-like particles are composed of an RNP complex in which a VLP vector encapsulates a Cas9 protein and a gRNA targeting the HTT gene; In the components of the VLP vector, the membrane protein includes a modified membrane protein with cell infection specificity, and the modified membrane protein with cell infection specificity is the NVR-G protein, and its amino acid sequence is SEQ ID NO.

8.

2. The lentivirus-like particle according to claim 1, wherein The gene sequences expressing the components of the VLP vector are respectively located on ① a plasmid expressing a membrane protein, ② a plasmid expressing a lentiviral GagPol long-chain protein containing an RNA-binding protein, ③ a plasmid expressing a lentiviral GagPol long-chain protein, ④ an auxiliary plasmid pRSV-Rev, ⑤ a plasmid expressing a Cas9 protein required for forming an RNP, and ⑥ a plasmid expressing a gRNA containing an RNA stem-loop structure recognized by an RNA-binding protein.

3. The lentivirus-like particle according to claim 1, wherein The amino acid sequence of the lentiviral GagPol long-chain protein containing an RNA-binding protein is SEQ ID NO.9; the gRNA backbone sequence containing an RNA stem-loop structure recognized by an RNA-binding protein is SEQ ID NO.

10.

4. The lentivirus-like particle according to claim 1, wherein The targeting site of the guide sequence carried on the gRNA targeting the HTT gene is any site on the HTT gene.

5. The lentivirus-like particle according to claim 1, wherein The gRNA sequence targeting the HTT gene is gRNA1: 5'-GGCCTTCATCAGCTTTTCCA-3', gRNA4: 5'-GAAGGACTTGAGGGACTCGA-3', gRNA7: 5'-GACCCTGGAAAAGCTGATGA-3', gRNA8: 5'-GGAGACCGCCATGGCGACCC-3', gRNA9: 5'-CAGCTTTTCCAGGGTCGCCA-3', gRNA10: 5'-CTTTTCCAGGGTCGCCATGG-3' or gRNA11: 5'-AGGCCTTCATCAGCTTTTCC-3'.

6. The lentivirus-like particle according to claim 1, wherein The gRNA sequences targeting the HTT gene are respectively selected from at least two of the following sequences: gRNA1: 5'-GGCCTTCATCAGCTTTTCCA-3', gRNA2: 5'-TGAGGAAGCTGAGGAGGCGG-3', gRNA3: 5'-GGCGGCGGCTGAGGAAGCTG-3', gRNA4: 5'-GAAGGACTTGAGGGACTCGA-3', gRNA5: 5'-TTCATTGCCCCGGTGCTGAG-3', gRNA6: 5'-GGCTGAGGAAGCTGAGGAGG-3', gRNA7: 5'-GACCCTGGAAAAGCTGATGA-3', gRNA8: 5'-GGAGACCGCCATGGCGACCC-3', gRNA9: 5'-CAGCTTTTCCAGGGTCGCCA-3', gRNA10: 5'-CTTTTCCAGGGTCGCCATGG-3', gRNA11: 5’-AGGCCTTCATCAGCTTTTCC-3’, gRNA12: 5’-GAGTCGGCCCGAGGCCTCCG-3’, gRNA13: 5’-GGCGGCTGAGGAAGCTGAGG-3’, gRNA14: 5’-AGCGGGCCCAAACTCACGGT-3’, gRNA15: 5’-AGCAGCGGCTGTGCCTGCGG-3’, gRNA16: 5’-GGAAGCTGAGGAGGCGGCGG-3’, gRNA17: 5’-GCCGGGACAGGGAGCTGCAG-3’, gRNA18: 5’-TGAGGAGGCGGCGGCGGCGG-3.

7. The lentivirus-like particle according to claim 6, wherein, The gRNA plasmid targeting the HTT gene is a plasmid combination of at least one of gRNA1, gRNA4, gRNA5, gRNA7-12 and at least one of gRNA2, gRNA3, gRNA6, gRNA13-18.

8. A method for preparing lentivirus-like particles according to claim 1, characterized in that, The method comprises the following steps: A plasmid expressing a membrane protein, a plasmid expressing a lentiviral GagPol long-chain protein containing an RNA-binding protein, a plasmid expressing a lentiviral GagPol long-chain protein, an auxiliary plasmid pRSV-Rev, are co-transfected into virus-producing cells with a plasmid expressing a Cas9 protein required for forming RNP and a plasmid expressing an HTT gRNA containing an RNA stem-loop structure recognized by the RNA-binding protein, or with a plasmid expressing a Cas9 protein required for forming RNP and an HTT gRNA plasmid containing an RNA stem-loop structure recognized by the RNA-binding protein, and the supernatant is collected, concentrated and purified to obtain the product.

9. Use of the lentivirus-like particle according to claim 1 in the preparation of a medicament for treating Huntington's disease.

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