Recombinant plasmid capable of autonomously secreting gRNA-loaded extracellular vesicles

Abstract

The invention relates to a recombinant plasmid capable of autonomously secreting gRNA-loaded extracellular vesicles, and belongs to the technical field of biology. The invention provides a recombinant plasmid capable of autonomously secreting gRNA-loaded extracellular vesicles, the recombinant plasmid uses a first promoter to carry out fusion expression on ARRDC1 protein and Tat cell penetrating peptide, and uses a second promoter to express gRNA and TAR sequences; or the recombinant plasmid uses the first promoter to perform fusion expression on the CD63 protein and the archaea ribosomal protein L7AE, and uses the second promoter to perform expression on the gRNA and the RNA C / D box sequence. After the recombinant plasmid disclosed by the invention is transfected into a host cell, the host cell can secrete the extracellular vesicles capable of automatically loading the gRNA, and the secreted extracellular vesicles have relatively high editing efficiency on target genes of the loaded gRNA.

Description

Technical Field

[0001] The invention relates to a recombinant plasmid capable of autonomously secreting an extracellular vesicle loaded with gRNA, and belongs to the field of biotechnology. Background Art

[0002] With the development and progress of genome sequencing technology, the occurrence of diseases is also closely related to genetic changes. Therefore, gene therapy (i.e., DNA therapy and RNA therapy) is also gradually developing and progressing. Gene therapy is a biomedical technology that treats diseases by modifying or manipulating gene expression. Since the concept was proposed in the 1960s, gene therapy has undergone decades of development and has made significant progress in the early 21st century, especially after the emergence of gene editing technology (such as the CRISPR / Cas9 system), gene therapy has entered a new era. However, with the continuous development of DNA editing technology, its persistence and irreversibility have become helpless in the face of off-target phenomena. The RNA targeting mode can achieve adjustability and reversibility. In specific therapeutic environments, the lack of permanent off-target provides unique advantages.

[0003] At present, the RNA targeting model mainly focuses on RNA editing technology, which is mainly based on adenosine deaminase (ADAR). By binding to RNA to form a double-stranded gRNA (i.e., the guide RNA that plays a role in RNA editing technology), adenine ribonucleotide deamination (A) is converted to inosine (I). Inosine is similar in structure to guanine ribonucleotide (G), and will be recognized as guanine at the transcriptome level, thereby exerting the RNA editing function. In addition to the great advantages of safety and reversibility, RNA editing technology also has a very broad prospect in disease treatment (such as the treatment of some genetic diseases). By precisely editing the mRNA in the patient's cells, diseases caused by gene mutations can be directly corrected, solving the problem at the source. In addition, some tumor-related diseases can also be treated with this technology. Through RNA editing technology, the expression of genes that promote cancer development in tumor cells can be reduced, or the expression of tumor suppressor genes can be enhanced, thereby inhibiting tumor growth and even inducing tumor cell death. In addition, RNA editing technology can also be used to enhance the ability of immune cells to recognize and eliminate tumors, thereby improving the effect of tumor immunotherapy.

[0004] However, RNA editing technology also faces huge challenges in clinical treatment. How to effectively and safely deliver RNA editing tools to target cells is the key to achieving RNA editing. At present, the delivery systems commonly used for RNA editing technology are mainly based on viral vectors and non-viral vectors. Among them, the delivery system based on viral vectors has a potential risk of immune response, and the delivery system based on non-viral vectors faces challenges in delivery efficiency and cell specificity. Extracellular vesicles (EVs) are considered to be a promising gene therapy vector. They participate in a variety of biological processes and can not only carry nucleic acids (such as mRNA, miRNA and other non-coding RNAs), but also carry a variety of biological macromolecules such as proteins to prevent the encapsulated materials from being degraded by enzymes in the circulatory system. They are relatively inert, non-immunogenic and biocompatible. In rodents, they can pass through various physical barriers, such as the blood-brain barrier. There are also studies reporting that through the specific interaction between peptides and RNA, specific mRNAs are encapsulated into extracellular vesicles and secreted outside the cells. The in vivo delivery of RNA editing technology through the construction of extracellular vesicles may overcome the bottleneck problem of existing RNA editing technology delivery systems. However, how to ensure that the extracellular vesicle-based delivery system can be stably delivered in the circulatory system, and how to ensure the editing efficiency of the extracellular vesicle-based delivery system is also a difficulty. Summary of the invention

[0005] To solve the above problems, the present invention provides a recombinant plasmid capable of autonomously secreting extracellular vesicles loaded with gRNA, wherein the recombinant plasmid uses a first promoter to express the ARRDC1 protein (a marker of plasma membrane budding microvesicles) and a Tat cell-penetrating peptide that can specifically bind to a TAR sequence, and uses a second promoter to express the gRNA and the TAR sequence (the binding site of the Tat cell-penetrating peptide);

[0006] Alternatively, the recombinant plasmid uses a first promoter to express a fusion of CD63 protein (a marker for extracellular vesicles) and the archaeal ribosomal protein L7AE that can specifically bind to the RNA C / D box sequence, and uses a second promoter to express gRNA and the RNA C / D box sequence (the binding site of the archaeal ribosomal protein L7AE).

[0007] In one embodiment of the present invention, the recombinant plasmid also expresses a central targeting peptide; the central targeting peptide is connected to the upstream (N-terminus) of the ARRDC1 protein or the CD63 protein.

[0008] In one embodiment of the present invention, the first promoter includes CMV promoter, U6 promoter, SV40 promoter, EF1α promoter, H1 promoter, CAG promoter and / or UBC promoter;

[0009] The second promoter includes U6 promoter, CMV promoter, SV40 promoter, EF1α promoter, H1 promoter, CAG promoter and / or UBC promoter.

[0010] In one embodiment of the present invention, the first promoter is a CMV promoter; the second promoter is a U6 promoter.

[0011] In one embodiment of the present invention, the nucleotide sequence of the CMV promoter is shown as SEQ ID NO.1; the nucleotide sequence of the U6 promoter is shown as SEQ ID NO.2.

[0012] In one embodiment of the present invention, the central targeting peptide includes brain-targeted RVG protein (chimeric rabies virus glycoprotein fragment peptide), brain-targeted Angiopep-2 (AP2), cell-penetrating peptide Transportan 10 (TP10) and / or brain-targeted MiniAp-4 (M4).

[0013] In one embodiment of the present invention, the amino acid sequence of the RVG protein is shown in SEQ ID NO.3.

[0014] In one embodiment of the present invention, the amino acid sequence of the ARRDC1 protein is shown as SEQ ID NO.4; the amino acid sequence of the Tat cell penetrating peptide is shown as SEQ ID NO.5; and the nucleotide sequence of the TAR sequence is shown as SEQ ID NO.6.

[0015] In one embodiment of the present invention, the amino acid sequence of the CD63 protein is shown as SEQ ID NO.7; the amino acid sequence of the archaeal ribosomal protein L7AE is shown as SEQ ID NO.8; and the nucleotide sequence of the RNA C / D box sequence is shown as SEQ ID NO.9.

[0016] In one embodiment of the present invention, the length of the gRNA is 91pb.

[0017] In one embodiment of the present invention, the method for constructing the recombinant plasmid comprises: linearizing the vector to obtain a linearized vector; connecting the linearized vector and a nucleic acid molecule to obtain a recombinant plasmid;

[0018] The nucleic acid molecule includes a first promoter, a gene fragment encoding an ARRDC1 protein, a gene fragment encoding a Tat cell-penetrating peptide, a second promoter, a gRNA, and a TAR sequence;

[0019] Alternatively, the nucleic acid molecule includes a first promoter, a gene fragment encoding CD63 protein, a gene fragment encoding archaeal ribosomal protein L7AE, a second promoter, gRNA and RNA C / D box sequence.

[0020] In one embodiment of the present invention, the nucleotide sequence of the gRNA is shown as SEQ ID NO.12 (P53 gene targeting GA mutation) or SEQ ID NO.13 (MECP2 gene targeting GA mutation).

[0021] In one embodiment of the present invention, when the nucleotide sequence of the gRNA is as shown in SEQ ID NO.12, the nucleic acid molecule consists of a first promoter, a gene fragment encoding the ARRDC1 protein, a gene fragment encoding the Tat cell penetrating peptide, a second promoter, the gRNA and the TAR sequence;

[0022] When the nucleotide sequence of gRNA is as shown in SEQ ID NO.13, the nucleic acid molecule consists of a first promoter, a gene fragment encoding a central targeting peptide, a gene fragment encoding an ARRDC1 protein, a gene fragment encoding a Tat cell penetrating peptide, a second promoter, gRNA and a TAR sequence.

[0023] In one embodiment of the present invention, when the nucleotide sequence of gRNA is as shown in SEQ ID NO.12, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.14; when the nucleotide sequence of gRNA is as shown in SEQ ID NO.13, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.15.

[0024] In one embodiment of the present invention, the vector comprises pcDNA 3.1 plasmid and / or pcDNA6.2 plasmid.

[0025] The present invention also provides a nucleic acid molecule for constructing an extracellular vesicle capable of autonomously loading gRNA, wherein the nucleic acid molecule comprises a first promoter, a gene fragment encoding an ARRDC1 protein, a gene fragment encoding a Tat cell-penetrating peptide, a second promoter, a gRNA, and a TAR sequence;

[0026] Alternatively, the nucleic acid molecule includes a first promoter, a gene fragment encoding CD63 protein, a gene fragment encoding archaeal ribosomal protein L7AE, a second promoter, gRNA and RNA C / D box sequence.

[0027] In one embodiment of the present invention, the nucleic acid molecule further comprises a gene fragment encoding a central targeting peptide; the gene fragment encoding the central targeting peptide is connected to the upstream (N-terminus) of the gene fragment encoding the ARRDC1 protein or the gene fragment encoding the CD63 protein.

[0028] In one embodiment of the present invention, the first promoter includes CMV promoter, U6 promoter, SV40 promoter, EF1α promoter, H1 promoter, CAG promoter and / or UBC promoter;

[0029] The second promoter includes U6 promoter, CMV promoter, SV40 promoter, EF1α promoter, H1 promoter, CAG promoter and / or UBC promoter.

[0030] In one embodiment of the present invention, the first promoter is a CMV promoter; the second promoter is a U6 promoter.

[0031] In one embodiment of the present invention, the nucleotide sequence of the CMV promoter is shown as SEQ ID NO.1; the nucleotide sequence of the U6 promoter is shown as SEQ ID NO.2.

[0032] In one embodiment of the present invention, the central targeting peptide includes brain-targeted RVG protein (chimeric rabies virus glycoprotein fragment peptide), brain-targeted Angiopep-2 (AP2), cell-penetrating peptide Transportan 10 (TP10) and / or brain-targeted MiniAp-4 (M4).

[0033] In one embodiment of the present invention, the amino acid sequence of the RVG protein is shown in SEQ ID NO.3.

[0034] In one embodiment of the present invention, the amino acid sequence of the ARRDC1 protein is shown as SEQ ID NO.4; the amino acid sequence of the Tat cell penetrating peptide is shown as SEQ ID NO.5; and the nucleotide sequence of the TAR sequence is shown as SEQ ID NO.6.

[0035] In one embodiment of the present invention, the amino acid sequence of the CD63 protein is shown as SEQ ID NO.7; the amino acid sequence of the archaeal ribosomal protein L7AE is shown as SEQ ID NO.8; and the nucleotide sequence of the RNA C / D box sequence is shown as SEQ ID NO.9.

[0036] In one embodiment of the present invention, the nucleotide sequence of the gene fragment encoding the Tat cell penetrating peptide is shown as SEQ ID NO.10; the nucleotide sequence of the gene fragment encoding the archaeal ribosomal protein L7AE is shown as SEQ ID NO.11.

[0037] In one embodiment of the present invention, the length of the gRNA is 91pb.

[0038] In one embodiment of the present invention, the nucleotide sequence of the gRNA is shown as SEQ ID NO.12 (P53 gene targeting GA mutation) or SEQ ID NO.13 (MECP2 gene targeting GA mutation).

[0039] In one embodiment of the present invention, when the nucleotide sequence of the gRNA is as shown in SEQ ID NO.12, the nucleic acid molecule consists of a first promoter, a gene fragment encoding the ARRDC1 protein, a gene fragment encoding the Tat cell penetrating peptide, a second promoter, the gRNA and the TAR sequence;

[0040] When the nucleotide sequence of gRNA is as shown in SEQ ID NO.13, the nucleic acid molecule consists of a first promoter, a gene fragment encoding a central targeting peptide, a gene fragment encoding an ARRDC1 protein, a gene fragment encoding a Tat cell penetrating peptide, a second promoter, gRNA and a TAR sequence.

[0041] In one embodiment of the present invention, when the nucleotide sequence of gRNA is as shown in SEQ ID NO.12, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.14; when the nucleotide sequence of gRNA is as shown in SEQ ID NO.13, the nucleotide sequence of the nucleic acid molecule is as shown in SEQ ID NO.15.

[0042] The present invention also provides a host cell for constructing an extracellular vesicle capable of autonomously loading gRNA, wherein the host cell carries the above-mentioned recombinant plasmid; or, the genome of the host cell is integrated with the above-mentioned nucleic acid molecule.

[0043] In one embodiment of the present invention, the method for preparing the host cell comprises: transfecting the above-mentioned recombinant plasmid into the host cell.

[0044] In one embodiment of the present invention, the host cells include 293T cells and / or umbilical cord mesenchymal stem cells.

[0045] The present invention also provides an extracellular vesicle capable of autonomously loading gRNA, wherein the extracellular vesicle is secreted by the above-mentioned host cell.

[0046] In one embodiment of the present invention, the method for preparing the extracellular vesicles comprises: inoculating the above-mentioned host cells into a culture medium for culturing, and then extracting the extracellular vesicles from the culture supernatant.

[0047] The present invention also provides the use of the above-mentioned recombinant plasmid or the above-mentioned nucleic acid molecule or the above-mentioned host cell or the above-mentioned extracellular vesicle in preparing a drug for preventing and / or treating a disease.

[0048] In one embodiment of the present invention, the disease includes a blood tumor caused by an abnormal mutation of the P53 gene or a Rett syndrome caused by an abnormal mutation of the MECP2 gene.

[0049] In one embodiment of the present invention, the blood tumor caused by abnormal mutation of the P53 gene refers to leukemia.

[0050] In one embodiment of the present invention, the ingredients of the drug further include pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include PBS buffer and / or water.

[0051] The present invention also provides a drug for preventing and / or treating a disease, wherein the drug comprises the above-mentioned recombinant plasmid, the above-mentioned nucleic acid molecule, the above-mentioned host cell and / or the above-mentioned extracellular vesicle.

[0052] In one embodiment of the present invention, the disease includes a blood tumor caused by an abnormal mutation of the P53 gene or a Rett syndrome caused by an abnormal mutation of the MECP2 gene.

[0053] In one embodiment of the present invention, the blood tumor caused by abnormal mutation of the P53 gene refers to leukemia.

[0054] In one embodiment of the present invention, the ingredients of the drug further include pharmaceutically acceptable excipients; the pharmaceutically acceptable excipients include PBS buffer and / or water.

[0055] The present invention also provides a gRNA for treating a disease, wherein the gRNA targets a GA mutated P53 gene or a GA mutated MECP2 gene; when targeting a GA mutated P53 gene, the nucleotide sequence of the gRNA is as shown in SEQ ID NO.12; when targeting a GA mutated MECP2 gene, the nucleotide sequence of the gRNA is as shown in SEQ ID NO.13.

[0056] In one embodiment of the present invention, the disease includes a blood tumor caused by an abnormal mutation of the P53 gene or a Rett syndrome caused by an abnormal mutation of the MECP2 gene.

[0057] In one embodiment of the present invention, the blood tumor caused by abnormal mutation of the P53 gene refers to leukemia.

[0058] The technical solution of the present invention has the following advantages:

[0059] The present invention provides a recombinant plasmid capable of autonomously secreting extracellular vesicles loaded with gRNA, wherein the recombinant plasmid uses a first promoter to express the ARRDC1 protein (a marker of plasma membrane budding microvesicles) and a Tat cell-penetrating peptide that can specifically bind to a TAR sequence, and uses a second promoter to express the gRNA and the TAR sequence (the binding site of the Tat cell-penetrating peptide);

[0060] Alternatively, the recombinant plasmid uses a first promoter to express a fusion of CD63 protein (a marker for extracellular vesicles) and the archaeal ribosomal protein L7AE that can specifically bind to the RNA C / D box sequence, and uses a second promoter to express gRNA and the RNA C / D box sequence (the binding site of the archaeal ribosomal protein L7AE).

[0061] Studies have shown that after the recombinant plasmid of the present invention is transfected into host cells, the host cells can secrete extracellular vesicles loaded with gRNA. At the same time, further studies have shown that the secreted extracellular vesicles can be stably delivered in the circulatory system, and the secreted extracellular vesicles have a high editing efficiency for the target gene of the gRNA loaded therein. Therefore, the recombinant plasmid of the present invention can realize the application of RNA editing technology in the treatment of diseases in vivo, and has great application prospects.

[0062] Furthermore, the gRNA has a length of 91 pb. A gRNA of this length can achieve higher editing efficiency.

[0063] Further, the nucleotide sequence of the gRNA is shown in SEQ ID NO.12 (P53 gene targeting GA mutation). The P53 gene of the human histiocytic lymphoma cell line (U937) changes its splicing site of Pre-mRNA due to the first nucleotide after codon 559 from G to A, which causes a frameshift mutation and causes the loss of P53 protein. Studies have shown that abnormal mutations of the P53 gene can lead to the occurrence of hematological tumors. If it is possible to find an RNA editing technology targeting abnormal mutations of the P53 gene, and to achieve its in vivo delivery through extracellular vesicles, it is very critical to treat hematological tumors caused by abnormal mutations of the P53 gene. At this stage, there is no report on RNA editing technology targeting abnormal mutations of the P53 gene and its extracellular vesicle delivery system. The recombinant plasmid of the present invention expresses the gRNA of the P53 gene targeting GA mutation. Studies have shown that after the recombinant plasmid of the present invention is transfected into host cells, the host cells can secrete extracellular vesicles that are loaded with gRNA targeting the GA mutant P53 gene. At the same time, further studies have shown that after the secreted extracellular vesicles are intravenously injected into the U937-SCID human M5 acute myeloid leukemia mouse model induced by U937 cells, the survival time of the U937-SCID human M5 acute myeloid leukemia mouse model can be significantly prolonged, and a good therapeutic effect is achieved. Therefore, the recombinant plasmid of the present invention can realize the application of RNA editing technology in the treatment of leukemia, and has great application prospects.

[0064] Further, the nucleotide sequence of the gRNA is shown in SEQ ID NO.13 (MECP2 gene targeting GA mutation). Rett syndrome is caused by a new mutation in the gene encoding the X-linked repressor methyl CpG binding protein 2 (MECP2). Among them, G>A missense and amber nonsense mutations lead to stop codons. C-terminal truncation caused by premature stop codons. Studies have shown that Rett syndrome is the main genetic cause of intellectual disability in women. During early development, women affected by Rett syndrome lose speech and purposeful hand movements, and have other hallmark neurological abnormalities, such as abnormal breathing, apnea, and epileptic seizures. If an RNA editing technology targeting abnormal mutations in the MECP2 gene can be found and its in vivo delivery can be achieved through extracellular vesicles, it is critical to the treatment of Rett syndrome caused by abnormal mutations in the MECP2 gene. At this stage, RNA editing technology targeting abnormal mutations in MECP2 and its extracellular vesicle delivery system have not been reported. Studies have shown that after the recombinant plasmid of the present invention is transfected into host cells, the host cells can secrete extracellular vesicles that are loaded with gRNA targeting the GA mutant MECP2 gene. At the same time, further studies have shown that the secreted extracellular vesicles can be injected intravenously into MECP2 G311ARett syndrome mouse model can significantly prolong MECP2 G311A The survival time of the Rett syndrome mouse model is prolonged, which has a good therapeutic effect. Therefore, the recombinant plasmid of the present invention can realize the application of RNA editing technology in the treatment of Rett syndrome and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 :The editing efficiency of gRNA was verified by the expression of green fluorescent protein (GFP).

[0066] Figure 2 :Flow cytometry quantification results of green fluorescent protein (GFP) expression.

[0067] Figure 3 : Statistical results of gRNA editing efficiency.

[0068] Figure 4 :Schematic diagram of the construction of the MVB extracellular vesicle secretion system.

[0069] Figure 5 :Schematic diagram of the construction of the ARMM extracellular vesicle secretion system.

[0070] Figure 6 : Enrichment of gRNA-91 in large vesicles secreted by cells.

[0071] Figure 7 : Enrichment of gRNA-91 in small vesicles secreted by cells.

[0072] Figure 8 : Detection of editing status after co-cultivation of extracellular vesicles containing gRNA-91 with 293 stable transfected cells expressing reporter plasmid.

[0073] Fig. 9 :Schematic diagram of the construction of the TP53-gRNA enriched extracellular vesicle system.

[0074] Fig.10 : Restoration of TP53 protein expression in U937 cells by TP53-gRNA.

[0075] Fig.11 :Effect of TP53-gRNA enriched extracellular vesicle system on the survival time of U937-SCID M5 acute myeloid leukemia model.

[0076] Fig.12 :Schematic diagram of the construction of the MECP2-gRNA enriched extracellular vesicle system.

[0077] Fig.13: Restoration of MECP2 protein expression by MECP2-gRNA in 293T cells with MECP2 gene G>A mutation.

[0078] Fig.14 : Enrichment of MECP2-gRNA extracellular vesicle system for MECP2 G311A Effects on survival time in a mouse model of Rett syndrome. DETAILED DESCRIPTION

[0079] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0080] If no specific experimental steps or conditions are specified in the following examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be purchased commercially.

[0081] Experimental Example 1: In vitro validation of RNA editing technology

[0082] This experiment validated RNA editing technology in vitro to determine whether gRNA can convert adenine ribonucleotides (A) to guanine ribonucleotides (G). The experimental process is as follows:

[0083] The gRNA with a length of 71 bp (hereinafter referred to as gRNA-71) and the gRNA with a length of 91 bp (hereinafter referred to as gRNA-91) were synthesized as shown in SEQ ID NO.16 and SEQ ID NO.17, respectively. 6After the cell suspension (cells suspended in serum medium) of 293T cells (purchased from Fuheng Bio) was inoculated into a 6-well plate with 2 mL of serum medium added to each well at an inoculation volume of 50 μL / well, the wells in the 6-well plate were divided into three groups, namely, the plasmid control group (reporter), gRNA intervention group A (reporter+arRNA71) and gRNA intervention group B (reporter+arRNA91); after the grouping, the reporter plasmid containing 1 μg / μL (synthesized by GenScript, the sequence can be found in the literature "Qu, L., et al. (2019)." Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs." Nature Biotechnology 37(9):1059-1069. ", the reporter plasmid contains dual luciferase mCherry and GFP, and the reporter plasmid has a stop codon of UAG at the junction) liposome transfection reagent (purchased from Shanghai Yisheng) was added to the wells of the plasmid control group at an addition volume of 5 μL / well, and 1 μg / μL reporter plasmid and 0.1 μM The liposome transfection reagent of gRNA-71 was added to the wells of gRNA intervention group A at an amount of 4 μL / well, and the liposome transfection reagent containing 1 μg / μL reporter plasmid and 0.1 μM gRNA-91 was added to the wells of gRNA intervention group B at an amount of 4 μL / well; after the addition, the 6-well plate was cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 hours; after the culture, the 6-well plate was photographed under a fluorescence microscope to observe whether green fluorescence was seen to determine whether the editing was successful; after the observation, the proportion of green fluorescent cells in red fluorescent cells was detected by flow cytometry to quantify the editing efficiency; wherein, the serum culture medium is a DMEM culture medium containing 10% (v / v) fetal bovine serum (FBS) (purchased from Yingjun Bio).

[0084] The results showed that gRNA was able to convert base A to base G in vitro, achieving RNA editing ( Figure 1 ). Moreover, the editing effect of gRNA-91 is better than that of gRNA-71 ( Figure 2-Figure 3 ), therefore, the gene expression sequence with a length of 91pb was also selected for subsequent vector optimization.

[0085] Experimental Example 2: Construction and verification of extracellular vesicles capable of autonomously loading gRNA

[0086] This experimental example constructed an extracellular vesicle that can autonomously load gRNA and verified the editing performance. The experimental process is as follows:

[0087] 1. Construction of target fragment

[0088] Construction plan 1: Extracellular vesicle system based on multivesicular body (MVB) secretion

[0089] In the protein expression part, CMV was first used as a promoter to express the extracellular vesicle marker CD63 protein and the archaeal ribosomal protein L7AE that can specifically bind to RNA C / Dbox. Then U6 was used as a promoter to express gRNA-91 and RNA C / D box sequence (the binding site of archaeal ribosomal protein L7AE). According to this design idea, the relevant gene elements were connected in series to obtain the target fragment A with a nucleotide sequence as shown in SEQ ID NO.18.

[0090] Construction scheme 2: A system for secreting extracellular vesicles based on the plasma membrane budding mediated by the arrestin protein structure 1 (ARRDC1)

[0091] In the protein expression part, CMV was first used as a promoter to express the plasma membrane budding microvesicle marker ARRDC1 protein and Tat cell penetrating peptide in fusion, and then U6 was used as a promoter to express gRNA-91 and TAR sequence (binding site of Tat cell penetrating peptide). According to this design idea, the relevant gene elements were connected in series to obtain the target fragment B with a nucleotide sequence as shown in SEQ ID NO.19.

[0092] 2. Construction of gene circuits

[0093] The pLV3ltr-ZsGreen-Puro-U6-xi (PHY-310) plasmid (purchased from Coris) was subjected to inverse PCR using an upstream primer with a nucleotide sequence as shown in SEQ ID NO.20 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.21 to obtain a linearized plasmid; the target fragment A and the target fragment B were subjected to inverse PCR using an upstream primer with a nucleotide sequence as shown in SEQ ID NO.22 and a downstream primer with a nucleotide sequence as shown in SEQ ID NO.23 to obtain gene fragment A and gene fragment B, respectively; the linearized plasmid was connected to gene fragment A and gene fragment B, respectively, using a recombinase (purchased from Novizan) to obtain a recombinant plasmid carrying the target fragment A (this recombinant plasmid is based on the gene loop A of the MVB extracellular vesicle secretion system, and the plasmid map is shown in Figure 4 ) and a recombinant plasmid carrying the target fragment (this recombinant plasmid is based on the gene loop B of the ARMM extracellular vesicle secretion system, the plasmid map is shown in Figure 5 ).

[0094] 3. Transfection of host cells

[0095] Containing 1×10 6After the cell suspension of 293T cells / mL (cells suspended in serum culture medium) was inoculated into T300 cell bottles with 8 mL of serum culture medium added to each bottle at an inoculation volume of 1000 μL / bottle, the T300 cell bottles were divided into four groups, namely, blank control group (mock), gRNA intervention group A (L7AE-gRNA-91), gRNA intervention group B (ARRDC1-gRNA-91) and gRNA intervention group C (U6-gRNA-91); after the grouping, the liposome transfection test The reagent was added to the T300 cell bottle of the blank control group at an addition volume of 200L / bottle, the liposome transfection reagent containing 1μg / μL gene loop A was added to the T300 cell bottle of the gRNA intervention group A at an addition volume of 100μL / bottle, the liposome transfection reagent containing 1μg / μL gene loop B was added to the T300 cell bottle of the gRNA intervention group B at an addition volume of 100μL / bottle, and the control gene loop (based on gene loop A, CD63 protein and RNA C / D were removed) was added to the T300 cell bottle of the gRNA intervention group B at an addition volume of 100μL / bottle. box sequence, and retain the rest) was added to the T300 cell flask of gRNA intervention group C at an addition volume of 100 μL / well; after the addition, the T300 cell flask was cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 6 hours; after the culture, the culture supernatant was discarded, the serum-free medium was replaced, and the T300 cell flask was continued to be cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 36 hours to secrete extracellular vesicles; after the culture, the culture supernatant was collected, first centrifuged at 3000g for 30 minutes to remove cell debris, and then centrifuged at 3000g for 30 minutes to remove impurities, and then ultracentrifuged at 10000g and 120000g for 90min to collect extracellular vesicles (ultracentrifugation at 10000g to harvest large vesicles, and ultracentrifugation at 120000g to harvest small vesicles); after centrifugation, discard the supernatant and take the precipitate; resuspend the precipitate in 200μL PBS buffer to obtain an extracellular vesicle resuspension; use q-PCR to detect the content of gRNA-91 in the extracellular vesicle resuspension; wherein the serum culture medium is a DMEM culture medium containing 10% (v / v) fetal bovine serum (FBS), and the serum-free culture medium is a DMEM culture medium without 10% (v / v) fetal bovine serum.

[0096] The results showed that compared with the blank control group, gRNA-91 was produced in the extracellular vesicle resuspensions obtained from gRNA intervention groups A and B, which were 3 nmol / g EV and 5 nmol / g EV, respectively (here, the total amount in large and small vesicles), while the extracellular vesicle resuspensions obtained from gRNA intervention group C contained almost no gRNA-91. It can be seen that the design of the extracellular vesicle system can significantly affect the autonomous loading of gRNA-91 in extracellular vesicles, and the ARRDC1-mediated plasma membrane budding secretion extracellular vesicle system has the highest content of gRNA-91 ( Figure 6-7 ).

[0097] 4. Verification of the editing effect of extracellular vesicles

[0098] 60 μL of transfection reagent lipo2000 (purchased from Thermo Fisher Scientific) was mixed with 750 μL of Opti-MEM medium (purchased from Thermo Fisher Scientific) and allowed to stand for 5 minutes to obtain mixture A; 12 μg of the reporter plasmid in Experimental Example 1, 6 μg of lentiviral packaging plasmid psPAX2 (purchased from GenScript), and 1.5 μg of lentiviral packaging plasmid pMD2G (purchased from GenScript) were mixed with 750 μL of Opti-MEM medium (purchased from GenScript) and allowed to stand for 5 minutes to obtain mixture B; mixture A and mixture B were mixed and allowed to stand for 20 minutes to obtain transfection solution; 293T cells were cultured at 3×10 5 The inoculum amount of each was inoculated into a 10 cm culture dish with 8 mL of serum culture medium added to each well, and cultured in a 37° C., 5% (v / v) CO2 incubator for 24 hours; after culturing for 24 hours, the cell culture medium in the 10 cm culture dish was discarded, and the transfection solution was added to the 10 cm culture dish, and cultured in a 37° C., 5% (v / v) CO2 incubator for 6 hours for transfection; after the transfection was completed, the cell culture medium in the 10 cm culture dish was discarded, and a new serum-free culture medium was replaced, and the transfection was continued in a 37° C., 5% (v / v) CO2 incubator for 48 hours; after the transfection was completed, the culture supernatant was collected in a biological safety cabinet; the culture supernatant was filtered with a 0.22 μm filter to obtain a cell culture fluid containing the lentivirus.

[0099] 2 μL of infection-assisting reagent Polybrene (purchased from Bio-Tech) was mixed with 2 mL of cell culture medium containing lentivirus to obtain infection solution (the amount for one well); 293T cells were plated at 1×10 5The inoculum was inoculated into a 6-well plate with 2 mL of serum culture medium added to each well, and cultured in a 37°C, 5% (v / v) CO2 incubator for 24 h; after culturing for 24 h, the culture supernatant was discarded, and the infection solution was added to the 6-well plate, and cultured in a 37°C, 5% (v / v) CO2 incubator for 48 h for infection; after the infection was completed, the infection solution in the 6-well plate was aspirated and discarded, and 2 mL of drug screen culture medium was added to each well of the 6-well plate, and cultured in a 37°C, 5% (v / v) CO2 incubator. The cells were cultured in a CO2 incubator for 48 hours for drug screening. After drug screening, 200 μL of trypsin digestion solution (purchased from Thermo Fisher Scientific) was added to a 6-well plate per well, and the plates were incubated in a 37°C, 5% (v / v) CO2 incubator for 30 seconds for digestion. After digestion, an equal amount of drug screening medium was added to the cell digestion solution obtained by digestion to terminate digestion, and then the cell digestion solution obtained by digestion was centrifuged to collect the cell precipitate. The cell precipitate was resuspended with 1 mL of drug screening medium. , and obtain a cell resuspension; take the cell resuspension obtained by digestion containing 450 cells and dilute it to 90mL with a drug screen medium to obtain a cell dilution; after inoculating the cell dilution into a 96-well plate at an inoculation volume of 200μL / well, culture it in a 37°C, 5% (v / v) CO2 incubator. During the culture process, observe whether it is necessary to change the medium every two days (if the culture medium turns yellow, replace it with a new drug screen medium; if the culture medium is still pink, there is no need to replace it with a new drug screen medium, and wait for the next replacement cycle). After two weeks of culture, observe the number of cells in each well of the 96-well plate under an inverted microscope, and mark the wells with 1 to 2 cells growing out; expand the marked positive cells until the required cell amount is obtained for monoclonal cell line sequencing verification, and if the verification is correct, the 293 stable strain expressing the reporter plasmid is obtained; wherein, the drug screen medium is an MSCM medium containing 1μg / mL puromycin, and the MSCM medium is purchased from Dakoway Company.

[0100] Containing 1×10 6After the serum culture medium of 293 stable strains was inoculated into a 6-well plate with 2 mL of serum culture medium added to each well at an inoculum volume of 50 μL / well, the wells in the 6-well plate were divided into three groups, namely, a blank control group (mock), a gRNA intervention group A (ARRDC1-gRNA-91), and a gRNA intervention group B (U6-gRNA-91); after the grouping, the extracellular vesicle resuspension obtained by collecting the blank control group (mock), gRNA intervention group B (ARRDC1-gRNA-91), and gRNA intervention group C (U6-gRNA-91) in the above host cell transfection step was Instead of testing, the large vesicles and small vesicles were harvested by ultracentrifugation at 120000g for 90 min. The vesicles were added to the blank control group, gRNA intervention group A and gRNA intervention group B of this experiment at an amount of 150 μL / well. After the addition, the 6-well plate was cultured in a cell culture incubator with 5% (v / v) CO2 and 37°C for 48 h. After the culture, the 6-well plate was photographed under a fluorescence microscope to observe whether green fluorescence was seen to determine whether the editing was successful. The serum culture medium is a DMEM culture medium containing 10% (v / v) fetal bovine serum (FBS), and the serum-free culture medium is a DMEM culture medium without 10% (v / v) fetal bovine serum.

[0101] The results showed that compared with the blank control group, the extracellular vesicles corresponding to gene loop A produced more green fluorescence after co-culture with the stable transgenic strain of the reporter gene, while the extracellular vesicles corresponding to gene loop B produced less green fluorescence after co-culture with the stable transgenic strain of the reporter gene. It can be seen that the extracellular vesicles secreted by the ARRDC1-mediated system played a better RNA editing effect ( Figure 8 ).

[0102] Experimental Example 3: Design of TP53-gRNA and verification of in vitro effects

[0103] In this experiment, TP53-gRNA was designed for the p53 gene with GA mutation based on RNA editing technology, and RNA editing of TP53-gRNA was tested. The experimental process is as follows:

[0104] The TP53-gRNA with the nucleotide sequence shown in SEQ ID NO.12 was transfected into U-937 cells (purchased from Fuheng) by electroporation (voltage 520V, capacitance 1275uF) to obtain transfected U-937 cells; the transfected U-937 cells were cultured at 1×10 5After adding 100 μg / well of the added amount to a 6-well plate with 2 mL of serum culture medium added to each well, the plate was cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 hours; after the culture, the expression of TP53 protein in the cells was detected by Western blot to verify the editing efficiency; wherein the serum culture medium is 1640 culture medium (purchased from Gibco) containing 10% (v / v) fetal bovine serum (FBS).

[0105] The results showed that the TP53 protein with U937 deletion mutation was re-expressed. It can be seen that TP53-gRNA corrected the GA mutation of the p53 gene at the transcriptome level and restored the missing p53 ( Fig.10 ).

[0106] Experimental Example 4: Verification of the therapeutic effect of TP53-gRNA-expressing extracellular vesicles on leukemia

[0107] This experimental example tested the therapeutic effect of TP53-gRNA-expressed extracellular vesicles on leukemia. The experimental process is as follows:

[0108] Referring to Experimental Example 2, on the basis of gene loop A, the gRNA with a length of 91 bp as shown in SEQ ID NO.17 was replaced with the TP53-gRNA as shown in SEQ ID NO.12, so as to assemble the TP53-gRNA with the ARDDC1-mediated extracellular vesicle skeleton vector system to construct a recombinant plasmid (this recombinant plasmid is the TP53-gRNA-ARDDC1 gene loop, the nucleotide sequence of the target fragment corresponding to this recombinant plasmid is shown in SEQ ID NO.14, and the plasmid map of this recombinant plasmid is shown in Fig. 9 ).

[0109] The 293T stable transfectant strain was constructed by referring to the verification steps of the extracellular vesicle editing effect in Experimental Example 2, and the extracellular vesicle resuspension rich in TP53-gRNA was obtained by referring to the transfection steps of the host cells in Experimental Example 2. Thirty SCID mice (purchased from Vitallife) were taken and U-937 cells were inoculated at 1×10 8The injection dose of 100 mg / mL (the solvent is PBS buffer) was injected into the mouse through the tail vein. After 15 days of injection, the U-937-SCID human M5 acute myeloid leukemia mouse model induced by U-937 cells was obtained. The U-937-SCID human M5 acute myeloid leukemia mouse model induced by U-937 cells was divided into three groups, two groups were model group, treatment group and control group, with 10 mice in each group. The extracellular vesicle resuspension rich in TP53-gRNA was injected into the mouse model of the treatment group through the tail vein at an injection dose of 0.2 mL, and the extracellular vesicle resuspension rich in gRNA-91 was injected into the mouse model of the control group through the tail vein at an injection dose of 0.2 mL. The mouse model of the model group was injected with an equal dose of normal saline. After the injection, normal SCID mice (10 mice) were used as controls, and the survival time of mice in different groups was continuously observed.

[0110] The survival cycle of model mice showed that TP53-gRNA-rich cells prolonged the survival time of U937-SCID human M5 acute myeloid leukemia mice ( Fig.11 ).

[0111] Experimental Example 5: Design of MECP2-gRNA and verification of in vitro effects

[0112] In this experiment, based on RNA editing technology, MECP2-gRNA was designed for the MECP2 gene with GA mutation, and RNA editing of MECP2-gRNA was tested. The experimental process is as follows:

[0113] The MECP2-gRNA with the nucleotide sequence shown in SEQ ID NO.13 was introduced into HEK293 cells (referred to as HEK293) with a G>A nonsense mutation in the MECP2 gene using CRISPR-Cas9 technology. MECP2(G>A) , purchased from Fuheng Biotechnology) to obtain treated HEK293 cells; the treated HEK293 cells were cultured at 1×10 5 After adding 1000 cells / well to a 6-well plate with 2 mL of serum culture medium added to each well, the cells were cultured in a 5% (v / v) CO2, 37°C cell culture incubator for 48 hours; after the culture, the expression of MECP2 protein in the cells was detected by Western blot to verify the editing efficiency; wherein the serum culture medium was 1640 culture medium (purchased from Gibco) containing 10% (v / v) fetal bovine serum (FBS).

[0114] The results showed that the MECP2 nonsense mutation caused a deletion mutation in the protein, and MECP2-gRNA restored the deletion of MECP2. It can be seen that MECP2-gRNA restored the full length and function of the MECP2 protein ( Fig.13 ).

[0115] Experimental Example 6: Verification of the therapeutic effect of extracellular vesicles expressed by MECP2-gRNA on Rett syndrome

[0116] This experimental example tested the therapeutic effect of MECP2-gRNA-expressed extracellular vesicles on Rett syndrome. The experimental process is as follows:

[0117] Referring to Experimental Example 2, on the basis of gene loop A, the gRNA with a length of 91 bp as shown in SEQ ID NO.17 was replaced with the MECP2-gRNA with a nucleotide sequence as shown in SEQ ID NO.13, so as to assemble the MECP2-gRNA with the ARDDC1-mediated extracellular vesicle skeleton carrier system. During the assembly process, since MECP2 gene deficiency is a neurological disease, extracellular vesicles need to pass through the blood-brain barrier. For this reason, a fused RVG short peptide (which can pass through the blood-brain barrier) is expressed at the N-terminus of ARDDC1, and a recombinant plasmid is constructed (this recombinant plasmid is the MECP2-gRNA-ARDDC1 gene loop, and the nucleotide sequence of the target fragment corresponding to this recombinant plasmid is shown in SEQ ID NO.15, and the plasmid map of this recombinant plasmid is shown in Fig.12 ).

[0118] Refer to the verification steps of the extracellular vesicle editing effect in Experimental Example 2 to construct a 293T stable transfectant strain, and refer to the host cell transfection steps in Experimental Example 2 to obtain an extracellular vesicle resuspension rich in MECP2-gRNA. G311A Rett syndrome mouse model (purchased from Vital River), the mouse model was divided into three groups, namely model group, treatment group and control group, with 10 mice in each group. The extracellular vesicle resuspension rich in MECP2-gRNA was injected into the mouse model of the treatment group through the tail vein at an injection dose of 0.2 mL, and the extracellular vesicle resuspension rich in gRNA-91 was injected into the mouse model of the control group through the tail vein at an injection dose of 0.2 mL. The mouse model of the control group was injected with an equal dose of normal saline. After the injection, normal mice (10 mice) were used as controls, and the survival time of mice in different groups was continuously observed.

[0119] The survival of model mice showed that extracellular vesicles enriched with MECP2-gRNA prolonged the expression of MECP2 G311A The survival time of Rett syndrome mice was significantly improved. Fig.14 ).

[0120] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A recombinant plasmid capable of autonomously secreting extracellular vesicles loaded with gRNA, characterized in that: The recombinant plasmid uses a first promoter to express the ARRDC1 protein and a Tat cell-penetrating peptide that can specifically bind to the TAR sequence, and uses a second promoter to express the gRNA and the TAR sequence; Alternatively, the recombinant plasmid uses a first promoter to express the CD63 protein and the archaeal ribosomal protein L7AE that can specifically bind to the RNA C / D box sequence, and uses a second promoter to express the gRNA and the RNA C / D box sequence.

2. The recombinant plasmid according to claim 1, characterized in that The recombinant plasmid also expresses a central targeting peptide; the central targeting peptide is connected to the upstream of the ARRDC1 protein or the CD63 protein.

3. The recombinant plasmid according to claim 1 or 2, characterized in that The method for constructing the recombinant plasmid comprises: linearizing the vector to obtain a linearized vector; connecting the linearized vector and a nucleic acid molecule to obtain a recombinant plasmid; The nucleic acid molecule includes a first promoter, a gene fragment encoding an ARRDC1 protein, a gene fragment encoding a Tat cell-penetrating peptide, a second promoter, a gRNA, and a TAR sequence; Alternatively, the nucleic acid molecule includes a first promoter, a gene fragment encoding CD63 protein, a gene fragment encoding archaeal ribosomal protein L7AE, a second promoter, gRNA and RNA C / D box sequence.

4. A nucleic acid molecule for constructing an extracellular vesicle capable of autonomously loading gRNA, characterized in that: The nucleic acid molecule includes a first promoter, a gene fragment encoding an ARRDC1 protein, a gene fragment encoding a Tat cell-penetrating peptide, a second promoter, a gRNA, and a TAR sequence; Alternatively, the nucleic acid molecule includes a first promoter, a gene fragment encoding CD63 protein, a gene fragment encoding archaeal ribosomal protein L7AE, a second promoter, gRNA and RNA C / D box sequence.

5. The nucleic acid molecule according to claim 4, characterized in that The nucleic acid molecule also includes a gene segment encoding a central targeting peptide; the gene segment encoding the central targeting peptide is connected to the upstream of the gene segment encoding the ARRDC1 protein or the gene segment encoding the CD63 protein.

6. A host cell for constructing an extracellular vesicle capable of autonomously loading gRNA, characterized in that: The host cell carries the recombinant plasmid according to any one of claims 1 to 3; or the genome of the host cell is integrated with the nucleic acid molecule according to claim 4 or 5.

7. An extracellular vesicle capable of autonomously loading gRNA, characterized in that: The extracellular vesicles are secreted from the host cell according to claim 6.

8. Use of the recombinant plasmid according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the host cell according to claim 6, or the extracellular vesicle according to claim 7 in the preparation of a drug for preventing and / or treating a disease.

9. A drug for preventing and / or treating a disease, characterized in that: The components of the drug include the recombinant plasmid according to any one of claims 1 to 3, the nucleic acid molecule according to claim 4 or 5, the host cell according to claim 6 and / or the extracellular vesicle according to claim 7.

10. A gRNA for treating a disease, characterized in that: The gRNA targets the GA mutated P53 gene or the GA mutated MECP2 gene; when targeting the GA mutated P53 gene, the nucleotide sequence of the gRNA is shown in SEQ ID NO.12; when targeting the GA mutated MECP2 gene, the nucleotide sequence of the gRNA is shown in SEQ ID NO.13.

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