Targeted gene editing enzyme engineered adeno-associated virus and pharmaceutical composition thereof

Abstract

The invention belongs to the field of biological medicine, and relates to a targeted gene editing enzyme engineered adeno-associated virus and a pharmaceutical composition thereof. Specifically, the invention relates to a modified adeno-associated virus, and the capsid protein of the modified adeno-associated virus is connected with a DNA cleavage enzyme. The invention also relates to a transformed cell. According to the method, knockin of the gene segment growing to 3.5-4K of the T cell can be achieved in one step, meanwhile, multi-gene knockout is conducted on the T cell, and the method has a good application prospect.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application is based on and claims priority to an application with CN application number 202311123533.4 and filing date September 1, 2023. The entire contents of the CN application are hereby introduced as a whole into this application. Technical Field

[0003] The present invention belongs to the field of biomedicine and relates to an adeno-associated virus engineered with a targeted gene editing enzyme and a pharmaceutical composition thereof. Background Art

[0004] Adeno-associated virus (AAV) is one of the simplest viruses discovered so far, consisting of only 60 capsid proteins and a single-stranded DNA of about 4.7K. It has extremely low immunogenicity and is not pathogenic to humans. It is a gene therapy drug approved by the FDA. Its characteristic is that it can directly deliver DNA into the cell nucleus for gene expression or DNA repair.

[0005] CRISPR / Cas gene editing technology originated from the study of the immune mechanism of bacteria against exogenous invading nucleic acids. After continuous development, it has become the most common gene editing tool. The most commonly used CRISPR / Cas proteins include Cas9 and Cas12a. CRISPR / Cas has DNA cutting activity, and gRNA can recognize the target region DNA on the genome. CRISPR / Cas protein combines with gRNA to form RNP to cut at this location, forming a DNA double-strand break (DSB). At this time, the cell will initiate a repair mechanism including two major categories: non-homologous end joining (NHEJ) and homologous recombination (HDR). If there is no repair template, the cell will form a mutation at the break using the NHEJ pathway. If the HDR template is contained at this time, the cell will start the HDR mode to repair the broken DNA. Therefore, specific genes can be knocked out through NHEJ, and exogenous genes can be knocked in at a specific point through HDR.

[0006] Therefore, the combination of AAV and CRISPR / Cas9 / 12a can achieve the insertion of customized DNA fragments at specific sites in the cell genome. At present, this process is generally completed in two steps: first, Cas9 / 12a RNP is electroporated into the cell using an electroporation device, and then AAV containing the target DNA fragment is added to infect the cell to achieve the targeted knock-in of the target DNA fragment in the genome. This operation is not only cumbersome, expensive, and heavily dependent on equipment, but also causes great damage to the cells.

[0007] Chimeric antigen receptors (CARs) are used to load T cells to form CAR T cells for killing tumor cells. The structure of CAR consists of three parts, namely the extracellular part that recognizes tumor antigens, the transmembrane region, and the intracellular cell conduction region; the extracellular part generally uses molecules containing antibody variable regions, such as ScFv. When the ScFv outside the CAR T cell recognizes the tumor antigen, it can transmit the signal to the inside of the CAR T cell, and the CAR T cell can play a role in killing tumor cells. The traditional CAR T preparation process uses lentivirus as a gene transduction tool. The genes carried during the lentiviral transduction process are randomly inserted into the genome of T cells, which has potential genotoxicity. Therefore, the development of site-specific insertion technology for the preparation of CAR T cells is one of the hot spots in the current research field of CAR T cell preparation. At present, the site-specific insertion preparation of CAR T cells is generally carried out by electroporation of Cas9 / 12RNP plus adeno-associated virus AAV infection, or direct electroporation of Cas9 / 12RNP plus DNA. The technical steps are cumbersome, the electric shock causes great damage to cells, and only site-specific insertion of cells in vitro can be performed.

[0008] Developing a gene editing technology that is easy to operate, economical, and harmless to cells will greatly improve the current technical shortcomings and promote the development of cell and gene therapy. Summary of the invention

[0009] After in-depth research and creative work, the inventors have deeply integrated CRISPR / Cas gene editing technology with AAV vectors, and developed a targeted gene editing enzyme engineered adeno-associated virus and its pharmaceutical composition. The present invention can achieve site-specific knock-in of large fragments in one step or in steps by non-electroporation, and simultaneously achieve multi-gene knockout. In addition to being used for the construction of CAR T, the present invention can also be used for the construction of TCR T, STAR T, and site-specific knock-in and knock-out editing of natural killer cells, dendritic cells, macrophages, hematopoietic stem cells, neurons and other cells. The following invention is thus provided:

[0010] One aspect of the present invention relates to a modified adeno-associated virus, whose capsid protein is linked to a DNA cutting enzyme.

[0011] In some embodiments of the present invention, in the modified adeno-associated virus, the DNA cutting enzyme is linked to a targeting molecule.

[0012] In some embodiments of the present invention, the modified adeno-associated virus, wherein:

[0013] The DNA shearing enzyme is directly connected to the capsid protein, or is connected to the capsid protein via a first linker; and / or

[0014] The DNA cutting enzyme is directly connected to the targeting molecule or connected to the targeting molecule through a second linker.

[0015] In some embodiments of the present invention, the modified adeno-associated virus, wherein:

[0016] The DNA shearing enzyme is directly linked to the capsid protein; and / or

[0017] The DNA cutting enzyme is directly linked to the targeting molecule.

[0018] In some embodiments of the present invention, the modified adeno-associated virus, wherein:

[0019] The DNA shearing enzyme is directly linked to the capsid protein; and / or

[0020] The DNA cutting enzyme is connected to the targeting molecule via a second linker.

[0021] In some embodiments of the present invention, the modified adeno-associated virus, wherein:

[0022] The DNA shearing enzyme is connected to the capsid protein via a first linker; and / or

[0023] The DNA cutting enzyme is directly linked to the targeting molecule.

[0024] In some embodiments of the present invention, the modified adeno-associated virus, wherein:

[0025] The DNA shearing enzyme is connected to the capsid protein via a first linker; and / or

[0026] The DNA cutting enzyme is connected to the targeting molecule via a second linker.

[0027] In some embodiments of the present invention, the modified adeno-associated virus, wherein the adeno-associated virus is AAV2 or AAV6.

[0028] In some embodiments of the present invention, the modified adeno-associated virus, wherein the DNA cutting enzyme is CAS12, Cas9, a base editing enzyme or a prime editing enzyme;

[0029] Preferably, the Cas12 is one selected from Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i and Cas12j;

[0030] Preferably, the Cas9 is one selected from spCas9, CjCas9 (984), Nme2Cas9 (1082), SauriCas9 (1061) and SauCas9 (1053).

[0031] In some embodiments of the present invention, in the modified adeno-associated virus, each adeno-associated virus is connected to 1-60 DNA shearing enzyme molecules. Theoretically, one adeno-associated virus has 60 AVI tags.

[0032] Without being limited by theory, adeno-associated virus has 60 capsid proteins. Taking into account the efficiency of connection, each capsid protein is connected to 1-60, 1-50, 1-40, 1-30, 1-20, 1-10, 10-50, 10-40, 10-30, 10 to 20, 20-50, 20-40, 20-30, 30 to 50, 30 to 40 or 40 to 50 DNA cutting enzyme molecules.

[0033] In some embodiments of the present invention, in the modified adeno-associated virus, the targeting molecule is a T cell targeting molecule.

[0034] In some embodiments of the present invention, the modified adeno-associated virus, wherein the targeting molecule is an antibody or ligand targeting T cells;

[0035] Preferably, the antibody is a single-chain antibody or a nanobody targeting T cells.

[0036] In some embodiments of the present invention, in the modified adeno-associated virus, the targeting molecule is a molecule targeting a T cell receptor.

[0037] In some embodiments of the present invention, the modified adeno-associated virus, wherein the targeting molecule is an antibody or ligand targeting a T cell receptor;

[0038] Preferably, the antibody is a single-chain antibody or a nanobody targeting a T cell receptor.

[0039] Antibody ScFvs that bind to TCR can be converted into ScFvs that target other cell types as needed, such as antiCD16 targeting NK cells, antiCD34 targeting HSC cells, and so on.

[0040] In some embodiments of the present invention, the modified adeno-associated virus, wherein the targeting molecule is an antibody or a ligand;

[0041] Preferably, the antibody is a single-chain antibody or a nanobody;

[0042] Preferably, the single-chain antibody is a single-chain antibody targeting a T cell receptor;

[0043] Preferably, the amino acid sequence of the single-chain antibody is as shown in SEQ ID NO:4.

[0044] In some embodiments of the present invention, the modified adeno-associated virus, wherein the first linker is selected from biotin-avidin, biotin-streptavidin, AAV receptor, SpyTag / SpyCatcher and anti-capsid protein antibody;

[0045] Preferably, the biotin is directly linked to the capsid protein via the AVI polypeptide; preferably, the AVI polypeptide is inserted into the VRVIII region of the AAV6 capsid triple axis (this region has little effect on AAV packaging);

[0046] Preferably, the amino acid sequence of the AAV receptor is as shown in SEQ ID NO:17.

[0047] In some embodiments of the present invention, the modified adeno-associated virus, wherein the second linker is selected from biotin-avidin, biotin-streptavidin, and paired proteins with binding effects such as: antibody-antigen, receptor-ligand, OsASI1-BAH-OsSUVH6 or SpyTag / SpyCatcher.

[0048] In some embodiments of the present invention, the modified adeno-associated virus comprises:

[0049] The plasmid pRepCap encoding AAV capsid and replicase, the adenovirus helper plasmid pHelp, and the pTransfer plasmid containing the target gene to be transferred.

[0050] In some embodiments of the present invention, the modified adeno-associated virus, wherein:

[0051] The target gene is a nucleic acid sequence encoding a chimeric antigen receptor (CAR), a coagulation factor gene or an SMN gene (spinal muscular atrophy lacks the SMN gene);

[0052] Preferably, the chimeric antigen receptor comprises a single chain antibody recognizing the CD19 antigen, a CD8 hinge region, a CD28 transmembrane region and an intracellular region, and a CD3 intracellular region.

[0053] In some embodiments of the present invention, the modified adeno-associated virus comprises, in sequence:

[0054] Adeno-associated virus, a first linker, a DNA cutting enzyme, a second linker and a targeting molecule;

[0055] AAV2 or AAV6, a first linker, Cas12a, a second linker, and a single-chain antibody targeting a T cell receptor;

[0056] AAV2 or AAV6, biotin-streptavidin, Cas12a, OsASI1-BAH-OsSUVH6, and a single-chain antibody targeting the T cell receptor;

[0057] AAV2 or AAV6, AAV receptor, Cas12 (e.g., Cas12a) or Cas9, a second linker (e.g., SpyTag / SpyCatcher), and a single-chain antibody targeting a T cell receptor; or

[0058] AAV2 or AAV6, and a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO:24 to SEQ ID NO:27.

[0059] In some embodiments of the present invention, the modified adeno-associated virus is composed of:

[0060] Adeno-associated virus, a first linker, a DNA cutting enzyme, a second linker and a targeting molecule;

[0061] AAV2 or AAV6, a first linker, Cas12a, a second linker, and a single-chain antibody targeting a T cell receptor;

[0062] AAV2 or AAV6, biotin-streptavidin, Cas12a, OsASI1-BAH-OsSUVH6, and a single-chain antibody targeting the T cell receptor;

[0063] AAV2 or AAV6, AAV receptor, Cas12 (e.g., Cas12a) or Cas9, a second linker (e.g., SpyTag / SpyCatcher), and a single-chain antibody targeting a T cell receptor; or

[0064] AAV2 or AAV6, and a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO:24 to SEQ ID NO:27.

[0065] In some embodiments of the present invention, the modified adeno-associated virus, wherein:

[0066] The N-terminus of the DNA shearing enzyme is connected to streptavidin, and the C-terminus of the DNA shearing enzyme is connected to a targeting molecule; or

[0067] The C-terminus of the DNA shearing enzyme is connected with streptavidin, and the N-terminus of the DNA shearing enzyme is connected with a targeting molecule.

[0068] In some embodiments of the present invention, the modified adeno-associated virus comprises the following components:

[0069] AAV2 or AAV6, AVI polypeptide, biotin-streptavidin, Cas12a, OsASI1-BAH-OsSUVH6 and single-chain antibody targeting T cell receptor. For example, it can be prepared by referring to the following steps:

[0070] 1) Inserting the AVI polypeptide into the VRVIII region of the AAV6 capsid tripartite axis to construct an engineered AAV6;

[0071] 2) Co-transfecting 293T cells with the AAV packaging plasmid and the plasmid expressing BirA, and adding D-biotin to the culture medium to produce biotin-site-labeled AAV6 at the AVI polypeptide (other AAVs are also applicable, such as AAV2);

[0072] 3) AsCas12a and antiTCR single-chain antibody ScFv were expressed in prokaryotic and eukaryotic cells, respectively. The N-terminus of AsCas12a was fused with Streptavidin, and the C-terminus was fused with OsASI1-BAH. The N-terminus of antiTCR ScFv was fused with the OsSUVH6 polypeptide.

[0073] As another embodiment, "Streptavidin is fused to the C-terminus of AsCas12a, and OsASI1-BAH is fused to the N-terminus. The C-terminus of antiTCR ScFv is fused to the polypeptide of OsSUVH6."

[0074] The two proteins are assembled into one protein through the polypeptides of OsASI1-BAH and OsSUVH6, and Streptavidin can bind to biotin on AAV6.

[0075] Alternatively, AAV receptors can be used to directly bind AAV without the need to biotinylate AAV.

[0076] 4) Assemble AAV6-AsCas12a-gRNA-antiTCR ScFv to form an editing tool that can specifically edit T cells to generate CAR T / STAR T / TCR T.

[0077] Another aspect of the present invention relates to a complex comprising the modified adeno-associated virus described in any one of the present invention and gRNA. The gRNA can be incubated with the modified adeno-associated virus described in any one of the present invention under appropriate conditions to obtain the complex.

[0078] Another aspect of the present invention relates to a transformed cell, which is obtained by transfecting a cell with the modified adeno-associated virus or the complex of the present invention as described in any one of the present invention;

[0079] Preferably, the cell is an animal cell, a plant cell or a microbial cell;

[0080] Preferably, the cell is a primary cell of a mammal; preferably, the cell is a primary cell of a human; preferably, the cell is one or more selected from human T cells, natural killer cells, dendritic cells, macrophages, hematopoietic stem cells and neural cells;

[0081] Preferably, the cell is a passage cell; preferably, the cell is a mammalian passage cell; preferably, the cell is a tumor cell of a human or non-human mammal; preferably, the cell is one or more selected from Jurkat cells, A549 cells, U87 cells, THP1 cells and Raji cells.

[0082] Another aspect of the present invention relates to a pharmaceutical composition comprising the modified adeno-associated virus, the complex or the transformed cell according to any one of the present invention, and one or more pharmaceutically acceptable excipients.

[0083] Another aspect of the present invention relates to a kit comprising:

[0084] The modified adeno-associated virus and gRNA according to any one of the present invention, or the complex of the present invention, and an optional transfection reagent;

[0085] Preferably, the kit further comprises one or more selected from TAT-HA2, protamine sulfate and a compound of formula I;

[0086] Preferably, the kit further comprises TAT-HA2 and a compound of formula I, or comprises protamine sulfate and a compound of formula I;

[0087]

[0088] Another aspect of the present invention relates to an isolated polypeptide comprising an AAV receptor, Cas12 (e.g., Cas12a) or Cas9, a linker (e.g., SpyTag / SpyCatcher), and a single-chain antibody targeting a T cell receptor;

[0089] Preferably, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:24 to SEQ ID NO:27.

[0090] Yet another aspect of the present invention relates to an isolated nucleic acid molecule encoding the isolated polypeptide of the present invention.

[0091] Another aspect of the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.

[0092] Another aspect of the present invention relates to a recombinant host cell comprising the recombinant vector of the present invention.

[0093] Another aspect of the present invention relates to the use of any of the modified adeno-associated viruses, the complexes, the transformed cells or the isolated polypeptides of the present invention in the preparation of a medicament for treating or preventing tumors, autoimmune diseases, AIDS, aging or asthma;

[0094] Preferably, the tumor is selected from one or more of leukemia, lymphoma, myeloma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, thyroid cancer, mesothelioma, liver cancer, gastric cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial cancer, prostate cancer, testicular cancer, skin cancer, melanoma, plasma cell cancer, vulvar cancer, bone cancer and osteosarcoma;

[0095] Preferably, the leukemia is acute lymphoblastic leukemia;

[0096] Preferably, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma;

[0097] Preferably, the myeloma is multiple myeloma;

[0098] Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer.

[0099] The modified adeno-associated virus, the complex, the transformed cell or the isolated polypeptide according to any one of the present invention, for use in treating or preventing tumors, autoimmune diseases, AIDS, aging or asthma;

[0100] Preferably, the tumor is selected from one or more of leukemia, lymphoma, myeloma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, thyroid cancer, mesothelioma, liver cancer, gastric cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial cancer, prostate cancer, testicular cancer, skin cancer, melanoma, plasma cell cancer, vulvar cancer, bone cancer and osteosarcoma;

[0101] Preferably, the leukemia is acute lymphoblastic leukemia;

[0102] Preferably, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma;

[0103] Preferably, the myeloma is multiple myeloma;

[0104] Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer.

[0105] Another aspect of the present invention relates to a method for treating or preventing tumors, autoimmune diseases, AIDS, aging or asthma, comprising the step of administering to a subject in need thereof an effective amount of the modified adeno-associated virus, the complex, the transformed cell or the isolated polypeptide of any one of the present invention;

[0106] Preferably, the tumor is selected from one or more of leukemia, lymphoma, myeloma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, thyroid cancer, mesothelioma, liver cancer, gastric cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial cancer, prostate cancer, testicular cancer, skin cancer, melanoma, plasma cell cancer, vulvar cancer, bone cancer and osteosarcoma;

[0107] Preferably, the leukemia is acute lymphoblastic leukemia;

[0108] Preferably, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma;

[0109] Preferably, the myeloma is multiple myeloma;

[0110] Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer.

[0111] The total daily dose of the drug (e.g., pharmaceutical composition) or active pharmaceutical ingredient (e.g., the modified adeno-associated virus, the complex, the transformed cell, or the isolated polypeptide of the present invention) of the present invention must be determined by the attending physician within the scope of sound medical judgment. For any specific patient, the specific therapeutically effective dosage level must be determined based on a variety of factors, including the specific tumor type and severity being treated; the form of the specific pharmaceutical preparation used; the patient's age, weight, general health, sex, and diet; administration time, route of administration, and excretion rate; duration of treatment; other drugs used simultaneously; and similar factors well known in the medical field. For example, the practice in the art is to start the administration of a dose lower than the level required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is obtained.

[0112] Without being bound by theory, the AVI polypeptide is a short peptide tag consisting of 15 amino acids (SEQ ID NO: 1). In the presence of ATP and biotin, BirA connects a biotin to the lysine residue of the AVI tag, thereby achieving biotin labeling of the target protein.

[0113] Without being limited by theory, BirA (biotin-protein ligase, BirA) can efficiently activate biotin (D-biotin) to form biotinyl-5' adenylate in the presence of ATP, and specifically connect biotin to the lysine residue of the AVI tag.

[0114] Without being bound by theory, the non-covalent interaction between biotin and avidin or streptavidin is known to be almost the strongest protein-ligand interaction. Avidin or streptavidin is a 4-mer protein that can bind to 4 biotin molecules at the same time with high specificity and is a naturally occurring biomolecular interaction system. The binding system of biotin and avidin or streptavidin is called the biotin-avidin system or the biotin-streptavidin system. This system is a signal amplification labeling technology with the advantages of high affinity, high sensitivity, strong specificity and good stability, and has been widely used in a variety of detection systems.

[0115] In the present invention, unless otherwise specified, AsCas12a and Cas12a have the same meaning.

[0116] In the present invention, unless otherwise specified, AsCas12 and Cas12 have the same meaning.

[0117] As used herein, the term "chimeric antigen receptor (CAR)" refers to a recombinant polypeptide construct comprising at least one extracellular antigen binding domain, an optional spacer domain, a transmembrane domain, and an intracellular signaling domain, which combines the antibody-based specificity for the target antigen with the immune effector cell activation intracellular domain to show specific immune activity for cells expressing the target antigen. In the present invention, the expression "immune effector cell expressing CAR" refers to an immune effector cell expressing CAR and having an antigen-specificity determined by the targeting domain of the CAR. Methods for making CARs (e.g., for cancer treatment) are known in the art, see, for example, Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT patent publications WO2012 / 079000, WO2013 / 059593; and U.S. Patent Publication 2012 / 0213783, all of which are incorporated herein by reference in their entirety.

[0118] In the present invention, the term "extracellular antigen binding domain" refers to a polypeptide that can specifically bind to an antigen or receptor of interest. This domain will be able to interact with cell surface molecules. For example, an extracellular antigen binding domain can be selected to recognize an antigen that is a cell surface marker of a target cell associated with a specific disease state. Typically, the extracellular antigen binding domain is an antibody-derived targeting domain.

[0119] In the present invention, the term "intracellular signaling domain" refers to a protein portion that conducts effector signaling function signals and guides cells to perform specialized functions. Therefore, the intracellular signaling domain has the ability to activate at least one normal effector function of an immune effector cell expressing CAR. For example, the effector function of a T cell can be cytolytic activity or auxiliary activity, including the secretion of cytokines.

[0120] In the present invention, the term "primary signaling domain" refers to a protein portion that can regulate the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary signaling domain that acts in a stimulatory manner generally contains a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). Non-limiting examples of the primary signaling domain include TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, DAP10, CD79a, CD79b, CD66d.

[0121] In the present invention, the term "costimulatory signaling domain" refers to the intracellular signaling domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules that provide a second signal required for efficient activation and function of T lymphocytes after binding to an antigen, except for antigen receptors or Fc receptors. Non-limiting examples of costimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1) CD270 (HVEM), CD278 (ICOS), DAP10.

[0122] In the present invention, the term "single chain antibody (ScFv)" refers to an antibody heavy chain variable region (V H ) and antibody light chain variable region (V L ) numerator. L and V H The domains are paired to form monovalent molecules via linkers that enable them to be generated as a single polypeptide chain (see, e.g., Bird et al, Science 1988; 242: 423-426 and Huston et al, Proc. Natl. Acad. Sci. USA 1988; 85: 5879-5883). Such scFv molecules may have the general structure: NH2-V L -Linker-V H -COOH or NH2-V H -Linker-V L -COOH.

[0123] In the present invention, the term "nanobody" has the meaning generally understood by those skilled in the art, and refers to an antibody fragment composed of a single monomeric variable antibody domain (e.g., a single heavy chain variable region), usually derived from the variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, a nanobody consists of four framework regions and three complementarity determining regions, with a structure of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. A nanobody may be truncated at the N-terminus or C-terminus so that it contains only part of FR1 and / or FR4, or lacks one or two of those framework regions, as long as it substantially maintains antigen binding and specificity. Nanobodies are also called single-domain antibodies (sdAbs), and the two can be used interchangeably.

[0124] As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with a subject and an active ingredient, which is well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH regulators, surfactants, adjuvants, and ionic strength enhancers. For example, pH regulators include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic or nonionic surfactants, such as Tween-80; ionic strength enhancers include, but are not limited to, sodium chloride.

[0125] As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient who already has the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an effective amount for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

[0126] As used herein, the term "isolated" or "isolated" refers to that obtained from the natural state by artificial means. If a certain "isolated" substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been separated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity separated from this natural state is called isolated. The term "isolated" or "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impure substances that do not affect the activity of the substance.

[0127] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC); bacteriophages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include but are not limited to retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0128] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, GS cells, BHK cells, HEK 293 cells or human cells.

[0129] In the present invention, unless otherwise specified, the "first" (e.g., the first linker, etc.) and "second" (e.g., the second linker, etc.) are for the purpose of distinguishing references or clarifying expressions, and do not have a typical order meaning.

[0130] Advantageous Effects of the Invention

[0131] The present invention achieves one or more of the following technical effects:

[0132] 1) Ability to achieve specific editing of T cells.

[0133] 2) It can knock in gene fragments as long as 3.5-4K into T cells in one step and simultaneously knock out multiple genes in T cells (such as TCR alpha, beta2m, regnase, roquin, etc.).

[0134] 3) CAR can be knocked into CD52 (gRNA can guide Cas protein to cut DNA at CD52, and use the homology arm at CD52 to accurately knock it into CD52. The current attempt is to knock it into TRAC. After knocking it into CD52, T cells will no longer express CD52). Anti-CD52 antibodies can be used for lymphocyte clearance, which can help CAR T to better expand in vivo.

[0135] 4) It can be used in vivo, that is, directly editing in vivo to generate CAR T / STAR T / TCR T cells (knocking into a certain gene site will destroy the gene at the same time. The advantage of the TRAC site is that it can utilize the endogenous TCR promoter to have a better expression regulation effect, so that the expressed CAR T is better).

[0136] 5) Ability to achieve permanent expression of CAR.

[0137] 6) Can be applied for gene therapy purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0138] Figure 1 : Biotin site-specific labeling of AAV packaging.

[0139] Figure 2 : Design of engineered AAV.

[0140] Figure 3 : Detection of biotinylated AAV virus.

[0141] Figure 4A : The streptavidin linker molecule binds to biotin.

[0142] Figure 4B :The antiTCR functional region of the linker molecule binds to TCR.

[0143] Figure 5 : Engineered AAV preferentially infects T cells expressing TCR.

[0144] Figure 6 :Engineering AAV can knock CAR into T cells at a specific site.

[0145] FIG. 7A to FIG. 7B :Design of adeno-associated virus (CAPA) engineered with targeted gene editing enzymes. Among them:

[0146] Fig. 7A, the design diagram of adeno-associated virus engineered with targeted gene editing enzyme, the upper part is the overall design, and the lower part is the design details of the modified structure. Among them: AAVR is AAV receptor, AsCas12a is gene editing enzyme, ST stands for SpyTag, SC stands for SpyCatcher, ST and SC can form a covalent link, αTCR stands for single-chain antibody that recognizes TCR (antiTCR ScFv-SC).

[0147] Figure 7B , Fig. 7A Protein expression and purification results of the structures shown in . Lane L1 is antiTCR-ScFv-SC, lane L2 is AAVR-Cas12a-ST, and lane L3 shows the complete adaptor protein AAVR-AsCas12a-ST-SC-antiTCR.

[0148] FIG. 8A to FIG. 8C :Biochemical detection of adeno-associated virus (CAPA) engineered with targeted gene editing enzymes. Among them:

[0149] Fig. 8A :Affinity determination of targeted gene editing enzyme and AAV. The curves in the figure are proteins of different concentrations;

[0150] Figure 8B :Capa binding assay with T cells;

[0151] Figure 8C : In vitro editing activity test of Capa.

[0152] FIG. 9A to FIG. 9B : Adeno-associated virus (Capa) engineered with targeted gene editing enzymes can be directly used for CAR T production. Among them:

[0153] Fig. 9A , Capa directly edits primary T cells to generate CAR T targeting CD19 (Capa-CD19-28CAR);

[0154] Fig. 9B , the results of the CAR T cell killing experiment produced by Capa on tumors, where the horizontal axis is the effector-target ratio, that is, the ratio of T cells to tumor cells (for example, 1:1 represents a T cell and a tumor), and the vertical axis represents the tumor lysis efficiency. The higher the vertical axis lysis efficiency, the stronger the killing of the tumor.

[0155] Fig.10:Optimization of adeno-associated virus (Capa) engineered with targeted gene editing enzymes. The synergistic effect of peptide TAT-HA2 and small molecule drug ADZ7648 with Capa can greatly improve the efficiency of gene editing. Among them: a is Capa, b is Capa+TAT-HA2, c is Capa+TAT-HA2+ADZ7648, and d is Capa+Protamine+ADZ7648.

[0156] Fig.11 : The horizontal axis represents the situation of TCR-CD3 complex on the surface of T cells, and the vertical axis represents the expression of CD19CAR. Q1 means that TCR is completely knocked out, and CD19CAR is precisely knocked into TRAC and expressed; Q2 means that T cells express CD19CAR and TCR at the same time, that is, only one TRAC is knocked into CD19CAR, and the other TRAC is intact; Q3 means T cells that have not been edited; Q4 means TCR is knocked out but CD19CAR is not knocked in.

[0157] FIG. 12A to FIG. 12B : Adeno-associated virus (Capa) engineered with targeted gene editing enzymes can be used for multiple gene editing. Fig. 12A This is a schematic diagram showing that Capa can carry multiple RNPs. Fig. 12B Showing the gene editing effects of various RNP-Capas.

[0158] Fig.13 : Comparison of the killing activity of CAR T prepared by adeno-associated virus (Capa) engineered with targeted gene editing enzymes. NC is the control group, Lentil is CAR T prepared by lentivirus, Electro is CAR T prepared by electroporation, and Capa is CAR T prepared by the present invention.

[0159] Some of the sequence information involved in this application is as follows:

[0160] AVI peptide

[0161] GLNDIFEAQKIEWHE (SEQ ID NO: 1)

[0162] OsASI1-BAH

[0163] GIKMDTQFYDSFTFDNVKYSLYDNVYLFKSGESEPYIGKIIKIWQQNQAKKVKILWFFLPDEIRKHLSGPVMEKEIFLACGEGVGLADINPLEAIGGKCTVLCISKDERNRQPSPRELAMADYIFYRFFDVNSCTLSEQLPEKIAGVEGNLLLNSKVEQVTSGGGGS(SEQ ID NO:2)

[0164] OsSUVH6

[0165] AAPPKRRAISAIRKFPRDCGAAGGSSGGGSASSG(SEQ ID NO:3)

[0166] antiTCR

[0167] QVQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWIGYINPYNDVTKYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVHYCARGSYYDYDGFVYWGQGTLVTVSSGGSGGSGGSGGSGGEIVLTQSPATLSLSPGERATLSCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPLTFGQGTKLEIKS(SEQ ID NO:4)

[0168] AsCas12a

[0169]

[0170] FMC-28CAR

[0171] DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSEFSHFVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:6)

[0172] AsCas12a

[0173]

[0174] antiTCR

[0175] CAGGTGCAGCTGGTGCAGTCCGGCGCCGAGGTGAAGAAGCCTGGCGCCAGCGTGAAGGTGTCCTGTAAGGCCAGCGGCTACAAGTTTACATCCTACGTGATGCACTGGGTGAGACAGGCCCCCGGCCAGGGACTGGAGTGGATCGGATACATCAACCCCTACAACGATGTGACAAAGTACAACGAGAAGTTCAAGGGCAGGGTGACCATCACCGCCGACAAGTCCACAAGCACAGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACAGCCGTGCACTACTGCGCCAGAGGCAGCTACTACGATTACGACGGCTTCGTGTACTGGGGCCAGGGCACACTGGTGACCGTGAGCTCCGGCGGCAGCGGAGGATCCGGAGGAAGCGGAGGCAGCGGCGGAGAGATCGTGCTGACCCAGAGCCCCGCCACCCTGTCCCTGAGCCCTGGAGAGAGGGCCACACTGAGCTGCTCCGCCACATCCTCCGTGAGCTACATGCACTGGTACCAGCAGAAGCCCGGCAAGGCCCCTAAGAGATGGATCTACGACACCAGCAAGCTGGCCTCCGGCGTGCCTTCCAGGTTCAGCGGCTCCGGCTCCGGCACCGAGTTCACACTGACAATCAGCTCCCTGCAGCCCGAGGACTTCGCCACATACTACTGTCAGCAGTGGAGCTCCAACCCCCTGACATTCGGCCAGGGCACCAAGCTGGAGATCAAGtct(SEQ ID NO:8)

[0176] OsSUVH6

[0177] GCtGCaCCtCCaAAGAGaAGaGCtATtAGCGCCATCAGGAAG TTCCCtAGGGACTGCGGtGCtGCtGGtGGtAGCAGCggtggaggcagc gcatcttcaggc(SEQ ID NO:9)

[0178] OsASI1-BAH

[0179] ATGGCCGCACCGAAAAAGAAACGCAAAGTGGGTGGCATTAAGATGGATACCCAGTTTTATGATAGCTTTACCTTTGATAACGTGAAATATAGTCTGTATGACAATGTTTACCTGTTTAAAAGTGGCGAAAGTGAACCGTATATTGGTAAAATTATCAAGATCTGGCAGCAGAATCAGGCAAAAAAGGTTAAAATTCTGTGGTTTTTCCTGCCGGATGAAATTCGTAAACATCTGAGCGGCCCGGTTATGGAAAAAGAAATTTTTCTGGCATGCGGCGAAGGCGTTGGTCTGGCCGATATTAATCCGCTGGAAGCCATTGGTGGTAAATGTACCGTTCTGTGCATTAGCAAAGATGAACGCAATCGTCAGCCGAGTCCGCGCGAACTGGCAATGGCAGATTATATTTTCTATCGTTTCTTTGACGTGAACAGTTGTACCCTGAGCGAACAGCTGCCGGAAAAAATTGCCGGTGTGGAAGGTAATCTGCTGCTGAATAGCAAAGTTGAACAGGTTACCAGCGGCGGCGGCGGTAGC(SEQ ID NO:10)

[0180] Substrate double-stranded DNA / target sequence to be knocked out

[0181]

[0182] AVI

[0183] GGCCTGAACGACATCTTCGAGGCCCAGAAGATCGAGTG GCACGAG(SEQ ID NO:12)

[0184] ITR

[0185] Cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct(SEQID NO:13)

[0186] TRAC 5'Homologous Arm

[0187] TTAATGCCAACATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACCC(SEQ ID NO:14)

[0188] FMC-28CAR

[0189]

[0190] TRAC 3' homologous arm

[0191] GAGAGACTCTAAATCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTTTGG(SEQ ID NO:16)

[0192] AAVR (AAV Receptor)

[0193] VSAGESVQITLPKNEVQLNAYVLQEPPKGETYTYDWQLITHPRDYSGEMEGKHSQILKLSKLTPGLYEFKVIVEGQNAHGEGYVNVTVKPEPRKNRPPIAIVSPQFQEISLPTTSTVIDGSQSTDDDKIVQYHWEELKGPLREEKISEDTAILKLSKLVPGNYTFSLTVVDSDGATNSTTANLTVNKAVGSGSGSGS(SEQ ID NO:17)

[0194] SpyTag

[0195] AHIVMVDAYKPTK(SEQ ID NO:18)

[0196] SpyCatcher

[0197] GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI(SEQ ID NO:19)

[0198] Amino acid sequence of AAVR-AsCas12a-ST:

[0199]

[0200] AAVR-AsCas12a-ST nucleic acid sequence:

[0201]

[0202] Amino acid sequence of antiTCR ScFv-SC:

[0203] QVQLVQSGAEVKKPGASVKVSCKASGYKFTSYVMHWVRQAPGQGLEWIGYINPYNDVTKYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVHYCARGSYYDYDGFVYWGQGTLVTVSSGGSGGSGGSGGSGGEIVLTQSPATLSLSPGERATLSCSATSSVSYMHWYQQKPGKAPKRWIYDTSKLASGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCQQWSSNPLTFGQGTKLEIKSSGSGGSGGGAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGSSVGFAGGSSHHHHHH(SEQ ID NO:22)

[0204] Nucleic acid sequence of antiTCR ScFv-SC:

[0205]

[0206] One of the amino acid sequences of AAVR-AsCas12a-ST-SC-antiTCR:

[0207]

[0208] Amino acid sequence 2 of AAVR-AsCas12a-ST-SC-antiTCR:

[0209]

[0210] Amino acid sequence 3 of AAVR-AsCas12a-ST-SC-antiTCR:

[0211]

[0212] Amino acid sequence 4 of AAVR-AsCas12a-ST-SC-antiTCR:

[0213] DETAILED DESCRIPTION

[0214] The embodiments of the present invention will be described in detail below in conjunction with the examples, but those skilled in the art will appreciate that the following examples are only used to illustrate the present invention and should not be considered to limit the scope of the present invention. If no specific conditions are specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0215] AAV6 virus was purchased from Paigen Biotechnology.

[0216] Plasmids were purchased from Addgene.

[0217] Cell lines such as 293F, Jurkat, T cells were purchased from ATCC.

[0218] gRNA was purchased from IDT.

[0219] FMC-28CAR includes:

[0220] FMC (single-chain antibody that recognizes CD19 antigen), CD8 hinge (hinge region, connecting single-chain antibody and transmembrane region), CD28 transmembrane region and intracellular region (transmitting CD28 signal) and CD3 intracellular region (transmitting CD3 signal).

[0221] Preparation Example 1: Preparation of engineered AAV that specifically targets and edits T cells (1)

[0222] 1.1 Design, modification and preparation of AAV containing CAR (FMC-28CAR)

[0223] The two ITRs (SEQ ID NO: 13) of the AAV6 genome are replaced with the DNA sequence of CAR (SEQ ID NO: 15, the target gene inserted into the pTransfer plasmid), and homology arms (SEQ ID NO: 14, SEQ ID NO: 16) are added to both ends of the CAR sequence, which targets the genomic site of TRAC and can insert CAR into the site. At the same time, the present invention inserts the VRVIII region of the AAV capsid into the AVI tag, which can be displayed on the surface of the AAV capsid without substantially affecting the AAV packaging.

[0224] The AVI tag is a substrate for the BirA enzyme, and the lysine (K) side chain in the sequence can be specifically modified with biotin by the BirA enzyme. Therefore, by co-expressing the BirA enzyme and adding D-biotin (1 μg / ml) to the culture medium, the AAV capsid can be site-specifically and specifically modified with biotin (see Figure 1 ).

[0225] AAV containing CAR (FMC-28CAR) was obtained.

[0226] 1.2 Design and preparation of linker molecules

[0227] Design an adaptor molecule with editing function for use with AAV. The adaptor molecule consists of three parts, in order from the N-terminus to the C-terminus:

[0228] 1) Streptavidin (SA) that binds to the biotin of the AAV capsid,

[0229] 2) CRISPR system (CRISPR / Cas12a+gRNA, i.e., RNP) that can cut TRAC at specific genomic sites, and

[0230] 3) ScFv that can specifically bind to T cell receptors.

[0231] Among them, after part 3) and part 1) are connected, gRNA is added to form RNP. Part 2) can also be connected to part 3) and part 1) after forming RNP. AsCas12a and gRNA in the linker molecule are mixed at a molar ratio of 1:2 and incubated at room temperature for 20 minutes.

[0232] The AsCas12a used in part 2) is obtained by purifying the coding sequence (SEQ ID NO: 7) through prokaryotic expression. The steps are as follows:

[0233] Inoculation: Pick a single clone or 1-10 ml of bacterial solution into 1L LB medium and culture at 37℃ in a shaking incubator until OD = 0.5-0.6;

[0234] Expression: Add 200 μL 1M IPTG and culture at 16°C in a shaking incubator for 12-16 h;

[0235] Centrifugation: centrifuge at 12000 rpm for 5 min, resuspend 1 g of precipitate in 7 ml 2XPBS and collect in a 100 ml beaker and place on ice;

[0236] Lysis: ultrasonic lysis 5 times (must be placed on ice, preferably ice-water mixture, each ultrasonic interval 2 minutes), centrifuge at 20000rpm for 1h after balancing;

[0237] Column: Wash the nickel column with 1×PBS, draw the supernatant with a syringe, and inject it into the nickel column. Be careful not to use too fast a flow rate (a peristaltic pump can be used);

[0238] Wash: Wash the nickel column with 20 ml 1× PBS;

[0239] Elution: elution with 5 mL 500 mM imidazole (1×PBS, pH=7.7);

[0240] Column: The sample was added to AKTA PurM with a syringe, passed through HiLoad 1; 6 / 600 Superdex 200 pg, and collected in a 96-well plate with a collector to obtain an AsCas12a protein sample;

[0241] Gel running: Pipette samples from the peak area of ​​the 96-well plate and run the gel to verify the protein size;

[0242] Cleaning: Soak the tubing and column in 0.2M NaOH for a day and night, and clean the tubing with 1× PBS the next day.

[0243] The ScFv used in part 3) is obtained by purifying the coding sequence (SEQ ID NO: 8) through eukaryotic expression. The steps are as follows:

[0244] Recovery: Recover 293F from the cell bank and culture in SMM 293-TII (1% PS) in a cell culture conical flask to 2×10 6 ~3×10 6 Pieces / ml;

[0245] Transfection: Take the same volume of culture medium, add PEI and plasmid respectively, let it stand at room temperature for 30 minutes, then add the mixture of PEI and PBS to the mixture of plasmid and PBS, add it to the cell suspension, and culture for 48 hours;

[0246] Centrifugation: centrifuge at 14000 rpm for 2 h, take the supernatant and filter it with a 0.22 μm filter;

[0247] Column: Use the injector to absorb the supernatant and put it into the nickel column to prevent bubbles from entering the pipeline;

[0248] Wash: Wash the nickel column with 10 ml 1× PBS;

[0249] Elution: elution with 500 mM imidazole (1×PBS, pH=7.7);

[0250] Concentration: Use a 10000 MCO concentrator tube, add 1× PBS and centrifuge at 3500 rpm to concentrate to 1 mL, repeat twice to obtain the ScFv protein sample;

[0251] Gel running: Gel running to verify protein size;

[0252] Cleaning: Soak the tubing and column in 0.2M NaOH for a day and night, and clean the tubing with 1× PBS the next day.

[0253] AsCas12a and ScFv were linked at a molar ratio of 1:1 by incubation on ice for 1 hour.

[0254] 1.3 Preparation of engineered AAVs that specifically target and edit T cells

[0255] The biotin-labeled AAV prepared in 1.1 and the linker molecule prepared in 1.2 were incubated in PBS buffer at room temperature at a molar ratio of 1:60 for 20 minutes, and purified by density gradient centrifugation to obtain an engineered AAV that specifically targets and edits T cells (see Figure 2 ), named AAV6-Cas12aRNP-antiTCR. It was used in the following experiments.

[0256] Example 1: The linker molecule can bind to biotin, bind to TCR, and specifically cleave the DNA substrate in vitro

[0257] (1) The packaged AAV (prepared in 1.1 of Preparation Example 1 above) was subjected to western blot (WB) detection. The results showed that the AAV capsid was indeed modified with biotin (see Figure 3 ).

[0258] (2) The linker molecule (prepared in 1.2 of the previous preparation example 1) was subjected to an in vitro functional test. The linker molecule carries a 6His tag and can be analyzed by flow cytometry using an anti-His flow cytometry antibody.

[0259] Biotin-labeled 293F cells were used to simulate the biotinylation labeling of AAV, and the binding of streptavidin and biotin was tested by flow cytometry. The flow cytometric results showed that the linker molecule was able to bind to the biotin-labeled 293F cells.

[0260] Among them, biotin-labeled 293F cells are used to simulate biotin-labeled AAV to detect whether streptavidin works. The preparation steps are as follows: mix an appropriate amount of biotin with 1E6 293F cells, incubate at room temperature for 30 minutes, rinse with 1XPBS+2% FBS, centrifuge at 400g for 5 minutes to remove excess biotin, and the labeling is completed.

[0261] (3) 1 μg of adaptor protein was added to every 1E6 human peripheral blood mononuclear cells (PBMCs), the adaptor protein was incubated with T cells on ice for 30 minutes, and the binding with T cells was detected by flow cytometry (see Figure 4A and Figure 4B ).

[0262] (4) The linker molecule prepared in 1.2 of Preparation Example 1 was incubated with the aforementioned gRNA at a molar ratio of 1:2 at room temperature for 30 minutes, and 500 ng of substrate double-stranded DNA complementary to the gRNA was added to test the enzyme activity of AsCas12a in the linker molecule (2 μg according to RNP). After the DNA substrate and the AAV assembled with the linker molecule were incubated at room temperature for 30 minutes, a DNA gel was run to observe whether the DNA was cut. The in vitro enzyme activity experiment showed that the linker molecule had complete activity in cutting the substrate DNA (see Figure 6 ).

[0263] Example 2: Testing the engineered AAV prepared in Example 1 that specifically targets and edits T cells at the T cell level

[0264] 2.1 Engineered AAV has the characteristic of preferentially infecting T cells

[0265] The engineered AAV containing the linker molecule, i.e., AAV6-Cas12aRNP-antiTCR, prepared in 1.3 of the previous preparation example 1 was used to infect Jurkat cells that naturally express TCR and Jurkat cells with TCR knocked out (named JC5 cells, the TCR in Jurkat cells was knocked out using CRISPR / Cas9, i.e., JC5 cells that do not express TCR) at an MOI of 1E4, respectively, to explore whether the engineered AAV has an infection preference.

[0266] The experimental results are as follows Figure 5 shown.

[0267] The results showed that the engineered AAV of the present invention mainly infected Jurkat cells, indicating that these AAVs bind to the surface of T cells with the help of TCR molecules, enter T cells through the endocytosis of TCR, and complete the infection process. Engineered AAV prefers to infect T cells expressing TCR.

[0268] 2.2 Engineered AAV can insert CAR into TRAC

[0269] The AAV6-Cas12aRNP-antiTCR prepared in the previous preparation example 1 was added to Jurkat cells (cell 1E5, AAV1E10). After culturing for 3 days (RPMI 1640 + 10% FBS, 37°C), 1 μl of cells was taken as a PCR template. A downstream primer was designed in the insert fragment and a downstream primer was designed on the genome of T cells outside the left homologous arm to identify the insert fragment. The target fragment size was 1.3K.

[0270] The results are as follows Figure 6 The results showed that FMC-28CAR was successfully inserted into the genome of T cells.

[0271] Preparation Example 2: Preparation of engineered AAV that specifically targets and edits T cells (2)

[0272] The linker molecule was designed as AAVR-AsCas12a-SA-AVI-antiTCR ScFv.

[0273] Among them, AAVR (AAV receptor, SEQ ID NO: 17) directly binds to AAV; SA (streptavidin) binds to biotinylated AVI-antiTCR ScFv.

[0274] AAVR and AAV were mixed at a molar ratio of 1:60 and incubated at room temperature for 30 minutes for binding.

[0275] The C-terminus of AsCas12a was fused with Streptavidin and the two were prepared together by prokaryotic expression.

[0276] When expressing the AVI-antiTCR ScFv protein, a plasmid expressing biotin ligase (BirA) and a final concentration of 1 μg / ml of D-biotin are added. The expressed AVI-antiTCR ScFv protein will be automatically labeled with biotin.

[0277] The other steps refer to the previous Preparation Example 1.

[0278] Preparation Example 3: Preparation of engineered AAV that specifically targets and edits T cells (3) (Capa)

[0279] The design of engineered AAV (Capa) that specifically targets and edits T cells Fig. 7A As shown. Cas12a / 9RNP binds to the capsid of AAV6 through the PKD1 and PKD2 domains of the fusion-expressed AAV receptor AAVR (AAVR has a total of 5 domains PKD1-5, only PKD1 and PKD2 bind to AAV, and can bind to various serotypes of AAV). At the same time, the ST short peptide designed at the C-terminus can covalently bind to the antiTCR-ScFv fusion-expressed SC domain, thereby forming an engineered AAV that specifically targets and edits T cells.

[0280] 1. Preparation of AAVR-AsCas12-ST

[0281] The amino acid sequence of AAVR-AsCas12-ST is shown in SEQ ID NO: 20, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO: 21. Wherein, AAVR represents AAV receptor, and ST represents SpyTag.

[0282] The preparation steps of AAVR-AsCas12-ST are as follows:

[0283] Inoculation: Pick a single clone of the recombinant host bacteria or 1-10 ml of bacterial solution in 1L LB medium and culture at 37°C in a shaking incubator until OD = 0.5-0.6;

[0284] The host bacteria were purchased from Shanghai Shenggong Biotechnology Co., Ltd. BL21 (DE3) strain, item number B528419-0010. SEQ ID NO: 21 was synthesized by General Biotechnology Co., Ltd. and directly constructed into the pet22b vector, and the plasmid was transformed into BL21 (DE3) for protein expression.

[0285] Expression: Add 200 μL 1M IPTG and culture at 16°C in a shaking incubator for 12-16 h;

[0286] Centrifugation: centrifuge at 12000 rpm for 5 min, resuspend 1 g of precipitate in 7 ml 2XPBS and collect in a 100 ml beaker and place on ice;

[0287] Lysis: ultrasonic lysis 5 times (must be placed on ice, preferably ice-water mixture, each ultrasonic interval 2 minutes), centrifuge at 20000rpm for 1h after balancing;

[0288] Column: Wash the nickel column with 1×PBS, draw the supernatant with a syringe, and inject it into the nickel column. Be careful not to use too fast a flow rate (a peristaltic pump can be used);

[0289] Wash: Wash the nickel column with 20 ml 1× PBS;

[0290] Elution: 5 mL 500 mM imidazole (1×PBS, pH=7.7)

[0291] Column: The sample was added to AKTA PurM with a syringe, passed through HiLoad 1; 6 / 600 Superdex 200 pg, and collected in a 96-well plate with a collector to obtain an AsCas12a protein sample;

[0292] Gel running: Pipette samples from the peak area of ​​the 96-well plate and run the gel to verify the protein size.

[0293] The results are as follows Figure 7B The results showed that the size of the protein (lane L2) was consistent with the expected value.

[0294] 2. Preparation of antiTCR ScFv-SC

[0295] The amino acid sequence of antiTCR ScFv-SC is shown in SEQ ID NO:22, and the encoding nucleic acid sequence thereof is shown in SEQ ID NO:23.

[0296] The preparation steps of antiTCR ScFv-SC are as follows:

[0297] Recovery: Recover 293F from the cell bank and culture in SMM 293-TII (1% PS) in a cell culture conical flask to 2×10 6 ~3×106 Pieces / ml;

[0298] Transfection: Take the same volume of culture medium, add PEI and pCDNA3.1 vector (Universal Biosynthesis) respectively, let it stand at room temperature for 30 minutes, then add the mixture of PEI and PBS to the mixture of plasmid and PBS, add it to the cell suspension, and culture for 48 hours;

[0299] Centrifugation: centrifuge at 14000 rpm for 2 h, take the supernatant and filter it with a 0.22 μm filter;

[0300] Column: Use the injector to absorb the supernatant and put it into the nickel column to prevent bubbles from entering the pipeline;

[0301] Wash: Wash the nickel column with 10 ml 1× PBS;

[0302] Elution: elution with 500 mM imidazole (1×PBS, pH=7.7);

[0303] Concentration: Use a 10000 MCO concentrator tube, add 1× PBS and centrifuge at 3500 rpm to concentrate to 1 mL, repeat twice to obtain the ScFv protein sample;

[0304] Gel running: Gel running is used to verify protein size.

[0305] The results are as follows Figure 7B The results showed that the size of the protein (lane L1) was consistent with the expected size.

[0306] 3. Preparation of AAVR-AsCas12a-ST-SC-antiTCR

[0307] AAVR-AsCas12a-ST and antiTCR ScFv-SC were incubated on ice overnight at a molar ratio of 1:1.2. The resulting ligation product was named AAVR-AsCas12a-ST-SC-antiTCR, where antiTCR represents antiTCR ScFv.

[0308] The ligation product AAVR-AsCas12a-ST-SC-antiTCR was run on a gel for verification. Figure 7B The results showed that the size of the protein (lane L3) was consistent with the expected size.

[0309] 4. Preparation of Capa

[0310] AAVR-AsCas12a-ST-SC-antiTCR was incubated with CrRNA at a molar ratio of 1:1.2 at room temperature for 15 minutes, and then mixed with AAV6 at a molar ratio of 100:1 and incubated at room temperature for 15 minutes. The resulting assembly was named Capa.

[0311] The Capa samples used in the following experiments, unless otherwise specified, are the Capa prepared in this step.

[0312] Preparation Example 4: Preparation of engineered AAV that specifically targets and edits T cells (Capa-Cas9)

[0313] The preparation method is the same as that of Preparation Example 3, except that Cas12 is replaced by Cas9.

[0314] The resulting assembly was named Capa-Cas9.

[0315] Example 3: Biochemical detection of adeno-associated virus (Capa) engineered with targeted gene editing enzymes

[0316] 1.SPR experiment:

[0317] AAV6 was immobilized on a chip (model CM5, Cytiva) at a concentration of 1E12 vg / ml by incubation at room temperature for 30 minutes.

[0318] The protein AAVR-AsCas12a-ST-SC-antiTCR prepared in Preparation Example 3 was incubated with a chip immobilized with AAV6 at different concentrations (78nM, 156nM, 312.5nM, 625nM, 1250nM, 2500nM and 5000nM), and the dynamic binding and dissociation were detected. The affinity values ​​of AAV6 and AAVR-AsCas12a-ST-SC-antiTCR were obtained after fitting. The results are shown in Fig. 8A As shown in the figure, the final affinity (dissociation constant) was 22.6 μM.

[0319] 2. Flow Cytometry Experiment

[0320] The two proteins AAVR-AsCas12a-ST-SC-antiTCR and AAVR-AsCas12a-ST prepared in Preparation Example 3 were incubated with T cells (human primary T cells) at 4 ° C for 30 minutes, and then centrifuged to remove excess protein (room temperature 3500rpm / 5min), incubated at 4 ° C for 30 minutes with a flow antibody containing 6his (biolegend: antihis-AF647), centrifuged to remove excess antibodies, and resuspended in 200μl 1X PBS for detection. The red peak is the binding of AAVR-AsCas12a-ST-SC-antiTCR to T cells, and the blue peak is the binding of AAVR-AsCas12a-ST to T cells.

[0321] The results are as follows Figure 8B The results showed that the red peak was significantly shifted to the right compared to the blue peak, indicating that the antiTCR in AAVR-AsCas12a-ST-SC-antiTCR can bind to the TCR molecule of T cells.

[0322] 3. In vitro enzyme activity test of Capa

[0323] 1E10 Capa (- / +CrRNA) (one with CrRNA as the experimental group, one without CrRNA as the negative control group) were incubated with 500 ng of DNA substrate at 37°C for 1 hour; then 1 μl of proteinase K was added and incubated at 37°C for 1 hour. The samples were run on a 1.5% DNA gel.

[0324] The results are as follows Figure 8C The results showed that Capa could efficiently cut the substrate DNA after adding guide RNA compared with without adding guide RNA.

[0325] Example 4: Adeno-associated virus (Capa) engineered with targeted gene editing enzymes can be directly used for the production of CART

[0326] 1. Capa directly edits primary T cells to generate CD19-targeted CART (Capa-CD19-28CAR)

[0327] After peripheral blood T cells from healthy donors were activated with CD3 / 28 antibodies for 48 hours, Capa was incubated with T cells at a MOI of 1E5 (100,000 Capa: 1 T cell) at room temperature for 30 minutes, and then the culture medium for T cells (Lonza brand X-vivo + 5% FBS + 200 IU of IL2) was added. After culturing for 7 days, the editing efficiency was detected by flow cytometry.

[0328] The results are as follows Fig. 9A shown.

[0329] Fig. 9A The horizontal axis represents the TCR-CD3 complex on the surface of T cells, and the vertical axis represents the expression of CD19 CAR. Q1 means that TCR is completely knocked out, and CD19CAR is precisely knocked into TRAC and expressed; Q2 means that T cells express CD19CAR and TCR at the same time, that is, only one TRAC is knocked into CD19CAR, and the other TRAC is intact; Q3, T cells that have not been edited; Q4, T cells that have been knocked out of TCR but not CD19CAR knocked in.

[0330] 2. Capa-CD19-28CAR has better killing activity than Lenti-CD19-28CAR

[0331] The construction of Capa-CD19-28CAR was the same as the previous steps.

[0332] Lenti-CD19-28CAR construction: After peripheral blood T cells from healthy donors were activated with CD3 / 28 antibodies for 48 hours, lentivirus encoding CD19-28CAR was added at MOI=5, and the CART preparation efficiency was detected after 3 days of continuous culture.

[0333] Tumor cells expressing luciferase can emit light when luciferase substrate is added. The intensity of the light emission can reflect the residual amount of the tumor, that is, the killing efficiency.

[0334] The constructed Capa-CD19-28CAR and Lenti-CD19-28CAR were co-incubated with CD19-expressing Raji cells at an effector-target ratio of 1:1 / 0.3:1 / 0.1:1 for 48 hours, and the killing of Raji cells was detected.

[0335] Cell killing calculation:

[0336] The specific operations are as follows:

[0337] 1) Substrate preparation: Melt the Buffer solution in the kit at 4°C or room temperature, add the melted buffer to the substrate, and gently invert for 3-5 times to fully dissolve the substrate.

[0338] 2) After the co-culture is completed, remove the well plate, gently blow the cell suspension evenly with a pipette, take 150 μL of cell suspension from each well and add it to the previously prepared white 96-well plate, take 2-3 replicates from each well, and then add luciferase substrate, 70 μL / well.

[0339] 3) Place the white 96-well plate on an oscillator at 300 rpm / min in the dark for 10 min, and then use a multifunctional microplate reader to detect the chemiluminescence value with a fixed gain value of 100.

[0340] 4) If the cytokine content in the co-culture supernatant is to be detected, the remaining cell suspension is centrifuged, the supernatant is taken, and frozen and stored at -80°C.

[0341] If the target cells are adherent cells:

[0342] Note: Lysis buffer and luciferase substrate need to be taken out from -40℃ in advance and thawed at room temperature.

[0343] 5) After the co-culture is completed, remove the well plate, centrifuge at 4000 rpm for 5 min, carefully aspirate all the co-culture cell suspension, and freeze and store at -80°C.

[0344] 6) Add 400 μL / well lysis solution to the well plate, pipette several times, and then place the well plate on an oscillator at 300 rpm / min and shake at room temperature for 10 min.

[0345] 7) Centrifuge the plate at 4000 rpm, 4°C for 5 min.

[0346] 8) Pipette 20 μL of supernatant and add it to the prepared white 96-well plate. Take 2-3 replicates for each well, add luciferase substrate, 50 μL / well, mix well, and use a multifunctional microplate reader to detect the chemiluminescence value, with the gain value fixed at 100.

[0347] The results are as follows Fig. 9B shown.

[0348] The results showed that Capa-CD19-28CAR (corresponding to Capa-1928CAR in the figure) had better killing activity than Lenti-CD19-28CAR (corresponding to Lenti-1928CAR in the figure).

[0349] Example 5: Effect of optimized adeno-associated virus (Capa) engineered with targeted gene editing enzyme (1)

[0350] Capa was incubated with T cells at an MOI of 1E4-1E5 (ratio of the number of AAV and T cells) to obtain a knock-in editing efficiency of 50%-85%.

[0351] Taking MOI (ratio of Capa to T cells) 1E5 as an example: Capa and 1E6 T cells (human primary T cells) were incubated at 37°C for 30 minutes, and then the peptide TAT-HA2 with a final concentration of 50 μM or protamine sulfate with a final concentration of 50 μM was added and incubated at 37°C for 30 minutes. Finally, the cells were added to X-vivo medium (containing 200 IU of IL2) containing ADZ7648 with a final concentration of 1 μM. The editing efficiency was detected after 5 days of culture.

[0352] The chemical formula of ADZ7648 (purchased from selleck) is C 18 H 20 N8O2, whose structural formula is shown in the following formula I:

[0353]

[0354] The results are as follows Fig.10 As shown in ad.

[0355] The horizontal axis represents the situation of TCR-CD3 complex on the surface of T cells, and the vertical axis represents the expression of CD19CAR. Q1 means that TCR is completely knocked out, and CD19CAR is precisely knocked into TRAC and expressed; Q2 means that T cells express CD19CAR and TCR at the same time, that is, only one TRAC is knocked into CD19CAR, and the other TRAC is intact; Q3, T cells that have not been edited; Q4, T cells with knocked out TCR but no CD19CAR knocked in.

[0356] The results showed that the addition of TAT-HA2, ADZ7648 and / or protamine could significantly improve the editing efficiency of Capa.

[0357] Example 6: Effect of optimized adeno-associated virus (Capa) engineered with targeted gene editing enzyme (2)

[0358] The experiment was performed according to the method of Example 5, except that Capa was replaced by Capa-Cas9, and the sample was Capa-Cas9+TAT-HA2+ADZ7648.

[0359] The results are as follows Fig.11 shown.

[0360] The results showed that the addition of TAT-HA2, ADZ7648 and / or protamine could significantly improve the editing efficiency of Capa-Cas9.

[0361] Example 7: Targeted gene editing enzyme engineered adeno-associated virus (Capa) can be used for multi-gene editing construction Universal CART cells (UCART)

[0362] Capa molecules of multiple Cas12aRNPs are used to infect T cells (schematic diagram as shown in Fig. 12A ).

[0363] Taking MOI (ratio of Capa to T cells) 1E5 as an example: Capa and 1E6 T cells (human primary T cells) were incubated at 37°C for 30 minutes, and then the peptide TAT-HA2 with a final concentration of 50 μM or protamine sulfate with a final concentration of 50 μM was added and incubated at 37°C for 30 minutes. Finally, the cells were added to X-vivo medium (containing 200 IU of IL2) containing ADZ7648 with a final concentration of 1 μM. The editing efficiency was detected after 5 days of culture.

[0364] The results are as follows Fig. 12B shown.

[0365] The results showed that the site-specific knock-in efficiency (KI) of CD19-28CAR was 70.44%, and the knock-out efficiency (KO) of TCR, HLA-A, HLA-B and MHCII was 93.5%, 96.55%, 66.99% and 74.9%, respectively; while retaining HLA-C / E / G, the occurrence of graft versus host disease (GVHD) and host versus graft reaction (HVGR) can be effectively avoided.

[0366] Example 8: Preparation of CAR by adeno-associated virus (Capa) engineered with targeted gene editing enzymes The killing activity ratio of T Comparison

[0367] NSG immunodeficient mice, 20, 6-week-old female mice, were purchased from Vital River Company.

[0368] Human B cell tumors were used to simulate leukemia. 1E6 / mouse Raji cells were injected into the tail vein of the above-mentioned immunodeficient mice, and the mice became B cell lymphoma models 5 days later.

[0369] Divide into 4 groups of 5 pieces each:

[0370] NC group, Lenti-CD19-28CAR group, Electro-CD 19-28CAR group and Capa-CD19-28CAR (prepared in the previous Example 4).

[0371] On Day-6, 1E6 / mouse Raji cells expressing CD19 molecules were injected into the tail vein (Raji cells express luciferase. After the luciferase substrate luciferin potassium salt was injected, the Raji tumor condition in the mouse could be observed in an imaging instrument).

[0372] On Day 0, 1E6 T cells (human peripheral blood T cells) were injected into the tail vein. The NC group was untreated T cells, and the others were Lenti-CD19-28CAR T, Electro-CD 19-28CAR T, and Capa-CD19-28CAR T.

[0373] in:

[0374] Lenti-CD19-28CAR process: 10 μl of lentivirus encoding CD19-28CAR was added to 1E6 human T cells activated for 24 hours, and the positive rate of CAR T was detected by flow cytometry after 36 hours of culture.

[0375] Preparation process of Electro-CD 19-28CAR: Cas12a-TRAC RNP and 1E6 T cells were placed in the electroporation cup, and CasRNP was electroporated into T cells using the Lonza preset Sti-human-T program. Then 1E11 AAV virus encoding CD19-28CAR was added, and the positive rate of CAR T was detected by flow cytometry after 5 days of culture.

[0376] On Day 1 / 4 / 7 / 14 / 19, 200 μl / mouse luciferin potassium salt solution was injected into the tail vein. After incubation for 5 minutes, the mice were placed in an imaging instrument and imaged for 5 minutes to collect fluorescence signals.

[0377] NC: Infusion of T cells without any treatment.

[0378] The results are as follows Fig.13As shown in the figure, the higher the brightness of the fluorescence value, the greater the tumor burden.

[0379] The results showed that in several groups of experiments, in order of killing activity from large to small, Capa-CD19-28CAR>Electro-CD19-28CAR>Lenti-CD19-28CAR.

[0380] Although the specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art. According to all the teachings disclosed, various modifications and replacements can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the attached claims and any equivalents thereof.

Claims

1. A modified adeno-associated virus whose capsid protein is linked to a DNA cutting enzyme.

2. The modified adeno-associated virus according to claim 1, wherein The DNA shearing enzyme is connected to a targeting molecule; Preferably, the adeno-associated virus is AAV2 or AAV6.

3. The modified adeno-associated virus according to any one of claims 1 to 2, wherein The DNA shearing enzyme is directly connected to the capsid protein, or is connected to the capsid protein via a first linker; and / or, The DNA cutting enzyme is directly connected to the targeting molecule or connected to the targeting molecule through a second linker.

4. The modified adeno-associated virus according to any one of claims 1 to 3, wherein The DNA shearing enzyme is CAS12, Cas9, a base editing enzyme or a lead editing enzyme; Preferably, the Cas12 is one selected from Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i and Cas12j; Preferably, the Cas9 is one selected from spCas9, CjCas9 (984), Nme2Cas9 (1082), SauriCas9 (1061) and SauCas9 (1053); Preferably, each capsid protein is linked to 1-60 DNA cutting enzyme molecules.

5. The modified adeno-associated virus according to any one of claims 2 to 4, wherein The targeting molecule is an antibody or a ligand; Preferably, the targeting molecule is a molecule targeting T cell receptor, CD34 or CD16; Preferably, the antibody is a single-chain antibody or a nanobody; Preferably, the single-chain antibody is a single-chain antibody targeting a T cell receptor; Preferably, the amino acid sequence of the single-chain antibody is as shown in SEQ ID NO: 4; Preferably, the Nanobody is a Nanobody targeting a T cell receptor.

6. The modified adeno-associated virus according to any one of claims 3 to 5, wherein The first linker is selected from the group consisting of biotin-avidin, biotin-streptavidin, AAV receptor, SpyTag / SpyCatcher, and anti-capsid protein antibody; Preferably, the biotin is directly linked to the capsid protein via the AVI polypeptide; Preferably, the amino acid sequence of the AAV receptor is as shown in SEQ ID NO:

17.

7. The modified adeno-associated virus according to any one of claims 3 to 6, wherein The second linker is selected from biotin-avidin, biotin-streptavidin, and paired proteins with binding effects such as antibody-antigen, receptor-ligand, OsASI1-BAH-OsSUVH6 or SpyTag / SpyCatcher.

8. The modified adeno-associated virus according to any one of claims 1 to 7, comprising: The plasmid pRepCap encoding AAV capsid and replicase, the adenovirus helper plasmid pHelp, and the pTransfer plasmid containing the target gene to be transferred; Preferably, the target gene is a nucleic acid sequence encoding a chimeric antigen receptor, a coagulation factor gene or an SMN gene; Preferably, the chimeric antigen receptor comprises a single chain antibody recognizing the CD19 antigen, a CD8 hinge region, a CD28 transmembrane region and an intracellular region, and a CD3 intracellular region.

9. The modified adeno-associated virus according to any one of claims 1 to 8, comprising: Adeno-associated virus, a first linker, a DNA cutting enzyme, a second linker and a targeting molecule; AAV2 or AAV6, a first linker, Cas12a, a second linker, and a single-chain antibody targeting a T cell receptor; AAV2 or AAV6, biotin-streptavidin, Cas12a, OsASI1-BAH-OsSUVH6, and a single-chain antibody targeting the T cell receptor; AAV2 or AAV6, AAV receptor, Cas12 (e.g., Cas12a) or Cas9, a second linker (e.g., SpyTag / SpyCatcher), and a single-chain antibody targeting a T cell receptor; or AAV2 or AAV6, and a polypeptide with an amino acid sequence as shown in any one of SEQ ID NO:24 to SEQ ID NO:

27.

10. A complex comprising the modified adeno-associated virus according to any one of claims 1 to 9, and gRNA.

11. A transformed cell obtained by transfecting a cell with the modified adeno-associated virus according to any one of claims 1 to 9 or the complex according to claim 10; Preferably, the cell is an animal cell, a plant cell or a microbial cell; Preferably, the cell is a primary cell of a mammal; preferably, the cell is a primary cell of a human; preferably, the cell is one or more selected from human T cells, natural killer cells, dendritic cells, macrophages, hematopoietic stem cells and neural cells; Preferably, the cell is a passage cell; preferably, the cell is a tumor cell of a human or non-human mammal; preferably, the cell is one or more selected from Jurkat cells, A549 cells, U87 cells, THP1 cells and Raji cells.

12. A pharmaceutical composition comprising the modified adeno-associated virus according to any one of claims 1 to 9, the complex according to claim 10 or the transformed cell according to claim 11, and one or more pharmaceutically acceptable excipients.

13. A kit comprising: The modified adeno-associated virus and gRNA according to any one of claims 1 to 9, or the complex according to claim 10, and an optional transfection reagent; Preferably, the kit further comprises one or more selected from TAT-HA2, protamine sulfate and a compound of formula I; Preferably, the kit further comprises TAT-HA2 and a compound of formula I, or comprises protamine sulfate and a compound of formula I; 14. An isolated polypeptide comprising an AAV receptor, Cas12 (eg, Cas12a) or Cas9, a linker (eg, SpyTag / SpyCatcher), and a single-chain antibody targeting a T cell receptor; Preferably, the amino acid sequence of the polypeptide is shown in any one of SEQ ID NO:24 to SEQ ID NO:

27.

15. An isolated nucleic acid molecule encoding the isolated polypeptide of claim 14.

16. A recombinant vector comprising the isolated nucleic acid molecule of claim 15.

17. A recombinant host cell comprising the recombinant vector according to claim 16.

18. Use of the modified adeno-associated virus according to any one of claims 1 to 9, the complex according to claim 10, the transformed cell according to claim 11 or the isolated polypeptide according to claim 14 in the preparation of a medicament for treating or preventing tumors, autoimmune diseases, AIDS, aging or asthma; Preferably, the tumor is selected from one or more of leukemia, lymphoma, myeloma, lung cancer, pancreatic cancer, breast cancer, ovarian cancer, colorectal cancer, head and neck cancer, brain cancer, pharyngeal cancer, nasopharyngeal cancer, thyroid cancer, mesothelioma, liver cancer, gastric cancer, biliary tract cancer, kidney cancer, fallopian tube cancer, endometrial cancer, cervical cancer, bladder cancer, urothelial cancer, prostate cancer, testicular cancer, skin cancer, melanoma, plasma cell cancer, vulvar cancer, bone cancer and osteosarcoma; Preferably, the leukemia is acute lymphoblastic leukemia; Preferably, the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma; Preferably, the myeloma is multiple myeloma; Preferably, the lung cancer is selected from one or more of non-small cell lung cancer, small cell lung cancer and squamous cell lung cancer.

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