Adeno-associated virus engineered with targeting gene editing enzyme and pharmaceutical composition thereof

Abstract

The present invention belongs to the field of biomedicine, and relates to an adeno-associated virus engineered with a targeting gene editing enzyme and a pharmaceutical composition thereof. Specifically, the present invention relates to a modified adeno-associated virus, a capsid protein of which is linked to a DNA cleavage enzyme. The present invention also relates to a transformed cell. The present invention can achieve in one step a knock-in of a gene fragment having a length up to 3.5-4K to a T cell and a lock-out of multiple genes from the T cell, and has good application prospects.

Description

Adeno-associated virus engineered with targeted gene editing enzyme and pharmaceutical composition thereof

[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 application date September 1, 2023. The entire contents of the CN application are hereby incorporated into this application as a whole. 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 to date, consisting of only 60 capsid proteins and a single-stranded DNA strand of approximately 4.7k. It has extremely low immunogenicity and is non-pathogenic to humans. It is an FDA-approved gene therapy drug. Its unique feature is its ability to deliver DNA directly into the cell nucleus for gene expression or DNA repair.

[0005] CRISPR / Cas gene editing technology originated from research on bacterial immunity to foreign invading nucleic acids and has evolved into the most versatile gene editing tool available today. The most commonly used CRISPR / Cas proteins include Cas9 and Cas12a. CRISPR / Cas possesses DNA cleavage activity. The guide RNA (gRNA) can recognize the target DNA region on the genome. The CRISPR / Cas protein binds to the gRNA to form an RNP that cuts the DNA at that location, creating a double-strand break (DSB). The cell then initiates two major repair mechanisms: non-homologous end joining (NHEJ) and homologous recombination (HDR). If a repair template is absent, the cell uses NHEJ, resulting in mutations at the break. However, if an HDR template is present, the cell initiates HDR to repair the DNA break. Therefore, NHEJ can be used to knock out specific genes, while HDR can be used to achieve site-specific knock-in of exogenous genes.

[0006] Therefore, combining AAV with CRISPR / Cas9 / 12a can insert customized DNA fragments into specific sites in the cell genome. Currently, this process is typically completed in two steps: first, Cas9 / 12a RNP is electroporated into the cell, and then AAV containing the target DNA fragment is added to infect the cell to achieve site-specific knock-in of the target DNA fragment into the genome. This operation is not only cumbersome, expensive, and heavily equipment-dependent, but also causes significant damage to the cells.

[0007] Chimeric antigen receptors (CARs) are loaded onto T cells to form CAR T cells, which are used to kill tumor cells. The CAR structure consists of three parts: an extracellular portion that recognizes tumor antigens, a transmembrane region, and an intracellular transduction domain. The extracellular portion typically comprises a molecule containing an antibody variable region, such as a scFv. Once the scFv outside the CAR T cell recognizes the tumor antigen, it transmits the signal to the CAR T cell, allowing the CAR T cell to kill the tumor cell. Traditional CAR T cell development uses lentivirus as a gene delivery tool. During lentiviral transduction, the gene carried by the lentivirus is randomly inserted into the T cell genome, potentially genotoxic. Therefore, developing site-specific insertion technologies for CAR T cell development is a hot topic in the field. Currently, site-specific insertion of CAR T cells is typically performed using electroporation of Cas9 / 12RNPs and adeno-associated virus (AAV) infection, or direct electroporation of Cas9 / 12RNPs and DNA. This technique is cumbersome, electroporation can cause significant cell damage, and can only be performed on cells in vitro.

[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.

[0009] Summary of the Invention

[0010] After in-depth research and creative work, the inventors have deeply integrated CRISPR / Cas gene editing technology with AAV vectors and developed adeno-associated viruses and pharmaceutical compositions engineered with targeted gene editing enzymes. The present invention can achieve site-specific knock-in of large fragments in one step or in steps by non-electrotransfer, 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:

[0011] One aspect of the present invention relates to a modified adeno-associated virus, the capsid protein of which is linked to a DNA cutting enzyme.

[0012] In some embodiments of the present invention, in the modified adeno-associated virus, the DNA cutting enzyme is linked to a targeting molecule.

[0013] In some embodiments of the present invention, the modified adeno-associated virus, wherein,

[0014] The DNA shearing enzyme is directly linked to the capsid protein, or is linked to the capsid protein via a first linker; and / or

[0015] The DNA shearing enzyme is directly connected to the targeting molecule or connected to the targeting molecule through a second linker.

[0016] In some embodiments of the present invention, the modified adeno-associated virus, wherein,

[0017] The DNA shearing enzyme is directly linked to the capsid protein; and / or

[0018] The DNA cutting enzyme is directly linked to the targeting molecule.

[0019] In some embodiments of the present invention, the modified adeno-associated virus, wherein,

[0020] The DNA shearing enzyme is directly linked to the capsid protein; and / or

[0021] The DNA cutting enzyme is connected to the targeting molecule via a second linker.

[0022] In some embodiments of the present invention, the modified adeno-associated virus, wherein,

[0023] The DNA shearing enzyme is connected to the capsid protein via a first linker; and / or

[0024] The DNA cutting enzyme is directly linked to the targeting molecule.

[0025] In some embodiments of the present invention, the modified adeno-associated virus, wherein,

[0026] The DNA shearing enzyme is connected to the capsid protein via a first linker; and / or

[0027] The DNA cutting enzyme is connected to the targeting molecule via a second linker.

[0028] In some embodiments of the present invention, the modified adeno-associated virus is AAV2 or AAV6.

[0029] 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;

[0030] Preferably, the Cas12 is one selected from Cas12a, Cas12b, Cas12d, Cas12e, Cas12f, Cas12g, Cas12h, Cas12i and Cas12j;

[0031] Preferably, the Cas9 is one selected from spCas9, CjCas9 (984), Nme2Cas9 (1082), SauriCas9 (1061) and SauCas9 (1053).

[0032] In some embodiments of the present invention, each modified adeno-associated virus is linked to 1-60 DNA shearing enzyme molecules. Theoretically, one adeno-associated virus has 60 AVI tags.

[0033] Without being bound 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.

[0034] In some embodiments of the present invention, in the modified adeno-associated virus, the targeting molecule is a T cell targeting molecule.

[0035] In some embodiments of the present invention, the modified adeno-associated virus, wherein the targeting molecule is an antibody or ligand targeting T cells;

[0036] Preferably, the antibody is a single-chain antibody or nanobody targeting T cells.

[0037] In some embodiments of the present invention, in the modified adeno-associated virus, the targeting molecule is a molecule targeting a T cell receptor.

[0038] 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;

[0039] Preferably, the antibody is a single-chain antibody or nanobody targeting T cell receptor.

[0040] TCR-binding antibody ScFv can be converted into ScFv targeting other cell types as needed, such as anti-CD16 targeting NK cells, anti-CD34 targeting HSC cells, etc.

[0041] In some embodiments of the present invention, the modified adeno-associated virus, wherein the targeting molecule is an antibody or a ligand;

[0042] Preferably, the antibody is a single-chain antibody or a nanobody;

[0043] Preferably, the single-chain antibody is a single-chain antibody targeting T cell receptor;

[0044] Preferably, the amino acid sequence of the single-chain antibody is shown in SEQ ID NO: 4.

[0045] In some embodiments of the present invention, the modified adeno-associated virus, wherein the first linker is selected from the group consisting of biotin-avidin, biotin-streptavidin, AAV receptor, SpyTag / SpyCatcher, and anti-capsid protein antibody;

[0046] 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 triplex (this region has little effect on AAV packaging);

[0047] Preferably, the amino acid sequence of the AAV receptor is shown in SEQ ID NO: 17.

[0048] 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.

[0049] In some embodiments of the present invention, the modified adeno-associated virus comprises:

[0050] The plasmid pRepCap encoding AAV capsid and replicase, the adenovirus helper plasmid pHelp, and the pTransfer plasmid containing the target gene to be transferred.

[0051] In some embodiments of the present invention, the modified adeno-associated virus, wherein,

[0052] 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);

[0053] Preferably, the chimeric antigen receptor comprises a single-chain antibody that recognizes the CD19 antigen, a CD8 hinge region, a CD28 transmembrane region and an intracellular region, and a CD3 intracellular region.

[0054] In some embodiments of the present invention, the modified adeno-associated virus comprises, in sequence:

[0055] Adeno-associated virus, a first linker, a DNA cutting enzyme, a second linker, and a targeting molecule;

[0056] AAV2 or AAV6, a first linker, Cas12a, a second linker, and a single-chain antibody targeting the T cell receptor;

[0057] AAV2 or AAV6, biotin-streptavidin, Cas12a, OsASI1-BAH-OsSUVH6, and a single-chain antibody targeting the T cell receptor;

[0058] AAV2 or AAV6, an 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

[0059] 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.

[0060] In some embodiments of the present invention, the modified adeno-associated virus comprises the following components:

[0061] Adeno-associated virus, a first linker, a DNA cutting enzyme, a second linker, and a targeting molecule;

[0062] AAV2 or AAV6, a first linker, Cas12a, a second linker, and a single-chain antibody targeting the T cell receptor;

[0063] AAV2 or AAV6, biotin-streptavidin, Cas12a, OsASI1-BAH-OsSUVH6, and a single-chain antibody targeting the T cell receptor;

[0064] AAV2 or AAV6, an 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

[0065] 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.

[0066] In some embodiments of the present invention, the modified adeno-associated virus, wherein:

[0067] 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

[0068] 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.

[0069] In some embodiments of the present invention, the modified adeno-associated virus comprises the following components:

[0070] AAV2 or AAV6, AVI polypeptide, biotin-streptavidin, Cas12a, OsASI1-BAH-OsSUVH6, and a single-chain antibody targeting the T cell receptor can be prepared, for example, by referring to the following steps:

[0071] 1) Inserting the AVI polypeptide into the VRVIII region of the AAV6 capsid tripartite axis to construct an engineered AAV6;

[0072] 2) Co-transfecting the AAV packaging plasmid and the BirA-expressing plasmid into 293T cells, while adding D-biotin to the culture medium, produces biotin-labeled AAV6 at the AVI polypeptide site (other AAVs are also applicable, such as AAV2);

[0073] 3) AsCas12a and anti-TCR single-chain antibody ScFv were expressed in prokaryotic and eukaryotic cells, respectively. AsCas12a was fused to Streptavidin at the N-terminus and OsASI1-BAH at the C-terminus. The anti-TCR ScFv was fused to the OsSUVH6 peptide at the N-terminus.

[0074] As another embodiment, "the C-terminus of AsCas12a is fused with Streptavidin, and the N-terminus is fused with OsASI1-BAH. The C-terminus of antiTCR ScFv is fused with the polypeptide of OsSUVH6."

[0075] The two proteins are assembled into one protein through the polypeptides of OsASI1-BAH and OsSUVH6, and Streptavidin can bind to biotin on AAV6.

[0076] Alternatively, AAV receptors can be used to directly bind to AAV without the need for biotin labeling of AAV.

[0077] 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.

[0078] Another aspect of the present invention relates to a complex comprising the modified adeno-associated virus according to any one of the present invention and a gRNA. The gRNA can be incubated with the modified adeno-associated virus according to any one of the present invention under appropriate conditions to obtain the complex.

[0079] 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;

[0080] Preferably, the cell is an animal cell, a plant cell or a microbial cell;

[0081] Preferably, the cell is a primary mammalian cell; preferably, the cell is a primary human cell; preferably, the cell is one or more selected from human T cells, natural killer cells, dendritic cells, macrophages, hematopoietic stem cells and neural cells;

[0082] 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.

[0083] 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.

[0084] Yet another aspect of the present invention relates to a kit comprising:

[0085] The modified adeno-associated virus and gRNA according to any one of the present invention, or the complex according to the present invention, and an optional transfection reagent;

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

[0087] Preferably, the kit further comprises TAT-HA2 and a compound of formula I, or comprises protamine sulfate and a compound of formula I;

[0088] Yet 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] Yet 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 containing the recombinant vector of the present invention.

[0093] Another aspect of the present invention relates to use of any one of the modified adeno-associated viruses, the complex of the present invention, the transformed cell of the present invention, or the isolated polypeptide 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 carcinoma, 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 carcinoma, 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 any one of the modified adeno-associated viruses, the complex of the present invention, the transformed cell of the present invention or the isolated polypeptide 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 carcinoma, 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 particular 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 formulation used; the patient's age, weight, general health, sex, and diet; the time of administration, route of administration, and excretion rate; the duration of treatment; other drugs used concomitantly; and similar factors well known in the medical field. For example, it is practiced in the art to start the administration at a dose lower than that required to obtain the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[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 bound by theory, BirA (biotin-protein ligase) can efficiently activate biotin (D-biotin) to form biotinyl-5' adenylate in the presence of ATP and specifically link 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 one of the strongest protein-ligand interactions. Avidin or streptavidin is a tetrameric protein that can simultaneously and highly specifically bind to four biotin molecules, representing a naturally occurring biomolecular interaction system. The biotin-avidin or streptavidin binding system, known as the biotin-streptavidin system, is a signal amplification labeling technology with advantages such as high affinity, high sensitivity, strong specificity, and good stability, and has been widely used in various 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 is directed against an antibody-based specificity for an antigen of interest and an immune effector cell activation intracellular domain combination to demonstrate specific immune activity for cells expressing the antigen of interest. In the present invention, the expression "immune effector cells expressing CAR" refers to immune effector cells 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 WO 2012 / 079000, WO 2013 / 059593; and U.S. Patent Publication 2012 / 0213783, all of which are incorporated herein by reference in their entirety.

[0118] As used herein, the term "extracellular antigen-binding domain" refers to a polypeptide that specifically binds to an antigen or receptor of interest. This domain will be capable of interacting 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 particular 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 the immune effector cell expressing CAR. For example, the effector function of T cells 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 or inhibitory manner. Primary signaling domains that act in a stimulatory manner generally contain 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, and 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 other than antigen receptors or Fc receptors that provide the second signal required for the efficient activation and function of T lymphocytes after binding to an antigen. 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 comprising a heavy chain variable region (V H ) and antibody light chain variable region (V L ) molecule. Where V L and V H The domains are paired to form monovalent molecules via linkers that enable their production 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 commonly 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 are 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 the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to, pH adjusters, surfactants, adjuvants, and ionic strength enhancers. For example, pH adjusters include, but are not limited to, phosphate buffers; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80; and 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 already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective 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 something that is obtained artificially from its natural state. If a substance or component is "isolated" in nature, it may be that its natural environment has been changed, or that the substance has been separated from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and a highly pure identical polynucleotide or polypeptide isolated 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 is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. 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, and papillomas (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. Additionally, a vector may contain an origin of replication.

[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 reference distinction or clarity of expression, 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 achieve the knock-in of gene fragments as long as 3.5-4K in T cells and the simultaneous knock-out of multiple genes in T cells (such as TCR alpha, beta2m, regnase, roquin, etc.) in one step.

[0134] 3) CAR can be knocked into CD52 (gRNA can guide Cas protein to cut DNA at CD52, and at the same time, the homology arm at CD52 can be used 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) Ability to be used in vivo, i.e., direct editing to generate CAR T / STAR T / TCR T cells in vivo (knocking into a certain gene site will simultaneously destroy the gene. The advantage of the TRAC site is that it can utilize the endogenous TCR promoter for better expression regulation, thus expressing better CAR T).

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

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

[0138] Figure 1: Packaging of biotin-targeted AAV.

[0139] Figure 2: Design of engineered AAV.

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

[0141] Figure 4A: Streptavidin linker molecule bound to biotin.

[0142] FIG4B : The antiTCR domain of the adaptor molecule binds to the TCR.

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

[0144] Figure 6: Engineered AAV can deliver CARs to T cells at specific locations.

[0145] Figures 7A to 7B: Design of adeno-associated virus (Capa) engineered with targeted gene editing enzymes.

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

[0147] Figure 7B shows protein expression and purification results of the constructs shown in Figure 7A. 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] Figures 8A to 8C: Biochemical detection of adeno-associated virus (Capa) engineered with targeted gene editing enzymes.

[0149] Figure 8A: Affinity determination of targeted gene editing enzymes and AAV, with the curves in the figure representing different protein concentrations;

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

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

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

[0153] Figure 9A, Capa directly edited primary T cells to generate CD19-targeted CAR T (Capa-CD19-28CAR);

[0154] Figure 9B shows the experimental results of tumor killing by Capa-generated CAR T cells. The horizontal axis represents the effector-target ratio, that is, the ratio of T cells to tumor cells (for example, 1:1 represents one T cell and one tumor), and the vertical axis represents the tumor lysis efficiency. The higher the vertical axis lysis efficiency, the stronger the tumor killing.

[0155] Figure 10: Optimization of adeno-associated virus (Capa) engineered with a targeted gene editing enzyme. The synergistic effect of the peptide TAT-HA2 and the small molecule drug ADZ7648 with Capa significantly improves gene editing efficiency. Where: a represents Capa, b represents Capa + TAT-HA2, c represents Capa + TAT-HA2 + ADZ7648, and d represents Capa + protamine + ADZ7648.

[0156] Figure 11: The horizontal axis represents the TCR-CD3 complex on the T cell surface, and the vertical axis represents the expression of CD19 CAR. Q1 indicates that the TCR is completely knocked out and the CD19 CAR is precisely knocked in and expressed at the TRAC; Q2 indicates that the T cell expresses both the CD19 CAR and the TCR, that is, only one TRAC is knocked in with the CD19 CAR, and the other TRAC is intact; Q3 indicates that the T cell has not been edited; Q4 indicates that the TCR is knocked out but the CD19 CAR is not knocked in.

[0157] Figures 12A-12B: Adeno-associated viruses (Capa) engineered with targeted gene editing enzymes can be used for multiple gene editing. Figure 12A is a schematic diagram showing that Capa can carry multiple RNPs, and Figure 12B shows the gene editing effects of various RNP-Capa.

[0158] Figure 13: Comparison of the cytotoxicity of CAR T cells prepared using adeno-associated viruses (Capa) engineered with targeted gene editing enzymes. NC represents the control group, Lentil represents CAR T cells prepared using lentivirus, Electro represents CAR T cells prepared using electroporation, and Capa represents CAR T cells prepared using the present invention.

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

[0160] AVI peptide

[0161] OsASI1-BAH

[0162] OsSUVH6

[0163] antiTCR

[0164] AsCas12a

[0165] FMC-28CAR

[0166] AsCas12a

[0167] antiTCR

[0168] OsSUVH6

[0169] OsASI1-BAH

[0170] Substrate double-stranded DNA / target sequence to be knocked out

[0171] AVI

[0172] ITR

[0173] TRAC 5' homology arm

[0174] FMC-28CAR

[0175] TRAC 3' homology arm

[0176] AAVR (AAV Receptor)

[0177] SpyTag

[0178] SpyCatcher

[0179] AAVR-AsCas12a-ST amino acid sequence:

[0180] AAVR-AsCas12a-ST nucleic acid sequence:

[0181] Anti-TCR ScFv-SC amino acid sequence:

[0182] Anti-TCR ScFv-SC nucleic acid sequence:

[0183] One of the amino acid sequences of AAVR-AsCas12a-ST-SC-antiTCR:

[0184] Amino acid sequence 2 of AAVR-AsCas12a-ST-SC-antiTCR:

[0185] Amino acid sequence 3 of AAVR-AsCas12a-ST-SC-antiTCR:

[0186] Amino acid sequence 4 of AAVR-AsCas12a-ST-SC-antiTCR: DETAILED DESCRIPTION

[0187] The embodiments of the present invention will be described in detail below with reference to the examples, but it will be understood by those skilled in the art that the following examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention. Where specific conditions are not specified in the examples, the methods were performed according to conventional conditions or the conditions recommended by the manufacturer. Where the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be obtained commercially.

[0188] AAV6 virus was purchased from Pigen Biotechnology.

[0189] Plasmids were purchased from Addgene.

[0190] Cell lines such as 293F, Jurkat, and T cells were purchased from ATCC.

[0191] gRNA was purchased from IDT.

[0192] The FMC-28CAR includes:

[0193] 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).

[0194] Preparation Example 1: Preparation of engineered AAV that specifically targets and edits T cells (1)

[0195] 1.1 Design, modification, and preparation of AAV containing CAR (FMC-28CAR)

[0196] 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.

[0197] 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 biotinylated (see Figure 1).

[0198] AAV containing CAR (FMC-28CAR) was obtained.

[0199] 1.2 Design and preparation of linker molecules

[0200] Design an adapter molecule with editing function for use with AAV. The adapter molecule consists of three parts, in order from the N-terminus to the C-terminus:

[0201] 1) Streptavidin (SA) that binds to the biotin of the AAV capsid,

[0202] 2) CRISPR system (CRISPR / Cas12a+gRNA, i.e., RNP) that can cut TRAC at specific genomic sites, and

[0203] 3) ScFv that can specifically bind to T cell receptors.

[0204] Among them, after part 3) is connected to part 1), 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.

[0205] The AsCas12a used in part 2) was obtained by purifying the coding sequence (SEQ ID NO: 7) through prokaryotic expression. The steps are as follows:

[0206] Inoculation: Pick a single colony or 1-10 ml of bacterial solution into 1 L of LB medium and culture at 37°C in a shaking incubator until OD = 0.5-0.6;

[0207] Expression: Add 200 μL 1 M IPTG and culture at 16°C in a shaking incubator for 12-16 h;

[0208] Centrifugation: centrifuge at 12000 rpm for 5 min, resuspend 1 g of pellet in 7 ml of 2X PBS and collect in a 100 ml beaker and place on ice;

[0209] Lysis: ultrasonic lysis 5 times (must be placed on ice, preferably ice-water mixture, with 2 minutes interval between each ultrasonication), centrifuge at 20000 rpm for 1 hour after balancing;

[0210] Column: Wash the nickel column with 1×PBS, draw up the supernatant with a syringe, and inject it into the nickel column. Be careful not to use a too fast flow rate (a peristaltic pump can be used).

[0211] Wash: Wash the nickel column with 20 ml of 1× PBS;

[0212] Elution: elution with 5 mL 500 mM imidazole (1×PBS, pH=7.7);

[0213] Column: The sample was added to AKTA PurM with a syringe, passed through HiLoad 1; 6 / 600 Superdex 200pg, and collected in a 96-well plate using a collector to prepare the AsCas12a protein sample;

[0214] Gel running: Pipette samples from the peak area of ​​the 96-well plate and run the gel to verify the protein size;

[0215] Cleaning: Soak the tubing and column in 0.2 M NaOH for one day and night, and clean the tubing with 1× PBS the next day.

[0216] The ScFv used in part 3) was obtained by eukaryotic expression and purification of the coding sequence (SEQ ID NO: 8). The steps are as follows:

[0217] Recovery: Recover 293F cells from the cell bank and culture them in SMM 293-TII (1% PS) in a cell culture conical flask to 2×10 6 ~3×10 6 pcs / ml;

[0218] 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;

[0219] Centrifugation: centrifuge at 14000 rpm for 2 h, take the supernatant and filter with a 0.22 μm filter;

[0220] Column: Use the injector to draw the supernatant and inject it into the nickel column to prevent bubbles from entering the pipeline;

[0221] Wash: Wash the nickel column with 10 ml of 1× PBS;

[0222] Elution: elution with 500 mM imidazole (1×PBS, pH=7.7);

[0223] Concentration: Use a 10,000 MCO concentrator tube, add 1× PBS, and centrifuge at 3,500 rpm to concentrate to 1 mL. Repeat twice to prepare the ScFv protein sample.

[0224] Gel running: Gel running to verify protein size;

[0225] Cleaning: Soak the tubing and column in 0.2 M NaOH for one day and night, and clean the tubing with 1× PBS the next day.

[0226] AsCas12a and ScFv were incubated on ice for 1 hour at a molar ratio of 1:1 for ligation.

[0227] 1.3 Preparation of engineered AAVs that specifically target and edit T cells

[0228] Incubate the biotin-labeled AAV prepared in 1.1 above with the linker molecule prepared in 1.2 in PBS buffer at room temperature at a molar ratio of 1:60 for 20 minutes. Purify by density gradient centrifugation to obtain an engineered AAV that specifically targets and edits T cells (see Figure 2). This was named AAV6-Cas12aRNP-antiTCR and used in subsequent experiments.

[0229] Example 1: The linker molecule can bind to biotin and TCR and specifically cleave the DNA substrate in vitro

[0230] (1) The packaged AAV (prepared in 1.1 of Preparation Example 1) was subjected to western blot (WB) detection. The results showed that the AAV capsid was indeed modified with biotin (see Figure 3).

[0231] (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.

[0232] Biotin-labeled 293F cells were used to mimic AAV biotinylation, and the binding of streptavidin to biotin was tested by flow cytometry. Flow cytometry results showed that the linker molecule was able to bind to the biotin-labeled 293F cells.

[0233] 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, and centrifuge at 400g for 5 minutes to remove excess biotin, thus completing the labeling.

[0234] (3) 1 μg of adaptor protein was added per 1E6 human peripheral blood mononuclear cells (PBMCs), and the adaptor protein was incubated with T cells on ice for 30 minutes. The binding to T cells was detected by flow cytometry (see Figures 4A and 4B).

[0235] (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 micrograms according to RNP). After incubating the DNA substrate with the AAV assembled with the linker molecule at room temperature for 30 minutes, the 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).

[0236] Example 2: Testing the Engineered AAV Prepared in Example 1 for Specific Targeting and Editing T Cells at the T Cell Level

[0237] 2.1 Engineered AAV has the characteristic of preferentially infecting T cells

[0238] The engineered AAV containing the linker molecule, 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, that is, JC5 cells that do not express TCR) at an MOI of 1E4 to explore whether the engineered AAV has an infection preference.

[0239] The experimental results are shown in Figure 5.

[0240] The results showed that the engineered AAVs of the present invention primarily infected Jurkat cells, indicating that these AAVs bind to the T cell surface via TCR molecules and enter the T cell through endocytosis of the TCR to complete the infection process. The engineered AAVs preferentially infect T cells expressing the TCR.

[0241] 2.2 Engineered AAV can insert CAR into TRAC

[0242] 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 the T cell outside the left homology arm to identify the insert fragment. The target fragment size was 1.3K.

[0243] The results are shown in Figure 6. The results showed that FMC-28CAR was successfully inserted into the genome of T cells.

[0244] Preparation Example 2: Preparation of engineered AAV that specifically targets and edits T cells (2)

[0245] The linker molecule was designed as AAVR-AsCas12a-SA-AVI-antiTCR ScFv.

[0246] Among them, AAVR (AAV receptor, SEQ ID NO: 17) directly binds to AAV; SA (streptavidin) binds to biotinylated AVI-antiTCR ScFv.

[0247] AAVR and AAV were mixed at a molar ratio of 1:60 and incubated at room temperature for 30 minutes for binding.

[0248] The C-terminus of AsCas12a was fused with Streptavidin, and the two were produced together by prokaryotic expression.

[0249] When expressing the AVI-antiTCR ScFv protein, add a plasmid expressing biotin ligase (BirA) and D-biotin at a final concentration of 1 μg / mL. The expressed AVI-antiTCR ScFv protein will be automatically labeled with biotin.

[0250] The other steps refer to the previous Preparation Example 1.

[0251] Preparation Example 3: Preparation of Engineered AAVs that Specifically Target and Edit T Cells (3) (Capa)

[0252] The design of the engineered AAV (Capa) that specifically targets and edits T cells is shown in Figure 7A. Cas12a / 9RNP binds to the AAV6 capsid through the fusion-expressed PKD1 and PKD2 domains of the 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 fused with the SC domain, thereby forming an engineered AAV that specifically targets and edits T cells.

[0253] 1. Preparation of AAVR-AsCas12-ST

[0254] The amino acid sequence of AAVR-AsCas12-ST is shown in SEQ ID NO: 20, and the encoding nucleic acid sequence is shown in SEQ ID NO: 21. Wherein, AAVR represents AAV receptor, and ST represents SpyTag.

[0255] The preparation steps of AAVR-AsCas12-ST are as follows:

[0256] Inoculation: Pick a single colony of recombinant host bacteria or 1-10 ml of bacterial solution into 1 L of LB medium and culture at 37°C in a shaking incubator until OD = 0.5-0.6;

[0257] The host bacteria were purchased from Shanghai Sangon Biotechnology Co., Ltd. as BL21(DE3) strain, catalog number B528419-0010. SEQ ID NO: 21 was synthesized by General Biotech and directly constructed into the pet22b vector. The resulting plasmid was then transformed into BL21(DE3) for protein expression.

[0258] Expression: Add 200 μL 1 M IPTG and culture at 16°C in a shaking incubator for 12-16 h;

[0259] Centrifugation: centrifuge at 12000 rpm for 5 min, resuspend 1 g of pellet in 7 ml of 2X PBS and collect in a 100 ml beaker and place on ice;

[0260] Lysis: ultrasonic lysis 5 times (must be placed on ice, preferably ice-water mixture, with 2 minutes interval between each ultrasonication), centrifuge at 20000 rpm for 1 hour after balancing;

[0261] Column: Wash the nickel column with 1×PBS, draw up the supernatant with a syringe, and inject it into the nickel column. Be careful not to use a too fast flow rate (a peristaltic pump can be used).

[0262] Wash: Wash the nickel column with 20 ml of 1× PBS;

[0263] Elution: 5 mL 500 mM imidazole (1×PBS, pH=7.7)

[0264] Column: The sample was added to AKTA PurM with a syringe, passed through HiLoad 1; 6 / 600 Superdex 200pg, and collected in a 96-well plate using a collector to prepare the AsCas12a protein sample;

[0265] Gel running: Pipette samples from the peak area of ​​96-well plate and run gel to verify protein size.

[0266] The results are shown in Figure 7B. The results show that the size of the protein (lane L2) is consistent with the expected size.

[0267] 2. Preparation of anti-TCR ScFv-SC

[0268] The amino acid sequence of antiTCR ScFv-SC is shown in SEQ ID NO: 22, and the encoding nucleic acid sequence is shown in SEQ ID NO: 23.

[0269] The preparation steps of antiTCR ScFv-SC are as follows:

[0270] Recovery: Recover 293F cells from the cell bank and culture them in SMM 293-TII (1% PS) in a cell culture conical flask to 2×10 6 ~3×10 6 pcs / ml;

[0271] 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;

[0272] Centrifugation: centrifuge at 14000 rpm for 2 h, take the supernatant and filter with a 0.22 μm filter;

[0273] Column: Use the injector to draw the supernatant and inject it into the nickel column to prevent bubbles from entering the pipeline;

[0274] Wash: Wash the nickel column with 10 ml of 1× PBS;

[0275] Elution: elution with 500 mM imidazole (1×PBS, pH=7.7);

[0276] Concentration: Use a 10,000 MCO concentrator tube, add 1× PBS, and centrifuge at 3,500 rpm to concentrate to 1 mL. Repeat twice to prepare the ScFv protein sample.

[0277] Gel running: Run gel to verify protein size.

[0278] The results are shown in Figure 7B. The results show that the size of the protein (lane L1) is consistent with the expected size.

[0279] 3. Preparation of AAVR-AsCas12a-ST-SC-antiTCR

[0280] 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.

[0281] The ligation product AAVR-AsCas12a-ST-SC-antiTCR was run on a gel for verification. The results are shown in Figure 7B. The results show that the protein (lane L3) size is consistent with expectations.

[0282] 4. Preparation of Capa

[0283] 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.

[0284] Unless otherwise specified, the Capa samples used in subsequent experiments are the Capa prepared in this step.

[0285] Preparation Example 4: Preparation of Engineered AAV (Capa-Cas9) that Specifically Targets and Edits T Cells

[0286] The preparation method is the same as that of Preparation Example 3, except that Cas12 is replaced by Cas9.

[0287] The resulting assembly was named Capa-Cas9.

[0288] Example 3: Biochemical Detection of Adeno-Associated Virus (Capa) Engineered with Targeted Gene Editing Enzymes

[0289] 1. SPR experiment:

[0290] AAV6 was immobilized on a chip (model CM5, Cytiva) at a concentration of 1E12 vg / ml by incubation at room temperature for 30 minutes.

[0291] 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 Figure 8A. The final affinity (dissociation constant) was 22.6μM.

[0292] 2. Flow Cytometry Experiment

[0293] The two proteins AAVR-AsCas12a-ST-SC-antiTCR and AAVR-AsCas12a-ST obtained 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), and incubated at 4 ° C for 30 minutes with a flow antibody containing 6his (biolegend:antihis-AF647), and the excess antibody was removed by centrifugation. 200μl 1X PBS was resuspended and then tested on the machine. 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.

[0294] The results are shown in Figure 8B. The results show that the red peak is 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.

[0295] 3. In vitro enzyme activity assay of Capa

[0296] 1E10 Capa (- / + CrRNA) cells (one with CrRNA as the experimental group and 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. Samples were run on a 1.5% DNA gel.

[0297] The results are shown in Figure 8C. The results show that Capa can efficiently cut the substrate DNA after adding guide RNA compared with without adding guide RNA.

[0298] Example 4: Adeno-associated virus (Capa) engineered with targeted gene editing enzymes can be directly used for CAR T production

[0299] 1. Capa directly edits primary T cells to generate CD19-targeted CAR T (Capa-CD19-28CAR)

[0300] After peripheral blood T cells from healthy donors were activated with CD3 / 28 antibodies for 48 hours, Capa was incubated with T cells at an MOI of 1E5 (100,000 Capa: 1 T cell) at room temperature for 30 minutes, and then T cell culture medium (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.

[0301] The results are shown in Figure 9A.

[0302] Figure 9A shows the TCR-CD3 complex on the T cell surface on the horizontal axis, and the CD19 CAR expression on the vertical axis. Q1 indicates that the TCR is completely knocked out, and the CD19CAR is precisely knocked in and expressed at the TRAC; Q2 indicates that the T cell expresses both the CD19CAR and the TCR, that is, only one TRAC is knocked in with the CD19CAR, and the other TRAC is intact; Q3, unedited T cells; Q4, T cells with knocked out TCR but no CD19CAR knocked in.

[0303] 2. Capa-CD19-28CAR has better killing activity than Lenti-CD19-28CAR

[0304] The construction of Capa-CD19-28CAR was the same as the previous steps.

[0305] Lenti-CD19-28CAR construction: Peripheral blood T cells from healthy donors were activated with CD3 / 28 antibodies for 48 hours, and lentivirus encoding CD19-28CAR was added at an MOI of 5. After further culture for 3 days, the CAR T preparation efficiency was tested.

[0306] Tumor cells expressing luciferase can emit light when luciferase substrate is added. The intensity of the luminescence can reflect the residual amount of tumor, that is, the killing efficiency.

[0307] 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.

[0308] Cell killing calculation:

[0309] The specific operations are as follows:

[0310] 1) Substrate preparation: Melt the buffer solution in the kit at 4°C or room temperature. Add the entire bottle of melted buffer to the substrate and gently invert 3-5 times to fully dissolve the substrate.

[0311] 2) After co-culture is complete, remove the well plate and gently pipette to evenly distribute the cell suspension. Take 150 μL of the cell suspension from each well and add it to a pre-prepared white 96-well plate. Draw 2-3 replicates from each well. Then add 70 μL of luciferase substrate per well.

[0312] 3) Place a white 96-well plate on an oscillator at 300 rpm / min in the dark for 10 minutes. Detect chemiluminescence using a multifunctional microplate reader with a fixed gain of 100.

[0313] 4) If the cytokine content in the co-culture supernatant is to be detected, the remaining cell suspension is centrifuged, the supernatant is collected, and frozen at -80°C for storage.

[0314] If the target cells are adherent cells:

[0315] Note: Lysate and luciferase substrate must be taken out from -40℃ in advance and thawed at room temperature.

[0316] 5) After co-culture is complete, remove the plate and centrifuge at 4000 rpm for 5 minutes. Carefully aspirate all the co-culture cell suspension and freeze it at -80°C.

[0317] 6) Add 400 μL / well of lysis buffer to the well plate, pipette up and down several times, then place the well plate on an oscillator at 300 rpm / min and shake at room temperature for 10 minutes.

[0318] 7) Centrifuge the plate at 4000 rpm and 4°C for 5 min.

[0319] 8) Pipette 20 μL of supernatant into a pre-prepared white 96-well plate, plate 2-3 times per well, add luciferase substrate (50 μL / well), mix thoroughly, and measure chemiluminescence using a multifunctional microplate reader with a fixed gain of 100.

[0320] The results are shown in Figure 9B.

[0321] 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).

[0322] Example 5: Effect of Optimized Adeno-Associated Virus (Capa) Engineered with Targeted Gene Editing Enzymes (1)

[0323] 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%.

[0324] For example, at an MOI (capa to T cell ratio) of 1E5, capa was incubated with 1E6 T cells (primary human T cells) at 37°C for 30 minutes. TAT-HA2 peptide or protamine sulfate was added at a final concentration of 50 μM and incubated at 37°C for 30 minutes. Finally, the cells were added to X-vivo medium (containing 200 IU of IL-2) containing ADZ7648 at a final concentration of 1 μM. Editing efficiency was measured after 5 days of culture.

[0325] The chemical formula of ADZ7648 (purchased from selleck) is C 18 H 20 N8O2, whose structural formula is shown in the following formula I:

[0326] The results are shown in Figures 10a-d.

[0327] The horizontal axis represents the TCR-CD3 complex on the T cell surface, and the vertical axis represents the expression of CD19 CAR. Q1 indicates that the TCR is completely knocked out, and the CD19CAR is precisely knocked in and expressed at the TRAC; Q2 indicates that the T cell expresses both CD19CAR and TCR, that is, only one TRAC is knocked in with CD19CAR, and the other TRAC is intact; Q3, unedited T cells; Q4, T cells with knocked out TCR but no CD19CAR knocked in.

[0328] The results showed that the addition of TAT-HA2, ADZ7648 and / or protamine could significantly improve the editing efficiency of Capa.

[0329] Example 6: Effect of Optimized Adeno-Associated Virus (Capa) Engineered with Targeted Gene Editing Enzymes (2)

[0330] 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.

[0331] The results are shown in Figure 11.

[0332] The results showed that the addition of TAT-HA2, ADZ7648 and / or protamine could significantly improve the editing efficiency of Capa-Cas9.

[0333] Example 7: Adeno-associated virus (Capa) engineered with targeted gene editing enzymes can be used for multi-gene editing to construct universal CAR T cells (U CAR T)

[0334] Multiple Capa molecules of Cas12aRNP are used to infect T cells (schematic diagram shown in FIG12A ).

[0335] For example, at an MOI (capa to T cell ratio) of 1E5, capa was incubated with 1E6 T cells (primary human T cells) at 37°C for 30 minutes. TAT-HA2 peptide or protamine sulfate was added at a final concentration of 50 μM and incubated at 37°C for 30 minutes. Finally, the cells were added to X-vivo medium (containing 200 IU of IL-2) containing ADZ7648 at a final concentration of 1 μM. Editing efficiency was measured after 5 days of culture.

[0336] The results are shown in FIG12B .

[0337] 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. At the same time, HLA-C / E / G was retained, which can effectively avoid the occurrence of graft versus host disease (GVHD) and host versus graft reaction (HVGR).

[0338] Example 8: Comparison of the killing activity of CAR T cells prepared by adeno-associated virus (Capa) engineered with targeted gene editing enzymes

[0339] Twenty NSG immunodeficient mice, 6 weeks old, female, were purchased from Vital River Company.

[0340] 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. Five days later, the mice became B cell lymphoma models.

[0341] Divide into 4 groups of 5 pieces as follows:

[0342] NC group, Lenti-CD19-28CAR group, Electro-CD 19-28CAR group and Capa-CD19-28CAR (prepared in the previous Example 4).

[0343] On Day 6, 1E6 / mouse of Raji cells expressing CD19 molecules were injected into the tail vein (Raji cells express luciferase. After the luciferase substrate luciferin potassium salt is injected, the Raji tumor status in the mouse can be observed on an imaging instrument).

[0344] 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.

[0345] in:

[0346] 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.

[0347] Preparation process of Electro-CD 19-28CAR: Cas12a-TRAC RNP and 1E6 T cells were placed in an electroporation cuvette, and Cas RNP was electroporated into T cells using Lonza's 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.

[0348] On Day 1 / 4 / 7 / 14 / 19, 200 μl / mouse fluorescein potassium salt solution was injected into the tail vein. After incubation for 5 minutes, the cells were placed in an imaging instrument and imaged for 5 minutes to collect fluorescence signals.

[0349] NC: Infusion of T cells without any treatment.

[0350] The results are shown in Figure 13. In the figure, the higher the fluorescence brightness, the greater the tumor burden.

[0351] The results showed that in several groups of experiments, in descending order of killing activity, Capa-CD19-28CAR>Electro-CD19-28CAR>Lenti-CD19-28CAR.

[0352] Although specific embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and substitutions may be made to those details in light of all the teachings disclosed herein, and such modifications are within the scope of the present invention. The full scope of the present invention is given by the appended 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.

19. 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, for use in 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.

20. 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 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; 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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