CN116515766A

CN116515766A - Natural killer cell, preparation method and application thereof

Info

Publication number: CN116515766A
Application number: CN202310786473.8A
Authority: CN
Inventors: 范文文; 徐天宏
Original assignee: Jikang Technology Zhuhai Co ltd; Shanghai Best Onco Biotechnology Co ltd
Current assignee: Jikang Technology Zhuhai Co ltd; Shanghai Best Onco Biotechnology Co ltd
Priority date: 2023-06-30
Filing date: 2023-06-30
Publication date: 2023-08-01

Abstract

The invention relates to the field of biotechnology, in particular to a natural killer cell subjected to gene editing, a preparation method and application thereof. According to the invention, natural killer cells, namely NK cells, safely and efficiently knock out TGFBR2 genes of the NK cells through a base editing system. The NK cells edited by the base have the effect of efficiently killing tumor cells, are expected to be developed into safe and effective anticancer drugs, and have wide clinical application prospect and development value.

Description

Natural killer cell, preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a natural killer cell subjected to gene editing, a preparation method and application thereof.

Background

Natural killer cells (Natural Killer Cell, NK cells) are one of the important components of the innate immune system of the body, which kill target cells without specific antigen stimulation, and have the functions of immune clearance and immune surveillance. NK is an important innate immune Cell in the human body, also the third type of lymphocyte found following B cells, T cells, is an important component of the innate immune response of the body, and is generally considered as the first line of defense against viral/bacterial infection, by the production of cytokines supporting helper T Cell polarization and T Cell activation, stimulating Dendritic Cell (DC) and B Cell maturation, bridging, coordinating innate and adaptive immune responses.

NK cells are mainly distributed in lymph nodes, spleens and bone marrow in peripheral blood, account for about 15% of peripheral blood lymphocytes, and play roles in immune monitoring, pathogen removal, aging resistance and tumor killing in organisms.

Malignant tumors remain one of the most feared killers threatening human life, and effective therapeutic strategies are urgently needed. Early significant research focused on the study of tumor cells and host cells, however with a continued understanding of tumor progression, tumor microenvironment (Tumor Micro Environment, TME) has been shown to play a key role in tumor progression, invasion and metastasis.

The concept of TME was first proposed by Lord in 1979. This theory symbolically symbolizes the tumor cells as "seeds", and other components (cells and cell matrix) that maintain the growth of tumor cells are called "soil", also called "seeds and soil" theory.

TME is mainly composed of oncolytic components such as tumor-associated fibroblasts (Cancer Associated Fibroblasts, CAFs), tumor blood vessels, M2-type tumor-associated macrophages (Tumor Associated Macrophages, TAMs), regulatory T-cells (Regulatory T cells, treg) cytokines, etc., and tumor-inhibiting components such as Cytotoxic T Lymphocytes (CTL), NK cells, M1-type macrophages, th1 cytokines, etc., and when the oncolytic components are overwhelmingly dominant in number and function, TME assumes a severe immunosuppressive state, which is one of the important causes of various anti-tumor failures including immunotherapy.

NK cells kill target cells by releasing granzyme and perforin or by mediating antibody-dependent cytotoxicity with their Fc-segment receptor, but TGF ⁃ β (Transforming Growth Factor beta, TGF- β) enriched in TME inhibits their killing activity. TGF-beta triggers signaling through TGF-beta type I receptors (TGF-beta RI or ALK 5) and TGF-beta type II receptors (TGF-beta RII), both of which are transmembrane serine/threonine kinase receptors. Upon binding to TGF- β, TGFBR2 recruits and phosphorylates the intracellular domain of TGF- β RI as a high affinity TGF- β receptor, forming heterotetramers, which subsequently activate downstream signaling SMAD proteins. In late stages of the tumor, TGF-beta has a tumorigenic effect by modulating genomic instability, epithelial-to-mesenchymal transition, neoangiogenesis, immune evasion and metastasis. When the pro-tumor function overwhelms the anti-tumor effect of TGF- β signaling in cells, it will act as a tumor promoter in a consistent manner.

TGFBR2 is a receptor to which TGF- β binds directly during this process, and therefore it acts as a gatekeeper for downstream signaling activation. TGFBR2 is a constitutively active independent kinase for ligand binding, phosphorylating itself, TGFBR1 or other receptors. Undoubtedly, the mutation of TGFBR2 can block TGF-beta signal path and reduce the immunosuppression of tumor TME to NK cells, thereby enhancing the tumor killing power and durability of NK cells, enhancing the immune response of tumors and reducing the invasion and migration of tumors, and has good clinical application prospect.

Although classical CRISPR/Cas9 gene editing techniques achieve efficient genetic manipulation of NK cells, genetic manipulation of classical CRISPR/Cas9 gene editing techniques based on DNA double strand breaks can lead to megabase-level chromosomal deletions affecting the integrity and stability of the human cell genome. In addition, double strand breaks can cause p 53-induced apoptosis, and positive edited cells are more prone to enrich for p 53-mutated cells, which clearly increase the risk of cancer when injected into the body. Therefore, the application of safer gene editing tools to the construction of gene editing NK cells with less safety risk is a problem that is currently in need of solution.

Disclosure of Invention

In view of the above-described drawbacks of the prior art, an object of the present invention is to provide a natural killer cell for solving the problems that NK cells subjected to DNA double strand break genetic manipulation in the prior art may cause chromosome deletion on the order of megabases and increase in risk of canceration.

To achieve the above and other related objects, the present invention provides a natural killer cell whose TGFBR2 gene is base-edited to perform gene editing without introducing a DNA double strand break.

Preferably, the base editing mutates base C to T or base A to G on the TGFBR2 gene in natural killer cells.

The invention also provides a preparation method of the natural killer cells, which is to transfer a base editing system into the natural killer cells to prepare the natural killer cells.

The present invention also provides a method of editing TGFBR2 gene by mutating base C to T or mutating base a to G on TGFBR2 gene by the aforementioned base editing system.

The present invention also provides the use of the aforementioned base editing system to mutate base C to T or base A to G on the TGFBR2 gene.

The invention also provides a composition, the main active ingredient of which is the natural killer cell or the base editing system.

The invention also provides a kit comprising the natural killer cell, the base editing system, or the composition.

The invention also provides the use of the natural killer cells, the base editing system, the composition, or the kit, the use selected from one or more of the following:

preparing a medicament for preventing and/or treating autoimmune diseases;

Preparing a medicament for preventing and/or treating tumors;

preparing a medicament for preventing and/or treating viral infectious diseases;

preparing the medicine for preventing and/or treating bacterial infectious diseases.

As described above, the natural killer cell, the preparation method and the application thereof have the following beneficial effects:

the research result of the invention shows that: by using modified sgRNA and base editing fusion protein, the TGFBR2 gene can BE knocked out efficiently by introducing the sgRNA/BE protein complex (RNP) into primary NK cells amplified in vitro by electroporation transfection. Meanwhile, the NK cells (TGFBR 2-NK cells) of the TGFBR2 are knocked out by base editing, and the untargeted DNA and RNA water are not obviously different from the wild type cells, so that the safety of the NK cells for cell therapy products is shown. The NK cells can be utilized to relieve the immunosuppression effect of the TGFBR2 high-expression tumor cells on the NK cells. Compared with wild NK cells, the NK cells of the invention have stronger anti-tumor activity in-vivo and in-vitro experiments. The NK cell can be used as a medicine and can be effectively applied to clinical immunotherapy in time, can provide a new choice for establishing base editing technology and combining with adoptive immunity in the treatment of tumors and virus infectious diseases (such as HIV/AIDS), can also provide support for the research of new effective gene targets, and simultaneously lays a solid technical foundation for the research of related disease treatment, so that the NK cell has obvious application prospect and clinical application value.

Drawings

FIG. 1 shows the editing efficiency corresponding to TGFBR2-sgRNA1-17 of the present invention.

FIG. 2 shows the effect of flow cytometry detection of different RNP mass ratios on editing efficiency according to the present invention.

FIG. 3 shows a comparison of the antitumor activity of different types of NK cells of the present invention.

FIG. 4 shows tumor volume changes following treatment with different types of NK cells of the present invention.

FIG. 5 shows tumor mass changes after treatment with different types of NK cells of the present invention.

FIG. 6 shows DNA off-targeting of base-edited NK for whole genome sequencing analysis of the present invention.

FIG. 7 shows RNA off-target for base-edited NK for full transcriptome sequencing analysis of the present invention.

FIG. 8 shows the electrical conversion procedure used for the Lonza instrument of the invention.

Fig. 9 shows the editing efficiency corresponding to the different Lonza electrical conversion procedures of the present invention.

FIG. 10 shows the cell proliferation fold corresponding to the different Lonza electrical transfer procedures of the present invention.

Fig. 11 shows tumor volumes of tumor-bearing mice of the present invention.

FIG. 12 shows a formula for calculating NK cell killing activity according to the present invention.

Description of the embodiments

The present invention provides a natural killer cell whose TGFBR2 gene is base-edited to perform gene editing without introducing a DNA double strand break.

The natural killer cells are derived from one or more of NK cells derived from peripheral blood cells, NK cells derived from umbilical cord blood, NK cells induced by embryonic stem cells or NK cells induced by induced pluripotent stem cells (ips).

Further, the base C of TGFBR2 gene in the natural killer cell is mutated to T and/or the base A is mutated to G; the mutation of the base C to the T generates a premature stop codon or causes the mutation of an initiation codon or causes the mutation of an intron splice site, and the mutation of the base A to the G causes the mutation of the initiation codon or causes the mutation of the intron splice site; the base C mutations to CAA, CAG, CGA, TGG in the T-occurrence CDS region; specifically, the premature stop codon in the natural killer cells is TAA, TAG, TGA or TGA; the base C is mutated to generate an initiation codon ATG mutation to ATA in T; the base C is mutated to T and the intronic splice sites GT and AG are mutated to AT and AA; the base A is mutated to G to generate an initiation codon ATG mutation to ACG or GTG; the base A is mutated to G and the intron splice site GT, A is mutated to GC, GG.

Further, mutating a base C of a base editing window corresponding to a target point shown in any one of SEQ ID NO.1-SEQ ID NO.17 in a TGFBR2 gene to T, and/or mutating a base A to G; preferably, the nucleotide sequence shown as any one of SEQ ID NO.1-SEQ ID NO.17 on the TGFBR2 gene is mutated to a nucleotide sequence shown as any one of SEQ ID NO. 47-SEQ ID NO. 65.

In the present application, increasing the activity of natural killer cells means increasing the tumor killing power and persistence of natural killer cells, enhancing the immune response of tumors, and reducing invasion and migration of tumors.

The invention also provides a preparation method of the natural killer cells, wherein the preparation method is to introduce a base editing system into the natural killer cells for base editing so as to obtain the natural killer cells.

In some embodiments, the aforementioned base editing system comprises I) a fusion protein or variant thereof or a nucleotide encoding a fusion protein or variant thereof; II) a guide nucleotide.

In some embodiments, the fusion protein or variant thereof is linked from N-terminus to C-terminus to a first nCas9 fragment, a deaminase fragment, and a second nCas9 fragment in sequence. More specifically, the amino acid sequence of the first nCas9 fragment is shown as SEQ ID NO. 39; the amino acid sequence of the second nCas9 fragment is shown as SEQ ID NO. 40.

In some embodiments, the aforementioned first nCas9 fragment and the aforementioned deaminase fragment are linked by a linker peptide a; the aforementioned second nCas9 fragment is linked to the aforementioned deaminase fragment by a linker peptide A; more specifically, the amino acid sequence of the connecting peptide A is shown as SEQ ID NO. 46.

In some embodiments, the fusion proteins described above further comprise one or more of a nuclear localization signal, a Uracil Glycosylase Inhibitor (UGI) fragment, or a GS peptide fragment. More specifically, the amino acid sequence of the nuclear localization signal is shown as SEQ ID NO. 43; the amino acid sequence of the uracil glycosylase inhibitor is shown as SEQ ID NO.44; the GS peptide fragment SEQ ID NO.45 is shown.

Preferably, the fusion protein has a structure of NH2- [ nuclear localization signal ] - [ first nCas9 fragment ] - [ connecting peptide A ] - [ cytosine deaminase fragment ] - [ connecting peptide A ] - [ second nCas9 fragment ] - [ GS peptide fragment ] - [ UGI peptide fragment ] - [ nuclear localization signal ] -COOH or NH2- [ nuclear localization signal ] - [ first nCas9 fragment ] - [ connecting peptide A ] - [ adenine deaminase fragment ] - [ connecting peptide A ] - [ second nCas9 fragment ] - [ GS peptide fragment ] - [ nuclear localization signal ] -COOH.

Further, the amino acid sequence of the fusion protein is shown in any one of SEQ ID No. 18-SEQ ID No. 19; the nucleotide sequence of the encoding fusion protein is shown in any one of SEQ ID No. 20-SEQ ID No. 21.

The nucleotides encoding the fusion proteins and variants thereof are in one or more of DNA or RNA form. The DNA is in the form of one or more of cDNA, genomic DNA, synthetic DNA, cDNA, genomic DNA or synthetic DNA in any single-stranded form; the RNA is in the form of one or more of a coding strand or a non-coding strand.

In some embodiments, the aforementioned fusion proteins and variants thereof are delivered directly into natural killer cells to edit the bases of nucleotides; or in the form of an RNA of the aforementioned nucleotide encoding the fusion protein and variants thereof, which is translated into the fusion protein or variants thereof after delivery into a host cell to edit the bases of the nucleotides; or in the form of a DNA comprising the aforementioned nucleotides encoding the fusion protein and variants thereof, said expression vector comprising a nucleotide sequence encoding one or more genes to express the fusion protein and variants thereof, which is transcribed, translated into said fusion protein or variants thereof to edit the bases of the nucleotides after delivery into a host cell.

In some embodiments, the aforementioned nucleotide sequences encoding the fusion proteins and variants thereof are one or more of the nucleotide sequences encoding only the fusion proteins and variants thereof, the nucleotide sequences encoding the fusion proteins and variants thereof and various additional nucleotide sequences or the nucleotide sequences encoding the fusion proteins and variants thereof and the nucleotide sequences other than the coding sequences; the nucleotide sequence of the aforementioned leader nucleotide is one or more of a nucleotide sequence encoding only the leader nucleotide sequence, a nucleotide sequence encoding the leader nucleotide and various additional nucleotide sequences, a nucleotide sequence encoding the leader nucleotide and non-coding sequences.

In some embodiments, the aforementioned base editing system is an expression vector. The expression vector is one or more. The foregoing expression vector comprises a first regulatory element comprising a nucleotide sequence encoding the foregoing fusion protein or variant thereof, and a second regulatory element; the second regulatory element comprises the nucleotide sequence encoding the leader nucleotide sequence described above. The first regulatory element and the second regulatory element are located on the same or different expression vectors. The first regulatory element and the second regulatory element in the expression vector are one or more.

The first regulatory element regulates transcription of the nucleotide sequence encoding the fusion protein and variants thereof. The first regulatory element has one or more nucleotide sequences encoding the fusion protein and variants thereof. The second regulatory element regulates transcription of the aforementioned nucleotide sequence encoding the leader nucleotide sequence. The second regulatory element has one or more nucleotide sequences encoding the leader nucleotide sequence.

In some embodiments, variants of the foregoing fusion proteins are fragments, derivatives, and analogs of the foregoing fusion proteins, which are proteins in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues are encoded by the genetic code or non-genetic code, or proteins having a substituent group in one or more amino acid residues, or proteins in which an additional amino acid sequence is fused to the protein sequence (e.g., a leader or secretory sequence or a sequence used to purify the protein or a proprotein sequence). The aforementioned fragments, derivatives and analogs fall within the scope of the present invention as defined by those skilled in the art. In some embodiments, the variant of the fusion protein refers to a protein that has 75% or more, or 85% or more, or 90% or more, or 95% or more identity to the amino acid sequence of the aforementioned fusion protein, and that has the same or similar function as the aforementioned fusion protein. The 75% or more identity may be 75%, 80%, 85%, 90% or more than 95% identity; in particular 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. The identity of 90% or more may be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity. By similar function is meant a function that retains 75% or more, or 85% or more, or 90% or more, or 95% or more of the original protein.

In some embodiments, the aforementioned deaminase fragment is a cytosine deaminase fragment or an adenine deaminase fragment. More specifically, the cytosine deaminase is selected from one of apobe 1, apobe 2, apobe 3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, activation-induced deaminase (AID) or pmCDA1One or more species; the adenine deaminase is selected from wild-type ectoda or mutant ectoda ^* One or more of (a) and (b).

Preferably, the cytosine deaminase is apodec 3A; the adenine deaminase is ectoda-ectoda ^* A complex.

In some embodiments, the nucleotide sequence of the target of the aforementioned guide nucleotide is set forth in any one of SEQ ID NO. 1-SEQ ID NO. 17; the nucleotide sequence of the guide nucleotide is shown in any one of SEQ ID NO. 22-SEQ ID NO. 38. More specifically, when the deaminase in the fusion protein and the variant thereof is APOBEC3A, the nucleotide sequence of the target point of the guide nucleotide is shown as any one of SEQ ID NO. 1-SEQ ID NO. 14; the nucleotide sequence of the guide nucleotide is shown in any one of SEQ ID NO. 22-SEQ ID NO. 35. Fusion protein and deaminase in variant thereof as ectoda-ectoda ^* When the target point of the guide nucleotide has a nucleotide sequence shown in any one of SEQ ID NO.11 and SEQ ID NO. 14-SEQ ID NO. 17; the nucleotide sequence of the guide nucleotide is shown as any one of SEQ ID NO. 32, SEQ ID NO. 35-SEQ ID NO. 38.

Preferably, when the fusion protein is a fusion protein with an amino acid sequence shown as SEQ ID No. 18, the nucleotide sequence of the target point of the guide nucleotide is shown as any one of SEQ ID NO. 1-SEQ ID NO. 14; the nucleotide sequence of the guide nucleotide is shown in any one of SEQ ID NO. 22-SEQ ID NO. 35. When the fusion protein is shown as SEQ ID No. 19, the nucleotide sequence of the target point of the guide nucleotide is shown as any one of SEQ ID No.11 and SEQ ID No. 14-SEQ ID No. 17; the nucleotide sequence of the guide nucleotide is shown as any one of SEQ ID NO. 32, SEQ ID NO. 35-SEQ ID NO.38

Preferably, the first 2-4 nucleotides at the 3 'and 5' ends of the aforementioned guide nucleotide are thio-and/or methoxy-modified nucleotides.

In some embodiments, the mass ratio of the guide nucleotide to the fusion protein or variant thereof in the editing system is 1 (2-20); more specifically, the mass ratio is 1 (2-4), 1 (2-6), 1 (2-8), 1 (2-10), 1 (2-12), 1 (2-14), 1 (2-16), 1 (2-18), 1 (2-20), 1: (4-6), 1 (4-8), 1 (4-10), 1 (4-12), 1 (4-14), 1 (4-16), 1 (4-18), 1 (4-20), 1 (8-10), 1 (8-12), 1 (8-14), 1 (8-16), 1 (8-18), 1 (8-20), 1 (10-12), 1 (10-14), 1 (10-16), 1 (10-18), 1 (10-20), 1 (12-14), 1 (14-16), 1 (16-18) or 1 (18-20). Preferably, the mass ratio is 1 (2-8). More preferably, the mass ratio is 1: (4-6).

In some embodiments, the method of the foregoing introduction is selected from one or more of electroporation, viral transduction, microinjection, particle bombardment, or gene gun transformation. More specifically, the method of introduction is electroporation.

Preferably, the electroporation system used in the foregoing electroporation method is selected from one or more of a LONZA system, a Thermo Neon transfection system, or a CTS Xenon electroporation system.

Further, when the electrotransport device system used in the electroporation method is a LONZA system, and the electrotransport device is a 4D-Nucleofector, the electrotransport program is selected from CM137, CM158 or CM189;

or the electrotransfection system used in the electroporation method is a Thermo electrotransfection system, a model Neon electrotransfection apparatus or a CTS Xenon electrotransfection apparatus, and the electrotransfection procedure is selected from any one of the following procedures:

1) Voltage 1650-1750v, pulse width 9-11ms, pulse times 1-3;

2) Voltage 1750-1850v, pulse width 9-11ms, pulse times 1-3;

3) Voltage 2150-2250v, pulse width 2-4ms, pulse number 3-5;

4) The voltage is 1550-1650v, the pulse width is 7-9ms, and the pulse number is 2-4.

The present invention also provides a method of editing TGFBR2 gene by mutating base C to T or mutating base a to G on TGFBR2 gene by a base editing system. The method for editing the TGFBR2 gene is a gene editing method of in vitro cells. The method for editing TGFBR2 gene is a non-therapeutic method or a non-diagnostic method.

In some embodiments, the mutation of base C to T, or the mutation of base a to G occurs in the coding nucleotide sequence of the TGFBR2 gene described previously; the mutation of C to T, or the mutation of base A to G, results in a mutation in the TGFBR2 gene.

Further, the mutation of the TGFBR2 gene is a loss-of-function mutation or a non-coding mutation. Specifically, the loss-of-function mutation is a splice site mutation that introduces a premature stop codon or introns in the TGFBR2 gene, which results in truncated or nonfunctional TGFBR2 protein production; or the non-coding mutation is by mutating the start codon ATG of the TGFBR2 gene, which results in elimination of TGFBR2 gene expression.

Further, the premature stop codon is TAA, TAG, TGA or TGA. For example, the early stop codon is generated via deamination of the first C on the coding strand from CAA to TAA conversion; the premature stop codon is generated by conversion of CAG to TAG via deamination of the first C on the coding strand; the premature stop codon is generated via deamination of the first C on the coding strand from CGA to TGA conversion; the premature stop codon is generated from TGG to TGA conversion via deamination of the third C on the complementary strand; the initiation codon mutation is generated by conversion from ATG to ATA via deamination of the third C on the complementary strand; the initiation codon mutation is generated by conversion of ATG to ACG via deamination of the second A on the complementary strand; the start codon mutation is generated by conversion of ATG to GTG via deamination of the first A on the coding strand; the intron splice site mutation is converted from CA to TA or TC to TT by deamination of C on the complementary strand; the intron splice site mutation is converted from CA to CG by deamination of A on the complementary strand; the intron splice site mutation is converted from AG to GG by deamination of A on the coding strand.

In some embodiments, the number of nucleotides of the mutation of the TGFBR2 gene is 1-17. More specifically, the number of the mutated nucleotides is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.

The present invention also provides a composition containing the base editing system or the natural killer cell as an active ingredient. Further, the composition further comprises a pharmaceutically acceptable carrier. Such as sterile or normal saline, stabilizers, excipients, antioxidants (ascorbic acid), buffers (phosphoric acid, citric acid, other organic acids), preservatives, surfactants (PEG, tween), chelating agents (EDTA), binders, and the like. Furthermore, low molecular weight polypeptides may also be included; serum albumin, gelatin or immunoglobulins; glycine, glutamine, asparagine, arginine or lysine; polysaccharides or monosaccharides; mannitol or sorbitol. When preparing an aqueous solution for injection, for example, physiological saline, isotonic solution containing glucose or other auxiliary drugs, such as D-sorbitol, D-mannose, D-mannitol, sodium chloride, an appropriate solubilizing agent such as alcohol (ethanol), polyol (propylene glycol, PEG), nonionic surfactant (Tween 80, HCO-50) may be used in combination.

The invention also provides a kit comprising the aforementioned base editing system, the aforementioned natural killer cells, or the aforementioned composition.

The invention also provides the use of the aforementioned base editing system, the aforementioned natural killer cells, the aforementioned composition, or the aforementioned kit, selected from one or more of the following:

preparing a medicament for preventing and/or treating autoimmune diseases;

preparing a medicament for preventing and/or treating tumors;

Wherein the autoimmune disease is selected from one or more of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, lupus nephritis, neuromyelitis optica, systemic sclerosis, dry mouth syndrome, polymyositis; .

Wherein the tumor is selected from lymphoma, hematological tumor or solid tumor; preferably, one or more selected from adrenocortical carcinoma, bladder urothelial carcinoma, breast carcinoma, cervical squamous cell carcinoma, cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid tumor, diffuse large B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, acute myelogenous leukemia, brain low glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian carcinoma, pancreatic carcinoma, pheochromocytoma and paraganglioma, prostate carcinoma, rectal carcinoma, malignant sarcoma, melanoma, gastric carcinoma, testicular germ cell tumor, thyroid carcinoma, thymus carcinoma, endometrial carcinoma, uterine sarcoma, uveal melanoma, multiple myeloma, acute gonomic leukemia, chronic myelogenous leukemia, T-cell lymphoma, B-cell lymphoma, lung carcinoma, anal carcinoma, intraocular melanoma, retinoblastoma. Preferably, the lung cancer is non-small cell lung cancer.

Wherein the virus is selected from one or more of influenza virus, parainfluenza virus, measles virus, mumps virus, herpes virus, adenovirus, respiratory syncytial virus, polio virus, coxsackie virus, or epstein barr virus.

Wherein the bacteria are selected from one or more of escherichia coli, lactobacillus casei, bacteroides fragilis, acinetobacter rouxii, fusobacterium nucleatum, bacteroides johnsonii, arabidopsis thaliana, lactobacillus rhamnosus, bacteroides massiliensis, bacteroides ovatus, campylobacter jejuni, staphylococcus saprophyticus, enterococcus faecalis, bacteroides thetaiotaomicron, bacteroides vulgare, bacteroides simplex, parabacteroides faecalis, fusobacterium mortiferum and bifidobacterium breve.

The present invention also provides a method of preventing and/or treating a disorder, the method comprising administering to a subject in need thereof a therapeutically effective amount of the aforementioned base editing system, the aforementioned natural killer cell, or the aforementioned composition; the condition is selected from one or more of the following: autoimmune diseases, tumors, viral infectious diseases, bacterial infectious diseases.

In the present invention, the aforementioned base editing system, the aforementioned natural killer cells, or the aforementioned composition may also be used in combination with other drugs. In some embodiments, the unedited natural killer cell is from a subject in need thereof. In some embodiments, the methods further comprise administering one or more cytokines, including, but not limited to, IL-2 and/or IL-15, and the like, to a subject in need thereof. The aforementioned natural killer cells subjected to base editing are more sensitive to cytokine stimulation and exhibit improved expansion, antitumor function, antiviral function, and the like, as compared with natural killer cells.

In the composition or use provided by the invention, the base editing system or the natural killer cells are single active ingredients or are combined with other active ingredients to form a combined preparation. The other active component can be other various medicines for treating autoimmune diseases, tumors, virus infection diseases and bacterial infection diseases. The content of the active ingredient in the composition should be generally a safe and effective amount which should be adjustable to those skilled in the art, for example, the amount of the active ingredient to be applied is generally dependent on the body weight of the patient, the type of application, the condition and severity of the disease, for example, the amount of the base editing system or NK cells to be applied as the active ingredient may be generally 1 to 1000 mg/kg/day, 20 to 200mg/kg/day, 1 to 3 mg/kg/day, 3 to 5 mg/kg/day, 5 to 10 mg/kg/day, 10 to 20 mg/kg/day, 20 to 30 mg/kg/day, 30 to 40 mg/kg/day, 40 to 60 mg/kg/day, 60 to 80 mg/kg/day, 80 to 100 mg/kg/day, 100 to 150 mg/kg/day, 150 to 200mg/kg/day, 200 to 300 mg/kg/day, 300 to 500 mg/kg/day, or 500 mg/day.

As used herein, "gene delivery," "gene transfer," "transduction," "transfer," and the like, refer to the introduction of an exogenous polynucleotide into a host cell, such as vector-mediated gene transfer (by, for example, viral infection/transfection, or various other protein-based or lipid-based gene delivery complexes) and techniques that facilitate delivery of a "naked" polynucleotide (such as electroporation, "gene gun" delivery and various other techniques for introducing polynucleotides). The introduced polynucleotide may be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide comprise an origin of replication compatible with the host cell or a replicon incorporated into the host cell, such as an extrachromosomal replicon (e.g., plasmid) or a nuclear or mitochondrial chromosome. Many "vectors" are known to be capable of mediating the transfer of genes to mammalian cells, as known in the art and described herein.

As used herein, the dosage form of the pharmaceutical composition is selected from: injection, sterile powder for injection, tablet, pill, capsule, lozenge, spirit, powder, granule, syrup, solution, tincture, aerosol, powder spray, or suppository. The skilled artisan can select a suitable formulation depending on the mode of administration, for example, a formulation suitable for oral administration can be a formulation including, but not limited to, pills, tablets, chews, capsules, granules, solutions, drops, syrups, aerosols or powder sprays and the like.

In the methods and uses of the invention, the active ingredient is co-administered with other therapeutic agents when used in combination therewith. By "co-administration" is meant simultaneous administration via the same or different routes, or sequential administration via the same or different routes, in the same formulation or in two different formulations. "sequential" administration means that there is a time difference in seconds, minutes, hours or days between administration of two or more different compounds.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.

As used herein, identity may be assessed with the naked eye or in computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to evaluate the identity between related sequences.

As used herein, "comprising," "including," and the like are to be construed as inclusive and not exclusive or exhaustive; i.e., the meaning of "including but not limited to".

As used herein, a "therapeutically effective amount" generally refers to an amount that, after a suitable period of administration, achieves the effect of treating the diseases listed above.

As used herein, "therapeutic" and "prophylactic" are to be understood in their broadest sense. The term "therapeutic" does not necessarily imply that the mammal is treated until complete recovery. Similarly, "prophylactic" does not necessarily mean that the subject will not ultimately be infected with a disease condition. Thus, treatment and prevention includes alleviation of symptoms of a particular disorder or prevention or reduction of risk of developing a particular disorder. The term "preventing" is understood to mean reducing the severity of the onset of a particular disorder. Treatment may also reduce the severity of existing conditions or the frequency of episodes.

As used herein, a subject or individual undergoing therapeutic or prophylactic treatment is preferably a mammal, such as, but not limited to, a human, primate, livestock (e.g., sheep, cow, horse, donkey, pig), companion animal (e.g., dog, cat), laboratory test animal (e.g., mouse, rabbit, rat, guinea pig, hamster) or wild animal (e.g., fox, deer) that is captured. The subject is preferably a primate. The subject is most preferably a person.

As used herein, the terms "nucleic acid" and "nucleic acid component" are used interchangeably to refer to a compound having a nucleobase and an acidic moiety, such as a nucleoside, nucleotide, or a polymer of nucleotides. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (e.g., nucleotide and/or nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more nucleotide residues. The terms "oligonucleotide" and "polynucleotide" are used interchangeably herein to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, "nucleic acid" includes RNA as well as single-and/or double-stranded DNA. The nucleic acid may be a naturally occurring or non-naturally occurring molecule.

As used herein, the term "expression" refers to the process by which a polynucleotide is transcribed into mRNA and/or the process by which the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of mRNA in eukaryotic cells. The expression level of a gene can be determined by measuring the amount of mRNA or protein in a cell or tissue sample.

The terms "protein," "peptide" and "polypeptide" are used interchangeably and in their broadest sense to refer to a compound of amino acids, amino acid analogs, or peptidomimetics of two or more subunits. The subunits may be linked by peptide bonds. In another aspect, the subunits may be linked by other linkages, e.g., esters, ethers, and the like. The protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids constituting the protein or peptide sequence. Proteins and peptides are known to have a C-terminus, which refers to the presence of an unbound carboxyl group at the terminal amino acid, and an N-terminus, which refers to the presence of an unbound amino group at the terminal amino acid. The term "amino acid" as used herein refers to natural and/or unnatural or synthetic amino acids, including glycine, as well as D and L optical isomers, amino acid analogs and peptidomimetics. The term "fusion" in the context of a protein or polypeptide refers to the attachment of two or more protein or polypeptide (or domains thereof) ends that form a fusion protein.

Before the embodiments of the invention are explained in further detail, it is to be understood that the invention is not limited in its scope to the particular embodiments described below; it is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention; in the description and claims of the invention, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Where numerical ranges are provided in the examples, it is understood that unless otherwise stated herein, both endpoints of each numerical range and any number between the two endpoints are significant both in the numerical range. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, materials used in the embodiments, any methods, devices, and materials of the prior art similar or equivalent to those described in the embodiments of the present invention may be used to practice the present invention according to the knowledge of one skilled in the art and the description of the present invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed in the present invention employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA techniques, and related arts. These techniques are well described in the prior art.

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

The nucleotide or amino acid sequences used in this application are as follows:

SEQ ID NO .1

ACGTTCAGAAGTCGGGTGAG

SEQ ID NO .2

GAAGCCACAGGAAGTCTGTG

SEQ ID NO .3

TTCAGAGCAGTTTGAGACAG

SEQ ID NO .4

ACTCCAGTTCCTGACGGCTG

SEQ ID NO .5

ACCTACAGGAGTACCTGACG

SEQ ID NO .6

GCAGACCGATGTCTACTCCA

SEQ ID NO .7

GTCCACAGGACGATGTGCAG

SEQ ID NO .8

TTCCCAGAGCACCAGAGCCA

SEQ ID NO .9

AGCCAGAAGCTGGGAATTTC

SEQ ID NO .10

CCAAGAGGCATACTCCTCAT

SEQ ID NO .11

CTCACCCGACTTCTGAACGTG

SEQ ID NO .12

CCTAGAGTGAAGAGATTCAT

SEQ ID NO .13

TCTGATGGGGAAACAAAACA

SEQ ID NO .14

TTACCTGCCCACTGTTAGCC

SEQ ID NO .15

CATGGGTCGGGGGCTGCTCA

SEQ ID NO .16

CCATGGGTCGGGGGCTGCTC

SEQ ID NO .17

ATGGGTCGGGGGCTGCTCAG

SEQ ID NO .18

MKRTADGSEFESPKKKRKVSSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKSGSETPGTSESATPESGSMEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIFDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGNSGSESGSGSETPGTSESATPESETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSGGSGGSTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKMLSGGSKRTADGSEFEPKKKRKV

SEQ ID NO .19

MKRTADGSEFESPKKKRKVSSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSKRTADGSEFEPKKKRKV

SEQ ID NO .20

ATGAAAAGGACAGCTGATGGATCAGAATTTGAGTCACCGAAGAAAAAGCGCAAGGTCAGCAGCGACAAAAAGTACTCGATTGGCCTGGCGATTGGTACGAATTCTGTCGGTTGGGCGGTCATCACGGATGAGTACAAGGTCCCGAGCAAGAAATTCAAAGTGCTGGGTAATACAGATCGTCACAGCATCAAAAAAAACTTAATTGGTGCACTGCTGTTCGATAGCGGTGAAACCGCGGAAGCTACCCGCCTGAAGCGTACCGCGCGTCGTCGTTACACCCGTCGCAAGAACCGCATTTGTTATCTGCAAGAAATATTCAGCAACGAAATGGCAAAGGTTGACGACAGCTTTTTTCATCGTCTGGAGGAGTCCTTCCTTGTGGAAGAGGACAAAAAGCACGAACGTCATCCGATCTTTGGTAACATTGTCGACGAAGTGGCATATCACGAAAAGTACCCGACCATCTATCATCTGAGAAAGAAGTTGGTTGACAGCACGGATAAGGCAGATCTGCGTCTGATTTACTTGGCTCTGGCGCACATGATTAAGTTTCGTGGTCATTTTCTGATCGAGGGTGATCTGAATCCGGATAACAGTGACGTTGACAAGCTTTTCATTCAGCTTGTGCAGACCTACAACCAGTTGTTCGAGGAAAACCCGATCAACGCCAGCGGGGTGGACGCGAAGGCGATTCTAAGCGCGCGTCTGTCCAAGAGCCGCCGTTTGGAGAACCTGATCGCTCAGTTGCCAGGTGAGAAGAAGAATGGCTTGTTCGGCAACCTCATCGCGCTGTCACTGGGTCTGACGCCGAATTTCAAATCAAACTTCGATTTAGCGGAGGATGCGAAACTGCAGCTGTCCAAAGATACCTATGATGATGACCTTGATAATCTCCTGGCCCAAATTGGCGACCAGTATGCAGATTTGTTCCTTGCCGCCAAGAACCTCTCTGACGCGATCCTGCTGTCTGATATCCTGCGCGTGAATACCGAAATAACCAAAGCCCCTCTCTCTGCGAGCATGATTAAACGTTACGACGAGCATCACCAAGATCTGACACTGCTCAAGGCCTTGGTGCGTCAGCAGCTACCGGAGAAGTATAAAGAGATCTTTTTCGACCAAAGCAAGAACGGCTACGCGGGTTATATCGATGGCGGCGCATCTCAAGAAGAGTTTTATAAGTTCATCAAGCCGATCCTCGAGAAAATGGACGGCACCGAGGAACTACTGGTCAAACTGAACCGAGAGGATTTATTACGTAAGCAACGTACCTTTGATAACGGCTCGATTCCGCATCAGATTCACCTGGGCGAGCTGCACGCCATTTTGCGCCGTCAGGAGGATTTCTATCCGTTTCTTAAAGACAACCGTGAAAAAATCGAAAAAATCTTGACTTTTCGTATCCCTTATTACGTGGGTCCGCTGGCCCGTGGCAATAGCCGCTTCGCGTGGATGACCCGTAAATCCGAAGAAACCATCACCCCGTGGAACTTTGAAGAAGTGGTTGACAAAGGAGCGTCAGCGCAATCCTTTATCGAGCGTATGACCAACTTCGATAAGAACCTGCCTAATGAAAAGGTGCTCCCGAAGCACAGCTTATTGTACGAATACTTTACCGTTTATAATGAACTGACGAAAGTGAAATACGTTACCGAGGGTATGCGTAAGCCGGCTTTTTTATCCGGCGAGCAGAAGAAGGCGATTGTTGACTTGCTGTTTAAAACCAACCGTAAAGTGACCGTGAAACAATTAAAGGAGGACTATTTCAAAAAAATTGAGTGCTTTGACTCTGTTGAGATCTCCGGGGTGGAAGATCGCTTTAACGCGAGCCTTGGCACCTATCATGATCTGCTGAAAATCATCAAGGACAAAGATTTTTTGGATAACGAGGAGAATGAAGACATTCTGGAGGACATTGTACTGACCCTGACGCTGTTCGAAGATCGTGAAATGATAGAAGAGCGTCTCAAAACCTATGCACATCTGTTTGACGACAAGGTTATGAAACAGTTGAAGCGTCGGCGCTACACCGGCTGGGGTCGCCTGTCTCGTAAATTGATCAATGGCATTCGCGACAAGCAGAGCGGCAAGACGATCCTGGACTTCCTGAAATCCGACGGTTTCGCGAATCGTAATTTTATGCAGTTGATCCACGACGACAGCCTGACCTTTAAAGAAGATATCCAAAAAGCTCAGGTTAGCGGCCAGGGTGACTCTCTTCACGAGCACATTGCAAATCTGGCCGGCAGCCCGGCAATTAAAAAAGGTATCTTGCAAACCGTTAAAGTGGTAGACGAGCTGGTGAAGGTGATGGGTAGGCACAAGCCGGAAAACATCGTGATCGAAATGGCTCGTGAAAATCAAACCACCCAGAAAGGCCAGAAGAACTCTCGTGAGCGCATGAAACGTATTGAAGAAGGTATCAAGGAGTTGGGCAGCCAAATTTTGAAGGAGCACCCGGTTGAAAACACCCAGCTGCAAAACGAGAAACTGTACTTGTACTATCTGCAAAACGGTAGAGATATGTATGTTGATCAAGAGCTCGACATCAACCGTTTGAGTGACTATGACGTTGACCACATCGTTCCGCAATCGTTCCTGAAAGACGACTCCATCGATAACAAAGTCTTAACCCGTAGCGATAAAAACCGCGGCAAGAGCGATAACGTGCCGTCCGAAGAAGTCGTCAAGAAAATGAAGAACTATTGGCGTCAGCTGCTGAATGCGAAGCTGATAACCCAGCGAAAGTTCGACAATCTGACCAAGGCGGAGCGTGGCGGCCTGTCGGAATTGGACAAAGCGGGCTTCATCAAACGCCAACTGGTCGAAACCCGTCAGATCACCAAACACGTTGCCCAGATCCTGGATTCCCGTATGAATACCAAGTACGATGAGAATGATAAGCTCATTCGTGAAGTTAAAGTGATTACGCTGAAATCTAAACTGGTGAGCGACTTCCGTAAGGATTTTCAGTTCTATAAAGTTCGCGAGATCAACAATTATCACCATGCCCACGATGCCTACTTGAACGCGGTGGTAGGTACGGCCCTGATCAAGAAGTACCCGAAGCTTGAGTCGGAGTTCGTGTACGGCGATTACAAAGTTTATGACGTTCGCAAAATGATTGCTAAGAGCGAGCAAGAAATCGGCAAGGCGACCGCGAAGTATTTTTTCTACTCTAACATCATGAACTTTTTCAAGTCTGGCTCGGAAACTCCGGGTACCAGTGAGTCGGCGACTCCCGAATCGGGGTCTATGGAAGCAAGTCCGGCTTCAGGACCGCGTCACCTGATGGACCCGCATATCTTCACCTCTAACTTCAATAACGGTATCGGCCGTCACAAGACCTACCTGTGCTACGAGGTCGAACGTCTGGATAACGGAACCAGCGTAAAGATGGACCAGCACCGCGGCTTCCTGCATAATCAGGCGAAGAACCTGCTGTGTGGTTTCTACGGTCGTCACGCAGAATTGCGTTTTCTGGACCTTGTACCGAGCCTCCAATTGGACCCGGCGCAAATCTATCGTGTGACCTGGTTCATAAGCTGGAGCCCTTGTTTTAGCTGGGGCTGCGCAGGCGAAGTTCGCGCATTTCTGCAGGAGAACACGCATGTTCGTCTGAGAATCTTCGCCGCGCGTATCTTTGACTATGACCCGCTGTACAAAGAGGCTCTTCAGATGCTGCGTGATGCGGGCGCGCAAGTGAGCATCATGACATACGACGAATTCAAGCACTGCTGGGACACCTTCGTGGATCATCAAGGTTGCCCGTTTCAGCCGTGGGACGGTCTGGACGAACATAGCCAGGCGTTGAGCGGACGTCTCCGTGCTATTCTGCAAAACCAGGGTAATAGCGGCTCCGAATCGGGTAGCGGTAGTGAAACTCCGGGTACATCGGAGAGCGCAACCCCGGAATCTGAAACGAACGGTGAAACAGGTGAGATCGTTTGGGATAAGGGCAGAGATTTTGCTACCGTTCGTAAAGTCTTAAGCATGCCGCAGGTTAATATTGTGAAAAAAACCGAGGTGCAGACCGGCGGTTTTAGCAAAGAGAGCATTCTGCCAAAGCGTAATAGCGACAAACTGATTGCACGTAAGAAGGACTGGGACCCGAAGAAATACGGCGGTTTTGATTCACCAACCGTTGCGTACAGCGTATTGGTGGTTGCTAAAGTGGAGAAGGGCAAGAGCAAGAAATTGAAAAGCGTTAAAGAGCTGCTGGGCATCACGATTATGGAAAGAAGCAGCTTCGAGAAGAATCCGATCGACTTCCTGGAGGCCAAAGGTTATAAAGAGGTAAAAAAGGATTTGATCATTAAGTTGCCAAAATATTCTCTATTCGAATTGGAAAACGGTCGCAAACGCATGCTGGCGTCTGCGGGCGAACTGCAAAAAGGTAACGAGCTGGCGCTGCCAAGCAAGTACGTCAACTTTTTGTACTTGGCGAGTCATTATGAAAAGCTGAAAGGTAGCCCGGAGGACAACGAGCAAAAACAACTGTTCGTTGAACAGCATAAACACTATCTGGACGAAATCATTGAGCAGATCAGCGAGTTTAGCAAGCGCGTTATTCTCGCTGACGCGAATCTGGACAAGGTTCTATCAGCGTATAACAAACATCGTGATAAACCGATTCGTGAACAAGCAGAGAACATCATCCATTTGTTCACCCTAACGAACCTGGGTGCGCCGGCGGCATTTAAATACTTCGATACGACCATTGATCGGAAGCGTTACACTAGCACCAAGGAAGTTCTGGACGCTACCCTCATCCACCAGAGCATCACGGGCCTGTACGAGACGCGCATCGACCTCAGCCAGTTAGGTGGTGACAGCGGTGGTTCCGGTGGTTCTGGCGGTAGCACCAATTTGAGCGATATTATCGAGAAGGAAACTGGCAAGCAATTAGTGATCCAGGAATCCATTCTGATGCTGCCGGAAGAAGTGGAAGAGGTTATTGGCAACAAGCCGGAAAGCGATATCCTGGTGCACACCGCTTACGACGAATCCACGGATGAGAACGTGATGCTGTTGACCAGCGACGCTCCGGAATATAAGCCGTGGGCACTGGTGATTCAGGATTCCAATGGCGAGAACAAGATTAAGATGCTGTCCGGTGGCTCCGGTGGCTCCGGCGGGTCGACGAACCTGTCCGACATTATCGAGAAAGAAACGGGTAAACAGTTAGTGATTCAAGAGTCCATTCTGATGCTCCCAGAGGAGGTTGAAGAGGTAATCGGTAACAAACCGGAAAGCGATATTCTAGTGCATACCGCTTACGACGAAAGCACCGATGAAAACGTTATGTTGCTGACTTCAGATGCGCCAGAATATAAGCCGTGGGCACTGGTTATTCAAGACAGTAACGGCGAAAATAAGATTAAGATGCTGTCTGGTGGTTCCAAAAGGACAGCCGATGGCTCCGAATTCGAACCAAAAAAAAAGAGAAAGGTT

SEQ ID NO .21

ATGAAAAGGACAGCTGATGGATCAGAATTTGAAAGCCCGAAAAAGAAGCGCAAAGTGAGCTCGGACAAGAAATACAGCATCGGGCTGGCGATTGGTACCAACAGCGTGGGTTGGGCAGTTATTACCGATGAATACAAAGTGCCGAGCAAAAAGTTCAAAGTTTTGGGCAATACCGATCGGCACAGCATTAAGAAGAACCTGATTGGCGCTTTACTCTTCGATTCCGGTGAGACGGCCGAGGCTACCCGTCTCAAGCGTACCGCTCGTCGTCGCTATACCCGTCGTAAGAACCGTATTTGTTATTTGCAGGAGATCTTTAGCAATGAAATGGCGAAGGTGGACGACTCCTTCTTTCACCGCCTGGAAGAGTCCTTCCTTGTCGAGGAAGATAAAAAGCACGAACGTCACCCGATCTTTGGTAACATTGTCGACGAAGTGGCCTATCATGAGAAATACCCGACCATTTATCATCTGCGTAAGAAGCTAGTGGACAGCACCGATAAAGCAGATCTGCGTTTGATCTATCTTGCGCTGGCACACATGATTAAGTTTCGTGGTCATTTTCTGATTGAGGGCGACCTGAATCCTGATAATAGCGACGTTGACAAACTCTTCATCCAGCTGGTTCAGACGTATAACCAGTTGTTCGAGGAAAACCCGATTAACGCGAGCGGTGTTGATGCGAAAGCGATTCTGAGTGCCCGTCTGAGCAAAAGCCGACGCCTGGAGAACCTGATCGCGCAACTGCCGGGTGAAAAGAAGAACGGTCTGTTCGGCAATCTGATCGCGCTGTCTCTGGGTCTGACGCCGAACTTCAAGTCTAACTTCGATCTCGCGGAAGACGCCAAATTACAGCTGTCCAAGGACACCTACGACGATGATCTGGACAACCTGTTGGCGCAGATTGGCGACCAATATGCTGACCTGTTTCTGGCTGCGAAAAACCTGTCCGACGCCATCTTGCTGAGCGACATTTTGAGAGTGAACACCGAGATCACCAAAGCGCCTCTGTCTGCAAGCATGATCAAGCGTTATGATGAGCATCACCAAGATTTGACTTTGCTCAAGGCTTTGGTTCGTCAGCAGTTGCCGGAGAAATATAAGGAAATCTTTTTCGATCAAAGTAAAAATGGTTATGCAGGCTACATTGATGGTGGTGCGAGCCAAGAAGAATTTTACAAGTTTATCAAACCGATTCTGGAGAAGATGGACGGTACTGAAGAGCTGCTGGTTAAATTGAACCGAGAAGATCTACTACGTAAGCAACGTACGTTCGACAACGGCTCCATCCCGCATCAGATCCACCTGGGTGAGCTGCACGCTATCCTGCGTCGGCAAGAGGATTTTTACCCGTTTCTGAAGGATAATCGTGAAAAGATTGAAAAGATTTTGACGTTTCGTATTCCGTATTACGTGGGTCCGCTGGCGCGGGGCAACTCGCGTTTCGCCTGGATGACCAGAAAGTCTGAAGAAACGATCACACCGTGGAACTTCGAGGAAGTAGTGGATAAAGGTGCCAGCGCCCAAAGCTTTATCGAGCGTATGACCAACTTCGACAAGAACCTGCCAAATGAAAAAGTGTTGCCAAAACATAGCCTGTTATATGAATACTTCACCGTTTATAACGAGTTAACCAAAGTCAAGTACGTGACCGAGGGTATGCGGAAGCCAGCGTTTCTATCAGGAGAGCAAAAGAAGGCGATTGTCGACCTGTTATTCAAAACCAACCGCAAAGTCACCGTTAAGCAACTTAAAGAAGATTACTTCAAGAAGATCGAATGTTTTGACAGCGTCGAGATCAGCGGTGTTGAAGACCGCTTTAATGCCAGCCTGGGTACCTATCATGACTTGTTGAAAATCATTAAGGATAAGGACTTTCTGGATAATGAGGAGAACGAGGATATCCTGGAGGACATCGTTTTGACCTTAACTCTGTTCGAGGACCGTGAAATGATCGAGGAACGACTGAAGACGTACGCGCATCTGTTCGACGACAAAGTTATGAAACAACTTAAGCGTCGCCGTTACACCGGTTGGGGTCGCTTGTCTCGCAAACTGATAAACGGCATCCGTGACAAACAGTCCGGAAAAACGATCCTAGATTTCCTGAAGAGCGACGGCTTCGCGAATCGTAACTTTATGCAGTTGATCCACGACGACTCGTTGACCTTTAAAGAAGATATCCAGAAGGCACAGGTTAGCGGACAGGGTGATTCGCTGCACGAGCACATTGCGAACTTGGCTGGTTCGCCGGCGATCAAGAAGGGTATTTTACAAACCGTGAAAGTGGTGGATGAGTTGGTCAAAGTGATGGGTCGTCACAAACCGGAAAACATTGTCATCGAAATGGCGCGCGAAAACCAAACGACCCAGAAGGGTCAAAAAAACTCTCGTGAGCGCATGAAACGTATCGAAGAGGGCATTAAAGAATTAGGCTCTCAGATTCTGAAAGAGCACCCGGTGGAAAACACCCAGTTACAGAATGAGAAACTTTACCTGTACTATCTGCAGAATGGTCGTGATATGTATGTTGATCAGGAACTGGACATTAACCGTCTGTCCGATTACGACGTGGACCATATTGTTCCGCAAAGCTTTCTGAAGGACGATTCAATCGACAACAAAGTCCTGACACGTAGCGACAAGAATCGTGGTAAGAGCGATAATGTTCCGAGCGAGGAAGTGGTTAAAAAGATGAAAAACTACTGGCGTCAACTGTTGAATGCAAAACTGATCACCCAGAGGAAATTCGACAACCTGACGAAGGCGGAACGTGGTGGCTTGTCAGAGTTGGATAAGGCTGGCTTCATCAAGCGCCAATTGGTCGAGACTCGTCAAATTACCAAACATGTTGCCCAAATTCTGGACTCCCGTATGAACACGAAATACGACGAGAACGATAAGCTCATCCGTGAGGTTAAAGTCATTACGCTGAAAAGTAAGCTGGTTAGTGACTTCCGCAAGGACTTCCAGTTTTACAAAGTTAGGGAGATCAACAACTATCATCACGCACACGATGCGTATCTTAATGCCGTTGTTGGCACCGCACTGATTAAAAAATACCCGAAATTGGAAAGCGAGTTTGTTTATGGCGACTATAAGGTGTATGACGTGCGCAAGATGATTGCTAAATCAGAACAGGAGATCGGTAAGGCGACTGCGAAATACTTCTTCTACAGCAATATCATGAATTTTTTCAAGTCCGGGGGTAGCAGTGGTGGCAGCTCCGGTTCTGAGACCCCGGGCACCTCCGAGTCTGCTACCCCGGAGAGTAGCGGCGGCTCCAGCGGTGGCAGCAGCGAGGTAGAGTTCAGCCACGAATACTGGATGCGTCACGCGCTGACCCTGGCCAAACGTGCATGGGACGAACGTGAAGTTCCGGTGGGCGCTGTGCTGGTGCATAACAACCGTGTGATCGGCGAGGGTTGGAATAGACCTATCGGCCGTCATGACCCTACGGCGCACGCTGAGATTATGGCCCTGCGTCAGGGTGGCCTGGTGATGCAGAACTACCGCCTGATCGATGCGACACTCTACGTTACCTTGGAACCGTGCGTCATGTGTGCCGGTGCAATGATTCACAGCCGTATCGGTCGTGTTGTATTTGGCGCGCGTGATGCGAAGACCGGTGCCGCAGGCAGCCTGATGGATGTTCTGCATCATCCCGGTATGAATCACCGTGTGGAAATCACTGAGGGTATCCTCGCGGATGAGTGCGCTGCGCTGCTCAGTGATTTCTTTCGGATGCGTCGCCAAGAGATTAAGGCGCAAAAAAAGGCGCAGAGCTCGACCGACTCCGGCGGATCCAGCGGTGGTTCTTCGGGCTCCGAGACCCCGGGCACCAGCGAGTCCGCAACCCCGGAATCTTCCGGAGGCTCGAGCGGTGGCAGCAGCGAAGTGGAATTTAGCCACGAGTACTGGATGAGACACGCCTTGACCCTAGCAAAGCGCGCACGAGATGAGAGAGAAGTTCCGGTTGGAGCCGTGCTTGTTCTGAACAACCGCGTAATTGGTGAGGGTTGGAATCGCGCGATCGGCCTGCACGACCCGACCGCTCACGCGGAGATCATGGCGCTGAGACAAGGCGGTCTGGTGATGCAAAACTATCGTCTGATTGACGCGACCCTGTATGTGACCTTTGAGCCGTGCGTTATGTGCGCTGGCGCGATGATCCACTCTCGTATTGGTCGCGTAGTGTTCGGTGTGCGCAACGCGAAGACCGGTGCAGCGGGCAGCCTGATGGATGTTCTGCACTATCCGGGCATGAATCATCGTGTCGAAATCACCGAAGGCATTCTGGCCGACGAGTGCGCAGCGCTGCTGTGCTATTTCTTCCGTATGCCGCGTCAAGTGTTCAACGCACAGAAAAAAGCGCAGAGCTCCACCGATAGCGGTGGTTCTAGCGGTGGTTCCTCGGGCTCCGAAACCCCGGGTACAAGCGAGAGCGCGACCCCGGAGAGCTCTGGTGGCAGTTCCGGTGGATCAGAGACGAATGGAGAGACGGGTGAAATCGTGTGGGATAAGGGTCGCGATTTCGCGACCGTTCGTAAGGTGCTGTCTATGCCGCAGGTAAATATCGTAAAGAAGACGGAGGTGCAGACCGGTGGTTTCAGCAAAGAGTCTATTCTGCCGAAACGCAATAGCGATAAACTGATAGCACGAAAGAAAGACTGGGATCCGAAGAAGTACGGCGGTTTTGACTCTCCGACCGTTGCGTACAGCGTTTTGGTAGTGGCAAAGGTGGAAAAAGGGAAGTCCAAGAAGCTGAAATCTGTTAAGGAATTGCTGGGCATCACCATCATGGAACGTTCCTCTTTTGAAAAAAACCCGATTGACTTCCTGGAAGCAAAAGGCTACAAAGAAGTCAAAAAAGACCTGATTATCAAGCTGCCGAAGTACAGCCTCTTCGAACTGGAGAACGGCCGGAAGCGTATGCTGGCGTCTGCGGGTGAACTGCAAAAAGGTAATGAACTCGCGCTGCCGTCAAAATACGTTAATTTCCTGTATCTTGCCTCCCATTACGAAAAACTGAAAGGCAGCCCGGAAGATAATGAGCAAAAACAGCTGTTCGTGGAGCAGCATAAACACTATTTGGACGAAATTATCGAACAGATTAGCGAATTTTCAAAGCGCGTTATCCTGGCTGATGCTAATCTGGATAAGGTCCTGTCCGCCTACAACAAGCACCGTGACAAACCGATTCGTGAGCAGGCAGAAAACATCATTCATTTGTTCACCCTCACTAACCTGGGGGCGCCAGCCGCATTTAAGTACTTCGACACCACCATAGATCGTAAACGCTACACCTCCACTAAAGAAGTTTTGGACGCGACTCTCATCCACCAGAGCATCACGGGCTTGTACGAAACTCGTATTGACCTGTCCCAGCTCGGCGGTGATAGCGGCGGATCAAAACGTACCGCTGATGGCTCTGAGTTCGAACCAAAAAAAAAGCGCAAGGTT

SEQ ID NO .22

ACGUUCAGAAGUCGGGUGAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .23 GAAGCCACAGGAAGUCUGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .24 UUCAGAGCAGUUUGAGACAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .25 ACUCCAGUUCCUGACGGCUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .26 ACCUACAGGAGUACCUGACGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .27 GCAGACCGAUGUCUACUCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .28 GUCCACAGGACGAUGUGCAGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .29 UUCCCAGAGCACCAGAGCCAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .30 AGCCAGAAGCUGGGAAUUUCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .31 CCAAGAGGCAUACUCCUCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .32 CUCACCCGACUUCUGAACGUGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .33 CCUAGAGUGAAGAGAUUCAUGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .34 UCUGAUGGGGAAACAAAACAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .35 UUACCUGCCCACUGUUAGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .36

CAUGGGUCGGGGGCUGCUCA

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .37

CCAUGGGUCGGGGGCUGCUC

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .38

AUGGGUCGGGGGCUGCUCAG

GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGG

CUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU

SEQ ID NO .39

IGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFK

SEQ ID NO .40

ETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

SEQ ID NO .41

MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIFDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN

SEQ ID NO .42

SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGTSESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

SEQ ID NO .43

MKRTADGSEFESPKKKRKV

SEQ ID NO .44

SEQ ID NO .45

SGGSGGSGGS

SEQ ID NO .46

SGSGSETPGTSESATPES

SEQ ID NO .47

ACGTTTAGAAGTCGGGTGAG

SEQ ID NO .48

GAAGCCATAGGAAGTCTGTG

SEQ ID NO .49

TTCAGAGTAGTTTGAGACAG

SEQ ID NO .50

ACTCTAGTTCCTGACGGCTG

SEQ ID NO .51

ACCTATAGGAGTACCTGACG

SEQ ID NO .52

GTAGACCGATGTCTACTCCA

SEQ ID NO .53

GTTTACAGGACGATGTGCAG

SEQ ID NO .54

TTCTTAGAGCACCAGAGCCA

SEQ ID NO .55

AGTTAGAAGCTGGGAATTTC

SEQ ID NO .56

TTAAGAGGCATACTCCTCAT

SEQ ID NO .57

CTCATCCGACTTCTGAACGTG

SEQ ID NO .58

CTCGCCCGACTTCTGAACGTG

SEQ ID NO .59

CTTAGAGTGAAGAGATTCAT

SEQ ID NO .60

TTTGATGGGGAAACAAAACA

SEQ ID NO .61

TTATCTGCCCACTGTTAGCC

SEQ ID NO .62

TTGCCTGCCCACTGTTAGCC

SEQ ID NO .63

CGTGGGTCGGGGGCTGCTCA

SEQ ID NO .64

CCGTGGGTCGGGGGCTGCTC

SEQ ID NO .65

GTGGGTCGGGGGCTGCTCAG

1. expansion and culture of primary NK cells

Frozen human Peripheral Blood Mononuclear Cells (PBMCs) were removed from liquid nitrogen and thawed rapidly in a 37 ℃ water bath.

9mL of RPMI-1640 complete medium containing 10% FBS was added to a new 15mL centrifuge tube, 1mL of PBMC suspension was transferred to the 15mL centrifuge tube, 20. Mu.L of the count was removed, and the remaining cells were centrifuged.

Centrifuge at 250 Xg for 5min at room temperature.

The supernatant was discarded and the cells were resuspended using 1mL of RPMI-1640 medium.

The cell suspension was transferred to a new 75mL cell culture flask by adding 19mL of RPMI-1640 medium.

Human recombinant IL-2 protein was added to the flask at a final concentration of 200U/mL.

The flask was placed in 5% CO at 37 ℃C ₂ The cells are cultivated in a flat way in a cell incubator.

The cryopreserved irradiated EK562 engineered cells were removed from the liquid nitrogen and rapidly thawed in a 37 ℃ water bath.

9mL of RPMI-1640 complete medium was added to a new 15mL centrifuge tube, and the EK562 cell suspension was transferred to the 15mL centrifuge tube.

Centrifuge at 250 Xg for 5min at room temperature.

The supernatant was discarded, and cells were resuspended and counted using 1mL of RPMI-1640 medium.

According to PBMC: EK 562=1: 1 into an EK562 cell in a targeting flask.

The cells were subjected to 5% CO at 37 ℃ ₂ Culturing in incubator, changing liquid every two days, and counting cells to control cell density at 0.5-1×10 ⁶ IL-2 was added in a final concentration of 100U/mL in the range of individual/mL depending on the volume of the culture medium.

On day 7, the cells in the flask were counted, and the purity of NK cells in the flask was up to 95% or more.

Preparation of sgRNA

By analysis of the coding and non-coding regions of the TGFBR2 gene, the present invention selects target sequences (SEQ ID NO. 1-SEQ ID NO. 17) that may be mutated to a stop codon or a mutated start codon or a mutated intron splice site. The corresponding sgrnas can be obtained synthetically by third party companies. The modification of the synthesized sgRNA terminal is helpful for improving the editing efficiency, and the sequence is shown as SEQ ID NO. 22-SEQ ID NO.38, wherein each sequence contains 3 thio groups and 3 methoxy groups at the 3 '-end and the 5' -end.

Wherein SEQ ID NO.1 and modified SEQ ID NO.22 are numbered TGFBR2-1, SEQ ID NO.2 and modified SEQ ID NO.23 are numbered TGFBR2-2, SEQ ID NO.3 and modified SEQ ID NO.24 are numbered TGFBR2-3, SEQ ID NO.4 and modified SEQ ID NO.25 are numbered TGFBR2-4, SEQ ID NO.5 and modified SEQ ID NO.26 are numbered TGFBR2-5, SEQ ID NO.6 and modified SEQ ID NO.27 are numbered TGFBR2-6, SEQ ID NO.7 and modified SEQ ID NO.28 are numbered TGFBR2-7, SEQ ID NO.8 and modified SEQ ID NO.29 are numbered TGFBR2-8, SEQ ID NO.9 and modified SEQ ID NO.30 are numbered TGFBR2-9, SEQ ID NO.10 and modified SEQ ID NO.31 are numbered TGR 2-10, SEQ ID NO.11 and modified SEQ ID NO.14 are numbered SEQ ID NO.16 and modified SEQ ID NO. 14-16 are numbered TGFBR2-15, SEQ ID NO.14 and modified SEQ ID NO.16 are numbered SEQ ID NO. 14-16.

3. Preparation and purification of base editing fusion proteins (BE proteins)

Codon optimization. And (3) performing escherichia coli codon optimization on the used cytosine base editing fusion protein and adenine base editing fusion protein, synthesizing a sequence by a Jinsrey company, and constructing the sequence into a pET28a expression plasmid skeleton. The optimized sequence is as follows: cytosine base editing fusion protein DNA sequence: SEQ ID NO. 20; adenine base editing fusion protein DNA sequence: SEQ ID NO. 21.

Plasmid transformation. BL21 competent is transformed respectively by plasmids, plates are coated, monoclonal is selected for shaking expression, 2L of culture medium is prepared for shaking by a shaking table at 37 ℃ and 220rpm, IPTG 2mL (2M concentration) is added when the OD value of bacterial liquid is between 0.6 and 0.8, and bacteria are harvested after induction culture is carried out for 24 hours.

And (5) bacterial collection. High-speed centrifugation of bacterial liquid: 4000rpm for 30min, the supernatant was discarded.

And (5) breaking bacteria. After the centrifuged cells were blown off with Buffer A, protease inhibitors were added, and E.coli was disrupted with a high-pressure disrupter. The supernatant was collected after high-speed centrifugation. Buffer A formulation: 25mM Tris pH=8.0, 500mM NaCl,10% (v/v) Glycerol, 0.22. Mu.M filter.

And (5) passing through a column. The disrupted supernatant was filtered through a 0.45 μm filter, and then a cobalt ion affinity column (Clontech, 635504) after Buffer a rinse was added to adsorb the His-tagged Cas9 protein.

Removing impurities. The column was run with 40ml Buffer A with 5mM imidazole added to remove the lower affinity impurities.

Eluting. The target protein was displaced by 30ml of BufferA with 500mM imidazole added thereto.

Concentrating. After Western blot identification, the eluted target protein is added into a protein concentration column at 3900rpm for 20min.

The concentrated protein was subjected to ion exchange chromatography (Ion exchange chromatography (IEC)) to remove nucleic acids bound to the protein. And concentrating again to obtain cytosine base editing protein (CBE protein) with an amino acid sequence shown as SEQ ID NO. 18 and adenine base editing fusion protein (ABE protein) with an amino acid sequence shown as SEQ ID NO. 19 respectively, measuring the concentration, sub-packaging and freezing for later use.

4. Electroporation transfection of NK cells

RNP was mixed with sgrnas, according to the ratio, to BE protein at 1:4, mixing the materials according to the mass ratio, and lightly blowing and uniformly mixing the materials by using a gun head.

NK cells were prepared. The NK cells prepared in step 1 were washed once with PBS and centrifuged.

Electroporation transfection.

In an alternative embodiment, the Lonza electrotransfection apparatus 4D-Nucleofector is used for electroporation transfection, NK cells and RNPs are resuspended in an electrotransfer solution and then added to an electrotransfer cup, and a suitable electrotransfer program is selected from the 135 candidate electrotransfer programs for electrotransfer. And sucking out liquid by using a gun head after electric conversion, putting the liquid into a culture medium preheated in advance for culture, and performing flow detection or sequencing analysis after 9 days of culture, thereby obtaining data related to editing efficiency.

In an alternative embodiment, electroporation transfection is performed using a Thermo electrotransfer device, and cells and RNP are resuspended in an electrotransfer solution and then added to an electrotransfer cup, and the appropriate electrotransfer program is selected from the candidate 4 electrotransfer programs for electrotransfer. And sucking out liquid by using a gun head after electric conversion, putting the liquid into a culture medium preheated in advance for culturing for 7 days, and performing flow detection or sequencing analysis to obtain data related to editing efficiency.

Currently there are already more mature electrotransport protocols for Cas9 proteins, but base editing fusion proteins are about 50kDa compared to Cas9, so the applicable electrotransport procedure is also different. In the embodiment, the sgRNA with the number of TGFBR2-2 is selected for electrotransformation condition optimization so as to further improve the base editing efficiency. The specific sequence is TGFBR2-2: SEQ ID NO. 2.

Centrifuge at 250 Xg for 5min at room temperature.

According to PBMC: EK 562=1: 1 into an EK562 cell in a targeting flask.

Cells in the flask were counted by day 7 of culture. At this time, the purity of NK cells in the culture flask can reach more than 95%.

The primary NK cells amplified in vitro as described above were collected and centrifuged at 250 Xg for 5min at room temperature.

The supernatant was discarded, and the cells were resuspended in 1 XPBS buffer and centrifuged at 250 Xg for 5min at room temperature.

Discarding the supernatant, and adopting an electrotransfer buffer solution matched with an electrotransfer instrument to resuspend the NK cells.

RNP was mixed, and the CBE protein prepared in example 1 and the sgRNA prepared in example 1 were mixed at a mass ratio of 4:1 according to the ratio.

Adding the RNP mixed solution into the prepared NK cell suspension, and carrying out electrotransformation on an electrotransformation instrument.

Electroporation transfection was performed using a Lonza electrotransfection apparatus 4D-Nucleofector, and 135 electrotransfection procedures were available for Lonza (FIG. 8). The aim of this example was to find an optimal combination of cell viability and electrotransformation efficiency. The present invention will utilize these 135 electrical switching sequences to individually switch to the same RNP.

Edit efficiency and cell proliferation were examined 7 days after electrotransformation. FIG. 9 shows the editing efficiency for the Lonza 135 electrotransformation program, and the proliferation ratio of cells at 7 days of culture in FIG. 10. Referring to fig. 9 and 10, it was found that both CM158 and CM137 electrical switching programs maintained high levels of both editing efficiency and cell proliferation factor, and that the CM137 electrical switching program had relatively better overall results.

The same method, the present invention is directed to four electrotransfer programs for a Neon electrotransfer instrument of a Thermo electrotransfer system: 1) Voltage 1650-1750v, pulse width 9-11ms, pulse times 1-3; 2) Voltage 1750-1850v, pulse width 9-11ms, pulse times 1-3; 3) Voltage 2150-2250v, pulse width 2-4ms, pulse number 3-5; 4) The voltage is 1550-1650v, the pulse width is 7-9ms, and the pulse number is 2-4. The editing efficiency of (2), 3), 4) was measured, wherein the editing efficiency was 40%, 65%, 68%, and 50% for each of the four electrical transfer procedures was 189, 234, 390, and 255, respectively. Comprehensive comparison 3) the electrical transfer procedure gave more ideal results in both the edit efficiency and the cell proliferation dimensions.

The TGFBR2 gene is knocked out by using a cytosine base editing tool, and the selected sgRNA needs to be mutated into a termination codon aiming at CAA, CAG, CGA or TGG triplet codons or mutated into ATA by acting on an initiation codon ATG. By analysis it was found that 10 sgrnas (SEQ ID No. 1-SEQ ID No. 10) could be mutated to stop codons, 3 sgrnas (SEQ ID No. 15-17) could be mutated to start codons, and 4 sgrnas (SEQ ID No. 11-14) could be mutated to intron splice sites.

In this example, the 17 groups of sgrnas (TGFBR 2-1 to 17) were analyzed for their corresponding editing efficiency, i.e., the editing efficiency of the sgrnas synthesized by the analysis company (SEQ ID nos. 12 to 20, each containing 3 thio groups and 3 methoxy modifications at the 3 'and 5' ends).

The corresponding RNP was electrotransformed using the CM137 electrotransformation program of Lonza, wherein the mass ratio of sgRNA to editing fusion protein was 1:4. Editing efficiency analysis was performed 7 days after the completion of the electric transfer culture. As a result, it was found that there was a large difference in editing efficiency of different sgRNAs, in which the editing efficiency of TGFBR2-2 sites shown in SEQ ID No.23, in which 3 thio groups and 3 methoxy groups were contained in each of the 3 '-end and the 5' -end, was highest, reaching 62%. TGFBR2-15, TGFBR2-16 and TGFBR2-17 shown in SEQ ID NO.36-38 with 3 thio groups and 3 methoxy groups at the 3 'end and the 5' end respectively act on sgRNA of the initiation codon, so that the relatively efficient knockout of TGFBR2 can be realized.

According to examples 2 and 3 results of the electrotransformation procedure and optimization of the sgrnas, the Lonza electrotransformer CM137 procedure was selected as the preferred electrotransformation procedure, and the high efficiency sgrnas used were TGFBR2-2 shown in SEQ ID No.23 with 3 thio groups and 3 methoxy modifications at the 3 'and 5' ends. On the basis, the proportion of the sgRNA and the base editing fusion protein is further optimized, so that the base editing efficiency is further improved.

1) Primary NK cells amplified in vitro in step 1 of example 1 were collected and centrifuged at 250 Xg for 5min at room temperature.

2) The supernatant was discarded, and the cells were resuspended in 1 XPBS buffer and centrifuged at 250 Xg for 5min at room temperature.

3) Discarding the supernatant, and adopting an electrotransfer buffer solution matched with an electrotransfer instrument to resuspend the NK cells.

4) RNP was mixed and the sgRNA prepared in step 2 of example 1 and the CBE protein prepared in step 3 of example 1 were mixed according to the ratio by mass of 1:2, 1:4, 1:6, 1:8.

5) Adding the RNP mixed solution into the prepared NK cell suspension, and carrying out electrotransformation on an electrotransformation instrument.

6) Electroporation transfection was performed using a Lonza electrotransfection apparatus, the electrotransfection procedure was set to CM137.

7) And sucking out liquid by using a gun head after electric rotation, putting the liquid into a culture medium preheated in advance for culturing, and performing flow detection after culturing for 7 days. As shown in FIG. 2, the editing efficiency reached the highest 75% when the sgRNA to protein mass ratio was 1:6.

1. Real-time dynamic method for detecting NK cell in-vitro killing activity

1) Tumor cells and NK cells were collected for counting, and tumor cells were added to a 96-well plate in a final volume of 50. Mu.L per 10000 tumor cells/well.

2) Collecting wild NK cells (NK-WT), cas9 gene edited NK cells (NK-Cas 9) [ construction steps are detailed in patent application No. 202011056575.7 ] and CBE base edited NK cells (NK-CBE) obtained when the mass ratio of sgRNA to protein is 1:6 in example 4, counting and then respectively pressing the NK cells: tumor cells = 1:1 number ratio, 50 μl NK cells were added to 96-well plates.

3) 3 replicate wells were made per group, and 3 tumor cells were established alone to culture control wells.

4) Cell plates were photographed on a real-time dynamic detector (IncuCyte S3, sartorius) for 48 hours.

5) After 48 hours, the fluorescent intensity of the cells is detected by a real-time dynamic detector.

6) NK cell killing activity was calculated by the following formula of FIG. 12:

NK _NK+Tumor : average luminous intensity of NK cells and tumor cells co-cultured group;

NK _Tumor : average luminous intensity of tumor cell alone cultures.

TGFBR2 is mainly combined with cytokines such as TGF beta 1 in a human body so as to achieve the effect of inhibiting NK activity, but in an in vitro experiment, the expression amount of TGF beta 1 in a tumor cell line can not reach the in vivo level, so that the experiment sets up a TGF beta 1 treatment group to simulate the in vivo TGF beta 1 level, thereby better reflecting the killing effect of NK cells after base editing TGFBR2, and the result is shown in figure 3, after the NK cells subjected to wild type, cas9 gene editing and CBE base editing are co-cultured with the tumor cells for 48 hours, the killing rate of the NK cells to target cells is dynamically detected in real time, and the result shows that the NK cells subjected to Cas9 gene editing and CBE base editing have stronger anti-tumor activity compared with wild type NK cells, and the anti-tumor effect is most remarkable after the treatment of TGFB 1.

2. In vivo detection of gene-edited NK cell anti-tumor Activity

1) Severe combined immunodeficiency (SCID-bg) mice with both T, B and NK cells deleted were selected as tumor bearing mice, the ordered mice were male, 5 weeks old, and the mice were housed in SPF-class animal housing, and tumor cells were inoculated to their backs after one week of adaptive housing.

2) H1975 lung cancer cells were digested and collected, and the cells were resuspended using serum-free RPMI-1640 medium.

3) H1975 cell suspension concentration was adjusted to 3X 10 ⁷ And each mL.

4) Male SCID-Bg mice of 6 weeks of age were weighed and placed in a respiratory anesthesia machine for isoflurane anesthesia.

5) The right back of the mice was shaved with a shaver, and 100 μl of the cell suspension was injected subcutaneously.

6) After 3 days of inoculation, mice were randomly divided into 4 groups by body weight, 4 in each group, and each group received adoptive immunotherapy with wild-type NK cells (NK-WT), cas9 gene-edited NK cells (NK-Cas 9) and CBE base-edited NK cells (NK-CBE) at a 1:6 sgRNA to protein mass ratio in example 4, and a blank Control group (Control) was injected with RPMI-1640 culture broth only. NK cells were administered by tail vein injection, 1X 10 per mouse ⁷ The NK cells were injected 1 time a week for 3 times of total treatment, starting with the first NK cell injection treatment on day 3 after tumor cell inoculation. The mouse tumor mass size was measured from day 3 after tumor cell inoculation, once every 2 days, while the mouse body weight was recorded.

As shown in fig. 4, 5 and 11, after gene editing (NK-Cas 9) or CBE base editing (NK-CBE) treatment, tumor growth of lung cancer tumor-bearing mice was significantly inhibited, and after 3 NK cell adoptive immunotherapy, the tumor volume of the gene editing (NK-Cas 9) or CBE base editing (NK-CBE) group was significantly reduced compared to the wild type (NK-WT) treatment group, and tumor weight was significantly reduced, indicating that both base-edited NK cells and gene-edited NK cells had stronger antitumor effect in vivo.

1. Analysis of DNA off-target using whole genome sequencing

By using the CM137 electrotransformation program of the optimized LONZA, TGFBR 2-2 sgRNA electrotransformation NK cells shown in SEQ ID No.23 with 3 thio groups and 3 methoxy groups respectively contained at the 3 'end and the 5' end are adopted, wherein the mass ratio of the sgRNA to the edited fusion protein is 1:4, the edited NK cells are collected after 7 days of electrotransformation culture, and unedited wild type NK cells are collected. Extracting NK cell genome DNA by using a Tiangen blood/cell/tissue genome DNA extraction kit (DP 304), and sending Beijing An Nuo to complete genome sequencing with the sequencing depth of 25-30x. The raw data sequenced were aligned to the human reference genome (GRCh 38/hg 38) by BWA v0.7.16. The SNP sites were analyzed by GATK HaplotypeCaller software and the off-target efficiency was calculated from the potential off-target sites.

FIG. 6 shows that DNA off-target was not found in NK cells treated with base editing by SNP associated with reference genome and wild type NK cell removal.

2. RNA off-target analysis using transcriptome

By using the optimized CM137 electrotransformation program of Lonza, TGFBR 2-2 sgRNA electrotransformation NK cells shown in SEQ ID No.23 with 3 thio groups and 3 methoxy groups respectively contained at the 3 'end and the 5' end, wherein the mass ratio of sgRNA to fusion protein is 1:4, collecting edited NK cells after electrotransformation culture is completed for 7 days, and collecting unedited wild type NK cells. Total RNA of edited NK and unedited NK cells was extracted using the Tiangen RNA Easy Fast animal tissue/cell total RNA extraction kit (DP 451), and was sent to Beijing An Nuo for optimization for RNA sequencing (Illumina Hiseq X10). Each sample was read to a depth of about 2000 ten thousand. Reads were mapped to the reference genome (hg 38) by STAR software (version 2.5.1) using notes from geneode v 30. After deletion of the duplicates, variants were identified by GATK HaplotypeCaller (version 4.1.2) and filtered with QDs (mass by depth), all variants were validated and quantified by bam-readcount, with the parameters-q 20-b 30. As shown in FIG. 7, NK cells knocked out of TGFBR2 gene by base editing system, no significant off-target was detected at the whole transcriptome level

The above can be seen in the following: compared with common NK cells, the base-edited NK cells have stronger anti-tumor activity in vivo and in vitro, and the base-edited NK cells have better safety compared with the gene-edited NK cells, so that the base-edited NK cells are expected to be developed into safe and effective anti-tumor biological agents, and have obvious application prospects and clinical application values.

The above examples are provided to illustrate the disclosed embodiments of the invention and are not to be construed as limiting the invention. Further, various modifications of the methods set forth herein, as well as variations of the methods of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been specifically described in connection with various specific preferred embodiments thereof, it should be understood that the invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the present invention. .

Claims

1. A natural killer cell, wherein a TGFBR2 gene of the natural killer cell is subjected to base editing to perform gene editing without introducing a DNA double strand break;

The base editing mutates base C to T and/or base a to G on the TGFBR2 gene in natural killer cells.

2. The natural killer cell of claim 1, wherein the natural killer cell is one or more of an NK cell derived from peripheral blood cells, an NK cell derived from umbilical cord blood, an NK cell induced by embryonic stem cells, or an NK cell induced by induced pluripotent stem cells.

3. The natural killer cell according to claim 1, wherein the nucleotide sequence of TGFBR2 gene is changed from base C to T and/or from base a to G in the base editing window corresponding to the target point shown in any one of SEQ ID No.1 to SEQ ID No. 17.

4. A natural killer cell according to claim 3, wherein the nucleotide sequence shown as any one of SEQ ID No.1 to SEQ ID No.17 on the TGFBR2 gene is mutated to the nucleotide sequence shown as any one of SEQ ID No.47 to SEQ ID No. 65.

5. The method for producing natural killer cells according to any one of claims 1 to 4, wherein the natural killer cells are obtained by introducing a base editing system into natural killer cells and editing the natural killer cells.

6. The method of claim 5, wherein the method of introducing the base editing system into natural killer cells is selected from one or more of electroporation, viral transduction, microinjection, particle bombardment, and gene gun transformation.

7. The method of claim 6, wherein the electroporation uses an electrotransport system selected from one or more of a LONZA system, a Thermo Neon transfection system, and a Gibco CTS Xenon electrotransfection system.

8. The method according to claim 7, wherein when the electrotransport device system used in the electroporation method is a LONZA system, a 4D-Nucleofector type electrotransport device, the electrotransport program is selected from CM137, CM158 and CM189;

or, when the electrotransfection system used in the electroporation method is a Thermo electrotransfection system, a model Neon electrotransfection apparatus or a CTS Xenon electrotransfection apparatus, the electrotransfection procedure is selected from any one of the following procedures:

1) Voltage 1650-1750v, pulse width 9-11ms, pulse times 1-3;

2) Voltage 1750-1850v, pulse width 9-11ms, pulse times 1-3;

3) Voltage 2150-2250v, pulse width 2-4ms, pulse number 3-5;

9. The method of claim 5, wherein the base editing system comprises I) a fusion protein or variant thereof or a nucleotide encoding a fusion protein or variant thereof; II) a guide nucleotide.

10. The method according to claim 9, wherein the mass ratio of the guide nucleotide to the fusion protein or the variant thereof in the base editing system is 1:2-1:20.

11. the preparation method according to claim 10, wherein the mass ratio 1:4-1:6.

12. the method of preparation of claim 9, wherein the fusion protein or variant thereof is linked from N-terminus to C-terminus to a first nCas9 fragment, a deaminase fragment and a second nCas9 fragment in sequence.

13. The method of preparation of claim 12, wherein the amino acid sequence of the first nCas9 fragment is set forth in SEQ ID No. 39; and/or, the amino acid sequence of the second nCas9 fragment is shown as SEQ ID NO. 40.

14. The method according to claim 12, wherein the deaminase fragment is a cytosine deaminase fragment or an adenine deaminase fragment.

15. The method of claim 14, wherein the cytosine deaminase is selected from one or more of aporec 1, aporec 2, aporec 3A, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, AID, or pmCDA 1.

16. The method of claim 15, wherein the cytosine deaminase is apodec 3A shown in SEQ ID No. 41.

17. The method of claim 14, wherein the adenine deaminase is selected from the group consisting of wild-type ectoda, mutant ectoda ^* Or ectoda-ectoda ^* One or more of the complexes.

18. The method of claim 17, wherein the adenine deaminase is ectoada-ectoada shown in SEQ ID No.42 ^* A complex.

19. The method of preparation of claim 9, wherein the fusion protein or variant thereof further comprises one or more of a nuclear localization signal, uracil glycosylase inhibitor fragment, or a GS peptide fragment.

20. The method of claim 19, wherein the amino acid sequence of the nuclear localization signal is as set forth in SEQ ID No. 43; and/or the uracil glycosylase inhibitor fragment has an amino acid sequence as shown in SEQ ID NO. 44; and/or the amino acid sequence of the GS peptide fragment is shown as SEQ ID NO. 45.

21. The method of claim 9, wherein the fusion protein has a structure of NH2- [ nuclear localization signal ] - [ first nCas9 fragment ] - [ connecting peptide a ] - [ cytosine deaminase fragment ] - [ connecting peptide a ] - [ second nCas9 fragment ] - [ GS peptide fragment ] - [ UGI peptide fragment ] - [ nuclear localization signal ] - [ first nCas9 fragment ] - [ connecting peptide a ] - [ adenine deaminase fragment ] - [ connecting peptide a ] - [ second nCas9 fragment ] - [ GS peptide fragment ] - [ nuclear localization signal ] -COOH.

22. The method according to claim 9, wherein the amino acid sequence of the fusion protein is shown in any one of SEQ ID No. 18 to SEQ ID No. 19; and/or the nucleotide sequence of the encoding fusion protein is shown in any one of SEQ ID No. 20-SEQ ID No. 21.

23. The method of claim 9, wherein the nucleotide sequence of the guide nucleotide is set forth in any one of SEQ ID No.22 to SEQ ID No. 38; and/or the nucleotide sequence of the target point of the guide nucleotide is shown as any one of SEQ ID NO. 1-SEQ ID NO. 17.

24. The method according to claim 23, wherein when the deaminase in the fusion protein is apodec 3A, the nucleotide sequence of the guide nucleotide is shown in any one of SEQ ID No.22 to SEQ ID No. 35; and/or the nucleotide sequence of the target point of the guide nucleotide is shown as any one of SEQ ID NO. 1-SEQ ID NO. 14.

25. The method according to claim 23, wherein the deaminase in the fusion protein is ectoda-ectoda ^* When the nucleotide sequence of the guide nucleotide is shown as any one of SEQ ID NO. 32, SEQ ID NO. 35-SEQ ID NO. 38; and/or the nucleotide sequence of the target point of the guide nucleotide is shown as any one of SEQ ID NO.11, SEQ ID NO. 14-SEQ ID NO. 17.

26. The method according to claim 9, wherein the first 2 to 4 nucleotides at the 3 'and 5' ends of the guide nucleotide are thio-and/or methoxy-modified nucleotides.

27. A method of editing a TGFBR2 gene, characterized in that the method is to mutate base C to T or to mutate base a to G on the TGFBR2 gene by the base editing system in the preparation method as claimed in any one of claims 5 to 26 to edit the TGFBR2 gene.

28. Use of the base editing system in the preparation method of any of claims 5-26 to mutate base C to T, or base a to G on the TGFBR2 gene.

29. A composition comprising the natural killer cell according to any one of claims 1 to 4 or the base editing system according to the production method of any one of claims 5 to 26 as an active ingredient.

30. A kit comprising a natural killer cell according to any one of claims 1 to 4, a base editing system according to the preparation method of any one of claims 5 to 26, or a composition according to claim 29.

31. Use of a natural killer cell according to any one of claims 1 to 4, a base editing system according to any one of claims 5 to 26 in a method of preparation, a composition according to claim 29 or a kit according to claim 30, said use being selected from one or more of the following:

1) Preparing a medicament for preventing and/or treating autoimmune diseases;

2) Preparing a medicament for preventing and/or treating tumors;

3) Preparing a medicament for preventing and/or treating viral infectious diseases;

4) Preparing the medicine for preventing and/or treating bacterial infectious diseases.

32. The use according to claim 31, wherein the autoimmune disease is selected from one or more of systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, lupus nephritis, neuromyelitis optica, systemic sclerosis, dry mouth syndrome, polymyositis;

and/or the tumor is selected from one or more of adrenal cortex cancer, bladder urothelial cancer, breast cancer, cervical squamous cell carcinoma, cervical adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, lymphoid tumor, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck squamous cell carcinoma, renal chromophobe cancer, renal clear cell carcinoma, renal papillary cell carcinoma, acute myelogenous leukemia, brain low grade glioma, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelial cell carcinoma, ovarian cancer, pancreatic cancer, pheochromocytoma and paraganglioma, prostate cancer, rectal cancer, malignant sarcoma, melanoma, gastric cancer, testicular germ cell tumor, thyroid cancer, thymus cancer, endometrial cancer, uterine sarcoma, uveal melanoma, multiple myeloma, acute gonomic leukemia, chronic myelogenous leukemia, T-cell lymphoma, B-cell lymphoma, lung cancer, anal carcinoma, intraocular melanoma, retinoblastoma;

And/or the virus is selected from one or more of influenza virus, parainfluenza virus, measles virus, mumps virus, herpes virus, adenovirus, respiratory syncytial virus, polio virus, coxsackie virus or epstein barr virus;

and/or the bacteria are selected from one or more of escherichia coli, lactobacillus casei, bacteroides fragilis, acinetobacter rouxii, fusobacterium nucleatum, bacteroides johnsonii, arabidopsis thaliana, lactobacillus rhamnosus, bacteroides massiliensis, bacteroides ovatus, campylobacter jejuni, staphylococcus saprophyticus, enterococcus faecalis, bacteroides thetaiotaomicron, bacteroides vulgare, bacteroides simplex, parabacteroides faecalis, fusobacterium mortiferum and bifidobacterium breve.