HK40006863A

HK40006863A - Gene editing t cell and use thereof

Info

Publication number: HK40006863A
Application number: HK19130222.3A
Authority: HK
Inventors: 袁鹏飞; 王飞; 于玲玲; 董曦
Original assignee: 博雅辑因（北京）生物科技有限公司
Priority date: 2017-09-18
Filing date: 2019-09-26
Publication date: 2020-05-29

Description

Gene editing T cell and application thereof

Cross-referencing

This application claims priority to chinese patent applications 201710842264.5 and 201710841323.7 filed 2017, 9, 18, the disclosure of which is incorporated by reference in its entirety.

Technical Field

The invention relates to a T cell, in particular to a single-gene, double-gene and three-gene knockout T cell subjected to gene editing, including universal T cells, CAR-T cells and TCR-T cells, and a preparation method and application thereof.

Background

Malignant tumor has become a serious disease threatening the health and life safety of human body, and the cure of tumor is always a dream for human beings. In recent years, tumor immunotherapy has gained widespread attention, and in particular the emergence of CAR-T (clinical antigen receptor T cells) technology has led to a milestone development in tumor control. The consensus recommendation of nova CAR-T drug CTL019 for the treatment of adolescent advanced B-cell acute lymphoblastic leukemia (r/rAll) was recommended from the first application of CAR-T technology in 1989, to EmilyWhitehead with a lead team for cure by professor Carl June, university of pennsylvania in 2012, to the consistent approval of nova CAR-T drug CTL019 for adolescent treatment with an overwhelming dominance vote of 10:0 in 2017.

Generally, the traditional CAR-T technology T cells are mainly derived from the patient himself, and are isolated in vitro, activated, CAR introduced, cultured and expanded in GMP environment, and finally returned to the patient through quality control. The problem of unsuitability for blood collection or difficulty in expansion after T cell isolation from blood collection due to the influence of the patient's own conditions may be accompanied. When the patient is critically ill, the waiting time of the whole process from the isolation of T cells to the reinfusion of CAR-T is also a significant problem to be faced with the reinfusion. These problems have limited the widespread use of CAR-T technology, and therefore an important current direction in CAR-T cell therapy is how to use T cells from a healthy donor to produce large numbers of CAR-T cells for clinical use in patients. The establishment of the technology can greatly reduce the cost of CAR-T therapy, better ensure the quality of uniformly prepared cells, and patients can be immediately treated by the CAR-T cells when needed.

Throughout this specification, several documents are cited. Each document herein (including any journal articles or abstracts, published or unpublished patent applications, issued patents, manufacturer's specifications, instructions for use, etc.) is incorporated by reference. However, there is no admission that the documents cited herein are in fact prior art to the present invention.

Summary of The Invention

The present invention has been made to solve the above-mentioned problems occurring in the art. The single gene (TRAC, B2M or PD-1), the double gene (TRAC and B2M) and the triple gene (TRAC, B2M and PD-1) are knocked out by using a CRISPR/Cas9 system, and the knocking-out efficiency is respectively up to 90 percent (single gene), 81 percent (double gene) and 67 percent (triple gene). The T cells subjected to gene editing can provide universal T cells for CAR or TCR aiming at different targets, provide a method for establishing a gene editing technology combined with adoptive immunity to treat tumor and virus infectious diseases (such as HIV/AIDS), and lay a solid technical foundation for the research of related disease treatment. Meanwhile, the genetically modified T cells (including universal T cells, CAR-T and TCR-T) can be used as medicaments to be applied to patients in need.

Thus, in one aspect, the invention provides methods for efficient single (TRAC, B2M or PD-1), double (TRAC and B2M) or triple (TRAC, B2M and PD-1) gene knock-out of T cells using gene editing techniques, such as the CRISPR/Cas9 system, to obtain universal T cells that do not express TCR, or TCR/HLA/PD-1. Meanwhile, the universal T cell can be combined with the required CAR or TCR at any time to prepare universal CAR-T or TCR-T, and the universal CAR-T or TCR-T can be used as a medicament for being applied to a patient in need at any time. In addition, the method can also provide support for the research of new effective gene targets and can be effectively applied to clinical immunotherapy in time.

In one aspect, the invention provides a method of making a genetically engineered T cell comprising: disrupting in the T cell by gene editing techniques:

(i) the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490 (shown in SEQ ID NO: 23);

(ii) the B2M genomic region from chromosome 15 from position 45003745 to position 45003788 (shown in SEQ ID NO: 24); and/or

(iii) The PD-1 genomic region from 242800936 th to 242800978 th of chromosome 2 (shown in SEQ ID NO: 25), or the PD-1 genomic region from 242795009 th to 242795051 th of chromosome 2 (shown in SEQ ID NO: 26). In some embodiments, the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region are all edited. In some embodiments, the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology, or a CRISPR/Cas gene editing technology, such as a CRISPR/Cas9 gene editing technology.

In some embodiments, the invention provides a method of making a genetically engineered T cell, comprising: disrupting in the T cell by gene editing techniques:

(i) and a polypeptide selected from the group consisting of SEQ ID NOs: 2-5, and a target nucleotide sequence of the TRAC genome which is complementary to the sequence of any one of the sequences;

(ii) and a polypeptide selected from the group consisting of SEQ ID NOs: 6-13, the target nucleotide sequence of the B2M genome being complementary in sequence; and/or

(iii) And a polypeptide selected from the group consisting of SEQ ID NOs: 14-22, and a target nucleotide sequence of the PD-1 genome that is complementary to the sequence of any one of claims 14-22. In some embodiments, the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region are all edited. In some embodiments, the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology, or a CRISPR/Cas gene editing technology, such as a CRISPR/Cas9 gene editing technology.

In some embodiments, the present invention provides a method of making a genetically engineered T cell in which disruption by CRISPR/Cas9 gene editing techniques:

(i) and a sequence selected from SEQ ID NO:2 or SEQ ID NO: 3, a target nucleotide sequence of the TRAC genome which is complementary to the sequence of any one of;

(ii) and a sequence selected from SEQ ID NO: 7 or SEQ ID NO: 8, the target nucleotide sequence of the B2M genome of which the sequences are complementary; and/or

(iii) And a sequence selected from SEQ ID NO: 14 or SEQ ID NO: 15, and a target nucleotide sequence of the PD-1 genome to which the sequence of any one of seq id nos is complementary.

(i) and SEQ ID NO:2, a target nucleotide sequence of the TRAC genome complementary to the sequence of seq id no;

(ii) and SEQ ID NO: 8, the target nucleotide sequence of the B2M genome complementary to the sequence of 8; and/or (iii) a sequence that is identical to SEQ ID NO: 14, and a target nucleotide sequence of the PD-1 genome that is complementary to the sequence of 14.

(ii) and SEQ ID NO: 8, the target nucleotide sequence of the B2M genome complementary to the sequence of 8; and/or (iii) a sequence that is identical to SEQ ID NO: 15, and a target nucleotide sequence of the PD-1 genome that is complementary to the sequence of seq id no.

(i) and SEQ ID NO: 3, a target nucleotide sequence of the TRAC genome complementary to the sequence of 3;

(ii) and SEQ ID NO: 7, the target nucleotide sequence of the B2M genome being complementary in sequence; and/or (iii) a sequence that is identical to SEQ ID NO: 15, and a target nucleotide sequence of the PD-1 genome that is complementary to the sequence of seq id no.

In some embodiments, the present invention provides a method of making a genetically engineered T cell by CRISPR/Cas9 gene editing technology, wherein:

(i) (ii) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5 to effect editing of the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490;

(ii) (ii) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-13 to effect editing of the B2M genomic region from chromosome 15 from position 45003745 to position 45003788; and/or

(iii) (ii) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-22 to effect editing of the PD-1 genomic region on chromosome 2 from position 242800936 to position 242800978, or the PD-1 genomic region on chromosome 2 from position 242795009 to position 242795051.

(i) (ii) a polypeptide comprising an amino acid sequence selected from SEQ ID NO:2 or SEQ ID NO: 3 to effect editing of the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490;

(ii) (ii) a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 7 or SEQ ID NO: 8 to effect editing of the B2M genomic region from chromosome 15 from position 45003745 to position 45003788; and/or

(iii) (ii) a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 14 or SEQ ID NO: 15 to effect editing of the PD-1 genomic region of chromosome 2 from position 242800936 to position 242800978, or the PD-1 genomic region of chromosome 2 from position 242795009 to position 242795051.

(i) converting a polypeptide comprising SEQ ID NO:2 to effect editing of the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490;

(ii) converting a polypeptide comprising SEQ ID NO: 8 to effect editing of the B2M genomic region of chromosome 15 from position 45003745 to position 45003788; and/or

(iii) Converting a polypeptide comprising SEQ ID NO: 15 to effect editing of the PD-1 genomic region of chromosome 2 from position 242800936 to position 242800978, or the PD-1 genomic region of chromosome 2 from position 242795009 to position 242795051.

(iii) Converting a polypeptide comprising SEQ ID NO: 14 to effect editing of the PD-1 genomic region of chromosome 2 from position 242800936 to position 242800978, or the PD-1 genomic region of chromosome 2 from position 242795009 to position 242795051.

(i) converting a polypeptide comprising SEQ ID NO: 3 to effect editing of the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490;

(ii) converting a polypeptide comprising SEQ ID NO: 7 to effect editing of the B2M genomic region of chromosome 15 from position 45003745 to position 45003788; and/or

In some embodiments, the TRAC-targeted sgRNA is introduced into the T cell alone. In some embodiments, the sgRNA targeting B2M is introduced separately to the T cell. In some embodiments, the sgRNA targeting PD-1 is introduced into the T cell alone. In some embodiments, the trc-targeted sgRNA and the B2M-targeted sgRNA are introduced simultaneously into the T cell. In some embodiments, the trc-targeted sgRNA and PD-1-targeted sgRNA are introduced simultaneously into the T cell. In some embodiments, the sgRNA targeting B2M and the sgRNA targeting PD-1 are introduced to the T cell simultaneously. In some embodiments, the trc-targeted sgRNA, the B2M-targeted sgRNA, and the PD-1-targeted sgRNA are introduced to the T cell simultaneously.

In some embodiments, the sgrnas (including a TRAC-targeted sgRNA, a B2M-targeted sgRNA, and/or a PD-1-targeted sgRNA) are modified with a2 '-O-methyl analog and/or an internucleotide 3' thio. In some embodiments, the chemical modification is a2 ' -O-methyl analog modification of the first, two, and/or three bases of the 5 ' end of the sgRNA (including a TRAC-targeted sgRNA, a B2M-targeted sgRNA, and/or a PD-1-targeted sgRNA) and/or the last base of the 3 ' end.

In some embodiments, the sgrnas described above (including a trc-targeted sgRNA, a B2M-targeted sgRNA, and/or a PD-1-targeted sgRNA) are introduced into the T cell by electroporation. In some embodiments, the sgrnas described above (including the sgRNA targeted to TRAC, the sgRNA targeted to B2M, and/or the sgRNA targeted to PD-1) are introduced into the T cell by electrotransformation with Cas9 encoding nucleotides (e.g., mRNA). In some embodiments, the electrotransfer conditions include any one selected from the group consisting of: 150-250V, 0.5-2 ms; 150V, 2 ms; 160V, 2 ms; 170V, 2 ms; 180V, 2 ms; 190V, 1 ms; 200V, 1 ms; 210V, 1 ms; 220V, 1 ms; 230V, 1 ms; 240V, 1 ms; 250V, 0.5 ms.

In some embodiments, the method further comprises screening the T cells with low expression of TRAC, B2M and/or PD-1 from the T cells subjected to gene editing. For example, the expression level of TRAC, B2M or PD-1 in a T cell with gene editing is 1/10 of the expression level of a T cell without gene editing

In some embodiments, the efficiency of a single gene knockout (TRAC, B2M, or PD-1) is 80% or greater, e.g., 80% -100%, 85% -100%, 90% -100%, 95% -100%, 81% or greater, 82% or greater, 83% or greater, 84% or greater, 85% or greater, 86% or greater, 87% or greater, 88% or greater, 89% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater; the knock-out efficiency (e.g., TRAC and B2M) of a dual gene is greater than 65%, e.g., 65% -100%, 70% -100%, 75% -100%, 80% -100%, 85% -100%, 90% -100%, 95% -100%, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more; the simultaneous knockout efficiency of TRAC, B2M, and PD-1 genes is 50% or more, for example, 55% -100%, 60% -100%, 65% -100%, 70% -100%, 75% -100%, 80% -100%, 85% -100%, 90% -100%, 95% -100%, 55% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more. All numbers herein encompass the number itself and each and every integer and decimal between the numbers. The knockout efficiency here encompasses the knockout efficiency in the case of one, two, or three knockout cases selected from the group consisting of TRAC gene, B2M gene, and PD-1 gene.

In some embodiments, the T cell is derived from a healthy subject, a tumor, or a virus-infected patient (e.g., an HIV-infected patient). In some embodiments, the T cell is a T cell differentiated from a stem cell or a precursor cell at a different differentiation stage.

In one aspect, the invention relates to genetically engineered T cells prepared by the above-described methods.

In one aspect, the invention relates to a genetically engineered T cell, wherein in the T cell:

(i) one or more sites of the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490 are disrupted by gene editing techniques;

(ii) one or more sites of the B2M genomic region from chromosome 15 from position 45003745 to position 45003788 were disrupted by gene editing techniques; and/or

(iii) One or more sites of the PD-1 genomic region from 242800936 th to 242800978 th on chromosome 2, or the PD-1 genomic region from 242795009 th to 242795051 th on chromosome 2 are disrupted by gene editing techniques. In some embodiments, the T cell is prepared by any of the methods described herein.

(i) the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490 having a change in the gene sequence as set forth in any one of tables D and E;

(ii) the B2M genomic region from chromosome 15 from position 45003745 to position 45003788 has a gene sequence modification of any one of the genes listed in table B and table C; and/or

(iii) The PD-1 genomic region of chromosome 2 from position 242800936 to position 242800978 has any of the gene sequence alterations described in Table F, or the PD-1 genomic region of chromosome 2 from position 242795009 to position 242795051 has any of the gene sequence alterations described in Table G. In some embodiments, the T cell is prepared by any of the methods described herein.

In one aspect, the invention relates to the use of the genetically modified T cell described above for the preparation of T cells for adoptive cell therapy. In some embodiments, the adoptive cell-treated T cell is a CAR-T cell or a TCR-T cell.

In one aspect, the invention relates to a method of making a CAR-T cell or a TCR-T cell comprising introducing into any of the genetically engineered T cells a Chimeric Antigen Receptor (CAR) or a nucleotide encoding thereof, or an engineered T Cell Receptor (TCR) or a nucleotide encoding thereof.

In one aspect, the invention relates to a method of making a CAR-T or TCR-T cell comprising:

(i) introducing into a T cell a sgRNA comprising a TRAC genome targeted to chromosome 14 from position 23016448 to position 23016490 to disrupt the TRAC genomic region; and/or

(ii) Introducing a sgRNA comprising a B2M genomic region targeted to chromosome 15 from position 45003745 to position 45003788 into the T cell to disrupt the B2M genomic region; and/or

(iii) Introducing into the T cell a sgRNA comprising a PD-1 genomic region targeted to chromosome 2 from position 242800936 to position 242800978, or to chromosome 2 from position 242795009 to position 242795051, to disrupt the B2M genomic region; and

(iv) introducing into the T cell a Chimeric Antigen Receptor (CAR) or a nucleotide encoding thereof, or an engineered T Cell Receptor (TCR) or a nucleotide encoding thereof. In some embodiments, the methods further comprise introducing CAS9, or a nucleotide encoding it, into the T cell.

In some embodiments, the method of making a CAR-T or TCR-T cell comprises:

(iii) (ii) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14-22 to effect editing of the PD-1 genomic region of chromosome 2 from position 242800936 to position 242800978, or the PD-1 genomic region of chromosome 2 from position 242795009 to position 242795051; and

In some embodiments, the method of making a CAR-T or TCR-T cell comprises:

(iii) (ii) a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 14 or SEQ ID NO: 15 to effect editing of the PD-1 genomic region of chromosome 2 from position 242800936 to position 242800978, or the PD-1 genomic region of chromosome 2 from position 242795009 to position 242795051; and

In some embodiments, the method of making a CAR-T or TCR-T cell comprises:

(iii) Converting a polypeptide comprising SEQ ID NO: 15 to effect editing of the PD-1 genomic region of chromosome 2 from position 242800936 to position 242800978, or the PD-1 genomic region of chromosome 2 from position 242795009 to position 242795051; and

In some embodiments, the method of making a CAR-T or TCR-T cell comprises:

(iii) Converting a polypeptide comprising SEQ ID NO: 14 to effect editing of the PD-1 genomic region of chromosome 2 from position 242800936 to position 242800978, or the PD-1 genomic region of chromosome 2 from position 242795009 to position 242795051; and

In some embodiments, the method of making a CAR-T or TCR-T cell comprises:

In some embodiments, the trc-targeted sgRNA and the B2M-targeted sgRNA are introduced simultaneously into T cells. In some embodiments, the trc-targeted sgRNA, the B2M-targeted sgRNA, and the PD-1-targeted sgRNA are introduced simultaneously into the T cell.

In some embodiments, the sgrnas described above (including a trc-targeted sgRNA, a B2M-targeted sgRNA, and/or a PD-1-targeted sgRNA) are introduced into the T cell by electroporation. In some embodiments, the sgrnas described above (including the sgRNA targeted to TRAC, the sgRNA targeted to B2M, and/or the sgRNA targeted to PD-1) are introduced into the T cell by electrotransformation with Cas9 encoding nucleotides (e.g., mRNA). In some embodiments, the electrotransfer conditions include any one selected from the group consisting of: 150-250V, 0.5-2 ms; 180 ℃ at 250V for 0.5-2 ms; 150V, 2 ms; 160V, 2 ms; 170V, 2 ms; 180V, 2 ms; 190V, 1 ms; 200V, 1 ms; 210V, 1 ms; 220V, 1 ms; 230V, 1 ms; 240V, 1 ms; 250V, 0.5 ms.

In some embodiments, the method comprises introducing a sgRNA targeted to TRAC, a sgRNA targeted to B2M, and/or a sgRNA targeted to PD-1 and a CAR or encoding nucleotides thereof, or an engineered T Cell Receptor (TCR) or encoding nucleotides thereof simultaneously into the T cell.

In some embodiments, the CAR or its encoding nucleotide, or the engineered T Cell Receptor (TCR) or its encoding nucleotide, is introduced into the T cell prior to the sgRNA targeting TRAC, the sgRNA targeting B2M, and/or the sgRNA targeting PD-1; or introducing the CAR or its encoding nucleotide, or the engineered T Cell Receptor (TCR) or its encoding nucleotide, into the T cell after introducing the sgRNA targeting B2M and/or the sgRNA targeting PD-1.

In one aspect, the invention relates to a CAR-T cell or TCR-T cell prepared by the above method.

In one aspect, the invention relates to a CAR-T cell comprising the above-described genetically engineered T cell expressing a Chimeric Antigen Receptor (CAR).

In one aspect, the invention relates to a CAR-T cell, wherein in the CAR-T cell:

(iii) The PD-1 genomic region of chromosome 2 from position 242800936 to position 242800978 has any of the gene sequence alterations described in Table F, or the PD-1 genomic region of chromosome 2 from position 242795009 to position 242795051 has any of the gene sequence alterations described in Table G.

In one aspect, the invention relates to a TCR-T cell comprising a genetically engineered T cell as described above expressing an engineered TCR.

In one aspect, the invention relates to a TCR-T cell, wherein in the TCR-T cell:

The above-mentioned positional information of the TRAC genomic region, B2M genomic region, PD-1 genomic region of the present invention is based on a reference database: the wild sequence position information of the gene in GRCh37(hg 19). The skilled person knows how to obtain the corresponding positional information of the above genomic regions with reference to other databases.

In some specific embodiments, the wild-type nucleotide sequence of the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490 is set forth in SEQ ID NO. 23 (tatccagaaccctgaccctgccgtgtaccagctgagagactct). In some specific embodiments, the wild-type nucleotide sequence of the B2M genomic region from position 45003745 to position 45003788 of chromosome 15 is set forth in SEQ ID NO. 24 (atgtctcgctccgtggccttagctgtgctcgcgctactctctct). In some specific embodiments, the wild-type nucleotide sequence of the PD-1 genomic region from position 242800936 to position 242800978 of chromosome 2 is shown in SEQ ID NO. 25 (agcccagttgtagcaccgcccagacgactggccagggcgcctg). In some specific embodiments, the wild-type nucleotide sequence of the PD-1 genomic region from position 242795009 to position 242795051 of chromosome 2 is shown in SEQ ID NO. 26 (cagtttagcacgaagctctccgatgtgttggagaagctgcagg).

In one aspect, the invention also relates to compositions (e.g., pharmaceutical combinations), kits, and medical articles of manufacture comprising the genetically engineered T cells, CAR-T cells, or TCR-T cells described above.

In one aspect, the invention relates to a method of treating a disease in a subject, comprising administering to the subject an effective amount of a CAR-T or TCR-T cell as described above. In some embodiments, the disease is a tumor. In some embodiments, the tumor is a hematological tumor. In some embodiments, the tumor is a lymphoma or leukemia. In some embodiments, the CAR targets a Tumor Specific Antigen (TSA) and/or a Tumor Associated Antigen (TAA), such as an antigen shown in table a herein (e.g., CD 19).

In one aspect, the invention relates to a polypeptide comprising SEQ ID NOs:2-22 or a vector thereof. In some embodiments, the sgRNA is chemically modified, e.g., a2 '-O-methyl analog and/or an internucleotide 3' thio modification. In some embodiments, the chemical modification is a modification of the 2 ' -O-methyl analog of the first, two, and/or three bases of the 5 ' end and/or the last base of the 3 ' end of the sgRNA.

Specifically, the present invention relates to:

a method of making a genetically engineered T cell, comprising: disrupting in the T cell by gene editing techniques:

(i) the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490;

(ii) the B2M genomic region from chromosome 15 from position 45003745 to position 45003788; and/or

(iii) The PD-1 genomic region from 242800936 th to 242800978 th on chromosome 2, or the PD-1 genomic region from 242795009 th to 242795051 th on chromosome 2.

The method of clause 2. clause 1, wherein the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region are all edited.

Item 3. the method of item 1 or 2, wherein the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology, or a CRISPR/Cas gene editing technology.

Item 4 the method of item 3, wherein the gene editing technology is CRISPR/Cas9 gene editing technology.

The method of any one of items 1-4, wherein:

(i) the target nucleotide sequence of the TRAC genome is identical to a sequence selected from SEQ ID NOs: 2-5;

(ii) the target nucleotide sequence of the B2M genome is identical to a nucleotide sequence selected from SEQ ID NOs: 6-13 is complementary to a sequence of any one of the above; and/or

(iii) The target nucleotide sequence of the PD-1 genome is identical to a nucleotide sequence selected from SEQ ID NOs: 14-22, respectively.

The method described in item 6 or item 4, wherein:

(i) introducing into the T cell a sgRNA comprising a region targeted to the TRAC genome to effect editing of the TRAC genome region;

(ii) introducing into the T cell a sgRNA comprising a gene targeting the B2M genome to effect editing of the B2M genomic region; and/or

(iii) Introducing a sgRNA comprising a region targeted to the PD-1 genome into the T cell to effect editing of the PD-1 genome region.

The method of item 7, item 6, comprising:

(i) (ii) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5 to effect editing of the TRAC genomic region;

(ii) (ii) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 6-14 is introduced into the T cell to effect editing of the B2M genomic region; and/or

(iii) (ii) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15-22 is introduced into the T cell to effect editing of the PD-1 genomic region.

The method of item 8, item 7 or 8, comprising simultaneously introducing into the T cell the sgRNA targeting TRAC, the sgRNA targeting B2M, and the sgRNA targeting PD-1.

The method of any one of items 6-8, 9, wherein the sgRNA is modified with a2 '-O-methyl analog and/or an internucleotide 3' thio.

The method of item 10, item 9, wherein the chemical modification is a2 ' -O-methyl analog modification of the 5 ' first, two, and/or three bases and/or the 3 ' last base of the sgRNA.

Item 11 the method of any one of items 6-10, wherein the sgRNA is co-introduced into the T cell with a Cas 9-encoding nucleotide sequence.

Item 12. the method of item 11, co-introducing the sgRNA into the T cell with a Cas 9-encoding nucleotide sequence by electroporation, preferably the electroporation conditions include any one selected from the group consisting of: 150-250V, 0.5-2 ms; 150V, 2 ms; 160V, 2 ms; 170V, 2 ms; 180V, 2 ms; 190V, 1 ms; 200V, 1 ms; 210V, 1 ms; 220V, 1 ms; 230V, 1 ms; 240V, 1 ms; and 250V, 0.5 ms.

The method of any one of items 1 to 12, further comprising screening the gene-edited T cells for T cells with low TRAC, B2M and/or PD-1 expression.

The method of any one of items 1-13, wherein the TRAC, B2M or PD-1 gene knockout efficiency is greater than 90%; the simultaneous knockout efficiency of the TRAC and the B2M gene is more than 75%; or the simultaneous knockout efficiency of the TRAC, B2M and PD-1 genes is more than 65%.

The method of any one of items 1-14, wherein the T cells are derived from a healthy subject.

A genetically engineered T cell prepared by the method of any one of claims 1-15.

A genetically engineered T cell, wherein in the T cell:

(iii) One or more sites of the PD-1 genomic region from 242800936 th to 242800978 th on chromosome 2, or the PD-1 genomic region from 242795009 th to 242795051 th on chromosome 2 are disrupted by gene editing techniques.

Use of the genetically modified T cell of item 16 or 17 to prepare a T cell for adoptive cell therapy.

The use of item 19 item 18, wherein the adoptive cell therapeutic T cell is a CAR-T cell or a TCR-T cell.

Item 20. a method of making a CAR-T cell or a TCR-T cell comprising introducing a Chimeric Antigen Receptor (CAR) or a nucleotide encoding thereof, or an engineered T Cell Receptor (TCR) or a nucleotide encoding thereof, into a genetically engineered T cell as described in item 16 or 17.

A method of making a CAR-T or TCR-T cell comprising:

(iv) introducing into the T cell a Chimeric Antigen Receptor (CAR) or a nucleotide encoding thereof, or an engineered T Cell Receptor (TCR) or a nucleotide encoding thereof.

The method of item 21, wherein a trc-targeted sgRNA, a B2M-targeted sgRNA, and/or a PD-1-targeted sgRNA and CAR or encoding nucleotides thereof or an engineered T Cell Receptor (TCR) or encoding nucleotides thereof are introduced into the T cell simultaneously.

Item 23 the method of item 21, wherein the CAR or its encoding nucleotide, or the engineered T Cell Receptor (TCR) or its encoding nucleotide, is introduced into the T cell prior to the sgRNA targeting the TRAC, the sgRNA targeting B2M and/or the sgRNA targeting PD-1; or introducing the CAR or its encoding nucleotide, or the engineered T Cell Receptor (TCR) or its encoding nucleotide into the T cell after introducing the grna targeting TRAC, the sgRNA targeting B2M, and/or the sgRNA targeting PD-1.

The method of any one of items 20-23, wherein the CAR-T is a universal CAR-T; or wherein the TCR-T is a universal TCR-T.

The CAR-T cell or TCR-T cell prepared by the method of any of claims 20-24.

A CAR-T cell comprising a genetically engineered T cell of item 16 or 17 that expresses a Chimeric Antigen Receptor (CAR).

A TCR-T cell comprising a genetically engineered T cell as described in item 16 or 17 which expresses an engineered TCR.

A composition comprising the genetically engineered T cell of item 16 or 17, the CAR-T cell of item 25 or 26, or the TCR-T cell of item 25 or 27.

A method of treating a disease in a subject, comprising administering to the subject an effective amount of a CAR-T cell of item 25 or 26, or a TCR-T cell of item 25 or 27.

The method of item 30, item 29, wherein the disease is a tumor.

The method of item 31, item 30, wherein the tumor is a hematological tumor.

The method of item 32, item 31, wherein the tumor is a lymphoma or leukemia.

The method of any of claims 29-32, wherein the CAR targets CD 19.

The method of any one of claims 29-33, wherein the T cells are not obtained from the subject.

Item 35. comprising SEQ ID NOs: 2-22.

The sgRNA of item 36, item 35, wherein the chemical modification is a2 '-O-methyl analog and/or an internucleotide 3' thio modification.

The sgRNA of item 37, item 36, wherein the chemical modification is a modification of a2 ' -O-methyl analog of the last base of the 5 ' end, two, and/or three bases and/or the 3 ' end of the sgRNA.

Brief Description of Drawings

Fig. 1 shows a comparison of the efficiency of the corresponding site knockout using chemically modified sgrnas and chemically unmodified sgrnas after 1 electrical transition.

Fig. 2A-2f fig. 2A shows B2M in HLA knockout using different sgrnas modified in combination with CRISPR/Cas9 gene knockout tools, and the knockout efficiency of B2M gene for each sgRNA was analyzed after knockout. Fig. 2B shows INDEL analysis of the T cell genome after B2M gene knock-out by INDEL analysis software on different modified sgrnas, resulting in the efficiency of INDEL generation in T cells. Fig. 2C shows the knock-out of TRAC in TCR using different sgrnas modified in combination with CRISPR/Cas9 gene knock-out tool, and the knock-out efficiency of each sgRNA for TRAC gene was analyzed after knock-out. Fig. 2D shows INDEL analysis of T cell genome after TRAC gene knock-out by INDEL analysis software on different modified sgrnas, resulting in the efficiency of INDEL generation in T cells. Fig. 2E shows the PD-1 knock-out using modified different sgrnas in combination with CRISPR/Cas9 gene knock-out tool, and the knock-out efficiency of PD-1 gene by each sgRNA was analyzed after knock-out. Fig. 2F shows INDEL analysis of the T cell genome after PD-1 gene knock-out by INDEL analysis software on different modified sgrnas, resulting in the efficiency of INDEL generation in T cells.

Fig. 2A shows that B2M in T cells is knocked out under the same electrotransfer condition by using an In Vitro Transcription (IVT) and modified and optimized sgRNA-combined CRISPR/Cas9 gene knock-out tool, and after knocking-out, the efficiency of knocking-out the B2M gene by each sgRNA is analyzed as follows: b1: 27.88%, B2: 86.17%, B3: 64.69%, B4: 1.06%, B5: 2.91%, B6: 0.17%, B7: 41.87% and B8: 3.14 percent. The sgRNA with better knockout efficiency can be selected according to the figure. Through 2 '-O-methyl analogue and/or 3' thio optimization modification among nucleotides to the selected sgRNA, a possibility is provided for high-efficiency double/triple gene knockout (as shown in figure 3). Fig. 2B shows INDEL analysis of T cell genome after B2M gene knock-out by INDEL analysis software on different sgrnas, resulting in efficiency of INDEL generation in T cells. Fig. 2C shows that In Vitro Transcription (IVT) and modified optimized sgrnas are combined with a CRISPR/Cas9 gene knockout tool to knock out TRAC in T cells under the same electrotransfer condition, and after knockout, the knockout efficiency of the TRAC gene by each sgRNA is analyzed as follows: t2: 77.84%, T3: 85.86%, T4: 2.59% and T6: 34.78 percent. The sgRNA with better knockout efficiency can be selected according to the figure. Through the optimized modification of the selected sgRNA, high-efficiency single/double/triple gene knockout (shown in figure 3) is possible. Fig. 2D shows INDEL analysis of T cell genome after TRAC gene knock-out by INDEL analysis software on different sgrnas, resulting in the efficiency of INDEL generation in T cells. Fig. 2E shows that sgrnas subjected to In Vitro Transcription (IVT) and optimized modification described above are combined with a CRISPR/Cas9 gene knockout tool to knock out PD-1 in T cells under the same electrotransfer condition, and after knockout, the knockout efficiency of PD-1 gene by each sgRNA is analyzed as follows: p1: 21.15%, P2: 36.99%, P4: 23.03%, P5: 25.6%, P6: 3.1%, P7: 22.49%, P8: 23.07%, P9: 31.18% and P10: 24.48 percent. The sgRNA with better knockout efficiency can be selected according to the figure. Through the optimized modification of the selected sgRNA, the possibility of high-efficiency three-gene knockout (shown in figure 3) is provided. Fig. 2F shows INDEL analysis of T cell genome after PD-1 gene knock-out by INDEL analysis software on different sgrnas, resulting in the efficiency of INDEL generation in T cells.

FIG. 3 shows the Knock-Out results of TRAC, TRAC/B2M (Double Knock-Out, DKO) and TRAC/B2M/PD-1(Triple Knock-Out, TKO) for T cells using optimized sgRNA and CRISPR/Cas9 gene editing techniques. And chemically modifying the sgRNA, and then delivering the chemically modified sgRNA and Cas9 to primary T cells through further optimized electrotransfer conditions to perform related gene knockout. As shown in the figure, the knockout efficiency of the single gene, TRAC, is improved to 90.42%; the knockout efficiency of the double genes, namely TRAC and B2M, is improved to 81.39 percent. The knockout efficiency of the three genes, i.e., TRAC, B2M and PD-1, was increased to 67.91% (82.70% + 15.76%) 68.98%).

FIG. 4 shows phenotypic analysis of T cells screened for purification by TRAC, TRAC/B2M (DKO) and TRAC/B2M/PD-1(TKO) knockouts. And (4) optimizing the later purification conditions, namely increasing the purification times (4-5 times) and the corresponding antibody amount (3 mg/mL). The results are shown in the figure, and the purity of the optimized double knockout, namely TRAC and B2M double gene knockout is improved to 99.72%. The purity of three genes, namely TRAC, B2M and PD-1 knockout is improved to 98.62%.

FIG. 5 shows the results of off-target detection of TRAC-sgRNA3(T2), B2M-sgRNA2(B3) and PD-1-sgRNA2 (P2). The figure shows that the INDEL efficiency in T cells after T2, B3, P2sgRNA binding CRISPR/Cas9 editing is obtained by combining INDEL analysis software after deep sequencing.

Fig. 6 shows phenotype analysis after TCR and/or B2M and/or PD-1 knockout of T cells and screening and purification of TCR and/or B2M and/or PD-1 knockout T cells using optimized sgRNA and CRISPR/Cas9 gene editing techniques. The abscissa is CD3 and the ordinate is TCR, B2M and PD-1.

FIG. 7 shows validation and comparison of killing function of T cells, CAR-T, TCRneg CAR-T, DKO CAR-T and TKO CAR-T cells. In the experiment, Raji and K562 are used as target cells, T, CAR-T, DKO CAR-T and TKO CAR-T are used as effector cells, and in-vitro killing experiments are carried out according to the effective-to-target ratio of 10:1, 5:1, 2.5:1, 1.25:1 and 0.625: 1.

FIG. 8 shows T cell, CAR-T, TCRneg CAR-T, DKO CAR-T and TKO CAR-T cytokine release. In the experiment, Raji and K562 are taken as target cells, T, CAR-T, DKO CAR-T and TKO CAR-T are taken as effector cells, and IL-2 and IFN-gamma in the cells are detected by taking supernatant after in vitro co-culture according to the effective target ratio of 10:1, 5:1, 2.5:1, 1.25:1 and 0.625: 1.

FIGS. 9A-9B show saline injection, CAR-T, TCR^negCAR-T、DKO CAR-T(TCR^neg\B2M^negCAR-T) and TKO CAR-T (TCR)^neg\B2M^neg\PD-1^negCAR-T) cells, their tumor suppression and killing effect in NPG mice was compared. FIG. 9A shows that NSG mice are randomly divided into 4 groups, saline, T cell, TCR/CD3 after tail vein injection of 5X10^5 tumor cells/mouse^negThe CD19-CAR-T cell group, the DKO CD19-CAR-T cell group and the TKOCD19-CAR-T cell group, and then four groups of mice were injected with 5X10^6 corresponding cells through caudal vein, and the saline group was used as a control group. The tumor burden in the mice was analyzed by pictures from a platinum-elmer imager. Figure 9B images obtained by platinum elmer imager analysis of tumor burden in mice with mouse cell feeding days on the abscissa and radiation signal emitted per second per unit body surface area on the ordinate.

FIG. 10 shows saline, CAR-T, TCR^negCAR-T、DKO CAR-T(TCR^neg\B2M^negCAR-T) and TKO CAR-T (TCR)^neg\B2M^neg\PD-1^negCAR-T) comparison of survival of mice in group 5. The abscissa is the number of days the mouse was raised after injection of effector cells, and the ordinate is the survival rate of the mouse.

FIG. 11 shows the injection of saline, CAR-T, TCR^negCAR-T、DKO CAR-T(TCR^neg\B2M^negCAR-T) and TKO CAR-T (TCR)^neg\B2M^neg\PD-1^negComparison of the results of the change in body weight of mice after CAR-T) cells. NPG mice were monitored for weight, with the abscissa being the number of days the mice were fed after injection of effector cells and the ordinate being the percentage of weight of the mice (n-4).

Detailed Description

Definition of

As used herein, "CRISPR/Cas" is a gene editing technique, including but not limited to various naturally occurring or artificially designed CRISPR/Cas systems, such as the CRISPR/Cas9 system. The Naturally occurring CRISPR/Cas system (Naturally acuurring CRISPR/Cas system) is an adaptive immune defense formed by bacteria and archaea during long-term evolution, and can be used to fight against invading viruses and foreign DNA. For example, the CRISPR/Cas9 works on the principle that crRNA (CRISPR-derived RNA) binds to tracrRNA (trans-activating RNA) by base pairing to form a tracrRNA/crRNA complex that directs the nuclease Cas9 protein to cleave double-stranded DNA at sequence target sites paired with the crRNA. By artificially designing tracrRNA and crRNA, sgrna (single guide rna) with guiding function can be engineered to guide Cas9 to cut DNA site-specifically. As an RNA-guided dsDNA binding protein, Cas9 effector nuclease is able to co-localize RNA, DNA and proteins, thus possessing tremendous engineering potential. CRISPR/Cas systems may use one, two or three classes of Cas proteins. In some embodiments of the invention, the method uses Cas 9. Other suitable CRISPR/Cas systems include, but are not limited to, the systems and methods described in WO2013176772, WO2014065596, WO2014018423, US8,697,359.

In the present invention, "sgrna (single guide RNA)" and "grna (guide RNA)" may be used interchangeably as "single guide RNA", "synthetic guide RNA" or "guide RNA". The sgrnas of the present invention comprise a guide sequence (guide sequence) that targets a target sequence.

The TCR is composed of α, β peptide chains, each of which can be divided into variable region (V region), constant region (C region), transmembrane region and cytoplasmic region, the TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region, the V region (V α, V β) has three hypervariable regions CDR1, CDR2 and CDR3, of which the variation with CDR3 is the largest and the antigen binding specificity of the TCR is directly determined, when the TCR recognizes the MHC-antigen peptide complex, CDR1, CDR2 recognizes and binds to the side wall of the MHC antigen binding groove of the MHC molecule, and CDR3 directly binds to the antigen peptide, the TCR1 and TCR2 are divided into two categories, TCR1 is composed of two of γ and δ, TCR2 is composed of two TCR α and β chains, TCR 90% -95% of T cells express one of TCR 585 and TCR1 and TCR2 express only one of TCR1 and TCR1 in peripheral blood.

"β 2 microglobulin (B2M)" is β chain (light chain) part of cell surface Human Leukocyte Antigen (HLA), and is a single-chain polypeptide with molecular mass of 11800 and composed of 99 amino acids.

"programmed death receptor 1 (PD-1)", a membrane protein of 268 amino acid residues, was originally cloned from apoptotic mouse T cell hybridoma 2B 4.11. PD-1 and PD-L1 bind to initiate programmed death of T cells, allowing tumor cells to gain immune escape, and thus, are important immunosuppressive molecules.

As used herein, "Indel" is collectively referred to as insertion/deletion, i.e., insertion and deletion mutations.

"graft versus host response" (GVHD) refers to a response that results from an immunogenetic difference between the donor and recipient, for example, in one aspect, when donor cells, such as T lymphocytes from an immunocompetent donor, enter the recipient patient and proliferate to a certain extent, the target is mistaken for normal cells or tissues of the recipient patient for attack. On the other hand, as allogeneic cells, the normal immune system in the recipient may also clear them to produce a "Host Versus Graft Response (HVGR)".

The HVGR and GVHR associated genes comprise TCR, HLA molecule associated genes, and the T lymphocytes knocked out simultaneously of these genes do not cause Graft Versus Host Disease (GVHD) when the T lymphocytes are reinfused into allogeneic patients, so the T lymphocytes can be called as 'universal T cells'.

"CAR-T" is a shorthand form of "chimeric antigen receptor T-cells", wherein the Chimeric Antigen Receptor (CAR) is the core component of CAR-T, conferring on T-cells the ability to recognize target cell (e.g., tumor) antigens in an HLA-independent manner, which enables CAR-engineered T-cells to recognize a broader range of targets than native T-cell surface receptor TCRs. In some implementations, a tumor-targeting CAR is designed to include a tumor-associated antigen (TAA) binding region (e.g., a scFV fragment that is typically derived from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The choice of antigen of interest is a key determinant for the specificity, efficacy of the CAR and safety of the genetically engineered T cells themselves.

By "universal CAR-T cell" is meant a CAR-T cell that is capable of targeting a specific target cell (e.g., tumor) associated marker and inactivating cell surface TCR and MHC functions, which can reduce immune rejection by allogeneic cell therapy.

The CAR-T treatment of autologous cells requires blood extraction and separation for the preparation of autologous T lymphocytes of patients, on one hand, the patients have more influence factors in the CAR-T production process with different patient conditions and T lymphocyte states, and the safety is influenced because standardized production cannot be realized, on the other hand, the activity and the quantity of autologous T lymphocytes of some patients are insufficient after chemotherapy, or the activity and the proliferation capacity of the T lymphocytes are limited because of the influence of tumor environment, the difficulty of the cells in the CAR-T preparation is often high, and the safety and the effectiveness of the treatment are influenced; or if the cells are prepared in a sudden situation and cannot be timely returned to the patient in the CAR-T cell preparation process, the treatment effect can be influenced, even the autologous T lymphocyte state influences that some tumor patients cannot receive autologous CAR-T cell adoptive therapy; the general CAR-T or general T lymphocytes available for allogeneic therapy are of great advantage in these cases.

TCR-T (T cell receptor (TCR) chimeric-T cell) refers to a T cell that expresses an engineered T cell receptor (engineerdTCR) or an artificial T cell receptor (artificial TCR). The engineered or artificial T cell receptor has been genetically modified to have a structure that targets an antigen of interest while also retaining the domains and/or accessory molecules in the TCR signaling pathway. In certain embodiments, the TCR-T retains all of the accessory molecules in the TCR signaling pathway and thus, upon stimulation with a small amount of antigen, a fully activated state can occur, resulting in a killing effect on the target cells. These TCR-Ts retain and utilize all accessory molecules on the TCR signaling pathway relative to CAR-T, and therefore TCR-T is more sensitive to recognition of low concentrations, low copy number antigens than some CAR-Ts and has a great therapeutic potential.

In certain embodiments, the TCR-T cells have improved the Affinity of the TCR for the corresponding antigen (e.g., TAA) by a partial gene modification approach, and thus the "gene modified TCR" technique is also referred to as "Affinity-Enhanced TCR" technique (Affinity-Enhanced TCR). For example, the "genetically modified TCR" developed by Adaptimune company reported in Nature-medicine journal, after several key amino acids have been modified, the genetically modified TCR has greatly improved affinity for a common cancer TAA, NY-ESO-1. And thus can be used to attack cancers with overexpression of NY-ESO-1, such as Multiple Myeloma (Multiple Myeloma).

Adoptive Cell Therapy (ACT), Adoptive immunotherapy (Adoptive immunotherapy), such as tumor Adoptive immunotherapy (tumor Adoptive immunotherapy), refers to a therapeutic method in which immune cells are treated in vitro, for example, specific antigens are added, molecules expressed by immune cells are modified or stimulated by cytokines, etc., target cells (e.g., tumors) with high specificity are screened and amplified in large quantities, and then the target cells (e.g., tumors) are killed, and is a passive immunotherapy.

Method for efficient editing of T cells

In one aspect of the invention, there is provided a method of making a gene-edited T cell (e.g., a universal T cell) comprising disrupting, by gene-editing techniques, in the T cell: (i) the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490 (shown in SEQ ID NO: 23); (ii) the B2M genomic region from chromosome 15 from position 45003745 to position 45003788 (shown as SEQ ID NO: 24); and/or (iii) the PD-1 genomic region from position 242800936 to position 242800978 of chromosome 2 (as shown in SEQ ID NO: 25), or the PD-1 genomic region from position 242795009 to position 242795051 of chromosome 2 (as shown in SEQ ID NO: 26). In some embodiments, the method the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology, or a CRISPR/Cas gene editing technology. In some embodiments, the TRAC genomic region, the B2M genomic region, or the PD-1 genomic region is edited. In some embodiments, wherein the TRAC genomic region, and the B2M genomic region are both edited. In some embodiments, wherein the TRAC genomic region and PD-1 genomic region are both edited. In some embodiments, wherein the B2M genomic region and the PD-1 genomic region are both edited. In some embodiments, wherein the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region are all edited.

In some embodiments, there is provided a method of making a gene-edited T cell (e.g., a universal T cell) comprising disrupting, by a gene-editing technique, in the T cell: (i) and a polypeptide selected from the group consisting of SEQ ID NOs: 2-5, a TRAC genomic target nucleotide sequence complementary to the sequence of any one of; (ii) and a peptide selected from the group consisting of SEQ ID NOs: 6-14, the target nucleotide sequence of the B2M genome being complementary in sequence; and/or (iii) to a polypeptide selected from SEQ ID NOs: 15-22, and a target nucleotide sequence of the PD-1 genome that is complementary to the sequence of any one of claims 15-22. In some embodiments, the method the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology, or a CRISPR/Cas gene editing technology. In some embodiments, the TRAC genomic region, the B2M genomic region, or the PD-1 genomic region is edited. In some embodiments, wherein the TRAC genomic region, and the B2M genomic region are both edited. In some embodiments, wherein the TRAC genomic region and PD-1 genomic region are both edited. In some embodiments, wherein the B2M genomic region and the PD-1 genomic region are both edited. In some embodiments, wherein the TRAC genomic region, the B2M genomic region, and the PD-1 genomic region are all edited.

In some embodiments, there is provided a method of making a gene-edited T cell (e.g., a universal T cell), comprising: the sgRNA targeting TRAC, the sgRNA targeting B2M, and/or the sgRNA of PD-1 are introduced into T cells to disrupt the TRAC, B2M, and/or PD-1 genes of the T cells. In some embodiments, the method comprises introducing Cas9 or its encoding nucleotide into a T cell.

In some embodiments, the sgRNA targets genes encoding the constant regions of the α and/or β chains of TCR2, thereby disrupting the structure of the T cell surface TCR, rendering the molecule non-functional.

In some embodiments, the sgRNA targets β 2 the gene encoding microglobulin (B2M), e.g., targets the first exon region of the B2M protein-encoding gene, thereby disrupting the structure of B2M, rendering the molecule non-functional.

In some embodiments, the sgRNA targets the first exon region of a gene encoding PD-1, e.g., a gene encoding a PD-1 protein, thereby disrupting the structure of PD-1 and rendering the molecule non-functional.

In some embodiments, there is provided a method of making a gene-edited T cell (e.g., a universal T cell), comprising: (i) (ii) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2-5 to effect editing of the TRAC genomic region; (ii) will comprise a sequence selected from SEQ id nos: 6-14 to effect editing of the B2M genomic region; and/or (iii) will comprise a nucleotide sequence selected from SEQ ID NOs: 15-22 is introduced into a T cell to effect editing of the PD-1 genomic region. In some embodiments, the method comprises introducing Cas9 or its encoding nucleotide into a T cell.

In some embodiments, the invention relates to introducing any one of the sgrnas selected from those shown in table 2 that targets TRAC into a T cell. In some embodiments of the invention, the invention relates to introducing into a T cell any one of the sgrnas shown in table 2 targeted to B2M. In some embodiments of the invention, the invention relates to introducing any one of the sgrnas selected from those shown in table 2 targeted to PD-1 into a T cell. In some embodiments, the method comprises introducing Cas9 or its encoding nucleotide into a T cell.

In some embodiments, the invention relates to introducing any sgRNA selected from those shown in table 2 that targets TRAC, and/or any sgRNA selected from those shown in table 2 that targets B2M, and/or any sgRNA selected from those shown in table 2 that targets PD-1, and Cas9 or its encoding nucleotide into a T cell.

In some embodiments, the invention relates to introducing any sgRNA selected from the group consisting of any sgRNA shown in table 2 that targets TRAC, and any sgRNA selected from the group consisting of any sgRNA shown in table 2 that targets B2M, and any sgRNA selected from the group consisting of any sgRNA shown in table 2 that targets PD-1 into a T cell. In some embodiments, the method comprises introducing Cas9 or its encoding nucleotide into a T cell.

In some embodiments of the invention, there is provided a method of preparing a universal T cell, comprising: (i) introducing a TRAC-sg3sgRNA into a T cell to effect editing of the TRAC genomic region; (ii) introducing a B2M-sg2sgRNA into a T cell to effect editing of the B2M genomic region; and (iii) introducing PD-1-sg2sgRNA into a T cell to effect editing of the PD-1 genomic region. In some embodiments, the method comprises introducing Cas9 or its encoding nucleotide into a T cell.

In some embodiments, there is provided a method of making a universal T cell, comprising: (i) converting a polypeptide comprising SEQ ID NO:2 or 3 into a T cell to effect editing of the TRAC genomic region; (ii) will comprise a sequence selected from seq id NOs: 7 or 11 into a T cell to effect editing of the B2M genomic region; and (iii) converting a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 14 or 15 into a T cell to effect editing of the PD-1 genomic region. In some embodiments, the method comprises introducing Cas9 or its encoding nucleotide into a T cell.

In some embodiments, the sgRNA is chemically modified. For example, 2 '-O-methyl analogs and/or internucleotide 3' thio modifications. In some embodiments, the chemical modification is a modification of the 2 ' -O-methyl analog of the first, two, and/or three bases of the 5 ' end and/or the last base of the 3 ' end of the sgRNA.

In general, a guide sequence in a sgRNA is any polynucleotide sequence that has sufficient complementarity to a target polynucleotide sequence to hybridize to the target sequence and direct sequence-specific binding of the CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is about or greater than about 80%, 85%, 90%, 95%, 97.5%, 99%, or more, when optimally aligned using an appropriate alignment algorithm. Optimal alignment may be determined using any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith-Waterman algorithm, Needleman-Wimsch algorithm, Burrows-Wheeler Transform-based algorithms (e.g., Burrows Wheeler Aligner), ClustalW, Clustai X, BLAT, Novoalign (Novocraft Technologies, ELAND ((Illumina, san diego, CA)), SOAP (available at SOAP. genomics. org. cn), and Maq (available at maq. sourceform. net.) in some embodiments, the guide sequence length may be about or greater than about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 65, 70, 75, or less than about the specific binding capacity of the guide sequence to a target sequence of any suitable CR sequence of the guide sequence of the SPR And (5) evaluating the determination method. For example, components of the CRISPR system (including the guide sequences to be tested) sufficient to form a CRISPR complex can be provided to a host cell having a corresponding target sequence, such as can be performed by transfection using a vector encoding the CRISPR sequence components followed by assessment of preferential cleavage within the target sequence. Likewise, cleavage of a target polynucleotide sequence can be assessed in a test tube by providing a package of the target sequence, the CRISPR complex (comprising the guide sequence to be tested and a control guide sequence different from the guide sequence), and comparing the binding or cleavage rates of the test and control guide sequences at the target sequence. Other assay methods known to those skilled in the art may also be used to perform the above assays and evaluations.

In some embodiments, the sgRNA targeting TRAC, the sgRNA targeting B2M, and/or the sgRNA targeting PD-1 and/or the Cas9 encoding nucleotide (e.g., mRNA) are introduced into the T cell by electrotransfer, e.g., by 150-250V, 0.5-2 ms; 180 ℃ at 250V for 0.5-2 ms; 150V, 2 ms; 160V, 2 ms; 170V, 2 ms; 180V, 2 ms; 190V, 1 ms; 200V, 1 ms; 210V, 1 ms; 220V, 1 ms; 230V, 1 ms; 240V, 1 ms; 250V, 0.5ms electrotransfer conditions were introduced into T cells. In some embodiments, the trc-targeted sgRNA, the B2M-targeted sgRNA, and the PD-1-targeted sgRNA are co-introduced into the T cell by electrotransfer with Cas 9-encoding nucleotides.

In some embodiments, the Cas9 encoding nucleotide is an mRNA, such as an mRNA containing an ARCA cap. In some embodiments, the Cas9 encoding nucleotide is in a viral vector, such as a lentiviral vector. In some embodiments, the Cas9 encoding nucleotide comprises the sequence set forth as SEQ ID NO: 1. In some embodiments, the trc-targeted sgRNA, B2M-targeted sgRNA, and/or PD-1-targeted sgRNA is in the same vector as the Cas 9-encoding nucleotide.

In some embodiments, a trc-targeted sgRNA, a B2M-targeted sgRNA, and a PD-1-targeted sgRNA are introduced into the T cell simultaneously. In particular embodiments, when a trc-targeted sgRNA, a B2M-targeted sgRNA, and a PD-1-targeted sgRNA are introduced into a T cell simultaneously, the amount between the TRAC-targeted sgRNA, the B2M-targeted sgRNA, and the PD-1-targeted sgRNA can be similar or equivalent. In some embodiments, the sgRNA targeting TRAC, the sgRNA targeting B2M, and the sgRNA targeting PD-1 are introduced into the T cell individually, in any suitable order. In some embodiments, a trc-targeted sgRNA, a B2M-targeted sgRNA, and/or a PD-1-targeted sgRNA is introduced into a T cell simultaneously with a Cas 9-encoding nucleotide. In some embodiments, the Cas9 encoding nucleotide is introduced into the T cell prior to the sgRNA targeting TRAC, the sgRNA targeting B2M, and/or the sgRNA targeting PD-1. In some embodiments, the T cell comprises a Cas9 encoding nucleotide or a Cas9 protein.

In some embodiments, the T cell is derived from a healthy human. In some embodiments, the T cells are derived from a patient, such as a cancer patient, e.g., a cancer patient prior to chemotherapy or radiation therapy. In some embodiments, the T cell source is umbilical cord blood, bone marrow, or Peripheral Blood Mononuclear Cells (PBMC). In some embodiments, the T cells are derived from stem cells, such as hematopoietic stem cells at various stages of differentiation. The preparation methods described herein may be used to knock out TRAC, B2M and/or PD-1 in, for example, PBMC or stem cells, and further culture, differentiate, and/or purify the corresponding genetically engineered T cells.

Compared with the prior art, the T cell gene knockout method provided by the invention has high gene knockout efficiency, for example, single gene (TRAC), double gene (TRAC and B2M) and triple gene (TRAC, B2M and PD-1) knockout efficiency is respectively at least 90%, 81% and 67%.

"knockout efficiency" can be expressed at the gene level as the efficiency of INDEL to produce a knockout, and at the cellular level as the percentage of cells in which the knockout results in the disappearance or significant reduction of the gene-expressed protein. In the present invention, "knockout efficiency" means knockout efficiency calculated based on the latter. As will be appreciated by those skilled in the art, high knockdown efficiency can increase the yield of cells of interest, reducing production and treatment costs.

In some embodiments, the gene-edited T cells (e.g., universal T cells) are further screened for higher purity single-gene (TRAC), double-gene (TRAC and B2M), and triple-gene (TRAC, B2M, and PD-1) knockout T cells. For example, FACS may be used to screen T cells (e.g., general purpose T cells) for gene editing with low expression of TRAC, B2M and/or PD-1.

In some embodiments, the TCR and \ or HLA and \ or PD-1 genes of the universal T cells of the invention are knocked out.

In some embodiments, the α chain constant coding region (TRAC) gene of the TCR is knocked out, B2M and/or the coding region of PD-1 are knocked out, and TRAC, B2M and PD-1 can be knocked out, one of the three can be knocked out, or two of the three can be knocked out.

For example, in particular embodiments, the TCR α chain constant coding region gene of the invention is knocked out by introducing one of the TRAC-sg2, 3, 4, 6 molecules (see Table 2) and the Cas9 molecule into the cell.

In other specific embodiments, the HLA constant coding region B2M gene is knocked out. For example, in a specific embodiment, the B2M constant coding region gene of the invention is introduced into one of the B2M-sg 1-8 molecules (see Table 2) and Cas9 molecule of the cell, preferably the B2M constant coding region gene is introduced into the B2M-sg2 or B2M-sg6 molecule and Cas9 molecule of the cell for knock-out.

In other specific embodiments, the gene encoding the constant coding region of PD-1 is knocked out. For example, in a specific embodiment, the PD-1 constant coding region gene of the invention is introduced into one of the PD-1-sg1-2, PD-1-sg4-10 molecules (see Table 2) and the Cas9 molecule of the cell, preferably the PD-1 constant coding region gene is introduced into the PD-1-sg1 or PD-1-sg2 molecule and the Cas9 molecule of the cell for knock-out.

In other specific embodiments, the α chain constant coding region (i.e., TRAC) gene and the B2M gene of the TCR are knocked out, for example, in specific embodiments, the TRAC and B2M genes of the invention are knocked out by TRAC-sg3 and B2M-sg2 molecules (see Table 2) and Cas9 molecules introduced into the cells.

In other specific embodiments, the α chain constant coding region (i.e., TRAC) gene, B2M gene, and the constant coding region gene of PD-1 of the TCR are knocked out, for example, in specific embodiments, the TCR, HLA, and PD-1 constant coding region genes of the invention are knocked out by TRAC-sg3, B2M-sg2, and PD-1-sg2 molecules (see Table 2) and Cas9 molecules introduced into the cells.

In some embodiments, the present invention provides a method for efficiently editing T cells, the method comprising the steps of:

introduction of sgRNA molecule and Cas9 molecule in T cells:

in some embodiments, the sgRNA molecule comprises a targeting domain that is complementary to a gene target region from the α chain constant coding region (i.e., TRAC) gene, the B2M gene, and the constant coding region of PD-1 of the TCR.

In some embodiments, the sgRNA molecule refers to a nucleic acid sequence comprising a targeting domain complementary to a target region of a gene to be knocked out, which recognizes the target DNA sequence and directs the Cas9 molecule to cleave the target site, which can achieve a one-step efficient (knock out efficiency of 85% or more) knock out of the corresponding site.

In some embodiments, the sgRNA molecule comprises a targeting domain having a sequence as set forth in one of table 2.

In some preferred embodiments, the targeting domain has the sequence shown as T2, B3, and P1.

In some preferred embodiments, the sgRNA molecule and mRNA encoding Cas9 molecule are introduced into the T cell by electrotransfer techniques.

In some specific embodiments, the T cells used in the above methods are from healthy humans, such as healthy adult peripheral blood, or umbilical cord blood of naturally delivered healthy humans.

In some embodiments, a third aspect of the invention provides sgRNA sequences with high efficiency editing after specific modifications (table 2).

In some specific embodiments, the sgrnas are chemically synthesized and modified to make the sgrnas more stable and more efficient to edit than sgrnas obtained by general In Vitro Transcription (IVT). Preferably, the sgRNA gene synthesized chemically and modified by one electroporation of T cells has an editing efficiency 10 times or more higher than that of sgRNA obtained by conventional IVT.

In some embodiments, there is provided the use of a universal T cell according to the invention for the preparation of a medicament for the treatment of a disease (such as a tumor).

In some specific embodiments, the α chain constant coding region (i.e., TRAC) gene, the HLA constant coding region B2M gene, and the PD-1 constant coding region gene of the TCR are knocked out, for example, in specific embodiments, the TCR α chain constant coding region gene of the invention is introduced into the TRAC-sg2 molecule of the cell, the B2M constant coding region gene of the invention is introduced into the B2M-sg6 molecule of the cell, the PD-1 constant coding region gene of the invention is introduced into the PD-1-sg1 (see Table 2) and the Cas9 molecule of the cell, preferably, the TCR α chain constant coding region gene is knocked out by the TRAC-sg2 of the cell, the B2M constant coding region gene is introduced into the B2M-sg6 of the cell, the PD-1-sg1 molecule and the Cas9 molecule of the cell.

Highly efficient editing T cells or universal T cells

The present invention relates to TRAC single gene knockout T cells (TRACs) prepared by the above-described method of the present invention^negative) TRAC/B2M Double Knockout (DKO) T cells and TRAC/B2M/PD-1 Triple Knockout (TKO) T cells.

Compared with the prior art, the single gene knockout T cell (TRAC) of the invention^negative) The gene knockout efficiency of TRAC/B2M double-gene knockout (DKO) T cells and TCR/B2M/PD-1 triple-gene knockout (TKO) T cells is greatly improved.

Single gene knockout T cells (TRACs) prepared by the invention^negative) TRAC/B2M Double Knockout (DKO) T cells and TCR/B2M/PD-1 Triple Knockout (TKO) T cells can be used as precursor cells for further modification of T cells or as universal T cells for preparing various genetically modified T cells, for example, can be used for preparing CAR-T cells or TCR-T cells.

In some embodiments of the invention there is provided a genetically engineered T cell (e.g. a universal T cell), wherein in the T cell: (i) one or more sites of the TRAC genomic region from chromosome 14 from position 23016448 to position 23016490 are disrupted by gene editing techniques; (ii) one or more sites of the B2M genomic region from chromosome 15 from position 45003745 to position 45003788 were disrupted by gene editing techniques; and/or (iii) the PD-1 genomic region from chromosome 2 from position 242800936 to position 242800978, or the PD-1 genomic region from chromosome 2 from position 242795009 to position 242795051, is disrupted by gene editing techniques. 2-5 by gene editing techniques; (ii) and a polypeptide selected from the group consisting of SEQ ID NOs: 6-14, the target nucleotide sequence of the B2M genome, which is complementary in sequence, is disrupted by gene editing techniques; and/or (iii) to a polypeptide selected from SEQ ID NOs: 15-22 is disrupted by gene editing techniques.

In some embodiments of the invention there is provided a genetically engineered T cell (e.g. a universal T cell), wherein in the T cell: (i) wherein the TRAC genome comprises the sequences set forth in any one of tables D and E;

(ii) wherein the B2M genome comprises the sequences of any one of tables B and C; and/or

(iii) The PD-1 genome comprises any one of the sequences shown in Table F or Table G.

In one aspect, the invention provides genetically engineered T cells (e.g., universal T cells), CAR-T cells, or TCR-T cell compositions, such as pharmaceutical compositions, comprising the same.

In one aspect, the invention also provides a kit or article of manufacture comprising a genetically engineered T cell (e.g., a universal T cell) according to the invention. The kit or article of manufacture can be used to prepare CAR-T, TCR-T or other adoptive cell therapeutic compositions.

Method for producing CAR-T cells

In one aspect, the invention provides methods of making CAR-T cells (e.g., universal CAR-T cells). In some embodiments, the method comprises introducing a CAR or a nucleotide encoding or vector thereof into any of the genetically engineered T cells described herein (e.g., universal T cells).

In some embodiments, there is provided a method of making a CAR-T cell, comprising:

(iv) introducing into the T cell a Chimeric Antigen Receptor (CAR) or a nucleic acid encoding the CAR.

The CAR or nucleic acid encoding it and the sgRNA targeting TRAC, the sgRNA targeting B2M and/or the sgRNA targeting PD-1 and the CAR or nucleotides encoding them can be introduced into the T cell in any suitable order. In some embodiments, a trc-targeted sgRNA, a B2M-targeted sgRNA, and/or a PD-1-targeted sgRNA and a CAR, or encoding nucleotides thereof, are introduced into the T cell simultaneously. In some embodiments, the CAR or its encoding nucleotide is introduced into the T cell prior to the sgRNA targeting TRAC, the sgRNA targeting B2M, and/or the sgRNA targeting PD-1. In some embodiments, the CAR or its encoding nucleotide is introduced into a T cell in which gene editing has been achieved, the TRAC, B2M and/or PD-1 genomic region of the T cell having been disrupted by editing. In some embodiments, the method further comprises introducing Cas9 or its encoding nucleotide into the T cell along with the sgRNA. In some embodiments, the CAR expressed by the universal CAR-T cells of the invention can be any CAR known in the art as long as it enables the T cells to recognize cell surface antigens in a human leukocyte antigen-independent manner, exerting a killing effect. For example, the CARs disclosed in U.S. patent application No. US20140271635a1 may be used, and the CARs used in specific embodiments of the present invention may be referred to the present patent application publication (US20140271635a 1). In some embodiments, the CAR in the CAR-T cells of the invention is a CAR that recognizes an antigen described in table a below tumor and related antigens.

TABLE A

In some embodiments, the CAR expressed in the CAR-T cell of the invention comprises a signal peptide, an extracellular binding region, a hinge region, a transmembrane region, and an intracellular signal region, connected in sequence. The term "signal peptide" as used herein refers to a short (e.g., 5-30 amino acids in length) peptide chain that directs the transfer of a newly synthesized protein to the secretory pathway. In the present invention, signal peptides of various proteins in the human body, for example, signal peptides of cytokine proteins secreted in the body, leukocyte differentiation antigen (CD molecule) can be used.

In some embodiments, the signal peptide is a CD8 signal peptide, for example the amino acid sequence of which is as shown in the patent application US20140271635a 1.

In some embodiments, the hinge region may employ the hinge region of a variety of different antibodies or antigen receptors, particularly the hinge region of a CD molecule. In a particular embodiment, the hinge region may be selected from the hinge region of proteins such as CD8 or CD 28. The CD8 or CD28 is a natural marker of the T cell surface.

In the present invention, transmembrane domains of various human proteins, particularly those of various antigen receptors, can be used. The preferred transmembrane region used is that of the CD molecule. In one embodiment, the transmembrane region may be selected from the transmembrane regions of proteins such as CD8 or CD28 or 4-1 BB.

In some embodiments, the hinge region is a CD8 α hinge region (CD8-hinge), the amino acid sequence of which is shown in the inventive patent application US20140271635a 1.

The "extracellular binding domain" refers to a domain that comprises a region that specifically recognizes a target antigen. In some embodiments, the extracellular binding region comprises a region that specifically recognizes a target tumor cell surface antigen. For example, this region may be an antigen-binding fragment of an scFv or other antibody. The term "scFv" as used herein refers to a recombinant protein of variable heavy chain regions (VH and light chain variable regions (VL)) linked by a linker region (linker) that allows the two domains to associate and ultimately form an antigen binding site.

The CARs and domains thereof used in the invention can be further modified by using conventional techniques known in the art, such as amino acid deletions, insertions, substitutions, additions, and/or recombinations and/or other modifications, alone or in combination. Methods for introducing such modifications into the DNA sequence of an antibody based on its amino acid sequence are well known to those skilled in the art (see, e.g., Sambrook molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y.). The modification is preferably carried out at the nucleic acid level.

The term "specifically recognizes" as used herein means that the antigen recognition region of the present invention does not cross-react or does not substantially cross-react with any polypeptide other than the antigen of interest. The degree of specificity can be determined by immunological techniques including, but not limited to, immunoblotting, immunoaffinity chromatography, flow cytometry, and the like.

In some embodiments, the extracellular binding region comprises an antigen binding region, such as an scFv, that specifically recognizes CD19, CEA, EGFR, GD2, CD7, or CD138, and the like.

In some embodiments, the extracellular binding region comprises a humanized engineered scFv that specifically recognizes CD 19. In some embodiments, the amino acid sequence of the scFv that specifically recognizes CD19 is as set forth in patent application US20140271635a 1.

In the present invention, intracellular signaling regions of various human proteins, particularly intracellular signaling regions of various antigen receptors, can be used. The intracellular signaling region preferably used is that of a CD molecule. In particular embodiments, the intracellular signaling region may be selected from the group consisting of CD3 ζ, Fc ε RI γ, CD28, CD137(4-1BB), the intracellular signaling region of CD134 protein, and combinations thereof. The CD3 molecule consists of five subunits, of which the CD3 ζ subunit (also known as CD3zeta, abbreviated ζ) contains 3 ITAM motifs, which are important signaling regions in the TCR-CD3 complex. Fc epsilon riy is distributed primarily on the surface of mast cells and basophils, contains an ITAM motif and is similar in structure, distribution and function to CD3 ζ. In addition, as mentioned above, CD28, CD137, and CD134 are costimulatory signaling molecules, and the costimulation by their intracellular signaling segments upon binding to their respective ligands results in sustained T cell proliferation and can increase the levels of cytokines such as IL-2 and IFN- γ secreted by T cells, as well as increase the survival cycle and antitumor effects of CAR-T cells in vivo.

In certain embodiments, the signal produced by the TCR alone is insufficient to fully activate native T cells, sequences that initiate antigen-dependent primary activation by the TCR (primary intracellular signaling domain) and sequences that act in an antigen-independent manner to provide co-stimulatory signals (co-stimulatory domain), the primary signaling domain regulating primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner the primary intracellular signaling domain acting in a stimulatory manner may contain a signaling motif, referred to as an Immunoreceptor Tyrosine Activation Motif (ITAM), examples of primary cytoplasmic signaling sequences containing ITAMs suitable for use in the present invention include CD3 ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, and cd66d.

The costimulatory signaling domain refers to the portion of the TCR that comprises the intracellular domain of the costimulatory molecule. Costimulatory molecules are cell surface molecules other than the antigen receptor or its ligand that are required for efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, and the like.

In some embodiments, the invention provides a method of making a CAR-T cell, e.g., a universal CAR-T cell, comprising the steps of:

1) introduction of sgRNA molecule and Cas9 molecule in T cells:

in some embodiments, the sgRNA molecule comprises a targeting domain complementary to a chain constant coding region (i.e., TRAC) gene from a TCR, an HLA constant coding region B2M gene, and a constant coding region gene target region of PD-1.

2) Introducing a CAR molecule in the T cell;

In some embodiments, the Cas9 molecule refers to Cas9mRNA that is capable of cleaving a target site under the guidance of the sgRNA.

In some specific embodiments, the sgRNA molecule comprises a targeting domain having a sequence as set forth in one of table 2.

In some preferred embodiments, the targeting domain has the sequence shown as T2, B3, and P1 (table 2).

In some embodiments, the CAR molecule is introduced into the T cell by, for example, lentiviral transfection techniques.

In some specific embodiments, comprising the step of isolating and/or activating T cells from peripheral blood or cord blood of a healthy person; preferably, the method further comprises the step of sorting the universal CAR-T cells after step 2) above; more preferably, the sorting is followed by functional validation of the resulting CAR-T cells, e.g., universal CAR-T cells.

In some embodiments, the invention provides the use of the CAR-T cell described above for the preparation of a medicament for the treatment of a disease (e.g., a tumor).

In one aspect, the invention provides a method of treating a disease in a subject, comprising administering to the subject an effective amount of a CAR-T cell of the invention. In some embodiments, the disease is a tumor, including a hematological tumor, such as a lymphoma or leukemia. In some embodiments, the CAR targets an antigen shown in table a and the disease is a tumor corresponding to the target antigen in table a. In some embodiments, the T cell is not obtained from a subject. For example, the T cells may be derived from a healthy donor.

The CAR-T cells to which the invention relates may be administered to a subject in need thereof by a route conventionally used for administering pharmaceutical preparations comprising a cellular component, such as the intravenous infusion route. The dosage administered may be specifically determined based on the condition and general health of the subject.

Method for preparing TCR-T cell

The invention provides T cells, also referred to as TCR-T cells, that express engineered TCRs. The invention also provides methods of making the TCR-T cells, comprising introducing an engineered TCR, or a nucleotide or vector encoding the same, into any of the genetically engineered T cells (e.g., universal T cells) described herein.

In some embodiments, a method of making a TCR-T cell is provided, comprising:

(iv) introducing an engineered TCR, or a nucleic acid encoding the same, into the T cell.

The TCR or nucleic acid encoding it and the sgRNA targeting TRAC, the sgRNA targeting B2M and/or the sgRNA targeting PD-1 and the TCR or nucleotides encoding them may be introduced into the T cell in any suitable order. In some embodiments, a trc-targeted sgRNA, a B2M-targeted sgRNA, and/or a PD-1-targeted sgRNA and TCR, or encoding nucleotides thereof, are introduced into the T cell simultaneously. In some embodiments, the TCR, or the nucleic acid encoding it, is introduced into the T cell prior to the sgRNA targeting TRAC, the sgRNA targeting B2M, and/or the sgRNA targeting PD-1. In some embodiments, the TCR, or the coding nucleotide thereof, is introduced into a T cell in which gene editing has been effected, the TRAC, B2M and/or PD-1 genomic region of the T cell having been disrupted by editing. In some embodiments, the method further comprises introducing Cas9 or its encoding nucleotide into the T cell along with the sgRNA.

In some embodiments, the engineered TCR expressed by the TCR-T cells of the invention can be any engineered TCR known in the art, so long as it is capable of causing T cells to recognize cell surface antigens in a human leukocyte antigen-independent manner, exerting a killing effect. For example, the engineered TCR in the TCR-T cells of the invention can be an engineered TCR that recognizes an antigen described in table a.

As used herein, an "engineered TCR molecule," "engineered TCR molecule," or "artificial TCR molecule" includes recombinant polypeptides derived from the various polypeptides that make up a TCR, which are generally capable of i) binding to a surface antigen on a target cell; and ii) interact with other polypeptide components of the intact TCR complex when co-localized in or on the surface of a T cell.

In particular embodiments, the engineered TCRs of the invention comprise a target-specific binding element, also known as an antigen-binding domain. The selectable antigen binding domains recognize target antigens that serve as cell surface markers on, for example, target cells associated with a particular disease state. In particular embodiments, the target antigen is, for example, an antigen in table a above. In particular embodiments, the target antigen is, for example, a target antigen associated with a viral infection, a systemic immune disorder. By way of genetic engineering, the antigen binding domain can be combined with various polypeptides derived from the constituent TCRs to direct a TCR-mediated T cell response to an antigen of interest.

In particular suitable transmembrane domains for use in the invention may include at least a transmembrane domain such as T cell receptor α, β or zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD 154.

In some cases, the transmembrane domain may be linked to an extracellular region of the engineered TCR, such as the antigen-binding domain of the engineered TCR, by a hinge, such as a hinge from a human protein. For example, in one embodiment, the hinge may be a human immunoglobulin (Ig) hinge, such as an IgG4 hinge, or a CD8a hinge.

In particular embodiments, the engineered TCRs of the invention comprise a linker connecting the transmembrane domain to the cytoplasmic region. Optionally, the linker is a short oligopeptide or polypeptide linker between 2 and 50 amino acids in length. The glycine-serine pair provides a particularly suitable linker.

In particular embodiments, the engineered TCRs of the invention comprise a cytoplasmic domain. The intracellular signaling domain is generally responsible for the activation of at least one of the normal effector functions of the immune cell into which the engineered TCR is introduced. The effector function of a T cell may for example be cytolytic activity or helper activity, including secretion of cytokines. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transduces effector function signals and directs a cell to perform a specialized function. Although it is generally possible to employ an intact intracellular signalling domain, in many cases it is not necessary to use the entire chain, and such a truncated portion may be used instead of the entire chain, provided that it transduces effector functional signals. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

In certain embodiments, the engineered TCR molecules comprise an engineered TCR α and a TCR β chain in certain embodiments, the engineered TCR molecules bind to CD3 molecules and zeta chains and/or other costimulatory molecules expressed in T cells.

In some embodiments, the invention provides the use of the above-described TCR-T cells for the preparation of a medicament for the treatment of a disease (e.g., a tumor).

In one aspect, the invention provides a method of treating a disease in a subject, comprising administering to the subject an effective amount of a TCR-T cell of the invention. In some embodiments, the disease is a tumor, including a hematological tumor, such as a lymphoma or leukemia. In some embodiments, the engineered TCR targets a target antigen shown in table a, and the disease is a tumor in table a corresponding to the target antigen. In some embodiments, the T cell is not obtained from a subject. For example, the T cells may be derived from a healthy donor.

The TCR-T cells involved in the present invention can be administered to a subject in need thereof by a route conventionally used for administering pharmaceutical preparations comprising a cellular component, such as an intravenous infusion route. The dosage administered may be specifically determined based on the condition and general health of the subject.

T cell source

Prior to amplification and genetic modification, a source of T cells is obtained from the subject. The term "subject" is intended to include living organisms (e.g., mammals) capable of eliciting an immune response. Examples of subjects include humans. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. The T cells of the invention may also be derived from hematopoietic stem cells at various stages of differentiation. Hematopoietic stem cells are differentiated into T cells under committed differentiation culture conditions. In certain aspects of the invention, a variety of T cell lines available in the art may be used.

In certain aspects of the invention, T cells may be obtained from blood collected from a subject using a variety of techniques known to the skilled artisan, such as Ficoll (TM) separation. Cells may also be obtained from the circulating blood of an individual by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one aspect, cells collected by apheresis may be washed to remove the plasma fraction and placed in an appropriate buffer or medium for subsequent processing steps.

Can be prepared by lysing erythrocytes and passing them, for example, through PERCOLL^TMDepletion of monocytes by gradient centrifugation or countercurrent centrifugation elutriation separates T cells from peripheral blood lymphocytes. Specific T cell subsets, such as CD3+, CD28+, CD4+, CD8+, CD45RA +, and CD45RO + T cells, can be further isolated by positive or negative selection techniques. For example, in one aspect, T cells are obtained by coupling to anti-CD 3/anti-CD 28 (e.g., 3x 28) beads, such as DYNABEADS^TMM-450CD3/CD28T were isolated by incubating together for a period of time sufficient to positively select for the desired T cells. Tumor Infiltrating Lymphocytes (TILs) can be isolated from tumor tissue.

sgRNA

In one aspect of the invention, a sgRNA targeted to TRAC, a sgRNA targeted to B2M, and a sgRNA targeted to PD-1 are provided. The sgRNA contains any nucleotide sequence selected from SEQ ID NOs: 2-22. In some embodiments, the sgRNA is chemically modified.

Also encompassed by the invention are sgRNA compositions, kits or articles of manufacture comprising the sgrnas or vectors thereof to which the invention relates. In some embodiments, the kit comprises: i) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 2-5; (ii) comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6-14; and/or (iii) comprises a sequence selected from SEQ ID NOs: 15-22. In some embodiments, the kit comprises: (i) comprises the amino acid sequence of SEQ ID NO: 3 sequence of sgRNA; (ii) comprises a sequence selected from SEQ ID NO: 16 sequence of sgRNA; and (iii) comprises SEQ ID NO: 7 sequence sgRNA. In some embodiments, the kit further comprises a Cas 6-encoding nucleic acid or a vector thereof. In some embodiments, the sgRNA is chemically modified.

In some embodiments, the T cell genetic engineering methods of the invention use chemically modified sgrnas. The chemically modified sgRNA employed by the present inventors is considered to have the following two advantages. First, because the sgRNA is a single-stranded form of RNA, its half-life is very short and degrades rapidly (up to 12 hours) after entering the cell, while at least 48hrs is required for the Cas9 protein to bind to the sgRNA for gene editing. Therefore, the chemically modified sgRNA is adopted, enters cells, is stably expressed, and can be efficiently subjected to gene editing to generate Indels after being combined with the Cas9 protein. Second, unmodified sgrnas have poor ability to penetrate cell membranes and cannot effectively enter cells or tissues to perform their functions. Whereas the ability of chemically modified sgrnas to penetrate cell membranes is generally enhanced. In the present invention, chemical modification methods commonly used in the art may be employed, as long as the stability (half-life extension) of sgRNA and the ability to enter cell membranes can be improved. In addition to the specific chemical modifications used in the examples, other modification methods are also contemplated, for example, Deleavey GF1, Damha MJ.design chemical modified oligonucleotides for targeted genetic engineering, chem biol.2012Aug 24; 937-54, and Hendel et al, chemical lymodified guide RNAs enhance CRISPR-Cas genome editing in human primarycells.Nat Biotechnol.2015Sep; 33(9) 985-989.

The invention allows for the electrotransformation of chemically modified sgrnas, one of the key factors in the invention, into T cells together with Cas 9-encoding genes, resulting in high efficiency of gene editing (e.g., expressed in Indels%). The data in the examples show that if the sgRNA without chemical modification is electroporated with Cas9mRNA, the Indels efficiency is much lower than that obtained when the chemically modified sgRNA is electroporated.

The present invention will be described with reference to specific examples. It should be understood that the specific embodiments are for illustrative purposes only and are not meant to limit the disclosure to only specific embodiments.

Example 1 preparation of Universal T cells

1.Isolation and activation of healthy donor T cells

Collecting cord blood of healthy donor by taking cord blood from blood bank, placing in 4 deg.C refrigerator, and standingWithin 24h, the cells were transported via a transport trolley equipped with thermostatic equipment to the GMP laboratory for separation of T cells.

1.1 preparing the mononuclear cells of the cord blood, namely, sucking normal saline by a pipette, adding the normal saline into the cord blood transported in the step (1), diluting the cord blood and the normal saline according to a ratio of 1:1(V/V), slowly adding a blood cell diluent into a lymphocyte separation tube, centrifuging for 20 minutes at 800g, sucking white membrane layer cells above the lymphocyte separation liquid, transferring into a new 50 ml centrifuge tube, adding a T cell culture medium, centrifuging for 5 minutes at 400g, discarding supernatant, and reserving cell precipitates at the bottom of the centrifuge tube to obtain the mononuclear cells of the peripheral blood.

1.2T cell separation and activation, namely counting the obtained cord blood mononuclear cells by using a cell counter and then carrying out T cell sorting, wherein the T cell sorting comprises the following specific steps:

1.2.1. the cell pellet was adjusted to a density of 5X10 by Easy buffer (manufacturer: StemCell, cat # 16F72331)⁷Per ml, cells were transferred to a 5ml flow tube with a 5ml pipette;

1.2.2. adding a T cell separation reagent with the concentration of 50ul/ml, and incubating at room temperature for 5 minutes after the addition;

1.2.3. adding sorting magnetic beads, mixing the magnetic beads evenly within 30 seconds, and adding the magnetic beads with the concentration of 40 mu l/ml;

1.2.4 using Easy buffer to supplement the cell sap to 2.5 ml, directly placing on a magnetic column for 3 minutes after the cell sap is added, and then pouring the cells into a 15 ml centrifuge tube to obtain the T cells;

1.2.5. after sorting, the mixture is mixed by a 1000-microliter pipette, counted, centrifuged (400G, 5min) and the supernatant is removed, thus obtaining the T cell sediment.

The T cell pellet was resuspended in T cell media. Then, adding a T cell activating factor according to the proportion of 1:1, wherein the T cells are in an activated state, and putting the T cells into an incubator to continue the expansion culture.

2.Optimization of electrotransfer conditions

The above-cultured T cells were collected in a 50-ml centrifuge tube, centrifuged at 300g for 7 minutes, the supernatant was discarded, washed 2 times with a DPBS solution (manufacturer: Gibco, cat # 1924294), and the cell density was adjusted to 2.5X10 with an electrotransfer reagent⁷Individual cells/mL. Using HISCRIBE^TMGFP mRNA (see 3.3 for details) prepared from T7ARCA mRNA Kit (tailed) (manufacturer: NEB, cat # E2060S) was mixed homogeneously with T cells to a final concentration of 2.5X10 per 100. mu.L⁶Individual cells and 6. mu.g GFP mRNA. GFP mRNA was introduced into T cells using an electrotransfer apparatus BTX Agile pulse MAX (manufacturer: BTX, model: 47-0200 NINT). The electrical swivel system was optimized according to the difference between the voltage and the pulse time, as shown in table 1, the voltage was sequentially increased from 180v to 400 v, and the pulse time was sequentially decreased from 2ms to 0.5 ms. The growth of the cells was observed every day, the number of the electroporated T cells was counted every other day and the phenotype of the gene-edited T cells was analyzed by a flow cytometry (manufacturer: Ason, type: ACEA novoCyte), and the cell survival rate and GFP electroporation efficiency were the best under the conditions of 150-250V, 0.5-2ms electroporation, as shown in Table 1.

Table 1: results of comparison of electroporation efficiency of GFP mRNA under different electroporation conditions and cell viability after electroporation

3.Gene knockout on T cells

The CRISPR/Cas9 gene editing technology is utilized to knock out TRAC, B2M and PD-1 genes in the T cells obtained in the step 1.2, and the specific operation steps are as follows:

3.1 sgRNA design and plasmid construction for the α chain constant coding region (i.e., TRAC) gene of TCR, the HLA constant coding region B2M gene, and the constant coding region gene of PD-1.

Sgrnas designed for all coding sequences of TRAC, B2M and PD-1 coding regions were designed by CRISPR rgentols, and sgRNA sequences selected according to the highest scores are shown in table 2.

Table 2: selected sgRNA sequences

Wherein T represents a sgRNA sequence for TRAC, P represents a sgRNA sequence for the PD-1 coding region, and B represents a sgRNA sequence for B2M.

3.2 the sgRNA is modified by 2 '-O-methyl analogue and/or 3' thio between nucleotides by a chemical method, so that the sgRNA with high knockout efficiency and stability is prepared.

3.3 Cas9 plasmid and GFP plasmid were taken, Xba1 (manufacturer: NEB, cat # R0145S), cutsmartbuffer (manufacturer: NEB, cat # B7204s) were linearized by digestion, and 50. mu.l reaction:

composition (I)	Volume of
		Cas9 plasmid	5μg
10 × cutsmart buffer	5μl
		Xba1	2μl
Adding water to the total reaction volume	50μl

And (3) carrying out water bath at 37 ℃ for 4 hours, carrying out agarose gel electrophoresis on 2 mu l of enzyme digestion product, setting the voltage to be 110U, carrying out electrophoresis for 30min, and observing a single band under a gel imager, wherein the condition indicates that the enzyme digestion is complete and all plasmids are linearized.

Taking the reaction product and cleaning and purifying the reaction product.

Collecting purified product, and purifying with HISCRIBE^TMT7ARCA mRNA Kit (manufacturer: NEB, cat # E2060S) was transcribed in vitro (i.e., IVT), 20. mu.l system:

composition (I)	Volume of
		2*ARCA Mix	10μl
Template DNA	0.5μg
		T7RNA Polymerase Mix	2μl
Nuclease-free water	To 20. mu.l
		Total reaction volume	20μl

The reaction system was placed on a PCR instrument and reacted at 37 ℃ for 4 hours. After 4 hours, 2. mu.l of DNase 1 was added to the reaction system and the reaction was carried out at 37 ℃ for 20 minutes.

Taking the reaction product, and carrying out the following operations:

composition (I)	Volume of
		Nuclease-free water	63μl
IVT reaction	22μl
		10 Poly (A) polymerase buffer	10μl
Poly (A) polymerase	5μl
		Total reaction volume	100μl

The reaction system is placed on a PCR instrument for reaction for 2 hours.

And cleaning and purifying the reaction product, and placing the product in a refrigerator at the temperature of-80 ℃ for later use.

3.4 using the electrotransfer technology to introduce sgRNA and Cas9mRNA into T cells, and using the antigen screening principle to screen T cells which are negative for TCR and/or B2M and/or PD1 and positive for CD4 and CD8, so as to obtain the universal T cells.

The cells were taken and subjected to cell genome extraction using TIANAmp Genomic DNA Kit (manufacturer: TIAN GEN, cat # DP 304-03). Using synthetic primers, the genome of the cells extracted AS described above was extracted, and the genomic regions of TRAC, B2M and PD-1 containing the corresponding sgRNA were PCR-amplified using 2 × Esay Taq SuperMix (+ dye) (manufacturer: TRANS, Code # AS111), 50. mu.l reaction:

the reaction conditions were as follows:

3.5 taking PCR products after reaction to carry out Sanger sequencing to verify the knockout efficiency of TCR, HLA and PD-1 from the molecular level, and the result is shown in figure 2, in the system, the CRISPR/Cas9 technology can be used for successfully editing the constant coding sequences of TRAC gene of TCR, B2M gene of HLA and PD-1, including insertion mutation and deletion mutation, and both cause frame shift mutation (see the following table B-G specifically), thereby inhibiting the expression of TCR, HLA and PD-1 from the gene level.

Table B: sequence changes made in chromosome 15 from position 45003745 to position 45003788 after introduction of B2:

note: in this table "-" is denoted "d" in the sequence listing "

Table C: sequence changes made in chromosome 15 from position 45003745 to position 45003788 after introduction of B3:

note: in this table "-" is denoted "d" in the sequence listing "

Table D: sequence changes made in chromosome 14 from position 23016448 to position 23016490 after introduction of T3:

note: in this table "-" is denoted "d" in the sequence listing "

Table E: sequence changes made in chromosome 14 from position 23016448 to position 23016490 after introduction of T2:

note: in this table "-" is denoted "d" in the sequence listing "

Table F: sequence changes made in chromosome 2 from position 242800936 to position 242800978 after introduction of P2:

note: in this table "-" is denoted "d" in the sequence listing "

Table G: sequence changes from position 242795009 to 242795051 on chromosome 2 after introduction of P1

Note: in this table "-" is denoted "d" in the sequence listing "

3.6 prediction of the potential off-target sites on the whole human genome of TRAC-sg3(T2, T3), B2M-sg2(B2, B3) and PD-1-sg2(P1, P2) and amplification analysis of the predicted off-target site regions that may affect the expression of other genes were performed simultaneously, in order to confirm from the molecular level that the knockout of TRAC, B2M and PD-1 did not introduce off-target (off-target) non-specific gene knockout, and the results are shown in Table 3 and FIG. 5.

Table 3: off-target assay results for TRAC, TRAC/B2M (DKO) and TRAC/B2M/PD-1(TKO) knock-out T cells

Note: all the above sequence position information is referred to the database: GRCh37(hg 19).

It can be seen from Table 3 that the TRAC, B2M and PD-1 genes did not have any mutation, indicating that the system satisfies the requirement for specificity of gene editing.

The above results show that the use of TRAC-sg2(T2), TRAC-sg3 (T3); the TRAC, B2M and PD-1 genes in the universal T cell obtained by completely knocking out the genes of B2M-sg2(B2), B2M-sg3(B3), PD-1-sg1(P1) and PD-1-sg2(P2) after finishing gene editing on the T cell as sgRNA and screening the T cell are not found, and meanwhile, no gene mutation at a potential target escape site is found.

4.Amplification of Gene editing T cells

Activation of sorted T cells with cytokines, followed by T cell media to 1X10 cell density⁶Individual cells/mL. Observing the state of cells after 72h, collecting cell suspension, centrifuging for 7min at 300g, discarding supernatant, washing with DPBS (Gibco) for 2 times, and adjusting the cell density to 2.5X10 with electrotransfer reagent culture medium⁷Individual cells/mL. Using HISCRIBE^TMT7ARCA mRNA Kit (manufacturer: NEB, cat # E2060S) prepared Cas9mRNA and synthetic sgRNA were mixed to a final concentration of 2.5X10 per 100. mu.L of T cells and RNA⁶Individual cells and 8 μ g RNA (Cas9mRNA and sgRNA 4 μ g each) and then BTX agill using a transtere pulseMAX introduced RNA into cells and cultured. The growth condition of the cells is observed every day, cell counting and fluid supplementing are carried out every other day, phenotype analysis is carried out on the T cells after gene editing, as shown in figure 3, the TCR knockout efficiency is about 90.42 percent, the TCR knockout efficiency and the B2M (DKO) knockout efficiency are about 81.39 percent, and the TKO knockout efficiency of the three genes is as high as 67.91. After 8d of cell culture, the resulting T cells were quality control monitored.

5.Detection of CRISPR/Cas9 gene editing efficiency

The cells are sampled after being cultured for 8 days, a human genome extraction kit (manufacturer: TIANGEN, cat # DP304-03) is utilized to extract genome from the sample, corresponding sequencing primers are designed at the same time, a target fragment is prepared by utilizing a PCR technology, and the target fragment and the corresponding primers are subjected to singer sequencing. The sequencing results were analyzed using the TIDE software and are shown in FIGS. 2A, 2C, and 2E. The selected sgRNA can efficiently knock out the corresponding gene.

6.Screening of cells of interest

T cells that were negative for TCR and/or B2M and/or PD-1, and positive for CD4 and CD8 were screened as follows.

T cells which are negative for TCR and/or B2M and/or PD-1 and positive for CD4 and CD8 are screened by an immunomagnetic bead technology, and the edited T cell state is monitored by the survival rate of the T cells.

In the first step, the T cells after electroporation were collected at day 12-14, 400G, centrifuged for 5min, the supernatant discarded, and the cells were lysed with Easybuffer to 1X10⁸And/ml, then transferring the cells into a 5ml flow tube, removing the cells which still express TCR, B2M and PD-1 from the T cells by using a screening reagent, and screening to obtain a final product, namely universal T cells.

And secondly, taking a small amount of T cells, carrying out flow detection, simultaneously staining cell surface biomarkers of TCR and/or B2M and/or PD-1, and carrying out the next step if the positive rate of TCR and/or B2M and/or PD-1 is less than 1%. In this example, the TCR positivity was 1%, the T cell positivity for TCR and B2M DKO was < 0.79%, and the T cell positivity for TCR, B2M, and PD-1TKO was < 1%, as shown in fig. 4. The survival rate of the purified T cells was not significantly affected, and was above 85%, as shown in Table 4.

Table 4: survival rates of TCR, TCR/B2M (DKO) and TCR/B2M/PD-1(TKO) knockout T cells after screening and purification

7. Off-target analysis

7.1. Collection of T cells 1X10⁶Then, the genome was extracted (manufacturer: QIAGEN, Cat # 69504).

7.2. Carrying out Cas9 in vitro enzyme digestion reaction on the extracted genome

The Cas9 in vitro enzyme digestion reaction system is as follows:

7.3. blowing and beating by using a gun head, gently mixing uniformly, and then incubating for 15min at 37 ℃;

7.4. adding 1. mu.l proteinase K (manufacturer: Tiangen, Cat # RT403), mixing, and incubating at room temperature for 10 min;

7.5. taking 5ul of samples for gel electrophoresis, and detecting the effect of the Cas9 in vitro enzyme digestion reaction;

7.6. the remaining samples were subjected to human heavy genome detection.

As a result, as shown in FIG. 5, no off-target phenomenon was observed.

Example 2 preparation of Universal T cells

Preparation and expansion of Universal CAR-T cells

Activation of sorted T cells with cytokines and subsequent adjustment of cell density to 1X10 with T cell media⁶Individual cells/mL. The state of the cells was observed after 72 hours, and the cell suspension was collected, centrifuged at 300g for 7min, the supernatant was discarded, washed 2 times with DPBS solution (manufacturer: Gibco; cat # 1924294), and the cell density was adjusted to 2.5X10 with the electrotransformation reagent medium⁷Individual cells/mL. Using HISCRIBE^TMT7ARCA mRNA Kit (tailed) (manufacturer: NEB, cat # E2060S) prepared Cas9mRNA and synthetic sgRNA were mixed to a final concentration of 2.5X10 per 100. mu.L of T cell and RNA⁶The cells and 8. mu.g of RNA (Cas9mRNA and sgRNA each 4. mu.g) were cultured after the RNA was introduced into the cells using a transter BTX Agile pulseMeAX. The growth of the cells was observed daily, cell counting was performed every other day, and lentiviruses comprising CAR (including anti-CD 19scFv, linking region (Linker), CD8alpha hinge region (CD8alpha hinge), CD8 transmembrane region (CD8 transmembrane domain), 4-11BB signaling domain (4-11BB signaling domain) and CD3zeta, for a specific structure, see US20140271635A1) were added at 5 days in a MOI of 2-10.

Screening of Universal CAR-T cells

T cells which are negative in TCR and/or B2M and/or PD-1 and positive in CD4 and CD8 are screened by an immunomagnetic bead technology, and the edited T cell state is monitored by the survival rate of the T cells, and the method comprises the following specific steps:

in the first step, the T cells after electroporation were collected at day 12-14, 400G, centrifuged for 5min, the supernatant discarded, and the cells were lysed with Easybuffer to 1X10⁸/ml, then the cells are transferredAnd transferring the cells to a 5ml flow tube, removing the cells which still express TCR, B2M and PD-1 in the T cells by using screening magnetic beads, and screening to obtain a final product, namely the universal T cells.

And secondly, taking a small amount of T cells, carrying out flow detection, simultaneously staining TCR and/or B2M and/or PD-1 cell surface biomarkers, and carrying out the next step if the positive rate of TCR and/or B2M and/or PD-1 is less than 1%. In this example, the TCR positivity was 1%, the T cell positivity for TCR and B2M DKO was < 0.79%, and the T cell positivity for TCR, B2M, and PD-1TKO was < 1%, as shown in table 5 and fig. 6. The survival rate of the purified T cells is not obviously influenced and is more than 85 percent.

Table 5:

example 3 Universal CAR-T functional validation

The killing effect of the universal CAR-T cells (i.e., effector cells) obtained in example 2 on B-cell type acute lymphoblastic leukemia cells was observed.

In vitro killing effect on specific tumor cells

The experimental steps of the invention are as follows:

first step, target cell labeling

Use CELL TRACE^TMThe Far Red Cell promotion Kit (manufacturer: Gibco; cat # 1888569) labeled target cells (human Burkitt's lymphoma cells Raji, K562, all from ATCC).

1. Cell TraceTM Far Red Cell promotion was diluted to a 1mmol solution with double distilled water;

2. take 1X10⁶Centrifuging 400g of each target cell for 5 minutes, and removing supernatant;

3. mu.l of Cell Trace TM Far Red Cell Proliferation solution was added and incubated at 37 ℃ for 20min in the absence of light.

4. Cells were added to T cell medium and incubated at 37 ℃ for 5 min.

5.400g after centrifugation for 5 minutes, the supernatant was removed and the labeling was complete.

Second step, detecting the killing of target cells by effector cells

The labeled target cells were cultured in R1640+ 10% FBS (Becton Dickinson) medium at 2X10⁵cells/mL density resuspend, 500. mu.L into 48-well plates. According to the appropriate effector-target ratio (2.5:1, 1.25:1, 0.6:1), 500. mu.L of effector cells were added to each well, and at the same time, as control cells, CAR-T cells prepared from lentivirus packed with CAR (see US20140271635A1 for specific structure) were added to T cells and healthy human cord blood CAR-T cells at MOI 2-10 when T cells were cultured up to day 2, 3 groups of individual target cells were designed in parallel, and their death rate was measured, 5% CO2 was cultured at 37 ℃ for 12-16h, 400g was centrifuged for 5min, cell pellets were taken, 150. mu.l of them was resuspended with DPBS (manufacturer: Gibco; cat No.: 1924294), after staining with PI (manufacturer: Sigma; cat No.: P4170), the death rate of target cells was measured by flow cytometry, and the results are shown in FIG. 7, general-T cells were kept substantially identical to the effect of other target-T cells on killing specific CAR cells, the effect is better than that of T cells. And almost no killing function to non-specific target cells.

Third step, ELISA detection of cytokine release

Labeled target cells were plated with RPMI1640 + 10% FBS at 2X10⁵Resuspend at density of one/mL, take 500. mu.L and add to 48-well plate. Adding 500 μ L effector cells per well according to appropriate effect-target ratio (10:1), simultaneously using T cells and healthy human umbilical cord blood CAR-T as control cells, each group having 3 parallel cells, designing individual target cell group, culturing at 37 deg.C with 5% CO2 for 12-16h, collecting culture supernatant 100 μ L per well, centrifuging at 400g for 5min to remove precipitate, collecting supernatant, and treating with LEGEND MAX^TMHumanIL-2/IFN-gama (manufacturer: Biolegend; cat nos.:431807, 430108) kit for detecting factor release according to instructions for use.

The results are shown in FIG. 8, and it can be seen that the killing ability of the general CAR-T on Raji cells is basically consistent with or slightly better than that of the common CAR-T, especially TCR^negThe release amount of CAR-T IFN-r is far higher than that of the common CAR-T and the killing power to K562 is lower, so that the factor release is relatively less.

Second, in vivo killing effect of universal CAR-T on specific tumor cells

1. Cell line human lymphoma cell line

Raji^{tg(luciferase-GFP)}the/Bcgen cell is a human Burkitt's lymphoma cell line, positive for CD19 expression, and can serve as a target cell for CAR-T cells. Raji^luc-GFPModified to express GFP and luciferase simultaneously. A human lymphoma model of mice can be constructed by tail vein injection, and the area of the tumor is counted by XenoLight D-Luciferin, Potasidum Salt (manufacturer: Perkinelmer; cat # 122799) in conjunction with the fluorescence presented by a small animal in vivo imager.

2.Raji^{tg(luciferase-GFP)}Bcgen cell culture

Raji^{tg(luciferase-GFP)}the/Bcgen cell line was a suspension cell line that grew rapidly in RPMI1640 medium containing 10% FBS. The cell density is 2-3X 10⁶at/mL, passage is required. At passage, the cell suspension was centrifuged at 300g for 6 minutes in a centrifuge tube, and the supernatant was discarded. Adjusting cell density to 1X10⁶Cells/ml, and culture was continued. Normal growth, alternate-day passage, cell density maintained at 0.8-1X 10⁶Individual cell^/Is required to be between mL.

3. Mouse model

7-10 weeks old NPG female mice 25, single tail vein injection of tumor cells (Raji)^{tg(luciferase-GFP)}/Bcgen)5X10⁵Weighing each cell every other day, observing once a day, and inoculatingAfter 3-5 days of tumor cells, the cells were randomly divided into 6 groups by XenoLight D-Luciferin, Potasssium Salt (manufacturer: PerkinElmer; cat # 122799) using the tumor area and tumor enrichment as indices in conjunction with a small animal in vivo imager: physiological saline group, T cell group, CAR-T cell group, TCR^neggroup-CAR-T, group DKO-CAR-T, group TKO-CAR-T.

4. Administration in mouse lymphoma model.

Record the day of molding as D0. The cells were transfused with 200. mu.L of physiological saline and 200. mu.L of human T cells (total 2X 10) by tail vein injection⁶Individual cell/individual), CAR-T cells 200. mu.L (2X 10 in total)⁶Per) TCR^neg200 μ L (2X 10 total) of CAR-T cells⁶DKO-CAR-T cells 200. mu.L (2X 10 in total)⁶/) TKO-CAR-T cells 200. mu.L (total 2X 10)⁶/only) all mice were dosed once. The results are shown in fig. 9A-9B and fig. 10, and it can be seen that the universal CAR-T prophase provided by the present invention has excellent inhibitory and tumor cell killing effects, almost the same as CAR-T cells; compared with common CAR-T, the later-stage CAR-T has obvious effect of inhibiting tumor cells.

5. Post-dose monitoring in mice

The mice after administration are monitored every day, and the weight of the mice is recorded every 2 days, including the weight, the skin integrity, the hair, the mental state, the activity frequency, the activity coordination and the like of the mice, the mice are continuously observed for 37 days, the elimination of the tumor area and the reduction of the tumor enrichment are used as the judgment indexes of the effector cell function, and the safety of the general CAR-T is judged according to the skin integrity, the hair, the mental state, the activity frequency and the activity coordination. As shown in FIG. 11, the mice did not lose weight, had intact skin and hair, were mentally active, and were coordinated to have no GVHD response.

Sequence listing

<110> Boya Yingyin (Beijing) Biotechnology Ltd

<120> gene editing T cell and use thereof

<130>PB00312

<150>201710842264.5

<151>2017-09-18

<150>201710841323.7

<151>2017-09-18

<160>164

<170>PatentIn version 3.5

<210>1

<211>4101

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: cas9mRNA

<400>1

gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc 60

accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac 120

agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc 180

acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat 240

ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg 300

gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac 360

atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa 420

ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg 480

atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg 540

gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc 600

aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg 660

ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg 720

attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat 780

gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag 840

atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg 900

ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg 960

atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag 1020

cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc 1080

tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa 1140

aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag 1200

cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc 1260

attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag 1320

aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga 1380

ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg 1440

gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac 1500

ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat 1560

aacgagctga ccaaagtgaa atacgtgacc gagggaatgagaaagcccgc cttcctgagc 1620

ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg 1680

aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc 1740

ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc 1800

aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg 1860

accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac 1920

ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg 1980

ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat 2040

ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc 2100

ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac 2160

gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg 2220

aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc 2280

gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg 2340

aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg 2400

gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat 2460

atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc 2520

gtgcctcaga gctttctgaa ggacgactcc atcgacaacaaggtgctgac cagaagcgac 2580

aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac 2640

tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc 2700

aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg 2760

gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact 2820

aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag 2880

ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac 2940

caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac 3000

cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg 3060

atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac 3120

atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct 3180

ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc 3240

accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag 3300

acaggcggct tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc 3360

agaaagaagg actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat 3420

tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa 3480

gagctgctgg ggatcaccat catggaaaga agcagcttcgagaagaatcc catcgacttt 3540

ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac 3600

tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag 3660

aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac 3720

tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag 3780

cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc 3840

ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc 3900

atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct 3960

gccgccttca agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag 4020

gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac 4080

ctgtctcagc tgggaggcga c 4101

<210>2

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: TRAC-sg2(T2)

<400>2

gctggtacac ggcagggtca 20

<210>3

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: TRAC-sg3(T3)

<400>3

ctctcagctg gtacacggca 20

<210>4

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: TRAC-sg 4 (T4)

<400>4

atttgtttga gaatcaaaat 20

<210>5

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: TRAC-sg 6 (T6)

<400>5

tctctcagct ggtacacggc 20

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: B2M-sg 1(B1)

<400>6

actctctctt tctggcctgg 20

<210>7

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: B2M-sg2(B2)

<400>7

gagtagcgcg agcacagcta 20

<210>8

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: B2M-sg3(B3)

<400>8

cgcgagcaca gctaaggcca 20

<210>9

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: B2M-sg 4(B4)

<400>9

tcacgtcatc cagcagagaa 20

<210>10

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: B2M-sg 5(B5)

<400>10

gctactctct ctttctggcc 20

<210>11

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: B2M-sg 6(B6)

<400>11

tttgactttc cattctctgc 20

<210>12

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: B2M-sg 7(B7)

<400>12

cgtgagtaaa cctgaatctt 20

<210>13

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: B2M-sg8(B8)

<400>13

ctcgcgctac tctctctttc 20

<210>14

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1-sg1(P1)

<400>14

ctgcagcttc tccaacacat 20

<210>15

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1-sg2(P2)

<400>15

gccctggcca gtcgtctggg 20

<210>16

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1-sg4(P4)

<400>16

gccctgctcg tggtgaccga 20

<210>17

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1-sg5(P5)

<400>17

gagaaggtgg gggggttcca 20

<210>18

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1-sg6(P6)

<400>18

ccctgctcgt ggtgaccgaa 20

<210>19

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1-sg7(P7)

<400>19

gaaggtggcg ttgtcccctt 20

<210>20

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1-sg8(P8)

<400>20

cctgctcgtg gtgaccgaag 20

<210>21

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1-sg9(P9)

<400>21

gtctgggcgg tgctacaact 20

<210>22

<211>20

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1-sg10(P10)

<400>22

cgatgtgttg gagaagctgc 20

<210>23

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: TRAC genomic region from 23016448 th to 23016490 th of chromosome 14

Domain

<400>23

tatccagaac cctgaccctg ccgtgtacca gctgagagac tct 43

<210>24

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: B2M genomic region from 45003745 th to 45003788 th of chromosome 15

Domain

<400>24

atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctct 44

<210>25

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1 genome from 242800936 th to 242800978 th of chromosome 2

Region(s)

<400>25

agcccagttg tagcaccgcc cagacgactg gccagggcgc ctg 43

<210>26

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: PD-1 genome from 242795009 th to 242795051 th of chromosome 2

Region(s)

<400>26

cagtttagca cgaagctctc cgatgtgttg gagaagctgc agg 43

<210>27

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: b2 as a guide RNA, and then edited on chromosome 45 of chromosome 15

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(22)..(22)

<223> base at position 22 was deleted after editing

<400>27

atgtctcgct ccgtggcctt adctgtgctc gcgctactct ctct 44

<210>28

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(8)..(32)

<223> 8 th to 32 th bases were deleted after editing

<400>28

atgtctcddd dddddddddd dddddddddd ddgctactct ctct 44

<210>29

<211>45

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(23)..(23)

<223> at position 23 indicating insertion of any base

<400>29

atgtctcgct ccgtggcctt agnctgtgct cgcgctactc tctct 45

<210>30

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(23)..(25)

<223> 23 st to 25 th bases were deleted after editing

<400>30

atgtctcgct ccgtggcctt agdddtgctc gcgctactct ctct 44

<210>31

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(17)..(27)

<223> 17 th to 27 th bases were deleted after editing

<400>31

atgtctcgct ccgtggdddd dddddddctc gcgctactct ctct 44

<210>32

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(17)..(35)

<223> 17 th to 35 th bases were deleted after editing

<400>32

atgtctcgct ccgtggdddd dddddddddd dddddactct ctct 44

<210>33

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(18)..(22)

<223> 18 th to 22 nd bases were deleted after editing

<400>33

atgtctcgct ccgtggcddd ddctgtgctc gcgctactct ctct 44

<210>34

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(13)..(35)

<223> bases at positions 13 to 35 were deleted after editing

<400>34

atgtctcgct ccdddddddd dddddddddd dddddactct ctct 44

<210>35

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(17)..(31)

<223> 17 th to 31 th bases were deleted after editing

<400>35

atgtctcgct ccgtggdddd dddddddddd dcgctactct ctct 44

<210>36

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(17)..(32)

<223> 17 th to 32 th bases were deleted after editing

<400>36

atgtctcgct ccgtggdddd dddddddddd ddgctactct ctct 44

<210>37

<211>46

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(23)..(24)

<223> n represents insertion of any one base

<400>37

atgtctcgct ccgtggcctt agnnctgtgc tcgcgctact ctctct 46

<210>38

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(17)..(26)

<223> bases at 17 th to 26 th positions were deleted after editing

<400>38

atgtctcgct ccgtggdddd ddddddgctc gcgctactct ctct 44

<210>39

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(20)..(31)

<223> 20 th to 31 th bases were deleted after editing

<400>39

atgtctcgct ccgtggcctd dddddddddd dcgctactct ctct 44

<210>40

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(22)..(23)

<223> bases at positions 22 to 23 were deleted after editing

<400>40

atgtctcgct ccgtggcctt addtgtgctc gcgctactct ctct 44

<210>41

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(8)..(37)

<223> 8 th to 37 th bases were deleted after editing

<400>41

atgtctcddd dddddddddd dddddddddd dddddddtct ctct 44

<210>42

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(19)..(35)

<223> 19 th to 35 th bases were deleted after editing

<400>42

atgtctcgct ccgtggccdd dddddddddd dddddactct ctct 44

<210>43

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(23)..(26)

<223> the 23 rd to 26 th bases were deleted after editing

<400>43

atgtctcgct ccgtggcctt agddddgctc gcgctactct ctct 44

<210>44

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(23)..(24)

<223> 23 rd to 24 th bases were deleted after editing

<400>44

atgtctcgct ccgtggcctt agddgtgctc gcgctactct ctct 44

<210>45

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(9)..(25)

<223> 9 th to 25 th bases were deleted after editing

<400>45

atgtctcgdd dddddddddd dddddtgctc gcgctactct ctct 44

<210>46

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(18)..(29)

<223> 18 th to 29 th bases were deleted after editing

<400>46

atgtctcgct ccgtggcddd dddddddddc gcgctactct ctct 44

<210>47

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(23)..(27)

<223> the 23 rd to 27 th bases were deleted after editing

<400>47

atgtctcgct ccgtggcctt agdddddctc gcgctactct ctct 44

<210>48

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(8)..(30)

<223> 8 th to 30 th bases were deleted after editing

<400>48

atgtctcddd dddddddddd dddddddddd gcgctactct ctct 44

<210>49

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(18)..(35)

<223> 18 th to 35 th bases were deleted after editing

<400>49

atgtctcgct ccgtggcddd dddddddddd dddddactct ctct 44

<210>50

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(18)..(24)

<223> 18 th to 24 th bases were deleted after editing

<400>50

atgtctcgct ccgtggcddd ddddgtgctc gcgctactct ctct 44

<210>51

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(16)..(35)

<223> 16 th to 35 th bases were deleted after editing

<400>51

atgtctcgct ccgtgddddd dddddddddd dddddactct ctct 44

<210>52

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(11)..(33)

<223> bases at 11 th to 33 th positions were deleted after editing

<400>52

atgtctcgct dddddddddd dddddddddd dddctactct ctct 44

<210>53

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: b3 as a guide RNA, and then edited on chromosome 45 of chromosome 15

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(17)..(17)

<223> base at position 17 was deleted after editing

<400>53

atgtctcgct ccgtggdctt agctgtgctc gcgctactct ctct 44

<210>54

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(16)..(17)

<223> 16 th to 17 th bases were deleted after editing

<400>54

atgtctcgct ccgtgddctt agctgtgctc gcgctactct ctct 44

<210>55

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(16)..(21)

<223> 16 th to 21 st bases were deleted after editing

<400>55

atgtctcgct ccgtgddddd dgctgtgctc gcgctactct ctct 44

<210>56

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(17)..(18)

<223> 17 th to 18 th bases were deleted after editing

<400>56

atgtctcgct ccgtggddtt agctgtgctc gcgctactct ctct 44

<210>57

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(7)..(29)

<223> bases at positions 7 to 29 were deleted after editing

<400>57

atgtctdddd dddddddddd dddddddddc gcgctactct ctct 44

<210>58

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(13)..(18)

<223> bases at positions 13 to 18 were deleted after editing

<400>58

atgtctcgct ccddddddtt agctgtgctc gcgctactct ctct 44

<210>59

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(16)..(18)

<223> 16 th to 18 th bases were deleted after editing

<400>59

atgtctcgct ccgtgdddtt agctgtgctc gcgctactct ctct 44

<210>60

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(2)..(23)

<223> bases at positions 2 to 23 were deleted after editing

<400>60

addddddddd dddddddddd dddtgtgctc gcgctactct ctct 44

<210>61

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(16)..(29)

<223> 16 th to 29 th bases were deleted after editing

<400>61

atgtctcgct ccgtgddddd dddddddddc gcgctactct ctct 44

<210>62

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(8)..(16)

<223> 8 th to 16 th bases were deleted after editing

<400>62

atgtctcddd ddddddcctt agctgtgctc gcgctactct ctct 44

<210>63

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(6)..(17)

<223> bases at positions 6 to 17 were deleted after editing

<400>63

atgtcddddd dddddddctt agctgtgctc gcgctactct ctct 44

<210>64

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(2)..(26)

<223> bases at positions 2 to 26 were deleted after editing

<400>64

addddddddd dddddddddd ddddddgctc gcgctactct ctct 44

<210>65

<211>44

<212>DNA

<213> Artificial sequence

<220>

The sequences generated from position 003745 to position 45003788 are shown in the table below

<220>

<221>misc_feature

<222>(16)..(20)

<223> 16 th to 20 th bases were deleted after editing

<400>65

atgtctcgct ccgtgddddd agctgtgctc gcgctactct ctct 44

<210>66

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(2)..(24)

<223> bases at 2 nd to 24 th positions were deleted after editing

<400>66

addddddddd dddddddddd ddddgtgctc gcgctactct ctct 44

<210>67

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(14)..(25)

<223> bases at positions 14 to 25 were deleted after editing

<400>67

atgtctcgct ccgddddddd dddddtgctc gcgctactct ctct 44

<210>68

<211>45

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(17)..(17)

<223> n represents insertion of any one base

<400>68

atgtctcgct ccgtggncct tagctgtgct cgcgctactc tctct 45

<210>69

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(16)..(19)

<223> 16 th to 19 th bases were deleted after editing

<400>69

atgtctcgct ccgtgddddt agctgtgctc gcgctactct ctct 44

<210>70

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(2)..(20)

<223> bases at 2 nd to 20 th positions were deleted after editing

<400>70

addddddddd dddddddddd agctgtgctc gcgctactct ctct 44

<210>71

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(13)..(16)

<223> bases at positions 13 to 16 were deleted after editing

<400>71

atgtctcgct ccddddcctt agctgtgctc gcgctactct ctct 44

<210>72

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(12)..(31)

<223> bases at positions 12 to 31 were deleted after editing

<400>72

atgtctcgct cddddddddd dddddddddd dcgctactct ctct 44

<210>73

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(13)..(19)

<223> bases at positions 13 to 19 were deleted after editing

<400>73

atgtctcgct ccdddddddt agctgtgctc gcgctactct ctct 44

<210>74

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(11)..(28)

<223> bases at 11 th to 28 th positions were deleted after editing

<400>74

atgtctcgct dddddddddd ddddddddtc gcgctactct ctct 44

<210>75

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 003745 th bit to 45003788 th bit

<220>

<221>misc_feature

<222>(7)..(25)

<223> bases at positions 7 to 25 were deleted after editing

<400>75

atgtctdddd dddddddddd dddddtgctc gcgctactct ctct 44

<210>76

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: t3 was edited as a guide RNA on chromosome 14, chromosome 23

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(22)..(22)

<223> n represents insertion of any one base

<400>76

tatccagaac cctgaccctg cncgtgtacc agctgagaga ctct 44

<210>77

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(20)..(30)

<223> 20 th to 30 th bases were deleted after editing

<400>77

tatccagaac cctgaccctd dddddddddd gctgagagac tct 43

<210>78

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(21)..(25)

<223> 21 st to 25 th bases were deleted after editing

<400>78

tatccagaac cctgaccctg dddddtacca gctgagagac tct 43

<210>79

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(9)..(32)

<223> 9 th to 32 th bases were deleted after editing

<400>79

tatccagadd dddddddddd dddddddddd ddtgagagac tct 43

<210>80

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(21)..(21)

<223> base at position 22 was deleted after editing

<400>80

tatccagaac cctgaccctg dcgtgtacca gctgagagac tct 43

<210>81

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(17)..(28)

<223> 17 th to 28 th bases were deleted after editing

<400>81

tatccagaac cctgacdddd ddddddddca gctgagagac tct 43

<210>82

<211>45

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(22)..(23)

<223> n represents insertion of any one base

<400>82

tatccagaac cctgaccctg cnncgtgtac cagctgagag actct 45

<210>83

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(16)..(26)

<223> 16 th to 26 th bases were deleted after editing

<400>83

tatccagaac cctgaddddd ddddddacca gctgagagac tct 43

<210>84

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(21)..(32)

<223> base at position 22 was deleted after editing

<400>84

tatccagaac cctgaccctg dddddddddd ddtgagagac tct 43

<210>85

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(18)..(23)

<223> 18 th to 23 rd bases were deleted after editing

<400>85

tatccagaac cctgaccddd dddtgtacca gctgagagac tct 43

<210>86

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(7)..(23)

<223> bases at positions 7 to 23 were deleted after editing

<400>86

tatccadddd dddddddddd dddtgtacca gctgagagac tct 43

<210>87

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(7)..(24)

<223> bases at positions 7 to 24 were deleted after editing

<400>87

tatccadddd dddddddddd ddddgtacca gctgagagac tct 43

<210>88

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(22)..(23)

<223> bases at positions 22 to 23 were deleted after editing

<400>88

tatccagaac cctgaccctg cddtgtacca gctgagagac tct 43

<210>89

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(9)..(22)

<223> 9 th to 22 nd bases were deleted after editing

<400>89

tatccagadd dddddddddd ddgtgtacca gctgagagac tct 43

<210>90

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(21)..(22)

<223> 21 st to 22 nd bases were deleted after editing

<400>90

tatccagaac cctgaccctg ddgtgtacca gctgagagac tct 43

<210>91

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(16)..(29)

<223> 16 th to 29 th bases were deleted after editing

<400>91

tatccagaac cctgaddddd ddddddddda gctgagagac tct 43

<210>92

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(16)..(35)

<223> 16 th to 35 th bases were deleted after editing

<400>92

tatccagaac cctgaddddd dddddddddd dddddgagac tct 43

<210>93

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(7)..(26)

<223> bases at positions 7 to 26 were deleted after editing

<400>93

tatccadddd dddddddddd ddddddacca gctgagagac tct 43

<210>94

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(12)..(23)

<223> bases at positions 12 to 23 were deleted after editing

<400>94

tatccagaac cddddddddd dddtgtacca gctgagagac tct 43

<210>95

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(21)..(23)

<223> 21 st to 23 rd bases were deleted after editing

<400>95

tatccagaac cctgaccctg dddtgtacca gctgagagac tct 43

<210>96

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(16)..(22)

<223> 16 th to 22 nd bases were deleted after editing

<400>96

tatccagaac cctgaddddd ddgtgtacca gctgagagac tct 43

<210>97

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(9)..(26)

<223> bases at positions 9 to 26 were deleted after editing

<400>97

tatccagadd dddddddddd ddddddacca gctgagagac tct 43

<210>98

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(18)..(22)

<223> 18 th to 22 nd bases were deleted after editing

<400>98

tatccagaac cctgaccddd ddgtgtacca gctgagagac tct 43

<210>99

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(21)..(34)

<223> bases at 21 st to 34 th positions were deleted after editing

<400>99

tatccagaac cctgaccctg dddddddddd ddddagagac tct 43

<210>100

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(15)..(32)

<223> 15 th to 32 th bases were deleted after editing

<400>100

tatccagaac cctgdddddd dddddddddd ddtgagagac tct 43

<210>101

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(15)..(25)

<223> bases at positions 15 to 25 were deleted after editing

<400>101

tatccagaac cctgdddddd dddddtacca gctgagagac tct 43

<210>102

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(17)..(29)

<223> 17 th to 29 th bases were deleted after editing

<400>102

tatccagaac cctgacdddd ddddddddda gctgagagac tct 43

<210>103

<211>43

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: t2 was edited as a guide RNA on chromosome 14, chromosome 23

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(15)..(20)

<223> bases at positions 15 to 20 were deleted after editing

<400>103

tatccagaac cctgdddddd ccgtgtacca gctgagagac tct 43

<210>104

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(16)..(21)

<223> 16 th to 21 st bases were deleted after editing

<400>104

tatccagaac cctgaddddd dcgtgtacca gctgagagac tct 43

<210>105

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(15)..(15)

<223> the 15 th base was deleted after editing

<400>105

tatccagaac cctgdccctg ccgtgtacca gctgagagac tct 43

<210>106

<211>44

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(16)..(16)

<223> n represents insertion of any one base

<400>106

tatccagaac cctganccct gccgtgtacc agctgagaga ctct 44

<210>107

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(15)..(16)

<223> bases at positions 15 to 16 were deleted after editing

<400>107

tatccagaac cctgddcctg ccgtgtacca gctgagagac tct 43

<210>108

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(2)..(25)

<223> bases at 2 nd to 25 th positions were deleted after editing

<400>108

tddddddddd dddddddddd dddddtacca gctgagagac tct 43

<210>109

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(15)..(18)

<223> bases at positions 15 to 18 were deleted after editing

<400>109

tatccagaac cctgddddtg ccgtgtacca gctgagagac tct 43

<210>110

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(7)..(23)

<223> bases at positions 7 to 23 were deleted after editing

<400>110

tatccadddd dddddddddd dddtgtacca gctgagagac tct 43

<210>111

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(5)..(15)

<223> bases at 5 th to 15 th positions were deleted after editing

<400>111

tatcdddddd dddddccctg ccgtgtacca gctgagagac tct 43

<210>112

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(9)..(26)

<223> bases at positions 9 to 26 were deleted after editing

<400>112

tatccagadd dddddddddd ddddddacca gctgagagac tct 43

<210>113

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(6)..(17)

<223> bases at positions 6 to 17 were deleted after editing

<400>113

tatccddddd dddddddctg ccgtgtacca gctgagagac tct 43

<210>114

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(5)..(29)

<223> bases at 5 th to 29 th positions were deleted after editing

<400>114

tatcdddddd dddddddddd ddddddddda gctgagagac tct 43

<210>115

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(9)..(15)

<223> 9 th to 15 th bases were deleted after editing

<400>115

tatccagadd dddddccctg ccgtgtacca gctgagagac tct 43

<210>116

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(15)..(21)

<223> bases at positions 15 to 21 were deleted after editing

<400>116

tatccagaac cctgdddddd dcgtgtacca gctgagagac tct 43

<210>117

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(21)..(25)

<223> 21 st to 25 th bases were deleted after editing

<400>117

tatccagaac dddddccctg ccgtgtacca gctgagagac tct 43

<210>118

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(2)..(24)

<223> bases at 2 nd to 24 th positions were deleted after editing

<400>118

tddddddddd dddddddddd ddddgtacca gctgagagac tct 43

<210>119

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(1)..(15)

<223> bases at 1-15 th positions were deleted after editing

<400>119

dddddddddd dddddccctg ccgtgtacca gctgagagac tct 43

<210>120

<211>58

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(15)..(30)

<223> bases at positions 15 to 30 were deleted after editing

<400>120

tatccagaac cctgannnnn nnnnnnnnnn ccctgccgtg taccagctga gagactct 58

<210>121

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(9)..(21)

<223> 9 st to 21 st bases were deleted after editing

<400>121

tatccagadd dddddddddd dcgtgtacca gctgagagac tct 43

<210>122

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(9)..(18)

<223> 9 th to 18 th bases were deleted after editing

<400>122

tatccagadd ddddddddtg ccgtgtacca gctgagagac tct 43

<210>123

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 016448 th bit to 23016490 th bit

<220>

<221>misc_feature

<222>(6)..(15)

<223> bases at positions 6 to 15 were deleted after editing

<400>123

tatccddddd dddddccctg ccgtgtacca gctgagagac tct 43

<210>124

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: p2 was edited as a guide RNA on chromosome 2, 242%

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(22)..(22)

<223> n represents insertion of any one base

<400>124

agcccagttg tagcaccgcc cnagacgact ggccagggcg cctg 44

<210>125

<211>45

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(22)..(23)

<223> n represents insertion of any one base

<400>125

agcccagttg tagcaccgcc cnnagacgac tggccagggc gcctg 45

<210>126

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(29)..(33)

<223> bases at positions 29 to 33 were deleted after editing

<400>126

agcccagttg tagcaccgdd dddacgactg gccagggcgc ctg 43

<210>127

<211>50

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(22)..(35)

<223> n represents insertion of any one base

<400>127

agcccagttg tagcaccgcc cnnnnnnnnn nnnnnagacg actggccagg 50

<210>128

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(13)..(29)

<223> bases at positions 13 to 29 were deleted after editing

<400>128

agcccagttg tadddddddd dddddddddg gccagggcgc ctg 43

<210>129

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(10)..(33)

<223> bases at 10 th to 33 th positions were deleted after editing

<400>129

agcccagttd dddddddddd dddddddddd dddagggcgc ctg 43

<210>130

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(7)..(28)

<223> bases at positions 7 to 28 were deleted after editing

<400>130

agcccadddd dddddddddd ddddddddtg gccagggcgc ctg 43

<210>131

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(21)..(21)

<223> base at position 21 was deleted after editing

<400>131

agcccagttg tagcaccgcc dagacgactg gccagggcgc ctg 43

<210>132

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(22)..(26)

<223> bases at positions 22 to 26 were deleted after editing

<400>132

agcccagttg tagcaccgcc cdddddactg gccagggcgc ctg 43

<210>133

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(10)..(24)

<223> bases at 10 th to 24 th positions were deleted after editing

<400>133

agcccagttd dddddddddd ddddcgactg gccagggcgc ctg 43

<210>134

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(22)..(23)

<223> bases at positions 22 to 23 were deleted after editing

<400>134

agcccagttg tagcaccgcc cddacgactg gccagggcgc ctg 43

<210>135

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(20)..(21)

<223> 20 th to 21 st bases were deleted after editing

<400>135

agcccagttg tagcaccgcd dagacgactg gccagggcgc ctg 43

<210>136

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(14)..(22)

<223> bases at positions 14-22 were deleted after editing

<400>136

agcccagttg tagddddddd ddgacgactg gccagggcgc ctg 43

<210>137

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(14)..(23)

<223> bases at positions 14-23 were deleted after editing

<400>137

agcccagttg tagddddddd dddacgactg gccagggcgc ctg 43

<210>138

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(9)..(32)

<223> 9 th to 32 th bases were deleted after editing

<400>138

agcccagtdd dddddddddd dddddddddd ddcagggcgc ctg 43

<210>139

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(7)..(36)

<223> bases at positions 7 to 36 were deleted after editing

<400>139

agcccadddd dddddddddd dddddddddd ddddddgcgc ctg 43

<210>140

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(19)..(22)

<223> bases at 19 th to 22 th positions were deleted after editing

<400>140

agcccagttg tagcaccgdd ddgacgactg gccagggcgc ctg 43

<210>141

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(21)..(22)

<223> 21 st to 22 nd bases were deleted after editing

<400>141

agcccagttg tagcaccgcc ddgacgactg gccagggcgc ctg 43

<210>142

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(11)..(25)

<223> bases at 11 th to 25 th positions were deleted after editing

<400>142

agcccagttg dddddddddd dddddgactg gccagggcgc ctg 43

<210>143

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(12)..(34)

<223> bases at positions 12 to 34 were deleted after editing

<400>143

agcccagttg tddddddddd dddddddddd ddddgggcgc ctg 43

<210>144

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(7)..(21)

<223> bases at positions 7 to 21 were deleted after editing

<400>144

agcccadddd dddddddddd dagacgactg gccagggcgc ctg 43

<210>145

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(12)..(26)

<223> bases at 12 th to 36 th positions were deleted after editing

<400>145

agcccagttg tddddddddd ddddddactg gccagggcgc ctg 43

<210>146

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(7)..(27)

<223> bases at positions 7 to 27 were deleted after editing

<400>146

agcccadddd dddddddddd dddddddctg gccagggcgc ctg 43

<210>147

<211>44

<212>DNA

<213> Artificial sequence

<220>

<223> description of artificial sequences: p1 was edited as a guide RNA on chromosome 2, 242%

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(26)..(26)

<223> n represents insertion of any one base

<400>147

cagtttagca cgaagctctc cgatgntgtt ggagaagctg cagg 44

<210>148

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(26)..(27)

<223> 26 th to 27 th bases were deleted after editing

<400>148

cagtttagca cgaagctctc cgatgddttg gagaagctgc agg 43

<210>149

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(25)..(25)

<223> base at position 25 was deleted after editing

<400>149

cagtttagca cgaagctctc cgatdtgttg gagaagctgc agg 43

<210>150

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(23)..(25)

<223> 23 st to 25 th bases were deleted after editing

<400>150

cagtttagca cgaagctctc cgdddtgttg gagaagctgc agg 43

<210>151

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(12)..(25)

<223> bases at positions 12 to 25 were deleted after editing

<400>151

cagtttagca cddddddddd dddddtgttg gagaagctgc agg 43

<210>152

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(21)..(25)

<223> 21 st to 25 th bases were deleted after editing

<400>152

cagtttagca cgaagctctc dddddtgttg gagaagctgc agg 43

<210>153

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(22)..(25)

<223> bases at positions 22 to 25 were deleted after editing

<400>153

cagtttagca cgaagctctc cddddtgttg gagaagctgc agg 43

<210>154

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(11)..(31)

<223> bases at 11 th to 31 th positions were deleted after editing

<400>154

cagtttagca dddddddddd dddddddddd dagaagctgc agg 43

<210>155

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(28)..(35)

<223> bases at positions 28 to 35 were deleted after editing

<400>155

cagtttagca cgaagctddd dddddtgttg gagaagctgc agg 43

<210>156

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(20)..(25)

<223> 20 th to 25 th bases were deleted after editing

<400>156

cagtttagca cgaagctctd dddddtgttg gagaagctgc agg 43

<210>157

<211>56

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(26)..(38)

<223> 26 th to 38 th bases were deleted after editing

<400>157

cagtttagca cgaagctctc cgatgnnnnn nnnnnnnntg ttggagaagc tgcagg 56

<210>158

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(21)..(36)

<223> bases at 21 st to 36 th positions were deleted after editing

<400>158

cagtttagca dddddddddd ddddddgttg gagaagctgc agg 43

<210>159

<211>45

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(26)..(27)

<223> n represents insertion of any one base

<400>159

cagtttagca cgaagctctc cgatgnntgt tggagaagct gcagg 45

<210>160

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(11)..(27)

<223> bases at 11 th to 27 th positions were deleted after editing

<400>160

cagtttagca dddddddddd dddddddttg gagaagctgc agg 43

<210>161

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(13)..(25)

<223> bases at positions 13 to 25 were deleted after editing

<400>161

cagtttagca cgdddddddd dddddtgttg gagaagctgc agg 43

<210>162

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(20)..(26)

<223> 20 th to 26 th bases were deleted after editing

<400>162

cagtttagca cgaagctctd ddddddgttg gagaagctgc agg 43

<210>163

<211>43

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(11)..(25)

<223> bases at 11 th to 25 th positions were deleted after editing

<400>163

cagtttagca dddddddddd dddddtgttg gagaagctgc agg 43

<210>164

<211>51

<212>DNA

<213> Artificial sequence

<220>

Sequence generated from 800936 th bit to 242800978 th bit

<220>

<221>misc_feature

<222>(26)..(33)

<223> 26 th to 33 th bases were deleted after editing

<400>164

cagtttagca cgaagctctc cgatgnnnnn nnntgttgga gaagctgcag g 51

Claims

1. A method of making a genetically engineered T cell comprising: disrupting in the T cell by gene editing techniques:

2. The method of claim 1 wherein said TRAC genomic region, B2M genomic region and PD-1 genomic region are edited.

3. The method of claim 1 or 2, wherein the gene editing technology is a zinc finger nuclease-based gene editing technology, a TALEN gene editing technology, or a CRISPR/Cas gene editing technology.

4. The method of claim 3, wherein the gene editing technique is a CRISPR/Cas9 gene editing technique.

5. The method of any one of claims 1-4, wherein:

6. The method recited in claim 4 wherein:

7. The method of claim 6, comprising:

8. The method of claim 7 or 8, comprising introducing the sgRNA targeting TRAC, the sgRNA targeting B2M, and the sgRNA targeting PD-1 simultaneously into the T cell.

9. The method of any one of claims 6-8, wherein the sgRNA is modified with a2 '-O-methyl analog and/or an internucleotide 3' thio.

10. The method of claim 9, wherein the chemical modification is a2 ' -O-methyl analog modification of the first, two, and/or three bases of the 5 ' end and/or the last base of the 3 ' end of the sgRNA.