CN117062831A

CN117062831A - Efficient TCR gene editing in T lymphocytes

Info

Publication number: CN117062831A
Application number: CN202280023611.0A
Authority: CN
Inventors: S·鲁茨; B·J·海利; S·麦迪雷迪; S·奥; D·肖; K·H·森杰
Original assignee: Genentech Inc
Current assignee: Genentech Inc
Priority date: 2021-03-24
Filing date: 2022-03-24
Publication date: 2023-11-14
Also published as: TW202309272A; EP4314039A1; AR125225A1

Abstract

The present disclosure relates to engineered T cells and methods of making and using the same, as well as reagents for making the engineered T cells.

Description

Efficient TCR gene editing in T lymphocytes

Cross Reference to Related Applications

The present application claims the benefit of priority from U.S. provisional application number 63/165,509, filed on 24 months 3 of 2021, and U.S. provisional application number 63/323,065, filed on 24 months 3 of 2022, each of which is incorporated herein by reference in its entirety and for all purposes.

Sequence listing

Background

T cell therapy is of increasing interest in the treatment of many different diseases, including cancer, infectious diseases and inflammatory diseases. However, the preparation of therapeutic T cells using current virus-based engineering methods is time consuming and expensive and presents certain safety issues. Furthermore, methods to date that use non-viral methods to produce engineered T cells have low editing efficiency and may limit cell yield.

Disclosure of Invention

The present technology relates generally to engineered T cells and related systems, methods, and kits. This technique solves the problem of low efficiency and low number of engineered T cells that are typically produced using known non-viral methods.

In one aspect, an engineered T cell is provided. Engineered T cells include nucleic acid sequences encoding polypeptides comprising exogenous T Cell Receptors (TCRs) or portions thereof. In embodiments, the exogenous TCR or portion thereof is exogenous T cell receptor β (TCR- β) and/or exogenous T cell receptor α (TCR- α) or portion thereof. In embodiments, the nucleic acid sequence is inserted into a TCR locus. In embodiments, the TCR locus is a TCR-a locus of an engineered T cell. In an embodiment, the TCR locus is the TCR- β locus of an engineered T cell.

In embodiments, the exogenous TCR-a comprises at least a portion of an endogenous TCR-a. In embodiments, the exogenous TCR-a comprises an exogenous TCR-a (VJ) domain and an endogenous TCR-a constant domain.

In embodiments, the nucleic acid sequence further comprises a polyadenylation (polyA) sequence. In embodiments, the nucleic acid sequence further comprises a stop codon. In an embodiment, the stop codon is 3 'of the exogenous TCR and polyA sequence 5'. In embodiments, the TCR knock-in (KI) construct further comprises a polyA sequence. In embodiments, SEQ ID NO:1 comprises a polyA sequence.

In another interrelated aspect, a composition comprising isolated T cells is provided, wherein at least 5% of the cells are engineered T cells. In embodiments, each engineered T cell includes a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR- β and an exogenous TCR- α. In embodiments, each engineered T cell includes a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR- β and an exogenous TCR- α. In embodiments, each engineered T cell comprises a nucleic acid sequence encoding a polypeptide comprising at least a portion of an exogenous TCR- β and at least a portion of an exogenous TCR- α. In embodiments, the portion of the exogenous TCR-alpha is the TCR-alpha VJ region. In embodiments, the nucleic acid sequence is inserted into the TCR-a locus of an engineered T cell.

In embodiments, the engineered T cells do not express a functional endogenous TCR- β protein. In embodiments, the engineered T cells do not express a functional endogenous TCR-a protein. In embodiments, the exogenous TCR-a (VJ) domain forms part of a heterologous TCR-a comprising at least a portion of an endogenous TCR-a of a T cell. In embodiments, the TCR-alpha locus is a TCR-alpha constant region. In embodiments, exogenous TCR- β and heterologous TCR- α are expressed from nucleic acids and form a functional TCR. In embodiments, the engineered T cells bind to an antigen. In embodiments, the engineered T cells bind to cancer cells. In an embodiment, the TCR binds to an antigen presented on a class I major histocompatibility complex (mhc I) molecule.

In embodiments, the antigen is a neoantigen or a Tumor Associated Antigen (TAA). In embodiments, the antigen is a neoantigen. In embodiments, the antigen is TAA. In embodiments, the neoantigen or TAA is selected from: WT1, JAK2, NY-ESO1, PRAME, KRAS, or antigens from table 1 or table 2. In embodiments, the antigen is WT1. The mhc i allele may be any allele known or found. In an embodiment, the MHCI is selected from HLA-A02:01, A02:03, A02:06, A02:07, A023:01, A26:01, A29:02, A30:01, A30:02, A31:01, A32:01, A68:01, A68:02, B18:01, B35:03, B40:01, B40:02, B40:06, B46:01, B51:01, B53:01, B57:01, B58:01, C01:02, C02:02, C03:03, C02:03, C02, C01, C02:12, C01, C02:08:08, C01, C12:12:08:01. In embodiments, the antigen is specific for cancer in a subject to whom the engineered T cells are to be administered.

Related methods, compositions and reagents are found in the following patent applications: US A1 "- -CELLS", WO A1 "", US A1 "", WO A1 "", HIGHOLY "; US A1" - -CELLS ", WO A2" ", WO A1" MONITORING "; WO A1" ", US A1" ", US A1" - ", US" - ", WO A2" ", WO A1" ", WO A2" ", WO A1" UNIQUELY ", WO A1" ", WO A1" UNIQUELY ", WO A1" LARGE-SEQUENCE TAGS ", WO A1" ", WO A1" METHODS FOR ", WO2015160439A2"QUANTIFICATION OF ADAPTIVE IMMUNE CELL GENOMES IN A COMPLEX MIXTURE OF CELLS", WO2016086029A1"CHARACTERIZATION OF ADAPTIVE IMMUNE RESPONSE TO VACCINATION OR INFECTION USING IMMUNE REPERTOIRE SEQUENCING", US20180282808A1"DETERMINING WT-1SPECIFIC T CELLS AND WT-1SPECIFIC T CELL RECEPTORS (TCRS)", each of which is incorporated herein by reference in its entirety for all purposes, including but not limited to all methods of preparation, methods of use, reagents, cells, proteins, nucleic acids, compositions, and the like.

In an embodiment, expression of the endogenous TCR- β gene is disrupted by gene editing. In embodiments, the endogenous TCR- β gene is disrupted in greater than about 80% of the cells. In embodiments, the endogenous TCR- β gene is disrupted in greater than about 90% of the cells. In embodiments, the endogenous TCR- β gene is disrupted in greater than about 95% of the cells.

In an embodiment, expression of the endogenous TCR-a gene is disrupted by gene editing. In embodiments, the endogenous TCR-a gene is disrupted in greater than about 80% of the cells. In embodiments, the endogenous TCR-a gene is disrupted in greater than about 90% of the cells. In embodiments, the endogenous TCR-a gene is disrupted in greater than about 95% of the cells.

In embodiments, the nucleic acid sequence further encodes a self-cleaving peptide. In an embodiment, the self-cleaving peptide is a self-cleaving viral peptide. In an embodiment, the self-cleaving viral peptides are T2A (SEQ ID NO: 49), P2A (SEQ ID NO: 50), E2A (SEQ ID NO: 51), F2A (SEQ ID NO: 52). In an embodiment, unique self-cleaving viral peptide sequences are labeled.

In an embodiment, the engineered T cells express CD45RO, type 7C-C chemokine receptor (CCR 7) and L-selectin (CD 62L). In embodiments, the engineered T cells have a Central Memory (CM) T cell phenotype. In embodiments, the engineered T cells have an initial T cell phenotype. In an embodiment, the engineered T cell having the initial T cell phenotype is cd45ra+cd45ro-cd27+cd95-. In an embodiment, the engineered T cells have a stem cell memory T cell phenotype. In an embodiment, the engineered T-cells with a stem cell memory T-cell phenotype are cd45ra+cd45ro-cd27+cd95+cd58+ccr7-Hi tcf1+. In embodiments, the engineered T cells have a central memory T cell phenotype. In an embodiment, the engineered T cells with a central memory T cell phenotype are cd45ro+cd45ra-cd27+cd95+cd58+. In embodiments, the engineered T cells have a progenitor depleted T cell phenotype. In an embodiment, the engineered T-cell having a progenitor cell depleted T-cell phenotype is PD-1+slamf6+tcf1+tim3-CD39-. In embodiments, an engineered T cell having a progenitor depleted T cell phenotype expresses PD-1 at a low or moderate level as compared to a PD-1 high depleted T cell. In an embodiment, an engineered T cell having a progenitor depleted T cell phenotype expresses PD-1 at a low or moderate level compared to a recently activated T cell. In embodiments, the T cells are autologous to the subject in need thereof.

In another interrelated aspect, a pharmaceutical composition is provided. The pharmaceutical composition comprises an engineered T cell population as described herein (including the examples) and a pharmaceutically acceptable excipient.

In an embodiment, at least 10% of the cells in the composition comprising isolated T cells are engineered T cells. In embodiments, at least 20% of the T cells are engineered T cells. In embodiments, at least 30% of the T cells are engineered T cells. In embodiments, at least 40% of the T cells are engineered T cells. In embodiments, at least 50% of the T cells are engineered T cells. In embodiments, at least 60% of the T cells are engineered T cells. In embodiments, at least 70% of the T cells are engineered T cells. In embodiments, at least 80% of the T cells are engineered T cells.

In an embodiment, the composition comprises about 0.1×10 ⁵ And about 1X 10 ¹¹ Engineered T cells between individuals. In embodiments, the composition comprises at least about 1x 10 ⁸ Engineering T cells. In an embodiment, the composition comprises about 1×10 ⁸ And about 1X 10 ¹¹ Engineered T cells between individuals. In embodiments, the composition comprises at least about 1x 10 ⁹ Engineering T cells. In embodiments, the composition comprises at least about 1x 10 ¹⁰ Engineering T cells. In embodiments, the composition comprises at least about 1x 10 ¹¹ Engineering T cells. In embodiments, the composition further comprises a pharmaceutically acceptable excipient.

In another interrelated aspect, a T cell comprising an RNA transcript having an mRNA transcript transcribed from a TCR transgene inserted into a TCR-a or TCR- β locus is provided.

In another interrelated aspect, a non-viral method for preparing engineered T cells is provided. In an embodiment, the method uses gene editing based on regularly spaced clustered short palindromic repeats (CRISPR). In embodiments, the method uses Cas9. In an embodiment, the method uses high fidelity Cas9. In an embodiment, the method uses SpyFi Cas9. In embodiments, the gene editing reagent is provided as Cas9 ribonucleoprotein particles (RNPs). The method comprises the following steps: a) Contacting a T cell with a first CRISPR/Cas9 RNP and a donor DNA under conditions that allow the RNP and the donor DNA to enter the cell, wherein the first Cas9/RNP comprises a first guide RNA that targets an endogenous TCR locus, and wherein the donor DNA comprises a nucleic acid sequence encoding a form of TCR specified herein; b) Incubating the T cells for a period of time; and c) culturing the cells in a medium for a period of time to allow the donor DNA to recombine into an endogenous TCR locus, thereby forming engineered T cells. In embodiments, the first guide RNA targets a TCR locus that is a TCR-a locus. In embodiments, the TCR locus targeted by the second guide RNA is a TCR- β locus. In embodiments, the first RNP comprises a first gene-editing protein and a molar excess of the first guide RNA relative to the guide RNA. In embodiments, the first RNP comprises a first gene-editing protein and the ratio of the first guide RNA to the first gene-editing protein is between 1:1 and 100:1.

In embodiments, the TCR locus targeted by the first guide RNA is a TCR-a locus, and the T cell is further contacted with a second RNP comprising a second guide RNA that targets an endogenous TCR- β locus. In embodiments, the T cell is further contacted with a second donor DNA, wherein the second donor DNA comprises a nucleic acid sequence encoding a TCR in the form specified herein. The cells may be contacted in any order. In embodiments, in step a), the T cells are contacted with a second RNP and optionally a second donor DNA. In embodiments, the T cell is contacted with a second RNP and a second donor DNA between steps a) and b). In embodiments, prior to step a), the T cell is contacted with a second RNP and a second donor DNA. In an embodiment, after step c), the T cells are contacted with a second RNP and a second donor DNA. In embodiments, the second RNP comprises a second gene-editing protein and a molar excess of the second guide RNA relative to the guide RNA. In embodiments, the second RNP comprises a second gene-editing protein and the ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 100:1. In embodiments, less than 10% of the engineered T cells express functional endogenous TCR- β as compared to the control. In embodiments, less than 1% of the engineered T cells express functional endogenous TCR- β as compared to the control. In embodiments, the engineered T cells do not express a functional endogenous TCR- β.

In an example, the first guide RNA is found by methods known to those of skill in the art such that it targets exon 1, exon 2, or exon 3 region of the TCR constant alpha region locus (TRAC). In embodiments, the first guide RNA comprises one or more of: TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21) or TRAC16 (SEQ ID NO: 22). In embodiments, the first guide RNA comprises one or more of: TRAC1, TRAC3, TRAC4, TRAC5, TRAC7, TRAC12 or TRAC15. In embodiments, the first guide RNA targets TRAC1. In embodiments, the first guide RNA comprises TRAC3. In an embodiment, the first guide RNA comprises the nucleic acid sequence in table 10.

In an example, the second guide RNA is found by methods known to those skilled in the art such that it targets exon 1 regions of two TCR constant β region loci (TRBC) in T cells. In embodiments, the second guide RNA comprises one or more of the following: TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 46) or TRBC 48 (SEQ ID NO: 48). In embodiments, the second guide RNA comprises one or more of the following: TRBC4, TRBC8, TRBC13, TRBC19, TRBC20, TRBC21, TRBC22, TRBC23 or TRBC26. In an embodiment, the second guide RNA comprises the nucleic acid sequence in table 11.

In embodiments, the conditions that allow RNP and donor DNA to enter the cell include electroporation. In an embodiment, the electroporation method comprises a commercial electroporation kit (e.g., NUCLEOFECTION (Lonza)). In an embodiment, in method step b), the T cells are incubated for at least 10 minutes. In an embodiment, T cells are incubated at about 37 ℃. In an embodiment, the T cells are incubated at less than about 37 ℃. In an embodiment, the medium comprises a cytokine. In embodiments, the cytokine comprises interleukin-2 (IL-2), interleukin-7 (IL-7), and/or interleukin-15 (IL-15). In embodiments, the cytokine comprises IL-2. In embodiments, the cytokine comprises IL-7. In embodiments, the cytokine comprises IL-15. In embodiments, cytokines include IL-7 and IL-15.

In an embodiment, method step a) is carried out in the presence of a negatively charged polymer. In embodiments, the polymer is poly (glutamic acid) (PGA) or a variant thereof, poly (aspartic acid), heparin, or poly (acrylic acid). In an embodiment, the PGA is poly (L-glutamic acid) or a variant thereof. In embodiments, the PGA is poly (D-glutamic acid) or a variant thereof. In an embodiment, the PGA or variant thereof has an average molecular weight between 15 kilodaltons (kDa) and 50 kDa. In an embodiment, about 2 μg/μl to about 15 μg/μl of polymer is added.

In embodiments, the amount of the first and/or second RNP is about 0.2 pmol/. Mu.L to about 10 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.5 fmol/. Mu.L to about 0.5 pmol/. Mu.L. In an embodiment, the amount of RNP is about 5pmol to about 200pmol. In an embodiment, the amount of donor DNA is from about 0.01pmol to about 10pmol. In embodiments, a portion of the donor DNA is recombined into the endogenous TCR-a locus. In embodiments, the TCR-alpha locus is a TCR-alpha constant region locus. In embodiments, the exogenous TCR-a (VJ) domain forms part of a heterologous TCR-a comprising at least a portion of an endogenous TCR-a of an engineered T cell. In an embodiment, the donor DNA comprises a left homology arm and a right homology arm.

In embodiments, the left and right homology arms are homologous to an endogenous TCR-a locus. In embodiments, the left homology arm is about 50 bases to about 2000 bases in length. In embodiments, the left homology arm is about 100 bases to about 1000 bases in length. In embodiments, the left homology arm is about 200 bases to about 800 bases in length. In embodiments, the right homology arm is about 200 bases to about 2000 bases in length. In embodiments, the right homology arm is from about 100 bases to about 1000 bases. In embodiments, the left and right homology arms are homologous to an endogenous TCR- β locus. In embodiments, the left homology arm is about 200 bases to about 800 bases in length. In embodiments, the left homology arm is about 250 bases to about 700 bases in length. In embodiments, the right homology arm is about 200 bases to about 800 bases in length. In embodiments, the right homology arm is about 250 bases to about 700 bases in length.

In an embodiment, the donor DNA comprises double stranded DNA (dsDNA). In embodiments, the donor DNA is on a plasmid, a nanoplasmid, or a microring. In an embodiment, the donor DNA is contained within a plasmid. In an embodiment, the donor DNA is on a nanoplasmid. In an embodiment, the donor DNA is on a micro-loop. In an embodiment, the donor DNA is linear. In an embodiment, the donor DNA is a PCR product. In an embodiment, the donor DNA comprises single stranded DNA (ssDNA). In an embodiment, the donor DNA is not chemically modified. In an embodiment, the donor DNA comprises a chemical modification. In embodiments, the modification comprises a 5 'phosphate or a 5' phosphorothioate.

In an embodiment, the donor DNA and RNP are incubated together prior to method step a). In embodiments, the gene-editing protein comprises at least one Nuclear Localization Signal (NLS).

In an embodiment, the T cells are activated prior to method step a). In embodiments, the T cells are activated for between 24 hours and 96 hours. In an embodiment, the T cells are activated in the presence of a cytokine. In embodiments, the cytokine includes IL-2, IL-7 and/or IL-15. In embodiments, T cells are activated in the presence of between about 1ng/mL and about 200ng/mL IL-2. In embodiments, T cells are activated in the presence of between about 0ng/mL and about 50ng/mL IL-2. In an embodiment, T cells are activated in the presence of about 10ng/mL IL-2. In embodiments, T cells are activated in the presence of between about 1ng/mL and about 100ng/mL IL-7. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 500ng/mL IL-15. In embodiments, the T cells are activated in the presence of an anti-CD 3 antibody and/or an anti-CD 28 antibody. In embodiments, the T cells are activated in the presence of a CD3 agonist and/or a CD28 agonist. In embodiments, the anti-CD 3 antibody and/or the anti-CD 28 antibody is conjugated to a substrate. In embodiments, the CD3 agonist and/or CD28 agonist is conjugated to a substrate. In an embodiment, method step a) is performed no more than about 24 hours after activation. In an embodiment, method step a) is performed between about 24 hours and about 72 hours after activation. In an embodiment, method step a) is performed between about 36 hours and about 60 hours after activation.

In another interrelated aspect, there is provided a method for preparing an engineered T cell population comprising: t cell populations are subjected to the methods described herein (including the examples). In embodiments, at least about 5% of the population of T cells are engineered T cells. In embodiments, about 5% to about 100% of the T cell population is engineered T cells. In embodiments, at least about 50% of the population of T cells are engineered T cells. In embodiments, at least about 60% of the population of T cells are engineered T cells. In an embodiment, at least about 25% of the T cell population is viable after method step c). In an embodiment, at least about 50% of the population of T cells is viable after method step c). In an embodiment, at least about 75% of the population of T cells is viable after method step c). Amounts may be any value or subrange within the exemplified ranges, including the endpoints.

In an embodiment, the method further comprises: contacting the T cell with a second RNP comprising a guide RNA targeting an endogenous TCR- β locus. In embodiments, at least about 5% of the population of T cells are engineered T cells, wherein less than about 20% of the engineered T cells express endogenous TCR- β. In embodiments, about 10% to about 100% of the T cell population is engineered T cells. The contacting of the T cell with the second RNP may be performed before, during or after the contacting with the first RNP. In embodiments, the first RNP comprises a guide RNA targeting an endogenous TCR-a locus.

In an embodiment, method step a) comprises the following steps in any order: (i) adding donor DNA to the chamber; (ii) adding RNP to the chamber; and (iii) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps in order: (i) adding RNP to the chamber; (ii) adding donor DNA to the chamber; and (iii) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps in order: (i) adding RNP to the chamber; (ii) adding T cells to the chamber; and (iii) adding donor DNA to the chamber. In an embodiment, method step a) comprises the following steps in any order: (i) adding donor DNA to the chamber; (ii) adding RNP to the chamber; (iii) adding a negatively charged polymer to the chamber; and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps in order: (i) adding donor DNA to the chamber; (ii) adding RNP to the chamber; (iii) adding a negatively charged polymer to the chamber; and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps in order: (I) Combining RNP and a negatively charged polymer to form an RNP-polymer mixture; (ii) adding donor DNA to the chamber; (iii) adding the RNP-polymer mixture to the chamber; and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps in order: (i) adding RNP to the chamber; (ii) adding donor DNA to the chamber; (iii) adding a negatively charged polymer to the chamber; and (iv) adding T cells to the chamber. In an embodiment, no pipetting of T cells is performed during method steps a) and b). In an embodiment, the negatively charged polymer comprises PGA. Where two or more RNPs and/or two or more donor DNAs are used, they may be added simultaneously or in any order. In an embodiment, no negatively charged polymer is added to the chamber.

In another interrelated aspect, an engineered T cell is provided. Engineered T cells are prepared by the methods described herein (including the examples). In another interrelated aspect, an engineered T cell population is provided. An engineered T cell population is prepared by the methods described herein (including the examples).

In another interrelated aspect, a method of treating a subject having cancer is provided. The method comprises the following steps: a) Providing a population of T cells; b) Engineering at least a subset of the population of T cells to express an exogenous T Cell Receptor (TCR) and knock-out an endogenous TCR- β, thereby forming an engineered population of T cells, wherein the exogenous TCR binds to an antigen expressed by the cancer; c) Expanding the population of engineered T cells; and d) administering the expanded population of engineered T cells to the subject.

In embodiments, the antigen is a neoantigen or a Tumor Associated Antigen (TAA). In embodiments, at least a portion of the genome and/or transcriptome of the cancer is sequenced to determine the presence of the antigen. In an embodiment, an engineered T cell is prepared using the methods described herein (including the examples). In embodiments, the antigen is Wilms tumor gene 1 (WT 1), janus kinase 2 (JAK 2), new york esophageal squamous cell carcinoma-1 (NY-ESO 1), PRAME nuclear receptor transcription regulator (PRAME), or mutant Kirsten rat sarcoma virus (KRAS). In embodiments, the antigen is specific for cancer. In an embodiment, the TCR binds to an antigen presented on a class I major histocompatibility complex (mhc I) molecule. In embodiments, the mhc i comprises an mhc i allele expressed by a subject. In an embodiment, the expanded population of engineered T cells comprises 1 x 10 ⁵ And 1X 10 ⁹ Engineered T cells between individuals. In an embodiment, the expanded population of engineered T cells comprises at least 1 x 10 ⁸ Engineering T cells. In an embodiment, the expanded population of engineered T cells comprises about 1 x 10 ⁸ And about 1X 10 ¹¹ Engineered T cells between individuals. In an embodiment, the expanded population of engineered T cells comprises at least 1 x 10 ⁹ Engineering T cells. In an embodiment, the expanded population of engineered T cells comprises at least 1 x 10 ¹⁰ Engineering T cells. In an embodiment, the expanded population of engineered T cells comprises at least 1 x 10 ¹¹ Engineering T cells. In embodiments, the T cells are autologous to the subject.

In another interrelated aspect, a method for treating a subject having cancer is provided. The method comprises the following steps: a) Providing a first population of T cells isolated from a subject; b) Engineering at least a subset of the first population of T cells to express a first exogenous T Cell Receptor (TCR) and knockout an endogenous TCR- β, thereby forming a first population of engineered T cells, wherein the exogenous TCR binds to a first antigen expressed by the cancer; c) Expanding the first engineered T cell; d) Administering an expanded first population of engineered T cells to the subject; e) Providing a second population of T cells isolated from the subject; f) Engineering at least a subset of the second population of T cells to express a second exogenous TCR and knock-out an endogenous TCR- β, thereby forming a second population of engineered T cells, wherein the exogenous TCR binds to a second antigen expressed by the cancer; g) Expanding the second engineered T cell; and h) administering the expanded second population of engineered T cells to the subject.

In embodiments, at least a portion of the genome or transcriptome of the cancer is sequenced to determine the presence of the first antigen and the second antigen. In embodiments, the first antigen is WT1, JAK2, NY-ESO1, PRAME, mutant KRAS or HPV. In embodiments, the first TCR and/or the second TCR binds an antigen presented on an mhc i molecule. In embodiments, the antigen is a neoantigen or TAA. In embodiments, the mhc i comprises an mhc i allele expressed by a subject.

In an embodiment, the first expanded population of engineered T cells comprises 1 x 10 ⁵ And 1X 10 ⁹ Engineered T cells between individuals. In an embodiment, the first expanded population of engineered T cells comprises at least 1 x 10 ⁸ Engineering T cells. In an embodiment, the first expanded population of engineered T cells comprises at least 1 x 10 ⁹ Engineering T cells. In an embodiment, the first expanded population of engineered T cells comprises about 1 x 10 ⁸ And about 1X 10 ¹¹ Engineered T cells between individuals. In an embodiment, the second expanded population of engineered T cells comprises 1 x 10 ⁵ And 1X 10 ⁹ Engineered T cells between individuals. In an embodiment, the second expanded population of engineered T cells comprises at least 1 x 10 ⁸ Engineering T cells. In an embodiment, the second expanded population of engineered T cells comprises at least 1 x 10 ⁹ Engineering T cells. In an embodiment, the second expanded population of engineered T cells comprises about 1 x 10 ⁸ And about 1X 10 ¹¹ Engineered T cells between individuals.

In embodiments, the T cells are autologous to the subject. In embodiments, an additional population of engineered T cells is administered to the patient, and the T cells in the additional population of engineered T cells express a third exogenous TCR that binds to a third antigen expressed by the cancer and do not express endogenous TCR- β. In embodiments, an additional population of engineered T cells is administered to the patient, and the T cells in the additional population of engineered T cells express up to five exogenous TCRs that bind (one at a time) to the antigen expressed by the cancer and do not express endogenous TCR- β. In embodiments, an additional population of engineered T cells is administered to the patient, and the T cells in the additional population of engineered T cells express up to ten exogenous TCRs that bind (one at a time) to the antigen expressed by the cancer and do not express endogenous TCR- β. In embodiments, a further plurality of engineered T cells are administered to the patient, and the T cells in these further plurality of engineered T cells express a further exogenous TCR that binds (one at a time) to a further antigen expressed by the cancer and do not express endogenous TCR- β.

In another interrelated aspect, a method of treating cancer is provided. The method comprises the following steps: t cells, compositions or pharmaceutical compositions as described herein (including examples) are administered to a patient suffering from cancer. In some embodiments, the method further comprises: an anti-cancer therapy is administered to a subject. In embodiments, the anti-cancer therapy includes immunotherapy, chemotherapy, and/or radiation therapy.

In another interrelated aspect, a guide RNA is provided. The guide RNA is found by methods known to those skilled in the art such that it targets exon 1, exon 2 or exon 3 regions of the TCR constant alpha region locus (TRAC). In embodiments, the guide RNA targets an endogenous TCR-a locus. In embodiments, the guide RNA targets one of the following sequences from the TCR-a locus: TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21) or TRAC16 (SEQ ID NO: 22). In an embodiment, the guide RNA targeting the endogenous TCR-a locus comprises the nucleic acid sequence of table 10. In embodiments, the endogenous TCR-a locus is an endogenous TCR-a constant region.

In another interrelated aspect, a guide RNA is provided. The guide RNA was found by methods known to those skilled in the art such that it targets the exon 1 region of two TCR constant β region loci (TRBC). In embodiments, the guide RNA targets an endogenous TCR- β locus. In embodiments, the guide RNA targets one of the following sequences from the TCR- β locus: TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 46) or TRBC 48 (SEQ ID NO: 48). In an embodiment, the guide RNA targeting the endogenous TCR- β locus comprises a nucleic acid sequence in table 11.

In another interrelated aspect, a nucleic acid is provided. The nucleic acid includes a nucleic acid sequence comprising an exogenous TCR- β encoding sequence and an exogenous TCR-a encoding sequence, wherein the nucleic acid sequence further comprises a first self-cleaving peptide encoding sequence. In embodiments, the nucleic acid further comprises a first homology arm and a second homology arm. In embodiments, the nucleic acid further comprises a second self-cleaving peptide coding sequence. In an embodiment, the nucleic acids comprise, in order from 5 'to 3': (i) a first homology arm; (ii) a first self-cleaving viral peptide coding sequence; (iii) an exogenous TCR- β encoding sequence; (iv) a second self-cleaving viral peptide coding sequence; (v) an exogenous TCR-a coding sequence; and (vi) a second homology arm. In embodiments, the nucleic acid further comprises an enzyme cleavage site. In embodiments, the enzyme cleavage site is located between the TCR- β encoding sequence and the second self-cleaving viral peptide encoding sequence. In an embodiment, the enzyme cleavage site is a furin cleavage site. In embodiments, the nucleic acid further comprises a GSG amino acid sequence. In embodiments, the GSG precedes one or both of the first self-cleaving viral peptide coding sequence and the second self-cleaving viral peptide coding sequence.

In embodiments, the nucleic acid further comprises a polyadenylation (polyA) sequence. In embodiments, the polyadenylation sequence may be positioned 3' immediately adjacent to the TCR coding sequence. In embodiments, the polyadenylation sequence may be positioned 5' immediately adjacent to the second homology arm. In an embodiment, the polyadenylation sequence is bovine growth hormone polyadenylation sequence (bgh-polyA).

In embodiments, the first homology arm is homologous to an endogenous TCR-a locus in a human T cell. In embodiments, the second homology arm is homologous to an endogenous TCR-a locus in a human T cell. In embodiments, the endogenous TCR-alpha locus is a TCR-alpha constant region. In embodiments, the first self-cleaving viral peptide is T2A, P2A, E a or F2A. In embodiments, the second self-cleaving viral peptide is T2A, P2A, E a or F2A. In embodiments, the first self-cleaving viral peptide and the second self-cleaving viral peptide are different. In embodiments, the first self-cleaving peptide coding sequence is 5' of the exogenous TCR- β coding sequence. In embodiments, the second self-cleaving peptide coding sequence is 5' of the exogenous TCR-alpha coding sequence. In embodiments, the second self-cleaving peptide coding sequence is 5' of the exogenous TCR-alpha coding sequence. In embodiments, the nucleic acid is a plasmid, a nanoplasmid, or a micro-loop.

In another interrelated aspect, a kit for producing engineered T cells is provided. The kit includes a TCR-a targeting guide RNA as described herein (including the examples). In an embodiment, the kit further comprises a TCR- β targeting guide RNA as described herein (including the embodiment). In embodiments, the kit further comprises a gene editing reagent or a nucleic acid sequence encoding a gene editing reagent. In an embodiment, the gene editing agent is a CRISPR system. In an embodiment, the kit further comprises donor DNA. In embodiments, the donor DNA comprises a nucleic acid sequence encoding a polypeptide comprising exogenous TCR- β and exogenous TCR- α. In embodiments, the donor DNA comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR- β and an exogenous TCR- α (VJ) domain. In embodiments, the exogenous TCR- β and the heterologous TCR- α form a TCR capable of binding to an antigen. In an embodiment, the TCR binds to an antigen presented on a class I major histocompatibility complex (mhc I) molecule. In embodiments, the antigen is WT1, JAK2, NY-ESO1, PRAME, mutant KRAS, or an antigen from table 1 or table 2. In embodiments, the kit further comprises poly (glutamic acid) (PGA) or a variant thereof. In an embodiment, the kit further comprises a nucleic acid as described herein (including the embodiments).

Drawings

FIGS. 1A and 1B are bar graphs showing the knock-in efficiency of TRAC1-mNEon templates according to different electroporation conditions. FIG. 1A shows the frequency of knockin positive mNaeon+TCRab+ cells, and FIG. 1B shows the frequency of residual TCRab expressing cells. Cells were electroporated with TRAC1 RNP (RNP) alone, NTC (non-targeted guide control without TCR knockout) RNP+4μg TRAC 1-mNaeon (4 μg) or 6 μg/4 μg/2 μg/1 μg TRAC 1-mNaeon+TRAC1 RNP (6 μg, 4 μg, 2 μg and 1 μg, respectively). All cells were electroporated with EH100 pulse code in P2 buffer, except for the two bars of the rightmost panel, which represent the group of cells electroporated with EH115 pulse code in P3 buffer. Following electroporation, cells were incubated at 37℃or 32 ℃.

Fig. 2A and 2B are bar graphs showing recovery of total cd8+ cells (fig. 2A) and knock-in positive cd8+ T cells (fig. 2B) according to the amount of template used for electroporation. The control group was cells electroporated with RNP (RNP) alone or NTC+4. Mu.g template (NTC+4. Mu.g). Following electroporation, cells were incubated at 37℃or 32 ℃.

Fig. 3 is a bar graph showing cell viability following electroporation for the data of fig. 2A-2B.

FIG. 4 is a flow cytometry analysis showing the knock-in of TRAC1-mNEon templates. The top row shows analysis of cells electroporated with EH100 pulse code in P2 buffer, and the bottom row shows analysis of cells electroporated with EH115 pulse code in P3 buffer. The green (top) box in each figure shows the detection of TRAC 1-mNaeon knockin cells, and the red (bottom) box shows the detection of residual TCRab expressing cells. T cells from three different donors were evaluated.

FIG. 5 is a flow cytometry plot showing the knockin of TRAC1-mNEon templates. The top and bottom rows are dot plots representing groups of cells electroporated with 6 μg or 4 μg template, respectively. The green (top) box in each figure shows the detection of TRAC 1-mNaeon knockin cells, and the red (bottom) box shows the detection of residual TCRab expressing cells. T cells from three different donors were evaluated.

FIG. 6 is a flow cytometry analysis showing the knock-in of TRAC1-mNEon templates. The top and bottom rows are dot plots representing cells electroporated with 2 μg or 1 μg template, respectively. The green (top) box in each figure shows the detection of TRAC 1-mNaeon knockin cells, and the red (bottom) box shows the detection of residual TCRab expressing cells. T cells from three different donors were evaluated.

Fig. 7 is a flow cytometry analysis demonstrating non-significant off-target integration. The top and bottom rows show dot plots representing groups of cells electroporated with RNP alone or NTC RNP+4 μg TRAC 1-mNaeon, respectively. The green (top) box in each figure shows the detection of TRAC 1-mNaeon knockin cells, and the red (bottom) box shows the detection of TCRab expressing cells. T cells from three different donors were evaluated.

FIGS. 8A and 8B show the knockin of TRAC 1-mNaeon (FIG. 8A) and Rab11A-YFP (FIG. 8B) templates in T cells. The left panel shows a flow cytometry analysis to detect knock-in positive cells, and the right panel is a graph showing the percentage of knockins as a function of the amount of template used for electroporation. T cells from three different donors were evaluated.

Fig. 8C is a flow cytometry graph demonstrating non-significant off-target integration with NTC RNP. Various donor templates and Cas9 enzymes were tested. T cells from three different donors were evaluated.

FIG. 8D is a flow cytometry analysis of T cells electroporated with TRAC1 RNP+4 μg TRAC1-mNEon template (right) and NTC RNP+4 μg TRAC1-mNEon template (left).

Fig. 9A and 9B are bar graphs showing knock-in efficiency according to the amount of TRAC locus for knockout and template DNA for electroporation. FIG. 9A shows the frequency of knockin positive mNaeon+TCRab+ cells generated with TRAC 1-mNaeon template, and FIG. 9B shows the frequency of knockin positive mNaeon+TCRab+ cells generated with TRAC 3-mNaeon template.

Fig. 10A to 10C are bar graphs showing TCRab knockout efficiency. The graph shows the frequency of TCR+Neon-cells after electroporation with varying amounts of TRAC 1-mNaeon template (FIG. 10A), TRAC 3-mNaeon template (FIG. 10B) or no template (FIG. 10C).

Fig. 11A to 11D are bar charts showing recovery of knock-in positive mneon+tcrab+t cells according to the amount of template used. FIGS. 11A and 11B show recovery of mNaeon+TCRab+ cells from T cells electroporated with different amounts of TRAC 1-mNaeon and TRAC 3-mNaeon templates, respectively. FIGS. 11C and 11D show the recovery of total CD8+ cells from cells electroporated with different amounts of TRAC 1-mNaeon and TRAC 3-mNaeon templates, respectively.

FIGS. 12A and 12B are bar graphs showing cell viability (percent viable cells) after electroporation with different amounts of TRAC 1-mNaeon (FIG. 14A) or TRAC 3-mNaeon (FIG. 14B) templates. Cell viability was assessed by the absence of 7AAD stain.

FIG. 13 is a flow cytometry analysis showing the knockin of TRAC 1-mNaeon (top row) and TRAC 3-mNaeon (bottom row) templates. 6 μg of template was used for electroporation. The green (top) box shows the detection of knocked-in cells, and the red (bottom) box shows the detection of residual TCRab expressing cells.

FIG. 14 is a flow cytometry plot showing the knockin of TRAC 1-mNaeon (top row) and TRAC 3-mNaeon (bottom row) templates. 4 μg per template was used for electroporation. The green (top) box shows the detection of knocked-in cells, and the red (bottom) box shows the detection of residual TCRab expressing cells.

FIG. 15 is a flow cytometry plot showing knockin of TRAC 1-mNaeon (top row) and TRAC 3-mNaeon (bottom row) templates; 2 μg per template was used for electroporation. The green (top) box shows the detection of knocked-in cells, and the red (bottom) box shows the detection of residual TCRab expressing cells.

FIG. 16 is a flow cytometry plot showing knockin of TRAC 1-mNaeon (top row) and TRAC 3-mNaeon (bottom row) templates; 1 μg per template was used for electroporation. The green (top) box shows the detection of knocked-in cells, and the red (bottom) box shows the detection of residual TCRab expressing cells.

FIG. 17 is a graph showing the knockin (top) of TRAC 3-mNaeon template in T cells and cell viability (bottom) after electroporation, depending on the amount of RNP used for electroporation. T cells were electroporated with varying amounts of TRAC3 RNP and fixed amounts of TRAC3-mNeon template. The cell group electroporated without template RNP (NT RNP) served as negative control.

FIG. 18 is a flow cytometry plot for samples from a first donor showing the knockin of TRAC 3-mNaeon template in T cells according to TRAC 3-mNaeon template and the amount of TRAC3 RNP used for electroporation.

FIG. 19 is a flow cytometry plot for samples from a second donor showing the knockin of TRAC 3-mNaeon template in T cells according to TRAC 3-mNaeon template and the amount of TRAC3 RNP used for electroporation.

FIG. 20 is a flow cytometry plot for samples from a third donor showing the knockin of TRAC 3-mNaeon template in T cells according to TRAC 3-mNaeon template and the amount of TRAC3 RNP used for electroporation.

Fig. 21 shows flow cytometry histograms demonstrating TCRab knockouts using different amounts of TRAC3 RNP (KO RNP).

FIG. 22 is an agarose gel image showing forward (pFW) and reverse (pRV) ssDNA products generated from a chain enzyme reaction with a dsDNA template. The chain enzyme reaction was completed with 20. Mu.g of double-stranded TRAC 3-mNaeon and TRAC3-NY-ESO 1. The leftmost lane shows molecular weight markers. The ssDNA yields from the reactions are shown on the right.

FIGS. 23A-23C show knock-in efficiency and cell viability following electroporation with ss or ds TRAC 3-mNaeon templates. FIG. 23A is a flow cytometry plot showing TRAC 3-mNaeon knockin in T cells. Cells were electroporated with TRAC3 NRP or control NT RNP and various amounts of TRAC3-mNEON template. dsDNA, forward ssDNA strand (ssDNA FW) or reverse ssDNA strand (ssDNA RV) templates are used. Fig. 23B is a graph showing cell viability, and fig. 23C is a graph showing the percent knockin under indicated conditions.

FIGS. 24A and 24B show the knock-in efficiency according to different TRANSACTTM titration and media conditions for T cell activation. The bar graph shows the frequency of knock-in positive T cells on day 4 (fig. 24A) or day 5 (fig. 24B) after electroporation with the TRAC3-mNeon template. The media and cytokine conditions tested were RPMI and X-VIVO (XV) media (2/7/15) with IL-2 (10 ng/mL), IL-7 (25 ng/mL) and IL-15 (50 ng/mL); or IL-7 (25 ng/mL) and IL-15 (50 ng/mL) (7/15).

FIGS. 25A and 25B are diagrams showing recovery and expansion of knock-in positive mNaeon+TCRab+ cells after electroporation with TRAC 3-mNaeon. Figure 25A shows recovery of cd8+ cells after electroporation of activated cells titrated with different TRANSACTTM. The media and cytokine conditions tested are as indicated for figure 24. FIG. 25B shows the expansion of mNaon+TCRab+ cells from day 4 to day 5 after electroporation with groups of cells activated in different media and TRANSACTTM titration conditions. TA 20, TA 50, TA 100 and TA 200 represent 1:20, 1:50, 1:100 and 1:200TRANSACTTM titrations, respectively. RPMI and XV represent RPMI medium and X-VIVO medium, respectively.

FIGS. 26A and 26B are diagrams showing recovery and expansion of CD8+ cells after electroporation with TRAC 3-mNEon. Figure 26A shows recovery of cd8+ cells from titration of activated cells with different TRANSACTTM. The media and cytokine conditions tested are as indicated for figure 24. Fig. 26B shows expansion of cd8+ cells from day 4 to day 5 after electroporation of activated cell groups with different media and TRANSACTTM titration conditions. TA 20, TA 50, TA 100 and TA 200 represent 1:20, 1:50, 1:100 and 1:200TRANSACTTM titrations, respectively. Cells were expanded in RPMI medium or X-VIVOTM (XV) medium.

FIGS. 27A and 27B are bar graphs showing the knock-in efficiency of TRAC3-mNEon for electroporation performed 48 hours or 72 hours post activation using different T cell activation conditions. FIGS. 27A and 27B show the percent of knock-in positive mNaeon+TCRab+ cells on day 4 or 5, respectively, after electroporation. The media and cytokine conditions tested are as indicated for figure 24.

Fig. 28 shows a diagram showing the following: expansion of knock-in positive (mNaon+TCRab+) CD8+ T cells after 72 hours of electroporation after activation (left), and comparison of cell expansion after 48 hours of activation to 72 hours of electroporation (right). Different media conditions and TRANSACTTM titration were tested for T cell activation. 48h and 72h represent data points at 48 hours and 72 hours, respectively, after activation; TA 20, TA 50, TA 100 and TA 200 represent 1:20, 1:50, 1:100 and 1:200TRANSACTTM titrations, respectively; RPMI and XV represent RPMI medium and X-VIVO medium, respectively.

FIGS. 29A and 29B are flow cytometry plots showing detection of mNaeon+TCRab+T cells activated in RPMI medium prior to electroporation. Fig. 29A shows an analysis of cells activated with 1:50transacttm and 1:200transacttm, and fig. 29B shows an analysis of cells activated by titration with 1:20 and 1:100 transacttm.

FIGS. 30A and 30B are flow cytometry plots showing detection of mNaeon+TCRab+T cells activated in X-VIVOTM medium prior to electroporation. Fig. 30A shows an analysis of cells activated with 1:50transacttm and 1:200transacttm, and fig. 30B shows an analysis of cells activated by titration with 1:20 and 1:100 transacttm.

FIGS. 31A and 31B are flow cytometry plots showing detection of knock-in positive mNaeon+TCRab+ cells activated in RPMI or X-VIVOTM medium prior to electroporation. Fig. 31A shows an analysis of cells activated with 1:50transacttm, and fig. 31B shows an analysis of cells activated by titration with 1:100 transacttm.

FIG. 32 is an agarose gel image showing forward (pFW) and reverse (pRV) ssDNA products generated by a chain enzyme reaction with a double stranded TRAC 3-mNaeon template. The leftmost lane of the agarose gel is the molecular weight marker. ssDNA yields from the reactions are shown on the right.

FIGS. 33A and 33B are flow cytometry analyses showing the knock-in efficiency of dsDNA, forward single stranded DNA (ssDNA FW) and reverse ssDNA (ssDNA RV) TRAC-mNaeon template DNA by detecting mNaeon+TCRab+T cells. Fig. 33A shows an analysis of cells from donor 1, and fig. 33B shows an analysis of cells from donor 2.

Fig. 34A and 34B are graphs showing the frequency (fig. 34A) and percentage of living cells (fig. 34B) of the mneon+tcrab+t cells of the TRAC-mNeon template according to different amounts of double-stranded DNA (ds), forward ssDNA (ssDNA-FW) or reverse ssDNA (ssDNA-RV) for electroporation.

Fig. 35A and 35B are diagrams showing TCRab knockdown according to interference of a template with knockdown efficiency. Fig. 35A shows TCRab expression percentages and fig. 35B shows TCRab knockout percentages. ds denotes dsDNA, ssFW denotes ssDNA forward strand, and ssRV denotes ssDNA reverse strand.

FIGS. 36A-36C show TRAC-mNaeon knock-in efficiencies according to various knock-in sites and different amounts of TRACsg RNP included in electroporation. FIG. 36A is a schematic representation of TRAC loci with various sgRNA sites. Fig. 36B is a flow cytometry analysis showing the knock-in efficiency using the TRACsg1, TRACsg3, TRACsg4, and TRACsg5 knock-in sites, and fig. 36C shows the knock-in efficiency using the TRACsg7, TRACsg12, and TRACsg15 knock-in sites.

FIG. 37 is a graph showing mNan knock-in efficiency by detecting mNan+TCR+ cells (top) and cell viability (bottom) after electroporation using different amounts of each of the templates indicated.

FIGS. 38A to 38C show the effect of media and supplements on mNaeon+ knock-in T cells after activation and electroporation. Fig. 38A is a bar graph showing cell recovery on day 5 after initial T cell activation, and fig. 38B is a bar graph showing cell recovery on day 7 after initial T cell activation. Fig. 38C is a graph showing T cell expansion between day 5 and day 7 for knock-in + cells. Cells were cultured and activated in RPMI, X-Vivo (XV), supplemented X-Vivo (sXV or Sup. XV), with IL-2+IL-7+IL-15 (2/7/15) or IL-7+IL-15 (7/15) added. TA 30, TA 100 and TA 200 represent TRANSACTTM titrations of 1:30, 1:100 and 1:200, respectively.

FIGS. 39A and 39B show the effect of medium and supplements on mNaeon knock-in after T cell activation and electroporation. Fig. 39A is a bar graph showing the frequency of knockin positive mneon+tcrab+ cells at day 5 after initial T cell activation, and fig. 39B is a bar graph showing the frequency of knockin positive mneon+tcrab+ cells at day 7 after initial T cell activation. Cells were cultured and activated under conditions as indicated for fig. 38.

FIGS. 40A and 40B show the effect of activation medium and supplements on recovery of CD8+ T cells after electroporation. Fig. 40A is a bar graph showing cell recovery on day 5 after initial T cell activation, and fig. 40B is a bar graph showing cell recovery on day 7 after initial T cell activation. Cells were cultured and activated under conditions as indicated for fig. 38.

Fig. 41 is a graph showing total cd8+ cell expansion between day 5 and day 7 after T cell activation. Cells were cultured and activated under the conditions as indicated for figure 40.

Fig. 42 is a bar graph showing the fold expansion of total cd8+ T cells (black bars) and knock-in positive neon+tcrab+ cells (red bars) from the time of electroporation to the fifth day after electroporation. Cells were cultured and activated under the conditions as indicated for figure 40.

FIG. 43 shows a flow cytometry plot demonstrating the effect of TRANSACTTM titration in RPMI medium on mNaeon knock-in efficiency. The medium includes IL-2, IL-7 and IL-15 cytokines.

FIG. 44 shows a flow cytometry plot demonstrating the effect of TRANSACTTM titration in X-VIVOTM medium on mNaeon knock-in efficiency of representative TRAC locus donor DNA. The medium includes IL-2, IL-7 and IL-15 cytokines.

FIG. 45 shows a flow cytometry plot demonstrating the effect of TRANSACTTM titration on mNaeon knock-in efficiency in X-VIVOTM medium. The medium includes IL-7 and IL-15 cytokines.

FIG. 46 shows a flow cytometry plot demonstrating the effect of addition of TRANSACTTM to mNaeon knock-in efficiency in supplemented X-VIVOTM medium. The medium includes IL-2, IL-7 and IL-15 cytokines.

FIG. 47 shows a flow cytometry plot demonstrating the effect of TRANSACTTM titration on mNaeon knock-in efficiency in supplemented X-VIVOTM medium. The medium includes IL-7 and IL-15 cytokines.

FIGS. 48A and 48B are flow cytometry plots demonstrating the effect of IL-2 on the frequency of knockin efficiency in T cells (FIG. 48A) and knockin positive mNaon+TCR+ cells using RAB11A-GFP (left) or TRAC3 (right) templates (FIG. 48B).

Fig. 49A and 49B show the effect of resting cells on knock-in efficiency after electroporation. FIG. 49A shows flow cytometry analysis three days after electroporation of cells electroporated using a mNaeon-TRAC 3 template; FIG. 49B shows flow cytometry analysis three days after electroporation of cells electroporated with Rab11A-YFP template. Following electroporation, the cells were subjected (from left to right) to the following conditions: add 100. Mu.L buffer and transfer immediately; add 20. Mu.L buffer and incubate at 37℃for 15 min; 100. Mu.L of buffer was added and incubated at 37℃for 15 min; incubation was carried out for 5 minutes, then 20. Mu.L of medium was added, followed by incubation at 37℃for 15 minutes; or 5 minutes of incubation, then 100 μl of medium was added, followed by 15 minutes of incubation at 37deg.C.

FIG. 50 is a graph showing the frequency of knock-in positive mNaeon+TCR+ cells under each of the conditions tested in FIGS. 49A and 49B.

FIGS. 51A and 51C show the efficiency of knock-in different amounts depending on the unmodified dsDNA template or the 5' -phosphate modified dsDNA PCR template used for electroporation. FIG. 51A is a flow cytometry plot showing detection of knockdown positive mNaeon+TCRab+ cells, FIG. 51B is a plot showing the frequency of mNaeon+TCRab+ cells, and FIG. 51C is a plot showing cell viability by analysis of PI negative cells after electroporation with dsDNA templates with (w/5 ' -P) or without (w/o 5' -P) 5' -phosphate.

FIGS. 52A and 52C show the knock-in efficiency of electroporation using different amounts of unmodified dsDNA template or 5' -phosphorothioate modified dsDNA template. FIG. 52A is a flow cytometry analysis showing detection of knockdown positive mNaeon+TCRab+ cells, FIG. 52B is a graph showing the frequency of mNaeon+TCRab+ cells, and FIG. 52C is a graph showing cell viability by analysis of PI negative cells after electroporation with dsDNA templates with (w/5 ' -Pthio) or without (w/o 5' -Pthio) 5' -phosphorothioate.

Fig. 53A-53C illustrate the efficiency of typing using a different amount of unmodified reverse strand (RV) ssDNA template or 5' -phosphorothioate modified reverse strand (RV) ssDNA template compared to the unmodified dsDNA template used for electroporation. FIG. 53A is a flow cytometry analysis showing detection of knockdown positive mNaeon+TCRab+ cells, FIG. 53B is a graph showing the frequency of mNaeon+TCRab+ cells, and FIG. 53C is a graph showing cell viability by analysis of PI-negative cells after electroporation with ssDNA templates with (w/5 ' -Pthio) or without (w/o 5' -Pthio) 5' -phosphorothioate.

FIG. 54 is a graph comparing the frequency of knockin positive mNEon+TCRab+ cells (top) and living cells (bottom) using different amounts of dsDNA template for electroporation, 5 '-phosphate modified dsDNA template (w/5' -P), 5 '-phosphorothioate modified dsDNA template (with 5' -Pthio), ssDNA template or 5 '-phosphorothioate modified ssDNA template (with 5' -Pthio).

Fig. 55 is a flow cytometry analysis showing the effect of electroporation time points on template knock-in efficiency. Electroporation was performed 60 (top row), 48 (middle row) or 36 (bottom row) hours after activation.

FIG. 56 is a flow cytometry analysis showing the effect of PGA on knockin efficiency by detecting knockin positive mNaeon+TCRab+ cells. Cells were electroporated with template DNA and 60pmol RNP (1:1 sgRNA: cas 9) in the presence (bottom row) or absence (top row) of PGA.

Fig. 57 is a flow cytometry analysis showing the effect of PGA on knockin efficiency by detecting knockin positive mneon+tcrab+ cells. Cells were electroporated with template DNA and 80pmol RNP at 3:1sgRNA:Cas9 in the presence (bottom row) or absence (top row) of PGA.

Fig. 58A and 58B are bar graphs showing cell viability of electroporated cd8+ T cells with or without PGA. Cell viability of samples electroporated with and without template DNA (control) and with or without PGA was assessed 3 days (fig. 58A) and 5 days (fig. 58B) after electroporation (on days 5 and 7 of cell culture). The amount of RNP (in pmol) used for electroporation is indicated on the x-axis.

FIGS. 59A and 59B are bar graphs showing the frequency of mNaeon+ cells of electroporated T cells in the presence or absence of PGA. The frequency of knockin positive cells was assessed 5 days (FIG. 59A) and 7 days (FIG. 59B) after electroporation. Cells were electroporated with or without template DNA and with or without PGA. The amount of RNP (in pmol) used for electroporation is indicated on the x-axis.

FIGS. 60A and 60B are bar graphs showing recovery of mNaeon+ cells of electroporated T cells in the presence or absence of PGA. Cell recovery was assessed 5 days (FIG. 60A) and 7 days (FIG. 60B) after electroporation. Cells were electroporated with or without template DNA and with or without PGA. The amount of RNP (in pmol) used for electroporation is indicated on the x-axis.

Fig. 61A and 61B are bar charts showing the effect of electroporation time on the efficiency of mNeon knock-in. Cells were electroporated 36, 48, or 60 hours after T cell activation, with cells that did not receive template during electroporation serving as controls. The mNaeon+ cell frequency was assessed 3 days after electroporation (FIG. 61A) and recovered (FIG. 61B).

Fig. 62A and 62B are bar graphs showing the effect of electroporation time on the efficiency of mn eon knockin to the TRAC locus. The mNaeon+ cell frequency was assessed 5 days after electroporation (FIG. 62A) and recovered (FIG. 62B). Cells were electroporated 36, 48 or 60 hours after T cell activation with tranact, with cells that did not receive template during electroporation serving as controls.

Fig. 63A to 63D show the effect of the reagent addition sequence on the knock-in efficiency. FIG. 63A is a flow cytometry analysis (bottom) of cells electroporated with different addition sequences of template, RNP and PGA to T cells, as indicated and numbered 1 to 6 (top). Knock-in efficiency was assessed by detection of mNaeon+TCRab+ cells. Fig. 63B is a graph showing the frequency of mneon+tcr+ cells, fig. 63C is a graph showing the frequency of TCR-knockout cells, and fig. 63D is a graph showing the frequency of living cells of a cell sample electroporated in a different order of addition (as indicated on the x-axis of the graph).

FIGS. 64A-64D show the efficiency of typing using a linear PCR product dsDNA template compared to a plasmid template used for electroporation. FIG. 64A shows a flow cytometry plot of mNan+TCRab+ cells detected from cells electroporated with different amounts of the PCR product dsDNA or pUC57 plasmid. FIG. 64B is a graph showing the frequency of mNaon+TCR+ cells, FIG. 64C is a graph showing the frequency of living cells, and FIG. 64D is a graph showing the frequency of TCR knockout cells of cell samples electroporated with different amounts of PCR product or pUC57 plasmid template (as indicated on the x-axis of the graph).

Fig. 65A to 65C show the effect of cell density and type of donor template for electroporation on knock-in efficiency. With 1. Mu.g or 2. Mu.g of the linear PCR product dsDNA (L) or plasmid (P) and 5X 10 ⁶ (5e6)、2x 10 ⁶ (2e6) Or 1x 10 ⁶ (1e6) Individual cells were electroporated T cells. Fig. 65A is a graph showing the frequency of the 5 th day of mneon+tcrab+ cells after activation, fig. 65B is a graph showing the frequency of the 7 th day of mneon+tcrab+ cells after activation, and fig. 65C is a graph showing the expansion of the 5 th day to the 7 th day of mneon+tcrab+ cells.

FIGS. 66A and 66B show mNaon+TCRab+ cell recovery according to electroporation with different amounts and types of donor templates at different cell densities. With 1. Mu.g or 2. Mu.g of the linear PCR product dsDNA (L) or plasmid (P) and 5X 10 ⁶ (5e6)、2x 10 ⁶ (2e6) Or 1x 10 ⁶ (1e6) Individual cells were electroporated T cells. Fig. 66A is a graph showing recovery of the mneon+tcrab+ cells on day 7 after activation, and fig. 66B is a graph showing expansion of the mneon+tcrab+ cells from day 5 to day 7. The 5e6 cell density condition was excluded to better visualize the expansion at lower cell densities.

FIG. 67 is a flow cytometry analysis showing detection of knock-in positive mNaeon+TCRab+T cells from cells electroporated with a linear dsDNA template. Electroporation was performed using different amounts of template and cell density.

FIG. 68 is a flow cytometry analysis of knock-in positive mNaon+TCRab+T cells from cells electroporated with a linear dsDNA template (top row) compared to cells electroporated with a plasmid template (bottom row). Either 1 μg or 2 μg template was used.

FIG. 69 is a flow cytometry spot diagram showing detection of total CD8+ cells, CD45RA+ cells, and CD45RO+ cells on day 0 of T cell activation for three donors.

FIG. 70 is a diagram showing T _SCM T cell differentiation schematic for the least differentiated cells (top), and table showing various cytokine combinations that may be advantageous for generating T cell subsets (bottom).

FIGS. 71A-71C show the efficiency of knockin using different TRAC loci and linear dsDNA or plasmid DNA as templates. Fig. 71A is a graph showing the frequency of knock-in positive mneon+tcrab+ cells on day 4 compared to day 7 after T cell activation, fig. 71B shows recovery of mneon+tcrab+ cells on days 4 and 7 after activation, and fig. 71C shows fold expansion of mneon+tcrab+ cells. Electroporation was performed using TRAC3, TRAC4, TRAC5, TRAC7, TRAC12 and TRAC15 templates in either the linear dsDNA (L) form or the plasmid DNA (P) form.

FIG. 72 is a flow cytometry analysis showing detection of knock-in positive mNaeon+TCRab+T cells electroporated with various templates. Cells were electroporated with RNP-only control (up, three panels on the left), TRAC3 linear dsDNA template (up, three panels on the right), TRAC3 plasmid (down, three panels on the left) or TRAC4 plasmid (down, three panels on the right).

FIG. 73 is a flow cytometry analysis showing detection of knock-in positive mNaeon+TCRab+T cells electroporated with various templates. Cells were electroporated using TRAC5 plasmid (up, three panels on the left), TRAC7 plasmid (up, three panels on the right), TRAC12 plasmid (down, three panels on the left) or TRAC15 plasmid (down, three panels on the right).

FIG. 74 is a flow cytometry analysis of total CD8+ (left column), CD45RA+ (middle column), and CD45RO+ (right column) T cells from donor samples used in the experiments shown in FIGS. 72 and 73.

FIG. 75 is a flow cytometry plot showing knock-in efficiency of NY-ESO1 TCR with gp100 TCR, MART 3TCR, WT1C TCR or MAGEAB3TCR, as indicated. Plasmid templates encoding NY-ESO1 TCRs were co-electroporated with templates encoding other TCRs. Flow cytometry data were collected 7 days after T cell activation. Cells were stained with Immulex peptide-MHC dextramers WB3247-APC (NY-ESO 1 TCR) and WB3469-PE (WT 1 TCR), WB2162-PE (MART TCR), WB3415_PE (MAGEA 3 TCR) or WB2156_PE (gp 100 TCR).

Fig. 76A to 76C are graphs showing the effects of linearity and plasmid template, the amount of template used, and cell density on knock-in efficiency. FIG. 76A is a graph showing frequency, and FIG. 76B is a graph showing recovery of knock-in positive mNaeon+TCRab+ cells on days 5 and 7 after activation. Fig. 76C is a graph showing recovery of cd8+ T cells on days 5 and 7 after activation. As indicated on the x-axis of the graph, 200 ten thousand (2M), 500 ten thousand (5M), or 1000 ten thousand (10M) cells are used; and electroporation was performed on each group of cells using 1. Mu.g or 2. Mu.g of linear dsDNA PCR product (L) or plasmid (P) template.

FIG. 77 is a flow cytometry analysis showing detection of knock-in positive mNaeon+TCRab+T cells. Electroporation was performed using 200 ten thousand (2M, top row) or 500 ten thousand (5M, bottom row) cells with 1 μg of linear dsDNA or 2 μg of plasmid DNA template.

FIG. 78 is a flow cytometry plot of detection of knock-in positive mNaeon+TCRab+T cells. Tens of millions of cells were electroporated using either 1 μg of linear dsDNA TRAC3-mNeon (top row) or 2 μg of TRAC3-mNeon plasmid (bottom row) template.

Fig. 79A to 79D are bar graphs showing the frequency of knock-in positive mneon+tcrab+ cells on day 5 (fig. 79A) and day 7 (fig. 79C) after activation and TCR-expressing mNeon-tcrab+ cells remaining on day 5 (fig. 79B) and day 7 (fig. 79D) after activation. All cell groups were electroporated in P3 buffer and tested for various pulse codes as indicated on the x-axis of the graph.

Fig. 80A to 80D are bar graphs showing recovery of knock-in positive mneon+tcrab+ cells at day 5 (fig. 80A) and day 7 (fig. 80C) after activation, and total cd8+ cells at day 5 (fig. 80B) and day 7 (fig. 80D) after activation. All cell groups were electroporated in P3 buffer and tested for various pulse codes as indicated on the x-axis of the graph.

Fig. 81A and 81B are flow cytometry analyses showing detection of knock-in positive mneon+tcrab+t cells on day 7 after activation using samples from donor 1. Fig. 81A shows data for cells electroporated using a pulse program, top row from left to right as follows: no program, DS150, DS120, EH100; and the bottom row is CA137, CM138, CM137 and CM150 from left to right. Fig. 81B shows data for cells electroporated using a pulse program, top row from left to right as follows: EO100, EN138, EN150, EN113; and the bottom row is DN100, DS138, DS137, DS130 from left to right.

Fig. 82A and 82B are flow cytometry analyses showing detection of knock-in positive mneon+tcrab+t cells on day 7 after activation using samples from donor 2. Fig. 82A shows data for cells electroporated using a pulse program, top row from left to right as follows: no program, DS150, DS120, EH100; and the bottom row is CA137, CM138, CM137 and CM150 from left to right. Fig. 82B shows data for cells electroporated using a pulse program, top row from left to right as follows: EO100, EN138, EN150, EN113; and the bottom row is DN100, DS138, DS137, DS130 from left to right.

Fig. 83A and 83B are flow cytometry analyses showing detection of knock-in positive mneon+tcrab+t cells on day 7 after activation using samples from donor 3. Fig. 83A shows data for cells electroporated using a pulse program, top row from left to right as follows: no program, DS150, DS120, EH100; and the bottom row is CA137, CM138, CM137 and CM150 from left to right. Fig. 83B shows data for cells electroporated using a pulse program, top row from left to right as follows: EO100, EN138, EN150, EN113; and the bottom row is DN100, DS138, DS137, DS130 from left to right.

Fig. 84 shows flow cytometry spot analysis of donor samples dissected for the experiments shown in fig. 81-83. Cells were analyzed on day 0 of activation. As indicated, a graph showing total cd8+ cells, cd45ra+ cells, cd45ro+ cells and CD45RA-CD45RO- (RA-RO-) cells is shown.

FIGS. 85A-85D are graphs showing the knock-in efficiency of different amounts of the linear dsDNA TRAC templates mNaon-TRACsg 3 (FIG. 85A), mNaon-TRACsg 4 (FIG. 85B), mNaon-TRACsg 5 (FIG. 85C) and mNaon-TRACsg 12 (FIG. 85D) in T cells. The rightmost column is a dot plot of electroporated cells with TRBC21 RNP added.

FIGS. 86A and 86B are diagrams showing knock-in of positive mNaeon+TCR+ cells. FIG. 86A is a graph showing the frequency of mNaeON+TCRab+ cells electroporated using varying amounts of mNaeON-TRAC 3, mNaeON-TRAC 4, mNaeON-TRAC 5 and mNaeON-TRAC 12 templates. FIG. 86B is a graph showing the frequency of mNaeON+TCRab+ cells using 1 μg mNaeON-TRAC 3, mNaeON-TRAC 4, mNaeON-TRAC 5 or mNaeON-TRAC 12 template for electroporation and with or without TRBC21 RNP.

Fig. 87 is a flow cytometry analysis showing detection of knock-in positive mneon+tcrab+t cells for a group of cells electroporated with either a CI unit electroporator (top row) or an ACE electroporator (bottom row). Cells were analyzed 5 days after activation and tested for various pulse codes.

FIG. 88 flow cytometry analysis of knock-in positive mNaeon+TCRab+T cells of cell groups electroporated with various pulse codes. Cells were analyzed 12 days after activation.

Fig. 89A and 89B are flow cytometry analyses showing the effect of different concentrations of PGA on knock-in efficiency. FIG. 89A is a flow cytometry plot of cells electroporated in the presence of 0, 12.5, or 254 μg PGA. Cells electroporated with NT (non-targeted guide control, i.e., TCR-free knockout) RNP were control groups. FIG. 89B is a flow cytometry plot of cells electroporated in the presence of 50, 100, 200, or 400 μg PGA.

Fig. 90A and 90B are graphs showing the frequency of knock-in positive mneon+tcrab+ cells (fig. 90A) and recovery of mneon+ cells (fig. 90B) according to the concentration of PGA used during electroporation.

Fig. 91 shows the effect of PGA concentration and PGA variants on knock-in efficiency. A graph showing the frequency of knockin positive mneon+tcrab+ cells (top) and recovery of mneon+ cells (bottom) is shown. PGA concentrations and variants are indicated on the x-axis.

FIGS. 92A to 92D are bar graphs showing the effect of electroporation pulse code on TRAC 3-mNaeon knock-in efficiency in T cells. The frequency of knock-in positive mNaeb + TCRab + cells on day 5 post-activation (FIG. 92A) and day 7 post-activation (FIG. 92C) and the frequency of TCRab + mNaeb-cells on day 5 post-activation (FIG. 92B) and day 7 post-activation (FIG. 92D) are shown.

FIGS. 93A and 93B are flow cytometry plots showing the effect of electroporation pulse code on TRAC 3-mNaeon knock-in efficiency in T cells. Fig. 93A shows flow cytometry analysis for cells electroporated with EH115, EN138, and EN158 pulse codes. Fig. 93B shows flow cytometry analysis for cells electroporated with EW113, EH111, and EO100 pulse codes. Percentages of mNaon+TCRab+ cells are indicated as the upper right hand corner of each plot.

FIGS. 94A and 94B are flow cytometry plots showing the effect of electroporation pulse code on TRAC 3-mNaeon knock-in efficiency in T cells. Fig. 94A shows flow cytometry analysis for cells electroporated with EO115, EO128 and EO151 pulse codes. FIG. 94B shows flow cytometry analysis for cells electroporated with DS130, DS137 and DS138 pulse codes. Percentages of mNaon+TCRab+ cells are indicated as the upper right hand corner of each plot.

Fig. 95A-95D are bar graphs showing the knock-in efficiency of TRAC3-mNeon in T cells according to the code of pulses without electroporation (as indicated on the x-axis of the graph). The frequency of knocking-in positive mNaeon+TCRab+ cells on day 5 after activation (FIG. 95A) and on day 7 after activation (FIG. 95C) and recovery of mNaeon+TCRab+ cells on day 5 after activation (FIG. 95B) and on day 7 after activation (FIG. 95D) are shown.

FIG. 96 is a flow cytometry analysis showing the efficiency of knockin of TRAC3-NY-ESO-4TCR plasmid templates in T cells. Cells electroporated with RNP only (no template) were control (three leftmost columns). From left to right, flow cytometry analysis showed detection of NY-ESO-4TCR+ cells in total CD8+ T cells, TCRab+ cells in total CD8+ T cells, and NY-ESO-4TCR+ cells in CD8+ TCRab+ cells for every three rows of the plot.

Fig. 97A and 97B are flow cytometry analyses showing the effectiveness of various cell staining methods. FIG. 97A is a flow cytometry detection of NY-ESO-4TCRab+ cells using Dextramer (Dex) stain (left) or using Dextramer stain (right) in the presence of other antibodies. FIG. 97B is a flow cytometry detection of NY-ESO-4TCRab+ with GNE tetramers. Dot plots for cells electroporated with RNP control are shown in the left column.

FIG. 98 is a flow cytometry analysis showing the knock-in efficiency of various TCRs using TRAC3 plasmid templates in HLA-A0201 CD8+ T cells. TRAC3-NY-ESO-4, TRAC3-WTIC-13, TRAC3-MART2 and TRAC3-MART3 templates were used for electroporation.

FIG. 99 is a flow cytometry analysis showing the knock-in efficiency of TRAC 3-mNaeon plasmid templates using two different electroporators. In each dot plot, the upper box shows knock-in positive mNaeon+TCRab+ cells, and the lower box shows residual TCRab expressing cells.

Graphs 100A and 100B are graphs of frequency of knock-in positive mneon+tcrab+t cells (graph 100A) and number of mneon+t cells (graph 100B) on day 6 after comparative activation according to pulse codes for electroporation. The pulse codes used are as indicated on the x-axis, and the four pulse codes on the far right are combined codes. Square and circular data markers represent data for cells electroporated with two different electroporators.

FIGS. 101A and 101B are graphs of frequency of knock-in positive mNaeon+TCRab+T cells (FIG. 101A) and number of mNaeon+T cells (FIG. 101B) at day 8 after comparative activation according to pulse codes for electroporation. The pulse codes used are as indicated on the x-axis, and the four pulse codes on the far right are combined codes. Square and circular data markers represent data for cells electroporated with two different electroporators.

FIGS. 102A to 102C show the effect of TRBC RNP on the efficiency of knock-in of the NY-ESO1TCR into the TRAC locus using the TRAC3-1G4 (NY-ESO 1) plasmid. FIG. 102A is a flow cytometry analysis using knock-in positive pMHC (NY-ESO 1) +TCRab+ cells of various RNPs as indicated above each dot plot. Fig. 102B is a graph showing the frequency of tcrab+pmhc+ cells, and fig. 102C is a graph showing the frequency of total pmhc+ cells. The RNP used for electroporation is indicated in the following figure.

FIGS. 103A and 103B show the effect of TRBC-different TBBC RNPs on the efficiency of NY-ESO1TCR knock-in of the combination of TRAC locus and TRAC3 RNP when using the TRAC3-1G4 (NY-ESO 1) plasmid. FIG. 103A is a flow cytometry analysis showing detection of biscationic pMHC (NY-ESO 1) +cells using various RNPs as indicated above each dot plot. Fig. 103B is a graph showing the frequency of biscationic pmhc+ cells. The RNP used for electroporation is indicated in the following figure.

FIGS. 104A and 104B show the effect of different homology arm lengths on the knock-in efficiency of TRACsg 3-mNaeon, TRACsg 4-mNaeon, TRACsg 5-mNaeon and TRACsg 12-mNaeon templates in T cells. Flow cytometry analysis showing knock-in efficiency as a function of left/right arms 300/300, 400/400 and 500/500 (fig. 104A) and longest/longest (different per template), longest/300 and 300/longest (fig. 104B) homology arm length (HA length) is shown.

FIGS. 105A to 105H are graphs showing the effect of different homology arm lengths on the knock-in efficiency of TRACsg 3-mNaeon, TRACsg 4-mNaeon, TRACsg 5-mNaeon and TRACsg 12-mNaeon templates in T cells. The frequency of knock-in positive mNaeon+TCRab+ cells electroporated using TRAC 3-mNaeon (FIG. 105A), TRAC 4-mNaeon (FIG. 105C), TRAC 5-mNaeon (FIG. 105E) or TRAC 12-mNaeon (FIG. 105G) templates is shown. The number of mNaeon+ cells electroporated using TRAC 3-mNaeon (FIG. 105B) and TRAC 4-mNaeon (FIG. 105D), TRAC 5-mNaeon (FIG. 105F) or TRAC 12-mNaeon (FIG. 105H) templates is shown. The different Homology Arm (HA) lengths of the left/right arms are indicated on the x-axis.

FIG. 106 is a graph showing T2 cytotoxicity of WT1 and gp100B TCR knock-in cells. T2 cells were electroporated with different concentrations of target peptide.

Fig. 107A to 107D are diagrams showing the overall effect of TRBC22 RNP titration. FIG. 107A shows the percentage of Neon positive TRAC+/-TRBC samples on day 8 after electroporation under the indicated conditions. Fig. 107B shows the percentage of Neon positive samples at day 8 after electroporation under the indicated conditions. FIG. 107C shows the percentage of Neon positive TRAC3/TRBC22 samples on day 7 after electroporation under the indicated conditions. FIG. 107D shows the percentage of Neon positive TRAC3/TRBC22 samples on day 7 after electroporation under the indicated conditions. The dashed box in fig. 107D shows a second electroporation with TRBC22 RNP after 24 hours, comparing EW113 and EO 100. 24w and 48w indicated whether cells were cultured in 24-well or 48-well plates after the first electroporation (2 x 10 ⁶ Individual cells).

Figures 108A and 108B are graphs of combined data from all experiments showing that the use of TRBC22 RNP appears to have minimal impact on Neon knock-in. Fig. 108A shows the percentage of Neon positive samples. Fig. 108B shows the number of Neon positive samples.

FIGS. 109A-109C are graphs showing the results of successive electroporation groups performed on TRAC3 RNP plus donor template and TRBC22 RNP after 24 hours. Fig. 109A shows the percentage (or frequency) of Neon positive samples. Figure 109B shows the number of Neon positive samples. Fig. 109C shows the frequency or percentage of tcrab+neon negative samples.

Fig. 110A to 110D are diagrams showing test results of the knock-in frequencies of various templates. Fig. 110A and 110B show the frequency of Neon positive samples from two separate experiments. Fig. 110C-110D show the combined data from two experiments, where fig. 110C shows the frequency of the Neon positive samples and fig. 110D shows the number of Neon positive samples.

FIG. 111 is day 7 flow cytometry data using TRAC3RNP alone (30 pmol or 60 pmol) using different donor templates, using plasmids or nanoplates.

FIG. 112 is day 7 flow cytometry data from titration experiments (as shown in FIG. 108) using 30pmol or 60pmol TRAC3RNP and 10pmol, 20pmol or 30pmol TRB22 RNP.

FIG. 113 is flow cytometry data for day 7 post electroporation using 30pmol TRAC3RNP and 30pmol TRBC22 RNP with electroporation code indicated.

FIGS. 114A and 114B are graphs showing data of pUC57 and nanoplasmids and micro-circular DNA at day 6 after electroporation. Fig. 114A shows the percentage (upper panel) and absolute number (lower panel) of the mneon+tcr+ samples. FIG. 114B shows the amount of DNA used in the experiment.

FIGS. 115A and 115B are graphs showing data of pUC57 and nanoplasmids and micro-circular DNA at day 8 after electroporation. Fig. 115A shows the percentage (upper panel) and absolute number (lower panel) of the mneon+tcr+ samples. FIG. 115B shows the amount of DNA used in the experiment.

FIGS. 116A and 116B are graphs showing the effect of equivalent molecules of each template on pUC57, a nanoplasmon or a microring on cell viability. Fig. 116A shows the percent cell viability at day 6 after electroporation, while fig. 116B shows the percent cell viability at day 8 after electroporation.

FIG. 117 is a graph of the variation in the number of knocked-in cells over time from day 6 to day 8 after electroporation based on the data from FIGS. 116A and 116B.

Fig. 118A and 118B are graphs summarizing cytokine reading monitoring. FIG. 118A shows the concentration of TNF- α, and FIG. 118B shows the concentration of IL-6 after electroporation with pUC57, a nanoplasmon or a microcircle.

FIG. 119 shows IFN-gamma (upper panel), IL-5 (middle panel) and IL-13 (lower panel) concentrations after electroporation with pUC57, a nanoplasmon or a microcirculatory.

FIGS. 120A and 120B are graphs showing data of pUC57 and nanoplasmms and micro-circular DNA on day 6. Figure 120A shows the percentage (upper panel) and absolute number (lower panel) of mneon+tcr+ samples on day 6. FIG. 120B shows the amount of DNA used in this experiment.

FIGS. 121A to 121B are graphs showing data of pUC57 and nanoplasmids and micro-circular DNA on day 8. Figure 121A shows the percentage (upper panel) and absolute number (lower panel) of the mneon+tcr+ samples on day 8. Fig. 121B shows the amount of DNA used in this experiment.

FIG. 122 is flow cytometry data from day 8 sample plots after electroporation of T cells electroporated with pUC57, nanoplasmms, microrings and PCR samples.

Fig. 123 is a pair of graphs showing the effect on cell viability following electroporation with equivalent molecules of the indicated templates, with data on day 6 following electroporation shown in the upper graph and data on day 8 shown in the lower graph.

Fig. 124 is a graph showing the change in the number of knocked-in cells over time from day 6 to day 8 based on the data in fig. 123.

Fig. 125 is a diagram showing that addition of TRBC RNP does not reduce the knock-in efficiency. The left plot shows the percentage of Neon-positive samples, while the right plot shows the number of Neon-positive samples after electroporation with template on plasmid or nanoplasmid.

Fig. 126A to 126B are diagrams showing that the knock-in efficiency increases with an increase in the amount of nanoplastlets. Fig. 126A shows the knock-in efficiency on day 5 (left panel) and on day 7 (right panel) after electroporation. Fig. 126B shows the change in number of knocked-in cells for day 5 and day 7 after electroporation.

FIG. 127 is a graph of day 7 electroporation flow cytometry data for TRBC19 or TRBC22 plasmid titration (10 pmol or 30 pmol) with 60pmol TRAC3.

FIG. 128 is a graph of day 7 flow cytometry data after electroporation for 30pmol TRAC3 alone (top row) or with TRBC22 (30 pmol, middle row) or TRBC19 (30 pmol, bottom row) on a plasmid (left 3 columns) or nanoplasmid (right 3 columns).

FIG. 129 is a graph of day 7 flow cytometry data after electroporation for 2 μg, 4 μg, 6 μg, or 8 μg GenScript plasmids.

Figure 130 is a graph of flow cytometry data at day 7 post electroporation for nanoplasmon titration.

Fig. 131A and 131B are graphs showing the results of Neon knockins at day 7 after electroporation using templates on plasmids or nanoplates, with or without PGA, and with different template addition sequences. Fig. 131A shows the percentage of Neon-positive cells, while fig. 131B shows the number of Neon-positive cells.

FIGS. 132A through 132C are graphs showing that codon optimization can significantly reduce the effects of TCR knock-in efficiency and template addition order on knock-in efficiency. Figure 132A shows the percentage of WT1+ (in total cd8 +) samples at day 7 post electroporation. Fig. 132B shows the percentage of cd3+ samples at day 7 post electroporation. Figure 132C shows the percentage of WT1+ (in total cd3+) samples at day 7 post electroporation.

Fig. 133 is a graph showing WT1 tcr+ cell numbers at day 7 post electroporation based on the data in fig. 132A-132C. The drawing conditions are as follows in sequence from left to right: first add template, last add template, and no PGA.

FIG. 134 is a graph of flow cytometry data knocked in by Neon template on day 7 after electroporation with or without PGA and with different template addition sequences.

FIG. 135 is a graph of flow cytometry data from day 7, WT 1-5213, after electroporation with or without PGA and with different template addition sequences.

FIG. 136 is a graph of flow cytometry data for WT 1-Ref at day 7 post-electroporation.

FIG. 137 is a graph of flow cytometry data for WT1 64_9 at day 7 post-electroporation.

Figure 138 is a cartoon representation of target cytotoxicity (upper panel) and graph of% poisoning against neo-TCR and TCR-free relative to control (lower panel).

FIG. 139 is a cartoon representation of the insertion of a DNA template described herein into a TCR-alpha locus. The DNA template (top) includes (from 5 'to 3'): left (5 ') homology arms, NEO-TCR (first self-cleaving peptide (e.g., T2A), TCR-. Beta.V segment, TCR-. Beta.D segment, TCR-. Beta.J segment, TCR-. Beta.constant region, second self-cleaving peptide (e.g., P2A), TCR-. Alpha.V segment, TCR-. Alpha.J segment, and a portion of TCR-. Alpha.constant region), and right (3') homology arms. A nuclease or gene editor (e.g., CRISPR/Cas 9) cleaves the endogenous TCR-a constant region in (a) T cell and promotes homologous recombination of the DNA template with the endogenous TCR-a locus within the constant region. The resulting edited TCR-a locus (bottom) includes the NEO-TCR in frame with a portion of the endogenous TCR-a constant region.

Fig. 140A to 140I. Plasmid-based donor templates enable efficient non-viral gene editing of the TRAC locus in primary T cells. (FIGS. 140A-140C) titration of Linear dsDNA donor templates (FIG. 140A) A plot of Linear dsDNA knock-in construct TRAC-mNG. (fig. 140B) depicts bar graphs (n=4) of three days post electroporation with 1, 2, 4, 6 or 8 μg of linear dsDNA donor template along with Cas9-RNP targeting the TRAC locus, knock-in efficiency, cell viability, total cell recovery and edit cell recovery (mNG positive cells). Circles represent individual donors; bars represent median values with ranges. (FIG. 140C) is a representative contour plot showing the frequency of CD8T cells expressing mNG. (fig. 140D to 140F): titration of pUC57 plasmid donor template; (fig. 140D): pUC57 knockin construct TRAC-mNG. (fig. 140E): a bar graph (n=4) showing frequency, cell viability, total cell recovery and edit cell recovery (mNG positive cells) of CD8T cells expressing mNG three days after electroporation with 1, 2, 4, 6 or 8 μg pUC57 plasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with ranges. (fig. 140F): a representative contour plot of the frequency of CD8T cells expressing mNG is shown. (fig. 140G to 140I): titration of the nanoplasmid donor template; (fig. 140G); graph of nanoplasmon knock-in construct TRAC-mNG. (fig. 140H): a bar graph (n=4) showing the frequency, total cell recovery and edit cell recovery (mNG positive cells) of CD8T cells expressing mNG three days after electroporation with 1, 2, 4, 6 or 8 μg nanoplasmon donor together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with ranges. (fig. 140I): a representative contour plot of the frequency of CD8T cells expressing mNG is shown.

Fig. 141A to 141F. CD4 and CD 8T cells optimized CRISPR/Cas9 mediated gene knock-in with plasmid-based donor DNA. (fig. 141A, fig. 141B): homology arm optimization for plasmid-based donor templates; (fig. 141A): a representative contour plot showing the frequency of CD 8T cells expressing mNG; (fig. 141B): bar graphs (n=2) of knock-in efficiency, cell viability, total cell recovery and edit cell recovery (mNG positive cells) three days after electroporation with pUC57 plasmid or nanoplasmon donor templates with homology arms length between 100bp and 2000bp (equimolar to the amount of 4 μg 2000bp construct) together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with ranges. (fig. 141C): frequency, cell viability, total cell recovery and edit cell recovery (mNG positive cells) of CD 8T cells expressing mNG three days after electroporation with the nanoplasmon donor template along with Cas9-RNP targeting the TRAC locus (24, 36, 48 or 72 hours of culture) (n=4). Circles represent individual donors; bars represent median values with ranges. (fig. 141D): nuclear transfection pulse code optimization in CD 8T cells electroporated with a nanoplasmid donor template and Cas9-RNP targeting the TRAC locus. The graph shows the cell frequency and edited cell recovery (mNG positive cells) of mNG expressed three days after electroporation. Each circle represents a different pulse code. Data represent three independent CD 8T cell donors. (fig. 141E, fig. 141F); gene editing targeting the TRAC locus in CD 4T cells; (fig. 141E): a representative contour plot showing the frequency of CD 4T cells expressing mNG; (fig. 141F): the bar graph (n=3) of five days post electroporation knock-in efficiency, cell viability, total cell recovery and edit cell recovery (mNG positive cells) of CD 4T cells with TRAC-mNG nanoplasmid donor template together with Cas9-RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with ranges.

Fig. 142A to 142J. Non-viral T cell receptor editing using plasmid DNA donors; (fig. 142A): map of TCR α and β loci. V gene (purple), D gene (red) and J gene (blue) and constant region (green) segments. The sgTRAC and sgTRBC targeting sites are indicated. (fig. 142B): graph of nanoplasmon knock-in constructs TRAC-1G4TCR, TRAC-TCR6-2 and TRAC-CD19 CAR. (fig. 142C, fig. 142E, fig. 142G): representative contour plots (left) and bar plots (right) (n=3) showing frequencies of CD 8T cells expressing NY-ESO 1-specific 1G4TCR (fig. 142C), CMV-specific pp65 6-2TCR (fig. 142E), CD19-CAR (fig. 142G) five days after electroporation with Cas9-RNP targeting the TRAC locus using a nanoplasmid donor template. Circles represent individual donors, and bars represent median values with ranges. (fig. 142D, fig. 142F, fig. 142H): bar graphs (n=3) showing cell viability, total cell recovery and edit cell recovery five days after electroporation with Cas9-RNP targeting the TRAC locus using nanoplasmid donor templates encoding NY-ESO 1-specific 1g4TCR (fig. 142D), CMV-specific pp65 6-2TCR (fig. 142F), CD19-CAR (fig. 142H). Circles represent individual donors; bars represent median values with ranges. (fig. 142I): lactate levels in culture supernatants analyzed by luminescence using the Lactate-Glo assay were measured one, three, five and seven days after transfection of CD 8T cells (as compared to non-transfected control T cells (no RNP)) with sgTRAC/sgTRBC Cas9-RNP (RNP only) or sgTRAC/sgTRBC Cas9-RNP and a nanoplasmid donor template targeting the TRAC locus (rnp+nanoplasmid). (fig. 142J): number of cells recovered from culture broth seven days after transfection of CD 8T cells with sgTRAC/sgTRBC Cas9-RNP (RNP only) or sgTRAC/sgTRBC Cas9-RNP and a nanoplasmon donor template targeting the TRAC locus (rnp+nanoplastomer) compared to non-transfected control T cells (RNP free).

Fig. 143A to 143I: TCR-engineered T cells recognize and poison target cells expressing an antigen. Representative histograms (fig. 143A) and bar graphs (fig. 143B) of CD137 frequency of IG4 TCR knockin cd8+ T cells activated with indicated doses of NY-ESO157-165 peptide (n=4) are shown. Circles represent individual donors, and bars represent median values with ranges. Representative histograms (fig. 143C) and bar charts (fig. 143D) of CD137 frequency of pp65 TCR knockin cd8+ T cells activated with indicated doses of pp65495-503 peptide (n=3) are shown. Circles represent individual donors and bars represent rangesMedian value. (fig. 143E): representative histograms showing the frequency of CFSE positive target cells and CFSE negative reference cells in co-cultures with IG4 TCR knockin cd8+ T cells in the absence or presence of cognate peptide. (fig. 143F): a graph showing calculated specific cleavage in the absence of peptide or in the presence of 0.1 μm NY-ESO157-165 peptide. Circles represent individual donors, and bars represent median values with ranges (n=4). The experiment was performed twice. (fig. 143G): showing TCR knockdown or 1g4 TCR knockin CD8 from three donors co-cultured with NY-ESO-1 antigen expressing a-375 cells ⁺ Bar graph of IFN- β, TNF- α and granzyme B (GzmB) produced by T cells. Circles represent technique replicates, and bars represent median values with range (n=3). The experiment was performed twice. (fig. 143H): representative images of A-375 cells expressing the NY-ESO-1 antigen and labeled with cytoplasmic dye and co-cultured with TCR knockout cells (left panel) or 1G4 TCR knockout cells (right panel) at 2 and 18 hours post-incubation in the presence of Caspase 3/7-green apoptotic agents. (fig. 143I): target cytotoxicity over time as measured by Cas3/7 positive object counts in co-cultures of a-375 cells expressing NY-ESO-1 antigen and labeled with cytoplasmic dye and co-cultured with TCR knockout cells (open circles) or 1g4 TCR knockout cells (filled circles). Mean ± standard deviation of six technical replicates. The experiment was performed twice. In RM one-way ANOVA corrected with Geisser-Greenhouse (fig. 143B-143D), paired t-test (fig. 143F), one-way ANOVA (G) or Tukey multiple comparison test, 2-way ANOVA (fig. 143I), p<0.05、**p<0.01、***p<0.001、****p<0.0001。

Fig. 144A to 144M. Generation of gene expression reports. (fig. 144A): graph of nanoplasmon knock-in construct RAB 11A-YFP. (fig. 144B): histograms of YFP expression frequency (left) and cell viability (right) ten days after electroporation of CD 8T cells transfected with RAB11A-YFP nanoplasmms with or without RAB11A targeting Cas9-RNP were superimposed. (fig. 144C): bar graphs showing YFP expression frequency (left) and cell viability (right) ten days after electroporation for CD 8T cells transfected with RAB11A-YFP nanoplasmms with or without RAB11A targeting Cas9-RNP (n=3). Circles represent individual donors, and bars represent median values with ranges. (fig. 144D): diagram of nanoplasmon knock-in construct AAVS 1-mNG. (fig. 144E): histograms of mNG expression ten days after electroporation of CD 8T cells transfected with AAVS1-mNG nanoplasmms of AAVS1 with or without Cas9-RNP targeting were superimposed. (fig. 144F): bar graph of mNG expression frequency (left) and cell viability (right) ten days after electroporation for CD 8T cells transfected with AAVS1-mNG nanoplasmms with or without AAVS1 targeting Cas9-RNP (n=4). Circles represent individual donors, and bars represent median values with ranges. (fig. 144G): graph of nanoplasmon knock-in construct CD 4-mNG. (fig. 144H): representative contour plots showing the frequencies of the nanoplasmon donor templates and CD4 and CD 8T cells expressing mNG ten days after Cas9/RNP electroporation targeting the CD4 locus. (fig. 144I): the frequency (top) and cell viability (bottom) bar graphs of CD4 and CD 8T cells expressing mNG ten days after Cas9/RNP electroporation targeting the CD4 locus are shown for the nanoplasmon donor template (n=4 for CD 4T cells; n=3 for CD 8T cells). Circles represent individual donors, and bars represent median values with ranges. (fig. 144J): histograms for CD4 expression in CD 4T cells transfected with CD4-mNG nanoplasmids and non-targeted control Cas9-RNP (sgNTC) or Cas9-RNP targeting the CD4 locus (sgCD 4) are superimposed. (fig. 144K): the graph of nanoplasmid knock-in constructs TNFRSF9-mNG and RAB11A-YFP (left) and a representative contour plot showing the frequency of CD 8T cells that express CD137 and mNG after electroporation with nanoplasmid mNG reporter constructs targeting the TNFRSF9 locus or constitutive YFP expression constructs targeting the RAB11A locus along with the respective Cas9-RNP without reactivation or 6h after reactivation with Transact (right). (fig. 144L): a bar graph showing the frequency of YFP (left) and mNG (right) expressing CD 8T cells over time after electroporation with the TNFRSF9 locus targeted nanoplasm mNG reporter construct or RAB11A locus targeted constitutive YFP expression construct together with the respective Cas9-RNP and reactivation with Transact at time 0h (n=4). Circles represent individual donors, and bars represent median values with ranges. (fig. 144M): bar graphs showing median fluorescence intensity (gmi) of CD137 expression in CD 8T cells over time after electroporation with the TNFRSF9 locus (right) -targeting nanoplasma mNG reporter construct or RAB11A locus (left) -targeting constitutive YFP expression construct together with the respective Cas9-RNP and reactivation with Transact at time 0h (n=4). Circles represent individual donors, and bars represent median values with ranges.

Fig. 145A to 145K. Multiple gene knockins in human T cells. (fig. 145A to 145C): a diagram of the nanoplasmon knock-in construct is provided at the top. Representative contours (left) and bar graphs (right) showing frequency of CD 8T cells expressing mNG (fig. 145A) ten days after electroporation with a nanoplasmid TRAC-mNG donor template and Cas9/RNP targeting the TRAC locus (n=3), mCherry (fig. 145B) ten days after electroporation with a nanoplasmid TRAC-mCherry donor template and Cas9/RNP targeting the TRAC locus (n=3), or mNG or mCherry (fig. 145C) (n=3) ten days after electroporation with two nanoplasmid donor templates (TRAC-mNG and TRAC-mCherry) and Cas9/RNP targeting the TRAC locus. The graph on the right (FIG. 145C) shows the proportion of transgenic expression cells expressing mNG (bottom right of the histogram), mCherry (top left), or both (top right). In the bar graph, circles represent individual donors, and bars represent median values with ranges. (fig. 145D): graph of nanoplasmids RAB11A-YFP and TRAC-mCherry used in the double targeting study. (fig. 145E): representative contour plots showing the frequency of CD 8T cells expressing YFP (bottom right), mCherry (top left), or both (top right) ten days after electroporation with nanoplasmon donors RAB11A-YFP and TRAC-mCherry and Cas9/RNP targeting RAB11A and TRAC loci. (fig. 145F): bar graphs (n=4) showing the efficiency of knockin (left), cell viability (middle) and total recovery (right) of CD 8T cells after electroporation with nanoplasmon donors RAB11A-YFP and TRAC-mCherry and Cas9/RNP targeting RAB11A and TRAC loci. Circles represent individual donors, and bars represent median values with ranges. (fig. 145G): proportion of transgenic expression cells co-transfected with nanoplasmon donors RAB11A-YFP and TRAC-mCherry and Cas9/RNP targeting RAB11A and TRAC loci expressing YFP, mCherry or both (n=4). Circles represent individual donors, and bars represent median values with ranges. (fig. 145H): graph of nanoplasmids AAVS1-mNG and TRAC-mCherry used in the double targeting study. (fig. 145I): representative contour plots showing the frequency of CD 8T cells expressing mNG, mCherry, or both ten days after electroporation with nanoplasmon donors AAVS1-mNG and TRAC-mCherry and Cas9/RNP targeting AAVS1 and TRAC loci. (fig. 145J): bar graphs (n=4) showing the efficiency of knockin (left), cell viability (middle) and total cell recovery (right) of CD 8T cells ten days after electroporation with nanoplasmon donors AAVS1-mNG and TRAC-mCherry and Cas9 targeting AAVS1 and TRAC loci. Circles represent individual donors, and bars represent median values with ranges. (fig. 145K): proportion of transgenic expression cells co-transfected with nanoplasmon donors AAVS1-mNG and TRAC-mCherry and Cas9/RNP targeting AAVS1 and TRAC loci expressing mNG, mCherry or both (n=4). Circles represent individual donors, and bars represent median values with ranges.

Fig. 146A to 146G: non-viral CRISPR gene editing with large payloads. (fig. 146A): diagram of nanoplasmon knock-in constructs TRAC_NotchICD_mNG, TRAC_NotchICD_1G4, and TRAC_THEMIS_1G4. (fig. 146B, 146D, 146F): representative contour plots of frequencies of CD8T cells expressing mNG (fig. 146B) or 1G4 TCR (fig. 146D, 146F) five days after electroporating a notchicd_ mNG (fig. 146B), notchicd_1g4 (fig. 146D), or themis_1g4 (fig. 146F) nanoplasmid donor template with Cas9/RNP targeting the TRAC locus; (fig. 146C, 146E, 146G): bar graphs showing frequency and cell viability of CD8T cells expressing mNG (fig. 146C) or 1G4 TCR (fig. 146E, fig. 146G) five days after electroporating with Cas9/RNP targeting the TRAC locus for the notchicd_ mNG (fig. 146C), notchicd_1g4 (fig. 146E) or themis_1g4 (fig. 146G) nanoplasmid donor templates (fig. 146G). Circles represent individual donors, and bars represent median values with ranges.

Fig. 147A to 147H: non-viral gene editing in primary T cells was optimized using plasmid-based donor templates. (fig. 147A to 147F); titration of linear dsDNA and nanoplasmid donor templates in RPMI/FBS medium. (fig. 147A): a plot of linear dsDNA knock-in construct TRAC-mNG. (fig. 147B): bar graphs (n=4) depicting knockin efficiency, cell viability, total cell recovery and edit cell recovery (mNG positive cells) of CD8T cells cultured in RPMI/10% FBS three days after electroporation with 1, 2, 4 μg of linear dsDNA donor template along with Cas9/RNP targeting the TRAC locus. (fig. 147C): representative contour plots showing frequency (left), viability (left two), total cell recovery (right two), and edit cell recovery (right) of CD8T cells expressing mNG. Circles represent individual donors; bars represent median values with ranges. (fig. 147D): graph of nanoplasmon knock-in construct TRAC-mNG. (fig. 147E): bar graphs (n=4) depicting knock-in efficiency, cell viability, total cell recovery and edit cell recovery (mNG positive cells) of CD8T cells cultured in RPMI/10% FBS three days after electroporation with 1, 2, 4 μg nano donor template together with Cas9/RNP targeting the TRAC locus. Circles represent individual donors; bars represent median values with ranges. (fig. 147F): representative contour plots showing frequency (left), viability (left two), total cell recovery (right two), and edit cell recovery (right) of CD8T cells expressing mNG. (fig. 147G): bar graphs depicting knock-in efficiency (left), cell viability (left two), total cell recovery (right two) and edit cell recovery (right) after electroporation with 2 μg of linear dsDNA or nano donor template along with Cas9/RNP targeting the TRAC locus. poly-L-glutamic acid (PGA) with or without (n=3). Circles represent individual donors; bars represent median values with ranges. (fig. 147H): bar graphs (n=3) depicting three days post electroporation with 2 μg of linear dsDNA or nanoplasmon donor templates with (ttsc) or without (w/o ttsc) truncated Cas9 target sequence together with Cas9-RNP targeting the TRAC locus (left), cell viability (left two), total cell recovery (right two) and edit cell recovery (right). Circles represent individual donors; bars represent median values with ranges.

Fig. 148A to 148H. Cytokine production and stress response induced in T cells following exposure to dsDNA donor templates. (fig. 148A): IFN- α as measured by SIMOA and IFN- γ, TNF- α and IL-2 as measured by Luminex from CD 8T cells compared to untransfected control T cells (RNP-free) at 18h after transfection with Cas9/RNP targeting the TRAC locus alone or together with a nanoplasmon donor template. Circles represent individual donors; bars represent median values with ranges. (n-3) (fig. 148B): GSEA enrichment analysis results from CD 8T cells after transfection with the TRAC-targeted Cas9/RNP and nanoplasmon donor templates compared to Cas9/RNP alone. The gene sets of interferon-gamma response, interferon-alpha response, tnfalpha signaling and inflammatory response are significantly enriched. (fig. 148C): GSEA enrichment analysis results of CD 8T cells after transfection with the TRAC-targeted Cas9/RNP and linear dsDNA donor templates compared to Cas9/RNP alone. The gene sets of interferon-gamma response, interferon-alpha response, tnfalpha signaling and inflammatory response are significantly enriched. (fig. 148B, fig. 148C): the y-axis represents enrichment fraction and genes represented in the genome (vertical black line) are on the x-axis. The color bars at the bottom represent the extent of differentially expressed genes (red for up-regulation and blue for down-regulation). (fig. 148D): GSEA was upregulated for all 375 genes in the nanoplasmon Cas9-RNP and linear dsDNA/Cas9-RNP relative to Cas9-RNP only using GSEA MSigDB Hallmark 2020. (fig. 148E to 148H): these upregulated genes are shown to contribute predominantly to the interferon- α response (fig. 148E), tnfα response (fig. 148F), apoptosis (fig. 148G) or (fig. 148H) inflammatory response (both MSigDB Hallmark) relative to the nanoplasmids of Cas9-RNP alone/Cas 9-RNP and linear dsDNA/Cas9-RNP upregulated genes. Color-coding is performed by RNA-seq count data normalized with Variance Stabilizing Transformation (VST).

Fig. 149A to 149O. Non-viral T cell receptor editing using plasmid DNA donors in cd4+ and cd8+ T cells. (fig. 149A): TCR expression on the cell surface by flow cytometry of CD 8T cells 48h after transfection with Cas9-RNP targeting the TRAC (sgTRAC) or TRBC (sgTRBC) loci; (fig. 149B to 149G): TCR editing in CD 4T cells. Representative contours showing the frequency of CD 8T cells expressing NY-ESO1 specific 1g4 TCR (fig. 149B), CMV specific pp65 6-2TCR (fig. 149D), CD19-CAR (fig. 149F) five days after electroporation with Cas9-RNP targeting the TRAC locus using a nanoplasmon donor template. Bar graphs (n=4) showing five days post electroporation with Cas9-RNP targeting the TRAC locus using nanoplasmid donor templates encoding NY-ESO 1-specific 1G4 TCR (fig. 149C), CMV-specific pp65 6-2TCR (fig. 149E), CD19-CAR (fig. 149G). Circles represent individual donors; bars represent median values with ranges. (fig. 149H) depicts a graph of all possible translocation events between TRAC, TRBC1 and TRBC2 genomic loci; (fig. 149I): showing in useBar graph of frequency of individual translocation events between the TRAC, TRBC1 and TRBC2 genomic loci quantified by ddPCR in Cas9-RNP co-transfected CD 8T cells or non-transfected control T cells targeting TRAC and TRBC loci (n=4). Circles represent individual donors; bars represent median values with ranges. The experiment was performed twice. Showing pp65 at the indicated concentrations _495-503 Peptide stimulated pp65 TCR knock-in CD8 ⁺ Representative histograms (fig. 149J) and bar charts (fig. 149K) of CD137 frequencies of T cells. Circles represent individual donors, and bars represent median values with ranges (n=3). The experiment was performed twice. (fig. 149L): showing pp65 at the indicated concentrations _495-503 Peptide stimulated pp65 TCR knock-in CD8 ⁺ IFN- β (left) and TNF- α (right) production by T cells. Circles represent individual donors, and bars represent median values with ranges (n=3). The experiment was performed twice. (fig. 149M): shows that a CD8 knock-in with pp65 TCR in the absence or presence of homologous peptide ⁺ Representative histograms of the frequency of CFSE positive target cells and CFSE negative reference cells in co-cultures of T cells. (fig. 149N): shows the presence of no peptide or with 0.1. Mu.M pp65 _495-503 Graph of calculated specific cleavage in case of peptide. Circles represent individual donors, and bars represent median values with ranges (n=3). The experiment was performed twice. (fig. 149O): showing the knock-in of TCR6-2 (unrelated TCR) or CD19-CAR from two donors (D1, D2) into CD4 ⁺ Bar graph of IFN- β and TNF- α produced by T cells in co-culture with CD 19B expressing cells. Circles represent technical repetitions; bars represent median values with ranges (n=9). The experiment was performed twice. RM one-way ANOVA corrected with Geisser-Greenhouse (FIG. 149A, FIG. 149K), paired t-test (FIG. 149N), one-way ANOVA (FIG. 149O), p <0.05、**p<0.01、***p<0.001、****p<0.0001。

Fig. 150A to 150I. Kinetics of gene expression following transient transfection of dsDNA, plasmids and nanoplates. (fig. 150A): graph of nanoplasmon knock-in construct RAB 11A-YFP. (fig. 150B): representative histograms of frequencies of CD 8T cells expressing YFP three, five or seven days after electroporation with a promoter-containing nanoplasmid donor template with (paired red/right bars) or without (paired blue/left bars) Cas9/RNP targeting the RAB11A locus are shown. (fig. 150C): graph depicting YFP expression frequency, cell viability, total cell recovery and edit total cell recovery three, five or seven days after electroporation with a promoter-containing nanoplasmid donor template with (red/right bar) or without (blue/left bar) Cas9/RNP targeting the RAB11A locus (n=3); (fig. 150D): FIG. of the linear dsDNA knock-in construct RAB 11A-YFP. (fig. 150E): representative histograms showing the frequency of CD 8T cells expressing YFP three, five or seven days after electroporation with Cas9/RNP with a linear dsDNA donor template containing a promoter with (top) or without (bottom) targeting the RAB11A locus are shown. (fig. 150F): plots (n=3) depicting YFP expression frequency, cell management, total cell recovery and editing total cell recovery three, five or seven days after electroporation with a linear dsDNA donor template containing a promoter with (paired red/right bars) or without (paired blue/left bars) Cas9/RNP targeting the RAB11A locus. (plot 150G): pUC57 plasmid knock-in construct RAB 11A-YFP. (plot 150H): representative histograms showing the frequency of CD 8T cells expressing YFP three, five or seven days after electroporation with pUC57 plasmid donor templates containing promoters with (top) or without (bottom) Cas9/RNP targeting the RAB11A locus. (fig. 150I): a graph (n=3) depicting YFP expression frequency, cell viability, total cell recovery and editing cell recovery three, five or seven days after electroporation with pUC57 plasmid donor templates containing promoters with (paired red/right bars) or without (paired blue/left bars) Cas9/RNP targeting the RAB11A locus.

Fig. 151A to 151G. Multiple gene knockins in human T cells. (fig. 151A to 151C): a diagram of the pUC57 plasmid knock-in construct is provided at the top. Representative contour plots (left) and bar graphs (right) showing frequency of CD 8T cells expressing mNG (fig. 151A) ten days after electroporation with pUC57 plasmid TRAC-mNG donor template and Cas9/RNP targeting the TRAC locus (n=3), mCherry (fig. 151B) ten days after electroporation with pUC57 plasmid TRAC-mCherry donor template and Cas9/RNP targeting the TRAC locus (n=3), or mNG or mCherry (fig. 151C) ten days after electroporation with two pUC57 plasmid donor templates (TRAC-mNG and TRAC-mCherry) and Cas9/RNP targeting the TRAC locus (n=3). The right hand graph of (fig. 151C) shows the proportion of transgenic expression cells expressing mNG (green/bottom right histogram), mCherry (red/top left), or both (blue/top right). Circles represent individual donors, and bars represent median values with ranges. (fig. 151D): map of pUC57 plasmids RAB11A-YFP and TRAC-mCherry used in the double targeting study. (fig. 151E): representative contour plots showing the frequency of CD 8T cells expressing YFP, mCherry, or both ten days after electroporation with pUC57 donor RAB11A-YFP and TRAC-mCherry and Cas9/RNP targeting RAB11A and TRAC loci. (fig. 151F): bar graphs showing efficiency of knockin, cell viability and total cell recovery of CD 8T cells ten days after electroporation with pUC57 donor RAB11A-YFP and TRAC-mCherry and Cas9/RNP targeting RAB11A and TRAC loci (n=4). Circles represent individual donors, and bars represent median values with ranges. (fig. 151G): proportion of transgenic expression cells co-transfected with pUC57 donor templates RAB11A-YFP and TRAC-mCherry, cas9/RNP targeting RAB11A and TRAC loci expressing YFP, mCherry or both (n=4). Circles represent individual donors, and bars represent median values with ranges. The experiment was performed three times. In RM one-way ANOVA corrected with Geisser-Greenhouse, p <0.05 p <0.01.

Graph 152: two TCR knock-in constructs with and without polyadenylation (bGHpA) sequences. The TRACg3_Wt1C13_pUC57 construct contains a truncated TCR- α sequence consisting of the entire variable domain plus a small constant region preceding the right homology arm. Once copied to the TRAC3 cleavage site within the genome, the truncated TCR- α within the construct becomes intact by in-frame fusion with genomic DNA, which provides the remainder of the TCR- α constant chain sequence. The construct named schober_tracg3_wt1c13_puc57 differs from tracg3_wt1c13_puc57 in that it contains the full length TCR-a sequence, followed by a stop codon, and then a polyadenylation sequence from the bovine growth hormone gene (bGHpA) preceding the right homology arm. It does not require in-frame fusion with the TCR-alpha constant chain sequence remaining in the genome for TCR-alpha expression.

Fig. 153: flow cytometry scatter plots of donor primary cells electroporated with two TCR knock-in constructs RNP with and without polyadenylation (bGHpA) sequences (schober_tracg3_wt1c13_puc57 and tracg3_wt1c13_puc57).

Fig. 154: comparison of the bar graphs of donor primary cells electroporated with two TCR knock-in constructs RNP with and without polyadenylation (bGHpA) sequences (schobaer_tracg3_wt1c13_puc57 and tracg3_wt1c13_puc57) (% pmhc+ and tcr+pmhc+).

Fig. 155: schematic representation of knock-in at the TCRB1 and TCRB2 locus nanoplasmon constructs.

Fig. 156: flow cytometry scatter plots of two knock-in electroporated donor primary cells at TCRB1 and TCRB2 locus nanoplasmid constructs without TCR alpha sequences.

Fig. 157: bar graph comparison (% pmhc+ and tcr+pmhc+) of two knock-in electroporated donor primary cells at TCRB1 and TCRB2 locus nanoplasmid constructs without TCR alpha sequence.

Fig. 158: scatter plots of cell expansion rates at day 5 and day 7 for two knock-in electroporated donor primary cells at TCRB1 and TCRB2 locus nanoplasmid constructs without TCR alpha sequences.

Fig. 159: flow cytometry scatter plots for non-viral TCR knockins were performed on day 7 using Jurkat cells with or without IL-2 activation.

Fig. 160: flow cytometry scatter plots for non-viral TCR knockins were performed using primary cd4+ cells without IL-2 activation.

Fig. 161: schematic of the overview of the use of TCRA1-TCRA16 and TCRB1-TCRB26 guide RNA to knock-out TCR-alpha and TCR-beta loci, including flow cytometry plots of TCR-alpha or beta negative T cells%.

Fig. 162: schematic of the overview of the use of TCRA1-TCRA16 and TCRB1-TCRB26 guide RNA to knock-out TCR-alpha and TCR-beta loci, including flow cytometry plots of TCR-alpha or beta negative T cells%. Boxed sequences represent guide RNA sequences with knockout efficiency > 90%.

Fig. 163: schematic overview of self-cleaving peptide (SCP) constructs. The SCP upstream of the TCRb gene is T2A, and the second SCP sequence upstream of the TCRa gene is P2A. The two consecutive SCPs in this example are not 100% identical to prevent erroneous recombination (i.e., each of the upstream or downstream P2A coding sequences may recombine with each other, resulting in a non-functional recombination locus). By using different nucleic acid sequences to encode each P2A site, the only homologous regions of the TCR donor and acceptor sites are the 5 'and 3' arms. In this example, different upstream and downstream SCP sequences (T2A and P2A, respectively, and not limited to only T2A and P2A sequences) are used. Alternatively, the nucleic acid sequence encoding each of the two SCPs may be codon-dispersed, but encode the same SCP peptide sequence. Short neutral GSG sequences are optionally added upstream of each SCP sequence.

Detailed Description

After reading this specification, it will become apparent to one of ordinary skill in the art how to implement the disclosure in various alternative embodiments and alternative applications. However, not all of the various embodiments of the invention will be described herein. It should be understood that the embodiments presented herein are presented by way of example only, and not limitation. Thus, the detailed description of various alternative embodiments should not be construed to limit the scope or breadth of the present disclosure as described herein.

Before the present technology is disclosed and described, it is to be understood that the aspects described below are not limited to particular compositions, methods of making such compositions, or uses thereof, as such may, of course, vary. In addition, it is to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

The detailed description divided into sections and the disclosure found in any section may be combined with the content in other sections for the convenience of the reader only. For the convenience of the reader, headings or sub-headings may be used in this specification and are not intended to affect the scope of this disclosure.

I. Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings:

the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

"optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

The term "about" as used prior to a numerical designation (e.g., temperature, time, amount, concentration, and such other designations, including ranges) indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subranges or sub-values therebetween. Preferably, the term "about" means that the amount can vary by +/-10%.

"comprising" or "including" is intended to mean that the compositions and methods include the recited elements, but not exclude other elements. When used to define compositions and methods, "consisting essentially of" means that for the stated purpose, other elements of any significance to the combination are excluded. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. "consisting of … …" means the parts and essential method steps excluding trace elements of other components. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

As used herein, a "T cell" or "T lymphocyte" is a type of lymphocyte (a subtype of white blood cell) that plays a central role in cell-mediated immunity. By the presence of T cell receptors on the cell surface, they can be distinguished from other lymphocytes (such as B cells and natural killer cells). T cells include, for example, natural Killer T (NKT) cells, cytotoxic T Lymphocytes (CTLs), regulatory T (Treg) cells, and T helper cells. Different types of T cells can be distinguished by the use of T cell detection agents.

"memory T cells" are T cells that have previously encountered and responded to their cognate antigen during a previous infection, encounter with cancer, or previous vaccination. Upon encountering a cognate antigen for the second time, memory T cells can proliferate (divide) to produce a faster, stronger immune response than when the immune system first responds to the pathogen.

"regulatory T cells" or "suppressor T cells" are lymphocytes that regulate the immune system, maintain tolerance to autoantigens, and prevent autoimmune diseases.

"nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-stranded, double-stranded or multi-stranded form, or the complement thereof; or a nucleoside (e.g., deoxyribonucleotide or ribonucleotide). In embodiments, "nucleic acid" does not include nucleosides. The terms "polynucleotide", "oligonucleotide" and the like refer to a linear sequence of nucleotides in a general and customary sense. The term "nucleoside" refers in a usual and customary sense to a glycosylamine comprising a nucleobase and a five-carbon sugar (ribose or deoxyribose). Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. In a general and customary sense, the term "nucleotide" refers to a polynucleotide and a single unit of nucleic acid, i.e., a monomer. The nucleotide may be a ribonucleotide, a deoxyribonucleotide or a modified form thereof. Examples of nucleic acids contemplated herein include single-and double-stranded DNA, single-and double-stranded RNA, and hybrid molecules having a mixture of single-and double-stranded DNA and RNA. Examples of nucleic acids (e.g., the nucleic acids contemplated herein) include any type of RNA, e.g., mRNA, siRNA, miRNA and guide RNAs, as well as any type of DNA, genomic DNA, plasmid DNA, and microloop DNA, and any fragments thereof. In the context of nucleic acids, the term "duplex" refers to double strand in the usual and customary sense. The nucleic acid may be linear or branched. For example, the nucleic acid may be a linear chain of nucleotides, or the nucleic acid may be branched, e.g., such that the nucleic acid comprises one or more nucleotide arms or branches. Optionally, the branched nucleic acid is repeatedly branched to form a higher order structure (such as dendrite, etc.).

Nucleic acids, including, for example, nucleic acids having a phosphorothioate oligodeoxynucleotide backbone, may include one or more reactive moieties. As used herein, the term reactive moiety includes any group capable of reacting with another molecule, such as a nucleic acid or polypeptide, through covalent, non-covalent, or other interactions. For example, a nucleic acid may include an amino acid reactive moiety that reacts with an amino acid on a protein or polypeptide by covalent, non-covalent, or other interactions.

The term also encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, but are not limited to, phosphodiester derivatives including, for example, phosphoramidates, phosphorodiamidates, phosphorothioates (also known as phosphorothioates with double bond sulfur substituted phosphates of oxygen), phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylate esters, phosphonoacetic acid, phosphonoformic acid, methylphosphonates, boron phosphonate or O-methylphosphinamide linkages (see Eckstein, oligonucleotides and analogs: methods of use, oxford university press), and modifications to nucleotide bases such as 5-methylcytidine or pseudouridine, and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those having a positive backbone; nucleic acids that are non-ionic backbones, modified sugars, and non-ribose backbones (e.g., phosphorodiamidate morpholino oligonucleotides or Locked Nucleic Acids (LNAs) as known in the art), including those described in U.S. patent nos. 5,235,033 and 5,034,506 and ASC Symposium Series, chapter 6 and chapter 7 CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH of 580, sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acid. The ribose-phosphate backbone can be modified for a variety of reasons, for example, to increase the stability and half-life of such molecules in physiological environments or as probes on biochips. Mixtures of naturally occurring nucleic acids and analogs can be prepared; alternatively, mixtures of different nucleic acid analogs can be prepared, as well as mixtures of naturally occurring nucleic acids and analogs. In embodiments, the internucleotide linkages in the DNA are phosphodiester, phosphodiester derivatives, or a combination of both.

As used herein, "left homology arm" and "right homology arm" are used to define 5 'and 3' genomic DNA fragments, respectively, flanking a DNA sequence of interest (a gene, portion of a gene, or other DNA segment to be introduced into the genome). For example, gene targeting via homologous recombination can include transfecting a cell with a targeting vector that is typically designed and constructed to contain a gene, transgene, gene fragment, or other DNA of interest flanked by two genomic DNA fragments (5 'or left and 3' or right homology arms). After transfection, the two arms are used to promote homologous recombination between the DNA donor and the endogenous target locus.

As used herein, "unmodified donor DNA" is defined as DNA that has not been altered from its original state. As used herein, "chemically modified donor DNA" is defined as DNA to which some form of chemical modification (e.g., addition or reduction of a bond or moiety) has been made. Examples of chemical modifications include, but are not limited to, phosphorothioate, phospho, methylate, acetylate, and the like.

As used herein, the term "ribonucleoprotein" or "RNP" is used to refer to a complex of ribonucleic acid and an RNA binding protein (such as Cas 9).

As used herein, the term "CRISPR" or "clustered regularly interspaced short palindromic repeats" is used in accordance with its ordinary meaning and refers to the use of bacteria as a genetic element for acquired immunity against viruses. CRISPR comprises short sequences derived from the viral genome and which have been incorporated into the bacterial genome. Cas (CRISPR-associated protein) processes these sequences and cleaves the matched viral DNA sequences. Thus, the CRISPR sequence serves as a guide for Cas recognition and cleavage of DNA at least partially complementary to the CRISPR sequence. By introducing a plasmid comprising a Cas gene and specifically constructed CRISPR into eukaryotic cells, the eukaryotic genome can be cleaved at any desired location.

As used herein, the term "Cas9" or "CRISPR-associated protein 9" is used in accordance with its ordinary meaning and refers to an enzyme that uses a CRISPR sequence as a guide to recognize and cleave a specific DNA strand that is at least partially complementary to the CRISPR sequence. The Cas9 enzyme forms the basis of a technique called CRISPR-Cas9, which can be used to edit genes in organisms, together with CRISPR sequences. Such editing processes have a wide range of applications, including basic biological research, development of biotechnology products, and treatment of diseases.

As referred to herein, "CRISPR-associated protein 9," "Cas9," "Csn1," or "Cas9 protein" includes any of the recombinant or naturally occurring forms of Cas9 endonuclease or variants or homologs thereof that maintain Cas9 endonuclease activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas 9). In some aspects, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 consecutive amino acid portions) as compared to the naturally occurring Cas9 protein. In some aspects, the Cas9 protein is substantially identical to a protein identified by UniProt reference Q99ZW2 or a variant or homolog thereof that has substantial identity thereto. In some aspects, the Cas9 protein has at least 75% sequence identity to the amino acid sequence of the protein identified by UniProt reference Q99ZW 2. In some aspects, the Cas9 protein has at least 80% sequence identity to the amino acid sequence of the protein identified by UniProt reference Q99ZW 2. In some aspects, the Cas9 protein has at least 85% sequence identity to the amino acid sequence of the protein identified by UniProt reference Q99ZW 2. In some aspects, the Cas9 protein has at least 90% sequence identity to the amino acid sequence of the protein identified by UniProt reference Q99ZW 2. In some aspects, the Cas9 protein has at least 95% sequence identity to the amino acid sequence of the protein identified by UniProt reference Q99ZW 2.

As referred to herein, "CRISPR-associated endonuclease Cas12a", "Cas12", or "Cas12 protein" includes any one of a recombinant or naturally occurring form of a Cas12 endonuclease or a variant or homolog thereof that maintains Cas12 endonuclease activity (e.g., within at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% activity compared to Cas 12). In some aspects, the variant or homologue has at least 90%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., 50, 100, 150 or 200 consecutive amino acid portions) as compared to the naturally occurring Cas12 protein. In some aspects, the Cas12 protein is substantially identical to a protein identified by UniProt reference A0Q7Q2 or a variant or homolog thereof that has substantial identity thereto.

"guide RNA" or "gRNA" as provided herein refers to an RNA sequence that has sufficient complementarity to a target nucleic acid sequence to hybridize to the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. For example, the gRNA can direct Cas to a target nucleic acid. In embodiments, the gRNA includes crRNA and tracrRNA. For example, the gRNA may include crRNA and tracrRNA hybridized by base pairing. Thus, in embodiments, the crRNA and tracrRNA are two RNA molecules that then form an RNA/RNA complex due to complementary base pairing between the crRNA and tracrRNA to form the gRNA. In an embodiment, the gRNA is a single gRNA (sgRNA), wherein both the crRNA and the tracrRNA are in a single RNA molecule. In some aspects, the degree of complementarity between a guide RNA sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. In some aspects, the degree of complementarity between a guide RNA sequence and its corresponding target sequence is at least about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% when optimally aligned using a suitable alignment algorithm.

Non-limiting examples of CRISPR enzymes include Cas1, cas1B, cas2, cas3, cas4, cas5, cas6, cas7, cas8, cas9 (also known as Csn1 and Csx 12), cas10, cas12, cas13, csy1, csy2, csy3, cse1, cse2, csc1, csc2, csa5, csn2, csm3, csm4, csm5, csm6, cmr1, cmr3, cmr4, cmr5, cmr6, csb1, csb2, csb3, csx17, csx14, csx10, csx16, csaX, csx3, csx1, csx15, csf1, csf2, csf3, csf4, their homologs, their fusion proteins, or modified versions thereof. In embodiments, the CRISPR enzyme is a Cas9 enzyme. In embodiments, the Cas9 enzyme is streptococcus pneumoniae, streptococcus pyogenes, or streptococcus thermophilus Cas9, or mutants derived from these organisms. In an embodiment, the CRISPR enzyme is codon optimized for expression in eukaryotic cells. In embodiments, the CRISPR enzyme directs cleavage of one or both strands at the target sequence position. In embodiments, the CRISPR enzyme lacks DNA strand cleavage activity.

As used herein, the terms "nuclear localization signal", "nuclear localization sequence" or "NLS" are used to refer to an amino acid sequence that labels a protein or tags it for transport into the nucleus by nuclear transfer. Typically, the signal consists of one or more short sequences of positively charged lysines or arginines exposed on the protein surface.

As used herein, the term "non-viral" or "non-viral gene therapy" refers to any nucleic acid sequence that does not comprise a virus, viral vector, or virus-mediated nucleic acid delivery. Examples of non-viral methods of nucleic acid delivery include, but are not limited to, needle injection, ballistic DNA, electroporation, acoustic perforation, optical perforation, magnetic transfection, water perforation, mechanical massage, chemical carriers, inorganic particles, cationic lipids, lipid emulsions, solid lipid nanoparticles, peptide-based complexes, polymer-based complexes, or mixtures thereof.

As used herein, the term "nanoplasmid" is used to refer to an engineered circular nucleic acid that contains at least a nucleic acid sequence of interest, a minireplication origin (e.g., R6K), and a selectable marker (e.g., a small RNA selectable marker, RNA-OUT). The nanoplasmms contain less than 500bp of prokaryotic DNA.

As used herein, the term "antigen" is used to describe a compound, composition, or chemical that is capable of inducing an immune response (e.g., a Cytotoxic T Lymphocyte (CTL) response, a T helper response, a B cell response (e.g., the production of antibodies that specifically bind an epitope), an NK cell response, or any combination thereof) when administered to an immunocompetent subject. Thus, an immunogenic or antigenic composition is a composition capable of eliciting an immune response in an immunocompetent subject.

As used herein, the term "neoantigen" is used to describe a newly formed antigen that has not been previously recognized by the immune system. The neoantigen may be derived from altered tumor proteins resulting from tumor mutations or from viral proteins. Non-limiting examples of novel antigens are listed in table 2.

As used herein, the term "tumor-associated antigen" or "TAA" is used to describe a protein that is significantly over-expressed in cancer compared to normal cells and thus is also presented in large amounts on the surface of cancer cells. Non-limiting examples of TAAs are listed in table 1.

TABLE 1 tumor associated antigens

WT1
	MAGEA1
MSLN
	PRAME
CTAG1A(NY-ESO)

TABLE 2 shared neoantigens

JAK2 V617F	PIK3CA H1047R	IDH2 R140Q
			BRAF V600E	EGFR L858R	FLT3 D835Y
BRAF V600M	EGFR E746_A750del	ERBB2 S310F
			KRAS G12V	TP53 R175H	FGFR3 S249C
KRAS G12C	TP53 R248Q	PTEN R130Q
			KRAS G12D	TP53 R273C	PTEN R130G
KRAS G12R	TP53 R273H	SF3B1 R625H
			KRAS G13D	TP53 R273L	SF3B1 R625C
NRAS Q61R	TP53 R282W	GTF2I L424H
			NRAS Q61K	MYD88 L265P	GNAQ Q209P
PIK3CA E542K	DNMT3A R882H	GNAQ Q209L
			PIK3CA E545K	IDH1 R132H	GNA11 Q209L

As used herein, "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavoring agent, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonizing agent, solvent, surfactant or emulsifier that has been approved by the U.S. food and drug administration as being acceptable for use in humans or livestock. Exemplary pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, cellulose acetate, etc.; tragacanth; malt; gelatin; talc; cocoa butter, waxes, animal and vegetable fats, waxes, silicones, bentonites, silicic acid, zinc oxide; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; diols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol and polyethylene glycol; esters such as ethyl oleate, ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; non-thermal raw water; isotonic saline; ringer's solution; ethanol; phosphate buffer solution; as well as any other compatible substances used in pharmaceutical formulations.

As used herein, the term "cell population" or "plurality of cells" may be used interchangeably and refer to more than one cell.

As used herein, the term "inflammatory disease" refers to a disease or disorder characterized by abnormal inflammation (e.g., elevated levels of inflammation as compared to a control such as a healthy person not suffering from the disease). Examples of inflammatory diseases include: autoimmune diseases, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic Lupus Erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, type 1 diabetes, gill-barre syndrome, hashimoto's encephalitis, hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, behcet's disease, crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, graves ' eye disease, inflammatory bowel disease, addison's disease, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, ischemia reperfusion injury, stroke, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, scleroderma and atopic dermatitis.

As used herein, the term "autoimmune disease" refers to a disease or disorder in which the immune system of a subject has an abnormal immune response against substances that would not normally elicit an immune response in a healthy subject. Examples of autoimmune diseases that can be treated with the compounds, pharmaceutical compositions, or methods described herein include: acute Disseminated Encephalomyelitis (ADEM), acute necrotizing hemorrhagic encephalomyelitis, addison's disease, agaropectinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM/anti-TBM nephritis, anti-phospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune Inner Ear Disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune Thrombocytopenic Purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axons or neuronal neuropathy, balosis Behcet's disease, bullous pemphigoid, cardiomyopathy, kaschmann's disease, celiac disease, chagas's disease, chronic fatigue syndrome, chronic Inflammatory Demyelinating Polyneuropathy (CIDP), chronic Recurrent Multifocal Osteomyelitis (CRMO), chargan-Schtreus syndrome, cicatricial pemphigoid/benign mucosa pemphigoid, crohn's disease, crohn's syndrome, crohn's disease, congenital heart disease, coxsackie myocarditis, CREST's disease, primary mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, devic's disease (neuromyelitis optica), discoid lupus, deschler syndrome, endometriosis, eosinophilic esophagitis, sarcoidosis, experimental allergic encephalomyelitis, atopic dermatitis, EWens syndrome, fibromyalgia, fibroalveolar inflammation, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, goldPascher's syndrome, granulomatous Polyangiitis (GPA) (formerly known as Wegener's granulomatosis), graves 'disease, grignard-Barlichia syndrome, hashimoto's encephalitis, hashimoto's thyroiditis, hemolytic anemia, allergic purpura, herpes gestation, hypoproteinemia, idiopathic Thrombocytopenic Purpura (ITP), igA nephropathy, igG 4-related sclerotic diseases, immunoregulatory lipoproteins, inclusion body myositis, interstitial cystitis, juvenile arthritis, juvenile diabetes (type 1 diabetes), juvenile myositis, kawasaki syndrome, lanbert-Eton syndrome, leukolytic vasculitis lichen planus, lichen sclerosus, lignan conjunctivitis, linear IgA disease (LAD), lupus (SLE), lyme disease, chronic, meniere's disease, microscopic polyangiitis, mixed Connective Tissue Disease (MCTD), silkworm ulcer, muha-Haeman disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatism, PANDAS (streptococcal related pediatric autoimmune neuropsychosis), paraneoplastic cerebellar degeneration, paroxysmal sleep hemoglobinuria (PNH), parry Romberg syndrome, parsonnage-Turner syndrome, flat panamate (peripheral uveitis), pemphigus, peripheral neuropathy, and the like, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyadenylic syndrome of type I, II, III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardial-incision syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, primary pulmonary fibrosis, pyoderma gangrene, pure red cell aplastic anemia, raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophia, leider's syndrome, recurrent polyadenylic osteomyelitis, restless leg syndrome, retroperitoneal fibrosis rheumatic fever, rheumatoid arthritis, sarcoidosis, schmitt syndrome, scleritis, scleroderma, sjogren's syndrome, sperm and testis autoimmunity, stiff person syndrome, subacute Bacterial Endocarditis (SBE), soxak's syndrome, sympathogenic ophthalmitis, large arteritis, temporal arteritis/giant cell arteritis, thrombocytopenic purpura (TTP), tolosa-Hunt syndrome, transverse myelitis, type 1 diabetes, ulcerative colitis, undifferentiated connective tissue disease (uccd), uveitis, vasculitis, bullous dermatoses, vitiligo, or wegener granulomatosis (i.e., granulomatous Polyangiitis (GPA)).

As used herein, the term "inflammatory disease" refers to a disease or disorder characterized by abnormal inflammation (e.g., elevated levels of inflammation as compared to a control such as a healthy person not suffering from the disease). Examples of inflammatory diseases include: traumatic brain injury, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic Lupus Erythematosus (SLE), myasthenia gravis, juvenile onset diabetes, type 1 diabetes, gill-barre syndrome, hashimoto's encephalitis, hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, behcet's disease, crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, graves ' eye disease, inflammatory bowel disease, addison's disease, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, graft rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

As used herein, the term "cancer" refers to all types of cancers, tumors or malignancies found in mammals (e.g., humans), including leukemia, lymphoma, carcinoma, and sarcoma. Exemplary cancers that may be treated with the compounds or methods provided herein include brain cancer, glioma, glioblastoma, neuroblastoma, prostate cancer, colorectal cancer, pancreatic cancer, medulloblastoma, melanoma, cervical cancer, gastric cancer, ovarian cancer, lung cancer, head cancer, hodgkin's disease, and non-hodgkin's lymphoma. Exemplary cancers that may be treated with the compounds or methods provided herein include thyroid cancer, endocrine system cancer, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, liver cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, rectal cancer, stomach cancer, and uterine cancer. Additional examples include thyroid cancer, cholangiocarcinoma, pancreatic cancer, cutaneous melanoma, colon adenocarcinoma, rectal adenocarcinoma, gastric adenocarcinoma, esophageal cancer, head and neck squamous cell carcinoma, breast invasive carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, non-small cell lung cancer, mesothelioma, multiple myeloma, neuroblastoma, glioma, glioblastoma multiforme, ovarian cancer, rhabdomyosarcoma, primary thrombocythemia, primary macroglobulinemia, primary brain tumor, malignant pancreatic insulinoma, malignant carcinoid, bladder cancer, pre-cancerous skin lesions, testicular cancer, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, endometrial cancer, adrenocortical carcinoma, endocrine or exocrine pancreatic tumor, medullary thyroid cancer, melanoma, colorectal cancer, papillary thyroid cancer, hepatocellular carcinoma, or prostate cancer.

Engineering T cells

In one aspect, an engineered T cell is provided. Engineered T cells include nucleic acid sequences encoding polypeptides comprising exogenous TCR- β and exogenous TCR- α. In embodiments, the engineered T cell comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR- β and an exogenous TCR- α (VJ) domain. In embodiments, the nucleic acid sequence is inserted into the TCR-a locus of an engineered T cell.

In another interrelated aspect, a composition comprising isolated T cells is provided, wherein at least 5% of the cells are engineered T cells, each engineered T cell comprising a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR- β and an exogenous TCR- β. In embodiments, the nucleic acid sequence is inserted into the TCR-a locus of an engineered T cell.

In embodiments, at least 10% of the T cells are engineered T cells. In embodiments, at least 15% of the T cells are engineered T cells. In embodiments, at least 20% of the T cells are engineered T cells. In embodiments, at least 25% of the T cells are engineered T cells. In embodiments, at least 30% of the T cells are engineered T cells. In embodiments, at least 40% of the T cells are engineered T cells. In embodiments, at least 50% of the T cells are engineered T cells. In embodiments, at least 60% of the T cells are engineered T cells. In embodiments, between about 5% and 100% of the cells are engineered T cells. In embodiments, between about 5% and about 50% of the cells are engineered T cells. In embodiments, between about 5% and about 25% of the cells are engineered T cells. In embodiments, between about 5% and about 20% of the cells are engineered T cells. In embodiments, between about 5% and about 15% of the cells are engineered T cells. Percentages may be any value or subrange within the exemplified ranges, including the endpoints.

In embodiments, the engineered T cells do not express a functional endogenous TCR- β protein. In embodiments, less than about 50% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 40% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 30% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 20% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 10% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 5% of the engineered T cells express a functional endogenous TCR- β. In embodiments, less than about 4% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 3% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 2% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 1% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, between about 0% and about 50% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, between about 0% and about 25% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, between about 1% and about 25% of the engineered T cells express a functional endogenous TCR- β protein. Percentages may be any value or subrange within the exemplified ranges, including the endpoints.

In embodiments, at least about 50% of the engineered T cells have a disrupted and nonfunctional TCR- β locus on all alleles. In embodiments, at least about 40% of the engineered T cells have a disrupted and nonfunctional TCR- β locus on all alleles. In embodiments, at least about 60%, 70%, 80% or 90% of the engineered T cells have a disrupted and non-functional TCR- β locus on all alleles. In embodiments, at least about 95%, 96%, 97%, 98% or 99% of the engineered T cells have a disrupted and nonfunctional TCR- β locus on all alleles. In embodiments, between about 50% and about 100% of the engineered T cells have a disrupted and nonfunctional TCR- β locus on all alleles. In embodiments, between about 60% and about 95% of the engineered T cells have a disrupted and nonfunctional TCR- β locus on all alleles. Percentages may be any value or subrange within the exemplified ranges, including the endpoints.

In embodiments, less than about 50% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, less than about 40% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, less than about 30% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, less than about 20% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, less than about 10% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, less than about 5% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, less than about 4% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, less than about 3% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, less than about 2% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, less than about 1% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, between about 0% and about 50% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, between about 0% and about 25% of the engineered T cells express a functional endogenous TCR-a protein. In embodiments, between about 1% and about 25% of the engineered T cells express a functional endogenous TCR-a protein. Percentages may be any value or subrange within the exemplified ranges, including the endpoints.

In embodiments, at least about 50% of the engineered T cells have a disrupted and nonfunctional TCR-a locus on all alleles. In embodiments, at least about 40% of the engineered T cells have a disrupted and nonfunctional TCR-a locus on all alleles. In embodiments, at least about 60%, 70%, 80% or 90% of the engineered T cells have a disrupted and non-functional TCR-a locus on all alleles. In embodiments, at least about 95%, 96%, 97%, 98% or 99% of the engineered T cells have a disrupted and nonfunctional TCR-a locus on all alleles. In embodiments, between about 50% and about 100% of the engineered T cells have a disrupted and nonfunctional TCR-a locus on all alleles. In embodiments, between about 60% and about 95% of the engineered T cells have a disrupted and nonfunctional TCR-a locus on all alleles. Percentages may be any value or subrange within the exemplified ranges, including the endpoints.

In embodiments, the exogenous TCR-a (VJ) domain forms part of a heterologous TCR-a comprising at least a portion of an endogenous TCR-a of a T cell. In embodiments, the TCR-alpha locus is a TCR-alpha constant region. In embodiments, exogenous TCR- β and heterologous TCR- α are expressed from nucleic acids and form a functional TCR. In embodiments, at least 40% of the engineered T cells express heterologous TCR- α. In embodiments, at least 50% of the engineered T cells express heterologous TCR- α. In embodiments, at least 60% of the engineered T cells express heterologous TCR- α. In embodiments, at least 70% of the engineered T cells express heterologous TCR- α. In embodiments, at least 80% of the engineered T cells express heterologous TCR- α. In embodiments, at least 90% of the engineered T cells express heterologous TCR- α.

In embodiments, the engineered T cells bind to an antigen. In embodiments, the engineered T cells bind to cancer cells. In an embodiment, the TCR binds to an antigen presented on a class I major histocompatibility complex (mhc I) molecule.

In embodiments, the antigen is a neoantigen or a Tumor Associated Antigen (TAA). In embodiments, the antigen is a neoantigen. In embodiments, the antigen is TAA. In embodiments, the neoantigen or TAA is selected from: WT1, JAK2, NY-ESO1, PRAME, KRAS, HPV, or an antigen from table 1 or table 2. In embodiments, the antigen is WT1. In embodiments, the antigen is specific for cancer in a subject to whom the engineered T cells are to be administered. In embodiments, the antigen is expressed by or associated with a cancer of the subject to which the engineered T cell is to be administered.

In embodiments, the nucleic acid sequence further encodes a self-cleaving peptide. In an embodiment, the self-cleaving peptide is a self-cleaving viral peptide. In an embodiment, the self-cleaving viral peptide is T2A. In an embodiment, the self-cleaving viral peptide is P2A. In an embodiment, the self-cleaving viral peptide is E2A. In an embodiment, the self-cleaving viral peptide is F2A.

In embodiments, the nucleic acid sequence further encodes a protease cleavage site. In an embodiment, the protease cleavage site is a furin cleavage site.

In an embodiment, the engineered T cells express CD45RO, type 7C-C chemokine receptor (CCR 7) and L-selectin (CD 62L). In embodiments, the engineered T cells have a Central Memory (CM) T cell phenotype. In embodiments, the engineered T cells have an initial T cell phenotype. In an embodiment, the engineered T-cells with the initial T-cell phenotype are cd45ra+cd45ro-cd27+cd95- (i.e., the cells express CD45RA and CD27 and do not express detectable levels of CD45RO and CD 95). In an embodiment, the engineered T cells have a stem cell memory T cell phenotype. In an embodiment, the engineered T-cells with a stem cell memory T-cell phenotype are cd45ra+cd45ro-cd27+cd95+cd58+ccr7-Hi tcf1+. In embodiments, the engineered T cells have a central memory T cell phenotype. In an embodiment, the engineered T cells with a central memory T cell phenotype are cd45ro+cd45ra-cd27+cd95+cd58+. In embodiments, the engineered T cells have a progenitor depleted T cell phenotype. In an embodiment, the engineered T-cell having a progenitor cell depleted T-cell phenotype is PD-1+slamf6+tcf1+tim3-CD39-. In embodiments, engineered T cells with a progenitor depleted T cell phenotype express PD-1 at low or moderate levels compared to PD-1 high depleted T cells or recently activated T cells. In an embodiment, an engineered T cell with a progenitor depleted T cell phenotype expresses PD-1 at a low or moderate level compared to the level of PD-1 expressed in a recently activated T cell. In embodiments, the T cells are autologous to the subject in need thereof.

In an embodiment, at least 10% of the cells in the composition comprising isolated T cells are engineered T cells. In embodiments, at least 20% of the T cells are engineered T cells. In embodiments, at least 30% of the T cells are engineered T cells. In embodiments, at least 40% of the T cells are engineered T cells. In embodiments, at least 50% of the T cells are engineered T cells. In embodiments, at least 60% of the T cells are engineered T cells. In embodiments, at least 70% of the T cells are engineered T cells. In embodiments, at least 80% of the T cells are engineered T cells. In embodiments, at least 90% of the T cells are engineered T cells.

In an embodiment, the composition comprises about 0.1×10 ⁵ And about 1X 10 ⁹ Engineered T cells between individuals. In embodiments, the composition comprises at least about 1x 10 ⁸ Engineering T cells. In embodiments, the composition comprises at least about 1x 10 ⁹ Engineering T cells. In an embodiment, the composition comprises 1×10 ⁹ Up to 1X 10 ¹¹ Engineering T cells. In embodiments, the composition comprises at least about 1x 10 ¹⁰ Engineering T cells. In embodiments, the composition comprises at least about 1x 10 ¹¹ Engineering T cells. Cell numbers can be any value or subrange within the enumerated ranges, including the endpoints.

In embodiments, the composition further comprises a pharmaceutically acceptable excipient. Methods of making such compositions or implants have been described in the art (see, e.g., remington's Pharmaceutical Sciences, 16 th edition, mack edit (1980)). Where appropriate, the engineered T-cells may be formulated in a semisolid or liquid form of formulation, such as a capsule, solution, injection, inhalant or aerosol, in the usual manner for their respective routes of administration. In embodiments, the excipient is a balanced salt solution, such as hanks balanced salt solution or physiological saline. It will also be appreciated that the compositions of the present invention may also be administered in combination with other agents, such as cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies or other various pharmaceutically active agents, if desired. There is virtually no limit to the other components that may also be included in the composition, so long as the additional agents do not adversely affect the ability of the composition to deliver the intended treatment.

In another interrelated aspect, there is provided a T cell comprising an RNA transcript having a structure as described herein. In embodiments, the T cell comprises an RNA transcript, wherein the mRNA transcript is transcribed from a TCR transgene inserted into a TCR- α and/or TCR- β locus.

III, manufacturing method

In another interrelated aspect, a method for preparing an engineered T cell is provided. The method comprises the following steps: a) Contacting a T cell with a first ribonucleoprotein particle (RNP) and a donor DNA under conditions that allow the RNP and the donor DNA to enter the cell, wherein the first RNP comprises a first guide RNA that targets an endogenous TCR-a locus, and wherein the donor DNA comprises a nucleic acid sequence of a gene encoding a polypeptide comprising an exogenous TCR- β and an exogenous TCR-a; b) Incubating the T cells for a period of time; c) The cells are cultured in a medium for a period of time to allow the donor DNA to insert into the endogenous TCR-alpha locus, thereby forming engineered T cells.

In embodiments, the first RNP comprises a first gene-editing protein and a molar excess of the first guide RNA. In embodiments, the first RNP comprises a first gene-editing protein and the ratio of the first guide RNA to the first gene-editing protein is between about 1:1 and about 100:1 molar ratio. In embodiments, the ratio of the first guide RNA to the first gene-editing protein is between about 1:1 and about 75:1 molar ratio. In embodiments, the ratio of the first guide RNA to the first gene-editing protein is between about 1:1 and about 50:1 molar ratio. In embodiments, the ratio of the first guide RNA to the first gene-editing protein is between about 1:1 and about 25:1 molar ratio. In embodiments, the ratio of the first guide RNA to the first gene-editing protein is between about 1:1 and about 10:1 molar ratio. In embodiments, the ratio of the first guide RNA to the first gene-editing protein is between about 1:1 and about 5:1 molar ratio. In embodiments, the ratio of the first guide RNA to the first gene-editing protein is between about 1:1 and about 4:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 1:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 2:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 3:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 4:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 5:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 6:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about 7:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 8:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 9:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 10:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 25:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 50:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 75:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 100:1 molar ratio. The molar ratio may be any value or subrange within the exemplified ranges, inclusive of the endpoints.

In embodiments, the T cell is contacted with a second RNP comprising a second guide RNA targeting an endogenous TCR- β locus. In embodiments, the second RNP comprises a second gene-editing protein and a molar excess of a second guide RNA. In embodiments, the second RNP comprises a second gene-editing protein and the molar ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 100:1. In embodiments, the second RNP comprises a second gene-editing protein and the molar ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 75:1. In embodiments, the second RNP comprises a second gene-editing protein and the molar ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 50:1. In embodiments, the second RNP comprises a second gene-editing protein and the molar ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 25:1. In embodiments, the second RNP comprises a second gene-editing protein and the molar ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 10:1. In embodiments, the second RNP comprises a second gene-editing protein and the molar ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 5:1. In embodiments, the molar ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 4:1. In embodiments, the molar ratio of the second guide RNA to the second gene-editing protein is about 1:1. In embodiments, the molar ratio of the second guide RNA to the second gene-editing protein is about 2:1. In embodiments, the molar ratio of the second guide RNA to the second gene-editing protein is about 3:1. In an embodiment, the molar ratio of the second guide RNA to the second gene-editing protein is about 4:1. In embodiments, the molar ratio of the second guide RNA to the second gene-editing protein is about 5:1. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 10:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 25:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 50:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 75:1 molar ratio. In an embodiment, the ratio of the first guide RNA to the first gene-editing protein is about a 100:1 molar ratio. The molar ratio may be any value or subrange within the exemplified ranges, inclusive of the endpoints.

In embodiments, the T cell is contacted with a second RNP comprising a second guide RNA while being contacted with a first RNP comprising a first guide RNA. In embodiments, the T cell is contacted with a second RNP comprising a second guide RNA prior to contacting with the first RNP comprising the first guide RNA. In embodiments, the T cell is contacted with a second RNP comprising a second guide RNA after contacting with a first RNP comprising a first guide RNA.

In an example, the first guide RNA is found by methods known to those of skill in the art such that it targets exon 1, exon 2, or exon 3 region of the TCR constant alpha region locus (TRAC). In embodiments, the first guide RNA targets one of the following sequences from the TCR-a locus: TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21) or TRAC16 (SEQ ID NO: 22). In embodiments, the first guide RNA targets one of the following sequences: TRAC1, TRAC3, TRAC4, TRAC5, TRAC7, TRAC12 or TRAC15. In embodiments, the first guide RNA targets sequence TRAC1. In an embodiment, the first guide RNA targets sequence TRAC3. In an embodiment, the first guide RNA comprises the nucleic acid sequence in table 10. Any one or more of the guide RNAs may be explicitly excluded.

In an example, the second guide RNA is found by methods known to those skilled in the art such that it targets exon 1 regions of two TCR constant β region loci (TRBC). In embodiments, the second guide RNA targets one of the following sequences from the TCR- β locus: TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 46) or TRBC 48 (SEQ ID NO: 48). In embodiments, the second guide RNA targets one of the following loci: TRBC4, TRBC8, TRBC13, TRBC19, TRBC20, TRBC21, TRBC22, TRBC23 or TRBC26. In an embodiment, the second guide RNA comprises the nucleic acid sequence in table 11. Any one or more of the guide RNAs may be explicitly excluded.

In embodiments, the RNP comprises a CRISPR-associated (CAS) protein. In embodiments, the CAS protein is CAS9. In embodiments, cas9 is streptococcus pyogenes (Sp) Cas9. In embodiments, spCas9 is wild-type SpCas9. In embodiments, spCas9 is a mutant SpCas9.

In embodiments, the RNP comprises a guide RNA. In embodiments, the guide RNA is a synthetic nucleic acid. In embodiments, the guide RNA contains non-naturally occurring bases and/or backbone linkages known in the art. In embodiments, the guide RNA is a single guide (sg) RNA. In an embodiment, the guide RNA comprises a tracer (tr) RNA and a crispr (cr) RNA. In embodiments, the guide RNA comprises a phosphodiester derivative including, but not limited to, phosphoramidate, phosphorodiamidate, phosphorothioate, phosphorodithioate, phosphonocarboxylic acid, phosphonocarboxylate, phosphonoacetic acid, phosphonoformic acid, methylphosphonate, boron phosphonate, or O-methylphosphonous amide linkages.

In embodiments, the conditions that allow RNP and donor DNA to enter the cell include electroporation. In an embodiment, in method step b), the T cells are incubated for at least 10 minutes. In an embodiment, T cells are incubated at about 37 ℃. In an embodiment, the T cells are incubated at less than about 37 ℃. In an embodiment, the medium comprises a cytokine. In embodiments, the cytokine includes IL-2, IL-7 and/or IL-15. In embodiments, the cytokine comprises IL-2. In embodiments, the cytokine comprises IL-7. In embodiments, the cytokine comprises IL-15. In embodiments, cytokines include IL-7 and IL-15. In embodiments, any one or more of the exemplified cytokines may be specifically excluded.

In an embodiment, method step a) is carried out in the presence of a negatively charged polymer. In embodiments, the polymer is poly (glutamic acid) (PGA) or a variant thereof, poly (aspartic acid), heparin, or poly (acrylic acid). In an embodiment, the PGA is poly (L-glutamic acid) or a variant thereof. In embodiments, the PGA is poly (D-glutamic acid) or a variant thereof. In an embodiment, the PGA or variant thereof has an average molecular weight between 15 kilodaltons (kDa) and 50 kDa. In embodiments, any one or more of the polymers may be explicitly excluded.

In an embodiment, about 0.4 μg/μl to about 20 μg/μl of polymer is added. In an embodiment, about 2 μg/μl to about 12 μg/μl of polymer is added. In an embodiment, about 2 μg/μl to about 8 μg/μl of polymer is added. In an embodiment, about 4 μg/μl to about 8 μg/μl of polymer is added. In an embodiment, about 6 μg/μl to about 8 μg/μl of polymer is added. In the examples, about 6. Mu.g/. Mu.L of polymer was added. In the examples, about 8. Mu.g/. Mu.L of polymer was added. The amount may be any value or subrange within the stated range, including the endpoints. In the examples, no polymer was added.

In an embodiment, the amount of RNP is about 0.04 pmol/. Mu.L to about 20 pmol/. Mu.L. In an embodiment, the amount of RNP is about 0.2 pmol/. Mu.L to about 8 pmol/. Mu.L. In an embodiment, the amount of RNP is about 0.6 pmol/. Mu.L to about 8 pmol/. Mu.L. In an embodiment, the amount of RNP is about 0.8 pmol/. Mu.L to about 8 pmol/. Mu.L. In an embodiment, the amount of RNP is about 1.2 pmol/. Mu.L to about 8 pmol/. Mu.L. In an embodiment, the amount of RNP is about 2 pmol/. Mu.L to about 8 pmol/. Mu.L. In an embodiment, the amount of RNP is about 3 pmol/. Mu.L to about 8 pmol/. Mu.L. In an embodiment, the amount of RNP is about 4 pmol/. Mu.L to about 8 pmol/. Mu.L. In an embodiment, the amount of RNP is about 0.2 pmol/. Mu.L to about 6 pmol/. Mu.L. In an embodiment, the amount of RNP is about 0.2 pmol/. Mu.L to about 4 pmol/. Mu.L. In an embodiment, the amount of RNP is about 0.2 pmol/. Mu.L to about 2 pmol/. Mu.L. In an embodiment, the amount of RNP is about 0.2 pmol/. Mu.L to about 1 pmol/. Mu.L. The amount may be any value or subrange within the stated range, including the endpoints.

In an embodiment, the amount of donor DNA is from about 0.0002 pmol/. Mu.L to about 2 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.0004 pmol/. Mu.L to about 0.4 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.0004 pmol/. Mu.L to about 0.2 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.0004 pmol/. Mu.L to about 0.04 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.0004 pmol/. Mu.L to about 0.02 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.0004 pmol/. Mu.L to about 0.004 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.0002 pmol/. Mu.L to about 0.4 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.004 pmol/. Mu.L to about 0.4 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.02 pmol/. Mu.L to about 0.4 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.04 pmol/. Mu.L to about 0.4 pmol/. Mu.L. In an embodiment, the amount of donor DNA is about 0.08 pmol/. Mu.L to about 0.4 pmol/. Mu.L. In an embodiment, the amount of donor DNA is from about 0.2 pmol/. Mu.L to about 0.4 pmol/. Mu.L. The amount may be any value or subrange within the stated range, including the endpoints.

The amounts of polymer, RNP, donor DNA, and any other components of the examples described herein can be scaled up as needed, for example, to prepare engineered T cells for clinical use.

In an embodiment, the donor DNA is recombined into an endogenous TCR-a locus. In embodiments, the TCR-alpha locus is a TCR-alpha constant chain. In an embodiment, the donor DNA is recombined into the endogenous TCR- β locus.

In an embodiment, the donor DNA comprises a left homology arm and a right homology arm. In embodiments, the left and right homology arms are homologous to an endogenous TCR-a locus. In embodiments, the left homology arm is about 50 bases to about 2000 bases in length. In embodiments, the left homology arm is about 100 bases to about 1000 bases in length. In embodiments, the left homology arm is about 200 bases to about 800 bases in length. In embodiments, the right homology arm is about 200 bases to about 2000 bases in length. In embodiments, the right homology arm is from about 100 bases to about 1000 bases. In embodiments, the left and right homology arms are homologous to an endogenous TCR- β locus. In embodiments, the left homology arm is about 200 bases to about 800 bases in length. In embodiments, the left homology arm is about 250 bases to about 700 bases in length. In embodiments, the right homology arm is about 200 bases to about 800 bases in length. In embodiments, the right homology arm is about 250 bases to about 700 bases in length. The length may be any value or subrange within the exemplified ranges, including the endpoints.

In an embodiment, the donor DNA comprises double stranded DNA (dsDNA). In embodiments, the donor DNA is on a plasmid, a nanoplasmid, or a microring. In an embodiment, the donor DNA is on a plasmid. In an embodiment, the donor DNA is on a nanoplasmid. In an embodiment, the donor DNA is on a micro-loop. In an embodiment, the donor DNA is linear. In an embodiment, the donor DNA comprises single stranded DNA (ssDNA). In an embodiment, the donor DNA is not chemically modified. In an embodiment, the donor DNA comprises a chemical modification. In embodiments, the modification comprises a 5 'phosphate or a 5' phosphorothioate.

In an embodiment, the donor DNA and RNP are incubated together prior to contacting the T cells with the first RNP and donor DNA. In an embodiment, the gene-editing protein of RNP comprises a Nuclear Localization Sequence (NLS).

In an embodiment, T cells are cultured in a medium. In embodiments, the medium is selected from RPMI, PRIME-XV, and/or X-VIVO.

In embodiments, the T cells are activated prior to contacting the T cells with the first RNP and the donor DNA. In embodiments, the T cells are activated for between 24 hours and 96 hours. In an embodiment, the T cells are activated in the presence of a cytokine. In embodiments, the cytokine includes IL-2, IL-7 and/or IL-15.

In an embodiment, the T cells are activated with IL-2. In embodiments, T cells are activated in the presence of between about 0ng/mL and about 50ng/mL IL-2. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 50ng/mL IL-2. In embodiments, T cells are activated in the presence of between about 1ng/mL and about 5ng/mL IL-2. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 25ng/mL IL-2. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 20ng/mL IL-2. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 15ng/mL IL-2. In an embodiment, T cells are activated in the presence of about 10ng/mL IL-2. The amount may be any value or subrange within the stated range, including the endpoints.

In an embodiment, the T cells are activated with IL-2. In embodiments, T cells are activated in the presence of between about 0 units/mL and about 1000 units/mL of IL-2. In embodiments, the T cells are activated in the presence of between about 25 units/mL and about 500 units/mL of IL-2. In embodiments, T cells are activated in the presence of between about 125 units/mL and about 500 units/mL of IL-2. In embodiments, T cells are activated in the presence of between about 125 units/mL and about 400 units/mL of IL-2. In embodiments, T cells are activated in the presence of between about 125 units/mL and about 300 units/mL of IL-2. In embodiments, T cells are activated in the presence of about 200 units/mL IL-2. The amount may be any value or subrange within the stated range, including the endpoints.

In an embodiment, IL-7 is used to activate T cells. In embodiments, T cells are activated in the presence of between about 0ng/mL and about 200ng/mL IL-7. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 200ng/mL IL-7. In embodiments, T cells are activated in the presence of between about 10ng/mL and about 200ng/mL IL-7. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 150ng/mL IL-7. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 100ng/mL IL-7. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 50ng/mL IL-7. In embodiments, T cells are activated in the presence of between about 10ng/mL and about 100ng/mL IL-7. In embodiments, T cells are activated in the presence of between about 10ng/mL and about 50ng/mL IL-7. In an embodiment, T cells are activated in the presence of about 25ng/mL IL-7. The amount may be any value or subrange within the stated range, including the endpoints.

In an embodiment, IL-7 is used to activate T cells. In embodiments, T cells are activated in the presence of IL-7 between about 0 units/mL and about 4000 units/mL. In embodiments, T cells are activated in the presence of IL-7 between about 20 units/mL and about 2000 units/mL. In embodiments, T cells are activated in the presence of between about 20 units/mL and about 1000 units/mL of IL-7. In embodiments, T cells are activated in the presence of between about 20 units/mL and about 500 units/mL of IL-7. In embodiments, T cells are activated in the presence of between about 100 units/mL and about 500 units/mL of IL-7. In embodiments, T cells are activated in the presence of about 500 units/mL IL-7. The amount may be any value or subrange within the stated range, including the endpoints.

In an embodiment, IL-15 activation of T cells. In embodiments, T cells are activated in the presence of between about 0ng/mL and about 500ng/mL IL-15. In embodiments, T cells are activated in the presence of between about 0ng/mL and about 200ng/mL IL-15. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 200ng/mL IL-15. In embodiments, T cells are activated in the presence of between about 10ng/mL and about 200ng/mL IL-15. In embodiments, T cells are activated in the presence of between about 15ng/mL and about 200ng/mL IL-15. In embodiments, T cells are activated in the presence of between about 25ng/mL and about 200ng/mL IL-15. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 150ng/mL IL-15. In embodiments, T cells are activated in the presence of between about 5ng/mL and about 100ng/mL IL-15. In embodiments, T cells are activated in the presence of between about 10ng/mL and about 100ng/mL IL-15. In embodiments, T cells are activated in the presence of between about 25ng/mL and about 100ng/mL IL-15. In embodiments, T cells are activated in the presence of about 50ng/mL IL-15. The amount may be any value or subrange within the stated range, including the endpoints.

In an embodiment, IL-15 activation of T cells. In embodiments, T cells are activated in the presence of between about 0 units/mL and about 500 units/mL of IL-15. In embodiments, the T cells are activated in the presence of between about 25 units/mL and about 500 units/mL of IL-15. In embodiments, T cells are activated in the presence of between about 125 units/mL and about 500 units/mL of IL-15. In embodiments, T cells are activated in the presence of between about 125 units/mL and about 400 units/mL of IL-15. In embodiments, T cells are activated in the presence of between about 125 units/mL and about 300 units/mL of IL-15. In embodiments, T cells are activated in the presence of about 200 units/mL IL-15. The amount may be any value or subrange within the stated range, including the endpoints.

In embodiments, the T cells are activated in the presence of an anti-CD 3 antibody and/or an anti-CD 28 antibody. In embodiments, the T cells are activated in the presence of a CD3 agonist and/or a CD28 agonist. In embodiments, the anti-CD 3 antibody and/or the anti-CD 28 antibody is conjugated to a substrate. In embodiments, the CD3 agonist and/or CD28 agonist is conjugated to a substrate. In an embodiment, the substrate is a colloidal polymer nanomatrix. In an embodiment, the substrate is a superparamagnetic particle. In an embodiment, the T cells are activated in the presence of T cells TRANSACTTM (Miltenyi Biotech). In an embodiment, at The human T activator CD3/CD28 (Thermo Fisher Scientific) activates T cells. In an embodiment, method step a) is performed no more than about 24 hours after activation. In an embodiment, method step a) is performed between about 24 hours and about 72 hours after activation. In an embodiment, method step a) is performed between about 36 hours and about 60 hours after activation.

In another interrelated aspect, there is provided a method for preparing an engineered T cell population comprising: t cell populations are subjected to the methods described herein (including the examples). In embodiments, at least about 5% of the population of T cells are recovered as engineered T cells. In embodiments, about 5% to about 100% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 50% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 60% of the population of T cells are recovered as engineered T cells. Percentages may be any value or subrange within the exemplified ranges, including the endpoints.

In an embodiment, the engineered T cells are expanded after genetic modification. In embodiments, the engineered T cells are expanded at least about 2-fold relative to the cell count on day 1 after electroporation. In embodiments, the engineered T cells are expanded at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 50-fold, at least about 100-fold, at least about 200-fold, at least about 500-fold, at least about 1000-fold relative to the cell count on day 1 after electroporation.

In an embodiment, about 5% to about 100% of the T cell population is viable after method step c). In an embodiment, about 10% to about 100% of the T cell population is viable after method step c). In an embodiment, about 15% to about 100% of the T cell population is viable after method step c). In an embodiment, about 20% to about 100% of the T cell population is viable after method step c). In an embodiment, about 25% to about 100% of the T cell population is viable after method step c). In an embodiment, at least about 5% of the T cell population is viable after method step c). In an embodiment, at least about 10% of the T cell population is viable after method step c). In an embodiment, at least about 15% of the T cell population is viable after method step c). In an embodiment, at least about 20% of the T cell population is viable after method step c). In an embodiment, at least about 25% of the T cell population is viable after method step c). In an embodiment, at least about 50% of the population of T cells is viable after method step c). In an embodiment, at least about 75% of the population of T cells is viable after method step c). Percentages may be any value or subrange within the exemplified ranges, including the endpoints.

In an embodiment, the method further comprises: contacting the cells with a second RNP comprising a guide RNA targeting an endogenous TCR- β locus, and wherein at least about 5% of the population of T cells are recovered as engineered T cells. In embodiments, less than about 30% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 20% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 10% of the engineered T cells express a functional endogenous TCR- β protein. In embodiments, less than about 5% of the engineered T cells express a functional endogenous TCR- β protein. Percentages may be any value or subrange within the exemplified ranges, including the endpoints.

In embodiments, about 10% to about 100% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 20% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 30% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 40% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 50% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 60% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 70% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 80% of the population of T cells are recovered as engineered T cells. In embodiments, at least about 90% of the population of T cells are recovered as engineered T cells. Percentages may be any value or subrange within the exemplified ranges, including the endpoints. In an embodiment, the percentage of engineered T cells is determined prior to expansion. In an embodiment, the percentage of engineered T cells is determined after expansion.

In an embodiment, method step a) comprises the following steps in any order: (i) adding donor DNA to the chamber; (ii) adding RNP to the chamber; (iii) adding a negatively charged polymer to the chamber; and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps in any order: (i) Combining RNP and a negatively charged polymer to form an RNP-PGA mixture; (ii) adding donor DNA to the chamber; (iii) adding the RNP-PGA mixture to the chamber; and (iv) adding T cells to the chamber. In an embodiment, method step a) comprises the following steps in any order: (i) adding RNP to the chamber; (ii) adding donor DNA to the chamber; and (iii) adding T cells to the chamber. In an embodiment, no pipetting of T cells is performed during method steps a) and b). In an embodiment, the method comprises: negatively charged polymer is added to the chamber. In an embodiment, the method explicitly excludes the addition of negatively charged polymers to the chamber.

IV method of use

The engineered T cells described herein may be used for any suitable purpose. For example, engineered T cells can be administered to treat a disease in a subject in need thereof. The disease may be a tumor, an infection or an inflammatory disease.

In embodiments, the tumor is a cancer. In embodiments, the infection is a viral infection, bacterial infection, fungal infection, protozoal infection, or helminth infection. In embodiments, the viral infection is caused by human immunodeficiency virus, hepatitis c virus, hepatitis b virus, human cytomegalovirus or coronavirus. In embodiments, the inflammatory disease is autoimmune disease, allergic reaction, arthritis, psoriasis, diabetes, gillin-barre syndrome, hashimoto's encephalitis, hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, behcet's disease, crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, graves ' eye disease, inflammatory bowel disease, addison's disease, vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, ischemia reperfusion injury, stroke, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, scleroderma, and atopic dermatitis.

In another interrelated aspect, a method for treating a subject having cancer is provided. The method comprises the following steps: a) Providing a first population of T cells isolated from a subject; b) Engineering at least a subset of the first population of T cells to express a first exogenous T Cell Receptor (TCR) and knockout an endogenous TCR- β, thereby forming a first population of engineered T cells, wherein the exogenous TCR binds to a first antigen expressed by the cancer; c) Expanding the first engineered T cell; d) Administering an expanded first population of engineered T cells to the subject; e) Providing a second population of T cells isolated from the subject; f) Engineering at least a subset of the second population of T cells to express a second exogenous TCR and knock-out an endogenous TCR- β, thereby forming a second population of engineered T cells, wherein the exogenous TCR binds to a second antigen expressed by the cancer; g) Expanding the second engineered T cell; and h) administering the expanded second population of engineered T cells to the subject. In embodiments, any number of a plurality of engineered T cells, each containing a different TCR, can be prepared and administered to a subject. In embodiments, each TCR binds a different antigen. In embodiments, two or more TCRs bind to the same antigen. Multiple T cells may be administered in any order, including simultaneously.

In another interrelated aspect, a method for treating a subject having cancer is provided. The method comprises the following steps: a) Providing a first population of T cells isolated from a subject; b) Engineering at least a subset of the first population of T cells to express a first exogenous T Cell Receptor (TCR) and knockout an endogenous TCR- α, thereby forming a first population of engineered T cells, wherein the exogenous TCR binds to a first antigen expressed by the cancer; c) Expanding the first engineered T cell; d) Administering an expanded first population of engineered T cells to the subject; e) Providing a second population of T cells isolated from the subject; f) Engineering at least a subset of the second population of T cells to express a second exogenous TCR and knock-out an endogenous TCR- α, thereby forming a second population of engineered T cells, wherein the exogenous TCR binds to a second antigen expressed by the cancer; g) Expanding the second engineered T cells; and h) administering the expanded second population of engineered T cells to the subject.

In embodiments, a method for treating a subject having cancer, wherein the subject is treated with any one or a combination of an engineered T cell population expressing exogenous TCR- β and/or TCR- α.

In an embodiment, the expanded population of engineered T cells comprises 1x 10 ⁵ To 1x 10 ¹¹ Engineering T cells. In an embodiment, the expanded population of engineered T cells comprises at least 1x 10 ⁸ Engineering T cells. In an embodiment, the expanded population of engineered T cells comprises at least 1x 10 ⁹ Engineering T cells. In an embodiment, the expanded population of engineered T cells comprises at least 1x 10 ¹⁰ Engineering T cells. In an embodiment, the expanded population of engineered T cells comprises at least 1x 10 ¹¹ Engineering T cells. Amounts may be any value or subrange within the exemplified ranges, including the endpoints.

In embodiments, the T cells are autologous to the subject. In embodiments, the T cells are allogeneic to the subject.

In embodiments, the antigen is a neoantigen or TAA. In embodiments, at least a portion of the genome and/or transcriptome of the cancer is sequenced to determine the presence of the antigen. In an embodiment, an engineered T cell is prepared using the methods described herein (including the examples). In embodiments, the antigen is WT1, JAK2, NY-ESO1, PRAME or mutant KRAS, HPV. In embodiments, the antigen is an antigen from table 1 or table 2. In embodiments, the antigen is specific for cancer. In an embodiment, the TCR binds to an antigen presented on a class I major histocompatibility complex (mhc I) molecule.

In an embodiment, the first expanded population of engineered T cells comprises 1 x 10 ⁵ And 1X 10 ¹¹ Engineered T cells between individuals. In an embodiment, the first expanded population of engineered T cells comprises at least 1 x 10 ⁸ Engineering T cells. In an embodiment, the first expanded population of engineered T cells comprises at least 1 x 10 ⁹ Engineering T cells. In an embodiment, the first expanded population of engineered T cells comprises at least 1 x 10 ¹⁰ Engineering T cells. In an embodiment, the first expanded population of engineered T cells comprises at least 1 x 10 ¹¹ Engineering T cells. In an embodiment, the second expanded population of engineered T cells comprises 1 x 10 ⁵ And 1X 10 ¹¹ Engineered T cells between individuals. In an embodiment, the second expanded population of engineered T cells comprises at least 1 x 10 ⁸ Engineering T cells. In an embodiment, the second expanded population of engineered T cells comprises at least 1 x 10 ⁹ Engineering T cells. In an embodiment, the second expanded population of engineered T cells comprises at least 1 x 10 ¹⁰ Engineering T cells. In an embodiment, the second expanded population of engineered T cells comprises at least 1 x 10 ¹¹ Engineering T cells. Amounts may be any value or subrange within the exemplified ranges, including the endpoints.

In embodiments, an additional population of engineered T cells is administered to the patient, and T cells in the additional population of engineered T cells express a third exogenous TCR that binds to a third antigen expressed by the cancer. In embodiments, an additional population of engineered T cells is administered to the patient, and T cells in the additional population of engineered T cells express a fourth exogenous TCR that binds to a fourth antigen expressed by the cancer. In embodiments, an additional population of engineered T cells is administered to the patient, and T cells in the additional population of engineered T cells express a fifth exogenous TCR that binds to a fifth antigen expressed by the cancer. In embodiments, an additional population of engineered T cells is administered to the patient, and T cells in the additional population of engineered T cells express a sixth exogenous TCR that binds to a sixth antigen expressed by the cancer. In embodiments, an additional population of engineered T cells is administered to the patient, and T cells in the additional population of engineered T cells express a seventh exogenous TCR that binds to a seventh antigen expressed by the cancer. In embodiments, an additional population of engineered T cells is administered to the patient, and T cells in the additional population of engineered T cells express an eighth exogenous TCR that binds to an eighth antigen expressed by the cancer. It will be appreciated by those of skill in the art that any number of additional T cells expressing any number of additional TCRs fall within the scope of the present disclosure.

In another interrelated aspect, a method of treating cancer is provided. The method comprises the following steps: t cells, compositions or pharmaceutical compositions as described herein (including examples) are administered to a patient suffering from cancer. In some embodiments, the method further comprises: an anti-cancer therapy is administered to a subject. In embodiments, T cells, compositions, or pharmaceutical compositions described herein are administered to a patient who has undergone anti-cancer treatment. In embodiments, a patient who has undergone anti-cancer treatment is selected to administer T cells, compositions, or pharmaceutical compositions described herein. In embodiments, the anti-cancer therapy includes immunotherapy, chemotherapy, and/or radiation therapy.

In embodiments, the patient undergoes lymphocyte depletion prior to administration of the T cells, compositions or pharmaceutical compositions described herein. In embodiments, T cells, compositions, or pharmaceutical compositions described herein are administered to a patient who has undergone lymphocyte depletion. In embodiments, a patient who has undergone lymphocyte depletion is selected for administration of a T cell, composition or pharmaceutical composition described herein.

V. nucleic acid and kit

In another interrelated aspect, a guide RNA is provided. The guide RNA is found by methods known to those skilled in the art such that it targets exon 1, exon 2 or exon 3 regions of the TCR constant alpha region locus (TRAC). In embodiments, the guide RNA targets an endogenous TCR-a locus at one of the following sites: TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21) or TRAC16 (SEQ ID NO: 22). In embodiments, the guide RNA targeting the endogenous TCR-a locus comprises a nucleic acid sequence as set forth in table 10. In embodiments, the endogenous TCR-a locus is an endogenous TCR-a constant region.

In another interrelated aspect, a guide RNA is provided. The guide RNA was found by methods known to those skilled in the art such that it targets the exon 1 region of two TCR constant β region loci (TRBC). In embodiments, the guide RNA targets an endogenous TCR- β locus that targets one of the following sequences: TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 46) or TRBC 48 (SEQ ID NO: 48). In embodiments, the guide RNA targeting the endogenous TCR- β locus comprises a nucleic acid sequence as set forth in table 11.

In another interrelated aspect, a nucleic acid is provided. The nucleic acid includes a nucleic acid sequence comprising an exogenous TCR- β encoding sequence and an exogenous TCR-a encoding sequence, wherein the nucleic acid sequence further comprises a first self-cleaving peptide encoding sequence. In embodiments, the nucleic acid further comprises a first homology arm and a second homology arm. In embodiments, the nucleic acid further comprises a second self-cleaving peptide coding sequence. In an embodiment, the nucleic acids comprise, in order from 5 'to 3': (i) a first homology arm; (ii) a first self-cleaving viral peptide coding sequence; (iii) an exogenous TCR- β encoding sequence; (iv) a second self-cleaving viral peptide coding sequence; (v) an exogenous TCR-a coding sequence; (vi) optionally, a polyA sequence; and (vii) a second homology arm. In embodiments, the exogenous TCR-a coding sequence encodes an exogenous TCR-a VJ domain.

In embodiments, the first homology arm is homologous to an endogenous TCR-a locus in a human T cell. In embodiments, the second homology arm is homologous to an endogenous TCR-a locus in a human T cell. In embodiments, the endogenous TCR-alpha locus is a TCR-alpha constant region. In embodiments, the first homology arm is homologous to an endogenous TCR- β locus in a human T cell. In embodiments, the second homology arm is homologous to an endogenous TCR- β locus in a human T cell.

In embodiments, the first self-cleaving viral peptide is T2A, P2A, E a or F2A. In embodiments, the second self-cleaving viral peptide is T2A, P2A, E a or F2A. In embodiments, the first self-cleaving viral peptide and the second self-cleaving viral peptide are different. In embodiments, the first self-cleaving viral peptide and the second self-cleaving viral peptide are the same. In embodiments, the first self-cleaving peptide coding sequence is 5' of an exogenous TCR-a coding sequence (e.g., an exogenous TCR-a VJ domain coding sequence). In embodiments, the second self-cleaving peptide coding sequence is 5' of the exogenous TCR- β coding sequence. In embodiments, the first self-cleaving peptide coding sequence is 5' of the exogenous TCR- β coding sequence. In embodiments, the second self-cleaving peptide coding sequence is 5' of an exogenous TCR-alpha coding sequence (e.g., an exogenous TCR-alpha VJ domain coding sequence). In embodiments, the nucleic acid sequence comprises a polyA signal. In an embodiment, the polyA signal is 3' of the full length TCR-alpha encoding sequence. In an embodiment, the polyA signal is 3' of the full length TCR- β coding sequence.

In embodiments, the nucleic acid is a plasmid, a nanoplasmid, or a micro-loop. In an embodiment, the nucleic acid is a plasmid. In an embodiment, the nucleic acid is a nanoplasmid. In embodiments, the nucleic acid is a micro-loop.

In another interrelated aspect, a kit for producing engineered T cells is provided. The kit includes a TCR-a targeting guide RNA as described herein (including the examples). In an embodiment, the kit further comprises a TCR- β targeting guide RNA as described herein (including the embodiment). In embodiments, the kit further comprises a gene editing reagent or a nucleotide encoding a gene editing reagent. In an embodiment, the gene editing agent is a CRISPR system. In an embodiment, the kit further comprises donor DNA. In embodiments, the donor DNA comprises a nucleic acid sequence encoding a polypeptide comprising an exogenous TCR- β and an exogenous TCR- α domain. In embodiments, the exogenous TCR- β and the heterologous TCR- α form a TCR capable of binding to an antigen. In an embodiment, the TCR binds to an antigen presented on a class I major histocompatibility complex (mhc I) molecule. In embodiments, the antigen is WT1, JAK2, NY-ESO1, PRAME, mutant KRAS, or an antigen from table 1 or table 2. In embodiments, the antigen is a neoantigen. In embodiments, the kit further comprises poly (glutamic acid) (PGA) or a variant thereof. In an embodiment, the kit further comprises a nucleic acid as described herein (including the embodiments).

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes.

Examples

Those skilled in the art will appreciate that the descriptions of making and using the particles described herein are for illustration purposes only and that the present disclosure is not limited by this illustration.

Example 1T cell purification and activation

To purify T cells, the following reagents were used:buffy coat CD8 microbeads (Miltenyi, catalog No. 130-114-978), MACS buffer (PBS/0.5% BSA/2mM EDTA), QUADROMACS separator (Miltenyi), X-VIVOTM15 without daptomycin and phenol red (Lonza, catalog No. 04-744Q) and erythrocyte lysis buffer.

Buffy coats from each donor were diluted to 80mL with PBS/0.5% BSA/2mM EDTA and 4mL was added to each buffy coatBuffy coat CD8 microbeads (i.e., 4mL of beads added per 80mL volume of blood). The tubes were inverted 5 to 8 times for mixing and incubated in a refrigerator (2 ℃ to 8 ℃) for 15 minutes.

For magnetic separation, the whole blood column is placed in the magnetic field of a quadtomacstm separator. The column was prepared by washing with 3mL of separation (MACS) buffer and the magnetically labelled cell suspension was applied to the prepared whole blood column. The effluent contains unlabeled cells. The whole blood column was washed twice with 2mL of separation buffer, then removed from the separator and placed in a new collection tube. Magnetically labeled cells are eluted by applying an elution buffer and then pushing the plunger firmly into the column. Cells were pelleted by centrifugation at 300g for 5 min and the supernatant removed. Cells were resuspended by pipetting up and down in 5mL of erythrocyte lysis buffer and incubated for 5 min at room temperature. The cells were washed with PBS and centrifuged again before resuspension of the resulting cell pellets by pipetting into 5mL of X-VIVOTM medium containing 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine. Counting cells; the culture target in the X-VIVOTM15 culture medium is 1X10 ⁶ Individual cells/mL.

To activate T cells, the following reagents were used: TRANSACTTM (Miltenyi, catalog No. 130-111-160), 25ng/mL research grade human IL-7 (Miltenyi, catalog No. 130-095-367), 50ng/mL research grade human IL-15 (Miltenyi, catalog No. 130-095-760), 10ng/mL research grade human IL-2 (Miltenyi, catalog No. 130-097-743), gentamicin and phenol red free X-VIVOTM15 (Lonza, catalog No. 04-744Q), N-acetyl-L-cysteine (catalog No. A9165) and b-mercaptoethanol (catalog No. 21985-023).

Plating was performed in tissue culture plates according to the following protocol. First, to 1X10 in a 12-well plate (4 mL/well) ⁶ Cells were plated at a concentration of 5×10≡6 cells per mL or in 6-well plates (5 mL/well) at X-VIVOTM15 containing 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine. T cell TRANSACTTM reagent (1:100), 10ng/mL IL-2, 25ng/mL IL-7, and 50ng/mL IL-15 were added to each well. Cells were incubated at 37℃under 5% CO2 for 36 to 48 hours under humid conditions.

EXAMPLE 2T cell electroporation

For electroporation of T cells, the following reagents and equipment were used: spyFiTMCas9 (Allevon, catalog No. 9214-5 MG), TCR-targeted sgRNA (see, e.g., tables 3 and 4; and NTC), donor DNA template, nuclease-free double-stranded buffer (IDT, catalog No. 1072570), poly-L-glutamic acid sodium salt of molecular weight 15,000-50,000 (Sigma, catalog No. P4761-100 MG), PCR tubes, P3 primary cell kit (Lonza V4SP-3096, 96-reactions), 4D-Nucleofectm core unit (Lonza, catalog No. AAF-1002B), and 4D-NucleofecttmX unit (Lonza, catalog No. AAF-1002X).

T cell electroporation. Unless otherwise indicated, sgrnas were reconstituted to 50 μm in nuclease-free duplex buffer, briefly vortexed, and incubated for 5 minutes at Room Temperature (RT), then vortexed again. If crRNA+tracrrna is used, each is reconstituted at 100. Mu.M. crRNA and tracRNA (20 μl each) were added to the tube, briefly vortexed and centrifuged. The RNA was allowed to anneal at 95℃for 5 minutes and slowly cooled to room temperature (about 5 minutes).

X-VIVOTM15 (900. Mu.L) containing 50. Mu.M 2-mercaptoethanol, 10. Mu. M N-acetyl-L-cysteine, 10ng/mL IL-2, 25ng/mL IL-7 and 50ng/mL IL-15 was dispensed into each well and incubated at 37℃to heat the medium.

Separately, the recovery medium (X-VIVOTM 15 medium containing 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine, without cytokine addition) was warmed up.

The RNP complex was prepared immediately prior to transfection as follows: 1. mu.l (180 pmol) of sgRNA was added to RNAse/DNAse-free PCR tubes; 2. mu.l of high fidelity Cas9 (60 pmol) was added and gently mixed by repeated pipetting of the solution (3:1 ratio); 3. incubate for 15 min at room temperature to obtain 4.5. Mu.l RNP mix ready for transfection.

Activated T cells were harvested and centrifuged at 300 g. Activated T CELLs were washed with PBS and counted (via Vi-CELL). The cells were centrifuged again at 300g, then resuspended with 1X10≡6 cells per 20. Mu.l of P3 nuclear transfection buffer with supplements, and mixed by pipetting 2-3 times. Donor template (2 μl containing a total of 1 μg) was added to the PCR tube. RNP (4.5. Mu.l) was added to the PCR tube and incubated for 1 min at room temperature. If used, 2. Mu.l of PGA (resuspended at 100 mg/mL) was added to the PCR tube and incubated for 1-2 minutes at room temperature. Cells (20. Mu.L) were added to the PCR tube and mixed by gentle pipetting up and down. The mixture was transferred to cuvette strips, capped and incubated in the cuvette strips for 3-5 minutes.

Cuvette strips were placed in a 4D-nucleic acid machine and electroporated using indicated procedures (e.g., primary cell P3 procedure and EH115 pulse code). Mu.l of pre-warmed X-VIVO 15 containing 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine (without cytokines) was added to each of the nuclear transfected cells and the cuvette was transferred to a 37℃incubator for at least 15 minutes. After 15 minutes of incubation, the cells were transferred to appropriate wells in a 48-well plate (X-VIVOTM 15 medium containing cytokines).

On day 3, cells were transferred to 24-well plates and 1mL fresh X-VIVO 15 medium containing 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine, 10ng/mL IL-2, 25ng/mL IL-7 and 50ng/mL IL-15 was added. On day 5, cells were resuspended by pipetting. Samples (100. Mu.L) were set aside for analysis, the remainder transferred to a 12-well plate, and 2mL fresh X-VIVO 15 medium containing 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine, 10ng/mL IL-2, 25ng/mL IL-7, and 50ng/mL IL-15 was added. On day 7, the cells were resuspended by pipetting and an additional 100 μl of sample was taken for analysis.

EXAMPLE 3 flow cytometry analysis

100 μl post-transfection cell culture was transferred to a 96-well V-bottom well. Mu.l of cold FACS buffer (0.5% Bovine Serum Albumin (BSA) +sodium azide in PBS) was added and the cells were centrifuged at 300x g for 5 min at 4 ℃. Cells were resuspended in 200 μl FACS buffer and centrifuged at 300x g for 5 min at 4 ℃. Cells were then resuspended in 100 μl of working solution of fixable vital dye (e.g., eBioscience Fixable Viability Dye eFluor 506,Thermo Fisher catalog No. 65-0866-14) generated by dilution at 1:1000 in PBS (azide-and serum/protein free). The cells were thoroughly mixed and incubated in the dark at 4℃for 30 min. Cells were washed twice with cold FACS buffer: first by adding 100 μl FACS buffer to the cell mixture in working solution where the vital dye can be immobilized and centrifuging at 300x g for 5 min at 4 ℃, and then by resuspension in 200 μl FACS buffer and centrifuging at 300x g for 5 min at 4 ℃. The cells are then resuspended in a working solution of the appropriate fluorochrome conjugated antibody. For most surface antibodies, cells were incubated in the dark at 4℃for 30 min. If exogenous T cell receptor expression is assessed by dextran or tetramer staining, cells are first incubated in the dark at room temperature with 50. Mu.L working solution of dextran/tetramer reagent for 15 minutes. After incubation, 50 μl of working solution of other fluorochrome conjugated antibodies (e.g., CD8, CD3, etc.) was added to the cells and the cells were incubated at 4 ℃ for an additional 15 minutes in the dark. The cells are then resuspended in a working solution of the appropriate fluorochrome conjugated antibody. For most surface antibodies, cells were incubated in the dark at 4℃for 30 min. If exogenous T cell receptor expression is assessed by dextran or tetramer staining, cells are first incubated in the dark at room temperature with 50. Mu.L working solution of dextran/tetramer reagent for 15 minutes. After incubation, 50 μl of working solution of other fluorochrome conjugated antibodies (e.g., CD8, CD3, etc.) was added to the cells and the cells were incubated at 4 ℃ for an additional 15 minutes in the dark. After incubation with antibody, the cells were washed twice with cold FACS buffer. After the second wash, the cells were resuspended in 100 μl FACS buffer and analyzed on a flow cytometer. To count cells, 10. Mu.L of CountBIght absolute count beads (molecular probe catalog number 402-ML-020) were added to the stained cells. At least 1000 beads were collected on a flow cytometer to determine accurate concentration values and cell counts were determined using calculations provided by the manufacturer.

EXAMPLE 4 donor DNA titration and electroporation conditions

The experimental conditions were determined using donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide and the TCRa VJ domain. T cells were activated using TRANSACTTM (1:30) in RPMI+10% FBS, glutamax, HEPES, non-essential amino acids, sodium pyruvate, beta-mercaptoethanol, and cytokines (10 ng/mL IL-2, 25ng/mL IL-7, and 50ng/mL IL-15)). Cells were electroporated 48 hours after activation using Lonza P2 buffer and pulse code EH100 or P3 buffer and EH115 pulse code.

RNP was prepared as follows: 1.6. Mu.L of PGA (15-50 kDa,100mg/mL in water) +2. Mu.L of TRAC1 sgRNA (100 pmol) +1. Mu.L of Cas9 (50 pmol); RNP was incubated for 10 min and with TRAC 1-mNaeon construct for an additional 5 min (both incubated at room temperature). Titration of the PCR product TRAC1-mNEon construct: 6 μg, 4 μg, 2 μg or 1 μg.

Electroporation conditions: TRAC1 RNP+TRAC1-mNEon titration amounts (6. Mu.g, 4. Mu.g, 2. Mu.g and 1. Mu.g); NTC (non-targeted guide control, i.e. TCR-free knockout) rnp+4 μg TRAC1-mNeon; TRAC1 RNP+2 μg TRAC 1-mNaeon+P3 buffer+EH115 pulses. After electroporation, the cells were divided into two parts: (1) 37 ℃ throughout the incubation period and (2) "cold shock": 32 ℃ for 24 hours, then transferred to 37 ℃.

The results are shown in fig. 1A to 7.

Example 5 targeting different TRAC loci and titrating donor DNA

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used.

T cells were activated using TRANSACTTM (1:30) in RPMI+10% FBS, glutamax, HEPES, non-essential amino acids, sodium pyruvate, beta-mercaptoethanol, and cytokines (10 ng/mL IL-2, 25ng/mL IL-7, and 50ng/mL IL-15)). Cells were electroporated 48 hours after activation using Lonza P3 buffer and EH115 pulse code. Cells were incubated at 37 ℃ immediately after electroporation.

RNP was prepared as follows: 1.6. Mu.L of PGA (15-50 kDa,100mg/mL in water) +2. Mu.L of TRAC1 or TRAC3 sgRNA+1. Mu.L of Cas9. RNPs were incubated for ten minutes and incubated with appropriate PCR product donor templates (TRAC 1-mNaeon or TRAC 3-mNaeon) for an additional 5 minutes; all incubations were performed at room temperature. Cells were electroporated with TRAC1 or TRAC3 RNP+ titrated corresponding TRAC-Neon templates (6. Mu.g, 4. Mu.g, 2. Mu.g, and 1. Mu.g) or TRAC1 RNP+ titrated NY-ESO TCR templates.

The results are provided in fig. 10A to 16.

EXAMPLE 6 titration of RNP

T cells were activated in RPMI+10% FBS, L-glutamine, nonessential amino acids, BME, and cytokines (10 ng/mL IL-2, 25ng/mL IL-7, and 50ng/mL IL-15) using TRANSACTTM (1:30). Cells were electroporated 48 hours after activation using P3 buffer and EH115 pulse code.

RNP was prepared as follows: 1.6. Mu.L of PGA (15-50 kDa,100mg/mL in water) +RNP+IDT double stranded buffer was used to equalize the volume between samples. RNPs were incubated for ten minutes at room temperature and 5 additional minutes with template (TRAC 3 mNaeon dsDNA (1. Mu.g or 2. Mu.g)). TRAC3 only (without TRBC), 0, 10, 20, 40, 80 or 120pmol RNA. On day 4 after electroporation, cells were stained with TCRa/b-APC (BioLegend clone IP 26) and analyzed by flow cytometry. The results are shown in fig. 7 to 21.

Example 7 Single-stranded DNA template and double-stranded DNA Donor template

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. T cells were activated in RPMI+10% FBS, L-glutamine, nonessential amino acids, BME, and cytokines (10 ng/mL IL-2, 25ng/mL IL-7, and 50ng/mL IL-15) using TRANSACTTM (1:30). Cells were electroporated 48 hours after activation using P3 buffer and EH115 pulse code.

RNP was prepared as follows: cas9+ gRNA (10 min, RT) followed by 1.6 μl PGA (15-50 kda,100mg/mL in water) was added, followed by template and incubation at RT for 5 min. The templates were TRAC3 mNaeon dsDNA (0.125,0.25,0.50 μg) or TRAC3 ssDNA Forward (FW) or Reverse (RV). RNP: NT or TRAC3 only (without TRBC), 10pmol. On day 4 after electroporation, cells were stained with TCRa/b-APC (BioLegend clone IP 26) and analyzed by flow cytometry. The results are provided in fig. 22-23C.

EXAMPLE 8 activation and electroporation conditions

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. T cells are activated in the following: RPMI+10% FBS, glutamax, HEPES, nonessential amino acids, sodium pyruvate, beta-mercaptoethanol, and cytokines (10 ng/mL IL-2, 25ng/mL IL-7, and 50ng/mL IL-15); X-VIVO 15 with 10ng/mL IL-2, 25ng/mL IL-7 and 50ng/mL IL-15; X-VIVO 15 with 25ng/mL IL-7 and 50ng/mL IL-15. For each set of media/cytokine conditions tested, titration was performed on TRANSACTTM (1:20, 1:50,1:100, 1:200). Cells were electroporated 48 or 72 hours after activation.

RNP was prepared as follows: RNPs were incubated for 10 min at room temperature (2. Mu.L of sgRNA+1. Mu.L of Cas9, 40pmol per reaction); incubation with 1.6. Mu.L of PGA (15-50 kDa,100mg/mL in water) +2. Mu.g of TRAC 3-mNaeon construct was carried out for an additional 5 minutes at room temperature.

The results are provided in fig. 24A to 31B.

Example 9 Single-stranded DNA templates (Forward and reverse) and double-stranded DNA

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. T cells were activated in X-VIVO medium+L-glutamine, non-essential amino acids, BME and cytokines (10 ng/mL IL-2, 25ng/mL IL-7 and 50ng/mL IL-15) using TRANSACTTM (1:30). Cells were electroporated 48 hours after activation using P3 buffer and EH115 pulse code.

RNP was prepared as follows: spyFi Cas9+sgrna+pga (1.6 μl/sample), for 10 min incubation. Templates (TRAC mNaeon dsDNA (0.5,1.0,1.5. Mu.g) and ssDNA (0.5, 1.0. Mu.g) were added, see FIG. 34) and incubated for an additional 5 minutes at RT. 40pmol RNP (TRBC free) was electroporated. On day 4 after electroporation, cells were stained with TCRa/b-APC (BioLegend clone IP 26) and analyzed by flow cytometry. The results are shown in fig. 33 to 35B.

Example 10 other TRAC loci for TCR knock-in

RNP was prepared as follows: spyFi Cas9+sgrna+pga (1.6 μl/sample), for 10 min incubation. Template (TRAC mNaeon dsDNA,0.25, 0.5 or 1.0 μg) was added and incubated for another 5 minutes at room temperature. 40pmol RNP (TRBC free) was electroporated. On day 4 after electroporation, cells were stained with TCRa/b-APC (BioLegend clone IP 26) and analyzed by flow cytometry. The corresponding gRNA site within the TCR-alpha locus is shown in FIG. 36A. The results are shown in fig. 36B, 36C, and 37.

EXAMPLE 11 TCR activated cytokine Condition and titration

T cells were activated using TRANSACTTM (1:30, 1:100 or 1:200) under the following media conditions:

the supplement added: glutamax, non-essential amino acids, sodium pyruvate and beta-mercaptoethanol. Cells were electroporated 48 hours after activation using P3 buffer and EH115 pulse code.

RNP was prepared as follows: spyFi Cas9+sgrna+pga (1.6 μl/sample) (sgRNA to Cas9 ratio 3:1) for 10 min incubation. Template (1. Mu.g TRAC 3-mNaeon) was added and incubated for an additional 5 minutes at room temperature. 40pmol RNP (TRBC free) was electroporated. On day 4 after electroporation, cells were stained with TCRa/b-APC (BioLegend clone IP 26) and analyzed by flow cytometry. The results are shown in fig. 38A to 47.

EXAMPLE 12 post electroporation cell handling

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. Activation was performed using X-VIVO medium with supplement, 1:100TRANSACTTM, 10ng/mL IL-2, 25ng/mL IL-7, and 50ng/mL IL-15. Electroporation was performed after 48 hours. Electroporation was performed as follows: 1) 2:1sgRNA, cas9 (SpyFi) plus PGA for 15 minutes; 2) Adding 0.5 μg mNaeon template for 5 min; 3) Adding cells; 4) The P3 buffer plus pulse code EH115; and 5) 100. Mu.L of preheated X-VIVO with cytokine is added and pipetted once for mixing.

Electroporation was followed by the following three conditions: 1) 100 μl of X-VIVO with cytokines was added and immediately transferred to 24-well plates; 2) Add 20 or 100 μl of X-VIVO with cytokine without pipetting, wait 15 minutes at 37 ℃ and then transfer to 24 well plate; 3) Wait 5 minutes at room temperature, add 20. Mu.L or 100. Mu.L of X-VIVO with cytokine without pipetting, wait 15 minutes at 37℃and transfer to 24 well plate. The results are shown in fig. 48A to 50.

Example 13 double-and Single-stranded DNA templates with 5' -phosphate and phosphorothioate

The PCR products that have been used as templates in the above experiments were generated using primers lacking the 5' phosphate. Here we use templates that add 5' phosphate or phosphorothioate to the ends of the primers. Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used.

The experimental setup included a new order of addition and RNP formulation. The plating was performed using X-VIVO with 10ng/mL IL-2, 25ng/mL IL-7, 50ng/mL IL-15 and TRANSACTTM 1:100. Electroporation was performed 42 hours after activation using P3 buffer and EH115 pulses. Templates used were 0.5. Mu.g, 1.0. Mu.g and 2.0. Mu.g TRAC3 mNaeon dsDNA and 40pmol RNP. RNP formulations involved SpyFi Cas9 and TRACsg3 at a ratio of 2:1 and incubated for 15 minutes at room temperature.

The addition sequence is as follows: 1) 2. Mu.L of template; 2) 2. Mu.L RNP; 3) 1.6 μL of 10mg/mL stock PGA; and 4) 20. Mu.L of cells. The time to pipette each component into all tubes is about 7-10 minutes. After electroporation, the samples were allowed to stand at room temperature for 5 minutes, then 20. Mu.L of ordinary X-VIVO was added, incubated at 37℃for 15 minutes without pipetting, and transferred to a 24-well plate containing 1mL of X-VIVO medium with cytokines. The staining conditions 4 days after electroporation included TCRa/b-APC (BioLegend clone IP 26) and PI. The results are shown in fig. 51A to 54.

Example 14. Time of transfection and RNP titration.

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. The activation conditions included X-VIVO medium, 1:100TRANSACTTM, 10ng/mL IL-2, 25ng/mL IL-7, and 50ng/mL IL 15. Cells were electroporated 36, 48 or 72 hours after activation. Use of approximately 1x 10 in P3 buffer under EH115 pulses ⁶ Individual cells. For RNP production, appropriate volumes of sgRNA and Cas9 were mixed and incubated for 10 minutes at room temperature. The Cas9 used is IDT HiFi Cas9. The order of addition of the reagents was as follows: 1) A donor template; 2) RNP; 3) PGA; and 4) cells. 80. Mu.L of pre-warmed X-VIVO medium without cytokines was added directly to the cuvette, without pipetting the cells, and incubated for 15 min at 37℃before transfer to larger plates.

Table 3. Samples of the experimental design used to test how the amount of RNP and PGA affect knock-in efficiency and cell recovery.

3:1 sgRNA and Cas9

1.80 pmol RNP only

2.60 pmol RNP only

3.40 pmol RNP only

4.20 pmol RNP only

5.80 pmol RNP+1ug donor template

6.60 pmol RNP+1ug donor template

7.40 pmol RNP+1ug donor template

8.20 pmol RNP+1ug donor template

PGA 80pmol RNP only

PGA 60pmol RNP only

PGA 40pmol RNP only

PGA 20pmol RNP only

13.PGA 80pmol RNP+1ug donor template

14.PGA 60pmol RNP+1ug donor template

15.PGA 40pmol RNP+1ug donor template

16.PGA 20pmol RNP+1ug donor template

1:1 sgRNA and Cas9

1.60 pmol RNP only

PGA 60pmol RNP only

3.60 pmol RNP+1ug donor template

4.PGA 60pmol RNP+1ug donor template

The results are shown in fig. 55 to 62B.

Example 15. Sequence of addition of reagents during transfection.

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. Experimental setup of the test sequence was added. The plating included X-VIVO with gentamicin, 10ng/mL IL-2, 25ng/mL IL-7, 50ng/mL IL-15, and TRANSACTTM 1:100. Electroporation was performed 48-52 hours after activation using P3 buffer and EH115 pulses. The template used was 0.5 μg of TRAC3 mNaeon dsDNA purified by SEC. The RNP formulation and order of addition are shown in fig. 63A. After electroporation, the samples were allowed to stand at room temperature for 5 minutes, then 20. Mu.L of ordinary X-VIVO without gentamicin was added, incubated at 37℃for 15 minutes without pipetting, and transferred to 24-well plates containing 1mL of X-VIVO medium with cytokines. The staining conditions on day 4 after electroporation included TCRa/b-APC (BioLegend clone IP 26) and PI. The results are shown in fig. 63B to 63D.

Example 16.DsPCR with plasmid templates.

The plating was performed using X-VIVO with gentamicin, 10ng/mL IL-2, 25ng/mL IL-7, 50ng/mL IL-15 and TRANSACTTM 1:100. Electroporation was performed 48-52 hours after activation using P3 buffer and EH115 pulses. The templates used were SEC purified TRAC3 mNaeon dsDNA or GenScript pUC57 TRAC3 mNaeon plasmid. RNP formulation was performed using SpyFi Cas9 plus TRACsg3 at a ratio of 2:1, followed by 15 minutes incubation at room temperature. The addition sequence is as follows: 1) 2. Mu.L of template; 2) 2. Mu.L RNP; 3) 1.6 μL of 10mg/mL stock PGA; and 4) 20. Mu.L of cells. After electroporation, the samples were allowed to stand at room temperature for 5 minutes, then 20. Mu.L of ordinary X-VIVO without gentamicin was added, incubated at 37℃for 15 minutes without pipetting, and transferred to a 24-well plate containing 1mL of X-VIVO medium with cytokines. The staining conditions 4 days after electroporation included TCRa/b-APC (BioLegend clone IP 26) and PI. The results are shown in fig. 64A to 64D.

Example 17 cell density and linearity with plasmid donor templates.

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. The parameters tested were: 1) Cell density of electroporation; and 2) PCR products with a plasmid as a donor template. The culture conditions included: X-VIVO medium without gentamicin or phenol red, supplemented with 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine and 1:100TRANSACTTM with 25ng/mL IL-7 and 50ng/mL IL-15 (no IL-2 added). Electroporation was performed 48 hours after activation using P3 buffer and EH115 pulse code using TRAC3-Neon template. The reagent addition sequence is as follows: 1) A donor template; 2) 60pmol RNP (sgRNA: spyFi Cas9 ratio of 3:1pmol, 15 min incubation at room temperature); and 3) 2. Mu.L of PGA (100 mg/mL). After electroporation, 80 μl of pre-warmed medium without cytokines was added to the electroporation cuvette and cells were incubated for 15 min at 37 ℃ before transfer to the larger cell culture plate. The test conditions are shown in table 4 below and include for 2x10 ⁶ Plasmid comparison of cell density conditions.

Table 4. Test conditions for testing the effect of cell density on knock-in efficiency.

The results are shown in fig. 65A to 70.

Example 18 targeting different TRAC loci with plasmid donor templates.

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. The parameters tested were: 1) Different TRAC loci for knock-in, and 2) PCR products with plasmids as donor templates. The culture conditions included: X-VIVO medium without gentamicin or phenol red, supplemented with 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine and 1:100TRANSACTTM with 25ng/mL IL-7 and 50ng/mL IL-15 (no IL-2 added). Electroporation was performed 48 hours after activation using P3 buffer and EH115 pulse code using TRAC3-mNeon template. The reagent addition sequence is as follows: 1) A donor template; 2) 60pmol RNP (sgRNA: spyFi Cas9 ratio of 3:1pmol, 15 min incubation at room temperature); and 3) 2. Mu.L of PGA (100 mg/mL). After electroporation, 80 μl of pre-warmed medium without cytokines was added to the electroporation cuvette and cells were incubated for 40 min at 37 ℃ before transfer to the larger cell culture plate. The test conditions included 1 μg of the following templates: TRAC 3L (TRAC 3 PCR product), TRAC3 plasmid, TRAC4 plasmid, TRAC5 plasmid, TRAC7 plasmid, TRAC12 plasmid and TRAC15 plasmid. The results are shown in fig. 71A to 74.

Example 19. Cotransfection with two TCR constructs.

The parameters tested were co-transfection of two different plasmid-based TCR templates. The culture conditions included: X-VIVO medium without gentamicin or phenol red, supplemented with 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine and 1:100TRANSACTTM with 25ng/mL IL-7 and 50ng/mL IL-15 (no IL-2 added). Electroporation was performed 44 hours after activation using P3 buffer and EH115 pulse code, using templates NYESO1_TRAC3 (SEQ ID NO: 2), eRT80_gp100B_8_TRAC3 (SEQ ID NO: 3), eRT76 _76_MART_3_TRAC3 (SEQ ID NO: 4), eJH52 _W1C_13_TRAC3 (SEQ ID NO: 5) and eRT76 _76_MAGEA3B_4_TRAC3 (SEQ ID NO: 6).

The reagent addition sequence is as follows: 1) 0.5 μg of each donor template; 2) 40pmol TRAC3 RNP (sgRNA: spyFi Cas9 ratio of 3:1 pmol), pre-assembled for 15 minutes at room temperature before adding 1 μL 100mg/mL PGA; 3) 40pmol TRBCg21 RNP (sgRNA: spyFi Cas9 ratio of 3:1 pmol), pre-assembled for 15 minutes at room temperature before adding 1. Mu.L of 100. Mu.g/. Mu.L PGA); 4) 100 ten thousand cells were resuspended in 20 μ L P3 buffer.

After electroporation, 80 μl of pre-heated medium without cytokines was added to the electroporation cuvette and the cells were incubated for 15 min at 37 ℃ before transfer to the larger cell culture plate. The results are shown in fig. 75.

Plasmids encoding NY-ESO1 TCRs were electroporated with several other TCRs and analyzed by flow cytometry on day 7 post-activation.

The results are shown in FIG. 75.

Example 20 cell density and electroporation conditions.

The culture conditions included: X-VIVO medium without gentamicin or phenol red supplemented with 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine and 1:100TRANSACTTM with 25ng/mL IL-7 and 50ng/mL IL-15. Electroporation was performed using TRAC3-mNEon template 48 hours after activation. The reagent addition sequence is as follows: 1) A donor template; 2) 60pmol RNP (sgRNA: aldevron SpyFi Cas ratio of 3:1pmol, 15 min incubation at room temperature) and 3) 2. Mu.L PGA (100 mg/mL). After electroporation, 80 μl of pre-warmed medium without cytokines was added to the electroporation cuvette and cells were incubated for 40 min at 37 ℃ before transfer to the larger cell culture plate. Cell densities were 2M, 5M and 10M cells. Knock-in efficiency was tested using 1. Mu.g of linear PCR product template or 2. Mu.g of plasmid template (TRAC 3-mNaeon) to normalize the number of molecules used per electroporation. Pulse optimization procedure was performed using P3 buffer and 2 μg of plasmid template TRAC3-mNeon (study grade), using 1.5M cells for each condition to allow testing of all conditions for each donor. The results are shown in fig. 76A to 84.

Example 21 knock-in at the TRAC locus, disruption of the endogenous TCR β chain.

Here, we compared the knock-in efficiency of the various TRAC templates as PCR products and tested whether the addition of TRBCsg21 RNP had any negative effect on the knock-in efficiency or staining pattern. Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. The parameters tested were titrations of four different PCR-based mNeon constructs targeting different regions of the TRAC locus, compared to TRAC RNP with TRBC RNP. The culture conditions included: X-VIVO medium without gentamicin or phenol red, supplemented with 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine and 1:100TRANSACTTM with 25ng/mL IL-7 and 50ng/mL IL-15 (no IL-2 added). Electroporation was performed 44 hours after activation using P3 buffer and EH115 pulse code. The template is the following double-stranded PCR product: TRAC3_mNEon, TRAC4_mNEon, TRAC5_mNEon and TRAC12_mNEon.

The reagent addition sequence is as follows: 1) 0.25, 0.5 or 1 μg of each donor template; 2) 60pmol TRACsg3 RNP (sgRNA: spyFi Cas9 ratio of 2.5:1 pmol), pre-assembled for 15 minutes at room temperature before adding 1. Mu.L 100mg/ml PGA; 3) Only 1 μg template set was replicated: 60pmol TRBCsg21 RNP (sgRNA: spyFi Cas9 ratio of 2.5:1 pmol), pre-assembled for 15 minutes at room temperature before adding 1. Mu.L of 100. Mu.g/uL PGA; 4) Cells were resuspended in 20 μ L P3 buffer. A duplicate set of template conditions was created for 1 μg, and only the set received both TRACsg3 and TRBCsg21 RNP. All other samples received only TRACsg3 RNP.

After electroporation, the cells were allowed to stand in an incubator at 37℃for 10 minutes. Then, 80 μl of pre-warmed medium without cytokines was added to the electroporation cuvette and the cells were incubated in a 37 ℃ incubator for an additional 10 minutes before transfer to a larger cell culture plate.

The results are shown in fig. 85A to 86B.

Example 22 Poly (L-glutamic acid) and Poly (D-glutamic acid) molecular weight variants.

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. We used a 2.5:1grna:cas9 ratio, and we titrated 15-50kDa PGA to better define its optimal range. The plating was performed using X-VIVO without gentamicin, with 25ng/mL IL-7, 50ng/mL IL-15, b-Me, L-Cys and 1:100 TRANSACTTM. Electroporation was performed in P3 buffer, EH115, 42 hours after activation. The template used was 0.75 μg TRAC3 mNaeon plasmid (GenScript Industrial grade, endotoxin-free maxiprep). The RNP used was 60pmol with a sgRNA3:Cas9 ratio of 2.5:1. After 1 hour cells were added to the master mix and then electroporated. The reagent addition sequence is as follows: 1) RNP pre-incubation for 15 min; 2) A template; 3) PGA; and 4) 150 ten thousand cells per cuvette. After electroporation, 80. Mu.L of ordinary X-VIVO was added without pipetting, then incubated at 37℃for 1 hour, and transferred to a 48-well plate containing 1mL of X-VIVO medium with cytokines. The staining conditions on days 6 and 8 after activation included TCRa/b-APC (BioLegend clone IP 26), 5. Mu.L stain and PI 1:200 for each stain.

The results are shown in fig. 89 to 90.

The structure of poly (L-glutamic acid) is shown below.

Other uses of PGA and its derivatives include: thermoplastics, fibers, films and film compositions; a cryoprotectant; superabsorbent polymers when crosslinked. PGA also has affinity for binding a variety of metal ions. Table 5 below lists the poly (L-glutamic acid) variants used in this study.

Table 5.Pga variants.

Variant number	Variant names	Variant size	Lot number
				1	PLE20	3000	000-E020-104
2	PLE50	7500	000-E050-105
				3	PLE100	15000
4	PLE200	30000	000-E200-106
				5	PLE300	45000	0000-E300-102
6	PLE400	60000	000-E400-107
				7	PLE800	120000	000-E800-105
8	PDE20	3000	000-DE020-102
				9	PDE100	15000	000-DE100-103
10	PDE400	60000	000-DE400-102

Example 23 tcr knockin.

The culture conditions included: X-VIVO medium without gentamicin or phenol red supplemented with 50. Mu.M 2-mercaptoethanol and 10. Mu. M N-acetyl-L-cysteine and 1:100TRANSACTTM with 25ng/mL IL-7 and 50ng/mL IL-15. Electroporation was performed using P3 buffer 48 hours after activation. The reagent addition sequence is as follows: 1) TRAC3-Neon or TRAC3-TCRs donor templates; 2) 60pmol RNP (sgRNA: aldevron SpyFi Cas ratio 3:1 pmol), 15 minutes incubation at room temperature); and 3) 2. Mu.L of PGA 100mg/mL. After electroporation, 80 μl of pre-warmed medium without cytokines was added to the electroporation cuvette and cells were incubated for 40 min at 37 ℃ before transfer to the larger cell culture plate. The results are shown in fig. 92A to 98.

Example 24 electroporation conditions.

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. The plating was performed using X-VIVO medium without gentamicin, 25ng/mL IL-7, 50ng/mL IL-15, b-Me, L-Cys and 1:100 TRANSACTTM. Electroporation was performed in P3 buffer at 44 hours after activation using 16 different pulse codes and two different machines. The template used was 0.75 μg TRAC3 mNaeon plasmid (GenScript, technical grade, endotoxin-free maxiprep). The RNP used was 60pmol with a sgRNA3:Cas9 ratio of 2.5:1. The reagent addition sequence is as follows: 1) RNP pre-incubation for 15 min; 2) A template; 3) PGA; and 4) 150 ten thousand cells per cuvette. A sufficient master mix was prepared to cover all reactions. Samples of the mixture of 4 reactions were pipetted into each tube of PCR 8 bands per donor (n=3, or one band per donor). Donor cells sufficient for 4 reactions were added to each tube. The cell mixture was pipetted from the PCR strip multichannel directly to two cuvettes for a total of 32 wells. Paired cuvettes were electroporated simultaneously on two different machines. After electroporation, 80. Mu.L of ordinary X-VIVO was added without pipetting, then incubated at 37℃for 1 hour, and transferred to a 48-well plate containing 1mL of X-VIVO medium with cytokines. The staining conditions on days 6 and 8 after activation included TCRa/b-APC (BioLegend clone IP 26), 5. Mu.L stain and PI 1200 per stain. Donor information is provided in table 6 below.

Table 6 donor information.

The results are shown in fig. 103 to 105B. Tables 7 and 8 below show the percent knockins versus the number of mNaeon+ cells on days 6 and 8.

Table 7. Comparison of percent knockins on day 6 with mNaeon+ cell number.

Table 8. Comparison of percent knockins on day 8 with mNaeon+ cell number.

Example 25 ny-ESO TCR knockins, disrupting endogenous TCR β chains.

The plating was performed using X-VIVO without gentamicin, 25ng/mL IL-7, 50ng/mL IL-15, b-Me, L-Cys and 1:100 TRANSACTTM. Electroporation was performed in P3 buffer, code EH115, 48 hours after activation. The template used was 0.75 μg TRAC3 1G4 (NY-ESO 1) plasmid (GenScript, technical grade, endotoxin free maxiprep) and RNP used was variable with a sgRNA3:Cas9 ratio of 2.5:1. The reagent addition sequence is as follows: 1) 0.75 μg template; 2) 60pmol NT or TRAC RNP (15 min pre-incubation); 3) 1 μL of PGA; 4) 15, 30 or 60pmol TRBC RNP (pre-incubated for 15 minutes to prepare RNP, respectively) and 5) 200 ten thousand cells per cuvette. After electroporation, 80. Mu.L of ordinary X-VIVO was added without pipetting, then incubated at 37℃for 20 min, and transferred to a 48-well plate containing 1mL of X-VIVO medium with cytokines. Dyeing conditions 8 days after activation included TCRa/b-BV421, 5. Mu.L/stain, each stain PI 1:200. The donor information is listed in table 9 below.

TABLE 9 Donor information

Example 26. Different homology arm lengths.

The plating was performed using X-VIVO without gentamicin, 25ng/mL IL-7, 50ng/mL IL-15, b-Me, L-Cys and 1:100 TRANSACTTM. Electroporation was performed in P3 buffer, code EH115, 43 hours after activation. The templates used were different numbers of double-stranded PCR products, which were used to obtain the same number of DNA molecules in the sample (3.34x10 ¹¹ ). The RNP used was 60pmol with a sgRNA3:Cas9 ratio of 2.5:1. The reagent addition sequence is as follows: 1) RNP (pre-incubation 15 min); 2) A template; 3) 0.5 μl of PGA per sample; and 4) one million cells per cuvette. After electroporation, 80. Mu.L of ordinary X-VIVO was added without pipetting, then incubated at 37℃for 20 min, and transferred to a 48-well plate containing 1mL of X-VIVO medium with cytokines. Dyeing conditions 7 days after activation include TCRa/b-BV421, 5. MuL/stain, each stain PI 1:200. The results are shown in fig. 104A to 105H.

Example 27. Titration of TRBC targeting guide RNAs and different donor DNA forms.

Donor DNA encoding the first 2A peptide, the mNeon fluorescent protein, the second 2A peptide, and the TCRa VJ domain was used. T cells were activated in Prime medium with TRANSACTTM (1:100) +25ng/mL IL-7 and 50ng/mL IL-15. TRAC3 and TRBC22 RNPs were generated by mixing TRAC3 or TRBC22 sgRNA and Cas9 at a 3:1 sgRNA to Cas9 molar ratio and incubating for 15 minutes at room temperature. 2. Mu.L of 100mg/mL PGA was used for each electroporation reaction. Master mixes of PGA, TRAC3 RNP, TRBC22 RNP and donor templates were generated for each reaction. Different amounts of TRAC3 RNP (60 or 30 pmol) and/or TRBC22 RNP (60, 30, 20 or 10 pmol) were used to determine the impact of TRBC22 RNP on the knockdown efficiency. An appropriate volume of master mix (varying according to pmol of TRAC3 and/or TRBC22 RNP used) was added per well, followed by 2X106 cells per reaction.

Electroporation conditions were all performed in Lonza P3 buffer under code EW113 at 48 hours post-activation. For continuous electroporation: (1) First, TRAC3 RNP and donor template (PUC 57 vector) were electroporated (48 hours after T cell activation) using the code EW 113; and (2) electroporating the TRBC22 RNP for 24 hours using the code EW113 or EO100 24 hours after the first electroporation. The RNP conditions used were as follows: TRAC3 RNP 60 alone or 30pmol, TRAC3 RNP/TRBC22 RNP used in combination: 60pmol/30pmol, 60pmol/20pmol, 60pmol/10pmol, 30pmol/30pmol or 30pmol/10pmol. After each electroporation, cells were incubated at 37 degrees celsius for 15 minutes (in P3 buffer only) activation.

The results are shown in fig. 111 to 123. Fig. 111A shows the percentage of Neon positive TRAC +/-TRBC samples on day 8 (left to right): 60pmol TRAC3 alone, 30pmol TRAC3 plus 30pmol TRBC21, and 30pmol TRAC3 plus 30pmol TRBC22. Fig. 111B shows the percentage of Neon positive samples on day 8 (left to right): 60pmol TRAC3, 60pmol TRAC3 plus 30pmol TRBC22, 60pmol TRAC3 plus 20pmol TRBC22, 30pmol TRAC3 plus 30pmol TRBC22, and 30pmol TRAC3 plus 10pmol TRBC22. Fig. 112 shows the percentage (fig. 108A) and number (fig. 108B) of Neon positive samples (left to right): 60pmol TRAC3-only RNP, 30pmol TRAC3-only RNP, 60pmol TRAC3-TRBC 22-RNP mixture, 60pmol/20pmol TRAC3-TRBC 22-RNP mixture, 60pmol/10pmol TRAC3-TRBC 22-RNP mixture, 30pmol/30pmol TRAC3-TRBC 22-RNP mixture, and 30pmol/10pmol TRAC3-TRBC 22-RNP mixture. The continuous electroporation test was performed by: the TRAC3 RNP and donor template (PUC 57 vector) were first electroporated using pulse code EW113, and then the TRBC22 RNP was electroporated 24 hours later using pulse code EW113 or EO 100. The data are shown in fig. 109A to 109C. Fig. 109A shows the percentage (or frequency) of Neon positive samples. Figure 109B shows the number of Neon positive samples. FIG. 109C shows the frequency of TCRab+Neon negative cells. Following continuous electroporation, cells were cultured in 24 well (24 w) or 48 well (48 w) plates.

Example 28 different donor DNA forms.

T cells were activated using Prime medium with TRANSACTTM (1:100) +25ng/mL IL-7 and 50ng/mL IL-15. TRAC3 and TRBC22 RNPs were generated by mixing TRAC3 sgRNA (IDT GMP grade) and Cas9 in a 3:1sgRNA:Cas9 molar ratio and incubating for 15 minutes at room temperature. 2. Mu.L of 100mg/mL PGA was used for each electroporation reaction. A master mix of PGA, TRAC3 RNP and donor template was generated for each reaction. The addition sequence is as follows: 1) RNP (pre-incubation for 15 min at room temperature); 2) A template; 3) PGA (1. Mu.L of 100. Mu.g/. Mu.L stock); 4) Cells (150 ten thousand per cuvette). Testing different template formats: PUC57 plasmids, nanoplasmms and microrings. The template amounts were normalized to yield a similar number of molecules for comparison. Electroporation conditions were all performed in Lonza P3 buffer under code EW113 at 48 hours post-activation. Following electroporation, the samples were left at 37℃for 15 min (either in P3 buffer alone or with the addition of 75. Mu.L of ordinary pre-warmed Prime-XV without pipetting), and after this incubation, the cells were transferred to 24 or 48 well plates containing Prime-XV medium with cytokines.

Fig. 110C and 11D show the frequency (fig. 110C) and number (fig. 110D) of Neon positive cells. Experiments were performed with TRAC3 RNP alone. Templates used were PUC57 vector (GS), nanoplasmon (nano) and microring (mini).

Data for pUC57 and nanoplasmids and micro-circular DNA on day 6 are shown in FIGS. 114A-114B. Fig. 114A shows the percentage (upper panel) and absolute number (lower panel) of the mneon+tcr+ samples. FIG. 114B shows the amount of DNA used in the experiment. Data for pUC57 and nanoplasmids and micro-circular DNA on day 8 are shown in FIGS. 115A-115B. Fig. 115A shows the percentage (upper panel) and absolute number (lower panel) of the mneon+tcr+ samples. FIG. 115B shows the amount of DNA used in the experiment. Fig. 116A shows the percent cell viability on day 6, while fig. 116B shows the percent cell viability on day 8. The variation of the number of knocked-in cells over time from day 6 to day 8 is shown in fig. 117.

Example 29 pUC57 plasmid, micro-loop (Mini), nano-plasmid (Nano) and PCR template.

Data for pUC57 and nanoplasmids and micro-circular DNA on day 6 are shown in FIGS. 120A-120B. Figure 120A shows the percentage (upper panel) and absolute number (lower panel) of mneon+tcr+ samples on day 6. FIG. 120B shows the amount of DNA used in this experiment. Data for pUC57 and nanoplasmids and micro-circular DNA on day 8 are shown in FIGS. 121A-121B. Figure 125A shows the percentage (upper panel) and absolute number (lower panel) of the mneon+tcr+ samples on day 8. Fig. 121B shows the amount of DNA used in this experiment. Day 8 sample graphs of pUC57, nanoplasmon, microring and PCR samples are shown in FIG. 122. Cell viability is shown in figure 123 with equivalent molecules for each template, with data on day 6 shown in the top panel and data on day 8 shown in the bottom panel. Fig. 124 shows the number of knocked-in cells over time from day 6 to day 8.

Example 30 targeting TCR- β of guide RNAs, plasmid and nanoplasmlet templates.

T cell activation conditions: prime, TRANSACTTM (1:100) +25ng/mL IL-7 and 50ng/mL IL-15. RNP was generated by mixing sgRNA and Cas9 at a ratio of 3:1sgrna:cas9 and incubating for 15 minutes at room temperature. TRAC3, TRBC22 and TRBC19 sgRNA were resuspended at 200. Mu.M; a master mix for electroporation was generated by mixing: (1) 2. Mu.L of 100mg/mL PGA per reaction; (2) TRBC22 or TRBC19 RNP; (3) TRAC3 RNP; and (4) a donor template. The exact amount of reagent varies based on pmol of RNP and the amount of donor template used per electroporation condition. The following RNP conditions were used: TRAC3 60 or 30pmol only; TRAC RNP/TRBC RNP 60pmol/30pmol, 60pmol/10pmol, 30pmol/30pmol (TRBC 22 or TRBC19 RNP). For titration of donor templates, the following amounts of PUC57 (GenScript) and nanoplasmid templates were used: PUC57 (GenScript): 8. 6, 4 and 2 μg; nano-plasmids: 10. 8, 6, 4 and 2 μg. The same number of nanoplasmic molecules was used for electroporation using the amount of PUC57 (GenScript) μg as reference. Cells were electroporated in Lonza P3 buffer 48 hours after activation under code EW 113. Following electroporation, cells were incubated at 37 ℃ for 15 minutes (in P3 buffer only) before transfer to plates containing pre-warmed PRIME medium and cytokines.

Fig. 125 (left) shows the percentage of Neon positive samples, and (right) shows the number of Neon positive samples (left to right): 60pmol TRAC3 RNP and PUC57 only template (GenScript), 60pmol TRBC22RNP and 30pmol TRBC19 RNP and PUC57 template (GenScript), 60pmol TRBC22RNP and 10pmol TRBC19 RNP and PUC57 template (GenScript), 30pmol TRAC3 RNP and PUC57 template (GenScript), 30pmol TRBC22RNP and 30pmol TRBC19 RNP and PUC57 template (GenScript), 30pmol TRAC3 RNP and nanoplasmid, and 30pmol TRAC3 RNP and 30pmol TRBC22RNP and 30pmol TRBC19 RNP and nanoplasmid.

Fig. 126A shows day 5 knock-in efficiency (left panel) and day 7 knock-in efficiency (right panel) for GenScript and nanoplasmon templates. Samples were electroporated with 30pmol TRAC3 RNP and 30pmol TRBC22RNP. Figure 126B shows the number of knocked-in cells on days 5 and 7.

Day 7 flow cytometry data from TRBC RNP titration (60 pmol) are shown in FIG. 127. Day 7 flow cytometry data from TRBC RNP titration (30 pmol) are shown in FIG. 128. Day 7 flow cytometry data of PUC57 (GenScript) plasmid titration is shown in fig. 129. Flow cytometry data at day 7 of nanoplasmon titration are shown in figure 130.

Example 31 TCR knockin with pUC57 and nanoplasmids.

T cell activation conditions: prime, TRANSACTTM (1:100) +25ng/mL IL-7 and 50ng/mL IL-15.RNP+/-PGA preparation includes: incubation at room temperature for 15 min was used to generate RNPs (3:1 sgRNAs: cas 9), where all sgRNAs were 200. Mu.M, and TRAC3 RNPs and TRBC22 RNPs were 30pmol each; and 2. Mu.L of 100mg/mL PGA. The templates used were PUC57 (Neon, WT1 TCR) or nanoplasmon (Neon). We used as reference an equal number of nanoplasmon molecules to 4 μg PUC 57. Following electroporation, incubation was carried out for 15 min at 37℃in P3 buffer only. Electroporation conditions (both in Lonza P3 buffer under code EW113 48 hours after activation) included: PGA requirement test (+/-PGA was used for templates of Neon PUC57, neon nanoplasmids and Wt1_5213PUC 57); template addition sequence test (first adding template with RNP/PGA mixture or last adding template); and initial (non-codon optimized) and codon optimized WT1 TCR (Wt1_Ref and Wt1_64-9) testing.

The results of the Neon knock-in are shown in fig. 131A to 131B. 4 μg of the PUC57 plasmid or an equal number of nanoplasmon molecules were used. Fig. 131A shows the percentage of Neon-positive samples on day 7, while fig. 131B shows the number of Neon-positive samples.

Figure 132A shows the percentage of WT1+ (in cd8+) samples on day 7. Figure 132B shows the percentage of cd3+ samples in cd8+ cells on day 7. Figure 132C shows the percentage of WT1+ in total cd3+cd8+ cells on day 7. For the "no PGA" condition, the template is added last. 4 μg of PUC57 plasmid was used.

FIG. 133 shows the number of WT1 TCR+ cells on day 7. The conditions illustrated are from left to right: first add template, last add template, and no PGA. For each condition, data of wt1_5213, wt1_ref_native, wt1_ref_codopt, wt1_64_9_native, and wt1_64_9_codopt are given from left to right.

Fig. 134 shows day 7 flow cytometry data from Neon template knockins. Fig. 135 shows day 7 flow cytometry data of wt1_5213. FIG. 136 shows day 7 flow cytometry data for WT1_Ref (all with PGA). FIG. 137 shows day 7 flow cytometry data (all with PGA) for WT1 64_9.

TABLE 10 TRAC sgRNA

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TABLE 11 TRBC sgRNA

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TABLE 12 neoantigens

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Example 32 functional characterization of tcr-engineered T cells.

The potency of TCR T cells is determined by measuring the ability of engineered T cells to induce lysis of specific target cells (as measured by% loss of specificity of target cells) using FACS. On the day of assay, target cell peptide pulses (3-4 hr) and bystander cell CellTrace staining were set in the morning. Killing assays were performed by incubating T cells with previously prepared target/bystander cells. Cell killing was measured after about 18hr using FACS. The assay diluent is T cell growth medium (X-VIVO or PRIME-XV plus IL-7 and IL-15).

And (3) preparing a reagent. Preparation of peptide stock solution: peptides were diluted to 10mM in DMSO. The CellTraceTM stock solution is prepared according to the manufacturer's protocol, i.e., the CellTraceTM solution is reconstituted by adding an appropriate volume (e.g., 20 μl) of DMSO (component B) to a vial of CellTraceTM reagent (component a) and mixing. Further dilutions were performed in DMSO and stored at < 20℃after reconstitution. LIVE/DEADTM stock solutions were prepared according to the manufacturer's protocol, i.e., a suitable volume (e.g., 50 μl) of DMSO (component B) was added to a bottle of LIVE/DEADTM reagent (component a) and mixed to reconstitute the LIVE/DEADTM stock solution immediately prior to use.

And (5) measuring the program. Peptide dilutions were prepared in target cell growth medium in deep well 96-well plates as shown in table 13.

Table 13.

Preparation of cells for assay: all steps are performed in a biosafety cabinet. Preparation of target cells: 0.05M target cells per well; 1) Cell viability and cell concentration were determined. Cells were centrifuged at 300x g for 5 min in a conical tube at ambient temperature and the supernatant discarded. The cell pellets were resuspended in target cell growth medium to a total volume of cell seeding suspension calculated for the assay, and 50 μl of the suspension was seeded into each well in a round bottom 96 well plate, covered with appropriate peptide dilutions, gently mixed by pipetting up and down, and incubated at 37 ℃ for 3-4hr.

Preparation of bystander cells: this format uses 0.05M bystander cells per well; cellTraceTM far red was used at a final concentration of 0.1 nM. The cells were centrifuged at 300x g for 5 min at ambient temperature andin 15 or 50mL conical tube at 1X10 ⁶ the/mL density was resuspended in PBS. CellTrace (1 μl of 100nM DMSO working solution) was added to each ml of PBS cell suspension. The cells were mixed and incubated at room temperature for 20 minutes in the dark. Five times the original staining volume of medium (containing at least 1% protein) was added to the cells and incubated for 5 minutes. This step removes any free dye remaining in the solution. Cells were pelleted by centrifugation (300 Xg at ambient temperature for 5 min) and dried at 1X10 ⁶ the/mL density was resuspended in fresh pre-warmed complete medium. Cells were incubated for at least 10 minutes prior to analysis to allow the CellTraceTM reagent to undergo acetate hydrolysis.

Washing the target cells: after 3-4 hours incubation, cells were washed 2 times with PBS to remove free peptides. Cells were resuspended in 50 μl of assay diluent and prepared to be mixed with bystander T cells and effector T cells.

Bystander cells were transferred to plates: bystander cell suspensions were resuspended in assay diluent (at 1x10 by centrifugation at 300x x g for 5 minutes at ambient temperature ⁶ density/mL); adding 50 μl to one or more plates containing 50 μl of peptide-loaded target cells; and mixed by pipetting up and down about 2 times.

Preparation of effector cells: KI TCR T cells recognizing specific peptides are provided after transfection. KI TCR T cells and untransfected (or mock) control cells were counted and the required cell suspension volumes were calculated (0.1x10 per reaction for 1:1T: e ratio ⁶ Individual T cells). Cells were centrifuged at 300x x g for 5 min at ambient temperature and resuspended in assay diluent (at 1x10 ⁶ density/mL)

Transfer of effector cells to plates: 100 μ L T cell suspension was added to the plate containing peptide-loaded target cells and bystander cells. One or more plates were incubated overnight at 37 ℃ in a humidified incubator.

Measurement reading: plates were centrifuged at 300x g for 5 min and 180 μl of supernatant was transferred to another 96 well plate and stored at-80 ℃ for later analysis. LIVE/DEAD reconstitution dye was diluted into FACS buffer at a ratio of 1:1000 (e.g., 5 μl LIVE/DEAD reconstitution dye +5mL FACS buffer), 100 μl LIVE/DEAD working solution was added to each well and pipetted up and down 2-3 times to mix gently. Plates were incubated at 4℃for 10 min in the dark. The FcR blocking solution was diluted into FACS buffer at a ratio of 1:20. To wash out the live/dead dye, 100 μ LFACS buffer was added to each well and the plate was centrifuged at 300x g for 5 minutes at room temperature. The supernatant was decanted and 50 μl FcR blocking solution was added to each well and mixed. Plates were incubated in the dark at 4℃for 10 min

A CD8 and CD137 antibody mixed solution was prepared by: CD8, CD137 and FACS buffer were mixed to prepare 2x stained antibody mixtures, e.g., for 80 wells: 4000. Mu.LFACS buffer+40. Mu.L CD8+400. Mu.L CD137. At the end of the FcR blocking incubation, 50 μl of antibody mixture was added to each well and mixed by pipetting. Plates were incubated in the dark at 4 ℃ for 30 min, then cells were washed twice to remove free antibodies. mu.L of FACS buffer was added to each well and the supernatant was decanted off the plate at 300x g at room temperature for 5 minutes. 200 μ LFACS buffer was added to each well and the plate was centrifuged at 300x g for 5 minutes at room temperature, and then the supernatant was decanted. mu.L of FACS buffer was added to each well and mixed by pipetting up and down about 2-3 times.

Samples were analyzed immediately using BD FACSLysic (gating strategy: lymphocytes > single cells > living cells > non-T (CD 8-) and T cells (CD8+): non-T cells: farred+/Farred-; T cells: CD 137). FlowJo was used for FACS gating, analysis.

Flow cytometry-based cell killing assays were used to determine the percent cell killing of T2 target cells when incubated with neo-TCR T cells. T2 cells (from ATCC, CRL-1992) were preloaded with target peptide WT1 (VLDFAPPGA, SEQ ID NO: 71) (0-100. Mu.M) by incubating the cells in the presence of the peptide for 3-4 hours at 37℃and washed twice with PBS before exposure to WT1 TCR-T cells in a cell killing assay. A target cell population pulsed with no peptide or irrelevant peptide was used as a negative control. The target cells were then mixed with neo-TCR T cells at a ratio of 1:1 for 20hrs to allow T cell-induced cell killing to occur.

To measure the percent cell killing, the cell mixtures from each assay well were stained with antibodies to label CD8+ (Anti-CD 8, biolegend: 406515) and CD137+ (Anti-CD 137, biolegend: 309822) cells and with reactive dyes (Fixable Aqua stain, thermosusher: L34965) to label dead cells. After selection of living cells, the cells are further divided into CD8+ T cells and CD 8-target cells. The% loss of target cells per response peptide pulse was calculated based on the negative control. Also, peptide-specific T cell activation during cell killing assays was assessed by determining the% cd137+ signal in the cd8+ T cell population.

FIG. 138 shows a cartoon representation of target cell killing and graph of percent killing against neo-TCR and TCR-free relative to control.

Example 33 electroporation pulse code.

Table 14 below shows a summary of the percent knockins for three donors for various buffers/codes.

Table 14. Summary of neot pulse codes.

EXAMPLE 34 knockout of TCR-alpha or TCR-beta

Purified (unactivated) cd8+ T cells were electroporated using Lonza P2 buffer and pulse code EH 100. RNP was generated by incubating sgRNA and Cas9 (ratio 3:1) for 15 minutes at room temperature. 60pmol RNP was added per electroporation. Following electroporation, cells were cultured in X-VIVO medium supplemented with IL-7 and IL-15. Two days after electroporation, cells were collected and analyzed for TCR surface expression by flow cytometry.

Example 35 efficient non-viral CRISPR/Cas9 mediated Gene editing Using plasmid donor DNA on human T cells

CRISPR-mediated gene knockout using Cas 9-Ribonucleoprotein (RNP) delivery to primary human T cells represents a rapid and versatile method of introducing loss-of-genetic perturbation in this clinically relevant cell type (Schumann et al, 2015; hendel et al, 2015; seki and Rutz,2018; oh et al, 2019). However, approaches to functional gain research and stable expression of therapeutic transgenes in T cells rely primarily on viral delivery techniques that do not allow for precise editing of genes.

Lentiviruses or retroviruses are widely used by the research community and are also applied to the introduction of Chimeric Antigen Receptors (CARs) or T Cell Receptors (TCRs) in the manufacture of adoptive T cell therapies (Wang and Rivi re,2016; zhang et al, 2017). Transposon-based gene delivery methods (e.g., piggyBac and sleep Beauty systems) have been developed as non-viral alternatives (Monjezi et al, 2017; kebriaei et al, 2016; hudecek and Ivics, 2018). While these methods allow efficient and stable gene delivery, they insert transgenes into the genome by random integration and are not suitable for precise gene editing. Furthermore, the randomness of the integration process is at risk of insertional mutagenesis (Hacein-Bey-Abina et al, 2008, 2003; modlich et al, 2009).

Homologous Directed Repair (HDR) of double strand breaks introduced by targeted gene editing methods such as transcription activator-like effector nucleases (TALENs), zinc Finger Nucleases (ZFNs) or CRISPR/Cas9 can be used to make intentional, specific alterations to genomic sequences, including the insertion of longer DNA fragments at defined locations in the T cell genome (Li et al 2020; singh et al 2017). Viral vectors, particularly adeno-associated viruses (AAV), have been used to deliver donor DNA templates for HDR-mediated target gene knock-in T cells (Sather et al, 2015; wang et al, 2016; eyquem et al, 2017; choi et al, 2019). This approach facilitates CAR integration into the T cell receptor alpha constant (TRAC) region locus (eyqm et al, 2017), which places the CAR under transcriptional control of the endogenous TCR promoter, resulting in improved CAR performance (eyqm et al, 2017). Multiple groups have subsequently reported high editing efficiency using AAV-based repair templates (Choi et al, 2019; vakulllskas et al, 2018; dai et al, 2019). However, AAV production and purification not only represents a significant clinical manufacturing challenge (Loo and Wright,2016; halbert et al, 2018; davidsson et al, 2020), but it also limits the wider use of this approach in the research community.

Recently, a series of papers have demonstrated that a linear double stranded (ds) DNA donor template can be co-delivered with Cas9-RNP for targeted insertion of full length coding sequences within the T cell genome (Nguyen et al, 2019; roth et al, 2018; schober et al, 2019), thus facilitating not only the generation of point mutants, but also targeted integration of one or more expression constructs (including CARs or TCRs). In particular, in addition to the modest knock-in efficiency observed with this donor DNA format, there is also a need to produce and purify linear dsDNA of sufficient quantity and quality, which severely limits the practicality and scalability of the method. Here, we address these challenges by developing an efficient and scalable solution for CRISPR/Cas 9-mediated non-viral gene editing in primary human T cells using an off-the-shelf plasmid-based donor template.

Plasmid-based homologous donors improve CRISPR-mediated gene knock-in efficiency and cell recovery compared to linear DNA templates.

Based on previous work, including protocols for CRISPR-Cas9 mediated gene perturbation in human and mouse T cells (Seki and Rutz,2018; oh et al, 2019) and reports describing the use of linear double stranded DNA as repair templates (Roth et al, 2018), we have set out to develop robust, efficient and scalable protocols for non-viral CRISPR/Cas9 mediated gene knock-in primary human T cells. To avoid the labor-intensive steps involved in the generation and purification of PCR-based linear dsDNA and to facilitate engineering using sequence-verified templates, we studied the use of plasmid DNA.

In addition to the conventional plasmid backbone of-2.5 kb in size (i.e., pUC 57), several smaller circular DNA backbones have been described for cell engineering applications, including micro-loops, midges and nanoplasmids (Hardee et al, 2017). The commercial nanoplaston consists of a <0.5kb backbone (Luke et al 2009; williams et al 2006). Because double-stranded DNA is toxic to T cells, the use of these minimal vectors maximizes the ratio of donor element to plasmid backbone sequences and reduces the total amount of DNA required for transfection. We designed the donor template to encode the alpha chain of NY-ESO1 specific T cell receptor 1G4 (Li et al 2005) and fluorescent protein mNeonGreen (mNG) targeting the T cell receptor alpha constant region (TRAC).

Here, the transgene and TRAC homology arms (. About.500 base pairs, bps) are encoded as linear dsDNA, pUC57 regular plasmids or nanoplasmids (FIGS. 1A, 1D, 1G). Similar to our previous approach to CRISPR/Cas9 knockout in T cells (Seki and Rutz, 2018), we optimized this process for CD4 and CD 8T cells, respectively, rather than using mixed cell populations. Here we isolated human CD8 in PRIME-XV medium ⁺ T cells, which are supplemented with cytokines IL-7 and IL-15, are activated with tranact (a beadless colloidal polymeric nanomatrix conjugated with humanized CD3 and CD28 agonists). CD8 is incubated prior to nuclear transfection with Cas9-RNP containing chemically synthesized single guide (sg) RNAs targeting the TRAC locus and the respective donor DNA templates (Lonza Biosciences D nuclear transfection system) ⁺ T cells for 48h. Importantly, all studies were performed using the R691A HiFi-Cas9 variant to minimize CRISPR/Cas9 off-target events (vakulskoas et al, 2018). Furthermore, we titrate a certain amount of linear DNA and plasmid DNA side by side and determine knock-in efficiency, cell viability and cell recovery by flow cytometry three days after electroporation.

Under these conditions, we found that 4 μg of linear DNA produced 28.8-32.1% of the maximum knock-in rate in four independent human T cell donors (fig. 140B, fig. 140C). However, this amount of DNA impaired cell viability and resulted in low T cell recovery (fig. 140B). When 1 μg of linear DNA was used instead, the knock-in rate was slightly lower, 13.9-20%, but cell viability and recovery was comparable to transfection with Cas9-RNP without DNA (fig. 140B, C). Titration of pUC57 plasmid DNA revealed a higher knock-in rate compared to linear dsDNA, 33.5-44% when up to 6. Mu.g plasmid was used (FIG. 140E, FIG. 140F). However, this condition resulted in suboptimal viability and cell recovery, whereas 2 μg of plasmid DNA resulted in 24-36.2% knock-in rate with minimal cell viability, resulting in optimal recovery of the edited cells (fig. 140E, fig. 140F). The nanoplasmon format was able to achieve a knock-in rate of 36.2-46.6% at 4 μg DNA with minimal impact on cell viability and recovery compared to Cas9-RNP alone conditions (fig. 140H, fig. 140I). We also assessed the efficiency of knockin, cell viability and recovery of T cells when cultured in RPMI-1640 supplemented with 10% FBS (R10), which is a more widely used and easy to use T cell medium. Experiments performed in R10 produced substantially comparable results to cultures in PRIME XV medium of linear dsDNA templates, but with lower knock-in rates of nanoplasmon templates (147A to 147F) (29.5-37.4%), indicating that R10 can be used as a surrogate for PRIME-XV medium. As previously described (Nguyen et al, 2019), the addition of negatively charged poly-L-glutamic acid (PGA) or a target sequence encoding truncated Cas9 (ttss) in the donor template did not affect targeting efficiency (147G, 147H).

Our preliminary studies indicate that nanoplasmids have advantageous qualities for gene targeting in T cells. However, regardless of form, higher amounts of donor DNA impair T cell viability. Thus, we sought to better characterize the stress response induced by T cells after exposure to nanoplasmids. We compared cytokine production after overnight culture of CD 8T cells that had been transfected with sgTRAC Cas9-RNP and nanoplasmon or Cas9-RNP alone, with non-edited T cells. Nanoplasmon transfection (but not Cas9-RNP alone) induced IFN- α, IFN-g, TNF- α and IL-2 levels (figure 148A). We also performed RNA sequencing, comparing Cas9-RNP plus nanoplasmids or Cas9-RNP transfected T cells alone. Interferon-alpha and interferon-beta and TNF-alpha and inflammatory response characteristics were up-regulated in nanoplasmid transfected cells (FIG. 148B), consistent with our cytokine data. Importantly, linear DNA and nanoplasmids induced qualitative and quantitative similar responses (fig. 148C-148H). In summary, we found that the plasmid DNA donor template was found to be in CD8 ⁺ Efficient CRISPR/Cas 9-mediated knock-in can be achieved in T cells, avoiding the need for linear dsDNA production and purification. While nanoplasmic vectors generally produce more consistent results with higher knock-in rates, conventional plasmid backbones (such as pUC 57) can be successfully used by careful titration.

Optimizing CRISPR/Cas9 mediated gene knock-in with plasmid-based donor DNA in CD4 and CD 8T cells

After confirming the beneficial properties of plasmid-based gene editing, we sought to further optimize the process and extend our protocol to enable cd4+ T cell modification. Thus, we used cd8+ T cells to compare pUC57 to a nanoplasmon donor template with a fixed transgene comprising a bicistronic mNG reporter and a 1g4 TCR a chain, and TRAC homology arms of varying lengths ranging from 0.1 to 2 kb. Transgene knock-in efficiency increased between 0.1 and 0.5kb homology arm length, slightly improved knock-in efficiency observed with nanoplasmms containing homology arms extending to 2kb, regardless of the backbone used (fig. 141A, fig. 141B). Importantly, cell viability and cell recovery were comparable to both backbones except for the 2kb homology arm pUC57 construct, which severely compromised cell viability (fig. 141A, fig. 141B). We conclude that a homology arm length of 0.5kb represents an optimal value for knock-in efficiency and cell recovery, but that longer arms can be used with nanoplasmms without compromising cell viability (fig. 141A, fig. 141B).

The time of Cas 9-RNP/nanoplasmon delivery after T cell activation is another optimization parameter. We found that transfection at 24h post T cell activation resulted in a significant decrease in knock-in efficiency compared to the later transfection time points (figure 141C). 48-72h Cas 9-RNP/nanoplasmon delivery resulted in maximum cell recovery (fig. 141C). Finally, we tested several nuclear transfection pulse codes with Cas9-RNP and nanoplasmon templates and found EH115 to result in the highest target gene editing efficiency. Other pulse codes such as EH111 further increased cell recovery and minimal reduction in knock-in efficiency occurred (fig. 141D). The desired application should drive the final choice of nuclear transfection conditions depending on whether efficiency or cell recovery has a higher priority. For subsequent studies, we used EH115.

Using the above conditions as baseline, we isolated and cultured human CD4 in PRIME XV medium supplemented with recombinant IL-7 and IL-15 ⁺ T cells. We also added IL-2 and activated with TransAct for 48h. We then performed nuclear transfection with TRAC-mNG nanoplasmon donor template and sgTRAC Cas9-RNP as before and assessed knock-in rate, cell viability and recovery by flow cytometry. We observed that the knock-in rate of three independent human T cell donors was 32.2-41.9, where the viability and recovery were comparable to that of our C D8 The data obtained for T cells were consistent (fig. 141E, fig. 141F). Our conclusion is that our method is equally applicable to human CD4 and CD 8T cells.

Efficient non-viral T cell receptor editing using plasmid DNA donors

Next, we applied our protocol to TCR editing in T cells. The introduction of a transgenic TCR with the desired antigen specificity also requires the knockout of the endogenous TCR in order to prevent mismatches with its alpha and beta chains. Using our targeting strategy, insertion of a transgenic TCR containing both alpha and beta chains into the TRAC locus on human chromosome 14 would disrupt the endogenous TCR alpha gene. However, the existing TCR β chain on human chromosome 7 requires a separate knockout. Thus, we designed a single sgTRBC sequence that simultaneously targeted T cell receptor β chain constant structures 1 and 2 (TRBC 1 and TRBC 2) (fig. 142A), resulting in complete loss of TCR expression as detected by flow cytometry (fig. 149A). Next, we designed a nanoplasmid donor template encoding TCR with known specificity, NY-ESO1 specific 1G4 and CMV A2/pp65495-503 specific TCR6-2 (Schober et al 2019) and human CD19 CAR (Bloemberg et al 2020), all targeting the TRAC locus (fig. 142B).

We activated and cultured CD8 as before ⁺ T cells were 48h and co-transfected with Cas9-RNP containing sgTRAC and sgTRBC and 2 μg TCR or CAR encoding nanoplasmid, and TCR expression was assessed five days later by flow cytometry. We detected 1g4TCR expression on the surface of 44.9-54% of T cells with minimal impact on cell viability (fig. 142C, 142D). From 2x10 ⁶ Starting with CD 8T cells we recovered 0.88-2.88x10 five days after electroporation ⁶ 1G4TCR positive cells in between (fig. 142D). Negative T cells for 1G4 expression did not express endogenous TCR complexes on their surface, demonstrating efficient gene knockout (fig. 142C). Transfection with nanoplasmms encoding pp65TCR and CD20 CAR constructs yielded similar results with knock-in rates of 44.4-53.3% and 46.3-57.9% (fig. 142E, 142G) and 1.7-3.1x10, respectively ⁶ Cell recovery of individual edited cells (FIGS. 142F, 142H). Likewise, endogenous T cell receptor knockouts were made in almost all T cells (fig. 142E, fig. 142G).

When we used the same targeting strategy and TCR or CAR donor templates for TCR editing in isolated CD 4T cells, we observed knock-out and knock-in rates comparable to our results with CD 8T cells (fig. 149B to 149G). When attempting simultaneous multiplex gene editing, such as the TRAC and TRBC knock-in/knock-out methods used herein, one must consider the occurrence of chromosomal translocations between cleavage sites. We designed a ddPCR assay to quantify all possible translocation events involving the TRAC, TRBC1 and TRBC2 loci (fig. 149H). Although translocation between TRAC and TRBC1 or TRBC2 sites occurs at a frequency of 0.01-4.4% (depending on translocation direction and donor, recombination (corresponding to deletion of 9.3 kb) adjacent to the TRBC1 and TRBC2 loci occurs at a frequency of 13.1-19% (FIG. 149I.) these numbers are consistent with or slightly higher than previously reported data (Stadtmauer et al 2020), possibly reflecting the higher editing efficiency of our approach.

Next, we wanted to determine how the gene editing process (knockout (Cas 9-RNP only condition) or the combination of knockout and knockout (Cas 9-rnp+nanoplasmid)) affected the overall expansion of T cells in culture over time. For this, we used a G-Rex culture system that allowed for high cell density and simple medium exchange without the need to split or re-plate in the course of one week. For minimal low interference cultures, we measured lactate levels representing cell metabolism and culture performance on the first, third, fifth and seventh days after electroporation, in addition to final cell recovery. Our data demonstrate that, as expected, cell growth and metabolic activity is compromised immediately after nuclear transfection; only RNPs and RNPs with n-nanoplasmid conditions showed reduced cell numbers compared to unedited cells (fig. 142E). However, cells were completely recovered on the third day and grown similarly to control cells for the remaining time of culture (fig. 142E). Cell recovery on day seven was comparable in control cells and in knockout-only conditions and about half compared to control cells in knockout/knockout conditions (fig. 142F). In summary, our approach enables efficient TCR editing, where endogenous TCRs from all T cells are almost completely removed, and transgenic TCRs are introduced in up to 60% of the cells with minimal impact on cell viability and growth kinetics.

TCR-engineered T cells recognize and kill target cells expressing an antigen

Having demonstrated effective TCR knockin, we next want to assess whether our TCR-engineered CD 8T cells are functional and able to respond to target cells expressing antigen. To this end, we harvested on day eight T cells engineered to express NY-ESO1 reactive 1G4TCR or CMVA2/pp65495-503 TCR6-2 and co-cultured them overnight with increasing concentrations of the NY-ESO1157-165 or pp65495-503 peptide pulsed HLA-A02:01 positive B cell line and measured the upregulation of the T cell activation marker CD137 (4-1 BB). No T cell activation was observed in the absence of exogenously added peptide, indicating that removal of endogenous TCR effectively prevented the alloreaction (fig. 143A-143D). We observed a peptide concentration-dependent up-regulation of CD137 expression for both TCRs (fig. 143A-143D). The EC50 concentration of 1G4TCR was 0.001 μm (fig. 143B), and the EC50 concentration of TCR6-2 was about 0.01 μm (fig. 143D).

To demonstrate antigen-specific target cell killing, we labeled B cells with CFSE and pulsed them with 0.1 μm peptide. Then, we co-cultured TCR-engineered T cells with CFSE-labeled antigen-positive and unlabeled antigen-negative B cells at a ratio of 1:1 overnight and determined the specific lysis of antigen-pulsed B cells by measuring the ratio of CFSE-positive to CFSE-negative cells. For both the NY-ESO1 antigen and pp65 antigen, we observed-80% target cell specific lysis at a 1:1 effector to target cell ratio (fig. 143E to 143H), demonstrating the highly potent cytotoxic potential of our TCR-editing cells.

To demonstrate activity against target cells with endogenous antigen expression, we fluorescently labeled a-375 cells expressing NY-ESO1 antigen and co-cultured with TCR knockdown or 1g4 TCR expressing T cells at a 1:1 ratio. Target cell lysis/apoptosis was captured via a real-time microscope and measured using a caspase lysis assay. Over the course of eighteen hours we detected robust target cell lysis in co-cultures with T cells expressing 1g4 TCR but not in control cultures with TCR-negative T cells (fig. 143I, fig. 143J). In summary, CD 8T cells that were TCR-edited using our non-viral plasmid-based approach were activated by their cognate antigen in a concentration-dependent manner and exhibited potent cytolytic activity at lower effector to target cell ratios.

Nanoplasmids containing promoters enable targeted gene knock-in and prolonged transient gene expression

To assess whether our optimized non-viral CRISPR knock-in method could achieve efficient integration beyond the TRAC locus, we first targeted the RAB11A locus using a homologous donor construct encoding the YFP-RAB11A fusion gene (Roth et al, 2018). Importantly, the construct contained the RAB11A promoter, indicating that YFP expression in transfected T cells may result from an integrated transgene or at least early after transfection from a non-integrated donor plasmid. To determine the appropriate time point to accurately assess knock-in efficiency (expression of integrated transgenes), we transfected T cells with YFP-RAB11A (fig. 150A, 150D, 150G) encoding a nanoplasmid, pUC57, or linear dsDNA, without Cas9-RNP (e.g., expression from only non-integrated templates) or with sgRNA/Cas9-RNP targeting RAB11A (e.g., expression from non-integrated and integrated templates). Three days after nuclear transfection, we observed CD8 expressing 66.9-91.3% YFP with the nanoplasmon donor template alone ⁺ T cells, 81.5-91.8% (fig. 150B, fig. 150C) were observed with nanoplastomers and sgRAB11A Cas9-RNP, demonstrating that expression is predominantly derived from non-integrated nanoplastomers. This transient YFP expression decreased over time to 8.6-15.4% on day seven post-transfection, while YFP expression was stable at 43.7-49.6% in cells transfected with nanoplasmon plus sgRAB11A Cas9-RNP (fig. 150B, fig. 150C). Expression from unincorporated YFP-RAB11A nanoplasmms stopped on day nine after nuclear transfection revealing 33.8-42.9% knock-in efficiency that was stable under the test conditions (fig. 143B, fig. 143C). Interestingly, this prolonged expression from donor templates containing non-integrated promoters is a unique feature of the nanoplasmid backbone, such as electroporation of constructs encoded by linear dsDNA (fig. 150D-150F) or pUC57 plasmids (fig. 150G-150I)The wells result in no transient YFP expression or substantially shorter transient YFP expression, respectively. Stable expression was observed in 18.6-36.2% of cells transfected with sgRAB11A-Cas9/RNP and pUC 57-based templates (FIG. 150H, FIG. 150I). These studies demonstrate that knock-in efficiency, as measured by transgene expression, can only be accurately assessed at least seven days after electroporation when using a nanoplasmid donor template containing a promoter. At the same time, these findings indicate that nanoplasmms have unique uses for transient open reading frame or reporter gene expression that are widely used in T cells.

In view of these results, we decided to evaluate the knock-in rate of the promoter-containing YFP-RAB11A construct compared to transient transfection of the nanoplasmon donor DNA nine days after transfection (fig. 144A-144C). Transient expression had stopped at this point and 33.8-42.9% knock-in rates were detected by flow cytometry (fig. 144A-144C). To extend this assessment, we targeted the AAVS1 safe harbor locus in intron 1 of the PPP1R12C (protein phosphatase 1 regulatory subunit 12C) gene, which is expected to not cause adverse physiological effects upon disruption and allow robust expression of the exogenous inserted gene (Smith et al, 2008; hockmeyer et al, 2009; chu et al, 2015). We designed an AAVS1 homologous donor construct (fig. 144D) expressing mNG under the control of the chicken/β actin hybrid intron (CBH) promoter (Gray et al, 2011). On day nine post-transfection, we observed knock-in efficiencies of the four test donors ranging from 32.5-45% (fig. 144E, 144F). In summary, our approach enables efficient targeting of transgenes to safe harbor loci.

T cell engineering with endogenous gene expression reporter genes

Next, we targeted the CD4 locus with a nanoplasmlet donor template designed to create a bicistronic transcript in which the existing CD4 gene is fused in-frame with the P2A peptide and mNG at the C-terminus, the locus being at CD4 ⁺ Active in T cells but in CD8 ⁺ T cells were inactive (fig. 144G). When we transfected CD 4T cells with sgCD4 Cas9-RNP and nanoplasmon template, we observed 43-51.8% of cells expressing both mNG and CD4, while at CD8 ⁺ No observation in T cellsTo mNG (FIG. 144H, FIG. 144I). The lower (on average about half) level of CD4 expression in CD 4T cells that had successfully integrated the mNG gene compared to control CD 4T cells transfected with non-targeted control guide RNAs (fig. 144J), suggests that only one CD4 allele was successfully recombined in most cells, while the second allele was modified by NHEJ, possibly resulting in loss-of-function mutations.

Next, we attempted to generate a reporter gene for T cell activation in primary CD 8T cells by targeting TNFRSF9 gene, which encodes CD137 (Ward-Kavanagh et al 2016), and was briefly up-regulated after TCR activation. We designed a nanoplasmon donor with a CD137 reporter construct that targets the first coding exon of the TNFRSF9 gene (exon 2) and inserts mNG in-frame with the N-terminus of CD137 followed by P2A, generating the CD137 reporter (fig. 144K). As a control we include a constitutively expressed RAB11A-YFP construct. After nuclear transfection, we will CD8 ⁺ T cells were cultured for ten days so that any CD137 expression resulting from initial T cell activation was resolved before reactivation with tranact. Then, we followed mNG expression by flow cytometry over a seven day period. RAB11A-YFP transfected cells constitutively expressed YFP, independent of TCR activation, and up-regulated CD137 expression 6h after reactivation (fig. 144K, 144L). In contrast, CD 8T cells transfected with TNFRSF9-mNG construct did not express mNG or CD137 without reactivation or 2h after reactivation, but up-regulated both as early as 6h after activation (fig. 144K, fig. 144L). mNG expression faithfully reproduced CD137 expression seven days after TCR activation (168 h) (fig. 144L), reaching a maximum at 24h and declining between the fourth and seventh days after activation. Similar to our observations of the CD4 reporter gene, we found that the expression level of CD137 itself was reduced by about half in cells expressing the mNG reporter gene (fig. 144M), again indicating that only one allele had incorporated the reporter gene and that the second TNFRSF9 allele had been disrupted. Our data demonstrate that the knock-in fusion construct reliably reports transcriptional activity of primary human T cells. However, due to incomplete (non-homozygous) gene editing, absolute target gene expression levels between transgenic and wt cells may be possible Different. Improved construct designs, targeting strategies, or transgenic cell selection methods may help minimize these effects.

Efficient multiplex gene knock-in human T cells

In view of the reduced target gene expression observed with our knock-in reporter, we wanted to further evaluate the potential for biallelic transgene integration with our method. To this end, we nuclear transfected CD 8T cells with TRAC-targeted nanoplasmid templates (identical homology arms) carrying either mNG or mCherry reporter gene alone (fig. 145A, fig. 145B) or an equivalent combination of reporter genes (fig. 145C). mNG and mCherry constructs alone resulted in 40.4-48.6% and 43.9-47.3% knock-in efficiency, respectively (fig. 145A, 145B). When the two donor templates were co-transfected, the overall knock-in rate remained 38.5-45% (FIG. 145C). Of all T cells in culture, 10.9-13% expressed only mNG,17.7-19.5% expressed only mCherry, and 9.9-12.5% expressed both reporter genes and thus underwent biallelic transgene integration (fig. 145C). Notably, this experimental strategy will underestimate the true rate of biallelic integration, given that a single positive population of cells detected by flow cytometry may carry one or two copies of the same donor template. However, at least 25% of cells that successfully integrate the donor template have this for both alleles. When the equivalent pUC57 plasmid template was used, the overall knock-in rate was slightly lower (22.9-39%) and only 3.5-5.8% of all cells had detectable biallelic integration (FIGS. 151A-151C).

Engineering of complex genetic circuits or multiplex reporter assays may require more than one gene edit at different loci to function adequately. Thus, we evaluated our protocol for the integration of two homologous donor templates at different genomic loci. We first tested the combination of a nanoplasmid donor containing the YFP-RAB11A transgene with a construct encoding mCherry-P2A as an in-frame fusion to the TRAC constant region (FIG. 145D). We transfected CD 8T cells with constructs alone or a combination of both and the respective sgRAB11a and sgTRAC Cas9-RNP and assessed reporter gene expression by flow cytometry ten days later. When transfected with YFP-RAB11A or TRAC-mCherry alone, 25.6-37.3% and 43-56.6% of the cells expressed the respective reporter genes (FIGS. 145E, 145F). In total 61.1-69.4% showed expression of either or both reporter genes in cells transfected with both constructs (fig. 145F). Although 7.3-12% of all cells expressed YFP only and 19.4-30.6% expressed mCherry only, 23.2-35% of all cells (corresponding to about 50% of transfected cells) co-expressed both transgenes (fig. 145G). Although we have doubled the total amount of nanoplastomer (4 μg) for these experiments, we observed only a small effect on cell viability and recovery (fig. 145F), consistent with our initial nanoplastomer titration study. pUC 57-based donor templates targeting the same locus produced comparable double knock-in rates (fig. 151D-151G). Similar results were obtained when targeting both AAVS1 and TRAC loci (figures 145H-145K). Taken together, these data demonstrate that successful multiple gene knockins occur without decreasing overall targeting efficiency and without interference between the two constructs.

Efficient non-viral CRISPR gene editing with large payloads

Finally, we sought to investigate whether our optimized targeting strategy could be used to integrate larger numbers of transgenic payloads, including those that exceeded AAV-based homologous donor limitations. Our NY-ESO1 TCR knock-in construct was 1.5kb in size. Using the 1G4 template as a framework, we designed a series of constructs with increasing burden sizes, targeting the 0.5kb homology arm of the following TRAC locus: the intracellular domain of human Notch1-P2A as an in-frame fusion at 3.8kb with mNG-P2A and 1G4 TCR alpha chain (TRAC_NotICD_ mNG); the intracellular domain of Notch1-P2A as a fusion in-frame with the full length 1G4 TCR at 4kb (TRAC_NotchICD_1G4); and a gene encoding THEMIS that plays a regulatory role in positive and negative T cell selection during the late thymic cell development period (Fu et al 2013) as a P2A in-frame fusion with a full-length 1G4 TCR at 5.45kb (trac_themis_1g4, fig. 146A). We transfected CD 8T cells with sgTRAC/Cas9-RNP and nanoplasmlets or pUC 57-based donor templates. For the 3.8kb TRAC_NotchICD_mNG construct, we obtained 38.9-40.6% and 44.1-49.9% knock-in frequencies with pUC57 and nanoplasmon templates, respectively (fig. 146B, fig. 1466C). Transfection of the 4kb TRAC_NotchICD_1G4 construct resulted in 27.1-30.5% (pUC 57) and 28.6-32.8% (nanoplasmid) knock-in rates (fig. 146D, fig. 146E). 5.45kB TRAC_Themis_1G4 donors produced 17.5-24.9% (pUC 57) and 16-25.7% (nanoplasmon) transgenic expression T cells (FIG. 146F, FIG. 146G). For two of the three constructs, a higher trend in cell recovery with the nanoplasmic DNA donor was observed, but statistical significance was not reached (fig. 146C, 146E, 146G). In general, the efficiency of knock-in and cell recovery for pUC57 and nanoplasmon forms was comparable for these longer donor templates, probably due to the lower percentage of plasmid backbone to total DNA delivered to T cells. The data demonstrate that with our non-viral plasmid based CRISPR gene editing approach we can increase the payload size more than three times up to more than 5kb and still achieve knock-in rates of over 20%.

Accurate gene editing in T cells is likely to rapidly enhance our understanding of basic T cell biology and bring about revolution for next generation engineered T cell therapies. Emerging methods utilizing DNA nuclease technology have enabled mutant gene correction, introduction of the entire gene or gene fusion into desired positions or manipulation of regulatory elements; this is not possible with existing retrovirus or lentivirus based methods (reference is made to the Marsen comment). Several groups have developed schemes for introducing CARs or TCRs into the TRAC locus using AAV vectors along with Cas9 or Cas12 a. It is generally demonstrated that ≡50% transgene integration is achieved by combined Cas9-RNP electroporation and viral infection, so AAV-based genetic modification is beneficial in generating T cell populations that can recognize and kill the desired target cell type without compromising functionality. Despite these obvious benefits, the use of AAV-based homologous donors requires cumbersome and expensive viral production methods (Bak et al, 2018), which currently limit their wider application in the research community. Although sufficient for CAR or TCR editing, the load size of AAV is limited to 4.8kb (Salganik et al 2015).

These limitations have stimulated interest in developing a completely non-viral precise gene editing method that is directed to increased versatility and ease of use, faster turn-around time, and reduced cost compared to viral gene delivery methods. The first breakthrough in this regard demonstrated that linear dsDNA donor templates (previously thought to be too toxic to T cells) could be successfully used to introduce longer DNA fragments, including TCRs (citations). However, the knock-in efficiency achieved when using linear dsDNA donors is applicable to TCR editing relative to AAV-based methods. Although the efficiency of linear dsDNA gene editing can be improved, editing a relatively small number of T cells, as we demonstrate here, requires large amounts of linear dsDNA to be produced and purified, which limits the versatility and scalability of the method.

Here we report a completely non-viral gene editing protocol that utilizes an off-the-shelf plasmid-based donor template that is co-delivered with high-fidelity Cas-RNP via electroporation into a T cell population. By this approach we achieved knock-in efficiency at multiple loci (alone or in combination) comparable to AAV-based methods, and maintained consistently high knock-out efficiency under multiple editing conditions, thus achieving all the potential of non-viral editing techniques with respect to versatility, turnaround time and cost savings. Plasmid donors with high purity can be designed and synthesized quickly and inexpensively. They provide the ability for sequence validation and are suitable for large-scale, good Manufacturing Practice (GMP) level qualification for use in cell therapies. Importantly, we demonstrate successful delivery of genetic loads greater than 5kb, with no dramatic drop in knock-in efficiency compared to smaller transgenes, suggesting that delivery of even larger constructs is possible. Furthermore, at the optimal parameters, the introduction of plasmid DNA had no effect on cell viability, and more importantly, the recovery of the edited cells was similar to the loss-of-function perturbation of Cas9-RNP alone. We believe that our approach will serve as a basis for releasing the full potential of accurate gene editing in primary human T cells for basic research and clinical applications, etc.

An antibody. All antibodies used for flow cytometry analysis are listed in table 16.

Table 16.

And (5) guiding RNA. Where applicable, custom sgRNA design tools were used to identify targeting sequences (20 mers) based on streptococcus pyogenes Cas 9. The guide RNAs were selected based on predicted target specificity using a cleavage frequency measurement (CFD) specificity score as a pretilt-specificity prediction algorithm (Doench et al, 2016) and two on-target cleavage efficiency scores (Azimuth algorithm, popular rule 2 on versions of target cleavage efficiency prediction algorithm (Doench et al, 2016) and deep cas9 algorithm (Wang et al, 2019). It is critical to select multiple guide RNAs around the desired target site and to empirically conduct the test. Guide RNAs targeting the TRAC and TRBC loci were previously described (Roth et al, 2018). All sgRNA sequences are listed in table 17. All guide RNAs were used asCRISPR-Cas9sgRNAs are ordered from Integrated DNA Technologies (IDT). />

TABLE 17

sgRNA	Sequence(s)
		sgTRAC	AGAGTCTCTCAGCTGGTACA
sgTRBC	GGAGAATGACGAGTGGACCC
		sgRAB11A	GGTAGTCGTACTCGTCGTCG
sgCD4	GGCAGGTCTTCTTCTCACTG
		sgTNFRSF9	GTTGAGGACCAGCAACAGAG
sgAAVS1	GGGACCACCTTATATTCCCA

HDR donor template design. Donor templates were designed in snap gene (GSL Biotech, LLC). To design a long homology directed repair template, the Cas9 cleavage site (3 nucleotides, nts, upstream of the Protospacer Adjacent Motif (PAM)) of the experimentally verified guide RNA near the desired knock-in site is identified within the genome, and the-0.5 kb regions 5 '-and 3' of this site are designated as left and right homology arms, respectively. Any native sequence between the actual guide RNA cleavage site and the desired knock-in site is included as part of the donor construct between the homology arms to avoid any offset and ensure perfect binding of the homology arms to the genomic sequence up to the cleavage site. In order to avoid nucleotide sequence duplication, this region should be codon optimized. Any loaded sequences are then included in the construct in frame with the target locus, if desired. If not required for other reasons, codon optimization should be avoided as it would reduce knock-in efficiency or affect transgene expression relative to endogenous equivalents. The guide RNA binding site in the donor template needs to be mutated as broadly as possible (preferably PAM mutation, followed by the largest mutation in the spacer binding site). If the existing gene transcript is used to express the exogenous protein, the cleavage site should be located within the coding sequence of the target gene. The GSG-2A site is placed downstream of the left homology arm in frame with the target gene, followed by the open reading frame of the exogenous gene. Multiple GSG-2A-Gene cassettes can then be added after the first. In case ribosome read-through to the next cassette is desired, the stop codon is excluded from all genes. At the end of the last gene in the series, but before the right homology arm, a stop codon, or another GSG-2A site or stop codon, may be inserted to polyaddite the adenylation sequence. Alternatively, the exogenous coding sequence may continue into the right homology arm to create an in-frame fusion with the target locus. When designing templates targeting non-coding regions of the genome, the left and right homology arms are selected as described above. Enhancers, promoters, and Kozak sequences are placed between homology arms, and then one or more genes of interest separated by GSG-2A sequences are placed as needed. The last gene in the series was terminated with a stop codon and polyadenylation sequence. Construct tissues are shown where LHA is the left homology arm (500 bp unless otherwise indicated), GSG is glycine-serine-glycine linker, T2A and P2A are ribosome cleavage sequences, furin is arginine-alanine-lysine-arginine endoprotease cleavage site, bGHpA is polyadenylation site from bovine growth hormone gene, pCBH is transcriptional regulatory element consisting of CMV enhancer and chicken β -actin promoter, and RHA is the right homology arm (500 bp unless otherwise indicated). the ttcts site is a truncated Cas9 targeting sequence with a PAM site having a 4bp mismatch at the 5' end. Templates with tts sites have one mismatch at each of the 5 'and 3' ends, both oriented inward and flanked by 16bp edge sequences (Nguyen et al, 2019). The donor template sequences are given in table 18.

HDR template fabrication. See sequence summary table 18. The nanoplasmids and pUC57 HDR templates were provided as primary cell transfection-grade material and resuspended in water at a concentration of 1mg/mL as provided by Nature Technology. The inc. Trac_1g4_500HA and trac_ mNG _500HA linear dsDNA donor DNA was prepared via PCR (Roth et al 2018). PCR products were generated using Q5 high fidelity polymerase (NEB, ipswitch, mass.) with 0.25. Mu.M forward primer (5'-AACATACCATAAACCTCCCATTCTG-3', SEQ ID NO: 57) and reverse primer (5'-TTGGAGAGACTGAGGCTGGGCCACG-3', SEQ ID NO: 58) each reacted with 10ng/mL plasmid DNA template. The cycle parameters were 98℃for 15 seconds, 60℃for 15 seconds and 72℃for 1 minute for a total of 30 cycles. Products from 96x 100 μl reactions were pooled and incubated in Qiagen buffer (Qiagen, germanntown, MD) and then purified by HiSpeed Plasmid Maxi Kit (Qiagen, germanntown, MD). The final product was eluted in nuclease-free water and the DNA concentration was adjusted to 1mg/mL. Isolation and culture of primary human T cells. According to the manufacturer's instructions (Miltenyi Biotec), use is made ofThe buffy coat CD8 microbead kit or CD4 microbead kit separates primary human cd8+ and cd4+ T cells from the buffy coat by forward selection. Residual erythrocytes were lysed prior to culture. Cells were plated at an initial concentration of one million cells per milliliter of activation medium. Unless otherwise indicated, the activation medium consists of +. >T cell CDM medium (Irvine Scientific) supplemented with 25ng/ml IL-7 (Miltenyi Biotec) and 50ng/ml IL-15 (Miltenyi Biotec) for CD8 ⁺ T cells and IL-7 (25 ng/ml), IL-15 (50 ng/ml) and IL-2 (Biolegend, 400U/ml) for CD4 ⁺ T cells. T Cell TransAct ^TM (Miltenyi Biotec) was added to the culture at a dilution of 1:100. In some experiments, the activation medium consisted of X-VIVO ^TM 15 serum-free hematopoietic cell culture medium (Lonza Bioscience) was composed supplemented with 5% heat-inactivated fetal bovine serum, 200U/mL IL-2, 5ng IL-7/mL, 5ng/mL IL-15, 50. Mu.M 2-mercaptoethanol, and 10. Mu. M N-acetyl-L-cysteine, to which was added the human T activator CD3/CD28 Dynabeads (Gibco) at a 1:1 bead to cell ratio. T cell media were prepared using the following ingredients: RPMI 1640 medium (Gibco, catalog No. 11875093), 10% FBS (Hyclone, catalog No. SH 30071.03), 2mM L-alanyl-L-glutamine (Glutamax; gibco), 1mM sodium pyruvate (Gibco), 0.1mM non-essential amino acid (Gibco), 55. Mu.M 2-mercaptoethanol (Gibco), 100U/mL penicillin (PenStrep), 100. Mu.g/mL streptomycin (Penstrep; gibco) and 10mM HEPES (Gibco). The medium was sterilized through a 0.22 μm filter. Unless otherwise indicated, T cells were cultured for 36 to 48 hours prior to electroporation. During the culture after electroporation, the culture volume is continuously expanded to Cells were kept at about 100 ten thousand cells per ml at all times.

RNP assembly. RNP is produced by combining target-specific Sgrnas (IDTs) and recombinant Cas9 (SpyFi, aldevron). Briefly, lyophilized sgrnas were reconstituted to a concentration of 200M in a nuclease-free duplex buffer (IDT). For each 60pmol of Cas9, 180pmol of sgRNA was added to obtain a 3:1sgRNA to cas9 ratio (conditions reported in the experiment (Roth et al 2018), 2:1sgRNA to cas9 ratio was used). The sgRNA: cas9 mixture was incubated for 15 minutes at room temperature to allow RNP formation. For the combined TCR-knock-in/TRBC knock-out experiments, 30pmol each of TRAC and TRBC RNP were assembled separately and then mixed together using equal volumes. A total of 60pmol of combined TRAC and TRBC RNP was used for a single nuclear transfection reaction. For knock-in experiments targeting other loci, 60pmol of total Cas9/RNP was used per nuclear transfection reaction.

Nuclear transfection. After 36 to 48 hours of activation, T cells were pelleted, washed with Phosphate Buffered Saline (PBS), and resuspended in P3 buffer with supplement (Lonza Bioscience) at 200 ten thousand cells per 20 l. Prior to the PBS wash step, cells activated with human T activator CD3/CD28 Dynabeads (Gibco) were magnetically isolated from the beads. The following components of the mononuclear transfection reaction were added to the PCR tube and gently mixed: preformed RNP (60 pmol total), HDR template (up to 8 g) and T cells resuspended in P3 buffer. In some cases, poly-L-glutamic acid (Sigma Aldrich,150 g) was also added to the mixture. The mixture was then transferred to one well of a 16-well 4D-nucleic acid cuvette (Lonza Bioscience) and pulsed with code EH 115. After electroporation, the 4D-Nucleofector cuvette was placed in a tissue incubator at 37℃for 15 minutes for cell recovery. After recovery, the cells were transferred to a cell line containing 2mL of IL-7 supplemented with 25ng/mL and 50ng/mL IL-15 (CD 8) ⁺ T cells) or 25ng/mL IL-7, 50ng/mL IL-15 and 400U/mL IL-2 (CD 4) ⁺ T cells) in a pre-warmed PRIME-XV medium. In some experiments, cells were cultured after electroporation in X-VIVOTM 15 serum-free hematopoietic cell medium (Lonza Bioscience) supplemented with 5% heat-inactivated fetal bovine serum, 50. Mu.M 2-mercaptoethanol, 10. Mu. M N-acetyl-L-cysteine, and 500U/mL IL-2。

Flow cytometry. Transfected cells at different time points were analyzed by flow cytometry to measure knock-in efficacy. The reagents were used as recommended by the manufacturer. Briefly, cells were pelleted, washed with phosphate buffered saline, and gently resuspended and incubated in pre-diluted, fixable, vital dye eFlur 780 or propidium iodide for 10 minutes at room temperature. After incubation, the cells were washed twice in FACS buffer and conjugated with fluorochromes for CD3 and/or anti-TCRa/b and anti-CD 4 (for CD4 ⁺ T cells) or anti-CD 8 (for CD8 ⁺ T cells) were surface stained. In some experiments, cells were also stained with IG4 or pp65 TCR dextran (PE or APC) for 10 minutes at room temperature in the absence of light prior to addition of surface antibodies. After addition of other surface antibodies, cells were incubated in the dark at 4℃for an additional 15 minutes. For CD19 CAR staining, cells were first stained with biotin anti-human CD19 CAR detection reagent (Miltenyi Biotec) and then with streptavidin PE. For staining cells with anti-CD 137 PE, the fluorescence intensity was amplified using FASER (fluorescence amplification by sequential use of reagents) kit-PE (Miltenyi Biotec). The stained cells were washed twice in FACS buffer prior to FACS acquisition. To calculate the absolute number of cells in some samples, countb right absolute count beads (Thermo Fisher Scientific) were added to the samples prior to FACS collection. Samples were collected using either FACSymphony or LSR Fortessa (both from BD Biosciences) equipped with FACSDiva software. Compensation was performed using a single staining control prepared with Ultra-comp ebeads (Thermo Fisher Scientific). Input Flow Cytometry Standard (FCS) 3.0 file and analyzed using FlowJo software version 3.0 (FlowJo). Aggregates were removed using conventional gating strategies and dead cells were excluded based on vital dye staining.

SIMOA measurements. IFN-a analysis in culture supernatants before and after electroporation was performed using Simoa IFN-a Advantage kit (HD-1/HD-X Item 100860) according to the manufacturer's protocol. Briefly, 200. Mu.L of IFN-a calibrator and experimental samples were added to each well in a 96-well plate. Bead reagents, detection reagents, SBG (streptavidin beta galactosidase) reagents and sample diluents provided by the kit are added to the reagent zone in Quanterix HD-X, and RGP (resorufin-D-galactopyranoside) is added to the sample zone. Following the IFN-a assay setup in Simoa software, plates containing calibrator and experimental samples were loaded into the sample area and analyzed on Quantix HD-X.

T cell activation. T cell activation cultures comprise CRISPR engineered T cells, HLA-A 02:01 ⁺ Target cell lines and non-target HLA-A 02:01 negative target cell lines (which serve as reference populations for calculation of target cell lysis). Both cell lines were obtained from Fred Hutch International Histocompatibility Working Group. Target and reference cell lines were labeled with CFSE and Cell Trace Violet (CTV) (Invitrogen), respectively, to distinguish populations during flow cytometry analysis. For peptide pulsing, CFSE labeled HLA-A 02:01+ target cells were incubated with various concentrations of the appropriate target peptide for 2 hours at 37 ℃. After the incubation period, the cells were washed twice with PBS and then resuspended in 10% fbs RPMI T cell medium. Peptide-loaded CFSE-labeled target cells were incubated with CTV-labeled reference populations at a 1:1 ratio, and CRISPR-engineered T cells were added at a 1:1 ratio of T cells to CFSE-labeled target cells. The condition without peptide added was included as a control. After about 24 hours, T cell activation was analyzed as follows: (1) Cells were collected and analyzed by flow cytometry to determine CD137 (Biolegend, clone 4B 4-1) up-regulation and target cell lysis, and (2) supernatant was collected for analysis of effector molecules by Luminex. To analyze target cell lysis, countBright absolute count beads (Thermo Fisher Scientific) were added to the flow cytometry analysis samples to quantify the number of CFSE-labeled target cells and CTV-labeled non-target cells during FACS acquisition. Specific target cell lysis was calculated using the following equation:

Percent specific lysis = [1- (no peptide control ratio/experimental ratio) ] ×100

The ratio was calculated by dividing the number of CTV-tagged reference populations by the number of CFSE-tagged HLA-A 02:01+ target cells.

In vitro killing assay. With 1. Mu.MCytolight quick dye (Cat 4706) labeled a375 (malignant human melanoma) cell line expressing nyso antigen and plated in 96 well plates at an seeding density of 50000 cells. Two hours after inoculation, caspase-3/7 green apoptosis reagent (2272582, invitrogen) and IG4 KI or KO control (50000 cells/well) were added to a375 cells. Cell killing was measured by assessing the number of caspase-3/7 agent-expressing a375 cells present in each well. The co-cultures were monitored for growth and apoptosis for 18hr using an intucyte imaging system. After co-culture, CD137 expression on CD8+ T cells was measured by flow cytometry (BioLegend, clone 4B 4-1).

T cell expansion culture/lactate measurement. Activated cd8+ T cells were electroporated with sgTRAC and sgTRBC RNP alone (knockdown) or with sgTRAC RNP, sgTRBC RNP and nanoplastomers encoding TCRs (knockin) at 48 hours. As a control without electroporation (RNP), cd8+ T cells were added only to the Lonza electroporation cuvette, but not subjected to electroporation pulse code. RNP-free, knocked-out and knocked-in T cells were cultured in 24-well G-Rex plates (Wilson-Wolf) following electroporation in PRIME-XV medium supplemented with 25ng/mL IL-7 (Miltenyi) and 50ng/mL IL-15 (Miltenyi). Supernatants were collected from RNP-free, knockout and knock-in conditions on the first day after electroporation, and then collected every 2-3 days for 7 days.

Extracellular Lactate content was analyzed as an alternative indicator of cell proliferation using the Lactate-Glo assay (Promega) according to the manufacturer's protocol (Grist et al, 2018). Briefly, after thawing, the fluorescein detection solution was brought to room temperature while all other kit components were maintained on ice. The lactate dehydrogenase was reconstituted with water and then placed on ice. Immediately before use, lactate detection reagents were prepared by mixing a fluorescein detection solution, reductase fifth, lactate dehydrogenase, and NAD in ratios specified by the manufacturer. Cell culture supernatants were diluted in PBS and 50 μl of sample or lactate control was added to 96-well plates followed by 50 μl lactate detection reagent. The plates were shaken for 30-60 seconds and incubated at room temperature for 60 minutes. Luminescence was recorded using a read plate luminometer.

Translocation assays. A set of ddPCR-based detection methods was developed to detect potential chromosomal translocations during a CRISPR-mediated editing process for three target sites (TRAC, TRBC1 and TRBC 2) simultaneously in engineered T cells (QX 200 ddPCR platform of Bio-Rad). These 6 translocations are specified as: TRAC-TRBC1, TRAC-TRBC2, TRBC1-TRAC, TRBC1-TRBC2, TRBC2-TRAC, TRBC2-TRBC1. The ratio of target sequence (copy number/. Mu.L) to RPP30 sequence was measured as a measure of chromosomal translocation at each DNA target site using a reference assay for detecting the RPP30 gene of interest. The primer and probe sequences are: TRAC-Forward: TGGGGCAAAGAGGGAAATGAG (SEQ ID NO: 59), TRAC-reverse: AGAACCTGGCCATTCCTGAAG (SEQ ID NO: 60), TRAC-probe: CATGTGCAAACGCCTTCAACAACAG (SEQ ID NO: 61), TRBC 1-Forward: CTGGGATGGTGACCCCAAAA (SEQ ID NO: 62), TRBC 1-reverse: GGCCACATAGAAAGGGGACC (SEQ ID NO: 63), TRBC 1-probe: ACCATGAAGGAGAATTGGGCACCT (SEQ ID NO: 64), TRBC 2-Forward: GGGGGATGGACAGACAATGG (SEQ ID NO: 65), TRBC 2-reversal: GCTGACCCTGTGAACCTTGA (SEQ ID NO: 66), TRBC 2-probe: ATCCAGGTAGCGGACAAGACTAGAT (SEQ ID NO: 67), RPP 30-forward: TCAGCCATATTGTCCCCTAAACT (SEQ ID NO: 68), RPP 30-reverse: TGGTCTGTCCATGGCATCTT (SEQ ID NO: 69), RPP 30-probe: CTGTATGGACACAGTGCCTA (SEQ ID NO: 70)

Whole genomic DNA isolated from T cells was tested using 7 ddPCR assays and translocations were reported as% ratio relative to reference detection.

RNASeq analysis. Human CD8+ T cells were isolated from five donors and activated as indicated above, and then electroporated with 60pmol TRAC RNP, either without or with 3ug TRAC-684 mNanGreen-500 HA template) in PCR or nanoplasmid form. Twenty hours after electroporation, RNA was isolated from cells using column deoxyribonuclease (DNase) I digestion according to the manufacturer's instructions using the RNeasy Mini kit (Qiagen). Differential expression analysis of transcriptome data was performed using R-package DESeq2 (Anders and Huber, 2010) (reference 1, see below). Heat maps were generated by converting RNA-seq read counts to normalized expression using Variance Stabilizing Transformation (VST). GSEA analysis was performed using an R Bioconductor package rich plot (Yu G (2021)). rich plot: visualization of Functional Enrichment Result, R package version 1.14.1, yulab-smu.top/biological-knowledges-mining-book /) and MSigDB (Subramannian et al 2005; liberzon et al, 2015). The MSigDB Hallmark 2020 genome was used for GSEA analysis.

Activation of cd4+ T cells with CD19 CAR constructs. Fifty thousand CD4+ T cells with CD19 specific CAR or pp65 specific 6-2TCR (control independent TCR) were plated at a 1:1E:T ratio with CD19 expressing Granta-519B cells and incubated for 24 hours. Culture supernatants were analyzed by Luminex for IFN-gamma and TNF-alpha production.

And (5) carrying out statistical analysis. GraphPad Prism software was used for drawing and statistical analysis. Unpaired t-tests or one-way anova were used to determine statistical significance.

Example 36 test construct design contains full length TCR-alpha ("Schober construct") and TRBC1/TRBC2 knock-in constructs.

Experimental conditions

The plating was performed by adding Prime-XV with IL-7 (25 ng/mL), IL-15 (50 ng/mL) and TransAct 1:100 (without Pen/Strep), electroporation using Haley Lonza 4D in P3 buffer, EW113 48 hours after activation, resting for 15min at 37℃after electroporation, then 75. Mu.L of ordinary Prime was added and transferred.

And (3) a template: TRAC3_Wt1C13_pUC57 (control); schober_trac3_wt1c13_puc57 (test); TRAC3_mNEon-NP (control); TRBCg22_TRBc1_mNEon_Noalpha-NP; TRBCg22_TRBc2_mNEon_Noalpha-NP.

RNP = for Schober test, 30pmol each of TRACsg3 and TRBCsg22 were used at a 3:1sgrna:cas9 ratio. For TRBC testing, a 3:1sgRNA:Cas9 ratio was used of 60pmol TRBCsg22.

The addition sequence is as follows: 1-pre-incubation of RNP for 15 min; 2-template (3 μg); 3-cells (200 ten thousand per cuvette).

Dyeing conditions (day 5 and day 7): live/read APC-Cy7 (1:1000 per stain), fc blocker (5 ul/stain), TCR-BV421 (5 ul/stain), pMHC Immudex dextramer (only for WT1C13 samples)

Example 37 lead test for non-viral TCR knockins using Jurkat and primary human cd4+ cells.

An experiment was performed to determine the conditions for non-viral TCR knockins using Jurkat and primary human cd4+ cells. The conditions were as follows: jurkat NF-kB-Luc medium: RPMI 1640+10%HI FBS+2mM L-glut+10mM HEPES+1mM NaPyr+10ug/ml blasticidin+100 ug/ml gecomycin with or without 10ng/ml rhIL-2.CD4 medium: prime-XV with or without 10ng/ml rhIL-2. Activation was performed using a TransAct@1:100. Electroporation was performed 44 hours after activation in P3 buffer, EW113, using Haley Lonza4D. Rnp=60 pmol TRAC3 RNP in 3:1sgrna:cas9 ratio in all samples. The addition sequence is as follows: 1-non-targeted or TRAC3 RNP (pre-incubated for 15 min at room temperature); 2-2ug TRAC3-mNaeon nanoplasmon template or water; 3-cells (150 ten thousand per cuvette). After electroporation: incubation was carried out at 37℃for 15 minutes, 75ul of normal pre-warmed medium without supplement was added and transferred to a 48-well plate. Dyeing conditions were (day 5 and day 7): TCRa/b-BV421, 5. Mu.l/stain, PI (1:200 per stain)

Culture conditions. Fig. 160 and 161 show flow cytometer scatter plots of Jurkat and donor CD4 cells. The following conditions are shown: unactivated Jurkat (no IL-2); unactivated Jurkat (no IL-2); unactivated Jurkat (with IL-2); unactivated CD4, donor No. 1 (no IL-2); aCD3/28 activated CD4, donor number 1 (with IL-2); unactivated CD4, donor number 1 (with IL-2); unactivated CD4, donor number 2 (no IL-2); aCD3/28 activated CD4, donor number 2 (with IL-2); unactivated CD4, donor number 2 (with IL-2).

Electroporation conditions. Electroporation conditions were as follows: RNP and non-targeting crRNA: tracrRNA; RNP and TRACsg3 (knockout only); TRACsg3 RNP with TRAC3-mNEon template (knock-in).

Jurkat data: at the stained antibodies we used, approximately 40% of Jurkats appeared to be TCR negative. Regardless of the activation conditions, the knockout performed well. A very small amount (0.5%) of "knock-in" expression occurs, regardless of the activation conditions. It may be desirable to classify the population and attempt to amplify the population.

Subsequent work may include: the sequence of the TRAC locus in Jurkats was checked for mutations that could inhibit HDR, the length/splice/sequence of transcripts produced at this locus was checked, and other gRNAs were tested in cases where the TRAC3 locus was not accessible in these cells. Furthermore, jurkats are known to be resistant to plasmid uptake. The use of oligonucleotides to generate gene knockins in these cells has been difficult.

Primary cd4+ data:

unactivated cells could not survive to day 5 and no evidence of knock-in was shown. Activated cells showed good TCR knockdown until day 5. Up to day 5, approximately 20% of gene knockins occurred in both donors. This increased to 40% until day 7. Between day 5 and day 7, a slight increase (-5%) in CD8 cells typically occurs.

Sequence(s)

Sequence used in eJH52_wt1c_13_trac3 construct:

WT1C13 alpha chain amino acid sequence: (underlined from endogenous sequence), SEQ ID NO:53:

MTRVSLLWAVVVSTCLESGMAQTVTQSQPEMSVQEAETVTLSCTYDTSENNYYLFWYKQPPSRQMILVIRQEAYKQQNATENRFSVNFQKAAKSFSLKISDSQLGDTAMYFCAFMGYYGGSQGNLIFGKGTKLSVKPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSAVAWSNKSDFACANAFNNSIIP EDTFFPSPESSCDVKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFNLLMTLRLWSS*

WT1C13 beta chain amino acid sequence (without stop codon) SEQ ID NO:54:

MDSWTFCCVSLCILVAKHTDAGVIQSPRHEVTEMGQEVTLRCKPISGHNSLFWYRQTMMRGLELLIYFNNNVPIDDSGMPEDRFSAKMPNASFSTLKIQPSEPRDSAVYFCASSSLQYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVSTDPQPLKEQPALNDSRYCLSSRLRVSATFWQNPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRADCGFTSESYQQGVLSATILYEILLGKATLYAVLVSALVLMAMVKRKDSRG

WT1C13 alpha chain nucleotide sequence: (text without underline is codon optimized; underlined part is endogenous sequence), SEQ ID NO:55:

ATGACCCGGGTGAGCCTGCTGTGGGCCGTGGTGGTGAGCACCTGCCTGGAGAGCGGCATGGCCCAGACCGTGACCCAGTCTCAGCCCGAGATGAGCGTGCAGGAGGCCGAGACCGTGACCCTGAGCTGTACCTACGACACCAGCGAGAACAACTACTACCTGTTCTGGTACAAGCAGCCCCCCAGCCGGCAGATGATCCTGGTGATCCGGCAGGAGGCCTACAAGCAGCAGAACGCCACCGAGAACAGATTCTCTGTGAACTTCCAGAAGGCCGCCAAGAGCTTCAGCCTGAAGATCAGCGACTCCCAGCTGGGCGATACCGCCATGTATTTCTGCGCCTTCATGGGCTACTACGGCGGCAGCCAGGGCAATCTGATCTTTGGCAAGGGCACAAAGCTGAGCGTGAAGCCCAACATCCAGAACCCCGACCCTGCCGTGTACCAGCTGAGGGACTCCAAGTCTAGCGATAAGAGCGTGTGCCTGTTCACCGACTTTGATTCCCAGACAAACGTGAGCCAGAGCAAGGACTCTGACGTGTACATCACCGACAAGACAGTGCTGGATATGAGGTCTATGGACTTCAAGAGCAACAGTGCTGTGGCCTGGAG CAACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAG AAAGTTCCTGTGATGTCAAGCTGGTCGAGAAAAGCTTTGAAACAGATACGAACCTAAACTTTCAAAACCTGTCAGTG ATTGGGTTCCGAATCCTCCTCCTGAAAGTGGCCGGGTTTAATCTGCTCATGACGCTGCGGCTGTGGTCCAGCTGA

WT1C13 β chain nucleotide sequence (all codons optimized, NO stop codon) SEQ ID NO 56:

ATGGACAGCTGGACCTTCTGCTGCGTGAGCCTGTGCATCCTGGTGGCCAAGCACACAGACGCAGGCGTGATCCAGAGCCCAAGGCACGAGGTGACAGAGATGGGCCAGGAGGTGACCCTGAGGTGTAAGCCCATCAGCGGCCACAACTCCCTGTTCTGGTATAGGCAGACCATGATGCGGGGACTGGAGCTGCTGATCTACTTCAACAACAACGTGCCCATCGATGACAGCGGCATGCCCGAGGACAGATTCAGCGCCAAGATGCCCAACGCCAGCTTCAGCACCCTGAAGATCCAGCCCAGCGAGCCCAGAGACTCCGCCGTGTATTTCTGCGCCAGCAGCAGCCTGCAGTATGAGCAGTACTTCGGCCCAGGCACACGCCTGACCGTGACAGAGGATCTGAAGAACGTGTTCCCCCCTGAGGTGGCCGTGTTTGAGCCTTCTGAGGCCGAGATCAGCCACACCCAGAAGGCCACCCTGGTGTGCCTGGCAACCGGCTTCTACCCAGACCACGTGGAGCTGAGCTGGTGGGTGAACGGCAAGGAGGTGCACAGCGGCGTGTCCACAGACCCACAGCCCCTGAAGGAGCAGCCCGCCCTGAATGATTCTAGATATTGCCTGTCTAGCCGGCTGAGAGTGAGCGCCACCTTTTGGCAGAACCCTAGGAATCACTTCCGCTGTCAGGTGCAGTTTTACGGCCTGAGCGAGAATGACGAGTGGACCCAGGATAGGGCCAAGCCTGTGACACAGATCGTGTCCGCCGAGGCATGGGGAAGGGCAGATTGCGGCTTCACAAGCGAGTCCTACCAGCAGGGCGTGCTGTCCGCCACCATCCTGTATGAGATCCTGCTGGGCAAGGCCACACTGTACGCCGTGCTGGTGAGCGCCCTGGTGCTGATGGCCATGGTGAAGAGGAAGGACTCCAGGGGC

TRAC_mNaneonGreen_500HA forward primer (SEQ ID NO: 105):

AACATACCATAAACCTCCCATTCTG

TRAC_mNaneonGreen_500HA reverse primer (SEQ ID NO: 106):

TTGGAGAGACTGAGGCTGGGCCACG

TRAC_tCTS_mNEonGreen_500HA forward primer (SEQ ID NO: 107):

TGGCGGGACTAGTGGCCACATCTCT

TRAC_tCTS_mNEonGreen_500HA reverse primer (SEQ ID NO: 108):

TGGCGGGACTAGTGGCCACATCTCT

reference to the literature

Marson et al, "A Cas9 nanoparticle system with truncated Cas9 target sequences on DNA repair templates enhances genome targeting in diverse human immune cell types" doi:10.1101/591719

Miller, B.C. et al Subsets of exhausted CD8+ T cells differentially mediate tumor control and respond to checkpoint blockade, nature immunology 20,326-336 (2019), doi 10.1038/s41590-019-0312-6

Sade-Feldman, M.et al, defining T Cell States Associated with Response to Checkpoint Immunotherapy in Melanoma, cell, volume 175, 4, 11.2018, 1.998-1013, doi 10.1016/j.cell.2018.10.038

Siddiqui, I et al, immunity, volume 50, stage 1, month 1, 15, 2019, pages 195-211, doi: 10.1016/j.immini.2018.12.021

Anders, S. And W.Huber,2010.Differential expression analysis for sequence count data, genome biology 11:R106, doi:10.1186/gb-2010-11-10-r106.

Bak, R.O. et al, 2018, CRISPR/Cas9 genome editing in human hematopoietic stem cells, nature-laboratory Manual, 13:358-376, doi:10.1038/nprot.2017.143.

Bloenberg, D et al 2020.A High-Throughput Method for Characterizing Novel Chimeric Antigen Receptors in Jurkat Cells, methods of molecular therapy and clinical development 16:238-254, doi:10.1016/j.omtm.2020.01.012.

Choi, B.D. et al 2019 CRISPR-Cas9 issue of PD-1 enhances activity of universal EGFRvIIICAR T cells in a preclinical model of human glioblastoma, J.cancer immunotherapy 7:304, doi:10.1186/s40425-019-0806-7.

Chu, V., T.et al 2015.Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells,33, doi:10.1038/nbt.3198.

Dai, X. Et al, 2019.One-step generation of modular CAR-T cells with AAV-Cpf1, nature methods 16:247-254, doi:10.1038/s41592-019-0329-7.

Dash, P. Et al 2011.Paired analysis of TCR. Alpha. And TCR. Beta. Chains at the single-cell level in mice, J.Clin.De.121:288-295, doi:10.1172/jci44752.

Davidsson, M.et al 2020.A comparison of AAV-vector production methods for gene therapy and preclinical assessment, science report (uk), 10:21532, doi:10.1038/s41598-020-78521-w.

Doench, j.g. et al 2016.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9, 34, doi:10.1038/nbt.3437.

Eyquem, J. Et al 2017.Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection, nature. 543:113-117, doi:10.1038/aperture 21405.

Fu, G. Et al 2013.Themis sets the signal threshold for positive and negative selection in T-cell development, nature. 504:441-445, doi:10.1038/aperture 12718.

Gray et al, 2011.Optimizing Promoters for Recombinant Adeno-Associated viruses-Mediated Gene Expression in the Peripheral and Central Nervous System Using Self-Complementary Vectors, human Gene therapy, 22:1143-1153, doi:10.1089/hum.2010.245.

Grist, J.T. et al 2018.Extracellular Lactate:A Novel Measure of T Cell Proliferation,J Immunol Author Choice,200:1220-1226, doi:10.4049/jimmunol.1700886.

Hacein-Bey-Abina, S. A.Garrigue, G.P.Wang, J.Soulier, A.Lim, E.Morillon, E.Clappier, L.Caccavelli, E.Delabesse, K.Beldjord, V.Asnafi, E.MacIntyre, L.D.Cortivo, I.Radford, N.Brousse, F.Sigaux, D.Moshous, J.Hauer, A.Borkhardt, B.H.Belohradsky, U.Wintergerst, M.C.Velez, L.Leiva, R.Sorensen, N.Wulffraat, S.Blanche, F.D.Bushman, A.Fischer and M.Cavazzana-Calvo,2008.Insertional oncogenesis in 4patients after retrovirus-mediated gene therapy of SCID-X1, J.Clin.De.S. 118:3132-3142, doi:10.1172/jci35700.

Hacein-Bey-Abina, S., C.V.Kalle, M.Schmidt, M.P.McCormack, N.Wulffraat, P.Leboulch, A.Lim, C.S.Osborne, R.Pawliuk, E.Morillon, R.Sorensen, A.Forster, P.Fraser, J.I.Cohen, G.de S.Basile, I.Alexander, U.Wintergerst, T.Frebourg, A.Aurias, D.Stoppa-Lyonnet, S.Romana, I.Radford-Weiss, F.Gross, F.Valensi, E.Delabesse, E.Macintyre, F.Sigaux, J.Soulier, L.E.Leiva, M.Wissler, C.Prinz, T.H.Rabbitts, F.L.Deist, A.Fischer and M.Cavazzana-Calvo,2003.LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene Therapy for SCID-X1, science 302:415-419, doi 10.1126/science 1088547.

Halbert, C.L., J.M. Allen and J.S.Chamberlain,2018.AAV6 Vector Production and Purification for Muscle Gene Therapy, methods of molecular biology (Clifton N J), 1687:257-266, doi:10.1007/978-1-4939-7374-3_18.

Hardee, C., L.Ar val-Soliz, B.Hornstein and L.Zechiedrich,2017.Advances in Non-Viral DNA Vectors for Gene Therapy, gene (basel), 8:65, doi:10.3390/genes8020065.

Hendel, a., R.O.Bak, J.T.Clark, A.B.Kennedy, D.E.Ryan, S.Roy, I.Steinfeld, B.D.Lunstad, R.J.Kaiser, A.B.Wilkens, R.Bacchetta, A.Tsalenko, D.Dellinger, L.Bruhn and M.H.Porteus,2015.Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells.33, doi:10.1038/nbt.3290.

Hockemeyer, D., F.Soldner, C.Beard, Q.Gao, M.Mitalipova, R.C.DeKelver, G.E.Katibah, R.Amora, E.A.Boydston, B.Zeitler, X.Meng, J.C.Miller, L.Zhang, E.J.Rebar, P.D.Gregory, F.D.Urnov and R.Jaenisch,2009.Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleic acids, nature Biotechnology, 27:851-857, doi:10.1038/nbt.1562.

Hudecek, M.and Z.Ivics,2018.Non-viral therapeutic cell engineering with the Sleeping Beauty transposon system, contemporary genetics and developmental views, 52:100-108, doi:10.1016/j.gde.2018.06.003.

Kebriaei, P., H.Singh, M.H.Huls, M.J.Figliola, R.Bassett, S.Olivares, B.Jena, M.J.Dawson, P.R.Kumaresan, S.Su, S.Maiti, J.Dai, B.Moriarity, M., A.A.Forget, V.Senyukov, A.Orozco, T.Liu, J.McCarty, R.N.Jackson, J.S.Moyes, G.Rondon, M.Qazilbash, S.Ciurea, A.Alousi, Y.Nieto, K.Rezvani, D.Marin, U.Popat, C.Hosing, E.J.Shpall, H.Kantarjian, M.Keating, W.Wierda, K.A.Do, D.A.Largaespada, D.A.Lee, P.B.Hackett, R.E.Champlin and L.J.Cooper,2016.Phase I trials using Sleeping Beauty to generate CD19-specific CAR T cells, J.Clinopodium.Dev.126:3363-76, doi:10.1172/JCI86721.

Li, H., Y.Yang, W.Hong, M.Huang, M.Wu and X.Zhao,2020.Applications of genome editing technology in the targeted therapy of human diseases:mechanisms,advances and prospects, signal transduction and Targeted therapy, 5:1, doi:10.1038/s41392-019-0089-y.

Li, Y., R.Moysey, P.E.Molloy, A., L.Vuidepot, T.Mahon, E.Baston, S.Dunn, N.Liddy, J.Jacob, B.K.Jakobsen and J.M.Boulter,2005.Directed evolution of human T-cell receptors with picomolar affinities by phage display, nature Biotechnology, 23:349-354, doi:10.1038/nbt1070.

Liberzon, A., C.Birger, H.Thorvaldsd, ttri, M.Ghandi, J.P.Mesirov and P.Tamayo,2015.The Molecular Signatures Database Hallmark Gene Set Collection, cell systems, 1:417-425, doi:10.1016/j.cells.2015.12.004.

29.Loo,J.C.M.van der and J.F.Wright,2016.Progress and challenges in viral vector manufacturing, human molecular genetics 25:R42-R52, doi 10.1093/hmg/ddv451.

Luke, j A.E.Carnes, C.P.Hodgson and j.a. williams,2009.Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system, vaccine, 27:6454-6459, doi:10.1016/j.vaccine.2009.06.017.

Mandal, P.K., L.M.Ferreira, R.Collins, T.B.Meissner, C.L.Boutwell, M.Friesen, V.Vrbanac, B.S.Garrison, A.Stortchevoi, D.Bryder, K.Musunuru, H.Brand, A.M.Tager, T.M.Allen, M.E.Talkowski, D.J.Rossi and C.A.Cowan,2014.Efficient ablation of genes in human hematopoietic stem and effector cells using CRISPR/Cas9, 15, doi:10.1016/j.stem.2014.10.004.

32.Mansilla-Soto,J.,J.Eyquem,S.Haubner,M.Hamieh,J.Feucht,N.Paillon,A.E.Zucchetti,Z.Li,M.P.L.Lindenbergh, M.Saetersmoen, A.Dobrin, M.Maurin, A.Iyer, A.G.Angus, M.M.Miele, Z.Zhao, T.Giavridis, S.J.C.van der Stegen, F.Tamzalit, I.Rivi re, M.Huse, R.C.Hendrickson, C.Hivroz and M.Sadelain,2022.HLA-independent T cell receptors for targeting tumors with low antigen density, nature medicine, 1-8, doi:10.1038/s41591-021-01621-1.

33.Modlich,U.,S.Navarro,D.Zychlinski,T.Maetzig,S.Knoess,M.H.Brugman,A.Schambach,S.Charrier,A.Galy,A.J.Thrasher,J.

Bueren and c.baum.2009.inserted Transformation of HematopoieticCells by Self-inactivating Lentiviral and Gammaretroviral Vectors, molecular therapy 17:1919-1928, doi:10.1038/mt.2009.179.

Monjezi, R., C.Miskey, T.Gogishvili, M.Schleef, M.Schmeer, H.Einsele, Z.Ivics and M.Hudecek,2017.Enhanced CAR T-cell engineering usingnon-viral Sleeping Beauty transposition from minicircle vectors, leukemia 31:186-194, doi:10.1038/leu.2016.180.

Nguyen, D.N., T.L.Roth, P.J.Li, P.A.Chen, R.Apathy, M.R.Mamedov, L.T.Vo, V.R.Tobin, D.Goodman, E.Shifrut, J.A.Bluestone, J.M.Puck, F.C.Szoka and A.Marson2019.Polymer-stabilized Cas9 nanoparticles andmodified repair templates increase genome editing efficiency, nature Biotechnology, 38:44-49, doi:10.1038/s41587-019-0325-6.

Oh, S.A., A.seki and S.Rutz,2019.Ribonucleoprotein Transfection forCRISPR/Cas9-Mediated Gene Knockout in Primary T Cells, contemporary immunology views, 124:e69, doi:10.1002/cpim.69.

Redmond, D., A.Portan and O.Elemento,2016.Single-cell TCRseq: pairedrecovery of entire T-cell alpha and beta chain transcripts in T-cell receptorsfrom single-cell RNAseq, genome Med,8:80, doi:10.1186/s13073-016-0335-7.

38.Roth,T.L.,C.Puig-Saus,R.Yu,E.Shifrut,J.Carnevale,P.J.Li,J.Hiatt,J.

Saco,P.Krystofinski,H.Li,V.Tobin,D.N.Nguyen,M.R.Lee,A.L.Putnam,A.L.Ferris,J.W.Chen,J.-N.Schickel,L.Pellerin,D.Carmody,G.Alkorta-

Aranburu,D.del Gaudio,H.Matsumoto,M.Morell,Y.Mao,M.Cho,

R.M.Quadros,C.B.Gurumurthy,B.Smith,M.Haugwitz,S.H.Hughes,J.S.Weissman,K.Schumann,J.H.Esensten,A.P.May,A.Ashworth,G.M.Kupfer,S.A.W.Greeley,R.Bacchetta,E.Meffre,M.G.Roncarolo,N.

Romberg, K.C.Herold, A.Ribas, M.D.Leonetti and A.Marson,

2018.Reprogramming human T cell function and specificity with non-viralgenome targeting, nature. 559:405-409, doi:10.1038/s41586-018-0326-5.

Salganik, M., M.L. Hirsch and R.J.Samulski,2015.Mobile DNA III,Microbiol Spectr.3:829-851, doi:10.1128/Microbiolspec.mdna3-0052-2014.

Sather, B.D., G.S.R.Ibarra, K.Sommer, G.Curinga, M.Hale, I.F.Khan, S.Singh, Y.Song, K.Gwiazda, J.Sahni, J.Jarjour, A.Astrakhan, T.A.Wagner, A.M.Scharenberg and D.J.Rawlings,2015.Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template, science conversion medicine, 7:307 ray 156-307 ray 156, doi:10.1126/scitranslmed.aac5530.

41.Schober,K.,T.R.Müller,F.S.Grassmann, M.Effenberger, M.Poltorak, C.Stemberger, K.Schumann, T.L.Roth, A.Marson and D.H.Busch,2019.Orthotopic replacement of T-cell receptor α -and β -chains with preservation of near-physiological T-cell function, nature biomedical engineering, 1-11, doi:10.1038/s41551-019-0409-0.

Schuldt, N.J. and B.A.Binstadt,2019.Dual TCR T Cells:Identity Crisis or MultitaskersJ Immunol,202:637-644, doi:10.4049/jimminol.1800904.

Schumann, K., S.Lin, E.Boyer, D.R.Simeonov, M.Subramaniam, R.E.Gate, G.E.Haliburton, C.J.Ye, J.A.Bluestone, J.A.Doudna and A.Marson,2015.Generation of knock-in primary human T cells using Cas, ribonucleoproteins,112, doi:10.1073/pnas.1512503112.

Seki, a., and s.rutz.2018.optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells, journal of experimental medicine, 215:985-997, doi:10.1084/jem.20171626.

Shaner, N.C., G.G.Lambert, A.Chammas, Y.Ni, P.J.Cranfill, M.A.Baird, B.R.Sell, J.R.Allen, R.N.Day, M.Israelsson, M.W.Davidson and J.Wang,2013.A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum, nature methods, 10:407-409, doi:10.1038/nmet.2413.

46.Simeonov,D.R.,and A.Marson.2019.CRISPR-Based Tools in Immunity.Annual review of immunology，37:1–27，doi:10.1146/annurev-immunol-042718-041522。

Singh, N., J.Shi, C.H.June and M.Ruella,2017.genome-Editing Technologies in Adoptive T Cell Immunotherapy for Cancer, report on malignancy of the blood System, 12:522-529, doi:10.1007/s11899-017-0417-7.

Smith, J.R., S.Maguire, L.A.Davis, M.Alexander, F.Yang, S.Chandran, C.ffrench-Constant and R.A.Pedersen,2008.Robust,Persistent Transgene Expression in Human Embryonic Stem Cells Is Achieved with AAVS1-Targeted Integration, stem cells, 26:496-504, doi:10.1634/stemcells.2007-0039.

Stadtmauer, E.A., J.A.Fraietta, M.M.Davis, A.D.Cohen, K.L.Weber, E.Lancaster, P.A.Mangan, I.Kulikovskaya, M.Gupta, F.Chen, L.Tian, V.E.Gonzalez, J.Xu, I.Jung, J.J.Melenhorst, G.Plesa, J.Shea, T.Matlawski, A.Cervini, A.L.Gaymon, S.Desjardins, A.Lamontagne, J.Salas-Mckee, A.Fesnak, D.L.Siegel, B.L.Levine, J.K.Jadlowsky, R.M.Young, A.Chew, W. -T.Hwang, E.O.Hexner, B.M.Carreno, C.L.Nobles, F.D.Bushman, K.R.Parker, Y.Qi, A.T.Satpathy, H.Y.Chang, Y.Zhao, S.F.Lacey and C.H.June,2020.CRISPR-engineered T cells in patients with refractory cancer, science (New York N Y), 367:eaba7365, doi:10.1126/science.aba7365.

Su, S., B.Hu, J.Shao, B.Shen, J.Du, Y.Du, J.Zhou, L.Yu, L.Zhang, F.Chen, H.Sha, L.Cheng, F.Meng, Z.Zou, X.Huang and B.Liu,2016.CRISPR-Cas9 mediated efficient PD-1disruption on human primary T cells from cancer patients,6,doi:10.1038/srep20070.

Subramannian, A., P.Tamayo, V.K.Mootha, S.Mukherjee, B.L.Ebert, M.A.Gillette, A.Paulovich, S.L.Pomeroy, T.R.Golub, E.S.Lander and J.P.Mesirov,2005.Gene set enrichment analysis:A knowledge-based approach for interpreting genome-wide expression profiles, proc. Natl. Acad. Sci. USA, 102:15545-15550, doi:10.1073/pnas.0506580102.

Vakulsskas, C.A., D.P.Dever, G.R.Rettig, R.Turk, A.M.Jacobi, M.A.Collingwood, N.M.Bode, M.S.McNeill, S.Yan, J.Camarena, C.M.Lee, S.Park, V.Wiebking, R.O.Bak, N.Gomez-Ospina, M.Patel-Dinu, W.Sun, G.Bao, M.H.Porteus and M.A.Behlke,2018.A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells, nature medicine, 24:1216-1224, doi:10.1038/s41591-018-0137-0.

Wang, D., C.Zhang, B.Wang, B.Li, Q.Wang, D.Liu, H.Wang, Y.Zhou, L.Shi, F.Lan and Y.Wang,2019.Optimized CRISPR guide RNA design for two high-fidelity Cas9 variants by deep learning, nature communication, 10:4284, doi:10.1038/s41467-019-12281-8.

Wang, J., J.J.DeClercq, S.B.Hayward, P.W. -L.Li, D.A.Shivak, P.D.Gregory, G.Lee and M.C. Holmes,2016.Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor release, nucleic acids research, 44:e30-e30, doi:10.1093/nar/gkv1121.

Wang, X. And I. Rivi re,2016.Clinical manufacturing of CAR T cells:foundation of a promising therapy, molecular therapy oncolytic Agents, 3:16015, doi:10.1038/mto.2016.15.

56.Ward-Kavanagh,L.K.,W.W.Lin,J.R.And C.F.Ware,2016.The TNF Receptor Superfamily in Co-activating and Co-inhibitory Responses, immunity 44:1005-1019, doi:10.1016/j.immini.2016.04.019.

Williams, J.A., J.Luke, L.Johnson and C.Hodgson,2006,pDNAVACCultra vector family:high throughput intracellular targeting DNA vaccine plasmids, vaccine 24:4671-4676, doi:10.1016/j.vaccine.2005.08.033.

Zhang, C., J.Liu, J.F.Zhong and X.Zhang,2017.Engineering CAR-T cells, biomarker study, 5:22, doi:10.1186/s40364-017-0102-y.

Zhang, L., J.A.Zuris, R.Viswanathan, J.N.Edelstein, R.Turk, B.Thommandru, H.T.Rube, S.E.Glenn, M.A.Collingwood, N.M.Bode, S.F.Beaudoin, S.Lele, S.N.Scott, K.M.Wasko, S.Sexton, C.M.Borges, M.S.Schubert, G.L.Kurgan, M.S.McNeill, C.A.Fernandez, V.E.Myer, R.A.Morgan, M.A.Behlke and C.A.Vakulskas,2021.AsCas12a ultra nuclease facilitates the rapid generation of therapeutic cell medicines, nature Communication 12:3908, doi:10.1038/s41467-021-24017-8.

All of the teachings of each of the references cited herein are incorporated by reference in their entirety.

Table 18.

/>

Sequence listing

<110> Gene Talck Co

S. Roots

B.j.hali

S.midehydic acid

S.ao

D-zodiac

K.H.Senjie

<120> efficient TCR Gene editing in T lymphocytes

<130> 048893-532001WO

<150> 63/323,065

<151> 2022-03-24

<150> 63/165,509

<151> 2021-03-24

<160> 108

<170> patent in version 3.5

<210> 1

<211> 3298

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(3298)

<400> 1

gagcaatctc ctggtaatgt gatagatttc ccaacttaat gccaacatac cataaacctc 60

ccattctgct aatgcccagc ctaagttggg gagaccactc cagattccaa gatgtacagt 120

ttgctttgct gggccttttt cccatgcctg cctttactct gccagagtta tattgctggg 180

gttttgaaga agatcctatt aaataaaaga ataagcagta ttattaagta gccctgcatt 240

tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac gttcactgaa atcatggcct 300

cttggccaag attgatagct tgtgcctgtc cctgagtccc agtccatcac gagcagctgg 360

tttctaagat gctatttccc gtataaagca tgagaccgtg acttgccagc cccacagagc 420

cccgcccttg tccatcactg gcatctggac tccagcctgg gttggggcaa agagggaaat 480

gagatcatgt cctaaccctg atcctcttgt cccacagata tccagaaccc tgaccctgcc 540

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 600

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 660

gtgctagacg gcagcggcgc caccaacttc agcctgctga agcaggccgg cgacgtggaa 720

gagaaccccg ggcccatgga cagctggacc ttctgctgcg tgagcctgtg catcctggtg 780

gccaagcaca cagacgcagg cgtgatccag agcccaaggc acgaggtgac agagatgggc 840

caggaggtga ccctgaggtg taagcccatc agcggccaca actccctgtt ctggtatagg 900

cagaccatga tgcggggact ggagctgctg atctacttca acaacaacgt gcccatcgat 960

gacagcggca tgcccgagga cagattcagc gccaagatgc ccaacgccag cttcagcacc 1020

ctgaagatcc agcccagcga gcccagagac tccgccgtgt atttctgcgc cagcagcagc 1080

ctgcagtatg agcagtactt cggcccaggc acacgcctga ccgtgacaga ggatctgaag 1140

aacgtgttcc cccctgaggt ggccgtgttt gagccttctg aggccgagat cagccacacc 1200

cagaaggcca ccctggtgtg cctggcaacc ggcttctacc cagaccacgt ggagctgagc 1260

tggtgggtga acggcaagga ggtgcacagc ggcgtgtcca cagacccaca gcccctgaag 1320

gagcagcccg ccctgaatga ttctagatat tgcctgtcta gccggctgag agtgagcgcc 1380

accttttggc agaaccctag gaatcacttc cgctgtcagg tgcagtttta cggcctgagc 1440

gagaatgacg agtggaccca ggatagggcc aagcctgtga cacagatcgt gtccgccgag 1500

gcatggggaa gggcagattg cggcttcaca agcgagtcct accagcaggg cgtgctgtcc 1560

gccaccatcc tgtatgagat cctgctgggc aaggccacac tgtacgccgt gctggtgagc 1620

gccctggtgc tgatggccat ggtgaagagg aaggactcca ggggccgcgc aaagagaggc 1680

tccggtgcta ccaatttctc actgttgaaa caagcgggcg atgttgaaga aaatcccggt 1740

ccaatgaccc gggtgagcct gctgtgggcc gtggtggtga gcacctgcct ggagagcggc 1800

atggcccaga ccgtgaccca gtctcagccc gagatgagcg tgcaggaggc cgagaccgtg 1860

accctgagct gtacctacga caccagcgag aacaactact acctgttctg gtacaagcag 1920

ccccccagcc ggcagatgat cctggtgatc cggcaggagg cctacaagca gcagaacgcc 1980

accgagaaca gattctctgt gaacttccag aaggccgcca agagcttcag cctgaagatc 2040

agcgactccc agctgggcga taccgccatg tatttctgcg ccttcatggg ctactacggc 2100

ggcagccagg gcaatctgat ctttggcaag ggcacaaagc tgagcgtgaa gcccaacatc 2160

cagaaccccg accctgccgt gtaccagctg agggactcca agtctagcga taagagcgtg 2220

tgcctgttca ccgactttga ttcccagaca aacgtgagcc agagcaagga ctctgacgtg 2280

tacatcaccg acaagacagt gctggatatg aggtctatgg acttcaagag caacagtgct 2340

gtggcctgga gcaacaaatc tgactttgca tgtgcaaacg ccttcaacaa cagcattatt 2400

ccagaagaca ccttcttccc cagcccagaa agttcctgtg atgtcaagct ggtcgagaaa 2460

agctttgaaa cagatacgaa cctaaacttt caaaacctgt cagtgattgg gttccgaatc 2520

ctcctcctga aagtggccgg gtttaatctg ctcatgacgc tgcggctgtg gtccagctga 2580

ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 2640

cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 2700

atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 2760

ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gaatgaggtc 2820

tatggacttc aagagcaaca gtgctgtggc ctggagcaac aaatctgact ttgcatgtgc 2880

aaacgccttc aacaacagca ttattccaga agacaccttc ttccccagcc caggtaaggg 2940

cagctttggt gccttcgcag gctgtttcct tgcttcagga atggccaggt tctgcccaga 3000

gctctggtca atgatgtcta aaactcctct gattggtggt ctcggcctta tccattgcca 3060

ccaaaaccct ctttttacta agaaacagtg agccttgttc tggcagtcca gagaatgaca 3120

cgggaaaaaa gcagatgaag agaaggtggc aggagagggc acgtggccca gcctcagtct 3180

ctccaactga gttcctgcct gcctgccttt gctcagactg tttgcccctt actgctcttc 3240

taggcctcat tctaagcccc ttctccaagt tgcctctcct tatttctccc tgtctgcc 3298

<210> 2

<211> 2781

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2781)

<400> 2

gagcaatctc ctggtaatgt gatagatttc ccaacttaat gccaacatac cataaacctc 60

ccattctgct aatgcccagc ctaagttggg gagaccactc cagattccaa gatgtacagt 120

ttgctttgct gggccttttt cccatgcctg cctttactct gccagagtta tattgctggg 180

gttttgaaga agatcctatt aaataaaaga ataagcagta ttattaagta gccctgcatt 240

tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac gttcactgaa atcatggcct 300

cttggccaag attgatagct tgtgcctgtc cctgagtccc agtccatcac gagcagctgg 360

tttctaagat gctatttccc gtataaagca tgagaccgtg acttgccagc cccacagagc 420

cccgcccttg tccatcactg gcatctggac tccagcctgg gttggggcaa agagggaaat 480

gagatcatgt cctaaccctg atcctcttgt cccacagata tccagaaccc tgaccctgcc 540

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 600

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 660

gtgctagacg gaagcggaga aggtagaggt tctctcctca cttgtggtga tgttgaagaa 720

aaccctggtc caatgagcat aggattgctg tgctgtgcag ccctgtccct tttgtgggca 780

gggccagtca acgcgggcgt tacgcagacc ccaaaattcc aagtcctcaa gacgggccaa 840

tccatgacat tgcaatgtgc gcaggatatg aatcacgaat acatgagttg gtaccgccaa 900

gaccccggaa tgggactccg gcttatacat tatagtgttg gcgctggaat cactgaccag 960

ggagaagtgc cgaatggata caacgtctcc aggagcacca cagaggactt tccgctgcgc 1020

ctcctgagcg cggctccgtc acaaaccagt gtttactttt gtgcatcaag ttatgtaggc 1080

aacacaggag aactcttttt tggcgaaggt tccaggttga ctgttctcga ggacctcaaa 1140

aatgtttttc caccagaggt cgcagtattt gagcctagtg aggctgaaat ttctcacact 1200

cagaaggcga ccctcgtctg tctggcgaca ggattttacc ccgatcatgt tgaactttcc 1260

tggtgggtca acgggaagga ggttcacagt ggggtatcaa ctgatcccca accactgaag 1320

gaacagccag cactcaatga ctcacggtat tgcctttctt ccaggctgag agtttctgct 1380

acgttctggc agaatcctag aaatcatttc cgatgccagg tccaattcta cggtcttagc 1440

gaaaatgacg agtggactca ggacagggca aagcccgtga cgcaaattgt gtcagccgag 1500

gcttggggca gagcggactg cggcttcacg tcagagagtt accagcaggg tgttctcagt 1560

gcgactatcc tgtacgaaat attgcttggc aaggcaacgt tgtatgcagt tctggtctct 1620

gctctcgtac tcatggcaat ggtaaagcgg aaagattcca gaggccgcgc caagcgcggc 1680

tccggtgcta ccaatttctc actgttgaaa caagcgggcg atgttgaaga aaatcccggt 1740

ccaatggaga cattgctcgg cttgttgatc ttgtggctcc agctgcaatg ggtatcatct 1800

aagcaagagg ttacgcaaat tcctgcagct cttagcgtac cggagggcga gaatttggtc 1860

cttaattgtt ctttcaccga ctcagcgatc tataatctcc aatggtttcg acaagacccc 1920

ggtaaaggcc tgacctcttt gttgctgata cagagttccc agcgcgagca gacgtccggt 1980

aggcttaatg caagtctgga taagagctct ggacgctcaa cactctacat agctgcttca 2040

caaccggggg atagtgcaac ttatctgtgt gctgtgcggc cactttatgg cggatcctac 2100

attcctactt tcgggagggg aactagtctc atcgtgcacc catacattca gaatccagac 2160

cctgcggtgt accagctgag ggactcaaaa agttctgata agtccgtctg cctgttcact 2220

gactttgact ctcaaacaaa tgtatcccag tctaaagatt ccgatgttta catcaccgac 2280

aagaccgtgc tcgatatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 2340

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 2400

ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg caggctgttt ccttgcttca 2460

ggaatggcca ggttctgccc agagctctgg tcaatgatgt ctaaaactcc tctgattggt 2520

ggtctcggcc ttatccattg ccaccaaaac cctcttttta ctaagaaaca gtgagccttg 2580

ttctggcagt ccagagaatg acacgggaaa aaagcagatg aagagaaggt ggcaggagag 2640

ggcacgtggc ccagcctcag tctctccaac tgagttcctg cctgcctgcc tttgctcaga 2700

ctgtttgccc cttactgctc ttctaggcct cattctaagc cccttctcca agttgcctct 2760

ccttatttct ccctgtctgc c 2781

<210> 3

<211> 2615

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2615)

<400> 3

atattgctgg ggttttgaag aagatcctat taaataaaag aataagcagt attattaagt 60

agccctgcat ttcaggtttc cttgagtggc aggccaggcc tggccgtgaa cgttcactga 120

aatcatggcc tcttggccaa gattgatagc ttgtgcctgt ccctgagtcc cagtccatca 180

cgagcagctg gtttctaaga tgctatttcc cgtataaagc atgagaccgt gacttgccag 240

ccccacagag ccccgccctt gtccatcact ggcatctgga ctccagcctg ggttggggca 300

aagagggaaa tgagatcatg tcctaaccct gatcctcttg tcccacagat atccagaacc 360

ctgaccctgc cgtgtaccag ctgagagact ctaaatccag tgacaagtct gtctgcctat 420

tcaccgattt tgattctcaa acaaatgtgt cacaaagtaa ggattctgat gtgtatatca 480

cagacaaaac tgtgctagac ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatgagca accaggtgct gtgctgcgtg gtgctgtgct 600

ttctgggagc caacaccgtg gacggcggca tcacccagag cccaaagtac ctgttccgga 660

aggagggcca gaacgtgacc ctgagctgtg agcagaacct gaaccacgac gccatgtact 720

ggtataggca ggaccccgga cagggactgc ggctgatcta ctacagccag atcgtgaacg 780

acttccagaa gggcgacatc gccgagggct acagcgtgag ccgggagaag aaggagagct 840

tccccctgac agtgaccagc gcccagaaga accccaccgc cttctatctg tgcgccagct 900

ccgtgaccgg cggcttctct tatgagcagt acttcggccc aggcacacgc ctgaccgtga 960

cagaggatct gaagaacgtg ttcccccctg aggtggccgt gtttgagcct tctgaggccg 1020

agatcagcca cacccagaag gccaccctgg tgtgcctggc aaccggcttc tacccagacc 1080

acgtggagct gagctggtgg gtgaacggca aggaggtgca cagcggcgtg tccacagacc 1140

cacagcccct gaaggagcag cccgccctga atgattctag atattgcctg tctagccggc 1200

tgagagtgag cgccaccttt tggcagaacc ctaggaatca cttccgctgt caggtgcagt 1260

tttacggcct gagcgagaat gacgagtgga cccaggatag ggccaagcct gtgacacaga 1320

tcgtgtccgc cgaggcatgg ggaagggcag attgcggctt cacaagcgag tcctaccagc 1380

agggcgtgct gtccgccacc atcctgtatg agatcctgct gggcaaggcc acactgtacg 1440

ccgtgctggt gagcgccctg gtgctgatgg ccatggtgaa gaggaaggac tccaggggcc 1500

gcgcaaagag aggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1560

aagaaaatcc cggtccaatg gccggcatcc gggccctgtt catgtacctg tggctgcagc 1620

tggactgggt gtcccggggc gagagcgtgg gcctgcacct gcccaccctg agcgtgcagg 1680

agggcgataa cagcatcatc aactgtgcct acagcaacag cgccagcgac tacttcatct 1740

ggtacaagca ggagagcggc aagggccccc agttcatcat cgacatccgg agcaacatgg 1800

acaagcggca gggccagaga gtgaccgtgc tgctgaataa gaccgtgaag cacctgagcc 1860

tgcagatcgc cgccacccag ccaggcgatt ctgccgtgta tttctgcgcc gagaacatcg 1920

cctacagcgg cagccggctg acctttggcg agggcacaca gctgaccgtg aaccccgaca 1980

tccagaaccc cgaccctgcc gtgtaccagc tgagggactc caagtctagc gataagagcg 2040

tgtgcctgtt caccgacttt gattcccaga caaacgtgag ccagagcaag gactctgacg 2100

tgtacatcac cgacaagaca gtgctggata tgaggtctat ggacttcaag agcaacagtg 2160

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 2220

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 2280

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 2340

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 2400

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 2460

aggtggcagg agagggcacg tggcccagcc tcagtctctc caactgagtt cctgcctgcc 2520

tgcctttgct cagactgttt gccccttact gctcttctag gcctcattct aagccccttc 2580

tccaagttgc ctctccttat ttctccctgt ctgcc 2615

<210> 4

<211> 2597

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2597)

<400> 4

atattgctgg ggttttgaag aagatcctat taaataaaag aataagcagt attattaagt 60

agccctgcat ttcaggtttc cttgagtggc aggccaggcc tggccgtgaa cgttcactga 120

aatcatggcc tcttggccaa gattgatagc ttgtgcctgt ccctgagtcc cagtccatca 180

cgagcagctg gtttctaaga tgctatttcc cgtataaagc atgagaccgt gacttgccag 240

ccccacagag ccccgccctt gtccatcact ggcatctgga ctccagcctg ggttggggca 300

aagagggaaa tgagatcatg tcctaaccct gatcctcttg tcccacagat atccagaacc 360

ctgaccctgc cgtgtaccag ctgagagact ctaaatccag tgacaagtct gtctgcctat 420

tcaccgattt tgattctcaa acaaatgtgt cacaaagtaa ggattctgat gtgtatatca 480

cagacaaaac tgtgctagac ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatgagcc tgggcctgct gtgctgcggc gccttcagcc 600

tgctgtgggc cggccccgtg aacgcaggcg tgacccagac cccaaagttc cgggtgctga 660

agaccggcca gagcatgacc ctgctgtgtg cccaggacat gaaccacgag tacatgtact 720

ggtataggca ggaccccgga atgggactgc ggctgatcca ctacagcgtg ggcgagggca 780

ccaccgccaa gggcgaggtg cccgacggct acaacgtgag ccggctgaag aagcagaact 840

tcctgctggg cctggagagc gccgccccca gccagaccag cgtgtatttc tgcgccagct 900

cctacggctt caaccagccc cagcacttcg gcgacggcac acgcctgagc atcctggagg 960

atctgaacaa ggtgttcccc cctgaggtgg ccgtgtttga gccttctgag gccgagatca 1020

gccacaccca gaaggccacc ctggtgtgcc tggcaaccgg cttcttccca gaccacgtgg 1080

agctgagctg gtgggtgaac ggcaaggagg tgcacagcgg cgtgtccaca gacccacagc 1140

ccctgaagga gcagcccgcc ctgaatgatt ctagatattg cctgtctagc cggctgagag 1200

tgagcgccac cttttggcag aaccctagga atcacttccg ctgtcaggtg cagttttacg 1260

gcctgagcga gaatgacgag tggacccagg atagggccaa gcctgtgaca cagatcgtgt 1320

ccgccgaggc atggggaagg gcagattgcg gcttcacaag cgtgtcctac cagcagggcg 1380

tgctgtccgc caccatcctg tatgagatcc tgctgggcaa ggccacactg tacgccgtgc 1440

tggtgagcgc cctggtgctg atggccatgg tgaagaggaa ggacttccgc gcaaagagag 1500

gctccggtgc taccaatttc tcactgttga aacaagcggg cgatgttgaa gaaaatcccg 1560

gtccaatgaa gagcctgcgg gtgctgctgg tgatcctgtg gctgcagctg agctgggtgt 1620

ggtcccagca gaaggaggtg gagcagaaca gcggccccct gagcgtgccc gagggcgcca 1680

tcgccagcct gaactgtacc tacagcgacc ggggcagcca gagcttcttc tggtacagac 1740

agtacagcgg caagagcccc gagctgatca tgttcatcta cagcaacggc gacaaggagg 1800

acggcagatt caccgcccag ctgaataagg ccagccagta cgtgagcctg ctgatccggg 1860

actcccagcc aagcgattct gccacctatc tgtgcgccgt gccctacaac aacaacgaca 1920

tgcggtttgg cgccggcaca agactgaccg tgaagcccaa catccagaac cccgaccctg 1980

ccgtgtacca gctgagggac tccaagtcta gcgataagag cgtgtgcctg ttcaccgact 2040

ttgattccca gacaaacgtg agccagagca aggactctga cgtgtacatc accgacaaga 2100

cagtgctgga tatgaggtct atggacttca agagcaacag tgctgtggcc tggagcaaca 2160

aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa gacaccttct 2220

tccccagccc aggtaagggc agctttggtg ccttcgcagg ctgtttcctt gcttcaggaa 2280

tggccaggtt ctgcccagag ctctggtcaa tgatgtctaa aactcctctg attggtggtc 2340

tcggccttat ccattgccac caaaaccctc tttttactaa gaaacagtga gccttgttct 2400

ggcagtccag agaatgacac gggaaaaaag cagatgaaga gaaggtggca ggagagggca 2460

cgtggcccag cctcagtctc tccaactgag ttcctgcctg cctgcctttg ctcagactgt 2520

ttgcccctta ctgctcttct aggcctcatt ctaagcccct tctccaagtt gcctctcctt 2580

atttctccct gtctgcc 2597

<210> 5

<211> 2790

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2790)

<400> 5

gagcaatctc ctggtaatgt gatagatttc ccaacttaat gccaacatac cataaacctc 60

ccattctgct aatgcccagc ctaagttggg gagaccactc cagattccaa gatgtacagt 120

ttgctttgct gggccttttt cccatgcctg cctttactct gccagagtta tattgctggg 180

gttttgaaga agatcctatt aaataaaaga ataagcagta ttattaagta gccctgcatt 240

tcaggtttcc ttgagtggca ggccaggcct ggccgtgaac gttcactgaa atcatggcct 300

cttggccaag attgatagct tgtgcctgtc cctgagtccc agtccatcac gagcagctgg 360

tttctaagat gctatttccc gtataaagca tgagaccgtg acttgccagc cccacagagc 420

cccgcccttg tccatcactg gcatctggac tccagcctgg gttggggcaa agagggaaat 480

gagatcatgt cctaaccctg atcctcttgt cccacagata tccagaaccc tgaccctgcc 540

gtgtaccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 600

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 660

gtgctagacg gaagcggaga aggtagaggt tctctcctca cttgtggtga tgttgaagaa 720

aaccctggtc caatggacag ctggaccttc tgctgcgtga gcctgtgcat cctggtggcc 780

aagcacacag acgcaggcgt gatccagagc ccaaggcacg aggtgacaga gatgggccag 840

gaggtgaccc tgaggtgtaa gcccatcagc ggccacaact ccctgttctg gtataggcag 900

accatgatgc ggggactgga gctgctgatc tacttcaaca acaacgtgcc catcgatgac 960

agcggcatgc ccgaggacag attcagcgcc aagatgccca acgccagctt cagcaccctg 1020

aagatccagc ccagcgagcc cagagactcc gccgtgtatt tctgcgccag cagcagcctg 1080

cagtatgagc agtacttcgg cccaggcaca cgcctgaccg tgacagagga tctgaagaac 1140

gtgttccccc ctgaggtggc cgtgtttgag ccttctgagg ccgagatcag ccacacccag 1200

aaggccaccc tggtgtgcct ggcaaccggc ttctacccag accacgtgga gctgagctgg 1260

tgggtgaacg gcaaggaggt gcacagcggc gtgtccacag acccacagcc cctgaaggag 1320

cagcccgccc tgaatgattc tagatattgc ctgtctagcc ggctgagagt gagcgccacc 1380

ttttggcaga accctaggaa tcacttccgc tgtcaggtgc agttttacgg cctgagcgag 1440

aatgacgagt ggacccagga tagggccaag cctgtgacac agatcgtgtc cgccgaggca 1500

tggggaaggg cagattgcgg cttcacaagc gagtcctacc agcagggcgt gctgtccgcc 1560

accatcctgt atgagatcct gctgggcaag gccacactgt acgccgtgct ggtgagcgcc 1620

ctggtgctga tggccatggt gaagaggaag gactccaggg gccgcgcaaa gagaggctcc 1680

ggtgctacca atttctcact gttgaaacaa gcgggcgatg ttgaagaaaa tcccggtcca 1740

atgacccggg tgagcctgct gtgggccgtg gtggtgagca cctgcctgga gagcggcatg 1800

gcccagaccg tgacccagtc tcagcccgag atgagcgtgc aggaggccga gaccgtgacc 1860

ctgagctgta cctacgacac cagcgagaac aactactacc tgttctggta caagcagccc 1920

cccagccggc agatgatcct ggtgatccgg caggaggcct acaagcagca gaacgccacc 1980

gagaacagat tctctgtgaa cttccagaag gccgccaaga gcttcagcct gaagatcagc 2040

gactcccagc tgggcgatac cgccatgtat ttctgcgcct tcatgggcta ctacggcggc 2100

agccagggca atctgatctt tggcaagggc acaaagctga gcgtgaagcc caacatccag 2160

aaccccgacc ctgccgtgta ccagctgagg gactccaagt ctagcgataa gagcgtgtgc 2220

ctgttcaccg actttgattc ccagacaaac gtgagccaga gcaaggactc tgacgtgtac 2280

atcaccgaca agacagtgct ggatatgagg tctatggact tcaagagcaa cagtgctgtg 2340

gcctggagca acaaatctga ctttgcatgt gcaaacgcct tcaacaacag cattattcca 2400

gaagacacct tcttccccag cccaggtaag ggcagctttg gtgccttcgc aggctgtttc 2460

cttgcttcag gaatggccag gttctgccca gagctctggt caatgatgtc taaaactcct 2520

ctgattggtg gtctcggcct tatccattgc caccaaaacc ctctttttac taagaaacag 2580

tgagccttgt tctggcagtc cagagaatga cacgggaaaa aagcagatga agagaaggtg 2640

gcaggagagg gcacgtggcc cagcctcagt ctctccaact gagttcctgc ctgcctgcct 2700

ttgctcagac tgtttgcccc ttactgctct tctaggcctc attctaagcc ccttctccaa 2760

gttgcctctc cttatttctc cctgtctgcc 2790

<210> 6

<211> 2612

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2612)

<400> 6

atattgctgg ggttttgaag aagatcctat taaataaaag aataagcagt attattaagt 60

agccctgcat ttcaggtttc cttgagtggc aggccaggcc tggccgtgaa cgttcactga 120

aatcatggcc tcttggccaa gattgatagc ttgtgcctgt ccctgagtcc cagtccatca 180

cgagcagctg gtttctaaga tgctatttcc cgtataaagc atgagaccgt gacttgccag 240

ccccacagag ccccgccctt gtccatcact ggcatctgga ctccagcctg ggttggggca 300

aagagggaaa tgagatcatg tcctaaccct gatcctcttg tcccacagat atccagaacc 360

ctgaccctgc cgtgtaccag ctgagagact ctaaatccag tgacaagtct gtctgcctat 420

tcaccgattt tgattctcaa acaaatgtgt cacaaagtaa ggattctgat gtgtatatca 480

cagacaaaac tgtgctagac ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatgggca cccggctgtt cttctacgtg gccctgtgcc 600

tgctgtgggc cggccaccgg gacgcaggca tcacccagag cccaaggtac aagatcacag 660

agaccggccg gcaggtgacc ctgatgtgtc accagacctg gagccacagc tacatgttct 720

ggtataggca ggacctggga cacggactgc ggctgatcta ctacagcgcc gccgccgaca 780

tcaccgataa gggcgaggtg cccgacggct acgtggtgag ccggagcaag accgagaact 840

tccccctgac actggagagc gccacccgga gccagaccag cgtgtatttc tgcgccagct 900

ccgagggcgg cctgaacacc gaggccttct tcggccaggg cacacgcctg accgtggtgg 960

aggatctgaa caaggtgttc ccccctgagg tggccgtgtt tgagccttct gaggccgaga 1020

tcagccacac ccagaaggcc accctggtgt gcctggcaac cggcttcttc ccagaccacg 1080

tggagctgag ctggtgggtg aacggcaagg aggtgcacag cggcgtgtcc acagacccac 1140

agcccctgaa ggagcagccc gccctgaatg attctagata ttgcctgtct agccggctga 1200

gagtgagcgc caccttttgg cagaacccta ggaatcactt ccgctgtcag gtgcagtttt 1260

acggcctgag cgagaatgac gagtggaccc aggatagggc caagcctgtg acacagatcg 1320

tgtccgccga ggcatgggga agggcagatt gcggcttcac aagcgtgtcc taccagcagg 1380

gcgtgctgtc cgccaccatc ctgtatgaga tcctgctggg caaggccaca ctgtacgccg 1440

tgctggtgag cgccctggtg ctgatggcca tggtgaagag gaaggacttc cgcgcaaaga 1500

gaggctccgg tgctaccaat ttctcactgt tgaaacaagc gggcgatgtt gaagaaaatc 1560

ccggtccaat gagcctgagc agcctgctga aggtggtgac cgccagcctg tggctgggcc 1620

ccggcatcgc ccagaagatc acccagaccc agcccggcat gttcgtgcag gagaaggagg 1680

ccgtgaccct ggactgtacc tacgacacca gcgaccagag ctacggcctg ttctggtaca 1740

agcagcccag cagcggcgag atgatcttcc tgatctacca gggcagctac gacgagcaga 1800

acgccaccga gggcagatac tctctgaact tccagaaggc ccggaagagc gccaacctgg 1860

tgatcagcgc ctcccagctg ggcgattctg ccatgtattt ctgcgccatc gccgagggca 1920

caggcttcca gaagctggtg tttggcaccg gcacaagact gctggtgagc cccaacatcc 1980

agaaccccga ccctgccgtg taccagctga gggactccaa gtctagcgat aagagcgtgt 2040

gcctgttcac cgactttgat tcccagacaa acgtgagcca gagcaaggac tctgacgtgt 2100

acatcaccga caagacagtg ctggatatga ggtctatgga cttcaagagc aacagtgctg 2160

tggcctggag caacaaatct gactttgcat gtgcaaacgc cttcaacaac agcattattc 2220

cagaagacac cttcttcccc agcccaggta agggcagctt tggtgccttc gcaggctgtt 2280

tccttgcttc aggaatggcc aggttctgcc cagagctctg gtcaatgatg tctaaaactc 2340

ctctgattgg tggtctcggc cttatccatt gccaccaaaa ccctcttttt actaagaaac 2400

agtgagcctt gttctggcag tccagagaat gacacgggaa aaaagcagat gaagagaagg 2460

tggcaggaga gggcacgtgg cccagcctca gtctctccaa ctgagttcct gcctgcctgc 2520

ctttgctcag actgtttgcc ccttactgct cttctaggcc tcattctaag ccccttctcc 2580

aagttgcctc tccttatttc tccctgtctg cc 2612

<210> 7

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 7

agagtctctc agctggtaca 20

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 8

tggatttaga gtctctcagc 20

<210> 9

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 9

acaaaactgt gctagacatg 20

<210> 10

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 10

cttcaagagc aacagtgctg 20

<210> 11

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 11

taaacccggc cactttcagg 20

<210> 12

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 12

ttaatctgct catgacgctg 20

<210> 13

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 13

gctggtacac ggcagggtca 20

<210> 14

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 14

ctctcagctg gtacacggca 20

<210> 15

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 15

taggcagaca gacttgtcac 20

<210> 16

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 16

aagttcctgt gatgtcaagc 20

<210> 17

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 17

gtcgagaaaa gctttgaaac 20

<210> 18

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 18

ttcggaaccc aatcactgac 20

<210> 19

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 19

ccgaatcctc ctcctgaaag 20

<210> 20

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 20

tcctcctcct gaaagtggcc 20

<210> 21

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 21

cgtcatgagc agattaaacc 20

<210> 22

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 22

ctgcggctgt ggtccagctg 20

<210> 23

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 23

gaaaaacgtg ttcccaccca 20

<210> 24

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 24

caaacacagc gaccttgggt 20

<210> 25

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 25

ccacacccaa aaggccacac 20

<210> 26

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 26

tgtggccagg cacaccagtg 20

<210> 27

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 27

gtggtcgggg tagaagcctg 20

<210> 28

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 28

aggcttctac cccgaccacg 20

<210> 29

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 29

cccaccagct cagctccacg 20

<210> 30

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 30

ccacgtggag ctgagctggt 20

<210> 31

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 31

gagctggtgg gtgaatggga 20

<210> 32

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 32

ctggtgggtg aatgggaagg 20

<210> 33

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 33

aatgggaagg aggtgcacag 20

<210> 34

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 34

tgggaaggag gtgcacagtg 20

<210> 35

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 35

tatctggagt cattgagggc 20

<210> 36

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 36

gtatctggag tcattgaggg 20

<210> 37

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 37

ggcagtatct ggagtcattg 20

<210> 38

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 38

aggtggccga gaccctcagg 20

<210> 39

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 39

gacagcggaa gtggttgcgg 20

<210> 40

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 40

cgtagaactg gacttgacag 20

<210> 41

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 41

ggctctcgga gaatgacgag 20

<210> 42

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 42

ggagaatgac gagtggaccc 20

<210> 43

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 43

cacccagatc gtcagcgccg 20

<210> 44

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 44

tggctcaaac acagcgacct 20

<210> 45

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 45

agagatctcc cacacccaaa 20

<210> 46

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 46

accacgtgga gctgagctgg 20

<210> 47

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 47

tgacagcgga agtggttgcg 20

<210> 48

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 48

atcgtcagcg ccgaggcctg 20

<210> 49

<211> 18

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> MISC_FEATURE

<222> (1)..(18)

<400> 49

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 50

<211> 19

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> MISC_FEATURE

<222> (1)..(19)

<400> 50

Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn

1 5 10 15

Pro Gly Pro

<210> 51

<211> 20

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> MISC_FEATURE

<222> (1)..(20)

<400> 51

Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser

1 5 10 15

Asn Pro Gly Pro

20

<210> 52

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> MISC_FEATURE

<222> (1)..(22)

<400> 52

Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Glu

1 5 10 15

Val Ser Asn Pro Gly Pro

20

<210> 53

<211> 278

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> MISC_FEATURE

<222> (1)..(278)

<400> 53

Met Thr Arg Val Ser Leu Leu Trp Ala Val Val Val Ser Thr Cys Leu

1 5 10 15

Glu Ser Gly Met Ala Gln Thr Val Thr Gln Ser Gln Pro Glu Met Ser

20 25 30

Val Gln Glu Ala Glu Thr Val Thr Leu Ser Cys Thr Tyr Asp Thr Ser

35 40 45

Glu Asn Asn Tyr Tyr Leu Phe Trp Tyr Lys Gln Pro Pro Ser Arg Gln

50 55 60

Met Ile Leu Val Ile Arg Gln Glu Ala Tyr Lys Gln Gln Asn Ala Thr

65 70 75 80

Glu Asn Arg Phe Ser Val Asn Phe Gln Lys Ala Ala Lys Ser Phe Ser

85 90 95

Leu Lys Ile Ser Asp Ser Gln Leu Gly Asp Thr Ala Met Tyr Phe Cys

100 105 110

Ala Phe Met Gly Tyr Tyr Gly Gly Ser Gln Gly Asn Leu Ile Phe Gly

115 120 125

Lys Gly Thr Lys Leu Ser Val Lys Pro Asn Ile Gln Asn Pro Asp Pro

130 135 140

Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser Val Cys

145 150 155 160

Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser Lys Asp

165 170 175

Ser Asp Val Tyr Ile Thr Asp Lys Thr Val Leu Asp Met Arg Ser Met

180 185 190

Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser Asp Phe

195 200 205

Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe

210 215 220

Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu Lys Ser

225 230 235 240

Phe Glu Thr Asp Thr Asn Leu Asn Phe Gln Asn Leu Ser Val Ile Gly

245 250 255

Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu Met Thr

260 265 270

Leu Arg Leu Trp Ser Ser

275

<210> 54

<211> 310

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> MISC_FEATURE

<222> (1)..(310)

<400> 54

Met Asp Ser Trp Thr Phe Cys Cys Val Ser Leu Cys Ile Leu Val Ala

1 5 10 15

Lys His Thr Asp Ala Gly Val Ile Gln Ser Pro Arg His Glu Val Thr

20 25 30

Glu Met Gly Gln Glu Val Thr Leu Arg Cys Lys Pro Ile Ser Gly His

35 40 45

Asn Ser Leu Phe Trp Tyr Arg Gln Thr Met Met Arg Gly Leu Glu Leu

50 55 60

Leu Ile Tyr Phe Asn Asn Asn Val Pro Ile Asp Asp Ser Gly Met Pro

65 70 75 80

Glu Asp Arg Phe Ser Ala Lys Met Pro Asn Ala Ser Phe Ser Thr Leu

85 90 95

Lys Ile Gln Pro Ser Glu Pro Arg Asp Ser Ala Val Tyr Phe Cys Ala

100 105 110

Ser Ser Ser Leu Gln Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu

115 120 125

Thr Val Thr Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val

130 135 140

Phe Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu

145 150 155 160

Val Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp

165 170 175

Trp Val Asn Gly Lys Glu Val His Ser Gly Val Ser Thr Asp Pro Gln

180 185 190

Pro Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Cys Leu Ser

195 200 205

Ser Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asn Pro Arg Asn His

210 215 220

Phe Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp

225 230 235 240

Thr Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala

245 250 255

Trp Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly

260 265 270

Val Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr

275 280 285

Leu Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys

290 295 300

Arg Lys Asp Ser Arg Gly

305 310

<210> 55

<211> 837

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(837)

<400> 55

atgacccggg tgagcctgct gtgggccgtg gtggtgagca cctgcctgga gagcggcatg 60

gcccagaccg tgacccagtc tcagcccgag atgagcgtgc aggaggccga gaccgtgacc 120

ctgagctgta cctacgacac cagcgagaac aactactacc tgttctggta caagcagccc 180

cccagccggc agatgatcct ggtgatccgg caggaggcct acaagcagca gaacgccacc 240

gagaacagat tctctgtgaa cttccagaag gccgccaaga gcttcagcct gaagatcagc 300

gactcccagc tgggcgatac cgccatgtat ttctgcgcct tcatgggcta ctacggcggc 360

agccagggca atctgatctt tggcaagggc acaaagctga gcgtgaagcc caacatccag 420

aaccccgacc ctgccgtgta ccagctgagg gactccaagt ctagcgataa gagcgtgtgc 480

ctgttcaccg actttgattc ccagacaaac gtgagccaga gcaaggactc tgacgtgtac 540

atcaccgaca agacagtgct ggatatgagg tctatggact tcaagagcaa cagtgctgtg 600

gcctggagca acaaatctga ctttgcatgt gcaaacgcct tcaacaacag cattattcca 660

gaagacacct tcttccccag cccagaaagt tcctgtgatg tcaagctggt cgagaaaagc 720

tttgaaacag atacgaacct aaactttcaa aacctgtcag tgattgggtt ccgaatcctc 780

ctcctgaaag tggccgggtt taatctgctc atgacgctgc ggctgtggtc cagctga 837

<210> 56

<211> 930

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(930)

<400> 56

atggacagct ggaccttctg ctgcgtgagc ctgtgcatcc tggtggccaa gcacacagac 60

gcaggcgtga tccagagccc aaggcacgag gtgacagaga tgggccagga ggtgaccctg 120

aggtgtaagc ccatcagcgg ccacaactcc ctgttctggt ataggcagac catgatgcgg 180

ggactggagc tgctgatcta cttcaacaac aacgtgccca tcgatgacag cggcatgccc 240

gaggacagat tcagcgccaa gatgcccaac gccagcttca gcaccctgaa gatccagccc 300

agcgagccca gagactccgc cgtgtatttc tgcgccagca gcagcctgca gtatgagcag 360

tacttcggcc caggcacacg cctgaccgtg acagaggatc tgaagaacgt gttcccccct 420

gaggtggccg tgtttgagcc ttctgaggcc gagatcagcc acacccagaa ggccaccctg 480

gtgtgcctgg caaccggctt ctacccagac cacgtggagc tgagctggtg ggtgaacggc 540

aaggaggtgc acagcggcgt gtccacagac ccacagcccc tgaaggagca gcccgccctg 600

aatgattcta gatattgcct gtctagccgg ctgagagtga gcgccacctt ttggcagaac 660

cctaggaatc acttccgctg tcaggtgcag ttttacggcc tgagcgagaa tgacgagtgg 720

acccaggata gggccaagcc tgtgacacag atcgtgtccg ccgaggcatg gggaagggca 780

gattgcggct tcacaagcga gtcctaccag cagggcgtgc tgtccgccac catcctgtat 840

gagatcctgc tgggcaaggc cacactgtac gccgtgctgg tgagcgccct ggtgctgatg 900

gccatggtga agaggaagga ctccaggggc 930

<210> 57

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(25)

<400> 57

aacataccat aaacctccca ttctg 25

<210> 58

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(25)

<400> 58

ttggagagac tgaggctggg ccacg 25

<210> 59

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<400> 59

tggggcaaag agggaaatga g 21

<210> 60

<211> 21

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(21)

<400> 60

agaacctggc cattcctgaa g 21

<210> 61

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(25)

<400> 61

catgtgcaaa cgccttcaac aacag 25

<210> 62

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 62

ctgggatggt gaccccaaaa 20

<210> 63

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 63

ggccacatag aaaggggacc 20

<210> 64

<211> 24

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(24)

<400> 64

accatgaagg agaattgggc acct 24

<210> 65

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 65

gggggatgga cagacaatgg 20

<210> 66

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 66

gctgaccctg tgaaccttga 20

<210> 67

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(25)

<400> 67

atccaggtag cggacaagac tagat 25

<210> 68

<211> 23

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(23)

<400> 68

tcagccatat tgtcccctaa act 23

<210> 69

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 69

tggtctgtcc atggcatctt 20

<210> 70

<211> 20

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(20)

<400> 70

ctgtatggac acagtgccta 20

<210> 71

<211> 9

<212> PRT

<213> artificial sequence

<220>

<223> synthetic polypeptide

<220>

<221> MISC_FEATURE

<222> (1)..(9)

<400> 71

Val Leu Asp Phe Ala Pro Pro Gly Ala

1 5

<210> 72

<211> 2503

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2503)

<400> 72

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatgagca tcggcctcct gtgctgtgca gccttgtctc 600

tcctgtgggc aggtccagtg aatgctggtg tcactcagac cccaaaattc caggtcctga 660

agacaggaca gagcatgaca ctgcagtgtg cccaggatat gaaccatgaa tacatgtcct 720

ggtatcgaca agacccaggc atggggctga ggctgattca ttactcagtt ggtgctggta 780

tcactgacca aggagaagtc cccaatggct acaatgtctc cagatcaacc acagaggatt 840

tcccgctcag gctgctgtcg gctgctccct cccagacatc tgtgtacttc tgtgccagca 900

gttacgtcgg gaacaccggg gagctgtttt ttggagaagg ctctaggctg accgtactgg 960

aggacctgaa aaacgtgttc ccacccgagg tcgctgtgtt tgagccatca gaagcagaga 1020

tctcccacac ccaaaaggcc acactggtat gcctggccac aggcttctac cccgaccacg 1080

tggagctgag ctggtgggtg aatgggaagg aggtgcacag tggggtcagc acagacccgc 1140

agcccctcaa ggagcagccc gccctcaatg actccagata ctgcctgagc agccgcctga 1200

gggtctcggc caccttctgg cagaaccccc gcaaccactt ccgctgtcaa gtccagttct 1260

acgggctctc agaaaacgat gaatggacac aagatagggc caaacccgtc acccagatcg 1320

tcagcgccga ggcctggggt agagcagact gtggcttcac ctccgagtct taccagcaag 1380

gggtcctgtc tgccaccatc ctctatgaga tcttgctagg gaaggccacc ttgtatgccg 1440

tgctggtcag tgccctcgtg ctgatggcta tggtcaagag aaaggattcc agaggccgcg 1500

ccaagcgctc cggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1560

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1620

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1680

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1740

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1800

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1860

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1920

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1980

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 2040

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2100

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2160

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 2220

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 2280

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 2340

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 2400

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 2460

aggtggcagg agagggcacg tggcccagcc tcagtctctc caa 2503

<210> 73

<211> 2503

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2503)

<400> 73

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatgagca tcggcctcct gtgctgtgca gccttgtctc 600

tcctgtgggc aggtccagtg aatgctggtg tcactcagac cccaaaattc caggtcctga 660

agacaggaca gagcatgaca ctgcagtgtg cccaggatat gaaccatgaa tacatgtcct 720

ggtatcgaca agacccaggc atggggctga ggctgattca ttactcagtt ggtgctggta 780

tcactgacca aggagaagtc cccaatggct acaatgtctc cagatcaacc acagaggatt 840

tcccgctcag gctgctgtcg gctgctccct cccagacatc tgtgtacttc tgtgccagca 900

gttacgtcgg gaacaccggg gagctgtttt ttggagaagg ctctaggctg accgtactgg 960

aggacctgaa aaacgtgttc ccacccgagg tcgctgtgtt tgagccatca gaagcagaga 1020

tctcccacac ccaaaaggcc acactggtat gcctggccac aggcttctac cccgaccacg 1080

tggagctgag ctggtgggtg aatgggaagg aggtgcacag tggggtcagc acagacccgc 1140

agcccctcaa ggagcagccc gccctcaatg actccagata ctgcctgagc agccgcctga 1200

gggtctcggc caccttctgg cagaaccccc gcaaccactt ccgctgtcaa gtccagttct 1260

acgggctctc agaaaacgat gaatggacac aagatagggc caaacccgtc acccagatcg 1320

tcagcgccga ggcctggggt agagcagact gtggcttcac ctccgagtct taccagcaag 1380

gggtcctgtc tgccaccatc ctctatgaga tcttgctagg gaaggccacc ttgtatgccg 1440

tgctggtcag tgccctcgtg ctgatggcta tggtcaagag aaaggattcc agaggccgcg 1500

ccaagcgctc cggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1560

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1620

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1680

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1740

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1800

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1860

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1920

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1980

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 2040

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2100

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2160

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 2220

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 2280

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 2340

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 2400

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 2460

aggtggcagg agagggcacg tggcccagcc tcagtctctc caa 2503

<210> 74

<211> 2503

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2503)

<400> 74

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatgagca tcggcctcct gtgctgtgca gccttgtctc 600

tcctgtgggc aggtccagtg aatgctggtg tcactcagac cccaaaattc caggtcctga 660

agacaggaca gagcatgaca ctgcagtgtg cccaggatat gaaccatgaa tacatgtcct 720

ggtatcgaca agacccaggc atggggctga ggctgattca ttactcagtt ggtgctggta 780

tcactgacca aggagaagtc cccaatggct acaatgtctc cagatcaacc acagaggatt 840

tcccgctcag gctgctgtcg gctgctccct cccagacatc tgtgtacttc tgtgccagca 900

gttacgtcgg gaacaccggg gagctgtttt ttggagaagg ctctaggctg accgtactgg 960

aggacctgaa aaacgtgttc ccacccgagg tcgctgtgtt tgagccatca gaagcagaga 1020

tctcccacac ccaaaaggcc acactggtat gcctggccac aggcttctac cccgaccacg 1080

tggagctgag ctggtgggtg aatgggaagg aggtgcacag tggggtcagc acagacccgc 1140

agcccctcaa ggagcagccc gccctcaatg actccagata ctgcctgagc agccgcctga 1200

gggtctcggc caccttctgg cagaaccccc gcaaccactt ccgctgtcaa gtccagttct 1260

acgggctctc agaaaacgat gaatggacac aagatagggc caaacccgtc acccagatcg 1320

tcagcgccga ggcctggggt agagcagact gtggcttcac ctccgagtct taccagcaag 1380

gggtcctgtc tgccaccatc ctctatgaga tcttgctagg gaaggccacc ttgtatgccg 1440

tgctggtcag tgccctcgtg ctgatggcta tggtcaagag aaaggattcc agaggccgcg 1500

ccaagcgctc cggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1560

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1620

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1680

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1740

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1800

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1860

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1920

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1980

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 2040

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 2100

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 2160

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 2220

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 2280

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 2340

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 2400

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 2460

aggtggcagg agagggcacg tggcccagcc tcagtctctc caa 2503

<210> 75

<211> 2263

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2263)

<400> 75

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggcctctc 600

tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg 660

tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca 720

agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc 780

agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg 840

gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc 900

gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc 960

ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga 1020

agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa 1080

atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg 1140

ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca 1200

agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg 1260

agctgtacaa gggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1320

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1380

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1440

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1500

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1560

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1620

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1680

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1740

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 1800

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 1860

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 1920

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 1980

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 2040

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 2100

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 2160

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 2220

aggtggcagg agagggcacg tggcccagcc tcagtctctc caa 2263

<210> 76

<211> 2263

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2263)

<400> 76

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggcctctc 600

tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg 660

tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca 720

agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc 780

agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg 840

gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc 900

gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc 960

ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga 1020

agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa 1080

atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg 1140

ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca 1200

agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg 1260

agctgtacaa gggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1320

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1380

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1440

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1500

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1560

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1620

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1680

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1740

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 1800

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 1860

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 1920

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 1980

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 2040

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 2100

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 2160

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 2220

aggtggcagg agagggcacg tggcccagcc tcagtctctc caa 2263

<210> 77

<211> 2263

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2263)

<400> 77

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggcctctc 600

tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg 660

tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca 720

agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc 780

agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg 840

gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc 900

gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc 960

ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga 1020

agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa 1080

atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg 1140

ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca 1200

agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg 1260

agctgtacaa gggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1320

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1380

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1440

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1500

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1560

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1620

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1680

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1740

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 1800

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 1860

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 1920

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 1980

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 2040

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 2100

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 2160

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 2220

aggtggcagg agagggcacg tggcccagcc tcagtctctc caa 2263

<210> 78

<211> 1463

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1463)

<400> 78

tctggactcc agcctgggtt ggggcaaaga gggaaatgag atcatgtcct aaccctgatc 60

ctcttgtccc acagatatcc agaaccctga ccctgccgtg ggaagcggag aaggtagagg 120

ttctctcctc acttgtggtg atgttgaaga aaaccctggt ccaatggtga gcaagggcga 180

ggaggataac atggcctctc tcccagcgac acatgagtta cacatctttg gctccatcaa 240

cggtgtggac tttgacatgg tgggtcaggg caccggcaat ccaaatgatg gttatgagga 300

gttaaacctg aagtccacca agggtgacct ccagttctcc ccctggattc tggtccctca 360

tatcgggtat ggcttccatc agtacctgcc ctaccctgac gggatgtcgc ctttccaggc 420

cgccatggta gatggctccg gataccaagt ccatcgcaca atgcagtttg aagatggtgc 480

ctcccttact gttaactacc gctacaccta cgagggaagc cacatcaaag gagaggccca 540

ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc 600

ggactggtgc aggtcgaaga agacttaccc caacgacaaa accatcatca gtacctttaa 660

gtggagttac accactggaa atggcaagcg ctaccggagc actgcgcgga ccacctacac 720

ctttgccaag ccaatggcgg ctaactatct gaagaaccag ccgatgtacg tgttccgtaa 780

gacggagctc aagcactcca agaccgagct caacttcaag gagtggcaaa aggcctttac 840

cgatgtgatg ggcatggacg agctgtacaa gggctccggt gctaccaatt tctcactgtt 900

gaaacaagcg ggcgatgttg aagaaaatcc cggtccaatg gagaccctct tgggcctgct 960

tatcctttgg ctgcagctgc aatgggtgag cagcaaacag gaggtgacgc agattcctgc 1020

agctctgagt gtcccagaag gagaaaactt ggttctcaac tgcagtttca ctgatagcgc 1080

tatttacaac ctccagtggt ttaggcagga ccctgggaaa ggtctcacat ctctgttgct 1140

tattcagtca agtcagagag agcaaacaag tggaagactt aatgcctcgc tggataaatc 1200

atcaggacgt agtactttat acattgcagc ttctcagcct ggtgactcag ccacctacct 1260

ctgtgctgtg aggcccctgt acggaggaag ctacatacct acatttggaa gaggaaccag 1320

ccttattgtt catccgtata tccagaaccc tgaccctgcg gtataccagc tgagagactc 1380

taaatccagt gacaagtctg tctgcctatt caccgatttt gattctcaaa caaatgtgtc 1440

acaaagtaag gattctgatg tgt 1463

<210> 79

<211> 1463

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1463)

<400> 79

tctggactcc agcctgggtt ggggcaaaga gggaaatgag atcatgtcct aaccctgatc 60

ctcttgtccc acagatatcc agaaccctga ccctgccgtg ggaagcggag aaggtagagg 120

ttctctcctc acttgtggtg atgttgaaga aaaccctggt ccaatggtga gcaagggcga 180

ggaggataac atggcctctc tcccagcgac acatgagtta cacatctttg gctccatcaa 240

cggtgtggac tttgacatgg tgggtcaggg caccggcaat ccaaatgatg gttatgagga 300

gttaaacctg aagtccacca agggtgacct ccagttctcc ccctggattc tggtccctca 360

tatcgggtat ggcttccatc agtacctgcc ctaccctgac gggatgtcgc ctttccaggc 420

cgccatggta gatggctccg gataccaagt ccatcgcaca atgcagtttg aagatggtgc 480

ctcccttact gttaactacc gctacaccta cgagggaagc cacatcaaag gagaggccca 540

ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc 600

ggactggtgc aggtcgaaga agacttaccc caacgacaaa accatcatca gtacctttaa 660

gtggagttac accactggaa atggcaagcg ctaccggagc actgcgcgga ccacctacac 720

ctttgccaag ccaatggcgg ctaactatct gaagaaccag ccgatgtacg tgttccgtaa 780

gacggagctc aagcactcca agaccgagct caacttcaag gagtggcaaa aggcctttac 840

cgatgtgatg ggcatggacg agctgtacaa gggctccggt gctaccaatt tctcactgtt 900

gaaacaagcg ggcgatgttg aagaaaatcc cggtccaatg gagaccctct tgggcctgct 960

tatcctttgg ctgcagctgc aatgggtgag cagcaaacag gaggtgacgc agattcctgc 1020

agctctgagt gtcccagaag gagaaaactt ggttctcaac tgcagtttca ctgatagcgc 1080

tatttacaac ctccagtggt ttaggcagga ccctgggaaa ggtctcacat ctctgttgct 1140

tattcagtca agtcagagag agcaaacaag tggaagactt aatgcctcgc tggataaatc 1200

atcaggacgt agtactttat acattgcagc ttctcagcct ggtgactcag ccacctacct 1260

ctgtgctgtg aggcccctgt acggaggaag ctacatacct acatttggaa gaggaaccag 1320

ccttattgtt catccgtata tccagaaccc tgaccctgcg gtataccagc tgagagactc 1380

taaatccagt gacaagtctg tctgcctatt caccgatttt gattctcaaa caaatgtgtc 1440

acaaagtaag gattctgatg tgt 1463

<210> 80

<211> 1663

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1663)

<400> 80

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 60

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 120

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 180

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 240

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggcctctc 300

tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg 360

tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca 420

agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc 480

agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg 540

gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc 600

gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc 660

ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga 720

agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa 780

atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg 840

ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca 900

agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg 960

agctgtacaa gggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1020

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1080

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1140

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1200

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1260

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1320

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1380

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1440

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 1500

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 1560

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 1620

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgc 1663

<210> 81

<211> 1663

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1663)

<400> 81

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 60

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 120

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 180

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 240

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggcctctc 300

tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg 360

tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca 420

agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc 480

agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg 540

gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc 600

gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc 660

ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga 720

agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa 780

atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg 840

ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca 900

agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg 960

agctgtacaa gggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1020

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1080

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1140

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1200

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1260

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1320

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1380

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1440

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 1500

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 1560

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 1620

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgc 1663

<210> 82

<211> 1863

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1863)

<400> 82

ggtttccttg agtggcaggc caggcctggc cgtgaacgtt cactgaaatc atggcctctt 60

ggccaagatt gatagcttgt gcctgtccct gagtcccagt ccatcacgag cagctggttt 120

ctaagatgct atttcccgta taaagcatga gaccgtgact tgccagcccc acagagcccc 180

gcccttgtcc atcactggca tctggactcc agcctgggtt ggggcaaaga gggaaatgag 240

atcatgtcct aaccctgatc ctcttgtccc acagatatcc agaaccctga ccctgccgtg 300

ggaagcggag aaggtagagg ttctctcctc acttgtggtg atgttgaaga aaaccctggt 360

ccaatggtga gcaagggcga ggaggataac atggcctctc tcccagcgac acatgagtta 420

cacatctttg gctccatcaa cggtgtggac tttgacatgg tgggtcaggg caccggcaat 480

ccaaatgatg gttatgagga gttaaacctg aagtccacca agggtgacct ccagttctcc 540

ccctggattc tggtccctca tatcgggtat ggcttccatc agtacctgcc ctaccctgac 600

gggatgtcgc ctttccaggc cgccatggta gatggctccg gataccaagt ccatcgcaca 660

atgcagtttg aagatggtgc ctcccttact gttaactacc gctacaccta cgagggaagc 720

cacatcaaag gagaggccca ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg 780

accaactcgc tgaccgctgc ggactggtgc aggtcgaaga agacttaccc caacgacaaa 840

accatcatca gtacctttaa gtggagttac accactggaa atggcaagcg ctaccggagc 900

actgcgcgga ccacctacac ctttgccaag ccaatggcgg ctaactatct gaagaaccag 960

ccgatgtacg tgttccgtaa gacggagctc aagcactcca agaccgagct caacttcaag 1020

gagtggcaaa aggcctttac cgatgtgatg ggcatggacg agctgtacaa gggctccggt 1080

gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg aagaaaatcc cggtccaatg 1140

gagaccctct tgggcctgct tatcctttgg ctgcagctgc aatgggtgag cagcaaacag 1200

gaggtgacgc agattcctgc agctctgagt gtcccagaag gagaaaactt ggttctcaac 1260

tgcagtttca ctgatagcgc tatttacaac ctccagtggt ttaggcagga ccctgggaaa 1320

ggtctcacat ctctgttgct tattcagtca agtcagagag agcaaacaag tggaagactt 1380

aatgcctcgc tggataaatc atcaggacgt agtactttat acattgcagc ttctcagcct 1440

ggtgactcag ccacctacct ctgtgctgtg aggcccctgt acggaggaag ctacatacct 1500

acatttggaa gaggaaccag ccttattgtt catccgtata tccagaaccc tgaccctgcg 1560

gtataccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 1620

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 1680

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 1740

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 1800

cccagcccag gtaagggcag ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg 1860

gcc 1863

<210> 83

<211> 1863

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1863)

<400> 83

ggtttccttg agtggcaggc caggcctggc cgtgaacgtt cactgaaatc atggcctctt 60

ggccaagatt gatagcttgt gcctgtccct gagtcccagt ccatcacgag cagctggttt 120

ctaagatgct atttcccgta taaagcatga gaccgtgact tgccagcccc acagagcccc 180

gcccttgtcc atcactggca tctggactcc agcctgggtt ggggcaaaga gggaaatgag 240

atcatgtcct aaccctgatc ctcttgtccc acagatatcc agaaccctga ccctgccgtg 300

ggaagcggag aaggtagagg ttctctcctc acttgtggtg atgttgaaga aaaccctggt 360

ccaatggtga gcaagggcga ggaggataac atggcctctc tcccagcgac acatgagtta 420

cacatctttg gctccatcaa cggtgtggac tttgacatgg tgggtcaggg caccggcaat 480

ccaaatgatg gttatgagga gttaaacctg aagtccacca agggtgacct ccagttctcc 540

ccctggattc tggtccctca tatcgggtat ggcttccatc agtacctgcc ctaccctgac 600

gggatgtcgc ctttccaggc cgccatggta gatggctccg gataccaagt ccatcgcaca 660

atgcagtttg aagatggtgc ctcccttact gttaactacc gctacaccta cgagggaagc 720

cacatcaaag gagaggccca ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg 780

accaactcgc tgaccgctgc ggactggtgc aggtcgaaga agacttaccc caacgacaaa 840

accatcatca gtacctttaa gtggagttac accactggaa atggcaagcg ctaccggagc 900

actgcgcgga ccacctacac ctttgccaag ccaatggcgg ctaactatct gaagaaccag 960

ccgatgtacg tgttccgtaa gacggagctc aagcactcca agaccgagct caacttcaag 1020

gagtggcaaa aggcctttac cgatgtgatg ggcatggacg agctgtacaa gggctccggt 1080

gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg aagaaaatcc cggtccaatg 1140

gagaccctct tgggcctgct tatcctttgg ctgcagctgc aatgggtgag cagcaaacag 1200

gaggtgacgc agattcctgc agctctgagt gtcccagaag gagaaaactt ggttctcaac 1260

tgcagtttca ctgatagcgc tatttacaac ctccagtggt ttaggcagga ccctgggaaa 1320

ggtctcacat ctctgttgct tattcagtca agtcagagag agcaaacaag tggaagactt 1380

aatgcctcgc tggataaatc atcaggacgt agtactttat acattgcagc ttctcagcct 1440

ggtgactcag ccacctacct ctgtgctgtg aggcccctgt acggaggaag ctacatacct 1500

acatttggaa gaggaaccag ccttattgtt catccgtata tccagaaccc tgaccctgcg 1560

gtataccagc tgagagactc taaatccagt gacaagtctg tctgcctatt caccgatttt 1620

gattctcaaa caaatgtgtc acaaagtaag gattctgatg tgtatatcac agacaaaact 1680

gtgctagaca tgaggtctat ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa 1740

tctgactttg catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc 1800

cccagcccag gtaagggcag ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg 1860

gcc 1863

<210> 84

<211> 2663

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2663)

<400> 84

ggaaggtgga tgaggcacca tattcatttt gcaggtgaaa ttcctgagat gtaaggagct 60

gctgtgactt gctcaaggcc ttatatcgag taaacggtag tgctggggct tagacgcagg 120

tgttctgatt tatagttcaa aacctctatc aatgagagag caatctcctg gtaatgtgat 180

agatttccca acttaatgcc aacataccat aaacctccca ttctgctaat gcccagccta 240

agttggggag accactccag attccaagat gtacagtttg ctttgctggg cctttttccc 300

atgcctgcct ttactctgcc agagttatat tgctggggtt ttgaagaaga tcctattaaa 360

taaaagaata agcagtatta ttaagtagcc ctgcatttca ggtttccttg agtggcaggc 420

caggcctggc cgtgaacgtt cactgaaatc atggcctctt ggccaagatt gatagcttgt 480

gcctgtccct gagtcccagt ccatcacgag cagctggttt ctaagatgct atttcccgta 540

taaagcatga gaccgtgact tgccagcccc acagagcccc gcccttgtcc atcactggca 600

tctggactcc agcctgggtt ggggcaaaga gggaaatgag atcatgtcct aaccctgatc 660

ctcttgtccc acagatatcc agaaccctga ccctgccgtg ggaagcggag aaggtagagg 720

ttctctcctc acttgtggtg atgttgaaga aaaccctggt ccaatggtga gcaagggcga 780

ggaggataac atggcctctc tcccagcgac acatgagtta cacatctttg gctccatcaa 840

cggtgtggac tttgacatgg tgggtcaggg caccggcaat ccaaatgatg gttatgagga 900

gttaaacctg aagtccacca agggtgacct ccagttctcc ccctggattc tggtccctca 960

tatcgggtat ggcttccatc agtacctgcc ctaccctgac gggatgtcgc ctttccaggc 1020

cgccatggta gatggctccg gataccaagt ccatcgcaca atgcagtttg aagatggtgc 1080

ctcccttact gttaactacc gctacaccta cgagggaagc cacatcaaag gagaggccca 1140

ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc 1200

ggactggtgc aggtcgaaga agacttaccc caacgacaaa accatcatca gtacctttaa 1260

gtggagttac accactggaa atggcaagcg ctaccggagc actgcgcgga ccacctacac 1320

ctttgccaag ccaatggcgg ctaactatct gaagaaccag ccgatgtacg tgttccgtaa 1380

gacggagctc aagcactcca agaccgagct caacttcaag gagtggcaaa aggcctttac 1440

cgatgtgatg ggcatggacg agctgtacaa gggctccggt gctaccaatt tctcactgtt 1500

gaaacaagcg ggcgatgttg aagaaaatcc cggtccaatg gagaccctct tgggcctgct 1560

tatcctttgg ctgcagctgc aatgggtgag cagcaaacag gaggtgacgc agattcctgc 1620

agctctgagt gtcccagaag gagaaaactt ggttctcaac tgcagtttca ctgatagcgc 1680

tatttacaac ctccagtggt ttaggcagga ccctgggaaa ggtctcacat ctctgttgct 1740

tattcagtca agtcagagag agcaaacaag tggaagactt aatgcctcgc tggataaatc 1800

atcaggacgt agtactttat acattgcagc ttctcagcct ggtgactcag ccacctacct 1860

ctgtgctgtg aggcccctgt acggaggaag ctacatacct acatttggaa gaggaaccag 1920

ccttattgtt catccgtata tccagaaccc tgaccctgcg gtataccagc tgagagactc 1980

taaatccagt gacaagtctg tctgcctatt caccgatttt gattctcaaa caaatgtgtc 2040

acaaagtaag gattctgatg tgtatatcac agacaaaact gtgctagaca tgaggtctat 2100

ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa 2160

cgccttcaac aacagcatta ttccagaaga caccttcttc cccagcccag gtaagggcag 2220

ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg gccaggttct gcccagagct 2280

ctggtcaatg atgtctaaaa ctcctctgat tggtggtctc ggccttatcc attgccacca 2340

aaaccctctt tttactaaga aacagtgagc cttgttctgg cagtccagag aatgacacgg 2400

gaaaaaagca gatgaagaga aggtggcagg agagggcacg tggcccagcc tcagtctctc 2460

caactgagtt cctgcctgcc tgcctttgct cagactgttt gccccttact gctcttctag 2520

gcctcattct aagccccttc tccaagttgc ctctccttat ttctccctgt ctgccaaaaa 2580

atctttccca gctcactaag tcagtctcac gcagtcactc attaacccac caatcactga 2640

ttgtgccggc acatgaatgc acc 2663

<210> 85

<211> 2663

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2663)

<400> 85

ggaaggtgga tgaggcacca tattcatttt gcaggtgaaa ttcctgagat gtaaggagct 60

gctgtgactt gctcaaggcc ttatatcgag taaacggtag tgctggggct tagacgcagg 120

tgttctgatt tatagttcaa aacctctatc aatgagagag caatctcctg gtaatgtgat 180

agatttccca acttaatgcc aacataccat aaacctccca ttctgctaat gcccagccta 240

agttggggag accactccag attccaagat gtacagtttg ctttgctggg cctttttccc 300

atgcctgcct ttactctgcc agagttatat tgctggggtt ttgaagaaga tcctattaaa 360

taaaagaata agcagtatta ttaagtagcc ctgcatttca ggtttccttg agtggcaggc 420

caggcctggc cgtgaacgtt cactgaaatc atggcctctt ggccaagatt gatagcttgt 480

gcctgtccct gagtcccagt ccatcacgag cagctggttt ctaagatgct atttcccgta 540

taaagcatga gaccgtgact tgccagcccc acagagcccc gcccttgtcc atcactggca 600

tctggactcc agcctgggtt ggggcaaaga gggaaatgag atcatgtcct aaccctgatc 660

ctcttgtccc acagatatcc agaaccctga ccctgccgtg ggaagcggag aaggtagagg 720

ttctctcctc acttgtggtg atgttgaaga aaaccctggt ccaatggtga gcaagggcga 780

ggaggataac atggcctctc tcccagcgac acatgagtta cacatctttg gctccatcaa 840

cggtgtggac tttgacatgg tgggtcaggg caccggcaat ccaaatgatg gttatgagga 900

gttaaacctg aagtccacca agggtgacct ccagttctcc ccctggattc tggtccctca 960

tatcgggtat ggcttccatc agtacctgcc ctaccctgac gggatgtcgc ctttccaggc 1020

cgccatggta gatggctccg gataccaagt ccatcgcaca atgcagtttg aagatggtgc 1080

ctcccttact gttaactacc gctacaccta cgagggaagc cacatcaaag gagaggccca 1140

ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc 1200

ggactggtgc aggtcgaaga agacttaccc caacgacaaa accatcatca gtacctttaa 1260

gtggagttac accactggaa atggcaagcg ctaccggagc actgcgcgga ccacctacac 1320

ctttgccaag ccaatggcgg ctaactatct gaagaaccag ccgatgtacg tgttccgtaa 1380

gacggagctc aagcactcca agaccgagct caacttcaag gagtggcaaa aggcctttac 1440

cgatgtgatg ggcatggacg agctgtacaa gggctccggt gctaccaatt tctcactgtt 1500

gaaacaagcg ggcgatgttg aagaaaatcc cggtccaatg gagaccctct tgggcctgct 1560

tatcctttgg ctgcagctgc aatgggtgag cagcaaacag gaggtgacgc agattcctgc 1620

agctctgagt gtcccagaag gagaaaactt ggttctcaac tgcagtttca ctgatagcgc 1680

tatttacaac ctccagtggt ttaggcagga ccctgggaaa ggtctcacat ctctgttgct 1740

tattcagtca agtcagagag agcaaacaag tggaagactt aatgcctcgc tggataaatc 1800

atcaggacgt agtactttat acattgcagc ttctcagcct ggtgactcag ccacctacct 1860

ctgtgctgtg aggcccctgt acggaggaag ctacatacct acatttggaa gaggaaccag 1920

ccttattgtt catccgtata tccagaaccc tgaccctgcg gtataccagc tgagagactc 1980

taaatccagt gacaagtctg tctgcctatt caccgatttt gattctcaaa caaatgtgtc 2040

acaaagtaag gattctgatg tgtatatcac agacaaaact gtgctagaca tgaggtctat 2100

ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa 2160

cgccttcaac aacagcatta ttccagaaga caccttcttc cccagcccag gtaagggcag 2220

ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg gccaggttct gcccagagct 2280

ctggtcaatg atgtctaaaa ctcctctgat tggtggtctc ggccttatcc attgccacca 2340

aaaccctctt tttactaaga aacagtgagc cttgttctgg cagtccagag aatgacacgg 2400

gaaaaaagca gatgaagaga aggtggcagg agagggcacg tggcccagcc tcagtctctc 2460

caactgagtt cctgcctgcc tgcctttgct cagactgttt gccccttact gctcttctag 2520

gcctcattct aagccccttc tccaagttgc ctctccttat ttctccctgt ctgccaaaaa 2580

atctttccca gctcactaag tcagtctcac gcagtcactc attaacccac caatcactga 2640

ttgtgccggc acatgaatgc acc 2663

<210> 86

<211> 3263

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(3263)

<400> 86

tttcctcctc aaaaggcagg aggtcggaaa gaataaacaa tgagagtcac attaaaaaca 60

caaaatccta cggaaatact gaagaatgag tctcagcact aaggaaaagc ctccagcagc 120

tcctgctttc tgagggtgaa ggatagacgc tgtggctctg catgactcac tagcactcta 180

tcacggccat attctggcag ggtcagtggc tccaactaac atttgtttgg tactttacag 240

tttattaaat agatgtttat atggagaagc tctcatttct ttctcagaag agcctggcta 300

ggaaggtgga tgaggcacca tattcatttt gcaggtgaaa ttcctgagat gtaaggagct 360

gctgtgactt gctcaaggcc ttatatcgag taaacggtag tgctggggct tagacgcagg 420

tgttctgatt tatagttcaa aacctctatc aatgagagag caatctcctg gtaatgtgat 480

agatttccca acttaatgcc aacataccat aaacctccca ttctgctaat gcccagccta 540

agttggggag accactccag attccaagat gtacagtttg ctttgctggg cctttttccc 600

atgcctgcct ttactctgcc agagttatat tgctggggtt ttgaagaaga tcctattaaa 660

taaaagaata agcagtatta ttaagtagcc ctgcatttca ggtttccttg agtggcaggc 720

caggcctggc cgtgaacgtt cactgaaatc atggcctctt ggccaagatt gatagcttgt 780

gcctgtccct gagtcccagt ccatcacgag cagctggttt ctaagatgct atttcccgta 840

taaagcatga gaccgtgact tgccagcccc acagagcccc gcccttgtcc atcactggca 900

tctggactcc agcctgggtt ggggcaaaga gggaaatgag atcatgtcct aaccctgatc 960

ctcttgtccc acagatatcc agaaccctga ccctgccgtg ggaagcggag aaggtagagg 1020

ttctctcctc acttgtggtg atgttgaaga aaaccctggt ccaatggtga gcaagggcga 1080

ggaggataac atggcctctc tcccagcgac acatgagtta cacatctttg gctccatcaa 1140

cggtgtggac tttgacatgg tgggtcaggg caccggcaat ccaaatgatg gttatgagga 1200

gttaaacctg aagtccacca agggtgacct ccagttctcc ccctggattc tggtccctca 1260

tatcgggtat ggcttccatc agtacctgcc ctaccctgac gggatgtcgc ctttccaggc 1320

cgccatggta gatggctccg gataccaagt ccatcgcaca atgcagtttg aagatggtgc 1380

ctcccttact gttaactacc gctacaccta cgagggaagc cacatcaaag gagaggccca 1440

ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc 1500

ggactggtgc aggtcgaaga agacttaccc caacgacaaa accatcatca gtacctttaa 1560

gtggagttac accactggaa atggcaagcg ctaccggagc actgcgcgga ccacctacac 1620

ctttgccaag ccaatggcgg ctaactatct gaagaaccag ccgatgtacg tgttccgtaa 1680

gacggagctc aagcactcca agaccgagct caacttcaag gagtggcaaa aggcctttac 1740

cgatgtgatg ggcatggacg agctgtacaa gggctccggt gctaccaatt tctcactgtt 1800

gaaacaagcg ggcgatgttg aagaaaatcc cggtccaatg gagaccctct tgggcctgct 1860

tatcctttgg ctgcagctgc aatgggtgag cagcaaacag gaggtgacgc agattcctgc 1920

agctctgagt gtcccagaag gagaaaactt ggttctcaac tgcagtttca ctgatagcgc 1980

tatttacaac ctccagtggt ttaggcagga ccctgggaaa ggtctcacat ctctgttgct 2040

tattcagtca agtcagagag agcaaacaag tggaagactt aatgcctcgc tggataaatc 2100

atcaggacgt agtactttat acattgcagc ttctcagcct ggtgactcag ccacctacct 2160

ctgtgctgtg aggcccctgt acggaggaag ctacatacct acatttggaa gaggaaccag 2220

ccttattgtt catccgtata tccagaaccc tgaccctgcg gtataccagc tgagagactc 2280

taaatccagt gacaagtctg tctgcctatt caccgatttt gattctcaaa caaatgtgtc 2340

acaaagtaag gattctgatg tgtatatcac agacaaaact gtgctagaca tgaggtctat 2400

ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa 2460

cgccttcaac aacagcatta ttccagaaga caccttcttc cccagcccag gtaagggcag 2520

ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg gccaggttct gcccagagct 2580

ctggtcaatg atgtctaaaa ctcctctgat tggtggtctc ggccttatcc attgccacca 2640

aaaccctctt tttactaaga aacagtgagc cttgttctgg cagtccagag aatgacacgg 2700

gaaaaaagca gatgaagaga aggtggcagg agagggcacg tggcccagcc tcagtctctc 2760

caactgagtt cctgcctgcc tgcctttgct cagactgttt gccccttact gctcttctag 2820

gcctcattct aagccccttc tccaagttgc ctctccttat ttctccctgt ctgccaaaaa 2880

atctttccca gctcactaag tcagtctcac gcagtcactc attaacccac caatcactga 2940

ttgtgccggc acatgaatgc accaggtgtt gaagtggagg aattaaaaag tcagatgagg 3000

ggtgtgccca gaggaagcac cattctagtt gggggagccc atctgtcagc tgggaaaagt 3060

ccaaataact tcagattgga atgtgtttta actcagggtt gagaaaacag ctaccttcag 3120

gacaaaagtc agggaagggc tctctgaaga aatgctactt gaagatacca gccctaccaa 3180

gggcagggag aggaccctat agaggcctgg gacaggagct caatgagaaa ggagaagagc 3240

agcaggcatg agttgaatga agg 3263

<210> 87

<211> 3263

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(3263)

<400> 87

tttcctcctc aaaaggcagg aggtcggaaa gaataaacaa tgagagtcac attaaaaaca 60

caaaatccta cggaaatact gaagaatgag tctcagcact aaggaaaagc ctccagcagc 120

tcctgctttc tgagggtgaa ggatagacgc tgtggctctg catgactcac tagcactcta 180

tcacggccat attctggcag ggtcagtggc tccaactaac atttgtttgg tactttacag 240

tttattaaat agatgtttat atggagaagc tctcatttct ttctcagaag agcctggcta 300

ggaaggtgga tgaggcacca tattcatttt gcaggtgaaa ttcctgagat gtaaggagct 360

gctgtgactt gctcaaggcc ttatatcgag taaacggtag tgctggggct tagacgcagg 420

tgttctgatt tatagttcaa aacctctatc aatgagagag caatctcctg gtaatgtgat 480

agatttccca acttaatgcc aacataccat aaacctccca ttctgctaat gcccagccta 540

agttggggag accactccag attccaagat gtacagtttg ctttgctggg cctttttccc 600

atgcctgcct ttactctgcc agagttatat tgctggggtt ttgaagaaga tcctattaaa 660

taaaagaata agcagtatta ttaagtagcc ctgcatttca ggtttccttg agtggcaggc 720

caggcctggc cgtgaacgtt cactgaaatc atggcctctt ggccaagatt gatagcttgt 780

gcctgtccct gagtcccagt ccatcacgag cagctggttt ctaagatgct atttcccgta 840

taaagcatga gaccgtgact tgccagcccc acagagcccc gcccttgtcc atcactggca 900

tctggactcc agcctgggtt ggggcaaaga gggaaatgag atcatgtcct aaccctgatc 960

ctcttgtccc acagatatcc agaaccctga ccctgccgtg ggaagcggag aaggtagagg 1020

ttctctcctc acttgtggtg atgttgaaga aaaccctggt ccaatggtga gcaagggcga 1080

ggaggataac atggcctctc tcccagcgac acatgagtta cacatctttg gctccatcaa 1140

cggtgtggac tttgacatgg tgggtcaggg caccggcaat ccaaatgatg gttatgagga 1200

gttaaacctg aagtccacca agggtgacct ccagttctcc ccctggattc tggtccctca 1260

tatcgggtat ggcttccatc agtacctgcc ctaccctgac gggatgtcgc ctttccaggc 1320

cgccatggta gatggctccg gataccaagt ccatcgcaca atgcagtttg aagatggtgc 1380

ctcccttact gttaactacc gctacaccta cgagggaagc cacatcaaag gagaggccca 1440

ggtgaagggg actggtttcc ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc 1500

ggactggtgc aggtcgaaga agacttaccc caacgacaaa accatcatca gtacctttaa 1560

gtggagttac accactggaa atggcaagcg ctaccggagc actgcgcgga ccacctacac 1620

ctttgccaag ccaatggcgg ctaactatct gaagaaccag ccgatgtacg tgttccgtaa 1680

gacggagctc aagcactcca agaccgagct caacttcaag gagtggcaaa aggcctttac 1740

cgatgtgatg ggcatggacg agctgtacaa gggctccggt gctaccaatt tctcactgtt 1800

gaaacaagcg ggcgatgttg aagaaaatcc cggtccaatg gagaccctct tgggcctgct 1860

tatcctttgg ctgcagctgc aatgggtgag cagcaaacag gaggtgacgc agattcctgc 1920

agctctgagt gtcccagaag gagaaaactt ggttctcaac tgcagtttca ctgatagcgc 1980

tatttacaac ctccagtggt ttaggcagga ccctgggaaa ggtctcacat ctctgttgct 2040

tattcagtca agtcagagag agcaaacaag tggaagactt aatgcctcgc tggataaatc 2100

atcaggacgt agtactttat acattgcagc ttctcagcct ggtgactcag ccacctacct 2160

ctgtgctgtg aggcccctgt acggaggaag ctacatacct acatttggaa gaggaaccag 2220

ccttattgtt catccgtata tccagaaccc tgaccctgcg gtataccagc tgagagactc 2280

taaatccagt gacaagtctg tctgcctatt caccgatttt gattctcaaa caaatgtgtc 2340

acaaagtaag gattctgatg tgtatatcac agacaaaact gtgctagaca tgaggtctat 2400

ggacttcaag agcaacagtg ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa 2460

cgccttcaac aacagcatta ttccagaaga caccttcttc cccagcccag gtaagggcag 2520

ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg gccaggttct gcccagagct 2580

ctggtcaatg atgtctaaaa ctcctctgat tggtggtctc ggccttatcc attgccacca 2640

aaaccctctt tttactaaga aacagtgagc cttgttctgg cagtccagag aatgacacgg 2700

gaaaaaagca gatgaagaga aggtggcagg agagggcacg tggcccagcc tcagtctctc 2760

caactgagtt cctgcctgcc tgcctttgct cagactgttt gccccttact gctcttctag 2820

gcctcattct aagccccttc tccaagttgc ctctccttat ttctccctgt ctgccaaaaa 2880

atctttccca gctcactaag tcagtctcac gcagtcactc attaacccac caatcactga 2940

ttgtgccggc acatgaatgc accaggtgtt gaagtggagg aattaaaaag tcagatgagg 3000

ggtgtgccca gaggaagcac cattctagtt gggggagccc atctgtcagc tgggaaaagt 3060

ccaaataact tcagattgga atgtgtttta actcagggtt gagaaaacag ctaccttcag 3120

gacaaaagtc agggaagggc tctctgaaga aatgctactt gaagatacca gccctaccaa 3180

gggcagggag aggaccctat agaggcctgg gacaggagct caatgagaaa ggagaagagc 3240

agcaggcatg agttgaatga agg 3263

<210> 88

<211> 5263

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(5263)

<400> 88

aaaaggtaca tgggaatgaa aggataaaaa ggctaaaaaa attaagtacc tctaactcag 60

cccctgttgc catttctcag agtcttgtgt tctgtggcat tgcgctttct agaccaacag 120

tgtccaatag aactttctgt ggcaatggaa atgtcctgtc aatctgcact gtcccataca 180

atagccacca gctacatgtg gctattgagc tcttgaaatg aagtttccat ttttaattga 240

aaacatttta tttcacattg actaattttt atttcaacag ccacatgtag ctagagacta 300

ttataccaga cagagcagcc tagatcttct ccagtctgac acccaccagc cccaggactt 360

gagtgagtgt ttaaccagga ctcaaagttg ggtttctgcc ccacaaggcc accccctttc 420

ctctttaaag ccaacctgca tctggtggcc cctgatcccc tgccttgagg atcggcactt 480

ccagactcct ctccccctct gcagtgctgt ccagtacccc cactgatgac taacaatcag 540

ggggatgtgt tggtagagct aatggctttc tgtctgtccc ttcccagcaa aggaactatg 600

ccttagggcc ttcacccaga gtgatgtcag gctgcccaag catgaggagg gaagtaggca 660

gaatcctctg gagccaaagc tctggatgtc tctcccctct gaccatggag cccacccctg 720

ctccactgct ccagggacag ccctatgctg caggcagctc tgcccccact cagcatccca 780

ggggctgatt tctttggttt tggatccagc tggatgtctg cattgccgag gccaccaggg 840

ctggctcagc aactgtcggg gaatcaccag ggtctgagaa atcttgtgcg catgtgaggg 900

gctgtgggag cagagaacca ctgggtggga aattctaatc cccaccctgc tggaaactct 960

ctgggtggcc ccaacatgct aatcctccgg caaacctctg tttcctcctc aaaaggcagg 1020

aggtcggaaa gaataaacaa tgagagtcac attaaaaaca caaaatccta cggaaatact 1080

gaagaatgag tctcagcact aaggaaaagc ctccagcagc tcctgctttc tgagggtgaa 1140

ggatagacgc tgtggctctg catgactcac tagcactcta tcacggccat attctggcag 1200

ggtcagtggc tccaactaac atttgtttgg tactttacag tttattaaat agatgtttat 1260

atggagaagc tctcatttct ttctcagaag agcctggcta ggaaggtgga tgaggcacca 1320

tattcatttt gcaggtgaaa ttcctgagat gtaaggagct gctgtgactt gctcaaggcc 1380

ttatatcgag taaacggtag tgctggggct tagacgcagg tgttctgatt tatagttcaa 1440

aacctctatc aatgagagag caatctcctg gtaatgtgat agatttccca acttaatgcc 1500

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 1560

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 1620

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 1680

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 1740

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 1800

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 1860

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 1920

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 1980

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 2040

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggcctctc 2100

tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg 2160

tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca 2220

agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc 2280

agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg 2340

gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc 2400

gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc 2460

ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga 2520

agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa 2580

atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg 2640

ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca 2700

agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg 2760

agctgtacaa gggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 2820

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 2880

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 2940

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 3000

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 3060

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 3120

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 3180

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 3240

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 3300

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 3360

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 3420

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 3480

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 3540

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 3600

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 3660

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 3720

aggtggcagg agagggcacg tggcccagcc tcagtctctc caactgagtt cctgcctgcc 3780

tgcctttgct cagactgttt gccccttact gctcttctag gcctcattct aagccccttc 3840

tccaagttgc ctctccttat ttctccctgt ctgccaaaaa atctttccca gctcactaag 3900

tcagtctcac gcagtcactc attaacccac caatcactga ttgtgccggc acatgaatgc 3960

accaggtgtt gaagtggagg aattaaaaag tcagatgagg ggtgtgccca gaggaagcac 4020

cattctagtt gggggagccc atctgtcagc tgggaaaagt ccaaataact tcagattgga 4080

atgtgtttta actcagggtt gagaaaacag ctaccttcag gacaaaagtc agggaagggc 4140

tctctgaaga aatgctactt gaagatacca gccctaccaa gggcagggag aggaccctat 4200

agaggcctgg gacaggagct caatgagaaa ggagaagagc agcaggcatg agttgaatga 4260

aggaggcagg gccgggtcac agggccttct aggccatgag agggtagaca gtattctaag 4320

gacgccagaa agctgttgat cggcttcaag caggggaggg acacctaatt tgcttttctt 4380

tttttttttt tttttttttt ttttttttga gatggagttt tgctcttgtt gcccaggctg 4440

gagtgcaatg gtgcatcttg gctcactgca acctccgcct cccaggttca agtgattctc 4500

ctgcctcagc ctcccgagta gctgagatta caggcacccg ccaccatgcc tggctaattt 4560

tttgtatttt tagtagagac agggtttcac tatgttggcc aggctggtct cgaactcctg 4620

acctcaggtg atccacccgc ttcagcctcc caaagtgctg ggattacagg cgtgagccac 4680

cacacccggc ctgcttttct taaagatcaa tctgagtgct gtacggagag tgggttgtaa 4740

gccaagagta gaagcagaaa gggagcagtt gcagcagaga gatgatggag gcctgggcag 4800

ggtggtggca gggaggtaac caacaccatt caggtttcaa aggtagaacc atgcagggat 4860

gagaaagcaa agaggggatc aaggaaggca gctggatttt ggcctgagca gctgagtcaa 4920

tgatagtgcc gtttactaag aagaaaccaa ggaaaaaatt tggggtgcag ggatcaaaac 4980

tttttggaac atatgaaagt acgtgtttat actctttatg gcccttgtca ctatgtatgc 5040

ctcgctgcct ccattggact ctagaatgaa gccaggcaag agcagggtct atgtgtgatg 5100

gcacatgtgg ccagggtcat gcaacatgta ctttgtacaa acagtgtata ttgagtaaat 5160

agaaatggtg tccaggagcc gaggtatcgg tcctgccagg gccaggggct ctccctagca 5220

ggtgctcata tgctgtaagt tccctccaga tctctccaca agg 5263

<210> 89

<211> 5263

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(5263)

<400> 89

aaaaggtaca tgggaatgaa aggataaaaa ggctaaaaaa attaagtacc tctaactcag 60

cccctgttgc catttctcag agtcttgtgt tctgtggcat tgcgctttct agaccaacag 120

tgtccaatag aactttctgt ggcaatggaa atgtcctgtc aatctgcact gtcccataca 180

atagccacca gctacatgtg gctattgagc tcttgaaatg aagtttccat ttttaattga 240

aaacatttta tttcacattg actaattttt atttcaacag ccacatgtag ctagagacta 300

ttataccaga cagagcagcc tagatcttct ccagtctgac acccaccagc cccaggactt 360

gagtgagtgt ttaaccagga ctcaaagttg ggtttctgcc ccacaaggcc accccctttc 420

ctctttaaag ccaacctgca tctggtggcc cctgatcccc tgccttgagg atcggcactt 480

ccagactcct ctccccctct gcagtgctgt ccagtacccc cactgatgac taacaatcag 540

ggggatgtgt tggtagagct aatggctttc tgtctgtccc ttcccagcaa aggaactatg 600

ccttagggcc ttcacccaga gtgatgtcag gctgcccaag catgaggagg gaagtaggca 660

gaatcctctg gagccaaagc tctggatgtc tctcccctct gaccatggag cccacccctg 720

ctccactgct ccagggacag ccctatgctg caggcagctc tgcccccact cagcatccca 780

ggggctgatt tctttggttt tggatccagc tggatgtctg cattgccgag gccaccaggg 840

ctggctcagc aactgtcggg gaatcaccag ggtctgagaa atcttgtgcg catgtgaggg 900

gctgtgggag cagagaacca ctgggtggga aattctaatc cccaccctgc tggaaactct 960

ctgggtggcc ccaacatgct aatcctccgg caaacctctg tttcctcctc aaaaggcagg 1020

aggtcggaaa gaataaacaa tgagagtcac attaaaaaca caaaatccta cggaaatact 1080

gaagaatgag tctcagcact aaggaaaagc ctccagcagc tcctgctttc tgagggtgaa 1140

ggatagacgc tgtggctctg catgactcac tagcactcta tcacggccat attctggcag 1200

ggtcagtggc tccaactaac atttgtttgg tactttacag tttattaaat agatgtttat 1260

atggagaagc tctcatttct ttctcagaag agcctggcta ggaaggtgga tgaggcacca 1320

tattcatttt gcaggtgaaa ttcctgagat gtaaggagct gctgtgactt gctcaaggcc 1380

ttatatcgag taaacggtag tgctggggct tagacgcagg tgttctgatt tatagttcaa 1440

aacctctatc aatgagagag caatctcctg gtaatgtgat agatttccca acttaatgcc 1500

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 1560

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 1620

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 1680

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 1740

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 1800

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 1860

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 1920

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 1980

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 2040

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggcctctc 2100

tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg 2160

tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca 2220

agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc 2280

agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg 2340

gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc 2400

gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc 2460

ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga 2520

agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa 2580

atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg 2640

ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca 2700

agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg 2760

agctgtacaa gggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 2820

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 2880

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 2940

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 3000

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 3060

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 3120

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 3180

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 3240

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 3300

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 3360

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 3420

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 3480

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 3540

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 3600

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 3660

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 3720

aggtggcagg agagggcacg tggcccagcc tcagtctctc caactgagtt cctgcctgcc 3780

tgcctttgct cagactgttt gccccttact gctcttctag gcctcattct aagccccttc 3840

tccaagttgc ctctccttat ttctccctgt ctgccaaaaa atctttccca gctcactaag 3900

tcagtctcac gcagtcactc attaacccac caatcactga ttgtgccggc acatgaatgc 3960

accaggtgtt gaagtggagg aattaaaaag tcagatgagg ggtgtgccca gaggaagcac 4020

cattctagtt gggggagccc atctgtcagc tgggaaaagt ccaaataact tcagattgga 4080

atgtgtttta actcagggtt gagaaaacag ctaccttcag gacaaaagtc agggaagggc 4140

tctctgaaga aatgctactt gaagatacca gccctaccaa gggcagggag aggaccctat 4200

agaggcctgg gacaggagct caatgagaaa ggagaagagc agcaggcatg agttgaatga 4260

aggaggcagg gccgggtcac agggccttct aggccatgag agggtagaca gtattctaag 4320

gacgccagaa agctgttgat cggcttcaag caggggaggg acacctaatt tgcttttctt 4380

tttttttttt tttttttttt ttttttttga gatggagttt tgctcttgtt gcccaggctg 4440

gagtgcaatg gtgcatcttg gctcactgca acctccgcct cccaggttca agtgattctc 4500

ctgcctcagc ctcccgagta gctgagatta caggcacccg ccaccatgcc tggctaattt 4560

tttgtatttt tagtagagac agggtttcac tatgttggcc aggctggtct cgaactcctg 4620

acctcaggtg atccacccgc ttcagcctcc caaagtgctg ggattacagg cgtgagccac 4680

cacacccggc ctgcttttct taaagatcaa tctgagtgct gtacggagag tgggttgtaa 4740

gccaagagta gaagcagaaa gggagcagtt gcagcagaga gatgatggag gcctgggcag 4800

ggtggtggca gggaggtaac caacaccatt caggtttcaa aggtagaacc atgcagggat 4860

gagaaagcaa agaggggatc aaggaaggca gctggatttt ggcctgagca gctgagtcaa 4920

tgatagtgcc gtttactaag aagaaaccaa ggaaaaaatt tggggtgcag ggatcaaaac 4980

tttttggaac atatgaaagt acgtgtttat actctttatg gcccttgtca ctatgtatgc 5040

ctcgctgcct ccattggact ctagaatgaa gccaggcaag agcagggtct atgtgtgatg 5100

gcacatgtgg ccagggtcat gcaacatgta ctttgtacaa acagtgtata ttgagtaaat 5160

agaaatggtg tccaggagcc gaggtatcgg tcctgccagg gccaggggct ctccctagca 5220

ggtgctcata tgctgtaagt tccctccaga tctctccaca agg 5263

<210> 90

<211> 2491

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2491)

<400> 90

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatgggct cctggaccct ctgctgtgtg tccctttgca 600

tcctggtagc aaagcacaca gatgctggag ttatccagtc accccggcac gaggtgacag 660

agatgggaca agaagtgact ctgagatgta aaccaatttc aggacacgac taccttttct 720

ggtacagaca gaccatgatg cggggactgg agttgctcat ttactttaac aacaacgttc 780

cgatagatga ttcagggatg cccgaggatc gattctcagc taagatgcct aatgcatcat 840

tctccactct gaagatccag ccctcagaac ccagggactc agctgtgtac ttctgtgcca 900

gcagttccgc taactatggc tacaccttcg gttcggggac caggttaacc gttgtagagg 960

acctgaaaaa cgtgttccca cccgaggtcg ctgtgtttga gccatcagaa gcagagatct 1020

cccacaccca aaaggccaca ctggtgtgcc tggccacagg cttctacccc gaccacgtgg 1080

agctgagctg gtgggtgaat gggaaggagg tgcacagtgg ggtcagcaca gacccgcagc 1140

ccctcaagga gcagcccgcc ctcaatgact ccagatactg cctgagcagc cgcctgaggg 1200

tctcggccac cttctggcag aacccccgca accacttccg ctgtcaagtc cagttctacg 1260

ggctctcaga aaacgatgaa tggacacaag atagggccaa acctgtcacc cagatcgtca 1320

gcgccgaggc ctggggtaga gcagactgtg gcttcacctc cgagtcttac cagcaagggg 1380

tcctgtctgc caccatcctc tatgagatct tgctagggaa ggccaccttg tatgccgtgc 1440

tggtcagtgc cctcgtgctg atggccatgg tcaagagaaa ggattccaga ggccgcgcca 1500

agcgctccgg ctccggtgct accaatttct cactgttgaa acaagcgggc gatgttgaag 1560

aaaatcccgg tccaatgctc cttgaacatt tattaataat cttgtggatg cagctgacat 1620

gggtcagtgg tcaacagctg aatcagagtc ctcaatctat gtttatccag gaaggagaag 1680

atgtctccat gaactgcact tcttcaagca tatttaacac ctggctatgg tacaagcagg 1740

accctgggga aggtcctgtc ctcttgatag ccttatataa ggctggtgaa ttgacctcaa 1800

atggaagact gactgctcag tttggtataa ccagaaagga cagcttcctg aatatctcag 1860

catccatacc tagtgatgta ggcatctact tctgtgctgg gccgatgaaa acctcctacg 1920

acaaggtgat atttgggcca gggacaagct tatcagtcat tccaaatatc cagaaccctg 1980

accctgcggt ataccagctg agagactcta aatccagtga caagtctgtc tgcctattca 2040

ccgattttga ttctcaaaca aatgtgtcac aaagtaagga ttctgatgtg tatatcacag 2100

acaaaactgt gctagacatg aggtctatgg acttcaagag caacagtgct gtggcctgga 2160

gcaacaaatc tgactttgca tgtgcaaacg ccttcaacaa cagcattatt ccagaagaca 2220

ccttcttccc cagcccaggt aagggcagct ttggtgcctt cgcaggctgt ttccttgctt 2280

caggaatggc caggttctgc ccagagctct ggtcaatgat gtctaaaact cctctgattg 2340

gtggtctcgg ccttatccat tgccaccaaa accctctttt tactaagaaa cagtgagcct 2400

tgttctggca gtccagagaa tgacacggga aaaaagcaga tgaagagaag gtggcaggag 2460

agggcacgtg gcccagcctc agtctctcca a 2491

<210> 91

<211> 2789

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2789)

<400> 91

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatgctcc ttttggtgac ttccttgctc ctgtgcgagc 600

tcccgcaccc cgcgttcctg ctcattcccg acgattataa agatgacgat gacaagatcc 660

agatgaccca gacgaccagc agcctgtctg cttcgctggg tgaccgcgtc accatctcat 720

gccgcgccag ccaggacatt tccaagtacc tcaactggta ccaacagaag ccagacggca 780

ccgtcaagct gctgatctac catacctctc gccttcatag tggtgtgcca tccaggtttt 840

cagggtctgg ctcaggcacc gactactccc tcactatctc taatctggaa caggaggaca 900

tcgctaccta tttctgtcaa cagggcaaca cgttacccta taccttcggc ggaggcacca 960

agttggagat caccggctcc acaagtggga gcggtaaacc gggctccggg gagggctcta 1020

caaaaggtga agtgaagttg caggagagcg ggcccggtct cgtagcacca tcccagagcc 1080

tgtcggtaac ctgcaccgtg tccggggtgt ccctgcccga ctacggcgtg agttggatcc 1140

gccagccacc caggaaagga ctggaatggc taggcgtgat ctggggctcc gagactacct 1200

actacaactc cgccctgaaa tctcgcctga ccataatcaa ggacaactct aagtcccagg 1260

tgttcctgaa gatgaattcc ctacagactg atgacaccgc catctactac tgtgccaagc 1320

actactacta cggtgggagc tacgccatgg attattgggg ccagggcacg tccgtgaccg 1380

tgtcgtctgc ggccgctatt gaggtgatgt atcccccgcc gtacctggac aacgagaagt 1440

ctaatggcac cattatccac gttaagggga agcacctgtg cccaagcccc ctgttccccg 1500

gcccttccaa gcccttctgg gtcctggtgg tggtcggggg tgtcctggcc tgttactctc 1560

tgttagtcac cgtggcattc atcatcttct gggtcagatc caagcgcagt cggctgctgc 1620

actccgacta catgaacatg accccccgcc ggcctggtcc tacccgcaag cattaccagc 1680

cgtacgcgcc gccccgggat tttgctgcct accgtagccg tgttaaattt tcacgctcgg 1740

cggacgcacc tgcgtatcag cagggacaga accagctgta caacgagctg aacctgggca 1800

ggcgtgagga gtacgacgtg ctggacaagc gccgcggccg cgaccccgag atgggcggca 1860

aacctcgtcg caagaaccct caggagggcc tttacaacga gctgcagaag gacaaaatgg 1920

ccgaggctta ttcggagatc ggaatgaagg gggagcgccg acgcggcaag ggccacgatg 1980

gcctgtacca gggtttgtcc actgccacta aggatacata tgatgcgctg cacatgcagg 2040

cccttcctcc tcgatagcta gagctcgctg atcagcctcg actgtgcctt ctagttgcca 2100

gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac 2160

tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat 2220

tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga atagcaggca 2280

tgctggggat accagctgag agactctaaa tccagtgaca agtctgtctg cctattcacc 2340

gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta tatcacagac 2400

aaaactgtgc tagacatgag gtctatggac ttcaagagca acagtgctgt ggcctggagc 2460

aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca gcattattcc agaagacacc 2520

ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg caggctgttt ccttgcttca 2580

ggaatggcca ggttctgccc agagctctgg tcaatgatgt ctaaaactcc tctgattggt 2640

ggtctcggcc ttatccattg ccaccaaaac cctcttttta ctaagaaaca gtgagccttg 2700

ttctggcagt ccagagaatg acacgggaaa aaagcagatg aagagaaggt ggcaggagag 2760

ggcacgtggc ccagcctcag tctctccaa 2789

<210> 92

<211> 1350

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1350)

<400> 92

ggtagctagg agttccagga ctcagtttcc cctttgagcc tcctttagcg actaaagctt 60

gaagccccac gcatctcgac tctcgcgcac accgcccttg ttgggctcag gggcggggcg 120

ccgcccccgg aagtacttcc ccttaaaggc tggggcctgc cggaaatggc gcagcggcag 180

ggaggggctc ttcacccagt ccggcagttg aagctcggcg ctcgggttac ccctgcagcg 240

acgccccctg gtcccacaga taccactgct gctcccgccc tttcgctcct cggccgcgca 300

atgggcggat cgggtgggac tagtggcagc aagggcgagg agctgttcac cggggtggtg 360

cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gcgcggcgag 420

ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac caccggcaag 480

ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 540

cgctaccccg accacatgaa gcgccacgac ttcttcaagt ccgccatgcc cgaaggctac 600

gtccaggagc gcaccatcag cttcaaggac gacggcacct acaagacccg cgccgaggtg 660

aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 720

gacggcaaca tcctggggca caagctggag tacaacttca acagccacaa cgtctatatc 780

accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacgtggag 840

gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 900

gtgctgctgc ccgacaacca ctacctgagc acccagtccg tgctgagcaa agaccccaac 960

gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactggaacc 1020

ggtgctggaa gtggtacacg cgacgacgag tacgactacc tctttaaagg tgaggccatg 1080

ggctctcgca ctctacacag tcctcgttcg gggacccggg ccactcccgg tggaccctcg 1140

tgccggccac ccctgcactg atataggcct ccctcagccc ttcctttttg tgcggttccg 1200

tctcctaccc agctcagcct cttctccccc gctcagacag gggtccccat cacatgccgc 1260

tctctgagcg acctctccat aggccttcgc tggcctcaga gcccctccct gcgtgtcctt 1320

cccctggcgg actgccttct cccacatcgt 1350

<210> 93

<211> 1350

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1350)

<400> 93

ggtagctagg agttccagga ctcagtttcc cctttgagcc tcctttagcg actaaagctt 60

gaagccccac gcatctcgac tctcgcgcac accgcccttg ttgggctcag gggcggggcg 120

ccgcccccgg aagtacttcc ccttaaaggc tggggcctgc cggaaatggc gcagcggcag 180

ggaggggctc ttcacccagt ccggcagttg aagctcggcg ctcgggttac ccctgcagcg 240

acgccccctg gtcccacaga taccactgct gctcccgccc tttcgctcct cggccgcgca 300

atgggcggat cgggtgggac tagtggcagc aagggcgagg agctgttcac cggggtggtg 360

cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gcgcggcgag 420

ggcgagggcg atgccaccaa cggcaagctg accctgaagt tcatctgcac caccggcaag 480

ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc 540

cgctaccccg accacatgaa gcgccacgac ttcttcaagt ccgccatgcc cgaaggctac 600

gtccaggagc gcaccatcag cttcaaggac gacggcacct acaagacccg cgccgaggtg 660

aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag 720

gacggcaaca tcctggggca caagctggag tacaacttca acagccacaa cgtctatatc 780

accgccgaca agcagaagaa cggcatcaag gccaacttca agatccgcca caacgtggag 840

gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc 900

gtgctgctgc ccgacaacca ctacctgagc acccagtccg tgctgagcaa agaccccaac 960

gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactggaacc 1020

ggtgctggaa gtggtacacg cgacgacgag tacgactacc tctttaaagg tgaggccatg 1080

ggctctcgca ctctacacag tcctcgttcg gggacccggg ccactcccgg tggaccctcg 1140

tgccggccac ccctgcactg atataggcct ccctcagccc ttcctttttg tgcggttccg 1200

tctcctaccc agctcagcct cttctccccc gctcagacag gggtccccat cacatgccgc 1260

tctctgagcg acctctccat aggccttcgc tggcctcaga gcccctccct gcgtgtcctt 1320

cccctggcgg actgccttct cccacatcgt 1350

<210> 94

<211> 2754

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2754)

<400> 94

agaaaggtga agagccaaag ttagaactca ggaccaactt attctgattt tgtttttcca 60

aactgcttct cctcttggga agtgtaagga agctgcagca ccaggatcag tgaaacgcac 120

cagacggccg cgtcagagca gctcaggttc tgggagaggg tagcgcaggg tggccactga 180

gaaccgggca ggtcacgcat cccccccttc cctcccaccc cctgccaagc tctccctccc 240

aggatcctct ctggctccat cgtaagcaaa ccttagaggt tctggcaagg agagagatgg 300

ctccaggaaa tgggggtgtg tcaccagata aggaatctgc ctaacaggag gtgggggtta 360

gacccaatat caggagacta ggaaggagga ggcctaagga tggggctttt ctgtcaccaa 420

tcctgtccct agtggcccca ctgtggggtg gaggggacag ataaaagtac ccagaaccag 480

agccacatta accggccctg cgttacataa cttacggtaa atggcccgcc tggctgaccg 540

cccaacgacc cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt 600

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 660

ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag 720

tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 780

accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca 840

cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg 900

gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg 960

agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg 1020

cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgacg 1080

ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 1140

gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 1200

gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta 1260

cctggagcac ctgcctgaaa tcactttttt tcaggttggt ctagagccac catggtgagc 1320

aagggcgagg aggataacat ggcctctctc ccagcgacac atgagttaca catctttggc 1380

tccatcaacg gtgtggactt tgacatggtg ggtcagggca ccggcaatcc aaatgatggt 1440

tatgaggagt taaacctgaa gtccaccaag ggtgacctcc agttctcccc ctggattctg 1500

gtccctcata tcgggtatgg cttccatcag tacctgccct accctgacgg gatgtcgcct 1560

ttccaggccg ccatggtaga tggctccgga taccaagtcc atcgcacaat gcagtttgaa 1620

gatggtgcct cccttactgt taactaccgc tacacctacg agggaagcca catcaaagga 1680

gaggcccagg tgaaggggac tggtttccct gctgacggtc ctgtgatgac caactcgctg 1740

accgctgcgg actggtgcag gtcgaagaag acttacccca acgacaaaac catcatcagt 1800

acctttaagt ggagttacac cactggaaat ggcaagcgct accggagcac tgcgcggacc 1860

acctacacct ttgccaagcc aatggcggct aactatctga agaaccagcc gatgtacgtg 1920

ttccgtaaga cggagctcaa gcactccaag accgagctca acttcaagga gtggcaaaag 1980

gcctttaccg atgtgatggg catggacgag ctgtacaagt agctagagct cgctgatcag 2040

cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 2100

tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 2160

attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 2220

aggattggga agagaatagc aggcatgctg gggaggaata taaggtggtc ccagctcggg 2280

gacacaggat ccctggaggc agcaaacatg ctgtcctgaa gtggacatag gggcccgggt 2340

tggaggaaga agactagctg agctctcgga cccctggaag atgccatgac agggggctgg 2400

aagagctagc acagactaga gaggtaaggg gggtagggga gctgcccaaa tgaaaggagt 2460

gagaggtgac ccgaatccac aggagaacgg ggtgtccagg caaagaaagc aagaggatgg 2520

agaggtggct aaagccaggg agacggggta ctttggggtt gtccagaaaa acggtgatga 2580

tgcaggccta caagaagggg aggcgggacg caagggagac atccgtcgga gaaggccatc 2640

ctaagaaacg agagatggca caggccccag aaggagaagg aaaagggaac ccagcgagtg 2700

aagacggcat ggggttgggt gagggaggag agatgcccgg agaggaccca gaca 2754

<210> 95

<211> 2754

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2754)

<400> 95

agaaaggtga agagccaaag ttagaactca ggaccaactt attctgattt tgtttttcca 60

aactgcttct cctcttggga agtgtaagga agctgcagca ccaggatcag tgaaacgcac 120

cagacggccg cgtcagagca gctcaggttc tgggagaggg tagcgcaggg tggccactga 180

gaaccgggca ggtcacgcat cccccccttc cctcccaccc cctgccaagc tctccctccc 240

aggatcctct ctggctccat cgtaagcaaa ccttagaggt tctggcaagg agagagatgg 300

ctccaggaaa tgggggtgtg tcaccagata aggaatctgc ctaacaggag gtgggggtta 360

gacccaatat caggagacta ggaaggagga ggcctaagga tggggctttt ctgtcaccaa 420

tcctgtccct agtggcccca ctgtggggtg gaggggacag ataaaagtac ccagaaccag 480

agccacatta accggccctg cgttacataa cttacggtaa atggcccgcc tggctgaccg 540

cccaacgacc cccgcccatt gacgtcaata gtaacgccaa tagggacttt ccattgacgt 600

caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg 660

ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag 720

tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt 780

accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca 840

cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg 900

gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg 960

agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg 1020

cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgacg 1080

ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact 1140

gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta 1200

gctgagcaag aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta 1260

cctggagcac ctgcctgaaa tcactttttt tcaggttggt ctagagccac catggtgagc 1320

aagggcgagg aggataacat ggcctctctc ccagcgacac atgagttaca catctttggc 1380

tccatcaacg gtgtggactt tgacatggtg ggtcagggca ccggcaatcc aaatgatggt 1440

tatgaggagt taaacctgaa gtccaccaag ggtgacctcc agttctcccc ctggattctg 1500

gtccctcata tcgggtatgg cttccatcag tacctgccct accctgacgg gatgtcgcct 1560

ttccaggccg ccatggtaga tggctccgga taccaagtcc atcgcacaat gcagtttgaa 1620

gatggtgcct cccttactgt taactaccgc tacacctacg agggaagcca catcaaagga 1680

gaggcccagg tgaaggggac tggtttccct gctgacggtc ctgtgatgac caactcgctg 1740

accgctgcgg actggtgcag gtcgaagaag acttacccca acgacaaaac catcatcagt 1800

acctttaagt ggagttacac cactggaaat ggcaagcgct accggagcac tgcgcggacc 1860

acctacacct ttgccaagcc aatggcggct aactatctga agaaccagcc gatgtacgtg 1920

ttccgtaaga cggagctcaa gcactccaag accgagctca acttcaagga gtggcaaaag 1980

gcctttaccg atgtgatggg catggacgag ctgtacaagt agctagagct cgctgatcag 2040

cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct 2100

tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc 2160

attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg 2220

aggattggga agagaatagc aggcatgctg gggaggaata taaggtggtc ccagctcggg 2280

gacacaggat ccctggaggc agcaaacatg ctgtcctgaa gtggacatag gggcccgggt 2340

tggaggaaga agactagctg agctctcgga cccctggaag atgccatgac agggggctgg 2400

aagagctagc acagactaga gaggtaaggg gggtagggga gctgcccaaa tgaaaggagt 2460

gagaggtgac ccgaatccac aggagaacgg ggtgtccagg caaagaaagc aagaggatgg 2520

agaggtggct aaagccaggg agacggggta ctttggggtt gtccagaaaa acggtgatga 2580

tgcaggccta caagaagggg aggcgggacg caagggagac atccgtcgga gaaggccatc 2640

ctaagaaacg agagatggca caggccccag aaggagaagg aaaagggaac ccagcgagtg 2700

aagacggcat ggggttgggt gagggaggag agatgcccgg agaggaccca gaca 2754

<210> 96

<211> 1841

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1841)

<400> 96

gcggtgcatt gagcacattt ctctcccttg cagttctgcc cacatggtcc accccggtgc 60

agccaatggc cctgattgtg ctggggggcg tcgccggcct cctgcttttc attgggctag 120

gcatcttctt ctgtgtcagg tgccggcacc gaagggtgag taaccccaca cctggtcccc 180

acaaggccct caaacccctg agtcctctac caggagatcc tgtatatggg aactgatttt 240

ggcccagctc cctctgccca ctcgtaagtt cccttgctgc cctgtcccag atcccactca 300

agggagagac aggaaggagc agagagttaa ttccaggata gatggcctgg gccatgtaac 360

tgcttctcct gtcgcagctt cccccactcc ccccaccaag gggcacctcc cttctggagg 420

cctgggaccc tcgtgactcc ctttcttgtc cctggacagc gccaagcaga gcggatgtct 480

cagatcaaga gactcctcag cgaaaaaaaa acatgtcagt gtcctcaccg gtttcagaag 540

acatgtagcc ccattcgcgc aaagagagga agcggagaag gtagaggttc tctcctcact 600

tgtggtgatg ttgaagaaaa ccctggtcca atggtgagca agggcgagga ggataacatg 660

gcctctctcc cagcgacaca tgagttacac atctttggct ccatcaacgg tgtggacttt 720

gacatggtgg gtcagggcac cggcaatcca aatgatggtt atgaggagtt aaacctgaag 780

tccaccaagg gtgacctcca gttctccccc tggattctgg tccctcatat cgggtatggc 840

ttccatcagt acctgcccta ccctgacggg atgtcgcctt tccaggccgc catggtagat 900

ggctccggat accaagtcca tcgcacaatg cagtttgaag atggtgcctc ccttactgtt 960

aactaccgct acacctacga gggaagccac atcaaaggag aggcccaggt gaaggggact 1020

ggtttccctg ctgacggtcc tgtgatgacc aactcgctga ccgctgcgga ctggtgcagg 1080

tcgaagaaga cttaccccaa cgacaaaacc atcatcagta cctttaagtg gagttacacc 1140

actggaaatg gcaagcgcta ccggagcact gcgcggacca cctacacctt tgccaagcca 1200

atggcggcta actatctgaa gaaccagccg atgtacgtgt tccgtaagac ggagctcaag 1260

cactccaaga ccgagctcaa cttcaaggag tggcaaaagg cctttaccga tgtgatgggc 1320

atggacgagc tgtacaagtg agaagaagac ctgccagtgt cctcagtaag gatctgggag 1380

gaggggttga gagaggggaa agggggaggg ggagggagtt agagaggagg gggaggaagg 1440

ggagcaaagg ggggcaggaa gggaggatgg agaggaggaa ggagttgagg aggaagagct 1500

gggaggggtg gaggtgagga gatgggggct aaaggggtgt ggtggagagg atagaggggt 1560

gggaaaagat ggccaggagc tagaaggagg cagaagtggg aggatggagc tgaaggagca 1620

gcaggccagg aaaggccctg ctggaaagcc actggagctg tgctgcgctg gaaaggccat 1680

tggaggtgct agaacgcaaa ggggttgcag tggggacaga cctgctcccc ttcttctttg 1740

ttcctgcagc cggtttcaga agacatgtag ccccatttga ggcacgaggc caggcagatc 1800

ccacttgcag cctccccagg tgtctgcccc gcgtttcctg c 1841

<210> 97

<211> 1882

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(1882)

<400> 97

ttgtctgatg cccctgacag attctctttg taaggagttt atttcagggg caataagtaa 60

ttggcattat tgctggttgg tactgcaaag tacctatgaa agtccccaaa agttcttgct 120

attgttattt ctgcattttg gcagaacatg atggaaaatg caccctcaaa ctttggcaaa 180

ccggcacaaa gctgtgtgtt taatcacgcc tgccttgtcc tagtggtttc tatgaatctg 240

ctacttttcc gtaatattgc atcattaatt gttcctgaaa aaccctgagt tatcctctta 300

tagaattgta taagtaatga ttgcaatata gataattttg aaaggagaaa ccacctttcc 360

ttggaaatgt ttatcttttg cagagtgaca tttgtgagac cagctaattt gattaaaatt 420

ctcttggaat cagctttgct agtatcatac ctgtgccaga tttcatcatg ggaaacagct 480

gttacaacat agtagccact ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggcctctc 600

tcccagcgac acatgagtta cacatctttg gctccatcaa cggtgtggac tttgacatgg 660

tgggtcaggg caccggcaat ccaaatgatg gttatgagga gttaaacctg aagtccacca 720

agggtgacct ccagttctcc ccctggattc tggtccctca tatcgggtat ggcttccatc 780

agtacctgcc ctaccctgac gggatgtcgc ctttccaggc cgccatggta gatggctccg 840

gataccaagt ccatcgcaca atgcagtttg aagatggtgc ctcccttact gttaactacc 900

gctacaccta cgagggaagc cacatcaaag gagaggccca ggtgaagggg actggtttcc 960

ctgctgacgg tcctgtgatg accaactcgc tgaccgctgc ggactggtgc aggtcgaaga 1020

agacttaccc caacgacaaa accatcatca gtacctttaa gtggagttac accactggaa 1080

atggcaagcg ctaccggagc actgcgcgga ccacctacac ctttgccaag ccaatggcgg 1140

ctaactatct gaagaaccag ccgatgtacg tgttccgtaa gacggagctc aagcactcca 1200

agaccgagct caacttcaag gagtggcaaa aggcctttac cgatgtgatg ggcatggacg 1260

agctgtacaa gcgcgcaaag agaggctccg gtgctaccaa tttctcactg ttgaaacaag 1320

cgggcgatgt tgaagaaaat cccggtccaa tgggaaacag ctgttacaac atagtagcaa 1380

cactgttgct ggtcctcaac tttgagagga caagatcatt gcaggatcct tgtagtaact 1440

gcccagctgg tgagtaccca gttatcatgt gcatttgatc tgctctgttg gaagtatggt 1500

tcagttagtc tagtagtcag ggctaacgag ctccctttta aggaaaggaa aatgaaaatt 1560

cattcattta caaatgttta ttggatgcta caacctagct gtgtgaacac agcaaagtca 1620

ttcaacctct tgtgccttga ctttctcatc tggggataat aagagaacct gttttatagg 1680

atggctggga ggatcaaatg aagggcttag aacagtgcat ggcacaaggc aagacttcaa 1740

taaatgttag ttttgtgtgt agggctttgt gctccgactg ggggcatagc agcgagtaag 1800

cgcgtagtaa agggcttaac agagtgggga cggtcagtcg catttaaatt ttagtgtagg 1860

acattgatgt cctcctggat cc 1882

<210> 98

<211> 2263

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2263)

<400> 98

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggccatca 600

tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg 660

agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg 720

tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg 780

gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc 840

ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga 900

cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca 960

acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg 1020

agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga 1080

aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc 1140

agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact 1200

acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg 1260

agctgtacaa gggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1320

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1380

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1440

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1500

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1560

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1620

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1680

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1740

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 1800

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 1860

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 1920

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 1980

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 2040

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 2100

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 2160

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 2220

aggtggcagg agagggcacg tggcccagcc tcagtctctc caa 2263

<210> 99

<211> 2263

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2263)

<400> 99

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatggtga gcaagggcga ggaggataac atggccatca 600

tcaaggagtt catgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg 660

agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg 720

tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg 780

gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc 840

ccgagggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga 900

cccaggactc ctccctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca 960

acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg 1020

agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga 1080

aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc 1140

agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact 1200

acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg 1260

agctgtacaa gggctccggt gctaccaatt tctcactgtt gaaacaagcg ggcgatgttg 1320

aagaaaatcc cggtccaatg gagaccctct tgggcctgct tatcctttgg ctgcagctgc 1380

aatgggtgag cagcaaacag gaggtgacgc agattcctgc agctctgagt gtcccagaag 1440

gagaaaactt ggttctcaac tgcagtttca ctgatagcgc tatttacaac ctccagtggt 1500

ttaggcagga ccctgggaaa ggtctcacat ctctgttgct tattcagtca agtcagagag 1560

agcaaacaag tggaagactt aatgcctcgc tggataaatc atcaggacgt agtactttat 1620

acattgcagc ttctcagcct ggtgactcag ccacctacct ctgtgctgtg aggcccctgt 1680

acggaggaag ctacatacct acatttggaa gaggaaccag ccttattgtt catccgtata 1740

tccagaaccc tgaccctgcg gtataccagc tgagagactc taaatccagt gacaagtctg 1800

tctgcctatt caccgatttt gattctcaaa caaatgtgtc acaaagtaag gattctgatg 1860

tgtatatcac agacaaaact gtgctagaca tgaggtctat ggacttcaag agcaacagtg 1920

ctgtggcctg gagcaacaaa tctgactttg catgtgcaaa cgccttcaac aacagcatta 1980

ttccagaaga caccttcttc cccagcccag gtaagggcag ctttggtgcc ttcgcaggct 2040

gtttccttgc ttcaggaatg gccaggttct gcccagagct ctggtcaatg atgtctaaaa 2100

ctcctctgat tggtggtctc ggccttatcc attgccacca aaaccctctt tttactaaga 2160

aacagtgagc cttgttctgg cagtccagag aatgacacgg gaaaaaagca gatgaagaga 2220

aggtggcagg agagggcacg tggcccagcc tcagtctctc caa 2263

<210> 100

<211> 4774

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(4774)

<400> 100

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatggtcc tgctgagccg caagcgccgc cggcagcacg 600

gccagctttg gttcccggag ggcttcaagg tgtctgaggc tagcaagaag aaacgcaggg 660

agcccctggg cgaagattct gttggactta agcctctcaa gaacgcatcc gatggcgccc 720

tgatggatga caaccagaat gagtggggcg acgaggatct ggagaccaag aagttccgct 780

tcgaggaacc ggtcgtgctg cccgacctgg acgaccagac cgaccatcgc cagtggacgc 840

agcagcacct ggacgcagcc gacctgcgga tgagtgccat ggcgccaacc ccccctcaag 900

gcgaggttga tgccgactgc atggatgtga acgtgcgggg ccccgacggg ttcacccctc 960

tgatgatcgc gtcgtgtagc ggcgggggcc tggagaccgg caactccgag gaagaggagg 1020

acgcccccgc cgtcatctct gatttcatct accagggtgc ttcccttcat aaccagactg 1080

accgcactgg ggagacggcc ctccacctgg cagcccgcta tagccgcagc gatgctgcga 1140

agcggttgtt ggaagccagc gccgacgcca acatacagga caatatgggc cgcactcctt 1200

tgcacgccgc ggtgtcggcc gacgctcagg gcgtgttcca gatcctaatt cgcaatcgcg 1260

ccaccgacct cgacgcccgc atgcacgacg gcaccacacc cctgatcctg gcggcgcgcc 1320

tcgccgtgga gggaatgttg gaggacctta ttaacagcca cgccgacgta aatgctgttg 1380

atgacctcgg gaaatcggcc ctccattggg ctgctgcagt caacaacgtg gacgctgccg 1440

tcgtcctgtt gaaaaatggg gcaaacaagg acatgcaaaa taaccgcgag gagactcccc 1500

tcttcctggc ggctagagag ggctcgtacg agactgccaa ggtgctcctc gatcacttcg 1560

ccaaccggga catcacggat catatggata ggctccctcg ggacatcgcg caggagcgaa 1620

tgcaccacga cattgtccgt ctgctggacg agtacaacct ggtgcgctcc ccacagctgc 1680

acggcgcccc gctggggggt actcccaccc tttccccgcc cctgtgcagc cccaacggct 1740

atctgggctc cctgaagccc ggcgtccagg gcaagaaagt ccgtaaacct agttccaagg 1800

gcttagcttg cgggtcgaag gaggctaagg atctgaaagc taggcggaag aagtcccaag 1860

acggcaaagg gtgcctgtta gattccagtg gcatgctgtc ccctgtcgac tctctggagt 1920

cgcctcacgg gtacctgtct gacgtggcct ccccccccct gcttccctcc ccgtttcagc 1980

agagcccctc cgtgcccctg aatcacctcc ctggtatgcc cgacacccac ctaggcatcg 2040

gccatttgaa cgtggctgca aagccagaga tggccgcttt ggggggcggg ggccgtctgg 2100

catttgagac aggcccgccc cgcctgagcc acctgcccgt ggcatctggg acctctaccg 2160

tgctgggttc ttcctccggc ggggccctta acttcaccgt gggcggctcc accagtctga 2220

acgggcagtg tgaatggcta tcacgactgc agagcggtat ggtgcctaac caatacaacc 2280

ccctgcgggg atccgtggcc ccgggccccc tctccactca ggctcctagc ttgcagcatg 2340

gtatggtggg ccctctacat tccagcctgg cggcgtccgc cctgtcccag atgatgtctt 2400

atcaggggct accgtctacc cgcctggcca cgcagccgca cctggtgcag acgcagcagg 2460

tgcagcctca gaacctgcag atgcagcagc agaacctgca gcccgccaac atccagcagc 2520

agcagtctct ccagccccca ccgccccccc ctcagccgca tctgggggtc agttccgcag 2580

cgtctggcca cctaggacgc tccttcctgt ctggagaacc atcgcaagct gatgtgcagc 2640

cccttggccc gtcctctctg gccgtgcaca ccatcttgcc tcaggagtcc cccgctctcc 2700

cgaccagcct gccatcaagc ttggtgccac ccgtgaccgc tgctcagttt ctgactcctc 2760

caagccagca ctcctattcc agccccgtgg ataacacccc cagccaccag ctgcaggtcc 2820

cggagcaccc ttttctcact cctagcccag agtcgcccga ccagtggtct tcgtcctccc 2880

cacacagcaa cgtgtccgat tggtccgagg gggtgagctc acctcccacg tccatgcagt 2940

cccagattgc ccggatcccg gaggcattta aggattataa agatgacgat gacaagcgcg 3000

ccaagcgctc cggaagcgga gaaggtagag gttctctcct cacttgtggt gatgttgaag 3060

aaaaccctgg tccaatggtg agcaagggcg aggaggataa catggcctct ctcccagcga 3120

cacatgagtt acacatcttt ggctccatca acggtgtgga ctttgacatg gtgggtcagg 3180

gcaccggcaa tccaaatgat ggttatgagg agttaaacct gaagtccacc aagggtgacc 3240

tccagttctc cccctggatt ctggtccctc atatcgggta tggcttccat cagtacctgc 3300

cctaccctga cgggatgtcg cctttccagg ccgccatggt agatggctcc ggataccaag 3360

tccatcgcac aatgcagttt gaagatggtg cctcccttac tgttaactac cgctacacct 3420

acgagggaag ccacatcaaa ggagaggccc aggtgaaggg gactggtttc cctgctgacg 3480

gtcctgtgat gaccaactcg ctgaccgctg cggactggtg caggtcgaag aagacttacc 3540

ccaacgacaa aaccatcatc agtaccttta agtggagtta caccactgga aatggcaagc 3600

gctaccggag cactgcgcgg accacctaca cctttgccaa gccaatggcg gctaactatc 3660

tgaagaacca gccgatgtac gtgttccgta agacggagct caagcactcc aagaccgagc 3720

tcaacttcaa ggagtggcaa aaggccttta ccgatgtgat gggcatggac gagctgtaca 3780

agggctccgg tgctaccaat ttctcactgt tgaaacaagc gggcgatgtt gaagaaaatc 3840

ccggtccaat ggagaccctc ttgggcctgc ttatcctttg gctgcagctg caatgggtga 3900

gcagcaaaca ggaggtgacg cagattcctg cagctctgag tgtcccagaa ggagaaaact 3960

tggttctcaa ctgcagtttc actgatagcg ctatttacaa cctccagtgg tttaggcagg 4020

accctgggaa aggtctcaca tctctgttgc ttattcagtc aagtcagaga gagcaaacaa 4080

gtggaagact taatgcctcg ctggataaat catcaggacg tagtacttta tacattgcag 4140

cttctcagcc tggtgactca gccacctacc tctgtgctgt gaggcccctg tacggaggaa 4200

gctacatacc tacatttgga agaggaacca gccttattgt tcatccgtat atccagaacc 4260

ctgaccctgc ggtataccag ctgagagact ctaaatccag tgacaagtct gtctgcctat 4320

tcaccgattt tgattctcaa acaaatgtgt cacaaagtaa ggattctgat gtgtatatca 4380

cagacaaaac tgtgctagac atgaggtcta tggacttcaa gagcaacagt gctgtggcct 4440

ggagcaacaa atctgacttt gcatgtgcaa acgccttcaa caacagcatt attccagaag 4500

acaccttctt ccccagccca ggtaagggca gctttggtgc cttcgcaggc tgtttccttg 4560

cttcaggaat ggccaggttc tgcccagagc tctggtcaat gatgtctaaa actcctctga 4620

ttggtggtct cggccttatc cattgccacc aaaaccctct ttttactaag aaacagtgag 4680

ccttgttctg gcagtccaga gaatgacacg ggaaaaaagc agatgaagag aaggtggcag 4740

gagagggcac gtggcccagc ctcagtctct ccaa 4774

<210> 101

<211> 5014

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(5014)

<400> 101

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatggtcc tgctgagccg caagcgccgc cggcagcacg 600

gccagctttg gttcccggag ggcttcaagg tgtctgaggc tagcaagaag aaacgcaggg 660

agcccctggg cgaagattct gttggactta agcctctcaa gaacgcatcc gatggcgccc 720

tgatggatga caaccagaat gagtggggcg acgaggatct ggagaccaag aagttccgct 780

tcgaggaacc ggtcgtgctg cccgacctgg acgaccagac cgaccatcgc cagtggacgc 840

agcagcacct ggacgcagcc gacctgcgga tgagtgccat ggcgccaacc ccccctcaag 900

gcgaggttga tgccgactgc atggatgtga acgtgcgggg ccccgacggg ttcacccctc 960

tgatgatcgc gtcgtgtagc ggcgggggcc tggagaccgg caactccgag gaagaggagg 1020

acgcccccgc cgtcatctct gatttcatct accagggtgc ttcccttcat aaccagactg 1080

accgcactgg ggagacggcc ctccacctgg cagcccgcta tagccgcagc gatgctgcga 1140

agcggttgtt ggaagccagc gccgacgcca acatacagga caatatgggc cgcactcctt 1200

tgcacgccgc ggtgtcggcc gacgctcagg gcgtgttcca gatcctaatt cgcaatcgcg 1260

ccaccgacct cgacgcccgc atgcacgacg gcaccacacc cctgatcctg gcggcgcgcc 1320

tcgccgtgga gggaatgttg gaggacctta ttaacagcca cgccgacgta aatgctgttg 1380

atgacctcgg gaaatcggcc ctccattggg ctgctgcagt caacaacgtg gacgctgccg 1440

tcgtcctgtt gaaaaatggg gcaaacaagg acatgcaaaa taaccgcgag gagactcccc 1500

tcttcctggc ggctagagag ggctcgtacg agactgccaa ggtgctcctc gatcacttcg 1560

ccaaccggga catcacggat catatggata ggctccctcg ggacatcgcg caggagcgaa 1620

tgcaccacga cattgtccgt ctgctggacg agtacaacct ggtgcgctcc ccacagctgc 1680

acggcgcccc gctggggggt actcccaccc tttccccgcc cctgtgcagc cccaacggct 1740

atctgggctc cctgaagccc ggcgtccagg gcaagaaagt ccgtaaacct agttccaagg 1800

gcttagcttg cgggtcgaag gaggctaagg atctgaaagc taggcggaag aagtcccaag 1860

acggcaaagg gtgcctgtta gattccagtg gcatgctgtc ccctgtcgac tctctggagt 1920

cgcctcacgg gtacctgtct gacgtggcct ccccccccct gcttccctcc ccgtttcagc 1980

agagcccctc cgtgcccctg aatcacctcc ctggtatgcc cgacacccac ctaggcatcg 2040

gccatttgaa cgtggctgca aagccagaga tggccgcttt ggggggcggg ggccgtctgg 2100

catttgagac aggcccgccc cgcctgagcc acctgcccgt ggcatctggg acctctaccg 2160

tgctgggttc ttcctccggc ggggccctta acttcaccgt gggcggctcc accagtctga 2220

acgggcagtg tgaatggcta tcacgactgc agagcggtat ggtgcctaac caatacaacc 2280

ccctgcgggg atccgtggcc ccgggccccc tctccactca ggctcctagc ttgcagcatg 2340

gtatggtggg ccctctacat tccagcctgg cggcgtccgc cctgtcccag atgatgtctt 2400

atcaggggct accgtctacc cgcctggcca cgcagccgca cctggtgcag acgcagcagg 2460

tgcagcctca gaacctgcag atgcagcagc agaacctgca gcccgccaac atccagcagc 2520

agcagtctct ccagccccca ccgccccccc ctcagccgca tctgggggtc agttccgcag 2580

cgtctggcca cctaggacgc tccttcctgt ctggagaacc atcgcaagct gatgtgcagc 2640

cccttggccc gtcctctctg gccgtgcaca ccatcttgcc tcaggagtcc cccgctctcc 2700

cgaccagcct gccatcaagc ttggtgccac ccgtgaccgc tgctcagttt ctgactcctc 2760

caagccagca ctcctattcc agccccgtgg ataacacccc cagccaccag ctgcaggtcc 2820

cggagcaccc ttttctcact cctagcccag agtcgcccga ccagtggtct tcgtcctccc 2880

cacacagcaa cgtgtccgat tggtccgagg gggtgagctc acctcccacg tccatgcagt 2940

cccagattgc ccggatcccg gaggcattta aggattataa agatgacgat gacaagcgcg 3000

ccaagcgctc cggaagcgga gaaggtagag gttctctcct cacttgtggt gatgttgaag 3060

aaaaccctgg tccaatgagc atcggcctcc tgtgctgtgc agccttgtct ctcctgtggg 3120

caggtccagt gaatgctggt gtcactcaga ccccaaaatt ccaggtcctg aagacaggac 3180

agagcatgac actgcagtgt gcccaggata tgaaccatga atacatgtcc tggtatcgac 3240

aagacccagg catggggctg aggctgattc attactcagt tggtgctggt atcactgacc 3300

aaggagaagt ccccaatggc tacaatgtct ccagatcaac cacagaggat ttcccgctca 3360

ggctgctgtc ggctgctccc tcccagacat ctgtgtactt ctgtgccagc agttacgtcg 3420

ggaacaccgg ggagctgttt tttggagaag gctctaggct gaccgtactg gaggacctga 3480

aaaacgtgtt cccacccgag gtcgctgtgt ttgagccatc agaagcagag atctcccaca 3540

cccaaaaggc cacactggta tgcctggcca caggcttcta ccccgaccac gtggagctga 3600

gctggtgggt gaatgggaag gaggtgcaca gtggggtcag cacagacccg cagcccctca 3660

aggagcagcc cgccctcaat gactccagat actgcctgag cagccgcctg agggtctcgg 3720

ccaccttctg gcagaacccc cgcaaccact tccgctgtca agtccagttc tacgggctct 3780

cagaaaacga tgaatggaca caagataggg ccaaacccgt cacccagatc gtcagcgccg 3840

aggcctgggg tagagcagac tgtggcttca cctccgagtc ttaccagcaa ggggtcctgt 3900

ctgccaccat cctctatgag atcttgctag ggaaggccac cttgtatgcc gtgctggtca 3960

gtgccctcgt gctgatggct atggtcaaga gaaaggattc cagaggccgc gccaagcgct 4020

ccggctccgg tgctaccaat ttctcactgt tgaaacaagc gggcgatgtt gaagaaaatc 4080

ccggtccaat ggagaccctc ttgggcctgc ttatcctttg gctgcagctg caatgggtga 4140

gcagcaaaca ggaggtgacg cagattcctg cagctctgag tgtcccagaa ggagaaaact 4200

tggttctcaa ctgcagtttc actgatagcg ctatttacaa cctccagtgg tttaggcagg 4260

accctgggaa aggtctcaca tctctgttgc ttattcagtc aagtcagaga gagcaaacaa 4320

gtggaagact taatgcctcg ctggataaat catcaggacg tagtacttta tacattgcag 4380

cttctcagcc tggtgactca gccacctacc tctgtgctgt gaggcccctg tacggaggaa 4440

gctacatacc tacatttgga agaggaacca gccttattgt tcatccgtat atccagaacc 4500

ctgaccctgc ggtataccag ctgagagact ctaaatccag tgacaagtct gtctgcctat 4560

tcaccgattt tgattctcaa acaaatgtgt cacaaagtaa ggattctgat gtgtatatca 4620

cagacaaaac tgtgctagac atgaggtcta tggacttcaa gagcaacagt gctgtggcct 4680

ggagcaacaa atctgacttt gcatgtgcaa acgccttcaa caacagcatt attccagaag 4740

acaccttctt ccccagccca ggtaagggca gctttggtgc cttcgcaggc tgtttccttg 4800

cttcaggaat ggccaggttc tgcccagagc tctggtcaat gatgtctaaa actcctctga 4860

ttggtggtct cggccttatc cattgccacc aaaaccctct ttttactaag aaacagtgag 4920

ccttgttctg gcagtccaga gaatgacacg ggaaaaaagc agatgaagag aaggtggcag 4980

gagagggcac gtggcccagc ctcagtctct ccaa 5014

<210> 102

<211> 6448

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(6448)

<400> 102

aacataccat aaacctccca ttctgctaat gcccagccta agttggggag accactccag 60

attccaagat gtacagtttg ctttgctggg cctttttccc atgcctgcct ttactctgcc 120

agagttatat tgctggggtt ttgaagaaga tcctattaaa taaaagaata agcagtatta 180

ttaagtagcc ctgcatttca ggtttccttg agtggcaggc caggcctggc cgtgaacgtt 240

cactgaaatc atggcctctt ggccaagatt gatagcttgt gcctgtccct gagtcccagt 300

ccatcacgag cagctggttt ctaagatgct atttcccgta taaagcatga gaccgtgact 360

tgccagcccc acagagcccc gcccttgtcc atcactggca tctggactcc agcctgggtt 420

ggggcaaaga gggaaatgag atcatgtcct aaccctgatc ctcttgtccc acagatatcc 480

agaaccctga ccctgccgtg ggaagcggag aaggtagagg ttctctcctc acttgtggtg 540

atgttgaaga aaaccctggt ccaatggatt ataaagatga cgatgacaag gcattatcac 600

tggaagaatt cgtccactcc cttgacctca ggaccctacc cagggttatg gcattatcac 660

tggaagaatt cgtccactcc cttgacctca ggaccctacc cagggttcta gaaatccagg 720

caggcatcta tcttgaaggc tctatttatg aaatgtttgg aaatgaacta gaaatccagg 780

caggcatcta tcttgaaggc tctatttatg aaatgtttgg aaatgaatgc tgtttttcaa 840

caggagaagt gattaaaatt actggtctca aagttaagaa gatcatatgc tgtttttcaa 900

caggagaagt gattaaaatt actggtctca aagttaagaa gatcatagct gaaatttgtg 960

agcagattga aggttgtgag tctctacagc catttgaact gcctatggct gaaatttgtg 1020

agcagattga aggttgtgag tctctacagc catttgaact gcctatgaat tttccaggtc 1080

tttttaagat tgtggctgat aaaactccat accttactat ggaagaaaat tttccaggtc 1140

tttttaagat tgtggctgat aaaactccat accttactat ggaagaaatc acaaggacca 1200

ttcatattgg accaagtaga ctagggcatc cttgcttcta tcatcagatc acaaggacca 1260

ttcatattgg accaagtaga ctagggcatc cttgcttcta tcatcagaag gatataaaac 1320

tagagaacct catcataaag cagggtgagc aaatcatgct caactcaaag gatataaaac 1380

tagagaacct catcataaag cagggtgagc aaatcatgct caactcagtt gaagagattg 1440

atggagaaat aatggtgagc tgtgcagtag caaggaatca tcaaactgtt gaagagattg 1500

atggagaaat aatggtgagc tgtgcagtag caaggaatca tcaaactcac tcatttaatt 1560

tgcctttgtc acaagaagga gaattctacg agtgtgaaga tgaacgtcac tcatttaatt 1620

tgcctttgtc acaagaagga gaattctacg agtgtgaaga tgaacgtatt tacactctaa 1680

aggagattgt tgaatggaag attcctaaga acagaacaag aactgtaatt tacactctaa 1740

aggagattgt tgaatggaag attcctaaga acagaacaag aactgtaaac cttacagatt 1800

tttcaaataa gtgggactca acgaatccat ttcctaaaga cttttataac cttacagatt 1860

tttcaaataa gtgggactca acgaatccat ttcctaaaga cttttatggt accctgattc 1920

tcaagcctgt ttatgaaatt caaggtgtga tgaaatttcg aaaagatggt accctgattc 1980

tcaagcctgt ttatgaaatt caaggtgtga tgaaatttcg aaaagatata atccgcatcc 2040

tccccagtct agatgtcgaa gtcaaagaca tcactgattc ttacgatata atccgcatcc 2100

tccccagtct agatgtcgaa gtcaaagaca tcactgattc ttacgatgct aactggtttc 2160

ttcagctgtt atcaacagaa gatctttttg aaatgactag taaagaggct aactggtttc 2220

ttcagctgtt atcaacagaa gatctttttg aaatgactag taaagagttc cccatagtga 2280

ctgaagtcat agaagcacct gaaggaaacc acctgcccca aagcattttc cccatagtga 2340

ctgaagtcat agaagcacct gaaggaaacc acctgcccca aagcatttta cagcctggga 2400

aaaccattgt gatccacaaa aagtaccagg catcaagaat cttagcttta cagcctggga 2460

aaaccattgt gatccacaaa aagtaccagg catcaagaat cttagcttca gaaattagaa 2520

gcaattttcc taaaagacac ttcttgatcc ccactagcta taaaggctca gaaattagaa 2580

gcaattttcc taaaagacac ttcttgatcc ccactagcta taaaggcaag ttcaagcggc 2640

gaccgaggga gttcccaacg gcctatgacc tagagatcgc taagagtaag ttcaagcggc 2700

gaccgaggga gttcccaacg gcctatgacc tagagatcgc taagagtgaa aaggagcctc 2760

ttcacgtggt ggccaccaaa gcgtttcatt cccctcatga caagctggaa aaggagcctc 2820

ttcacgtggt ggccaccaaa gcgtttcatt cccctcatga caagctgtca tccgtatctg 2880

ttggggacca gtttctggtg catcagtcag agacgactga agtcctctca tccgtatctg 2940

ttggggacca gtttctggtg catcagtcag agacgactga agtcctctgt gagggaataa 3000

aaaaagtggt gaatgttctg gcctgtgaaa aaatcctcaa aaagtcctgt gagggaataa 3060

aaaaagtggt gaatgttctg gcctgtgaaa aaatcctcaa aaagtcctat gaggctgcgc 3120

tgctcccttt gtacatggaa ggaggttttg tagaggtgat tcatgattat gaggctgcgc 3180

tgctcccttt gtacatggaa ggaggttttg tagaggtgat tcatgataag aaacagtacc 3240

cgatttctga gctctgtaaa cagttccgtt tgcccttcaa tgtgaagaag aaacagtacc 3300

cgatttctga gctctgtaaa cagttccgtt tgcccttcaa tgtgaaggtg tctgtcaggg 3360

atctttccat tgaagaggac gtgttggctg ccacaccagg actgcaggtg tctgtcaggg 3420

atctttccat tgaagaggac gtgttggctg ccacaccagg actgcagttg gaggaggaca 3480

ttacagactc ttacctactc ataagtgact ttgccaaccc cacggagttg gaggaggaca 3540

ttacagactc ttacctactc ataagtgact ttgccaaccc cacggagtgc tgggaaattc 3600

ctgtgggccg cttgaatatg actgttcagt tagttagtaa tttctcttgc tgggaaattc 3660

ctgtgggccg cttgaatatg actgttcagt tagttagtaa tttctctagg gatgcagaac 3720

catttctagt caggactctg gtagaagaga tcactgaaga gcaatatagg gatgcagaac 3780

catttctagt caggactctg gtagaagaga tcactgaaga gcaatattac atgatgcgga 3840

gatatgaaag ctcagcctca catcccccac ctcgccctcc gaaacactac atgatgcgga 3900

gatatgaaag ctcagcctca catcccccac ctcgccctcc gaaacacccc tcagtagagg 3960

aaacaaagtt aaccctgcta accttagcag aagaaaggac ggtagacccc tcagtagagg 4020

aaacaaagtt aaccctgcta accttagcag aagaaaggac ggtagacctg cccaagtctc 4080

ccaagcgtca tcacgtagac ataaccaaga aacttcaccc aaatcaactg cccaagtctc 4140

ccaagcgtca tcacgtagac ataaccaaga aacttcaccc aaatcaagct ggcctggatt 4200

caaaagtact gattggtagt cagaatgatt tggtggatga agagaaagct ggcctggatt 4260

caaaagtact gattggtagt cagaatgatt tggtggatga agagaaagaa aggagcaacc 4320

gtggggccac agcaatagca gaaacattca aaaatgaaaa acatcaagaa aggagcaacc 4380

gtggggccac agcaatagca gaaacattca aaaatgaaaa acatcaaaaa cgcgccaagc 4440

gctccggaag cggagaaggt agaggttctc tcctcacttg tggtgatgtt gaagaaaacc 4500

ctggtccaat gagcatcggc ctcctgtgct gtgcagcctt gtctctcctg tgggcaggtc 4560

cagtgaatgc tggtgtcact cagaccccaa aattccaggt cctgaagaca ggacagagca 4620

tgacactgca gtgtgcccag gatatgaacc atgaatacat gtcctggtat cgacaagacc 4680

caggcatggg gctgaggctg attcattact cagttggtgc tggtatcact gaccaaggag 4740

aagtccccaa tggctacaat gtctccagat caaccacaga ggatttcccg ctcaggctgc 4800

tgtcggctgc tccctcccag acatctgtgt acttctgtgc cagcagttac gtcgggaaca 4860

ccggggagct gttttttgga gaaggctcta ggctgaccgt actggaggac ctgaaaaacg 4920

tgttcccacc cgaggtcgct gtgtttgagc catcagaagc agagatctcc cacacccaaa 4980

aggccacact ggtatgcctg gccacaggct tctaccccga ccacgtggag ctgagctggt 5040

gggtgaatgg gaaggaggtg cacagtgggg tcagcacaga cccgcagccc ctcaaggagc 5100

agcccgccct caatgactcc agatactgcc tgagcagccg cctgagggtc tcggccacct 5160

tctggcagaa cccccgcaac cacttccgct gtcaagtcca gttctacggg ctctcagaaa 5220

acgatgaatg gacacaagat agggccaaac ccgtcaccca gatcgtcagc gccgaggcct 5280

ggggtagagc agactgtggc ttcacctccg agtcttacca gcaaggggtc ctgtctgcca 5340

ccatcctcta tgagatcttg ctagggaagg ccaccttgta tgccgtgctg gtcagtgccc 5400

tcgtgctgat ggctatggtc aagagaaagg attccagagg ccgcgccaag cgctccggct 5460

ccggtgctac caatttctca ctgttgaaac aagcgggcga tgttgaagaa aatcccggtc 5520

caatggagac cctcttgggc ctgcttatcc tttggctgca gctgcaatgg gtgagcagca 5580

aacaggaggt gacgcagatt cctgcagctc tgagtgtccc agaaggagaa aacttggttc 5640

tcaactgcag tttcactgat agcgctattt acaacctcca gtggtttagg caggaccctg 5700

ggaaaggtct cacatctctg ttgcttattc agtcaagtca gagagagcaa acaagtggaa 5760

gacttaatgc ctcgctggat aaatcatcag gacgtagtac tttatacatt gcagcttctc 5820

agcctggtga ctcagccacc tacctctgtg ctgtgaggcc cctgtacgga ggaagctaca 5880

tacctacatt tggaagagga accagcctta ttgttcatcc gtatatccag aaccctgacc 5940

ctgcggtata ccagctgaga gactctaaat ccagtgacaa gtctgtctgc ctattcaccg 6000

attttgattc tcaaacaaat gtgtcacaaa gtaaggattc tgatgtgtat atcacagaca 6060

aaactgtgct agacatgagg tctatggact tcaagagcaa cagtgctgtg gcctggagca 6120

acaaatctga ctttgcatgt gcaaacgcct tcaacaacag cattattcca gaagacacct 6180

tcttccccag cccaggtaag ggcagctttg gtgccttcgc aggctgtttc cttgcttcag 6240

gaatggccag gttctgccca gagctctggt caatgatgtc taaaactcct ctgattggtg 6300

gtctcggcct tatccattgc caccaaaacc ctctttttac taagaaacag tgagccttgt 6360

tctggcagtc cagagaatga cacgggaaaa aagcagatga agagaaggtg gcaggagagg 6420

gcacgtggcc cagcctcagt ctctccaa 6448

<210> 103

<211> 2341

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2341)

<400> 103

tggcgggact agtggccaca tctctcagct ggtacacgga acataccata aacctcccat 60

tctgctaatg cccagcctaa gttggggaga ccactccaga ttccaagatg tacagtttgc 120

tttgctgggc ctttttccca tgcctgcctt tactctgcca gagttatatt gctggggttt 180

tgaagaagat cctattaaat aaaagaataa gcagtattat taagtagccc tgcatttcag 240

gtttccttga gtggcaggcc aggcctggcc gtgaacgttc actgaaatca tggcctcttg 300

gccaagattg atagcttgtg cctgtccctg agtcccagtc catcacgagc agctggtttc 360

taagatgcta tttcccgtat aaagcatgag accgtgactt gccagcccca cagagccccg 420

cccttgtcca tcactggcat ctggactcca gcctgggttg gggcaaagag ggaaatgaga 480

tcatgtccta accctgatcc tcttgtccca cagatatcca gaaccctgac cctgccgtgg 540

gaagcggaga aggtagaggt tctctcctca cttgtggtga tgttgaagaa aaccctggtc 600

caatggtgag caagggcgag gaggataaca tggcctctct cccagcgaca catgagttac 660

acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc accggcaatc 720

caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc cagttctccc 780

cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc taccctgacg 840

ggatgtcgcc tttccaggcc gccatggtag atggctccgg ataccaagtc catcgcacaa 900

tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac gagggaagcc 960

acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt cctgtgatga 1020

ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc aacgacaaaa 1080

ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc taccggagca 1140

ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg aagaaccagc 1200

cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc aacttcaagg 1260

agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag ggctccggtg 1320

ctaccaattt ctcactgttg aaacaagcgg gcgatgttga agaaaatccc ggtccaatgg 1380

agaccctctt gggcctgctt atcctttggc tgcagctgca atgggtgagc agcaaacagg 1440

aggtgacgca gattcctgca gctctgagtg tcccagaagg agaaaacttg gttctcaact 1500

gcagtttcac tgatagcgct atttacaacc tccagtggtt taggcaggac cctgggaaag 1560

gtctcacatc tctgttgctt attcagtcaa gtcagagaga gcaaacaagt ggaagactta 1620

atgcctcgct ggataaatca tcaggacgta gtactttata cattgcagct tctcagcctg 1680

gtgactcagc cacctacctc tgtgctgtga ggcccctgta cggaggaagc tacataccta 1740

catttggaag aggaaccagc cttattgttc atccgtatat ccagaaccct gaccctgcgg 1800

tataccagct gagagactct aaatccagtg acaagtctgt ctgcctattc accgattttg 1860

attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca gacaaaactg 1920

tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg agcaacaaat 1980

ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac accttcttcc 2040

ccagcccagg taagggcagc tttggtgcct tcgcaggctg tttccttgct tcaggaatgg 2100

ccaggttctg cccagagctc tggtcaatga tgtctaaaac tcctctgatt ggtggtctcg 2160

gccttatcca ttgccaccaa aaccctcttt ttactaagaa acagtgagcc ttgttctggc 2220

agtccagaga atgacacggg aaaaaagcag atgaagagaa ggtggcagga gagggcacgt 2280

ggcccagcct cagtctctcc aaccgtgtac cagctgagag atgtggccac tagtcccgcc 2340

a 2341

<210> 104

<211> 2341

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(2341)

<400> 104

tggcgggact agtggccaca tctctcagct ggtacacgga acataccata aacctcccat 60

tctgctaatg cccagcctaa gttggggaga ccactccaga ttccaagatg tacagtttgc 120

tttgctgggc ctttttccca tgcctgcctt tactctgcca gagttatatt gctggggttt 180

tgaagaagat cctattaaat aaaagaataa gcagtattat taagtagccc tgcatttcag 240

gtttccttga gtggcaggcc aggcctggcc gtgaacgttc actgaaatca tggcctcttg 300

gccaagattg atagcttgtg cctgtccctg agtcccagtc catcacgagc agctggtttc 360

taagatgcta tttcccgtat aaagcatgag accgtgactt gccagcccca cagagccccg 420

cccttgtcca tcactggcat ctggactcca gcctgggttg gggcaaagag ggaaatgaga 480

tcatgtccta accctgatcc tcttgtccca cagatatcca gaaccctgac cctgccgtgg 540

gaagcggaga aggtagaggt tctctcctca cttgtggtga tgttgaagaa aaccctggtc 600

caatggtgag caagggcgag gaggataaca tggcctctct cccagcgaca catgagttac 660

acatctttgg ctccatcaac ggtgtggact ttgacatggt gggtcagggc accggcaatc 720

caaatgatgg ttatgaggag ttaaacctga agtccaccaa gggtgacctc cagttctccc 780

cctggattct ggtccctcat atcgggtatg gcttccatca gtacctgccc taccctgacg 840

ggatgtcgcc tttccaggcc gccatggtag atggctccgg ataccaagtc catcgcacaa 900

tgcagtttga agatggtgcc tcccttactg ttaactaccg ctacacctac gagggaagcc 960

acatcaaagg agaggcccag gtgaagggga ctggtttccc tgctgacggt cctgtgatga 1020

ccaactcgct gaccgctgcg gactggtgca ggtcgaagaa gacttacccc aacgacaaaa 1080

ccatcatcag tacctttaag tggagttaca ccactggaaa tggcaagcgc taccggagca 1140

ctgcgcggac cacctacacc tttgccaagc caatggcggc taactatctg aagaaccagc 1200

cgatgtacgt gttccgtaag acggagctca agcactccaa gaccgagctc aacttcaagg 1260

agtggcaaaa ggcctttacc gatgtgatgg gcatggacga gctgtacaag ggctccggtg 1320

ctaccaattt ctcactgttg aaacaagcgg gcgatgttga agaaaatccc ggtccaatgg 1380

agaccctctt gggcctgctt atcctttggc tgcagctgca atgggtgagc agcaaacagg 1440

aggtgacgca gattcctgca gctctgagtg tcccagaagg agaaaacttg gttctcaact 1500

gcagtttcac tgatagcgct atttacaacc tccagtggtt taggcaggac cctgggaaag 1560

gtctcacatc tctgttgctt attcagtcaa gtcagagaga gcaaacaagt ggaagactta 1620

atgcctcgct ggataaatca tcaggacgta gtactttata cattgcagct tctcagcctg 1680

gtgactcagc cacctacctc tgtgctgtga ggcccctgta cggaggaagc tacataccta 1740

catttggaag aggaaccagc cttattgttc atccgtatat ccagaaccct gaccctgcgg 1800

tataccagct gagagactct aaatccagtg acaagtctgt ctgcctattc accgattttg 1860

attctcaaac aaatgtgtca caaagtaagg attctgatgt gtatatcaca gacaaaactg 1920

tgctagacat gaggtctatg gacttcaaga gcaacagtgc tgtggcctgg agcaacaaat 1980

ctgactttgc atgtgcaaac gccttcaaca acagcattat tccagaagac accttcttcc 2040

ccagcccagg taagggcagc tttggtgcct tcgcaggctg tttccttgct tcaggaatgg 2100

ccaggttctg cccagagctc tggtcaatga tgtctaaaac tcctctgatt ggtggtctcg 2160

gccttatcca ttgccaccaa aaccctcttt ttactaagaa acagtgagcc ttgttctggc 2220

agtccagaga atgacacggg aaaaaagcag atgaagagaa ggtggcagga gagggcacgt 2280

ggcccagcct cagtctctcc aaccgtgtac cagctgagag atgtggccac tagtcccgcc 2340

a 2341

<210> 105

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(25)

<400> 105

aacataccat aaacctccca ttctg 25

<210> 106

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(25)

<400> 106

ttggagagac tgaggctggg ccacg 25

<210> 107

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(25)

<400> 107

tggcgggact agtggccaca tctct 25

<210> 108

<211> 25

<212> DNA

<213> artificial sequence

<220>

<223> Synthesis of Polynucleotide

<220>

<221> misc_feature

<222> (1)..(25)

<400> 108

tggcgggact agtggccaca tctct 25

Claims

1. A method for preparing an engineered T cell, the method comprising:

a) Contacting a T cell with a first ribonucleoprotein particle (RNP) and a donor DNA under conditions that allow the T cell to enter the first RNP and the donor DNA, wherein the first RNP comprises a first guide RNA that targets an endogenous TCR-locus, and wherein the donor DNA comprises a nucleic acid sequence comprising a gene encoding a polypeptide comprising an exogenous TCR- β and an exogenous TCR- α or a portion thereof;

b) Incubating the T cells for a period of time; and

c) Culturing the cells in a medium for a period of time to allow insertion of the donor DNA into the endogenous TCR-a locus, thereby forming engineered T cells.

2. The method of claim 1, wherein the exogenous TCR-a comprises full-length TCR-a.

3. The method of claim 1, wherein the exogenous TCR-a or portion thereof comprises a TCR-a (VJ) domain.

4. A method according to any one of claims 1 to 3, wherein the endogenous TCR locus is an endogenous TCR-a locus.

5. A method according to any one of claims 1 to 3, wherein the endogenous TCR locus is an endogenous TCR- β locus.

6. The method of claim 1, wherein the TCR locus is a TCR-a locus and the T cell is contacted with a second RNP comprising a second guide RNA targeting an endogenous TCR- β locus.

7. The method of any one of claims 1 to 3, wherein the first RNP comprises a first gene-editing protein and the ratio of the first guide RNA to the first gene-editing protein is between 1:1 and 100:1.

8. The method of claim 6, wherein the second RNP comprises a second gene-editing protein and the ratio of the second guide RNA to the second gene-editing protein is between 1:1 and 100:1.

9. The method of any one of claims 1-3, wherein the engineered T cell does not express an endogenous TCR- β protein.

10. The method of any one of claims 1 to 3, wherein the first guide RNA targets exon 1, 2 or 3 of TRAC: TRAC1 (SEQ ID NO: 7), TRAC2 (SEQ ID NO: 8), TRAC3 (SEQ ID NO: 9), TRAC4 (SEQ ID NO: 10), TRAC5 (SEQ ID NO: 11), TRAC6 (SEQ ID NO: 12), TRAC7 (SEQ ID NO: 13), TRAC8 (SEQ ID NO: 14), TRAC9 (SEQ ID NO: 15), TRAC10 (SEQ ID NO: 16), TRAC11 (SEQ ID NO: 17), TRAC12 (SEQ ID NO: 18), TRAC13 (SEQ ID NO: 19), TRAC14 (SEQ ID NO: 20), TRAC15 (SEQ ID NO: 21) or TRAC16 (SEQ ID NO: 22).

11. The method of claim 10, wherein the first guide RNA target comprises one of: TRAC1, TRAC3, TRAC4, TRAC5, TRAC7, TRAC12 or TRAC15.

12. The method of claim 10 or 11, wherein the first guide RNA comprises a nucleic acid sequence in table 10.

13. A method according to any one of claims 1 to 3, wherein the first guide RNA targets exon 1, 2 or 3 of TRAC.

14. The method of any one of claims 1 to 3, wherein the second guide RNA targets exon 1 of TRBC: TRBC1 (SEQ ID NO: 23), TRBC2 (SEQ ID NO: 24), TRBC3 (SEQ ID NO: 25), TRBC4 (SEQ ID NO: 26), TRBC5 (SEQ ID NO: 27), TRBC6 (SEQ ID NO: 28), TRBC7 (SEQ ID NO: 29), TRBC8 (SEQ ID NO: 30), TRBC9 (SEQ ID NO: 31), TRBC10 (SEQ ID NO: 32), TRBC11 (SEQ ID NO: 33), TRBC12 (SEQ ID NO: 34), TRBC13 (SEQ ID NO: 35), TRBC14 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 37), TRBC16 (SEQ ID NO: 38), TRBC17 (SEQ ID NO: 39), TRBC18 (SEQ ID NO: 40), TRBC19 (SEQ ID NO: 41), TRBC20 (SEQ ID NO: 42), TRBC21 (SEQ ID NO: 43), TRBC22 (SEQ ID NO: 44), TRBC23 (SEQ ID NO: 45), TRBC24 (SEQ ID NO: 36), TRBC15 (SEQ ID NO: 46) or TRBC 48 (SEQ ID NO: 48).

15. The method of claim 14, wherein the second guide RNA targets one of the following loci: TRBC4, TRBC8, TRBC13, TRBC19, TRBC20, TRBC21, TRBC22, TRBC23 or TRBC26.

16. The method of any one of claims 14 or 15, wherein the second guide RNA targets exon 1 of TRBC1 and TRBC 2.

17. The method of claim 14 or 15, wherein the second guide RNA comprises a nucleic acid sequence in table 11.

18. The method of claim 16, wherein the second guide RNA targets exon 1 of TRBC.

19. The method of any one of claims 1 to 18, wherein the condition that allows the RNP and the donor DNA to enter the cell comprises electroporation.

20. The method according to any one of claims 1 to 19, wherein in step b) the T cells are incubated for at least 10 minutes.

21. The method of claim 20, wherein the T cells are incubated at about 37 ℃.

22. The method of any one of claims 1 to 21, wherein the medium comprises a cytokine.

23. The method of claim 22, wherein the cytokine comprises IL-2, IL-7 and/or IL-15.

24. The method of claim 23, wherein the cytokine comprises IL-2.

25. The method of claim 23, wherein the cytokine comprises IL-7.

26. The method of claim 23, wherein the cytokine comprises IL-15.

27. The method of claim 23, wherein the cytokine comprises IL-7 and IL-15.

28. The method according to any one of claims 1 to 27, wherein step a) is performed in the presence of a negatively charged polymer.

29. The method of claim 28, wherein the polymer is poly (glutamic acid) (PGA) or a variant thereof, poly (aspartic acid), heparin, or poly (acrylic acid).

30. The method of claim 29, wherein the PGA is poly (L-glutamic acid) or a variant thereof.

31. The method of claim 29, wherein the PGA is poly (D-glutamic acid) or a variant thereof.

32. The method of any one of claims 29 to 31, wherein the PGA or variant thereof has an average molecular weight between 15 kilodaltons (kDa) and 50 kDa.

33. The method of any one of claims 28 to 32, wherein about 2 μg/μl to about 12 μg/μl of the polymer is added.

34. The method of any one of claims 1 to 33, wherein the amount of RNP is from about 0.2pmol/μl to about 8pmol/μl.

35. The method of any one of claims 1 to 34, wherein the amount of donor DNA is about 0.0004pmol/μl to about 0.4pmol/μl.

36. The method of any one of claims 1 to 35, wherein the donor DNA is recombined into an endogenous TCR-a locus.

37. The method of claim 36, wherein the TCR-a locus is a TCR-a constant chain.

38. The method of claim 36, wherein the exogenous TCR-a (VJ) domain forms part of a heterologous TCR-a comprising at least a portion of the endogenous TCR-a of the engineered T cell.

39. The method of any one of claims 1 to 38, wherein the donor DNA comprises a left homology arm and a right homology arm.

40. The method of claim 39, wherein the left homology arm and the right homology arm are homologous to the endogenous TCR locus.

41. The method of claim 40, wherein the left homology arm is about 50 bases to about 2000 bases in length.

42. The method of claim 40, wherein the left homology arm is about 100 bases to about 1000 bases in length.

43. The method of claim 40, wherein the left homology arm is about 200 bases to about 800 bases in length.

44. The method of any one of claims 39 to 43, wherein the right homology arm is about 200 bases to about 800 bases in length.

45. The method of claim 44, wherein the right homology arm is about 250 bases to about 700 bases in length.

46. The method of claim 40, wherein the left homology arm and the right homology arm are homologous to an endogenous TCR- β locus.

47. The method of claim 46, wherein the left homology arm is about 200 bases to about 800 bases in length.

48. The method of claim 47, wherein the left homology arm is about 250 bases to about 700 bases in length.

49. The method of claim 47, wherein the right homology arm is about 200 bases to about 800 bases in length.

50. The method of claim 47, wherein the right homology arm is about 250 bases to about 700 bases in length.

51. The method of any one of claims 1 to 50, wherein the donor DNA comprises double-stranded DNA (dsDNA).

52. The method of claim 51, wherein the donor DNA is on a plasmid, a nanoplasmid, or a microring.

53. The method of claim 52, wherein the donor DNA is contained within a nanoplasmset.

54. The method of claim 51, wherein the donor DNA is linear.

55. The method of any one of claims 1-54, wherein the donor DNA comprises single-stranded DNA (ssDNA).

56. The method of any one of claims 1 to 55, wherein the donor DNA is not chemically modified.

57. The method of any one of claims 1 to 55, wherein the donor DNA comprises a chemical modification.

58. The method of claim 57, wherein the modification comprises a 5 'phosphate or a 5' phosphorothioate.

59. The method of any one of claims 1 to 58, wherein the donor DNA and RNP are incubated prior to step a).

60. The method of any one of claims 1 to 59, wherein the RNP comprises at least one Nuclear Localization Signal (NLS).

61. The method of any one of claims 1 to 60, wherein the T cells are activated prior to step a).

62. The method of claim 61, wherein the T cells are activated for a period of between 24 hours and 96 hours.

63. The method of claim 62, wherein the T cells are activated in the presence of a cytokine.

64. The method of claim 63, wherein the cytokine comprises IL-2, IL-7 and/or IL-15.

65. The method of claim 64, wherein the T cells are activated in the presence of between about 5ng/mL and about 200ng/mL of IL-2.

66. The method of claim 65, wherein the T cells are activated in the presence of between about 5ng/mL and about 50ng/mL of IL-2.

67. The method of claim 65, wherein the T cells are activated in the presence of about 10ng/mL IL-2.

68. The method of any one of claims 61-67, wherein the T cells are activated in the presence of an anti-CD 3 antibody and/or an anti-CD 28 antibody.

69. The method of claim 68, wherein the T cells are activated in the presence of a CD3 agonist and/or a CD28 agonist.

70. The method of claim 68, wherein the anti-CD 3 antibody and/or anti-CD 28 antibody is conjugated to a substrate.

71. The method of claim 69, wherein the CD3 agonist and/or CD28 agonist is conjugated to a substrate.

72. The method of any one of claims 61 to 71, wherein step a) is performed no more than about 24 hours after activation.

73. The method of any one of claims 61-71, wherein step a) is performed between about 24 hours and about 72 hours after activation.

74. The method of claim 73, wherein step a) is performed between about 36 hours and about 60 hours after activation.

75. A method for preparing an engineered T cell population, comprising: subjecting a population of T cells to the method of any one of claims 1 to 75.

76. The method of claim 76, wherein at least about 5% of the population of T cells are recovered as engineered T cells.

77. The method of claim 77, wherein about 5% to about 100% of said population of T cells are recovered as engineered T cells.

78. The method of claim 77, wherein at least about 50% of said population of T cells are recovered as engineered T cells.

79. The method of claim 77, wherein at least about 60% of said population of T cells are recovered as engineered T cells.

80. The method of any one of claims 75 to 79, wherein at least about 25% of the population of T cells is viable after step c).

81. The method of claim 80, wherein at least about 50% of the population of T cells is viable after step c).

82. The method of claim 80, wherein at least about 75% of the population of T cells are viable after step c).

83. The method of any one of claims 75 to 137, further comprising: contacting the cells with a second RNP comprising a guide RNA targeting an endogenous TCR- β locus, and wherein at least about 5% of the population of T cells are recovered as engineered T cells, wherein the engineered T cells express less than about 20% of the endogenous TCR- β.

84. The method of claim 83, wherein about 10% to about 100% of the population of T cells are recovered as engineered T cells.

85. The method of claim 84, wherein at least about 50% of the population of T cells are recovered as engineered T cells.

86. The method of claim 84, wherein at least about 60% of the population of T cells are recovered as engineered T cells.

87. The method of claim 85 or 86, wherein at least about 25% of the population of T cells is viable after step c).

88. The method of claim 87, wherein at least about 50% of the population of T cells are viable after step c).

89. The method according to any one of claims 1 to 88, wherein step a) comprises the following steps in any order: (i) adding the donor DNA to a chamber; (ii) adding the RNP to the chamber; and (iii) adding one or more of the T cells to the chamber.

90. The method according to any one of claims 1 to 88, wherein step a) comprises the following steps in any order: (i) Optionally combining the RNP and a negatively charged polymer to form an RNP-PGA mixture; (ii) adding the donor DNA to a chamber; (iii) Adding the RNP or the RNP-PGA mixture to the chamber; and (iv) adding one or more of the T cells to the chamber.

91. The method according to any one of claims 1 to 88, wherein step a) comprises the following steps in any order: (i) adding the RNP to a chamber; (ii) adding the donor DNA to the chamber; (iii) Optionally adding poly (glutamic acid) to the chamber; and

(iv) The T cells are added to the chamber.

92. The method of any one of claims 1-91, wherein during step a and step b, the T cells are not pipetted.

93. An engineered T cell prepared by the method of any one of claims 1 to 92.

94. An engineered T cell population prepared by the method of any one of claims 1 to 92.

95. A method for treating a subject having cancer, the method comprising:

a) Providing a population of T cells;

b) Engineering at least a subset of the population of T cells to express an exogenous T Cell Receptor (TCR) and knockout an endogenous TCR- β, thereby forming an engineered population of T cells, wherein the exogenous TCR binds to an antigen expressed by the cancer;

c) Expanding the population of engineered T cells; and

d) Administering an expanded population of engineered T cells to the subject.

96. The method of claim 95, wherein the antigen is a neoantigen or TAA.

97. The method of claim 95 or 96, wherein at least a portion of the genome and/or transcriptome of the cancer is sequenced to determine the presence of the antigen.

98. The method of any one of claims 95 to 97, wherein the engineered T-cells are prepared using the method of any one of claims 1 to 92.

99. The method of any one of claims 95-98, wherein said antigen is WT1, JAK2, NY-ESO1, PRAME, mutant KRAS, or HPV.

100. The method of any one of claims 95-99, wherein the antigen is specific for the cancer.

101. The method of any one of claims 95-100, wherein the TCR binds to the antigen presented on a class I major histocompatibility complex (mhc I) molecule.

102. The method of claim 101, wherein the mhc i comprises an mhc i allele expressed by the subject.

103. The method of any one of claims 95-102, wherein said expanded population of engineered T cells comprises 1 x 10 ⁵ And 1X 10 ¹¹ Engineered T cells between individuals.

104. The method of any one of claims 95-102, wherein said expanded population of engineered T-cells comprises at least 1 x 10 ⁸ Engineering T cells.

105. The method of any one of claims 95-102, wherein said expanded population of engineered T-cells comprises at least 1 x 10 ¹¹ Engineering T cells.

106. The method of any one of claims 95-105, wherein said T cells are autologous to the subject.

107. A method for treating a subject having cancer, the method comprising:

a) Providing a first population of T cells isolated from the subject;

b) Engineering at least a subset of the first population of T cells to express a first exogenous T Cell Receptor (TCR) and knock-out an endogenous TCR- β, thereby forming a first population of engineered T cells, wherein the exogenous TCR binds to a first antigen expressed by the cancer;

c) Expanding the first population of engineered T cells;

d) Administering an expanded first population of engineered T cells to the subject;

e) Providing a second population of T cells isolated from the subject;

f) Engineering at least a subset of the second population of T cells to express a second exogenous TCR and knock out endogenous TCR- β, thereby forming a second population of engineered T cells, wherein the exogenous TCR binds to a second antigen expressed by the cancer;

g) Expanding the second population of engineered T cells; and

h) Administering an expanded second population of engineered T cells to the subject.

108. The method of claim 107, wherein at least a portion of the genome or transcriptome of the cancer is sequenced to determine the presence of the first antigen and the second antigen.

109. The method of claim 107 or 108, wherein the first antigen is WT1, JAK2, NY-ESO1, PRAME, or mutant KRAS.

110. The method of any one of claims 107-109, wherein the first TCR and/or second TCR binds to an antigen presented on a class I major histocompatibility complex (mhc I) molecule.

111. The method of claim 110, wherein the antigen is a neoantigen or TAA.

112. The method of claim 110 or 111, wherein the mhc i comprises an mhc i allele expressed by the subject.

113. The method of any one of claims 107-112, wherein the first expanded population of engineered T cells comprises 1 x 10 ⁵ And 1X 10 ¹¹ Engineered T cells between individuals.

114. The method of any one of claims 107-113, wherein the first expanded population of engineered T cells comprises at least 1 x 10 ⁸ Engineering T cells.

115. The method of claim 114, wherein the first expanded population of engineered T cells comprises at least 1 x 10 ¹¹ Engineering T cells.

116. The method of any one of claims 107-115, wherein the second expanded population of engineered T cells comprises 1 x 10 ⁵ And 1X 10 ¹¹ Engineered T cells between individuals.

117. The method of any one of claims 107-116, wherein the second expanded population of engineered T cells comprises at least 1 x 10 ⁸ Engineering T cells.

118. The method of claim 117, wherein the second expanded population of engineered T cells comprises at least 1 x 10 ¹¹ Engineering T cells.

119. The method of any one of claims 107-118, wherein the T cells are autologous to the subject.

120. The method of any one of claims 107-119, wherein an additional population of engineered T cells is administered to the patient, and wherein T cells in the additional population of engineered T cells express a third exogenous TCR that binds to a third antigen expressed by the cancer and do not express endogenous TCR- β.

121. A method of treating cancer, comprising: administering the T cell, composition or pharmaceutical composition of any of claims 124-177 to a patient suffering from cancer.

122. The method of any one of claims 107-121, further comprising: administering an anti-cancer therapy to the subject.

123. The method of claim 122, wherein the anti-cancer therapy comprises immunotherapy, chemotherapy, and/or radiation.

124. An engineered T cell comprising a nucleic acid sequence encoding a polypeptide comprising exogenous TCR- β and/or exogenous TCR- α or a portion thereof, wherein the nucleic acid sequence is inserted into a TCR- α or TCR- β locus of the engineered T cell.

125. The engineered T cell of claim 124, wherein the T cell does not express a functional endogenous TCR- β protein.

126. The engineered T cell of claim 124 or 125, wherein the exogenous TCR-a is full-length TCR-a.

127. The engineered T cell of claim 124 or 125, wherein the exogenous TCR-a or portion thereof comprises a TCR-a (VJ) domain.

128. The engineered T cell of claim 127, wherein an exogenous TCR-a (VJ) domain forms part of a heterologous TCR-a comprising at least a portion of the endogenous TCR-a of the T cell.

129. The engineered T cell of claim 124 or 125, wherein the TCR-a locus is a TCR-a constant region.

130. The engineered T cell of any one of claims 124-129, wherein the exogenous TCR- β and the exogenous or heterologous TCR-a are expressed from the nucleic acid and form a functional TCR.

131. The engineered T-cell of any one of claims 124-130, which binds to an antigen.

132. The engineered T-cell of any one of claims 124-131, which binds to a cancer cell.

133. The engineered T-cell of claim 131 or 132, wherein the TCR binds to the antigen presented on a class I major histocompatibility complex (mhc I) molecule.

134. The engineered T-cell of any one of claims 131-133, wherein the antigen is a neo-antigen or a tumor-associated antigen (TAA).

135. The engineered T-cell of claim 134, wherein the antigen is a neo-antigen.

136. The engineered T-cell of claim 134, wherein the antigen is TAA.

137. The engineered T-cell of claim 134, wherein the neoantigen or tumor-associated antigen (TAA) is selected from the group consisting of: WT1, JAK2, NY-ESO1, PRAME, mutant KRAS, or antigens from table 1, table 2, or table 12.

138. The engineered T-cell of claim 137, wherein the antigen is WT1.

139. The engineered T-cell according to claim 318, wherein said mhc is selected from the group consisting of HLA-A02:01, a 02:03, a 02:06, a 02:07, a 023:01, a 26:01, a 29:02, a 30:01, a 30:02, a 31:01, a 32:01, a 68:01, a 68:02, B18:01, B35:03, B40:01, B40:02, B40:06, B46:01, B51:01, B53:01, B57:01, B58:01, C01:02, C02:02, C03:02, C03, C03:03, C02:03, C02:08, C02:08:01, C02:12:08:01.

140. The engineered T-cell according to any one of claims 133 to 139, wherein said antigen is specific for cancer in a subject to whom said engineered T-cell is to be administered.

141. The engineered T cell of any one of claims 133-140, wherein expression of an endogenous TCR- β gene is disrupted by gene editing.

142. An engineered T cell population comprising a plurality of engineered T cells of claim 141, wherein expression of the endogenous TCR- β gene is disrupted in greater than about 80% of the cells.

143. The population of claim 142, wherein expression of a functional endogenous TCR- β protein is disrupted in greater than about 80% of the cells.

144. The population of claim 142, wherein expression of a functional endogenous TCR- β protein is disrupted in greater than about 90% of the cells.

145. The population of claim 142, wherein expression of a functional endogenous TCR- β protein is disrupted in greater than about 95% of the cells.

146. The engineered T-cell of any one of claims 124-141, wherein the nucleic acid sequence further encodes a self-cleaving peptide or enzyme cleavage site.

147. The engineered T-cell of claim 146, wherein the self-cleaving peptide is a self-cleaving viral peptide.

148. The engineered T cell of claim 147, wherein the self-cleaving viral peptide is T2A, P2A, E2A, F2A.

149. The engineered T-cell of claim 147, wherein the enzyme cleavage site is a furin cleavage site.

150. The engineered T cell of any one of claims 124-149, having an initial T cell phenotype.

151. The engineered T-cell of any one of claims 124-150, having a stem cell memory T-cell phenotype.

152. The engineered T cell of any one of claims 124-151, having a progenitor cell depleted T cell phenotype.

153. The engineered T-cell of any one of claims 124-152, wherein the T-cell is autologous to a subject in need thereof.

154. A pharmaceutical composition comprising the population of engineered T cells of any one of claims 124-153, and a pharmaceutically acceptable excipient.

155. A composition comprising a population of isolated T cells, wherein at least 5% of the cells in the population are engineered T cells, each engineered T cell comprising a nucleic acid sequence encoding a polypeptide comprising exogenous TCR- β and/or exogenous TCR- α or a portion thereof, wherein the nucleic acid sequence is inserted into a TCR locus of the engineered T cell, and wherein the engineered T cell does not express endogenous TCR β.

156. The composition of claim 155, wherein the exogenous TCR-a is full length TCR-a.

157. The composition of claim 155, wherein the exogenous TCR-a or portion thereof is a TCR-a (VJ) domain.

158. The composition of claim 155, wherein at least 10% of the cells in the population are engineered T cells.

159. The composition of claim 155, wherein at least 20% of the cells in the population are engineered T cells.

160. The composition of claim 155, wherein at least 30% of the cells in the population are engineered T cells.

161. The composition of claim 155, wherein at least 40% of the cells in the population are engineered T cells.

162. The composition of claim 155, wherein at least 50% of the cells in the population are engineered T cells.

163. The composition of any one of claims 155-162, wherein at least 10% of T cells in the population do not express a functional endogenous TCR- β protein.

164. The composition of any one of claims 155 to 163, wherein the exogenous TCR-a or portion thereof comprises a TCR-avj domain.

165. The composition of claim 164, wherein an exogenous TCR-a (VJ) domain forms part of a heterologous TCR-a comprising at least a portion of the endogenous TCR-a of the T cell.

166. The composition of any one of claims 155 to 163, wherein the TCR-a locus is a TCR-a constant region.

167. The composition of any one of claims 155 to 166, wherein the exogenous TCR- β and the exogenous or heterologous TCR-a are expressed from the nucleic acid and form a functional TCR.

168. The composition of any one of claims 155 to 167, wherein the exogenous TCR- β and the full length TCR-a are expressed from the nucleic acid and form a functional TCR.

169. The composition of claim 167 or 168, wherein the functional TCR binds an antigen.

170. The composition of claim 169, wherein the functional TCR binds to the antigen presented on a class I major histocompatibility complex (mhc I) molecule.

171. The composition of any one of claims 169-170, wherein said antigen is a neo-antigen or a tumor-associated antigen (TAA).

172. The composition of claim 171, wherein the antigen is a neoantigen.

173. The composition of claim 171, wherein the neoantigen or Tumor Associated Antigen (TAA) is selected from the group consisting of: WT1, JAK2, NY-ESO1, PRAME, mutant KRAS, or antigens from table 1, table 2, or table 12.

174. The composition of claim 173, wherein the antigen is WT1.

175. The composition of any one of claims 170-174, wherein said mhc is selected from HLA-A 02:01, a 02:03, a 02:06, a 02:07, a 023:01, a 26:01, a 29:02, a 30:01, a 30:02, a 31:01, a 32:01, a 68:01, a 68:02, B18:01, B35:03, B40:01, B40:02, B40:06, B46:01, B51:01, B53:01, B57:01, B58:01, C01:02, C02:02, C02, C03, C03:03, C02, C02:08:01, C02.

176. The composition of any one of claims 169-175, wherein the antigen is specific for cancer in a subject to whom the composition is to be administered.

177. The composition of any one of claims 155 to 176, wherein expression of an endogenous TCR- β gene in each engineered T cell is disrupted by gene editing.

178. The composition of claim 177, wherein expression of the endogenous TCR- β gene is disrupted in greater than about 80% of the engineered T cells.

179. The composition of claim 178, wherein greater than about 90% of the expression of the endogenous TCR- β gene is disrupted.

180. The composition of claim 178, wherein greater than about 95% of the expression of the endogenous TCR- β gene is disrupted.

181. The composition of any one of claims 155 to 180, wherein said nucleic acid sequence further encodes a self-cleaving peptide.

182. The composition of claim 181, wherein the self-cleaving peptide is a self-cleaving viral peptide.

183. The composition of claim 182, wherein the self-cleaving viral peptide is T2A, P2A, E a or F2A.

184. The composition of any one of claims 155 to 183, wherein at least a subset of the T cells have a Central Memory (CM) T cell phenotype.

185. The composition of any one of claims 155-184, wherein said T cells are autologous to a subject in need thereof.

186. The composition of any one of claims 155 to 186, comprising about 0.1 x 10 ⁵ And about 1X 10 ¹¹ Engineered T cells between individuals.

187. The composition of any one of claims 155 to 186, comprising at least 1 x 10 ⁸ Engineering T cells.

188. The composition of any one of claims 155 to 186, comprising at least 1 x 10 ¹¹ Engineering T cells.

189. The composition of any one of claims 155 to 188, further comprising a pharmaceutically acceptable excipient.

190. A guide RNA that targets a sequence within the endogenous exon 1, 2 or 3 sequence of a TCR-alpha constant (TRAC) domain locus.

191. The guide RNA of claim 190, wherein the guide RNA that targets an endogenous TCR-a locus comprises a nucleic acid sequence of table 10.

192. The guide RNA of claim 190 or 191, wherein the endogenous TCR-a locus is an endogenous TCR-a constant region.

193. A guide RNA that targets a polypeptide consisting essentially of SEQ ID NO:23-48, and an endogenous TCR- β locus consisting of a nucleic acid sequence of any one of claims 23-48.

194. The guide RNA of claim 193, wherein the guide RNA that targets an endogenous TCR- β locus comprises a nucleic acid sequence in table 1.

195. A nucleic acid comprising a nucleic acid sequence comprising an exogenous TCR- β encoding sequence and an exogenous TCR-a encoding sequence, or a portion thereof, and wherein the nucleic acid sequence further comprises a first self-cleaving peptide encoding sequence.

196. The nucleic acid of claim 195, wherein the exogenous TCR-a or portion thereof comprises full length TCR-a.

197. The nucleic acid of claim 195, wherein the exogenous TCR-a or portion thereof comprises a TCR-a (VJ) domain.

198. The nucleic acid of any one of claims 195-197, further comprising a first homology arm and a second homology arm.

199. The nucleic acid of any one of claims 195-198, further comprising a second self-cleaving peptide coding sequence.

200. The nucleic acid of claim 199, comprising, in order from 5 'to 3': (i) the first homology arm; (ii) a first self-cleaving viral peptide coding sequence; (iii) the exogenous TCR- β encoding sequence; (iv) a second self-cleaving viral peptide coding sequence; (v) an exogenous TCR-a (VJ) domain coding sequence; and (vi) the second homology arm.

201. The nucleic acid of claim 198 or 200, wherein the first homology arm is homologous to an endogenous TCR-a locus in a human T cell.

202. The nucleic acid of claim 198, 200 or 201, wherein the second homology arm is homologous to an endogenous TCR-a locus in a human T cell.

203. The nucleic acid of claim 202, wherein the endogenous TCR-a locus is a TCR-a constant region.

204. The nucleic acid of any one of claims 195-203, wherein the first self-cleaving viral peptide is T2A, P2A, E a or F2A.

205. The nucleic acid of any one of claims 195-204, wherein the second self-cleaving viral peptide is T2A, P2A, E a or F2A.

206. The nucleic acid of any one of claims 195-205, wherein the first self-cleaving viral peptide and the second self-cleaving viral peptide are different.

207. The nucleic acid of any one of claims 195-206, wherein the first self-cleaving peptide encoding sequence is 5' of an exogenous TCR-a domain encoding sequence.

208. The nucleic acid of any one of claims 195-207, wherein the second self-cleaving peptide coding sequence is 5' to the exogenous TCR- β coding sequence.

209. The nucleic acid of claims 195-208, which is a plasmid, a nanoplasmlet, or a micro-loop.

210. The engineered T-cell or population of any one of claims 124-153, wherein the nucleic acid sequence comprises the nucleic acid of any one of claims 195-209.

211. The method of any one of claims 1-123, wherein the nucleic acid sequence comprises the nucleic acid of any one of claims 195-209.

212. A kit for producing an engineered T cell comprising the guide RNA of any one of claims 190 to 193.

213. The kit of claim 212, further comprising the guide RNA of claim 194 or 195.

214. The kit of claim 212 or 213, further comprising a gene editing reagent or a nucleic acid encoding a gene editing reagent.

215. The kit of claim 214, wherein the gene editing reagent is a CRISPR system.

216. The kit of any one of claims 213-215, further comprising donor DNA.

217. The kit of claim 216, wherein the donor DNA comprises a nucleic acid sequence encoding a polypeptide comprising exogenous TCR- β and exogenous TCR- α or portions thereof.

218. The kit of claim 217, wherein the exogenous TCR-a or portion thereof comprises full-length TCR-a.

219. The kit of claim 217, wherein the exogenous TCR-a or portion thereof comprises a TCR-a (VJ) domain.

220. The kit of claim 219, wherein the exogenous TCR- β and the heterologous TCR-a form a TCR capable of binding to an antigen.

221. The kit of claim 220, wherein the TCR binds to an antigen presented on a class I major histocompatibility complex (mhc I) molecule.

222. The kit of claim 220 or 221, wherein the antigen is: WT1, JAK2, NY-ESO1, PRAME, KRAS, or antigens from table 1, table 2, or table 12.

223. The kit of any one of claims 212-222, further comprising poly (glutamic acid) (PGA) or a variant thereof.

224. The kit of any one of claims 212-223, further comprising the nucleic acid of any one of claims 195-209.