RU2781083C2 - Options, compositions, and methods for use of homing-endonuclease pd-1 - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to the field of biotechnology, in particular to polypeptide containing variant I-OnuI of homing-endonuclease (HE), as well as to polynucleotide encoding it. A vector containing the above-mentioned polynucleotide is also disclosed. The invention also relates to a cell containing the above-mentioned polypeptide or polynucleotide, as well as to a composition containing the above-mentioned cell.

EFFECT: invention is effective for adoptive cell therapy.

16 cl, 24 dwg, 2 tbl, 14 ex

Description

CROSS REFERENCES TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) based on U.S. Provisional Application No. 62/414,279 filed October 28, 2016 and U.S. Provisional Application No. 62/385,079 filed September 8 2016, each of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING THE SEQUENCE LISTING

The sequence listing associated with this application is provided in text format instead of a paper copy and is hereby incorporated by reference into this specification. The name of the text file containing the sequence listing is BLBD_076_02WO_ST25.txt. The text file is 266 kB in size and was created on September 8, 2017 and submitted electronically via EFS-Web at the same time as this description was submitted.

State of the art

Technical field

The present invention relates to improved genome editing compositions. In particular, the invention relates to nuclease variants, compositions, and methods of using them to edit the human programmed cell death 1 (PD-1) gene.

Description of the Prior Art

The global burden of cancer doubled between 1975 and 2000. Cancer is the second leading cause of morbidity and mortality worldwide, with approximately 14.1 million new cases and 8.2 million cancer deaths since 2012. The most common cancers are breast cancer, lung and bronchial cancer, prostate cancer, colon and rectal cancer, bladder cancer, skin melanoma, non-Hodgkin's lymphoma, thyroid cancer, kidney and renal pelvis cancer, endometrial cancer, leukemia, and pancreatic cancer. glands. An increase in the number of new cancer cases is projected to reach 22 million over the next two decades.

The immune system plays a key role in detecting and fighting human cancer. Most transformed cells are quickly detected by immune sentinels and destroyed through the activation of antigen-specific T cells through clonally expressed T cell receptors (TCRs). Accordingly, cancer can be considered an immunological disorder, the inability of the immune system to develop the necessary antitumor response to sustainably suppress and eliminate the disease. In order to more effectively fight cancer, certain immunotherapeutic interventions developed in the past few decades have been specifically directed at enhancing T-cell immunity. These treatments have only resulted in sporadic disease remissions and have not had significant overall success. More recent therapies using molecules that target monoclonal antibodies that inhibit T cell activation, such as CTLA-4 or PD-1, have shown a more significant antitumor effect; however, these treatments are also associated with significant toxicity due to systemic immune activation.

More recently, adoptive cellular immunotherapy strategies based on the isolation, modification, expansion and reinfusion of T cells have been studied and tested in early clinical trials. T cells are often the effector cells of choice for cancer immunotherapy due to their selective recognition and powerful effector mechanisms. These therapies have shown mixed success rates, but a small number of patients have experienced long-term remission, indicating an as-yet untapped potential for T cell-based immunotherapy.

Successful recognition of tumor cell-associated antigens by cytolytic T cells initiates targeted tumor lysis and underpins any effective cancer immunotherapy approach. Tumor-infiltrating T cells (TILs) express TCRs specifically targeting tumor-associated antigens; however, the presence of significant amounts of TIL is limited to only a few human cancers. Engineered T cell receptors (TCRs) and chimeric antigen receptors (CARs) have the potential to increase the utility of T cell immunotherapy for many cancers and other immune disorders.

In addition, today's engineered T cells are still regulated by a complex immunosuppressive microenvironment that consists of cancer cells, inflammatory cells, stromal cells, and cytokines. Among these components, cancer cells, inflammatory cells, and suppressive cytokines regulate T cell phenotype and function. Collectively, the tumor microenvironment causes T cells to terminally differentiate into depleted T cells.

T cell depletion is a state of T cell dysfunction in a chronic environment that is characterized by increased expression of inhibitory receptors or an increase in their signals; decreased production of effector cytokines; and a decrease in the ability to survive and eliminate cancer. Depleted T cells also show loss of function in a hierarchical manner: decreased IL-2 production and ex vivo killing capacity are lost in the early depletion stage, TNF-α production is lost in the intermediate stage, and IFN-γ and GzmB production are lost in the late depletion stage. Most T cells in the tumor microenvironment differentiate into depleted T cells, lose their ability to eliminate the cancer, and are eventually eliminated from the game.

Programmed cell death protein 1 (PD-1) is expressed on T cells and mediates immunosuppression by binding to immunosuppressive factors such as PD-L1 and PD-L2 present in the tumor microenvironment. Expression of PD-L1 and PD-L2 correlates with prognosis in some human cancers. The PD-L1/PD-1 signaling pathway is one of the important regulatory pathways for T cell depletion. PD-L1 is widely expressed in cancer cells and stromal cells, and blockade of PD-L1/PD-1 by monoclonal antibodies enhances the antitumor function of T cells. PD-L2 also binds to PD-1 and negatively regulates T cell function.

Short description

In general, the present invention relates in particular to compositions containing homing endonuclease and megaTAL variants that cleave a target site in the human PD-1 gene, and methods of using them.

In various embodiments, the present invention specifically provides a polypeptide comprising a homing endonuclease (HE) variant that cleaves a target site in the human programmed cell death 1 (PD-1) gene.

In particular embodiments, the HE variant is a homing endonuclease (LHE) LAGLIDADG variant.

In some embodiments, said polypeptide comprises a biologically active fragment of said HE variant.

In some embodiments, the biologically active fragment lacks 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids compared to the corresponding wild-type HE.

In further embodiments, the biologically active fragment lacks the 4 N-terminal amino acids compared to the corresponding wild-type HE.

In some embodiments, the biologically active fragment is missing 8 N-terminal amino acids compared to the corresponding wild-type HE.

In particular embodiments, the biologically active fragment lacks 1, 2, 3, 4, or 5 C-terminal amino acids compared to the corresponding wild-type HE.

In particular embodiments, the biologically active fragment lacks a C-terminal amino acid compared to the corresponding wild-type HE.

In some embodiments, the biologically active fragment lacks 2 C-terminal amino acids compared to the corresponding wild-type HE.

In further embodiments, the HE variant is an LHE variant selected from the group consisting of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I- HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI and I-Vdi141I.

In particular embodiments, the HE variant is an LHE variant selected from the group consisting of: I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, and SmaMI.

In further embodiments, the HE variant is the I-OnuI LHE variant.

In additional embodiments, the HE variant contains one or more amino acid substitutions at the surface DNA recognition site at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193 , 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238 and 240 of the I-OnuI LHE amino acid sequence shown in SEQ ID NOs: 1-5, or corresponding biologically active fragment.

In particular embodiments, the HE variant contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76 77 , 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232 , 234, 236, 238 and 240 amino acid sequence of I-OnuI LHE, presented in SEQ ID NO: 1-5, or the corresponding biologically active fragment.

In particular embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acids. substitutions in at least one position selected from the group of positions consisting of positions: 26, 28, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72 , 75, 76, 78, 80, 138, 143, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 197, 199, 201, 203, 207, 223 , 224, 225, 227, 229, 232, 236 and 238 any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In some embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more, or all of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40K, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72Q, S72R, N75S, A76Y, S78K K80R, L138M, T143N, S159P, F168L, E1788D, C180H, F182G, N184I, I186A, S188RR, K189R, S190P, K191A, L192S, G193P, K197R, V199RA, S201M, T203G, K207M, K207M, K207M, K207M, T203G, K207M, T203G, K207M. , K229I, F232K, D236E and V238E any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In some embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more, or all of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40K, E42R, G44R, Q46E, T48D, V68K, A70Y, S72Q, N75S, A76Y, S78K, K80R, L138M, T143N , S159P, F168L, C180H, F182G, N184I, I186A, S188R, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201A, T203G, K207R, Y223R, K225R, K229I, F236K IDE and any of V236K IDE NO: 1-5, or the corresponding biologically active fragment.

In particular embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more or all of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72R, N75S, A76Y, K80R, L138M T143N, S159P, F168L, C180H, F182G, N184I, I186A, S188RR, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201A, T207G, K207R, Y223R, I224T, K229KA, K229K any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In additional embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more, or all of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72Q, N75S, A76Y, K80R, L138M, T143N S159P, F168L, E1788D, C180H, F182G, N184I, I186A, S188RR, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201A, K207R, Y223R, I224T, K229KA, K229K any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In additional embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more or all of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72Q, N75S, A76Y, K80R, L138M, T143N, S159P, F168L, E178D, C180H, F182G, N184I, I186A, S188R, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201A, T203G, K207R, Y223R, K225R, F232K, D236E и V238E любой from SEQ ID NO: 1-5, or the corresponding biologically active fragment.

In particular embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more or all of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72Q, N75S, A76Y, K80R, L138M, T143N, S159P, F168L, E178D, C180H, F182G, N184I, I186A, S188R, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201M, T203G, Y223R, K232KE any of SE2, F233R ID NO: 1-5, or the corresponding biologically active fragment.

In particular embodiments, the HE variant cleaves the target site of exon 1 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acids. substitutions in at least one position selected from the group of positions consisting of positions: 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72 , 75, 76, 78, 80, 100, 132, 138, 143, 155, 159, 178, 180, 184, 186, 189, 190, 191, 192, 193, 195, 201, 203, 207, 223, 225 , 227, 232, 236, 238 and 240 any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In some embodiments, the HE variant cleaves the target site of exon 1 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acids. substitutions in at least one position selected from the group of positions consisting of positions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, V37G, G38R, S40H, E42R, G44S, Q46A, Q46T, T48V, T48M , V68I, V68S, A70T, A70Y, A70L, S72D, S72N, N75R, N75H, A76Y, S78R, S78T, K80R, K80C, K80E, K80V, T82F, T82Y, I100V, V132A, L138M, T143N, S155G, E18DP , C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R and T240E any of SEQ ID NOs: 1-5 biologically active fragment.

In additional embodiments, the HE variant cleaves the target site of exon 1 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of or all of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46A, T48V, V68I, A70T, S72D, N75R, A76Y, S78R K80R, I100V, L138M, T143N, S159P, E1788D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223R, K225Y, K2277G, F2326R, F2326r, K227G any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In some embodiments, the HE variant cleaves the target site of exon 1 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of or all of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, V37G, G38R, S40H, E42R, G44S, Q46T, T48V, V68I, A70T, S72D, N75R, A76Y , S78R, K80C, I100V, V132A, L138M, T143N, S155G, S159P, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R , D236Q, V238R and T240E any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In some embodiments, the HE variant cleaves the target site of exon 1 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of or all of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68I, A70T, S72N, N75H, A76Y, S78T K80R, I100V, L138M, T143N, S159P, E1788D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223R, K225Y, K2277G, F2326R, F2326r, K227G any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In some embodiments, the HE variant cleaves the target site of exon 1 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of or all of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70Y, S72N, N75H, A76Y, K80E , T82F, L138M, T143N, S159P, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, T230QR, any of the SEQ ID NO: 1-5, or the corresponding biologically active fragment.

In some embodiments, the HE variant cleaves the target site of exon 1 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of or all of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70L, S72N, N75H, A76Y, K80V . SEQ ID NO: 1-5, or the corresponding biologically active fragment.

In some embodiments, the HE variant cleaves the target site of exon 1 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of , or all of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37G, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70T, S72N, N75H, A76Y, K80V, T82Y, L138M, T143N, S159P, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D23R Q : 1-5, or the corresponding biologically active fragment.

In particular embodiments, the HE variant cleaves the target site of exon 2 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acids. substitutions in at least one position selected from the group of positions consisting of positions: 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 41, 42, 44, 46, 48, 68 , 70, 72, 74, 75, 76, 78, 80, 82, 116, 138, 143, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195 , 199, 203, 207, 225, 227, 229, 232, 236 and 238 any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In additional embodiments, the HE variant cleaves the target site of exon 2 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acids. substitutions in at least one position selected from the group of positions consisting of positions: S24C, L26Q, R28Y, R28H, R30S, N32V, N32L, K34N, K34R, S35N, S35T, S36R, V37S, V37T, G38R, G38K, S40R . , C180N, F182Y, N184H, I186K, K189G, S190R, K191T, L192T, G193R, Q195Y, V199R, T203A, T203S, K207R, K225N, K225T, K227W, K227S, K229A, K229P, F232R, W234A, W234D, D236E и V238R any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In particular embodiments, the HE variant cleaves the PD-1 exon 2 target site and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of , or all of the following amino acid substitutions: L26Q, R28Y, R30S, N32V, K34N, S35N, S36R, V37S, G38R, S40R, T41A, E42R, G44R, Q46A, T48E, A70N, S72I, N75T, A76S, S78R, K80S, T82G, L138M, T143N, S159P, F168L, E178D, C180N, F182Y, N184H, I186K, K189G, S190R, K191T, L192T, G193R, Q195Y, V199R, T203A, K207R, K225N, K227W, K229A, F232R, W234A, D236E и V238R any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In some embodiments, the HE variant cleaves the target site of exon 2 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of , or all of the following amino acid substitutions: S24C, R28H, N32L, K34R, S35T, V37T, G38K, S40R, E42R, G44S, Q46E, T48E, V68I, A70N, S72I, D74N, N75R, A76R, S78R, K80S, T82R, V116L, L138M, T143N, S159P, F168L, E178D, C180N, F182Y, N184H, I186K, K189G, S190R, K191T, L192T, G193R, Q195Y, V199R, T203S, K207R, K225T, K227S, K229P, F232R, W234D, D236E и V238R any of SEQ ID NOs: 1-5, or the corresponding biologically active fragment.

In additional embodiments, the HE variant contains an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid the sequence presented in any of SEQ ID NO: 6-14, 60-63 or its corresponding biologically active fragment.

In private embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 6 or a corresponding biologically active fragment.

In private embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 7 or the corresponding biologically active fragment.

In some embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 8 or a corresponding biologically active fragment.

In private embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 9 or a corresponding biologically active fragment.

In private embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 10 or a corresponding biologically active fragment.

In private embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 11 or a corresponding biologically active fragment.

In some embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 12 or a corresponding biologically active fragment.

In private embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 13 or a corresponding biologically active fragment.

In some embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 14 or a corresponding biologically active fragment.

In some embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 60 or a corresponding biologically active fragment.

In some embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 61 or a corresponding biologically active fragment.

In some embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 62 or a corresponding biologically active fragment.

In some embodiments, the HE variant contains the amino acid sequence shown in SEQ ID NO: 63 or a corresponding biologically active fragment.

In particular embodiments, the polypeptide binds the polynucleotide sequence shown in SEQ ID NO: 25.

In particular embodiments, the polypeptide binds the polynucleotide sequence shown in SEQ ID NO: 30.

In particular embodiments, the polypeptide binds the polynucleotide sequence shown in SEQ ID NO: 35.

In further embodiments, the polypeptide further comprises a DNA binding domain.

In some embodiments, the DNA binding domain is selected from the group consisting of: the TALE DNA binding domain and the zinc fingers DNA binding domain.

In some embodiments, a TALE DNA binding domain contains from about 9.5 TALE repeat regions to about 15.5 TALE repeat regions.

In further embodiments, the TALE DNA binding domain binds a polynucleotide sequence within the PD-1 gene.

In particular embodiments, the TALE DNA binding domain binds the polynucleotide sequence shown in SEQ ID NO: 26.

In some embodiments, the polypeptide binds and cleaves the polynucleotide sequence shown in SEQ ID NO: 27.

In particular embodiments, the TALE DNA binding domain binds the polynucleotide sequence shown in SEQ ID NO: 31.

In some embodiments, the polypeptide binds and cleaves the polynucleotide sequence shown in SEQ ID NO: 32.

In particular embodiments, the TALE DNA binding domain binds the polynucleotide sequence shown in SEQ ID NO: 36.

In certain embodiments, the polypeptide binds and cleaves the polynucleotide sequence shown in SEQ ID NO: 37.

In some embodiments, the zinc finger DNA binding domain contains 2, 3, 4, 5, 6, 7, or 8 zinc finger motifs.

In further embodiments, the polypeptide further comprises a peptide linker and an end restructuring enzyme or an appropriate biologically active fragment.

In private embodiments, the polypeptide further comprises a viral self-cleaving peptide 2A and an end-reversing enzyme or a corresponding biologically active fragment.

In additional embodiments, the end-reversing enzyme or corresponding biologically active fragment has a 5'-3'-exonuclease, 5'-3' alkaline exonuclease, 3'-5', 5'-flap exonuclease, helicase, or template-independent DNA polymerase activity.

In private embodiments, the end-altering enzyme comprises the Trex2 protein or an appropriate biologically active fragment.

In some embodiments, the polypeptide contains the amino acid sequence shown in any of SEQ ID NOs: 15-23 and 64, or a corresponding biologically active fragment.

In additional embodiments, the implementation of the polypeptide contains the amino acid sequence presented in SEQ ID NO: 15, or the corresponding biologically active fragment.

In private embodiments, the polypeptide contains the amino acid sequence shown in SEQ ID NO: 16, or the corresponding biologically active fragment.

In various embodiments, the polypeptide contains the amino acid sequence shown in SEQ ID NO: 17, or the corresponding biologically active fragment.

In private embodiments, the polypeptide contains the amino acid sequence shown in SEQ ID NO: 18, or the corresponding biologically active fragment.

In private embodiments, the polypeptide contains the amino acid sequence shown in SEQ ID NO: 19, or the corresponding biologically active fragment.

In some embodiments, the polypeptide contains the amino acid sequence shown in SEQ ID NO: 20 or a corresponding biologically active fragment.

In some embodiments, the polypeptide contains the amino acid sequence shown in SEQ ID NO: 21 or a corresponding biologically active fragment.

In additional embodiments, the implementation of the polypeptide contains the amino acid sequence presented in SEQ ID NO: 22, or the corresponding biologically active fragment.

In additional embodiments, the implementation of the polypeptide contains the amino acid sequence shown in SEQ ID NO: 23, or the corresponding biologically active fragment.

In additional embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NO: 64 or a corresponding biologically active fragment.

In additional embodiments, the implementation of the polypeptide considered in this application contains the amino acid sequence presented in SEQ ID NO: 24, or the corresponding biologically active fragment.

In other embodiments, the polypeptide cleaves the human PD-1 gene at the polynucleotide sequence shown in SEQ ID NOS: 25, 27, 30, 32, 35, or 37.

In various embodiments, the present invention specifically provides a polynucleotide encoding a polypeptide contemplated in this application.

In particular embodiments of the present invention, in particular, provides mRNA encoding the polypeptide discussed in this application.

In private embodiments, the mRNA contains the sequence shown in SEQ ID NOs: 40-42 and 65-68.

In various embodiments, the present invention provides, in particular, cDNA encoding the polypeptide discussed in this application.

In certain embodiments of the present invention, in particular, provides a vector containing a polynucleotide encoding a polypeptide discussed in this application.

In various embodiments, the present invention specifically provides a cell containing the polypeptide discussed in this application.

In some embodiments, the present invention specifically provides a cell containing a polynucleotide encoding a polypeptide contemplated in this application.

In various embodiments, the present invention specifically provides a cell containing the vector discussed in this application.

In additional embodiments, the present invention specifically provides a cell containing one or more genome modifications introduced by the polypeptide discussed in this application.

In particular embodiments, the cell contains a polynucleotide encoding one or more groups consisting of: an immunopotentiation enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

In some embodiments, the polynucleotide further comprises an RNA polymerase II promoter operably linked to a polynucleotide encoding an immunopotentiation enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

In particular embodiments, the RNA polymerase II promoter is selected from the group consisting of EF1α short promoter, EF1α long promoter, human ROSA 26 locus, ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer/β-actin promoter chicken (CAG), promoter of β-actin and enhancer of myeloproliferative sarcoma virus with a deletion of the downregulatory site, with a replacement for the primer-binding site from dl587rev (MND).

In some embodiments, the polynucleotide further encodes one or more self-cleaving viral peptides operably linked, interspersed between, and/or flanking an immunopotentiation enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

In some embodiments, the self-cleaving viral peptide is a 2A peptide.

In certain embodiments, the polynucleotide further comprises a heterologous polyadenylation signal.

In some embodiments, an immunosuppressive signal damper includes an enzymatic function that counteracts an immunosuppressive factor.

In some embodiments, the immunosuppressive signal damper includes kynureninase activity.

In particular embodiments, the immunosuppressive signal damper comprises: an exodomain that binds an immunosuppressive factor, optionally said exodomain being an antibody or an antigen-binding fragment thereof; an exodomain that binds an immunosuppressive factor and a transmembrane domain; or an exodomain that binds an immunosuppressive factor, a transmembrane domain, and a modified endodomain that is unable to transduce immunosuppressive signals into the cell.

In some embodiments, the immunosuppressive signal damper is a dominant negative TGFβRII receptor.

In some embodiments, the immunopotentiator enhancer is selected from the group consisting of: a bispecific T cell activator (BiTE) molecule, an immunopotentiating factor, and a flip receptor.

In particular embodiments, the immunopotentiating factor is selected from the group consisting of a cytokine, a chemokine, a cytotoxin, a cytokine receptor, and variants thereof.

In particular embodiments, the cytokine receptor is selected from the group consisting of IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, and IL-21 receptor.

In a preferred embodiment, the cell comprises a polynucleotide encoding a cytokine receptor selected from the group consisting of an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, an IL-18 receptor, and an IL-21 receptor, functionally associated with the endogenous PD-1 promoter.

In another preferred embodiment, the cell contains a polynucleotide encoding an IL-12 cytokine receptor operably linked to an endogenous PD-1 promoter.

In particular embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-21.

In a preferred embodiment, the cell contains a polynucleotide encoding a cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-21 operably linked to the endogenous PD-1 promoter.

In another preferred embodiment, the cell contains a polynucleotide encoding IL-12 operably linked to an endogenous PD-1 promoter.

In further embodiments, the flip receptor comprises a PD-1 exodomain and a transmembrane domain; and an endodomain of CD28, CD134, CD137, CD278 and/or CD3ζ fused in frame to the C-terminal end of the PD-1 transmembrane domain.

In certain embodiments, the flip receptor comprises a PD-1 exodomain; a transmembrane domain isolated from a CD3, CD4, CD8α, CD28, CD134 or CD137 polypeptide; and an endodomain of CD28, CD134, CD137, CD278 and/or CD3ζ fused in frame to the C-terminal end of the PD-1 exodomain.

In private embodiments, the flip receptor contains a PD-1 exodomain; as well as a transmembrane domain and an endodomain isolated from a CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide fused in frame to the C-terminal end of the PD-1 exodomain.

In further embodiments, the engineered antigen receptor is selected from the group consisting of: engineered TCR, CAR, Daric, or zetakin.

In particular embodiments, the engineered receptor is not integrated into the PD-1 gene.

In some embodiments, a polynucleotide encoding one or more of an immunopotentiation enhancer, an immunosuppressive signal damper, or an engineered antigen receptor is inserted into the PD-1 gene.

In other embodiments, a donor repair template comprising a polynucleotide encoding one or more of an immunopotentiation enhancer, an immunosuppressive signal damper, or an engineered antigen receptor is inserted into the PD-1 gene at a DNA double-strand break site introduced by the polypeptide of this application.

In particular embodiments, a donor repair template containing a polynucleotide encoding a cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21 is inserted into the PD-1 gene. at the DNA double-strand break site introduced by the polypeptide of the present application. In preferred embodiments, the cytokine is inserted into the PD-1 gene in operable association with the endogenous PD-1 promoter.

In particular embodiments, a repair donor template containing a polynucleotide encoding the cytokine IL-12 is inserted into the PD-1 gene at the DNA double-strand break site introduced by the polypeptide of this application. In preferred embodiments, the IL-12 cytokine is inserted into the PD-1 gene in operable association with the endogenous PD-1 promoter.

In particular embodiments, the donor repair template contains a polynucleotide encoding a cytokine receptor selected from the group consisting of an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, an IL-18 receptor, and an IL-21 receptor, inserted into the PD-1 gene at the DNA double-strand break site introduced by the polypeptide of the present application. In preferred embodiments, the cytokine receptor is inserted into the PD-1 gene in operable association with the endogenous PD-1 promoter.

In particular embodiments, a repair donor template containing a polynucleotide encoding the IL-12 cytokine receptor is inserted into the PD-1 gene at a DNA double-strand break site introduced by the polypeptide of the present application. In preferred embodiments, the IL-12 cytokine receptor is inserted into the PD-1 gene in operable association with the endogenous PD-1 promoter.

In some embodiments, the cell is a hematopoietic cell.

In further embodiments, the cell is a T cell.

In particular embodiments, the cell is a CD3+, CD4+, and/or CD8+ cell.

In particular embodiments, the cell is an immune effector cell.

In other embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

In some embodiments, the cell is a natural killer (NK) or natural killer T (NKT) cell.

In particular embodiments, the cell source is peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, or tumor.

In private embodiments, the present invention provides, in particular, a set of cells containing one or more of the cells discussed in this application.

In various embodiments, the present invention provides, in particular, a composition containing one or more of the cells discussed in this application.

In certain embodiments, the present invention provides, in particular, a composition containing one or more of the cells discussed in this application, and a physiologically acceptable carrier.

In various embodiments, the present invention provides, in particular, a method for editing a human PD-1 gene in a cell, comprising introducing a polynucleotide encoding a polypeptide contemplated in this application into a cell, in which the expression of this polypeptide creates a double strand break at a target site in human PD-1 gene.

In some embodiments, the present invention specifically provides a method for editing a human PD-1 gene in a cell, comprising: introducing a polynucleotide encoding a polypeptide of the present application into the cell, wherein expression of the polypeptide creates a double-strand break at a target site in the human PD-1 gene, said gap being repaired by non-homologous end joining (NHEJ).

In some embodiments, the present invention specifically provides a method for editing a human PD-1 gene in a cell, comprising introducing a polynucleotide encoding a polypeptide of the present application and a repair donor template into the cell, wherein expression of the polypeptide creates a double-strand break at the site- targets in the human PD-1 gene, and the repair donor template is incorporated into the human PD-1 gene via homologous repair (HDR) at a double-strand break site (DSB).

In other embodiments, the cell is a hematopoietic cell.

In particular embodiments, the cell is a T cell.

In particular embodiments, the cell is a CD3 ⁺ , CD4 ⁺ and/or CD8 ⁺ cell.

In certain embodiments, the cell is an immune effector cell.

In some embodiments, the cell is a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL), or a helper T cell.

In particular embodiments, the cell is a natural killer (NK) cell or a natural killer T (NKT) cell.

In some embodiments, the cell source is peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, or tumor.

In particular embodiments, the polynucleotide encoding the polypeptide is an mRNA.

In further embodiments, a polynucleotide encoding a 3'-5' exonuclease is introduced into the cell.

In some embodiments, a polynucleotide encoding Trex2 or a corresponding biologically active fragment is introduced into the cell.

In other embodiments, the donor repair template encodes a PD-1 gene or a portion thereof containing one or more mutations compared to the wild-type PD-1 gene.

In particular embodiments, the donor repair template encodes one or more of an immunopotentiation enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

In further embodiments, the donor repair template further comprises an RNA polymerase II promoter operably linked to an immunopotentiation enhancer, immunosuppressive signal damper, or engineered antigen receptor.

In other embodiments, the RNA polymerase II promoter is selected from the group consisting of EF1α short promoter, EF1α long promoter, human ROSA 26 locus, ubiquitin C (UBC) promoter, phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer/chicken β-actin promoter (CAG), promoter of β-actin and enhancer of myeloproliferative sarcoma virus with a deletion of the downregulatory site, with a replacement for the primer-binding site from dl587rev (MND).

In certain embodiments, the donor repair template further encodes one or more self-cleaving viral peptides operably linked, interspersed between, and/or flanking an immunopotentiation enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

In further embodiments, the self-cleaving viral peptide is a 2A peptide.

In some embodiments, the repair donor matrix further comprises a heterologous polyadenylation signal.

In some embodiments, an immunosuppressive signal damper includes an enzymatic function that counteracts an immunosuppressive factor.

In other embodiments, the immunosuppressive signal damper includes kynureninase activity.

In particular embodiments, the immunosuppressive signal damper comprises: an exodomain that binds an immunosuppressive factor, optionally said exodomain being an antibody or an antigen-binding fragment thereof; an exodomain that binds an immunosuppressive factor and a transmembrane domain; or an exodomain that binds an immunosuppressive factor, a transmembrane domain, and a modified endodomain that is unable to transduce immunosuppressive signals into the cell.

In further embodiments, the immunosuppressive signal damper is a dominant negative TGFβRII receptor.

In some embodiments, the immunopotentiator enhancer is selected from the group consisting of: a bispecific T cell activator (BiTE) molecule, an immunopotentiating factor, and a flip receptor.

In other embodiments, the immunopotentiating factor is selected from the group consisting of a cytokine, a chemokine, a cytotoxin, a cytokine receptor, and variants thereof.

In particular embodiments, the immunopotentiating factor is selected from the group consisting of a cytokine, a chemokine, a cytotoxin, a cytokine receptor, and variants thereof.

In particular embodiments, the cytokine receptor is selected from the group consisting of IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, IL-18 receptor, and IL-21 receptor.

In a preferred embodiment, the cell comprises a polynucleotide encoding a cytokine receptor selected from the group consisting of an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, an IL-18 receptor, and an IL-21 receptor, functionally associated with the endogenous PD-1 promoter.

In another preferred embodiment, the cell contains a polynucleotide encoding an IL-12 cytokine receptor operably linked to an endogenous PD-1 promoter.

In particular embodiments, the cytokine is selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-21.

In a preferred embodiment, the cell contains a polynucleotide encoding a cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18 and IL-21 operably linked to the endogenous PD-1 promoter.

In another preferred embodiment, the cell contains a polynucleotide encoding IL-12 operably linked to an endogenous PD-1 promoter.

In private embodiments, the flip receptor contains a PD-1 exodomain and a transmembrane domain; and an endodomain of CD28, CD134, CD137, CD278 and/or CD3ζ fused in frame to the C-terminal end of the PD-1 transmembrane domain.

In further embodiments, the flip receptor comprises a PD-1 exodomain; a transmembrane domain isolated from a CD3, CD4, CD8α, CD28, CD134 or CD137 polypeptide; and an endodomain of CD28, CD134, CD137, CD278 and/or CD3ζ fused in frame to the C-terminal end of the PD-1 exodomain.

In other embodiments, the flip receptor comprises a PD-1 exodomain; as well as a transmembrane domain and an endodomain isolated from a CD3, CD4, CD8α, CD28, CD134, or CD137 polypeptide fused in frame to the C-terminal end of the PD-1 exodomain.

In further embodiments, the engineered antigen receptor is selected from the group consisting of: engineered TCR, CAR, Daric, or zetakin.

In further embodiments, the donor repair template comprises a 5' homologous arm homologous to the 5' human DSB PD-1 gene sequence and a 3' homologous arm homologous to the 3' human PD-1 DSB gene sequence.

In private embodiments, the lengths of the 5' and 3' homologous arms are independently chosen from about 100 to about 2500 bp.

In some embodiments, the lengths of the 5' and 3' homologous arms are independently selected from about 600 to about 1500 bp.

In some embodiments, the 5' homologous arm is about 1500 bp and the 3' homologous arm is about 1000 bp.

In some embodiments, the 5' homologous arm is approximately 600 bp and the 3' homologous arm is approximately 600 bp.

In private embodiments, a viral vector is used to introduce the donor repair template into the cell.

In further embodiments, the viral vector is a recombinant adeno-associated viral vector (rAAV) or a retrovirus.

In other embodiments, said rAAV has one or more ITRs from AAV2.

In some embodiments, rAAV has a serotype selected from the group consisting of: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10.

In additional implementations, rAAV has the serotype AAV2 or AAV6.

In some embodiments, the retrovirus is a lentivirus.

In particular embodiments, the lentivirus is an integrase-deficient lentivirus (IDLV).

In various embodiments, the present invention specifically provides a method of treating, preventing, or alleviating at least one symptom of cancer, an infectious disease, an autoimmune disease, an inflammatory disease, and an immunodeficiency or condition associated therewith, comprising administering to a subject an effective amount of a composition contemplated in this application.

In various embodiments, the present invention specifically provides a method of treating solid cancer comprising administering to a subject an effective amount of a composition of the present application.

In other embodiments, solid cancer includes liver cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, prostate cancer, testicular cancer, bladder cancer, brain cancer, sarcoma, head and neck cancer, bone cancer, cancer thyroid, kidney cancer, or skin cancer.

In various embodiments, the present invention specifically provides a method of treating a hematological malignancy comprising administering to a subject an effective amount of a composition of the present application.

In further embodiments, said hematologic malignancy is leukemia, lymphoma, or multiple myeloma.

Brief description of several types of drawings

Figure 1A. Diagram illustrating the positions of the IgV domain and the ITIM and ITSM motifs of PD-1 relative to the position of the megaTAL target site in the exon.

Figure 1B. The PD-1 gene and target site sequence in the ITSM motif encoded by exon 5 (SEQ ID NO: 106 - 108) isolated the central-4 HE motif located in the codon for the ITSM phosphotyrosine residue at position 248.

Figure 2. I-OnuI was reprogrammed by constructing NTD (N-terminal domain) and CTD (C-terminal domain) for chimeric "semi-sites" (SEQ ID NOS: 109 and 110) using two rounds of sorting followed by fusion of the reprogrammed domains (SEQ ID NO: 111) and screening for the full exon of the PD-1 5 target site to isolate the fully reprogrammed HE.

Figure 3A. Initial screening of the exon 5 variant of the PD-1 gene for activity in a chromosomal reporter assay.

Figure 3B. The HE PD-1 variant (PD-1.ITSM.ex5_RD1_CV3-08) had moderate DNA binding affinity properties as measured by equilibrium substrate titration.

Figure 4. Secondary screening of HE PD-1 for activity in the chromosomal reporter assay following isolation of improved variants from stream sorting based on display of a randomly mutagenized library of PD-1.ITSM.ex5_RD1_CV3-08 variants performed under more stringent cleavage and affinity conditions for isolating variants with improved activity.

Figure 5. Results of bisulfite sequencing analysis of exon 5 of PD-1 (SEQ ID NO: 112) in activated primary human T cells to determine the methylation status of CpG motifs (SEQ ID NO: 113) present in exon 5 of HE PD-1.

Figure 6. DNA binding and cleavage affinity analysis results for the PD-1.ITSM.ex5_RD5_CV23MK HE variant compared to partially and fully methylated PD-1 exon 5 substrates.

Figure 7A. Co-delivery of megaTAL PD-1.ITSM.ex5_RD5_CV23MK to T cells along with TREX2 edits the target locus at approximately 60% as measured by TIDE analysis.

Figure 7B. Distribution of indels according to the PD-1 ITSM consensus motif during cleavage of megaTAL PD-1.ITSM.ex5_RD5_CV23MK in the presence of Trex2.

Figure 8. Results of HE PD-1 exon 5 central-4 specificity profiling.

Figure 9A. Diagram illustrating the positions of the IgV, ITIM, and ITSM PD-1 domains relative to the positions of exons 1, 2, and 5.

Figure 9B. PD-1 gene and target site location in exons 1 (SEQ ID NO: 114-116), 2 (SEQ ID NO: 117-119) and 5 (SEQ ID NO: 106-108).

Figure 10. Results of chromosomal reporter assays after stream sorting based on display for catalytic activity for initially reprogrammed I-OnuI targeting exon 1 of PD-1 (PD-1.ile3.exon1_RD1_B1, upper panel); enrichment of PD-1.ile3.exon1_RD1_B1 by mutagenization and screening under more stringent catalytic conditions to identify mutations that promote target cleavage to identify more active variants (PD-1.ile3.exon1_RD2_B1H8 and PD-1.ile3.exon1_RD2_B1G2, middle panels ); and originally reprogrammed I-OnuI targeting PD-1 exon 2 (PD-1.IgV.exon2_RD1_G5, bottom panel). Results are shown for nuclease in the presence of Trex2 (left panels) and formatted as megaTAL (right panels).

Figure 11A. DNA binding affinity values for the PD-1.ile3.exon1_RD2_B1H8 and PD-1.IgV.exon2_RD1_G5 variants as measured by equilibrium substrate titration using their respective target sequences.

Figure 11B. Comparison of DNA cleavage activity among specificity-rich PD-1.ile3.exon1 nucleases (RD2_B1G2, RD3_B1G2C4, RD3_B1G2C11, RD3_B1G2C5) against 64 target DNAs, with variations in base pairs -8, -7 and -6. The heat map represents the non-split coefficient: the median split values are obtained from the scatter plots.

Figure 12. Results of bisulfite sequencing analysis of exon 1 (SEQ ID NO: 120) and exon 2 (SEQ ID NO: 122) of PD-1 in activated primary human T cells (left panels), demonstrating that the CpG motif of exon 1 of PD- 1 (SEQ ID NO: 121) remains unmethylated, while the CpG motifs of exon 2 of PD-1 (SEQ ID NO: 123) are methylated. Figure 12 also shows that PD-1.IgV.exon2_RD1_G5 efficiently cleaves both the unmethylated and methylated target site (top right and bottom right panels, respectively).

Figure 13A. Surface expression of PD-1 in CAR T cells electroporated with blank vehicle, CFP, megaTALCCR5, megaTALPD-1.ITSM.ex5_RD5_CV23MK or megaTAL PD-1.ile3.exon1_RD2_B1H8 and then stimulated with blank vehicle or phorbol-12-myristate 13-acetate (PMA)/ionomycin (P/I).

Figure 13B. Surface expression of PD-1 in T cells electroporated with improved versions of megaTAL PD-1.ile3.exon1 (RD2_B1G2, RD3_B1G2C4, RD3_B1G2C11, RD3_B1G2C5)

Figure 14. Simultaneous delivery of megaTAL PD-1.ITSM.ex5_RD5_CV23MK and megaTAL PD-1.ile3.exon1_RD2_B1H8 with or without Trex2 significantly reduces cell surface expression of PD-1.

Figure 15A. Anti-BCMA CAR T cells electroporated with mRNA encoding megaTAL PD-1.ITSM.ex5_RD5_CV23MK or PD-1.ile3.exon1_RD2_B1H8 show reduced PD-L1-mediated cytokine suppression compared to anti-BCMA CAR T cells electroporated blank carrier or mRNA encoding megaTAL CFP or CCR5.

Figure 15B. Anti-BCMA CAR T cells electroporated with mRNA encoding megaTAL PD-1.ITSM.ex5_RD5_CV23MK or PD-1.ile3.exon1_RD2_B1H8 show reduced PD-L1-mediated cytokine suppression compared to anti-BCMA CAR T cells electroporated blank carrier or mRNA encoding catalytically inactive TCRα-megaTAL (raw). Addition of PD-1 antibody to cultures abolishes cytokine suppression in anti-BCMA CAR T cells electroporated with blank media or mRNA encoding catalytically inactive megaTAL TCRα.

Figure 16A. Strategy for introducing different expression cassettes (GFP, upper panel; anti-CD19 CAR, middle panel and anti-BCMA CAR, lower panel) into exon 1 of PD-1 by homologous recombination.

Figure 16B. Representative results of flow cytometry assays to determine long-term expression of chromosomally integrated cassettes in T cells treated with megaTAL PD-1.ile3.exon1_RD2_B1H8 and rAAV-targeted vectors containing the GFP expression cassette, anti-CD19 CAR, or anti-BCMA CAR.

Figure 17. Strategy for introducing the mCherry reporter gene at the PD-1 start codon in exon 1 and flow cytometry analysis of mCherry expression in T cells electroporated with blank vehicle or megaTAL PD-1.ile3.exon1_RD2_B1H8 and transduced with blank vehicle or targeted to rAAV a vector encoding mCherry both in the presence and absence of a 24 hour PMA/ionomycin treatment.

Figure 18. Strategy for introducing the MND-promoter-BFP expression cassette into the ITSM motif in exon 5 of PD-1 and flow cytometry to analyze BFP expression in T cells electroporated with blank vehicle or PD-1.ITSM.ex5_RD5_CV23MK and transduced with blank vehicle or targeted on rAAV with a vector containing the pMND-BFP expression cassette.

Figure 19. Strategy for introducing the MND-promoter-BFP expression cassette into the ITSM motif in exon 5 of PD-1 and flow cytometry analysis for PD-1 and BFP expression in T cells electroporated with blank vehicle or PD-1.ITSM.ex5_RD5_CV23MK megaTAL and transduced with a blank carrier or an rAAV-targeted vector containing the pMND-BFP expression cassette.

Figure 20. Strategy for introducing the MND-promoter-BFP expression cassette into the ITSM motif in exon 5 of PD-1 when the homologous arms contain single nucleotide polymorphisms (SNPs) (upper panel). The bottom panel shows flow cytometry analysis of T cells electroporated with megaTAL PD-1.ITSM.ex5_RD5_CV23MK and transduced with an rAAV-targeted vector containing a pMND-BFP expression cassette with wild-type homologous arms, with the 5' homologous arm containing SNPs, or 3 The '-homologous arm contains a SNP.

Figure 21. Strategy for introducing the MND-promoter-PD-1.CD28 flip receptor expression cassette into the ITSM motif in exon 5 of PD-1 and analysis by flow cytometry of PD-1 and BFP expression in T cells electroporated with blank vehicle or megaTAL PD-1.ITSM.ex5_RD5_CV23MK and transduced with an empty carrier or an rAAV-targeted vector containing the pMND-PD-1.CD28 flip receptor expression cassette.

Figure 22. Strategy for creating a large-scale deletion in the PD-1 gene by delivering the megaTAL PD-1.ITSM.ex5_RD5_CV23MK and rAAV cassette containing homologous arms over a kbp apart. upstream from exon 5 of the target site.

Figure 23. Cytokine insertion strategy into the PD-1 gene when expression is under the control of the endogenous PD-1 promoter (upper panel). After 24 hours of PMA/ionomycin treatment, anti-BCMA CAR T cells electroporated with megaTAL PD-1 mRNA and transduced with rAAV encoding IL-12 showed reduced expression of PD-1 compared to control-treated cells (lower panel).

Figure 24A. After 24 hours of PMA/ionomycin treatment, anti-BCMA CAR T cells electroporated with megaTAL PD-1 mRNA and transduced with rAAV encoding IL-12 showed increased production of IL-12 compared to control-treated cells (left panel). Anti-BCMA CAR T cells electroporated with PD-1 mRNA and transduced with rAAV encoding IL-12 and cultured in the presence of K562-BCMA target cells showed increased IL-12 production compared to control-treated cells (right panel) .

Figure 24B. Results of the analysis of serial stimulation. K52-BCMA cells and anti-BCMA CAR T cells were mixed, cultured for 7 days, and additional K562-BCMA target cells were added to mimic restimulation. After restimulation with anti-BCMA CAR, T cells treated with recombinant IL-12 or treated with both megaTAL PD-1 and HDR IL-12 matrix showed increased IFNγ production and cytotoxicity compared to control-treated cells.

SUMMARY OF SEQUENCE IDENTIFIERS

SEQ ID NO: 1 - Amino acid sequence of wild-type homing endonuclease I-OnuI LAGLIDADG (LHE).

SEQ ID NO: 2 - Wild type LHE I-OnuI amino acid sequence.

SEQ ID NO: 3 - amino acid sequence of the biologically active wild-type LHE I-OnuI fragment.

SEQ ID NO: 4 - amino acid sequence of the biologically active wild-type LHE I-OnuI fragment.

SEQ ID NO: 5 - Amino acid sequence of the biologically active wild-type LHE I-OnuI fragment.

SEQ ID NO: 6 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene.

SEQ ID NO: 7 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene.

SEQ ID NO: 8 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene.

SEQ ID NO: 9 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene.

SEQ ID NO: 10 is the amino acid sequence of the LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene.

SEQ ID NO: 11 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene.

SEQ ID NO: 12 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene.

SEQ ID NO: 13 is the amino acid sequence of the LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene.

SEQ ID NO: 14 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene.

SEQ ID NO: 15 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene.

SEQ ID NO: 16 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene.

SEQ ID NO: 17 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene.

SEQ ID NO: 18 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene.

SEQ ID NO: 19 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene.

SEQ ID NO: 20 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene.

SEQ ID NO: 21 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene.

SEQ ID NO: 22 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene.

SEQ ID NO: 23 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene.

SEQ ID NO: 24 - Amino acid sequence encoding mouse Trex2.

SEQ ID NO: 25 - Target site of the LHE I-OnuI variant in exon 5 of the human PD-1 gene.

SEQ ID NO: 26 - Target site of the TALE DNA-binding domain in exon 5 of the human PD-1 gene.

SEQ ID NO: 27 - MegaTAL target site in exon 5 of the human PD-1 gene.

SEQ ID NO: 28 - Target site of the I-OnuI LHE variant of the N-terminal domain in exon 5 of the human PD-1 gene.

SEQ ID NO: 29 - Target site of the I-OnuI LHE variant of the C-terminal domain in exon 5 of the human PD-1 gene.

SEQ ID NO: 30 - Target site of the I-OnuI LHE variant in exon 1 of the human PD-1 gene.

SEQ ID NO: 31 - Target site of the TALE DNA-binding domain in exon 1 of the human PD-1 gene.

SEQ ID NO: 32 - MegaTAL target site in exon 1 of the human PD-1 gene.

SEQ ID NO: 33 - Target site of the I-OnuI LHE variant of the N-terminal domain in exon 1 of the human PD-1 gene.

SEQ ID NO: 34 - Target site of the I-OnuI LHE variant of the C-terminal domain in exon 1 of the human PD-1 gene.

SEQ ID NO: 35 - Target site of the I-OnuI LHE variant in exon 2 of the human PD-1 gene.

SEQ ID NO: 36 - Target site of the TALE DNA-binding domain in exon 2 of the human PD-1 gene.

SEQ ID NO: 37 - MegaTAL target site in exon 2 of the human PD-1 gene.

SEQ ID NO: 38 - Target site of the I-OnuI LHE variant of the N-terminal domain in exon 2 of the human PD-1 gene.

SEQ ID NO: 39 - Target site of the I-OnuI LHE variant of the C-terminal domain in exon 2 of the human PD-1 gene.

SEQ ID NO: 40 - mRNA encoding megaTAL PD-1.

SEQ ID NO: 41 - mRNA encoding megaTAL PD-1.

SEQ ID NO: 42 - mRNA encoding megaTAL PD-1.

SEQ ID NO: 43 - mRNA encoding mouse Trex2 protein.

SEQ ID NO: 44 - Polynucleotide sequence encoding rAAV targeting vector with homologous arms PD-1 and expression cassette pMND-BFP-SV40polyA.

SEQ ID NO: 45 - polynucleotide encoding the 5' homologous arm of SEQ ID NO: 44.

SEQ ID NO: 46 - polynucleotide encoding the 3' homologous arm of SEQ ID NO: 44.

SEQ ID NO: 47 - Polynucleotide sequence encoding rAAV targeting vector with homologous arms PD-1 and expression cassette pMND-BFP-SV40polyA. Arm 3' contains a single nucleotide polymorphism (SNP) relative to the wild-type genomic sequence.

SEQ ID NO: 48 - Polynucleotide sequence encoding rAAV targeting vector with homologous arms PD-1 and expression cassette pMND-BFP-SV40polyA. The 5' arm contains a single nucleotide polymorphism (SNP) relative to the wild-type genomic sequence.

SEQ ID NO: 49 - polynucleotide encoding the 5' homologous arm of SEQ ID NO: 48.

SEQ ID NO: 50 - polynucleotide encoding the 3' homologous arm of SEQ ID NO: 47.

SEQ ID NO: 51 - Polynucleotide sequence encoding rAAV targeting vector with pMND-PD-1.CD28 expression cassette. switch receptor SV40polyA.

SEQ ID NO: 52 - Polynucleotide sequence encoding rAAV-targeted vector with homologous arms PD-1 and expression cassette pMND-BFP.SV40polyA with 5' homologous arm ~1.3 kb. in the opposite direction from the ITSM motif in exon 5 of PD-1.

SEQ ID NO: 53 - Polynucleotide sequence encoding rAAV targeting vector with homologous arms PD-1 and expression cassette pMND-GFP-SV40polyA.

SEQ ID NO: 54 - polynucleotide encoding the 5' homologous arm of SEQ ID NO: 53.

SEQ ID NO: 55 - polynucleotide encoding the 3' homologous arm of SEQ ID NO: 53.

SEQ ID NO: 56 - Polynucleotide sequence encoding rAAV targeting vector with homologous arms PD-1 and pMND-anti-CD19 expression cassette CAR-SV40polyA.

SEQ ID NO: 57 - Polynucleotide sequence encoding rAAV targeting vector with homologous arms PD-1 and pMND-anti-BCMA expression cassette CAR-SV40polyA.

SEQ ID NO: 58 - Polynucleotide sequence encoding rAAV targeting vector with homologous arms PD-1 and cDNA encoding mCherry.

SEQ ID NO: 60 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene

SEQ ID NO: 61 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene

SEQ ID NO: 62 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene

SEQ ID NO: 63 - Amino acid sequence of LHE I-OnuI variant reprogrammed to bind and cleave a target site in the human PD-1 gene

SEQ ID NO: 64 - Amino acid sequence of megaTAL that binds and cleaves a target site in the human PD-1 gene

SEQ ID NO: 65 - mRNA encoding megaTAL PD-1

SEQ ID NO: 66 - mRNA encoding megaTAL PD-1

SEQ ID NO: 67 - mRNA encoding megaTAL PD-1

SEQ ID NO: 68 - mRNA encoding megaTAL PD-1

SEQ ID NO: 69-79 - amino acid sequences of various linkers.

SEQ ID NO: 80-104 - amino acid sequences of protease cleavage sites and self-cleaving polypeptide cleavage sites.

In the above sequences, the symbol X, where present, denotes any amino acid or the absence of an amino acid.

Detailed description

A. OVERVIEW

The present invention generally relates, in particular, to improved genome editing compositions and methods for their use. Without wishing to be bound by any particular theory, the genome editing compositions contemplated in various embodiments can be used to prevent or treat cancer, an infectious disease, an autoimmune disease, an inflammatory disease, and an immunodeficiency or a condition associated therewith, or to alleviate at least at least one of their symptoms. One limitation or problem that is a weak point of current adoptive cell therapy is the hyporeactivity of immune effector cells due to depletion mediated by the tumor microenvironment. Depleted T cells have a unique molecular signature that is markedly different from naïve, effector, or memory T cells. Depleted T cells are defined as T cells with reduced cytokine expression and effector function. PD-1 is a marker of T cell depletion; overexpression of PD-1 is associated with reduced T cell proliferation and reduced production of IL-2, TNF and IFN-γ.

In particular embodiments, the genome-edited immune effector cells of the present application become more resistant to depletion by eliminating, reducing, or downregulating PD-1 expression and/or signaling.

Genome editing compositions and methods contemplated in various embodiments comprise nuclease variants designed to bind and cleave a target site in the programmed cell death 1 (PD-1) gene. Nuclease variants contemplated in particular embodiments can be used to introduce a double-strand break in a target polynucleotide sequence that can be repaired by non-homologous end joining (NHEJ) in the absence of a polynucleotide template, such as a repair donor template, or homologous directed repair (HDR), those. homologous recombination, in the presence of a donor repair template. Nuclease variants contemplated in some embodiments can also be engineered as nicases that generate single-stranded DNA breaks that can be repaired using a cellular base excision repair (BER) mechanism or homologous recombination in the presence of a donor repair template. NHEJ is an error-prone process that often results in small insertions and deletions that impair gene function. Homologous recombination requires homologous DNA as a template for repair and can be used to create an unlimited variety of modifications defined by the introduction of a donor DNA containing the desired sequence at the target site, flanked on both sides by sequences bearing homology to the regions flanking the target site.

In one preferred embodiment, the genome editing compositions contemplated herein comprise a homing endonuclease or megaTAL variant that targets the human PD-1 gene.

In one preferred embodiment, the genome editing compositions contemplated herein comprise a homing endonuclease or megaTAL variant and an end restructuring enzyme such as Trex2.

In various embodiments, genome-edited cells are contemplated. Genome-edited cells contain an edited PD-1 gene, wherein the editing strategy is designed to reduce or eliminate PD-1 expression and/or co-opt PD-1 to act as a dominant negative by expressing the extracellular ligand-binding domain of PD-1, in violation of its ability to transduce immunosuppressive intracellular signals.

In various embodiments, a DNA break is created at the target site of the PD-1 gene in a T cell, such as an immune effector cell, and the NHEJ ends of the cleaved genomic sequence can result in a cell with little or no expression of PD-1 and preferably T- a cell that lacks or substantially lacks functional expression and/or signaling of PD-1, eg lacks the ability to increase T cell depletion. Without wishing to be bound by any particular theory, T cells that lack functional PD-1 expression are more resistant to immunosuppression and T cell depletion and therefore more resistant and therapeutically effective.

In various other embodiments, a donor template is provided for the repair of a cleaved PD-1 genomic sequence. The PD-1 gene is repaired using the sequence of the specified template by homologous recombination at the DNA break site. In particular embodiments, the repair template contains a polynucleotide sequence that disrupts, and preferably substantially reduces or abolishes, functional expression of PD-1.

In particular embodiments, the PD-1 gene is repaired with a template encoding a PD-1 exodomain with increased affinity for its ligands.

In particular embodiments, the PD-1 gene is repaired with a polynucleotide encoding an immunopotentiation enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

In particular embodiments, the PD-1 gene is repaired with a polynucleotide encoding an immunopotentiation enhancer, immunosuppressive signal damper, or engineered antigen receptor and introduced into the PD-1 gene to co-opt the endogenous PD-1 promoter for transcriptional control of expression of the immunopotentiation enhancer, immunosuppressive signal damper, or engineered antigen receptor. antigen receptor.

In preferred embodiments, the genome compositions and editing methods discussed herein are used to edit the human PD-1 gene.

Accordingly, the methods and compositions contemplated in this application represent a fundamental improvement over existing adaptive cellular therapies.

In the practice of private embodiments, unless otherwise indicated, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology and cell biology, which are known to specialists in this field, many of which are described below with purpose of illustration. Such techniques are described in detail in the literature. See, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Maniatis et al., Molecular Cloning: A Laboratory Manual (1982); Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008); Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985); Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992); Transcription and Translation (B. Hames & S. Higgins, Eds., 1984); Perbal, A Practical Guide to Molecular Cloning (1984); Harlow and Lane, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1998) Current Protocols in Immunology Q. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; as well as contributions to journals such as Advances in Immunology.

B. DEFINITIONS

Unless otherwise indicated, all technical and scientific terms used in this application have the same meaning as generally understood by experts in the field of technology to which the invention pertains. Although any methods and materials similar or equivalent to those described in this application can be used in the practice or testing of particular embodiments, this application describes the preferred embodiments of the compositions, methods and materials. For the purposes of the present invention, the following terms are defined below.

The definite and indefinite articles are used in this application to refer to one or more than one (ie at least one or one or more) grammatical entities. For example, "element" means one element or one or more elements.

The use of alternatives (eg, "or") should be understood to mean either one or both, or any combination of these alternatives.

The term "and/or" should be understood to mean either one or both of the alternatives.

As used herein, the term "about" or "about" refers to an amount, level, value, number, frequency, percentage, size, dimension, amount, weight, or length that varies by 15%, 10%, 9.%, 8 %, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to the control amount, level, value, number, frequency, percentage, size, dimension, amount, weight, or length. In one embodiment, the term "about" or "about" refers to a range of amount, level, value, number, frequency, percentage, size, size, amount, weight, or length of ±15%, ±10%, ±9%, ±8 %, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of Reference, Level, Value, Number, Frequency, Percent, Size, Size, Quantity, Weight or length.

In one embodiment, a range, such as 1 to 5, about 1 to 5, or about 1 to about 5, refers to each numeric value covered by that range. For example, in one non-limiting and purely illustrative implementation, the range from 1 to 5 is equivalent to the expression 1, 2, 3, 4, 5; or 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, or 5.0; or 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4 , 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, or 5.0.

As used herein, the term "substantially" refers to an amount, level, value, number, frequency, percentage, size, dimension, amount, weight, or length that is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher than the reference value, level, value, number, frequency, percentage, gauge, size, quantity, weight, or length. In one embodiment, "substantially the same" refers to an amount, level, value, number, frequency, percentage, size, size, amount, weight, or length that produces an effect, such as a physiological effect that is approximately the same as as reference value, level, value, number, frequency, percentage, dimension, size, quantity, weight, or length.

Throughout the description, unless the context otherwise requires, the words "comprise", "comprises" and "comprising" will be understood to mean the inclusion of a specified step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps. or elements. The term "consisting of" means inclusive of and limited to everything following the phrase "consisting of". Thus, the phrase "consisting of" means that the listed elements are mandatory or necessary and that no other elements may be present. The term "consisting essentially of" means including any of the elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the description for the listed elements. Thus, the phrase "consisting primarily of" indicates that the listed elements are mandatory or necessary, but that there are no other elements that significantly affect the activity or operation of the listed elements.

Reference throughout the specification to "one implementation", "an implementation", "a particular implementation", "a related implementation", "some implementation", "an additional implementation", or "another implementation", or combinations thereof means, that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment. Thus, the occurrences of the above phrases in various places in the present specification do not necessarily all refer to the same embodiment. In addition, particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. It is also understood that the positive inclusion of a feature in one embodiment warrants the exclusion of said feature in a particular implementation.

The term "ex vivo" generally refers to activities that take place outside the body, such as experiments or measurements performed in or on living tissues in an artificial environment outside the body, preferably with minimal alteration of natural conditions. In particular embodiments, ex vivo procedures include living cells or tissues taken from the body and cultured or modified in a laboratory apparatus, usually under sterile conditions and usually for several hours or up to about 24 hours, but including up to 48 or 72 hours. hours, depending on the circumstances. In some embodiments, such tissues or cells can be harvested and frozen and then thawed for ex vivo processing. Tissue culture experiments or procedures that last longer than a few days using living cells or tissues are generally considered "in vitro", although the term may be used interchangeably with "ex vivo" in some embodiments.

The term "in vivo" generally refers to activities that take place within the body. In one embodiment, the implementation of cellular genomes are designed, edited or modified in vivo.

The terms "enhance" or "facilitate" or "increase", "expand" or "potentiate" generally refer to the ability of the nuclease variant, genome-editing composition, or genome-edited cell contemplated herein to produce or elicit a greater response (i.e. , physiological response) compared to the response elicited by vehicle blank or control. A measurable response may include an increase in catalytic activity, binding affinity, stability, cytolytic activity, and/or an increase in pro-inflammatory cytokines, among other effects evident from the understanding in the art and the description in this application. An "increased" or "expanded" amount is usually a "statistically significant" amount and may include an increase of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 15, 20, 30 or more times (e.g. 500, 1000 times) (including all integers and decimals in between and above 1, e.g. 1.5, 1.6, 1.7, 1.8, etc. .e) compared to the response produced by blank vehicle or control.

The terms "reduce" or "reduce" or "reduce" or "attenuate" or "inhibit" or "suppress" or "absorb" generally refers to the ability of the nuclease variant, genome-editing composition, or genome-edited cell contemplated herein to elicits, produces, or elicits a smaller response (i.e., a physiological response) compared to the response elicited by the blank vehicle or control. A measurable response may include a reduction in off-target binding affinity, off-target cleavage specificity, T cell depletion, and the like. A "reduced" or "reduced" amount is usually a "statistically significant" amount and may include a reduction of 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10 , 15., 20, 30 or more times (e.g. 500, 1000 times) (including all integers and decimal places in between and above 1, e.g. 1.5, 1.6, 1.7, 1.8, etc. .e) compared to the response (reference response) produced by blank vehicle or control.

The terms "maintain", "maintain", "maintain", "no change", "no significant change" or "no significant reduction" generally refer to the ability of the nuclease variant, genome-editing composition or genome-edited cell contemplated herein to produces, produces, or elicits a substantially similar or comparable physiological response (ie, downstream effects) as compared to a response elicited by either a blank vehicle or a control. A comparable response is a response that is not significantly different or does not differ measurably from a control response.

The terms "specific binding affinity" or "specifically binds" or "specifically bound" or "specific binding" or "specifically targeted" as used in this application describe the binding of one molecule to another, for example, the DNA-binding domain of a polypeptide, binding to DNA, with a higher binding affinity than background binding. A binding domain "specifically binds" to a target site if it binds to or binds to the target site with an affinity or K _a (i.e., the equilibrium association constant of a particular binding interaction with units of 1/M) of a value, e.g., greater than or equal to approximately 105 M ^-1 . In some embodiments, the binding domain binds to a target site with a K _a greater than or equal to approximately 10 ⁶ M ^-1 , 10 ⁷ M ^-1 , 10 ⁸ M ^-1 , 10 ⁹ M ^-1 , 10 ¹⁰ M ^-1 , 10 ¹¹ M ^-1 , 10 ¹² M ^-1 or 10 ¹³ M ^-1 . "High affinity" binding domains refer to binding domains with a K _a value of at least 10 ⁷ M ^-1 , at least 10 ⁸ M ^-1 , at least 10 ⁹ M ^-1 , at least 10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , at least 10 ¹³ M ^-1 , or more.

Alternatively, affinity can be defined as the equilibrium dissociation constant (K _d ) of a particular binding interaction with M units (eg, 10 ^-5 M to 10 ^-13 M, or less). The affinity of nuclease variants comprising one or more DNA-binding domains for DNA target sites considered in particular embodiments can be easily determined using conventional methods, such as surface presentation on yeast cells, or binding association analysis or displacement analysis using labeled ligands.

In one embodiment, specific binding affinity is about 2 times background binding, about 5 times background binding, about 10 times background binding, about 20 times background binding, about 50 times more than background binding, about 100 times more than background binding, or about 1000 times more than background binding, or even more.

The terms "selectively binds" or "selectively bound" or "selectively bound" or "selectively targeted" describe the preferential binding of one molecule to a target molecule (target binding) in the presence of a plurality of off-target molecules. In particular embodiments, HE or megaTAL selectively binds to a target DNA binding site approximately 5, 10, 15, 20, 25, 50, 100, or 1000 times more likely than HE or megaTAL to bind to an off-target DNA binding site.

"Target" refers to the sequence of the target site.

"Off-target" refers to a sequence that is similar, but not identical to the sequence of the target site.

A "target site" or "target sequence" is a chromosomal or extrachromosomal nucleic acid sequence that defines the portion of the nucleic acid that the binding molecule will bind and/or cleave, provided there are sufficient conditions for binding and/or cleavage. When referring to a polynucleotide sequence or SEQ ID NO with reference to only one strand of a target site or target sequence, it should be understood that the target site or target sequence linked and/or cleaved by a variant nuclease is double-stranded and contains a reference sequence and her complement. In a preferred embodiment, the target site is a sequence in the human PD-1 gene.

"Recombination" refers to the process of exchanging genetic information between two polynucleotides, including, but not limited to, non-homologous end-joining (NHEJ) donor capture and homologous recombination. For the purposes of the present invention, the term "homologous recombination (HR)" refers to a specialized form of such an exchange that occurs, for example, during the repair of double-strand breaks in cells through homologous repair (HDR) mechanisms. This process requires nucleotide sequence homology, uses a "donor" molecule as a template to repair the "target" molecule (i.e., the one that has experienced a double-strand break), and is variously known as "no crossover gene conversion" or "short tract gene conversion" because it results in the transfer of genetic information from the donor to the target. Without wishing to be bound by any particular theory, such transfer may involve correcting the heteroduplex DNA mismatch that forms between the damaged target and the donor, and/or "synthesis dependent strand annealing", which uses the donor to resynthesize genetic information. that will become part of the goal and/or related processes. Such specialized HR often results in a change in the sequence of the target molecule such that part or all of the donor polynucleotide sequence is incorporated into the target polynucleotide.

The term "NHEJ" or "non-homologous end joining" refers to the resolution of a double strand break in the absence of a repair donor template or homologous sequence. NHEJ can lead to insertions and deletions at the site of the break. NHEJ is mediated by several sub-pathways, each of which has clear mutational consequences. The classic NHEJ (cNHEJ) pathway requires the KU/DNA-PKcs/Lig4/XRCC4 complex, re-ligates the ends together with minimal processing, and often results in accurate tear repair. Alternative NHEJ pathways (altNHEJ) are also active in resolving dsDNA breaks, but these pathways are significantly more mutagenic and often result in inaccurate break repair marked by insertions and deletions. Without wishing to be bound by any particular theory, it is believed that modification of dsDNA breaks with end-reshaping enzymes, such as, for example, exonucleases such as Trex2, may increase the likelihood of inaccurate repair.

"Cleavage" refers to the destruction of the covalent backbone of the DNA molecule. Cleavage can be initiated in a variety of ways, including, but not limited to, enzymatic or chemical hydrolysis of the phosphodiester bond. Possible as single strand cleavage and double strand cleavage. Double-strand cleavage can occur as a result of two separate single-strand cleavage events. DNA cleavage can result in either blunt ends or stepped ends. In some embodiments, polypeptides and nuclease variants, such as homing endonuclease variants, megaTAL, and others, discussed in this application, are used for directed double-stranded DNA cleavage. Recognition sites for endonuclease cleavage can be on any strand of DNA.

An "exogenous" molecule is one that is not normally present in a cell, but that is introduced into the cell by one or more genetic, biochemical, or other methods. Typical exogenous molecules include, among others, small organic molecules, protein, nucleic acid, carbohydrate, lipid, glycoprotein, lipoprotein, polysaccharide, any modified derivative of the above molecules, or any complex comprising one or more of the above molecules. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, among others, lipid-mediated transfer (i.e., liposomes including neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, biopolymer nanoparticles, co-precipitation calcium phosphate, DEAE-dextran mediated transfer and viral vector mediated transfer.

An "endogenous" molecule is one that is normally present in a particular cell at a particular developmental stage under certain environmental conditions. Additionally, endogenous molecules may include proteins.

"Gene" refers to the DNA region encoding the gene product, as well as all DNA regions that regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. The gene contains, among others, promoter sequences, enhancers, silencers, insulators, boundary elements, terminators, polyadenylation sequences, post-transcriptional response elements, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, origins of replication, template attachment sites, and regions loci of control.

"Gene expression" refers to the transformation of the information contained in a gene into a gene product. A gene product may be a direct transcription product of a gene (eg, mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein obtained by translation of an mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, and proteins modified by, for example, methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristylation, and glycosylation.

Used in this application, the term "engineered" or "genetically modified" refers to the chromosomal or extrachromosomal addition of additional genetic material in the form of DNA or RNA to the total genetic material in the cell. Genetic modifications may or may not target a specific site in the cell's genome. In one embodiment, the genetic modification is site-specific. In another embodiment, the genetic modification is not site-specific.

As used herein, the term "genome editing" refers to the replacement, deletion, and/or introduction of genetic material at a target site in the genome of a cell that restores, repairs, disrupts, and/or modifies the expression and/or function of a gene or gene product. Genome editing provided in particular embodiments involves introducing one or more nuclease variants into a cell to generate DNA damage at or near a target site in the cell's genome, optionally in the presence of a repair donor template.

As used herein, the term "gene therapy" refers to the introduction of additional genetic material into the total genetic material in a cell that restores, corrects, or modifies the expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide. In particular embodiments, the introduction of genetic material into the genome of a cell by genome editing that restores, corrects, disrupts, or alters the expression of a gene or gene product, or for the purpose of expressing a therapeutic polypeptide, is considered gene therapy.

"Immune disorder" refers to a disease that causes an immune system response. In particular embodiments, the term "immune disorder" refers to cancer, graft-versus-host disease, autoimmune disease, or immunodeficiency. In one embodiment, immune disorders encompass an infectious disease.

As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide out of control and can invade nearby tissues.

As used herein, the term "malignant" refers to a cancer in which a group of tumor cells exhibits one or more of: uncontrolled growth (i.e., dividing beyond normal limits), invasion (i.e., invasion of and destruction of adjacent tissues), and metastasis (i.e. spread to other parts of the body through the lymph or blood).

Used in this application, the term "metastasis" refers to the spread of cancer from one part of the body to another. A tumor formed by spreading cells is called a "metastatic tumor" or "metastasis". A metastatic tumor contains cells similar to those in the original (primary) tumor.

Used in this application, the term "benign" or "non-cancerous" refers to tumors that may increase in size, but not spread to other parts of the body. Benign tumors are self-limited and usually do not invade other sites or metastasize.

"Cancer cell" or "tumor cell" refers to a single cell of a cancerous growth or tissue. "Tumor" generally refers to an edema or lesion formed by abnormal growth of cells, which may be benign, premalignant, or malignant. Most cancers form tumors, but some, such as leukemia, do not necessarily form tumors. For those cancers that form tumors, the terms cancer (cancer cell) and tumor (tumor cell) are used interchangeably. The amount of tumor in an individual is the "tumor burden", which can be measured as the number, volume or weight of the tumor.

The term "graft-versus-host disease" or "GVHD" refers to complications that can occur after transplantation of a cell, tissue, or solid organ. GVHD can occur after a stem cell or bone marrow transplant, in which the transplanted donor cells attack the transplant recipient's body. Acute GVHD in humans occurs within approximately 60 days after transplantation and results in damage to the skin, liver, and intestines by cytolytic lymphocytes. Chronic GVHD occurs later and is a systemic autoimmune disease that primarily affects the skin, resulting in polyclonal B-cell activation and overproduction of Ig and autoantibodies. Solid organ transplant graft-versus-host disease (SOT-GVHD) occurs in two forms. The more common type is antibody-mediated, in which antibodies from a donor with blood type O attack the recipient's red blood cells in recipients with blood type A, B, or AB, resulting in mild transient hemolytic anemias. The second form of SOT-GVHD is a cell type associated with high mortality, where donor T cells mount an immunological attack against immunologically incompatible host tissue, most commonly in the skin, liver, gastrointestinal tract, and bone marrow, leading to complications in these organs.

The term "anti-leukemia graft" or "GVL" refers to an immune response to human leukemia cells by immune cells present in transplanted donor tissue, such as bone marrow or peripheral blood.

"Autoimmune disease" refers to a disease in which the body mounts an immunogenic response (ie, immune system response) against some part of its own tissue. In other words, the immune system loses the ability to recognize some tissue or system in the body as "self" and targets and attacks it as if it were foreign. Illustrative examples of autoimmune diseases include, but are not limited to: arthritis, inflammatory bowel disease, Hashimoto's thyroiditis, Graves' disease, lupus, multiple sclerosis, rheumatoid arthritis, hemolytic anemia, anti-immune thyroiditis, systemic lupus erythematosus, celiac disease, Crohn's disease, colitis, diabetes, scleroderma, psoriasis and the like.

"Immunodeficiency" means the condition of a patient whose immune system has been compromised as a result of disease or exposure to chemicals. This condition makes the system deficient in the number and type of blood cells needed to protect against foreign substances. Immune deficiency conditions or diseases are known in the art and include, for example, AIDS (acquired immunodeficiency syndrome), SCID (severe combined immunodeficiency disease), selective IgA deficiency, common variable immunodeficiency, X-linked agammaglobulinemia, chronic granulomatous disease, hyper-IgM Wiskott syndrome -Aldrich (WAS) and diabetes.

"Infectious disease" refers to a disease that can be transmitted from person to person or from body to body and is caused by a microbial or viral agent (eg, the common cold virus). Infectious diseases are known in the art and include, for example, hepatitis, sexually transmitted diseases (eg, chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria, hepatitis B, hepatitis C, cholera, and influenza.

As used herein, the terms "individual" and "subject" are often used interchangeably and refer to any animal that exhibits a symptom of an immune disorder that can be treated with nuclease variants, genome editing compositions, gene therapy vectors, genome editing vectors. , genome-edited cells, and other methods contemplated in this application. Suitable subjects (eg, patients) include laboratory animals (such as a mouse, rat, rabbit, or guinea pig), farm animals, and pets (such as a cat or dog). Non-human primates and preferably humans are included. Typical subjects are human patients who have been diagnosed with an immune disorder or are at risk of developing one.

As used herein, the term "patient" refers to a subject diagnosed with an immune disorder that can be treated with nuclease variants, genome editing compositions, gene therapy vectors, genome editing vectors, genome edited cells, and others contemplated herein. application methods.

As used herein, the term "treatment" or "treat" includes any beneficial or desirable effect on the symptoms or pathology of the disease or condition, and may include even a minimal reduction in one or more measurable markers of the disease or condition being treated, e.g., cancer, GVHD, infectious diseases, autoimmune diseases, inflammatory diseases and immunodeficiency. Treatment may include delaying the progression of the disease or condition. "Treatment" does not necessarily indicate the complete eradication or cure of a disease or condition or its associated symptoms.

As used in this application, the terms "prevent" and similar words such as "prevention", "prevented", "prevention", etc. indicate an approach to preventing, inhibiting or reducing the likelihood of occurrence or recurrence of a disease or condition, for example, cancer, GVHD, infectious diseases, autoimmune diseases, inflammatory diseases and immunodeficiency. It also refers to delaying the onset or recurrence of a disease or condition, or delaying the onset or recurrence of symptoms of said disease or condition. As used herein, the term "prevention" and similar words also includes reducing the intensity, effect, symptoms and/or burden of a disease or condition prior to the onset or recurrence of said disease or condition.

As used herein, the expression "alleviation of at least one symptom" refers to the reduction of one or more symptoms of the disease or condition for which the subject is being treated, for example, cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, and immunodeficiency. In particular embodiments, the disease or condition being treated is cancer, in which one or more relieved symptoms include, but are not limited to, weakness, fatigue, shortness of breath, tendency to bruise and bleed, frequent infections, swollen lymph nodes, bloated or painful abdomen ( due to enlarged abdominal organs), bone or joint pain, fractures, unplanned weight loss, poor appetite, night sweats, persistent mild fever, and decreased urination (due to impaired kidney function)).

As used herein, the term "amount" refers to an "amount effective" or "effective amount" of a nuclease variant, genome-editing composition, or genome-edited cells sufficient to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results.

A "prophylactically effective amount" refers to an amount of the nuclease variant, genome-editing composition, or genome-edited cells sufficient to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of the disease, the prophylactically effective amount is less than the therapeutically effective amount.

The "therapeutically effective amount" of the nuclease variant, genome editing composition, or genome edited cells may vary depending on factors such as disease status, age, sex, and weight of the individual, and the ability to elicit the desired response in that individual. A therapeutically effective amount is also one in which any toxic or deleterious effects are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" includes an amount that is effective to "treat" a subject (eg, patient). When a therapeutic amount is indicated, the precise amount of compositions contemplated in particular embodiments to be administered may be determined by the clinician, taking into account the specification and taking into account individual differences in age, weight, tumor size, degree of infection or metastasis, as well as the condition of the patient (subject).

C. NUCLEASE VARIANTS

The nuclease variants contemplated herein in particular embodiments are suitable for genomic editing of a target site in the PD-1 gene and contain one or more DNA-binding domains and one or more DNA cleavage domains (e.g., one or more endonuclease and/or exonuclease) and, optionally, one or more linkers considered in this application. The terms "reprogrammed nuclease", "engineered nuclease" or "nuclease variant" are used interchangeably and refer to a nuclease containing one or more DNA-binding domains and one or more DNA cleavage domains, and such a nuclease has been designed and/or modified from a parent or a naturally occurring nuclease to bind and cleave a double-stranded DNA target sequence in the PD-1 gene.

In particular embodiments, the nuclease variant binds and cleaves a target sequence in exon 5 of the PD-1 gene, preferably SEQ ID NO: 25 in exon 5 of the PD-1 gene, and more preferably in the "ATAC" sequence in SEQ ID NO: 25 in exon 5 gene PD-1.

In particular embodiments, a variant nuclease binds and cleaves a target sequence in exon 1 of the PD-1 gene, preferably at SEQ ID NO: 30 in exon 1 of the PD-1 gene, and more preferably at "ATCC" in SEQ ID NO: 30 in exon 1 PD-1 gene.

In particular embodiments, a variant nuclease binds and cleaves a target sequence in exon 2 of the PD-1 gene, preferably at SEQ ID NO: 35 in exon 2 of the PD-1 gene, and more preferably at "ACTT" in SEQ ID NO: 35 in exon 2 PD-1 genes.

Such a nuclease variant may be constructed and/or modified from a naturally occurring nuclease or from a prior nuclease variant. Nuclease variants contemplated in particular embodiments may further comprise one or more additional functional domains, e.g., the end-processing enzymatic domain of an end-reversing enzyme that exhibits a 5'-3'-exonuclease, 5'-3'-basic exonuclease, 3 '-5' exonuclease (eg Trex2), 5'-flap endonuclease, helicase, template dependent DNA polymerase or template independent DNA polymerase activity.

Illustrative examples of nuclease variants that bind and cleave a target sequence in the PD-1 gene include, among others, homing endonuclease (meganuclease) and megaTAL variants.

1. Homing endonuclease (meganuclease) variants

In various embodiments, the homing endonuclease or meganuclease is reprogrammed to introduce a double strand break (DSB) at a target site in the PD-1 gene. In particular embodiments, a homing endonuclease variant introduces a double-strand break in exon 5 of the PD-1 gene, preferably in SEQ ID NO: 25 in exon 5 of the PD-1 gene, and more preferably in the "ATAC" sequence in SEQ ID NO: 25 in exon 5 gene PD-1. In particular embodiments, a homing endonuclease variant introduces a double-strand break in exon 1 of the PD-1 gene, preferably in SEQ ID NO: 30 in exon 1 of the PD-1 gene, and more preferably in the "ATCC" sequence in SEQ ID NO: 30 in exon 1 gene PD-1. In particular embodiments, a homing endonuclease variant introduces a double-strand break in exon 2 of the PD-1 gene, preferably in SEQ ID NO: 35 in exon 2 of the PD-1 gene, and more preferably in the "ACTT" sequence in SEQ ID NO: 35 in exon 2 gene PD-1.

The terms "homing endonuclease" and "meganuclease" are used interchangeably and refer to natural homing endonucleases that recognize cleavage sites of 12-45 nucleotide pairs and are generally grouped into five families based on sequence and structure motifs: LAGLIDADG, GIY-YIG HNH, box His-Cys and PD-(D/E)XK.

The terms "reference homing endonuclease" or "reference meganuclease" refer to wild-type homing endonuclease or homing endonuclease found in nature. In one embodiment, a "reference homing endonuclease" refers to a wild-type homing endonuclease that has been modified to increase basal activity.

The terms “engineered homing endonuclease,” “reprogrammed homing endonuclease,” “variant homing endonuclease,” “engineered meganuclease,” “reprogrammed meganuclease,” or “variant meganuclease” refer to a homing endonuclease containing one or more DNA-binding domains. and one or more DNA cleavage domains, wherein said homing endonuclease has been engineered and/or modified from a parental or natural homing endonuclease to bind and cleave a target DNA sequence within the PD-1 gene. A homing endonuclease variant may be constructed and/or modified from a natural homing endonuclease or from another homing endonuclease variant. Homing endonuclease variants contemplated in particular embodiments may further comprise one or more additional functional domains, e.g. , 3'-5'-exonuclease (eg, Trex2), 5'-flap-endonuclease, helicase, template-dependent DNA polymerase or template-independent DNA polymerase activity.

Homing endonuclease (HE) variants do not exist in nature and can be generated using recombinant DNA technology or random mutagenesis. HE variants can be generated by making one or more amino acid changes, such as mutation, substitution, addition or deletion of one or more amino acids in a naturally occurring HE or HE variant. In particular embodiments, the HE variant contains one or more amino acid substitutions in the DNA recognition interface.

Variants of HE contemplated in particular embodiments may further include one or more linkers and/or additional functional domains, e.g. 3'-alkaline exonuclease, 3'-5'-exonuclease (for example, Trex2), 5'-flap-endonuclease, helicase, template-dependent DNA polymerase or template-independent DNA polymerase activity. In particular embodiments, HE variants are introduced into the T cell with a terminating enzyme that exhibits a 5'-3'-exonuclease, 5'-3'-alkaline exonuclease, 3'-5'-exonuclease (e.g., Trex2), 5 '-flap-endonuclease, helicase, template-dependent DNA polymerase or template-independent DNA polymerase activity. The HE variant and the 3' processing enzyme can be introduced separately, eg in different vectors or separate mRNAs, or together, eg as a fusion protein or in a polycistronic construct separated by a viral self-cleaving peptide or IRES element.

The terms "DNA recognition interface" refers to HE amino acid residues that interact with target nucleic acid bases as well as immediately adjacent residues. For each NOT, the DNA recognition interface includes an extensive network of side-chain-to-side-chain and side-chain-to-DNA contacts, most of which are necessarily unique to recognize a particular nucleic acid target sequence. Thus, the amino acid sequence of the DNA recognition interface corresponding to a particular nucleic acid sequence varies greatly and is a feature of any natural or HE variant. As a non-limiting example, a HE variant considered in particular embodiments can be generated by constructing HE variant libraries in which one or more amino acid residues located at the DNA recognition interface of native HE (or a previously generated HE variant) are varied. Libraries can be screened for target cleavage activity against each predicted PD-1 target site using cleavage assays (see, for example, Jarjour et al., 2009. Nuc. Acids Res. 37(20): 6871-6880).

Homing endonucleases LAGLIDADG (LHE) are the best studied family of homing endonucleases, encoded mainly in archaea and in the organelle DNA of green algae and fungi, and have the highest overall DNA recognition specificity. LHEs contain one or two LAGLIDADG catalytic motifs per protein chain and function as homodimers or single chain monomers, respectively. Structural studies of LAGLIDADG proteins revealed a highly conserved nuclear structure (Stoddard 2005) characterized by the αββαββα fold, with the LAGLIDADG motif belonging to the first helix of this fold. Highly efficient and specific cleavage of LHE provides a protein scaffold to generate new highly specific endonucleases. However, LHE gene engineering to bind and cleave a non-natural or non-canonical target site requires the selection of an appropriate LHE scaffold, exploration of the target locus, selection of putative target sites, and extensive alteration of the LHE to change the contact points. and the specificity of its DNA cleavage, up to two-thirds of the base pair positions at the target site.

In one embodiment, LHEs from which reprogrammed LHEs or variants of LHEs can be constructed include, but are not limited to, I-CreI and I-SceI.

Illustrative examples of LHEs from which reprogrammed LHEs or LHE variants can be constructed include, among others, I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I- CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I- OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I-SscMI and I-Vdi141I.

In one embodiment, the reprogrammed LHE or LHE variant is selected from the group consisting of: I-CpaMI variant, I-HjeMI variant, I-OnuI variant, I-PanMI variant, and I-SmaMI variant.

In one embodiment, the reprogrammed LHE or LHE variant is an I-OnuI variant. See, for example, SEQ ID NOs: 6-14 and 60-63.

In one embodiment, reprogrammed I-OnuI LHEs or I-OnuI variants targeting the PD-1 gene were constructed from natural I-OnuI or a biologically active fragment thereof (SEQ ID NOS: 1-5). In a preferred embodiment, reprogrammed I-OnuI LHEs or I-OnuI variants targeting the human PD-1 gene were constructed from an existing I-OnuI variant. In one embodiment, the reprogrammed LHE I-OnuI were engineered against the target site of the human PD-1 gene set forth in SEQ ID NO: 25. In one embodiment, the reprogrammed LHE I-OnuI OnuI were engineered against the target site of the PD-1 gene. 1 of the human indicated in SEQ ID NO: 30. In one embodiment, reprogrammed I-OnuI LHEs were engineered against the target site of the human PD-1 gene indicated in SEQ ID NO: 35.

In a particular embodiment, the reprogrammed I-OnuI LHE or I-OnuI variant that binds and cleaves the human PD-1 gene contains one or more amino acid substitutions in the DNA recognition interface. In particular embodiments, the I-OnuI LHE that binds and cleaves the human PD-1 gene contains at least 70% sequence, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 8% 4%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95 % at least 96%, at least 97%, at least 98%, or at least 99% identical to the I-OnuI DNA recognition interface sequence (Taekuchi et al. 2011. Proc Natl Acad Sci USA 2011 Aug 9; 108 (32): 13077-13082) or option I-OnuI LHE, specified in any of SEQ ID NO: 6-14 and 60-63, their biologically active fragments and/or their additional options.

In one embodiment, the I-OnuI LHE that binds and cleaves the human PD-1 gene contains the sequence for at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99% sequence identical to the I-OnuI DNA recognition contact region (Taekuchi et al 2011. Proc Natl Acad Sci U. S. A. 2011 Aug 9;108(32): 13077-13082) or the I-OnuI LHE variant listed in any of SEQ ID NOs: 6-14 and 60-63, biologically active fragments thereof, and /or their additional options.

In a particular embodiment, the LHE I-OnuI variant that binds and cleaves the human PD-1 gene contains one or more amino acid substitutions or modifications in the I-OnuI DNA recognition interface as set forth in any of SEQ ID NOs: 1-14 and 60- 63, their biologically active fragments and/or their additional variants.

In a particular embodiment, the LHE I-OnuI variant that binds and cleaves the human PD-1 gene contains one or more amino acid substitutions or modifications in the DNA recognition interface, particularly in submotifs located at positions 24-50, 68. 82, 180 -203 and 223-240 I-OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 6-14 and 60-63, biologically active fragments thereof and/or additional variants thereof .

In a particular embodiment, the I-OnuI LHE that binds and cleaves the human PD-1 gene contains one or more amino acid substitutions or modifications in the DNA recognition interface at amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186 , 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238 and 240 in I-OnuI (SEQ ID NO : 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 6-14 and 60-63, their biologically active fragments and/or their additional variants.

In a particular embodiment, an LHE I-OnuI variant that binds and cleaves the human PD-1 gene contains 5, 10, 15, 20, 25, 30, 35, or 40 or more amino acid substitutions or modifications in the DNA recognition interface. in particular, in submotives located at positions 24-50, 68-82, 180-203 and 223-240 I-OnuI (SEQ ID NO: 1-5) or the I-OnuI variant specified in any of SEQ ID no: 6-14 and 60-63, biologically active fragments thereof and/or additional variants thereof.

In a particular embodiment, an LHE I-OnuI variant that binds and cleaves the human PD-1 gene contains 5, 10, 15, 20, 25, 30, 35, or 40 or more amino acid substitutions or modifications in the DNA recognition interface. At amino acid positions selected from the group consisting of: 19, 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72 , 75, 76 77, 78, 80, 82, 168, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 195, 197, 199, 201, 203, 223, 225, 227, 229, 231, 232, 234, 236, 238 and 240 in I-OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant listed in any of SEQ ID NOs: 6-14 and 60-63, their biologically active fragments and/or their additional variants.

In one embodiment, the LHE I-OnuI variant that binds and cleaves the human PD-1 gene contains one or more amino acid substitutions or modifications at additional positions located anywhere within the entire I-OnuI sequence. Residues that can be substituted and/or modified include, among others, amino acids that bind to the target nucleic acid or that interact with the nucleic acid backbone or with nucleotide bases, either directly or via a water molecule. In one non-limiting example, the LHE I-OnuI variant considered herein, which binds and cleaves the human PD-1 gene, contains one or more substitutions and/or modifications, preferably at least 5, preferably at least 10, preferably at least 15, preferably at least 20, more preferably at least 25, more preferably at least 30, even more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions at least one position selected from group of positions, consisting of positions: 26, 28, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 59, 68, 70, 72, 75, 76, 78, 80, 138 , 143, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192, 193, 197, 199, 201, 203, 207, 223, 224, 225, 227, 229, 232 , 236 and 238 in I-OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant listed in any of SEQ ID NOs: 6-14 and 60-63, their biologically active fragments and/or their additional options.

In some embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40K, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72Q, S72R, N75S, A76Y, S78K K80R, L138M, T143N, S159P, F168L, E1788D, C180H, F182G, N184I, I186A, S188RR, K189R, S190P, K191A, L192S, G193P, K197R, V199RA, S201M, T203G, K207M, K207M, K207M, K207M, T203G, K207M, T203G, K207M. , K229I, F232K, D236E and V238E in I-OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 6-10, their biologically active fragments and/or their additional variants .

In some embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40K, E42R, G44R, Q46E, T48D, V68K, A70Y, S72Q, N75S, A76Y, S78K, K80R, L138M, T143N , S159P, F168L, C180H, F182G, N184I, I186A, S188R, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201A, T203G, K207R, Y223R, K225R, K229I, V2362E (SEQ ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 6-10, their biologically active fragments and/or their additional variants.

In particular embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72R, N75S, A76Y, K80R, L138M, T143N , S159P, F168L, C180H, F182G, N184I, I186A, S188R, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201A, T203G, K207R, Y223R, I224T, K225R, K2329I -OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 6-10, their biologically active fragments and/or their additional variants.

In additional embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72Q, N75S, A76Y, K80R, L138M, T143N, S159P , F168L, E178D, C180H, F182G, N184I, I186A, S188R, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201A, T203G, K207R, Y223R, I224T, K225R, K2329I -OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 6-10, their biologically active fragments and/or their additional variants.

In other embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72Q, N75S, A76Y, K80R, L138M, T143N, S159P , F168L, E178D, C180H, F182G, N184I, I186A, S188R, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201A, T203G, K207R, Y223R, K225R, F232K, D236E in IQ ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 6-10, their biologically active fragments and/or their additional variants.

In particular embodiments, the HE variant cleaves the target site of exon 5 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26Q, R28E, N32V, K34P, S35N, S36I, V37P, G38R, S40R, E42R, G44R, Q46E, T48D, N59D, V68K, A70Y, S72Q, N75S, A76Y, K80R, L138M, T143N, S159P, F168L, E178D, C180H, F182G, N184I, I186A, S188R, K189R, S190P, K191A, L192S, G193P, Q197R, V199R, S201M, T203G, Y223R, K225R, F232K, D236E and V238E 1-5) or an I-OnuI variant as set forth in any of SEQ ID NOs: 6-10, biologically active fragments thereof, and/or additional variants thereof.

In particular embodiments, the HE variant cleaves the target site of exon 1 of PD-1 and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions at at least one position selected from the group of positions consisting of positions: 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 42, 44, 46, 48, 68, 70, 72, 75, 76, 78, 80, 100, 132, 138, 143, 155, 159, 178, 180, 184, 186, 189, 190, 191, 192, 193, 195, 201, 203, 207, 223, 225, 227, 232, 236, 238 and 240 in I-OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant listed in any of SEQ ID NOs: 11-12 and 60-63, their biologically active fragments and/or their additional variants.

In some embodiments, the HE variant cleaves the target site of exon 1 of the PD-1 gene and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46A, Q46T, T48V, V68I, A70T, S72D, N75R, A76Y, S78R, K80R, K80C I100V, V132A, L138M, T143N, S155g, S159P, E1780S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227H, K2227H, K2227H, K2227H, K22227H, K225YUA, K2227H, K22227H, K225 and T240E in I-OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 11-12 and 60-63, biologically active fragments thereof, and/or additional variants thereof.

In other embodiments, the HE variant cleaves the target site of exon 1 of the PD-1 gene and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46A, T48V, V68I, A70T, S72D, N75R, A76Y, S78R, K80R, I100V, L138M, T143N, S159P, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D2386Q (in SEQ ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 11-12 and 60-63, their biologically active fragments and/or their additional variants.

In some embodiments, the HE variant cleaves the target site of exon 1 of the PD-1 gene and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48V, V68I, A70T, S72D, N75R, A76Y, S78R, K80C, I100V, V132A , L138M, T143N, S155G, S159P, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K22R7G, V236G, V23QE -OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 11-12 and 60-63, their biologically active fragments and/or their additional variants.

In some embodiments, the HE variant cleaves the target site of exon 1 of the PD-1 gene and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68I, A70T, S72N, N75H, A76Y, S78T, K80R, I100V, L138M , T143N, S159P, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D23 R ID NOs: 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 11-12 and 60-63, their biologically active fragments and/or their additional variants.

In some embodiments, the HE variant cleaves the target site of exon 1 of the PD-1 gene and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70L, S72N, N75H, A76Y, K80V, T82Y, L138M, T143N , S159P, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R and T240E in ID : 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 11-12 and 60-63, their biologically active fragments and/or their additional variants.

In some embodiments, the HE variant cleaves the target site of exon 1 of the PD-1 gene and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37G, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70T, S72N, N75H, A76Y, K80V, T82Y, L138M, T143N , S159P, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R and T240E in ID : 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 11-12 and 60-63, their biologically active fragments and/or their additional variants.

In some embodiments, the HE variant cleaves the target site of exon 1 of the PD-1 gene and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70Y, S72N, N75H, A76Y, K80E, T82F, L138M, T143N , S159P, E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R and T240E in ID : 1-5) or the I-OnuI variant specified in any of SEQ ID NOs: 11-12 and 60-63, their biologically active fragments and/or their additional variants.

In particular embodiments, the HE variant cleaves the target site of exon 2 of the PD-1 gene and contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more amino acid substitutions in at least one position selected from the group of positions consisting of positions: 24, 26, 28, 30, 32, 34, 35, 36, 37, 38, 40, 41, 42, 44, 46 , 48, 68, 70, 72, 74, 75, 76, 78, 80, 82, 116, 138, 143, 159, 168, 178, 180, 182, 184, 186, 188, 189, 190, 191, 192 , 193, 195, 199, 203, 207, 225, 227, 229, 232, 236 and 238 in I-OnuI (SEQ ID NOs: 1-5) or the I-OnuI variant listed in any of SEQ ID NO: 13 -14, their biologically active fragments and/or their additional variants.

In further embodiments, the HE variant contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: S24C, L26Q, R28Y, R28H, R30S, N32V, N32L, K34N, K34R, S35N, S35T, S36R, V37S, V37T, G38R, G38K, S40R, T41A, E42R, G44S, G44R, Q46E, Q46A, T48E, V68I, A70N, S72I, D74N, N75T, N75R, A76S, A76R, S78R, K80S, T82G, T82R, V116L, L138M, T143N, S159P, F168L, E178D, C180N, F182Y, N184H, I186K, K189G, S193, Q195Y, V199R, T203A, T203S, K207R, K225N, K225T, K227W, K227S, K229A, K229P, F232R, W234A, W234D, D236E and V238R in I-OnuI (SEQ ID NO: 1-5) or I-OnuI option, specified in any of SEQ ID NO: 13-14, their biologically active fragments and/or their additional variants.

In particular embodiments, the HE variant contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: L26Q, R28Y . , C180N, F182Y, N184H, I186K, K189G, S190R, K191T, L192T, G193R, Q195Y, V199R, T203A, K207R, K225N, K227W, K229A, F232R, W234A, D236E and V238R in I-OnuI (ID: NO 1 -5) or the I-OnuI variant specified in any of SEQ ID NOs: 13-14, their biologically active fragments and/or their additional variants.

In some embodiments, the HE variant contains at least 5, at least 15, preferably at least 25, more preferably at least 35, or even more preferably at least 40 or more of the following amino acid substitutions: S24C, R28H, N32L, K34R, S35T, V37T, G38K, S40R, E42R, G44S, Q46E, T48E, V68I, A70N, S72I, D74N, N75R, A76R, S78R, K80S, T82R, V116L, L138M, T143N, S159P, F178D, E1 C180N, F182Y, N184H, I186K, K189G, S190R, K191T, L192T, G193R, Q195Y, V199R, T203S, K207R, K225T, K227S, K229P, F232R, W234D, D236E and V238R in I-OnuI (SEQ 5) or the I-OnuI variant specified in any of SEQ ID NOs: 13-14, their biologically active fragments and/or their additional variants.

In particular embodiments, the I-OnuI LHE variant that binds and cleaves the human PD-1 gene contains an amino acid sequence that is at least 80%, preferably at least 85%, more preferably at least 90%, or even more preferably at least 95% identical to the amino acid sequence specified in any of SEQ ID NOs: 6-14 and 60-63, or the corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in any of SEQ ID NOs: 6-14 and 60-63, or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 6 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 7 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 8 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 9 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 10 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 11 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 12 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 13 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 14 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 60 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 61 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 62 or a corresponding biologically active fragment.

In private embodiments, the I-OnuI LHE variant contains the amino acid sequence shown in SEQ ID NO: 63 or a corresponding biologically active fragment.

MegaTAL

In various embodiments, megaTAL containing a homing endonuclease variant is reprogrammed to introduce a double strand break (DSB) at a target site in the PD-1 gene. In particular embodiments, megaTAL introduces DSB in exon 5 of the PD-1 gene, preferably in SEQ ID NO: 27 in exon 5 of the PD-1 gene, and more preferably in the "ATAC" sequence in SEQ ID NO: 27 in exon 5 of the PD-1 gene . In particular embodiments, megaTAL introduces a double strand break in exon 1 of the PD-1 gene, preferably in SEQ ID NO: 32 in exon 1 of the PD-1 gene, and more preferably in the "ATCC" sequence in SEQ ID NO: 32 in exon 1 of the PD gene -one. In particular embodiments, megaTAL introduces a double strand break in exon 2 of the PD-1 gene, preferably in SEQ ID NO: 37 in exon 2 of the PD-1 gene, and more preferably in the "ACTT" sequence in SEQ ID NO: 37 in exon 2 of the PD- gene. one.

The term "megaTAL" refers to a polypeptide comprising a TALE DNA binding domain and a homing endonuclease variant that binds and cleaves a target DNA sequence in the PD-1 gene and optionally contains one or more linkers and/or additional functional domains, e.g., an enzymatic domain final processing of an end-reversing enzyme that exhibits 5'-3'-exonuclease, 5'-3'-alkaline exonuclease, 3'-5'-exonuclease (e.g. Trex2), 5'-flap endonuclease, helicase, template -dependent DNA polymerase or template-independent DNA polymerase activity.

In particular embodiments, megaTAL can be introduced into the cell along with an end-reversing enzyme that exhibits a 5'-3'-exonuclease, 5'-3'-alkaline exonuclease, 3'-5'-exonuclease (e.g., Trex2), 5'-flap-endonuclease, helicase, template-dependent DNA polymerase or template-independent DNA polymerase activity. MegaTAL and the 3' processing enzyme can be administered separately, eg in separate vectors or separate mRNAs, or together, eg as a fusion protein or in a polycistronic construct separated by a viral self-cleaving peptide or IRES element.

A "TALE DNA binding domain" is a DNA binding portion of transcription activator-like effectors (TALE or TAL effectors) that mimic plant transcription activators to manipulate the plant transcriptome (see, for example, Kay et al., 2007. Science 318 : 648-651). TALE DNA binding domains contemplated in particular embodiments are engineered de novo or from naturally occurring TALEs, eg AvrBs3 from Xanthomonas campestris pv. vesicatoria, Xanthomonas gardneri, Xanthomonas translucens, Xanthomonas axonopodis, Xanthomonas perforans, Xanthomonas alfalfa, Xanthomonas citri, Xanthomonas euvesicatoria and Xanthomonas oryzae and brg11 and hpx17 from Ralstonia solanacearum. Illustrative examples of TALE proteins for the production and construction of DNA binding domains are disclosed in US Pat. No. 9,019,767 and the references cited therein, all of which are incorporated herein by reference in their entirety.

In particular embodiments, megaTAL comprises a TALE DNA binding domain containing one or more repeat units that are involved in binding the TALE DNA binding domain to the corresponding target DNA sequence. A single "repeating unit" (also called a "repeat") is typically 33-35 amino acids long. Each repeating unit of the TALE DNA-binding domain contains 1 or 2 DNA-binding residues constituting a repeat variable di-residue (RVD), usually at positions 12 and/or 13 repetitions. The natural (canonical) code for DNA recognition of these TALE DNA-binding domains has been defined such that the HD sequence at positions 12 and 13 results in binding to cytosine (C), NG binds to T, NI to A, NN binds to G, or A, and NG binds to T. In some cases, non-canonical (atypical) RVDs are provided.

Illustrative examples of non-canonical RVDs suitable for use in specific megaTALs discussed in particular embodiments include, among others, HH, KH, NH, NK, NQ, RH, RN, SS, NN, SN, KN for guanine (G) recognition; NI, KI, RI, HI, SI for adenine recognition (A); NG, HG, KG, RG for thymine recognition (T); RD, SD, HD, ND, KD, YG for cytosine recognition (C); NV, HN for A or G recognition; and H*, HA, KA, N*, NA, NC, NS, RA, S* for A or T or G or C recognition, where (*) means that the amino acid at position 13 is missing. for use in particular in megaTAL, considered in particular implementation options further include the examples given in US patent No. 8614092, which is incorporated by reference in its entirety.

In private embodiments, the megaTAL discussed in this application contains a TALE DNA-binding domain containing from 3 to 30 repeating units. In some cases, megaTAL consists of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 repeat units of the TALE DNA-binding domain. In a preferred embodiment, the implementation of megaTAL, considered in this application, contains a DNA binding domain TALE containing 5-15 repeat units, more preferably 7-15 repeat units, more preferably 9-15 repeat units, and more preferably 9, 10 , 11, 12, 13, 14 or 15 repeating units.

In particular embodiments, the megaTAL considered in this application contains a TALE DNA-binding domain containing from 3 to 30 repeat units, and an additional single truncated TALE repeat unit containing 20 amino acids located at the C-terminus of the TALE repeat unit set, i.e. e. an additional repeating unit of the C-terminal half-DNA-binding domain of TALE (amino acids from -20 to -1 C-cap, disclosed below in this application). Thus, considered in this application megaTAL contains a DNA-binding domain TALE containing from 3.5 to 30.5 repeating units. In some cases, megaTAL contains 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14 .5, 15.5, 16.5, 17.5, 18.5, 19.5, 20.5, 21.5, 22.5, 23.5, 24.5, 25.5, 26.5 , 27.5, 28.5, 29.5, or 30.5 repeat units of the TALE DNA-binding domain. In a preferred embodiment, the implementation of megaTAL, considered in this application, contains a TALE DNA-binding domain containing 5.5-15.5 repeat units, more preferably 7.5-15.5 repeat units, more preferably 9.5-15.5 repeating units and more preferably 9.5, 10.5, 11.5, 12.5, 13.5, 14.5 or 15.5 repeating units.

In particular embodiments, megaTAL comprises a TAL effector structure comprising an N-terminal domain (NTD) polypeptide, one or more TALE repeat domains/units, a C-terminal domain (CTD) polypeptide, and a homing endonuclease variant. In some embodiments of the NTD, the TALE repeats and/or the CTD domains belong to the same species. In other embodiments, one or more of the NTD, TALE, and/or CTD domains are of different species.

As used herein, the term "N-terminal domain (NTD) polypeptide" refers to a sequence that flanks the N-terminal region or fragment of the naturally occurring DNA-binding domain of TALE. The NTD sequence, if present, may be of any length, as long as the repeat units of the TALE DNA-binding domain retain the ability to bind DNA. In particular embodiments, the NTD polypeptide contains at least 120 to at least 140 or more amino acids N-terminal to the TALE DNA-binding domain (0 is amino acid 1 of most N-terminal repeat units). In particular embodiments, the NTD polypeptide comprises at least about 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139 or at least 140 amino acids N-terminal to the DNA-binding domain of TALE. In one embodiment, megaTAL as contemplated herein comprises an NTD polypeptide with at least about +1 to +122 amino acids and at least about +1 to +137 amino acids of the Xanthomonas TALE protein (0 is amino acid 1 of the most N-terminal repeating unit). In particular embodiments, the NTD polypeptide contains at least about 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids N-terminal to DNA-binding domain TALE of the Xanthomonas TALE protein. In one embodiment, megaTAL as contemplated herein comprises an NTD polypeptide with at least amino acids +1 to +121 of the Ralstonia TALE protein (0 is amino acid 1 of the most N-terminal repeat unit). In particular embodiments, the NTD polypeptide contains at least about 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, or 137 amino acids, N-terminal relation to the DNA-binding domain TALE of the Ralstonia TALE protein.

As used herein, the term "C-terminal domain (CTD) polypeptide" refers to a sequence that flanks the C-terminal region or fragment of the naturally occurring DNA-binding domain of TALE. The CTD sequence, if present, may be of any length as long as the TALE repeat units of the DNA binding domain retain the ability to bind DNA. In particular embodiments, the CTD polypeptide contains at least 20 to at least 85 or more amino acids C-terminal to the last complete repeat of the TALE DNA-binding domain (the first 20 amino acids are a semi-repeating unit, C-terminal to last complete C-terminal repeat unit). In particular embodiments, the CTD polypeptide contains at least about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 443, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 , 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or at least 85 amino acids, C- terminal with respect to the last complete repeat of the TALE DNA-binding domain. In one embodiment, the implementation of megaTAL, considered in this application, contains a CTD polypeptide containing at least amino acids approximately from -20 to -1 of the Xanthomonas TALE protein (-20 is amino acid 1 of the semi-repeating unit, C-terminal to the last complete C- terminal repeating unit). In particular embodiments, the CTD polypeptide contains at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, C-terminal to the last complete repeat of the Xanthomonas TALE DNA-binding domain. In one embodiment, the implementation of megaTAL, considered in this application, contains a CTD polypeptide containing at least amino acids approximately from -20 to -1 of the Ralstonia TALE protein (-20 is amino acid 1 of the semi-repeating unit, C-terminal to the last complete C- terminal repeating unit). In particular embodiments, the CTD polypeptide contains at least about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid, C-terminal to the last complete repeat of the TALE DNA-binding domain of the Ralstonia TALE protein.

In private embodiments, the megaTAL discussed in this application contains a fusion polypeptide containing a TALE DNA-binding domain designed to bind a target sequence, a homing endonuclease reprogrammed to bind and cleave the target sequence, and optionally an NTD polypeptide and/ or CTD optionally linked to each other by one or more linker polypeptides discussed elsewhere in this application. Without wishing to be bound by any particular theory, it is believed that megaTAL containing the TALE DNA binding domain and optionally an NTD and/or CTD polypeptide is fused to a linker polypeptide which is then fused to a homing endonuclease variant. Thus, the TALE DNA-binding domain binds a DNA target sequence that is within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nucleotides of the sequence target bound by the DNA-binding domain of the homing endonuclease variant. Thus, the megaTALs contemplated herein increase the specificity and efficiency of genome editing.

In one embodiment, megaTAL contains a homing endonuclease variant and a TALE DNA binding domain that binds a nucleotide sequence that is within about 2, 3, 4, 5, or 6 nucleotides, preferably 2 or 4 nucleotides upstream. from the binding site of the reprogrammed homing endonuclease.

In one embodiment, megaTAL comprises a homing endonuclease variant and a TALE DNA binding domain that binds the nucleotide sequence shown in SEQ ID NO: 26, which is 5 nucleotides upstream of the nucleotide sequence linked and cleaved by the homing variant. -endonuclease (SEQ ID NO: 25). In preferred embodiments, the megaTAL target sequence is SEQ ID NO: 27.

In one embodiment, megaTAL contains a homing endonuclease variant and a TALE DNA binding domain that binds the nucleotide sequence shown in SEQ ID NO: 31, which is 2 nucleotides upstream from the nucleotide sequence bound and cleaved by the homing variant. -endonuclease (SEQ ID NO: 30). In preferred embodiments, the megaTAL target sequence is SEQ ID NO: 32.

In one embodiment, megaTAL contains a homing endonuclease variant and a TALE DNA binding domain that binds the nucleotide sequence shown in SEQ ID NO: 36, which is 5 nucleotides upstream of the nucleotide sequence associated with the nucleotide sequence, associated and cleaved variant of homing endonuclease (SEQ ID NO: 35). In preferred embodiments, the megaTAL target sequence is SEQ ID NO: 37.

In private embodiments, the implementation of megaTAL, considered in this application, contains one or more repeating units of DNA-binding TALE and an LHE variant constructed or reprogrammed from an LHE selected from the group consisting of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I- GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I- SmaMI, I-SscMI, I-Vdi141I and variants thereof, or preferably I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI and variants thereof, or more preferably I-OnuI and variants thereof.

In private embodiments, the implementation of megaTAL considered in this application contains NTD, one or more repeating units of DNA-binding TALE, CTD and an LHE variant selected from the group consisting of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI , I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I-EjeMI, I-GpeMI, I -GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI , I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I-ScuMI, I-SmaMI, I -SscMI, I-Vdi141I and variants thereof, or preferably I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI and variants thereof, or more preferably I-OnuI and variants thereof.

In private embodiments, the implementation of megaTAL, considered in this application, contains NTD, from about 9.5 to about 15.5 repeating units of DNA-binding TALE, and an LHE variant selected from the group consisting of: I-AabMI, I-AaeMI, I-AniI, I-ApaMI, I-CapIII, I-CapIV, I-CkaMI, I-CpaMI, I-CpaMII, I-CpaMIII, I-CpaMIV, I-CpaMV, I-CpaV, I-CraMI, I- EjeMI, I-GpeMI, I-GpiI, I-GzeMI, I-GzeMII, I-GzeMIII, I-HjeMI, I-LtrII, I-LtrI, I-LtrWI, I-MpeMI, I-MveMI, I-NcrII, I-Ncrl, I-NcrMI, I-OheMI, I-OnuI, I-OsoMI, I-OsoMII, I-OsoMIII, I-OsoMIV, I-PanMI, I-PanMII, I-PanMIII, I-PnoMI, I- ScuMI, I-SmaMI, I-SscMI, I-Vdi141I and variants thereof, or preferably I-CpaMI, I-HjeMI, I-OnuI, I-PanMI, SmaMI and variants thereof, or more preferably I-OnuI and variants thereof.

In particular embodiments, the implementation of MegaTAL, considered in this application, contains NTD length from about 122 amino acids to 137 amino acids, about 9.5, about 10.5, about 11.5, about 12.5, about 13.5, about 14, 5 or about 15.5 repeat binding units, a CTD of about 20 amino acids to about 85 amino acids in length, and an LHE I-OnuI variant. In particular embodiments, any, two or all of the NTD, DNA binding domain, and CTD may be constructed from the same species or from different species in any suitable combination.

In private embodiments, the implementation of megaTAL, considered in this application, contains the amino acid sequence specified in any of SEQ ID NOS: 15-23 and 64.

In some embodiments, the implementation of megaTAL, considered in this application, is encoded by the mRNA sequence presented in any of SEQ ID NO: 40-42 and 65-68.

In particular embodiments, the megaTAL-Trex2 fusion protein of this application comprises the amino acid sequence shown in SEQ ID NO: 24.

In some embodiments, megaTAL contains the TALE DNA-binding domain and the LHE I-OnuI variant binds and cleaves the nucleotide sequence shown in SEQ ID NO: 27. In particular embodiments, megaTAL that binds and cleaves the nucleotide sequence shown in SEQ ID. NO: 27 contains the amino acid sequence shown in any of SEQ ID NOs: 15-19.

In some embodiments, megaTAL contains the TALE DNA-binding domain and the I-OnuI LHE variant binds and cleaves the nucleotide sequence shown in SEQ ID NO: 32. In particular embodiments, megaTAL that binds and cleaves the nucleotide sequence shown in SEQ ID NO: : 32, contains the amino acid sequence shown in any of SEQ ID NOs: 20-21 and 64.

In some embodiments, megaTAL contains the TALE DNA-binding domain and the LHE I-OnuI variant binds and cleaves the nucleotide sequence shown in SEQ ID NO: 37. In particular embodiments, megaTAL binds and cleaves the nucleotide sequence shown in SEQ ID. NO: 37 contains the amino acid sequence shown in any of SEQ ID NOs: 22-23.

2. Enzymes that change the structure of the ends

Genome editing compositions and methods contemplated in particular embodiments include editing cellular genomes using a variant nuclease and one or more copies of an end-altering enzyme. In particular embodiments, one polynucleotide encodes a homing endonuclease variant and a restructuring enzyme separated by a linker, a self-cleaving peptide sequence, such as a 2A sequence, or an IRES sequence. In particular embodiments, genome-editing compositions comprise a polynucleotide encoding a nuclease variant and a single polynucleotide encoding an end-reshaping enzyme. In particular embodiments, the genome editing compositions comprise a polynucleotide encoding a homing endonuclease variant enzyme that restructures the ends of a single fusion polypeptide, in addition to a tandem copy of the restructuring enzyme separated by a self-cleaving peptide.

The term "end restructuring enzyme" refers to an enzyme that modifies the open ends of a polynucleotide chain. The polynucleotide can be double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), RNA, double-stranded DNA/RNA hybrids, and synthetic DNA (eg, those containing bases other than A, C, G, and T). An end-reversing enzyme can modify the open ends of a polynucleotide chain by adding one or more nucleotides, removing one or more nucleotides, removing or modifying a phosphate group, and/or removing or modifying a hydroxyl group. The end-reversing enzyme can modify the ends at endonuclease cut sites or at ends created by other chemical or mechanical means such as shearing (e.g., through a fine needle, heat, sonication, mini-bead drumming, and spray), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis and chemotherapeutic agents.

In particular embodiments, genome editing compositions and methods contemplated in particular embodiments include editing cellular genomes using a variant of homing endonuclease or megaTAL and a DNA end restructuring enzyme.

The term "DNA end restructuring enzyme" refers to an enzyme that modifies the open ends of DNA. An enzyme that restructures DNA ends can modify blunt ends or stepped ends ("sticky" 5' or 3' ends). The DNA end restructuring enzyme can modify single-stranded or double-stranded DNA. The DNA end restructuring enzyme can modify the ends at endonuclease cut sites or at ends generated by other chemical or mechanical means, such as shearing (e.g., shearing (e.g., by passing through a fine needle, heat, sonication, drumming). beads and spray), ionizing radiation, ultraviolet radiation, oxygen radicals, chemical hydrolysis, and chemotherapeutic agents.DNA end-reshaping enzyme can modify the open ends of DNA by adding one or more nucleotides, deleting one or more nucleotides, removing or modifying the phosphate group and/or removing or modifying the hydroxyl group.

Illustrative examples of DNA end restructuring enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to: 5'-3' exonucleases, 5'-3' alkaline exonucleases, 3'-5' exonucleases, 5 'flap endonucleases, helicases, phosphatases, hydrolases, and template-independent DNA polymerases.

Other illustrative examples of DNA end restructuring enzymes suitable for use in particular embodiments contemplated herein include, but are not limited to, Trex2, Trex1, Trex1 without a transmembrane domain, Apollo, Artemis, DNA2, Exo1, ExoT, ExoIII, Fen1 , Fan1, MreII, Rad2, Rad9, TdT (terminal deoxynucleotidyl transferase), PNKP, RecE, RecJ, RecQ, lambda exonuclease, Sox, Vaccinia DNA polymerase, exonuclease I, exonuclease III, exonuclease VII, NDK1, NDK5, NDK7, NDR , NDR7, NRK T7 exonuclease Gene 6, avian myeloblastosis virus (IN) integration protein, Bloom, Antarctic phosphatase, alkaline phosphatase, polynucleotide kinase (PNK), ApeI, mung bean nuclease, Hex1, TTRAP (TDP2), Sgs1, Sae2, CUP , Pol Mu, Pol Lambda, MUS81, EME1, EME2, SLX1, SLX4 and UL-12.

In particular embodiments, genome editing compositions and methods for editing cellular genomes contemplated herein include polypeptides containing a homing endonuclease or megaTAL variant and an exonuclease. The term "exonuclease" refers to enzymes that cleave phosphodiester bonds at the end of a polynucleotide chain through a hydrolysis reaction that breaks phosphodiester bonds at the 3' or 5' end.

Illustrative examples of exonucleases suitable for use in the particular embodiments discussed herein include, among others: hExoI, Yeast ExoI, E. coli ExoI, hTREX2, mouse TREX2, rat TREX2, hTREX1, mouse TREX1, rat TREX1, and Rat TREX1 .

In particular embodiments, the end-reversing DNA enzyme is a 3' to 5' exonuclease, preferably Trex 1 or Trex2, more preferably Trex2, and even more preferably human or mouse Trex2.

B. Target sites

Nuclease variants contemplated in particular embodiments may be designed to bind to any suitable target sequence and may have new binding specificity compared to the native nuclease. In particular embodiments, the target site is a regulatory region of a gene, including but not limited to promoters, enhancers, repressor elements, and the like. In private embodiments, the target site is a coding region of a gene or a splicing site. In some embodiments, nuclease variants are designed to suppress or reduce gene expression. In particular embodiments, the nuclease variant and the repair donor template may be designed to repair or remove the desired target sequence.

In various embodiments, nuclease variants bind and cleave a target sequence in the programmed death receptor 1 (PD-1) gene. PD-1 is also referred to as programmed cell death receptor 1 (PDCD1), susceptibility to systemic lupus erythematosus 2 (SLEB2), CD279, HPD1, PD1, HPD-L, and HSLE1. PD-1 is a member of the B7/CD28 costimulatory receptor family. The PD-1 molecule consists of an IgV extracellular ligand-binding domain, a transmembrane domain, and an intracellular domain that has potential phosphorylation sites located with an immune tyrosine inhibitory motif (ITIM) and an immunoreceptor tyrosine inhibitory receptor (ITSM) inhibitory motif. PD-1 is an inhibitory co-receptor expressed on T cells, Tregs, depleted T cells, B cells, activated monocytes, dendritic cells (DC), natural killer (NK) cells and natural killer T (NKT) cells. PD-1 negatively regulates T cell activation through binding to its ligands, programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2). Binding of PD-1 inhibits T cell proliferation and production of interferon-γ (IFN-γ), tumor necrosis factor-α, and IL-2 and reduces T cell survival. Expression of PD-1 is a hallmark of "depleted" T cells that have experienced a high level of stimulation. This state of exhaustion, which occurs during chronic infections and cancer, is characterized by T-cell dysfunction resulting in poor control of infections and tumors.

In particular embodiments, a variant of homing endonuclease or megaTAL introduces a double strand break (DSB) at a target site in the PD-1 gene. In particular embodiments, a homing endonuclease or megaTAL variant introduces DSB in exon 5 of the PD-1 gene, preferably in SEQ ID NO: 25 (or SEQ ID NO: 27) in exon 5 of the PD-1 gene, and more preferably in the sequence "ATAC » in SEQ ID NO: 25 (or SEQ ID NO: 27) in exon 5 of the PD-1 gene.

In a preferred embodiment, a homing endonuclease or megaTAL variant cleaves double stranded DNA and introduces DSB into the polynucleotide sequence shown in SEQ ID NO: 25 or 27.

In particular embodiments, a homing endonuclease or megaTAL variant introduces DSB in exon 1 of the PD-1 gene, preferably in SEQ ID NO: 30 (or SEQ ID NO: 32) in exon 1 of the PD-1 gene, and more preferably in the sequence "ATCC » in SEQ ID NO: 30 (or SEQ ID NO: 32) in exon 1 of the PD-1 gene.

In a preferred embodiment, a homing endonuclease or megaTAL variant cleaves double stranded DNA and introduces DSB into the polynucleotide sequence shown in SEQ ID NO: 30 or 32.

In particular embodiments, a homing endonuclease or megaTAL variant introduces DSB in exon 2 of the PD-1 gene, preferably in SEQ ID NO: 35 (or SEQ ID NO: 37) in exon 2 of the PD-1 gene, and more preferably in the sequence "ACTT » in SEQ ID NO: 35 (or SEQ ID NO: 37) in exon 2 of the PD-1 gene.

In a preferred embodiment, a homing endonuclease or megaTAL variant cleaves double stranded DNA and introduces DSB into the polynucleotide sequence shown in SEQ ID NO: 35 or 37.

In a preferred embodiment, the PD-1 gene is a human PD-1 gene.

C. Donor repair matrices

Nuclease variants can be used to introduce DSB into the target sequence; DSB can be repaired using homologous repair (HDR) mechanisms in the presence of one or more donor repair templates.

In various embodiments, the repair donor template contains one or more polynucleotides encoding an immunopotentiation enhancer, an immunosuppressive signal damper, or an engineered antigen receptor.

In various embodiments, administration of engineered nuclease into a cell in the presence of multiple donor repair templates independently encoding immunopotentiation enhancers and/or immunosuppressive signal dampers targeting different immunosuppressive pathways is contemplated to produce genome-edited T cells with increased therapeutic efficacy and viability. For example, in particular embodiments, immunopotentiation enhancers or immunosuppressive signal dampers targeting combinations of the PD-1, LAG-3, CTLA-4, TIM3, IL-10R, TIGIT, and TGFβRII pathways may be preferred.

In particular embodiments, a repair donor template is used to insert the sequence into the genome. In particular preferred embodiments, a repair donor template is used to repair or modify a sequence in the genome.

In various embodiments, a repair donor template is introduced into a hematopoietic cell, such as a T cell, by transducing the cell with an adeno-associated virus (AAV) vector, a retrovirus, such as lentivirus, IDLV, etc., herpes simplex virus, adenovirus, or bovine virus. smallpox containing a donor repair matrix.

In particular embodiments, the donor repair template contains one or more homology arms that flank the DSB site.

As used herein, the term "homologous arms" refers to a nucleotide sequence in the donor repair template that is identical or nearly identical to the DNA sequence flanking the DNA break introduced by the nuclease at the target site. In one embodiment, the repair donor template contains a 5' homologous arm that contains a nucleotide sequence that is identical or nearly identical to the DNA sequence of the 5' DNA break site. In one embodiment, the repair donor template contains a 3' homologous arm that contains a nucleotide sequence that is identical or nearly identical to the DNA sequence of the 3' DNA break site. In a preferred embodiment, the repair donor template contains a 5' homologous arm and a 3' homologous arm. The repair donor template may contain homology to a genomic sequence immediately adjacent to the DSB site, or homology to a genomic sequence within any number of base pairs from the DSB site. In one embodiment, the repair donor template contains a nucleotide sequence that is homologous to a genomic sequence of about 5 bp, about 10 bp, about 25 bp, about 50 bp, about 100 bp. , approximately 250 bp, approximately 500 bp, approximately 1000 bp, approximately 2500 bp, approximately 5000 bp, approximately 10000 bp or more, including any intermediate homologous length sequences.

Illustrative examples of suitable lengths of homologous arms considered in particular embodiments can be independently selected and include, among others: homologous arms about 100 bp long, about 200 bp long, about 300 bp long, about 400 bp long. about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1000 bp, about 1100 bp ., about 1200 bp, about 1300 bp, about 1400 bp, about 1500 bp, about 1600 bp, about 1700 bp, about 1800 bp , approximately 1900 approximately 2000 bp, approximately 2100 bp, approximately 2200 bp, approximately 2300 bp, approximately 2400 bp, approximately 2500 bp, approximately 2600 bp ., about 2700 bp, about 2800 bp, about 2900 bp or approximately 3000 bp or more, including all intermediate lengths of homologous arms.

Additional illustrative examples of suitable homologous arm length include, among others: about 100 to about 3000 bp, about 200 to about 3000 bp, about 300 to about 3000 bp, about 400 to about 3000 bp, about 500 about 3000 to about 2500 bp, from about 500 to about 2500 bp, from about 500 to about 2000 bp, from about 750 to about 2000 bp ., from about 750 to about 1500 b.p. or from about 1000 to about 1500 bp, including all intermediate lengths of homologous arms.

In a particular embodiment, the lengths of the 5' and 3' homologous arms are independently selected from about 500 to about 1500 bp. In one embodiment, the 5' homologous arm is about 1500 bp long and the 3' homologous arm is about 1000 bp long. In one embodiment, the 5' homologous arm is 200 to 600 bp in length and the homologous arm is 200 to 600 bp in length. In one embodiment, the 5' homologous arm is about 200 bp long and the 3' homologous arm is about 200 bp long. In one embodiment, the 5' homologous arm is about 300 bp long and the 3' homologous arm is about 300 bp long. In one embodiment, the 5' homologous arm is about 400 bp long and the 3' homologous arm is about 400 bp long. In one embodiment, the 5' homologous arm is about 500 bp long and the 3' homologous arm is about 500 bp long. In one embodiment, the 5' homologous arm is about 600 bp long and the 3' homologous arm is about 600 bp long.

The repair donor template may further comprise one or more polynucleotides such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRES) , recombinase recognition sites. (eg, LoxP, FRT, and Att sites), termination codons, transcription termination signals, and polynucleotides encoding self-cleaving polypeptides, epitope tags, as discussed elsewhere in this application.

In one embodiment, the donor repair template contains a polynucleotide containing the PD-1 gene, or a portion thereof, and is designed to introduce one or more mutations into the PD-1 genomic sequence so as to express the product of the mutated PD-1 gene. In one embodiment, the PD-1 mutant has reduced ligand binding and/or reduced intracellular signaling.

In various embodiments, the repair donor template comprises a 5' homologous arm, an RNA polymerase II promoter, one or more polynucleotides encoding an immunopotentiator enhancer, an immunosuppressive signal damper, or an engineered antigen receptor, and a 3' homologous arm.

In various embodiments, the target site is modified with a repair donor template comprising a 5' homologous arm, one or more polynucleotides encoding a self-cleaving viral peptide, e.g. a signal or self-cleaving peptide; and a 3' homologous arm, wherein the expression of one or more polynucleotides is regulated by the endogenous PD-1 promoter.

1. Enhancers of immunopotency

In particular embodiments, the genome-edited immune effector cells contemplated herein are made more active and/or resistant to immunosuppressive factors by introducing DSB into the PD-1 gene in the presence of a donor repair template encoding an immunopotentiator enhancer. As used herein, the term "immunopotency enhancer" refers to non-naturally occurring molecules that stimulate and/or potentiate T cell activation and/or function, immunopotentiating factors, and non-naturally occurring polypeptides that convert immunosuppressive signals from the tumor microenvironment into an immunostimulatory one. signal in a T cell or other immune cells.

In particular embodiments, the immunopotentiator enhancer is selected from the group consisting of a bispecific T cell activator (BiTE) molecule; an immunostimulatory factor including, but not limited to, cytokines, chemokines, cytotoxins, and/or cytokine receptors; and flip receptor.

In some embodiments, the immunopotentiator enhancer, immunopotentiating factor, or flip receptor are fusion polypeptides containing a protein destabilization domain.

a. Bispecific T-cell activator (BiTE) molecules

In particular embodiments, the genome-edited immune effector cells of the present application are made more effective by introducing DSB into the PD-1 gene in the presence of a donor repair template encoding bispecific T-cell activator (BiTE) molecules. BiTE molecules are two-part molecules containing a first binding domain that binds a target antigen, linker or spacer discussed elsewhere in this application and a second binding domain that binds a stimulatory or co-stimulatory molecule on an immune effector cell. The first and second binding domains can be independently selected from ligands, receptors, antibodies or antigen-binding fragments thereof, lectins, and carbohydrates.

In particular embodiments, the first and second binding domains are antigen binding domains.

In particular embodiments, the first and second binding domains are antibodies or antigen-binding fragments thereof. In one embodiment, the first and second binding domains are single chain variable fragments (scFv).

Illustrative examples of target antigens that can be recognized and bound by the first binding domain in particular embodiments include, but are not limited to: alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, Fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO -1, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEMs, VEGFR2 and WT-1.

Other exemplary target antigen embodiments include MHC-peptide complexes, optionally wherein the peptide is processed from alpha-folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30 , CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR families including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP , Fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), MAGE1, NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, Mesothelin, Muc1, Muc16, NCAM, ligands NKG2D, PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEMs, VEGFR2 and WT-1.

Illustrative examples of stimulatory or costimulatory molecules on immune effector cells recognized and bound by the second binding domain in particular embodiments include, but are not limited to: CD3γ, CD3δ, CD3ε, CD3ζ, CD28, CD134, CD137, and CD278.

In particular embodiments, DSB in the PD-1 gene is induced with a engineered nuclease, and a repair donor template containing BiTE is introduced into the cell and inserted into the PD-1 gene by homologous recombination.

b. Immunopotentiating factors

In particular embodiments, the genome-edited immune effector cells contemplated herein become more active by increasing immunopotentiating factors either in the genome-edited cells or in cells in the tumor microenvironment. "Immunopotentiating factors" refers to specific cytokines, chemokines, cytotoxins, and cytokine receptors that enhance the immune response in immune effector cells. In one embodiment, T cells are constructed by introducing a DSB into the PD-1 gene in the presence of a donor repair template encoding a cytokine, chemokine, cytotoxin, or cytokine receptor.

In particular embodiments, the donor repair template encodes a cytokine selected from the group consisting of: IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15, and TNF-α .

In a preferred embodiment, the donor repair template encodes a cytokine selected from the group consisting of IL-2, IL-7, IL-12, IL-15, IL-18, and IL-21, which when inserted into a target site in the PD gene -1 operably links the cytokine to the endogenous PD-1 promoter, thereby placing the cytokine's transcriptional control under the control of the endogenous PD-1 promoter.

In another preferred embodiment, the donor repair template encodes IL-12 which, when inserted into a target site in the PD-1 gene, operably links the cytokine to the endogenous PD-1 promoter, thereby placing the transcriptional control of the cytokine under the control of the endogenous PD-1 promoter. .

In particular embodiments, the donor repair template encodes a chemokine selected from the group consisting of: MIP-1α, MIP-1β, MCP-1, MCP-3, and RANTES.

In particular embodiments, the donor repair template encodes a cytotoxin selected from the group consisting of perforin, granzyme A, and granzyme B.

In particular embodiments, the donor repair template encodes a cytokine receptor selected from the group consisting of: IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, and IL-21 receptor.

In a preferred embodiment, the repair donor template encodes a cytokine receptor selected from the group consisting of an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, an IL-18 receptor, and an IL-21 receptor, which, being inserted into a target site in the PD-1 gene operably links the cytokine receptor to the endogenous PD-1 promoter, thereby placing transcriptional control of the cytokine receptor under the control of the endogenous PD-1 promoter.

In another preferred embodiment, the donor repair template encodes an IL-12 receptor that, when inserted into a target site in the PD-1 gene, operably binds the cytokine receptor to the endogenous PD-1 promoter, thereby placing cytokine receptor transcriptional control under endogenous control. control of the endogenous PD-1 promoter.

c. Flip receptors

In other embodiments, the donor repair template encodes a flip receptor, or a portion thereof. As used herein, the term "flip receptor" refers to a non-naturally occurring polypeptide that converts immunosuppressive signals from the tumor microenvironment into an immunostimulatory signal in a T cell. In particular embodiments, "PD-1 flip receptor" refers to a polypeptide that contains a PD-1 exodomain or ligand binding domain or variant thereof, a transmembrane domain, and an endodomain that transduces an immunostimulatory signal into a T cell. In particular embodiments, "PD-1 flip receptor" refers to a polypeptide that contains a PD-1 exodomain or ligand binding domain or variant thereof, a PD-1 transmembrane domain, and an endodomain that transduces an immunostimulatory signal into a T cell. In particular embodiments, "PD-1 flip receptor" refers to a polypeptide that contains a PD-1 exodomain or ligand binding domain or variant thereof and a transmembrane domain and an endodomain from a protein that transduces an immunostimulatory signal into a T cell. In some embodiments, the PD-1 exodomain variant has increased binding affinity for PD-L1 and/or PD-L2.

In one embodiment, the transmembrane is isolated from CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152 , CD154, AMN and PD-1.

In one embodiment, the transmembrane is isolated from CD4, CD8α, CD8β, CD27, CD28, CD134, CD137, CD3 polypeptide, IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, or IL-21 receptor.

In one embodiment, the endodomain is isolated from an IL-2 receptor, an IL-7 receptor, an IL-12 receptor, an IL-15 receptor, or an IL-21 receptor.

In one embodiment, the repair donor matrix comprises a PD-1 flip receptor, an exodomain or PD-1 ligand binding domain, a transmembrane domain, and one or more intracellular co-stimulatory signaling domains and/or primary signaling domains. Transmembrane and endodomains can be isolated from the same protein or different proteins.

In one embodiment, the repair donor matrix comprises a PD-1 flip receptor, a PD-1 exodomain or ligand binding domain, a PD-1 transmembrane domain, and one or more intracellular co-stimulatory signaling domains and/or primary signaling domains.

2. Immunosuppressive signal dampers

One limitation or problem that is a weak point of current adoptive cell therapy is the hyporeactivity of immune effector cells due to depletion mediated by the tumor microenvironment. Depleted T cells have a unique molecular signature that is markedly different from naïve, effector, or memory T cells. Depleted T cells are defined as T cells with reduced cytokine expression and effector function.

In particular embodiments, the genome-edited immune effector cells of the present application become more resistant to depletion by reducing or damping signaling by immunosuppressive factors. In one embodiment, T cells are constructed by introducing a DSB into the PD-1 gene in the presence of a donor repair template encoding an immunosuppressive signal damper.

As used herein, the term "immunosuppressive signal damper" refers to a non-naturally occurring polypeptide that reduces the transmission of immunosuppressive signals from the tumor microenvironment to the T cell. In one embodiment, an "immunosuppressive signal damper" is an antibody, or antigen-binding fragment thereof, that binds an immunosuppressive factor. In preferred embodiments, an "immunosuppressive signal damper" refers to a polypeptide that has a suppressive, damping, or dominant negative effect on a particular immunosuppressive factor or signal transduction pathway, since the damper contains an exodomain that binds the immunosuppressive factor and optionally a transmembrane domain, and optionally , modified endodomain (eg, intracellular signaling domain).

In private embodiments, the exodomain is an extracellular binding domain that recognizes and binds an immunosuppressive factor.

In particular embodiments, such a modified endodomain is mutated to reduce or inhibit immunosuppressive signals. Suitable mutation strategies include, inter alia, substitution, addition or deletion of an amino acid. Suitable mutations further include, but are not limited to, shortening the endodomain to remove signaling domains, mutating endodomains to remove residues important for signaling motif activity, and mutating endodomains to block receptor cycling. In particular embodiments, the endodomain, when present, does not transduce immunosuppressive signals or has a substantially reduced ability to transduce immunosuppressive signals.

Thus, in some embodiments, the immunosuppressive signal damper acts as a scavenger for one or more immunosuppressive factors from the tumor microenvironment and inhibits the appropriate immunosuppressive signaling pathways in the T cell.

One immunosuppressive signal is mediated by tryptophan catabolism. Tryptophan catabolism by indolamine 2,3-dioxygenase (IDO) in cancer cells results in the production of kynurenins, which have been shown to have an immunosuppressive effect on T cells in the tumor microenvironment. See, for example, Platten et al. (2012) Cancer Res. 72(21):5435-40.

In one embodiment, the repair donor matrix contains an enzyme with kynureninase activity.

Illustrative examples of enzymes having kynureninase activity suitable for use in private embodiments include, among others, L-kynurenine hydrolase.

In one embodiment, the donor repair template contains one or more polynucleotides encoding an immunosuppressive signal damper that reduces or blocks immunosuppressive signaling mediated by an immunosuppressive factor.

Illustrative examples of immunosuppressive factors targeted by immunosuppressive signal dampers contemplated in particular embodiments include, but are not limited to: programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), transforming growth factor β (TGFβ), macrophage colony-stimulating factor 1 (M-CSF1), tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), cancer receptor-binding antigen expressed on cell ligand SiSo (RCAS1), Fas ligand (FasL), CD47, interleukin-4 (IL -4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and interleukin-13 (IL-13).

In various embodiments, the immunosuppressive signal damper comprises an antibody, or an antigen-binding fragment thereof, that binds an immunosuppressive factor.

In various embodiments, the immunosuppressive signal damper comprises an exodomain that binds an immunosuppressive factor.

In particular embodiments, the immunosuppressive signal damper comprises an exodomain that binds the immunosuppressive factor and the transmembrane domain.

In another embodiment, the immunosuppressive signal damper comprises an exodomain that binds an immunosuppressive factor, a transmembrane domain, and a modified endodomain that does not transduce or that has a substantially reduced ability to transduce immunosuppressive signals.

Used in this application, the term "exodomain" refers to the antigen-binding domain. In one embodiment, the exodomain is an extracellular ligand-binding domain of an immunosuppressive receptor that transduces immunosuppressive signals from the tumor microenvironment into a T cell. In particular embodiments, "exodomain" refers to the extracellular ligand-binding domain of a receptor that contains an immunoreceptor tyrosine inhibitory motif (ITIM) and/or an immunoreceptor tyrosine switch motif (ITSM).

Illustrative examples of exodomains suitable for use in particular embodiments of immunosuppressive signal dampers include, among others, antibodies or antigen-binding fragments or extracellular ligand-binding domains isolated from the following polypeptides: programmed cell death protein 1 (PD-1), gene protein lymphocyte activation 3 (LAG-3), immunoglobulin T-cell domain and mucin domain protein 3 (TIM3), cytotoxic T-lymphocyte antigen 4 4 (CTLA-4), B- and T-lymphocyte attenuator (BTLA), T-cell immunoglobulin and immunoreceptor domain inhibitory motif tyrosine (TIGIT), transforming growth factor β receptor II (TGFβRII), macrophage colony stimulating factor 1 receptor (CSF1R), interleukin 4 receptor (IL4R), interleukin 6 receptor (IL6R), chemokine receptor (C-X-C motif ) 1 (CXCR1), chemokine receptor 2 (C-X-C motif) (CXCR2), interleukin 10 receptor alpha subunit (IL10R), interleukin 13 receptor subunit alpha-2 (IL13Rα2), TNF family apoptosis-inducing ligand receptor (TRAILR1), SiSo cell-expressed cancer antigen receptor-binding (RCAS1R), and cell surface death receptor Fas (FAS).

In one embodiment, the exodomain comprises an extracellular ligand-binding receptor domain selected from the group consisting of: PD-1, LAG-3, TIM3, CTLA-4, IL10R, TIGIT, CSF1R, and TGFβRII.

In private embodiments, a number of transmembrane domains may be used. Illustrative examples of transmembrane domains suitable for use in particular embodiments of immunosuppressive signal dampers contemplated in particular embodiments include, but are not limited to, transmembrane domains of the following proteins: T cell receptor alpha or beta chain, CDδ, CD3ε, CDγ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154 and PD-1.

In particular embodiments, the adoptive cell therapy contemplated herein comprises an immunosuppressive signal damper that inhibits or blocks the transduction of TGFβ immunosuppressive signals from the tumor microenvironment via TGFβRII. In one embodiment, the immunosuppressive signal damper comprises an exodomain that contains TGFβRII extracellular ligand binding, a TGFβRII transmembrane domain, and a truncated non-functional TGFβRII endodomain. In another embodiment, the immunosuppressive signal damper comprises an exodomain that contains TGFβRII extracellular ligand binding, a TGFβRII transmembrane domain, and no endodomain.

3. Engineered antigen receptors

In particular embodiments, the genome-edited immune effector cells contemplated herein comprise an engineered antigen receptor. In one embodiment, the T cells are engineered by introducing a DSB into one or more PD-1 genes in the presence of a donor repair template encoding the engineered antigen receptor.

In particular embodiments, the engineered antigen receptor is a T cell engineered receptor (TCR), a chimeric antigen receptor (CAR), a Daric receptor or components thereof, or a chimeric cytokine receptor.

a. Engineered TCRs

In particular embodiments, the genome-edited immune effector cells contemplated herein comprise an engineered TCR. In one embodiment, T cells are constructed by introducing a DSB into one or more PD-1 genes in the presence of a donor repair template encoding the engineered TCR. In a particular embodiment, the engineered TCR is inserted into a DSB in one PD-1 gene. In another embodiment, the engineered TCR alpha chain is inserted into a DSB in one PD-1 gene and the engineered TCR beta chain is inserted into a DSB in another PD-1 gene.

In one embodiment, the engineered T cells of this application comprise an engineered TCR that is not integrated into the PD-1 gene and one or more of: an immunosuppressive signal damper, a flip receptor, an alpha and/or beta chain of the engineered A T cell receptor (TCR), a chimeric antigen receptor (CAR), the Daric receptor or components thereof, or a chimeric cytokine receptor is inserted into the DSB at one or more PD-1 genes.

Naturally occurring T cell receptors contain two subunits, an alpha chain subunit and a beta chain subunit, each of which is a unique protein produced by recombination in the genome of each T cell. TCR libraries can be screened for their selectivity for specific target antigens. Thus, natural TCRs that have high avidity and reactivity towards target antigens can be selected, cloned and subsequently introduced into the T cell population used for adoptive immunotherapy.

In one embodiment, T cells are modified by introducing a repair donor template containing a polynucleotide encoding a TCR subunit of the DSB in one or more PD-1 genes, said TCR subunit having the ability to form TCRs that confer specificity for T cells against tumor cells, expressing the target antigen. In particular embodiments, these subunits have one or more amino acid substitutions, deletions, insertions, or modifications compared to the naturally occurring subunit, provided that the subunits retain the ability to form TCRs and confer the ability of the transfected T cells to hom to target cells, and involved in immunologically relevant cytokine signaling. These engineered TCRs preferably also bind target cells, displaying the respective tumor-associated peptide with high avidity, and optionally ensure efficient killing of target cells presenting the respective peptide in vivo.

Nucleic acids encoding the engineered TCR are preferably isolated from their natural context on the (naturally occurring) chromosome of the T cell and can be inserted into suitable vectors as described elsewhere in this application. Both the nucleic acids and the vectors that contain them can be transferred into a cell, preferably a T cell in particular embodiments. Such modified T cells are then capable of expressing one or more TCR strands encoded by the transduced nucleic acid or nucleic acids. In preferred embodiments, the engineered TCR is an exogenous TCR because it is introduced into T cells that do not normally express that particular TCR. An essential aspect of these engineered TCRs is that it has high avidity for a tumor antigen represented by a major histocompatibility complex (MHC) or similar immunological component. Unlike engineered TCR, CARs are engineered to bind target antigens in an MHC-independent manner.

The TCR may be expressed by additional polypeptides attached to the amino-terminal or carboxyl-terminal portion of the alpha or beta chain of the present TCR unless the attached additional polypeptide interferes with the ability of the alpha or beta chain to form a functional T cell receptor and MHC-dependent antigen recognition.

Antigens that are recognized by the engineered TCR considered in particular embodiments include, among others, cancer antigens, including antigens in both hematologic cancer and solid tumors. Illustrative antigens include, among others, alpha folate receptor, alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/ 8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR family including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, Fetal AchR, FRα, GD2, GD3 , glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+NY-ESO-1, HLA-A3+ NY-ESO-1, IL-11Rα, IL-13Rα2, lambda, Lewis-Y, Kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME, PSCA, PSMA, ROR1, SSX, survivin , TAG72, TEMs, VEGFR2 and WT-1.

In one embodiment, the repair donor template comprises a polynucleotide encoding an RNA polymerase II promoter or a first self-cleaving viral peptide and a polynucleotide encoding the alpha and/or beta chain of an engineered TCR inserted into a single modified and/or non-functional PD-1 gene. .

In one embodiment, the repair donor template comprises a polynucleotide encoding an RNA polymerase II promoter or a first self-cleaving viral peptide and a polynucleotide encoding an engineered TCR alpha chain and beta chain inserted into a single modified and/or non-functional PD-1 gene.

In a particular embodiment, the repair donor template comprises, in the 5' to 3' direction, a polynucleotide encoding the first self-cleaving viral peptide, a polynucleotide encoding the alpha chain of the engineered TCR, a polynucleotide encoding the second self-cleaving viral peptide, and a polynucleotide encoding the beta chain of the engineered TCR. TCRs inserted into a single modified and/or non-functional PD-1 gene. In such a case, the other PD-1 gene may be functional, or may be down-functional or de-functionalized by DSB and NHEJ repair. In one embodiment, another PD-1 gene has been modified with the engineered nuclease of this application and may have reduced or no functionality.

In a certain embodiment, both PD-1 genes are modified and have reduced function or are non-functional: the first modified PD-1 gene contains a nucleic acid comprising a polynucleotide encoding a first self-cleaving viral peptide and a polynucleotide encoding an engineered TCR alpha chain, and a second modified the PD-1 gene contains a polynucleotide encoding a second self-cleaving viral peptide and a polynucleotide encoding the engineered TCR beta chain.

b. Chimeric antigen receptors (CAR)

In private embodiments, the engineered immune effector cells contemplated herein comprise one or more chimeric antigen receptors (CARs). In one embodiment, T cells are constructed by introducing DSB into one or more PD-1 genes in the presence of a donor repair template encoding CAR. In a particular embodiment, CARs are inserted into the DSB in one PD-1 gene.

In one embodiment, the engineered T cells of this application contain a CAR that is not integrated into the PD-1 gene and one or more of: an immunosuppressive signal damper, a flip receptor, an engineered T-alpha and/or beta chain. a cellular receptor (TCR), a chimeric cytokine receptor (CAR), a Daric receptor or components thereof, or a chimeric antigen receptor is integrated into the DSB in one or more PD-1 genes.

In various embodiments, genome-edited T cells express CARs that redirect cytotoxicity to tumor cells. CARs are molecules that combine antibody-based specificity for a target antigen (eg, tumor antigen) with an intracellular T cell receptor activating domain to generate a chimeric protein that exhibits specific antitumor cellular immune activity. Used in this application, the term "chimeric" indicates that the protein consists of parts of different proteins or DNA of different origin.

In various embodiments, the CAR contains an extracellular domain that binds to a specific target antigen (also called a binding domain or an antigen-specific binding domain), a transmembrane domain, and an intracellular signaling domain. The main characteristics of CARs are their ability to redirect the specificity of immune effector cells, thereby inducing proliferation, cytokine production, phagocytosis, or the production of molecules that can mediate cell death of an antigen-expressing target cell in a major histocompatibility complex (MHC) independent manner using specific targeting abilities. on the cell of monoclonal antibodies, soluble ligands or cell-specific co-receptors.

In particular embodiments, CARs comprise an extracellular binding domain that specifically binds to a target polypeptide, eg, a target antigen expressed in a tumor cell. As used herein, the terms "binding domain", "extracellular domain", "extracellular binding domain", "antigen binding domain", "antigen-specific binding domain", and "extracellular antigen-specific binding domain" are used interchangeably and refer to a chimeric receptor, e.g., CAR or Daric, with the ability to specifically bind to the target antigen in question. The binding domain may comprise any protein, polypeptide, oligopeptide, or peptide that has the ability to specifically recognize and bind to a biological molecule (e.g., a cell surface receptor or tumor protein, lipid, polysaccharide, or other target cell surface molecule, or component thereof). The binding domain includes any naturally occurring synthetic, semi-synthetic or recombinantly produced binding partner for the biological molecule in question.

In particular embodiments, the extracellular binding domain comprises an antibody or an antigen-binding fragment thereof.

The term "antibody" refers to a binding agent that is a polypeptide containing at least an immunoglobulin light or heavy chain variable region that specifically recognizes and binds an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant. , for example, recognized by an immune cell. Antibodies include antigen-binding fragments, e.g., Camel Ig (camelid antibody or VHH fragment thereof), NAR Ig, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain variable antibody fragment Fv ("scFv"), bis-scFv, (scFv) 2, minibody, diabody, triatebody, tetrabody, disulfide stabilized Fv protein ("dsFv") and single domain antibody (sdAb, Nanobody) or other appropriate antibody fragments. The term also includes engineered forms such as chimeric antibodies (eg, humanized mouse antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.

In one preferred embodiment, the binding domain is scFv.

In another preferred embodiment, the binding domain is a camelid antibody.

In particular embodiments, the CAR contains an extracellular domain that binds an antigen selected from the group consisting of: alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR families including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, fetal AchR, FRα, GD2, GD3, glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA-A2+ NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1, PRAME , PSCA, PSMA, ROR1, SSX, Survivin, TAG72, TEMs, VEGFR2 and WT-1.

In particular embodiments, CARs comprise an extracellular binding domain, such as an antibody or antigen-binding fragment thereof, that binds an antigen, said antigen being an MHC-peptide complex, such as an MHC class I-peptide complex or an MHC class II-peptide complex.

In some cases, CARs contain linker residues between different domains. A "variable region binding sequence" is an amino acid sequence that links a heavy chain variable region to a light chain variable region and provides a spacer function compatible with the interaction of two binding domains such that the resulting polypeptide retains a specific binding affinity for the same target molecule that and an antibody that contains the same light and heavy chain variable regions. In private embodiments, CARs contain one, two, three, four, or five or more linkers. In particular embodiments, the linker length is from about 1 to about 25 amino acids, from about 5 to about 20 amino acids, or from about 10 to about 20 amino acids, or any intermediate amino acid length. In some embodiments, the linker length is 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 23, 24, 25 or more amino acids.

In particular embodiments, the CAR binding domain is followed by one or more "spacer domains", ie. regions that move the antigen binding domain away from the surface of effector cells to allow proper intercellular contact, antigen binding and activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The spacer domain can be derived from a natural, synthetic, semi-synthetic or recombinant source. In some instances, the spacer domain is part of an immunoglobulin, including but not limited to one or more heavy chain constant regions, such as CH2 and CH3. The spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

In one embodiment, the spacer domain contains the CH2 and CH3 of an IgG1, IgG4, or IgD immunoglobulin.

In one embodiment, the CAR binding domain is associated with one or more "hinge domains" that play their role in positioning the antigen binding domain away from the surface of effector cells to ensure proper intercellular contact, antigen binding, and activation. A CAR typically contains one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain can be obtained from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.

Exemplary hinge domains suitable for use in the CARs described herein include a hinge region derived from extracellular regions of type 1 membrane proteins such as CD8α and CD4, which may be wild-type hinge regions from these molecules or may be altered. In another embodiment, the hinge domain contains the CD8α hinge region.

In one embodiment, the hinge is a PD-1 hinge or a CD152 hinge.

The "transmembrane domain" is the portion of the CAR that integrates the extracellular binding portion and the intracellular signaling domain and anchors the CAR to the plasma membrane of an immune effector cell. The TM domain may be derived from a natural, synthetic, semi-synthetic, or recombinant source.

Exemplary TM domains can be derived from (i.e., contain at least the transmembrane region(s)) T cell receptor alpha or beta chain, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16 , CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN θ PD-1.

In one embodiment, the CAR contains a TM domain derived from CD8α. In another embodiment, the CAR under consideration herein comprises a CD8α-derived TM domain and a short oligo- or polypeptide linker, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids long, that binds a TM domain; and a CAR intracellular signaling domain. A particularly suitable linker is a glycine-serine linker.

In particular embodiments, the CAR contains an intracellular signaling domain. The term "intracellular signaling domain" refers to the portion of the CAR that is involved in relaying the message of effective binding of the CAR to the target antigen to the interior of the immune effector cell to induce effector cell function, such as activation, cytokine production, proliferation, and cytotoxic activity, including release of cytotoxic factors into the CAR-bound target cell or other cellular responses caused by antigen binding to the extracellular domain of CAR.

The term "effector function" refers to a specialized function of a cell. For example, the effector function of a T cell may be cytolytic activity or help or activity, including the secretion of a cytokine. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transduces an effector function signal and that directs the cell to perform a specialized function. Although it is usually possible to use the entire intracellular signaling domain, in many cases it is not necessary to use the entire domain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used instead of the entire domain, provided that it transduces an effector function signal. The term "intracellular signaling domain" includes any truncated portion of an intracellular signaling domain sufficient to transform an effector function signal.

It is known that signals generated only through the TCR are not sufficient for complete T cell activation and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation via the TCR (e.g., the TCR/CD3 complex), and costimulatory signaling domains that act on antigen- independently providing a secondary or costimulatory signal. In preferred embodiments, the CAR contains an intracellular signaling domain that contains one or more "co-stimulatory signaling domains" and a "primary signaling domain".

Primary signaling domains regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary signaling domains that act in a stimulatory manner may contain signaling motifs known as tyrosine-based immunoreceptor activation motifs or ITAMs.

Illustrative examples of ITAMs containing primary signaling domains suitable for use in CARs considered in particular embodiments include domains derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In particular preferred embodiments, the CAR comprises a CD3ζ primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains may be linked in any order in tandem with the carboxyl terminus of the transmembrane domain.

In particular embodiments, the CAR contains one or more co-stimulatory signaling domains to enhance the efficiency and expansion of T cells expressing CAR receptors. As used herein, the term "costimulatory signaling domain" or "costimulatory domain" refers to the intracellular signaling domain of a costimulatory molecule.

Illustrative examples of such co-stimulatory molecules suitable for use in CAR, considered in particular embodiments, include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM and ZAP70. In one embodiment, the CAR contains one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134 and a CD3ζ primary signaling domain.

In various embodiments, the CAR contains: an extracellular domain that binds an antigen selected from the group consisting of: BCMA, CD19, CSPG4, PSCA, ROR1, and TAG72; a transmembrane domain isolated from a polypeptide selected from the group consisting of: CD4, CD8α, CD154 and PD-1; one or more intracellular co-stimulatory signaling domains derived from a polypeptide selected from the group consisting of: CD28, CD134 and CD137; and a signal domain isolated from a polypeptide selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

c. Daric receptors

In particular embodiments, the engineered immune effector cells contain one or more Daric receptors. The term "Daric receptor" as used herein refers to a multi-chain engineered antigen receptor. In one embodiment, T cells are constructed by introducing DSB into one or more PD-1 genes in the presence of a donor repair template encoding one or more Daric components. In a particular embodiment, Daric or one or more of its components is integrated into the DSB in a single PD-1 gene.

In one embodiment, engineered T cells contain Daric, which is not integrated into the PD-1 gene, and one or more of the following: immunosuppressive signal damper, flip receptor, engineered T cell receptor (TCR) alpha and/or beta chain ), a chimeric antigen receptor (CAR), or the Daric receptor or components thereof are integrated into the DSB in one or more PD-1 genes.

Illustrative examples of Daric structures and components are disclosed in PCT Publication No. WO2015/017214 and US Patent Publication No. 20150266973, each of which is incorporated herein by reference in its entirety.

In one embodiment, the donor repair matrix contains the following Daric components: a signal polypeptide comprising a first multimerization domain, a first transmembrane domain, and one or more intracellular co-stimulatory signaling domains and/or primary signaling domains; and a binding polypeptide comprising a binding domain, a second multimerization domain, and optionally a second transmembrane domain. Functional Daric contains a bridging factor that promotes the formation of the Daric receptor complex on the cell surface, the bridging factor being associated with and located between the multimerization domains of the signal polypeptide and the binding polypeptide.

In particular embodiments, the first and second multimerization domains are associated with a bridging factor selected from the group consisting of: rapamycin or its rapalogue, cumermycin or its derivative, gibberellin or its derivative, abscisic acid (ABA) or its derivative, methotrexate or its derivative, cyclosporin A or its derivative, FKCsA or its derivative, trimethoprim (Tmp) - a synthetic ligand for FKBP (SLF) or its derivative, and any combination thereof.

Illustrative examples of rapamycin analogs (rapalogues) include those disclosed in US Patent No. 6649595, and the structures of these rapalogues are included in the present description by reference in their entirety. In some cases, the bridging factor is rapalogue with a significantly reduced immunosuppressive effect compared to rapamycin. "Significantly reduced immunosuppressive effect" refers to rapalogue having an immunosuppressive effect at least 0.1-0.005 times less than the immunosuppressive effect observed or expected for an equimolar amount of rapamycin, measured either clinically or in an appropriate in vitro (e.g., inhibition of T cell proliferation) or an in vivo surrogate for human immunosuppressive activity. In one embodiment, "significantly reduced immunosuppressive effect" refers to rapalogue having an EC 50 value in such an in vitro assay that is at least 10-250 times the EC 50 value observed for rapamycin in the same assay.

Other illustrative examples of rapalogues include, among others, everolimus, novolimus, pimecrolimus, ridaforolimus, tacrolimus, temsirolimus, umirolimus, and zotarolimus.

In some instances of the invention, the multimerization domains will be associated with a bridging factor that is rapamycin or its rapalogue. For example, the first and second multimerization domains are a pair selected from FKBP and FRB. FRB domains are polypeptide regions (protein "domains") that are capable of forming a tripartite complex with the FKBP protein and rapamycin or its rapalogue. FRB domains are present in many naturally occurring proteins, including mTOR proteins (also referred to in the literature as FRAP, RAPT1, or RAFT) from humans and other species; yeast proteins, including Tor1 and Tor2; and homologue of Candida FRAP. Information about the nucleotide sequences, cloning and other aspects of these proteins is already known in the art. For example, the protein sequence accession number for human mTOR is GenBank Accession No. L34075.1 (Brown et al., Nature 369:756, 1994).

FRB domains suitable for use in the private embodiments discussed herein typically contain at least about 85 to about 100 amino acid residues. In some embodiments, the FRB amino acid sequence for use in the fusion proteins of the present invention will comprise the 93 amino acid sequence Ile-2021 to Lys-2113 and the T2098L mutation based on the GenBank amino acid sequence accession number L34075. 1. The FRB domain for use in Daric, contemplated in particular embodiments, will be capable of binding to the FKBP protein complex associated with rapamycin or its rapalogue. In some cases, the peptide sequence of the FRB domain contains (a) a naturally occurring peptide sequence spanning at least the specified 93-amino acid region of human mTOR or corresponding regions of homologous proteins; (b) a variant of a naturally occurring FRB in which up to about ten amino acids, or from about 1 to about 5 amino acids, or from about 1 to about 3 amino acids, or in some embodiments, only one amino acid of the naturally occurring peptide has been removed, inserted or substituted; or (c) a peptide encoded by a nucleic acid molecule capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FRB domain, or a DNA sequence that would, were it not for the degeneracy of the genetic code, selectively hybridizing to a DNA molecule encoding a naturally occurring FRB domain. nature of the FRB domain.

FKBPs (FK506 binding proteins) are cytosolic macrolide receptors such as FK506, FK520 and rapamycin and are highly conserved across species. FKBPs are proteins or protein domains that are capable of binding to rapamycin or its rapalogue and additionally forming a tripartite complex with an FRB-containing protein or fusion protein. The FKBP domain may also be referred to as the "rapamycin binding domain". Information regarding nucleotide sequences, cloning, and other aspects of various FKBP species is known in the art (see, for example, Staendart et al., Nature 346: 671, 1990 (human FKBP12); Kay, Biochem. J. 314: 361, 1996). Homologous FKBP proteins from other mammalian species, yeast and other organisms are also known in the art and can be used in the fusion proteins disclosed in this application. The FKBP domain, considered in particular embodiments, will be able to bind to rapamycin or its rapalogue and participate in a tripartite complex with an FRB-containing protein (which can be determined by any means, direct or indirect, to detect such binding).

Illustrative examples of FKBP domains suitable for use in Daric, considered in particular embodiments, include, among others: a naturally occurring FKBP peptide sequence, preferably isolated from the human FKBP12 protein (GenBank number AAA58476.1) or a peptide sequence isolated from it, from FKBP of another human, from FKBP of a mouse or other mammal, or from FKBP of any other animal, yeast or fungal origin; a variant of a naturally occurring FKBP sequence in which up to about ten amino acids, or from about 1 to about 5 amino acids, or from about 1 to about 3 amino acids, or in some embodiments, only one amino acid of the naturally occurring peptide has been deleted, inserted, or replaced ; or a peptide sequence encoded by a nucleic acid molecule capable of selectively hybridizing to a DNA molecule encoding a naturally occurring FKBP domain, or a DNA sequence that would, were it not for the degeneracy of the genetic code, selectively hybridizing to a DNA molecule encoding a naturally occurring FKBP domain .

Other illustrative examples of multimerization domain pairs suitable for use in Daric, considered in particular embodiments, include, among others, domains from FKBP and FRB, FKBP and calcineurin, FKBP and cyclophilin, FKBP and bacterial DHFR, calcineurin and cyclophilin, PYL1 and ABI1 or GIB1 and GAI, or variants thereof.

In other embodiments, the anti-bridge factor blocks the association of the signal polypeptide and the binding polypeptide with the bridging factor. For example, cyclosporine or FK506 can be used as anti-bridge factors to titrate rapamycin and therefore stop signaling because only one multimerization domain is bound. In some instances, the anti-bridge factor (eg, cyclosporine, FK506) is an immunosuppressive agent. pathological inflammatory response in the clinical setting.

In one embodiment, the first multimerization domain contains FRB T2098L, the second multimerization domain contains FKBP12, and the bridging factor is rapalogue AP21967.

In another embodiment, the first multimerization domain contains FRB, the second multimerization domain contains FKBP12, and the binding factor is rapamycin, temsirolimus, or everolimus.

In particular embodiments, the signal polypeptide comprises a first transmembrane domain and the binding polypeptide comprises a second transmembrane domain or GPI anchor. Illustrative examples of the first and second transmembrane domains are isolated from a polypeptide independently selected from the group consisting of: CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, AMN and PD-1.

In one embodiment, the signal polypeptide comprises one or more intracellular costimulatory signaling domains and/or primary signaling domains.

Illustrative examples of primary signaling domains suitable for use in Daric signaling components considered in particular embodiments include domains that are derived from FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In particular preferred embodiments of the invention, the Daric signaling component comprises a primary CD3 signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling and costimulatory signaling domains may be linked in any order in tandem with the carboxyl terminus of the transmembrane domain.

Illustrative examples of such co-stimulatory molecules suitable for use in Daric signaling components considered in particular embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM and ZAP70. In one embodiment, the Daric signaling component comprises one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137 and CD134 and a CD3ζ primary signaling domain.

In private embodiments, the Daric-binding component contains a binding domain. In one embodiment, such a binding domain is an antibody or an antigen-binding fragment thereof.

An antibody, or antigen-binding fragment thereof, comprises at least an immunoglobulin light or heavy chain variable region that specifically recognizes and binds an epitope of a target antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, such as those recognized by an immune cell. Antibodies include antigen-binding fragments, e.g., Camel Ig (camelid antibody or VHH fragment thereof), NAR Ig, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, single chain variable antibody fragment Fv ("scFv"), bis-scFv, (scFv) 2, minibody, diabody, triatebody, tetrabody, disulfide stabilized Fv protein ("dsFv") and single domain antibody (sdAb, Nanobody) or other appropriate antibody fragments. The term also includes engineered forms such as chimeric antibodies (eg, humanized mouse antibodies), heteroconjugate antibodies (such as bispecific antibodies), and antigen-binding fragments thereof. See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., W. H. Freeman & Co., New York, 1997.

In one preferred embodiment, the binding domain is scFv.

In another preferred embodiment, the binding domain is a camelid antibody.

In particular embodiments, the Daric binding component comprises an extracellular domain that binds an antigen selected from the group consisting of: alpha folate receptor, 5T4, αvβ6 integrin, BCMA, B7-H3, B7-H6, CAIX, CD16, CD19, CD20, CD22, CD30, CD33, CD44, CD44v6, CD44v7/8, CD70, CD79a, CD79b, CD123, CD138, CD171, CEA, CSPG4, EGFR, EGFR families including ErbB2 (HER2), EGFRvIII, EGP2, EGP40, EPCAM, EphA2, EpCAM, FAP, Fetal AchR, FRα, GD2, GD3, Glypican-3 (GPC3), HLA-A1+MAGE1, HLA-A2+MAGE1, HLA-A3+MAGE1, HLA-A1+NY-ESO-1, HLA- A2+NY-ESO-1, HLA-A3+NY-ESO-1, IL-11Rα, IL-13Rα2, Lambda, Lewis-Y, Kappa, mesothelin, Muc1, Muc16, NCAM, NKG2D ligands, NY-ESO-1 , PRAME, PSCA, PSMA, ROR1, SSX, survivin, TAG72, TEMs, VEGFR2 and WT-1.

In one embodiment, the Daric binding component comprises an extracellular domain, such as an antibody or antigen-binding fragment thereof, that binds an MHC-peptide complex, such as an MHC class I-peptide complex or an MHC class II-peptide complex.

In particular embodiments, the Daric components contemplated herein comprise a linker or spacer that connects two proteins, polypeptides, peptides, domains, regions, or motifs. In some embodiments, the linker contains from about two to about 35 amino acids, or from about four to about 20 amino acids, or from about eight to about 15 amino acids, or from about 15 to about 25 amino acids. In other embodiments, the spacer may have a specific structure, such as a CH2CH3 antibody domain, a hinge domain, or the like. In one embodiment, the spacer contains the CH2 and CH3 domains of an IgG1, IgG4, or IgD protein.

In particular embodiments, the Daric components contemplated herein comprise one or more "hinge domains" that play a role in domain positioning for proper intercellular contact, antigen binding, and activation. Daric may contain one or more hinge domains between the binding domain and the multimerization domain and/or the transmembrane domain (TM) or between the multimerization domain and the transmembrane domain. The hinge domain can be obtained from a natural, synthetic, semi-synthetic or recombinant source. The hinge domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. In a particular embodiment, the hinge is a CD8α hinge or a CD4 hinge.

In one embodiment, Daric comprises a signal polypeptide comprising the first T2098L FRB multimerization domain, CD8 transmembrane domain, 4-1BB co-stimulatory domain, and CD3ζ primary signaling domain; the binding polypeptide contains an scFv that binds CD19, the second multimerization domain of FKBP12, and the transmembrane domain of CD4; and the bridging factor is rapalogue AP21967.

In one embodiment, Daric comprises a signal polypeptide comprising a first FRB multimerization domain, a CD8 transmembrane domain, a 4-1BB co-stimulatory domain, and a primary CD3ζ signaling domain; the binding polypeptide contains an scFv that binds CD19, the second multimerization domain of FKBP12, and the transmembrane domain of CD4; and the bridging factor is rapamycin, temsirolimus, or everolimus.

d. Zetakins

In particular embodiments, the engineered immune effector cells of this application comprise one or more chimeric cytokine receptors. In one embodiment, T cells are constructed by introducing DSB into one or more PD-1 genes in the presence of a donor repair template encoding CAR. In a particular embodiment, the chimeric cytokine receptor is inserted into the DSB in a single PD-1 gene.

In one embodiment, the engineered T cells of this application contain a chimeric cytokine receptor that is not integrated into the PD-1 gene and one or more of: an immunosuppressive signal dampener, a flip receptor, an engineered alpha and/or beta chain. A T cell receptor (TCR), a chimeric antigen receptor (CAR), the Daric receptor or components thereof, or a chimeric cytokine receptor is integrated into the DSB in one or more PD-1 genes.

In various embodiments, the genome-edited T cells express a chimeric cytokine receptor that redirects cytotoxicity to tumor cells. Zetakines are chimeric transmembrane immunoreceptors that contain an extracellular domain containing a soluble receptor ligand associated with an accessory region capable of binding the extracellular domain to the cell surface, transmembrane region, and intracellular signaling domain. Zetakines, when expressed on the surface of T lymphocytes, direct T cell activity to those cells that express the receptor for which the soluble receptor ligand is specific. Zetakin chimeric immunoreceptors redirect T cell antigen specificity with potential for use in the treatment of various cancers, in particular through the autocrine/paracrine cytokine systems used by human cancers.

In particular embodiments, the chimeric cytokine receptor comprises an immunosuppressive cytokine or a cytokine receptor-binding variant thereof, a linker, a transmembrane domain, and an intracellular signaling domain.

In particular embodiments, the cytokine or cytokine receptor binding variant thereof is selected from the group consisting of: interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL -10) and interleukin-13 (IL-13).

In some embodiments, the linker contains a CH2CH3 domain, a hinge domain, or the like. In one embodiment, the implementation of the linker contains the CH2 and CH3 domains of IgG1, IgG4 or IgD. In one embodiment, the linker contains a CD8α or CD4 hinge domain.

In particular embodiments, the transmembrane domain is selected from the group consisting of: T cell receptor alpha or beta chain, CD3δ, CD3ε, CD3γ, CD3ζ, CD4, CD5, CD8α, CD9, CD 16, CD22, CD27, CD28, CD33 , CD37, CD45, CD64, CD80, CD86, CD 134, CD137, CD152, CD154, AMN θ PD-1.

ITAM containing a primary signaling domain and/or a co-stimulatory domain.

In particular embodiments, the intracellular signaling domain is selected from the group consisting of: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

In particular embodiments, the intracellular signaling domain is selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 ( ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, NKD2C, SLP76, TRIM and ZAP70.

In one embodiment, the chimeric cytokine receptor contains one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134 and a CD3ζ primary signaling domain.

D. Polypeptides

This application contemplates various polypeptides, including but not limited to homing endonuclease variants, megaTAL, and fusion polypeptides. In preferred embodiments, the polypeptide comprises the amino acid sequence shown in SEQ ID NOs: 1-24 and 60-64. "Polypeptide", "polypeptide fragment", "peptide", and "protein" are used interchangeably, unless otherwise indicated, and according to the conventional meaning, i.e. as an amino acid sequence. In one embodiment, "polypeptide" includes fusion polypeptides and other variants . The polypeptides can be obtained using any of a variety of well known recombinant and/or synthetic techniques. Polypeptides are not limited to a specific length, for example, they may contain a full length protein sequence, a fragment of a full length protein, or a fusion protein, and may include post-translational modifications of the polypeptide, such as glycosylation, acetylation, phosphorylation, and the like, as well as other modifications known in the art, such as found in nature and not found in nature.

The terms "isolated protein", "isolated peptide" or "isolated polypeptide" and the like, as used in this application, refer to the synthesis, isolation and / or purification of a peptide or polypeptide molecule in vitro from the cellular environment and from association with other components of the cell, those. it is not significantly associated with substances in vivo.

Illustrative examples of polypeptides contemplated in particular embodiments include, but are not limited to, homing endonuclease variants, megaTAL, restructuring nuclease ends, fusion polypeptides, and variants thereof.

Polypeptides include "variant polypeptides". Variant polypeptides may differ from naturally occurring polypeptides by substitutions, deletions, additions, and/or insertions of one or more amino acids. Such variants may occur naturally or may be produced synthetically, for example, by modifying one or more amino acids from the above polypeptide sequences. For example, in particular embodiments, it may be desirable to improve the biological properties of a homing endonuclease, megaTAL, or the like, that binds and cleaves a target site in the human PD-1 gene by introducing one or more substitutions, deletions, additions, and/or insertions into the polypeptide . In particular embodiments, the polypeptides include polypeptides having at least about 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80 amino acid identity. %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% with any of the reference sequences considered in this application, usually when the specified option retains at least one type of biological activity of the reference sequence.

Polypeptide variants include biologically active "polypeptide fragments". Illustrative examples of biologically active polypeptide fragments include DNA binding domains, nuclease domains, and the like. Used in this application, the term "biologically active fragment" or "minimum biologically active fragment" refers to a polypeptide fragment that retains at least 100%, at least 90%, at least 80%, at least 70%, at least at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% of the naturally occurring polypeptide activity. In preferred embodiments, the biological activity is a binding affinity and/or a cleavage activity for a target sequence. In some embodiments, the polypeptide fragment may contain an amino acid chain of at least 5 to 1700 amino acids in length. It is understood that in some embodiments, fragments are at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 in length. , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 , 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250 . embodiments, the polypeptide contains a biologically active fragment of a homing endonuclease variant. In private embodiments, the implementation of the polypeptides described in this application may contain one or more amino acids, designated as "X". "X", if present in the amino acid of SEQ ID NO, refers to any amino acid. One or more "X" residues may be present at the N- and C-terminus of the amino acid sequence shown in the specific SEQ ID NOs contemplated herein. If "X" amino acids are missing, the remaining amino acid sequence shown in SEQ ID NO can be considered a biologically active fragment.

In particular embodiments, the polypeptide comprises a biologically active fragment of a homing endonuclease variant, for example, SEQ ID NOs: 3-14 and 60-63, or megaTAL (SEQ ID NOs: 15-23 and 64). The biologically active fragment may contain an N-terminal truncation and/or a C-terminal truncation. In a particular embodiment, the biologically active fragment does not contain or contains a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 N-terminal amino acids of the homing endonuclease variant compared to the sequence of the corresponding wild-type homing endonuclease, more preferably the deletion The 4 N-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence. In a particular embodiment, the biologically active fragment does not contain or contains a deletion of the 1, 2, 3, 4, or 5 C-terminal amino acids of the homing endonuclease variant compared to the corresponding wild-type homing endonuclease sequence, more preferably a deletion of the 2 C-terminal amino acids of the homing variant α-endonuclease compared to the corresponding wild-type homing endonuclease sequence. In a particular preferred embodiment, the biologically active fragment lacks or contains a deletion of the 4 N-terminal amino acids and 2 C-terminal amino acids of the homing endonuclease variant compared to the corresponding sequence of the wild-type homing endonuclease.

In a private embodiment, the I-OnuI variant contains a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or deletion of the following 1, 2, 3, 4 or 5 C-terminal amino acids: R, G, S, F, V.

In a private embodiment, the I-OnuI variant contains a deletion or substitution of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or deletion or substitution of the following 1, 2, 3, 4, or 5 C-terminal amino acids: R, G, S, F, V.

In a private embodiment, the I-OnuI variant contains a deletion of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or deletion of the following 1 or 2 C-terminal amino acids: F, V.

In a private embodiment, the I-OnuI variant contains a deletion or substitution of 1, 2, 3, 4, 5, 6, 7, or 8 of the following N-terminal amino acids: M, A, Y, M, S, R, R, E; and/or deletion or substitution of the following 1 or 2 C-terminal amino acids: F, V.

As noted above, polypeptides can be altered in a variety of ways, including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known in the art. For example, variants of the amino acid sequence of a reference polypeptide can be generated by mutations in the DNA. Methods for mutagenesis and alteration of nucleotide sequences are well known in the art. See for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al. , J. D. et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin/Cummings, Menlo Park, Calif., 1987) and references cited there. Guidance on suitable amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, D.C.).

In some embodiments, the variant will contain one or more conservative substitutions. A "conservative substitution" is the substitution of an amino acid for another amino acid that has similar properties, such that one skilled in the art of peptide chemistry would expect the secondary structure and hydropathic nature of the polypeptide to be substantially unchanged. Modifications can be made to the structure of polynucleotides and polypeptides considered in particular variants, and the polypeptides include polypeptides having, at least approximately, and at the same time obtain functional molecules that encode a variant or derivative of a polypeptide with the desired characteristics. When it is desired to change the amino acid sequence of a polypeptide to create an equivalent or even improved variant of the polypeptide, one skilled in the art can, for example, change one or more codons of the DNA coding sequence, for example, in accordance with Table 1.

Table 1 - Amino acid codons

Руководство по определению того, какие именно аминокислотные остатки могут быть замещены, вставлены или удалены без потери биологической активности, можно найти с помощью компьютерных программ, хорошо известных в данной области, таких как программное обеспечение DNASTAR, DNA Strider, Geneious, Mac Vector или Vector NTI. Предпочтительно аминокислотные замены в вариантах белка, раскрытых в данном описании, представляют собой консервативные аминокислотные замены, то есть замены одинаково заряженных или незаряженных аминокислот.Консервативная замена аминокислот включает замену одной из семейства аминокислот, которые родственны в их боковых цепях. Встречающиеся в природе аминокислоты, как правило, делятся на четыре семейства: кислые (аспартат, глутамат), основные (лизин, аргинин, гистидин), неполярные (аланин, валин, лейцин, изолейцин, пролин, фенилаланин, метионин, триптофан) и незаряженные полярные (глицин, аспарагин, глутамин, цистеин, серин, треонин, тирозин) аминокислоты. Фенилаланин, триптофан и тирозин иногда классифицируют как ароматические аминокислоты. В пептиде или белке подходящие консервативные замены аминокислот известны специалистам в данной области и обычно могут быть сделаны без изменения биологической активности получающейся в результате молекулы. Специалисты в данной области признают, что, как правило, единичные аминокислотные замены в несущественных областях полипептида существенно не изменяют биологическую активность (см., например, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p.224).

Guidance on which amino acid residues can be substituted, inserted, or removed without loss of biological activity can be found using computer programs well known in the art, such as DNASTAR, DNA Strider, Geneious, Mac Vector, or Vector NTI software. . Preferably, amino acid substitutions in the protein variants disclosed herein are conservative amino acid substitutions, that is, substitutions of identically charged or uncharged amino acids. A conservative amino acid substitution includes substitution of one of a family of amino acids that are related in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), non-polar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids. In a peptide or protein, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. It will be recognized by those skilled in the art that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not significantly alter biological activity (see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin/Cummings Pub. Co., p.224).

In one embodiment, where expression of two or more polypeptides is desired, the polynucleotide sequences encoding them may be separated by an IRES sequence, as described elsewhere in this application.

Polypeptides contemplated in particular embodiments include fusion polypeptides. In particular embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides are provided. Fusion polypeptides and fusion proteins refer to a polypeptide having at least two, three, four, five, six, seven, eight, nine, or ten polypeptide segments.

In another embodiment, two or more polypeptides can be expressed as a fusion protein that contains one or more self-cleaving polypeptide sequences, as described elsewhere in this application.

In one embodiment, the fusion protein of the present application contains one or more DNA-binding domains and one or more nucleases, as well as one or more linkers and/or self-cleaving polypeptides.

In one embodiment, the fusion protein of the present application contains a nuclease variant; a linker or self-cleaving peptide; and an end-altering enzyme including but not limited to 5'-3' exonuclease, 5'-3' alkaline exonuclease, and 3'-5' exonuclease (eg, Trex2).

Fusion polypeptides may contain one or more polypeptide domains or segments, including, but not limited to, signal peptides, cell penetrating peptide (CPP) domains, DNA binding domains, nuclease domains, etc., epitope tags (e.g., maltose-binding protein ( "MBP"), glutathione S-transferase (GST), HIS6, MYC, FLAG, V5, VSV-G and HA), polypeptide linkers and polypeptide cleavage signals. Fusion polypeptides are usually linked C-terminus to N-terminus, although they can also be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. In particular embodiments, the fusion protein polypeptides may be in any order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and cross-species homologues, so long as the desired activity of the fusion polypeptide is maintained. Fusion polypeptides may be prepared by chemical synthesis or by chemical bonding between two fragments, or generally may be prepared using other standard techniques. Ligated DNA sequences containing the fusion polypeptide are operably linked to appropriate transcription or translation control elements as described elsewhere in this application.

Fusion polypeptides may optionally contain a linker that can be used to link one or more polypeptides or domains within the polypeptide. A peptide linker sequence can be used to separate any two or more polypeptide components by a distance sufficient for each polypeptide to fold into its respective secondary and tertiary structures so as to allow the polypeptide domains to perform their desired functions. Such a peptide linker sequence is incorporated into the fusion polypeptide using standard techniques in the art. Suitable peptide linker sequences can be selected based on the following factors: (1) their ability to adopt a flexible extended conformation; (2) their inability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides; and (3) the absence of hydrophobic or charged residues that could react with polypeptide functional epitopes. Preferred peptide linker sequences contain Gly, Asn and Ser residues. Other nearly neutral amino acids such as Thr and Ala can also be used in the linker sequence. Amino acid sequences that can be effectively used as linkers include those disclosed in Maratea et al., Gene 40:39-46, 1985; Murphy et al., Proc. Natl. Acad. sci. USA 83:8258-8262, 1986; US Patent No. 4,935,233 and US Patent No. 4,751,180. Linker sequences are not required when the private segment of the fusion polypeptide contains non-essential N-terminal amino acid regions that can be used to separate functional domains and prevent steric influence. Preferred linkers are usually flexible amino acid subsequences that are synthesized as part of a recombinant fusion protein. Linker polypeptides can be 1 to 200 amino acids in length, 1 to 100 amino acids in length, or 1 to 50 amino acids in length, including all integers in between.

Typical linkers include, among others, the following amino acid sequences: glycine polymers (G) _n ; glycine-serine polymers (G ₁ - ₅ S ₁ - ₅ ) _n , where n is an integer of at least one, two, three, four, or five; glycine-alanine polymers; alanine-serine polymers; GGG (SEQ ID NO: 69); DGGGS (SEQ ID NO: 70); TGEKP (SEQ ID NO: 71) (see, for example, Liu et al., PNAS 5525-5530 (1997)); GGRR (SEQ ID NO: 72) (Pomerantz et al. 1995, supra); (GGGGS) _n where n=1, 2, 3, 4 or 5 (SEQ ID NO: 73) (Kim et al., PNAS 93, 1156-1160 (1996.); EGKSSGSGSESKVD (SEQ ID NO: 74) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); NO: 76) LRQRDGERP (SEQ ID NO: 77) LRQKDGGGSERP (SEQ ID NO: 78) LRQKD(GGGS) ₂ ERP (SEQ ID NO: 79) Alternatively, flexible linkers can be intelligently designed using a computer program capable of modeling both DNA binding sites and peptides themselves (Desjarlais & Berg, PNAS 90:2256-2260 (1993), PNAS 91:11099-11103 (1994) or phage display methods.

The fused polypeptides may further comprise a polypeptide cleavage signal between each of the polypeptide domains described herein, or between an endogenous open reading frame and a polypeptide encoded by a donor repair template. In addition, a polypeptide cleavage site can be introduced into any linker peptide sequence. Exemplary polypeptide cleavage signals include polypeptide cleavage recognition sites such as protease cleavage sites, nuclease cleavage sites (eg, rare restriction enzyme recognition sites, self-cleaving ribozyme recognition sites), and self-cleaving viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5( 8); 616-26).

Suitable protease cleavage sites and self-cleaving peptide are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Exemplary protease cleavage sites include, but are not limited to, cleavage sites for potyvirus NIa proteases (e.g., tobacco etch virus proteases), potyvirus HC proteases, potyvirus P1 (P35) proteases, biovirus NIa proteases, biovirus-encoded RNA-2 proteases, aphthovirus L proteases, Enterovirus 2A proteases, Rhinovirus 2A proteases, Picornavirus 3C proteases, Comovirus 24K proteases, Nepovirus 24K proteases, RTSV 3C-like protease (spherical tungro rice virus), PYVF 3C-like protease (parsnip yellow spot virus), heparin, thrombin and enterokinase. Due to the high stringency of cleavage, TEV (tobacco etch virus) protease cleavage sites are preferred in one embodiment, e.g., EXXYXQ (G/S) (SEQ ID NO: 80), e.g. (SEQ ID NO: 82) where X is any amino acid (TEV cleavage occurs between Q and G or Q and S).

In some embodiments, the self-cleaving polypeptide site contains a 2A or 2A-like site, sequence, or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041). In a particular embodiment, the 2A viral peptide is an aphthovirus 2A peptide, a potyvirus 2A peptide, or a cardiovirus 2A peptide.

In one embodiment, the viral 2A peptide is selected from the group consisting of: foot and mouth disease virus (FMDV) 2A peptide, rhinitis A virus (ERAV) 2A peptide, Thosea asigna virus (TaV) 2A peptide, porcine teshovirus 2A peptide. 1 (PTV-1), theilovirus 2A peptide and encephalomyocarditis virus 2A peptide.

Illustrative examples of 2A sites are shown in Table 2.

TABLE 2: Representative 2A sites include the following sequences:

E. Polynucleotides

In particular embodiments, provided are polynucleotides encoding one or more variants of the homing endonuclease, megaTAL, end restructuring enzymes, and fusion polypeptides of the present application. As used herein, the terms "polynucleotide" or "nucleic acid" refer to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and DNA/RNA hybrids. Polynucleotides can be single or double stranded, as well as recombinant, synthetic or isolated. Polynucleotides include, but are not limited to: mRNA precursor (pre-mRNA), messenger RNA (mRNA), RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (siRNA), ribozymes, genomic RNA (gRNA), RNA plus strand (RNA (+)), minus strand RNA (RNA (-)), tracrRNA, crRNA, single guide RNA (sgRNA), synthetic RNA, synthetic mRNA, genomic DNA (gDNA), amplified PCR DNA, complementary DNA (cDNA), synthetic DNA or recombinant DNA. The term "polynucleotides" refers to the polymeric form of nucleotides at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50 long, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000, at least 5000, at least 10000 or at least 15000 or more nucleotides or ribonucleotides or deoxyribonucleotides, or modified forms of nucleotides of any type, and all lengths in between. It is understood that "intermediate lengths" in this context means any length between the specified values, such as 6, 7, 8, 9, etc., 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc. In particular embodiments, the polynucleotides or variants have a sequence identity that is at least or about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99%, or 100% with a reference sequence.

In particular embodiments, the polynucleotides may be codon-optimized. Used in this application, the term "codon-optimized" refers to the replacement of codons in the polynucleotide encoding the polypeptide, to increase the expression, stability and/or activity of the polypeptide. Factors that influence codon optimization include, but are not limited to, one or more of the following: (i) variation in codon bias between two or more organisms or genes or synthetically engineered bias tables, (ii) variation in the degree of codon bias within an organism, gene or a set of genes, (iii) systematic codon variation, including context, (iv) codon variation according to their decoding tRNAs, (v) codon variation according to GC%, either in total or in one triplet position, (vi) change in degree of similarity to a reference sequence, such as a naturally occurring sequence, (vii) variation in codon frequency threshold, (viii) structural properties of mRNAs transcribed from a DNA sequence, (ix) prior knowledge of the function of DNA sequences on which to base the design set of codon substitutions, (x) systematic variation of codon sets for each amino acid, and/or (xi) isolated deletion of l different translation initiation sites.

As used herein, the term "nucleotide" refers to a heterocyclic nitrogenous base in an N-glycosidic bond to a phosphorylated sugar. It is understood that nucleotides include natural bases and a wide range of modified bases accepted in the art. Such bases are usually located at the 1' position of the nucleotide sugar group. Nucleotides typically contain a base, a sugar, and a phosphate group. In ribonucleic acid (RNA), the sugar is ribose, and in deoxyribonucleic acid (DNA), the sugar is deoxyribose, that is, a sugar that lacks the hydroxyl group present in ribose. Representative natural nitrogenous bases include purines, adenosine (A) and guanidine (G), as well as pyrimidines, cytidine (C) and thymidine (T) (or in the context of RNA, uracil (U)). The C-1 atom of deoxyribose is linked to the N-1 of the pyrimidine or the N-9 of the purine. Nucleotides are usually mono-, di-, or triphosphates. Nucleotides may be unmodified or modified at a sugar, phosphate and/or backbone group (also referred to interchangeably as "nucleotide analogs", "nucleotide derivatives", "modified nucleotides", "non-natural nucleotides", and "non-standard nucleotides"; see, for example, WO 92)./07065 and WO 93/15187). Examples of modified nucleic acid bases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).

The nucleotide can also be considered as the phosphate ester of the nucleoside, with the esterification occurring at the hydroxyl group attached to the C-5 of the sugar. As used herein, the term "nucleoside" refers to a heterocyclic nitrogenous base in an N-glycosidic bond to a sugar. Nucleosides are known in the art to include naturally occurring bases as well as well-known modified bases. Such bases are usually located at the 1' position of the nucleoside sugar moiety. Nucleosides usually contain a base and a sugar group. Nucleosides may be unmodified or modified at the sugar and/or base group (interchangeably referred to as "nucleoside analogs", "nucleoside derivatives", "modified nucleosides", "non-natural nucleosides" or "non-standard nucleosides"). As also noted above, examples of modified nucleic acid bases are summarized by Limbach et al., (1994, Nucleic Acids Res. 22, 2183-2196).

Illustrative examples of polynucleotides include, among others, polynucleotides encoding SEQ ID NOs: 1-24 and 60-64 and polynucleotide sequences shown in SEQ ID NOs: 25-59 and 65-68.

In various illustrative embodiments, the polynucleotides contemplated herein include, among others, polynucleotides encoding homing endonuclease variants, megaTAL, end-reversing enzymes, fusion polypeptides, and expression vectors, viral vectors, and transport plasmids containing the polynucleotides. considered in this application.

As used herein, the terms "polynucleotide variant" and "variant" and the like refer to polynucleotides that exhibit substantial sequence identity with a reference polynucleotide sequence, or polynucleotides that hybridize to a reference sequence under stringent conditions, as defined below. The terms also cover polynucleotides that differ from a reference polynucleotide by the addition, deletion, substitution, or modification of at least one nucleotide. Accordingly, the terms "polynucleotide variant" and "variant" include polynucleotides in which one or more nucleotides have been added or removed, or modified or replaced with other nucleotides. In this regard, it is well known in the art that certain changes can be made to a reference polynucleotide, including mutations, additions, deletions, and substitutions, in which the altered polynucleotide retains the biological function or activity of the reference polynucleotide.

In one embodiment, the polynucleotide comprises a nucleotide sequence that hybridizes to a target nucleotide sequence under stringent conditions. "Stringent condition" hybridization describes hybridization protocols in which nucleotide sequences at least 60% identical to each other remain hybridized. Generally, stringent conditions are chosen to be approximately 5° C. lower than the melting point (Tm) for a particular sequence at a particular ionic strength and pH. Tm is the temperature (at a given ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are usually present in excess, at Tm 50% of the probes are occupied in the equilibrium state.

As used herein, "sequence identity" or, for example, "comprise 50% sequence identity" refers to the extent to which sequences are identical based on a comparison of each nucleotide or based on a comparison of each amino acid in the comparison window. Thus, "percent sequence identity" can be calculated by comparing two optimally aligned sequences in a comparison window, determining the number of positions at which an identical nucleic acid base (e.g. A, T, C, G, I) or an identical amino acid residue (e.g. , Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occur in both sequences to obtain the number of matches positions, then dividing the number of matching positions by the total number of positions in the comparison window (i.e. the size of the window) and multiplying the result by 100 to get the percent sequence identity. Included are nucleotides and polypeptides having a sequence identity of at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% , 99% or 100% with any of the reference sequences described in this application, while as a rule, the polypeptide variant retains at least one biological activity of the reference polypeptide.

Terms used to describe sequence relatedness between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence identity", "percent sequence identity" and "substantial identity". A "reference sequence" is at least 12, but often 15 to 18, and often at least 25 monomeric units in length, including nucleotides and amino acid residues. Because each of the two polynucleotides may contain (1) a sequence (i.e., only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that diverges between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of two polynucleotides in a "comparison window" to identify and compare local areas of sequence similarity. The term "comparison window" refers to a conceptual segment of at least 6 contiguous positions, typically from about 50 to about 100, more typically from about 100 to about 150, in which the sequence is compared to a reference sequence of the same number of contiguous positions after how the two sequences are optimally aligned. The comparison window may contain additions or deletions (ie, gaps) of approximately 20% or less compared to the reference sequence (which contains no additions or deletions) to optimally align the two sequences. Optimal sequence alignment for comparison window alignment can be performed using computerized implementations of the algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wisconsin, USA) or visual comparison and best alignment (ie, leading to the highest percent homology in the comparison window) obtained by any of the various methods chosen. Reference may also be made to the BLAST family of programs, such as described by Altschul et al., 1997, Nucl. Acids Res. 25:3389. A detailed discussion of sequence analysis can be found in section 19.3 of Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994-1998, Chapter 15.

The term "isolated polynucleotide" as used herein refers to a polynucleotide that has been purified from sequences that flanking it in its natural state, such as a DNA fragment that has been removed from sequences that would normally be adjacent to that fragment. In particular embodiments, the term "isolated polynucleotide" refers to complementary DNA (cDNA), recombinant polynucleotide, synthetic polynucleotide, or other polynucleotide that does not exist in nature and has been created by human hand.

In various embodiments, the polynucleotide comprises an mRNA encoding the polypeptide of the present application, including, but not limited to, a homing endonuclease variant, megaTAL, and an end-reversing enzyme. In some cases, the mRNA contains a cap, one or more nucleotides, and a poly(A) tail.

As used herein, the terms "5' cap" or "5' cap structure" or "5' cap fragment" refer to a chemical modification that has been inserted into the 5' end of an mRNA. The 5' cap is involved in nuclear export, mRNA stability and translation.

In private embodiments, the mRNA considered in this application contains a 5'-cap containing a 5'-ppp-5'-triphosphate bond between the terminal residue of the guanosine cap and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. Said 5'-guanylate cap can then be methylated to form an N7-methyl guanylate residue.

Illustrative examples of the 5'cap suitable for use in particular embodiments of the mRNA polynucleotides contemplated herein include, among others: unmethylated 5'cap analogs, e.g., G(5')ppp(5')G, G( 5')ppp(5')C, G(5')ppp(5')A; methylated 5'-cap analogs, e.g. m ⁷ G(5')ppp(5')G, m ⁷ G(5')ppp(5')C and m ⁷ G(5')ppp(5')A ; dimethyl analogs of the 5'-cap, e.g. m ^2.7 G(5')ppp(5')G, m ^2.7 G(5')ppp(5')C and m ^2.7 G(5') ppp(5')A; ^{trimethylated analogs} of the 5'-cap ^, e.g. ^7G (5')ppp(5')A; dimethylated symmetrical analogs of the 5'-cap, for example, m ⁷ G(5')pppm ⁷ (5')G, m ⁷ G(5')pppm ⁷ (5')C and m ⁷ G(5')pppm ⁷ ( 5')A; and anti-reverse 5'-cap analogs, e.g. Anti-Reverse Cap Analog (ARCA), specialized 3'O-Me-m ⁷ G(5')ppp(5')G, 2'O-Me-m ⁷ G(5')ppp(5')G, 2'O-Me-m ⁷ G(5')ppp(5')C, 2'O-Me-m ⁷ G(5')ppp(5' )A, m ⁷ 2'd(5')ppp(5')G, m ⁷ 2'd(5')ppp(5')C, m ⁷ 2'd(5')ppp(5')A , 3'O-Me-m ⁷ G(5')ppp(5')C, 3'O-Me-m ⁷ G(5')ppp(5')A, m ⁷ 3'd(5') ppp(5')G, m ⁷ 3'd(5')ppp(5')C, m ⁷ 3'd(5')ppp(5')A and their tetraphosphate derivatives) (see, for example, Jemielity et al., RNA, 9: 1108-1122 (2003)).

In particular embodiments, the mRNAs contain a 5' cap which is 7-methyl guanylate ("m ⁷ G") linked via a triphosphate bridge to the 5' end of the first transcribed nucleotide, resulting in m ⁷ G(5')ppp(5' )N, where N is any nucleoside.

In some embodiments, the mRNAs contain a 5'-cap, where the cap is a Cap0 structure (Cap0 structures do not have a 2'-O-methyl ribose residue attached to bases 1 and 2), a Cap1 structure (Cap1 structures have a 2'-O- methyl residue at base 2) or a Cap2 structure (Cap2 structures have a 2'-O-methyl residue attached to both bases 2 and 3).

In one embodiment, the mRNA contains an m ⁷ G(5')ppp(5')G cap.

In one embodiment, the mRNA contains an ARCA cap.

In private embodiments, the mRNA discussed in this application contains one or more modified nucleosides.

In one embodiment, the mRNA contains one or more modified nucleosides selected from the group consisting of the following: pseudouridine, pyridin-4-one ribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseudouridine . -taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thiouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropsevduridine, 2-thio-dihydrouridine, 2-thio-dihydropsevduridine, 2-methoxyuridine, 2-methoxy-4- thiouridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisoisitidine, pyrrolocytide in, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1- methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methylzebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4- methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-azaadenine, 7-deaza-2-aminopurine, 7-deaza- 8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl ) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, inosine, 1-methylinosine, viosine, vibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaz a-guanosine, 6-thio-7-deaza-8-azaguanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2,N2-dimethyl-6-thio-guanosine.

In one embodiment, the mRNA contains one or more modified nucleosides selected from the group consisting of the following: pseudouridine, pyridin-4-one-ribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio- pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyluridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1-taurinomethyl-4-thiouridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-tidihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxy-pseudouridine and 4-methoxy-2-thio-pseudouridine.

In one embodiment, the mRNA contains one or more modified nucleosides selected from the group consisting of the following: 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl- pseudoisocytidine, pyrrolocytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza- pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methylzebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxycytidine, 2-methoxy-5- methylcytidine, 4-methoxy-pseudoisocytidine and 4-methoxy-1-methylpseudoisocytidine.

In one embodiment, the mRNA contains one or more modified nucleosides selected from the group consisting of the following: 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza- 2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6, N6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine and 2-methoxyadenine.

In one embodiment, the mRNA contains one or more modified nucleosides selected from the group consisting of the following: inosine, 1-methylinosine, viosine, vibutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-azaguanosine, 7-methylguanosine, 6-thio-7-methylguanosine, 7-methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2- methylguanosine, N2, N2-dimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine and N2, N2-dimethyl-6-thio- guanosine.

In one embodiment, the mRNA contains one or more pseudouridines, one or more 5-methylcytosines, and/or one or more 5-methylcytidines.

In one embodiment, the mRNA contains one or more pseudouridines.

In one embodiment, the mRNA contains one or more 5-methylcytidines.

In one embodiment, the mRNA contains one or more 5-methylcytosines.

In particular embodiments, the mRNA of this application contains a poly(A) tail to help protect the mRNA from exonuclease degradation, stabilize the mRNA, and facilitate translation. In some embodiments, the mRNA contains a 3' poly(A) tail structure.

In particular embodiments, the length of the poly(A) tail is at least about 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, or at least about 500 or more adenine nucleotides, or any intermediate number of adenine nucleotides. In particular embodiments, the length of the poly(A) tail is at least about 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192 193 194 195 196 197 198 199 200 201 202 202 203 205 206 207 208 209 210 211 212 213 214 215 216 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274 or 275 or more adenine nucleotides.

In particular embodiments, the length of the poly(A) tail is from about 10 to about 500 adenine nucleotides, from about 50 to about 500 adenine nucleotides, from about 100 to about 500 adenine nucleotides, from about 150 to about 500 adenine nucleotides, from about 200 to about 500 adenine nucleotides, from about 250 to about 500 adenine nucleotides, from about 300 to about 500 adenine nucleotides, from about 50 to about 450 adenine nucleotides, from about 50 to about 400 adenine nucleotides, from about 100 to about 450 adenine nucleotides, about 100 to about 400 adenine nucleotides, about 100 to about 350 adenine nucleotides, about 100 to about 300 adenine nucleotides, about 150 to about 500 adenine nucleotides, about 150 to about 450 adenine nucleotides, about 150 to about 400 adenine nucleotides, about 150 to about 350 adenine nucleotides, about 150 to about 300 adenine nucleotides, about 150 to about 250 adenine nucleotides, from about 150 to about 200 adenine nucleotides, about 200 to about 500 adenine nucleotides, about 200 to about 450 adenine nucleotides, about 200 to about 400 adenine nucleotides, about 200 to about 350 adenine nucleotides, about 200 to about 300 adenine nucleotides, from about 250 to about 500 adenine nucleotides, about 250 to about 450 adenine nucleotides, about 250 to about 400 adenine nucleotides, about 250 to about 350 adenine nucleotides otides or from about 250 to about 300 adenine nucleotides or any intermediate range of adenine nucleotides.

Terms that describe the orientation of polynucleotides include: "5'" (usually the end of the polynucleotide having a free phosphate group) and "3'" (usually the end of the polynucleotide having a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in the "5' to 3'" or "3' to 5'" orientation. For DNA and mRNA, the 5' to 3' strand is referred to as the "sense", "plus" or "coding" strand because its sequence is identical to that of the pre-mRNA [except for uracil (U) in RNA instead of thymine (T) in DNA ]. For DNA and mRNA, the 3' to 5' complementary strand, which is the strand transcribed by RNA polymerase, is referred to as "template", "antisense", "minus", or "non-coding" strand. As used herein, the term "reverse orientation" refers to a 5' to 3' sequence written in a 3' to 5' orientation, or a 3' to 5' sequence written in a 5' to 3' orientation.

The terms "complementary" and "complementarity" refer to polynucleotides (ie, sequences of nucleotides) linked by base pairing rules. For example, the complementary strand for the DNA sequence 5' A G T C A T G 3' would be 3' T C A G T A C 5'. The last sequence is often written as a reverse complement with a 5' end on the left and a 3' end on the right, 5' C A T G A C T 3'. A sequence that is identical to its reverse complement is called a palindromic sequence. Complementarity can be "partial" in which only some nucleic acid bases match according to base pairing rules. Or there can be "complete" complementarity between nucleic acids.

As used herein, the term "nucleic acid cassette" or "expression cassette" refers to genetic sequences within a vector that can express an RNA and then a polypeptide. In one embodiment, the nucleic acid cassette contains the gene(s) of interest, eg, the polynucleotide(s) of interest. In another embodiment, the nucleic acid cassette contains one or more expression control sequences, eg, a promoter, an enhancer, a poly(A) sequence, and a gene(s) of interest, eg, a polynucleotide(s) of interest. The vectors may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more nucleic acid cassettes. The nucleic acid cassette is positionally and sequentially oriented within the vector so that the nucleic acid in the cassette can be transcribed into RNA and, if necessary, translated into a protein or polypeptide, undergo the appropriate post-translational modifications necessary for activity in the transformed cell, and be moved to the appropriate compartment for biological activity by targeting appropriate intracellular compartments or secretions into extracellular compartments. Preferably, the cassette has 3' and 5' ends adapted for ready insertion into the vector, for example, it has restriction endonuclease sites at each end. In a preferred embodiment, the nucleic acid cassette contains a therapeutic gene sequence used to treat, prevent, or ameliorate a genetic disorder. The cassette can be removed and inserted into a plasmid or viral vector as a whole.

Polynucleotides include the polynucleotide(s) of interest. The term "polynucleotide of interest" as used herein refers to a polynucleotide encoding a polypeptide or a fusion polypeptide or a polynucleotide that serves as a template for transcription of an inhibitory polynucleotide as provided herein.

In addition, those skilled in the art will appreciate that, as a result of the degeneracy of the genetic code, there are many nucleotide sequences that can encode a polypeptide or variant fragment thereof, as contemplated herein. Some of these polynucleotides share minimal homology with the nucleotide sequence of any native gene. However, polynucleotides that vary due to differences in codon usage are specifically contemplated in particular embodiments, eg, polynucleotides that are optimized for human and/or primate codon selection. In one embodiment, polynucleotides containing specific allelic sequences are provided. Alleles are endogenous polynucleotide sequences that change as a result of one or more mutations such as deletions, additions and/or substitutions of nucleotides.

In a particular embodiment, the polynucleotide of interest comprises a repair donor template.

In a particular embodiment, the polynucleotide of interest comprises an inhibitory polynucleotide, including, but not limited to, a siRNA, siRNA, shRNA, ribozyme, or other inhibitory RNA.

In one embodiment, the inhibitory RNA-containing repair donor template contains one or more regulatory sequences such as, for example, a strong constitutive pol III, such as the human or mouse U6 nRNA promoter, the human and mouse RNA H1 promoter, or the human tRNA-val promoter. or a strong constitutive pol II promoter as described elsewhere herein.

Polynucleotides considered in particular embodiments, regardless of the length of the coding sequence itself, can be combined with other DNA sequences such as promoters and/or enhancers, untranslated regions (UTRs), Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites , internal ribosome entry sites (IRES), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, post-transcriptional response elements, and polynucleotides encoding a self-cleaving polypeptide, epitope tags described elsewhere in the present application or as known in the art, so that their overall length can vary considerably. Therefore, in particular embodiments, it is contemplated that a polynucleotide fragment of substantially any length can be used, the overall length being preferably limited by ease of preparation and use in the intended recombinant DNA protocol.

Polynucleotides can be generated, processed, expressed and/or delivered using any of a variety of well known methods known and available in the art. To express the desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into an appropriate vector. The desired polypeptide can also be expressed by delivering mRNA encoding the polypeptide into the cell.

Illustrative examples of vectors include, among others, a plasmid, autonomously replicating sequences, and transposable elements, eg Sleeping Beauty, PiggyBac.

Additional illustrative examples of vectors include, without limitation, plasmids, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC), bacteriophages such as lambda phage or M13 phage and animal viruses.

Illustrative examples of viruses useful as vectors include, without limitation, retrovirus (including lentivirus), adenovirus, adeno-associated virus, herpesvirus (eg, herpes simplex virus), poxvirus, baculovirus, papillomavirus, and papovavirus (eg, SV40).

Illustrative examples of expression vectors include, inter alia, pClneo (Promega) vectors for expression in mammalian cells; pLenti4/V5-DEST™, pLenti6/V5-DEST™ and pLenti6.2/V5-GW/lacZ (Invitrogen) for lentivirus-mediated gene transfer and expression in mammalian cells. In particular embodiments, the coding sequences for the polypeptides disclosed herein may be ligated into such expression vectors for expression of the polypeptides in mammalian cells.

In particular embodiments, the vector is an episomal vector or a vector that is supported extrachromosomally. As used herein, the term "episomal" refers to a vector that is capable of replicating without integration into the host's chromosomal DNA and without gradual loss from the dividing host cell, and also means that said vector replicates extrachromosomal or episomal.

"Expression control sequences", "control elements", or "regulatory sequences" present in an expression vector are the non-translated regions of the vector: source of replication, selection cassettes, promoters, enhancers, translation initiation signal introns (Shine Dalgarno sequence or Kozak sequence) , post-transcriptional regulatory elements, polyadenylation sequence, 5'- and 3'-untranslated regions that interact with host cell proteins to effect transcription and translation. Such elements may differ in their strength and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements may be used, including ubiquitous promoters and inducible promoters.

In particular embodiments, the polynucleotide comprises a vector, including but not limited to expression vectors and viral vectors. The vector may contain one or more exogenous, endogenous or heterologous control sequences such as promoters and/or enhancers. An "endogenous control sequence" is a sequence that is naturally associated with a given gene in the genome. An "exogenous control sequence" is a sequence that is placed into alignment with a gene through genetic manipulation (ie, molecular biological techniques) such that transcription of said gene is directed by an associated enhancer/promoter. A "heterologous control sequence" is an exogenous sequence that belongs to a different species than the genetically manipulated cell. A "synthetic" control sequence may contain elements of yet another endogenous and/or exogenous sequence and/or in vitro or in silico determined sequences that provide optimal promoter and/or enhancer activity for a particular therapy.

As used herein, the term "promoter" refers to a polynucleotide recognition site (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes polynucleotides operably linked to a promoter. In particular embodiments, promoters functional in mammalian cells comprise an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated and/or another sequence located 70 to 80 bases upstream from the start of transcription, CNCAAT region, where N can be any nucleotide.

The term "enhancer" refers to a segment of DNA that contains sequences capable of providing enhanced transcription, and in some cases capable of functioning independently of their orientation relative to another control sequence. The enhancer may function in conjunction with or in addition to promoters and/or other enhancer elements. The term "promoter/enhancer" refers to a segment of DNA that contains sequences capable of providing both promoter and enhancer functions.

The term "operably linked" refers to an association in which the described components are in a relationship that allows them to function as intended. In one embodiment, the term refers to a functional relationship between a nucleic acid expression control sequence (such as a promoter and/or enhancer) and a second polynucleotide sequence, e.g., a polynucleotide of interest, when the expression control sequence directs transcription of expression of the nucleic acid corresponding to the second sequence.

As used herein, the term "constitutive expression control sequence" refers to a promoter, enhancer, or promoter/enhancer that sustainably or continuously transcribes an operably linked sequence. A constitutive expression control sequence can be a "ubiquitous" promoter, enhancer, or promoter/enhancer that allows for expression in a wide variety of cell types and tissues, or "cell specific", "cell type specific", "cell line specific" or " "tissue-specific" promoter, enhancer or promoter/enhancer that allows expression in a limited variety of cell types and tissues, respectively.

Illustrative ubiquitary expression control sequences suitable for use in particular embodiments include, but are not limited to, cytomegalovirus (CMV) immediate-early promoter, simian virus 40 (SV40) promoter (e.g., early or late), Moloney murine leukemia virus LTR promoter ( MoMLV), Rous sarcoma virus (RSV) LTR promoter, Herpes simplex virus (HSV) promoter (thymidine kinase), H5, P7.5 and P11 promoters from vaccinia virus, short elongation factor 1-alpha (EF1a-short) promoter, long elongation factor 1-alpha (EF1a-long), early growth response 1 (EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), protein heat shock protein 5 70 kDa (HSPA5), heat shock protein beta 90 kDa member 1 (HSP90B1), heat shock protein 70 kDa (HSP70), β-kinesin (β-KIN), human ROSA 26k locus (Irions et al., Nature Biotechnology 25, 1477-1482 (2007)), about ubiquitin C motor (UBC), phosphoglycerate kinase-1 (PGK) promoter, chicken cytomegalovirus/β-actin (CAG) enhancer promoter, β-actin promoter, and myeloproliferative sarcoma virus enhancer promoter, with negative control site removed, with primer binding site substituted dl587rev (MND) (Challita et al., J Virol. 69(2):748-55 (1995)).

In a particular embodiment, it may be desirable to use a cell, cell type, cell line, or tissue specific expression control sequence to achieve cell type, lineage, or tissue specific expression of the desired polynucleotide sequence (e.g., to express a particular nucleic acid encoding polypeptide only in a certain subset of cell types, cell lines or tissues, or at certain developmental stages).

"Conditional expression" in the present application may refer to any type of conditional expression, including, but not limited to, inducible expression; repressible expression; expression in cells or tissues having a specific physiological, biological or disease state, etc. This definition is not intended to exclude cell or tissue type specific expression. Some embodiments provide for conditional expression of the polynucleotide in question, for example, expression is controlled by acting on a cell, tissue, organism, and so on. a treatment or condition that causes the expression of a polynucleotide or that causes an increase or decrease in the expression of a polynucleotide encoded by the polynucleotide in question.

Illustrative examples of inducible promoters/systems include, among others, steroid-inducible promoters such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the appropriate hormone), metallothionein promoter (inducible by treatment with various heavy metals), MX-1 promoter (inducible interferon), mifepristone-regulated GeneSwitch system (Sirin et al., 2003, Gene, 323:67), cumat-induced gene switch (WO 2002/088346), tetracycline-dependent regulatory systems, etc.

Conditional expression can also be achieved using site-specific DNA recombinase. In some embodiments, the polynucleotides contain at least one (typically two) site(s) for recombination mediated by a site-specific recombinase. As used herein, the terms "recombinase" or "site-specific recombinase" include excisive or integrative proteins, enzymes, cofactors, or associated proteins that participate in recombination reactions involving one or more recombination sites (e.g., two, three, four , five, six, seven, eight, nine, ten, or more), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3: 699-707 (1993)) or mutants, derivatives (e.g., fusion proteins, containing sequences of recombination proteins or their fragments), fragments and their variants. Illustrative examples of recombinases suitable for use in private embodiments include, among others: Cre, Int, IHF, Xis, Flp, Fis, Hin, Gin, C31, Cin, Tn3 resolvase, TndX, XerC, XerD, TnpX, Hjc, Gin , SpCCE1 and ParA.

Polynucleotides may contain one or more recombination sites for any of a variety of site-specific recombinases. It should be understood that the target site for a site-specific recombinase is in addition to any site(s) required for vector integration, eg a retroviral vector or a lentiviral vector. As used herein, the terms "recombination sequence", "recombination site", or "site-specific recombination site" refer to a particular nucleotide sequence that the recombinase recognizes and binds to.

For example, one recombination site for Cre recombinase is loxP, which is a 34 base pair sequence containing two 13 base pair inverted repeats (serving as recombinase binding sites) flanking an 8 base pair core sequence (see Figure 1 from Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, among others: lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995) and lox66 (Albert et al., 1995).

Suitable recognition sites for FLP recombinase include, among others: FRT (McLeod, et al., 1996), F _1, F _2, F ₃ (Schlake and Bode, 1994), F _4, F ₅ (Schlake and Bode, 1994) , FRT(LE) (Senecoff et al., 1988), FRT(RE) (Senecoff et al., 1988).

Other examples of recognition sequences are the attB, attP, attL and attR sequences, which are recognized by the λ-integrase recombinase enzyme, eg phi-c31. The φC31 SSR allows recombination only between the heterotypic sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). attB and attP, named after phage integrase attachment sites on the bacterial and phage genomes, respectively, both contain imperfect inverted repeats that are probably linked by φC31 homodimers (Groth et al., 2000). The product sites, attL and attR, are effectively inert to further φC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. It was found that, to catalyze insertion, attB-carrying DNA is inserted into the attP genomic site more easily than the attP site into the attB genomic site (Thyagarajan et al., 2001; Belteki et al., 2003). Thus, exemplary strategies place, by homologous recombination, an attP-carrying "docking site" into a specific locus, which is then fused to an incoming attB-carrying sequence for insertion.

In one embodiment, the polynucleotide of this application comprises a polynucleotide repair donor template flanked by a pair of recombinase recognition sites. in particular embodiments, the repair template polynucleotide is flanked by LoxP sites, FRT sites, or att sites.

In private embodiments, the polynucleotides contemplated in this application include one or more polynucleotides of interest that encode one or more polypeptides. In particular embodiments, in order to achieve efficient translation of each of the plurality of polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding a self-cleaving polypeptide.

As used herein, the term "internal ribosome entry site" or "IRES" refers to an element that facilitates direct internal entry of the ribosome into an initiation codon, such as ATG, of a cistron (protein-coding region), thereby leading to cap-independent gene translation . See, for example, Jackson et al., 1990. Trends Biochem Sci 15 (12): 477-83) and Jackson and Kaminski. 1995. RNA 1(10): 985-1000. Examples of IRES commonly used by those skilled in the art include those described in US Patent No. 6,692,736. Additional examples of "IRES" known in the art include, among others, IRES derived from picornavirus (Jackson et al., 1990 ), and IRES derived from viral or cellular mRNA sources such as, for example, immunoglobulin heavy chain binding protein (BiP), vascular endothelial growth factor (VEGF) (Huez et al. 1998. Mol. Cell. Biol. 18 ( 11): 6178-6190), fibroblast growth factor 2 (FGF-2) and insulin-like growth factor (IGFII), translation initiation factor eIF4G and yeast transcription factors TFIID and HAP4, encephalomycarditis virus (EMCV) marketed by Novagen (Duke et al., 1992. J. Virol 66 (3): 1602-9) and VEGF IRES (Huez et al., 1998. Mol Cell Biol 18 (11): 6178-90). IRES have also been reported in the viral genomes of Picornaviridae, Dicistroviridae and Flaviviridae species and in HCV, Friend mouse leukemia virus (FrMLV) and Moloney mouse leukemia virus (MoMLV).

In one embodiment, the implementation of the IRES used in the polynucleotides discussed in this application is an EMCV IRES.

In particular embodiments, the polynucleotides comprise polynucleotides having a Kozak consensus sequence and encoding the desired polypeptide. As used herein, the term "Kozak sequence" refers to a short nucleotide sequence that greatly facilitates the initial binding of an mRNA to a small subunit of the ribosome and increases translation. Kozak's consensus sequence is (GCC) RCCATGG (SEQ ID NO: 105) where R is a purine (A or G) (Kozak, 1986. Cell. 44(2):283-92 and Kozak, 1987. Nucleic Acids Res 15(20):8125-48).

Elements directing efficient termination and polyadenylation of heterologous nucleic acid transcripts increase the expression of heterologous genes. Transcription termination signals are usually in the forward direction from the polyadenylation signal. In particular embodiments, the vectors contain a 3' polyadenylation sequence of a polynucleotide encoding a polypeptide to be expressed. As used herein, the terms "polyA site", "polyA sequence", "poly(A) site", or "poly(A) sequence" refer to a DNA sequence that directs both termination and polyadenylation of a nascent RNA transcript by RNA- polymerase II. Polyadenylation sequences can contribute to mRNA stability by adding a poly(A) tail to the 3' end of the coding sequence and thus improve translation efficiency. Efficient polyadenylation of a recombinant transcript is desirable because transcripts lacking a poly(A) tail are unstable and rapidly degrade. , ATTAAA, AGTAAA), bovine growth hormone poly(A) sequence (BGHpA), rabbit β-globin poly(A) sequence (rβgpA), or other suitable heterologous or endogenous poly(A) sequence known in the art. technology.

In some embodiments, the polynucleotide, or the cell carrying the polynucleotide, uses a suicide gene, including an inducible suicide gene, to reduce the risk of direct toxicity and/or uncontrolled proliferation. In particular embodiments, the suicide gene is not immunogenic to the host carrying said polynucleotide or cell. A specific example of a suicide gene that can be used is caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 can be activated using a special chemical dimerization inducer (CID).

In some aspects, the polynucleotides contain gene segments that render the genetically modified cells of the present application susceptible to negative selection in vivo. The term "negative selection" refers to an infused cell that can be eliminated as a result of a change in the in vivo state of the individual. A negative selectable phenotype may result from the insertion of a gene that confers sensitivity to the administered agent, such as a compound. Negative selection genes are known in the art and include, among others: the herpes simplex virus type I (HSV-I TK) thymidine kinase gene, which confers sensitivity to ganciclovir; a cellular hypoxanthine phosphoribosyl transferase (HPRT) gene, a cellular adenine phosphoribosyl transferase (APRT) gene, and a bacterial cytosine deaminase gene.

In some embodiments, the genetically modified cells comprise a polynucleotide further containing a positive marker that allows selection of cells with a negative selectable phenotype in vitro. A positive selectable marker may be a gene which, when introduced into a host cell, expresses a dominant phenotype allowing positive selection of cells carrying said gene. Genes of this type are known in the art and include, inter alia, the hygromycin B phosphotransferase (hph) gene which confers resistance to hygromycin B, the aminoglycoside phosphotransferase (neo or aph) gene from Tn5 which encodes resistance to the antibiotic G418, the gene dihydrofolate reductase (DHFR), adenosine deaminase (ADA) gene and multidrug resistance (MDR) gene.

In one embodiment, the positive selectable marker and the negative selectable element are linked such that the loss of the negative selectable element is necessarily also accompanied by the loss of the positive selectable marker. In a particular embodiment, the positive and negative selectable markers are fused such that the loss of one necessarily leads to the loss of the other. An example of a fusion polynucleotide that yields as an expression product a polypeptide that has the desired positive and negative selection features described above is the thymidine kinase hygromycin phosphotransferase (HyTK) fusion gene. Expression of this gene produces a polypeptide that confers resistance to hygromycin B for positive selection in vitro and sensitivity to ganciclovir for negative selection in vivo. See also PCT Publications US91/08442 and PCT/US94/05601 by S.D. Lupton describing the use of bifunctional selectable fusion genes resulting from the fusion of dominant positive selectable markers with negative selectable markers.

Preferred positive selectable markers are from genes selected from the group consisting of hph, nco and gpt and preferred negative selectable markers are from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT and gpt. Exemplary bifunctional selectable fusion genes contemplated in particular embodiments include, inter alia, genes in which the positive selectable marker is derived from hph or neo and the negative selectable marker is derived from cytosine deaminase or the TK gene or selectable marker.

In particular embodiments, polynucleotides encoding one or more variants of nucleases, megaTALs, end-reversing enzymes, or fusion polypeptides can be introduced into hematopoietic cells, such as T cells, by both non-viral and viral methods. In particular embodiments, delivery of one or more nuclease-encoding polynucleotides and/or repair donor template may be provided by the same or different methods and/or the same vector or different vectors.

The term "vector" is used in this application to refer to a nucleic acid molecule capable of transferring or transporting another nucleic acid molecule. The transferred nucleic acid is typically associated with, for example, embedded in, the nucleic acid vector molecule. The vector may include sequences that direct autonomous replication in the cell, or may include sequences sufficient to integrate into the DNA of the host cell. In private embodiments, non-viral vectors are used to deliver one or more of the polynucleotides contemplated herein to a T cell.

Illustrative examples of non-viral vectors include, among others, plasmids (eg, DNA plasmids or RNA plasmids), transposons, cosmids, and bacterial artificial chromosomes.

Exemplary methods for non-viral delivery of polynucleotides contemplated in particular embodiments include, but are not limited to: electroporation, sonoporation, lipofection, microinjection, biolisting, virosomes, liposomes, immunoliposomes, nanoparticles, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, DEAE-dextran-mediated transfer, gene gun and heat shock.

Illustrative examples of polynucleotide delivery systems suitable for use in the particular embodiments contemplated herein include those provided by Amaxa Biosystems, Maxcyte, Inc., BTX Molecular Delivery Systems, ThermoFisher Scientific, and Copernicus Therapeutics Inc., among others. Lipofection reagents are commercially available (eg Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient lipofection of polynucleotides with receptor recognition are described in the literature. See, for example, Liu et al. (2003) Gene Therapy. 10:180-187; and Balazs et al. (2011) Journal of Drug Delivery. 2011:1-12. In particular embodiments, antibody-targeted delivery based on non-living nanocells derived from bacteria is also contemplated.

Viral vectors containing polynucleotides contemplated in particular embodiments can be delivered in vivo by administration to an individual patient, usually by systemic administration (eg, intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial infusion) or topical application, as described below. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., mobilized peripheral blood, lymphocytes, bone marrow aspirates, tissue biopsy, etc.), or universal donor hematopoietic stem cells, followed by reimplantation cells in the patient.

In one embodiment, viral vectors containing nuclease variants and/or a repair donor template are administered directly to an organism for in vivo cell transduction. Alternatively, you can enter naked DNA. Administration is by any route commonly used to bring the molecule into final contact with blood or tissue cells, including, but not limited to, injection, infusion, topical application, and electroporation. Suitable routes for administering such nucleic acids are available and well known to those skilled in the art, and while more than one route may be used to administer a particular composition, a particular route can often provide a more immediate and more efficient response than another route.

Illustrative examples of viral vector systems suitable for use in the particular embodiments contemplated herein include, among others, adeno-associated virus (AAV), retrovirus, herpes simplex virus, adenovirus, and vaccinia vectors.

In various embodiments, one or more polynucleotides encoding a nuclease variant and/or a repair donor template is introduced into a hematopoietic cell, such as a T cell, by transducing the cell with a recombinant adeno-associated virus (rAAV) containing one or more polynucleotides.

AAV is a small (~26 nm) replication-defective, mostly episomal, non-enveloped virus. AAV can infect both dividing and non-dividing cells and can integrate its genome into the host cell's genome. Recombinant AAVs (rAAVs) typically consist of at least a transgene and its regulatory sequences, as well as AAV 5' and 3' inverted terminal repeats (ITRs). The ITR sequences are approximately 145 bp in length. rAAV vectors containing two ITRs have a payload of approximately 4.4 kb.

Self-complementary rAAV vectors contain a third ITR and pack two strands of the recombinant portion of the vector, leaving only about 2.1 kb for the polynucleotides contemplated here. In one embodiment, the AAV vector is an scAAV vector.

Expanded packaging capabilities that are approximately double the packaging capacity of rAAV (approximately 9 kb) have been achieved using rAAV dual vector strategies. Dual vector strategies useful for obtaining rAAV, discussed in this application include, among others, splicing (trans-splicing), homologous recombination (overlap) or a combination of these two strategies (hybrid). In the double AAV trans splicing strategy, the splicing donor (SD) signal is placed at the 3' end of the 5' half vector and the splicing acceptor (SA) signal is placed at the 5' end of the 3' half vector. When coinfecting the same cell with dual AAV vectors and head-to-tail concatemerization of the two halves mediated by inverted terminal repeat (ITR), trans-splicing results in the formation of mature mRNA and full-length protein (Yan et al, 2000). Transplication has been successfully used to express large genes in muscle and retina (Reich et al, 2003; Lai et al, 2005). Alternatively, the two halves of the large transgene expression cassette contained in the twin AAV vectors may contain homologous overlapping sequences (at the 3'end of the 5'half vector and at the 5'end of the 3'half vector, the overlap of the double AAVs) such that will promote the restoration of one large genome by homologous recombination (Duan et al, 2001). This strategy depends on the recombinogenic properties of transgenic overlapping sequences (Ghosh et al, 2006). A third double AAV strategy (hybrid) is based on the addition of a highly recombinogenic region from an exogenous gene (ie, alkaline phosphatase; Ghosh et al., 2008, Ghosh et al, 2011)) to trans-splicing vectors. This added region is located upstream of the SD signal in the 5' half vector and upstream of the S A signal in the 3' half vector to increase recombination between double AAVs.

The terms "hybrid AAV", "hybrid rAAV", "chimeric AAV", or "chimeric rAAV" refer to the rAAV genome packaged with the capsid of another AAV serotype (and preferably another serotype from one or more AAV ITRs), which may otherwise referred to as pseudo-typed rAAV. For example, the rAAV type 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 genome can be encapsidated into an AAV type 1, 2, 3, 4 capsid. , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16, or a variant thereof, provided that the AAV capsid and genome (and preferably one or more AAV ITRs)) are different serotypes. In some cases, a pseudotyped rAAV particle can be designated as type "x/y", where "x" indicates the source of the ITR and "y" indicates the capsid serotype, for example, 2/5 rAAV. the particle has an ITR from AAV2 and a capsid from AAV6.

In particular embodiments, rAAV contains ITRs and capsid sequences derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, AAV13, AAV 14, AAV15, and AAV16.

In some embodiments, a chimeric rAAV is used, where the ITR sequences are derived from one AAV serotype and the capsid sequences are derived from another AAV serotype. For example, rAAV with ITR sequences derived from AAV2 and capsid sequences derived from AAV6 are referred to as AAV2/AAV6. In particular embodiments, the rAAV vector may comprise ITRs from AAV2 and capsid proteins from any of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, or AAV10. In a preferred embodiment, the rAAV contains ITR sequences derived from AAV2 and capsid sequences derived from AAV6. In a preferred embodiment, the rAAV contains ITR sequences derived from AAV2 and capsid sequences derived from AAV2.

In some embodiments, engineered and breeding techniques can be applied to AAV capsids to make them more capable of transducing the cells of interest.

The construction of rAAV vectors, their production and purification are described, for example, in US patent No. 9169494; 9169492; 9012224; 8889641; 8809058; and 8,784,999, each of which is incorporated herein by reference in its entirety.

In various embodiments, one or more polynucleotides encoding a nuclease variant and/or a repair donor template are introduced into a hematopoietic cell by transducing the cell with a retrovirus, such as a lentivirus, containing one or more polynucleotides.

As used herein, the term "retrovirus" refers to an RNA virus that reverse-transcribes its genomic RNA into a linear double-stranded DNA copy and subsequently covalently integrates its genomic DNA into the host genome. Illustrative retroviruses suitable for use in particular embodiments include, but are not limited to: Moloney mouse leukemia virus (M-MuLV), Moloney mouse sarcoma virus (MoMSV), Harvey mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV) , gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, Friend mouse leukemia virus, mouse stem cell virus (MSCV) and Rous sarcoma virus (RSV) and lentivirus.

Used in this application, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, among others: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); visna maedi virus (VMV); ovine and goat arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). In one embodiment, HIV-based vector backbone chains (ie, HIV cis-acting sequence elements) are preferred.

In various embodiments, the lentiviral vector discussed herein contains one or more LTRs and one or more or all of the following accessory elements: cPPT/FLAP, Psi (Ψ) packing signal, export element, poly(A) sequences, and may, optionally, contain a WPRE or HPRE, an insulator element, a selectable marker and a cell suicide gene, as discussed elsewhere in this application.

In particular embodiments, the lentiviral vectors contemplated herein may be an integrating or non-integrating or integration-defective lentivirus. As used herein, the term "integration-defective lentivirus" or "IDLV" refers to a lentivirus having an integrase that does not have the ability to integrate the viral genome into the genome of host cells. Integration-incompetent viral vectors have been described in patent application WO 2006/010834, which is incorporated herein by reference in its entirety.

Illustrative mutations in the HIV-1 pol gene suitable for reducing integrase activity include, among others: H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A E87A, D116N, D1161, D116A, N120G, N120G, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K160A, R1667A, E170A, E173AA, K186T, K173AA, K186T, K173AA, K186T, K173AA, K186T, K173AA, K186T, K173AA, K186T .

In one embodiment, the HIV-1 integrase-deficient pol gene contains the D64V, D116I, D116A, E152G, or E152A mutation; mutations D64V, D116I and E152G; or mutations D64V, D116A and E152A.

In one embodiment, the HIV-1 integrase-deficient pol gene contains the D64V mutation.

The term "long terminal repeat (LTR)" refers to the base pair domains located at the ends of retroviral DNA, which in the context of their natural sequence are direct repeats and contain regions U3, R and U5.

As used herein, the term "FLAP element" or "cPPT/FLAP" refers to a nucleic acid whose sequence includes the central polypurine tract and central terminal sequences (cPPT and CTS) of a retrovirus, such as HIV-1 or HIV-2. Suitable FLAP elements are described in US Pat. No. 6,682,907 and Zennou et al., 2000, Cell, 101:173. In another embodiment, the lentiviral vector contains a FLAP element with one or more mutations in the cPPT and/or CTS elements. In yet another embodiment, the lentiviral vector contains either a cPPT or a CTS element. In another embodiment, the lentiviral vector does not contain a cPPT or a CTS element.

As used herein, the term "packaging signal" or "packing sequence" refers to the psi [Ψ] sequences located in the retroviral genome that are required for the insertion of viral RNA into a viral capsid or particle, see, for example, Clever et al., 1995. J. of Virology, Vol.69, No. four; pp.2101-2109.

The term "export element" refers to a cis-acting post-transcriptional regulatory element that regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, among others, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65:1053; and Cullen et al., 1991. Cell 58: 423), and the hepatitis B virus post-transcriptional regulatory element (HPRE).

In particular embodiments, the expression of heterologous sequences in viral vectors is increased by inserting post-transcriptional regulatory elements, effective polyadenylation sites, and optionally transcription termination signals into the vectors. A variety of post-transcriptional regulatory elements can increase the expression of a heterologous nucleic acid in a protein, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and the like (Liu et al., 1995, Genes Dev., 9:1766).

Lentiviral vectors preferably contain several safety improvements as a result of the LTR modification. The term "self-inactivating" (SIN) vectors refers to replication-defective vectors, e.g., in which the right (3') region of the LTR enhancer promoter, known as the U3 region, has been modified (e.g., by deletion or substitution) to prevent viral transcription after first round of viral replication. An additional increase in safety is provided by replacing the U3 region of the 5'-LTR with a heterologous promoter to drive transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that can be used include, for example, simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney mouse leukemia virus (MoMLV), Rous sarcoma virus (RSV) and herpes simplex virus (HSV) (thymidine kinase).

As used herein, the terms "pseudotype" or "pseudotyping" refer to a virus whose viral envelope proteins have been replaced with proteins from another virus that have preferred characteristics. For example, HIV can be pseudotyped by vesicular stomatitis virus G protein (VSV-G) envelope proteins, which allow HIV to infect a wider range of cells, since HIV envelope proteins (encoded by the env gene) typically target the virus to CD4 ⁺ presenting cells.

In some cases, lentiviral vectors are prepared according to known methods. See, for example, Kutner et al., BMC Biotechnol. 2009;9:10. doi: 10.1186/1472-6750-9-10; Kutner et al. Nat. Protocol. 2009;4(4):495-505. doi: 10.1038/nprot.2009.22.

In certain specific embodiments contemplated herein, most or all of the viral vector backbone sequences are derived from a lentivirus, such as HIV-1. However, it should be understood that many different sources of retroviral and/or lentiviral sequences can be used or combined, and numerous substitutions and changes can be accommodated in some of the lentiviral sequences without compromising the transfer vector's ability to perform the functions described herein. In addition, many lentiviral vectors are known in the art, see Naldini et al., (1996a, 1996b and 1998); Zufferey et al., (1997); Dull et al., 1998, US Pat. No. 6,013,516; and 5994136, many of which can be adapted to produce the viral vector or transfer plasmid discussed in this application.

In various embodiments, one or more polynucleotides encoding a nuclease variant and/or a repair donor template are introduced into a hematopoietic cell by transducing the cell with an adenovirus containing one or more polynucleotides.

Adenovirus-based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, a high titer and a high level of expression were obtained. The specified vector can be obtained in large quantities in a relatively simple system. Most adenoviral vectors are designed such that the transgene replaces the Ad E1a, E1b and/or E3 genes; subsequently, the replication-deficient vector propagates in human 293 cells that provide the functions of the deleted gene in trans. Adenovirus vectors can transduce various tissue types in vivo, including non-dividing, differentiated cells such as liver, kidney, and muscle cells. Conventional adenoviral vectors can carry a large payload.

When generating and propagating modern adenoviral vectors that have a replication defect, it is possible to use a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells with Ad5 DNA fragments and constitutively expresses E1 proteins (Graham et al., 1977). Because the E3 region can be removed from the adenovirus genome (Jones & Shenk, 1978), existing adenovirus vectors, using 293 cells, carry foreign DNA in the E1, D3 or both regions (Graham & Prevec, 1991). Adenovirus vectors have already been used for eukaryotic gene expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and vaccine development (Grunhaus & Horwitz, 1992; Graham & Prevec, 1992). Studies on the introduction of recombinant adenovirus into various tissues include tracheal instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injection (Herz & Gerard, 1993) and stereotaxic brain inoculation (Le Gal La Salle et al., 1993). An example of the use of an Ad vector in a clinical trial included polynucleotide therapy for antitumor immunization with intramuscular injection (Sterman et al., Hum. Gene Ther. 7:1083-9 (1998)).

In various embodiments, one or more polynucleotides encoding a nuclease variant and/or a repair donor template are introduced into a hematopoietic cell by transducing the cell with a herpes simplex virus, e.g., HSV-1, HSV-2, containing one or more polynucleotides.

The mature HSV virion consists of an enveloped icosahedral capsid with a viral genome consisting of a 152 kb linear double-stranded DNA molecule. In one embodiment, the HSV viral vector is deficient in one or more major or minor HSV genes. In one embodiment, the HSV-based viral vector is replication defective. Most replication-defective HSV vectors contain a deletion of one or more intermediate-early, early, or late HSV genes to prevent replication. For example, the HSV vector may be defective in an immediate early gene selected from the group consisting of: ICP4, ICP22, ICP27, ICP47, and combinations thereof. The advantages of the HSV vector are its ability to enter a latent stage, which can lead to long-term DNA expression, and its large viral DNA genome, which can accommodate exogenous DNA inserts up to 25 kb. HSV-based vectors are described, for example, in US patents. Nos. 5837532, 5846782 and 5844513, as well as in international patent applications WO 91/02788, WO 96/04394, WO 98/15637 and WO 99/06583, each of which is fully incorporated into this description by reference.

F. Genome-edited cells

Genome-edited cells obtained by methods provided in private embodiments contain one or more gene modifications in the PD-1 gene and allow the creation of improved cellular therapeutics for the prevention, treatment, or amelioration of at least one symptom of cancer, GVHD, infectious disease, autoimmune disease, immunodeficiency or a condition associated with them. Without being bound by any particular theory, we believe that the compositions and methods discussed in this application increase the effectiveness of adoptive cell therapy, in particular, making therapeutic cells more resistant to immunosuppressive signals and depletion.

Genome-edited cells considered in particular embodiments may be autologous/autologous ("self") or non-autologous ("non-self", eg, allogeneic, syngeneic, or xenogeneic). The term "autologous" in this application refers to cells from the same subject. The term "allogeneic", as used herein, refers to cells of the same species that are genetically different from the cell being compared. The term "syngeneic" in this application refers to cells of another subject that are genetically identical to the compared cell. The term "xenogenic", in this application, refers to cells of a different kind compared to the compared cell. In preferred embodiments, the cells are obtained from a mammalian subject. In a more preferred embodiment, the cells are obtained from a primate subject, optionally a non-human primate. In the most preferred embodiment, the cells are obtained from a human.

The term "isolated cell" refers to a non-naturally occurring cell, such as a cell that does not exist in nature, a modified cell, an engineered cell, a recombinant cell, etc., that has been obtained from a tissue or organ in vivo and is substantially free of extracellular matrix.

Used in this application, the term "cell population" refers to a set of cells, which may consist of any number and/or combination of homogeneous or heterogeneous cell types, as described elsewhere in this application. For example, for transduction of T cells, a population of cells can be isolated or obtained from peripheral blood. The cell population may contain about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the target cell type for editing. In some embodiments, T cells can be isolated or purified from a population of heterogeneous cells using methods known in the art.

Illustrative examples of cell types whose genome can be edited using the compositions and methods provided in this application include, among others, cell lines, primary cells, stem cells, progenitor cells and differentiated cells, and mixtures thereof.

In a preferred embodiment, genome editing compositions and methods are used to edit hematopoietic cells, more preferably immune cells, and even more preferably T cells.

The terms "T cell" or "T lymphocyte" are recognized in the art and include thymocytes, immune effector cells, regulatory T cells, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T-lymphocytes. The T cell may be a T helper (Th), such as T helper 1 (Th1) or T helper 2 (Th2). The T cell may be a helper T cell (HTL; CD4⁺T cell) CD4⁺T cell, cytotoxic T cell (CTL; CD8⁺T-cell) infiltrating the tumor with a cytotoxic T-cell (TIL; CD8⁺T cell), CD4⁺CD8⁺T cell, CD4-CD8- T cell or any other subgroup of T cells. In one embodiment, the T cell is an immune effector cell. In one embodiment, the T cell is a NKT cell. Other exemplary T cell populations suitable for use in particular embodiments include naive T cells and memory T cells.

In various embodiments, the genome-edited cells comprise immune effector cells containing the PD-1 gene edited by the compositions and methods provided herein. An "immune effector cell" is any cell of the immune system that performs one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). Exemplary immune effector cells contemplated in particular embodiments are T lymphocytes, specifically cytotoxic T cells (CTL; CD8 ⁺ T cells), TILs, and helper T cells (HTL; CD4 ⁺ T cells). In one embodiment, the immune effector cells include natural killer (NK) cells. In one embodiment, the immune effector cells include natural killer T cells (NKT).

"Active T cells" and "young T cells" are used interchangeably in particular embodiments and refer to T cell phenotypes where the T cell is capable of proliferating and concomitantly decreasing differentiation. In particular embodiments, the young T cell has a "naive T cell" phenotype. In particular embodiments, young T cells contain one or more or all of the following biological markers: CD62L, CCR7, CD28, CD27, CD122, CD127, CD197, and CD38. In one embodiment, young T cells contain one or more or all of the following biological markers: CD62L, CD127, CD197, and CD38. In one embodiment, young T cells lack the expression of CD57, CD244, CD160, PD-1, CTLA4, PD-1, and LAG3.

T cells can be obtained from a number of sources including, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors.

In particular embodiments, the cell population, including immune effector cells or T cells, contains an edited PD-1 gene, where the edit is DSB repaired with NHEJ. In particular embodiments, the immune effector cell or T cell contains an edited PD-1 gene, where the edit is DSB repaired by NHEJ. In particular implementations, the edit is an insertion or deletion (INDEL) of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18., 19, 20, 21, 22, 23, 24, 25 or more nucleotides in the PD-1 gene coding sequence, preferably in exon 5, exon 1 or exon 2 of the PD-1 gene, more preferably in SEQ ID NO: 25 (or SEQ ID NO: 27) in exon 5 of the PD-1 gene, in SEQ ID NO: 30 (or SEQ ID NO: 32) in exon 1 of the PD-1 gene, or in SEQ ID NO: 35 (or SEQ ID NO: 37) in exon 2 of the PD-1 gene.

In a particular embodiment, the edit is a deletion of +1, -1, -2, -3 or -4 nucleotides in the coding sequence of the PD-1 gene, preferably in exon 5, exon 1 or exon 2 of the PD-1 gene, more preferably in SEQ ID NO: 25 (or SEQ ID NO: 27) in exon 5 of the PD-1 gene, in SEQ ID NO: 30 (or SEQ ID NO: 32) in exon 1 of the PD-1 gene, or SEQ ID NO: 35 (or SEQ ID NO: 37) in exon 2 of the PD-1 gene.

In particular embodiments, the cell population, including immune effector cells or T cells, contains an edited PD-1 gene containing a repair donor template inserted into a DSB, a repaired HDR.

In particular embodiments, the cell population, including immune effector cells or T cells, contains an edited PD-1 gene containing a donor repair template containing the PD-1 gene or a portion thereof, and is designed to introduce one or more mutations into genomic PD-1 a sequence to alter PD-1 expression or signaling, and preferably to reduce or eliminate PD-1 expression and/or signaling.

In various embodiments, the genome-edited cell comprises an edit in the PD-1 gene and further comprises a polynucleotide encoding the PD-1 flip receptor, a bispecific T cell activator (BiTE) molecule; cytokine (eg IL-2, insulin, IFN-γ, IL-7, IL-21, IL-10, IL-12, IL-15 and TNF-α), chemokine (eg MIP-1α, MIP-1β , MCP-1, MCP-3, and RANTES), a cytotoxin (eg, perforin, granzyme A, and granzyme B), a cytokine receptor (eg, IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-15 receptor, and IL-21 receptor) or an engineered antigen receptor (eg, engineered T cell receptor (TCR), chimeric antigen receptor (CAR), Daric receptor or components thereof, or chimeric cytokine receptor). In one embodiment, a repair donor template containing a polynucleotide and a nuclease variant is inserted into the cell, and the polynucleotide is inserted into the cell's genome at the DSB site in the PD-1 gene by HDR repair. The polynucleotide can also be introduced into the cell at a site other than the PD-1 gene, for example by transducing the cell with a vector containing the polynucleotide.

G. Compositions and formulations

Compositions contemplated in particular embodiments may comprise one or more polypeptides, polynucleotides, vectors containing them, as well as compositions for editing the genome and compositions with a genome-edited cell, as provided in this application. Genome editing compositions and methods contemplated in particular embodiments are useful for editing a target site in the human programmed cell death 1 (PD-1) gene in a cell or population of cells. In preferred embodiments, the genome editing composition is used to edit the PD-1 gene in a hematopoietic cell, such as a T cell or an immune effector cell.

In various embodiments, the compositions contemplated herein comprise a variant nuclease and optionally an end-reshaping enzyme, such as 3'-5' exonuclease (Trex2). The nuclease variant may be in the form of an mRNA that is introduced into the cell using the polynucleotide delivery methods described above, eg, electroporation, lipid nanoparticles, etc. In one embodiment, a composition comprising mRNA encoding a homing endonuclease or megaTAL variant. and optionally a 3'-5' exonuclease is introduced into the cell using the polynucleotide delivery methods described above. Such a composition can be used to create a genome-edited cell or population of genome-edited cells using error-prone NHEJ.

In various embodiments, the compositions contemplated herein comprise a repair donor matrix. The composition can be delivered to a cell that expresses or will express a variant nuclease, and optionally an end-reversing enzyme. In one embodiment, the composition can be delivered to a cell that expresses or will express a variant homing endonuclease or megaTAL and optionally a 3'-5' exonuclease. To create a genome-edited cell or population of genome-edited cells, the expression of gene-editing enzymes in the presence of a donor repair template by HDR can be used.

In particular embodiments, the compositions contemplated herein comprise a population of cells, a nuclease variant, and, optionally, a repair donor template. In private embodiments, the compositions contemplated in this application contain a population of cells, a variant nuclease, an enzyme that changes the structure of the ends and, optionally, a repair donor template. The nuclease variant and/or end-reversing enzyme may be in the form of mRNA that is introduced into the cell using the polynucleotide delivery methods described above.

In particular embodiments, the compositions contemplated herein comprise a population of cells, a homing endonuclease or megaTAL variant, and optionally a repair donor template. In particular embodiments, the compositions contemplated herein comprise a population of cells, a homing endonuclease or megaTAL variant, a 3'-5' exonuclease, and optionally a repair donor template. The homing endonuclease, megaTAL and/or 3'-5' exonuclease variant may be in the form of mRNA that is introduced into the cell using the polynucleotide delivery methods described above.

In private embodiments, the cell population contains genetically modified immune effector cells.

Compositions include, inter alia, pharmaceutical compositions. "Pharmaceutical composition" refers to a composition formulated as pharmaceutically acceptable or physiologically acceptable solutions for administration to a cell or animal, either alone or in combination with one or more other therapies. It should also be understood that, if desired, the compositions may be administered in combination with other agents such as, for example, cytokines, growth factors, hormones, small molecules, chemotherapeutic agents, prodrugs, drugs, antibodies, or others. various pharmaceutically active substances. For other components that can also be included in the compositions, there are practically no restrictions, provided that additional agents do not adversely affect the composition.

The phrase "pharmaceutically acceptable" is used in this application to refer to those compounds, materials, compositions and/or dosage forms that, within the framework of sound medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic response, or other problems. or complications commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or carrier with which therapeutic cells are administered. Illustrative examples of pharmaceutical carriers can be sterile liquids such as cell culture media, water, and oils, including oils of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Saline solutions and aqueous solutions of dextrose and glycerol can also be used as liquid carriers, especially for injectable solutions. Suitable pharmaceutical excipients in particular embodiments include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skimmed milk powder, glycerin, propylene, glycol, water, ethanol and the like. Unless any conventional carrier or agent is incompatible with the active ingredient, its use in such therapeutic compositions is contemplated. Additional active ingredients may also be included in the compositions.

In one embodiment, a composition containing a pharmaceutically acceptable carrier is suitable for administration to a subject. In private embodiments, the composition containing the carrier is suitable for parenteral administration, for example, intravascular (intravenous or intra-arterial), intraperitoneal or intramuscular administration. In private embodiments, the composition containing a pharmaceutically acceptable carrier is suitable for intraventricular, intraspinal, or intrathecal administration. Pharmaceutically acceptable carriers include sterile aqueous solutions, cell culture media or dispersions. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional carrier or agent is incompatible with the transduced cells, its use in such pharmaceutical compositions is contemplated.

In private embodiments, the compositions discussed in this application contain genetically modified T cells and a pharmaceutically acceptable carrier. The composition containing the composition based on the cells considered in this application, you can enter alone by enteral or parenteral routes of administration or in combination with other suitable compounds to achieve the desired goals of treatment.

The pharmaceutically acceptable carrier must be of sufficiently high purity and low enough toxicity to be suitable for administration to the person being treated. In addition, it must maintain or increase the stability of the composition. The pharmaceutically acceptable carrier may be liquid or solid and is selected with regard to the intended route of administration to provide the desired weight, consistency, etc. when combined with other components of the composition. For example, a pharmaceutically acceptable carrier may include, but is not limited to, a binding agent (for example, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.), a bulking agent (for example, lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates, calcium hydrogen phosphate, etc.), lubricant (e.g. magnesium stearate, talc, silicon dioxide, colloidal silicon dioxide, stearic acid, metal stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.) , a disintegrant (eg, sodium starch glycolate, etc.) or a wetting agent (eg, sodium lauryl sulfate, etc.). Other suitable pharmaceutically acceptable carriers for the compositions herein include, inter alia, water, saline solutions, alcohols, polyethylene glycols, gelatins, amyloses, magnesium stearates, talcs, silicic acids, viscous paraffins, hydroxymethylcelluloses, polyvinyl pyrrolidones, and the like.

Such carrier solutions may also contain buffers, diluents and other suitable additives. Used in this application, the term "buffer" refers to a solution or liquid, the chemical composition of which neutralizes acids or bases without a significant change in pH. Examples of buffers contemplated herein include Dulbecco's phosphate buffered saline (PBS), Ringer's solution, 5% dextrose in water (D5W), normal/physiological saline (0.9% NaCl), among others.

Pharmaceutically acceptable carriers may be present in amounts sufficient to maintain the pH of the composition at about 7. Alternatively, the composition has a pH in the range of about 6.8 to about 7.4, such as 6.8, 6.9, 7.0, 7 ,1, 7,2. 7.3 and 7.4. In yet another embodiment, the composition has a pH of about 7.4.

The compositions contemplated in this application may contain a non-toxic pharmaceutically acceptable environment. The compositions may be a suspension. Used in this application, the term "suspension" refers to conditions without sticking, in which the cells are not attached to a solid substrate. For example, cells maintained in suspension can be agitated or agitated without sticking to a support such as a culture dish.

In particular embodiments, the compositions contemplated herein are formulated as a suspension in which genome-edited T cells are dispersed in an acceptable liquid medium or solution, such as saline or serum-free medium, in an intravenous (IV) infusion bag, or the like. similar. Suitable diluents include, among others, water, PlasmaLyte, Ringer's solution, isotonic sodium chloride solution (saline), serum-free cell culture medium, and cryogenic storage medium such as Cryostor® medium.

In some embodiments, the pharmaceutically acceptable carrier is substantially free of naturally occurring proteins of human or animal origin and is suitable for holding a composition containing a population of genome-edited T cells. The therapeutic composition is intended to be administered to a human patient and is therefore substantially free of cell culture components such as bovine serum albumin, equine serum and fetal bovine serum.

In some embodiments, the compositions are formulated in a pharmaceutically acceptable cell culture medium. Such compositions are suitable for administration to human subjects. In particular embodiments, the pharmaceutically acceptable cell culture medium is a serum-free medium.

Serum-free media has several advantages over serum-containing media, including simplified and more well-defined composition, less contamination, elimination of a potential source of infectious agents, and lower cost. In various embodiments, the serum-free medium is animal-free and optionally protein-free. Optionally, the medium may contain biopharmaceutically acceptable recombinant proteins. The term "animal-free" environment refers to an environment in which the components are derived from non-animal sources. In an animal-free environment, recombinant proteins replace native animal proteins and nutrients are obtained from synthetic, plant or microbial sources. "Protein-free" medium, in contrast, is defined as essentially free of protein.

Illustrative examples of serum-free media used in specific formulations include, among others, QBSF-60 (Quality Biological, Inc.), StemPro-34 (Life Technologies), and X-VIVO 10.

In a preferred embodiment, compositions containing genome-edited T cells are formulated in a PlasmaLyte solution.

In various embodiments, compositions containing genome-edited T cells are prepared in a cryopreservative medium. For example, cryopreservation medium with cryopreservation agents can be used to maintain a high yield of viable cells after thawing. Illustrative examples of cryopreservation media used in specific compositions include, among others, CryoStor CS10, CryoStor CS5 and CryoStor CS2.

In one embodiment, the compositions are formulated in a solution containing a 50:50 mixture of PlasmaLyte A with CryoStor CS10.

In particular embodiments, the composition is substantially free of mycoplasma, endotoxin, and microbial contaminants. By "substantially free" in relation to endotoxin, it is meant that there is less endotoxin per dose of cells than is allowed by the FDA for the biologic, i.e. a total amount of endotoxin of 5 EU/kg of body weight per day, which is an average of 350 per person weighing 70 kg. EU per total cell dose. In private embodiments, compositions containing hematopoietic stem or progenitor cells transduced with a retroviral vector of this application contain from about 0.5 EU/ml to about 5.0 EU/ml or about 0.5 EU/ml, 1, 0 EU/ml, 1.5 EU/ml, 2.0 EU/ml, 2.5 EU/ml, 3.0 EU/ml, 3.5 EU/ml, 4.0 EU/ml, 4.5 EU/ml or 5.0 EU/ml.

In some embodiments, compositions and formulations are provided suitable for the delivery of polynucleotides, including, inter alia, one or more mRNAs encoding one or more reprogrammed nucleases, and optionally, end-altering enzymes.

Illustrative formulations for ex vivo delivery may also include the use of various transfection agents known in the art, such as calcium phosphate, electroporation, heat shock, and various liposome formulations (ie, for lipid-mediated transfection). Liposomes, as described in more detail below, are lipid bilayers that capture a fraction of an aqueous liquid. DNA spontaneously binds to the outer surface of cationic liposomes (due to its charge) and these liposomes will interact with the cell membrane.

In particular embodiments, the formulation of pharmaceutically acceptable carrier solutions is well known to those skilled in the art, as is the development of suitable dosing and treatment regimens for use of the specific compositions described herein in a variety of treatment regimens, including, for example, enteral and parenteral, for example, intravascular. , intravenous, intracardiac, intraosseous, intraventricular, intracerebral, intracranial, intraspinal, intrathecal and intramedullary administration and composition. The person skilled in the art will appreciate that the specific embodiments contemplated herein may contain other formulations, such as those well known in the pharmaceutical field and described, for example, in Remington: The Science and Practice of Pharmacy, t .I and vol.II. 22nd edition, edited by Loyd V. Allen Jr. Philadelphia, PA: Pharmaceutical Press; 2012, which is incorporated into this application by reference in its entirety.

H. Treatment methods with genome-edited cells

Genome-edited cells made using the compositions and methods provided herein provide improved drug products for use in the prevention, treatment, or amelioration of at least one symptom of cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease, or immunodeficiency. Used in this application, the term "drug product" refers to genetically modified cells obtained using the compositions and methods provided in this application. In private embodiments, the drug product contains genetically edited immune effector cells or T cells. In addition, genome-edited T cells contemplated in particular embodiments provide a safer and more effective adoptive cell therapy because they are resistant to T cell depletion and show increased persistence and viability in the tumor microenvironment, which may allow long-term therapy.

In particular embodiments, an effective amount of immune effector cells or genome-edited T cells comprising the edited PD-1 gene is administered to a subject to prevent, treat, or ameliorate at least one symptom of cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or immunodeficiency.

In particular embodiments, edited PD-1 cells do not substantially or completely express PD-1 and therefore lack or substantially lack functional PD-1 expression, e.g., lack the ability to enhance T cell depletion and inhibit the expression of pro-inflammatory cytokines. . In particular embodiments, genome-edited immune effector cells lacking PD-1 are more resistant to immunosuppressive signals from the tumor microenvironment and exhibit increased viability and resistance to T cell depletion.

In particular embodiments, a method for preventing, treating, or alleviating at least one symptom of cancer comprises administering to a subject an effective amount of genome-edited immune effector cells or T cells containing an edited PD-1 gene and engineered TCR, CAR, or Daric, or other therapeutic a transgene to retarget those cells to a tumor or cancer. Genetically modified cells represent a longer lasting drug product because these cells are more resistant to immunosuppressive signals from the tumor microenvironment due to editing the PD-1 gene to reduce or eliminate PD-1 expression.

In particular embodiments, the genome-edited cells of this application are used in the treatment of solid tumors or cancers.

In particular embodiments, the genome-edited cells of this application are used in the treatment of solid tumors or cancers including, but not limited to, the following: adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, atypical teratoid/rhabdoid tumor, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, brain/CNS cancer, breast cancer, bronchial tumors, heart tumors, cervical cancer, cholangiocarcinoma, chondrosarcoma, chordomas, colon cancer, colorectal cancer, craniopharyngioma , ductal carcinoma (DCIS), endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing's sarcoma, extracranial germinoma, extragonadal germinoma, eye cancer, fallopian tube cancer, fibrous histiosarcoma, fibrosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumors , gastrointestinal stromal tumor (GIST), germinal tumors cell carcinoma, glioma, glioblastoma, head and neck cancer, hemangioblastoma, hepatocellular carcinoma, hypopharyngeal cancer, intraocular melanoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, leiomyosarcoma, lip cancer, liposarcoma, liver cancer, lung cancer, non-small cell lung cancer, lung cancer, malignant mesothelioma, medullary carcinoma, medulloblastoma, menangioma, melanoma, Merkel's carcinoma, median carcinoma, oral cancer, myxosarcoma, myelodysplastic syndrome, myeloproliferative neoplasms, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal cancer, neuroblastoma, oligodendroglioma, oral cancer , oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, pancreatic islet cell tumors, papillary cancer, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pinealoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer, reti noblastoma, renal cell carcinoma, renal pelvis and pelvis, rhabdomyosarcoma, salivary gland cancer, sebaceous gland carcinoma, skin cancer, soft tissue sarcoma, squamous cell carcinoma, small cell lung cancer, small intestine cancer, gastric cancer, sweat gland carcinoma, synovioma, cancer testicular cancer, throat cancer, thymus cancer, thyroid cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vascular cancer, vulvar cancer, and Wilms tumor.

In particular embodiments, the genome-edited cells of this application are used in the treatment of solid tumors or cancers, including, but not limited to, liver cancer, pancreatic cancer, lung cancer, breast cancer, bladder cancer, brain cancer, cancer bone, thyroid cancer, kidney cancer, or skin cancer.

In particular embodiments, the genome-edited cells of this application are used to treat a variety of cancers, including, but not limited to, pancreatic, bladder, and lung cancer.

In private embodiments, the genome-edited cells of this application are used to treat hematological or hematological cancers.

In particular embodiments, the genome-edited cells of this application are used to treat B-cell malignancies, including but not limited to leukemias, lymphomas, and multiple myeloma.

In particular embodiments, the genome-edited cells of this application are used to treat hematological malignancies, including but not limited to leukemias, lymphomas, and multiple myelomas: acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), myeloblastic, promyelocytic, myelomonocytic , monocytic, erythroleukemia, hairy cell leukemia (PVC), chronic lymphocytic leukemia (CLL) and chronic myeloid leukemia (CML), chronic myelomonocytic leukemia (CMML) and polycythemia vera, Hodgkin's lymphoma, Hodgkin's lymphoma with a predominance of nodular lymphocytes, Burkitt's lymphoma, lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, progenitor B-lymphoblastic lymphoma, mantle cell lymphoma, marginal zone lymphoma, mycosis fungoides, anaplastic large cell lymphoma, Cesari syndrome, T-lymphoblastic lymphoma from progenitor cells, multiple mi aloma, overt multiple myeloma, indolent multiple myeloma, plasma cell leukemia, nonsecretory myeloma, IgD myeloma, osteosclerotic myeloma, solitary bone plasmacytoma, and extramedullary plasmacytoma.

Preferred cells for use in the genome editing methods contemplated herein include autologous/autologous (“self”) cells, preferably hematopoietic cells, more preferably T cells, and more preferably immune effector or Treg cells.

In private embodiments, methods are provided that include administering a therapeutically effective amount of the genome-edited cells contemplated herein, or a composition containing them, to a patient in need thereof, as monotherapy or in combination with one or more therapeutic agents. According to some reports, such cells are used in the treatment of patients at risk of developing cancer, GVHD, infectious disease, autoimmune disease, inflammatory disease or immunodeficiency. Thus, specific embodiments include treating, preventing, or ameliorating at least one symptom of a cancer, infectious disease, autoimmune disease, inflammatory disease, or immunodeficiency, comprising administering to a subject in need thereof a therapeutically effective amount of the genome-edited cells contemplated herein. .

In one embodiment, a method for treating cancer, GVHD, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency in a subject in need thereof comprises administering an effective amount, e.g., a therapeutically effective amount, of a composition comprising the genome-edited cells of the present application. The amount and frequency of administration will be determined by such factors as the condition of the patient and the type and severity of the patient's illness, although appropriate doses may be determined by clinical trials.

In one exemplary embodiment, the effective number of genome-edited cells provided to a subject is at least 2 x 10 ⁶ cells/kg, at least 3 x 10 ⁶ cells/kg, at least 4 x 10 ⁶ cells/kg, by at least 5 x 10 ⁶ cells/kg, at least 6 x 10 ⁶ cells/kg, at least 7 x 10 ⁶ cells/kg, at least 8 x 10 ⁶ cells/kg, at least 9 x 10 ⁶ cells/kg, or at least 10 x 10 ⁶ cells/kg, or more cells/kg, including all intermediate doses of cells.

In another exemplary embodiment, the effective number of genome-edited cells provided to a subject is about 2 x 10 ⁶ cells/kg, about 3 x 10 ⁶ cells/kg, about 4 x 10 ⁶ cells/kg, about 5 x 10 ⁶ cells/kg. kg, approximately 6 x 10 ⁶ cells/kg, approximately 7 x 10 ⁶ cells/kg, approximately 8 x 10 ⁶ cells/kg, approximately 9 x 10 ⁶ cells/kg, or approximately 10 x 10 ⁶ cells/kg, or more cells/kg including all intermediate cell doses.

In another exemplary embodiment, the effective number of genome-edited cells provided to a subject is from about 2 x 10 ⁶ cells/kg to about 10 x 10 ⁶ cells/kg, from about 3 x 10 ⁶ cells/kg to about 10 x 10 ⁶ cells/kg, from about 4 x 10 ⁶ cells/kg to about 10 x 10 ⁶ cells/kg, from about 5 x 10 ⁶ cells/kg to about 10 x 10 ⁶ cells/kg, from 2 x 10 ⁶ cells/kg up to about 6 x 10 ⁶ cells/kg, from 2 x 10 ⁶ cells/kg to about 7 x 10 ⁶ cells/kg, from 2 x 10 ⁶ cells/kg to about 8 x 10 ⁶ cells/kg, from 3 x 10 ⁶ cells/kg to approx. 6 x 10 ⁶ cells/kg, 3 x 10 ⁶ cells/kg to approx. 7 x 10 ⁶ cells/kg, 3 x 10 ⁶ cells/kg to approx. 8 x 10 ⁶ cells/kg , 4 x 10 ⁶ cells/kg to about 6 x 10 ⁶ cells/kg, 4 x 10 ⁶ cells/kg to about 7 x 10 ⁶ cells/kg, 4 x 10 ⁶ cells/kg to about 8 x 106 cells/kg, from 5 x 10 ⁶ cells cells/kg to about 6 x 10 ⁶ cells/kg, from 5 x 10 ⁶ cells/kg to about 7 x 10 ⁶ cells/kg, from 5 x 10 ⁶ cells/kg to about 8 x 10 ⁶ cells/kg, or from 6 x 10 ⁶ cells/kg up to about 8 x 10 ⁶ cells/kg including all intermediate cell doses.

One skilled in the art will appreciate that multiple administrations of the compositions contemplated in particular embodiments may be required to effect the desired therapy. For example, the composition can be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times within 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 5 years, 10 years or more.

In some embodiments, it may be desirable to administer activated T cells to a subject and then subsequently bleed (or apheresis), activate the T cells from it, and reinject the activated and expanded T cells to the patient. This process can be performed several times every few weeks. In some cases, T cells from 10 to 400 cm3 ^of collected blood can be used. In some embodiments, T cells are activated from a drawn blood volume of 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, or 400 cm ³ or more. Without being bound by any particular theory, the use of such a multiple blood draw/multiple reinfusion protocol may serve to select specific T cell populations.

Administration of the compositions contemplated in particular embodiments may be by any convenient means, including aerosol inhalation, injection, ingestion, transfusion, implantation, or transplantation. In a preferred embodiment, the compositions are administered parenterally. The phrases "parenteral administration" and "introduced parenteral", as used in this application, refer to methods of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravascular, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intratumoral, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrasternal injections and infusions. In one embodiment, the compositions of this application are administered to a subject by direct injection into a tumor, lymph node, or site of infection.

In one embodiment, the method of treating a subject diagnosed with cancer comprises removing the immune effector cells from the subject, editing the genome of said immune effector cells and obtaining a genome-edited immune effector cell population, and introducing the genome-edited immune effector cell population to the same subject. In a preferred embodiment, the immune effector cells include T cells.

Methods of administering cell compositions contemplated in particular embodiments include any method that is effective to result in the ex vivo reintroduction of genome-edited immune effector cells or the reintroduction of genome-edited immune effector cell progenitors that, when administered to a subject, differentiate into mature immune effector cells. One method involves editing the genome of peripheral blood T cells ex vivo and returning these transduced cells to the subject.

All publications, patent applications, and granted patents cited in the specification are incorporated herein by reference, as if each individual publication, patent application, or granted patent were expressly and individually indicated as being incorporated by reference.

While for the purposes of clarity of understanding, the foregoing embodiments have been described in some detail by way of illustration and example, it will be readily apparent to one skilled in the art in light of the teachings contemplated in this application that certain changes and modifications can be made without departing from the spirit or the scope of the appended claims. The following examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize many non-critical parameters that can be changed or modified to produce substantially similar results.

EXAMPLES

Example 1

Reprogramming of I-OnuI to Destroy an Intracellular Signaling Motif in the Programmed Death-1 (pd-1) Receptor Gene

PD-1 is expressed on the plasma membrane of T cells after stimulation and activation of antigen receptors. PD-1 contains a signal peptide, an extracellular IgV-like domain, a transmembrane domain, and an intracellular tail that contains an immunoreceptor tyrosine-based motif (ITIM, consensus sequence S/I/V/LxYxxI/V/L) and an immunoreceptor tyrosine-based switch motif (ITSM, consensus sequence TxYxxV/I). Figure 1A. Tyrosine at amino acid position 248 in ITSM PD-1 becomes phosphorylated upon binding of the PD-L1/2 ligand concurrently with T cell activation, creating a binding substrate for the SH2 domain containing protein tyrosine phosphatase-2 (SHP2, see Chemnitz JM) . et. al. J. Immunol. 2004 Jul 15; 173(2):945-54.). Recruitment of SHP2 to the plasma membrane counteracts phosphotyrosine-driven activation signals in T cells (Yokosuka T et al., J Exp Med. 2012 Jun 4; 209 (6): 1201-17) and suppresses the duration of the activated state of T cells. The codon for ITSM phosphorylated tyrosine is encompassed by the canonical "central-4" I-OnuI cleavage motif, ATAC. Figure 1B. A variant of the homing endonuclease was designed to target the 22 bp target sequence. (SEQ ID NO: 25) centered on the indicated central -4 motif in exon 5 of the PD-1 gene.

Without being bound by any particular theory, it is assumed that all possible insertion/deletion events (“indels”) in and near the “central-4” ATAC sequence will completely eliminate one or more essential features of the ITSM motif, with the most possible indels eliminating the tyrosine codon itself is 248. These indels are likely to generate dominant-negative PD-1 proteins containing a normal extracellular domain and a non-functional intracellular signaling domain. Dominant negative PD-1 proteins can act as a "sink" for PD-1 ligands, thereby reducing or eliminating immunosuppressive signaling. In addition, since activated T cells upregulate PD-L1 expression; since PD-1:PD-L1 interactions occur among T cells, both in cis and trans; and because these interactions are important for T cell function, gene editing to disrupt PD-1 signaling without affecting expression may preserve possible PD-L1-driven functions.

Thus, I-OnuI was reprogrammed to target the ITSM coding region by constructing modular libraries containing variable amino acid residues in the DNA recognition interface. To construct these variants, degenerate codons were inserted into the DNA-binding domains of I-OnuI using oligonucleotides. Oligonucleotides encoding these degenerate codons were used as PCR templates to generate variant libraries by recombination of the break in the yeast strain S. cerevisiae. Each variant library spanned either the N- or C-terminal I-OnuI DNA recognition domain and contained ~10 ⁷ -10 ⁸ unique transformants. The resulting surface display libraries were screened by flow cytometry for cleavage activity against a target site containing "half sites" of the respective domains (SEQ ID NOs: 28-29) as shown in FIG. 2.

Yeasts having HE I-OnuI with reprogrammed N- and C-terminal domains were purified and plasmid DNA was extracted. PCR reactions were performed to amplify the reprogrammed domains, which were subsequently transformed into S. cerevisiae to create a library of reprogrammed domain combinations. From this library, fully reprogrammed I-OnuI variants that recognize the complete target site (SEQ ID NO: 25) present in the ITSM coding region in exon 5 of the PD-1 gene were identified and purified.

The activity of reprogrammed I-OnuI HEs that target the ITSM coding region of PD-1 in exon 5 was measured using a chromosome-integrated fluorescent reporter system (Certo et. Al., 2011). Fully reprogrammed I-OnuI HEs that bind and cleave the ITSM PD-1 target sequence were cloned into mammalian expression plasmids and then individually transfected into the HEK 293T fibroblast cell line containing the upstream PD-1 exon 5 target sequence. from a gene outside the translation frame encoding the mCherry fluorescent protein. Cleavage of the inserted target site of this HE and accumulation of indels after DNA repair by non-homologous end joining (NHEJ) results in approximately one in three repaired loci placing the fluorescent reporter gene back in-frame. Therefore, the percentage of HEK 293T cells with fluorescent mCherry is used as an indicator of endonuclease activity in the chromosomally inserted target sequence. Fully reprogrammed I-OnuI HE (PD-1.ITSM.ex5_RD1_CV3-08, SEQ ID NO: 6) targeted at the exon 5 site of PD-1 showed a very modest efficiency of mCherry expression in a cellular chromosomal context. Figure 3A. The HE variant had moderate DNA affinity properties as measured by substrate equilibrium titration (FIG. 3B).

A library of I-OnuI secondary variants was then generated by performing random mutagenesis on the PD-1.ITSM.ex5_RD1_CV3-08 HE variant. In order to isolate variants with improved catalytic efficiency, display-based stream sorting was performed under more stringent splitting conditions. This process made it possible to identify the I-OnuI variant (PD-1.ITSM.ex5_RD2_73, SEQ ID NO: 7), which contained four amino acid mutations relative to the RD1 variant and had a several times higher percentage of cells expressing mCherry compared to the RD1 variant. . Figure 4. To increase the efficiency of gene editing at the target site of exon 5, three additional rounds of screening for activity enrichment were performed (PD-1.ITSM.ex5_RD3_03, SEQ ID NO: 8; PD-1.ITSM.ex5_RD4_CV23, SEQ ID NO: 9 and PD-1.ITSM.ex5_RD5_CV23MK, SEQ ID NO: 10).

Example 2

Characterization of I-OnuI and megaTAL variant targeting exon 5 of PD-1

The PD-1 exon 5 target site contains CpG dinucleotide motifs both at the meganuclease binding site and at the adjacent TAL array binding site (FIG. 1B). The methylation status of these dinucleotides was assessed by bisulfite sequencing in primary human T cells activated with CD3 and CD28 and cultured in complete medium supplemented with IL-2. After 3 days, genomic DNA was isolated and treated with bisulfite to convert any unmethylated cytosine bases to uracil. The exon 5 target site was then sequenced to identify the unmethylated (converted to thymine) versus methylated (remaining cytosine) status of each cytosine. The results show that both cytosines with a CpG motif at the target site were predominantly methylated (Figure 5), consistent with typical "gene body" methylation patterns.

To confirm that the homing endonuclease (HE) variant PD-1.ITSM.ex5_RD5_CV23MK recognizes and cleaves the methylated target site of exon 5, DNA binding and cleavage affinity assays were performed using substrates containing 5-methylcytosine at position p5 of the target site, or reverse complement of a guanine base at p6 or both strands of the target sequence, which is indicative of the methylation status of the target site in activated T cells. Figure 6. The HE variant bound and cleaved methylated DNA substrates and unmodified substrate in a similar manner.

The PD-1.ITSM.ex5_RD5_CV23MK HE variant was formatted as megaTAL (SEQ ID NO: 19) by adding a 5-methylcytosine-tolerant TAL array of 10.5 units, which corresponds to the target site of the 11 bp TAL array (SEQ ID NO: : 26), to the N-terminus of the meganuclease domain (as described by Boissel et al., 2013). The sequence of the megaTAL target site is described in SEQ ID NO: 27. MegaTAL was tested to match the methylated CpG dinucleotide present in the target site of the TAL array. The TAL array was designed to be methylated base tolerant by including "N*" RVD at the appropriate array position.

The efficiency of megaTAL editing was assessed by prestimulating primary human T cells with anti-CD3 and anti-CD28 antibodies in cytokine-supplemented medium for 48-72 hours, and then subjecting the cells to electroporation with in vitro transcribed (IVT), capped, and polyadenylated mRNA ( SEQ ID NO: 40) encoding megaTAL PD-1.ITSM.ex5_RD5_CV23MK. In addition, an IVT mRNA encoding Trex2 3'-5' exonuclease was added to enhance break processing by non-homologous end attachment (NHEJ) (see Certo et al., 2012). After electroporation, the cells were cultured for 7-10 days in a medium with cytokines, during which aliquots were removed for isolation of genomic DNA, followed by PCR amplification at the target site of exon 5 of PD-1.

Insertion/deletion rates were measured by Tracking of Indels DEcomposition (TIDE, see Brinkman et al., 2014) or high throughput sequencing. The editing efficiency of megaTAL PD-1.ITSM.ex5_RD5_CV23MK in the presence of Trex2 was approximately 60%. Figure 7A. The predominant types of insertions/deletions observed at the target site were +1, -1, -2, -3 or -4 nucleotides. Figure 7B. This analysis confirmed that the PD-1.ITSM.ex5_RD5_CV23MK variant disrupted the ITSM PD-1 consensus motif in a significant proportion of megaTAL-treated human T cells.

Example 3

Reprogramming of I-OnuI meganuclease domains to disrupt extracellular domains in the programmed death receptor-1 (pd-1) gene

The central-4-specificity of the I-OnuI HE variant that targets the target site of exon 5 of PD-1 (SEQ ID NO: 25) was characterized using high-throughput yeast surface display in vitro endonuclease assays (Jarjour, West-Foyle et al. al., 2009). A plasmid encoding an HE variant that targets exon 5 of PD-1 was transformed into S. cerevisiae for surface display and then tested for cleavage activity against PCR-generated double-stranded DNA substrates containing the DNA sequence of the target site of exon 5 of PD- 1, which contains each of the 256 possible central-4 sequences. The resulting specificity profile showed that this reprogrammed I-OnuI is highly selective for central-4 target sites (Figure 8), but can also cleave additional non-canonical central-4 target sites. Since very few canonical central-4 target sites of I-OnuI are located elsewhere in the PD-1 gene, non-canonical central-4 target sites in exon 1 and exon 2 of PD-1 were used to reprogram additional homing endonucleases.

These non-canonical central-4 target sites in exon 1 and exon 2 of PD-1 are in regions that encode the signal peptide and the IgV domain, respectively. Without being bound by any particular theory, it is hypothesized that targeting these regions by gene editing indels abolishes or destabilizes PD-1 protein expression creating a clear inactivated phenotype. In addition, having access to these sites for more complex gene editing operations , including but not limited to the targeted insertion of transgenic cassettes, phenotypes can be created that are under unique regulatory control.

I-OnuI was reprogrammed to target two non-canonical central-4 target sites (SEQ ID NOs: 30 and 35), one in each of these two exons/motifs, and extending to target sites of the corresponding TAL array ( SEQ ID NOs: 26 and 31) and complete megaTAL target sites (SEQ ID NOs: 31 and 37). Figures 9A and 9B. I-OnuI was reprogrammed to target either exon 1 or exon 2 of the PD-1 gene by constructing modular libraries containing variable amino acid residues in the DNA recognition interface. To construct variants, degenerate codons were inserted into the DNA-binding domains of I-OnuI using oligonucleotides. Oligonucleotides encoding degenerate codons were used as PCR templates to generate variant libraries by recombining breaks in the yeast strain S. cerevisiae. Each variant library spanned the N- or C-terminal I-OnuI DNA recognition domain and contained ~10 ⁷ -10 ⁸ unique transformants. The resulting surface display libraries were screened by flow cytometry for cleavage activity against target sites containing "half sites" of the respective domains (exon 1: SEQ ID NOS: 34 and 35; exon 2: SEQ ID NOS: 38 and 39).

Yeasts having I-OnuI HE with reprogrammed N- and C-terminal domains were purified and plasmid DNA was extracted. PCR reactions were performed to amplify the reprogrammed domains, which were subsequently transformed into S. cerevisiae to create libraries of reprogrammed domain combinations. Fully reprogrammed I-OnuI variants active against the entire target site in the region encoding the signal peptide in exon 1 of PD-1 and in the region encoding the IgV domain in exon 2 of PD-1 were identified and isolated from these libraries.

Reprogrammed I-OnuI HEs targeting PD-1 exon 1 and exon 2 target sites were cloned into mammalian expression plasmids and then individually transfected into the HEK 293T fibroblast cell line containing the appropriate target sequences upstream of the out-of-frame gene. coding for the fluorescent protein iRFP.

Reprogrammed HE I-OnuI targeting the PD-1 exon 1 site (PD-1.ile3.exon1_RD1_B1, SEQ ID NO: 11) showed moderate efficiency in iRFP expression in the context of the cellular chromosome, either as a standalone HE variant or after formatting as megaTAL (SEQ ID NO: 20). A secondary library of I-OnuI variants was generated by random mutagenesis on the RD1 variant of exon 1 of PD-1 identified during the screening of the original library. Display-based stream sorting was performed under more stringent splitting conditions to highlight variants with improved catalytic efficiency. This process revealed two variants of I-OnuI (PD-1.ile3.exon1_RD2_B1H8, PD-1.ile3.exon1_RD2_B1G2, SEQ ID NOS: 12 and 60, respectively). These variants contained four (PD-1.ile3.exon1_RD2_B1H8) or five (PD-1.ile3.exon1_RD2_B1G2) amino acid mutations relative to the RD1 variant and had a significantly higher level of iRFP-expressing cells compared to the RD1 variant of exon 1PD-1, as in as a standalone HE variant, and after formatting as megaTAL (SEQ ID NO: 21 and SEQ ID NO: 64). Figure 10.

Reprogrammed HE I-OnuI targeting the PD-1 exon 2 site (PD-1.IgV.exon2_RD1_G5; SEQ ID NO: 13) showed high efficiency of iRFP expression when delivered either as a standalone HE variant or formatted as megaTAL (SEQ ID NO: 22).

HE variants targeting exon 1 and exon 2 exhibited strong DNA affinity properties as measured by equilibrium substrate titration using their respective target sequences. Figure 11A.

Exon 1 nuclease was further purified to improve its specificity in the region in contact with base pair -8, -7 and -6. Microlibraries were constructed by randomizing amino acid residues 68, 70, 78, 80 and 82, and after 6 rounds of flow cytometer sorting three clones (PD-1.ile3.exon1_RD2_B1G2C4, PD-1.ile3.exon1_RD2_B1G2C11 and PD-1.ile3 .exon1_RD2_B1G2C5; SEQ ID NO: 61, 62 and 63, respectively) showed higher specificity than the parent nuclease (PD-1.ile3.exon1_RD2_B1G2; SEQ ID No: 60). Figure 11B.

Bisulfite sequencing was used to assess the methylation status of CpG motifs present in exon 1 and exon 2 target sites of PD-1. Figure 12. The CpG motif at the PD-1 target site of exon 1 in activated T cells was shown to be unmethylated, while both CpG motifs at the target site of exon 2 of PD-1 were methylated. Display-based activity analysis was performed to confirm that the fully methylated CpG target sequence of exon 2 of PD-1 was efficiently cleaved by the corresponding HE variant.

In addition, a fully methylated exon 2 CpG target site was used to identify an I-OnuI variant (PD-1.IgV.exon2_RD1_PS3, SEQ ID NO: 14) with improved binding and cleaving activity for the CpG-methylated target site.

Example 4

Targeted disruption of the pd-1 gene in primary human t cells rescues pd-l1-mediated suppression of t cell function

The functional impact of megaTAL reprogrammed to cleave various target sequences in the PD-1 gene was evaluated in primary human T cells activated with CD3 and CD28 and cultured in complete medium supplemented with IL-2. Activated PBMCs were transduced with a lentiviral vector encoding anti-BCMA CAR. Anti-BCMA CAR T cells were electroporated with in vitro transcribed mRNA encoding megaTAL targeting either exon 5 of PD-1 or exon 1 of PD-1 (SEQ ID NOs: 40 and 41, respectively) and mRNA encoding Trex2 (SEQ ID NO: 43). Control cells included untreated T cells or T cells treated with mRNA encoding blue fluorescent protein (CFP) or megaTAL targeting CCR5 (see Sather et. al., Sci Transl Med. 2015 Sep 30;7(307): 307ra156). After 10 days of expansion, T cells were stimulated with the polyclonal activating reagent phorbol-12-myristate-13-acetate (PMA)/ionomycin (P/I). PD-1 is naturally activated on the cell surface after T cell activation. PD-1 activation was downregulated following transfection of PD-1 exon 1 megaTAL mRNA, indicating that indels in this region interfere with normal PD-1 protein production. In contrast, treatment with mRNA encoding the control CCR5-megaTAL or targeting PD-1 exon 5, megaTAL, had no effect on surface expression of PD-1, despite the high level of indels induced in ITSM by this megaTAL. Figure 13A. Further experiments repeated under similar conditions with specificity enriched versions of PD-1 exon 1 mRNA (SEQ ID NOs: 65, 66, 67 and 68) also showed a similar disruption of PD-1 expression in T cells. Figure 13B

Simultaneous delivery of megaTAL exon 1 and exon 5 of PD-1 significantly improved the disruption of PD-1 expression on the cell surface, independent of the delivery of Trex2 exonuclease. Figure 14. This indicates that the simultaneous formation of a proximal DNA break is a mechanism that promotes large gene deletion events with high efficiency, independent of indel exonuclease amplification.

The effect of disrupting PD-1 signaling in T cells by targeting either its expression or its signaling functions was analyzed based on cytokine production in the CAR-T cell in response to tumor cells modified to express the PD-1 ligand, PD-L1 . Co-culture of anti-BCMA CAR T cells with BCMA-expressing tumor cell lines resulted in T cell activation and subsequent secretion of inflammatory cytokines, exemplified by high levels of TNFα and IL-17A measured in the supernatant. Co-expression of PD-L1 on BCMA-expressing tumor cells suppressed the production of inflammatory cytokines. However, transfection of anti-BCMA CAR T cells with mRNA encoding either exon 1 or exon 5 PD-1 megaTALs reduces PD-L1 mediated cytokine suppression as inflammatory cytokine production in these samples is restored to baseline levels. Figure 15A.

Example 5

Homologous recombination of the transgene into exon 1 of the PD-1 gene

A recombinant adeno-associated virus (rAAV) plasmid was designed and constructed containing a promoter-transgene cassette containing a heterologous promoter, a transgene encoding a fluorescent protein, and a polyadenylation signal located between regions of homology targeting genes. The integrity of the AAV ITR elements was verified using the XmaI lysate. The transgene cassette was placed between two homologous regions, approximately 600-700 bp long, flanking the megaTAL cleavage site of exon 1 of PD-1 (SEQ ID NO: 27). The 5' homologous arm (SEQ ID NO: 54) contained part of the first exon of PD-1 and other sequences upstream from the megaTAL cleavage site. The 3' homologous arm (SEQ ID NO: 55) contained a portion of the PD-1 exon and other sequences downstream of the megaTAL cleavage site. None of the homologous regions contained the complete megaTAL target site. Said exemplary expression cassette contained a myeloproliferative sarcoma virus enhancer promoter with a deletion of the downregulation-mediating region, replaced by a primer-binding site from dl587rev (MND) operably linked to a polynucleotide encoding a fluorescent polypeptide, e.g. blue fluorescent protein (BFP), red fluorescent protein (RFP), blue fluorescent protein (CFP), green fluorescent protein (GFP), etc. In addition, the expression cassette contained the SV40 late polyadenylation signal located downstream of the transgene termination codon. Figure 16A. Recombinant AAV (rAAV) was generated by transient co-transfection of HEK 293T cells with plasmids providing the replication, capsid, and adenoviral helper elements required for virus production. Recombinant rAAV was isolated from a co-transfected HEK 293T cell culture using iodixanol gradient ultracentrifugation.

MegaTAL-induced homologous recombination was evaluated in primary human T cells activated by CD3 and CD28 and cultured in complete medium supplemented with IL-2. After 3 days of culture, T cells were washed and electroporated with in vitro transcribed PD-1 exon 1 megaTAL mRNA (SEQ ID NO: 41) and then transduced with a purified transgenic cassette encoding recombinant AAV, MND-GFP (SEQ ID NO: 53) . The control group included T cells treated with megaTAL mRNA or a vector targeted to rAAV. Flow cytometry was used to measure, at multiple time points, the proportion of T cells expressing the fluorescent protein and to differentiate transient expression of fluorescent protein from an episomal rAAV-targeted vector from long-term expression of a chromosomally integrated cassette. Figure 16B. MegaTAL-mediated disruption of the PD-1 gene was detected by sequencing and loss of PD-1 expression after polyclonal T cell activation.

Long-term expression of the transgene was observed in 20-60% of T cells that were treated with a vector that targeted both megaTAL and rAAV. In controls, rAAV-only treatment resulted in variable levels of transient fluorescent protein expression and very low levels (<1%) of long-term fluorescent protein expression in treated T cells, consistent with a lack of genome integration. The results were confirmed in experiments carried out on T cells isolated from several independent donors.

Example 6

Homologous recombination of a transgene encoding a chimeric antigen receptor (CAR) into the PD-1 gene

Recombinant adeno-associated virus (rAAV) plasmids were designed, constructed and verified as described above, except that the transgenic cassette encoding the anti-CD19 CAR (SEQ ID NO: 56) or anti-BCMA CAR (SEQ ID NO: 57 ) was placed between homologous regions directed to exon 1 of PD-1. The CAR expression cassettes contained an MND promoter operably linked to a polynucleotide encoding a CAR containing a signal peptide derived from CD8α, a single chain variable fragment (scFv) targeted either at the CD19 antigen or at the B cell maturation antigen (BCMA) derived from CD8α. a hinge region and a transmembrane domain, an intracellular co-stimulatory domain 4-1BB, a CD3 zeta signaling domain, and homologous arms designed to target the target site of exon 1 of PDCD1 (SEQ ID NOS: 54 and 55).

Primary human T cells were activated with CD3 and CD28 and grown in cytokine supplemented media as described above. Homologous recombination of CAR transgenes into PD-1 exon 1 target site was assessed using activated primary human T cells electroporated in vitro with transcribed PD-1 exon 1 mRNA (SEQ ID NO: 41) and then transduced with anti-CD19 coding rAAV or anti-BCMA CAR. Flow cytometry to determine CAR expression was performed 10 days later, i.e. 7 days after electroporation and transduction. Control samples included T cells treated with either megaTAL mRNA or a vector targeted to rAAV. Expression of CD19-CAR and BCMA-CAR was analyzed using recombinant PE-conjugated CD19-Fc or PE-conjugated BCMA-Fc staining reagents. T cells treated with megaTAL mRNA and rAAV-CAR showed CD19-CAR expression in 30-60% of T cells and BCMA-CAR expression in 10-20% of T cells. Figure 16B. Similar T cell proliferation rates and indistinguishable T cell phenotypes were observed between untreated, rAAV-treated and megaTAL/rAAV CAR-treated T cells.

Example 7

Homologous recombination of a promoter-deprived transgene into the pd-1 gene

A recombinant adeno-associated virus (rAAV) plasmid was designed, constructed, and verified containing a fluorescent reporter transgene (mCherry) and a polyadenylation signal, but without an exogenous promoter (SEQ ID NO: 58). Figure 17. The mCherry start codon is fused and overlapped with the endogenous PD-1 start codon, replacing the rest of exon 1 of PD-1 with the cDNA encoding the mCherry protein. This strategy stimulates the expression of a fluorescent protein from the endogenous PD-1 promoter and also disrupts the normal expression of the PD-1 protein.

Primary human T cells were activated, electroporated with transcribed megaTAL mRNA in vitro, and transduced with rAAV as described above. Control cells included T cells treated with megaTAL mRNA or a vector targeted to rAAV. Fluorescent reporter expression was analyzed by flow cytometry at various time points after transfection, in the presence or absence of polyclonal T cell activation, using phorbol-12-myristate-13-acetate (PMA)/ionomycin (P/I). In T cells treated with megaTAL mRNA alone or with a vector targeted to rAAV, reporter expression was not observed. Similar measures of megaTAL activity were observed with or without rAAV transduction. Low-level expression of the fluorescent reporter was observed in T cells that received both megaTAL and the rAAV-targeting vector and were activated by P/I for 48 hours. Expression of the fluorescent reporter driven by the endogenous PD-1 promoter was lower than that of the receptor driven by the heterologous promoter (~5-fold decrease in fluorescence intensity).

Example 8

Constitutive expression of PD-1 after homologous recombination of a transgene encoding a fluorescent protein into exon 5 of the PD-1 gene

A recombinant adeno-associated virus (rAAV) plasmid containing a heterologous promoter, a blue fluorescent protein (BFP) transgene, and a polyadenylation signal (SEQ ID NO: 44) was designed, constructed, and verified for introduction into exon 5 of PD-1. The transgenic cassette was placed between 1.3 kb. and 1.0 kb homologous arms flanking the megaTAL cleavage site of exon 5 of PD-1. The 5' homologous arm (SEQ ID NO: 45) included a portion of exon 5 of PD-1 upstream from the megaTAL site, and other sequences upstream. The 3' homologous arm (SEQ ID NO: 46) contained the terminal coding sequence for exon 5 of PD-1 and other sequences downstream of transcription. None of the homologous regions contained the complete megaTAL target site. Figure 18.

Primary human T cells were activated with CD3 and CD28, electroporated in vitro with transcribed PD-1 exon 5 megaTAL mRNA (SEQ ID NO: 40), then transduced with rAAV encoding the fluorescent reporter transgene. Control cells included untreated T cells and T cells treated with rAAV targeting vector only. Fluorescent reporter expression was analyzed by flow cytometry at various time points after transfection. Cells treated with rAAV alone showed transient expression of BFP, which decreased to background levels during cell expansion. T cells that received both PD-1 exon 5 megaTAL mRNA and the rAAV vector showed a stable population of BFP-expressing T cells that persisted throughout the ex vivo culture period.

T cells treated with PD-1 exon 5 megaTAL mRNA and rAAV vector had a distinct PD-1 expression pattern, resulting in almost all BFP-expressing cells also expressing PD-1 protein in the absence of activation. This indicates that the targeting strategy changed the normal regulation of the PD-1 gene to a constitutive rather than an inducible expression pattern. Moreover, by targeting exon 5 of PD-1, the constitutively expressed protein in this targeting strategy is a PD-1 variant that has impaired inhibitory signaling and that can act as a dominant negative receptor. PD-1 expression was not upregulated in PD-1 exon 5 megaTAL mRNA-treated T cells or in megaTAL and rAAV mRNA-treated T cells containing homologous arms to exon 1. Figure 19.

Example 9

Homologous recombination of transgene into PD-1 gene is independent of single nucleotide polymorphism (SNP)

The PD-1 locus is genetically heterogeneous, with a high abundance of SNPs present in both intron and exon regions. The presence of divergent SNPs near the megaTAL cleavage site could potentially influence the efficiency of homologous recombination at the PD-1 locus. Recombinant adeno-associated virus (rAAV) plasmids comprising a heterologous promoter, a fluorescent protein transgene and an SV40 late polyadenylation signal were designed to assess SNP exposure, then constructed and verified as described above (SEQ ID NOS: 47 and 48). The homologous arms were either identical to the PD-1 consensus sequence, or they were designed to include point mutations from normal SNPs proximal to the megaTAL targeting site of exon 5 of PD-1. In these constructs, the 5' homologous arm (L-SNP, SEQ ID NO: 49) contains an intron G/A SNP (rs6705653) located at 220 bp. upstream of the megaTAL cleavage site, while the 3' homologous arm (R-SNP, SEQ ID NO: 50) contains a silent C/T SNP (rs2227981) located at 68 bp. downstream from the megaTAL PD-1 cleavage site. Figure 20.

Primary human T cells were activated with CD3 and CD28, electroporated with in vitro transcribed megaTAL mRNA, and transduced with rAAV encoding consensus homologous arms or rAAV carrying either L-SNP or R-SNP homologous arms. Control cells included T cells transduced with the targeted rAAV vector only. Fluorescent protein expression was analyzed by flow cytometry at different time points after transduction. Fluorescent protein expression decreased to background levels in samples that received rAAV alone, while T cells treated with megaTAL in combination with rAAV showed stable levels of fluorescent protein expression. All samples showed similar levels of fluorescence despite being treated with either non-SNP-containing rAAV vectors or vectors containing L- or R-variant SNPs, indicating that SNPs proximal to the megaTAL target site do not affect or have no significant effect on HR at the target site.

Example 10

Homologous recombination of a transgene encoding the switch receptor into the PDCD1 locus

Switch receptors are engineered chimeric molecules capable of converting extracellular inhibitory signals into intracellular activation signals, but their effectiveness often correlates with their ability to downregulate native expression of natural receptors. One way to get around this limitation is to insert a switch receptor into the native locus and disrupt the natural receptor. Recombinant adeno-associated virus (rAAV) plasmid containing a heterologous promoter, a transgene encoding the PD-1 extracellular ligand-binding domain and the CD28 intracellular signaling domain (PD-1 switch receptor, SEQ ID NO: 59), and the SV40 polyadenylation late signal , was designed to target exon 5 of PD-1, constructed and verified. The 5' homologous arm included a portion of exon 5 of PD-1 upstream from the megaTAL cleavage site containing ITIM and other sequences upstream. The 3' homologous arm contained the terminal coding sequence for exon 5 of PD-1 and part of the UTR region and was shortened to ~650 bp. to host the switch receptor cDNA. None of the homologous regions contained the complete megaTAL target site. Figure 21.

Primary human T cells were activated with CD3 and CD28, electroporated in vitro with transcribed mRNA of PD-1 exon 5 megaTAL exon 5, and transduced with an rAAV-targeted vector encoding the PD-1 switch receptor. Control cells included samples that received only megaTAL or rAAV. Expression of the PD-1 switch receptor was analyzed by anti-PD-1 antibody staining in the absence of T cell stimulation. High levels of expression were observed in T cells treated with PD-1 exon 5 megaTAL mRNA and transduced with rAAV.

Example 11

Long-range excision of the PD-1 intracellular signaling region using HDR

It is known that the mechanism of homologous recombination includes the mechanisms of "search" for homology, which can cover distant genomic sequences. Without wishing to be bound by any particular theory, it is believed that more significant portions of target genes or chromosomal regions can be effectively manipulated using a combination of megaTAL and rAAV delivery. A recombinant adeno-associated virus (rAAV) plasmid was designed, constructed, and verified, comprising a heterologous promoter, a BFP transgene, and an SV40 late polyadenylation signal flanked by homologous arms to assess distal HR events (SEQ ID NO: 52). The transgene was flanked by a 1.3 kb 5' homologous arm that started upstream from exon 3 of PD-1 and ended just before the start of exon 4. The 3' homologous arm contained the terminal coding sequence for exon 5 of PD-1. 1 and part of the UTR area. None of the homologous regions contained the complete megaTAL target site. Successful homologous recombination is designed to eliminate the entire intracellular signaling region of PD-1, leaving only the extracellular and transmembrane portion of PD-1 on the treated cell. Figure 22.

Primary human T cells activate CD3 and CD28, are electroporated with in vitro transcribed megaTAL mRNA, and transduced with BFP encoding an rAAV-targeted vector containing a distal 5' homologous arm. Control cells include T cells treated with a vector targeted to megaTAL or rAAV. Stable expression of BFP was observed only in samples that received mRNA of megaTAL targeting exon 5 and rAAV and PDCD1.

Example 12

Destruction of the PD-1 gene and antibody-mediated blockade of PD1

in primary human T cells in vitro

The functional impact of megaTAL reprogrammed to cleave various target sequences in the PD-1 gene was evaluated in primary human T cells activated by CD3 and CD28 and cultured in complete medium supplemented with IL-2. Activated PBMCs were transduced with a lentiviral vector encoding an anti-BCMA CAR. Anti-BCMA CAR T cells were electroporated with in vitro transcribed mRNA encoding megaTAL targeting either exon 5 of PD-1 or exon 1 of PD-1 (SEQ ID NOS: 40 and 41, respectively) and mRNA encoding Trex2 (SEQ ID NO: 43). Control cells included T cells electroporated without mRNA (mock EP mocks) or T cells electroporated with mRNA encoding TCRα-specific megaTAL, which has no catalytic activity.

After 10 days of expansion, T cells were co-cultured with A549 cells transduced with a lentiviral vector encoding BCMA-GFP. CAR T cells were co-cultured with A549 target cells alone or in the presence of 20 μg/ml anti-PD-1 antibody. Cells were co-cultured for 72 hours and cytokine levels in the supernatants were analyzed using a microbead assay (Intellicyt QBeads). In the absence of anti-PD-1 antibody, cells that were treated with megaTAL with either PD1-Ex1 or PD1-Ex5 showed increased production of IFNγ and TNFα compared to control Mock EP and TCRα mock cells. The addition of the αPD-1 antibody eliminates this difference, resulting in an equivalent level of IFNγ and TNFα secretion in supernatants derived from control and PD1 megaTAL-treated cells (Figure 15B).

Example 13

Homologous recombination of a transgene lacking a promoter into the PD-1 gene

A recombinant adeno-associated virus (rAAV) plasmid containing a fluorescent reporter transgene (mCherry) and a polyadenylation signal but without an exogenous promoter was designed, constructed and verified (SEQ ID NO: 58). Figure 17. The mCherry start codon fuses and overlaps with the endogenous PD-1 start codon, while replacing the rest of exon 1 of PD-1 with the cDNA encoding the mCherry protein. This strategy stimulates the expression of a fluorescent protein from the endogenous PD-1 promoter and also disrupts the normal expression of the PD-1 protein.

Primary human T cells were activated, electroporated with in vitro transcribed megaTAL mRNA, and transduced with rAAV as described above. Control samples included T cells treated with megaTAL mRNA or a vector targeted to rAAV. Fluorescent reporter expression was analyzed by flow cytometry 24 hours after stimulation in the presence or absence of polyclonal T cell activation using phorbol-12-myristate-13-acetate (PMA)/ionomycin (P/I). In untreated T cells, megaTAL expression was not observed. Prior to P/I stimulation, low basal levels of fluorescent reporter expression were observed in T cells that received both megaTAL and the rAAV-targeted vector for HDR. Twenty-four hours of P/I stimulation increased endogenous PD-1-induced fluorescent reporter expression from 3% to 27.3% (compare upper and lower right panels in Figure 17).

Example 14

Homologous recombination of a promoter-deficient cytokine transgene into the PD-1 locus

A recombinant adeno-associated virus (rAAV) plasmid containing IL-12 or IL-15 transgenes and a polyadenylation signal but without an exogenous promoter was designed, constructed and verified (SEQ ID NO: 58). The start codon of the transgene fuses and overlaps with the endogenous start codon of PD-1, replacing the rest of exon 1 of PD-1 with cDNA encoding the cytokine IL-12 or IL-15 (Figure 23, upper panel). This strategy stimulates transgene expression from the endogenous PD-1 promoter and also disrupts normal PD-1 expression.

Primary human T cells were activated and transduced with a lentiviral vector encoding anti-BCMA CAR 24 hours after activation. Cells were expanded for 2 days, electroporated with in vitro transcribed megaTAL mRNA, and transduced with rAAV as described above. Control samples included non-transduced T cells, T cells treated with anti-BCMA CAR alone, or T cells treated with anti-BCMA CAR and cultured in the presence of recombinant IL-12. megaTAL PD-1 activity was determined by flow cytometry analysis of PD-1 expression on T cells stimulated for 24 hours with PMA/ionomycin. The combination of megaTAL PD-1 with IL-12 AAV showed a decrease in the expression of PD-1 by approximately 40% compared with controls (Figure 23, bottom panel).

IL-12 expression levels were measured by ELISA after activation with PMA/ionomycin or after co-culture of T cells with the antigen-expressing K562-BCMA cell line. Unstimulated T cells showed minimal IL-12 production, and PMA/ionomycin stimulation when co-cultured with antigen positive K562 cells resulted in IL-12 secretion (Figure 24A). Restimulation assays were used to measure functional depletion of BCMA-CAR T cells. Tumor cells and anti-BCMA CAR T cells were mixed, cultured for 7 days, and additional K562-BCMA target cells were added to mimic restimulation. After the 1st stimulation, the levels of IFNγ production and cytotoxicity were the same in all T cell samples. After 2nd stimulation, IFNγ production and cytotoxicity increased in anti-BCMA-CAR T cells treated with recombinant IL-12 or treated with both megaTAL PD-1 and HDR matrix IL-12 compared to control treated cells (Figure 24B) .

In general, in the following claims, the terms used are not to be construed as limiting the claims to the specific embodiments disclosed in the specification and claims, but are to be construed as including all possible embodiments together with the full scope of equivalents implied by such claims. Accordingly, the claims are not limited to the above description.

--->

SEQUENCE LIST

<110>BLUEBIRD BIO, INC.

MANN, Jasdeep

GUY, Joel

JARJUR, Jordan

CHANG, Joy

<120>VARIANTS, COMPOSITIONS AND METHODS FOR USING PD-1 HOMING-ENDONUCLEASE

<130>BLBD-076/02WO

<150>USA 62/414 279

<151>2016-10-28

<150>USA 62/385 079

<151>2016-09-08

<160>123

<170>PatentIn version 3.5

<210>1

<211>303

<212>PROTEIN

<213>Ophiostoma novo-ulmi subsp.americana (mitochondrion)

<400>1

Met Ala Tyr Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn

20 25 30

Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Met Leu Phe Lys Gln

100 105 110

Ala Phe Cys Val Met Glu Asn Lys Glu His Leu Lys Ile Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Ile Ile Ser Lys Glu Arg Ser Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu

180 185 190

Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile

210 215 220

Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe

290 295 300

<210>2

<211>303

<212>PROTEIN

<213>Ophiostoma novo-ulmi subsp.americana (mitochondrion)

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Met Ala Tyr Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn

20 25 30

Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu

180 185 190

Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile

210 215 220

Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe

290 295 300

<210>3

<211>303

<212>PROTEIN

<213>Ophiostoma novo-ulmi subsp.americana (mitochondrion)

<220>

<221>MOD_RES

<222>(1)..(3)

<223>Any or missing amino acid

<400>3

Xaa Xaa Xaa Met Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn

20 25 30

Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu

180 185 190

Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile

210 215 220

Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Val Phe

290 295 300

<210>4

<211>303

<212>PROTEIN

<213>Ophiostoma novo-ulmi subsp.americana (mitochondrion)

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>4

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn

20 25 30

Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu

180 185 190

Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile

210 215 220

Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>5

<211>303

<212>PROTEIN

<213>Ophiostoma novo-ulmi subsp.americana (mitochondrion)

<220>

<221>MOD_RES

<222>(1)..(8)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>5

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Leu Leu Arg Ile Arg Asn Asn

20 25 30

Asn Lys Ser Ser Val Gly Tyr Ser Thr Glu Leu Gly Phe Gln Ile Thr

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Val Ile Ala Asn Ser Gly Asp Asn Ala Val Ser Leu Lys

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Leu Asn Trp Gly Leu Thr Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Ser Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Glu Gly Cys Phe Phe Val Asn Leu Ile Lys Ser Lys Ser Lys Leu

180 185 190

Gly Val Gln Val Gln Leu Val Phe Ser Ile Thr Gln His Ile Lys Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile

210 215 220

Lys Glu Lys Asn Lys Ser Glu Phe Ser Trp Leu Asp Phe Val Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>6

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI PD-1.ITSM.ex5_RD1_CV3-08

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>6

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gln Leu Glu Ile Arg Asn Val

20 25 30

Asn Pro Asn Ile Pro Arg Tyr Lys Thr Arg Leu Arg Phe Glu Ile Asp

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Lys Ile Tyr Asn Gln Gly Asp Ser Tyr Val Lys Leu Arg

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Glu Gly His Phe Gly Val Ile Leu Ala Lys Arg Arg Pro Ala Ser

180 185 190

Pro Val Gln Val Arg Leu Arg Phe Ala Ile Gly Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg Ile

210 215 220

Arg Glu Lys Asn Ile Ser Glu Lys Ser Trp Leu Glu Phe Glu Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>7

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ITSM.ex5_RD2_73

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>7

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gln Leu Glu Ile Arg Asn Val

20 25 30

Asn Pro Asn Ile Pro Arg Tyr Arg Thr Arg Leu Arg Phe Glu Ile Asp

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asp Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Lys Ile Tyr Asn Arg Gly Asp Ser Tyr Val Lys Leu Arg

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Glu Gly His Phe Gly Val Ile Leu Ala Lys Arg Arg Pro Ala Ser

180 185 190

Pro Val Gln Val Arg Leu Arg Phe Ala Ile Gly Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg Thr

210 215 220

Arg Glu Lys Asn Ile Ser Glu Lys Ser Trp Leu Glu Phe Glu Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>8

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ITSM.ex5_RD3_03

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>8

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gln Leu Glu Ile Arg Asn Val

20 25 30

Asn Pro Asn Ile Pro Arg Tyr Arg Thr Arg Leu Arg Phe Glu Ile Asp

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asp Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Lys Ile Tyr Asn Gln Gly Asp Ser Tyr Val Lys Leu Arg

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly His Phe Gly Val Ile Leu Ala Lys Arg Arg Pro Ala Ser

180 185 190

Pro Val Gln Val Arg Leu Arg Phe Ala Ile Gly Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg Thr

210 215 220

Arg Glu Lys Asn Ile Ser Glu Lys Ser Trp Leu Glu Phe Glu Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>9

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ITSM.ex5_RD4_CV23

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>9

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gln Leu Glu Ile Arg Asn Val

20 25 30

Asn Pro Asn Ile Pro Arg Tyr Arg Thr Arg Leu Arg Phe Glu Ile Asp

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asp Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Lys Ile Tyr Asn Gln Gly Asp Ser Tyr Val Lys Leu Arg

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly His Phe Gly Val Ile Leu Ala Lys Arg Arg Pro Ala Ser

180 185 190

Pro Val Gln Val Arg Leu Arg Phe Ala Ile Gly Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg Ile

210 215 220

Arg Glu Lys Asn Lys Ser Glu Lys Ser Trp Leu Glu Phe Glu Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>10

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ITSM.ex5_RD5_CV23MK

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>10

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gln Leu Glu Ile Arg Asn Val

20 25 30

Asn Pro Asn Ile Pro Arg Tyr Arg Thr Arg Leu Arg Phe Glu Ile Asp

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asp Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Lys Ile Tyr Asn Gln Gly Asp Ser Tyr Val Lys Leu Arg

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly His Phe Gly Val Ile Leu Ala Lys Arg Arg Pro Ala Ser

180 185 190

Pro Val Gln Val Arg Leu Arg Phe Met Ile Gly Gln His Ile Lys Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg Ile

210 215 220

Arg Glu Lys Asn Lys Ser Glu Lys Ser Trp Leu Glu Phe Glu Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>11

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ile3.exon1_RD1_B1

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>11

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile Leu Asn Arg

20 25 30

Asn Arg Gly Thr Ala Arg Tyr His Thr Arg Leu Ser Phe Ala Ile Val

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Ile Ile Thr Asn Asp Gly Asp Arg Tyr Val Arg Leu Arg

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Val Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn Val Asn Ala

180 185 190

Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly His Ile

210 215 220

Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe Arg Val Glu

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>12

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ile3.exon1_RD2_B1H8

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>12

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile Leu Asn Arg

20 25 30

Asn Arg Gly Thr Ala Arg Tyr His Thr Arg Leu Ser Phe Thr Ile Val

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Ile Ile Thr Asn Asp Gly Asp Arg Tyr Val Arg Leu Cys

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Val Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Ala Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Gly Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn Val Asn Ala

180 185 190

Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly His Ile

210 215 220

Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe Arg Val Glu

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>13

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.IgV.exon2_RD1_G5

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>13

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gln Leu Tyr Ile Ser Asn Val

20 25 30

Asn Asn Asn Arg Ser Arg Tyr Arg Ala Arg Leu Arg Phe Ala Ile Glu

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Val Ile Asn Asn Ile Gly Asp Thr Ser Val Arg Leu Ser

65 70 75 80

Val Gly Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly Asn Phe Tyr Val His Leu Lys Lys Ser Gly Arg Thr Thr

180 185 190

Arg Val Tyr Val Gln Leu Arg Phe Ser Ile Ala Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile

210 215 220

Asn Glu Trp Asn Ala Ser Glu Arg Ser Ala Leu Glu Phe Arg Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>14

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.IgV.exon2_RD1_PS3

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>14

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Cys Phe Leu Leu His Ile Arg Asn Leu

20 25 30

Asn Arg Thr Ser Thr Lys Tyr Arg Thr Arg Leu Ser Phe Glu Ile Glu

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Ile Ile Asn Asn Ile Gly Asn Arg Arg Val Arg Leu Ser

65 70 75 80

Val Arg Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Leu Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly Asn Phe Tyr Val His Leu Lys Lys Ser Gly Arg Thr Thr

180 185 190

Arg Val Tyr Val Gln Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile

210 215 220

Thr Glu Ser Asn Pro Ser Glu Arg Ser Asp Leu Glu Phe Arg Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>15

<211>870

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized megaTAL PD-1.ITSM.ex5_RD1_CV3-08

<400>15

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

225 230 235 240

Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val

245 250 255

Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

260 265 270

Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln

275 280 285

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

290 295 300

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro

305 310 315 320

Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu

325 330 335

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu

340 345 350

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

355 360 365

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His

370 375 380

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

385 390 395 400

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

405 410 415

Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn

420 425 430

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu

435 440 445

Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser

450 455 460

Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

465 470 475 480

Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile

485 490 495

Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln

500 505 510

Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu

515 520 525

Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val Lys

530 535 540

Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg Arg

545 550 555 560

Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Ser Arg Arg

565 570 575

Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe Ala Asp Ala Glu Gly

580 585 590

Ser Phe Gln Leu Glu Ile Arg Asn Val Asn Pro Asn Ile Pro Arg Tyr

595 600 605

Lys Thr Arg Leu Arg Phe Glu Ile Asp Leu His Asn Lys Asp Lys Ser

610 615 620

Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys Val Gly Lys Ile Tyr Asn

625 630 635 640

Gln Gly Asp Ser Tyr Val Lys Leu Arg Val Thr Arg Phe Glu Asp Leu

645 650 655

Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro Leu Ile Thr Gln Lys

660 665 670

Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe Ser Val Met Glu Asn

675 680 685

Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu Leu Val Arg Ile Lys

690 695 700

Ala Lys Met Asn Trp Gly Leu Asn Asp Glu Leu Lys Lys Ala Phe Pro

705 710 715 720

Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn Lys Asn Ile Pro Asn

725 730 735

Leu Lys Trp Leu Ala Gly Phe Thr Ser Gly Glu Gly His Phe Gly Val

740 745 750

Ile Leu Ala Lys Arg Arg Pro Ala Ser Pro Val Gln Val Arg Leu Arg

755 760 765

Phe Ala Ile Gly Gln His Ile Arg Asp Lys Asn Leu Met Asn Ser Leu

770 775 780

Ile Thr Tyr Leu Gly Cys Gly Arg Ile Arg Glu Lys Asn Ile Ser Glu

785 790 795 800

Lys Ser Trp Leu Glu Phe Glu Val Thr Lys Phe Ser Asp Ile Asn Asp

805 810 815

Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu Ile Gly Val Lys Leu

820 825 830

Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys Leu Ile Glu Glu Lys

835 840 845

Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile Lys Lys Ile Lys Leu

850 855 860

Asn Met Asn Lys Gly Arg

865 870

<210>16

<211>870

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized megaTAL PD-1.ITSM.ex5_RD2_73

<400>16

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

225 230 235 240

Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val

245 250 255

Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

260 265 270

Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln

275 280 285

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

290 295 300

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro

305 310 315 320

Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu

325 330 335

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu

340 345 350

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

355 360 365

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His

370 375 380

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

385 390 395 400

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

405 410 415

Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn

420 425 430

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu

435 440 445

Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser

450 455 460

Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

465 470 475 480

Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile

485 490 495

Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln

500 505 510

Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu

515 520 525

Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val Lys

530 535 540

Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg Arg

545 550 555 560

Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Ser Arg Arg

565 570 575

Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe Ala Asp Ala Glu Gly

580 585 590

Ser Phe Gln Leu Glu Ile Arg Asn Val Asn Pro Asn Ile Pro Arg Tyr

595 600 605

Arg Thr Arg Leu Arg Phe Glu Ile Asp Leu His Asn Lys Asp Lys Ser

610 615 620

Ile Leu Glu Asp Ile Gln Ser Thr Trp Lys Val Gly Lys Ile Tyr Asn

625 630 635 640

Arg Gly Asp Ser Tyr Val Lys Leu Arg Val Thr Arg Phe Glu Asp Leu

645 650 655

Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro Leu Ile Thr Gln Lys

660 665 670

Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe Ser Val Met Glu Asn

675 680 685

Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu Leu Val Arg Ile Lys

690 695 700

Ala Lys Met Asn Trp Gly Leu Asn Asp Glu Leu Lys Lys Ala Phe Pro

705 710 715 720

Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn Lys Asn Ile Pro Asn

725 730 735

Leu Lys Trp Leu Ala Gly Phe Thr Ser Gly Glu Gly His Phe Gly Val

740 745 750

Ile Leu Ala Lys Arg Arg Pro Ala Ser Pro Val Gln Val Arg Leu Arg

755 760 765

Phe Ala Ile Gly Gln His Ile Arg Asp Lys Asn Leu Met Asn Ser Leu

770 775 780

Ile Thr Tyr Leu Gly Cys Gly Arg Thr Arg Glu Lys Asn Ile Ser Glu

785 790 795 800

Lys Ser Trp Leu Glu Phe Glu Val Thr Lys Phe Ser Asp Ile Asn Asp

805 810 815

Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu Ile Gly Val Lys Leu

820 825 830

Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys Leu Ile Glu Glu Lys

835 840 845

Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile Lys Lys Ile Lys Leu

850 855 860

Asn Met Asn Lys Gly Arg

865 870

<210>17

<211>870

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized megaTAL PD-1.ITSM.ex5_RD3_03

<400>17

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

225 230 235 240

Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val

245 250 255

Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

260 265 270

Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln

275 280 285

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

290 295 300

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro

305 310 315 320

Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu

325 330 335

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu

340 345 350

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

355 360 365

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His

370 375 380

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

385 390 395 400

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

405 410 415

Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn

420 425 430

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu

435 440 445

Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser

450 455 460

Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

465 470 475 480

Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile

485 490 495

Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln

500 505 510

Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu

515 520 525

Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val Lys

530 535 540

Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg Arg

545 550 555 560

Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Ser Arg Arg

565 570 575

Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe Ala Asp Ala Glu Gly

580 585 590

Ser Phe Gln Leu Glu Ile Arg Asn Val Asn Pro Asn Ile Pro Arg Tyr

595 600 605

Arg Thr Arg Leu Arg Phe Glu Ile Asp Leu His Asn Lys Asp Lys Ser

610 615 620

Ile Leu Glu Asp Ile Gln Ser Thr Trp Lys Val Gly Lys Ile Tyr Asn

625 630 635 640

Gln Gly Asp Ser Tyr Val Lys Leu Arg Val Thr Arg Phe Glu Asp Leu

645 650 655

Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro Leu Ile Thr Gln Lys

660 665 670

Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe Ser Val Met Glu Asn

675 680 685

Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu Leu Val Arg Ile Lys

690 695 700

Ala Lys Met Asn Trp Gly Leu Asn Asp Glu Leu Lys Lys Ala Phe Pro

705 710 715 720

Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn Lys Asn Ile Pro Asn

725 730 735

Leu Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp Gly His Phe Gly Val

740 745 750

Ile Leu Ala Lys Arg Arg Pro Ala Ser Pro Val Gln Val Arg Leu Arg

755 760 765

Phe Ala Ile Gly Gln His Ile Arg Asp Lys Asn Leu Met Asn Ser Leu

770 775 780

Ile Thr Tyr Leu Gly Cys Gly Arg Thr Arg Glu Lys Asn Ile Ser Glu

785 790 795 800

Lys Ser Trp Leu Glu Phe Glu Val Thr Lys Phe Ser Asp Ile Asn Asp

805 810 815

Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu Ile Gly Val Lys Leu

820 825 830

Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys Leu Ile Glu Glu Lys

835 840 845

Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile Lys Lys Ile Lys Leu

850 855 860

Asn Met Asn Lys Gly Arg

865 870

<210>18

<211>870

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized megaTAL PD-1.ITSM.ex5_RD4_CV23

<400>18

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

225 230 235 240

Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val

245 250 255

Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

260 265 270

Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln

275 280 285

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

290 295 300

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro

305 310 315 320

Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu

325 330 335

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu

340 345 350

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

355 360 365

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His

370 375 380

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

385 390 395 400

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

405 410 415

Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn

420 425 430

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu

435 440 445

Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser

450 455 460

Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

465 470 475 480

Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile

485 490 495

Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln

500 505 510

Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu

515 520 525

Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val Lys

530 535 540

Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg Arg

545 550 555 560

Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Ser Arg Arg

565 570 575

Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe Ala Asp Ala Glu Gly

580 585 590

Ser Phe Gln Leu Glu Ile Arg Asn Val Asn Pro Asn Ile Pro Arg Tyr

595 600 605

Arg Thr Arg Leu Arg Phe Glu Ile Asp Leu His Asn Lys Asp Lys Ser

610 615 620

Ile Leu Glu Asp Ile Gln Ser Thr Trp Lys Val Gly Lys Ile Tyr Asn

625 630 635 640

Gln Gly Asp Ser Tyr Val Lys Leu Arg Val Thr Arg Phe Glu Asp Leu

645 650 655

Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro Leu Ile Thr Gln Lys

660 665 670

Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe Ser Val Met Glu Asn

675 680 685

Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu Leu Val Arg Ile Lys

690 695 700

Ala Lys Met Asn Trp Gly Leu Asn Asp Glu Leu Lys Lys Ala Phe Pro

705 710 715 720

Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn Lys Asn Ile Pro Asn

725 730 735

Leu Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp Gly His Phe Gly Val

740 745 750

Ile Leu Ala Lys Arg Arg Pro Ala Ser Pro Val Gln Val Arg Leu Arg

755 760 765

Phe Ala Ile Gly Gln His Ile Arg Asp Lys Asn Leu Met Asn Ser Leu

770 775 780

Ile Thr Tyr Leu Gly Cys Gly Arg Ile Arg Glu Lys Asn Lys Ser Glu

785 790 795 800

Lys Ser Trp Leu Glu Phe Glu Val Thr Lys Phe Ser Asp Ile Asn Asp

805 810 815

Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu Ile Gly Val Lys Leu

820 825 830

Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys Leu Ile Glu Glu Lys

835 840 845

Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile Lys Lys Ile Lys Leu

850 855 860

Asn Met Asn Lys Gly Arg

865 870

<210>19

<211>870

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized megaTAL PD-1.ITSM.ex5_RD5_CV23MK

<400>19

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

Ile Ala Ser Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu

225 230 235 240

Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val

245 250 255

Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln

260 265 270

Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln

275 280 285

Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr

290 295 300

Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro

305 310 315 320

Asp Gln Val Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu

325 330 335

Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu

340 345 350

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

355 360 365

Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His

370 375 380

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

385 390 395 400

Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln

405 410 415

Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn

420 425 430

Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu

435 440 445

Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser

450 455 460

Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro

465 470 475 480

Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile

485 490 495

Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln

500 505 510

Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu

515 520 525

Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val Lys

530 535 540

Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg Arg

545 550 555 560

Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Ser Arg Arg

565 570 575

Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe Ala Asp Ala Glu Gly

580 585 590

Ser Phe Gln Leu Glu Ile Arg Asn Val Asn Pro Asn Ile Pro Arg Tyr

595 600 605

Arg Thr Arg Leu Arg Phe Glu Ile Asp Leu His Asn Lys Asp Lys Ser

610 615 620

Ile Leu Glu Asp Ile Gln Ser Thr Trp Lys Val Gly Lys Ile Tyr Asn

625 630 635 640

Gln Gly Asp Ser Tyr Val Lys Leu Arg Val Thr Arg Phe Glu Asp Leu

645 650 655

Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro Leu Ile Thr Gln Lys

660 665 670

Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe Ser Val Met Glu Asn

675 680 685

Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu Leu Val Arg Ile Lys

690 695 700

Ala Lys Met Asn Trp Gly Leu Asn Asp Glu Leu Lys Lys Ala Phe Pro

705 710 715 720

Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn Lys Asn Ile Pro Asn

725 730 735

Leu Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp Gly His Phe Gly Val

740 745 750

Ile Leu Ala Lys Arg Arg Pro Ala Ser Pro Val Gln Val Arg Leu Arg

755 760 765

Phe Met Ile Gly Gln His Ile Lys Asp Lys Asn Leu Met Asn Ser Leu

770 775 780

Ile Thr Tyr Leu Gly Cys Gly Arg Ile Arg Glu Lys Asn Lys Ser Glu

785 790 795 800

Lys Ser Trp Leu Glu Phe Glu Val Thr Lys Phe Ser Asp Ile Asn Asp

805 810 815

Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu Ile Gly Val Lys Leu

820 825 830

Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys Leu Ile Glu Glu Lys

835 840 845

Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile Lys Lys Ile Lys Leu

850 855 860

Asn Met Asn Lys Gly Arg

865 870

<210>20

<211>841

<212>PRT

<213>Artificial sequence

<220>

<223>Synthesized megaTAL PD-1.ile3.exon1_RD1 B1

<400>20

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg

225 230 235 240

Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val

245 250 255

Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val

260 265 270

Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp

275 280 285

Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu

290 295 300

Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr

305 310 315 320

Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala

325 330 335

Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly

340 345 350

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

355 360 365

Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp

370 375 380

His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly

385 390 395 400

Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys

405 410 415

Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

420 425 430

Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

435 440 445

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

450 455 460

Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln Leu

465 470 475 480

Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val

485 490 495

Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val Lys Lys

500 505 510

Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg Arg Ile

515 520 525

Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val Gly Gly Ser

530 535 540

Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe Ala Asp

545 550 555 560

Ala Glu Gly Ser Phe Gly Leu Ser Ile Leu Asn Arg Asn Arg Gly Thr

565 570 575

Ala Arg Tyr His Thr Arg Leu Ser Phe Ala Ile Val Leu His Asn Lys

580 585 590

Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys Val Gly Ile

595 600 605

Ile Thr Asn Asp Gly Asp Arg Tyr Val Arg Leu Arg Val Thr Arg Phe

610 615 620

Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro Leu Val

625 630 635 640

Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe Ser Val

645 650 655

Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu Leu Val

660 665 670

Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu Leu Lys Lys

675 680 685

Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu Ile Asn Lys Asn

690 695 700

Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp Gly Ser

705 710 715 720

Phe Phe Val Arg Leu Arg Lys Ser Asn Val Asn Ala Arg Val Arg Val

725 730 735

Gln Leu Val Phe Glu Ile Ser Gln His Ile Arg Asp Lys Asn Leu Met

740 745 750

Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly His Ile Tyr Glu Gly Asn

755 760 765

Lys Ser Glu Arg Ser Trp Leu Gln Phe Arg Val Glu Lys Phe Ser Asp

770 775 780

Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu Ile Gly

785 790 795 800

Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys Leu Ile

805 810 815

Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile Lys Lys

820 825 830

Ile Lys Leu Asn Met Asn Lys Gly Arg

835 840

<210>21

<211>841

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized megaTAL PD-1.ile3.exon1_RD2_B1H8

<400>21

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg

225 230 235 240

Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val

245 250 255

Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val

260 265 270

Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp

275 280 285

Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu

290 295 300

Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr

305 310 315 320

Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala

325 330 335

Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly

340 345 350

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

355 360 365

Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp

370 375 380

His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly

385 390 395 400

Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys

405 410 415

Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

420 425 430

Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

435 440 445

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

450 455 460

Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Ser Ile Val Ala Gln Leu

465 470 475 480

Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn Asp His Leu Val

485 490 495

Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp Ala Val Lys Lys

500 505 510

Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val Asn Arg Arg Ile

515 520 525

Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg Val Gly Gly Ser

530 535 540

Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr Gly Phe Ala Asp

545 550 555 560

Ala Glu Gly Ser Phe Gly Leu Ser Ile Leu Asn Arg Asn Arg Gly Thr

565 570 575

Ala Arg Tyr His Thr Arg Leu Ser Phe Thr Ile Val Leu His Asn Lys

580 585 590

Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp Lys Val Gly Ile

595 600 605

Ile Thr Asn Asp Gly Asp Arg Tyr Val Arg Leu Cys Val Thr Arg Phe

610 615 620

Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys Tyr Pro Leu Val

625 630 635 640

Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln Ala Phe Ser Val

645 650 655

Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile Lys Glu Leu Ala

660 665 670

Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp Glu Leu Lys Lys

675 680 685

Ala Phe Pro Glu Asn Ile Gly Lys Glu Arg Pro Leu Ile Asn Lys Asn

690 695 700

Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser Gly Asp Gly Ser

705 710 715 720

Phe Phe Val Arg Leu Arg Lys Ser Asn Val Asn Ala Arg Val Arg Val

725 730 735

Gln Leu Val Phe Glu Ile Ser Gln His Ile Arg Asp Lys Asn Leu Met

740 745 750

Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly His Ile Tyr Glu Gly Asn

755 760 765

Lys Ser Glu Arg Ser Trp Leu Gln Phe Arg Val Glu Lys Phe Ser Asp

770 775 780

Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn Thr Leu Ile Gly

785 790 795 800

Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val Ala Lys Leu Ile

805 810 815

Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp Glu Ile Lys Lys

820 825 830

Ile Lys Leu Asn Met Asn Lys Gly Arg

835 840

<210>22

<211>909

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized megaTAL PD-1.IgV.exon2_RD1_G5

<400>22

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

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

225 230 235 240

Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val

245 250 255

Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val

260 265 270

Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp

275 280 285

Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu

290 295 300

Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr

305 310 315 320

Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala

325 330 335

Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly

340 345 350

Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys

355 360 365

Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp

370 375 380

His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly

385 390 395 400

Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys

405 410 415

Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

420 425 430

Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

435 440 445

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

450 455 460

Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

465 470 475 480

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

485 490 495

Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg

500 505 510

Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val

515 520 525

Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Ser Ile

530 535 540

Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn

545 550 555 560

Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp

565 570 575

Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val

580 585 590

Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg

595 600 605

Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

610 615 620

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gln Leu Tyr Ile Ser Asn Val

625 630 635 640

Asn Asn Asn Arg Ser Arg Tyr Arg Ala Arg Leu Arg Phe Ala Ile Glu

645 650 655

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

660 665 670

Lys Val Gly Val Ile Asn Asn Ile Gly Asp Thr Ser Val Arg Leu Ser

675 680 685

Val Gly Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

690 695 700

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

705 710 715 720

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

725 730 735

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

740 745 750

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

755 760 765

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

770 775 780

Gly Asp Gly Asn Phe Tyr Val His Leu Lys Lys Ser Gly Arg Thr Thr

785 790 795 800

Arg Val Tyr Val Gln Leu Arg Phe Ser Ile Ala Gln His Ile Arg Asp

805 810 815

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile

820 825 830

Asn Glu Trp Asn Ala Ser Glu Arg Ser Ala Leu Glu Phe Arg Val Thr

835 840 845

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

850 855 860

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

865 870 875 880

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

885 890 895

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg

900 905

<210>23

<211>909

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized megaTAL PD-1.IgV.exon2_RD1_PS3

<400>23

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

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

225 230 235 240

Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val

245 250 255

Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala Leu Glu Thr Val

260 265 270

Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp

275 280 285

Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu

290 295 300

Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr

305 310 315 320

Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly Gly Lys Gln Ala

325 330 335

Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly

340 345 350

Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys

355 360 365

Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp

370 375 380

His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn Ile Gly

385 390 395 400

Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys

405 410 415

Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

420 425 430

Ile Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

435 440 445

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

450 455 460

Ser His Asp Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

465 470 475 480

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

485 490 495

Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg

500 505 510

Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val

515 520 525

Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Ser Ile

530 535 540

Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu Thr Asn

545 550 555 560

Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala Met Asp

565 570 575

Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg Arg Val

580 585 590

Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile Ser Arg

595 600 605

Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

610 615 620

Gly Phe Ala Asp Ala Glu Gly Cys Phe Leu Leu His Ile Arg Asn Leu

625 630 635 640

Asn Arg Thr Ser Thr Lys Tyr Arg Thr Arg Leu Ser Phe Glu Ile Glu

645 650 655

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

660 665 670

Lys Val Gly Ile Ile Asn Asn Ile Gly Asn Arg Arg Val Arg Leu Ser

675 680 685

Val Arg Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

690 695 700

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

705 710 715 720

Ala Phe Ser Leu Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

725 730 735

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

740 745 750

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

755 760 765

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

770 775 780

Gly Asp Gly Asn Phe Tyr Val His Leu Lys Lys Ser Gly Arg Thr Thr

785 790 795 800

Arg Val Tyr Val Gln Leu Arg Phe Ser Ile Ser Gln His Ile Arg Asp

805 810 815

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Tyr Ile

820 825 830

Thr Glu Ser Asn Pro Ser Glu Arg Ser Asp Leu Glu Phe Arg Val Thr

835 840 845

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

850 855 860

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

865 870 875 880

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

885 890 895

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg

900 905

<210>24

<211>236

<212>PROTEIN

<213>Mus musculus

<400>24

Met Ser Glu Pro Pro Arg Ala Glu Thr Phe Val Phe Leu Asp Leu Glu

1 5 10 15

Ala Thr Gly Leu Pro Asn Met Asp Pro Glu Ile Ala Glu Ile Ser Leu

20 25 30

Phe Ala Val His Arg Ser Ser Leu Glu Asn Pro Glu Arg Asp Asp Ser

35 40 45

Gly Ser Leu Val Leu Pro Arg Val Leu Asp Lys Leu Thr Leu Cys Met

50 55 60

Cys Pro Glu Arg Pro Phe Thr Ala Lys Ala Ser Glu Ile Thr Gly Leu

65 70 75 80

Ser Ser Glu Ser Leu Met His Cys Gly Lys Ala Gly Phe Asn Gly Ala

85 90 95

Val Val Arg Thr Leu Gln Gly Phe Leu Ser Arg Gln Glu Gly Pro Ile

100 105 110

Cys Leu Val Ala His Asn Gly Phe Asp Tyr Asp Phe Pro Leu Leu Cys

115 120 125

Thr Gly Leu Gln Arg Leu Gly Ala His Leu Pro Gln Asp Thr Val Cys

130 135 140

Leu Asp Thr Leu Pro Ala Leu Arg Gly Leu Asp Arg Ala His Ser His

145 150 155 160

Gly Thr Arg Ala Gln Gly Arg Lys Ser Tyr Ser Leu Ala Ser Leu Phe

165 170 175

His Arg Tyr Phe Gln Ala Glu Pro Ser Ala Ala His Ser Ala Glu Gly

180 185 190

Asp Val His Thr Leu Leu Leu Ile Phe Leu His Arg Ala Pro Glu Leu

195 200 205

Leu Ala Trp Ala Asp Glu Gln Ala Arg Ser Trp Ala His Ile Glu Pro

210 215 220

Met Tyr Val Pro Pro Asp Gly Pro Ser Leu Glu Ala

225 230 235

<210>25

<211>22

<212>DNA

<213>Homo sapiens

<400>25

aatggtggca tactccgtct gc 22

<210>26

<211>11

<212>DNA

<213>Homo sapiens

<400>26

tccgctagga a 11

<210>27

<211>37

<212>DNA

<213>Homo sapiens

<400>27

tccgctagga aagacaatgg tggcatactc cgtctgc 37

<210>28

<211>22

<212>DNA

<213>Homo sapiens

<400>28

aatggtggca tacaaccttt ta 22

<210>29

<211>22

<212>DNA

<213>Homo sapiens

<400>29

tttccactta tactccgtct gc 22

<210>30

<211>22

<212>DNA

<213>Homo sapiens

<400>30

ggcatgcaga tcccacaggc gc 22

<210>31

<211>14

<212>DNA

<213>Homo sapiens

<400>31

ggtggggctg ctcc 14

<210>32

<211>37

<212>DNA

<213>Homo sapiens

<400>32

ggtggggctg ctccaggcat gcagatccca caggcgc 37

<210>33

<211>22

<212>DNA

<213>Homo sapiens

<400>33

ggcatgcaga tccaaccttt ta 22

<210>34

<211>22

<212>DNA

<213>Homo sapiens

<400>34

tttccactta tcccacaggc gc 22

<210>35

<211>22

<212>DNA

<213>Homo sapiens

<400>35

acgggcgtga cttccacatg ag 22

<210>36

<211>12

<212>DNA

<213>Homo sapiens

<400>36

gtcacacaac tg 12

<210>37

<211>38

<212>DNA

<213>Homo sapiens

<400>37

gtcacacaac tgcccaacgg gcgtgacttc cacatgag 38

<210>38

<211>22

<212>DNA

<213>Homo sapiens

<400>38

acggggcgtga cttaaccttt ta 22

<210>39

<211>22

<212>DNA

<213>Homo sapiens

<400>39

tttccactta cttccacatg ag 22

<210>40

<211>2650

<212>RNA

<213>Artificial sequence

<220>

<223>Synthesized mRNA megaTAL RD5_CV23MK exon 5 PD-1

<400>40

augggauccg ccccccccaa gaagaagcgc aagguggugg accugagaac ccugggguac 60

agccagcagc aacaggagaa gaucaaaccg aaggugcgca gcacgguggc ccagcaccau 120

180

gccgcccugg gcaccguggc ggugaccuac cagcacauca ucaccgcccu gcccgaagcc 240

acccacgagg acaucguggg agugggcaag cagugguccg gagcacgcgc ccuggaggcc 300

cugcugaccg acgccgggga acugcgcggc ccgccucugc agcuggauac cggccaacug 360

gucaagaucg ccaagagg cggagugacc gcgauggagg ccguccacgc cucccggaac 420

gcucugaccg gugccccgcu caaucugacu ccggaccagg ugguggcuau cgccagcaac 480

ggaggaggaa aacaggcccu cgaaacagug cagaggcugc ugccuguccu uugucaagau 540

cacggggcuga cucccgacca ggucguggcc auugccagcc acgacggcgg caagcaggcu 600

uuggagacug ugcagcggcu ccugccagug cugugccaag aucacggucu gaccccagau 660

caggucgucg ccauugcuuc caacgggaggc aaacaagcgc uggaaacggu ccaacgccug 720

cugccggugc uuugucagga ucacggccug accccugauc aggugguggc caucgcgucc 780

aauaacgggg ggaagcaggc acucgagacu guccagaggc uccugccugu gcucugccag 840

gaccacgggc ucacacccga ucaggucguc gcuaucgcgu cgcacgacgg uggaaagcag 900

gcccuggaaa ccgugcagcg ccuguugccg gugcuguguc aggaccaugg ccuuucuccg 960

gaucaggucg ucgcgaucgc aucuaauggu ggaggaaagc aggcccugga gacaguccag 1020

cgccugcucc cgguguugug ccaagaccau ggucuuaccc cugaccaggu ggucgcuauu 1080

1140

ugcggauc auggauugac cccagaccag gugguggcga uugccagcaa caacggcggg 1200

1260

accccggacc aagucgucgc caucgcuucc aacaacggag ggaagcaggc acucgaaacu 1320

1380

gccaucgcaa gcaacaucgg uggcaaacag gcucuggaaa cuguccaaag acugcugccc 1440

gugcuuugcc aggaccacgg acugacuccu gaccaagugg uggcaauugc cuccaacauc 1500

ggaggcaagc aagcgcucga auccaucgug gcgcagcuca gccggccaga ccccgcccug 1560

gccgcccuga cuaacgauca ccugguggcc cuggcgugcc ucggcggucg ccccgcuaug 1620

gacgcgguga agaaggggcu gccccgcc cccgagcuca uucggcgggu gaaccgccgg 1680

1740

cccuggaucc ugacuggcuu ugccgacgcc gagggauccu uccagcucga aauccggaac 1800

gugaacccaa acaucccccg guaucgcacc agacugcggu ucgagaucga ccuucacaac 1860

aaggacaagu ccauucugga ggacauccag ucaaccugga aagugggaaa gaucuacaac 1920

cagggggacu cauacgugaa gcugcgggug acccgcuucg aagaucucaa agugaucauc 1980

2040

2100

gugcgaauua aggcgaaaau gaacugggga uugaacgaug agcugaagaa ggcguucccg 2160

gaaaacauuu ccaaggagcg cccgcucauc aacaagaaca ucccuaaucu gaaguggcuc 2220

gcgggguuua ccucuggcga cggccauuuc ggagugauuc ucgcaaagcg caggcccgcc 2280

agcccuguc aagugcggcu gcgguucaug aucggccagc acauuaagga caagaaccug 2340

augaacucgc ucaucaccua ccuuggaugc ggccggauuc gcgaaaagaa caagagcgag 2400

uucagcgaca ucaacgacaa gaucauuccg 2460

guguuccagg aaaacacccu gauuggcgug aagcuggagg acuucgagga uuggugcaag 2520

guggccaagc ucaucgaaga aaagaagcac cugacugaau ccggcuugga cgagaucaag 2580

aagauuaagc ugaauuugaa caagggaagg ugauagcgcg cuagccguac ggaaaaaaaa 2640

aaaaaaaaaa 2650

<210>41

<211>2526

<212>RNA

<213>Artificial sequence

<220>

<223>Synthesized mRNA megaTAL RD2_B1H8 exon 1 PD-1

<400>41

augggaagcg ccccgccgaa gaagaagcgc aagguggugg aucugagaac ccugggauac 60

agccagcagc agcaggagaa gaucaagccg aagguccggu cuaccguggc ccagcaccau 120

gaggcccuug ugggccacgg cuucacacau gcacacaucg ucgcccuguc gcagcauccc 180

gccgcccugg ggaccguggc cgugaccuau caacacauca uuaccgcccu gccgggaggcc 240

acccacgagg acaucguggg uguggggaag caguggagcg gagccagggc acucgaagcc 300

cuccucacug acgcuggaga acugcgcgga ccgccucucc agcuggacac cggacagcug 360

gugaaaaucg ccaagcgggg aggagugacc gccauggaag ccgugcacgc cucgaggaac 420

gcgcugacug gcgccccucu gaaccugacc ccugaucagg ucguggcuau cgccucaaac 480

aacgggggua agcaggcgcu ggagacagug caacgacuuc ugccagugcu uugucaggac 540

cauggucuga cccccgacca ggucgucgcc auugcaucca acaauggugg caagcaggca 600

660

caaguggucg ccaucgccuc caacggagga ggaaaacaag cucuggagac ugugcaacgc 720

780

ucgaacaacg gaggaaagca agcgcuggaa accgugcagc gccuccugcc cguccucugc 840

caggaucacg gccugacucc ggaccaggug gucgcgaucg ccagcaauaa cggggggaag 900

caagcccucg agacugugca gcgguugcug cccgugcucu gccaagauca uggccuuacc 960

ccagaccaag ucguggccau ugcuuccaac aacgguggca aacaggcgcu cgaaaccguc 1020

1080

auugcgucca acaacggugg aaagcaggcc cuggaaacgg ugcagcggcu gcuuccgguc 1140

cugugucagg aucaugggcu gacuccgac caggucgucg ccauugcauc ccacgauggg 1200

1260

cuuaccccgg aucagguggu ggccauagcc ucgaacggcg gcgggaaaca ggcucuggaa 1320

1380

guggcuauug cccucuaacaa cggcggcaag caagcacucg aaagcaucgu ggcccaguug 1440

ucacgccccg accccgcacu ggcugcccug acgaaugacc aucugguggc gcuggccugc 1500

cugggaggga ggccagcgau ggaugcggug aagaagggac ugccccaugc uccggagcug 1560

1620

gugggcggca gcucccggcg cgaguccauu aaccccugga uccugaccgg cuuugccgac 1680

gccgaagggu ccuucggccu cucgauccug aaccggaacc gggguaccgc ucgguaccac 1740

accagacugu ccuucaccau cgugcugcac aacaaggaca agagcauccu cgaaaacauu 1800

cagucaacgu ggaagguggg aauuauuacu aacgacggcg acagauacgu gcgccugugc 1860

gugacccggu uugaggaccu gaaggucauu aucgaccacu ucgagaagua cccccucgug 1920

acucagaagc ugggagacua caagcuguuc aagcaggcgu ucucggugau ggaaaacaag 1980

gagcaccuga aggagaacgg caucaaggag cucgccggga ucaaggccaa gaugaacugg 2040

ggccugaaug augaacucaa gaaggcguuc ccugaaaaca ucgguaaaga acggccccug 2100

aucaacaaga acaucccgaa cuucaagugg cuugccggau ucaccuccgg cgacggaucc 2160

2220

gaaaucucac agcacaucag ggacaagaau uugaugaacu cccucaucac cuaccugggu 2280

ugcggacaca ucuacgaagg caauaagucg gagcggagcu ggcugcaguu ucgcguggaa 2340

aaguucuccg acauuaacga caagaucauc ccaguguucc aggaaaacac ucugauuggc 2400

2460

caccugaccg aguccggccu ggacgaaauc aagaaaauca agcugaacau gaacaaggga 2520

cgguga 2526

<210>42

<211>2730

<212>RNA

<213>Artificial sequence

<220>

<223>Synthesized mRNA megaTAL RD1_G5 exon 2 PD-1

<400>42

augggaagcg ccccuccgaa gaagaagcgc aaggucgugg accucagaac ccuggguuac 60

ucccagcagc agcaggagaa aauuaagccg aaggugcgcu ccaccguggc ucaacaccac 120

180

gccgcccugg gcaccguggc ggugaccuac cagcacauca ucaccgcgcu gccugaagcc 240

acccacgagg acaucgucgg uguggguaag cagugguccg gagccagagc ccuggaggcu 300

cugcugaccg acgccggaga acucagaggc ccgccucugc agcuggacac cggacagcug 360

gugaagauag ccaagagagg cggcgugacc gccauggaag ccgugcaugc gucccgcaac 420

gcacugaccg gggccccccu gaaccugacu ccagaccaag ugguggcuau cgccagcaac 480

aauggaggaa agcaagccuu ggaaaccgug cagcggcugc ucccgguccu uugccaagac 540

cacggccuga cacccgauca ggugguggca aucgcaucga auggcggcgg gaagcaggcc 600

cucgagacug ugcagaggcu ccugccugug cugugccagg accaugggcu gaccccagac 660

cagguggucg caaucgccuc gcacgacgga ggcaagcaag cccuggaaac uguccagcgc 720

cugcucccug uccuguguca agaucauggg cucacuccug aucaggucgu cgccaucgcc 780

ucgaacauug guggcaagca ggcgcucgaa accguccagc gguugcugcc agugcuuugc 840

caggaccaug gucugacccc cgaucaagug gucgcgauug ccucacacga uggcgguaag 900

caggcuuugg aaaccgugca acggcuguug ccuguccucu gccaggacca cggcuugacu 960

cccgaccagg ugguggccau agccucaaac aucggaggga aacaagcccu cgaaaccguc 1020

cagaggcugc ugccgguguu gugccaggau cacggauuga ccccagacca gguggucgcc 1080

1140

cugugucagg aucauggacu uacccccgac caagucgugg cgauugcuuc cauauucggga 1200

1260

1320

1380

guggcuaucg caucccauga uggugggaaa caggcucuug aaacugugca acgccugcug 1440

ccggugcugu gccaagacca cggacugacu ccggaccagg ucguggccau cgcuucaaac 1500

1560

cacggguuga ccccggacca ggucguggcu auugccucga acaacggggg gaagcaagcg 1620

cucgagucca uuguggccca gcugagccgg ccugaucccg cacuggccgc gcugaccaac 1680

1740

ggacugccgc acgcgcccga gcugauccgc cgcgugaaca ggcggauugg agaacgcacc 1800

ucccaccggg uggccaucuc gagaguggga ggauccucua gacgggaguc cauuaacccg 1860

uggauccuga cuggauucgc cgacgccgag ggguccuucc agcuguacau cuccaacgug 1920

aacaacaacc gcagcagaua cagggcccgg cugcgguucg cgaucgaacu gcacaauaag 1980

2040

ggcgauacca gcgugcggcu cuccgugggg cgguucgaag aucucaaggu gaucaucgac 2100

cacuucgaga aguacccgcu gaucacccag aagcugggag acuacaagcu guucaagcaa 2160

gcuuucagcg ugauggaaaa caaggaacau cugaaagaga acgguaucaa ggaacuggug 2220

cggauuaagg ccaagaugaa cuggggucug aacgaugaac ugaagaaggc guucccggag 2280

2340

ggcuuuacuu ccggagaugg uaacuucuac gugcaccuga agaaguccgg aagaaccacc 2400

2460

aacucccuga ucaccuaccu cgguugcgga uacaucaacg aauggaacgc cagcgagaga 2520

ucggcccugg aguucagggu gacuaaguuc uccgacauca acgacaagau uauccccgug 2580

uuucaggaaa auacccucau cggcgugaag cuggaggacu uugaggacug gugcaaggug 2640

gccaagcuga ucgaggaaaa gaaacaucug accgagagcg gccuggacga aaucaagaag 2700

auuaagcuga acaugaacaa gggacgauga 2730

<210>43

<211>711

<212>RNA

<213>Mus musculus

<400>43

augucugagc caccucgggc ugagaccuuu guauuccugg accuagaagc cacugggcuc

ccaaacaugg acccugagau ugcagagaua ucccuuuuug cuguucaccg cucuucccug 120

gagaacccag aacgggauga uucugguucc uuggugcugc cccguguucu ggacaagcuc 180

acacugugca ugugcccgga gcgccccuuu acugccaagg ccagugagau uacugguuug 240

agcagcgaaa gccugaugca cugcgggaag gcugguuuca auggcgcugu gguaaggaca 300

cugcagggcu uccuaagccg ccaggagggc cccaucugcc uuguggccca caauggcuuc 360

gauuaugacu uccacugcu gugcacgggg cuacaacguc ugggugccca ucugccccaa 420

gacacugucu gccuggacac acugccugca uugcggggcc uggaccgugc ucacagccac 480

ggcaccagggg cucaaggccg caaaagcuac agccuggcca gucucuucca ccgcuacuuc 540

caggcugaac ccagugcugc ccauucagca gaaggugaug ugcacacccu gcuucugauc 600

uuccugcauc gugcuccuga gcugcucgcc ugggcagaug agcaggcccg cagcugggcu 660

cauauugagc ccauguacgu gccaccugau gguccaagcc ucgaagccug a 711

<210>44

<211>7601

<212>DNA

<213>Artificial sequence

<220>

<223>Exon5_MND-BFP rAAV PD-1 synthesized construct

<400>44

cagctgcgcg ctcgctcgct cactgaggcc gcccggggcaa agcccggggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagaattca 180

gtccagggct ctgtcctgca cctggggaat ggtgaccggc atctctgtcc tctagctctg 240

gaagcacccc agcccctcta gtctgccctc acccctgacc ctgaccctcc accctgaccc 300

cgtcctaacc cctgaccttt gtgcccttcc agagagaagg gcagaagtgc cccagccca 360

ccccagcccc tcacccaggc cagccggcca gttccaaacc ctggtggttg gtgtcgtggg 420

cggcctgctg ggcagcctgg tgctgctagt ctgggtcctg gccgtcatct gctcccggggc 480

cgcacgaggt aacgtcatcc cagcccctcg gcctgccctg ccctaaccct gctggcggcc 540

ctcactcccg ccctccccttc ctccaccctt ccctcacccc accccacctc cccccatctc 600

cccgccaggc taagtccctg atgaaggccc ctggactaag accccccacc taggagcacg 660

gctcagggtc ggcctggtga ccccaagtgt gtttctctgc agggacaata ggagccaggc 720

gcaccggcca gcccctggtg agtctcactc ttttcctgca tgatccactg tgccttcctt 780

cctgggtggg cagaggtgga aggacaggct gggaccacac ggcctgcagg actcacattc 840

tattatagcc aggaccccac ctccccagcc cccaggcagc aacctcaatc cctaaagcca 900

tgatctgggg ccccagccca cctgcggtct ccggggggtgc ccggcccatg tgtgtgcctg 960

1020

ccggcccaca tatgtgcctg cctgcggtct ccaggtgtgc ccggcccatg cgtgtgccca 1080

cctgcgaggg cgtggggtgg gcttggtcat ttcttatctt acattggaga caggaagct 1140

tgaaaagtca cattttggaa tcctaaatct gcaagaatgc cagggacatt tcagagggggg 1200

1260

gctcagggta agcagctcat agtggggggc ccaggttcgg tgccggtact gcagccaggc 1320

tgtggagccg cgggcctcct tcctgcggtg ggccgtgggg ctgactccct ctccctttct 1380

cctcaaagaa ggaggacccc tcagccgtgc ctgtgttctc tgtggactat ggggagctgg 1440

atttccagtg gcgagagaag accccggagc cccccgtgcc ctgtgtccct gagcagacgg 1500

agtaatgcat gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca 1560

gttcctgccc cggctcaggg ccaagaacag ttggaacagc agaatatgggg ccaaacagga 1620

tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1680

ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 1740

tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 1800

gcgcttctgc tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatcgcc 1860

tggagacgcc atccacgctg ttttgacttc catagaagga tctcgaggcc accatggcta 1920

gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc 1980

atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga 2040

gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct 2100

tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc 2160

agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaggac gggggcgtgc 2220

tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca 2280

gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg 2340

ccttcaccga gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc 2400

tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat agatccaaga 2460

aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa 2520

tcaaggaggc caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact 2580

gcgacctccc tagcaaactg gggcacaagc taaattgaaa gctttgcttt atttgtgaaa 2640

tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gtttaacaac 2700

2760

cgacccacca ttgtctttcc tagcggaatg ggcacctcat cccccgcccg caggggctca 2820

gctgacggcc ctcggagtgc ccagccactg aggcctgagg atggacactg ctcttggccc 2880

ctctgaccgg cttccttggc caccagtgtt ctgcagaccc tccaccatga gcccgggtca 2940

gcgcatttcc tcaggagaag caggcagggt gcaggccatt gcaggccgtc caggggctga 3000

gctgcctggg ggcgaccggg gctccagcct gcacctgcac caggcacagc cccaccacag 3060

gactcatgtc tcaatgccca cagtgagccc aggcagcagg tgtcaccgtc ccctacaggg 3120

agggccagat gcagtcactg cttcaggtcc tgccagcaca gagctgcctg cgtccagctc 3180

cctgaatctc tgctgctgct gctgctgctg ctgctgctgc ctgcggcccg gggctgaagg 3240

cgccgtggcc ctgcctgacg ccccggagcc tcctgcctga acttgggggc tggttggaga 3300

tggccttgga gcagccaagg tgcccctggc agtggcatcc cgaaacgccc tggacgcagg 3360

gcccaagact gggcacagga gtgggaggta catggggctg gggactcccc aggagttatc 3420

tgctccctgc aggcctagag aagtttcagg gaaggtcaga agagctcctg gctgtggtgg 3480

gcagggcagg aaacccctcc acctttacac atgcccaggc agcacctcag gccctttgtg 3540

gggcagggaa gctgaggcag taagcgggca ggcagagctg gaggcctttc aggcccagcc 3600

agcactctgg ccctcctgccg ccgcattcca ccccagcccc tcacaccact cgggagaggg 3660

acatcctacg gtcccaaggt caggagggca gggctggggt tgactcaggc ccctcccagc 3720

tgtggccacc tgggtgttgg gagggcagaa gtgcaggcac ctagggcccc ccatgtgccc 3780

accctgggag ctctccttgg aacccattcc tgaaattatt taaaaggggtt ggccggacta 3840

gttacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg 3900

agttggccac tccctctctg cgcgctcgct cgctcactga ggccggggcga ccaaaggtcg 3960

cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc cagctggcgt 4020

aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 4080

tggcgattcc gttgcaatgg ctggcggtaa tattgttctg gatattacca gcaaggccga 4140

tagtttgagt tcttctactc aggcaagtga tgttattact aatcaaagaa gtattgcgac 4200

aacggttaat ttgcgtgatg gacagactct tttactcggt ggcctcactg attataaaaa 4260

cacttctcag gattctggcg taccgttcct gtctaaaatc cctttaatcg gcctcctgtt 4320

tagctcccgc tctgattcta acgaggaaag cacgttatac gtgctcgtca aagcaaccat 4380

agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4440

4500

ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4560

ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 4620

ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4680

gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4740

tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4800

ttaacgcgaa ttttaacaaa atattaacgt ttacaattta aatatttgct tatacaatct 4860

4920

tacgattacc gttcatcgat tctcttgttt gctccagact ctcaggcaat gacctgatag 4980

cctttgtaga gacctctcaa aaatagctac cctctccggc atgaatttat cagctagaac 5040

ggttgaatat catattgatg gtgatttgac tgtctccggc ctttctcacc cgtttgaatc 5100

tttacctaca cattactcag gcattgcatt taaaatatat gagggttcta aaaattttta 5160

tccttgcgtt gaaataaagg cttctcccgc aaaagtatta cagggtcata atgtttttgg 5220

tacaaccgat ttagctttat gctctgaggc tttattgctt aattttgcta attctttgcc 5280

ttgcctgtat gatttattgg atgttggaat cgcctgatgc ggtattttct ccttacgcat 5340

ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca 5400

tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 5460

ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 5520

ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 5580

taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 5640

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 5700

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 5760

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 5820

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 5880

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 5940

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 6000

6060

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 6120

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 6180

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 6240

6300

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 6360

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 6420

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 6480

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 6540

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 6600

6660

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 6720

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 6780

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 6840

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 6900

agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 6960

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 7020

gccagtggcg ataagtcgtg tcttaccgggg ttggactcaa gacgatagtt accggataag 7080

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 7140

tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 7200

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 7260

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 7320

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 7380

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 7440

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 7500

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata 7560

cgcaaaccgc ctctccccgc gcgttggccg attcattaat g 7601

<210>45

<211>1325

<212>DNA

<213>Homo sapiens

<400>45

agtccagggc tctgtcctgc acctggggaa tggtgaccgg catctctgtc ctctagctct 60

ggaagcaccc cagcccctct agtctgccct cacccctgac cctgaccctc caccctgacc 120

ccgtcctaac ccctgacctt tgtgcccttc cagagagaag ggcagaagtg cccacagccc 180

accccagccc ctcacccagg ccagccggcc agttccaaac cctggtggtt ggtgtcgtgg 240

gcggcctgct gggcagcctg gtgctgctag tctgggtcct ggccgtcatc tgctcccggg 300

ccgcacgagg taacgtcatc ccagcccctc ggcctgccct gccctaaccc tgctggcggc 360

cctcactccc gcctcccctt cctccaccct tccctcaccc caccccacct ccccccatct 420

ccccgccagg ctaagtccct gatgaaggcc cctggactaa gaccccccac ctaggagcac 480

ggctcagggt cggcctggtg accccaagtg tgtttctctg cagggacaat aggagccagg 540

cgcaccggcc agcccctggt gagtctcact cttttcctgc atgatccact gtgccttcct 600

tcctgggtgg gcagaggtgg aaggacaggc tgggaccaca cggcctgcag gactcacatt 660

ctattatagc caggacccca cctccccagc ccccaggcag caacctcaat ccctaaagcc 720

atgatctggg gccccagccc acctgcggtc tccgggggtg cccggcccat gtgtgtgcct 780

gcctgcggtc tccaggggtg cctggcccac gcgtgtgccc gcctgcggtc tctgggggtg 840

cccggcccac atatgtgcct gcctgcggtc tccaggtgtg cccggcccat gcgtgtgccc 900

acctgcgagg gcgtggggtg ggcttggtca tttcttatct tacattggag acaggagagc 960

ttgaaaagtc acattttgga atcctaaatc tgcaagaatg ccagggacat ttcagagggg 1020

gacattgagc cagagaggag gggtggtgtc cccagatcac acagagggca gtggtgggac 1080

agctcagggt aagcagctca tagtgggggg cccaggttcg gtgccggtac tgcagccagg 1140

ctgtggagcc gcgggcctcc ttcctgcggt gggccgtggg gctgactccc tctccctttc 1200

1260

gatttccagt ggcgagagaa gaccccggag ccccccgtgc cctgtgtccc tgagcagacg 1320

gagta 1325

<210>46

<211>1072

<212>DNA

<213>Homo sapiens

<400>46

ccaccattgt ctttcctagc ggaatgggca cctcatcccc cgcccgcagg ggctcagctg 60

acggccctcg gagtgcccag ccactgaggc ctgaggatgg acactgctct tggcccctct 120

gaccggcttc cttggccacc agtgttctgc agaccctcca ccatgagccc gggtcagcgc 180

atttcctcag gagaagcagg cagggtgcag gccattgcag gccgtccagg ggctgagctg 240

cctgggggcg accggggctc cagcctgcac ctgcaccagg cacagcccca ccacaggact 300

catgtctcaa tgcccacagt gagcccaggc agcaggtgtc accgtcccct acagggaggg 360

ccagatgcag tcactgcttc aggtcctgcc agcacagagc tgcctgcgtc cagctccctg 420

aatctctgct gctgctgctg ctgctgctgc tgctgcctgc ggcccggggc tgaaggcgcc 480

gtggccctgc ctgacgcccc ggagcctcct gcctgaactt gggggctggt tggagatggc 540

cttggagcag ccaaggtgcc cctggcagtg gcatcccgaa acgccctgga cgcagggccc 600

aagactgggc acaggagtgg gaggtacatg gggctgggga ctccccagga gttatctgct 660

ccctgcaggc ctagagaagt ttcagggaag gtcagaagag ctcctggctg tggtgggcag 720

ggcaggaaac ccctccacct ttacacatgc ccaggcagca cctcaggccc tttgtggggc 780

agggaagctg aggcagtaag cgggcaggca gagctggagg cctttcaggc cggccagca 840

ctctggcctc ctgccgccgc attccacccc agcccctcac accactcggg agagggacat 900

cctacggtcc caaggtcagg agggcagggc tggggttgac tcaggcccct cccagctgtg 960

gccacctggg tgttgggagg gcagaagtgc aggcacctag ggccccccat gtgcccaccc 1020

tgggagctct ccttgggaacc cattcctgaa attatttaaa ggggttggcc gg 1072

<210>47

<211>7601

<212>DNA

<213>Artificial sequence

<220>

<223>Synthesized SNP construct of the 3' homologous arm of rAAV PD-1

Exon5_MND-BFP

<400>47

cagctgcgcg ctcgctcgct cactgaggcc gcccggggcaa agcccggggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagaattca 180

gtccagggct ctgtcctgca cctggggaat ggtgaccggc atctctgtcc tctagctctg 240

gaagcacccc agcccctcta gtctgccctc acccctgacc ctgaccctcc accctgaccc 300

cgtcctaacc cctgaccttt gtgcccttcc agagagaagg gcagaagtgc cccagccca 360

ccccagcccc tcacccaggc cagccggcca gttccaaacc ctggtggttg gtgtcgtggg 420

cggcctgctg ggcagcctgg tgctgctagt ctgggtcctg gccgtcatct gctcccggggc 480

cgcacgaggt aacgtcatcc cagcccctcg gcctgccctg ccctaaccct gctggcggcc 540

ctcactcccg ccctccccttc ctccaccctt ccctcacccc accccacctc cccccatctc 600

cccgccaggc taagtccctg atgaaggccc ctggactaag accccccacc taggagcacg 660

gctcagggtc ggcctggtga ccccaagtgt gtttctctgc agggacaata ggagccaggc 720

gcaccggcca gcccctggtg agtctcactc ttttcctgca tgatccactg tgccttcctt 780

cctgggtggg cagaggtgga aggacaggct gggaccacac ggcctgcagg actcacattc 840

tattatagcc aggaccccac ctccccagcc cccaggcagc aacctcaatc cctaaagcca 900

tgatctgggg ccccagccca cctgcggtct ccggggggtgc ccggcccatg tgtgtgcctg 960

1020

ccggcccaca tatgtgcctg cctgcggtct ccaggtgtgc ccggcccatg cgtgtgccca 1080

cctgcgaggg cgtggggtgg gcttggtcat ttcttatctt acattggaga caggaagct 1140

tgaaaagtca cattttggaa tcctaaatct gcaagaatgc cagggacatt tcagagggggg 1200

1260

gctcagggta agcagctcat agtggggggc ccaggttcgg tgccggtact gcagccaggc 1320

tgtggagccg cgggcctcct tcctgcggtg ggccgtgggg ctgactccct ctccctttct 1380

cctcaaagaa ggaggacccc tcagccgtgc ctgtgttctc tgtggactat ggggagctgg 1440

atttccagtg gcgagagaag accccggagc cccccgtgcc ctgtgtccct gagcagacgg 1500

agtaatgcat gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca 1560

gttcctgccc cggctcaggg ccaagaacag ttggaacagc agaatatgggg ccaaacagga 1620

tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1680

ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 1740

tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 1800

gcgcttctgc tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatcgcc 1860

tggagacgcc atccacgctg ttttgacttc catagaagga tctcgaggcc accatggcta 1920

gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc 1980

atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga 2040

gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct 2100

tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc 2160

agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaggac gggggcgtgc 2220

tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca 2280

gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg 2340

ccttcaccga gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc 2400

tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat agatccaaga 2460

aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa 2520

tcaaggaggc caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact 2580

gcgacctccc tagcaaactg gggcacaagc taaattgaaa gctttgcttt atttgtgaaa 2640

tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gtttaacaac 2700

2760

cgacccacca ttgtctttcc tagcggaatg ggcacctcat cccccgcccg caggggctca 2820

gccgacggcc ctcggagtgc ccagccactg aggcctgagg atggacactg ctcttggccc 2880

ctctgaccgg cttccttggc caccagtgtt ctgcagaccc tccaccatga gcccgggtca 2940

gcgcatttcc tcaggagaag caggcagggt gcaggccatt gcaggccgtc caggggctga 3000

gctgcctggg ggcgaccggg gctccagcct gcacctgcac caggcacagc cccaccacag 3060

gactcatgtc tcaatgccca cagtgagccc aggcagcagg tgtcaccgtc ccctacaggg 3120

agggccagat gcagtcactg cttcaggtcc tgccagcaca gagctgcctg cgtccagctc 3180

cctgaatctc tgctgctgct gctgctgctg ctgctgctgc ctgcggcccg gggctgaagg 3240

cgccgtggcc ctgcctgacg ccccggagcc tcctgcctga acttgggggc tggttggaga 3300

tggccttgga gcagccaagg tgcccctggc agtggcatcc cgaaacgccc tggacgcagg 3360

gcccaagact gggcacagga gtgggaggta catggggctg gggactcccc aggagttatc 3420

tgctccctgc aggcctagag aagtttcagg gaaggtcaga agagctcctg gctgtggtgg 3480

gcagggcagg aaacccctcc acctttacac atgcccaggc agcacctcag gccctttgtg 3540

gggcagggaa gctgaggcag taagcgggca ggcagagctg gaggcctttc aggcccagcc 3600

agcactctgg ccctcctgccg ccgcattcca ccccagcccc tcacaccact cgggagaggg 3660

acatcctacg gtcccaaggt caggagggca gggctggggt tgactcaggc ccctcccagc 3720

tgtggccacc tgggtgttgg gagggcagaa gtgcaggcac ctagggcccc ccatgtgccc 3780

accctgggag ctctccttgg aacccattcc tgaaattatt taaaaggggtt ggccggacta 3840

gttacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg 3900

agttggccac tccctctctg cgcgctcgct cgctcactga ggccggggcga ccaaaggtcg 3960

cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc cagctggcgt 4020

aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 4080

tggcgattcc gttgcaatgg ctggcggtaa tattgttctg gatattacca gcaaggccga 4140

tagtttgagt tcttctactc aggcaagtga tgttattact aatcaaagaa gtattgcgac 4200

aacggttaat ttgcgtgatg gacagactct tttactcggt ggcctcactg attataaaaa 4260

cacttctcag gattctggcg taccgttcct gtctaaaatc cctttaatcg gcctcctgtt 4320

tagctcccgc tctgattcta acgaggaaag cacgttatac gtgctcgtca aagcaaccat 4380

agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4440

4500

ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4560

ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 4620

ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4680

gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4740

tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4800

ttaacgcgaa ttttaacaaa atattaacgt ttacaattta aatatttgct tatacaatct 4860

4920

tacgattacc gttcatcgat tctcttgttt gctccagact ctcaggcaat gacctgatag 4980

cctttgtaga gacctctcaa aaatagctac cctctccggc atgaatttat cagctagaac 5040

ggttgaatat catattgatg gtgatttgac tgtctccggc ctttctcacc cgtttgaatc 5100

tttacctaca cattactcag gcattgcatt taaaatatat gagggttcta aaaattttta 5160

tccttgcgtt gaaataaagg cttctcccgc aaaagtatta cagggtcata atgtttttgg 5220

tacaaccgat ttagctttat gctctgaggc tttattgctt aattttgcta attctttgcc 5280

ttgcctgtat gatttattgg atgttggaat cgcctgatgc ggtattttct ccttacgcat 5340

ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca 5400

tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 5460

ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 5520

ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 5580

taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 5640

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 5700

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 5760

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 5820

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 5880

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 5940

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 6000

6060

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 6120

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 6180

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 6240

6300

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 6360

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 6420

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 6480

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 6540

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 6600

6660

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 6720

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 6780

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 6840

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 6900

agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 6960

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 7020

gccagtggcg ataagtcgtg tcttaccgggg ttggactcaa gacgatagtt accggataag 7080

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 7140

tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 7200

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 7260

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 7320

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 7380

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 7440

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 7500

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata 7560

cgcaaaccgc ctctccccgc gcgttggccg attcattaat g 7601

<210>48

<211>7601

<212>DNA

<213>Artificial sequence

<220>

<223>Synthesized SNP construct rAAV PD-1 Exon5_MND-BFP 5'-

homologous shoulder

<400>48

cagctgcgcg ctcgctcgct cactgaggcc gcccggggcaa agcccggggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagaattca 180

gtccagggct ctgtcctgca cctggggaat ggtgaccggc atctctgtcc tctagctctg 240

gaagcacccc agcccctcta gtctgccctc acccctgacc ctgaccctcc accctgaccc 300

cgtcctaacc cctgaccttt gtgcccttcc agagagaagg gcagaagtgc cccagccca 360

ccccagcccc tcacccaggc cagccggcca gttccaaacc ctggtggttg gtgtcgtggg 420

cggcctgctg ggcagcctgg tgctgctagt ctgggtcctg gccgtcatct gctcccggggc 480

cgcacgaggt aacgtcatcc cagcccctcg gcctgccctg ccctaaccct gctggcggcc 540

ctcactcccg ccctccccttc ctccaccctt ccctcacccc accccacctc cccccatctc 600

cccgccaggc taagtccctg atgaaggccc ctggactaag accccccacc taggagcacg 660

gctcagggtc ggcctggtga ccccaagtgt gtttctctgc agggacaata ggagccaggc 720

gcaccggcca gcccctggtg agtctcactc ttttcctgca tgatccactg tgccttcctt 780

cctgggtggg cagaggtgga aggacaggct gggaccacac ggcctgcagg actcacattc 840

tattatagcc aggaccccac ctccccagcc cccaggcagc aacctcaatc cctaaagcca 900

tgatctgggg ccccagccca cctgcggtct ccggggggtgc ccggcccatg tgtgtgcctg 960

1020

ccggcccaca tatgtgcctg cctgcggtct ccaggtgtgc ccggcccatg cgtgtgccca 1080

cctgcgaggg cgtggggtgg gcttggtcat ttcttatctt acattggaga caggaagct 1140

tgaaaagtca cattttggaa tcctaaatct gcaagaatgc cagggacatt tcagagggggg 1200

1260

gctcagggta agcagctcgt agtggggggc ccaggttcgg tgccggtact gcagccaggc 1320

tgtggagccg cgggcctcct tcctgcggtg ggccgtgggg ctgactccct ctccctttct 1380

cctcaaagaa ggaggacccc tcagccgtgc ctgtgttctc tgtggactat ggggagctgg 1440

atttccagtg gcgagagaag accccggagc cccccgtgcc ctgtgtccct gagcagacgg 1500

agtaatgcat gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca 1560

gttcctgccc cggctcaggg ccaagaacag ttggaacagc agaatatgggg ccaaacagga 1620

tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1680

ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 1740

tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 1800

gcgcttctgc tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatcgcc 1860

tggagacgcc atccacgctg ttttgacttc catagaagga tctcgaggcc accatggcta 1920

gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc gtggacaacc 1980

atcacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc cagaccatga 2040

gaatcaaggt ggtcgagggc ggccctctcc ccttcgcctt cgacatcctg gctactagct 2100

tcctctacgg cagcaagacc ttcatcaacc acacccaggg catccccgac ttcttcaagc 2160

agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaggac gggggcgtgc 2220

tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac gtcaagatca 2280

gaggggtgaa cttcacatcc aacggccctg tgatgcagaa gaaaacactc ggctgggagg 2340

ccttcaccga gacgctgtac cccgctgacg gcggcctgga aggcagaaac gacatggccc 2400

tgaagctcgt gggcgggagc catctgatcg caaacatcaa gaccacatat agatccaaga 2460

aacccgctaa gaacctcaag atgcctggcg tctactatgt ggactacaga ctggaaagaa 2520

tcaaggaggc caacaacgag acctacgtcg agcagcacga ggtggcagtg gccagatact 2580

gcgacctccc tagcaaactg gggcacaagc taaattgaaa gctttgcttt atttgtgaaa 2640

tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gtttaacaac 2700

2760

cgacccacca ttgtctttcc tagcggaatg ggcacctcat cccccgcccg caggggctca 2820

gctgacggcc ctcggagtgc ccagccactg aggcctgagg atggacactg ctcttggccc 2880

ctctgaccgg cttccttggc caccagtgtt ctgcagaccc tccaccatga gcccgggtca 2940

gcgcatttcc tcaggagaag caggcagggt gcaggccatt gcaggccgtc caggggctga 3000

gctgcctggg ggcgaccggg gctccagcct gcacctgcac caggcacagc cccaccacag 3060

gactcatgtc tcaatgccca cagtgagccc aggcagcagg tgtcaccgtc ccctacaggg 3120

agggccagat gcagtcactg cttcaggtcc tgccagcaca gagctgcctg cgtccagctc 3180

cctgaatctc tgctgctgct gctgctgctg ctgctgctgc ctgcggcccg gggctgaagg 3240

cgccgtggcc ctgcctgacg ccccggagcc tcctgcctga acttgggggc tggttggaga 3300

tggccttgga gcagccaagg tgcccctggc agtggcatcc cgaaacgccc tggacgcagg 3360

gcccaagact gggcacagga gtgggaggta catggggctg gggactcccc aggagttatc 3420

tgctccctgc aggcctagag aagtttcagg gaaggtcaga agagctcctg gctgtggtgg 3480

gcagggcagg aaacccctcc acctttacac atgcccaggc agcacctcag gccctttgtg 3540

gggcagggaa gctgaggcag taagcgggca ggcagagctg gaggcctttc aggcccagcc 3600

agcactctgg ccctcctgccg ccgcattcca ccccagcccc tcacaccact cgggagaggg 3660

acatcctacg gtcccaaggt caggagggca gggctggggt tgactcaggc ccctcccagc 3720

tgtggccacc tgggtgttgg gagggcagaa gtgcaggcac ctagggcccc ccatgtgccc 3780

accctgggag ctctccttgg aacccattcc tgaaattatt taaaaggggtt ggccggacta 3840

gttacgtaga taagtagcat ggcgggttaa tcattaacta caaggaaccc ctagtgatgg 3900

agttggccac tccctctctg cgcgctcgct cgctcactga ggccggggcga ccaaaggtcg 3960

cccgacgccc gggctttgcc cgggcggcct cagtgagcga gcgagcgcgc cagctggcgt 4020

aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa 4080

tggcgattcc gttgcaatgg ctggcggtaa tattgttctg gatattacca gcaaggccga 4140

tagtttgagt tcttctactc aggcaagtga tgttattact aatcaaagaa gtattgcgac 4200

aacggttaat ttgcgtgatg gacagactct tttactcggt ggcctcactg attataaaaa 4260

cacttctcag gattctggcg taccgttcct gtctaaaatc cctttaatcg gcctcctgtt 4320

tagctcccgc tctgattcta acgaggaaag cacgttatac gtgctcgtca aagcaaccat 4380

agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga 4440

4500

ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat 4560

ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 4620

ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata 4680

gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt 4740

tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat 4800

ttaacgcgaa ttttaacaaa atattaacgt ttacaattta aatatttgct tatacaatct 4860

4920

tacgattacc gttcatcgat tctcttgttt gctccagact ctcaggcaat gacctgatag 4980

cctttgtaga gacctctcaa aaatagctac cctctccggc atgaatttat cagctagaac 5040

ggttgaatat catattgatg gtgatttgac tgtctccggc ctttctcacc cgtttgaatc 5100

tttacctaca cattactcag gcattgcatt taaaatatat gagggttcta aaaattttta 5160

tccttgcgtt gaaataaagg cttctcccgc aaaagtatta cagggtcata atgtttttgg 5220

tacaaccgat ttagctttat gctctgaggc tttattgctt aattttgcta attctttgcc 5280

ttgcctgtat gatttattgg atgttggaat cgcctgatgc ggtattttct ccttacgcat 5340

ctgtgcggta tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca 5400

tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 5460

ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 5520

ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 5580

taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 5640

gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 5700

agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 5760

catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 5820

ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 5880

atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 5940

ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 6000

6060

ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 6120

ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 6180

gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 6240

6300

gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 6360

ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 6420

gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt 6480

gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 6540

caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 6600

6660

ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 6720

taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 6780

tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 6840

gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 6900

agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 6960

aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 7020

gccagtggcg ataagtcgtg tcttaccgggg ttggactcaa gacgatagtt accggataag 7080

gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 7140

tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 7200

agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 7260

cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 7320

gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 7380

gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg 7440

ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc 7500

cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata 7560

cgcaaaccgc ctctccccgc gcgttggccg attcattaat g 7601

<210>49

<211>1325

<212>DNA

<213>Homo sapiens

<400>49

agtccagggc tctgtcctgc acctggggaa tggtgaccgg catctctgtc ctctagctct 60

ggaagcaccc cagcccctct agtctgccct cacccctgac cctgaccctc caccctgacc 120

ccgtcctaac ccctgacctt tgtgcccttc cagagagaag ggcagaagtg cccacagccc 180

accccagccc ctcacccagg ccagccggcc agttccaaac cctggtggtt ggtgtcgtgg 240

gcggcctgct gggcagcctg gtgctgctag tctgggtcct ggccgtcatc tgctcccggg 300

ccgcacgagg taacgtcatc ccagcccctc ggcctgccct gccctaaccc tgctggcggc 360

cctcactccc gcctcccctt cctccaccct tccctcaccc caccccacct ccccccatct 420

ccccgccagg ctaagtccct gatgaaggcc cctggactaa gaccccccac ctaggagcac 480

ggctcagggt cggcctggtg accccaagtg tgtttctctg cagggacaat aggagccagg 540

cgcaccggcc agcccctggt gagtctcact cttttcctgc atgatccact gtgccttcct 600

tcctgggtgg gcagaggtgg aaggacaggc tgggaccaca cggcctgcag gactcacatt 660

ctattatagc caggacccca cctccccagc ccccaggcag caacctcaat ccctaaagcc 720

atgatctggg gccccagccc acctgcggtc tccgggggtg cccggcccat gtgtgtgcct 780

gcctgcggtc tccaggggtg cctggcccac gcgtgtgccc gcctgcggtc tctgggggtg 840

cccggcccac atatgtgcct gcctgcggtc tccaggtgtg cccggcccat gcgtgtgccc 900

acctgcgagg gcgtggggtg ggcttggtca tttcttatct tacattggag acaggagagc 960

ttgaaaagtc acattttgga atcctaaatc tgcaagaatg ccagggacat ttcagagggg 1020

gacattgagc cagagaggag gggtggtgtc cccagatcac acagagggca gtggtgggac 1080

agctcagggt aagcagctcg tagtgggggg cccaggttcg gtgccggtac tgcagccagg 1140

ctgtggagcc gcgggcctcc ttcctgcggt gggccgtggg gctgactccc tctccctttc 1200

1260

gatttccagt ggcgagagaa gaccccggag ccccccgtgc cctgtgtccc tgagcagacg 1320

gagta 1325

<210>50

<211>1072

<212>DNA

<213>Homo sapiens

<400>50

ccaccattgt ctttcctagc ggaatgggca cctcatcccc cgcccgcagg ggctcagccg 60

acggccctcg gagtgcccag ccactgaggc ctgaggatgg acactgctct tggcccctct 120

gaccggcttc cttggccacc agtgttctgc agaccctcca ccatgagccc gggtcagcgc 180

atttcctcag gagaagcagg cagggtgcag gccattgcag gccgtccagg ggctgagctg 240

cctgggggcg accggggctc cagcctgcac ctgcaccagg cacagcccca ccacaggact 300

catgtctcaa tgcccacagt gagcccaggc agcaggtgtc accgtcccct acagggaggg 360

ccagatgcag tcactgcttc aggtcctgcc agcacagagc tgcctgcgtc cagctccctg 420

aatctctgct gctgctgctg ctgctgctgc tgctgcctgc ggcccggggc tgaaggcgcc 480

gtggccctgc ctgacgcccc ggagcctcct gcctgaactt gggggctggt tggagatggc 540

cttggagcag ccaaggtgcc cctggcagtg gcatcccgaa acgccctgga cgcagggccc 600

aagactgggc acaggagtgg gaggtacatg gggctgggga ctccccagga gttatctgct 660

ccctgcaggc ctagagaagt ttcagggaag gtcagaagag ctcctggctg tggtgggcag 720

ggcaggaaac ccctccacct ttacacatgc ccaggcagca cctcaggccc tttgtggggc 780

agggaagctg aggcagtaag cgggcaggca gagctggagg cctttcaggc cggccagca 840

ctctggcctc ctgccgccgc attccacccc agcccctcac accactcggg agagggacat 900

cctacggtcc caaggtcagg agggcagggc tggggttgac tcaggcccct cccagctgtg 960

gccacctggg tgttgggagg gcagaagtgc aggcacctag ggccccccat gtgcccaccc 1020

tgggagctct ccttgggaacc cattcctgaa attatttaaa ggggttggcc gg 1072

<210>51

<211>7880

<212>DNA

<213>Artificial sequence

<220>

<223>Synthesized construction of the rAAV PD-1 switch receptor

Exon5_MND-PD-1-CD28

<400>51

cagctgcgcg ctcgctcgct cactgaggcc gcccggggcaa agcccggggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagaattca 180

gtccagggct ctgtcctgca cctggggaat ggtgaccggc atctctgtcc tctagctctg 240

gaagcacccc agcccctcta gtctgccctc acccctgacc ctgaccctcc accctgaccc 300

cgtcctaacc cctgaccttt gtgcccttcc agagagaagg gcagaagtgc cccagccca 360

ccccagcccc tcacccaggc cagccggcca gttccaaacc ctggtggttg gtgtcgtggg 420

cggcctgctg ggcagcctgg tgctgctagt ctgggtcctg gccgtcatct gctcccggggc 480

cgcacgaggt aacgtcatcc cagcccctcg gcctgccctg ccctaaccct gctggcggcc 540

ctcactcccg ccctccccttc ctccaccctt ccctcacccc accccacctc cccccatctc 600

cccgccaggc taagtccctg atgaaggccc ctggactaag accccccacc taggagcacg 660

gctcagggtc ggcctggtga ccccaagtgt gtttctctgc agggacaata ggagccaggc 720

gcaccggcca gcccctggtg agtctcactc ttttcctgca tgatccactg tgccttcctt 780

cctgggtggg cagaggtgga aggacaggct gggaccacac ggcctgcagg actcacattc 840

tattatagcc aggaccccac ctccccagcc cccaggcagc aacctcaatc cctaaagcca 900

tgatctgggg ccccagccca cctgcggtct ccggggggtgc ccggcccatg tgtgtgcctg 960

1020

ccggcccaca tatgtgcctg cctgcggtct ccaggtgtgc ccggcccatg cgtgtgccca 1080

cctgcgaggg cgtggggtgg gcttggtcat ttcttatctt acattggaga caggaagct 1140

tgaaaagtca cattttggaa tcctaaatct gcaagaatgc cagggacatt tcagagggggg 1200

1260

gctcagggta agcagctcat agtggggggc ccaggttcgg tgccggtact gcagccaggc 1320

tgtggagccg cgggcctcct tcctgcggtg ggccgtgggg ctgactccct ctccctttct 1380

cctcaaagaa ggaggacccc tcagccgtgc ctgtgttctc tgtggactat ggggagctgg 1440

atttccagtg gcgagagaag accccggagc cccccgtgcc ctgtgtccct gagcagacgg 1500

agtaatgcat gaacagagaa acaggagaat atgggccaaa caggatatct gtggtaagca 1560

gttcctgccc cggctcaggg ccaagaacag ttggaacagc agaatatgggg ccaaacagga 1620

tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc 1680

ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc 1740

tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc 1800

gcgcttctgc tccccgagct ctatataagc agagctcgtt tagtgaaccg tcagatcgcc 1860

tggagacgcc atccacgctg ttttgacttc catagaagga tctcgaggcc accatgcaga 1920

tcccgcaagc gccctggcca gtcgtctggg cggtgctaca actgggctgg cggccaggat 1980

ggttcttaga ctccccagac aggccctgga acccccccac cttctcccca gccctgctcg 2040

tggtgaccga aggggacaac gccaccttca cctgcagctt ctccaacaca tcggagagct 2100

tcgtgctaaa ctggtaccgc atgagcccca gcaaccagac ggacaagctg gccgccttcc 2160

ccgaggaccg cagccagccc ggccaggact gccgcttccg tgtcacacaa ctgcccaacg 2220

ggcgtgactt ccacatgagc gtggtcaggg cccggcgcaa tgacagcggc acctacctct 2280

gtggggccat ctccctggcc cccaaggcgc agatcaaaga gagcctgcgg gcagagctca 2340

gggtgacaga gagaagggca gaagtgccca cagcccaccc cagcccctca cccaggccag 2400

ccggccagtt ccaaaccctg gtggttggtg tcgtgggcgg cctgctgggc agcctggtgc 2460

tgctagtctg ggtcctggcc gtcatcagga gtaagaggag caggctcctg cacagtgact 2520

acatgaacat gactccccgc cgccccgggc cccaccgcaa gcattaccag ccctatgccc 2580

caccacgcga cttcgcagcc tatcgctccg gtgaggggcag aggaagtctt ctaacatgcg 2640

gtgacgtgga ggagaatccg ggccctgtga gcaagggcga ggaggataac tccgccatca 2700

tcaaggagtt cctgcgcttc aaggtgcaca tggagggctc cgtgaacggc cacgagttcg 2760

agatcgaggg cgagggcgag ggccgcccct acgagggcac ccagaccgcc aagctgaagg 2820

tgaccaaggg tggccccctg cccttcgcct gggacatcct gtcccctcag ttcatgtacg 2880

gctccaaggc ctacgtgaag caccccgccg acatccccga ctacttgaag ctgtccttcc 2940

ccgaggggctt caagtgggag cgcgtgatga acttcgagga cggcggcgtg gtgaccgtga 3000

cccaggactc ctctctgcag gacggcgagt tcatctacaa ggtgaagctg cgcggcacca 3060

acttcccctc cgacggcccc gtaatgcaga agaagaccat gggctgggag gcctcctccg 3120

agcggatgta ccccgaggac ggcgccctga agggcgagat caagcagagg ctgaagctga 3180

aggacggcgg ccactacgac gctgaggtca agaccaccta caaggccaag aagcccgtgc 3240

agctgcccgg cgcctacaac gtcaacatca agttggacat cacctcccac aacgaggact 3300

acaccatcgt ggaacagtac gaacgcgccg agggccgcca ctccaccggc ggcatggacg 3360

agctgtacaa gtgatgaaag ctttgcttta tttgtgaaat ttgtgatgct attgctttat 3420

ttgtaaccat tataagctgc aataaacaag

tttcaggttc agggggaggt gtgggaggtt ttttaaagtc gacgtggccc tgcctgacgc 3540

cccggagcct cctgcctgaa cttgggggct ggttggagat ggccttggag cagccaaggt 3600

gcccctggca gtggcatccc gaaacgccct ggacgcaggg cccaagactg ggcacaggag 3660

tgggaggtac atggggctgg ggactcccca ggagttatct gctccctgca ggcctagaga 3720

agtttcaggg aaggtcagaa gagctcctgg ctgtggtggg cagggcagga aacccctcca 3780

cctttacaca tgcccaggca gcacctcagg ccctttgtgg ggcagggaag ctgaggcagt 3840

aagcgggcag gcagagctgg aggcctttca ggcccagcca gcactctggc ctcctgccgc 3900

cgcattccac cccagcccct cacaccactc gggagaggga catcctacgg tcccaaggtc 3960

aggagggcag ggctggggtt gactcaggcc cctcccagct gtggccacct gggtgttggg 4020

agggcagaag tgcaggcacc tagggccccc catgtgccca ccctggggagc tctccttgga 4080

acccattcct gaaattattt aaaggggttg gccggactag ttacgtagat aagtagcatg 4140

gcgggttaat cattaactac aaggaacccc tagtgatgga gttggccact ccctctctgc 4200

gcgctcgctc gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc 4260

gggcggcctc agtgagcgag cgagcgcgcc agctggcgta atagcgaaga ggcccgcacc 4320

gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcgattccg ttgcaatggc 4380

tggcggtaat attgttctgg atattaccag caaggccgat agtttgagtt cttctactca 4440

ggcaagtgat gttatacta atcaaagaag tattgcgaca acggttaatt tgcgtgatgg 4500

acagactctt ttactcggtg gcctcactga ttataaaaac acttctcagg attctggcgt 4560

accgttcctg tctaaaatcc ctttaatcgg ccctcctgttt agctcccgct ctgattctaa 4620

4680

cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 4740

tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 4800

gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 4860

accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 4920

tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 4980

gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 5040

cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 5100

tattaacgtt tacaatttaa atatttgctt atacaatctt cctgtttttg gggctttttct 5160

gattatcaac cggggtacat atgattgaca tgctagtttt acgattaccg ttcatcgatt 5220

ctcttgtttg ctccagactc tcaggcaatg acctgatagc ctttgtagag acctctcaaa 5280

aatagctacc ctctccggca tgaatttatc agctagaacg gttgaatatc atattgatgg 5340

tgatttgact gtctccggcc tttctcaccc gtttgaatct ttacctacac attactcagg 5400

cattgcattt aaaatattg agggttctaa aaatttttat ccttgcgttg aaataaaggc 5460

ttctcccgca aaagtattac agggtcataa tgtttttggt acaaccgatt tagctttatg 5520

ctctgaggct ttattgctta attttgctaa ttctttgcct tgcctgtatg atttattgga 5580

tgttggaatc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc 5640

atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac 5700

ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga 5760

caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa 5820

cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata 5880

atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 5940

ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 6000

6060

cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 6120

aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 6180

ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 6240

gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc 6300

cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 6360

6420

gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 6480

aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 6540

ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta 6600

ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 6660

gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 6720

aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 6780

aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 6840

aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 6900

gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aggatctag 6960

gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 7020

tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 7080

gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 7140

caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 7200

actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 7260

acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 7320

cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 7380

gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 7440

cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcggga caggtatccg 7500

7560

tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 7620

tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 7680

gccttttgct ggccttttgc tcacatgttc ttttcctgcgt tatcccctga ttctgtggat 7740

aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc 7800

agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg 7860

cgttggccga ttcattaatg 7880

<210>52

<211>7593

<212>DNA

<213>Artificial sequence

<220>

<223>Synthesized construct rAAV PD-1 Exon5_MND-BFP_distal 5'-

homologous shoulder

<400>52

cagctgcgcg ctcgctcgct cactgaggcc gcccggggcaa agcccggggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagaattcg 180

gccccactgc ccactgccca gggcagcaat gcccatacca cgtggtccca gctccgagct 240

tgtcctgaaa agggggcaaa gactggaccc tgagcctgcc aaggggccac actcctccca 300

gggctggggt ctccatgggc agccccccac ccacccagac cagttacact cccctgtgcc 360

agagcagtgc agacaggacc aggccaggat gcccaagggt caggggctgg ggatgggtag 420

cccccaaaca gccctttctg ggggactggc ctcaacgggg aagggggtga aggctcttag 480

taggaaatca gggagaccca agtcagagcc aggcgctgtg cagaagctgc agcctcacgt 540

agaaggaaga gcctctgcag tggaggccag tgcccatccc cgggtggcag aggccccagc 600

agagacttct caatgacatt ccagctgggg tggcccttcc agagcccttg ctgcccgagg 660

gatgtgagca ggtggccggg gaggctttgt ggggccaccc agccccttcc tcacctctct 720

ccatctctca gactccccag acaggccctg gaaccccccc accttctccc cagccctgct 780

cgtggtgacc gaaggggaca acgccacctt cacctgcagc ttctccaaca catcggagag 840

cttcgtgcta aactggtacc gcatgagccc cagcaaccag acggacaagc tggccgcctt 900

ccccgaggac cgcagccagc ccggccagga ctgccgcttc cgtgtcacac aactgcccaa 960

cgggcgtgac ttccacatga gcgtggtcag ggcccggcgc aatgacagcg gcacctacct 1020

ctgtggggcc atctccctgg cccccaaggc gcagatcaaa gagagcctgc gggcagagct 1080

cagggtgaca ggtgcggcct cggaggcccc ggggcagggg tgagctgagc cggtcctggg 1140

gtgggtgtcc cctcctgcac aggatcagga gctccagggt cgtagggcag ggacccccca 1200

gctccagtcc agggctctgt cctgcacctg gggaatggtg accggcatct ctgtcctcta 1260

gctctggaag caccccagcc cctctagtct gccctcaccc ctgaccctga ccctccaccc 1320

tgaccccgtc ctaacccctg acctttgtgc ccttccagag agaagggcag aagtgcccac 1380

agcccacccc agcccctcac ccaggccagc cggccagttc caaaccctgg tggttggtgt 1440

cgtgggcggc ctgctgggca gcctggtgct gctagtctgg gtcctggccg tcatctatgc 1500

1560

cccggctcag ggccaagaac agttggaaca gcagaatatg ggccaaacag gatatctgtg 1620

gtaagcagtt cctgccccgg ctcaggggcca agaacagatg gtccccagat gcggtcccgc 1680

cctcagcagt ttctagagaa ccatcagatg tttccagggt gccccaagga cctgaaatga 1740

ccctgtgcct tatttgaact aaccaatcag ttcgcttctc gcttctgttc gcgcgcttct 1800

1860

ccatccacgc tgttttgact tccatagaag gatctcgagg ccaccatggc tagcgagctg 1920

attaaggaga acatgcacat gaagctgtac atggagggca ccgtggacaa ccatcacttc 1980

aagtgcacat ccgagggcga aggcaagccc tacgaggca cccagaccat gagaatcaag 2040

gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc tggctactag cttcctctac 2100

ggcagcaaga ccttcatcaa ccacacccag ggcatccccg acttcttcaa gcagtccttc 2160

cctgagggct tcacatggga gagagtcacc acatacgagg acggggggcgt gctgaccgct 2220

acccaggaca ccagcctcca ggacggctgc ctcatctaca acgtcaagat cagaggggtg 2280

aacttcacat ccaacggccc tgtgatgcag aagaaaacac tcggctggga ggccttcacc 2340

gagacgctgt accccgctga cggcggcctg gaaggcagaa acgacatggc cctgaagctc 2400

gtgggcggga gccatctgat cgcaaacatc aagaccacat atagatccaa gaaacccgct 2460

aagaacctca agatgcctgg cgtctactat gtggactaca gactggaaag aatcaaggag 2520

gccaacaacg agacctacgt cgagcagcac gaggtggcag tggccagata ctgcgacctc 2580

cctagcaaac tggggcacaa gctaaattga aagctttgct ttatttgtga aatttgtgat 2640

gctattgctt tatttgtaac cattataagc tgcaataaac aagtttaaca acaacaattg 2700

cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa gtcgacccac 2760

cattgtcttt cctagcggaa tgggcacctc atcccccgcc cgcaggggct cagctgacgg 2820

ccctcggagt gcccagccac tgaggcctga ggatggacac tgctcttggc ccctctgacc 2880

ggcttccttg gccaccagtg ttctgcagac cctccaccat gagcccgggt cagcgcattt 2940

cctcaggaga agcaggcagg gtgcaggcca ttgcaggccg tccaggggct gagctgcctg 3000

ggggcgaccg gggctccagc ctgcacctgc accaggcaca gccccaccac aggactcatg 3060

tctcaatgcc cacagtgagc ccaggcagca ggtgtcaccg tcccctacag ggagggccag 3120

atgcagtcac tgcttcaggt cctgccagca cagagctgcc tgcgtccagc tccctgaatc 3180

tctgctgctg ctgctgctgc tgctgctgct gcctgcggcc cggggctgaa ggcgccgtgg 3240

ccctgcctga cgccccggag ccctcctgcct gaacttgggg gctggttgga gatggccttg 3300

gagcagccaa ggtgcccctg gcagtggcat cccgaaacgc cctggacgca gggcccaaga 3360

ctgggcacag gagtgggagg tacatggggc tggggactcc ccaggagtta tctgctccct 3420

gcaggcctag agaagtttca gggaaggtca gaagagctcc tggctgtggt gggcagggca 3480

ggaaacccct ccaccttttac acatgcccag gcagcacctc aggccctttg tggggcaggg 3540

aagctgaggc agtaagcggg caggcagagc tggaggcctt tcaggcccag ccagcactct 3600

ggcctcctgc cgccgcattc caccccagcc cctcacacca ctcggggagag ggacatccta 3660

cggtcccaag gtcaggaggg cagggctggg gttgactcag gcccctccca gctgtggcca 3720

cctgggtgtt gggagggcag aagtgcaggc acctagggcc ccccatgtgc ccaccctggg 3780

agctctcctt ggaacccatt cctgaaatta tttaaagggg ttggccggac tagttacgta 3840

gataagtagc atggcggtt aatcattaac tacaaggaac ccctagtgat ggagttggcc 3900

actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgccgacgc 3960

ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gccagctggc gtaatagcga 4020

agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgatt 4080

ccgttgcaat ggctggcggt aatattgttc tggatattac cagcaaggcc gatagtttga 4140

gttcttctac tcaggcaagt gatgttatta ctaatcaaag aagtattgcg acaacggtta 4200

atttgcgtga tggacagact cttttactcg gtggcctcac tgattataaa aacacttctc 4260

aggattctgg cgtaccgttc ctgtctaaaa tccctttaat cggcctcctg tttagctccc 4320

gctctgattc taacgaggaa agcacgttat acgtgctcgt caaagcaacc atagtacgcg 4380

ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 4440

cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 4500

gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 4560

ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg 4620

ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 4680

ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg 4740

attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 4800

aattttaaca aaatattaac gtttacaatt taaatatttg cttatacaat cttcctgttt 4860

ttggggcttt tctgattatc aaccggggta catatgattg acatgctagt tttacgatta 4920

ccgttcatcg attctcttgt ttgctccaga ctctcaggca atgacctgat agcctttgta 4980

gagacctctc aaaaatagct accctctccg gcatgaattt atcagctaga acggttgaat 5040

atcatattga tggtgatttg actgtctccg gcctttctca cccgtttgaa tctttaccta 5100

cacattactc aggcattgca tttaaaatat atgagggttc taaaaattttt tatccttgcg 5160

ttgaaataaa ggcttctccc gcaaaagtat tacagggtca taatgttttt ggtacaaccg 5220

5280

atgatttatt ggatgttgga atcgcctgat gcggtatttt ctccttacgc atctgtgcgg 5340

tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg catagttaag 5400

ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 5460

atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 5520

gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt tataggttaa 5580

tgtcatgata ataatggttt cttagacgtc aggtggcact tttcggggaa atgtgcgcgg 5640

aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 5700

accctgataa atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg 5760

tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac 5820

gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact 5880

ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat 5940

gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg cggggcaaga 6000

gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac 6060

agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat 6120

gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac 6180

cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttgggg aaccggagct 6240

gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac 6300

gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga 6360

ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg 6420

6480

ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac 6540

tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta 6600

actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt 6660

6720

gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc 6780

tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt 6840

ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc 6900

gcagatacca aatactgtcc ttctagtgta gccgtagtta ggccaccact tcaagaactc 6960

tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg 7020

cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg 7080

gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga 7140

actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc 7200

ggacaggtat ccggtaagcg gcagggtcgg aacaggag cgcacgaggg agcttccagg 7260

gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg 7320

atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt 7380

tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc 7440

tgattctgtg gtaaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg 7500

aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc 7560

gcctctcccc gcgcgttggc cgattcatta atg 7593

<210>53

<211>6718

<212>DNA

<213>Artificial sequence

<220>

<223>Synthesized construct rAAV PD-1 Exon1_MND-GFP

<400>53

cagctgcgcg ctcgctcgct cactgaggcc gcccggggcaa agcccggggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtaaggctgt 180

tgcaggcatc acacggtgga aagatctgga actgtggcca tggtgtgagg ccatccacaa 240

ggtggaagct ttgaggggga gccgattagc catggacagt tgtcattcag tagggtcacc 300

tgtgccccag cgaaggggga tgggccggga aggcagaggc caggcacctg cccccagcag 360

gggcagaggc tgtgggcagc cgggaggctc ccagaggctc cgacagaatg ggagtggggt 420

tgagcccacc ccctcactgca gccaggaac ctgagcccag agggggccac ccaccttccc 480

caggcaggga ggcccggccc ccagggagat gggggggatg ggggaggaga agggcctgcc 540

cccacccggc agcctcagga ggggcagctc gggcgggata tggaaagagg ccacagcagt 600

gagcagagac acagaggagg aaggggccct gagctgggga gacccccacg gggtagggcg 660

tgggggccac gggccccacct cctccccatc tcctctgtct ccctgtctct gtctctctct 720

ccctccccca ccctctcccc agtcctaccc cctcctcacc cctcctcccc cagcactgcc 780

tctgtcactc tcgcccacgt ggatgtggag gaagaggggg cgggagcaag gggcgggcac 840

cctcccttca acctgacctg ggacagtttc ccttccgctc acctccgcct gagcagtgga 900

gaaggcggca ctctggtggg gctgctccaa cgcgtgaaca gagaaacagg agaatatggg 960

ccaaacagga tatctgtggt aagcagttcc tgccccggct cagggccaag aacagttgga 1020

acagcagaat atgggccaaa caggatatct gtggtaagca gttcctgccc cggctcaggg 1080

ccaagaacag atggtcccca gatgcggtcc cgccctcagc agtttctaga gaaccatcag 1140

atgtttccag ggtgccccaa ggacctgaaa tgaccctgtg ccttatttga actaaccaat 1200

cagttcgctt ctcgcttctg ttcgcgcgct tctgctcccc gagctctata taagcagagc 1260

tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg acttccatag 1320

aaggatctcg aggccaccat ggtgagcaag ggcgaggagc tgttcaccgg ggtggtgccc 1380

atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc cggcgagggc 1440

gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg 1500

cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc 1560

taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc 1620

caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc cgaggtgaag 1680

ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac 1740

ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt ctatatcatg 1800

gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa catcgaggac 1860

ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg 1920

ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga ccccaacgag 1980

aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac tctcggcatg 2040

gacgagctgt acaagtaagc ggccgcgctt tatttgtgaa atttgtgatg ctattgcttt 2100

2160

gtttcaggtt cagggggaga tgtgggaggt tttttaaagc ctcaccggtt ctgggcggtg 2220

ctacaactgg gctggcggcc aggatggttc ttaggtaggt ggggtcggcg gtcaggtgtc 2280

ccagagccag gggtctggag ggaccttcca ccctcagtcc ctggcaggtc ggggggtgct 2340

gaggcggggcc tggccctggc agcccagggg tcccggagcg aggggtctgg agggaccttt 2400

cactctcagt ccctggcagg tcggggggtg ctgtggcagg cccagccttg gcccccagct 2460

ctgcccctta ccctgagctg tgtggctttg ggcagctcga actcctgggt tcctctctgg 2520

gccccaactc ctcccctggc ccaagtcccc tctttgctcc tgggcaggca ggacctctgt 2580

cccctctcag ccggtccttg gggctgcgtg tttctgtaga atgacgggtc aggctggcca 2640

gaaccccaaa ccttggccgt ggggagtctg cgtggcggct ctgccttgcc caggcatcct 2700

tggtcctcac tcgagttttc ctaaggatgg gatgagcccc atgtgggact aaccttggct 2760

ttacgacgtc aaagtttaga tgagctggtg atatttttct cattatatcc aaagtgtacc 2820

tgttcgagtg aggacagttc ttctgtctcc aggatccctc ctgggtgggg attgtgcccg 2880

cctgggtctc tgcccagatt ccagggctct ccccgagccc tgttcagacc atccgtgggg 2940

gaggccttgg cctcactctt acgtagataa gtagcatggc gggttaatca ttaactacaa 3000

ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc 3060

cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg 3120

agcgcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 3180

gcagcctgaa tggcgaatgg cgattccgtt gcaatggctg gcggtaatat tgttctggat 3240

attaccagca aggccgatag tttgagttct tctactcagg caagtgatgt tattactaat 3300

caaagaagta ttgcgacaac ggttaatttg cgtgatggac agactctttt actcggtggc 3360

ctcactgatt ataaaaacac ttctcaggat tctggcgtac cgttcctgtc taaaatccct 3420

ttaatcggcc tcctgtttag ctcccgctct gattctaacg aggaaagcac gttatacgtg 3480

3540

ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt 3600

cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct 3660

ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg 3720

tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga 3780

gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc 3840

ggtctattct ttttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga 3900

3960

atttgcttat acaatcttcc tgtttttggg gcttttctga ttatcaaccg gggtacatat 4020

gattgacatg ctagttttac gattaccgtt catcgattct cttgtttgct ccagactctc 4080

aggcaatgac ctgatagcct ttgtagagaac ctctcaaaaa tagctaccct ctccggcatg 4140

aatttatcag ctagaacggt tgaatatcat attgatggtg atttgactgt ctccggcctt 4200

tctcacccgt ttgaatcttt acctacacat tactcaggca ttgcatttaa aatatatgag 4260

ggttctaaaa atttttatcc ttgcgttgaa ataaaggctt ctcccgcaaa agtattacag 4320

ggtcataatg tttttggtac aaccgattta gctttatgct ctgaggcttt attgcttaat 4380

tttgctaatt ctttgccttg cctgtatgat ttattggatg ttggaatcgc ctgatgcggt 4440

attttctcct tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa 4500

tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc 4560

cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga 4620

gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg 4680

tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg 4740

gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 4800

atatgtatcc gctcatgaga caataaccct gtaaatgct tcaataatat tgaaaaagga 4860

agagtatgag tattcaacat ttccgtgtcg cccttattcc ctttttttgcg gcattttgcc 4920

ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 4980

gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 5040

gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat 5100

tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg 5160

acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 5220

aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 5280

cgatcgggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 5340

gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 5400

cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc 5460

tagcttcccg gcaacaatta atagactgga tggaggcggga taaagttgca ggaccacttc 5520

tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 5580

ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 5640

tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 5700

gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 5760

ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 5820

tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 5880

agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 5940

aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 6000

6060

agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 6120

tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccggggttg gactcaagac 6180

gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 6240

gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 6300

ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 6360

gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 6420

ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 6480

6540

acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 6600

6660 gagctgatac cgctcgccgc agccgaacga ccgagcgcag

cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatg 6718

<210>54

<211>756

<212>DNA

<213>Homo sapiens

<400>54

aggctgttgc aggcatcaca cggtggaaag atctggaact gtggccatgg tgtgaggcca 60

tccacaaggt ggaagctttg agggggagcc gattagccat ggacagttgt cattcagtag 120

ggtcacctgt gccccagcga agggggatgg gccgggaagg cagaggccag gcacctgccc 180

ccagcagggg cagaggctgt gggcagccgg gaggctccca gaggctccga cagaatggga 240

gtggggttga gcccacccct cactgcagcc caggaacctg agcccagagg gggccaccca 300

ccttccccag gcagggaggc ccggccccca gggagatggg ggggatgggg gaggagaagg 360

gcctgccccc acccggcagc ctcaggaggg gcagctcggg cgggatatgg aaagaggcca 420

cagcagtgag cagagacaca gaggaggaag gggccctgag ctggggagac ccccacgggg 480

tagggcgtgg gggccacggg cccacctcct ccccatctcc tctgtctccc tgtctctgtc 540

tctctctccc tccccccaccc tctccccagt cctaccccct cctcacccct cctcccccag 600

cactgcctct gtcactctcg cccacgtgga tgtggaggaa gagggggcgg gagcaagggg 660

cgggcaccct cccttcaacc tgacctggga cagttttccct tccgctcacc tccgcctgag 720

cagtggagaa ggcggcactc tggtggggct gctcca 756

<210>55

<211>750

<212>DNA

<213>Homo sapiens

<400>55

tctgggcggt gctacaactg ggctggcggc caggatggtt cttaggtagg tggggtcggc 60

ggtcaggtgt cccagagcca ggggtctgga gggaccttcc accctcagtc cctggcaggt 120

cggggggtgc tgaggcgggc ctggccctgg cagcccaggg gtcccggagc gaggggtctg 180

gagggacctt tcactctcag tccctggcag gtcggggggt gctgtggcag gcccagcctt 240

ggcccccagc tctgcccctt accctgagct gtgtggcttt gggcagctcg aactcctggg 300

ttcctctctg ggccccaact ccctcccctgg cccaagtccc ctctttgctc ctgggcaggc 360

aggacctctg tcccctctca gccggtcctt ggggctgcgt gtttctgtag aatgacgggt 420

caggctggcc agaaccccaa accttggccg tggggagtct gcgtggcggc tctgccttgc 480

ccaggcatcc ttggtcctca ctcgagtttt cctaaggatg ggatgagccc catgtgggac 540

taaccttggc tttacgacgt caaagtttag atgagctggt gatatttttc tcattatatc 600

caaagtgtac ctgttcgagt gaggacagtt cttctgtctc caggatccct cctgggtggg 660

gattgtgccc gcctgggtct ctgcccagat tccagggctc tccccgagcc ctgttcagac 720

catccgtggg ggaggccttg gcctcactct 750

<210>56

<211>7609

<212>DNA

<213>Artificial sequence

<220>

<223>Synthesized construct rAAV PD-1 Exon1_MND-CD19CAR

<400>56

cagctgcgcg ctcgctcgct cactgaggcc gcccggggcaa agcccggggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtaaggctgt 180

tgcaggcatc acacggtgga aagatctgga actgtggcca tggtgtgagg ccatccacaa 240

ggtggaagct ttgaggggga gccgattagc catggacagt tgtcattcag tagggtcacc 300

tgtgccccag cgaaggggga tgggccggga aggcagaggc caggcacctg cccccagcag 360

gggcagaggc tgtgggcagc cgggaggctc ccagaggctc cgacagaatg ggagtggggt 420

tgagcccacc ccctcactgca gccaggaac ctgagcccag agggggccac ccaccttccc 480

caggcaggga ggcccggccc ccagggagat gggggggatg ggggaggaga agggcctgcc 540

cccacccggc agcctcagga ggggcagctc gggcgggata tggaaagagg ccacagcagt 600

gagcagagac acagaggagg aaggggccct gagctgggga gacccccacg gggtagggcg 660

tgggggccac gggccccacct cctccccatc tcctctgtct ccctgtctct gtctctctct 720

ccctccccca ccctctcccc agtcctaccc cctcctcacc cctcctcccc cagcactgcc 780

tctgtcactc tcgcccacgt ggatgtggag gaagaggggg cgggagcaag gggcgggcac 840

acctcccttca acctgacctg ggacagtttc ccttccgctc acctccgcct gagcagtgga 900

gaaggcggca ctctggtggg gctgctccaa cgcgtgatcc atcgattagt ccaatttgtt 960

aaagacagga tatcagtggt ccaggctcta gttttgactc aacaatatca ccagctgaag 1020

cctatagagt acgagccata gatagaataa aagattttat ttagtctcca gaaaaagggg 1080

ggaatgaaag accccacctg taggtttggc aagctaggat caaggttagg aacagagaga 1140

cagcagaata tggggccaaac aggatatctg tggtaagcag ttcctgcccc ggctcaggggc 1200

1260

gccccggctc agggccaaga acagatggtc cccagatgcg gtcccgccct cagcagtttc 1320

1380

ttgaactaac caatcagttc gcttctcgct tctgttcgcg cgcttctgct ccccgagctc 1440

aataaaagag cccacaaccc ctcactcggc gcgacgcgtc atagccacca tggccttacc 1500

agtgaccgcc ttgctcctgc cgctggcctt gctgctccac gccgccaggc cggacatcca 1560

gatgacacag actacatcct ccctgtctgc ctctctggga gacagagtca ccatcagttg 1620

cagggcaagt caggacatta gtaaatattt aaattggtat cagcagaaac cagatggaac 1680

tgttaaactc ctgatctacc atacatcaag attacactca ggagtcccat caaggttcag 1740

tggcagtggg tctggaacag attattctct caccattagc aacctggagc aagaagatat 1800

tgccacttac ttttgccaac agggtaatac gcttccgtac acgttcggag gggggaccaa 1860

gctggagatc acaggtggcg gtggctccgg cggtggtggg tctggtggcg gcggaagcga 1920

ggtgaaactg caggagtcag gacctggcct ggtggcgccc tcacagagcc tgtccgtcac 1980

atgcactgtc tcaggggtct cattacccga ctatggtgta agctggattc gccagcctcc 2040

acgaaagggt ctggagtggc tgggagtaat atggggtagt gaaaccacat actataattc 2100

agctctcaaa tccagactga ccatcatcaa ggacaactcc aagagccaag ttttcttaaa 2160

aatgaacagt ctgcaaactg atgacacagc catttactac tgtgccaaac attattacta 2220

cggtggtagc tatgctatgg actactgggg tcaaggaacc tcggtcaccg tctcctcaac 2280

cacgacgcca gcgccgcgac caccaacacc ggcgcccacc atcgcgtcgc agcccctgtc 2340

cctgcgccca gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga 2400

cttcgcctgt gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct 2460

gtcactggtg atcacccttt actgcaaacg gggcagaaag aaactcctgt atatattcaa 2520

acaaccattt atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt 2580

tccagaagaa gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc 2640

ccccgcgtac cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga 2700

ggagtacgat gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag 2760

aaggaagaac cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcgggaggc 2820

ctacagtgag attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta 2880

ccagggtctc agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc 2940

ccctcgctaa gcggccgcgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa 3000

3060

ttcaggggga gatgtgggag gttttttaaa gctcaccggt tctgggcggt gctacaactg 3120

ggctggcggc caggatggtt cttaggtagg tggggtcggc ggtcaggtgt cccagagcca 3180

ggggtctgga gggaccttcc accctcagtc cctggcaggt cggggggtgc tgaggcggggc 3240

ctggccctgg cagcccaggg gtcccggagc gaggggtctg gagggacctt tcactctcag 3300

tccctggcag gtcggggggt gctgtggcag gcccagcctt ggcccccagc tctgcccctt 3360

accctgagct gtgtggcttt gggcagctcg aactcctggg ttcctctctg ggccccaact 3420

cctcccctgg cccaagtccc ctctttgctc ctgggcaggc aggacctctg tcccctctca 3480

gccggtcctt ggggctgcgt gtttctgtag aatgacgggt caggctggcc agaaccccaa 3540

accttggccg tggggagtct gcgtggcggc tctgccttgc ccaggcatcc ttggtcctca 3600

3660

3720

gaggacagtt cttctgtctc caggatccct cctgggtggg gattgtgccc gcctgggtct 3780

ctgccgat tccagggctc tccccgagcc ctgttcagac catccgtggg ggaggccttg 3840

gcctcactct tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct 3900

agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg cggggcgacc 3960

aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcca 4020

gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 4080

atggcgaatg gcgattccgt tgcaatggct ggcggtaata ttgttctgga tattaccagc 4140

aaggccgata gtttgagttc ttctactcag gcaagtgatg ttattactaa tcaaagaagt 4200

attgcgacaa cggttaattt gcgtgatgga cagactcttt tactcggtgg cctcactgat 4260

tataaaaaca cttctcagga ttctggcgta ccgttcctgt ctaaaatccc tttaatcggc 4320

ctcctgttta gctcccgctc tgattctaac gaggaaagca cgttatacgt gctcgtcaaa 4380

gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 4440

cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 4500

ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 4560

gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 4620

acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 4680

ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 4740

ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 4800

4860

tacaatcttc ctgtttttgg ggcttttctg attatcaacc ggggtacata tgattgacat 4920

gctagtttta cgattaccgt tcatcgattc tcttgtttgc tccagactct caggcaatga 4980

cctgatagcc tttgtagaga cctctcaaaa atagctaccc tctccggcat gaatttatca 5040

gctagaacgg ttgaatatca tattgatggt gatttgactg tctccggcct ttctcacccg 5100

tttgaatctt tacctacaca ttactcaggc attgcattta aaatatatga gggttctaaa 5160

aatttttatc cttgcgttga aataaaggct tctcccgcaa aagtattaca gggtcataat 5220

gtttttggta caaccgattt agctttatgc tctgaggctt tattgcttaa ttttgctaat 5280

tctttgcctt gcctgtatga tttattggat gttggaatcg cctgatgcgg tattttctcc 5340

ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg 5400

atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg 5460

cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt 5520

gtcagaggtt ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc 5580

tatttttata ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc 5640

ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 5700

cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 5760

gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 5820

ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 5880

tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 5940

aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 6000

ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 6060

agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 6120

gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 6180

gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 6240

gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 6300

tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 6360

ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 6420

cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 6480

gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 6540

6600

6660

aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 6720

aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 6780

gatcttcttg agatcctttt ttttgcgcg taatctgctg cttgcaaaca aaaaaaccac 6840

cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 6900

ctggcttcag tagttaggcc 6960

accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 7020

tggctgctgc cagtggcgat aagtcgtgtc ttaccggggtt ggactcaaga cgatagttac 7080

cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 7140

gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 7200

aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 7260

cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 7320

tctgacttga gcgtcgattt ttgtgatgct cgtcagggggg gcggagccta tggaaaaacg 7380

ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 7440

ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 7500

ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 7560

gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatg 7609

<210>57

<211>7492

<212>DNA

<213>Artificial sequence

<220>

<223>Synthesized construct rAAV PD-1 Exon1_MND-BCMACAR

<400>57

cagctgcgcg ctcgctcgct cactgaggcc gcccggggcaa agcccggggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtaaggctgt 180

tgcaggcatc acacggtgga aagatctgga actgtggcca tggtgtgagg ccatccacaa 240

ggtggaagct ttgaggggga gccgattagc catggacagt tgtcattcag tagggtcacc 300

tgtgccccag cgaaggggga tgggccggga aggcagaggc caggcacctg cccccagcag 360

gggcagaggc tgtgggcagc cgggaggctc ccagaggctc cgacagaatg ggagtggggt 420

tgagcccacc ccctcactgca gccaggaac ctgagcccag agggggccac ccaccttccc 480

caggcaggga ggcccggccc ccagggagat gggggggatg ggggaggaga agggcctgcc 540

cccacccggc agcctcagga ggggcagctc gggcgggata tggaaagagg ccacagcagt 600

gagcagagac acagaggagg aaggggccct gagctgggga gacccccacg gggtagggcg 660

tgggggccac gggccccacct cctccccatc tcctctgtct ccctgtctct gtctctctct 720

ccctccccca ccctctcccc agtcctaccc cctcctcacc cctcctcccc cagcactgcc 780

tctgtcactc tcgcccacgt ggatgtggag gaagaggggg cgggagcaag gggcgggcac 840

cctcccttca acctgacctg ggacagtttc ccttccgctc acctccgcct gagcagtgga 900

gaaggcggca ctctggtggg gctgctccaa cgcgtaatga aagaccccac ctgtaggttt 960

ggcaagctag gatcaaggtt aggaacagag agacagcaga atatggggcca aacaggatat 1020

ctgtggtaag cagttcctgc cccggctcag ggccaagaac agttggaaca gcagaatatg 1080

ggccaaacag gatatctgtg gtaagcagtt cctgccccgg ctcaggggcca agaacagatg 1140

1200

gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag ttcgcttctc 1260

gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa cccctcactc 1320

ggcgcgattc acctgacgcg tctacgccac catggcactc cccgtcaccg cccttctctt 1380

gcccctcgcc ctgctgctgc atgctgccag gcccgacatt gtgctcactc agtcacctcc 1440

cagcctggcc atgagcctgg gaaaaagggc caccatctcc tgtagagcca gtgagtccgt 1500

cacaatcttg gggagccatc ttattcactg gtatcagcag aagcccgggc agcctccaac 1560

ccttcttatt cagctcgcgt caaacgtcca gacgggtgta cctgccagat tttctggtag 1620

cgggtcccgc actgatttta cactgaccat agatccagtg gaagaagacg atgtggccgt 1680

gtattattgt ctgcagagca gaacgattcc tcgcacattt ggtgggggta ctaagctgga 1740

gattaaggga agcacgtccg gctcagggaa gccgggctcc ggcgagggaa gcacgaaggg 1800

gcaaattcag ctggtccaga gcggacctga gctgaaaaaa cccggcgaga ctgttaagat 1860

cagttgtaaa gcatctggct ataccttcac cgactacagc ataaattggg tgaaacgggc 1920

ccctggaaag ggcctcaaat ggatgggttg gatcaatacc gaaactaggg agcctgctta 1980

tgcatatgac ttccgcggga gattcgcctt ttcactcgag acatctgcct ctactgctta 2040

cctccaaata aacaacctca agtatgaaga tacagccact tacttttgcg ccctcgacta 2100

tagttacgcc atggactact ggggacaggg aacctccgtt accgtcagtt ccgcggccgc 2160

aaccacaaca cctgctccaa ggccccccac acccgctcca actatagcca gccaaccatt 2220

gagcctcaga cctgaagctt gcaggcccgc agcaggaggc gccgtccata cgcgaggcct 2280

ggacttcgcg tgtgatattt atatttgggc ccctttggcc ggaacatgtg gggtgttgct 2340

tctctccctt gtgatcactc tgtattgtaa gcgcgggaga aagaagctcc tgtacatctt 2400

caagcagcct tttatgcgac ctgtgcaaac cactcaggaa gaagatgggt gttcatgccg 2460

cttccccgag gaggaagaag gagggtgtga actgagggtg aaattttcta gaagcgccga 2520

tgctcccgca tatcagcagg gtcagaatca gctctacaat gaattgaatc tcggcaggcg 2580

agaagagtac gatgttctgg acaagagacg gggcagggat cccgagatgg ggggaaagcc 2640

ccggagaaaa aatcctcagg aggggttgta caatgagctg cagaaggaca agatggctga 2700

agcctatagc gagatcggaa tgaaaggcga aagacgcaga ggcaaggggc atgacggtct 2760

gtaccagggt ctctctacag ccaccaagga cacttatgat gcgttgcata tgcaagcctt 2820

gccaccccgc taagcggccg cgctttattt gtgaaatttg tgatgctatt gctttatttg 2880

taaccattat aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc 2940

aggttcaggg ggagatgtgg gaggtttttt aaagctcacc ggttctggggc ggtgctacaa 3000

ctgggctggc ggccaggatg gttcttaggt aggtggggtc ggcggtcagg tgtcccagag 3060

ccaggggtct ggagggacct tccaccctca gtccctggca ggtcgggggg tgctgaggcg 3120

ggcctggccc tggcagccca ggggtcccgg agcgaggggt ctggagggac ctttcactct 3180

cagtccctgg caggtcgggg ggtgctgtgg caggcccagc cttggccccc agctctgccc 3240

cttaccctga gctgtgtggc tttggggcagc tcgaactcct gggttcctct ctgggcccca 3300

actcctcccc tggcccaagt cccctctttg ctcctgggca ggcaggacct ctgtcccctc 3360

tcagccggtc cttggggctg cgtgtttctg tagaatgacg ggtcaggctg gccagaaccc 3420

caaaccttgg ccgtggggag tctgcgtggc ggctctgcct tgcccaggca tccttggtcc 3480

tcactcgagt ttttcctaagg atgggatgag ccccatgtgg gactaacctt ggctttacga 3540

3600

3660

tctctgccca gattccaggg ctctccccga gccctgttca gaccatccgt gggggaggcc 3720

ttggcctcac tcttacgtag ataagtagca tggcgggtta atcattaact acaaggaacc 3780

cctagtgatg gagttggcca ctccctctct gcgcgctcgc tcgctcactg aggccgggcg 3840

accaaaggtc gcccgacgcc cgggctttgc ccgggcggcc tcagtgagcg agcgagcgcg 3900

ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc 3960

tgaatggcga atggcgattc cgttgcaatg gctggcggta atattgttct ggatattacc 4020

agcaaggccg atagtttgag ttcttctact caggcaagtg atgttattac taatcaaaga 4080

agtattgcga caacggttaa tttgcgtgat ggacagactc ttttactcgg tggcctcact 4140

gattataaaa acacttctca ggattctggc gtaccgttcc tgtctaaaat ccctttaatc 4200

ggcctcctgt ttagctcccg ctctgattct aacgaggaaa gcacgttata cgtgctcgtc 4260

aaagcaacca tagtacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac 4320

gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc 4380

ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt 4440

agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg 4500

ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac 4560

4620

ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat 4680

ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt aaatatttgc 4740

ttatacaatc ttcctgtttt tggggctttt ctgattatca accggggtac atatgattga 4800

catgctagtt ttacgattac cgttcatcga ttctcttgtt tgctccagac tctcaggcaa 4860

tgacctgata gcctttgtag agacctctca aaaatagcta ccctctccgg catgaattta 4920

tcagctagaa cggttgaata tcatattgat ggtgatttga ctgtctccgg cctttctcac 4980

ccgtttgaat ctttacctac acattactca ggcattgcat ttaaaatata tgagggttct 5040

aaaaattttt atccttgcgt tgaaataaag gcttctcccg caaaagtatt acagggtcat 5100

5160

aattctttgc cttgcctgta tgatttattg gatgttggaa tcgcctgatg cggtattttc 5220

tccttacgca tctgtgcggt atttcacacc gcatatggtg cactctcagt acaatctgct 5280

ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 5340

gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca 5400

tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 5460

gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt 5520

ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt 5580

atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta 5640

tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg 5700

tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac 5760

gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg 5820

aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc 5880

gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg 5940

ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat 6000

gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg 6060

gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg 6120

atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc 6180

ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt 6240

6300

6360

gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca 6420

cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct 6480

cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt 6540

taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga 6600

ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca 6660

aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac 6720

6780

taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag 6840

gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac 6900

6960

taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg 7020

agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc 7080

ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc 7140

gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc 7200

acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa 7260

acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt 7320

tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg 7380

ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag 7440

agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tg 7492

<210>58

<211>6147

<212>DNA

<213>Artificial sequence

<220>

<223>Synthesized construct rAAV PD-1 Exon1_ATG-mCherry

<400>58

cagctgcgcg ctcgctcgct cactgaggcc gcccggggcaa agcccggggcg tcgggcgacc 60

tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc 120

actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtaaggctgt 180

tgcaggcatc acacggtgga aagatctgga actgtggcca tggtgtgagg ccatccacaa 240

ggtggaagct ttgaggggga gccgattagc catggacagt tgtcattcag tagggtcacc 300

tgtgccccag cgaaggggga tgggccggga aggcagaggc caggcacctg cccccagcag 360

gggcagaggc tgtgggcagc cgggaggctc ccagaggctc cgacagaatg ggagtggggt 420

tgagcccacc ccctcactgca gccaggaac ctgagcccag agggggccac ccaccttccc 480

caggcaggga ggcccggccc ccagggagat gggggggatg ggggaggaga agggcctgcc 540

cccacccggc agcctcagga ggggcagctc gggcgggata tggaaagagg ccacagcagt 600

gagcagagac acagaggagg aaggggccct gagctgggga gacccccacg gggtagggcg 660

tgggggccac gggccccacct cctccccatc tcctctgtct ccctgtctct gtctctctct 720

ccctccccca ccctctcccc agtcctaccc cctcctcacc cctcctcccc cagcactgcc 780

tctgtcactc tcgcccacgt ggatgtggag gaagaggggg cgggagcaag gggcgggcac 840

cctcccttca acctgacctg ggacagtttc ccttccgctc acctccgcct gagcagtgga 900

gaaggcggca ctctggtggg gctgctccag gcatgcaggt gagcaagggc gaggaggata 960

actccgccat catcaaggag ttcctgcgct tcaaggtgca catggagggc tccgtgaacg 1020

gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc acccagaccg 1080

ccaagctgaa ggtgaccaag ggtggccccc tgcccttcgc ctgggacatc ctgtcccctc 1140

agttcatgta cggctccaag gcctacgtga agcaccccgc cgacatcccc gactacttga 1200

agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag gacggcggcg 1260

tggtgaccgt gacccaggac tcctctctgc aggacggcga gttcatctac aaggtgaagc 1320

tgcgcggcac caacttcccc tccgacggcc ccgtaatgca gaagaagacc atgggctggg 1380

aggcctcctc cgagcggatg taccccgagg acggcgccct gaagggcgag atcaagcaga 1440

ggctgaagct gaaggacggc ggccactacg acgctgaggt caagaccacc tacaaggcca 1500

agaagcccgt gcagctgccc ggcgcctaca acgtcaacat caagttggac atcacctccc 1560

acaacgagga ctacaccatc gtggaacagt acgaacgcgc cgagggccgc cactccaccg 1620

gcggcatgga cgagctgtac aagtgatgaa agctttgctt tatttgtgaa atttgtgatg 1680

ctatgcttta tttgtaacca ttataagctg caataaacaa gtttaacaac aacaattgca 1740

ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagt aggtggggtc 1800

ggcggtcagg tgtccgag ccaggggtct ggagggacct tccaccctca gtccctggca 1860

ggtcgggggg tgctgaggcg ggcctggccc tggcagccca ggggtcccgg agcgaggggt 1920

ctggagggac ctttcactct cagtccctgg caggtcgggg ggtgctgtgg caggcccagc 1980

cttggccccc agctctgccc cttaccctga gctgtgtggc tttgggcagc tcgaactcct 2040

gggttcctct ctgggcccca actcctcccc tggcccaagt cccctctttg ctcctgggca 2100

ggcaggacct ctgtcccctc tcagccggtc cttggggctg cgtgtttctg tagaatgacg 2160

ggtcaggctg gccagaaccc caaaccttgg ccgtggggag tctgcgtggc ggctctgcct 2220

2280

2340

atccaaagtg tacctgttcg agtgaggaca gttcttctgt ctccaggata cgtagataag 2400

tagcatggcg ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc 2460

tctctgcgcg ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccggggc 2520

tttgcccggg cggcctcagt gagcgagcga gcgcgccagc tggcgtaata gcgaagaggc 2580

ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gattccgttg 2640

caatggctgg cggtaatatt gttctggata ttaccagcaa ggccgatagt ttgagttctt 2700

ctactcaggc aagtgatgtt attactaatc aaagaagtat tgcgacaacg gttaatttgc 2760

gtgatggaca gactctttta ctcggtggcc tcactgatta taaaaacact tctcaggatt 2820

ctggcgtacc gttcctgtct aaaatccctt taatcggcct cctgtttagc tcccgctctg 2880

2940

agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc 3000

agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc 3060

tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg 3120

cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga 3180

tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc 3240

3300

ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt 3360

3420

cttttctgat tatcaaccgg ggtacatatg attgacatgc tagttttacg attaccgttc 3480

atcgattctc ttgtttgctc cagactctca ggcaatgacc tgatagcctt tgtagagacc 3540

tctcaaaaat agctaccctc tccggcatga atttatcagc tagaacggtt gaatatcata 3600

3660

actcaggcat tgcatttaaa atatatgagg gttctaaaaa tttttatcct tgcgttgaaa 3720

taaaggcttc tcccgcaaaa gtattacagg gtcataatgt ttttggtaca accgatttag 3780

ctttatgctc tgaggcttta ttgcttaatt ttgctaattc tttgccttgc ctgtatgatt 3840

tattggatgt tggaatcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc 3900

acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc 3960

ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc 4020

ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc 4080

accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat 4140

gtaataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc 4200

tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg 4260

ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc 4320

ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt 4380

gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct 4440

caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac 4500

ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccggggc aagagcaact 4560

cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa 4620

gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga 4680

taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt 4740

tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga 4800

agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg 4860

caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat 4920

ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat 4980

tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc 5040

agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga 5100

tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc 5160

agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag 5220

gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 5280

gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 5340

tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 5400

gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 5460

accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 5520

accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 5580

gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 5640

ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 5700

atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 5760

5820

5880

gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 5940

gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc 6000

tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac 6060

cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct 6120

ccccgcgcgt tggccgattc attaatg 6147

<210>59

<211>232

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Lab made

<400>59

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Arg

180 185 190

Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro

195 200 205

Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro

210 215 220

Arg Asp Phe Ala Ala Tyr Arg Ser

225 230

<210>60

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ile3.exon1_RD2_B1G2

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>60

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gln Leu Glu Ile Arg Asn Val

20 25 30

Asn Pro Asn Ile Pro Arg Tyr Lys Thr Arg Leu Arg Phe Glu Ile Asp

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Lys Ile Tyr Asn Gln Gly Asp Ser Tyr Val Lys Leu Arg

65 70 75 80

Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Leu Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Glu Gly His Phe Gly Val Ile Leu Ala Lys Arg Arg Pro Ala Ser

180 185 190

Pro Val Gln Val Arg Leu Arg Phe Ala Ile Gly Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly Arg Ile

210 215 220

Arg Glu Lys Asn Ile Ser Glu Lys Ser Trp Leu Glu Phe Glu Val Thr

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>61

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ile3.exon1_RD3_B1G2C4

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>61

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile Leu Asn Arg

20 25 30

Asn Arg Gly Thr Ala Arg Tyr His Thr Arg Leu Ser Phe Thr Ile Met

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Ser Ile Leu Asn Asn Gly Asp His Tyr Val Ser Leu Val

65 70 75 80

Val Tyr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn Val Asn Ala

180 185 190

Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly His Ile

210 215 220

Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe Arg Val Glu

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>62

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ile3.exon1_RD3_B1 G2C11

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>62

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile Leu Asn Arg

20 25 30

Asn Arg Gly Thr Gly Arg Tyr His Thr Arg Leu Ser Phe Thr Ile Met

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Ser Ile Thr Asn Asn Gly Asp His Tyr Val Ser Leu Val

65 70 75 80

Val Tyr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn Val Asn Ala

180 185 190

Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly His Ile

210 215 220

Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe Arg Val Glu

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>63

<211>303

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized version of I-OnuI, PD-1.ile3.exon1_RD3_B1 G2C5

<220>

<221>MOD_RES

<222>(1)..(4)

<223>Any or missing amino acid

<220>

<221>MOD_RES

<222>(302)..(303)

<223>Any or missing amino acid

<400>63

Xaa Xaa Xaa Xaa Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile Leu Thr

1 5 10 15

Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile Leu Asn Arg

20 25 30

Asn Arg Gly Thr Ala Arg Tyr His Thr Arg Leu Ser Phe Thr Ile Met

35 40 45

Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser Thr Trp

50 55 60

Lys Val Gly Ser Ile Tyr Asn Asn Gly Asp His Tyr Val Ser Leu Glu

65 70 75 80

Val Phe Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe Glu Lys

85 90 95

Tyr Pro Leu Ile Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe Lys Gln

100 105 110

Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn Gly Ile

115 120 125

Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu Asn Asp

130 135 140

Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg Pro Leu

145 150 155 160

Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe Thr Ser

165 170 175

Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn Val Asn Ala

180 185 190

Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His Ile Arg Asp

195 200 205

Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly His Ile

210 215 220

Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe Arg Val Glu

225 230 235 240

Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln Glu Asn

245 250 255

Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys Lys Val

260 265 270

Ala Lys Leu Ile Glu Glu Lys Lys His Leu Thr Glu Ser Gly Leu Asp

275 280 285

Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg Xaa Xaa

290 295 300

<210>64

<211>943

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Synthesized construct megaTAL PD-1.ile3.exon1_RD1_B1G2

<400>64

Met Gly Ser Ala Pro Pro Lys Lys Lys Arg Lys Val Val Asp Leu Arg

1 5 10 15

Thr Leu Gly Tyr Ser Gln Gln Gln Gln Glu Lys Ile Lys Pro Lys Val

20 25 30

Arg Ser Thr Val Ala Gln His His Glu Ala Leu Val Gly His Gly Phe

35 40 45

Thr His Ala His Ile Val Ala Leu Ser Gln His Pro Ala Ala Leu Gly

50 55 60

Thr Val Ala Val Thr Tyr Gln His Ile Ile Thr Ala Leu Pro Glu Ala

65 70 75 80

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

85 90 95

Ala Leu Glu Ala Leu Leu Thr Asp Ala Gly Glu Leu Arg Gly Pro Pro

100 105 110

Leu Gln Leu Asp Thr Gly Gln Leu Val Lys Ile Ala Lys Arg Gly Gly

115 120 125

Val Thr Ala Met Glu Ala Val His Ala Ser Arg Asn Ala Leu Thr Gly

130 135 140

Ala Pro Leu Asn Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

145 150 155 160

Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

165 170 175

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

180 185 190

Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

195 200 205

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

210 215 220

Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg

225 230 235 240

Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val

245 250 255

Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val

260 265 270

Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp

275 280 285

Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu

290 295 300

Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr

305 310 315 320

Pro Asp Gln Val Val Ala Ile Ala Ser Asn Asn Gly Gly Lys Gln Ala

325 330 335

Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly

340 345 350

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

355 360 365

Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Asp

370 375 380

His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser His Asp Gly

385 390 395 400

Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys

405 410 415

Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala Ser Asn

420 425 430

Gly Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val

435 440 445

Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala Ile Ala

450 455 460

Ser Asn Asn Gly Gly Lys Gln Ala Leu Glu Thr Val Gln Arg Leu Leu

465 470 475 480

Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val Val Ala

485 490 495

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

500 505 510

Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp Gln Val

515 520 525

Val Ala Ile Ala Ser Asn Gly Gly Gly Lys Gln Ala Leu Glu Thr Val

530 535 540

Gln Arg Leu Leu Pro Val Leu Cys Gln Asp His Gly Leu Thr Pro Asp

545 550 555 560

Gln Val Val Ala Ile Ala Ser His Asp Gly Gly Lys Gln Ala Leu Glu

565 570 575

Ser Ile Val Ala Gln Leu Ser Arg Pro Asp Pro Ala Leu Ala Ala Leu

580 585 590

Thr Asn Asp His Leu Val Ala Leu Ala Cys Leu Gly Gly Arg Pro Ala

595 600 605

Met Asp Ala Val Lys Lys Gly Leu Pro His Ala Pro Glu Leu Ile Arg

610 615 620

Arg Val Asn Arg Arg Ile Gly Glu Arg Thr Ser His Arg Val Ala Ile

625 630 635 640

Ser Arg Val Gly Gly Ser Ser Arg Arg Glu Ser Ile Asn Pro Trp Ile

645 650 655

Leu Thr Gly Phe Ala Asp Ala Glu Gly Ser Phe Gly Leu Ser Ile Leu

660 665 670

Asn Arg Asn Arg Gly Thr Ala Arg Tyr His Thr Arg Leu Ser Phe Thr

675 680 685

Ile Met Leu His Asn Lys Asp Lys Ser Ile Leu Glu Asn Ile Gln Ser

690 695 700

Thr Trp Lys Val Gly Ile Ile Thr Asn Asn Gly Asp His Tyr Val Thr

705 710 715 720

Leu Arg Val Thr Arg Phe Glu Asp Leu Lys Val Ile Ile Asp His Phe

725 730 735

Glu Lys Tyr Pro Leu Val Thr Gln Lys Leu Gly Asp Tyr Lys Leu Phe

740 745 750

Lys Gln Ala Phe Ser Val Met Glu Asn Lys Glu His Leu Lys Glu Asn

755 760 765

Gly Ile Lys Glu Leu Val Arg Ile Lys Ala Lys Met Asn Trp Gly Leu

770 775 780

Asn Asp Glu Leu Lys Lys Ala Phe Pro Glu Asn Ile Ser Lys Glu Arg

785 790 795 800

Pro Leu Ile Asn Lys Asn Ile Pro Asn Phe Lys Trp Leu Ala Gly Phe

805 810 815

Thr Ser Gly Asp Gly Ser Phe Phe Val Arg Leu Arg Lys Ser Asn Val

820 825 830

Asn Ala Arg Val Arg Val Gln Leu Val Phe Glu Ile Ser Gln His Ile

835 840 845

Arg Asp Lys Asn Leu Met Asn Ser Leu Ile Thr Tyr Leu Gly Cys Gly

850 855 860

His Ile Tyr Glu Gly Asn Lys Ser Glu Arg Ser Trp Leu Gln Phe Arg

865 870 875 880

Val Glu Lys Phe Ser Asp Ile Asn Asp Lys Ile Ile Pro Val Phe Gln

885 890 895

Glu Asn Thr Leu Ile Gly Val Lys Leu Glu Asp Phe Glu Asp Trp Cys

900 905 910

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

915 920 925

Leu Asp Glu Ile Lys Lys Ile Lys Leu Asn Met Asn Lys Gly Arg

930 935 940

<210>65

<211>2832

<212>RNA

<213>Artificial sequence

<220>

<223>Synthesized mRNA construct megaTAL PD-1 Exon1 RD2_B1G2

<400>65

augggaagcg cgccaccuaa gaagaaacgc aaagucgugg aucuacgcac gcucggcuac 60

agucagcagc agcaagagaa gaucaaaccg aaggugcguu cgacaguggc gcagcaccac 120

gaggcacugg ugggccaugg guuuacacac gcgcacaucg uugcgcucag ccaacacccg 180

gcagcguuag ggaccgucgc ugucacguau cagcacauaa ucacggcguu gccagaggcg 240

acacacgaag acaucguugg cgucggcaaa cagugguccg gcgcacgcgc ccuggaggcc 300

uugcucacgg augcggggga guugagaggu ccgccguuac aguuggacac aggccaacuu 360

gugaagauug caaaacgugg cggcgugacc gcaauggagg cagugcaugc aucgcgcaau 420

gcacugacgg gugccccccu gaaccuaacc ccugaucagg uagucgcuau agcuucaaac 480

aacgggggca agcaagcacu ggagaccguu caacgacucc ugccagugcu cugccaagac 540

cacggacuua cgccagauca ggugguugcu auugccucca acaauggcgg gaaacaagcg 600

660

caggugguag ccauagcguc gaauggaggu gguaagcaag cccuugaaac gguccagcgu 720

cuucugccgg uguugugcca ggaccacggga cuaacgccgg aucaggucgu agccauugcu 780

ucaaauaacg gcggcaaaca ggcgcuagag acaguccagc gccucuugcc uguguuaugc 840

caggaucacg gcuuaacccc agaccaaguu guggcuauug cauuaacaa ugguggcaaa 900

caagccuugg agacagugca acgauuacug ccugucuuau gucaggauca uggccugacg 960

cccgaucagg uaguggcaau cgcaucuaau aauggaggua agcaagcacu ggagacuguc 1020

cagagauugu uacccguacu augucaagau caugguuuga cgccugauca gguuguugcg 1080

1140

uugugccaag aucacgggcu uacuccugau caagucguag cuaucgccag ccacgacggu 1200

gggaaacagg cccuggaaac cguacaacgu cuccucccag uacuuuguca agaccacggg 1260

uugacuccgg aucaagucgu cgcgaucgcg agcaauggag gggggaagca ggcgcuggaa 1320

acuguucaga gacugcugcc uguacuuugu caggaccaug gucugacacc ugaccaaguu 1380

guggcgauag ccaguaacaa ugggggaaaa caggcacuag agacgguuca aagguuguug 1440

cccguucugu gccaggacca cggcuugaca ccggaucagg ugguagcuau cgcuucacac 1500

1560

cacggauuga cuccagauca agucguugcu auugcaagua augguggugg uaagcaagcu 1620

1680

caagugguag caauugcauc ucaugaugga ggaaaacaag cucuggaaag cauuguggcc 1740

cagcugagcc ggccugaucc ggcguuggcc gcguugacca acgaccaccu cgucgccuug 1800

gccugccucg gcggacgucc ugccauggau gcagugaaaa agggauugcc gcacgcgccg 1860

gaauugauca gaagagucaa ucgccguauu ggcgaacgca cgucccaucg cguugcgaua 1920

ucuagagugg gaggaagcuc ucgcagagag uccaucaacc cauggauucu gacugguuuc 1980

gcugaugccg aaggaucauu cgggcuaagc auccucaaca gaaacagagg uacugcuaga 2040

uaccacacuc gacugucauu cacaaucaug cugcacaaca aggacaaauc gauucuggag 2100

aauauccagu cgacuuggaa ggucggcaua aucaccaaca acggcgacca cuacgucacc 2160

cugcgcguca cccguuucga agauuugaaa gugauuaucg accacuucga gaaauauccg 2220

cugguaacac agaaauuggg cgauuacaag uuguuuaaac aggcauucag cgucauggag 2280

aacaaagaac aucuuaagga gaaugggauu aaggagcucg

aauugggguc ucaugacga auugaaaaaa gcauuuccag agaacauuag caaagagcgc 2400

ccccuuauca auaagaacau uccgaauuuc aaauggcugg cuggauucac aucggugau 2460

ggcuccuucu ucgugcgccu aagaaagucu aauguuaaug cuagaguacg ugugcaacug 2520

guauucgaga ucucacagca caucagagac aagaaccuga ugaauucauu gauaacauac 2580

cuaggcugug gucacaucua cgagggaaac aaauucugagc gcaguuggcu ccaauucaga 2640

guagaaaaau ucagcgauau caacgacaag aucauuccgg uauuccagga aaauacucug 2700

2760

aagaaacacc ugaccgaauc cgguuuggau gagauuaaga aaaucaagcu gaacaugaac 2820

aaaggucguu ga 2832

<210>66

<211>2832

<212>RNA

<213>Artificial sequence

<220>

<223>Synthesized mRNA construct PD-1 Exon1 RD2_B1G2C4 megaTAL

<400>66

augggaagcg cgccaccuaa gaagaaacgc aaagucgugg aucuacgcac gcucggcuac 60

agucagcagc agcaagagaa gaucaaaccg aaggugcguu cgacaguggc gcagcaccac 120

gaggcacugg ugggccaugg guuuacacac gcgcacaucg uugcgcucag ccaacacccg 180

gcagcguuag ggaccgucgc ugucacguau cagcacauaa ucacggcguu gccagaggcg 240

acacacgaag acaucguugg cgucggcaaa cagugguccg gcgcacgcgc ccuggaggcc 300

uugcucacgg augcggggga guugagaggu ccgccguuac aguuggacac aggccaacuu 360

gugaagauug caaaacgugg cggcgugacc gcaauggagg cagugcaugc aucgcgcaau 420

gcacugacgg gugccccccu gaaccuaacc ccugaucagg uagucgcuau agcuucaaac 480

aacgggggca agcaagcacu ggagaccguu caacgacucc ugccagugcu cugccaagac 540

cacggacuua cgccagauca ggugguugcu auugccucca acaauggcgg gaaacaagcg 600

660

caggugguag ccauagcguc gaauggaggu gguaagcaag cccuugaaac gguccagcgu 720

cuucugccgg uguugugcca ggaccacggga cuaacgccgg aucaggucgu agccauugcu 780

ucaaauaacg gcggcaaaca ggcgcuagag acaguccagc gccucuugcc uguguuaugc 840

caggaucacg gcuuaacccc agaccaaguu guggcuauug cauuaacaa ugguggcaaa 900

caagccuugg agacagugca acgauuacug ccugucuuau gucaggauca uggccugacg 960

cccgaucagg uaguggcaau cgcaucuaau aauggaggua agcaagcacu ggagacuguc 1020

cagagauugu uacccguacu augucaagau caugguuuga cgccugauca gguuguugcg 1080

1140

uugugccaag aucacgggcu uacuccugau caagucguag cuaucgccag ccacgacggu 1200

gggaaacagg cccuggaaac cguacaacgu cuccucccag uacuuuguca agaccacggg 1260

uugacuccgg aucaagucgu cgcgaucgcg agcaauggag gggggaagca ggcgcuggaa 1320

acuguucaga gacugcugcc uguacuuugu caggaccaug gucugacacc ugaccaaguu 1380

guggcgauag ccaguaacaa ugggggaaaa caggcacuag agacgguuca aagguuguug 1440

cccguucugu gccaggacca cggcuugaca ccggaucagg ugguagcuau cgcuucacac 1500

1560

cacggauuga cuccagauca agucguugcu auugcaagua augguggugg uaagcaagcu 1620

1680

caagugguag caauugcauc ucaugaugga ggaaaacaag cucuggaaag cauuguggcc 1740

cagcugagcc ggccugaucc ggcguuggcc gcguugacca acgaccaccu cgucgccuug 1800

gccugccucg gcggacgucc ugccauggau gcagugaaaa agggauugcc gcacgcgccg 1860

gaauugauca gaagagucaa ucgccguauu ggcgaacgca cgucccaucg cguugcgaua 1920

ucuagagugg gaggaagcuc ucgcagagag uccaucaacc cauggauucu gacugguuuc 1980

gcugaugccg aaggaucauu cgggcuaagc auccucaaca gaaacagagg uacugcuaga 2040

uaccacacuc gacugucauu cacaaucaug cugcacaaca aggacaaauc gauucuggag 2100

aauauccagu cgacuuggaa ggucggcagc auccucaaca auggcgacca cuacgucucg 2160

cuggggucu accguuucga agauuugaaa gugauuuaucg accacuucga gaaauauccg 2220

cugauaacac agaaauuggg cgauuacaag uuguuuaaac aggcauucag cgucauggag 2280

aacaaagaac aucuuaagga gaaugggauu aaggagcucg

aauugggguc ucaugacga auugaaaaaa gcauuuccag agaacauuag caaagagcgc 2400

ccccuuauca auaagaacau uccgaauuuc aaauggcugg cuggauucac aucggugau 2460

ggcuccuucu ucgugcgccu aagaaagucu aauguuaaug cuagaguacg ugugcaacug 2520

guauucgaga ucucacagca caucagagac aagaaccuga ugaauucauu gauaacauac 2580

cuaggcugug gucacaucua cgagggaaac aaauucugagc gcaguuggcu ccaauucaga 2640

guagaaaaau ucagcgauau caacgacaag aucauuccgg uauuccagga aaauacucug 2700

2760

aagaaacacc ugaccgaauc cgguuuggau gagauuaaga aaaucaagcu gaacaugaac 2820

aaaggucguu ga 2832

<210>67

<211>2832

<212>RNA

<213>Artificial sequence

<220>

<223>Synthesized mRNA construct megaTAL PD-1 Exon1 RD2_B1G2C11

<400>67

augggaagcg cgccaccuaa gaagaaacgc aaagucgugg aucuacgcac gcucggcuac 60

agucagcagc agcaagagaa gaucaaaccg aaggugcguu cgacaguggc gcagcaccac 120

gaggcacugg ugggccaugg guuuacacac gcgcacaucg uugcgcucag ccaacacccg 180

gcagcguuag ggaccgucgc ugucacguau cagcacauaa ucacggcguu gccagaggcg 240

acacacgaag acaucguugg cgucggcaaa cagugguccg gcgcacgcgc ccuggaggcc 300

uugcucacgg augcggggga guugagaggu ccgccguuac aguuggacac aggccaacuu 360

gugaagauug caaaacgugg cggcgugacc gcaauggagg cagugcaugc aucgcgcaau 420

gcacugacgg gugccccccu gaaccuaacc ccugaucagg uagucgcuau agcuucaaac 480

aacgggggca agcaagcacu ggagaccguu caacgacucc ugccagugcu cugccaagac 540

cacggacuua cgccagauca ggugguugcu auugccucca acaauggcgg gaaacaagcg 600

660

caggugguag ccauagcguc gaauggaggu gguaagcaag cccuugaaac gguccagcgu 720

cuucugccgg uguugugcca ggaccacggga cuaacgccgg aucaggucgu agccauugcu 780

ucaaauaacg gcggcaaaca ggcgcuagag acaguccagc gccucuugcc uguguuaugc 840

caggaucacg gcuuaacccc agaccaaguu guggcuauug cauuaacaa ugguggcaaa 900

caagccuugg agacagugca acgauuacug ccugucuuau gucaggauca uggccugacg 960

cccgaucagg uaguggcaau cgcaucuaau aauggaggua agcaagcacu ggagacuguc 1020

cagagauugu uacccguacu augucaagau caugguuuga cgccugauca gguuguugcg 1080

1140

uugugccaag aucacgggcu uacuccugau caagucguag cuaucgccag ccacgacggu 1200

gggaaacagg cccuggaaac cguacaacgu cuccucccag uacuuuguca agaccacggg 1260

uugacuccgg aucaagucgu cgcgaucgcg agcaauggag gggggaagca ggcgcuggaa 1320

acuguucaga gacugcugcc uguacuuugu caggaccaug gucugacacc ugaccaaguu 1380

guggcgauag ccaguaacaa ugggggaaaa caggcacuag agacgguuca aagguuguug 1440

cccguucugu gccaggacca cggcuugaca ccggaucagg ugguagcuau cgcuucacac 1500

1560

cacggauuga cuccagauca agucguugcu auugcaagua augguggugg uaagcaagcu 1620

1680

caagugguag caauugcauc ucaugaugga ggaaaacaag cucuggaaag cauuguggcc 1740

cagcugagcc ggccugaucc ggcguuggcc gcguugacca acgaccaccu cgucgccuug 1800

gccugccucg gcggacgucc ugccauggau gcagugaaaa agggauugcc gcacgcgccg 1860

gaauugauca gaagagucaa ucgccguauu ggcgaacgca cgucccaucg cguugcgaua 1920

ucuagagugg gaggaagcuc ucgcagagag uccaucaacc cauggauucu gacugguuuc 1980

gcugaugccg aaggaucauu cgggcuaagc auccucaaca gaaacagagg uacugguaga 2040

uaccacacuc gacugucauu cacaaucaug cugcacaaca aggacaaauc gauucuggag 2100

aauauccagu cgacuuggaa ggucggcucg aucacgaaca acggcgacca cuacgucagc 2160

cuggucgucu accguuucga agauuugaaa gugauuaucg accacuucga gaaauauccg 2220

cugauaacac agaaauuggg cgauuacaag uuguuuaaac aggcauucag cgucauggag 2280

aacaaagaac aucuuaagga gaaugggauu aaggagcucg

aauugggguc ucaugacga auugaaaaaa gcauuuccag agaacauuag caaagagcgc 2400

ccccuuauca auaagaacau uccgaauuuc aaauggcugg cuggauucac aucggugau 2460

ggcuccuucu ucgugcgccu aagaaagucu aauguuaaug cuagaguacg ugugcaacug 2520

guauucgaga ucucacagca caucagagac aagaaccuga ugaauucauu gauaacauac 2580

cuaggcugug gucacaucua cgagggaaac aaauucugagc gcaguuggcu ccaauucaga 2640

guagaaaaau ucagcgauau caacgacaag aucauuccgg uauuccagga aaauacucug 2700

2760

aagaaacacc ugaccgaauc cgguuuggau gagauuaaga aaaucaagcu gaacaugaac 2820

aaaggucguu ga 2832

<210>68

<211>2832

<212>RNA

<213>Artificial sequence

<220>

<223>Synthesized mRNA construct megaTAL PD-1 Exon1 RD2_B1G2C5

<400>68

augggaagcg cgccaccuaa gaagaaacgc aaagucgugg aucuacgcac gcucggcuac 60

agucagcagc agcaagagaa gaucaaaccg aaggugcguu cgacaguggc gcagcaccac 120

gaggcacugg ugggccaugg guuuacacac gcgcacaucg uugcgcucag ccaacacccg 180

gcagcguuag ggaccgucgc ugucacguau cagcacauaa ucacggcguu gccagaggcg 240

acacacgaag acaucguugg cgucggcaaa cagugguccg gcgcacgcgc ccuggaggcc 300

uugcucacgg augcggggga guugagaggu ccgccguuac aguuggacac aggccaacuu 360

gugaagauug caaaacgugg cggcgugacc gcaauggagg cagugcaugc aucgcgcaau 420

gcacugacgg gugccccccu gaaccuaacc ccugaucagg uagucgcuau agcuucaaac 480

aacgggggca agcaagcacu ggagaccguu caacgacucc ugccagugcu cugccaagac 540

cacggacuua cgccagauca ggugguugcu auugccucca acaauggcgg gaaacaagcg 600

660

caggugguag ccauagcguc gaauggaggu gguaagcaag cccuugaaac gguccagcgu 720

cuucugccgg uguugugcca ggaccacggga cuaacgccgg aucaggucgu agccauugcu 780

ucaaauaacg gcggcaaaca ggcgcuagag acaguccagc gccucuugcc uguguuaugc 840

caggaucacg gcuuaacccc agaccaaguu guggcuauug cauuaacaa ugguggcaaa 900

caagccuugg agacagugca acgauuacug ccugucuuau gucaggauca uggccugacg 960

cccgaucagg uaguggcaau cgcaucuaau aauggaggua agcaagcacu ggagacuguc 1020

cagagauugu uacccguacu augucaagau caugguuuga cgccugauca gguuguugcg 1080

1140

uugugccaag aucacgggcu uacuccugau caagucguag cuaucgccag ccacgacggu 1200

gggaaacagg cccuggaaac cguacaacgu cuccucccag uacuuuguca agaccacggg 1260

uugacuccgg aucaagucgu cgcgaucgcg agcaauggag gggggaagca ggcgcuggaa 1320

acuguucaga gacugcugcc uguacuuugu caggaccaug gucugacacc ugaccaaguu 1380

guggcgauag ccaguaacaa ugggggaaaa caggcacuag agacgguuca aagguuguug 1440

cccguucugu gccaggacca cggcuugaca ccggaucagg ugguagcuau cgcuucacac 1500

1560

cacggauuga cuccagauca agucguugcu auugcaagua augguggugg uaagcaagcu 1620

1680

caagugguag caauugcauc ucaugaugga ggaaaacaag cucuggaaag cauuguggcc 1740

cagcugagcc ggccugaucc ggcguuggcc gcguugacca acgaccaccu cgucgccuug 1800

gccugccucg gcggacgucc ugccauggau gcagugaaaa agggauugcc gcacgcgccg 1860

gaauugauca gaagagucaa ucgccguauu ggcgaacgca cgucccaucg cguugcgaua 1920

ucuagagugg gaggaagcuc ucgcagagag uccaucaacc cauggauucu gacugguuuc 1980

gcugaugccg aaggaucauu cgggcuaagc auccucaaca gaaacagagg uacugcuaga 2040

uaccacacuc gacugucauu cacaaucaug cugcacaaca aggacaaauc gauucuggag 2100

aauauccagu cgacuuggaa ggucggcucg aucuacaaca acggcgacca cuacgucucg 2160

cuggaggucu uccguuucga agauuugaaa gugauuaucg accacuucga gaaauauccg 2220

cugauaacac agaaauuggg cgauuacaag uuguuuaaac aggcauucag cgucauggag 2280

aacaaagaac aucuuaagga gaaugggauu aaggagcucg

aauugggguc ucaugacga auugaaaaaa gcauuuccag agaacauuag caaagagcgc 2400

ccccuuauca auaagaacau uccgaauuuc aaauggcugg cuggauucac aucggugau 2460

ggcuccuucu ucgugcgccu aagaaagucu aauguuaaug cuagaguacg ugugcaacug 2520

guauucgaga ucucacagca caucagagac aagaaccuga ugaauucauu gauaacauac 2580

cuaggcugug gucacaucua cgagggaaac aaauucugagc gcaguuggcu ccaauucaga 2640

guagaaaaau ucagcgauau caacgacaag aucauuccgg uauuccagga aaauacucug 2700

2760

aagaaacacc ugaccgaauc cgguuuggau gagauuaaga aaaucaagcu gaacaugaac 2820

aaaggucguu ga 2832

<210>69

<211>3

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>69

Gly Gly Gly

one

<210>70

<211>5

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>70

Asp Gly Gly Gly Ser

fifteen

<210>71

<211>5

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>71

Thr Gly Glu Lys Pro

fifteen

<210>72

<211>4

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>72

Gly Gly Arg Arg

one

<210>73

<211>5

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>73

Gly Gly Gly Gly Ser

fifteen

<210>74

<211>14

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>74

Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Val Asp

1 5 10

<210>75

<211>18

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>75

Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser

1 5 10 15

Leu Asp

<210>76

<211>8

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>76

Gly Gly Arg Arg Gly Gly Gly Ser

fifteen

<210>77

<211>9

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>77

Leu Arg Gln Arg Asp Gly Glu Arg Pro

fifteen

<210>78

<211>12

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>78

Leu Arg Gln Lys Asp Gly Gly Gly Ser Glu Arg Pro

1 5 10

<210>79

<211>16

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Exemplary linker sequence

<400>79

Leu Arg Gln Lys Asp Gly Gly Gly Ser Gly Gly Gly Ser Glu Arg Pro

1 5 10 15

<210>80

<211>7

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Cleavage sequence by TEV protease

<220>

<221>other_characteristic

<222>(2)..(3)

<223>Xaa - any amino acid

<220>

<221>other_characteristic

<222>(5)..(5)

<223>Xaa - any amino acid

<220>

<221>OTHER_SIGNIFICANT

<222>(7)..(7)

<223>Xaa=Gly or Ser

<400>80

Glu Xaa Xaa Tyr Xaa Gln Xaa

fifteen

<210>81

<211>7

<212>PROTEIN

<213>Artificial sequence

<220>

<223>TEV protease cleavage site sequence

<400>81

Glu Asn Leu Tyr Phe Gln Gly

fifteen

<210>82

<211>7

<212>PROTEIN

<213>Artificial sequence

<220>

<223>TEV protease cleavage site sequence

<400>82

Glu Asn Leu Tyr Phe Gln Ser

fifteen

<210>83

<211>22

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>83

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

1 5 10 15

Glu Glu Asn Pro Gly Pro

twenty

<210>84

<211>19

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>84

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

1 5 10 15

Pro Gly Pro

<210>85

<211>14

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>85

Leu Leu Lys Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro

1 5 10

<210>86

<211>21

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>86

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

1 5 10 15

Glu Asn Pro Gly Pro

twenty

<210>87

<211>18

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>87

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

1 5 10 15

GlyPro

<210>88

<211>13

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>88

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

1 5 10

<210>89

<211>23

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>89

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

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

twenty

<210>90

<211>20

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>90

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

1 5 10 15

Asn Pro Gly Pro

twenty

<210>91

<211>14

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>91

Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro

1 5 10

<210>92

<211>25

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>92

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

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210>93

<211>22

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>93

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

1 5 10 15

Glu Ser Asn Pro Gly Pro

twenty

<210>94

<211>14

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>94

Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro

1 5 10

<210>95

<211>19

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>95

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

1 5 10 15

Pro Gly Pro

<210>96

<211>19

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>96

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

1 5 10 15

Pro Gly Pro

<210>97

<211>14

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>97

Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro

1 5 10

<210>98

<211>17

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>98

Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly

1 5 10 15

Pro

<210>99

<211>20

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>99

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

1 5 10 15

Asn Pro Gly Pro

twenty

<210>100

<211>24

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>100

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

1 5 10 15

Asp Val Glu Ser Asn Pro Gly Pro

twenty

<210>101

<211>40

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>101

Val Thr Glu Leu Leu Tyr Arg Met Lys Arg Ala Glu Thr Tyr Cys Pro

1 5 10 15

Arg Pro Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys

20 25 30

Ile Val Ala Pro Val Lys Gln Thr

35 40

<210>102

<211>18

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>102

Leu Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro

1 5 10 15

GlyPro

<210>103

<211>40

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>103

Leu Leu Ala Ile His Pro Thr Glu Ala Arg His Lys Gln Lys Ile Val

1 5 10 15

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

20 25 30

Asp Val Glu Ser Asn Pro Gly Pro

35 40

<210>104

<211>33

<212>PROTEIN

<213>Artificial sequence

<220>

<223>Self-cleaving polypeptide containing the 2A site

<400>104

Glu Ala Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Thr Leu

1 5 10 15

Asn Phe Asp Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly

20 25 30

Pro

<210>105

<211>10

<212>DNA

<213>Artificial sequence

<220>

<223>Kozak consensus sequence

<400>105

gccrccatgg 10

<210>106

<211>8

<212>PROTEIN

<213>Homo sapiens

<400>106

Glu Gln Thr Glu Tyr Ala Thr Ile

fifteen

<210>107

<211>43

<212>DNA

<213>Homo sapiens

<400>107

tgagcagacg gagtatgcca ccattgtctt tcctagcgga atg 43

<210>108

<211>43

<212>DNA

<213>Artificial sequence

<220>

<223>Lab made

<400>108

cattccgcta ggaaagacaa tggtggcata ctccgtctgc tca 43

<210>109

<211>22

<212>DNA

<213>Artificial sequence

<220>

<223>Lab made

<220>

<221>other_characteristic

<222>(14)..(22)

<223>n is A, C, G, or T

<400>109

aatggtggca tacnnnnnnn nn 22

<210>110

<211>22

<212>DNA

<213>Artificial sequence

<220>

<223>Lab made

<220>

<221>other_characteristic

<222>(1)..(9)

<223>n is a, c, g, or t

<400>110

nnnnnnnnna tactccgtct gc 22

<210>111

<211>22

<212>DNA

<213>Artificial sequence

<220>

<223>Lab made

<400>111

aatggtggca tactccgtct gc 22

<210>112

<211>43

<212>DNA

<213>Homo sapiens

<400>112

attccgctag gaaagacaat ggtggcatac tccgtctgct cag 43

<210>113

<211>43

<212>DNA

<213>Artificial sequence

<220>

<223>Lab made

<400>113

atttcgttag gaaagataat ggtggtatat ttcgtttgtt tag 43

<210>114

<211>7

<212>PROTEIN

<213>Homo sapiens

<400>114

Met Gln Ile Pro Gln Ala Pro

fifteen

<210>115

<211>44

<212>DNA

<213>Homo sapiens

<400>115

ctctggtggg gctgctccag gcatgcagat cccacaggcg ccct 44

<210>116

<211>44

<212>DNA

<213>Artificial sequence

<220>

<223>Lab made

<400>116

agggcgcctg tgggatctgc atccctggag cagccccacc agag 44

<210>117

<211>8

<212>PROTEIN

<213>Homo sapiens

<400>117

Asn Gly Arg Asp Phe His Met Ser

fifteen

<210>118

<211>43

<212>DNA

<213>Homo sapiens

<400>118

gtgtcacaca actgcccaac gggcgtgact tccacatgag cgt 43

<210>119

<211>43

<212>DNA

<213>Artificial sequence

<220>

<223>Lab made

<400>119

acgctcatgt ggaagtcacg cccgttggggc agttgtgtga cac 43

<210>120

<211>32

<212>DNA

<213>Homo sapiens

<400>120

gctccaggca tgcagatccc acaggcgccc tg 32

<210>121

<211>32

<212>DNA

<213>Artificial sequence

<220>

<223>Lab Made

<400>121

actccaaaca tacaaatccc acaaacaccc ta 32

<210>122

<211>31

<212>DNA

<213>Homo sapiens

<400>122

gcccaacggg cgtgacttcc acatgagcgt g 31

<210>123

<211>31

<212>DNA

<213>Artificial sequence

<220>

<223>Lab Made

<400>123

acccaacgaa cgtaacttcc acataaacgt a 31

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Claims (22)

1. A polypeptide containing an I-OnuI homing endonuclease (HE) variant that binds and cleaves a target site in the human programmed cell death 1 (PD-1) gene, characterized in that the I-OnuI variant does NOT contain the amino acid sequence at least 95% identical to the amino acid sequence shown in any of SEQ ID NOs: 60-63. 2. The polypeptide of claim 1, wherein the I-OnuI variant does NOT cleave the target site of exon 1 of the PD-1 gene and contains the following amino acid substitutions relative to the amino acid sequence shown in SEQ ID NO: 4: (a) L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46A, T48V, V68I, A70T, S72D, N75R, A76Y, S78R, K80R, I100V, L138M, T143N . (b) L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, V37G, G38R, S40H, E42R, G44S, Q46T, T48V, V68I, A70T, S72D, N75R, A76Y, S78R, K80C, I100V, V132A . (c) L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68I, A70T, S72N, N75H, A76Y, S78T, K80R, I100V, L138M, T143N . (d) L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70Y, S72N, N75H, A76Y, K80E, T82F, L138M, T143N, S159P , E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R and T240E; (e) L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37A, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70L, S72N, N75H, A76Y, K80V, T82Y, L138M, T143N, S159P , E178D, C180S, N184R, I186R, K189N, S190V, K191N, L192A, G193R, Q195R, S201E, T203S, K207R, Y223H, K225Y, K227G, F232R, D236Q, V238R and T240E; or (f) L26G, R28S, R30L, N32R, K34R, S35G, S36T, V37G, G38R, S40H, E42R, G44S, Q46T, T48M, V68S, A70T, S72N, N75H, A76Y, K80V, T82Y, L138M, T143N, S159P . 3. A polypeptide according to claim 1 or 2, wherein said polypeptide binds the polynucleotide sequence shown in SEQ ID NO: 30. 4. A polypeptide containing an I-OnuI homing endonuclease (HE) variant that binds and cleaves a target site in the human programmed cell death 1 (PD-1) gene, characterized in that the I-OnuI variant does NOT contain the amino acid sequence at least 95% identical to the amino acid sequence shown in any of SEQ ID NOs: 60-63, further containing a TALE DNA binding domain that contains from about 9.5 TALE repeat units to about 15.5 TALE repeat units. 5. The polypeptide of claim 4, wherein the TALE DNA binding domain binds the polynucleotide sequence shown in SEQ ID NO: 31. 6. The polypeptide of claim 5, wherein said polypeptide binds and cleaves the polynucleotide sequence shown in SEQ ID NO: 32. 7. A polypeptide containing an I-OnuI homing endonuclease (HE) variant that binds and cleaves a target site in the human programmed cell death 1 (PD-1) gene, characterized in that the I-OnuI variant does NOT contain the amino acid sequence at least 95% identical to the amino acid sequence shown in any of SEQ ID NOs: 60-63, additionally containing a peptide linker or viral self-cleaving peptide 2A and 3'-5' Trex2 exonuclease, or an appropriate biologically active fragment. 8. Polynucleotide encoding a polypeptide according to any one of paragraphs. 1-7. 9. The polynucleotide of claim 8, wherein the polynucleotide is mRNA. 10. The polynucleotide of claim 8, wherein the polynucleotide is cDNA. 11. An expression vector containing a polynucleotide encoding a polypeptide according to any one of paragraphs. 1-10. 12. Cell for adoptive cell therapy containing a polypeptide according to any one of paragraphs. 1-7, the polynucleotide according to claims. 8-10 or an expression vector according to claim 11, wherein the cell is a hematopoietic cell, a T cell, a CD3+ cell, a CD4+ cell, a CD8+ cell, an immune effector cell, a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL) , a helper T cell, a natural killer (NK), or a natural killer T cell (NKT). 13. Cell for adoptive cell therapy containing a polypeptide according to any one of paragraphs. 1-7, a polynucleotide according to any one of paragraphs. 8-10 or an expression vector according to claim 11, wherein said cell contains a polynucleotide encoding an engineered TCR, CAR, Daric receptor, or zetakin. 14. An adoptive cell therapy cell according to claim 13, wherein said cell is a hematopoietic cell, a T cell, a CD3+ cell, a CD4+ cell, a CD8+ cell, an immune effector cell, a cytotoxic T lymphocyte (CTL), a tumor infiltrating lymphocyte (TIL). ), a helper T cell, a natural killer (NK) cell, or a natural killer T cell (NKT). 15. Composition for adoptive cell therapy, containing an effective number of cells according to any one of paragraphs. 12-14. 16. Composition for adoptive cell therapy, containing an effective number of cells according to any one of paragraphs. 12-14 and a physiologically acceptable carrier.

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