Methods and Compositions for Modulating CAR-T Activity

Abstract

Disclosed herein are engineered and / or hypoimmunogenic cells, including engineered and / or hypoimmunogenic stem cells, engineered and / or hypoimmunogenic cells differentiated therefrom, engineered and / or hypoimmunogenic CAR-T cells (differentiated from primary or engineered and / or hypoimmunogenic stem cells), and related methods of their use and production. Provided herein are engineered and / or hypoimmunogenic cells that exhibit reduced expression of MHC class I and / or MHC class II human leukocyte antigens and T cell receptors. In some embodiments, such cells also exogenously express one or more tolerogenic factors, such as CD47 and one or more chimeric antigen receptors (CARs).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Nos. 63 / 133,171 (filed December 31, 2020); 63 / 136,172 (filed January 11, 2021); 63 / 175,003 (filed April 14, 2021); 63 / 255,795 (filed October 14, 2021); and 63 / 288,477 (filed December 10, 2021), the disclosures of which are incorporated herein by reference in their entireties. Summary of the Invention

[0002] Off-the-shelf CAR-T cells and other therapeutic cells may offer advantages over autologous cell-based strategies, including ease of manufacturing, quality control, and avoidance of harmful contamination and T cell dysfunction. However, robust host-versus-graft immune responses against histo-incompatible T cells hinder the expansion and persistence of allogeneic CAR-T cells, reducing the effectiveness of this approach.

[0003] There is strong evidence in both animal models and human patients that hypoimmunogenic cell transplantation is a scientifically feasible and clinically promising approach to the treatment of numerous disorders, conditions, and diseases.

[0004] There remains a need for novel approaches, compositions, and methods for generating cell-based therapies that avoid detection by the recipient's immune system.

[0005] In some embodiments, provided herein are engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to wild-type or control cells, wherein the engineered cells further comprise a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), wherein the first and / or second exogenous polynucleotides are inserted into a specific locus of at least one allele of the cell. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the exogenous polynucleotides. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis virus glycoprotein (VSV-G) envelope and carries an exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0006] In some embodiments, the specific locus is selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In many embodiments, a first exogenous polynucleotide encoding CD47 is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, a second exogenous polynucleotide encoding a CAR is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0007] In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at different loci. In many embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the same locus. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the B2M locus. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the CIITA locus. In many embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the TRAC locus. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at the TRB locus. In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted at a safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C (also known as AAVS1) locus, the albumin locus, the SHS231 locus, the CLYBL locus, the ROSA locus (e.g., the ROSA26 locus), the F3 locus (also known as CD142), the MICA locus, the MICB locus, the LRP1 locus (also known as the CD91 locus), the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus. In various embodiments, the safe harbor or target locus is selected from the group consisting of the AAVS1 locus, the CCR5 locus, and the ROSA26 locus. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the exogenous polynucleotide.In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope, and carries an exogenous polynucleotide.In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell, for example, by using vector-based viral transduction.In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell using a lentiviral-based viral vector.

[0008] In some embodiments, the CAR is selected from the group consisting of a CD19-specific CAR and a CD22-specific CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the CAR is a CD19-specific CAR. In some embodiments, the CAR is a CD22-specific CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the CAR is a CD19 / CD22 bispecific CAR.

[0009] In many embodiments, the engineered cells do not express HLA-A, HLA-B, and / or HLA-C antigens. In some embodiments, the engineered cells do not express B2M. In other embodiments, the engineered cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens. In some embodiments, the engineered cells do not express CIITA. In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells do not express TCR-alpha and / or TCR-beta.

[0010] In many embodiments, the engineered cells are pluripotent stem cells, hi some embodiments, the engineered cells are induced pluripotent stem cells.

[0011] In some embodiments, the engineered cells are differentiated cells derived from induced pluripotent stem cells. In various embodiments, the differentiated cells are selected from the group consisting of NK cells and T cells.

[0012] In some embodiments, the engineered cells are cells derived from primary T cells. In many embodiments, the cells derived from primary T cells are derived from a pool of T cells that includes primary T cells from one or more donor subjects that are different from the recipient subject.

[0013] In some embodiments, the engineered cells are cells derived from primary NK cells. In many embodiments, the cells derived from primary NK cells are derived from a pool of NK cells that includes primary NK cells from one or more donor subjects that are different from the recipient subject.

[0014] In some embodiments, the engineered cells retain pluripotency and / or retain differentiation potential.

[0015] In many embodiments, after transfer into a first subject, the engineered cells exhibit one or more responses selected from the group consisting of: (a) a T cell response, (b) a NK cell response, and (c) a macrophage response that are reduced compared to wild-type cells after transfer into a second subject. In some cases, the first subject and the second subject are different subjects. In some cases, the macrophage response is phagocytosis. In various embodiments, after transfer into a subject, the engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in the subject, (b) reduced NK cell killing in the subject, and (c) reduced killing by total PBMCs in the subject compared to wild-type cells after transfer into the subject. In many embodiments, after transfer into a subject, the engineered T cells elicit one or more selected from the group consisting of: (a) reduced donor-specific antibodies in the subject, (b) reduced IgM or IgG antibodies in the subject, and (c) reduced complement-dependent cytotoxicity (CDC) in the subject compared to wild-type cells after transfer into the subject.

[0016] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In some embodiments, the engineered cell is a B2M cell. In some embodiments, the first and / or second exogenous polynucleotide is inserted into at least one allele of the T cell using viral transduction. In some embodiments, the first and / or second exogenous polynucleotide is inserted into at least one allele of the T cell using a lentivirus-based viral vector. In some embodiments, the engineered cell is selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In many embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル, C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, wherein the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In many embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, wherein the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデルIn many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル The exogenous polynucleotide is a cell. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0017] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus.インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, wherein the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデルIn some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, wherein the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル The exogenous polynucleotide is a cell. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0018] In some embodiments, the engineered cells comprise a B2M comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデルIn many embodiments, the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are B2M cells that contain a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In some embodiments, the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are B2M cells that contain a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In many embodiments, the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In some embodiments, the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRBインデル / インデル In many embodiments, the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル The exogenous polynucleotide is a cell. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0019] In some embodiments, engineered cells are provided that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to wild-type or control cells.

[0020] In some embodiments, the engineered cells do not express HLA-A, HLA-B and / or HLA-C antigens. In many embodiments, the engineered cells do not express CIITA.

[0021] In many embodiments, the engineered cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens, hi some embodiments, the engineered cells do not express B2M.

[0022] In many embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells do not express TCR-alpha. In many embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells do not express TCR-beta.

[0023] In various embodiments, the engineered cells overexpress CD47 relative to wild-type or control cells.

[0024] In some embodiments, the engineered cells are pluripotent stem cells. In many embodiments, the engineered cells are induced pluripotent stem cells.

[0025] In many embodiments, the engineered cells are differentiated cells derived from induced pluripotent stem cells, hi some embodiments, the differentiated cells are selected from the group consisting of NK cells and T cells.

[0026] In many embodiments, the engineered cells are cells derived from primary T cells. In some embodiments, the cells derived from primary T cells are derived from a pool of T cells that includes primary T cells from one or more donor subjects that are different from the recipient subject.

[0027] In various embodiments, the engineered cells retain pluripotency and / or retain differentiation potential.

[0028] In some embodiments, after transfer into a subject, the engineered cells elicit one or more responses selected from the group consisting of: (a) a T cell response, (b) a NK cell response, and (c) a macrophage response that is reduced compared to wild-type cells after transfer into a second subject. In some cases, the first subject and the second subject are different subjects. In some cases, the macrophage response is phagocytosis.

[0029] In various embodiments, after transfer into a subject, the engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in the subject, (b) reduced NK cell killing in the subject, and (c) reduced killing by total PBMCs in the subject, compared to wild-type cells after transfer into the subject. In many embodiments, after transfer into a subject, the engineered T cells elicit one or more selected from the group consisting of: (a) reduced donor-specific antibodies in the subject, (b) reduced IgM or IgG antibodies in the subject, and (c) reduced complement-dependent cytotoxicity (CDC) in the subject, compared to wild-type cells after transfer into the subject.

[0030] In some embodiments, the engineered cells are B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In some cases, the engineered cells are B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル Primary T cells. In some cases, the engineered cells are B2M cells differentiated from hypoimmunogenic induced pluripotent stem cells. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are selected from the group consisting of B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some cases, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are selected from the group consisting of B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデルIn some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are selected from the group consisting of B2M インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル In some cases, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are selected from the group consisting of B2M インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル Primary T cells. In some cases, the engineered cells are B2M cells differentiated from hypoimmunogenic induced pluripotent stem cells. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRAC インデル / インデル In some embodiments, the engineered cells are B2M T cells. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル In some cases, the engineered cells are B2M インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル Primary T cells. In some cases, the engineered cells are B2M cells differentiated from hypoimmunogenic induced pluripotent stem cells. インデル / インデル , C.I.T.A. インデル / インデル , and / or TRB インデル / インデル T cells.

[0031] In some embodiments, the engineered cells are hypoimmunogenic cells.

[0032] In some embodiments, a pharmaceutical composition is provided comprising any population of engineered cells described herein and a pharmaceutically acceptable excipient, carrier, diluent, or excipient.

[0033] In some embodiments, the pharmaceutically acceptable additive, carrier, diluent, or excipient comprises one or more selected from the group consisting of Plasma-Lyte A®, dextrose, dextran, sodium chloride, human serum albumin (HSA), dimethyl sulfoxide (DMSO), and combinations thereof. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer. In some embodiments, the pharmaceutically acceptable buffer is neutral buffered saline or phosphate buffered saline.

[0034] In some embodiments, pharmaceutical compositions are provided that include any population of engineered cells described herein, a base solution of CryoStor® CSB at a concentration of about 70-80% w / w, and one or more of about 20-30% w / w PlasmaLyte-A™, about 0.3-5.3% w / v human serum albumin (HSA), about 0-20% v / v dimethyl sulfoxide (DMSO), and about 100-400 mM trehalose.

[0035] In some embodiments, pharmaceutical compositions are provided that include any population of engineered cells described herein, a base solution of PlasmaLyte-A™ at a concentration of about 20-30% w / w, and one or more of about 70-80% w / w CryoStor® CSB, about 0.3-5.3% w / v human serum albumin (HSA), about 0-20% v / v dimethyl sulfoxide (DMSO), and about 100-400 mM trehalose.

[0036] In some embodiments, pharmaceutical compositions are provided that include any population of engineered cells described herein, about 0.3-5.3% w / v human serum albumin (HSA), and one or more of about 70-80% w / w CryoStor® CSB, about 20-30% w / w PlasmaLyte-A™, about 0-20% v / v dimethyl sulfoxide (DMSO), and about 100-400 mM trehalose.

[0037] In some embodiments, pharmaceutical compositions are provided that include any population of engineered cells described herein, about 0-20% v / v dimethyl sulfoxide (DMSO), and one or more of about 70-80% w / w CryoStor® CSB, about 20-30% w / w PlasmaLyte-A™, about 0.3-5.3% w / v human serum albumin (HSA), and about 100-400 mM trehalose.

[0038] In some embodiments, pharmaceutical compositions are provided that include any population of engineered cells described herein, about 100-400 mM trehalose, and one or more of about 70-80% w / w CryoStor® CSB, about 20-30% w / w PlasmaLyte-A™, about 0.3-5.3% w / v human serum albumin (HSA), and about 0-20% v / v dimethyl sulfoxide (DMSO).

[0039] In some embodiments, the pharmaceutical composition comprises about 75% w / w CryoStor® CSB. In some embodiments, the pharmaceutical composition comprises about 25% w / w PlasmaLyte-A™. In some embodiments, the pharmaceutical composition comprises about 0.3% w / v HSA. In some embodiments, the pharmaceutical composition comprises about 7.5% v / v DMSO.

[0040] In some embodiments, a pharmaceutical composition is provided that includes any population of engineered cells described herein, a base solution of CryoStor® CSB at a concentration of about 75% w / w, about 25% w / w PlasmaLyte-A™, about 0.3% w / v human serum albumin (HSA), and about 7.5% v / v dimethyl sulfoxide (DMSO).

[0041] In some embodiments, the population of engineered cells comprises at most about 8.0 x 10 8 In many embodiments, the population of engineered cells is at most about 6.0 x 10 cells. 8In other embodiments, the population of engineered cells is about 1.0 x 10 cells. 6 ~about 2.5x10 8 In some embodiments, the population of engineered cells is about 2.0 x 10 cells. 6 ~about 2.0x10 8 Each cell is an individual cell.

[0042] In various embodiments, the population of engineered cells ranges from about 5 ml to about 80 ml. In many embodiments, the population of engineered cells ranges from about 10 ml to about 70 ml. In some embodiments, the population of engineered cells ranges from about 10 ml to about 50 ml.

[0043] In some embodiments, the composition is formulated for administration in a single dose. In many embodiments, the composition is formulated for administration in up to three doses.

[0044] In some embodiments, the compositions are formulated so that administration of a single dose to a subject requires a duration of about 60 minutes or less. In many embodiments, the compositions are formulated so that administration of a single dose to a subject requires a duration of about 30 minutes or less.

[0045] In some embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 40% survival in the subject 10 days after administration. In various embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 80% survival in the subject about 2 weeks after administration. In some embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 100% survival in the subject about 3 weeks after administration. In many embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 150% survival in the subject about 4 weeks after administration.

[0046] In another embodiment, there is provided a dosing regimen for treating a disease or disorder in a subject, the dosing regimen comprising administering a pharmaceutical composition comprising any population of engineered cells described herein and a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in about 1 to 3 doses.

[0047] In some embodiments, the pharmaceutical composition administered is up to about 6.0x10 in about 1-3 doses. 8 In some embodiments, the pharmaceutical composition administered is about 0.6x10 cells in about 1-3 doses. 6 ~about 6.0x10 8 In some embodiments, the pharmaceutical composition administered is about 0.2 x 10 cells per kg of subject's body weight in about 1 to 3 doses when the subject weighs 50 kg or less. 6 ~about 5.0x10 6 In some embodiments, the pharmaceutical composition administered is about 0.1 x 10 cells in about 1 to 3 doses if the subject weighs more than 50 kg. 8 ~about 2.5x10 8 In some embodiments, the pharmaceutical composition administered is about 2.0 x 10 cells per kg of the subject's body weight in about 1-3 doses. 6 cells up to approximately 2.x10 8 Each cell is an individual cell.

[0048] In some embodiments, administration of a single dose to a subject takes about 60 minutes or less in duration. In some embodiments, administration of a single dose to a subject takes about 30 minutes or less in duration.

[0049] In some embodiments, the pharmaceutically acceptable additive, carrier, diluent, or excipient comprises one or more selected from the group consisting of Plasma-Lyte A®, dextrose, dextran, sodium chloride, human serum albumin (HSA), dimethyl sulfoxide (DMSO), and combinations thereof.

[0050] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer, hi some embodiments, the pharmaceutically acceptable buffer is neutral buffered saline or phosphate buffered saline.

[0051] In some embodiments, the population of cells or their progeny are present in the subject for up to 9 months after administration of the pharmaceutical composition, hi some embodiments, the population of cells or their progeny are present in the subject for at least 2 years or more after administration of the pharmaceutical composition.

[0052] In some embodiments, after administration of the pharmaceutical composition, the population of engineered cells or their progeny exhibit at least 40% survival in the subject about 10 days after administration. In some embodiments, after administration of the pharmaceutical composition, the population of engineered cells or their progeny exhibit at least 80% survival in the subject about 2 weeks after administration. In some embodiments, after administration of the pharmaceutical composition, the population of engineered cells or their progeny exhibit at least 100% survival in the subject about 3 weeks after administration. In some embodiments, after administration of the pharmaceutical composition, the population of engineered cells or their progeny exhibit at least 150% survival in the subject about 4 weeks after administration.

[0053] In some embodiments, the subject is administered two to three doses, with each dose administered 1 to 24 hours apart. In some embodiments, the subject is administered two to three doses, with each dose administered 1 to 28 days apart. In some embodiments, the subject is administered two to three doses, with each dose administered 1 to 6 weeks apart. In some embodiments, the subject is administered two to three doses, with each dose administered 1 to 12 months or more apart.

[0054] Provided herein are dosing regimens for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition comprising (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, wherein the engineered cells further comprise a set of exogenous polynucleotides encoding CD47 and a chimeric antigen receptor (CAR). In some embodiments, the set of exogenous polynucleotides is inserted into at least one allele of a T cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the set of exogenous polynucleotides. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carrying the set of exogenous polynucleotides. In some embodiments, the set of exogenous polynucleotides is inserted into at least one allele of a T cell using a lentiviral-based viral vector. In some embodiments, the set of exogenous polynucleotides is inserted into a safe harbor or target locus of at least one allele of the cell; and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient, the pharmaceutical composition comprises up to about 6.0x10 8The cell includes cells. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentiviral-based viral vector.

[0055] Provided herein are dosing regimens for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition comprising: (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, wherein the engineered cells further comprise a set of exogenous polynucleotides encoding CD47 and a chimeric antigen receptor (CAR). In some embodiments, the set of exogenous polynucleotides is inserted into at least one allele of a T cell using viral transduction. In some embodiments, the set of exogenous polynucleotides is inserted into at least one allele of a T cell using a lentivirus-based viral vector. In some embodiments, the set of exogenous polynucleotides is inserted into a safe harbor or target locus of at least one allele of a cell; and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient. The pharmaceutical composition is administered in one to three doses. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell using a lentiviral-based viral vector.

[0056] Provided herein is a dosing regimen for treating a disease or disorder in a subject, the dosing regimen comprising: (i) an engineered cell comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, the engineered cell further comprising a set of exogenous polynucleotides encoding CD47 and a chimeric antigen receptor (CAR), the set of exogenous polynucleotides being inserted into a safe harbor or target locus of at least one allele of the cell; and (ii) a pharmaceutical composition comprising a pharmaceutically acceptable additive, carrier, diluent, or excipient, the pharmaceutical composition being administered for a duration of about 60 minutes or less. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope, and carries an exogenous polynucleotide.In some embodiments, the exogenous polynucleotide is inserted into at least one allele of cells using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into at least one allele of cells using lentiviral-based viral vectors.

[0057] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition comprising: (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta; and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in a dose range of up to about 6.0x10 8 A dosing regimen comprising the cells is provided.

[0058] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition comprising: (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta; and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in one to three doses.

[0059] Provided herein is a dosing regimen for treating a disease or disorder in a subject, the dosing regimen comprising administering a pharmaceutical composition comprising: (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta; and (ii) a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered for a duration of 60 minutes or less.

[0060] In some embodiments, methods of treating cancer in a subject are provided, comprising administering to the subject any of the engineered cells described herein, any of the pharmaceutical compositions described herein, or any of the dosing regimens described herein. In some embodiments, the cancer is CD19 + It's cancer.

[0061] In some embodiments, methods are provided for preventing T cell depletion or treating a disease in a subject, the methods comprising administering to the subject any of the engineered cells described herein, wherein the CAR is a CD19 / CD22 bispecific CAR.

[0062] In some embodiments, provided herein are methods of preventing T cell depletion or treating a disease in a subject, the methods comprising: (i) administering to the subject at a first time a first dosage regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosage regimen comprising a second population of any of the engineered cells described herein, wherein the first dosage regimen and the second dosage regimen are different.

[0063] In some embodiments, provided herein are methods of preventing T cell depletion or treating a disease in a subject, the methods comprising: (i) administering to the subject at a first time a first dosage regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosage regimen comprising a second population of any of the engineered cells described herein, wherein both the first population of engineered cells and the second population of engineered cells comprise the same chimeric antigen receptor.

[0064] In some embodiments, provided herein are methods of preventing T cell depletion or treating a disease in a subject, the methods comprising: (i) administering to the subject at a first time a first dosage regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosage regimen comprising a second population of any of the engineered cells described herein, wherein both the first population of engineered cells and the second population of engineered cells comprise different chimeric antigen receptors.

[0065] In some embodiments, provided herein are methods of preventing T cell depletion or treating a disease in a subject, the methods comprising: (i) administering to the subject at a first time a first dosage regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosage regimen comprising a second population of any of the engineered cells described herein, wherein the engineered cells of the first population comprise a first chimeric antigen receptor that binds a first antigen, and the engineered cells of the second population comprise a second chimeric antigen receptor that binds a second antigen, and wherein the first antigen and the second antigen are the same.

[0066] In some embodiments, provided herein are methods of preventing T cell depletion or treating a disease in a subject, the methods comprising: (i) administering to the subject at a first time a first dosing regimen comprising a first population of any of the engineered cells described herein; and (ii) administering to the subject at a second time a second dosing regimen comprising a second population of any of the engineered cells described herein, wherein the engineered cells of the first population comprise a first chimeric antigen receptor that binds a first antigen, and the engineered cells of the second population comprise a second chimeric antigen receptor that binds a second antigen, and wherein the first antigen and the second antigen are different.

[0067] Provided herein are resting T cells comprising a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR), and reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to a wild-type T cell. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentiviral-based viral vector.

[0068] In some embodiments, the resting T cells are primary T cells. In other embodiments, the resting T cells are differentiated from engineered cells of the present technology.

[0069] In some embodiments, the T cells are CD8 + T cells.

[0070] In some embodiments, the resting T cells have not been treated with an anti-CD3 antibody, an anti-CD28 antibody, a T cell activating cytokine, or a soluble T cell costimulatory molecule.

[0071] In some embodiments, the anti-CD3 antibody is OKT3. In some embodiments, the anti-CD28 antibody is CD28.2. In some embodiments, the T cell activating cytokine is selected from the group of T cell activating cytokines consisting of IL-2, IL-7, IL-15, and IL-21. In some embodiments, the soluble T cell costimulatory molecule is selected from the group of soluble T cell costimulatory molecules consisting of anti-CD28 antibody, anti-CD80 antibody, anti-CD86 antibody, anti-CD137L antibody, and anti-ICOS-L antibody.

[0072] In some embodiments, the resting T cells do not express activation markers.

[0073] In some embodiments, the resting T cells express CD3 and CD28, and the CD3 and / or CD28 are resting.

[0074] In some embodiments, the first exogenous polynucleotide is carried by a lentiviral vector comprising a CD8 binding agent.

[0075] In some embodiments, the resting T cells further comprise a second exogenous polynucleotide encoding CD47.

[0076] In some embodiments, the first and / or second exogenous polynucleotides are inserted into a specific locus of at least one allele of a T cell. In some embodiments, the first and / or second exogenous polynucleotides are inserted into at least one allele of a T cell using viral transduction. In some embodiments, the first and / or second exogenous polynucleotides are inserted into at least one allele of a T cell using a lentivirus-based viral vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the first and / or second exogenous polynucleotides. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carries the set of first and / or second exogenous polynucleotides. In some embodiments, the specific locus is selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 is inserted at a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the first exogenous polynucleotide encoding a CAR is inserted at a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at different loci. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the same locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the B2M locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the CIITA locus.In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the TRAC locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into a safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, CXCR4 locus, PPP1R12C (also known as AAVS1) locus, albumin locus, SHS231 locus, CLYBL locus, ROSA locus (e.g., ROSA26 locus), F3 locus (also known as CD142), MICA locus, MICB locus, LRP1 locus (also known as CD91 locus), HMGB1 locus, ABO locus, RHD locus, FUT1 locus, and KDM5D locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of the AAVS1 locus, CCR5 locus, and ROSA26 locus. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope, and carries an exogenous polynucleotide.In some embodiments, the exogenous polynucleotide is inserted into at least one allele of cells using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into at least one allele of cells using lentiviral-based viral vectors.

[0077] In some embodiments, the CAR is selected from the group consisting of a CD19-specific CAR and a CD22-specific CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the bispecific CAR is a CD19 / CD22 bispecific CAR.

[0078] In some embodiments, the resting T cells do not express HLA-A, HLA-B, and / or HLA-C antigens. In some embodiments, the resting T cells do not express B2M. In some embodiments, the resting T cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens. In some embodiments, the resting T cells do not express CIITA. In some embodiments, the resting T cells do not express TCR-alpha and TCR-beta.

[0079] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0080] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 inserted into the TRAC locus and a first exogenous polynucleotide encoding a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0081] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0082] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0083] In some embodiments, the resting T cells comprise a B2M gene encoding a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0084] In some embodiments, the resting T cells comprise a B2M gene encoding a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0085] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0086] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0087] In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0088] Provided herein are engineered T cells that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta relative to wild-type T cells, the engineered T cells further comprising a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) delivered by a lentiviral vector. Provided herein are engineered T cells that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta relative to wild-type T cells, the engineered T cells further comprising a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) delivered by a lentiviral vector comprising a CD8 binding agent. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0089] In some embodiments, the engineered T cells are primary T cells. In other embodiments, the engineered T cells are differentiated from engineered cells of the present technology. In some embodiments, the T cells are CD8 + T cells.

[0090] In some embodiments, the engineered T cells have not been treated with an anti-CD3 antibody, an anti-CD28 antibody, a T cell activating cytokine, or a soluble T cell costimulatory molecule. In some embodiments, the anti-CD3 antibody is OKT3, the anti-CD28 antibody is CD28.2, the T cell activating cytokine is selected from the group of T cell activating cytokines consisting of IL-2, IL-7, IL-15, and IL-21, and the soluble T cell costimulatory molecule is selected from the group of soluble T cell costimulatory molecules consisting of an anti-CD28 antibody, an anti-CD80 antibody, an anti-CD86 antibody, an anti-CD137L antibody, and an anti-ICOS-L antibody.

[0091] In some embodiments, the engineered T cells do not express activation markers, hi some embodiments, the engineered T cells express CD3 and CD28, and CD3 and / or CD28 are inactive.

[0092] In some embodiments, the engineered T cell further comprises a second exogenous polynucleotide encoding CD47. In some embodiments, the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the T cell. In some embodiments, the specific locus is selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the first exogenous polynucleotide encoding a CAR is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into different loci. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the same locus. In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the B2M locus, the CIITA locus, the TRAC locus, the TRB locus, or a safe harbor or target locus. In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C (also known as AAVS1) locus, the albumin locus, the SHS231 locus, the CLYBL locus, the Rosa locus (e.g., the ROSA26 locus), the F3 locus (also known as CD142), the MICA locus, the MICB locus, the LRP1 locus (also known as the CD91 locus), the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus.In some embodiments, the safe harbor or target locus is selected from the group consisting of the AAVS1 locus, the CCR5 locus, and the ROSA26 locus. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentiviral-based viral vector.

[0093] In some embodiments, the CAR is selected from the group consisting of a CD19-specific CAR and a CD22-specific CAR.

[0094] In some embodiments, the engineered T cells do not express HLA-A, HLA-B, and / or HLA-C antigens; the engineered T cells do not express B2M; the engineered T cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens; the engineered T cells do not express CIITA; and / or the engineered T cells do not express TCR-alpha and TCR-beta.

[0095] In some embodiments, the engineered T cells comprise a B2M comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the TRAC locus, the TRB locus, the B2M locus, or the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデルThe exogenous polynucleotide is a cell. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0096] In some embodiments, the rested T cells and / or engineered T cells of the present technology are in a subject, hi other embodiments, the rested T cells and / or engineered T cells of the present technology are in vitro.

[0097] In some embodiments, the resting T cells and / or engineered T cells of the present technology express a CD8 binding agent. In some embodiments, the CD8 binding agent is an anti-CD8 antibody. In some embodiments, the anti-CD8 antibody is selected from the group consisting of a mouse anti-CD8 antibody, a rabbit anti-CD8 antibody, a human anti-CD8 antibody, a humanized anti-CD8 antibody, a camelid (e.g., llama, alpaca, camel) anti-CD8 antibody, and a fragment thereof. In some embodiments, the fragment is an scFV or VHH. In some embodiments, the CD8 binding agent binds to the CD8 alpha chain and / or the CD8 beta chain.

[0098] In some embodiments, the CD8 binding agent is fused to a transmembrane domain incorporated into the viral envelope. In some embodiments, the lentiviral vector is pseudotyped with a viral fusion protein. In some embodiments, the viral fusion protein contains one or more modifications to reduce binding to its native receptor.

[0099] In some embodiments, the viral fusion protein is fused to a CD8 binding factor. In some embodiments, the viral fusion protein comprises a Nipah virus F glycoprotein and a Nipah virus G glycoprotein fused to a CD8 binding factor. In some embodiments, the lentiviral vector does not comprise a T cell activation molecule or a T cell costimulatory molecule. In some embodiments, the lentiviral vector encodes a first exogenous polynucleotide and / or a second exogenous polynucleotide.

[0100] In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0101] In some embodiments, after transfer into a first subject, the resting T cells or engineered T cells exhibit one or more responses selected from the group consisting of: (a) a T cell response, (b) a NK cell response, and (c) a macrophage response that is reduced compared to wild-type cells after transfer into a second subject. In some embodiments, the first subject and the second subject are different subjects. In some embodiments, the macrophage response is phagocytosis.

[0102] In some embodiments, after transfer into a subject, the resting T cells or engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in the subject; (b) reduced NK cell killing in the subject; and (c) reduced killing by total PBMCs in the subject, compared to wild-type cells after transfer into a subject.

[0103] In some embodiments, after transfer into a subject, the rested T cells or engineered T cells elicit one or more selected from the group consisting of: (a) reduced donor-specific antibodies in the subject; (b) reduced IgM or IgG antibodies in the subject; and (c) reduced complement-dependent cytotoxicity (CDC) in the subject, compared to wild-type cells after transfer into a subject.

[0104] In some embodiments, resting T cells or engineered T cells are transduced in a subject with a lentiviral vector comprising a CD8 binding agent. In some embodiments, the lentiviral vector carries a gene encoding a CAR and / or CD47.

[0105] Provided herein are pharmaceutical compositions comprising a population of rested T cells and / or engineered T cells of the present technology and a pharmaceutically acceptable additive, carrier, diluent, or excipient.

[0106] Provided herein are methods that include administering to a subject a population of rested T cells and / or engineered T cells of the present technology, or a composition comprising one or more pharmaceutical compositions of the present technology.

[0107] In some embodiments, the subject does not receive a T cell activation treatment before, after, and / or concomitantly with administration of the composition, hi some embodiments, the T cell activation treatment comprises lymphodepletion.

[0108] Provided herein are methods of treating a subject suffering from cancer, comprising administering to the subject a composition comprising a population of rested T cells and / or engineered T cells of the present technology, or one or more pharmaceutical compositions of the present technology, wherein the subject does not receive a T cell activation treatment before, after, and / or simultaneously with administration of the composition. In some embodiments, the T cell activation treatment comprises lymphodepletion.

[0109] Provided herein are methods for expanding T cells capable of recognizing and killing tumor cells internally in a subject in need thereof, the method comprising administering to the subject a composition comprising a population of rested T cells and / or engineered T cells of the present technology, or one or more pharmaceutical compositions of the present technology, wherein the subject does not receive a T cell activation treatment before, after, and / or simultaneously with administration of the composition. In some embodiments, the T cell activation treatment comprises lymphodepletion.

[0110] Provided herein are dosing regimens for treating a disease or disorder in a subject, comprising administering a pharmaceutical composition comprising a population of rested T cells and / or engineered T cells of the present technology, or one or more pharmaceutical compositions of the present technology, and a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in about 1 to 3 therapeutically effective doses.

[0111] Provided herein are engineered cells that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to a wild-type or control cell, the engineered cell further comprising a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), wherein the first and / or second exogenous polynucleotides are inserted at a specific locus of at least one allele of the cell.

[0112] In some embodiments, the specific locus is selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0113] In some embodiments, the first exogenous polynucleotide encoding CD47 is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0114] In some embodiments, the second exogenous polynucleotide encoding a CAR is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus.

[0115] In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding the CAR are inserted at different loci.

[0116] In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding the CAR are inserted at the same locus.

[0117] In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted into the B2M locus.

[0118] In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding a CAR are inserted into the CIITA locus.

[0119] In some embodiments, a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR are inserted into the TRAC locus.

[0120] In some embodiments, a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR are inserted into the TRB locus.

[0121] In some embodiments, the first exogenous polynucleotide encoding CD47 and the second exogenous polynucleotide encoding the CAR are inserted into a safe harbor or target locus.

[0122] In some embodiments, the safe harbor locus is selected from the group consisting of the CCR5 locus, the PPP1R12C locus, the CLYBL locus, and the Rosa locus, and the target locus is selected from the group consisting of the CXCR4 locus, the albumin locus, the SHS231 locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus.

[0123] In some embodiments, the CAR is selected from the group consisting of a CD19-specific CAR and a CD22-specific CAR.

[0124] In some embodiments, the CD19-specific CAR is substantially equivalent to the CD19-specific CAR of any one of the CAR-T cell-based therapies selected from the group consisting of axicabtagene ciloreucel, lisocabtagene malareucel, brexcabtagene outrucel, and tisagenlecleucel.

[0125] In some embodiments, the CAR is a bispecific CAR.

[0126] In some embodiments, the CAR is a CD19 / CD22 bispecific CAR.

[0127] In some embodiments, the engineered cells do not express HLA-A, HLA-B and / or HLA-C antigens.

[0128] In some embodiments, the engineered cells do not express B2M.

[0129] In some embodiments, the engineered cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens.

[0130] In some embodiments, the engineered cells do not express CIITA.

[0131] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells do not express TCR-alpha and / or TCR-beta.

[0132] In some embodiments, the engineered cells are pluripotent stem cells.

[0133] In some embodiments, the engineered cells are induced pluripotent stem cells.

[0134] In some embodiments, the engineered cells are differentiated cells derived from induced pluripotent stem cells.

[0135] In some embodiments, the differentiated cells are selected from the group consisting of NK cells and T cells.

[0136] In some embodiments, the engineered cells are cells derived from primary T cells.

[0137] In some embodiments, the cells derived from primary T cells are derived from a pool of T cells that includes primary T cells from one or more donor subjects that are different from the recipient subject.

[0138] In some embodiments, the engineered cells retain pluripotency and / or retain differentiation potential.

[0139] In some embodiments, after transfer into a first subject, the engineered cells exhibit one or more responses selected from the group consisting of: (a) a T cell response, (b) an NK cell response, and (c) a macrophage response that are reduced compared to wild-type cells after transfer into a second subject.

[0140] In some embodiments, the first subject and the second subject are different subjects.

[0141] In some embodiments, the macrophage response is phagocytosis.

[0142] In some embodiments, after transfer into a subject, the engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in the subject; (b) reduced NK cell killing in the subject; and (c) reduced killing by total PBMCs in the subject, compared to wild-type cells after transfer into a subject.

[0143] In some embodiments, after transfer into a subject, the engineered T cells elicit one or more selected from the group consisting of: (a) reduced donor-specific antibodies in the subject; (b) reduced IgM or IgG antibodies in the subject; and (c) reduced complement-dependent cytotoxicity (CDC) in the subject, compared to wild-type cells after transfer into a subject.

[0144] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0145] In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0146] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0147] In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell comprises a B2M gene encoding a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0148] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0149] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0150] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are B2M cells comprising a first exogenous polynucleotide encoding CD47 and / or a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0151] In some embodiments, the engineered cell is selected from the group consisting of a pluripotent stem cell, an induced pluripotent stem cell, a T cell differentiated from an induced pluripotent stem cell, a primary T cell, and a cell derived from a primary T cell, and the engineered cell is a B2M cell comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0152] Provided herein are engineered cells that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to wild-type or control cells.

[0153] In some embodiments, the engineered cells do not express HLA-A, HLA-B and / or HLA-C antigens.

[0154] In some embodiments, the engineered cells do not express CIITA.

[0155] In some embodiments, the engineered cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens.

[0156] In some embodiments, the engineered cells do not express B2M.

[0157] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells do not express TCR-alpha.

[0158] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells do not express TCR-beta.

[0159] In some embodiments, the engineered cells overexpress CD47 relative to wild-type or control cells.

[0160] In some embodiments, the engineered cells are pluripotent stem cells.

[0161] In some embodiments, the engineered cells are induced pluripotent stem cells.

[0162] In some embodiments, the engineered cells are differentiated cells derived from induced pluripotent stem cells.

[0163] In some embodiments, the differentiated cells are selected from the group consisting of NK cells and T cells.

[0164] In some embodiments, the engineered cells are cells derived from primary T cells.

[0165] In some embodiments, the cells derived from primary T cells are derived from a pool of T cells that includes primary T cells from one or more donor subjects that are different from the recipient subject.

[0166] In some embodiments, the engineered cells retain pluripotency and / or retain differentiation potential.

[0167] In some embodiments, after transfer into a first subject, the engineered cells exhibit one or more responses selected from the group consisting of: (a) a T cell response, (b) an NK cell response, and (c) a macrophage response that are reduced compared to wild-type cells after transfer into a second subject.

[0168] In some embodiments, the first subject and the second subject are different subjects.

[0169] In some embodiments, the macrophage response is phagocytosis.

[0170] In some embodiments, after transfer into a subject, the engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in the subject; (b) reduced NK cell killing in the subject; and (c) reduced killing by total PBMCs in the subject, compared to wild-type cells after transfer into a subject.

[0171] In some embodiments, after transfer into a subject, the engineered T cells elicit one or more selected from the group consisting of: (a) reduced donor-specific antibodies in the subject; (b) reduced IgM or IgG antibodies in the subject; and (c) reduced complement-dependent cytotoxicity (CDC) in the subject, compared to wild-type cells after transfer into a subject.

[0172] In some embodiments, the engineered cells are selected from the group consisting of pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, and the engineered cells are selected from the group consisting of B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , and / or TRB インデル / インデル It is a cell.

[0173] In some embodiments, the engineered cells are hypoimmunogenic cells.

[0174] In some embodiments, wild-type or control cells are the starting material.

[0175] In some embodiments, the first and / or second exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction.

[0176] In some embodiments, viral transduction is via a lentiviral-based viral vector.

[0177] In some embodiments, the lentiviral-based viral vector is a pseudotyped, self-inactivating lentiviral vector carrying the first and / or second exogenous polynucleotide.

[0178] In some embodiments, the lentiviral-based viral vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carries a set of first and / or second exogenous polynucleotides.

[0179] Provided herein are pharmaceutical compositions comprising the population of engineered cells described herein and a pharmaceutically acceptable excipient, carrier, diluent, or vehicle.

[0180] In some embodiments, the pharmaceutically acceptable additive, carrier, diluent, or excipient comprises one or more selected from the group consisting of Plasma-Lyte A®, dextrose, dextran, sodium chloride, human serum albumin (HSA), dimethyl sulfoxide (DMSO), and combinations thereof.

[0181] In some embodiments of the composition, the composition further comprises a pharmaceutically acceptable buffer.

[0182] In some embodiments, the pharmaceutically acceptable buffer is neutral buffered saline or phosphate buffered saline.

[0183] Provided herein are pharmaceutical compositions comprising a population of engineered cells as described herein, a base solution of CryoStor® CSB at a concentration of about 70-80% w / w, and one or more of about 20-30% w / w PlasmaLyte-A™, about 0.3-5.3% w / v human serum albumin (HSA), about 0-20% v / v dimethyl sulfoxide (DMSO), and about 100-400 mM trehalose.

[0184] Provided herein are pharmaceutical compositions comprising a population of engineered cells as described herein, a base solution of PlasmaLyte-A™ at a concentration of about 20-30% w / w, and one or more of about 70-80% w / w CryoStor® CSB, about 0.3-5.3% w / v human serum albumin (HSA), about 0-20% v / v dimethyl sulfoxide (DMSO), and about 100-400 mM trehalose.

[0185] Provided herein are pharmaceutical compositions comprising a population of engineered cells described herein, about 0.3-5.3% w / v human serum albumin (HSA), and one or more of about 70-80% w / w CryoStor® CSB, about 20-30% w / w PlasmaLyte-A™, about 0-20% v / v dimethyl sulfoxide (DMSO), and about 100-400 mM trehalose.

[0186] Provided herein are pharmaceutical compositions comprising a population of engineered cells described herein, about 0-20% v / v dimethyl sulfoxide (DMSO), and one or more of about 70-80% w / w CryoStor® CSB, about 20-30% w / w PlasmaLyte-A™, about 0.3-5.3% w / v human serum albumin (HSA), and about 100-400 mM trehalose.

[0187] Provided herein are pharmaceutical compositions comprising a population of engineered cells described herein, about 100-400 mM trehalose, and one or more of about 70-80% w / w CryoStor® CSB, about 20-30% w / w PlasmaLyte-A™, about 0.3-5.3% w / v human serum albumin (HSA), and about 0-20% v / v dimethyl sulfoxide (DMSO).

[0188] In some embodiments, the pharmaceutical composition comprises about 75% w / w CryoStor® CSB.

[0189] In some embodiments, the pharmaceutical composition comprises about 25% w / w PlasmaLyte-A™.

[0190] In some embodiments, the pharmaceutical composition comprises about 0.3% w / v HSA.

[0191] In some embodiments, the pharmaceutical composition comprises about 7.5% v / v DMSO.

[0192] Provided herein is a pharmaceutical composition comprising a population of engineered cells as described herein, a base solution of CryoStor® CSB at a concentration of about 75% w / w, about 25% w / w PlasmaLyte-A™, about 0.3% w / v human serum albumin (HSA), and about 7.5% v / v dimethyl sulfoxide (DMSO).

[0193] In some embodiments, the population of engineered cells comprises at most about 8.0 x 10 8 Each cell is an individual cell.

[0194] In some embodiments, the population of engineered cells comprises at most about 6.0 x 10 8 Each cell is an individual cell.

[0195] In some embodiments, the population of engineered cells comprises about 1.0x10 6 ~about 2.5x10 8 Each cell is an individual cell.

[0196] In some embodiments, the population of engineered cells comprises about 2.0x10 6 ~about 2.0x10 8 Each cell is an individual cell.

[0197] In some embodiments, the population of engineered cells ranges from about 5 ml to about 80 ml.

[0198] In some embodiments, the population of engineered cells ranges from about 10 ml to about 70 ml.

[0199] In some embodiments, the population of engineered cells ranges from about 10 ml to about 50 ml.

[0200] In some embodiments, the composition is formulated for administration in a single dose.

[0201] In some embodiments, the composition is formulated for administration in up to three doses.

[0202] In some embodiments, the compositions are formulated so that administration of a single dose to a subject requires a duration of about 60 minutes or less.

[0203] In some embodiments, the compositions are formulated so that administration of a single dose to a subject requires a duration of about 30 minutes or less.

[0204] In some embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 40% survival in the subject 10 days after administration.

[0205] In some embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 80% survival in the subject about 2 weeks after administration.

[0206] In some embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 100% survival in the subject about 3 weeks after administration.

[0207] In some embodiments, the population of engineered cells of the pharmaceutical composition, or their progeny, exhibit at least 150% survival in the subject about 4 weeks after administration.

[0208] Provided herein is a dosing regimen for treating a disease or disorder in a subject, the dosing regimen comprising administering a pharmaceutical composition comprising a population of engineered cells described herein and a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in about 1 to 3 doses.

[0209] In some embodiments, the pharmaceutical composition administered is up to about 6.0x10 in about 1-3 doses. 8 Each cell is an individual cell.

[0210] In some embodiments, the pharmaceutical composition administered is about 0.6x10 in about 1-3 doses. 6 ~about 6.0x10 8 Each cell is an individual cell.

[0211] In some embodiments, the pharmaceutical composition administered is about 0.2 x 10 per kg of subject body weight in about 1 to 3 doses when the subject weighs 50 kg or less. 6 ~about 5.0x10 6 Each cell is an individual cell.

[0212] In some embodiments, the pharmaceutical composition administered is about 0.1 x 10 in about 1 to 3 doses when the subject weighs more than 50 kg. 8 ~about 2.5x10 8 Each cell is an individual cell.

[0213] In some embodiments, the pharmaceutical composition administered is about 2.0 x 10 per kg of subject body weight in about 1-3 doses. 6 cells up to approximately 2.x10 8 Each cell is an individual cell.

[0214] In some embodiments, administration of a single dose to a subject takes about 60 minutes or less in duration.

[0215] In some embodiments, administration of a single dose to a subject takes about 30 minutes or less in duration.

[0216] In some embodiments, the pharmaceutically acceptable additive, carrier, diluent, or excipient comprises one or more selected from the group consisting of Plasma-Lyte A®, dextrose, dextran, sodium chloride, human serum albumin (HSA), dimethyl sulfoxide (DMSO), and combinations thereof.

[0217] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable buffer.

[0218] In some embodiments, the pharmaceutically acceptable buffer is neutral buffered saline or phosphate buffered saline.

[0219] In some embodiments, administration of the pharmaceutical composition, population of cells or progeny thereof is present in the subject for up to 9 months.

[0220] In some embodiments, administration of the pharmaceutical composition, population of cells or the progeny thereof is present in the subject for at least two years or more.

[0221] In some embodiments, administration of the pharmaceutical composition, population of engineered cells or progeny thereof exhibits at least 40% survival in the subject about 10 days after administration.

[0222] In some embodiments, administration of the pharmaceutical composition, population of engineered cells or progeny thereof exhibits at least 80% survival in the subject about 2 weeks after administration.

[0223] In some embodiments, administration of the pharmaceutical composition, population of engineered cells or progeny thereof exhibits at least 100% survival in the subject about 3 weeks after administration.

[0224] In some embodiments, administration of the pharmaceutical composition, population of engineered cells or progeny thereof exhibits at least 150% survival in the subject about 4 weeks after administration.

[0225] In some embodiments, the subject receives two to three doses, with each dose administered between 1 and 24 hours apart.

[0226] In some embodiments, the subject receives two to three doses, with each dose administered between 1 and 28 days apart.

[0227] In some embodiments, the subject receives two to three doses, with each dose administered between one and six weeks apart.

[0228] In some embodiments, the subject receives two to three doses, with each dose being administered between one to twelve months or more apart.

[0229] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising: (i) an engineered cell comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, wherein the engineered cell further comprises a set of exogenous polynucleotides encoding CD47 and a chimeric antigen receptor (CAR), wherein the set of exogenous polynucleotides is inserted into a safe harbor or target locus of at least one allele of the cell; and (ii) a pharmaceutically acceptable additive, carrier, diluent, or excipient; and administering a pharmaceutical composition comprising at most about 6.0x10 8 A dosing regimen comprising the cells is provided.

[0230] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising: (i) an engineered cell comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, wherein the engineered cell further comprises a set of exogenous polynucleotides encoding CD47 and a chimeric antigen receptor (CAR), wherein the set of exogenous polynucleotides is inserted into a safe harbor or target locus of at least one allele of the cell; and (ii) a pharmaceutically acceptable additive, carrier, diluent, or excipient; wherein the pharmaceutical composition is administered in 1 to 3 doses.

[0231] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising: (i) an engineered cell comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta, wherein the engineered cell further comprises a set of exogenous polynucleotides encoding CD47 and a chimeric antigen receptor (CAR), wherein the set of exogenous polynucleotides is inserted into a safe harbor or target locus of at least one allele of the cell; and (ii) a pharmaceutically acceptable additive, carrier, diluent, or excipient; wherein the pharmaceutical composition is administered for a duration of about 60 minutes or less.

[0232] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising: (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta; and (ii) a pharmaceutically acceptable additive, carrier, diluent, or excipient; and administering a pharmaceutical composition comprising at most about 6.0x10 8 A dosing regimen comprising the cells is provided.

[0233] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising: (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta; and (ii) a pharmaceutically acceptable additive, carrier, diluent, or excipient; wherein the pharmaceutical composition is administered in 1 to 3 doses.

[0234] Provided herein is a dosing regimen for treating a disease or disorder in a subject, comprising: (i) engineered cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta; and (ii) a pharmaceutically acceptable additive, carrier, diluent, or excipient; wherein the pharmaceutical composition is administered for a duration of 60 minutes or less.

[0235] Provided herein are methods of treating cancer in a subject, the methods comprising administering to the subject an engineered cell described herein, a pharmaceutical composition described herein, or a dosing regimen described herein.

[0236] In some embodiments, the cancer is a CD19+ cancer.

[0237] Provided herein are methods for preventing T cell depletion in a subject, comprising administering to the subject the engineered cells described herein. wherein the CAR is a CD19-specific CAR or a CD22-specific CAR.

[0238] In some embodiments, the CD19-specific CAR is substantially equivalent to the CD19-specific CAR of any one of the CAR-T cell-based therapies selected from the group consisting of axicabtagene ciloreucel, lisocabtagene malareucel, brexcabtagene outrucel, and tisagenlecleucel.

[0239] Provided herein is a method of preventing T cell depletion or treating a disease in a subject, comprising: (i) administering to a subject at a first time point a first dosing regimen comprising a first population of engineered cells described herein; and (ii) administering to the subject at a second time point a second dosing regimen comprising a second population of engineered cells described herein. wherein the first dosing regimen and the second dosing regimen are different.

[0240] 1. A method of preventing T cell depletion or treating a disease in a subject, comprising: (i) administering to a subject at a first time point a first dosing regimen comprising a first population of engineered cells described herein; and (ii) administering to the subject at a second time point a second dosing regimen comprising a second population of engineered cells described herein. wherein the first population of engineered cells and the second population of engineered cells both comprise the same chimeric antigen receptor.

[0241] Provided herein is a method of preventing T cell depletion or treating a disease in a subject, comprising: (i) administering to a subject at a first time point a first dosing regimen comprising a first population of engineered cells described herein; and (ii) administering to the subject at a second time point a second dosing regimen comprising a second population of engineered cells described herein. wherein the first population of engineered cells and the second population of engineered cells both comprise different chimeric antigen receptors.

[0242] Provided herein is a method of preventing T cell depletion or treating a disease in a subject, comprising: (i) administering to a subject at a first time point a first dosing regimen comprising a first population of engineered cells described herein; and (ii) administering to the subject at a second time point a second dosing regimen comprising a second population of engineered cells described herein. wherein the first population of engineered cells comprises a first chimeric antigen receptor that binds to a first antigen and the second population of engineered cells comprises a second chimeric antigen receptor that binds to a second antigen, and the first antigen and the second antigen are the same.

[0243] Provided herein is a method of preventing T cell depletion or treating a disease in a subject, comprising: (i) administering to a subject at a first time point a first dosing regimen comprising a first population of engineered cells described herein; and (ii) administering to the subject at a second time point a second dosing regimen comprising a second population of engineered cells described herein. wherein the first population of engineered cells comprises a first chimeric antigen receptor that binds to a first antigen and the second population of engineered cells comprises a second chimeric antigen receptor that binds to a second antigen, and the first antigen and the second antigen are different.

[0244] In some embodiments, the engineered cells are not activated.

[0245] Provided herein are resting T cells that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta relative to a wild-type T cell, and a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR).

[0246] In some embodiments, the resting T cells are primary T cells.

[0247] In some embodiments, the resting T cells are differentiated from the engineered cells described herein.

[0248] In some embodiments, the T cells are CD8 + T cells.

[0249] In some embodiments, the resting T cells have not been treated with an anti-CD3 antibody, an anti-CD28 antibody, a T cell activating cytokine, or a soluble T cell costimulatory molecule.

[0250] In some embodiments, the anti-CD3 antibody is OKT3.

[0251] In some embodiments, the anti-CD28 antibody is CD28.2.

[0252] In some embodiments, the T cell activating cytokine is selected from the group consisting of IL-2, IL-7, IL-15, and IL-21.

[0253] In some embodiments, the soluble T cell costimulatory molecule is selected from the group consisting of an anti-CD28 antibody, an anti-CD80 antibody, an anti-CD86 antibody, an anti-CD137L antibody, and an anti-ICOS-L antibody.

[0254] In some embodiments, the resting T cells do not express activation markers.

[0255] In some embodiments, the resting T cells express CD3 and CD28, and the CD3 and / or CD28 are resting.

[0256] In some embodiments, the first exogenous polynucleotide is carried by a lentiviral vector comprising a CD8 binding agent.

[0257] In some embodiments, the resting T cells further comprise a second exogenous polynucleotide encoding CD47.

[0258] In some embodiments, the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the T cell.

[0259] In some embodiments, the specific locus is selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0260] In some embodiments, the second exogenous polynucleotide encoding CD47 is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0261] In some embodiments, the first exogenous polynucleotide encoding a CAR is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0262] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding the CAR are inserted at different loci.

[0263] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the same locus.

[0264] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the B2M locus.

[0265] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the CIITA locus.

[0266] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding the CAR are inserted into the TCR locus.

[0267] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the TRAC locus.

[0268] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the TRB locus.

[0269] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding the CAR are inserted into a safe harbor or target locus.

[0270] In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the albumin locus, the SHS231 locus, the CLYBL locus, the Rosa locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus.

[0271] In some embodiments, the CAR is selected from the group consisting of a CD19-specific CAR and a CD22-specific CAR.

[0272] In some embodiments, the CD19-specific CAR is substantially equivalent to the CD19-specific CAR of any one of the CAR-T cell-based therapies selected from the group consisting of axicabtagene ciloreucel, lisocabtagene malareucel, brexcabtagene outrucel, and tisagenlecleucel.

[0273] In some embodiments, the CAR is a bispecific CAR.

[0274] In some embodiments, the bispecific CAR is a CD19 / CD22 bispecific CAR.

[0275] In some embodiments, the resting T cells do not express HLA-A, HLA-B, and / or HLA-C antigens.

[0276] In some embodiments, the resting T cells do not express B2M.

[0277] In some embodiments, the resting T cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens.

[0278] In some embodiments, the resting T cells do not express CIITA.

[0279] In some embodiments, the resting T cells do not express TCR-alpha and / or TCR-beta.

[0280] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the TRAC locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0281] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 inserted into the TRAC locus and a first exogenous polynucleotide encoding a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0282] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0283] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the TRB locus. インデル / インデル , C.I.T.A. インデル / インデル,TRAC インデル / インデル It is a cell.

[0284] In some embodiments, the resting T cells comprise a B2M gene encoding a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0285] In some embodiments, the resting T cells comprise a B2M gene encoding a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the B2M locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0286] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0287] In some embodiments, the resting T cells are B2M T cells comprising a second exogenous polynucleotide encoding CD47 and a first exogenous polynucleotide encoding a CAR inserted into the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0288] Provided herein are engineered T cells that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to a wild-type T cell, wherein the engineered T cell further comprises a first exogenous polynucleotide encoding a chimeric antigen receptor (CAR) delivered by a lentiviral vector comprising a CD8 binding agent.

[0289] In some embodiments, the engineered T cells are primary T cells.

[0290] In some embodiments, the engineered T cells are differentiated from the engineered cells described herein.

[0291] In some embodiments, the T cells are CD8 + T cells.

[0292] In some embodiments, the engineered T cells have not been treated with an anti-CD3 antibody, an anti-CD28 antibody, a T cell activating cytokine, or a soluble T cell costimulatory molecule.

[0293] In some embodiments, the anti-CD3 antibody is OKT3, the anti-CD28 antibody is CD28.2, the T cell activating cytokine is selected from the group consisting of IL-2, IL-7, IL-15, and IL-21, and the soluble T cell costimulatory molecule is selected from the group consisting of an anti-CD28 antibody, an anti-CD80 antibody, an anti-CD86 antibody, an anti-CD137L antibody, and an anti-ICOS-L antibody.

[0294] In some embodiments, the engineered T cells do not express activation markers.

[0295] In some embodiments, the engineered T cells express CD3 and CD28, and CD3 and / or CD28 are inactive.

[0296] In some embodiments, the engineered T cells further comprise a second exogenous polynucleotide encoding CD47.

[0297] In some embodiments, the first and / or second exogenous polynucleotide is inserted into a specific locus of at least one allele of the T cell.

[0298] In some embodiments, the specific locus is selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0299] In some embodiments, the second exogenous polynucleotide encoding CD47 is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0300] In some embodiments, the first exogenous polynucleotide encoding a CAR is inserted into a specific locus selected from the group consisting of a safe harbor or target locus, a B2M locus, a CIITA locus, a TRAC locus, and a TRB locus.

[0301] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding the CAR are inserted at different loci.

[0302] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted at the same locus.

[0303] In some embodiments, the second exogenous polynucleotide encoding CD47 and the first exogenous polynucleotide encoding a CAR are inserted into the B2M locus, the CIITA locus, the TRAC locus, the TRB locus, or a safe harbor or target locus.

[0304] In some embodiments, the safe harbor or target locus is selected from the group consisting of the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the albumin locus, the SHS231 locus, the CLYBL locus, the Rosa locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus.

[0305] In some embodiments, the CAR is selected from the group consisting of a CD19-specific CAR and a CD22-specific CAR.

[0306] In some embodiments, the CD19-specific CAR is substantially equivalent to the CD19-specific CAR of any one of the CAR-T cell-based therapies selected from the group consisting of axicabtagene ciloreucel, lisocabtagene malareucel, brexcabtagene outrucel, and tisagenlecleucel.

[0307] In some embodiments, the engineered T cells do not express HLA-A, HLA-B, and / or HLA-C antigens, the engineered T cells do not express B2M, the engineered T cells do not express HLA-DP, HLA-DQ, and / or HLA-DR antigens, the engineered T cells do not express CIITA, and / or the engineered T cells do not express TCR-alpha and / or TCR-beta.

[0308] In some embodiments, the engineered T cells comprise a B2M comprising a second exogenous polynucleotide encoding CD47 and / or a first exogenous polynucleotide encoding a CAR inserted into the TRAC locus, the TRB locus, the B2M locus, or the CIITA locus. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル It is a cell.

[0309] In some embodiments, the resting T cells are as described herein, or the engineered T cells are as described herein, and the resting T cells or engineered T cells are in the subject.

[0310] In some embodiments, the resting T cells are as described herein, or the engineered T cells are as described herein, and the resting T cells or engineered T cells are in vitro.

[0311] In some embodiments, the resting T cells are as described herein or the engineered T cells are as described herein and the CD8 binding agent is an anti-CD8 antibody.

[0312] In some embodiments, the resting T cells or engineered T cells are as described herein and the anti-CD8 antibody is selected from the group consisting of a mouse anti-CD8 antibody, a rabbit anti-CD8 antibody, a human anti-CD8 antibody, a humanized anti-CD8 antibody, a camelid anti-CD8 antibody, and fragments thereof.

[0313] In some embodiments, the resting T cell or engineered T cell is as described herein and the fragment is an scFV or a VHH.

[0314] In some embodiments, the resting T cells or engineered T cells are as described herein and the CD8 binding agent binds to the CD8 alpha chain and / or the CD8 beta chain.

[0315] In some embodiments, the resting T cells or engineered T cells are as described herein, and the CD8 binding agent is fused to a transmembrane domain incorporated into the viral envelope.

[0316] In some embodiments, the resting T cells or engineered T cells are as described herein and the lentiviral vector is pseudotyped with a viral fusion protein.

[0317] In some embodiments, the resting T cells or engineered T cells are as described herein and the viral fusion protein comprises one or more modifications to reduce binding to its native receptor.

[0318] In some embodiments, the resting T cells or engineered T cells are as described herein and the viral fusion protein is fused to a CD8 binding agent.

[0319] In some embodiments, the resting T cells or engineered T cells are as described herein and the viral fusion protein comprises a Nipah virus F glycoprotein and a Nipah virus G glycoprotein fused to a CD8 binding agent.

[0320] In some embodiments, the resting T cells or engineered T cells are as described herein and the lentiviral vector does not comprise a T cell activation molecule or a T cell costimulatory molecule.

[0321] In some embodiments, the resting T cells or engineered T cells are as described herein and the lentiviral vector encodes the first exogenous polynucleotide and / or the second exogenous polynucleotide.

[0322] In some embodiments, the resting T cells or engineered T cells are as described herein, and after transfer into a first subject, the resting T cells or engineered T cells exhibit one or more responses selected from the group consisting of: (a) a T cell response, (b) an NK cell response, and (c) a macrophage response that is reduced compared to wild-type cells after transfer into a second subject.

[0323] In some embodiments, the resting T cells or engineered T cells are as described herein, and the first subject and second subject are different subjects.

[0324] In some embodiments, the resting T cells or engineered T cells are as described herein and the macrophage response is phagocytosis.

[0325] In some embodiments, the resting T cells or engineered T cells are as described herein, and after transfer into a subject, the resting T cells or engineered T cells exhibit one or more selected from the group consisting of: (a) reduced TH1 activation in the subject; (b) reduced NK cell killing in the subject; and (c) reduced killing by total PBMCs in the subject, compared to wild-type cells after transfer into the subject.

[0326] In some embodiments, the rested T cells or engineered T cells are as described herein, and after transfer into a subject, the rested T cells or engineered T cells elicit one or more selected from the group consisting of: (a) reduced donor-specific antibodies in the subject; (b) reduced IgM or IgG antibodies in the subject; and (c) reduced complement-dependent cytotoxicity (CDC) in the subject, compared to wild-type cells after transfer into the subject.

[0327] In some embodiments, the resting T cells or engineered T cells are as described herein, and the resting T cells or engineered T cells are transduced in the subject with a lentiviral vector comprising a CD8 binding agent.

[0328] In some embodiments, the resting T cells or engineered T cells are as described herein and the lentiviral vector carries a gene encoding a CAR and / or CD47.

[0329] Provided herein are pharmaceutical compositions comprising a population of rested T cells or engineered T cells described herein and a pharmaceutically acceptable excipient, carrier, diluent or vehicle.

[0330] Provided herein are methods that include administering to a subject a composition comprising a rested T cell described herein, an engineered T cell described herein, or a pharmaceutical composition described herein.

[0331] In some embodiments, the subject is not administered a T cell activation treatment before, after, and / or simultaneously with administration of the composition.

[0332] In some embodiments, the T cell activation treatment comprises lymphodepletion.

[0333] Provided herein are methods of treating a subject suffering from cancer, the methods comprising administering to the subject a composition comprising a rested T cell described herein, an engineered T cell described herein, or a pharmaceutical composition described herein, wherein the subject is not administered a T cell activating treatment before, after, and / or concomitantly with administration of the composition.

[0334] In some embodiments, the T cell activation treatment comprises lymphodepletion.

[0335] A method for expanding T cells capable of recognizing and killing tumor cells internally in a subject in need thereof, the method comprising administering to the subject a composition comprising a rested T cell described herein, an engineered T cell described herein, or a pharmaceutical composition described herein, wherein the subject is not administered a T cell activation treatment before, after, and / or simultaneously with administration of the composition.

[0336] In some embodiments, the T cell activation treatment comprises lymphodepletion.

[0337] Provided herein is a dosing regimen for treating a disease or disorder in a subject, the dosing regimen comprising administering to a population of subjects a pharmaceutical composition comprising a composition comprising the rested T cells described herein and / or the engineered T cells described herein and a pharmaceutically acceptable excipient, carrier, diluent, or excipient, wherein the pharmaceutical composition is administered in about 1 to 3 doses.

[0338] This disclosure is related to U.S. Provisional Application No. 112864-5057-PR filed December 31, 2020, and U.S. Provisional Application No. 18615-30046.00 filed January 11, 2021, filed by Morrison and Foerester, the contents of which are incorporated herein by reference in their entireties. Engineered and / or hypoimmunogenic cells, methods for their production, and methods for their use can be found in U.S. Provisional Application No. 63 / 065,342, filed August 13, 2020; WO2016 / 183041, filed May 9, 2015; WO2018 / 132783, filed January 14, 2018; WO2020 / 018615, filed July 17, 2019; WO2020 / 018620, filed July 17, 2019; and WO2020 / 168317, filed February 16, 2020, the disclosures of which, including examples, sequence listings, and figures, are incorporated herein by reference in their entireties. [Brief explanation of the drawings]

[0339] [Figure 1]

[0023] Figure 1 shows the characterization of hypoimmunogenic T cells described herein. Such cells are HLA-I and HLA-II knockout and CD47 knock-in cells. [Figure 2]This demonstrates the absence of NK cell-mediated killing of hypoimmunogenic T cells. In contrast, blocking CD47 with an anti-CD47 antibody resulted in NK cell-mediated killing of the cells. Mock T cells were not killed by allogeneic NK cells (as expected). T cells lacking HLA-I / II were killed by NK cells. HLA-I / II knockout and CD47 knockin cells were not killed by NK cells. Blocking CD47 with magrolimab (e.g., an anti-CD47 antibody) resulted in killing of HLA-I / II knockout, CD47 knockin cells, thus highlighting the protection provided by CD47. [Figure 3] This demonstrates the absence of macrophage-mediated killing of low-immunogenic T cells. Control T cells were not killed by allogeneic macrophages, and T cells lacking HLA-I / II were not killed by macrophages. HLA-I / II knockout and CD47 knockin cells were not killed by macrophages. Blockade of CD47 with magrolimab (e.g., an anti-CD47 antibody) resulted in the killing of HLA-I / II knockout, CD47 knockin cells, thus highlighting the protection provided by CD47. [Figure 4]

[0023] Figure 1 shows the expression of CD19-specific CAR and CD47 constructs in exemplary low-immunogenic CAR-T cells. CD19-specific CAR-CD47 T cells expressed high levels of exogenous CD47. As used herein, the term "CD19-specific CAR-CD47 T cells" refers to T cells that exogenously express a CD19-specific CAR and CD47. [Figure 5A] Figure 5 shows an example of dose-dependent in vitro killing of CD19+ tumor cells by hypoimmunogenic CAR-T cells (CD19-specific CAR-CD47 T cells). CD47 overexpression did not appear to affect CD19-specific CAR activity. CD19-specific CAR-CD47 T cells exhibited similar killing to control CD19-specific CAR-T cells ("CAR-low" (Figure 5A) and "CAR-high" (Figure 5B) cells). [Figure 5B]Figure 5 shows an example of dose-dependent in vitro killing of CD19+ tumor cells by hypoimmunogenic CAR-T cells (CD19-specific CAR-CD47 T cells). CD47 overexpression did not appear to affect CD19-specific CAR activity. CD19-specific CAR-CD47 T cells exhibited similar killing to control CD19-specific CAR-T cells ("CAR-low" (Figure 5A) and "CAR-high" (Figure 5B) cells). [Figure 6A] Figure 6 shows flow data confirming real-time cell analysis data demonstrating dose-dependent killing of CD19+ tumor cells by exemplary hypoimmunogenic CAR-T cells (e.g., CD19-specific CAR-CD47 T cells) in vitro ("CAR-low" (Figure 6A) and "CAR-high" (Figure 6B) cells). CD47 overexpression did not affect CD19-specific CAR activity in in vitro assays. [Figure 6B] Figure 6 shows flow data confirming real-time cell analysis data demonstrating dose-dependent killing of CD19+ tumor cells by exemplary hypoimmunogenic CAR-T cells (e.g., CD19-specific CAR-CD47 T cells) in vitro ("CAR-low" (Figure 6A) and "CAR-high" (Figure 6B) cells). CD47 overexpression did not affect CD19-specific CAR activity in in vitro assays. [Figure 7] Figure 1 shows the killing of CD19+ tumor cells by FACS-sorted exemplary hypoimmunogenic CAR-T cells (CD19-specific CAR-CD47 T cells). The target cell:effector cell ratio was 1:3, and killing was analyzed for 48 hours using a real-time quantitative microelectronic biosensor system for cell analysis (xCELLigence® RTCA System, Agilent) and by flow cytometry. The data show that CD19-specific CAR-T cells with endogenous CD47 expression killed tumor cells. CD19-specific CAR-T cells with exogenous CD47 expression also killed tumor cells. Control T cells (mock T cells) did not kill tumor cells. CD47 overexpression did not appear to affect CD19-specific CAR activity in in vitro assays. [Figure 8] 1 shows the CD47-dependent and CD47-independent growth of the cells described herein, particularly CD19-specific CAR-T cells and CD19-specific CAR-CD47 T cells. [Figure 9A] We demonstrate the efficacy of exemplary low-immunogenic CAR-T cells in a mouse model bearing human CD19+ tumors. Whole-animal scans demonstrate the effects of CD19-specific CAR-T cells, CD19-specific CAR-CD47 T cells, and mock T cells on CD19+ tumor cells. [Figure 9B] We demonstrate the efficacy of exemplary low-immunogenic CAR-T cells in a mouse model bearing human CD19+ tumors. Whole-animal scans demonstrate the effects of CD19-specific CAR-T cells, CD19-specific CAR-CD47 T cells, and mock T cells on CD19+ tumor cells. [Figure 10] 1 shows the efficacy of exemplary low-immunogenic CAR-T cells at various effector to Nalm6 target ratios. [Figure 11] Figure 1 shows cell viability and frequency of TRAC, B2M, and CIITA triple knockout in low-immunogenic CD19-specific CAR-CD47 T cells and control T cells (CD19-specific CAR-EGFRt T cells and mock T cells) 4 days after nucleofection to introduce the CRISPR / Cas9-based gene editing system into CD19-specific CAR-T cells and CD19-specific CAR-CD47 T cells. [Figure 12A] The frequency (FIG. 12A) and MFI (FIG. 12B) of exemplary CD19-specific CARs in low-immunogenic CD19-specific CAR-CD47 T cells and control T cells (CD19-specific CAR-EGFRt T cells, tisagenlecleucel biosimilar / surrogate cells, and mock T cells) are shown. [Figure 12B]The frequency (FIG. 12A) and MFI (FIG. 12B) of exemplary CD19-specific CARs in low-immunogenic CD19-specific CAR-CD47 T cells and control T cells (CD19-specific CAR-EGFRt T cells, tisagenlecleucel biosimilar / surrogate cells, and mock T cells) are shown. [Figure 13] Shown are the frequencies of both exemplary CD19-specific CAR and CD47 molecules in low-immunogenic CD19-specific CAR-CD47 T cells and control T cells (CD19-specific CAR-EGFRt T cells, tisagenlecleucel biosimilar / surrogate cells, CD47-expressing T cells, and mock T cells). [Figure 14] Shown are vector copy numbers in low-immunogenic CD19-specific CAR-CD47 T cells and control T cells (CD19-specific CAR-EGFRt T cells, tisagenlecleucel biosimilar / surrogate cells, and mock T cells) at day 8 after activation. [Figure 15] We show the expression of CD47 molecules in low-immunogenic CD19-specific CAR-CD47 T cells using a method for flow cytometry estimation of antibody per cell (e.g., QuantiBRITE™, BD Biosciences). In this assay, exogenous CD47 expression exceeds 200,000 molecules per cell. [Figure 16A] We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 16B] We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 16C]We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 16D] We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 17A] We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 17B] We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 17C] We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 17D] We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 18A]We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 18B] We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 18C] We show that the presence of triple gene inactivation of the TRAC, B2M, and CIITA genes and overexpression of CD47 protein did not affect the activity of the exemplary CD19-specific CAR in the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein. [Figure 19] This paper provides the efficacy of the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein in a mouse model bearing CD19+ tumor cells. Whole animal scans demonstrate tumor cell killing by these hypoimmunogenic CD19-specific CAR-T cells. The killing activity appeared to be dose-dependent. [Figure 20] This paper provides the efficacy of the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein in a mouse model bearing CD19+ tumor cells. Whole animal scans demonstrate tumor cell killing by these hypoimmunogenic CD19-specific CAR-T cells. The killing activity appeared to be dose-dependent. [Figure 21] This paper provides the efficacy of the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein in a mouse model bearing CD19+ tumor cells. Whole animal scans demonstrate tumor cell killing by these hypoimmunogenic CD19-specific CAR-T cells. The killing activity appeared to be dose-dependent. [Figure 22]This paper provides the efficacy of the hypoimmunogenic CD19-specific CAR-CD47 T cells described herein in a mouse model bearing CD19+ tumor cells. Whole animal scans demonstrate tumor cell killing by these hypoimmunogenic CD19-specific CAR-T cells. The killing activity appeared to be dose-dependent. [Figure 23] Shows efficacy of low-immunogenic CD19-specific CAR-CD47 T cells at various effector:tumor cell ratios over a range of days 0-28. [Figure 24]

[0033] Figure 1 shows whole animal scans demonstrating tumor cell killing by low-immunogenic CD19-specific CAR-CD47 T cells over a 27-day range. The effector:tumor cell ratio used was 7:1. [Figure 25] Exemplary test and control cells used in the study are shown. Exemplary test cells include hypoimmunogenic CD19-specific CAR-CD47 T cells with genome editing of B2M, CIITA, and TRAC genes and overexpressing CD47 molecules and CD19-specific chimeric antigen receptors. Control cells include immunogenic CD19-specific CAR-T cells co-expressing CD47 and EGFR, as well as tisagenlecleucel biosimilars or surrogates. [Figure 26] 1 shows FACS analysis of exemplary low-immunogenic CD19-specific CAR-CD47 T cells and the absence of CD3, B2M, HLA-DR / HLA-DP / HLA-DQ, and HLA-A / HLA-B / HLA-C expression. [Figure 27] 1 shows FACS analysis of exemplary low-immunogenic CD19-specific CAR-CD47 T cells and the absence of CD3, B2M, HLA-DR / HLA-DP / HLA-DQ, and HLA-A / HLA-B / HLA-C expression. [Figure 28] 1 shows FACS analysis of CD47 and CD19-CAR expression in low-immunogenic CD19-specific CAR-CD47 T cells. [Figure 29]1 shows the expression of CD3, B2M, TRAC, HLA-DR / HLA-DP / HLA-DQ, and HLA-A / HLA-B / HLA-C as determined by FACS and ICE in hypoimmunogenic CD19-specific CAR-CD47 T cells, CD19-specific CAR-EGFRt T cells, and mock T cells. [Figure 30] We demonstrate that the hypoimmunogenic CD19-specific CAR-CD47 T cells were able to kill tumor cells in vitro as effectively as control CD19-specific CAR-T cells. B-cell leukemia killing kinetics and total B-cell leukemia killing are shown. [Figure 31] FIG. 1 provides a schematic diagram of the experimental approach of Example 3 to study cytokine-independent proliferation of TRAC, B2M, and CIITA triple knockout CAR-T cells expressing a CD47 transgene (B2M− / −, CIITA− / −, TRAC− / −, CD47tg CD19-specific CAR-T cells (also known as tKO / CD47 CAR-T cells or HIP CD19-CAR-T cells)). [Figure 32] A and B show graphs showing the proliferation of B2M- / -, CIITA- / -, TRAC- / -, CD47tg CD19-specific CAR-T cells (tKO / CD47 CAR-T cells or HIP CD19-CAR-T cells) cultured in medium either supplemented with IL-2 or not supplemented with IL-2. [Figure 33] 1 provides a schematic diagram of the experimental approach of Example 4. [Figure 34] Figure 1 shows in vivo bioluminescence imaging for detecting Nalm6-luc tumor cells in xenografted mice. Mice were administered either tKO / CD47 CAR-T cells (HIP CD19-CAR-T cells), control CAR-T cells (CD19-CAR-T cells), unedited T cells, or saline. Bioluminescence images show tumor progression on days 35, 42, 49, and 56 of the study. [Figure 35]Graphs are provided showing Nalm6-luc tumor cells detected in xenografted mice. Images of Nalm6-luc-bearing mice show delayed tumor growth in mice treated with tKO / CD47 CAR-T cells (HIP CD19-CAR-T cells) compared to mice treated with control CAR-T (CD19-CAR-T), unedited T cells, and saline. [Figure 36] Figure 1 shows a schematic diagram of the experimental approach for generating B2M indel / indel, CIITA indel / indel, TRAC indel / indel, CD47tg T cells expressing a CD19-specific CAR, also referred to as tKO / CD47 CAR-T cells or HIP CD19-CAR-T cells. These cells are gene-edited T cells transduced with a CD47-CAR lentivirus (bottom row; HIP CAR-T cells). The diagram also shows the generation of two types of control cells: untransduced and unedited cells (top row; unedited T cells) and unedited cells transduced with a CAR-EGFRt lentivirus (middle row; control CAR-T cells). [Figure 37] FIG. 1 shows a flow chart of an exemplary sequential positive immunomagnetic cell selection strategy for isolating CD8 and CD4 T cells from enriched leukapheresis products collected from normal healthy peripheral blood. [Figure 38] Figure 36 provides a table of flow cytometry data characterizing HIP CAR-T cells (HIP CD19-CAR-T cells), control CAR-T cells (CD19-CAR-T cells), and unedited T cells generated according to the methods outlined in Figure 36. [Figure 39] Figures A to D show the experimental results of Example 6. Figures A to B show Elispot analysis of Th1 (IFNg) responses, and Figures C to D show the results of a killing assay using CD19-CAR-T cells (C) or HIP CD19-CAR-T cells (D). [Figure 40]Panels A and B show the viability and killing efficacy of HIP cells formulated with the exemplary formulations described in Table 20. Panel A shows the viability of HIP cells prepared with the formulations before freezing (blank marker) or after thawing (solid marker), and Panel B shows the NALM-6 killing efficacy of HIP cells prepared with the formulations. [Figure 41A] Th1 (IFNg) responses as determined by Elispot for unsorted and sorted T cells are shown. [Figure 41B] Th1 (IFNg) responses as determined by Elispot for unsorted and sorted T cells are shown. [Figure 41C] Th1 (IFNg) responses as determined by Elispot for unsorted and sorted T cells are shown. [Figure 41D] Th1 (IFNg) responses as determined by Elispot for unsorted and sorted T cells are shown. [Figure 42A] Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 42B] Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 42C] Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 42D] Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 42E] Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 42F] Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 42G] Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 42H] Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 42I]Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 42J] Quantification of tumor burden over time for the study described in Example 9 is shown. [Figure 43A] Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood and CD47 expression for the study described in Example 9. Specifically, Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood assessed at the time of first bleed and sacrifice, and Figure 43G shows the CD47 MFI of CD19-CAR-T cells in blood at day 108 in the group reloaded with 5x10 HIP CD19-CAR-T cells and the tKO CD19-CAR-T (HIP CD19-CAR-T)-treated group. A one-way ANOVA with Tukey's multiple comparison test was performed on the CAR+ cell frequency data, and a two-way ANOVA with Bonferroni's multiple comparison test was performed on the blood CD47 MFI data. [Figure 43B] Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood and CD47 expression for the study described in Example 9. Specifically, Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood assessed at the time of first bleed and sacrifice, and Figure 43G shows the CD47 MFI of CD19-CAR-T cells in blood at day 108 in the group reloaded with 5x10 HIP CD19-CAR-T cells and the tKO CD19-CAR-T (HIP CD19-CAR-T)-treated group. A one-way ANOVA with Tukey's multiple comparison test was performed on the CAR+ cell frequency data, and a two-way ANOVA with Bonferroni's multiple comparison test was performed on the blood CD47 MFI data. [Figure 43C]Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood and CD47 expression for the study described in Example 9. Specifically, Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood assessed at the time of first bleed and sacrifice, and Figure 43G shows the CD47 MFI of CD19-CAR-T cells in blood at day 108 in the group reloaded with 5x10 HIP CD19-CAR-T cells and the tKO CD19-CAR-T (HIP CD19-CAR-T)-treated group. A one-way ANOVA with Tukey's multiple comparison test was performed on the CAR+ cell frequency data, and a two-way ANOVA with Bonferroni's multiple comparison test was performed on the blood CD47 MFI data. [Figure 43D] Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood and CD47 expression for the study described in Example 9. Specifically, Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood assessed at the time of first bleed and sacrifice, and Figure 43G shows the CD47 MFI of CD19-CAR-T cells in blood at day 108 in the group reloaded with 5x10 HIP CD19-CAR-T cells and the tKO CD19-CAR-T (HIP CD19-CAR-T)-treated group. A one-way ANOVA with Tukey's multiple comparison test was performed on the CAR+ cell frequency data, and a two-way ANOVA with Bonferroni's multiple comparison test was performed on the blood CD47 MFI data. [Figure 43E]Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood and CD47 expression for the study described in Example 9. Specifically, Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood assessed at the time of first bleed and sacrifice, and Figure 43G shows the CD47 MFI of CD19-CAR-T cells in blood at day 108 in the group reloaded with 5x10 HIP CD19-CAR-T cells and the tKO CD19-CAR-T (HIP CD19-CAR-T)-treated group. A one-way ANOVA with Tukey's multiple comparison test was performed on the CAR+ cell frequency data, and a two-way ANOVA with Bonferroni's multiple comparison test was performed on the blood CD47 MFI data. [Figure 43F] Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood and CD47 expression for the study described in Example 9. Specifically, Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood assessed at the time of first bleed and sacrifice, and Figure 43G shows the CD47 MFI of CD19-CAR-T cells in blood at day 108 in the group reloaded with 5x10 HIP CD19-CAR-T cells and the tKO CD19-CAR-T (HIP CD19-CAR-T)-treated group. A one-way ANOVA with Tukey's multiple comparison test was performed on the CAR+ cell frequency data, and a two-way ANOVA with Bonferroni's multiple comparison test was performed on the blood CD47 MFI data. [Figure 43G]Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood and CD47 expression for the study described in Example 9. Specifically, Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood assessed at the time of first bleed and sacrifice, and Figure 43G shows the CD47 MFI of CD19-CAR-T cells in blood at day 108 in the group reloaded with 5x10 HIP CD19-CAR-T cells and the tKO CD19-CAR-T (HIP CD19-CAR-T)-treated group. A one-way ANOVA with Tukey's multiple comparison test was performed on the CAR+ cell frequency data, and a two-way ANOVA with Bonferroni's multiple comparison test was performed on the blood CD47 MFI data. [Figure 43H] Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood and CD47 expression for the study described in Example 9. Specifically, Figures 43A-F show the frequency of HIP CD19-CAR-T cells in blood assessed at the time of first bleed and sacrifice, and Figure 43G shows the CD47 MFI of CD19-CAR-T cells in blood at day 108 in the group reloaded with 5x10 HIP CD19-CAR-T cells and the tKO CD19-CAR-T (HIP CD19-CAR-T)-treated group. A one-way ANOVA with Tukey's multiple comparison test was performed on the CAR+ cell frequency data, and a two-way ANOVA with Bonferroni's multiple comparison test was performed on the blood CD47 MFI data. [Figure 44] 1 shows the cell count results of an exploratory autonomous growth assay for the study described in Example 10.

[0340] Other objects, advantages and embodiments of the present disclosure will become apparent from the following detailed description. DETAILED DESCRIPTION OF THE INVENTION

[0341] I. Introduction Described herein are engineered or modified immune evasive cells based in part on the hypoimmunediating platform described in WO2018132783, including, but not limited to, human immune evasive cells. To address the issue of immune rejection in subjects of these primary and / or stem cell-derived transplants, the inventors have developed and described herein hypoimmunogenic cells (e.g., hypoimmunogenic pluripotent cells, differentiated cells derived therefrom, and primary cells) that represent a viable source for any transplantable cell type. Such cells are protected from adaptive and / or innate immune rejection after administration to a recipient subject. Advantageously, the cells disclosed herein are protected from adaptive and innate immune rejection after administration to a recipient subject, regardless of the subject's genetic makeup, and therefore are not rejected by the recipient subject's immune system. In some embodiments, the engineered and / or hypoimmunogenic cells do not express major histocompatibility complex (MHC) class I and class II antigens and / or T cell receptors. In certain embodiments, the engineered and / or hypoimmunogenic cells do not express MHC I and II antigens and / or T cell receptors and overexpress CD47 protein. In certain embodiments, the engineered and / or hypoimmunogenic cells, e.g., engineered and / or hypoimmunogenic T cells, do not express MHC I and II antigens and / or T cell receptors, overexpress CD47 protein, and express an exogenous CAR.

[0342] In some embodiments, the hypoimmunogenic cells outlined herein are not subject to innate immune cell rejection. In some cases, hypoimmunogenic cells are not susceptible to NK cell-mediated lysis. In some cases, hypoimmunogenic cells are not susceptible to macrophage phagocytosis. In some embodiments, hypoimmunogenic cells are useful as a source of universally compatible cells or tissues (e.g., universal donor cells or tissues) that can be transplanted into a recipient subject with little to no need for immunosuppressive agents. Such hypoimmunogenic cells retain cell-specific properties and characteristics after transplantation, including, for example, pluripotency and the ability to engraft and function similarly to the corresponding native cells.

[0343] The technology disclosed herein utilizes expression of tolerogenic factors and modulation (e.g., reduction or elimination) of MHC I, MHC II, and / or TCR expression in human cells. In some embodiments, genome editing techniques utilizing rare-cutting endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) are used to also reduce or eliminate expression of genes involved in immune responses in cells (e.g., by deleting genomic DNA of genes involved in immune responses or by inserting genomic DNA into such genes such that gene expression is affected). In some embodiments, genome editing or other gene modulation techniques are used to insert tolerance-inducing (tolerogenic) factors in human cells, enabling the cells and their progeny (including any differentiated cells prepared therefrom) to avoid immune recognition upon transplantation into a recipient subject. Thus, the cells described herein exhibit regulated expression of one or more genes and factors that affect MHC I, MHC II, and / or TCR expression and evade the immune system of a recipient subject.

[0344] Genome editing technology allows for double-stranded DNA breaks at desired gene loci. These controlled double-stranded breaks promote homologous recombination at specific gene loci. This process focuses on targeting specific sequences of nucleic acid molecules, such as chromosomes, with endonucleases that recognize and bind to the sequence and induce double-stranded breaks in the nucleic acid molecule. The double-stranded breaks are repaired by either error-prone non-homologous end joining (NHEJ) or homologous recombination (HR).

[0345] The practice of many embodiments will employ, unless otherwise indicated to the contrary, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA techniques, genetics, immunology, and cell biology, which are within the skill of those in the art, many of which are described below by way of illustration, and such techniques are explained more fully in the literature. 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, NYSee monographs in publications such as (1998) Current Protocols in Immunology QE Coligan, A.M. Kruisbeek, D.H. Margulies, E.M. Shevach and W. Strober, eds., 1991); Annual Review of Immunology; and Advances in Immunology.

[0346] II. Definition As described in this disclosure, the following terms are used and are defined as set forth below.

[0347] The term "autoimmune disease" refers to any disease or disorder in which a subject mounts an immune response against its own tissues and / or cells. Autoimmune disorders can affect almost every organ system in a subject (e.g., a human), including, but not limited to, diseases of the nervous system, gastrointestinal system, and endocrine system, as well as skin and other connective tissues, eyes, blood, and blood vessels. Examples of autoimmune diseases include, but are not limited to, Hashimoto's thyroiditis, systemic lupus erythematosus, Sjogren's syndrome, Graves' disease, scleroderma, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and diabetes.

[0348] The term "cancer," as used herein, is defined as the hyperproliferation of cells whose intrinsic traits (e.g., loss of normal control) result in uncontrolled growth, lack of differentiation, local tissue invasion, and metastasis. In the context of the methods of the present invention, cancer includes acute lymphocytic carcinoma, acute myeloid leukemia, alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal, or anorectum, eye cancer, intrahepatic bile duct cancer, cancer of the joints, cancer of the neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, oral cancer, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid carcinoma, colon cancer, esophageal cancer, cervical cancer, fibrosarcoma, gastrointestinal carcinoid tumor, Hodgkin's lymphoma, and the like. The tumor may be any cancer, including any of the following: ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, renal cancer, skin cancer, small intestine cancer, soft tissue cancer, solid tumor, stomach cancer, testicular cancer, thyroid cancer, ureteral cancer, and / or bladder cancer. As used herein, the term "tumor" refers to an abnormal growth of cells or tissue of a malignant type, and does not include benign type tissue, unless specifically indicated otherwise.

[0349] The term "chronic infectious disease" refers to a disease caused by an infectious agent in which the infection persists. Such diseases may include hepatitis (A, B, or C), herpes viruses (e.g., VZV, HSV-1, HSV-6, HSV-II, CMV, and EBV), and HIV / AIDS. Non-viral examples may include chronic fungal diseases such as aspergillosis, candidiasis, coccidioidomycosis, and diseases associated with cryptococcosis and histoplasmosis. Non-limiting examples of chronic bacterial infectious agents may be Chlamydia pneumoniae, Listeria monocytogenes, and Mycobacterium tuberculosis. In some embodiments, the disorder is human immunodeficiency virus (HIV) infection. In some embodiments, the disorder is acquired immune deficiency syndrome (AIDS).

[0350] As used herein, a "clinically effective amount" refers to an amount sufficient to provide a clinical benefit in the treatment and / or management of a disease, disorder, or condition. In some embodiments, a clinically effective amount is an amount shown to produce at least one improved clinical endpoint relative to the standard of care for the disease, disorder, or condition. In some embodiments, a clinically effective amount is an amount demonstrated, for example, in a clinical trial, to be sufficient to provide statistically significant and meaningful efficacy for treating a disease, disorder, or condition. In some embodiments, a clinically effective amount is also a therapeutically effective amount. In other embodiments, a clinically effective amount is not a therapeutically effective amount.

[0351] In some embodiments, the changes or modifications described herein (including, for example, genetic changes or modifications) result in reduced expression of a target or selected polynucleotide sequence. In some embodiments, the changes or modifications described herein result in reduced expression of a target or selected polypeptide sequence. In some embodiments, the changes or modifications described herein result in increased expression of a target or selected polynucleotide sequence. In some embodiments, the changes or modifications described herein result in increased expression of a target or selected polypeptide sequence.

[0352] In additional or alternative embodiments, the present disclosure contemplates altering the target polynucleotide sequence by any method available to those skilled in the art, for example, using a TALEN system or RNA-guided transposase. Although examples of methods using CRISPR / Cas (e.g., Cas9 and Cas12a) and TALEN are detailed herein, it should be understood that the present disclosure is not limited to the use of these methods / systems. Other methods known to those skilled in the art, such as targeting B2M, can be used herein to reduce or eliminate expression in target cells.

[0353] The terms "decreasing," "reduced," "reducing," and "decreasing" are all used herein to generally mean a statistically significant amount of reduction. However, for the avoidance of doubt, "decreasing," "reduced," "reducing," and "decreasing" refer to a reduction of at least 10% compared to a reference level, e.g., a reduction of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or a reduction of up to and including 100% (i.e., absent levels compared to a reference sample), or any reduction between 10-100%. In some embodiments, cells are engineered to have reduced expression of one or more targets compared to unaltered or unmodified wild-type cells.

[0354] In some embodiments, the engineered and hypoimmunogenic cells described are derived from iPSCs or their progeny. As used herein, the term "derived from iPSCs or their progeny" encompasses the initial iPSC generated and any subsequent progeny thereof. As used herein, the term "progeny" encompasses, for example, first generation progeny. That is, progeny are derived, obtained, obtainable, or derivable directly from the initial iPSC, for example, by conventional propagation methods. The term "progeny" also encompasses additional generations, for example, second, third, fourth, fifth, sixth, seventh, or later generations, that is, generations of cells derived, obtained, obtainable, or derivable from previous generations, for example, by conventional propagation methods. The term "progeny" also encompasses modified cells resulting from modification or alteration of the initial iPSC or its progeny.

[0355] The term "donor subject" refers to an animal, e.g., a human, from which cells can be obtained. "Non-human animal" and "non-human mammal," used interchangeably herein, include mammals, e.g., rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "donor subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the donor subject is a mammal, e.g., a human, or other mammal, e.g., a domesticated mammal, e.g., a dog, cat, horse, etc., or a production mammal, e.g., a cow, sheep, pig, etc. "Donor subject" also refers to multiple donors, e.g., one or more human or non-human animals or non-human mammals.

[0356] The term "endogenous" refers to a molecule or polypeptide of interest that is naturally present in a cell. Similarly, when used in reference to the expression of an encoding nucleic acid, the term refers to the expression of an encoding nucleic acid that is naturally contained within a cell and not exogenously introduced. Similarly, when used in reference to a promoter sequence, the term refers to a promoter sequence that is naturally contained within a cell and not exogenously introduced.

[0357] The term "engineered cell," as used herein, refers to a cell that has been altered in at least some way by human intervention, including, for example, a genetic change or modification that causes the engineered cell to differ from a wild-type cell.

[0358] As used herein, the term "exogenous" in the context of polynucleotide or polypeptide expression is intended to mean that the reference molecule or polypeptide is introduced into a cell of interest. A polypeptide can be introduced, for example, by introducing the encoding nucleic acid into the genetic material of a cell, such as by integration into a chromosome or as non-chromosomal genetic material, such as a plasmid or expression vector. Thus, when used in reference to the expression of an encoding nucleic acid, the term refers to the introduction of the encoding nucleic acid into a cell in an expressible form. An exogenous polynucleotide can be inserted into at least one allele of a cell, for example, using a vector via viral transduction. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell via viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.In some embodiments, the exogenous polynucleotide is inserted into the safe harbor or target locus of at least one allele of the cell.

[0359] An "exogenous" molecule is a molecule, construct, factor, etc. that is not normally present in a cell but that can be introduced into the cell by one or more genetic, biochemical, or other methods. "Normal presence in a cell" is determined relative to the particular developmental stage and environmental conditions of the cell. Thus, for example, a molecule that is present only during embryonic development of a neuron is an exogenous molecule relative to an adult neuronal cell. Exogenous molecules can include, for example, a functional version of a dysfunctional endogenous molecule or a dysfunctional version of a normally functioning endogenous molecule.

[0360] Exogenous molecules or factors can be, for example, small molecules produced by combinatorial chemistry processes, or macromolecules such as proteins, nucleic acids, carbohydrates, lipids, glycoproteins, lipoproteins, polysaccharides, any modified derivatives of the above molecules, or any complex containing one or more of the above molecules. Nucleic acids include DNA and RNA, can be single-stranded or double-stranded, linear, branched, or circular, and can be of any length. Nucleic acids include nucleic acids capable of forming duplexes as well as triplex-forming nucleic acids. See, for example, U.S. Patent Nos. 5,176,996 and 5,422,251. Proteins include, but are not limited to, DNA-binding proteins, transcription factors, chromatin-remodeling factors, methylated DNA-binding proteins, polymerases, methylases, demethylases, acetylases, deacetylases, kinases, phosphatases, integrases, recombinases, ligases, topoisomerases, gyrases, and helicases.

[0361] The exogenous molecule or construct may be the same type of molecule as the endogenous molecule, such as an exogenous protein or nucleic acid. In such cases, the exogenous molecule is introduced into the cell at a concentration higher than the concentration of the endogenous molecule in the cell. In some cases, the exogenous nucleic acid may include an infectious viral genome, a plasmid or episome introduced into the cell, or a chromosome not normally present in the cell. Methods for introducing exogenous molecules into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate coprecipitation, DEAE-dextran-mediated transfer, and viral vector-mediated transfer.

[0362] For purposes of this disclosure, a "gene" includes a DNA region that encodes a gene product, as well as all DNA regions that control the production of that gene product (whether or not such control sequences flank the coding and / or transcribed sequence). Thus, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translation control sequences, e.g., ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, origins of replication, matrix attachment sites, and / or locus control regions.

[0363] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. A gene product can be the direct transcription product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA, or any other type of RNA) or a protein produced by translation of mRNA. Gene products also include RNAs that are modified by processes such as capping, polyadenylation, methylation, and editing, as well as proteins that are modified, for example, by methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, myristoylation, and / or glycosylation.

[0364] As used herein, the term "genetic modification" and its grammatical equivalents may refer to one or more changes in a nucleic acid, e.g., a nucleic acid in the genome of an organism. For example, a genetic modification may refer to a change, addition, and / or deletion of a gene or a portion of a gene or other nucleic acid sequence. A genetically modified cell may also refer to a cell having an addition, deletion, and / or change of a gene or a portion of a gene. A genetically modified cell may also refer to a cell having an addition of a nucleic acid sequence that is not a gene or a portion of a gene. Genetic modifications include, for example, both transient knock-in or knock-down mechanisms and mechanisms that result in permanent knock-in, knock-down, or knock-out of a target gene or a portion of a gene or nucleic acid sequence. Genetic modifications also include, for example, both transient knock-in and mechanisms that result in permanent knock-in of a nucleic acid sequence. Genetic modifications also include, for example, reduced or increased transcription, reduced or increased mRNA stability, reduced or increased translation, and reduced or increased protein stability.

[0365] As used herein, the terms "grafting," "administering," "introducing," "implanting," and "transplanting," as well as grammatical variations thereof, are used interchangeably in the context of placing cells (e.g., cells described herein) into a subject by a method or route that results in localization or at least partial localization at a desired site or systemic introduction (e.g., into the circulation) of the introduced cells. Cells can be directly implanted at a desired site within a subject, or alternatively, can be administered by any suitable route that results in delivery to a desired location, where at least a portion of the implanted cells or components of the cells remain viable. The survival period of cells after administration to a subject can be as short as a few hours, e.g., 24 hours, to as long as several days or years. In some embodiments, cells can also be administered (e.g., injected) to a site other than the desired site, such as intracerebrally or subcutaneously, e.g., in a capsule to maintain the implanted cells at the site of implantation and avoid migration of the implanted cells.

[0366] The "HLA" or "human leukocyte antigen" complex is a complex of genes encoding MHC proteins in humans. These cell surface proteins, which make up the HLA complex, are responsible for regulating immune responses to antigens. In humans, there are two types of MHC: class I and class II, HLA-I and HLA-II. HLA-I contains three proteins, HLA-A, HLA-B, and HLA-C, which present peptides from the inside of cells. Antigens presented by the HLA-I complex attract killer T cells (also known as CD8+ T cells or cytotoxic T cells). HLA-I proteins associate with beta-2 microglobulin (B2M). HLA-II contains five proteins, HLA-DP, HLA-DM, HLA-DOB, HLA-DQ, and HLA-DR, which present antigens from the outside of cells to T lymphocytes. These stimulate CD4+ cells (also known as helper T cells). It should be understood that the use of either "MHC" or "HLA" is not meant to be limiting, as it depends on whether the gene is human (HLA) or murine (MHC). Thus, these terms may be used interchangeably herein when it comes to mammalian cells.

[0367] The term "low immunogenicity," as used herein to characterize cells, generally means that the cells are less likely to be subject to innate or adaptive immune rejection by a subject receiving the cells, e.g., the cells are less likely to be subject to allogeneic rejection by a subject receiving the cells. For example, compared to cells of the same cell type that do not contain modifications, such low immunogenic cells may be about 2.5%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99% or more less prone to innate or adaptive immune rejection by a subject receiving the cells. In some embodiments, genome editing techniques are used to regulate the expression of MHC I and MHC II genes, thereby contributing to the generation of low immunogenic cells. In some embodiments, the low immunogenic cells avoid immune rejection in an MHC-mismatched allogeneic recipient. In some cases, differentiated cells generated from the hypoimmunogenic stem cells outlined herein avoid immune rejection when administered (e.g., transplanted or grafted) to an MHC-mismatched allogeneic recipient. In some embodiments, the hypoimmunogenic cells are protected from T cell-mediated adaptive immune rejection and / or innate immune cell rejection. Detailed descriptions of hypoimmunogenic cells, methods for their production, and methods for their use are found in WO2016183041, filed May 9, 2015; WO2018132783, filed January 14, 2018; WO2018176390, filed March 20, 2018; WO2020018615, filed July 17, 2019; WO2020018620, filed July 31, 2020; and US 63 / 052,360, filed July 15, 2020, the disclosures of which, including the Examples, sequence listings, and figures, are incorporated herein by reference in their entireties.

[0368] The low immunogenicity of cells can be determined by assessing the immunogenicity of the cells, such as their ability to induce or avoid inducing adaptive and innate immune responses. Such immune responses can be measured using assays recognized by those skilled in the art. In some embodiments, immune response assays measure the effect of hypoimmunogenic cells on T cell proliferation, T cell activation, T cell killing, donor-specific antibody production, NK cell proliferation, NK cell activation, and macrophage activity. In some cases, hypoimmunogenic cells and their derivatives undergo reduced killing by T cells and / or NK cells after administration to a subject. In some cases, the cells and their derivatives exhibit reduced macrophage phagocytosis compared to unmodified or wild-type cells. In some embodiments, hypoimmunogenic cells induce a reduced or attenuated immune response in a recipient subject compared to corresponding unmodified wild-type cells. In some embodiments, hypoimmunogenic cells are non-immunogenic or unable to induce an immune response in a recipient subject.

[0369] The term "percent identity" in the context of two or more nucleic acid or polypeptide sequences refers to two or more sequences or subsequences that have a specified percentage of nucleotide or amino acid residues that are the same when compared and aligned for maximum correspondence, as determined using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection. Depending on the application, the percent "identity" can exist over a region of the sequences being compared, e.g., over a functional domain, or alternatively, over the entire length of the two sequences being compared. For sequence comparison, typically, one sequence serves as a reference sequence, and test sequences are compared to this sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, and sequence algorithm program parameters are designated, if necessary. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence(s) relative to the reference sequence based on the designated program parameters.

[0370] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the similarity search method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).

[0371] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0372] As used herein, "immune signaling factor" refers, in some cases, to a molecule, protein, peptide, etc. that activates an immune signaling pathway.

[0373] As used herein, "immunosuppressive factors" or "immunoregulatory factors" or "tolerogenic factors" include hypoimmune factors, complement inhibitors, and other factors that modulate or affect the ability of cells to be recognized by the immune system of a host or recipient subject upon administration, transplantation, or engraftment, optionally in combination with additional genetic modifications.

[0374] The terms "increased," "increase," or "enhance" or "activate" are all used herein to generally mean an increase by a statistically significant amount. For the avoidance of doubt, the terms "increased," "increase," or "enhance" or "activate" mean an increase of at least 10% compared to a reference level, e.g., an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a reference level, or an increase of up to and including 100%, or any increase between 10 and 100%, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and more than 10-fold compared to a reference level. In some embodiments, the reference level, also referred to as the basal level, is 0.

[0375] In some embodiments, the change is an indel. As used herein, "indel" refers to a mutation resulting from an insertion, deletion, or a combination thereof. As will be understood by those skilled in the art, an indel in the coding region of a genome sequence will result in a frameshift mutation unless the length of the indel is a multiple of three. In some embodiments, the change is a point mutation. As used herein, "point mutation" refers to a substitution that replaces one nucleotide. The gene editing (e.g., CRISPR / Cas) system of the present disclosure can be used to induce indels or point mutations of any length in a target polynucleotide sequence.

[0376] As used herein, "knockdown" refers to the reduction of the expression of target mRNA or corresponding target protein.Knockdown is generally reported relative to the level present after the administration or expression of a non-control molecule (e.g., non-targeting control shRNA, siRNA, or miRNA) that does not mediate the reduction of RNA expression levels.In some embodiments, the knockdown of target gene is achieved by conditional or inducible shRNA, conditional or inducible siRNA, conditional or inducible miRNA, or conditional or inducible CRISPR interference (CRISPRi) method.In some embodiments, the knockdown of target gene is achieved by protein-based method, for example, conditional or inducible degron method.In some embodiments, the knockdown of target gene is achieved by genetic modification, including shRNA, siRNA, miRNA, or by using gene editing system (e.g., CRISPR / Cas).

[0377] Knockdown is generally assessed by measuring mRNA levels using quantitative polymerase chain reaction (qPCR) amplification, or by measuring protein levels by Western blot or enzyme-linked immunosorbent assay (ELISA). Protein level analysis provides an assessment of both mRNA cleavage and translation inhibition. Additional techniques for measuring knockdown include RNA solution hybridization, nuclease protection, Northern hybridization, gene expression monitoring using microarrays, antibody binding, radioimmunoassay, and fluorescence-activated cell analysis. Those skilled in the art can easily understand how to use the gene editing system (e.g., CRISPR / Cas) of the present disclosure to knock out a target polynucleotide sequence or a portion thereof based on the details described herein.

[0378] As used herein, "knock-in" or "knock-in" refers to a genetic modification resulting from the insertion of a DNA sequence into a chromosomal locus in a host cell. This results in the initiation or increased level of expression of the knocked-in gene, portion of a gene, or nucleic acid sequence insertion product, e.g., increased RNA transcript levels and / or encoded protein levels. As will be understood by those skilled in the art, this can be accomplished in several ways, including inserting or adding one or more additional copies of a gene or portion thereof to the host cell, or altering the regulatory components of an endogenous gene to increase expression of the protein being produced, or inserting a specific nucleic acid sequence whose expression is desired. This may be accomplished by modifying the promoter, adding a different promoter, adding enhancers, adding other regulatory elements, or modifying other gene expression sequences.

[0379] As used herein, "knock out" or "knock-out" includes deleting all or a portion of a target polynucleotide sequence to prevent translation or function of the target polynucleotide sequence. For example, knockout can be achieved by altering the target polynucleotide sequence by introducing an insertion or deletion ("indel") into the target polynucleotide sequence, including within a functional domain (e.g., a DNA-binding domain) of the target polynucleotide sequence. Those skilled in the art will readily understand how to use the gene editing system (e.g., CRISPR / Cas) of the present disclosure to knock out a target polynucleotide sequence or a portion thereof based on the details described herein.

[0380] In some embodiments, genetic modification or alteration results in knockout or knockdown of a target polynucleotide sequence or a portion thereof.Knocking out a target polynucleotide sequence or a portion thereof using the gene editing system (e.g., CRISPR / Cas) of the present disclosure can be useful for various applications.For example, knocking out a target polynucleotide sequence in a cell can be performed in vitro for research purposes.For ex vivo purposes, knocking out a target polynucleotide sequence in a cell can be useful for treating or preventing disorders associated with the expression of a target polynucleotide sequence, or for changing the genotype or phenotype of a cell (for example, by knocking out a mutant allele in a cell ex vivo and introducing the cells containing the knocked-out mutant allele into a subject).

[0381] "Modulation" of gene expression refers to a change in the expression level of a gene. Modulation of expression can include, but is not limited to, gene activation and gene repression. Modulation can also be complete (i.e., gene expression is completely inactivated or activated to above wild-type levels), or it can be partial (gene expression is partially reduced or partially activated to a fraction of wild-type levels).

[0382] In additional or alternative aspects, the present disclosure contemplates altering a target polynucleotide sequence by any method available to one of skill in the art, for example, using a nuclease system, such as a TAL effector nuclease (TALEN) or zinc finger nuclease (ZFN) system. While exemplary methods utilizing CRISPR / Cas (e.g., Cas9 and Cas12a) and TALEN are detailed herein, it should be understood that the present disclosure is not limited to the use of these methods / systems. Other targeting methods known to those of skill in the art can be utilized herein to reduce or eliminate expression in target cells. The methods provided herein can be used to alter a target polynucleotide sequence within a cell. The present disclosure contemplates altering a target polynucleotide sequence within a cell for any purpose. In some embodiments, a target polynucleotide sequence within a cell is altered to produce a mutant cell. As used herein, a "mutant cell" refers to a cell whose resulting genotype differs from its original genotype. In some cases, a "mutated cell" exhibits a mutant phenotype, for example, when a normally functioning gene is altered using a gene editing system (e.g., CRISPR / Cas) system of the present disclosure. In other cases, a "mutated cell" exhibits a wild-type phenotype, for example, when a gene editing system (e.g., CRISPR / Cas) system of the present disclosure is used to correct a mutant gene type. In some embodiments, a target polynucleotide sequence in a cell is altered to correct or repair a genetic mutation (e.g., to restore a normal phenotype to the cell). In some embodiments, a target polynucleotide sequence in a cell is altered to induce a genetic mutation (e.g., to disrupt the function of a gene or genomic element).

[0383] The term "native cell," as used herein, refers to a cell that has not been otherwise modified (e.g., engineered). In some embodiments, a native cell is a naturally occurring wild-type or control cell.

[0384] The terms "operably linked" or "operably linked" are used interchangeably in reference to the juxtaposition of two or more components (such as sequence elements) that allow for the potential for both components to function normally and for at least one of the components to mediate a function on at least one of the other components. By way of illustration, a transcription control sequence, such as a promoter, is operably linked to a coding sequence if it controls the level of transcription of the coding sequence in response to the presence or absence of one or more transcription factors. A transcription control sequence is usually operably linked in cis with a coding sequence, but need not be directly adjacent to it. For example, an enhancer is a transcription control sequence that is operably linked to a coding sequence, even if they are not contiguous.

[0385] As used herein, "pluripotent stem cells" have the potential to differentiate into any of three germ layers: endoderm (e.g., stomach wall, digestive tract, lungs, etc.), mesoderm (e.g., muscle, bone, blood, urogenital tissue, etc.), or ectoderm (e.g., epithelial tissue and nervous system tissue). The term "pluripotent stem cells" as used herein also encompasses "induced pluripotent stem cells" or "iPSCs," a type of pluripotent stem cell derived from a non-pluripotent cell. In some embodiments, pluripotent stem cells are produced or generated from a cell that is not a pluripotent cell. In other words, pluripotent stem cells can be the direct or indirect progeny of a non-pluripotent cell. Examples of parent cells include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means. Such "iPS" or "iPSC" cells can be generated by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for the derivation of iPS cells are known in the art and are further described below (see, e.g., Zhou et al., Stem Cells 27(11):2667-74(2009); Huangfu et al., Nature Biotechnol. 26(7):795(2008); Woltjen et al., Nature 458(7239):766-770(2009); and Zhou et al., Cell Stem Cell 8:381-384(2009), each of which is incorporated herein by reference in its entirety). The generation of induced pluripotent stem cells (iPSCs) is outlined below. As used herein, "hiPSCs" refers to human induced pluripotent stem cells. In some embodiments, "pluripotent stem cells," as used herein, also encompass mesenchymal stem cells (MSCs) and / or embryonic stem cells (ESCs).

[0386] As used herein, a "promoter," "promoter sequence," or "promoter region" refers to a DNA regulatory region / sequence that is capable of binding RNA polymerase and is involved in initiating transcription of a downstream coding or non-coding sequence. In some instances, a promoter sequence includes a transcription initiation site and extends upstream to include the minimum number of bases or elements necessary to initiate transcription at a level detectable above background. In some embodiments, a promoter sequence includes a transcription initiation site and a protein binding domain that contributes to the binding of RNA polymerase. Eukaryotic promoters often, but not always, contain "TATA" boxes and "CAT" boxes.

[0387] In some embodiments, the described engineered and hypoimmunogenic cells are expanded from primary T cells or their progeny. As used herein, the term "expanded from primary T cells or their progeny" includes primary T cells isolated from a donor subject and any subsequent progeny thereof. As used herein, the term "progeny" includes, for example, first generation progeny. That is, progeny are derived, obtained, obtainable, or derivable directly from the primary T cell, for example, by conventional propagation methods. The term "progeny" also includes additional generations, for example, second, third, fourth, fifth, sixth, seventh, or later generations, that is, generations of cells derived, obtained, obtainable, or derivable from previous generations, for example, by conventional propagation methods. The term "progeny" also includes modified cells resulting from the modification or alteration of primary T cells or their progeny.

[0388] The term "recipient patient" refers to an animal, e.g., a human, to which treatment, including prophylactic treatment, with the cells described herein is provided. For treatment of those infections, conditions, or disease states specific to a particular animal, e.g., a human patient, the term patient refers to that particular animal. The term "recipient patient" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the recipient patient is a mammal, e.g., a human, or other mammal, e.g., a domesticated mammal, e.g., a dog, cat, horse, etc., or a production mammal, e.g., a cow, sheep, pig, etc.

[0389] As used herein, the terms "regulatory sequence," "regulatory element," and "control element" are used interchangeably and refer to polynucleotide sequences that are upstream (5' non-coding sequences), within, or downstream (3' untranslated sequences) of a polynucleotide target to be expressed. Regulatory sequences influence, for example, but not limited to, the timing of transcription, the amount or level of transcription, RNA processing or stability, and / or translation of associated structural nucleotide sequences. Regulatory sequences can include activator binding sequences, enhancers, introns, polyadenylation recognition sequences, promoters, repressor binding sequences, stem-loop structures, translation initiation sequences, translation leader sequences, transcription termination sequences, translation termination sequences, primer binding sites, and the like. Since in most cases the exact boundaries of regulatory sequences have not been completely defined, it will be recognized that nucleotide sequences of different lengths can have identical regulatory or promoter activity.

[0390] As used herein, "safe harbor loci" refers to loci that allow expression of a transgene or exogenous gene in a manner that allows the newly inserted genetic element to function as expected and that may not alter the host genome in a manner that poses a risk to the host cell. Exemplary "safe harbor" loci include, but are not limited to, the CCR5 gene, the PPP1R12C (also known as AAVS1) gene, the CLYBL gene, and / or the Rosa gene (e.g., ROSA26).

[0391] As used herein, a "target locus" refers to a locus capable of expressing a transgene or exogenous gene. Exemplary "target loci" include, but are not limited to, the CXCR4 gene, albumin gene, SHS231 locus, F3 gene (also known as CD142), MICA gene, MICB gene, LRP1 gene (also known as CD91), HMGB1 gene, ABO gene, RHD gene, FUT1 gene, and / or KDM5D gene (also known as HY). An exogenous polynucleotide gene encoding an exogenous polynucleotide may be inserted into the CDS region of B2M, CIITA, TRAC, TRBC, CCR5, F3 (i.e., CD142), MICA, MICB, LRP1, HMGB1, ABO, RHD, FUT1, KDM5D (i.e., HY), PDGFRa, OLIG2, and / or GFAP. An exogenous polynucleotide encoding an exogenous gene may be inserted into intron 1 or 2 of PPP1R12C (i.e., AAVS1) or CCR5. An exogenous polynucleotide encoding an exogenous gene may be inserted into exon 1, 2, or 3 of CCR5. An exogenous polynucleotide encoding an exogenous gene may be inserted into intron 2 of CLYBL. An exogenous polynucleotide encoding an exogenous gene may be inserted into a 500 bp window in Ch-4:58,976,613 (i.e., SHS231). An exogenous polynucleotide encoding an exogenous gene may be inserted into any suitable region of the aforementioned safe harbor or target locus that allows expression of the exogenous gene, including, for example, an intron, exon, or coding sequence region within the safe harbor or target locus.

[0392] As used herein, "target" may refer to a gene, portion of a gene, portion of a genome, or protein that is subject to controllably reduced expression by the methods described herein.

[0393] As used herein, a "therapeutically effective amount" refers to an amount sufficient to provide a therapeutic benefit in the treatment and / or management of a disease, disorder, or condition. In some embodiments, a therapeutically effective amount is an amount sufficient to ameliorate, alleviate, stabilize, reverse, slow, attenuate, or delay the progression of a disease, disorder, or condition, or the symptoms or side effects of a disease, disorder, or condition. In some embodiments, a therapeutically effective amount is also a clinically effective amount. In other embodiments, a therapeutically effective amount is not a clinically effective amount.

[0394] As used herein, the terms "treating" and "treatment" include administering a therapeutically or clinically effective amount of the cells described herein to a subject so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired therapeutic or clinical result. For purposes of the present technology, a beneficial or desired therapeutic or clinical result includes, but is not limited to, alleviation of one or more symptoms, whether detectable or undetectable, attenuation of the extent of the disease, a stabilized (i.e., not worsening) disease state, a delay or slowing of disease progression, an improvement or palliation of the disease state, and remission (whether partial or complete). Treating may also refer to extending survival compared to the expected survival in the absence of treatment. Thus, those skilled in the art recognize that treatment may improve a condition but may not be a complete cure for the disease. In some embodiments, one or more symptoms of a condition, disease, or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50% upon treatment of the condition, disease, or disorder.

[0395] For purposes of the present technology, beneficial or desired therapeutic or clinical results of disease treatment include, but are not limited to, alleviation of one or more symptoms, whether detectable or undetectable, attenuation of the extent of the disease, a stabilized (i.e., not worsening) disease state, delay or slowing of disease progression, improvement or palliation of the disease state, and remission (whether partial or complete).

[0396] A "vector" or "construct" is capable of transferring a gene sequence into a target cell. Typically, "vector construct," "expression vector," and "gene transfer vector" refer to any nucleic acid construct capable of directing the expression of a gene of interest and transferring a gene sequence into a target cell. Thus, the term includes cloning and expression vehicles as well as integrating vectors. Methods for introducing vectors or constructs into cells are known to those skilled in the art and include, but are not limited to, lipid-mediated transfer (i.e., liposomes containing neutral and cationic lipids), electroporation, direct injection, cell fusion, particle bombardment, calcium phosphate co-precipitation, DEAE-dextran-mediated transfer, and / or viral vector-mediated transfer.

[0397] In some embodiments, cells are engineered to have reduced or increased expression of one or more targets compared to unaltered or unmodified wild-type cells. In some embodiments, cells are engineered to have constitutively reduced or increased expression of one or more targets compared to unaltered or unmodified wild-type cells. In some embodiments, cells are engineered to have controllably reduced or increased expression of one or more targets compared to unaltered or unmodified wild-type cells. In some embodiments, cells comprise increased expression of CD47 compared to wild-type or control cells of the same cell type. "Wild-type" or "wild-type (wt)" or "control" in the context of cells refers to any cell found in nature. Examples of wild-type or control cells include naturally occurring primary cells and T cells. However, by way of example, in the context of engineered cells, "wild-type" or "control" as used herein can also refer to engineered cells that may contain nucleic acid alterations that result in reduced expression of MHC I and / or II and / or T cell receptors, but that have not undergone a gene editing procedure to result in overexpression of CD47 protein. For example, as used herein, "wild-type" or "control" refers to engineered cells that contain reduced or knocked-out expression of B2M, CIITA, and / or TRAC. Also, as used herein, "wild-type" or "control" refers to engineered cells that contain reduced or knocked-out expression of B2M, CIITA, TRAC, and / or TRBC. As used herein, "wild-type" or "control" also refers to engineered cells that may contain nucleic acid alterations that result in overexpression of CD47 protein but have not undergone a gene editing procedure to achieve reduced expression of MHC I and / or II and / or T cell receptors. In the context of iPSCs or their progeny, "wild-type" or "control" also refers to iPSCs or their progeny that may contain nucleic acid alterations that result in pluripotency and have not undergone a gene editing procedure of the present disclosure to achieve reduced expression of MHC I and / or II and / or T cell receptors and / or overexpression of CD47 protein.For example, as used herein, "wild-type" or "control" refers to iPSCs or their progeny that contain reduced or knocked-out expression of B2M, CIITA, and / or TRAC. Also, as used herein, "wild-type" or "control" refers to iPSCs or their progeny that contain reduced or knocked-out expression of B2M, CIITA, TRAC, and / or TRBC. In the context of primary T cells or their progeny, "wild-type" or "control" can also refer to primary T cells or their progeny that may contain nucleic acid alterations that result in reduced expression of MHC I and / or II and / or T cell receptors, but that have not undergone a gene editing procedure to result in overexpression of CD47 protein. For example, as used herein, "wild-type" or "control" refers to primary T cells or their progeny that contain reduced or knocked-out expression of B2M, CIITA, and / or TRAC. Also, as used herein, "wild-type" or "control" refers to a primary T cell or its progeny that contains reduced or knocked-out expression of B2M, CIITA, TRAC, and / or TRBC. In the context of a primary T cell or its progeny, "wild-type" or "control" also refers to a primary T cell or its progeny that may contain a nucleic acid alteration that results in overexpression of CD47 protein, but that has not undergone a gene editing procedure to result in reduced expression of MHC I and / or II and / or T cell receptors. In some embodiments, the cells are engineered to have controllably reduced or increased expression of one or more targets compared to cells of the same cell type that do not contain the modification. In some embodiments, wild-type or control cells are the starting material. In some embodiments, the starting material is primary cells harvested from a donor. In some embodiments, the starting material is primary blood cells harvested from a donor, e.g., via leukopaque. For example, unmodified T cells obtained from a donor are the starting material that are considered wild-type or control cells as contemplated herein. In another example, the iPSC cell line starting material is the starting material that is considered a wild-type or control cell as contemplated herein.In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate an engineered cell.

[0398] It should be noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as a predicate for the use of exclusive terminology such as "solely," "only," and the like in connection with the recitation of claim elements, or for the use of a "negative" limitation. As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features that may be readily separated from or combined with features of any of the other embodiments without departing from the scope or spirit of the present disclosure. Any recited method may be carried out in the order of events recited or in any other order that is logically possible. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, representative exemplary methods and materials are now described.

[0399] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limits of that range, to one-tenth of the unit of the lower limit, and any other stated or intervening value in that stated range, is encompassed within the disclosure unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and may also be encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. Certain ranges are presented herein with the term "about" preceding the numerical value. The term "about" is used herein to provide literal support for the number it precedes, as well as for a number that is near or approximately the number it precedes. When determining whether a number is near or approximately a specifically recited number, the near or approximately unrecited number may be a number that, in the context provided, provides a substantial equivalent value to the specifically recited number. The term "about" is used herein to mean plus or minus ten percent (10%) of a value. For example, "about 100" refers to any number between 90 and 110.

[0400] All publications, patents, and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Furthermore, each cited publication, patent, or patent application is incorporated by reference herein to disclose and describe the subject matter in connection with which the publication is cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the technology described herein is not entitled to antedate such publication by virtue of prior art. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0401] Before further describing the present technology, it should be understood that the present technology is not limited to the particular embodiments described, and thus may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims. It should also be understood that the headings used herein are not limiting, but are intended only to provide orientation to the reader, but that their content generally applies to the technology disclosed herein.

[0402] III. Detailed Description of the Embodiments A. Low immunogenic cells In some embodiments, the present disclosure is directed to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., but not limited to, T cells and NK cells), and primary cells (e.g., but not limited to, primary T cells and primary NK cells). In some embodiments, the pluripotent stem cells, differentiated cells derived therefrom, e.g., T cells and NK cells, and primary cells, e.g., primary T cells and primary NK cells, are engineered for reduced or absent expression of MHC class I and / or MHC class II human leukocyte antigens, and in some cases, reduced or absent expression of the T cell receptor (TCR) complex. In some embodiments, hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a chimeric antigen receptor (CAR), and have reduced or absent expression of the T cell receptor (TCR) complex, in addition to reduced or absent expression of MHC class I and / or MHC class II human leukocyte antigens. In some embodiments, the CAR comprises an antigen-binding domain that binds to any one selected from the group consisting of CD19, CD22, CD38, CD123, CD138, and BCMA. In some embodiments, the CAR is a CD19-specific CAR. In some embodiments, the CAR is a CD22-specific CAR. In some cases, the CAR is a CD38-specific CAR. In some embodiments, the CAR is a CD123-specific CAR. In some embodiments, the CAR is a CD138-specific CAR. In some cases, the CAR is a BCMA-specific CAR. In some embodiments, the CAR is a bispecific CAR. In some embodiments, the bispecific CAR is a CD19 / CD22 bispecific CAR. In some embodiments, the bispecific CAR is a BCMA / CD38 bispecific CAR. In some embodiments, the described cells express a CD19-specific CAR and a different CAR, for example, but not limited to, a CD22-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR.In some embodiments, the described cells express a CD22-specific CAR and a different CAR, for example, but not limited to, a CD19-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. In some embodiments, the described cells express a CD38-specific CAR and a different CAR, for example, but not limited to, a CD22-specific CAR, a CD18-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. In some embodiments, the described cells express a CD123-specific CAR and a different CAR, for example, but not limited to, a CD22-specific CAR, a CD38-specific CAR, a CD19-specific CAR, a CD138-specific CAR, and a BCMA-specific CAR. In some embodiments, the described cells express a CD138-specific CAR and a different CAR, for example, but not limited to, a CD22-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD19-specific CAR, and a BCMA-specific CAR. In some embodiments, the described cells express a BCMA-specific CAR and a different CAR, for example, but not limited to, a CD22-specific CAR, a CD38-specific CAR, a CD123-specific CAR, a CD138-specific CAR, and a CD19-specific CAR. In some embodiments, the cells are modified or engineered relative to wild-type or control cells, including unchanged or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is primary cells collected from a donor. In some embodiments, the starting material is primary blood cells collected from a donor, for example, via leukopaque. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate engineered cells.

[0403] In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a chimeric antigen receptor (CAR) and comprise a genomic modification of the B2M gene. In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and comprise a genomic modification of the CIITA gene. In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR and comprise a genomic modification of the TRAC gene. In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR and comprise a genomic modification of the TRB gene. In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and a CAR and comprise one or more genomic modifications selected from the group consisting of the B2M, CIITA, TRAC, and TRB genes. In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells overexpress CD47 and CAR and contain genomic modifications of the B2M, CIITA, TRAC, and TRB genes. In some embodiments, the cells also express CAR. - / - , C.I.T.A. - / - ,TRAC - / - , CD47tg cells. In some embodiments, engineered and / or hypoimmune (HIP) T cells are generated by differentiating induced pluripotent stem cells, e.g., engineered and / or hypoimmunogenic induced pluripotent stem cells. In some embodiments, the cells are modified or engineered relative to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is primary cells collected from a donor. In some embodiments, the starting material is primary blood cells collected from a donor, e.g., via leukopaque. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate the engineered cells.

[0404] In some embodiments, the engineered and / or hypoimmune (HIP) T cells and primary T cells are B2M T cells that also express a CAR. - / - , C.I.T.A. - / - , TRB - / - In some embodiments, the cells are B2M, CD47tg cells that also express a CAR. - / - , C.I.T.A. - / - ,TRAC - / - , TRB - / - In certain embodiments, the cells are B2M, CD47tg cells that also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In certain embodiments, the cells are B2M, CD47tg cells that also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル In certain embodiments, the cells are B2M, CD47tg cells that also express a CAR. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , TRB インデル / インデル, CD47tg cells. In some embodiments, the engineered or modified cells described are pluripotent stem cells, induced pluripotent stem cells, NK cells differentiated from such pluripotent stem cells and induced pluripotent stem cells, T cells differentiated from such pluripotent stem cells and induced pluripotent stem cells, or primary T cells. Non-limiting examples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, T follicular helper (Tfh) cells, cytotoxic T lymphocytes (CTL), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue tolerance memory (Trm) cells, virtual memory T cells, natural memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cell. In some embodiments, the primary T cells are selected from the group including cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, tumor-infiltrating lymphocytes, and combinations thereof. Non-limiting examples of NK cells and primary NK cells include immature NK cells and mature NK cells. In some embodiments, the cells are modified or engineered relative to wild-type or control cells, including unchanged or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is primary cells collected from a donor. In some embodiments, the starting material is primary blood cells collected from a donor, e.g., via leukopaque. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate the engineered cells.

[0405] In some embodiments, the primary T cells are from a pool of primary T cells from one or more donor subjects different from the recipient subject (e.g., the patient receiving the cells). The primary T cells can be obtained and pooled together from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, or more donor subjects. The primary T cells can be obtained and pooled together from 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 50 or more, or 100 or more donor subjects. In some embodiments, the primary T cells are harvested from one or more individuals, and in some cases, the primary T cells or pool of primary T cells are cultured in vitro. In some embodiments, the primary T cells or pool of primary T cells are engineered to exogenously express CD47 and cultured in vitro.

[0406] In certain embodiments, primary T cells or a pool of primary T cells are engineered to express a chimeric antigen receptor (CAR). The CAR can be any known to those skilled in the art. Useful CARs include those that bind to an antigen selected from the group consisting of CD19, CD20, CD22, CD38, CD123, CD138, and BCMA. In some cases, the CAR is the same as or equivalent to those used in FDA-approved CAR-T cell therapies, such as, but not limited to, tisagenlecleucel and axicabtageneciloreucel, or others under investigation in clinical trials.

[0407] In some embodiments, primary T cells or pools of primary T cells are engineered to exhibit reduced expression of endogenous T cell receptors compared to unmodified primary T cells. In certain embodiments, primary T cells or pools of primary T cells are engineered to exhibit reduced expression of CTLA-4, PD-1, or both CTLA-4 and PD-1 compared to unmodified primary T cells. Methods for genetically modifying cells, including T cells, are detailed, for example, in WO2020 / 018620 and WO2016 / 183041, the disclosures of which are incorporated by reference in their entirety, including tables, appendices, sequence listings, and figures.

[0408] In some embodiments, the CAR-T cells comprise a CAR selected from the group comprising: (a) a first generation CAR comprising an antigen binding domain, a transmembrane domain, and a signaling domain; (b) a second generation CAR comprising an antigen binding domain, a transmembrane domain, and at least two signaling domains; (c) a third generation CAR comprising an antigen binding domain, a transmembrane domain, and at least three signaling domains; and (d) a fourth generation CAR comprising an antigen binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful signaling of the CAR.

[0409] In some embodiments, the CAR-T cells comprise a CAR comprising an antigen-binding domain, a transmembrane, and one or more signaling domains. In some embodiments, the CAR also comprises a linker. In some embodiments, the CAR comprises a CD19 antigen-binding domain. In some embodiments, the CAR comprises a CD28 or CD8α transmembrane domain. In some embodiments, the CAR comprises a CD8α signal peptide. In some embodiments, the CAR comprises the Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15). In some embodiments, the antigen-binding domain of the CAR is selected from the group including, but not limited to, (a) an antigen-binding domain that targets an antigen specific to neoplastic cells, (b) an antigen-binding domain that targets an antigen specific to T cells, (c) an antigen-binding domain that targets an antigen specific to an autoimmune or inflammatory disorder, (d) an antigen-binding domain that targets an antigen specific to senescent cells, (e) an antigen-binding domain that targets an antigen specific to an infectious disease, and (f) an antigen-binding domain that binds to a cell surface antigen of a cell.

[0410] In some embodiments, the CAR further comprises one or more linkers. The scFv format generally comprises two variable domains linked by a flexible peptide sequence or "linker" in either a VH-linker-VL or VL-linker-VH orientation. Any suitable linker known in the art in light of this specification can be used in a CAR. Examples of suitable linkers include, but are not limited to, GS-based linker sequences and the Whitlow linker GSTSGSGKPGSGEGSTKG (SEQ ID NO: 15). In some embodiments, the linker is a GS or gly-ser linker. An exemplary gly-ser polypeptide linker has the amino acid sequence Ser (Gly4Ser) n , and (Gly4Ser) n and / or (Gly4Ser3) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3, i.e., Ser(Gly4Ser)3. In some embodiments, n=4, i.e., Ser(Gly4Ser)4. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary gly-ser polypeptide linker comprises the amino acid sequence Ser(Gly4Ser) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In other embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly4Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly3Ser)n. In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker comprises (Gly3Ser)n. nIn some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In other embodiments, n=5. In yet other embodiments, n=6. Another exemplary gly-ser polypeptide linker is (Gly4Ser3) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary gly-ser polypeptide linker is (Gly3Ser) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In other embodiments, n=5. In yet other embodiments, n=6.

[0411] In some embodiments, the antigen-binding domain is selected from the group including an antibody, an antigen-binding portion or fragment thereof, an scFv, and a Fab. In some embodiments, the antigen-binding domain binds to CD19, CD20, CD22, CD38, CD123, CD138, or BCMA. In some embodiments, the antigen-binding domain is an anti-CD19 scFv, such as, but not limited to, FMC63.

[0412] In some embodiments, the transmembrane domain comprises one selected from the group comprising the transmembrane region of TCRα, TCRβ, TCRζ, CD3ε, CD3γ, CD3δ, CD3ζ, CD4, CD5, CD8α, CD8β, CD9, CD16, CD28, CD45, CD22, CD33, CD34, CD37, CD40, CD40L / CD154, CD45, CD64, CD80, CD86, OX40 / CD134, 4-1BB / CD137, CD154, FcεRIγ, VEGFR2, FAS, FGFR2B, and functional variants thereof.

[0413] In some embodiments, the signaling domain(s) of the CAR comprise costimulatory domain(s). For example, the signaling domain can contain a costimulatory domain. Or, the signaling domain can contain one or more costimulatory domains. In certain embodiments, the signaling domain comprises one costimulatory domain. In other embodiments, the signaling domain comprises multiple costimulatory domains. In some cases, when a CAR comprises two or more costimulatory domains, the two costimulatory domains are not the same. In some embodiments, the costimulatory domain comprises two costimulatory domains that are not the same. In some embodiments, the costimulatory domain enhances cytokine production during T cell activation, CAR-T cell proliferation, and / or CAR-T cell persistence. In some embodiments, the costimulatory domain enhances cytokine production during T cell activation, CAR-T cell proliferation, and / or CAR-T cell persistence.

[0414] As described herein, a fourth-generation CAR may contain an antigen-binding domain, a transmembrane domain, three or four signaling domains, and a domain that induces expression of a cytokine gene upon successful CAR signaling. In some cases, the cytokine gene is an endogenous or exogenous cytokine gene of a low-immunogenic cell. In some cases, the cytokine gene encodes a proinflammatory cytokine. In some embodiments, the proinflammatory cytokine is selected from the group including IL-1, IL-2, IL-9, IL-12, IL-18, TNF, IFN-gamma, and functional fragments thereof. In some embodiments, the domain that induces expression of a cytokine gene upon successful CAR signaling comprises a transcription factor or a functional domain or fragment thereof.

[0415] In some embodiments, the CAR comprises a CD3 zeta (CD3ζ) domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof. In some embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; and (ii) a CD28 domain or a 4-1BB domain, or a functional variant thereof. In other embodiments, the CAR comprises (i) a CD3 zeta domain or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof, (ii) a CD28 domain, or a functional variant thereof, and (iii) a 4-1BB domain or a CD134 domain, or a functional variant thereof. In certain embodiments, the CAR comprises (i) a CD3 zeta domain, or an immunoreceptor tyrosine-based activation motif (ITAM), or a functional variant thereof; (ii) a CD28 domain, or a functional variant thereof; (iii) a 4-1BB domain, or a CD134 domain, or a functional variant thereof; and (iv) a cytokine or costimulatory ligand transgene. In some embodiments, the CAR comprises (i) an anti-CD19 scFv, (ii) a CD8α hinge and transmembrane domain, or a functional variant thereof, (iii) a 4-1BB costimulatory domain, or a functional variant thereof, and (iv) a CD3ζ signaling domain, or a functional variant thereof.

[0416] Methods for introducing CAR constructs or producing CAR-T cells are well known to those skilled in the art. Detailed descriptions can be found, for example, in Vormittag et al., Curr Opin Biotechnol., 2018, 53, 162-181, and Eyquem et al., Nature, 2017, 543, 113-117.

[0417] In some embodiments, the cells derived from the primary T cells comprise reduced expression of an endogenous T cell receptor, e.g., by disruption of an endogenous T cell receptor gene (e.g., the T cell receptor alpha constant region ("TRAC") or the T cell receptor beta constant region ("TRB"). In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the disrupted T cell receptor gene. In some embodiments, the exogenous nucleic acid encoding a polypeptide is inserted at the TRAC or TRB locus.

[0418] In some embodiments, cells derived from primary T cells comprise reduced expression of cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and / or programmed cell death (PD1). Methods for reducing or eliminating expression of CTLA4, PD1, or both CTLA4 and PD1 can include any of those recognized by those skilled in the art, such as, but not limited to, genetic modification techniques utilizing rare-cutting endonucleases, and RNA silencing or RNA interference techniques. Non-limiting examples of rare-cutting endonucleases include any Cas protein, TALEN, zinc finger nuclease, meganuclease, and / or homing endonuclease. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted at the CTLA4 and / or PD1 locus. In some embodiments, the cells are modified or engineered compared to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, wild-type cells or control cells are the starting material. In some embodiments, the starting material is primary cells collected from a donor. In some embodiments, the starting material is primary blood cells collected from a donor, for example, via leukopaque. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate engineered cells. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cells using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carries the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cells using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cells using a lentiviral-based viral vector.

[0419] In some embodiments, the CD47 transgene is inserted into a preselected locus of the cell. In some embodiments, the CD47 transgene is inserted into a random locus of the cell. In some embodiments, the transgene encoding a CAR is inserted into a preselected locus of the cell. In some embodiments, the transgene encoding a CAR is inserted into a random locus of the cell. In certain embodiments, the CD47 transgene and the transgene encoding a CAR are inserted into a preselected locus of the cell. In some embodiments, the transgene encoding a CAR is inserted into a random or preselected locus of the cell, comprising a safe harbor locus, via viral vector transduction / integration. In some embodiments, the CD47 transgene and the transgene encoding a CAR are inserted into a random or preselected locus of the cell, comprising a safe harbor locus, via viral vector transduction / integration. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with the vesicular stomatitis VSV-G envelope. In some embodiments, the transgene encoding a CAR is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell using a lentivirus-based viral vector. Random and / or preselected loci can be safe harbor or target loci. Non-limiting examples of safe harbor loci include, but are not limited to, the CCR5 locus, the PPP1R12C (also known as AAVS1) locus, the CLYBL locus, and the Rosa locus (e.g., the ROSA26 locus). Non-limiting examples of target loci include, but are not limited to, the CXCR4 locus, the albumin locus, the SHS231 locus, the F3 locus (also known as CD142), the MICA locus, the MICB locus, the LRP1 locus (also known as the CD91 locus), the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus.The CD47 transgene may be inserted into intron 1 or 2 of PPP1R12C (i.e., AAVS1) or CCR5. The CD47 transgene may be inserted into exon 1, 2, or 3 of CCR5. The CD47 transgene may be inserted into intron 2 of CLYBL. The CD47 transgene may be inserted into a 500 bp window in Ch-4:58,976,613 (i.e., SHS231). The CD47 transgene may be inserted into any suitable region of the aforementioned safe harbor or target locus that allows for expression of the exogenous polynucleotide, including, for example, an intron, exon, or coding sequence region within the safe harbor or target locus. In some embodiments, the preselected locus is selected from the group consisting of the B2M locus, the CIITA locus, the TRAC locus, and the TRB locus. In some embodiments, the preselected locus is the B2M locus. In some embodiments, the preselected locus is the CIITA locus. In some embodiments, the preselected locus is the TRAC locus. In some embodiments, the preselected locus is the TRB locus. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, for example, using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentiviral-based viral vector.

[0420] In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted at the same locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted at different loci. In many cases, the CD47 transgene is inserted at a safe harbor or target locus. In many cases, the transgene encoding the CAR is inserted at a safe harbor or target locus. In some cases, the CD47 transgene is inserted at the B2M locus. In some cases, the transgene encoding the CAR is inserted at the B2M locus. In certain cases, the CD47 transgene is inserted at the CIITA locus. In certain cases, the transgene encoding the CAR is inserted at the CIITA locus. In certain cases, the CD47 transgene is inserted at the TRAC locus. In certain cases, the transgene encoding the CAR is inserted at the TRAC locus. In many other cases, the CD47 transgene is inserted at the TRB locus. In many other cases, the transgene encoding the CAR is inserted at the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into a safe harbor or target locus (e.g., the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the albumin locus, the SHS231 locus, the CLYBL locus, the Rosa locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus).

[0421] In certain embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into a safe harbor or target locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into a safe harbor or target locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into a safe harbor or target locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the TRAC locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the TRAC locus. In certain embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the TRAC locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the TRB locus. In some embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the TRB locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the B2M locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the B2M locus. In other embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the B2M locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are inserted into the CIITA locus. In various embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by a single promoter and inserted into the CIITA locus.In various embodiments, the CD47 transgene and the transgene encoding the CAR are controlled by their own promoters and inserted into the CIITA locus. In some cases, the promoter controlling expression of any of the described transgenes is a constitutive promoter. In other cases, the promoter for any of the described transgenes is an inducible promoter. In some embodiments, the promoter is an EF1α promoter. In some embodiments, the promoter is a CAG promoter. In some embodiments, the CD47 transgene and the transgene encoding the CAR are both controlled by constitutive promoters. In some embodiments, the CD47 transgene and the transgene encoding the CAR are both controlled by inducible promoters. In some embodiments, the CD47 transgene is controlled by a constitutive promoter and the transgene encoding the CAR is controlled by an inducible promoter. In some embodiments, the CD47 transgene is controlled by an inducible promoter and the transgene encoding the CAR is controlled by a constitutive promoter. In various embodiments, the CD47 transgene is controlled by an EF1α promoter and the transgene encoding the CAR is controlled by an EF1α promoter. In some embodiments, the CD47 transgene is controlled by a CAG promoter and the transgene encoding the CAR is controlled by a CAG promoter. In some embodiments, the CD47 transgene is controlled by a CAG promoter and the transgene encoding the CAR is controlled by an EF1α promoter. In some embodiments, the CD47 transgene is controlled by an EF1α promoter and the transgene encoding the CAR is controlled by a CAG promoter. In some embodiments, the expression of both the CD47 transgene and the transgene encoding the CAR is controlled by a single EF1α promoter. In some embodiments, the expression of both the CD47 transgene and the transgene encoding the CAR is controlled by a single CAG promoter.

[0422] In another embodiment, the disclosure disclosed herein is directed to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., hypoimmune (HIP) T cells), and primary T cells that overexpress CD47 (e.g., exogenously express CD47 protein), have reduced or absent expression of MHC class I and / or MHC class II human leukocyte antigens, and have reduced or absent expression of the T cell receptor (TCR) complex. In some embodiments, the hypoimmune (HIP) T cells and primary T cells overexpress CD47 (e.g., exogenously express CD47 protein), have reduced or absent expression of MHC class I and / or MHC class II human leukocyte antigens, and have reduced or absent expression of the T cell receptor (TCR) complex.

[0423] In some embodiments, pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), differentiated cells derived from such pluripotent stem cells (e.g., hypoimmune (HIP) T cells), and primary T cells overexpress CD47 and comprise a genomic modification in the B2M gene. In some embodiments, pluripotent stem cells, differentiated cells derived from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise a genomic modification in the CIITA gene. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise a genomic modification in the TRAC gene. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise a genomic modification in the TRB gene. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise one or more genomic modifications selected from the group consisting of B2M, CIITA, TRAC, and TRB genes. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise genomic modifications in B2M, CIITA, and TRAC genes. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise genomic modifications in B2M, CIITA, and TRB genes. In some embodiments, pluripotent stem cells, T cells differentiated from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise genomic modifications in B2M, CIITA, TRAC, and TRB genes. In certain embodiments, pluripotent stem cells, differentiated cells derived from such pluripotent stem cells, and primary T cells overexpress CD47 and comprise genomic modifications in B2M, CIITA, TRAC, and TRB genes. - / - , C.I.T.A. - / - ,TRAC - / - In certain embodiments, the cells are B2M, CD47tg cells. - / - , C.I.T.A. - / - , TRB - / - In certain embodiments, the cells are B2M, CD47tg cells. - / - , C.I.T.A. - / - ,TRAC - / - , TRB - / - In some embodiments, the cells are B2M, CD47tg cells.インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル In some embodiments, the cells are B2M, CD47tg cells. インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル In some embodiments, the cells are B2M, CD47tg cells. インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , TRB インデル / インデル , CD47tg cells. In some embodiments, the engineered or modified cells described are pluripotent stem cells, T cells differentiated from such pluripotent stem cells, or primary T cells. Non-limiting examples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, T follicular helper (Tfh) cells, cytotoxic T lymphocytes (CTLs), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue tolerance memory (Trm) cells, virtual memory T cells, natural memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cell. In some embodiments, the cells are modified or engineered relative to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is primary cells collected from a donor. In some embodiments, the starting material is primary blood cells collected from a donor, for example, via leukopaque. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate engineered cells.

[0424] In some embodiments, the CD47 transgene is inserted into a preselected locus of the cell. The preselected locus can be a safe harbor or a target locus. Non-limiting examples of safe harbor or target loci include the CCR5 locus, CXCR4 locus, PPP1R12C locus, albumin locus, SHS231 locus, CLYBL locus, Rosa locus, F3 (CD142) locus, MICA locus, MICB locus, LRP1 (CD91) locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus, and KDM5D locus. In some embodiments, the preselected locus is the TRAC locus. In some embodiments, the CD47 transgene is inserted into a safe harbor or target locus (e.g., the CCR5 locus, the CXCR4 locus, the PPP1R12C locus, the albumin locus, the SHS231 locus, the CLYBL locus, the ROSA locus, the F3 (CD142) locus, the MICA locus, the MICB locus, the LRP1 (CD91) locus, the HMGB1 locus, the ABO locus, the RHD locus, the FUT1 locus, and the KDM5D locus). In certain embodiments, the CD47 transgene is inserted into the B2M locus. In certain embodiments, the CD47 transgene is inserted into the B2M locus. In certain embodiments, the CD47 transgene is inserted into the TRAC locus. The CD47 transgene is inserted into a locus. In certain embodiments, the CD47 transgene is inserted into the TRB locus. In some embodiments, the CD47 transgene is inserted into a preselected locus of a cell, including a safe harbor locus, via viral vector transduction / integration. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with the vesicular stomatitis VSV-G envelope. In some embodiments, the CD47 transgene is inserted into at least one allele of a cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell using a lentiviral-based viral vector.

[0425] In some cases, expression of the CD47 transgene is controlled by a constitutive promoter. In other cases, expression of the CD47 transgene is controlled by an inducible promoter. In some embodiments, the promoter is an EF1 alpha (EF1α) promoter. In some embodiments, the promoter is a CAG promoter.

[0426] In yet another embodiment, the disclosure disclosed herein is directed to pluripotent stem cells (e.g., pluripotent stem cells and induced pluripotent stem cells (iPSCs)), T cells derived from such pluripotent stem cells (e.g., hypoimmune (HIP) T cells), and primary T cells that have reduced or absent expression of MHC class I and / or MHC class II human leukocyte antigens and reduced or absent expression of the T cell receptor (TCR) complex. In some embodiments, the cells have reduced or absent expression of MHC class I antigens, MHC class II antigens, and the TCR complex.

[0427] In some embodiments, the pluripotent stem cells (e.g., iPSCs), differentiated cells derived therefrom (e.g., T cells differentiated therefrom), and primary T cells comprise a genomic modification in the B2M gene. In some embodiments, the pluripotent stem cells (e.g., iPSCs), differentiated cells derived therefrom (e.g., T cells differentiated therefrom), and primary T cells comprise a genomic modification in the CIITA gene. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated therefrom, and primary T cells comprise a genomic modification in the TRAC gene. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated therefrom, and primary T cells comprise a genomic modification in the TRB gene. In some embodiments, the pluripotent stem cells (e.g., iPSCs), T cells differentiated therefrom, and primary T cells comprise one or more genomic modifications selected from the group consisting of the B2M, CIITA, and TRAC genes. In some embodiments, pluripotent stem cells (e.g., iPSCs), T cells differentiated therefrom, and primary T cells comprise one or more genomic modifications selected from the group consisting of B2M, CIITA, and TRB genes. In some embodiments, pluripotent stem cells (e.g., iPSCs), T cells differentiated therefrom, and primary T cells comprise one or more genomic modifications selected from the group consisting of B2M, CIITA, TRAC, and TRB genes. In certain embodiments, cells, including iPSCs, T cells differentiated therefrom, and primary T cells, comprise one or more genomic modifications selected from the group consisting of B2M, CIITA, TRAC, and TRB genes. - / - , C.I.T.A. - / - ,TRAC - / - In certain embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are B2M - / - , C.I.T.A. - / - , TRB - / - In some embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデルIn some embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are B2M インデル / インデル , C.I.T.A. インデル / インデル , TRB インデル / インデル In some embodiments, the cells, including iPSCs, T cells differentiated therefrom, and primary T cells, are B2M インデル / インデル , C.I.T.A. インデル / インデル ,TRAC インデル / インデル , TRB インデル / インデル In some embodiments, the modified cells described are pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from such pluripotent stem cells and induced pluripotent stem cells, or primary T cells. Non-limiting examples of primary T cells include CD3+ T cells, CD4+ T cells, CD8+ T cells, naive T cells, regulatory T (Treg) cells, non-regulatory T cells, Th1 cells, Th2 cells, Th9 cells, Th17 cells, T follicular helper (Tfh) cells, cytotoxic T lymphocytes (CTLs), effector T (Teff) cells, central memory T (Tcm) cells, effector memory T (Tem) cells, effector memory T cells expressing CD45RA (TEMRA cells), tissue tolerance memory (Trm) cells, virtual memory T cells, natural memory T cells, memory stem cells (Tsc), γδ T cells, and any other subtype of T cell. In some embodiments, the cells are modified or engineered relative to wild-type or control cells, including unaltered or unmodified wild-type or control cells. In some embodiments, the wild-type or control cells are the starting material. In some embodiments, the starting material is primary cells collected from a donor. In some embodiments, the starting material is primary blood cells collected from a donor, for example, via leukopaque. In some embodiments, the starting material is otherwise modified or engineered to have altered expression of one or more genes to generate engineered cells.

[0428] The cells of the present disclosure have reduced or absent expression of MHC class I antigens, MHC class II antigens, and / or TCR complexes. Reduced MHC I and / or MHC II expression can be achieved, for example, by one or more of the following: (1) direct targeting of polymorphic HLA alleles (HLA-A, HLA-B, HLA-C) and MHC-II genes, (2) ablation of B2M to block surface trafficking of all MHC-I molecules, (3) ablation of CIITA to block surface trafficking of all MHC-II molecules, and / or (4) deletion of components of the MHC enhanceosome, such as LRC5, RFX5, RFXANK, RFXAP, IRF1, NF-Y (including NFY-A, NFY-B, NFY-C), and CIITA, which are essential for HLA expression.

[0429] In some embodiments, HLA expression is disrupted by targeting individual HLAs (e.g., knocking out, knocking down, or reducing expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and / or HLA-DR), targeting transcriptional regulators of HLA expression (e.g., knocking out, knocking down, or reducing expression of NLRC5, CIITA, RFX5, RFXAP, RFXANK, NFY-A, NFY-B, NFY-C, and / or IRF-1), blocking surface trafficking of MHC class I molecules (e.g., knocking out, knocking down, or reducing expression of B2M and / or TAP1), and / or targeting with HLA-Razor (see, e.g., WO2016183041).

[0430] In some embodiments, the cells disclosed herein, including but not limited to pluripotent stem cells, induced pluripotent stem cells, differentiated cells derived from such stem cells, and primary T cells, do not express one or more human leukocyte antigens corresponding to MHC-I and / or MHC-II (e.g., HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, and / or HLA-DR), and are therefore characterized as being hypoimmunogenic. For example, in certain embodiments, the disclosed pluripotent stem cells and induced pluripotent stem cells have been modified such that the stem cells or differentiated stem cells prepared therefrom do not express or exhibit reduced expression of one or more of the following MHC-I molecules: HLA-A, HLA-B, and HLA-C. In some embodiments, one or more of HLA-A, HLA-B, and HLA-C can be "knocked out" from the cells. Cells in which the HLA-A, HLA-B, and / or HLA-C genes have been knocked out can exhibit reduced or eliminated expression of the respective gene that has been knocked out.

[0431] In some embodiments, the guide RNA, shRNA, siRNA or miRNA that targets the conserved region in HLA gene and allows simultaneous deletion of all MHC class I alleles is identified as HLA Razor.In some embodiments, gRNA is part of CRISPR system.In alternative embodiments, gRNA is part of TALEN system.In some embodiments, the HLA Razor that targets the identified conserved region in HLA is described in WO2016183041.In some embodiments, multiple HLA Razors that target the identified conserved region in HLA are utilized.It is generally understood that any guide, siRNA, shRNA or miRNA molecule that targets the conserved region in HLA can act as HLA Razor.

[0432] The provided methods are useful for inactivating or eliminating MHC class I and / or MHC class II expression in cells, including, but not limited to, pluripotent stem cells, differentiated cells, and primary T cells. In some embodiments, genome editing techniques utilizing rare-cutting endonucleases (e.g., CRISPR / Cas, TALEN, zinc finger nucleases, meganucleases, and homing endonuclease systems) are also used to reduce or eliminate expression of genes involved in immune responses in cells (e.g., by deleting genomic DNA of genes involved in immune responses or by inserting genomic DNA into such genes so that gene expression is affected). In certain embodiments, genome editing techniques or other gene regulation techniques are used to insert tolerance-inducing factors in human cells, thereby rendering them and differentiated cells prepared therefrom hypoimmunogenic. In this way, hypoimmunogenic cells have reduced or eliminated expression of MHC I and MHC II expression. In some embodiments, the cells are non-immunogenic (e.g., do not induce innate and / or adaptive immune responses) in recipient subjects.

[0433] In some embodiments, the cells comprise a modification to increase expression of CD47 and one or more factors selected from the group consisting of DUX4, CD24, CD27, CD35, CD46, CD55, CD59, CD200, HLA-C, HLA-E, HLA-E heavy chain, HLA-G, PD-L1, IDO1, CTLA4-Ig, C1-inhibitor, IL-10, IL-35, IL-39, FasL, CCL21, CCL22, Mfge8, CD16, CD52, H2-M3, CD16 Fc receptor, IL15-RF, and / or serpin b9.

[0434] In some embodiments, the cells comprise genomic modifications of one or more target polynucleotide sequences that control the expression of either an MHC class I molecule, an MHC class II molecule, or both an MHC class I and an MHC class II molecule. In some embodiments, a gene editing system is used to modify the one or more target polynucleotide sequences. In some embodiments, the target polynucleotide sequences are one or more selected from the group including B2M, CIITA, and NLRC5. In some embodiments, the cells comprise a gene editing modification to the B2M gene. In some embodiments, the cells comprise a gene editing modification to the CIITA gene. In some embodiments, the cells comprise a gene editing modification to the NLRC5 gene. In some embodiments, the cells comprise a gene editing modification to the B2M and CIITA genes. In some embodiments, the cells comprise a gene editing modification to the B2M and NLRC5 genes. In some embodiments, the cells comprise a gene editing modification to the CIITA and NLRC5 genes. In many embodiments, the cells comprise a gene editing modification to the B2M, CIITA, and NLRC5 genes. In certain embodiments, the genome of the cell is altered to reduce or delete key components of HLA expression. In some embodiments, the cell is modified or engineered compared to a wild-type or control cell, including an unaltered or unmodified wild-type cell or control cell. In some embodiments, the wild-type cell or control cell is the starting material. In some embodiments, the starting material is a primary cell collected from a donor. In some embodiments, the starting material is a primary blood cell collected from a donor, for example, via leukopaque. In some embodiments, the starting material is also modified or engineered to have altered expression of one or more genes to generate engineered cells.

[0435] In some embodiments, the present disclosure provides cells (e.g., stem cells, induced pluripotent stem cells, differentiated cells, e.g., primary NK cells, CAR-NK cells, primary T cells, or CAR-T cells) or populations thereof comprising a genome that has been edited to delete a continuous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cells or populations thereof. In certain embodiments, the present disclosure provides cells (e.g., stem cells, induced pluripotent stem cells, differentiated cells, e.g., primary NK cells, CAR-NK cells, primary T cells, or CAR-T cells) or populations thereof comprising a genome that has been edited to delete a continuous stretch of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cells or populations thereof. In many embodiments, the disclosure provides cells (e.g., stem cells, induced pluripotent stem cells, differentiated cells, hematopoietic stem cells, primary T cells, or CAR-T cells) or populations thereof comprising a genome in which one or more genes have been edited to delete continuous stretches of genomic DNA, thereby reducing or eliminating surface expression of MHC class I and II molecules in the cells or populations thereof.

[0436] In certain embodiments, expression of MHC I and / or MHC II molecules is regulated by targeted deletion of continuous stretches of genomic DNA, thereby reducing or eliminating expression of a target gene selected from the group consisting of B2M, CIITA, and NLRC5. In some embodiments, described herein are gene-edited cells (e.g., modified human cells) comprising an exogenous CD47 protein and an inactivated or modified CIITA gene sequence, and in some cases, an additional genetic modification that inactivates or modifies the B2M gene sequence. In some embodiments, described herein are gene-edited cells comprising an exogenous CD47 protein and an inactivated or modified CIITA gene sequence, and in some cases, an additional genetic modification that inactivates or modifies the NLRC5 gene sequence. In some embodiments, described herein are gene-edited cells comprising an exogenous CD47 protein and an inactivated or modified B2M gene sequence, and in some cases, an additional genetic modification that inactivates or modifies the NLRC5 gene sequence. In some embodiments, described herein are gene-edited cells that include an exogenous CD47 protein and an inactivated or modified B2M gene sequence, and in some cases, additional genetic modifications that inactivate or modify the CIITA and NLRC5 gene sequences.

[0437] Provided herein are cells that exhibit modification of one or more targeting polynucleotide sequences that control expression of any one of the following: (a) MHC I antigen, (b) MHC II antigen, (c) TCR complex, (d) both MHC I and II antigens, and (e) MHC I and II antigens and TCR complex. In certain embodiments, the modification includes increasing expression of CD47. In some embodiments, the cell includes an exogenous or recombinant CD47 polypeptide. In certain embodiments, the modification includes expression of a chimeric antigen receptor. In some embodiments, the cell includes an exogenous or recombinant chimeric antigen receptor polypeptide.

[0438] In some embodiments, the cells include genomic modifications of one or more targeting polynucleotide sequences that control expression of MHC I antigens, MHC II antigens, and / or TCR complexes. In some embodiments, a gene editing system is used to modify the one or more targeting polynucleotide sequences. In some embodiments, the polynucleotide sequences target one or more genes selected from the group consisting of B2M, CIITA, TRAC, and TRB. In certain embodiments, the genome of T cells (e.g., T cells differentiated from hypoimmunogenic iPSCs and primary T cells) has been altered to reduce or delete key components of HLA and TCR expression, such as HLA-A antigens, HLA-B antigens, HLA-C antigens, HLA-DP antigens, HLA-DQ antigens, HLA-DR antigens, TCR-alpha, and TCR-beta.

[0439] In some embodiments, the present disclosure provides a cell or population thereof comprising a genome edited with genes to delete continuous stretches of genomic DNA, thereby reducing or eliminating surface expression of MHC class I molecules in the cell or population thereof. In certain embodiments, the present disclosure provides a cell or population thereof comprising a genome edited with genes to delete continuous stretches of genomic DNA, thereby reducing or eliminating surface expression of MHC class II molecules in the cell or population thereof. In certain embodiments, the present disclosure provides a cell or population thereof comprising a genome edited with genes to delete continuous stretches of genomic DNA, thereby reducing or eliminating surface expression of TCR molecules in the cell or population thereof. In many embodiments, the present disclosure provides a cell or population thereof comprising a genome edited with one or more genes to delete continuous stretches of genomic DNA, thereby reducing or eliminating surface expression of MHC class I and II molecules and TCR complex molecules in the cell or population thereof.

[0440] In some embodiments, the cells and methods described herein include genomically editing a human cell to cleave a CIITA gene sequence and editing the genome of such a cell to alter one or more additional target polynucleotide sequences, such as, but not limited to, B2M, TRAC, and TRB. In some embodiments, the cells and methods described herein include genomically editing a human cell to cleave a B2M gene sequence and editing the genome of such a cell to alter one or more additional target polynucleotide sequences, such as, but not limited to, CIITA, TRAC, and TRB. In some embodiments, the cells and methods described herein include genomically editing a human cell to cleave a TRAC gene sequence and editing the genome of such a cell to alter one or more additional target polynucleotide sequences, such as, but not limited to, B2M, CIITA, and TRB. In some embodiments, the cells and methods described herein include genomically editing a human cell to cleave a TRB gene sequence and editing the genome of such a cell to alter one or more additional target polynucleotide sequences, such as, but not limited to, B2M, CIITA, and TRAC.

[0441] Provided herein are hypoimmunogenic stem cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to wild-type stem cells, wherein the hypoimmunogenic stem cells further comprise a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), wherein the first and / or second exogenous polynucleotides are inserted into a specific locus of at least one allele of the cell. Also provided herein are hypoimmunogenic primary T cells comprising any subtype of primary T cells comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to wild-type primary T cells, wherein the hypoimmunogenic stem cells further comprise a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), wherein the first and / or second exogenous polynucleotides are inserted into a specific locus of at least one allele of the cell. Further provided herein are hypoimmunogenic T cells differentiated from hypoimmunogenic induced pluripotent stem cells that comprise reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to wild-type primary T cells, wherein the hypoimmunogenic stem cells further comprise a set of exogenous polynucleotides comprising a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), and the first and / or second exogenous polynucleotides are inserted into a specific locus of at least one allele of the cell.

[0442] In some embodiments, the described population of engineered cells avoids NK cell-mediated cytotoxicity upon administration to a recipient patient. In some embodiments, the population of engineered cells avoids NK cell-mediated cytotoxicity by one or more subpopulations of NK cells. In some embodiments, the population of engineered cells are protected from cytolysis by NK cells, including immature and / or mature NK cells, after administration to a recipient patient. In some embodiments, the population of engineered cells avoids macrophage phagocytosis upon administration to a recipient patient. In some embodiments, the population of engineered cells do not induce an innate and / or adaptive immune response against the cells upon administration to a recipient patient.

[0443] In some embodiments, the cells described herein comprise a safety switch. As used herein, the term "safety switch" refers to a system for controlling the expression of a gene or protein of interest that, when down- or up-regulated, triggers elimination or death of the cell, for example, through recognition by the host's immune system. Safety switches can be designed to be activated by exogenous molecules in the event of an adverse clinical event. Safety switches can be manipulated by controlling expression at the DNA, RNA, and protein levels. Safety switches include proteins or molecules that allow for the control of cellular activity in response to adverse events. In one embodiment, the safety switch is a "kill switch" that is expressed in an inactivated state; activation of the switch by an externally provided selective agent is lethal to the cell expressing the safety switch. In one embodiment, the safety switch gene is cis-acting relative to the gene of interest in the construct. Activation of the safety switch causes the cell to kill itself alone, or itself and neighboring cells by apoptosis or necrosis. In some embodiments, the cells described herein, for example, stem cells, induced pluripotent stem cells, hematopoietic stem cells, primary cells, or differentiated cells, including but not limited to, T cells, CAR-T cells, NK cells, and / or CAR-NK cells, comprise a safety switch.

[0444] In some embodiments, the safety switch comprises a therapeutic agent that inhibits or blocks the interaction of CD47 and SIRPα. In some aspects, the CD47-SIRPα blocking agent is an agent that neutralizes, blocks, antagonizes, or interferes with cell surface expression of CD47, SIRPα, or both. In some embodiments, the CD47-SIRPα blocking agent inhibits or blocks the interaction of CD47, SIRPα, or both. In some embodiments, the CD47-SIRPα blocking agent (e.g., a CD47-SIRPα blocking, inhibiting, reducing, antagonizing, neutralizing, or interfering agent) comprises an agent selected from the group including an antibody or fragment thereof that binds CD47, a bispecific antibody that binds CD47, an immunocytokine fusion protein that binds CD47, a CD47-containing fusion protein, an antibody or fragment thereof that binds SIRPα, a bispecific antibody that binds SIRPα, an immunocytokine fusion protein that binds SIRPα, a SIRPα-containing fusion protein, and combinations thereof.

[0445] In some embodiments, the cells described herein comprise a "suicide gene" (or "suicide switch") that can cause the death of hypoimmunogenic cells if they are allowed to grow and divide in an undesired manner. The "suicide gene" ablation approach comprises a suicide gene in a gene transfer vector that encodes a protein that results in cell killing only when activated by a specific compound. The suicide gene may encode an enzyme that selectively converts non-toxic compounds into highly toxic metabolites. In some embodiments, the cells described herein, for example, stem cells, induced pluripotent stem cells, hematopoietic stem cells, primary cells, or differentiated cells, including, but not limited to, T cells, CAR-T cells, NK cells, and / or CAR-NK cells, comprise a suicide gene.

[0446] In some embodiments, the populations of engineered cells described elicit reduced levels or no immune activation upon administration to a recipient subject. In some embodiments, the cells elicit reduced levels or no systemic TH1 activation in the recipient subject. In some embodiments, the cells elicit reduced levels or no peripheral blood mononuclear cell (PBMC) immune activation in the recipient subject. In some embodiments, the cells elicit reduced levels or no donor-specific IgG antibodies against the cells upon administration to a recipient subject. In some embodiments, the cells elicit reduced levels or no IgM and IgG antibody production against the cells in the recipient subject. In some embodiments, the cells elicit reduced levels of cytotoxic T cell killing of the cells upon administration to a recipient subject.

[0447] B.CIITA In some embodiments, the technology disclosed herein regulates (e.g., reduces or eliminates) expression of MHC II genes by targeting and modulating (e.g., reducing or eliminating) the expression of class II transactivator (CIITA). In some embodiments, the regulation is achieved using a CRISPR / Cas system. CIITA is a member of the LR, or nucleotide-binding domain (NBD) leucine-rich repeat (LRR) family of proteins, which regulates MHC II transcription by associating with the MHC enhanceosome.

[0448] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of CIITA. In some embodiments, the target polynucleotide sequence is a homolog of CIITA. In some embodiments, the target polynucleotide sequence is an ortholog of CIITA.

[0449] In some embodiments, the reduced expression or elimination of CIITA reduces or eliminates expression of one or more of the following MHC class II: HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0450] In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a CIITA protein. In other words, the cells comprise a genetic modification at the CIITA locus. In some cases, the nucleotide sequence encoding the CIITA protein is set forth in reference sequence number NM_000246.4 and NCBI GenBank number U18259. In some cases, the CIITA locus is set forth in NCBI Gene ID number 4261. In certain cases, the amino acid sequence of CIITA is set forth as NCBI GenBank number AAA88861.1. Additional descriptions of the CIITA protein and locus can be found in Uniprot number P33076, HGNC reference number 7067, and OMIM reference number 600005.

[0451] In some embodiments, the hypoimmunogenic cells outlined herein comprise a genetic modification targeting the CIITA gene. In some embodiments, the genetic modification targeting the CIITA gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the CIITA gene is selected from the group consisting of SEQ ID NOs: 5184-36352 in Table 12 of WO2016183041, which is incorporated herein by reference. In some embodiments, the cells have a reduced ability to induce innate and / or adaptive immune responses in a recipient subject. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted into the CIITA gene.

[0452] Assays for testing whether the CIITA gene is inactivated are known and described herein. In some embodiments, the resulting genetic modification of the CIITA gene can be assayed by PCR, and the reduction in HLA-II expression can be assayed by FACS analysis. In another embodiment, CIITA protein expression is detected using Western blot of cell lysates probed with an antibody against the CIITA protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR). In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using, for example, viral transduction using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with the vesicular stomatitis VSV-G envelope and carrying the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentivirus-based viral vector.

[0453] C.B2M In some embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of MHC-I genes by targeting or modulating (e.g., reducing or eliminating) the expression of the accessory chain B2M. In some embodiments, the modulation is achieved using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) the expression of B2M, surface trafficking of MHC-I molecules is blocked, rendering the cells hypoimmunogenic. In some embodiments, the cells have a reduced ability to induce innate and / or adaptive immune responses in a recipient subject.

[0454] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of B2M. In some embodiments, the target polynucleotide sequence is a homolog of B2M. In some embodiments, the target polynucleotide sequence is an ortholog of B2M.

[0455] In some embodiments, reducing or eliminating the expression of B2M reduces or eliminates the expression of one or more of the following MHC I molecules: HLA-A, HLA-B, and HLA-C.

[0456] In some embodiments, the cells described herein comprise a genetic modification at a locus encoding a B2M protein. In other words, the cells comprise a genetic modification at the B2M locus. In some cases, the nucleotide sequence encoding the B2M protein is set forth in reference SEQ ID NO: NM_004048.4 and GenBank number AB021288.1. In some cases, the B2M locus is set forth in NCBI Gene ID number 567. In certain cases, the amino acid sequence of B2M is set forth as NCBI GenBank number BAA35182.1. Additional descriptions of the B2M protein and locus can be found in Uniprot number P61769, HGNC reference number 914, and OMIM reference number 109700.

[0457] In some embodiments, the hypoimmunogenic cells outlined herein comprise a genetic modification targeting the B2M gene. In some embodiments, the genetic modification targeting the B2M gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein and at least one guide ribonucleic acid sequence for specifically targeting the B2M gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the B2M gene is selected from the group consisting of SEQ ID NOs: 81240-85644 in Table 15 of WO2016183041, which is incorporated herein by reference. In some embodiments, an exogenous nucleic acid encoding a polypeptide as disclosed herein (e.g., a chimeric antigen receptor, CD47, or another tolerogenic factor disclosed herein) is inserted into the B2M gene. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector that carries an exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector that is pseudotyped with vesicular stomatitis VSV-G envelope and carries an exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using a lentiviral-based viral vector.

[0458] Assays for testing whether the B2M gene is inactivated are known and described herein. In some embodiments, the resulting genetic modification of the B2M gene can be assayed by PCR, and the reduction in HLA-I expression can be assayed by FACS analysis. In another embodiment, B2M protein expression is detected using Western blot of cell lysates probed with an antibody against the B2M protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0459] D.NLRC5 In many embodiments, the technology disclosed herein regulates (e.g., reduces or eliminates) the expression of MHC-I genes by targeting or modulating (e.g., reducing or eliminating) the expression of the NLR family, CARD domain-containing 5 / NOD27 / CLR16.1 (NLRC5). In some embodiments, the modulation is achieved using the CRISPR / Cas system. NLRC5 is a key regulator of MHC-I-mediated immune responses; like CIITA, NLRC5 is highly inducible by IFN-γ and can translocate to the nucleus. NLRC5 activates the promoters of MHC-I genes, inducing the transcription of not only MHC-I but also related genes involved in MHC-I antigen presentation.

[0460] In some embodiments, the target polynucleotide sequence is a variant of NLRC5. In some embodiments, the target polynucleotide sequence is a homolog of NLRC5. In some embodiments, the target polynucleotide sequence is an ortholog of NLRC5.

[0461] In some embodiments, reducing or eliminating the expression of NLRC5 reduces or eliminates the expression of one or more of the following MHC I molecules: HLA-A, HLA-B, and HLA-C.

[0462] In some embodiments, the cells described herein comprise a genetic modification targeting the NLRC5 gene. In some embodiments, the genetic modification targeting the NLRC5 gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein and at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the NLRC5 gene is selected from the group consisting of SEQ ID NOs: 36353 to 81239 in Appendix 3 or Table 14 of WO2016183041, the disclosure of which is incorporated by reference in its entirety.

[0463] Assays for testing whether the NLRC5 gene is inactivated are known and described herein. In some embodiments, the resulting genetic modification of the NLRC5 gene can be assayed by PCR, and the reduction in HLA-I expression can be assayed by FACS analysis. In another embodiment, NLRC5 protein expression is detected using Western blot of cell lysates probed with an antibody against the NLRC5 protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0464] E.TRAC In many embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of the TRAC gene TCR gene by targeting or modulating (e.g., reducing or eliminating) the expression of the constant region of the T cell receptor alpha chain. In some embodiments, the modulation is achieved using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) the expression of TRAC, surface trafficking of TCR molecules is blocked. In some embodiments, the cells also have a reduced ability to induce innate and / or adaptive immune responses in a recipient subject.

[0465] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of TRAC. In some embodiments, the target polynucleotide sequence is a homolog of TRAC. In some embodiments, the target polynucleotide sequence is an ortholog of TRAC.

[0466] In some embodiments, reducing or eliminating the expression of TRAC reduces or eliminates the surface expression of a TCR.

[0467] In some embodiments, cells, such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, comprise a genetic modification at a locus encoding a TRAC protein. In other words, the cells comprise a genetic modification at the TRAC locus. In some cases, the nucleotide sequence encoding the TRAC protein is set forth in Genbank number X02592.1. In some cases, the TRAC locus is set forth in reference sequence number NG_001332.3 and NCBI gene ID number 28755. In certain instances, the amino acid sequence of TRAC is set forth as Uniprot number P01848. Additional descriptions of the TRAC protein and locus can be found in Uniprot number P01848, HGNC reference number 12029, and OMIM reference number 186880.

[0468] In some embodiments, the hypoimmunogenic cells described herein comprise a genetic modification targeting the TRAC gene. In some embodiments, the genetic modification targeting the TRAC gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein, and at least one guide ribonucleic acid sequence for specifically targeting the TRAC gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the TRAC gene is selected from the group consisting of SEQ ID NOs: 532-609 and 9102-9797 of US20160348073, which is incorporated herein by reference.

[0469] Assays for testing whether the TRAC gene is inactivated are known and are described herein. In some embodiments, the resulting genetic modification of the TRAC gene can be assayed by PCR, and the reduction in TCR expression can be assayed by FACS analysis. In another embodiment, TRAC protein expression is detected using Western blot of cell lysate probed with an antibody against TRAC protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0470] F.TRB In many embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) expression of TCR genes, including genes encoding the T cell antigen receptor, beta chain (e.g., TRB, TRBC, or TCRB genes), by targeting or modulating (e.g., reducing or eliminating) the expression of the constant region of the T cell receptor beta chain. In some embodiments, the modulation is achieved using a CRISPR / Cas system. By modulating (e.g., reducing or deleting) the expression of TRB, surface trafficking of TCR molecules is blocked. In some embodiments, the cells also have a reduced ability to induce innate and / or adaptive immune responses in a recipient subject.

[0471] In some embodiments, the target polynucleotide sequence of the present disclosure is a variant of TRB. In some embodiments, the target polynucleotide sequence is a homolog of TRB. In some embodiments, the target polynucleotide sequence is an ortholog of TRB.

[0472] In some embodiments, reducing or eliminating expression of a TRB reduces or eliminates surface expression of a TCR.

[0473] In some embodiments, cells, such as, but not limited to, pluripotent stem cells, induced pluripotent stem cells, T cells differentiated from induced pluripotent stem cells, primary T cells, and cells derived from primary T cells, comprise a genetic modification at a locus encoding a TRB protein. In other words, the cells comprise a genetic modification at the TRB locus. In some cases, the nucleotide sequence encoding the TRB protein is set forth in UniProt number P0DSE2. In some cases, the TRB locus is set forth in reference SEQ ID NO: NG_001333.2 and NCBI Gene ID number 6957. In certain cases, the amino acid sequence of TRB is set forth as Uniprot number P01848. Additional descriptions of TRB proteins and loci can be found in GenBank number L36092.2, Uniprot number P0DSE2, and HGNC reference number 12155.

[0474] In some embodiments, the hypoimmunogenic cells described herein comprise a genetic modification targeting the TRB gene. In some embodiments, the genetic modification targeting the TRB gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein and at least one guide ribonucleic acid sequence for specifically targeting the TRB gene. In some embodiments, the at least one guide ribonucleic acid sequence for specifically targeting the TRB gene is selected from the group consisting of SEQ ID NOs: 610-765 and 9798-10532 of US20160348073, which is incorporated herein by reference.

[0475] Assays for testing whether the TRB gene is inactivated are known and are described herein. In some embodiments, the resulting genetic modification of the TRB gene can be assayed by PCR, and the reduction in TCR expression can be assayed by FACS analysis. In another embodiment, TRB protein expression is detected using Western blot of cell lysate probed with an antibody against the TRB protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0476] G.CD142 In many embodiments, the technology disclosed herein modulates (e.g., reduces or eliminates) the expression of CD142, also known as tissue factor, factor III, and F3. In some embodiments, modulation is achieved using a gene editing system (e.g., CRISPR / Cas).

[0477] In some embodiments, the target polynucleotide sequence is CD142 or a variant of CD142. In some embodiments, the target polynucleotide sequence is a homolog of CD142. In some embodiments, the target polynucleotide sequence is an ortholog of CD142.

[0478] In some embodiments, the cells described herein comprise a genetic modification targeting the CD142 gene. In some embodiments, the genetic modification targeting the CD142 gene by a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein, and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the CD142 gene. Useful methods for identifying gRNA sequences targeting CD142 are described below.

[0479] Assays for testing whether the CD142 gene is inactivated are known and described herein. In some embodiments, the resulting genetic modification of the CD142 gene can be assayed by PCR, and the reduction of CD142 expression can be assayed by FACS analysis. In another embodiment, CD142 protein expression is detected using Western blot of cell lysate probed with an antibody against CD142 protein. In another embodiment, the presence of the inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0480] Useful genome, polynucleotide, and polypeptide information for human CD142 is provided, for example, under GeneCard identifier GC01M094530, HGNC number 3541, NCBI gene ID 2152, NCBI reference sequence numbers NM_001178096.1, NM_001993.4, NP_001171567.1, and NP_001984.1, UniProt number P13726, etc.

[0481] H.CTLA-4 In some embodiments, the target polynucleotide sequence is CTLA-4 or a variant of CTLA-4. In some embodiments, the target polynucleotide sequence is a homolog of CTLA-4. In some embodiments, the target polynucleotide sequence is an ortholog of CTLA-4.

[0482] In some embodiments, the cells outlined herein comprise a genetic modification targeting the CTLA-4 gene. In certain embodiments, primary T cells comprise a genetic modification targeting the CTLA-4 gene. The genetic modification may reduce expression of CTLA-4 polynucleotides and CTLA-4 polypeptides in T cells, including primary T cells and CAR-T cells. In some embodiments, the genetic modification targeting the CTLA-4 gene using a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding the Cas protein and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the CTLA-4 gene. Useful methods for identifying gRNA sequences targeting CTLA-4 are described below.

[0483] Assays for testing whether the CTLA-4 gene is inactivated are known and are described herein. In some embodiments, the resulting genetic modification of the CTLA-4 gene can be assayed by PCR, and the reduction of CTLA-4 expression can be assayed by FACS analysis. In another embodiment, CTLA-4 protein expression is detected using Western blot of cell lysate probed with an antibody against CTLA-4 protein. In another embodiment, the presence of an inactivating genetic modification is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0484] Useful genomic, polynucleotide, and polypeptide information for human CTLA-4 is provided, for example, under GeneCard identifier GC02P203867, HGNC number 2505, NCBI gene ID 1493, NCBI reference sequence numbers NM_005214.4, NM_001037631.2, NP_001032720.1, and NP_005205.2, UniProt number P16410, etc.

[0485] I.PD-1 In some embodiments, the target polynucleotide sequence is PD-1 or a variant of PD-1. In some embodiments, the target polynucleotide sequence is a homolog of PD-1. In some embodiments, the target polynucleotide sequence is an ortholog of PD-1.

[0486] In some embodiments, the cells outlined herein comprise a gene encoding the programmed cell death protein 1 (PD-1) protein or a genetic modification targeting the PDCD1 gene. In certain embodiments, primary T cells comprise a genetic modification targeting the PDCD1 gene. The genetic modification may reduce expression of PD-1 polynucleotides and PD-1 polypeptides in T cells, including primary T cells and CAR-T cells. In some embodiments, the genetic modification targeting the PDCD1 gene with a rare-cutting endonuclease comprises a Cas protein or a polynucleotide encoding a Cas protein and at least one guide ribonucleic acid (gRNA) sequence for specifically targeting the PDCD1 gene. Useful methods for identifying gRNA sequences targeting PD-1 are described below.

[0487] Assays for testing whether the PDCD1 gene is inactivated are known and described herein. In some embodiments, the resulting genetic alteration of the PDCD1 gene can be assayed by PCR, and the reduction in PD-1 expression can be assayed by FACS analysis. In another embodiment, PD-1 protein expression is detected using Western blot of cell lysates probed with an antibody against PD-1 protein. In another embodiment, the presence of the inactivating genetic alteration is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0488] Useful genomic, polynucleotide, and polypeptide information for human PD-1, including the PDCD1 gene, is provided, for example, under GeneCard identifier GC02M241849, HGNC number 8760, NCBI gene ID 5133, Uniprot number Q15116, and NCBI reference sequence numbers NM_005018.2 and NP_005009.2.

[0489] J.CD47 In some embodiments, the present disclosure provides a cell or population thereof modified to express a tolerogenic factor (e.g., an immunomodulatory polypeptide) CD47. In some embodiments, the present disclosure provides a method for altering the genome of a cell to express CD47. In some embodiments, the stem cell expresses exogenous CD47. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD47 polypeptide. In some embodiments, the cell is genetically modified using homology-directed repair to contain an integrated exogenous polynucleotide encoding CD47. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide such that the nucleotide sequence is inserted into at least one allele of a safe harbor or target locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted into at least one allele of the AAVS1 locus. In some cases, the cell expresses a nucleotide sequence encoding a human CD47 polypeptide, wherein the nucleotide sequence is inserted into at least one allele of the CCR5 locus. In some cases, the cells express a nucleotide sequence encoding a human CD47 polypeptide, and the nucleotide sequence is inserted into at least one allele of a safe harbor or target locus, for example, but not limited to, the CCR5 locus, CXCR4 locus, PPP1R12C locus, albumin locus, SHS231 locus, CLYBL locus, ROSA locus, F3 (CD142) locus, MICA locus, MICB locus, LRP1 (CD91) locus, HMGB1 locus, ABO locus, RHD locus, FUT1 locus, and KDM5D locus. In some cases, the cells express a nucleotide sequence encoding a human CD47 polypeptide, and the nucleotide sequence is inserted into at least one allele of the TRAC locus.

[0490] CD47 is a leukocyte surface antigen that plays a role in cell adhesion and regulation of integrins. It is expressed on the surface of cells and signals circulating macrophages to prevent them from phagocytosing the cells.

[0491] In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in NCBI reference SEQ ID NOs: NP_001768.1 and NP_942088.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence set forth in NCBI reference SEQ ID NOs: NP_001768.1 and NP_942088.1. In some embodiments, the cells comprise a nucleotide sequence for CD47 having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the sequence set forth in NCBI reference numbers NM_001777.3 and NM_198793.2. In some embodiments, the cells comprise a nucleotide sequence for CD47 set forth in NCBI reference numbers NM_001777.3 and NM_198793.2. In some embodiments, the nucleotide sequence encoding the CD47 polynucleotide is a codon-optimized sequence. In some embodiments, the nucleotide sequence encoding the CD47 polynucleotide is a human codon-optimized sequence.

[0492] In some embodiments, the cells comprise a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in NCBI reference SEQ ID NOs: NP_001768.1 and NP_942088.1. In some embodiments, the cells outlined herein comprise a CD47 polypeptide having the amino acid sequence set forth in NCBI reference SEQ ID NOs: NP_001768.1 and NP_942088.1.

[0493] Exemplary amino acid sequences of human CD47 with and without the signal sequence are provided in Table 1.

[0494] [Table 1]

[0495] In some embodiments, the cells comprise a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the cells comprise a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the cells comprise a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the cells comprise a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 14.

[0496] In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 13. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99%, or more) to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the cell comprises a nucleotide sequence encoding a CD47 polypeptide having the amino acid sequence of SEQ ID NO: 14. In some embodiments, the nucleotide sequence is codon-optimized for expression in a particular cell.

[0497] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system, or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CD47 into a genomic locus of a hypoimmunogenic cell. In some cases, the polynucleotide encoding CD47 is inserted into a safe harbor or target locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding CD47 is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, the polynucleotide encoding CD47 is inserted into any one of the loci set forth in Table 15 provided herein. In certain embodiments, the polynucleotide encoding CD47 is operably linked to a promoter.

[0498] In some embodiments, a polynucleotide encoding CD47 is inserted into at least one allele of a T cell using viral transduction. In some embodiments, a polynucleotide encoding CD47 is inserted into at least one allele of a T cell using a lentiviral-based viral vector. In some embodiments, the lentiviral-based viral vector is a pseudotyped, self-inactivating lentiviral vector carrying a polynucleotide encoding CD47. In some embodiments, the lentiviral-based viral vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carrying a polynucleotide encoding CD47.

[0499] In another embodiment, CD47 protein expression is detected using Western blots of cell lysates probed with an antibody against CD47 protein, hi another embodiment, the presence of exogenous CD47 mRNA is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0500] K.CD24 In some embodiments, the present disclosure provides cells or populations thereof modified to express a tolerogenic factor (e.g., an immunomodulatory polypeptide) CD24. In some embodiments, the present disclosure provides methods for altering the genome of a cell to express CD24. In some embodiments, a stem cell expresses exogenous CD24. In some cases, the cell expresses an expression vector comprising a nucleotide sequence encoding a human CD24 polypeptide. In some embodiments, an exogenous polynucleotide is inserted into at least one allele of a cell using viral transduction, for example, with a vector. In some embodiments, the vector is a pseudotyped self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with a vesicular stomatitis VSV-G envelope and carries the exogenous polynucleotide. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell using viral transduction. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of a cell using a lentiviral-based viral vector.

[0501] CD24, also known as heat-stable antigen or small cell lung cancer cluster 4 antigen, is a glycosylated glycosylphosphatidylinositol-anchored surface protein (Pirruccello et al., J Immunol, 1986, 136, 3779-3784; Chen et al., Glycobiology, 2017, 57, 800-806). It binds to Siglec-10 on innate immune cells. Recently, CD24, via Siglec-10, has been shown to act as an innate immune checkpoint (Barkal et al., Nature, 2019, 572, 392-396).

[0502] In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide having at least 95% sequence identity (e.g., 95%, 96%, 97%, 98%, 99% or more) to the amino acid sequence set forth in NCBI reference numbers NP_001278666.1, NP_001278667.1, NP_001278668.1, and NP_037362.1. In some embodiments, the cells outlined herein comprise a nucleotide sequence encoding a CD24 polypeptide having the amino acid sequence set forth in NCBI reference numbers NP_001278666.1, NP_001278667.1, NP_001278668.1, and NP_037362.1.

[0503] In some embodiments, the cells comprise a nucleotide sequence having at least 85% sequence identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to a sequence set forth in NCBI reference numbers NM_00129737.1, NM_00129738.1, NM_001291739.1, and NM_013230.3. In some embodiments, the cells comprise a nucleotide sequence set forth in NCBI reference numbers NM_00129737.1, NM_00129738.1, NM_001291739.1, and NM_013230.3.

[0504] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system, or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CD24 into a genomic locus of a hypoimmunogenic cell. In some cases, the polynucleotide encoding CD24 is inserted into a safe harbor or target locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding CD24 is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, the polynucleotide encoding CD24 is inserted into any one of the loci set forth in Table 15 provided herein. In certain embodiments, the polynucleotide encoding CD24 is operably linked to a promoter.

[0505] In another embodiment, CD24 protein expression is detected using Western blots of cell lysates probed with an antibody against CD24 protein, hi another embodiment, the presence of exogenous CD24 mRNA is confirmed using reverse transcriptase polymerase chain reaction (RT-PCR).

[0506] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system, or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding CD24 into a genomic locus of a hypoimmunogenic cell. In some cases, the polynucleotide encoding CD24 is inserted into a safe harbor or target locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (also known as CD142), MICA, MICB, LRP1 (also known as CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding CD24 is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, the polynucleotide encoding CD24 is inserted into any one of the loci set forth in Table 15 provided herein. In certain embodiments, the polynucleotide encoding CD24 is operably linked to a promoter.

[0507] L.DUX4 In some embodiments, the present disclosure provides a cell (e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary T cell, or a CAR-T cell) or population thereof comprising a genome modified to increase expression of a tolerogenic or immunosuppressive factor, such as DUX4. In some embodiments, the present disclosure provides a method for altering the genome of a cell to provide increased expression of DUX4, including via an exogenous polynucleotide. In some embodiments, the present disclosure provides a cell or population thereof comprising exogenously expressed DUX4 protein. In some embodiments, increased expression of DUX4 suppresses, reduces, or eliminates expression of one or more of the following MHC I molecules: HLA-A, HLA-B, and HLA-C. In some embodiments, the exogenous polynucleotide is inserted into at least one allele of the cell using viral transduction, e.g., using a vector. In some embodiments, the vector is a pseudotyped, self-inactivating lentiviral vector carrying the exogenous polynucleotide. In some embodiments, the vector is a self-inactivating lentiviral vector pseudotyped with vesicular stomatitis VSV-G envelope, and carries an exogenous polynucleotide.In some embodiments, the exogenous polynucleotide is inserted into at least one allele of cells using viral transduction.In some embodiments, the exogenous polynucleotide is inserted into at least one allele of cells using lentiviral-based viral vectors.

[0508] DUX4 is a transcription factor active in embryonic tissues and induced pluripotent stem cells but silent in normal, healthy somatic tissues (Feng et al., 2015, ELife4; De Iaco et al., 2017, Nat Genet., 49, 941-945; Hendrickson et al., 2017, Nat Genet., 49, 925-934; Snider et al., 2010, PLoS Genet., e1001181; Whiddon et al., 2017, Nat Genet.). DUX4 expression acts to block IFN-gamma-mediated induction of major histocompatibility complex (MHC) class I gene expression (e.g., B2M, HLA-A, HLA-B, and HLA-C). DUX4 expression has been implicated in the suppression of antigen presentation by MHC class I (Chew et al., Developmental Cell, 2019, 50:1-14). DUX4 functions as a transcription factor in the cleavage-stage gene expression (transcription) program. Its target genes include, but are not limited to, coding genes, non-coding genes, and repetitive elements.

[0509] At least two isoforms of DUX4 exist, the longest of which contains a transcriptional activation domain at the C-terminus of DUX4. These isoforms are generated by alternative splicing. See, e.g., Geng et al., 2012, Dev Cell, 22, 38-51; Snider et al., 2010, PLoS Genet., e1001181. The active isoform of DUX4 contains a DNA-binding domain at its N-terminus and an activation domain at its C-terminus. See, e.g., Choi et al., 2016, Nucleic Acid Res., 44, 5161-5173.

[0510] It has been shown that reducing the number of CpG motifs in DUX4 reduces silencing of the DUX4 transgene (Jagannathan et al., Human Molecular Genetics, 2016, 25(20):4419-4431). The nucleic acid sequence provided in Jagannathan et al. (see above) represents a codon-altered sequence of DUX4 containing one or more base substitutions that reduce the total number of CpG sites while preserving the DUX4 protein sequence. This nucleic acid sequence is commercially available from Addgene (Catalog No. 99281).

[0511] In many embodiments, at least one or more polynucleotides may be utilized to facilitate exogenous expression of DUX4 by a cell, e.g., a stem cell, an induced pluripotent stem cell, a differentiated cell, a hematopoietic stem cell, a primary T cell, or a CAR-T cell.

[0512] In some embodiments, a suitable gene editing system (e.g., a CRISPR / Cas system, or any of the gene editing systems described herein) is used to facilitate insertion of a polynucleotide encoding DUX4 into a genomic locus of a hypoimmunogenic cell. In some cases, the polynucleotide encoding DUX4 is inserted into a safe harbor or target locus, such as, but not limited to, the AAVS1, CCR5, CLYBL, ROSA26, SHS231, F3 (CD142), MICA, MICB, LRP1 (CD91), HMGB1, ABO, RHD, FUT1, or KDM5D locus. In some embodiments, the polynucleotide encoding DUX4 is inserted into the B2M locus, CIITA locus, TRAC locus, or TRB locus. In some embodiments, the polynucleotide encoding DUX4 is inserted into any one of ...

Claims

[Claim 1] 1. An engineered cell comprising reduced expression of HLA-A, HLA-B, HLA-C, HLA-DP, HLA-DQ, HLA-DR, B2M, CIITA, TCR-alpha, and / or TCR-beta compared to a wild type or control cell, said engineered cell further comprising a set of a first exogenous polynucleotide encoding CD47 and a second exogenous polynucleotide encoding a chimeric antigen receptor (CAR), wherein the first and / or second exogenous polynucleotides are inserted into a specific locus of at least one allele of the cell.

Images