Cell type-selective immunoprotection of cells

Abstract

The present disclosure relates to a preparation of one or more cells, wherein the cells of the preparation have been modified to conditionally express (i) an increased level of one or more immune checkpoint proteins relative to corresponding wild-type cells, (ii) a decreased level of one or more HLA-I proteins relative to corresponding wild-type cells, or a combination of (i) and (ii). The disclosure further relates to methods and constructs for making the cell preparation, and methods for administering the cell preparation to a subject in need thereof.

Description

Technical Field

[0001] This application claims the benefit of priority of U.S. Provisional Patent Application No. 62 / 875,883, filed Jul. 18, 2019, the entire disclosure of which is incorporated herein by reference.

[0002] Field of Disclosure The present disclosure relates to methods for selectively inducing immunoprotection of terminally differentiated cells and to cell preparations that can be selectively immunoprotected.

Background Art

[0003] Background The acute phase of transplant rejection can occur within about 1 to 3 weeks and typically involves the action of host T cells on donor tissue due to sensitization of the host system to donor human leukocyte antigen class I (HLA-I) and human leukocyte antigen class II (HLA-II) molecules. In most cases, the trigger antigen is the HLA-I protein. To achieve the best success, non-self donor cells are classified for HLA and matched to the transplant recipient as completely as possible. However, even among family members who can share a high percentage of HLA identity, allogeneic donation often ends in failure. To prevent rejection, allogeneic transplant recipients are often subjected to intensive immunosuppressive therapies that can lead to complications due to opportunistic infections and significant morbidity. Thus, the recognition of non-self HLA-I and non-self HLA-II proteins is a major obstacle in allogeneic cell transplantation and cell replacement therapies.

[0004] The surface expression of HLA-I or HLA-II genes can be regulated by tumor cells and viral pathogens. For example, the downregulation of β2-microglobulin (B2M), which forms a heterodimer with the HLA-I α chain, is a widespread mechanism used by tumor cells to escape the anti-tumor mediated immune response (Nomura et al., "β2-Microglobulin-mediated Signaling as a Target for Cancer Therapy," Anticancer Agents Med Chem. 14(3)343-352(2014) (Non-Patent Document 1), which is hereby incorporated by reference in its entirety). In another example, infection of specific cell types by alpha or beta herpesviruses such as HSV and HCMV results in a decrease in the surface expression of HLA-I and HLA-II complexes via proteasomal degradation of the HLA-I heavy chain and the HLA-II α chain (HLA-DRα and HLA-DMα) (Wiertz et al., "Herpesvirus Interference with Major Histocompatibility Complex Class II-Restricted T-Cell Activation," J. Virology 81(9):4389-4386(2007) (Non-Patent Document 2)).

[0005] Importantly, in the context of non-self cell transplantation, the downregulation or absence of HLA-I and HLA-II molecules on the surface of donor cells can make such cells more susceptible to clearance by the innate immune system. For example, natural killer (NK) cells recognize cells that do not express HLA-I molecules and monitor for infection in the host by inducing apoptosis of the cells. Similarly, macrophages resident in the spleen and liver target self cells that are unable to present "self" proteins for clearance by programmed phagocytosis (Krysoko et al., "Macrophages Regulate the Clearance of Living Cells by Calreticulin," Nature Comm. 9, Article Number: 4644(2018) (Non-Patent Document 3)).

[0006] Another consideration for cell transplantation and cell replacement therapy is the use of non-terminally differentiated cells such as pluripotent (e.g., embryonic stem cells and induced pluripotent stem cells) or multipotent stem cells. Such cells can be transplanted as allogeneic (donor-derived) stem cells or autologous (self-derived) stem cells. Undifferentiated stem cells are characterized by rapid proliferative ability with a slow spontaneous differentiation rate, so there are concerns regarding the risk of tumor formation both immediately and long-term after stem cell transplantation (Mousavinejad et al., "Current Biosafety Considerations in Stem Cell Therapy," Cell J. 18(2):281-287(2016) (Non-Patent Document 4)).

[0007] The present disclosure relates to overcoming deficiencies in the art.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

[0009] [Abstract] One aspect of the present disclosure relates to a recombinant gene construct comprising a first gene sequence that is cell-type specifically expressed, one or more nucleotide sequences encoding immune checkpoint proteins located 3' to the first gene sequence, and a second gene sequence that is cell-type specifically expressed located 3' to the one or more nucleotide sequences encoding immune checkpoint proteins.

[0010] Another aspect of the present disclosure relates to a recombinant gene construct comprising a first gene sequence that is cell-type specifically expressed, a nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules located 3' to the first gene sequence, and a second gene sequence that is cell-type specifically expressed located 3' to the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules.

[0011] Another aspect of the disclosure relates to a recombinant gene construct comprising a first gene sequence that is expressed in a cell-type specific manner, a nucleotide sequence encoding one or more immune checkpoint protein coding nucleotide sequences, and a nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules, wherein the immune checkpoint protein coding nucleotide sequence and the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules are located 3' to the first gene sequence. The recombinant gene construct further comprises a second gene sequence that is expressed in a cell-type specific manner, and the second gene sequence is located 3' to the nucleotide sequence encoding one or more immune checkpoint protein coding nucleotide sequences and the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules.

[0012] Another aspect of the disclosure relates to a preparation of one or more cells comprising the recombinant gene construct of the disclosure.

[0013] A further aspect relates to a method comprising the step of administering a preparation of one or more cells comprising the recombinant gene construct of the disclosure to a subject in need thereof.

[0014] Yet another aspect of the disclosure relates to a method of treating a subject having a condition mediated by a decrease in myelin or oligodendrocyte dysfunction or decrease. The method comprises the step of administering to the subject a preparation of one or more cells comprising the recombinant gene construct described herein under conditions effective to treat the condition.

[0015] Another aspect relates to a method of treating a subject having a condition mediated by astrocyte dysfunction or decrease. The method comprises the step of administering to the subject a preparation of one or more cells comprising the recombinant gene construct described herein under conditions effective to treat the condition.

[0016] Another aspect relates to a method of treating a subject having a condition mediated by neuronal dysfunction or reduction. The method includes administering to the subject a preparation of one or more cells comprising the recombinant gene construct described herein under conditions effective to treat the condition.

[0017] A further aspect relates to a preparation of one or more cells, wherein the cells of the preparation are conditionally expressing increased levels of one or more immune checkpoint proteins as compared to the corresponding wild-type cells, conditionally expressing decreased levels of one or more endogenous HLA-I proteins as compared to the corresponding wild-type cells, or are modified to conditionally express increased levels of one or more immune checkpoint proteins and express decreased levels of one or more endogenous HLA-I proteins as compared to the corresponding wild-type cells.

[0018] Yet another aspect relates to a method of generating conditionally immunoprotected cells. The method includes modifying the cells to conditionally express increased levels of one or more immune checkpoint proteins, modifying the cells to conditionally express one or more substances that reduce the expression of one or more endogenous HLA proteins, or modifying the cells to conditionally express increased levels of one or more immune checkpoint proteins and conditionally express one or more substances that reduce the expression of one or more endogenous HLA proteins. [The present invention 1001] A first gene sequence that is specifically expressed in a cell type, One or more nucleotide sequences encoding immune checkpoint proteins, which are located on the 3' side of the first gene sequence, and A second gene sequence that is specifically expressed in a cell type, which is located on the 3' side of the nucleotide sequence encoding the immune checkpoint protein A recombinant gene construct comprising. [The present invention 1002] A nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules, the nucleotide sequence being linked to one or more nucleotide sequences encoding immune checkpoint proteins The recombinant gene construct of the present invention 1001, further comprising. [The present invention 1003] A first gene sequence that is specifically expressed in a cell type, A nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules, which is located on the 3' side of the first cell-specific gene sequence, and A second gene sequence that is specifically expressed in a cell type, which is located on the 3' side of the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules A recombinant gene construct comprising. [The present invention 1004] The recombinant gene construct of the present invention 1001 or the present invention 1002, wherein one or more immune checkpoint proteins are selected from programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), CD47, CD200, CTLA-4, HLA-E, and any combination thereof. [The present invention 1005] The recombinant gene construct according to any one of the present inventions 1002 to 1004, wherein one or more substances that reduce the expression of one or more HLA-I molecules are selected from the group consisting of shRNA, miRNA, and siRNA. [The present invention 1006] The recombinant gene construct according to any one of the present inventions 1002 to 1004, wherein one or more substances that reduce the expression of one or more HLA-I molecules are nuclease-deficient Cas9 or zinc finger nuclease. [The present invention 1007] One or more substances that reduce the expression of one or more HLA-I molecules are substances that reduce the expression of β 2 M, the recombinant gene construct according to any one of the present inventions 1002 to 1006. [The present invention 1008] One or more HLA-I molecules are selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and combinations thereof, any of the recombinant gene constructs of the present invention 1002 to 1006. [The present invention 1009] Any of the recombinant gene constructs of the present invention 1001 to 1008, wherein the first and second gene sequences of the recombinant gene construct are derived from genes that are specifically expressed in one or more terminally differentiated cells. [The present invention 1010] The recombinant gene construct of the present invention 1009, wherein the terminally differentiated cell is an oligodendrocyte. [The present invention 1011] The recombinant gene construct of the present invention 1010, wherein the first and second gene sequences are derived from genes selected from the group consisting of SOX10, MYRF, MAG, and MBP. [The present invention 1012] The recombinant gene construct of the present invention 1009, wherein the terminally differentiated cell is an astrocyte. [The present invention 1013] The recombinant gene construct of the present invention 1012, wherein the first and second gene sequences are derived from genes selected from GFAP and AQP4. [The present invention 1014] The recombinant gene construct of the present invention 1009, wherein the terminally differentiated cell is a neuron. [The present invention 1015] The recombinant gene construct of the present invention 1014, wherein the first and second gene sequences are derived from genes selected from the group consisting of SYN1, MAP2, and ELAV4. [The present invention 1016] The recombinant gene construct of the present invention 1014, wherein the terminally differentiated cell is a dopaminergic neuron, and the first and second gene sequences are derived from genes selected from TH and DDC. [The present invention 1017] The recombinant gene construct of the present invention 1014, wherein the terminally differentiated cells are medium spiny neurons and cortical interneurons, and the first and second gene sequences are derived from genes selected from GAD65 and GAD67. [The present invention 1018] The recombinant gene construct of the present invention 1014, wherein the terminally differentiated cell is a cholinergic neuron, and the first and second gene sequences are derived from CHAT. [The present invention 1017] A further nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-II molecules, the further nucleotide sequence being linked to a nucleotide sequence encoding one or more immune checkpoint protein encoding nucleotide sequences and / or a nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules The recombinant gene construct according to any one of 1001 to 1016 of the present invention, further comprising the same. [The present invention 1018] The recombinant gene construct according to 1017 of the present invention, wherein the one or more substances that reduce the expression of one or more HLA-II molecules are selected from the group consisting of shRNA, miRNA, and siRNA. [The present invention 1019] The recombinant gene construct according to 1017 of the present invention, wherein the one or more substances that reduce the expression of one or more HLA-II molecules are nuclease-deficient Cas9 protein or zinc finger nuclease. [The present invention 1020] The recombinant gene construct according to 1017 of the present invention, wherein the one or more substances that reduce the expression of one or more HLA-II molecules are substances that reduce the expression of class II major histocompatibility complex transactivator (CIITA). [The present invention 1021] A nucleotide sequence encoding one or more self-cleaving peptides, arranged within the construct in a manner effective to mediate the translation of one or more immune checkpoint proteins The recombinant gene construct according to any one of 1001 to 1020 of the present invention, further comprising the same. [The present invention 1022] The recombinant gene construct according to 1021 of the present invention, wherein the self-cleaving peptide is selected from the group consisting of porcine teschovirus 1 2A (P2A), thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), cytoplasmic polyhedrosis virus (BmCPV 2A), and flacherie virus (BmIFV 2A). [The present invention 1023] An inducible cell death gene arranged within the construct in a manner effective to achieve inducible cell suicide The recombinant gene construct according to any one of 1001 to 1022 of the present invention, further comprising the same. [The present invention 1024] The recombinant gene construct according to 1022 of the present invention, wherein the inducible cell death gene is selected from caspase 3, caspase 9, and thymidine kinase. [The present invention 1025] A preparation of one or more cells, wherein the cells of the preparation comprise any one of the recombinant gene constructs of 1001 to 1024 of the present invention. [1026 of the present invention] The preparation of 1025 of the present invention, wherein the cells of the preparation are mammalian cells. [1027 of the present invention] The preparation of 1025 of the present invention, wherein the cells of the preparation are human cells. [1028 of the present invention] The preparation of 1025 of the present invention, wherein the cells of the preparation are pluripotent cells. [1029 of the present invention] The preparation of 1028 of the present invention, wherein the pluripotent cells are induced pluripotent stem cells. [1030 of the present invention] The preparation of 1028 of the present invention, wherein the pluripotent cells are embryonic stem cells. [1031 of the present invention] The preparation of 1025 of the present invention, wherein the cells of the preparation are progenitor cells. [1032 of the present invention] The preparation of 1031 of the present invention, wherein the progenitor cells are glial progenitor cells. [1033 of the present invention] The preparation of 1031 of the present invention, wherein the progenitor cells are progenitor cells biased towards oligodendrocytes. [1034 of the present invention] The preparation of 1031 of the present invention, wherein the progenitor cells are progenitor cells biased towards astrocytes. [1035 of the present invention] The preparation of 1031 of the present invention, wherein the progenitor cells are neuronal progenitor cells. [1036 of the present invention] The preparation of 1025 of the present invention, wherein the cells of the preparation are terminally differentiated cells. [1037 of the present invention] The preparation of 1036 of the present invention, wherein the terminally differentiated cells are neurons, oligodendrocytes, or astrocytes. [1038 of the present invention] A method comprising the step of administering a preparation of any one of 1025 to 1037 of the present invention to a subject in need thereof thereof. [1039 of the present invention] A method for treating a subject having a condition mediated by a decrease in myelin or a dysfunction or decrease in oligodendrocytes, comprising administering to the subject a preparation of 1032 or 1033 of the present invention under conditions effective to treat the condition thereof. [1040 of the present invention] A method for treating a subject having a condition mediated by a dysfunction or decrease in astrocytes, comprising administering to the subject a preparation of 1032 or 1034 of the present invention under conditions effective to treat the condition thereof. [1041 of the present invention] A method for treating a subject having a condition mediated by a dysfunction or decrease in neurons, comprising administering to the subject a preparation of 1031 or 1035 of the present invention under conditions effective to treat the condition thereof. [1042 of the present invention] The method of any one of 1039 to 1041 of the present invention, wherein the preparation is administered to one or more sites of the brain, brainstem, spinal cord, or a combination thereof. [The present invention 1043] The method of the present invention 1042, wherein the preparation is administered into the cerebral ventricle, the corpus callosum, or the parenchyma. [The present invention 1044] A preparation of one or more cells, wherein the cells of the preparation (i) one or more immune checkpoint proteins at increased levels compared to the corresponding wild-type cells, (ii) one or more HLA-I proteins at decreased levels compared to the corresponding wild-type cells, or (iii) a combination of (i) and (ii) is modified to conditionally express. [The present invention 1045] The preparation of the present invention 1044, wherein the modified cells of the preparation are terminally differentiated cells. [The present invention 1046] The preparation of the present invention 1044, wherein one or more HLA-I proteins are selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and combinations thereof. [The present invention 1047] The preparation of the present invention 1044, wherein one or more immune checkpoint proteins are selected from programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), CD47, CD200, CTL4A, HLE-1, and any combination thereof. [The present invention 1048] The preparation according to any one of the present inventions 1044 to 1047, wherein the modified cells of the preparation conditionally express one or more HLA-II proteins at decreased levels compared to the corresponding wild-type cells. [The present invention 1049] The preparation of the cells of the present invention 1048, wherein one or more HLA-II proteins are selected from the group consisting of HLA-DM, HLA-DO, HLA-DP, HLA-DQ, HLA-DR, and combinations thereof. [The present invention 1050] (i) one or more immune checkpoint proteins at increased levels, (ii) one or more substances that reduce the expression of one or more HLA-I proteins, or (iii) both (i) and (ii) A step of modifying the cells to conditionally express A method for producing conditionally immunoprotected cells, comprising. [The present invention 1051] The method of the present invention 1050, wherein the conditional expression of one or more immune checkpoint proteins and the conditional expression of one or more substances that reduce the expression of one or more HLA-1 molecules are operably linked to a gene that is limitedly expressed in terminally differentiated cells. [The present invention 1052] The method of the present invention 1051, wherein the terminally differentiated cell is an oligodendrocyte. [The present invention 1053] The method of the present invention 1052, wherein the gene that is specifically expressed in oligodendrocytes is selected from the group consisting of SOX10, MYRF, MAG, and MBP. [The present invention 1054] The method of the present invention 1051, wherein the terminally differentiated cell is an astrocyte. [The present invention 1055] The method of the present invention 1054, wherein the gene that is specifically expressed in astrocytes is GFAP or AQP4. [The present invention 1056] The method of the present invention 1051, wherein the terminally differentiated cell is a neuron. [The present invention 1057] The method of the present invention 1056, wherein the gene that is specifically expressed in neurons is selected from the group consisting of SYN1, MAP2, and ELAV4. [The present invention 1058] The recombinant gene construct of the present invention 1051, wherein the terminally differentiated cell is a dopaminergic neuron, and the gene that is specifically expressed in dopaminergic neurons is TH or DDC. [The present invention 1059] The recombinant gene construct of the present invention 1051, wherein the terminally differentiated cells are medium spiny neurons and cortical interneurons, and the gene that is specifically expressed in medium spiny neurons and cortical interneurons is GAD65 or GAD67. [The present invention 1060] The recombinant gene construct of the present invention 1051, wherein the terminally differentiated cell is a cholinergic neuron, and the gene that is specifically expressed in cholinergic neurons is acetylcholine transferase. [The present invention 1061] The method of the present invention 1050, wherein one or more immune checkpoint proteins are selected from programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), CD47, CD200, CTLA4, HLA-A, and any combination thereof. [The present invention 1062] The method of the present invention 1050, wherein one or more HLA-I proteins are selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and combinations thereof. [The present invention 1063] The method of the present invention 1050, wherein one or more substances that reduce the expression of one or more HLA-I proteins are selected from the group consisting of shRNA, miRNA, and siRNA. [The present invention 1064] The method of the present invention 1050, wherein the one or more substances that reduce the expression of one or more HLA-I proteins are nuclease-deficient CRISPR-Cas9 proteins or zinc finger nucleases. [The present invention 1065] The method of the present invention 1050, wherein the one or more substances that reduce the expression of one or more HLA-I molecules are substances that reduce the expression of β 2 M. [The present invention 1066] The method of the present invention 1050, further comprising the step of modifying a cell to conditionally express one or more substances that reduce the expression of one or more HLA-II molecules . [The present invention 1067] The method of the present invention 1066, wherein the one or more substances that reduce the expression of one or more HLA-II molecules are substances that reduce the expression of class II major histocompatibility complex transactivator (CIITA). [The present invention 1068] The method of the present invention 1062, wherein the one or more substances that reduce the expression of one or more HLA-II molecules are selected from the group consisting of shRNA, miRNA, and siRNA. [The present invention 1069] The method of the present invention 1062, wherein the one or more substances that reduce the expression of one or more HLA-II proteins are nuclease-deficient CRISPR-Cas9 proteins or zinc finger nucleases. [The present invention 1070] The method according to any one of the present inventions 1050 to 1069, wherein the conditionally immunoprotected cell is a mammalian cell. [The present invention 1071] The method of the present invention 1070, wherein the conditionally immunoprotected mammalian cell is a human cell. [The present invention 1072] The method according to any one of the present inventions 1050 to 1069, wherein the conditionally immunoprotected cell is a pluripotent cell. [The present invention 1073] The method of the present invention 1072, wherein the conditionally immunoprotected pluripotent cell is an induced pluripotent stem cell. [The present invention 1074] The method of the present invention 1073, wherein the conditionally immunoprotected pluripotent cell is an embryonic stem cell. [The present invention 1075] The method according to any one of the present inventions 1050 to 1069, wherein the conditionally immunoprotected cell is a progenitor cell. [The present invention 1076] The method of the present invention 1075, wherein the conditionally immunoprotected progenitor cell is a glial progenitor cell. [The present invention 1077] The method of the present invention 1075, wherein the conditionally immunoprotected progenitor cell is a progenitor cell biased towards oligodendrocytes. [The present invention 1078] The method of the 1075th invention of the present invention, wherein the precursor cells immunoprotected conditionally are precursor cells biased towards astrocytes. [The 1079th invention of the present invention] The modification is (i) introducing into the cell a sequence-specific nuclease that cleaves the target gene at a position upstream of its 3' untranslated region (UTR), wherein the target gene is a gene specifically expressed in the cell, and (ii) introducing into the cell a recombinant gene construct comprising (a) one or more nucleotide sequences encoding immune checkpoint protein(s), (b) one or more nucleotide sequences encoding one or more substances that reduce the expression of one or more HLA-I molecules, or (c) both (a) and (b) and the recombinant gene construct is inserted into the target gene at the nuclease cleavage site by homologous recombination, according to the method of the 1050th invention of the present invention. [The 1080th invention of the present invention] The method of the 1079th invention of the present invention, wherein the sequence-specific nuclease is selected from the group consisting of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and RNA-guided nuclease. [The 1081st invention of the present invention] The method of the 1080th invention of the present invention, wherein the sequence-specific nuclease is an RNA-guided nuclease in the form of Cas9. [The 1082nd invention of the present invention] The method of the 1079th invention of the present invention, wherein the sequence-specific nuclease is introduced into the cell as a protein, mRNA, or cDNA.

Brief Description of the Drawings

[0019]

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BRIEF DESCRIPTION OF THE DRAWINGS

[0020] DETAILED DESCRIPTION The present disclosure relates to recombinant gene constructs, preparations of one or more cells comprising the recombinant gene constructs described herein, and methods of treating a subject using the disclosed preparations of cells.

[0021] One aspect of the present disclosure relates to recombinant gene constructs designed to provide cell-type selective immune protection to cells expressing the construct.

[0022] In one embodiment, the recombinant gene construct comprises a first gene sequence that is cell-type specifically expressed, one or more nucleotide sequences encoding immune checkpoint proteins located 3' to the first cell-specific gene sequence, and a second gene sequence that is cell-type specifically expressed and located 3' to the nucleotide sequence encoding the immune checkpoint protein.

[0023] In another aspect, the recombinant gene construct is a first nucleotide sequence that is cell-type specifically expressed, a nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules, the nucleotide sequence being located 3' to the first cell-specific gene sequence, and a second nucleotide sequence that is cell-type specifically expressed, the second nucleotide sequence being located 3' to the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules.

[0024] In another aspect, the recombinant gene construct comprises a first nucleotide sequence that is cell-type specifically expressed. The recombinant gene construct further comprises one or more immune checkpoint protein-encoding nucleotide sequences linked to a nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules, the immune checkpoint protein-encoding nucleotide sequence and the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules being located 3' to the first gene sequence. This construct further comprises a second nucleotide sequence that is cell-type specifically expressed, the second nucleotide sequence being located 3' to the immune checkpoint protein-encoding nucleotide sequence and the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules.

[0025] As described in more detail below, any one of the above recombinant gene constructs may further comprise an additional nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-II molecules. This additional nucleotide sequence may be linked to one or more immune checkpoint protein-encoding nucleotide sequences, a nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules, or both.

[0026] As used herein, the “recombinant gene construct” of the present disclosure refers to a nucleic acid molecule comprising a combination of two or more genetic elements that do not naturally occur together. The recombinant gene construct can be in the form of linear DNA, circular DNA, i.e., can be disposed within a vector (e.g., a bacterial vector, a viral vector), or can be incorporated into the genome, and includes unnatural nucleotide sequences.

[0027] As described in more detail below, the recombinant gene construct is introduced into the genome of a cell of interest to achieve the expression of one or more immune checkpoint proteins or peptides and / or one or more substances that reduce the expression of one or more HLA-I proteins. In some embodiments, the one or more substances that reduce the expression of one or more HLA-I proteins function to reduce the surface expression of the one or more HLA-I proteins.

[0028] As used herein, the terms "nucleotide sequence" and "nucleic acid sequence" are used interchangeably to refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, the term includes, but is not limited to, single-stranded, double-stranded, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA / RNA hybrids, or polymers comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. In the context of the recombinant gene constructs of the present disclosure, a nucleotide sequence can be a nucleotide sequence that "encodes" a protein if, in its native state or when manipulated by methods well known to those skilled in the art, the nucleotide sequence can be transcribed and / or translated to produce mRNA of the protein and / or a fragment thereof. The nucleotide sequence of a recombinant gene construct can also "encode" a substance having an effector function (e.g., shRNA, siRNA, microRNA, guide RNA, etc.) if, in its native state or when manipulated by methods well known in the art, it can be transcribed to produce a substance having the desired effector function.

[0029] The immune checkpoint protein encoded by the nucleotide sequence of the recombinant gene construct of the present disclosure can be any protein or peptide thereof that is involved in downregulating the immune system and / or promoting immune self-tolerance. In one aspect, the immune checkpoint protein or peptide thereof suppresses the activity of the adaptive immune response. In one aspect, the immune checkpoint protein or peptide thereof suppresses the activity of the innate immune response.

[0030] In one aspect, the immune checkpoint protein encoded by the recombinant gene construct is programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), or a functionally active peptide thereof that binds to inhibitory programmed cell death protein 1 (PD-1). PD-1 is mainly expressed on mature T cells in peripheral tissues and the tumor microenvironment. It is also expressed on other non-T cell subsets including B cells, professional APCs, and natural killer (NK) cells. PD-1 signaling is mediated through its interaction with its ligands PD-L1 (also known as B7-H1 and CD274) and PD-L2 (also known as B7-DC and CD273). The interaction of PD-1 with either of its ligands, namely PD-L1 and PD-L2, transmits an inhibitory signal that reduces the proliferation of CD8 + T cells in lymph nodes, thereby suppressing the immune response.

[0031] Appropriate nucleotide sequences encoding human PD-L1 and PD-L2 for inclusion in the recombinant gene constructs described herein are shown in Table 1 below. Appropriate nucleotide sequences also include nucleotide sequences having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% sequence identity to the PD-L1 and PD-L2 coding sequences (i.e., SEQ ID NO: 1-4) provided in Table 1 below.

[0032] (Table 1) Appropriate PD-L1 and PD-L2 coding sequences TIFF0007708737000001.tif173166TIFF0007708737000002.tif197166

[0033] Additional suitable nucleotide sequences encoding human PD-L1 that can be incorporated into the recombinant gene constructs described herein are known in the art and are described, for example, in GenBank accession numbers BC113734.1, BC113736.1, BC074984.2, and BC069381.1, which are hereby incorporated by reference in their entirety.

[0034] Additional suitable nucleotide sequences encoding human PDL-2 that can be incorporated into the recombinant gene constructs described herein are known in the art and are described, for example, in GenBank accession numbers BC113680.1, BC113678.1, and BC074766.2, which are hereby incorporated by reference in their entirety.

[0035] In another aspect, the immune checkpoint protein or peptide encoded by the recombinant gene construct of the present disclosure is the differentiation cluster 47 (CD47; integrin-associated protein (IAP)), a cell surface antigen. The phagocytic activity of macrophages is regulated by activation (“eat”) and inhibition (“do not eat”) signals. Under normal physiological conditions, ubiquitously expressed CD47 suppresses phagocytosis by binding to signal regulatory protein alpha (SIRPα) on macrophages. SIRPα, also known as Src homology 2 domain-containing protein tyrosine phosphatase substrate 1 / tyrosine-based activation motif-containing brain Ig-like molecule / differentiation cluster antigen-like family member A (SHPS-1 / BIT / CD172a), is another membrane protein of the immunoglobulin superfamily that is particularly abundant in myeloid hematopoietic cells such as macrophages and dendritic cells. Ligation of SIRPα on phagocytes by CD47 expressed on adjacent cells results in phosphorylation of the SIRPα cytoplasmic immunoreceptor tyrosine-based inhibition (ITIM) motif, leading to the recruitment of SHP-1 and SHP-2 phosphatases. One resulting downstream effect is the prevention of myosin IIA accumulation at the phagocytic synapse and thus inhibition of phagocytosis. Thus, the CD47-SIRPα interaction functions as a negative immune checkpoint that sends a “don't eat me” signal to ensure that healthy self-cells are not inappropriately phagocytosed (Lui et al., “Is CD47 an Innate Immune Checkpoint for Tumor Evasion?” J. Hematol. Oncol. 10:12 (2017), which is hereby incorporated by reference in its entirety).

[0036] An appropriate nucleotide sequence encoding human CD47 for inclusion in the recombinant gene constructs described herein is shown in Table 2 below. Appropriate nucleotide sequences also include nucleotide sequences having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% sequence identity to the CD47 coding sequences provided in Table 2 below (i.e., SEQ ID NO: 5-8).

[0037] (Table 2) Exemplary CD47 Coding Sequences TIFF0007708737000003.tif220164TIFF0007708737000004.tif211164

[0038] In another aspect, the immune checkpoint protein encoded by the recombinant gene construct is CD200. CD200 (also known as OX-2 membrane glycoprotein) is a 45 kDa transmembrane immune checkpoint protein. The CD200 receptor (CD200R) is expressed on cells of the monocyte / macrophage lineage as well as subsets of B and T cells. Signaling by CD200 inhibits the normal activation of myeloid cells bearing CD200R and results in an immunosuppressive cascade that includes the induction of regulatory T cells (T reg )(Gaiser et al., “Merke Cell Carcinoma Expresses the Immunoregulatory Ligand CD200 and Induces Immunosuppressive Macrophages and Regulatory T Cells,” Oncoimmunology 7(5):e1426517 (2018), which is incorporated herein by reference in its entirety). For example, CD200 signaling inhibits classical macrophage activation (M1 polarization) and supports an immunosuppressive M2 polarization state that secretes high levels of IL-10, thereby inducing T reg . Thus, cellular expression of CD200 via the recombinant gene constructs described herein confers protection to cells from macrophage- and T cell-mediated responses.

[0039] An appropriate nucleotide sequence encoding human CD200 for inclusion in the recombinant gene constructs described herein is shown in Table 3 below. The appropriate nucleotide sequences also include nucleotide sequences having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% sequence identity to the CD200 coding sequences provided in Table 3 below (i.e., SEQ ID NOs: 9-12).

[0040] (Table 3) Exemplary CD200 Coding Sequences TIFF0007708737000005.tif105166TIFF0007708737000006.tif225166

[0041] In another aspect, the immune checkpoint protein encoded by the recombinant gene construct is CTLA-4. In the process of immune recognition, two signals are required for the expansion, proliferation, and differentiation of T lymphocytes: the T cell receptor (TCR) that binds to the HLA molecule-peptide complex and the antigen-independent costimulatory signal provided by the B7 (CD80 and Cd86) / CD28 interaction. Cytotoxic T lymphocyte antigen (CTLA-4) is a homologous molecule of CD28, a competitive antagonist of B7. CTLA-4 has a greater affinity and binding activity for B7 than CD28, and its translocation to the cell surface after T cell activation results in the transmission of a negative signal involved in the sequestration of B7 and the inactivation of T cells (Perez-Garcia et al., "CTLA-4 Polymorphisms and Clinical Outcome after Allogeneic Stem Cell Transplantation from HLA-Identical Sibling Donors," Blood 110(1):461-7(2007), which is incorporated herein by reference in its entirety). Thus, the cellular expression of CTLA-4 via the recombinant gene constructs described herein confers protection to cells from cytotoxic T cell-mediated lysis.

[0042] The CTLA-4 gene is translated into two isoforms: a full-length protein (flCLTA-4) and a soluble counterpart (sCTLA-4) that lacks exon 3 (which encodes the transmembrane domain) due to alternative splicing. flCTLA-4 downregulates T cell responses by inducing cell cycle arrest and blocking cytokine production. Thus, in some embodiments, the immune checkpoint protein encoded by the recombinant gene construct is full-length CTLA-4 (flCTLA-4).

[0043] Appropriate nucleotide sequences encoding human CTLA-4 for inclusion in the recombinant gene constructs described herein are shown in Table 4 below. Appropriate nucleotide sequences also include nucleotide sequences having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% sequence identity to the CTLA-4 coding sequences provided in Table 4 below (i.e., SEQ ID NO: 13-14 and 44).

[0044] (Table 4) Exemplary CTLA-4 Coding Sequences TIFF0007708737000007.tif226166

[0045] In another embodiment, the immune checkpoint protein encoded by the recombinant gene construct is HLA-E (major histocompatibility complex, class I, E). Natural killer (NK) cells detect infected cells (primarily virus-infected), foreign cells, or malignant cells in which the expression of MHC molecules is reduced, altered, absent, or non-existent. NK cells discriminate normal host cells via killer cell immunoglobulin-like receptors (KIRs) that recognize MHC class I expressed on the surface of normal host cells and the CD94-NKG2A inhibitory receptor. In particular, CD94-NKG2A recognizes HLA-E on the surface of NK cells and CD8 + T cells. Engagement of these receptors inhibits lysis and cytokine secretion by NK cells. KIRs also recognize CD8 +It is also expressed on T cells and APCs. Therefore, cellular expression of HLA-E via the recombinant gene constructs described herein confers protection to cells from NK cell lysis.

[0046] Similar to other HLA class I proteins, HLA-E is a heterodimer consisting of a heavy chain (α-chain) and a light chain (β2-microglobulin). In one aspect, the recombinant gene construct can include a nucleotide sequence encoding HLA-E (α-chain E) and a nucleotide sequence encoding the β2-microglobulin chain. Alternatively, the recombinant gene construct can include a nucleotide sequence encoding a fusion construct, i.e., a single-chain fusion protein comprising at least a portion of β2-microglobulin covalently attached to at least a portion of HLA-E. In other aspects, the HLA-E / β2M fusion protein is syβ2M-HLA-E, and syB2M (synthetic B2M) is expressed as a complex with HLA-E. syB2M contains several silent mutations in the target sequence of the shRNA that targets endogenous B2M. Thus, syB2M encodes exactly the same protein as wild-type B2M but is resistant to shRNAs that target only endogenous B2M.

[0047] Exemplary nucleotide sequences encoding human HLA-E (alpha chain) are provided in Table 5 below. Suitable nucleotide sequences also include nucleotide sequences having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% sequence identity to the HLA-E coding sequences (i.e., SEQ ID NOs: 15 - 17) provided in Table 5 below.

[0048] (Table 5) Exemplary HLA-E coding sequences TIFF0007708737000008.tif245166TIFF0007708737000009.tif134166

[0049] Exemplary nucleotide sequences encoding human β2M are provided in Table 6 below. Suitable nucleotide sequences also include nucleotide sequences having about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 98%, about 99%, or about 100% sequence identity to the β2M coding sequences (i.e., SEQ ID NOs: 18 - 21) provided in Table 6 below.

[0050] (Table 6) Suitable β2M coding sequences TIFF0007708737000010.tif208158

[0051] The single-chain HLA-E / β2M fusion protein can comprise an HLA-E heavy chain covalently fused to β2M via a flexible linker. In some embodiments, the flexible linker is a glycine - serine linker, such as a G4S4 linker (Gornalusse et al., "HLA-E-Expressing Pluripotent Stem Cells Escape Allogenic Responses and Lysis by NK Cells," Nat. Biotechnol. 35(8):765 - 772(2017), which is incorporated herein by reference in its entirety).

[0052] The signal sequence of HLA-G contains the peptide sequence normally presented by HLA-E, which inhibits NK cell-dependent lysis by binding to CD94 / NKG2A (Lee et al., "HLA-E is a Major Ligand for the Natural Killer Inhibitory Receptor CD94 / NKG2A," Proc. Natl. Acad. Sci. USA 95:5199-5204 (1998), which is hereby incorporated by reference in its entirety). Thus, in some embodiments, the single-chain HLA-E / β2M fusion protein further comprises an additional glycine-serine linker fused to a non-polymorphic peptide derived from the signal sequence of HLA-G (Gornalusse et al., "HLA-E-Expressing Pluripotent Stem Cells Escape Allogenic Responses and Lysis by NK Cells," Nat. Biotechnol. 35(8):765-772 (2017), which is hereby incorporated by reference in its entirety).

[0053] As described above, the recombinant gene constructs disclosed herein may alternatively or additionally comprise a nucleotide sequence encoding one or more substances that reduce the expression of one or more major histocompatibility complex class I molecules, particularly one or more HLA-I molecules. In one embodiment, this nucleotide sequence exists alone in the recombinant gene construct and is located between the first gene sequence and the second gene sequence. In another embodiment, this nucleotide sequence is present in the recombinant gene construct in combination with one or more immune checkpoint protein-encoding nucleotide sequences. In this embodiment, the aforementioned combination of nucleotide sequences is located between the first gene sequence and the second gene sequence. The nucleotide sequence encoding one or more substances that reduce the expression of HLA-I molecules may be located 5' or 3' to one or more immune checkpoint protein-encoding nucleotide sequences.

[0054] The recombinant gene constructs of the present disclosure may include additional nucleotide sequences encoding one or more substances that reduce the expression of one or more HLA-II molecules. In some embodiments, the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-II molecules is ligated to the nucleotide sequence encoding one or more immune checkpoint protein coding nucleotide sequences and / or the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules.

[0055] Suitable substances that reduce the expression of one or more HLA-I and / or HLA-II molecules are described in detail below and include, without limitation, inhibitory oligonucleotide molecules such as small hairpin RNA (shRNA), microRNA (miRNA), small interfering RNA (siRNA), and / or antisense oligonucleotides.

[0056] The human leukocyte antigen (HLA) system is the major histocompatibility complex (MHC) in humans. Thus, for the purposes of the present disclosure, the terms HLA and MHC are used interchangeably to refer to the human genes and proteins of the major histocompatibility complex. In other embodiments, the recombinant gene constructs may include nucleotide sequences encoding one or more substances that reduce the expression of one or more MHC class I or II molecules of non-human mammals, such as MHC class I or II molecules of mice, rats, pigs, horses, monkeys.

[0057] Class I MHC proteins (e.g., HLA-I proteins) are heterodimers of two proteins, an α chain, which is a transmembrane protein encoded by the MHC class I gene (chromosome 6 in humans; chromosome 17 in mice), and a β2-microglobulin (β2M) chain (chromosome 15 in humans; chromosome 2 in mice). The α chain is folded into three globular domains - α1, α2, and α3. The α1 domain lies on top of the β2M unit. The α3 domain is transmembrane and anchors the MHC class I molecule to the cell membrane. The MHC class I complex presents foreign peptides / molecules to cells of the immune system. The peptide / molecule presented is held by a peptide-binding groove in the central region of the MHC α1 / α2 heterodimer. Classical MHC class I molecules are highly polymorphic and present epitopes to the T cell receptors (TCRs) of CD8 + T cells, while non-classical MHC class I molecules show limited polymorphism, expression patterns, and presented antigens.

[0058] The class I HLA gene cluster in humans encodes the heavy chains of classical (HLA-A, HLA-B, and HLA-C) and non-classical (HLA-E, HLA-F, HLA-G) class I molecules. Thus, in one aspect, the recombinant gene constructs disclosed herein include nucleotide sequences encoding one or more substances that reduce the expression of one or more HLA-I molecules endogenous to the cell in which the recombinant gene construct is expressed, i.e., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, or combinations thereof. In another aspect, the recombinant gene constructs disclosed herein include nucleotide sequences encoding a substance that reduces the expression of β2M, thereby reducing the expression of all class I HLA in the cell.

[0059] Class II HLA molecules, i.e., the human form of class II MHC proteins, are heterodimers of two transmembrane proteins, an α-chain and a β-chain, encoded by the class II genes (the HLA-II genes on human chromosome 6; the MHC-II genes on mouse chromosome 17). Each of the α-chain and β-chain contains two domains - α1 and α2 and β1 and β2, respectively. The α2 and β2 domains are the transmembrane domains of the α-chain and β-chain, respectively, which anchor the MHC / HLA class II molecule to the membrane. Classical MHC / HLA class II molecules are expressed on the surface of dendritic cells, monocytes, and B lymphocytes and present peptides to CD4 + T cells, while non-classical MHC / HLA class II molecules are not exposed on the cell membrane but are exposed on the inner membrane of lysosomes. The expression of MHC / HLA class II is induced by IFN-γ via the production of MHC class II transactivator (CIITA). Thus, in one aspect, the nucleotide sequence of the recombinant gene construct encodes a substance that inhibits CIITA expression, thereby reducing the expression of all class II HLA in cells.

[0060] The human HLAs corresponding to MHC class II include three gene families that encode the α-chain and β-chain of class II molecules, respectively. The DR gene family consists of a single DRA gene and up to nine DRB genes (DRB1 - DRB9). The DRA gene encodes an invariant α-chain that binds to various β-chains encoded by the DRB genes. The DP and DQ families each have one expressed gene for the α-chain and β-chain and additional unexpressed pseudogenes. The DQA1 and DQB1 gene products associate to form DQ molecules, and the DPA1 and DPB1 products form DP molecules.

[0061] As described above, the inhibitory oligonucleotide molecule is a suitable substance encoded by a recombinant gene construct for reducing the expression of one or more HLA-I or HLA-II molecules. Exemplary inhibitory oligonucleotide molecules include, without limitation, small hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), and / or antisense oligonucleotides.

[0062] siRNA is a double-stranded synthetic RNA molecule about 20-25 nucleotides in length, with short 2-3 nucleotide 3' overhangs at both ends. The double-stranded siRNA molecule represents the sense and antisense strands of a portion of a target mRNA molecule, in this case HLA-I and / or HLA-II mRNA, β2M mRNA (e.g., SEQ ID NO: 18-21), and / or CIITA mRNA (SEQ ID NO: 22-23). The sequences of various HLA-I (HLA-A, HLA-B, HLA-C) mRNAs and HLA-II (HLA-E, HLA-F, HLA-G) mRNAs are readily known in the art and are available to those skilled in the art for designing siRNA and shRNA oligonucleotides. siRNA molecules are typically designed to target regions of the mRNA target approximately 50-100 nucleotides downstream from the start codon. Methods and online tools for designing appropriate siRNA sequences based on the target mRNA sequence are readily available in the art (see, e.g., Reynolds et al., "Rational siRNA Design for RNA Interference," Nat. Biotech. 2:326-330 (2004); Chalk et al., "Improved and Automated Prediction of Effective siRNA," Biochem. Biophys. Res. Comm. 319(1):264-274 (2004); Zhang et al., "Weak Base Pairing in Both Seed and 3' Regions Reduces RNAi Off-targets and Enhances si / shRNA Designs," Nucleic Acids Res. 42(19):12169-76 (2014), which are incorporated herein by reference in their entirety). When introduced into cells, the siRNA complex induces the endogenous RNA interference (RNAi) pathway, resulting in cleavage and degradation of the target mRNA molecule.Various improvements to siRNA compositions are described, such as the incorporation of modified nucleosides or motifs into one or both strands of the siRNA molecule to enhance stability, specificity, and efficacy, and are suitable for use according to this aspect of the invention (see, e.g., International Publication No. WO 2004 / 015107 by Giese et al.; International Publication No. WO 2003 / 070918 by McSwiggen et al.; International Publication No. WO 1998 / 39352 by Imanishi et al.; U.S. Patent Application Publication No. 2002 / 0068708 by Jesper et al.; U.S. Patent Application Publication No. 2002 / 0147332 by Kaneko et al.; U.S. Patent Application Publication No. 2008 / 0119427 by Bhat et al., each of which is incorporated herein by reference). Methods for constructing DNA vectors for siRNA expression in mammalian cells are known in the art, see, e.g., Sui et al., "A DNA Vector-Based RNAi Technology to Suppress Gene Expression in Mammalian Cells," Proc. Nat'l Acad. Sci. USA 99(8):5515-5520 (2002), which is incorporated herein by reference.

[0063] (Table 7) Human CIITA mRNA sequence TIFF0007708737000011.tif155170TIFF0007708737000012.tif250166TIFF0007708737000013.tif250166TIFF0007708737000014.tif250166TIFF0007708737000015.tif250166TIFF0007708737000016.tif54166

[0064] Short or small hairpin RNA (shRNA) molecules have functions similar to siRNA molecules but contain longer RNA sequences that make tight hairpin turns. shRNAs are cleaved into siRNAs by cellular machinery, and gene expression is suppressed via the cellular RNA interference pathway. Methods and tools for designing appropriate shRNA sequences based on target mRNA sequences (e.g., β2M, CIITA, and other HLA-I and HLA-II mRNA sequences) are readily available in the art (see, e.g., Taxman et al., "Criteria for Effective Design, Constructions, and Gene Knockdown shRNA Vectors," BMC Biotech. 6:7 (2006) and Taxman et al., "Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown," Meth. Mol. Biol. 629:139-156 (2010), which are incorporated herein by reference in their entirety). Methods for constructing DNA vectors for shRNA expression and gene silencing in mammalian cells are described herein and are known in the art, see, e.g., Cheng and Chang, "Construction of Simple and Efficient DNA Vector-based Short Hairpin RNA Expression Systems for Specific Gene Silencing in Mammalian Cells," Methods Mol. Biol. 408:223-41 (2007), which is incorporated herein by reference in its entirety).

[0065] Other suitable substances that may be encoded by the recombinant constructs disclosed herein for the purpose of inhibiting HLA-I or HLA-II molecules include microRNAs (miRNAs). miRNAs are small regulatory non-coding RNA molecules that mainly control the expression of their target mRNAs by binding to the 3' untranslated region (UTR). A single UTR may have binding sites for multiple miRNAs, or multiple binding sites for a single miRNA, suggesting the complex post-transcriptional regulation of gene expression exerted by these regulatory RNAs (Shulka et al., "MicroRNAs: Processing, Maturation, Target Recognition and Regulatory Functions," Mol. Cell. Pharmacol. 3(3):83-92(2011), which is incorporated herein by reference in its entirety). Mature miRNAs are first expressed as primary transcripts known as pre-miRNAs, which are processed in the cell nucleus by the microprocessor complex into 70-nucleotide stem-loop structures called pre-miRNAs. The dsRNA portion of the pre-miRNA is bound and cleaved by Dicer to generate mature 22bp double-stranded miRNA molecules that can be incorporated into the RISC complex; thus, miRNAs and siRNAs share the same cellular machinery downstream of their initial processing.

[0066] MicroRNAs known to inhibit the expression of MHC class I molecules are known in the art and are suitable for incorporation into the recombinant gene constructs described herein. For example, miR-148a is known to regulate the expression of HLA-C (O'Huigin et al., "The Molecular Origin and Consequences of Escape from miRNA Regulation by HLA-C Alleles," Am. J. Hum. Genet. 89(3):424-431(2011), which is incorporated herein by reference in its entirety); miR-148 and miR-152 downregulate HLA-G expression (Manaster et al., "miRNA-mediated Control of HLA-G Expression and Function," PLoS ONE 7(3):e33395(2012), which is incorporated herein by reference in its entirety); miR-9 regulates the expression of β2-microglobulin, HLA-B, and other class I MHC molecules (Gao et al., "miR-9 Modulates the Expression of Interferon-Regulated Genes and MHC Class I Molecules in Human Nasopharyngeal Carcinoma Cells," Biochem. Biophys. Res. Commun. 4313:610-616(2013), which is incorporated herein by reference in its entirety); miR-181a regulates the expression of HLA-A (Liu et al., "Altered Expression Profiles of microRNAs in a Stable Hepatitis B Virus-Expressing Cell Line," Chin. Med J. 1221:10-14(2009), which is incorporated herein by reference in its entirety).Methods for constructing DNA vectors for miRNA expression and gene silencing in mammalian cells are known in the art, see, for example, Yang N., "An Overview of Viral and Non-Viral Delivery Systems for microRNA," Int.J.Pharm.Investig.5(4):179-181(2015).

[0067] Other suitable substances that can be encoded by the recombinant constructs disclosed herein for the purpose of inhibiting HLA-I or HLA-II molecules include antisense nucleotides. The use of antisense methods for inhibiting in vivo translation of genes and subsequent protein expression are well known in the art (e.g., U.S. Patent No. 7,425,544 to Dobie et al., U.S. Patent No. 7,307,069 to Karras et al., U.S. Patent No. 7,288,530 to Bennett et al., U.S. Patent No. 7,179,796 to Cowsert et al., which are hereby incorporated by reference in their entirety). Antisense nucleic acids are nucleic acid molecules that are complementary to or hybridize to at least a portion of a specific nucleic acid molecule such as an mRNA molecule (e.g., DNA nucleotides, RNA nucleotides, or modified (e.g., modifications that increase the stability of the molecule such as 2'-O-alkyl (e.g., methyl) substituted nucleotides) or molecules comprising combinations thereof) (see, e.g., Weintraub, H.M., "Antisense DNA and RNA," Scientific Am. 262:40-46 (1990), which is hereby incorporated by reference in its entirety). The antisense nucleic acid molecule hybridizes to a corresponding target nucleic acid molecule such as HLA-I or HLA-II mRNA, β2M mRNA, or CIITA mRNA to form a double-stranded molecule, which prevents translation of the mRNA since the cell does not translate double-stranded mRNA. The antisense nucleic acids used in the methods of the present invention are typically at least 10-15 nucleotides in length, e.g., at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75 nucleotides in length, or greater than 75 nucleotides in length. The antisense nucleic acid can also be the same length as its target nucleic acid, which is intended to form an inhibitory double-stranded.

[0068]

[0068] In some embodiments, the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I or HLA-II molecules encodes multiple (e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or more) RNA molecules.

[0069] In some embodiments, one or more substances encoded by the recombinant gene constructs disclosed herein that inhibit one or more HLA-I and / or HLA-II molecules comprise a CRISPR / Cas9 system or a zinc finger nuclease.

[0070] The CRISPR / CRISPR-associated (Cas) system uses single-guide RNAs to target and cleave DNA elements in a sequence-specific manner. The CRISPR / Cas system is well-known in the art and includes, for example, the type II CRISPR system from Streptococcus pyogenes (Qi et al, "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression," Cell 152(5):1173-1183 (2013), which is hereby incorporated by reference in its entirety). The type II CRISPR system from Streptococcus pyogenes includes a single gene encoding the Cas9 protein and two RNAs, a mature CRISPR RNA (crRNA) and a partially complementary trans-activating RNA (tracrRNA). The maturation of crRNA requires tracrRNA and RNase II. However, this requirement can be circumvented by using an engineered small guide RNA (sgRNA) containing a designed hairpin that mimics the tracrRNA-crRNA complex. Base pairing between the sgRNA and the target DNA causes a double-strand break (DSB) due to the endonuclease activity of Cas9. The binding specificity is determined by both the sgRNA-DNA base pairing and a short DNA motif (protospacer adjacent motif (PAM) sequence: NGG) juxtaposed to the complementary region of the DNA.

[0071] In some embodiments, the CRISPR / Cas9 system encoded by a recombinant gene construct includes the Cas9 protein and the sgRNA.

[0072] The Cas9 protein can include a wild-type Cas9 protein or a nuclease-deficient Cas9 protein. When wild-type Cas9 binds to the sgRNA, a protein-RNA complex is formed that mediates cleavage of the target DNA by the Cas9 nuclease. When nuclease-deficient Cas9 binds to the sgRNA, a protein-RNA complex is formed that mediates transcriptional regulation of the target DNA by the nuclease-deficient Cas9 (Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression," Cell 152(5):1173-1183 (2013); Maeder et al., "CRISPR RNA-Guided Activation of Endogenous Human Genes," Nat. Methods 10(10):977-999 (2013); and Gilbert et al., "CRISPR-Mediated Modular RNA-Guided Regulation of Transcription in Eukaryotes," Cell 154(2):442-451 (2013), which are hereby incorporated by reference in their entirety).

[0073] The sgRNA includes a region complementary to a specific DNA sequence (e.g., a region of the HLA-I or HLA-II gene), a hairpin for Cas9 binding, and / or a transcription terminator (Qi et al., "Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression," Cell 152(5):1173-1183(2013), which is hereby incorporated by reference in its entirety). Methods for designing sgRNAs for the purpose of targeting specific gene sequences are well known in the art and are described in more detail, for example, in International Publication No. WO 2015 / 089364, International Publication No. WO 2014 / 191521, and International Publication No. WO 2015 / 065964, which are hereby incorporated by reference in their entirety).

[0074] In another aspect, one or more substances encoded by the recombinant gene constructs disclosed herein for the purpose of inhibiting HLA-I or HLA-II molecules are zinc finger nucleases. A zinc finger nuclease (ZFN) is a synthetic enzyme that contains three (or more) zinc finger domains linked together to create an artificial DNA-binding protein that binds to DNA of 9 bp or more. To cleave DNA, the zinc finger domain is fused to half of the FokI nuclease domain so that when two ZFNs bind to two unique 9 bp sites separated by an appropriate spacer, cleavage can occur within the spacer to generate a DSB. Methods for designing zinc finger nucleases that recognize a desired target are well known in the art and are described in more detail, for example, in U.S. Patent No. 7,163,824 to Cox III; U.S. Patent Application Publication No. 2017 / 0327795 to Kim et al.; and Harrison et al., "A Beginner's Guide to Gene Editing," Exp. Physiol. 103(4):439-448(2018), which are hereby incorporated by reference in their entirety.

[0075] In some embodiments, one or more substances that reduce the expression of one or more endogenous HLA-I and / or HLA-II molecules reduce the expression of all HLA-I and / or HLA-II molecules. In some embodiments, one or more substances can reduce the expression of one or more HLA-I and / or HLA-II molecules on the surface of cells by 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.5%, 99.9%, or 100% compared to the wild-type expression level.

[0076] The recombinant gene constructs described herein further include first and second "gene sequences", also referred to herein as "homology arms". These cell-type specifically expressed gene sequences direct the insertion of the recombinant construct into a gene of interest (i.e., the target gene) within a cell population, for example, by homologous recombination. Thus, the recombinant gene construct includes a first gene sequence that is cell-type specifically expressed and is located 5' of one or more nucleotide sequences encoding an immune checkpoint protein nucleotide sequence and / or one or more substances (one or more) for reducing the expression of HLA-I and / or HLA-II molecules, and a second gene sequence that is cell-type specifically expressed in the same manner as the first gene sequence. The second gene sequence is located 3' of one or more nucleotide sequences encoding an immune checkpoint protein nucleotide sequence and / or one or more substances (one or more) for reducing the expression of HLA-I and / or HLA-II molecules.

[0077] The first and second gene sequences (one or more) of the recombinant gene construct described herein are nucleotide sequences that are the same as or closely homologous to (i.e., share significant sequence identity with) the nucleotide sequence of a specific region of the target gene, i.e., the gene into which the recombinant gene construct is inserted. Preferably, the first and second gene sequences of the recombinant construct are the same as or similar to (e.g., the same as the sense strand of the target gene) the target gene sequences immediately upstream and downstream of the insertion cleavage site.

[0078] In some embodiments, the percent identity between the first gene sequence (i.e., the 5' homology arm) located at the 5' end of the recombinant construct and the corresponding sequence (e.g., the sense strand) of the target gene is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100%. In some embodiments, the percent identity between the second gene sequence (i.e., the 3' homology arm) located at the 3' end of the recombinant construct and the corresponding sequence (e.g., the sense strand) of the target gene is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or at least about 100%.

[0079] In some embodiments, the first and second gene sequences (e.g., the 5' and 3' homology arms) are of a length greater than about 30 nucleotide residues, such as greater than about 50 nucleotide residues, greater than about 100 nucleotide residues, greater than about 200 nucleotide residues, greater than about 300 nucleotide residues, greater than about 500 nucleotide residues, greater than about 800 nucleotide residues, greater than about 1,000 nucleotide residues, greater than about 1,500 nucleotide residues, greater than about 2,000 nucleotide residues, and greater than about 5,000 nucleotide residues.

[0080] The recombinant gene constructs disclosed herein can be circular or linear. When the recombinant gene construct is linear, the first and second gene sequences (e.g., 5' and 3' homology arms) are proximal to the 5' and 3' ends of the linear nucleic acid, respectively, i.e., about 200 bp away from the 5' and 3' ends of the linear nucleic acid. In some embodiments, the first gene sequence (e.g., 5' homology arm) is about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, or about 200 nucleotide residues away from the 5' end of the linear DNA. In some embodiments, the second gene sequence (e.g., 3' homology arm) is about 1, about 2, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 40, about 45, about 50, about 55, about 60, about 70, about 80, about 90, about 100, about 120, about 140, about 160, about 180, or about 200 nucleotide residues away from the 3' end of the linear DNA.

[0081] The first and second gene sequences of the recombinant gene construct are designed to mimic the sequence of the "target gene" to facilitate insertion of the construct into the target gene. According to various aspects of the present disclosure, the "target gene" is a gene that is expressed cell-type specifically. In some embodiments, the "target gene" is a gene that is selectively and / or restrictedly expressed in terminally differentiated cells. "Terminally differentiated cells" refer to specialized cells that have acquired a specialized function, are restricted to that specialized function, and have irreversibly lost the ability to divide and proliferate.

[0082] In some embodiments, the target gene is a gene that is expressed in terminally differentiated cells of the central nervous system. Exemplary terminally differentiated brain cells include, without limitation, oligodendrocytes, astrocytes, and neurons, including cholinergic neurons, medium spiny neurons and interneurons, and dopaminergic neurons. Exemplary terminally differentiated brain cells and gene targets selectively expressed in these cells are identified in Table 8 and discussed in more detail below.

[0083] (Table 8) Exemplary CNS Cells and Gene Targets Selectively Expressed Therein TIFF0007708737000017.tif179148

[0084] In one aspect, the target gene is a gene that is selectively expressed in oligodendrocytes. Oligodendrocytes are the terminally differentiated myelinating cells of the vertebrate central nervous system (CNS) that are involved in the myelination of recipient neuron axons, which is essential for the rapid propagation of nerve impulses. The differentiation of oligodendrocyte progenitor cells (OPCs) into oligodendrocytes and subsequent myelination of axons are highly regulated processes. Genes that are selectively or specifically expressed in oligodendrocytes include, without limitation, the transcription factor SRY-box 10 (SOX10) (Stolt et al., "Terminal Differentiation of Myelin-Forming Oligodendrocytes Depends on the Transcription Factor Sox10," Genes and Dev. 16:165 - 170 (2002), which is hereby incorporated by reference in its entirety); the myelin regulatory factor (MYRF), a membrane-associated transcription factor (Bujalka et al., "MYRF is a Membrane-Associated Transcription Factor that Autoproteolytically Cleaves to Directly Activate Myelin Genes," PLoS Biol. 11(8):e1001625 (2013), which is hereby incorporated by reference in its entirety); myelin-associated glycoprotein (MAG); and myelin basic protein (MBP).

[0085] In one aspect, the recombinant gene constructs described herein are designed for insertion into any one of the SOX10, MYRF, MAG, or MBP genes such that the expression of the recombinant construct is linked to the expression of the gene in oligodendrocytes. According to this aspect, the first and second gene sequences are derived from the SOX10, MYRF, MAG, or MBP gene.

[0086] In one aspect, the recombinant gene construct is designed to be inserted into or around the 3' untranslated region of any one of the aforementioned genes, and the first and second gene sequences of the recombinant gene construct are homologous to regions of the selected gene that are 5' and 3' of the selected insertion site, respectively. The specific position of the insertion site can vary, and thus the specific sequences of the first and second gene sequences of the recombinant construct can also vary. However, the selection of these parameters is well within the level of those skilled in the art, for example, using the known sequences and structures of these genes readily available in the art via, for example, the NCBI gene database and gene ID numbers.

[0087] In another aspect, the target gene is a gene that is specifically expressed in astrocytes. Astrocytes are the most abundant terminally differentiated cell type in the CNS and play various roles, from axon guidance and synapse support to blood-brain barrier and blood flow control.

[0088] Terminally differentiated astrocytes can be identified by the presence of various cell surface markers, including, for example, glial fibrillary acidic protein (GFAP) and aquaporin 4 (AQP4). Thus, genes that are selectively expressed in astrocytes into which the recombinant construct can be inserted include, without limitation, GFAP and AQP4. According to this aspect, the first and second gene sequences are derived from GFAP and AQP4.

[0089] In one aspect, the recombinant gene construct described herein is inserted into GFAP or AQP4 such that the expression of the recombinant construct is linked to the expression of GFAP or AQP4. In one aspect, the recombinant gene construct is inserted into or around the 3' untranslated region of GFAP or AQP4, and the first and second gene sequences of the recombinant gene construct are homologous to regions of GFAP or AQP4 that are 5' and 3' of the selected insertion site, respectively. The specific location of the insertion site can vary, and thus the specific sequences of the first and second cell-specific gene sequences of the recombinant construct can also vary. However, the selection of these parameters is well within the level of those skilled in the art using the known sequences and structures of these genes, which are readily available in the art.

[0090] In another aspect, the target gene is a gene that is expressed exclusively in neurons. Neurons are electrically excitable cells in the central and peripheral nervous systems that function to process and transmit information. Differentiated neurons can be identified, for example, by the presence of various cell surface markers, including synapsin 1 (SYN1), microtubule-associated protein 2 (MAP2), and ELAV-like RNA-binding protein 4 (ELAV4). Thus, in one aspect, the recombinant gene construct described herein is inserted into any one of SYN1, MAP2, or ELAV4 such that the expression of the recombinant construct is linked to the expression of any one of the SYN1, MAP2, or ELAV4 genes in neurons. According to this aspect, the first and second gene sequences are derived from the SYN1, MAP2, or ELAV4 gene.

[0091] In a preferred embodiment where the expression of the recombinant gene construct is limited to a specific type of neuron, such as dopaminergic neurons, the recombinant gene construct is inserted into a gene that is specifically expressed in the desired neuron population. Thus, in one embodiment, the recombinant gene constructs described herein are designed for insertion into the tyrosine hydroxylase gene (TH) or dopa decarboxylase gene (DDC), which are genes that are selectively expressed in dopaminergic neurons. In another embodiment, the recombinant gene construct is designed for insertion into the gene encoding glutamate decarboxylase 2 (GAD2, also known as GAD65) or glutamate decarboxylase 1 (GAD1, also known as GAD67), which are genes that are selectively expressed in medium spiny neurons and cortical interneurons. In another embodiment, the recombinant gene constructs described herein are inserted into the choline O-acetyltransferase gene (CHAT), which is selectively expressed in cholinergic neurons.

[0092] In one embodiment, the recombinant gene construct is inserted into the 3' untranslated region or its vicinity of any one of the above neuron-specific genes (i.e., SYN1, MAP2, ELAV4, TH, DDC, GAD65, GAD67, or CHAT), and the first and second gene sequences of the recombinant gene construct are homologous to regions that are respectively on the 5' side and 3' side of the selected insertion site. The specific position of the insertion site can vary, and thus the specific sequences of the first and second gene sequences of the recombinant construct can also vary. However, the selection of these parameters is well within the level of those skilled in the art using the known sequences and structures of these genes that are readily available in the art.

[0093] In another aspect, the target gene is a gene that is expressed in terminally differentiated cells outside the central nervous system (CNS). Exemplary terminally differentiated non-CNS cells include, without limitation, adipocytes, chondrocytes, endothelial cells, epithelial cells (keratinocytes, melanocytes), osteocytes (osteoblasts, osteoclasts), hepatocytes (cholangiocytes, hepatocytes), muscle cells (cardiomyocytes, skeletal muscle cells, smooth muscle cells), retinal cells (ganglion cells, Müller cells, photoreceptor cells), retinal pigment epithelial cells, renal cells (podocytes, proximal tubule cells, collecting duct cells, distal tubule cells), adrenal cells (adrenal cortical cells, adrenal medullary cells), pancreatic cells (alpha cells, beta cells, delta cells, epsilon cells, pancreatic polypeptide-producing cells, exocrine cells), lung cells, bone marrow cells (early B cell development, early T cell development, macrophages, monocytes), urothelial cells, fibroblasts, parathyroid cells, thyroid cells, hypothalamic cells, pituitary cells, salivary gland cells, ovarian cells, and testicular cells. Exemplary terminally differentiated non-CNS cells and gene targets selectively expressed in these cells are identified in Table 9 below.

[0094] (Table 9) Exemplary non-CNS cells and gene targets selectively expressed therein TIFF0007708737000018.tif240164TIFF0007708737000019.tif249164TIFF0007708737000020.tif255164TIFF0007708737000021.tif245164TIFF0007708737000022.tif154164

[0095] In one aspect, the recombinant gene construct described herein is designed for insertion into any one of the genes provided in Table 9 such that expression of the recombinant construct is linked to expression of a particular gene in a desired cell. In one aspect, the recombinant gene construct is inserted into or around the 3' untranslated region of any one of the aforementioned genes, and the first and second gene sequences of the recombinant gene construct are homologous to regions of the selected gene that are 5' and 3' of the selected insertion site, respectively. The specific location of the insertion site can vary, and thus the specific sequences of the first and second cell-specific gene sequences of the recombinant construct can likewise vary. However, the selection of these parameters is well within the level of those skilled in the art using, for example, the known sequences and structures of these genes readily available in the art via the NCBI gene database and the provided gene ID numbers.

[0096] In some embodiments, the recombinant gene construct further comprises one or more self-cleaving peptide-encoding nucleotide sequences, and the self-cleaving peptide-encoding nucleotide sequences are arranged within the construct in a manner effective to mediate the translation of one or more immune checkpoint proteins in vivo. A “self-cleaving peptide” is a viral oligopeptide sequence 18-22 amino acids in length that mediates ribosome skipping during translation in eukaryotic cells (Liu et al., “Systemic Comparison of 2A peptides for Cloning Multi-Genes in a Polycistronic Vector,” Scientific Reports 7: Article Number 2193 (2017), which is hereby incorporated by reference in its entirety). A non-limiting example of such a self-cleaving peptide is peptide 2A, a short protein sequence first discovered in picornaviruses. Peptide 2A functions by skipping the synthesis of a peptide bond at the C-terminus of the 2A element by the ribosome, resulting in a separation between the end of the 2A sequence and the peptide downstream thereof. This “cleavage” occurs between the C-terminal glycine and proline residues. Thus, successful ribosome skipping and resumption of translation results in individual “cleaved” proteins, with the protein upstream of the 2A element binding to the full 2A peptide except for the C-terminal proline, and the protein downstream of the 2A element binding to one N-terminal proline (Liu et al., “Systemic Comparison of 2A peptides for Cloning Multi-Genes in a Polycistronic Vector,” Scientific Reports 7: Article Number 2193 (2017), which is hereby incorporated by reference in its entirety).

[0097] Exemplary self-cleaving peptides that can be incorporated into recombinant gene constructs include, without limitation, porcine teschovirus 1 2A (P2A), foot-and-mouth disease virus 2A (F2A), thosea asigna virus 2A (T2A), equine rhinitis A virus 2A (E2A), cytoplasmic polyhedrosis virus (BmCPV 2A), and flacherie virus (BmIFV 2A). Nucleotide sequences encoding these self-cleaving peptides suitable for inclusion in the recombinant gene constructs described herein are provided in Table 10 below.

[0098] (Table 10) Appropriate self-cleaving peptide coding nucleotide sequences TIFF0007708737000023.tif127161*See Wang et al., "2A Self-Cleaving Peptide-Based Multi-Gene Expression System in the Silkworm Bombyx mori," Sci.Rep. 5:16273 (2015) and U.S. Patent Application Publication No. 2018 / 0369280 to Schmitt et al., which are hereby incorporated by reference in their entirety.

[0099] In some embodiments, the recombinant gene construct further includes an inducible cell death gene disposed within the construct in a manner effective to achieve inducible cell suicide. An inducible cell death gene refers to a genetically encoded element that allows for the selective destruction of expressing cells in the face of unacceptable toxicity due to the administration of an activating agent.

[0100] Several inducible cell death genes are well known in the art and suitable for inclusion in the recombinant gene constructs described herein (see Stavrou et al., "A Rapamycin-Activated Caspase 9-Based Suicide Gene," Mol. Ther. 26(5):1266-1276(2018), which is incorporated herein by reference in its entirety). Exemplary suicide genes include, without limitation, RQR8 and huEGFRt, surface proteins recognized by therapeutic monoclonal antibodies (mAbs); herpes simplex virus thymidine kinase (HSV-TK), an inducible cell death gene activated by ganciclovir; inducible caspase 9 (iCasp9), a fusion of the catalytic domain of caspase 9 and mutant FKBP12 that enables docking of a small molecule chemical inducer of dimerization (CID, AP1903 / AP20187); rapamycin-activated caspase 9 (rapaCasp9), an inducible cell death gene activated by rapamycin (see Stavrou et al., "A Rapamycin-Activated Caspase 9-Based Suicide Gene," Mol. Ther. 26(5):1266-1276(2018), which is incorporated herein by reference in its entirety); and inducible caspase 3 (iCasp3), a fusion of caspase 3 and a mutant FK506 binding domain that enables docking of CID (AP20187) (see Ono et al., "Exposure to Sequestered Self-Antigens in vivo is not sufficient for the Induction of Autoimmune Diabetes," PLos One 12(3):e0173176(2017) and MacCorkle et al., "Synthetic Activation of Caspases:Artificial Death Switches," PNAS 95(7):3655-3660(1998), which are incorporated herein by reference in their entirety).In another aspect, the recombinant gene construct comprises an inducible cell death gene linked to the expression of a cell division gene, such as cyclin-dependent kinase 1 (CDK1) (Liang et al., “Linking a Cell-Division Gene and a Suicide Gene to Define and Improve Cell Therapy Safety,” Nature 563:701-704 (2018), which is incorporated herein by reference in its entirety).

[0101] In some aspects, the recombinant gene construct further comprises a selectable marker. Suitable selectable markers for mammalian cells are known in the art and include, for example, thymidine kinase, dihydrofolate reductase (in combination with methotrexate as a DHFR amplifying agent), aminoglycoside phosphotransferase, hygromycin B phosphotransferase, asparagine synthetase, adenosine deaminase, metallothionein, and antibiotic resistance genes such as the puromycin resistance gene or the neomycin resistance gene. Exemplary antibiotic resistance gene sequences that can be used as selectable markers in the recombinant gene constructs described herein are provided in Table 11 below.

[0102] (Table 11) Suitable selectable marker gene sequences TIFF0007708737000024.tif141163TIFF0007708737000025.tif145163

[0103] When the recombinant gene construct comprises a mammalian selectable marker, the selectable marker can be operably linked to a constitutive mammalian promoter.

[0104] Exemplary constitutive mammalian promoters suitable for inclusion in the recombinant constructs described herein are well known in the art and are shown in Table 12 below (Qin et al., "Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter," PLoS One 5(5):e10611(2010), which is incorporated herein by reference in its entirety).

[0105] (Table 12) Appropriate promoter sequences TIFF0007708737000026.tif30163TIFF0007708737000027.tif246163TIFF0007708737000028.tif246163TIFF0007708737000029.tif102163*See Qin et al., "Systematic Comparison of Constitutive Promoters and the Doxycycline-Inducible Promoter," PLoS One 5(5):e10611(2010), which is incorporated herein by reference in its entirety.

[0106] In some embodiments, the recombinant gene construct further encodes at least one marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, and epitope tags.

[0107] In some instances, the marker domain can be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent protein (e.g., GFP, GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1), yellow fluorescent protein (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent protein (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-Sapphire), cyan fluorescent protein (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent protein (mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-monomer, HcRed-Tandem, HcRed1, AsRed2, mRasberry, mStrawberry, Jred), and orange fluorescent protein (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein.

[0108] In other instances, the marker domain can be a purification tag and / or an epitope tag. Exemplary tags include, but are not limited to, glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein, thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, biotin carboxyl carrier protein (BCCP), and calmodulin.

[0109] The marker domain can be operably linked to a constitutive mammalian promoter. For example, in some embodiments, the constitutive mammalian promoter is EF1a, and the marker domain is operably linked to EF1a. According to this embodiment, the marker domain can be CopGFP. Exemplary nucleotide sequences encoding suitable marker domain sequences are shown in Table 13 below.

[0110] (Table 13) Suitable marker domain sequences TIFF0007708737000030.tif174166TIFF0007708737000031.tif150166

[0111] In some embodiments, the recombinant gene constructs of the present disclosure are incorporated into a delivery vector. Suitable delivery vectors include, without limitation, plasmid vectors, without limitation, vaccinia vectors, lentiviral vectors (integrating competent or integrating defective lentiviral vectors), adenoviral vectors, viral vectors including adeno-associated viral vectors, baculovirus expression vectors, transposon-based vectors, or any other vector suitable for introducing the recombinant gene constructs described herein into cells by any means that promotes gene / cell-selective expression of the recombinant construct.

[0112] Another aspect of the present disclosure relates to a preparation of one or more cells comprising the recombinant gene constructs described herein. The preparation can be a preparation of cells from any organism. In some embodiments, the preparation is a preparation of mammalian cells, e.g., a preparation of rodent cells (i.e., mouse or rat cells), rabbit cells, guinea pig cells, cat cells, dog cells, pig cells, horse cells, bovine cells, sheep cells, monkey cells, or human cells. In one embodiment, the preparation is a preparation of human cells. Suitable cells comprising the recombinant gene constructs described herein include primary or immortalized embryonic cells, fetal cells, or adult cells at any stage of the lineage, e.g., totipotent cells, pluripotent cells, multipotent cells, or differentiated cells.

[0113] In some embodiments, the preparation is a preparation of pluripotent stem cells. Pluripotent stem cells can give rise to cells of any of the three germ layers (i.e., endoderm, mesoderm, and ectoderm). In one embodiment, the preparation of cells comprising a recombinant gene construct is a preparation of induced pluripotent stem cells (iPSCs). In another embodiment, the preparation of cells comprising a recombinant gene construct is a preparation of pluripotent embryonic stem cells.

[0114] In another embodiment, the preparation of one or more cells can be a preparation of multipotent stem cells. Multipotent stem cells can develop into a limited number of cell types of a particular lineage. Examples of multipotent stem cells include progenitor cells, such as neural progenitor cells that give rise to cells of the central nervous system, such as neurons, astrocytes, and oligodendrocytes. Progenitor cells are an immature or undifferentiated population of cells that have the potential to mature and differentiate into more specialized, differentiated cell types. Progenitor cells can also proliferate to generate more progenitor cells that are likewise immature or undifferentiated. Suitable preparations of progenitor cells comprising a recombinant gene construct include, without limitation, preparations of neural progenitor cells, neuronal progenitor cells, glial progenitor cells, progenitor cells biased towards oligodendrocytes, and progenitor cells biased towards astrocytes. Other suitable progenitor cell populations include, without limitation, bone marrow progenitor cells, cardiac progenitor cells, endothelial progenitor cells, epithelial progenitor cells, hematopoietic progenitor cells, liver progenitor cells, bone progenitor cells, muscle progenitor cells, pancreatic progenitor cells, lung progenitor cells, kidney progenitor cells, vascular progenitor cells, and retinal progenitor cells.

[0115] Preparations of cells containing the recombinant gene constructs described herein can also be preparations of terminally differentiated cells. In one aspect, the preparation of one or more cells can be a preparation of terminally differentiated neurons, oligodendrocytes, or astrocytes. In another aspect, the preparation of one or more cells containing the recombinant gene construct is an adipocyte, chondrocyte, endothelial cell, epithelial cell (keratinocyte, melanocyte), bone cell (osteoblast, osteoclast), hepatocyte (cholangiocyte, hepatocyte), muscle cell (cardiomyocyte, skeletal muscle cell, smooth muscle cell), retinal cell (ganglion cell, Müller cell, photoreceptor cell), retinal pigment epithelial cell, kidney cell (podocyte, proximal tubule cell, collecting duct cell, distal tubule cell), adrenal cell (adrenal cortical cell, adrenal medullary cell), pancreatic cell (alpha cell, beta cell, delta cell, epsilon cell, pancreatic polypeptide-producing cell, exocrine cell), lung cell, bone marrow cell (early B cell development, early T cell development, macrophage, monocyte), urothelial cell, fibroblast, parathyroid cell, thyroid cell, hypothalamic cell, pituitary cell, salivary gland cell, ovarian cell, and testicular cell preparation.

[0116] Additional exemplary cell types that may contain the recombinant gene constructs described herein include, without limitation, placental cells, keratinocytes, basal epidermal cells, urothelial cells, salivary gland cells, mucosal cells, serous cells, von Ebner gland cells, mammary gland cells, lacrimal gland cells, eccrine sweat gland cells, apocrine sweat gland cells, MpH gland cells, sebaceous gland cells, Bowman gland cells, Brunner gland cells, seminal vesicle cells, prostate cells, bulbourethral gland cells, Bartholin gland cells, Littre gland cells, endometrial cells, goblet cells of the airway or digestive tract, gastric mucosal cells, pepsinogen cells of the gastric gland, acid-secreting cells of the gastric gland, insulin-producing P cells, glucagon-producing α cells, somatostatin-producing δ cells, pancreatic polypeptide-producing cells, pancreatic duct cells, Paneth cells of the small intestine, type II pneumocytes of the lung, Clara cells of the lung, anterior pituitary cells, intermediate pituitary cells, posterior pituitary cells, hormone-secreting cells of the digestive tract or airway, gonadal cells, juxtaglomerular cells of the kidney, macula densa cells of the kidney, perivascular pole cells of the kidney, mesangial cells of the kidney, brush border cells of the intestine, striated duct cells of exocrine glands, gallbladder epithelial cells, brush border cells of the proximal renal tubule of the kidney, distal renal tubule cells of the kidney, intercalated cells of the collecting duct, principal cells of the parathyroid gland, basal cells of the parathyroid gland, hepatocytes, adipocytes, type I pneumocytes, pancreatic duct cells, non-striated duct cells of sweat glands, non-striated duct cells of salivary glands, non-striated duct cells of mammary glands, parietal cells of renal glomeruli, podocytes of renal glomeruli, cells of the thin part of the loop of Henle, collecting duct cells, duct cells of seminal vesicles, duct cells of the prostate, vascular endothelial cells, synovial cells, serosal cells, squamous epithelial cells covering the outer lymphatic cavity of the ear, cells covering the inner lymphatic cavity of the ear, choroid plexus cells, squamous epithelial cells of the leptomeninges, ciliary body epithelial cells of the eye, corneal endothelial cells, ciliated cells with propulsive function, ameloblasts, maculae cells of the vestibular organ of the ear, interdental cells of the organ of Corti, fibroblasts, pericytes of capillaries, nucleus pulposus cells of intervertebral discs, cementoblasts, cementocytes, odontoblasts, odontoblasts, chondrocytes, osteocytes, osteoprogenitor cells, vitreous cells of the vitreous body of the eye, stellate cells of the outer lymphatic cavity of the ear, skeletal muscle cells, cardiomyocytes, smooth muscle cells, myoepithelial cells, platelets, megakaryocytes, monocytes, connective tissue macrophages, Langerhans cells, osteoclasts, dendritic cells, microglial cells, neutrophils, eosinophils, basophils, mast cells, plasma cells, helper T cells, suppressor T cells, killer T cells, killer cells, rod cells, cone cells, inner hair cells of the organ of Corti, outer hair cells of the organ of Corti, type I hair cells, cells of the vestibular organ of the ear,Type II cells of the inner ear vestibular apparatus, type II taste bud cells, olfactory neurons, basal cells of the olfactory epithelium, type I carotid body cells, type II carotid body cells, Merkel cells, primary sensory neurons, cholinergic neurons of the autonomic nervous system, adrenergic neurons of the autonomic nervous system, peptidergic neurons of the autonomic nervous system, inner pillar cells of the Corti organ, outer pillar cells of the Corti organ, inner supporting cells of the Corti organ, outer supporting cells of the Corti organ, border cells, Hensen cells, supporting cells of the vestibular apparatus, supporting cells of taste buds, supporting cells of the olfactory epithelium, Schwann cells, satellite cells, enteric glial cells, neurons of the central nervous system, astrocytes of the central nervous system, oligodendrocytes of the central nervous system, anterior lens epithelial cells, lens fiber cells, melanocytes, retinal pigment epithelial cells, iris pigment epithelial cells, oogonia, oocytes, spermatocytes, spermatogonia, ovarian cells, Sertoli cells, and thymic epithelial cells are included.

[0117] According to this aspect of the present disclosure, the recombinant gene construct is integrated into the chromosome of one or more cells in the preparation. The term "integrated," when used in connection with the recombinant gene constructs of the present disclosure, means that the recombinant gene construct is inserted into the genome or genomic sequence of one or more cells in the preparation. When integrated, the integrated recombinant gene construct is replicated and passed on to daughter cells of dividing cells in the same manner as the original genome of the cell.

[0118] According to the design of the recombinant gene construct, genomic integration of the construct targets the desired gene of interest in order to achieve cell-selective expression of one or more immune checkpoint protein-encoding nucleotide sequences and / or nucleotide sequences encoding one or more substances that reduce the expression of one or more HLA-I and / or HLA-II molecules. In some embodiments, the gene of interest is a gene that is expressed limitedly in terminally differentiated cells. In some embodiments, the recombinant gene construct is integrated into a gene that is selectively expressed in oligodendrocytes such as SOX10, MYRF, MAG or MBP. In some embodiments, the recombinant gene construct is integrated into a gene that is selectively expressed in astrocytes such as GFAP or AQP4. In some embodiments, the recombinant gene construct is a gene that is selectively expressed in neurons such as SYN1, MAP2 and ELAV4; a gene that is selectively expressed in dopaminergic neurons such as TH or DDC; a gene that is selectively expressed in medium spiny neurons and interneurons such as GAD65 or GAD67; or a gene that is selectively expressed in cholinergic neurons such as CHAT. According to these embodiments, the nucleotide sequence encoding one or more immune checkpoint proteins and / or the nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I and HLA-II molecules are expressed conditionally (i.e., transcribed and / or translated) in terminally differentiated cells. Expression of the recombinant gene constructs described herein in preparations of terminally differentiated cells makes those cells less susceptible to attack by immune cells in the in vivo environment. Thus, when cells containing the recombinant gene construct are transplanted into a host subject, as described in more detail below, the cells, in their differentiated state, are protected from attack by the host immune system as a result of the expression of one or more immune checkpoint proteins and / or the expression of one or more substances that inhibit one or more HLA-I / HLA-II proteins.

[0119] Another aspect of the disclosure relates to a method of administering a preparation of cells comprising the recombinant gene constructs described herein to a subject in need thereof.

[0120] As used herein, a "subject" or "patient" suitable for administering a preparation of cells comprising the recombinant gene constructs described herein encompasses any animal, preferably a mammal. Suitable subjects include, without limitation, domesticated and non-domesticated animals such as rodents (mice or rats), cats, dogs, rabbits, horses, sheep, pigs, and monkeys. In one aspect, the subject is a human subject. Suitable human subjects include, without limitation, infants, children, adults, and elderly subjects.

[0121] In one aspect, the subject requires a terminally differentiated cell type. For example, the subject has a condition mediated by a reduction or dysfunction of a differentiated cell population. Thus, a preparation of cells comprising the recombinant gene construct is administered to such a subject in an amount sufficient to restore normal levels and / or function of the differentiated cell population in the selected subject, thereby treating the condition. In some aspects, the preparation of cells comprising the recombinant gene construct administered to the subject is a preparation of a differentiated cell population that is reduced or dysfunctional in the subject. In another aspect, the preparation of cells comprising the recombinant gene construct administered to the subject is a preparation of precursor cells or progenitor cells of the differentiated cell population. According to this aspect, the precursor cells or progenitor cells comprising the recombinant gene construct mature or differentiate into the desired differentiated cell population after administration to a subject in need thereof.

[0122] In practicing the methods of the disclosure, "treating" or "treatment" includes inhibiting, preventing, ameliorating, or delaying the onset of a particular condition. Treating and treatment also encompass the amelioration of one or more symptoms of a condition or disorder. Treating and treatment encompass a modification of the course of a condition or disease progression as compared to the condition or disease in the absence of a therapeutic intervention.

[0123] In some embodiments, administration is effective to reduce at least one symptom of a disease or condition associated with a decrease or dysfunction of a differentiated cell type. In another embodiment, administration is effective to mediate an improvement of a disease or condition associated with a decrease or insufficiency of a differentiated cell type. In another embodiment, administration is effective to extend the survival period of a subject as compared to the survival period expected if the administration were not performed.

[0124] According to this aspect of the disclosure, a preparation of one or more cells comprising a recombinant gene construct can be autologous / self ( "self") for a recipient subject. In another embodiment, a preparation of cells comprising a recombinant gene construct is non - autologous ( "non - self", e.g., allogeneic, syngeneic or xenogeneic) to the recipient subject.

[0125] In practicing the methods of the disclosure, administration can be carried out in the absence of immunosuppression or in a modified course of immunosuppressive therapy. For example, in one embodiment, administration can be followed by an initial series of immunosuppressive therapies, but long - term administration of immunosuppressive therapy is not required.

[0126] In one embodiment, a method of treating a subject in need of a preparation of the cells described herein comprises treating a subject having a condition mediated by a decrease or insufficiency of oligodendrocytes or a decrease or insufficiency of myelin produced by oligodendrocytes. This method comprises the step of administering to the subject a preparation of cells comprising the recombinant gene construct described herein, wherein the preparation of cells is a preparation of glial progenitor cells or progenitor cells biased towards oligodendrocytes. According to this method, the cells are administered in an amount sufficient and under conditions effective to treat a condition mediated by a decrease or insufficiency of oligodendrocytes or a decrease or insufficiency of myelin.

[0127] Oligodendrocytes produce myelin, an insulating sheath necessary for the saltatory conduction of electrical impulses along axons (Goldman et al., "How to Make an Oligodendrocyte," Development 142(23):3983-3985 (2015), which is hereby incorporated by reference in its entirety). As described herein, a decrease in oligodendrocytes results in demyelination, which leads to neurological dysfunction in a wide range of diseases ranging from pediatric leukodystrophies and cerebral palsy to multiple sclerosis and white matter stroke.

[0128] States mediated by a decrease in myelin or oligodendrocyte dysfunction or decrease that can be treated according to the methods and cell preparations containing the recombinant gene constructs described herein include hypomyelination disorders and demyelination disorders. In one aspect, the state is an autoimmune demyelinating state such as, for example, multiple sclerosis, Schilder's disease, neuromyelitis optica, transverse myelitis, and optic neuritis. In another aspect, the myelin-related disorder is a vascular white matter encephalopathy such as, for example, subcortical stroke, diabetic leukoencephalopathy, hypertensive leukoencephalopathy, age-related white matter disease, and spinal cord injury. In another aspect, the myelin-related state is a radiation-induced demyelinating state. In another aspect, the myelin-related disorder is a pediatric leukodystrophy such as, for example, Pelizaeus-Merzbacher disease, Tay-Sachs disease, Sandhoff gangliosidosis, Krabbe disease, metachromatic leukodystrophy, mucopolysaccharidosis (e.g., Sly disease), Niemann-Pick disease type A, adrenoleukodystrophy, Canavan disease, vanishing white matter disease, and Alexander disease. In yet another aspect, the myelin-related state is periventricular leukomalacia or cerebral palsy.

[0129] Methods for generating glial progenitor cells or progenitor cells biased towards oligodendrocytes suitable for treating a subject having a condition mediated by a reduction or dysfunction of oligodendrocytes or myelin are known in the art, see, for example, U.S. Patent No. 9,790,553 to Goldman et al., U.S. Patent No. 10,190,095 to Goldman et al., and U.S. Patent Application Publication No. 2015 / 0352154 to Goldman et al., each of which is incorporated herein by reference in its entirety. These cells are modified according to the present disclosure to contain a recombinant gene vector at any point prior to transplantation. For example, in one aspect, the recombinant gene construct is introduced into glial progenitor cells or progenitor cells biased towards oligodendrocytes immediately prior to transplantation. In another aspect, the recombinant gene construct is introduced into progenitor cells biased towards glial progenitor cells or oligodendrocytes, such as neural progenitor cells or pluripotent stem cells.

[0130] In another aspect, a method of treating a subject in need of a preparation of the cells described herein comprises treating a condition mediated by a reduction or dysfunction of astrocytes. The method comprises administering to the subject a preparation of cells comprising the recombinant gene construct described herein, the preparation of cells being a preparation of glial progenitor cells or progenitor cells biased towards astrocytes. The cells are administered in an amount sufficient to treat a condition mediated by a reduction or dysfunction of astrocytes and under conditions effective to treat the condition.

[0131] As noted above, astrocytes are the largest and most common type of glial cell in the central nervous system. Astrocytes contribute to the formation of the blood-brain barrier, are involved in the maintenance of extracellular ionic and chemical homeostasis, are involved in the response to injury, and affect neuron development and plasticity.

[0132] Thus, in some aspects, the condition mediated by the reduction or dysfunction of astrocytes is a neurodegenerative disorder. Neurodegenerative disorders associated with the reduction of astrocytes that can be treated according to the methods and cell preparations of the present disclosure include, without limitation, Parkinson's disease (PD), Alzheimer's disease (AD) and other dementias, degenerative nerve diseases, encephalitis, epilepsy, hereditary brain disorders, head and brain malformations, hydrocephalus, multiple sclerosis, amyotrophic lateral sclerosis (ALS or Lou Gehrig's disease), Huntington's disease (HD), prion disease, frontotemporal dementia, Lewy body dementia, progressive supranuclear palsy, corticobasal degeneration, multiple system atrophy, hereditary spastic paraplegia, spinocerebellar atrophy, amyloidosis, motor neuron disease (MND), spinocerebellar ataxia (SCA), and stroke and spinal muscular atrophy (SMA).

[0133] Methods for generating glial progenitor cells or astrocyte-biased progenitor cells suitable for treating a subject having a condition mediated by the reduction or dysfunction of astrocytes are known in the art, see, for example, U.S. Patent Application Publication No. 2015 / 0352154 to Goldman et al., which is hereby incorporated by reference in its entirety. These cells are modified according to the present disclosure to contain a recombinant gene vector at any point in time prior to transplantation into a subject in need of these cells. For example, in one aspect, the recombinant gene construct is introduced into glial progenitor cells or astrocyte-biased progenitor cells immediately prior to transplantation. In another aspect, the recombinant gene construct is introduced into glial progenitor cells or astrocyte-biased progenitor cells, such as progenitor cells of neural progenitor cells or pluripotent stem cells.

[0134] In another aspect, a method of treating a subject in need of a cell preparation described herein includes treating a condition mediated by the reduction or dysfunction of neurons. The method includes administering to the subject a cell preparation containing a recombinant gene construct described herein, wherein the cell preparation is a preparation of neuronal progenitor cells. The cells are administered in an amount sufficient to treat a condition mediated by the reduction or dysfunction of neurons and under conditions effective to treat the condition.

[0135] According to this aspect, the condition to be treated can be a condition mediated by a decrease or dysfunction of a specific type of neuron. For example, in one aspect, the condition to be treated is a condition mediated by a decrease or dysfunction of cholinergic neurons. Exemplary conditions mediated by a decrease or dysfunction of cholinergic neurons include Alzheimer's disease, corticobasal degeneration, Lewy body dementia, frontotemporal dementia, multiple system atrophy, Parkinson's disease, Parkinson's disease dementia, and progressive supranuclear palsy (Roy et al., "Cholinergic Imaging in Dementia Spectrum Disorders," Eur. J. Nucl. Med. Mol. Imaging 43:1376-1386 (2016), which is hereby incorporated by reference in its entirety).

[0136] In another aspect, the condition to be treated is a condition mediated by a decrease or dysfunction of dopaminergic neurons. Exemplary conditions mediated by a decrease or dysfunction of dopaminergic neurons include Parkinson's disease, Parkinsonian disorders (e.g., juvenile parkinsonism, Ramsay-Hunt paralysis syndrome), and mental disorders (e.g., schizophrenia, depression, drug dependence).

[0137] In another aspect, the condition to be treated is a condition mediated by a decrease or dysfunction of medium spiny neurons and / or cortical interneurons. Exemplary conditions mediated by a decrease or dysfunction of medium spiny neurons and / or cortical interneurons include Huntington's disease, epilepsy, anxiety, and depression (Powell et al., "Genetic Disruption of Cortical Interneuron Development Causes Region-and GABA Cell Type-Specific Deficits, Epilepsy, and Behavioral Dysfunction," J. Neurosci. 23(2):622-631 (2003), which is hereby incorporated by reference in its entirety).

[0138] Methods for generating neuron progenitor cells suitable for treating a subject having a condition mediated by neuron loss or dysfunction are known in the art, for example, Goldman, SA., "Transplanted Neural Progenitors Bridge Gaps to Benefit Cord-Injured Monkeys." Nat. Med. 24(4):388-390 (2018); Roy et al., "Functional Engraftment of Human ES Cell-Derived Dopaminergic Neurons Enriched by Coculture with Telomerase-Immortalized Midbrain Astrocytes," Nat. Med. 12(11):1259-1268 (2006); Nunes et al., "Identification and Isolation of Multipotential Neural Progenitor Cells from the Subcortical White Matter of the Adult Human Brain," Nat. Med. 9(4):439-447 (2003), U.S. Patent No. 6,812,027 to Goldman et al.; U.S. Patent No. 7,150,989 to Goldman et al.; U.S. Patent No. 7,468,277 to Goldman et al.; U.S. Patent No. 7,785,882 to Goldman; U.S. Patent No. 8,263,406 to Goldman et al.; U.S. Patent No. 8,642,332 to Goldman et al.; and U.S. Patent No. 8,945,921 to Goldman et al., each of which is incorporated herein by reference in its entirety. These cells are modified according to the present disclosure to contain a recombinant gene vector at any point in time prior to transplantation into a subject in need thereof. For example, in one aspect, the recombinant gene construct is introduced into the neuron progenitor cells immediately prior to transplantation. In another aspect, the recombinant gene construct is introduced into neuron progenitor cells, such as progenitor cells of neural progenitor cells or pluripotent stem cells.

[0139] When practicing the methods of the invention that include cell replacement in the central nervous system, the cell preparations described herein can be administered systemically into the circulation or directly to one or more sites of the brain, brainstem, spinal cord, or combinations thereof.

[0140] When the cell preparation is injected systemically into the circulation, the cell preparation can be placed in a syringe, cannula, or other injection device in order to be accurately positioned at a preselected site. The term "injectable" means that the cell preparation can be dispensed from a syringe under normal conditions at atmospheric pressure.

[0141] Methods for directly administering various nerve tissues / cells to the host brain (i.e., transplantation) are well known in the art. In some embodiments, the preparation is administered into the ventricles, the corpus callosum, or the parenchyma.

[0142] Parenchymal administration, i.e., administration into the host brain, is achieved (as compared to extracerebral or extraparenchymal transplantation) by injection or deposition of cells into the brain parenchyma at the time of administration. Intrapenchymal transplantation can be performed using two approaches: (i) injecting the cell preparation into the host brain parenchyma, or (ii) preparing a cavity by surgical means to expose the host brain parenchyma and then depositing the cell preparation into the cavity. Both methods provide parenchymal deposition between the cell preparation and the host brain tissue at the time of administration and both facilitate anatomical integration between the graft (i.e., the cell preparation) and the host brain tissue.

[0143] Alternatively, the cell graft can be placed into the ventricles, such as the cerebral ventricles, or subdural, i.e., on the surface of the host brain separated from the host brain parenchyma by the intervening pia or arachnoid and pia mater. Transplantation into the ventricles can be achieved by injection of donor cells or by growing the cells in a matrix such as 3% collagen and then forming a solid tissue plug that can be transplanted into the ventricles to prevent dislocation of the graft. In subdural transplantation, after forming a slit in the dura, cells can be injected near the surface of the brain.

[0144] For transplantation into the cavity, which may be preferred for spinal cord transplantation, the bone covering the brain is removed and bleeding is stopped with a material such as Gelfoam, and tissue is removed from the area close to the outer surface of the CNS to form a transplantation cavity. The cavity can be formed using suction. Then, a preparation of cells is placed into the cavity. Multiple preparations of cells may be placed into the same cavity. In some embodiments, the site of transplantation is determined by the CNS disorder being treated.

[0145] Injection into a selected region of the host brain can be performed by drilling a hole to allow insertion of the needle of a microsyringe and puncturing the dura mater. The microsyringe is preferably attached to a stereotaxic frame and three-dimensional stereotaxic coordinates are selected to position the needle at the desired location in the brain or spinal cord. Cells can also be introduced into the putamen, basal ganglia, hippocampal cortex, striatum, substantia nigra or caudal region of the brain, and into the spinal cord.

[0146] The number of cells in a given volume can be determined by well-known routine procedures and equipment. The percentage of cells in a cell mixture of a given volume can be determined by substantially the same procedures. Cells can be easily counted manually or by using an automated cell counter. Specific cells can be determined in a given volume using specific staining and visual inspection, as well as by automated methods using specific binding reagents, typically antibodies, fluorescent tags, and fluorescence-activated cell sorters.

[0147] Cell preparations can be administered by dosages and techniques well known to those of ordinary skill in the medical and veterinary arts, taking into account factors such as the age, gender, weight and condition of a particular patient, as well as the formulation to be administered. Appropriate dosages for use in accordance with the various aspects described herein depend on a number of factors. This can vary quite significantly depending on the circumstances. Parameters for determining the optimal dosages administered for primary and adjuvant therapies generally include some or all of the following: the disease being treated and its stage; the species of the subject, its health status, gender, age, weight; the immune competence of the subject; other therapies being administered; and potential complications anticipated from the subject's medical history or genotype. The parameters also can include whether the cells are autologous, syngeneic, allogeneic, or xenogeneic; their potency (specific activity); the site and / or distribution to which the cell / media must be targeted to be effective; and such characteristics of the site such as accessibility to the cell / media and / or engraftment of the cells. Additional parameters include co - administration with other factors (such as growth factors and cytokines). The optimal dosage in a given situation also takes into account how the cell / media are formulated, how they are administered, and the extent to which the cell / media localize to the target site after administration. Finally, determination of the optimal dosage necessarily provides an effective dosage that does not fall below the threshold of maximum beneficial effect and does not exceed the threshold above which the disadvantages of dosages with increased harmful effects related to the dosage.

[0148] In the case of a fairly pure preparation of cells, the optimal dosage in various aspects will be in the range of about 10 4 ~ about 10 9 cells per administration. In some aspects, the optimal dosage per administration will be about 10 5 ~ about 10 7 cells. In many aspects, the optimal dosage per administration will be about 5×10 5 ~ about 5×10 6 cells.

[0149] It should be understood that the single dose can be delivered once, divided, or continuously over a period of time. The total dose may also be delivered to a single location or spread out divided among several locations.

[0150] Human subjects are generally treated for longer than experimental animals; however, the treatment generally has a length proportional to the length of the disease process and the effectiveness of the treatment. One of ordinary skill in the art will consider this in determining the appropriate dose for humans using the results of other procedures conducted in animals such as humans and / or rats, mice, non-human primates, etc. Such determination is enabled for one of ordinary skill in the art to make it without undue experimentation based on these considerations and taking into account the guidance provided by this disclosure and the prior art.

[0151] The regimens suitable for the initial administration and further doses or continuous administration may all be the same or variable. Appropriate regimens can be ascertained by one of ordinary skill in the art from this disclosure, the documents cited herein, and the knowledge in the art.

[0152] In some embodiments, the cell preparation is administered to the subject in a single dose. In other embodiments, the cell preparation is administered to the subject in a series of two or more consecutive doses. In some other embodiments where the cell preparation is administered in a single dose, two doses, and / or more than two doses, the doses may be the same or different and are administered at equal or unequal intervals therebetween.

[0153] Preparations of cells can be administered at many frequencies over a wide range of times. In some embodiments, they are administered over a period of less than one day. In other embodiments, they are administered over a period of 2, 3, 4, 5, or 6 days. In some embodiments, they are administered once or multiple times a week over a period of several weeks. In other embodiments, they are administered over a period of several weeks to several months. In various embodiments, they can be administered over a period of several months. In other embodiments, they can be administered over a period of one year or multiple years. Generally, the length of treatment is proportional to the length of the disease process, the effectiveness of the applied therapy, and the condition and response of the subject being treated.

[0154] The selection of a formulation for administering a composition for a given use depends on a variety of factors. Among these are prominent the species of the subject, the disorder being treated, the nature of the dysfunction or disease and its state and distribution in the subject, the nature of other therapies and agents being administered, the optimal route for administration, the viability by the route, the dosing regimen, as well as other factors that will be apparent to those skilled in the art. In particular, for example, the selection of appropriate carriers and other additives depends on the exact route of administration and the nature of the particular dosage form.

[0155] For example, cell survival can be an important determinant of the effectiveness of cell-based therapies. This applies to both primary and adjuvant therapies. When the target site is not suitable for cell seeding and cell proliferation, another concern arises. This can prevent access of the therapeutic cells to the site and / or engraftment there. Thus, measures can be taken to increase cell survival and / or to overcome problems posed by barriers to seeding and / or proliferation.

[0156] The final formulation may contain a cell / media and optionally an aqueous suspension of a protein and / or a small molecule, and typically involves adjusting the ionic strength of the suspension to isotonicity (i.e., about 0.1 to about 0.2) and physiological pH (i.e., pH about 6.8 to about 7.5). The final formulation also typically contains a fluid lubricant such as maltose that must be tolerated by the body. Exemplary lubricant components include glycerol, glycogen, maltose, etc. Organic polymer substrates such as polyethylene glycol and hyaluronic acid, as well as non-fibrillar collagen such as succinylated collagen, can also function as lubricants. Such lubricants are generally used to improve the injectability, invasiveness, and dispersibility of the injected substance at the injection site and to reduce the amount of spiking by changing the viscosity of the composition. This final formulation is, by definition, the cells described herein in a pharmaceutically acceptable carrier.

[0157] Multiple preparations of cells can be administered simultaneously at different locations, such as intrathecal and intravenous co-administration, to maximize the potential for targeting the affected area.

[0158] A further aspect relates to a preparation of one or more cells, wherein the cells of the preparation are modified to conditionally express one or more immune checkpoint proteins at increased levels compared to the corresponding wild-type cells. In one embodiment, the cells of the preparation are further modified to conditionally express one or more endogenous HLA-I proteins at reduced levels compared to the corresponding wild-type cells. In some embodiments, the cells of the preparation are further modified to conditionally express one or more HLA-II proteins at reduced levels compared to the corresponding wild-type cells.

[0159] Another aspect relates to a preparation of one or more cells, wherein the cells of the preparation are modified to conditionally express one or more endogenous HLA-I proteins at a reduced level compared to the corresponding wild-type cells. In some embodiments, the cells of the preparation are further modified to conditionally express one or more HLA-II proteins at a reduced level compared to the corresponding wild-type cells.

[0160] Exemplary immune checkpoint proteins that are conditionally expressed in the modified cells of the preparation are described in detail above and include, for example, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), CD47, HLA-E, CD200, and CTLA-4.

[0161] Similarly, exemplary HLA-I proteins whose expression is conditionally reduced in the modified cells of the preparation are described above and include, for example, one or more of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and combinations thereof. Exemplary HLA-II proteins whose expression is conditionally reduced in the modified cells of the preparation include any one or more of HLA-DM, HLA-DO, HLA-DP, HLA-DQ, HLA-DR.

[0162] Yet another aspect of the disclosure relates to a method of producing conditionally immune-protected cells. The method includes the step of modifying a cell to (i) conditionally express one or more immune checkpoint proteins at an increased level or (ii) conditionally express one or more substances that reduce the surface expression of one or more endogenous HLA proteins. In another embodiment, the method includes the step of modifying a cell to (i) conditionally express one or more immune checkpoint proteins at an increased level and (ii) conditionally express one or more substances that reduce the surface expression of one or more endogenous HLA proteins.

[0163] According to this aspect of the disclosure, the conditional expression of one or more immune checkpoint proteins and / or the conditional expression of one or more substances that reduce the expression of one or more endogenous HLA proteins are functionally linked to the expression of genes that are expressed limitedly in terminally differentiated cells. Suitable terminally differentiated cells and the genes selectively expressed therein are described in detail above.

[0164] Cells that can be modified according to this aspect of the disclosure include cells from any organism. In some embodiments, the preparation is a preparation of mammalian cells, such as a preparation of rodent cells (i.e., mouse or rat cells), rabbit cells, guinea pig cells, cat cells, dog cells, pig cells, horse cells, bovine cells, ovine cells, monkey cells, or human cells. Suitable cells include primary or immortalized embryonic cells, fetal cells, or adult cells at any stage of the lineage, such as totipotent cells, pluripotent cells, multipotent cells, or differentiated cells.

[0165] In some embodiments, modifying the cell of interest involves introducing into the cell a sequence-specific nuclease that cleaves the target gene at or within the 3'UTR of the gene or at a position immediately upstream of the 3'UTR. As described in detail above, suitable target genes are genes that are expressed cell-specifically, selectively, or limitedly. When the target gene is cleaved by the sequence-specific nuclease, the method further includes introducing into the target gene any of the recombinant gene constructs described herein, for example, by homologous recombination.

[0166] Suitable sequence-specific nucleases for cleaving the target gene and introducing the recombinant gene construct include, without limitation, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases. In some embodiments, the sequence-specific nuclease is introduced into the cell as a protein, mRNA, or cDNA.

[0167] Zinc finger nucleases are a class of engineered DNA-binding proteins that facilitate targeted editing of DNA by introducing sequence-specific double-strand DNA breaks. Each ZFN contains two functional domains: a DNA-binding domain composed of strands of a two-finger module that each recognize a unique hexameric sequence of DNA, and a DNA cleavage domain composed of the nuclease domain of Fok I. ZFNs suitable for targeted cleavage of target genes described herein to facilitate insertion of recombinant gene constructs are known in the art; see, for example, U.S. Patent No. 8,106,255 to Carroll et al., U.S. Patent No. 9,428,756 to Cai et al., U.S. Patent Application Publication No. 20110281306 to Soo and Joo, and U.S. Patent Application Publication No. 20050130304 to Cox et al., each of which is incorporated herein by reference in its entirety.

[0168] In another aspect, DNA editing via transcription activator-like effector nucleases (TALENs) is utilized to introduce the recombinant gene constructs described herein into a target gene of interest. Functional TALENs consist of a DNA binding domain derived from a transcription activator-like effector (TALE) protein and a nuclease catalytic domain derived from the DNA nuclease, FokI. The DNA binding domain of TALE is characterized by an array of 33-34 amino acid repeats. Each repeat is conserved except for the repeat variable diresidue (RVD) at amino acid positions 12 and 13 that determines which nucleotide of the target DNA sequence each repeat recognizes. Methods for customizing TALE proteins to bind to a target site using canonical or non-canonical RVDs within the repeat units are known in the art and suitable for use according to the present disclosure (see, e.g., U.S. Patent No. 8,586,526 to Philip et al. and U.S. Patent No. 9,458,205 to Philip et al., which are incorporated herein by reference in their entireties). Similarly, methods for using TALENs for gene editing that are suitable for use according to the present disclosure are also known in the art and, for example, see U.S. Patent No. 9,393,257 to Osborn et al., which is incorporated herein by reference in its entirety).

[0169] In another aspect, the sequence-specific nuclease used to introduce the recombinant gene construct described herein into a target gene of interest is an RNA-guided nuclease in the form of Cas9. Cas9 is a CRISPR-associated protein that contains two nuclease domains and can achieve site-specific DNA recognition and double-strand cleavage when complexed with CRISPR RNA (cRNA) and trans-activating rRNA. CRISPR-Cas9 systems and methods for gene editing suitable for use according to the present disclosure are well known in the art, for example, Jinek, M., et al. "A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity," Science 337:816-821 (2012); Doench et al., "Rational Design of Highly Active sgRNAs for CRISPR-mediated Gene Inactivation," Nature Biotechnol. 32(12):1262-7(2014); U.S. Patent No. 9,970,001 to Miller; U.S. Patent Application Publication No. 20180282762 to Gori et al. and U.S. Patent Application Publication No. 20160201089 to Gersbach et al., which are hereby incorporated by reference in their entireties.

Example

[0170] The following examples are provided to illustrate aspects of the invention but are in no way intended to limit its scope.

[0171] Example 1 - Recombinant Gene Knock-In Constructs for Targeted Expression in Terminally Differentiated Cells The design of various recombinant gene constructs containing immune inhibitory protein knock-in vectors targeting cell-specific genes (e.g., MYRF, SYN1, or GFAP) is shown in FIGS. 1-14.

[0172] Figure 1 shows the general design of a recombinant gene construct comprising a first gene sequence (i.e., 5' homology arm) that is specifically expressed in a cell type, a self-cleaving peptide-encoding nucleotide sequence (e.g., P2a), a first nucleotide sequence encoding one or more immune inhibitory proteins (e.g., HLA-E / syB2M, CD47 or PD-L1), a stop codon, a second nucleotide sequence encoding one or more substances that reduce the surface expression of one or more endogenous HLA-I molecules (i.e., shRNA), a selection marker, and a second gene sequence (i.e., 3' homology arm) that is specifically expressed in a cell type.

[0173] Figures 2-4 show the general design of knock-in vectors comprising 5' and 3' homology arms. The knock-in vectors encode immune inhibitory proteins, namely HLA-E / syB2M (Figure 2), CD47 (Figure 3) or PD-L1 (Figure 4), self-cleaving peptide (P2a), HLA-E / syB2M, anti-B2M shRNA, anti-CIITA shRNA, and puromycin. The expression of puromycin is operably linked to the EF1a promoter for constitutive expression in mammalian cells.

[0174] Figure 5 is a matrix showing combinations of various target cells and protective signals (i.e., immune inhibitory proteins or peptides thereof).

[0175] Figures 6-8 show general exemplary designs of knock-in vectors targeting the SYN1 locus to achieve neuron-specific expression. Each SYN1-targeted knock-in vector comprises 5' and 3' homology arms and encodes immune inhibitory proteins, namely HLA-E / syB2M (Figure 6), CD47 (Figure 7), or PD-L1 (Figure 8), self-cleaving peptide (P2a), HLA-E / syB2M, anti-B2M shRNA, anti-CIITA shRNA, and puromycin. The expression of puromycin is operably linked to the EF1a promoter for constitutive expression in mammalian cells.

[0176] Figures 9-11 show the general design of knock-in vectors targeting the MYRF locus to achieve specific expression in oligodendrocytes. Each MYRF-targeted knock-in vector contains 5' and 3' homology arms and encodes an immune inhibitory protein, namely HLA-E / syB2M (Figure 9), CD47 (Figure 10), or PD-L1 (Figure 11), a self-cleaving peptide (P2a), HLA-E / syB2M, anti-B2M shRNA, anti-CIITA shRNA, and puromycin. The expression of puromycin is functionally linked to the EF1a promoter for constitutive expression in mammalian cells.

[0177] Figures 12-14 show the general design of knock-in vectors targeting the GFAP locus to achieve specific expression in astrocytes. Each GFAP-targeted knock-in vector contains 5' and 3' homology arms and encodes an immune inhibitory protein, namely HLA-E / syB2M (Figure 12), CD47 (Figure 13), or PD-L1 (Figure 14), a self-cleaving peptide (P2a), HLA-E / syB2M, anti-B2M shRNA, anti-CIITA shRNA, and puromycin. The expression of puromycin is functionally linked to the EF1a promoter for constitutive expression in mammalian cells.

[0178] Preparation of a recombinant gene knock-in construct expressing CD47 cDNA with a target sequence at the MYRF locus in a predicted example 2 A schematic diagram of a recombinant gene construct containing a CD47 knock-in vector targeting the MYRF locus is shown in Figure 15. The recombinant gene construct includes a 5' homology arm (HAL), a self-cleaving peptide-encoding nucleotide sequence (P2A), a first nucleotide sequence encoding CD47, a second nucleotide sequence encoding anti-β2M shRNA, a third nucleotide sequence encoding anti-CIITA shRNA, a nucleotide sequence encoding GFP functionally linked to the EF1a promoter, and a 3' homology arm (HAR). The recombinant gene construct in Figure 15 is prepared as follows.

[0179] β2-Microglobulin and CIITA knockdown shRNAs for β2M and CIITA are generated using an online tool (e.g., Thermofisher's iRNA designer). The shRNAs are inserted immediately downstream of the puromycin gene in the lentiviral vector pTANK-EF1a-copGFP-Puro-WPRE. Viral particles pseudotyped with vesicular stomatitis virus G glycoprotein are generated, concentrated by ultracentrifugation, and titrated in 293HEK cells.

[0180] hGPC derived from HAD100 is transduced with lentivirus carrying shRNA against β2M or CIITA (MOI = 1). The knockdown efficiency is evaluated by QPCR. shRNAs with knockdown efficiency exceeding 80% are further verified by immunostaining and expression of each protein by western blot.

[0181] sgRNA design and CRSPR / Cas9 vector construction Single guide RNAs are designed to enable double nicking using the CRISPR / Cas9 design tool (crispr.mit.edu) developed by the Zhang lab at MIT. The sgRNAs are selected in the coding sequence just prior to the codon stop (e.g., TIFF0007708737000032.tif4128. The sgRNAs are verified by transfection of HEK-29 cells using Surveyor Mutation Detection Kits (IDT inc).

[0182] Cloning of homologous arms Extract genomic DNA from cells using the DNeasy Blood and Tissue Kit (QIAGEN) according to the manufacturer's instructions. Amplify the homology arms from the genomic DNA of the HAD100 cell line using AmpliTaq Gold 360 (Thermo Fisher Scientific) (primers TBD). Subclone both homology arms into pCR2.1-TOPO and verify the sequences. The left homology arm (HAL) contains the last exon within the target gene.

[0183] Transfection and Selection of hESCs Amplify the knock-in and sgRNA-CRIPR / Cas9 plasmids using an endotoxin-free Maxi-prep kit (Qiagen). Transfect both plasmids (3 μg each) into hESCs (800,000 cells) using an Amaxa 4D-Nucleofector (Lonza; program CA-137 was used according to the manufacturer's instructions). Twenty-four hours after electroporation, grow the cells in medium containing puromycin (1 μg / mL).

[0184] Isolate single colonies and expand them. Verify transgenic clones by PCR for both the correct integration of the knock-in cassette and the absence of the sgRNA-CRISPR / Cas9 plasmid.

[0185] The sequences suitable for generating a recombinant gene knock-in construct expressing CD47 cDNA with the target sequence of the MYRF locus are shown in Table 14 below.

[0186] (Table 14) Exemplary sequences of a recombinant gene knock-in construct expressing CD47 cDNA with the target sequence of the MYRF locus TIFF0007708737000033.tif127164TIFF0007708737000034.tif246164TIFF0007708737000035.tif213164

[0187] Human U251 glioma cells expressing PD-L1 and CD47 expand and preferentially survive in immunodeficient humanized hosts. Materials and Methods Construction of targeting plasmids: Targeting vectors were generated using basic molecular cloning techniques with PCR-generated inserts. The coding sequences of human PD-L1 (NCBI Reference Sequence: NM_014143.4, which is incorporated herein by reference in its entirety), human CD47 (NCBI Reference Sequence: NM_001777.3, which is incorporated herein by reference in its entirety), or EGFP were cloned immediately downstream of the internal ribosome entry site (IRES) in pIRES-hPGK-Puro-WPRE-BGHpa. Two shRNAs targeting CIITA and B2M were also cloned immediately after PDL1 or CD47 (Table 15).

[0188] (Table 15) shRNA sequences TIFF0007708737000036.tif41164

[0189] Homology arms overlapping with the last coding exon were cloned from HEK293 cell genomic DNA. The left homology arm consisted of 842 bp (NCBI Reference Sequence: NC_000004.12 spanning 54294436 - 54295277, which is incorporated herein by reference in its entirety), while the right homology arm consisted of 875 bp (NCBI Reference Sequence: NC_000004.12 spanning 54295286 - 54296160, which is incorporated herein by reference in its entirety).

[0190] sgRNA TIFF0007708737000037.tif4128 was cloned downstream of the U6 promoter in pU6-PDGFRA2-CBh-Cas9-T2A-mCherry (Addgene plasmid No. 64324) and verified using the Surveyor nuclease assay in HEK293 cells (Surveyor Mutation Detection Kit, IDT).

[0191] Cell Transfection and Selection U251 human glioblastoma cells were maintained at 37 °C in 5% CO2 in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 units / mL penicillin and 100 μg / mL streptomycin).

[0192] Using the SE Cell Line 4D-Nucleofector™ X transfection kit with 4D Nucleofector™ (Lonza), 2 μg of a DNA mixture of the targeting plasmid and the sgRNA / Cas9 plasmid (1:1 ratio) was transfected into U251 cells (5×10 5 cells) according to the DS-126 protocol and the instructions supplied by the manufacturer. Three days after transfection, the cells were passaged and cultured in puromycin-containing (1.5 μg / ml; Sigma) medium for selection. Individual clones were expanded and genotyped for correct integration, transgene integrity, and the absence of the donor bacterial plasmid.

[0193] The selected clones were transduced with a lentivirus expressing luciferase (pTANK-CMV-luciferase-IRES-mCherry-WPRE; MOI = 5). For transplantation, the cells were collected by trypsinization and concentrated to 1×10 7 cells / ml in Hank's balanced salt solution.

[0194] Animals, Cell Transplantation, and Imaging Female huPBMC-NOG mice (NOD.Cg-Prkdc scid Il2rg tm1Sug / JicTac) were purchased from Taconic. The mice were housed in a sterile environment (3 - 4 mice per cage). Transplantation was performed under 2.5% isoflurane anesthesia. A total of 1×10 6 cells in 100 μl of HBSS were subcutaneously injected into one side of the flank of the mice.

[0195] In Vivo Bioluminescence Imaging Under 2.5% isoflurane anesthesia, bioluminescence imaging was performed using an IVIS® Spectrum imaging station (PerkinElmer). At the time of imaging the mice, D-luciferin (150 mg / kg body weight, i.p.; Sigma) was injected 10 minutes before imaging. Luminescence was calculated using IVIS® Spectrum software.

[0196] Results: Generation of a Recombinant Gene Knock-In Construct Expressing PD-L1, CD47, and EGFP cDNAs with Target Sequences at the PDGFRA Locus A schematic diagram of a recombinant gene construct containing a PD-L1 or CD47 knock-in vector targeting the PDGFRA locus is shown in FIG. 16A. The PD-L2 and CD47 knock-in vectors contain, in the 5'→3' direction, a 5' homology arm, a stop codon, an internal ribosome entry site (IRES), a nucleotide sequence encoding CD47 or PD-L1, a nucleotide sequence encoding an anti-B2M shRNA, a nucleotide sequence encoding an anti-CIITA shRNA, a puromycin selection marker, and a 3' homology arm. The EGFP vector (control vector) contains, in the 5'→3' direction, a 5' homology arm, a stop codon, an IRES, a nucleotide sequence encoding enhanced green fluorescent protein (EGFP), a stop codon, a puromycin selection marker, and a 3' homology arm. The puromycin selection marker in these constructs contains a phosphoglycerate kinase (PGK) promoter and a polyadenylation signal (PA) for constitutive expression in mammalian cells. The CD47 and PD-L1 knock-in vectors enable knockdown of class I and II major histocompatibility gene complexes via shRNAi suppression of β2-microglobulin and class 2 transactivator CIITA (FIG. 16A, upper construct). The EGFP knock-in vector (control vector) expresses only EGFP instead of CD47 or PDL1 and does not express any shRNA (FIG. 16A, lower construct). FIGS. 16B-16D show verification by immunostaining of clones generated by CRISPR-mediated knock-in of the recombinant gene construct of FIG. 16A into the PDGFRA locus after puromycin selection and clonal expansion.

[0197] Human U251 glioma cells expressing PD-L1 and CD47 expand and preferentially survive in an immunodeficient humanized host. Similar to those related glial progenitor cells, U251 cells express PDGFRA. Based on this, gene-edited U251 knock-in (KI) cells expressing PD-L1 or CD47 or EGFP (control) at the PDGFRA locus were subcutaneously injected into the flanks of huPBMC-NOG mice (human peripheral blood mononuclear cell chimeric immunodeficient NOG mice). Tumor growth was monitored by in vivo bioluminescence imaging on day 1, 5, or 9 after transplantation (Figure 17A). By 9 days after transplantation, U251 cells expressing CD47 had expanded and survived to a significantly greater extent than EGFP-expressing control cells, consistent with the avoidance of graft rejection by the humanized host immune system (Figure 17B).

[0198] Preferred embodiments have been illustrated and described in detail herein, but various modifications, additions, substitutions, etc. can be made without departing from the spirit of the present invention, and thus it will be apparent to those skilled in the art that these are considered to be within the scope of the present invention as defined by the following claims.

Claims

1. a first gene sequence that is specifically expressed in a cell type, one or more nucleotide sequences encoding immune checkpoint protein(s) located on the 3'-side of the first gene sequence, and / or one or more nucleotide sequences encoding one or more substances that reduce the expression of one or more HLA-I molecules located on the 3'-side of the first gene sequence, a second gene sequence that is specifically expressed in a cell type and is located on the 3'-side of the nucleotide sequence encoding the immune checkpoint protein(s) and / or the nucleotide sequence encoding the one or more substances that reduce the expression of the one or more HLA-I molecules, a recombinant gene construct comprising the same.

2. The recombinant gene construct according to Claim 1, wherein the first and second gene sequences of the recombinant gene construct are derived from genes that are specifically expressed in one or more terminally differentiated cells, and the terminally differentiated cells are oligodendrocytes, astrocytes, or neurons.

3. (i) one or more immune checkpoint proteins are selected from programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), CD47, CD200, CTLA-4, HLA-E, and any combination thereof; (ii)One or more substances that reduce the expression of one or more HLA-I molecules are (a) selected from the group consisting of shRNA, miRNA, and siRNA, (b) nuclease-deficient Cas9 or zinc finger nuclease, and / or (c) a substance that reduces the expression of β 2 M, and / or (iii) one or more HLA-I molecules are selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and combinations thereof; The recombinant gene construct according to Claim 1 or 2.

4. a further nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-II molecules, the further nucleotide sequence being linked to the nucleotide sequence encoding the one or more immune checkpoint proteins and / or the nucleotide sequence encoding the one or more substances that reduce the expression of the one or more HLA-I molecules The recombinant gene construct according to any one of Claims 1 to 3, further comprising the same.

5. one or more self-cleaving peptide-encoding nucleotide sequences arranged in the construct in a manner effective to mediate the translation of one or more immune checkpoint proteins The recombinant gene construct according to any one of Claims 1 to 4, further comprising the same.

6. an inducible cell death gene arranged in the construct in a manner effective to achieve inducible cell suicide The recombinant gene construct according to any one of claims 1 to 5, further comprising

7. A preparation of one or more cells, wherein the cells of the preparation comprise the recombinant gene construct according to any one of claims 1 to 6.

8. The preparation of one or more cells according to claim 7 for use in a method of treating a subject in need thereof.

9. The preparation of one or more cells according to claim 7 for use in a method of treating a condition mediated by a decrease in myelin or a dysfunction or decrease in oligodendrocytes in a subject, the method comprising administering the preparation to a subject having a condition mediated by a decrease in myelin or a dysfunction or decrease in oligodendrocytes, under conditions effective to treat the condition in the subject, wherein the cells of the preparation are glial progenitor cells or progenitor cells biased towards oligodendrocytes. The preparation comprising

10. The preparation of one or more cells according to claim 7 for use in a method of treating a condition mediated by a dysfunction or decrease in astrocytes in a subject, the method comprising administering the preparation to a subject having a condition mediated by a dysfunction or decrease in astrocytes, under conditions effective to treat the condition in the subject, wherein the cells of the preparation are glial progenitor cells or progenitor cells biased towards astrocytes. The preparation comprising

11. The preparation of one or more cells according to claim 7 for use in a method of treating a condition mediated by a dysfunction or decrease in neurons in a subject, the method comprising administering the preparation to a subject having a condition mediated by a dysfunction or decrease in neurons, under conditions effective to treat the condition in the subject, wherein the cells of the preparation are progenitor cells or neuronal progenitor cells. The preparation comprising

12. The preparation according to any one of claims 8 to 11, wherein the preparation is administered to one or more sites of the brain, brainstem, spinal cord, or a combination thereof.

13. A preparation of one or more cells, wherein when the cells of the preparation are finally differentiated, the cells (i) express one or more immune checkpoint proteins at increased levels compared to corresponding wild-type cells, (ii) express one or more HLA-I proteins at decreased levels compared to corresponding wild-type cells, or (iii) a combination of (i) and (ii) and A preparation, wherein the one or more cells are derived from the one or more cells according to claim 7.

14. The preparation according to claim 13, wherein the modified cells of the preparation are terminally differentiated cells.

15. The preparation according to claim 13, wherein the modified cells of the preparation conditionally express one or more HLA-II proteins at a reduced level compared to the corresponding wild-type cells.

16. A method for preparing a preparation of one or more cells according to claim 13, the method comprising: (i) one or more immune checkpoint proteins at increased levels, (ii) one or more substances that reduce the expression of one or more HLA-I proteins, or (iii) both (i) and (ii) a step of modifying the cells to conditionally express A method comprising.

17. The method according to claim 16, wherein the terminally differentiated cells are oligodendrocytes, astrocytes, or neurons.

18. (i) the one or more immune checkpoint proteins are selected from the group consisting of CD47, programmed death ligand 1 (PD-L1), programmed death ligand 2 (PD-L2), CD200, CTLA4, HLE-A, and any combination thereof, (ii)One or more substances that reduce the expression of one or more HLA-I proteins are (a) selected from the group consisting of shRNA, miRNA, and siRNA, (b) nuclease-deficient CRISPR-Cas9 protein or zinc finger nuclease, and / or a substance that reduces the expression of β 2 M, and / or (iii) the one or more HLA-I molecules are selected from the group consisting of HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and combinations thereof, The method according to claim 16 or 17.

19. a step of further modifying the cells to conditionally express one or more substances that reduce the expression of one or more HLA-II molecules The method according to any one of claims 16 to 18, further comprising.

20. The method according to any one of claims 16 to 19, wherein the conditionally immunoprotected cells are mammalian cells, pluripotent cells, or progenitor cells.

21. The modification is (i) introducing into the cell a sequence-specific nuclease that cleaves the target gene at a position upstream of its 3' untranslated region (UTR), wherein the target gene is a gene that is cell-specifically expressed, and (ii) (a) one or more immune checkpoint protein coding nucleotide sequences, (b) a nucleotide sequence encoding one or more substances that reduce the expression of one or more HLA-I molecules, or (c) both (a) and (b) introducing into the cell a recombinant gene construct comprising The method according to claim 16, comprising, wherein the recombinant gene construct is inserted into the target gene at the nuclease cleavage site by homologous recombination. [

22. ] The sequence-specific nuclease is (i) selected from the group consisting of zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and RNA-guided nuclease, (ii) an RNA-guided nuclease in the form of Cas9, or (iii) introduced into the cell as a protein, mRNA, or cDNA, The method according to claim 21.

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