Genetically modified cells for enhancing immune evasion in allogeneic cell therapy

Abstract

This disclosure provides methods and cell populations engineered to modulate the expression of selected genes and thereby reduce natural killer cell-mediated cytotoxicity. For example, this disclosure provides engineered cells equipped with one or more heterologous nucleic acid sequences encoding polypeptides, which, when expressed, inhibit typical cytotoxicity of natural killer cells compared to comparable cells lacking the heterologous nucleic acid sequences.

Description

[0001] Cross-references to related applications This application claims priority to U.S. Provisional Application No. 63 / 598,521, filed November 13, 2023; U.S. Provisional Application No. 63 / 572,755, filed April 1, 2024; and U.S. Provisional Application No. 63 / 679,512, filed August 5, 2024, the entire contents of which are incorporated herein by reference.

[0002] sequence list This application includes a sequence list, which has been filed through the Patent Center. The sequence list, titled 213661-016004_PCT_SL.xml, was created on November 12, 2024, and is 38,042 bytes in size, and is incorporated herein by reference in its entirety. Technical Field

[0003] This disclosure generally relates to the field of cell therapy. More specifically, this disclosure relates to genetically modified cells that exhibit enhanced immune evasion properties. Background Technology

[0004] In recent years, cell therapy has gained attention as a promising approach to treating certain diseases and conditions. One of the major challenges in achieving successful treatment outcomes, especially with allogeneic cells, is the host's immune response to the transplanted cells. This immune response can lead to rejection of the transplanted cells, impairing therapeutic efficacy and potentially causing adverse reactions in the recipient.

[0005] Several strategies have been developed to address the problem of immune rejection. These include immunosuppressive drugs, donor-receptor matching, and co-stimulation blockade. However, these methods have limitations, such as side effects, partial efficacy, and potential complications. The limitations of immune escape persist and severely hinder the realization of true allogeneic therapy, forcing patients to rely on potentially harmful immunosuppressive drugs and risk adverse reactions after treatment. Summary of the Invention

[0006] This disclosure provides methods and compositions for engineered cells, wherein the engineered cells contain modifications enabling effective immune evasion. Supported by experimental results, this disclosure demonstrates the efficacy of regulating the expression of specific genes (e.g., CLEC2D, SERPINB9, and TRAIL) in reducing or preventing natural killer cell immunotoxicity in engineered cells. In some cases, the engineered cells are engineered human pluripotent stem cells, and the experimental results disclosed herein demonstrate that these engineered cells can differentiate into specific cell types, including cardiomyocytes, while retaining reduced natural killer cell immunotoxicity and cellular function. Therefore, this disclosure provides a promising pathway to reduce dependence on external immunosuppression, ultimately allowing for safer and more effective allogeneic cell therapies.

[0007] In one aspect, this disclosure provides an engineered cell comprising at least one heterologous nucleic acid sequence encoding a polypeptide, wherein the polypeptide is expressed at a level sufficient to inhibit immune cell-mediated cytotoxicity (e.g., natural killer cell-mediated cytotoxicity) of the engineered cell, and wherein the polypeptide comprises one or more of the following: C-type lectin domain family 2 member D (CLEC2D), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), serine protease inhibitor family B member 9 (SERPINB9), and human leukocyte antigen-C (HLA-C), or variants thereof.

[0008] In some embodiments, the polypeptide comprises one or more of CLEC2D, TRAIL, and SERPINB9 or variants thereof. In some embodiments, the polypeptide comprises CLEC2D or a variant thereof.

[0009] In some embodiments, the engineered cells comprise at least two distinct heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the at least two distinct heterologous nucleic acid sequences encode CLEC2D or a variant thereof; and SERPINB9 or a variant thereof. In some embodiments, the at least two distinct heterologous nucleic acid sequences encode CLEC2D or a variant thereof; and TRAIL or a variant thereof. In some embodiments, the engineered cells comprise at least three distinct heterologous nucleic acid sequences, each encoding a different polypeptide.

[0010] In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the at least one heterologous nucleic acid sequence comprises a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO: 13, 15, 17, and 22.

[0011] In some embodiments, the at least one heterologous nucleic acid sequence is integrated into a sustained transgenic expression locus (STEL) or a sustained transcriptional activity payload region (STAPLR). In some embodiments, the STEL comprises human glyceraldehyde-3-phosphate dehydrogenase (GD2). GAPDH The gene locus within the gene. In some embodiments, the expression of the polypeptide is driven by an endogenous gene promoter.

[0012] In some implementations, the engineered cell includes a kill switch.

[0013] In some embodiments, the engineered cells further comprise genetic modifications that result in reduced T cell-mediated killing compared to wild-type cells. In some embodiments, the genetic modifications include: (i) β2 microglobulin ( B2M (ii) Deletion, damage, or attenuation of genes; or HLA-A Gene, HLA-B Genes and HLA-C One or more genes are deleted, disrupted, or weakened. In some embodiments, the engineered cells further comprise a second genetic modification containing a class II major histocompatibility complex transactivator (MHC). CIITA The deletion, disruption, or attenuation of the gene or regulatory factor X5 (RFX5) gene.

[0014] In some embodiments, the engineered cells comprise stem cells. In some embodiments, the engineered cells include cardiac cells, nerve cells, myeloid cells, T cells, endocrine cells, epithelial cells, muscle cells, or retinal cells. In some embodiments, the engineered cells are human cells.

[0015] On the other hand, this disclosure provides engineered cell populations derived from the engineered cells described herein. In some embodiments, the engineered cell populations are at least about 10% less susceptible to natural killer cell-mediated cytotoxicity compared to cell populations not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell populations are at least about 20% less susceptible to natural killer cell-mediated cytotoxicity compared to cell populations not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, at least 95% of the engineered cells in the engineered cell population are positive for cardiac troponin T (cTnT).

[0016] In some embodiments, the engineered cell population is present in a composition formulated for administration to a subject.

[0017] On the other hand, this disclosure provides a method for treating a subject with a disease or condition, the method comprising administering an engineered cell population as described herein. In some embodiments, the disease or condition includes heart failure, Parkinson's disease, multiple sclerosis, irritable bowel syndrome, type 1 diabetes, rheumatoid arthritis, cancer, liver disease, or neuroinflammation. In some embodiments, the subject is a human being.

[0018] In another aspect, this disclosure provides a method for achieving immune evasion, the method comprising: genetically engineering cells to increase the expression of at least one polypeptide, wherein the at least one polypeptide comprises one or more of the following: C-type lectin domain family 2 member D (CLEC2D), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), serine protease inhibitor family B member 9 (SERPINB9), and human leukocyte antigen-C (HLA-C), or variants thereof; and thereby the genetically engineered cells exhibit increased survival upon contact with natural killer cells compared to cells that have not been genetically engineered to increase the expression of said at least one polypeptide.

[0019] In some embodiments, the polypeptide comprises one or more of CLEC2D, TRAIL, and SERPINB9 or variants thereof. In some embodiments, the polypeptide comprises CLEC2D or a variant thereof.

[0020] In some embodiments, the genetic modification includes integrating a heterologous nucleic acid sequence encoding a polypeptide into a genomic locus of the cell. In some embodiments, the genomic locus is a sustained transgenic expression locus (STEL) or a sustained transcriptional activity payload region (STAPLR). In some embodiments, the STEL comprises human glyceraldehyde-3-phosphate dehydrogenase (GD2). GAPDH (The locus within the gene.)

[0021] In some embodiments, the method further includes amplifying the cells that have been genetically engineered to produce an engineered cell population. In some embodiments, the method further includes differentiating the engineered cell population into cardiac cells, nerve cells, T cells, retinal cells, endocrine cells, epithelial cells, muscle cells, or myeloid cell populations. In some embodiments, the cells are human cells.

[0022] On the other hand, this disclosure provides a pharmaceutical composition comprising an engineered cell population as described herein and a pharmaceutically acceptable carrier, transporter, or diluent.

[0023] In another aspect, this disclosure provides a kit comprising a dosage form suitable for administration to a subject and instructional materials for using the dosage form, the dosage form comprising an engineered cell population as described herein.

[0024] In another aspect, this disclosure provides a kit comprising a dosage form suitable for administration to a subject and instructional materials for using the dosage form, the dosage form comprising a pharmaceutical composition as described herein. Attached Figure Description

[0025] Figure 1 A schematic diagram illustrates an exemplary method for engineering induced pluripotent stem cells (iPSCs) to express a target peptide. A heterologous nucleic acid encoding the target peptide is integrated into a sustained transgenic expression locus (STEL; e.g., GAPDH) in the iPSC genome. Expression of the target peptide is then driven by an endogenous gene promoter.

[0026] Figure 2 Exemplary results of a natural killer (NK) cell cytotoxicity assay evaluating iPSCs expressing VISTA, CLEC2D, FASL, TRAIL, SERPINB9, CTLA4, CD47, or HLA-C challenged with NK cells from three different healthy human donors (pNK1, pNK2, and pNK3) are shown. The x-axis shows different iPSC modifications, including wild-type (WT) cells with various gene knock-ins. The y-axis represents mean NK-specific cytotoxicity as a percentage, showing data for each healthy NK cell donor separately (pNK1: black bar, pNK2: medium gray bar, pNK3: light gray bar). Error bars with p-values ​​(<0.0001, <0.0001, <0.0002, and <0.001) indicate statistical significance.

[0027] Figure 3 Exemplary results of an NK cell cytotoxicity assay assessment of iPSCs expressing VISTA, CLEC2D, FASL, TRAIL, SERPINB9, CTLA4, CD47, or HLA-C are shown. Within a column of this figure, each circle represents NK cell-mediated cytotoxicity from one of three different healthy donor NK cell populations (pNK1: black circle, pNK2: light gray triangle, pNK3: dark gray circle). The x-axis shows different engineered iPSCs, including wild-type (WT) control cells modified with various gene knock-in modifications identified along this axis. The y-axis represents NK-specific cytotoxicity as a percentage. Data points are shown as mean ± SEM.

[0028] Figure 4A and 4BExemplary results are shown in the natural killer (NK) cell cytotoxicity assays of engineered cardiomyocytes expressing FasL, TRAIL, HLA-C*04:01, CLEC2D, CTLA4, SERPINB9, HLA-C*05:01, or VISTA, challenged with NK cells from four different healthy donors (pNK1, pNK2, pNK3, and pNK4). Figure 4A This is a bar graph of exemplary results. The x-axis represents the gene target. The y-axis represents the percentage of NK cell-specific cytotoxicity. Error bars with p-values ​​(<0.0001, <0.0001, <0.0002, and <0.001) indicate statistical significance. Figure 4B yes Figure 4A The results are shown in the table (NS: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001).

[0029] Figures 5A-5C Exemplary results of in vivo NK cell toxicity assessments from engineered cardiomyocytes expressing CLEC2D or SERPINB9 are shown. Figure 5A The total photon flux of control cells (luciferase-positive cardiomyocytes) and control cells with either a healthy donor 1 NK cell or a donor 2 NK cell is shown over an 11-day period. Figure 5B The total photon flux from luciferase-positive cardiomyocytes engineered to express CLEC2D is shown over an 11-day period. Figure 5C The total photon flux from luciferase-positive cardiomyocytes engineered to express SERPINB9 is shown over an 11-day period. Total proton flux is represented along the y-axis. Time (days) is represented along the x-axis.

[0030] Figure 6 Exemplary results of in vitro NK cell cytotoxicity assays of iPSCs engineered with CLEC2D, SERPINB9, or TRAIL alone or in combination (i.e., CLEC2D+SERPINB9, CLEC2D+TRAIL) are shown. The x-axis shows different PSC modifications, including wild-type (WT) and B2M knockout (KO) cells with various gene knock-ins. The y-axis represents NK-specific cytotoxicity as a percentage. The horizontal dashed line represents the baseline cytotoxicity level of B2M-KO iPSCs. Each bar represents the mean cytotoxicity of results from all four NK cells from different healthy donors, where the mean result from each donor NK cell is represented by a black triangle.

[0031] Figure 7Exemplary results from in vitro NK cell cytotoxicity assays with different NK cell to iPSC ratios are shown. The x-axis displays different engineered iPSCs, including B2M KO iPSCs and iPSCs engineered with B2M KO in combination with various gene knock-in combinations. Engineered iPSCs are grouped along the x-axis according to their NK:PSC ratios (i.e., 2:1 and 1:1, respectively). The y-axis represents NK-specific cytotoxicity as a percentage. Each bar represents the average cytotoxicity of the results from all three NK cells from different healthy donors, where the average result from each donor NK cell is represented by a black triangle.

[0032] Figure 8 Exemplary results from in vitro NK cell cytotoxicity assays with different NK cell to engineered cardiomyocyte (CM) ratios are shown. The x-axis displays different engineered CMs, including B2M KO CMs and CMs engineered with B2M KO and various gene knock-in combinations. Engineered CMs are grouped along the x-axis according to their NK:CM ratios (i.e., 2:1 and 1:1, respectively). The y-axis represents NK-specific cytotoxicity as a percentage. Each column represents the mean cytotoxicity of results from all four NK cells from different healthy donors, where the mean result from each donor NK cell is represented as a triangle, rhombus, or circle / dot.

[0033] Figure 9 Exemplary results of an in vitro NK cell cytotoxicity assay of cardiomyocytes engineered with TRAIL alone or in combination with CLEC2D are shown. The x-axis displays different engineered cardiomyocytes, including baseline control cells, cells expressing TRAIL, and cells expressing both CLEC2D and TRAIL. The y-axis represents NK-specific cytotoxicity as a percentage. Dashed lines represent baseline cytotoxicity levels. Each bar represents mean cytotoxicity, with individual data points shown as black triangles. Results were obtained at a 1:1 NK cell to cardiomyocyte ratio, demonstrating that the combination of CLEC2D and TRAIL provides enhanced protection against NK cell-mediated cytotoxicity compared to TRAIL alone or baseline conditions. Detailed Implementation

[0034] This disclosure relates to methods, engineered cell populations, pharmaceutical compositions, and kits comprising engineered cells, wherein the engineered cells exhibit increased survival upon challenge with immune cells (e.g., natural killer cells). Specifically, the methods, engineered cell populations, pharmaceutical compositions, and kits comprise engineered cells including heterologous nucleic acids encoding polypeptides expressed at levels sufficient to inhibit natural killer cell-mediated cytotoxicity.

[0035] The immune response plays a crucial role in recognizing and eliminating foreign substances (such as pathogens or foreign antigens). For example, the presence of foreign antigens triggers an immune response against them. Foreign antigens presented on the major histocompatibility complex (MHC) molecule target cells for elimination by natural killer (NK) cells and cytotoxic T cells, thereby clearing the foreign antigens. While NK and T cell cytotoxicity typically eliminates pathogen-infected cells, tumor cells, and other harmful substances, they can also target and eliminate therapeutic agents (such as cell therapies), thus reducing the therapeutic benefit of these treatments. Therefore, designing therapies that overcome NK and T cell cytotoxicity is key to maintaining optimal therapeutic efficacy.

[0036] In the embodiments described herein, engineered cells with increased immune evasion are provided. In some embodiments, the engineered cells express peptides at levels sufficient to inhibit natural killer (NK) cell-mediated cytotoxicity. By inhibiting the cytotoxicity of natural killer cells, the engineered cells exhibit increased survival after administration to a subject. In some aspects of the embodiments described herein, the engineered cells may further include additional modifications to reduce T cell-mediated cell killing, thereby further increasing their survival in the subject. When administered to a subject, the increased survival of the engineered cells described herein results in an increased cell half-life. By increasing the half-life of the engineered cells in a subject, the engineered cells may confer greater therapeutic benefit in treating a disease or condition in the subject.

[0037] Therefore, the implication of this disclosure is that the expression levels of one or more specific target genes within the modified cells can modulate the cells' susceptibility to NK cell-mediated cytotoxicity. Specifically, this disclosure provides experimental data demonstrating that increased expression of specific target genes (including CLEC2D, TRAIL, SERPINB9, and HLA-C or variants thereof) can partially or completely resistant cells to NK cell recognition and subsequent cell lysis. This disclosure provides methods and compositions for engineering enhanced cell survival through the genetic manipulation of target genes, wherein overexpression of at least one target gene (such as CLEC2D) in human pluripotent stem cells leads to reduced NK cell-mediated killing of the stem cells. Furthermore, this disclosure provides experimental data demonstrating that the stem cells can differentiate into downstream cell types, such as cardiomyocytes, while retaining cellular function.

[0038] Furthermore, this disclosure provides cells engineered to increase the expression of a combination of at least two target genes. The at least two target genes may be selected from CLEC2D, TRAIL, and SERPINB9 or variants thereof. In some embodiments, at least one of the target genes is CLEC2D or a variant thereof. Exemplary combinations include, but are not limited to, cells modified to increase the expression of both CLEC2D or a variant thereof and SERPINB9 or a variant thereof, or cells modified to increase the expression of both CLEC2D or a variant thereof and TRAIL or a variant thereof. In some embodiments, the combination includes at least three target genes, including CLEC2D, SERPINB9, and TRAIL or variants thereof. This disclosure contemplates that such modifications can be implemented in various cell types, including human pluripotent stem cells and differentiated cell types derived therefrom, such as cardiomyocytes.

[0039] This disclosure further provides experimental validation of reducing NK cell immune cytotoxicity by introducing heterologous nucleic acids encoding one or more target genes (including CLEC2D or variants thereof) into target cells. In a particular embodiment, the expression of the at least two target genes is increased by integrating heterologous nucleic acids encoding the target genes into the genome of the target cells. This integration can be accomplished by various methods known in the art, including genome editing systems, such as CRISPR-based methods, or by lentiviral transduction. While the exemplary embodiments described herein utilize heterologous gene introduction, those skilled in the art will readily understand that alternative methods for increasing target gene expression fall within the scope of this disclosure. Such alternative methods include, but are not limited to, manipulating endogenous gene expression by modifying regulatory elements, such as promoters, enhancers, or other control elements associated with the target genes disclosed herein. Therefore, this disclosure contemplates that any method resulting in increased expression of the identified target genes, whether heterologous or endogenous, can be used to achieve the NK cell resistance phenotype described herein.

[0040] While this disclosure describes various exemplary alternatives and implementations as provided herein, it should be understood that the various features, aspects, and functions described in one or more individual alternatives are not limited to their applicability to the particular alternative to which they are described. Rather, they may be applied individually or in various combinations to one or more other alternatives to this disclosure, whether or not said alternative is described or whether a feature is presented as part of a described alternative. The breadth and scope of this disclosure should not be limited by any exemplary alternatives described or illustrated herein.

[0041] I. Definition The following definitions supplement those in the art and are specific to this disclosure. The following definitions should not be extrapolated to any related or irrelevant circumstances, such as any jointly owned patents or patent applications. While some methods and materials similar or equivalent to those described herein can be used to practice the features of this disclosure, some preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0042] Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural forms, and plural terms shall include singular forms.

[0043] It should be understood that the present invention is not limited to the specific methods, schemes, and reagents described herein, and therefore variations are possible. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims.

[0044] As used herein, the articles “a,” “an,” and “the” refer to one or more (i.e., at least one) grammatical objects of the article. For example, “an element” means one or more elements.

[0045] The use of alternatives (e.g., "or") should be understood to mean one, two, or any combination of alternatives.

[0046] As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In some cases, the term "about" or "approximately" refers to a range of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

[0047] As used herein, the terms “administration,” “administering,” and variations thereof refer to the introduction of a composition or therapeutic agent (e.g., a cell population) into a subject. Administration includes simultaneous and sequential introduction of the composition or therapeutic agent. The composition or therapeutic agent (e.g., a cell population) is administered to a subject via any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, or local routes. A suitable route of administration allows the composition or agent to perform its intended function. Administration also includes self-administration and administration by another person. Administration can also be systemic or local. For example, the composition or therapeutic agent (e.g., a cell population) can be administered locally, such as by local injection into a tissue.

[0048] As used herein, the term “and / or” should be understood to mean one or both or any combination of alternatives.

[0049] As used herein, the terms “β2 microglobulin” and “B2M” are used interchangeably to refer to… B2M A gene-encoded polypeptide. B2M is a component of the major histocompatibility complex (MHC) class I and forms a complex with human leukocyte antigen (HLA) class I of MHC. B2M is also known as IMD43; MHC1D4; AMYLD6; β-2-microglobulin; and B2 microglobulin. In some cases, the B2M is human B2M. Exemplary B2M The gene is available as Gene ID: 567 (e.g., at www.ncbi.nlm.nih.gov / gene / 567). It can be used to generate B2Ms or for modification. B2M An exemplary nucleotide sequence of the gene is available as Transcript ID NM_004048.4 (e.g., available at www.ncbi.nlm.nih.gov / nuccore / NM_004048.4).

[0050] As used herein, the term "cardiac cell" means any cell present in the heart that provides cardiac function such as cardiac contraction or blood supply, or otherwise serves to maintain the structure of the heart. Cardiac cells can be cells of the epicardium, myocardium, or endocardium of the heart. Cardiac cells also include, but are not limited to, cardiac muscle cells or cardiomyocytes, and cells of the cardiac vascular system, such as cells of the coronary arteries or veins. Other non-limiting examples of cardiac cells include epithelial cells, endothelial cells, fibroblasts, cardiac stem cells or progenitor cells, cardiac conduction cells, and cardiac pacemaker cells that make up the myocardium, blood vessels, and supporting structures of the heart cells.

[0051] As used herein, the terms "C-type lectin domain family 2 member D" and "CLEC2D" are used interchangeably to refer to... CLEC2D A gene-encoded polypeptide. CLEC2D is also known as natural killer cell receptor C-type lectin; CLAX; LLT1; osteoclast inhibitory lectin (OCIL); C-type lectin domain family 2 member D; and C-type lectin domain family 2 member D. In some cases, the CLEC2D is human CLEC2D. Exemplary CLEC2DThe gene is available as Gene ID: 29121 (e.g., available at www.ncbi.nlm.nih.gov / gene / 29121). An exemplary nucleotide sequence that can be used to generate CLEC2D is available as Transcript ID NM_001004419.5 (e.g., available at www.ncbi.nlm.nih.gov / nuccore / NM_001004419.5). Transcript ID NM_001197317.3 (e.g., obtainable from https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001197317.3), Transcript ID NM_001197318.3 (e.g., obtainable from https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001197318.3), Transcript ID NM_001197319.3 (e.g., obtainable from https: / / www.ncbi.nlm.nih.gov / nuccore / NM_001197319.3), Transcript ID NM_013269.6 (e.g., obtainable from https: / / www.ncbi.nlm.nih.gov / nuccore / NM_013269.6), Transcript ID ENST00000261340 (e.g., obtainable from https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_cDNA?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000261340), Transcript ID ENST00000430909 (e.g., obtainable from https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_cDNA?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000430909), Transcript ID ENST00000466035 (e.g., available on the website https: / / useast.ensembl).The Transcript ID is ENST00000460309 (e.g., it can be obtained from https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_cDNA?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000466035). ENST00000261339 (e.g., obtainable from https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_cDNA?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000261339), Transcript ID ENST00000543300 (e.g., obtainable from https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_cDNA?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000543300), Transcript ID ENST00000544322 (e.g., available at https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_cDNA?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000544322), and Transcript IDENST00000545918 (e.g., available at https: / / useast.ensembl.org).(obtained from org / Homo_sapiens / Transcript / Sequence_cDNA?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000545918); and the nucleotide sequence provided herein, such as SEQ ID NO: 13. Exemplary amino acid sequences of CLEC2D are available from Protein ID NP_001004419.1 (e.g., available at www.ncbi.nlm.nih.gov / protein / NP_001004419.1), Protein ID NP_001184246.1 (e.g., available at https: / / www.ncbi.nlm.nih.gov / protein / NP_001184246.1), Protein ID NP_001184247.1 (e.g., available at https: / / www.ncbi.nlm.nih.gov / protein / NP_001184247.1), Protein ID NP_001184248.1 (e.g., available at https: / / www.ncbi.nlm.nih.gov / protein / NP_001184248.1), Protein ID NP_037401.1 (e.g., obtainable from https: / / www.ncbi.nlm.nih.gov / protein / NP_037401.1), Protein ID ENST00000261340 (e.g., obtainable from https: / / useast.14nsemble.org / Homo_sapiens / Transcript / Sequence_Protein?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000261340), Protein ID ENST00000430909 (e.g., can be obtained from the website https: / / useast.14nsemble.org / Homo_sapiens / Transcript / Sequence_Protein?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000430909), Protein ID ENST00000466035 (e.g., can be obtained from the website https: / / useast.14nsemble).The Protein ID is ENST00000460309 (e.g., it can be obtained from https: / / useast.14nsemble.org / Homo_sapiens / Transcript / Sequence_Protein?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000466035). ENST00000261339 (e.g., obtainable from https: / / useast.14nsemble.org / Homo_sapiens / Transcript / Sequence_Protein?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000261339), Protein ID ENST00000543300 (e.g., obtainable from https: / / useast.14nsemble.org / Homo_sapiens / Transcript / Sequence_Protein?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000543300), Protein ID ENST00000544322 (e.g., available at https: / / useast.14nsemble.org / Homo_sapiens / Transcript / Sequence_Protein?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000544332) and Protein ID ENST00000545918 (e.g., available at https: / / useast.nsemble.org / Homo_sapiens / Transcript / Sequence_Protein?db=core;g=ENSG00000069493;r=12:9664969-9699553;t=ENST00000545918); and the amino acid sequence as provided herein, such as SEQ ID NO: 2.

[0052] As used herein, the terms “Class II Major Histocompatibility Complex Trans-activator” and “CIITA” are used interchangeably to refer to… CIITA Genetically encoded polypeptides. CIITA is also known as C2TA; CIITAIV; MHC2TA; the NLR family, containing an acid domain; NLRA; and a nucleotide-binding oligomerization domain containing leucine-enriched repeat sequences and an acid domain. In some cases, the CIITA is human CIITA. Exemplary CIITA The gene is available as Gene ID: 4261 (e.g., at www.ncbi.nlm.nih.gov / gene / 4261). It can be used to generate CIITA or for modification. CIITA An exemplary nucleotide sequence of the gene is available as Transcript ID NM_000246.4 (e.g., available at www.ncbi.nlm.nih.gov / nuccore / NM_000246.4).

[0053] As used herein, the terms "cluster of differentiation 47" and "CD47" are used interchangeably to refer to... CD47 A gene-encoded polypeptide. CD47 is also known as integrin-associated protein; IAP; MER6; OA3; CD47 molecule; CD47 antigen; Rh-associated antigen; integrin-associated signal transduction; antigen identified by monoclonal antibody 1D8; antigenic surface determinant protein OA3; leukocyte surface antigen CD47; and CD47 glycoprotein. In some cases, said CD47 is human CD47. Exemplary CD47 The gene is available as Gene ID: 961 (e.g., available at www.ncbi.nlm.nih.gov / gene / 961). Exemplary nucleotide sequences that can be used to generate CD47 are available as Transcript ID NM_001382306.1 (e.g., available at www.ncbi.nlm.nih.gov / nuccore / NM_001382306.1), as well as the nucleotide sequences provided herein, such as SEQ ID NO: 19. Exemplary amino acid sequences of CD47 are available as Protein ID NP_001369235.1 (e.g., available at www.ncbi.nlm.nih.gov / protein / NP_001369235.1), as well as the amino acid sequences provided herein, such as SEQ ID NO: 8.

[0054] As used herein, the terms “reduction” and “inhibition” are interchangeable and refer to any statistically significant reduction in the biological activity of a reference protein or cell (e.g., natural killer cell-mediated toxicity). For example, inhibition of biological activity may refer to a reduction of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of biological activity compared to a control.

[0055] As used herein, the term "differentiation" and its grammatical equivalents refer to the process by which stem cells or progenitor cells change from one cell type to a more specialized cell type. Each specialized cell type in an organism can express a subset of all the genes that make up the cell's genome. Each cell type can be defined by its specific regulated gene expression patterns. Therefore, cell differentiation can be described as the transformation of cells from one cell type to another, consistent with the conversion from one gene expression pattern to another.

[0056] As used herein, the term "dosage form" refers to a composition comprising a discrete amount of a predetermined amount of an active ingredient (e.g., a cell population). The amount of the active ingredient is typically equal to the dose of the active ingredient to be administered to the subject, or a convenient portion of such a dose, such as half or one-third of such a dose. The relative amounts of the active ingredient, pharmaceutically acceptable excipients, and any other components in the pharmaceutical composition will vary depending on the identity, size, and condition of the subject being treated, and further depend on the route of administration of the composition. The dosage form may further include one or more additional pharmaceutically active agents. In some cases, the dosage form is intended for administration by injection to the subject.

[0057] As used herein, the term "encoding" refers to the properties of a specific nucleotide sequence in a nucleic acid (e.g., a gene, cDNA, or mRNA) and the resulting biological properties, which serves as a template for the synthesis of other polymers and macromolecules having defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences in biological processes. Thus, if the transcription and translation of mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene, or at least its exons, encodes a protein. Both the coding strand (the nucleotide sequence identical to the mRNA sequence) and the non-coding strand (serving as a template for gene or cDNA transcription) can be referred to as encoding a protein or other product of that gene or cDNA.

[0058] As used herein, the term "endogenous" refers to genes, nucleic acids, polypeptides, etc., that are typically present in a particular cell. For example, endogenous genes can be genes that are typically present in the cell's genome.

[0059] As used herein, the term “expression” refers to the transcription and / or translation of a specific nucleotide sequence in a cell.

[0060] As used herein, the terms “enhancement” and “increase” are used interchangeably and refer to any statistically significant increase in the biological activity (e.g., survival) of a reference protein or cell. For example, an increase in biological activity may refer to an increase of approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% compared to a control.

[0061] As used herein, the terms “genetic modification” or “genetic alteration” refer to changes at the cellular DNA level. Genetic modifications include insertions, deletions, or substitutions, typically within defined sequences or genomic loci. In some cases, the genetic modification may be the deletion, disruption, or attenuation of a gene that results in reduced or lost expression of the gene product. In other cases, the genetic modification includes the integration of a heterologous nucleotide sequence into a genomic locus. The genetic modification may occur at a single nucleotide position or at multiple nucleotide positions, such as two, three, four, five, or more nucleotides, typically very close to each other, such as consecutive nucleotides. Depending on the nature of the genetic modification, the expression of the gene may be upregulated or downregulated.

[0062] As used herein, when used in the context of cells, the terms "genetically modified" or "engineered" refer to cells that include one or more genetic modifications and are not found in nature. Such engineered cells can be prepared by any method known in the art, such as by manipulating the cell's genome or inserting new nucleic acids into the cell. For example, cells can be modified by integrating nucleic acids encoding a target gene into the cell using genome editing technologies such as the CRISPR / Cas9 system or the CRISPR / Cas12a system. In some cases, cells can be modified by integrating nucleic acids encoding a target gene into the cell using lentiviral transduction.

[0063] As used in this article, the term "heart failure" refers to a condition characterized by the heart's inability to pump enough blood to meet the needs of the body's tissues and organs. Heart failure can be caused by abnormally low cardiac output.

[0064] As used herein, when referring to nucleic acids and / or peptides introduced into host cells, the term "heterologous" means nucleic acids and / or peptides that are not naturally present in host cells or at a given location in the host cell genome. For example, a construct is heterologous to host cells if it contains some homologous sequences arranged in a manner not found in host cells and / or contains some heterologous sequences not found in host cells.

[0065] As used herein, the terms “human leukocyte antigen” and “HLA” are used interchangeably to refer to subunits of the major histocompatibility complex (MHC). MHCs are glycoproteins that present antigens to immune cells, such as T cells. Type I MHCs are expressed in most nucleated cells and interact with the CD8 receptor on the surface of T cells. Type I MHCs consist of type I HLA molecules and β2-microglobulin (B2M). Type II MHCs are typically found only in antigen-presenting cells and interact with the CD4 receptor on the surface of T cells. Type II MHCs are heterodimers of two type II HLAs.

[0066] As used herein, the terms "human leukocyte antigen-A" and "HLA-A" are used interchangeably, referring to... HLA-A Genetically encoded polypeptide. HLA-A is a class I HLA. HLA-A is also known as HLAA.

[0067] As used herein, the terms “human leukocyte antigen-B” and “HLA-B” are used interchangeably to refer to… HLA-B Genetically encoded polypeptide. HLA-B is a class I HLA. HLA-B is also known as AS, HLAB, and B-4901. Example HLA-B The gene can be obtained as Gene ID: 3106 (e.g., available at www.ncbi.nlm.nih.gov / gene / 3106).

[0068] As used herein, the terms “human leukocyte antigen-C” and “HLA-C” are used interchangeably to refer to… HLA-C Genetically encoded polypeptide. HLA-C is a class I HLA. HLA-C is also known as D6S204; HLA-JY3; HLAC; HLC-C; and PSORS1. Examples HLA The HLA-C gene is available as Gene ID: 3107 (e.g., available at www.ncbi.nlm.nih.gov / gene / 3107). Exemplary nucleic acid sequences that can be used to generate HLA-C, such as any one of SEQ ID NO: 20-22, are provided herein. Exemplary amino acid sequences of HLA-C, such as any one of SEQ ID NO: 9-11, are provided herein.

[0069] As used herein, the term "immune cell" refers to a hematopoietic cell that is functionally involved in the initiation and / or execution of an immune response in an organism. Immune cells can be part of the innate and / or adaptive immune system. Exemplary immune cells include, but are not limited to, myeloid cells (e.g., neutrophils, dendritic cells, eosinophils, mast cells, basophils, monocytes, microglia, and their precursors) and lymphoid cells (e.g., T cells, B cells, natural killer cells, and their precursors).

[0070] As used herein, the term "instructional material" refers to a publication, record, chart, or other medium of expression that can be used to convey the usefulness of the composition and the method of using the composition associated with the publication, record, chart, or other medium of expression. Instructional materials for kits of this disclosure may, for example, be attached to or shipped with a container containing a cell population and / or pharmaceutical composition of this disclosure. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the composition be used synergistically by the recipient.

[0071] As used herein, the term "kill switch" refers to a nucleic acid sequence that, when expressed in a host cell under certain conditions, causes the host cell to undergo apoptosis. Exemplary kill switch nucleic acid sequences include, but are not limited to, nucleotide sequences encoding herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (iCasp9), CD20, and mutant human thymidine kinase (mTMPK). In some cases, the kill switch gene is inducible, wherein the kill switch is activated only upon the addition of an activator (e.g., a small molecule drug).

[0072] As used herein, the term "myeloid cell" refers to cells of the myeloid lineage. Exemplary myeloid cell types include, but are not limited to, monocytes, microglia, macrophages, dendritic cells, basophils, eosinophils, erythrocytes, mast cells, or neutrophils and any of their precursor cells or intermediate progenitor cells.

[0073] As used herein, the term "natural killer cell" refers to a type of cytotoxic lymphocyte that is a subset of innate lymphoid cells that expresses CD56 (also known as NCAM1) and / or any natural cytotoxicity triggering receptor and killer cell lectin-like receptor F1 (also known as NKp80); and lacks CD3 expression. NK cells are involved in the clearance of cells infected by viruses and other intracellular pathogens, as well as in the clearance of tumor cells and senescent cells. "Natural killer cell-mediated cytotoxicity" refers to the killing of cells by NK cells.

[0074] As used herein, the term "nerve cell" refers to any cell belonging to the nervous system. Exemplary nerve cells include, but are not limited to, neurons and neuronal precursor cells (without regard to any particular neuronal subtype, such as dopaminergic neurons, cortical neurons, spinal or oculomotor neurons, enteric neurons, interneurons, and trigeminal or sensory neurons), microglia and microglia precursor cells, glial cells and glial precursor cells (without regard to any particular glial subtype, such as oligodendrocytes, astrocytes, dedicated oligodendrocyte precursor cells, and bienergetic glial precursor cells that can generate astrocytes and oligodendrocytes), basal plate-derived cells, Schwann cells, and satellite cells.

[0075] As used herein, the term "neuritis" refers to inflammation of the neural tissues of the central nervous system, such as the brain or spinal cord. Neuroinflammation can be caused by any means, including but not limited to infection, traumatic brain injury, toxic metabolites, ischemia-reperfusion injury, or autoimmune disorders. Neuroinflammation can be acute, chronic, or both.

[0076] As used herein, the term "nucleic acid" or "nucleic acid molecule" refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments produced by polymerase chain reaction (PCR), and fragments produced by any of ligation, cleavage, endonuclease action, and exonuclease action. Nucleic acid molecules may consist of monomers of naturally occurring nucleotides (e.g., DNA and RNA) or analogs of naturally occurring nucleotides (e.g., enantiomers of naturally occurring nucleotides), or combinations thereof. Modified nucleotides may have alterations in the sugar moiety and / or pyrimidine or purine base moiety. Sugar modifications include, for example, substitution of one or more hydroxyl groups with halogens, alkyl groups, amines, and azides, or functionalization of the sugar into an ether or ester. Furthermore, the entire sugar moiety may be replaced by spatially and electronically similar structures (e.g., azahesaccharides and carbocyclic sugar analogs). Examples of modifications in the base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substituents. Nucleic acid monomers may be linked by phosphodiester bonds or analogs of such bonds. Phosphodiester analogues include thiophosphates, dithiophosphates, selenophosphates, diselenophosphates, aniline thiophosphates, aminophosphates, etc.

[0077] As used herein, the term "Parkinson's disease" refers to a neurodegenerative disorder characterized by widespread degeneration of dopaminergic neurons in the substantia nigra region of the brain. Parkinson's disease manifests as alterations in both motor and nonmotor functions. Exemplary changes in motor function as a symptom of Parkinson's disease include, but are not limited to, tremor, hypokinesis, postural instability, gait abnormalities, and dysphagia. Exemplary changes in nonmotor function as a symptom of Parkinson's disease include, but are not limited to, autonomic and neuropsychiatric disorders, such as anosmia or sleep disturbances. Parkinson's disease can present as early-stage Parkinson's disease or late-stage Parkinson's disease, with more severe symptoms in the later stages of the disease.

[0078] As used herein, the terms “patient,” “subject,” “individual,” etc., are used interchangeably and refer to any animal or its cells, whether in vitro or in situ, suitable for the compositions and methods described herein. In some cases, the patient, subject, or individual is a human being.

[0079] As used herein, the term "pharmaceutically acceptable excipient, carrier, or diluent" means any material that, when combined with an active ingredient (e.g., cell population), allows the ingredient to retain its biological activity and is unresponsive to the immune system of a subject. Examples include, but are not limited to, any standard pharmaceutical excipient, carrier, or diluent, such as phosphate-buffered saline solutions, physiological saline, water, emulsions (e.g., oil / water emulsions), and various wetting agents.

[0080] As used herein, the terms “peptide” and “protein” refer to polymers of any length of amino acid residues. Peptides may include naturally occurring modifications of one or more amino acids in the polymer. For example, peptides may include disulfide bond formation, glycosylation, esterification, acetylation, or phosphorylation. Peptides may also include amino acid analogs or non-naturally occurring amino acids. Peptides may exist as single chains or as related chains.

[0081] As used herein, the term "promoter" and its grammatical equivalents refer to a nucleic acid region located upstream of a gene, in which a related protein (such as RNA polymerase and transcription factor) binds to initiate gene transcription. The promoter can be an endogenous gene (such as...) GAPDHThe promoter can be a tissue-specific promoter, such as a promoter for a gene that is turned on or off in certain cell or tissue types. The promoter can be an engineered promoter, for example, engineered to include one or more elements that can enhance or de-encode gene expression. These one or more elements may include enhancers. The promoter can be a synthetic promoter, for example, containing a non-naturally occurring sequence engineered to express a target gene at a desired expression level in one or more cell types. The promoter can be a heterologous promoter.

[0082] As used herein, the terms “regulation factor X5” and “RFX5” are used interchangeably to refer to… RFX5 A gene-encoded polypeptide. RFX5 is also known as MHC2D3; and MHC2D5. In some cases, the RFX5 is human RFX5. Example RFX5 The gene can be obtained as Gene ID: 5993 (e.g., available at https: / / www.ncbi.nlm.nih.gov / gene / 5993). It can be used for modification. RFX5 An exemplary nucleotide sequence of the gene is available as Transcript ID NM_000449.4 (e.g., available at https: / / www.ncbi.nlm.nih.gov / nuccore / NM_000449.4).

[0083] As used herein, the terms "serine protease inhibitor family A member 3" and "SERPINA3" are used interchangeably to refer to... SERPINA3 A gene-encoded polypeptide. SERPINA3 is also known as α-1-antichymotrypsin. In some cases, the SERPINA3 is human SERPINA3. Example SERPINA3 The gene is available as Gene ID: 12 (e.g., available at www.ncbi.nlm.nih.gov / gene / 12). An exemplary SERPINA3 amino acid sequence is provided herein as SEQ ID NO: 5. An exemplary SERPINA3 nucleotide sequence is provided herein as SEQ ID NO: 16.

[0084] As used herein, the terms "serine protease inhibitor family B member 9" and "SERPINB9" are used interchangeably, referring to [the specific term used in this context]. SERPINB9A gene-encoded polypeptide. SERPINB9 is also known as CAP-3; CAP3; PI-9; PI9; serine (or cysteine) protease inhibitor, clade B (ovalbumin), member 9; serine protease inhibitor, clade B (ovalbumin), member 9; and cytoplasmic antiprotease 3. In some cases, said SERPINB9 is human SERPINB9. Exemplary SERPINB9 The gene is available as Gene ID: 5272 (e.g., available at www.ncbi.nlm.nih.gov / gene / 5272). Exemplary nucleotide sequences that can be used to generate SERPINB9 are available as Transcript ID NM_004155.6 (e.g., available at www.ncbi.nlm.nih.gov / nuccore / NM_004155.6), as well as the nucleotide sequences provided herein, such as SEQ ID NO: 17. Exemplary SERPINB9 amino acid sequences are available as Protein ID NP_004146.1 (e.g., available at www.ncbi.nlm.nih.gov / protein / NP_004146.1), as well as the amino acid sequences provided herein, such as SEQ ID NO: 6.

[0085] As used herein, the term "stem cell" refers to a cell that has the ability to divide indefinitely in a culture and produce specialized cells.

[0086] As used herein, the terms "substantially" or "essentially" refer to a quantity, level, value, number, frequency, percentage, size, volume, weight, or length that is approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher than a reference quantity, level, value, number, frequency, percentage, size, volume, weight, or length. In some cases, the terms "substantially identical" or "substantially identical" refer to a range of quantities, levels, values, numbers, frequencies, percentages, sizes, volumes, weights, or lengths that are substantially the same as a reference quantity, level, value, number, frequency, percentage, size, volume, weight, or length.

[0087] As used herein, the term “continuously active transcriptional payload region” or “STAPLR” and its grammatical equivalents refer to intergenic regions in the mammalian genome that allow for consistent levels of transgene expression, including as cells undergo changes in their differentiation state. STAPLRs extend the genomic safe harbor library, where transgenes can be stably integrated and their expression can be maintained across multiple passages and as cells alter their phenotype. In the context of STAPLRs, the term “payload” or “genomic payload” refers to a foreign or heterologous nucleotide sequence introduced into the region. STAPLRs can refer to intergenic regions existing between essential genes or between genes expressed in different cellular states. Additional information regarding STAPLRs is provided in International Patent Application WO2023212722A1, which is incorporated herein by reference.

[0088] As used herein, the term "sustained transgenic expression locus" or "STEL" refers to a locus in an organism's genome that is resistant to gene expression silencing. For example, an STEL may be resistant to silencing over time or after a change in cell fate (e.g., differentiation), thus maintaining the expression of the gene contained in the STEL. Exemplary STELs include, but are not limited to, genes encoding ribosomal subunits, mitochondrial proteins, actin, eukaryotic translation factors, and histones. Further exemplary STELs are described in WO2021072329A1, which is incorporated herein by reference.

[0089] As used herein, the term "T cell" refers to a type of lymphocyte that plays a central role in cell-mediated immunity. T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells (NK cells), by the presence of T cell receptors (TCRs) on their cell surface. Exemplary T cell types include, but are not limited to, conventional adaptive T cells, which include helper CD4+ T cells (also known as helper T cells), CD8+ T cells (also known as cytotoxic T cells), memory and naive T cells of both CD4+ and CD8+ T cells, and regulatory T cells (also known as T regs), as well as innate-like T cells, including natural killer T cells (also known as NKT cells), mucosa-associated invariant T cells, and γδ T cells. T cells can be naturally occurring or non-natural, such as modified T cells, like CAR-T cells.

[0090] As used herein, the terms “tumor necrosis factor-associated apoptosis-inducing ligand” and “TRAIL” are used interchangeably to refer to the ligands involved in apoptosis. TRAILGenetically encoded polypeptides. TRAIL is also known as TNFSF10; APO2L; Apo-2L; differentiation cluster 253; CD253; TL2; TNLG6A; tumor necrosis factor superfamily member 10; tumor necrosis factor (ligand) superfamily member 10; TNF superfamily member 10; and TANCR. In some cases, the TRAIL is human TRAIL. Exemplary TRAILThe gene is available as Gene ID: 8743 (e.g., available at www.ncbi.nlm.nih.gov / gene / 8743). Exemplary nucleotide sequences that can be used to generate TRAIL are available from Transcript ID NM_001190942.2 (e.g., available at www.ncbi.nlm.nih.gov / nuccore / NM_001190942.2), Transcript ID NM_001190943.2 (e.g., available at https: / / www.ncbi.nlm.gov / nuccore / NM_001190943.2), and Transcript ID NM_003810.4 (e.g., available at https: / / www.nih.gov / nuccore / NM_003810.4). ENST00000420541 (e.g., available at https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_cDNA?db=core;g=ENSG00000121858;r=3:172505508-172523475;t=ENST00000420541) and Transcript ID ENST00000430881 (e.g., available at https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_cDNA?db=core;g=ENSG00000121858;r=3:172505508-172523475;t=ENST00000430881); and the nucleotide sequence provided herein, such as SEQ ID NO: 15.Exemplary amino acid sequences for TRAIL are available from Protein ID NP_001177871.1 (e.g., available at www.ncbi.nlm.nih.gov / protein / NP_001177871.1), Protein ID NP_001177872.1 (e.g., available at https: / / www.ncbi.nlm.gov / protein / NP_001177872.1), Protein ID NP_003801.1 (e.g., available at https: / / www.nih.gov / protein / NP_003801.1), and Protein ID ENST00000420541 (e.g., available at https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_Protein?db=core;g=ENSG00000121858;r=3:172505508-172523475;t=ENST00000420541) and Protein ID ENST00000430881 (e.g., available at https: / / useast.ensembl.org / Homo_sapiens / Transcript / Sequence_Protein?db=core;g=ENSG00000121858;r=3:172505508-172523475;t=ENST00000430881); and the amino acid sequence provided herein, such as SEQ ID NO: 4.

[0091] As used herein, the term “treatment” or its grammatical equivalents refer to means of reducing the frequency or severity of at least one sign or symptom of a disease or condition experienced by a subject.

[0092] As used herein, when used in the context of polypeptides, proteins, nucleic acids, or polynucleotides, the term "variant" refers to a polypeptide, protein, nucleic acid, or polynucleotide with respect to the amino acid sequence of a parent polypeptide or protein (e.g., an endogenous polypeptide) or the nucleotide sequence of a parent nucleic acid or polynucleotide (e.g., an endogenous gene), including at least one altered (e.g., substitution, deletion, or addition of one or more amino acids or nucleotides), but the resulting variant polypeptide, protein, nucleic acid, or polynucleotide retains substantially the same function as the parent polypeptide, protein, nucleic acid, or polynucleotide. The parental sequence of amino acids or nucleic acids can be, for example, a wild-type sequence or its homolog, or a variant of a wild-type sequence or its homolog.

[0093] As used herein, the terms “V-domain Ig repressor of T cell activation” and “VISTA” are used interchangeably to refer to… VISTA Genetically encoded polypeptide. VISTA is also known as VSIR; B7-H5; B7H5; GI24; PP2135; SISP1; DD1alpha; VISTA; C10orf54; chromosome 10 open reading frame 54; PD-1H; V-set immunomodulatory receptor; Dies1; PDCD1 homolog; stress-induced secretory protein 1; and SISP1. In some cases, the VISTA is human VISTA. Exemplary VISTA The gene is available as Gene ID: 64115 (e.g., available at www.ncbi.nlm.nih.gov / gene / 64115). Exemplary nucleotide sequences that can be used to generate VISTA are available as Transcript ID NM_022153.2 (e.g., available at www.ncbi.nlm.nih.gov / nuccore / NM_022153.2), as well as the nucleotide sequences provided herein, such as SEQ ID NO: 12. Exemplary VISTA amino acid sequences are available as Protein ID NP_071436.1 (e.g., available at www.ncbi.nlm.nih.gov / protein / NP_071436.1), as well as the amino acid sequences provided herein, such as SEQ ID NO: 1.

[0094] Throughout this disclosure, various aspects of the invention may be expressed in terms of scope. It should be understood that this scope-based description is merely for convenience and brevity and should not be construed as an inflexible limitation of the scope of the invention. Therefore, the description of scope should be considered as having specifically disclosed all possible sub-scopes and individual numerical values ​​within those scopes. For example, a description of a scope such as 1 to 6 should be considered as having specifically disclosed sub-scopes, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and individual numbers within that scope, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the width of the scope.

[0095] II. Engineered Cells Certain aspects of this disclosure provide an engineered cell. In some embodiments, the engineered cell comprises at least one heterologous nucleic acid sequence encoding a polypeptide or combination of peptides. In some embodiments, the polypeptide or combination of peptides is expressed at a level sufficient to inhibit natural killer (NK) cell-mediated cytotoxicity.

[0096] In some embodiments, the polypeptide comprises one or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the polypeptide comprises one or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of the polypeptide comprises two or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of the polypeptide comprises two or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of the polypeptide comprises two or more of CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of the polypeptide comprises three or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of the polypeptide comprises three or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of peptides includes CLEC2D, TRAIL, and SERPINB9 or variants thereof. In some embodiments, the combination of peptides includes four or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47 or variants thereof. In some embodiments, the combination of peptides includes CLEC2D, TRAIL, SERPINB9, and HLA-C or variants thereof. In some embodiments, the combination of peptides includes five or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47 or variants thereof. In some embodiments, the combination of peptides includes VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47 or variants thereof. In some embodiments, the combination of peptides includes one or more of VISTA, CLEC2D, TRAIL, and SERPINB9 or variants thereof. In some embodiments, the combination of peptides includes CLEC2D or a variant thereof and TRAIL or a variant thereof. In some embodiments, the combination of peptides includes CLEC2D or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the polypeptide comprises VISTA or a variant thereof. In some embodiments, the polypeptide comprises human CLEC2D or a variant thereof. In some embodiments, the polypeptide comprises human TRAIL or a variant thereof. In some embodiments, the polypeptide comprises human SERPINB9 or a variant thereof. In some embodiments, the polypeptide comprises human HLA-C or a variant thereof.In some embodiments, the polypeptide includes human CD47 or a variant thereof.

[0097] In some embodiments, the polypeptide (including variants of the polypeptide described herein) comprises an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence that is identical to any one of SEQ ID NO: 1-11.

[0098] In some embodiments, the polypeptide (including variants of the polypeptide described herein) comprises an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NO: 2, 4, 5, and 11.

[0099] In some embodiments, the polypeptide (including variants of the polypeptide described herein) contains immune evasion activity. For example, in some embodiments, the polypeptide or variants of the polypeptide produce immune evasion activity when expressed by cells at appropriate levels. In some embodiments, the immune evasion activity includes inhibition of natural killer cell-mediated cytotoxicity by engineered cells. In some embodiments, the immune evasion activity includes reduced T cell-mediated killing of engineered cells. In some embodiments, a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11 contains immune evasion activity. In some embodiments, a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11 contains immune evasion activity.

[0100] In some embodiments, engineered cells comprising at least one heterologous nucleic acid sequence encoding a polypeptide or a combination of peptides or a polypeptide variant contain the bioactivity of said polypeptide. For example, in some embodiments, engineered cells comprising at least one heterologous nucleic acid sequence encoding any one of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47 contain bioactivity associated with VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, respectively. In some embodiments, engineered cells comprising at least one heterologous nucleic acid sequence encoding a variant of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, or CD47 contain the bioactivity of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, or CD47, respectively. In some embodiments, engineered cells comprising at least one heterologous nucleic acid sequence encoding a polypeptide or combination of polypeptides having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs: 1-11 contain polypeptide-related biological activity, wherein the polypeptide has the same sequence as any one of SEQ ID NOs: 1-11. In some embodiments, engineered cells comprising at least one heterologous nucleic acid sequence encoding a polypeptide or combination of polypeptides having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with any one of SEQ ID NOs: 2, 4, 5, and 11 contain polypeptide-related biological activity, wherein the polypeptide has the same sequence as any one of SEQ ID NOs: 2, 4, 5, and 11.

[0101] In some embodiments, the engineered cells contain at least two different heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the engineered cells contain at least three different heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the engineered cells contain at least four different heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the engineered cells contain at least five different heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the engineered cells contain at least six different heterologous nucleic acid sequences, each encoding a different polypeptide.

[0102] In some embodiments, the engineered cells comprise at least two distinct heterologous nucleic acid sequences, each encoding CLEC2D or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the engineered cells comprise at least two distinct heterologous nucleic acid sequences, each encoding CLEC2D or a variant thereof and TRAIL or a variant thereof. In some embodiments, the engineered cells comprise at least two distinct heterologous nucleic acid sequences, each encoding CLEC2D or a variant thereof and HLA or a variant thereof. In some embodiments, the engineered cells comprise at least two distinct heterologous nucleic acid sequences, each encoding TRAIL or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the engineered cells comprise at least two distinct heterologous nucleic acid sequences, each encoding TRAIL or a variant thereof and HLA-C or a variant thereof. In some embodiments, the engineered cells comprise at least two distinct heterologous nucleic acid sequences, each encoding SERPINB9 or a variant thereof and HLA or a variant thereof.

[0103] In some embodiments, the at least one heterologous nucleic acid sequence has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 65% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 75% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 85% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 90% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 95% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 98% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 99% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has the same sequence as any one of SEQ ID NO: 12-22.

[0104] In some embodiments, the at least one heteronucleotide sequence has at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heteronucleotide sequence has at least 65% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heteronucleotide sequence has at least 75% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heteronucleotide sequence has at least 85% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heteronucleotide sequence has at least 90% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 95% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 98% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 99% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has the same sequence as any one of SEQ ID NO: 13, 15, 17, and 22.

[0105] In some embodiments, the at least one heterologous nucleic acid sequence is integrated into a genomic locus. In some embodiments, the at least one heterologous nucleic acid sequence is integrated into a sustained transgenic expression locus (STEL) in the engineered cell genome. In some embodiments, the STEL is a locus encoding a protein involved in one or more of the following: ribonucleoprotein complex formation, focal adhesion, cell-substrate adhesion junctions, cell-substrate junctions, cell anchoring, extracellular exosomes, extracellular vesicles, intracellular organelles, anchoring junctions, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. In some embodiments, the STEL is glyceraldehyde-3-phosphate dehydrogenase (… GAPDH In some embodiments, the STEL is a ribosomal protein locus, for example... RPL or RPS Gene locus. RPL An example of a gene is RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPLP0, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4 and RPL22 . RPS An example of a gene is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20 and RPSII In some embodiments, the STEL is a locus encoding mitochondrial proteins (e.g., MT-Co1, MT-CO2, MT-ND4, MT-ND1, and MT-ND2). In some embodiments, the STEL is a locus encoding actin (e.g., ACTG1 and ACTB). In some embodiments, the STEL is a locus encoding eukaryotic translation elongation factors (e.g., EEF1A1 and EEF2) or eukaryotic translation initiation factors (e.g., EIEI). In some embodiments, the STEL is a locus encoding histones (e.g., H3F3A and H3F3B). In some embodiments, the STEL is selected from... FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2 and SRP14 The locus. Compositions and methods for integrating heterologous nucleic acids into STELs are described in International Patent Application Publications WO 2021 / 072329 and WO 2024 / 145653, which are incorporated herein by reference.

[0106] Advantageously, by integrating at least one heterologous nucleic acid encoding a polypeptide into the STEL in the cell genome, the polypeptide encoded by at least one heterologous nucleic acid can be generated by an endogenous gene promoter (e.g., GAPDH The expression of the polypeptide is controlled by a promoter. Therefore, the expression of the polypeptide can be associated with the expression of endogenous genes. The sustained activity of endogenous genes in the engineered cells will then mean that the polypeptide will maintain sustained and constitutive expression. By utilizing the inherent regulatory mechanisms of endogenous genes, this disclosure provides a useful mechanism for maintaining polypeptide expression at levels sufficient to suppress natural killer cell-mediated cytotoxicity.

[0107] In some embodiments, the heterologous nucleic acid is integrated into the genome of the engineered cell in a sustained transcriptional activity payload region (STAPLR). In some embodiments, the STAPLR is selected from the following: RPL34 Genes and OSTC Intergenic spacer regions between genes; ACTB Genes and FSCN1 Intergenic spacer regions between genes; AKIRIN1 Genes and NDUFS5 Intergenic spacer regions between genes; PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes; PTGES3 Genes and NACA Intergenic spacer regions between genes; MLF2 Genes and PTMS Intergenic spacer regions between genes; RAB13 Genes and RPS27 Intergenic spacer regions between genes; JTB Genes and RAB13 Intergenic spacer regions between genes; AKR1A1 Genes and NASP Intergenic spacer regions between genes; NDUFS5 Genes and MACF1 Intergenic spacer regions between genes; SRSF9 Genes and DYNLL1 Intergenic spacer regions between genes; MYL6B Genes and MYL6 Intergenic spacer regions between genes; GPX1 Genes and RHOA Intergenic spacer regions between genes; HNRNPA2B1 Genes and CBX3 Intergenic spacer regions between genes; ROMO Genes and RBM39 Intergenic spacer regions between genes; and PA2G4 Genes and RPL41 A gene spacer region between genes. In some embodiments, the STAPLR is... PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes. In some embodiments, the heteronucleotide is integrated at a location at least 100-5000 base pairs from the nearest gene. Compositions and methods for integrating heteronucleotides into STAPLR are described in International Patent Application Publication No. WO 2024 / 145653, which is incorporated herein by reference.

[0108] In some embodiments, the engineered cells include a kill switch. Including a kill switch can be used to increase the safety of the engineered cells. For example, activation of the kill switch can remove the engineered cells without interfering with the subject's own cells. In some embodiments, the kill switch is a gene encoding herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (also known as incasep 9 and iCasp9), CD20, or mutant human thymidine kinase (mTMPK). In some embodiments, the kill switch is under the control of an inducible promoter. In some embodiments, the kill switch is encoded by a heterologous nucleic acid. In some embodiments, at least one heterologous nucleic acid encoding the kill switch is integrated into a different locus than at least one heterologous nucleic acid encoding the polypeptide. In some embodiments, at least one heterologous nucleic acid encoding the kill switch is integrated into the same locus as at least one heterologous nucleic acid encoding the polypeptide.

[0109] In some embodiments, the engineered cells further comprise genetic modifications that reduce T cell-mediated killing compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that reduce T cell-mediated killing by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that reduce T cell-mediated killing by at least 20% compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that reduce T cell-mediated killing by at least 50% compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that reduce T cell-mediated killing by at least 80% compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that reduce T cell-mediated killing by at least 90% compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that result in a reduction of at least 95% in T cell-mediated killing compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that result in a reduction of at least 100% in T cell-mediated killing compared to wild-type cells.

[0110] In some implementations, the genetic modifications that result in reduced T cell-mediated killing in engineered cells compared to wild-type cells include: β 2 microglobulin ( B2M The deletion, disruption, or attenuation of genes. In some implementations, genetic modifications that reduce T cell-mediated killing in engineered cells compared to wild-type cells include... HLA-A Gene, HLA-B Genes and HLA-C One or more genes are missing, damaged, or weakened.

[0111] In some embodiments, the engineered cells further comprise a class II major histocompatibility complex transactivator (MHC). CIITA ) gene or regulatory factor 5 ( RFX5 The engineered cells contain the deletion, disruption, or attenuation of genes. Therefore, in some embodiments, the engineered cells include genetic modifications, as described herein, that result in reduced T cell-mediated killing in the engineered cells compared to wild-type cells (e.g., [missing information]). B2M , HLA-A , HLA-B and / or HLA-C (loss, damage or weakening) and CIITAThe engineered cells contain gene deletions, disruptions, or attenuations. In some embodiments, the engineered cells include genetic modifications, as described herein, that result in reduced T cell-mediated killing compared to wild-type cells (e.g., B2M , HLA-A , HLA-B and / or HLA- C (loss, damage or weakening) and RFX5 The engineered cells contain gene deletions, disruptions, or attenuations. In some embodiments, the engineered cells include genetic modifications, as described herein, that result in reduced T cell-mediated killing compared to wild-type cells (e.g., B2M , HLA-A , HLA-B and / or HLA-C (loss, damage or weakening) and CIITA Genes and RFX5 The loss, damage, or weakening of genes.

[0112] In some embodiments, the engineered cells further comprise at least one selectable marker gene. The selectable marker can be used to detect or select the engineered cells, and its expression can be detected by any suitable method known in the art, such as FACS or culture in a selective medium. Non-limiting examples of selectable marker genes include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein (GFP), blue fluorescent proteins (EBFP, EBFP2, Azurite, mKalamal), cyan fluorescent proteins (ECFP, Cerulean, CyPet, mTurquoise2), and yellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, EYFP), β-galactosidase (LacZ), chloramphenicol acetyltransferase (cat), neomycin phosphotransferase (neo), puromycin N-acetyltransferase (PAC), enzymes (e.g., oxidases and peroxidases), and antigen molecules. In some embodiments, the selectable marker gene may be driven by an endogenous gene promoter.

[0113] In some embodiments, the engineered cells comprise engineered stem cells. In some embodiments, the engineered stem cells comprise pluripotent stem cells. In some embodiments, the pluripotent stem cells comprise embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the engineered stem cells comprise pluripotent stem cells.

[0114] In some embodiments, the engineered cells include cardiac cells, nerve cells, T cells (e.g., regulatory T cells), retinal cells, endocrine cells, epithelial cells, muscle cells, or myeloid cells. In some embodiments, the engineered cells include cardiac cells. In some embodiments, the cardiac cells are cells of the epicardium, myocardium, or endocardium of the heart. In some embodiments, the cardiac cells are cardiomyocytes, cardiac fibroblasts, cardiac smooth muscle cells, epicardial cells, cardiac endothelial cells, Purkinje fibers, or pacemaker cells. In some embodiments, the engineered cells include nerve cells. In some embodiments, the nerve cells are neurons or glial cells. In some embodiments, the engineered cells include myeloid cells. In some embodiments, the myeloid cells are monocytes, microglia, macrophages, dendritic cells, basophils, eosinophils, erythrocytes, mast cells, neutrophils, megakaryocytes, or platelets or any of their precursor progenitor cells. In some embodiments, the cardiac cells, nerve cells, or myeloid cells are derived from engineered stem cells (e.g., engineered stem cells as described herein). In some embodiments, the T cells are helper T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), memory T cells, regulatory T cells (T reg cells), innate-like T cells (e.g., natural killer T cells), mucosa-associated invariant T cells, or γδ T cells, or modified T cells (e.g., CAR-T cells). In some embodiments, the retinal cells are photoreceptors, retinal horizontal cells, retinal bipolar cells, retinal amacrine cells, or retinal ganglion cells. In some embodiments, the endocrine cells are hypothalamic endocrine cells, pituitary endocrine cells, pineal endocrine cells, thyroid endocrine cells (e.g., follicular cells), parathyroid endocrine cells, thymic endocrine cells, adrenal endocrine cells, pancreatic endocrine cells (e.g., α cells, β cells, δ cells, or F cells), ovarian endocrine cells (e.g., granulosa cells), or testicular endocrine cells (e.g., interstitial cells of the testes). In some embodiments, the epithelial cells are squamous epithelial cells, cuboidal epithelial cells, columnar epithelial cells, pseudostratified epithelial cells, or stratified epithelial cells. In some embodiments, the muscle cells are skeletal muscle cells, cardiomyocytes (e.g., cardiac muscle cells), or smooth muscle cells.

[0115] In some implementations, the engineered cells are human engineered cells.

[0116] Therefore, in one aspect, this disclosure provides cells and methods engineered to reduce or inhibit natural killer (NK) cell-mediated cytotoxicity, focusing on target genes involved in specific cell-NK cell interactions. In some embodiments, the engineering strategy is designed to upregulate the expression of proteins that interact with receptors on NK cells, such as CD161. For example, one revelation of this disclosure is that overexpression of the gene CLEC2D can modulate NK cell activity by binding to CD161 on these cells, thereby leading to a reduction in NK cell-induced killing rates.

[0117] In another aspect, this disclosure provides cells and methods thereof engineered to inhibit or reduce natural killer (NK) cell-mediated cytotoxicity by modulating the expression of one or more proteins involved in apoptosis. In some embodiments, this disclosure provides cells engineered to overexpress one or more of SERPINB9, SERPINA3, TRAIL, or FASL. For example, in some embodiments, this disclosure provides cells engineered to increase TRAIL expression, thereby providing the cells with the ability to induce apoptosis in target cells through association with TRAIL receptors, particularly TRAIL-R1 or TRAIL-R2. This strategic increase in TRAIL expression serves as a countermeasure against the cytotoxic effects of NK cells, potentially improving the survival and efficacy of these engineered cells in therapeutic applications where NK cell activity poses a significant challenge.

[0118] On the other hand, this disclosure provides cells and methods thereof engineered to inhibit or reduce natural killer (NK) cell-mediated cytotoxicity by modulating the expression of one or more proteins involved in the serine protease inhibitor family. For example, in some embodiments, this disclosure provides cells engineered to overexpress proteins belonging to the intracellular serine protease inhibitor subfamily that inhibits granzyme B (GrzB) activity, which is a mechanism by which T and NK cells kill target cells. Thus, in some embodiments, this disclosure provides cells engineered to overexpress SERPINB9.

[0119] cell population In some aspects of this disclosure, engineered cell populations (e.g., engineered cell populations described herein) are provided.

[0120] In some embodiments, engineered cell populations exhibit reduced susceptibility to natural killer cell-mediated cytotoxicity compared to cell populations not engineered to have at least one heterologous nucleic acid sequence. The susceptibility of said engineered cell population to natural killer cell-mediated cytotoxicity can be measured by any suitable method known in the art. For example, susceptibility to natural killer cell-mediated cytotoxicity can be measured by a natural killer cell cytotoxicity assay, such as the natural killer cell cytotoxicity assay described in Example 2. In some embodiments, the engineered cell population exhibits reduced susceptibility to natural killer cell-mediated cytotoxicity by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% compared to cell populations not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population exhibits reduced susceptibility to natural killer cell-mediated cytotoxicity by at least about 10% compared to cell populations not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population is at least about 20% less susceptible to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population is at least about 30% less susceptible to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population is at least about 50% less susceptible to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence.

[0121] In some embodiments, the engineered cells in the engineered cell population are positive for cardiac troponin T (cTNT). In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 70% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 80% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 90% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 95% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 98% of the engineered cells in the engineered cell population are positive for cTNT. In some implementations, at least 99% of the engineered cells in the engineered cell population are positive for cTnT.

[0122] In some embodiments, the engineered cell population is present in a composition formulated for administration to a subject. The engineered cell population can be formulated for any suitable administration method, such as administration via local injection into a tissue.

[0123] Preparation of engineered cells In some aspects of this disclosure, methods for preparing any of the engineered cells described herein are provided.

[0124] In some embodiments, the method of preparing the engineered cells described herein includes genetically modifying the cells to increase the expression of a peptide or combination of peptides. In some embodiments, the peptide comprises one or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of peptides comprises two or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises two or more of CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of peptides comprises three or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises three or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of peptides comprises CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of peptides comprises four or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of peptides comprises five or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises VISTA, CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of peptides comprises CLEC2D or a variant thereof and TRAIL or a variant thereof. In some embodiments, the combination of peptides comprises CLEC2D or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the peptide comprises VISTA or a variant thereof. In some embodiments, the peptide comprises CLEC2D or a variant thereof.

[0125] In some embodiments, the genetically modified cells comprise a heterologous nucleic acid sequence encoding a polypeptide integrated into the cell's genome. In some embodiments, the genetically modified cells exhibit increased survival when attacked with natural killer cells compared to wild-type cells.

[0126] In some implementations, at least one heterologous nucleic acid sequence encoding a polypeptide is integrated into the genome of the cell. Any suitable technique for integrating nucleic acids into the host cell genome can be used. For example, at least one heterologous nucleic acid sequence can be integrated into the cell genome using, for example, known gene editing systems, such as those utilizing genome-targeting elements comprising DNA-binding domains (e.g., zinc finger DNA-binding proteins or TALE DNA-binding domains), guide RNA elements (e.g., CRISPR guide RNA), and guide DNA elements (e.g., NgAgo guide DNA). Programmable gene-targeting elements and nuclease elements enable precise targeting of the integration of heterologous nucleic acids. In some cases, at least one heterologous nucleic acid is integrated into the cell genome using systems based on a wide range of nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-based systems, or NgAgo-based systems.

[0127] In some embodiments, a CRISPR / Cas system is used to integrate at least one heterologous nucleic acid sequence encoding a polypeptide into the genome of a cell. The CRISPR / Cas system has been used to introduce genetic modifications and gene regulation in various species. Without being theoretically limited, target nucleic acids can be modified by the interaction of the CRISPR / Cas system with sequences present in the target nucleic acid, for example, to induce cleavage of the target nucleic acid (e.g., hydrolysis of one or more phosphodiester bonds) and introduce genetic modifications. In some embodiments, a CRISPR / Cas9 system or a CRISPR / Cas12 system is used to integrate at least one heterologous nucleic acid sequence encoding a polypeptide into the genome of a cell.

[0128] In some embodiments, at least one heterologous nucleic acid sequence encoding a polypeptide is integrated into an STEL in the cellular genome. In some embodiments, the STEL is a gene locus encoding a protein involved in one or more of the following: ribonucleoprotein complex formation, focal adhesion, cell-substrate adhesion, cell-substrate junctions, cell anchoring, extracellular exosomes, extracellular vesicles, intracellular organelles, anchoring junctions, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. In some embodiments, the STEL is glyceraldehyde-3-phosphate dehydrogenase (… GAPDH In some embodiments, the STEL is a ribosomal protein locus, for example... RPL or RPS Gene locus. RPL An example of a gene is RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPL30, RPL24, RPL39, RPL37, RPL14,RPL27A, RPLP2, RPLP0, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4 and RPL22 . RPS An example of a gene is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20 and RPSII In some embodiments, the STEL is a locus encoding mitochondrial proteins (e.g., MT-Co1, MT-CO2, MT-ND4, MT-ND1, and MT-ND2). In some embodiments, the STEL is a locus encoding actin (e.g., ACTG1 and ACTB). In some embodiments, the STEL is a locus encoding eukaryotic translation elongation factors (e.g., EEF1A1 and EEF2) or eukaryotic translation initiation factors (e.g., EIEI). In some embodiments, the STEL is a locus encoding histones (e.g., H3F3A and H3F3B). In some embodiments, the STEL is selected from... FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2 and SRP14 The locus. Compositions and methods for integrating heterologous nucleic acids into STELs are described in International Patent Application Publications WO 2021 / 072329 and WO 2024 / 145653, which are incorporated herein by reference.

[0129] In some embodiments, the heterologous nucleic acid is integrated into the genome of the engineered cell in a sustained transcriptional activity payload region (STAPLR). In some embodiments, the STAPLR is selected from the following: RPL34 Genes and OSTC Intergenic spacer regions between genes; ACTB Genes and FSCN1 Intergenic spacer regions between genes; AKIRIN1 Genes and NDUFS5 Intergenic spacer regions between genes; PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes; PTGES3 Genes and NACA Intergenic spacer regions between genes; MLF2 Genes and PTMS Intergenic spacer regions between genes; RAB13 Genes and RPS27 Intergenic spacer regions between genes; JTB Genes and RAB13 Intergenic spacer regions between genes; AKR1A1 Genes and NASP Intergenic spacer regions between genes; NDUFS5 Genes and MACF1 Intergenic spacer regions between genes; SRSF9 Genes and DYNLL1 Intergenic spacer regions between genes; MYL6B Genes and MYL6Intergenic spacer regions between genes; GPX1 Genes and RHOA Intergenic spacer regions between genes; HNRNPA2B1 Genes and CBX3 Intergenic spacer regions between genes; ROMO Genes and RBM39 Intergenic spacer regions between genes; and PA2G4 Genes and RPL41 A gene spacer region between genes. In some embodiments, the STAPLR is... PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes. In some embodiments, the heteronucleotide is integrated at a location at least 100-5000 base pairs from the nearest gene. Compositions and methods for integrating heteronucleotides into STAPLR are described in International Patent Application Publication No. WO 2024 / 145653, which is incorporated herein by reference.

[0130] In some embodiments, at least one heterologous nucleic acid sequence encoding the polypeptide further comprises one or more regulatory elements. Such regulatory elements may comprise regulatory sequences, which are any DNA sequences responsible for regulating gene expression, such as promoters and operons. The regulatory element may be a segment of a nucleic acid molecule capable of increasing or decreasing the expression of a specific gene in an organism.

[0131] In some embodiments, the regulatory element is a promoter. A promoter is a nucleotide sequence that directs the transcription of a structural gene. In some alternative embodiments, the promoter is located in the 5' uncoding region of the gene, near the transcription start site of the structural gene. Sequence elements within a promoter that perform transcription initiation are typically characterized by a consistent nucleotide sequence. However, these promoter elements may include, but are not limited to, RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSE; McGehee et al., Mol. Endocrinol. 7:551 (1993);), cyclic AMP response elements (CRE), serum response elements (SREs; Treisman et al., Seminars in Cancer Biol. 1:47 (1990); incorporated in whole by reference), glucocorticoid response elements (GRE), and binding sites for other transcription factors, such as CRE / ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992); incorporated in whole by reference), AP2 (Ye et al., J. Biol. Chem. 269:25728 (1994); incorporated in whole by reference), SPI, and cAMP response element-binding proteins (CREB; Loeken et al., Gene Expr. 3:253). (1993); incorporated by reference) and octamer factor (see Watson et al., eds., Molecular Biology of the Gene, 4th ed. (The Benjamin / Cummings Publishing Company, Inc. 1987; incorporated by reference) and Lemaigre and Rousseau, Biochem. J. 303:1 (1994); incorporated by reference).

[0132] In some alternatives, the promoters used herein may be inducible or constitutive promoters. However, they are not limited to, inducible promoters may include, for example, tamoxifen-inducible promoters, tetracycline-inducible promoters, or doxocycline-inducible promoters (e.g., tre promoters). Constitutive promoters may include, for example, SV40, CMV, UBC, EFLα, PGK, or CAGG. Any suitable promoter known in the art for expressing genes in engineered cell populations as described herein may be used. In some embodiments, at least one heterologous nucleic acid sequence encoding the polypeptide includes a promoter. In some embodiments, expression of the polypeptide is driven by an endogenous gene promoter.

[0133] At least one heterologous nucleic acid sequence encoding the polypeptide can be introduced into cells using any suitable method known in the art. For example, at least one heterologous nucleic acid can be introduced via electroporation, sonoporation, lipid transfection, microinjection, gene gun methods, virions, liposomes, immunoliposomes, polycationic or lipid:nucleic acid conjugates, naked DNA, artificial viral particles, viral vector systems (such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, vaccinia virus, and herpes simplex virus vectors), and agent-enhanced DNA uptake. In some embodiments, at least one heterologous nucleic acid encoding the polypeptide can be introduced into cells as RNA (e.g., mRNA or self-replicating RNA).

[0134] In some embodiments, at least one heterologous nucleic acid sequence encoding the polypeptide is introduced into a cell via a vector. The vector may be a plasmid, a virus, or another vector designed to introduce the target nucleic acid into the cell. Viral vectors include DNA and RNA viruses that have a free genome or an integrated genome after delivery to the cell. Exemplary viral vectors that can be used include adeno-associated virus, lentivirus, retrovirus, herpes simplex virus, vaccinia virus, or adenovirus. The vector is used to introduce a target gene into a host cell, wherein the vector will interact with a polymerase in the cell to express the protein encoded in the vector. The vector may be present outside the chromosome in the cell or integrated into the genome of the host cell. In some embodiments, the use of a viral vector will result in the integration of the heterologous nucleic acid into the genome of the cell.

[0135] In some embodiments, methods for preparing the engineered cells described herein include introducing a heterologous nucleic acid encoding a killer switch into the cells. In some embodiments, the killer switch is a gene encoding herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (also known as incasep 9 and iCasp9), CD20, or mutant human thymidine kinase (mTMPK). In some embodiments, the killer switch is under the control of an inducible promoter.

[0136] In some implementations, a CRISPR / Cas system is used to integrate a nucleic acid encoding a kill switch into the cell's genome. The nucleic acid encoding the kill switch can be integrated into a locus that is the same as or different from at least one heterologous nucleic acid sequence encoding the polypeptide.

[0137] In some embodiments, the nucleic acid encoding the kill switch is introduced into the cell via a vector. The nucleic acid encoding the kill switch may be introduced into the cell via a vector that is the same as or different from at least one heterologous nucleic acid sequence encoding the polypeptide.

[0138] In some embodiments, the method for preparing engineered cells described herein further includes introducing a genetic modification that results in a reduction in T cell-mediated killing of the engineered cells compared to wild-type cells. In some embodiments, the genetic modification results in a reduction of at least 20% in T cell-mediated killing of the engineered cells compared to wild-type cells. In some embodiments, the genetic modification results in a reduction of at least 50% in T cell-mediated killing of the engineered cells compared to wild-type cells. In some embodiments, the genetic modification results in a reduction of at least 80% in T cell-mediated killing of the engineered cells compared to wild-type cells.

[0139] In some embodiments, the engineered cells in the engineered cell population are positive for cardiac troponin T (cTNT). In some embodiments, at least 90% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 95% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 98% of the engineered cells in the engineered cell population are positive for cTNT.

[0140] In some implementations, the genetic modifications that result in reduced T cell-mediated killing in engineered cells compared to wild-type cells include: β 2 microglobulin ( B2M The deletion, disruption, or attenuation of genes. In some implementations, genetic modifications that reduce T cell-mediated killing in engineered cells compared to wild-type cells include... HLA - A Gene, HLA - B Genes and HLA - COne or more genes are missing, damaged, or weakened.

[0141] In some embodiments, the method for preparing engineered cells described herein further includes introducing... CIITA Gene or RFX5 The deletion, disruption, or attenuation of genes. Therefore, in some embodiments, the method includes preparing engineered cells containing genetic modifications, as described herein, that result in reduced T-cell-mediated killing in the engineered cells compared to wild-type cells (e.g., B2M , HLA - A , HLA - B and / or HLA - C (loss, damage or weakening) and CIITA The deletion, disruption, or attenuation of genes. In some embodiments, the method includes preparing engineered cells containing genetic modifications, as described herein, that result in reduced T cell-mediated killing in the engineered cells compared to wild-type cells (e.g., B2M , HLA - A , HLA - B and / or HLA - C (loss, damage or weakening) and RFX5 The deletion, disruption, or attenuation of genes. In some embodiments, the method includes preparing engineered cells containing genetic modifications, as described herein, that result in reduced T cell-mediated killing in the engineered cells compared to wild-type cells (e.g., B2M , HLA - A , HLA - B and / or HLA - C (loss, damage or weakening) and CIITA Genes and RFX5 The loss, damage, or weakening of genes.

[0142] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs). Methods for obtaining iPSCs are known in the art, and the iPSCs are used in methods for preparing engineered cells as described herein. For example, the iPSCs can be prepared by inducing the expression of one or more genes, such as... [[ID=7)2]]POU5F1 / OCT4 With, but not limited to, SOX2, KLF, c - MYC, NANOG and / or LIN28 / LIN28A Combination. Reprogramming factors can be delivered in various ways (e.g., viral, non-viral, RNA, DNA, or protein delivery). Alternatively, endogenous genes can be activated by using, for example, a CRISPR / Cas system to reprogram non-pluripotent cells into iPSCs.

[0143] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells and differentiating the stem cells into the engineered cells. Methods for inducing stem cells to differentiate into cells of various lineages are well known in the art. For example, methods for inducing stem cells to differentiate into myeloid cells or neural cells are described, for example, in U.S. Patent Nos. 11,525,119,B2 and 10,260,044 B1 and Slukvin et al., J Imm. (2006) 176:2924-32; and Su et al., Clin Cancer Res. (2008) 14(19):6207-17; WO2021072329A1; and Tseng et al., Regen Med. (2009) 4(4):513-26, the disclosures of which are incorporated herein by reference. In some embodiments, differentiating stem cells into engineered cells includes contacting the stem cells with one or more differentiation factors. The specific combination of differentiation factors used will depend on the desired cell type. In some implementations, differentiating stem cells into engineered cells includes contacting iPSCs with one or more differentiation factors that drive their development and / or differentiation into myeloid progenitor cells.

[0144] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells and differentiating the genetically modified stem cells into cardiac cells. In some embodiments, the stem cells are pluripotent stem cells. Any suitable methods known in the art for differentiating stem cells into cardiac cells can be used in conjunction with the methods of this disclosure. Various methods for differentiating stem cells into cardiac cells are described in, for example, Kattman et al., Cell Stem Cell (2011) 8(2):228-40, WO2016131137, WO2018098597, U.S. Patents 9,453,201, WO2020227232, WO2020227232A2; and WO2021072329, the disclosures of which are incorporated herein by reference.

[0145] In some embodiments, differentiating the stem cells into cardiac cells includes incubating the stem cells in one or more cardiac differentiation media. For example, the cardiac differentiation media may contain varying concentrations of bone morphogenetic proteins (BMPs; such as BMP4) and activins (such as activin A). Titration of the differentiation factor concentrations can be performed to determine the optimal concentrations required to achieve differentiation into the desired cardiac cell type. For example, the differentiation factors can be selected to guide differentiation into cardiomyocytes. In some embodiments, differentiating the stem cells into cardiac cells involves modulating Wnt / β-catenin signaling under fully defined conditions. For example, in some embodiments, modulation of Wnt signaling can be achieved by contacting the stem cells with a Wnt activator (e.g., CHIR) followed by inactivation with a small molecule IWR1. More information on methods for differentiating stem cells into cardiomyocytes can be found in Lian, Xiaojun, et al “Al Xiaojun, more information on methods for myocyte differentiation can be found at the optimal concentration required. For example, the differentiation factor can be selected to guide differentiation into cardiomyocytes. In some embodiments, differentiating the stem cells into cardiac cells involves complete Wnt / β-catenin signaling under fully defined conditions.” Nature protocols 8.1 (2013): 162-175; and Lee, Jee Hoon, et al “Human pluripotent stem cell-derived atrial and ventricular cardiomyocytes develop from distinct mesoderm populations.” Cell stem cell21.2 (2017): 179-194, each incorporated by reference.

[0146] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells and differentiating the genetically modified stem cells into neural cells. In some embodiments, the stem cells are pluripotent stem cells. Any suitable methods known in the art for differentiating stem cells into neural cells may be used in conjunction with the methods of this disclosure. Exemplary methods for differentiating stem cells into neural cells are described, for example, in NatBiotechnol; 27(3): 275-280; Cell Stem Cell, 28(2): 343–355; WO2016196661A1; and WO2010096496A2, the disclosures of which are incorporated herein by reference.

[0147] For many neuronal cell types, stem cells can be pre-differentiated to adopt primitive neuronal cell fates through a neural induction process involving dual SMAD inhibition (Chambers et al., Nat Biotechnol. (2009) 27(3):275-80). Primitive neurons possess prior characteristics, and therefore the lack of additional signals will yield anterior / precordial cells. Caudalizing signals can be blocked to prevent paracrine signaling, which would otherwise produce cultures with more posterior characteristics (e.g., XAV939 can block WNT and SU5402 can block FGF signaling). Dorsal cortical neurons can be prepared by blocking SHH activation, while ventral cortical neurons can be prepared by SHH activation. By caudating cultures with the addition of FGF and / or WNT signaling, more tailed cell types, such as serotonergic neurons or spinal motor neurons, can be prepared. For some neuronal cell types, retinoic acid (another tailing agent) can be added to post-treatment of the culture. The generation of glial cell types generally follows the same pattern as primitive neurons before expansion culture in media containing FGF2 and / or EGF. Peripheral nervous system cell types can follow the same general principles, but exhibit timely WNT signaling early in the differentiation process.

[0148] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells and differentiating the genetically modified stem cells into myeloid cells. In some embodiments, the stem cells are pluripotent stem cells. Any suitable methods known in the art for differentiating stem cells into myeloid cells may be used in conjunction with the methods of this disclosure. Exemplary methods for differentiating stem cells into myeloid cells are described, for example, in Muffat et al., Nat Med. 2016 Nov; 22(11): 1358–1367, Pandaya et al., Nat Neurosci. 2017 May;20(5): 753–759, Abud et al., Neuron 2017 Apr 19;94(2):278-293, Douvaras et al., Stem Cell Reports, Volume 8, Issue 6, P1516-1524, June 06, 2017, VanWilgenburg PLOS ONE, https: / / doi.org / 10.1371 / journal.pone.0071098 — Aug2013, Haenseler et al., Stem Cell Reports 2017 Jun The contents of these publications are incorporated herein by reference in 6;8(6):1727-1742 and Takataet.al, Immunity 2017 Jul 18;47(1):183-198.

[0149] In some embodiments, differentiating the genetically modified stem cells into myeloid cells includes culturing the stem cells under conditions that induce bone marrow differentiation to generate myeloid cells. In some embodiments, the stem cells are cultured under conditions that induce bone marrow differentiation to generate CX3CR1+ myeloid cells. In some embodiments, the stem cells are cultured under conditions that induce bone marrow differentiation to generate CD14+ myeloid cells. In some embodiments, the stem cells are cultured under conditions that induce bone marrow differentiation to generate CD45+ / CD14+ / CX3CR1+ myeloid cells.

[0150] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells and differentiating the genetically modified stem cells into retinal cells. In some embodiments, differentiating the genetically modified stem cells into myeloid cells includes culturing stem cells under conditions that induce retinal cell differentiation to generate retinal cells.

[0151] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells and differentiating the genetically modified stem cells into T cells. In some embodiments, differentiating the genetically modified stem cells into myeloid cells includes culturing stem cells under conditions that induce T cell differentiation to generate T cells.

[0152] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells and differentiating the genetically modified stem cells into endocrine cells. In some embodiments, differentiating the genetically modified stem cells into myeloid cells includes culturing stem cells under conditions that induce endocrine cell differentiation to generate endocrine cells.

[0153] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells and differentiating the genetically modified stem cells into epithelial cells. In some embodiments, differentiating the genetically modified stem cells into myeloid cells includes culturing stem cells under conditions that induce epithelial cell differentiation to generate epithelial cells.

[0154] In some embodiments, the methods for preparing engineered cells described herein include genetically modifying stem cells and differentiating the genetically modified stem cells into muscle cells. In some embodiments, differentiating the genetically modified stem cells into myeloid cells includes culturing stem cells under conditions that induce muscle cell differentiation to generate muscle cells.

[0155] In some embodiments, the stem cells are cultured in a bioreactor. In some embodiments, the stem cells are cultured in a cell factory under activated gassing. “Active gassing” refers to the application of a pressure gradient of a gas mixture in one or more cell factories.

[0156] In some embodiments, a multi-step approach is used to differentiate the stem cells into myeloid cells, wherein the stem cells are cultured with different combinations of cytokines and tissue culture media at each stage. For example, in some embodiments, differentiating the stem cells into myeloid cells includes performing one or more of the following steps: first, contacting the cell culture with a first composition containing BMP4 in the culture medium, wherein the cell culture contains stem cells when the cell culture is initially contacted with the first composition. The process can involve: first, using small molecules capable of activating the same pathway as BMP4; second, contacting the cell culture with a second composition in hematopoietic cell culture medium, the second composition comprising one or more of SCF and VEGF, and optionally bFGF (e.g., each of SCF and VEGF, with or without bFGF); third, contacting the cell culture with a third composition in hematopoietic cell culture medium, the third composition comprising one or more of SCF, IL-3, TPO, M-CSF, and FLT3 ligand (e.g., each of SCF, IL-3, TPO, M-CSF, and FLT3 ligand); and fourth, contacting the cell culture with a fourth composition in hematopoietic cell culture medium, the fourth composition comprising one or more of M-CSF, FLT3 ligand, and GM-CSF (e.g., each of M-CSF, FLT3 ligand, and GM-CSF). In some embodiments, all four steps are performed sequentially. In some such embodiments, the culture medium used for any of the four steps is a serum-free culture medium. In some such embodiments, the culture medium used for any of the four steps is a chemically defined culture medium. In some embodiments, a tissue culture medium suitable for stem cell maintenance is used in the first step of the four steps described above, while in other embodiments, a tissue culture medium suitable for stem cell differentiation is used. In the last three steps of the four steps described above, any suitable hematopoietic cell culture medium can be used.

[0157] In some embodiments, the method for preparing engineered cells described herein further includes amplifying the engineered cells to generate an engineered cell population. Amplifying the engineered cells may include culturing the engineered cells or contacting the engineered cells with a culture medium containing a mixture of cytokines and growth factors that allow the engineered cells to amplify.

[0158] In some embodiments, the method for preparing engineered cells described herein further includes preserving the engineered cell population after expansion. For example, the engineered cell population can be cryopreserved. The cryopreserved engineered cell population can be thawed at a later time and can be diluted for downstream applications.

[0159] In some embodiments, the engineered cell populations generated by the methods described herein are used to prepare compositions for treating a disease or condition in a subject.

[0160] III. Composition Certain aspects of this disclosure provide pharmaceutical compositions comprising engineered cell populations (such as those described herein) and pharmaceutically acceptable carriers, transporters, or diluents.

[0161] In some embodiments, the pharmaceutical composition comprises a cell population containing at least one heterologous nucleic acid sequence encoding a polypeptide or combination of polypeptides. In some embodiments, the polypeptide or combination of polypeptides is expressed at a level sufficient to inhibit natural killer cell-mediated cytotoxicity.

[0162] In some embodiments, the polypeptide comprises one or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of the polypeptide comprises two or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of the polypeptide comprises two or more of CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of the polypeptide comprises three or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of the polypeptide comprises three or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of the polypeptide comprises CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of peptides comprises four or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of peptides comprises five or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises VISTA, CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of peptides comprises CLEC2D or a variant thereof and TRAIL or a variant thereof. In some embodiments, the combination of peptides comprises CLEC2D or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the peptide comprises VISTA or a variant thereof. In some embodiments, the peptide comprises human CLEC2D or a variant thereof. In some embodiments, the polypeptide comprises human TRAIL or a variant thereof. In some embodiments, the polypeptide comprises human SERPINB9 or a variant thereof. In some embodiments, the polypeptide comprises human HLA-C or a variant thereof. In some embodiments, the polypeptide comprises human CD47 or a variant thereof. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding a different polypeptide.In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least three distinct heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least four distinct heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least five distinct heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least six distinct heterologous nucleic acid sequences, each encoding a different polypeptide.

[0163] In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding CLEC2D or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding CLEC2D or a variant thereof and TRAIL or a variant thereof. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding CLEC2D or a variant thereof and HLA-C or a variant thereof. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding TRAIL or a variant thereof and HLA-C or a variant thereof. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding SERPINB9 or a variant thereof and HLA-C or a variant thereof.

[0164] In some embodiments, the polypeptide (including variants of the polypeptide described herein) comprises an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence that is identical to any one of SEQ ID NO: 1-11.

[0165] In some embodiments, the polypeptide (including variants of the polypeptide described herein) comprises an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NO: 2, 4, 5, and 11.

[0166] In some embodiments, peptides comprising variants of the peptides described herein possess immune evasion activity. For example, in some embodiments, the peptide or variants of the peptide produce immune evasion activity when expressed by cells at appropriate levels. In some embodiments, the immune evasion activity includes inhibition of natural killer cell-mediated cytotoxicity by engineered cells. In some embodiments, the immune evasion activity includes reduced T cell-mediated killing by engineered cells. In some embodiments, peptides having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11 possess immune evasion activity.

[0167] In some embodiments, the pharmaceutical composition comprises engineered cell populations that contain polypeptides or combinations of polypeptides, or variants of polypeptides containing the biological activity of said polypeptides. For example, in some embodiments, engineered cell populations containing at least one heterologous nucleic acid sequence encoding any one of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47 contain biological activities associated with VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, respectively. In some embodiments, variants of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, or CD47 contain the biological activities of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, or CD47, respectively. In some embodiments, a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11 contains polypeptide-related biological activity, said polypeptide having the same sequence as any one of SEQ ID NO: 1-11. In some embodiments, a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11 contains polypeptide-related biological activity, said polypeptide having the same sequence as any one of SEQ ID NO: 2, 4, 5, and 11.

[0168] In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least one heterologous nucleic acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 65% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 75% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 85% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 90% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 95% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 98% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 99% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has the same sequence as any one of SEQ ID NO: 12-22.

[0169] In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least one heterologous nucleic acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 65% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 75% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 85% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 90% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 95% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 98% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 99% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has the same sequence as any one of SEQ ID NO: 13, 15, 17, and 22.

[0170] In some embodiments, the pharmaceutical composition comprises an engineered cell population containing at least one heterologous nucleic acid sequence integrated into the genome of the engineered cells. In some embodiments, at least one heterologous nucleic acid sequence is integrated into a persistent transgenic expression locus (STEL) in the genome of the engineered cells. In some embodiments, the STEL is a locus encoding a protein involved in one or more of the following: ribonucleoprotein complex formation, focal adhesion, cell-substrate adhesion junctions, cell-substrate junctions, cell anchoring, extracellular exosomes, extracellular vesicles, intracellular organelles, anchoring junctions, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. In some embodiments, the STEL is glyceraldehyde-3-phosphate dehydrogenase (… GAPDH In some embodiments, the STEL is a ribosomal protein locus, for example... RPL or RPS Locus. An example of the RPL gene is... RPL10, RPL13, RPS18, RPL3,RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPLP0, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4 and RPL22 . RPS An example of a gene is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20 and RPSII In some embodiments, the STEL is a locus encoding mitochondrial proteins (e.g., MT-Co1, MT-CO2, MT-ND4, MT-ND1, and MT-ND2). In some embodiments, the STEL is a locus encoding actin (e.g., ACTG1 and ACTB). In some embodiments, the STEL is a locus encoding eukaryotic translation elongation factors (e.g., EEF1A1 and EEF2) or eukaryotic translation initiation factors (e.g., EIEI). In some embodiments, the STEL is a locus encoding histones (e.g., H3F3A and H3F3B). In some embodiments, the STEL is selected from... FTL, FTH1, TPT1, IMSB10 GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2 and SRP14 The locus. Compositions and methods for integrating heterologous nucleic acids into STELs are described in International Patent Application Publications WO 2021 / 072329 and WO 2024 / 145653, which are incorporated herein by reference.

[0171] In some embodiments, the heterologous nucleic acid is integrated into the genome of the engineered cell in a sustained transcriptional activity payload region (STAPLR). In some embodiments, the STAPLR is selected from the following: RPL34 Genes and OSTC Intergenic spacer regions between genes; ACTB Genes and FSCN1 Intergenic spacer regions between genes; AKIRIN1 Genes and NDUFS5 Intergenic spacer regions between genes; PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes; PTGES3 Genes and NACA Intergenic spacer regions between genes; MLF2 Genes and PTMS Intergenic spacer regions between genes; RAB13 Genes and RPS27 Intergenic spacer regions between genes; JTB Genes and RAB13 Intergenic spacer regions between genes; AKR1A1 Genes and NASP Intergenic spacer regions between genes; NDUFS5 Genes and MACF1 Intergenic spacer regions between genes; SRSF9 Genes and DYNLL1 Intergenic spacer regions between genes; MYL6B Genes and MYL6 Intergenic spacer regions between genes; GPX1 Genes and RHOA Intergenic spacer regions between genes; HNRNPA2B1 Genes and CBX3 Intergenic spacer regions between genes; ROMO Genes and RBM39 Intergenic spacer regions between genes; and PA2G4 Genes and RPL41 A gene spacer region between genes. In some embodiments, the STAPLR is... PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes. In some embodiments, the heteronucleotide is integrated at a location at least 100-5000 base pairs from the nearest gene. Compositions and methods for integrating heteronucleotides into STAPLR are described in International Patent Application Publication No. WO 2024 / 145653, which is incorporated herein by reference.

[0172] In some embodiments, the pharmaceutical composition comprises an engineered cell population that further comprises a kill switch. In some embodiments, the kill switch is a gene encoding herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (also known as incasep 9 and iCasp9), CD20, or mutant human thymidine kinase (mTMPK). In some embodiments, the kill switch is under the control of an inducible promoter. In some embodiments, the kill switch is encoded by a heterologous nucleic acid. In some embodiments, at least one heterologous nucleic acid encoding the kill switch is integrated into a different locus than at least one heterologous nucleic acid encoding the polypeptide. In some embodiments, at least one heterologous nucleic acid encoding the kill switch is integrated into the same locus as at least one heterologous nucleic acid encoding the polypeptide.

[0173] In some embodiments, the pharmaceutical composition comprises an engineered cell population that further comprises genetic modifications resulting in a reduction in T-cell-mediated killing compared to wild-type cells. In some embodiments, the engineered cell population further comprises genetic modifications resulting in a reduction of T-cell-mediated killing by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to wild-type cells. In some embodiments, the engineered cell population further comprises genetic modifications resulting in a reduction of T-cell-mediated killing by at least 20% compared to wild-type cells. In some embodiments, the engineered cell population further comprises genetic modifications resulting in a reduction of T-cell-mediated killing by at least 50% compared to wild-type cells. In some embodiments, the engineered cell population further comprises genetic modifications resulting in a reduction of T-cell-mediated killing by at least 80% compared to wild-type cells. In some embodiments, the genetic modifications that reduce T-cell-mediated killing of the engineered cells compared to wild-type cells include… β 2. Knockout of the microglobulin (B2M) gene. In some embodiments, the genetic modifications that reduce T cell-mediated killing in the engineered cells compared to wild-type cells include the knockout of HLA-A, B, and C molecules.

[0174] In some embodiments, the pharmaceutical composition comprises an engineered cell population, the engineered cell population further comprising genetic modifications that result in a reduction in T cell-mediated killing compared to wild-type cells. In some embodiments, the pharmaceutical composition comprises an engineered cell population, the engineered cell population further comprising genetic modifications that result in a reduction in T cell-mediated killing by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to wild-type cells. In some embodiments, the pharmaceutical composition comprises an engineered cell population, the engineered cell population further comprising genetic modifications that result in a reduction in T cell-mediated killing by at least 20% compared to wild-type cells. In some embodiments, the pharmaceutical composition comprises an engineered cell population, the engineered cell population further comprising genetic modifications that result in a reduction in T cell-mediated killing by at least 50% compared to wild-type cells. In some embodiments, the pharmaceutical composition comprises an engineered cell population, which further comprises genetic modifications that result in at least an 80% reduction in T-cell-mediated killing compared to wild-type cells.

[0175] In some implementations, the genetic modifications that result in reduced T cell-mediated killing in engineered cells compared to wild-type cells include... β 2 microglobulin ( B2MThe deletion, disruption, or attenuation of genes. In some implementations, genetic modifications that reduce T cell-mediated killing in engineered cells compared to wild-type cells include... HLA-A Gene, HLA-B Genes and HLA-C One or more genes are missing, damaged, or weakened.

[0176] In some embodiments, the pharmaceutical composition comprises an engineered cell population, the engineered cell population further comprising... CIITA Gene or RFX5 Deletion, disruption, or attenuation in genes. Therefore, in some embodiments, the pharmaceutical composition comprises an engineered cell population containing genetic modifications, as described herein, that result in reduced T-cell-mediated killing of the engineered cells compared to wild-type cells (e.g., B2M , HLA-A , HLA-B and / or HLA-C (loss, damage or weakening) and CIITA The deletion, disruption, or attenuation of genes. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing genetic modifications, as described herein, that result in reduced T-cell-mediated killing of the engineered cells compared to wild-type cells (e.g., B2M , HLA-A , HLA-B and / or HLA-C (loss, damage or weakening) and RFX5 The deletion, disruption, or attenuation of genes. In some embodiments, the pharmaceutical composition comprises an engineered cell population containing genetic modifications, as described herein, that result in reduced T-cell-mediated killing of the engineered cells compared to wild-type cells (e.g., B2M , HLA-A , HLA-B and / or HLA-C (loss, damage or weakening) and CIITA Genes and RFX5 The loss, damage, or weakening of genes.

[0177] In some embodiments, the pharmaceutical composition comprises an engineered cell population, which further comprises at least one selectable marker gene.

[0178] In some embodiments, the pharmaceutical composition comprises an engineered cell population that is less susceptible to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population exhibits at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, or at least a 90% reduction in susceptibility to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population exhibits at least a 10% reduction in susceptibility to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population exhibits at least a 20% reduction in susceptibility to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population is at least about 30% less susceptible to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population is at least about 50% less susceptible to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence.

[0179] In some embodiments, the pharmaceutical composition comprises an engineered cell population, which includes engineered stem cells. In some embodiments, the engineered stem cells comprise pluripotent stem cells. In some embodiments, the pluripotent stem cells comprise embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the engineered stem cells comprise pluripotent stem cells.

[0180] In some embodiments, the pharmaceutical composition comprises an engineered cell population including cardiac cells, nerve cells, T cells, retinal cells, endocrine cells, epithelial cells, muscle cells, or myeloid cells. In some embodiments, the cardiac cells are cardiomyocytes, cardiac fibroblasts, cardiac smooth muscle cells, epicardial cells, cardiac endothelial cells, Purkinje fibers, or pacemaker cells. In some embodiments, the nerve cells are neurons or glial cells. In some embodiments, the T cells are helper T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), memory T cells, regulatory T cells (T reg cells), innate-like T cells (e.g., natural killer T cells), mucosa-associated invariant T cells, or γδ T cells, or modified T cells (e.g., CAR-T cells). In some embodiments, the retinal cells are photoreceptors, retinal horizontal cells, retinal bipolar cells, retinal amacrine cells, or retinal ganglion cells. In some embodiments, the endocrine cells are hypothalamic endocrine cells, pituitary endocrine cells, pineal endocrine cells, thyroid endocrine cells (e.g., follicular cells), parathyroid endocrine cells, thymic endocrine cells, adrenal endocrine cells, pancreatic endocrine cells (e.g., α cells, β cells, δ cells, or F cells), ovarian endocrine cells (e.g., granulosa cells), or testicular endocrine cells (e.g., testicular interstitial cells). In some embodiments, the epithelial cells are squamous epithelial cells, cuboidal epithelial cells, columnar epithelial cells, pseudostratified epithelial cells, or stratified epithelial cells. In some embodiments, the muscle cells are skeletal muscle cells, cardiomyocytes (e.g., cardiomyocytes), or smooth muscle cells. In some embodiments, the myeloid cells are monocytes, microglia, macrophages, dendritic cells, basophils, eosinophils, erythrocytes, mast cells, neutrophils, T cells, or any of their precursor progenitor cells.

[0181] In some embodiments, the pharmaceutical composition comprises a population of human engineered cells.

[0182] In some embodiments, the pharmaceutical composition is formulated for administration to a subject. The pharmaceutical composition is formulated for any suitable method of administration, such as administration via local injection into a tissue.

[0183] The pharmaceutically acceptable excipient, carrier, or diluent may be any excipient, carrier, or diluent known in the art. For example, the pharmaceutically acceptable excipient, carrier, or diluent may be cell culture medium (e.g., a medium optionally lacking any animal-derived components), sterile water, physiological saline, general buffers (e.g., phosphoric acid, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants, surfactants, suspensions, isotonic agents, and / or preservatives, which may be included in the pharmaceutical composition described herein. The specific excipient, carrier, or diluent will depend on the intended route of administration for the pharmaceutical composition. In some embodiments, the pharmaceutically acceptable excipient, carrier, or diluent is an excipient, carrier, or diluent suitable for treating a tumor in a subject. In some embodiments, the pharmaceutically acceptable excipient, carrier, or diluent is an excipient, carrier, or diluent suitable for administering the pharmaceutical composition by injection into a subject.

[0184] The pharmaceutical compositions described herein may be used to treat a disease or condition in a subject. In some embodiments, the pharmaceutical composition is used to treat Parkinson's disease in a subject. In some embodiments, the Parkinson's disease is early-stage Parkinson's disease. In some embodiments, the Parkinson's disease is late-stage Parkinson's disease. In some embodiments, the pharmaceutical composition is used to treat heart failure. In some embodiments, the pharmaceutical composition is used to treat neuroinflammation.

[0185] In some embodiments, the pharmaceutical composition comprises an allogeneic engineered cell population of the subject receiving the pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises an autologous engineered cell population of the subject receiving the pharmaceutical composition.

[0186] IV. Methods Certain aspects of this disclosure provide methods for treating a disease or condition in a subject or for achieving cellular immune escape.

[0187] Methods for treating the subject's disease or condition In some implementations, methods of treating a subject’s disease or condition include administering an engineered cell population (e.g., the engineered cell population described herein) or a pharmaceutical composition comprising said engineered cell population.

[0188] In some embodiments, a method of treating a subject's disease or condition includes administering a cell population containing at least one heterologous nucleic acid sequence encoding a polypeptide. In some embodiments, the polypeptide is expressed at a level sufficient to inhibit natural killer cell-mediated cytotoxicity.

[0189] In some embodiments, the method includes administering a cell population containing at least one heterologous nucleic acid sequence encoding a polypeptide, said polypeptide including one or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47 or variants thereof.

[0190] In some embodiments, the polypeptide comprises one or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of the polypeptide comprises two or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of the polypeptide comprises two or more of CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of the polypeptide comprises three or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of the polypeptide comprises three or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of the polypeptide comprises CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of peptides comprises four or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of peptides comprises five or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises VISTA, CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of peptides comprises CLEC2D or a variant thereof and TRAIL or a variant thereof. In some embodiments, the combination of peptides comprises CLEC2D or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the peptide comprises VISTA or a variant thereof. In some embodiments, the peptide comprises human CLEC2D or a variant thereof. In some embodiments, the polypeptide comprises human TRAIL or a variant thereof. In some embodiments, the polypeptide comprises human SERPINB9 or a variant thereof. In some embodiments, the polypeptide comprises human HLA-C or a variant thereof. In some embodiments, the polypeptide comprises human CD47 or a variant thereof.

[0191] In some embodiments, the method includes administering a cell population comprising at least one heterologous nucleic acid sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 99% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NO: 1-11.

[0192] In some embodiments, the method includes administering a cell population comprising at least one heterologous nucleic acid sequence encoding a polypeptide, the polypeptide comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NO: 2, 4, 5, and 11.

[0193] In some embodiments, the method includes administering a cell population containing at least one heterologous nucleic acid sequence encoding a variant of the polypeptide described herein, the variant of which contains immune evasion activity. For example, in some embodiments, the polypeptide or a variant of the polypeptide produces immune evasion activity when expressed by cells at appropriate levels. In some embodiments, the immune evasion activity includes inhibition of natural killer cell-mediated cytotoxicity by engineered cells. In some embodiments, the immune evasion activity includes reduced T cell-mediated killing by engineered cells. In some embodiments, the polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11 contains immune evasion activity. In some embodiments, the polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11 contains immune evasion activity.

[0194] In some embodiments, the method includes administering a cell population containing at least one heterologous nucleic acid sequence encoding a polypeptide or a variant of said polypeptide, said variant containing the biological activity of said polypeptide. For example, in some embodiments, VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47 each contain biological activities associated with VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, respectively. In some embodiments, variants of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, or CD47 each contain the biological activity of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, or CD47, respectively. In some embodiments, a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11 contains polypeptide-related biological activity, said polypeptide having the same sequence as any one of SEQ ID NO: 1-11. In some embodiments, a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11 contains polypeptide-related biological activity, said polypeptide having the same sequence as any one of SEQ ID NO: 2, 4, 5, and 11.

[0195] In some embodiments, the method includes applying an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the method includes applying an engineered cell population containing at least three distinct heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the method includes applying an engineered cell population containing at least four distinct heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the method includes using an engineered cell population containing at least five distinct heterologous nucleic acid sequences, each encoding a different polypeptide. In some embodiments, the method includes applying an engineered cell population containing at least six distinct heterologous nucleic acid sequences, each encoding a different polypeptide.

[0196] In some embodiments, the method includes applying an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding CLEC2D or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the method includes applying an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding CLEC2D or a variant thereof and TRAIL or a variant thereof. In some embodiments, the method includes applying an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding CLEC2D or a variant thereof and HLA-C or a variant thereof. In some embodiments, the method includes applying an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding TRAIL or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the method includes applying an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding TRAIL or a variant thereof and HLA-C or a variant thereof. In some embodiments, the method includes applying an engineered cell population containing at least two distinct heterologous nucleic acid sequences, each encoding SERPINB9 or a variant thereof and HLA-C or a variant thereof.

[0197] In some embodiments, the method includes applying an engineered cell population comprising at least one heterologous nucleic acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 65% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 75% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 85% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 90% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 95% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 98% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 99% identity with any one of SEQ ID NO: 12-22. In some embodiments, the at least one heterologous nucleic acid sequence has the same sequence as any one of SEQ ID NO: 12-22.

[0198] In some embodiments, the method includes applying an engineered cell population comprising at least one heterologous nucleic acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 65% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 75% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 85% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 90% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 95% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 98% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 99% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has the same sequence as any one of SEQ ID NO: 13, 15, 17, and 22.

[0199] In some embodiments, the method includes administering an engineered cell population comprising at least one heterologous nucleic acid sequence integrated into the genome of the engineered cells. In some embodiments, at least one heterologous nucleic acid sequence is integrated into a persistent transgenic expression locus (STEL) in the genome of the engineered cells. In some embodiments, the STEL is a locus encoding a protein involved in one or more of the following: ribonucleoprotein complex formation, focal adhesion, cell-substrate adhesion junctions, cell-substrate junctions, cell anchoring, extracellular exosomes, extracellular vesicles, intracellular organelles, anchoring junctions, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. In some embodiments, the STEL is glyceraldehyde-3-phosphate dehydrogenase (… GAPDH In some embodiments, the STEL is a ribosomal protein locus, for example... RPL or RPS Gene locus. RPLAn example of a gene is RPL10, RPL13 RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPLP0, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4 and RPL22 . RPS An example of a gene is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20 and RPSII In some embodiments, the STEL is a locus encoding mitochondrial proteins (e.g., MT-Co1, MT-CO2, MT-ND4, MT-ND1, and MT-ND2). In some embodiments, the STEL is a locus encoding actin (e.g., ACTG1 and ACTB). In some embodiments, the STEL is a locus encoding eukaryotic translation elongation factors (e.g., EEF1A1 and EEF2) or eukaryotic translation initiation factors (e.g., EIEI). In some embodiments, the STEL is a locus encoding histones (e.g., H3F3A and H3F3B). In some embodiments, the STEL is selected from... FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2 and SRP14 The locus. Compositions and methods for integrating heterologous nucleic acids into STELs are described in International Patent Application Publications WO 2021 / 072329 and WO 2024 / 145653, which are incorporated herein by reference.

[0200] In some embodiments, the heterologous nucleic acid is integrated into the genome of the engineered cell in a sustained transcriptional activity payload region (STAPLR). In some embodiments, the STAPLR is selected from the following: RPL34 Genes and OSTC Intergenic spacer regions between genes; ACTB Genes and FSCN1 Intergenic spacer regions between genes; AKIRIN1 Genes and NDUFS5 Intergenic spacer regions between genes; PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes; PTGES3 Genes and NACA Intergenic spacer regions between genes; MLF2 Genes and PTMS Intergenic spacer regions between genes; RAB13 Genes and RPS27 Intergenic spacer regions between genes; JTB Genes and RAB13 Intergenic spacer regions between genes; AKR1A1 Genes and NASP Intergenic spacer regions between genes; NDUFS5 Genes and MACF1Intergenic spacer regions between genes; SRSF9 Genes and DYNLL1 Intergenic spacer regions between genes; MYL6B Genes and MYL6 Intergenic spacer regions between genes; GPX1 Genes and RHOA Intergenic spacer regions between genes; HNRNPA2B1 Genes and CBX3 Intergenic spacer regions between genes; ROMO Genes and RBM39 Intergenic spacer regions between genes; and PA2G4 Genes and RPL41 A gene spacer region between genes. In some embodiments, the STAPLR is... PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes. In some embodiments, the heteronucleotide is integrated at a location at least 100-5000 base pairs from the nearest gene. Compositions and methods for integrating heteronucleotides into STAPLR are described in International Patent Application Publication No. WO 2024 / 145653, which is incorporated herein by reference.

[0201] In some embodiments, the method includes applying an engineered cell population, the engineered cell population further comprising a kill switch. In some embodiments, the kill switch is a gene encoding herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (also known as incasep 9 and iCasp9), CD20, or mutant human thymidine kinase (mTMPK). In some embodiments, the kill switch is under the control of an inducible promoter. In some embodiments, the kill switch is encoded by a heterologous nucleic acid. In some embodiments, at least one heterologous nucleic acid encoding the kill switch is integrated into a different locus than at least one heterologous nucleic acid encoding the polypeptide. In some embodiments, at least one heterologous nucleic acid encoding the kill switch is integrated into the same locus as at least one heterologous nucleic acid encoding the polypeptide.

[0202] In some embodiments, the method includes applying an engineered cell population, the engineered cell population further comprising genetic modifications that result in a reduction in T cell-mediated killing compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that result in a reduction of T cell-mediated killing by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that result in a reduction of T cell-mediated killing by at least 20% compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that result in a reduction of T cell-mediated killing by at least 50% compared to wild-type cells. In some embodiments, the engineered cells further comprise genetic modifications that result in a reduction of T cell-mediated killing by at least 80% compared to wild-type cells. In some embodiments, the genetic modifications that result in a reduction in T cell-mediated killing of the engineered cells compared to wild-type cells include… β 2 microglobulin ( B2M The deletion, disruption, or attenuation of genes. In some implementations, genetic modifications that reduce T cell-mediated killing in engineered cells compared to wild-type cells include... HLA - A Gene, HLA - B Genes and HLA - C One or more genes are missing, damaged, or weakened.

[0203] In some embodiments, the method includes applying an engineered cell population, the engineered cell population further comprising... CIITA Gene or RFX5 Deletion, disruption, or attenuation in genes. Therefore, in some embodiments, the method includes administering an engineered cell population containing genetic modifications, as described herein, that result in reduced T-cell-mediated killing of the engineered cells compared to wild-type cells (e.g., [missing information]). B2M , HLA - A , HLA - B and / or HLA - C (loss, damage or weakening) and CIITA The deletion, disruption, or attenuation of genes. In some embodiments, the method includes administering an engineered cell population containing genetic modifications, as described herein, that result in reduced T cell-mediated killing of the engineered cells compared to wild-type cells (e.g., B2M , HLA - A , HLA - B and / or HLA - C (loss, damage or weakening) and RFX5The deletion, disruption, or attenuation of genes. In some embodiments, the method includes administering an engineered cell population containing genetic modifications, as described herein, that result in reduced T cell-mediated killing of the engineered cells compared to wild-type cells (e.g., B2M , HLA - A , HLA - B and / or HLA - C (loss, damage or weakening) and CIITA Genes and RFX5 The loss, damage, or weakening of genes.

[0204] In some embodiments, the method includes applying an engineered cell population, the engineered cell population further comprising at least one selectable marker gene.

[0205] In some embodiments, the method includes administering an engineered cell population that is less susceptible to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population exhibits at least a 10%, at least a 20%, at least a 30%, at least a 40%, at least a 50%, at least a 60%, at least a 70%, at least a 80%, or at least a 90% reduction in susceptibility to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population exhibits at least a 10% reduction in susceptibility to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population exhibits at least a 20% reduction in susceptibility to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population exhibits at least a 30% reduction in susceptibility to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the engineered cell population exhibits at least a 50% reduction in susceptibility to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence. In some embodiments, the method includes administering an engineered cell population comprising engineered stem cells. In some embodiments, the engineered stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some embodiments, the engineered stem cells are pluripotent stem cells.

[0206] In some embodiments, the method includes administering an engineered cell population that is positive for cardiac troponin T (cTNT). In some embodiments, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 70% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 80% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 90% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 95% of the engineered cells in the engineered cell population are positive for cTNT. In some embodiments, at least 98% of the engineered cells in the engineered cell population are positive for cTNT. In some implementations, at least 99% of the engineered cells in the engineered cell population are positive for cTnT.

[0207] In some embodiments, the method includes applying an engineered cell population comprising cardiac cells, nerve cells, T cells, retinal cells, endocrine cells, epithelial cells, muscle cells, or myeloid cells. In some embodiments, the cardiac cells are cardiomyocytes, cardiac fibroblasts, cardiac smooth muscle cells, epicardial cells, cardiac endothelial cells, Purkinje fibers, or pacemaker cells. In some embodiments, the nerve cells are neurons or glial cells. In some embodiments, the T cells are helper T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), memory T cells, regulatory T cells (T reg cells), innate-like T cells (e.g., natural killer T cells), mucosa-associated invariant T cells, or γδ T cells, or modified T cells (e.g., CAR-T cells). In some embodiments, the retinal cells are photoreceptors, retinal horizontal cells, retinal bipolar cells, retinal amacrine cells, or retinal ganglion cells. In some embodiments, the endocrine cells are hypothalamic endocrine cells, pituitary endocrine cells, pineal endocrine cells, thyroid endocrine cells (e.g., follicular cells), parathyroid endocrine cells, thymic endocrine cells, adrenal endocrine cells, pancreatic endocrine cells (e.g., α cells, β cells, δ cells, or F cells), ovarian endocrine cells (e.g., granulosa cells), or testicular endocrine cells (e.g., testicular interstitial cells). In some embodiments, the epithelial cells are squamous epithelial cells, cuboidal epithelial cells, columnar epithelial cells, pseudostratified epithelial cells, or stratified epithelial cells. In some embodiments, the muscle cells are skeletal muscle cells, cardiomyocytes (e.g., cardiomyocytes), or smooth muscle cells. In some embodiments, the myeloid cells are monocytes, microglia, macrophages, dendritic cells, basophils, eosinophils, erythrocytes, mast cells, neutrophils, or any of their precursor progenitor cells. In some embodiments, the cardiac cells, nerve cells, T cells, retinal cells, endocrine cells, epithelial cells, muscle cells, or myeloid cells are derived from stem cells.

[0208] In some embodiments, the method includes administering a human-engineered cell population to a subject. In some embodiments, the subject is a human being. In some embodiments, the cell population is allogeneic to the subject. In some embodiments, the cell population is autologous to the subject.

[0209] In some embodiments, the disease or condition treated by the methods described herein is Parkinson's disease, multiple sclerosis, irritable bowel syndrome, type 1 diabetes, rheumatoid arthritis, heart failure, liver disease, cancer, inflammation, or neuroinflammation. In some embodiments, the method is used to treat a subject with Parkinson's disease. In some embodiments, the Parkinson's disease is early-stage Parkinson's disease. In some embodiments, the Parkinson's disease is late-stage Parkinson's disease. In some embodiments, the method is used to treat a subject with heart failure. In some embodiments, the method is used to treat a subject with neuroinflammation.

[0210] In some embodiments, the application of the engineered cell population results in the improvement of at least one symptom associated with the disease or condition, as determined by responsiveness / non-responsiveness or other indicators known in the art. The choice of techniques and methods, as well as the timing and frequency of examinations, can be determined and / or adjusted by those skilled in the art based on the specific condition of the subject.

[0211] In some embodiments, the method further includes examining the responsiveness of a subject to the engineered cell population. The subject's responsiveness can be determined as an improvement in at least one parameter of disease progression. The subject may have a partial or complete response to treatment. Response to treatment can be determined based on methods known in the art. Those skilled in the art can determine appropriate methods based on the type of disease being evaluated.

[0212] In some embodiments, the method further includes administering a killer switch activator after the application of the engineered cell population. The killer switch activator can be used to remove the engineered cell population after a period of time. The killer switch activator can be administered at any time after the application of the engineered cell population, for example, after the subject has achieved a level of responsiveness as measured by improvement in at least one symptom associated with the disease or condition. In some embodiments, the method includes administering a killer switch activator selected from genes encoding herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (also known as incasep 9 and iCasp9), CD20, or mutant human thymidine kinase (mTMPK). 。

[0213] The engineered cell populations described herein can be administered to a subject via any suitable route of administration. Methods for administering the engineered cell populations described herein are known to those skilled in the art. In some embodiments, the methods include oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, or local administration of the engineered cell populations. In some embodiments, the engineered cell populations are administered by direct injection into tissues. For example, in some embodiments, the engineered cell populations are administered by direct injection into the heart of a subject, such as intracoronary, intramyocardial, or transendocardial administration. Administration of the engineered cell populations to a subject can be performed by any suitable means, such as by injection, via catheter, via an implantable device, or via any other suitable delivery device. For example, the engineered cell populations can be introduced into the heart using a catheter inserted via the femoral vein, subclavian vein, jugular vein, or axillary vein, or via transendocardial transplantation into the ventricular or atrial region. The engineered cell populations can also be transplanted into the ventricular or atrial region via an epicardial route using a needle inserted through the chest.

[0214] The engineered cell population can be administered to the subject in a single dose or through a series of administration procedures, and can be administered to the patient at any time from the onset of diagnosis of the disease or condition. For example, the engineered cell population can be administered as one or more doses during treatment. The doses can be administered using the same or different routes of administration and can contain the same or different amounts of the engineered cell population.

[0215] The duration of application may depend on the route of application of the engineered cell population.

[0216] In some embodiments, the method further includes administering additional therapy to the subject. The additional therapy will depend on the disease or condition being treated by the method described herein.

[0217] Methods for achieving immune escape In some aspects of this disclosure, this paper provides methods for achieving cellular immune escape.

[0218] In some embodiments, the method for achieving immune evasion includes genetically modifying cells to increase the expression of at least one polypeptide. In some embodiments, the polypeptide is expressed at a level sufficient to inhibit natural killer cell-mediated cytotoxicity. In some embodiments, the genetically modified cells exhibit increased survival when attacked with natural killer cells compared to wild-type cells.

[0219] In some embodiments, the method includes genetically modifying cells to increase the expression of one or more peptides, including VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47 or variants thereof.

[0220] In some embodiments, the polypeptide comprises one or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of the polypeptide comprises two or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of the polypeptide comprises two or more of CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of the polypeptide comprises three or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of the polypeptide comprises three or more of CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of the polypeptide comprises CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of peptides comprises four or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises CLEC2D, TRAIL, SERPINB9, and HLA-C, or variants thereof. In some embodiments, the combination of peptides comprises five or more of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, or variants thereof. In some embodiments, the combination of peptides comprises VISTA, CLEC2D, TRAIL, and SERPINB9, or variants thereof. In some embodiments, the combination of peptides comprises CLEC2D or a variant thereof and TRAIL or a variant thereof. In some embodiments, the combination of peptides comprises CLEC2D or a variant thereof and SERPINB9 or a variant thereof. In some embodiments, the peptide comprises VISTA or a variant thereof. In some embodiments, the peptide comprises human CLEC2D or a variant thereof. In some embodiments, the polypeptide comprises human TRAIL or a variant thereof. In some embodiments, the polypeptide comprises human SERPINB9 or a variant thereof. In some embodiments, the polypeptide comprises human HLA-C or a variant thereof. In some embodiments, the polypeptide comprises human CD47 or a variant thereof.

[0221] In some embodiments, the method includes genetically modifying cells to increase the expression of at least one polypeptide, the polypeptide comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 99% identity with any one of SEQ ID NO: 1-11. In some embodiments, the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NO: 1-11.

[0222] In some embodiments, the method includes genetically modifying cells to increase the expression of at least one polypeptide, the polypeptide comprising an amino acid sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 65% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 70% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 75% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 80% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 85% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 90% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 95% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence having at least 98% identity with any one of SEQ ID NO: 2, 4, 5, and 11. In some embodiments, the polypeptide comprises an amino acid sequence identical to any one of SEQ ID NO: 2, 4, 5, and 11.

[0223] In some embodiments, peptides comprising variants of the peptides described herein possess immune evasion activity. For example, in some embodiments, the peptide or variants of the peptide produce immune evasion activity when expressed by cells at appropriate levels. In some embodiments, the immune evasion activity comprises inhibition of natural killer cell-mediated cytotoxicity by engineered cells. In some embodiments, the immune evasion activity comprises reduced T cell-mediated killing by engineered cells. In some embodiments, peptides having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 1-11 possess immune evasion activity. In some embodiments, peptides having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NOs: 2, 4, 5, and 11 possess immune evasion activity.

[0224] In some embodiments, the polypeptide or variants of the polypeptide contain the biological activity of the polypeptide. For example, in some embodiments, VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47 each contain biological activities associated with VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, and CD47, respectively. In some embodiments, variants of VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, or CD47 each contain biological activities associated with VISTA, CLEC2D, TRAIL, SERPINB9, HLA-C, or CD47, respectively. In some embodiments, polypeptides having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11 contain polypeptide-related biological activities, and the polypeptides each have the same sequence as any one of SEQ ID NO: 1-11. In some embodiments, a polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 2, 4, 5, and 11 contains polypeptide-related biological activity, said polypeptide having the same sequence as any one of SEQ ID NO: 2, 4, 5, and 11, respectively.

[0225] In some embodiments, the genetically modified cell comprises integrating at least one heterologous nucleic acid sequence encoding a polypeptide into the cell's genome. In some embodiments, the genetically modified cell comprises integrating at least one heterologous nucleic acid sequence encoding a polypeptide into the cell's genome, said polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 1-11. In some embodiments, the at least one heterologous nucleic acid sequence has at least 65% identity with any one of SEQ ID NO: 1-11. In some embodiments, the at least one heterologous nucleic acid sequence has at least 75% identity with any one of SEQ ID NO: 1-11. In some embodiments, the at least one heterologous nucleic acid sequence has at least 85% identity with any one of SEQ ID NO: 1-11. In some embodiments, the at least one heterologous nucleic acid sequence has at least 90% identity with any one of SEQ ID NO: 1-11. In some embodiments, the at least one heterologous nucleic acid sequence has at least 95% identity with any one of SEQ ID NO: 1-11. In some embodiments, the at least one heterologous nucleic acid sequence has at least 98% identity with any one of SEQ ID NO: 1-11. In some embodiments, the at least one heterologous nucleic acid sequence has at least 99% identity with any one of SEQ ID NO: 1-11. In some embodiments, the at least one heterologous nucleic acid sequence has the same sequence as any one of SEQ ID NO: 1-11.

[0226] In some embodiments, the genetically modified cell comprises integrating at least one heterologous nucleic acid sequence encoding a polypeptide into the cell's genome. In some embodiments, the genetically modified cell comprises integrating at least one heterologous nucleic acid sequence encoding a polypeptide into the cell's genome, said polypeptide having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, said at least one heterologous nucleic acid sequence has at least 65% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, said at least one heterologous nucleic acid sequence has at least 75% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, said at least one heterologous nucleic acid sequence has at least 85% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 90% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 95% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 98% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has at least 99% identity with any one of SEQ ID NO: 13, 15, 17, and 22. In some embodiments, the at least one heterologous nucleic acid sequence has the same sequence as any one of SEQ ID NO: 13, 15, 17, and 22.

[0227] In some embodiments, at least one heterologous nucleic acid sequence encoding a polypeptide is integrated into the genome of the cell. The CRISPR / Cas system has been used to introduce genetic modifications and gene regulation in various species. Without being theoretically limited, target nucleic acids can be modified by the interaction of the CRISPR / Cas system with sequences present in the target nucleic acid, for example, to induce cleavage of the target nucleic acid (e.g., hydrolysis of one or more phosphodiester bonds) and introduce genetic modifications. In some embodiments, at least one heterologous nucleic acid sequence encoding a polypeptide is integrated into the genome of the cell using a CRISPR / Cas9 or CRISPR / Cas12 system.

[0228] In some embodiments, methods for achieving immune escape include integrating at least one heterologous nucleic acid sequence encoding the polypeptide into an STEL in the cell genome. In some embodiments, the STEL is a gene locus encoding a protein involved in one or more of the following: ribonucleoprotein complex formation, focal adhesion, cell-substrate adhesion junctions, cell-substrate linkages, cell anchoring, extracellular exosomes, extracellular vesicles, intracellular organelles, anchoring junctions, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. In some embodiments, the STEL is glyceraldehyde-3-phosphate dehydrogenase (… GAPDH In some embodiments, the STEL is a ribosomal protein locus, for example... RPL or RPS Locus. An example of the RPL gene is... RPL10, RPL13, RPS18, RPL3, RPLP1, RPL13A, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPLP0, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4 and RPL22 . RPS An example of a gene is RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20 and RPSII In some embodiments, the STEL is a locus encoding mitochondrial proteins (e.g., MT-Co1, MT-CO2, MT-ND4, MT-ND1, and MT-ND2). In some embodiments, the STEL is a locus encoding actin (e.g., ACTG1 and ACTB). In some embodiments, the STEL is a locus encoding eukaryotic translation elongation factors (e.g., EEF1A1 and EEF2) or eukaryotic translation initiation factors (e.g., EIEI). In some embodiments, the STEL is a locus encoding histones (e.g., H3F3A and H3F3B). In some embodiments, the STEL is selected from... FTL, FTH1, TPT1, IMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2 and SRP14 The locus. Compositions and methods for integrating heterologous nucleic acids into STELs are described in International Patent Application Publications WO 2021 / 072329 and WO 2024 / 145653, which are incorporated herein by reference.

[0229] In some embodiments, the heterologous nucleic acid is integrated into the genome of the engineered cell in a sustained transcriptional activity payload region (STAPLR). In some embodiments, the STAPLR is selected from the following: RPL34 Genes and OSTC Intergenic spacer regions between genes; ACTB Genes and FSCN1 Intergenic spacer regions between genes; AKIRIN1 Genes and NDUFS5Intergenic spacer regions between genes; PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes; PTGES3 Genes and NACA Intergenic spacer regions between genes; MLF2 Genes and PTMS Intergenic spacer regions between genes; RAB13 Genes and RPS27 Intergenic spacer regions between genes; JTB Genes and RAB13 Intergenic spacer regions between genes; AKR1A1 Genes and NASP Intergenic spacer regions between genes; NDUFS5 Genes and MACF1 Intergenic spacer regions between genes; SRSF9 Genes and DYNLL1 Intergenic spacer regions between genes; MYL6B Genes and MYL6 Intergenic spacer regions between genes; GPX1 Genes and RHOA Intergenic spacer regions between genes; HNRNPA2B1 Genes and CBX3 Intergenic spacer regions between genes; ROMO Genes and RBM39 Intergenic spacer regions between genes; and PA2G4 Genes and RPL41 A gene spacer region between genes. In some embodiments, the STAPLR is... PRDX1 Genes and AKR1A1 Intergenic spacer regions between genes. In some embodiments, the heteronucleotide is integrated at a location at least 100-5000 base pairs from the nearest gene. Compositions and methods for integrating heteronucleotides into STAPLR are described in International Patent Application Publication No. WO 2024 / 145653, which is incorporated herein by reference.

[0230] In some embodiments, at least one heterologous nucleic acid sequence encoding the polypeptide further comprises one or more regulatory elements. In some embodiments, the regulatory element is a promoter. In some embodiments, the expression of the polypeptide is driven by an endogenous gene promoter.

[0231] In some embodiments, at least one heterologous nucleic acid sequence encoding the polypeptide is introduced into a cell via a vector. The vector may be a plasmid, a virus, or other nucleic acid designed to introduce the target nucleic acid into the cell. The vector is used to introduce a target gene into a host cell, wherein the vector interacts with a polymerase in the cell to express the protein encoded in the vector. The vector may exist outside the chromosome within the cell or be integrated into the genome of the host cell.

[0232] In some embodiments, the method for achieving immune evasion further includes introducing at least one heterologous nucleic acid encoding a killer switch into the cell. In some embodiments, the killer switch is a gene encoding herpes simplex virus thymidine kinase (HSV-TK), inducible caspase 9 (also known as incasep 9 and iCasp9), CD20, or mutant human thymidine kinase (mTMPK). In some embodiments, the killer switch is under the control of an inducible promoter. In some embodiments, at least one heterologous nucleic acid encoding the killer switch is integrated into the genome of the cell using a CRISPR / Cas system. The at least one heterologous nucleic acid encoding the killer switch may be integrated into a locus that is the same as or different from the sequence of at least one heterologous nucleic acid encoding the polypeptide.

[0233] In some embodiments, at least one heterologous nucleic acid encoding the kill switch is introduced into cells in a vector. The at least one heterologous nucleic acid encoding the kill switch may be introduced into cells in a vector with the same or different sequences as at least one heterologous nucleic acid encoding the polypeptide.

[0234] In some embodiments, the method for achieving immune evasion further includes introducing a genetic modification into cells that results in reduced T-cell-mediated killing of the genetically modified cells compared to wild-type cells. In some embodiments, the genetic modification results in a reduction of at least 20% in T-cell-mediated killing of the genetically modified cells compared to wild-type cells. In some embodiments, the genetic modification results in a reduction of at least 50% in T-cell-mediated killing of the genetically modified cells compared to wild-type cells. In some embodiments, the genetic modification results in a reduction of at least 80% in T-cell-mediated killing of the genetically modified cells compared to wild-type cells.

[0235] In some implementations, the genetic modifications that result in reduced T cell-mediated killing in engineered cells compared to wild-type cells include... β 2 microglobulin ( B2M The deletion, disruption, or attenuation of genes. In some implementations, genetic modifications that reduce T cell-mediated killing in engineered cells compared to wild-type cells include... HLA-A Gene, HLA-B Genes and HLA-C One or more genes are missing, damaged, or weakened.

[0236] In some implementations, the method for achieving immune escape further includes CIITA Genes and RFX5Deletion, disruption, or attenuation is introduced into the gene. Therefore, in some embodiments, methods for achieving immune evasion include the introduction of genetic modifications, such as those described herein, that result in reduced T-cell-mediated killing in engineered cells compared to wild-type cells (e.g., B2M , HLA-A , HLA-B and / or HLA-C (loss, damage or weakening) and CIITA The deletion, disruption, or attenuation of genes. In some implementations, methods for achieving immune evasion include the introduction of genetic modifications, such as those described herein, that result in reduced T-cell-mediated killing in engineered cells compared to wild-type cells (e.g., B2M , HLA-A , HLA-B and / or HLA-C (loss, damage or weakening) and RFX5 The deletion, disruption, or attenuation of genes. In some implementations, methods for achieving immune evasion include the introduction of genetic modifications, such as those described herein, that result in reduced T-cell-mediated killing in engineered cells compared to wild-type cells (e.g., B2M , HLA-A , HLA-B and / or HLA-C (loss, damage or weakening) and CIITA Genes and RFX5 The loss, damage, or weakening of genes.

[0237] In some embodiments, methods for achieving immune escape include genetically modifying stem cells. In some embodiments, the stem cells are pluripotent stem cells. In some embodiments, the pluripotent stem cells are embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). In some embodiments, methods for achieving immune escape include genetically modifying stem cells and differentiating the stem cells into engineered cells. In some embodiments, differentiating the stem cells into engineered cells includes contacting the stem cells with one or more differentiation factors. The specific combination of differentiation factors used will depend on the desired cell type. In some embodiments, differentiating stem cells includes contacting the stem cells with one or more differentiation factors that drive orientation and / or differentiation into cardiac cells, nerve cells, T cells, retinal cells, endocrine cells, epithelial cells, muscle cells, or myeloid cells.

[0238] In some embodiments, methods for achieving immune evasion include genetically modified cardiac cells, nerve cells, T cells, retinal cells, endocrine cells, epithelial cells, muscle cells, or myeloid cells. In some embodiments, the cardiac cells are cells of the epicardium, myocardium, or endocardium of the heart. In some embodiments, the cardiac cells are cardiomyocytes, cardiac fibroblasts, cardiac smooth muscle cells, epicardial cells, cardiac endothelial cells, Purkinje fibers, or pacemaker cells. In some embodiments, the T cells are helper T cells (CD4+ T cells), cytotoxic T cells (CD8+ T cells), memory T cells, regulatory T cells (T reg cells), innate-like T cells (e.g., natural killer T cells), mucosa-associated invariant T cells, or γδ T cells, or modified T cells (e.g., CAR-T cells). In some embodiments, the retinal cells are photoreceptors, retinal horizontal cells, retinal bipolar cells, retinal amacrine cells, or retinal ganglion cells. In some embodiments, the endocrine cells are hypothalamic endocrine cells, pituitary endocrine cells, pineal endocrine cells, thyroid endocrine cells (e.g., follicular cells), parathyroid endocrine cells, thymic endocrine cells, adrenal endocrine cells, pancreatic endocrine cells (e.g., α cells, β cells, δ cells, or F cells), ovarian endocrine cells (e.g., granulosa cells), or testicular endocrine cells (e.g., testicular interstitial cells). In some embodiments, the epithelial cells are squamous epithelial cells, cuboidal epithelial cells, columnar epithelial cells, pseudostratified epithelial cells, or stratified epithelial cells. In some embodiments, the muscle cells are skeletal muscle cells, cardiomyocytes (e.g., cardiomyocytes), or smooth muscle cells. In some embodiments, the nerve cells are neurons or glial cells. In some embodiments, the myeloid cells are monocytes, microglia, macrophages, dendritic cells, basophils, eosinophils, erythrocytes, mast cells, neutrophils, megakaryocytes, or platelets or any of their precursor progenitor cells.

[0239] In some implementations, methods for achieving immune evasion include genetically modified human cells.

[0240] In some embodiments, the method for achieving immune escape further includes measuring the immune escape of the genetically modified cells. In some embodiments, the immune escape includes reducing susceptibility to or inhibiting natural killer cell-mediated cytotoxicity. In some embodiments, the immune escape includes reducing susceptibility to or inhibiting T cell-mediated cytotoxicity. The immune escape can be measured, for example, by attacking the genetically modified cells with NK cells or T cells and measuring cell death, apoptosis, or proliferation of the genetically modified cells in the presence of NK cells or T cells. Exemplary methods that can be used to measure natural killer cell cytotoxicity include, but are not limited to, cell viability assays, NK cell migration assays, degranulation assays, flow cytometry (FC)-based NK cell cytotoxicity assays, and any other suitable NK cell-mediated cytotoxicity assays known in the art. Exemplary methods for measuring T cell-mediated cytotoxicity include, but are not limited to, cell viability assays, chromium (… 51 Cr) release cytotoxicity assay, IFNγ production assay, annexin V binding cytotoxicity assay, and any other suitable T cell-mediated cytotoxicity assay known in the art.

[0241] In some implementations, the method for achieving immune evasion further includes amplifying the genetically modified cells to generate a population of genetically modified cells.

[0242] V. Reagent kits and dosage forms Certain aspects of this disclosure provide kits for treating diseases or conditions, said kits comprising engineered cell populations, or pharmaceutical compositions comprising engineered cell populations suitable for administration to a subject, including any of the engineered cell populations and pharmaceutical compositions described herein.

[0243] In some embodiments, the kit includes instruction materials for using the engineered cell population or the pharmaceutical composition. In some embodiments, the instruction materials include instructions for preparing the pharmaceutical composition or engineered cell population for administration to a subject. Such instructions may include, but are not limited to, instructions for preparing or storing the engineered cell population or pharmaceutical composition, adding an additive to a treatment, or combining the cell population or pharmaceutical composition with another therapeutic agent. In some embodiments, the instruction materials include instructions for administering the engineered cell population or pharmaceutical composition to a subject.

[0244] In some embodiments, the kit further includes an applicator for administering engineered cell populations or pharmaceutical compositions containing engineered cell populations as described herein. The applicator can be any device suitable for administering the engineered cell populations or compositions described herein to a subject, including but not limited to hypodermic syringes, needles, balloon dilation catheters, pipettes, etc. The applicator can be a single-use or multiple-use application device and can be included in the kit as a pre-filled delivery system, for example, for use with a pharmaceutical composition containing cell populations.

[0245] Certain aspects of this disclosure also provide dosage forms of pharmaceutical compositions comprising engineered cell populations suitable for administration to a subject. The dosage form can be any form suitable for administration to a subject via any suitable route, including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, intracranial, intracerebral, or local administration. In some embodiments, the dosage form is suitable for administration to a subject by injection.

[0246] Example To better understand this disclosure, the following embodiments are described. These embodiments are for illustrative purposes only and should not be construed as limiting the scope of this disclosure in any way.

[0247] Example 1: iPSC Screening To identify innovative targets for achieving natural killer (NK) cell immune escape, a comprehensive literature search was conducted across various fields known to be related to immune escape, including the tumor microenvironment, immune privileged sites, stem cells, infection, microbiome, peripheral tolerance, CRISPR screening, and transplantation science. From this comprehensive exploration, 78 potential gene targets were initially identified. Through rigorous categorization based on the strength of supporting evidence, this list was refined into a final selection of 12 gene targets, including both cytoplasmic and membrane-bound genes. These gene targets were intentionally selected to cover a wide range of biological functions, including the initiation of lymphocyte apoptosis, defense against lymphocyte-mediated apoptosis, and the initiation of inhibitory signaling pathways. This diverse array of selected gene targets significantly enhances the potential for downstream multi-pathway immune escape strategies (i.e., engineering cells with combinations of gene targets as described herein), providing benefits such as synergistic combination and resistance prophylaxis.

[0248] From these efforts, specific gene targets listed in Table 1-2 were selected for experimental research.

[0249] Table 1. Amino acid sequences of gene targets used to achieve immune escape. Table 2. Nucleic acid sequences of gene targets used to achieve immune evasion. Based on the selection process and without being bound by theory, it was anticipated that if one or more gene targets were upregulated in induced pluripotent stem cells (iPSCs), they might inhibit or suppress NK cell-mediated cytotoxicity. To test this, iPSCs were genetically engineered to express each gene target by introducing nucleic acids encoding the stated gene targets, and the engineered iPSCs were subjected to an NK cell cytotoxicity assay.

[0250] Figure 1 This paper illustrates a strategy for genetically engineering iPSCs using potential gene targets that escape from NK cells. In short, a CRISPR gene-editing system is used to introduce heterologous nucleic acids encoding the gene targets listed in Tables 1 and 2 into the iPSCs. Specifically, the system comprises a Cas protein pre-complexed with a guide RNA specific to the GAPDH locus to form ribonucleic acid particles (RNPs), which are then electroporated into the iPSCs along with a homology-directed DNA repair (HDR) template. This strategy enables the heterologous nucleic acid encoding each gene target to integrate downstream of the endogenous STEL promoter.

[0251] Before integrating the heterologous nucleic acid encoding the gene target into the iPSC genome, the optimal integration site was analyzed. One factor considered was the transgene's ability to exhibit stable expression (e.g., >5 cell divisions) and differentiation into downstream cell types (e.g., cardiomyocytes) throughout extended cell culture. As a result, the GAPDH STEL site was selected based on previously described work (WO2021072329A1, which is incorporated in its entirety by reference).

[0252] After integration, the expression of each immune escape gene target is regulated by an endogenous gene promoter, particularly the GAPDH promoter. Therefore, the expression of these gene targets is correlated with the expression of the endogenous GAPDH gene. This design ensures that the immune escape gene targets maintain sustained and constitutive expression, as the GAPDH gene is essential for cellular metabolism and is continuously expressed. Flow cytometry analysis confirmed the surface expression of each immune escape gene target.

[0253] The nucleic acid sequences of each gene target identified in Tables 1-2 were individually integrated into separate iPSC lines to prevent cross-interference and to allow for individual assessment of the role of each gene in immune escape. The engineered cells were then amplified and cryopreserved for further research.

[0254] Example 2: Immunoassay of engineered iPSCs using natural killer cells To assess the impact of the gene targets identified in Tables 1-2 on NK cell escape, a series of in vitro immunoassays were performed in which the engineered iPSCs of Example 1 were directly exposed to NK cells from different healthy donors. The percentage of engineered iPSCs that remained viable upon encountering NK cells was determined and used as a measure of NK cell-mediated cytotoxicity. Higher survival rates indicate more effective immune escape and demonstrate the utility of the introduced gene targets in treating cellular immune escape.

[0255] In these NK cell cytotoxicity assays, engineered iPSCs were co-cultured with NK cells from three different healthy donors (i.e., pNK1, pNK2, and pNK3). Each batch of NK cells used was derived from a different donor to account for potential inter-individual variability in NK cell behavior and reactivity. This diversity among donors also allowed for assessment of the robustness and consistency of immune escape strategies across a range of genetic backgrounds, helping to ensure their broader applicability and effectiveness.

[0256] Figure 2 and 3 Exemplary experimental results from an NK cell cytotoxicity assay are shown. Figure 2 The data are presented in a bar chart, showing that iPSCs expressing CLEC2D, TRAIL, SERPINB9, or CD47 exhibit the ability to suppress NK cell-mediated cytotoxicity, as demonstrated by the reduced NK cell-mediated cytotoxicity (represented along the y-axis) compared to control cells (iPSCs not engineered with the gene targets described). Figure 3The data is presented as a line graph to visualize the consistency of responses across different NK cell populations. The line graph format highlights the robustness of certain gene targets (particularly CLEC2D, TRAIL, and SERPIB9) in enabling the escape of natural killer cells from various NK cell populations.

[0257] The results of NK cell cytotoxicity assays indicate that upregulating the gene targets listed in Tables 1-2 is a feasible and novel strategy for treating immune evasion in cells.

[0258] Example 3: Differentiation of engineered iPSCs into cardiomyocytes and attack by NK cells To evaluate the ability of engineered iPSCs to differentiate into cardiomyocytes after genetic manipulation (including upregulation of CLEC2D, TRAIL, SERPINB9, and two HLA-C variants (HLA-C*05:01 (IIDKSGIPV (SEQ ID NO: 25)) and HLA-C*04:01 (QYDDAVYKL (SEQ ID NO: 24))), engineered iPSCs were subjected to a cardiomyocyte differentiation protocol. Gene expression profiling of the resulting cardiomyocytes was performed to confirm successful differentiation, focusing on specific markers of cardiomyocyte identity and function (e.g., cardiac troponin T (cTNT)). For example, flow cytometry analysis confirmed that over 99% of cardiomyocytes differentiated from engineered iPSCs edited to upregulate CLEC2D were positive for cTNT expression.

[0259] Following successful differentiation, the engineered cardiomyocytes were evaluated by flow cytometry to confirm that the engineered cells maintained positive surface expression of gene targets (CLEC2D, TRAIL, SERPINB9, and two HLA-C variants (HLA-C*05:01 (IIDKSGIPV (SEQ ID NO: 25)) or HLA-C*04:01 (QYDDAVYKL (SEQ ID NO: 24))). Subsequently, the engineered cardiomyocytes were challenged with NK cells from multiple healthy donors, and the immunoassay detailed in Example 2 was repeated. This assay is important for assessing whether the immune escape characteristics observed in engineered iPSCs extend to differentiated cardiomyocytes.

[0260] Figures 4A-4BExemplary experimental results of NK cell cytotoxicity assays performed using engineered cardiomyocytes are shown. Data showed that engineered cardiomyocytes expressing CLEC2D, SERPINB9, and HLA-C*05:01 (IIDKSGIPV (SEQ ID NO: 25)) demonstrated the ability to inhibit NK cell-mediated cytotoxicity in all four pNK donors. This was demonstrated by reduced NK cell-mediated cytotoxicity in these cell populations compared to control cells (i.e., cardiomyocytes not engineered with the stated gene targets) (expressed along the y-axis).

[0261] Notably, some genes that effectively reduce cytotoxicity in engineered iPSCs did not exhibit the same level of efficacy in differentiated cardiomyocytes. This discrepancy underscores the importance of the assays, highlighting that success of a gene target in one cellular context does not guarantee similar results in another, thus reinforcing the unique and context-dependent nature of the insights presented herein. Furthermore, the expression levels of these gene targets may influence their ability to confer protection against NK cell-mediated cytotoxicity.

[0262] Example 4: Successful in vivo escape of NK cell-mediated blast furnace cardiomyocytes engineered with CLEC2D or SERPINB9. Rejection reaction This example describes experiments conducted to evaluate the ability of cardiomyocytes engineered to express novel gene targets (CLEC2D or SERPINB9) to escape NK cell-mediated rejection responses in vivo. Heteronucleotides encoding CLEC2D (SEQ ID NO: 2) or SERPINB9 (SEQ ID NO: 6) were incorporated into separate iPSC lines previously engineered to express luciferase. As described in Example 1, iPSCs were engineered to express CLEC2D or SERPINB9 using a CRISPR gene editing system to integrate the corresponding heteronucleotides into the GAPDH STEL site. Luciferase expression in the engineered cells enabled the visualization, quantification, and tracking of CLEC2D and SERPINB9 engineered cardiomyocytes in vivo.

[0263] Following engineering, engineered iPSC populations expressing CLEC2D or SERPINB9, along with luciferase-positive control iPSCs, differentiated into cardiomyocytes as described herein. Flow cytometry analysis confirmed successful differentiation of iPSCs into cardiomyocytes (data not shown). These engineered cardiomyocytes were then transplanted into the hind limbs of humanized mice that had depleted their NK cells. Transplantation was performed either alone with engineered cells or together with NK cells derived from two different donors (donor 1 and donor 2).

[0264] Figures 5A-5CThis is an exemplary experimental result demonstrating the ability of engineered cardiomyocytes expressing CLEC2D or SERPINB9 to escape NK cell-mediated toxicity in vivo. Co-transplantation of control cardiomyocytes with donor 1 NK cells or donor 2 NK cells in vivo resulted in a significant reduction in the amount of transplanted cardiomyocytes over time, as indicated by a decrease in total photon flux from luciferase-positive cells. Figure 5A Conversely, engineered cardiomyocytes expressing CLEC2D or SERPINB9 exhibited consistent photon flux in both cases of co-transplantation with and without donor NK cells. Figure 5B and 5C This study demonstrated the ability of these gene targets to help cardiomyocytes escape NK cell-mediated toxicity from two different donors in vivo.

[0265] In summary, these data demonstrate that, compared with control cells, cardiomyocytes engineered with increased CLEC2D or SERPINB9 expression exhibit the ability to escape NK-mediated multi-donor killing in vivo.

[0266] Example 5: Evaluation of NK cell cytotoxicity against iPSCs engineered with multiple immune escape genes This embodiment describes experiments conducted to evaluate the ability of iPSCs engineered to express multiple immune escape genes to escape NK cell-mediated cytotoxicity in vitro.

[0267] As described in Example 1, the corresponding heterologous nucleic acid was integrated into the GAPDHSTEL site using the CRISPR gene editing system, and the iPSCs were engineered to express CLEC2D, SERPINB9, or TRAIL alone or in combination (CLEC2D+SERPINB9 and CLEC2D+TRAIL). Control iPSCs without any genetic modifications and iPSCs with B2M knockout were also included.

[0268] As described in Example 2, the engineered iPSCs were subjected to in vitro NK cell cytotoxicity assays. These cells were co-cultured with NK cells from multiple healthy donors, and the percentage of NK-specific cytotoxicity was measured.

[0269] Figure 6 and 7Exemplary results from NK cell cytotoxicity assays are shown. These results indicate that individual expression of CLEC2D, SERPINB9, or TRAIL in wild-type iPSCs and B2M KO iPSCs reduced NK cell-mediated cytotoxicity to varying degrees. In B2M KO-engineered iPSCs, combining CLEC2D with SERPINB9 or TRAIL further reduced NK cell-mediated cytotoxicity compared to single gene expression. Although higher levels of NK cell-mediated cytotoxicity were observed at higher NK cell to iPSC ratios, a reduction in NK cell-mediated cytotoxicity was observed with individual expression of CLEC2D, SERPINB9, or TRAIL. Figure 7 These results demonstrate the effectiveness of combining specific gene targets in protecting iPSCs from NK cell-mediated cytotoxicity. Example 6: Evaluation of NK cell cytotoxicity against cardiomyocytes engineered with multiple immune escape genes This embodiment describes an experiment conducted to evaluate the ability of cardiomyocytes engineered to express multiple immune escape genes to escape NK cell-mediated cytotoxicity in vitro.

[0270] B2M knockout iPSCs were engineered to express CLEC2D alone and in combination with SERPINB9 (CLEC2D+SERPINB9). The engineered iPSCs were then differentiated into cardiomyocytes. Control cardiomyocytes with only B2M knockout were also included.

[0271] As described in Example 2, in vitro NK cell cytotoxicity assays were performed on engineered cardiomyocytes. These cells were co-cultured with NK cells at two different NK cell to cardiomyocyte ratios (2:1 and 1:1), and the percentage of NK-specific cytotoxicity was measured.

[0272] Figure 8 Exemplary results from NK cell cytotoxicity assays are shown. These results demonstrate that CLEC2D expression in B2M KO cardiomyocytes reduces NK cell-mediated cytotoxicity compared to B2M KO cardiomyocytes alone. In B2M KO-engineered cardiomyocytes, the combination of CLEC2D and SERPINB9 further reduces NK cell-mediated cytotoxicity compared to CLEC2D expression alone. This protective effect was observed at both tested NK cell to cardiomyocyte ratios (2:1 and 1:1), although higher levels of NK cell-mediated cytotoxicity were observed at higher NK cell to cardiomyocyte ratios. These results demonstrate the effectiveness of combining specific gene targets in protecting cardiomyocytes from NK cell-mediated cytotoxicity, including in the context of B2M knockout cells.

[0273] Further multiple experiments were conducted using methods similar to those described herein to evaluate the potential synergistic effect of TRAIL and CLEC2D in protecting cardiomyocytes from NK cell-mediated cytotoxicity. Figure 9 Exemplary results of an NK cell cytotoxicity assay performed at a 1:1 NK cell to cardiomyocyte ratio are shown. Cardiomyocytes expressing TRAIL alone showed moderate protection against NK cell-mediated killing compared to baseline control cells. However, cardiomyocytes expressing both CLEC2D and TRAIL showed enhanced protection, with NK-specific cytotoxicity reduced to approximately 25% compared to approximately 45% of baseline levels. These results further support the effectiveness of combined immune escape gene targets, including CLEC2D and TRAIL, in achieving greater protection against NK cell-mediated cytotoxicity in cardiomyocytes.

[0274] Equivalent schemes and scopes, incorporated by reference Those skilled in the art will be able to recognize, or determine through routine experiments, many equivalents of the specific embodiments described herein. It should be understood that modifications that do not materially affect the activity of the various embodiments of this disclosure are also included in the description of the disclosure provided herein. The scope of this disclosure is not intended to be limited to the foregoing description, but rather as set forth in the appended claims.

[0275] In the claims, articles such as “a,” “an,” and “the” may indicate one or more members unless otherwise stated or clearly understood from the context. If a claim or specification uses “or” to connect two or more members of a group, it is deemed to conform to the statement as long as one, several, or all of these members are present in, used in, or otherwise associated with a given product or method, unless otherwise stated or clearly understood from the context. This disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or method. This disclosure also includes embodiments in which more than one or all members are present in, used in, or otherwise associated with a given product or method.

[0276] Furthermore, it should be understood that this disclosure covers all variations, combinations, and arrangements in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more claims or from a relevant portion of the specification are incorporated into another claim. For example, any claim dependent on another claim may be modified to include one or more limitations found in any other claim dependent on the same basic claim. Moreover, in the case of a composition described in the claims, it should be understood that this includes methods of using the composition for any purpose disclosed herein, and methods of preparing the composition according to any preparation method disclosed herein or other methods known in the art, unless otherwise stated or unless contradictory or inconsistent to a person skilled in the art.

[0277] When elements are presented as a list, such as in a Markush group format, it should be understood that each subgroup of said elements is also disclosed, and any element can be removed from that group. It should be understood that, generally, where an aspect of this disclosure or embodiment is referred to as including a particular element, feature, step, etc., certain embodiments or aspects of this disclosure consist of or are substantially composed of such elements, features, steps, etc. Therefore, for each embodiment of this disclosure that includes one or more elements, features, steps, etc., this disclosure also provides embodiments that consist of or are substantially composed of those elements, features, steps, etc.

[0278] In the case of a given range, the endpoints are included. Furthermore, it should be understood that, unless otherwise stated or clearly understood from the context and / or by one of ordinary skill in the art, in different embodiments of this disclosure, a numerical value represented as a range may take any specific value within the range, up to one-tenth of the unit of the lower limit of the range, unless the context clearly specifies otherwise. It should also be understood that, unless otherwise stated or clearly understood from the context and / or by one of ordinary skill in the art, a numerical value represented as a range may take any subrange within the given range, wherein the endpoints of said subranges express the same precision as one-tenth of the unit of the lower limit of the range.

[0279] Furthermore, it should be understood that any particular embodiment of this disclosure may be expressly excluded from any one or more claims. Where a scope is given, any value within that scope may be expressly excluded from any one or more claims. Any embodiment, element, feature, application, or aspect of the composition and / or method of this disclosure may be excluded from any one or more claims. For the sake of brevity, not all embodiments in which one or more elements, features, objects, or aspects are explicitly excluded herein.

[0280] Throughout this disclosure, references are made to various publications, patents, and serial database entries. The disclosures of these publications, patents, and serial database entries, including those listed above, are incorporated herein by reference in their entirety, as if each individual publication or patent were specifically and individually indicated by reference. In case of conflict, this application (including any definitions herein) shall prevail.

[0281] Although the present disclosure has been described with reference to the embodiments provided above, it should be understood that various modifications can be made without departing from the scope of the present disclosure. Therefore, the above embodiments are intended to illustrate, not limit, the present disclosure.

Claims

1. An engineered cell, said engineered cell comprising: The polypeptide comprises at least one heterologous nucleic acid sequence encoding a polypeptide, wherein the polypeptide is expressed at a level sufficient to inhibit immune cell-mediated cytotoxicity of the engineered cells, and wherein the polypeptide comprises one or more of the following: C-type lectin domain family 2 member D (CLEC2D), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), serine protease inhibitor family B member 9 (SERPINB9), and human leukocyte antigen-C (HLA-C), or variants thereof.

2. The engineered cell of claim 1, wherein the polypeptide comprises one or more of CLEC2D, TRAIL, and SERPINB9 or variants thereof.

3. The engineered cell according to claim 1 or claim 2, wherein the engineered cell comprises at least two different heterologous nucleic acid sequences, each nucleic acid sequence encoding a different polypeptide.

4. The engineered cell according to claims 1 to 3, wherein the engineered cell comprises at least three different heterologous nucleic acid sequences, each nucleic acid sequence encoding a different polypeptide.

5. The engineered cell according to any one of claims 1 to 4, wherein the polypeptide comprises CLEC2D or a variant thereof.

6. The engineered cell of claim 3, wherein the at least two different heterologous nucleic acid sequences encode CLEC2D or a variant thereof; and SERPINB9 or a variant thereof.

7. The engineered cell of claim 3, wherein the at least two different heterologous nucleic acid sequences encode CLEC2D or a variant thereof; and TRAIL or a variant thereof.

8. The engineered cell according to any one of claims 1-7, wherein the polypeptide comprises an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO: 2, 4, 5 and 11.

9. The engineered cell according to any one of claims 1-8, wherein the at least one heterologous nucleic acid sequence comprises a nucleotide sequence having at least 90% sequence identity with any one of SEQ ID NO: 13, 15, 17 and 22.

10. The engineered cell according to any one of claims 1-9, wherein the at least one heterologous nucleic acid sequence is integrated into a sustained transgenic expression locus (STEL) or a sustained transcriptional activity payload region (STAPLR).

11. The engineered cell of claim 10, wherein the STEL comprises human glyceraldehyde-3-phosphate dehydrogenase (… GAPDH (The locus within the gene.) 12. The engineered cell according to any one of claims 1-11, wherein the expression of the polypeptide is driven by an endogenous gene promoter.

13. The engineered cell according to any one of claims 1-12, wherein the engineered cell comprises a kill switch.

14. The engineered cell according to any one of claims 1-13, wherein the engineered cell further comprises a genetic modification that results in reduced T cell-mediated killing compared to wild-type cells.

15. The engineered cell of claim 14, wherein the genetic modification comprises: (i)β2 microglobulin ( B2M The deletion, damage, or weakening of genes; Or (ii) HLA-A Gene, HLA-B Genes and HLA-C One or more genes are missing, damaged, or weakened.

16. The engineered cell of claim 15, wherein the engineered cell further comprises a second genetic modification, the second genetic modification comprising class II major histocompatibility complex transactivator (MHC). CIITA ) gene or regulatory factor X5 ( RFX5 ) The deletion, damage, or weakening of genes.

17. The engineered cell according to any one of claims 1-16, wherein the engineered cell comprises stem cells.

18. The engineered cell according to any one of claims 1-17, wherein the engineered cell comprises cardiac cells, nerve cells, myeloid cells, T cells, endocrine cells, epithelial cells, muscle cells, or retinal cells.

19. The engineered cell according to any one of claims 1-18, wherein the engineered cell is a human cell.

20. An engineered cell population derived from the engineered cells of any one of claims 1-19.

21. The engineered cell population of claim 20, wherein the engineered cell population is at least about 10% less susceptible to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence.

22. The engineered cell population of claim 21, wherein the engineered cell population is at least about 20% less susceptible to natural killer cell-mediated cytotoxicity compared to a cell population not engineered to have at least one heterologous nucleic acid sequence.

23. The engineered cell population according to any one of claims 18-22, wherein at least 95% of the engineered cells in the engineered cell population are positive for cardiac troponin T (cTnT).

24. The engineered cell population according to any one of claims 18-23, wherein the engineered cell population is present in a composition formulated for administration to a subject.

25. A method for treating a disease or ailment of a subject, the method comprising administering an engineered cell population according to any one of claims 18-24.

26. The method of claim 25, wherein the disease or condition includes heart failure, Parkinson's disease, multiple sclerosis, irritable bowel syndrome, type 1 diabetes, rheumatoid arthritis, liver disease, cancer, inflammation, or neuroinflammation.

27. The method of claim 25 or claim 26, wherein the subject is a human.

28. A method for achieving immune escape, the method comprising: Cells are genetically engineered to increase the expression of at least one polypeptide, wherein the at least one polypeptide comprises one or more of the following: C-type lectin domain family 2 member D (CLEC2D), tumor necrosis factor-associated apoptosis-inducing ligand (TRAIL), serine protease inhibitor family B member 9 (SERPINB9), and human leukocyte antigen-C (HLA-C) or variants thereof; and Thus, compared with cells that have not been genetically engineered to increase the expression of the at least one polypeptide, genetically engineered cells exhibit increased survival upon contact with natural killer cells.

29. The method of claim 28, wherein the polypeptide comprises one or more of CLEC2D, TRAIL, and SERPINB9 or variants thereof.

30. The method of claim 29, wherein the polypeptide comprises CLEC2D or a variant thereof.

31. The method according to any one of claims 27 to 30, wherein the cells are genetically engineered to increase the expression of at least two polypeptides, and wherein the at least two polypeptides comprise CLEC2D and SERPINB9.

32. The method according to any one of claims 27 to 30, wherein the cells are genetically engineered to increase the expression of at least two polypeptides, wherein the at least two polypeptides comprise CLEC2D and TRAIL.

33. The method according to any one of claims 28 to 32, wherein the genetic modification comprises integrating a heterologous nucleic acid sequence encoding the polypeptide into a genomic locus of the cell.

34. The method of claim 33, wherein the genomic locus is a sustained transgene expression locus (STEL) or a sustained transcriptional active payload region (STAPLR).

35. The method of claim 34, wherein the STEL comprises human glyceraldehyde-3-phosphate dehydrogenase (… GAPDH (The locus within the gene.) 36. The method according to any one of claims 28-35, further comprising amplifying the cells that have been genetically engineered to produce an engineered cell population.

37. The method of claim 36, further comprising differentiating the engineered cell population into cardiac cells, nerve cells, T cells, retinal cells, endocrine cells, epithelial cells, muscle cells, or myeloid cell populations.

38. The method according to any one of claims 29-37, wherein the cell is a human cell.

39. A pharmaceutical composition comprising an engineered cell population according to any one of claims 20 to 23 and a pharmaceutically acceptable carrier, transporter or diluent.

40. A kit comprising a dosage form suitable for administration to a subject and instructional materials for using the dosage form, the dosage form comprising an engineered cell population according to any one of claims 20-23.

41. A kit comprising a dosage form suitable for administration to a subject and instructional materials for using the dosage form, the dosage form comprising the pharmaceutical composition of claim 39.

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