Cells with sustained transgene expression

Abstract

To provide cells with sustained transgene expression.SOLUTION: There are provided herein genetically engineered mammalian cells comprising a transgene at a sustained transgene expression locus (STEL) in the genome, where the transgene is expressed at a detectable level, optionally where the mammalian cell is a human cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 913,062, filed October 9, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on October 9, 2020, is named 025450_WO009_SL.txt and is 29,071 bytes in size. [Background technology]

[0003] Background of the Invention Cell therapy offers great promise for the treatment of various diseases and conditions. In cell therapy, autologous or allogeneic cells are transplanted into patients to replace or repair missing or damaged tissues or cells. Many different types of cells can be used, such as pluripotent stem cells (PSCs), multipotent stem cells (e.g., hematopoietic stem cells and mesenchymal stem cells), or differentiated cells (e.g., dopaminergic neurons, lymphocytes, cardiomyocytes, and pancreatic islet cells). Potential applications of cell therapy include the treatment of cancer, autoimmune diseases, and the regeneration of damaged tissues, for example, in the joints, heart, and central and / or peripheral nervous system.

[0004] Therapeutic cells in cell therapy can be genetically modified using transgenes that are stably integrated into their genome.When transgenes are expressed, they can introduce new features, such as proteins that are not normally present, into modified cells.However, stable long-term transgene expression in cells or organisms remains difficult in the field.Transgenes can be subjected to the gene expression patterns that exist in advance or are developed in target cells.This pattern can override the signal from transgenic regulatory factors, for example, by DNA methylation and histone modification of genome, which leads to chromatin remodeling and transgene silencing.

[0005] Similar problems exist with the integration of transgenes into the locus of certain ubiquitously expressed genes, such as housekeeping genes. Some genes are ubiquitously expressed in all human tissues. Due to this uniformity of expression, promoters from these genes may be considered prime candidates for gene engineering when sustained transgene expression is desired (Non-Patent Document 1). However, some of these genes have been found not to be uniformly expressed in all known cell phenotypes as previously thought (Non-Patent Document 2). Therefore, using the promoters of these housekeeping genes for transgene expression may ultimately result in low or negligible levels of transgene expression. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Kao et al., Stem Cell Rep. (2016) 9(3):518-26 [Non-patent document 2] de Jonge et al., PLoS One (2007) 2(9):e898 Summary of the Invention [Problem to be solved by the invention]

[0007] Therefore, there remains a need to identify transgene integration sites that allow sustained transgene expression in PSCs and PSC-derived cells. [Means for solving the problem]

[0008] Summary of the Invention The present disclosure provides genetically modified mammalian cells comprising a transgene at a sustained transgene expression locus (STEL) in the genome, wherein the transgene is expressed at a detectable level. In some embodiments, the expression level of the transgene does not change by more than 40%, more than 30%, more than 20%, or more than 10% (i) over 5 or more, 10 or more, or 15 or more passages, or (ii) upon a change in cell state, wherein the cell state is optionally a pluripotent and / or differentiated state.

[0009] The STEL site can be, for example, one of the loci listed in Table 1 below. In some embodiments, the STEL is a locus with an average normalized expression greater than 3.30, greater than 3.50, greater than 3.75, greater than 4.00, greater than 4.10, greater than 4.20, greater than 4.30, greater than 4.50, greater than 4.60, or greater than 4.70 as shown in the table.

[0010] In some embodiments, the STEL is a genetic locus that encodes a protein involved in one or more of ribonucleoprotein complex formation, focal adhesions, cell-substrate adherens 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.

[0011] In some embodiments, STEL is a transcription factor (e.g., RPL13A, RPLP0, RPL10, RPL13, RPS18, RPL3, RPLP1, RPL15, RPL41, RPL11, RPL32, RPL18A, RPL19, RPL28, RPL29, RPL9, RPL8, RPL6, RPL18, RPL7, RPL7A, RPL21, RPL37A, RPL12, RPL5, RPL34, RPL 35A, RPL30, RPL24, RPL39, RPL37, RPL14, RPL27A, RPLP2, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4 and RPL22) or RPS genes (e.g. RPS2, RPS19, RPS14, RPS3A, RPS12, RPS3, RPS6, RPS23, RPS27A, RPS8, RPS4X, RPS7, RPS24, RPS27, genes encoding ribosomal proteins such as RPS15A, RPS9, RPS28, RPS13, RPSA, RPS5, RPS16, RPS25, RPS15, RPS20 and RPS11; genes encoding mitochondrial proteins (e.g., MT-CO1, MT-CO2, MT-ND4, MT-ND1 and MT-ND2), genes encoding actin proteins (e.g., ACTG1 and ACTB); genes encoding eukaryotic translation factors (e.g., EEF1A1, EEF2 and EIF1); genes encoding histones (e.g., H3F3A and H3F3B); or genes selected from FTL, FTH1, TPT1, TMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2 and SRP14. In certain embodiments, the STEL is the GAPDH, RPL13A, RPL7 or RPLP0 locus.

[0012] In some embodiments, the transgene is inserted into the 3' untranslated region of the locus. In some embodiments, the transgene sequence is linked in-frame to the STEL gene sequence via a coding sequence for a self-cleaving peptide. In some embodiments, the transgene sequence is linked to the STEL gene sequence via an internal ribosome entry site (IRES).

[0013] In some embodiments, the transgene encodes a therapeutic protein, an immunomodulatory protein, a reporter protein, or a safety switch signal (e.g., a suicide gene).

[0014] In some embodiments, the genetically modified mammalian cells are human cells, such as PSCs (e.g., embryonic stem cells or induced PSCs) or differentiated cells. In some embodiments, the differentiated cells are (i) immune cells, optionally selected from T cells, chimeric antigen receptor (CAR)-expressing T cells, inhibitory T cells, myeloid cells, dendritic cells, and immunosuppressive macrophages; (ii) cells of the nervous system, optionally selected from dopaminergic neurons, microglial cells, oligodendrocytes, astrocytes, cortical neurons, spinal cord or oculomotor neurons, enteric neurons, placode-derived cells, Schwann cells, and trigeminal or sensory neurons; (iii) cells of the cardiovascular system, optionally selected from cardiac myocytes, endothelial cells, and nodal cells; or (iv) cells of the metabolic system, optionally selected from hepatocytes, bile duct cells, and pancreatic beta cells.

[0015] In another aspect, the present disclosure provides a method of treating a human patient in need of treatment, comprising introducing the genetically modified human cells. Also provided are genetically modified human cells for use in treating a human patient in need of treatment and the use of genetically modified human cells for the manufacture of a medicament for treating a human in need of treatment.

[0016] In yet another aspect, the present disclosure provides a method for producing genetically modified mammalian cells as described herein, comprising providing cultured mammalian cells and introducing a target transgene into the STEL site in the genome of the cultured cells.In some embodiments, the transgene is introduced into the genome of the cell by CRISPR gene editing (for example, CRISPR-Cas9 gene editing).In some embodiments, the genetically engineered cell of the present disclosure is a pluripotent stem cell (PSC), for example, embryonic stem cell (for example, human embryonic stem cell) or induced PSC (for example, human induced PSC). In some embodiments, the engineered cell is a differentiated cell, such as an immune cell (e.g., a T cell, a T cell expressing a chimeric antigen receptor (CAR), a myeloid cell, or a dendritic cell), an immunosuppressive cell (e.g., an inhibitory T cell or an immunosuppressive macrophage), a cell of the nervous system (e.g., a dopaminergic neuron, a microglial cell, an oligodendrocyte, an astrocyte, a cortical neuron, a spinal cord or oculomotor neuron, an enteric neuron, a placode-derived cell, a Schwann cell, or a trigeminal or sensory neuron), a cell of the cardiovascular system (e.g., a cardiomyocyte, an endothelial cell, or a nodule cell), a cell of the metabolic system (e.g., a hepatocyte, a bile duct cell, or a pancreatic beta cell), or a cell of the human ocular system, optionally selected from a retinal pigment epithelial cell, a photoreceptor cone cell, a photoreceptor rod cell, a bipolar cell, and a ganglion cell.

[0017] In another aspect, the present disclosure provides a method of treating a human patient in need of treatment, comprising introducing into the human patient a genetically modified human cell of the present disclosure. In some embodiments, where the introduced genetically engineered cell contains a suicide gene, the method can further comprise administering an activator of the suicide gene at a desired time.

[0018] In some embodiments, the human patient requires immunosuppression, and the genetically modified immune cells are immunosuppressive cells, suppressive T cells, or immunosuppressive macrophages. In some embodiments, the human patient requires transplantation or has inflammation (e.g., neuroinflammation), autoimmune disease, or cancer. In some embodiments, the human patient requires cell therapy for, for example, damaged or degenerated tissue (e.g., brain tissue, heart tissue, muscle tissue, joints, or tissue involved in metabolism).

[0019] In yet another aspect, the present disclosure provides a method for producing genetically modified recombinant human cells as described herein, comprising providing cultured human cells and introducing an exogenous sequence and / or suicide gene into the genome of the cultured human cells.In some embodiments, the introducing step is carried out by homologous recombination with or without nuclease-mediated gene editing (for example, ZFN, TALEN, or CRISPR-Cas9 or CRISPR-cpf1).Non-homologous end joining can also be used to target transgenes.

[0020] Also provided herein are genetically modified human cells as described herein for use in treating a human patient in need of treatment in one of the present therapeutic methods. Also provided are uses of genetically modified human cells as described herein for the manufacture of a medicament for treating a human in need of treatment in one of the present therapeutic methods. Also provided are articles of manufacture, such as kits, that include the genetically modified human cells described herein.

[0021] Other features, objects, and advantages of the present invention will become apparent in the detailed description that follows. It should be understood, however, that the detailed description, while illustrating embodiments and aspects of the present invention, is given by way of illustration only and not by way of limitation. Various changes and modifications within the scope of the present invention will become apparent to those skilled in the art from the detailed description.

[0022] That is, the gist of the present invention relates to the following. Item 1 A genetically modified mammalian cell comprising a transgene at a persistent transgene expression locus (STEL) in the genome, wherein the transgene is expressed at a detectable level, and optionally the mammalian cell is a human cell. [Effects of the Invention]

[0023] The present invention can provide cells with persistent transgene expression. [Brief explanation of the drawings]

[0024] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 is a panel of UMAP plots showing the ubiquity of expression of four different putative STEL genes in the context of the cell types included in the analysis. Cell types: dopaminergic neurons, microglia, pluripotent stem cells, and ventricular cardiomyocytes. Panel a: UMAP plot showing the identity and clustering of the four cell types included in the analysis. Panel b: UMAP plot showing the expression profiles of GAPDH, RPL7, RPLP0, and RPL13A. [Figure 2] Figure 2 is a diagram illustrating the integration of an enhanced green fluorescent protein (EGFP) transgene into the human GAPDH, RPL13A, RPLPO, or RPL7 locus. The coding sequence of the targeted endogenous gene was linked to the EGFP coding sequence via the coding sequence for the self-cleaving PQR peptide. [Figure 3] Figure 3 shows cytometry plots showing EGFP expression levels in PSCs homozygous or heterozygous for EGFP transgenes targeted to the GAPDH or RPL13A locus. Non-edited PSCs (PSCs without the transgene) served as negative controls. [Figure 4]Figure 4 is a cytometry plot showing EGFP expression levels in PSCs heterozygous for an EGFP transgene targeted to the RPLP0 locus. Non-edited PSCs (PSCs without the transgene) were used as a negative control. [Figure 5] Figure 5 is a qPCR histogram showing that EGFP expression was detected in GAPDH-targeted EGFP-edited heterozygous and homozygous PSCs on a weekly basis for up to 8 weeks, but not in non-edited PSCs (PSCs without the transgene). [Figure 6] Figure 6 is a qPCR histogram showing that EGFP expression was detected in RPL13A-targeted EGFP-edited heterozygous and homozygous PSCs on a weekly basis for up to 8 weeks, but not in non-edited PSCs (PSCs without the transgene). [Figure 7] Figure 7 shows cytometry plots showing EGFP expression levels in PSC-derived cells homozygous or heterozygous for EGFP transgenes targeted to the GAPDH or RPL13A locus. After gene editing, cells were assayed 16 days after differentiation into dopaminergic neurons. [Figure 8] Figure 8 is a set of cytometry plots showing EGFP expression levels in PSCs or PSC-derived cells heterozygous for EGFP transgenes targeted to the GAPDH or RPL13A locus. After gene editing, cells were assayed 12 days after differentiation into cardiomyocytes. Non-edited PSCs (PSCs without transgenes) served as negative controls. [Figure 9] Figure 9 is a diagram illustrating integration of an HLA-G6 transgene into the human GAPDH or RPL13A locus. The coding sequence of the targeted endogenous gene was linked to the HLA-G6 coding sequence via the coding sequence for the self-cleaving PQR peptide. [Figure 10]Figure 10 is a Western blot photograph showing that HLA-G6 was detected by an HLA-G5 / G6-specific antibody in cell culture supernatants of GAPDH-targeted, HLA-G6-edited PSCs and JEG-3 cells (positive control). Non-edited ("wild-type") PSCs were used as a negative control. [Figure 11] Figure 11 is a fluorescence resonance energy transfer (FRET) assay histogram showing that HLA-G6 was detected in cell culture supernatants of GAPDH-targeted HLA-G6-edited PSCs and JEG-3 cells (positive control). Non-edited ("wild-type") PSCs were used as a negative control. [Figure 12] Figure 12 is a FRET assay histogram showing that HLA-G6 was detected in cell culture supernatants of RPL13A-targeted HLA-G6-edited PSCs, but not in unedited ("wild-type") PSCs. [Figure 13] 13 is a panel of cytometry plots showing HLA-G expression in PSCs edited for HLA-G6 transgenes targeted to the GAPDH or RPL13A locus and B2M knockout (KO) mice. HLA-G expression is detectable in edited PSCs at 1 and 8 weeks after analysis, but not in non-edited PSCs (PSCs without the transgene). [Figure 14] Figure 14 is a diagram illustrating integration of an anti-τ scFv transgene into the human GAPDH locus. The coding sequence of the targeted endogenous gene was linked to the scFv coding sequence via the coding sequence for the self-cleaving PQR peptide. SP: signal peptide coding sequence. PL: peptide linker coding sequence. HA: hemagglutinin A tag coding sequence. [Figure 15] Figure 15 is a Western blot photograph showing that anti-τ scFv was detected in straight and concentrated cell culture supernatants and cell lysates of GAPDH-targeting scFv-edited PSCs. Non-edited ("wild-type") PSCs were used as a negative control. [Figure 16]Figure 16 illustrates the integration of two components of the RapaCasp9 transgene into the human GAPDH locus. The coding sequence of the targeted endogenous gene is linked to the respective RapaCasp9 coding sequence via the coding sequence for the self-cleaving PQR peptide. L1: FRB peptide linker coding sequence. L2: FKBP12 peptide linker coding sequence. truncCasp9: cleaved caspase 9 with the CARD domain removed. [Figure 17] Figure 17 is a panel of cytometry dot plots showing the detection of cleaved caspase 3 following the addition of 5 nM or 10 nM rapamycin to PSCs biallelically edited for a RapaCasp9 transgene targeted to the GAPDH locus. Cells were analyzed after 1, 2, 4, or 24 hours of rapamycin treatment and compared to untreated edited PSCs, which served as a negative control. [Figure 18] Figure 18 is a panel of two cytometry dot plots showing the detection of PD-L1 and CD47 costaining in PSCs biallelically edited for PD-L1- and CD47-based transgenes targeted to the human GAPDH locus. [Figure 19] FIG. 19 is an ELISA immunoassay histogram showing that CSF1 was detected in cell culture supernatants of three different GAPDH-targeted, CSF1-edited human PSC lines, but not in non-edited PSCs. [Figure 20] Figure 20A is a diagram showing the transgene integration site at the AAVS1 locus. The transgene encodes PD-L1 and HSV-TK. The coding sequences for the two proteins are separated in frame by the P2A coding sequence. The transgene is under the control of the EF1α promoter. Figure 20B is a panel of two cytometry plots showing PD-L1 expression levels from the transgenes shown in Figure 20A in undifferentiated edited human PSCs and cardiomyocytes differentiated from PSCs. DETAILED DESCRIPTION OF THE INVENTION

[0025] Detailed Description of the Invention The present invention is based on the discovery that certain loci in the genome, referred to herein as "sustained transgene expression loci" (STELs), are more resistant to silencing than non-STEL loci. Resistance to silencing can be observed, for example, when cells engineered with STELs are cultured over time (e.g., over days of culture, optionally including one or more cell passages) or when cell fate is changed (e.g., differentiation from pluripotent stem cells to lineage-specific cells). When a transgene is inserted into such a locus, transgene expression is sustained, making transgene-dependent cell therapy highly effective.

[0026] Thus, the present disclosure provides methods for obtaining genetically modified mammalian cells (e.g., human) in which an exogenously introduced transgene is expressed at a stable and sustained level over a period of time or when the cells differentiate. These methods are particularly advantageous when applied to genetically engineered PSCs for use in cell therapy. The genetically modified PSCs obtained by the present methods do not lose transgene expression over time in culture and / or when the cells differentiate into one or more cell types.

[0027] In some embodiments, the expression level of the transgene in the modified cells does not change by more than 50%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, or more than 5% over one or more cell culture passages compared to the expression level of the transgene before one or more passages. The one or more passages can be, for example, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or fifteen or more passages.

[0028] In some embodiments, the expression level of the transgene in the modified cell does not change by more than 50%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, or more than 5% when the cell state is changed in the cell compared to the expression level of the transgene before the cell state change. The cell state can be, for example, the pluripotency, biological activity, phenotype, or differentiation state of the cell.

[0029] The expression level of a gene (e.g., a transgene or endogenous gene) can be determined by any method appropriate for the particular gene. For example, the levels of RNA (e.g., by RT-PCR) or protein (e.g., by FRET, ELISA, cytometric analysis, and Western blot) expressed from the gene can be measured.

[0030] To date, transgenes are most commonly targeted to safe harbor sites in the genome, such as the AAVS1 locus. High-level transgene expression from safe harbor loci typically requires the inclusion of an external promoter sequence. However, different promoters vary in their ability to maintain transgene expression in specific cell populations. Increasing evidence suggests that transgene expression at AAVS1 and other safe harbor sites is not supported in some cell lineages (e.g., dopaminergic neurons, microglia, macrophages, or T cells) and may be subject to promoter silencing. Genetically modified human pluripotent stem cells have been observed to exhibit impaired transgene expression during lineage-directed differentiation (see, e.g., Klatt et al., Hum Gene Ther. (2020) 31(3-4):199-210; Ordovas et al., Stem Cell Rep. (2015) 5:918-31). The present disclosure provides a method of transgene expression that circumvents this problem and greatly facilitates the development of cell therapies.

[0031] I. Persistent transgene expression loci Sustained transgene expression loci (STELs) of the present disclosure include, but are not limited to, specific housekeeping genes that are active in multiple cell types, such as those involved in gene expression (e.g., transcription factors and histones), cellular metabolism (e.g., GAPDH and NADH dehydrogenase), or cellular structure (e.g., actin), or those encoding ribosomal proteins (e.g., large or small ribosomal subunits, e.g., RPL13A, RPLP0, and RPL7). Further examples of STELs are listed in Table 1 below. These proteins include those that form ribonucleoprotein complexes, focal adhesions, cell-matrix adherens junctions, cell-matrix binding, cell anchoring, extracellular exosomes, extracellular vesicles, intracellular organelles, or anchoring bonds. Some of the proteins are involved in RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), or protein binding.

[0032] In some embodiments, the STEL site is the locus of an endogenous gene that is robustly and consistently expressed in the pluripotent state and during differentiation (e.g., as tested by single-cell RNA sequencing (scRNAseq) analysis). For example, the expression level of the endogenous gene does not change (e.g., decrease) by more than 50%, more than 40%, more than 35%, more than 30%, more than 25%, more than 20%, more than 15%, more than 10%, or more than 5% over 5 or more passages, or when the cell state changes (e.g., pluripotency and / or differentiation state).

[0033] In some embodiments, STEL is ribosomal protein locus, for example, RPL or RPS locus.The example of 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. Examples of RPS genes are 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 RPS11.

[0034] In some embodiments, the STEL is a locus encoding a mitochondrial protein. Examples of such loci are MT-CO1, MT-CO2, MT-ND4, MT-ND1, and MT-ND2.

[0035] In some embodiments, STEL is a genetic locus that encodes actin proteins such as ACTG1 and ACTB.

[0036] In some embodiments, the STEL is a genetic locus encoding a eukaryotic translation elongation factor, such as EEF1A1 and EEF2, or a eukaryotic translation initiation factor, such as EIF1.

[0037] In some embodiments, the STEL is a locus encoding a histone, such as H3F3A and H3F3B.

[0038] In other embodiments, the STEL is a locus selected from FTL, FTH1, TPT1, TMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2 and SRP14.

[0039] To introduce a transgene construct into a host cell, chemical methods (e.g., calcium phosphate transfection or lipofection), non-chemical methods (e.g., electroporation or nucleofection), particle-based methods (e.g., magnetofection), or viral delivery (e.g., by using viral vectors such as lentiviral vectors, adeno-associated viral (AAV) vectors, retroviral vectors, and hybrid viral vectors) can be used. The transgene can be integrated into the STEL site in a site-specific manner through single- or double-stranded DNA breaks caused by, for example, ZFN, TALEN, CRISPR-cas9, CRISPR / cpf1, or another nuclease. For example, various types of homologous recombination gene editing systems can be used, in which the edited allele is generated by homologous recombination between the host genome and a double-stranded DNA donor molecule. Homologous recombination can be facilitated by introducing a double-stranded DNA break at a targeted homologous locus in the host genome, resulting in the exchange of the exogenous DNA donor sequence with the endogenous host genome sequence. See, e.g., Hoshijima et al., Methods Cell Biol. (2016) 135:121-47. However, double-stranded DNA breaks are not required for homologous recombination.

[0040] Other well-known gene editing systems can also be used, such as those using genome targeting factors such as DNA binding domains (e.g., zinc finger DNA binding proteins or TALE DNA binding domains), guide RNA factors (e.g., CRISPR guide RNA), and guide DNA factors (e.g., NgAgo guide DNA). Programmable gene targeting and nuclease factors enable precise genome editing by introducing DNA breaks, such as double-strand breaks, at specific genomic loci. In some embodiments, the genome editing system is a meganuclease-based system, a zinc finger nuclease (ZFN)-based system, a transcription activator-like effector nuclease (TALEN)-based system, a CRISPR-based system, or an NgAgo-based system. In some embodiments, exogenously introduced DNA can be used to introduce transgenes into the genome via homologous recombination, utilizing cellular repair mechanisms.

[0041] In certain embodiments, the genome editing system is a CRISPR-based system. A CRISPR-based system comprises one or more guide RNA factors and one or more RNA-guided nucleases.

[0042] In another embodiment, the CRISPR-based system is a CRISPR-Cas system. " CRISPR-Cas system " comprises: (a) at least one guide RNA factor or a nucleic acid comprising the nucleotide sequence (s) encoding the guide RNA factor, the guide RNA factor comprises a targeter RNA comprising a nucleotide sequence substantially complementary to the nucleotide sequence in one or more target genome regions, and an activator RNA comprising a nucleotide sequence that can hybridize with the guide RNA; and (b) a Cas protein factor comprising a Cas protein or a nucleic acid comprising a nucleotide sequence encoding the Cas protein. Guide RNA and activator RNA can be separate or can be fused together into a single RNA.

[0043] In some embodiments, the CRISPR-based system includes class 1 CRISPR and / or class 2 CRISPR systems. Class 1 systems use several Cas proteins together with CRISPR RNA (crRNA) as targeter RNA to assemble a functional endonuclease. Class 2 CRISPR systems use a single Cas protein and crRNA as targeter RNA. Class 2 CRISPR systems, such as type II Cas9-based systems, contain a single Cas protein to mediate cleavage rather than the multi-subunit complex used in class 1 systems. CRISPR-based systems also include class 2, type V CRISPR systems, which use Cpf1 protein and crRNA as targeter RNA.

[0044] The Cas protein is a CRISPR-associated (Cas) double-stranded DNA nuclease. In some embodiments, the CRISPR-Cas system comprises a Cas9 protein. In some embodiments, the Cas9 protein is SaCas9, SpCas9, SpCas9n, Cas9-HF, Cas9-H840A, FokI-dCas9, or D10A nickase. The term "Cas protein," such as a Cas9 protein, includes wild-type Cas proteins or functional derivatives thereof (e.g., truncated versions or variants of wild-type Cas proteins that have nuclease activity).

[0045] In some embodiments, the CRISPR-based system is a CRISPR-Cpf system. A "CRISPR-Cpf system" includes: (a) at least one guide RNA factor or a nucleic acid comprising a nucleotide sequence(s) encoding the guide RNA factor, the guide RNA comprising a targeting factor RNA having a nucleotide sequence complementary to the nucleotide sequence at the locus of the target nucleic acid; and (b) a Cpf protein (e.g., cpf1) factor or a nucleic acid comprising a nucleotide sequence encoding the Cpf protein factor.

[0046] II. Transgenes The transgene encodes a payload, which may be, for example, a therapeutic protein or a gene product that imparts desired characteristics to the modified cell. In some embodiments, the transgene encodes a reporter protein, such as a fluorescent protein (e.g., green fluorescent protein, red fluorescent protein, cyan fluorescent protein, yellow fluorescent protein, blue fluorescent protein, DsRED, mCherry, mKate2 and tdTomato) and an enzyme (e.g., luciferase and lacZ). The reporter gene can help track the therapeutic cell when it is implanted into a patient.

[0047] In some embodiments, the transgene encodes a therapeutic protein, such as a protein that is deficient in a patient. Examples of such therapeutic proteins include, but are not limited to, those that are deficient in lysosomal storage disorders, such as α-L-iduronidase, arylsulfatase A, β-glucocerebrosidase, acid sphingomyelinase, and α- and β-galactosidase; and those that are deficient in hemophilia, such as factor VIII and factor IX. Other examples of therapeutic proteins include, but are not limited to, antibodies or antibody fragments (e.g., scFv), such as those that target pathogen proteins (e.g., tau, α-synuclein, and β-amyloid protein) and those that target cancer cells (e.g., CD19, CD20, and chimeric antigen receptors (CARs) that target tumor antigens).

[0048] In some embodiments, the transgene encodes a protein involved in immune regulation or immunomodulation. Examples of such proteins are HLA-G, HLA-E, CD47, PD-L1, CTLA-4, M-CSF, IL-4, IL-6, IL-10, IL-11, IL-13, TGF-β1, and various isoforms thereof. By way of example, the transgene may encode an isoform of HLA-G (e.g., HLA-G1, -G2, -G3, -G4, -G5, -G6, or -G7) or HLA-E; allogeneic cells expressing such non-classical MHC class I molecules may be less immunogenic and better tolerated when transplanted into a human patient who is not the source of the cells, enabling "universal" cell therapy. See also the detailed description below.

[0049] In some embodiments, the transgene encodes a safety switch signal. In cell therapy, a safety switch can be used to stop the growth of genetically modified cells when their presence in a patient is undesirable, such as when the cells do not function properly or when the therapeutic goal has been achieved. For example, the safety switch can be a so-called suicide gene, which is activated or inactivated to cause cells to enter apoptosis upon administration of a pharmaceutical compound to a patient. The suicide gene can encode an enzyme not found in humans (e.g., a bacterial or viral enzyme) that converts harmless substances into toxic metabolites in human cells. Examples of suicide genes include, but are not limited to, genes for thymidine kinase, cytosine deaminase, intracellular antibodies, telomerase, toxins, caspases (e.g., iCaspase 9) and HSV-TK, and DNase. See, for example, Zarogoulidis et al., J Genet Syndr Gene Ther. (2013) doi:10.4172 / 2157-7412.1000139. In some embodiments, the suicide gene can be a thymidine kinase (TK) gene from herpes simplex virus (HSV), which becomes toxic to cells upon administration of ganciclovir, valganciclovir, famciclovir, etc. to a patient.

[0050] In some embodiments, the safety switch can be a rapamycin-inducible human caspase-9 system (RapaCasp9) cellular suicide switch, in which a cleaved caspase-9 gene with its CADR domain removed is linked to either the FRB (FKBP12-rapamycin binding) domain of mTOR or FKBP12 (FK506-binding protein 12) domain. Addition of the drug rapamycin allows heterodimerization of FRB and FKBP12, which then leads to homodimerization of cleaved caspase-9 and induction of apoptosis.

[0051] In some embodiments, the transgene encodes a payload that is not a polypeptide. For example, the transgene can encode miRNA, which can selectively eliminate cells based on gene expression patterns. The transgene can also encode lncRNA or other RNA switches, which can control cell behavior in a desired manner.

[0052] III. Transgene Expression at the STEL Site A transgene can be transcribed into a single mRNA together with an endogenous gene at the STEL site under the transcriptional control of an endogenous promoter, and the RNA sequences for each gene can then be translated separately using internal ribosome entry sites (IRES) in the mRNA. In yet another approach, a transgene can be inserted in-frame with an endogenous gene, for example at the 3' end of the gene, but separated from the endogenous gene sequence by a coding sequence for a self-cleaving peptide, resulting in ribosomal skipping during translation. This arrangement results in the production of two separate polypeptides—the payload encoded by the transgene and the polypeptide encoded by the endogenous gene. An example of a self-cleaving peptide is the 2A peptide, a virus-derived peptide typically 18-22 amino acids long. 2A peptides include T2A, P2A, E2A, F2A, and PQR (Lo et al., Cell Reports (2015) 13:2634-2644). By way of example, P2A is a 19-amino acid peptide; after cleavage, several amino acid residues from P2A remain on the upstream gene and a proline remains at the beginning of the second gene. See also the example below for the use of the PQR peptide. In other embodiments, the STEL gene and transgene are transcribed into a single mRNA and expressed as a fusion protein.

[0053] In some embodiments, the transgene construct may introduce additional regulatory sequences into the targeted locus, such as a transcription termination sequence (e.g., a polyadenylation (polyA) site, e.g., an SV40 polyA site) and a sequence that enhances gene expression or RNA stability (e.g., a WPRE element). To further ensure sustained expression of the transgene, appropriate transcriptional regulatory elements may also be introduced into the STEL site targeted via the transgene construct. Such elements include, but are not limited to, ubiquitous chromatin opening elements (UCOEs) located upstream of the promoter and chromatin insulators that create functional boundaries. Chromatin insulators (e.g., chicken β-globin gene culture (cHS4) and ArsI) may be enhancer-blocking or barrier insulators that prevent heterochromatin silencing from spreading to the transgene.

[0054] IV. Genetically Modified Cells The present disclosure provides mammalian (e.g., human, non-human primate, rodent, or mouse) cells containing one or more transgenes at one or more STEL sites in the genome. Cells, such as human cells, can be genetically engineered in vitro, in vivo, or ex vivo using gene editing methods such as those described herein. Various human cell types can be genetically engineered to express a transgene of interest. In some embodiments, the genetically engineered cells are pluripotent stem cells, such as human embryonic stem cells (hESCs) or human induced pluripotent stem cells (iPSCs), which can then be induced to differentiate into a desired cell type and may be referred to herein as PSC derivatives, PSC-derived cells, or PSC-derived cells. In yet other embodiments, the genetically engineered cells are differentiated cells (e.g., partially or terminally differentiated cells). Partially differentiated cells can be, for example, tissue-specific progenitor or stem cells, such as hematopoietic progenitor or stem cells, skeletal muscle progenitor or stem cells, cardiac progenitor or stem cells, neural progenitor or stem cells, and mesenchymal stem cells.

[0055] As used herein, the terms "pluripotent" or "pluripotency" refer to the ability of a cell to self-renew and differentiate into cells of any of the three embryonic layers: endoderm, mesoderm, or ectoderm. "Pluripotent stem cells" or "PSCs" include ESCs derived from, for example, the inner cell mass of a blastocyst or from somatic cell nuclear transfer, and iPSCs derived from non-pluripotent cells.

[0056] As used herein, the terms "embryonic stem," "ES" cell, and "ESC" refer to pluripotent stem cells obtained from early embryos. In some embodiments, the terms exclude stem cells involved in the destruction of human embryos; i.e., ESCs are obtained from previously established ESC lines.

[0057] The term "induced pluripotent stem cells" or "iPSCs" refers to a type of pluripotent stem cell that is inductively prepared from a non-pluripotent cell, such as an adult somatic cell, a partially differentiated cell, or a terminally differentiated cell, such as a fibroblast, a cell of the hematopoietic system, a muscle cell, a neuron, an epidermal cell, etc., by introducing or contacting the cell with one or more reprogramming factors. Methods for generating iPSCs are known in the art and include, for example, inducing expression of one or more genes (such as, but not limited to, SOX2 (Gene ID: 6657), KLF4 (Gene ID: 9314), c-MYC (Gene ID: 4609, POU5F1 / OCT4 (Gene ID: 5460) in combination with NANOG (Gene ID: 79923) and / or LIN28 / LIN28A (Gene ID: 79727)). Reprogramming factors can be delivered by various means (e.g., viral, non-viral, RNA, DNA, or protein delivery); alternatively, endogenous genes can be activated, for example, by using CRISPR tools to reprogram non-pluripotent cells into PSCs.

[0058] Methods for inducing PSCs to differentiate into various lineage cells are well known in the art.For example, methods for inducing PSCs to differentiate into dendritic cells are described in Slukvin et al., J Imm. (2006) 176:2924-32; and Su et al., Clin Cancer Res. (2008) 14(19):6207-17; and Tseng et al., Regen Med. (2009) 4(4):513-26.Methods for inducing PSCs to hematopoietic progenitor cells, myeloid cells and T lymphocytes are described in, for example, Kennedy et al., Cell Rep. (2012) 2:1722-35.

[0059] In addition to incorporating a transgene of interest into the STEL site, the genetically modified human cells (e.g., iPSCs or ESCs) herein can be further engineered to improve their therapeutic potential, including making them less immunogenic in allogeneic cell therapy, by knocking out one or more of their MHC class I genes (e.g., B2M genes). The human cells can optionally contain a safety switch signal (e.g., a suicide gene) at the STEL site.

[0060] Methods for isolating and maintaining PSCs, such as ESCs and iPSCs, are well known in the art.See, for example, Thomson et al., Science (1998) 282(5391):1145-7; Hovatta et al., Human Reprod. (2003) 18(7):1404-09; Ludwig et al., Nature Methods (2006) 3:637-46; Kennedy et al., Blood (2007) 109:2679-87; Chen et al., Nature Methods (2011) 8:424-9; and Wang et al., Stem Cell Res. (2013) 11(3):1103-16.

[0061] In some embodiments, either the PSCs or PSC-derived mature or intermediate cell types can be further engineered (before, simultaneously with, or after the engineering of the STEL site) for additional functions, such as, for example, payload delivery and safety control.

[0062] In some embodiments, PSCs can be differentiated into the cell type of interest for cell therapy. In some embodiments, the cells that are genetically engineered are already differentiated into the cell type of interest. Non-limiting examples of differentiated cell types are described below.

[0063] A. Immune cells The genetically modified human cells can be immune cells such as PSC-derived immune cells, e.g., lymphoid cells and lymphoid progenitor cells (e.g., T cells and T cell progenitors (regardless of any particular T cell subtype, including, e.g., regulatory T cells and T effector cells), B cells and NK cells), myeloid and myeloid progenitor cells (e.g., granulocytes, monocytes / macrophages and microglial cells), and dendritic cells and dendritic progenitor cells (e.g., myeloid dendritic cells and plasmacytoid dendritic cells). In some embodiments, the genetically modified cells are T cells or CAR T cells expressing a chimeric antigen receptor (CAR). The genetically modified immune cells can also express immunoregulatory transgenes, such as those described herein.

[0064] Genetically engineered immune cells, such as immunosuppressive immune cells (e.g., regulatory T cells and immunosuppressive macrophages), can be transplanted into patients with autoimmune diseases such as, but not limited to, rheumatoid arthritis, multiple sclerosis, chronic lymphocytic thyroiditis, insulin-dependent diabetes mellitus, myasthenia gravis, chronic ulcerative colitis, ulcerative colitis, Crohn's disease, inflammatory bowel disease, Goodpasture's syndrome, systemic lupus erythematosus, systemic vasculitis, scleroderma, autoimmune hemolytic anemia, and autoimmune thyroid disease. Immune cell-based therapy can also be used to treat graft rejection in transplants, including the treatment of transplant-related conditions such as fibrosis.

[0065] B. Nerve cells The genetically modified human cells may be neural cells such as PSC-derived neural cells, including, but not limited to, neurons and neuronal progenitor cells (regardless of any particular neuronal subtype, including, for example, dopaminergic neurons, cortical neurons, spinal or oculomotor neurons, enteric neurons, interneurons, and trigeminal or sensory neurons), microglia and microglial progenitor cells, glial cells and glial progenitor cells (regardless of any particular glial subtype, including, for example, oligodendrocytes, astrocytes, oligodendrocyte precursor cells of particular interest that can become astrocytes and oligodendrocytes, and bipotent glial precursors), placode-derived cells, Schwann cells.

[0066] Genetically engineered neurons can be transplanted into patients with neurodegenerative diseases, including, but not limited to, Parkinson's disease, Alzheimer's disease, dementia, epilepsy, Lewy body syndrome, Huntington's disease, spinal muscular atrophy, Friedreich's ataxia, amyotrophic lateral sclerosis, Batten disease, and multiple system atrophy, among others.

[0067] For many of these diseases, PSCs can be initially directed to adopt a progenitor neuronal fate by dual SMAD inhibition (Chambers et al., Nat Biotechnol. (2009) 27(3):275-80). Because progenitor neurons adopt anterior characteristics, the absence of additional signals provides anterior / forebrain cortical cells. Caudalizing signals can be blocked to prevent paracrine signals that would otherwise result in cultures with more posterior characteristics (e.g., XAV939 can block WNT, and SU5402 can block FGF signaling). Dorsal cortical neurons can be generated by blocking SHH activation, while ventral cortical neurons can be generated by SHH activation. More caudal cell types, such as serotonergic neurons or spinal motor neurons, can be generated by caudalizing the culture through the addition of FGF and / or WNT signals. For some cell types, retinoic acid (another caudalizing factor) can be added to posteriorize the culture. The production of glial cell types may generally follow the same patterning of primary neural cells prior to expansion in FGF2- and / or EGF-containing medium. PNS cell types may follow the same general principles, but with well-timed WNT signaling early in the differentiation process.

[0068] Genetically modified neural cells can be introduced into patients through a cannula placed in the damaged tissue in question. The cell preparation can be placed in a support medium and loaded into a syringe or pipette-like device that can precisely deliver the preparation. The cannula can then be placed into the patient's nervous system, usually using a stereotactic method to precisely target delivery. The cells can then be released into the tissue at a suitable rate.

[0069] C. Cardiovascular cells The genetically modified human cells can be PSC-derived cardiovascular cells, e.g., cells of the cardiovascular system such as cardiomyocytes, cardiac fibroblasts, cardiac smooth muscle cells, epicardial cells, cardiac endothelial cells, Purkinje fibers, and pacemaker cells.

[0070] In some embodiments, the cardiomyocytes prepared, enriched or isolated by the method of the present disclosure are derived from PSCs, such as iPSCs.For example, as shown in Kattman et al., Cell Stem Cell (2011) 8(2):228-40, and as shown in WO2016131137, WO2018098597 and U.S. Patent No. 9,453,201, there are many methods for differentiating PSCs into cardiomyocytes.Any suitable method in the art can be used together with the method of the present disclosure to obtain PSC-derived cardiomyocytes modified to express transgenes in STEL.

[0071] In some embodiments, PSCs are incubated in one or more cardiac differentiation mediums.For example, this medium can contain varying concentrations of bone morphogenetic protein (BMP; for example, BMP4) and activin (for example, activin A).The concentration of differentiation factor can be titrated to determine the optimal concentration required to achieve desired cardiomyocyte differentiation.

[0072] In some embodiments, the differentiated cardiomyocytes express one or more of cardiac troponin T (cTnT) and / or myosin light chain 2v (MLC2v). In some embodiments, the immature cardiomyocytes express one or more of troponin T, cardiac troponin I, alpha actinin, and / or beta myosin heavy chain.

[0073] D. Metabolic cells Genetically modified human cells can be involved in human metabolic system.For example, said cells can be gastrointestinal cells (for example, hepatocytes, bile duct cells and pancreatic β cells), hematopoietic cells and central nervous system cells (for example, pituitary hormone-releasing cells).For example, to generate pituitary hormone-releasing cells, PSCs are cultured with BMP4 and SB431542 (blocking activin signal transduction), and then SHH / FGF8 and FGF10 are added; then, cells are subjected to SHH / FGF8 and FGF10 only for a prolonged period, and then FGF8 or BMP (or both), and cells are induced to become specific hormone-releasing cells.For example, see Zimmer et al., Stem Cell Reports (2016) 6:858-72.

[0074] E. Cells of the ocular system The genetically modified human cell can be an ocular cell.For example, the cell can be a retinal progenitor cell, a retinal pigment epithelium (RPE) progenitor cell, an RPE cell, a neural retinal progenitor cell, a photoreceptor progenitor cell, a photoreceptor cell, a bipolar cell, a horizontal cell, a ganglion cell, an amacrine cell, a Müller glial cell, a cone cell, or a rod cell.The method for differentiating iPSCs into RPE cells is described, for example, in WO 2017 / 044483.The method for isolating RPE cells is described, for example, in WO 2017 / 044488.The method for differentiating iPSCs into neural retinal progenitor cells is described, for example, in WO 2019 / 204817.The method for identifying and isolating retinal progenitor cells and RPE cells is described, for example, in WO 2011 / 028524.

[0075] V. Pharmaceutical Compositions and Uses The genetically engineered cells described herein can be provided in a pharmaceutical composition comprising the cells and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier can be a cell culture medium that optionally does not contain any animal-derived components. For storage and transportation, the cells can be cryopreserved at <-70°C (e.g., on dry ice or in liquid nitrogen). Before use, the cells can be thawed and diluted in a sterile cell culture medium that supports the desired cell type.

[0076] Cells can be administered to a patient systemically (e.g., by intravenous injection or infusion) or locally (e.g., by direct injection into local tissues, such as the heart, brain, and sites of damaged tissue). Various methods are known in the art for administering cells to a patient's tissues or organs, including, but not limited to, intracoronary, intramyocardial, intracardiac, or intracranial administration.

[0077] A therapeutically effective number of genetically engineered cells is administered to a patient. As used herein, the term "therapeutically effective" refers to the number of cells or the amount of pharmaceutical composition that, when administered to a human subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, prevent, and / or delay the onset or progression of the symptom(s) of the disease, disorder, and / or condition. Those skilled in the art will understand that a therapeutically effective amount is typically administered by a dosing regimen comprising at least one unit dose.

[0078] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure have the meanings commonly understood by those skilled in the art. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein may also be used in practicing or testing this disclosure. In the case of conflict, the present specification, including definitions, will control. Generally, the nomenclature used in and techniques related to cell and tissue culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medical and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. Furthermore, unless otherwise required by context, singular terms include pluralities, and plural terms include the singular. Throughout this specification and embodiments, the words "have" and "comprise" or variations such as "has," "having," "comprises," or "comprising" are understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although several documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.

[0079] In order that this invention may be better understood, the following examples are set forth. These examples are illustrative only and are not intended to limit the scope of the invention in any way. [Example]

[0080] Example In the following examples, gene editing was performed as follows.

[0081] Guide RNA and validation In the following experiments, CRISPR-Cas9 gene editing was performed to insert a transgene into the intended STEL site. Three guide RNAs (gRNAs) were computationally designed to target the 3'UTR of GAPDH adjacent to the stop codon. Five gRNAs were computationally designed to target the 3'UTR of RPL13A adjacent to the stop codon. These gRNAs were designed to have few off-target sites and high predicted activity against the target sequence.

[0082] To test gRNA cleavage efficiency, gRNA complexed with Cas9 nuclease was separately delivered as ribonucleoprotein (RNP) into human PSCs by nucleofection. Seventy-two hours after nucleofection, gDNA was extracted from each pool of nucleofected cells. The region surrounding the intended cleavage site in the GAPDH or RPL13A locus was amplified using the following primers: GAPDH F: 5'-TGGACCTGACCTGCCGTCTA-3' (SEQ ID NO: 1) and GAPDH R: 5'-CCCCAGACCCTAGAATAAGACAGG-3' (SEQ ID NO: 2) (amplicon size = 619 bp) and RPL13A F: 5'-AACAGTTGCATTATGATATGCCCAG-3' (SEQ ID NO: 3), RPL13A R: 5'-TGCTTTCAAGCAACTTCGGGA-3' (SEQ ID NO: 4) (amplicon size = 696 bp) was amplified by PCR using The PCR product was purified and ligated with the following primers: GAPDH: 5'-AAAACCTGCCAAATATGATGACA-3' (SEQ ID NO: 5), and RPL13A: 5'-AAGTACCAGGCAGTGACAGC-3' (SEQ ID NO: 6) Sanger sequencing was performed using

[0083] The overall cleavage efficiency of each gRNA was determined by Inference of CRISPR Edits (ICE) analysis by comparing Sanger sequencing chromatograms from unedited cells with those from each gRNA condition. ICE analysis determined that the GAPDH gRNA, with the RNA sequence 5'-CUUCCUCUUGUGCUCUUGCU-3' (SEQ ID NO: 7), and the RPL13A gRNA, with the RNA sequence 5'-GGAAGGGCAGGCAACGCAUG-3' (SEQ ID NO: 8), had the highest relative cleavage efficiency of all gRNAs tested for each locus.

[0084] Knock-in production Chemically modified gRNAs for each selected STEL site were resuspended in nuclease-free TE buffer provided by the manufacturer and nucleofected into human iPSCs as RNPs in complex with S. pyogenes Cas9 nuclease 2NLS (Synthego) and GAPDH- or RPL13A-targeting donor plasmids using a Lonza 4D Nucleofector for transfection. TM X-Unit was used (P3 Nucleofector solution and Nucleofector program CA-137). Individual colonies were then transferred under sterile conditions by pick-to-keep to 96-well plates coated with recombinant truncated vitronectin and expanded for genetic screening and freezing (in Essential 8 complete medium + 10% DMSO). Care was taken to limit the number of passages during characterization and screening to provide the lowest possible number of passages.

[0085] Clones were screened for relevant knock-ins by 5' and 3' junction PCR using one primer set per pair external to the targeting construct and one primer per pair internal to the targeting construct. Clones positive for both the 5' and 3' junction PCR products were expanded and cryopreserved. gDNA from each 5' and 3' positive clone was used as a template to generate PCR products (including homology arms) that fully spanned the integrated construct. These PCR products were then used to Sanger sequence the length of the integrated construct in its genomic context.

[0086] Cell Culture Platform iPSCs were maintained using Essential 8 Medium (Thermo Fisher Scientific; catalog #A1517001) and recombinant human vitronectin (VTN-N) (N-terminally truncated vitronectin polypeptide). Y-27632 ROCK inhibitor was used during single-cell passaging and cloning procedures. iPSCs were fed daily and double-fed once a week. Cell cultures were maintained at 37°C and 5% CO2. No significant changes in morphology were observed between the knockout clones and the parental wild-type cells during culture.

[0087] Clonality Immediately after electroporation for the desired genetic modification, iPSCs were plated at low density to ensure that single cells adhered and proliferated independently. Individual cells were allowed to grow into colonies. Once the colonies reached an optimal size, each individual colony was picked and placed in a separate well. Each clone was sequenced for gene editing events and G-banded karyotyping was performed.

[0088] Clonal HLA-G protein characterization Flow cytometry was performed using a pan-HLA-G antibody (clone 4H84) from BD Biosciences to confirm cell surface expression of HLA-G. Secretion of HLA-G6 and HLA-G5 into the cell culture medium was assessed by Western blot using an HLA-G5 / G6-specific antibody (clone 5A6G7) from Thermo Fisher Scientific. Specifically, 4 mL of medium was concentrated and reduced to 100 μl and then tested by Western blot for the presence of HLA-G6 and -G5.

[0089] Example 1: Identification of STEL sites In this study, we evaluated single-cell RNA sequencing (scRNA-seq) data collected from human PSCs and their differentiated derivatives to conduct a site search for STEL candidates. We hypothesized that putative STEL sites could be discovered using scRNA-seq data from multiple cell types. This approach enabled the direct interrogation of hundreds of thousands of available individual transcriptomes. In the current study, single-cell RNA-seq data were collected from PSCs and three PSC-derived cell types: microglia, dopaminergic neurons, and ventricular cardiomyocytes. Data were collected from 267,058 cells with a transcriptomic depth of 28,387 unique genes. The first key characteristic of STEL sites is ubiquity of expression. Genes were ranked by ubiquity of expression by first binarizing the transcript count data and then summing across cells. The sum for each gene was then divided by the total number of cells to obtain a percentage reflecting the gene's prevalence in all data.

[0090] A total of 98 genes had a proportional representation of greater than 99% and were subsequently selected for further analysis. The selected genes were then sorted by the standard deviation of the non-binarized expression data. Genes with a standard deviation higher than 1 were removed. The remaining 94 genes were then sorted by mean expression, which were primarily ribosomal genes but also included some known housekeeping genes such as GAPDH and ACTB (Table 1). [Table 1-1] [Table 1-2] [Table 1-3]

[0091] Several of these genes (GAPDH, RPLP0, RPL7, and RPL13A) are visualized in the UMAP plot shown in Figure 1. These four loci were selected as STELs for the experiments described below. Other criteria we considered when finalizing the selection of STEL sites from those listed above included genomic distance from cancer genes (as far as possible), published studies for proof-of-concept, and the number of pseudogenes at the locus (fewer is better to minimize off-target primer binding). The RNA-seq methods described above can be used to discover STEL sites, but can also be used to disqualify potential sites.

[0092] Furthermore, our scRNAseq analysis of gene expression indicates that not all endogenous genes commonly used as controls for gene expression analysis are STEL sites. For example, the genes encoding peptidyl prolyl isomerase A (PPIA; or cyclophilin A), tubulin β polypeptide (TUBB), and β-2-microglobulin (B2M) are generally considered reliable housekeeping genes whose expression levels are used to normalize references for RT-PCR assays of mammalian cells. However, based on our data, these genes are not STEL sites because their expression levels are significantly more variable across cell types than the STEL genes listed in Table 1 above. Similar observations were made using other housekeeping genes commonly used as normalization controls for RNA analysis, such as the genes encoding ALAS1, GUSB, HMBS, HPRT, SDHA, TBP, and TFRC. In contrast, ribosomal protein genes, such as the RPL13A and RPLP0 genes, have robust expression across cell types, making them suitable STEL sites for transgene integration.

[0093] In order to reduce the risk of abnormal integration, STEL is preferably not adjacent to oncogene or tumor suppressor gene.For example, TUBB gene is near the MDC1 gene, which is a mediator of DNA repair and a known tumor suppressor gene.For this additional reason, TUBB gene is not selected as STEL site.STEL site can have splicing variants, if any, and an appropriate distance from adjacent genes that are amenable to gene editing.It may also be preferable that STEL site does not have many pseudogenes, which may reduce transgene targeting efficiency due to the sequence homologous to the targeted gene.

[0094] Example 2: Expression of EGFP at STEL sites in PSCs Based on the above studies, we selected four STEL sites (GAPDH, RPL13A, RPLP0, and RPL7) for testing payload candidate expression. The expression cassettes of the payload candidates were under the control of the endogenous STEL promoter. Consequently, expression of the payload candidates was linked to expression of the endogenous STEL gene. If the STEL promoter remained active in the cells, expression of the associated payload transgene was expected to be sustained and constitutive. We used CRISPR-cas9 gene editing to insert an enhanced green fluorescent protein (EGFP) expression construct into the GAPDH, RPL13A, RPLP0, or RPL7 locus (Figure 2). The EGFP coding sequence is shown below. [Table 2]

[0095] The inserted EGFP transgene was linked in-frame to the endogenous STEL gene by a DNA sequence encoding the PQR sequence (Lo et al., supra) (Figure 2). The PQR sequence is a modified 2A self-cleaving peptide that causes ribosomal skipping during translation, resulting in bicistronic expression of EGFP and the endogenous STEL gene once the PQR sequence is cleaved. The PQR nucleotide and amino acid sequences are shown below. [Table 3]

[0096] Each PQR / EGFP insertion construct was also flanked by an 800-bp left and an 800-bp right homology arm containing sequences homologous to the endogenous STEL locus, which allowed integration of the targeting construct in the 3'UTR of the STEL gene immediately after the last amino acid codon.

[0097] The sequences of the left and right homology arms for targeting the GAPDH locus are shown below as SEQ ID NOs: 12 and 13, respectively. [Table 4]

[0098] The sequences of the left and right homology arms for targeting the RPL13A locus are shown below as SEQ ID NOs: 14 and 15, respectively. [Table 5-1] [Table 5-2]

[0099] The sequences of the left and right homology arms for targeting the RPLP0 locus are shown below as SEQ ID NOs: 16 and 17, respectively. [Table 6]

[0100] Flow cytometry analysis was performed on either undifferentiated, non-edited PSCs, undifferentiated, GAPDH-targeted, EGFP-edited PSCs, or undifferentiated, RPL13A-targeted, edited PSCs (Figure 3). For both GAPDH-targeted and RPL13A-targeted, EGFP-edited PSC lines, we tested one homozygous targeted line (with gene edits in both alleles) and one heterozygous targeted line (with gene edits in one allele). The data show higher EGFP fluorescence signals from all four edited PSC lines compared to non-edited PSC lines. These results indicate that insertion of EGFP constructs at the GAPDH and RPL13A loci enabled high levels of transgene expression in edited PSCs.

[0101] Flow cytometry analysis was performed on three different undifferentiated clonal PSC lines: undifferentiated, unedited PSCs or RPLP0-targeted, EGFP-edited PSCs (Figure 4). All three RPLP0-targeted, EGFP PSC lines were heterozygous, with gene editing in one allele. The data show higher EGFP fluorescence signals from all three edited PSC lines compared to unedited PSC lines. These results indicate that insertion of the EGFP construct at the RPLP0 locus enabled high levels of transgene expression in edited PSCs.

[0102] qPCR analysis was performed on RNA recovered from unedited PSCs and GAPDH-targeted EGFP-edited PSCs on a weekly basis for cell lines cultured for 8 weeks (Figure 5). Cell lines were routinely passaged 2-3 times each week on average. The average Cq range of 15-20 cycles indicates very high amounts of target RNA and transgene expression. Cq values ​​are inversely related to the amount of target RNA in the sample; the lower the Cq value, the higher the amount of transgene expression. Both heterozygous GAPDH-targeted EGFP PSC lines (with gene edits in one allele) and homozygous GAPDH-targeted EGFP PSC lines (with gene edits in both alleles) showed high transgene expression compared to unedited PSC lines that did not express EGFP. The homozygous GAPDH-targeted EGFP PSC lines showed slightly lower Cq values ​​than the heterozygous GAPDH-targeted EGFP PSC lines, indicating higher transgene expression from the homozygous GAPDH-targeted EGFP PSC lines. Both edited PSC lines expressed high levels of EGFP expression every week for up to 8 weeks, indicating that high levels of transgene expression were maintained after up to 8 weeks of conventional PSC culture.

[0103] qPCR analysis was performed on RNA recovered from unedited PSCs and RPL13A-targeted EGFP-edited PSCs on a weekly basis for cell lines cultured for 8 weeks (Figure 6). Cell lines were routinely passaged 2-3 times each week on average. An average Cq range of 15-25 cycles indicates very high amounts of target RNA and transgene expression. Cq values ​​are inversely related to the amount of target RNA in the sample; the lower the Cq value, the higher the amount of transgene expression. Both heterozygous RPL13A-targeted EGFP PSC lines (with gene edits in one allele) and homozygous RPL13A-targeted EGFP PSC lines (with gene edits in both alleles) showed higher transgene expression compared to unedited PSC lines that did not express EGFP. The heterozygous RPL13A-targeted EGFP PSC lines showed slightly lower Cq values ​​than the homozygous RPL13A-targeted EGFP PSC lines, indicating higher transgene expression from the heterozygous RPL13A-targeted EGFP PSC lines. Both edited PSC lines expressed high levels of EGFP every week for up to 8 weeks, indicating that high levels of transgene expression were maintained after up to 8 weeks of conventional PSC culture.

[0104] Flow cytometry analysis was also performed on non-edited PSCs, GAPDH-targeted EGFP-edited PSCs, and RPL13A-targeted EGFP-edited PSCs differentiated into dopaminergic neurons at day 16 (Figure 7) (see, e.g., Chambers et al., supra). For both GAPDH-targeted and RPL13A-targeted EGFP-edited PSC lines, we tested one homozygous targeted line (with gene edits in both alleles) and one heterozygous targeted line (with gene edits in one allele).

[0105] The data show that after 16 days of differentiation into dopaminergic neurons, all four edited PSC lines showed higher EGFP fluorescence signals compared to unedited PSC lines. These results indicate that insertion of EGFP constructs at the GAPDH and RPL13A loci enabled high levels of transgene expression in edited PSCs and that high levels of transgene expression were maintained after lineage-directed differentiation of the edited PSCs.

[0106] Flow cytometry analysis was also performed on unedited PSCs, heterozygous GAPDH-targeted EGFP lines (with gene edits in one allele) that did or did not differentiate into cardiomyocytes at day 12, and heterozygous RPL13A-targeted EGFP lines (with gene edits in one allele) that did or did not differentiate into cardiomyocytes at day 12 (Figure 8) (see, e.g., Lian et al., Nat. Protoc. (2013) 8(1):162-75). The data show higher EGFP fluorescence from both the GAPDH-targeted EGFP lines and the RPL13A-targeted EGFP lines compared to unedited PSC lines after 12 days of differentiation into cardiomyocytes. The level of fluorescence in the differentiated edited lines was slightly lower compared to the undifferentiated edited lines, but remained high. The results indicate that high levels of transgene expression were maintained after directed differentiation of edited PSCs into the cardiomyocyte lineage.

[0107] Example 3: Expression of HLA-G6 at the GAPDH and RPL13A loci in iPSCs In this study, constructs expressing HLA-G6 were edited into either the GAPDH locus or the RPL13A locus in iPSCs. The HLA-G6 coding sequence is shown below. [Table 7-1] [Table 7-2]

[0108] The inserted HLA-G6 transgene was linked in-frame to the endogenous housekeeping gene by the PQR sequence as described above (Figure 9). Each PQR / HLA-G6 insertion construct was also flanked by an 800-bp left homology arm and an 800-bp right homology arm containing sequences homologous to the endogenous STEL locus (either GAPDH or RPL13A), as described above.

[0109] Secretion of HLA-G6 into the cell culture medium was assessed by Western blot using an HLA-G5 / G6-specific antibody (clone 5A6G7) from Thermo Fisher Scientific. Western blot analysis was performed on cell culture supernatants from unedited wild-type PSCs, control JEG-3 choriocarcinoma cells (derived from human placenta, in which HLA-G is normally expressed), and the GAPDH-targeted HLA-G6 PSC line (Figure 10). The primary antibody used was specific for soluble HLA-G isoforms, such as HLA-G5 and HLA-G6. The predicted protein size of HLA-G6 is approximately 30 kDa. The data show that HLA-G6 was detected at comparable levels in the cell culture supernatants of GAPDH-targeted HLA-G6-edited PSCs and control JEG-3 cells, but was absent in the cell culture supernatant of unedited PSCs. These results indicate that insertion of the HLA-G6 construct at the GAPDH locus enabled edited PSCs to secrete high levels of HLA-G6.

[0110] We also performed a fluorescence resonance energy transfer (FRET) detection assay on cell culture supernatants from unedited wild-type PSCs, control JEG-3 cells, and GAPDH-targeted HLA-G6 PSCs (Figure 11). FRET involves the transfer of energy between two fluorophores, a donor and an acceptor, when they come into close proximity. The donor molecule was linked to a pan-HLA-G antibody (BD Biosciences; clone 4H84), and the acceptor molecule was linked to an antibody that detects soluble HLA-G isoforms, including HLA-G5 and HLA-G6 (Thermo Fisher Scientific; clone 5A6G7). Both antibodies bind to secreted HLA-G6 protein, allowing FRET to occur between the donor and acceptor molecules. The higher the FRET signal, the greater the amount of protein detected. The data show a high FRET signal in the cell culture supernatant of control JEG-3 cells and an even higher FRET signal from the GAPDH-targeted HLA-G6-edited PSCs, but no signal from the unedited PSCs. These results confirm that insertion of the HLA-G6 construct at the GAPDH locus enabled the edited PSCs to secrete high levels of HLA-G6.

[0111] In another study, FRET detection assays were performed on cell culture supernatants of unedited PSCs and RPL13A-targeted HLA-G6 PSC lines ( FIG. 12 ). The data show high FRET signals in cell culture supernatants of RPL13A-targeted HLA-G6-edited PSCs, but little signal from unedited PSCs. These results indicate that insertion of the HLA-G6 construct at the RPL13A locus also enabled edited PSCs to secrete high levels of HLA-G6.

[0112] We used CRISPR / Cas9 gene editing to knock out the B2M gene in both GAPDH-targeted and RPL13A-targeted HLA-G6 lines, generating three different B2M knockout (KO) clones for each HLA-G6-edited PSC line. Flow cytometry analysis was performed on all six edited clones using a pan-HLA-G antibody (BD Biosciences; clone 4H84) (Figure 13). Analysis was repeated after one week of conventional PSC culture and eight weeks of conventional PSC culture. The data show higher HLA-G expression in edited PSC lines compared to unedited PSC lines, and HLA-G expression is maintained across all edited clonal cell lines up to eight weeks of conventional PSC culture, even after B2M gene knockout. HLA-G expression from GAPDH-targeted PSC lines was higher than that from RPL13A-targeted PSC lines, indicating higher transgene expression from the GAPDH locus.

[0113] Example 4: Expression of anti-τscFv at the GAPDH locus in PSCs In this study, a construct expressing a single-chain variable fragment (scFv) antibody against human tau (Ising et al., J. Exp. Med. (2017) 214(5):1227-1238) was inserted into the GAPDH locus. The anti-tau scFv insertion construct consisted of a secretory signal peptide (SP), the light chain variable region (VL) and heavy chain variable region (VH) of the anti-tau antibody HJ8.5 linked by an S(GGGGS)3 (SEQ ID NO: 19) peptide linker (PL) (WO 2016 / 126993 and WO 2014 / 008404), and a sequence encoding a human influenza hemagglutinin (HA) peptide tag (FIG. 14). The coding sequence for the anti-τscFv is shown below, where the coding sequence for the secretory signal peptide is boldfaced and underlined, the coding sequence for the VL is italicized, the coding sequence for the peptide linker is boldfaced, the coding sequence for the VH is underlined, and the coding sequence for the HA tag is boldfaced and italicized. [Table 8-1] [Table 8-2]

[0114] A TGA stop codon was incorporated after the transgene coding sequence to terminate translation. Expression of the scFv was coupled to that of GAPDH by the PQR sequence, as described above. Each PQR / anti-τ scFv insert construct was also flanked by an 800 bp left homology arm and an 800 bp right homology arm, as described above.

[0115] Western blot analysis was performed on cell culture supernatants of non-edited PSCs, GAPDH-targeting anti-τscFv PSC lines (either straight supernatant or enriched by anti-HA agarose immunoprecipitation), and cell lysates of GAPDH-targeting anti-τscFv PSC lines (Figure 15). The primary antibody used was an anti-HA monoclonal antibody that recognizes the nine amino acid sequence YPYDVPDYA (SEQ ID NO: 21) derived from the HA peptide tag. The predicted protein size of anti-τscFv is approximately 30 kDa.

[0116] The data show that anti-τscFv was detected in the cell culture supernatants of both straight and concentrated GAPDH-targeted anti-τscFv-edited PSC lines and in the cell lysates of GAPDH-targeted anti-τscFv-edited PSC lines, but was absent from the cell culture supernatants of unedited PSC lines. These results indicate that insertion of the anti-τscFv construct at the GAPDH locus enabled edited PSCs to secrete high levels of scFv.

[0117] Example 5: Expression of the RapaCasp9 cellular suicide switch at the GAPDH locus in PSCs In this study, two different constructs (Stavrou et al., Mol. Ther. (2018) 26(5):1266-76) that together comprise the rapamycin-inducible human caspase 9 system (RapaCasp9) cellular suicide switch were inserted into each allele of the GAPDH locus. One RapaCasp9 construct consisted of a sequence encoding the FRB (FKBP12 rapamycin-binding) domain of mTOR linked by an SGGGS (SEQ ID NO: 22) peptide linker (L1) to a truncated caspase 9 gene (truncCasp9) with its CARD domain removed. The other RapaCasp9 construct consisted of a sequence encoding the FKBP12 (FK506-binding protein 12) gene linked by an SGGGS (SEQ ID NO: 22) peptide linker (L2) to a truncated caspase 9 gene (truncCasp9) with its CARD domain removed (Figure 16). Addition of the drug rapamycin allows heterodimerization of FRB and FKBP12, which subsequently leads to homodimerization of cleaved caspase-9 and induction of apoptosis.

[0118] The coding sequence for the FRB-L1-truncCasp9 component of RapaCasp9 is shown below, where the coding sequence for FRB is shown in bold, the coding sequence for the peptide linker (L1) is underlined, and the coding sequence for cleaved caspase 9 is shown in italics. [Table 9]

[0119] The coding sequence for the FKBP12-L2-truncCasp9 component of RapaCasp9 is shown below, where the coding sequence for FKBP12 is shown in bold, the coding sequence for the peptide linker (L2) is underlined, and the coding sequence for cleaved caspase 9 is shown in italics. [Table 10-1] [Table 10-2]

[0120] A TGA stop codon was incorporated after each transgene coding sequence to terminate translation. Expression of both the FRB-L1-truncCasp9 and FKBP12-L2-truncCasp9 components of RapaCasp9 was linked to GAPDH expression by the PQR sequence, as described above. Each PQR / RapaCasp9 construct was also flanked by an 800-bp left homology arm and an 800-bp right homology arm, as described above.

[0121] The GAPDH-targeted RapaCasp9 PSC line was treated with either 5 nM or 10 nM rapamycin for 1, 2, 4, or 24 hours, and cells were harvested for flow cytometry analysis after each time point (Figure 17). The primary antibody used was anti-human / mouse cleaved caspase 3 conjugated to an Alexa Fluor® 488 secondary antibody. The primary antibody detects human and mouse caspase 3 cleaved at Asp175. Caspase 3 is an executioner caspase that functions downstream of the initiator caspase, caspase 9, in the apoptotic cascade. Human procaspase 3 is normally an inactive homodimer. Upon induction of apoptosis by either cellular stress or activation, it undergoes proteolysis into the cleaved caspase 3 subunit. The data show that after treatment of GAPDH-targeted RapaCasp9 PSC lines with either 5 nM or 10 nM rapamycin, cleaved caspase 3 staining is readily detectable 4 hours after treatment, and almost all cells (>99%) stain for cleaved caspase 3 24 hours after treatment. For edited PSCs not treated with rapamycin, there was negligible detection of cleaved caspase 3. These results indicate that biallelic insertion of the FRB-L1-truncCasp9 and FKBP12-L2-truncCasp9 RapaCasp9 constructs at the GAPDH locus caused edited PSCs to undergo apoptosis upon induction with rapamycin.

[0122] Example 6: Expression of PD-L1 and CD47 immunoregulatory molecules at the GAPDH locus in PSCs In this study, two different constructs, each containing both an immune modulator and an HSV-TK.007 (herpes simplex thymidine kinase) cellular suicide switch, were inserted into each allele of the GAPDH locus. The PD-L1-based construct consisted of the coding sequence for PD-L1 (programmed death ligand 1) linked via an internal ribosome entry site (IRES) sequence to the coding sequence for HSV-TK.007 linked via a P2A sequence to the coding sequence for puroR (puromycin resistance gene). The CD47-based construct consisted of the coding sequence for CD47 linked via an IRES sequence to the coding sequence for HSV-TK.007. Upon addition of ganciclovir, cells containing these constructs convert ganciclovir into a toxic nucleotide analog, causing DNA replication failure and cell death in actively proliferating cells.

[0123] The coding sequences for the PD-L1 based constructs are shown below, with the coding sequence for PD-L1 in bold, the coding sequence for the IRES underlined, the coding sequence for HSV-TK.007 in italics, the coding sequence for P2A (including the GSG linker) in bold and underlined, and the coding sequence for puroR in standard script. [Table 11-1] [Table 11-2]

[0124] The coding sequences for the CD47-based constructs are shown below, where the coding sequence for CD47 is shown in bold, the coding sequence for the IRES is underlined, and the coding sequence for HSV-TK.007 is shown in italics. [Table 12-1] [Table 12-2]

[0125] A stop codon was incorporated after each transgene coding sequence to terminate translation. Expression of the PD-L1-based construct was linked to GAPDH expression by the PQR sequence, as described above, and flanked by an 800 bp left homology arm and an 800 bp right homology arm, as described above. Expression of the CD47-based construct was linked to GAPDH expression by the P2A sequence, where the GSG linker is shown in bold in the sequence below, and flanked by an 800 bp left homology arm and an 800 bp right homology arm, as described above. [Table 13]

[0126] Flow cytometry analysis was performed on unedited PSCs or GAPDH-targeted PSCs that contained one allele edited with a PD-L1-based construct and the other allele edited with a CD47-based construct (Figure 18). The data show detection of dual PD-L1 and CD47 co-staining in GAPDH-targeted PSCs, but no staining in unedited PSCs, indicating that biallelic insertion of PD-L1- and CD47-based constructs at the GAPDH locus enabled edited PSCs to express PD-L1 and CD47.

[0127] Example 7: Expression of CSF1 at the GAPDH locus in PSCs In this study, a construct containing the coding sequence for CSF1 (colony-stimulating factor 1) was inserted into either one or both alleles of the GAPDH locus. CSF1 is a cytokine that regulates macrophage survival, differentiation, and function. The coding sequence for CSF1 is shown below. [Table 14-1] [Table 14-2]

[0128] A TAG stop codon was incorporated after the transgene coding sequence to terminate translation. Expression of CSF1 was linked to that of GAPDH by the PQR sequence as described above. Each PQR / CSF1 insertion construct was also flanked by an 800 bp left homology arm and an 800 bp right homology arm as described above.

[0129] An ELISA immunoassay was performed on cell culture supernatants of non-edited PSCs and three different GAPDH-targeted CSF1 PSC lines (Figure 19). The data show that secreted CSF1 was detected in the cell culture supernatants of all three GAPDH-targeted CSF1-edited PSC lines, but was absent from the cell culture supernatant of the non-edited PSC line. These results indicate that insertion of the CSF1 construct at the GAPDH locus enabled edited PSCs to secrete readily detectable levels of CSF1.

[0130] Example 8: Transgene silencing of PD-L1 at the AAVS1 locus in differentiated PSCs In this study, we used CRISPR-Cas9 gene editing to insert a construct expressing PD-L1 at the AAVS1 safe harbor locus (Figure 20A). The insertion construct contained an external EF1a promoter to drive expression of the transgene construct. Additionally, HSV-TK, a suicide gene that can be induced to eliminate proliferating cells by small molecule treatment, was linked to PD-L1 by the P2A sequence, which allows bicistronic expression of both PD-L1 and HSV-TK upon P2A cleavage. The insertion construct was also flanked by left and right homology arms containing sequences homologous to the endogenous AAVS1 locus, allowing integration of the construct at its intended target site. Flow cytometry analysis was performed on either undifferentiated wild-type PSCs or undifferentiated AAVS1-targeted PD-L1 / HSV-TK-edited PSCs (Figure 20B). Cells were stained with an anti-PD-L1 primary antibody.

[0131] The data show that the majority (99.9%) of PSCs with PD-L1 / HSV-TK editing express PD-L1 by flow cytometry, whereas wild-type PSCs do not. Both PSC lines were subsequently differentiated into cardiomyocytes and analyzed by flow cytometry followed by staining with an anti-PD-L1 primary antibody. The data show that only 49% of PSCs with PD-L1 / HSV-TK editing express PD-L1 by flow cytometry. These results indicate that insertion of a PD-L1 / HSV-TK construct at the AAVS1 locus results in transgene silencing of PD-L1 expression during lineage-committed differentiation of PSCs into cardiomyocytes. Similar transgene silencing was observed at the B2M locus.

[0132] In conclusion, the above data demonstrate that the GAPDH, RPL13A, and RPLP0 loci enable sustained, high-level expression of various transgenes integrated therein. Our data also demonstrate that transgenes integrated at the commonly used AAVS1 and B2M loci (e.g., those encoding PD-L1) lost their expression in edited cells once the cells differentiated from PSCs to cardiomyocytes.

[0133] The present invention includes the following aspects. Item 1 A genetically modified mammalian cell comprising a transgene at a persistent transgene expression locus (STEL) in the genome, wherein the transgene is expressed at a detectable level, and optionally the mammalian cell is a human cell. Section 2 2. The genetically modified cell of paragraph 1, wherein the expression level of the transgene does not change by more than 40%, more than 30%, more than 20%, or more than 10% over (i) 5 or more, 10 or more, or 15 or more passages, or (ii) more than 40%, more than 30%, more than 20%, or more than 10% upon a change in cell state, wherein the cell state is optionally a pluripotent and / or differentiated state. Section 3 3. The genetically modified cell of paragraph 1 or 2, wherein the STEL is selected from the loci listed in Table 1. Section 4 The genetically modified cell of paragraph 3, wherein the STEL is a locus having an average normalized expression of greater than 3.30, greater than 3.50, greater than 3.75, greater than 4.00, greater than 4.10, greater than 4.20, greater than 4.30, greater than 4.50, greater than 4.60, or greater than 4.70, as set forth in Table 1. Section 5 4. The genetically modified cell of paragraph 3, wherein the STEL is in a gene encoding a protein involved in one or more of ribonucleoprotein complex formation, focal adhesions, cell-substrate adherens junctions, cell-substrate binding, cell anchoring, extracellular exosomes, extracellular vesicles, intracellular organelles, anchoring binding, RNA binding, nucleic acid binding (e.g., rRNA or mRNA binding), and protein binding. Section 6 STEL, Genes encoding ribosomal proteins, optionally (i) RPL13A, RPLP0, RPL10, RPL13, RPS18, RPL3, RPLP1, 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, RPL (ii) an RPL gene selected from P2, RPL23A, RPL26, RPL36, RPL35, RPL23, RPL4, and RPL22; or (ii) an RPS gene selected from 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 RPS11; a gene encoding a mitochondrial protein, optionally selected from MT-CO1, MT-CO2, MT-ND4, MT-ND1 and MT-ND2; a gene encoding an actin protein, optionally selected from ACTG1 and ACTB; a gene encoding a eukaryotic translation factor, optionally selected from EEF1A1, EEF2, and EIF1; genes encoding histones, such as H3F3A and H3F3B; or A gene selected from FTL, FTH1, TPT1, TMSB10, GAPDH, PTMA, GNB2L1, NACA, YBX1, NPM1, FAU, UBA52, HSP90AB1, MYL6, SERF2, and SRP14 4. The genetically modified cell according to item 3, wherein Section 7 4. The genetically modified cell of paragraph 3, wherein the STEL is the GAPDH gene. Section 8 4. The genetically modified cell of paragraph 3, wherein STEL is a ribosomal protein gene. Section 9 9. The genetically modified cell of paragraph 8, wherein the STEL is a ribosomal protein L (RPL) gene optionally selected from the RPL13A, RPL7, and RPLP0 genes. Item 10 10. The genetically modified cell of any one of paragraphs 1 to 9, wherein the cell is a pluripotent stem cell (PSC). Section 11 11. The genetically modified mammalian cell of paragraph 10, wherein the PSC is a human embryonic stem cell (ESC) or a human induced PSC (iPSC). Item 12 10. The genetically modified cell according to any one of items 1 to 9, wherein the cell is a differentiated cell. Item 13 13. The genetically modified cell of paragraph 12, wherein the differentiated cell is derived from a human PSC, optionally selected from a human ESC and a human iPSC. Section 14 The differentiated cells human immune cells optionally selected from T cells, T cells expressing a chimeric antigen receptor (CAR), inhibitory T cells, myeloid cells, dendritic cells, and immunosuppressive macrophages; a cell of the human nervous system optionally selected from a dopaminergic neuron, a microglial cell, an oligodendrocyte, an astrocyte, a cortical neuron, a spinal cord or oculomotor neuron, an enteric neuron, a placode-derived cell, a Schwann cell, and a trigeminal or sensory neuron; cells of the human cardiovascular system optionally selected from cardiomyocytes, endothelial cells and nodal cells; cells of the human metabolic system, optionally selected from hepatocytes, bile duct cells and pancreatic beta cells; or Cells of the human ocular system, optionally selected from retinal pigment epithelial cells, photoreceptor cone cells, photoreceptor rod cells, bipolar cells, and ganglion cells. 14. The genetically modified mammalian cell of claim 12 or 13, wherein the cell is Section 15 Item 10. The genetically modified cell of any preceding paragraph, wherein the transgene is inserted into the 3' untranslated region of the locus. Section 16 A genetically modified cell described in any one of the preceding paragraphs, wherein the transgene sequence is linked in frame to the STEL gene sequence via a coding sequence for a self-cleaving peptide or is linked to the STEL gene sequence via an internal ribosome entry site (IRES). Section 17 Item 10. The genetically modified cell of any preceding item, wherein the transgene encodes a therapeutic protein, an immunomodulatory protein, a reporter protein, or a safety switch signal. Section 18 The genetically modified cell of any one of the preceding paragraphs, wherein the genome of the cell further comprises an exogenous suicide gene, optionally within a STEL locus in the genome, which, once activated, causes apoptosis of the cell. Section 19 19. The genetically modified cell of paragraph 18, wherein the suicide gene is the herpes simplex virus (HSV) thymidine kinase (TK) gene. Section 20 A pharmaceutical composition comprising the genetically modified cell according to any one of items 1 to 19 and a pharmaceutically acceptable carrier. Section 21 20. A method for treating a human patient in need of treatment, comprising the step of introducing into the patient the genetically modified cells according to any one of items 1 to 19, wherein the cells are human cells. Section 22 Human patients needing a graft transplant, or having inflammation, optionally neuroinflammation, autoimmune disease or cancer, Item 22. The method described in item 21. Section 23 23. The genetically modified mammalian cell of any one of paragraphs 1 to 19, for use in the method of paragraph 21 or 22. Section 24 23. Use of the genetically modified mammalian cell of any one of items 1 to 19 for the manufacture of a medicament for use in the method of item 21 or 22. Section 25 Providing cultured mammalian cells; and introducing the transgene into the STEL site in the genome of the cultured cells; 20. A method for producing a genetically modified mammalian cell according to any one of items 1 to 19, comprising: Section 26 The method of paragraph 25, wherein the introducing step is performed by CRISPR gene editing.

Claims

[Claim 1] A genetically modified mammalian cell comprising a transgene at a persistent transgene expression locus (STEL) in the genome, wherein the transgene is expressed at a detectable level, and optionally the mammalian cell is a human cell.

Images