Engineered cells and implantable elements for disease treatment

Abstract

Described herein are modified mammalian cells, and methods of making and using the same, that comprise reduced levels or function of one or more of a major histocompatibility complex (MHC) class I protein complex component, optionally an MHC class II protein complex component, and / or CIITA, and one of a pro-inflammatory cytokine and a pro-fibrotic factor.

Description

[Technical Field]

[0001] Priority claims This application claims priority to U.S. Provisional Patent Application No. 63 / 415,272, filed October 11, 2022, and U.S. Provisional Patent Application No. 63 / 415,273, filed October 11, 2022. [Background technology]

[0002] Treating chronic and genetic diseases by transplanting cells modified to produce therapeutic substances capable of treating such diseases has the tantalizing potential to improve the health of patients with such diseases. To fully realize the potential of such therapies, the transplanted cells must be capable of producing therapeutic levels of the desired therapeutic substance for weeks, months, or longer periods without resulting in overstimulation of the host immune response. Summary of the Invention

[0003] Described herein are modified mammalian cells comprising reduced levels or reduced function of a major histocompatibility complex (MHC) class I protein complex and one of a pro-inflammatory cytokine or a pro-fibrotic factor, as well as related devices (e.g., implantable elements), compositions, and methods of making and using them. In one embodiment, the modified mammalian cells comprise reduced levels or reduced function of one or more proteins selected from human leukocyte antigen (HLA) A, HLA-B, HLA-C, and beta-2-microglobulin (beta-2M). The reduced levels or reduced function of the MHC class I protein complex may result from mutations in components of the MHC class I protein complex or from silencing or knockdown of components of the MHC class I protein complex. In one embodiment, the pro-inflammatory cytokine comprises IL-6, IL-8, or MCP-1. In one embodiment, the pro-fibrotic factor comprises FGF-2, PDGF, or VEGFA.

[0004] In one aspect, the present disclosure may also feature a modified mammalian cell further comprising a reduced level or reduced function of an MHC class II protein complex. In one embodiment, the modified mammalian cell comprises a reduced level or reduced function of one or more proteins selected from HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. The reduced level or reduced function of an MHC class II protein complex may result from a mutation in a component of the MHC class II protein complex or from silencing or knockdown of a component of the MHC class II protein complex. In another aspect, the modified mammalian cell described herein may also comprise a reduced level or reduced function of class II major histocompatibility complex transactivator (CIITA).

[0005] In another aspect, the present disclosure features a transplantable element comprising a modified mammalian cell or a plurality of modified mammalian cells described herein. The modified mammalian cell may comprise an embryonic stem cell (ESC) or an induced pluripotent stem cell (iPSC). The modified mammalian cell may comprise a retinal pigment epithelial (RPE) cell, a CCD-33Lu cell, an MRC-5 cell, an MRC-9 cell, an MCF10a cell, or a cell derived therefrom. In one embodiment, the modified mammalian cell comprises a modified RPE cell (e.g., a modified ARPE-19 cell) or is derived from an RPE cell (e.g., an ARPE-19 cell). In one embodiment, the modified mammalian cell comprises a modified ARPE-19 cell or is derived from an ARPE-19 cell. In one embodiment, the transplantable element comprises at least one cell-containing compartment comprising a modified mammalian cell or a plurality of modified mammalian cells described herein. In one embodiment, the implantable element comprises one cell-containing compartment containing a modified mammalian cell or cells described herein and a second compartment surrounding the cell-containing compartment. In one embodiment, the implantable element further comprises at least one means for mitigating a foreign body reaction (FBR) upon implantation of the implantable element (e.g., a compound of Formula (I) described herein) into a subject. In one embodiment, the implantable element comprises a polymer selected from alginate, hyaluronate, and chitosan. In one embodiment, the implantable element comprises a cell-containing compartment surrounded by a barrier compartment comprising an alginate hydrogel disposed on the outer surface of the barrier compartment and, optionally, a compound of Formula (I) (e.g., a compound of Formula (I) described herein). In one embodiment, the implantable element is formulated for implantation into a subject (e.g., into the intraperitoneal (IP) space, peritoneal cavity, omentum, lesser omental pouch, subcutaneous fat). In one embodiment, the implantable element is configured to protect the modified mammalian cell or cells from the recipient's immune system and to reduce foreign body reaction (FBR) (as defined herein) to the implanted device.In one embodiment, the implantable element is capable of delivering a therapeutic agent (e.g., a protein) for a sustained period of time (e.g., from one to several months up to one to several years) after implantation into a subject.

[0006] In another aspect, the disclosure features a method of treating a disease or disorder in a subject, the method including administering to the subject a modified mammalian cell described herein or an implantable element comprising a plurality of modified mammalian cells described herein, wherein the modified mammalian cell or plurality of modified mammalian cells comprises a reduced level or reduced function of an MHC class I protein complex. In one embodiment, the modified mammalian cell or plurality of modified mammalian cells further comprises a reduced level or reduced function of an MHC class II protein complex and / or a reduced level or reduced function of CIITA. In one embodiment, the modified mammalian cell or plurality of modified mammalian cells further comprises a reduced level or reduced function of a pro-inflammatory cytokine or a pro-fibrotic factor. In one embodiment, the disease or disorder is a lysosomal storage disease. In one embodiment, the disease or disorder is a metabolic disease.

[0007] In one embodiment, an implantable element described herein or a plurality of implantable elements described herein are combined with a pharmaceutically acceptable excipient to prepare an implantable element preparation or composition that can be administered to a subject (e.g., intraperitoneally) when in need of treatment with a therapeutic agent produced by the device. In one embodiment, the subject is human, the modified mammalian cells are derived from human cells (e.g., RPE cells, ARPE-19 cells), and the implantable element preparation or composition is capable of continuously delivering an effective amount of a therapeutic agent to the subject for a sustained period of time, for example, at least 3 months, 6 months, 1 year, 2 years, or more. In one embodiment, the modified mammalian cells are derived from human RPE cells, e.g., ARPE-19 cells. In one embodiment, the modified mammalian cells are derived from human ARPE-19 cells. [Brief explanation of the drawings]

[0008] [Figure 1A] 1 is a graph showing protein expression levels in ARPE-19 transduced with shRNA-containing lentiviral particles targeting beta-2-microglobulin (beta-2M). [Figure 1B] 1 is a graph showing protein expression levels in ARPE-19 cells transduced with shRNA-containing lentiviral particles targeting monocyte chemoattractant protein 1 (MCP-1), also known as chemokine (CC motif) ligand 2 (CCL2). [Figure 1C] 1 is a graph showing protein expression levels in ARPE-19 cells transduced with lentiviral particles containing shRNA targeting fibroblast growth factor 2 (FGF2). [Figure 1D] 1 is a graph showing protein expression levels in ARPE-19 cells transduced with lentiviral particles containing shRNA targeting interleukin-6 (IL-6). [Figure 1E] 1 is a graph showing protein expression levels in ARPE-19 cells transduced with lentiviral particles containing shRNA targeting interleukin-8 (IL-8). [Figure 2] This graph shows that beta-2M protein expression in alpha-L-iduronidase (IDUA)-expressing ARPE-19 cells containing beta-2M shRNA is significantly (89%) lower than that in IDUA-expressing ARPE-19 cells containing scrambled control shRNA. [Figure 3A] 1 is a graph showing protein expression levels in IDUA-expressing ARPE-19 cells transduced with lentiviral particles containing shRNA targeting beta-2M. [Figure 3B] 1 is a graph showing protein expression levels in IDUA-expressing ARPE-19 cells transduced with lentiviral particles containing shRNA targeting MCP-1 (CCL2). [Figure 3C]1 is a graph showing protein expression levels in IDUA-expressing ARPE-19 cells transduced with lentiviral particles containing shRNA targeting IL-6. [Figure 4] 1 is a graph showing that expression levels of Beta-2M were reduced by 99% in ARPE-19 cells using CRISPR and gRNA targeting Beta-2M compared to ARPE-19 cells modified with scrambled gRNA. [Figure 5A] 5A-5D are a series of graphs showing that reduced beta-2M protein expression in ARPE-19 cells (FIG. 5A) results in reduced human leukocyte antigen (HLA) expression (FIG. 5B) compared to beta-2M expression in wild-type ARPE-19 cells (FIG. 5C). [Figure 5B] 5A-5D are a series of graphs showing that reduced beta-2M protein expression in ARPE-19 cells (FIG. 5A) results in reduced human leukocyte antigen (HLA) expression (FIG. 5B) compared to beta-2M expression in wild-type ARPE-19 cells (FIG. 5C). [Figure 5C] 5A-5D are a series of graphs showing that reduced beta-2M protein expression in ARPE-19 cells (FIG. 5A) results in reduced human leukocyte antigen (HLA) expression (FIG. 5B) compared to beta-2M expression in wild-type ARPE-19 cells (FIG. 5C). [Figure 5D] 5A-5D are a series of graphs showing that reduced beta-2M protein expression in ARPE-19 cells (FIG. 5A) results in reduced human leukocyte antigen (HLA) expression (FIG. 5B) compared to beta-2M expression in wild-type ARPE-19 cells (FIG. 5C). DETAILED DESCRIPTION OF THE INVENTION

[0009] The present disclosure features mammalian cells (e.g., human RPE cells) modified to modulate the level or function of a major histocompatibility complex (MHC) class I protein complex or a component thereof (e.g., beta-2-microglobulin (beta-2M)). In one embodiment, the mammalian cells are modified to reduce expression of an MHC class I protein complex or a component thereof, such as beta-2M. The mammalian cells may be modified to produce a low-functioning or non-functional variant of an MHC class I protein complex or a component thereof (e.g., beta-2M), or the expression of an MHC class I protein complex or a component thereof (e.g., beta-2M) may be silenced, knocked down, or knocked out. The present disclosure also features mammalian cells further modified to modulate the level or function of an MHC class II protein complex or a component thereof, and / or the level or function of class II major histocompatibility complex transactivator (CIITA). In one embodiment, the mammalian cells are modified to reduce expression of an MHC class II protein complex or a component thereof, and / or the level or function of CIITA. Mammalian cells may be modified to produce low-functioning or non-functional variants of the MHC class II protein complex or its components, and / or CIITA, or expression of the MHC class II protein complex or its components, and / or CIITA may be silenced or knocked down or knocked out.

[0010] Abbreviations and Definitions The following abbreviations are used throughout the detailed description and examples of this disclosure: CM-Alg Chemically modified alginate CM-LMW-Alg Chemically modified low molecular weight alginate CM-LMW-Alg-101: Low molecular weight alginate chemically modified with compound 101 shown in Table 4 CM-HMW-Alg Chemically modified high molecular weight alginate CM-HMW-Alg-101 High molecular weight alginate chemically modified with compound 101 shown in Table 4 CM-MMW-Alg Chemically modified medium molecular weight alginate CM-MMW-Alg-101 Medium molecular weight alginate chemically modified with compound 101 shown in Table 4 HMW-Alg High molecular weight alginate MMW-Alg Medium molecular weight alginate U-Alg unmodified alginate U-HMW-Alg Unmodified high molecular weight alginate U-LMW-Alg Unmodified low molecular weight alginate U-MMW-Alg Unmodified medium molecular weight alginate 70:30 CM-Alg:U-Alg A 70:30 mixture (V:V) of chemically modified and unmodified alginate, as described, for example, in WO2020069429.

[0011] In order that the present disclosure may be more readily understood, certain technical and scientific terms used herein are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0012] As used in this specification, including the appended claims, the singular forms of terms such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0013] "About" or "approximately," when used herein to modify a numerically defined parameter (e.g., the amount of a therapeutic agent secreted by modified cells, a physical description of a device (e.g., hydrogel capsule), e.g., diameter, sphericity, number of cells encapsulated therein, number of devices in a preparation), means that the quoted numerical value is within an acceptable functional range for the defined parameter as determined by one of ordinary skill in the art, which depends in part on how the measurement or determination was made, e.g., the limits of the measurement system (including the acceptable error range for that measurement system). For example, "about" can mean a range of ±20% of the quoted numerical value. As a non-limiting example, a hydrogel capsule defined as having a diameter of about 1.5 millimeters (mm) and encapsulating about 5 million (M) cells may have a diameter of 1.2-1.8 mm and encapsulate 4-6 M cells. As another non-limiting example, a preparation of about 100 devices (e.g., hydrogel capsules) includes a preparation with 80-120 devices. In some embodiments, the term "about" means that the modified parameter can vary by as much as 15%, 10%, or 5% above or below the numerical value stated for that parameter.

[0014] As used herein, "obtain" or "obtaining" refers to obtaining a value, e.g., a numerical value, or an image, or a physical entity (e.g., a sample), either by "directly obtaining" or "indirectly obtaining" the value or physical entity. "Directly obtaining" means performing a process (e.g., running an analytical method or protocol) to obtain the value or physical entity. "Indirectly obtaining" refers to receiving a value or physical entity from another party or source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a value or physical entity includes performing a process that involves a physical change in a physical substance, or using a machine or device. An example of directly obtaining a value includes obtaining a sample from a human subject. Directly obtaining a value includes performing a process using a machine or device, e.g., using a fluorescence microscope to obtain fluorescence microscopy data.

[0015] "Administer," "administering," or "administration," as used herein, refers to implanting, absorbing, ingesting, injecting, placing, or otherwise introducing into a subject an entity described herein (e.g., a device or preparation of a device), or providing such an entity to a subject for administration.

[0016] "Non-fibrotic," as used herein, refers to a compound or material that reduces foreign body reaction (FBR). For example, implantation of a device (e.g., a hydrogel capsule) containing a non-fibrotic compound (e.g., a hydrogel capsule comprising a polymer covalently modified with a compound listed in Table 4) into a biological tissue induces a lower amount of FBR in the tissue than the FBR induced by implantation of a non-fibrotic null reference capsule lacking any non-fibrotic compound but of substantially the same composition (e.g., same cell type(s)) and structure (e.g., size, shape, number of compartments). In one embodiment, the degree of FBR is assessed by an immune response in the tissue containing the implanted device (e.g., hydrogel capsule), which may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis, using assays known in the art, for example, as described in WO 2017 / 075630, or as described in Vegas, A., et al., Nature 101:101-102. The assays / methods used may include one or more of the following: subcutaneous cathepsin measurement of implanted capsules, Masson's Trichrome staining (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cell staining and confocal microscopy of macrophages (CD68 or F4 / 80), myofibroblasts (alpha-muscle actin, SMA), or general cellular deposition; quantification of 79 RNA sequences of known inflammatory factors and immune cell markers; or FACS analysis of macrophage and neutrophil cells in the intraperitoneal space of suitable test subjects, e.g., immunocompetent mice, on devices (e.g., capsules) retrieved after 14 days. In one embodiment, FBR is assessed by measuring the levels of one or more biomarkers of immune response, e.g., cathepsins, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4, in tissues containing the implant.In some embodiments, the FBR induced by a device of the invention (e.g., a hydrogel capsule comprising a non-fibrous compound disposed on its exterior surface) is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the FBR induced by an FBR-null reference device, e.g., a device that is substantially identical to the test or claimed device except for lacking a means for mitigating FBR (e.g., a hydrogel capsule that does not comprise a non-fibrous compound but is otherwise substantially identical to the claimed capsule). In some embodiments, the FBR (e.g., the level of biomarker(s)) is measured after about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or more.

[0017] As used herein, "cell" refers to a modified or unmodified cell. In one embodiment, the cell is an immortalized cell or a modified cell derived from an immortalized cell. In one embodiment, the cell is a viable cell, e.g., viable as measured by any technique described herein or known in the art.

[0018] "Cell-binding peptide (CBP)," as used herein, refers to a linear or cyclic peptide comprising an amino acid sequence derived from the cell-binding domain of a ligand of a cell adhesion molecule (CAM) (e.g., mediating cell-matrix or cell-cell connections). In one embodiment, the CBP is any of the CBPs described in International Patent Publication No. 2020069429. In one embodiment, the CBP is a linear peptide comprising RGD and is less than 6 amino acids in length. In one embodiment, the CBP is a linear peptide consisting essentially of RGD or RGDSP.

[0019] As used herein, "CBP-polymer" refers to a polymer comprising at least one cell-binding peptide molecule covalently attached to the polymer via a linker. In one embodiment, the polymer in the CBP-polymer is a synthetic or naturally occurring polysaccharide, such as an alginate, e.g., sodium alginate. In one embodiment, the linker is an amino acid linker (i.e., consisting essentially of a single amino acid or a peptide of several identical or different amino acids), which is connected to the N-terminus or C-terminus of CBP via a peptide bond. In one embodiment, the CBP-polymer is any of the CBP-alginates defined in WO2020069429.

[0020] As used herein, "cell-binding substance (CBS)" refers to any chemical, biological, or other type of substance (e.g., small organic compound, peptide, polypeptide) capable of mimicking at least one activity of a ligand for a cell-adhesion molecule (CAM) or other cell-surface molecule that mediates cell-matrix or cell-cell connections or other receptor-mediated signaling. In one embodiment, when present in a polymer composition that encapsulates viable cells, the CBS is capable of forming a transient or permanent bond or contact with one or more of the cells. In one embodiment, the CBS facilitates interaction between two or more viable cells encapsulated in the polymer composition. In one embodiment, the presence of a CBS in a polymer composition that encapsulates multiple cells (e.g., viable cells) correlates with one or both of increased cell productivity (e.g., expression of a therapeutic agent) and increased cell viability when the encapsulated cells are transplanted into a test subject, e.g., a mouse. In one embodiment, the CBS is physically bound to one or more polymer molecules in the polymer composition. In one embodiment, the CBS is a cell-binding peptide as defined herein or in WO2020069429.

[0021] "Conservatively modified variants," or "conservative substitutions," as used herein, refer to variants of a reference peptide or polypeptide that are identical to the reference molecule except for one or more conservative amino acid substitutions in its amino acid sequence. In one embodiment, a conservatively modified variant consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the reference amino acid sequence. A conservative amino acid substitution refers to the substitution of an amino acid with an amino acid that has similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) and has minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution tables for functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional characteristics are provided in Table 1 below. [Table 1] "Consists essentially of" and variations such as "consist essentially of" or "consisting essentially of," as used throughout this specification and claims, indicate the inclusion of any recited element or group of elements, and the optional inclusion of other elements of similar or different nature to the recited elements, which do not substantially alter the basic or novel properties of the identified molecule, composition, device, or method. As a non-limiting example, a therapeutic protein agent secreted by an engineered mammalian cell described herein consisting essentially of a recited amino acid sequence may also include one or more amino acids, each of which comprises a substitution in the recited amino acid sequence of one or more amino acid residues that does not substantially affect the relevant biological activity of the therapeutic protein agent.

[0022] "Derived from," as used herein with respect to a cell or cells, refers to cells obtained from a cell, cell line, or tissue, and optionally then cultured, passaged, immortalized, differentiated, and / or induced, etc., to produce a derivative cell(s).

[0023] "Device" and "implantable element," as used herein, refer to any implantable object (e.g., particle, hydrogel capsule, graft, medical device) that contains a modified cell or cells (e.g., living cells) that can express and secrete a therapeutic agent after implantation of the device and has a configuration that supports cell survival by allowing cellular nutrients to enter the device. The terms "device" and "implantable element" are used interchangeably herein.

[0024] "Differential volume," as used herein, refers to the volume of one compartment in a device described herein, excluding the space occupied by another compartment(s). For example, in a two-compartment device having an inner and outer compartment, the differential volume of the second (e.g., outer) compartment refers to the volume within the second compartment, excluding the space occupied by the first (inner) compartment.

[0025] "Effective amount," as used herein, refers to an amount of any of the following: engineered cells secreting a protein, a device preparation producing a protein, or a component of a device (e.g., the amount of a therapeutic agent co-expressed with another therapeutic agent by cells in a device, the number of engineered cells in a device, the amount of CBS and / or non-fibrous compound in a device) sufficient to elicit a desired biological response. In some embodiments, the term "effective amount" refers to the amount of a component of a device (e.g., the number of cells in a device, the density of non-fibrous compound disposed on the surface and / or in the barrier compartment of a device, the density of CBS in the cell-containing compartment).

[0026] In one embodiment, the desired biological response upon implantation of the implantable element in a subject is a reduced amount of pericapsular fibrotic overgrowth (PFO) compared to the amount of PFO observed in a control implantable element (e.g., defined as an identical implantable element except that the cells lack a reduction in MHC class I protein complexes). An effective amount can include the amount of a therapeutic agent secreted by the modified mammalian cells described herein. An effective amount includes therapeutic and prophylactic treatment.

[0027] As used herein, "effective amount" refers to an amount of genetically modified cells (e.g., derived from human cells (e.g., epithelial cells)) that produce an exogenous polypeptide or a polypeptide-producing device preparation sufficient to elicit a desired biological response. In one embodiment, the desired biological response is an increase in the level of the exogenous polypeptide in cells, or, in the case of a secreted polypeptide, an increase in the level in a tissue sample removed from a subject treated (e.g., implanted) with the genetically modified cells, device, or device preparation containing such cells. As will be understood by those skilled in the art, an effective amount can vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the exogenous polypeptide, composition, or device, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment.

[0028] An "endogenous nucleic acid," as used herein, is a nucleic acid that is naturally present in a subject's cell.

[0029] An "endogenous polypeptide," as used herein, is a polypeptide that is naturally present in the cells of a subject.

[0030] The terms "modified human cell" and "genetically modified human cell" can be used interchangeably herein, and each term refers to a human cell (e.g., an epithelial cell) that has a non-naturally occurring genetic alteration (e.g., in the cellular genome) and typically contains an exogenous nucleic acid sequence (e.g., DNA or RNA) that is not present (or is present at different levels) in an otherwise similar human cell (e.g., an epithelial cell). In one embodiment, the modified human cell (e.g., a modified RPE cell) contains an exogenous nucleic acid encoding a polypeptide, e.g., a therapeutic protein. In one embodiment, the exogenous nucleic acid sequence is chromosomal (e.g., the exogenous nucleic acid sequence is an exogenous sequence placed into an endogenous chromosomal sequence) or extrachromosomal (e.g., a non-integrated expression vector). In one embodiment, the exogenous nucleic acid sequence comprises an RNA sequence, e.g., mRNA. In one embodiment, the exogenous nucleic acid sequence comprises a chromosomal or extrachromosomal exogenous nucleic acid sequence that contains a sequence that is expressed as RNA, e.g., mRNA or regulatory RNA. In one embodiment, the exogenous nucleic acid sequence comprises a first chromosomal or extrachromosomal exogenous nucleic acid sequence that regulates the conformation or expression of a second nucleic acid sequence, and the second nucleic acid sequence can be exogenous or endogenous. For example, the modified cell can comprise an exogenous nucleic acid that controls the expression of an endogenous sequence. In one embodiment, the modified cell comprises an exogenous nucleic acid sequence that comprises a codon-optimized coding sequence for a polypeptide of interest, achieving higher expression of the polypeptide than the native coding sequence. Codon-optimized coding sequences can be generated using commercially available algorithms, such as GeneOptimizer (ThermoFisher Scientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), or the Java® Codon Adaptation Tool (JCat, www.jcat.de; Grote, A. et al., Nucleic Acids Research, Vol. 33, Issue suppl 2, pp. W526-W531 (2005)).In one embodiment, the modified cells (e.g., modified epithelial cells, e.g., modified RPE cells, e.g., modified ARPE-19 cells) are cultured from a monoclonal cell line. In some embodiments, the modified cells are not pancreatic islet cells, as defined herein.

[0031] An "exogenous nucleic acid," as used herein, is a nucleic acid that does not naturally occur in a subject cell.

[0032] An "exogenous polypeptide," as used herein, is a polypeptide encoded by an exogenous nucleic acid of a subject cell. Reference to an amino acid position of a particular sequence refers to the position of that amino acid in a reference amino acid sequence, e.g., the sequence of a full-length mature (after signal peptide cleavage) wild-type protein (unless otherwise stated), and does not exclude the presence of variations, e.g., deletions, insertions, and / or substitutions, at other positions in the reference amino acid sequence.

[0033] "Factor VII protein" or "FVII protein," as used herein, unless otherwise specified, refers to a polypeptide comprising the amino acid sequence of a naturally occurring Factor VII protein or a variant thereof having FVII biological activity (e.g., promoting blood clotting) as determined by art-recognized assays. Naturally occurring FVII exists as a single-chain zymogen, a zymogen-like two-chain polypeptide, and a fully activated two-chain form (FVIIa). In some embodiments, reference to FVII includes both single-chain and its two-chain forms (including zymogen-like and FVIIa). FVII proteins that can be produced by the genetically modified cells described herein (e.g., derived from a human epithelial cell line, e.g., the ARPE-19 cell line) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, and variants having one or more amino acid substitutions and / or deletions. In some embodiments, the variant FVII protein is capable of being activated to a fully activated two-chain form (Factor VIIa) having at least 50%, 75%, 90% or more (including >100%) of the activity of wild-type Factor VIIa. Variants of FVII and FVIIa are known, such as marzeptacog alfa (activated) (MarzAA) and the variants described in European Patent No. 1373493, U.S. Patent No. 7771996, U.S. Patent No. 9476037, and U.S. Published Application No. US20080058255.

[0034] Factor VII biological activity may be quantified by art-recognized assays unless otherwise specified. For example, FVII biological activity in a sample of biological fluid, e.g., plasma, may be quantified by (i) measuring the amount of factor Xa and factor X produced in a system containing tissue factor (TF) embedded in a lipid membrane (Persson et al., J. Biol. Chem. 272:19919-19924, 1997), (ii) measuring factor X hydrolysis in an aqueous system, (iii) measuring its physical binding to TF using a surface plasmon resonance-based device (Persson, FEBS Letts. 413:359-363, 1997), or (iv) measuring the hydrolysis of a synthetic substrate, and / or (v) measuring thrombin generation in a TF-independent in vitro system. In one embodiment, FVII activity is assessed by a commercially available chromogenic assay (BIOPHEN FVII, HYPHEN BioMed Neuville sur Oise, France) in which a biological sample containing FVII is mixed with thromboplastin calcium, factor X, and SXa-11 (a chromogenic substrate specific for factor Xa).

[0035] "Factor VIII protein" or "FVIII protein," as used herein, unless otherwise specified, refers to a polypeptide comprising the amino acid sequence of a naturally occurring factor VIII polypeptide or a variant thereof having FVIII biological activity, e.g., clotting activity, as determined by art-recognized assays. FVIII proteins that can be expressed by the genetically modified cells described herein (e.g., derived from a human epithelial cell line, e.g., the ARPE-19 cell line) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, and variants having one or more amino acid substitutions and / or deletions, B-domain deleted (BDD) variants, single-chain variants, and fusions of any of the foregoing wild-type or variants with a half-life-extending polypeptide. In one embodiment, the cells are engineered to encode a precursor factor VIII polypeptide (e.g., having a signal sequence) having a complete or partial deletion of the B domain. In one embodiment, the cells are modified to encode a single-chain factor VIII polypeptide, preferably containing a variant FVIII protein having at least 50%, 75%, 90%, or more (including >100%) of the clotting activity of the corresponding wild-type factor VIII. Assays for measuring the clotting activity of FVIII proteins include one- or two-stage clotting assays (Rizza et al., 1982, Coagulation assay of FVIII:C and FIXa in Bloom ed. The Hemophelias. NY Churchill Livingston 1992) or chromogenic substrate FVIII:C assays (Rosen, S. 1984, Scand J Haematol 33:139-145, suppl.).

[0036] Numerous FVIII-BDD variants are known, including, for example, those described in the following U.S. Patent Nos.: 4,868,112 (e.g., column 2, line 2 to column 19, line 21 and Table 2); 5,112,950 (e.g., column 2, lines 55-68, Figure 2, and Example 1); 5,171,844 (e.g., column 4, line 22 to column 5, line 36); 5,543,502 (e.g., column 2, lines 17-46); 5,595,886; 5,610,278; 5,789,203 (e.g., column 2, lines 26-51 and Examples 5-8); and 5,972,885 (e.g., column 1, lines 25 to column 2, line 40). 6,048,720 (e.g., column 6, lines 1-22 and Example 1); 6,060,447; 6,228,620; 6,316,226 (e.g., column 4, lines 4-5, line 28 and Examples 1-5); 6,346,513; 6,458,563 (e.g., column 4, lines 25-53) and 7,041,635 (e.g., column 2, lines 1-3, line 19; column 3, lines 40-4, line 67; column 7, lines 43-8, line 26; and column 11, lines 5-13, line 39).

[0037] In some embodiments, the FVIII-BDD protein produced by the genetically modified cells described herein (e.g., derived from a human epithelial cell line, e.g., the ARPE-19 cell line) has one or more of the following deletions of amino acids in the B-domain: (i) most of the B-domain except for the amino-terminal B-domain sequence essential for intracellular processing of the primary translation product into two polypeptide chains (WO 91 / 09122); (ii) amino acids 747-1638 (Hoeben RC, et al. J. Biol. Chem. 265(13):7318-7323 (1990)); amino acids 771-1666 or amino acids 868-1562 (Meulien P., et al. Protein Eng. 2(4):301-6 (1988)); amino acids 982-1562 or amino acids 760-1639 (Toole et al. al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)); amino acids 797 to 1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)); 741 to 1646 (Kaufman, WO 87 / 04187)), 747 to 1560 (Sarver et al., DNA 6:553-564 (1987)); amino acids 741 to 1648 (Pasek, WO 88 / 00831)), amino acids 816 to 1598 or 741 to 1689 (Lagner (Behring Inst. Mitt. (1988) No. 82:16-25, EP295597); deletions involving one or more residues in the Furin protease recognition sequence, including any of the specific deletions described in column 10, line 65 to column 11, line 36 in U.S. Patent No. 9,956,269.

[0038] In other embodiments, the FVIII-BDD protein retains any of the following B-domain amino acids or amino acid sequences: (i) one or more N-linked glycosylation sites in the B-domain, e.g., residues 757, 784, 828, 900, 963, or optionally 943, the first 226 amino acids, or the first 163 amino acids (Miao, HZ, et al., Blood 103(a):3412-3419 (2004), Kasuda, A., et al., J. Thromb. Haemost. 6:1352-1359 (2008), and Pipe, SW, et al., J. Thromb. Haemost. 9:2235-2242 (2011)).

[0039] In some embodiments, the FVIII-BDD protein is a single-chain variant generated by substitution or deletion of one or more amino acids in the Furin protease recognition sequence LKRHQR (including any of the substitutions at positions R1645 and / or R1648 described in U.S. Patent Nos. 10,023,628, 9,394,353, and 9,670,267) that prevents proteolytic cleavage at this site.

[0040] In some embodiments, any of the above FVIII-BDD proteins may further comprise one or more of the following variations: an F309S substitution to improve expression of the FVIII-BDD protein (Miao, HZ, et al., Blood 103(a):3412-3419 (2004)); an albumin fusion (WO2011 / 020866); and an Fc fusion (WO04 / 101740).

[0041] All FVIII-BDD amino acid positions referenced herein refer to positions in full-length human FVIII unless otherwise specified.

[0042] "Factor IX protein" or "FIX protein," as used herein, unless otherwise specified, refers to a polypeptide comprising the amino acid sequence of a naturally occurring factor IX protein or a variant thereof having FIX biological activity, e.g., clotting activity, as determined by art-recognized assays. FIX is produced as an inactive zymogen that is converted to its active form by factor XIa excision of the activation peptide to generate heavy and light chains held together by one or more disulfide bonds. FIX proteins that can be produced by the genetically modified cells described herein (e.g., derived from RPE cell lines, e.g., the ARPE-19 cell line) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, and variants having one or more amino acid substitutions and / or deletions, and fusions of any of the aforementioned wild-type or variant proteins with a half-life-extending polypeptide. In one embodiment, the cells are engineered to encode a full-length wild-type human factor IX polypeptide (e.g., with a signal sequence) or a functional variant thereof. The variant FIX protein preferably has at least 50%, 75%, 90% or more (including >100%) of the clotting activity of wild-type factor VIX. Assays for measuring the clotting activity of FIX proteins include the Biophen Factor IX Assay (Hyphen BioMed) and one-stage clotting assays (activated partial thromboplastin time (aPTT) (e.g., as described in EP 2032 607), thrombin generation time assay (TGA) and rotational thromboelastometry (e.g., as described in WO 2012 / 006624).

[0043] Numerous functional FIX variants are known and can be expressed by modified cells encapsulated in the devices described herein, including any of the functional FIX variants described in the following international patent publications: WO 02 / 040544, page 4, lines 9-30 and page 15, lines 6-31; WO 03 / 020764, Tables 2 and 3, pages 14-24, and page 12, lines 1-27. WO2007 / 149406, page 4, line 1 to page 19, line 11; WO2007 / 149406A2, page 19, line 12 to page 20, line 9; WO08 / 118507, page 5, line 14 to page 6, line 5; WO09 / 051717, page 9, line 11 to page 20, line 2; WO09 / 137254, page 2, paragraph

[0006] to page 5, paragraph

[0011] and page 16, paragraph

[0044] ~Page 24, paragraph

[0057] ; WO09 / 130198A2, page 4, line 26 ~ page 12, line 6; WO09 / 140015, page 11, paragraph

[0043] ~ page 13, paragraph

[0053] ; WO2012 / 006624; WO2015 / 086406.

[0044] In certain embodiments, the FIX polypeptide comprises a wild-type or variant sequence fused to a heterologous polypeptide or non-polypeptide moiety that extends the half-life of the FIX protein. Exemplary half-life extending moieties include Fc, albumin, PAS sequence, transferrin, CTP (the 28-amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) with its four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin-binding polypeptides, albumin-binding small molecules, or any combination thereof. An exemplary FIX polypeptide is the rFIXFc protein described in WO 2012 / 006624, which is a FIXFc single chain (FIXFc-sc) and an Fc single chain (Fc-sc) linked together via two disulfide bonds in the hinge region of Fc.

[0045] FIX variants also include gain and loss of function variants. An example of a gain of function variant is the "Padua" variant of human FIX, which has L (leucine) instead of R (arginine) at position 338 of the mature protein (corresponding to amino acid position 384 of SEQ ID NO: 20) and has higher catalytic and aggregation activity compared to wild-type human FIX (Chang et al., J. Biol. Chem., 273:12089-94 (1998)). An example of a loss-of-function variant is an alanine substituted for lysine at the fifth amino acid position from the start of the mature protein, which results in a protein with reduced binding to collagen IV (e.g., loss of function).

[0046] "Pancreatic islet cells," as used herein, refer to cells, including any naturally occurring or synthetically produced or modified cells, intended to partially or wholly reproduce, mimic, or otherwise express the function of a cell of a pancreatic islet of Langerhans. The term "pancreatic islet cells" includes glucose-responsive insulin-producing cells derived from stem cells, e.g., induced pluripotent stem cell lines.

[0047] A "polymer composition," as used herein, is a composition (e.g., a solution, a mixture) that includes one or more polymers. As a class, "polymers" include homopolymers, heteropolymers, copolymers, block polymers, and block copolymers, and can be both natural and synthetic. Homopolymers contain one type of building block, or monomer, while copolymers contain multiple types of monomers.

[0048] "Polypeptide," as used herein, refers to a polymer comprising amino acid residues linked via peptide bonds and having at least 2, and in some embodiments at least 3, 4, 5, 10, 50, 75, 100, 150, or 200 amino acid residues.

[0049] "Prevention," "prevent," and "preventing," as used herein, refer to treatments that involve administering a composition (or preparation) of a device encapsulating genetically modified cells that express an exogenous polypeptide prior to the onset of one or more symptoms of a disease or condition amenable to treatment with the exogenous polypeptide, to prevent the physical manifestation of the symptom(s). In some embodiments, "prevention," "prevent," and "preventing" require that signs or symptoms of the disease or condition have not yet occurred or been observed.

[0050] "RPE cells," as used herein, refer to cells having one or more of the following characteristics: a) retinal pigment epithelial cells (RPE) (e.g., the ARPE-19 cell line (ATCC OCRL-2302O), or cells cultured from, for example, the ARPE-19 cell line, derived from or modified by stably transfecting with an exogenous sequence encoding a polypeptide of interest or by inserting an exogenous sequence into one of the specific OCR insertion sites described herein, cells derived from primary cell cultures of RPE cells, naturally occurring RPE cells, including, for example, cells isolated directly from a human or other mammal (without long-term culture, e.g., less than 5 or 10 passages or rounds of cell division since isolation), transformed, immortalized, or derived from long-term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell culture; b) undifferentiated cells, e.g., RPE cells, or naturally occurring RPE cells or RPE cells except for any genetic manipulation. or c) cells obtained from cells that have been developed, programmed, or reprogrammed (e.g., in vitro) into cells that are substantially similar to one or more of the following: cells from primary or long-term cultures of RPE cells (e.g., the cells may be derived from IPS cells); or c) cells that have one or more of the following characteristics: i) express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) do not express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; iii) are naturally found in the retina and form a monolayer over the choroidal blood vessels in Bruch's membrane; or iv) are responsible for epithelial transport, light absorption, secretion, and immunoregulation in the retina; or v) are immortalized RPE cell lines (e.g., the ARPE-19 cell line (ATCC OCRL-2302O). Other exemplary RPE cell lines include ARPE-19-SEAP-2-neo cells, RPE-J cells, and hTERT RPE-1 cells. In one embodiment, the RPE described herein is engineered to have new properties, e.g., the cells are genetically modified by inserting at least one exogenous transcription unit into one or more of the OCR locations described herein.

[0051] "Sequence identity," or "percent identical," as used herein to refer to two nucleotide sequences or two amino acid sequences, means that the two sequences are identical within a specified region, or that when the two sequences are compared and aligned for maximum correspondence over a comparison window, or designated region, they have identical nucleotides or amino acids at a specified percentage of nucleotide or amino acid positions within a specified region. Sequence identity can be determined using standard techniques known in the art, including, but not limited to, any of the algorithms described in U.S. Patent Application Publication No. 2017 / 02334455 A1. In one embodiment, the specified percentage of identical nucleotide or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more.

[0052] As used herein, "spherical" refers to a device (e.g., a hydrogel capsule or other particle) with a curved surface that forms a sphere (e.g., a perfectly round ball) or sphere-like shape, which may have, for example, waves and undulations on the surface. Spheres and sphere-like objects can be mathematically defined by the rotation of a circle, an ellipse, or a combination thereof around each of three orthogonal axes a, b, and c. In a sphere, the three axes are of equal length. Typically, a sphere-like shape is an ellipsoid (about its average surface) with semi-major axes within 10%, 5%, or 2.5% of each other. The diameter of a sphere or sphere-like shape is the average diameter, e.g., the average of the semi-major axes.

[0053] "Ellipsoid," as the term is used herein to refer to a device (e.g., a hydrogel capsule, or other particle), means that the device (i) has a perfect, or classical, oblate, or prolate ellipsoid shape, or (ii) has a surface that roughly forms an ellipsoid, e.g., may have waves and undulations, and / or may be ellipsoidal (about its average surface) with semimajor axes within 100% of each other.

[0054] "Subject," as used herein, refers to a human or a non-human animal. In one embodiment, the subject is a human (i.e., male or female) of any age group, e.g., a pediatric human subject (e.g., infancy, childhood, adolescence) or an adult subject (e.g., young adult, middle-aged adult, or elderly adult). In one embodiment, the subject is a non-human animal, e.g., a mammal (e.g., a mouse, a dog, a primate (e.g., a cynomolgus monkey or a rhesus monkey)). In one embodiment, the subject is a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog) or a bird (e.g., a commercially relevant bird, e.g., a chicken, a duck, a goose, or a turkey). In certain embodiments, the animal is a mammal. The animal can be male or female and at any stage of development. The non-human animal can be a transgenic animal.

[0055] "Treatment," "treat," and "treating," as used herein, refer to one or more of reducing, reversing, alleviating, delaying the onset of, or inhibiting the progression of one or more of the symptoms, signs, or underlying causes of a disease (e.g., hemophilia A). In one embodiment, treating includes reducing, reversing, alleviating, delaying the onset of, or inhibiting the progression of a symptom or pathology associated with a disease. In one embodiment, treating includes increasing the level of a therapeutic polypeptide in at least one tissue of a subject in need thereof, e.g., in one or more of the plasma, liver, kidney, and heart. In some embodiments, "treatment," "treat," and "treating" require that a sign or symptom associated with a disease or condition has developed or been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease or condition, e.g., in prophylactic treatment. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., due to a history of symptoms and / or genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treatment includes prevention, while in other embodiments it does not. "Wild-type" (wt) refers to the naturally occurring form, including the sequence, of a polynucleotide, polypeptide, or protein in a species. The wild-type form is distinguished from mutant forms of a polynucleotide, polypeptide, or protein that result from genetic mutation(s).

[0056] Selected Chemical Definitions Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are listed in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 thEd. (inside cover), and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0057] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0058] When a range of values ​​is listed, it is intended to encompass each value and sub-range within the range. For example, "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0059] As used herein, "alkyl" refers to the radical of a straight- or branched-chain saturated hydrocarbon group having from 1 to 24 carbon atoms ("C1-C 24 In some embodiments, an alkyl group is a group having 1 to 12 carbon atoms ("C-C 12 alkyl), 1 to 10 carbon atoms ("C1-C12 In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C-C alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C-C alkyl"). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Each example of an alkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents; for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl").

[0060] As used herein, "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ("C2-C 24 In some embodiments, an alkenyl group refers to an alkyl group having 2 to 10 carbon atoms ("C-C"). 10C2-C4 alkenyl) has 2 to 8 carbon atoms ("C2-C8 alkenyl"), 2 to 6 carbon atoms ("C2-C6 alkenyl"), 2 to 5 carbon atoms ("C2-C5 alkenyl"), 2 to 4 carbon atoms ("C2-C4 alkenyl"), 2 to 3 carbon atoms ("C2-C3 alkenyl"), or 2 carbon atoms ("C2 alkenyl"). The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the C2-C6 alkenyl groups described above. 2-4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Each example of an alkenyl group independently may be optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted with one or more substituents; for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted alkenyl").

[0061] As used herein, the term "alkynyl" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 24 carbon atoms and one or more carbon-carbon triple bonds ("C2-C 24 In some embodiments, an alkynyl group refers to an alkyl group having 2 to 10 carbon atoms ("C-C"). 10C-C alkynyl groups have 2 to 8 carbon atoms ("C-C alkynyl"), 2 to 6 carbon atoms ("C-C alkynyl"), 2 to 5 carbon atoms ("C-C alkynyl"), 2 to 4 carbon atoms ("C-C alkynyl"), 2 to 3 carbon atoms ("C-C alkynyl"), or 2 carbon atoms ("C alkynyl"). The one or more carbon-carbon triple bonds can be internal (e.g., as in 2-butynyl) or terminal (e.g., as in 1-butynyl). Examples of C-C alkynyl groups include ethynyl (C), 1-propynyl (C), 2-propynyl (C), 1-butynyl (C), 2-butynyl (C), and the like. Each instance of an alkynyl group independently may be optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents; for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted alkynyl").

[0062] As used herein, the term "heteroalkyl" refers to an acyclic, stable, straight or branched chain, or combination thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms can be optionally oxidized and the nitrogen heteroatom can be optionally quaternized. The heteroatom(s) O, N, P, S, and Si can be substituted at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, and -O-CH2-CH3. Up to two or three heteroatoms may be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si(CH3)3. A "heteroalkyl" may be recited followed by a particular heteroalkyl group, e.g., -CHO, -NRC R D When the term heteroalkyl is used, the term -CH2O or -NR C R D It is understood that the terms "heteroalkyl" and "heteroalkyl-" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" refers to specific heteroalkyl groups, such as, for example, -CHO, -NR C R D and the like. Each example of a heteroalkyl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroalkyl") or substituted with one or more substituents; for example, with 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted heteroalkyl").

[0063] The terms "alkylene," "alkenylene," "alkynylene," or "heteroalkylene," by themselves or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively. An alkylene, alkenylene, alkynylene, or heteroalkylene group may be described, for example, as a C-C membered alkylene, a C-C membered alkenylene, a C-C membered alkynylene, or a C-C membered heteroalkylene, where the term "membered" refers to a non-hydrogen atom within the moiety. In the case of heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- can represent both -C(O)2R'- and -R'C(O)2-.

[0064] As used herein, "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array) having 6 to 14 ring carbon atoms and 0 heteroatoms provided in the aromatic ring system ("C6-C14 aryl"). In some embodiments, an aryl group has 6 ring carbon atoms ("C6 aryl", e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 aryl," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl. In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 Aryl, e.g., anthracyl). Aryl groups include, for example, C-C 10 The term "membered" refers to a non-hydrogen ring atom within a moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may independently be optionally substituted, i.e., unsubstituted (an "unsubstituted aryl") or substituted with one or more substituents (a "substituted aryl").

[0065] As used herein, "heteroaryl" refers to a radical of a 5-10-membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared by the cyclic array) having ring carbon atoms and 1 to 4 ring heteroatoms provided to the aromatic ring system ("5-10-membered heteroaryl"), where each heteroatom is independently selected from nitrogen, oxygen, and sulfur. In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, where valence permits. Heteroaryl bicyclic ring systems can contain one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems in which a heteroaryl ring, as defined above, is fused with one or more aryl groups, with the point of attachment being on either the aryl or heteroaryl ring; in such instances, the number of ring members refers to the number of ring members in the fused (aryl / heteroaryl) ring system. In bicyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., on the ring bearing the heteroatom (e.g., 2-indolyl) or on the ring that does not contain a heteroatom (e.g., 5-indolyl). Heteroaryl groups may be described, for example, as 6- to 10-membered heteroaryl, where the term "member" refers to a non-hydrogen ring atom within the moiety.

[0066] In some embodiments, heteroaryl groups are 5-10 membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms provided by the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In some embodiments, heteroaryl groups are 5-8 membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms provided by the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some embodiments, heteroaryl groups are 5-6 membered aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms provided by the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl"). In some embodiments, 5-6 membered heteroaryls have 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, 5-6 membered heteroaryls have 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has one ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of a heteroaryl group independently can be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroaryl") or substituted with one or more substituents (a "substituted heteroaryl").

[0067] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cissonolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.

[0068] As used herein, the terms "arylene" and "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.

[0069] As used herein, "cycloalkyl" refers to a group having 3 to 10 ring carbon atoms in a non-aromatic ring system ("C3-C 10 "cycloalkyl" refers to the radical of a non-aromatic cyclic hydrocarbon group having no heteroatoms. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C-C cycloalkyl"), 3 to 6 ring carbon atoms ("C-C cycloalkyl"), or 5 to 10 ring carbon atoms ("C-C 10 Cycloalkyl groups may be described, for example, as C4-C7 membered cycloalkyl, where the term "membered" refers to a non-hydrogen ring atom within the moiety. Exemplary C3-C6 cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 cycloalkyl groups include, but are not limited to, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), and the like. Exemplary C3-C 10 Cycloalkyl groups include, but are not limited to, the aforementioned C3-C8 cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10), and the like. As the foregoing examples illustrate, in certain embodiments, a cycloalkyl group is monocyclic ("monocyclic cycloalkyl") or contains fused, bridged, or spiro ring systems, e.g., bicyclic systems ("bicyclic cycloalkyl"), and can be saturated or partially unsaturated. "Cycloalkyl" also includes ring systems in which a cycloalkyl ring, as defined above, is fused to one or more aryl groups, and the point of attachment is on the cycloalkyl ring; in such cases, the number of carbons continues to refer to the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group independently can be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl").

[0070] "Heterocyclyl," as used herein, is the radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each heteroatom independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, where valence permits. Heterocyclyl groups may be either monocyclic ("monocyclic heterocyclyl") or fused, bridged, or spiro ring systems, e.g., bicyclic systems ("bicyclic heterocyclyl"), and may be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring, as defined above, is fused to one or more cycloalkyl groups, with the point of attachment being on either the cycloalkyl or heterocyclyl ring, or in which a heterocyclyl ring, as defined above, is fused to one or more aryl or heteroaryl groups, with the point of attachment being on the heterocyclyl ring; in such cases, the number of ring members continues to refer to the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described, for example, as a 3- to 7-membered heterocyclyl, where the term "member" refers to the non-hydrogen ring atoms in the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of heterocyclyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 10-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 10-membered heterocyclyl.

[0071] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (a "5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur (a "5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, each independently selected from nitrogen, oxygen, and sulfur (a "5- to 6-membered heterocyclyl"). In some embodiments, a 5- to 6-membered heterocyclyl has 1 to 3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur, hi some embodiments, the 5- to 6-membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0072] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl or thiomorpholinyl-1,1-dioxide. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl. Exemplary 5-membered heterocyclyl groups (also referred to herein as 5,6-bicyclic heterocyclic rings) fused to a C6 aryl ring include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, etc. Exemplary 6-membered heterocyclyl groups (also referred to herein as 6,6-bicyclic heterocyclic rings) fused to an aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[0073] "Amino," as used herein, refers to the radical -NR 70 R 71 (In the formula, R 70 and R 71 are each independently hydrogen, C1-C8 alkyl, C3-C 10 Cycloalkyl, C4-C 10 Heterocyclyl, C6-C 10 Aryl and C5-C 10 In some embodiments, amino refers to NH2.

[0074] As used herein, "cyano" refers to the group --CN.

[0075] As used herein, "halo," or "halogen," independently or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0076] As used herein, "hydroxy" refers to the group --OH.

[0077] Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" cycloalkyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl groups). Typically, the term "substituted," whether preceded by the term "optionally," means that at least one hydrogen present on the group (e.g., a carbon or nitrogen atom) is replaced with an acceptable substituent, e.g., a substituent that results in a stable compound (e.g., a compound that does not undergo spontaneous transformation by rearrangement, cyclization, elimination, or other reaction). Unless otherwise specified, a "substituted" group has a substituent at one or more substitutable positions of the group, and when multiple positions in any given structure are substituted, the substituents can be the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds, including any of the substituents described herein, that result in the formation of stable compounds. The present disclosure contemplates all such combinations in order to arrive at stable compounds. For purposes of this disclosure, heteroatoms such as nitrogen can have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0078] Two or more substituents may optionally be linked to form an aryl, heteroaryl, cycloalkyl, or heterocyclyl group. Such so-called ring-forming substituents are typically, but not necessarily, seen to be attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0079] The compounds of formula (I) described herein may contain one or more asymmetric centers and therefore may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers, or geometric isomers, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure additionally encompasses the compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0080] As used herein, a pure enantiomer compound is substantially free of other enantiomers, or stereoisomers, of the compound (i.e., in enantiomeric excess). That is, the "S" form of a compound is substantially free of the "R" form of the compound and is thus in enantiomeric excess of the "R" form. The terms "enantiomerically pure" or "pure enantiomer" indicate that a compound contains greater than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% of an enantiomer by weight. In certain embodiments, weights are based on the total weight of all enantiomers or stereoisomers of a compound.

[0081] The compounds of formula (I) described herein may also contain one or more isotopic substitutions. For example, H may be: 1 H, 2 H (D or deuterium), and 3 H can be in any isotopic form, including T or tritium; C can be in any isotopic form, including T or tritium; 12 C. 13 C, and 14 C can be in any isotopic form; O can be in any isotopic form, including 16 and 18 It can be any isotopic form containing O; and the like.

[0082] The term "pharmaceutically acceptable salt" is intended to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compound of formula (I) used to prepare the device of the present disclosure contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. When the compound used in the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphate, dihydrogenphosphate, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, etc. Also included are salts of amino acids, e.g., alginates, and salts of organic acids such as glucuronic acid or galacturonic acid (see, e.g., Berge et al., Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds used in the devices (e.g., particles, hydrogel capsules) of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. These salts can be prepared by methods known to those of ordinary skill in the art. Other pharmaceutically acceptable carriers known to those of ordinary skill in the art are suitable for use in the present disclosure.

[0083] The device of the present disclosure may contain a compound of formula (I) in a prodrug form. Prodrugs are compounds that readily undergo chemical changes under physiological conditions to provide compounds useful for preparing devices in the present disclosure. In addition, prodrugs can be converted to useful compounds of formula (I) by chemical or biochemical methods in an ex vivo environment.

[0084] Certain compounds of formula (I) described herein may exist in unsolvated and solvated forms, including hydrated forms. Generally, solvated forms are equivalent to unsolvated forms and are included within the scope of the present disclosure. Certain compounds of formula (I) described herein may exist in polycrystalline or amorphous forms. Generally, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0085] The term "solvate" refers to a form of a compound associated with a solvent, usually by solvolysis. This physical association may involve hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, for example, in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates, further including both stoichiometric and non-stoichiometric solvates.

[0086] The term "hydrate" refers to a compound associated with water. Typically, the number of water molecules contained in the hydrate of a compound is in a certain ratio to the number of compound molecules in the hydrate. Therefore, the hydrate of a compound can be represented by, for example, the general formula R×x H2O, where R is a compound and x is a number greater than 0.

[0087] As used herein, the term "tautomer" refers to an interchangeable form of a compound structure, where there is a change in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium between the displacement of π electrons and atoms (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomeric forms may be relevant to achieving optimal chemical reactivity and biological activity of a compound of interest.

[0088] Symbols used herein [ka] refers to a connection to an entity, e.g., a polymer (e.g., a hydrogel-forming polymer such as alginate) or to the surface of an implantable device, e.g., a particle, hydrogel capsule. [ka] The connection represented by may refer to a direct bond with an entity, e.g., a polymer or an implantable element, or may refer to a linkage with an entity via a linking group. As described herein, a "linking group" refers to a moiety for linking a compound of Formula (I) with an entity (e.g., a polymer or an implantable element (e.g., a device) described herein) and may include any linking chemistry known in the art. A list of exemplary linking groups can be found in Bioconjugate Techniques (3 rd ed. Greg T. Hermanson, Waltham, MA: Elsevier, Inc., 2013), which is incorporated herein by reference in its entirety. In some embodiments, the linking group is selected from alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)-, -OC(O)-, -N(R C )-, -N(R C )C(O)-, -C(O)N(R C )-, -N(R C )N(R D)-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F )y -, -Si(OR A) 2-, -Si(R G )(OR A )-, -B(OR A )-, or containing metal, R A , R C , R D , R F , R G , x, and y are each independently as described herein. In some embodiments, the linking group comprises an amine, a ketone, an ester, an amide, or an alkyl. In some embodiments, the linking group is a cross-linker. In some embodiments, the linking group is a -C(O)(C1-C6 1 and R 1 is as described herein. In some embodiments, the linking group is -C(O)(C1-C6-alkylene)-, where the alkylene is substituted with 1 to 2 alkyl groups (e.g., 1 to 2 methyl groups). In some embodiments, the linking group is -C(O)C(CH 3)2 In some embodiments, the linking group is -C(O)(methylene)-, where the alkylene is substituted with 1 to 2 alkyl groups (e.g., 1 to 2 methyl groups). In some embodiments, the linking group is -C(O)CH(CH3)-. In some embodiments, the linking group is -C(O)C(CH3)-.

[0089] Modified mammalian cells The present disclosure provides modified mammalian cells capable of modulating the level or function of MHC class I protein complexes, and optionally MHC class II protein complexes and / or CIITA, and inflammatory cytokines or profibrotic factors. In one embodiment, the modified mammalian cells reduce the level or function of MHC class I protein complexes, and optionally MHC class II protein complexes and / or CIITA, and inflammatory cytokines or profibrotic factors.

[0090] The MHC class I protein complex is a class of molecules present on the surface of nucleated cells that informs the host's immune system of the status of a particular antigen as self or non-self. MHC class I molecules present peptide fragments of cytotoxic proteins on the cell surface, which can trigger an immune response in the host if the cytotoxic protein is derived from a non-self source. Generally, MHC class I molecules are heterodimeric proteins consisting of two polypeptide chains. The alpha chain is polymorphic and is encoded by human leukocyte antigens (HLA) (including one of HLA-A, HLA-B, or HLA-C). The beta chain contains a beta-2-microglobulin (beta-2M) domain. The alpha and beta chains of each MHC class I molecule are non-covalently linked through an interaction between beta-2M and one of the transmembrane domains of the alpha chain (alpha-3). The alpha chain also contains two other domains, alpha-1 and alpha-2. In one embodiment, the modified mammalian cell of the present disclosure comprises a reduction in the level or function of an MHC class I protein complex or a component thereof, such as HLA-A, HLA-B, HLA-C, or Beta-2M.

[0091] Between alpha-1 and alpha-2 is a peptide-binding groove that binds peptides derived from cytosolic proteins. This groove consists of an eight-beta pleated sheet at the base and two alpha helices at the sides. Tyrosine residues flanking this groove create a closed end, limiting the size of peptides that can bind within the groove. Peptides within the groove remain bound for the lifetime of the class I molecule and are typically 8–9 amino acids long. Self or foreign cytosolic proteins are degraded via the proteasome and transported into the lumen of the ER. In the ER, peptides are loaded onto MHC class I with the assistance of a chaperone protein called tapasin. The peptide-bound MHC class I is then transported to the cell's plasma membrane, where it presents the peptide to CD8+ T cell receptors (Becar M et al. (2022) Physiology, MHC Class I. In: StatPearls [Internet]. Treasure Island, FL: StatPearls Publishing; 2023 Jan-).

[0092] In addition to interacting with beta-2M, the transmembrane alpha-3 domain interacts with the T cell receptor (TCR) co-receptor CD8, promoting antigen-specific activation. Although MHC class I binding to CD8 is approximately 100-fold weaker than TCR binding to MHC class I, alpha-3-CD8 binding enhances the affinity of TCR binding (Wooldridge et al. (2010) MHC Class I Molecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184:3357-3366).

[0093] Beta-2M is a non-glycosylated 12-kDa protein whose function is to stabilize the MHC class I alpha chain. Unlike the alpha chain, beta-2M does not span the membrane. The human beta-2M gene locus is located on chromosome 15. The beta-2M gene consists of four exons and three introns. Circulating forms of beta-2M are present in serum, urine, and other body fluids; therefore, non-covalently bound MHC I-associated beta-2M can exchange with circulating beta-2M under physiological conditions. Beta-2M binds not only to the alpha chain of MHC class I molecules, but also to class I-like molecules such as CD1 (five genes in humans), MR1, neonatal Fc receptor (FcRn), and Qa-1 (a form of alloantigen).

[0094] In some embodiments, the modified mammalian cell (e.g., a modified RPE cell, e.g., a modified ARPE-19 cell) comprises a reduced level or function of an MHC class I protein complex or a component thereof, such as HLA-A, HLA-B, HLA-C, or beta-2M. In some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduction in the alpha and / or beta chain of an MHC class I protein complex or a component thereof. For example, in some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduced level or function of the alpha-1 domain. In some embodiments, the level or function of the alpha-1 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the alpha-1 domain is reduced by about 20%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 30%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 40%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 50%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 60%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 70%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 80%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 90%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 100%.

[0095] In some embodiments, the level or function of the alpha-1 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the alpha-1 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-1 domain is reduced by more than 99.9%.

[0096] In some embodiments, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in the level or function of the alpha-2 domain. In some embodiments, the level or function of the alpha-2 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the alpha-2 domain is reduced by about 20%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 30%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 40%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 50%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 60%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 70%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 80%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 90%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 100%.

[0097] In some embodiments, the level or function of the alpha-2 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the alpha-2 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-2 domain is reduced by more than 99.9%.

[0098] In some embodiments, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in the level or function of the alpha-3 domain. In some embodiments, the level or function of the alpha-3 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the alpha-3 domain is reduced by about 20%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 30%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 40%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 50%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 60%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 70%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 80%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 90%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 100%.

[0099] In some embodiments, the level or function of the alpha-3 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the alpha-3 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-3 domain is reduced by more than 99.9%.

[0100] In some embodiments, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in the level or function of the beta-2M domain. In some embodiments, the level or function of the beta-2M domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the beta-2M domain is reduced by about 20%. In some embodiments, the level or function of the beta-2M domain is reduced by about 30%. In some embodiments, the level or function of the beta-2M domain is reduced by about 40%. In some embodiments, the level or function of the beta-2M domain is reduced by about 50%. In some embodiments, the level or function of the beta-2M domain is reduced by about 60%. In some embodiments, the level or function of the beta-2M domain is reduced by about 70%. In some embodiments, the level or function of the beta-2M domain is reduced by about 80%. In some embodiments, the level or function of the beta-2M domain is reduced by about 90%. In some embodiments, the level or function of the beta-2M domain is reduced by about 100%.

[0101] In some embodiments, the level or function of the beta-2M domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the beta-2M domain is reduced by at least 10%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 20%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 30%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 40%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 50%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 60%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 70%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 80%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 90%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 95%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 99%. In some embodiments, the level or function of the beta-2M domain is reduced by at least 99.9%. In some embodiments, the level or function of the beta-2M domain is reduced by more than 99.9%.

[0102] In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of beta-2M with an MHC class I protein complex or component thereof, or an MHC class I-like molecule or component (e.g., CD1, MR1, FcRn, and Qa-1) by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of beta-2M with an MHC class I protein complex or component thereof. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of beta-2M with the alpha-1 domain of an MHC class I protein. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of beta-2M with the alpha-2 domain of an MHC class I protein. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of Beta-2M to the alpha-3 domain of an MHC class I protein. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of Beta-2M to an MHC class I-like molecule or component thereof (e.g., CD1, MR1, FcRn, and Qa-1). In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of Beta-2M to CD1. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of Beta-2M to MR1. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of Beta-2M to FcRn.In some embodiments, the decrease in level or function substantially reduces, prevents, or inhibits the interaction (eg, binding) of beta-2M to Qa-1.

[0103] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha-3 domain to the TCR co-receptor CD8, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more.

[0104] HLA-A interacts with calnexin, calreticulin, transporter associated with antigen processing (TAP), tapasin, thiol-disulfide oxidoreductase ERp57 enzyme, and any cytosolic peptides bound within its peptide-binding groove. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A with calnexin, calreticulin, TAP, tapasin, ERp57 enzyme, and / or any cytosolic peptides bound within its peptide-binding groove, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A with calnexin. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A to calreticulin. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A to TAP. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A to TAP-1. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A to TAP-2. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A to tapasin. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A to ERp57. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A to cytosolic peptides bound within its peptide-binding groove.

[0105] HLA-C interacts with killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1) and the leukocyte immunoglobulin-like receptor family (e.g., leukocyte immunoglobulin-like receptor subfamily A member 1 (LILRA1) and LILRA3). In some embodiments, reducing the level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C to KIR2DL1 and the leukocyte immunoglobulin-like receptor family (e.g., LILRA1 and LILRA3), for example, by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, reducing the level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C to KIR2DL1. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C to the leukocyte immunoglobulin-like receptor family. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C to LILRA1. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C to LILRA3.

[0106] In some embodiments, a decrease in the level or function of the MHC class I protein complex or its components, such as HLA-A, HLA-B, HLA-C, or beta-2M, reduces antigen presentation, thereby reducing and / or abrogating the recruitment of immune cells, such as T cells and NK cells.

[0107] The HLA-A gene, located on the short arm of chromosome 6, encodes the larger alpha chain component of HLA-A. Variation in the HLA-A alpha chain is important for HLA function. This variation promotes genetic diversity within a population. Because each HLA has a different affinity for peptides of specific structures, more diverse HLAs mean that a greater variety of antigens can be "presented" on the cell surface, potentially increasing the likelihood that a subset of the population will be resistant to a given foreign invader. This reduces the likelihood that a single pathogen will have the ability to wipe out an entire human population.

[0108] Each individual can express up to two types of HLA-A, one from each of their parents. While some individuals inherit the same HLA-A from both parents, reducing individual HLA diversity, the majority of individuals receive two different copies of HLA-A. This same pattern applies to all HLA groups (Fix et al. (1998). HLA Matching, Antibodies, and Youth. Kidney Transplantation: Past, Present, and Future. University of Michigan Medical Center / Stanford University). In other words, every human can express only one or two of the 2,432 known HLA-A alleles.

[0109] The HLA-B gene is located on the short (p) arm of chromosome 6 at cytoband 21.3 and encodes the larger alpha chain component of HLA-B. As with HLA-A, variation in the HLA-B alpha chain is important for HLA function. HLA-C, a locus on chromosome 6, encodes many HLA-C alleles, a class-I MHC receptor. HLA-C, located proximal to the HLA-B locus, is located at the distal end of the HLA region. Most HLA-C:B haplotypes are in strong linkage disequilibrium, and many are as old as the human species itself.

[0110] In some embodiments, reducing the level or function of an MHC class I protein complex or a component thereof, e.g., HLA-A, HLA-B, HLA-C, or Beta-2M, comprises mutating one or more nucleotides in the nucleotide sequence of one or more genes selected from HLA-A, HLA-B, HLA-C, or Beta-2M. Nucleotide mutations can include nucleotide deletions, additions, and / or substitutions. As described herein, such mutations can result in reduced expression of the gene, for example, by reducing, altering, or abolishing transcription and / or splicing of the nucleotide sequence. For example, in some embodiments, reducing the level or function of an MHC class I protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of a Beta-2M gene. In some embodiments, reducing the level or function of an MHC class I protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of an HLA-A gene. In some embodiments, reducing the level or function of an MHC class I protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of an HLA-B gene. In some embodiments, reducing the level or function of an MHC class I protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of an HLA-C gene.

[0111] In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise a sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence identity to a nucleotide sequence provided in Table 5. For example, in some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise a sequence having at least 65% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise a sequence having at least 70% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 75% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 95% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 99% sequence identity to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5.

[0112] In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence identity to the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 65% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 70% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 75% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 95% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 99% sequence identity to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes comprise sequences having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5.

[0113] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in expression of MHC class I components, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except that it does not comprise a reduced level of MHC class I components. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in expression of MHC class I components, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell but for which the levels of MHC class I components are not reduced. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in expression of MHC class I components by about 50%, 75%, or greater than 90%, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell but for which the levels of MHC class I components are not reduced. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0114] In one embodiment, a modified mammalian cell (e.g., a modified RPE cell, e.g., a modified ARPE-19 cell) described herein comprises about 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in MHC class I component function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical except that it does not comprise a reduction in MHC class I component function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in MHC class I component function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell but for lack of a reduction in MHC class I component function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 50%, 75%, or greater than 90% reduction in MHC class I component function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell but for lack of a reduction in MHC class I component function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0115] In one embodiment, a modified mammalian cell (e.g., a modified RPE cell, e.g., a modified ARPE-19 cell) described herein comprises about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in HLA-A levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, or is identical, e.g., does not comprise a reduction in HLA-A levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in HLA-A levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the lack of a reduction in HLA-A levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in HLA-A levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the lack of a reduction in HLA-A levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0116] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in HLA-B levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, or is identical to the modified mammalian cell, except for, e.g., not comprising a reduction in HLA-B levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in HLA-B levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in HLA-B levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in HLA-B levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in HLA-B levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0117] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in HLA-C levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, or is identical to the modified mammalian cell, except for, e.g., not comprising a reduction in HLA-C levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in HLA-C levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in HLA-C levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in HLA-C levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in HLA-C levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0118] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in beta-2M levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, or is identical to the modified mammalian cell, except for, e.g., not comprising a reduction in beta-2M levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in beta-2M levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in beta-2M levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in beta-2M levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in beta-2M levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0119] In some embodiments, the MHC class I protein complex or a component thereof, such as HLA-A, HLA-B, HLA-C, or Beta-2M, is encoded by one or more nucleotide sequences provided in Table 5, or fragments thereof.

[0120] In some embodiments, the decrease in the level or function of an MHC class I protein complex or a component thereof, such as HLA-A, HLA-B, HLA-C, or beta-2M, persists for at least 15 minutes (e.g., 30 minutes, 1 hour, 12 hours, 24 hours, 48 ​​hours, 72 hours, 1 week, 1 month, or 1 year). For example, in some embodiments, the decrease in the level or function of an MHC class I protein complex or a component thereof persists for at least 30 minutes. In some embodiments, the decrease in the level or function of an MHC class I protein complex or a component thereof persists for at least 1 hour. In some embodiments, the decrease in the level or function of an MHC class I protein complex or a component thereof persists for at least 12 hours. In some embodiments, the decrease in the level or function of an MHC class I protein complex or a component thereof persists for at least 24 hours. In some embodiments, the decrease in the level or function of an MHC class I protein complex or a component thereof persists for at least 48 hours. In some embodiments, the decrease in the level or function of an MHC class I protein complex or a component thereof persists for at least 72 hours. In some embodiments, the reduction in the level or function of an MHC class I protein complex or a component thereof persists for at least one week. In some embodiments, the reduction in the level or function of an MHC class I protein complex or a component thereof persists for at least one month. In some embodiments, the reduction in the level or function of an MHC class I protein complex or a component thereof persists for at least one year.

[0121] The MHC class II protein complex is a class of molecules present on the surface of antigen-presenting cells within a subject, such as dendritic cells, mononuclear phagocytes, certain endothelial cells, and B cells. One important distinguishing feature between MHC class II and MHC class I protein complexes is that the antigens presented by MHC class II protein complexes are derived from extracellular proteins, unlike the cytosolic antigens presented by MHC class I protein complexes. Like MHC class I protein complexes, MHC class II protein complexes are heterodimeric proteins consisting of two polypeptide chains, an alpha chain and a beta chain. Unlike MHC class I protein complexes, the alpha and beta chains of MHC class II protein complexes contain identical peptides. The alpha-peptides contain alpha-1 and beta-1 domains, which together form a membrane-distal peptide-binding groove, while the beta-peptides contain alpha-2 and beta-2 domains, which form a membrane-proximal immunoglobulin-like domain. The peptide-binding groove is composed of two alpha-helical walls and a beta sheet. The antigen-binding groove of MHC class II molecules is open at both ends, whereas the corresponding groove of class I molecules is closed at both ends, so antigens presented by MHC class II molecules are longer, generally 15–24 amino acid residues in length.

[0122] Exemplary MHC class II protein complex components include HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. In some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduced level or function of an MHC class II protein complex or a component thereof, e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR. In some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduction in the alpha and / or beta chain of an MHC class II protein complex or a component thereof. For example, in some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduced level or function of the alpha-1 domain. In some embodiments, the level or function of the alpha-1 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the alpha-1 domain is reduced by about 20%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 30%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 40%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 50%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 60%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 70%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 80%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 90%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 100%.

[0123] In some embodiments, the level or function of the alpha-1 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the alpha-1 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-1 domain is reduced by more than 99.9%.

[0124] In some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of the alpha-2 domain. In some embodiments, the level or function of the alpha-2 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the alpha-2 domain is reduced by about 20%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 30%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 40%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 50%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 60%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 70%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 80%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 90%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 100%.

[0125] In some embodiments, the level or function of the alpha-2 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the alpha-2 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-2 domain is reduced by more than 99.9%.

[0126] In some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of the beta-1 domain. In some embodiments, the level or function of the beta-1 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the beta-1 domain is reduced by about 20%. In some embodiments, the level or function of the beta-1 domain is reduced by about 30%. In some embodiments, the level or function of the beta-1 domain is reduced by about 40%. In some embodiments, the level or function of the beta-1 domain is reduced by about 50%. In some embodiments, the level or function of the beta-1 domain is reduced by about 60%. In some embodiments, the level or function of the beta-1 domain is reduced by about 70%. In some embodiments, the level or function of the beta-1 domain is reduced by about 80%. In some embodiments, the level or function of the beta-1 domain is reduced by about 90%. In some embodiments, the level or function of the beta-1 domain is reduced by about 100%.

[0127] In some embodiments, the level or function of the beta-1 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the beta-1 domain is reduced by at least 10%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 20%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 30%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 40%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 50%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 60%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 70%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 80%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 90%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 95%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 99%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 99.9%. In some embodiments, the level or function of the beta-1 domain is reduced by more than 99.9%.

[0128] In some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of the beta-2 domain. In some embodiments, the level or function of the beta-2 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the beta-2 domain is reduced by about 20%. In some embodiments, the level or function of the beta-2 domain is reduced by about 30%. In some embodiments, the level or function of the beta-2 domain is reduced by about 40%. In some embodiments, the level or function of the beta-2 domain is reduced by about 50%. In some embodiments, the level or function of the beta-2 domain is reduced by about 60%. In some embodiments, the level or function of the beta-2 domain is reduced by about 70%. In some embodiments, the level or function of the beta-2 domain is reduced by about 80%. In some embodiments, the level or function of the beta-2 domain is reduced by about 90%. In some embodiments, the level or function of the beta-2 domain is reduced by about 100%.

[0129] In some embodiments, the level or function of the beta-2 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the beta-2 domain is reduced by at least 10%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 20%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 30%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 40%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 50%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 60%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 70%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 80%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 90%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 95%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 99%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 99.9%. In some embodiments, the level or function of the beta-2 domain is reduced by more than 99.9%.

[0130] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha chain of an MHC class II protein complex or a component thereof with a beta chain, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of an alpha-1 domain with an alpha-2 or beta-2 domain. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of a beta-1 domain with an alpha-2 or beta-2 domain. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of an alpha-2 domain with an alpha-1 or beta-1 domain. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of the beta-2 domain with the alpha-1 domain or the beta-1 domain. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha-1 domain with the beta-1 domain. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha-2 domain with the beta-2 domain.

[0131] HLA-DP is a protein / peptide antigen receptor and a graft-versus-host disease antigen composed of two subunits, DPα and DPβ. DPα and DPβ are encoded by two loci, HLA-DPA1 and HLA-DPB1, found in the MHC class II (or HLA-D) region on human chromosome 6 within the HLA complex. HLA-DP is an αβ-heterodimeric cell surface receptor. Each DP subunit (α subunit, β subunit) consists of an α-helical N-terminal domain, an IgG-like β-sheet, a transmembrane domain, and a cytoplasmic domain. The α-helical domain forms the sides of the peptide-binding groove. The β-sheet region forms the base of the binding groove and the majority of the molecule, as well as the intersubunit (noncovalent) binding region. The peptide-bound HLA-DP complex interacts with the TCR on CD4+ T cells.

[0132] In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits, e.g., 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more, the interaction (e.g., binding) of HLA-DP to the TCR of CD4+ T cells and / or any peptide (e.g., antigenic peptide) bound within its peptide-binding groove. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DP to the TCR of CD4+ T cells. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DP to peptides bound within its peptide-binding groove.

[0133] HLA-DM, a non-classical MHC molecule, is an intracellular protein involved in the mechanism of antigen presentation and is encoded by the HLA-DMA and HLA-DMB genes. Like HLA-DP, the HLA-DM gene is located in the MHC II region of human chromosome 6. HLA-DM is a molecular chaperone that functions in lysosomes and endosomes within cells of the immune system. It functions by interacting with MHC class II molecules on antigen-specific receptors (APCs) such as macrophages, dendritic cells, and B cells (Arndt et al. (2000) "Functional HLA-DM on the surface of B cells and immature dendritic cells". The EMBO Journal 19 6):1241-51. doi:10.1093 / emboj / 19.6.1241; Pashine et al. (2003) "Interaction of HLA-DR with an acidic face of HLA-DM disrupts sequence-dependent interactions with peptides". Immunity. 19(2):183-92. doi:10.1016 / S1074-7613(03)00200-0). HLA-DM not only protects MHC class II molecules from degradation but also controls which proteins or peptides bind to them. This controls when and how peptides act as antigens to initiate immune responses. HLA-DM is required to release CLIP (fragments resulting from cathepsin S- or cathepsin D-mediated cleavage of CD74) from MHC class II molecules, chaperone empty MHC molecules from denaturation, facilitate antigen-antigen exchange (e.g., by releasing weakly bound peptides from the peptide-binding groove to load peptides with higher affinity binding), and regulate proper loading and release of peptides in the peptide-binding groove.To release peptides from the MHC groove, HLA-DM binds to the N-terminus of the groove, causing a conformational change and disrupting hydrogen bonds, resulting in the loss of peptide binding and subsequent excretion (Yin et al. (2015) "Evaluating the Role of HLA-DM in MHC Class II-Peptide Association Reactions". Journal of Immunology. 195(2):706-16. doi:10.4049 / jimmunol.1403190). HLA-DM helps catalyze peptide exchange not only in late endosomes exiting the ER, but also on the plasma membrane and in early endosomes. While much of this pathway is still being explored, it is known that HLA-DM can load exogenous peptides onto MHC Class II molecules when they are expressed on the cell surface. Loading can also occur in rapidly recycling early endosomes. HLA-DM does not have the capacity to bind peptides as it lacks a deep peptide-binding groove, but instead contains a shallow, negatively charged cavity with two disulfide bonds.

[0134] HLA-DM also strongly interacts with another non-classical MHC molecule, the chaperone protein HLA-DO. HLA-DO begins to bind to DM in early endosomes but is less expressed in late endosomes / lysosomes. The binding between HLA-DM and HLA-DO is weaker at low pH, but overall is much stronger than HLA-DM binding to MHC molecules. Both HLA-DM and HLA-DO lack the N-terminal trafficking signal. Before antigen contact, DO acts as a chaperone for DM, stabilizing it against denaturation and directing it to lysosomes. It binds to HLA-DM at the same position as MHC class II molecules, thereby preventing HLA-DM from binding to MHC class II molecules. This inhibits peptide exchange catalysis and retains CLIP in the MHC groove until antigen-containing lysosomes fuse with DM / DO / MHC-containing lysosomes, facilitating degradation of HLA-DO molecules. The alpha chain of HLA-DO (HLA-DOA) is encoded by the HLA-DOA gene, and the beta chain (HLA-DOB) is encoded by the HLA-DOB gene.

[0135] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DM with CLIP, HLA-DO, and the beta chain of MHC class II molecules, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DM with CLIP. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DM with HLA-DO. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DO with HLA-DM. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (eg, binding) of HLA-DM to the beta chain of an MHC class II molecule.

[0136] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DO to HLA-DM or any peptide bound in its peptide-binding groove, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DO to HLA-DM. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DO to peptides bound in its peptide-binding groove.

[0137] HLA-DQ is a cell surface receptor protein found on antigen-presenting cells. It is an αβ heterodimer of the MHC class II type. The alpha and beta chains are encoded by two adjacent loci, HLA-DQA1 and HLA-DQB1, on chromosome band 6p21.3. Both the alpha and beta chains contain a large number of variants. Humans often produce two alpha and two beta chain variants, resulting in four isoforms of HLA-DQ. The HLA-DQ locus is closely linked genetically to HLA-DR and less closely linked to HLA-DP, non-classical MHC class II molecules (HLA-DM and HLA-DO), and MHC class I molecules.

[0138] Different isoforms of HLA-DQ can bind to T cells and present different antigens to them. During this process, T cells are stimulated to proliferate and can signal B cells to produce antibodies. HLA-DQ functions in the recognition and presentation of foreign antigens (proteins derived from potential pathogens). For example, peptide-bound HLA-DP complexes interact with the TCR on CD4+ T cells. HLA-DQ is also involved in recognizing common self-antigens and presenting them to the immune system from a very early age to develop tolerance. Loss of tolerance to self-proteins can contribute to autoimmune diseases. Two autoimmune diseases involving HLA-DQ are celiac disease and type 1 diabetes. HLA-DQ mediates autoimmunity by skewing the TCR repertoire during thymic selection (Rubio et al. (2021) "HLA class II mediates type 1 diabetes risk by anti-insulin repertoire selection". bioRxiv:2021.09.06.458974.doi:10.1101 / 2021.09.06.458974). Carriers of risk serotypes, such as HLA-DQ8, have a high proportion of circulating T cell receptors capable of binding insulin, a major autoantigen in type 1 diabetes.

[0139] In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more, the interaction (e.g., binding) of HLA-DQ to the TCR of CD4+ T cells and / or any peptide bound within its peptide-binding groove. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DQ to the TCR of CD4+ T cells. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DQ to peptides bound within its peptide-binding groove.

[0140] HLA-DR is a cell surface receptor, an αβ heterodimer, each subunit of which contains two extracellular domains, a transmembrane domain, and a cytoplasmic tail. Both the α and β chains are membrane-anchored. The N-terminal domain of the mature protein forms an alpha helix that constitutes the exposed portion of the binding groove, while the C-terminal cytoplasmic region interacts with the other chain to form a beta sheet below the binding groove that extends to the cell membrane. Most of the peptide contact sites are within the first 80 residues of each chain. HLA-DR is encoded by several loci and several "genes" with different functions at each locus. The DR α-chain is encoded by the HLA-DRA locus. Unlike other DR loci, there is no functional variation in the mature DRA gene product. The DRβ-chain is encoded by four loci, but no more than three functional loci exist in a single individual, and no more than two functional loci exist on a single chromosome (Marsh et al. (2010) "Nomenclature for factors of the HLA system, 2010". Tissue Antigens. 75(4):291-455. doi:10.1111 / j.1399-0039.2010.01466.x). In some cases, individuals may have only two copies of the same locus, DRB1. The HLA-DRB1 locus is widely distributed and encodes a large number of functionally variable gene products (HLA-DR1 to HLA-DR17). The HLA-DRB3 locus, encoding HLA-DR52, is moderately variable and variably associated with certain HLA-DRB1 types. The HLA-DRB4 locus encodes HLA-DR53, which is somewhat variable and associated with certain HLA-DRB1 types. The HLA-DRB5 locus encodes HLA-DR51, which is typically invariant and linked to HLA-DR2 types. HLA-DR interacts with CD74, HLA-DM, the TCR of CD4+ T cells, and / or any peptide bound within its peptide-binding groove.

[0141] In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits, e.g., 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more, the interaction (e.g., binding) of HLA-DR with CD74, HLA-DM, the TCR of CD4+ T cells, and / or any peptide bound within its peptide-binding groove. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DR with CD74. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DR with HLA-DM. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DR with the TCR of CD4+ T cells. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DR to peptides bound within its peptide-binding groove.

[0142] In some embodiments, a decrease in the level or function of the MHC class II protein complex or its components, such as HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR, reduces antigen presentation, thereby reducing and / or abrogating the recruitment of immune cells, such as T cells and NK cells.

[0143] In some embodiments, reducing the level or function of an MHC class II protein complex or a component thereof, e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR, comprises mutating one or more nucleotides in the nucleotide sequence of one or more genes selected from HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5. Nucleotide mutations can include nucleotide deletions, additions, and / or substitutions. As described herein, such mutations can result in reduced expression of the gene by, for example, reducing, altering, or abrogating transcription and / or splicing of the nucleotide sequence. For example, in some embodiments, reducing the level or function of an MHC class II protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DP. In some embodiments, reducing the level or function of an MHC class II protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DM. In some embodiments, reducing the level or function of an MHC class II protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DOA. In some embodiments, reducing the level or function of an MHC class II protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DOB. In some embodiments, reducing the level or function of an MHC class II protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DP. In some embodiments, reducing the level or function of an MHC class II protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DM.In some embodiments, reducing the level or function of an MHC class II protein complex or its component comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DOA. In some embodiments, reducing the level or function of an MHC class II protein complex or its component comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DOB. In some embodiments, reducing the level or function of an MHC class II protein complex or its component comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DQA1. In some embodiments, reducing the level or function of an MHC class II protein complex or its component comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DQB1. In some embodiments, reducing the level or function of an MHC class II protein complex or its component comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DRA. In some embodiments, reducing the level or function of an MHC class II protein complex or its component comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DRB1. In some embodiments, reducing the level or function of an MHC class II protein complex or its component comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DRB3. In some embodiments, reducing the level or function of an MHC class II protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DRB4. In some embodiments, reducing the level or function of an MHC class II protein complex or a component thereof comprises mutating one or more nucleotides in the nucleotide sequence of HLA-DRB5.

[0144] In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence identity to the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 65% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 70% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 75% sequence identity to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 95% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 99% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5.

[0145] In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence identity to the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 65% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 70% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 75% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 80% sequence identity to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 95% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 99% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprise sequences having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5.

[0146] In one embodiment, the modified mammalian cell of the present disclosure comprises a reduced level or function of an MHC class II protein complex or a component thereof, such as HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR. In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in expression of MHC class II components, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except that it does not comprise a reduced level of MHC class II components. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in expression of MHC class II components, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell but for which the levels of MHC class II components are not reduced. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in expression of MHC class II components by about 50%, 75%, or more than 90%, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell but for which the levels of MHC class II components are not reduced. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0147] In one embodiment, a modified mammalian cell (e.g., a modified RPE cell, e.g., a modified ARPE-19 cell) described herein comprises about 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in MHC class II component function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except that it does not comprise a reduction in MHC class II component function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in MHC class II component function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell but for lack of a reduction in MHC class II component function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 50%, 75%, or greater than 90% reduction in MHC class I component function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell but for lack of a reduction in MHC class II component function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0148] In some embodiments, the MHC class II protein complex or a component thereof, such as HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR, is encoded by one or more nucleotide sequences provided in Table 5, or fragments thereof.

[0149] In some embodiments, the decreased level or function of an MHC class II protein complex or its component lasts for at least 15 minutes (e.g., 30 minutes, 1 hour, 12 hours, 24 hours, 48 ​​hours, 72 hours, 1 week, 1 month, or 1 year). For example, in some embodiments, the decreased level or function of an MHC class II protein complex or its component lasts for at least 30 minutes. In some embodiments, the decreased level or function of an MHC class II protein complex or its component lasts for at least 1 hour. In some embodiments, the decreased level or function of an MHC class II protein complex or its component lasts for at least 12 hours. In some embodiments, the decreased level or function of an MHC class II protein complex or its component lasts for at least 24 hours. In some embodiments, the decreased level or function of an MHC class II protein complex or its component lasts for at least 48 hours. In some embodiments, the decreased level or function of an MHC class II protein complex or its component lasts for at least 72 hours. In some embodiments, the decreased level or function of an MHC class II protein complex or its component lasts for at least 1 week. In some embodiments, the reduction in the level or function of an MHC class II protein complex or a component thereof persists for at least one month, hi some embodiments, the reduction in the level or function of an MHC class II protein complex or a component thereof persists for at least one year.

[0150] The class II major histocompatibility complex transactivator (CIITA) is a gene involved in the regulation of the expression of the MHC class II protein complex. The CIITA gene is located on chromosome 16 and encodes the CIITA protein, which plays a role in enhancing the transcription of MHC class I genes. The CIITA protein contains an acidic transcription activation domain, four leucine-rich repeats, and a GTP-binding domain. The protein uses GTP binding to promote its own transport into the nucleus. Once inside the nucleus, the protein acts as a positive regulator of class II major histocompatibility complex gene transcription and is often referred to as the "master regulator" of the expression of these genes (Harton et al. (2000) "Class II transactivator: mastering the art of major histocompatibility complex expression" Molecular and Cellular Biology. 20(17):6185-94. doi:10.1128 / MCB.20.17.6185-6194.2000; LeibundGut-Landmann et al. (2004) "Mini-review: Specificity and expression of CIITA, the master regulator of MHC class II genes" European Journal of Immunology. 34(6):1513-25. doi:10.1002 / eji.200424964). CIITA expression is induced by interferon-gamma (Heuberger et al. (2021) “Why do intestinal epithelial cells express MHC class II?” Immunology. 162(4):357-367. doi:10.1111 / imm.13270).

[0151] In one embodiment, the modified mammalian cell of the present disclosure comprises a reduction in the level or function of a CIITA protein. In some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of a domain of the CIITA protein, wherein the domain is selected from a transcription activation domain, a leucine-rich repeat domain, and a GTP-binding domain. For example, in some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of the transcription activation domain. In some embodiments, the level or function of the transcription activation domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the transcription activation domain is reduced by about 20%. In some embodiments, the level or function of the transcription activation domain is reduced by about 30%. In some embodiments, the level or function of the transcription activation domain is reduced by about 40%. In some embodiments, the level or function of the transcription activation domain is reduced by about 50%. In some embodiments, the level or function of the transcription activation domain is reduced by about 60%. In some embodiments, the level or function of the transcription activation domain is reduced by about 70%. In some embodiments, the level or function of the transcription activation domain is reduced by about 80%. In some embodiments, the level or function of the transcription activation domain is reduced by about 90%. In some embodiments, the level or function of the transcription activation domain is reduced by about 100%.

[0152] In some embodiments, the level or function of the transcription activation domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the transcription activation domain is reduced by at least 10%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 20%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 30%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 40%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 50%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 60%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 70%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 80%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 90%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 95%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 99%. In some embodiments, the level or function of the transcription activation domain is reduced by at least 99.9%. In some embodiments, the level or function of the transcription activation domain is reduced by more than 99.9%.

[0153] In some embodiments, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in the level or function of a leucine-rich repeat domain. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 20%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 30%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 40%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 50%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 60%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 70%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 80%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 90%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by about 100%.

[0154] In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 10%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 20%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 30%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 40%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 50%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 60%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 70%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 80%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 90%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 95%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 99%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 99.9%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by more than 99.9%.

[0155] In some embodiments, the modified mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of a GTP-binding domain. In some embodiments, the level or function of the GTP-binding domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the GTP-binding domain is reduced by about 20%. In some embodiments, the level or function of the GTP-binding domain is reduced by about 30%. In some embodiments, the level or function of the GTP-binding domain is reduced by about 40%. In some embodiments, the level or function of the GTP-binding domain is reduced by about 50%. In some embodiments, the level or function of the GTP-binding domain is reduced by about 60%. In some embodiments, the level or function of the GTP-binding domain is reduced by about 70%. In some embodiments, the level or function of the GTP-binding domain is reduced by about 80%. In some embodiments, the level or function of the GTP-binding domain is reduced by about 90%. In some embodiments, the level or function of the GTP-binding domain is reduced by about 100%.

[0156] In some embodiments, the level or function of the GTP-binding domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or more). For example, in some embodiments, the level or function of the GTP-binding domain is reduced by at least 10%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 20%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 30%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 40%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 50%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 60%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 70%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 80%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 90%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 95%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 99%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 99.9%. In some embodiments, the level or function of the GTP-binding domain is reduced by more than 99.9%.

[0157] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of a CIITA protein domain with another CIITA protein domain, for example, by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of a transcription activation domain with a leucine-rich repeat domain or a GTP-binding domain. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of a leucine-rich repeat domain with a transcription activation domain, a leucine-rich repeat domain, or a GTP-binding domain. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of a GTP-binding domain with a transcription activation domain or a leucine-rich repeat domain.

[0158] The CIITA protein interacts with mitogen-activated protein kinase 1 (MAPK1), nuclear receptor coactivator 1 (NCOA1), DNA-binding protein RFX5 (RFX5), DNA-binding protein RFXANK (RFXANK), exportin 1 (XPO1), and zinc finger, X-linked, duplicated family member C (ZXDC). In some embodiments, reducing the level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA with MAPK1, NCOA1, RFX5, RFXANK, XPO1, and / or ZXDC, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, reducing the level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA with MAPK1. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA to NCOA1. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA to RFX5. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA to RFXANK. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA to XPO1. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA to ZXDC.

[0159] In some embodiments, a decrease in the level or function of CIITA protein (e.g., by a decrease in the level of transcription) results in a decrease in the level of expression of the MHC class II protein complex or its components, such as HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR.

[0160] In some embodiments, reducing the level or function of the CIITA protein comprises mutating one or more nucleotides in the nucleotide sequence of the CIITA gene. The nucleotide mutation may include a deletion, addition, and / or substitution of a nucleotide. As described herein, such a mutation may result in reduced expression of the gene, for example, by reducing, altering, or abrogating the transcription and / or splicing of the nucleotide sequence.

[0161] In some embodiments, the nucleotide sequence of the CIITA gene comprises a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence identity to a nucleotide sequence provided in Table 5. For example, in some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 65% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 70% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 75% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 80% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 85% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 90% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 95% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 99% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 99.9% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having more than 99.9% sequence identity to the nucleotide sequence provided in Table 5.

[0162] In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence homology to a nucleotide sequence provided in Table 5. For example, in some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 65% sequence homology to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 70% sequence homology to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 75% sequence homology to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 80% sequence homology to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 85% sequence homology to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 90% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 95% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 99% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 99.9% sequence identity to a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having more than 99.9% sequence identity to a nucleotide sequence provided in Table 5.

[0163] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise a reduction in expression of CIITA of about 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except that it does not comprise a reduced level of CIITA. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in CIITA expression, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, but for the lack of a reduced level of CIITA. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in CIITA expression of about 50%, 75%, or greater than 90%, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, but for the lack of a reduced level of CIITA. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0164] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in CIITA function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical except that it does not comprise a reduction in CIITA function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in CIITA function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, but for lack of CIITA reduction. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 50%, 75%, or greater than 90% reduction in CIITA function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, but for lack of CIITA reduction. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0165] In some embodiments, CIITA is encoded by a nucleotide sequence provided in Table 5, or a fragment thereof.

[0166] In some embodiments, the decrease in CIITA level or function persists for at least 15 minutes (e.g., 30 minutes, 1 hour, 12 hours, 24 hours, 48 ​​hours, 72 hours, 1 week, 1 month, or 1 year). For example, in some embodiments, the decrease in CIITA level or function persists for at least 30 minutes. In some embodiments, the decrease in CIITA level or function persists for at least 1 hour. In some embodiments, the decrease in CIITA level or function persists for at least 12 hours. In some embodiments, the decrease in CIITA level or function persists for at least 24 hours. In some embodiments, the decrease in CIITA level or function persists for at least 48 hours. In some embodiments, the decrease in CIITA level or function persists for at least 72 hours. In some embodiments, the decrease in CIITA level or function persists for at least 1 week. In some embodiments, the decrease in CIITA level or function persists for at least 1 month. In some embodiments, the decrease in CIITA level or function persists for at least 1 year.

[0167] In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduced level or function of MHC class I protein complexes and a reduced level or function of MHC class II protein complexes and / or CIITA. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduced level or function of HLA-A and a reduced level or function of MHC class II protein complexes and / or CIITA. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduced level or function of HLA-B and a reduced level or function of MHC class II protein complexes and / or CIITA. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduced level or function of HLA-C and a reduced level or function of MHC class II protein complexes and / or CIITA. In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise a reduced level or function of beta-2M and a reduced level or function of the MHC class II protein complex and / or CIITA. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0168] Modified mammalian cells containing proinflammatory cytokines and / or profibrotic factors The present disclosure further features modified mammalian cells comprising pro-inflammatory cytokines and / or pro-fibrotic factors. Pro-inflammatory cytokines are signaling molecules secreted by immune cells that can promote or induce inflammation in a host. Exemplary pro-inflammatory cytokines include interleukin 6 (IL-6), IL-8, IL-10, IL-1-beta, monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor alpha (TNF-alpha). In one embodiment, the modified mammalian cells comprise a reduced level or function of a pro-inflammatory cytokine selected from, for example, IL-6, IL-8, IL-10, IL-1-beta, MCP-1, and TNF-alpha.

[0169] IL-6, encoded by the IL-6 gene, is a cytokine characterized by pleiotropic activities. It induces the synthesis of acute-phase proteins, such as CRP, serum amyloid A, fibrinogen, and hepcidin, while inhibiting albumin production. IL-6 also plays an important role in adaptive immune responses by stimulating antibody production and the development of effector T cells. Furthermore, IL-6 can promote the differentiation or proliferation of several non-immune cells. Continuous production of IL-6 can lead to the onset or development of various diseases (Tanaka et al. (2014). IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol. 2014 Sep 4;6(10):a016295. doi:10.1101 / cshperspect.a016295.). IL-6 interacts with its receptor, IL-6R, as well as IL-7 and IL-15.

[0170] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-6R, IL-7, and / or IL-15, for example, by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-6R, for example, by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-6R, for example, by 10% or more. In some embodiments, the reduction in level or function results in a substantial reduction, prevention, or inhibition of IL-6 interaction (e.g., binding) to IL-6R, for example, by 20% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of IL-6 interaction (e.g., binding) to IL-6R, for example, by 30% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of IL-6 interaction (e.g., binding) to IL-6R, for example, by 40% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of IL-6 interaction (e.g., binding) to IL-6R, for example, by 50% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of IL-6 interaction (e.g., binding) to IL-6R, for example, by 75% or more. The reduction in the level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-6R, e.g., by 90% or more. The reduction in the level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-6R, e.g., by 95% or more.

[0171] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-7, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-7, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-7, e.g., by 20% or more. The reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-7, e.g., by 30% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-7, for example, by 40% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-7, for example, by 50% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-7, for example, by 75% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-7, for example, by 90% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-7, for example, by 95% or more.

[0172] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-15 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-15 by, for example, 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-15 by, for example, 20% or more. The reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-6 to IL-15 by, for example, 30% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-15, for example, by 40% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-15, for example, by 50% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-15, for example, by 75% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-15, for example, by 90% or more. The reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-6 to IL-15, for example, by 95% or more.

[0173] IL-8, encoded by the IL-8 gene, is a cytokine that induces chemotaxis in target cells (mainly neutrophils, but also other granulocytes) to move toward the site of infection and further stimulates phagocytosis after the target cells reach the site of infection. IL-8 also induces a series of physiological responses in target cells that are necessary for migration and phagocytosis, such as intracellular Ca2+. 2+IL-8 is also known to be a potent promoter of angiogenesis by inducing IL-8 production, exocytosis (e.g., histamine release), and increased respiratory burst. IL-8 can be secreted by any cell bearing Toll-like receptors (TLRs) involved in the innate immune response and has been demonstrated to be the signature chemokine of complement receptor 2 (CR2)+ naive T cells, also known as recent thymic emigrants (Pekalski et al. (2017). "Neonatal and adult recent thymic emigrants produce IL-8 and express complement receptors CR1 and CR2". JCI Insight. 2(16). doi:10.1172 / jci.insight.93739). Both the monomeric and homodimeric forms of IL-8 have been reported to be potent inducers of the chemokine receptors C-X-C motif chemokine receptor 1 (CXCR1) and C-X-C motif chemokine receptor 2 (CXCR2).

[0174] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR1 and / or CXCR2, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR1, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR1, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR1, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR1, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR1, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR1, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR1, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR1, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (eg, binding) of IL-8 to CXCR1, eg, by 95% or more.

[0175] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR2, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR2, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR2, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR2, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR2, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR2, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR2, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR2, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-8 to CXCR2, e.g., by 95% or more.

[0176] IL-10, encoded by the IL-10 gene, is a cytokine with pleiotropic effects in immunoregulation and inflammation. This downregulates the expression of Th1 cytokines (e.g., IFN-γ, IL-2, IL-3, TNFα, and GM-CSF), MHC class II antigens, and costimulatory molecules on macrophages, suppressing antigen presentation and CD4+ T cell activation (Moore et al. (2001). “Interleukin-10 and the interleukin-10 receptor”. Annual Review of Immunology. 19(1):683-765. doi:1146 / annurev.immunol.19.1.6 83; de Waal Malefyt et al. (1991). “Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes”. The Journal of Experimental Medicine. 174(5):1209-20. doi:10.1084 / jem.174.5.1209; de Waal Malefyt et al(1991). “Interleukin 10 (IL-10) and viral IL-10 strongly reduce antigen-specific human T cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class II major histocompatibility complex expression”. The Journal of Experimental Medicine.174(4):915-24.doi:10.1084 / jem.174.4.915;Akdis et al(2000).“A molecular basis for T cell suppression by IL-10:CD28-associated IL-10 receptor inhibits CD28 tyrosine phosphorylation and phosphatidylinositol 3-kinase binding”.FASEB Journal.14(12):1666-8.doi:10.1096 / fj.99-0874fje;Joss et al(2000).“IL-10 directly acts on T cells by specifically altering the CD28 co-stimulation pathway”.European Journal of Immunology.30(6):1683-90.doi:10.1002 / 1521-4141(200006)30:6<1683::AID-IMMU1683>3.0.CO;2-A). On the other hand, IL-10 enhances B cell survival, maturation, proliferation, and antibody production, and is stimulatory for Th2 cells. Furthermore, IL-10 can block NF-κB activity and is involved in regulating the JAK-STAT signaling pathway. IL-10 signaling is induced when IL-10 binds to its receptor, IL-10R.

[0177] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-10 to IL-10R, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-10 to IL-10R, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-10 to IL-10R, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-10 to IL-10R, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-10 to IL-10R, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-10 to IL-10R, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-10 to IL-10R, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-10 to IL-10R, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-10 to IL-10R, e.g., by 95% or more.

[0178] IL-1-beta is a cytokine encoded by the IL-1-beta gene and produced by activated macrophages, monocytes, and a subset of dendritic cells known as slanDCs (Yaseen et al. (2023). The role of IL-1β during human immunodeficiency virus type 1 infection. Reviews in Medical Virology.33(1):e2400.doi:10.1002 / rmv.2400) precursor protein, which is proteolytically processed by caspase 1 (CASP1 / ICE) to its active form. IL-1-beta is a key mediator of the inflammatory response and is involved in various cellular activities, including cell proliferation, differentiation, and apoptosis. IL-1-beta induction of cyclooxygenase-2 (PTGS2 / COX2) in the central nervous system (CNS) has been shown to contribute to inflammatory pain hypersensitivity. IL-1-beta, in combination with IL-23, induces the expression of IL-17, IL-21, and IL-22 by gamma delta T cells. This induction of expression is in the absence of additional signals, suggesting that IL-1-beta is involved in the regulation of autoimmune inflammation (Sutton et al. (2009) "Interleukin-1 and IL-23 induce innate IL-17 production from gamma delta T cells." cells, amplifying Th17 responses and autoimmunity”. Immunity. 31(2):331-341. doi:10.1016 / j.immuni.2009.08.001). IL-1-beta signaling is mediated by binding of IL-1-beta to its receptor, IL-1 receptor-1 (IL-1R1), and IL-1 receptor accessory protein (IL-1RAcP).

[0179] In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1R1 and / or IL-1RAcP, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1R1, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1R1, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1R1, e.g., by 10% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1R1, e.g., by 20% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1R1, e.g., by 30% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1R1, e.g., by 40% or more. For example, in some embodiments, the reduction in level or function results in a substantial reduction, prevention, or inhibition of the interaction (e.g., binding) of IL-1-beta to IL-1R1, e.g., by 50% or more.For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1R1, e.g., by 75% or more. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1R1, e.g., by 90% or more. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1R1, e.g., by 95% or more.

[0180] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1RACP, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1RACP, e.g., by 10% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1RACP, e.g., by 20% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1 beta to IL-1RACP, e.g., by 30% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1-beta to IL-1RACP, e.g., by 40% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1-beta to IL-1RACP, e.g., by 50% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1-beta to IL-1RACP, e.g., by 75% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1-beta to IL-1RACP, e.g., by 90% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1-beta to IL-1RAcP, e.g., by 95% or more.

[0181] MCP-1, also known as chemokine (CC motif) ligand 2 (CCL2), is a small cytokine that belongs to the CC chemokine family and is encoded by the CCL2 gene. CCL2 tightly regulates cellular mechanisms, thereby recruiting monocytes, memory T cells, and dendritic cells to sites of inflammation resulting from either tissue injury or infection (Evers et al. (2022) "Single-cell analysis reveals chemokine-mediated differential regulation of monocyte mechanics". iScience. 25(1):103555. Bibcode: 2022iSci...25j3555E. doi:10.1016 / j.sci.2021.103 555; Carr et al. (1994) "Monocyte chemoattractant protein 1 acts as a T-lymphocyte chemoattractant". Proceedings of the National Academy of Sciences of the United States of America America. 91(9):3652-6. Bibcode: 1994PNAS…91.3652C. doi:10.1073 / pnas.91.9.3652; Xu et al. (1996) "Human recombinant monocyte chemotactic protein and other CC chemokines bind and induce directional migration of dendritic cells in vitro." Journal of Leukocyte Biology. 60(3):365-71. doi:10.1002 / jlb.60.3.365). MCP-1 signaling is mediated by binding of MCP-1 to its receptor, CC chemokine receptor type 2 (CCR2).

[0182] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of MCP-1 to CCR2, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of MCP-1 to CCR2, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of MCP-1 to CCR2, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of MCP-1 to CCR2, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of MCP-1 to CCR2, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of MCP-1 to CCR2, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of MCP-1 to CCR2, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of MCP-1 to CCR2, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of MCP-1 to CCR2, e.g., by 95% or more.

[0183] TNF-alpha is an adipokine and cytokine encoded by the TNF gene. As an adipokine, TNF promotes insulin resistance and is associated with obesity-induced type 2 diabetes (Sethi et al. (2021). “Metabolic Messengers: tumor necrosis factor”. Nature Metabolism. 3(10):1302-1312. doi:10.1038 / s42255-021-00470-z). As a cytokine, TNF is used by the immune system for cell signaling. Macrophages release TNF to alert other immune system cells as part of an inflammatory response. TNF signaling occurs through two receptors, TNFR1 and TNFR2. While TNFR1 is constitutively expressed in most cell types, TNFR2 is primarily restricted to subsets of endothelial, epithelial, and immune cells (Heir et al. (2020) “TNF-Mediated Homeostatic Synaptic Plasticity: From in vitro to in vivo Models”. Frontiers in Cellular Neuroscience. 14:565841. doi:10.3389 / fncel.2020.565841; Gough et al. (2020) “Tumor Necrosis Factor Receptors: Pleiotropic Signaling Complexes and Their Differential Effects”. Frontiers in Immunology. 11:585880. doi:10.3389 / fimmu.2020.585880). TNFR1 signaling tends to be pro-inflammatory and apoptotic, whereas TNFR2 signaling is anti-inflammatory and promotes cell proliferation. Inhibition of TNFR1 signaling is important for the treatment of autoimmune diseases, while TNFR2 signaling promotes wound healing.

[0184] In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR1 and / or TNFR2, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR1, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR1, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR1, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR1, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR1, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR1, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR1, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (eg, binding) of TNF-alpha to TNFR1, eg, by 90% or more.In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (eg, binding) of TNF-alpha to TNFR1, eg, by 95% or more.

[0185] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR2, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR2, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR2, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR2, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR2, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR2, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR2, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR2, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-alpha to TNFR2, e.g., by 95% or more.

[0186] In some embodiments, a decrease in the level or function of a pro-inflammatory cytokine selected from, for example, IL-6, IL-8, IL-10, IL-1-beta, MCP-1, and TNF-alpha is determined by: (a) reducing cytokine release from immune cells, e.g., T cells and B cells, and non-immune cells, e.g., endothelial cells, fibroblasts, adipocytes, and stromal cells; and (b) reduces oxidative stress; Thereby, inflammation is prevented, reduced and / or inhibited.

[0187] In some embodiments, reducing the level or function of a pro-inflammatory cytokine, e.g., selected from IL-6, IL-8, IL-10, IL-1-beta, MCP-1, and TNF-alpha, comprises mutating one or more nucleotides in the nucleotide sequence of one or more genes selected from IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF. Nucleotide mutations can include nucleotide deletions, additions, and / or substitutions. As described herein, such mutations can result in reduced expression of the gene by, for example, reducing, altering, or preventing transcription and / or splicing of the nucleotide sequence. For example, in some embodiments, reducing the level or function of a pro-inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of IL-6. In some embodiments, reducing the level or function of a pro-inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of IL-8. In some embodiments, reducing the level or function of a pro-inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of IL-10. In some embodiments, reducing the level or function of a proinflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of IL-1-beta. In some embodiments, reducing the level or function of a proinflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of CCL2. In some embodiments, reducing the level or function of a proinflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of TNF.

[0188] In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence identity to the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 65% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 70% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 75% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 95% sequence identity to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 99% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5.

[0189] In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence identity to the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 65% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 70% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 75% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 95% sequence identity to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 99% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the IL-6, IL-8, IL-10, IL-1-beta, CCL2, and TNF genes comprise sequences having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5.

[0190] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in expression of a pro-inflammatory cytokine, e.g., selected from IL-6, IL-8, IL-10, IL-1-beta, MCP-1, and TNF-alpha, compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except that it does not comprise a reduced level of the pro-inflammatory cytokine. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in expression of inflammatory cytokines, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, except for the absence of a reduced level of inflammatory cytokines. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in expression of inflammatory cytokines by about 50%, 75%, or greater than 90%, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, except for the absence of a reduced level of inflammatory cytokines. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0191] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in inflammatory cytokine function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except that the modified mammalian cell does not comprise a reduction in inflammatory cytokine function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in inflammatory cytokine function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, except for the absence of the reduction in inflammatory cytokine function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in inflammatory cytokine function of about 50%, 75%, or greater than 90%, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, except for the absence of the reduction in inflammatory cytokine function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0192] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in IL-6 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except for, e.g., not comprising a reduction in IL-6 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in IL-6 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in IL-6 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in IL-6 levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in IL-6 levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0193] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in IL-8 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except, for example, that does not comprise a reduction in IL-8 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in IL-8 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in IL-8 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in IL-8 levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in IL-8 levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0194] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in IL-10 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except, for example, that does not comprise a reduction in IL-10 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in IL-10 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in IL-10 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in IL-10 levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in IL-10 levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0195] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in IL-1 beta levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell, e.g., except that the modified mammalian cell does not comprise a reduction in IL-1 beta levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in IL-1 beta levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., but for lack of a reduction in IL-1 beta levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in IL-1 beta levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., but for lack of a reduction in IL-1 beta levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0196] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in MCP-1 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, or is identical to the modified mammalian cell, except for, e.g., not comprising a reduction in MCP-1 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in MCP-1 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in MCP-1 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in MCP-1 levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in MCP-1 levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0197] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in TNF-alpha levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, or is identical to the modified mammalian cell, except for, e.g., not comprising a reduction in TNF-alpha levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in TNF-alpha levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in TNF-alpha levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in TNF-alpha levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in TNF-alpha levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0198] In some embodiments, the proinflammatory cytokine, e.g., selected from IL-6, IL-8, IL-10, IL-1-beta, MCP-1, and TNF-alpha, is encoded by one or more nucleotide sequences provided in Table 5, or fragments thereof.

[0199] In some embodiments, the reduction in the level or function of a pro-inflammatory cytokine, e.g., selected from IL-6, IL-8, IL-10, IL-1-beta, MCP-1, and TNF-alpha, persists for at least 15 minutes (e.g., 30 minutes, 1 hour, 12 hours, 24 hours, 48 ​​hours, 72 hours, 1 week, 1 month, or 1 year). For example, in some embodiments, the reduction in the level or function of a pro-inflammatory cytokine persists for at least 30 minutes. In some embodiments, the reduction in the level or function of a pro-inflammatory cytokine persists for at least 1 hour. In some embodiments, the reduction in the level or function of a pro-inflammatory cytokine persists for at least 12 hours. In some embodiments, the reduction in the level or function of a pro-inflammatory cytokine persists for at least 24 hours. In some embodiments, the reduction in the level or function of a pro-inflammatory cytokine persists for at least 48 hours. In some embodiments, the reduction in the level or function of a pro-inflammatory cytokine persists for at least 72 hours. In some embodiments, the reduction in the level or function of a pro-inflammatory cytokine persists for at least 1 week. In some embodiments, the reduction in the level or function of a pro-inflammatory cytokine persists for at least 1 month. In some embodiments, the reduction in inflammatory cytokine levels or function persists for at least one year.

[0200] A profibrotic factor is a molecule that stimulates a host fibrotic response, e.g., fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor A (VEGFA), or platelet-derived growth factor (PDGF). In one embodiment, the modified mammalian cells comprise a reduced level or function of a profibrotic factor selected from, e.g., FGF-2, VEGFA, and PDGF.

[0201] FGF-2, also known as basic FGF, heparin-binding growth factor-2, and endothelial growth factor-2, is a growth factor and signaling protein that binds to and exerts its effects through specific fibroblast growth factor receptor (FGFR) proteins. FGF-2 induces and mediates angiogenesis. It is synthesized and secreted by adipocytes and stimulates proliferation by binding to FGFR1, thereby activating phosphorinositide 3-kinase. FGF-2 is encoded by the FGF2 gene and interacts with casein kinase 1, alpha 1, 60S ribosomal protein L6 (RPL6), ribosomal protein S19, and inhibitor of apoptosis 5 (API5).

[0202] In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1, alpha 1, RPL6, S19, and / or API5, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1, e.g., by 10% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to casein kinase 1, e.g., by 20% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to casein kinase 1, e.g., by 30% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to casein kinase 1, e.g., by 40% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to casein kinase 1, e.g., by 50% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to casein kinase 1, e.g., by 75% or more. In some embodiments, the reduction in level or function results in a substantial reduction, eg, by 90% or more, prevention, or inhibition of the interaction (eg, binding) of FGF-2 to casein kinase 1.In some embodiments, the reduction in level or function results in a substantial reduction, eg, by 95% or more, prevention, or inhibition of the interaction (eg, binding) of FGF-2 to casein kinase 1.

[0203] In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to alpha 1, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to alpha 1, e.g., by 10% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to alpha 1, e.g., by 20% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to alpha 1, e.g., by 30% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to alpha 1, e.g., by 40% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to alpha 1, e.g., by 50% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to alpha 1, e.g., by 75% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to alpha 1, e.g., by 90% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to alpha 1, e.g., by 95% or more.

[0204] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to RPL6, for example, by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to RPL6, for example, by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to RPL6, for example, by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to RPL6, for example, by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to RPL6, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to RPL6, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to RPL6, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to RPL6, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to RPL6, e.g., by 95% or more.

[0205] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to S19, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to S19, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to S19, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to S19, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to S19, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to S19, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to S19, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to S19, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to S19, e.g., by 95% or more.

[0206] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to API 5, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to API 5, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to API 5, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to API 5, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to API 5, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to API 5, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to API 5, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to API 5, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of FGF-2 to API 5, e.g., by 95% or more.

[0207] VEGFA is a glycosylated mitogen that specifically acts on endothelial cells and has various effects, including mediating increased vascular permeability, inducing angiogenesis, neovasculogenesis, and endothelial cell proliferation, promoting cell migration, and inhibiting apoptosis. It is considered a major and dominant inducer of vascular growth and is essential in adults during organ remodeling and in vascular diseases, such as wound healing, tumor angiogenesis, diabetic retinopathy, and age-related macular degeneration. VEGFA is also chemotactic for macrophages and granulocytes and indirectly induces vasodilation, for example, through the release of NO. VEGFA is encoded by the VEGFA gene and interacts with a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), connective tissue growth factor (CTGF), and neuropilin-1 (NRP1).

[0208] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to ADAMTS1, CTGF, and / or NRP1, for example, by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to ADAMTS1, for example, by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to ADAMTS1, for example, by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to ADAMTS1, for example, by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to ADAMTS1, for example, by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to ADAMTS1, for example, by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to ADAMTS1, for example, by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to ADAMTS1, for example, by 75% or more. In some embodiments, the reduction in level or function results in a substantial reduction, eg, by 90% or more, prevention, or inhibition of VEGFA interaction (eg, binding) to ADAMTS1.In some embodiments, the reduction in level or function results in a substantial reduction, eg, by 95% or more, prevention, or inhibition of VEGFA interaction (eg, binding) to ADAMTS1.

[0209] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to CTGF, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to CTGF, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to CTGF, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to CTGF, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to CTGF, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to CTGF, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to CTGF, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to CTGF, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to CTGF, e.g., by 95% or more.

[0210] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to NRP1, for example, by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to NRP1, for example, by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to NRP1, for example, by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to NRP1, for example, by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to NRP1, for example, by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to NRP1, for example, by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to NRP1, for example, by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to NRP1, for example, by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits the interaction (e.g., binding) of VEGFA to NRP1, for example, by 95% or more.

[0211] PDGF is a growth factor that plays a key role in angiogenesis, the growth of blood vessels from preexisting vascular tissue; mitogenesis, i.e., the proliferation of mesenchymal cells such as fibroblasts, osteoblasts, tenocytes, vascular smooth muscle cells, and mesenchymal stem cells; and chemotaxis, the directional migration of mesenchymal cells. Platelet-derived growth factor (PDGF) is a dimeric glycoprotein that can be composed of two A subunits (PDGF-AA), two B subunits (PDGF-BB), or one of each (PDGF-AB). Furthermore, PDGF is a potent mitogen for cells of mesenchymal origin, including fibroblasts, smooth muscle cells, and glial cells. In both mice and humans, the PDGF signaling network consists of five ligands, PDGF-AA (encoded by the PDGFA gene), -BB (encoded by the PDGFB gene), -CC (encoded by the PDGFC gene), and -DD (encoded by the PDGFD gene), and -AB (a PDGFA and PDGFB heterodimer), and two receptors, PDGFR-alpha and PDGFR-beta. All PDGFs function as secreted disulfide-linked homodimers, but only PDGFs A and B can form functional heterodimers. In some embodiments, PDGFs comprise PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and / or PDGF-AB. In some embodiments, PDGFs comprise PDGF-AA. In some embodiments, PDGFs comprise PDGF-BB. In some embodiments, PDGFs comprise PDGF-CC. In some embodiments, PDGFs comprise PDGF-DD. In some embodiments, PDGFs comprise PDGF-AB.

[0212] In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-alpha and / or PDGFR-beta, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-alpha, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-alpha, e.g., by 10% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-alpha, e.g., by 20% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-alpha, e.g., by 30% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-alpha, e.g., by 40% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-alpha, e.g., by 50% or more. In some embodiments, the reduction in levels or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-alpha, e.g., by 75% or more. In some embodiments, the reduction in level or function results in a substantial reduction, eg, by 90% or more, prevention, or inhibition of PDGF interaction (eg, binding) to PDGFR-alpha.In some embodiments, the reduction in level or function results in a substantial reduction, eg, by 95% or more, prevention, or inhibition of PDGF interaction (eg, binding) to PDGFR-alpha.

[0213] In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-beta, e.g., by 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-beta, e.g., by 10% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-beta, e.g., by 20% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-beta, e.g., by 30% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-beta, e.g., by 40% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-beta, e.g., by 50% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-beta, e.g., by 75% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-beta, e.g., by 90% or more. In some embodiments, the reduction in level or function substantially reduces, prevents, or inhibits PDGF interaction (e.g., binding) to PDGFR-beta, e.g., by 95% or more.

[0214] In some embodiments, a decrease in the level or function of a profibrotic factor selected from, for example, FGF-2, VEGFA, and PDGF is (i) inhibition of fibroblast-related signaling pathways, such as the AKT / mTOR and SMAD pathways; and (ii) further resulting in inhibition of fibroblast expression, proliferation, and activation; Thereby, inflammation is prevented, reduced and / or inhibited.

[0215] In some embodiments, reducing the level or function of a pro-fibrotic factor selected from, for example, FGF-2, VEGFA, and PDGF comprises mutating one or more nucleotides in the nucleotide sequence of one or more genes selected from FGF-2, VEGFA, and PDGF. Nucleotide mutations can include nucleotide deletions, additions, and / or substitutions. As described herein, such mutations can result in reduced expression of the gene by, for example, reducing, altering, or preventing transcription and / or splicing of the nucleotide sequence. For example, in some embodiments, reducing the level or function of a pro-inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of FGF-2. In some embodiments, reducing the level or function of a pro-inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of VEGFA. In some embodiments, reducing the level or function of a pro-inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of PDGF.

[0216] In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence identity to the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 65% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 70% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 75% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 95% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 99% sequence identity to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5.

[0217] In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9%, or more) sequence identity to the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 65% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 70% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 75% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 95% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 99% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequences of the FGF-2, VEGFA, and PDGF genes comprise sequences having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5. In one embodiment, a modified mammalian cell (e.g., a modified RPE cell, e.g., a modified ARPE-19 cell) described herein comprises about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in expression of a pro-fibrotic factor, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except that it does not comprise a reduced level of the pro-fibrotic factor. In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in expression of a profibrotic factor compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., but does not comprise a reduced level of a profibrotic factor. In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise a reduction in expression of a profibrotic factor by about 50%, 75%, or greater than 90%, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., but does not comprise a reduced level of a profibrotic factor. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0218] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in pro-fibrotic factor function, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell or is identical to the modified mammalian cell except that it does not comprise a reduction in pro-fibrotic factor function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in profibrotic factor function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of the reduction in profibrotic factor function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in profibrotic factor function of about 50%, 75%, or greater than 90%, e.g., compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of the reduction in profibrotic factor function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0219] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in FGF-2 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, or is identical to the modified mammalian cell, except for, e.g., not comprising a reduction in FGF-2 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in FGF-2 levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in FGF-2 levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in FGF-2 levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in FGF-2 levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0220] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in PDGF levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, or is identical to the modified mammalian cell, except for, e.g., not comprising a reduction in PDGF levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in PDGF levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in PDGF levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in PDGF levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in PDGF levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0221] In one embodiment, the modified mammalian cells (e.g., modified RPE cells, e.g., modified ARPE-19 cells) described herein comprise about a 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in VEGFA levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, or is identical to the modified mammalian cell, except for, e.g., not comprising a reduction in VEGFA levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% reduction in VEGFA levels or function compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in VEGFA levels or function. In one embodiment, the modified mammalian cells described herein (e.g., modified RPE cells, e.g., modified ARPE-19 cells) comprise a reduction in VEGFA levels or function of about 50%, 75%, or greater than 90% compared to a modified mammalian cell that is substantially identical to the modified mammalian cell, e.g., except for the absence of a reduction in VEGFA levels or function. In one embodiment, the modified mammalian cells are modified RPE cells (e.g., modified ARPE-19 cells). In one embodiment, the modified mammalian cells are modified ARPE-19 cells.

[0222] In some embodiments, the profibrotic factor, eg, selected from FGF-2, PDGF, and VEGFA, is encoded by one or more nucleotide sequences provided in Table 5, or fragments thereof.

[0223] In some embodiments, the reduced level or function of a profibrotic factor, e.g., selected from FGF-2, PDGF, and VEGFA, persists for at least 15 minutes (e.g., 30 minutes, 1 hour, 12 hours, 24 hours, 48 ​​hours, 72 hours, 1 week, 1 month, or 1 year). For example, in some embodiments, the reduced level or function of a profibrotic factor persists for at least 30 minutes. In some embodiments, the reduced level or function of a profibrotic factor persists for at least 1 hour. In some embodiments, the reduced level or function of a profibrotic factor persists for at least 12 hours. In some embodiments, the reduced level or function of a profibrotic factor persists for at least 24 hours. In some embodiments, the reduced level or function of a profibrotic factor persists for at least 48 hours. In some embodiments, the reduced level or function of a profibrotic factor persists for at least 72 hours. In some embodiments, the reduced level or function of a profibrotic factor persists for at least 1 week. In some embodiments, the reduced level or function of a profibrotic factor persists for at least 1 month. In some embodiments, the reduction in profibrotic factor level or function persists for at least one year. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 2-38

Table 2-39

Table 2-40

Table 2-41

Table 2-42

Table 2-43

Table 2-44

Table 2-45

Table 2-46

Table 2-47

Table 2-48

Table 2-49

Table 2-50

Table 2-51

Table 2-52

Table 2-53

Table 2-54

Table 2-55

Table 2-56

Table 2-57

Table 2-58

Table 2-59

Table 2-60

Table 2-61

Table 2-62

Table 2-63

Table 2-64

Table 2-65

Table 2-66

Table 2-67

Table 2-68

Table 2-69

Table 2-70

Table 2-71

Table 2-72

Table 2-73

Table 2-74

Table 2-75

Table 2-76

Table 2-77

Table 2-78

Table 2-79

Table 2-80

Table 2-81

Table 2-82

Table 2-83

Table 2-84

Table 2-85

Table 2-86

Table 2-87

Table 2-88

Table 2-89

Table 2-90

Table 2-91

Table 2-92

Table 2-93

Table 2-94

Table 2-95

Table 2-96

Table 2-97

Table 2-98

Table 2-99

Table 2-100

Table 2-101

Table 2-102

Table 2-103

Table 2-104

Table 2-105

Table 2-106

Table 2-107

Table 2-108

Table 2-109

Table 2-110

Table 2-111

Table 2-112

Table 2-113

Table 2-114

Table 2-115

Table 2-116

Table 2-117

Table 2-118

Table 2-119

Table 2-120

Table 2-121

Table 2-122

Table 2-123

Table 2-124

Table 2-125

Table 2-126

Table 2-127

Table 2-128

Table 2-129

Table 2-131

Table 2-132

Table 2-133

Table 2-134

Table 2-135

Table 2-136

Table 2-137

Table 2-138

Table 2-139

Table 2-140

Table 2-141

Table 2-142

Table 2-143

Table 2-144

Table 2-145

Table 2-146

Table 2-147

Table 2-148

Table 2-149

Table 2-150

Table 2-151

Table 2-152

Table 2-153

Table 2-154

Table 2-155

Table 2-156

Table 2-157

Table 2-158

Table 2-159

Table 2-160

Table 2-161

Table 2-162

Table 2-163

Table 2-164

Table 2-165

Table 2-166

Table 2-167

Table 2-169

Table 2-170

Table 2-171

Table 2-172

Table 2-173

Table 2-174

Table 2-175

Table 2-176

Table 2-177

Table 2-178

Table 2-179

Table 2-181

Table 2-182

Table 2-183

Table 2-184

Table 2-186

Table 2-187

Table 2-189

Table 2-190

Table 2-191

Table 2-192

Table 2-193

Table 2-194

Table 2-195

Table 2-196

Table 2-197

Table 2-199

Table 2-200

Table 2-201

Table 2-202

Table 2-203

Table 2-204

Table 2-205

Table 2-206

Table 2-207

Table 2-208

Table 2-209

Table 2-210

Table 2-211

Table 2-212

Table 2-213

Table 2-214

Table 2-215

Table 2-216

Table 2-217

Table 2-218

Table 2-219

Table 2-220

Table 2-221

Table 2-222

Table 2-223

Table 2-224

Table 2-225

Table 2-226

[0224] Genetic manipulation and modification of mammalian cells or modified mammalian cells can be carried out using any known method in the art, including gene silencing, gene knockdown, gene knockout, and gene editing techniques. For example, gene mutants can be generated at desired sites (multiple sites) within the target OCR using targeted genome editing techniques. Targeted genome editing techniques can be any known technique in the art, such as CRISPR-Cas, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and techniques using site-specific nucleases such as meganucleases.

[0225] The modified mammalian cells described herein can be derived from a variety of different mammalian cell types (e.g., human cells), including adipocytes, epidermal cells, epithelial cells, endothelial cells, fibroblasts, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, pericytes, keratinocyte cells, subtypes of any of the above, and cells derived from any of the above. Exemplary cell types include those listed in WO2017 / 075631. In some embodiments, the cells are derived from the cell lines set forth in Table 2 below. [Table 3]

[0226] In one embodiment, any of the modified mammalian cells described herein is derived from an RPE cell, e.g., an ARPE-19 cell. In one embodiment, the modified RPE cell (e.g., a modified ARPE-19 cell) comprises any of the expression cassettes, transposons, and polynucleotides described herein.

[0227] Modified mammalian cells for use in the devices, compositions, and methods described herein can be in various stages of the cell cycle, for example, as a plurality of modified cells contained or encapsulated in a hydrogel capsule. In some embodiments, at least one modified cell in the plurality of modified cells is undergoing cell division. Cell division can be measured using any method known in the art, for example, as described in DeFazio A et al. (1987) J Histochem Cytochem 35:571-577 and Dolbeare F et al. (1983) Proc Natl Acad Sci USA 80:5573-5577, each of which is incorporated by reference in its entirety. In one embodiment, at least 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, as determined, for example, by a 5-ethynyl-2'-deoxyuridine (EdU) assay or a 5-bromo-2'-deoxyuridine (BrdU) assay. In some embodiments, cell proliferation is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry). In some embodiments, none of the modified cells in the plurality of modified cells are undergoing cell division and are quiescent. In one embodiment, less than 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, 5-ethynyl-2'deoxyuridine (EdU) assay, 5-bromo-2'-deoxyuridine (BrdU) assay, microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation), or flow cytometry).

[0228] In one embodiment, at least 50%, 60%, 70%, 80%, 90%, or more of the modified cells in the plurality are viable. Cell viability can be measured using any method known in the art, for example, as described in Riss, T. et al. (2013) "Cell Viability Assays" in Assay Guidance Manual (Sittapalam, G.S. et al., eds.). For example, cell viability can be measured or quantified by ATP assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, 5-bromo-2'-deoxyuridine (BrdU) assay. In some embodiments, cell viability is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse photography or evaluation of spindle formation) or flow cytometry). In one embodiment, at least 80% of the plurality of modified cells are viable, as determined, for example, by an ATP assay, a 5-ethynyl-2'-deoxyuridine (EdU) assay, a 5-bromo-2'-deoxyuridine (BrdU) assay, microscopy (e.g., fluorescence microscopy (e.g., time-lapse photography or assessment of spindle formation)), or flow cytometry.

[0229] Any of the parameters described herein can be assessed using standard techniques known to those skilled in the art, such as histology, microscopy, and various functional assays.

[0230] In some embodiments, the exogenous transcription unit encodes a therapeutic polypeptide (e.g., a protein), such as a clotting factor, growth factor, hormone, enzyme, cytokine (e.g., a pro-inflammatory cytokine or an anti-inflammatory cytokine), cytokine receptor, chimeric protein, fusion protein, or lipoprotein. The polypeptide encoded by the exogenous transcription unit may have a naturally occurring amino acid sequence or may contain a variant of a naturally occurring sequence. The variant may be a non-naturally occurring or naturally occurring amino acid substitution, mutation, deletion, or addition compared to a reference (e.g., naturally occurring) sequence. The naturally occurring amino acid sequence may be a polymorphic variant. The naturally occurring amino acid sequence may be a human or non-human amino acid sequence. In some embodiments, the naturally occurring amino acid sequence is a human sequence. In some embodiments, the therapeutic polypeptide has fewer than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the polypeptides have an average molecular weight of 5 kD, 10 kD, 25 kD, 50 kD, 100 kD, 150 kD, 200 kD, 250 kD, 500 kD, or more.

[0231] In some embodiments, the polypeptide is a hormone. Exemplary hormones include antidiuretic hormone (ADH), oxytocin, growth hormone (GH), prolactin, growth hormone-releasing hormone (GHRH), thyroid-stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), luteinizing hormone-releasing hormone (LHRH), thyroxine, calcitonin, parathyroid hormone (PTH), aldosterone, cortisol, epinephrine, glucagon, insulin, estrogen, progesterone, and testosterone. In some embodiments, the polypeptide is insulin (e.g., insulin A-chain, insulin B-chain, or proinsulin). In some embodiments, the polypeptide is a growth hormone, such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methionine-human growth hormone, des-phenylalanine-human growth hormone, and porcine growth hormone.

[0232] In some embodiments, the polypeptide is a growth factor, such as vascular endothelial growth factor (VEGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), and insulin-like growth factor-I and -II (IGF-I and IGF-II).

[0233] In some embodiments, the polypeptide is a clotting factor or coagulation factor, e.g., a blood clotting factor or blood coagulation factor. In some embodiments, the polypeptide is involved in coagulation, the process by which blood is converted from a liquid to a solid or gel. Exemplary clotting factors include Factor I (e.g., fibrinogen), Factor II (e.g., prothrombin), Factor III (e.g., tissue factor), Factor V (e.g., proaccelerin, labile factor), Factor VI, Factor VII (e.g., stable factor, proconvertin), Factor VIII (e.g., antihemophilic factor A), Factor VIIIC, Factor IX (e.g., antihemophilic factor B), Factor X (e.g., Stuart-Prower factor), Factor XI (e.g., plasma thromboplastin precursor), Factor XII (e.g., Hageman factor), Factor XIII (e.g., fibrin-stabilizing factor), von Willebrand factor (vWF), prekallikrein, heparin cofactor II, high molecular weight kininogen (e.g., Fitzgerald factor), antithrombin III, and fibronectin. In some embodiments, the polypeptide is an anticoagulant, e.g., protein C.

[0234] In some embodiments, the polypeptide is an immunoglobulin chain (heavy or light) or a fragment thereof comprising at least one immunoglobulin variable domain sequence, and optionally comprising an immunoglobulin Fc region. In one embodiment, the polypeptide is a full-length immunoglobulin chain.

[0235] In some embodiments, the polypeptide is selected from the group consisting of, for example, tumor necrosis factors alpha and beta, their receptors and their derivatives, renin; lipoproteins; colchicine; corticotrophin; vasopressin; somatostatin; lypressin; pancreozymin; leuprolide; alpha-1-antitrypsin; atrial natriuretic factor; pulmonary surfactant; plasminogen activators other than tissue-type plasminogen activator (t-PA), such as urokinase; bombesin; thrombin; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, e.g., human serum albumin; Müllerian inhibitory substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-related peptide; chorionic gonadotropin; microbial proteins, e.g., beta-lactamase; DNase; inhibin; activin; receptors for hormones or growth factors; integrins; protein A or D; rheumatoid factor; platelet-derived growth factor (PDGF); epidermal growth factor (EGF); transforming factor Transforming growth factors (TGFs), such as TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5); insulin-like growth factors-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins, such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors; antitoxins; interferons, such as interferon-alpha (e.g., interferon alpha).2A), -beta, -gamma, -lambda, and consensus interferons; colony-stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), such as IL-1, IL-2, and IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; degradation-promoting factors; transport proteins; homing receptors; addressins; conception inhibitors, such as prostaglandins; conception promoters; regulatory proteins; cytokines or cytokine receptors, or chimeric proteins comprising cytokines or their receptors, including antibodies (including fragments thereof), and chimeric proteins, such as immunoadhesins. Suitable polypeptides can be native or recombinant, including, for example, fusion proteins.

[0236] Examples of polypeptides that can be encoded by exogenous transcription units also include CCL1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1 (KC), CXCL2 (SDF1a), CXCL3 (SEQ ID NO: 1), CXCL4 (SEQ ID NO: 1), CXCL5 (SEQ ID NO: 1), CXCL6 (SEQ ID NO: 1), CXCL7 (SEQ ID NO: 1), CXCL8 (SEQ ID NO: 1), CXCL9 (SEQ ID NO: 1), CXCL10 (SEQ ID NO: 1), CXCL11 (SEQ ID NO: 1), CXCL12 (SEQ ID NO: 1), CXCL13 (SEQ ID NO: 1), CXCL14 (SEQ ID NO: 1), CXCL15 (SEQ ID NO: 1), CXCL16 (SEQ ID NO: 1), CXCL17 (SEQ ID NO: 1), CXCL18 (SEQ ID NO: 1), CXCL19 (SEQ ID NO: 1), CXCL20 (SEQ ID NO: 1), CXCL21 (SEQ ID NO: 1), CXCL22 (SEQ ID NO: 1), CXCL23 (SEQ ID NO: 1), CXCL24 (SEQ ID NO: 1), CXCL25 (SEQ ID NO: 1), CXCL3 (SEQ ID NO: 3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8(IL8), CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, TNFA, TNF B(LTA), TNFC(LTB), TNFSF4, TNFSF5(CD40LG), TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF13B, EDA, IL2, IL15, IL4, IL13, IL7, IL9, IL2 1, IL3, IL5, IL6, IL11, IL27, IL30, IL31, OSM, LIF, CNTF, CTF1, IL12a, IL12b, IL23, IL27, IL35, IL14, IL16, IL32, IL34, IL10, IL22, IL19, IL20, IL24, IF NK, IFNW1, IFNG, IL1A (IL1F1), IL1B (IL1F2), IL1Ra (IL1F3), IL1F5 (IL36RN), IL1F6 (IL36A), IL1F7 (IL37), IL1F8 (IL36B), IL1F9 (IL36G), IL1F10 (IL 38), IL33 (IL1F11), IL18 (IL1G), IL17, KITLG, IL25 (IL17E), CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), SPP1, TGFB1, TGFB2, TGFB3, CCL3L1, CCL3L2,CCL3L3, CCL4L1, CCL4L2, IL17B, IL17C, IL17D, IL17F, AIMP1(SCYE1), MIF, Areg, BC096441, Bmp1, Bmp10, Bmp15, Bmp2, Bmp3, Bmp4, Bmp5, Bmp6, Bmp7, Bm p8a, Bmp8b, C1qTNF4, Ccl21a, Ccl27a, Cd70, Cer1, Cklf, Clcf1, Cmtm2a, Cmtm2b, Cmtm3, Cmtm4, Cmtm5, Cmtm6, Cmtm7, Cmtm8, Crlf1, Ctf2, Ebi3, Edn1, F am3b, Fasl, Fgf2, Flt3l, Gdf10, Gdf11, Gdf15, Gdf2, Gdf3, Gdf5, Gdf6, Gdf 7, Gdf9, Gm12597, Gm13271, Gm13275, Gm13276, Gm13280, Gm13283, Gm2564, G pi1, Grem1, Grem2, Grn, Hmgb1, Ifna11, Ifna12, Ifna9, Ifnab, Ifne, Il17a, Il23a, Il25, Il31, Iltifb, Inhba, Lefty1, Lefty2, Mstn, Nampt, Ndp, Noda l, Pf4, Pglyrp1, Prl7d1, Scg2, Scgb3a1, Slurp1, Spp1, Thpo, TNFsf10, TNFsf11, TNFsf12, TNFsf13, TNFsf13b, TNFsf14, TNFsf15, TNFsf18, TNFsf4, TNFsf8, TNFsf9, Tslp, Vegfa, Wnt1, Wnt2, Wnt5a, Wnt7a, Xcl1, epinephrine, melatonin, triiodothyronine, prostaglandins, leukotrienes, prostacyclin, thromboxane, islet amyloid polypeptide, myosin duct inhibitory factors or hormones, adiponectin, corticotropin, angiotensin, vasopressin, arginine vasopressin, atriopeptin, brain natriuretic peptide, calcitonin, cholecystokinin, cortistatin, enkephalin, endothelin, erythropoietin, follicle-stimulating hormone, galanin, gastric inhibitory polypeptide, gastrin, ghrelin, glucagon, glucagon-like peptide-1, gonadotropin-releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, inhibin, somatomedin, leptin,These include lipotropin, melanocyte-stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, pituitary adenylate cyclase-activating peptide, relaxin, renin, secretin, somatostatin, thrombopoietin, thyrotropin, thyrotropin-releasing hormone, vasoactive intestinal peptide, androgens, alpha-glucosidase (also known as acid maltase), glycogen phosphorylase, glycogen debranching enzyme, phosphofructokinase, phosphoglycerate kinase, phosphoglycerate mutase, lactate dehydrogenase, carnitine parmethyltransferase, carnitine, and myoadenylate deaminase.

[0237] In some embodiments, the polypeptide is a replacement therapy or replacement protein.

[0238] In some embodiments, the replacement therapy or replacement protein is a clotting factor or coagulation factor, for example, Factor VII, Factor VIII, or Factor IX.

[0239] In some embodiments, the replacement therapy or protein is an enzyme, such as alpha-galactosidase A (GLA), alpha-L-iduronidase (IDUA), glucocerebrosidase, or N-sulfoglucosamine sulfohydrolase (SGSH). In one embodiment, the modified mammalian cell comprises an exogenous nucleic acid encoding IDUA.

[0240] In one embodiment, the modified mammalian cell is not a pancreatic islet cell as defined herein. In one embodiment, the modified mammalian cell has one or more of the following characteristics: (i) is unable to produce insulin (e.g., insulin A-chain, insulin B-chain, or proinsulin) in an amount effective to treat diabetes or another disease or condition that can be treated with insulin, (ii) is unable to produce insulin in a glucose-responsive manner, or (iii) is not derived from an induced pluripotent stem cell that has been modified or differentiated into an insulin-producing pancreatic beta cell.

[0241] Portable element features The modified mammalian cell or cells described herein can be incorporated into an implantable element for use in treating a disease or disorder in a subject, as well as to reduce the level of pericapsular fibrotic overgrowth on the implantable element when implanted in a subject.

[0242] The implantable elements of the present disclosure include at least one barrier that prevents immune cells from contacting cells contained within the device. At least a portion of the barrier should be sufficiently porous to allow therapeutic agents expressed and secreted by the cells to exit the device. A variety of device configurations known in the art are suitable.

[0243] The device (e.g., particle) can have any configuration and shape suitable for supporting the viability and productivity of contained cells after implantation at the intended target site. By way of non-limiting example, the device shape can be cylindrical, rectangular, disc-shaped, oval, star-shaped, or spherical. The device can be comprised of a mesh-like or nested structure. In some embodiments, the device can prevent materials over a certain size from passing through pores or openings. In some embodiments, the device (e.g., particle) can prevent materials over 50 kD, 75 kD, 100 kD, 125 kD, 150 kD, 175 kD, 200 kD, 250 kD, 300 kD, 400 kD, 500 kD, 750 kD, or 1,000 kD from passing through.

[0244] In one embodiment, the device is a macroencapsulation device. Non-limiting examples of macroencapsulation devices are described in WO2019 / 068059, WO2019 / 169089, U.S. Patent Nos. 9,526,880, 9,724,430, and 8,278,106, European Patent No. 742818B1, and Sang, S. and Roy, S., Biotechnol. Bioeng. 113(7):1381-1402 (2016).

[0245] In one embodiment, the device is a macrodevice having one or more cell-containing compartments. A device having two or more cell-containing compartments can be configured to produce two or more proteins, for example, cells expressing a first therapeutic agent are placed in one compartment and cells expressing a different protein (e.g., a therapeutic protein) are placed in another compartment. WO Publication No. 2018 / 232027 describes a device having multiple cell-containing compartments formed in a microassembly and covered by a porous membrane.

[0246] In one embodiment, the device is configured as a thin, flexible strand, as described in U.S. Patent No. 10,493,107. The strand includes a substrate, an inner polymeric coating surrounding the substrate, and an outer hydrogel coating surrounding the inner polymeric coating. Protein-expressing cells are disposed within the outer coating.

[0247] In some embodiments, the devices (e.g., particles) have a largest linear dimension (LLD), e.g., average diameter, or size, that is at least about 0.5 millimeters (mm), preferably about 1.0 mm, about 1.5 mm, or more. In some embodiments, the devices can be as large as 10 mm in diameter or size. For example, the devices or particles described herein may be sized to have a diameter of 0.5 mm to 10 mm, 1 mm to 10 mm, 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm, 1.5 mm to 3 mm, 1.5 mm to 2 mm, 2 mm to 8 mm, 2 mm to 7 mm, 2 mm to 6 mm, 2 mm to 5 mm, 2 mm to 4 mm, 2 mm to 3 mm, 2.5 mm to 8 mm, 2.5 mm to 7 mm, 2.5 mm to 6 mm, 2.5 mm to 5 mm, 2.5 mm to 4 mm, 2.5 mm to 3 mm, 3 mm to 8 mm Available in sizes ranging from m, 3mm to 7mm, 3mm to 6mm, 3mm to 5mm, 3mm to 4mm, 3.5mm to 8mm, 3.5mm to 7mm, 3.5mm to 6mm, 3.5mm to 5mm, 3.5mm to 4mm, 4mm to 8mm, 4mm to 7mm, 4mm to 6mm, 4mm to 5mm, 4.5mm to 8mm, 4.5mm to 7mm, 4.5mm to 6mm, 4.5mm to 5mm, 5mm to 8mm, 5mm to 7mm, 5mm to 6mm, 5.5mm to 8mm, 5.5mm to 7mm, 5.5mm to 6mm, 6mm to 8mm, 6mm to 7mm, 6.5mm to 8mm, 6.5mm to 7mm, 7mm to 8mm, or 7.5mm to 8mm.

[0248] In some embodiments, a device (e.g., particle, capsule) of the present disclosure comprises at least one pore or opening, e.g., to allow free flow of material. In some embodiments, the average pore size of the device is about 0.1 μm to about 10 μm. For example, the average pore size can be 0.1 μm to 10 μm, 0.1 μm to 5 μm, 0.1 μm to 2 μm, 0.15 μm to 10 μm, 0.15 μm to 5 μm, 0.15 μm to 2 μm, 0.2 μm to 10 μm, 0.2 μm to 5 μm, 0.25 μm to 10 μm, 0.25 μm to 5 μm, 0.5 μm to 10 μm, 0.75 μm to 10 μm, 1 μm to 10 μm, 1 μm to 5 μm, 1 μm to 2 μm, 2 μm to 10 μm, 2 μm to 5 μm, or 5 μm to 10 μm. In some embodiments, the average pore size of the device is about 0.1 μm to 10 μm. In some embodiments, the average pore size of the device is about 0.1 μm to 5 μm. In some embodiments, the average pore size of the device is between about 0.1 μm and 1 μm.

[0249] In some embodiments, the device includes a semipermeable, biocompatible membrane surrounding genetically modified cells encapsulated in a polymeric composition (e.g., alginate hydrogel). The membrane pore size is selected to allow oxygen and other molecules important to cell survival and function to migrate through the semipermeable membrane while preventing immune cells from crossing through the pores. In one embodiment, the semipermeable membrane has a molecular weight cutoff of less than 1000 kD, or 50-700 kD, 70-300 kD, 70-150 kD, or 70-130 kD.

[0250] In one embodiment, the device may contain a cell-containing compartment surrounded by a barrier compartment formed from a cell-free biocompatible material, such as the core-shell microcapsules described in Ma, M et al., Adv. Healthc Mater., 2(5):667-672 (2012). Such barrier compartments may be used with or without a semi-permeable membrane.

[0251] The cells in the cell-containing compartment(s) of the device of the present disclosure may be encapsulated in a polymeric composition. The polymeric composition may include one or more hydrogel-forming polymers. In addition to the polymeric composition in the cell-containing compartment(s), the device (e.g., macrodevice, particle, hydrogel capsule) may include or be formed from materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials, and combinations thereof. The device may be made entirely of one type of material, or may include other materials in the cell-containing compartment and any other compartments.

[0252] In some embodiments, the device comprises a metal or metal alloy. In one embodiment, one or more of the compartments within the device (e.g., the first compartment, the second compartment, or all compartments) comprise a metal or metal alloy. Exemplary metals or metal alloys include titanium and titanium-based alloys (e.g., nitinol, nickel-titanium alloys, thermal memory alloy materials), platinum, platinum-based alloys, stainless steel, tantalum, palladium, zirconium, niobium, molybdenum, nickel-chromium, chromium-molybdenum alloys, or certain cobalt alloys (e.g., cobalt-chromium and cobalt-chromium-nickel alloys, e.g., ELGILOY® and PHYNOX®). For example, the metal material can be stainless steel grade 316 (SS316L) (composed of Fe, <0.3% C, 16-18.5% Cr, 10-14% Ni, 2-3% Mo, <2% Mn, <1% Si, <0.45% P, and <0.03% S). In metal-containing devices, the amount of metal (e.g., weight %, actual weight) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0253] In some embodiments, the device comprises a ceramic. In one embodiment, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all compartments) comprise a ceramic. Exemplary ceramic materials include oxides, carbides, or nitrides of transition elements, such as titanium oxide, hafnium oxide, iridium oxide, chromium oxide, aluminum oxide, and zirconium oxide. Silicon-based materials, such as silica, can also be used. In ceramic-containing devices, the amount of ceramic (e.g., weight %, actual weight) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0254] In some embodiments, the device has two hydrogel compartments, with the inner cell-containing compartment completely surrounded by a second, outer (e.g., barrier) compartment. In one embodiment, the inner boundary of the second compartment forms an interface with the outer boundary of the first compartment. In such embodiments, the thickness of the second (outer) compartment refers to the average distance between the outer boundary of the second compartment and the interface between the two compartments, e.g., the average distance measured at the thinnest and thickest points visually observed in the outer compartment. In some embodiments (e.g., the device is about 1.5 mm in diameter), the thinnest and thickest distances in the outer compartment are 25-110 micrometers (μm) and 270-480 μm, respectively. In some embodiments, the thickness of the outer compartment is greater than about 10 nanometers (nm), preferably 100 nm or greater, and can be as large as 1 millimeter (mm). For example, the thickness (e.g., average distance) of the outer compartment in the hydrogel capsule devices described herein can be 10 nm to 1 mm, 100 nm to 1 mm, 500 nm to 1 millimeter, 1 micrometer (μm) to 1 mm, 1 μm to 1 mm, 1 μm to 500 μm, 1 μm to 250 μm, 1 μm to 1 mm, 5 μm to 500 μm, 5 μm to 250 μm, 10 μm to 1 mm, 10 μm to 500 μm, or 10 μm to 250 μm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is 100 nm to 1 mm, 1 μm to 1 mm, 1 μm to 500 μm, or 5 μm to 1 mm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is about 50 μm to about 100 μm. In some embodiments (eg, the device is about 1.5 mm in diameter), the thickness (eg, average distance) of the outer section is about 180 μm to 260 μm or about 310 μm to 440 μm.

[0255] In some embodiments of a two-compartment hydrogel capsule device, the average pore size of the cell-containing inner ...

Claims

1. 1. An implantable element comprising modified mammalian cells, (i) (a) the major histocompatibility complex (MHC) class I protein complex; (b) MHC class II protein complexes; (c) a decrease in the level or function of one or more of the class II major histocompatibility complex transactivators (CIITAs); and (ii) (d) inflammatory cytokines, and (e) a decrease in the level or function of one or more of the profibrotic factors; the modified mammalian cell comprises an exogenous nucleic acid encoding a therapeutic agent; said implantable element comprising a polymer and a compound of formula (I): 【Chemical 1】 or a pharmaceutically acceptable salt thereof, wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —O—, —C(O)O—, —C(O)—, —OC(O)—, —N(R Ｃ ) -, -N(R Ｃ )C(O)-, -C(O)N(R Ｃ ) -, -N(R Ｃ )C(O)(C １ -C ６ -alkylene)-, -N(R Ｃ )C(O)(C １ -C ６ -alkenylene)-, -N(R Ｃ ) N (R Ｄ )-, -NCN-, -C(=N(R Ｃ ) (R Ｄ )) O-, -S-, -S(O) ｘ -, -OS (O) ｘ -, -N(R Ｃ ) S (O) ｘ -, -S(O) ｘ N (R Ｃ ) -, -P(R Ｆ ) ｙ -, -Si(OR Ａ ) ２ -, -Si(R Ｇ ) (OR Ａ ) -, -B(OR Ａ )-, or a metal, each of which is optionally bonded to a linking group (e.g., a linking group described herein) and one or more R １ and optionally substituted by L １ and L ３ is independently a bond, alkyl, or heteroalkyl, and each alkyl and heteroalkyl is selected from one or more R ２ and optionally substituted by L ２ is a bond, M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from the group consisting of one or more R ３ and optionally substituted by P is absent, cycloalkyl, heterocyclyl, or heteroaryl, each of which is selected from the group consisting of one or more R ４ and optionally substituted by Z is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -OR Ａ , -C(O)R Ａ , -C(O)OR Ａ , -C(O)N(R Ｃ ) (R Ｄ ), -N(R Ｃ ) CFv Ａ , cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally joined to one or more R ５ and optionally substituted by Each R Ａ , R Ｂ , R Ｃ , R Ｄ , R Ｅ , R Ｆ , and R Ｇ are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of one or more R ６ or optionally replaced by or R Ｃ and R Ｄ together with the nitrogen atom to which they are attached, form one or more R ６ forming a ring (e.g., a 5- to 7-membered ring) optionally substituted with Each R １ , R ２ , R ３ , R ４ , R ５ , and R ６ are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR Ａ１ , -C(O)OR Ａ１ , -C(O)R Ｂ１ , -OC(O)R Ｂ１ , -N(R Ｃ１ ) (R Ｄ１ ), -N(R Ｃ１ ) C(O)R Ｂ１ , -C(O)N(R Ｃ１ ), S.R. Ｅ１ , S(O) ｘ R Ｅ１ , -OS(O) ｘ R Ｅ１ , -N(R Ｃ１ ) S (O) ｘ R Ｅ１ , -S(O) ｘ N (R Ｃ１ ) (R Ｄ１ ), -P(R Ｆ１ ) ｙ , cycloalkyl, heterocyclyl, aryl, heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl may be selected from one or more R ７ and optionally substituted by Each R Ａ１ , R Ｂ１ , R Ｃ１ , R Ｄ１ , R Ｅ１ , and R Ｆ１ are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is selected from the group consisting of one or more R ７ and optionally substituted by Each R ７ is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is 1 or 2; The implantable element, wherein y is 2, 3, or 4.

2. The implantable element of claim 1 comprising: (i)(a).

3. 10. An implantable element according to any one of the preceding claims, comprising (i)(b).

4. 10. An implantable element according to any one of the preceding claims, comprising (i)(c).

5. 10. An implantable element according to any one of the preceding claims, comprising (ii)(d).

6. 10. An implantable element according to any one of the preceding claims, comprising (ii)(e).

7. 10. An implantable element according to any one of the preceding claims, comprising (i)(a) and one of (ii)(d) and (ii)(e).

8. 10. An implantable element according to any one of the preceding claims, comprising (i)(a) and both (ii)(d) and (ii)(e).

9. 10. An implantable element according to any one of the preceding claims, comprising (i)(b) and one of (ii)(d) and (ii)(e).

10. 10. An implantable element according to any one of the preceding claims, comprising both (i)(b) and (ii)(d) and (ii)(e).

11. 10. An implantable element according to any one of the preceding claims, comprising (i)(c) and one of (ii)(d) and (ii)(e).

12. 10. An implantable element according to any one of the preceding claims, comprising (i)(c) and both (ii)(d) and (ii)(e).

13. (i) the decrease in the level or function in (a) is (ai) human leukocyte antigen (HLA) A; (a-ii) HLA-B, (a-iii) HLA-C, and (a-iv) beta-2-microglobulin (beta-2M).

14. The implantable element of claim 13 comprising (ai).

15. An implantable element according to any one of claims 13 to 14, comprising (a-ii).

16. An implantable element according to any one of claims 13 to 15, comprising (a-iii).

17. An implantable element according to any one of claims 13 to 16, comprising (a-iv).

18. An implantable element according to any one of the preceding claims, wherein the modified mammalian cells comprise a mutation that results in a reduced level of a component of the MHC class I complex, e.g., compared to a reference standard.

19. An implantable element according to any one of the preceding claims, wherein the modified mammalian cells comprise low-functioning or non-functional variants of components of the MHC class I complex, e.g., compared to a reference standard.

20. 10. An implantable element according to any one of the preceding claims, wherein the levels of a component of the MHC class I complex are silenced or knocked down, e.g., compared to a reference standard.

21. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise about 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more reduction in levels of MHC class I components compared to modified mammalian cells that are substantially identical to, or identical to, the modified mammalian cells except for not comprising reduced levels of MHC class I components, e.g., compared to a reference standard.

22. (i) the decrease in the level or function in (b) (b-i) human leukocyte antigen (HLA) DP; (b-ii) HLA-DM, (b-iii) HLA-DOA, (b-iv) HLA-DOB, (b-v) HLA-DQ, and (b-vi) HLA-DR.

23. The implantable element of claim 22, wherein the MHC class II complex comprises (bi).

24. The implantable element of any one of claims 22 to 23, wherein the MHC class II complex comprises (b-ii).

25. The implantable element of any one of claims 22 to 24, wherein the MHC class II complex comprises (b-iii).

26. The implantable element of any one of claims 22 to 25, wherein the MHC class II complex comprises (b-iv).

27. The implantable element of any one of claims 22 to 26, wherein the MHC class II complex comprises (bv).

28. The implantable element of any one of claims 22 to 27, wherein the MHC class II complex comprises (b-vi).

29. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise a mutation that results in a reduced level of a component of the MHC class II complex, e.g., compared to a reference standard.

30. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise low-functioning or non-functional variants of components of the MHC class II components, e.g., compared to a reference standard.

31. 10. An implantable element according to any one of the preceding claims, wherein the expression of a component of the MHC class II complex is silenced or knocked down, e.g., compared to a reference standard.

32. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise a reduction in the level of MHC class II components of about 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, e.g., compared to modified mammalian cells that do not comprise a reduction in the level of MHC class II components, e.g., compared to a reference standard.

33. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise a reduced function or expression of class II major histocompatibility complex transactivator (CIITA).

34. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise a mutation that results in reduced expression of the CIITA component, e.g., compared to a reference standard.

35. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise hypo- or non-functional variants of components of the CIITA, e.g., compared to a reference standard.

36. 10. The implantable element of any one of the preceding claims, wherein expression of CIITA is silenced or knocked down, e.g., compared to a reference standard.

37. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise a reduction in the level of CIITA of about 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to modified mammalian cells that are substantially identical to the modified mammalian cells except for not comprising the reduction in the level of CIITA, e.g., compared to a reference standard.

38. The inflammatory cytokine (di) IL-6 (d-ii) IL-8, and An implantable element according to any one of the preceding claims, selected from (d-iii) MCP-1.

39. The implantable element of claim 38, wherein the proinflammatory cytokine is (di).

40. The implantable element according to any one of claims 38 to 39, wherein the pro-inflammatory cytokine is (d-ii).

41. The implantable element of any one of claims 38 to 40, wherein the pro-inflammatory cytokine is (d-iii).

42. 10. An implantable element according to any one of the preceding claims, wherein the modified mammalian cells comprise a mutation that results in a reduced expression of a pro-inflammatory cytokine, for example compared to a reference standard.

43. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise low-functioning or non-functioning variants of inflammatory cytokines, e.g., compared to a reference standard.

44. 10. An implantable element according to any one of the preceding claims, wherein the expression of inflammatory cytokines is silenced or knocked down, e.g. compared to a reference standard.

45. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise a reduction in the level of the inflammatory cytokine of about 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to modified mammalian cells that are substantially identical to the modified mammalian cells, or that are identical to the modified mammalian cells except that they do not comprise a reduction in the level of the inflammatory cytokine, e.g., compared to a reference standard.

46. The pro-fibrosis factor is (ei) FGF-2, (e-ii) PDGF, and An implantable element according to any one of the preceding claims, selected from (e-iii) VEGFA.

47. The implantable element of claim 46, wherein the profibrotic factor is (ei).

48. The implantable element of any one of claims 46 to 47, wherein the pro-fibrotic factor is (e-ii).

49. The implantable element of any one of claims 46 to 48, wherein the pro-fibrotic factor is (e-iii).

50. 10. An implantable element according to any one of the preceding claims, wherein the modified mammalian cells comprise a mutation that results in a reduced expression of the pro-fibrotic factor, e.g., compared to a reference standard.

51. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise a low-functioning or non-functioning variant of the pro-fibrotic factor, e.g., compared to a reference standard.

52. 10. An implantable element according to any one of the preceding claims, wherein expression of said pro-fibrotic factor is silenced or knocked down, e.g., compared to a reference standard.

53. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise a reduction in the level of the pro-fibrotic factor of about 0.05%, 0.1%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to modified mammalian cells that are substantially identical to the modified mammalian cells, or are identical to the modified mammalian cells except that they do not comprise a reduction in the level of the pro-fibrotic factor, e.g., compared to a reference standard.

54. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells are human cells.

55. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise (a) embryonic stem cells (ESCs) or cells derived therefrom, or (b) induced pluripotent stem cells (iPSCs) or cells derived therefrom.

56. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise retinal pigment epithelial (RPE) cells, CCD-33Lu cells, MRC-5 cells, MRC-9 cells, MCF10a cells, or cells derived from any of the foregoing cells.

57. 10. The implantable element of any one of the preceding claims, wherein the modified mammalian cells comprise RPE cells (eg ARPE-19 cells).

58. 10. The implantable element of claim 1, wherein the exogenous nucleotide sequence is extrachromosomal.

59. 10. An implantable element according to any one of the preceding claims, wherein the exogenous nucleotide sequence is inserted into at least one location in the genome of the mammalian cell.

60. 10. An implantable element according to any one of the preceding claims, wherein the polymer comprises a polymer selected from alginate, hyaluronate, and chitosan.

61. 61. The implantable element of any of claims 60, wherein the polymer comprises alginate.

62. 62. The implantable element of claim 61, wherein the alginate is a high guluronic acid (G) alginate or a high mannuronic acid (M) alginate.

63. 10. An implantable element according to any one of the preceding claims, wherein said polymer composition comprises at least one polymer covalently modified with a peptide.

64. 64. The implantable element of claim 63, wherein the peptide comprises, consists essentially of, or consists of GRGDSP, GGRGDSP, or GGGRGDSP.

65. 10. The implantable element of any one of the preceding claims, wherein the cell-containing compartment is surrounded by a barrier compartment comprising an alginate hydrogel disposed on the outer surface of the barrier compartment, and optionally a compound of formula (I) (e.g., a compound of formula (I) described herein).

66. The polymer composition comprises alginate covalently modified with a peptide, the peptide consisting essentially of or consisting of GRGDSP or GGRGDSP, and the barrier compartment comprises: 【Chemistry 2】 10. An implantable element according to any one of the preceding claims, comprising an alginate chemically modified with PEG-100 or a pharmaceutically acceptable salt thereof.

67. 10. An implantable element according to any one of the preceding claims, wherein the implantable element is spherical.

68. 10. An implantable element according to any one of the preceding claims, wherein the implantable element comprises a two-compartment hydrogel capsule.

69. 10. An implantable element according to any one of the preceding claims, wherein the implantable element is spherical with a diameter of about 0.75 mm to about 2 mm.

70. 10. An implantable element according to any one of the preceding claims, wherein the therapeutic agent is a protein, such as a hormone, a clotting factor, an antibody, or an enzyme.

71. (i) Beta-2M or CIITA, and (a) an inflammatory cytokine selected from IL-16, IL-8, and MCP-1; and (b) modified ARPE cells capable of reducing the expression of one of the pro-fibrotic factors selected from FGF-2, PDGF, and VEGFA; (ii) (c) a compound of formula (I) (e.g., as described herein), and (d) a polymeric composition comprising alginate covalently modified with one or more peptides.

72. (i) Beta-2M or CIITA, and (a) an inflammatory cytokine selected from IL-16, IL-8, and MCP-1; and (b) modified ARPE cells capable of reducing the expression of one of the pro-fibrotic factors selected from FGF-2, PDGF, and VEGFA; (ii) 【Chemistry 3】 or a pharmaceutically acceptable salt thereof, and (d) a polymer composition comprising alginate covalently modified with one or more of GRGDSP or peptides comprising or consisting of GGRGDSP.

73. 10. An implantable element according to any one of the preceding claims, formulated for implantation into a subject (e.g., into the intraperitoneal (IP) space, peritoneal cavity, omentum, lesser omental pouch, subcutaneous fat).

74. 74. The implantable element of claim 73, formulated for implantation into the IP space of a subject.

75. 10. A preparation of implantable elements, wherein each implantable element in said preparation is an implantable element according to any one of the preceding claims.

76. 76. A composition for use in treating a disease or disorder in a subject, said composition comprising an implantable element according to any one of claims 1 to 74 or a preparation according to claim 75, thereby treating said disease or disorder in said subject.

77. 77. The composition for use of claim 76, wherein the disease or disorder is a lysosomal storage disease or metabolic disorder.

78. 78. The composition for use according to any one of claims 76 to 77, wherein the subject is a human.

Images