Engineered cells and implantable elements for treating disease

Abstract

Described herein are engineered mammalian cells comprising a reduction in the level or function of one or more of the major histocompatibility complex (MHC) class I protein complex component, optionally an MHC class II protein complex component and / or CIITA, and one of an inflammatory cytokine and a pro-fibrotic factor; and methods of making and using the engineered mammalian cells.

Description

[0001] Priority claim

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 415,272 filed on October 11, 2022 and U.S. Provisional Patent Application No. 63 / 415,273 filed on October 11, 2022. Background Art

[0003] Treating chronic and genetic diseases by implanting cells engineered to produce therapeutic substances capable of treating such diseases holds exciting potential for improving the health of patients suffering from such diseases. To fully realize the potential of such therapies, the implanted cells must be able to produce therapeutic levels of the desired substance for weeks, months, or even longer without overstimulating the host immune response. Summary of the Invention

[0004] Described herein are engineered mammalian cells comprising a reduced level or reduced function of a major histocompatibility complex (MHC) class I protein complex and an inflammatory cytokine or one of a pro-fibrotic factor, as well as related devices (e.g., implantable elements), compositions, and methods of making and using the same. In one embodiment, the engineered mammalian cells comprise a reduced level 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 level or reduced function of the MHC class I protein complex may be due to a mutation in a component of the MHC class I protein complex, or may be due to silencing or knocking down a component of the MHC class I protein complex. In one embodiment, the inflammatory cytokine comprises IL-6, IL-8, or MCP-1. In one embodiment, the pro-fibrotic factor comprises FGF-2, PDGF, or VEGFA.

[0005] In one aspect, the present disclosure may also be characterized by an engineered mammalian cell further comprising a reduced level or reduced function of an MHC class II protein complex. In one embodiment, the engineered 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 the MHC class II protein complex may be due to a mutation in a component of the MHC class II protein complex, or may be due to silencing or knocking down a component of the MHC class II protein complex. In another aspect, the engineered mammalian cell described herein may also comprise a reduced level or reduced function of a class II major histocompatibility complex transactivator (CIITA).

[0006] On the other hand, the present disclosure is characterized in that a kind of implantable element, it comprises mammalian cell through engineered approaches described herein or a plurality of mammalian cells through engineered approaches.This mammalian cell through engineered approaches can include embryonic stem cell (ESC) or induced pluripotent stem cell (iPSC).This mammalian cell through engineered approaches can include retinal pigment epithelium (RPE) cell, CCD-33Lu cell, MRC-5 cell, MRC-9 cell, MCF10a cell, or by their derived cell. In one embodiment, this mammalian cell through engineered approaches includes RPE cell (for example, ARPE-19 cell through engineered approaches), or is derived from RPE cell (for example, ARPE-19 cell). In one embodiment, this mammalian cell through engineered approaches includes ARPE-19 cell through engineered approaches or is derived from ARPE-19 cell. In one embodiment, implantable element includes at least one cell-containing compartment, and this compartment includes mammalian cell through engineered approaches described herein or a plurality of mammalian cells through engineered approaches. In one embodiment, implantable element includes a cell-containing compartment, which includes engineered mammalian cells or multiple engineered mammalian cells as described herein; and a second compartment around the cell-containing compartment. In one embodiment, implantable element further includes at least one device (for example, formula (I) compound as described herein) for alleviating foreign body reaction (FBR) when implantable element is implanted into a subject. In one embodiment, implantable element includes a polymer selected from alginate, hyaluronate and chitosan. In one embodiment, implantable element includes a cell-containing compartment surrounded by a barrier compartment, which includes alginate hydrogel and optional formula (I) compound (for example, formula (I) compound described herein) arranged on the outer surface of the barrier compartment. In one embodiment, implantable element is formulated for implantation into a subject (for example, implanted into intraperitoneal (IP) space, peritoneal cavity, omentum, lesser sac, subcutaneous fat). In one embodiment, the implantable element is configured to protect the engineered mammalian cell or cells from the recipient's immune system and to mitigate a 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) over a sustained period of time (e.g., one to several months up to one to several years) after implantation into a subject.

[0007] In another aspect, the disclosure is characterized in that a method for treating a disease or condition in a subject is provided, the method comprising administering an implantable element to the subject, the implantable element comprising an engineered mammalian cell as described herein or a plurality of engineered mammalian cells as described herein, wherein the engineered mammalian cell or the plurality of engineered mammalian cells comprise a reduced level or reduced functional MHC class I protein complex. In one embodiment, the engineered mammalian cell or the plurality of engineered mammalian cells further comprise a reduced level or reduced functional MHC class II protein complex and / or a reduced level or reduced functional CIITA. In one embodiment, the engineered mammalian cell or the plurality of engineered mammalian cells further comprise a reduced level or reduced functional inflammatory cytokine or a pro-fibrotic factor. In one embodiment, the disease or condition is a lysosomal storage disease. In one embodiment, the disease or condition is a metabolic disease.

[0008] In one embodiment, implantable element described herein or multiple implantable elements described herein are combined with a pharmaceutically acceptable excipient, to prepare the preparation or the composition of implantable element, the preparation of this implantable element or composition can be applied to the experimenter (for example, being administered in the intraperitoneal cavity) of the therapeutic agent treatment that needs to be produced by this equipment.In one embodiment, experimenter is people, and the mammalian cell through engineered approaches is derived from human cell (for example RPE cell, ARPE-19 cell), and the preparation of implantable element or composition can continue to deliver the therapeutic agent of effective dose to experimenter in the time period (for example 3 months, 6 months, 1 year, 2 years or more) that continues.In one embodiment, the mammalian cell through engineered approaches is derived from human RPE cell, for example ARPE-19 cell.In one embodiment, the mammalian cell through engineered approaches is derived from people ARPE-19 cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A-1E is a set of graphs showing protein expression levels in ARPE-19 transduced with shRNA-containing lentiviral particles targeting one of the following: beta-2-microglobulin (β-2M) ( Figure 1A ), monocyte chemoattractant protein-1 (MCP-1) (also known as chemokine (CC motif) ligand 2 (CCL2)) ( Figure 1B ), fibroblast growth factor 2 (FGF2) ( Figure 1C ), interleukin-6 (IL-6) ( Figure 1D ) and interleukin-8 (IL-8) ( Figure 1E ).

[0010] Figure 2 is a graph showing that β-2M protein expression in α-L-iduronidase (IDUA)-expressing ARPE-19 cells containing β-2M shRNA was significantly (89%) lower than that in IDUA-expressing ARPE-19 cells containing a scrambled control shRNA.

[0011] Figure 3A -C is a set of graphs showing protein expression levels in IDUA-expressing ARPE-19 cells transduced with shRNA-containing lentiviral particles targeting one of the following: β-2M ( Figure 3A )、MCP-1(CCL2)( Figure 3B ) and IL-6 ( Figure 3C ).

[0012] Figure 4 is a graph showing that β-2M expression levels were reduced by 99% in ARPE-19 cells treated with CRISPR and β-2M-targeting gRNA compared to ARPE-19 cells modified with scrambled gRNA.

[0013] Figure 5A -D is a set of graphs showing β-2M expression in wild-type ARPE-19 cells ( Figure 5C ) compared to those with decreased β-2M protein expression ( Figure 5A ) decreased β-2M protein expression in ARPE-19 cells ( Figure 5B ) leads to decreased expression of human leukocyte antigen (HLA) ( Figure 5D ). DETAILED DESCRIPTION

[0014] The present disclosure is characterized in that mammalian cell (for example, human RPE cell), it is engineered to regulate the level or function of major histocompatibility complex (MHC) I class protein complex or its component (for example, beta-2-microglobulin (β-2M)).In one embodiment, mammalian cell is engineered to reduce the expression of MHC I class protein complex or its component (for example, beta-2M).Mammalian cell can be engineered to produce the function lower or non-functional variant of MHC I class protein complex or its component (for example, beta-2M), or the expression of MHC I class protein complex or its component (for example, beta-2M) can be silenced or strike low or knock out.The present disclosure is also characterized in that it is further engineered to regulate the level or function of MHC II class protein complex or its component, and / or the level or function of II class major histocompatibility complex transactivator (CIITA).In one embodiment, mammalian cell is engineered to reduce the expression of MHC II class protein complex or its component, and / or the level or function of CIITA. Mammalian cells can be engineered to produce less functional or non-functional variants of the MHC class II protein complex, or components thereof, and / or CIITA, or the expression of the MHC class II protein complex, or components thereof, and / or CIITA can be silenced or knocked down or knocked out.

[0015] Abbreviations and definitions

[0016] Throughout the detailed description and examples of the present disclosure, the following abbreviations will be used.

[0017] CM-Alg Chemically modified alginate

[0018] CM-LMW-Alg Chemically modified low molecular weight alginate

[0019] CM-LMW-Alg-101 Low molecular weight alginate chemically modified with compound 101 shown in Table 4

[0020] CM-HMW-Alg Chemically modified high molecular weight alginate

[0021] CM-HMW-Alg-101 High molecular weight alginate chemically modified with compound 101 shown in Table 4

[0022] CM-MMW-Alg Chemically modified medium molecular weight alginate

[0023] CM-MMW-Alg-101 Medium molecular weight alginate chemically modified with compound 101 shown in Table 4

[0024] HMW-Alg High molecular weight alginate

[0025] MMW-Alg Medium molecular weight alginate

[0026] U-Alg unmodified alginate

[0027] U-HMW-Alg Unmodified high molecular weight alginate

[0028] U-LMW-Alg Unmodified low molecular weight alginate

[0029] U-MMW-Alg Unmodified medium molecular weight alginate

[0030] 70:30 CM-Alg:U-Alg A 70:30 mixture (V:V) of chemically modified and unmodified alginate, e.g. as described in WO2020069429.

[0031] In order to make the present disclosure more easily 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 those of ordinary skill in the art to which the present disclosure belongs.

[0032] As used herein, including the appended claims, singular forms such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0033] "About" or "approximately," when used herein to modify a numerically defined parameter (e.g., the amount of therapeutic agent secreted by engineered cells, a physical description of a device (e.g., a hydrogel capsule) such as diameter, sphericity, the number of cells encapsulated therein, the number of devices in a formulation), means that the recited value is within an acceptable functional range for the defined parameter as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system, including the acceptable error range for the measurement system. For example, "about" can mean a range of 20% above and below the recited value. As a non-limiting example, a hydrogel capsule defined as having a diameter of approximately 1.5 millimeters (mm) and encapsulating approximately 5 million (M) cells can have a diameter of 1.2 to 1.8 mm and can encapsulate 4M to 6M cells. As another non-limiting example, a formulation of approximately 100 devices (e.g., hydrogel capsules) includes a formulation having 80 to 120 devices. In some embodiments, the term "about" means that the modified parameter may vary by as much as 15%, 10%, or 5% above and below the stated value of that parameter.

[0034] As used herein, the term "acquire" or "acquiring" refers to the process of obtaining a value (e.g., a numerical value) or a physical entity (e.g., a sample) by "directly obtaining" or "indirectly obtaining" the value or image or physical entity. "Directly obtaining" means performing a process (e.g., performing an analytical method or protocol) to obtain a value or physical entity. "Indirectly obtaining" refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly obtains the physical entity or value). Directly obtaining a value or physical entity includes performing a process that includes a physical change of a physical substance or the use of a machine or device. Examples of directly obtaining a value include obtaining a sample from a human subject. Directly obtaining a value includes performing a process that uses a machine or device, such as using a fluorescence microscope to obtain fluorescence microscopy data.

[0035] As used herein, "Administer," "administering," or "administration" refers to implanting, absorbing, ingesting, injecting, placing, or otherwise introducing an entity described herein (e.g., a device or a formulation of a device) into a subject, or providing such an entity to a subject for administration.

[0036] As used herein, "defibrotic" means a compound or material that reduces the foreign body response (FBR). For example, by implanting a device (e.g., a hydrogel capsule) comprising a defibrotic compound (e.g., a hydrogel capsule comprising a polymer covalently modified with a compound listed in Table 4) into a biological tissue, the amount of FBR induced in the tissue is lower than the FBR induced by implanting a reference device without defibrotic (afibrotic-null), i.e., a device lacking any defibrotic compound but having substantially the same composition (e.g., one or more identical cell types) and structure (e.g., size, shape, number of compartments). In one embodiment, the FBR is induced using an assay known in the art, such as described in WO 2017 / 075630 or using Vegas, A. et al., Nature 2017. One or more of the assays / methods described in Biotechnol (above) (e.g., measurement of subcutaneous tissue proteases 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 (α-muscle actin, SMA), or general cell deposition, quantification of 79 RNA sequences for known inflammatory factors and immune cell markers, or FACS analysis of macrophages and neutrophils on the device (e.g., capsule) retrieved after 14 days in the intraperitoneal space of a suitable test subject (e.g., immunocompetent mouse)) assesses the extent of FBR through an immune response in 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. In one embodiment, FBR is assessed by measuring the level of one or more immune response biomarkers (e.g., cathepsins, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4) in tissue containing the implant. In some embodiments, the FBR induced by a device of the invention (e.g., a hydrogel capsule comprising a defibrotic compound disposed on its outer surface) is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the FBR induced by a reference device without FBR, e.g., a device that is substantially identical to the device being tested or claimed except for the lack of a device for reducing FBR (e.g., a hydrogel capsule that does not comprise a defibrotic compound but is otherwise substantially identical to the claimed capsule). In some embodiments, the FBR (e.g., the level of one or more biomarkers) 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.

[0037] As used herein, "cell" refers to an engineered cell or a non-engineered cell. In one embodiment, the cell is an immortalized cell or an engineered cell derived from an immortalized cell. In one embodiment, the cell is a living cell, e.g., viable as measured by any technique described herein or known in the art.

[0038] As used herein, "cell binding peptide (CBP)" means a linear or cyclic peptide comprising an amino acid sequence of a cell binding domain of a ligand derived from a cell adhesion molecule (CAM) (e.g., a cell adhesion molecule that mediates cell-matrix attachment or cell-cell attachment). In one embodiment, CBP is any CBP described in International Patent Publication WO2020069429. In one embodiment, CBP is a linear peptide comprising RGD and is less than 6 amino acids in length. In one embodiment, CBP is a linear peptide consisting essentially of RGD or RGDSP.

[0039] As used herein, "CBP-polymer" refers to a polymer comprising at least one cell-binding peptide molecule covalently attached to a polymer by a linker. In one embodiment, the polymer in the CBP-polymer is a synthetic or naturally occurring polysaccharide, such as an alginate, such as sodium alginate. In one embodiment, the linker is an amino acid linker (i.e., substantially consisting of a single amino acid, or a peptide of several identical or different amino acids), which is coupled to the N-terminus or C-terminus of the CBP via a peptide bond. In one embodiment, the CBP-polymer is any CBP-alginate defined in WO2020069429.

[0040] As used herein, " cell binding substance (CBS) " means any chemical, biological or other type of substance (for example, small organic compounds, peptides, polypeptides) that can simulate at least one active part of the cell adhesion molecule (CAM) or other cell surface molecules that mediates cell-matrix connection or cell-cell connection or other receptor-mediated signal transduction. In one embodiment, when present in the polymer composition encapsulating living cells, CBS can form a temporary or permanent bond or contact with one or more cells. In one embodiment, CBS promotes the interaction between two or more living cells encapsulated in a polymer composition. In one embodiment, the presence of CBS in the polymer composition encapsulating multiple cells (for example, living cells) is related to one or both of the cell productivity (for example, expression of therapeutic agent) and the cell viability increased when the encapsulated cells are implanted in a test subject (for example, mouse). In one embodiment, CBS is physically attached to one or more polymer molecules in a polymer composition. In one embodiment, CBS is a cell binding peptide as defined herein or in WO2020069429.

[0041] As used herein, "conservatively modified variants" or "conservative substitutions" refer to variants of reference peptides or polypeptides that are identical to a reference molecule except for having one or more conservative amino acid substitutions in the amino acid sequence. In one embodiment, the 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. Conservative amino acid substitutions refer to substitutions of amino acids with amino acids that have similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, main chain conformation and rigidity, etc.) and that have minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution tables of functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional characteristics are listed in Table 1 below.

[0042] Table 1. Exemplary conservative amino acid substitution groups.

[0043]

[0044]

[0045] As used throughout the specification and claims, "consisting essentially of" and variations such as "consist essentially of" or "consisting essentially of" means that any recited element or group of elements is included, and optionally other elements having similar or different properties from the recited elements, which other elements do not materially alter the basic or novel characteristics of the specified molecule, composition, device, or method. As a non-limiting example, a therapeutic protein agent secreted by the engineered mammalian cells described herein that consists essentially of a recited amino acid sequence may further comprise one or more amino acid substitutions, including substitutions of one or more amino acid residues within the recited amino acid sequence, each of which does not materially affect the relevant biological activity of the therapeutic protein agent.

[0046] As used herein, "derived from" with respect to one or more cells refers to cells obtained from a tissue, cell line, or cells that are then optionally cultured, passaged, immortalized, differentiated, and / or induced, etc. to produce one or more derived cells.

[0047] As used herein, "device" and "implantable element" refer to any implantable object (e.g., a particle, a hydrogel capsule, an implant, a medical device) that contains one or more engineered cells (e.g., living cells) capable of expressing and secreting a therapeutic agent upon implantation of the device, and has a configuration that supports the viability of the cells by allowing cellular nutrients to enter the device. The terms "device" and "implantable element" are used interchangeably herein.

[0048] As used herein, "differential volume" refers to the volume of a compartment within a device described herein, excluding the space occupied by another compartment or compartments. For example, in a 2-compartment device with an inner compartment and an outer compartment, the differential volume of the second compartment (e.g., the outer compartment) refers to the volume within the second compartment excluding the space occupied by the first compartment (the inner compartment).

[0049] As used herein, "effective amount" refers to the amount of any of the following: engineered cells that secrete a protein, a device formulation that produces the protein, or a component of the device that is sufficient to elicit a desired biological response (e.g., the amount of a therapeutic agent co-expressed by cells in the device along with another therapeutic agent, the number of engineered cells in the device, the amount of CBS and / or defibrotic compound in the device). In some embodiments, the term "effective amount" refers to the amount of a component of the device (e.g., the number of cells in the device, the density of the defibrotic compound disposed on the surface of the device and / or in the barrier compartment, the density of the CBS in the cell-containing compartment).

[0050] In one embodiment, the desired biological response after implantation of the implantable element into a subject is a lower amount of pericytic fibrotic overgrowth (PFO) compared to the amount of PFO observed in a control implantable element (e.g., defined as an implantable element that is otherwise identical except that the cells do not have a reduction in MHC class I protein complexes). An effective amount can include the amount of therapeutic agent secreted by the engineered mammalian cells described herein. An effective amount encompasses both therapeutic and prophylactic treatments.

[0051] As used herein, an "effective amount" refers to an amount of a genetically modified cell (e.g., derived from a human cell (e.g., an epithelial cell)) that produces an exogenous polypeptide, or a device formulation that produces the polypeptide, sufficient to elicit a desired biological response. In one embodiment, the desired biological response is an increase in the level of an exogenous polypeptide or secreted polypeptide in a tissue sample removed from a subject treated with (e.g., implanted with) the genetically modified cell, a device containing such a cell, or a device formulation. As will be understood by one of ordinary skill in the art, the 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 both therapeutic and prophylactic treatments.

[0052] As used herein, "endogenous nucleic acid" is a nucleic acid that naturally occurs in the cells of a subject.

[0053] As used herein, an "endogenous polypeptide" is a polypeptide that naturally occurs in the cells of a subject.

[0054] " people's cell through engineered approaches " and " people's cell through genetic modification " can be used interchangeably in this article, and each term all means the people's cell (for example, epithelial cell) with non-natural gene change (for example, in cellular genome), and generally comprises the exogenous nucleic acid sequence (for example, DNA or RNA) that is not present in (or is present in with different levels) unengineered otherwise similar people's cell (for example, epithelial cell).In one embodiment, the people's cell through engineered approaches (for example, RPE cell through engineered approaches) comprises the exogenous nucleic acid of coding polypeptide (for example, therapeutic protein).In one embodiment, exogenous nucleotide sequence is chromosomal (for example, exogenous nucleic acid sequence is the exogenous sequence arranged in endogenous chromosome sequence) or extrachromosomal (for example, non-integrated expression vector).In one embodiment, exogenous nucleic acid sequence comprises RNA sequence, for example mRNA.In one embodiment, exogenous nucleic acid sequence comprises chromosome or extrachromosomal exogenous nucleic acid sequence, and this chromosome or extrachromosomal exogenous nucleic acid sequence comprises the sequence that is expressed as RNA, for example 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, which can be exogenous or endogenous. For example, an engineered cell may comprise an exogenous nucleic acid that controls the expression of an endogenous sequence. In one embodiment, the engineered cell comprises an exogenous nucleic acid sequence that comprises a codon-optimized coding sequence for a polypeptide of interest and achieves higher polypeptide expression than a naturally occurring coding sequence. Codon-optimized coding sequences can be optimized using commercially available algorithms, such as GeneOptimizer (ThermoFisher Scientific), OptimumGene TM (GenScript,Piscataway,NJ USA)、 (ATUM, Newark, CA USA) or Java Codon Adaptation Tool (JCat, www.jcat.de, Grote, A. et al., Nucleic Acids Research, Vol. 33, Supplement No. 2, pp. W526-W531 (2005) production. In one embodiment, the engineered cell (e.g., engineered epithelial cell, e.g., engineered RPE cell, e.g., engineered ARPE-19 cell) is cultivated from a monoclonal cell line. In some embodiments, the engineered cell is not an islet cell as defined herein.

[0055] As used herein, "exogenous nucleic acid" is a nucleic acid that does not naturally occur in the cells of a subject.

[0056] As used herein, an "exogenous polypeptide" is a polypeptide encoded by an exogenous nucleic acid in a subject's cells. Reference to an amino acid position in a particular sequence refers to the position of the amino acid in a reference amino acid sequence, e.g., the sequence of a full-length mature (after cleavage of the signal peptide) wild-type protein (unless otherwise indicated), and does not exclude the presence of variations (e.g., deletions, insertions, and / or substitutions) at other positions in the reference amino acid sequence.

[0057] Unless otherwise indicated, "Factor VII protein" or "FVII protein" as used herein refers to a polypeptide comprising the amino acid sequence of a naturally occurring Factor VII protein or a variant thereof, which polypeptide has a FVII biological activity, such as promoting blood coagulation, as determined by an assay recognized in the art. 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 single-chain and two-chain forms thereof, including zymogen-like and FVIIa. The FVII protein that can be produced by genetically modified cells described herein (e.g., derived from a human epithelial cell line, such as an ARPE-19 cell line) includes wild-type primates (e.g., humans), porcine, canine, and mouse proteins, as well as variants of such wild-type proteins, including fragments, mutants, variants with one or more amino acid substitutions and / or deletions. In some embodiments, the variant FVII protein can be 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 α (activated) (MarzAA) and variants described in European Patent No. 1373493, U.S. Patent No. 7771996, U.S. Patent No. 9476037 and U.S. Published Application No. US20080058255.

[0058] Unless otherwise indicated, otherwise factor VII bioactivity can be quantified by art-recognized determination.For example, the FVII bioactivity in the sample of biological fluid (such as blood plasma) can be measured by the following manner: (i) measuring the amount of factor Xa produced in the system comprising tissue factor (TF) and factor X embedded in lipid membrane (Persson et al., J.Biol.Chem.272:19919-19924,1997);(ii) measuring the factor X hydrolysis in aqueous system;(iii) using surface plasmon resonance-based instrument measurement of its physical binding to TF (Persson, FEBS Letts.413:359-363,1997);Or (iv) measuring the hydrolysis of synthetic substrate;And / or (v) measuring the generation of thrombin in the in vitro system independent of TF. 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).

[0059] Unless otherwise indicated, as used herein, "Factor VIII protein" or "FVIII protein" refers to a polypeptide comprising the amino acid sequence of a naturally occurring Factor VIII polypeptide or a variant thereof, which polypeptide has FVIII biological activity, such as coagulation activity, as determined by an assay recognized in the art. FVIII proteins that can be expressed by the genetically modified cells described herein (e.g., derived from a human epithelial cell line, such as an 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, 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., with a signal sequence) having a complete or partial deletion of the B domain. In one embodiment, the cell is engineered to encode a single-chain Factor VIII polypeptide comprising a variant FVIII protein, preferably 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 the FVIII protein include a one-stage or two-stage clotting assay (Rizza et al., 1982, Coagulation assay of FVIII:C and FIXa in Bloom ed. The Hemophelias. NY Churchill Livingston 1992) or a chromogenic substrate FVIII:C assay (Rosen, S. 1984. Scand J Haematol 33:139-145, suppl).

[0060] Many FVIII-BDD variants are known and include, for example, variants with complete or partial B domain deletions disclosed in any of 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 2and Example 1); 5,171,844 (e.g., Col. 4, line 22 to Col. 5, line 36); 5,543,502 (e.g., Col. 2, lines 17-46); 5,595,886; 5,610,278; 5,789,203 (e.g., Col. 2, lines 26-51 and Examples 5-8); 5,972,885 (e.g., Col. 1, line 25 to Col. 2, line 40); 6,048,720 (e.g., Col. 6, lines 1-22 and Example 1); ); 6,060,447; 6,228,620; 6,316,226 (e.g., column 4, line 4 to column 5, line 28 and Examples 1-5); 6,346,513; 6,458,563 (e.g., column 4, line 25-53) and 7,041,635 (e.g., column 2, line 1 to column 3, line 19, column 3, line 40 to column 4, line 67, column 7, line 43 to column 8, line 26, and column 11, line 5 to column 13, line 39).

[0061] In some embodiments, the FVIII-BDD protein produced by the genetically modified cells described herein (e.g., derived from a human epithelial cell line, such as an ARPE-19 cell line) has one or more of the following amino acid deletions in the B-domain: (i) most of the B domain except for the amino-terminal B domain sequence necessary 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 760-1639 (Toole et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)); amino acids 797-1562 (Eaton et al., Biochemistry 25:8343-8347 (1986)); 741-1646 (Kaufman, WO 87 / 04187); amino acids 747-1560 (Sarver et al., DNA 6:553-564 (1987)); amino acids 741-1648 (Pasek, WO 88 / 00831)); amino acids 816-1598 or 741-1689 (Lagner (Behring Inst. Mitt. (1988) No. 82:16-25, EP 295597); including deletions of one or more residues in the furin recognition sequence, including any of the specific deletions cited in U.S. Patent No. 9,956,269 at column 10, line 65 to column 11, line 36.

[0062] 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).

[0063] 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 recognition sequence LKRHQR that prevents proteolytic cleavage at that site, including any of the substitutions at positions R1645 and R1648 described in U.S. Patent Nos. 10,023,628, 9,394,353, and 9,670,267.

[0064] 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 (WO 2011 / 020866); and an Fc fusion (WO 04 / 101740).

[0065] Unless otherwise indicated, all FVIII-BDD amino acid positions referred to herein refer to positions in full-length human FVIII.

[0066] Unless otherwise indicated, " factor IX protein " or " FIX protein " as used herein refer to the polypeptide comprising the amino acid sequence of naturally occurring factor IX protein or its variant, as determined by art-recognized mensuration, and the polypeptide has FIX biological activity, such as coagulation activity. FIX is produced as an inactive zymogen, and this inactive zymogen is converted into active form by the factor XIa excision of activating peptide, thus producing heavy chain and light chain linked together by one or more disulfide bonds. The FIX protein that can be produced by genetically modified cells described herein (for example, derived from RPE cell line, such as ARPE-19 cell line) includes wild-type primates (such as people), pigs, dogs and rat proteins, and variants of this type of wild-type protein, including fragments, mutants, variants with one or more amino acid substitutions and / or deletions and any one of the aforementioned wild-type or variant proteins and the fusion of a half-life-extending polypeptide. In one embodiment, cell is engineered to encode full-length wild-type human factor IX polypeptide (for example, with signal sequence) or its functional variant. Variant FIX protein preferably has at least 50%, 75%, 90% or more (including>100%) of the clotting activity of wild-type factor VIX.The mensuration for measuring the clotting activity of FIX protein includes Biophen Factor IX assay (Hyphen BioMed) and primary clotting assay (activated partial thromboplastin time (aPTT) (e.g., as described in EP 2 032 607), thrombin generation time assay (TGA) and rotational thromboelastometry (e.g., as described in WO 2012 / 006624).

[0067] Many functional FIX variants are known and can be expressed by the engineered 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; WO 2007 / 149406, page 4, line 1 to page 19, line 11; WO 2007 / 149406A2, page 19, lines 12 to page 20, line 9; WO 08 / 118507, page 5, line 14 to page 6, line 5; WO 09 / 051717, page 9, lines 11 to page 20, line 2; WO 09 / 137254, page 2, paragraph

[006] to page 5, paragraph

[011] and page 16, paragraph

[044] to page 24, paragraph

[057] ; WO 09 / 130198A2, page 4, line 26 to page 12, line 6; WO 09 / 140015, page 11, paragraph

[0043] to page 13, paragraph

[0053] ; WO 2012 / 006624; WO 2015 / 086406.

[0068] 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, a PAS sequence, transferrin, CTP (a 28-amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) with four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), an albumin-binding polypeptide, an albumin-binding small molecule, 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) bound together by two disulfide bonds in the hinge region of the Fc.

[0069] FIX variant also includes gain of function and loss of function variant.An example of gain of function variant is " Padua " variant of people FIX, it has L (leucine) instead of R (arginine) (corresponding to SEQ ID NO:20 amino acid position 384) at the position 338 of mature protein, and has larger catalysis and coagulation activity (Chang et al., J.Biol.Chem., 273:12089-94 (1998)) compared with wild-type people FIX.An example of loss of function variant is to replace lysine with alanine in the 5th amino acid position starting from mature protein, and this produces the protein (for example, loss of function) that combines with collagen IV and reduces.

[0070] As used herein, "islet cells" are intended to include any naturally occurring or synthetically produced or modified cells that are intended to partially or completely reproduce, mimic, or otherwise express some or all of the functions of the islet cells of Langerhans. The term "islet cells" includes glucose-responsive insulin-producing cells derived from stem cells (e.g., derived from an induced pluripotent stem cell line).

[0071] As used herein, a "polymer composition" is a composition (e.g., a solution, a mixture) comprising one or more polymers. As a class, "polymers" include homopolymers, heteropolymers, copolymers, block polymers, block copolymers and can be natural and synthetic. Homopolymers contain one type of structural unit or monomer, while copolymers contain more than one type of monomer.

[0072] As used herein, "polypeptide" refers to a polymer comprising amino acid residues linked by peptide bonds and having at least two, and in some embodiments at least 3, 4, 5, 10, 50, 75, 100, 150, or 200 amino acid residues.

[0073] As used herein, "Prevention," "prevent," and "preventing" refer to a treatment that includes administering a composition (or formulation) that encapsulates a device that encapsulates genetically modified cells expressing an exogenous polypeptide prior to the onset of one or more symptoms of a disease or condition that can be treated with the exogenous polypeptide, so as to prevent the physical manifestation of the one or more symptoms. In some embodiments, "Prevention," "prevent," and "preventing" require that signs or symptoms of the disease or condition have not yet developed or been observed.

[0074] As used herein, "RPE cells" refer to cells having one or more of the following characteristics: a) they comprise retinal pigment epithelial cells (RPE) (e.g., using an RPE cell line such as the ARPE-19 cell line) a) cells derived from or engineered (e.g., cells cultivated from the ARPE-19 cell line by stable transfection of exogenous sequences encoding a polypeptide of interest or cells derived or engineered by inserting an exogenous sequence into one of the specific OCR insertion sites described herein), cells derived from primary cell cultures of RPE cells, cells directly isolated from naturally occurring RPE cells (e.g., from humans or other mammals) (without long-term culture, e.g., less than 5 or 10 generations or rounds of cell division since separation), cells derived from transformed, immortalized or long-term (e.g., more than 5 or 10 generations or rounds of cell division) RPE cell cultures; b) cells obtained from less differentiated cells, e.g., cells developed, programmed or reprogrammed (e.g., in vitro) into RPE cells, or cells derived from naturally occurring RPE cells or c) a cell that is substantially similar to one or more cells from a primary or long-term culture of RPE cells (e.g., the cell may be derived from an IPS cell); i) it expresses one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) it does not express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; iii) it occurs naturally in the retina and forms a monolayer above the choroidal vessels in Bruch's membrane; or iv) it is responsible for epithelial transport, light absorption, secretion, and immunomodulation in the retina; or v) it is synthetically produced or modified from a naturally occurring cell to have properties similar to those of an immortalized RPE cell line (e.g., an ARPE-19 cell line). ) identical or substantially identical genetic content and optional identical or substantially identical epigenetic content.Other exemplary strains of RPE cell comprise ARPE-19-SEAP-2-neo cell, RPE-J cell and hTERT RPE-1 cell.In one embodiment, RPE described herein is engineered to for example have novel characteristics, for example, by at least one exogenous transcription unit is inserted into one or more in OCR position described herein cell is carried out genetic modification.

[0075] When used in this article to refer to two nucleotide sequences or two amino acid sequences, "sequence identity" or "percentage identity" means that when two sequences are compared and aligned on a comparison window or a specified region to obtain maximum correspondence, the two sequences are identical within the specified region, or there are identical nucleotides or amino acids at the nucleotide or amino acid positions of a specified percentage within the specified region. Sequence identity can be determined using standard techniques known in the art (including but not limited to any algorithm described in U.S. Patent Application Publication No. 2017 / 02334455A1). 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 higher.

[0076] As used herein, "spherical" means a device (e.g., a hydrogel capsule or other particle) having a curved surface that forms a sphere (e.g., a perfectly round sphere) or a sphere-like shape, which may have, for example, waves and undulations on the surface. Spheres and sphere-like objects can be mathematically defined by rotating a circle, ellipse, or combination about each of three perpendicular axes a, b, and c. For a sphere, the three axes are the same length. In general, a sphere-like shape is an ellipsoid whose semi-major axes are within 10%, 5%, or 2.5% of each other (with respect to its average surface). The diameter of a sphere or sphere-like shape is the average diameter, such as the average of the semi-major axes.

[0077] The term "spheroid" as used herein to refer to a device (e.g., a hydrogel capsule or other particle) means that the device has (i) a perfect or classic oblate spheroid or prolate spheroid shape, or (ii) has a surface that is generally spheroidal, e.g., may have corrugations and undulations and / or may be an ellipsoid with semi-major axes within 100% of each other (with respect to its average surface).

[0078] As used herein, "subject" refers to a human or non-human animal. In one embodiment, the subject is a human (i.e., male or female) of any age group, such as a pediatric human subject (e.g., infant, child, teenager) or an adult subject (e.g., young, middle-aged, or elderly). In one embodiment, the subject is a non-human animal, such as a mammal (e.g., mouse, dog, primate (e.g., cynomolgus monkey or rhesus monkey). In one embodiment, the subject is a commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog) or bird (e.g., commercially relevant bird, such as chicken, duck, goose, or 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.

[0079] As used herein, "treatment," "treat," and "treating" refer to alleviating, reversing, mitigating a disease (e.g., hemophilia A), delaying the onset of the disease, or inhibiting the progression of one or more of the symptoms, manifestations, or underlying causes of the disease. In one embodiment, treatment includes reducing, reversing, mitigating a symptom or condition associated with the disease, delaying its onset, or inhibiting its progression. In one embodiment, treatment includes increasing the level of a therapeutic polypeptide in at least one tissue of a subject in need thereof, such as one or more of plasma, liver, kidney, and heart. In some embodiments, "treatment," "treat," and "treating" require that signs or symptoms associated with the disease or disorder have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease or disorder, such as in preventive treatment. For example, treatment may be administered to a susceptible individual before the onset of symptoms (e.g., due to a history of symptoms and / or genetic or other susceptibility factors). Treatment may also continue after symptoms have resolved, for example, to delay or prevent relapse. In some embodiments, treatment includes prevention, while in other embodiments it does not. "Wild type" (wt) refers to the native form, including 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 resulting from one or more genetic mutations.

[0080] Selected chemical definitions

[0081] Definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition, 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.

[0082] Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.The chemical structures and formulas shown herein are constructed according to standard rules of chemical valence known in the chemical arts.

[0083] When a range of values ​​is listed, it is intended to encompass each value and subrange within that range. For example, "C1-C6 alkyl" is intended to encompass 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.

[0084] As used herein, "alkyl" refers to a group having a straight chain or branched saturated hydrocarbon group of 1 to 24 carbon atoms ("C1-C 24 In some embodiments, an alkyl group has 1 to 12 carbon atoms ("C1-C 12 alkyl”), 1 to 10 carbon atoms (“C1-C 12In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), and n-propyl (C1). ), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), isobutyl (C4), n-pentyl (C5), 3-pentyl (C5), pentyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tert-pentyl (C5), and n-hexyl (C6). Additional examples of alkyl include n-heptyl (C7), n-octyl (C8), and the like. Each example of alkyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl"), or substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl").

[0085] As used herein, "alkenyl" refers to 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 has 2 to 10 carbon atoms ("C2-C 10 The carbon-carbon double bond(s) may be internal (as in 2-butenyl) or terminal (as 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 aforementioned C 2-4 alkenyl as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Each instance of alkenyl can independently be optionally substituted, i.e., unsubstituted (“unsubstituted alkenyl”) or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (“substituted alkenyl”).

[0086] As used herein, the term "alkynyl" refers to a group having a straight chain or branched hydrocarbon group of 2 to 24 carbon atoms, one or more carbon-carbon triple bonds ("C2-C24 In some embodiments, an alkynyl group has 2 to 10 carbon atoms ("C2-C 10 The carbon-carbon triple bond(s) may be internal (as in 2-butynyl) or terminal (as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), 2-butynyl (C4), and the like. Each example of an alkynyl group may independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted alkynyl").

[0087] As used herein, the term "heteroalkyl" refers to a non-cyclic stable straight or branched chain or a combination thereof comprising at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si and S, and wherein the nitrogen and sulfur atoms are optionally oxidized and the nitrogen heteroatom is optionally quaternized. One or more heteroatoms O, N, P, S and Si can be placed 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)2-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. In the description of "heteroalkyl", the specific heteroalkyl group is followed, such as -CH2O, -NR C R D etc., it will be understood that the term heteroalkyl and -CH2O or -NR C R D are not redundant or mutually exclusive. Rather, specific heteroalkyl groups are recited to increase clarity. Therefore, the term "heteroalkyl" should not be interpreted herein to exclude specific heteroalkyl groups such as -CH2O, -NR C R DEach instance of heterocyclyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted heteroalkyl") or substituted with one or more substituents, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted heteroalkyl").

[0088] Unless otherwise indicated, the terms "alkylene," "alkenylene," "alkynylene," or "heteroalkylene," alone or as part of another substituent, mean a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkylene, respectively. An alkylene, alkenylene, alkynylene, or heteroalkylene group may be described, for example, as a C1-C6-membered alkylene, a C2-C6-membered alkenylene, a C2-C6-membered alkynylene, or a C1-C6-membered heteroalkylene group, where the term "membered" refers to the non-hydrogen atoms within the group. In the case of heteroalkylene groups, heteroatoms may also occupy one or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied by the direction in which the chemical formula of the linking group is written. For example, the formula -C(O)2R'- may represent both -C(O)2R'- and -R'C(O)2-.

[0089] As used herein, "aryl" refers to 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 the ring array) having 6-14 ring carbon atoms and zero heteroatoms ("C6-C 14 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 In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 "aryl"; for example, anthracenyl). Aryl can be described as, for example, C6-C 10 The term "membered" refers to a non-hydrogen ring atom within the moiety. Aryl includes phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted aryl") or substituted (a "substituted aryl") with one or more substituents.

[0090] 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 in the ring array) having ring carbon atoms and 1-4 ring heteroatoms in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom when valence permits. Heteroaryl bicyclic ring systems may include one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the aryl or heteroaryl ring, and in such cases, the number of ring members indicates the number of ring members in the fused (aryl / heteroaryl) ring system. Bicyclic heteroaryl groups, where one ring does not contain heteroatoms (e.g., indolyl, quinolinyl, carbazolyl, etc.), can be attached at either ring, i.e., the ring bearing the heteroatom (e.g., 2-indolyl) or the ring containing no heteroatoms (e.g., 5-indolyl). Heteroaryl groups can be described, for example, as 6-10 membered heteroaryl groups, where the term "membered" refers to non-hydrogen ring atoms within the moiety.

[0091] In some embodiments, heteroaryl is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, heteroaryl is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, heteroaryl is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl group has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of a heteroaryl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroaryl") or substituted (a "substituted heteroaryl") with one or more substituents.

[0092] 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, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms 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, azepine, oxazepine, and thiazepine. Exemplary 5,6-bicyclic heteroaryls include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothienyl, isobenzothienyl, benzofuranyl, benzisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzothiazolyl, benzisothiazolyl, benzothiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryls include, but are not limited to, naphthyridinyl, pteridinyl, quinolyl, isoquinolyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryls include heme and heme derivatives.

[0093] As used herein, the terms "arylene" and "heteroarylene," by themselves or as part of another substituent, refer to a divalent radical derived from an aryl and heteroaryl group, respectively.

[0094] As used herein, "cycloalkyl" refers to a non-aromatic ring system having 3 to 10 ring carbon atoms ("C3-C 10 In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"), 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"), or 5 to 10 ring carbon atoms ("C5-C8 cycloalkyl"). 10Cycloalkyl"). Cycloalkyl can be described as, for example, C4-C7 membered cycloalkyl, where the term "membered" refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyls 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 cycloalkyls include but are not limited to Not limited to the aforementioned C3-C6 cycloalkyl and cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), etc. Exemplary C3-C 10 Cycloalkyl includes but is not limited to the aforementioned C3-C8 cycloalkyl and cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C9), 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ) and the like. As illustrated by the foregoing examples, in certain embodiments, a cycloalkyl group is monocyclic (a "monocyclic cycloalkyl") or contains a fused, bridged, or spiro ring system, such as a bicyclic ring system (a "bicyclic cycloalkyl"), and may be saturated or may be partially unsaturated. "Cycloalkyl" also includes ring systems in which a cycloalkyl ring as defined above is fused to one or more aryl groups, wherein the point of attachment is on the cycloalkyl ring, and 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 may independently be optionally substituted, i.e., unsubstituted (an "unsubstituted cycloalkyl"), or substituted (a "substituted cycloalkyl") with one or more substituents.

[0095] As used herein, "heterocyclyl" refers to a group of a 3 to 10 membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon ("3-10 membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, as long as valence permits. The heterocyclyl group may be monocyclic ("monocyclic heterocyclyl") or fused, bridged or spirocyclic ring systems, such as bicyclic systems ("bicyclic heterocyclyl"), and may be saturated or may be partially unsaturated. The heterocyclyl bicyclic system may include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which the heterocyclyl ring as defined above is fused to one or more cycloalkyl groups, wherein the point of attachment is on the cycloalkyl or heterocyclyl ring; or ring systems in which the heterocyclyl ring as defined above is fused to one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such cases, the number of ring members continues to represent the number of ring members in the heterocyclyl ring system. Heterocyclyl can be described as, for example, a 3-7 membered heterocyclyl, wherein the term "member" refers to the non-hydrogen ring atoms within the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of a heterocyclyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl"), or substituted ("substituted heterocyclyl") by one or more substituents. In certain embodiments, a heterocyclyl is an unsubstituted 3-10 membered heterocyclyl. In certain embodiments, a heterocyclyl is a substituted 3-10 membered heterocyclyl.

[0096] In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, a 5-6 membered heterocyclyl group has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, a 5-6 membered heterocyclyl group has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0097] Exemplary 3-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, aziridinyl, oxiranyl, and thioranyl. Exemplary 4-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclic groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrophenylthio, dihydrophenylthio, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclic groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxathiolanyl, dithiolanyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclic groups containing 3 heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclic groups containing 2 heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclic groups containing two heteroatoms include, but are not limited to, triazinanyl or thiomorpholinyl-1,1-dioxide. Exemplary 7-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azocanyl, oxepanyl, and thiecanyl. Exemplary 5-membered heterocyclic groups fused to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocycles) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinone, and the like. Exemplary 6-membered heterocyclic groups fused to an aryl ring (also referred to herein as 6,6-bicyclic heterocycles) include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.

[0098] As used herein, "amino" refers to the group -NR 70 R 71 , where R 70 and R 71 Each independently represents hydrogen, C1-C8 alkyl, C3-C 10 Cycloalkyl, C4-C 10 Heterocyclic group, C6-C 10 Aryl and C5-C 10 In some embodiments, amino refers to NH2.

[0099] As used herein, "cyano" refers to -CN.

[0100]

[0046] As used herein, "halo" or "halogen," by itself or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0101] As used herein, "hydroxy" refers to the group -OH.

[0102] As defined herein, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups 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). In general, the term "substituted," whether preceded by the term "optionally" or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, for example, a substituent that, upon substitution, results in a stable compound, for example, a compound that does not spontaneously undergo a transformation, such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise stated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the substituent is the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of an organic compound, any substituent that produces the formation of a stable compound as described herein. The present disclosure contemplates any and all such combinations to achieve stable compounds. For purposes of this disclosure, heteroatoms (such as nitrogen) may have hydrogen substituents and / or any suitable substituents that satisfy the valence of the heteroatoms and produce the formation of a stable moiety as described herein.

[0103] Two or more substituents may optionally be joined to form an aryl, heteroaryl, cycloalkyl or heterocyclyl group. Such so-called ring-forming substituents are typically, but not necessarily, attached to a cyclic basic structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the basic structure. For example, two ring-forming substituents attached to adjacent members of the cyclic basic structure form a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the basic structure. For example, two ring-forming substituents attached to a single member of the cyclic basic structure form a spirocyclic structure. In another embodiment, the ring-forming substituents are attached to non-adjacent members of the basic structure.

[0104] The compounds of formula (I) described herein may contain one or more asymmetric centers and may therefore exist in various isomeric forms (e.g., 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 stereoisomer mixtures (including racemic mixtures and mixtures enriched in one or more stereoisomers). Isomers may be separated from the mixture 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 may be prepared by asymmetric synthesis. See, e.g., 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 (E.L. Eliel, ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure further encompasses the compounds described herein as individual isomers substantially free of other isomers, as well as alternatively as mixtures of different isomers.

[0105] As used herein, a pure enantiomer compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, the "S" form of the compound is substantially free of the "R" form of the compound, and is therefore in enantiomeric excess of the "R" form. The term "enantiomerically pure" or "pure enantiomer" means that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight of the enantiomer. In certain embodiments, weight is the total weight of all enantiomers or stereoisomers based on the compound.

[0106] The compounds of formula (I) described herein may also contain one or more isotopic substitutions. For example, H may be in any isotopic form, including 1 H. 2H (D or deuterium) and 3 H (or tritium); C can be in any isotopic form, including 12 C. 13 C and 14 C; O can be in any isotopic form, including 16 O and 18 O etc.

[0107] The term "pharmaceutically acceptable salt" is intended to include salts of the active compounds prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When the compounds of formula (I) used to prepare the devices of the present disclosure contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds 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 compounds used in the present disclosure contain relatively basic functional groups, acid addition salts can be obtained by contacting the neutral form of such compounds 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, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid or phosphorous acid; and 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-toluenesulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids such as arginine salts; and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al., Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds used in the devices of the present disclosure (e.g., particles, hydrogel capsules) contain both basic and acidic functional groups that allow the compound to be converted into base addition salts or acid addition salts. These salts can be prepared by methods known to those skilled in the art. Other pharmaceutically acceptable carriers known to those skilled in the art are also suitable for use in the present disclosure.

[0108] The devices of the present disclosure may contain compounds of formula (I) in the form of prodrugs. Prodrugs are compounds that readily undergo chemical changes under physiological conditions to provide compounds useful in preparing the devices of the present disclosure. Additionally, prodrugs can be converted into useful compounds of formula (I) by chemical or biochemical methods in an ex vivo environment.

[0109] Some of the compounds of formula (I) described herein can exist in unsolvated form and solvated form (including hydrated form). In general, solvated form is equivalent to unsolvated form and is encompassed within the scope of the present disclosure. Some of the compounds of formula (I) described herein can exist in multiple crystals or amorphous forms. In general, all physical forms are equivalent for the purposes encompassed by the present disclosure and are intended to fall within the scope of the present disclosure.

[0110] The term "solvate" refers to a form of a compound that is associated with a solvent, typically by a solvolysis reaction. This physical association may include hydrogen bonding. Commonly used solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein can be prepared, for example, in crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates.

[0111] The term "hydrate" refers to a compound that is associated with water. Typically, the number of water molecules contained in a hydrate of a compound is in a certain ratio to the number of compound molecules in the hydrate. Thus, a hydrate of a compound can be represented, for example, by the general formula R x x H2O, where R is a compound and x is a number greater than 0.

[0112] As used herein, the term "tautomer" refers to a compound structure that is an interchangeable form and that varies in hydrogen atom and electron displacement. Thus, by the movement of π electrons and atoms (usually H), the two structures can be in equilibrium. For example, enols and ketones are tautomers because they can be rapidly converted to each other by treatment with an acid or base. Tautomeric forms can be relevant to obtaining the optimal chemical reactivity and biological activity of the target compound.

[0113] Notation used herein Refers to attachment to the surface of an entity, such as a polymer (e.g., a hydrogel-forming polymer such as alginate) or an implantable device (e.g., a particle, a hydrogel capsule). The connection represented can refer to direct attachment to an entity (e.g., a polymer or an implantable element) or can refer to a linkage to an entity through an attachment group. An "attachment group" as described herein refers to a portion used to link a compound of formula (I) to an entity (e.g., a polymer or an implantable element (e.g., a device) as described herein) and can include any attachment chemistry known in the art. A list of exemplary attachment groups is summarized in Bioconjugate Techniques (3rd Edition, Greg T. Hermanson, Waltham, MA: Elsevier, Inc, 2013), which is incorporated herein by reference in its entirety. In some embodiments, the attachment group includes 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 metal, where R A 、R C 、R D 、R F 、R G Each of x, x, and y is independently as described herein. In some embodiments, the attachment group comprises an amine, a ketone, an ester, an amide, an alkyl group. In some embodiments, the attachment group is a cross-linking agent. In some embodiments, the attachment group is -C(O)(C1-C6 1 , and R 1 As described herein. In some embodiments, the attachment group is -C(O)(C1-C6-alkylene)-, wherein the alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attachment group is -C(O)C(CH 3)2-. In some embodiments, the attachment group is -C(O)(methylene)-, wherein the alkylene group is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attachment group is -C(O)CH(CH3)-. In some embodiments, the attachment group is -C(O)C(CH3)-.

[0114] Engineered mammalian cells

[0115] The present disclosure provides an engineered mammalian cell that is capable of regulating the level or function of an MHC class I protein complex, and optionally an MHC class II protein complex and / or CIITA, and an inflammatory cytokine or a pro-fibrotic factor. In one embodiment, the engineered mammalian cell has reduced the level or function of an MHC class I protein complex, and optionally an MHC class II protein complex and / or CIITA, and an inflammatory cytokine or a pro-fibrotic factor.

[0116] MHC class I protein complexes are molecules present on the surface of nucleated cells that inform the host's immune system of the self or non-self status of specific antigens. MHC class I molecules display peptide fragments of cytotoxic proteins on the cell surface, triggering an immune response in the host if the cytotoxic protein is from a non-self source. Generally, MHC class I molecules are heterodimeric proteins composed of two polypeptide chains. The α chain is polymorphic and is encoded by human leukocyte antigens (HLAs) containing one of the HLA-A, HLA-B, or HLA-C. The β chain contains the β-2-microglobulin (β-2M) domain. The α and β chains of each MHC class I molecule are non-covalently linked through the interaction of the β-2M with one of the α chain's transmembrane domains (α-3). The α chain also contains two additional domains: α-1 and α-2. In one embodiment, the engineered mammalian cells of the present disclosure comprise reduced levels or function of an MHC class I protein complex or a component thereof (eg, HLA-A, HLA-B, HLA-C, or beta-2M).

[0117] Between α-1 and α-2 is a peptide binding groove that binds peptides derived from cytoplasmic proteins. The groove consists of eight β-pleated sheets located on the bottom and two α-helices that constitute the sides. The groove is flanked by tyrosine residues and forms a closed end that limits the size of the peptide that can be bound to the groove. The peptide in the groove remains bound during the life of the class I molecule and is typically 8-9 amino acids in length. Self- or foreign cytoplasmic proteins are degraded via the proteasome and transported to the ER cavity. In the ER, the peptide is loaded onto the MHC class 1 with the help of a chaperone protein called tapasin. The peptide-bound MHC class I is then transported to the plasma membrane of the cell, where the peptide is presented to the CD8+ T cell receptor (Becar M et al. (2022) Physiology, MHC Class I. See: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; January 2023-).

[0118] In addition to interacting with β-2M, the transplasma membrane α-3 domain also interacts with the T cell receptor (TCR) co-receptor CD8, thereby promoting antigen-specific activation. Although the binding of MHC class I to CD8 is approximately 100 times weaker than the binding of TCR to MHC class I, α-3-CD8 binding enhances the affinity of TCR binding (Wooldridge et al. (2010) MHC Class IMolecules with Superenhanced CD8Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184: 3357-3366).

[0119] β-2M is a non-glycosylated 12 kDa protein; one of its functions is to stabilize the MHC class I α chain. Unlike the α chain, β-2M does not span membranes. The human β-2M locus is located on chromosome 15. The β-2M gene consists of four exons and three introns. Circulating forms of β-2M are present in serum, urine, and other body fluids; thus, non-covalently MHC class I-associated β-2M can be exchanged with circulating β-2M under physiological conditions. β-2M associates not only with the α chain of MHC class I molecules but also with class I-like molecules such as CD1 (five genes in humans), MR1, the neonatal Fc receptor (FcRn), and Qa-1 (a form of alloantigen).

[0120] In some embodiments, the level or function of engineered mammalian cells (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprising MHC class I protein complexes or their components (e.g., HLA-A, HLA-B, HLA-C or β-2M) is reduced. In some embodiments, the level or function of engineered mammalian cells (e.g., ARPE-19) comprising MHC class I protein complexes or their components is reduced. For example, in some embodiments, the level or function of engineered mammalian cells (e.g., ARPE-19) comprising α and / or β chains of α-1 domains is reduced. In some embodiments, the level or function of α-1 domains 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 α-1 domains is reduced by about 20%. In some embodiments, the level or function of α-1 domains 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%.

[0121] 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 greater than 99.9%.

[0122] In some embodiments, the level or function of α-2 domain is reduced by about 10% (for example, about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). In some embodiments, the level or function of α-2 domain is reduced by about 20%. In some embodiments, the level or function of α-2 domain is reduced by about 30%. In some embodiments, the level or function of α-2 domain is reduced by about 40%. In some embodiments, the level or function of α-2 domain is reduced by about 50%. In some embodiments, the level or function of α-2 domain is reduced by about 60%. In some embodiments, the level or function of α-2 domain is reduced by about 70%. In some embodiments, the level or function of α-2 domain is reduced by about 80%. In some embodiments, the level or function of α-2 domain is reduced by about 90%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 100%.

[0123] 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 greater than 99.9%.

[0124] In some embodiments, the level or function of the α-3 structural domain included in the mammalian cell through engineering (for example, the RPE cell through engineering, for example, the ARPE-19 cell through engineering) is reduced. In some embodiments, the level or function of the α-3 structural domain is reduced by about 10% (for example, about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). For example, in some embodiments, the level or function of the α-3 structural domain is reduced by about 20%. In some embodiments, the level or function of the α-3 structural domain is reduced by about 30%. In some embodiments, the level or function of the α-3 structural domain is reduced by about 40%. In some embodiments, the level or function of the α-3 structural domain is reduced by about 50%. In some embodiments, the level or function of the α-3 structural domain is reduced by about 60%. In some embodiments, the level or function of the α-3 structural domain is reduced by about 70%. In some embodiments, the level or function of the α-3 structural domain is reduced by about 80%. In some embodiments, the level or function of the α-3 structural domain is reduced by about 90%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 100%.

[0125] 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 greater than 99.9%.

[0126] In some embodiments, the level or function of β-2M domains included in engineered mammalian cells (for example, engineered RPE cells, for example, engineered ARPE-19 cells) is reduced. In some embodiments, the level or function of β-2M domains are reduced by about 10% (for example, about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). For example, in some embodiments, the level or function of β-2M domains are reduced by about 20%. In some embodiments, the level or function of β-2M domains are reduced by about 30%. In some embodiments, the level or function of β-2M domains are reduced by about 40%. In some embodiments, the level or function of β-2M domains are reduced by about 50%. In some embodiments, the level or function of β-2M domains are reduced by about 60%. In some embodiments, the level or function of β-2M domains are reduced by about 70%. In some embodiments, the level or function of β-2M domains are 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%.

[0127] In some embodiments, the level or function of the β-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 β-2M domain is reduced by at least 10%. In some embodiments, the level or function of the β-2M domain is reduced by at least 20%. In some embodiments, the level or function of the β-2M domain is reduced by at least 30%. In some embodiments, the level or function of the β-2M domain is reduced by at least 40%. In some embodiments, the level or function of the β-2M domain is reduced by at least 50%. In some embodiments, the level or function of the β-2M domain is reduced by at least 60%. In some embodiments, the level or function of the β-2M domain is reduced by at least 70%. In some embodiments, the level or function of the β-2M domain is reduced by at least 80%. In some embodiments, the level or function of the β-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 greater than 99.9%.

[0128] In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of Beta-2M with an MHC class I protein complex or a 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, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of Beta-2M with an MHC class I protein complex or a component thereof. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of Beta-2M with an alpha-1 domain of an MHC class I protein. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of Beta-2M with an alpha-2 domain of an MHC class I protein. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between β-2M and the α-3 domain of MHC class I proteins. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between β-2M and MHC class I molecules or components (e.g., CD1, MR1, FcRn and Qa-1). In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between β-2M and CD1. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between β-2M and MR1. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between β-2M and FcRn. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between β-2M and Qa-1.

[0129] In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha-3 domain with the TCR co-receptor CD8 by, e.g., 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more.

[0130] HLA-A interacts with calnexin, calreticulin, transporter associated with antigen processing (TAP), tapasin, thiol-disulfide oxidoreductase ERp57 enzyme, and any cytoplasmic peptide bound within its peptide binding groove. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A with calnexin, calreticulin, TAP, tapasin, ERp57 enzyme, and / or any cytoplasmic peptide bound within its peptide binding groove by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A with calnexin. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A with calreticulin. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A with calreticulin. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-A with TAP. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between HLA-A and TAP-1. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between HLA-A and TAP-2. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between HLA-A and tapasin. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between HLA-A and ERp57. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) between HLA-A and a cytoplasmic peptide bound to its peptide binding groove.

[0131] 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 significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C with 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 significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C with KIR2DL1. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C with the leukocyte immunoglobulin-like receptor family. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C with LILRA1. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-C with LILRA3.

[0132] 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) results in decreased antigen presentation, thereby reducing and / or eliminating the recruitment of immune cells (e.g., T cells and NK cells).

[0133] The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger α-chain component of HLA-A. Variation in the HLA-A α-chain is crucial for HLA function. This variation contributes to genetic diversity within the human population. Because each HLA has different affinities for certain peptide structures, a greater number of HLA types means a wider variety of antigens can be presented on the cell surface, increasing the likelihood that a subset of the population will be able to defend against a specific foreign invader. This reduces the likelihood that a single pathogen will wipe out the entire human population.

[0134] Each person can express up to two types of HLA-A, one from each parent. Some individuals inherit the same HLA-A from both parents, reducing their individual HLA diversity; however, most individuals acquire two different copies of HLA-A. The same pattern applies to all HLA groups (Fix et al. (1998). HLA Matching, Antibodies, and You. Kidney Transplantation: Past, Present, and Future. University of Michigan Medical Center / Stanford University). In other words, each individual can only express one or two of the 2432 known HLA-A alleles.

[0135] The HLA-B gene is located on the short arm (p) of chromosome 6, at cytoband 21.3, and encodes the larger α-chain component of HLA-B. Similar to HLA-A, variations in the HLA-B α-chain are key to HLA function. HLA-C is a locus on chromosome 6 that encodes many HLA-C alleles, which are class I MHC receptors. HLA-C, located proximal to the HLA-B locus, is distal to the HLA region. Most HLA-C:B haplotypes are in strong linkage disequilibrium, and many are as old as the human species itself.

[0136] 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. Such mutations, as described herein, can result in reduced expression of the gene, for example, by reducing, altering, or eliminating 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.

[0137] In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and Beta-2M genes include 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 include 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 include 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 include 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.

[0138] 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 homology 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 homology 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 homology 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 homology 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 homology 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.

[0139] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a reduction in expression of MHC class I components, e.g., by 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 an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except that it does not comprise a reduction in the level of MHC class I components. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the expression of MHC I class components for example with except the reduction of the level not comprising MHC I class components with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the expression of MHC I class components for example with except the reduction of the level not comprising MHC I class components with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0140] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a reduction in the function of an MHC class I component, e.g., 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 of the reduction in the function of an MHC class I component compared to an engineered mammalian cell that is substantially the same or identical to the engineered mammalian cell except that it does not comprise a reduction in the function of an MHC class I component. In one embodiment, the function of engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise MHC I class components for example with except the reduction of the function of not comprising MHC I class components with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, the function of engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise MHC I class components for example with except the reduction of the function of not comprising MHC I class components with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0141] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level of HLA-A or a reduction in function, e.g., 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 an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in level or function of HLA-A. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of HLA-A or function for example with except not comprising the level of HLA-A or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of HLA-A or function for example with except not comprising the level of HLA-A or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0142] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level of HLA-B or a reduction in function, e.g., 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 an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except that it does not comprise a reduction in level or function of HLA-B. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of HLA-B or function for example with except not comprising the level of HLA-B or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of HLA-B or function for example with except not comprising the level of HLA-B or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0143] In one embodiment, an engineered mammalian cell described herein (e.g., an engineered RPE cell, e.g., an engineered ARPE-19 cell) comprises a level or function of HLA-C that is, for example, 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the level or function of HLA-C. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of HLA-C or function for example with except not comprising the level of HLA-C or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of HLA-C or function for example with except not comprising the level of HLA-C or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0144] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of β-2M that is, for example, 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 of a reduction compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the level or function of β-2M. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of β-2M or function for example with except not comprising the level of β-2M or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of β-2M or function for example with except not comprising the level of β-2M or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0145] 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 of the nucleotide sequences provided in Table 5, or a fragment thereof.

[0146] In some embodiments, the reduction in 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) 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 an MHC class I protein complex or a component thereof persists for at least 30 minutes. 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 1 hour. 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 12 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 24 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 48 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 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 1 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 1 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 1 year.

[0147] MHC class II protein complexes are a class of molecules present on the surface of antigen-presenting cells (such as dendritic cells, mononuclear phagocytes, certain endothelial cells, and B cells) in a subject. A key distinguishing feature between MHC class II protein complexes and MHC class I protein complexes is that the antigens presented by MHC class II protein complexes are derived from extracellular proteins, which are different from the cytoplasmic antigens presented by MHC class I protein complexes. Like MHC class I protein complexes, MHC class II protein complexes are also heterodimeric proteins composed of two polypeptide chains (α chain and β chain). Unlike MHC class I protein complexes, the α chain and β chain of MHC class II protein complexes contain homomeric peptides. The α peptide contains α-1 and β-1 domains, which combine to form a membrane-distal peptide binding groove, while the β peptide contains α-2 and β-2 domains, which form a membrane-proximal immunoglobulin-like domain. The peptide binding groove is composed of two α-helical walls and a β sheet. Because the antigen-binding groove of MHC class II molecules is open at both ends, while the corresponding groove on class I molecules is closed at each end, the antigens presented by MHC class II molecules are longer, generally between 15 and 24 amino acid residues in length.

[0148] 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 engineered mammalian cells (e.g., ARPE-19) comprise a reduction in the level or function of an MHC class II protein complex or its components (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR). In some embodiments, the engineered mammalian cells (e.g., ARPE-19) comprise a reduction in the α and / or β chains of an MHC class II protein complex or its components. For example, in some embodiments, the engineered mammalian cells (e.g., ARPE-19) comprise a reduction in the level or function of the α-1 domain. In some embodiments, the level or function of the α-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 α-1 domain is reduced by about 20%. In some embodiments, the level or function of the α-1 domain is reduced by about 30%. In some embodiments, the level or function of the α-1 domain is reduced by about 40%. In some embodiments, the level or function of the α-1 domain is reduced by about 50%. In some embodiments, the level or function of the α-1 domain is reduced by about 60%. In some embodiments, the level or function of the α-1 domain is reduced by about 70%. In some embodiments, the level or function of the α-1 domain is reduced by about 80%. In some embodiments, the level or function of the α-1 domain is reduced by about 90%. In some embodiments, the level or function of the α-1 domain is reduced by about 100%.

[0149] 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 greater than 99.9%.

[0150] In some embodiments, the engineered mammalian cells (e.g., ARPE-19) comprise a reduction in the level or function of the α-2 domain. In some embodiments, the level or function of the α-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 α-2 domain is reduced by about 20%. In some embodiments, the level or function of the α-2 domain is reduced by about 30%. In some embodiments, the level or function of the α-2 domain is reduced by about 40%. In some embodiments, the level or function of the α-2 domain is reduced by about 50%. In some embodiments, the level or function of the α-2 domain is reduced by about 60%. In some embodiments, the level or function of the α-2 domain is reduced by about 70%. In some embodiments, the level or function of the α-2 domain is reduced by about 80%. In some embodiments, the level or function of the α-2 domain is reduced by about 90%. In some embodiments, the level or function of the alpha-2 domain is reduced by about 100%.

[0151] 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 greater than 99.9%.

[0152] In some embodiments, the engineered mammalian cells (e.g., ARPE-19) comprise 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%.

[0153] 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 greater than 99.9%.

[0154] In some embodiments, the engineered mammalian cells (e.g., ARPE-19) comprise 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%.

[0155] 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 greater than 99.9%.

[0156] In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha chain and beta chain of the MHC class II protein complex or its components by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha-1 domain with the alpha-2 or beta-2 domain. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of the beta-1 domain with the alpha-2 or beta-2 domain. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha-2 domain with the alpha-1 or beta-1 domain. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of the beta-2 domain with the alpha-1 domain or beta-1 domain. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha-1 domain with the beta-1 domain. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of the alpha-2 domain with the beta-2 domain.

[0157] HLA-DP is a protein / peptide antigen receptor and 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, which are found in the MHC class II (or HLA-D) region of the HLA complex on human chromosome 6. HLA-DP is an αβ-heterodimer cell surface receptor. Each DP subunit (α, β) is composed of an α-helical N-terminal domain, an IgG-like β sheet, a transmembrane domain, and a cytoplasmic domain. The α-helical domains flank the peptide-binding groove. The β sheet region forms the bottom of the binding groove and the main body of the molecule, as well as the intersubunit (non-covalent) binding region. The peptide-bound HLA-DP complex interacts with the TCR on CD4+ T cells.

[0158] In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DP with the CD4+ T cell TCR and / or any peptide (e.g., antigenic peptide) bound within its peptide binding groove by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DP with the CD4+ T-cell TCR. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DP with a peptide bound within its peptide binding groove.

[0159] HLA-DM is a nonclassical MHC molecule, an intracellular protein involved in antigen presentation. It is encoded by the genes HLA-DMA and HLA-DMB. Like HLA-DP, the gene for HLA-DM is located in the MHC II region of human chromosome 6. HLA-DM is a molecular chaperone that functions in the lysosomes and endosomes of immune system cells. It plays a role in APCs such as macrophages, dendritic cells, and B cells by interacting with MHC class II molecules (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 protects MHC class II molecules from being broken down and also regulates which proteins or peptides bind to them. This regulates how and when peptides act as antigens that trigger immune responses. HLA-DM is required to release CLIP (fragment generated by cathepsin S or cathepsin D-mediated CD74 cleavage) from MHC class II molecules, to provide a chaperone for the empty MHC molecule to resist denaturation, to promote antigen-antigen exchange (e.g., by releasing weakly bound peptides from the peptide binding groove to allow loading of peptides with higher affinity), and to control the correct loading and release of peptides at the peptide binding groove. To release peptides from the MHC groove, HLA-DM binds to the N-terminus of the groove, thereby changing its conformation and disrupting hydrogen bonds, so that peptides interacting with the MHC groove are no longer bound and are ejected (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 traveling from the ER, but also on the cell membrane and in early endosomes.Much of this pathway is still under investigation, but 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 early endosomes, which are rapidly recycled. HLA-DM lacks the ability to bind peptides because it lacks a deep peptide-binding groove; instead, it contains a shallow, negatively charged indentation with two disulfide bonds.

[0160] HLA-DM also interacts extensively with another nonclassical MHC molecule, the chaperone protein HLA-DO. HLA-DO initially binds to DM in early endosomes but is expressed to a lesser extent in late endosomes / lysosomes. At low pH, the binding between HLA-DM and HLA-DO is less strong, but overall much stronger than the binding of HLA-DM to MHC molecules. Both HLA-DM and HLA-DO lack an N-terminal trafficking signal. Prior to antigen encounter, DO acts as a chaperone to DM, stabilizing it against denaturation and directing it to lysosomes. It binds to HLA-DM at the same site where it binds to 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 the antigen-containing lysosome fuses with the DM / DO / MHC-containing lysosome, prompting degradation of the HLA-DO molecule. The α chain (HLA-DOA) of HLA-DO is encoded by the HLA-DOA gene, and the β chain (HLA-DOB) is encoded by the HLA-DOB gene.

[0161] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DM with CLIP, HLA-DO and the β chain of MHC class II molecules by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DM with CLIP. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DM with HLA-DO. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DM with HLA-DO. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DM with the β chain of MHC class II molecules.

[0162] In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DO with HLA-DM or any peptide bound within its peptide binding groove by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DO with HLA-DM. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DO with a peptide bound within its peptide binding groove.

[0163] HLA-DQ is a cell surface receptor protein found on antigen-presenting cells. It is an αβ heterodimer of MHC class II. The α and β chains are encoded by two adjacent loci, HLA-DQA1 and HLA-DQB1, on chromosome band 6p21.3. Both the α and β chains contain a large number of variants. A single individual typically produces two α and two β chain variants, resulting in four HLA-DQ isoforms. The HLA-DQ locus is closely genetically linked to HLA-DR, but less closely linked to HLA-DP, nonclassical MHC class II molecules (HLA-DM and HLA-DO), and MHC class I molecules.

[0164] Different isoforms of HLA-DQ can bind to different antigens and present different antigens to T cells. In this process, T cells are stimulated to grow and send signals to B cells to produce antibodies. The role of HLA-DQ is to recognize and present foreign antigens (proteins derived from potential pathogens). For example, peptide-bound HLA-DP complexes interact with TCRs on CD4+ T cells. HLA-DQ is also involved in recognizing common self-antigens and presenting these antigens to the immune system in order to develop tolerance from a very young age. When tolerance to self-proteins is lost, HLA-DQ may become involved in autoimmune diseases. Two autoimmune diseases involved in 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 higher proportion of circulating T cell receptors that are likely to bind insulin, the major autoantigen in type 1 diabetes.

[0165] In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DQ with the CD4+ T-cell TCR and / or any peptide bound within its peptide binding groove by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95%, or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DQ with the CD4+ T-cell TCR. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of HLA-DQ with a peptide bound within its peptide binding groove.

[0166] HLA-DR is a cell surface receptor αβ heterodimer, each subunit of which contains two extracellular domains, a transmembrane domain, and a cytoplasmic tail. Both the α and β chains are anchored to the membrane. The N-terminal domain of the mature protein forms an α-helix that constitutes the exposed portion of the binding groove, and the C-terminal cytoplasmic region interacts with the other chain to form a β-sheet that spans the cell membrane below the binding groove. Most peptide contacts are located in 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). Sometimes an individual may have only two copies of the same locus, DRB1. The HLA-DRB1 locus is ubiquitous and encodes a very large number of functionally variable gene products (HLA-DR1 to HLA-DR17). The HLA-DRB3 locus encodes HLA-DR52, which has moderate variability and is variably associated with certain HLA-DRB1 types. The HLA-DRB4 locus encodes HLA-DR53, which has some variability and is associated with certain HLA-DRB1 types. The HLA-DRB5 locus encodes HLA-DR 51, which is usually invariant and associated with the HLA-DR2 type. HLA-DR interacts with CD74, HLA-DM, the CD4+ T cell TCR, and / or any peptide that binds within its peptide-binding groove.

[0167] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DR with CD74, HLA-DM, CD4+ T-cell TCR and / or any peptide bound within its peptide binding groove by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DR with CD74. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DR with HLA-DM. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DR with CD4+ T-cell TCR. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DR with a peptide bound within its peptide binding groove.

[0168] 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) results in decreased antigen presentation, thereby reducing and / or eliminating the recruitment of immune cells (e.g., T cells and NK cells).

[0169] 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. Such mutations, as described herein, can result in reduced expression of the gene, for example, by reducing, altering, or eliminating 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 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-DQA1. 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-DQB1. 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-DRA.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-DRB1. 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-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.

[0170] In some embodiments, the nucleotide sequence 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 comprises the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 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 the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequence 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 comprises a sequence having at least 65% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 70% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 75% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 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 HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQA1, HLA-DQB1, HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5 genes comprises a sequence having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 95% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 99% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having greater than 99.9% sequence identity to the nucleotide sequence provided in Table 5.

[0171] In some embodiments, the nucleotide sequence 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 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 the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequence 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 comprises a sequence having at least 65% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 70% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 75% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 80% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 85% sequence homology to the nucleotide sequences provided in Table 5.In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 90% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 95% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 99% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having at least 99.9% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence 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 comprises a sequence having greater than 99.9% sequence homology to the nucleotide sequence provided in Table 5.

[0172] In one embodiment, the engineered mammalian cells of the present disclosure comprise reduced levels 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 one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a reduction in expression of MHC class II components, e.g., by 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 an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except that it does not comprise a reduction in the level of MHC class II components. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the expression of MHC II class components for example with except the reduction of the level not comprising MHC II class components with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the expression of MHC II class components for example with except the reduction of the level not comprising MHC II class components with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0173] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a reduction in the function of an MHC class II component, e.g., 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 of the reduction in the function of an MHC class II component compared to an engineered mammalian cell that is substantially the same or identical to the engineered mammalian cell except that it does not comprise a reduction in the function of an MHC class II component. In one embodiment, the function of engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise MHC II class components for example with except the reduction of the function of not comprising MHC II class components with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, the function of engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise MHC I class components for example with except the reduction of the function of not comprising MHC II class components with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0174] In some embodiments, the MHC class II protein complex or a component thereof (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, or HLA-DR) is encoded by one or more of the nucleotide sequences provided in Table 5, or a fragment thereof.

[0175] In some embodiments, the reduction in the level or function of an MHC class II protein complex or a component thereof 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 reduction in the level or function of an MHC class II protein complex or a component thereof lasts for at least 30 minutes. In some embodiments, the reduction in the level or function of an MHC class II protein complex or a component thereof lasts for at least 1 hour. In some embodiments, the reduction in the level or function of an MHC class II protein complex or a component thereof lasts for at least 12 hours. In some embodiments, the reduction in the level or function of an MHC class II protein complex or a component thereof lasts for at least 24 hours. In some embodiments, the reduction in the level or function of an MHC class II protein complex or a component thereof lasts for at least 48 hours. In some embodiments, the reduction in the level or function of an MHC class II protein complex or a component thereof lasts for at least 72 hours. In some embodiments, the reduction in the level or function of an MHC class II protein complex or a component thereof 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 lasts for at least 1 month. In some embodiments, the reduction in the level or function of the MHC class II protein complex or a component thereof persists for at least 1 year.

[0176] The class II major histocompatibility complex transactivator (CIITA) is a gene involved in regulating the expression of the MHC class II protein complex. The CIITA gene, located on chromosome 16, encodes the CIITA protein, which enhances the transcription of MHC class I genes. The CIITA protein contains an acidic transcriptional activation domain, four leucine-rich repeats, and a GTP-binding domain. This protein uses GTP binding to facilitate its translocation into the cell nucleus. Once in the nucleus, the protein acts as a positive regulator of transcription of class II major histocompatibility complex genes and is often referred to as a "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).

[0177] In one embodiment, the engineered mammalian cells of the present disclosure include a reduction in the level or function of a CIITA protein. In some embodiments, the engineered mammalian cells (e.g., ARPE-19) include a reduction in the level or function of a domain of a CIITA protein, wherein the domain is selected from a transcriptional activation domain, a leucine-rich repeat domain, and a GTP-binding domain. For example, in some embodiments, the engineered mammalian cells (e.g., ARPE-19) include a reduction in the level or function of a transcriptional activation domain. In some embodiments, the level or function of a transcriptional 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 a transcriptional activation domain is reduced by about 20%. In some embodiments, the level or function of a transcriptional activation domain is reduced by about 30%. In some embodiments, the level or function of a transcriptional activation domain is reduced by about 40%. In some embodiments, the level or function of a transcriptional activation domain is reduced by about 50%. In some embodiments, the level or function of the transcriptional activation domain is reduced by about 60%. In some embodiments, the level or function of the transcriptional activation domain is reduced by about 70%. In some embodiments, the level or function of the transcriptional activation domain is reduced by about 80%. In some embodiments, the level or function of the transcriptional activation domain is reduced by about 90%. In some embodiments, the level or function of the transcriptional activation domain is reduced by about 100%.

[0178] In some embodiments, the level or function of the transcriptional 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 transcriptional activation domain is reduced by at least 10%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 20%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 30%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 40%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 50%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 60%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 70%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 80%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 90%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 95%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 99%. In some embodiments, the level or function of the transcriptional activation domain is reduced by at least 99.9%. In some embodiments, the level or function of the transcriptional activation domain is reduced by greater than 99.9%.

[0179] In some embodiments, the level or function of engineered mammalian cells (for example, engineered RPE cells, for example, engineered ARPE-19 cells) comprising leucine-rich repeat domains is reduced. In some embodiments, the level or function of leucine-rich repeat domains is reduced by about 10% (for example, about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). For example, in some embodiments, the level or function of leucine-rich repeat domains is reduced by about 20%. In some embodiments, the level or function of leucine-rich repeat domains is reduced by about 30%. In some embodiments, the level or function of leucine-rich repeat domains is reduced by about 40%. In some embodiments, the level or function of leucine-rich repeat domains is reduced by about 50%. In some embodiments, the level or function of leucine-rich repeat domains is reduced by about 60%. In some embodiments, the level or function of leucine-rich repeat domains is reduced by about 70%. In some embodiments, the level or function of leucine-rich repeat domains 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%.

[0180] 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 greater than 99.9%.

[0181] In some embodiments, the engineered mammalian cells (e.g., ARPE-19) comprise 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%.

[0182] 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 greater than 99.9%.

[0183] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of a CIITA protein domain with another CIITA protein domain by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of a transcriptional activation domain with a leucine-rich repeat domain or a GTP-binding domain. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of a leucine-rich repeat domain with a transcriptional activation domain, a leucine-rich repeat domain, or a GTP-binding domain. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of a GTP-binding domain with a transcriptional activation domain or a leucine-rich repeat domain.

[0184] 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 repeat family member C (ZXDC). In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA with MAPK1, NCOA1, RFX5, RFXANK, XPO1, and / or ZXDC by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA with MAPK1. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA with NCOA1. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA with RFX5. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA with RFXANK. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA with XPO1. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of CIITA with ZXDC.

[0185] In some embodiments, reducing the level or function of the CIITA protein results in a decrease in the expression level of an MHC class II protein complex or a component thereof (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR) (e.g., by reducing the transcriptional level of the MHC class II protein complex or a component thereof).

[0186] 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. Nucleotide mutations can include nucleotide deletions, additions, and / or substitutions. Such mutations as described herein can, for example, lead to a decrease in the expression of the gene by reducing, altering, or eliminating transcription and / or splicing of the nucleotide sequence.

[0187] In some embodiments, the nucleotide sequence of the CIITA gene comprises the 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 higher) sequence identity to the 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 the 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 the 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 the 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 the 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 greater than 99.9% sequence identity to the nucleotide sequence provided in Table 5.

[0188] 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 higher) sequence homology to the 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 the 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 the 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 the 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 the 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 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 homology 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 homology 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 homology 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 homology to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having greater than 99.9% sequence homology to the nucleotide sequence provided in Table 5.

[0189] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise expression of CIITA, e.g., 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising a reduction in the level of CIITA. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise expression of CIITA for example with except the reduction of the level not comprising CIITA substantially identical with this engineered mammalian cell or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise expression of CIITA for example with except the reduction of the level not comprising CIITA substantially identical with this engineered mammalian cell or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0190] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a function of CIITA, e.g., 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 of the reduction compared to an engineered mammalian cell that is substantially the same or identical to the engineered mammalian cell except that it does not comprise a reduction in the function of CIITA. In one embodiment, the function of engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise CIITA for example with except the reduction of the function not comprising CIITA substantially identical with this engineered mammalian cell or the reduction compared to identical engineered mammalian cell between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, the function of engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise CIITA for example with except the reduction of the function not comprising CIITA substantially identical with this engineered mammalian cell or the reduction compared to identical engineered mammalian cell greater than about 50%, 75% or 90%.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0191] In some embodiments, CIITA is encoded by the nucleotide sequence provided in Table 5 or a fragment thereof.

[0192] In some embodiments, the reduction in the level or function of CIITA continues 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 CIITA continues for at least 30 minutes. In some embodiments, the reduction in the level or function of CIITA continues for at least 1 hour. In some embodiments, the reduction in the level or function of CIITA continues for at least 12 hours. In some embodiments, the reduction in the level or function of CIITA continues for at least 24 hours. In some embodiments, the reduction in the level or function of CIITA continues for at least 48 hours. In some embodiments, the reduction in the level or function of CIITA continues for at least 72 hours. In some embodiments, the reduction in the level or function of CIITA continues for at least 1 week. In some embodiments, the reduction in the level or function of CIITA continues for at least 1 month. In some embodiments, the reduction in the level or function of CIITA continues for at least 1 year.

[0193] In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprises the level of MHC I class protein complex or the reduction of function and the level of MHC II class protein complex and / or CIITA or the reduction of function.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprises the level of HLA-A or the reduction of function and the level of MHC II class protein complex and / or CIITA or the reduction of function.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprises the level of HLA-B or the reduction of function and the level of MHC II class protein complex and / or CIITA or the reduction of function.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprises the level of HLA-C or the reduction of function and the level of MHC II class protein complex and / or CIITA or the reduction of function. In one embodiment, the engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprises the level of β-2M or the reduction of function and MHC II class protein complex and / or the level of CIITA or the reduction of function.In one embodiment, the engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell).In one embodiment, the engineered mammalian cell is engineered ARPE-19 cell.

[0194] Engineered mammalian cells containing inflammatory cytokines and / or profibrotic factors

[0195] The present disclosure is further characterized by an engineered mammalian cell comprising inflammatory cytokines and / or pro-fibrotic factors. Inflammatory cytokines are signaling molecules secreted by immune cells that can promote or induce inflammation in the host. Exemplary inflammatory cytokines include interleukin 6 (IL-6), IL-8, IL-10, IL-1-β, monocyte chemoattractant protein 1 (MCP-1), and tumor necrosis factor alpha (TNF-α). In one embodiment, the engineered mammalian cell comprises a reduction in the level or function of an inflammatory cytokine, for example, selected from IL-6, IL-8, IL-10, IL-1-β, MCP-1, and TNF-α.

[0196] IL-6, encoded by the IL-6 gene, is a cytokine characterized by pleiotropic activity; 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 acquired immune responses by stimulating antibody production and effector T cell development. In addition, IL-6 can promote the differentiation or proliferation of several non-immune cells. Continuous production of IL-6 can lead to the occurrence 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 receptors IL-6R, IL-7, and IL-15.

[0197] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 with IL-6R, IL-7 and / or IL-15 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 with IL-6R by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 with IL-6R by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 with IL-6R by, for example, 20% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-6R by, for example, 30% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-6R by, for example, 40% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-6R by, for example, 50% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-6R by, for example, 75% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-6R by, for example, 90% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-6R by, for example, 95% or more.

[0198] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-7 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-7 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-7 by, for example, 20% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-7 by, for example, 30% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-7 by, for example, 40% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-7 by, for example, 50% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-7 by, for example, 75% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-7 by, for example, 90% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-7 by, for example, 95% or more.

[0199] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-15 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-15 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-15 by, for example, 20% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-15 by, for example, 30% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-15 by, for example, 40% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-15 by, for example, 50% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-15 by, for example, 75% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-15 by, for example, 90% or more. Reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-6 and IL-15 by, for example, 95% or more.

[0200] IL-8 is encoded by the Il-8 gene and is a cytokine that induces chemotaxis of target cells (primarily neutrophils, but also other granulocytes), causing the target cells to migrate to the site of infection and, once the target cells have arrived at the site of infection, stimulates phagocytosis. IL-8 is also known to induce a series of physiological responses required for migration and phagocytosis in target cells, such as intracellular Ca 2+IL-8 is a potent angiogenesis promoter that increases cytokines and exocytosis (e.g., histamine release and respiratory burst). IL-8 can be secreted by any cell with toll-like receptors that participates in the innate immune response and has been shown to be a marker chemokine for 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 CR1and CR2". JCI Insight. 2(16). doi:10.1172 / jci.insight.93739). It has been reported that both monomeric and homodimeric forms of IL-8 are potent inducers of the chemokine receptors CXC motif chemokine receptor 1 (CXCR1) and CXCR2.

[0201] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 with CXCR1 and / or CXCR2 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 with CXCR1 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 with CXCR1 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 with CXCR1 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., combination) of IL-8 and CXCR1 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., combination) of IL-8 and CXCR1 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., combination) of IL-8 and CXCR1 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., combination) of IL-8 and CXCR1 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., combination) of IL-8 and CXCR1 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., combination) of IL-8 and CXCR1 by, for example, 95% or more.

[0202] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 and CXCR2 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 and CXCR2 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 and CXCR2 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 and CXCR2 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 and CXCR2 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 and CXCR2 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 and CXCR2 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 and CXCR2 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-8 and CXCR2 by, for example, 95% or more.

[0203] IL-10, encoded by the IL-10 gene, is a cytokine with pleiotropic effects in immune regulation and inflammation. It downregulates the expression of Th1 cytokines (e.g., IFN-γ, IL-2, IL-3, TNFα, and GM-CSF), MHC class II antigens, and co-stimulatory molecules on macrophages, and inhibits 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 cellproliferation 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-associatedIL-10 receptor inhibits CD28tyrosine phosphorylation and phosphatidylinositol3-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 has a stimulatory effect on Th2 cells. In addition, IL-10 can block NF-κB activity and participate in the regulation of the JAK-STAT signaling pathway. IL-10 signaling is induced after IL-10 binds to its receptor IL-10R.

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

[0205] IL-1-β is a cytokine encoded by the IL-1-β gene that is produced by activated macrophages, monocytes, and a subset of dendritic cells called slanDCs (Yaseen et al. (2023). "The role of IL-1βduring human immunodeficiency virus type 1 infection". Reviews in Medical Virology. 33(1): e2400. doi: 10.1002 / rmv.2400) as a proprotein that is proteolytically processed by caspase 1 (CASP1 / ICE) into its active form. IL-1-β is an important mediator of inflammatory responses and is involved in a variety of cellular activities including cell proliferation, differentiation, and apoptosis. IL-1-β has been found to induce cyclooxygenase-2 (PTGS2 / COX2) in the central nervous system (CNS), leading to inflammatory pain hypersensitivity. The combination of IL-1-β and IL-23 can induce γδ T cells to express IL-17, IL-21, and IL-22. This induction of expression occurs in the absence of additional signals, suggesting that IL-1-β is involved in regulating autoimmune inflammation (Sutton et al. (2009). "Interleukin-1 and IL-23 induce innate IL-17 production from gammadelta T cells, amplifying Th17 responses and autoimmunity". Immunity. 31(2): 331–341. doi: 10.1016 / j.immuni.2009.08.001). IL-1-β signaling is mediated by the binding of IL-1-β to its receptors IL-1-receptor-1 (IL-1R1) and IL-1-receptor accessory protein (IL-1RAcP).

[0206] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1R1 and / or IL-1RAcP by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1R1 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1R1 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β and IL-1R1 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β and IL-1R1 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β and IL-1R1 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β and IL-1R1 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β and IL-1R1 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β and IL-1R1 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1R1 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1R1 by, for example, 95% or more.

[0207] In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1RAcP by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1RACP by, for example, 10% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1RACP by, for example, 20% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1RACP by, for example, 30% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of IL-1-β with IL-1RACP by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1-β with IL-1RACP by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1-β with IL-1RACP by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1-β with IL-1RACP by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of IL-1-β with IL-1RACP by, for example, 95% or more.

[0208] MCP-1, also known as chemokine (CC motif) ligand 2 (CCL2), is a small cytokine belonging to the CC chemokine family and is encoded by the CCL2 gene. CCL2 strictly regulates cell mechanics and thereby recruits monocytes, memory T cells, and dendritic cells to sites of inflammation caused by tissue damage 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; Car et al. (1994). "Monocyte chemoat tractant protein 1 acts as a T-lymphocyte chemoattractant". Proceedings of the National Academy of Sciences of the United States of America. 91(9):3652–6. Bibcode: 1994 PNAS…91.3652C. doi: 10.1073 / pnas.91.9.3652; Xu et al. (1996). "Human recombinant monocytechemotactic 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 the binding of MCP-1 to its receptor, CC chemokine receptor type 2 (CCR2).

[0209] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of MCP-1 and CCR2 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of MCP-1 and CCR2 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of MCP-1 and CCR2 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of MCP-1 and CCR2 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of MCP-1 and CCR2 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of MCP-1 and CCR2 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of MCP-1 and CCR2 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of MCP-1 and CCR2 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of MCP-1 and CCR2 by, for example, 95% or more.

[0210] TNF-α is an adipokine and a 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. As part of the inflammatory response, macrophages release TNF to alert other immune system cells. TNF signaling occurs through two receptors: TNFR1 and TNFR2. TNFR1 is constitutively expressed in most cell types, while TNFR2 is primarily restricted to endothelial cells, epithelial cells, and immune cell subsets (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 have pro-inflammatory and apoptotic effects, while TNFR2 signaling has anti-inflammatory effects and promotes cell proliferation. Inhibition of TNFR1 signaling is important for the treatment of autoimmune diseases, while TNFR2 signaling promotes wound healing.

[0211] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 and / or TNFR2 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α with TNFR1 by, for example, 95% or more.

[0212] In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α to TNFR2 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α to TNFR2 by, for example, 10% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α to TNFR2 by, for example, 20% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α to TNFR2 by, for example, 30% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of TNF-α to TNFR2 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-α with TNFR2 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-α with TNFR2 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-α with TNFR2 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents, or inhibits the interaction (e.g., binding) of TNF-α with TNFR2 by, for example, 95% or more.

[0213] In some embodiments, reducing the level or function of an inflammatory cytokine, e.g., selected from IL-6, IL-8, IL-10, IL-1-β, MCP-1, and TNF-α, results in:

[0214] (a) decreased release of cytokines from immune cells (e.g., T cells and B cells) and non-immune cells (e.g., endothelial cells, fibroblasts, adipocytes, and stromal cells), and

[0215] (b) reduced oxidative stress,

[0216] thereby preventing, reducing and / or eliminating inflammation.

[0217] In some embodiments, reducing the level or function of an inflammatory cytokine, for example, selected from IL-6, IL-8, IL-10, IL-1-β, MCP-1, and TNF-α, 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-β, CCL2, and TNF. Nucleotide mutations may include nucleotide deletions, additions, and / or substitutions. Such mutations as described herein may, for example, result in a reduction in the expression of the gene by reducing, altering, or eliminating the transcription and / or splicing of the nucleotide sequence. For example, in some embodiments, reducing the level or function of an inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of IL-6. In some embodiments, reducing the level or function of an inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of IL-8. In some embodiments, reducing the level or function of an inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of IL-10. In some embodiments, reducing the level or function of an inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of IL-1-β. In some embodiments, reducing the level or function of an inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of CCL2. In some embodiments, reducing the level or function of an inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of TNF.

[0218] In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes 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 the nucleotide sequence provided in Table 5. For example, in some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 65% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 70% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 75% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 95% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 99% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5.

[0219] In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes 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 the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 65% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 70% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 75% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 80% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 85% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 90% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 95% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 99% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having at least 99.9% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the IL-6, IL-8, IL-10, IL-1-β, CCL2, and TNF genes comprises a sequence having greater than 99.9% sequence homology to the nucleotide sequences provided in Table 5.

[0220] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise, for example, expression of an inflammatory cytokine selected from the group consisting of IL-6, IL-8, IL-10, IL-1-β, MCP-1, and TNF-α, e.g., 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 of the reduction in expression of an inflammatory cytokine selected from the group consisting of IL-6, IL-8, IL-10, IL-1-β, MCP-1, and TNF-α, as compared to an engineered mammalian cell that is substantially the same or identical to the engineered mammalian cell except that it does not comprise a reduction in the level of the inflammatory cytokine. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the expression of inflammatory cytokines for example with except the reduction of the level not comprising inflammatory cytokines substantially identical with this engineered mammalian cell or the reduction compared to identical engineered mammalian cell between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the expression of inflammatory cytokines for example with except the reduction of the level not comprising inflammatory cytokines substantially identical with this engineered mammalian cell or the reduction compared to identical engineered mammalian cell greater than about 50%, 75% or 90%.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0221] In one embodiment, the engineered mammalian cell described herein (e.g., engineered RPE cell, e.g., engineered ARPE-19 cell) comprises a function of an inflammatory cytokine, e.g., 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 of the reduction compared to an engineered mammalian cell that is substantially the same or identical with the engineered mammalian cell except that it does not comprise a reduction in the function of an inflammatory cytokine. In one embodiment, the function of engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise inflammatory cytokine for example with except the reduction of the function not comprising inflammatory cytokine substantially identical with this engineered mammalian cell or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, the function of engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise inflammatory cytokine for example with except the reduction of the function not comprising inflammatory cytokine substantially identical with this engineered mammalian cell or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0222] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of IL-6 that is, for example, 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in level or function of IL-6. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of IL-6 or function for example with except not comprising the level of IL-6 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of IL-6 or function for example with except not comprising the level of IL-6 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0223] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of IL-8 that is, for example, 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in level or function of IL-8. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of IL-8 or function for example with except not comprising the level of IL-8 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of IL-8 or function for example with except not comprising the level of IL-8 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0224] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of IL-10 that is, for example, 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 an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the level or function of IL-10. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of IL-10 or function for example with except not comprising the level of IL-10 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of IL-10 or function for example with except not comprising the level of IL-10 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0225] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of IL-1-β that is, for example, 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 of the reduction compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except that it does not comprise the level or function of IL-1-β. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of IL-1-β or function for example with except not comprising the level of IL-1-β or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of IL-1-β or function for example with except not comprising the level of IL-1-β or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction. In one embodiment, the engineered mammalian cell is an engineered RPE cell (eg, an engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0226] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of MCP-1 that is, for example, 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in level or function of MCP-1. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of MCP-1 or function for example with except not comprising the level of MCP-1 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of MCP-1 or function for example with except not comprising the level of MCP-1 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0227] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of TNF-α that is, for example, 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 of the reduction compared to an engineered mammalian cell that is substantially the same or identical to the engineered mammalian cell except that it does not comprise a reduction in the level or function of TNF-α. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of TNF-α or function for example with except not comprising the level of TNF-α or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of TNF-α or function for example with except not comprising the level of TNF-α or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0228] In some embodiments, an inflammatory cytokine, for example, selected from IL-6, IL-8, IL-10, IL-1-β, MCP-1, and TNF-α, is encoded by one or more nucleotide sequences provided in Table 5, or fragments thereof.

[0229] In some embodiments, the reduction in the level or function of an inflammatory cytokine, for example, selected from IL-6, IL-8, IL-10, IL-1-β, MCP-1, and TNF-α, 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 reduction in the level or function of an inflammatory cytokine lasts for at least 30 minutes. In some embodiments, the reduction in the level or function of an inflammatory cytokine lasts for at least 1 hour. In some embodiments, the reduction in the level or function of an inflammatory cytokine lasts for at least 12 hours. In some embodiments, the reduction in the level or function of an inflammatory cytokine lasts for at least 24 hours. In some embodiments, the reduction in the level or function of an inflammatory cytokine lasts for at least 48 hours. In some embodiments, the reduction in the level or function of an inflammatory cytokine lasts for at least 72 hours. In some embodiments, the reduction in the level or function of an inflammatory cytokine lasts for at least 1 week. In some embodiments, the reduction in the level or function of an inflammatory cytokine lasts for at least 1 month. In some embodiments, the reduction in the level or function of an inflammatory cytokine lasts for at least 1 year.

[0230] Profibrotic factors are molecules that stimulate the host's fibrotic response, such as fibroblast growth factor 2 (FGF-2), vascular endothelial growth factor A (VEGFA), or platelet-derived growth factor (PDGF). In one embodiment, the engineered mammalian cells comprise a reduction in the level or function of a profibrotic factor, such as selected from FGF-2, VEGFA, and PDGF.

[0231] 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 acts 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 phosphoinositide 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).

[0232] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1, α1, RPL6, S19 and / or API5 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with casein kinase 1 by, for example, 95% or more.

[0233] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and α1 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and α1 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and α1 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and α1 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and α1 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and α1 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and α1 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and α1 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and α1 by, for example, 95% or more.

[0234] In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with RPL6 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with RPL6 by, for example, 10% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with RPL6 by, for example, 20% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with RPL6 by, for example, 30% or more. In some embodiments, the reduction in level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with RPL6 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with RPL6 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with RPL6 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with RPL6 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with RPL6 by, for example, 95% or more.

[0235] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and S19 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and S19 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and S19 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and S19 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and S19 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and S19 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and S19 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and S19 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and S19 by, for example, 95% or more.

[0236] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and API5 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and API5 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and API5 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and API5 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 and API5 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with API5 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with API5 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with API5 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of FGF-2 with API5 by, for example, 95% or more.

[0237] VEGFA is a glycosylated mitogen that acts specifically on endothelial cells and has multiple effects, including mediating increased vascular permeability, inducing angiogenesis, vasculogenesis and endothelial cell growth, promoting cell migration and inhibiting apoptosis. It is considered to be the main, leading inducer of vascular growth and is crucial for adults during organ remodeling and diseases involving blood vessels (such as wound healing, tumor angiogenesis, diabetic retinopathy and age-related macular degeneration). VEGFA also has a chemotactic effect on macrophages and granulocytes and indirectly (for example, through NO release) induces vasodilation. VEGFA is encoded by the VEGFA gene and interacts with a disintegrin and metalloproteinase with thrombospondin motif 1 (ADAMTS1), connective tissue growth factor (CTGF) and neuropilin-1 (NRP1).

[0238] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA with ADAMTS1, CTGF and / or NRP1 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA with ADAMTS1 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA with ADAMTS1 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA with ADAMTS1 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and ADAMTS1 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and ADAMTS1 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and ADAMTS1 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and ADAMTS1 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and ADAMTS1 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and ADAMTS1 by, for example, 95% or more.

[0239] In some embodiments, the reduction in the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and CTGF by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, the reduction in the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and CTGF by, for example, 10% or more. In some embodiments, the reduction in the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and CTGF by, for example, 20% or more. In some embodiments, the reduction in the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and CTGF by, for example, 30% or more. In some embodiments, the reduction in the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and CTGF by, for example, 40% or more. In some embodiments, the reduction in the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and CTGF by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and CTGF by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and CTGF by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and CTGF by, for example, 95% or more.

[0240] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and NRP1 by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and NRP1 by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and NRP1 by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and NRP1 by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA and NRP1 by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA with NRP1 by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA with NRP1 by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA with NRP1 by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of VEGFA with NRP1 by, for example, 95% or more.

[0241] PDGF is a growth factor that plays an important role in angiogenesis, the growth of blood vessels from existing vascular tissue, mitogenesis (i.e., the proliferation of mesenchymal cells such as fibroblasts, osteoblasts, tenocytes, vascular smooth muscle cells, and mesenchymal stem cells), and the chemotaxis (directional migration) of mesenchymal cells. Platelet-derived growth factor 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). In addition, 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 is composed of five ligands: PDGF-AA (encoded by the PDGFA gene), -BB (encoded by the PDGFB gene), -CC (encoded by the PDGFC gene), -DD (encoded by the PDGFD gene), and -AB (a heterodimer of PDGFA and PDGFB); and two receptors: PDGFR-α and PDGFR-β. All PDGFs function as secreted disulfide-linked homodimers, but only PDGFA and B can form functional heterodimers. In some embodiments, PDGF comprises PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and / or PDGF-AB. In some embodiments, PDGF comprises PDGF-AA. In some embodiments, PDGF comprises PDGF-BB. In some embodiments, PDGF comprises PDGF-CC. In some embodiments, PDGF comprises PDGF-DD. In some embodiments, PDGF comprises PDGF-AB.

[0242] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF with PDGFR-α and / or PDGFR-β by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF with PDGFR-α by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF with PDGFR-α by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF with PDGFR-α by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-α by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-α by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-α by, for example, 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-α by, for example, 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-α by, for example, 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-α by, for example, 95% or more.

[0243] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-β by, for example, 10%, 20%, 30%, 40%, 50%, 75%, 90%, 95% or more. For example, in some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-β by, for example, 10% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-β by, for example, 20% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-β by, for example, 30% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of PDGF and PDGFR-β by, for example, 40% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (for example, in combination) of PDGF and PDGFR-β by for example 50% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (for example, in combination) of PDGF and PDGFR-β by for example 75% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (for example, in combination) of PDGF and PDGFR-β by for example 90% or more. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (for example, in combination) of PDGF and PDGFR-β by for example 95% or more.

[0244] In some embodiments, reducing the level or function of a pro-fibrotic factor, e.g., selected from FGF-2, VEGFA, and PDGF, results in:

[0245] (i) Inhibit fibroblast-related signaling pathways, such as the AKT / mTOR and SMAD pathways, and

[0246] (ii) further inhibiting fibroblast expression, proliferation and activation, thereby preventing, reducing and / or eliminating inflammation.

[0247] In some embodiments, reducing the level or function of a profibrotic factor, such as one selected from 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. Such mutations as described herein can, for example, result in a reduction in the expression of the gene by reducing, altering, or eliminating the transcription and / or splicing of the nucleotide sequence. For example, in some embodiments, reducing the level or function of an inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of FGF-2. In some embodiments, reducing the level or function of an inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of VEGFA. In some embodiments, reducing the level or function of an inflammatory cytokine comprises mutating one or more nucleotides in the nucleotide sequence of PDGF.

[0248] In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes 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 the nucleotide sequence provided in Table 5. For example, in some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 65% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 70% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 75% sequence identity to the nucleotide sequence provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 80% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 85% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 90% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF F genes comprises a sequence having at least 95% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 99% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 99.9% sequence identity to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having greater than 99.9% sequence identity to the nucleotide sequence provided in Table 5.

[0249] In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes 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 the nucleotide sequences provided in Table 5. For example, in some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 65% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 70% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 75% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 80% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 85% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 90% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 95% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 99% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having at least 99.9% sequence homology to the nucleotide sequences provided in Table 5. In some embodiments, the nucleotide sequence of the FGF-2, VEGFA, and PDGF genes comprises a sequence having greater than 99.9% sequence homology to the nucleotide sequences provided in Table 5.In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a reduction in expression of a pro-fibrotic factor, e.g., by 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 an engineered mammalian cell that is substantially the same or identical to the engineered mammalian cell except that it does not comprise a reduction in the level of the pro-fibrotic factor. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the expression of pro-fibrotic factors for example with except the reduction of the level not comprising pro-fibrotic factors with the 1-25% that is substantially the same or the same engineered mammalian cell compared to this engineered mammalian cell, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the expression of pro-fibrotic factors for example with except the reduction of the level not comprising pro-fibrotic factors with the 1-25% that is substantially the same or the same engineered mammalian cell compared to this engineered mammalian cell, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the expression of pro-fibrotic factors for example with except the reduction of the level not comprising pro-fibrotic factors with the reduction that is substantially the same or the same engineered mammalian cell compared to this engineered mammalian cell, greater than about 50%, 75% or 90%.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0250] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a function of a pro-fibrotic factor, e.g., 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 the function of a pro-fibrotic factor compared to an engineered mammalian cell that is substantially the same or identical to the engineered mammalian cell except that it does not comprise a reduction in the function of a pro-fibrotic factor. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the function of pro-fibrotic factor for example with except the reduction of the function of not comprising pro-fibrotic factor with the 1-25% that is substantially identical with this engineered mammalian cell or the reduction compared to identical engineered mammalian cell, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the function of pro-fibrotic factor for example with except the reduction of the function of not comprising pro-fibrotic factor with the 1-25% that is substantially identical with this engineered mammalian cell or the reduction compared to identical engineered mammalian cell, greater than about 50%, 75% or 90%.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0251] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of FGF-2 that is, for example, 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in level or function of FGF-2. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of FGF-2 or function for example with except not comprising the level of FGF-2 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of FGF-2 or function for example with except not comprising the level of FGF-2 or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0252] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of PDGF that is, for example, 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 of a reduction compared to an engineered mammalian cell that is substantially the same or identical to the engineered mammalian cell except that it does not comprise a reduction in the level or function of PDGF. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of PDGF or function for example with except the level that does not comprise PDGF or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of PDGF or function for example with except the level that does not comprise PDGF or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0253] In one embodiment, the engineered mammalian cells described herein (e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) comprise a level or function of VEGFA that is, for example, 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 of a reduction compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except that it does not comprise a reduction in the level or function of VEGFA. In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of VEGFA or function for example with except not comprising the level of VEGFA or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or the reduction between 75-100%.In one embodiment, engineered mammalian cell described herein (for example, engineered RPE cell, for example engineered ARPE-19 cell) comprise the level of VEGFA or function for example with except not comprising the level of VEGFA or the reduction of function with this engineered mammalian cell substantially identical or identical engineered mammalian cell compared greater than about 50%, 75% or 90% reduction.In one embodiment, engineered mammalian cell is engineered RPE cell (for example, engineered ARPE-19 cell). In one embodiment, the engineered mammalian cell is an engineered ARPE-19 cell.

[0254] In some embodiments, a pro-fibrotic factor, eg, selected from FGF-2, PDGF, and VEGFA, is encoded by one or more nucleotide sequences provided in Table 5, or fragments thereof.

[0255] In some embodiments, the reduction in the level or function of a profibrotic factor, such as one selected from FGF-2, PDGF, and VEGFA, 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 reduction in the level or function of a profibrotic factor lasts for at least 30 minutes. In some embodiments, the reduction in the level or function of a profibrotic factor lasts for at least 1 hour. In some embodiments, the reduction in the level or function of a profibrotic factor lasts for at least 12 hours. In some embodiments, the reduction in the level or function of a profibrotic factor lasts for at least 24 hours. In some embodiments, the reduction in the level or function of a profibrotic factor lasts for at least 48 hours. In some embodiments, the reduction in the level or function of a profibrotic factor lasts for at least 72 hours. In some embodiments, the reduction in the level or function of a profibrotic factor lasts for at least 1 week. In some embodiments, the reduction in the level or function of a profibrotic factor lasts for at least 1 month. In some embodiments, the reduction in the level or function of a profibrotic factor lasts for at least 1 year.

[0256] Table 5: Exemplary sequences

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

[0280]

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288]

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295]

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303]

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323]

[0324]

[0325]

[0326]

[0327]

[0328]

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358]

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367]

[0368]

[0369]

[0370]

[0371]

[0372]

[0373]

[0374]

[0375]

[0376]

[0377]

[0378]

[0379]

[0380]

[0381]

[0382]

[0383]

[0384]

[0385]

[0386]

[0387]

[0388]

[0389]

[0390] Mammalian cells or engineered mammalian cells can be genetically manipulated and modified using any method known in the art, including gene silencing, gene knockdown, gene knockout, and gene editing techniques. For example, targeted genome editing techniques can be used to generate gene mutants at one or more desired sites in the target OCR. Targeted genome editing techniques can be any technique known in the art, such as the use of site-directed nucleases (such as CRISPR-Cas), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and meganucleases.

[0391] The engineered 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, keratinocytes, subtypes of any of the foregoing, and cells derived from any of the foregoing. Exemplary cell types include those described in WO 2017 / 075631. In some embodiments, the cell is derived from the cell lines shown in Table 2 below.

[0392] Table 2: Exemplary cell lines

[0393]

[0394] In one embodiment, any one in engineered mammalian cell described herein is all derived from RPE cell, for example, ARPE-19 cell.In one embodiment, engineered RPE cell (for example, engineered ARPE-19 cell) comprises any one in expression cassette described herein, transposon and polynucleotide.

[0395] The engineered mammalian cells used in the devices, compositions and methods described herein, for example, as a plurality of engineered cells contained in or encapsulated in a hydrogel capsule, may be in various stages of the cell cycle. In some embodiments, at least one engineered cell in the plurality of engineered cells is undergoing cell division. Cell division can be measured using any known method 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, for example, as determined by 5-ethynyl-2'-deoxyuridine (EdU) assay or 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 engineered cells in the plurality of engineered cells are undergoing cell division and are in a quiescent state. 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).

[0396] In one embodiment, at least 50%, 60%, 70%, 80%, 90% or more of the engineered cells in the plurality of engineered cells are viable. Cell viability can be measured using any known method in the art, for example, as described in Riss, T. et al. (2013) "Cell Viability Assays" in Assay Guidance Manual (Sittapalam, GS 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, such as fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry. In one embodiment, at least 80% of the engineered cells in the plurality of genetically modified cells are viable, e.g., as determined 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 or assessment of spindle formation), or flow cytometry.

[0397] Any of the parameters described herein can be assessed using standard techniques known to those of skill in the art, such as histology, microscopy, and various functional assays.

[0398] In some embodiments, the exogenous transcription unit encodes a therapeutic polypeptide (e.g., a protein) such as a coagulation factor, a growth factor, a hormone, an enzyme, a cytokine (e.g., a proinflammatory cytokine or an anti-inflammatory cytokine), a cytokine receptor, a chimeric protein, a fusion protein, or a 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 relative 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 about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, or less than 50 amino acids. In some embodiments, the polypeptide has 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.

[0399] In some embodiments, polypeptide is 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, polypeptide is insulin (for example, insulin A chain, insulin B chain or proinsulin). In some embodiments, polypeptide is growth hormone, such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methionine-human growth hormone, dephenylalanine human growth hormone and porcine growth hormone.

[0400] 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).

[0401] In some embodiments, the polypeptide is a clotting factor or coagulation factor, such as a blood clotting factor or blood coagulation factor. In some embodiments, the polypeptide is involved in coagulation, the process by which blood changes from a liquid to a solid or gel. Exemplary coagulation factors and clotting factors include factor I (e.g., fibrinogen), factor II (e.g., prothrombin), factor III (e.g., tissue factor), factor V (e.g., prothrombin, unstable 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., Hagemann 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 anti-coagulation factor, such as protein C.

[0402] 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 an immunoglobulin Fc region. In one embodiment, the polypeptide is a full-length immunoglobulin chain.

[0403] In some embodiments, the polypeptide is a cytokine or cytokine receptor, or a chimeric protein comprising a cytokine or its receptor, including, for example, tumor necrosis factor alpha and beta, their receptors and their derivatives, renin; lipoproteins; colchicine; adrenocorticotropic hormone; vasopressin; somatostatin; lysine vasopressin; cholecystokinin; leuprolide; alpha-1-antitrypsin; atrial natriuretic factor; pulmonary surfactant; plasminogen activators other than tissue-type plasminogen activator (t-PA), such as urokinase; bombesin; thrombin; brain peptidase; RANTES (regulated activation, normal T cell expression and secretion); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, such as human serum albumin; Müllerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; chorionic gonadotropin; microbial proteins, such as beta-lactamase; DNA enzymes; inhibins; activins; hormone or growth factor receptors; integrins; proteins A or D; rheumatoid factor; platelet-derived growth factor (PDGF); epidermal growth factor ( EGF); transforming growth factors (TGF), 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 protein; CD proteins, such as CD-3, CD-4, CD-8 and CD-19; erythropoietin; osteoinductive factors; immunotoxins; interferons, Such as interferon-α (e.g., interferon.α.2A), -β, -γ, -λ and consensus interferon; colony stimulating factor (CSF), such as M-CSF, GM-CSF and G-CSF; interleukin (IL), such as IL-1, IL-2 to IL-10; superoxide dismutase; T cell receptor; surface membrane protein; decay accelerating factor; transport protein; homing receptor; addressin; fertility inhibitors, such as prostaglandins; fertility promoters; regulatory proteins; antibodies (including fragments thereof) and chimeric proteins, such as immunoadhesins. Suitable polypeptides can be natural or recombinant and include, for example, fusion proteins.

[0404] Examples of polypeptides that can be encoded by an exogenous transcription unit 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, CXCL4, CXCL5 (KC), and CXCL6 (SDF1b). 5. CXCL6, CXCL7, CXCL8(IL8), CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, TNFA, TNFB(LTA), TNFC(L TB), TNFSF4, TNFSF5(CD40LG), TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF13B, EDA, IL2, IL15, IL4, IL13, IL7, IL9, IL21, 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, IL26, IL29, IFNL 1. IFNL2, IFNL3, IL28, IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, IFNA21, IFNB1, IFNK, IFNW1, IFNG, IL1A(IL1F1), IL1B(IL1F2), IL1Ra(IL1F3), IL1F5(IL36RN), IL1F6(IL36A), IL1F7(IL37), IL1F8(IL36B), IL1F9(IL36G), IL1F10(IL38), IL33(IL1F1 1), 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, Bmp8a, Bmp8b, C1qt nf4, Ccl21a, Ccl27a, Cd70, Cer1, Cklf, Clcf1, Cmtm2a, Cmtm2b, Cmtm3, Cmtm4, Cmtm5, Cmtm6, Cmtm7, Cmtm8, Crlf1, Ctf2, Ebi3, Edn1, Fam3b, Fasl, Fgf2 , Flt3l, Gdf10, Gdf11, Gdf15, Gdf2, Gdf3, Gdf5, Gdf6, Gdf7, Gdf9, Gm12597 , Gm13271, Gm13275, Gm13276, Gm13280, Gm13283, Gm2564, Gpi1, Grem1, Grem 2. Grn, Hmgb1, Ifna11, Ifna12, Ifna9, Ifnab, Ifne, Il17a, Il23a, Il25, Il31, Iltifb, Inhba, Lefty1, Lefty2, Mstn, Nampt, Ndp, Nodal, 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 amylin, Müllerian inhibitory factor or hormone, adiponectin, adrenocorticotropic hormone, angiotensin, vasopressin, arginine vasopressin, atrial natriuretic peptide, 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, 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 palmitoyltransferase, carnitine and myoadenylate deaminases.

[0405] In some embodiments, the polypeptide is a replacement therapy or replacement protein.

[0406] In some embodiments, the replacement therapy or replacement protein is a coagulation factor, such as Factor VII, Factor VIII, or Factor IX.

[0407] In some embodiments, the replacement therapy or replacement protein is an enzyme, such as α-galactosidase A (GLA), α-L-iduronidase (IDUA), glucocerebrosidase, or N-sulfoglucosamine sulfohydrolase (SGSH). In one embodiment, the engineered mammalian cell comprises an exogenous nucleic acid encoding IDUA.

[0408] In one embodiment, the engineered mammalian cell is not a pancreatic islet cell as defined herein. In one embodiment, the engineered 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 treatable 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 is engineered or differentiated into an insulin-producing pancreatic beta cell.

[0409] Characteristics of implantable devices

[0410] The engineered mammalian cells or a plurality of such cells described herein can be incorporated into implantable elements for treating a disease or condition in a subject, as well as for reducing the level of pericranial fibrotic overgrowth on the implantable element after implantation in a subject.

[0411] The implantable element of the present disclosure includes at least one barrier that prevents immune cells from contacting cells contained within the device. At least a portion of the barrier needs to be sufficiently porous to allow therapeutic agents expressed and secreted by the cells to leave the device. A variety of device configurations known in the art are suitable.

[0412] The device (e.g., particle) can have any structure and shape suitable for supporting the vitality and productivity of the contained cells after being implanted in the expected target location. As a non-limiting example, the device shape can be cylindrical, rectangular, disc-shaped, oval, star-shaped or spherical. The device can be composed of a mesh or nested structure. In some embodiments, the device can prevent materials exceeding a certain size from passing through a pore or opening. In some embodiments, the device (e.g., particle) can prevent materials greater than 50kD, 75kD, 100kD, 125kD, 150kD, 175kD, 200kD, 250kD, 300kD, 400kD, 500kD, 750kD or 1,000kD from passing through.

[0413] In one embodiment, the device is a macroencapsulation device. Non-limiting examples of macrodevices are described in WO 2019 / 068059, WO 2019 / 169089, U.S. Patent Nos. 9,526,880, 9,724,430, and 8,278,106; European Patent No. EP742818B1, and Sang, S. and Roy, S., Biotechnol. Bioeng. 113(7):1381-1402 (2016).

[0414] In one embodiment, the device is a macro device 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 will be placed in one compartment, and cells expressing a different protein (e.g., a therapeutic protein) will be placed in separate compartments. WO 2018 / 232027 describes a device having multiple cell-containing compartments formed within a micro-fabricated body and covered by a porous membrane.

[0415] 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 polymer coating surrounding the substrate, and an outer hydrogel coating surrounding the inner polymer coating. Protein-expressing cells are located in the outer coating.

[0416] In some embodiments, the device (e.g., particle) has a maximum linear dimension (LLD), such as an average diameter or size of at least about 0.5 millimeters (mm), preferably about 1.0 mm, about 1.5 mm, or more. In some embodiments, the diameter or size of the device can be as large as 10 mm. For example, the devices or particles described herein may have a range 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 , 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.

[0417] In some embodiments, the device of the present disclosure (e.g., particles, capsules) includes at least one pore or opening, such as to allow material to flow freely. In some embodiments, the average pore size of the device is between about 0.1 μm and about 10 μm. For example, the average pore size can be between 0.1 μm and 10 μm, 0.1 μm and 5 μm, 0.1 μm and 2 μm, 0.15 μm and 10 μm, 0.15 μm and 5 μm, 0.15 μm and 2 μm, 0.2 μm and 10 μm, 0.2 μm and 5 μm, 0.25 μm and 10 μm, 0.25 μm and 5 μm, 0.5 μm and 10 μm, 0.75 μm and 10 μm, 1 μm and 10 μm, 1 μm and 5 μm, 1 μm and 2 μm, 2 μm and 10 μm, 2 μm and 5 μm or 5 μm and 10 μm. In some embodiments, the average pore size of the device is between about 0.1 μm and 10 μm. In some embodiments, the average pore size of the device is between about 0.1 μm and 5 μm. In some embodiments, the average pore size of the device is between about 0.1 μm and 1 μm.

[0418] In some embodiments, equipment includes semipermeability, biocompatible membrane, and this membrane is around the genetically modified cell that is encapsulated in polymer composition (for example, alginate hydrogel).Select membrane pore size to allow oxygen and other important molecules to cell survival and function to move through semipermeable membrane, while stopping immunocyte from passing through hole.In one embodiment, semipermeable membrane has and is less than 1000kD or between 50-700kD, between 70-300kD, or between 70-150kD, or the molecular weight cut-off between 70 and 130kD.

[0419] In one embodiment, the device may contain a cell-containing compartment surrounded by a barrier compartment formed of 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 a barrier compartment may be used with or without a semipermeable membrane.

[0420] The cells in one or more cell-containing compartments of the device of the present disclosure can be encapsulated in a polymer composition. The polymer composition can include one or more polymers that form a hydrogel. In addition to the polymer composition in one or more cell-containing compartments, the device (e.g., macro device, particle, hydrogel capsule) can also include materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials, and combinations thereof or be formed of the material. The device can be made entirely of one type of material, or can include other materials in the cell-containing compartment and any other compartment.

[0421] In some embodiments, the device comprises a metal or metal alloy. 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 metal or metal alloy. Exemplary metals or metal alloys include titanium and titanium group alloys (e.g., nitinol, nickel titanium alloy, heat memory alloy materials), platinum, platinum group 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., and ). For example, the metal material can be 316 grade stainless steel (SS 316L) (including 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 the metal-containing device, the amount of metal (e.g., in weight %, actual weight) can be at least 5%, such as at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, such as w / w; less than 20%, such as less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1% or less.

[0422] In some embodiments, the device includes ceramics. In one embodiment, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all compartments) include ceramics. 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 silicon dioxide can also be used. In devices containing ceramics, the amount of ceramic (e.g., in wt. %, actual weight) can be at least 5%, such as at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, such as w / w; less than 20%, such as less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1% or less.

[0423] In some embodiments, the device has two hydrogel compartments, wherein the interior cell-containing compartment is completely surrounded by the second outer (for example, 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 means the average distance between the outer boundary of the second compartment and the interface between the two compartments, for example, the mean value of the distance measured at each of the thinnest point and the thickest point visually observed in the outer compartment. In some embodiments (for example, the diameter of the device is about 1.5mm), the thinnest distance and the thickest distance of the outer compartment are respectively between 25 and 110 microns (μm) and between 270 μm and 480 μm. In some embodiments, the thickness of the outer compartment is greater than about 10 nanometers (nm), preferably 100nm or larger, 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 device 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, between 1 μm and 1 mm, between 1 μm and 500 μm, or between 5 μm and 1 mm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is between about 50 μm and about 100 μm. In some embodiments (eg, the diameter of the device is about 1.5 mm), the thickness (eg, average distance) of the outer compartment is between about 180 μm and 260 μm or between about 310 μm and 440 μm.

[0424] In some embodiments of the two-compartment hydrogel capsule device, the average pore size of the cell-containing inner compartment and the outer compartment is substantially the same. In some embodiments, the average pore size of the inner compartment and the second compartment differs by about 1.5%, 2%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or more. In some embodiments, the average pore size of the device (e.g., the average pore size of the first compartment and / or the average pore size of the second compartment) depends on many factors, such as the presence and density of the one or more materials and the compound of Formula (I) in each compartment.

[0425] In some embodiments, the polymer composition in one or more cell-containing compartments includes a polysaccharide or other polymer (e.g., alginate, hyaluronate or chondroitin) forming a hydrogel. In some embodiments, the polymer is an alginate, which is a polysaccharide consisting of β-D-mannuronic acid (M) and α-L-guluronic acid (G). In some embodiments, alginate has a low molecular weight (e.g., <75kD approximate molecular weight) and a G:M ratio of ≥1.5, (ii) a medium molecular weight alginate, such as an approximate molecular weight of...

Claims

1. An implantable element comprising an engineered mammalian cell comprising: (i) A decrease in the level or function of one or more of the following: (a) Major histocompatibility complex (MHC) class I protein complex; (b) MHC class II protein complex; (c) class II major histocompatibility complex transactivator (CIITA); and (ii) a decrease in the level or function of one or more of the following: (d) inflammatory cytokines; and (e) profibrotic factors, wherein the engineered mammalian cell comprises an exogenous nucleic acid encoding a therapeutic agent; and The implantable element comprises a polymer and a compound of formula (I): 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)(C1-C6-alkylene)–、-N(R C )C(O)(C1-C6-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 linked to an attachment group (e.g., an attachment group described herein) and optionally surrounded by one or more R 1 replace; L 1 and L 3 Each of the R 2 replace; L 2 It is a key; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally replaced by one or more R 3 replace; P is absent, cycloalkyl, heterocyclyl, or heteroaryl, each of which is optionally replaced by one or more R 4 replace; 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, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally replaced by one or more R 5 replace; 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, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally replaced by one or more R 6 replace; or R C and R D Together with the nitrogen atom to which they are attached, they form a 6 substituted rings (e.g., 5-7 membered rings); 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 ), SR 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, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally replaced by one or more R 7 replace; 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, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally replaced by one or more R 7 replace; Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, oxo, hydroxy, cycloalkyl, or heterocyclyl; x is 1 or 2; and y is 2, 3, or 4.

2. The implantable device of claim 1, comprising (i)(a).

3. The implantable element of any one of claims 1-2, comprising (i)(b).

4. The implantable element of any one of claims 1 to 3, comprising (i) (c).

5. The implantable element of any one of claims 1 to 4, comprising (ii) (d).

6. The implantable element of any one of claims 1 to 5, comprising (ii) (e).

7. The implantable element of any one of claims 1 to 6, comprising (i)(a) and one of (ii)(d) and (ii)(e).

8. The implantable element of any one of claims 1 to 7, comprising (i)(a) and both (ii)(d) and (ii)(e).

9. The implantable element of any one of claims 1 to 8, comprising (i)(b) and one of (ii)(d) and (ii)(e).

10. The implantable element of any one of claims 1 to 9, comprising (i)(b) and both (ii)(d) and (ii)(e).

11. The implantable element of any one of claims 1 to 10, comprising (i)(c) and one of (ii)(d) and (ii)(e).

12. The implantable element of any one of claims 1 to 11, comprising (i)(c) and both (ii)(d) and (ii)(e).

13. The implantable device of any one of claims 1 to 12, wherein the reduction in level or function in (i)(a) occurs in one or more of the MHC class I protein components selected from: (ai) human leukocyte antigen (HLA) A; (a-ii)HLA-B; (a-iii) HLA-C; and (a-iv) β-2-microglobulin (β-2M).

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

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

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

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

18. The implantable element of any one of the preceding claims, wherein the engineered mammalian cell comprises a mutation that results in a decrease in the level of a component of the MHC class I complex, eg, compared to a reference standard.

19. The implantable element of any one of the preceding claims, wherein the engineered mammalian cells comprise a less functional or non-functional variant of a component of the MHC class I complex, eg, compared to a reference standard.

20. The implantable element of any one of the preceding claims, wherein the level of a component of the MHC class I complex is silenced or knocked down, eg, compared to a reference standard.

21. The implantable element of any of the preceding claims, wherein the engineered mammalian cells comprise a reduction in the levels of MHC class I components, e.g., compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduced levels of MHC class I components, e.g., 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 a reference standard.

22. An implantable element according to any one of the preceding claims, wherein the reduction in level or function in (i)(b) occurs in one or more of the MHC class I protein components selected from: (bi) human leukocyte antigen (HLA) DP; (b-ii)HLA-DM; (b-iii) HLA-DOA; (b-iv)HLA-DOB; (bv) 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 22-23, 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. The implantable element of any one of the preceding claims, wherein the engineered mammalian cell comprises a mutation that results in a decrease in the level of a component of the MHC class II complex, eg, compared to a reference standard.

30. The implantable element of any one of the preceding claims, wherein the engineered mammalian cells comprise a less functional or non-functional variant of a component of the MHC class II component, eg, compared to a reference standard.

31. The implantable element of any of the preceding claims, wherein expression of a component of the MHC class II complex is silenced or knocked down, eg, compared to a reference standard.

32. An implantable element as claimed in any of the preceding claims, wherein the engineered mammalian cells comprise a reduction in the level of MHC class II components, e.g., by 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 a reference standard, e.g., compared to engineered mammalian cells that do not comprise reduced levels of MHC class II components.

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

34. The implantable element of any one of the preceding claims, wherein the engineered mammalian cell comprises a mutation that results in reduced expression of a component of CIITA, eg, compared to a reference standard.

35. The implantable element of any one of the preceding claims, wherein the engineered mammalian cells comprise a less functional or non-functional variant of a component of CIITA, eg, compared to a reference standard.

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

37. An implantable element as described in any of the preceding claims, wherein the engineered mammalian cell comprises a reduction in the level of CIITA, e.g., compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduced level of CIITA, e.g., 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 a reference standard.

38. The implantable element of any one of the preceding claims, wherein the inflammatory cytokine is selected from the group consisting of: (di)IL-6 (d-ii) IL-8; and (d-iii)MCP-1.

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

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

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

42. The implantable element of any one of the preceding claims, wherein the engineered mammalian cells comprise a mutation that results in reduced expression of an inflammatory cytokine, eg, compared to a reference standard.

43. The implantable element of any one of the preceding claims, wherein the engineered mammalian cells comprise a less functional or non-functional variant of an inflammatory cytokine, eg, compared to a reference standard.

44. The implantable element of any one of the preceding claims, wherein expression of an inflammatory cytokine is silenced or knocked down, eg, compared to a reference standard.

45. An implantable element as described in any of the preceding claims, wherein the engineered mammalian cells comprise a level of an inflammatory cytokine that is, e.g., compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduced level of the inflammatory cytokine, e.g., 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 a reference standard.

46. ​​The implantable element of any one of the preceding claims, wherein the profibrotic factor is selected from the group consisting of: (ei) FGF-2; (e-ii)PDGF; and (e-iii)VEGFA.

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

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

49. The implantable element of any one of claims 47-49, wherein the profibrotic factor is (e-iii).

50. The implantable element of any one of the preceding claims, wherein the engineered mammalian cells comprise a mutation that results in reduced expression of the profibrotic factor, eg, compared to a reference standard.

51. The implantable element of any one of the preceding claims, wherein the engineered mammalian cells comprise a less functional or non-functional variant of the pro-fibrotic factor, eg, compared to a reference standard.

52. The implantable element of any one of the preceding claims, wherein expression of the profibrotic factor is silenced or knocked down, eg, compared to a reference standard.

53. An implantable element as described in any of the preceding claims, wherein the engineered mammalian cells comprise a level of a pro-fibrotic factor that is, for example, 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 a reference standard, e.g., compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduced level of the pro-fibrotic factor.

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

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

56. The implantable element of any of the preceding claims, wherein the engineered 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. The implantable element of any one of the preceding claims, wherein the engineered mammalian cells comprise RPE cells (e.g., ARPE-19 cells).

58. An implantable element as claimed in any one of the preceding claims, wherein the exogenous nucleotide sequence is extrachromosomal.

59. The implantable element of 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. The implantable member of any one of the preceding claims, wherein the polymer comprises a polymer selected from the group consisting of alginate, hyaluronate, and chitosan.

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

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

63. The implantable element of any one of the preceding claims, wherein the polymer composition comprises at least one polymer covalently modified with a peptide.

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

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

66. An implantable element as claimed in any one of the preceding claims, wherein the polymer composition comprises alginate covalently modified with a peptide, wherein the peptide consists essentially of or consists of GRGDSP or GGRGDSP, and wherein the barrier compartment comprises alginate covalently modified with a peptide. or a chemically modified alginate or a pharmaceutically acceptable salt thereof.

67. The implantable element of any of the preceding claims, wherein the implantable element is spherical.

68. The implantable element of any one of the preceding claims, wherein the implantable element comprises a two-compartment hydrogel capsule.

69. The implantable element of 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. An implantable element as claimed in any one of the preceding claims, wherein the therapeutic agent is a protein, such as a hormone, a blood clotting factor, an antibody or an enzyme.

71. An implantable element comprising: (i) Engineered ARPE cells capable of reducing the expression of β-2M or CIITA and one of: (a) an inflammatory cytokine selected from the group consisting of IL-16, IL-8, and MCP-1; and (b) a profibrotic factor selected from the group consisting of FGF-2, PDGF, and VEGFA; (ii) a polymer composition comprising alginate covalently modified with one or more of: (c) a compound of formula (I) (eg, as described herein); and (d) Peptides.

72. An implantable element comprising: (i) Engineered ARPE cells capable of reducing the expression of β-2M or CIITA and one of: (a) an inflammatory cytokine selected from the group consisting of IL-16, IL-8, and MCP-1; and (b) a profibrotic factor selected from the group consisting of FGF-2, PDGF, and VEGFA; (ii) a polymer composition comprising alginate covalently modified with one or more of: (c) or a pharmaceutically acceptable salt thereof; and (d) A peptide comprising or consisting of GRGDSP or GGRGDSP.

73. The implantable device of any of the preceding claims, formulated for implantation into a subject (e.g., into the intraperitoneal (IP) space, peritoneal cavity, omentum, bursa, subcutaneous fat).

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

75. A preparation of implantable elements, wherein each implantable element in the preparation is the implantable element of any one of claims 1-74.

76. A composition for treating a disease or condition in a subject, comprising the implantable element of any one of claims 1-74 or the formulation of claim 75, thereby treating the disease or condition in the subject.

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

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

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