Engineered cells and implantable elements for treating disease

Abstract

Described herein are engineered mammalian cells comprising a major histocompatibility complex (MHC) class I protein complex component and, optionally, an MHC class II protein complex component and / or a reduction in the level or function of CIITA; 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 / 415,271 filed on October 11, 2022. Background Art

[0003] Treating chronic and genetic diseases by implanting cells that are engineered to produce therapeutic substances capable of treating such diseases has 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 therapeutic substance for weeks, months, or even longer without overstimulating the host's immune response. Summary of the invention

[0004] Described herein are engineered mammalian cells comprising reduced levels or reduced function of major histocompatibility complex (MHC) class I protein complexes, 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 reduced levels or reduced function of one or more proteins selected from human leukocyte antigen (HLA) A, HLA-B, HLA-C, and beta-2-microglobulin (β-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.

[0005] In one aspect, the 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 of a component of the MHC class II protein complex, or may be due to silencing or knocking down of 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 comprises engineered mammalian cells described herein or multiple engineered mammalian cells.The engineered mammalian cells may include embryonic stem cells (ESC) or induced pluripotent stem cells (iPSC).The engineered mammalian cells may include retinal pigment epithelium (RPE) cells, CCD-33Lu cells, MRC-5 cells, MRC-9 cells, MCF10a cells, or cells derived from them.In one embodiment, the engineered mammalian cells include engineered retinal pigment epithelium (RPE) cells (for example, engineered ARPE-19 cells), or derived from retinal pigment epithelium (RPE) cells (for example, ARPE-19 cells).In one embodiment, implantable element includes at least one cell-containing compartment, and the compartment includes engineered mammalian cells described herein or multiple engineered mammalian cells.In one embodiment, implantable element includes a cell-containing compartment, and the compartment includes engineered mammalian cells described herein or multiple engineered mammalian cells; And a second compartment around the cell-containing compartment. In one embodiment, the implantable element further includes at least one device (e.g., a compound of formula (I) as described herein) for alleviating foreign body reaction (FBR) when the implantable element is implanted into a subject. In one embodiment, the implantable element includes a polymer selected from alginate, hyaluronate and chitosan. In one embodiment, the implantable element includes a cell-containing compartment surrounded by a barrier compartment, the barrier compartment includes an alginate hydrogel and an optional compound of formula (I) (e.g., a compound of formula (I) described herein) arranged on the outer surface of the barrier compartment. In one embodiment, the implantable element is formulated for implantation into a subject (e.g., implanted into intraperitoneal (IP) space, peritoneal cavity, omentum, lesser sac, subcutaneous fat). In one embodiment, the implantable element is configured to protect engineered mammalian cells or multiple engineered mammalian cells from the influence of the recipient's immune system, and to alleviate foreign body reaction (FBR) (as defined herein) to implanted devices. In one embodiment, the implantable element is capable of delivering a therapeutic agent (eg, a protein) over a sustained period of time (eg, one to several months up to one to several years) following implantation into a subject.

[0007] In another aspect, the present disclosure is characterized in that a method for treating a disease or condition of a subject, the method comprising administering an implantable element to the subject, the implantable element comprising an engineered mammalian cell described herein or a plurality of engineered mammalian cells 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 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 composition of implantable element, the preparation or composition of this implantable element can be applied to the experimenter (for example, being applied to the intraperitoneal cavity) of the therapeutic agent treatment that needs to be produced by this equipment.In one embodiment, the experimenter is people, the mammalian cell derived from human cell (for example RPE cell, ARPE-19 cell) through engineered approaches, and the preparation or composition of implantable element can continue to deliver the therapeutic agent of effective dose to the experimenter in the time period (for example 3 months, 6 months, one year, two years or longer time at least any one) that lasts. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 is a graph showing that β-2M protein expression in IDUA-expressing ARPE-19 cells containing β-2M shRNA is significantly lower (89% lower) compared to β-2M protein expression in IDUA-expressing ARPE-19 cells containing scrambled control shRNA.

[0010] Figure 2 is a graph showing that β-2M expression levels in ARPE-19 cells treated with CRISPR and β-2M targeting gRNA were reduced by 99% compared to ARPE-19 cells modified with scrambled gRNA.

[0011] Figure 3A -D is a set of graphs showing β-2M expression in wild-type ARPE-19 cells ( Figure 3C ) compared to those with decreased β-2M protein expression ( Figure 3A ) in ARPE-19 cells. Figure 3B ) leads to decreased expression of HLA class I ( Figure 3D ). DETAILED DESCRIPTION

[0012] The disclosure is characterized in that mammalian cells (for example, human RPE cells) are engineered to regulate the level or function of major histocompatibility complex (MHC) class I protein complex or its component (for example, beta-2-microglobulin (β-2M)). In one embodiment, mammalian cells are engineered to reduce the expression of MHC class I protein complex or its component (for example, beta-2M). Mammalian cells can be engineered to produce a variant with a lower or non-functional function of MHC class I protein complex or its component (for example, beta-2M), or the expression of MHC class I protein complex or its component (for example, beta-2M) can be silenced or knocked down or knocked out. The disclosure is also characterized in that it is further engineered to regulate the level or function of MHC class II protein complex or its component, and / or the level or function of class II major histocompatibility complex transactivator (CIITA) of mammalian cells. In one embodiment, mammalian cells are engineered to reduce the expression of MHC class II protein complex or its component, and / or the level or function of CIITA. Mammalian cells may be engineered to produce less functional or non-functional variants of MHC class II protein complexes or components thereof, and / or CIITA, or the expression of MHC class II protein complexes or components thereof, and / or CIITA may be silenced or knocked down or knocked out.

[0013] Abbreviations and definitions

[0014] Throughout the detailed description and examples of the present disclosure, the following abbreviations will be used.

[0015] CM-Alg Chemically modified alginate

[0016] CM-LMW-Alg Chemically modified low molecular weight alginate

[0017] CM-LMW-Alg-101 Low molecular weight alginate chemically modified with compound 101 shown in Table 4

[0018] CM-HMW-Alg Chemically modified high molecular weight alginate

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

[0020] CM-MMW-Alg Chemically modified medium molecular weight alginate

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

[0022] HMW-Alg High Molecular Weight Alginate

[0023] MMW-Alg Medium Molecular Weight Alginate

[0024] U-Alg Unmodified alginate

[0025] U-HMW-Alg Unmodified high molecular weight alginate

[0026] U-LMW-Alg Unmodified low molecular weight alginate

[0027] U-MMW-Alg Unmodified medium molecular weight alginate

[0028] 70:30 CM-Alg:U-Alg A 70:30 mixture (V:V) of chemically modified and unmodified alginate, e.g. as described in WO2020069429.

[0029] In order to more easily understand the present disclosure, some 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 ordinary technicians in the field to which the present disclosure belongs.

[0030] 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.

[0031] "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 of 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" may 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 about 1.5 millimeters (mm) and encapsulating about 5 million (M) cells may have a diameter of 1.2 to 1.8 mm and may encapsulate 4M to 6M cells. As another non-limiting example, a formulation of about 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.

[0032] As used herein, the term "acquire" or "acquiring" refers to the process of obtaining a value (e.g., a numerical value) or an image or physical entity (e.g., a sample) by "directly obtaining" or "indirectly obtaining" a value 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 a 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 using a machine or device, such as using a fluorescence microscope to obtain fluorescence microscopy data.

[0033] 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.

[0034] "Defibrotic," as used herein, means a compound or material that reduces the foreign body response (FBR). For example, the amount of FBR in a tissue induced by implanting a device (e.g., a hydrogel capsule) containing a defibrotic compound (e.g., a hydrogel capsule containing a polymer covalently modified with a compound listed in Table 4) into the biological tissue is lower than that induced by implanting a reference device that is fibrotic-null, i.e., lacks any defibrotic compound but has substantially the same composition (e.g., one or more of the same cell types) and structure (e.g., size, shape, number of compartments). In one embodiment, the degree of FBR is evaluated by an immune response in a tissue containing an implanted device (e.g., a hydrogel capsule) using, for example, assays known in the art as described in WO 2017 / 075630 or one or more of the assays / methods described in Vegas, A. et al., Nature Biotechnol (supra) (e.g., subcutaneous cathepsin measurement of the implanted capsule, Masson's trichrome staining (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cell staining of macrophages (CD68 or F4 / 80), myofibroblasts (α-smooth muscle actin, SMA), or general cell deposition, and confocal microscopy, quantification of 79 RNA sequences of known inflammatory factors and immune cell markers, or FACS analysis of macrophages and neutrophils on a device (e.g., a capsule) retrieved 14 days later from the peritoneal cavity of a suitable test subject (e.g., an immunocompetent mouse)), and the immune response may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis. In one embodiment, FBR is evaluated by measuring the levels of one or more immune response biomarkers (e.g., cathepsin, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4) in the tissue containing the implant. In some embodiments, the FBR induced by the device of the present invention (e.g., a hydrogel capsule containing 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 that is FBR-null, e.g., a device that is substantially identical to the tested or claimed device except that it lacks a device for reducing FBR (e.g., a hydrogel capsule that does not contain a defibrotic compound but is otherwise substantially identical to the claimed capsule). In some embodiments, FBR (e.g., the level of one or more biomarkers) is measured at 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 longer.

[0035] 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.

[0036] 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 connection or cell-cell connection). 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.

[0037] 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 composed 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 CBP via a peptide bond. In one embodiment, the CBP-polymer is any CBP-alginate defined in WO2020069429.

[0038] As used herein, "cell binding substance (CBS)" means any chemical, biological or other type of material (e.g., small organic compounds, peptides, polypeptides) that can simulate at least one activity of a ligand of a cell adhesion molecule (CAM) or other cell surface molecule that mediates cell-matrix connection or cell-cell connection or other receptor-mediated signaling. In one embodiment, when present in a 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 a polymer composition encapsulating multiple cells (e.g., living cells) is related to one or both of the cell productivity (e.g., expression of therapeutic agents) and increased cell viability when the encapsulated cells are implanted in a test subject (e.g., 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.

[0039] 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 that they have 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 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.

[0040] Table 1. Exemplary conservative amino acid substitution groups.

[0041]

[0042] As used throughout the specification and claims, "consisting essentially of" and variations such as "consisting essentially of" or "consisting essentially of" means including any recited elements or groups of elements, and optionally including other elements of similar or different properties to the recited elements, which other elements do not substantially change the basic or novel characteristics of the specified molecule, composition, device, or method. As a non-limiting example, a therapeutic protein agent secreted by an engineered mammalian cell described herein that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues in a recited amino acid sequence, each of which does not substantially affect the relevant biological activity of the therapeutic protein agent.

[0043] "Derived from" as used herein 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.

[0044] 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 after implantation of the device, and having 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.

[0045] As used herein, "differential volume" refers to the volume of a compartment in the device described herein, which does not include the space occupied by another one or more 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 of the space in the second compartment that does not include the space occupied by the first compartment (inner compartment).

[0046] As used herein, "effective amount" refers to the following: an amount of an engineered cell that secretes a protein, a device formulation that produces the protein, or any of the components 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 compounds 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 defibrotic compounds disposed on the surface of the device and / or in the barrier compartment, the density of CBS in the cell-containing compartment).

[0047] In one embodiment, the desired biological response after implanting the implantable element into a subject is a lower amount of pericranial 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 an amount of a therapeutic agent secreted by an engineered mammalian cell as described herein. An effective amount encompasses both therapeutic and prophylactic treatments.

[0048] 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 preparation 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 cells removed from a tissue sample removed from a subject treated with (e.g., implanted with) a genetically modified cell, a device or a device preparation containing such a cell. As will be appreciated by one of ordinary skill in the art, an effective amount may 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.

[0049] As used herein, "endogenous nucleic acid" is a nucleic acid that naturally occurs in the cells of a subject.

[0050] As used herein, an "endogenous polypeptide" is a polypeptide that naturally occurs in the cells of a subject.

[0051] "Engineered human cell" and "genetically modified human cell" can be used interchangeably herein, and each term means a human cell (e.g., epithelial cell) with a non-naturally occurring gene change (e.g., in a cell genome), and generally includes an exogenous nucleic acid sequence (e.g., DNA or RNA) that is not present in (or is present in at different levels in) an otherwise similar human cell (e.g., epithelial cell) that is not engineered. In one embodiment, an engineered human cell (e.g., an engineered RPE cell) includes an exogenous nucleic acid encoding a polypeptide (e.g., a therapeutic protein). In one embodiment, an exogenous nucleotide sequence is chromosomal (e.g., an exogenous nucleic acid sequence is an exogenous sequence arranged in an endogenous chromosomal sequence) or extrachromosomal (e.g., a non-integrated expression vector). In one embodiment, an exogenous nucleic acid sequence includes an RNA sequence, such as an mRNA. In one embodiment, an exogenous nucleic acid sequence includes a chromosome or an extrachromosomal exogenous nucleic acid sequence, and the chromosome or the extrachromosomal exogenous nucleic acid sequence includes a sequence expressed as RNA, such as an mRNA or a 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, engineered cells (e.g., engineered epithelial cells, e.g., engineered RPE cells, e.g., engineered ARPE-19 cells) are cultured from a monoclonal cell line. In some embodiments, the engineered cells are not pancreatic islet cells as defined herein.

[0052] As used herein, "exogenous nucleic acid" is a nucleic acid that does not naturally occur in the cells of a subject.

[0053] As used herein, "exogenous polypeptide" is a polypeptide encoded by an exogenous nucleic acid in a subject's cells. Reference to an amino acid position of a particular sequence means the position of the amino acid in a reference amino acid sequence, e.g., the sequence of a full-length mature (after signal peptide cleavage) wild-type protein (unless otherwise 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.

[0054] Unless otherwise indicated, "factor VII protein" or "FVII protein" as used herein refers to a polypeptide comprising an amino acid sequence of a naturally occurring factor VII protein or a variant thereof, which 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. FVII proteins 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) include 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 marzeptacogα (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.

[0055] Unless otherwise indicated, factor VII biological activity can be quantified by art-recognized determination. For example, the FVII biological activity in a sample of a biological fluid (e.g., blood plasma) can be measured in the following manner: (i) measuring the amount of factor Xa produced in a system comprising tissue factor (TF) and factor X embedded in a lipid membrane (Persson et al., J.Biol.Chem.272:19919-19924, 1997); (ii) measuring the factor X hydrolysis in an aqueous system; (iii) using an instrument based on surface plasmon resonance to measure its physical binding to TF (Persson, FEBS Letts.413:359-363, 1997); or (iv) measuring the hydrolysis of a synthetic substrate; and / or (v) measuring the generation of thrombin in an 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).

[0056] 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 a 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 extension polypeptide. In one embodiment, the cell is 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 containing 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, Supplement).

[0057] 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., Column 4, Line 22 to Column 5, Line 36); 5,543,502 (e.g., Column 2, Lines 17-46); 5,595,886; 5,610,278; 5,789,203 (e.g., Column 2, Lines 26-51 and Examples 5-8); 5,972,885 (e.g., Column 1, Line 25 to Column 2, Line 40); 6,048,720 (e.g., Column 6, Lines 1-22 and Example 1 ); 6,060,447; 6,228,620; 6,316,226 (e.g., column 4, 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).

[0058] 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 required for intracellular processing of the primary translation product into two polypeptide chains (WO 91 / 09122); (ii) amino acids 747-1638 (Hoebene 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. Pat. No. 9,956,269, column 10, line 65 to column 11, line 36.

[0059] 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).

[0060] In some embodiments, the FVIII-BDD protein is a single-chain variant produced by substitution or deletion of one or more amino acids in the furin recognition sequence LKRHQR, which substitution or deletion prevents proteolytic cleavage at that site, including any substitutions at positions R1645 and R1648 described in U.S. Pat. Nos. 10,023,628, 9,394,353, and 9,670,267.

[0061] In some embodiments, any of the above FVIII-BDD proteins may further comprise one or more of the following variations: F309S substitution to improve expression of FVIII-BDD protein (Miao, HZ, et al., Blood 103(a):3412-3419 (2004); albumin fusion (WO 2011 / 020866); and Fc fusion (WO 04 / 101740).

[0062] Unless otherwise indicated, all FVIII-BDD amino acid positions referred to herein refer to positions in full-length human FVIII.

[0063] Unless otherwise indicated, "factor IX protein" or "FIX protein" as used herein refers to a polypeptide comprising an amino acid sequence of a naturally occurring factor IX protein or a variant thereof, as determined by an art-recognized assay, and the polypeptide has a FIX biological activity, such as coagulation activity. FIX is produced as an inactive zymogen, which is converted into an active form by excision of the factor XIa of an activating peptide, thereby producing a heavy chain and a light chain linked together by one or more disulfide bonds. The FIX protein produced by genetically modified cells described herein (e.g., derived from RPE cell lines, such as ARPE-19 cell lines) includes wild-type primates (e.g., humans), pigs, dogs and rat proteins, and variants of such wild-type proteins, 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 a fusion of a half-life-extending polypeptide. In one embodiment, the cell is engineered to encode a full-length wild-type human factor IX polypeptide (e.g., with a 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. Assays for measuring the clotting activity of FIX protein include the 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).

[0064] 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, lines 1 to page 19, line 11; WO 2007 / 149406A2, page 19, lines 12 to page 20, line 9; WO 08 / 118507, page 5, lines 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.

[0065] 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 4 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.

[0066] FIX variants also include gain-of-function and loss-of-function variants. An example of gain-of-function variants is the "Padua" variant of people FIX, which has L (leucine) instead of R (arginine) at position 338 of mature protein, and has greater 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 variants is to replace lysine with alanine in the fifth amino acid position starting from mature protein, which produces a protein (e.g., loss of function) with reduced combination with collagen IV.

[0067] As used herein, "islet cells" are intended to include any naturally occurring or any synthetically produced or modified cells that are intended to partially or completely reproduce, simulate or otherwise express some or all of the functions of Langerhans islet cells. The term "islet cells" includes glucose-responsive insulin-producing cells derived from stem cells (e.g., derived from an induced pluripotent stem cell line).

[0068] As used herein, a "polymer composition" is a composition (e.g., solution, mixture) comprising one or more polymers. As a class, "polymers" include homopolymers, heteropolymers, copolymers, block polymers, block copolymers and may be natural and synthetic. Homopolymers contain one type of structural unit or monomer, while copolymers contain more than one type of monomer.

[0069] 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.

[0070] As used herein, "Prevention", "prevent" and "preventing" refer to a treatment that includes administering a composition (or formulation) of a device encapsulating 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 an 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 a disease or condition have not yet developed or have not yet been observed.

[0071] 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 ( CRL-2302 TM) cells that are cultured, or derived or engineered therefrom (e.g., by stable transfection with an exogenous sequence encoding a polypeptide of interest of cells cultured from the ARPE-19 cell line or by inserting an exogenous sequence into one of the specific OCR insertion sites described herein), cells of a primary cell culture derived from RPE cells, cells directly isolated from naturally occurring RPE cells (e.g., from a human or other mammal) without long-term culture (e.g., less than 5 or 10 passages or rounds of cell division since isolation), cells derived from a transformed, immortalized or long-term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell culture; b) cells obtained from less differentiated cells, such as cells that are developed, programmed or reprogrammed (e.g., in vitro) into RPE cells, or cells that are substantially similar to one or more of naturally occurring RPE cells or cells from a primary or long-term culture of RPE cells apart from any genetic engineering (e.g., the cells may be derived from iPS cells); c) cells having one or more of the following characteristics: i) that express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1 or αB-crystallin; ii) that do not express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1 or αB-crystallin; iii) that naturally occur in the retina and form a monolayer above the choroidal blood vessels in Bruch's membrane; or iv) that are responsible for epithelial transport, light absorption, secretion and immune regulation in the retina; or v) that are synthetically produced or modified from naturally occurring cells to have the same or substantially the same genetic content and optionally the same or substantially the same epigenetic content as an immortalized RPE cell line (e.g., the ARPE-19 cell line ( CRL-2302 TM ))). Other exemplary strains of RPE cells include ARPE-19-SEAP-2-neo cells, RPE-J cells and hTERT RPE-1 cells. In one embodiment, the RPE described herein is engineered to have, for example, new properties, such as by genetically modifying the cells by inserting at least one exogenous transcription unit into one or more of the OCR positions described herein.

[0072] When used herein 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 in a specified region, or have the same nucleotides or amino acids at a specified percentage of nucleotides or amino acid positions in a 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 the same nucleotide or amino acid position is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher.

[0073] 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 ball) or a sphere-like shape, which may have waves and undulations on the surface, for example. 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.

[0074] The term "spheroid" when 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 roughly spheroidal, e.g., may have corrugations and undulations and / or may be an ellipsoid whose semi-major axes are within 100% of each other (with respect to their average surface).

[0075] 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, pigs, horses, sheep, goats, cats, or dogs) or a bird (e.g., a commercially relevant bird, such as a 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.

[0076] As used herein, "treatment", "treat" and "treating" refer to reducing, reversing, alleviating 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, alleviating symptoms or conditions 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, 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 when there are no signs or symptoms of the disease or disorder, such as in preventive treatment. For example, treatment may be administered to susceptible individuals 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, in order to delay or prevent recurrence. In some embodiments, treatment includes prevention, while in other embodiments it does not. "Wild type" (wt) refers to the native form of a polynucleotide, polypeptide, or protein in a species, including the sequence. The wild type form is distinguished from a mutant form of a polynucleotide, polypeptide, or protein resulting from one or more genetic mutations.

[0077] Selected chemical definitions

[0078] The 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, cover, and specific functional groups are generally defined as described therein. In addition, the general principles of organic chemistry as well as specific functional moieties and reactivity 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.

[0079] Abbreviations used herein have their conventional meanings in the fields of chemistry and biology.The chemical structures and formulae shown herein are constructed according to standard rules of chemical valency known in the chemical arts.

[0080] When a range of values ​​is listed, it is intended to include every value and sub-range within that range. For example, "C 1 -C 6 "Alkyl" is intended to cover C 1 , C 2 , C 3 , C 4 , C 5 , C 6 , C 1 -C 6 , C 1 -C 5 , C 1 -C 4 , C 1 -C 3 , C 1 -C 2 , C 2 -C 6 , C 2 -C 5 , C 2 -C 4 , C 2 -C3 , C 3 -C 6 , C 3 -C 5 , C 3 -C 4 , C 4 -C 6 , C 4 -C 5 , and C 5 -C 6 alkyl.

[0081] As used herein, "alkyl" refers to a straight chain or branched saturated hydrocarbon group having 1 to 24 carbon atoms ("C 1 -C 24 In some embodiments, an alkyl group has 1 to 12 carbon atoms (“C 1 -C 12 alkyl”), 1 to 10 carbon atoms (“C 1 -C 12 alkyl”), 1 to 8 carbon atoms (“C 1 -C 8 alkyl”), 1 to 6 carbon atoms (“C 1 -C 6 alkyl”), 1 to 5 carbon atoms (“C 1 -C 5 alkyl”), 1 to 4 carbon atoms (“C 1 -C 4 alkyl”), 1 to 3 carbon atoms (“C 1 -C 3 alkyl”), 1 to 2 carbon atoms (“C 1 -C 2 alkyl") or 1 carbon atom ("C 1 In some embodiments, an alkyl group has 2 to 6 carbon atoms (“C 2 -C 6 C 1 -C 6 Examples of alkyl groups include methyl (C 1 ), ethyl (C 2 ), n-propyl (C 3 ), isopropyl (C 3 ), n-butyl (C 4 ), tert-butyl (C 4 ), sec-butyl (C 4 ), isobutyl (C 4 ), n-pentyl (C 5 ), 3-pentyl (C 5 ), pentyl (C 5 ), neopentyl (C5 ), 3-methyl-2-butane (C 5 ), tert-amyl (C 5 ) and n-hexyl (C 6 Other examples of alkyl groups include n-heptyl (C 7 ), n-octyl (C 8 ) etc. Each example of alkyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl"), or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl").

[0082] 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 ("C 2 -C 24 In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C 2 -C 10 alkenyl”), 2 to 8 carbon atoms (“C 2 -C 8 alkenyl”), 2 to 6 carbon atoms (“C 2 -C 6 alkenyl”), 2 to 5 carbon atoms (“C 2 -C 5 alkenyl”), 2 to 4 carbon atoms (“C 2 -C 4 alkenyl”), 2 to 3 carbon atoms (“C 2 -C 3 alkenyl") or 2 carbon atoms ("C 2 The one or more carbon-carbon double bonds may be internal (as in 2-butenyl) or terminal (as in 1-butenyl). 2 -C 4 Examples of alkenyl groups include vinyl (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadiene (C 4 ) etc. 2 -C 6 Examples of alkenyl groups include the aforementioned C 2-4 Alkenyl and pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6) etc. Each example 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”).

[0083] As used herein, the term "alkynyl" refers to a straight chain or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon triple bonds ("C 2 -C 24 In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C 2 -C 10 alkynyl”), 2 to 8 carbon atoms (“C 2 -C 8 alkynyl”), 2 to 6 carbon atoms (“C 2 -C 6 alkynyl”), 2 to 5 carbon atoms (“C 2 -C 5 alkynyl”), 2 to 4 carbon atoms (“C 2 -C 4 alkynyl”), 2 to 3 carbon atoms (“C 2 -C 3 Alkynyl") or 2 carbon atoms ("C 2 The carbon-carbon triple bond(s) may be internal (as in 2-butynyl) or terminal (as in 1-butynyl). 2 -C 4 Examples of alkynyl groups include ethynyl (C 2 ), 1-propynyl (C 3 ), 2-propynyl (C 3 ), 1-butynyl (C 4 ), 2-butynyl (C 4 ) etc. Each instance of alkynyl can independently be optionally substituted, i.e., unsubstituted (“unsubstituted alkynyl”) or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (“substituted alkynyl”).

[0084] As used herein, the term "heteroalkyl" refers to a non-cyclic stable straight or branched chain or 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 may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. One or more heteroatoms O, N, P, S and Si may be placed at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to: -CH 2 -CH 2 -O-CH 3 、-CH 2-CH 2 -NH-CH 3 、-CH 2 -CH 2 -N(CH 3 )-CH 3 、-CH 2 -S-CH 2 -CH 3 、-CH 2 -CH 2 、-S(O)-CH 3 、-CH 2 -CH 2 -S(O) 2 -CH 3 、-CH=CH-O-CH 3 、-Si(CH 3 ) 3 、-CH 2 -CH=N-OCH 3 、-CH=CH-N(CH 3 )-CH 3 、-O-CH 3 and -O-CH 2 -CH 3 Up to two or three heteroatoms may be consecutive, such as, for example, -CH 2 -NH-OCH 3 and -CH 2 -O-Si(CH 3 ) 3 When describing "heteroalkyl", it is followed by a specific heteroalkyl group such as -CH 2 O, -NR C R D etc., it will be understood that the terms heteroalkyl and -CH 2 O or -NR C R D The terms "heteroalkyl" and "heteroalkyl" are not redundant or mutually exclusive. Rather, specific heteroalkyl groups are described to increase clarity. Therefore, the term "heteroalkyl" should not be interpreted herein to exclude specific heteroalkyl groups, such as -CH 2 O, -NR C R D Etc. Each instance of heterocyclyl can independently be optionally substituted, i.e., unsubstituted (“unsubstituted heteroalkyl”) or substituted with one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (“substituted heteroalkyl”).

[0085] Unless otherwise indicated, the terms "alkylene", "alkenylene", "alkynylene" or "heteroalkylene" by themselves or as part of another substituent mean a divalent radical derived from an alkyl, alkenyl, alkynyl or heteroalkyl group, respectively. Alkylene, alkenylene, alkynylene or heteroalkylene can be described, for example, as C 1 -C 6 Alkylene, C 2 -C 6 Alkenylene, C 2 -C 6 Alkynylidene or C 1 -C 6 Membered heteroalkylene groups, wherein the term "membered" refers to non-hydrogen atoms within the moiety. In the case of heteroalkylene groups, heteroatoms may also occupy one or both chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Further, 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) 2 R'- can represent -C(O) 2 R'- and -R'C(O) 2 -Both.

[0086] 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) radical having 6-14 ring carbon atoms and zero heteroatoms ("C 6 -C 14 In some embodiments, an aryl group has 6 ring carbon atoms (“C 6 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, C 6 -C 10 The term "aryl" refers to the non-hydrogen ring atoms within the moiety. Aryl includes phenyl, naphthyl, indenyl and tetrahydronaphthyl. Each example of aryl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted aryl"), or substituted ("substituted aryl") with one or more substituents.

[0087] 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 contains no heteroatoms (e.g., indolyl, quinolyl, carbazolyl, etc.), the point of attachment can be on 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.

[0088] In some embodiments, heteroaryl is a 5-10 membered aromatic ring system 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 some embodiments, heteroaryl is a 5-8 membered aromatic ring system 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 (“5-8 membered heteroaryl”). In some embodiments, heteroaryl is a 5-6 membered aromatic ring system 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 (“5-6 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 has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of heteroaryl can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroaryl") or substituted (a "substituted heteroaryl") with one or more substituents.

[0089] Exemplary 5-membered heteroaryls containing 1 heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryls containing 2 heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryls containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryls containing 4 heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryls containing 1 heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryls containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryls containing 3 or 4 heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryls containing 1 heteroatom include, but are not limited to, aza-zepta ... 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.

[0090] 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.

[0091] As used herein, "cycloalkyl" refers to a non-aromatic ring system having 3 to 10 ring carbon atoms ("C 3 -C 10 In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms ("C 3 -C 8 cycloalkyl”), 3 to 6 ring carbon atoms (“C 3 -C 6 cycloalkyl”) or 5 to 10 ring carbon atoms (“C 5 -C 10 Cycloalkyl"). Cycloalkyl can be described as, for example, C 4 -C 7 The term "membered" refers to the non-hydrogen ring atoms within the moiety. 3 -C 6 Cycloalkyl includes, but is not limited to, cyclopropyl (C 3 ), cyclopropenyl (C 3 ), cyclobutyl (C 4 ), cyclobutenyl (C 4), cyclopentyl (C 5 ), cyclopentenyl (C 5 ), cyclohexyl (C 6 ), cyclohexenyl (C 6 ), cyclohexadienyl (C 6 ) etc. Exemplary C 3 -C 8 Cycloalkyl includes but is not limited to the aforementioned C 3 -C 6 Cycloalkyl and cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), cyclooctyl (C 8 ), cyclooctenyl (C 8 ), cubic alkyl (C 8 ), bicyclo[1.1.1]pentyl (C 5 ), bicyclo[2.2.2]octyl (C 8 ), bicyclo[2.1.1]hexane (C 6 ), bicyclo[3.1.1]heptyl (C 7 ) etc. Exemplary C 3 -C 10 Cycloalkyl includes but is not limited to the aforementioned C 3 -C 8 Cycloalkyl and cyclononyl (C 9 ), cyclononenyl (C 9 ), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C 9 ), decahydronaphthyl (C 10 ), spiro[4.5]decyl (C 10 ) and the like. As illustrated by the foregoing examples, in certain embodiments, the cycloalkyl is monocyclic ("monocyclic cycloalkyl") or contains a fused, bridged or spiro ring system (such as a bicyclic ring system ("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 ("unsubstituted cycloalkyl"), or substituted ("substituted cycloalkyl") with one or more substituents.

[0092] As used herein, "heterocyclyl" refers to a group of a 3-10 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 heterocyclyls containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom as long as valence permits. The heterocyclyl may be a monocyclic ("monocyclic heterocyclyl") or a fused, bridged or spirocyclic system, such as a bicyclic system ("bicyclic heterocyclyl"), and may be saturated or may be partially unsaturated. A 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, 3-7 membered heterocyclyl, wherein the term "member" refers to non-hydrogen ring atoms within the portion, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each example of heterocyclyl can be independently optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl"), or substituted ("substituted heterocyclyl") by one or more substituents. In certain embodiments, heterocyclyl is an unsubstituted 3-10 membered heterocyclyl. In certain embodiments, heterocyclyl is a substituted 3-10 membered heterocyclyl.

[0093] In some embodiments, heterocyclyl 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, heterocyclyl 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, heterocyclyl 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, 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen and sulfur. In some embodiments, 5-6 membered heterocyclyl 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.

[0094] Exemplary 3-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, aziridine, oxirane and thioethane. Exemplary 4-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azetidinyl, oxetane 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, oxepane, and thiepanyl. Exemplary 8-membered heterocyclic groups containing 1 heteroatom include, but are not limited to, azocanyl, oxepane, and thiecanyl. Fused to C 6 Exemplary 5-membered heterocyclic groups (also referred to herein as 5,6-bicyclic heterocycles) of an aryl ring include, but are not limited to, indolinyl, isoindolyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, etc. Exemplary 6-membered heterocyclic groups (also referred to herein as 6,6-bicyclic heterocycles) fused to an aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[0095] As used herein, "amino" refers to the group -NR 70 R 71 , where R 70 and R 71 are independently hydrogen, C 1 -C 8 Alkyl, C 3 -C 10 Cycloalkyl, C 4 -C 10 Heterocyclic group, C 6 -C 10 Aryl and C 5 -C 10 In some embodiments, amino refers to NH 2 .

[0096] As used herein, "cyano" refers to -CN.

[0097]

[0043] 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.

[0098] As used herein, "hydroxy" refers to the group -OH.

[0099] 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, such as a substituent that, upon substitution, results in a stable compound, such as 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 the present disclosure, a heteroatom (such as nitrogen) may have a hydrogen substituent and / or any suitable substituent that satisfies the valence of the heteroatom and produces the formation of a stable moiety as described herein.

[0100] Two or more substituents may optionally be joined to form an aryl, heteroaryl, cycloalkyl or heterocyclic radical. Such so-called ring-forming substituents are usually 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 condensed 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 spiro ring structure. In another embodiment, the ring-forming substituents are attached to non-adjacent members of the basic structure.

[0101] 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 (ELEliel, 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.

[0102] 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, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0103] 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.

[0104] 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 (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, 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, etc.; 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, etc. Also included are salts of amino acids such as arginine salts, etc.; and salts of organic acids such as glucuronic acid or galacturonic acid, etc. (see, e.g., Berge et al., Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds used in the disclosed devices (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.

[0105] The devices of the present disclosure may contain compounds of formula (I) in the form of prodrugs. Prodrugs are those compounds that readily undergo chemical changes under physiological conditions to provide compounds that can be used to prepare the devices of the present disclosure. In addition, prodrugs can be converted into useful compounds of formula (I) by chemical or biochemical methods in an ex vivo environment.

[0106] Some of the compounds of formula (I) described herein can exist in non-solvated form and solvated form (including hydrated form). In general, the solvated form is equivalent to the non-solvated form and is included in 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 covered by the present disclosure and are intended to fall within the scope of the present disclosure.

[0107] The term "solvate" refers to a form of a compound associated with a solvent, typically by a solvolysis reaction. Such physical association may include hydrogen bonding. Commonly used solvents include water, methanol, ethanol, acetic acid, DMSO, THF, ether, and the like. The compounds described herein may be prepared, for example, in crystalline form and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include stoichiometric solvates and non-stoichiometric solvates.

[0108] The term "hydrate" refers to a compound 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 H2O, where R is a compound and where x is a number greater than 0.

[0109] 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 interconverted 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.

[0110] Symbols used in this article 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 by can refer to direct attachment to an entity (e.g., a polymer or an implantable element), and can refer to a linkage to an entity through an attachment group. As described herein, an "attachment group" 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 may include any attachment chemistry known in the art. A list of exemplary attachment groups is summarized in Bioconju gate 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 and y is independently as described herein. In some embodiments, the attachment group comprises an amine, a ketone, an ester, an amide, an alkyl. In some embodiments, the attachment group is a cross-linking agent. In some embodiments, the attachment group is -C(O)(C 1 -C 6 1 , and R 1 As described in this article. In some embodiments, the attachment group is -C(O)(C 1 -C 6-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(CH 3 )-. In some embodiments, the attachment group is -C(O)C(CH 3 )-.

[0111] Engineered mammalian cells

[0112] 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. In one embodiment, the engineered mammalian cell reduces the level or function of an MHC class I protein complex, and optionally an MHC class II protein complex and / or CIITA.

[0113] MHC class I protein complexes are a class of molecules present on the surface of nucleated cells that inform the host's immune system of the status of a particular antigen as self or non-self. MHC class I molecules display peptide fragments of cytotoxic proteins on the cell surface, which trigger an immune response in the host if the cytotoxic protein is from a non-self source. In general, MHC class I molecules are heterodimeric proteins composed of two polypeptide chains. The alpha chain is polymorphic and is encoded by human leukocyte antigens (HLA) containing one of the HLA-A, HLA-B, or HLA-C. The beta chain contains the beta-2-microglobulin (β-2M) domain. The alpha and beta chains of each MHC class I molecule are non-covalently linked by the interaction of the beta-2M with one of the three plasma membrane-spanning domains of the alpha chain (α-3). The alpha chain also contains two additional domains: alpha-1 and alpha-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 β-2M).

[0114] The groove between the α-1 and α-2 domains is a peptide binding groove in which peptides derived from cytoplasmic proteins are displayed. The groove contains eight β-pleated sheets located on the bottom and two α-helices constituting the sides. The groove is flanked by tyrosine residues and forms a closed end, which limits the size of the peptide that can be bound to the groove. The peptide in the groove remains essentially bound there during the life of the class I molecule, and is typically 8-9 amino acids in length. Self or foreign cytoplasmic proteins are degraded and transported to the ER cavity via proteasomes. In the ER, peptides are loaded onto MHC class 1 with the help of a chaperone protein called tapasin. Then, the peptide-bound MHC class I is 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-).

[0115] In addition to the interaction 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 about 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 I Molecules with Superenhanced CD8 Binding Properties Bypass the Requirement for Cognate TCR Recognition and Nonspecifically Activate CTLs, J. Immunol. 184: 3357-3366).

[0116] 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 cross membranes. The human β-2M locus is located on chromosome 15. The β-2M gene consists of 4 exons and 3 introns. Circulating forms of β-2M are present in serum, urine, and other body fluids; therefore, non-covalently MHC 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 (5 genes in humans), MR1, neonatal Fc receptor (FcRn), and Qa-1 (a form of alloantigen).

[0117] In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises a 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 β-2M). In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises a reduction in the α and / or β chains of an MHC class I protein complex or a component thereof. For example, in some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of an 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 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%.

[0118] In some embodiments, the level or function of the alpha-1 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more). For example, in some embodiments, the level or function of the alpha-1 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-1 domain is reduced by more than 99.9%.

[0119] In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises 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%.

[0120] In some embodiments, the level or function of the alpha-2 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more). For example, in some embodiments, the level or function of the alpha-2 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-2 domain is reduced by more than 99.9%.

[0121] In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of the alpha-3 domain. In some embodiments, the level or function of the alpha-3 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). For example, in some embodiments, the level or function of the alpha-3 domain is reduced by about 20%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 30%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 40%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 50%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 60%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 70%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 80%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 90%. In some embodiments, the level or function of the alpha-3 domain is reduced by about 100%.

[0122] In some embodiments, the level or function of the alpha-3 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more). For example, in some embodiments, the level or function of the alpha-3 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-3 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-3 domain is reduced by more than 99.9%.

[0123] In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of the β-2M domain. In some embodiments, the level or function of the β-2M domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). For example, in some embodiments, the level or function of the β-2M domain is reduced by about 20%. In some embodiments, the level or function of the β-2M domain is reduced by about 30%. In some embodiments, the level or function of the β-2M domain is reduced by about 40%. In some embodiments, the level or function of the β-2M domain is reduced by about 50%. In some embodiments, the level or function of the β-2M domain is reduced by about 60%. In some embodiments, the level or function of the β-2M domain is reduced by about 70%. In some embodiments, the level or function of the β-2M domain is reduced by about 80%. In some embodiments, the level or function of the β-2M domain is reduced by about 90%. In some embodiments, the level or function of the Beta-2M domain is reduced by about 100%.

[0124] 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 β-2M domain is reduced by at least 95%. In some embodiments, the level or function of the β-2M domain is reduced by at least 99%. In some embodiments, the level or function of the β-2M domain is reduced by at least 99.9%. In some embodiments, the level or function of the β-2M domain is reduced by more than 99.9%.

[0125] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of β-2M with MHC class I protein complexes or components thereof or MHC class I-like molecules or components (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 β-2M with MHC class I protein complexes or components thereof. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of β-2M with the α-1 domain of MHC class I proteins. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of β-2M with the α-2 domain of MHC class I proteins. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of β-2M with the α-3 domain of MHC I class proteins. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of β-2M with MHC class I-like 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) of β-2M with CD1. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of β-2M with MR1. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of β-2M with FcRn. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of β-2M with Qa-1.

[0126] 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.

[0127] HLA-A interacts with calnexin, calreticulin, antigen processing-related transporter (TAP), tapasin, thiol disulfide oxidoreductase ERp57 enzyme, and any cytoplasmic peptides bound to 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 to its peptide binding groove, 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 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 TAP. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-A with TAP-1. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-A with TAP-2. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-A with tapasin. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-A with ERp57. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-A with a cytoplasmic peptide bound to its peptide binding groove.

[0128] 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 to LILRA1. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-C to LILRA3.

[0129] 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 β-2M) results in reduced antigen presentation, thereby reducing and / or eliminating the recruitment of immune cells (e.g., T cells and NK cells).

[0130] The HLA-A gene is located on the short arm of chromosome 6 and encodes the larger alpha chain component of HLA-A. Variation in the HLA-A alpha chain is key to HLA function. This variation promotes genetic diversity in the population. Since each HLA has a different affinity for peptides of certain structures, more HLA types mean a greater variety of antigens can be "presented" on the cell surface, increasing the likelihood that a subset of the population will be able to resist a specific foreign invader. This reduces the likelihood that a single pathogen will wipe out an entire human population.

[0131] Each person can express up to two types of HLA-A, one from each parent. Some individuals will inherit the same HLA-A from both parents, thereby reducing their individual HLA diversity; however, most individuals will obtain 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.

[0132] The HLA-B gene is located on the short arm (p) of chromosome 6 at cytoband 21.3 and encodes the larger alpha chain component of HLA-B. Similar to HLA-A, variations in the HLA-B alpha 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, which is 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.

[0133] 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 β-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 β-2M. 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 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 the β-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 the 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 the 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 the HLA-C gene.

[0134] In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and β-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 β-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 β-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 β-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 include 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 include 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 include 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 include 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 include 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 include 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 include 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 include sequences having greater than 99.9% sequence identity to the nucleotide sequences provided in Table 5.

[0135] In some embodiments, the nucleotide sequences of the HLA-A, HLA-B, HLA-C, and β-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 homology 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 β-2M genes include sequences having at least 65% 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 β-2M genes include sequences having at least 70% 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 β-2M genes include sequences having at least 75% 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 include 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 include 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 include 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 include 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 include 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 include sequences having at least 99.9% 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 include sequences having greater than 99.9% sequence homology to the nucleotide sequences provided in Table 5.

[0136] In one embodiment, an engineered mammalian cell described herein (e.g., ARPE-19) comprises a reduction in expression 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 an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising a reduction in the levels of MHC class I components. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise expression of MHC class I components, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100% 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 MHC class I components. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise expression of MHC class I components, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100% 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 MHC class I components.

[0137] In one embodiment, an engineered mammalian cell described herein (e.g., ARPE-19) comprises 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in the function of the MHC class I component. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction of 1-25%, 5-25%, 10-25%, 25-50%, 25-75%, 50-75%, or 75-100% of the function of the MHC class I component, for example, compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for the reduction in the function of the MHC class I component. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction of greater than about 50%, 75%, or 90% of the function of the MHC class I component, for example, compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for the reduction in the function of the MHC class I component.

[0138] In one embodiment, an engineered mammalian cell described herein (e.g., ARPE-19) comprises a decrease in the level or function of HLA-A, 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 decrease in the level or function of HLA-A. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the level or function of HLA-A, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100% compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in the level or function of HLA-A. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the level or function of HLA-A, e.g., greater than about 50%, 75%, or 90% compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in the level or function of HLA-A.

[0139] In one embodiment, an engineered mammalian cell described herein (e.g., ARPE-19) comprises a decrease in the level or function of HLA-B, 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 decrease in the level or function of HLA-B. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the level or function of HLA-B, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100% compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in the level or function of HLA-B. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the level or function of HLA-B, e.g., greater than about 50%, 75%, or 90% compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in the level or function of HLA-B.

[0140] In one embodiment, an engineered mammalian cell described herein (e.g., ARPE-19) comprises a decrease in the level or function of HLA-C, 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 decreased level or function of HLA-C. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the level or function of HLA-C, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100% compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduced level or function of HLA-C. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the level or function of HLA-C, e.g., greater than about 50%, 75%, or 90% compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduced level or function of HLA-C.

[0141] In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a decrease in the level or function of β-2M, for example, 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 for not comprising a decrease in the level or function of β-2M. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the level or function of β-2M, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100% 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 or function of β-2M. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the level or function of β-2M, e.g., greater than about 50%, 75%, or 90% 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 or function of β-2M.

[0142] In some embodiments, the MHC class I protein complex or a component thereof, such as HLA-A, HLA-B, HLA-C or β-2M, is encoded by one or more nucleotide sequences provided in Table 5, or a fragment thereof.

[0143] 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 β-2M) 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 I 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 I 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 I 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 I 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 I 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 I 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 I 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 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.

[0144] 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 antigen presented by MHC class II protein complexes is derived from extracellular proteins, which is 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 homogeneous 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. Since both ends of the antigen binding groove of MHC class II molecules are open, while each end of the corresponding groove on class I molecules is closed, the antigens presented by MHC class II molecules are longer, generally between 15 and 24 amino acid residues in length.

[0145] 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 cell (e.g., ARPE-19) comprises 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 cell (e.g., ARPE-19) comprises 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 cell (e.g., ARPE-19) comprises a reduction in the level or function of an 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 alpha-1 domain is reduced by about 20%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 30%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 40%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 50%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 60%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 70%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 80%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 90%. In some embodiments, the level or function of the alpha-1 domain is reduced by about 100%.

[0146] In some embodiments, the level or function of the alpha-1 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more). For example, in some embodiments, the level or function of the alpha-1 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-1 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-1 domain is reduced by more than 99.9%.

[0147] In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises 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%.

[0148] In some embodiments, the level or function of the alpha-2 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more). For example, in some embodiments, the level or function of the alpha-2 domain is reduced by at least 10%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 20%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 30%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 40%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 50%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 60%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 70%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 80%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 90%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 95%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 99%. In some embodiments, the level or function of the alpha-2 domain is reduced by at least 99.9%. In some embodiments, the level or function of the alpha-2 domain is reduced by more than 99.9%.

[0149] In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of the beta-1 domain. In some embodiments, the level or function of the beta-1 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). For example, in some embodiments, the level or function of the beta-1 domain is reduced by about 20%. In some embodiments, the level or function of the beta-1 domain is reduced by about 30%. In some embodiments, the level or function of the beta-1 domain is reduced by about 40%. In some embodiments, the level or function of the beta-1 domain is reduced by about 50%. In some embodiments, the level or function of the beta-1 domain is reduced by about 60%. In some embodiments, the level or function of the beta-1 domain is reduced by about 70%. In some embodiments, the level or function of the beta-1 domain is reduced by about 80%. In some embodiments, the level or function of the beta-1 domain is reduced by about 90%. In some embodiments, the level or function of the beta-1 domain is reduced by about 100%.

[0150] In some embodiments, the level or function of the beta-1 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more). For example, in some embodiments, the level or function of the beta-1 domain is reduced by at least 10%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 20%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 30%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 40%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 50%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 60%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 70%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 80%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 90%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 95%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 99%. In some embodiments, the level or function of the beta-1 domain is reduced by at least 99.9%. In some embodiments, the level or function of the beta-1 domain is reduced by more than 99.9%.

[0151] In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of the beta-2 domain. In some embodiments, the level or function of the beta-2 domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). For example, in some embodiments, the level or function of the beta-2 domain is reduced by about 20%. In some embodiments, the level or function of the beta-2 domain is reduced by about 30%. In some embodiments, the level or function of the beta-2 domain is reduced by about 40%. In some embodiments, the level or function of the beta-2 domain is reduced by about 50%. In some embodiments, the level or function of the beta-2 domain is reduced by about 60%. In some embodiments, the level or function of the beta-2 domain is reduced by about 70%. In some embodiments, the level or function of the beta-2 domain is reduced by about 80%. In some embodiments, the level or function of the beta-2 domain is reduced by about 90%. In some embodiments, the level or function of the beta-2 domain is reduced by about 100%.

[0152] In some embodiments, the level or function of the beta-2 domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more). For example, in some embodiments, the level or function of the beta-2 domain is reduced by at least 10%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 20%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 30%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 40%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 50%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 60%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 70%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 80%. In some embodiments, the level or function of the beta-2 domain is reduced by at least 90%. In some embodiments, the level or function of the Beta-2 domain is reduced by at least 95%. In some embodiments, the level or function of the Beta-2 domain is reduced by at least 99%. In some embodiments, the level or function of the Beta-2 domain is reduced by at least 99.9%. In some embodiments, the level or function of the Beta-2 domain is reduced by more than 99.9%.

[0153] 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 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-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.

[0154] HLA-DP is a protein / peptide-antigen receptor and graft-versus-host disease antigen, which consists 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 subunit, β subunit) consists of an α-helical N-terminal domain, an IgG-like β sheet, a transmembrane domain, and a cytoplasmic domain. The α-helical domain forms the sides of the peptide binding groove. The β sheet region forms the bottom of the binding groove and the main body of the molecule as well as the inter-subunit (non-covalent) binding region. The peptide-bound HLA-DP complex interacts with the TCR on CD4+T cells.

[0155] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DP with CD4+T cell TCR and / or any peptide (e.g., antigenic peptide) bound in its peptide binding groove, 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-DP 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-DP with a peptide bound in its peptide binding groove.

[0156] HLA-DM is a nonclassical MHC molecule, an intracellular protein involved in the antigen presentation mechanism, and is encoded by the genes HLA-DMA and HLA-DMB. Like HLA-DP, the gene for HLA-DM is located in the MHCII 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 empty MHC molecules to resist denaturation, to promote antigen-antigen exchange (e.g., by releasing weakly bound peptides from the peptide binding groove to load peptides with higher affinity), and to control the correct loading and release of peptides at the peptide binding groove. In order 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 no longer bind 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 travelling 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 foreign 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 has no ability to bind peptides due to the lack of a deep peptide binding groove, instead it contains a shallow negatively charged indentation with two disulfide bonds.

[0157] HLA-DM also interacts extensively with another nonclassical MHC molecule, the chaperone protein HLA-DO. HLA-DO begins binding 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 a transport signal N-terminus. Prior to encountering antigen, DO acts as a chaperone to DM to stabilize DM against denaturation and direct it to lysosomes. It binds to HLA-DM at the same location as it binds to MHC class II molecules, thereby preventing HLA-DM from binding to MHC class II molecules. This inhibits peptide exchange catalysis and holds CLIP in the MHC groove until antigen-containing lysosomes fuse with DM / DO / MHC-containing lysosomes, 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.

[0158] 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, 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-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-DO with HLA-DM. 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.

[0159] 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 in its peptide binding groove, for example, by 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 in its peptide binding groove.

[0160] HLA-DQ is a cell surface receptor protein found on antigen presenting cells. It is an αβ heterodimer of MHC type II. The α chain and β chain are encoded by two loci, HLA-DQA1 and HLA-DQB1, located adjacent to each other on chromosome band 6p21.3. Both the α chain and the β chain contain a large number of variants. A person usually produces two α chain and two β chain variants, and thus produces 4 HLA-DQ isoforms. The HLA-DQ locus has a close genetic linkage with HLA-DR, and a less close linkage with HLA-DP, non-classical MHC class II molecules (HLA-DM and HLA-DO), and MHC class I molecules.

[0161] 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 signal 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 that HLA-DQ is involved in 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, which is the major autoantigen in type 1 diabetes.

[0162] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of HLA-DQ with a CD4+T-cell TCR and / or any peptide bound within its peptide binding groove, 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-DQ with a 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.

[0163] 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 chain and the β chain are anchored to the membrane. The N-terminal domain of the mature protein forms an α helix that constitutes the exposed part 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 contact positions 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 DR A 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, CD4+ T cell TCR and / or any peptide that binds within its peptide binding groove.

[0164] 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 in 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 in its peptide binding groove.

[0165] 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 reduced antigen presentation, thereby reducing and / or eliminating the recruitment of immune cells (e.g., T cells and NK cells).

[0166] 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 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 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.

[0167] 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 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 70% 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 75% 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 80% 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 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.

[0168] 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 homology to the nucleotide sequence 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 homology 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 70% sequence homology 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 75% sequence homology 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 80% sequence homology 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 85% sequence homology 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 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.

[0169] 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., ARPE-19) comprise a reduction in expression of MHC class II 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 an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising a reduction in the levels of MHC class II components. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a decrease in expression of MHC class II components, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100%, compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising a decrease in the level of MHC class II components. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a decrease in expression of MHC class II components, e.g., greater than about 50%, 75%, or 90%, compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising a decrease in the level of MHC class II components.

[0170] In one embodiment, an engineered mammalian cell described herein (e.g., ARPE-19) comprises 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in the function of the MHC class II component. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the function of an MHC class II component, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100% compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for the reduction in the function of the MHC class II component. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the function of an MHC class I component, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100% compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for the reduction in the function of the MHC class II component.

[0171] 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 nucleotide sequences provided in Table 5, or a fragment thereof.

[0172] 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.

[0173] 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 is located on chromosome 16 and encodes the CIITA protein, which plays a role in enhancing the transcription of MHC class I genes. The CIITA protein contains an acidic transcriptional activation domain, four leucine-rich repeats, and a GTP-binding domain. The protein uses GTP binding to facilitate its own transport 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 (He uberger et al. (2021). "Why do intestinal epithelial cells express MHC class II?". Immunology. 162(4): 357–367. doi: 10.1111 / imm.13270).

[0174] In one embodiment, the engineered mammalian cells of the present disclosure include a reduced level or function of 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 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 the 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 the transcriptional activation domain is reduced by about 20%. In some embodiments, the level or function of the transcriptional activation domain is reduced by about 30%. In some embodiments, the level or function of the transcriptional activation domain is reduced by about 40%. In some embodiments, the level or function of the 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%.

[0175] 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 more than 99.9%.

[0176] In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of a leucine-rich repeat domain. In some embodiments, the level or function of a leucine-rich repeat domain is reduced by about 10% (e.g., about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%). For example, in some embodiments, the level or function of a leucine-rich repeat domain is reduced by about 20%. In some embodiments, the level or function of a leucine-rich repeat domain is reduced by about 30%. In some embodiments, the level or function of a leucine-rich repeat domain is reduced by about 40%. In some embodiments, the level or function of a leucine-rich repeat domain is reduced by about 50%. In some embodiments, the level or function of a leucine-rich repeat domain is reduced by about 60%. In some embodiments, the level or function of a leucine-rich repeat domain is reduced by about 70%. In some embodiments, the level or function of a leucine-rich repeat domain is reduced by about 80%. In some embodiments, the level or function of a 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%.

[0177] In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more). For example, in some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 10%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 20%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 30%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 40%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 50%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 60%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 70%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 80%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 90%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 95%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 99%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by at least 99.9%. In some embodiments, the level or function of the leucine-rich repeat domain is reduced by more than 99.9%.

[0178] In some embodiments, the engineered mammalian cell (e.g., ARPE-19) comprises a reduction in the level or function of a GTP binding domain. In some embodiments, the level or function of a 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 a GTP binding domain is reduced by about 20%. In some embodiments, the level or function of a GTP binding domain is reduced by about 30%. In some embodiments, the level or function of a GTP binding domain is reduced by about 40%. In some embodiments, the level or function of a GTP binding domain is reduced by about 50%. In some embodiments, the level or function of a GTP binding domain is reduced by about 60%. In some embodiments, the level or function of a GTP binding domain is reduced by about 70%. In some embodiments, the level or function of a GTP binding domain is reduced by about 80%. In some embodiments, the level or function of a 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%.

[0179] In some embodiments, the level or function of the GTP-binding domain is reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or more). For example, in some embodiments, the level or function of the GTP-binding domain is reduced by at least 10%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 20%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 30%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 40%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 50%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 60%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 70%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 80%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 90%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 95%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 99%. In some embodiments, the level or function of the GTP-binding domain is reduced by at least 99.9%. In some embodiments, the level or function of the GTP-binding domain is reduced by more than 99.9%.

[0180] In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of the 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 the transcriptional activation domain with the leucine-rich repeat domain or the GTP-binding domain. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of the leucine-rich repeat domain with the transcriptional activation domain, the leucine-rich repeat domain, or the GTP-binding domain. In some embodiments, reducing the level or function significantly reduces, prevents or inhibits the interaction (e.g., binding) of the GTP-binding domain with the transcriptional activation domain or the leucine-rich repeat domain.

[0181] The CIITA protein interacts with mitogen-activated protein kinase 1 (MAPK1), nuclear receptor coactivator 1 (NCOA1), DNA-binding protein RFX5 (RFX5), DNA-binding protein RFXANK (RFXANK), exportin 1 (XPO1), and zinc finger X-linked 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.

[0182] In some embodiments, reducing the level or function of the CIITA protein results in a decrease in the expression level of the MHC class II protein complex or its components (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 its components).

[0183] In some embodiments, reducing the level or function of the CIITA protein includes 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 lead to a decrease in the expression of the gene, for example, by reducing, altering, or eliminating the transcription and / or splicing of the nucleotide sequence.

[0184] 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with 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 with the nucleotide sequence provided in Table 5.

[0185] In some embodiments, the nucleotide sequence of the CIITA gene comprises a sequence having at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, 99.9% or more) sequence homology to 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.

[0186] In one embodiment, an engineered mammalian cell described herein (e.g., ARPE-19) comprises a reduction in 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising a reduced level of CIITA. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise expression of CIITA, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100% reduction compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for the reduction in levels of CIITA. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise expression of CIITA, e.g., greater than about 50%, 75%, or 90% reduction compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for the reduction in levels of CIITA.

[0187] In one embodiment, an engineered mammalian cell described herein (e.g., ARPE-19) comprises a reduction in the 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in the function of CIITA. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the function of CIITA, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75%, or between 75-100%, compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for the reduction in the function of CIITA. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) comprise a reduction in the function of CIITA, e.g., greater than about 50%, 75%, or 90%, compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for the reduction in the function of CIITA.

[0188] In some embodiments, CIITA is encoded by the nucleotide sequence provided in Table 5 or a fragment thereof.

[0189] In some embodiments, the reduction of the level or function of CIITA 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 of the level or function of CIITA lasts for at least 30 minutes. In some embodiments, the reduction of the level or function of CIITA lasts for at least 1 hour. In some embodiments, the reduction of the level or function of CIITA lasts for at least 12 hours. In some embodiments, the reduction of the level or function of CIITA lasts for at least 24 hours. In some embodiments, the reduction of the level or function of CIITA lasts for at least 48 hours. In some embodiments, the reduction of the level or function of CIITA lasts for at least 72 hours. In some embodiments, the reduction of the level or function of CIITA lasts for at least 1 week. In some embodiments, the reduction of the level or function of CIITA lasts for at least 1 month. In some embodiments, the reduction of the level or function of CIITA lasts for at least 1 year.

[0190] In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) include a reduction in the level or function of MHC class I protein complexes and a reduction in the level or function of MHC class II protein complexes and / or CIITA. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) include a reduction in the level or function of HLA-A and a reduction in the level or function of MHC class II protein complexes and / or CIITA. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) include a reduction in the level or function of HLA-B and a reduction in the level or function of MHC class II protein complexes and / or CIITA. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) include a reduction in the level or function of HLA-C and a reduction in the level or function of MHC class II protein complexes and / or CIITA. In one embodiment, the engineered mammalian cells described herein (e.g., ARPE-19) include a reduction in the level or function of β-2M and a reduction in the level or function of MHC class II protein complexes and / or CIITA.

[0191] Table 5: Exemplary sequences

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[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] 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 techniques using site-directed nucleases (such as CRISPR-Cas), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and giant nucleases.

[0286] 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 the cell types described in WO 2017 / 075631. In some embodiments, the cell is derived from the cell lines shown in Table 2 below.

[0287] Table 2: Exemplary cell lines

[0288]

[0289]

[0290] In one embodiment, any one of the engineered mammalian cells described herein is derived from RPE cells, such as ARPE-19 cells. In one embodiment, engineered RPE cells (e.g., engineered ARPE-19 cells) include any one of expression cassettes, transposons, and polynucleotides described herein.

[0291] 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).

[0292] 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 evaluation of spindle formation), or flow cytometry.

[0293] 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.

[0294] In some embodiments, the exogenous transcription unit encodes a therapeutic polypeptide (e.g., 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.

[0295] In some embodiments, the polypeptide is a hormone. Exemplary hormones include antidiuretic hormone (ADH), oxytocin, growth hormone (GH), prolactin, growth hormone-releasing hormone (GHRH), thyroid stimulating hormone (TSH), thyrotropin-releasing hormone (TRH), adrenocorticotropic hormone (ACTH), follicle stimulating hormone (FSH), luteinizing hormone (LH), luteinizing hormone-releasing hormone (LHRH), thyroxine, calcitonin, parathyroid hormone (PTH), aldosterone, cortisol, adrenaline, glucagon, insulin, estrogen, progesterone and testosterone. In some embodiments, the polypeptide is insulin (e.g., insulin A chain, insulin B chain or proinsulin). In some embodiments, the polypeptide is 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.

[0296] 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).

[0297] 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., prothrombinogen, 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.

[0298] In some embodiments, the polypeptide is an immunoglobulin chain (heavy or light chain) or a fragment thereof, which comprises at least one immunoglobulin variable domain sequence, and optionally comprises an immunoglobulin Fc region. In one embodiment, the polypeptide is a full-length immunoglobulin chain.

[0299] 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; lipoprotein; colchicine; adrenocorticotropic hormone; vasopressin; somatostatin; lysine vasopressin; pancreatin; 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 expressed and secreted); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, such as human serum albumin; Müllerian inhibiting substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; chorionic gonadotropin; microbial proteins, such as beta-lactamase; DNA enzymes; inhibins; activins; receptors for hormones or growth factors; 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 factor-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 common 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.

[0300] Examples of polypeptides that may 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 (RANTES), CCL6, CCL7, CCL8, CCL9 (CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, 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, adrenaline, 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 polypeptide, androgen, alpha-glucosidase (also known as acid maltase), glycogen phosphorylase, glycogen debranching enzyme, phosphofructokinase,Phosphoglycerate kinase, phosphoglycerate mutase, lactate dehydrogenase, carnitine palmitoyltransferase, carnitine and myoadenylate deaminase.

[0301] In some embodiments, the polypeptide is a replacement therapy or replacement protein.

[0302] In some embodiments, the replacement therapy or replacement protein is a coagulation factor, such as Factor VII, Factor VIII, or Factor IX.

[0303] 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.

[0304] 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) inability 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) inability to produce insulin in a glucose-responsive manner; or (iii) not derived from an induced pluripotent stem cell engineered or differentiated into a pancreatic beta cell that produces insulin.

[0305] Characteristics of implantable components

[0306] The engineered mammalian cells or a plurality of such cells described herein can be incorporated into an implantable element for use in 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 of the implantable element in a subject.

[0307] 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.

[0308] The device (e.g., particle) may have any configuration and shape suitable for supporting the vigor and productivity of the contained cells after being implanted in the expected target position. As a non-limiting example, the device shape may be cylindrical, rectangular, disc-shaped, oval, star-shaped or spherical. The device may 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.

[0309] 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. Patents 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).

[0310] 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 a separate compartment. WO 2018 / 232027 describes a device having multiple cell-containing compartments formed in a micro-fabricated body and covered by a porous membrane.

[0311] In one embodiment, the device is configured as a thin, flexible strand, as described in U.S. Pat. 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. Cells expressing the protein are located in the outer coating.

[0312] 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 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.

[0313] In some embodiments, the devices of the present disclosure (e.g., particles, capsules) include at least one pore or opening, e.g., to allow materials 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.

[0314] In some embodiments, the device includes a semipermeable, biocompatible membrane that surrounds genetically modified cells encapsulated in a polymer composition (e.g., alginate hydrogel). The membrane pore size is selected to allow oxygen and other molecules important for cell survival and function to move through the semipermeable membrane while preventing immune cells from exiting the pores. In one embodiment, the semipermeable membrane has a molecular weight cut-off of less than 1000 kD or between 50 - 700 kD, between 70 - 300 kD, or between 70 - 150 kD, or between 70 and 130 kD.

[0315] In one embodiment, the device can contain a cell-containing compartment that is surrounded by a barrier compartment formed from a cell-free biocompatible material, such as the core-shell microcapsules described in Ma, M et al., Adv. Healthc Mater., 2(5):667 - 672 (2012). Such a barrier compartment can be used with or without a semipermeable membrane.

[0316] The cells in one or more cell-containing compartments of the devices 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., macrodevice, particle, hydrogel capsule) can also include materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials, and combinations thereof or be formed from such materials. The device can be made entirely of one type of material or can include other materials in the cell-containing compartment and any other compartments.

[0317] In some embodiments, the device comprises a metal or a 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 a metal alloy. Exemplary metals or metal alloys include titanium and titanium group alloys (e.g., nitinol, nickel titanium alloys, 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, such as 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.

[0318] In some embodiments, the device includes ceramics. In one embodiment, one or more compartments (e.g., the first compartment, the second compartment, or all compartments) in the compartments in the device 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 a device containing ceramics, the amount of ceramics (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.

[0319] In some embodiments, the device has two hydrogel compartments, wherein the inner cell-containing compartment is completely surrounded by the second outer (e.g., barrier) compartment. In one embodiment, the inner boundary of the second compartment forms an interface with the outer boundary of the first compartment. In such embodiments, the thickness of the second (outer) compartment means the average distance between the outer boundary of the second compartment and the interface between the two compartments, for example, the average value of the distance measured at each of the thinnest point and the thickest point visually observed in the outer compartment. In some embodiments (e.g., 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 10nm to 1mm, 100nm to 1mm, 500nm to 1 millimeter, 1 micrometer (μm) to 1mm, 1μm to 1mm, 1μm to 500μm, 1μm to 250μm, 1μm to 1mm, 5μm to 500μm, 5μm to 250μm, 10μm to 1mm, 10μm to 500μm or 10μm to 250μm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is 100nm to 1mm, between 1μm and 1mm, between 1μm and 500μm or between 5μm and 1mm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is between about 50μm and about 100μm. 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.

[0320] 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 one or more materials and the compound of Formula (I) in each compartment.

[0321] In some embodiments, one or more polymer compositions containing cell compartments include polysaccharides or other polymers (for example, alginate, hyaluronate or chondroitin) forming hydrogels. In some embodiments, polymer is alginate, which is a polysaccharide made of β-D-mannuronic acid (M) and α-L-guluronic acid (G). In some embodiments, alginate has low molecular weight (for example, <75kD approximate molecular weight) and ≥1.5 G:M ratio, (ii) medium molecular weight alginate, for example, with 75-150kDa approximate molecular weight about and ≥1.5 G:M ratio, (iii) high molecular weight alginate, for example, with 150kDa-250kDa approximate MW and ≥1.5 G:M ratio, (iv) or two or more mixtures in these alginates. In some embodiments, one or more cell compartments further include at least one cell binding material (CBS), such as cell binding peptide (CBP) or cell binding polypeptide (CBPP) described in WO2020069429.

[0322] In some embodiments, one or more cell-containing compartments include alginate covalently modified with a joint-cell binding peptide moiety (e.g., GRGD or GRGDSP). In one embodiment, the cell binding peptide density (such as nitrogen % determined by combustion analysis such as described in WO2020198695) in one or more cell-containing compartments is at least 0.05%, 0.1%, 0.2% or 0.3%, but less than 4%, 3%, 2% or 1%. In one embodiment, the total density of joint-CBP in the cell-containing compartment is about 0.1 to about 1.0 micromolar CBP / g CBP- polymer (e.g., MMW-alginate covalently modified with GRGD or GRGDSP in solution), such as determined by quantitative peptide conjugation, such as the determination described in WO2020198695. In one embodiment, joint-CBP is GRGDSP and alginate has a molecular weight of 75kDa to 150kDa and a G:M ratio greater than or equal to 1.5. In one embodiment, the cell-containing compartment further comprises unmodified alginate having a molecular weight of 75 kDa to 150 kDa and a G:M ratio greater than or equal to 1.5.

[0323] The device may form part of a plurality of substantially identical devices in a formulation (e.g., a composition). In some embodiments, the device (e.g., particle, hydrogel capsule) in the formulation has an average diameter or size between about 0.5 mm and about 8 mm. In some embodiments, the average diameter or size of the device in the formulation is between about 0.5 mm and about 4 mm or between about 0.5 mm and about 2 mm. In some embodiments, the device in the formulation is a two-compartment hydrogel capsule and has an average diameter or size of about 0.7 mm to about 1.3 mm, or about 1.2 mm to about 1.8 mm.

[0324] In some embodiments, the surface of the device comprises a compound capable of reducing FBR after implantation in a subject, such as a defibrotic compound as described herein below. For devices comprising a barrier compartment surrounding a cell-containing compartment, the defibrotic compound can covalently modify a polymer disposed throughout the barrier compartment and optionally throughout the cell-containing compartment.

[0325] In some embodiments, one or more compartments in the device include a defibrotizing polymer, e.g., a defibrotizing compound of formula (I) covalently attached to a polymer. In one embodiment, some or all monomers in the defibrotizing polymer are modified with the same compound of formula (I). In some embodiments, some or all monomers in the defibrotizing polymer are modified with different compounds of formula (I). In some embodiments where the device is a 2-compartment hydrogel capsule, the defibrotizing polymer is present only in the outer barrier compartment.

[0326] One or more compartments in the device may include an unmodified polymer that is the same or different than the polymer in any defibrillating polymer present in the device. In one embodiment, the first compartment, the second compartment, or all compartments in the device include an unmodified polymer.

[0327] Each of the modified polymers and the unmodified polymers in the device can be a linear, branched or cross-linked polymer, or a polymer with a selected molecular weight range, degree of polymerization, viscosity or melt flow rate. Branched polymers can include one or more of the following types: star polymers, comb polymers, brush polymers, dendritic polymers, ladder polymers and dendrimers. The polymer can be a thermosensitive polymer, such as a gel (e.g., a solid or liquid after exposure to heat or a certain temperature) or a photocross-linkable polymer. Exemplary polymers include polystyrene, polyethylene, polypropylene, polyacetylene, poly(vinyl chloride) (PVC), polyolefin copolymers, poly(polyurethane), polyacrylates and polymethacrylates, polyacrylamides and polymethacrylamides, poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), polyesters, polysiloxanes, polydimethylsiloxanes (PDMS), polyethers, poly(orthoesters), poly(carbonates), poly(hydroxyalkanoates), polyfluorocarbons, (polytetrafluoroethylene, PTFE), PEEK, silicone, epoxy resin, (condensation polymer obtained from ethylene glycol and terephthalic acid), polyethylene glycol, nylon, polyolefins, phenolic resins, natural and synthetic elastomers, adhesives and sealants, polyolefins, polysulfones, polyacrylonitrile, biopolymers such as polysaccharides and natural latex, collagen, cellulosic polymers (e.g., alkyl cellulose, etc.), polyethylene glycol and 2-hydroxyethyl methacrylate (HEMA), polysaccharides, poly(glycolic acid), poly(L-lactic acid) (PLLA), poly(lactic-glycolic acid) (PLGA), polydioxanone (PDA), or racemic poly(lactic acid), polycarbonates (e.g., polyamides (e.g., nylon)), fluoroplastics, carbon fibers, agarose, alginate, chitosan, and blends or copolymers thereof. In a polymer-containing device, the amount of polymer (e.g., actual weight of polymer as a % by weight of the device) 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.

[0328] In some embodiments, one or more of the modified polymer and the unmodified polymer in the device comprises polyethylene. Exemplary polyethylenes include ultra low density polyethylene (ULDPE) (e.g., having a density in the range of 0.890 to 0.905 g / cm 3 Very low density polyethylene (VLDPE) (e.g., having a density in the range of 0.905 to 0.915 g / cm 3A polymer having a density of 0.915 to 0.935 g / cm 3 A polymer having a density of about 0.915 to 0.935 g / m 3 medium density polyethylene (MDPE) (e.g., having a density in the range of 0.926 to 0.940 g / cm 3 Polymers with a density of 0.940 to 0.970 g / cm 3 A polymer having a density of , which may or may not contain a comonomer) and polyethylene glycol.

[0329] In some embodiments, one or more of the modified polymer and the unmodified polymer in the device comprises polypropylene. Exemplary polypropylenes include homopolymers, random copolymers (homophase copolymers) and impact copolymers (heterogeneous copolymers), for example, as described in McKeen, Handbook of Polymer Applications in Medicine and Medical Devices, 3-Plastics Used in Medical Devices, (2014): 21-53.

[0330] In some embodiments, one or more of the modified polymer and the unmodified polymer in the device comprises polypropylene.Exemplary polystyrenes include general purpose or crystalline polystyrene (PS or GPPS), high impact polystyrene (HIPS), and syndiotactic polystyrene (SPS).

[0331] In some embodiments, one or more of the modified polymer and the unmodified polymer comprises a thermoplastic elastomer (TPE). Exemplary TPEs include (i) TPA-polyamide TPEs, which comprise block copolymers of alternating hard segments and soft segments, wherein there are amide chemical linkages in the hard blocks and ether and / or ester linkages in the soft blocks; (ii) TPC-copolyester TPEs, which consist of block copolymers of alternating hard segments and soft segments, wherein the chemical linkages in the backbone are esters and / or ethers; (iii) TPO-olefin TPEs, which consist of a blend of polyolefins and conventional rubbers, wherein the rubber phase in the blend has little or no crosslinking; (iv) TPS-styrenic TPEs, which consist of at least a triblock copolymer of styrene and a specific diene, wherein the two end blocks (hard blocks) are polystyrene, and and the interior blocks (soft block(s)) are polydiene or hydrogenated polydiene; (v) TPU - urethane TPE, which consists of block copolymers of alternating hard segments and soft segments with urethane chemical linkages in the hard blocks and ether, ester or carbonate linkages or mixtures thereof in the soft blocks; (vi) TPV - thermoplastic rubber vulcanizate, which consists of a blend of thermoplastic material and conventional rubber, wherein the rubber is crosslinked by a dynamic vulcanization process during the blending and mixing steps; and (vii) TPZ - unclassified TPE, which contains any composition or structure other than those grouped in TPA, TPC, TPO, TPS, TPU and TPV.

[0332] In some embodiments, the unmodified polymer is an unmodified alginate. In some embodiments, alginate is a high guluronic acid (G) alginate, and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more guluronic acid (G). In some embodiments, alginate is a high mannuronic acid (M) alginate, and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more mannuronic acid (M). In some embodiments, the ratio of M:G is about 1. In some embodiments, the ratio of M:G is less than 1. In some embodiments, the ratio of M:G is greater than 1. In one embodiment, unmodified alginate has a molecular weight of 150kDa–250kDa and a G:M ratio of ≥1.5.

[0333] In some embodiments, defibration polymer comprises alginate chemically modified with formula (I) compound. The alginate in defibration polymer can be identical or different with any unmodified alginate present in equipment. In one embodiment, the density (e.g., amount conjugated) of formula (I) compound in defibration alginate is between about 4.0% and about 8.0% nitrogen, between about 5.0% and about 7.0% nitrogen or between about 6.0% and about 7.0% nitrogen (e.g., as determined by combustion analysis for nitrogen percentage). In one embodiment, the amount of compound 101 produces about 0.5% to 2%2% to 4%N, about 4% to 6%N, about 6% to 8% or about 8% to 10%N) N% increase (compared with unmodified alginate), wherein N% is determined by combustion analysis and corresponds to the amount of compound 101 in modified alginate.

[0334] In other embodiments, the density (e.g., concentration) of the compound of Formula (I) (e.g., Compound 101) in the defibrotized alginate is defined as % w / w, for example, as a % of the weight of amine in a solution (e.g., saline) / weight of the defibrotized alginate as determined by a suitable quantitative amine conjugation assay (e.g., by the assay described in WO2020069429), and in certain embodiments, the density of the compound of Formula (I) (e.g., Compound 101) is between about 1.0% w / w and about 3.0% w / w, between about 1.3% w / w and about 2.5% w / w, or between about 1.5% w / w and 2.2% w / w.

[0335] In an alginate-containing device, the amount of modified and unmodified alginate (e.g., actual weight of alginate as a % by weight of the device) 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.

[0336] The alginate in the defibrinated polymer can be chemically modified with a compound of formula (I) using any suitable method known in the art. For example, the alginate carboxylic acid moiety can be activated for coupling with one or more amine-functionalized compounds to obtain an alginate modified with a compound of formula (I). The alginate polymer can be dissolved in water (30 mL / gram of polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 equivalent) and N-methylmorpholine (1 equivalent). A solution of the compound of formula (I) in acetonitrile (0.3 M) can be added to this mixture. The reaction can be warmed to 55 °C and maintained for 16 hours, then cooled to room temperature and gently concentrated by rotary evaporation, and then the residue can be dissolved in, for example, water. The mixture can then be filtered, for example, through a bed of cyanide-modified silica gel (Silicycle), and the filter cake can be washed with water. The resulting solution can then be dialyzed against water (10,000 MWCO membrane) for 24 hours, for example, changing the water twice. The resulting solution can be concentrated, for example, by lyophilization, to obtain the desired chemically modified alginate.

[0337] In one embodiment, the modified polymer described herein can be covalently bound to a photoactive crosslinker. A photoactive crosslinker is a part that is activated when exposed to light. The light can include light of any wavelength from infrared to X-ray energy. In some embodiments, the light includes ultraviolet light (e.g., between 360nm and 400nm, for example, 370nm to 390nm, for example, 380nm to 400nm, for example, 390nm to 400nm). In some embodiments, the light includes visible light (e.g., between 400nm and 700nm). Photoactive crosslinkers often include at least one unsaturated functional group that can undergo free radical polymerization. In one embodiment, a photoactive crosslinker includes an alkenyl group (e.g., C2-C12 alkenyl, C2-C8 alkenyl). In one embodiment, a photoactive crosslinker includes an alkynyl group (e.g., C2-C12 alkynyl, C2-C8 alkynyl). The part that can be activated after exposure to irradiation includes an aromatic group, an alkenyl group, an alkynyl group, and an azide group. Exemplary alkenyl compounds that can serve as photoactive crosslinking agents include alkenyl acids, such as acrylates, methacrylates, acrylamides and methacrylamides and their corresponding acyl chlorides and anhydrides. Other exemplary alkenyl compounds include enols (e.g., 2-propylene-1-ol), alkenyl halides (such as allyl chloride, etc.), organometallic alkenyl compounds (such as vinyl magnesium bromide), aryl compounds (e.g., styrene). Exemplary photoactive crosslinking agents include acrylates, methacrylates, ethylene glycol dimethacrylate, divinylbenzene, 1,3-diisopropylbenzene and N,N'-methylenebisacrylamide. In one embodiment, the photoactive crosslinking agent is a bifunctional crosslinking agent, i.e., has two reactive functional groups. In one embodiment, the photoactive covalent crosslinking agent has alkenyl and amide functional groups. In one embodiment, the photoactive crosslinking agent has alkenyl and carboxylate functional groups. In one embodiment, the photoactive crosslinking agent has alkenyl and amide functional groups.

[0338] In one embodiment, the modified polymer described herein comprises a photoactive crosslinker having the structure of Formula (IV):

[0339] or a pharmaceutically acceptable salt or tautomer thereof, wherein X 1 Does not exist, O, NR 33 or C(R 34a )(R 34b );R 30a , R 30b , R 31 , R 32 , R 33 , R 34a and R 34bEach of the above is independently hydrogen, 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 , cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; and each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl.

[0340] In one embodiment, X 1 For O, R 30a , R 30b , R 31 and R 32 Each of is hydrogen, and R 32 is a heteroalkyl group (e.g., propylamine, e.g., -CH 2 CH 2 CH 2 NH 2 In one embodiment, X 1 For O, R 30a , R 30b , R 31 and R 32 Each of is hydrogen, and R 32 is a heteroalkyl group (e.g., ethylamine, e.g., -CH 2 CH 2 NH 2 In one embodiment, the photoactive crosslinker of formula (IV) is a methacrylate. 1 Does not exist; R 32 is a halogen group (eg, a chloro group); and R 30a , R 30b and R 31Each of is hydrogen. In one embodiment, the photoactive cross-linking agent of formula (IV) is acryloyl chloride.

[0341] In one embodiment, X 1 NR 33 (e.g., NH), and R 30a , R 30b , R 31 and R 32 Each of is hydrogen. In one embodiment, the photoactive cross-linker of formula (IV) is acrylamide.

[0342] In one embodiment, the modified polymer described herein comprises a photoactive crosslinker having the structure of Formula (IV-a):

[0343] or a pharmaceutically acceptable salt or tautomer thereof, wherein R 30a , R 30b , R 31 , R 32 and R 35 Each of the above is independently hydrogen, 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 , cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; and each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl.

[0344] In one embodiment, the modified polymer described herein comprises a photoactive cross-linker having the structure of formula (IV-b):

[0345] or a pharmaceutically acceptable salt or tautomer thereof, wherein R 30a , R 30b , R 31 , R 32 , R 36a and R 36b Each of the above is independently hydrogen, 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 , cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; each R 7 R is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; 35 is hydrogen, alkyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl; and n is 1, 2, 3, 4, 5 or 6.

[0346] In one embodiment, the modified polymer described herein comprises a photoactive cross-linker having the structure of formula (IV-c):

[0347] or a pharmaceutically acceptable salt or tautomer thereof, wherein R 30a , R 30b and R 31 Each of the above is independently hydrogen, 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)RB1 , –C(O)N(R C1 ), SR E1 , cycloalkyl, heterocyclyl, aryl or heteroaryl; R 32 is alkyl, alkenyl, alkynyl, heteroalkyl, –C(O)OR A1 , –C(O)R B1 , cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; and each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl.

[0348] In one embodiment, the modified polymer described herein comprises a photoactive cross-linker having the structure of formula (IV-d):

[0349] or a pharmaceutically acceptable salt or tautomer thereof, wherein R 30a , R 30b , R 31 , R 32 , R 36a and R 36b Each of the above is independently hydrogen, 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 , cycloalkyl, heterocyclyl, aryl or heteroaryl; R 32 is alkyl, alkenyl, alkynyl, heteroalkyl, –C(O)OR A1 , –C(O)R B1 , cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; each R 7 R is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; and n is 1, 2, 3, 4, 5 or 6.

[0350] Photoactive crosslinking agents can be used alone or preferably in the presence of a photoinitiator. As used herein, "photoinitiator" refers to a molecule that can absorb radiation (e.g., light, such as photons) and form an excited state of an active species. Various free radical initiators that can be readily identified by those skilled in the art can be employed in the practice of the present invention. In one embodiment, the photoinitiator is an ultraviolet (UV) photoinitiator. Exemplary UV photoinitiators include lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), camphorquinone, benzoin methyl ether, 1-hydroxy-cyclohexyl-phenyl-ketone (i.e., Irgacure 184), 2-hydroxy-2-methyl-1-phenyl-1-propanone (i.e., Darocur 1173), 2-hydroxy-1-[4-(2-hydroxyethoxy)phenyl]-2-methylpropan-1-one (i.e., Irgacure 2959), 2-benzyl-2-(dimethylamino)-1-(4-morpholin-4-ylphenyl)butan-1-one (i.e., Irgacure 369), 2-methyl-1-(4-methylthiophenyl)-2-morpholin-4-ylpropan-1-one (i.e., Irgacure 907), diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (e.g., Darocur The invention relates to a photoinitiator for the polymerization of visible light. The photoinitiator for the polymerization of visible light includes 2-(2,4,5,7-tetrabromo-3-hydroxy-6-oxoxanthene-9-yl)benzoic acid (i.e., eosin Y), erythrosine, riboflavin, rose Bengal, methylene blue and thionine. A small amount of comonomer can be optionally added to the crosslinking reaction to increase the polymerization rate. Examples of suitable comonomers include vinyl pyrrolidone, acrylamide, methacrylamide, acrylic acid, methacrylic acid, sodium acrylate, sodium methacrylate, hydroxyethyl acrylate, hydroxyethyl methacrylate (HEMA), ethylene glycol diacrylate, ethylene glycol dimethacrylate, pentaerythritol triacrylate, pentaerythritol trimethacrylate, trimethylolpropane triacrylate, trimethylolpropane trimethacrylate, tripropylene glycol diacrylate, tripropylene glycol dimethacrylate, glyceryl acrylate, glyceryl methacrylate, etc. In some embodiments, the photoinitiator is a thermally activated photoinitiator.

[0351] The photoactive crosslinking agent can be used in the presence of a single photoinitiator or a plurality of photoinitiators. The plurality of photoinitiators can include 2, 3, 4, 5, 6, 7, 8 or more photoinitiators. In one embodiment, the covalent crosslinking moiety is present on the polysaccharide polymer at a density of at least 1%, for example 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10% or more, for example as determined by LC-UV assay.

[0352] The photoactive cross-linking agent can be covalently bound to a polysaccharide (e.g., alginate). The modified polysaccharide polymer (e.g., modified alginate polymer) may be cross-linked with another polymer. In one embodiment, the polysaccharide polymer is modified by more than one type of photoactive cross-linking agent.

[0353] In one embodiment, the modified polysaccharide is a compound of formula (V):

[0354] or a pharmaceutically acceptable salt or tautomer thereof, wherein each of T and U is independently C(R 40 )(R 41 ), O or N (R 42 );R 38a , R 38b , R 39a , R 39b , R 40 , R 41 and R 42 Each of the above is independently hydrogen, 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 , cycloalkyl, heterocyclyl, aryl or heteroaryl; R 32 and R 35 Each of is hydrogen, alkyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl or heterocyclic; and the photoactive crosslinker has a structure of formula (IV), (IV-a), (IV-b), (IV-c) or (IV-d).

[0355] In one embodiment, the photoactive crosslinker of formula (V) has the structure of formula (Va):

[0356] or a pharmaceutically acceptable salt or tautomer thereof, wherein each of T and U is independently C(R 40 )(R 41 ), O or N (R 42 );R 30a , R 30b , R 31 , R 32 , R 38a , R 38b , R 39a , R 39b , R 40 , R 41 and R 42 Each of the above is independently hydrogen, 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 , cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; and each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl.

[0357] In one embodiment, the modified polymer described herein has the structure of Formula (Vb):

[0358] or a pharmaceutically acceptable salt or tautomer thereof, wherein each of U and T is independently C(R 40 )(R 41 ), O or N (R 42 );R 30a , R30b , R 31 , R 35 , R 38a , R 38b , R 39a , R 39b , R 40 , R 41 , R 42 , R 43a and R 43b Each of the above is independently hydrogen, 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 , cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; each R 7 R is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; and n is 1, 2, 3, 4, 5 or 6.

[0359] In one embodiment, the modified polymer described herein has the structure of Formula (Vc):

[0360] or a pharmaceutically acceptable salt or tautomer thereof, wherein U is C(R 40 )(R 41 ), O or N (R 42 );R 30a , R 30b , R 31 , R 35 , R 38a , R 38b , R 39a , R 39b , R 40 , R 41 , R 42 , R43a , R 43b and R 44 Each of the above is independently hydrogen, 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 , cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; and each R 7 R is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; and n is 1, 2, 3, 4, 5 or 6.

[0361] In one embodiment, the modified polymer described herein has the structure of Formula (Vd):

[0362] or a pharmaceutically acceptable salt or tautomer thereof, wherein U is C(R 40 )(R 41 ), O or N (R 42 );R 30a , R 30b , R 31 , R 38a , R 38b , R 39a , R 39b , R 40 , R 41 , R 42 , R 43a and R 43b Each of the above is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , –C(O)OR A1 , –C(O)R B1 、–OC(O)R B1 , –N(RC1 )(R D1 ),–N(R C1 )C(O)R B1 , –C(O)N(R C1 ), SR E1 , cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; each R 7 R is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; and n is 1, 2, 3, 4, 5 or 6.

[0363] In one embodiment, the modified polymer described herein comprises the structure of Formula (VI):

[0364] or a pharmaceutically acceptable salt or tautomer thereof, wherein W, T 1 , T 2 , U 1 and U 2 Each of which is independently C(R 40 )(R 41 ), O or N (R 42 );R 38a , R 38b , R 38c , R 38d R 39a , R 39b , R 39a , R 39b , R 40 , R 41 and R 42 Each of the above is independently hydrogen, 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, cycloalkyl, heterocyclyl, aryl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; p is an integer from 1 to 100; afibromer has the structure of formula (I) or a subtype of formula (I) as described herein.

[0365] In one embodiment, the device includes at least one cell-containing compartment, and in some embodiments contains two, three, four or more cell-containing compartments. In one embodiment, each cell-containing compartment includes a plurality of cells (e.g., living cells), and when the device is implanted in a subject, the cells in at least one of these compartments are capable of expressing and secreting a mammalian protein.

[0366] In one embodiment, all cells in a cell-containing compartment are derived from a single parent cell type or a mixture of at least two different parent cell types. In one embodiment, all cells in a cell-containing compartment are derived from the same parent cell type. In a device having two or more cell-containing compartments, the cells and the one or more proteins produced thereby may be the same or different in each cell-containing compartment. In some embodiments, all cell-containing compartments are surrounded by a single barrier compartment. In some embodiments, the barrier compartment is substantially free of cells.

[0367] In one embodiment, cells to be incorporated into a device described herein (e.g., a hydrogel capsule) are prepared in the form of a cell suspension prior to being encapsulated in the device. The cells in the suspension may be in the form of a single cell (e.g., from a monolayer cell culture), or provided in another form, such as disposed on a microcarrier (e.g., a bead or matrix) or as a three-dimensional aggregate of cells (e.g., a cell cluster or spheroid). The cell suspension may contain a plurality of cell clusters (e.g., as spheroids) or microcarriers.

[0368] In one embodiment, the implantable element described herein includes a plurality of engineered mammalian cells (e.g., engineered ARPE-19 cells), which are, for example, at a specific cell density. For example, the implantable element can include more than 1 million cells / mL, 2.5 million cells / mL, 5 million cells / mL, 7.5 million cells / mL, 10 million cells / mL, 15 million cells / mL, 20 million cells / mL, 25 million cells / mL, 30 million cells / mL, 40 million cells / mL, 50 million cells / mL or more engineered mammalian cells. In one embodiment, the implantable element includes a plurality of engineered ARPE-19 cells capable of expressing proteins (e.g., hormones, blood coagulation factors, antibodies or enzymes) at a cell density of 1-5 million cells / mL, 5-10 million cells / mL, or 10-20 million cells / mL. In one embodiment, the implantable element comprises a plurality of engineered ARPE-19 cells capable of expressing a protein (e.g., insulin) at a cell density of 1-5 million cells / ml, 5-10 million cells / ml, or 10-20 million cells / ml. In one embodiment, the implantable element comprises a plurality of engineered ARPE-19 cells capable of expressing a protein (e.g., IDUA) at a cell density of 1-5 million cells / ml, 5-10 million cells / ml, or 10-20 million cells / ml. In one embodiment, the implantable element comprises a plurality of engineered ARPE-19 cells capable of expressing a protein (e.g., insulin) at a cell density of 1-5 million cells / ml, 5-10 million cells / ml, or 10-20 million cells / ml. In one embodiment, the implantable element comprises a plurality of engineered ARPE-19 cells capable of expressing a protein (eg, insulin) at a cell density of between 1-5 million cells / ml, 5-10 million cells / ml, or 10-20 million cells / ml.

[0369] The device (e.g., capsule, particle) may contain one or more exogenous agents that are not expressed by the cell, and may contain, for example, nucleic acids (e.g., RNA or DNA molecules), proteins (e.g., hormones, enzymes (e.g., glucose oxidase, kinase, phosphatase, oxygenase, hydrogenase, reductase), antibodies, antibody fragments, antigens or epitopes)), active or inactive fragments of proteins or polypeptides, small molecules, or drugs. In one embodiment, the device is configured to release such exogenous agents.

[0370] In one embodiment, the implantable elements described herein, when implanted into a mammalian host, result in a lesser amount of pericranial fibrotic overgrowth (PFO) than implantation of 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 complexes). In one embodiment, the implantable elements described herein comprise engineered mammalian cells that are capable of expressing a therapeutic agent within at least any one of two months, three months, four months, or more after the implantable element is implanted into a mammalian subject. In one embodiment, the implantable elements described herein comprise engineered mammalian cells that express a therapeutic agent detectable in the plasma of a mammalian subject within at least any one of two months, three months, four months, or more after the implantable element is implanted into the subject.

[0371] Defibrillating (e.g., FBR-mitigating) compounds

[0372] In some embodiments, the devices described herein include at least one compound of formula (I):

[0373]

[0374] or a pharmaceutically acceptable salt thereof, wherein:

[0375] A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, –O–, –C(O)O–, –C(O)–, –OC(O)–, –N(R C )–、–N(R C )C(O)–,–C(O)N(R C )–、-N(R C )C(O)(C 1 -C 6 -alkylene)–, -N(R C )C(O)(C 1 -C 6 -alkenylene)–, –N(R C )N(R D )–, –NCN–, –C(=N(R C )(R D ))O–, –S–, –S(O) x –, –OS(O) x –、–N(R C )S(O) x –, –S(O) x N(R C )–,–P(R F ) y –、–Si(ORA ) 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;

[0376] L 1 and L 3 Each of the R is independently a bond, an alkyl group or a heteroalkyl group, wherein each alkyl group and heteroalkyl group is optionally substituted by one or more R 2 replace;

[0377] L 2 is the key;

[0378] M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally replaced by one or more R 3 replace;

[0379] P is absent, cycloalkyl, heterocyclyl or heteroaryl, each of which is optionally replaced by one or more R 4 replace;

[0380] 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 substituted by one or more R 5 replace;

[0381] 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 substituted by one or more R 6 replace;

[0382] or R C and R DTogether with the nitrogen atom to which they are attached, they form a 6 substituted rings (e.g., 5-7 membered rings);

[0383] 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;

[0384] 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 substituted by one or more R 7 replace;

[0385] Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl;

[0386] x is 1 or 2; and

[0387] y is 2, 3 or 4.

[0388] In some embodiments, the compound of formula (I) is a compound of formula (I-a):

[0389]

[0390] or a pharmaceutically acceptable salt thereof, wherein:

[0391] A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocycloalkyl, 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 )N(R D )-, N(R C )C(O)(C 1 -C 6 -alkylene)-, -N(R C )C(O)(C 1 -C 6 -alkenylene)-, -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 attached to an attachment group (e.g., an attachment group as described herein) and is optionally substituted with one or more R 1 ;

[0392] L 1 and L 3 each independently is a bond, alkyl or heteroalkyl, each alkyl and heteroalkyl being optionally substituted with one or more R 2 ;

[0393] L 2 is a bond;

[0394] M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally replaced by one or more R 3 replace;

[0395] P is optionally replaced by one or more R 4 substituted heteroaryl;

[0396] Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 replace;

[0397] 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 substituted by one or more R 6 replace;

[0398] 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);

[0399] 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 substituted by one or more R 7 replace;

[0400] 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 substituted by one or more R 7 replace;

[0401] Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl;

[0402] x is 1 or 2; and

[0403] y is 2, 3, or 4.

[0404] In some embodiments, for Formula (I) and (Ia), A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )C(O)-、-N(R C )C(O)(C 1 -C 6 -alkylene)-, -N(R C )C(O)(C 1 -C 6 -alkenylene)-or-N(R C )-. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C )-. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C )-. In some embodiments, A is alkyl, -O-, -C(O)O-, -C(O)-, -OC(O), or -N(R C)-. In some embodiments, A is -N(R C )C(O)-、-N(R C )C(O)(C 1 -C 6 -alkylene)- or -N(R C )C(O)(C 1 -C 6 -alkenylene)-. In some embodiments, A is -N(R C )-. In some embodiments, A is -N(R C )-, and R C and R D is independently hydrogen or alkyl. In some embodiments, A is -NH-. In some embodiments, A is -N(R C )C(O)(C 1 -C 6 -alkylene)-, wherein the alkylene is R 1 In some embodiments, A is -N(R C )C(O)(C 1 -C 6 -alkylene)-, and R 1 In some embodiments, A is -NHC(O)C(CH 3 ) 2 -. In some embodiments, A is -N(R C )C(O)(methylene)-, and R 1 is alkyl (eg, methyl). In some embodiments, A is -NHC(O)CH(CH 3 )-. In some embodiments, A is -NHC(O)C(CH 3 )-.

[0405] In some embodiments, for Formula (I) and (Ia), L 1 is a bond, alkyl or heteroalkyl. 1 is a bond or an alkyl group. In some embodiments, L 1 is a bond. In some embodiments, L 1 In some embodiments, L 1 It is C 1 -C 6 In some embodiments, L 1 Yes-CH 2 -、-CH(CH 3 )-、-CH 2 CH 2 CH 2 or -CH 2 CH2 -. In some embodiments, L 1 Yes-CH 2 -or-CH 2 CH 2 -.

[0406] In some embodiments, for Formula (I) and (Ia), L 3 is a bond, alkyl or heteroalkyl. 3 is a bond. In some embodiments, L 3 In some embodiments, L 3 It is C 1 -C 12 In some embodiments, L 3 It is C 1 -C 6 In some embodiments, L 3 Yes-CH 2 -. In some embodiments, L 3 In some embodiments, L 3 is optionally replaced by one or more R 2 (e.g., oxo) substituted C 1 -C 12 In some embodiments, L 3 is optionally replaced by one or more R 2 (e.g., oxo) substituted C 1 -C 6 In some embodiments, L 3 Yes -C(O)OCH 2 -、-CH 2 (OCH 2 CH 2 ) 2 -、-CH 2 (OCH 2 CH 2 ) 3 -、CH 2 CH 2 O- or -CH 2 In some embodiments, L 3 Yes-CH 2 O-.

[0407] In some embodiments, for Formula (I) and (Ia), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, for Formula (I) and (Ia), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, M is heteroalkyl, aryl, or heteroaryl. In some embodiments, M is absent. In some embodiments, M is alkyl (e.g., C 1 -C 6 In some embodiments, M is -CH 2 -. In some embodiments, M is heteroalkyl (e.g., C 1 -C 6 In some embodiments, M is (—OCH 2 CH 2 -) z , wherein z is an integer selected from 1 to 10. In some embodiments, z is an integer selected from 1 to 5. In some embodiments, M is -(OCH 2 ) 2 -、(-OCH 2 CH 2- ) 2 、(-OCH 2 CH 2- ) 3 、(-OCH 2 CH 2 -) 4 or (-OCH 2 CH 25 In some embodiments, M is -OCH 2 CH 2 -、(-OCH 2 CH 2 -) 2 、(-OCH 2 CH 2 -) 3 or (-OCH 2 In some embodiments, M is -(-OCH 2 -) 3 In some embodiments, M is aryl. In some embodiments, M is phenyl. In some embodiments, M is unsubstituted phenyl. In some embodiments, M is In some embodiments, M is In some embodiments, M is replaced by 1-4 R 3 (For example, 1 R 3 ) substituted phenyl. In some embodiments, R 3 It is CF 3 .

[0408] In some embodiments, for formula (I) and (Ia), P is absent, heterocyclyl, or heteroaryl. In some embodiments, for formula (I) and (Ia), P is absent, heterocyclyl, or heteroaryl. In some embodiments, P is absent. In some embodiments, for formula (I) and (Ia), P is a tricyclic, bicyclic, or monocyclic heteroaryl. In some embodiments, P is a monocyclic heteroaryl. In some embodiments, P is a nitrogen-containing heteroaryl. In some embodiments, P is a monocyclic nitrogen-containing heteroaryl. In some embodiments, P is a 5-membered heteroaryl. In some embodiments, P is a 5-membered nitrogen-containing heteroaryl. In some embodiments, P is a tetrazolyl, imidazolyl, pyrazolyl, or triazolyl or pyrrolyl. In some embodiments, P is imidazolyl. In some embodiments, P is 1,2,3-triazolyl. In some embodiments, P is In some embodiments, P is In some embodiments, P is

[0409] In some embodiments, P is heterocyclyl. In some embodiments, P is heterocyclyl. In some embodiments, P is 5-membered heterocyclyl. In some embodiments, P is imidazolidinone. In some embodiments, P is In some embodiments, P is thiomorpholinyl-1,1-dioxide. In some embodiments, P is

[0410] In some embodiments, for formula (I) and (Ia), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl. In some embodiments, for formula (I) and (Ia), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl. In some embodiments, Z is heterocyclyl. In some embodiments, Z is monocyclic or bicyclic heterocyclyl, 5-membered heterocyclyl or 6-membered heterocyclyl. In some embodiments, Z is 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is In some embodiments, Z is a 4-membered oxygen-containing heterocyclic group. In some embodiments, Z is

[0411] In some embodiments, Z is a bicyclic oxygen-containing heterocyclic group. In some embodiments, Z is a bicyclic oxygen-containing heterocyclic group. In some embodiments, Z is a phthalic anhydride group. In some embodiments, Z is a sulfur-containing heterocyclic group. In some embodiments, Z is a 6-membered sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered heterocyclic group containing a nitrogen atom and a sulfur atom. In some embodiments, Z is thiomorpholinyl-1,1-dioxyl. In some embodiments, Z is In some embodiments, Z is a nitrogen-containing heterocyclic group. In some embodiments, Z is a 6-membered nitrogen-containing heterocyclic group. In some embodiments, Z is

[0412] In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is optionally substituted with one or more R 5 In some embodiments, Z is 2-oxa-7-azaspiro[3.5]nonyl. In some embodiments, Z is In some embodiments, Z is 1-oxa-3,8-diazaspiro[4.5]decan-2-one. In some embodiments, Z is

[0413] In some embodiments, for Formula (I) and (Ia), Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is (e.g., with 1 R 5 ) is a monosubstituted phenyl. In some embodiments, Z is a monosubstituted phenyl, wherein 1 R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 Yes NH 2 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 OCH 3 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 In some embodiments, Z is a monosubstituted phenyl group, wherein one R 5 Located in the opposite position.

[0414] In some embodiments, for Formula (I) and (Ia), Z is alkyl. In some embodiments, Z is C 1 -C 12 In some embodiments, Z is C 1 -C 10 In some embodiments, Z is C 1 -C8 In some embodiments, Z is substituted with 1-5 R 5 Substituted C 1 -C 8 In some embodiments, Z is replaced by 1 R 5 Substituted C 1 -C 8 In some embodiments, Z is replaced by 1 R 5 Substituted C 1 -C 8 Alkyl, where R 5 is alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 、-C(O)R B1 、-OC(O)R B1 or -N(R C1 )(R D1 In some embodiments, Z is replaced by 1 R 5 Substituted C 1 -C 8 Alkyl, where R 5 Yes-OR A1 or-C(O)OR A1 In some embodiments, Z is replaced by 1 R 5 Substituted C 1 -C 8 Alkyl, where R 5 Yes-OR A1 or -C(O)OH. In some embodiments, Z is -CH 3 .

[0415] In some embodiments, for Formula (I) and (Ia), Z is heteroalkyl. In some embodiments, Z is C 1 -C 12 In some embodiments, Z is C 1 -C 10 In some embodiments, Z is C 1 -C 8 In some embodiments, Z is C 1 -C 6 In some embodiments, Z is optionally replaced by one or more R 5 In some embodiments, Z is substituted with 1-5 R 5 Substituted nitrogen and sulfur containing heteroalkyl. In some embodiments, Z is N-methyl-2-(methylsulfonyl)eth-1-amino free radical (aminyl).

[0416] In some embodiments, Z is -ORA or-C(O)OR A In some embodiments, Z is -OR A (e.g., -OH or -OCH 3 In some embodiments, Z is -OCH 3 In some embodiments, Z is -C(O)OR A (e.g., -C(O)OH).

[0417] In some embodiments, Z is hydrogen.

[0418] In some embodiments, L 2 is a key and P and L 3 In some embodiments, L 2 is a bond, P is a heteroaryl, L 3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl.

[0419] In some embodiments, the compound of formula (I) is a compound of formula (Ib):

[0420]

[0421] or a pharmaceutically acceptable salt thereof, wherein ring M 1 is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1-5 R 3 Substitution; Ring Z 1 is optionally replaced by 1-5 R 5 Substituted cycloalkyl, heterocyclyl, aryl or heteroaryl; R 2a , R 2b , R 2c and R 2d Each of is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl, or R 2a and R 2b or R 2c and R 2d Each of the groups together form an oxo group; X is absent, N(R 10 )(R 11 ), O or S; R C is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally substituted by 1-6 R 6 Replace; each R 3 , R 5 and R 6are 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 , cycloalkyl, heterocyclyl, aryl or heteroaryl; R 10 and R 11 Each of the above is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 、-C(O)R B1 、-OC(O)R B1 、-C(O)N(R C1 ), cycloalkyl, heterocyclyl or heteroaryl; each R A1 , R B1 , R C1 , R D1 and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted by 1-6 R 7 Replace; each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; each m and n are independently 1, 2, 3, 4, 5 or 6; and Refers to a connection to an attachment group or polymer described herein. In some embodiments, for each R 3 and R 5 , each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl is optionally and independently substituted with halogen, oxo, cyano, cycloalkyl or heterocyclyl.

[0422] In some embodiments, the compound of formula (Ib) is a compound of formula (Ibi):

[0423]

[0424] or a pharmaceutically acceptable salt thereof, wherein ring M 2 is optionally replaced by one or more R 3 Substituted aryl or heteroaryl; Ring Z 2 is a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2a , R2b , R 2c and R 2d each independently is hydrogen, alkyl or heteroalkyl, or R 2a and R 2b or R 2c and R 2d each together form an oxo group; X is absent, O or S; each R 3 and R 5 are each independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; or two R 5 together form a 5- to 6-membered ring fused to ring Z 2 ; each R A1 and R B1 are independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4, 5 or 6; and refers to a connection to an attachment group or polymer described herein.

[0425] In some embodiments, the compound of formula (I-b-i) is a compound of formula (I-b-ii):

[0426]

[0427] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclic, aryl or heteroaryl; each of R 2c and R 2d independently is hydrogen, alkyl or heteroalkyl, or R 2c and R together form an oxo group; each of R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; each of R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; each of p and q is independently 0, 1, 2, 3, 4, 5 or 6; and refers to a connection to an attachment group or polymer described herein.

[0428] In some embodiments, the compound of formula (I) is a compound of formula (I-c):

[0429]

[0430] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2c and R 2d Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2c and R 2d together to form an oxo group; each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR, -C(O)OR or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; each R A1 and R B1 are each independently hydrogen, alkyl or heteroalkyl; m is 1, 2, 3, 4, 5 or 6; each of p and q is independently 0, 1, 2, 3, 4, 5 or 6; and Refers to linkage to an attachment group or polymer as described herein.

[0431] In some embodiments, the compound of formula (I) is a compound of formula (Id):

[0432]

[0433] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, O or S; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b or R 2c and R 2d Each of the R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 , wherein each alkyl and heteroalkyl is optionally substituted with halogen; each R A1 and R are each independently hydrogen, alkyl or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4, 5 or 6; and Refers to linkage to an attachment group or polymer as described herein.

[0434] In some embodiments, the compound of formula (I) is a compound of formula (Ie):

[0435]

[0436] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, O or S; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b or R 2c and R 2d Each of the R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; Each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4, 5 or 6; and Refers to linkage to an attachment group or polymer as described herein.

[0437] In some embodiments, the compound of formula (I) is a compound of formula (If):

[0438]

[0439] or a pharmaceutically acceptable salt thereof, wherein M is optionally replaced by one or more R 3 substituted alkyl; ring P is optionally substituted with one or more R 4 Substituted heteroaryl; L 3 is optionally replaced by one or more R 2 substituted alkyl or heteroalkyl; Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R 5 Replacement; R 2a and R 2b Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b together to form an oxo group; each R 2 , R 3 , R 4 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; Each RA1 and R B1 is independently hydrogen, alkyl or heteroalkyl; n is independently 1, 2, 3, 4, 5 or 6; and Refers to linkage to an attachment group or polymer as described herein.

[0440] In some embodiments, the compound of formula (I) is a compound of formula (II):

[0441]

[0442] or a pharmaceutically acceptable salt thereof, wherein M is a bond, an alkyl group or an aryl group, wherein the alkyl group and the aryl group are optionally replaced by one or more R 3 Replacement; L 3 is optionally replaced by one or more R 2 substituted alkyl or heteroalkyl; Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl or -OR, wherein alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl are optionally substituted by one or more R 5 Replacement; R A is hydrogen; R 2a and R 2b Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R 2b together to form an oxo group; each R 2 , R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 or -C(O)R B1 ; Each R A1 and R B1 are independently hydrogen, alkyl or heteroalkyl; n is independently 1, 2, 3, 4, 5 or 6; and Refers to linkage to an attachment group or polymer as described herein.

[0443] In some embodiments, the compound of formula (II) is a compound of formula (II-a):

[0444]

[0445] or a pharmaceutically acceptable salt thereof, wherein L 3 is alkyl or heteroalkyl, each of which is optionally substituted by one or more R 2 substituted; Z is hydrogen, alkyl, heteroalkyl or -OR A , heteroalkyl is optionally replaced by one or more R 5 Replacement; R 2a and R 2b Each of R is independently hydrogen, alkyl or heteroalkyl, or R 2a and R2b Each of the R 2 , R 3 and R 5 are independently heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 ; R A is hydrogen; each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; n is independently 1, 2, 3, 4, 5 or 6; and Refers to linkage to an attachment group or polymer as described herein.

[0446] In some embodiments, the compound of formula (I) is a compound of formula (III):

[0447]

[0448] or a pharmaceutically acceptable salt thereof, wherein Z 1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1-5 R 5 Replacement; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl; or R 2a and R 2b or R 2c and R 2d Each of the R C is hydrogen, alkyl, alkenyl, alkynyl or heteroalkyl, wherein each of the alkyl, alkenyl, alkynyl or heteroalkyl is optionally substituted by 1-6 R 6 Replacement; R 3 , R 5 and R 6 Each of the following is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; Each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; q is an integer from 0 to 25; and Refers to linkage to an attachment group or polymer as described herein.

[0449] In some embodiments, the compound of formula (III) is a compound of formula (III-a):

[0450]

[0451] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is a cycloalkyl group, a heterocyclyl group, an aryl group or a heteroaryl group; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, heteroalkyl, halo; or 2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 and R 5 Each of is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , –C(O)OR A1 or –C(O)R B1 ; Each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; o and p are each independently 0, 1, 2, 3, 4 or 5; q is an integer from 0 to 25; and Refers to linkage to an attachment group or polymer as described herein.

[0452] In some embodiments, the compound of formula (III-a) is a compound of formula (III-b):

[0453]

[0454] or a pharmaceutically acceptable salt thereof, wherein ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1 to 5 R 5 Replacement; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, heteroalkyl, halo; or 2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 and R 5 Each of the following is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; Each R A1 and R B1is independently hydrogen, alkyl or heteroalkyl; m and n are 1, 2, 3, 4, 5 or 6; o and p are each independently 0, 1, 2, 3, 4 or 5; q is an integer from 0 to 25; and Refers to linkage to an attachment group or polymer as described herein.

[0455] In some embodiments, the compound of formula (III-a) is a compound of formula (III-c):

[0456]

[0457] or a pharmaceutically acceptable salt thereof, wherein X is C(R')(R"), N(R') or S(O) x ; R' and R" are each independently hydrogen, alkyl, halogen or cycloalkyl; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, heteroalkyl or halo; or 2a and R 2b or R 2c and R 2d Together they form an oxo group; R 3 and R 5 Each of the following is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; Each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4; q is an integer from 0 to 25; x is 0, 1 or 2; and Refers to linkage to an attachment group or polymer as described herein.

[0458] In some embodiments, the compound of formula (III-c) is a compound of formula (III-d):

[0459]

[0460] or a pharmaceutically acceptable salt thereof, wherein X is C(R')(R"), N(R') or S(O) x ; R' and R" are each independently hydrogen, alkyl, halogen or cycloalkyl; R 2a , R 2b , R 2c and R 2d Each of R is independently hydrogen, alkyl, heteroalkyl or halo; or 2a and R 2b or R2c and R 2d Together they form an oxo group; R 3 and R 5 Each of the following is independently alkyl, heteroalkyl, halogen, oxo, -OR A1 、-C(O)OR A1 or -C(O)R B1 ; Each R A1 and R B1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4; q is an integer from 0 to 25; x is 0, 1 or 2; and Refers to linkage to an attachment group or polymer as described herein.

[0461] In some embodiments, the compound is a compound of formula (I). 2 is a key and P and L 3 Does not exist independently.

[0462] In some embodiments, the compound is a compound of formula (Ia). In some embodiments of formula (II-a), L 2 is a bond, P is a heteroaryl, L 3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl. 2 is a key and P and L 3 In some embodiments, L 2 is a bond, P is a heteroaryl, L 3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L 3 is heteroalkyl, and Z is alkyl.

[0463] In some embodiments, the compound is a compound of formula (Ib). In some embodiments, P is absent, L 1 Yes-NHCH 2 , L 2 is a bond, M is an aryl group (e.g., phenyl), L 3 Yes-CH 2 O, and Z is a heterocyclyl (eg, a nitrogen-containing heterocyclyl such as thiomorpholinyl-1,1-dioxide). In some embodiments, the compound of Formula (Ib) is Compound 116.

[0464] In some embodiments of Formula (Ib), P is absent, L 1 Formula -NHCH 2 , L 2 is a key, M does not exist, L3 is a key, and Z is a heterocyclic group (e.g., an oxygen-containing heterocyclic group such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of formula (I-b) is compound 105.

[0465] In some embodiments, the compound is a compound of formula (I-b-i). In some embodiments of formula (I-b-i), R 2a and R 2b each independently is hydrogen or CH 3 , R 2c and R 2d each independently is hydrogen, m is 1 or 2, n is 1, X is O, p is 0, M 2 is a phenyl optionally substituted with one or more R 3 , R 3 is -CF 3 , and Z 2 is a heterocyclic group (e.g., an oxygen-containing heterocyclic group such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of formula (I-b-i) is compound 100, compound 106, compound 107, compound 108, compound 109, or compound 111.

[0466] In some embodiments, the compound is a compound of formula (I-b-ii). In some embodiments of formula (I-b-ii), R 2a , R 2b , R 2c and R 2d each independently is hydrogen, q is 0, p is 0, m is 1, and Z 2 is a heterocyclic group (e.g., an oxygen-containing heterocyclic group such as tetrahydropyranyl). In some embodiments, the compound of formula (I-b-ii) is compound 100.

[0467] In some embodiments, the compound is a compound of formula (I-c). In some embodiments of formula (I-c), R 2c and R 2d each independently is hydrogen, m is 1, p is 1, q is 0, R 5 is -CH 3 , and Z is a heterocyclic group (e.g., a nitrogen-containing heterocyclic group such as piperazinyl). In some embodiments, the compound of formula (I-c) is compound 113.

[0468] In some embodiments, the compound is a compound of formula (I-d). In some embodiments of formula (I-d), R 2a , R 2b , R 2c and R2d In some embodiments, the compound of formula (Id) is compound 110 or compound 114.

[0469] In some embodiments, the compound is a compound of formula (If). In some embodiments of formula (If), R 2a and R 2b Each of the above is independently hydrogen, n is 1, and M is -CH 2 -, P is a nitrogen-containing heteroaryl group (e.g., imidazolyl), L 3 Yes -C(O)OCH 2 -, and Z is CH 3 In some embodiments, the compound of formula (If) is compound 115.

[0470] In some embodiments, the compound is a compound of formula (II-a). In some embodiments of formula (II-a), R 2a and R 2b Each of the above is independently hydrogen, n is 1, q is 0, L 3 Yes-CH 2 (OCH 2 CH 2 ) 2 , and Z is -OCH 3 In some embodiments, the compound of formula (II-a) is Compound 112.

[0471] In some embodiments of Formula (II-a), R 2a and R 2b Each of the above is independently hydrogen, n is 1, L 3 is a bond or –CH 2 , and Z is hydrogen or -OH. In some embodiments, the compound of formula (II-a) is Compound 103 or Compound 104.

[0472] In some embodiments, the compound is a compound of formula (III). In some embodiments of formula (III), R 2a , R 2b , R 2c and R 2d Each of the above is independently hydrogen, m is 1, n is 2, q is 3, p is 0, R C is hydrogen, and Z 1 is optionally replaced by R 5 Substituted heteroalkyl (e.g., -N(CH 3 )(CH 2CH 2 )S(O) 2 CH 3 In some embodiments, the compound of formula (III) is compound 120.

[0473] In some embodiments, the compound is a compound of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c and R 2d Each of is independently hydrogen, m is 0, n is 2, q is 3, p is 0, and Z 2 It is 1 R 5 (e.g. -NH 2 In some embodiments, the compound of formula (III-b) is compound 102.

[0474] In some embodiments, the compound is a compound of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c and R 2d Each of the above is independently hydrogen, m is 1, n is 2, q is 3, p is 0, R C is hydrogen, and Z 2 is a heterocyclyl (eg, a nitrogen-containing heterocyclyl, such as a nitrogen-containing spiro heterocyclyl, such as 2-oxa-7-azaspiro[3.5]nonyl). In some embodiments, the compound of formula (III-b) is Compound 121.

[0475] In some embodiments, the compound is a compound of formula (III-d). In some embodiments of formula (III-d), R 2a , R 2b , R 2c and R 2d Each of the above is independently hydrogen, m is 1, n is 2, q is 1, 2, 3 or 4, p is 0, and X is S(O) 2 In some embodiments of Formula (III-d), R 2a and R 2b Each of the above is independently hydrogen, m is 1, n is 2, q is 1, 2, 3 or 4, p is 0, and X is S(O) 2 In some embodiments, the compound of formula (III-d) is Compound 101, Compound 117, Compound 118, or Compound 119.

[0476] In some embodiments, the compound is a compound of formula (Ib), (Id) or (Ie). In some embodiments, the compound is a compound of formula (Ib), (Id) or (II). In some embodiments, the compound is a compound of formula (Ib), (Id) or (If). In some embodiments, the compound is a compound of formula (Ib), (Id) or (III).

[0477] In some embodiments, the compound of formula (I) is not a compound disclosed in WO2012 / 112982, WO2012 / 167223, WO2014 / 153126, WO2016 / 019391, WO 2017 / 075630, US2012-0213708, US2016-0030359, or US2016-0030360.

[0478] In some embodiments, the compound of formula (I) comprises a compound shown in Table 4, or a pharmaceutically acceptable salt thereof. In some embodiments, the external surface and / or one or more compartments within the devices described herein comprise a small molecule compound shown in Table 4, or a pharmaceutically acceptable salt thereof.

[0479] Table 4: Exemplary Defiberizing (FBR Reducing) Compounds

[0480]

[0481]

[0482]

[0483] Conjugation of any of the compounds in Table 4 to a polymer (e.g., alginate) can be performed as described in Example 2 of WO 2019 / 195055 or any other suitable chemical reaction.

[0484] In some embodiments, the compound is a compound of Formula (I) (e.g., Formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (II), (II-a), (III), (III-a), (III-b), (III-c), or (III-d)), or a pharmaceutically acceptable salt thereof, and is selected from:

[0485]

[0486] or a pharmaceutically acceptable salt thereof.

[0487] In some embodiments, the devices described herein comprise a compound

[0488] or a pharmaceutically acceptable salt of either compound.

[0489] In some embodiments, the compound of formula (I) (e.g., compound 101 in Table 4) is covalently attached to alginate (e.g., alginate having an approximate MW <75 kDa, a G:M ratio ≥1.5) with a conjugation density of at least 2.0% and less than 9.0%, or 3.0% to 8.0%, 4.0-7.0, 5.0 to 7.0, or 6.0 to 7.0, or about 6.8, as determined by combustion analysis of nitrogen percentage as described in WO 2020 / 069429. In one embodiment, the conjugation density of compound 101 in the modified alginate is determined by quantitative free amine analysis, for example as described in WO2020198695, wherein the determined conjugation density is 1.0% w / w to 3.0% w / w, 1.3% w / w to 2.8% w / w, 1.3% w / w to 2.6% w / w, 1.5% w / w to 2.4% w / w, 1.5% w / w to 2.2% w / w, or 1.7% w / w to 2.2% w / w.

[0490] The device, device preparation, or device composition may be configured for implantation, or implanted or disposed in or on any part or portion of the body. In some embodiments, the implantable device or device preparation is configured for implantation in the peritoneal cavity (e.g., the omental bursa, also known as the omental bursa or the bursalis omentum). The device, device preparation, or device composition may be implanted in the peritoneal cavity (e.g., the omentum, such as the omentum) or disposed on a surface within the peritoneal cavity (e.g., the omentum, such as the omentum) via injection or catheter. Additional considerations for implanting or disposing a device, device preparation, or device composition in the omentum (e.g., the omentum) are provided in M. Pellicciaro et al. (2017) Cell R4 5(3): e2410.

[0491] Equipment Manufacturing

[0492] Engineered mammalian cells (e.g., engineered ARPE-19 cells) for making devices (e.g., implantable elements) described herein can be produced and cultured using methods known in the art. For example, stably transfected ARPE-19 cells can be cultured in vitro substantially as described in WO2020198695.

[0493] Compounds of formula (I) and alginates modified with such compounds may be obtained using procedures known in the art, for example essentially as described in WO2020198695.

[0494] The alginate solution used to prepare the two-compartment hydrogel capsules can be obtained using procedures known in the art, for example essentially as described in WO2020198695.

[0495] Two-compartment hydrogel capsules encapsulating the engineered mammalian cells described herein can be obtained using procedures known in the art, for example essentially as described in WO2020198696.

[0496] Treatment

[0497] Described herein is a method for preventing or treating a disease, disorder or condition of a subject by administering to the subject an implantable element comprising an engineered mammalian cell (e.g., an engineered ARPE-19 cell), wherein the engineered mammalian cell comprises (i) a reduction in the level or function of an MHC class I protein complex and optionally an MHC class II protein complex and / or CIITA; and (ii) an exogenous nucleic acid encoding, for example, a therapeutic agent for treating the disease, disorder or condition. In some embodiments, after administration, the engineered mammalian cell comprises a reduction in antigenicity or immunogenicity, which is, for example, 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 an MHC class I protein complex and optionally an MHC class II protein complex and / or CIITA. In some embodiments, the reduction in antigenicity or immunogenicity includes a reduction in (a) release of particles or components of the engineered mammalian cells described herein into the bloodstream of a subject, and / or (b) antigen presentation on cells of the subject comprising particles or components of the engineered mammalian cells described herein, for example, 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 an MHC class I protein complex and optionally an MHC class II protein complex and / or CIITA. Such reduction can be characterized by standard methods known in the art, for example, by obtaining a blood sample from a subject and quantifying, for example, protein expression and / or RNA expression.

[0498] The cell can be applied by implanting the preparation of the implantable element or such equipment containing the cell as described herein into the subject.In one embodiment, the preparation of the implantable element or the implantable element is implanted (for example, implanted via laparoscopy) into the intraperitoneal space, for example, in the large sac of the peritoneal cavity.In one embodiment, the engineered mammalian cell is an engineered RPE cell, and the method includes the composition of the two-compartment alginate hydrogel capsule of applying (for example, implanting) an effective amount, the capsule includes an engineered RPE cell and a cell binding polymer as described herein in the internal compartment, and includes formula (I) compound, for example compound 101, on the outer capsule surface.In some embodiments, the method of treating directly or indirectly alleviates or alleviates at least one symptom of a disease, illness or disease, and / or the method prevents or slows down the onset of a disease, illness or disease.In some embodiments, the subject is a person.

[0499] In some embodiments, the disease, disorder, or condition affects a system of the body, such as the nervous system (e.g., peripheral nervous system (PNS) or central nervous system (CNS)), vascular system, skeletal system, respiratory system, endocrine system, lymphatic system, reproductive system, or gastrointestinal tract. In some embodiments, the disease, disorder, or condition affects a part of the body, such as the blood, eyes, brain, skin, lungs, stomach, mouth, ears, legs, feet, hands, liver, heart, kidneys, bones, pancreas, spleen, large intestine, small intestine, spinal cord, muscle, ovaries, uterus, vagina, or penis.

[0500] In some embodiments, the disease, disorder or condition is a neurodegenerative disease, diabetes, heart disease, an autoimmune disease, cancer, liver disease, a lysosomal storage disease, a blood coagulation disorder or a coagulation disorder, an orthopedic disorder, an amino acid metabolism disorder.

[0501] In some embodiments, the disease, disorder or condition is a neurodegenerative disease. Exemplary neurodegenerative diseases include Alzheimer's disease, Huntington's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and cerebral palsy (CP), dentatorubral nucleus-globus pallidus Lewy body atrophy (DRPLA), neuronal intranuclear hyaline inclusion disease (NIHID), Lewy body dementia, Down syndrome, Hallervorden-Spatz disease, prion disease, argyrophilic grain dementia, corticobasal degeneration, dementia pugilistica, diffuse neurofibrillary tangles, Gerstmann-Strauss-Scheinker disease, Jacob-Kreutzfeldt disease, Niemann-Pick disease type 3, progressive supranuclear palsy, subacute sclerosing panencephalitis, spinocerebellar ataxia, Pick's disease and dentatorubral nucleus-globus pallidus Lewy body atrophy.

[0502] In some embodiments, the disease, disorder, or condition is an autoimmune disease, such as scleroderma, multiple sclerosis, lupus, or allergy.

[0503] In some embodiments, the disease is a liver disease, such as hepatitis B, hepatitis C, cirrhosis, NASH.

[0504] In some embodiments, the disease, disorder or condition is cancer. Exemplary cancers include leukemia, lymphoma, melanoma, lung cancer, brain cancer (e.g., glioblastoma), sarcoma, pancreatic cancer, kidney cancer, liver cancer, testicular cancer, prostate cancer, or uterine cancer.

[0505] In some embodiments, the disease, disorder, or condition is an orthopedic condition. Exemplary orthopedic conditions include osteoporosis, osteonecrosis, Paget's disease, or a bone fracture.

[0506] In some embodiments, the disease, disorder or condition is a lysosomal storage disease. Exemplary lysosomal storage diseases include Gaucher disease (e.g., type I, type II, type III), Tay-Sachs disease, Fabry disease, Farber disease, mucopolysaccharidosis type I (MPSI) (also known as Hurler syndrome), Hunter syndrome, lysosomal acid lipase deficiency, Niemann-Pick disease, sialic acid storage disease, Sanfilippo syndrome (also known as mucopolysaccharidosis type IIIA (MPS3A)), multiple sulfatase deficiency, Mara-La syndrome, metachromatic leukodystrophy, Krabbe disease, Scheher-Ellison syndrome, Hurler-Schwein syndrome, Sly syndrome, hyaluronidase deficiency, Pompe disease, Danon disease, gangliosidosis, or Morquio syndrome.

[0507] In some embodiments, the disease, disorder or condition is a blood coagulation disorder or coagulation disorder. Exemplary blood coagulation disorders or coagulation disorders include hemophilia (e.g., hemophilia A or hemophilia B), von Willebrand's disease, thrombocytopenia, uremia, Bernard-Soulier syndrome, factor XII deficiency, vitamin K deficiency, or afibrinogenemia.

[0508] In some embodiments, the disease, condition, or disorder is an amino acid metabolism disorder, such as phenylketonuria, tyrosinemia (eg, type 1 or type 2), alkaptonuria, homocystinuria, hyperhomocysteinemia, maple syrup urine disease.

[0509] In some embodiments, the disease, disorder or condition is a fatty acid metabolism disorder, such as hyperlipidemia, hypercholesterolemia, galactosemia.

[0510] In some embodiments, the disease, disorder, or condition is a disorder of purine or pyrimidine metabolism, such as Lesch-Nyhan syndrome.

[0511] In some embodiments, the disease, disorder or condition is diabetes (e.g., type I or type II diabetes). In some embodiments, the disease, disorder or condition is not diabetes. In some embodiments, the disease, disorder or condition is not type I diabetes. In some embodiments, the disease, disorder or condition is not type II diabetes.

[0512] List of implementation plans

[0513] 1. An implantable element comprising engineered mammalian cells, wherein:

[0514] (i) the engineered mammalian cell comprises a reduction in the level or function of a major histocompatibility complex (MHC) class I protein complex; and

[0515] (ii) The engineered mammalian cell comprises an exogenous nucleic acid encoding a therapeutic agent.

[0516] 2. The implantable element of embodiment 1, wherein the MHC class I protein complex comprises one or more of the following:

[0517] (i) human leukocyte antigen (HLA) A;

[0518] (ii) HLA-B;

[0519] (iii) HLA-C; and

[0520] (iv) β-2-microglobulin (β-2M).

[0521] 3. The implantable element of embodiment 2, comprising (i).

[0522] 4. The implantable element of any one of embodiments 2-3, comprising (ii).

[0523] 5. The implantable element of any one of embodiments 2-4, comprising (iii).

[0524] 6. The implantable element of any one of embodiments 2-5, comprising (iv).

[0525] 7. The implantable element of any of the preceding embodiments, wherein the engineered mammalian cells comprise a mutation that results in a reduction in the expression of a component of the MHC class I complex.

[0526] 8. The implantable element of any of the preceding embodiments, wherein the engineered mammalian cells comprise a less functional or non-functional variant of a component of the MHC class I component.

[0527] 9. The implantable element of any one of the preceding embodiments, wherein the expression of a component of the MHC class I complex is silenced or knocked down.

[0528] 10. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cells comprise a reduction in the 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 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.

[0529] 11. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of the MHC class I component HLA-A of, 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 reduced level of the MHC class I component HLA-A.

[0530] 12. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of the MHC class I component HLA-B, for example, 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 for not comprising the reduced level of the MHC class I component HLA-B.

[0531] 13. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cells comprise a reduction in the level of the MHC class I component HLA-C of, 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 reduced level of the MHC class I component HLA-C.

[0532] 14. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cells comprise a reduction in the level of MHC class I component β-2M, for example, 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 for not comprising the reduced level of MHC class I component β-2M.

[0533] 15. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cells comprise a reduction in the level of MHC class I components, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, or between 50-75% 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 MHC class I components.

[0534] 16. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of MHC class I components, e.g., between 1-25% 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 MHC class I components.

[0535] 17. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of MHC class I components, e.g., between 5-25% 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 MHC class I components.

[0536] 18. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of MHC class I components, e.g., between 10-25% 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 MHC class I components.

[0537] 19. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of MHC class I components, e.g., between 25-50% 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 MHC class I components.

[0538] 20. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of MHC class I components, e.g., between 25-75% 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 MHC class I components.

[0539] 21. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of MHC class I components, e.g., between 50-75% 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 MHC class I components.

[0540] 22. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cells comprise a reduction in the level of MHC class I components, e.g., greater than 50%, greater than 75%, or greater than 90% 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 MHC class I components.

[0541] 23. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of MHC class I components by greater than 50%, 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 MHC class I components.

[0542] 24. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of MHC class I components by greater than 75%, 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 MHC class I components.

[0543] 25. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the level of MHC class I components by greater than 90%, 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 MHC class I components.

[0544] 26. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises 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 compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in the function of the MHC class I component.

[0545] 27. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the function of HLA-A, 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 reduction in the function of HLA-A.

[0546] 28. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the function of HLA-B, 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 the function of HLA-B.

[0547] 29. An implantable element as described in any of the preceding embodiments, wherein the engineered mammalian cell comprises a reduction in the function of HLA-C, 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 reduction in the function of HLA-C.

[0548] 30. An implantable ele...

Claims

1. An implantable element comprising engineered mammalian cells, in: (i) the engineered mammalian cell comprises a reduction in the level or function of a major histocompatibility complex (MHC) class I protein complex; and (ii) the engineered mammalian cell comprises an exogenous nucleic acid encoding a therapeutic agent; 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)(C 1 -C 6 -alkylene)–, -N(R C )C(O)(C 1 -C 6 -alkenylene)–, –N(R C )N(R D )–, –NCN–, –C(=N(R C )(R D ))O–, –S–, –S(O) x –, –OS(O) x –、–N(R C )S(O) x –, –S(O) x N(R C )–,–P(R F ) y –、–Si(OR A ) 2 –、–Si(R G )(OR A )–、–B(OR A )- or a metal, each of which is optionally 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 is independently a bond, an alkyl group or a heteroalkyl group, wherein each alkyl group and heteroalkyl group is optionally substituted by one or more R 2 replace; L 2 is the 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 substituted 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 substituted 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 substituted 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 substituted by one or more R 7 replace; Each R 7 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxy, cycloalkyl or heterocyclyl; x is 1 or 2; and y is 2, 3, or 4.

2. The implantable element of claim 1, wherein the MHC class I protein complex comprises one or more of: (i) human leukocyte antigen (HLA) A; (ii) HLA-B; (iii) HLA-C; and (iv) β-2-microglobulin (β-2M).

3. The implantable element of claim 2, comprising (i).

4. The implantable element of any one of claims 2 to 3, comprising (ii).

5. The implantable element of any one of claims 2 to 4, comprising (iii).

6. The implantable element of any one of claims 2 to 5, comprising (iv).

7. An implantable element as claimed in any one of the preceding claims, wherein the engineered mammalian cell comprises a mutation that results in a decrease in expression of a component of the MHC class I complex, e.g., compared to a reference standard.

8. An implantable element as claimed in 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 component, e.g. compared to a reference standard.

9. An implantable element as claimed in any one of the preceding claims, wherein the expression of a component of the MHC class I complex is silenced or knocked down, e.g. compared to a reference standard.

10. 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 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 a reduced level 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.

11. 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 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 a reduced level of MHC class I components, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, or between 50-75% compared to a reference standard.

12. An implantable element as claimed in any of the preceding claims, wherein the engineered mammalian cell comprises a reduction in the level 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 level of MHC class I components, e.g., greater than 50%, greater than 75% or greater than 90% compared to a reference standard.

13. An implantable element as claimed in any of the preceding claims, wherein the engineered mammalian cell comprises a decrease in the function of an MHC class I component, e.g., compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the decrease in the function of the 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 compared to a reference standard.

14. An implantable element as claimed in any of the preceding claims, wherein the engineered mammalian cell comprises a decrease in the function of an MHC class I component, e.g., compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising a decrease in the function of an MHC class I component, e.g., between 1-25%, between 5-25%, between 10-25%, between 25-50%, between 25-75%, between 50-75% or between 75-100% compared to a reference standard.

15. An implantable element as claimed in any of the preceding claims, wherein the engineered mammalian cell comprises a reduction in the function of an MHC class I component, e.g., compared to an engineered mammalian cell that is substantially identical or identical to the engineered mammalian cell except for not comprising the reduction in the function of an MHC class I component, e.g., greater than 50%, greater than 75% or greater than 90% compared to a reference standard.

16. The implantable element of any of the preceding claims, wherein the engineered mammalian cells further comprise a reduction in the level or function of the MHC class II complex.

17. The implantable element of claim 16, wherein the MHC class II complex comprises one or more of: (v) human leukocyte antigen (HLA) DP; (vi) HLA-DM; (vii) HLA-DOA; (viii) HLA-DOB; (ix) HLA-DQ; and (x)HLA-DR.

18. The implantable element of claim 17, wherein the MHC class II complex comprises (v).

19. The implantable element of any one of claims 17-18, wherein the MHC class II complex comprises (vi).

20. The implantable element of any one of claims 17-19, wherein the MHC class II complex comprises (vii).

21. The implantable element of any one of claims 17-20, wherein the MHC class II complex comprises (viii).

22. The implantable element of any one of claims 17-21, wherein the MHC class II complex comprises (ix).

23. The implantable element of any one of claims 17-22, wherein the MHC class II complex comprises (x).

24. The implantable element of any one of claims 17-23, wherein the cells comprise a reduction in the function or expression of class II major histocompatibility complex transactivator (CIITA).

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

26. The implantable element of any of the preceding claims, wherein the engineered mammalian cells comprise embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

27. An implantable element as claimed in 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 therefrom.

28. The implantable element of any of the preceding claims, wherein the engineered mammalian cells comprise engineered retinal pigment epithelial (RPE) cells (eg, engineered ARPE-19 cells).

29. The implantable element of any one of the preceding claims, wherein the exogenous nucleotide sequence is extrachromosomal.

30. The implantable element of any of the preceding claims, wherein the exogenous nucleotide sequence is inserted into at least one location in the genome of the mammalian cell.

31. The implantable element of any of the preceding claims, comprising at least one cell-containing compartment comprising the engineered mammalian cells of any of claims 1-30.

32. An implantable element as claimed in any one of the preceding claims, wherein the polymer is selected from the group consisting of alginate, hyaluronate and chitosan.

33. The implantable element of claim 32, wherein the polymer comprises alginate.

34. The implantable element of any one of claims 32-33, wherein the alginate is high guluronic acid (G) alginate or high mannuronic acid (M) alginate.

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

36. The implantable element of claim 35, wherein the peptide comprises, consists essentially of, or consists of GRGDSP, GGRGDSP, or GGGRGDSP.

37. An implantable element as described in any one of claims 31-36, wherein the cell-containing compartment is surrounded by a barrier compartment, the 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.

38. An implantable element as described in any one of claims 31-37, wherein the polymer 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 Or a chemically modified alginate or a pharmaceutically acceptable salt thereof.

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

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

41. The implantable element of any of the preceding claims, wherein the implantable element is spherical with a diameter of about 0.75 mm to about 2 mm.

42. 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.

43. An implantable element as claimed in any one of the preceding claims, comprising: (i) engineered ARPE cells capable of reducing the expression of β-2M; (ii) A polymer composition comprising alginate covalently modified with one or more of: (a) a compound of formula (I) (eg, as described herein); and (b) Peptides.

44. The implantable element of claim 43, wherein the engineered ARPE cells are further capable of reducing expression of CIITA.

45. An implantable element as claimed in any one of the preceding claims, comprising: (i) engineered ARPE cells capable of reducing the expression of β-2M; (ii) A polymer composition comprising alginate covalently modified with one or more of: (a) or a pharmaceutically acceptable salt thereof; and (b) A peptide comprising or consisting of GRGDSP or GGRGDSP.

46. ​​The implantable element of claim 47, wherein the engineered ARPE cells are further capable of reducing expression of CIITA.

47. The implantable device of any of the preceding claims, formulated for implantation into a subject (eg, into the intraperitoneal (IP) space, peritoneal cavity, omentum, fossa, subcutaneous fat).

48. An implantable element as claimed in any preceding claim, which is configured for implantation into the IP space of a subject.

49. A preparation of implantable elements, wherein each implantable element in the preparation is an implantable element as claimed in any one of claims 1 to 48.

50. A composition for treating a disease or condition in a subject comprising the implantable element of any one of claims 1-48 or the formulation of claim 49.

51. The composition for use of claim 50, wherein the disease or disorder is a lysosomal storage disease or a metabolic disorder.

52. A composition for use as described in any one of claims 50-51, wherein the subject is a human.

Images