Compositions and Related Methods for Cell-Based Therapy - Patent application

Abstract

Described herein is a hydrogel capsule composition comprising a population of hydrogel capsules and a pharma- ceutically acceptable solution, wherein the hydrogel capsules in the population comprise a hydrogel-forming polymer and encapsulate a plurality of cells.

Description

[Technical field]

[0001] Claiming priority This application claims priority to U.S. Provisional Application No. 63 / 273,678, filed October 29, 2021. The entire disclosure of the aforementioned application is incorporated by reference in its entirety. [Background technology]

[0002] Treating chronic and genetic diseases by transplanting viable cells that produce therapeutic substances capable of treating such diseases has the attractive potential to improve the health of patients with such diseases. To fully achieve this potential, the transplanted cells must be protected from the patient's immune response so that they remain viable, and the transplanted cells must be capable of producing therapeutic levels of the desired therapeutic substance for weeks, months, or even longer. One exploratory approach to deliver such cell-based therapies is to encapsulate the therapeutic-producing cells in alginate hydrogel capsules, with the goal that the capsule structure will isolate the therapeutic-producing cells from immune system cells while allowing the entry of nutrients for the therapeutic-producing cells and the exit of the therapeutic substance from the capsule. Once such hydrogel capsules are generated, they need to be stored in a manner that preserves the integrity of the capsule structure and the viability of the encapsulated cells. Summary of the Invention

[0003] In one aspect, the disclosure provides a hydrogel capsule composition comprising a population of hydrogel capsules and a pharma- ceutically acceptable solution, each hydrogel capsule in the population comprising an ionically crosslinked alginate and encapsulating a plurality of viable mammalian cells. In one embodiment, the solution comprises a calcium salt at an elemental calcium concentration of about 1.0 mM to about 10 mM. In one embodiment, the elemental calcium concentration is about 1.2 mM to about 3 mM. In one embodiment, the calcium salt is calcium chloride. In one embodiment, the solution has an osmolality of about 250 mOsm / kg to about 350 mOsm / kg at a temperature of 12° C. to 30° C. and a pH of 6.0 to 9.0.

[0004] In some embodiments, the solution further comprises at least one buffer capable of maintaining the pH of the solution within a desired range (e.g., 6.0-9.0) when the composition is stored at a temperature between 12° C. and 30° C. (e.g., about 15-25° C.) In one embodiment, the buffer comprises one or more of an acetate (e.g., sodium acetate), a gluconate (e.g., sodium gluconate), a bicarbonate (e.g., sodium bicarbonate), a lactate (e.g., sodium lactate), or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

[0005] In one embodiment, the solution further comprises a carbon source (e.g., a sugar (e.g., dextrose, glucose, galactose, hexose, fructose, maltose), glycerol, glutamine, pyruvate) to support viability of the encapsulated cells. In one embodiment, the carbon source is glucose. In one embodiment, the solution comprises about 1.5 mM to about 2.5 mM calcium chloride, about 5 mM to about 25 mM D-glucose, and about 40 mM to about 50 mM sodium bicarbonate.

[0006] In some embodiments, the solution further comprises minerals, amino acids and vitamins that support the viability of the encapsulated cells.

[0007] In one embodiment, the mineral includes a magnesium salt (eg, magnesium chloride or magnesium sulfate) or a potassium salt (eg, potassium chloride).

[0008] In one embodiment, the amino acids include histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine.

[0009] In one embodiment, the vitamins include a vitamin B1 compound (e.g., thiamine or a thiamine salt, e.g., thiamine hydrochloride), a vitamin B3 compound (e.g., nicotinic acid or niacinamide), and a vitamin B6 compound (e.g., pyroxidine or a pyroxidine salt, e.g., pyroxidine hydrochloride).

[0010] In some embodiments, the amino acids include L-arginine, L-cystine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

[0011] In some embodiments, vitamins include choline or a choline salt (e.g., choline chloride), a vitamin B1 compound (e.g., thiamine or a thiamine salt, e.g., thiamine hydrochloride), a vitamin B3 compound (e.g., nicotinic acid or niacinamide), a vitamin B5 compound (e.g., pantothenic acid or calcium pantothenate), a vitamin B6 compound (e.g., pyroxidine or a pyroxidine salt, e.g., pyroxidine hydrochloride), a folate compound (e.g., folic acid), riboflavin, and i-inositol.

[0012] In one embodiment, the hydrogel capsules in the population of hydrogel capsules are sphere-like or spherical in shape. In one embodiment, the hydrogel capsules have an average capsule diameter of about 500 μm to about 5000 μm (e.g., about 500 μm to about 4000 μm, about 500 μm to about 3000 μm, about 500 μm to about 2500 μm, about 500 μm to about 2000 μm, about 500 μm to about 1500 μm, about 500 μm to about 1000 μm, about 1000 μm to about 2500 μm). In one embodiment, the hydrogel capsules are not sphere-like or spherical in shape. In one embodiment, each capsule comprises a cell-containing compartment surrounded by a barrier compartment comprising ionically crosslinked alginate. In one embodiment, the crosslinked alginate comprises alginate covalently modified with at least one non-fibrous compound as defined herein. In one embodiment, the crosslinking agent comprises barium ions. In some embodiments, the cell-containing compartment encapsulates live mammalian cells in a first polymer composition comprising alginate that is optionally ionically crosslinked (e.g., with barium ions). In some embodiments, the alginate in the first polymer composition is covalently modified with a cell contact peptide as defined herein. In one embodiment, the average capsule diameter of the hydrogel capsule is 1400-2000 μm.

[0013] In some embodiments, the encapsulated live mammalian cells are derived from human cells, e.g., RPE cells (e.g., ARPE-19 cells). In one embodiment, the cells are modified to express and secrete an exogenous protein, e.g., any of the therapeutic proteins described herein.

[0014] In another aspect, the present disclosure provides a sealed container comprising any of the capsule compositions described herein. Each interior surface of the container that contacts the composition consists essentially of a biocompatible material, such as a medical grade plastic (e.g., fluorinated ethylene propylene (FEP) or polyethylene terephthalate glycol (PETG)). In one embodiment, the bottom interior surface of the container has a rectangular or round shape.

[0015] In one embodiment, substantially all of the hydrogel capsules in the composition (e.g., at least 90%, 95%, 98% or more) are substantially uniformly distributed across the bottom interior surface of the container in a capsule layer, and the volume of solution (VS) is disposed above the top of the layer. In one embodiment, the capsule layer has a depth equal to about 1.00 to about 1.25 times the average diameter of the capsules in the composition.

[0016] In yet another aspect, the disclosure features a method of making a sealed container comprising a hydrogel capsule composition described herein. In one embodiment, the method includes providing a population of hydrogel capsules encapsulating viable mammalian cells, combining the population of capsules with a pharma- ceutically acceptable aqueous solution described herein, and placing a desired volume of the composition in a biocompatible, sealable container in a manner to produce a capsule layer, where in the capsule layer, substantially all of the capsules in the volume of the composition are substantially uniformly distributed across the bottom of the container at a depth equivalent to about 1.00 to about 1.25 times the average diameter of the capsules in the composition. In one embodiment, the method further includes adding a desired volume of a pharma- ceutically acceptable solution in a manner to form a solution layer on top of the capsule layer, and sealing the container. In one embodiment, the method further includes storing the sealed container for a desired period of time at a temperature of 2°C to 30°C or about 12°C to 30°C (e.g., about 15-25°C) and evaluating the viability of the encapsulated cells in the composition at one or more time points during the desired period.

[0017] In yet a further aspect, the disclosure features the use of a hydrogel capsule composition described herein to treat a mammalian subject (e.g., a human subject) in need of treatment with a substance (e.g., a protein) produced by cells encapsulated in the composition. In one embodiment, the subject is treated by a method that includes administering to the subject a desired amount of the composition, for example, by implanting the desired amount into the peritoneal cavity of the subject. In one embodiment, the method includes providing the hydrogel capsule composition in a sealed container described herein, opening the container, and removing the desired amount. In one embodiment, the encapsulated cells in the hydrogel capsule composition are genetically modified to produce one of the exogenous proteins described herein. [Brief description of the drawings]

[0018] [Figure 1] A graph of the viability of cells encapsulated in alginate hydrogel spheres and stored for up to 3 days in aqueous solution lacking calcium or in the same solution with added calcium is shown, with error bars representing the standard deviation (SD) between two replicates per time point. [Diagram 2] Graph of plasma FVIII levels in mice implanted with alginate hydrogel spheres encapsulating FVIII-expressing cells after the spheres were stored in different preservation solutions; error bars represent standard deviation (SD) among four mice per storage condition. [Figure 3A] FIG. 1 shows a graph of plasma FVIII levels in mice implanted with alginate hydrogel spheres encapsulating FVIII-expressing cells after the spheres were stored in different preservation solutions for the indicated periods of time; error bars represent the standard deviation (SD) among four mice per storage condition. [Figure 3B] FIG. 1 shows a graph of plasma FVIII levels in mice implanted with alginate hydrogel spheres encapsulating FVIII-expressing cells after the spheres were stored in different preservation solutions for the indicated periods of time; error bars represent the standard deviation (SD) among four mice per storage condition. [Figure 4] Shown are FVIII levels secreted into conditioned medium from alginate hydrogel spheres encapsulating FVIII-expressing cells after the spheres were stored as monolayers, bilayers, or triple layers for up to 7 days, with the average of two replicate samples graphed as a single point. [Diagram 5] Graph of plasma FVIII levels in mice implanted with alginate hydrogel spheres encapsulating FVIII-expressing cells after the spheres were stored in different preservation solutions; error bars represent standard deviation (SD) among four mice per storage condition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present disclosure features a hydrogel capsule composition comprising a population of viable mammalian cells (e.g., human RPE cells) in a pharma- ceutically acceptable solution, a sealed container comprising the hydrogel capsule composition, and its use to treat a mammalian subject in need of treatment with a substance produced by the encapsulated cells. Various embodiments are described below.

[0020] Abbreviations and Definitions The following abbreviations are used throughout the detailed description and examples of this disclosure:

[0021] CM-Alg Chemically modified alginate CM-LMW-Alg Chemically modified low molecular weight alginate CM-LMW-Alg-101 Low molecular weight alginate chemically modified with compound 101 shown in Table 4 CM-HMW-Alg Chemically modified high molecular weight alginate CM-HMW-Alg-101 High molecular weight alginate chemically modified with compound 101 shown in Table 4 CM-MMW-Alg Chemically modified medium molecular weight alginate CM-MMW-Alg-101 Medium molecular weight alginate chemically modified with compound 101 shown in Table 4 HMW-Alg High molecular weight alginate MMW-Alg Medium molecular weight alginate U-Alg Unmodified alginate U-HMW-Alg Unmodified high molecular weight alginate U-LMW-Alg Unmodified low molecular weight alginate U-MMW-Alg Unmodified medium molecular weight alginate 70:30 CM-Alg:U-Alg A 70:30 mixture (V:V) of chemically modified and unmodified alginates, as described, for example, in WO2020069429.

[0022] definition In order that the present disclosure may be more readily understood, certain technical and scientific terms used herein are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0023] As used in this specification, including the appended claims, the singular forms of terms such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0024] "About" or "approximately," when used herein to modify a numerically defined parameter (e.g., physical description of a hydrogel capsule, e.g., diameter, sphericity, number of cells encapsulated therein, number of capsules in a preparation), means that the stated numerical value is within an acceptable functional range for the defined parameter as determined by one of ordinary skill in the art, which depends in part on how the measurement system's limits (including the acceptable error range for that measurement system) were measured or determined. For example, "about" can mean a range of 20% above and below the stated numerical value. As a non-limiting example, a hydrogel capsule defined as having a diameter of about 1.5 millimeters (mm) and encapsulating about 5 million (M) cells can have a diameter of 1.2-1.8 mm and encapsulate 4M-6M cells. As another non-limiting example, a preparation of about 100 hydrogel capsules includes a preparation having 80-120 capsules. In some embodiments, the term "about" means that the modified parameter may vary by as much as 15%, 10% or 5% above or below the numerical value stated for that parameter.

[0025] "Obtain" or "obtaining", as used herein, refers to obtaining a possession of a value, e.g., a numerical value, or an image, or a physical entity (e.g., a sample), by "directly obtaining" or "indirectly obtaining" the value or physical entity. "Directly obtaining" means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. "Indirectly obtaining" refers to obtaining a value or physical entity from another person or other source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a value or physical entity includes performing a process or using a machine or device that involves a physical change of a physical substance. An example of directly obtaining a value includes obtaining a sample from a human subject. Directly obtaining a value includes performing a process using a machine or device, e.g., using a fluorescent microscope to obtain fluorescent microscopy data.

[0026] "Administer," "administering," or "administration," as used herein, refers to implanting, absorbing, ingesting, injecting, or otherwise introducing into a subject an entity described herein (e.g., a hydrogel capsule composition or an aliquot thereof), or providing such an entity to a subject for administration.

[0027] "Nonfibrotic," as used herein with respect to a compound, polymer, or other substance, means that the substance reduces foreign body response (FBR). For example, the amount of FBR induced in a biological tissue by implantation of a hydrogel capsule containing a nonfibrotic compound (e.g., a polymer covalently modified with a compound listed in Table 4) is lower than the FBR induced in that tissue by implantation of a nonfibrotic null reference capsule lacking any nonfibrotic compound but of substantially the same composition (e.g., same cell type(s)) and structure (e.g., size, shape, number of compartments). In one embodiment, the degree of FBR is assessed by immunological responses in tissues containing the implanted hydrogel capsules, which may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis, using assays known in the art, for example, as described in WO2017 / 075630, WO2021 / 119522, or using one or more of the assays / methods described in Vegas, A., et al., Nature Biotechnol (supra). In one embodiment, the non-fibrous compound is a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, 1 , M, L 2 , P., L. 3 and Z, and the associated subvariables, are as defined herein.

[0028] "Alpha-galactosidase A," "α-Gal A," "alpha-D-galactosidase-A," alpha-galactoside galactohydrolase, "galactosidase alpha," and "GLA protein" may be used interchangeably herein and refer to a protein that includes the mature amino acid sequence of mature wild-type mammalian ARSB or any fragment, mutant, variant, or derivative thereof that has within 80-120%, 85-115%, 90-110%, or 95-105% of the enzymatic activity of the corresponding wild-type mammalian mature GLA protein as measured by any art-recognized GLA activity assay. GLA is a glycosphingolipid that binds glycosphingolipids, particularly globotriaosylceramide (Gb 3 ) hydrolyzes the terminal alpha-D-galactose residues in the GLA gene. The wild-type human GLA gene encodes a 429 amino acid polypeptide, the N-terminal 31 amino acids of which constitute a signal peptide (GenBank Accession No. CAA29232.1). In one embodiment, the GBA protein is part of a fusion protein that further comprises one or more amino acid sequences from one or more heterologous polypeptides. In one embodiment, the encapsulated cell comprises an exogenous nucleotide sequence encoding the GLA fusion protein shown in Figure 4A of WO2020 / 198685.

[0029] GLA activity can be measured directly by obtaining blood leukocytes from a subject, lysing the leukocytes, and determining the enzyme activity in the lysate after adding an enzyme substrate such as 4-methylumbelliferal alpha-D-galactoside. Immunoassays for measuring GLA activity and protein to determine the concentration of alpha-galactosidase in blood and plasma are described in Clin Chem.2004;50(11):1979-85. Indirect assessment of GLA activity is based on measuring the levels of substrates such as Gb3 and biomarker lysoGb3 in plasma and / or urine samples collected from a subject or tissues of interest such as liver, kidney, heart biopsies. Gb3 and lysoGb3 levels can be measured using any art-recognized assay. For example, methods for measuring Gb3 levels in plasma and urine of humans with Fabry disease are described, for example, in Boscaro et al., Rapid Commun Mass Spectrom. 2002; 16(16): 1507-14. Gb3 accumulation in skin biopsies obtained using a "punch" device can be detected using immunoelectron microscopic methods as described in Kanekura et al., Br J Dermatol. 2005, 153(3): 544-8. Various biopsy techniques and assays for detecting Gb3 and other surrogate biomarkers are described in US Patent Application Publication US2010 / 0113517. GLA activity and / or other plasma surrogate biomarkers of Fabry disease progression (e.g., various inflammatory and cardiac remodeling biomarkers) are described in Yogasundaram, H. et al., J Am Heart Assoc. 2018; 7: e009098.

[0030] "Alpha-L-iduronidase protein" and "IDUA protein" are used interchangeably herein and may refer to a protein that (i) is capable of hydrolyzing non-reducing terminal alpha-L-iduronic acid residues in glycosaminoglycans (GAGs) (e.g., dermatan sulfate and heparan sulfate) and (ii) comprises the mature amino acid sequence of a mature wild-type mammalian IDUA protein or any fragment, mutant, variant, or derivative thereof that has an enzymatic activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature IDUA protein as measured by any art-recognized IDUA activity assay (e.g., hydrolysis of the substrate 4-methylumbelliferyl-α-L-iduronide) (4MU-iduronide), see, e.g., Ou, L. et al., Mol Genet Metab. 2014 Feb:111(2):113-115). The wild-type human IDUA gene encodes a 653 amino acid precursor protein, the N-terminal 26 amino acids of which constitute a signal peptide (GenBank Accession No. AAA81589.1, GenBank Accession No. AAA51698.1). In one embodiment, the IDUA protein is part of a fusion protein that further comprises one or more amino acid sequences from one or more heterologous polypeptides. "Arylsulfatase B protein" and "ARSB protein" may be used interchangeably herein and refer to a protein that (i) is capable of hydrolyzing the 4-sulfate group of the N-acetyl-D-galactosamine 4-sulfate units of chondroitin sulfate and dermatan sulfate, and (ii) comprises the amino acid sequence of mature wild-type mammalian ARSB or any fragment, mutant, variant or derivative thereof that has an enzymatic activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature ARSB protein as measured by any ARSB activity assay known in the art. The wild-type human ARSB gene encodes a precursor polypeptide of 533 amino acids, the N-terminal 36 or 38 amino acids of which constitute a signal peptide (UniProtKB-P15848).In one embodiment, the ARSB protein is part of a fusion protein that further comprises one or more amino acid sequences from one or more heterologous polypeptides.

[0031] "Beta-glucosidase protein", "acid beta-glucocerebrosidase protein", "glucosylceramidase beta protein" and "GBA protein" may be used interchangeably herein to refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian GBA gene or any fragment, mutant, variant or derivative thereof having within 80-120%, 85-115%, 90-110% or 95-105% of the enzymatic activity of the corresponding wild-type mammalian mature GBA protein as measured by any GBA assay known in the art. GBA catalyzes the degradation of the glycolipid glucosylceramide (GlcCer) to ceramide and glucose. The wild-type human GBA gene encodes a 536 amino acid precursor polypeptide (UniProtKB-P04062-1). In one embodiment, the GBA protein is part of a fusion protein further comprising one or more amino acid sequences from one or more heterologous polypeptides.

[0032] "Cell" as used herein refers to a genetically modified or non-genetically modified cell. In one embodiment, the cell is an immortalized cell or a genetically modified cell derived from an immortalized cell. In one embodiment, the cell is a viable cell, e.g., viable as measured by any technique described herein or known in the art.

[0033] "Cell-binding peptide (CBP)" as used herein means a linear or cyclic peptide comprising an amino acid sequence derived from a cell-binding domain of a ligand of a cell adhesion molecule (CAM) (e.g., mediating cell-matrix or cell-cell connections). In one embodiment, the CBP is any of the CBPs described in International Patent Publication WO2020 / 069429. In one embodiment, the CBP is a linear peptide comprising RGD and is less than 6 amino acids in length. In one embodiment, the CBP is a linear peptide consisting essentially of RGD or RGDSP.

[0034] "CBP-polymer" as used herein means a polymer comprising at least one cell-binding peptide molecule covalently bound to the polymer via a linker. In one embodiment, the polymer in the CBP-polymer is a synthetic or naturally occurring polysaccharide, such as an alginate, e.g., sodium alginate. In one embodiment, the linker is an amino acid linker (i.e., consisting essentially of a single amino acid, or a peptide of several identical or different amino acids), which is connected to the N-terminus or C-terminus of CBP via a peptide bond. In one embodiment, the CBP-polymer is any of the CBP-alginates defined in WO2020 / 069429.

[0035] "Cell-binding substance (CBS)" as used herein means any chemical, biological, or other type of substance (e.g., small organic compounds, peptides, polypeptides) capable of mimicking at least one activity of a ligand of a cell-adhesion molecule (CAM) or other cell-surface molecule that mediates cell-matrix or cell-cell connections or other receptor-mediated signaling. In one embodiment, when present in a polymer composition that encapsulates viable cells, the CBS is capable of forming a transient or permanent bond or contact with one or more of the cells. In one embodiment, the CBS facilitates an interaction between two or more viable cells encapsulated in the polymer composition. In one embodiment, the presence of the CBS in a polymer composition that encapsulates a plurality of cells (e.g., viable cells) correlates with one or both of increased cell productivity (e.g., expression of a therapeutic agent) and increased cell viability when the encapsulated cells are implanted into a test subject, e.g., a mouse. In one embodiment, the CBS is physically bound to one or more polymer molecules in the polymer composition. In one embodiment, the CBS is a cell-binding peptide as defined herein or in WO2020 / 069429.

[0036] "Conservatively modified variants" or "conservative substitutions", as used herein, refer to variants of a reference peptide or polypeptide that are identical to the reference molecule except for having one or more conservative amino acid substitutions in its amino acid sequence. In one embodiment, a conservatively modified variant consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the reference amino acid sequence. A conservative amino acid substitution refers to the replacement of an amino acid with an amino acid that has similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) and has minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution tables of functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional characteristics are shown in Table 1 below. [Table 1]

[0037] "Consists essentially of" and variants such as "consist essentially of" or "consisting essentially of," as used throughout this specification and claims, refer to the inclusion of any recited element or group of elements, and the optional inclusion of other elements of similar or different nature to the recited elements that do not substantially alter the basic or novel properties of the identified molecule, composition, hydrogel capsule, or method. As a non-limiting example, a therapeutic agent consisting essentially of a recited amino acid sequence can also include one or more amino acids that include a substitution in the recited amino acid sequence of one or more amino acid residues that does not substantially affect the relevant biological activity of the therapeutic agent.

[0038] "Derived from," as used herein with respect to a cell, refers to a cell obtained from a tissue, cell line, or cell, and optionally then cultured, passaged, differentiated, induced, etc. to produce a derived cell. For example, mesenchymal stem cells are derived from mesenchymal tissue and can then be differentiated into a variety of cell types.

[0039] An "exogenous nucleic acid," as used herein, is a nucleic acid that does not naturally occur in a subject cell.

[0040] An "exogenous polypeptide," as used herein, is a polypeptide encoded by an exogenous nucleic acid introduced into a cell. Reference to an amino acid position of a particular sequence refers to the position of said amino acid in the reference amino acid sequence, e.g., the sequence of the full-length mature (after signal peptide cleavage) wild-type protein (unless otherwise stated), and does not exclude the presence of variations, e.g., deletions, insertions and / or substitutions, at other positions in the reference amino acid sequence.

[0041] "Factor VII protein" or "FVII protein", as used herein, unless otherwise specified, refers to a polypeptide comprising the amino acid sequence of a naturally occurring Factor VII protein or a variant thereof having FVII biological activity (e.g., promoting blood clotting) as determined by art-recognized assays. Naturally occurring FVII exists as a single-chain zymogen, a zymogen-like two-chain polypeptide, and a fully activated two-chain form (FVIIa). In some embodiments, reference to FVII includes single-chain and its two-chain forms (including zymogen-like and FVIIa). FVII proteins that can be produced by the genetically modified cells described herein (e.g., derived from a human epithelial cell line, e.g., ARPE-19 cell line) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, and variants having one or more amino acid substitutions and / or deletions. In some embodiments, the variant FVII protein is capable of being activated to a fully activated two-chain form (Factor VIIa) having at least 50%, 75%, 90% or more (including >100%) of the activity of wild-type Factor VIIa. Variants of FVII and FVIIa are known, such as Marzeptacog alfa (activated) (MarzAA) and the variants described in European Patent No. 1373493, U.S. Patent No. 7771996, U.S. Patent No. 9476037 and U.S. Published Application No. US2008 / 0058255.

[0042] Factor VII biological activity can be quantified by art-recognized assays unless otherwise specified.For example, FVII biological activity in a sample of biological fluid, such as plasma, can be quantified by (i) measuring the amount of factor Xa and factor X produced in a system containing tissue factor (TF) embedded in lipid membrane (Persson et al., J.Biol.Chem.272:19919-19924,1997);(ii) measuring factor X hydrolysis in an aqueous system;(iii) measuring its physical binding to TF using a surface plasmon resonance-based device (Persson, FEBS Letts.413:359-363,1997);or (iv) measuring the hydrolysis of a synthetic substrate;and / or (v) measuring the generation of thrombin in a TF-independent in vitro system. In one embodiment, FVII activity is assessed by a commercially available chromogenic assay (BIOPHEN FVII, HYPHEN BioMed Neuville sur Oise, France) in which a biological sample containing FVII is mixed with thromboplastin calcium, factor X and SXa-11 (a chromogenic substrate specific for factor Xa).

[0043] "Factor VIII protein" or "FVIII protein", as used herein, unless otherwise specified, refers to a polypeptide comprising the amino acid sequence of a naturally occurring factor VIII polypeptide or a variant thereof having FVIII biological activity, e.g., coagulation activity, as determined by art-recognized assays. FVIII proteins that may be expressed by the genetically modified cells described herein (e.g., derived from a human epithelial cell line, e.g., ARPE-19 cell line) include wild-type primate (e.g., human), porcine, canine, and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, variants having one or more amino acid substitutions and / or deletions, B-domain deleted (BDD) variants, single-chain variants, and fusions of any of the foregoing wild-type or variants with a half-life extending polypeptide. In one embodiment, the cell comprises an exogenous sequence encoding a precursor factor VIII polypeptide (e.g., having a signal sequence) having a complete or partial deletion of the B domain. In one embodiment, the cell comprises an exogenous sequence encoding a single-chain factor VIII polypeptide. In one embodiment, the expressed FVIII protein is a variant FVIII protein having at least 50%, 75%, 90% or more (including >100%) of the clotting activity of the corresponding wild-type factor VIII, e.g., human wild-type FVIII. Assays for measuring the clotting activity of FVIII proteins include one-stage or two-stage clotting assays (Rizza et al., 1982, Coagulation assay of FVIII:C and FIXa in Bloom ed. The Hemophelias. NY Churchill Livingston 1992) or chromogenic substrate FVIII:C assay (Rosen, S. 1984. Scand J Haematol 33:139-145, suppl.).

[0044] Numerous FVIII-BDD variants are known, including, for example, those described in the following U.S. Patent Nos. 4,868,112 (e.g., column 2, lines 2 to column 19, lines 21 and Table 2); 5,112,950 (e.g., column 2, lines 55 to 68, Figure 2, and Example 1); 5,171,844 (e.g., column 4, lines 22 to column 5, lines 36); 5,543,502 (e.g., column 2, lines 17 to 46); 5,595,886; 5,610,278; 5,789,203 (e.g., column 2, lines 26 to 51 and Examples 5 to 8); and 5,972,885 (e.g., column 1, lines 25 to column 2, lines 40). ); 6,048,720 (e.g., column 6, lines 1-22 and Example 1); 6,060,447; 6,228,620; 6,316,226 (e.g., column 4, lines 4-5, line 28 and Examples 1-5); 6,346,513; 6,458,563 (e.g., column 4, lines 25-53) and 7,041,635 (e.g., column 2, lines 1-3, lines 19; column 3, lines 40-4, line 67; column 7, lines 43-8, line 26; and column 11, lines 5-13, line 39). In one embodiment, the encapsulated cells comprise an exogenous nucleotide sequence encoding the mature FVII-BDD amino acid sequence shown in Figure 3 of WO2019 / 067766.

[0045] In some embodiments, the FVIII-BDD protein produced by the genetically modified cells described herein (e.g., derived from a human epithelial cell line, e.g., the ARPE-19 cell line) has one or more of the following deletions of amino acids in the B-domain: (i) most of the B-domain except for the amino-terminal B-domain sequence essential for intracellular processing of the primary translation product into two polypeptide chains (WO 91 / 09122); (ii) amino acids 747-1638 (Hoeben RC, et al. J. Biol. Chem. 265(13):7318-7323 (1990)); amino acids 771-1666 or amino acids 868-1562 (Meulien P., et al. Protein Eng. 2(4):301-6 (1988); amino acids 982-1562 or 760-1639 (Toole et al. 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)), 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); deletions involving one or more residues in the Furin protease recognition sequence, including any of the specific deletions described in U.S. Patent No. 9,956,269 at column 10, line 65 to column 11, line 36.

[0046] 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).

[0047] In some embodiments, the FVIII-BDD protein is a single-chain variant generated by substitution or deletion of one or more amino acids in the Furin protease recognition sequence LKRHQR (including any of the substitutions at positions R1645 and / or R1648 described in U.S. Patent Nos. 10,023,628, 9,394,353 and 9,670,267) that prevents proteolytic cleavage at this site.

[0048] 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 the FVIII-BDD protein (Miao, HZ, et al., Blood 103(a):3412-3419 (2004); albumin fusion (WO2011 / 020866); and Fc fusion (WO04 / 101740).

[0049] All FVIII-BDD amino acid positions referenced herein refer to positions in full-length human FVIII unless otherwise specified.

[0050] "Factor IX protein" or "FIX protein", as used herein, unless otherwise specified, refers to a polypeptide comprising the amino acid sequence of a naturally occurring factor IX protein or a variant thereof having FIX biological activity, e.g., clotting activity, as determined by art-recognized assays. FIX is produced as an inactive zymogen that is converted to an active form by factor XIa excision of the activation peptide to generate heavy and light chains held together by one or more disulfide bonds. FIX proteins that can be produced by the genetically modified cells described herein (e.g., derived from RPE cell lines, e.g., ARPE-19 cell lines) 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, and fusions of any of the foregoing wild-type or variant proteins with a half-life extending polypeptide. In one embodiment, the cells are engineered to encode a full-length wild-type human factor IX polypeptide (e.g., with a signal sequence) or a functional variant thereof. The variant FIX protein preferably has at least 50%, 75%, 90% or more (including >100%) of the clotting activity of wild-type factor VIX. Assays for measuring the clotting activity of FIX proteins include the Biophen Factor IX Assay (Hyphen BioMed) and one-stage clotting assays (activated partial thromboplastin time (aPTT) (e.g. as described in EP 2032 607), thrombin generation time assay (TGA) and rotational thromboelastometry (e.g. as described in WO 2012 / 006624).

[0051] Numerous functional FIX variants are known and may be expressed by engineered cells encapsulated in the devices described herein, including any of the functional FIX variants described in the following international patent publications: WO02 / 040544, page 4, lines 9-30 and page 15, lines 6-31; WO03 / 020764, Tables 2 and 3, pages 14-24 and page 12, lines 1-27; WO2007 / 149406, page 4, line 1-19, line 11; WO2007 / 149406A2, page 19, lines 12-20 p., line 9; WO08 / 118507, p. 5, line 14 to p. 6, line 5; WO09 / 051717, p. 9, line 11 to p. 20, line 2; WO09 / 137254, p. 2, paragraph

[0006] to p. 5, paragraph

[0011] and p. 16, paragraph

[0044] to p. 24, paragraph

[0057] ; WO09 / 130198A2, p. 4, line 26 to p. 12, line 6; WO09 / 140015, p. 11, paragraph

[0043] to p. 13, paragraph

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

[0052] 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 extension moieties include Fc, albumin, PAS sequence, transferrin, CTP (the 28 amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) with its four O-glycans), polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin-binding polypeptides, albumin-binding small molecules, or any combination thereof. An exemplary FIX polypeptide is the rFIXFc protein described in WO2012 / 006624, which is a FIXFc single chain (FIXFc-sc) and a Fc single chain (Fc-sc) linked together via two disulfide bonds in the hinge region of Fc.

[0053] FIX variants also include gain and loss of function variants. An example of a gain of function variant is the "Padua" variant of human FIX, which has L (leucine) instead of R (arginine) at position 338 (corresponding to amino acid position 384 of SEQ ID NO: 20) of the mature protein and has higher catalytic and aggregation activity compared to wild-type human FIX (Chang et al., J. Biol. Chem., 273:12089-94 (1998)). An example of a loss of function variant is an alanine substituted for lysine at the 5th amino acid position from the start of the mature protein, which results in a protein with reduced binding (e.g., loss of function) to collagen IV.

[0054] "Pancreatic islet cell," as used herein, refers to any cell, naturally occurring or synthetically produced or modified, and intended to partially or wholly recapitulate, mimic, or otherwise express the function of a cell of a pancreatic islet of Langerhans. The term "pancreatic islet cell" includes glucose-responsive insulin-producing cells derived from stem cells, e.g., induced pluripotent stem cell lines.

[0055] A "genetically modified cell," as used herein, is a cell (e.g., an RPE cell) that has a non-naturally occurring modification, typically comprising a nucleic acid sequence (e.g., exogenous DNA or RNA) or polypeptide that is not present in other similar cells under similar conditions that are not genetically modified (e.g., lacking the exogenous nucleic acid sequence). In one embodiment, the genetically modified cell comprises an exogenous nucleic acid (e.g., a vector or modified chromosomal sequence). In one embodiment, the genetically modified cell comprises an exogenous polypeptide. In one embodiment, the genetically modified cell comprises an exogenous nucleic acid sequence, e.g., a sequence, e.g., DNA or RNA, that is not present in similar cells that are not genetically modified. In one embodiment, the exogenous nucleic acid sequence is chromosomal, e.g., the exogenous nucleic acid sequence is an exogenous sequence located within an endogenous chromosomal sequence. In one embodiment, the exogenous nucleic acid sequence is chromosomal or extrachromosomal, e.g., a non-integrated vector. In one embodiment, the exogenous nucleic acid sequence comprises an RNA sequence, e.g., an mRNA. In one embodiment, the exogenous nucleic acid sequence comprises a chromosomal or extrachromosomal exogenous nucleic acid sequence comprising a sequence that is expressed as an RNA, e.g., an mRNA or a regulatory RNA. In one embodiment, the exogenous nucleic acid sequence comprises a chromosomal or extrachromosomal exogenous nucleic acid sequence comprising a sequence that encodes or is expressed as a polypeptide. In one embodiment, the exogenous nucleic acid sequence comprises a first chromosomal or extrachromosomal exogenous nucleic acid sequence that regulates the conformation or expression of a second nucleic acid sequence, and the second amino acid sequence can be exogenous or endogenous. For example, the genetically modified cell can comprise an exogenous nucleic acid that controls the expression of an endogenous sequence. In one embodiment, the genetically modified cell comprises a polypeptide that is present at a level or distribution that differs from that found in a similar cell that is not genetically modified. In one embodiment, the genetically modified cell comprises a RPE that has been genetically modified to produce an RNA or a polypeptide. For example, the genetically modified cell can comprise an exogenous nucleic acid sequence comprising a chromosomal or extrachromosomal exogenous nucleic acid sequence comprising a sequence that is expressed as an RNA, e.g., an mRNA or a regulatory RNA.In one embodiment, the genetically modified cell (e.g., RPE cell) comprises an exogenous nucleic acid sequence comprising a chromosomal or extrachromosomal nucleic acid sequence comprising a sequence encoding or expressing a polypeptide. In one embodiment, the polypeptide is encoded by a codon-optimized sequence to achieve higher expression of the polypeptide than the naturally occurring coding sequence. The codon-optimized sequence can be generated using commercially available algorithms, such as GeneOptimizer (ThermoFisher Scientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), or Java Codon Adaptation Tool (JCat, www.jcat.de, Grote, A. et al., Nucleic Acids Research, Vol 33, Issue suppl_2, pp. W526-W531 (2005)). In one embodiment, the genetically modified cell (e.g., RPE cell) comprises an exogenous nucleic acid sequence that regulates the conformation or expression of an endogenous sequence. In one embodiment, the genetically modified cells (e.g., RPE cells) are cultured from a population of stably transfected cells or from a monoclonal cell line.

[0056] A "peptide," as used herein, is a polypeptide of fewer than 50 amino acids, typically fewer than 25 amino acids.

[0057] "Iduronate-2-sulfatase protein," "IDS protein," "I2S protein," and "alpha-L-iduronate sulfate sulfatase protein" may be used interchangeably herein to refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) IDS gene or any fragment, mutant, variant, or derivative thereof having an IDS enzymatic activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature IDS protein as measured by any art-recognized IDS assay. IDS hydrolyzes the 2-sulfate group of the L-iduronate 2-sulfate units of dermatan sulfate, heparan sulfate, and heparan. The wild-type human IDS gene encodes a precursor propolypeptide of 550 amino acids, the N-terminal 25 amino acids of which constitute a signal peptide and the remaining amino acids constitute the propolypeptide, which is processed to the mature polypeptide by removal of the propeptide from amino acids 26 to 33, followed by cleavage into two chains formed by amino acids 34 to 455 and amino acids 456 to 550. (UniProtKB-P22304). In one embodiment, the GBA protein is part of a fusion protein further comprising one or more amino acid sequences from one or more heterologous polypeptides.

[0058] "Osmolality" and "mOsm" are used herein to refer to a measure of the osmotic pressure of dissolved solute particles in an aqueous solution. Solute particles include both ions and non-ionized molecules. Osmolality is typically expressed as the concentration of osmotically active particles (i.e., osmoles) dissolved in 1 kg of solution. "Osmolarity" on the other hand refers to the number of solute particles dissolved in 1 liter of solution. For aqueous solutions, osmolarity is temperature dependent because water changes its volume with temperature. Therefore, osmolarity is the preferred measure for aqueous solutions because it is not temperature dependent. When the concentration of solutes is very low, osmolarity and osmolarity are considered equivalent. As used herein, the abbreviation "mOsm" means "milliosmoles / kg solution."

[0059] A "peptide," as used herein, is a polypeptide of fewer than 50 amino acids, typically fewer than 25 amino acids.

[0060] "Poly A" signal, as used herein, refers to any contiguous sequence of adenylic acids that terminates transcription of a coding sequence into RNA and induces the direct addition of a poly A tail to the RNA. The length of the poly A sequence is 10-200 nucleotides and can be variously controlled depending on the allowable size of the backbone of the expression vector. Examples of poly A signals are rabbit binding globulin (rBG) poly A signal, SV40 late poly A signal, SV50 poly A signal, bovine growth hormone (BGH) poly A signal, human growth hormone (HGH) poly A signal, and synthetic poly A signals known in the art.

[0061] A "polymer composition," as used herein, is a composition (e.g., solution, mixture) that includes one or more polymers. As a class, "polymers" include homopolymers, heteropolymers, copolymers, block polymers, and block copolymers, and can be both natural and synthetic. Homopolymers contain one type of building block, or monomer, while copolymers contain multiple types of monomers.

[0062] A "polypeptide," as used herein, is a polymer comprising amino acid residues linked via peptide bonds and having at least 2, and in some embodiments at least 10, 50, 75, 100, 150 or 200 amino acid residues.

[0063] "Prevention," "prevent," and "preventing," as used herein, refer to treatments that include administering or applying a therapy, e.g., administering a hydrogel capsule composition described herein, prior to the onset of a disease, disorder, or condition to eliminate physical signs of said disease, disorder, or condition. In some embodiments, "prevention," "prevent," and "preventing" require that no signs or symptoms of a disease, disorder, or condition have developed or been observed. In some embodiments, treatment includes prevention, while in other embodiments it does not include prevention.

[0064] "Promoter sequence" as used herein refers to a nucleotide sequence capable of inducing expression in a mammalian cell, e.g., a human cell, e.g., an ARPE-19 cell. In some embodiments, the promoter sequence is from a strong mammalian promoter, e.g., a human promoter sequence. Non-limiting examples of strong promoters for use in the genetically modified cells described herein include the EF-1 alpha (EF1A) promoter, the CAG promoter, the PGK (phosphoglycerate kinase) promoter, and the ACTB (human beta-actin) promoter. In one embodiment, the promoter sequence can be from a medium-strength promoter, e.g., the EFS promoter sequence (which is a shortened form of the EF1A promoter sequence).

[0065] A "protein," as used herein, includes one or more polypeptide chains at least 50 amino acids in length. In one embodiment, a protein has two or more polypeptide chains having identical or non-identical amino acid sequences at least 50 amino acids in length. In one embodiment, the polypeptide chains in a protein are non-covalently associated or covalently connected, for example, via disulfide bond(s).

[0066] "RPE cells," as used herein, refer to cells having one or more of the following characteristics: a) retinal pigment epithelial cells (RPE) (e.g., cultured using the ARPE-19 cell line (ATCC® CRL-2302™)) or cells derived therefrom, e.g., by stably transfecting cultured cells from the ARPE-19 cell line with an exogenous sequence encoding a therapeutic agent or by otherwise engineering such cultured ARPE-19 cells to express an exogenous protein or other exogenous substance; cells derived from primary cell cultures of RPE cells; naturally occurring (b) RPE cells that have been developed, programmed, or reprogrammed (e.g., in vitro fertilization) into one or more of the following: RPE cells that have been transformed, immortalized, or derived from long-term (e.g., more than 5 or 10 passages or rounds of cell division since isolation) RPE cells isolated directly from a human or other mammal (without long-term culture, e.g., less than 5 or 10 passages or rounds of cell division since isolation); (c) RPE cells that have been transformed, immortalized, or derived from long-term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell cultures; (d) undifferentiated cells, e.g., RPE cells or cells that, except for any genetic manipulation, are substantially similar to one or more of naturally occurring RPE cells or cells from primary or long-term cultures of RPE cells (e.g., the cells can be derived from IPS cells). or c) cells obtained from cells grown in vitro; or c) cells with one or more of the following characteristics: i) express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) do not express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; iii) are naturally found in the retina and form a monolayer over the choroidal blood vessels in Bruch's membrane; iv) are responsible for epithelial transport, light absorption, secretion, and immunoregulation in the retina; or v) immortalized RPE cell lines (e.g., the ARPE-19 cell line (ATCC 61221) OThe RPE cells may be 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 RPE cells derived from human RPE cells (e.g., RPE cells derived from human RPE cells 100-1400 cells). In one embodiment, the RPE cells described herein are genetically modified, e.g., to have new properties, e.g., the cells are genetically modified to express and secrete one or more therapeutic agents. In other embodiments, the RPE cells are not genetically modified.

[0067] "Sequence identity" or "percent identity," as used herein to refer to two nucleotide sequences or two amino acid sequences, means that the two sequences are the same within a specified region, or have the same nucleotides or amino acids at a specified percentage of nucleotide or amino acid positions within a specified region when the two sequences are compared and aligned for maximum correspondence over a comparison window or designated region. Sequence identity may be determined using standard techniques known in the art, including, but not limited to, any of the algorithms described in U.S. Patent Application Publication No. 2017 / 02334455. In one embodiment, the specified percentage of identical nucleotide or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0068] "Spherical" as used herein means a hydrogel capsule with a curved surface, forming a sphere (e.g., a perfectly round ball) or a sphere-like shape, which may have, for example, waves and undulations on the surface. Spheres and sphere-like objects can be mathematically defined by the rotation of a circle, an ellipse, or a combination around each of the three orthogonal axes a, b, and c. For a sphere, the three axes are of equal length. Typically, a sphere-like shape is an ellipsoid (about its average surface) with semi-major axes within 10%, or 5%, or 2.5% of each other. The diameter of a sphere or sphere-like shape is the average diameter, e.g., the average of the semi-major axes.

[0069] "Ellipsoidal," as the term is used herein to refer to a hydrogel capsule, means that the capsule (i) has a perfect or classical oblate or prolate ellipsoid shape, or (ii) has a surface that roughly forms an ellipsoid, e.g., may have waves and undulations and / or may be ellipsoidal (about its averaged surface) with semi-major axes within 100% of each other.

[0070] "Subject" as used herein refers to a human or a non-human animal. In one embodiment, the subject is, for example, a human (i.e., male or female) of any age group, a pediatric subject (e.g., infant, childhood, adolescent) or an adult subject (e.g., young adult, middle-aged adult, or elderly adult). In one embodiment, the subject is a non-human animal, for example, a mammal (e.g., mouse, dog, primate (e.g., cynomolgus or rhesus monkey)). In one embodiment, the subject is a commercially relevant mammal (e.g., cow, pig, horse, sheep, goat, cat, or dog) or bird (e.g., commercially relevant bird, e.g., 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.

[0071] "Transcription unit" refers to a DNA sequence, e.g., present in an exogenous nucleic acid, that includes at least a promoter sequence operably linked to a coding sequence, and may also include one or more additional elements that control or enhance transcription of the coding sequence into an RNA molecule or translation of an RNA molecule into a polypeptide molecule. In some embodiments, a transcription unit also includes a polyadenylation (polyA) signal sequence and a polyA site.

[0072] "Treatment", "treat", and "treating" as used herein refer to one or more of reducing, reversing, alleviating, delaying the onset, or inhibiting the progression of one or more of the symptoms, signs, or underlying causes of a disease, disorder, or condition. In one embodiment, treating includes reducing, reversing, alleviating, delaying the onset, or inhibiting the progression of a symptom of a disease, disorder, or condition. In one embodiment, treating includes reducing, reversing, alleviating, delaying the onset, or inhibiting the progression of a sign of a disease, disorder, or condition. In one embodiment, treating includes reducing, reversing, alleviating, reducing, or delaying the onset of an underlying cause of a disease, disorder, or condition. In some embodiments, "treatment", "treat", and "treating" require that a sign or symptom of a disease, disorder, or condition has developed or been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of a disease or condition, e.g., in a prophylactic treatment. For example, therapy (e.g., a hydrogel capsule composition) may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in view of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, e.g., to delay or prevent recurrence. In some embodiments, treatment includes prevention, while in other embodiments it does not include prevention.

[0073] Selected Chemical Definitions Definitions of certain functional groups and chemical terms are described in more detail below. Chemical elements are defined in the Handbook of Chemistry and Physics, 75 thEd. (back cover), and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, as well as specific functional sites and reactivities, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 th 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, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0074] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulas depicted herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0075] When a range of values ​​is listed, it is intended to encompass each value and subrange within the range. For example, "C 1 -C 6 Alkyl" is 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 -C 3 , 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 is intended to be included.

[0076] As used herein, "alkyl" refers to the radical of a linear or branched saturated hydrocarbon group having 1 to 24 carbon atoms ("C 1 -C 24 In some embodiments, an alkyl group is an alkyl group having 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 one carbon atom ("C 1 In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C 2 -C 6 "Alkyl"). C 1 -C 6 Examples of alkyl groups include methyl (C 1 ), ethyl (C 2 ), n-propyl (C3 ), isopropyl (C 3 ), n-Butyl (C 4 ), tert-Butyl (C 4 ), sec-Butyl (C 4 ), iso-butyl (C 4 ), n-pentyl (C 5 ), 3-pentanyl (C 5 ), Amyl (C 5 ), neopentyl (C 5 ), 3-methyl-2-butanyl (C 5 ), tertiary amyl (C 5 ), and n-hexyl (C 6 Additional examples of alkyl groups include n-heptyl (C 7 ), n-octyl (C 8 ), and the like. Each example of an alkyl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkyl") or substituted with one or more substituents; for example, with, for example, 1-5 substituents, 1-3 substituents, or 1 substituent (a "substituted alkyl").

[0077] As used herein, "alkenyl" refers to a radical of a straight or branched chain 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 refers to an alkyl group having 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 two carbon atoms ("C 2The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). 2 -C 4 Examples of alkenyl groups include ethenyl (C 2 ), 1-propenyl (C 3 ), 2-propenyl (C 3 ), 1-butenyl (C 4 ), 2-butenyl (C 4 ), butadienyl (C 4 ) etc. 2 -C 6 Examples of alkenyl groups include the above-mentioned C 2-4 Alkenyl and pentenyl (C 5 ), pentadienyl (C 5 ), hexenyl (C 6 ), and the like. Each instance of an alkenyl group may independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or substituted with one or more substituents; for example, with, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted alkenyl").

[0078] As used herein, the term "alkynyl" refers to a radical of a linear or branched hydrocarbon group having 2 to 24 carbon atoms and one or more carbon-carbon triple bonds ("C 2 -C 24 In some embodiments, an alkynyl group refers to an alkynyl group having 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 two carbon atoms ("C2 The one or more carbon-carbon triple bonds can be internal (such as in 2-butynyl) or terminal (such 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 ), and the like. Each example of an alkynyl group independently may be optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents; for example, with, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted alkynyl").

[0079] As used herein, the term "heteroalkyl" refers to an acyclic stable straight or branched chain, or combinations thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, where the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) O, N, P, S, and Si may be substituted at any position of the heteroalkyl group. Exemplary heteroalkyl groups include -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, e.g., -CH 2 -NH-OCH 3 and -CH 2 -O-Si(CH 3 ) 3 etc. "Heteroalkyl" may be described followed by a particular heteroalkyl group, such as, for example, -CH 2 O, -NR C R D etc., the term heteroalkyl and -CH 2 -O or -NR C R D It is understood that the terms "heteroalkyl" and "heteroalkyl-alkyl" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to provide clarity. Thus, the term "heteroalkyl" refers to a specific heteroalkyl group, such as, for example, -CH 2 O, -NR C R D and the like. Each instance of a heteroalkyl group may independently be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroalkyl") or substituted with one or more substituents; for example, substituted with, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted heteroalkyl").

[0080] The terms “alkylene,” “alkenylene,” “alkynylene,” or “heteroalkylene,” by themselves or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively. An alkylene, alkenylene, alkynylene, or heteroalkylene group can be, for example, C 1 -C 6 Alkylene, C2 -C 6 Alkenylene, C 2 -C 6 C 1 -C 6 Heteroalkylene may be described as a heteroalkylene having a 1-membered heteroatom, where the term "membered" refers to a non-hydrogen atom in the moiety. In the case of heteroalkylene groups, heteroatoms may also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, a group of the formula -C(O) 2 R'- is -C(O) 2 R'- and -R'C(O) 2 - can represent both.

[0081] As used herein, "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared by the cyclic array) having 6 to 14 ring carbon atoms and 0 heteroatoms provided in the aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared by the cyclic array). 6 -C 14 In some embodiments, an aryl group has six ring carbon atoms ("C 6 aryl"; e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 Aryl"; for example, naphthyl, e.g., 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 aryl"; e.g., anthracyl). The aryl group is, e.g., C 6 -C 10 The term "membered" refers to a non-hydrogen ring atom in the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl").

[0082] 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 pi electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being 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 at a carbon or nitrogen atom, if valence permits. Heteroaryl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused with one or more aryl groups with the point of attachment being either on the aryl or heteroaryl ring, and in such instances the number of ring members refers to the number of ring members in the fused (aryl / heteroaryl) ring system. In bicyclic heteroaryl groups where one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., on the ring bearing a heteroatom (e.g., 2-indolyl) or on the ring that does not contain a heteroatom (e.g., 5-indolyl). Heteroaryl groups can be described as, for example, 6- to 10-membered heteroaryl, with the term "member" referring to the non-hydrogen ring atoms in the moiety.

[0083] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided by the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided by the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided by the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has one ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of a heteroaryl group independently may be optionally substituted, i.e., unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl").

[0084] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridazinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.

[0085] The terms "arylene" and "heteroarylene," as used herein, alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.

[0086] As used herein, "cycloalkyl" refers to alkyl groups having 3 to 10 ring carbon atoms in a non-aromatic ring system ("C 3 -C 10 Cycloalkyl refers to a radical of a non-aromatic cyclic hydrocarbon group having 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 groups include, for example, C 4 -C 7 cycloalkyl, where the term "membered" refers to a non-hydrogen ring atom within the moiety. 3 -C 6 Cycloalkyl groups include, but are 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 ) and others. 3 -C 8 Cycloalkyl groups include, but are not limited to, those described above. 3 -C 6 Cycloalkyl groups and cycloheptyl (C 7 ), cycloheptenyl (C 7 ), cycloheptadienyl (C 7 ), cycloheptatrienyl (C 7 ), cyclooctyl (C 8 ), cyclooctenyl (C 8 ), cubanyl (C 8 ), bicyclo[1.1.1]pentanyl (C 5 ), bicyclo[2.2.2]octanyl (C 8), bicyclo[2.1.1]hexanyl (C 6 ), bicyclo[3.1.1]heptanyl (C 7 ) and others. 3 -C 10 Cycloalkyl groups include, but are not limited to, those described above. 3 -C 8 Cycloalkyl groups and cyclononyl groups (C 9 ), cyclononenyl (C 9 ), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C 9 ), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10 ), and the like. As the preceding examples illustrate, in certain embodiments, the cycloalkyl group is monocyclic ("monocyclic cycloalkyl") or contains fused, bridged, or spiro ring systems, e.g., bicyclic systems ("bicyclic cycloalkyl"), and can be saturated or partially unsaturated. "Cycloalkyl" also includes ring systems in which a cycloalkyl ring, as defined above, is fused with one or more aryl groups, with the point of attachment being on the cycloalkyl ring, and in such instances the number of carbons continues to designate 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 with one or more substituents ("substituted cycloalkyl").

[0087] "Heterocyclyl," as used herein, is a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, where valence permits. Heterocyclyl groups may be either monocyclic ("monocyclic heterocyclyl") or fused, bridged, or spiro ring systems, e.g., bicyclic systems ("bicyclic cycloalkyl"), and may be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring as defined above is fused with one or more cycloalkyl groups with the point of attachment either on the cycloalkyl or heterocyclyl ring, or a heterocyclyl ring as defined above is fused with one or more aryl or heteroaryl groups with the point of attachment on the heterocyclyl ring, in such instances the number of ring members continues to designate the number of ring members in the heterocyclyl ring system. Heterocyclyl groups may be described, for example, as 3- to 7-membered heterocyclyl, with the term "member" referring to the non-hydrogen ring atoms in the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of heterocyclyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 10-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 10-membered heterocyclyl.

[0088] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 6-membered heterocyclyl"). In some embodiments, a 5- to 6-membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0089] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl or thiomorpholinyl-1,1-dioxide.

[0090] Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl. 6Exemplary 5-membered heterocyclyl groups (also referred to herein as 5,6-bicyclic heterocyclic rings) fused to an aryl ring include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, etc. Exemplary 6-membered heterocyclyl groups (also referred to herein as 6,6-bicyclic heterocyclic rings) fused to an aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[0091] "Amino" as used herein refers to the radical -NR 70 R 71 (In the formula, R 70 and R 71 are each independently hydrogen, C 1 -C 8 Alkyl, C 3 -C 10 Cycloalkyl, C 4 -C 10 Heterocyclyl, C 6 -C 10 Aryl and C 5 -C 10 In some embodiments, amino refers to NH 2 Refers to...

[0092] As used herein, "cyano" refers to the radical --CN.

[0093] As used herein, "halo" or "halogen," independently or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0094] As used herein, "hydroxy" refers to the radical --OH.

[0095] Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" cycloalkyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl groups). Typically, the term "substituted," whether preceded by the term "optionally," means that at least one hydrogen present on the group (e.g., a carbon or nitrogen atom) is replaced with an acceptable substituent, e.g., a substituent that provides for a stable compound (e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reaction). Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when multiple positions in any given structure are substituted, the substituents are the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein, that result in the formation of a stable compound. The present disclosure contemplates all such combinations to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0096] Two or more substituents may be optionally connected to form an aryl, heteroaryl, cycloalkyl, or heterocyclyl group. Such so-called ring-forming substituents are typically, but not necessarily, found to be attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure generate a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure generate a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.

[0097] The compounds of formula (I) described herein may contain one or more asymmetric centers and therefore may exist in various isomeric forms, such as enantiomers and / or diastereomers.For example, the compounds described herein may be in the form of individual enantiomers, diastereomers or geometric isomers, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers.Isomers may be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure additionally encompasses the compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0098] As used herein, a pure enantiomer compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). That is, the "S" form of the compound is substantially free of the "R" form of the compound, and is thus in enantiomeric excess of the "R" form. The term "enantiomerically pure" or "pure enantiomer" indicates that the compound contains 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.

[0099] The compounds of formula (I) described herein may also contain one or more isotopic substitutions. For example, H is: 1 H, 2 H (D or deuterium), and 3 H can be in any isotopic form, including T or tritium; 12 C. 13 C, and 14 It can be in any isotopic form, including C; O is 16 O and 18 It can be any isotopic form including O; and so forth.

[0100] The term "pharmaceutically acceptable salts" is meant to include salts of active compounds prepared with relatively non-toxic acids or bases, depending on the specific substituents found in the compounds described herein. When the compounds of formula (I) used to prepare the hydrogel capsules of the present disclosure contain a relatively acidic functionality, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. When the compounds used in the present disclosure contain a relatively basic functionality, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monohydrogencarbonic acid, phosphoric acid, monohydrogenphosphoric acid, dihydrogenphosphoric acid, sulfuric acid, monohydrogensulfuric acid, hydroiodic acid, or phosphorous acid, and those derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, methanesulfonic acid, and the like. Also included are salts of amino acids, such as alginates, and salts of organic acids, such as glucuronic acid or galacturonic acid (see, e.g., Berge et al, Journal of Pharmaceutical Science 66:1-19 (1977)). Certain compounds used in the hydrogel capsules of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. These salts may be prepared by methods known to those of ordinary skill in the art. Other pharma- ceutically acceptable carriers known to those of ordinary skill in the art are suitable for use in the present disclosure.

[0101] The hydrogel capsules in the composition of the present disclosure, for example, a population of hydrogel capsules in the composition, may contain the compound of formula (I) in prodrug form. Prodrugs are those compounds that easily undergo chemical changes under physiological conditions to provide compounds useful for preparing capsules in the present disclosure. Also, prodrugs can be converted to useful compounds of formula (I) by chemical or biochemical methods in an ex vivo environment.

[0102] Certain compounds of formula (I) described herein may exist in solvated forms, including unsolvated forms and hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are included within the scope of the present disclosure. Certain compounds of formula (I) described herein may exist in polycrystalline or amorphous forms. In general, all physical forms are equivalent for the use contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0103] The term "solvate" refers to a form of a compound associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, for example, in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates, and further include both stoichiometric and non-stoichiometric solvates.

[0104] The term "hydrate" refers to a compound associated with water. Typically, the number of water molecules contained in a hydrate of a compound is within a defined ratio to the number of compound molecules in the hydrate. Thus, a hydrate of a compound may be, for example, a compound having the general formula R×xH 2 O, where R is a compound and x is a number greater than 0.

[0105] symbol [ka] As used herein, refers to the connection of an entity, e.g., a polymer (e.g., a hydrogel-forming polymer, e.g., alginate) or surface of a hydrogel capsule. [ka] The connection represented by may refer to a direct bond to an entity, such as a polymer or capsule surface, or may refer to a link to an entity via a linking group. "Linking group," as described herein, refers to a site for linking a compound of formula (I) to an entity (e.g., a polymer or hydrogel capsule described herein) and may include any linking chemistry known in the art. A list of exemplary linking groups can be found in Bioconjugate Techniques (3 rd ed. Greg T. Hermanson, Waltham, MA: Elsevier, Inc, 2013), which is incorporated herein by reference in its entirety. In some embodiments, the linking group is an 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-containing, R A , R C , R D , RF , R G Each of x, x, and y is independently as described herein. In some embodiments, the linking group comprises an amine, a ketone, an ester, an amide, an alkyl. In some embodiments, the linking group is a crosslinker. In some embodiments, the linking group is a -C(O)(C 1 -C 6 1 and R 1 is as described herein. In some embodiments, the linking group is -C(O)(C 1 -C 6 -alkylene)-, where the alkylene is substituted with 1 to 2 alkyl groups (e.g., 1 to 2 methyl groups). In some embodiments, the linking group is -C(O)C(CH 3)2 In some embodiments, the linking group is -C(O)(methylene)-, where the alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the linking group is -C(O)CH(CH 3 In some embodiments, the linking group is -C(O)C(CH 3 )-.

[0106] Characteristics of Hydrogel Capsule Composition The hydrogel capsule composition of the present disclosure comprises a population of hydrogel capsules disposed in a pharma- ceutically acceptable aqueous solution. The hydrogel capsules in the population encapsulate cells (e.g., mammalian cells) and comprise a hydrogel-forming polymer, e.g., a naturally occurring or synthetic polysaccharide. Exemplary polysaccharides include, e.g., alginate, agar, agarose, carrageenan, cellulose and amylose, chitin, chitosan, and hyaluronate. The hydrogel-forming polymer may be crosslinked, e.g., crosslinked by diacrylates, or may comprise a polysaccharide or derivative / modification thereof, e.g., as described in Laurienzo (2010), Mar. Drugs. 8.9:2435-65. In one embodiment, the capsule comprises a mixture of two or more hydrogel-forming polymers, e.g., a mixture of two alginates having different G:M contents and / or different average molecular weights. In one embodiment, the hydrogel capsules comprise an ionically crosslinked alginate(s) (e.g., crosslinked with a divalent cation, e.g., barium, calcium, magnesium, or strontium). In one embodiment, the capsules in the population comprise an ionically crosslinked alginate.

[0107] The hydrogel capsules in the composition may have any of a variety of shapes: cylinders, cylinders with hemispherical ends (also known as spherocylinders), disks, noodles (e.g., as described in WO2015 / 191547), spheres (e.g., as defined herein), or ellipsoids (e.g., as defined herein). In one embodiment, the hydrogel capsules are spheres, as defined herein.

[0108] In one embodiment, the composition includes one or more additional capsules that do not have all of the characteristics of the population of capsules, e.g., the additional non-populated capsules may be composed of a different hydrogel, may be of a different shape or size, may encapsulate a different cell type and / or average number of cells, or may not encapsulate any cells.

[0109] aqueous solution In some embodiments, the solution in the hydrogel capsule composition comprises elemental calcium at a concentration of at least about 1.0 mM but not more than about 10 mM. In one embodiment, the elemental calcium concentration is less than about 6 mM. In one embodiment, the elemental calcium concentration is about 1.2 mM to about 2.0 mM. In one embodiment, the elemental calcium concentration is about 1.5 mM to about 2.5 mM. In one embodiment, the elemental calcium concentration is about 2.0 mM. The calcium source can be any pharma- ceutically acceptable calcium salt, such as one or more of calcium acetate, calcium carbonate, calcium citrate, calcium chloride, or calcium gluconate. In one embodiment, the calcium salt is calcium chloride.

[0110] Calcium may be measured by the amount of calcium salt (mg of cation and anion, or mL of a specified concentration) or the amount of elemental calcium in milligrams (mg), milliequivalents (mEq), or millimoles (mmol). Since calcium has a valence of +2, an mEq is equal to twice the number of mM. A calcium equivalent calculator is publicly available on the Cornell University Medical College website at http: / / www-users.med.cornell.edu / ~spon / picu / calc / cacalc.htm. The amount of elemental Ca in 1 gram of common calcium salts is shown in Table 2 below. [Table 2]

[0111] In some embodiments, the aqueous solution has an osmolality of about 250 mOsm to about 350 mOsm, a pH of 6.0 to 9.0 (e.g., 6.5 to 9.0) at 12° C. to 30° C. (e.g., about 15 to 25° C.), and includes a calcium salt at an elemental calcium concentration of at least about 1.0 millimolar (mM) to about 10 mM. The components of the solution may be pharma- ceutical acceptable for administration to a mammal (e.g., a human). The components of the solution may also be capable of supporting the viability of encapsulated cells.

[0112] Cell viability can be affected by the osmolality of the aqueous solution. The osmolality of the solution can be maintained between the desired ranges by including in the solution an appropriate amount of one or more osmotically active agents known in the art, such as ions (e.g., sodium, potassium, chloride, bicarbonate, calcium, phosphate), monosaccharides (e.g., glucose, fructose), oligosaccharides (e.g., sucrose, lactose, dextrose, mannitol), amino acids, etc. The osmolality of the solution can be determined by methods known in the art using an osmometer, e.g., a freezing point depression osmometer. In one embodiment, the osmolality of the solution can be, for example, 250, 260, 265, 270, 280, 290, 300, 310, 320, 330, 340, or 350 mOsm / kg, or any number between about 250 and 350 mOsm / kg.

[0113] Since the pH of the aqueous solution may also affect cell viability and / or productivity, the pH may typically be controlled between 6.5 and 9.0 within a temperature range of 12° C. to 30° C. by including one or more pharma- ceutically acceptable buffers having an appropriate pKa and compatible with cell viability. Exemplary buffers suitable for inclusion in the aqueous solution include, but are not limited to, bicarbonate, acetate, phosphate, gluconate, and lactate. In one embodiment, the buffer is not HEPES. In one embodiment, the buffer includes sodium acetate and sodium gluconate. In one embodiment, the buffer includes sodium bicarbonate and sodium phosphate.

[0114] In some embodiments, the aqueous solution in the hydrogel capsule composition may also include at least one carbon source to help support cell viability. In one embodiment, the carbon source may be provided by an osmotically active agent or buffer in the solution. In another embodiment, each carbon source is different from the other components in the solution. Exemplary carbon sources include gluconate, sugars (e.g., dextrose, glucose, galactose, hexose, fructose, maltose), glycerol, glutamine, and pyruvate (and pharma- ceutically acceptable salts of pyruvate). In one embodiment, sodium gluconate is present in the solution to provide a carbon source and may also act as a buffer. In one embodiment, the solution includes glucose as a carbon source. In one embodiment, the solution includes about 5 mM to about 25 mM D-glucose.

[0115] In one embodiment, the aqueous solution in the hydrogel capsule composition comprises, consists essentially of, or consists of calcium chloride, sodium chloride, sodium acetate, sodium gluconate, potassium chloride, and magnesium chloride. In one embodiment, the solution consists essentially of the components listed in Tables 3A or 3B below. [Table 3] [Table 4-1] [Table 4-2]

[0116] In one embodiment, the solution of Table 3B further comprises D-glucose in an amount selected from the group consisting of about 1 mM to about 50 mM; about 2 mM to about 40 mM; about 3 mM to about 30 mM; about 4 mM to about 20 mM; about 5 mM to about 10 mM; about 10 mM to about 40 mM; about 15 mM to about 35 mM; about 20 mM to about 30 mM; and about 25 mM.

[0117] Hydrogel Capsules The composition includes a population of hydrogel capsules that encapsulate cells. All of the hydrogel capsules in the population have substantially similar composition (e.g., alginate(s) and any other polymers used in the hydrogel) and configuration (e.g., shape, permeability, number of cell-containing compartments) that allows retention of cells within the capsule, preventing immune cell infiltration while allowing passage of nutrients into the capsule and egress of cell-consumed substances and cell-produced therapeutic substances(s) from the capsule. Exemplary capsules that encapsulate cells are described in U.S. Patent Nos. 9,867,781; 10,292,936; 10,786,446; 10,898,443; and PCT International Publication Nos. WO2019 / 169245; WO2019 / 195055; WO2021 / 113751; and WO2021 / 113751.

[0118] In one embodiment, each capsule in the population of hydrogel capsules is configured as a two-compartment hydrogel capsule, with an inner compartment comprising a first alginate hydrogel that encapsulates cells, and an outer barrier hydrogel compartment (also referred to herein as the outer layer) completely surrounding the inner compartment and substantially free of encapsulated cells. The barrier compartment comprises an ionically crosslinked alginate hydrogel, optionally comprising a mixture of two or more alginates having different average molecular weights.

[0119] In one embodiment, the inner boundary of the barrier compartment forms an interface with the outer boundary of the inner compartment. In such an embodiment, the thickness of the barrier compartment refers to the average distance between the outer boundary of the barrier compartment and the interface between the two compartments, e.g., the average of the distances measured at each of the thinnest and thickest points visually observed in the barrier compartment. In some embodiments (e.g., the hydrogel capsule is about 1.5 mm in diameter), the thinnest and thickest distances for the barrier compartment are 25-110 μm and 270-480 μm, respectively. In some embodiments, the thickness of the barrier compartment is greater than about 10 nm, preferably 100 nm or more, and can be as large as 1 mm. For example, the thickness (e.g., average distance) of the barrier compartment in the hydrogel capsules described herein can be 10 nm to 1 mm, 100 nm to 1 mm, 500 nm to 1 mm, 1 μm to 1 mm, 1 μm to 1 mm, 1 μm to 1 mm, 1 μm to 500 μm, 1 μm to 250 μm, 1 μm to 1 mm, 5 μm to 500 μm, 5 μm to 250 μm, 10 μm to 1 mm, 10 μm to 500 μm, or 10 μm to 250 μm. In some embodiments, the thickness (e.g., average distance) of the barrier compartment can be 100 nm to 1 mm, 1 μm to 1 mm, 1 μm to 500 μm, or 5 μm to 1 mm. In some embodiments, the thickness (e.g., average distance) of the barrier compartment is about 50 μm to about 100 μm. In some embodiments (eg, the capsule is about 1.5 mm in diameter), the thickness (eg, average distance) of the barrier compartment is about 180 μm to 260 μm or about 310 μm to 440 μm.

[0120] In some embodiments, the average pore size of the cell-containing inner compartment and the cell-free barrier compartment is substantially the same. In some embodiments, the average pore size of the inner compartment and the barrier 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 capsule (e.g., the average pore size of the inner compartment and / or the average pore size of the barrier compartment) depends on a number of factors, such as the material(s) in each compartment and the presence and density of the compound of formula (I).

[0121] In some embodiments, the average capsule diameter or size of the dual compartment hydrogel capsules in a population may be in any of the following ranges: from about 0.5 mm to about 8 mm, from about 0.5 mm to about 4 mm, from about 0.5 mm to about 2 mm, from about 0.7 mm to about 1.3 mm, or from about 1.2 mm to about 1.8 mm.

[0122] In some embodiments, the outer surface of each hydrogel capsule in the population comprises a compound capable of reducing the FBR, such as a non-fibrous compound of formula (I) described herein below, such as compound 101. For capsules that include a barrier compartment surrounding a cell-containing compartment, the non-fibrous compound can be covalently bonded to a polymer (e.g., alginate) disposed across the barrier compartment. In one embodiment, some or all of the monomers in the polymer are modified with the same compound of formula (I). In some embodiments, some or all of the monomers in the polymer are modified with different compounds of formula (I).

[0123] In some embodiments, the alginate hydrogel in the barrier compartment comprises a non-fibrous alginate, e.g., an alginate chemically modified with a compound of formula (I). The alginate in the modified alginate can be the same or different from any unmodified alginate present in the capsule. In one embodiment, the density (e.g., amount of conjugation) of the compound of formula (I) in the modified alginate is about 4.0% to about 8.0%, about 5.0% to about 7.0%, or about 6.0% to about 7.0% nitrogen (e.g., as determined by combustion analysis for nitrogen percentage). In one embodiment, the amount of compound 101 results in an increase in % of N (as compared to unmodified alginate) of about 0.5% to 2%, 2% to 4% N, about 4% to 6% N, about 6% to 8%, or about 8% to 10% N), where % N corresponds to the amount of compound 101 in the modified alginate, as determined by combustion analysis.

[0124] In other embodiments, the density (e.g., concentration) of a compound of formula (I) (e.g., Compound 101) in a non-fibrous alginate, as determined by a suitable quantitative amine conjugation assay (e.g., by an assay described in WO2020069429), defined as % w / w in solution (e.g., saline), e.g., % weight of amine / weight of non-fibrous alginate, in certain embodiments, the density of a compound of formula (I) (e.g., Compound 101) is from about 1.0% w / w to about 3.0% w / w, from about 1.3% w / w to about 2.5% w / w, or from about 1.5% w / w to 2.2% w / w.

[0125] The alginate in the non-fibrous polymer can be chemically modified with a compound of formula (I) using any suitable method known in the art. For example, alginate carboxylic acid sites can be activated for coupling to one or more amine-functionalized compounds to achieve an alginate modified with a compound of formula (I). The alginate polymer can be dissolved in water (30 mL / gram polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 eq) and N-methylmorpholine (1 eq). To this mixture can be added a solution of the compound of formula (I) (0.3 M) in acetonitrile. The reaction can be warmed to 55° C. for 16 hours, then cooled to room temperature and gently concentrated via rotary evaporation, and the residue can then be dissolved, for example, in water. The mixture can then be filtered, for example, through a bed of cyano-modified silica gel (Silicycle), and the filter cake washed with water. The resulting solution can then be dialyzed (10,000 MWCO membrane), for example, against two changes of water for 24 hours. The resulting solution can then be concentrated, for example, via lyophilization, to provide the desired chemically modified alginate.

[0126] The alginate hydrogel in the barrier compartment may also include unmodified alginate. In some embodiments, the alginate is a high guluronic acid (G) alginate, comprising about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more guluronic acid (G). In some embodiments, the alginate is a high mannuronic acid (M) alginate, comprising 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 or the ratio of G:M is greater than 1. In one embodiment, the unmodified alginate in the barrier compartment has a molecular weight of 150 kDa to 250 kDa and a G:M ratio of ≧1.5.

[0127] In some embodiments, the alginate hydrogel in the inner compartment comprises an alginate with a G:M ratio greater than 1, e.g., a low molecular weight alginate, e.g., with an approximate molecular weight of <75 kDa and a G:M ratio of ≧1.5, (ii) a medium molecular weight alginate, e.g., with an approximate molecular weight of 75-150 kDa and a G:M ratio of ≧1.5, (iii) a high molecular weight alginate, e.g., with an approximate MW of 150 kDa-250 kDa and a G:M ratio of ≧1.5, (iv) or a blend of two or more of these alginates. In some embodiments, the inner compartment further comprises at least one cell-binding substance (CBS), e.g., a cell-binding peptide (CBP) or cell-binding polypeptide (CBPP) as described in WO2020069429.

[0128] In some embodiments, the inner compartment comprises alginate covalently modified with a linker-cell-binding peptide moiety, e.g., GRGD or GRGDSP. In one embodiment, the cell-binding peptide density (e.g., % nitrogen determined by combustion analysis as described in WO2020198695) in the inner compartment 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 linker-CBP in the inner compartment is about 0.1 to about 1.0 micromoles of CBP per gram of CBP-alginate (e.g., MMW-alginate covalently modified with GRGD or GRGDSP in solution), as determined by a quantitative peptide conjugation assay, e.g., the assay described in WO2020198695. In one embodiment, the linker-CBP is GRGDSP and the alginate has a molecular weight of 75 kDa to 150 kDa and a G:M ratio of 1.5 or greater. In one embodiment, the inner compartment also comprises an unmodified alginate having a molecular weight of 75 kDa to 150 kDa and a G:M ratio of 1.5 or greater.

[0129] In addition to the alginate hydrogel, either or both of the inner and outer compartments may further comprise a non-alginate polymer, which may be a linear, branched, or crosslinked polymer, or a polymer of a selected molecular weight range, degree of polymerization, viscosity, or melt flow rate. The branched polymer may include one or more of the following types: star polymer, comb polymer, brush polymer, dendrimerized polymer, ladder, and dendrimer. The non-alginate polymer may be a thermoresponsive polymer, for example, a gel (e.g., becomes solid or liquid upon exposure to heat or a selected temperature) or a photocrosslinkable polymer. Exemplary polymers include polystyrene, polyethylene, polypropylene, polyacetylene, poly(vinyl chloride) (PVC), polyolefin copolymers, poly(urethanes), polyacrylates and polymethacrylates, polyacrylamides and polymethacrylamides, poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), polyesters, polysiloxanes, polydimethylsiloxanes (PDMS), polyethers such as polyetherketones (PEEK), poly(orthoesters), poly(carbonates), poly(hydroxyalkanoates), polyfluorocarbons, polytetrafluoroethylene (PTFE), silicones, epoxy resins, polyethylene glycols, nylons, Included are polyalkenes, phenolic resins, natural and synthetic elastomers, adhesives and sealants, polyolefins, polysulfones, polyacrylonitriles, biopolymers such as polysaccharides and natural latex, collagen, cellulose polymers (such as alkyl celluloses), 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, chitosan, and blends or copolymers thereof.

[0130] In some embodiments, each capsule in the population of hydrogel capsules comprises a plurality of cells (e.g., viable cells) capable of expressing and secreting at least one therapeutic agent (e.g., a peptide or protein) when the hydrogel capsule is placed into a subject, e.g., as a portion of the total volume of the capsule composition or an aliquot thereof. In some embodiments, the cells express two or more therapeutic agents, e.g., proteins with complementary activities useful for treating a particular disease of interest.

[0131] In one embodiment, each hydrogel capsule of the population contains cells (e.g., in the inner compartment) derived from a single parent cell type or a mixture of at least two different parent cell types. In one embodiment, all of the cells are derived from the same parent cell type, but a first plurality of derived cells are genetically modified to express a first therapeutic agent and a second plurality of derived cells are genetically modified to express a second therapeutic agent. In hydrogel capsule compositions comprising two or more populations of hydrogel capsules, the cells and the therapeutic agent(s) produced thereby can be the same or different for each capsule population.

[0132] In one embodiment, the cells to be incorporated into the hydrogel capsules described herein are prepared in the form of a cell suspension before being encapsulated. The cells in the suspension can be in the form of single cells (e.g., from a monolayer cell culture) or can be provided in another form, for example, arranged 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 can include multiple cell clusters (e.g., as spheroids) or microcarriers.

[0133] In addition to the therapeutic substance(s) expressed by the encapsulated cells, each hydrogel capsule of a capsule population may contain one or more exogenous agents not expressed by the cells, such as, for example, nucleic acids (e.g., RNA or DNA molecules), proteins (e.g., hormones, enzymes (e.g., glucose oxidase, kinases, phosphatases, oxygenases, hydrogenases, reductases), antibodies, antibody fragments, antigens, or epitopes), active or inactive fragments of proteins or polypeptides, small molecules, or drugs. In one embodiment, the capsule is configured to release such exogenous agents.

[0134] Non-fibrous compounds In some embodiments, the hydrogel capsules in the population of hydrogel capsules described herein comprise a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, 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 2 -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 a linking group (e.g., a linking group described herein), and one or more R 1 is optionally replaced by; L 1 and L 3 is independently a bond, an alkyl, or a heteroalkyl, and each alkyl and heteroalkyl is selected from one or more R 2 is optionally replaced by; L 2 is a bond; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from the group consisting of one or more R 3 is optionally replaced by; P is absent, cycloalkyl, heterocyclyl, or heteroaryl, each of which is selected from the group consisting of one or more R 4 is optionally replaced by; Z is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -OR A , -C(O)R A , -C(O)OR A , -C(O)N(R C )(R D ), -N(R C )C(O)R A , cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which may be selected from the group consisting of one or more R 5 is optionally replaced by; Each R A , R B , R C , R D , R E , R F , and R Gis independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of one or more R 6 is optionally substituted with; or R C and R D together with the nitrogen atom to which they are attached, form one or more R 6 forming an optionally substituted ring (e.g., a 5- to 7-membered ring); Each R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), S.R. E1 , S(O) x R E1 , -OS(O) x R E1 , -N(R C1 )S(O) x R E1 , -S(O) x N(R C1 )(R D1 ), -P(R F1 ) y , cycloalkyl, heterocyclyl, aryl, and heteroaryl, each of which may be selected from the group consisting of one or more R 7 is optionally replaced by; Each R A1 , R B1 , RC1 , R D1 , R E1 , and R F1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from the group consisting of one or more R 7 is optionally replaced by; Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is 1 or 2; and y is 2, 3, or 4.

[0135] In some embodiments, the compound of formula (I) is a compound of formula (Ia): [ka] or a 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 )N(R D )-, -NCN-, -N(R C )C(O)(C 1 -C 6 -alkylene)-, -N(R C )C(O)(C 2 -C 6 -alkenylene)-, -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 a linking group (e.g., a linking group described herein), and one or more R 1 is optionally replaced by; L 1 and L 3 is independently a bond, an alkyl, or a heteroalkyl, and each alkyl and heteroalkyl is selected from one or more R 2 is optionally replaced by; L 2 is a bond; M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from the group consisting of one or more R 3 is optionally replaced by; P is one or more R 4 is heteroaryl optionally substituted by Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which may be selected from one or more R 5 is optionally replaced by; Each R A , R B , R C , R D , R E , R F , and R G is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of one or more R 6 is optionally substituted with; or RC and R D together with the nitrogen atom to which they are attached, form one or more R 6 forming an optionally substituted ring (e.g., a 5- to 7-membered ring); Each R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), S.R. E1 , S(O) x R E1 , -OS(O) x R E1 , -N(R C1 )S(O) x R E1 , -S(O) x N(R C1 )(R D1 ), -P(R F1 ) y , cycloalkyl, heterocyclyl, aryl, and heteroaryl, each of which may be selected from the group consisting of one or more R 7 is optionally replaced by; Each R A1 , R B1 , R C1 , R D1 , R E1 , and R F1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from the group consisting of one or more R7 is optionally replaced by; Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is 1 or 2; and y is 2, 3, or 4.

[0136] In some embodiments, for formula (I) or (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 2 -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 In some embodiments, A is -N(R CIn 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)-, where alkylene is R 1 In some embodiments, A is substituted with -N(R C )C(O)(C 1 -C 6 -alkylene)-, and R 1 is alkyl (e.g., methyl). 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 (e.g., methyl). In some embodiments, A is -NHC(O)CH(CH 3 In some embodiments, A is -NHC(O)C(CH 3 )-.

[0137] In some embodiments, for formula (I) or (Ia), L 1 is a bond, alkyl, or heteroalkyl. In some embodiments, L 1 is a bond or alkyl. In some embodiments, L 1 is a bond. In some embodiments, L 1 is alkyl. In some embodiments, L 1 is C 1 -C 6 In some embodiments, L 1 is -CH 2 -, -CH(CH 3 )-, -CH 2 CH 2 CH 2 , or -CH 2 CH 2In some embodiments, L 1 is -CH 2 -or-CH 2 CH 2 -It is.

[0138] In some embodiments, for formula (I) or (Ia), L 3 is a bond, alkyl, or heteroalkyl. In some embodiments, L 3 is a bond. In some embodiments, L 3 is alkyl. In some embodiments, L 3 is C 1 -C 12 In some embodiments, L 3 is C 1 -C 6 In some embodiments, L 3 is -CH 2 In some embodiments, L 3 is heteroalkyl. In some embodiments, L 3 is one or more R 2 (e.g., oxo) optionally substituted C 1 -C 12 In some embodiments, L is heteroalkyl. 3 is one or more R 2 (e.g., oxo) optionally substituted C 1 -C 6 In some embodiments, L is heteroalkyl. 3 is -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 is -CH 2 It is O-.

[0139] In some embodiments, for formula (I) or (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, where 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 CH 2 -, (-OCH 2 CH 2 -) 2 , (-OCH 2 CH 2 -) 3 , (-OCH 2 CH 2 -) 4 , or (-OCH 2 CH 2 -) 5 In some embodiments, M is -OCH 2 CH 2 -, (-OCH 2 CH 2 -) 2 , (-OCH 2 CH 2 -) 3 , or (-OCH 2 CH 2 -) 4 In some embodiments, M is (-OCH 2 CH 2 -) 3In some embodiments, M is aryl. In some embodiments, M is phenyl. In some embodiments, M is unsubstituted phenyl. In some embodiments, M is [ka] In some embodiments, M is R 7 (For example, one R 7 In some embodiments, M is phenyl substituted with [ka] In some embodiments, R 7 CF 3 It is.

[0140] In some embodiments, for formula (I) or (Ia), P is absent, heterocyclyl, or heteroaryl. In some embodiments, P is absent. In some embodiments, for formula (I) and (Ia), P is tricyclic, bicyclic, or monocyclic heteroaryl. In some embodiments, P is monocyclic heteroaryl. In some embodiments, P is nitrogen-containing heteroaryl. In some embodiments, P is monocyclic, nitrogen-containing heteroaryl. In some embodiments, P is 5-membered heteroaryl. In some embodiments, P is 5-membered nitrogen-containing heteroaryl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, pyrrolyl, oxazolyl, or thiazolyl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, or pyrrolyl. In some embodiments, P is imidazolyl. In some embodiments, P is [ka] In some embodiments, P is triazolyl. In some embodiments, P is 1,2,3-triazolyl. In some embodiments, P is [ka] It is.

[0141] In some embodiments, P is heterocyclyl. In some embodiments, P is 5-membered heterocyclyl or 6-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some embodiments, P is [ka] In some embodiments, P is thiomorpholinyl-1,1-dioxidyl.

[0142] In some embodiments, P is [ka] It is.

[0143] In some embodiments, P is one or more R 4 In some embodiments, R is a triazolyl substituted with 4 is deuterium, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), -S(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, and heteroaryl, each of which may be selected from the group consisting of one or more R 7 In some embodiments, R 4 is deuterium, alkyl, heteroalkyl, halogen, cyano, or azido, and each alkyl and heteroalkyl is selected from one or more R 7 (e.g., halogen). In some embodiments, P is optionally substituted by [ka] In some embodiments, P is R 4 (e.g., halogen). In some embodiments, R 4 is deuterium, alkyl, or halogen. In some embodiments, R 4 is halogen (e.g., fluoro, chloro, bromo). In some embodiments, R 4 is an alkyl group (e.g., -CH 3 , -CH 2 CH 3 , -CF 3 , -CH 2 F, -CHF 2 In some embodiments, R 4 In some embodiments, P is [ka] In some embodiments, P is [ka] In some embodiments, P is [ka] In some embodiments, P is [ka] In some embodiments, P is [ka] In some embodiments, P is [ka] In some embodiments, P is [ka] In some embodiments, P is [ka] In some embodiments, P is [ka] It is.

[0144] In some embodiments, for formula (I) or (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. In some embodiments, Z is oxygen-containing heterocyclyl. In some embodiments, Z is 4-membered heterocyclyl, 5-membered heterocyclyl, or 6-membered heterocyclyl. In some embodiments, Z is 6-membered heterocyclyl. In some embodiments, Z is 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is [ka] In some embodiments, Z is a 4-membered oxygen-containing heterocyclyl. In some embodiments, Z is [ka] It is.

[0145] In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments, Z is phthalic anhydride. In some embodiments, Z is a sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered heterocyclyl containing a nitrogen atom and a sulfur atom. In some embodiments, Z is thiomorpholinyl-1,1-dioxidyl. In some embodiments, Z is [ka] In some embodiments, Z is a nitrogen-containing heterocyclyl. In some embodiments, Z is a 6-membered nitrogen-containing heterocyclyl. In some embodiments, Z is [ka] It is.

[0146] In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is selected from one or more R 5 In some embodiments, Z is a bicyclic nitrogen-containing heterocyclyl optionally substituted with [ka] In some embodiments, Z is 1-oxa-3,8-diazaspiro[4.5]decan-2-one. In some embodiments, Z is [ka] It is.

[0147] In some embodiments, for Formula (I) or (Ia), Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is a monosubstituted phenyl (e.g., one R 5 In some embodiments, Z has one R 5 In some embodiments, Z is a monosubstituted phenyl, wherein R 5 NH 2 In some embodiments, Z is a monosubstituted phenyl having one R 5 In some embodiments, Z is a monosubstituted phenyl, wherein R 5 In some embodiments, Z is an oxygen-containing heteroalkyl, where R 5 OCH 3 In some embodiments, Z is a monosubstituted phenyl having one R 5 In some embodiments, Z is a monosubstituted phenyl having one R 5 In some embodiments, Z is a monosubstituted phenyl having one R 5 is a monosubstituted phenyl in the para position.

[0148] In some embodiments, for formula (I) or (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 -C 8 In some embodiments, Z is selected from 1 to 5 R 5 C replaced with 1 -C8 In some embodiments, Z is one R 5 C replaced with 1 -C 8 In some embodiments, Z is one R 5 C replaced with 1 -C 8 is alkyl, 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 one R 5 C replaced with 1 -C 8 is alkyl, R 5 -OR A1 OR -C(O)OR A1 In some embodiments, Z is one R 5 C replaced with 1 -C 8 is alkyl, R 5 -OR A1 or -C(O)OH. In some embodiments, Z is -CH 3 It is.

[0149] In some embodiments, for formula (I) or (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 one or more R 5 In some embodiments, Z is a nitrogen-containing heteroalkyl optionally substituted with 1 to 5 R5 In some embodiments, Z is N-methyl-2-(methylsulfonyl)ethane-1-aminyl.

[0150] In some embodiments, Z is -OR A OR -C(O)OR A In some embodiments, Z is -OR A (e.g., -OH or -OCH 3 ).

[0151] In some embodiments, Z is -OCH 3 In some embodiments, Z is -C(O)OR A (e.g., -C(O)OH).

[0152] In some embodiments, Z is hydrogen.

[0153] In some embodiments, L 2 is a bond, and P and L 3 is independently absent. In some embodiments, L 2 is a bond, P is heteroaryl, and L 3 is a bond and Z is hydrogen. In some embodiments, P is heteroaryl and L 3 is heteroalkyl and Z is alkyl.

[0154] In some embodiments, the compound of formula (I) is a compound of formula (Ib): [ka] or a salt thereof (wherein, ring M 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 3 and optionally substituted with ring Z 1 is 1 to 5 R 5cycloalkyl, heterocyclyl, aryl or heteroaryl optionally substituted with; R 2a , R 2b , R 2c , and R 2d each 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 taken together form an oxo group; X is absent, N(R 10 ), O, or S;R C is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 6 R 6 and each R 3 , R 5 , and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), S.R. E1 , cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 10 is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl, or heteroaryl; each R A1 , R B1 , RC1 , R D1 , and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from 1 to 6 R 7 and each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; each m and n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to a connection to a linking group or polymer as described herein. In some embodiments, each R 3 and R 5 wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally and independently substituted with halogen, oxo, cyano, cycloalkyl, or heterocyclyl.

[0155] In some embodiments, the compound of formula (Ib) is a compound of formula (Ibi): [ka] or a pharma- ceutically acceptable salt thereof (wherein, ring M 2 is one or more R 3 aryl or heteroaryl optionally substituted with 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R2b or R 2c and R 2d each of R taken together form an oxo group; X is absent, O, or S; 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; or two R 5 Together, they form the ring Z 2 form a 5- to 6-membered ring fused to 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, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein).

[0156] In some embodiments, the compound of formula (Ibi) is a compound of formula (Ib-ii): [ka] or a pharma- ceutically acceptable salt thereof (wherein, ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; R 2c and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R 2d taken together form an oxo group; each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1wherein each alkyl and heteroalkyl is optionally substituted with halogen; 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; m is 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein).

[0157] In some embodiments, the compound of formula (I) is a compound of formula (Ic): [ka] or a pharma- ceutically acceptable salt thereof (wherein, ring Z 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; R 2c and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R 2d taken together form an oxo group; each 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; A1 and R B1 is 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; [ka] refers to the connection to a linking group or polymer as described herein).

[0158] In some embodiments, the compound of formula (I) is a compound of formula (Id): [ka] or a pharma- ceutically 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 is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b or R 2c and R 2d taken together form an oxo group; each 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; 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; [ka] refers to the connection to a linking group or polymer as described herein).

[0159] In some embodiments, the compound of formula (I) is a compound of formula (Ie): [ka] or a pharma- ceutically acceptable salt thereof (wherein, ring Z 2is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, O or S; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b or R 2c and R 2d taken together form an oxo group; each R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and 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; [ka] refers to the connection to a linking group or polymer as described herein).

[0160] In some embodiments, the compound of formula (I) is a compound of formula (If): [ka] or a pharma- ceutically acceptable salt thereof, 3 ring P is an alkyl optionally substituted with one or more R 4 is heteroaryl optionally substituted with 3 is one or more R 2 Z is an alkyl or heteroalkyl optionally substituted with one or more of R 5 Optionally substituted with; R 2a and R2b each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b taken together 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 and each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein).

[0161] In some embodiments, the compound of formula (I) is a compound of formula (II): [ka] or a pharma- ceutically acceptable salt thereof, wherein M is a bond, alkyl, or aryl, and alkyl and aryl are each independently selected from one or more R 3 Optionally substituted with; L 3 is one or more R 2 Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or -OR A and alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently one or more R 5 Optionally substituted with; R A is hydrogen; R 2a and R 2b each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R2b taken together form an oxo group; each R 2 , R 3 , and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein).

[0162] In some embodiments, the compound of formula (II) is a compound of formula (II-a): [ka] or a pharma- ceutically acceptable salt thereof, 3 is alkyl or heteroalkyl, each of which may be one or more R 2 and Z is hydrogen, alkyl, heteroalkyl, or -OR A and the alkyl and heteroalkyl are each independently one or more R 5 Optionally substituted with; R 2a and R 2b each is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b taken together form an oxo group; each R 2 , R 3 , and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 ;R A is hydrogen; each R A1 and RB1 is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein).

[0163] In some embodiments, the compound of formula (I) is a compound of formula (III): [ka] or a pharma- ceutically acceptable salt thereof, 1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 Optionally substituted with; R 2a , R 2b , R 2c , and R 2d each 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 together form an oxo group; R C is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, each of which is selected from 1 to 6 R 6 Optionally substituted with; R 3 , R 5 , and R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and each R A1 and R B1is 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; [ka] refers to the connection to a linking group or polymer as described herein).

[0164] In some embodiments, the compound of formula (III) is a compound of formula (III-a): [ka] or a pharma- ceutically acceptable salt thereof (wherein, ring Z 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 Optionally substituted with; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and 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; [ka] refers to the connection to a linking group or polymer as described herein).

[0165] In some embodiments, the compound of formula (III-a) is a compound of formula (III-b): [ka] or a pharma- ceutically acceptable salt thereof (wherein, ring Z 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 Optionally substituted with; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and 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; [ka] refers to the connection to a linking group or polymer as described herein).

[0166] In some embodiments, the compound of formula (III-a) is a compound of formula (III-c): [ka] or a pharma- ceutically acceptable salt thereof, wherein X is C(R')(R"), N(R'), or S(O). x each of R' and R" is independently hydrogen, alkyl, halogen, or cycloalkyl; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, or halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and 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, or 5; q is an integer from 0 to 25; x is 0, 1, or 2; [ka] refers to the connection to a linking group or polymer as described herein).

[0167] In some embodiments, the compound of formula (III-c) is a compound of formula (III-d): [ka] or a pharma- ceutically acceptable salt thereof, wherein X is C(R')(R"), N(R'), or S(O). x each of R' and R" is independently hydrogen, alkyl, halogen, or cycloalkyl; R 2a , R 2b , R 2c , and R2d each is independently hydrogen, alkyl, heteroalkyl, or halo; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 and R 5 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and 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, or 5; q is an integer from 0 to 25; x is 0, 1, or 2; [ka] refers to the connection to a linking group or polymer as described herein).

[0168] In some embodiments, the compound of formula (I) is a compound of formula (IV-a): [ka] or a pharma- ceutically acceptable salt thereof (wherein, ring Z 1 is 1 to 5 R 5 R is a heterocyclyl optionally substituted with C is hydrogen, alkyl, alkenyl, -C(O)(C 1 -C 6 -alkyl), or -C(O)(C 1 -C 6 -alkenyl), each alkyl and alkenyl being 1 to 6 R 6 Optionally substituted with; R 2a , R 2b , R 2c , and R 2deach is independently hydrogen, alkyl, heteroalkyl, halo, or amino; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 3 , R 5 and R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -SR E1 , -S(O) x R E1 , or -OS(O) x R E1 and each R 10 is independently deuterium, alkyl, haloalkyl, heteroalkyl, halo, cyano, nitro, or amino; A1 , R B1 , and R E1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; w is 0, 1, or 2; q and p are each independently an integer from 0 to 25; and x is 0, 1, or 2).

[0169] In some embodiments, ring Z 1 In some embodiments, the ring Z is heterocyclyl. 1 is a nitrogen-containing heterocyclyl. 1 is a 4-membered heterocyclyl or a 6-membered heterocyclyl. 1 is one R 5 In some embodiments, R is a heterocyclyl substituted with 5 -S(O) x R E1 In some embodiments, R E1 is an alkyl group (e.g., -CH 3 In some embodiments, x is 2. In some embodiments, R 5 -S(O)2 (CH 3 ).

[0170] In some embodiments, R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen.

[0171] In some embodiments, R C is hydrogen, -C(O)(C 1 -C 6 -alkyl), or -C(O)(C 1 -C 6 -alkenyl). In some embodiments, R C is hydrogen. In some embodiments, n is 1. In some embodiments, q is 2, 3, 4, or 5. In some embodiments, q is 3. In some embodiments, m is 1. In some embodiments, p is 0. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, R 10 is halo (e.g., Cl).

[0172] In some embodiments, the compound of formula (I) is a compound of formula (IV-b): [ka] or a pharma- ceutically acceptable salt thereof, C is hydrogen, alkyl, -N(R C )C(O)R B , -N(R C )C(O)(C 1 -C 6 -alkyl), or -N(R C )C(O)(C 1 -C 6 -alkenyl), each of the alkyl and alkenyl is selected from 1 to 6 R 6 Optionally substituted with; R 2a , R 2b , R 2c , and R2d each is independently hydrogen or alkyl; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R 5 and R 6 Each of is independently alkyl, heteroalkyl, halogen, oxo, -S(O) x R E1 , or -OS(O) x R E1 and each R 10 is independently deuterium, alkyl, haloalkyl, heteroalkyl, halo, cyano, nitro, or amino; R E1 are independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; w is 0, 1, or 2; q is an integer from 0 to 25; x is 0, 1, or 2; and z is 0, 1, 2, 3, 4, 5, or 6).

[0173] In some embodiments, R 5 -S(O) x R E1 In some embodiments, R E1 is an alkyl group (e.g., -CH 3 In some embodiments, x is 2. In some embodiments, R 5 -S(O) 2 (CH 3 In some embodiments, z is 1. In some embodiments, R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen.

[0174] In some embodiments, R C is hydrogen, -C(O)(C 1 -C 6 -alkyl), or -C(O)(C 1 -C 6 -alkenyl). In some embodiments, RC is hydrogen.

[0175] In some embodiments, n is 1. In some embodiments, q is 2, 3, 4, or 5. In some embodiments, q is 3. In some embodiments, m is 1. In some embodiments, w is 0.

[0176] In some embodiments, the compound of formula (I) is a compound of formula (IV-c): [ka] or a pharma- ceutically acceptable salt thereof, wherein X is C(R')(R"), N(R'), or S(O). x each of R' and R" is independently hydrogen, alkyl, or halogen; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, or halogen; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R C is hydrogen, alkyl, -N(R C )C(O)R B , -N(R C )C(O)(C 1 -C 6 -alkyl), or -N(R C )C(O)(C 1 -C 6 -alkenyl), each of the alkyl and alkenyl is selected from 1 to 6 R 6 Optionally substituted with; R 3 , R 5 , and R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -SR E1 , -S(O)x R E1 , or -OS(O) x R E1 and each R 10 is independently deuterium, alkyl, haloalkyl, heteroalkyl, halo, cyano, nitro, or amino; A1 , R B1 , and R E1 are each independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; w is 1; q and p are each independently an integer from 0 to 25; and x is 0, 1, or 2).

[0177] In some embodiments, X is S(O) x In some embodiments, x is 2. In some embodiments, X is S(O) 2 It is.

[0178] In some embodiments, R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen.

[0179] In some embodiments, R C are independently -C(O)(C 1 -C 6 -alkyl), or -C(O)(C 1 -C 6 -alkenyl). In some embodiments, R C is hydrogen.

[0180] In some embodiments, n is 1. In some embodiments, q is 2, 3, 4, or 5. In some embodiments, q is 3. In some embodiments, m is 1. In some embodiments, p is 0. In some embodiments, R 10 is halo (e.g., Cl).

[0181] In some embodiments, the compound of formula (I) is a compound of formula (IV-d): [ka] or a pharma- ceutically acceptable salt thereof, wherein X is C(R')(R"), N(R'), or S(O). x each of R' and R" is independently hydrogen, alkyl, or halogen; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, or halogen; or R 2a and R 2b or R 2c and R 2d together form an oxo group; R C is hydrogen, alkyl, -N(R C )C(O)R B , -N(R C )C(O)(C 1 -C 6 -alkyl), or -N(R C )C(O)(C 1 -C 6 -alkenyl), each of the alkyl and alkenyl is selected from 1 to 6 R 6 Optionally substituted with; R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 and each R 10 is independently deuterium, alkyl, haloalkyl, heteroalkyl, halo, cyano, nitro, or amino; A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; n is 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; and x is 0, 1, or 2).

[0182] In some embodiments, X is S(O) xIn some embodiments, x is 2. In some embodiments, X is S(O) 2 It is.

[0183] In some embodiments, R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen.

[0184] In some embodiments, R C is hydrogen, -C(O)(C 1 -C 6 -alkyl), or -C(O)(C 1 -C 6 -alkenyl). In some embodiments, R C is hydrogen.

[0185] In some embodiments, n is 1. In some embodiments, q is 2, 3, 4, or 5. In some embodiments, q is 3. In some embodiments, m is 1. In some embodiments, p is 0. In some embodiments, R 10 is halo (e.g., Cl).

[0186] In some embodiments, the compound of formula (I) is a compound of formula (IV-e): [ka] or a pharma- ceutically acceptable salt thereof, wherein X is C(R')(R"), N(R'), or S(O). x each of R' and R" is independently hydrogen, alkyl, or halogen; R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, heteroalkyl, or halogen; or R 2a and R 2b or R 2c and R 2dtogether form an oxo group; R C are independently hydrogen, alkyl, -N(R C )C(O)R B , -N(R C )C(O)(C 1 -C 6 -alkyl), or -N(R C )C(O)(C 1 -C 6 -alkenyl), each of the alkyl and alkenyl is selected from 1 to 6 R 6 Optionally substituted with; R 3 , R 5 , and R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -SR E1 , -S(O) x R E1 , or -OS(O) x R E1 and each R 10 is independently deuterium, alkyl, haloalkyl, heteroalkyl, halo, cyano, nitro, or amino, and each alkyl or heteroalkyl is selected from one or more R 11 and each R 11 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; each R A1 , R B1 , and R E1 are each independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; w is 1; q and p are each independently an integer from 0 to 25; and x is 0, 1, or 2).

[0187] In some embodiments, X is S(O) x In some embodiments, x is 2. In some embodiments, X is S(O) 2 It is.

[0188] In some embodiments, R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen.

[0189] In some embodiments, R 10 is deuterium, alkyl, heteroalkyl, halogen, cyano, or azido, and each alkyl and heteroalkyl is selected from one or more R 11 (e.g., halogen). In some embodiments, R 10 is deuterium, alkyl, or halogen. In some embodiments, R 10 is halogen (e.g., fluoro, chloro, bromo). In some embodiments, R 10 is an alkyl group (e.g., -CH 3 , -CH 2 CH 3 , -CF 3 , -CH 2 F, -CHF 2 ).

[0190] In some embodiments, R C is hydrogen, -C(O)(C 1 -C 6 -alkyl), or -C(O)(C 1 -C 6 -alkenyl). In some embodiments, R C is hydrogen.

[0191] In some embodiments, n is 1. In some embodiments, q is 2, 3, 4, or 5. In some embodiments, q is 3. In some embodiments, m is 1. In some embodiments, p is 0.

[0192] In some embodiments, the compound is a compound of formula (I). 2 is a bond, and P and L 3 is, independently, non-existent.

[0193] 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 heteroaryl, and L 3 is a bond and Z is hydrogen. In some embodiments, P is heteroaryl and L 3 is heteroalkyl and Z is alkyl. In some embodiments, L 2 is a bond, and P and L 3 is independently absent. In some embodiments, L 2 is a bond, P is heteroaryl, and L 3 is a bond and Z is hydrogen. In some embodiments, P is heteroaryl and L 3 is heteroalkyl and Z is alkyl.

[0194] In some embodiments, the compound is a compound of formula (Ib). In some embodiments, P is absent and L 1 -NHCH 2 And L 2 is a bond, M is aryl (e.g., phenyl), and L 3 is -CH 2 O and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, such as thiomorpholinyl-1,1-dioxide). In some embodiments, the compound of Formula (Ib) is compound 116.

[0195] In some embodiments of Formula (Ib), P is absent and L 1 -NHCH 2 And L 2 is a bond, M is absent, and L 3 is a bond and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl, such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (Ib) is compound 105.

[0196] In some embodiments, the compound is a compound of formula (Ibi). In some embodiments of formula (Ibi), R 2a and R 2b Each of 3 and R 2c and R 2d are each independently hydrogen, m is 1 or 2, n is 1, X is O, p is 0, and M 2 is one or more R 3 phenyl optionally substituted with R 3 -CF 3 and Z 2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of formula (Ibi) is Compound 100, Compound 106, Compound 107, Compound 108, Compound 109, or Compound 111.

[0197] In some embodiments, the compound is a compound of formula (Ib-ii). In some embodiments of formula (Ib-ii), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, q is 0, p is 0, m is 1, and Z 2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, such as tetrahydropyranyl). In some embodiments, the compound of formula (Ib-ii) is compound 100.

[0198] In some embodiments, the compound is a compound of formula (Ic). In some embodiments of formula (Ic), R 2c and R 2d is independently hydrogen, m is 1, p is 1, q is 0, and R 5 is -CH 3and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, such as piperazinyl). In some embodiments, the compound of Formula (Ic) is compound 113.

[0199] In some embodiments, the compound is a compound of formula (Id). In some embodiments of formula (Id), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 3, X is O, p is 0, and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl, such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of formula (Id) is compound 110 or compound 114.

[0200] In some embodiments, the compound is a compound of formula (If). In some embodiments of formula (If), R 2a and R 2b is independently hydrogen, n is 1, and M is -CH 2 -, P is a nitrogen-containing heteroaryl (e.g., imidazolyl), and L 3 is -C(O)OCH 2 - and Z is CH 3 In some embodiments, the compound of formula (If) is compound 115.

[0201] In some embodiments, the compound is a compound of formula (II-a). In some embodiments of formula (II-a), R 2a and R 2b is independently hydrogen, n is 1, q is 0, and L 3 is -CH 2 (OCH 2 CH 2 ) 2 and Z is -OCH 3 In some embodiments, the compound of Formula (II-a) is compound 112.

[0202] In some embodiments of Formula (II-a), R 2a and R 2b is independently hydrogen, n is 1, and 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.

[0203] 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 is independently hydrogen, m is 1, n is 2, q is 3, p is 0, and R C is hydrogen, and Z 1 is R 5 (For example, -N(CH 3 )(CH 2 CH 2 )S(O) 2 CH 3 In some embodiments, the compound of Formula (III) is compound 120.

[0204] In some embodiments, the compound is of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 0, n is 2, q is 3, p is 0, and Z 2 is one R 5 (For example, -NH 2 In some embodiments, the compound of Formula (III-b) is compound 102.

[0205] In some embodiments, the compound is of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 2, q is 3, p is 0, and R C is hydrogen, and Z 2 is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, such as a nitrogen-containing spiroheterocyclyl, such as 2-oxa-7-azaspiro[3.5]nonanyl). In some embodiments, the compound of Formula (III-a) is compound 121.

[0206] In some embodiments, the compound is of formula (III-d). In some embodiments of formula (III-d), R 2a , R 2b , R 2c , and R 2d 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 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.

[0207] 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).

[0208] In some embodiments, the compound is a compound of formula (IV-a) or (IV-b). In some embodiments, the compound is a compound of formula (IV-a), (IV-c), (IV-d), or (IV-e). In some embodiments, R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 1, q is 1, 2, 3, or 4, w is 1, and X is S(O). 2 In some embodiments, the compound is any one of compounds 122-154. In some embodiments, the compound of formula (I) includes deuterium (e.g., R 4 or R 10 is deuterium). In some embodiments, the compound of formula (I) does not contain deuterium (e.g., R 4 or R 10 is not deuterium).

[0209] Exemplary compounds of formula (I) may be prepared as described in WO2019 / 169333, WO2021 / 119522, or by any other method known to one of skill in the art.

[0210] In some embodiments, the compound of formula (I) is not a compound disclosed in WO2012 / 112982, WO2012 / 167223, WO2014 / 153126, WO2016 / 019391, WO2017 / 075630, US2012 / 0213708, US2016 / 0030359, or US2016 / 0030360.

[0211] In some embodiments, the compound of formula (I) comprises a compound shown in Table 3, or a pharma- ceutically acceptable salt thereof. In some embodiments, the device described herein comprises a compound shown in Table 4, or a pharma- ceutically acceptable salt thereof. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6] [Table 5-7] [Table 5-8]

[0212] In some embodiments, the compound is a compound of Formula (I) (e.g., Formula (Ia), (Ib), (Ibi), (Ib-ii), (Ic), (Id), (Ie), (If), (II), (II-a), (III), (III-a), (III-b), (III-c), (III-d), (IV-a), (IV-b), (IV-c), (IV-d), or (IV-e)), or a pharma- ceutically acceptable salt thereof; [ka] or a pharma- ceutically acceptable salt thereof.

[0213] In some embodiments, the hydrogel capsules in the population of hydrogel capsules described herein are [ka] or a pharma- ceutically acceptable salt of any one of these compounds.

[0214] In some embodiments, the hydrogel capsules in the population of hydrogel capsules described herein are [ka] This includes compounds of the formula:

[0215] In some embodiments, the compound of formula (I) (e.g., compound 101 in Table 4) is covalently attached to the alginate (e.g., an alginate having an approximate MW<75 kDa, G:M ratio ≧1.5) with a conjugation density of at least 2.0% and less than 9.0%, or between 3.0% and 8.0%, between 4.0 and 7.0, between 5.0 and 7.0, or between 6.0 and 7.0, or about 6.8, as determined by combustion analysis for nitrogen percentage as described in WO2020 / 069429. In one embodiment, the conjugation density of compound 101 in the modified alginate is determined by quantitative free amine analysis, e.g., as described in WO2020198695, and the determined conjugation density is between 1.0% w / w and 3.0% w / w, between 1.3% w / w and 2.8% w / w, between 1.3% w / w and 2.6% w / w, between 1.5% w / w and 2.4% w / w, between 1.5% w / w and 2.2% w / w, or between 1.7% w / w and 2.2% w / w.

[0216] In one embodiment, the hydrogel capsules in the population of hydrogel capsules described herein comprise a compound of formula (I) (e.g., a compound shown in Table 4) covalently bonded to an alginate polymer. The alginate polymer can be chemically modified with a compound of formula (I) using any suitable method known in the art, for example, as described in WO2019 / 195055.

[0217] Cells and Therapeutic Agents For example, the cells contained in the hydrogel capsules described herein in a population of hydrogel capsules may be derived from a variety of different cell types (e.g., human cells), including epithelial cells, endothelial cells, fibroblasts, pancreatic islet cells (as defined herein), mesenchymal stem cells, induced pluripotent stem cells (iPSCs), and keratinocyte cells. Exemplary cell types include those described in WO2017 / 075631. In one embodiment, the cells are not pancreatic islet cells (as defined herein). In one embodiment, the cells are pancreatic islet cells. In some embodiments, the cells are derived from the cell lines shown in Table 5. [Table 6]

[0218] Cells can be genetically modified to express and secrete a therapeutic agent of interest using any of a variety of genetic engineering techniques known in the art. For example, cells can be transfected with an expression vector containing a nucleotide sequence encoding a desired polypeptide operably linked to control elements necessary or useful for gene expression, e.g., promoters, ribosome binding sites, enhancers, polyA signals, etc.

[0219] In some embodiments, the expression vector encodes a polypeptide intended to provide a therapeutic effect when the hydrogel capsule is placed in a subject in need thereof, such as a clotting factor, growth factor, hormone, enzyme, cytokine (e.g., pro-inflammatory or anti-inflammatory cytokine), cytokine receptor, chimeric protein, fusion protein, or lipoprotein. The polypeptide encoded by the expression vector may have a naturally occurring amino acid sequence or may contain a variant of a naturally occurring sequence. The variant may be a non-naturally occurring or naturally occurring amino acid substitution, mutation, deletion, or addition compared to a reference (e.g., naturally occurring) sequence. The naturally occurring amino acid sequence may be a polymorphic variant. The naturally occurring amino acid sequence may be a human or non-human amino acid sequence. In some embodiments, the naturally occurring amino acid sequence is a human sequence. In some embodiments, the therapeutic polypeptide has less than about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, the polypeptides have an average molecular weight of 5 kD, 10 kD, 25 kD, 50 kD, 100 kD, 150 kD, 200 kD, 250 kD, 500 kD, or more.

[0220] In some embodiments, the polypeptide is a hormone. Exemplary hormones include antidiuretic hormone (ADH), oxytocin, growth hormone (GH), prolactin, growth hormone releasing hormone (GHRH), thyroid stimulating hormone (TSH), thyrotropin releasing hormone (TRH), adrenocorticotropic hormone (ACTH), follicle stimulating hormone (FSH), luteinizing hormone (LH), luteinizing hormone releasing hormone (LHRH), thyroxine, calcitonin, parathyroid hormone (PTH), aldosterone, cortisol, epinephrine, glucagon, insulin (INS), estrogen, progesterone, and testosterone. In some embodiments, the polypeptide is an INS (e.g., insulin A-chain, insulin B-chain, or proinsulin). In some embodiments, the polypeptide is a growth hormone, such as human growth hormone (hGH), recombinant human growth hormone (rhGH), bovine growth hormone, methionine-human growth hormone, des-phenylalanine human growth hormone, and porcine growth hormone.

[0221] 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).

[0222] In some embodiments, the polypeptide is a clotting factor or coagulation factor, e.g., a blood clotting factor or blood coagulation factor. In some embodiments, the polypeptide is involved in coagulation, the process by which blood is converted from a liquid to a solid or gel. Exemplary clotting and coagulation factors include Factor I (e.g., fibrinogen), Factor II (e.g., prothrombin), Factor III (e.g., tissue factor), Factor V (e.g., proaccelerin, labile factor), Factor VI, Factor VII (e.g., stable factor, proconvertin), Factor VIII (e.g., antihemophilic factor A), Factor VIII C, Factor IX (e.g., antihemophilic factor B), Factor X (e.g., Stuart-Prower factor), Factor XI (e.g., plasma thromboplastin precursor), Factor XII (e.g., Hageman factor), Factor XIII (e.g., fibrin stabilizing factor), von Willebrand factor (vWF), prekallikrein, heparin cofactor II, high molecular weight kininogen (e.g., Fitzgerald factor), antithrombin III, and fibronectin. In some embodiments, the polypeptide is an anticoagulant, e.g., protein C.

[0223] In some embodiments, the polypeptide is an immunoglobulin chain (heavy or light) or a fragment thereof that comprises at least one immunoglobulin variable domain sequence, and optionally an immunoglobulin Fc region. In one embodiment, the polypeptide is a full-length immunoglobulin chain.

[0224] In some embodiments, the polypeptide is selected from the group consisting of, for example, tumor necrosis factors alpha and beta, their receptors and their derivatives, renin; lipoproteins; colchicine; corticotrophin; vasopressin; somatostatin; lypressin; pancreozymin; leuprolide; alpha-1-antitrypsin; atrial natriuretic factor; pulmonary surfactant; plasminogen activators other than tissue-type plasminogen activator (t-PA), such as urokinase; bombesin; thrombin; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted);human macrophage inflammatory protein (MIP-1-alpha);serum albumins, e.g., human serum albumin;Mullerian inhibitory substance;relaxin A-chain;relaxin B-chain;prorelaxin;mouse gonadotropin-related peptide;chorionic gonadotropin;microbial proteins, e.g., beta-lactamase;DNase;inhibin;activin;receptors for hormones or growth factors;integrins;protein A or D;rheumatoid factor;platelet-derived growth factor (PDGF);epidermal growth factor (EGF);transformation factor Transforming growth factors (TGFs), such as TGF-α and TGF-β (including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5); insulin-like growth factors-I and -II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins, such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; osteoinductive factors; antitoxins; interferons, such as interferon-alpha (e.g., interferon.alpha.2A), -beta, -gamma, -lambda and consensus interferons; colony stimulating factors (CSFs), such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), such as IL-1, IL-2 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; degradation-promoting factors; transport proteins; homing receptors; addressins; conception inhibitors, such as prostaglandins; conception promoters; regulatory proteins; cytokines or cytokine receptors, or chimeric proteins comprising cytokines or their receptors, including antibodies (including fragments thereof) and chimeric proteins, such as immunoadhesins. Suitable polypeptides may be native or recombinant, including, for example, fusion proteins.

[0225] Examples of polypeptides that can be encoded by an exogenous transcription unit also include CCL1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP-1β), CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1 (KC), CXCL2 (SDF1a), CXCL 3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8(IL8), CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, CX3CL1, XCL1, XCL2, TNFA, TNF B(LTA), TNFC(LTB), TNFSF4, TNFSF5(CD40LG), TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF13B, EDA, IL2, IL15, IL4, IL13, IL7, IL9, IL2 1, IL3, IL5, IL6, IL11, IL27, IL30, IL31, OSM, LIF, CNTF, CTF1, IL12a, IL12b, IL23, IL27, IL35, IL14, IL16, IL32, IL34, IL10, IL22, IL19, IL20, IL24, IF NK, IFNW1, IFNG, IL1A (IL1F1), IL1B (IL1F2), IL1Ra (IL1F3), IL1F5 (IL36RN), IL1F6 (IL36A), IL1F7 (IL37), IL1F8 (IL36B), IL1F9 (IL36G), IL1F10 (IL 38), IL33 (IL1F11), IL18 (IL1G), IL17, KITLG, IL25 (IL17E), CSF1 (M-CSF), CSF2 (GM-CSF), CSF3 (G-CSF), SPP1, TGFB1, TGFB2, TGFB3, CCL3L1, CCL3L2,CCL3L3, CCL4L1, CCL4L2, IL17B, IL17C, IL17D, IL17F, AIMP1(SCYE1), MIF, Areg, BC096441, Bmp1, Bmp10, Bmp15, Bmp2, Bmp3, Bmp4, Bmp5, Bmp6, Bmp7, Bm p8a, Bmp8b, C1qtnf4, Ccl21a, Ccl27a, Cd70, Cer1, Cklf, Clcf1, Cmtm2a, Cmtm2b, Cmtm3, Cmtm4, Cmtm5, Cmtm6, Cmtm7, Cmtm8, Crlf1, Ctf2, Ebi3, Edn1, F am3b, Fasl, Fgf2, Flt3l, Gdf10, Gdf11, Gdf15, Gdf2, Gdf3, Gdf5, Gdf6, Gdf 7, Gdf9, Gm12597, Gm13271, Gm13275, Gm13276, Gm13280, Gm13283, Gm2564, G pi1, Grem1, Grem2, Grn, Hmgb1, Ifna11, Ifna12, Ifna9, Ifnab, Ifne, Il17a, Il23a, Il25, Il31, Iltifb, Inhba, Lefty1, Lefty2, Mstn, Nampt, Ndp, Noda l, Pf4, Pglyrp1, Prl7d1, Scg2, Scgb3a1, Slurp1, Spp1, Thpo, Tnfsf10, Tnfsf11, Tnfsf12, Tnfsf13, Tnfsf13b, Tnfsf14, Tnfsf15, Tnfsf18, Tnfsf4, Tnfsf8, Tnfsf9, Tslp, Vegfa, Wnt1, Wnt2, Wnt5a, Wnt7a, Xcl1, epinephrine, melatonin, triiodothyronine, prostaglandins, leukotrienes, prostacyclin, thromboxane, islet amyloid polypeptide, myosin, ductal inhibitory factor or hormone, adiponectin, corticotropin, angiotensin, vasopressin, arginine vasopressin, atriopeptin, brain natriuretic peptide, calcitonin, cholecystokinin, cortistatin, enkephalin, endothelin, erythropoietin, follicle-stimulating hormone, galanin, gastric inhibitory polypeptide, gastrin, ghrelin, glucagon, glucagon-like peptide-1, gonadotropin-releasing hormone, hepcidin, human chorionic gonadotropin, human placental lactogen, inhibin, somatomedin, leptin,These include lipotropin, melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, pituitary adenylate cyclase activating peptide, relaxin, renin, secretin, somatostatin, thrombopoietin, thyrotropin, thyrotropin releasing hormone, vasoactive intestinal peptide, androgens, alpha-glucosidase (also known as acid maltase), glycogen phosphorylase, glycogen debranching enzyme, phosphofructokinase, phosphoglycerate kinase, phosphoglycerate mutase, lactate dehydrogenase, carnitine parmethyltransferase, carnitine, and myoadenylate deaminase.

[0226] In some embodiments, the polypeptide is a replacement therapy or protein.

[0227] In some embodiments, the replacement therapy or protein is an enzyme, such as alpha-galactosidase A (GLA), alpha-L-iduronidase (IDUA), arylsulfatase B (ARSB), glucocerebrosidase, or N-sulfoglucosamine sulfohydrolase (SGSH).

[0228] In some embodiments, the replacement therapy or replacement protein is a clotting factor or coagulation factor, such as factor VII, factor VIII (e.g., comprising a naturally occurring human factor VIII amino acid sequence or a variant thereof), or factor IX (e.g., comprising a naturally occurring human factor IX amino acid sequence or a variant thereof).

[0229] In one embodiment, the genetically modified cells have one or more of the following characteristics: (i) are not capable of producing INS (e.g., insulin A-chain, insulin B-chain, or proinsulin) in amounts effective to treat diabetes or another disease or condition that can be treated with INS; (ii) are not capable of producing INS in a glucose-responsive manner; or (iii) are not derived from induced pluripotent stem cells that have been engineered or differentiated into INS-producing pancreatic beta cells.

[0230] In one embodiment, the genetically modified cells have one or more of the following characteristics: (i) are capable of producing INS (e.g., insulin A-chain, insulin B-chain, or proinsulin) in an amount effective to treat diabetes or another disease or condition that can be treated with INS; (ii) are capable of producing INS in a glucose-responsive manner; or (iii) are derived from induced pluripotent stem cells that have been engineered or differentiated into INS-producing pancreatic beta cells.

[0231] Storage and transportation The hydrogel capsule composition may be provided in a sealed container. In some embodiments, all interior container surfaces in contact with the composition consist essentially of or consist of chemically and biologically inert materials. Suitable materials include pharmaceutical or medical grade plastics, such as polycarbonate, polyetherimide (PEI), polyethylene, polypropylene, polyvinyl chloride, PEEK, polysulfone, polyurethane, fluorinated ethylene propylene (FEP) or polyethylene terephthalate glycol (PETG).

[0232] In one embodiment, the container can be of any size and shape that allows the container to be stored in a manner that causes a substantially uniform capsule layer containing substantially all of the hydrogel capsules in the composition to form over the bottom interior surface of the stored container, and a layer of aqueous solution substantially free of hydrogel capsules to form on top of the capsule layer. In some embodiments, the capsule layer has a depth equal to about 1.00 to about 1.25 times the average diameter of the capsules in the composition. In some embodiments, the total volume (VS) of the aqueous solution in the container (e.g., the amount of residual solution in the capsule layer combined with the amount in the solution layer) is equal to or greater than the volume of the hydrogel capsules in the container. In some embodiments, the ratio of the total volume of the aqueous solution to the total volume of the hydrogel capsules in the container is about 1, 1.1, 1.2, 1.3, 1.4, or 1.5 to about 100, about 2 to about 75, about 3 to about 50, about 4 to about 40, about 5 to about 30, and about 10 to about 20. In one embodiment, the average capsule diameter of the hydrogel capsules in the container is about 1500 μm and the ratio of VHC to VS is at least about 1:1, about 1:2, or about 1:3, and less than about 1:40, e.g., any of about 1:1, 1:2, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, and 1:35.

[0233] In some embodiments, the configuration (size / shape) of the container and the volume of the composition placed within the container are selected to allow an air space between the top of the solution layer and the upper interior surface of the stored container. In some embodiments, the air space is at least about 0.5 inches to 2.5 inches, about 1.0 inches to 2.0 inches, or about 1.5 inches to 2.0 inches.

[0234] In one embodiment, the storage container is a flexible rectangular bag made from FEP with one or more ports configured to allow addition of the composition or other substance(s) to the bag (e.g., via a syringe) and one or more ports configured to remove the solution, with or without hydrogel capsules, from the bag (e.g., via a syringe). An exemplary FEP bag is about 400 cm2 with a maximum fill volume of about 400 milliliters (mL) to about 700 mL. 2 ~about 600cm 2 In one embodiment, the amount of hydrogel capsule composition in the FEP bag is about 200 ml to 500 ml, e.g., about 300-500 ml, 400-500 ml, 200-400 ml, 200-300 ml, or 300-400 ml. Other exemplary containers for storing the hydrogel capsule compositions described herein include round-bottomed bottles formed from PETG sealed with a cap and rectangular trays formed from PETG and sealed with a foil lid.

[0235] The selection of the amount of hydrogel capsules and aqueous solution to be added to a particular size container depends on the size of the capsule, the number of capsules per mL of capsule composition, the desired VHC to VS ratio, and any desired air space above the solution layer.These amounts can be determined by those skilled in the art using formulas known in the art.An exemplary approach for determining the volume of spherically shaped capsules ("spheres") to be added to a container is shown below.

[0236] a. Calculate the surface area covered by the spheres by multiplying the surface area of ​​the container base by the expected sphere envelopment target, e.g., 0.64 for random envelopment to 0.74 for complete envelopment; b. This formula: (average sphere diameter / 2) 2 *Calculate the surface area of ​​the sphere using Pi c. Calculate the number of spheres to add to the vessel by dividing the result of step a (the surface area covered by the spheres) by the result of step b (the surface area of ​​the spheres); and d. Divide the result of step c by the number of spheres per mL of composition.

[0237] The table directly below is about 500cm 2 1 shows an exemplary volume of sphere preparation that would be added to an exemplary FEP bag having an internal surface area of ​​1000 μm, 1400 μm, and 2000 μm and a random surrounding target (0.64) for three different average sphere diameters (1000 micrometers (μm), 1400 micrometers (μm), and 2000 μm) and two different sphere concentrations for each diameter. [Table 7]

[0238] In some embodiments of the capsule composition where the population of hydrogel capsules consists essentially of spherically shaped capsules ("spheres"), the average sphere diameter in the population represents the average of the diameters of at least 10 spheres. In one embodiment, the diameters of the at least 10 spheres are obtained by analysis of a microscopic image of an aliquot of the hydrogel capsule composition.

[0239] In one embodiment, after the capsule composition is added to the container, the container is sealed and then stored at about 2° C. to room temperature (15° C. to 25° C.; 59° F. to 77° F.) for a desired period of time, which may include the time required to transport the container to a patient implantation site, e.g., an operating room in a hospital. In one embodiment, the desired period of time is either 12 hours, 24 hours, 36 hours, 2 days, 3 days, 4 days, or 5 days. In one embodiment, at the end of the desired period of time, the encapsulated cells in the sealed container retain at least 80%, 85%, 90%, 95% or up to 100% of their starting viability and / or productivity, which may be assessed using any method known in the art or described herein.

[0240] Treatment The hydrogel capsule composition described herein may be administered to a patient in need of treatment with a therapeutic agent secreted by the encapsulated cells. In one embodiment, the capsule composition, or a therapeutically effective amount thereof, is administered, implanted, or otherwise placed in the peritoneal cavity (e.g., omentum), and the site of administration may include one or more of the lesser sac, also known as the omental bursa or bursalis omentum. The omental bursa refers to a cavity located in the abdomen formed by the omentum, and is in close proximity to, for example, the greater omentum, the lesser omentum, the stomach, the small intestine, the large intestine, the liver, the spleen, the gastrosplenic ligament, the adrenal glands, and the pancreas. Typically, the omental bursa is connected to the greater sac via a greater omental foramen (i.e., the foramen of Winslow). A therapeutically effective amount of the hydrogel capsule composition may be implanted into the peritoneal cavity (e.g., omentum, e.g., omental bursa) or placed on a surface within the peritoneal cavity (e.g., omentum, e.g., omental bursa) via injection or catheter. Additional considerations for transplantation into the omentum (e.g., omental pouch) are provided in M. Pellicciaro et al. (2017) CellR4 5(3):e2410, which is incorporated herein by reference in its entirety.

[0241] Enumerated embodiments 1. A composition comprising a population of hydrogel capsules disposed in a pharma- ceutically acceptable aqueous solution, each hydrogel capsule in the population encapsulating a plurality of viable mammalian cells, the solution having a pH of 6.0 to 9.0 at 12° C. to 30° C. (e.g., about 15 to 25° C.) and comprising a calcium salt at an elemental calcium concentration of about 1.0 millimolar (mM) to about 10 mM.

[0242] 2. The composition of embodiment 1, wherein the solution has an osmolarity of about 250 mOsmol / kg of solution to about 350 mOsmol / kg of solution.

[0243] 3. The composition of any one of the preceding embodiments, wherein each hydrogel capsule in the population comprises an ionically crosslinked alginate.

[0244] 4. The composition of any one of the preceding embodiments, wherein the pH of the solution is 6.5 to 9.0 at 15° C. to 25° C.

[0245] 5. The calcium element concentration is an x ​​value to a y value, and the x and y values ​​are (i) x = about 1.1 mM and y = about 8.0 mM, 6.0 mM, 4.0 mM, or 2.0 mM; (ii) x = about 1.2 mM and y = about 5 mM, 4 mM, 3 mM, or 2.0 mM; (iii) x = about 1.2 mM and y = about 2.0 mM or 1.5 mM; (iv) x = about 1.3 mM and y = about 1.4 mM; (v) x=about 1.4 mM and y=about 4.0 mM, 3.0 mM, or 2.0 mM; and (vi) x = about 1.5 mM and y = about 2.5 mM The composition of any one of the preceding embodiments, selected from the group consisting of:

[0246] 6. The composition of any one of the preceding embodiments, wherein the solution further comprises at least one carbon source (e.g., a sugar (e.g., dextrose, glucose, galactose, hexose, fructose, maltose), glycerol, glutamine, pyruvate or a salt thereof).

[0247] 7. The composition of any one of the preceding embodiments, wherein the solution further comprises a buffer comprising one or more of an acetate salt (e.g., sodium acetate), a gluconate salt (e.g., sodium gluconate), a phosphate salt (e.g., sodium monobasic phosphate), a bicarbonate salt (e.g., sodium bicarbonate), and a lactate salt (e.g., sodium lactate).

[0248] 8. The composition of embodiment 7, wherein the buffer comprises sodium acetate and sodium gluconate.

[0249] 9. The composition of embodiment 8, wherein the buffer consists essentially of about 0.5-5 g / L sodium acetate (e.g., 2 g / L, e.g., 2.29 g / L sodium acetate) and about 0.5-10 g / L sodium gluconate (e.g., 5 g / L, e.g., 5.18 g / L sodium gluconate).

[0250] 10. The composition of embodiment 6, wherein the carbon source is glucose and the solution does not contain any added glutamine or phenol red.

[0251] 11. The composition of embodiment 7, wherein the buffer comprises sodium bicarbonate and sodium phosphate, and the solution does not contain any added HEPES or sodium pyruvate.

[0252] 12. The composition of any one of the preceding embodiments, wherein the calcium salt is calcium chloride.

[0253] 13. The composition of any one of the preceding embodiments, wherein the solution comprises about 1.5 mM to about 2.5 mM calcium chloride, about 5 mM to about 25 mM D-glucose, and about 40 mM to about 50 mM sodium bicarbonate.

[0254] 14. The solution is (i) magnesium compounds (e.g., magnesium chloride or magnesium sulfate); (ii) potassium compounds (e.g., potassium chloride); (iii) sodium chloride; and (iv) a set of amino acids that includes histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine; and (v) The composition of any one of the preceding embodiments, further comprising a set of vitamins including a vitamin B1 compound (e.g., thiamine or a thiamine salt, e.g., thiamine hydrochloride), a vitamin B3 compound (e.g., nicotinic acid or niacinamide), and a vitamin B6 compound (e.g., pyroxidine or a pyroxidine salt, e.g., pyroxidine hydrochloride).

[0255] 15. The composition of claim 14, wherein the set of amino acids also includes arginine, glycine, cystine, serine, and tyrosine, and the set of vitamins also includes choline or a choline salt (e.g., choline chloride), a vitamin B5 compound (e.g., pantothenic acid or calcium pantothenate), a folate compound (e.g., folic acid), riboflavin, and i-inositol.

[0256] 16. The composition of any one of the preceding embodiments, wherein some or all of the amino acids in the set are L-amino acids.

[0257] 17. The solution comprises or consists essentially of the components and concentrations set forth immediately below: [Table 8]

[0258] 18. The composition of any one of the preceding embodiments, wherein the solution comprises or consists essentially of the components and concentrations set forth immediately below: [Table 9-1] [Table 9-2] Optionally, the solution contains D-glucose Approximately 1mM to approximately 50mM; About 2 mM to about 40 mM; About 3mM~about 30mM; About 4mM~about 20mM; About 5mM~about 10mM; About 10 mM to about 40 mM; Approximately 15mM to approximately 35mM; about 20 mM to about 30 mM; and Approximately 25mM The composition of any one of the preceding embodiments, further comprising an amount selected from the group consisting of:

[0259] 19. Each hydrogel capsule in the population has a sphere-like or spherical shape; (a) a cell-containing compartment comprising said plurality of viable cells encapsulated in a first polymer composition; and (b) a barrier compartment surrounding the cell-containing compartment and comprising a second polymer composition comprising the ionically crosslinked alginate, wherein the average diameter of the hydrogel capsules in the population is from about 500 micrometers (μm) to about 5000 μm, from about 1000 (μm) to about 3000 μm, from about 1100 μm to about 2500 μm, from about 1200 μm to about 2300 μm, from about 1300 μm to about 2100 μm, from about 1400 μm to about 2000 μm, from about 1400 μm to about 1900 μm, or from about 1400 μm to about 1800 μm.

[0260] 20. The composition of embodiment 19, wherein the average capsule diameter of the hydrogel capsules in the population is 1400 to 2000 μm.

[0261] 21. The composition of any one of the preceding embodiments, wherein the average thickness of the barrier compartment is from about 10 to about 300 microns, from about 20 to about 150 microns, or from about 40 to about 75 microns.

[0262] 22. The composition of any one of the preceding embodiments, wherein the first polymer composition comprises an alginate covalently modified with a cell contacting peptide via a linker, and the crosslinked alginate in the barrier compartment comprises an alginate covalently modified with at least one non-fibrous compound, optionally a compound selected from the compounds shown in the table below. [Table 10-1] [Table 10-2] [Table 10-3] [Table 10-4] [Table 10-5] [Table 10-6] [Table 10-7] [Table 10-8]

[0263] 23. The composition of embodiment 21, wherein the ionically cross-linked alginate in the barrier compartment comprises barium ions as at least one cross-linking agent.

[0264] 24. The composition of any one of the preceding embodiments, wherein the ionically crosslinked alginate in the barrier compartment comprises a mixture of the covalently modified alginate and an unmodified alginate.

[0265] 25. A composition according to any one of the preceding embodiments, wherein the covalently modified alginate in the cell-containing compartment is ionically crosslinked with at least one barium ion as a crosslinking agent.

[0266] 26. (a) the average diameter of the hydrogel capsules in the population is between 1400 μm and 2000 μm, or between 1400 μm and 1600 μm, or between 1000 μm and 1200 μm; (b) the alginate in the first polymer composition has a molecular weight of 150 to 250 kDa and a G:M ratio of 1.5 or greater; (c) the cell contacting peptide consists of RGDSP and the linker is a single glycine residue attached to the N-terminus of the cell contacting peptide; (d) the alginate in the covalently modified alginate in the barrier compartment has a molecular weight of <75 kDa and a G:M ratio of 1.5 or greater; (e) the non-fibrous compound is [ka] and; (f) the unmodified alginate in the barrier compartment has a molecular weight of 150 kDa to 250 kDa and a G:M ratio of 1.5 or greater; The composition of any one of the preceding embodiments.

[0267] 27. The composition of any one of the preceding embodiments, wherein the live mammalian cell is a human cell.

[0268] 28. The composition of any one of the preceding embodiments, wherein the cells are derived from induced pluripotent stem cells.

[0269] 29. The composition of any one of the preceding embodiments, wherein the cells are derived from RPE cells, optionally from ARPE-19 cells.

[0270] 30. The composition of any one of the preceding embodiments, wherein the encapsulated cell comprises a single cell.

[0271] 31. The composition of any one of the preceding embodiments, wherein the encapsulated cells comprise one or more cell clusters.

[0272] 32. The composition of any one of the preceding embodiments, wherein the encapsulated cells comprise cells disposed on microbeads.

[0273] 33. The composition of any one of the preceding embodiments, wherein the live mammalian cells are genetically modified to express and secrete a therapeutic substance, e.g., a therapeutic polypeptide.

[0274] 34. The composition of any one of the preceding embodiments, wherein the mammalian cell comprises an exogenous nucleotide sequence encoding a therapeutic polypeptide, and optionally the therapeutic polypeptide is a growth factor, a blood clotting factor, an enzyme, a cytokine, a cytokine receptor, an antibody or an antigen-binding fragment thereof.

[0275] 35. The composition of embodiment 34, wherein the therapeutic polypeptide is a FVIII protein (e.g., a FVIII BDD protein), a FIX protein, or a FVII protein.

[0276] 36. The composition of embodiment 34, wherein the therapeutic polypeptide is a GLA protein, an IDUA protein, an IDS protein, an ARSB protein, or a GBA protein.

[0277] 37. The composition of any one of embodiments 19 to 36, wherein the plurality of live mammalian cells is about 5,000 to about 250,000 cells, about 10,000 to about 125,000 cells, about 20,000 to about 75,000 cells, about 12,500 to about 40,000 cells, or about 15,000 to about 30,000 cells.

[0278] The composition of any one of the preceding embodiments, comprising about 200 to about 400 of the hydrogel capsules per 38.1 milliliters of the pharma- ceutically acceptable solution.

[0279] 39. A closed container containing a composition according to any one of the preceding claims.

[0280] 40. The sealed container of claim 39, wherein the volume of the aqueous solution in the container (VS) is approximately equal to or greater than the volume of the hydrogel capsule in the container (VHC).

[0281] 41. The ratio of VS to VHC is (i) about 1.5 to about 100, (ii) about 2 to about 75, (iii) about 3 to about 50, (iv) about 4 to about 40, (v) about 5 to about 30, and (vi) About 10 to about 20 40. The sealed container of embodiment 39, selected from the group consisting of:

[0282] 42. A sealed container described in any one of embodiments 39 to 41, configured to be stored in a manner such that substantially all of the hydrogel capsules in the composition are substantially uniformly distributed across the bottom inner surface of the stored container in a capsule layer having a depth equivalent to about 1.00 to about 1.25 times the average diameter of the capsules in the composition.

[0283] 43. The sealed container of any one of embodiments 39-42, wherein all interior surfaces of the container consist essentially of fluorinated ethylene propylene (FEP) or polyethylene terephthalate glycol (PETG).

[0284] 44. The sealed container of any one of embodiments 39 to 43, which is a flexible rectangular bag comprising a first port configured to allow addition of the composition to the bag and a second port configured to allow removal of a desired volume of the composition from the bag.

[0285] 45. The hydrogel capsules in the container have an average capsule diameter of about 1500 μm and an internal surface area of ​​about 500 cm 2and a total volume of the composition in the container is about 200 mL to about 500 mL.

[0286] 46. ​​A sealed container described in any one of embodiments 39 to 45, wherein the average capsule diameter of the hydrogel capsules in the container is about 1500 μm and the ratio of VHC to VS is at least about 1:1, 1:2, 1:3, and less than about 1:40, for example, any of about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, and 1:35.

[0287] 47. A method of treating a subject in need of a therapeutic substance, comprising providing a composition according to any one of embodiments 33 to 38 and administering a therapeutically effective amount of the composition to the subject.

[0288] 48. The method of embodiment 47, wherein said administering comprises placing said effective amount intraperitoneally in said subject.

[0289] 49. The method of embodiment 47 or 48, wherein the subject is a human.

[0290] 50. A method of making a sealed container containing a hydrogel capsule composition, the method comprising: (i) providing a population of hydrogel capsules encapsulating viable mammalian cells, optionally the mammalian cells being genetically modified to express and secrete a therapeutic substance, e.g., a therapeutic polypeptide; (ii) combining the population of hydrogel capsules with a pharma- ceutically acceptable aqueous solution; (iii) placing a desired volume of the capsule composition into a biocompatible, sealable container in a manner that produces a capsule layer, in which substantially all of the capsules in the volume of the composition are substantially uniformly distributed across a bottom of the container at a depth equivalent to about 1.00 to about 1.25 times the average diameter of the capsules in the composition; and (iv) sealing said container.

[0291] 51. The method of embodiment 50, wherein the aqueous solution has a pH of 6.0 to 9.0 at 12°C to 30°C (e.g., about 15 to 25°C) and contains a calcium salt at an elemental calcium concentration of about 1.0 millimolar (mM) to about 10 mM.

[0292] 52. The method of embodiment 50 or 51, comprising adding a desired volume of the pharma- ceutically acceptable aqueous solution to the container prior to the sealing step to form a solution layer on top of the capsule layer.

[0293] 53. The method of any one of embodiments 50 to 52, further comprising storing the sealed container for a desired period of time at a temperature between 2°C and 30°C or between about 12°C and 30°C (e.g., between about 15°C and 25°C) and evaluating the viability of the encapsulated cells in the composition at one or more time points during the desired period. EXAMPLES

[0294] In order that the disclosure set forth herein may be more fully understood, the following examples are set forth. The examples described in this application are provided to illustrate the genetically modified cells, compositions and implantable devices and methods provided herein and should not be construed in any way as limiting the scope thereof.

[0295] Example 1: Cultivation of Exemplary Genetically Modified ARPE-19 Cells for Encapsulation Genetically modified ARPE-19 cells expressing one or more therapeutic agents described herein can be cultured to generate compositions of cells suitable for encapsulation in a two-compartment hydrogel capsule. The genetically modified cells can be cultured in a 150 cm 2Cells are grown in complete growth medium (DMEM:F12 with 10% FBS) in cell culture flasks or CellSTACK® culture chambers (Corning Inc., Corning, NY). To passage cells, the medium in the culture flask is aspirated and the cell layer is resuspended in phosphate-buffered saline (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8 mM NaCl, 1.0 mM NaCl, 0.01 ... 2 HPO 4 , and 2 mM KH 2 PO 4 Rinse briefly with 10 mL of 0.05% (w / v) trypsin / 0.53 mM EDTA solution ("TrypsinEDTA") to the flask and observe the cells under an inverted microscope until the cell layer is dispersed, usually 3-5 min. To avoid clumping, handle the cells carefully and minimize tapping or shaking the flask during the dispersion period. If the cells do not detach, leave the flask at 37 °C to facilitate dispersion. Once the cells are dispersed, add 10 mL of complete growth medium and aspirate the cells by gentle pipetting. Transfer the cell suspension to a centrifuge tube and spun down at approximately 125 x g for 5-10 min to remove the TrypsinEDTA. Discard the supernatant and resuspend the cells in fresh growth medium. Add an appropriate aliquot of the cell suspension to a new culture vessel and incubate this at 37 °C. Change the medium weekly.

[0296] Example 2: Preparation of exemplary modified polymers Chemically modified polymers. Polymeric materials can be chemically modified with a compound of formula (I) (or a pharma- ceutically acceptable salt thereof) prior to the formation of a device (e.g., a hydrogel capsule) as described herein. For example, in the case of alginate, the alginate carboxylic acid is activated for coupling to one or more amine-functionalized compounds to achieve a non-fibrous compound, e.g., an alginate modified with a compound of formula (I). The alginate polymer is dissolved in water (30 mL / gram polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 eq) and N-methylmorpholine (1 eq). To this mixture is added a solution (0.3 M) of a compound of interest (e.g., compound 101 shown in Table 4) in acetonitrile.

[0297] The amount of compound and coupling reagent added depends on the desired concentration of compound bound to the alginate, e.g., conjugation density. Medium conjugation density of compound 101 typically ranges from 2% to 5% N, whereas high conjugation density of compound 101 typically ranges from 5.1% to 8% N. To prepare a solution of low molecular weight alginate chemically modified with medium conjugation density compound 101 (CM-LMW-Alg-101-medium polymer), dissolved unmodified low molecular weight alginate (approximate MW<75 kDa, G:M ratio≧1.5) is treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (5.1 mmol / g alginate) and N-methylmorpholine (10.2 mmol / g alginate) and compound 101 (5.4 mmol / g alginate). To prepare a solution of low molecular weight alginate chemically modified with compound 101 (CM-LMW-Alg-101-high polymer) with high conjugation density, dissolved unmodified low molecular weight alginate (approximate MW < 75 kDa, G:M ratio ≥ 1.5) was treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (5.1 mmol / g alginate) and N-methylmorpholine (10.2 mmol / g alginate) and compound 101 (10.5 mmol / g alginate).

[0298] The reaction is warmed to 55 °C for 16 h, then cooled to room temperature and gently concentrated via rotary evaporation, then the residue is dissolved in water. The mixture is filtered through a bed of cyano-modified silica gel (Silicycle) and the filter cake is washed with water. The resulting solution is then dialyzed extensively (10,000 MWCO membrane) and the alginate solution is concentrated via lyophilization to provide the desired chemically modified alginate as a solid, or concentrated using any suitable technique to provide a chemically modified alginate solution with a viscosity of 25 cP to 35 cP.

[0299] The conjugation density of the chemically modified alginate is measured by combustion analysis for nitrogen percentage. Samples are prepared by dialysis (10,000 MWCO membrane) of a solution of the chemically modified alginate against two changes of water for 24 hours, followed by lyophilization to constant weight.

[0300] CBP-alginate. Polymeric materials can be covalently modified with cell-binding peptides prior to formation of the devices described herein (e.g., hydrogel capsules described herein) using methods known in the art. See, e.g., Jeon O, et al., Tissue Eng Part A. 16:2915-2925 (2010) and Rowley, JA et al., Biomaterials 20:45-53 (1999).

[0301] For example, in the case of alginate, an alginate solution (1%, w / v) is prepared using 50 mM 2-(N-morpholino)-ethanesulfonic acid hydrate buffer solution (pH 6.5) containing 0.5 M NaCl, and mixed sequentially with N-hydroxysuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). The molar ratio of N-hydroxysuccinimide to EDC is 0.5:1.0. The peptide of interest is added to the alginate solution. The amount of peptide and coupling reagent added depends on the desired concentration of peptide to be bound to alginate, e.g., peptide conjugation density. Higher conjugation density can be obtained by increasing the amount of peptide and coupling reagent. After reacting for 24 hours, the reaction is purified by dialysis against ultrapure deionized water (diH2O) (MWCO 3500) for 3 days, treated with activated charcoal for 30 minutes, filtered (0.22 mm filter), and concentrated to the desired viscosity.

[0302] The conjugation density of peptide-modified alginate is measured by combustion analysis for nitrogen percentage. Samples are prepared by dialysis (10,000 MWCO membrane) of a solution of chemically modified alginate against two changes of water for 24 hours, followed by lyophilization to constant weight.

[0303] Example 3: Preparation of an exemplary alginate solution for making hydrogel capsules A 70:30 mixture of chemically modified and unmodified alginates. A low molecular weight alginate (PRONOVA™ VLVG alginate, NovaMatrix, Sandvika, Norway, cat. #4200506, approximate molecular weight <75 kDa; G:M ratio >= 1.5) is chemically modified with compound 101 to produce a chemically modified low molecular weight alginate (CM-LMW-Alg-101) solution with a viscosity of 25 cP to 35 cP and a conjugation density of 5.1% to 8% N (as determined by combustion analysis for nitrogen percentage). A solution of high molecular weight unmodified alginate (U-HMW-Alg) is prepared by dissolving unmodified alginate (PRONOVA™ SLG100, NovaMatrix, Sandvika, Norway, cat.#4202106, approximate molecular weight of 150 kDa-250 kDa) in 0.9% saline at 3% w / v. The CM-LMW-Alg solution is blended with the U-HMW-Alg solution in a volume ratio of 70% CM-LMW-Alg to 30% U-HMW-Alg (referred to herein as 70:30 CM-Alg:UM-Alg solution).

[0304] Unmodified alginate solution. Prepare U-MMW-Alg solution by dissolving unmodified medium molecular weight alginate (SLG20, NovaMatrix, Sandvika, Norway, cat.#4202006, approximate molecular weight of 75-150 kDa) at 1.4 w / v% in 0.9% saline.

[0305] Unmodified alginate solution. Prepare U-MMW-Alg solution by dissolving unmodified medium molecular weight alginate (SLG20, NovaMatrix, Sandvika, Norway, cat.#4202006, approximate molecular weight of 75-150 kDa) at 1.4 w / v% in 0.9% saline.

[0306] Alginate solution containing cell-binding sites. A solution of SLG20 alginate is modified with a peptide consisting of GRGDSP as described above and concentrated to a viscosity of approximately 100 cP. The amounts of peptide and coupling reagents used are selected to achieve a target peptide conjugation density of approximately 0.2-0.3, as measured by combustion analysis.

[0307] Example 4: Formation of an exemplary two-compartment hydrogel capsule The genetically modified cells can be encapsulated in a two-compartment hydrogel capsule according to processes known in the art, for example, as described in WO2021 / 113751. An exemplary protocol is described below.

[0308] Prior to making the hydrogel capsules, the buffer and alginate solutions are sterilized by filtration through a 0.2 μm filter using an aseptic process.

[0309] Immediately prior to encapsulation, a desired volume of the composition containing cells (e.g., from the culture of cells described in Example 1) is centrifuged at 1,400 rpm for 1 minute and resuspended in calcium-free Krebs-Henseleit (KH) buffer (4.7 mM KCl, 25 mM HEPES, 1.2 mM KH 2 PO 4 , 1.2 mM MgSO 4 x7H 2 Wash the cells with 100 mM NaCl, 135 mM NaCl, pH ~7.4, ~290 mOsm). After washing, centrifuge the cells again and aspirate all of the supernatant. Resuspend the cell pellet in the GRGDSP modified alginate solution described in Example 3 at the desired cell density (e.g., ~50-150 million suspended single cells per ml of alginate solution).

[0310] To prepare two-compartment hydrogel millicapsules with a diameter of approximately 1.5 mm, the electrostatic droplet generator is set up as follows: ES series 0-100-kV, 20 watt high voltage generator (EQ series, Matsuda, NC, USA) is connected to the top and bottom of the coaxial needle. For capsules without immunosuppressants, a suitable needle has an inner lumen of 22 G, an outer lumen of 18 G (Rame-Hart Instrument Co., Succasunna, NJ, USA). To prepare capsules that co-encapsulate immunosuppressant particles in the inner compartment, the lumen of the coaxial needle may need to have a larger diameter to avoid needle clogging by the immunosuppressant particles, for example, a useful coaxial needle has an inner lumen of 21 G and an outer lumen of 17 G (Rame-Hart Instrument Co., Succasunna, NJ, USA).

[0311] The inner lumen is attached to a first 5 ml Luer-lock syringe (BD, NJ, USA) connected to a vertically oriented syringe pump (Pump 11 Pico Plus, Harvard Apparatus, Holliston, MA, USA). The outer lumen is connected via a Luer coupling to a second 5 ml Luer-lock syringe connected to a horizontally oriented second syringe pump (Pump 11 Pico Plus). A first alginate solution containing genetically modified cells (as single cells) suspended in a GRGDSP-modified alginate solution is placed in the first syringe, and a cell-free alginate solution containing a mixture containing chemically modified and unmodified alginate (e.g., the 70:30 mixture described in Example 3) is placed in the second syringe. Two syringe pumps move the first and second alginate solutions from the syringes through both lumens of the coaxial needle, and a single droplet containing both alginate solutions is extruded from the needle into the glass dish containing the crosslinking solution. The setting for each Pico Plus syringe pump is 12.06 mm diameter, and the flow rate of each pump is adjusted to achieve a 1:1 flow rate ratio for the two alginate solutions. Thus, with the total flow rate set at 10 ml / h, the flow rate for each alginate solution was approximately 5 mL / h. Control (empty) capsules are prepared in the same manner, except that the alginate solution used for the inner compartment is a cell-free solution.

[0312] After extrusion of the desired volume of alginate solution, the alginate droplets are resuspended in 25 mM HEPES buffer, 20 mM BaCl 2 The capsules are crosslinked for 5 minutes in a crosslinking solution that contained 0.2 M mannitol and 0.01% poloxamer 188. The capsules that fall to the bottom of the crosslinking vessel are collected in a conical tube by pipetting. After the capsules settle in the tube, the crosslinking buffer is removed and the capsules are washed multiple times with an aqueous solution and stored at a desired temperature, e.g., room temperature.

[0313] Example 5: Composition of the aqueous preservative solutions used in Examples 6-10 [Table 11] [Table 12]

[0314] Example 6: The viability of encapsulated cells is improved by the presence of calcium in the capsule storage solution. Two-compartment hydrogel capsules encapsulating ARPE-19 cells genetically modified to secrete FVIII protein ("FVIII-Spheres") were prepared substantially as described in Example 4. Five milliliters (mL) of spheres were suspended in 63 mL of Solution A or Solution B, and the resulting suspension was placed in a horizontally placed 125 mL square sterile Nalgene™ PETG bottle (ThermoFisher Scientific Cat No. 342020-0125), allowing the spheres to settle as a 1.25x layer across the bottom of the bottle while the remaining storage solution formed a solution layer on top of the sphere layer. The sphere-containing bottles were stored in a horizontal position at room temperature (e.g., at about 25°C) for 3 days, and cell viability was assessed at several time points. An aliquot of the sphere composition was removed from each bottle, the aliquot was combined with an alginate lyase solution to digest the alginate hydrogel and release the cells from the spheres, and cell viability was assessed by incubating the released cells with trypan blue stain and counting the number of dead cells (i.e., stained with trypan blue) and viable cells using an automated cell counter.

[0315] As shown in Figure 1, cell viability in spheres stored in solution A decreased continuously over the 3 days of storage, whereas cell viability in capsules stored in solution B remained essentially constant during the 3 days of storage. Since the only difference between these two solutions is the presence of 2 mM calcium chloride in solution B, the inventors of the present application hypothesized that calcium is important for maintaining the viability of ARPE-19 cells encapsulated in hydrogel capsules.

[0316] Based on the results of additional experiments in which FVIII-spheres were stored in aqueous solutions containing various amounts of calcium chloride (data not shown), the inventors of the present application believe that increased in vitro viability of encapsulated ARPE-19 cells may be achieved when spheres containing the cells are stored in aqueous solutions containing elemental calcium concentrations of at least about 1.0 millimolar (mM) to about 10 mM, compared to storage of the same spheres in the same solutions lacking calcium.

[0317] Example 7: Osmolarity and pH of storage solutions affect productivity of encapsulated cells.

[0318] FVIII-spheres were prepared substantially as described in Example 4. A 1 milliliter (mL) suspension of spheres in 30 mL of solution A (Ca-Sol A) containing 2-5 mM calcium was placed in a horizontally oriented 30 mL rectangular sterile Nalgene™ PETG bottle (ThermoFisher Scientific, Cat No. 342020-030), and a 5 milliliter (mL) suspension of spheres in 63 mL of aqueous storage solution (Solution C or Solution D) was placed in a horizontally oriented 125 mL rectangular sterile Nalgene™ PETG bottle (ThermoFisher Scientific Cat No. 342020-0125). The spheres in both bottle sizes were allowed to settle as a monolayer across the bottom of the bottle, while the remaining storage solution formed a solution layer on top of the sphere layer. After storing the sphere-containing bottles in a horizontal position at room temperature (e.g., at about 25°C) for 4 days, 0.25 mL of spheres were removed from each bottle and implanted into the intraperitoneal (IP) space of NSG™ mice (The Jackson Laboratory, Ellsworth, ME USA). Four mice were implanted per storage solution. As a control, 0.25 ml of FVIII-spheres suspended in solution A containing 2-5 mM calcium (Ca-Sol A) were implanted into the IP space of each of four NSG mice 20-30 hours after sphere preparation. Seven days after implantation, the amount of plasma FVIII was determined for each cohort using an enzyme-linked immunoassay (ELISA) and the results are shown in Figure 2.

[0319] Similar levels of FVIII were secreted from implanted spheres stored in Ca-Sol A or solution C, whereas no plasma FVIII was detected in spheres stored in solution D. Because the osmolality and pH of solutions A and C were similar, with solution D having a significantly higher osmolality and a significantly lower pH, the inventors of the present application hypothesized that the osmolality and pH of the aqueous storage solution are factors that may affect the productivity of cells encapsulated in hydrogel capsules.

[0320] Example 8: Productivity of encapsulated cells can be improved by including amino acids and vitamins in the storage solution.

[0321] FVIII-spheres were prepared substantially as described in Example 4. A suspension of 20 milliliters (mL) of spheres in 400 mL of aqueous storage solution (solution B or solution E) was placed in a rectangular PETG tray, the spheres were allowed to settle as a monolayer across the bottom of the tray, and the remaining storage solution formed a solution layer on top of the sphere layer. After storing the sphere-containing trays at room temperature (e.g., at about 25° C.) for up to 120 hours, 0.5 mL of spheres were removed from each tray and implanted into the intraperitoneal (IP) space of NSG mice. Three mice were implanted per storage solution and storage time, with spheres stored in solution B implanted after 96 hours (t96) and spheres stored in solution E implanted after 96 or 120 hours. As a control, 0.5 ml of FVIII-spheres suspended in the same storage solution (solution E or solution B) was implanted into the IP space of each of three NSG mice within 4 hours after sphere preparation. Six days after transplantation, plasma FVIII levels were determined for each cohort using enzyme-linked immunoassay (ELISA), and the results are shown in Figure 3 .

[0322] Example 9: The depth of the sphere layer in the storage vessel can affect the productivity of encapsulated cells.

[0323] FVIII-spheres were prepared substantially as described in Example 4. Three sphere suspensions containing different volumes of spheres in the same volume of Ca-Sol A were placed in rectangular PETG trays. The sphere volumes in each suspension were selected to provide sphere:solution ratios of 1:20, 1:10, and 1:5 and the spheres settled across the bottom of the tray as a monolayer, bilayer, or triple layer, respectively. The trays were stored at room temperature (e.g., at about 25° C.) for 7 days. At the end of 1, 5, and 7 days, five spheres were removed from each tray and incubated in 100 microliters of medium at 37° C. for 4 to 20 hours, and then the amount of FVIII secreted into the medium was determined by ELISA. The results are shown in FIG. 4, with each bar representing the average of replicate samples for each tray configuration and storage time.

[0324] The results shown in Figure 4 indicate that the thickness of the sphere layer on the bottom of the storage container can affect the in vitro productivity of encapsulated cells during storage, with productivity maintained in spheres stored as monolayers for 7 days, whereas productivity decreased in spheres stored as double or triple layers.

[0325] Example 10: The ratio of spheres to storage solution in the storage container has minimal effect on the productivity of encapsulated cells.

[0326] FVIII-spheres were prepared substantially as described in Example 4. Four sphere suspensions containing different volumes of spheres and volumes of Ca-Sol A were placed in 125 mL rectangular sterile Nalgene™ PETG bottles (ThermoFisher Scientific Cat. No. 342020-0125). The sphere and storage solution volumes in each suspension were selected to produce four different storage configurations by sphere sedimentation: (1) a sphere monolayer with a sphere:solution ratio of 1:18; (2) a sphere monolayer with a sphere:solution ratio of 1:38; (3) a sphere 1.25x layer with a sphere:solution ratio of 1:6, and (4) a sphere 1.25x layer with a sphere:solution ratio of 1:30. The bottles were then stored at room temperature (e.g., at about 25° C.) for up to 4 days.

[0327] After the desired storage period (1 day for storage configuration 1; 4 days for 2-4), 0.5 mL of spheres were removed from the bottle and implanted into the IP space of NSG mice (four mice were implanted per storage configuration). Seven days after implantation, the amount of plasma FVIII was determined using an enzyme-linked immunoassay (ELISA) for each mouse cohort, and the results are shown in Figure 5. The results show that substantially similar amounts of FVIII were secreted from implanted spheres stored as a monolayer or 1.25 layers in different amounts of storage solution.

[0328] Equivalents and Scope This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference in their entirety. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. Also, any particular embodiment of the present disclosure that falls within the prior art may be expressly excluded from any one or more of the claims. Since such embodiments are deemed known to those skilled in the art, they may be excluded even if the exclusion is not expressly set forth herein. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether related to the existence of prior art or not.

[0329] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above specification, drawings, or examples, but is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to the specification may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

1. 1. A composition comprising a population of hydrogel capsules disposed in a pharmaceutically acceptable aqueous solution, wherein each hydrogel capsule in the population encapsulates a plurality of viable mammalian cells, the aqueous solution having a pH of 6.0 to 9.0 at 12°C to 30°C (e.g., about 15 to 25°C) and comprising a calcium salt at an elemental calcium concentration of about 1.0 millimolar (mM) to about 10 mM. (i) the aqueous solution has an osmolality of about 250 mOsmol / kg solution to about 350 mOsmol / kg solution; and / or (ii) The composition of claim 1, wherein the pH of the aqueous solution is 6.5 to 9.0 at 15°C to 25°C.

3. The composition of claim 1 , wherein each hydrogel capsule in the population comprises an ionically cross-linked alginate.

4. The calcium element concentration is an x ​​value to a y value, and the x value and the y value are (i) x = about 1.1 mM and y = about 8.0 mM, 6.0 mM, 4.0 mM, or 2.0 mM; (ii) x = about 1.2 mM and y = about 5 mM, 4 mM, 3 mM, or 2.0 mM; (iii) x = about 1.2 mM and y = about 2.0 mM or 1.5 mM; (iv) x = about 1.3 mM and y = about 1.4 mM; (v) x = about 1.4 mM and y = about 4.0 mM, 3.0 mM, or 2.0 mM; and (vi) x = about 1.5 mM and y = about 2.5 mM 10. The composition of claim 1, selected from the group consisting of:

5. 10. The composition of claim 1, wherein the aqueous solution further comprises at least one carbon source (e.g., a sugar (e.g., dextrose, glucose, galactose, hexose, fructose, maltose), glycerol, glutamine, pyruvate, or a salt thereof).

6. The aqueous solution is (a) a buffer comprising one or more of an acetate (e.g., sodium acetate), a gluconate (e.g., sodium gluconate), a phosphate (e.g., monobasic sodium phosphate), a bicarbonate (e.g., sodium bicarbonate), and a lactate (e.g., sodium lactate); or (b) a buffer comprising sodium acetate and sodium gluconate; or (c) a buffer consisting essentially of about 0.5-5 g / L sodium acetate (e.g., 2 g / L, e.g., 2.29 g / L sodium acetate) and about 0.5-10 g / L sodium gluconate (e.g., 5 g / L, e.g., 5.18 g / L sodium gluconate). The composition of claim 1 further comprising:

7. 6. The composition of claim 5, wherein the carbon source is glucose and the aqueous solution does not contain any added glutamine or phenol red.

8. 7. The composition of claim 6, wherein the buffer comprises sodium bicarbonate and sodium phosphate, and the aqueous solution does not contain any added HEPES or sodium pyruvate.

9. 10. The composition of claim 1, wherein the calcium salt is calcium chloride.

10. 10. The composition of claim 1, wherein the aqueous solution comprises about 1.5 mM to about 2.5 mM calcium chloride, about 5 mM to about 25 mM D-glucose, and about 40 mM to about 50 mM sodium bicarbonate.

11. The aqueous solution is (i) a magnesium compound (e.g., magnesium chloride or magnesium sulfate); (ii) potassium compounds (e.g., potassium chloride); (iii) sodium chloride; (iv) a set of amino acids comprising histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine; (v) a set of amino acids comprising arginine, glycine, cystine, serine, and tyrosine; (vi) a set of vitamins including choline or a choline salt, a vitamin B5 compound, a folic acid compound, riboflavin, and i-inositol; and / or (vii) a set of vitamins including a vitamin B1 compound (e.g., thiamine or a thiamine salt, e.g., thiamine hydrochloride), a vitamin B3 compound (e.g., nicotinic acid or niacinamide), and a vitamin B6 compound (e.g., pyridoxine or a pyridoxine salt, e.g., pyridoxine hydrochloride). The composition of claim 1 further comprising:

12. each hydrogel capsule in the population has a spherical or globular shape; (a) a cell-containing compartment comprising said plurality of viable mammalian cells encapsulated in a first polymer composition; and (b) a barrier compartment surrounding the cell-containing compartment and comprising a second polymer composition comprising ionically cross-linked alginate; 10. The composition of claim 1, wherein the hydrogel capsules in the population have an average diameter of from about 500 micrometers (μm) to about 5000 μm, from about 1000 μm to about 3000 μm, from about 1100 μm to about 2500 μm, from about 1200 μm to about 2300 μm, from about 1300 μm to about 2100 μm, from about 1400 μm to about 2000 μm, from about 1400 μm to about 1900 μm, or from about 1400 μm to about 1800 μm.

13. the first polymer composition comprises alginate covalently modified with a cell contacting peptide via a linker; the ionically cross-linked alginate in the barrier compartment comprises alginate covalently modified with at least one non-fibrous compound; Optionally, the non-fibrous compound is a compound selected from compounds 100 to 154 shown below. 【Chemistry 1】 【change】 【change】 【change】 【change】 【change】 【change】 【change】

14. The ionically cross-linked alginate in the barrier compartment is (a) barium ions as at least one cross-linking agent; and / or (b) a mixture of the covalently modified alginate and an unmodified alginate. The composition of claim 13 comprising:

15. 15. The composition of claim 14, wherein the covalently modified alginate in the cell-containing compartment is ionically crosslinked with at least one barium ion as a crosslinking agent.

16. (a) the average diameter of the hydrogel capsules in the population is between 1400 μm and 2000 μm, or between 1400 μm and 1600 μm, or between 1000 μm and 1200 μm; (b) the alginate in the first polymer composition has a molecular weight of 150 to 250 kDa and a G:M ratio of 1.5 or greater; (c) the cell contacting peptide consists of RGDSP and the linker is a single glycine residue attached to the N-terminus of the cell contacting peptide; (d) the alginate in the covalently modified alginate in the barrier compartment has a molecular weight of <75 kDa and a G:M ratio of 1.5 or greater; (e) the non-fibrous compound is 【Chemistry 2】 and (f) the unmodified alginate in the barrier compartment has a molecular weight of 150 kDa to 250 kDa and a G:M ratio of 1.5 or greater; 15. The composition of claim 14.

17. The live mammalian cells (a) human cells; (b) a cell derived from an induced pluripotent stem cell; or (c) Cells derived from RPE cells 2. The composition of claim 1, wherein:

18. The encapsulated cells are (a) Single cell; (b) one or more cell clusters; and / or (c) Cells arranged on microbeads The composition of claim 1 comprising:

19. The composition of claim 1 , wherein the live mammalian cells are genetically modified to express and secrete a therapeutic substance, e.g., a therapeutic polypeptide.

20. 2. The composition of claim 1, wherein the mammalian cell comprises an exogenous nucleotide sequence encoding a therapeutic polypeptide, optionally wherein the therapeutic polypeptide is a growth factor, a blood clotting factor, an enzyme, a cytokine, a cytokine receptor, an antibody, or an antigen-binding fragment thereof.

21. 21. The composition of claim 20, wherein the therapeutic polypeptide is a FVIII protein, a FIX protein, or a FVII protein.

22. 21. The composition of claim 20, wherein the therapeutic polypeptide is a GLA protein, an IDUA protein, an IDS protein, an ARSB protein, or a GBA protein.

23. 10. The composition of claim 1, wherein the plurality of viable mammalian cells is about 5,000 to about 250,000 cells, about 10,000 to about 125,000 cells, about 20,000 to about 75,000 cells, about 12,500 to about 40,000 cells, or about 15,000 to about 30,000 cells.

24. 10. The composition of claim 1, comprising about 200 to about 400 of said hydrogel capsules per milliliter of said pharmaceutically acceptable aqueous solution.

25. A sealed container containing the composition of any one of claims 1 to 24.

26. (a) the volume of the aqueous solution in the container (V) is approximately equal to or greater than the volume of the hydrogel capsule in the container (V); or (b) The ratio of VS to VHC is (i) from about 1.5 to about 100; (ii) about 2 to about 75, (iii) about 3 to about 50, (iv) from about 4 to about 40, (v) from about 5 to about 30, and (vi) About 10 to about 20 26. The sealed container of claim 25, selected from the group consisting of:

27. 27. The sealed container of claim 26, wherein the container is configured to be stored in a manner such that substantially all of the hydrogel capsules in the composition are substantially uniformly distributed across the bottom interior surface of the stored container in a capsule layer having a depth equivalent to about 1.00 to about 1.25 times the average diameter of the capsules in the composition.

28. 27. The sealed container of claim 26, wherein all interior surfaces of the container consist essentially of fluorinated ethylene propylene (FEP) or polyethylene terephthalate glycol (PETG).

29. 27. The sealed container of claim 26, wherein the bag is a flexible rectangular bag, the bag including a first port configured to allow addition of the composition to the bag and a second port configured to allow removal of a desired volume of the composition from the bag.

30. 27. The sealed container of claim 26, wherein the average diameter of the hydrogel capsules in the container is about 1500 μm and the ratio of VHC to VS is at least about 1:1, 1:2, 1:3, and less than about 1:40, e.g., any of about 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, and 1:

35.

31. 10. The composition of claim 1 for use in treating a subject in need of a therapeutic agent, comprising: the treatment comprises administering to the subject a therapeutically effective amount of the composition; said administering comprising placing said therapeutically effective amount of said composition intraperitoneally in said subject; The composition, wherein the subject is a human.

32. 1. A method of making a sealed container containing a hydrogel capsule composition, the method comprising: (i) providing a population of hydrogel capsules encapsulating viable mammalian cells, optionally wherein the mammalian cells have been genetically modified to express and secrete a therapeutic substance, e.g., a therapeutic polypeptide; (ii) combining the population of hydrogel capsules with a pharmaceutically acceptable aqueous solution; (iii) placing a desired volume of the capsule composition into a biocompatible, sealable container in a manner to produce a capsule layer, wherein in the capsule layer, substantially all of the capsules in the volume of the composition are substantially uniformly distributed across the bottom of the container at a depth equivalent to about 1.00 to about 1.25 times the average diameter of the capsules in the composition; and (iv) sealing the container The method comprising:

33. 33. The method of claim 32, wherein the aqueous solution has a pH of 6.0 to 9.0 at 12° C. to 30° C. (e.g., about 15 to 25° C.) and comprises a calcium salt at an elemental calcium concentration of about 1.0 millimolar (mM) to about 10 mM.

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