Treatment of mucopolysaccharidosis type 6

Abstract

Described herein are ARSB fusion proteins, polynucleotides encoding the fusion proteins, and mammalian cells genetically modified to express and secrete the fusion proteins, as well as compositions comprising the fusion proteins or genetically modified cells that secrete the fusion proteins, implantable devices and device preparations, and methods of making and using the same to treat MPS-6.

Description

Detailed Description of the Invention

[0001] Claiming priority This application claims priority to U.S. Patent Application No. 63 / 304,382, filed January 28, 2022. The entire contents of the aforementioned application are incorporated herein by reference in their entirety. [Background technology]

[0002] Mucopolysaccharidosis type 6 (MPS-6) is a rare autosomal genetic disorder characterized by deficient activity of the lysosomal enzyme arylsulfatase B (ARSB) (also known as N-acetylglucosamine 4-sulfatase), leading to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan sulfate (DS) and chondroitin sulfate (CS). Lysosomal storage of DS and CS causes many problems, including bone dysplasia, joint restriction, organomegaly, heart disease, and corneal opacity. MPS-6 typically manifests in one of two forms: an aggressive form that results in severe disease in the majority of patients, and a slow progressive form that shows more attenuated symptoms in a minority of patients.

[0003] The most widely used specific treatment for MPS-6 disease is enzyme replacement therapy (ERT), which is currently a lifelong therapy that requires weekly or twice-weekly intravenous infusions of recombinant ARSB. Therefore, novel treatment modalities for MPS-6 are desirable. Summary of the Invention

[0004] Described herein is an ARSB fusion protein that includes a human serum albumin (HSA) binding domain and an amino acid sequence of a mammalian ARSB protein, such as the amino acid sequence of human mature ARSB. The HSA binding domain is located N-terminal to the ARSB amino acid sequence.

[0005] In one aspect, the disclosure features an ARSB fusion protein comprising a primary structure defined by the formula F:AB-L-ARSB, where AB is an HSA binding domain, L is a linker moiety, and ARSB comprises the amino acid sequence of a mature mammalian ARSB protein, e.g., from amino acid 37 or 39 to amino acid 533 of FIG. 1 (SEQ ID NO: 1). In one embodiment, AB has a molecular weight of less than about 75 kDa, about 50 kDa, or about 25 kDa. In one embodiment, AB comprises, consists essentially of, or consists of an amino acid sequence from a single chain Fab (scFab), a single chain Fv (scFv), or a single domain antibody (sdAb). In one embodiment, the linker moiety is a linker peptide that is less than 50 amino acids in length.

[0006] In one embodiment, the ARSB fusion protein binds to domain 1 (DI) or domain 2 (DII) of HSA via AB and does not substantially inhibit binding of human FcRn (h-FcRn) to HSA. In one embodiment, the fusion protein has a K of less than about 1 nM to about 100 nM within a pH range of about 5.0 to about 7.4 as determined by surface plasmon resonance at 25° C. D The fusion protein binds to HSA via AB with high affinity. In some embodiments, the fusion protein also binds to at least one non-human mammalian serum albumin ortholog via AB at 25° C. within a pH range of about 5.5 to about 7.4. In one embodiment, the albumin ortholog is from a mouse, rat, hamster, rabbit, guinea pig, pig, cat, dog, or non-human primate (e.g., a cynomolgus or rhesus monkey).

[0007] In one embodiment, AB comprises a first, second, and third amino acid sequence corresponding to the three complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of an anti-HSA antibody (e.g., a conventional antibody having two heavy chains and two light chains, scFab, scFv, sdAb). In one embodiment, the CDR1, CDR2, and CDR3 amino acid sequences in AB are GRTFIAYA (SEQ ID NO:3), or a conservatively substituted variant thereof, ITNFAGGTT (SEQ ID NO:4), or a conservatively substituted variant thereof, and AADRSAQTMRQVRPVLPY (SEQ ID NO:5), or a conservatively substituted variant thereof.

[0008] In one embodiment, AB consists essentially of or consists of QVQLVESGGGLVQAGGSLRLSCVASGRTFIAYAMGWFRQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNAKTTVYLQMNSLKPEDTALYYCAADRSAQTMRQVRPVLPYWGQGTQVTVSS (SEQ ID NO: 6), or a conservatively substituted variant thereof.

[0009] In one embodiment, the AB consists essentially of, or consists of, QVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWFRQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNAKTTVYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPVLPYWGQGTLVTVSS (SEQ ID NO: 7), or a conservatively substituted variant thereof. In one embodiment, the AB consists essentially of, or consists of, the amino acid sequence of the heavy chain variable region of an antibody that cross-competes with an sdAb consisting of SEQ ID NO: 6 or SEQ ID NO: 7 for binding to HSA.

[0010] The linker moiety L, in some embodiments, is a peptide that is at least 5 amino acids in length and no more than about 50 amino acids in length, e.g., about 45, about 40, about 35, about 30, about 25, about 20, about 15, or about 10 amino acids in length. In one embodiment, L is (GGGGS) n(SEQ ID NO: 8), where n is equal to 3, 4 or 5. In one embodiment, L is (GGGGS) 3 (SEQ ID NO:9)

[0011] In one embodiment, the ARSB fusion protein comprises, consists essentially of, or consists of the amino acid sequence shown in Figure 3A (SEQ ID NO:10) or Figure 3B (SEQ ID NO:11).

[0012] In another aspect, the disclosure provides a polynucleotide (e.g., an isolated polynucleotide) comprising a nucleotide sequence encoding an ARSB fusion protein described herein. In one embodiment, the nucleotide sequence is operably linked to a promoter sequence and a polyA signal sequence. In one embodiment, the promoter sequence is identical or substantially identical to one of the promoter sequences of Figures 4A-4C. In one embodiment, the polyA signal sequence is identical or substantially identical to one of the polyA signal sequences shown in Figure 5. In one embodiment, the ARSB fusion protein consists of the amino acid sequence shown in Figure 6A and the polynucleotide comprises, consists essentially of, or consists of the nucleotide sequence shown in Figures 6B-1 and 6B-2.

[0013] In yet another aspect, the disclosure provides mammalian cells (e.g., mouse cells, Chinese Hamster Ovary (CHO) cells, monkey cells, human cells (e.g., RPE cells) that have been genetically modified to express and secrete an ARSB fusion protein described herein. In one embodiment, the mammalian cells are genetically modified by transfection with a polynucleotide (e.g., an expression vector) described herein that encodes an ARSB fusion protein. In one embodiment, the mammalian cells are derived from RPE cells (e.g., ARPE-19 cells) by transfection with an expression vector that consists essentially of, or consists of, the nucleotide sequence depicted in Figures 7-1 to 7-5 (SEQ ID NO:20).

[0014] The present disclosure also provides a composition comprising a plurality of genetically modified cells as described herein, and a method for producing the composition. In one embodiment, the composition comprises a cell culture medium or a storage medium. In one embodiment, the composition comprises a polymer solution in which the cells are suspended, for example, a polymer solution as described herein comprises, for example, an alginate and a cell binding agent, for example, as defined herein. In one embodiment, the method for producing the composition comprises culturing a plurality of genetically modified cells as described herein until a desired number of cultured cells is produced, and combining the desired number of cultured cells with a cell culture medium, a storage medium, or a polymer solution.

[0015] In yet another aspect, the disclosure features a device comprising at least one cell-containing compartment comprising a genetically modified mammalian cell or a plurality of such cells as described herein. The device is configured to protect the cell(s) from the recipient's immune system and reduce the foreign body response (FBR) (as defined herein) to the implanted device. In one embodiment, the surface of the device comprises a compound or polymer that reduces the FBR (as defined herein) to the device (e.g., a non-fibrous compound or a non-fibrous polymer). In one embodiment, the non-fibrous polymer comprises a biocompatible, zwitterionic polymer, e.g., as described in WO2017 / 218507, WO2018 / 140834, or Liu et al., Zwitterionically modified alginates mitigate cellular overgrowth for cell encapsulation, Nature Communications (2019) 10:5262. In one embodiment, the compound is a compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein the variables A, L 1 , M, L 2 , P., L. 3, and Z, and associated subvariables, are defined herein. In some embodiments, the compound of Formula (I) or a pharma- ceutically acceptable salt thereof (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)) is a compound described herein, including, for example, one of the compounds shown in Table 5 herein, e.g., Compound 100, Compound 101, Compound 102, or Compound 122 shown in Table 5.

[0016] 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, optionally ionically (e.g., with barium ions) crosslinked. 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 hydrogel capsules have an average capsule diameter of 1400-2000 μm.

[0017] In one aspect, the device of the present disclosure is a two-compartment hydrogel capsule (e.g., a microcapsule (diameter less than 1 mm) or millicapsule (diameter at least 1 mm)) in which a cell-containing compartment (e.g., an inner compartment) comprising a plurality of live genetically modified cells (and optionally one or more cell-binding substances) as described herein is surrounded by a barrier compartment comprising a non-fibrous polymer (e.g., an outer compartment, e.g., a hydrogel layer). In one embodiment, the non-fibrous polymer comprises a non-fibrous compound. In one embodiment, the non-fibrous compound is a compound of formula (I).

[0018] In another aspect, the disclosure features a preparation (e.g., a composition) that includes a plurality (3, 6, 12, 25, 50, or more) of the cell-containing devices described herein, e.g., a hydrogel capsule encapsulating genetically modified cells, such as genetically modified ARPE-19 cells. In some embodiments, the preparation is a pharma- ceutically acceptable composition.

[0019] In another aspect, the disclosure features a method of making or manufacturing a device comprising a genetically modified cell as described herein. In some embodiments, the method includes providing a genetically modified cell, or a plurality of such cells, and disposing the cell(s) in a surrounding component, e.g., a cell-containing compartment of a device as described herein. In some embodiments, the surrounding component includes a flexible polymer (e.g., PLA, PLG, PEG, CMC, or a polysaccharide, e.g., alginate). In some embodiments, the surrounding component includes a non-flexible polymer or metal housing. In some embodiments, the surface of the device is chemically modified, e.g., with a compound of formula (I) as described herein.

[0020] In one embodiment, the device, or devices, described herein are combined with a pharma- ceutically acceptable excipient to prepare a device preparation or composition that can be administered (e.g., intraperitoneally) to a subject in need of treatment with the ARSB fusion protein produced by the device. In one embodiment, the genetically modified cells are derived from human cells (e.g., RPE cells, ARPE-19 cells), and the device preparation or composition can continuously deliver an effective amount of the ARSB fusion protein to the subject for a sustained period of time, e.g., at least any of 3 months, 6 months, 1 year, 2 years, or more.

[0021] In another aspect, the disclosure features a method of evaluating a composition, device, or device preparation described herein. In some embodiments, the method includes providing the composition, device, or device preparation and evaluating a functional parameter of the composition, device, or device preparation. In one embodiment, the functional parameter is the amount of ARSB fusion protein produced by cells in the composition, device, or device preparation in vitro (e.g., when placed in a suitable culture medium) and / or in vivo (e.g., after implantation into a subject, e.g., a non-human subject or a human subject).

[0022] In another aspect, the disclosure features a method of treating a subject in need of therapy with an ARSB fusion protein described herein, e.g., a human subject diagnosed with MPS-6. In one embodiment, the method includes administering to the subject genetically modified cells that express and secrete the fusion protein, or a device or device preparation comprising a plurality of such cells. In some embodiments, the administering step includes placing in the subject a pharma- ceutically acceptable preparation comprising a plurality of devices, each capable of producing the ARSB fusion protein. In some embodiments, the implantable element is administered, placed, or injected into the peritoneal cavity (e.g., the lesser pouch), omentum, or subcutaneous fat of the subject. In one embodiment, the method further includes measuring the amount of ARSB fusion protein present in a tissue sample removed from the subject, e.g., in plasma separated from a blood sample. In one embodiment, the tissue sample is removed 15, 30, 60, or 120 days after administration, implantation, or placement of the device or device preparation.

[0023] The details of one or more embodiments of the disclosure are set forth herein. Other features, objects, and advantages of the disclosure will become apparent from the detailed description, drawings, examples, and claims. [Brief description of the drawings]

[0024] [Figure 1] The amino acid sequence of precursor human ARSB (SEQ ID NO:1) is shown. [Diagram 2] The amino acid sequence of precursor human serum albumin (SEQ ID NO:2) is shown with the signal peptide underlined. [Diagram 3]

[0023] Figures 1A-1D show the amino acid sequences of exemplary ARSB fusion proteins of the present disclosure. Fusion protein A (SEQ ID NO: 10) contains the parent anti-HSA sdAb sequence, and fusion protein B (SEQ ID NO: 11) contains a humanized variant of the anti-HSA sdAb sequence shown in A, with underlining indicating the anti-HSA sdAb sequence, italic font in each sequence indicating the linker peptide, and bold font indicating the human mature ARSB sequence. [Figure 4A] An exemplary promoter sequence that is useful in expression constructs for the ARSB fusion proteins of the disclosure is the pCAG promoter sequence (SEQ ID NO: 12). [Figure 4B] An exemplary promoter sequence that is useful in expression constructs for the ARSB fusion proteins of the present disclosure is the EF1α promoter sequence (SEQ ID NO:13). [Figure 4C] An exemplary promoter sequence that is useful in expression constructs for the ARSB fusion proteins of the present disclosure is the EFS promoter sequence (SEQ ID NO:14). [Diagram 5] Exemplary polyA signal sequences that are useful in expression constructs for the ARSB fusion proteins of the present disclosure are shown: rBG polyA signal sequence (A, SEQ ID NO:15), SV40 late polyA signal sequence (B, SEQ ID NO:16), and BGH polyA signal sequence (C, SEQ ID NO:17). [Figure 6A] Shown is the amino acid coding sequence of an exemplary precursor ARSB fusion protein of the present disclosure (SEQ ID NO: 18), where single underlining indicates the consensus VHH signal peptide, double underlining indicates the R28 sdAb, italics indicate the linker peptide, and bold font indicates human mature ARSB. [Figure 6B-1] 1 shows the nucleotide coding sequence of an exemplary precursor ARSB fusion protein of the present disclosure (SEQ ID NO: 19), where single underlining indicates the consensus VHH signal peptide, double underlining indicates the R28 sdAb, italics indicate the linker peptide, and bold font indicates human mature ARSB. [Figure 6B-2] This is a continuation of Figure 6B-1. [Figure 7-1] FIG. 6B shows the nucleotide sequence (SEQ ID NO:20) of an exemplary transposon expression vector that is useful for genetically modifying human cells to express the ARSB fusion protein shown in FIG. 6A. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 7-3] This is a continuation of Figure 7-2. [Figure 7-4] This is a continuation of Figure 7-3. [Figure 7-5] This is a continuation of Figure 7-4. [Figure 8] 1 shows the assessment of ARSB activity of ARSB fusion proteins. A shows a standard curve generated using unfused recombinant human ARSB (galsulfase), and B compares the ARSB activity of fusion proteins with anti-HSA sdAb either N-terminal or C-terminal to the ARSB amino acid sequence. [Figure 9] We show that the activity of native and recombinant HL-hARSB fusion proteins exhibit similar biochemical properties as measured by their ability to reduce the amount of chondroitin sulfate / dermatan sulfate (CS / DS) compared to untreated controls. [Figure 10A] 4 shows that total hARSB activity in the liver is highest in mice treated with hydrogel capsules containing cells engineered to express the HL-hARSB enzyme. [Figure 10B] 4 shows that total hARSB activity in the heart is highest in mice treated with hydrogel capsules containing cells engineered to express the HL-hARSB enzyme. [Figure 10C] 2 shows that total hARSB activity in the spleen is highest in mice treated with hydrogel capsules containing cells engineered to express the HL-hARSB enzyme. [Figure 11A] 1 shows that total CS / DS activity in the liver was comparable between HL-hARSB and the galsulfase control in all test conditions. [Figure 11B] 1 shows that total CS / DS activity in the heart is comparable between HL-hARSB and galsulfase control in all conditions tested. [Figure 11C] 1 shows that total CS / DS activity in the spleen was comparable between HL-hARSB and the galsulfase control in all test conditions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present disclosure features ARSB fusion proteins comprising an HSA binding domain located N-terminal to an enzymatic domain comprising a mammalian ARSB protein, mammalian cells (e.g., human RPE cells) genetically modified to express and secrete these ARSB fusion proteins, and compositions and devices comprising the genetically modified cells. In some embodiments, the devices comprise a cell-containing compartment comprising a cell-binding agent and the genetically modified cells. In some embodiments, the devices are configured to reduce FBR when placed within a subject, e.g., a human subject. In some embodiments, the fusion proteins, genetically modified cells, compositions, and devices are useful for treating MPS-6.g.

[0026] Abbreviations and Definitions The following abbreviations are used throughout the detailed description and examples of this disclosure: CS: chondroitin sulfate DS: Dermatan sulfate GAG: glycosaminoglycan MPS: Mucopolysaccharidosis

[0027] 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.

[0028] 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.

[0029] "About" or "approximately," when used herein to modify a numerically defined parameter (e.g., the amount of fusion protein secreted by engineered cells, the physical description of a device (e.g., hydrogel capsule), e.g., diameter, sphericity, number of cells encapsulated therein, number of devices in a preparation), means that the stated numerical value is within an acceptable functional range for the defined parameter as determined by one of 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 devices (e.g., hydrogel capsules) includes a preparation having 80-120 devices. 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.

[0030] As used herein, "obtain" or "obtaining" refers to obtaining a value, e.g., a numerical value, or an image, or a physical entity (e.g., a sample), by "directly obtaining" or "indirectly obtaining" the value or physical entity. "Directly obtaining" refers to performing a process (e.g., performing an analytical method or protocol) to obtain a value or physical entity. "Indirectly obtaining" refers to receiving a value or physical entity from another entity or source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a value or physical entity includes performing a process that involves a physical change in a physical substance or using a machine or device. An example of directly obtaining a value includes obtaining a sample from a human subject. Directly obtaining a value includes performing a process using a machine or device, e.g., using a fluorescent microscope to obtain fluorescent microscopy data.

[0031] "Administer," "administering," or "administration," as used herein, refers to implanting, absorbing, ingesting, injecting, placing, or otherwise introducing into a subject an entity described herein (e.g., a device or preparation of a device), or providing such an entity to a subject for administration.

[0032] "Non-fibrotic" as used herein means a compound or material that mitigates foreign body response (FBR). For example, the amount of FBR induced in a biological tissue by implantation of a device (e.g., a hydrogel capsule) containing a non-fibrotic compound (e.g., a hydrogel capsule containing a polymer covalently modified with a compound listed in Table 5) is lower than the FBR induced by implantation of a non-fibrotic null reference capsule lacking any non-fibrotic compound but of substantially the same composition (e.g., same cell type(s)) and structure (e.g., size, shape, number of compartments). In one embodiment, the degree of FBR is assessed by an immunological response in the tissue containing the implanted device (e.g., hydrogel capsule), which may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis, using assays known in the art, for example, as described in WO2017 / 075630, or as described in Vegas, A., et al., Nature 1999, 10, 141-145, 1999, 10, 142-143, 1999, 10, 143-144, 1999, 10, 144-145, 1999, 10, 145-146, 1999, 10, 146-147, 1999, 10, 147-148, 1999, 10, 148-149, 1999, 10, 149 ... Using one or more of the assays / methods described in Biotechnol (ibid.) (e.g., subcutaneous cathepsin measurement of implanted capsules, Masson's trichrome staining (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cell staining and confocal microscopy of macrophages (CD68 or F4 / 80), myofibroblasts (alpha-muscle actin, SMA), or general cellular deposits, quantification of 79 RNA sequences of known inflammatory factors and immune cell markers, or FACS analysis of macrophages and neutrophil cells in devices (e.g., capsules) retrieved after 14 days in the intraperitoneal space of suitable test subjects, e.g., immunocompetent mice). In one embodiment, FBR is assessed by measuring the levels of one or more biomarkers of immune response, e.g., cathepsin, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4, in tissues containing the implant.In some embodiments, the FBR induced by a device of the invention (e.g., a hydrogel capsule comprising a non-fibrous compound disposed on its exterior surface) is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the FBR induced by an FBR null reference device, e.g., a device that is substantially identical to the tested or claimed device except for lacking a means for mitigating the FBR (e.g., a hydrogel capsule that does not comprise a non-fibrous compound but is otherwise substantially identical to the claimed capsule). In some embodiments, the FBR (e.g., the level of biomarker(s)) is measured after about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or more.

[0033] "Arylsulfatase B protein" and "ARSB protein" may be used interchangeably herein and refer to a protein comprising the amino acid sequence of a mature wild-type mammalian ARSB, or any fragment, mutant, variant, or derivative thereof having an enzymatic activity (e.g., sulfatase activity) within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature ARSB protein, as measured by the ARSB activity assay described herein. ARSB hydrolyzes sulfates in the body by metabolizing the sulfate moieties of dermatan sulfate and chondroitin sulfate. 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. The amino acid sequence of wild-type human precursor ARSB is shown in FIG. 1 (SEQ ID NO:1). In one embodiment, the mature human ARSB sequence is amino acids 37-533 of FIG. 1. In another embodiment, the mature human ARSB sequence is amino acids 39-533 of Figure 1. In some embodiments, the term "ARSB protein" refers to a polypeptide comprising the wild-type mature amino acid sequence, optionally followed by the ARSB signal peptide or a signal peptide of a different secreted protein (e.g., a protein secreted by human cells, such as ARPE-19 cells).

[0034] "Cell" as used herein refers to an engineered cell (e.g., a genetically modified cell) or a non-engineered cell. In one embodiment, the cell is an immortalized cell or an engineered cell derived from an immortalized cell. In one embodiment, the cell is a viable cell, e.g., viable as measured by any technique described herein or known in the art.

[0035] "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 No. WO2020069429. In one embodiment, the CBP is a linear peptide comprising RGD (SEQ ID NO: 56) and is less than 10 amino acids in length. In one embodiment, the CBP is a linear peptide consisting essentially of GRGD (SEQ ID NO: 57) or GRGDSP (SEQ ID NO: 58).

[0036] "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, such as sodium alginate. In one embodiment, the linker is an amino acid linker (i.e., consisting essentially of a single amino acid, or a peptide of several identical or different amino acids), which is connected to the N-terminus or C-terminus of CBP via a peptide bond. In one embodiment, the CBP-polymer is any of the CBP-alginates defined in WO2020069429.

[0037] "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 WO2020069429.

[0038] "Conservatively modified variant" or "conservative substitution" as used herein refers to a variant of a reference peptide or polypeptide that is 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. In some embodiments, a conservatively modified variant of an HSA binding domain sequence does not include any amino acid substitutions in the CDRs. 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]

[0039] "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 any 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, device, or method. As a non-limiting example, an HSA binding domain or ARSB domain consisting essentially of a recited amino acid sequence can also include one or more amino acids that include substitutions in the recited amino acid sequence of one or more amino acid residues that do not substantially affect the relevant biological activity of the HSA binding domain or ARSB portion, respectively.

[0040] "Derived from," as used herein with respect to a cell or cells, refers to cells obtained from a tissue, cell line, or cells, which are then optionally cultured, passaged, immortalized, differentiated, and / or induced to result in a derived cell(s).

[0041] "Device," as used herein, refers to any implantable object (e.g., particle, hydrogel capsule, implant, medical device) that contains an engineered cell or cells (e.g., living cells) that are capable of expressing and secreting a fusion protein after implantation of the device and has a configuration that supports cell survival by allowing cellular nutrients to enter the device.

[0042] "Effective amount," as used herein, refers to any of the following: genetically modified cells secreting an ARSB fusion protein, a device preparation producing a fusion protein, the number of genetically modified cells in the device, the amount of CBS and / or non-fibrous compound in the device sufficient to elicit a desired biological response. In some embodiments, the term "effective amount" refers to the amount of a component of the device (e.g., the number of cells in the device, the density of non-fibrous compound disposed on the surface and / or in the barrier compartment of the device, the density of CBS in the cell-containing compartment).

[0043] In one embodiment, the desired biological response is an increase in the level of ARSB protein in a tissue sample removed from a subject treated (e.g., implanted) with genetically modified cells, a device containing such cells, or a device preparation. As will be appreciated by those skilled in the art, the effective amount may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the secreted ARSB fusion protein, composition, or device, the condition being treated, the mode of administration, and the age and health of the subject. Effective amounts include therapeutic and prophylactic treatments.

[0044] In one embodiment, an effective amount of a compound of formula (I) disposed on or within the device is an amount that reduces the FBR for the implanted device compared to a reference device, e.g., reduces the amount of fibrosis or fibrous tissue on or near the implanted device.

[0045] In one embodiment, an effective amount of CBS placed with the engineered cells in the cell-containing compartment is an amount that enhances the viability of the cells (e.g., number of viable cells) compared to a reference device and / or increases the production of ARSB fusion protein by the cells (e.g., increased levels of fusion protein in the plasma of a subject in which the device is implanted) compared to a reference device. The effective amount of a device, composition, or component (e.g., non-fibrous compound, CBS, engineered cells) may be determined by any technique known in the art as described herein.

[0046] An "engineered cell" or "genetically modified cell," as used herein, is a mammalian cell (e.g., a human cell, e.g., an RPE cell, a cell derived from a cell line (e.g., ARPE-19 or other cell line), a stem cell, a cell differentiated from an iPSC) that has at least one non-naturally occurring mutation and typically comprises an exogenous nucleotide sequence (e.g., a vector or a mutated chromosomal sequence) that encodes an ARSB fusion protein as described herein. In one embodiment, the exogenous nucleotide sequence is chromosomal (e.g., the exogenous sequence is placed in an endogenous chromosomal sequence) or extrachromosomal (e.g., a non-integrating expression vector). In one embodiment, the exogenous nucleotide sequence in the genetically modified cell comprises a codon-optimized coding sequence for one or both of the AB and ARSB domains in the fusion protein, which achieves increased expression of the fusion protein by the modified cell compared to the naturally occurring coding sequences for each of these domains. Codon optimized sequences can be generated using commercially available algorithms, such as GeneOptimizer (ThermoFisher Scientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), or the Java Codon Adaptation Tool (JCat, www.jcat.de, Grote, A. et al., Nucleic Acids Research, Vol 33, Issue suppl_2, pp. W526-W531 (2005). In one embodiment, the cells are also genetically modified to reduce or eliminate expression of one or more proteins naturally expressed by the parent cell. In one embodiment, the genetically modified cells (e.g., modified RPE cells, modified ARPE-19 cells) are cultured from a population of stably transfected cells or from a monoclonal cell line.

[0047] An "exogenous nucleic acid," as used herein, is a nucleotide sequence that does not naturally occur in a subject cell.

[0048] An "exogenous polypeptide," as used herein, is a polypeptide that does not naturally occur in a subject cell, e.g., an engineered cell. Reference to an amino acid position of a particular sequence refers to the position of that 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.

[0049] "Expression vector" as used herein refers to a recombinant polynucleotide comprising one or more expression constructs encoding one or more proteins to be expressed. Each expression construct comprises an expression control sequence operably linked to one or more nucleotide sequences to be expressed. An expression vector comprises sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. A vector can comprise additional sequence elements used for expression and / or integration of the expression cassette(s) into the genome of a mammalian cell. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides. Expression vectors suitable for use in engineering mammalian cells to express any of the fusion proteins described herein can also contain a nucleotide sequence encoding a marker for selection of cells containing such a vector. Examples of suitable markers are genes encoding resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.

[0050] "High molecular weight alginate" or "HMW-Alg" as used herein means an alginate having an approximate molecular weight of 150 kDa to 250 kDa.

[0051] "Low molecular weight alginate" or "LMS-Alg" as used herein means an alginate having an approximate molecular weight of <75 kDa.

[0052] As used herein, "medium molecular weight alginate" or "MMW-Alg" refers to an alginate having an approximate molecular weight of 75 kDa to 150 kDa.

[0053] As used herein, "MPS-6 patient" refers to an individual who has been diagnosed with or is suspected of having MPS-6 disease. In one embodiment, the MPS-6 patient has a mutant ARSB gene. The patient may be diagnosed using any method known in the art, including one or more of the clinical, biochemical, or genetic methods for diagnosing MPS-6 described in Vairo et al., The Application of Clinical Genetics Vol.8, pp.245-255 (2015).

[0054] A "peptide," as used herein, is a polypeptide of fewer than 50 amino acids, typically fewer than 25 amino acids.

[0055] "Poly A" signal, as used herein, refers to any contiguous sequence that terminates transcription of a coding sequence into RNA and directs the addition of a poly A tail to the RNA. Examples of poly A signals are the rabbit binding globulin (rBG) poly A signal, the SV40 late poly A signal, the SV50 poly A signal, the bovine growth hormone (BGH) poly A signal, the human growth hormone (HGH) poly A signal, and synthetic poly A signals known in the art.

[0056] 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.

[0057] 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.

[0058] "Prevention," "prevent," and "preventing," as used herein, refer to treatments that include administering or applying an ARSB fusion protein as described herein, e.g., administering a composition of a device encapsulating modified cells expressing a fusion protein (e.g., as described herein) prior to the onset of one or more symptoms of MPS-6 to prevent the physical manifestation of the symptom(s). In some embodiments, "prevention," "prevent," and "preventing" require that a sign or symptom of MPS-6 has not yet developed or has not yet been observed. In some embodiments, treatment includes prevention, and in other embodiments does not include prevention.

[0059] "Promoter sequence" as used herein refers to a nucleotide sequence capable of driving expression in mammalian cells, e.g., human cells, e.g., cells derived from mammalian cell lines (e.g., RPE cell lines, iPSC cell lines). In some embodiments, for example, to drive expression of the ARSB fusion proteins described herein, 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 expression cassettes described herein include the EF1A promoter, the CAG promoter, the PGK (phosphoglycerate kinase) promoter, and the ACTB (human beta-actin) promoter. In one embodiment, a promoter sequence useful for driving expression of the ST proteins described herein can be from a medium-strength promoter, e.g., the EFS promoter sequence, which is a shortened form of the EF1A promoter sequence.

[0060] "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 or modified therefrom, e.g., by stably transfecting cells cultured from the ARPE-19 cell line with an exogenous sequence encoding an ARSB fusion protein, cells derived from primary cell cultures of RPE cells, naturally occurring RPE cells, e.g., human or other mammalian RPE cells, including cells isolated directly from an animal (without long-term culture, e.g., less than 5 or 10 passages or rounds of cell division from isolation), transformed, immortalized, or derived from long-term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell cultures; b) undifferentiated cells, e.g., RPE cells or cells that, except for any genetic manipulation, have been developed, programmed, or otherwise 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 may be derived from IPS cells). or c) cells obtained from cells that have been transformed or reprogrammed (e.g., in vitro); or c) cells that have one or more of the following characteristics: i) express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) do not express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; iii) are naturally found in the retina and form a monolayer over the choroidal blood vessels in Bruch's membrane; iv) are involved in epithelial transport, light absorption, and / or cellular functions in the retina; or v) have been synthetically produced or have been modified from naturally occurring cells to have the same or substantially the same genetic content, and optionally the same or substantially the same epigenetic content, as an immortalized RPE cell line (e.g., the ARPE-19 cell line (ATCC® CRL-2302™). In one embodiment, the RPE cells described herein are genetically modified, e.g., to have new properties, e.g., the cells are modified to express and secrete a fusion protein described herein.In other embodiments, the RPE cells are not genetically modified.

[0061] "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 A1. In one embodiment, the specified percentage of identical nucleotide or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0062] As used herein, "spherical" refers to a device with a curved surface (e.g., a hydrogel capsule or other particle) that forms 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 three orthogonal axes a, b, and c. In a sphere, the three axes are of equal length. Typically, a sphere-like shape is an ellipsoid (about its average surface) with semi-major axes within 10%, 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.

[0063] "Subject" as used herein refers to a human or a non-human animal. In one embodiment, the subject is a human (i.e., male or female) of any age group, e.g., a pediatric human subject (e.g., 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, e.g., a mammal (e.g., a mouse, a dog, a primate (e.g., a cynomolgus or rhesus monkey)). In one embodiment, the subject is a commercially relevant mammal (e.g., a cow, a pig, a horse, a sheep, a goat, a cat, or a dog) or a bird (e.g., a commercially relevant bird, e.g., a chicken, a duck, a goose, or a turkey). In certain embodiments, the animal is a mammal. The animal can be male or female and at any stage of development. The non-human animal can be a transgenic animal.

[0064] "Treatment", "treat", and "treating" as used herein refer to one or more of reducing, reversing, alleviating, delaying the onset of, or inhibiting the progression of one or more of the symptoms, signs, or underlying causes of MPS-6. In one embodiment, treating includes reducing, reversing, alleviating, delaying the onset of, or inhibiting the progression of symptoms associated with MPS-6. In some embodiments, "treatment", "treat", and "treating" require that a sign or symptom associated with MPS-6 has developed or been observed. In other embodiments, treatment may be performed in the absence of signs or symptoms of disease, e.g., in prophylactic treatment. For example, treatment may be performed in a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and / or in light 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, and in other embodiments does not include prevention.

[0065] "Wild type" (wt) refers to the naturally occurring form, including sequence, of a polynucleotide, polypeptide, or protein in a species. The wild type form is distinguished from mutant forms of the polynucleotide, polypeptide, or protein that result from genetic mutation(s).

[0066] Selected Chemical Definitions Definitions of certain functional groups and chemical terms are described in more detail below. Chemical elements are defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 th Ed. (inside cover), and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0067] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae depicted herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0068] When a range of values ​​is listed, it is intended to encompass each value and subrange within the range. For example, "C 1 -C 6Alkyl" 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.

[0069] 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 4alkyl"), 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 alkyl).

[0070] 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 (C 3 ), 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 ) are included.

[0071] 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").

[0072] 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 -C24 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 2 alkenyl).

[0073] The one or more carbon-carbon double bonds can be internal (e.g., in 2-butenyl) or terminal (e.g., 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.

[0074] C 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 can 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").

[0075] 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 ("C 2 alkynyl).

[0076] 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 ) etc.

[0077] Each instance of an alkynyl group may independently 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").

[0078] 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 These include, but are not limited to:

[0079] Up to two or three heteroatoms, e.g., -CH 2 -NH-OCH 3 and -CH 2 -O-Si(CH 3 ) 3 It may be continuous like this.

[0080] "Heteroalkyl" is described followed by a particular heteroalkyl group, e.g., -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 can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroalkyl") or substituted with one or more substituents; for example, substituted with, for example, 1-5 substituents, 1-3 substituents, or 1 substituent (a "substituted heteroalkyl").

[0081] 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, C 2 -C 6 Alkenylene, C 2 -C 6 C 1 -C 6 and heteroalkylenes, where the term "membered" refers to a non-hydrogen atom within the moiety. In the case of heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, a group of the formula -C(O) 2R'- is -C(O) 2 R'- and -R'C(O) 2 - can represent both.

[0082] 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 “aryl”

[0083] In some embodiments, an aryl group has 6 ring carbon atoms ("C 6 aryl" (e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 aryl," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 Aryl" e.g., anthracyl). The aryl group is e.g., C 6 -C 10 The term "membered" refers to the non-hydrogen ring atoms in the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of the aryl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl").

[0084] 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.

[0085] 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").

[0086] 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.

[0087] 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.

[0088] As used herein, "cycloalkyl" refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms in the non-aromatic ring system ("C 3 -C 10 “Cycloalkyl” refers to a cyclic alkyl group.

[0089] In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms ("C 3 -C 8 cycloalkyl"), 3 to 6 ring carbon atoms ("C 3 -C 6 cycloalkyl"), or 5 to 10 ring carbon atoms ("C 5 -C 10 Cycloalkyl 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 to one or more aryl groups, and the point of attachment is on the cycloalkyl ring, in which case the number of carbons continues to refer to the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl").

[0090] "Heterocyclyl," as used herein, is a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and one to four ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl").

[0091] In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, if valence permits. Heterocyclyl groups may be either monocyclic ("monocyclic heterocyclyl") or fused, bridged or spiro ring systems, e.g., bicyclic systems ("bicyclic heterocyclyl"), and may be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring as defined above is fused with one or more cycloalkyl groups, with the point of attachment being on either the cycloalkyl ring or the heterocyclyl ring, or ring systems in which a heterocyclyl ring as defined above is fused with one or more aryl or heteroaryl groups, with the point of attachment being on the heterocyclyl ring, in which case the number of ring members still refers to the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described, for example, as a 3- to 7-membered heterocyclyl, where the term "member" refers to the non-hydrogen ring atoms in the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of heterocyclyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, a heterocyclyl group is an unsubstituted 3- to 10-membered heterocyclyl. In certain embodiments, a heterocyclyl group is a substituted 3- to 10-membered heterocyclyl.

[0092] In some embodiments, heterocyclyl groups are 5-10 membered non-aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("5-10 membered heterocyclyl"). In some embodiments, heterocyclyl groups are 5-8 membered non-aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heterocyclyl"). In some embodiments, heterocyclyl groups are 5-6 membered non-aromatic ring systems having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heterocyclyl").

[0093] In some embodiments, the 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 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0094] 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.

[0095] Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl or thiomorpholinyl-1,1-dioxide. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl. 6 Exemplary 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.

[0096] "Amino" as used herein refers to the radical -NR 70 R 71 (In the formula, R 70 and R 71 are each independently hydrogen, C1 -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...

[0097] As used herein, "cyano" refers to the radical --CN.

[0098] 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.

[0099] As used herein, "hydroxy" refers to the radical --OH.

[0100] 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.

[0101] Two or more substituents may be optionally bonded to form an aryl, heteroaryl, cycloalkyl, or heterocyclyl group. Such so-called ring-forming substituents are typically, but not necessarily, found to be bonded to a cyclic base structure. In one embodiment, the ring-forming substituents are bonded to adjacent members of the base structure. For example, two ring-forming substituents bonded to adjacent members of a cyclic base structure generate a fused ring structure. In another embodiment, the ring-forming substituents are bonded to a single member of the base structure. For example, two ring-forming substituents bonded to a single member of a cyclic base structure generate a spirocyclic structure. In yet another embodiment, the ring-forming substituents are bonded to non-adjacent members of the base structure.

[0102] 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.

[0103] 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.

[0104] 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 the like.

[0105] The term "pharmaceutically acceptable salt" is meant to include salts of active compounds prepared using relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compound of formula (I) used to prepare the device of the present disclosure contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. When the compound used in the present disclosure contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphate, dihydrogenphosphate, sulfuric, monohydrogensulfuric, hydroiodic, or phosphorous acids, and the like, as well as salts derived from organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids, e.g., arginates, and the like, and salts of organic acids, such as glucuronic or galacturonic acid (see, e.g., Berge et al, Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds used in the devices (e.g., particles, hydrogel capsules) of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. These salts can be prepared by methods known to those of ordinary skill in the art. Other pharma- ceutically acceptable carriers known to those of ordinary skill in the art are suitable for use in the present disclosure.

[0106] The device of the present disclosure 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 devices in the present disclosure. In addition, prodrugs can be converted into useful compounds of formula (I) by chemical or biochemical methods in an ex vivo environment.

[0107] 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.

[0108] The term "solvate" refers to a form of a compound associated with a solvent, usually by solvolysis. 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.

[0109] 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.

[0110] As used herein, the term "tautomer" refers to a compound structure that is an interchangeable form, with changes in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium between the movement of π electrons and atoms (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomeric forms may be relevant to achieving optimal chemical reactivity and biological activity of a compound of interest.

[0111] Symbols used herein [ka] refers to a connection to an entity, e.g., a polymer (e.g., a hydrogel-forming polymer such as alginate) or to the surface of an implantable device, e.g., a particle, a hydrogel capsule. [ka] The connection represented by may refer to a direct bond with an entity, e.g., a polymer or an implantable element, or may refer to a linkage with an entity via a linking group. A "linking group" as described herein refers to a moiety for linking a compound of formula (I) with an entity (e.g., a polymer or an implantable element (e.g., a device) as 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.

[0112] 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 , R F , 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 cross-linker. In some embodiments,

number

number

[0113] Characteristics of ARSB fusion proteins The ARSB fusion protein of the present disclosure comprises an HSA binding domain (AB) located upstream of the amino acid sequence of a mature mammalian ARSB protein (ARSB) having ARSB enzymatic activity. In some embodiments, a linker moiety (e.g., a linker peptide) is located between the AB domain and the ARSB domain.

[0114] HSA binding domain AB The serum half-life of the ARSB protein is longer than that of an otherwise identical fusion protein lacking the AB domain. This increased half-life is primarily due to the binding of the fusion protein to HSA via the AB domain. Mature human serum albumin (HSA) is a monomeric protein of 585 amino acids (GenBank accession number AAA98797.1) that contains three partially overlapping domains: DI (amino acids 25-221 in Figure 1), DII (amino acids 213-409 in Figure 1), and DIII (amino acids 405-609 in Figure 1) (Dockal, M., et al., 2003). al., J.Biol.Chem., Vol.274, No.41, pp.29303-29310(1999). The long half-life of albumin in blood is mainly driven by two features: (i) the large size (65 kDa) of albumin limits its glomerular filtration, and (ii) albumin binds to FcRn at low pH (pH 6), which returns it from early endosomes to the extracellular environment, thereby protecting it from degradation in lysosomes after passive endocytosis in endothelial and epithelial cells. In one embodiment, the AB domain confers on the ARSB fusion protein a serum half-life in humans (expressed as t1 / 2-beta) that exceeds any of 6 hours, 12 hours, 24 hours, 72 hours, 1 week, 2 weeks, or up to the half-life of HSA in humans (estimated to be about 19 days).

[0115] In some embodiments, the AB domain is specific for serum albumin, i.e., it does not substantially bind to any non-albumin protein. In some embodiments, the fusion protein does not substantially inhibit the binding of FcRn to HSA, and its binding site is in DIII. In one embodiment, less than about 20%, 15%, 10%, 5%, or 1% inhibition of FcRn binding to HSA occurs in the presence of the fusion protein of the present disclosure, which can be determined by any competitive binding assay known in the art, such as the SPR competitive binding assay described in

[0095] of WO2019 / 204925 or in Example 3 of US2019 / 0367596A1. The possibility of interference with FcRn binding can be reduced by deriving the AB domain from an anti-HSA antibody that does not bind to DIII. In one embodiment, the fusion protein binds to the DI of HSA via the AB domain. In one embodiment, the fusion protein cross-competes via AB for HSA binding with an sdAb consisting of the amino acid sequences of the single domain antibodies (sdAbs) described in WO2019 / 204925, namely R11, R28, M75, or M79.

[0116] In some embodiments, AB (as part of the fusion protein) also binds (e.g., cross-reacts with) serum albumin from any combination of two, three, four, or more of one, two, three, four, or more other mammalian species, such as mouse, rat, guinea pig, hamster, rabbit, cat, dog, pig, sheep, horse, cow, and monkey (e.g., rhesus and / or cynomolgus). In one embodiment, the fusion protein binds serum albumin from at least mouse, rat, monkey (rhesus or cynomolgus), and human via the AB domain. In one embodiment, the fusion protein binds serum albumin from at least mouse, dog, and human via the AB domain.

[0117] In some embodiments, the AB domain (as part of a fusion protein) binds to HSA, and optionally at least one other mammalian serum albumin, with a desired affinity within a pH range of about 5.0 or about 5.5 up to about 7.4. In one embodiment, the desired affinity is a dissociation constant (K) in any of the ranges of about 0.1 nM to about 1,000 nM, about 0.5 nM to about 500 nM, about 1 nM to about 250 nM, about 5 nM to about 50 nM, about 10 nM to about 25 nM, or about 0.5 nM to about 1 nM. D In one embodiment, the affinity is determined by surface plasmon resonance (SPR) at 25° C. and a pH range of about 5.5 to about 7.4. In one embodiment, the affinity of the fusion protein for serum albumin from mouse, rat, and monkey is similar to the affinity for HSA, e.g., K D is within 70%-130%, 80%-120%, or 90%-110% of the

[0118] K of anti-HSA antibody and fusion protein D An exemplary SPR assay for measuring affinity in the pH range of 5.0-7.4 is described in US2019 / 0367597A1. The K of anti-HSA antibodies and fusion proteins to HSA and mammalian orthologs is described in US2019 / 0367597A1. D may also be determined substantially as described in WO2019 / 246003 at

[0994] to

[0995] .

[0119] The AB domain of the ARSB fusion protein of the present disclosure comprises the amino acid sequence of the heavy chain variable region (HCVR) of an anti-HSA antibody. To facilitate expression of the fusion protein by genetically engineered cells, in some embodiments, the AB domain has a molecular weight of less than about 75 kDa, about 50 kDa, or about 25 kDa. The AB domain can be used in the construction of a conventional four-chain antibody, an antigen-binding fragment, Fab, Fab', F(ab') 2The AB may be derived from any anti-HSA antibody molecule known in the art, including Fvs (double-chain and single-chain (scFv)), minibodies, diabodies, and sdAbs. In one embodiment, the AB consists essentially of or consists of the HCVR amino acid sequence of an sdAb.

[0120] In one embodiment, the AB domain comprises a set of three CDR amino acid sequences of one of the anti-HSA sdAbs listed in Table I of WO2006 / 122787. In one embodiment, the AB consists essentially of or consists of the amino acid sequences of one of the anti-HSA sdAbs listed in Table II of WO2006 / 122787 (e.g., Alb-1), or one of the humanized variants of Alb-1 listed in Table III of WO2006 / 122787 (e.g., Alb-8). In one embodiment, the AB consists essentially of or consists of any of the Alb-23 sequences listed in WO2012 / 175400 (e.g., Alb-23D). In one embodiment, the ARSB fusion protein of the present disclosure cross-competes with any of the anti-HSA sdAbs described in WO2006 / 122787 (e.g., Alb-8) or WO2012 / 175400 (e.g., Alb-23D) for binding to HSA. In one embodiment, the ARSB fusion protein does not include the amino acid sequence of Alb-1. In one embodiment, the ARSB fusion protein does not include the amino acid sequence of Alb-8.

[0121] In one embodiment, AB comprises (e.g., or consists of) an amino acid sequence of one of the following sequences, e.g., as set forth in Table II of WO2006 / 122787: (i) PMP6A8 (ALB2): AVQLVESGGGLVQGGGSLRLACAASERIFDLNLMGWYRQGPGNERELVATCITVGDSTNYADSVKGRFTISMDYTKQTVYLHMNSLRPEDTGLYYCKIRRTWHSELWGQGTQVTVSS (SEQ ID NO: 62) (ii) PMP6B4 - EVQLVESGGGLVQEGGSLRLACAASERIWDINLLGWYRQGPGNERELVATITVGDSTSYADSVKGRFTISRDYDKNTLYLQMNSLRPEDTGLYYCKIRRTWHSELWGQGTQVTVSS (SEQ ID NO: 63) (iii) PMP6A6 (ALBl) - AVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSS (SEQ ID NO: 64) (iv) PMP6Cl AVQLVDSGGGLVQPGGSLRLSCAASGFSFGSFGMSWVRQYPGKEPEWVSSINGRGDDTRYADSVKGRFSISRDNAKNTLYLQMNSLKPEDTAEYYCTIGRSVSRSRTQGTQVTVSS (SEQ ID NO: 65) (v) PMP6G8 - AVQLVESGGGLVQPGGSRLTCTASGFTFRSFGMSWVRQAPGKDQEWVSAISADSSTKNYADSVKGRFTISRDNAKKMLYLEMNSLKPEDTAVYYCVIGRGSPSSPGTQVTVSS (SEQ ID NO: 66) (vi) PMP6AS - QVQLAESGGGLVQPGGSLRLTCTASGFTFGSFGMSWVRQAPGEGLEWVSAISADSSDKRYADSVKGRFTISRDNAKKMLYLEMNSLKSEDTA VYYCVIGRGSPASQGTQVTVSS (SEQ ID NO: 67) (vii) PMP6G7 - QVQLVESGGGLVQPGGSLRLSCAASGFTFSNYWMYWVRVAPGKGLERISRDISTGGGYSYYADSVKGRFTISRDNAKNTLYLQMNSLKPEDTALYYCAKDREAQVDTLDFDYRGQGTQVTVSS (sequence number 68).

[0122] In one embodiment, AB comprises (e.g., or consists of) an amino acid sequence of one of the following sequences, e.g., as set forth in Table III of WO2006 / 122787: (i) ALB3 (ALB1 HUM1): EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS (SEQ ID NO: 69) (ii) ALB4 (ALB1 HUM2): EVQLVESGGGLVQPGGSLRLSCAASGFTFSSFGMSWVRQAPGKEPEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTQVTVSS (SEQ ID NO: 70) (iii) ALB5 (ALB1 HUM3): EVQLVESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTA VYYCTIGGSLSRSSQGTQVTVSS (SEQ ID NO: 71) (iv) ALB6 (ALB1 HUM1): EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLKPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 72) (v) ALB7 (ALB1 HUM2): EVQLVESGGGLVQPGNSLRLSCAASGFTFRSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 73) (vi)ALB8(ALB1 HUM3): EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 74) (vii) ALB9 (ALB1 HUM4): EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS (SEQ ID NO: 75) (viii) ALB10 (ALB1 HUM5): EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSGQGTLVTVSS (sequence number 76).

[0123] In another embodiment, the AB domain consists essentially of, or consists of, the amino acid sequence of an albumin binding domain set forth in Table 7 of WO2019 / 246003, e.g., LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP (SEQ ID NO: 21), and has a K of about 1.2 nM for HSA. D In one embodiment, the AB domain of the ARSB fusion protein comprises the three CDRs of an anti-HSA P367 antibody as set forth in Table 14 of WO2019 / 2460003. In one embodiment, the AB consists essentially of or consists of the amino acid sequence of an anti-HSA P367 antibody or its humanized variant P494, each of which are listed in Table 14 of WO2019 / 2460003. In one embodiment, the ARSB fusion protein of the present disclosure cross-competes with the P494 sdAb as set forth in WO2019 / 2460003 for binding to HSA.

[0124] In one embodiment, the AB domain comprises a set of three heavy chain CDR amino acid sequences of one of the anti-HSA sdAbs listed in Table 5 of WO2021 / 119551. In one embodiment, the AB consists essentially of, or consists of, the VH amino acid sequence of one of the anti-HSA sdAbs listed in Table 5 of WO2021 / 119551. In one embodiment, the ARSB fusion protein of the present disclosure cross-competes for binding to HSA with one or more of the sdAbs listed in Table 5 of WO2021 / 119551.

[0125] In another embodiment, the AB domain comprises a set of three CDR amino acid sequences found in the T0235002C06 sdAb as set forth in Table B of US20190367597A1. In one embodiment, the AB consists essentially of or consists of the amino acid sequence of T0235002C06 as set forth in Table B of US2019 / 0367597A1. In one embodiment, the ARSB fusion protein of the present disclosure cross-competes with the T0235002C06 sdAb as set forth in Table B of US20190367597A1 for binding to HSA.

[0126] In another embodiment, the AB domain comprises a set of three CDR amino acid sequences found in the T0235005D04 sdAb described in Table B of US20190367596A1. In one embodiment, the AB consists essentially of or consists of the amino acid sequence of T0235005D04 described in Table B of US2019 / 0367596A1. In one embodiment, the ARSB fusion protein of the present disclosure cross-competes with the T0235002D04 sdAb described in Table B of US20190367596A1 for binding to HSA.

[0127] In yet another embodiment, the AB domain comprises a set of three CDR amino acid sequences found in the T0235005G01 sdAb or T023500043 sdAb as set forth in Table B of US20190367598A1. In one embodiment, the AB consists essentially of or consists of the amino acid sequence of T0235005G01 or T023500043 as set forth in Table B of US20190367598A1. In one embodiment, the ARSB fusion protein of the present disclosure cross-competes with the T0235005G01 or T023500043 sdAb as set forth in Table B of US20190367598A1 for binding to HSA.

[0128] In one embodiment, the AB domain comprises a set of three CDR amino acid sequences found in the R28, R11, M75, or M79 sdAbs described in WO2019 / 204925. These CDR sequences are shown in Table 2A below. [Table 2]

[0129] In one embodiment, AB consists essentially of or consists of the amino acid sequence of R28, R11, M75, or M79 sdAb as described in WO2019 / 204925. In one embodiment, AB consists essentially of or consists of the amino acid sequence of one of the humanized variants of R28, R11, M75, or M79 as described in WO2019 / 204925. The amino acid sequences of the parent and humanized variants of R28, R11, M75, and M79 are shown in the sequence listing on pages 46-48 of WO2019 / 204925. In one embodiment, AB consists essentially of or consists of an amino acid sequence selected from the parent and humanized sequences shown in Table 2B herein below. In one embodiment, AB consists essentially of or consists of the parent or humanized amino acid sequence of R28 as shown in Table 2B below. [Table 3]

[0130] Generation of HSA-binding domain AB The AB portion of the ARSB fusion protein may be derived from any antibody or antigen-binding fragment thereof having the desired properties described herein. The antibody or antigen-binding fragment may be known in the art or may be identified by any approach known in the art.

[0131] In some embodiments, the AB is derived from a single domain Ab (sdAb). For example, sdAbs of camelid origin lack light chains and therefore their antigen binding site is V H It consists of one domain, designated H. sdAbs have also been observed in sharks, and NAR Other sdAbs can be engineered based on human Ig heavy and light chain sequences. As used herein, the term "sdAb" refers to VGAs of any origin, such as those derived from human Ig heavy and light chains, through phage display or other techniques. H , V H H, V L , or V NAR These include sdAbs directly isolated from a reservoir, recombinantly produced sdAbs, as well as those sdAbs generated by further modification of such sdAbs by humanization, affinity maturation, stabilization, solubilization, or other methods of antibody engineering. Also encompassed by the present disclosure are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the parent sdAb.

[0132] V binds to HSA H To generate H sdAbs, one of skill in the art can generate phage-displayed V sdAbs from a repertoire of heavy chain-only antibodies from a llama or other camelid immunized with a desired antigen using any approach known in the art, such as, for example, substantially as described in WO2019 / 204925. H A H library can be generated and screened.

[0133] The HSA binding domain derived from the sdAb may comprise the parent framework regions, or alternatively the parent CDRs may be the V domains of other sdAbs. NAR , V HH, V H , or V L The framework regions, or framework regions of other types of antibody fragments or antibody-like molecules (Fv, scFv, Fab) of any source (e.g., human), or proteins of similar size and nature onto which CDRs can be grafted (see, e.g., Nicaise, M. et al, Protein Sci 13:1882-91 2004).

[0134] In some embodiments, the amino acid sequence in AB is a humanized version (humanized variant) of the parent variable region. Humanization of an antibody or antibody fragment involves replacing amino acids in the sequence with human counterparts, as found in the human consensus sequence, without losing antigen-binding ability or specificity; this approach reduces the immunogenicity of the antibody or fragment thereof when introduced into a human subject. The parent sequence can be humanized using any suitable method known in the art, such as, but not limited to, CDR grafting and veneering.

[0135] In the process of CDR grafting, one or more of the CDRs defined herein are substituted with human variable regions (V H , or V L ), to another human antibody (IgA, IgD, IgE, IgG, and IgM), to an antibody fragment framework region (Fv, scFv, Fab), or to a protein of similar size and nature onto which the CDRs can be grafted. CDR grafting is known in the art and is described in at least the following: U.S. Pat. Nos. 6,180,370, 5,693,761, 6,054,297, and European Patent No. 626390.

[0136] Veneering, also referred to as "variable region resurfacing", involves humanizing the solvent exposed positions of an antibody or antibody fragment, thus preserving buried non-human residues that may be important for CDR conformation, while minimizing potential immunological reactions to the solvent exposed regions. Veneering is known in the art and described at least in U.S. Pat. Nos. 5,869,619, 5,766,886, and 5,821,123, and European Patent No. 519596.

[0137] Linker The ARSB fusion protein may include a linker between the AB and ARSB domains. The linker should be of sufficient length to allow the linked polypeptides to fold individually into a three-dimensional structure that has the desired functional activity, e.g., binding or therapeutic activity. Also, the linker should not be cleaved by any proteases or other enzymes present in serum.

[0138] In some embodiments, the linker is a peptide linker that includes at least 2, 3, or 4 amino acids and less than about 30 amino acids, such as less than about 25, about 20, about 15, or about 10. Peptide linkers are known in the art and non-limiting examples are described herein.

[0139] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO1996 / 34103.

[0140] Suitable linker peptides typically contain G and / or S residues in various forms, and exemplary linkers include GGGG (SEQ ID NO:39), TGGGG (SEQ ID NO:40), GGSSGGSGSSSGSGGSGSSG (SEQ ID NO:41), (GGSS)n (SEQ ID NO:42), (GGGGS)n (SEQ ID NO:8), (SGGGG)n (SEQ ID NO:43), and GGGG(SGGGG)n (SEQ ID NO:62), where "n" in each case is generally an integer between 1 and 7 (inclusive), but not exceeding a maximum length of about 30 amino acids. Another exemplary peptide linker is SKPTCPPPELLGGPSVFIFPPK (SEQ ID NO:45).

[0141] In one embodiment, the fusion protein comprises a peptide linker of about 15-20 amino acids, or about 20-25 amino acids in length. In one embodiment, the linker is (GGGS) 3 (SEQ ID NO: 9), (GGGS) 4 (SEQ ID NO: 46), or (GGGS) 5 (SEQ ID NO: 47). In one embodiment, the linker consists essentially of, or consists of, (GGGS) 3 It consists of:

[0142] Genetically modified cells Any of the above ARSB fusion proteins can be expressed by a mammalian cell(s) that has been genetically modified to express and secrete the fusion protein. The genetically modified cell(s) can be derived from a variety of different mammalian cell types (e.g., human cells), including epithelial cells, endothelial cells, fibroblasts, mesenchymal stem cells, keratinocyte cells, and stem cells, e.g., embryonic stem cells or induced pluripotent stem cells. Exemplary cell types include those described in WO2017 / 075631. In some embodiments, the cells are derived from the cell lines shown in Table 3. [Table 4]

[0143] In one embodiment, any of the genetically modified mammalian cells described herein are derived from RPE cells, e.g., ARPE-19 cells. In one embodiment, the genetically modified RPE cells comprise any of the expression cassettes, transposons, and polynucleotides described herein.

[0144] Cells can be genetically modified to express and secrete the desired ARSB fusion protein using any of a variety of genetic engineering techniques known in the art. For example, cells can be transfected with an expression vector that contains an exogenous nucleotide sequence(s) encoding the desired fusion protein operably linked to control elements necessary or useful for gene expression, e.g., promoters, ribosome binding sites, enhancers, polyA signals, etc. In some embodiments, the exogenous nucleotide sequence is part of a transcription unit that is stably integrated into the genome of the parent cell.

[0145] In one embodiment, the exogenous sequence comprises a nucleotide sequence encoding a secretory signal sequence for the fusion protein. In one embodiment, the signal sequence is from a naturally secreted protein. In one embodiment, the signal sequence is MELGLSWVVLAALLQGVQA (SEQ ID NO: 48). In some embodiments, the signal sequence consists essentially of the amino acid sequence shown in Table 4 below. [Table 5]

[0146] Genetically modified mammalian cells for use in the devices, compositions, and methods described herein may be in various stages of the cell cycle, for example, as a plurality of cells in a hydrogel capsule. In some embodiments, at least one cell in the plurality of genetically modified cells is undergoing cell division. Cell division may be measured using any method known in the art, for example, as described in DeFazio A et al (1987) J Histochem Cytochem 35:571-577 and Dolbeare F et al (1983) Proc Natl Acad Sci USA 80:5573-5577, each of which is incorporated by reference in its entirety. In one embodiment, at least 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, as determined, for example, by 5-ethynyl-2'deoxyuridine (EdU) assay or 5-bromo-2'-deoxyuridine (BrdU) assay. In some embodiments, cell proliferation is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry). In some embodiments, none of the cells in the plurality of genetically modified cells are undergoing cell division and are quiescent. In one embodiment, less than 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, 5-ethynyl-2'deoxyuridine (EdU) assay, 5-bromo-2'-deoxyuridine (BrdU) assay, microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation), or flow cytometry).

[0147] In one embodiment, at least 50%, 60%, 70%, 80%, 90% or more of the genetically modified cells in the plurality are viable. Cell viability can be measured using any method known in the art, for example, as described in Riss, T. et al (2013) "Cell Viability Assays" in Assay Guidance Manual (Sittapalam, GSet al, eds). For example, cell viability can be measured or quantified by ATP assay, 5-ethynyl-2'deoxyuridine (EdU) assay, 5-bromo-2'deoxyuridine (BrdU) assay. In some embodiments, cell viability is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry). In one embodiment, at least 80% of the plurality of engineered cells are viable, as determined, for example, by ATP assay, 5-ethynyl-2'deoxyuridine (EdU) assay, 5-bromo-2'deoxyuridine (BrdU) assay, microscopy (e.g., fluorescence microscopy (e.g., time-lapse or assessment of spindle formation), or flow cytometry.

[0148] Any of the parameters described herein may be assessed using standard techniques known to those of skill in the art, such as histology, microscopy, and various functional assays.

[0149] Measurement of ARSB activity The activity of ARSB secreted by the genetically modified cells or devices described herein can be measured by any direct or indirect ARSB activity assay known in the art. In one embodiment, ARSB activity can be measured using one or both of the in vitro fluorogenic activity assay and the cell-based functional assay described in the Examples below.

[0150] Implantable cell encapsulation devices A genetically modified cell or a plurality of such cells as described herein may be incorporated into an implantable device for use in providing ARSB protein to a subject in need thereof, e.g., a human subject diagnosed with MPS-6.

[0151] The implantable devices of the present disclosure include at least one barrier that prevents immune cells from contacting cells contained within the device. At least a portion of the barrier must be sufficiently porous to allow proteins (e.g., fusion proteins) expressed and secreted by the cells to exit the device. A variety of device configurations known in the art are suitable.

[0152] The device (e.g., particle) can have any configuration and shape suitable to support the viability and productivity of contained cells after implantation at the intended target site. As non-limiting examples, the device shape can be cylindrical, rectangular, disk-shaped, oval, star-shaped, or spherical. The device can be composed of mesh-like or nested structures. In some embodiments, the device can prevent materials over a certain size from passing through the pores or openings. In some embodiments, the device (e.g., particle) can prevent materials over 50 kD, 75 kD, 100 kD, 125 kD, 150 kD, 175 kD, 200 kD, 250 kD, 300 kD, 400 kD, 500 kD, 750 kD, or 1,000 kD from passing.

[0153] The device may include one hydrogel capsule, or multiple hydrogel capsules. The hydrogel capsule(s) in the device may have any of a variety of shapes: cylinder, cylinder with hemispherical ends (also known as spherocylinder), disk, noodle (e.g., as described in WO2015 / 191547), sphere (e.g., as defined herein), or ellipsoid (e.g., as defined herein). In one embodiment, the hydrogel capsule(s) in the device is a sphere as defined herein.

[0154] In one embodiment, the device is a macroencapsulation device. Non-limiting examples of macrodevices are described in WO2019 / 068059, WO2019 / 169089, U.S. Patent Nos. 9,526,880, 9,724,430, and 8,278,106, European Patent No. 742818B1, and Sang, S. and Roy, S., Biotechnol. Bioeng. 113(7):1381-1402 (2016).

[0155] In one embodiment, the device is a macro device with one or more cell-containing compartments. A device with two or more cell-containing compartments can be configured to produce two or more proteins, for example, cells expressing an ARSB fusion protein are placed in one compartment, and cells expressing a different protein (e.g., a therapeutic protein that can alleviate one or more symptoms of MPS-6) are placed in another compartment. WO2018 / 232027 describes a device with multiple cell-containing compartments formed in a microassembly and covered by a porous membrane.

[0156] In one embodiment, the device is configured as a thin, flexible strand as described in U.S. Patent No. 10,493,107. The strand includes a substrate, an inner polymeric coating surrounding the substrate, and an outer hydrogel coating surrounding the inner polymeric coating. The protein-expressing cells are disposed within the outer coating.

[0157] In some embodiments, the devices (e.g., particles) have a largest linear dimension (LLD), e.g., average diameter, or size, that is at least about 0.5 millimeters (mm), preferably about 1.0 mm, about 1.5 mm, or more. In some embodiments, the devices can be as large as 10 mm in diameter or size. For example, the devices or particles described herein may be 0.5mm to 10mm, 1mm to 10mm, 1mm to 8mm, 1mm to 6mm, 1mm to 5mm, 1mm to 4mm, 1mm to 3mm, 1mm to 2mm, 1mm to 1.5mm, 1.5mm to 8mm, 1.5mm to 6mm, 1.5mm to 5mm, 1.5mm to 4mm, 1.5mm to 3mm, 1.5mm to 2mm, 2mm to 8mm, 2mm to 7mm, 2mm to 6mm, 2mm to 5mm, 2mm to 4mm, 2mm to 3mm, 2.5mm to 8mm, 2.5mm to 7mm, 2.5mm to 6mm, 2.5mm to 5mm, 2.5mm to 4mm, 2.5mm to 3mm, 3mm to 8mm m, in size range of 3mm~7mm, 3mm~6mm, 3mm~5mm, 3mm~4mm, 3.5mm~8mm, 3.5mm~7mm, 3.5mm~6mm, 3.5mm~5mm, 3.5mm~4mm, 4mm~8mm, 4mm~7mm, 4mm~6mm, 4mm~5mm, 4.5mm~8mm, 4.5mm~7mm, 4.5mm~6mm, 4.5mm~5mm, 5mm~8mm, 5mm~7mm, 5mm~6mm, 5.5mm~8mm, 5.5mm~7mm, 5.5mm~6mm, 6mm~8mm, 6mm~7mm, 6.5mm~8mm, 6.5mm~7mm, 7mm~8mm, or 7.5mm~8mm.

[0158] In some embodiments, the devices (e.g., particles, capsules) of the present disclosure include at least one hole or opening, e.g., to allow free flow of material. In some embodiments, the average pore size of the device is about 0.1 μm to about 10 μm. For example, the average pore size may be 0.1 μm to 10 μm, 0.1 μm to 5 μm, 0.1 μm to 2 μm, 0.15 μm to 10 μm, 0.15 μm to 5 μm, 0.15 μm to 2 μm, 0.2 μm to 10 μm, 0.2 μm to 5 μm, 0.25 μm to 10 μm, 0.25 μm to 5 μm, 0.5 μm to 10 μm, 0.75 μm to 10 μm, 1 μm to 10 μm, 1 μm to 5 μm, 1 μm to 2 μm, 2 μm to 10 μm, 2 μm to 5 μm, or 5 μm to 10 μm. In some embodiments, the average pore size of the device is about 0.1 μm to 10 μm. In some embodiments, the average pore size of the device is about 0.1 μm to 5 μm. In some embodiments, the average pore size of the device is between about 0.1 μm and 1 μm.

[0159] In some embodiments, the device includes a semi-permeable biocompatible membrane that surrounds the genetically modified cells that are encapsulated in a polymeric composition (e.g., an alginate hydrogel). The membrane pore size is selected to allow oxygen and other molecules important to cell survival and function to migrate through the semi-permeable membrane while preventing immune cells from traversing through the pores. In one embodiment, the semi-permeable membrane has a molecular weight cutoff of less than 1000 kD, or between 50 and 700 kD, between 70 and 300 kD, or between 70 and 150 kD, or between 70 and 130 kD.

[0160] In one embodiment, the device may contain a cell-containing compartment surrounded by a barrier compartment formed from a cell-free biocompatible material, such as the core-shell microcapsules described in Ma, M et al., Adv. Healthc Mater., 2(5):667-672 (2012). Such barrier compartments may be used with or without a semi-permeable membrane.

[0161] The cells in the cell-containing compartment(s) of the device of the present disclosure may be encapsulated in a polymeric composition. The polymeric composition may include one or more hydrogel-forming polymers. In addition to the polymeric composition in the cell-containing compartment(s), the device (e.g., macrodevice, particle, hydrogel capsule) may include or be formed from materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials, and combinations thereof. The device may be made entirely of one type of material, or may include other materials in the cell-containing compartment and any other compartments.

[0162] In some embodiments, the device comprises a metal or metal alloy. In one embodiment, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all of the compartments) comprise a metal or metal alloy. Exemplary metals or metal alloys include titanium and titanium-based alloys (e.g., Nitinol, nickel-titanium alloys, thermal memory alloy materials), platinum, platinum-based alloys, stainless steel, tantalum, palladium, zirconium, niobium, molybdenum, nickel-chromium, chromium-molybdenum alloys, or certain cobalt alloys (e.g., cobalt-chromium and cobalt-chromium-nickel alloys, e.g., ELGILOY® and PHYNOX®). For example, the metal material can be stainless steel grade 316 (SS316L) (composed of Fe, <0.3% C, 16-18.5% Cr, 10-14% Ni, 2-3% Mo, <2% Mn, <1% Si, <0.45% P, and <0.03% S). In metal-containing devices, the amount of metal (e.g., weight %, actual weight) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0163] In some embodiments, the device comprises a ceramic. In one embodiment, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all of the compartments) comprise a ceramic. Exemplary ceramic materials include oxides, carbides, or nitrides of transition elements, such as titanium oxide, hafnium oxide, iridium oxide, chromium oxide, aluminum oxide, and zirconium oxide. Silicon-based materials, such as silica, may also be used. In ceramic-containing devices, the amount of ceramic (e.g., weight %, actual weight) may be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0164] In some embodiments, the device has two hydrogel compartments, an inner cell-containing compartment completely surrounded by a second, outer (e.g., barrier) compartment. In one embodiment, the inner boundary of the second compartment forms an interface with the outer boundary of the first compartment. In such embodiments, the thickness of the second (outer) compartment refers to the average distance between the outer boundary of the second compartment and the interface between the two compartments, e.g., the average of the distances measured at each of the thinnest and thickest points visually observed in the outer compartment. In some embodiments (e.g., the device is about 1.5 mm in diameter), the thinnest and thickest distances in the outer compartment are 25-110 micrometers (μm) and 270-480 μm, respectively. In some embodiments, the thickness of the outer compartment is greater than about 10 nanometers (nm), preferably 100 nm or more, and can be as large as 1 millimeter (mm). For example, the thickness (e.g., average distance) of the outer compartment in the hydrogel capsule device described herein can be 10 nm to 1 mm, 100 nm to 1 mm, 500 nm to 1 millimeter, 1 micrometer (μm) to 1 mm, 1 μm to 1 mm, 1 μm to 500 μm, 1 μm to 250 μm, 1 μm to 1 mm, 5 μm to 500 μm, 5 μm to 250 μm, 10 μm to 1 mm, 10 μm to 500 μm, or 10 μm to 250 μm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is 100 nm to 1 mm, 1 μm to 1 mm, 1 μm to 500 μm, or 5 μm to 1 mm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is about 50 μm to about 100 μm. In some embodiments (eg, the device is about 1.5 mm in diameter), the thickness (eg, average distance) of the outer section is about 180 μm to 260 μm or about 310 μm to 440 μm.

[0165] In some embodiments of the two-compartment hydrogel capsule device, the average pore size of the cell-containing inner compartment and the outer compartment are substantially the same. In some embodiments, the average pore size of the inner compartment and the second compartment differs by about 1.5%, 2%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some embodiments, the average pore size of the device (e.g., the average pore size of the first compartment and / or the average pore size of the second compartment) depends on a number of factors, such as the material(s) in each compartment and the presence and density of the compound of formula (I).

[0166] In some embodiments, the polymer composition in the cell-containing compartment(s) comprises a polysaccharide or other hydrogel-forming polymer (e.g., alginate, hyaluronic acid, or chondroitin). In some embodiments, the polymer is alginate, a polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). In some embodiments, the alginate has a low molecular weight (e.g., an approximate molecular weight of <75 kDa) and a G:M ratio of ≧1.5, (ii) a medium molecular weight alginate, e.g., an approximate molecular weight of 75-150 kDa and a G:M ratio of ≧1.5, (iii) a high molecular weight alginate, e.g., an approximate MW of 150 kDa-250 kDa and a G:M ratio of ≧1.5, or (iv) a blend of two or more of these alginates. In some embodiments, the cell-containing compartment(s) further comprises at least one cell-binding substance (CBS), such as a cell-binding peptide (CBP) or cell-binding polypeptide (CBPP) described in WO2020069429.

[0167] In some embodiments, the cell-containing compartment(s) comprises alginate covalently modified with a linker-cell-binding peptide moiety, e.g., GRGD (SEQ ID NO: 57) or GRGDSP (SEQ ID NO: 58). In one embodiment, the cell-binding peptide density (e.g., % nitrogen determined by combustion analysis as described in WO2020198695) in the cell-containing compartment(s) 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 cell-containing compartment is about 0.1 to about 1.0 micromoles of CBP per gram of CBP polymer in solution (e.g., MMW-alginate covalently modified with GRGD (SEQ ID NO: 57) or GRGDSP (SEQ ID NO: 58)) 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 cell-containing compartment also comprises unmodified alginate having a molecular weight of 75 kDa to 150 kDa and a G:M ratio of 1.5 or greater.

[0168] The device may form part of multiple substantially identical devices in the preparation (e.g., composition). In some embodiments, the devices (e.g., particles, hydrogel capsules) in the preparation have an average diameter or size of about 0.5 mm to about 8 mm. In some embodiments, the devices in the preparation have an average diameter or size of about 0.5 mm to about 4 mm or about 0.5 mm to about 2 mm. In some embodiments, the devices in the preparation are two-compartment hydrogel capsules and have an average diameter or size of about 0.7 mm to about 1.3 mm or about 1.2 mm to about 1.8 mm.

[0169] In some embodiments, the surface of the device comprises a compound capable of mitigating the FBR upon implantation in a subject, a non-fibrous compound described below. In devices that include a barrier compartment surrounding a cell-containing compartment, the non-fibrous compound may covalently modify polymers disposed throughout the barrier compartment and optionally throughout the cell-containing compartment.

[0170] In some embodiments, one or more compartments in the device comprise a non-fibrous polymer, e.g., a non-fibrous compound of formula (I) covalently bonded to the polymer. In one embodiment, some or all of the monomers in the non-fibrous polymer are modified with the same compound of formula (I). In some embodiments, some or all of the monomers in the non-fibrous polymer are modified with different compounds of formula (I). In some embodiments, where the device is a two-compartment hydrogel capsule, the non-fibrous polymer is present only in the outer barrier compartment.

[0171] One or more compartments within the device may contain an unmodified polymer that is the same or different from the polymer in any non-fibrous polymer present in the device. In one embodiment, the first compartment, the second compartment, or all compartments in the device contain an unmodified polymer.

[0172] Each of the modified and unmodified polymers in the device can be linear, branched, or crosslinked polymers, or polymers of a selected molecular weight range, degree of polymerization, viscosity, or melt flow rate. Branched polymers can include one or more of the following types: star polymers, comb polymers, brush polymers, dendrimerized polymers, ladder polymers, and dendrimers. The polymers can be thermoresponsive polymers, such as gels (e.g., that become solid or liquid upon exposure to heat or a selected temperature) or photocrosslinkable polymers. 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, poly(orthoesters), poly(carbonates), poly(hydroxyalkanoates), polyfluorocarbons, PEEK®, Teflon® (polytetrafluoroethylene, PTFE), PEEK, silicones, epoxy resins, Kevlar®, Dacron® (made from ethylene glycol and terephthalic acid), and the like. Poly(lactic 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, alginates, chitosan, and blends or copolymers thereof.In a polymer-containing device, the amount of polymer (e.g., % by weight of the device, actual weight of polymer) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0173] In some embodiments, one or more of the modified and unmodified polymers in the device comprises polyethylene. Exemplary polyethylenes include very low density polyethylene (ULDPE) (e.g., 0.890-0.905 g / cm, containing comonomers). 3 with polymers having densities in the range of 0.905 to 0.915 g / cm 3 with polymers having densities in the range of 0.915 to 0.935 g / cm 3 with polymers having densities in the range of about 0.915 to 0.935 g / m 3 with polymers having densities in the range of 0.926 to 0.940 g / cm, with or without comonomers; 3 with polymers having densities in the range of 0.940 to 0.970 g / cm, with or without comonomers; 3 with polymers having densities in the range of 0.1 to 100 MPa, and polyethylene glycol.

[0174] In some embodiments, one or more of the modified and unmodified polymers in the device comprises polypropylene. Exemplary polypropylenes include homopolymers, random copolymers (homophase copolymers), and impact copolymers (heterophase copolymers), as described, for example, in McKeen, Handbook of Polymer Applications in Medicine and Medical Devices, 3-Plastics Used in Medical Devices, (2014): 21-53.

[0175] In some embodiments, one or more of the modified and unmodified polymers in the device comprises polypropylene. Exemplary polystyrenes include general purpose, or crystalline (PS or GPPS), high impact (HIPS), and syndiotactic (SPS) polystyrene.

[0176] In some embodiments, one or more of the modified and unmodified polymers comprises a thermoplastic elastomer (TPE). Exemplary TPEs include: (i) TPA-polyamide TPEs, which comprise block copolymers of alternating hard and soft segments with amide chemical bonds in the hard blocks and ether and / or ester bonds in the soft blocks; (ii) TPC-co-polyester TPEs, which consist of block copolymers of alternating hard and soft segments, where the chemical bonds in the backbone are ester and / or ether; (iii) TPO-olefin TPEs, which consist of blends of polyolefins and conventional rubbers, where the rubber phase in the blend has little or no crosslinking; (iv) at least triblock copolymers of styrene and certain dienes, where the two end blocks (hard blocks) are polystyrene and the inner (v) TPU-urethane TPEs consisting of alternating hard and soft segment block copolymers having urethane chemical bonds in the hard blocks and ether, ester, or carbonate bonds, or mixtures thereof, in the soft blocks; (vi) TPV-thermoplastic rubber vulcanizates consisting of blends of thermoplastic materials and conventional rubbers where the rubber is crosslinked by a process of dynamic vulcanization during the blending and mixing steps; and (vii) TPZ unclassified TPEs which include any composition or structure other than those classified as TPA, TPC, TPO, TPS, TPU, and TPV.

[0177] In some embodiments, the unmodified polymer is an 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. In one embodiment, the unmodified alginate has a molecular weight of 150 kDa to 250 kDa and a G:M ratio of ≧1.5.

[0178] In some embodiments, the non-fibrous polymer comprises an alginate chemically modified with a compound of formula (I). The alginate in the modified non-fibrous polymer can be the same or different from any unmodified alginate present in the device. In one embodiment, the density (e.g., amount of conjugation) of the compound of formula (I) in the non-fibrous 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.

[0179] 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.

[0180] In an alginate-containing device, the amount of modified and unmodified alginate (e.g., % by weight of the device, actual weight of alginate) can be at least 5%, e.g., at least 5%, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0181] 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, the alginate carboxylic acid moiety 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 an activating agent (e.g., 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 eq)) and a base (e.g., 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 allowed to cool 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) against water for 24 hours, for example, with two changes of water. The resulting solution can be concentrated, for example, via lyophilization, to provide the desired chemically modified alginate. The alginate in the non-fibrous polymer can be chemically modified with the compound of formula (I) using any suitable method known in the art, for example, as described in any of WO2021 / 119522, WO2019 / 195055, WO2018 / 067615, WO2017 / 075631, WO2016 / 019391, and WO2012 / 167223.

[0182] In one embodiment, the device comprises at least one cell-containing compartment, and in some embodiments, two, three, four or more cell-containing compartments, hi one embodiment, each cell-containing compartment comprises a plurality of cells (e.g., living cells), and the cells in at least one of the compartments are capable of expressing and secreting an ARSB fusion protein when the device is implanted in a subject.

[0183] In one embodiment, all cells in the cell-containing compartment are derived from a single parent cell type or a mixture of at least two different parent cell types. In one embodiment, all cells in the cell-containing compartment are derived from the same parent cell type, but a first plurality of derived cells are engineered to express an ARSB fusion protein and a second plurality of derived cells are engineered to express a different therapeutic protein. In devices with two or more cell-containing compartments, the cells and the protein(s) produced thereby may be the same or different in each cell-containing compartment. In some embodiments, all cell-containing compartments are surrounded by a single barrier compartment. In some embodiments, the barrier compartment is substantially free of cells.

[0184] In one embodiment, the cells to be incorporated into the device described herein, e.g., hydrogel capsules, are prepared in the form of a cell suspension before being encapsulated in the device. 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 a plurality of cell clusters (e.g., as a spheroid) or microcarriers.

[0185] In addition to the fusion protein secreted by the encapsulated cells, the device (e.g., capsule, particle) may contain one or more exogenous substances not expressed by the cells, such as, for example, a nucleic acid (e.g., an RNA or DNA molecule), a protein (e.g., a hormone, an enzyme (e.g., glucose oxidase, kinase, phosphatase, oxygenase, hydrogenase, reductase) antibody, antibody fragment, antigen, or epitope), an active or inactive fragment of a protein or polypeptide, a small molecule, or a drug. In one embodiment, the device is configured to release such exogenous substances.

[0186] Non-fibrotic (e.g., FBR-reducing) compounds In some embodiments, the devices described herein comprise at least one compound of formula (I): [ka] or a pharma- ceutically acceptable salt thereof, wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-, -N(R C )C(O)-, -C(O)N(R C )-, -N(R C )C(O)(C 1 -C 6 -alkylene)-, -N(R C )C(O)(C 1 -C 6 -alkenylene)-, -N(R C )N(R D )-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F ) y -, -Si(OR A ) 2 -, -Si(R G )(OR A )-, -B(OR A )-, or a metal, each of which is optionally linked to 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 L2 is a bond, M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 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 , -N(R C )(R D ), 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 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 and is optionally replaced by or R C and R D together with the nitrogen atom to which they are attached, form one or more R 6 forming an optionally substituted ring (e.g., a 5- to 7-membered ring) with Each R 1, R 2 , R 3 , R 4 , R 5 , and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), S.R. E1 , S(O) x R E1 , -OS(O) x R E1 , -N(R C1 )S(O) x R E1 , -S(O) x N(R C1 )(R D1 ), -P(R F1 ) y , cycloalkyl, heterocyclyl, aryl, 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 R 7 is optionally replaced by Each R 7is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is 1 or 2; y is 2, 3, or 4.

[0187] In some embodiments, the compound of formula (I) is a compound of formula (Ia): [ka] or a pharma- ceutically acceptable salt thereof, wherein A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-, -N(R C )C(O)-, -C(O)N(R C )-, -N(R C )N(R D )-, N(R C )C(O)(C 1 -C 6 -alkylene)-, -N(R C )C(O)(C 1 -C 6 -Alkenylene)-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F ) y -, -Si(OR A ) 2 -, -Si(R G )(OR A )-, -B(OR A )-, or a metal, each of which is optionally linked to a linking group (e.g., a linking group described herein), and one or more R 1is 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 one or more R 3 is optionally replaced by P is one or more R 4 is a 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 and is optionally replaced by or R C and R D together with the nitrogen atom to which they are attached, form one or more R 6 forming an optionally substituted ring (e.g., a 5- to 7-membered ring) with Each R 1 , R 2 , R 3 , R 4 , R 5 , and R 6are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), 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 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; y is 2, 3, or 4.

[0188] In some embodiments, for formula (I) and (Ia), A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )C(O)-, -N(R C )C(O)(C 1 -C 6 -alkylene)-, -N(R C )C(O)(C 1 -C 6 -alkenylene)- or -N(R C In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C In some embodiments, A is alkyl, -O-, -C(O)O-, -C(O)-, -OC(O), or -N(R C In some embodiments, A is -N(R C )C(O)-, -N(R C )C(O)(C 1 -C 6 -alkylene)- or -N(R C )C(O)(C 1 -C 6 In some embodiments, A is -N(R C In some embodiments, A is -N(R C )- and R C and R D is independently hydrogen or alkyl. In some embodiments, A is -NH-. In some embodiments, A is -N(R C )C(O)(C 1 -C 6 -alkylene)-, where alkylene is R1 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 )-.

[0189] In some embodiments, for Formulas (I) and (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 -CH 2 -, -CH(CH 3 )-, -CH 2 CH 2 CH 2 , or -CH 2 CH 2 In some embodiments, L 1 -CH 2 -or-CH 2 CH 2 In some embodiments, for formula (I) and (Ia), L 3 is a bond, alkyl, or heteroalkyl. In some embodiments, L 3 is a bond. In some embodiments, L3 is alkyl. In some embodiments, L 3 is C 1 -C 12 It is an alkyl.

[0190] In some embodiments, L 3 is C 1 -C 6 In some embodiments, L 3 -CH 2 In some embodiments, L 3 is heteroalkyl. In some embodiments, L 3 is one or more R 2 C optionally substituted with (e.g., oxo) 1 -C 12 In some embodiments, L is heteroalkyl. 3 is one or more R 2 C optionally substituted with (e.g., oxo) 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 -CH 2 It is O-.

[0191] In some embodiments, for formula (I) and (Ia), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, for formula (I) and (Ia), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, M is heteroalkyl, aryl, or heteroaryl. In some embodiments, M is absent. In some embodiments, M is alkyl (e.g., C 1 -C 6 In some embodiments, M is -CH 2 In some embodiments, M is heteroalkyl (e.g., C 1 -C 6 In some embodiments, M is (-OCH 2 CH 2 -) z and z is an integer selected from 1 to 10. In some embodiments, z is an integer selected from 1 to 5. In some embodiments, M is -(OCH 2 ) 2 -, (-OCH 2 CH 2- ) 2 , (-OCH 2 CH 2- ) 3 , (-OCH 2 CH 2 -) 4 , or (-OCH 2 CH 25 In some embodiments, M is -OCH 2 CH 2-、 (-OCH 2 CH 2-)2 , (-OCH 2 CH 2 -) 3 , or (-OCH 2 CH 2-)4 In some embodiments, M is (-OCH 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 [ka] In some embodiments, M is 1 to 4 R 3 (For example, one R 3 In some embodiments, R 3 CF 3 It is.

[0192] In some embodiments, for formulae (I) and (Ia), P is absent, heterocyclyl, or heteroaryl. In some embodiments, for formulae (I) and (Ia), P is absent, heterocyclyl, or heteroaryl. In some embodiments, P is absent. In some embodiments, for formulae (I) and (Ia), P is a tricyclic, bicyclic, or monocyclic heteroaryl. In some embodiments, P is a monocyclic heteroaryl. In some embodiments, P is a nitrogen-containing heteroaryl. In some embodiments, P is a monocyclic, nitrogen-containing heteroaryl. In some embodiments, P is a 5-membered heteroaryl. In some embodiments, P is a 5-membered nitrogen-containing heteroaryl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, or pyrrolyl. In some embodiments, P is imidazolyl. In some embodiments, P is 1,2,3-triazolyl. In some embodiments, P is [ka] In some embodiments, P is [ka] In some embodiments, P is [ka] It is.

[0193] In some embodiments, P is heterocyclyl. In some embodiments, P is heterocyclyl. In some embodiments, P is 5-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some embodiments, P is [ka] In some embodiments, P is thiomorpholinyl-1,1-dioxydyl. In some embodiments, P is [ka] It is.

[0194] In some embodiments, for formula (I) or (Ia), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 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, 5-membered heterocyclyl, or 6-membered heterocyclyl. In some embodiments, Z is 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is [ka] In some embodiments, Z is a 4-membered oxygen-containing heterocyclyl. In some embodiments, Z is [ka] It is.

[0195] In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. 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. [ka] In some embodiments, Z is a 6-membered sulfur-containing heterocyclyl. [ka] 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-dioxydyl. 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.

[0196] In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is selected from one or more R 5In 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.

[0197] In some embodiments, for formula (I) and (Ia), Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is 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.

[0198] In some embodiments, for formula (I) and (Ia), Z is alkyl. In some embodiments, Z is C 1 -C 12 In some embodiments, Z is C 1 -C 10 In some embodiments, Z is C 1 -C 8 In some embodiments, Z is selected from 1 to 5 R 5 C replaced with 1 -C 8 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.

[0199] In some embodiments, for formula (I) and (Ia), Z is heteroalkyl. In some embodiments, Z is C 1 -C 12In 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 R 5 In some embodiments, Z is N-methyl-2-(methylsulfonyl)ethane-1-aminyl.

[0200] 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 In some embodiments, Z is -OCH 3 In some embodiments, Z is -C(O)OR A (e.g., -C(O)OH).

[0201] In some embodiments, Z is hydrogen.

[0202] 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.

[0203] In some embodiments, the compound of formula (I) is a compound of formula (Ib): [ka] or a pharma- ceutically acceptable 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 5 cycloalkyl, 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 each taken together forms an oxo group, X is absent, N(R 10 )(R 11 ), O, or S, and 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 and R 11 each independently represents hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -C(O)N(R C1 ), cycloalkyl, heterocyclyl, or heteroaryl, and each R A1 , R B1 , R C1 , 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.

[0204] 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 and the ring Z is an aryl or heteroaryl optionally substituted with 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b Or R 2c and R 2d taken together form an oxo group, X is absent, O, or S, and 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, or two R 5 Together, they form the ring Z 2 A 5- or 6-membered ring is formed by condensing 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; and p is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0205] In some embodiments, the compound of formula (Ibi) is a compound of formula (Ib-ii): [ka] or a pharma- ceutically acceptable salt thereof, 2is cycloalkyl, heterocyclyl, aryl or heteroaryl; R 2c and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R together form an oxo group, and 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; and each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; each of p and q is independently 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0206] In some embodiments, the compound of formula (I) is a compound of formula (Ic): [ka] or a pharma- ceutically acceptable salt thereof, 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 2c and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R 2d are taken together to form an oxo group, and each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR, -C(O)OR, or -C(O)R B1 wherein each alkyl and heteroalkyl is optionally substituted with halogen; and each R A1 and R B1is independently hydrogen, alkyl, or heteroalkyl; m is 1, 2, 3, 4, 5, or 6; and 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.

[0207] In some embodiments, the compound of formula (I) is a compound of formula (Id): [ka] or a pharma- ceutically acceptable salt thereof, 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; X is absent, O, or S; R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b Or R 2c and R 2d taken together form an oxo group, and 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; and each R A1 and R is independently hydrogen, alkyl, or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5, or 6; and p is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0208] In some embodiments, the compound of formula (I) is a compound of formula (Ie): [ka] or a pharma- ceutically acceptable salt thereof, 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl, X is absent, O or S, and R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b Or R 2c and R 2d taken together form an oxo group, and 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; and p is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0209] In some embodiments, the compound of formula (I) is a compound of formula (If): [ka] or a pharma- ceutically acceptable salt thereof, wherein M is one or more R 3 and ring P is an alkyl optionally substituted with one or more R 4 and L is heteroaryl optionally substituted with3 is one or more R 2 and Z is an alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from the group consisting of one or more R 5 Optionally replaced by R 2a and R 2b Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b are taken together to form an oxo group, and 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; and n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0210] 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 the alkyl and aryl are each independently selected from one or more R 3 and optionally substituted with L 3 is one or more R 2Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or -OR, and the alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently selected from one or more R 5 Optionally replaced by R A is hydrogen and R 2a and R 2b Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b are taken together to form an oxo group, and each R 2 , R 3 , and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , or -C(O)R B1 And each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; and n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0211] In some embodiments, the compound of formula (II) is a compound of formula (II-a): [ka] or a pharma- ceutically acceptable salt thereof, wherein L 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 heteroalkyl is one or more R 5 Optionally replaced by R 2a and R 2bEach of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b are taken together to form an oxo group, and each R 2 , R 3 , and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 and R A is hydrogen, and each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; and n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0212] In some embodiments, the compound of formula (I) is a compound of formula (II): [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 replaced by 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, R Cis hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, each of which is selected from 1 to 6 R 6 Optionally replaced by 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 12 is independently deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; and w is 0 or 1; [ka] refers to the connection to a linking group or polymer as described herein.

[0213] In some embodiments, the compound of formula (III) is a compound of formula (III-a): [ka] or a pharma- ceutically acceptable salt thereof, 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 Optionally replaced by R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen, alkyl, heteroalkyl, halo, or R 2a and R 2b Or R 2c and R 2d together form an oxo group, and R Cis hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, each of which is selected from 1 to 6 R 6 Optionally replaced by 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 12 is independently deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; and w is 0 or 1; [ka] refers to the connection to a linking group or polymer as described herein.

[0214] In some embodiments, the compound of formula (III) is a compound of formula (III-b): [ka] or a pharma- ceutically acceptable salt thereof, 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 Optionally replaced by 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 replaced by R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen or alkyl, or R 2a and R 2b Or R 2c and R 2d together form an oxo group, and 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 12 is independently hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; A1 , R B1 , and R E1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; and x is 0, 1, or 2; [ka] refers to the connection to a linking group or polymer as described herein.

[0215] In some embodiments, the compound of formula (III) is a compound of formula (III-c): [ka] or a pharma- ceutically acceptable salt thereof, wherein 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 replaced by R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen or alkyl, or R 2a and R 2b Or R 2c and R 2d together form an oxo group, and 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 R 12 is hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino, and each R A1 , R B1 , and R E1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; x is 0, 1, or 2; z is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0216] In some embodiments, the compound of formula (III) 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). xeach of R′ and R″ is independently hydrogen, alkyl, or halogen; 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 replaced by R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen or alkyl, or R 2a and R 2b Or R 2c and R 2d together form an oxo group, and 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 12 is independently hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; A1 , R B1 , and R E1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; x is 0, 1, or 2; z is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0217] In some embodiments, X is S(O) xIn some embodiments, x is 2. In some embodiments, X is S(O) 2 It is.

[0218] In some embodiments, R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen.

[0219] 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, each of the alkyl and alkenyl is selected from one R 6 (For example, -CH 3 In some embodiments, R C is hydrogen.

[0220] 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 12 is halo (e.g., Cl).

[0221] In some embodiments, the compound is a compound of formula (I). 2 is a bond, and P and L 3 is, independently, non-existent.

[0222] 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 3is 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.

[0223] 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 -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.

[0224] 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.

[0225] In some embodiments, the compound is of formula (Ibi). In some embodiments of formula (Ibi), R 2a and R 2b Each of 3 and R 2c and R 2dare 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.

[0226] In some embodiments, the compound is 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.

[0227] 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 -CH 3 and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, such as piperazinyl). In some embodiments, the compound of Formula (Ic) is compound 113.

[0228] 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.

[0229] 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.

[0230] 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 -CH 2 (OCH 2 CH 2 ) 2 and Z is -OCH 3 In some embodiments, the compound of Formula (II-a) is compound 112.

[0231] 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.

[0232] 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.

[0233] 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.

[0234] 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 2is 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-b) is compound 121.

[0235] 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.

[0236] 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).

[0237] 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.

[0238] In some embodiments, the compound of formula (I) comprises a compound set forth herein below in Table 5, or a pharma- ceutically acceptable salt thereof. In some embodiments, the exterior surface and / or one or more compartments within the devices described herein comprise a small molecule compound set forth in Table 5, or a pharma- ceutically acceptable salt thereof. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7]

[0239] Conjugation of any of the compounds in Table 5 with a polymer (e.g., alginate) can be carried out as described in Example 2 of WO2019 / 195055, or any other suitable chemical reaction.

[0240] In some embodiments, the compound is a compound of Formula (I) (e.g., Formula (Ia), (Ib), (Ic), (Id), (Ie), (If), (II), (II-a), (III), (III-a), (III-b), (III-c), or (III-d)), or a pharma- ceutically acceptable salt thereof; [ka] or a pharma- ceutically acceptable salt thereof.

[0241] In some embodiments, the devices described herein include [ka] or a pharma- ceutically acceptable salt of either compound.

[0242] In some embodiments, a compound of formula (I) (e.g., compound 101 in Table 5) is covalently attached to an alginate (e.g., an alginate having approximately a 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 WO2020069429. 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.

[0243] The device, device preparation, or device composition may be configured for implantation or may be implanted or placed in any part or portion of the body. In some embodiments, the implantable device or device preparation is configured for implantation in the peritoneal cavity (e.g., the omental bursa, also known as the omental bursa or bursalis omentum). The device, device preparation, or device composition may be implanted in the peritoneal cavity (e.g., the omentum, e.g., the bursa) or placed on a surface in the peritoneal cavity (e.g., the omentum, e.g., the bursa) via injection or catheter. Additional considerations regarding the implantation or placement of the device, device preparation, or device composition in the omentum (e.g., the bursa) are provided in M. Pellicciaro et al. (2017) CellR4 5(3):e2410.

[0244] Device fabrication Genetically modified ARPE-19 cells for use in manufacturing the devices described herein can be generated and cultured using methods known in the art, for example, stably transfected ARPE-19 cells can be cultured in vitro substantially as described in WO2020198695.

[0245] The compounds of formula (I) and alginates modified with such compounds can be obtained using procedures known in the art, for example those substantially as described in WO2020198695.

[0246] Alginate solutions for making non-fibrous two-compartment hydrogel capsules can be obtained using procedures known in the art, for example substantially as described in WO2020198695.

[0247] Two-compartment hydrogel capsules ("shielded capsules") encapsulating genetically modified mammalian cells in the inner compartment and non-fibrous alginate in the outer layer can be produced using procedures known in the art, e.g., substantially as described in WO2020198696.

[0248] Treatment Described herein are methods for preventing or treating MPS-6 in a subject by administration or implantation of a pharmaceutical composition or genetically modified cells described herein. In one embodiment, the pharmaceutical composition comprises an ARSB fusion protein described herein and is formulated for intravenous or subcutaneous administration. In another embodiment, the pharmaceutical composition comprises a plurality of cells that have been genetically modified to express an ARSB fusion protein. In one embodiment, the cells are encapsulated in a hydrogel capsule described herein. In another embodiment, the cells are encapsulated in a macrodevice described herein. In some embodiments, the methods described herein directly or indirectly reduce or alleviate at least one symptom of MPS-6 or prevent or delay the onset of the disease. In one embodiment, the method comprises administering (e.g., implanting) an effective amount of a composition of a two-compartment alginate hydrogel capsule comprising genetically modified RPE cells and a cell-binding polymer described herein in the inner compartment and a compound of formula (I), e.g., compound 101, in the outer capsule surface and optionally in the outer compartment. EXAMPLES

[0249] In order that the disclosure set forth herein may be more fully understood, the following examples are set forth. The examples set forth in this application are provided to illustrate the ARSB fusion proteins and genetically modified cells provided herein and should not be construed in any way as limiting the scope thereof.

[0250] Example 1: Effect of fusion of an exemplary HSA binding domain to human mature ARSB on ARSB activity in vitro. The DNA expression vector was engineered to express G4S. 3The expression vectors were designed to encode ARSB fusion enzymes containing the anti-HSA sdAb (R28) fused via a linker unit to the N- or C-terminus of the mature human ARSB open reading frame. ARPE-19 cells were transfected with these expression vectors, and polyclonal colonies stably expressing the encoded ARSB fusion enzymes were generated for each transfection.

[0251] The in vitro ARSB activity of the ARSB fusion proteins secreted from the two polyclonal colonies was assessed by seeding approximately 400,000 cells per well of a 6-well plate from each polyclonal colony in 2 ml of high serum medium (DMEM / F12 / 10% FBS + 1ug / ml puromycin) and incubated in a temperature-controlled (TC) incubator (37°C, 5% CO). 2 ) Twenty to 24 hours after seeding, conditioned medium was collected and assessed for ARSB activity as described below.

[0252] The conditioned medium was diluted 8-fold with assay buffer (50 mM sodium acetate, pH 5.6). 50 microliters (ul) of the diluted conditioned medium was placed into wells of a 96-well black plate. Galsulfase, an exemplary recombinant human ARSB (rhARSB), was used as an activity standard. rhARSB was serially diluted in assay matrix (composed of 1 part sterile high serum medium, 7 parts assay buffer) to generate a 7-point standard curve, with the highest point set at 10 ng rhARSB in 50 ul of assay matrix. The standard curve is shown in Figure 8A.

[0253] Fifty microliters of 5 mM 4-methylumbelliferyl sulfate (4-MUS, Sigma Aldrich model no. M7133-500MG) substrate was mixed with 50 ul of diluted conditioned medium or 50 ul of rhARSB standard in a 96-well black plate. The reaction was incubated for 1 hour in the dark at ambient temperature. The reaction was then quenched with 100 ul of glycine carbonate buffer (12.8 grams of glycine and 18 grams of sodium carbonate in 200 ml of molecular biology grade water). The blue fluorescence of the assay plate was scanned in endpoint mode on a microplate reader with excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively.

[0254] As shown in Fig. 8B , the ARSB activity produced by cells transfected with a vector encoding the R28-ARSB fusion protein was significantly higher than the ARSB activity produced by cells transfected with a vector encoding the ARSB-R28 fusion protein.

[0255] Example 2: Exemplary cell-based ARSB functional assay: Quantification of dermatan sulfate substrate levels MPS-6 patient fibroblasts (Coriell GM00538) were seeded at 250,000 cells per well of a 6-well plate in 2 ml of high serum medium (EMEM / 15% FBS) and incubated in a TC incubator (37°C, 5% CO 2 Engineered ARPE-19 cells secreting ARSB fusion proteins were seeded at 400,000 cells per well of a 6-well plate in 2 ml of non-selective high serum medium (DMEM / F12 / 10% FBS, no puromycin) and then incubated in a TC incubator.

[0256] 20-24 hours after seeding, collect conditioned medium from engineered cells and assess for hARSB activity as described above. Dilute conditioned medium containing the ARSB fusion protein described herein ("ARSB fusion") to 500 ng of ARSB fusion per ml of sterile EMEM / 15% FBS. Use 2 milliliters of the 500 ng / ml solution of ARSB fusion to replace the conditioned medium of MPS-6 patient fibroblasts seeded from the previous day. Incubate the fibroblasts with 2 ml of the 500 ng / ml solution of ARSB fusion for 3 days in a TC incubator. After 3 days, rinse the fibroblasts with 1x PBS (pH 7.4), collect by cell scraping, and lyse in 0.1% Triton-X 100 solution.

[0257] 100mM Tris-Cl (pH7.5), 100mM NaOAc, 10mM CaCl 2 , 20 mM MgCl 2 Ten microliters of cell lysate is combined with an equal volume of chondroitinase cocktail (0.8 ng / ul chondroitinase B (R&D Systems #6974-GH) and 2 ng / ul chondroitinase AC (R&D Systems)) in a buffer containing 0.1% Triton-X 100. The reaction is incubated for 3 days in a thermocycler set at 20° C. After 3 days, the entire reaction (20 ul) is combined with 180 ul of aqueous acetonitrile [83.3 parts acetonitrile:16.7 parts water] and clarified by centrifugation at 12,000 rpm for 10 minutes at 4° C. A 100 microliter sample is analyzed for dermatan sulfate levels by LCMS.

[0258] Example 3: Exemplary cell-based ARSB functional assay: Comparison of native and half-life extended hARSB fusion enzymes In this example, the ability of native hARSB, extended half-life hARSB (HL-hARSB), and recombinant enzyme (rhARSB) to reduce chondroitin sulfate and dermatan sulfate was compared. Conditioned media was prepared from cells expressing native hARSB, HL-hARSB, and rhARSB, and tested on fibroblasts from MPS VI patients according to the method of Example 2. As shown in Figure 9, the HL-hARSB fusion protein exhibited similar biochemical properties to the native and recombinant enzymes, and successfully reduced the amount of chondroitin sulfate / dermatan sulfate (CS / DS) compared to untreated controls.

[0259] Example 4: Exemplary in vivo functional ARSB functional assay In this example, ARPE-19 cells engineered to express hARSB and HL-hARSB were encapsulated in non-fibrous, two-compartment alginate hydrogel capsules as described in WO2020198695. The hydrogel capsules were implanted into MPS VI mice. After one week, organs were isolated from the mice and activity was assessed. As shown in Figure 10A-C, total hARSB activity in the liver, heart, and spleen was highest in mice treated with hydrogel capsules containing cells engineered to express the HL-hARSB enzyme.

[0260] Example 5: Exemplary in vivo functional ARSB function assay - Evaluation of reduction of CS and DS substrates in MPS VI mouse tissues In this example, the ability of encapsulated HL-hARSB expressing cells to reduce CS / DS in mouse tissues was evaluated and compared to a positive control of recombinant protein (galsulfase). Hydrogel capsules were prepared as outlined in Example 4 and implanted into MPS VI mice. Mice were sacrificed at 2-week and 4-week time intervals and CS / DS activity was measured in liver, heart, and spleen. As shown in Figure 11A-C, total CS / DS activity in liver, heart, and spleen is comparable between HL-hARSB and galsulfase groups in all test conditions.

[0261] 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 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.

[0262] 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

[Claim 1] A fusion protein comprising an N-terminal to C-terminal structure defined by formula (F): AB-L-ARSB, wherein: AB comprises a domain that binds to human serum albumin (HSA); L may be present or absent and comprises a linker amino acid sequence; and ARSB comprises the amino acid sequence of a mature mammalian ARSB protein; The fusion protein.

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