Compositions, devices, and methods for treating Fabry disease

Abstract

Described herein are RPE cells engineered to secrete GLA protein, as well as compositions, pharmaceutical formulations, and implantable devices comprising the engineered RPE cells, and methods of making and using them to treat Fabry disease.

Description

Detailed description of the invention

[0001] [Claiming priority] This application claims priority to U.S. Provisional Patent Application No. 62 / 824,969, filed on 27 March 2019, and U.S. Provisional Patent Application No. 62 / 907,380, filed on 27 September 2019. The disclosures of each of the aforementioned provisional applications are incorporated herein by reference in their entirety.

[0002] [Sequence List] This application includes a sequence listing filed electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on 25 May 2020, is named S2225-7028WO_SL.txt and has a size of 79,658 bytes. [Background technology]

[0003] Fabry disease is a rare X-linked lysosomal storage disorder caused by a deficiency in the activity of the enzyme α-galactosidase A (GLA or GALA), impairing the accumulation of the sphingoglycolipid globotriaosylceramide (Gb3) in various tissues and organs. The GLA gene encodes a homodimeric glycoprotein that hydrolyzes the terminal α-galactosyl moieties of glycolipids and glycoproteins, with globotriaosylceramide (Gb3, ceramide trihexoside) as its primary substrate. More than 370 mutations in the GLA gene have been identified in people with Fabry disease, many of which are specific to a single family. Mutations that eliminate GLA activity lead to the severe, classic form of Fabry disease, which typically begins in childhood. Milder, later-onset forms of Fabry disease are associated with mutations that reduce but do not eliminate GLA activity.

[0004] Recombinantly produced GLA proteins are approved for use in enzyme replacement therapy for Fabry disease. Gene therapy is being investigated as an alternative approach to delivering GLA enzymes to Fabry disease patients. However, ERT and gene therapy approaches to treat Fabry disease present various manufacturing and efficacy challenges. Therefore, novel treatment modalities for Fabry disease are desirable. [Overview of the project]

[0005] This specification describes retinal pigment epithelial (RPE) cells recombinant to express and secrete GLA, as well as compositions, pharmaceuticals, and medical devices comprising recombinant RPE cells, and methods for preparing and using them. In some embodiments, compositions, products, and devices comprising recombinant RPE cells are configured to mitigate foreign body reactions when administered to a mammalian subject, for example, when placed internally therein.

[0006] In one embodiment, the disclosure features an isolated polynucleotide comprising a promoter operably ligated to a precursor GLA coding sequence. In embodiments, the promoter sequence essentially consists of a nucleotide sequence identical or substantially identical to nucleotides 337-2069 of the sequence shown in Figures 8B-1 and 8B-2 (referred to herein as SEQ ID NO: 18). In embodiments, the precursor GLA coding sequence encodes a GLA fusion protein. In embodiments, the GLA fusion protein comprises a signal peptide from a secreted protein operably ligated to the N-terminus of a mature human GLA amino acid sequence. In embodiments, the precursor or mature GLA coding sequence is codon-optimized for expression in mammalian cells. In embodiments, the precursor GLA codon-optimized coding sequence is SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 6. In embodiments, the signal peptide is derived from HSPG2 (e.g., amino acids 1-21 in Figure 4A). In embodiments, the GLA fusion protein comprises an amino acid sequence encoding a signal peptide (e.g., derived from GLA or HSPG2) operably ligated to the N-terminus of a mature human GLA amino acid sequence, and a non-GLA polypeptide operably ligated to the C-terminus of the GLA amino acid sequence. In embodiments, the non-GLA polypeptide confers beneficial properties to the fusion protein, such as increasing the amount of protein expressed and / or secreted, extending the in vivo half-life, or enhancing the distribution or tissue uptake of the fusion protein. In embodiments, the isolated polynucleotide comprises SEQ ID NO: 47. In embodiments, the isolated polynucleotide is provided as an isolated double-stranded DNA molecule, and in embodiments, the DNA molecule comprises SEQ ID NO: 48.

[0007] In another embodiment, the disclosure provides recombinant RPE cells comprising an exogenous nucleotide sequence including a promoter sequence operably linked to a precursor GLA coding sequence. In an embodiment, the exogenous nucleotide sequence comprises an extrachromosomal expression vector. In an embodiment, the exogenous nucleotide sequence is incorporated at at least one position in the genome of an RPE cell, e.g., an ARPE19 cell.

[0008] In yet another embodiment, the Disclosure provides a device comprising at least one cell-containing compartment containing recombinant RPE cells or a plurality of such cells as described herein. In some embodiments, the composition, product, and device comprises a polymer composition for encapsulating recombinant RPE cells. In embodiments, the encapsulating polymer composition is at least one cell-binding substance (CBS), e.g., a cell-binding peptide, e.g., RGD (SEQ ID NO: 28) or RGSP (SEQ ID NO: 49). In embodiments, the encapsulating polymer composition comprises an alginate covalently modified with GRGDSP (SEQ ID NO: 44). In some embodiments, the device further comprises at least one means for mitigating foreign body reaction (FBR) when the device is placed inside a subject. In embodiments, the means for mitigating FBR comprises an anti-fibrous compound as defined herein, disposed on the outer surface of the device and / or within a barrier compartment surrounding the cell-containing compartment. In some embodiments, the anti-fibrous compound is of formula (I): [ka] A compound of or a pharmaceutically acceptable salt thereof, in which variables A and L 1 M, L 2 P, L 3 , and Z, and related sub-variables are defined herein. In some embodiments, the compound of formula (I) or a pharmaceutically acceptable salt thereof (e.g., formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), (II), (II-a), (III), (III-a), (III-b), (III-c), or (III-d)) is a compound described herein, for example, one of the compounds shown in Table 3 herein. In some embodiments, the antifibrotic compound is compound 100, compound 101, or compound 102 shown in Table 3.

[0009] In one embodiment, the device of the present disclosure is a two-compartment hydrogel capsule (e.g., a microcapsule (less than 1 mm in diameter) or a millicapsule (at least 1 mm in diameter)) in which a cell-containing compartment (e.g., inner compartment) containing a plurality of recombinant living RPE cells (and optionally one or more cell-binding substances) is surrounded by a barrier compartment (e.g., outer compartment) containing an anti-fibrous polymer. In one embodiment, the anti-fibrous compound is a compound of formula (I). In the embodiment, the hydrogel capsule is a spherical capsule.

[0010] In another embodiment, the present disclosure features a preparation (e.g., a composition) comprising a plurality (at least one of 3, 6, 12, 25, 50, or more) of RPE cell-containing devices as described herein. In some embodiments, the preparation is a pharmaceutically acceptable composition.

[0011] In another embodiment, the disclosure features a method for preparing or manufacturing a device comprising a plurality of recombinant RPE cells that express and secrete GLA. In some embodiments, the method comprises providing a plurality of recombinant RPE cells and arranging the plurality of RPE cells within an encapsulation component, e.g., a cell-containing compartment of the device described herein. In some embodiments, the encapsulation component comprises a flexible polymer (e.g., PLA, PLG, PEG, CMC, or a polysaccharide, e.g., alginate). In some embodiments, the encapsulation component comprises a non-flexible polymer or a metal housing. In some embodiments, the surface of the device is chemically modified with, for example, a compound of formula (I) described herein.

[0012] In another embodiment, the Disclosure features a method for evaluating recombinant RPE cells or devices as described herein. In some embodiments, the method includes providing recombinant RPE cells or devices and evaluating structural or functional parameters of the RPE cells or devices. In some embodiments, the method includes evaluating the recombinant RPE cells or devices for one or more of a) cell viability and b) the amount of GLA produced. In some embodiments, this evaluation is performed at least 1, 5, 10, 20, 30, 60, 90, or 120 days after (i) the formation of the device (or device preparation) or (ii) the administration of the device (or device preparation) to a subject. In some embodiments, this evaluation further includes evaluating the amount of fibrosis and / or structural integrity of the device (or device in the preparation) at least 30, 60, 90, or 120 days after administration to a subject. In some embodiments, the subject is a mammal (e.g., mouse, human).

[0013] In another embodiment, the Disclosure features a method for treating a subject with Fabry disease, comprising administering a device or device preparation comprising RPE cells recombinant to express and secrete GLA as described herein. In some embodiments, the administration step comprises placing a pharmaceutically acceptable preparation comprising a plurality of devices into the subject, each of which has the ability to produce GLA. In some embodiments, the device or device preparation is administered, placed, or provided to a site other than the central nervous system, brain, spinal column, eye, or retina. In some embodiments, the implantable element is administered, placed, or injected into the peritoneal cavity (e.g., membrane sac), omentum, or subcutaneous fat of the subject. In embodiments, the method further comprises measuring the amount or activity of GLA present in tissue samples taken from the subject, e.g., plasma separated from a blood sample, liver biopsy. In embodiments, tissue samples are taken at 15, 30, 60, or 120 days. In some embodiments, the subject is human.

[0014] Details of one or more embodiments of this disclosure are described herein. Other features, subjects, and advantages of this disclosure will be apparent from the modes for carrying out the invention, drawings, examples, and claims. [Brief explanation of the drawing]

[0015] [Figure 1A] The amino acid sequence of the wild-type human precursor GLA protein expressed by exemplary recombinant human RPE cells (Figure 1A, Sequence ID No. 1) is shown. The underlined amino acids and coding sequence of the GLA signal peptide are shown. [Figure 1B] The nucleotide coding sequence of the wild-type human precursor GLA protein expressed by exemplary recombinant human RPE cells is shown (Figure 1B, Sequence ID No. 2). The underlined amino acids and coding sequence of the GLA signal peptide are shown. [Figure 2] Figure 1A shows an exemplary codon-optimized nucleotide sequence (SEQ ID NO: 3) encoding the wild-type precursor human GLA protein. The underlined part indicates the coding sequence for the GLA signal peptide. [Figure 3] Figure 1A shows another exemplary codon-optimized nucleotide sequence (SEQ ID NO: 4) encoding the wild-type precursor human GLA protein. The underlined part indicates the coding sequence for the GLA signal peptide. [Figure 4A] The amino acid sequence of the GLA fusion protein expressed by exemplary recombinant human RPE cells (Figure 4A, SEQ ID NO: 5) is shown. Here, the HSPG2 signal peptide is fused to the human wild-type mature GLA amino acid sequence. The underlined amino acids and coding sequences of the HSPG2 signal peptide (SEQ ID NOs: 15 and 16, respectively) are shown. [Figure 4B] The codon-optimized nucleotide coding sequence of a GLA fusion protein expressed by an exemplary recombinant human RPE cell (Figure 4B, SEQ ID NO: 6) is shown. Here, the HSPG2 signal peptide is fused to the human wild-type mature GLA amino acid sequence. The underlined amino acids and coding sequences of the HSPG2 signal peptide (SEQ ID NOs: 15 and 16, respectively) are shown. [Figure 5A]The amino acid and nucleotide coding sequences of exemplary GLA-IgG fusion proteins expressed by exemplary recombinant human RPE cells are shown. Figure 5A (SEQ ID NO: 7) shows the HSPG2 signal peptide (underlined) fused to the N-terminus of the human wild-type GLA mature amino acid sequence, fused to the amino acid sequence of the constant region of the IgG (bold) light chain via a linker amino acid linker sequence (italicized). [Figure 5B] The amino acid and nucleotide coding sequences of exemplary GLA-IgG fusion proteins expressed by exemplary recombinant human RPE cells are shown. Figure 5B (SEQ ID NO: 8) shows the HSPG2 signal peptide (underlined) fused to the amino acid sequence of the IgG1 heavy chain constant region. [Figure 5C] The amino acid and nucleotide coding sequences of an exemplary GLA-IgG fusion protein expressed by exemplary recombinant human RPE cells are shown. Figure 5C (SEQ ID NO: 9) shows the nucleotide coding sequence of the amino acid sequence in Figure 5A. The coding sequence of the HSPG2 signal peptide is underlined, the coding sequence of the linker is italicized, and the coding sequence of the IgG light chain is bold. [Figure 5D] The amino acid and nucleotide coding sequences of an exemplary GLA-IgG fusion protein expressed by exemplary recombinant human RPE cells are shown. Figure 5D (SEQ ID NO: 10) shows the nucleotide coding sequence of the amino acid sequence in Figure 5B. The coding sequence of the HSPG2 signal peptide is underlined, the coding sequence of the linker is italicized, and the coding sequence of the IgG light chain is bold. [Figure 6A] The amino acid and nucleotide coding sequences of an exemplary GLA-nanobody-Fc fusion protein expressed by exemplary recombinant human RPE cells are shown. Figure 6A (SEQ ID NO: 11) shows the HSPG2 signal peptide (underlined) fused to the N-terminus of the human wild-type GLA mature amino acid sequence, fused to the amino acid sequences of the FC5 nanobody (bold) and the Fc region of human IgG1 (bold underlined) via the linker amino acid sequence (italicized). [Figure 6B-1]The amino acid and nucleotide coding sequences of an exemplary GLA-nanobody-Fc fusion protein expressed by exemplary recombinant human RPE cells are shown. Figures 6B-1 and 6B-2 (SEQ ID NO: 12) show the nucleotide coding sequences of the amino acid sequence in Figure 6A. The coding sequence of the HSPG2 signal peptide is underlined, the coding sequence of the linker is italicized, and the coding sequences of the FC5 and IgG1 Fc regions are bolded. [Figure 6B-2] This is a continuation of Figure 6B-1. [Figure 7A] The amino acid and nucleotide coding sequences of exemplary GLA-Fc fusion proteins expressed by exemplary recombinant human RPE cells are shown. Figure 7A (SEQ ID NO: 13) shows the HSPG2 signal peptide (underlined) fused to the N-terminus of the human wild-type GLA mature amino acid sequence, fused to the Fc region (bold) of human IgG1 via a linker (italicized). [Figure 7B-1] The amino acid and nucleotide coding sequences of an exemplary GLA-Fc fusion protein expressed by exemplary recombinant human RPE cells are shown. Figures 7B-1 and 7B-2 (SEQ ID NO: 14) show the nucleotide coding sequences of the amino acid sequence in Figure 6A. The coding sequence of the HSPG2 signal peptide is underlined, the coding sequence of the linker is italicized, and the coding sequence of the IgG1 Fc region is bold. [Figure 7B-2] This is a continuation of Figure 7B-1. [Figure 8A] This specification shows an exemplary PiggyBac transposon expression vector useful for generating recombinant RPE cells expressing the GLA protein described herein. Figure 8A shows the vector map. [Figure 8B-1] An exemplary PiggyBac transposon expression vector useful for generating recombinant RPE cells expressing the GLA protein described herein is shown. Figures 8B-1 and 8B-2 show the nucleotide sequence of the vector (SEQ ID NO: 17), with the promoter sequence underlined (SEQ ID NO: 18). [Figure 8B-2] This is a continuation of Figure 8B-1. [Figure 9]Figure 9 shows an exemplary two-compartment hydrogel capsule of the present disclosure, where lines indicate: recombinant RPE cells encapsulated in a first inner compartment formed from a mixture of a hydrogel-forming polymer and a hydrogel-forming polymer covalently attached to a cell-binding peptide; a second compartment; and an anti-fibrous compound disposed both within the second compartment and on the surface of the capsule. Figure 9 discloses “GRGDSP” as Sequence ID No. 44. [Figure 10] Figures 1A-7B-2 are bar graphs showing the amount of GLA protein secreted in vitro by RPE cells recombinant with various GLA nucleotide sequence constructs: wild type (GLA1), codon-optimized (GLA2, GLA3), and codon-optimized GLA fusion (GLA4-1, GLA4-2, GLA5, GLA6, and GLA7). [Figure 11A] The two-compartment alginate hydrogel capsules, formed from an alginate solution including a blend of unmodified alginate with or without a suspension of GLA4 construct (GLA) transfected clonal cell lines, exhibit human GLA activity in tissue samples (liver (Figure 11A)) obtained from Fabry mice 14 days after transplantation. The outer compartment is formed from a chemically modified alginate solution, and the inner compartment from GRGDSP-modified alginate ("GRGDSP" is disclosed as SEQ ID NO: 44). [Figure 11B] The two-compartment alginate hydrogel capsules, formed from an alginate solution including a blend of unmodified alginate with or without a suspension of GLA4 construct (GLA) transfected clonal cell lines, exhibit human GLA activity in tissue samples (plasma (Figure 11B)) obtained from Fabry mice 14 days after transplantation. The outer compartment is formed from a chemically modified alginate solution, and the inner compartment from GRGDSP-modified alginate ("GRGDSP" is disclosed as SEQ ID NO: 44). [Figure 12A](i) Gb3 levels in various tissue samples (plasma (Figure 12A)) obtained from the same Fabry mice described in Figures 11A and 11B 14 days after transplantation of a control capsule or a GLA-producing capsule, or (ii) from wild-type mice (WT) that were not transplanted. [Figure 12B] (i) Gb3 levels in various tissue samples (liver (Figure 12B)) obtained from the same Fabry mice described in Figures 11A and 11B, 14 days after transplantation of a control capsule or a GLA-producing capsule, or (ii) from wild-type mice (WT) that were not transplanted. [Figure 12C] (i) Gb3 levels in various tissue samples (kidney (Figure 12C)) obtained from the same Fabry mice described in Figures 11A and 11B 14 days after transplantation of a control capsule or a GLA-producing capsule, or (ii) from wild-type mice (WT) that did not receive transplantation. [Figure 12D] (i) Gb3 levels in various tissue samples (heart (Figure 12D)) obtained from the same Fabry mice described in Figures 11A and 11B, 14 days after transplantation of a control capsule or a GLA-producing capsule, or (ii) from wild-type mice (WT) that did not receive the transplant. [Figure 13A] Figures 12A-12D show the amount of Lyso-Gb3 reduction in the same tissue samples as those described. Figure 13A shows Lyso-Gb3 levels in the liver. [Figure 13B] Figures 12A-12D show the amount of Lyso-Gb3 reduction in the same tissue samples as described. Figure 13B shows Lyso-Gb3 levels in the kidney. [Figure 13C] Figures 12A-12D show the amount of Lyso-Gb3 reduction in the same tissue samples as described. Figure 13C shows Lyso-Gb3 levels in the heart. [Figure 13D] Figures 12A-12D show the amount of Lyso-Gb3 decrease in the same tissue samples as described. Figure 13D shows Lyso-Gb3 levels in plasma. [Figure 14A] Shows the amount of Gb3 reduction in various tissue samples (plasma (Figure 14A)) obtained from Fabry mice 10 days after transplantation of low-dose, medium-dose, or high-dose GLA-producing capsules, or high-dose control capsules as described in Figures 11A and 11B. [Figure 14B] Shows the amount of Gb3 reduction in various tissue samples (liver (Figure 14B)) obtained from Fabry mice 10 days after transplantation of low-dose, medium-dose, or high-dose GLA-producing capsules, or high-dose control capsules as described in Figures 11A and 11B. [Figure 14C] Shows the amount of Gb3 reduction in various tissue samples (kidney (Figure 14C)) obtained from Fabry mice 10 days after transplantation of low-dose, medium-dose, or high-dose GLA-producing capsules, or high-dose control capsules as described in Figures 11A and 11B. [Figure 14D] Shows the amount of Gb3 reduction in various tissue samples (heart (Figure 14D)) obtained from Fabry mice 10 days after transplantation of low-dose, medium-dose, or high-dose GLA-producing capsules, or high-dose control capsules as described in Figures 11A and 11B. [Figure 15A] Shows the amount of Lyso-Gb3 reduction in the same tissue samples as described in Figures 14A - 14D. Figure 15A shows the Lyso-GB3 levels in plasma. [Figure 15B] Shows the amount of Lyso-Gb3 reduction in the same tissue samples as described in Figures 14A - 14D. Figure 15B shows the Lyso-GB3 levels in the liver. [Figure 15C] Shows the amount of Lyso-Gb3 reduction in the same tissue samples as described in Figures 14A - 14D. Figure 15C shows the Lyso-GB3 levels in the kidney. [Figure 15D] Shows the amount of Lyso-Gb3 reduction in the same tissue samples as described in Figures 14A - 14D. Figure 15D shows the Lyso-GB3 levels in the heart.

Mode for Carrying Out the Invention

[0016] The present disclosure features retinal pigment epithelial (RPE) cells that have been recombinantly engineered to express and secrete GLA (e.g., a GLA cell therapy agent), compositions thereof, devices comprising such recombinant RPE cells, and device preparations comprising the same. In some embodiments, the device comprises a cell-containing compartment that comprises a cell-binding substance and recombinant RPE cells. In some embodiments, the device is configured to reduce FBR when placed inside a subject, e.g., a human subject. In some embodiments, the recombinant RPE cells, compositions, and devices are useful for treating Fabry disease.

[0017] [Abbreviations and Definitions] Throughout the detailed description and examples of the present disclosure, the following abbreviations are used. CBP: Cell-binding peptide CBPP: Cell-binding polypeptide CBP-polymer: A polymer covalently modified with CBP via a linker CBS: Cell-binding substance CM-Alg: Chemically modified alginate CM-LMW-Alg: Chemically modified, low molecular weight alginate CM-LMW-Alg-101: Low molecular weight alginate chemically modified with compound 101 shown in Table 3 CM-HMW-Alg: Chemically modified, high molecular weight alginate CM-HMW-Alg-101: High molecular weight alginate chemically modified with compound 101 shown in Table 3 CM-MMW-Alg: Chemically modified, medium molecular weight alginate CM-MMW-Alg-101: Medium molecular weight alginate chemically modified with compound 101 shown in Table 3 Gb3 or GL3 globotriaosylceramide LysoGb3 or Lyso-Gb3 globotriaosylsphingosine HMW-Alg: High molecular weight alginate MMW-Alg: Medium molecular weight alginate RGD-Alginate: Alginate covalently modified with a peptide containing the amino acid sequence RGD (disclosed as "RGD" in SEQ ID NO: 28). U-Alg: Unmodified alginate U-HMW-Alg: Unmodified high molecular weight alginate U-LMW-Alg: Unmodified low molecular weight alginate U-MMW-Alg: Unmodified intermediate molecular weight alginate A 70:30 mixture (V:V) of CM-Alg:U-Alg:chemically modified alginate and unmodified alginate, for example, as described in the examples below.

[0018] To make this disclosure more easily understandable, some technical and scientific terms used herein are specifically defined below. Unless otherwise defined herein, all other technical and scientific terms used herein have meanings that are generally understood by those skilled in the art to which this disclosure belongs.

[0019] In this specification, including in the attached claims, singular terms such as "a," "an," and "the" refer to the plurality of their corresponding subjects unless otherwise specified in the context.

[0020] When "approximately" or "about" is used herein to modify a numerically defined parameter (e.g., the amount of GLA secreted by RPE cells, the physical properties of a device (e.g., a hydrogel capsule), e.g., diameter, sphericity, the number of cells encapsulated in the capsule, the number of devices in a preparation), it means that the cited numerical value is within the acceptable functional range of the defined parameter as determined by a person skilled in the art, and this functional range depends in part on how the numerical value is measured or determined, and in part on the limits of the measurement system, such as the acceptable error range of the measurement system. For example, "approximately" may mean a range of 20% above or below the cited numerical value. As a non-limiting example, a device defined as having a diameter of approximately 1.5 millimeters (mm) and encapsulating approximately 5 million (M) cells may have a diameter of 1.2 to 1.8 mm and encapsulate 4 to 6 M cells. As another non-limiting example, a preparation having 80 to 120 devices may be a preparation of approximately 100 devices (e.g., hydrogel capsules). In some embodiments, the term “about” means that the modified parameter may vary by only 15%, 10%, or 5% above or below the numerical value stated for that parameter. Alternatively, in particular with respect to certain properties of the devices described herein (e.g., cell productivity, or density of CBP or anti-fibrous compounds), the term “about” may mean within one order of magnitude of the cited value (e.g., within 5, 4, 3, 2, or 1).

[0021] As used herein, “acquire” or “acquiring” means achieving the acquisition of a value (e.g., a number or image) or a physical entity (e.g., a sample) by “directly acquiring” or “indirectly acquiring” the value or physical entity. “Directly acquiring” means obtaining a value or physical entity by performing a process (e.g., performing an analytical method or protocol). “Indirectly acquiring” means receiving a value or physical entity from another entity or provider (e.g., a third-party laboratory that directly acquired the physical entity or value). Steps of directly acquiring a value or physical entity include performing a process that involves physical modification of a physical substance or the use of instruments or devices. An example of a step of directly acquiring a value is obtaining a sample from a human subject. Steps of directly acquiring a value include performing a process that involves the use of instruments or devices, such as the use of a fluorescence microscope to obtain fluorescence microscopy data.

[0022] When used herein, “administer,” “administering,” or “administer” means to implant, absorb, ingest, inject, position, or otherwise introduce an entity (e.g., a device or a preparation of a device) described herein into a subject, or to provide such an entity to a subject for administration.

[0023] As used herein, "anti-fibrous" means a compound or substance that reduces foreign body reactions (FBR). For example, the amount of FBR induced in biological tissue by implantation of a device containing an anti-fibrous compound (e.g., a hydrogel capsule) (e.g., a hydrogel capsule containing a polymer covalently modified with the compounds listed in Table 3) is less than the amount of FBR induced by implantation of a reference device without anti-fibrous properties (i.e., a device lacking any anti-fibrous compounds but having substantially the same composition (e.g., the same CBP-polymer, the same cell type) and structure (e.g., size, shape, number of compartments)). In embodiments, the degree of FBR is determined, for example, using assays known in the art as described in International Publication No. 2017 / 075630, or assays / methods as described in Vegas, A., et al., Nature Biotechnol (cited above) (e.g., subcutaneous cathepsin measurement of implanted capsules, Masson's trichrome (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, macrophage (CD68 or F4 / 80) cell staining and confocal microscopy, myofibroblast (alpha-smooth muscle actin (alpha-muscle) FBR is evaluated by the immune response in tissues including a transplanted device (e.g., a hydrogel capsule) which may include, for example, protein adsorption, macrophages, multinucleated xenobody giant cells, fibroblasts, and angiogenesis, using one or more of the following methods: quantification of 79RNA sequences of actin), SMA), or general cell adhesion, known inflammatory factors, and immune cell markers, or FACS analysis of macrophages and neutrophils in a device (e.g., a capsule) recovered intraperitoneally after 14 days in a suitable subject, e.g., an immune-responsive mouse. In embodiments, FBR is evaluated by measuring the levels of one or more biomarkers of the immune response in transplanted tissues, e.g., cathepsin, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4.In some embodiments, FBR induced by the device of the present invention (e.g., a hydrogel capsule containing an anti-fibrous compound placed on its outer surface) is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than FBR induced by a reference device without FBR (e.g., a device substantially identical to the test device or the claimed device except lacking means for reducing FBR (e.g., a hydrogel capsule that does not contain an anti-fibrous compound but is otherwise substantially identical to the claimed capsule)). In some embodiments, FBR (e.g., biomarker levels) 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 about longer.

[0024] "Alpha-galactosidase A," "α-GalA," "α-D-galactosidase-A," "α-galactoside galactohydrolase," "galactosidase α," and "GLA protein" may be used interchangeably herein and refer to homodimeric proteins comprising the mature amino acid sequence encoded by the wild-type mammalian GLA gene or an amino acid sequence having its conserved substitutions. In embodiments, the conservedly substituted GLA protein, when measured by the GLA activity assay described herein, has enzymatic activity within 80–120%, 85–115%, 90–110%, or 95–105% of the corresponding wild-type mammalian mature GLA protein. The wild-type human GLA gene encodes a polypeptide of 429 amino acids, of which the N-terminal 31 amino acids constitute a signal peptide. The complete DNA sequence of the wild-type human GLA gene, including introns and exons, is available under GenBank accession number X14448.1. The amino acid sequence of wild-type human precursor α-Gal A is available under GenBank accession numbers X14448.1 and U78027, and is shown in Figure 1A (SEQ ID NO: 1). In some embodiments, the term “GLA protein” (and any of the aforementioned synonyms) refers to a polypeptide comprising the wild-type mature amino acid sequence and optionally preceded by a signal peptide of a GLA signal peptide or a signal peptide of a different secreted protein (e.g., a protein secreted by RPE cells, e.g., the signal peptide of HSPG2).

[0025] As used herein, “cell” means either a recombinant cell or a non-recombinant cell. In some embodiments, a cell is an immortalized cell or a recombinant cell derived from an immortalized cell. In some embodiments, a cell is a living cell and is viable, for example, as measured by any technique described herein or known in the art.

[0026] As used herein, "cell-binding peptide (CBP)" means a linear or cyclic peptide comprising an amino acid sequence derived from the cell-binding domain of a ligand of a cell-adherence molecule (CAM) (e.g., a CAM mediating cell-matrix or cell-cell binding). CBPs are 50, 40, 30, 25, 20, 15, or less than 10 amino acids in length. In some embodiments, CBPs are 3 to 12 amino acids, 4 to 10 amino acids in length, or 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length. The amino acid sequence of a CBP may be identical to a naturally occurring binding domain sequence or a conserved variant thereof. In some embodiments, the CAM ligand is a mammalian protein. In some embodiments, the CAM ligand is a human protein selected from the group of proteins listed in Table 1 below. In some embodiments, CBPs comprise, are essentially, or consist of the cell-binding sequences listed in Table 1 below, or conserved variants thereof. In one embodiment, CBP is an RGD peptide, meaning that this peptide contains the amino acid sequence RGD (SEQ ID NO: 28) and optionally contains one or more additional amino acids located at either the N-terminus or C-terminus, or both. In one embodiment, CBP is a cyclic peptide containing RGD (SEQ ID NO: 28), such as one of the cyclic RGD peptides described in, for example, Vilaca, H. et al., Tetrahedron 70(35):5420-5427 (2014). In one embodiment, CBP is a linear peptide containing RGD (SEQ ID NO: 28) and having a length of less than six amino acids. In one embodiment, CBP is a linear peptide essentially consisting of RGD (SEQ ID NO: 28) or RGSP (SEQ ID NO: 49).

[0027] [Table 1]

[0028] "CBP-polymer", as used herein, means a polymer that contains at least one cell-binding peptide molecule covalently attached to this polymer via a linker. In certain embodiments, the polymer in the CBP-polymer is neither a peptide nor a polypeptide. In certain embodiments, the polymer in the CBP-polymer is a synthetic or naturally occurring polysaccharide, such as alginate, such as sodium alginate. In certain embodiments, 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), and this amino acid linker is linked via a peptide bound to the N-terminus or C-terminus of the CBP. In certain embodiments, the C-terminus of the amino acid linker is linked to the N-terminus of the CBP, and the N-terminus of the amino acid linker is linked to at least one pendant carboxyl group in the polysaccharide via an amide bond. In certain embodiments, the linker-CBP structure means that the linker has 1, 2, 3, or 4 glycine residues, represented as G (1~4) -CBP. In certain embodiments, one or more of the monosaccharide moieties in the CBP-polysaccharide, such as CBP-alginate), are not modified by the CBP. For example, this unmodified portion has a free carboxyl group or lacks a pendant carboxyl group that can be modified. In certain embodiments, the number of polysaccharide moieties to which the CBP is covalently attached is less than any of the following values: 99%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%.

[0029] In one embodiment, the density of CBP modification in the CBP-polymer is estimated by combustion analysis for nitrogen percentage, as described in the examples below, for example. In one embodiment, the CBP-polymer is an RGD-polymer (e.g., RGD-alginate), which is a polymer (e.g., alginate) covalently modified with linker RGD molecules (e.g., peptides essentially consisting of GRGD (SEQ ID NO: 43) or GRGDSP (SEQ ID NO: 44)), and the density of modification with linker RGD molecules is about 0.05% nitrogen (N) to 1.00% N, about 0.10% N to about 0.75% N, about 0.20% N to about 0.50% N, or about 0.30% N to about 0.40% N, as determined using the assays described herein. In one embodiment, the conjugation density of linker-RGD modifications in RGD-alginates (e.g., MMW alginates covalently modified with GRGDSP (SEQ ID NO: 44)) is 0.2-2.0, 0.2-1.5, 0.2-1.0, 0.3-0.7, 0.3-0.6, or 0.4-0.6 micromoles of linker-RGD portions per gram of RGD-polymer in a solution (e.g., physiological saline) with a viscosity of 80-120 cP, when determined by any assay capable of quantifying the amount of peptides conjugated to the polymer (e.g., the quantitative peptide conjugation assay described herein). Unless otherwise explicitly stated or evident from the context, any specifically listed numerical concentrations, concentration ranges, densities, or density ranges relating to CBP in CBP-polymers refer to the concentrations of conjugated CBP molecules in the CBP-polymer composition, i.e., do not include any residual free (e.g., unconjugated) CBP that may be present in the CBP-polymer.

[0030] As used herein, "cell-binding polypeptide (CBPP)" means a polypeptide having a length of at least 50, at least 75, or at least 100 amino acids and comprising the amino acid sequence of the cell-binding domain of a CAM ligand or a conservedly substituted variant thereof. In some embodiments, the CAM ligand is a mammalian protein. In some embodiments, the CBPP amino acids comprise the naturally occurring amino acid sequence of a full-length CAM ligand, and include, for example, one of the proteins listed in Table 1 below or a conservedly substituted variant thereof.

[0031] When used herein, "CBP density" refers to the CBP-polymer composition (e.g., G) unless otherwise expressly stated herein. 1~3 RGD (Sequence ID 50) or G 1~3 This refers to the concentration of the linker-CBP moiety in alginate modified with RGDSP (SEQ ID NO: 51).

[0032] "Cell-binding substance (CBS)" as used herein means any chemical, biological, or other type of substance (e.g., small organic compounds, peptides, polypeptides) that can mimic the activity of at least one ligand of a cell adhesion molecule (CAM) or other cell surface molecule that mediates cell-matrix junction or cell-cell junction or other receptor-mediated signaling. In some embodiments, when present in a polymer composition encapsulating living cells, a CBS may form transient or permanent binding or contact with one or more cells. In some embodiments, a CBS facilitates interaction between two or more living cells encapsulated in a polymer composition. In some embodiments, the presence of a CBS in a polymer composition encapsulating multiple cells (e.g., living cells) correlates with one or both of the following when the encapsulated cells are transplanted into a test subject (e.g., a mouse): increased cell productivity (e.g., expression of therapeutic agents) and / or increased cell viability. In some embodiments, a CBS is physically attached to one or more polymer molecules in a polymer composition. In some embodiments, a CBS is a cell-binding peptide or cell-binding polypeptide as defined herein.

[0033] As used herein, “conservatively modified variant” or “conservative substitution” refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule except that it has one or more conservative amino acid substitutions in its amino acid sequence. In embodiments, a conservatively modified variant consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% identical to the reference amino acid sequence. A conservative amino acid substitution refers to an amino acid substitution by an amino acid that has similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilic, skeletal structure, and rigidity) and has minimal effect on the biological activity of the resulting substituted peptide or polypeptide. A table of functionally similar conservative amino acid substitutions is well known in the art, and exemplary substitutions classified by functional characteristics are listed in Table 2 below.

[0034] [Table 2]

[0035] As used throughout this specification and the claims, “consists essentially of,” and variations such as “consist essentially of” or “consisting essentially of,” indicate the inclusion of any described element or group of elements, and the inclusion of any selection of other elements of similar or different nature to the described element, without substantially altering the basic or novel nature of the specified molecule, composition, device, or method. As a non-limiting example, a cell-binding peptide or GLA protein essentially consisting of an enumerated amino acid sequence may also include one or more amino acids, including substitutions of one or more amino acid residues in the enumerated amino acid sequence, each of which does not substantially affect the relevant biological activity of the cell-binding peptide or GLA protein.

[0036] When used herein in relation to cells, "derived from" means cells obtained from tissue, cell line, or other cells which are then optionally cultured, passaged, immortalized, differentiated, and / or induced to produce derived cells.

[0037] "Device," as used herein, refers to any implantable object (e.g., particles, hydrogel capsules, grafts, medical devices) that contains living recombinant RPE cells capable of expressing and secreting GLA proteins after implantation of the device and has a structure that supports the viability of these RPE cells by allowing cellular nutrients to enter the device. In some embodiments, the device is capable of releasing metabolic byproducts produced by living cells.

[0038] As used herein, “differential volume” refers to the volume of one compartment within the device described herein, excluding the space occupied by another compartment. For example, the differential volume of a second (e.g., outer) compartment in a two-compartment device having an inner and an outer compartment refers to the volume within the second compartment, excluding the space occupied by the first (inner) compartment.

[0039] As used herein, “effective amount” refers to any amount of: recombinant RPE cells secreting GLA, a GLA-producing device preparation, or a component of the device sufficient to elicit a desired biological response (e.g., the number of recombinant RPE cells in the device, the amount of CBS and / or non-fibrous compounds in the device). In some embodiments, the term “effective amount” refers to the amount of components of the device, e.g., the number of cells in the device, the density of anti-fibrous compounds on the surface of the device and / or in the barrier compartment, the density of CBS in the cell-containing compartment. In embodiments, the desired biological response is an increase in GLA levels in a tissue sample taken from a subject treated (e.g., transplanted) with recombinant RPE cells, the device, or a device preparation containing such cells. As will be recognized by those skilled in the art, the effective amount may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of secreted GLA, the composition or device, the condition being treated, the mode of administration, and the age and health status of the subject. The effective amount encompasses both therapeutic and prophylactic treatments. In embodiments, the effective amount of the compound of formula (I) placed on or within the device is an amount that reduces FBR to the implanted device compared to a reference device, for example, by reducing the amount of fibrosis or fibrous tissue on or near the implanted device. In embodiments, the effective amount of CBS placed with recombinant RPE cells in a cell-containing compartment is an amount that enhances cell viability (e.g., the number of living cells) and / or increases GLA production by RPE cells compared to a reference device (e.g., by increasing GLA levels in the plasma of the subject to whom the device was implanted). The effective amounts of the device, composition, or components (e.g., anti-fibrous compounds, CBS, recombinant cells) may be determined by any techniques known in the art or described herein.

[0040] In one embodiment, the CBS (e.g., alginate modified with RGD peptide, e.g., GRGDSP-alginate) in the cell-containing compartment is present in an amount effective to increase cell viability and / or cell productivity at the time after the device has been transplanted into an immunodeficient or immunocompetent animal (e.g., an immunocompetent mouse (e.g., the C57BL / 6J mouse strain available from Jackson Laboratory, Bar Harbor, ME USA)) compared to a reference device without CBS as defined below herein. In one embodiment, the increase in cell viability and / or productivity is detectable at a desired time after transplantation, for example, at one or more of 1 day, 3 days, 5 days, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, 24 weeks, 36 weeks, and 48 weeks. In one embodiment, an effective amount of CBS results in either (i) an increase of at least 10%, 25%, 50%, or 100% in cell viability as measured at 1, 2, 4, or 12 weeks post-transplant, and (ii) an increase of at least 1.25, 1.5, 2, 5, 8, or 10 times in cell productivity as measured at 1, 2, 4, or 12 weeks post-transplant. In one embodiment, the effective amount of CBS in the cell-containing compartment is within a range between the minimum effective amount and a higher amount that results in decreased cell viability and / or productivity compared to a reference device without CBS or compared to a device containing the maximum effective amount (e.g., the optimal amount) of CBS in the cell-containing compartment. In one embodiment, the amount of CBS in the cell-containing compartment is 50%, 25%, 10%, or 5% or less above or below the optimal amount (e.g., the amount that results in the maximum increase in cell viability and / or productivity compared to a reference device without CBS).

[0041] The number of viable cells (and optionally dead cells) in the devices described herein can be estimated using any technique known in the art (e.g., an assay in which live and dead cells are separately labeled with two fluorescent dyes, and then the labeled cells are detected and optionally quantified using a fluorescence microscope). Cell viability can also be assessed by evaluating other indicators of cell viability (e.g., by measuring esterase activity or quantifying the amount of ATP in the cells).

[0042] In one embodiment, the post-transplant increase in cell productivity is detected by assaying the level of GLA protein expressed in vivo by cells or ex vivo by cells (e.g., cell expression after the device is recovered from the animal). GLA expression may be measured extracellularly but internally within the device, and / or externally (e.g., in tissue samples extracted from animals treated with the device or device preparation described herein (e.g., non-human animals)). In one embodiment, cell productivity is expressed as a measured amount of GLA protein or GLA activity, divided by the number of administered devices (e.g., the number of capsules placed in the animal) and / or the number of administered recombinant RPE cells (e.g., the approximate number per capsule in the administered capsule preparation). In one embodiment, the increase in cell productivity is further normalized by dividing the determined amount or activity of GLA by the time between two point in time of interest (e.g., the time between administration and measurement, e.g., several hours, several days, or several weeks). In one embodiment, the amount and / or activity of GLA protein in a tissue sample extracted from an animal (e.g., plasma separated from a blood sample taken from an animal) is measured, and this measured amount and / or activity is divided by the number of administered devices (e.g., the number of implanted 2-compartment capsules), and optionally, the increase in productivity is determined by dividing this result by the number of days between administration and tissue sample extraction.

[0043] As used herein, "endogenous nucleic acid" refers to nucleic acid that occurs naturally within the cells in question.

[0044] As used herein, "endogenous polypeptide" refers to a polypeptide that occurs naturally within the target cell.

[0045] As used herein, “recombinant RPE cells” refers to RPE cells having non-natural modifications, which typically include nucleic acid sequences (e.g., DNA or RNA) or polypeptides (exogenous nucleic acid sequences) that are not present (or present at different levels) in other similar RPE cells that have not been recombinant under similar conditions. In embodiments, recombinant RPE cells include exogenous nucleic acids encoding GLA protein (e.g., vectors or modified chromosomal sequences). In embodiments, recombinant RPE cells secrete GLA protein containing human wild-type GLA amino acid sequences or variants thereof. In embodiments, the exogenous nucleic acid sequence is chromosomal (e.g., the exogenous nucleic acid sequence is an exogenous sequence placed within an endogenous chromosomal sequence) or extrachromosomal (e.g., a non-integrated expression vector). In embodiments, the exogenous nucleic acid sequence includes RNA sequences, e.g., mRNA. In embodiments, the exogenous nucleic acid sequence includes chromosomal or extrachromosomal exogenous nucleic acid sequences containing sequences expressed as RNA, e.g., mRNA or regulatory RNA. In embodiments, the exogenous nucleic acid sequence includes a second nucleic acid sequence, for example, a first chromosomal or extrachromosomal exogenous nucleic acid sequence that modulates the three-dimensional structure or expression of a GLA coding sequence, where the second amino acid sequence can be exogenous or endogenous. For example, recombinant RPE cells may contain an exogenous nucleic acid that controls the expression of an endogenous sequence. In embodiments, recombinant RPE cells include an exogenous nucleic acid sequence that codes for GLA and achieves higher GLA expression than that of a naturally occurring GLA coding sequence, comprising a codon-optimized sequence.This codon-optimized sequence can be generated using commercially available algorithms, for example, 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, recombinant RPE cells (e.g., recombinant ARPE-19 cells) are cultured from a population of stably transfected cells or from a monoclonal cell line.

[0046] As used herein, "exogenous nucleic acid" refers to nucleic acid that is not naturally present in the cells of interest.

[0047] As used herein, "exogenous polypeptide" refers to a polypeptide that is not naturally present in the cells of interest (e.g., recombinant cells). References to specific amino acid positions in a sequence mean (unless otherwise stated) the position of the amino acid in a reference amino acid sequence (e.g., the sequence of the full-length mature (after signal peptide cleavage) wild-type protein) and do not exclude the presence of mutations (e.g., deletions, insertions, and / or substitutions) at other positions in the reference amino acid sequence.

[0048] "Fabry disease," "GLA deficiency," "alpha-galactosidase A deficiency," "Fabry's disease," "Anderson-Fabry disease," "diffuse angiokeratomas," "diffuse angiokeratomas," and "hereditary ectopic lipidosis" can be used interchangeably and refer to a rare, X-linked hereditary lysosomal storage disorder caused by a deficiency of the lysosomal enzyme galactosidase alpha (GLA). This enzyme cleaves the terminal α-D-galactose residue from glycolipids. GLA deficiency results in systemic and lifelong lysosomal storage of sphingoglycolipids, mainly globotriaosylceramide (Gb3), in the vascular endothelium and other tissues. This leads to multi-organ pathology that primarily affects the kidneys, heart, and cerebrovascular system. Patients with Fabry disease suffer from a wide range of symptoms, including gastrointestinal disorders, pain, stroke, and heart and kidney failure, and often die prematurely from complications such as stroke, heart disease, or kidney failure.

[0049] Classical Fabry disease, occurring in men with α-Gal A enzyme activity less than 1%, typically develops in childhood or adolescence and is accompanied by severe extremity pain (acrotactile dyspareunia), the appearance of vascular lesions (angiokeratoma), abnormal sweating (anhidrosis, hypohidrosis, rarely hyperhidrosis), characteristic corneal and lens opacity, and periodic acute onsets of proteinuria. The gradual deterioration of renal function to end-stage renal disease (ESRD) usually occurs in men between their 30s and 50s. In middle age, the majority of men who have successfully treated ESRD develop cardiovascular and / or cerebrovascular disease, which is the leading cause of morbidity and mortality. In contrast, men with α-Gal A activity exceeding 1% may have: (1) a cardiac variant phenotype that typically appears in the 60s to 80s, with left ventricular hypertrophy, cardiomyopathy and arrhythmias, and proteinuria, but without ESRD; or (2) a renal variant phenotype associated with ESRD, but without skin lesions or pain; or (3) cerebrovascular disease that manifests as stroke or transient ischemic attack. In embodiments, patients with “cardiac variant” Fabry disease have approximately 5–15% of normal α-Gal A activity and present with left ventricular hypertrophy or cardiomyopathy. Heterozygous women typically have milder symptoms and an older onset than men. Rarely, they may be relatively asymptomatic throughout their normal lifespan, or they may have symptoms as severe as those observed in men with the classic phenotype.

[0050] Signs and symptoms that can provide a predictive diagnosis of Fabry disease include angiokeratoma and itchy cornea. Further support can be provided by examining family history and paying attention to other family members with symptoms such as early renal disease, early stroke, and early cardiac problems. A definitive diagnosis in males can be made by testing for GLA enzyme deficiency in biological samples such as plasma, leukocytes, cultured dermal fibroblasts, biopsy tissue, or dried blood. In females, heterozygous mutation carriers can be identified by mutation or linkage analysis. Many female carriers (with or without symptoms) exhibit below-normal GLA activity and / or characteristic corneal opacity.

[0051] As used herein, “Fabry disease patient” refers to an individual diagnosed with or suspected of having Fabry disease. In embodiments, a Fabry disease patient has a mutated GLA gene. Characteristic markers of Fabry disease may occur in male hemizygotes and female carriers of the same prevalence, although females are typically less severely affected. Female carriers have one X chromosome with the defective α-Gal A gene and one X chromosome with the normal gene, and X chromosome inactivation of the normal allele is present in one or more cell types. Carriers are often diagnosed with Fabry disease.

[0052] As used herein, "polymer composition" refers to a composition (e.g., a solution, a mixture) comprising one or more polymers. As a class, "polymer" includes homopolymers, heteropolymers, copolymers, block polymers, and block copolymers, and can be both natural and synthetic. Homopolymers contain one type of building block (i.e., monomer), while copolymers contain multiple types of monomers.

[0053] As used herein, "polypeptide" refers to a polymer comprising amino acid residues linked via peptide bonds, having at least 2, and in some embodiments, at least 3, 4, 5, 10, 50, 75, 100, 150, or 200 amino acid residues.

[0054] As used herein, “prevention,” “prevent,” and “preventing” refer to a procedure including the administration or application of a GLA replacement therapy agent, which includes, for example, a step of administering a device composition for encapsulating recombinant RPE cells (e.g., one described herein) to prevent physical signs relating to one or more symptoms of Fabry disease before the onset of those symptoms. In some embodiments, “prevention,” “prevent,” and “preventing” require that no signs or symptoms of Fabry disease have yet occurred or been measured. In some embodiments, the procedure includes prevention, while in other embodiments it does not.

[0055] When used herein in reference to the claimed device (e.g., a hydrogel capsule), “reference device” means a device (e.g., a hydrogel capsule) that (i) lacks certain features of the claimed device (e.g., certain exogenous nucleotide sequences, e.g., elements that enhance mRNA or protein expression, e.g., promoter sequences, signal peptide sequences), FBR mitigation means (e.g., a barrier compartment containing an anti-fibrous compound (as defined herein) or CBS (as defined herein) (e.g., an RGD polymer)), (ii) encapsulates substantially the same amount of cells of the same cell type as in the claimed device in a cell-containing compartment, and (iii) has a polymer composition and structure substantially similar to that of the claimed device, except that it lacks certain features (e.g., an anti-fibrous compound or CBS). In one embodiment, the number of live recombinant RPE cells in the cell-containing compartment of the reference device is within 80-120% or 90-110% of the number of live recombinant RPE cells in the cell-containing compartment of the claimed device. In one embodiment, recombinant cells in the reference device and recombinant cells in the claimed device are obtained from the same cell culture. In one embodiment, substantially similar polymer composition means that all polymers in the reference device and the claimed device (e.g., polymer components of any CBP-polymer and anti-fibrous polymer) are identical in chemical class and molecular weight class, where applicable (e.g., alginates with high G content and the same molecular weight range). For example, in one embodiment, the cell-containing compartment of a CBP-free reference device is formed from an unmodified version of the polymer (e.g., alginate) in the CBP-polymer used to form the cell-containing compartment of the claimed device.In some embodiments, the claimed two-compartment hydrogel millicapsule has (i) an inner compartment formed from a CBP-polymer that encapsulates multiple cells, and (ii) an outer compartment formed from a mixture of a chemically modified polymer (e.g., CM-LMW-alginate as described herein) and an unmodified polymer (e.g., U-HMW-alginate as described herein), wherein the outer compartment of the reference capsule and the claimed capsule are formed from the same polymer mixture, but the inner compartment of the reference capsule is formed from a suspension of cells in the same polymer mixture used for the outer compartment. In some embodiments, substantially similar structures mean that the reference device and the claimed device have the same number of compartments (e.g., one, two, three, etc.) as well as nearly identical size and shape.

[0056] When used herein, “RPE cells” refers 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 from such RPE, for example, by stably transfecting cells cultured from the ARPE-19 cell line with an exogenous sequence encoding the GLA protein, or by otherwise recombining such cultured ARPE-19 cells to express the GLA protein. a) Cells derived from recombinant cells, cells derived from primary cell cultures of RPE cells, cells directly isolated from naturally occurring RPE cells (e.g., from humans or other mammals) (without long-term culture, e.g., fewer 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) Cells obtained from poorly differentiated cells, e.g., cells that have developed into RPE cells, programmed, or (e.g., in vitro) (b) Reprogrammed cells, or cells that are substantially identical to one or more naturally occurring RPE cells except for any genetic recombination, or cells from primary or long-term cultures of RPE cells (for example, these cells may be derived from iPS cells); or (c) Cells having one or more of the following characteristics: i) Expressing one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) Expressing the biomarkers CRALBP, RPE-65, RLBP, BEST1, or α iii) not expressing one or more of the β-crystallins; or iv) being naturally found in the retina and forming a single layer on the choroidal blood vessels in Bruch's membrane; or iv) being involved in epithelial transport, light absorption, secretion, and immunomodulation in the retina; or v) being synthesized or modified from naturally occurring cells to have the same or substantially the same genetic content as immortalized RPE cell lines (e.g., ARPE-19 cell line (ATCC® CRL-2302®)) and optionally the same or substantially the same epigenetic content.In one embodiment, the RPE described herein is recombinant to have, for example, novel properties, such as being recombinant to express and secrete GLA. In other embodiments, the RPE cells are not recombinant.

[0057] As used herein, "physiological saline" means ordinary physiological saline unless otherwise specified, i.e., water containing 0.9% NaCl.

[0058] When “sequence identity” or “percent identical” is used herein to refer to two nucleotide sequences or two amino acid sequences, it means that, when the two sequences are compared and aligned for maximum match across a comparison window or a specified region, the two sequences are identical within a specified region or have a specified percentage of identical nucleotides or amino acids at a specified percentage of nucleotide or amino acid positions within that specified region. Sequence identity can be determined using standard techniques known in the art, including, but not limited to, any algorithm described in U.S. Patent Application Publication No. 2017 / 02334455A1. In embodiments, a 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.

[0059] As used herein, “spherical” means a sphere (e.g., a perfectly circular sphere) or a device (e.g., a hydrogel capsule or other particle) having a curved surface that forms a spherical shape, which may have waves and undulations on its surface. Spheres and spherical-like objects can be mathematically defined by the rotation of circles, ellipses, or combinations of rotations around three vertical axes, a, b, and c, respectively. For a sphere, the three axes are of equal length. In general, a spherical-like shape is an ellipsoid (with respect to its mean plane) with semi-principal axes that are no more than 10%, 5%, or 2.5% of each other. The diameter of a sphere or spherical-like shape is the mean diameter, such as the mean of the semi-principal axes.

[0060] When the term "spheroid" is used herein to refer to a device (e.g., a hydrogel capsule or other particle), it means that the device (i) has the shape of a perfect or classical flattened or elongated spheroid, or (ii) has a surface that roughly forms a spheroid, which may have, for example, waves and ridges, and / or be an ellipsoid (average surface) whose semi-principal axes are 100% in each other.

[0061] As used herein, “subject” refers to a human or a non-human animal. In embodiments, the subject is, for example, a human of any age group (i.e., male or female), for example, a pediatric human subject (e.g., infant, child, adolescent) or an adult human subject (e.g., young adult, middle-aged adult, or elderly). In embodiments, the subject is a non-human animal, for example, a mammal (e.g., mouse, dog, primate (e.g., crab-eating macaque or rhesus macaque). In embodiments, the subject is a commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog)) or a bird (e.g., a commercially relevant bird such as a chicken, duck, goose, or turkey). In certain embodiments, the animal is a mammal. The animal may be male or female and may be at any stage of development. The non-human animal may be a genetically modified animal.

[0062] As used herein, “total volume” refers to the volume within one compartment of a multi-compartment device, including space occupied by another compartment. For example, the total volume of the second (e.g., outer) compartment of a two-compartment device refers to the volume within the second compartment, including space occupied by the first compartment.

[0063] A “transcription unit” means a DNA sequence that, for example, is present in an exogenous nucleic acid and includes at least one promoter sequence operably linked to a coding sequence, and may also include one or more additional elements that control or enhance the transcription of the coding sequence into an RNA molecule or the translation of the RNA molecule into a polypeptide molecule. In some embodiments, the transcription unit also includes a polyadenylation (polyA) signal sequence and a polyA site. In embodiments, the transcription unit is present in an exogenous extrachromosomal expression vector, for example, as shown in Figure 8A, or as an exogenous sequence incorporated into the chromosome of a recombinant RPE cell as described herein.

[0064] As used herein, “treatment,” “treat,” and “treating” refer to one or more reductions, reversals, mitigations, or delays in the onset of Fabry disease, or inhibition of the progression of one or more symptoms, signs, or underlying causes of the disease, disorder, or condition. In embodiments, treatment includes reductions, reversals, mitigations, delays in onset, or inhibition of progression of symptoms or conditions associated with Fabry disease. In embodiments, treatment includes increasing GLA levels in at least one tissue of the subject requiring it, e.g., one or more of the plasma, liver, kidney, and heart. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms associated with Fabry disease have occurred or been measured. In other embodiments, treatment may be administered even in the absence of signs or symptoms of Fabry disease, for example, as a prophylactic measure. For example, treatment may be administered to a susceptible individual before 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 subsided, for example, to delay or prevent recurrence. In some embodiments, treatment includes prevention, while in other embodiments it does not.

[0065] Selected chemical definitions The definitions of specific functional groups and chemical terms are described in more detail below. Chemical elements are defined in Handbook of Chemistry and Physics, 75. th Identification is based on the Periodic Table of the Elements, CAS version, on the inside cover of the ed., and specific functional groups are defined as usual, as described here. Furthermore, general principles of organic chemistry, as well as specific functional groups and reactivity, are based on Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5 thEdition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3 rd Edition, Cambridge University Press, Cambridge, 1987.

[0066] The abbreviations used in this specification have their conventional meanings in the technical fields of chemistry and biology. The chemical structures and formulas described in this specification are constructed according to the standard rules regarding chemical valences known in the technical field of chemistry.

[0067] When a range of values is recited, each value and sub-range within the range is intended to be included. For example, "C1-C6 alkyl" is intended to include C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0068] As used herein, "alkyl" refers to a radical of a straight-chain or branched saturated hydrocarbon group having 1 to 24 carbon atoms ("C1-C 24 alkyl"). In some embodiments, the alkyl group has 1 to 12 carbon atoms ("C1-C 12 alkyl"), 1 to 10 carbon atoms ("C1-C 10Alkyl groups include those with 1 to 8 carbon atoms ("C1-C8 alkyl"), 1 to 6 carbon atoms ("C1-C6 alkyl"), 1 to 5 carbon atoms ("C1-C5 alkyl"), 1 to 4 carbon atoms ("C1-C4 alkyl"), 1 to 3 carbon atoms ("C1-C3 alkyl"), 1 to 2 carbon atoms ("C1-C2 alkyl"), or 1 carbon atom ("C1 alkyl"). In some embodiments, the alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7) and n-octyl (C8). Each example of an alkyl group can be independently and optionally substituted, i.e., either unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents (e.g., 1-5 substituents, 1-3 substituents, or 1 substituent) ("substituted alkyl").

[0069] As used herein, "alkenyl" refers to a radical of a linear or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds ("C2- 24 The term "alkenyl" refers to an alkenyl group consisting of 2 to 12 carbon atoms ("C2-C2"). In some embodiments, the alkenyl group consists of 2 to 12 carbon atoms ("C2-C2"). 12 "Alkenyl"), 2 to 10 carbon atoms ("C2~C 10Alkenyl groups include those with 2-8 carbon atoms ("C2-C8 alkenyl"), 2-6 carbon atoms ("C2-C6 alkenyl"), 2-5 carbon atoms ("C2-C5 alkenyl"), 2-4 carbon atoms ("C2-C4 alkenyl"), 2-3 carbon atoms ("C2-C3 alkenyl"), or 2 carbon atoms ("C2 alkenyl"). One or more carbon-carbon double bonds can be intermediate (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), and butadienyl (C4). Examples of C2-C6 alkenyl groups include the aforementioned C 2~4 Examples include alkenyl groups, as well as pentenyl (C5), pentadienyl (C5), and hexenyl (C6). Each example of an alkenyl group can be independently and arbitrarily substituted, that is, it can be unsubstituted ("unsubstituted alkenyl") or substituted with one or more substituents (for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent) ("substituted alkenyl").

[0070] As used herein, the term "alkynyl" refers to a radical of a linear or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon triple bonds ("C2-C24"). 24 The term "alkenyl" refers to an alkenyl group. In some embodiments, the alkynyl group consists of 2 to 12 carbon atoms ("C2-C2"). 12 Alkynyl), 2 to 10 carbon atoms ("C2-C2") 10Alkynyl groups include those with 2-8 carbon atoms ("C2-C8 alkynyl"), 2-6 carbon atoms ("C2-C6 alkynyl"), 2-5 carbon atoms ("C2-C5 alkynyl"), 2-4 carbon atoms ("C2-C4 alkynyl"), 2-3 carbon atoms ("C2-C3 alkynyl"), or 2 carbon atoms ("C2 alkynyl"). One or more carbon-carbon triple bonds can be intermediate (such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples of C2-C4 alkynyl groups include ethynyl (C2), 1-propynyl (C3), 2-propynyl (C3), 1-butynyl (C4), and 2-butynyl (C4). Each example of an alkynyl group can be independently and optionally substituted, i.e., either unsubstituted ("unsubstituted alkynyl") or substituted with one or more substituents (e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent) ("substituted alkynyl").

[0071] As used herein, the term "heteroalkyl" means an acyclic, stable linear or branched chain, or a combination thereof, comprising at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen heteroatom may be optionally quaternized. The heteroatoms O, N, P, S, and Si may be positioned at any position in the heteroalkyl group. Examples of heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, and -O-CH2-CH3. Two or fewer heteroatoms may be consecutive (e.g., -CH2-NH-OCH3 and -CH2-O-Si(CH3)3). Whenever "heteroalkyl" is cited followed by a specific heteroalkyl group (-CH2O, -NR), it may be cited. C R D (etc.), terms such as heteroalkyl and -CH2O or -NR C R D It will be understood that these are not redundant or mutually exclusive. Rather, certain heteroalkyl groups are cited for additional clarity. Therefore, the term "heteroalkyl" is -CH2O, -NR C R D This specification should not be construed as excluding certain heteroalkyl groups. Each example of a heteroalkyl group may be independently and optionally substituted, i.e., unsubstituted ("unsubstituted heteroalkyl") or substituted with, for example, one or more substituents, e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted heteroalkyl").

[0072] The terms "alkylene," "alkenylene," "alkynylene," or "heteroalkylene," unless otherwise specified, refer to divalent radicals derived from alkyl, alkenyl, alkynyl, or heteroalkyl groups, either alone or as part of other substituents. Alkylene, alkenylene, alkynylene, or heteroalkylene groups may be written, for example, as C1-C6 member alkylene, C2-C6 member alkenylene, C1-C6 member alkynylene, or C1-C6 member heteroalkylene, where "member" refers to a non-hydrogen atom in the part. In the case of heteroalkylene groups, the heteroatom may occupy one or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not indicated by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- can represent both -C(O)2R'- and -R'C(O)2-.

[0073] As used herein, "aryl" has 6 to 14 ring carbon atoms and zero heteroatoms in an aromatic ring system ("C6~C 14 The term "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π electrons shared in a cyclic array). In some embodiments, the aryl group has 6 ring carbon atoms ("C6 aryl"; e.g., phenyl). In some embodiments, the aryl group has 10 ring carbon atoms ("C6 aryl"). 10 "Aryl"; for example, naphthyl groups such as 1-naphthyl and 2-naphthyl). In some embodiments, the aryl group has 14 ring carbon atoms ("C"). 14 "Aryl" (e.g., anthracyl). Aryl groups are, for example, C6~C 10These may be described as member aryls, where the term "member" refers to the non-hydrogen ring atom in the moiety. Examples of aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each example of an aryl group may be independently and optionally substituted, i.e., either unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl").

[0074] As used herein, “heteroaryl” refers to a 5- to 10-membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic array) having a ring carbon atom and 1 to 4 ring heteroatoms in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5- to 10-membered heteroaryl”). In a heteroaryl group containing one or more nitrogen atoms, the bond site can be a carbon or nitrogen atom, as valence-acceptable. A heteroaryl bicyclic system can contain one or more heteroatoms in one or both rings. “Heteroaryl” also includes a ring system in which the heteroaryl ring as defined above is fused with one or more aryl groups, and the bond site is in either an aryl or heteroaryl ring, in such examples, the number of ring members indicates the number of ring members in the fused (aryl / heteroaryl) ring system. In bicyclic heteroaryl groups in which one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl), the bond site can be on either ring, i.e., the bond site can be on either the ring containing a heteroatom (e.g., 2-indolyl) or the ring not containing a heteroatom (e.g., 5-indolyl). Heteroaryl groups may be described, for example, as 6- to 10-membered heteroaryls, where the term "member" refers to a non-hydrogen ring atom in the part.

[0075] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is 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 a ring carbon atom and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is 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 a ring carbon atom and 1-4 ring heteroatoms in the aromatic ring system, where each heteroatom is 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 group has one ring heteroatom selected from nitrogen, oxygen, and sulfur. Each example of the heteroaryl group can be independently and optionally substituted, i.e., unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl").

[0076] Examples of five-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Examples of five-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Examples of five-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Examples of five-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Examples of six-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridinyl. Examples of six-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Examples of six-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetradinyl, respectively. Examples of seven-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Examples of 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranil, benzoisofuranil, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolidinyl, and prinyl. Examples of 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthylidinyl, pteridinyl, quinolinyl, isoquinolinyl, sinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other examples of heteroaryl groups include heme and heme derivatives.

[0077] As used herein, the terms "arylene" and "heteroarylene" mean divalent radicals derived from aryl and heteroaryl, respectively, either alone or as part of other substituents.

[0078] As used herein, "cycloalkyl" refers to a ring of carbon atoms (3 to 10 C3-C3) 10 The term "cycloalkyl" refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"), 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"), or 5 to 10 ring carbon atoms ("C5-C6 cycloalkyl"). In some embodiments, the cycloalkyl group has 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"), 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"), or 5 to 10 ring carbon atoms ("C5-C6 cycloalkyl"). 10 Cycloalkyl groups are, for example, C4-C7 member cycloalkyl groups, where the term "member" refers to a non-hydrogen ring atom in the group. Examples of C3-C6 cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), and cyclohexadienyl (C6). Examples of C3-C8 cycloalkyl groups include, but are not limited to, the aforementioned C3-C6 cycloalkyl groups, as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), and bicyclo[3.1.1]heptanyl (C7). 10 The cycloalkyl groups are not limited to the aforementioned C3-C8 cycloalkyl groups, as well as cyclononyl (C9), cyclononenyl (C9), and cyclodecyl (C9). 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C9) 10), spiro[4.5]decanil(C 10 Examples include the above. As illustrated in the examples above, in certain embodiments, the cycloalkyl group may be monocyclic ("monocyclic cycloalkyl") or contain a condensed, crosslinked, or spirocyclic system such as a bicyclic system ("bicyclic cycloalkyl"), and may be saturated or partially unsaturated. "Cycloalkyl" also includes a ring system in which the cycloalkyl ring defined above is condensed with one or more aryl groups and the bonding site is on the cycloalkyl ring, in which case the number of carbons still indicates the number of carbons in the cycloalkyl ring system. Each example of a cycloalkyl group may be independently and optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl").

[0079] As used herein, "heterocyclyl" refers to a radical of a 3- to 10-membered non-aromatic ring system having a ring carbon atom and 1 to 4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl"). In a heterocyclyl group containing one or more nitrogen atoms, the bond site can be a carbon or nitrogen atom, as valence-acceptable. A heterocyclyl group can be monocyclic ("monocyclic heterocyclyl") or a condensed, bridged, or spirocyclic system such as a bicyclic system ("bicyclic heterocyclyl"), and can be saturated or partially unsaturated. A heterocyclyl bicyclic system can contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes a ring system in which the heterocyclyl ring as defined above is fused with one or more cycloalkyl groups, and the bond site is located on either the cycloalkyl or heterocyclyl ring, or a ring system in which the heterocyclyl ring as defined above is fused with one or more aryl or heteroaryl groups, and the bond site is located on the heterocyclyl ring, in which case the number of ring members still indicates 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 part, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each example of a heterocyclyl may be independently optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 10-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 10-membered heterocyclyl.

[0080] In some embodiments, the heterocyclyl group is a 5-10 member non-aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("5-10 member heterocyclyl"). In some embodiments, the heterocyclyl group is a 5-8 member non-aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-8 member heterocyclyl"). In some embodiments, the heterocyclyl group is a 5-6 member non-aromatic ring system having a ring carbon atom and 1-4 ring heteroatoms, where each heteroatom is independently selected from nitrogen, oxygen, and sulfur ("5-6 member heterocyclyl"). In some embodiments, the 5-6 member heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclil has one or two ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclil has one ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0081] Examples of three-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azirdinyl, oxylanil, and thiorenyl. Examples of four-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanil, and thietanil. Examples of five-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranil, dihydrofuranil, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Examples of five-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanil, oxasulfuranil, disulfuranil, and oxazolidine-2-one. Examples of five-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Examples of six-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranil, dihydropyridinyl, and thianil. Examples of six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianil, and dioxanil. Examples of six-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinyl or thiomorpholinyl-1,1-dioxide. Examples of seven-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Examples of eight-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azokanyl, oxecanyl, and thiokanyl. Examples of five-membered heterocyclyl groups condensed to a C6 aryl ring (also referred to herein as 5,6-bicyclic heterocycles) include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, and benzoxazolinol.Examples of six-membered heterocyclyl groups condensed to an aryl ring (also referred to herein as 6,6-bicyclic heterocycles) include, but are not limited to, tetrahydroquinolinyl and tetrahydroisoquinolinyl.

[0082] As used herein, "amino" means radical-NR 70 R 71 This refers to R 70 and R 71 Each of these is independently hydrogen, C1-C8 alkyl, and C3-C 10 Cycloalkyl, C4~C 10 Heterocycline, C6~C 10 Aryl, and C5~C 10 It is a heteroaryl compound. In some embodiments, amino refers to NH2.

[0083] As used herein, "cyano" refers to the radical-CN.

[0084] As used herein, "halo" or "halogen" means, unless otherwise specified, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom, either independently or as part of another substituent.

[0085] As used herein, "hydroxy" refers to the radical -OH.

[0086] As defined herein, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups are optionally substituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" cycloalkyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl groups). Generally, the term "substituted," whether preceded by the term "optionally," means that at least one hydrogen atom present on a group (e.g., a carbon or nitrogen atom) is replaced by an acceptable substituent, such that the substitution results in a stable compound (e.g., a compound that does not undergo spontaneous transformation by rearrangement, cyclization, elimination, or other reactions). Unless otherwise specified, a “substituted” group has substituents at one or more substituted positions of the group, and if two or more positions in any given structure are substituted, these substituents are either identical or different at each position. The term “substituted” is intended to include substitution by all acceptable substituents of an organic compound, such as any substituent described herein that forms a stable compound. This disclosure assumes any such combination to obtain a stable compound. For the purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any preferred substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0087] Two or more substituents can be optionally bonded to form aryl, heteroaryl, cycloalkyl, or heterocyclyl groups. Such so-called ring-forming substituents are typically found bonded to the ring base structure, although this is not essential. 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 the ring base structure form 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 the ring base structure form a spirocyclic structure. In yet another embodiment, the ring-forming substituents are bonded to non-adjacent members of the base structure.

[0088] The compounds of formula (I) described herein may contain one or more chiral centers and therefore may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may exist in the form of individual enantiomers, diastereomers or geometric isomers, or in the form of a mixture of stereoisomers, including a racemic mixture and a mixture enriched with one or more stereoisomers. The isomers can be isolated from the mixture by methods known to those skilled in the art, including chiral high-pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p.268 (ELEliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). This disclosure further includes the compounds described herein as individual isomers substantially free from other isomers, and instead as mixtures of various isomers.

[0089] As used herein, a pure enantiomer compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., an enantiomer excess). In other words, the "S" form of the compound is substantially free of the "R" form, and therefore the "R" form is an enantiomer excess. The terms "enantiomerically pure" or "pure enantiomer" indicate 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 enantiomers. In certain embodiments, weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0090] The compounds of formula (I) described herein may also include one or more isotopic substitutions. For example, H is 1 H, 2 H (D or deuterium), and 3 It may be any isotopic form containing H (T or tritium); C is 12 C, 13 C, and 14 It can be any isotopic form containing C; O is, 16 O and 18 It may be any isotopic form, including O.

[0091] The term "pharmaceutically acceptable salt" means that the effective salt of the compound may be prepared with a relatively non-toxic acid or base, depending on the specific substituent found in the compound described herein. If the compound of formula (I) used in providing the devices of this disclosure contains a relatively acidic functional group, the base addition salt may be obtained by contacting such a neutral compound with a sufficient amount of the desired base, either in its original form 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. If the compound used in this disclosure contains a relatively basic functional group, the acid addition salt may be obtained by contacting such a neutral compound with a sufficient amount of the desired acid, either in its original form or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, monocarbonate, phosphoric acid, monohydrogen-phosphoric acid, dihydrogen-phosphoric acid, sulfuric acid, monohydrogen-sulfuric acid, hydroiodic acid, or phosphorous acid, as well as salts derived from organic acids such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-tolylsulfonic acid, citric acid, tartaric acid, and methanesulfonic acid. Also included are salts of amino acids such as arginates, and salts of organic acids such as glucuronic acid or galacturonic acid (see, for example, Berge et al, Journal of Pharmaceutical Science 66:1-19 (1977)). Compounds used in certain specific devices of this disclosure (e.g., particles, hydrogel capsules) contain both basic and acidic functional groups that enable the conversion of the compound to a base or acid addition salt. These salts can be prepared by methods known to those skilled in the art. Other pharmaceutically acceptable carriers known to those skilled in the art are suitable for use in this disclosure.

[0092] The devices of this disclosure may include a compound of formula (I) in prodrug form. The prodrug is a compound that readily undergoes a chemical change under physiological conditions to provide a compound used to reduce FBR to the devices of this disclosure. Furthermore, the prodrug can be converted to a useful compound of formula (I) by chemical or biochemical methods in an exovivotive environment.

[0093] Certain compounds of formula (I) can exist in non-solvated and solvated forms, including hydrated forms. Generally, the solvated forms are equivalent to the non-solvated forms and are included within the scope of this disclosure. Certain compounds of formula (I) can exist in a number of crystalline or amorphous forms. Generally, all physical forms are intended to be equivalent for the uses envisioned by this disclosure and are within the scope of this disclosure.

[0094] The term "solvate" usually refers to the form of a compound associated with a solvent, typically resulting from solvolysis. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, and diethyl ether. The compounds described herein may be prepared, for example, in crystalline form and then solvated. Preferred solvates include pharmaceutically acceptable solvates, and further include both stoichiometric and non-stoichiometric solvates.

[0095] The term "hydrate" refers to a compound that is accompanied by water. Typically, the number of water molecules contained in a compound hydrate is a fixed proportion of the number of compound molecules in the hydrate. Thus, a compound hydrate can be represented, for example, by the general formula R·x H₂O (where R is the compound and x is a number greater than 0).

[0096] The term "tautomer," as used herein, refers to a compound whose structure is interconvertible, with varying displacements of hydrogen atoms and electrons. Thus, two structures can be in equilibrium via the transfer of π electrons and atoms (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either an acid or a base. Tautomerism may be relevant to achieving optimal chemical reactivity and biological activity of the compound in question.

[0097] In this specification, the symbol [ka] This refers to the bonding of an entity, such as a polymer (e.g., a hydrogel-forming polymer such as alginate) or an implantable element (e.g., a particle, a device (e.g., a hydrogel capsule) or a substance) to a surface. [ka] The bond represented by may refer to a direct bond to an entity, such as a polymer or a portable element (e.g., a device), or to a linkage to an entity via a bonding group. As used herein, “bonding group” refers to the portion for linking the compound of formula (I) to an entity (e.g., a polymer or portable element as described herein), and may include any bonding chemistry known in the art. An enumeration of exemplary bonding groups is incorporated herein by reference in its entirety from Bioconjugate Techniques (3 rd This is outlined in (ed., Greg T. Hermanson, Waltham, MA: Elsevier, Inc., 2013). In some embodiments, the binding group is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)-, -OC(O)-, -N(R C )-,-N(R C )C(O)-, -C(O)N(RC )-,-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 including metal, where R A , R C , R D , R F , R G Each of x and y is independently as described herein. In some embodiments, the binding group comprises an amine, ketone, ester, amide, alkyl, alkenyl, alkynyl, or thiol. In some embodiments, the binding group is a crosslinking agent. In some embodiments, the binding group is -C(O)(C1~C6-alkylene)-, where alkylene is R 1 Replaced with R 1 The following is described herein. In some embodiments, the bonding group is -C(O)(C1-C6-alkylene)-, where the alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the bonding group is -C(O)C(CH3)2-. In some embodiments, the bonding group is -C(O)(methylene)-, where the alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the bonding group is -C(O)CH(CH3)-. In some embodiments, the bonding group is -C(O)C(CH3)-.

[0098] [GLA expression construct] This disclosure provides isolated polynucleotides comprising a promoter operably ligated to a nucleotide sequence encoding a human GLA precursor protein or a variant thereof, such as a GLA fusion protein.

[0099] In embodiments, the promoter is selected to achieve higher expression of GLA mRNA in RPE cells (e.g., ARPE-19 cells) compared to the same GLA coding sequence operably linked to the promoter of the human GLA gene. In embodiments, the promoter essentially consists of or comprises a nucleotide sequence of SEQ ID NO: 18, or substantially identical to SEQ ID NO: 18, for example, at least 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 18. In embodiments, the promoter comprises SEQ ID NO: 18.

[0100] In embodiments, the GLA precursor protein comprises a mature amino acid sequence from wild-type human GLA protein, for example, sequences 32-429 of SEQ ID NO: 1, or a conservedly substituted variant thereof. In embodiments, the conservedly substituted variant has 10 or fewer, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer conserved substitutions. In embodiments, the GLA precursor protein consists of SEQ ID NO: 1.

[0101] In the embodiments, the nucleotide sequence encoding the precursor GLA protein is codon-optimized for GLA expression in mammalian cells. In the embodiments, the codon-optimized sequence is a nucleotide sequence identical to SEQ ID NO: 3, or at least 95%, 96%, 97%, 98%, 99%, or more. In the embodiments, the codon-optimized sequence is a nucleotide sequence identical to SEQ ID NO: 4, or at least 95%, 96%, 97%, 98%, 99%, or more.

[0102] In this embodiment, the nucleotide sequence encodes a GLA fusion protein.

[0103] In embodiments, the GLA fusion protein comprises a signal peptide from a secretory protein other than GLA, operably linked to the amino acid sequence of mature human GLA or a conservedly substituted variant thereof. In embodiments, the signal peptide comprises or is essentially derived from SEQ ID NO: 15 or a conservedly substituted variant thereof. In embodiments, a conservedly substituted variant of SEQ ID NO: 15 has three or fewer, two or fewer, or one or fewer conservative substitutions. In embodiments, the coding sequence of the signal peptide (SEQ ID NO: 15) is the wild-type coding sequence of human HSPG2. In embodiments, the coding sequence of the HSPG2 signal peptide is a nucleotide sequence that is codon-optimized for expression in mammalian cells (e.g., SEQ ID NO: 16) or substantially identical to SEQ ID NO: 16, e.g., at least 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 16. In embodiments, the fusion protein comprises SEQ ID NO: 5. In embodiments, the nucleotide sequence includes SEQ ID NO: 6, or a nucleotide sequence that is substantially identical to SEQ ID NO: 6, for example, at least 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 6.

[0104] In embodiments, the GLA fusion protein comprises a GLA wild-type or mutant amino acid sequence operably linked to an amino acid sequence encoding a non-GLA polypeptide. The non-GLA polypeptide may be any protein or protein domain that the fusion protein confers to a long half-life or other desired properties, such as albumin, IgG Fc, a constant domain from the IgG light chain, one, two, or three constant domains from the IgG heavy chain, a nanobody, transferrin, CTP (a 28-amino acid C-terminal peptide (CTP) of human chorionic gonadotropin (hCG) containing four O-glycans), XTEN, homoamino acid polymer (HAP), proline-alanine-serine (PAS), or any combination thereof. In embodiments, the GLA fusion protein comprises one of SEQ ID NOs: 7, SEQ ID NOs: 11, or SEQ ID NOs: 13. In embodiments, the nucleotide sequence encoding the GLA fusion protein comprises one of SEQ ID NOs: 9, 12, or 14.

[0105] In embodiments, the isolated polynucleotide further comprises a transcription unit comprising a Kozak translation sequence located immediately upstream of the ATG start codon of the polypeptide coding sequence. In embodiments, the Kozak translation sequence essentially consists of or comprises a nucleotide sequence substantially identical to nucleotides 2094-2099 of SEQ ID NO: 17 (referred to herein as SEQ ID NO: 19), for example, being at least 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 19. In embodiments, the transcription unit further comprises a polyA sequence essentially consisting of or comprising a nucleotide sequence substantially identical to nucleotides 2163-2684 of SEQ ID NO: 17 (referred to herein as SEQ ID NO: 20), for example, being at least 95%, 96%, 97%, 98%, 99%, or more identical to SEQ ID NO: 20. In embodiments, the isolated polynucleotide comprises SEQ ID NO: 17 having one of SEQ ID NOs: 3, 4, 6, 9, 12, or 14 inserted between nucleotides 2100 and 2101 of SEQ ID NO: 17. In embodiments, the isolated polynucleotide comprises two, three, or more transcription units. In embodiments, the transcription units are located between pairs of inverted terminal repeat sequences, for example, 5'ITR and 3'ITR.

[0106] [Recombinant RPE cells] The isolated polynucleotides described above are useful for generating retinal pigment epithelial (RPE) cells, or cells derived from RPE cells recombinant to express and secrete GLA protein. In embodiments, the recombinant (engineered) (e.g., recombinant) RPE cells include a nucleotide sequence that is substantially identical to one or more of SEQ ID NOs: 3, 4, 6, 9, 12, and 14, or any of these specific sequences, for example, having at least 95%, 96%, 97%, 98%, 99%, or more identity with respect to a specific sequence. In embodiments, the recombinant RPE cells produce a GLA-IgG fusion protein and include a first transcription unit including SEQ ID NO: 9 and a second transcription unit including SEQ ID NO: 10. In embodiments, the recombinant RPE cells include the transcription units described herein, which may be present in an extrachromosomal expression vector or incorporated into one or more chromosomal sites in the cell nucleus. In the embodiment, the recombinant cell contains two, three, four, or more copies of the transcription unit that are tandem-integrated into the same genomic region within the cell nucleus.

[0107] The recombinant RPE cells described herein may be derived from any of a variety of cell lines. Exemplary RPE cell lines include ARPE-19 cells, ARPE-19-SEAP-2-neo cells, RPE-J cells, and hTERTRPE-1 cells. In some embodiments, the recombinant cells are derived from the ARPE-19 (ATCC CRL®-2302) cell line. In some embodiments, the recombinant RPE (e.g., ARPE-19) cells are grown from a monoclonal cell line.

[0108] In embodiments, the recombinant cells described herein express biomarkers characteristic of RPE cells, such as naturally occurring RPE cells, e.g., antigens. In some embodiments, the biomarker (e.g., antigen) is a protein. Exemplary biomarkers include CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin. In embodiments, the recombinant cells express at least one of CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin. In embodiments, the recombinant cells express at least one of CRALBP and RPE-65.

[0109] For example, recombinant RPE cells for use in the devices, compositions, and methods described herein may be at various stages of the cell cycle, such as multiple recombinant cells contained in or encapsulated within a hydrogel capsule. In some embodiments, at least one of the multiple recombinant cells is undergoing cell division. Cell division can be measured using any known method in the art, such as those described, for example, 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 in whole by reference). In embodiments, at least 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, as determined, for example, by a 5-ethynyl-2'-deoxyuridine (EdU) assay or a 5-bromo-2'-deoxyuridine (BrdU) assay. In some embodiments, cell proliferation is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation)) or flow cytometry. In some embodiments, none of the recombinant cells in a group of recombinant cells are undergoing cell division and are in a quiescent state. In embodiments, less than 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division and are evaluated by 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.

[0110] In some embodiments, at least 1, 2, 3, 4, 5, 10, 20, 40, or 80% of a plurality of recombinant RPE cells 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 the Assay Guidance Manual (Sittapalam, G. Set al., eds.). For example, cell viability can be measured or quantified by ATP assays, 5-ethynyl-2'-deoxyuridine (EdU) assays, or 5-bromo-2'-deoxyuridine (BrdU) assays. 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 embodiments, at least 1, 2, 3, 4, 5, 10, 20, 40, or 80% of a plurality of RPE cells are viable, as determined by, for example, an ATP assay, a 5-ethinyl-2'-deoxyuridine (EdU) assay, a 5-bromo-2'-deoxyuridine (BrdU) assay, microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation)) or flow cytometry.

[0111] Any of the parameters described herein can be evaluated using standard techniques known to a person skilled in the art, such as histological examination, microscopic examination, and various functional assays.

[0112] [Measurement of GLA activity] The activity of GLA secreted by recombinant cells or devices described herein can be measured by direct or indirect GLA activity assays known in the art.

[0113] For example, GLA activity can be directly measured in blood leukocytes from a subject by measuring the enzyme activity of the lysate obtained by lysing cells and adding an enzyme substrate such as 4-methylumbelliferal α-D-galactosamine and / or N-acetylgalactosamine (see U.S. Patent No. 6,274,597). Immunoassays for measuring GLA activity and protein to determine the concentration of α-galactosidase in blood and plasma are described in Fuller et al., Clin Chem. 2004;50(11):1979-85. In embodiments, GLA activity is measured in culture medium or tissue samples (e.g., homogenates of plasma, liver, kidney, or cardiac tissue samples separated from blood) using the enzyme assays described in the following examples.

[0114] Indirect assessment of GLA activity is based on the measurement of levels of Gb3 and / or lysoGb3 (and optionally its six related analogues) in surrogate biomarkers, such as plasma and / or urine samples taken from a subject, or in biopsies of the tissue of interest, such as liver, kidney, or heart. Gb3 and lysoGb3 levels can be measured using the assays described in the examples herein or any assay known in the art. For example, a method for measuring Gb3 levels in plasma and urine from a person who has developed Fabry disease is described, for example, in Boscaro et al., Rapid Commun Mass Spectrom. 2002;16(16):1507-14. In this method, the analysis is performed using flow injection analysis-electrospray ionization-tandem mass spectrometry (FIA-ESI-MS / MS). Accumulation of Gb3 in skin biopsies obtained using a "punch" device can be detected using immunoelectron microscopy as described in Kanekura et al., Br J Dermatol. 2005, 153(3):544-8. Various biopsy techniques and assays for detecting Gb3 and other surrogate biomarkers are described in U.S. Patent Application Publication No. 2010 / 0113517. Other plasma surrogate biomarkers for GLA activity and / or Fabry disease progression (e.g., various inflammatory and cardiac remodeling biomarkers) are described in Yogasundaram, H. et al., J Am Heart Assoc. 2018;7:e009098.

[0115] [device] The recombinant RPE cells or a plurality of such cells described herein may be incorporated into an implantable device for use in providing GLA protein to a target, for example, a patient with Fabry disease.

[0116] Exemplary implantable devices include materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials, and combinations thereof. The device (e.g., a particle) may have any configuration and shape suitable for supporting the viability and productivity of encapsulated cells after implantation at the intended target site. In some embodiments, the device is a hydrogel capsule, e.g., a millicapsule or microcapsule (e.g., a hydrogel millicapsule or hydrogel microcapsule). The device (e.g., a capsule, a particle) may contain (and be configured to selectively release) one or more exogenous agents not expressed by recombinant RPE cells, such as nucleic acids (e.g., RNA or DNA molecules), proteins (e.g., hormones, enzymes (e.g., glucose oxidase, kinase, phosphatase, oxygenase, hydrogenase, reductase), antibodies, antibody fragments, antigens, or epitopes), small molecules, lipids, drugs, vaccines, or any derivatives thereof, active or inactive fragments of small molecules, proteins, or polypeptides. In some embodiments, the device includes at least one means for mitigating foreign body reaction (FBR) (for example, for mitigating FBR when the device is implanted in or on a subject).

[0117] The devices described herein may be provided as preparations or compositions (i.e., device preparations or device compositions) for implantation or administration to a subject. In some embodiments, the device preparation or device composition comprises at least 2, 4, 8, 16, 32, 64, or more devices, wherein at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% of the devices in the preparation or composition have the characteristics described herein (e.g., average capsule diameter or number of cells in a cell-containing compartment).

[0118] Devices, device preparations, or device compositions may be configured for implantation, transplantation, or placement inside or on any part or section of the body. In some embodiments, implantable devices or device preparations are configured for implantation into the peritoneal cavity (e.g., the omental bursa or lesser sac, also known as the omental bursa or bursalis omentum). Devices, device preparations, or device compositions may be implanted into the peritoneal cavity (e.g., the reticular bursa, e.g., the omental sac) or placed on the surface within the peritoneal cavity (e.g., the reticular bursa, e.g., the omental sac) by injection or catheterization. Further discussion of implantation or placement of devices, device preparations, or device compositions into the reticular bursa is presented in M. Pellicciaro et al. (2017) CellR4 5(3):e2410.

[0119] In some embodiments, the implantable device includes at least one cell-containing compartment containing multiple living cells encapsulated by a polymer composition. In some embodiments, the device includes two, three, four, or more cell-containing compartments. Each cell-containing compartment contains multiple living cells, and cells in at least one of these compartments may express and secrete GLA proteins when the device is implanted in a subject.

[0120] In some embodiments, the polymer composition within the cell-containing compartment comprises a polysaccharide or other hydrogel-forming polymer (e.g., alginate, hyaluronic acid, or chondroitin). In some embodiments, the polymer is an alginate, which is a polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). In some embodiments, the alginate is either (ii) a low molecular weight (e.g., less than approximately 75 kDa) with a G:M ratio ≥ 1.5, (ii) a medium molecular weight alginate, e.g., with an approximately molecular weight of 75-150 kDa and a G:M ratio ≥ 1.5, (iv) a high molecular weight alginate, e.g., with an approximately MW of 150-250 kDa and a G:M ratio ≥ 1.5, or (iv) a blend of two or more of these alginates.

[0121] In some embodiments, the cell-containing compartment further comprises at least one cell-binding substance (CBS), e.g., a cell-binding peptide (CBP) or a cell-binding polypeptide (CBPP). In some embodiments, the CBS comprises a CBP ("CBP-polymer") covalently attached to a polymer molecule in the polymer composition via a linker. In embodiments, the polymer in the CBP-polymer is a polysaccharide (e.g., alginate) or another hydrogel-forming polymer. Various cell-binding peptides for use in the devices of this disclosure are described herein. In some embodiments, the cell-binding peptide has a length of 25 amino acids or less (e.g., 20, 15, 10, or fewer) and includes a cell-binding sequence of a ligand for a cell-adhering molecule (CAM). In some embodiments, the cell-binding peptide is essentially derived from the cell-binding sequences shown in Table 1 herein. In some embodiments, the cell-binding sequence is RGD (SEQ ID NO: 28) or RGSP (SEQ ID NO: 49). In some embodiments, the amino terminus of the cell-binding peptide is covalently attached to the polymer via an amino acid linker. In one embodiment, this amino acid linker essentially consists of 1 to 3 glycine residues. In another embodiment, this cell-binding peptide essentially consists of RGD (SEQ ID NO: 28) or RGSP (SEQ ID NO: 49), and this linker essentially consists of a single glycine residue.

[0122] In embodiments, each CBP polymer present in the first compartment has a cell-binding peptide density (% nitrogen, as determined by combustion analysis as described in the examples herein) of at least 0.05%, 0.1%, 0.2%, or 0.3%, but less than 4%, 3%, 2%, or 1%. In embodiments, 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: 43) or GRGDSP (SEQ ID NO: 44)) as determined by a quantitative peptide conjugation assay (e.g., the assay described herein). In one embodiment, the CBP is RGSP (SEQ ID NO: 49), the linker is G, and the polymer is an alginate with a molecular weight of 75 kDa to 150 kDa and a G:M ratio of 1.5 or higher. In one embodiment, the cell-containing compartment also includes an unmodified hydrogel-forming polymer that is identical to or different from the polymer in the CBP-polymer. In one embodiment, the polymers in the CBP-polymer and the unmodified polymer are alginates having a molecular weight of 75 kDa to 150 kDa and a G:M ratio of 1.5 or higher.

[0123] In one embodiment, the quantitative peptide conjugation assay includes subjecting a sample of the CBP-polymer to acid hydrolysis to generate individual amino acids from the conjugated peptide (and any residual non-conjugated peptides in the CBP-polymer), quantifying these individual amino acids, averaging the molar concentrations of each amino acid, and calculating the total peptide concentration in the sample. In one embodiment, the quantitative peptide conjugation assay is carried out substantially in the same manner as the process described in the following examples of this specification. In one embodiment, the quantitative peptide conjugation assay also includes subtracting the concentration of any residual non-conjugated peptides in the sample from the total peptide concentration. The concentration of non-conjugated peptides in the CBP-polymer composition may be determined using any suitable assay known in the art, for example, by LC-MS as described in the following examples of this specification. Typically, the quantitative peptide conjugation assay is performed on a sample of a saline solution of the CBP-polymer used to prepare the device, but it may also be performed on a lyophilized sample of the CBP-polymer.

[0124] In some embodiments, the device further includes at least one means for reducing foreign body reaction (FBR) (for example, for reducing FBR when the device is implanted in or on a subject). Various means for reducing the FBR of a device are described herein, but any biological, chemical or physical element that can reduce FBR to the device compared to a reference device is considered herein.

[0125] For example, means for reducing FBR in the devices disclosed herein may include surrounding cells with a semipermeable biocompatible membrane having a pore size selected to allow oxygen and other molecules important for cell survival and function to move through the semipermeable membrane, but to prevent immune cells from passing through the pores. In one embodiment, the semipermeable membrane has a molecular weight cutoff of less than 1000 kD, or 50-700 kD, 70-300 kD, 70-150 kD, or 70-130 kD.

[0126] Another FBR mitigation method involves surrounding a cell-containing compartment with a barrier compartment formed from a cell-free biocompatible material (e.g., core-shell microcapsules as described in Ma, M et al., Adv. Healthc Mater., 2(5):667-672 (2012)). Such barrier compartments may be used with or without semipermeable membrane means. FBR mitigation methods may include placing anti-inflammatory agents released from the implanted device on or within the device to inhibit FBR, as described, for example, in U.S. Patent No. 9,867,781. Other FBR mitigation methods utilize CSF-1R inhibitors placed on or encapsulated within the device, as described in International Publication Nos. 2017 / 176792 and 2017 / 176804. Other FBR mitigation methods involve configuring the device in a spherical shape with a diameter greater than 1 mm, as described in Veiseh, O., et al., Nature Materials 14:643-652 (2015). In some embodiments, means for mitigating FBR include placing an anti-fibrous compound on the outer surface of the device and / or within a barrier compartment surrounding the cell-containing compartment. Exemplary anti-fibrous compounds include compounds of formula (I) as described below herein. In some embodiments, the device may include a combination of two or more of the above FBR mitigation means.

[0127] In some embodiments, the device has two hydrogel compartments, in which an inner cell-containing compartment is completely surrounded by a second outer (e.g., barrier) compartment. In some embodiments, the inner boundary of the second compartment forms an interface with the outer boundary of the first compartment, as shown, for example, in Figure 9. In such embodiments, the thickness of the second (outer) compartment means the average distance between the outer boundary of the second compartment and the interface between these two compartments. In some embodiments, the thickness of the outer compartment is greater than about 10 nanometers (nm), preferably 100 nm or more, and may be on the order of 1 millimeter (mm). For example, the thickness of the outer compartment in the hydrogel capsule device described herein may be 10 nm to 1 mm, 100 nm to 1 mm, 500 nm to 1 mm, 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 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 of the outer compartment is about 50 μm to about 100 μm.

[0128] In some embodiments, one or more compartments in the device contain an anti-fibrous polymer, for example, an anti-fibrous compound of formula (I) covalently attached to a polymer that is identical to or different from the polymer in the CBP polymer. In some embodiments, some or all monomers in this anti-fibrous polymer are modified with the same compound of formula (I). In some embodiments, some or all monomers in this anti-fibrous polymer are modified with various compounds of formula (I). In some embodiments, the device is a two-compartment hydrogel capsule, and this anti-fibrous polymer is present only in the outer barrier compartment, including the outer surface.

[0129] One or more compartments in the device may contain an unmodified polymer that is identical or different from the polymer in the CBP polymer and the polymer in any anti-fibrous polymer present in the device. In some embodiments, the first compartment, the second compartment, or all compartments in the device contain an unmodified polymer. In some embodiments, the unmodified polymer is an unmodified alginate. In some embodiments, the unmodified alginate has a molecular weight of 150 kDa to 250 kDa and a G:M ratio of ≥1.5.

[0130] In some embodiments, the anti-fibrous polymer comprises an alginate chemically modified with the compound of formula (I). The alginate in this anti-fibrous polymer may be identical to or different from any unmodified alginate present in the device. In some embodiments, the compound of formula (I) (e.g., compound 101 in Table 3) is covalently bonded to an alginate (e.g., an alginate having an approximate MW < 75 kDa and a G:M ratio ≥ 1.5) at a conjugation density of at least 2.0% and less than 9.0% nitrogen, or 2.0% to 5% nitrogen, 3.0% to 8.0% nitrogen, 5% to 8.0% nitrogen, 4.0% to 7.0% nitrogen, or about 6.0% to about 7.0% nitrogen or about 6.8% nitrogen, as determined by combustion analysis of nitrogen percentages as described in the following examples. In the embodiment, the amount of compound 101 results in an increase in %N (compared to the 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 is determined by combustion analysis and corresponds to the amount of compound 101 in the modified alginate.

[0131] In other embodiments, the density (e.g., concentration) of the compound of formula (I) (e.g., compound 101) in the antifibrillating alginate is defined as w / w%, for example, as the weight of the amine / % of the amount of antifibrillating alginate in solution (e.g., physiological saline) as determined by a suitable quantitative amine conjugation assay (e.g., the assay described herein), and in certain embodiments, the density of the compound of formula (I) (e.g., compound 101) is about 1.0 w / w% to about 3.0 w / w%, about 1.3 w / w% to about 2.5 w / w%, or about 1.5 w / w% to 2.2 w / w%. In one embodiment, the quantitative amine conjugation assay includes subjecting a sample of a chemically modified polymer (e.g., alginate modified with the compound of formula (I), e.g., CM-LMW-Alg-101) to acidic hydrolysis to produce free amines, and quantifying the total free amines in the sample. In one embodiment, the quantitative amine conjugation assay also includes subtracting the concentration of the unconjugated amine (e.g., the compound of formula (I)) in the non-hydrolyzed sample from the total amine concentration. The quantitative amine conjugation assay is typically performed on a sample of saline solution of the chemically modified alginate used to prepare the device, but may also be performed on a lyophilized sample of the chemically modified alginate. In one embodiment, the quantitative amine conjugation assay is performed substantially in the same manner as the process described in Example 9 of this specification. In one embodiment, the compound of formula (I) is compound 101 shown in Table 3.

[0132] Alginates in anti-fibrous polymers can be chemically modified with the compound of formula (I) using any suitable method known in the art. For example, the alginate carboxylic acid moiety can be activated for binding to one or more amine-functionalized compounds to obtain an alginate modified with the compound of formula (I). The alginate polymer can be dissolved in water (30 mL per gram of polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 equivalents) and N-methylmorpholine (1 equivalent). A solution of the compound of formula (I) in acetonitrile (0.3 M) can be added to this mixture. The reaction mixture can be heated to 55°C for, for example, 16 hours, then cooled to room temperature and gradually concentrated by rotary evaporation. The residue can then be dissolved in, for example, water. The mixture can then be filtered, for example, through a bed of cyano-modified silica gel (Silicycle), and the filtered cake can be washed with water. Next, the obtained solution can be dialyzed against water over 24 hours (using a 10,000 MWCO membrane), for example, by changing the water twice. The obtained solution can then be concentrated, for example, by freeze-drying, to obtain the desired chemically modified alginate.

[0133] [Compounds of formula (I)] In some embodiments, the device described herein is based on formula (I): [ka] The compound or a pharmaceutically acceptable salt thereof comprises, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-,-N(R C )C(O)-, -C(O)N(R C )-,-N(R C )C(O)(C1~C6-alkylene)-,-N(R C )C(O)(C1~C6-alkenylene)-,-N(R C )N(R D)-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F ) y -, -Si(OR A )2-, -Si(R G )(OR A )-, -B(OR A )-, or a metal, each of which is optionally bonded to a linking group (e.g., a linking group described herein) and is optionally substituted with one or more R 1 ; L 1 and L 3 each is independently a bond, alkyl, or heteroalkyl, where each of the alkyl and heteroalkyl is optionally substituted with one or more R 2 ; L 2 is a bond; M is absent or is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each optionally substituted with one or more R 3 ; P is absent or is cycloalkyl, heterocyclyl, or heteroaryl, each optionally substituted with one or more R 4 ; Z is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -OR A , -C(O)R A , -C(O)OR A , -C(O)N(R C )(R D ), -N(R C )C(O)R A, cycloalkyl, heterocyclyl, aryl, or heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl contains one or more R 5 It is replaced by optional selection; R A , R B , R C , R D , R E , R F , and R G Each of these is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azide, cycloalkyl, heterocyclyl, aryl, or heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is one or more R 6 It is replaced by optional selection; or R C and R D These, together with the nitrogen atom to which they are bonded, form one or more R 6 This forms a ring (e.g., a 5- to 7-membered ring) which is substituted by arbitrary selection; R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 Each of these can independently be alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, or -OR. A1 , -C(O)OR A1 , -C(O)R B1 -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 ,-C(O)N(R C1 ), SR E1 , S(O) x R E1 , -OS(O) x R E1 , -N(R C1 )S(O) x RE1 , -S(O) x N(R C1 )(R D1 ), -P(R F1 ) y , cycloalkyl, heterocyclyl, aryl, heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl contains one or more R 7 It is replaced by optional selection; R A1 , R B1 , R C1 , R D1 , R E1 , and R F1 Each of them is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is one or more R 7 It is replaced by optional selection; Each R 7 These are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is either 1 or 2; y is 2, 3, or 4.

[0134] In some embodiments, the compound of formula (I) is formula (Ia): [ka] A compound or salt thereof, in which, 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(RD )-, -NCN-, -N(R C )C(O)(C1~C6-alkylene)-,-N(R C )C(O)(C1~C6-alkenylene)-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-,-P(R F ) y -, -Si(OR A )2-,-Si(R G )(OR A )-, -B(OR A )-, or a metal, each of which is optionally bonded to a bonding group (e.g., a bonding group as described herein), and one or more R 1 It is replaced by optional selection; L 1 and L 3 Each of these is independently a bond, alkyl, or heteroalkyl, where each alkyl and heteroalkyl is one or more R 2 It is replaced by optional selection; L 2 is a combination; M is either absent or each has one or more R 3 The alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl elements are optionally substituted; P is one or more R 4 It is a heteroaryl that has been optionally substituted; Z is each one or more R 5 The alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl elements are optionally substituted; R A , R B , R C , R D , R E , R F, and R G Each of these is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azide, cycloalkyl, heterocyclyl, aryl, or heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is one or more R 6 It is replaced by optional selection; or R C and R D These, together with the nitrogen atom to which they are bonded, form one or more R 6 This forms a ring (e.g., a 5- to 7-membered ring) which is substituted by arbitrary selection; R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 Each of these can independently be alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, or -OR. A1 , -C(O)OR A1 , -C(O)R B1 -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 ,-C(O)N(R C1 ), SR E1 , S(O) x R E1 , -OS(O) x R E1 , -N(R C1 )S(O) x R E1 , -S(O) x N(R C1 )(R D1 ), -P(R F1 ) y , cycloalkyl, heterocyclyl, aryl, heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl contains one or more R 7 It is replaced by optional selection; R A1 , R B1 , R C1 , R D1 , R E1 , and R F1 Each of them is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is one or more R 7 It is replaced by optional selection; Each R 7 These are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is either 1 or 2; y is 2, 3, or 4.

[0135] In some embodiments, with respect to formula (I) or (Ia), A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )C(O)-, -N(R C )C(O)(C1~C6-alkylene)-,-N(R C )C(O)(C2~C6-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)(C1~C6-alkylene)-, or -N(R C )C(O)(C2~C6-alkenylene)-. In some embodiments, A is -N(R C )-. In some embodiments, A is -N(R C )- and R C and R D A is independently hydrogen or alkyl. In some embodiments, A is -NH-. In some embodiments, A is -N(R C )C(O)(C1~C6-alkylene)-, where alkylene is R 1 It is replaced by -N(R C )C(O)(C1~C6-alkylene)-, R 1 is alkyl (e.g., methyl). In some embodiments, A is -NHC(O)CH(CH3)2-. In some embodiments, A is -N(R C )C(O)(methylene)-, R 1 A is alkyl (e.g., methyl). In some embodiments, A is -NHC(O)CH(CH3)-. In some embodiments, A is -NHC(O)C(CH3)-.

[0136] In some embodiments, with respect to formula (I) or (Ia), L 1 L is a bond, alkyl, or heteroalkyl. In some embodiments, L 1 is a bond or alkyl. In some embodiments, L 1 L is a bond. In some embodiments, L 1 is alkyl. In some embodiments, L 1 L is a C1-C6 alkyl group. In some embodiments, L 1 is -CH2-, -CH(CH3)-, -CH2CH2CH2, or -CH2CH2-. In some embodiments, L 1It is either -CH2- or -CH2CH2-.

[0137] In some embodiments, with respect to formula (I) or (Ia), L 3 L is a bond, alkyl, or heteroalkyl. In some embodiments, L 3 L is a bond. In some embodiments, L 3 is alkyl. In some embodiments, L 3 is C1~C 12 It is alkyl. In some embodiments, L 3 L is a C1-C6 alkyl group. In some embodiments, L 3 is -CH2-. In some embodiments, L 3 L is a heteroalkyl. In some embodiments, L 3 is one or more R 2 C1~C are replaced by arbitrary selection (for example, oxo). 12 It is heteroalkyl. In some embodiments, L 3 is one or more R 2 It is a C1-C6 heteroalkyl that is optionally substituted with (for example, oxo). In some embodiments, L 3 is -C(O)OCH2-, -CH2(OCH2CH2)2-, -CH2(OCH2CH2)3-, CH2CH2O-, or -CH2O-. In some embodiments, L 3 It is -CH2O-.

[0138] In some embodiments, for formula (I) or (Ia), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, M is heteroalkyl, aryl, or heteroaryl. In some embodiments, M is absent. In some embodiments, M is alkyl (e.g., C1-C6 alkyl). In some embodiments, M is -CH2-. In some embodiments, M is heteroalkyl (e.g., C1-C6 heteroalkyl). In some embodiments, M is (-OCH2CH2-)z, where z is an integer selected from 1 to 10. In some embodiments, z is an integer selected from 1 to 5. In some embodiments, M is -OCH2CH2-, (-OCH2CH2-)2, (-OCH2CH2-)3, (-OCH2CH2-)4, or (-OCH2CH2-)5. In some embodiments, M is -OCH2CH2-, (-OCH2CH2-)2, (-OCH2CH2-)3, or (-OCH2CH2-)4. In some embodiments, M is (-OCH2CH2-)3. In some embodiments, M is aryl. In some embodiments, M is phenyl. In some embodiments, M is unsubstituted phenyl. In some embodiments, M is [ka] In some embodiments, M is R 7 (For example, one R 7 It is a phenyl substituted with ). In some embodiments, M is [ka] In some embodiments, R 7 This is CF3.

[0139] In some embodiments, for formula (I) or (Ia), P is absent, a heterocyclyl, or a heteroaryl. In some embodiments, P is absent. In some embodiments, for formulas (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 five-membered heteroaryl. In some embodiments, P is a five-membered nitrogen-containing heteroaryl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, pyrrolyl, oxazolyl, or thiazolyl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, or pyrrolyl. In some embodiments, P is imidazolyl. In some embodiments, P is [ka] In some embodiments, P is triazolyl. In some embodiments, P is 1,2,3-triazolyl. In some embodiments, P is [ka] That is the case.

[0140] In some embodiments, P is a heterocyclyl. In some embodiments, P is a 5-membered heterocyclyl or a 6-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some embodiments, P is [ka] That is the case.

[0141] In some embodiments, P is thiomorpholinyl-1,1-dioxydyl. In some embodiments, P is [ka] That is the case.

[0142] In some embodiments, for formula (I) or (Ia), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, Z is a heterocyclyl. In some embodiments, Z is a monocyclic or bicyclic heterocyclyl. In some embodiments, Z is an oxygen-containing heterocyclyl. In some embodiments, Z is a 4-membered heterocyclyl, a 5-membered heterocyclyl, or a 6-membered heterocyclyl. In some embodiments, Z is a 6-membered heterocyclyl. In some embodiments, Z is a 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is [ka] In some embodiments, Z is a four-membered oxygen-containing heterocycline. In some embodiments, Z is [ka] That is the case.

[0143] In some embodiments, Z is a bicyclic oxygen-containing heterocycline. In some embodiments, Z is phthalic anhydride. In some embodiments, Z is a sulfur-containing heterocycline. In some embodiments, Z is a six-membered sulfur-containing heterocycline. In some embodiments, Z is a six-membered heterocycline 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 heterocycline. In some embodiments, Z is a 6-membered nitrogen-containing heterocycline. In some embodiments, Z is [ka] That is the case.

[0144] In some embodiments, Z is a bicyclic heterocycline. In some embodiments, Z is one or more R 5 It is a bicyclic nitrogen-containing heterocycline that is optionally substituted with . In some embodiments, Z is 2-oxa-7-azaspiro[3.5]nonanyl. In some embodiments, Z is [ka] In some embodiments, Z is 1-oxa-3,8-diazaspiro[4.5]decan-2-one. In some embodiments, Z is [ka] That is the case.

[0145] In some embodiments, Z is an aryl compound in formula (I) or (Ia). In some embodiments, Z is a monocyclic aryl compound. In some embodiments, Z is a phenyl compound. In some embodiments, Z is a monosubstituted phenyl compound (e.g., one R compound). 5 (by) In some embodiments, Z is a monosubstituted phenyl, where one R 5 is a nitrogen-containing group. In some embodiments, Z is a monosubstituted phenyl, where one R 5 is NH2. In some embodiments, Z is a monosubstituted phenyl, where one R 5 is an oxygen-containing group. In some embodiments, Z is a monosubstituted phenyl, where one R 5 is an oxygen-containing heteroalkyl. In some embodiments, Z is a monosubstituted phenyl, where one R 5 is OCH3. In some embodiments, Z is a monosubstituted phenyl, where one R 5 It is in the ortho position. In some embodiments, Z is a monosubstituted phenyl, where one R 5 It is in the meta position. In some embodiments, Z is a monosubstituted phenyl, where one R 5 It is in the para position.

[0146] In some embodiments, Z is alkyl in formula (I) or (Ia). In some embodiments, Z is C1-C 12 It is alkyl. In some embodiments, Z is C1-C 10 It is alkyl. In some embodiments, Z is C1-C8 alkyl. In some embodiments, Z is 1-5 R 5 It is a C1-C8 alkyl group substituted with R. In some embodiments, Z is one R 5 It is a C1-C8 alkyl group substituted with R. In some embodiments, Z is one R 5 These are C1-C8 alkyl groups substituted with R, where R 5These include alkyl, heteroalkyl, halogen, oxo, and -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 These are C1-C8 alkyl groups substituted with R, where R 5 is -OR A1 or -C(O)OR A1 In some embodiments, Z is one R 5 These are C1-C8 alkyl groups substituted with R, where R 5 is -OR A1 Alternatively, it is -C(O)OH. In some embodiments, Z is -CH3.

[0147] In some embodiments, Z is a heteroalkyl group in formula (I) or (Ia). In some embodiments, Z is C1-C 12 It is heteroalkyl. In some embodiments, Z is C1-C 10 It is a heteroalkyl group. In some embodiments, Z is a C1-C8 heteroalkyl group. In some embodiments, Z is a C1-C6 heteroalkyl group. In some embodiments, Z is a group of one or more R groups. 5 It is a nitrogen-containing heteroalkyl that is optionally substituted with R. In some embodiments, Z is 1 to 5 R 5 It is a nitrogen- and sulfur-containing heteroalkyl substituted with . In some embodiments, Z is N-methyl-2-(methylsulfonyl)ethane-1-aminyl.

[0148] In some embodiments, Z is -OR A or -C(O)OR A In some embodiments, Z is -OR A (For example, -OH or -OCH3). In some embodiments, Z is OCH3. In some embodiments, Z is -C(O)ORA (For example, -C(O)OH).

[0149] In some embodiments, Z is hydrogen.

[0150] In some embodiments, L 2 This is a bond between P and L 3 In some embodiments, L 2 is a bond, P is a heteroaryl, and L 3 is a bond, and Z is hydrogen. In some embodiments, P is a heteroaryl, and L 3 Z is a heteroalkyl group, and Z is an alkyl group.

[0151] In some embodiments, the compound of formula (I) is formula (Ib): [ka] A compound or salt thereof, wherein ring M 1 Each of these has 1 to 5 R 3 A cycloalkyl, heterocyclyl, aryl, or heteroaryl ring Z is optionally substituted; 1 This is 1 to 5 R 5 R is a cycloalkyl, heterocyclyl, aryl, or heteroaryl molecule that is optionally substituted; 2a , R 2b , R 2c , and R 2d Each of them independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R 2a and R 2b or R 2c and R 2d Each of them together forms an oxo group; X is absent, N(R) 10 )(R 11 ), O, or S; R CR is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl has 1 to 6 R 6 It is replaced by choice; R 3 , R 5 , and R 6 Each of these can independently be alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azide, oxo, or -OR. A1 , -C(O)OR A1 , -C(O)R B1 -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 ,-C(O)N(R C1 ), SR E1 , cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 10 and R 11 Each of these can independently be hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 , -C(O)R B1 -OC(O)R B1 ,-C(O)N(R C1 ), cycloalkyl, heterocyclyl, aryl, or heteroaryl; R A1 , R B1 , R C1 , R D1 , and R E1 Each of these is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, where each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl contains 1 to 6 R 7 It is replaced by arbitrary selection in each R 7m and n are 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] This refers to bonding to a bonding group or polymer as described herein. In some embodiments, each R 3 and R 5 Each of the alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl groups is optionally and independently substituted with a halogen, oxo, cyano, cycloalkyl, or heterocyclyl group.

[0152] In some embodiments, the compound of formula (Ib) is of formula (Ibi): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein the ring M 2 is one or more R 3 It is an aryl or heteroaryl that is optionally substituted in ring Z; 2 is a cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 2a , R 2b , R 2c , and R 2d Each of them is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b or R 2c and R 2d Each of them together forms an oxo group; X is absent, O, or S; each R 3 and R 5 These are independently alkyl, heteroalkyl, halogen, oxo, and -OR A1 , -C(O)OR A1 , or -C(O)R B1Here, each of the alkyl and heteroalkyl groups is optionally substituted with a halogen; or two R groups 5 They come together in the ring Z 2 It forms a condensed 5-6 member ring; each R A1 and R B1 m and n are independently hydrogen, alkyl, or heteroalkyl; m and n are independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; [ka] This refers to bonding to a binding group or polymer as described herein.

[0153] In some embodiments, the compound of formula (Ibi) is of formula (Ib-ii): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein the ring Z 2 R is a cycloalkyl, heterocyclyl, aryl, or heteroaryl compound; 2c and R 2d Each of them is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R 2d They combine to form an oxo group; each R 3 and R 5 These are independently alkyl, heteroalkyl, halogen, oxo, and -OR A1 , -C(O)OR A1 , or -C(O)R B1 Here, each of the alkyl and heteroalkyl groups is optionally substituted with a halogen; each R A1 and R B1 p is independently hydrogen, alkyl, or heteroalkyl; p and q are independently 0, 1, 2, 3, 4, 5, or 6; [ka] This refers to bonding to a binding group or polymer as described herein.

[0154] In some embodiments, the compound of formula (I) is of formula (Ic): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein the ring Z 2 R is a cycloalkyl, heterocyclyl, aryl, or heteroaryl compound; 2c and R 2d Each of them is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R 2d They combine to form an oxo group; each R 3 and R 5 These are independently alkyl, heteroalkyl, halogen, oxo, and -OR A1 , -C(O)OR A1 , or -C(O)R B1 Here, each of the alkyl and heteroalkyl groups is optionally substituted with a halogen; each R A1 and R B1 m is independently hydrogen, alkyl, or heteroalkyl; m is 1, 2, 3, 4, 5, or 6; p and q are independently 0, 1, 2, 3, 4, 5, or 6; [ka] This refers to bonding to a binding group or polymer as described herein.

[0155] In some embodiments, the compound of formula (I) is of formula (Id): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein the ring Z 2 is a cycloalkyl, heterocyclyl, aryl, or heteroaryl; X is absent, O, or S; R 2a, R 2b , R 2c , and R 2d Each of them is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b or R 2c and R 2d Each of them together forms an oxo group; each R 5 These are independently alkyl, heteroalkyl, halogen, oxo, and -OR A1 , -C(O)OR A1 , or -C(O)R B1 Here, each of the alkyl and heteroalkyl groups is optionally substituted with a halogen; each R A1 and R B1 m and n are independently hydrogen, alkyl, or heteroalkyl; m and n are independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; [ka] This refers to bonding to a binding group or polymer as described herein.

[0156] In some embodiments, the compound of formula (I) is of formula (Ie): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein the ring Z 2 is a cycloalkyl, heterocyclyl, aryl, or heteroaryl; X is absent, O, or S; R 2a , R 2b , R 2c , and R 2d Each of them is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b or R 2c and R 2d Each of them together forms an oxo group; each R 5 These are independently alkyl, heteroalkyl, halogen, oxo, and -OR A1, -C(O)OR A1 , or -C(O)R B1 and; each R A1 and R B1 m and n are independently hydrogen, alkyl, or heteroalkyl; m and n are independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; [ka] This refers to bonding to a binding group or polymer as described herein.

[0157] In some embodiments, the compound of formula (I) is of formula (If): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein M is one or more R 3 The alkyl group is optionally substituted with; ring P has one or more R 4 It is a heteroaryl that is optionally substituted; L 3 is one or more R 2 The alkyl or heteroalkyl group is optionally substituted with; Z is each one or more R groups. 5 The alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl elements are optionally substituted; R 2a and R 2b Each of them is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b They combine to form an oxo group; R 2 , R 3 , R 4 , and R 5 Each of these can independently be alkyl, heteroalkyl, halogen, oxo, or -OR. A1 , -C(O)OR A1 , or -C(O)R B1 and; each R A1 and R B1n is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; [ka] This refers to bonding to a binding group or polymer as described herein.

[0158] In some embodiments, the compound of formula (I) is of formula (II): [ka] A compound of or a pharmaceutically acceptable salt thereof, where M is a bond, alkyl, or aryl, where alkyl and aryl are one or more R 3 It is replaced by optional selection; L 3 is one or more R 2 Z is an alkyl or heteroalkyl group that is optionally substituted; Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or -OR A Here, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are defined as having one or more R 5 It is replaced by choice; R A is hydrogen; R 2a and R 2b Each of them is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b They combine to form an oxo group; R 2 , R 3 , and R 5 Each of these can independently be alkyl, heteroalkyl, halogen, oxo, or -OR. A1 , -C(O)OR A1 , or -C(O)R B1 and; each R A1 and R B1 n is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; [ka] This refers to bonding to a binding group or polymer as described herein.

[0159] In some embodiments, the compound of formula (II) is formula (II-a): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein L 3 Each of them has one or more R 2 Z is an alkyl or heteroalkyl group that is optionally substituted; Z is hydrogen, alkyl, heteroalkyl, or -OR A Here, alkyl and heteroalkyl are defined as having one or more R 5 It is replaced by choice; R 2a and R 2b Each of them is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b They combine to form an oxo group; R 2 , R 3 , and R 5 Each of these can independently be alkyl, heteroalkyl, halogen, oxo, or -OR. A1 , -C(O)OR A1 , or -C(O)R B1 And; R A is hydrogen; each R A1 and R B1 n is independently hydrogen, alkyl, or heteroalkyl; n is independently 1, 2, 3, 4, 5, or 6; [ka] This refers to bonding to a binding group or polymer as described herein.

[0160] In some embodiments, the compound of formula (I) is formula (III): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein Z 1 Each of these has 1 to 5 R 5 The alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl elements are optionally substituted; R 2a , R 2b , R 2c , and R 2d Each of them independently is 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 They combine to form an oxo group; R C R is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, where each alkyl, alkenyl, alkynyl, or heteroalkyl has 1 to 6 R 6 It is replaced by choice; R 3 , R 5 , and R 6 Each of these can independently be alkyl, heteroalkyl, halogen, oxo, or -OR. A1 , -C(O)OR A1 , or -C(O)R B1 and; each R A1 and R B1 m and n are independently hydrogen, alkyl, or heteroalkyl; m and n are independently 1, 2, 3, 4, 5, or 6; q is an integer between 0 and 25; [ka] This refers to bonding to a binding group or polymer as described herein.

[0161] In some embodiments, the compound of formula (III) is formula (III-a): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein the ring Z 2 Each of these has 1 to 5 R 5 A cycloalkyl, heterocyclyl, aryl, or heteroaryl that is optionally substituted; R 2a , R 2b , R 2c , and R 2d Each of them independently is hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b or R 2c and R 2d They combine to form an oxo group; R 3 and R 5 Each of these can independently be alkyl, heteroalkyl, halogen, oxo, or -OR. A1 , -C(O)OR A1 , or -C(O)R B1 and; each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m and n are independently 1, 2, 3, 4, 5, or 6; o and p are independently 0, 1, 2, 3, 4, or 5; q is an integer between 0 and 25; [ka] This refers to bonding to a binding group or polymer as described herein.

[0162] In some embodiments, the compound of formula (III-a) is formula (III-b): [ka] A compound of or a pharmaceutically acceptable salt thereof, wherein the ring Z 2 Each of these has 1 to 5 R 5 A cycloalkyl, heterocyclyl, aryl, or heteroaryl that is optionally substituted; R 2a , R 2b , R 2c, and R 2d Each of them independently is hydrogen, alkyl, heteroalkyl, halo; or R 2a and R 2b or R 2c and R 2d They combine to form an oxo group; R 3 and R 5 Each of these can independently be alkyl, heteroalkyl, halogen, oxo, or -OR. A1 , -C(O)OR A1 , or -C(O)R B1 and; each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m and n are independently 1, 2, 3, 4, 5, or 6; o and p are independently 0, 1, 2, 3, 4, or 5; q is an integer between 0 and 25; [ka] This refers to bonding to a binding group or polymer as described herein.

[0163] In some embodiments, the compound of formula (III-a) is: [ka] A compound of or a pharmaceutically acceptable salt thereof, where X is C(R')(R”), N(R'), or S(O) x R' and R'' are each independently hydrogen, alkyl, halogen, or cycloalkyl; R 2a , R 2b , R 2c , and R 2d Each of them is independently hydrogen, alkyl, heteroalkyl, or halo; or R 2a and R 2b or R 2c and R 2d They combine to form an oxo group; R 3 and R 5 Each of these can independently be alkyl, heteroalkyl, halogen, oxo, or -OR.A1 , -C(O)OR A1 , or -C(O)R B1 and; each R A1 and R B1 m and n are independently hydrogen, alkyl, or heteroalkyl; m and n are independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, or 5; q is an integer between 0 and 25; x is 0, 1, or 2; [ka] This refers to bonding to a binding group or polymer as described herein.

[0164] In some embodiments, the compound of formula (III-c) is formula (III-d): [ka] A compound of or a pharmaceutically acceptable salt thereof, where X is C(R')(R”), N(R'), or S(O) x R' and R'' are each independently hydrogen, alkyl, halogen, or cycloalkyl; R 2a , R 2b , R 2c , and R 2d Each of them is independently hydrogen, alkyl, heteroalkyl, or halo; or R 2a and R 2b or R 2c and R 2d They combine to form an oxo group; R 3 and R 5 Each of these can independently be alkyl, heteroalkyl, halogen, oxo, or -OR. A1 , -C(O)OR A1 , or -C(O)R B1 and; each R A1 and R B1 m and n are independently hydrogen, alkyl, or heteroalkyl; m and n are independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, or 5; q is an integer between 0 and 25; x is 0, 1, or 2; [ka] This refers to bonding to a binding group or polymer as described herein.

[0165] In some embodiments, the compound is a compound of formula (I). In some embodiments, L 2 This is a bond between P and L 3 It is independent and absent.

[0166] In some embodiments, the compound is the compound of formula (Ia). In some embodiments of formula (II-a), L 2 is a bond, P is a heteroaryl, and L 3 is a bond, and Z is hydrogen. In some embodiments, P is a heteroaryl, and L 3 In some embodiments, L 2 This is a bond between P and L 3 In some embodiments, L 2 is a bond, P is a heteroaryl, and L 3 is a bond, and Z is hydrogen. In some embodiments, P is a heteroaryl, and L 3 Z is a heteroalkyl group, and Z is an alkyl group.

[0167] In some embodiments, the compound is the compound of formula (Ib). In some embodiments, P is absent, and L 1 is -NHCH2, L 2 is a bond, M is an aryl (e.g., phenyl), and L 3 is -CH2O, and Z is a heterocycline (e.g., nitrogen-containing heterocycline, e.g., thiomorpholinyl-1,1-dioxide). In some embodiments, the compound of formula (Ib) is compound 116.

[0168] In some embodiments of formula (Ib), P is absent, and L 1 is -NHCH2, L 2 is a combination, M is absent, L 3 is a bond, and Z is a heterocyclyl (e.g., oxygen-containing heterocyclyl, e.g., tetrahydropyranil, tetrahydrofuranil, oxetanil, or oxyranil). In some embodiments, the compound of formula (Ib) is compound 105.

[0169] In some embodiments, the compound is a compound of formula (Ibi). In some embodiments of formula (Ibi), R 2a and R 2b Each of them is independently hydrogen or CH3, and R 2c and R 2d Each of them is independently a hydrogen atom, m is either 1 or 2, n is 1, X is O, p is 0, M 2 is one or more R 3 It is a phenyl that is optionally substituted with R 3 is -CF3, Z 2 is a heterocycline (e.g., oxygen-containing heterocycline, e.g., tetrahydropyranil, tetrahydrofuranil, oxetanil, or oxyranil). In some embodiments, the compound of formula (Ibi) is compound 100, compound 106, compound 107, compound 108, compound 109, or compound 111.

[0170] In some embodiments, the compound is the compound of formula (Ib-ii). In some embodiments of formula (Ib-ii), R 2a , R 2b , R 2c , and R 2d Each of them is independently a hydrogen, q is 0, p is 0, m is 1, Z 2 is a heterocycline (e.g., oxygen-containing heterocycline, e.g., tetrahydropyranil). In some embodiments, the compound of formula (Ib-ii) is compound 100.

[0171] In some embodiments, the compound is a compound of formula (Ic). In some embodiments of formula (Ic), R 2c and R 2d Each of them is independently a hydrogen atom, m is 1, p is 1, q is 0, R 5 is -CH3, and Z is a heterocycline (e.g., nitrogen-containing heterocycline, e.g., piperazinyl). In some embodiments, the compound of formula (Ic) is compound 113.

[0172] 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 Each of the elements is independently hydrogen, m is 1, n is 3, X is O, p is 0, and Z is a heterocyclyl (e.g., oxygen-containing heterocyclyl, e.g., tetrahydropyranil, tetrahydrofuranil, oxetanil, or oxyranil). In some embodiments, the compound of formula (Id) is compound 110 or compound 114.

[0173] In some embodiments, the compound is a compound of formula (If). In some embodiments of formula (If), R 2a and R 2b Each of them is independently hydrogen, n is 1, M is -CH2-, P is a nitrogen-containing heteroaryl (e.g., imidazolyl), and L 3 is -C(O)OCH2- and Z is CH3. In some embodiments, the compound of formula (If) is compound 115.

[0174] In some embodiments, the compound is the compound of formula (II-a). In some embodiments of formula (II-a), R 2a and R 2b Each of them is independently a hydrogen, n is 1, q is 0, L 3is -CH2(OCH2CH2)2 and Z is -OCH3. In some embodiments, the compound of formula (II-a) is compound 112.

[0175] In some embodiments of equation (II-a), R 2a and R 2b Each of them is independently a hydrogen, n is 1, L 3 is a bond or -CH2, and Z is hydrogen or -OH. In some embodiments, the compound of formula (II-a) is compound 103 or compound 104.

[0176] In some embodiments, the compound is the compound of formula (III). In some embodiments of formula (III), R 2a , R 2b , R 2c , and R 2d Each of them is independently a hydrogen atom, m is 1, n is 2, q is 3, p is 0, R C is hydrogen, Z 1 R 5 It is a heteroalkyl group optionally substituted with (for example, -N(CH3)(CH2CH2)S(O)2CH3). In some embodiments, the compound of formula (III) is compound 120.

[0177] In some embodiments, the compound is the compound of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c , and R 2d Each of them is independently hydrogen, m is 0, n is 2, q is 3, p is 0, Z 2 This is one R 5 The compound is an aryl (e.g., phenyl) substituted with (e.g., -NH2). In some embodiments, the compound of formula (III-b) is compound 102.

[0178] In some embodiments, the compound is the compound of formula (III-b). In some embodiments of formula (III-b), R 2a , R 2b , R 2c , and R 2d Each of them is independently a hydrogen atom, m is 1, n is 2, q is 3, p is 0, R C is hydrogen, Z 2 is a heterocycline (e.g., nitrogen-containing heterocycline, e.g., nitrogen-containing spiroheterocycline, e.g., 2-oxa-7-azaspiro[3.5]nonanyl). In some embodiments, the compound of formula (III-a) is compound 121.

[0179] In some embodiments, the compound is the compound of formula (III-d). In some embodiments of formula (III-d), R 2a , R 2b , R 2c , and R 2d Each of them is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O)2. In some embodiments of formula (III-d), R 2a and R 2b Each of them 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.

[0180] 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).

[0181] In some embodiments, the compound of formula (I) is not a compound disclosed in International Publication Brochure 2012 / 112982, International Publication Brochure 2012 / 167223, International Publication Brochure 2014 / 153126, International Publication Brochure 2016 / 019391, International Publication Brochure 2017 / 075630, U.S. Patent Application Publication 2012-0213708, U.S. Patent Application Publication 2016-0030359, or U.S. Patent Application Publication 2016-0030360.

[0182] In some embodiments, the compound of formula (I) comprises the compounds shown in Table 3 or pharmaceutically acceptable salts thereof. In some embodiments, the outer surface and / or one or more compartments within the device described herein comprises the compounds shown in Table 3 or pharmaceutically acceptable salts thereof.

[0183] [Table 3]

[0184] [Table 4]

[0185] [Table 5]

[0186] In some embodiments, the compound is a compound of formula (I) (e.g., formula (Ib), (Ic), (Id), (Ie), (If), (II), (II-a), (III), (III-a), (III-b), (III-c), or (III-d)) or a pharmaceutically acceptable salt thereof, and [ka] or selected from any pharmaceutically acceptable salt of the above compound.

[0187] In some embodiments, the devices described herein are [ka] It contains the compound, or a pharmaceutically acceptable salt of either compound.

[0188] In embodiments, the devices described herein include a compound of formula (I) (e.g., the compounds shown in Table 3) covalently bonded to an alginate polymer. In embodiments, the particles described herein include a compound of formula (I) (e.g., the compounds shown in Table 3, e.g., compound 101) covalently bonded to one or more guluronic acid and / or mannuronic acid monomers in an alginate polymer, for example, by an amide bond.

[0189] In some embodiments, the compound of formula (I) (e.g., compound 101 in Table 3) is covalently bonded to an alginate (e.g., an alginate having an approximate MW < 75 kDa and a G:M ratio ≥ 1.5) at a conjugation density of at least 2.0% and less than 9.0% nitrogen, or 2.0% to 5% nitrogen, 3.0% to 8.0% nitrogen, 5% to 8.0% nitrogen, 4.0% to 7.0% nitrogen, 5.0% to 7.0% nitrogen, or 6.0% to 7.0% nitrogen or about 6.8% nitrogen, as determined by combustion analysis of nitrogen percentage as described in the following examples.

[0190] [Treatment Method] Methods for preventing or treating a target Fabry disease by administering or transplanting a plurality of recombinant RPE cells capable of expressing and secreting the GLA protein described herein are described herein. In embodiments, the plurality of RPE cells are contained in an implantable device described herein. In some embodiments, the methods described herein directly or indirectly reduce or mitigate at least one symptom of Fabry disease, or prevent or delay the onset of Fabry disease. In embodiments, the method comprises administering (e.g., transplanting) an effective amount of a composition of a two-compartment alginate hydrogel capsule, comprising the recombinant RPE cells and cell-binding polymer described herein in the inner compartment, and a compound of formula (I), e.g., compound 101, on the outer surface of the capsule and optionally in the outer compartment.

[0191] [Examples of listed embodiments] 1. An isolated polynucleotide comprising a promoter operably ligated to a precursor GLA coding sequence, wherein the polynucleotide has the following characteristics: (a) The promoter essentially consists of a nucleotide sequence that is identical or substantially identical to Sequence ID No. 18; (b) The precursor GLA coding sequence is codon-optimized for expression in mammalian cells; (c) The precursor GLA coding sequence codes for a GLA fusion protein, and the GLA fusion protein has one or more of the following characteristics: (i) The GLA fusion protein contains a signal peptide from a non-GLA secretory protein (e.g., mammalian HSPG2 protein, e.g., human HSPG2) operably ligated to the N-terminus of a mature human GLA amino acid sequence; (ii) The GLA fusion protein comprises an amino acid sequence encoding a signal peptide (e.g., derived from GLA or HSPG2) operably linked to the N-terminus of a mature human GLA amino acid sequence, and an amino acid sequence encoding a non-GLA polypeptide operably linked to the C-terminus of the GLA amino acid sequence. A polynucleotide having at least one or more of the following.

[0192] 2. An isolated polynucleotide according to Embodiment 1, comprising features (a) and (c)(i); features (a) and (c)(ii); features (b) and (c)(i); features (b) and (c)(ii); or a combination of features selected from the group consisting of features (a), (b), (c)(i) or features (a), (b), (c)(ii).

[0193] 3. Multiple recombinant RPE cells capable of secreting GLA protein (e.g., human GLA protein or its variants), wherein each of the multiple cells contains an exogenous nucleotide sequence comprising a promoter operably linked to a precursor GLA coding sequence, and the recombinant RPE cells have the following characteristics: a) By choice, the promoter shall consist essentially of a nucleotide sequence that is identical or substantially identical to sequence number 18. b) The precursor GLA coding sequence is codon-optimized for expression in mammalian cells; c) The precursor GLA coding sequence encodes a GLA fusion protein, and the GLA fusion protein has one or more of the following characteristics: i) GLA fusion proteins contain a signal peptide from a non-GLA secretory protein (e.g., mammalian HSPG2 protein, e.g., human HSPG2) operably ligated to the N-terminus of a mature human GLA amino acid sequence; ii) The GLA fusion protein comprises an amino acid sequence encoding a signal peptide (e.g., derived from GLA or HSPG2) operably linked to the N-terminus of a mature human GLA amino acid sequence, and a non-GLA polypeptide operably linked to the C-terminus of the GLA amino acid sequence. Multiple recombinant RPE cells having at least one or more of the following:

[0194] 4. Multiple recombinant RPE cells according to Embodiment 3, wherein the exogenous nucleotide sequence includes features (a) and (c)(i); features (a) and (c)(ii); features (b) and (c)(i); features (b) and (c)(ii); features (a), (b), (c)(i) or a combination of features selected from the group consisting of features (a), (b), (c)(ii).

[0195] 5. Multiple recombinant RPE cells according to Embodiment 4, wherein the exogenous nucleotide sequence contains an extrachromosomal expression vector or is incorporated at least one position in the nuclear genome of the RPE cells.

[0196] 6. Multiple recombinant RPE cells according to any one of Embodiments 3 to 5, derived from ARPE-19 cells transfected with a transcription unit containing an exogenous nucleotide sequence.

[0197] 7. Multiple recombinant RPE cells according to Embodiment 6, provided as a polyclonal cell culture or as a monoclonal cell line.

[0198] 8. Isolated polynucleotide or multiple recombinant RPE cells according to any one of Embodiments 1 to 7, wherein the precursor GLA codon-optimized coding sequence is SEQ ID NO: 3 or SEQ ID NO: 4.

[0199] 9. The signal peptide is essentially a polynucleotide or a plurality of recombinant RPE cells as described in any one of Embodiments 1 to 8, derived from SEQ ID NO: 15.

[0200] 10. The GLA fusion protein is an isolated polynucleotide or multiple recombinant RPE cell according to any one of Embodiments 1 to 9, comprising SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 13.

[0201] 11. Isolated polynucleotide or recombinant RPE cells according to any one of Embodiments 1 to 9, comprising one or more of Sequence IDs 3, 4, 6, 9, 12, and 14.

[0202] 12. The following characteristics: a) Multiple recombinant RPE cells secrete GLA protein for at least 5 days, at least 10 days, at least 1 month, or at least 2 months, for example, in in vitro cell culture or when transplanted into a subject (for example, as evaluated by the reference method described herein); or b) Multiple recombinant RPE cells secrete at least 2, 3, 4, 5, or 10 times more GLA protein than multiple reference recombinant RPE cells transfected with wild-type human nucleotide sequences encoding precursor GLA. Multiple recombinant RPE cells according to any one of embodiments 3 to 11, having one or more of the above.

[0203] 13. An implantable device comprising at least one cell-containing compartment containing a plurality of recombinant RPE cells as described in any of embodiments 3 to 12, and at least one means for mitigating a foreign body reaction (FBR) when the device is implanted in a subject.

[0204] 14. The device according to Embodiment 13, wherein at least one cell-containing compartment comprises a polymer composition that encapsulates a plurality of recombinant RPE cells, and optionally comprises at least one cell-binding substance (CBS).

[0205] 15. The device according to Embodiment 13 or 14, wherein the cell-containing compartment comprises an alginate hydrogel and is surrounded by a barrier compartment, the barrier compartment comprising an alginate hydrogel and optionally comprising a compound of formula (I), for example, compound 101 of Table 3, disposed on the outer surface of the barrier compartment.

[0206] 16. The device according to either Embodiment 14 or 15, wherein the polymer composition comprises an alginate covalently modified with a peptide, the peptide essentially consisting of or comprising GRGDSP (SEQ ID NO: 44), GGGRGDSP (SEQ ID NO: 45), or GGGRGDSP (SEQ ID NO: 46).

[0207] 17. The barrier compartment contains a mixture of unmodified alginate and alginate modified with compound 101. (a) Unmodified alginates have a molecular weight of 150 kDa to 250 kDa and a G:M ratio of 1.5 or higher. (b) The alginate in the modified alginate has a molecular weight of less than 75 kDa and a G:M ratio of 1.5 or higher. The device according to Embodiment 15 or 16.

[0208] 18. The device of Embodiment 17, wherein the conjugation density of compound 101 in the modified alginate is determined by quantitative free amine analysis, as described in Example 10 below, for example, and the determined conjugation density is 1.0 w / w% to 3.0 w / w%, 1.3 w / w% to 2.8 w / w%, 1.3 w / w% to 2.6 w / w%, 1.5 w / w% to 2.4 w / w%, 1.5 w / w% to 2.2 w / w%, or 1.7 w / w% to 2.2 w / w%.

[0209] 19. Hydrogel capsule, (a) An inner cell-containing compartment comprising a plurality of recombinant cells according to any one of Embodiments 3 to 12, encapsulated in a first polymer composition comprising a first RGD-polymer, wherein optionally, the concentration of the plurality of cells is 40 million cells per ml of the first polymer composition; 40 million to 100 million cells per ml of the first polymer composition, 60 million to 100 million cells per ml, or 80 million to 100 million cells per ml; and (b) A barrier compartment surrounding a cell-containing compartment and comprising a second polymer composition, wherein the second polymer composition comprises a mixture of an unmodified alginate and an alginate covalently modified with at least one compound selected from the group consisting of compound 100, compound 101, compound 110, compound 112, compound 113, and compound 114 shown in Table 3, The hydrogel capsule contains a spherical shape with a diameter of 0.5 mm to 5 mm, and optionally, the average thickness of the barrier compartment is approximately 10 to 300 microns, approximately 20 to 150 microns, or approximately 40 to 75 microns.

[0210] 20. A hydrogel capsule according to Embodiment 19, comprising an amount effective for increasing the secretion of GLA protein, wherein the first RGD polymer essentially consists of an alginate covalently modified with an RGD peptide via a linker, the cell-containing compartment substantially free of any anti-fibrotic compounds, the barrier compartment substantially free of cells and RGD peptide, and optionally, the effective amount of the RGD polymer is an optimal amount.

[0211] 21. The hydrogel capsule according to Embodiment 19 or 20, wherein the RGD peptide essentially consists of the amino acid sequence of RGD (SEQ ID NO: 28) or RGSP (SEQ ID NO: 49), and the linker is a single glycine residue or a single beta-alanine residue attached to the N-terminus of the RGD peptide.

[0212] twenty two. (a) The polymer in the first RGD polymer is an alginate with a molecular weight of 150-250 kDa and a G:M ratio of 1.5 or higher, and optionally, the cell-containing compartment is formed from an alginate solution with a viscosity of approximately 90 cP and between approximately 230 cP and approximately 300, 350, or 400 cP, or between approximately 80 and approximately 120 cP; (b) The alginates in the covalently modified alginates within the barrier compartment have a molecular weight of <75 kDa and a G:M ratio of 1.5 or greater; (c) The unmodified alginate in the barrier compartment has a molecular weight of 150 kDa to 250 kDa and a G:M ratio of 1.5 or higher. (d) Optionally, the barrier compartment is formed from an alginate solution comprising a mixture of covalently modified alginate and unmodified alginate and having a viscosity of 250-350 cP. A hydrogel capsule according to Embodiment 19 or 21.

[0213] twenty three. (a) The capsules are approximately 1.0 mm to 2.0 mm in diameter; (b) The conjugation density of the RGD peptide on the alginate is the nitrogen percentage determined by combustion analysis (e.g., as described in Example 1B herein) of a certain amount of lyophilized RGD polymer, where the determined nitrogen percentage is approximately 0.10% nitrogen (N) to 1.00% N, approximately 0.20% N to approximately 0.80% N, approximately 0.30% N to approximately 0.60% N, approximately 0.30% to approximately 0.50%, or 0.33% N to 0.46% N; (c) The covalently modified alginate in the barrier compartment is modified with compound 101 only, and the density of compound 101 in the covalently modified alginate is the nitrogen percentage determined by combustion analysis of a certain amount of lyophilized covalently modified alginate, as described, for example, in Example 1A of this specification, and the determined nitrogen percentage is at least 2.0% and less than 9.0%, or 3.0% to 8.0%, 4.0% to 7.0%, 5.0% to 7.0%, or 6.0% to 7.0%, or about 6.8%. A hydroxy capsule according to Embodiment 22.

[0214] twenty four. (a) The capsule has a diameter of approximately 1.5 millimeters; (b) The RGD peptide is essentially composed of the amino acid sequence of RGSP (SEQ ID NO: 49), and the linker is a single glycine residue; (c) The conjugation density of RGD peptide on alginate is, (i) an amount sufficient to increase the viability of recombinant RPE cells, as determined by, for example, the assay described herein; (ii) an amount sufficient to increase the productivity of recombinant RPE cells, as determined by assays such as those described herein; (iii) A nitrogen percentage determined by combustion analysis (as described, for example, in Example 1B herein) of a RGD polymer freeze-dried to a certain weight, which is approximately 0.10% to 1.00% N, approximately 0.20% to 0.80% N, approximately 0.30% to 0.60% N, approximately 0.30% to 0.50%, or 0.33% to 0.46% N; (iv) 0.1 to 1.0, 0.2 to 0.8, 0.3 to 0.7, or 0.3 to 0.6 micromoles of GRGDSP (SEQ ID NO: 44) per gram of RGD-polymer in solution, as described in Example 7 and optionally in Example 8 of this specification; and (v) Any combination of two or more of c(i), c(ii), c(iii), and c(iv) Selected from the group consisting of, (d) The covalently modified alginate in the barrier compartment is modified only with compound 101, and the density of compound 101 in the covalently modified alginate is (i) a nitrogen percentage determined by combustion analysis of a covalently modified alginate that has been freeze-dried to a certain volume, as described in Example 1A of this specification, wherein the determined nitrogen percentage is at least 2.0% and less than 9.0%, or 3.0% to 8.0%, 4.0% to 7.0%, 5.0% to 7.0%, or 6.0% to 7.0%, or about 6.8%; or (ii) a weight / weight-based % amine determined by quantitative free amine analysis, for example, as described in Example 9 of this specification, where the determined densities are 1.0 w / w% to 3.0 w / w%, 1.3 w / w% to 2.8 w / w%, 1.3 w / w% to 2.6 w / w%, 1.5 w / w% to 2.4 w / w%, 1.5 w / w% to 2.2 w / w%, or 1.7 w / w% to 2.2 w / w%. A hydrogel capsule according to Embodiment 23.

[0215] 25. The hydrogel capsule according to Embodiment 24, wherein the conjugation density of RGD peptide on the alginate in the RGD polymer is 0.3 to 0.6 micromoles of GRGDSP (SEQ ID NO: 44) per gram of RGD polymer in solution.

[0216] 26. A device preparation, wherein each device in the preparation is a device described in any one of embodiments 13 to 18.

[0217] 27. A composition comprising a plurality of hydrogel capsules, wherein each capsule in the composition is a hydrogel capsule according to any one of embodiments 19 to 25.

[0218] 28. The composition according to Embodiment 27, wherein each capsule is a spherical hydrogel capsule selected from the group consisting of diameters of 0.5 mm to 2 mm; 0.7 mm to 1.8 mm; 1.0 mm to 1.8 mm; 1.2 mm to 1.7 mm; 1.3 mm to 1.7 mm; and 1.4 mm to 1.6 mm.

[0219] 29. The composition of Embodiment 27 or 28, which is a pharmaceutically acceptable composition.

[0220] 30. A method for treating a patient with Fabry disease, comprising administering to the patient an effective amount of the device preparation described in Embodiment 26 or the composition described in any one of Embodiments 27 to 29.

[0221] 31. A method for recombining multiple RPE cells to produce a GLA protein (e.g., human GLA protein or a variant thereof) having the characteristics described herein, comprising stably transfecting RPE cells with a polynucleotide as defined in any one of Embodiments 1, 2, or 8-11, and optionally isolating a monoclonal cell line expressing a GLA protein.

[0222] 32. The method of Embodiment 31, wherein multiple RPE cells are derived from ARPE-19 cells.

[0223] 33. Recombinant mammalian cells capable of expressing and secreting GLA protein, wherein the cells have the following characteristics: (a) Codon-optimized for expression in mammalian cells; (b) Encodes a GLA fusion protein including the following: (i) A signal peptide from a secretory protein other than GLA, operably ligated to the N-terminus of a mature human GLA amino acid sequence; or (ii) A GLA fusion protein comprising an amino acid sequence encoding a signal peptide operably ligated to the N-terminus of a mature human GLA amino acid sequence, and an amino acid sequence encoding a non-GLA polypeptide operably ligated to the C-terminus of the GLA amino acid sequence. Recombinant mammalian cells comprising an exogenous nucleotide sequence including a promoter operably ligated to one or more of the precursor GLA coding sequences.

[0224] 34. Recombinant cells according to Embodiment 33, wherein the mammalian cells are derived from RPE cells and, by optional selection, the promoter is essentially made up of a nucleotide sequence that is identical or substantially identical to SEQ ID NO: 18.

[0225] 35. Recombinant cells according to Embodiment 33 or 34, wherein the precursor GLA coding sequence includes SEQ ID NO: 3 or SEQ ID NO: 4.

[0226] 36. Recombinant cells according to Embodiment 33 or 34, wherein the signal peptide is derived from the mammalian HSPG2 protein, and optionally, the signal peptide is essentially derived from SEQ ID NO: 15.

[0227] 37. Recombinant cells according to Embodiment 36, wherein the precursor GLA coding sequence includes SEQ ID NO: 16.

[0228] 38. Recombinant cells according to Embodiment 33 or 34, wherein the GLA fusion protein comprises SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 13.

[0229] 39. Recombinant cells according to Embodiment 38, wherein the precursor GLA coding sequence includes SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO: 14.

[0230] 40. Recombinant cells according to any one of embodiments 33 to 39, wherein the mammalian cells are derived from ARPE-19 cells transfected with a transcription unit containing an exogenous nucleotide sequence.

[0231] 41. Recombinant cells according to any one of embodiments 33 to 40, wherein the exogenous nucleotide sequence includes SEQ ID NO: 47.

[0232] 42. Recombinant cell according to any one of embodiments 33 to 41, wherein the exogenous nucleotide sequence includes an extrachromosomal vector or is incorporated into at least one chromosomal location within a mammalian cell.

[0233] 43. A composition comprising recombinant mammalian cells according to any one of embodiments 33 to 42.

[0234] 44. The composition according to Embodiment 43, which is a polyclonal cell culture or a monoclonal cell line culture.

[0235] 45. An isolated double-stranded DNA molecule containing a nucleotide sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 47, or SEQ ID NO: 48.

[0236] 46. ​​An isolated DNA molecule according to Embodiment 45, essentially derived from Sequence ID No. 48.

[0237] 47. An implantable device comprising at least one cell-containing compartment containing recombinant cells as described in any one of embodiments 33 to 42, and at least one means for mitigating a foreign body reaction (FBR) when the device is implanted in a subject.

[0238] 48. The implantable device according to Embodiment 47, wherein at least one cell-containing compartment comprises a polymer composition for encapsulating recombinant cells and optionally comprises at least one cell-binding substance (CBS).

[0239] 49. The implantable device according to Embodiment 47 or 48, wherein the cell-containing compartment is surrounded by a barrier compartment comprising an alginate hydrogel and optionally a compound of formula (I) disposed on the outer surface of the barrier compartment.

[0240] 50. The polymer composition comprises an alginate covalently modified with a peptide, the peptide essentially consisting of or derived from GRGDSP (SEQ ID NO: 44) or GGGRGDSP (SEQ ID NO: 45), and the barrier compartment is [ka] An implantable device according to Embodiment 48 or 49, comprising an alginate modified with or a pharmaceutically acceptable salt thereof.

[0241] 51. Hydrogel capsule, (a) an inner compartment comprising recombinant cells according to any one of embodiments 33 to 42, encapsulated in a first polymer composition, wherein the first polymer composition comprises a hydrogel-forming polymer; and (b) A barrier compartment surrounding an inner compartment and comprising a second polymer composition, wherein the second polymer composition comprises an alginate covalently modified with at least one compound selected from the group consisting of compound 100, compound 101, compound 110, compound 112, compound 113, and compound 114 shown in Table 3. A hydrogel capsule containing [the specified ingredient].

[0242] 52. The selected compound is [ka] The hydrogel capsule according to Embodiment 51.

[0243] 53. The hydrogel capsule according to Embodiment 51 or 52, wherein the inner compartment contains a plurality of recombinant cells as described in Embodiment 41, and optionally, the concentration of recombinant cells in the inner compartment is at least 40 million cells per ml of the first polymer composition.

[0244] 54. A composition comprising one or more hydrogel capsules from any one of embodiments 51 to 53.

[0245] 55. A method for treating a patient with Fabry disease, comprising administering to the patient an implantable device according to any one of Embodiments 15 to 18, a hydrogel capsule according to any one of Embodiments 51 to 53, or a composition according to Embodiment 54. [Examples]

[0246] The following examples are provided to allow for a more complete understanding of the disclosures described herein. The examples described herein are provided to illustrate the recombinant RPE cells, implantable devices, and compositions and methods provided herein and should not be construed as limiting their scope in any way.

[0247] [Example 1: Generation and culture of exemplary recombinant ARPE-19 cells] GLA-secreting cells were created using the expression vector shown in Figure 8A, where the exogenous nucleotide sequence encoding the precursor GLA protein was inserted using standard cloning methods. To achieve this, ARPE-19 cells were cotransfected with the GLA expression vector using PiggyBac containing a transposase plasmid, and the stably transfected cells were cultured in full growth medium containing puromycin.

[0248] Stable transfected ARPE-19 cells were cultured according to the following protocol. The cells were cultured at 150 cm². 2 Cells were grown in full growth medium (10% FBS and 1x penicillin-streptomycin-neomycin antibiotics, Gibco, DMEM:F12) in cell culture flasks. To subculture the cells, the medium in the culture flask was aspirated and the cell layer was briefly rinsed with phosphate-buffered saline (pH 7.4, 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, Gibco). 5–10 mL of 0.25% (w / v) trypsin / 0.53 mM EDTA solution ("TrypsinEDTA") was added to the flask, and the cells were observed under an inverted microscope for 3–5 minutes, usually until the cell layer dispersed. To avoid aggregation, the cells were handled carefully, and the flask was minimized from being bumped or shaken during the dispersion period. If the cells did not disperse, the flask was placed at 37°C to promote dispersion. Once the cells were dispersed, 10 mL of full growth medium was added, and the cells were aspirated by gentle pipetting. The cell suspension was transferred to a centrifuge tube and centrifuged at approximately 125 × g for 5–10 minutes to remove Trypsin EDTA. The supernatant was discarded, and the cells were resuspended in fresh growth medium. An appropriate amount of cell suspension was added to a new culture vessel and incubated at 37°C. The medium was changed weekly.

[0249] [Example 2: GLA secretion from exemplary recombinant ARPE-10 cells] To quantify intracellular GLA expression, ARPE-19 cells recombinant with various GLA expression constructs were trypsin-treated as described above, and 400,000 cells were added twice to the wells of a 6-well plate with 2 mL of complete medium. The plates were incubated with 5% CO2 at 37°C for 16 hours, and the amount of GLA protein secreted in vitro by various recombinant cell cultures was quantified by an enzymatic assay using 4-methylumbelliferyl-α-D-galactopyranoside, a blue fluorescence-generating substrate. This assay measures GLA activity by measuring the formation of free 4-methylumbelliferyl as an increase in fluorescence at 460 nm emission when excited with 360 nm light. The concentration of active GLA was determined by comparing this activity in cell culture medium with a standard curve created using commercially available recombinant GLA protein (agalsidase beta, Sanofi Genzyme).

[0250] The results are shown in Figure 10. GLA4-1 is a polyclonal culture of cells transfected with a transcription unit containing SEQ ID NO: 6, which is the coding sequence of a human HSPG2 signal peptide fused to the codon-optimized coding sequence of the human wild-type mature GLA amino acid sequence. The codon-optimized sequence of GLA-1 is identical to the corresponding portion of the codon-optimized sequence of the GLA3 construct. GLA4-2 is a culture of a clonal cell line isolated from the GLA4-1 polyclonal culture using limiting dilution cloning.

[0251] [Example 3: Synthesis of an exemplary compound of formula (I)] [Common protocols] The following procedure describes how to prepare exemplary compounds for preparing the portable devices described herein. The compounds provided herein can be prepared from readily available starting materials using modifications to the specific synthesis protocols described below, which are well known to those skilled in the art. Where typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are given, it will be understood that other process conditions may also be used unless otherwise specified. Optimal reaction conditions may vary depending on the specific reactants or solvent used, but such conditions can be determined by routine optimization procedures for those skilled in the art.

[0252] Furthermore, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesirable reactions. The selection of suitable protecting groups for specific functional groups, as well as suitable conditions for protection and deprotection, are well known in the art. For example, numerous protecting groups, and their introduction and removal, are described in Greene et al., *Protecting Groups in Organic Synthesis*, Second Edition, Wiley, New York, 1991 and the references cited therein.

[0253] [Husgen cyclization to obtain 1,4-substituted triazoles] Triazole-based compounds and their compositions, devices, and materials were prepared using a copper-catalyzed hysgen[3+2] cycloaddition reaction. The scope and typical protocols have been the subject of numerous reviews (e.g., Meldal, M. and Tornoe, CWChem. Rev. (2008) 108:2952-3015; Hein, JE and Fokin, VVChem. Soc. Rev. (2010) 39(4):1302-1315; both of which are incorporated herein by reference). [ka]

[0254] In the example above, the azide is the reactive portion of the fragment containing binding element A, and the alkyne is the reactive component of the pendant group Z. These functional handles can be swapped to produce structurally related triazole products, as shown below. The preparation of these substitutes is similar and requires no special considerations. [ka]

[0255] The following outlines a typical hysgene cycloaddition procedure starting from an iodide. In some cases, for safety reasons, the iodide is converted to an azide during the reaction. [ka]

[0256] A solution of sodium azide (1.1 equivalents), sodium ascorbate (0.1 equivalents), trans-N,N'-dimethylcyclohexane-1,2-diamine (0.25 equivalents), and copper(I) iodide in methanol solution (1.0 M, limiting reagent) was degassed by bubbling nitrogen and treated with acetylene (1 equivalent) and aryl iodide (1.2 equivalents). The mixture was stirred at room temperature for 5 minutes and then heated to 55°C for 16 hours. The reaction mixture was then cooled to room temperature, filtered through a funnel, and the filter cake was washed with methanol. The combined filtrate was concentrated and purified by flash chromatography in silica gel (120 g silica, 0-40% gradient (3% ammonium hydroxide aqueous solution, 22% methanol, remainder dichloromethane)) to obtain the desired target substance.

[0257] The following is an outline of a typical hysgen cyclization procedure starting from azide. [ka]

[0258] A solution of tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine (0.2 equivalents), triethylamine (0.5 equivalents), and copper(I) iodide (0.06 equivalents) in methanol (0.4 M, limiting reagent) was treated with acetylene (1.0 equivalent) and cooled to 0°C. The reaction mixture was heated to room temperature over 30 minutes, then heated to 55°C for 16 hours. The reaction mixture was cooled to room temperature, concentrated, and purified by HPLC (C18 column, 0-100% gradient (3% ammonium hydroxide aqueous solution, 22% methanol, remainder dichloromethane)) in dichloromethane to obtain the desired target material.

[0259] [Hyusgen cyclization to obtain 1,5-substituted triazoles] Hysgen[3+2] cycloaddition was also carried out using a ruthenium catalyst, and 1,5-disubstituted products were preferentially obtained (as described, for example, in Zhang et al, J.Am.Chem.Soc., 2005, 127, 15998-15999; Boren et al, J.Am.Chem.Soc., 2008, 130, 8923-8930, which are respectively incorporated herein by reference in their entirety). [ka]

[0260] As mentioned above, by exchanging the azide group and the alkyne group, a triazole similar to the one shown below can be formed. [ka]

[0261] A typical procedure is as follows: A solution of alkyne (1 equivalent) and azide (1 equivalent) in dioxane (0.8 M) was added dropwise to a solution of pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride (0.02 equivalents) in dioxane (0.16 M). The vial was purged with nitrogen, sealed, and the mixture was heated to 60°C for 12 hours. The resulting mixture was concentrated and purified by flash chromatography on silica gel to obtain the desired compound.

[0262] [(4-(4-((4-methylpiperazine-1-yl)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methanamine (3) experimental procedure] [ka] A mixture of (4-iodophenyl)methaneamine (1,843 mg, 3.62 mmol, 1.0 equivalent), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (74 μL, 0.47 mmol, 0.13 equivalents), sodium ascorbate (72 mg, 0.36 mmol, 0.1 equivalent), copper iodide (69 mg, 0.36 mmol, 0.1 equivalent), sodium azide (470 mg, 7.24 mmol, 2.0 equivalents), and 1-methyl-4-(prop-2-in-1-yl)piperazine (2,0.5 g, 3.62 mmol, 1.0 equivalent) in methanol (9 mL) and water (1 mL) was purged with nitrogen for 5 minutes and heated overnight to 55°C. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, and the brownish slurry was extracted with dichloromethane. Celite was added to the combined dichloromethane phase, and the solvent was removed under reduced pressure. The crude product was purified on silica gel (80 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 7.5% to obtain (4-(4-((4-methylpiperazine-1-yl)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methaneamine (3, 0.45 g, 43%). LCMS m / z:[M+H]+ C 15 H 22 Calculated value for N6: 287.2; measured value: 287.1.

[0263] [Experimental procedure for N-(4-(4-((4-methylpiperazine-1-yl)methyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (4)] [ka] A solution of (4-(4-((4-methylpiperazine-1-yl)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methanamine (3, 1.2 g, 4.19 mmol, 1.0 equivalent) and triethylamine (0.70 mL, 5.03 mmol, 1.2 equivalents) in CH2Cl2 (50 mL) was cooled to 0°C in an ice bath, and methacryloyl chloride (0.43 mL, 4.40 mmol, 1.05 eq in 5 mL of CH2Cl2) was added. The reaction mixture was stirred for 1 day while cooling in an ice bath. 10 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (80 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 7.5%. The solvent was removed under reduced pressure, the resulting solid was pulverized with diethyl ether, filtered, and washed multiple times with diethyl ether to obtain N-(4-(4-((4-methylpiperazine-1-yl)methyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (4, 0.41 g, 28% yield) as a white solid. LCMS m / z:[M+H] + C 19 H 26 Calculated value of N6O: 355.2; measured value: 355.2.

[0264] [(4-(4-((2-(2-methoxyethoxy)ethoxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methanamine (6) experimental procedure] [ka] A mixture of (4-iodophenyl)methaneamine (1, 2.95 g, 12.64 mmol, 1.0 equivalent), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (259 μL, 1.64 mmol, 0.13 equivalents), sodium ascorbate (250 mg, 1.26 mmol, 0.1 equivalent), copper iodide (241 mg, 1.26 mmol, 0.1 equivalent), sodium azide (1.64 g, 25.29 mmol, 2.0 equivalents), and 1-methyl-4-(prop-2-in-1-yl)piperazine (5, 2.0 g, 12.64 mmol, 1.0 equivalent) in methanol (40 mL) and water (4 mL) was purged with nitrogen for 5 minutes and heated overnight at 55°C. The reaction mixture was cooled to room temperature and concentrated under reduced pressure. The residue was dissolved in dichloromethane, filtered, and concentrated on Celite (10 g). The crude product was purified by silica gel chromatography (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 6.25% to obtain (4-(4-((2-(2-methoxyethoxy)ethoxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methaneamine (6, 1.37 g, 35%). LCMS m / z:[M+H] + C 15 H 22 Calculated value of N4O3: 307.2; measured value: 307.0.

[0265] [Experimental procedure for N-(4-(4-((2-(2-methoxyethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)benzyl)methacrylamide (7)] [ka] A solution of 4-(4-((2-(2-methoxyethoxy)ethoxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methanamine (6, 1.69 g, 5.52 mmol, 1.0 equivalent) and triethylamine (0.92 mL, 6.62 mmol, 1.2 equivalents) in CH2Cl2 (50 mL) was cooled to 0°C in an ice bath, and methacryloyl chloride (0.57 mL, 5.79 mmol, 1.05 equivalents) was added dropwise. The reaction mixture was stirred at room temperature for 4 hours. 10 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel (80 g) chromatography using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 1.25% to obtain N-(4-(4-((2-(2-methoxyethoxy)ethoxy)methyl)-1H-1,2,3-triazol-1-yl)benzyl)methacrylamide (7, 1.76 g, 85% yield) as a white solid. LCMS m / z:[M+H] + C 19 H 26 Calculated value of N4O4: 375.2; measured value: 375.0.

[0266] [Experimental procedure for 3-(prop-2-in-1-yloxy)oxetane (9)] [ka] A suspension of sodium hydride (27.0 g, 675 mmol, 60% purity) in THF (200 mL) was cooled in an ice bath. Oxetane-3-ol (8, 25 g, 337 mmol) was added dropwise, and the mixture was stirred at 0°C for 30 minutes. Next, 3-bromoprop-1-in (9, 41.2 mL, 371 mmol, 80% purity) was added dropwise. The mixture was stirred overnight while being warmed to room temperature. The mixture was filtered through Celite, washed with THF, and concentrated on Celite under reduced pressure. The crude product was purified over silica gel (220 g) and eluted with hexane / siRNA. The concentration of siRNA in the mobile phase was increased from 0 to 25% to obtain a yellow oil (9, 18.25 g, 48%).

[0267] [Experimental procedure for 3-(4-((oxetane-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)propan-1-amine (11)] [ka] A mixture of 3-(prop-2-in-1-yloxy)oxetane (9, 7.96 g, 71 mmol, 1.0 equivalent), 3-azidopropan-1-amine (10, 7.82 g, 78 mmol, 1.1 equivalent), tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine (8.29 g, 15.6 mmol, 0.22 equivalents), copper iodide (1.35 g, 7.1 mmol, 0.1 equivalent), and triethylamine (2.47 mL, 17.8 mmol, 0.25 equivalents) in methanol (80 mL) was heated to 55°C and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, Celite (20 g) was added, and the mixture was concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 15% to obtain 3-(4-((oxetane-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)propan-1-amine (11, 11.85 g, 79%) as a yellow oil. LCMS m / z:[M+H] + C9H 16 Calculated value of N4O2: 213.1; measured value: 213.0.

[0268] [Experimental procedure for N-(3-(4-((oxetane-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)propyl)methacrylamide (12)] [ka] A solution of 3-(4-((oxetane-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)propan-1-amine (11, 3.94 g, 18.56 mmol, 1.0 equivalent) and triethylamine (3.1 mL, 22.28 mmol, 1.2 equivalents) in CH2Cl2 (100 mL) was cooled to 0°C in an ice bath, and methacryloyl chloride (1.99 mL, 20.42 mmol, 1.1 equivalents) was added dropwise. The reaction mixture was stirred overnight while warming to room temperature. 20 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (220 g) using dichloromethane / methanol as the mobile phase. The methanol concentration was gradually increased from 0% to 5% to obtain N-(3-(4-((oxetane-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)propyl)methacrylamide (12, 3.22 g, 62% yield) as a solid. LCMS m / z:[M+H] + C 13 H 20 Calculated value of N4O3: 281.2; measured value: 281.0.

[0269] [Experimental procedure for N-(4-(1H-1,2,3-triazol-1-yl)benzyl)methacrylamide (14)] [ka] To a solution of (4-(1H-1,2,3-triazole-1-yl)phenyl)methaneamine (13, obtained from WuXi, 1.2 g, 5.70 mmol, 1.0 equivalent) and triethylamine (15 mL, 107.55 mmol, 18.9 equivalents) in CH2Cl2 (100 mL), methacryloyl chloride (893 mg, 8.54 mmol, 1.5 equivalents) was slowly added dropwise. The reaction mixture was stirred overnight. 20 grams of Celite were added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 1.25% to obtain N-(4-(1H-1,2,3-triazol-1-yl)benzyl)methacrylamide (14, 1.38 g, 40% yield).

[0270] [(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methanamine (15) experimental procedure] [ka] (4-iodophenyl)methaneamine hydrochloride (5.0 g, 18.55 mmol, 1.0 equivalent), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (0.59 mL, 3.71 mmol, 0.2 equivalents), sodium ascorbate (368 mg, 1.86 mmol, 0.1 equivalent), copper iodide (53) in methanol (50 mL) and water (12 mL). A mixture of 0 mg (2.78 mmol, 0.15 equivalents), sodium azide (2.41 g, 37.1 mmol, 2.0 equivalents), Et3N (3.11 mL, 22.26 mmol, 1.2 equivalents), and 2-(prop-2-in-1-yloxy)tetrahydro-2H-pyran (2.6 g, 18.55 mmol, 1.0 equivalent) was purged with nitrogen for 5 minutes and heated overnight to 55°C. The reaction mixture was cooled to room temperature and filtered through 413 filter paper. Celite was added, the solvent was removed under reduced pressure, and the residue was purified on silica gel (120 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 6.25% to obtain (4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methaneamine (15, 3.54 g, 66%) as a white solid. LCMS m / z:[M+H] + C 15 H 20 Calculated value of N4O2: 289.2; measured value: 289.2.

[0271] [Experimental procedure for N-(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (16)] [ka] A solution of (4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methaneamine (15, 3.46 g, 12.00 mmol, 1.0 equivalent) and triethylamine (2.01 mL, 14.40 mmol, 1.2 equivalents) in CH2Cl2 (40 mL) was cooled to 0°C in an ice bath, and methacryloyl chloride (1.23 mL, 12.60 mmol, 1.05 equivalents, diluted in 5 mL of CH2Cl2) was added dropwise. The cooling bath was removed, and the reaction mixture was stirred for 4 hours. 20 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (80 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 3.75% to obtain N-(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (16, 2.74 g, 64% yield) as a white solid. LCMS m / z:[M+H] + C 19 H 24 Calculated value of N4O3: 357.2; measured value: 357.3.

[0272] [Experimental procedure for N-(4-(4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)benzyl)methacrylamide (17)] [ka] A solution of N-(4-(4-(hydroxymethyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (16, 1.2 g, 3.37 mmol, 1.0 equivalent) was dissolved overnight at room temperature in methanol (6 mL) and HCl (1N aqueous solution, 9 mL). Celite was added, and the solvent was removed under reduced pressure. The crude product was purified by silica gel chromatography (24 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 12.5% ​​to obtain N-(4-(4-(hydroxymethyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (17, 0.85 g, 92% yield) as a white solid. LCMS m / z:[M+H] + C 14 H 16 Calculated value of N4O2: 273.1; measured value: 273.1.

[0273] [(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)benzyl)carbamate (19) experimental procedure] [ka] Benzyl(4-(hydroxymethyl)benzyl)carbamate (2.71 g, 10 mmol, 1 equivalent), 3,4-dihydro-2H-pyran (1.81 mL, 20 mmol, 2 equivalents), and p-toluenesulfonic acid monohydrate (285 mg, 1.5 mmol, 0.15 equivalents) were stirred overnight at room temperature in dichloromethane (100 mL). Celite was added, and the solvent was removed under reduced pressure. The crude product was purified over silica gel (24 g) using hexane / siRNA as the eluent, starting with 100% hexane and gradually increasing the siRNA concentration to 100%, to obtain benzyl(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)benzyl)carbamate (19, 2.4 g, 68%) as a colorless oil. LCMS m / z:[M+Na] + C 21 H 25Calculated value for NO4: 378.17; measured value: 378.17.

[0274] [(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)phenyl)methanamine (20) experimental procedure] [ka] (4-(((tetrahydro-2H-pyran-2-yl)oxymethyl)benzyl)carbamate (19, 1.5 g, 4.2 mmol, 1 equivalent) and palladium carbon (160 mg, 10 wt%) in EtOH were briefly evacuated, then hydrogen was added via balloon, and the mixture was stirred at room temperature for 1 hour. Celite was added, and the solvent was removed under reduced pressure. The crude product was purified on silica gel (12 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 25% to obtain (4-(((tetrahydro-2H-pyran-2-yl)oxymethyl)phenyl)methaneamine (20, 890 mg, 95%) as a colorless oil. LCMS m / z:[M+H] + C 13 H 19 Calculated value of NO2: 222.15; measured value: 222.14.

[0275] [Experimental procedure for N-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)benzyl)-methacrylamide (21)] [ka] A solution of (4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)phenyl)methaneamine (20, 0.5 g, 2.26 mmol, 1.0 equivalent) and triethylamine (0.47 mL, 3.39 mmol, 1.5 equivalent) in CH2Cl2 (10 mL) was briefly evacuated and flushed with nitrogen. Methacryloyl chloride (0.33 mL, 3.39 mmol, 1.5 equivalent) was added dropwise. The reaction mixture was stirred overnight at room temperature. 10 grams of Celite was added, and the solvent was removed under reduced pressure. The residue was subjected to silica gel chromatography (12 g) using hexane / SiO as the eluent, starting with 100% hexane and gradually increasing the SiO concentration to 100%, to obtain N-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)benzyl)methacrylamide (21, 0.47 g, 72% yield) as a colorless solid. LCMS m / z:[M+Na] + C 17 H 23 Calculated value for NO3: 312.16; measured value: 312.17.

[0276] [(4-(4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-1-yl)phenyl)methanamine (22) experimental procedure] [ka] A mixture of (4-iodophenyl)methaneamine (5.0 g, 21.45 mmol, 1.0 equivalent), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (0.44 mL, 2.79 mmol, 0.13 equivalents), sodium ascorbate (425 mg, 2.15 mmol, 0.1 equivalent), copper iodide (409 mg, 2.15 mmol, 0.1 equivalent), sodium azide (2.79 g, 42.91 mmol, 2.0 equivalents), and 2-(buto-3-in-1-yloxy)tetrahydro-2H-pyran (3.36 mL, 21.45 mmol, 1.0 equivalent) in methanol (20 mL) and water (5 mL) was purged with nitrogen for 5 minutes and heated overnight at 55°C. The reaction mixture was cooled to room temperature and filtered through 413 filter paper. Celite (10 g) was added, the solvent was removed under reduced pressure, and the residue was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 5% to obtain (4-(4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-1-yl)phenyl)methaneamine (22, 3.15 g, 49%) as a solid. LCMS m / z:[M+H] + C 16 H 22 Calculated value of N4O2: 303.18; measured value: 303.18.

[0277] [Experimental procedure for N-(4-(4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (23)] [ka] A solution of (4-(4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-1-yl)phenyl)methanamine (22, 3.10 g, 10.25 mmol, 1.0 equivalent) and triethylamine (1.71 mL, 12.30 mmol, 1.2 equivalents) in CH2Cl2 (55 mL) was cooled to 0°C in an ice bath, and methacryloyl chloride (1.05 mL, 12.30 mmol, 1.2 equivalents, diluted in 5 mL of CH2Cl2) was added dropwise. The cooling bath was removed, and the reaction mixture was stirred for 4 hours. 8 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (80 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 2.5% to obtain N-(4-(4-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (23, 2.06 g, 54% yield) as a white solid. LCMS m / z:[M+H] + C 20 H 26 Calculated value of N4O3: 371.2078; measured value: 371.2085.

[0278] [(4-(1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-4-yl)phenyl)methanamine (24) experimental procedure] [ka] A mixture of (4-ethynylphenyl)methaneamine (2.36 g, 18.00 mmol, 1.0 equivalent), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (0.56 mL, 3.60 mmol, 0.2 equivalents), sodium ascorbate (357 mg, 1.80 mmol, 0.1 equivalent), copper iodide (514 mg, 2.70 mmol, 0.15 equivalents), and 2-(2-azidoethoxy)tetrahydro-2H-pyran (3.08 mg, 18.00 mmol, 1.0 equivalent) in methanol (24 mL) and water (6 mL) was purged with nitrogen for 5 minutes and heated overnight to 55°C. The reaction mixture was cooled to room temperature, filtered through Celite, and rinsed with MeOH (3 × 50 mL). The solvent was removed under reduced pressure, the residue was redissolved in dichloromethane, Celite (20g) was added, the solvent was removed under reduced pressure, and the residue was purified on silica gel (120g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 25% to obtain (4-(1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-4-yl)phenyl)methaneamine (24, 3.51g, 64%) as a yellowish oil. LCMS m / z:[M+H] + C 16 H 22 Calculated value of N4O2: 303.1816; measured value: 303.1814.

[0279] [Experimental procedure for N-(4-(1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-4-yl)benzyl)methacrylamide (25)] [ka] A solution of (4-(1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-4-yl)phenyl)methanamine (24, 1.5 g, 4.96 mmol, 1.0 equivalent) and triethylamine (1.04 mL, 7.44 mmol, 1.5 equivalent) in CH2Cl2 (30 mL) was briefly evacuated and flushed with nitrogen. Methacryloyl chloride (0.72 mL, 7.44 mmol, 1.5 equivalent) was added dropwise. The reaction mixture was stirred at room temperature for 2 hours. 10 grams of Celite was added, and the solvent was removed under reduced pressure. The residue was subjected to silica gel chromatography (40 g) using hexane / siRNA as the eluent, starting with 100% hexane and gradually increasing the siRNA concentration to 100%, to obtain N-(4-(1-(2-((tetrahydro-2H-pyran-2-yl)oxy)ethyl)-1H-1,2,3-triazole-4-yl)benzyl)methacrylamide (25, 0.9 g, 49% yield) as a colorless solid. LCMS m / z:[M+Na] + C 20 H 26 Calculated value of N4O3: 371.2078; measured value: 371.2076.

[0280] [Experimental procedure for 1-(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)ethane-1-amine (26)] [ka] A mixture of 1-(4-iodophenyl)ethane-1-amine hydrochloride (1.0 g, 4.05 mmol, 1.0 equivalent), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (0.08 mL, 0.53 mmol, 0.13 equivalents), sodium ascorbate (80 mg, 0.40 mmol, 0.1 equivalent), copper iodide (77 mg, 0.40 mmol, 0.1 equivalent), sodium azide (526 g, 8.09 mmol, 2.0 equivalents), and 2-(prop-2-in-1-yloxy)tetrahydro-2H-pyran (0.57 g, 4.05 mmol, 1.0 equivalent) in methanol (9 mL) and water (1 mL) was purged with nitrogen for 5 minutes and heated overnight to 55°C. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was redissolved in dichloromethane and filtered through a Celite plug. Celite was added to the filtrate and the solvent was removed under reduced pressure. The residue was purified on silica gel (40 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 5% to obtain 1-(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)ethane-1-amine (26, 0.62 g, 51%) as a yellowish solid. LCMS m / z:[M+H] + C 16 H 22 Calculated value of N4O2: 303.2; measured value: 303.2.

[0281] [Experimental procedure for N-(1-(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)ethyl)methacrylamide (27)] [ka] A solution of 1-(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)ethane-1-amine (26, 0.52 g, 1.7 mmol, 1.0 equivalent) and triethylamine (0.29 mL, 2.1 mmol, 1.2 equivalents) in CH2Cl2 (11 mL) was cooled to 0°C in an ice bath, and methacryloyl chloride (0.18 mL, 1.8 mmol, 1.05 equivalents, diluted in 11 mL of CH2Cl2) was added dropwise. The cooling bath was removed, and the reaction mixture was stirred for 4 hours. 5 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (40 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 2.5% to obtain N-(1-(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)ethyl)methacrylamide (27, 0.49 g, 76% yield) as a white solid. LCMS m / z:[M+H] + C 20 H 26 Calculated value of N4O3: 371.2078; measured value: 371.2087.

[0282] [(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)-2-(trifluoromethyl)phenyl)methanamine (28) experimental procedure] [ka] (4-iodo-2-(trifluoromethyl)phenyl)methaneamine (3.0 g, 9.97 mmol, 1.0 equivalent), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (0.31 mL, 1.99 mmol, 0.2 equivalents), sodium ascorbate (197 mg, 1.00 mmol, 0.1 equivalent), iodide in methanol (24 mL) and water (6 mL) A mixture of copper (285 mg, 1.49 mmol, 0.15 equivalents), sodium azide (1.30 g, 19.93 mmol, 2.0 equivalents), Et3N (1.67 mL, 11.96 mmol, 1.2 equivalents), and 2-(prop-2-in-1-yloxy)tetrahydro-2H-pyran (1.40 g, 9.97 mmol, 1.0 equivalent) was purged with nitrogen for 5 minutes and heated overnight to 55°C. The reaction mixture was cooled to room temperature, filtered through a Celite plug, and rinsed with methanol (3 × 50 mL). Celite was added to the filtrate, and the solvent was removed under reduced pressure. The residue was purified on silica gel (120 g) using dichloromethane / (methanol containing a 12% (v / v) aqueous solution of ammonium hydroxide) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 25% to obtain (4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)-2-(trifluoromethyl)phenyl)methaneamine (28, 2.53 g, 71%) as a green oil. LCMS m / z:[M+H] + C 16 H 19 Calculated value of N4O2F3: 357.2; measured value: 357.1.

[0283] [Experimental procedure for N-(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)-2(trifluoromethyl)benzyl)methacrylamide (29)] [ka] A solution of (4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)-2-(trifluoromethyl)phenyl)methanamine (28, 1.0 g, 2.81 mmol, 1.0 equivalent) and triethylamine (0.59 mL, 4.21 mmol, 1.5 equivalents) in CH2Cl2 (25 mL) was briefly evacuated and flushed with nitrogen. Methacryloyl chloride (0.41 mL, 4.21 mmol, 1.5 equivalents) was added dropwise. The reaction mixture was stirred at room temperature for 6 hours. 10 grams of Celite was added, and the solvent was removed under reduced pressure. The residue was subjected to silica gel chromatography (40 g) using hexane / siRNA as the eluent, starting with 100% hexane and gradually increasing the siRNA concentration to 100%, to obtain N-(4-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)-2(trifluoromethyl)benzyl)methacrylamide (29, 0.65 g, 55% yield) as a colorless solid. LCMS m / z:[M+H] + C 20 H 23 The calculated value for N4O3F3 is 425.2; the measured value is 425.1.

[0284] [Experimental procedure for 3-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)propan-1-amine (30)] [ka] A mixture of 3-azidopropan-1-amine hydrochloride (1.5 g, 14.98 mmol, 1.0 equivalent), tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine (1.99 g, 3.75 mmol, 0.25 equivalent), copper iodide (0.29 g, 1.50 mmol, 0.1 equivalent), and triethylamine (0.52 mL, 3.75 mmol, 0.25 equivalent) in methanol (50 mL) and water (6 mL) was purged with nitrogen for 5 minutes and cooled to 0°C. 2-(prop-2-in-1-yloxy)tetrahydro-2H-pyran (2.10 g, 14.98 mmol, 1.0 equivalent) was added, and the reaction mixture was warmed to 55°C and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, filtered through a Celite plug, and rinsed with methanol (3 × 50 mL). Celite (20 g) was added to the filtrate, and the solvent was removed under reduced pressure. The residue was purified on silica gel (120 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 20% to obtain 3-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)propan-1-amine (30, 2.36 g, 66%). LCMS m / z:[M+H] + C 11 H 20 Calculated value of N4O2: 241.2; measured value: 241.2.

[0285] [Experimental procedure for N-(3-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)propyl)methacrylamide (31)] [ka] A solution of 3-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)propan-1-amine (30, 1.0 g, 4.16 mmol, 1.0 equivalent) and triethylamine (0.58 mL, 4.16 mmol, 1.0 equivalent) in CH2Cl2 (20 mL) was briefly evacuated and flushed with nitrogen. Methacryloyl chloride (0.40 mL, 4.16 mmol, 1.0 equivalent) was added dropwise. The reaction mixture was stirred overnight at room temperature. 10 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (40 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 20% to obtain N-(3-(4-(((tetrahydro-2H-pyran-2-yl)oxy)methyl)-1H-1,2,3-triazole-1-yl)propyl)methacrylamide (31, 0.96 g, 75% yield) as a colorless oil. LCMS m / z:[M+H] + C 15 H 24 Calculated value of N4O3: 309.2; measured value: 309.4.

[0286] [(4-(4-((oxetane-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methanamine (32) experimental procedure] [ka] (4-iodophenyl)methaneamine hydrochloride (2.64 g, 9.80 mmol, 1.0 equivalent), (1S,2S)-N1,N2-dimethylcyclohexane-1,2-diamine (0.31 mL, 1.96 mmol, 0.2 equivalents), sodium ascorbate (198 mg, 0.98 mmol, 0.1 equivalent), copper iodide ( A mixture of 279 mg (1.47 mmol, 0.15 equivalents) of sodium azide (1.27 g, 19.59 mmol, 2.0 equivalents), Et3N (1.64 mL, 11.75 mmol, 1.2 equivalents), and 3-(prop-2-in-1-yloxy)oxetane (9, 1.10 g, 9.80 mmol, 1.0 equivalent) was purged with nitrogen for 5 minutes and heated overnight to 55°C. The reaction mixture was cooled to room temperature, filtered through a Celite plug, and rinsed with methanol (3 × 50 mL). Celite was added to the filtrate, and the solvent was removed under reduced pressure. The residue was purified on silica gel (120 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 25% to obtain (4-(4-((oxetane-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methaneamine (32, 1.43 g, 56%) as an oil. LCMS m / z:[M+H] + C 13 H 16 Calculated value of N4O2: 261.1346; measured value: 261.1342.

[0287] [Experimental procedure for N-(4-(4-((oxetane-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (33)] [ka] A solution of (4-(4-((oxetane-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)phenyl)methanamine (32, 0.58 g, 2.23 mmol, 1.0 equivalent) and triethylamine (0.47 mL, 3.34 mmol, 1.5 equivalent) in CH2Cl2 (20 mL) was briefly evacuated and flushed with nitrogen. Methacryloyl chloride (0.32 mL, 3.34 mmol, 1.5 equivalent) was added dropwise. The reaction mixture was stirred at room temperature for 6 hours. 10 grams of Celite was added, and the solvent was removed under reduced pressure. The residue was subjected to silica gel chromatography (24 g) using hexane / siRNA as the eluent, starting with 100% hexane and gradually increasing the siRNA concentration to 100%, to obtain N-(4-(4-((oxatan-3-yloxy)methyl)-1H-1,2,3-triazole-1-yl)benzyl)methacrylamide (33, 0.48 g, 66% yield) as a colorless solid. LCMS m / z:[M+H] + C 17 H 20 Calculated value of N4O3: 329.1608; measured value: 329.1611.

[0288] [Experimental procedure for 1-(2-methacrylamidoethyl)-1H-imidazole-4-carboxylate ethyl(35)] [ka] A solution of ethyl 1-(2-aminoethyl)-1H-imidazole-4-carboxylate (34, 2.0 g, 10.91 mmol, 1.0 equivalent) and triethylamine (3.80 mL, 27.29 mmol, 2.5 equivalents) in CH2Cl2 (20 mL) was briefly evacuated and flushed with nitrogen. Methacryloyl chloride (1.60 mL, 16.37 mmol, 1.5 equivalents) was added dropwise. The reaction mixture was stirred at room temperature for 3 hours. 15 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (40 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 25% to obtain ethyl 1-(2-methacrylamideethyl)-1H-imidazole-4-carboxylate (35, 1.28 g, 47% yield) as a colorless solid. LCMS m / z:[M+H] + C 12 H 17 Calculated value of N3O3: 252.1; measured value: 252.1.

[0289] [Experimental procedure for N-(4-(1,1-dioxidedothiomorpholino)benzyl)methacrylamide (37)] [ka] A solution of 4-(4-(aminomethyl)phenyl)thiomorpholine 1,1-dioxide hydrochloride (36 g, 1.15 g, 4.15 mmol, 1.0 equivalent) and triethylamine (1.39 mL, 9.97 mmol, 2.4 equivalents) in CH2Cl2 (80 mL) was to be added dropwise with a solution of methacryloyl chloride (0.43 mL, 4.36 mmol, 1.05 equivalents in 5 mL of CH2Cl2). The reaction mixture was stirred at room temperature for 22 hours. 8 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (80 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 3.75% to obtain N-(4-(1,1-dioxide thiomorpholino)benzyl)methacrylamide (37, 0.32 g, 25% yield) as a solid.

[0290] [Experimental procedure for N-methyl-N-(2-(methylsulfonyl)ethyl)prop-2-in-1-amine (38)] [ka] A mixture of 1-methylsulfonylethylene (4.99 g, 47.03 mmol, 4.13 mL) and Amberlyst-15 ((30% w / w)) was mixed with N-methylprop-2-in-1-amine (2.6 g, 37.62 mmol) added dropwise. The mixture was stirred at room temperature for 12 hours. The catalyst was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain N-methyl-N-(2-(methylsulfonyl)ethyl)prop-2-in-1-amine (38, 6.43 g, 98%) as oil. LCMS m / z:[M+H] + C7H 13 Calculated value of NSO2: 176.11; measured value: 176.1.

[0291] [Experimental procedure for N-((1-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)-N-methyl-2-(methylsulfonyl)ethane-1-amine (40)] [ka] A mixture of N-methyl-N-(2-(methylsulfonyl)ethyl)prop-2-in-1-amine (38, 5.02 g, 28.64 mmol, 1.25 equivalents), tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine (3.04 g, 5.73 mmol, 0.25 equivalents), copper iodide (436 mg, 2.29 mmol, 0.1 equivalent), and triethylamine (0.8 mL, 5.7 mmol, 0.25 equivalents) in methanol (50 mL) and water (6 mL) was evacuated, flushed with nitrogen (3 times), and cooled in an ice bath. 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane-1-amine (39, 5.02 g, 22.91 mmol, 1.0 equivalent) was added dropwise, the cooling bath was removed, and the mixture was stirred for 5 minutes. The reaction mixture was heated to 55°C and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, Celite (20 g) was added, and the mixture was concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 25% to obtain N-((1-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazole-4-yl)methyl)-N-methyl-2-(methylsulfonyl)ethane-1-amine (40, 4.98 g, 55%) as an oil. LCMS m / z:[M+H] + C 15 H 31 Calculated value of N5O5S: 394.2; measured value: 394.2.

[0292] [Experimental procedure for N-(2-(2-(2-(2-(4-((methyl(2-(methylsulfonylethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)methacrylamide (41)] [ka] A solution of N-((1-(2-(2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazole-4-yl)methyl)-N-methyl-2-(methylsulfonyl)ethane-1-amine (40, 1.0 g, 2.54 mmol, 1.0 equivalent) and triethylamine (0.43 mL, 3.05 mmol, 1.2 equivalents) was added dropwise to a solution of methacryloyl chloride (0.30 mL, 3.05 mmol, 1.5 equivalents). The reaction mixture was stirred at room temperature for 5 hours. Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (40 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 12.5% ​​to obtain N-(2-(2-(2-(2-(4-((methyl(2-(methylsulfonylethyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)methacrylamide (41, 0.86 g, 73% yield) as oil. LCMS m / z:[M+H] + C 19 H 35 Calculated value of N5O6S: 462.2; measured value: 462.2.

[0293] [Experimental procedure for 7-(prop-2-in-1-yl)-2-oxa-7-azaspiro[3.5]nonan(42)] [ka] 3-bromoprop-1-yin (4.4 mL, 39.32 mmol, 1.0 equivalent) was added to a mixture of 2-oxa-7-azaspiro[3.5]nonane (8.54 g, 39.32 mmol, 1.0 equivalent) and potassium carbonate (17.9 g, 129.7 mmol, 3.3 equivalents) in methanol (200 mL), and stirred overnight at room temperature. The mixture was filtered, Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (220 g) using dichloromethane / methanol as the mobile phase. The methanol concentration was gradually increased from 0% to 5% to obtain 7-(prop-2-yin-1-yl)-2-oxa-7-azaspiro[3.5]nonane (42, 4.44 g, 68%) as oil.

[0294] [Experimental procedure for 2-(2-(2-(2-(4-((2-oxa-7-azaspiro[3.5]nonan-7-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethane-1-amine (43)] [ka] A mixture of 7-(prop-2-in-1-yl)-2-oxa-7-azaspiro[3.5]nonane (42, 2.5 g, 15.13 mmol, 1.0 equivalent), tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine (1.77 g, 3.33 mmol, 0.22 equivalents), copper iodide (288 mg, 1.51 mmol, 0.1 equivalent), and triethylamine (0.53 mL, 3.8 mmol, 0.25 equivalents) in methanol (50 mL) was cooled in an ice bath. 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane-1-amine (39, 3.86 g, 17.70 mmol, 1.17 equivalents) was added dropwise, the cooling bath was removed, and the mixture was stirred for 5 minutes. The reaction mixture was heated to 55°C and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, Celite (10 g) was added, and the mixture was concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 10% to obtain 2-(2-(2-(2-(4-((2-oxa-7-azaspiro[3.5]nonan-7-yl)methyl)-1H-1,2,3-triazole-1-yl)ethoxy)ethoxy)ethoxy)ethane-1-amine (43, 4.76 g, 82%) as oil. LCMS m / z:[M+H] + C 18 H 33 Calculated value of N5O4: 384.3; measured value: 384.2.

[0295] [Experimental procedure for N-(2-(2-(2-(2-(4-((2-oxa-7-azaspiro[3.5]nonan-7-yl)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethoxy)ethyl)methacrylamide (44)] [ka] A solution of 2-(2-(2-(2-(4-((2-oxa-7-azaspiro[3.5]nonan-7-yl)methyl)-1H-1,2,3-triazole-1-yl)ethoxy)ethoxy)ethoxy)ethane-1-amine (43, 2.65 g, 6.91 mmol, 1.0 equivalent) and triethylamine (1.16 mL, 8.29 mmol, 1.2 equivalents) in CH2Cl2 (100 mL) was cooled in an ice bath under a nitrogen atmosphere. Methacryloyl chloride (0.74 mL, 7.6 mmol, 1.1 equivalents) was added dropwise. The cooling bath was removed and the reaction mixture was stirred at room temperature for 4 hours. 10 g of Celite was added and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (120 g) using dichloromethane / methanol as the mobile phase. The methanol concentration was gradually increased from 0% to 10% to obtain N-(2-(2-(2-(2-(4-((2-oxa-7-azaspiro[3.5]nonan-7-yl)methyl)-1H-1,2,3-triazole-1-yl)ethoxy)ethoxy)ethoxy)ethyl)methacrylamide (44, 1.50 g, 48% yield) as a colorless oil. LCMS m / z:[M+H] + C 22 H 37 Calculated value of N5O5: 452.29; measured value: 452.25.

[0296] [Experimental procedure for 4-((1-(2-(2-aminoethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)thiomorpholine 1,1-dioxide (45)] [ka] A mixture of 4-(prop-2-in-1-yl)thiomorpholine 1,1-dioxide (1.14 g, 6.58 mmol, 1.0 equivalent), tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine (768 mg, 1.45 mmol, 0.22 equivalents), copper iodide (125 mg, 0.66 mmol, 0.1 equivalent), and triethylamine (0.23 mL, 1.65 mmol, 0.25 equivalents) in methanol (20 mL) was cooled in an ice bath. 2-(2-azidoethoxy)ethane-1-amine (1.00 g, 7.70 mmol, 1.17 equivalents) was added dropwise, the cooling bath was removed, and the mixture was stirred for 5 minutes. The reaction mixture was warmed to 55°C and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, 10 g of Celite was added, and the mixture was concentrated under reduced pressure. The crude product was purified on silica gel (40 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 9.5% to obtain 4-((1-(2-(2-aminoethoxy)ethyl)-1H-1,2,3-triazole-4-yl)methyl)thiomorpholine 1,1-dioxide (45, 1.86 g, 93%) as a white solid. LCMS m / z:[M+H] + C 11 H 21 Calculated value of N5O4S: 304.1438; measured value: 304.1445.

[0297] [Experimental procedure for N-(2-(2-(4-((1,1-dioxidethiomorpholino)methyl)-1H-1,2,3-triazole-1-yl)ethoxyethyl)methacrylamide (46)] [ka] A solution of 4-((1-(2-(2-aminoethoxy)ethyl)-1H-1,2,3-triazole-4-yl)methyl)thiomorpholine 1,1-dioxide (45, 1.32 g, 4.35 mmol, 1.0 equivalent) and triethylamine (0.73 mL, 5.22 mmol, 1.2 equivalents) in CH2Cl2 (100 mL) was cooled in an ice bath under a nitrogen atmosphere. Methacryloyl chloride (0.47 mL, 4.8 mmol, 1.1 equivalents) was added dropwise. The cooling bath was removed, and the reaction mixture was stirred at room temperature for 4 hours. 10 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (120 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of methanol (containing a 12% (v / v) aqueous solution of ammonium hydroxide) was gradually increased from 0% to 1.25% to obtain N-(2-(2-(4-((1,1-dioxidethiomorpholino)methyl)-1H-1,2,3-triazole-1-yl)ethoxy)ethyl)-methacrylamide (46, 0.90 g, 56% yield) as a colorless oil. LCMS m / z:[M+H] + C 15 H 25 Calculated value of N5O4S: 372.17; measured value: 372.15.

[0298] [Experimental procedure for 4-((1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)methyl)thiomorpholine 1,1-dioxide (47)] [ka] A mixture of 4-(prop-2-in-1-yl)thiomorpholine 1,1-dioxide (4.6 g, 26.55 mmol, 1.0 equivalent), tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine (3.1 g, 5.84 mmol, 0.22 equivalents), copper iodide (506 mg, 2.66 mmol, 0.1 equivalent), and triethylamine (0.93 mL, 6.64 mmol, 0.25 equivalents) in methanol (80 mL) was cooled in an ice bath. 2-(2-(2-azidoethoxy)ethoxy)ethane-1-amine (5.00 g, 28.68 mmol, 1.08 equivalents) was added dropwise, the cooling bath was removed, and the mixture was stirred for 5 minutes. The reaction mixture was warmed to 55°C and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, Celite was added, and the mixture was concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 10% to obtain 4-((1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-1,2,3-triazole-4-yl)methyl)thiomorpholine 1,1-dioxide (47, 5.26 g, 57%) as a yellowish oil. LCMS m / z:[M+H] + C 13 H 25 Calculated value of N5O4S: 348.1700; measured value: 348.1700.

[0299] [Experimental procedure for N-(2-(2-(2-(4-((1,1-dioxidethiomorpholino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)ethoxy)ethyl)methacrylamide (48)] [ka] A solution of 4-((1-(2-(2-(2-aminoethoxy)ethoxy)ethyl)-1H-1,2,3-triazole-4-yl)methyl)thiomorpholine 1,1-dioxide (47, 1.49 g, 4.29 mmol, 1.0 equivalent) and triethylamine (0.72 mL, 5.15 mmol, 1.2 equivalents) in CH2Cl2 (50 mL) was cooled in an ice bath under a nitrogen atmosphere. Methacryloyl chloride (0.46 mL, 4.7 mmol, 1.1 equivalents) was added dropwise. The cooling bath was removed, and the reaction mixture was stirred at room temperature for 4 hours. 10 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (80 g) using dichloromethane / methanol as the mobile phase. The methanol concentration was gradually increased from 0% to 5% to obtain N-(2-(2-(2-(4-((1,1-dioxidethiomorpholino)methyl)-1H-1,2,3-triazole-1-yl)ethoxy)ethoxy)ethyl)-methacrylamide (48, 0.67 g, 38% yield) as a colorless oil. LCMS m / z:[M+H] + C 17 H 29 Calculated value of N5O5S: 416.20; measured value: 416.20.

[0300] [Experimental procedure for 4-((1-(14-amino-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazole-4-yl)methyl)thiomorpholine 1,1-dioxide (49)] [ka] A mixture of 4-(prop-2-in-1-yl)thiomorpholine 1,1-dioxide (5.0 g, 28.86 mmol, 1.0 equivalent), tris[(1-benzyl-1H-1,2,3-triazole-4-yl)methyl]amine (3.37 g, 6.35 mmol, 0.22 equivalents), copper iodide (550 mg, 2.89 mmol, 0.1 equivalent), and triethylamine (1.01 mL, 7.22 mmol, 0.25 equivalents) in methanol (90 mL) was cooled in an ice bath. 14-azido-3,6,9,12-tetraoxatetradecane-1-amine (8.86 g, 33.77 mmol, 1.17 equivalents) was added dropwise, the cooling bath was removed, and the mixture was stirred for 5 minutes. The reaction mixture was heated to 55°C and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, 15 g of Celite was added, and the mixture was concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide solution) was gradually increased from 0% to 10% to obtain 4-((1-(14-amino-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazole-4-yl)methyl)thiomorpholine 1,1-dioxide (49, 7.56 g, 60%) as oil. LCMS m / z:[M+H] + C 17 H 33 Calculated value of N5O6S: 436.2224; measured value: 436.2228.

[0301] [Experimental procedure for N-(14-(4-((1,1-dioxidethiomorpholino)methyl)-1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxatetradecyl)methacrylamide (50)] [ka] A solution of 4-((1-(14-amino-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-triazole-4-yl)methyl)thiomorpholine 1,1-dioxide (49, 1.95 g, 4.79 mmol, 1.0 equivalent) and triethylamine (0.80 mL, 5.74 mmol, 1.2 equivalents) in CH2Cl2 (50 mL) was cooled in an ice bath under a nitrogen atmosphere. Methacryloyl chloride (0.51 mL, 5.26 mmol, 1.1 equivalents) was added dropwise. The cooling bath was removed, and the reaction mixture was stirred at room temperature for 4 hours. 10 g of Celite was added, and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (80 g) using dichloromethane / methanol as the mobile phase. The methanol concentration was gradually increased from 0% to 5% to obtain N-(14-(4-((1,1-dioxidethiomorpholino)methyl)-1H-1,2,3-triazole-1-yl)-3,6,9,12-tetraoxatetradecyl)methacrylamide (50, 0.76 g, 32% yield) as a colorless oil. LCMS m / z:[M+H] + C 21 H 37 Calculated value of N5O7S: 504.25; measured value: 504.20.

[0302] [Example 4A: Preparation of Biographic Modified Polymers] 1A. Chemically Modified Polymers. Polymer materials may be chemically modified with compounds of formula (I) (or pharmaceutically acceptable salts thereof) before the formation of the devices described herein (e.g., hydrogel capsules). Exemplary synthesis protocols of compounds for modifying polymer materials are outlined in Example 3 above. These compounds or other compounds may be used to chemically modify any polymer material.

[0303] For example, in the case of alginates, the alginate carboxylic acid is activated for coupling with one or more amine-functionalized compounds to obtain alginates modified with an afibrotic compound, such as the compound of formula (I). The alginate polymer is dissolved in water (30 mL / gram polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 equivalents) and N-methylmorpholine (1 equivalent). To this mixture, a solution of the target compound (e.g., compound 101 shown in Table 3) in acetonitrile (0.3 M) is added.

[0304] The amounts of compound and coupling reagent added are determined according to the desired concentration of the compound to be conjugated to the alginate, e.g., the conjugation density. A moderate conjugation density of compound 101 is typically in the range of 2% to 5%N, while a high conjugation density of compound 101 is typically in the range of 5.1% to 8%N. To prepare the CM-LMW-Alg-101-Medium polymer solution, dissolved unmodified low molecular weight alginate (approximate molecular weight < 75 kDa, G:M ratio ≥ 1.5) is treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (5.1 mmol / g alginate), N-methylmorpholine (10.2 mmol / g alginate), and compound 101 (5.4 mmol / g alginate). To prepare the CM-LMW-Alg-101-High polymer solution, dissolved unmodified low molecular weight alginates (approximate molecular weight < 75 kDa, G:M ratio ≥ 1.5) are treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (5.1 mmol / g alginate), N-methylmorpholine (10.2 mmol / g alginate), and compound 101 (10.5 mmol / g alginate).

[0305] The reaction is heated to 55°C for 16 hours, then cooled to room temperature, gently concentrated by rotary evaporation, and the residue is dissolved in water. The mixture is filtered through a cyano-modified silica gel (Silicycle) bed, and the filter cake is washed with water. The resulting solution is then thoroughly dialyzed (10,000 MWCO membrane), and the alginate solution is concentrated by lyophilization to obtain the desired chemically modified alginate as a solid, or concentrated using any technique suitable for producing a chemically modified alginate solution with a viscosity of 25 cP to 35 cP.

[0306] The binding density of chemically modified alginates is measured by combustion analysis of nitrogen percentage. Samples are prepared by dialyzing a solution of chemically modified alginates in water for 24 hours (10,000 MWCO membrane), changing the water twice, and then freeze-drying until a constant weight is reached.

[0307] For use in the production of hydrogel capsules as described in the following examples, a chemically modified alginate polymer is prepared with compound 101 (shown in Table 3), which is conjugated to a low molecular weight alginate (approximately MW < 75 kDa, G:M ratio ≥ 1.5) at a medium (2% to 5% N) or high (5.1% to 8% N) density as determined by combustion analysis for nitrogen percentage, and is referred to herein as CM-LMW-Alg-101-Medium and CM-LMW-Alg-101-High.

[0308] 1B. CBP-alginate. Polymer materials may be covalently modified with cell-binding peptides using methods known in the art before the formation of the devices described herein (e.g., the hydrogel capsules described herein) (see, for example, Jeon O, et al., Tissue Eng Part A.16:2915-2925 (2010) and Rowley, JA et al., Biomaterials 20:45-53 (1999)).

[0309] For example, in the case of alginate, an alginate solution (1%, w / v) is prepared at pH 6.5 with a 50 mM 2-(N-morpholino)-ethanesulfonic acid hydrate buffer solution containing 0.5 M NaCl, and then sequentially mixed with N-hydroxysuccinimide and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDC). The molar ratio of N-hydroxysuccinimide to EDC is 0.5:1.0. The target peptide is added to this alginate solution. The amount of peptide and coupling reagent added depends on the desired concentration of the peptide bound to the alginate (e.g., peptide conjugation density). By increasing the amount of peptide and coupling reagent, a higher conjugation density can be obtained. After a 24-hour reaction, the reaction is purified by dialysis against ultra-high purity deionized water (diH2O) (MWCO 3500) for 3 days, treated with activated carbon for 30 minutes, filtered (0.22 mm filter), and concentrated to the desired viscosity.

[0310] The conjugation density of peptide-modified alginates is measured by combustion analysis for nitrogen percentage. Samples are prepared by dialyzing a solution of chemically modified alginates in water over 24 hours (10,000 MWCO membrane), changing the water twice, and then freeze-drying to a certain weight.

[0311] In another embodiment, the conjugation density of the peptide-modified alginate is measured using the quantitative peptide conjugation assay described in Example 7 and optionally in Example 8.

[0312] [Example 4B: Preparation of exemplary alginate solution for creating hydrogel capsules] A 70:30 mixture of chemically modified and unmodified alginates. Low molecular weight alginates (PRONOVA® VLVG alginate, NovaMatrix, Sandvika, Norway, cat.#4200506, approximate molecular weight <75 kDa; G:M ratio ≥1.5) were chemically modified with compound 101 in Table 3 to produce chemically modified low molecular weight alginate (CM-LMW-Alg-101) solutions with a viscosity of 25 cp to 35 cP and a conjugation density of 5.1% to 8% N as determined by combustion analysis for nitrogen percentage. A solution of high molecular weight unmodified alginate (U-HMW-Alg) was prepared by dissolving unmodified alginate (PRONOVA® SLG100, NovaMatrix, Sandvika, Norway, cat.#4202106, with an approximate molecular weight of 150kDa to 250kDa) in 0.9% physiological saline at a weight-to-volume ratio of 3%. The CM-LMW-Alg solution was mixed with a U-HMW-Alg solution (referred to herein as a 70:30 CM-Alg:UM-Alg solution) in a volume ratio of 70% CM-LMW-Alg to 30% U-HMW-Alg.

[0313] Unmodified alginate solution. An unmodified intermediate molecular weight alginate (SLG20, NovaMatrix, Sandvika, Norway, cat.#4202006, approximate molecular weight of 75-150 kDa) was dissolved in 0.9% physiological saline at a weight-to-volume ratio of 1.4% to prepare a U-MMW-Alg solution.

[0314] An alginate solution containing a cell binding site. The SLG20 alginate solution was modified with a peptide consisting of GRGDSP (SEQ ID NO: 44) and concentrated to a viscosity of approximately 100 cP as described in Example 4A above. In the embodiment, the amounts of GRGDSP peptide (SEQ ID NO: 44) and coupling reagent used were selected to achieve a target peptide conjugation density of approximately 0.2 to 0.3, as measured by the combustion analysis described in Example 4A above. In another embodiment, the amounts of medium molecular weight alginate (approximate molecular weight 75-150 kDa, G:M ratio ≥ 1.5), GRGDSP peptide, and coupling reagent used are selected to prepare a GRGDSP-MMW-Alg solution ("GRGDSP" disclosed as SEQ ID NO: 44) having a target peptide conjugation density of 0.3-0.6 micromoles of GRGDSP ("GRGDSP" disclosed as SEQ ID NO: 44) per gram of GRGDSP-MMW-Alg ("GRGDSP" disclosed as SEQ ID NO: 44) in physiological saline having a viscosity of 80-120 cP.

[0315] [Example 5: Formation of a 2-compartment hydrogel capsule] The recombinant ARPE-19 cell (i.e., GLA 4-2 cell) suspension was encapsulated as single cells in a two-compartment hydrogel capsule according to the protocol described below.

[0316] Immediately before encapsulation, recombinant ARPE-19 cells were centrifuged at 1,400 rpm for 1 minute and washed with calcium-free Krebs-Henseleit (KH) buffer (4.7 mM KCl, 25 mM HEPES, 1.2 mM KH2PO4, 1.2 mM MgSO4 × 7H2O, 135 mM NaCl, pH ≈ 7.4, approximately 290 mOsm). After washing, the cells were centrifuged again and all of the supernatant was aspirated. Next, the cell pellet was resuspended in the GRGDSP-modified alginate solution described in Example 4B at a density of approximately 100 million suspended single cells per ml of alginate solution.

[0317] Before preparing the hydrogel capsules, the buffer and alginate solution were sterilized by filtration through a 0.2 μm filter using a sterile process.

[0318] To prepare particles consisting of two-compartment hydrogel millicapsules with a diameter of approximately 1.5 mm, the electrostatic droplet generator was configured as follows: An ES series 0-100kV, 20-watt high-voltage generator (EQ series, Matsusada, NC, USA) was connected to the upper and lower parts of a concentric needle (22G lumen, 18G lumen, Rame-Hart Instrument Co., Succasunna, NJ, USA). The lumen was attached to a first BD disposable 5 ml syringe equipped with a BD Luer-Lock syringe (BD, NJ, USA), and this syringe was connected to a vertically oriented syringe pump (Pump 11 Pico Plus, Harvard Apparatus, Holliston, MA, USA). The lumen was connected to a second 5 ml Luer-lock syringe via a Luer coupling, and this syringe was connected to a second horizontally oriented syringe pump (Pump 11 Pico Plus). A first alginate solution containing recombinant GLA-ARPE-19 cells (as single cells) suspended in a GRGDSP-modified alginate solution ("GRGDSP" is disclosed as SEQ ID NO: 44) was placed in a first syringe, and a cell-free alginate solution containing a mixture of chemically modified and unmodified alginates was placed in a second syringe. Two syringe pumps moved the first and second alginate solutions from the syringes through both lumens of a concentric needle, and a single droplet containing both alginate solutions was pushed from the needle into a glass dish containing the crosslinking solution. Each Pico Plus syringe pump was set to a diameter of 12.06 mm, and the flow rate of each pump was adjusted to achieve a 1:1 flow rate ratio for the two alginate solutions. Thus, with a total flow rate set at 10 ml / hour, the flow rate of each alginate solution was approximately 5 ml / hour.

[0319] After extruding the desired volume of alginate solution, the alginate droplets were crosslinked for 5 minutes in a crosslinking solution containing 25 mM HEPES buffer, 20 mM BaCl2, 0.2 M mannitol, and 0.01% poloxamer 188. The capsules that fell to the bottom of the crosslinking container were collected by pipetting into a conical tube. After the capsules settled in the tube, the crosslinking buffer was removed and the capsules were washed. Capsules free of cells were washed four times with HEPES buffer (15.428 g NaCl, 0.70 g KCl, 0.488 g MgCl2·6H2O, 0 ml HEPES (1M) buffer in 2 liters of deionized water (Gibco, Life Technologies, California, USA)) and stored at 4°C until use. The capsules containing the cells were washed four times with HEPES buffer, twice with 0.9% physiological saline, and twice with culture medium, and then stored in an incubator at 37°C.

[0320] The quality of capsules with a two-compartment configuration can be examined. For example, an aliquot containing at least 200 capsules can be taken from the composition, transferred to a well plate, and the entire aliquot can be inspected for quality by optical microscopy by counting the number of spherical capsules in total.

[0321] [Example 6: Evaluation of a device containing GLA recombinant RPE cells in an animal model of Fabry disease] The biological activity of exemplary GLA-producing implantation devices for treating Fabry mice was evaluated in single-dose and dose-response experiments described below.

[0322] The Fabry mice used in each experiment were 8-11 week old male mice purchased from Jackson Labs (https: / / www.jax.org / strain / 003535). This mouse strain contains a neocassette that replaces exon 3 and intron 3 of the galactosidase, alpha (Gla) gene, thus rendering gene expression inactive. All mice were housed in the animal facility under pathogen-free conditions, following protocols approved by IACUC. Procedures involving mice followed guidelines established by the Association for Assessment of Accreditation of Laboratory Animal Care (AAALAC).

[0323] As described in Example 5 above, two-compartment GLA-producing or control hydrogel capsules were prepared. The GLA-producing capsules were prepared by encapsulating cells from a GLA 4-2 clone (e.g., ARPE-19 cells recombinant with a GLA 4 expression construct). In single-dose experiments, approximately 140 GLA-producing capsules or control capsules were intraperitoneally implanted into two groups of three Fabry mice. In dose-response experiments, approximately 40, 70, or 140 GLA-producing capsules or approximately 140 control capsules were implanted into four groups of five Fabry mice.

[0324] Mice were sacrificed either 14 days after transplantation (single dose) or 10 days after transplantation (dose-response), and blood, liver, kidney, and heart samples were collected from the sacrificed mice. Human GLA activity and Gb3 and Lyso-Gb3 levels were measured in the samples using the following assays.

[0325] [Enzyme assay of GLA activity] Blood samples were collected in EDTA solution, and plasma was separated according to a standard protocol.

[0326] Liver, kidney, and heart tissue samples were homogenized with matrix D using an MP Bio FastPrep-24 tissue homogenizer in 50 mM citrate, 176 mM K2PO4, pH 5.0. The samples were centrifuged at 12,000xg for 10 minutes at 4°C. The supernatant was diluted 5-fold with assay buffer (50 mM citrate, 176 mM K2PO4, 0.01% Tween-20, pH 5.0). 40 μl of the diluted homogenate or plasma was added to a Greiner black 96-well plate containing 40 μL of 1 mM 4-methylumbelliferyl β-D-galactopyranoside substrate and incubated at 37°C for 60 minutes. The reaction was stopped with 200 μL of 0.5 M sodium hydroxide and 0.5 M glycine (pH 11.6). Fluorescence intensity was measured using Biotek Synergy LX (excitation: 360 / 40, emission: 460 / 40). Data were normalized to the total protein concentration of the homogenate. Enzyme activity levels were compared to standard curves prepared for agalsidase beta and 4-methylumbelliferone.

[0327] [Detection of LysoGb3 and Gb3] Liver, kidney, and heart tissue samples were homogenized using MP Biomedicals FastPrep-25 5G Grinder in 2 mL Matrix D homogenization tubes provided by MP Biomedicals. The homogenates were then centrifuged at 14,000 g for 10 minutes, and the supernatant was transferred directly to HPLC vials for LC-MS / MS analysis.

[0328] For plasma analysis, plasma was diluted 20-fold with methanol, vortexed, and vigorously sonicated. The plasma extract was centrifuged at 14,000 g for 10 minutes, the supernatant was removed, and the extract was dried under a gentle stream of nitrogen. The dried extract was reconstituted with 100 uL of methanol and transferred to an HPLC vial for LC-MS / MS analysis.

[0329] [LCMS analysis] For the measurement of Lyso GB3 and GB3 isoforms, a Thermo Vanquish UHPLC and a Thermo Q-Exactive mass spectrometer were used. Chromatographic separation was performed using a Waters 2.1 × 100 mm BEH amide column packed with 1.7 μm particles. The column was maintained at 60°C and a flow rate of 0.3 mL / min. All separations were performed in gradient mode using mobile phase A consisting of 95:5 acetonitrile:H2O and aqueous mobile phase B, both containing 10 mM ammonium formate. Mass spectrometry was performed at a resolution of 70,000 using a full scan mode from 750 to 1200 m / z. Accurate mass was used for the quantification of 13GB3 isoforms and LysoGB3. To confirm the identity of each analyte, data-dependent MS2 scans were obtained and fragment ions indicating each species were examined. Quantification of GB3 isoforms and Lyso GB3 was performed by spiking C17 GB3 and Lyso GB3 for each tissue type and creating standard curves.

[0330] The results of these direct and indirect GLA activity assays for single-dose experiments are shown in Figures 11A–13D, and the results of indirect GLA activity assays for dose-response experiments are shown in Figures 14A–15D. Human GLA activity was detected in both liver and plasma samples (Figures 11A and 11B). Gb3 was readily detectable in plasma, liver, kidney, and cardiac tissue obtained from control Fabry mice at 14 days (Figures 12A–12D) or 10 days (Figures 14A–14D), with higher Gb3 levels observed in the kidney than in the liver, and in the heart, which was consistent with the original description of this Fabry disease model. In Fabry mice transplanted with a single dose of GLA-producing hydrogel capsules, a significant and equivalent decrease in accumulated Gb3 and Lyso-Gb3 levels was observed in plasma, liver, kidney, and cardiac samples 14 days after transplantation (Figures 12A–12D and 13A–13D). Furthermore, the decrease in Gb3 and Lyso-Gb3 levels in plasma, liver, and cardiac tissue over 10 days increased with increasing doses of the GLA-producing capsule (Figures 14A-14D and 15A-14D). These results demonstrate that the device preparations described herein can produce biologically active and therapeutically effective levels of human GLA when implanted in Fabry mice.

[0331] [Example 7: Exemplary quantitative peptide conjugation assay] In this assay, the amount of peptides in a CBP-polymer is determined by subjecting a CBP-polymer sample to acidic hydrolysis, which cleaves CBP into individual amino acids. Individual amino acids in this hydrolyzed sample are separated and quantified using amino acid standards by pre-column online derivatization and reverse-phase liquid chromatography-ultraviolet fluorescence (LC-UV-FLR) (Source: Agilent Biocolumns Amino Acid Analysis “How-To” Guide, Agilent Technologies, Inc., 5991-7694EN, published March 1, 2018). Primary AAs (e.g., all of the 20 standard L-alpha amino acids except proline) are derivatized with ortho-phthalaldehyde (OPA), and secondary AAs (e.g., proline) are derivatized with 9-fluorenylmethylchloroformate (FMOC). The molar concentrations of each amino acid are then averaged to calculate the total peptide concentration in the sample. For example, the amount of peptide in a non-hydrolyzed sample of the CBP-polymer can be determined using any suitable analytical technique described in Example 8, and this amount can be subtracted from the total peptide amount to correct for the presence of any residual non-conjugate CBP in the CBP-polymer.

[0332] This assay is further described below as being applied to determine the peptide conjugation density in GRGDSP-alginate (SEQ ID NO: 44), but those skilled in the art can readily modify this assay to determine the peptide concentration in GRGDSP-alginate (SEQ ID NO: 44) or other peptide-modified polymers if the unmodified polymer contains no amino acids. Similarly, those skilled in the art can readily replace any of the instruments, apparatus, or chemicals shown below with other instruments, apparatus, or chemicals that can perform or perform substantially the same function or role in this assay.

[0333] [Table 6]

[0334] [Equipment, instruments, and chemical substances] [device] • Agilent 1260 LC System • Agilent diode array detector (G1315D): 13 μL / 10 mm flow cell • Agilent fluorescence detector (G1321B) • AdvanceBio AA LC column, 2.7 μm, 4.6 × 100 mm, Agilent 655950-80 • AdvanceBio AAA Guard Column, 2.7 μm, 4.6 × 5 mm, Agilent 820750-931

[0335] [Table 7]

[0336] [procedure] Prepare 10 mM Na2HPO4 / Na2B4O7 / pH8.2 (aqueous mobile phase) and 45 / 45 / 10 ACN / MeOH / water (organic mobile phase) for use in LC / MS procedures.

[0337] [Acidic hydrolysis of exemplary peptide-alginate conjugate samples] Weigh 12-16 mg of freeze-dried peptide-alginate conjugate into a microwave reaction vial, ensuring that the sample is not agitated. • Hydrolysis is performed according to steps 3.3.2 to 3.3.19 below.

[0338] [Acidic hydrolysis of exemplary peptide-alginate conjugate samples in physiological saline] Weigh 1000±50 mg of peptide-alginate conjugate solution in physiological saline into a microwave reaction vial. Using a 10 mL transfer pipette or volumetric pipette and a stirring bar, add 10.0 mL of 6N HCl to the sample and seal the PTFE-lined cap with a crimper. Place each sample vial in the corresponding heat block on the heating / stirring plate, heat at 120°C, and stir at 400 rpm for 6 hours. Remove from the heat source and cool to ambient temperature. Remove the cap and transfer the entire solution from the reaction vial to a 20 mL volumetric flask using a disposable transfer pipette. Pipette 2 mL of LCMS-grade water into the empty reaction vial, thoroughly rinse the inner wall with the same disposable transfer pipette, and completely transfer this rinse solution into the same 20 mL volumetric flask. Repeat the above steps twice. • Use LCMS-grade water to adjust the volumetric flask to the markings. • Cap this flask and invert it several times to mix thoroughly. Transfer completely to a 50 mL centrifuge tube. • Centrifugation at 5000 rpm for 10 minutes • Accurately pipette 1 mL of supernatant into an LC vial and store at 2-8°C until dry (e.g., until the next day). (Store any remaining supernatant at 2-8°C for any retesting as needed.) • Completely dry 1 mL of the supernatant under nitrogen at 60°C, ensuring that the needle does not touch the sample but is low enough to dry the sample quickly. Pipette 0.25 mL of the 0.1 μmol / mL internal standard mixture into the vial containing the dried sample. • Vortex thoroughly • Transfer the contents of the LC vial insert to the LC vial using a pipette. • Store at 2-8°C until HPLC analysis (Step 3.6)

[0339] [Standard preparation] [AA standard stock solution: 10 μmol / mL] For each AA, calculate the weight required to prepare a 10 μmol / mL stock solution based on MW. See the example below:

[0340] [Table 8]

[0341] • Weigh the calculated weight into a 50 mL volumetric flask. • Dissolve in 0.1N HCl and adjust to the scale. Mix thoroughly by capping and inverting or vortexing. • Store at 2-8°C until HPLC analysis (Step 3.6)

[0342] [Internal standard mixture: 1μmol / mL] Accurately pipette 1.0 mL of norvaline stock solution (10 μmol / mL) and 1.0 mL of sarcosin stock solution (10 μmol / mL) into the same 10 mL volumetric flask. • Align scale with 0.1N HCl Mix thoroughly by capping and inverting or vortexing. • Store at 2-8°C until HPLC analysis (Step 3.6)

[0343] [Internal standard mixture: 0.1μmol / mL] Note: This solution is for reconstituting the sample after drying. Accurately pipette 1.0 mL of the internal standard mixture (1 μmol / mL) into a 10 mL volumetric flask. • Align scale with 0.1N HCl Mix thoroughly by capping and inverting or vortexing. • Store at 2-8°C until HPLC analysis (Step 3.6)

[0344] [5AA mixture (+iSTD0.1):0.025 / 0.1 / 0.25μmol / mL] Accurately pipette xx μL each of D, S, G, R, N-iSTD, S-iSTD, and P (10 μmol / mL) solutions (see "AA STD" in the table below) into the same 10 mL volumetric flask. • Align scale with 0.1N HCl Mix thoroughly by capping and inverting or vortexing. • Store at 2-8°C until HPLC analysis (Step 3.6)

[0345] [Table 9]

[0346] [17AA standard (+iSTD0.1) mixture: 0.1μmol / mL] • Open the ampoule of 0.1 μmol / mL 17AA standard solution. • Accurately pipette 0.9 mL of this AA standard mixture into an LC vial. Accurately pipette 100 μL of the iSTD mixture (1 μmol / mL) into the same LC vial. Mix thoroughly by vortexing. Dispense 100 μL of aliquot NLT into the LC vial containing the LC vial insert. • Store at 2-8°C until HPLC analysis (Step 3.6)

[0347] [Table 10]

[0348] [Table 11]

[0349] [System conformance criteria] The retention time and peak area of ​​each target AA (D / S / G / R) used for quantification, as well as internal standards (norvaline and sarcosine) in standard injection (both UV and FLR), will be analyzed.

[0350] [Table 12]

[0351] [Data Analysis - Samples will only be analyzed if the system's suitability is approved.] [Identifying the target AA] • The RT of the internal standard in the target AA and 17AA(+iSTD) standard mixture should match the RT of the 5AA(+iSTD) standard mixture. • The RT of AA in each sample should match the RT of the standard (UV / FLR). Relative area (D, S, G, R) = Area (D, S, G, R) / Area (norvaline) Relative area (P) = Area (P) / Area (Sarcosine)

[0352] [Standard Calibration Curve] • Calculate the concentration of the standard solution using the value reported in the certificate of analysis, or using the actual weight adjusted with the dilution factor used during standard preparation. Plot the mean area or mean relative area for each target AA concentration from the standard injections below: 0.025, 0.1, and 0.25 μmol / mL. Perform linear regression. concentration=m * Area or relative area + b (In the equation, m is the slope of the linear approximation curve, and b is the Y-intercept of the linear approximation curve.) • RSQ should be 0.99 or higher. If linearity is compromised, prepare a new derivatization reagent / standard solution, resolve the system malfunction, and repeat the test.

[0353] [Sample Analysis] • Quantitative analysis can be performed using both UV and FLR. • Quantitative analysis is performed using relative area (area ratio to internal standard). • Use a linear approximation standard curve to calculate the concentrations of AA:G, R, D, and S in each sample: Concentration, AA (μmol / mL) = (m * Area or relative area + b) • The total concentration of GRGDSP (SEQ ID NO: 44) is calculated by averaging the concentrations of each AA: Concentration, total GRGDSP (SEQ ID NO: 44) (μmol / mL) = (concentration, G / 2+ concentration, R+ concentration, D+ concentration, S+ concentration, P) / 5 μmol (total GRGDSP) (SEQ ID NO: 44) / g (conjugate) = concentration, total GRGDSP (μmol / mL) × 0.25 mL / 1.0 mL × 20 mL / weight (g) μmol(conjugate GRGDSP(SEQ ID NO: 44)) / g(conjugate) = μmol(total GRGDSP(SEQ ID NO: 44)) / g(conjugate) - μmol(residual free GRGDSP(SEQ ID NO: 44)) / g(conjugate)

[0354] [Example 8: Exemplary assay for determining residual free peptides in a CBP-polymer composition] This assay uses liquid chromatography-mass spectrometry (LC-MS) to determine the amount of residual non-conjugate peptides in a composition containing a peptide-polymer conjugate after removing a relative proportion of the non-conjugate peptide (e.g., more than 95%, 98%, 99%, or more) by, for example, typically one or more purification steps. Briefly, a sample of the conjugate in physiological saline is placed in an MWCO tube having a molecular weight cutoff (MWCO) higher than the molecular weight of the peptide, and the tube is centrifuged to separate the residual peptide from the conjugate. The amount of peptide is then quantified by LC-MS using a reference composition containing the same peptide at a known concentration as a standard.

[0355] [Example 9: Exemplary quantitative amine assay for determining amine conjugation density in antifibrous polymers modified with compound (I)] This assay determines the amount of amine-containing compounds (e.g., compounds of formula I, e.g., compound 101 in Table 3) in polymers chemically modified with amine compounds. A sample of the chemically modified polymer is subjected to acidic hydrolysis, which cleaves the conjugated amines. The weight percentage of the total amines in the hydrolyzed sample is quantified by reversed-phase liquid chromatography (LC-UV) with UV detection, using a non-conjugated amine compound as a standard. The identity of the LC peaks can be further confirmed by mass spectrometry. The weight percentage of the total amines can be used as the percentage conjugation of the amine compounds in the chemically modified polymer. More accurate results can be obtained by determining the amount of any residual non-conjugated amine compounds in the non-hydrolyzed sample of the chemically modified polymer using any suitable method (e.g., the method described below) and subtracting this amount from the total peptide amount.

[0356] This assay is further described below as being applied to determine the % conjugation density in an alginate chemically modified with compound 101 (i.e., CM-LMW-Alg-101), but those skilled in the art can readily modify this assay to determine the conjugation density of any compound of formula I used to chemically modify an amine-free polysaccharide (e.g., alginate) or another polymer. Similarly, those skilled in the art can readily replace any of the instruments, apparatus, or chemicals shown below with other instruments, apparatus, or chemicals that can perform or perform substantially the same function or role in this assay.

[0357] [Table 13]

[0358] [device] • Agilent 1260 LC system (DAD: 13μL / 10mm flow cell) • Agilent SQ MS detector (G1956B) · XBridge C18, 2.5μm, 4.6×50mm, Waters 186006037 • Small molecule reference material (unconjugate, free amine version of compound 101 in Table 3; purity over 98.0%)

[0359] [procedure] Prepare 0.1% ammonia in aqueous solution (aqueous mobile phase) and 0.1% ammonia in ACN solution (organic mobile phase) for use in the LC procedure.

[0360] [Exemplary solid alginate-small molecule conjugate sample acid hydrolysis] Weigh 50±5 mg of freeze-dried small molecule alginate conjugate solid into a microwave reaction vial, ensuring that the sample is not agitated. Add 10.0 mL of 2N HCl using a 10 mL transfer pipette or volumetric pipette and a stirring bar, then seal the PTFE-lined cap with a crimper. Place each sample vial in the corresponding heat block on the heating / stirring plate, heat to 120°C, and stir at 400 rpm for 120 minutes. Remove from the heat source and cool to ambient temperature. • Transfer the entire solution from the reaction vial to a 25 mL volumetric flask using a disposable transfer pipette. Pipette 5 mL of LCMS-grade water into the empty reaction vial, thoroughly rinse the inner wall with the same disposable transfer pipette, and completely transfer this rinse solution into the same 25 mL volumetric flask. Repeat the above steps twice. • Use LCMS-grade water to adjust the volumetric flask to the markings. Transfer completely to a 50 mL centrifuge tube. • Centrifugation at 3000 rpm for 10 minutes • The supernatant is subjected to HPLC analysis. Store at 2-8°C.

[0361] [Acidic hydrolysis of exemplary small alginate conjugate samples in physiological saline] Weigh 1000 ± 50 mg of a saline solution containing small molecule alginate conjugate into a microwave reaction vial. Add 10.0 mL of 2N HCl using a 10 mL transfer pipette or volumetric pipette and a stirring bar, then seal the PTFE-lined cap with a crimper. • Place each sample vial into a suitable heating block on the hot / stirring plate. Heat at 120°C for 120 minutes, stirring at 400 rpm. Remove from the heat source and cool to ambient temperature. • Transfer the entire solution from the reaction vial to a 25 mL volumetric flask using a disposable transfer pipette. Pipette 5 mL of LCMS-grade water into the empty reaction vial, thoroughly rinse the inner wall with the same disposable transfer pipette, and completely transfer this rinse solution into the same 25 mL volumetric flask. Repeat the above steps twice. • Use LCMS-grade water to adjust the volumetric flask to the markings. Transfer completely to a 50 mL centrifuge tube. • Centrifugation at 3000 rpm for 10 minutes • The supernatant is subjected to HPLC analysis. Store at 2-8°C.

[0362] [Sample preparation of residual free amines in solid small molecule alginate conjugates] Weigh 50±5 mg of freeze-dried small molecule alginate conjugate solid into a scintillation vial. Pipette 5.0 mL of physiological saline into this scintillation vial. Completely dissolve by shaking or vortexing for 10 minutes. Transfer completely to MWCO tubes. Centrifugal separation is performed at 5000 rpm for 60 minutes. Remove the top part of this MWCO tube and discard it. Completely transfer the sample from the bottom to a 5 mL volumetric flask. Fill the container with water or saline solution to the marked level, then invert the container and mix thoroughly. Transfer to a scintillation vial and store at 2-8°C. Transfer the aliquot to HPLC analysis.

[0363] [Sample preparation of residual free amines in small alginate conjugates in physiological saline] Weigh 1000±50 mg of small molecule alginate conjugate (or a blend with unmodified alginate) in physiological saline into a WMCO tube. Pipette 4.0 mL of physiological saline into this MWCO tube. Invert or vortex this tube five times, or until the solution is thoroughly mixed, to completely extract the free amines. Centrifugal separation is performed at 5000 rpm for 90 minutes. Remove the top part of this MWCO tube and discard it. Completely transfer the sample from the bottom to a 5 mL volumetric flask. Add water to the marked level, then invert and mix thoroughly. Transfer to a scintillation vial and store at 2-8°C. Transfer the aliquot to HPLC analysis.

[0364] [Standard preparation] [Standard solution: 1mg / mL] Weigh 50.00 ± 5.00 mg of small molecule reference material standard into a scintillation vial. Add approximately 10 mL of LCMS-grade water and shake or vortex to completely dissolve the solid. Using a disposable transfer pipette, completely transfer this scintillation vial to a 50 mL volumetric flask by rinsing it twice with LC-MS grade water. Add LCMS-grade water to the correct volume and mix thoroughly. Store at 2-8°C.

[0365] [Standard solution: 0.01mg / mL] Pipette 100 μL of this 1 mg / mL solution into a 10 mL volumetric flask. • Use LCMS grade water to match the volume. • Mix thoroughly by turning the device over. Store at 2-8°C.

[0366] [Table 14]

[0367] [Table 15]

[0368] [Data Analysis - Samples will only be analyzed if the system's suitability is approved.] [Identification of free amines] • (Optional) The m / z of the free amine peak in each sample should be within 392.1 ± 0.5. • The UV RT of the amine peak in each sample is consistent with the standard RT. Concentration, standard (mg / mL) = Weight (mg) / 50 mL / Dilution factor Here, in the case of a 1.0 mg / mL standard, the dilution factor = 1; For a 0.01 mg / mL standard, the dilution factor is 100. Concentration, free amine (mg / mL) = Area, non-hydrolyzed sample / area, 0.01 standard × concentration, 0.01 standard % Residual Free Amine = Concentration, free amine (mg / mL) × 5mL / Weight, non-hydrolyzed conjugate (mg) x 100 Concentration, total amine (mg / mL) = Area, Hydrolysis sample / area, 1.0 standard × concentration, 1.0 standard % Total amines = Concentration, total amine (mg / mL) × 25 mL / weight, hydrolyzed conjugate (mg) × 100

[0369] [Equivalents and ranges] This application references various published patents, published patent applications, scholarly articles, books, manuals and other publications, which are incorporated herein by reference in their entirety. In the event of any discrepancy between any of the incorporated documents and this specification, this specification shall prevail. In addition, any particular embodiment of this disclosure belonging to the prior art may be expressly excluded from any one or more claims. Such embodiments may be excluded even if not expressly excluded herein, as they are considered to be known to those skilled in the art. Any particular embodiment of this disclosure may be excluded from any claim for any reason, whether or not it relates to the existence of the prior art.

[0370] Those skilled in the art will be able to recognize or verify equivalents of the specific embodiments described herein without performing many routine experiments. The scope of the embodiments described herein is not intended to be limited to the above description, drawings, or examples, but is intended to be as set forth in the appended claims. Those skilled in the art will acknowledge that various changes and modifications to this description may be made without departing from the spirit or scope of this disclosure, as defined in the following claims.

Claims

1. Recombinant mammalian cells capable of expressing and secreting GLA protein, wherein the mammalian cells have the following characteristics: (a) Codon-optimized for expression in the mammalian cells; and (b) Encodes a GLA fusion protein including the following: (i) A signal peptide from a secretory protein other than GLA, operably ligated to the N-terminus of a mature human GLA amino acid sequence, comprising the signal peptide of SEQ ID NO: 15; or (ii) A GLA fusion protein comprising a signal peptide operably linked to the N-terminus of a mature human GLA amino acid sequence, and an amino acid sequence encoding a non-GLA polypeptide operably linked to the C-terminus of the GLA amino acid sequence, wherein the signal peptide is sequence number 15. Recombinant mammalian cells, which are retinal pigment epithelial cells transfected with a transcription unit comprising an exogenous nucleotide sequence containing a promoter operably linked to a precursor GLA coding sequence having [a certain characteristic].

2. The recombinant cell according to claim 1, wherein the promoter comprises a nucleotide sequence that is identical to, or at least 95% identical to, SEQ ID NO:

18.

3. The recombinant cell according to claim 1, wherein the precursor GLA coding sequence includes SEQ ID NO: 3 or SEQ ID NO:

4.

4. The recombinant cell according to claim 1, wherein the precursor GLA coding sequence includes sequence number 16.

5. The recombinant cell according to claim 1, wherein the GLA fusion protein comprises SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO:

13.

6. The recombinant cell according to claim 5, wherein the precursor GLA coding sequence includes SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, or SEQ ID NO:

14.

7. The recombinant cell according to claim 1, wherein the exogenous nucleotide sequence includes sequence number 47.

8. The recombinant cell according to claim 1, wherein the exogenous nucleotide sequence includes an extrachromosomal vector or is incorporated into at least one chromosomal location within the mammalian cell.

9. A composition comprising recombinant mammalian cells as described in claim 1.

10. The composition according to claim 9, wherein the composition is a polyclonal cell culture or a monoclonal cell line culture.

11. An implantable device comprising at least one cell-containing compartment containing recombinant cells as described in claim 1, and at least one means for reducing the foreign body reaction (FBR) when the device is implanted in a subject.

12. The implantable device according to claim 11, wherein the at least one cell-containing compartment contains a polymer composition that encapsulates the recombinant cells.

13. The implantable device according to claim 12, wherein the cell-containing compartment is surrounded by a barrier compartment containing an alginate hydrogel.

14. The polymer composition comprises an alginate covalently modified with a peptide, the peptide being GRGDSP (SEQ ID NO: 44) or GGRGDSP (SEQ ID NO: 45), and the barrier compartment is 【Chemistry 1】 The implantable device according to claim 13, comprising an alginate modified with or a pharmaceutically acceptable salt thereof.

15. It is a hydrogel capsule, (a) an inner compartment comprising recombinant cells according to claim 1, encapsulated in a first polymer composition, wherein the first polymer composition comprises a hydrogel-forming polymer; and (b) A barrier compartment surrounding the inner compartment and comprising a second polymer composition, wherein the second polymer composition comprises an alginate covalently modified with at least one compound selected from the group consisting of compound 100, compound 101, compound 110, compound 112, compound 113, and compound 114 shown in the following table. Table 1 Or a hydrogel capsule containing a pharmaceutically acceptable salt of the compound.

16. The selected compound is 【Chemistry 2】 The hydrogel capsule according to claim 15.

17. The hydrogel capsule according to claim 15, wherein the inner compartment contains a plurality of recombinant cells expressing an exogenous nucleotide sequence including sequence number 47.

18. A composition comprising a plurality of hydrogel capsules as described in claim 15.

19. An implantable device according to claim 11, a hydrogel capsule according to claim 15, or a composition according to claim 18 for treating Fabry disease.

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