manipulated active cell, implantable element, and pharmaceutical composition.

专利摘要:
Cell compositions comprising an active cell (e.g., a manipulated active cell, e.g., a manipulated RPE cell) or derivatives thereof, as well as compositions, pharmaceuticals, and implantable elements comprising an active cell, and methods of manufacturing and using them. The cells and compositions can express a therapeutic agent useful for the treatment of a disease, dysfunction or condition described herein.
公开号:BR112020006149A2
申请号:R112020006149-2
申请日:2018-09-27
公开日:2020-10-20
发明作者:Guillaume Carmona；Jered A. Sewell；Francisco Caballerro Gonzalez；Richard Heidebrecht；Robert James Miller；Matthias Alexander Oberli；David Peritt；Devyn McKinley Smith；Omid Veiseh；Paul Kevin Wotton
申请人:Sigilon Therapeutics, Inc.；
IPC主号:

专利说明:

[001] [001] This order claims priority over U.S. Provisional Order No. 62 / 563,877, filed September 27, 2017; U.S. Order No. 62 / 652,881, filed April 4, 2018; and U.S. Order No. 62 / 652,882, filed April 4, 2018. Disclosure for each of the previous orders is incorporated herein by reference in its entirety. SEQUENCE LISTING
[002] [002] The present application contains a Sequence Listing that was submitted electronically in ASCII format and is thus incorporated by reference in its entirety. Said ASCII copy, created on September 26, 2018, is called S2225-7015WO_SL.txt and is 205,145 bytes in size. BACKGROUND
[003] [003] The function of cells, tissues and implanted devices depends on numerous factors including the ability to provide a product and the recipient's biological immune response pathway (Anderson et al., Semin Immunol (2008) 20: 86–100; Langer , Adv Mater (2009) 21: 3235– 3236). The selection of cells and the modulation of the immune response can confer a beneficial effect on the fidelity and function of cells, tissues and implanted devices. SUMMARY
[004] [004] Cell compositions comprising an active cell, eg, a manipulated active cell, eg, a manipulated retinal pigment epithelial cell (RPE) or derivatives, as well as compositions, pharmaceuticals and implantable elements comprising an active cell, and methods of making and using them.
[005] [005] In one aspect, the present disclosure features an implantable element comprising an engineered active cell (e.g., a engineered RPE cell) that produces (e.g., or is capable of producing) a therapeutic agent. The therapeutic agent can be a biological substance, such as a nucleic acid (eg, a nucleotide, DNA or RNA), a polypeptide, a lipid, a sugar (eg, a monosaccharide, disaccharide, oligosaccharide or polysaccharide ) or a small molecule. In some embodiments, the therapeutic agent is a polypeptide and the engineered active cell comprises a promoter operably linked to a nucleotide sequence encoding the polypeptide, where the promoter consists essentially of a nucleotide sequence that is identical to, or substantially identical to, SEQ ID NO: 23. In some embodiments, the therapeutic agent is a replacement therapy or a replacement protein, e.g., useful for treating a blood clotting disorder or a lysosomal storage disease in a subject.
[006] [006] In some embodiments, the implantable element comprises a single manipulated active cell (eg, manipulated RPE cell). In some embodiments, the implantable element comprises a plurality of active manipulated cells (e.g., manipulated RPE cells), e.g., provided as a cluster or arranged in a microcarrier. In some embodiments, the active cell or active cells
[007] [007] In another aspect, the present disclosure provides a method of treating a subject comprising administering to the subject an implantable element comprising a manipulated active cell (e.g., a manipulated RPE cell). In some embodiments, the implantable element comprises a plurality of active manipulated cells (e.g., manipulated RPE cells). In some modalities, the subject is a human. In some embodiments, the manipulated active cell (eg, a manipulated active cell) is a human cell (eg, a human RPE cell). In some embodiments, the implantable element comprises a manipulated active cell (e.g., a manipulated RPE cell) that produces (e.g., or is capable of producing) a therapeutic agent, such as a nucleic acid (e.g. ., a nucleotide, DNA or RNA), a polypeptide, a lipid, a sugar (eg, a monosaccharide, disaccharide, oligosaccharide or polysaccharide) or a small molecule. In some embodiments, the therapeutic agent is a replacement therapy or a
[008] [008] In another aspect, the present disclosure presents a method of preparing or fabricating an implantable element comprising a manipulated active cell (e.g., a manipulated RPE cell). In some embodiments, the method comprises providing a manipulated active cell (eg, a manipulated RPE cell) and arranging the manipulated active cell (eg, the manipulated RPE cell) in an enveloping component, eg, as described here. In some embodiments, the implantable element comprises a plurality of active manipulated cells (e.g., manipulated RPE cells). In some embodiments, the implantable element comprises a plurality of active manipulated cells (e.g., manipulated RPE cells), e.g., provided as a cluster or arranged in a microcarrier. In some embodiments, the surrounding component is formed in situ in or surrounding a manipulated active cell (eg, manipulated RPE cell), a plurality of manipulated active cells (eg, manipulated RPE cells) or a microcarrier (eg eg a spherule or matrix) comprising an active cell or active cells. In some embodiments, the surrounding component is preformed prior to the combination with the manipulated active cell (eg, manipulated RPE cell) involved, a plurality of manipulated active cells (eg, manipulated RPE cells) or a microcarrier ( eg a spherule or
[009] [009] In another aspect, the present disclosure presents a method of evaluating an implantable element comprising a manipulated active cell (e.g., a manipulated RPE cell). In some embodiments, the method comprises providing a manipulated active cell (e.g., a manipulated RPE cell) and evaluating a structural or functional parameter of the encapsulated RPE cell. In some embodiments, the method comprises evaluating the manipulated active cell or a plurality of manipulated active cells for one or more of: a) viability; b) the production of a therapeutic agent (e.g., an engineered RNA or polypeptide); c) the capture of a nutrient or oxygen; or d) the production of a waste product. In some modalities, the evaluation is carried out at least 1, 5, 10, 20, 30 or 60 days after formation of the implantable element or administration of the implantable element to a subject.
[0010] [0010] In another aspect, the present disclosure presents a method of monitoring an implantable element comprising a manipulated active cell (e.g., a manipulated RPE cell). In some modalities, the method comprises obtaining, for example, by testing the subject or a sample of him, the level of a parameter; and comparing, for example, by testing the subject or a sample of him, the value obtained with that of a reference value. In some embodiments, the parameter comprises a) cell viability; b) level of production of a therapeutic agent (eg, an engineered RNA or polypeptide); c) the capture of a nutrient or oxygen; or d) the
[0011] [0011] In another aspect, the present disclosure features a plurality of active manipulated cells (e.g., manipulated RPE cells). In some embodiments, the plurality has a pre-selected form factor or a form factor described here, eg, a grouping of active manipulated cells (eg, manipulated RPE cells). In some embodiments, the cluster of engineered active cells (eg, engineered RPE cells) comprises at least about 5, 10, 25, 50, 75, 100, 200, 250, 300, 400, 500 or more active cells manipulated. In some modalities, the grouping is globular or spherical. In some modalities, the grouping is not a monolayer. In some embodiments, the cluster has a density of about 500 cells / cm2 or more. In some embodiments, the plurality of active manipulated cells (eg, manipulated RPE cells) is arranged in a microcarrier (eg, a spherule or matrix).
[0012] [0012] In another aspect, the present disclosure presents a substrate comprising a plurality of chambers, each chamber comprising a manipulated active cell (e.g., a manipulated RPE cell). In some embodiments, each chamber comprises a plurality of manipulated active cells (e.g., manipulated RPE cells). In some embodiments, the plurality comprises a cluster of manipulated active cells (eg, manipulated RPE cells) and / or is arranged in a microcarrier (eg, a spherule or matrix).
[0013] [0013] In another aspect, the present disclosure features a microcarrier, e.g., a spherule or matrix, having a manipulated active cell (e.g., a manipulated RPE cell) disposed therein.
[0014] [0014] In another aspect, the present disclosure presents a
[0015] [0015] Details of one or more embodiments of the invention are presented here. Other characteristics, objectives and advantages of the disclosure will be apparent from the Detailed Description, Figures, Examples and Claims. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] [0016] FIG. 1 is a graph illustrating the amount of an exemplary polypeptide released from encapsulated implantable elements comprising engineered active cells (e.g., engineered RPE cells) compared to active cells not encapsulated at various time points.
[0017] [0017] FIGS. 2A-2B are microscopic images of exemplary encapsulated implantable elements comprising manipulated active cells (e.g., manipulated RPE cells). As shown, implantable elements comprising active cells expressing Factor VIII-BDD show high viability over the duration of the experiment.
[0018] [0018] FIG. 3 shows the amino acid sequence of the human Factor VII-BDD protein encoded by an exemplary engineered RPE cell (SEQ ID NO: 1), with the underlined signal sequence.
[0019] [0019] FIG. 4 shows the amino acid sequence of a human wild type Factor IX protein (SEQ ID NO: 2).
[0020] [0020] FIGS. 5A-5H show the effect of cell architecture on cell packaging density, cell viability and quality of capsules for prepared implantable elements (eg hydrogel capsules)
[0021] [0021] FIGS. 6A-6G show the effect of cell architecture on cell packaging density, cell viability and quality of capsules for implantable elements (eg, hydrogel capsules) prepared using spheroid cell capsule suspensions. FIGS. 6A-6E are microscopy images of exemplary encapsulated implantable elements comprising manipulated active cells (eg, manipulated RPE cells) prepared from spheroidal suspensions of 30, 40, 50, 75 and 100 million cells / ml of solution alginate (M / mL), showing cell viability through live / dead staining. FIG. 6F illustrates the effect of spheroid concentration on the overall quality of the implantable element, and FIG. 6G represents the relationship between the number of cells contained within the implantable element and its overall quality.
[0022] [0022] FIGS. 7A-7H show the effect of cell architecture on cell packaging density, cell viability and quality of capsules for implantable elements (eg, hydrogel capsules) prepared using cell suspensions adhered to Cytodex® microcarriers. FIGS. 7A-7F are microscopy images of exemplary encapsulated implantable elements comprising manipulated active cells (eg, manipulated RPE cells) prepared from cell suspensions with Cytodex® microcarriers with 1: 8, 1: 4 volume ratios, 1: 2,
[0023] [0023] FIGS. 8A-8H show the effect of cell architecture on cell packaging density, cell viability and quality of capsules for implantable elements (eg, hydrogel capsules) prepared using cell suspensions adhered to CultiSpher® microcarriers. FIGS. 8A-8F are microscopy images of exemplary encapsulated implantable elements comprising manipulated active cells (eg, manipulated RPE cells) prepared from cell suspensions with CultiSpher® microcarriers with 1:14, 1:10 volume ratios, 1: 8, 1: 6, 1: 4 and 1: 2 (mL of pelleted microcarriers: mL of alginate solution), showing cell viability through live / dead staining. FIG. 8G illustrates the effect of the concentration of CultiSpher® microcarriers on the overall quality of the implantable element, and FIG. 8H represents the relationship between the number of cells contained within the implantable element and its overall quality.
[0024] [0024] FIG. 9 shows the levels of in vitro expression of a human Factor IX polypeptide (F9: hFIX, wild type; F9p: hFIX-Padua) directed by different exogenous promoters (CMV, CAP or Ubc) in engineered RPE cells or HS27 cells.
[0025] [0025] FIG. 10 is a schematic of a PiggyBac transposon expression vector useful for generating engineered RPE cells.
[0026] [0026] FIG. 11 shows the in vitro expression levels of the Factor VIII-BDD protein shown in FIG. 1 by RPE cells manipulated with coding sequence with codon optimization (CO2, CO3 or CO6)
[0027] [0027] FIG. 12 shows the levels of in vitro expression of different Factor VIII-BDD variant proteins by RPE cells manipulated with or without an FVIII-BDD coding sequence with codon optimization in relation to the expression level of the Factor VIII-BDD protein shown in FIG. 1 by RPE cells engineered with the BDD version of a naturally occurring (native) FVIII nucleotide sequence.
[0028] [0028] FIG. 13 shows the levels of in vitro expression of a human Factor IX protein (FIX-Padua) by RPE cells manipulated with a coding sequence of FIX-Padua with codon optimization (CO2, CO3 or CO5) in relation to the expression of FIX- Padua by RPE cells manipulated with a non-optimized coding sequence (Native).
[0029] [0029] FIG. 14 shows the levels of in vitro expression of human FIX-Padua by RPE cells manipulated with a transcription unit comprising a non-optimized (Native) FIX coding sequence or with one or two copies of the same transcription unit except that they comprise a coding sequence of FIX-Padua with codon optimization. DETAILED DESCRIPTION
[0030] [0030] The present disclosure presents cell therapy compositions comprising active cells, e.g., retinal pigment epithelial cells (RPE) (e.g., engineered RPE cells) or their cell derivatives, as well as their cell compositions. implantable elements comprising them. In some embodiments, active cells, compositions and implantable elements are useful for the prevention or treatment of a disease, dysfunction or condition. The active cells described here exhibit advantageous properties, such as maintaining the density of
[0031] [0031] The following terms are intended to have the meanings presented below and are useful in understanding the description and intended scope of this disclosure.
[0032] [0032] "Acquire" or "acquisition", as used here, refers to obtaining possession of a value, eg, a numerical value or image or a physical entity (eg, a sample), for example “Direct acquisition” or “indirect acquisition” of the value or physical entity. “Direct acquisition” means carrying out a process (eg, carrying out an analytical method or protocol) to obtain the physical value or entity. “Indirect acquisition ”Refers to the receipt of the value or physical entity from another party or source (eg, a third party laboratory that directly purchased the physical entity or value). The direct acquisition of a value or physical entity includes carrying out a process that includes a physical change to a physical substance or the use of a machine or device. Examples of direct acquisition of a value include taking a sample from a human subject. Direct acquisition of a value includes performing a process using a machine or device, eg fluorescence microscope for to acquire fluorescence microscopy data.
[0033] [0033] "Active cell" as used herein refers to a cell having one or more of the following characteristics: a) comprises a retinal pigment epithelial cell (RPE) or a cell derived therefrom, including a cell derived from a cell culture primary cells of RPE cells, a
[0034] [0034] In some embodiments, an active cell, including a manipulated active cell, is not an islet cell. An islet cell as defined here is a cell that comprises any naturally occurring cell or any cell synthetically created, or modified, which is intended to recapitulate, mimic or otherwise express, in part or in whole, the functions, in part or altogether, from the cells of the pancreatic islets of Langerhans. An active cell, including a manipulated active cell, is unable to produce insulin (eg, insulin A chain, insulin B chain
[0035] [0035] "Administer", "administering" or "administration", as used here, refers to the implantation, absorption, ingestion, injection or otherwise introducing an entity (eg, an active cell, eg ., a manipulated RPE cell, or a composition thereof, or an implantable element comprising an active cell) or providing it to a subject.
[0036] [0036] "Cell", as used here, refers to a manipulated cell, eg, a manipulated active cell, or a cell that is not manipulated, eg, an unhandled active cell.
[0037] [0037] "Conservatively modified variants" or "conservative substitution", as used here, 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 one embodiment, a conservatively modified variant of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the reference amino acid sequence. A conservative amino acid substitution refers to the replacement of an amino acid by an amino acid having similar characteristics (eg, charge, size of the side chain, hydrophobicity / hydrophilicity, skeletal conformation and rigidity, etc.) and which has minimal impact on biological activity of the resulting substituted peptide or polypeptide. Conservative substitution tables of functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional characteristics are presented in
[0038] [0038] "Consists essentially of" and variations such as "consist essentially of" or "consisting essentially of", as used throughout the specification and claims, indicate the inclusion of any elements or group of elements recited, and the optional inclusion other elements, of a similar or different nature to the recited elements, which do not materially change the basic or new properties of the specified molecule, composition, device or method. As a non-limiting example, a therapeutic protein consisting essentially of a recited amino acid sequence can also include one or more amino acids, including additions at the N-terminus, C-terminus or within the recited amino acid sequence, of one or more amino acid residues, that do not materially affect the relevant biological activity of the therapeutic protein, respectively. As another non-limiting example, a promoter consisting essentially of a recited nucleotide sequence may contain one or more additional nucleotides that do not change
[0039] [0039] "Effective amount" as used herein refers to an amount of an active cell composition, eg, engineered RPE cells, or an agent, eg, a therapeutic agent, produced by an active cell, eg, a manipulated RPE cell, sufficient to elicit a biological response, eg, to treat a disease, dysfunction or condition. As will be appreciated by those skilled in the art, the effective amount may vary depending on such factors as the desired biological end point, the pharmacokinetics of the therapeutic agent, composition or implantable element, the condition being treated, the mode of administration and the age and health of the patient. subject. An effective amount includes prophylactic and therapeutic treatment. For example, to treat a fibrotic condition, an effective amount of a compound can reduce fibrosis or stop the growth or spread of fibrotic tissue.
[0040] [0040] An "endogenous nucleic acid", as used here, is a naturally occurring nucleic acid in a cell in question.
[0041] [0041] An "endogenous polypeptide", as used here, is a polypeptide that occurs naturally in a cell in question.
[0042] [0042] "Manipulated cell", as used here, is a cell, eg, an active cell, having a change that does not occur naturally and typically comprises a sequence of nucleic acids (eg, DNA or RNA) or a polypeptide not present (or present at a different level than that) in an otherwise similar cell under similar similar conditions that is not manipulated (an exogenous nucleic acid sequence). In one embodiment, a genetically modified cell comprises an exogenous nucleic acid (for example, a vector or chromosomal sequence
[0043] [0043] An "exogenous nucleic acid", as used here, is a nucleic acid that does not occur naturally in a cell in question.
[0044] [0044] An "exogenous polypeptide", as used here, is a polypeptide that does not occur naturally in a cell in question.
[0045] [0045] "Factor VII protein" or "FVII protein", as used here, means a polypeptide that comprises the amino acid sequence of a naturally occurring factor VII protein or its variant that has a biological FVII activity, e.g. promoting blood coagulation, as determined by a test recognized in the art, unless otherwise specified. Naturally occurring FVII exists as a
[0046] [0046] The biological activity of Factor VII can be quantified by an assay recognized in the art, unless otherwise specified. For example, the biological activity of FVII in a sample of a biological fluid, eg, plasma, can be quantified by (i) measuring the amount of Factor Xa produced in a system comprising TF embedded in a lipid membrane and Factor X. (Persson et al., J. Biol. Chem. 272: 19919- 19924, 1997); (ii) measurement of Factor X hydrolysis in an aqueous system; (iii) measuring its physical connection to TF using an instrument based on surface plasmon resonance (Persson, FEBS Letts. 413: 359-363, 1997); or (iv) measuring the hydrolysis of a synthetic substrate; and / or (v) measurement of thrombin generation in an in vitro TF-independent system. In one embodiment, FVII activity is assessed by a commercially available chromogenic assay (BIOPHEN FVII, HYPHEN
[0047] [0047] "Factor VIII protein" or "FVIII protein", as used herein, means a polypeptide that comprises the amino acid sequence of a naturally occurring factor VIII polypeptide or its variant that has a biological FVIII activity, e.g. coagulation activity, as determined by a test recognized in the art, unless otherwise specified. FVIII proteins that can be expressed by active cells described here, eg, engineered RPE cells, include primate (eg, human) wild-type, porcine, canine and murine proteins, as well as variants of such wild-type proteins, including fragments, mutants, variants with one or more substitutions and / or deletions of amino acids, B domain deletion (BDD) variants, single chain variants and fusions of any of the previous wild types or variants with a half-life-prolonging polypeptide. In one embodiment, the active cells are engineered to encode a precursor factor VIII polypeptide (eg, with the signal sequence) with a total or partial deletion of domain B. In one embodiment, the active cells are engineered to encode a single chain factor VIII polypeptide containing A variant A FVIII protein preferably has at least 50%, 75%, 90% or more (including> 100%) of the clotting activity of the corresponding wild type factor VIII. Assays for measuring the clotting activity of FVIII proteins include the one-step or two-step clotting assay (Rizza et al., 1982, Coagulation assay of FVIII: C and FIXa in Bloom ed. The Hemophelias. NY Churchill Livingston 1992) or the FVIII: C chromogenic substrate assay (Rosen, S. 1984. Scand J Haematol 33: 139-145, suppl.)
[0048] [0048] A number of FVIII-BDD variants are known and include,
[0049] [0049] In some embodiments, an FVIII-BDD protein expressed by RPE cells, eg, manipulated ARPE-19 cells, has one or more of the following amino acid deletions in the B domain: (i) most of the B domain except amino-terminal B domain sequences essential for intracellular processing of the primary translation product into two polypeptide chains (WO 91/09122); (ii) a deletion of amino acids 747-1638 (Hoeben R. C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990)); amino acids 771-1666 or amino acids 868-1562 (Meulien P., et al. Protein Eng. 2 (4): 301-6 (1988); amino acids 982-1562 or 760-1639 (Toole et al., Proc. Natl. Acad. Sci. USA 83: 5939-5942 (1986)); amino acids 797-1562 (Eaton et al., Biochemistry 25: 8343-8347 (1986)); 741-1646 (Kaufman, WO 87/04187)), 747 -1560 (Sarver et al., DNA 6: 553-564 (1987)); amino acids 741-1648 (Pasek, WO 88/00831)), amino acids 816-1598 or 741-1689 (Lagner (Behring Inst. Mitt. (1988) No. 82: 16-25, EP 295597); a deletion that includes one or more residues in a furin protease recognition sequence, eg, LKRHQR at amino acids 1643-1648, including any of the specific deletions recited in U.S. Patent No. 9,956,269 in col. 10, line 65 through col. 11, line 36.
[0050] [0050] In other embodiments, an FVIII-BDD protein retains any of the following B domain amino acids or amino acid sequences: (i) one or more N-linked glycosylation sites in the B domain, e.g., residues 757 , 784, 828, 900, 963 or, optionally, 943, the first 226 amino acids or the first 163 amino acids (Miao, HZ, et al., Blood 103 (a): 3412-3419 (2004), Kasuda, A., et al., J. Thromb. Haemost. 6: 1352-1359 (2008) and Pipe, SW, et al., J. Thromb. Haemost. 9: 2235-2242 (2011)).
[0051] [0051] In some embodiments, the FVIII-BDD protein is a single chain variant generated by replacing one or more amino acids in the furin protease recognition sequence (LKRHQR at amino acids 1643-1648) that prevents proteolytic cleavage at this location , including any of the substitutions in heading R1645 and / or R1648 described in U.S. Patent Nos. 10,023,628, 9,394,353 and 9,670,267.
[0052] [0052] In some embodiments, any of the above FVIII-BDD proteins may additionally comprise one or more of the following variations: an F309S substitution to improve expression of the FVIII-BDD protein (Miao, HZ, et al., Blood 103 (a): 3412-3419 (2004); albumin fusions (WO 2011/020866); and Fc fusions (WO 04/101740).
[0053] [0053] All FVIII-BDD amino acid positions referenced herein refer to positions in full-length human FVIII, unless otherwise specified.
[0054] [0054] "Factor IX protein" or "FIX protein", as used here, means a polypeptide comprising the amino acid sequence of a naturally occurring factor IX protein or its variant that has a biological FIX activity, e.g. coagulation activity, as determined by a test recognized in the art, unless otherwise specified. FIX is produced as an inactive zymogen, which is converted to an active form by excision with factor XIa of the activation peptide to produce a heavy chain and a light chain held together by a
[0055] [0055] A number of functional FIX variants are known and can be expressed by active cells of the present disclosure, including any of the functional FIX variants described in the following international patent publications: WO 02/040544 A3 on page 4, lines 9 -30 and page 15, lines 6-31; WO 03/020764 A2 in Tables 2 and 3 on pages 14-24 and on page 12, lines 1-27; WO 2007/149406 A2 on page 4, line 1 through page 19, line 11; WO 2007/149406 A2 on page 19, line 12 through page 20, line 9; WO 08/118507 A2 on page 5, line 14 through page 6, line 5; WO 09/051717 A2 on page 9, line 11 through page 20, line 2; WO 09/137254 A2 on page 2, paragraph [006] to page 5, paragraph [011] and page 16, paragraph [044] to page 24, paragraph [057]; WO 09/130198 A2 on page
[0043] [0043] up to page 13, paragraph [0053]; WO 2012/006624; WO 2015/086406.
[0056] [0056] In certain embodiments, the FIX polypeptide comprises a wild-type or variant sequence fused to a heterologous polypeptide or different polypeptide fraction extending the half-life of the FIX protein. Exemplary half-life extending fractions include Fc, albumin, a PAS sequence, transferrin, CTP (28-amino acid C-terminal peptide (CTP)) of human chorionic gonadotropin (hCG) with its 4 O-glycans, polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin-binding polypeptide, small albumin-binding molecules or any combination thereof. An exemplary FIX polypeptide is the rFIXFc protein described in WO 2012/006624, which is a single chain of FIXFc (FIXF c-sc) and a single chain of Fc (Fc-sc) linked together via two disulfide bonds in the hinge region of Fc.
[0057] [0057] The FIX variants also include variants of gain and loss of function. An example of a function gain variant is the “Padua” variant of human FIX, which has an L (leucine) at position 338 of the mature protein instead of an R (arginine) (corresponding to the amino acid position 384 of SEQ ID NO: 2) and has greater catalytic and coagulant activity compared to wild-type human FIX (Chang et al., J. Biol. Chem., 273: 12089-94 (1998)). An example of a loss of function variant is an alanine substituted by lysine in the fifth amino acid position from the beginning of the mature protein, which results in a protein with reduced binding to collagen IV (eg loss of function)
[0058] [0058] "Form factor", as used here, refers to one or more of: the number of active cells present in a plurality of active cells, the shape of the plurality of active cells, the level of contact between the active cells plurality or the level of junctions formed between the active cells of the
[0059] [0059] "Interleukin 2 protein" or "IL-2 protein", as used here, means a polypeptide comprising the amino acid sequence of a naturally occurring IL-2 protein or its variant that has biological activity of IL- 2, e.g., activate IL-2 receptor signaling in Treg cells, as determined by a test recognized in the art, unless otherwise specified. IL-2 proteins that can be expressed by active cells described here, e.g., engineered RPE cells, include primate (e.g., human) wild-type, porcine, canine, and murine proteins, as well as variants of such wild-type proteins. A variant IL-2 protein preferably has at least 50%, 75%, 90% or more (including> 100%) of the biological activity of the corresponding wild-type IL-2. Biological activity assays for IL-2 proteins are described in U.S. Patent No. 10,035,836 and include, for example, measurement of STAT5 protein levels
[0060] [0060] An "implantable element", as used here, comprises an active cell, eg, a plurality of active cells, eg, a grouping of active cells, in which the active cell or active cells are entirely or partially arranged within an enveloping component (a component surrounding it that is different from an active cell), eg, the enveloping component comprises a non-cellular component. In one embodiment, the surrounding component inhibits an immune attack, or effect of the immune attack, on the active cell or active cells involved. In one embodiment, the surrounding component comprises a semipermeable membrane or a semipermeable polymer matrix or coating. Typically, the surrounding component allows small molecules, eg nutrients and waste products, to pass through. Typically, the surrounding component allows for the passage of a product (e.g., a therapeutic polypeptide) released by an active cell disposed within the surrounding component. In one embodiment, placement within an enveloping component minimizes an effect of an immune response, eg, a fibrotic response, from the subject directed on the implantable element, eg, against an active cell within an implantable element, eg in comparison with a similar active cell that is not arranged in an implantable element. In one embodiment, the surrounding component comprises a fraction, e.g., a fraction described here (e.g., a compound in the Compound Table 1), which minimizes an effect of an immune response, e.g., a response fibrotic, of the subject directed at the implantable element, eg, against the surrounding component or an active cell within the implantable element,
[0061] [0061] In one embodiment, an implantable element comprises an enveloping component that is formed, or could be formed, in situ in or surrounding an active cell, e.g., a plurality of active cells, e.g., a cluster of active cells, or cells in a microcarrier, eg, a spherule, or a matrix comprising an active cell or active cells (referred to here as an “in situ encapsulated implantable element”).
[0062] [0062] In one embodiment, the implantable element comprises an enclosing component comprising a flexible polymer, eg, alginate (eg, a chemically modified alginate), PLA, PLG, PEG, CMC or mixtures thereof (referred to here as a “polymer-encapsulated implantable device”).
[0063] [0063] Implantable devices encapsulated in situ and implantable devices encapsulated in polymer (categories that are not mutually exclusive) are collectively referred to here as implanted encapsulated elements.
[0064] [0064] An exemplary encapsulated implantable element comprises an active cell, e.g., a plurality of active cells, e.g.,
[0065] [0065] In one embodiment, an implantable element comprises an enveloping component that is preformed prior to combining with the active cell involved, e.g., a plurality of active cells, e.g., a grouping of active cells, or a microcarrier, e.g., a spherule or a matrix comprising an active cell (referred to here as implantable element based on device or implantable element DB). In one embodiment, an implantable device element comprises an enclosing component comprising a polymer or metal. An implantable element in an exemplary device comprises an active cell, e.g., a plurality of active cells, e.g., a cluster of active cells, or a microcarrier, e.g., a spherule comprising an active cell or cells , arranged within a surrounding component comprising a preformed housing, e.g., a polymeric or inflexible metal housing, or a flexible housing, e.g., a semipermeable membrane. In embodiments, an implantable device element has a linear dimension greater than at least 1.5 mm, 2 mm, 3 mm, 4 mm, 5 mm, 6 mm, 7 mm or 8 mm.
[0066] [0066] "Parathyroid hormone protein" or "PTH protein", as used here, means a polypeptide that comprises the amino acid sequence of a naturally occurring parathyroid hormone polypeptide or its variant that has a biological PTH activity, eg as determined by a test recognized in the art. PTH polypeptides that can be expressed by active cells described here (e.g., cells
[0067] [0067] "Polypeptide", as used herein, refers to a polymer comprising amino acid residues linked via peptide bonds and having at least two and, in some embodiments, at least 10, 50, 75, 100, 150 or 200 amino acid residues. The term "polypeptide" is intended to include any chain or chains of two or more amino acids and includes without limitation peptides, dipeptides, tripeptides, oligopeptides and proteins, and the term "polypeptide" can be used instead of, or interchangeably with, any one of these terms. The term "polypeptide" is also intended to refer to the products of post-translational modifications of a polypeptide encoded by an exogenous nucleotide sequence within the engineered cell, including, without limitation: proteolytic cleavage
[0068] [0068] "Prevention", "prevent" and "preventing" as used herein refers to a treatment that comprises administration or application of a therapy, eg, administration of an active cell, eg, an RPE cell manipulated (eg, as described here), before the onset of a disease, dysfunction or condition in order to prevent the physical manifestation of said disease, dysfunction or condition. In some modalities, "prevention", "prevent" and "preventing" require that signs or symptoms of the disease, dysfunction or condition have not yet developed or have not yet been observed. In some modalities, treatment comprises prevention and in other modalities it does not.
[0069] [0069] A "replacement therapy" or "replacement protein" is a therapeutic protein or its functional fragment that replaces or increases a protein that is decreased, is present in insufficient quantity, is altered (eg, mutated) or it is absent in a subject having a decreased, altered or absent protein-related disease or condition. Examples are certain blood clotting factors in certain blood clotting disorders or certain lysosomal enzymes in certain diseases of lysosomal storage. In one embodiment, a replacement therapy or replacement protein provides the function of an endogenous protein. In one embodiment, a replacement therapy or replacement protein has the same amino acid sequence as a naturally occurring variant, eg, a wild-type allele or an allele not associated with a dysfunction, of the replaced protein. In one embodiment, a replacement therapy or a replacement protein differs in the amino acid sequence of a naturally occurring variant, eg, a wild-type allele or an allele not associated with a dysfunction, eg, the allele
[0070] [0070] "Sequence Identity" or "Percent Identity", when used here to refer to two nucleotide sequences or two amino acid sequences, means that the two sequences are the same within a specified region or have the same nucleotides or amino acids to a specified percentage of nucleotide or amino acid positions within the specified when the two sequences are compared and aligned for maximum match across a designated comparison window or region. Sequence identity can be determined using standard techniques known in the art including, but not limited to, any of the algorithms described in US 2017/02334455 A1. In one embodiment, the specified percentage of identical nucleotide or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or higher.
[0071] [0071] "Subject" as used here refers to a human or non-human animal. In one embodiment, the subject is a human (ie, a man or woman, eg, of any age group, a pediatric subject (eg, baby, child, teenager) or adult subject (eg , young adult, middle age adult or senior adult)). In one embodiment, the subject is a non-human animal, for example, a mammal (eg, a primate (eg, a cynomologist monkey or a rhesus monkey). In one embodiment, the subject is a commercially mammal relevant such as cattle, pig, horse, sheep, goat, cat or dog or a bird (eg a commercially relevant bird such as a chicken, duck, goose or turkey). In certain embodiments, the animal is a mammal The animal can be a male or female individual at any stage of development A non-human animal can be a transgenic animal In one embodiment, the subject is a human.
[0072] [0072] "Transcription unit" means a DNA sequence, eg, present in an exogenous nucleic acid, which comprises at least one promoter sequence operably linked to a coding sequence and may also comprise one or more additional elements that control or enhance the transcription of the coding sequence into RNA molecules or translation of the RNA molecules into polypeptide molecules. In some embodiments, a transcription unit also comprises polyadenylation signal sequence (polyA) and polyA site. In one embodiment, a transcription unit is an extrachromosomal, exogenous expression vector, e.g., as shown in FIG. 5, or is present as an exogenous sequence integrated into a chromosome of a manipulated active cell described here.
[0073] [0073] "Treatment", "treating" and "treating" as used herein refers to one or more of reducing, reversing, relieving, delaying the onset of or inhibiting the progress of one or more of an underlying symptom, manifestation or cause of a disease, dysfunction or condition. In one embodiment, treatment comprises reducing, reversing, relieving, delaying the onset of, or inhibiting the progress of, a symptom of a disease, dysfunction or condition. In one embodiment, treatment comprises reducing, reversing, relieving, delaying the onset of, or inhibiting the progress of, a manifestation of a symptom of a disease, dysfunction or condition. In one embodiment, treatment comprises reducing, reversing, relieving, reducing, or delaying the onset of, an underlying cause of a disease, dysfunction or condition. In some modalities, "treatment", "treating" and "treating" require that signs or symptoms of the disease, dysfunction or condition have developed or been observed. In other modalities, treatment can be administered in the absence of signs or symptoms of the disease or condition, eg, in preventive treatment. For example, treatment can be administered to a susceptible individual before the onset of symptoms
[0074] [0074] "von Willebrand factor protein" or "vWF protein", as used here, means a polypeptide that comprises the amino acid sequence of a naturally occurring vWF polypeptide or its variant that has biological vWF activity, e.g. , FVIII binding activity, as determined by a test recognized in the art, unless otherwise specified. The vWF proteins that can be expressed by engineered active cells described herein include primate (e.g., human), porcine, canine and murine wild-type proteins, as well as variants of such wild-type proteins. Active cells (eg, ARPE-19 cells) can be engineered to encode any of the following vWF polypeptides: vWF 2813 amino acid precursor, a vWF lacking the 22 amino acid signal peptide and optionally the pre-pro -741 amino acid peptide, mature 2050 amino acid vWF protein, and its truncated variants, such as a sufficient vWF fragment to stabilize endogenous levels of FVIII in vWF-deficient mice, eg, a truncated variant containing the region D´D3 (amino acids 764-1247) or the D1D2D´D3 region; and vWF variants with one or more amino acid substitutions, e.g., in the D 'region as described in U.S. Patent No. 9458223. A variant vWF protein preferably has at least 50%, 75%, 90% or more (including> 100%) of a biological activity of the corresponding wild-type vWF protein. Tests recognized in the art for determining the biological activity of a vWF include ristocetin cofactor activity (Federici A B et al. 2004. Haematologica 89: 77-85), binding of vWF to GP Ibα of
[0075] [0075] In some embodiments, the vWF protein produced by an engineered active cell of the disclosure comprises a naturally occurring vWF amino acid sequence or variant fused to a heterologous polypeptide or fraction other than polypeptide extending the half-life of the vWF protein. Exemplary half-life extending fractions include Fc, albumin, a PAS sequence, transferrin, CTP (28-amino acid C-terminal peptide (CTP)) of human chorionic gonadotropin (hCG) with its 4 O-glycans, polyethylene glycol (PEG), hydroxyethyl starch (HES), albumin-binding polypeptide, small albumin-binding molecules or any combination thereof. Selected Chemical Definitions
[0076] [0076] Definitions of functional groups and specific chemical terms are described in more detail below. Chemical elements are identified according to the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., Back cover, and specific functional groups are generally defined as described there. In addition, the general principles of organic chemistry, as well as functional fractions and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
[0077] [0077] The abbreviations used here have their conventional meaning within chemical and biological techniques. The chemical structures and formulas presented here are built according to standard valence rules
[0078] [0078] When a range of values is listed it is intended to encompass each value and sub-range within the range. For example, "C1-C6 alkyl" is intended to encompass C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5 and C5-C6.
[0079] [0079] As used herein, "alkyl" refers to a radical of a saturated straight or branched chain hydrocarbon group having 1 to 24 carbon atoms ("C1-C24 alkyl"). In some embodiments, an alkyl group has 1 to 12 carbon atoms ("C1-C12 alkyl"), 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, an 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) en –Hexyl (C6). Additional examples of alkyl groups include n – heptyl (C7), n – octyl (C8) and the like. Each case of an alkyl group can be independently optionally substituted, i.e., unsubstituted (an "unsubstituted alkyl") or substituted (an "substituted alkyl") with one or more substituents; eg, for 1 to 5 substituents, 1 to 3 substituents or 1 substituent.
[0080] [0080] As used here, “alkenyl” refers to a radical of a straight or branched chain hydrocarbon group having 2 to 24 carbon atoms, one or more carbon – carbon double bonds and no triple bonds (“C2 alkenyl -C24 ”). In some embodiments, an alkenyl group has 2 to 10 carbon atoms (“C2-C10 alkenyl”), 2 to 8 carbon atoms (“C2-C8 alkenyl”), 2 to 6 carbon atoms (“alkenyl
[0081] [0081] As used herein, the term "alkynyl" refers to a radical of a group of straight or branched chain hydrocarbons having from 2 to 24 carbon atoms, one or more carbon-carbon triple bonds ("C2-C24 alkenyl ”). In some embodiments, an alkynyl group has 2 to 10 carbon atoms (“C2-C10 alkynyl”), 2 to 8 carbon atoms (“C2-C8 alkynyl”), 2 to 6 carbon atoms (“C2-C6 alkynyl) ”), 2 to 5 carbon atoms (“ C2-C5 alkynyl ”), 2 to 4 carbon atoms (“ C2-C4 alkynyl ”), 2 to 3 carbon atoms (“ C2-C3 alkynyl ”) or 2 atoms carbon (“C2 alkynyl”). The one or more carbon-carbon triple bonds can be internal (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), 2 – butynyl (C4) and the like. Each case of an alkynyl group can be independently optionally substituted, ie, unsubstituted (an "unsubstituted alkynyl") or substituted (an "substituted alkynyl") by one or more substituents, eg, from 1 to 5 substituents, 1 to 3 substituents or 1 substituent.
[0082] [0082] As used herein, the term "heteroalkyl" refers to a stable non-cyclic straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P , Si and S, and in which the nitrogen and sulfur atoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized. The O, N, P, S and Si heteroatom (s) can be placed in any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to: -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N (CH3) -CH3, -CH2-S-CH2-CH3, -CH2-CH2, -S (O) -CH3, -CH2-CH2-S (O) 2-CH3, -CH = CH-O-CH3, -Si (CH3) 3, -CH2-CH = N-OCH3 , -CH = CH-N (CH3) -CH3, -O-CH3 and -O- CH2-CH3. Up to two or three hetero atoms can be consecutive, such as, for example, -CH2-NH-OCH3 and -CH2-O-Si (CH3) 3. Where "heteroalkyl” is recited, followed by recitations from specific heteroalkyl groups, such as –CH2O, –NRCRD or similar, it will be understood that the terms heteroalkyl and - CH2O or –NRCRD are not redundant or mutually exclusive. Instead, heteroalkyl groups specifics are recited to add clarity, so the term “heteroalkyl” should not be interpreted here as excluding specific heteroalkyl groups, such as –CH2O, –NRCRD or similar.
[0083] [0083] The terms "alkylene", "alkenylene", "alkynylene" or "heteroalkylene", alone or as part of another substituent, mean, unless otherwise stated, a divalent radical derived from an alkyl, alkenyl, alkynyl or heteroalkyl, respectively. An alkylene, alkenylene, alkylene or heteroalkylene group can be described as, for example, a C1-C6 membered alkylene, C1-C6 membered alkenylene, C1-C6 membered alkylene or C1-C6 membered heteroalkylene, where the term "members" refers to atoms other than hydrogen within the fraction. In the case of heteroalkylene groups, heteroatoms can
[0084] [0084] As used here, "aryl" refers to a radical of a 4n + 2 monocyclic or polycyclic aromatic ring system (eg, bicyclic or tricyclic) (eg, having 6, 10 or 14 π electrons) shared in a cyclic arrangement) having 6-14 ring carbon atoms and zero hetero atoms provided in the aromatic ring system (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms ("C6 aryl"; eg, phenyl). In some embodiments, an aryl group has ten ring carbon atoms ("C10 aryl"; eg, naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms ("C14 aryl"; eg, anthracyl). An aryl group can be described as, for example, a C6-C10-membered aryl, where the term "members" refers to the different ring atoms of hydrogen within the fraction. Aryl groups include phenyl, naphthyl, indenyl and tetrahydronaphthyl. Each case of an aryl group can be independently optionally substituted, i.e., unsubstituted (an "unsubstituted aryl") or replaced (an "substituted aryl") with one or more substituents.
[0085] [0085] As used here, “heteroaryl” refers to a radical of an aromatic 4n + 2 monocyclic or bicyclic ring system with 5-10 members (eg, having 6 or 10 π electrons shared in a cyclic arrangement) having ring carbon atoms and 1-4 ring hetero atoms provided in the aromatic ring system, where each hetero atom is independently selected from nitrogen, oxygen and sulfur ("5-10 membered heteroaryl"). In heteroaryl groups that contain one or more atoms of
[0086] [0086] In some embodiments, a heteroaryl group is an aromatic ring system with 5-10 members having carbon ring atoms and 1-4 ring heteroatoms provided in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen and sulfur (“Heteroaryl with 5-10 members”). In some embodiments, a heteroaryl group is an aromatic ring system with 5-8 members having carbon ring atoms and 1-4 ring heteroatoms provided in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen and sulfur (“heteroaryl with 5-8 members ”). In some embodiments, a heteroaryl group is an aromatic ring system with 5-6 members having carbon ring atoms and 1-4 ring heteroatoms provided in the aromatic ring system, where each heteroatom is independently selected from nitrogen, oxygen and sulfur (“heteroaryl with 5-6 members ”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from
[0087] [0087] 5-membered heteroaryl groups containing an exemplary heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. 5-membered heteroaryl groups containing two exemplary heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolia, thiazolyl and isothiazolyl. 5-membered heteroaryl groups containing three exemplary heteroatoms include, without limitation, triazolyl, oxadiazolyl and thiadiazolyl. 5-membered heteroaryl groups containing four exemplary heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing an exemplary heteroatom include, without limitation, pyridinyl. 6-membered heteroaryl groups containing two exemplary heteroatoms include, without limitation, pyridazinyl, pyrimidinyl and pyrazinyl. 6-membered heteroaryl groups containing three or four exemplary heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing an exemplary heteroatom include, without limitation, azepinyl, oxepinyl and tiepinyl. Exemplary 5,6 – bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benziazolol, benziazolazol, benziazol, zinc Exemplary 6,6 – bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, crinoline, quinoxalinyl, phthalazinyl and
[0088] [0088] As used here, the terms "arylene" and "heteroarylene", alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.
[0089] [0089] As used here, "cycloalkyl" refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms ("C3-C10 cycloalkyl") and zero heteroatom in the non-aromatic ring system. In some embodiments, a 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 (“cycloalkyl C5-C10 ”). A cycloalkyl group can be described as, for example, a cycloalkyl with C4-C7 members, where the term "members" refers to the ring atoms other than hydrogen within the fraction. Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclo- hexenyl (C6), cyclohexadienyl (C6) and the like. Exemplary C3-C8 cycloalkyl groups include, without limitation, the C3-C6 cycloalkyl groups mentioned above as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclohexyl -octyl (C8), cyclooctenyl (C8), cubanyl (C8), bicycle [1.1.1] pentanyl (C5), bicycle [2.2.2] octanyl (C8), bicycle [2.1.1] hexanyl (C6) , bicycle [3.1.1] heptanyl (C7) and the like. Exemplary C3-C10 cycloalkyl groups include, without limitation, the C3-C8 cycloalkyl groups mentioned above as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1H – indenyl (C9 ), decahydronaphthalenyl (C10), spiro [4.5] decanyl (C10) and the like. As the previous examples illustrate, in certain embodiments, the cycloalkyl group is monocyclic (“monocyclic cycloalkyl”) or contains a fused ring system,
[0090] [0090] "Heterocyclyl" as used here refers to a radical of a non-aromatic ring system with 3 to 10 members having ring carbon atoms and 1 to 4 ring hetero atoms, where each hetero atom is independently selected from nitrogen, oxygen, sulfur , boron, phosphorus and silicon ("3-10 membered heterocyclyl"). In heterocyclyl groups that contain one or more nitrogen atoms, the attachment point can be a carbon or nitrogen atom, as valence allows. A heterocyclyl group can be monocyclic ("monocyclic heterocyclic") or a fused, bridged or spiro ring system such as a bicyclic system ("bicyclic heterocyclic") and can be saturated or partially unsaturated. Bicyclic heterocyclyl ring systems can include one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups in which the attachment point is in the cycloalkyl or heterocyclyl ring or ring systems in which the heterocyclyl ring, such as defined above, is fused to one or more aryl or heteroaryl groups, where the attachment point is on the heterocyclyl ring and, in such cases, the number of ring members continues to designate the number of ring members in the heterocyclyl ring system.
[0091] [0091] In some embodiments, a heterocyclyl group is a non-aromatic ring system with 5-10 members having ring carbon atoms and 1-4 ring hetero atoms, where each hetero atom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus and silicon ("5-10 membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring hetero atoms, where each hetero atom is independently selected from nitrogen, oxygen and sulfur ("5-8 ring heterocyclyl" members ”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring hetero atoms, where each hetero atom is independently selected from nitrogen, oxygen and sulfur ("5-6 ring heterocyclyl" members ”). In some modalities, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen and sulfur. In some modalities, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen and sulfur. In some modalities, the 5-6 membered heterocyclyl has an annular heteroatom selected from nitrogen, oxygen and sulfur.
[0092] [0092] 3-membered heterocyclyl groups containing one
[0093] [0093] “Amino” as used here refers to the radical –NR70R71, in
[0094] [0094] As used here, "cyan" refers to the radical -CN.
[0095] [0095] As used herein, "halo" or "halogen", independently or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br ) or iodine (I).
[0096] [0096] As used here, "hydroxy" refers to the radical -OH.
[0097] [0097] Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl groups, as defined here, are optionally substituted (eg, "substituted" or "unsubstituted", "substituted" alkenyl or "Unsubstituted", alkynyl "substituted" or "unsubstituted", heteroalkyl "substituted" or "unsubstituted", cycloalkyl "substituted" or "unsubstituted", heterocyclyl "substituted" or "unsubstituted", "substituted" “Unsubstituted” or heteroaryl “substituted” or “unsubstituted”). In general, the term "substituted", whether preceded by the term "optionally" or not, means that at least one hydrogen present in a group (eg, a carbon or nitrogen atom) is replaced by a permissible substituent, eg, a substituent that after replacement results in a stable compound, eg, a compound that does not undergo spontaneous transformation such as rearrangement, cyclization, elimination or other reaction. Unless otherwise indicated, a "substituted" group has a substituent in one or more substitutable positions in the group, and when a position in any given structure is replaced, the substituent is the same or different in each position. The term "substituted" is contemplated to include substitution by all permissible substituents on organic compounds, such as any of the substituents described herein that result in the formation of a stable compound. This disclosure contemplates
[0098] [0098] Two or more substituents can optionally be joined to form aryl, heteroaryl, cycloalkyl or heterocyclyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic-based structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic-based structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic-based structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.
[0099] [0099] The compounds described here can comprise one or more asymmetric centers and thus can exist in various isomeric forms, e.g., enantiomers and / or diastereoisomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereoisomer or geometric isomer, or it can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers can be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers can be prepared by syntheses
[00100] [00100] As used herein, a pure enantiomeric compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). In other words, an "S" form of the compound is substantially free from the "R" form of the compound and is thus in enantiomeric excess of the "R" form. The term "enantiomerically pure" or "pure enantiomer" denotes that the compound comprises more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97 % by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight or more than 99.9% by weight of the enantiomer. In certain embodiments, weights are based on the total weight of all enantiomers or stereoisomers of the compound.
[00101] [00101] The compounds described here can also comprise one or more isotopic substitutions. For example, H can be in any isotropic form, including 1H, 2H (D or deuterium) and 3H (T or tritium); C can be in any isotropic form, including 12C, 13C and 14C; O can be in any isotropic form, including 16O and 18O; and the like.
[00102] [00102] The term "pharmaceutically acceptable salt" is intended to include salts of the active compounds that are prepared with acids or bases
[00103] [00103] In addition to salt forms, this disclosure
[00104] [00104] Certain compounds of the present disclosure may exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, solvated forms are equivalent to unsolvated forms and are included within the scope of this disclosure. Certain compounds of the present disclosure can exist in multiple crystalline and amorphous forms. In general, all physical forms are equivalent to the uses contemplated by this disclosure and are intended to be within the scope of this disclosure.
[00105] [00105] The term "solvate" refers to forms of the compound that are associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether and the like. The compounds described here can be prepared, for example, in crystalline form and can be solvated. Suitable solvates include pharmaceutically acceptable solvates and additionally include both stoichiometric and non-stoichiometric solvates.
[00106] [00106] The term "hydrate" refers to a compound that is associated with water. Typically, the number of water molecules contained in a hydrate of a compound is in a defined ratio to the number of compound molecules in the hydrate. Therefore, a hydrate of a compound can be represented, for example, by the general formula R⋅x H2O, where R is the compound and where x is a number greater than 0.
[00107] [00107] The term "tautomer" as used here refers to compounds
[00108] [00108] The symbol “” as used here refers to a connection to an entity, eg, a polymer (eg, hydrogel-forming polymer such as alginate) or an implantable element (eg, device or material). The connection represented by "" can refer to the direct attachment to the entity, eg, a polymer or an implantable element, can refer to the connection to the entity through an attachment group. An "attachment group", as described here, refers to a fraction for attaching a compound of Formula (II) to an entity (eg, a polymer or an implantable element as described here) and can comprise any chemistry of attachment known in the art. A list of exemplary attachment groups is outlined in Bioconjugate Techniques (3rd ed, Greg T. Hermanson, Waltham, MA: Elsevier, Inc, 2013), which is incorporated here by reference in its entirety. In some embodiments, an attachment group comprises alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, –C (O) -, –OC (O) -, –N (RC) -, - N (RC ) C (O) -, –C (O) N (RC) -, –N (RC) N (RD) -, –NCN–, –C (= N (RC) (RD)) O–, - S -, –S (O) x–, –OS (O) x–, –N (RC) S (O) x–, –S (O) xN (RC) -, –P (RF) y–, - Si (ORA) 2 -, –Si (RG) (ORA) -, –B (ORA) - or a metal, where each of RA, RC, RD, RF, RG, x and y is independently as described here. In some embodiments, an attachment group comprises an amine, ketone, ester, amide, alkyl, alkenyl, alkynyl or thiol. In some embodiments, an attachment group is a crosslinker. In some embodiments, the attachment group is -
[00109] [00109] Cell compositions comprising active cells, e.g., retinal pigment epithelial cells (RPE) or cells derived from RPE cells, including manipulated RPE cells or manipulated cells derived from RPE cells, their compositions, implantable elements are disclosed herein comprising the same and methods of preparation or manufacture and use of such cells, compositions and implantable elements. In one embodiment, an active cell, eg, an RPE cell, is a manipulated active cell, eg, a manipulated RPE cell.
[00110] [00110] As naturally occurring in the body, RPE cells constitute the base layer of the epithelium in the eye, constituting a monolayer of cuboidal cells within or on Bruch's membrane directly behind the photoreceptor cells in the retina. RPE cells play a critical role in maintaining subretinal space by nutrient traffic and regulating ion balance, as well as preventing damage to surrounding retinal tissue by capturing scattered light and facilitating retinoid storage (Sparrow, JR et al ( 2010) Curr Mol Med 10: 802-823). The aberrant function of RPE cells is implicated in the pathology of several diseases, such as macular degeneration, central serous chorioretinopathy and retinitis pigmentosa (Sato, R. et al. (2013) Invest Ophthalmol Vis Sci
[00111] [00111] Active engineered cells, e.g., engineered RPE cells or engineered cells derived from RPE cells, are described here and have advantageous properties that can be exploited for use in the present disclosure. For example, in modalities, active cells may exhibit contact inhibition and in modalities they are capable of phagocytosis from neighboring cells or both. In embodiments, either or both of these properties provide a homeostatic function; for example, in modalities, contact inhibition prevents or inhibits unwanted growth that could compromise the function or integrity of encapsulated active cells while the phagocytosis ability allows for a more permissive environment for cell division and replacement of dead active cells. In one embodiment, the encapsulated active cells maintain a density or number of cells that does not vary by more than 10, 20, 30, 40 or 50% over a pre-selected period of time, in vitro culture, or implanted in a subject, e.g., more than about 1, 2, 3, 4, 5, 10, 20, 30, 45, 60 or 90 days.
[00112] [00112] In one embodiment, the active cell is an autologous, allogeneic or xenogenic cell (these terms refer to the relationship between the cell and a subject to which the cell is administered).
[00113] [00113] In one embodiment, the active cell is an immortalized cell or is derived from an immortalized cell.
[00114] [00114] In one embodiment, the active cell is either a non-immortalized cell or is derived from a non-immortalized cell.
[00115] [00115] In one embodiment, an active cell is a cell derived from a less differentiated cell (eg, less differentiated than an RPE cell), eg, a pluripotent cell, multipotent cell, a stem cell, an embryonic stem cell, a mesenchymal stem cell, an induced pluripotent stem cell; a reprogrammed cell, a stem cell
[00116] [00116] In one embodiment, an active cell is derived from a naturally derived source, xenotyped, allotized, a corpse, a cell line or a primary cell.
[00117] [00117] An active cell can be a engineered cell, such as a cell engineered to express a protein or nucleic acid, or a cell engineered to produce a metabolic product. An active cell can be a mammalian cell, e.g., a human cell. A manipulated active cell can be a mammalian cell, e.g., a human cell.
[00118] [00118] In one embodiment, an active manipulated cell is an RPE cell (or is derived from an RPE cell) that comprises at least one exogenous transcription unit, which can be present in an extrachromosomal expression vector or integrated into one or more chromosomal sites in the cell. In one embodiment, the transcription unit comprises a promoter operably linked to a sequence encoding a polypeptide, wherein the promoter consists essentially of or consists of SEQ ID NO: 23 or a nucleotide sequence that is substantially identical to SEQ ID NO: 23 , e.g., is at least 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 23. In one embodiment, the promoter consists of SEQ ID NO: 23. In one embodiment, the polypeptide coding sequence is a naturally occurring sequence (eg, wild-type or native) or a codon-optimized sequence. In one embodiment, the transcription unit further comprises a Kozak translation sequence immediately upstream of the ATG start codon in the polypeptide coding sequence (e.g., the Kozak sequence shown in nucleotides 2094-2099 of SEQ ID NO: 26). In a
[00119] [00119] In one embodiment, an active cell is derived from a culture in which at least 10, 20, 30, 40, 50, 60, 79, 80, 90, 95, 98 or 99% of the cells in the culture are active cells , eg, RPE cells or active manipulated cells, eg, manipulated RPE cells. In one embodiment, a culture comprises active cells, e.g., RPE cells, or engineered RPE cells, and a second type of cells, e.g., a feeder cell or a contaminating cell. In one embodiment, an active cell is an RPE cell, eg, a manipulated or unhandled RPE cell derived from an individual, eg, the same individual or a different individual to which the cells are administered.
[00120] [00120] An active cell can be derived from any one of a variety of strains. Exemplary strains of RPE cells include ARPE-19 cells, ARPE-19-SEAP-2-neo cells, RPE-J cells and hTERT RPE-1 cells. In some embodiments, the active cell is an ARPE-19 cell or derived from an ARPE-19 cell. In some embodiments, the active cell is a manipulated ARPE-19 cell, which is derived from the ARPE-19 cell line (ATCC CRL-2302).
[00121] [00121] In one embodiment, an active cell expresses a biomarker, eg, an antigen, which is characteristic of an RPE cell,
[00122] [00122] In one embodiment, a plurality of active cells (eg, RPE cells), eg, manipulated active cells (eg, manipulated RPE cells), have or are provided in a form factor pre-selected or a form factor described here. In one embodiment, the form factor is a monolayer or grouping. A "cluster of active cells, e.g., a cluster of RPE cells", as used here, refers to a plurality of active cells or an aggregate of active cells typically having a ratio between cells and surface area of the form factor which is lower than that of a monolayer. In some embodiments, a cluster of active cells comprises at least about 2, 3, 4, 5, 10, 50, 100, 200, 300, 400, 500, 1,000, 2,000, 3,000, 4,000, or 5,000 active cells. In some embodiments, the cluster of active cells comprises between 2 and 5,000 cells, 2 and 1,000 cells, 5 and 1,000 cells, 5 and 500 cells, 10 and 500 cells. In some embodiments, the cluster of active cells comprises between 2 and 10 cells, 5 and 10 cells, about 5 and 20 cells, 5 and 50 cells or 10 and 100 cells. In some embodiments, the cluster of active cells comprises 50 to 100 cells, 50 to 250 cells, 100 to 500 cells, 100 to 1,000 cells or 500 to 1,000 cells. In one embodiment, the lower, upper, or both end points of a cell number range are averaged and can vary by 5%. In one embodiment, the lower, upper, or both end points of a cell number range are averaged and can vary by 10%.
[00123] [00123] In one embodiment, a grouping of active cells has
[00124] [00124] In one embodiment, a grouping of active cells comprises certain dimensions, eg, with a range of sizes in each of dimension x, dimension y or dimension z. In some embodiments, the length of at least one of the dimensions x, y or z is independently greater than about 10 µm (eg, greater than about 15 µm, about 20 µm, about 30 µm, about 40 µm, about 50 µm, about 75 µm, about 100 µm, about 250 µm, about 500 µm, about 750 µm, about 1 mm, about 1.1 mm, about 1 , 2 mm, about 1.3 mm, about 1.4 mm, about 1.5 mm or more). In some embodiments, the length of at least one of the x, y, or z dimensions of the cluster of active cells is independently less than about 2 mm (e.g., less than about 1.5 mm, about 1 , 4 mm, about 1.3 mm, about 1.2 mm, about 1.1 mm, about 1.0 mm, about 750 µm, about 500 µm, about 250 µm, about 100 µm, about 75 µm, about 50 µm, about 40 µm, about 30 µm, about 20 µm or less).
[00125] [00125] In some embodiments, the length of at least one of the x, y, or z dimensions of the cluster of active cells is independently between about 10 µm and about 5 mm in size (eg, between about 20 µm and about 4 mm, about 50 µm and about 2 mm or about 100 µm and about 1.5 mm). In some embodiments, the length of at least two of the x, y, or z dimensions of the cluster of active cells is independently between about 10 µm and about 5 mm in size (eg, between about 20 µm and about 4 mm, about 50 µm and about 2 mm or about 100 µm and about 1.5 mm). In some embodiments, the length of all three of the x, y, or z dimensions of the cluster of active cells is independently between about 10 µm and about 5 mm in size (eg, between about 20 µm and about 4 mm, about 50 µm and about 2 mm or about 100 µm and about 1.5 mm).
[00126] [00126] In some embodiments, each of the dimensions of the active cell pool is independently within about 5% (e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90% or about 95%) other dimensions. For example, the x dimension of the RPE cell pool can be about 5% (eg, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90% or about 95%) of both dimension y and dimension z . In some embodiments, the y dimension of the cluster of active cells can be about 5% (e.g., about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 60%, about 70%, about 80%, about 90% or about 95%) of both dimension x and dimension z. In other embodiments, the z dimension of the cluster of active cells can be about 5% (e.g., about 10%,
[00127] [00127] The grouping of active cells can be embedded in a matrix, eg, an extracellular matrix secreted by an active cell (eg, a cluster of embedded cells). In some embodiments, the cluster of active cells is encapsulated by a matrix, eg, an extracellular matrix secreted by an active cell (eg, a cluster of encapsulated active cells). In some embodiments, the extracellular matrix comprises proteins, eg, collagen (eg, structural collagen or angiostatic collagen, eg, collagen IV, collagen III, collagen V, collagen VI, collagen XVIII) , laminin, elastin, integrin or fibronectin. The extracellular matrix or a component thereof can be naturally occurring or not naturally occurring. In some embodiments, the extracellular matrix or a component thereof is naturally occurring and is supplemented by a non-naturally occurring component. In other embodiments, the extracellular matrix or a component thereof is non-naturally occurring and is supplemented by a naturally occurring component.
[00128] [00128] Active cells for use in compositions and methods described here, eg, for use in a plurality of active cells encapsulated in a hydrogel capsule or having a pre-selected form factor or form factor described here , eg, a grouping of active cells, can be at various stages of the cell cycle. In some embodiments, at least one cell active in the plurality or cluster of active cells is undergoing cell division. Cell division can be measured using any method known in the art, e.g., as described in DeFazio A et al. (1987) J Histochem Cytochem 35: 571-577 and Dolbeare F et al. (1983) Proc Natl Acad Sci USA 80: 5573-5577, each of which is
[00129] [00129] In some embodiments, cells active in the plurality or grouping of active cells are capable of autophagy. Autophagy can be measured using any method known in the art, e.g., as described in Barth et al. (2010) J. Pathol 221: 117-124 or Zhang, Z. et al. (2016) Curr Protoc Toxicol. 69: 20.12.1-20.1.26, each of which is incorporated by reference in its entirety. For example, autophagy can be determined or quantified by a 5-ethinyl-2'deoxyuridine (EdU) assay, a 5-bromo-2'-deoxyuridine (BrdU) assay, a cationic amphiphile tracer (CAT) assay, in which the dye divides rapidly in the cells and selectively marks vacuoles associated with the autophagy pathway. In some embodiments, autophagy is visualized or quantified by microscopy (eg, fluorescence microscopy (eg, time lapse or evaluation of spindle formation)). In some modalities, autophagy is analyzed by one or more immunoblot analysis of LC3 and p62, detection of autophagosome formation by fluorescence microscopy and monitoring of autophagosome maturation by mRFP- fluorescence microscopy.
[00130] [00130] In some embodiments, RPE cells in the plurality or cluster of RPE cells are capable of phagocytosis. Phagocytosis can be measured using any method known in the art, e.g., as described in Oda T and Maeda H (1986) J Immunol Methods 88: 175-183 and Nuutila J and Lilius EM (2005) Cytometry A (2005) 65: 93-102, each of which is incorporated by reference in its entirety. For example, phagocytosis can be measured by a fluorescein-labeled antibody assay, in which the uptake of a labeled substance via the phagocytic pathway is monitored. In some embodiments, phagocytosis is visualized or quantified by microscopy (eg, fluorescence microscopy (eg, time lapse or evaluation of spindle formation)) or flow cytometry. In one embodiment, at least 1, 2, 3, 4, 5, 10 or 20% of the cells are capable of phagocytosis, eg, as determined by a fluorescein-labeled antibody assay, microscopy (eg, fluorescence microscopy (eg, time lapse or evaluation of spindle formation)) or flow cytometry.
[00131] [00131] In one embodiment, at least 1, 2, 3, 4, 5, 10, 20, 40 or 80% of the RPE cells in the plurality or cluster are viable. Cell viability can be measured using a method known in the art, e.g., as described in Riss, T. et al. (2013) “Cell Viability Assays” in Assay Guidance Manual (Sittapalam, G.S. et al., Eds). For example, cell viability can be measured or quantified by an ATP assay, 5-ethynyl-2'deoxyuridine (EdU) assay, 5-bromo-2'- assay
[00132] [00132] Any of the parameters described here can be evaluated using standard techniques known to a person skilled in the art, such as histology, microscopy and various functional assays.
[00133] [00133] In some embodiments, the active cells having a form factor, eg, in a cluster of active cells, form close junctions with each other. In one embodiment, at least 1, 2, 3, 4, 5, 10 or 20% of the cells have a close junction with at least one other active form factor cell, e.g., as determined by methods known in the art , e.g., staining and microscopy assays known in the art. In some embodiments, active cells having a form factor, eg, in a cluster of active cells, do not form tight junctions with each other. In one embodiment, at least 1, 2, 3, 4, 5, 10 or 20% of the active cells do not have a close junction with another active form factor cell, e.g., as determined by methods known in the art, e.g., staining and microscopy assays known in the art. In some embodiments, active cells having a form factor, eg, in a cluster of active cells, exhibit polarity. For example, active cells having a form factor may exhibit polarity characteristics in situ in the eye (eg, the retina). In one embodiment, at least 1, 2, 3, 4, 5, 10 or 20% of active cells exhibit polarity, e.g., as determined by known methods
[00134] [00134] An active cell, eg, an RPE cell (eg, a manipulated RPE cell) can be arranged on a non-cellular carrier (eg, a microcarrier). In some embodiments, the microcarrier is a spherule. In some embodiments, the microcarrier comprises a polymer, e.g., plastic (e.g., polystyrene, polyethylene, polyester, polypropylene), glass, acrylamide, silica, silicone rubber, cellulose, dextran, collagen (e.g. ., gelatin) or a glycosaminoglycan. The microcarrier can have any shape or configuration, including a sphere (eg, a spherule), flat disc, fiber, woven disc or cube. In some embodiments, the microcarrier may have a polar surface or a charged surface (eg, a negative charge or a positive charge). In some embodiments, the microcarrier may have a smooth surface or a textured surface. In some embodiments, an active cell (eg, a manipulated active cell) is attached to a microcarrier by adsorption of cell surface proteins (eg, glycoproteins, eg, fibronectin) to the surface of the cell. microcarrier. The microcarrier can vary in size from about 10 µm to about 5 mm (eg, between about 10 µm and about 3 mm, 10 µm and about 1 mm, 50 µm and about 1 mm, 100 µm and about 1 mm, 100 µm and about 500 µm).
[00135] [00135] An active cell (eg, an RPE cell) can be arranged in a microcarrier (eg, a bead, eg, a polystyrene bead, eg, a Cytodex microcarrier ® 1) using any method known in the art (see, eg, Nilsson, K. (1988) Biotechnol
[00136] [00136] The present disclosure features an active cell (e.g., an RPE cell) that produces or is capable of producing a therapeutic agent for the prevention or treatment of the disease, dysfunction or condition described here. In one embodiment, the active cell (eg, the RPE cell) is a manipulated active cell (eg, a manipulated RPE cell, a manipulated ARPE-19 cell). The therapeutic agent can be any biological substance, such as a nucleic acid (eg, a nucleotide, DNA or RNA), a polypeptide, a lipid, a sugar (eg, a monosaccharide, disaccharide, oligosaccharide or polysaccharide ) or a small molecule, each of which is further elaborated below.
[00137] [00137] In some embodiments, active cells (eg, engineered RPE cells) produce a nucleic acid. A nucleic acid produced by an active cell described here can vary in size and contain one or more nucleosides or nucleotides, e.g., greater than 2, 3, 4, 5, 10, 25, 50 or more nucleosides or nucleotides. In some embodiments, acid
[00138] [00138] In some embodiments, the therapeutic agent is a peptide or polypeptide (eg, a protein), such as a hormone, enzyme, cytokine (eg, a pro-inflammatory cytokine or an anti-inflammatory cytokine ), growth factor, clotting factor or lipoprotein. A peptide or polypeptide (e.g., a protein) produced by an RPE cell can have a naturally occurring amino acid sequence or it can contain a mutation, deletion or addition of amino acids in relation to the naturally occurring sequence. In addition, a peptide or polypeptide (e.g., a protein) for use with the present disclosure can be modified in some way, e.g., by chemical or enzymatic modification (e.g., glycosylation, phosphorylation). In some embodiments, the peptide has about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 45 or 50 amino acids. In some embodiments, the protein has an average molecular weight of about 5 kD, 10 kD, 25 kD, 50 kD, 100 kD, 150 kD, 200 kD, 250 kD, 500 kD or more.
[00139] [00139] In some modalities, protein is a hormone. Exemplary hormones include antidiuretic hormone (ADH), oxytocin, growth hormone (GH), prolactin, growth hormone releasing hormone (GHRH), thyroid stimulating hormone (TSH), thyrotropin releasing hormone (TRH), adrenocorticotropic hormone (ACTH) ), follicle stimulating hormone (FSH), luteinizing hormone
[00140] [00140] In some embodiments, protein is a growth factor, eg, vascular endothelial growth factor (VEGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth (FGF), epidermal growth factor (EGF), transforming growth factor (TGF) and insulin-like growth factor-I and -II (IGF-I and IGF-II).
[00141] [00141] In some embodiments, the protein is either a clotting factor or a clotting factor, eg, a blood clotting factor or a blood clotting factor. In some embodiments, protein is a protein involved in coagulation, i.e., the process by which blood is converted from a liquid to a solid or gel. Exemplary coagulation factors and coagulation factors include Factor I (eg, fibrinogen), Factor II (eg, prothrombin), Factor III (eg, tissue factor), Factor V (eg ., proacelerin, labile factor), Factor VI, Factor VII (e.g., stable factor, proconvertin), Factor VIII (e.g., anti-hemophilic factor A), Factor VIIIC, Factor IX (e.g. , anti-hemophilic factor B), Factor X (eg, Stuart-Prower factor), Factor XI (eg, history of plasma thromboplastin), Factor XII (eg, Hagerman factor) , Factor XIII (eg, fibrin stabilizing factor),
[00142] [00142] In some embodiments, the protein is an antibody molecule. As used herein, the term "antibody molecule" refers to a protein, e.g., an immunoglobulin chain or fragment thereof, comprising at least one immunoglobulin variable domain sequence. The term "antibody molecule" includes, for example, a monoclonal antibody (including a full-length antibody that has an immunoglobulin Fc region). In one embodiment, an antibody molecule comprises a full-length antibody or a full-length immunoglobulin chain. In one embodiment, an antibody molecule comprises an antigen-binding or functional fragment of a full-length antibody or a full-length immunoglobulin chain. In one embodiment, an antibody molecule is a monospecific antibody molecule and binds to a single epitope, e.g., a monospecific antibody molecule having a plurality of sequences of immunoglobulin variable domains, each of which binds to the same epitope.
[00143] [00143] In one embodiment, an antibody molecule is a multispecific antibody molecule, e.g., comprises a plurality of immunoglobulin variable domain sequences, wherein a plurality first immunoglobulin variable domain sequence has binding specificity by a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity by a second epitope. In one embodiment, the first and second epitopes are on the same antigen, eg, on the same protein (or subunit of a multimeric protein). In one embodiment, a molecule of
[00144] [00144] Various types of antibody molecules can be produced by the active cells described here, including whole immunoglobulins of any class, their fragments and synthetic proteins containing at least the antigen-binding variable domain of an antibody. The antibody molecule can be an antibody, e.g., an antibody against IgG, such as IgG1, IgG2, IgG3 or IgG4. An antibody molecule can be in the form of an antigen-binding fragment including a Fab fragment, F (ab ') 2 fragment, a single-chain variable region and the like. Antibodies can be polyclonal or monoclonal (mAb). Monoclonal antibodies can include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequences in antibodies derived from a specific species or belonging to a particular class or subclass of antibodies, while the remainder of ( s) chain (s) is identical or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, as long as they specifically bind to the target antigen and / or exhibit activity desired biological value. In some embodiments, the antibody molecule is a single domain antibody (eg, a nanobody). The described antibodies can also be modified by recombinant means, for example by deletions, additions or substitutions of amino acids, to increase the effectiveness of the antibody in mediating the desired function. Exemplary antibodies include anti-beta-galactosidase, anti-collagen, anti-CD14, anti-CD20, anti-CD40, anti-HER2, anti-IL-1, anti-IL-4, anti-IL6, anti-IL- 13, anti-IL17, anti-IL18, anti-IL-23, anti-IL-28, anti-IL-29, anti-IL-33, anti-EGFR, anti-VEGF, anti-
[00145] [00145] In some embodiments, the protein is a cytokine or cytokine receptor, or a chimeric protein including cytokines or their receptors, including, for example, tumor necrosis factor alpha and beta, its receptors and derivatives, renin; lipoproteins; colchicine; corticotrophin; vasopressin; somatostatin; lipressin; pancreozimine; leuprolide; alpha-1-antitrypsin; atrial natriuretic factor; pulmonary xigenasse67; a plasminogen activator other than a tissue-type plasminogen activator (t-PA), for example a urokinase; bombesin; thrombin; encephalinase; RANTES (regulated after activation normally expressed and secreted by T cells); inflammatory human macrophage protein (MIP-1-alpha); a serum albumin such as human serum albumin; Mullerian inhibiting substance; relaxin chain A; relaxin B chain; prelaxin; mouse gonadotropin-associated peptide; chorionic gonadotropin; a microbial protein, such as beta-lactamase; Dnase; inhibin; activin; receptors for hormones or growth factors; integrin; protein A or D; rheumatoid factors; platelet-derived growth factor (PDGF); epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and -II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor-binding proteins; CD proteins such as CD-3, CD-4, CD-8 and CD-19; erythropoietin; osteoinductive factors; immunotoxins; an interferon such as interferon-alpha (eg, interferon.alpha.2nd), -beta, -gama, -lambda and consensus interferon; factors
[00146] [00146] Examples of polypeptide (e.g., protein) produced by an active cell (e.g., an RPE cell) include CCL1, CCL2 (MCP-1), CCL3 (MIP-1α), CCL4 (MIP- 1β), CCL5 (RANTES), CCL6, CCL7, CCL8, CCL9 (CCL10), CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL24 , CCL27, CCL28, CXCL1 (KC), CXCL2 (SDF1a), CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8 (IL8), CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CCLCL, CXCL XCL1, XCL2, TNFA, TNFB (LTA), TNFC (LTB), TNFSF4, TNFSF5 (CD40LG), TNFSF6, TNFSF7, TNFSF8, TNFSF9, TNFSF10, TNFSF11, TNFSF13B, EDA, IL2, IL15, IL4, IL13, IL7, IL9, IL7, IL13, IL7 , IL21, IL3, IL5, IL6, IL11, IL27, IL30, IL31, OSM, LIF, CNTF, CTF1, IL12a, IL12b, IL23, IL27, IL35, IL14, IL16, IL32, IL34, IL10, IL22, IL19, IL20 , IL24, IL26, IL29, IFNL1, IFNL2, IFNL3, IL28,
[00147] [00147] In some embodiments, the protein is a replacement therapy or a replacement protein. In some embodiments, replacement therapy or replacement protein is either a coagulation factor or a coagulation factor, eg, vWF (comprises a naturally occurring human vWF factor or a variant thereof), Factor VII (eg ., comprises a naturally occurring human Factor VII amino acid sequence or a variant thereof, Factor VIII (e.g., comprises a naturally occurring human Factor VIII amino acid sequence or a variant thereof) or Factor IX (e.g. ., comprises a naturally occurring human Factor IX amino acid sequence or a variant thereof.
[00148] [00148] In some embodiments, the active cell (e.g., RPE cell) is engineered to express a human Factor VIII protein, e.g., a recombinant Factor VIII protein. In some embodiments, the recombinant Factor VIII protein is a recombinant Factor VIII protein with domain B deletion (FVIII-BDD) or a variant thereof. In some embodiments, the active cell is a manipulated RPE cell (e.g., derived from the ARPE-19 cell line) and comprises an exogenous nucleic acid sequence that encodes the FVIII-BDD amino acid sequence shown in FIG. 3 (SEQ ID NO: 1) or encodes one of the single chain FVIII-BDD amino acid sequences shown in SEQ ID NO: 3, 4, 5 and 6.
[00149] [00149] In some embodiments, the active cell (eg, ARPE-cell
[00150] [00150] In some embodiments, the active cell (eg, ARPE-19 cell) is manipulated to express a gain-of-function (GIF) variant of a wild type FIX protein (FIX-GIF), wherein the GIF variant has higher specific activity than the corresponding wild-type FIX. In some embodiments, the active cell is a manipulated RPE cell (eg, derived from the ARPE-19 cell line) and comprises an exogenous nucleic acid sequence that encodes the variant amino acid sequence (Factor IX Padua) shown in SEQ ID NO: 2.
[00151] [00151] In some embodiments, the active cell (eg, ARPE-19 cell) is manipulated to express a truncated vWF variant, eg, consisting of the D1-D3 domains (eg, SEQ ID NO: 33) or consisting of D´D3 (eg, SEQ ID NO: 32).
[00152] [00152] In some embodiments, replacement therapy or replacement protein is an enzyme, eg, alpha-galactosidase, alpha-L-iduronidase (IDUA) or N-sulfoglucosamine sulfo-hydrolase (SGSH). In some embodiments, the replacement therapy or replacement protein is an enzyme, eg, alpha-galactosidase (eg, alpha-galactosidase A) In some embodiments, the replacement therapy or replacement protein is a cytokine (e.g., interleukin 2, e.g., SEQ ID NO: 29) or an antibody. In some embodiments, replacement therapy or replacement protein is a parathyroid hormone polypeptide (eg, SEQ ID NO: 30 or SEQ ID NO: 31).
[00153] [00153] In some modalities, the therapeutic agent is a sugar,
[00154] [00154] In some embodiments, the therapeutic agent is a lipid. A lipid can be hydrophobic or amphiphilic and can form a tertiary structure such as a liposome, vesicle or membrane or insert itself into a liposome, vesicle or membrane. A lipid can comprise a fatty acid, glycerolipid, glycerophospholipid, sterol lipid, prenol lipid, sphingolipid, saccharolipid, polyketide or sphingolipid. Examples of lipids produced by encapsulated cells include anandamide, docosahexaenoic acid, a prostaglandin, a leukotriene, a thromboxane, an eicosanoid, a triglyceride, a cannabinoid, phosphatidylcholine, phosphatidylethanolamine, a phosphatidylinositol, a phosphatidyl acid, a phosphatididine, a cerebroside, a ganglioside, estrogen, androsterone, testosterone, cholesterol, a carotenoid, a quinone, a hydroquinone or a ubiquinone.
[00155] [00155] In some modalities, the therapeutic agent is a
[00156] [00156] In some embodiments, the active cell (eg, RPE cell) is manipulated to synthesize a small molecule different from protein or different from peptide. For example, in one embodiment, an active cell (eg, RPE cell) can produce a statin (eg, taurostatin, pravastatin, fluvastatin or atorvastatin).
[00157] [00157] In some modalities, the therapeutic agent is an antigen
[00158] [00158] Active cells, e.g., manipulated active cells, e.g., manipulated RPE cells described here, can produce a single therapeutic agent or a plurality of therapeutic agents. In some embodiments, the active cells (eg, RPE cells) produce a single therapeutic agent. In some embodiments, a cluster of active cells (eg, RPE cells) comprises active cells that produce a single therapeutic agent. In some modalities, at least about 1%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of active cells (eg, RPE cells) in a cluster produce a single therapeutic agent (eg, a therapeutic agent described here). In some embodiments, the active cells (e.g., RPE cells) produce a plurality of therapeutic agents, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 therapeutic agents. In some embodiments, a cluster of active cells (e.g., RPE cells) comprises active cells that produce a plurality of therapeutic agents. In some modalities, at least about 1%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of active cells (eg, RPE cells) in a pool produce a plurality of therapeutic agents (eg, a therapeutic agent described here).
[00159] [00159] Therapeutic agents can be related or can form a complex. In some embodiments, the therapeutic agent secreted or released from an active cell (eg, RPE cell) in a
[00160] [00160] The present disclosure comprises active cells (e.g., manipulated active cells, e.g., manipulated RPE cells) entirely or partially arranged within or on top of an implantable element. The implantable element may comprise an enclosing element that encapsulates or covers an active cell (eg, an RPE cell), in part or in whole. In one embodiment, an implantable element comprises an enveloping component that is formed, or could be formed, in situ in or surrounding an active cell, e.g., a plurality of active cells, e.g., a cluster of active cells , or in a microcarrier, e.g., a spherule, or a matrix comprising an active cell or active cells (referred to here as an “in situ encapsulated implantable element”).
[00161] [00161] Implantable elements and exemplary surrounding components comprise materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials and their combinations. An implantable element can be used to encapsulate an active cell (eg, a manipulated active cell, eg, a manipulated RPE cell) or a cluster of active cells (eg, manipulated active cells, eg. manipulated RPE cells). An implantable element can be completely
[00162] [00162] In some embodiments, the material is a metal or a metal alloy. Exemplary metals or metal alloys include comprising titanium and titanium group alloys (eg, nitinol, nickel and titanium alloys, thermally memory alloy materials), platinum, platinum group alloys, stainless steel, tantalum, palladium , zirconium, niobium, molybdenum, nickel-chromium, chromium and molybdenum alloys or certain cobalt alloys (eg, cobalt-chromium and cobalt-chromium-nickel alloys, eg, Elgiloy® and Phynox®) . For example, a metallic material may be grade 316 stainless steel (SS 316L) (comprised of Fe, <0.3% C, 16-18.5% Cr, 10-14% Ni, 2-3% Mo, <2 % Mn, <1% Si, <0.45% P and <0.03% S).
[00163] [00163] In some modalities, the material is a ceramic. Exemplary ceramic materials include oxides, carbides or nitrides of the transition elements, such as titanium oxides, hafnium oxides, iridium oxides, chromium oxides, aluminum oxides and zirconium oxides. Silicon-based materials, such as silica, can also be used.
[00164] [00164] In some embodiments, the material is a polymer. A polymer can be a linear, branched or crosslinked polymer or a polymer from selected molecular weight ranges, degree of polymerization, viscosity or melt index. Branched polymers can include one or more of the following types: star polymers, comb polymers, brush polymers, dendronized polymers, ladders and dendrimers. A polymer can be a heat-responsive polymer, eg, gel (eg, becomes a solid or liquid after exposure to heat or a certain temperature) or a photoreticulable polymer. Exemplary polymers include polystyrene, polyethylene, polypropylene, polyacetylene, poly (vinyl chloride) (PVC),
[00165] [00165] In some embodiments, the material is polyethylene. Exemplary polyethylenes include ultra-low density polyethylene (ULDPE) (eg, with polymers with densities ranging from 0.890 to 0.905 g / cm3, containing comonomer); very low density polyethylene (VLDPE) (eg, with polymers with densities ranging from 0.905 to 0.915 g / cm3, containing comonomer); linear low density polyethylene (LLDPE) (eg, with polymers with densities ranging from 0.915 to 0.935 g / cm3, containing comonomer); low density polyethylene (LDPE) (eg, with polymers with densities ranging from about 0.915 to 0.935 g / m3); medium density polyethylene (MDPE) (eg, with polymers with densities ranging from 0.926 to 0.940 g / cm3, may or may not contain a comonomer); high density polyethylene (HDPE) (eg, with polymers with densities ranging from 0.940 to 0.970 g / cm3, may or may not
[00166] [00166] In some embodiments, the material is a polypropylene. Exemplary polypropylenes include homopolymers, random copolymers (homophasic copolymers) and impact copolymers (heterophasic copolymers), e.g., as described in McKeen, Handbook of Polymer Applications in Medicine and Medical Devices, 3- Plastics Used in Medical Devices, (2014 ): 21-53, which is incorporated herein by reference in its entirety.
[00167] [00167] In some embodiments, the material is polystyrene. Exemplary polystyrenes include general purpose or crystalline polystyrene (SER or GPPS), high impact (HIPS) and syndiotactic (SPS).
[00168] [00168] In some embodiments, the material is a thermoplastic elastomer (TPE). Exemplary TPEs (i) TPA — TPE of polyamide, comprising a block copolymer of alternating hard and soft segments with chemical amide bonds in the hard blocks and ether and / or ester bonds in the soft blocks; (ii) TPC — copolyester TPE, consisting of a block copolymer of alternating hard segments and soft segments, with chemical bonds in the main chain ester and / or ether; (iii) TPO — olefinic TPE, consisting of a combination of a polyolefin and a conventional rubber, with the rubber phase in the combination having little or no crosslinking; (iv) TPS — styrenic TPE, consisting of at least one copolymer in styrene triblock and a specific diene, where the two end blocks (hard blocks) are polystyrene and the inner block (block or soft blocks) is a hydrogenated polydene or polydiene. ; (v) TPU — urethane TPE, consisting of a block copolymer of alternating hard and soft segments with chemical urethane bonds in the hard blocks and ether, ester or carbonate bonds or mixtures thereof in the soft blocks; (vi) TPV — vulcanized thermoplastic rubber consisting of a combination of a thermoplastic material and a conventional rubber in the
[00169] [00169] In some embodiments, the material is a polymer, and the polymer is alginate. Alginate is a polysaccharide consisting of β-D-manuronic acid (M) and α-L-guluronic acid (G). In some embodiments, alginate is an alginate rich in guluronic acid (G) and comprises more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than guluronic acid (G). In some embodiments, alginate is an alginate rich in manuronic acid (M) and comprises more than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of manuronic acid (M). In some modalities, the M: G ratio is about 1. In some modalities, the M: G ratio is less than 1. In some modalities, the M: G ratio is greater than 1.
[00170] [00170] The polymer can be covalently or non-covalently associated with a surrounding component of the implantable element (eg, the surface). In some embodiments, the polymer is covalently associated with a surrounding component of the implantable element (eg, on the inner or outer surface of an implantable element). In some embodiments, the polymer is not covalently associated with a surrounding component of the implantable element (eg, on the inner or outer surface of an implantable element). The polymer can be applied by a variety of techniques in the art including, but not limited to, spraying, wetting, dipping, dipping, such as dip coating (eg, intraoperative dip coating), painting or otherwise application from a hydrophobic polymer to a surface of the surrounding component or the implantable element itself.
[00171] [00171] The active cells (eg, RPE cells) described here may be encapsulated or contained, in part or in whole, within an implantable component or device comprising a material or a number of components or materials. Exemplary components or materials can be purely structural, therapeutic, or both. An enveloping component or implantable element may comprise a biomolecule component, e.g., a carbohydrate, e.g., a polysaccharide, e.g., a marine polysaccharide, e.g., alginate, agar, agarose, carrageenans , cellulose and amylose, chitin and chitosan; cross-linked polysaccharides, e.g., cross-linked by diacrylates; or a polysaccharide or its derivative / modification described in, eg, Laurienzo (2010), Mar. Drugs. 8.9: 2435-65.
[00172] [00172] In one embodiment, the implantable element comprises an envelope component comprising a flexible polymer, eg, alginate (eg, a chemically modified alginate), PLA, PLG, PEG, CMC or mixtures thereof (referred to here as a “polymer-encapsulated implantable device”).
[00173] [00173] In one embodiment, an implantable element comprises an enveloping component that is formed, or could be formed, in situ in or surrounding an active cell, e.g., a plurality of active cells, e.g., a cluster of active cells, or in a microcarrier, eg, a spherule, or a matrix comprising an active cell or active cells (referred to here as an “in situ encapsulated implantable element”).
[00174] [00174] In one embodiment, an implantable element comprises an enveloping component that is preformed prior to combining with the active cell involved, e.g., a plurality of active cells, e.g., a grouping of active cells, or a microcarrier, e.g., a spherule or matrix comprising an active cell (referred to here as a device-based implantable element).
[00175] [00175] An implantable element can include a protein or polypeptide, e.g., an antibody, protein, enzyme or growth factor. An implantable element can include an active or inactive fragment of a protein or polypeptide, such as glucose oxidase (e.g., for glucose sensor), kinase, phosphatase, xygenase, hydrogenase, reductase.
[00176] [00176] The implantable elements can include any material, such as a material described here. In many embodiments, an implantable element consists of one material or many types of materials. In some embodiments, an implantable element comprises a polymer component (eg, hydrogel, plastic). Exemplary polymers include polyethylene, polypropylene, polystyrene, polyester (eg, PLA, PLG, or PGA, polyhydroxyalkanoates (PHAs) or other bioabsorbable plastic), polycarbonate, polyvinyl chloride (PVC), polyethersulfone (PES), polyacrylate (e.g., acrylic or PMMA), hydrogel (e.g., acrylic polymer or combination of acrylic and silicone polymers), polysulfone, polyetheretherketone, thermoplastic elastomers (TPE or TPU), thermoset elastomer (e.g. silicone (eg, silicone elastomer)), poly-p-xylene (Parylene), fluoropolymers (eg, PTFE) and polyacrylics such as poly (acrylic acid) and / or poly (acrylamide) or mixtures thereof .
[00177] [00177] The implantable elements may comprise non-organic or metal components or materials, eg, steel (eg, stainless steel), titanium, another metal or alloy. The implantable elements may include components or materials other than metal, e.g., ceramic, or hydroxyapatite elements.
[00178] [00178] The implantable elements may include components or materials that are made of a conductive material (eg, gold, platinum, palladium, titanium, copper, aluminum, silver, metals, any combinations thereof, etc.).
[00179] [00179] Implantable elements may include more than one component, e.g., more than one component disclosed here, e.g., more
[00180] [00180] In implantable elements containing metal, the amount of metal (eg, by weight%, actual weight) can be at least 5%, eg, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, e.g., w / w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1% or less.
[00181] [00181] In implantable elements containing plastic, the amount of plastic (eg, by weight%, actual weight) can be at least 5%, eg, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, w / w; or less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1% or less.
[00182] [00182] In implantable elements containing ceramic, the amount of ceramic (eg, by weight%, actual weight) can be at least 5%, eg, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, w / w; or less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1% or less.
[00183] [00183] The implantable elements included here include implantable elements that are configured with a lumen, e.g., a lumen having one, two or more openings, e.g., tubular devices. A typical endoprosthesis is an example of a device configured with a lumen and having two openings. Other examples include shunts.
[00184] [00184] The implantable elements included here include flexible implantable elements, eg, that are configured to conform to the shape of the body.
[00185] [00185] The implantable elements included here include components that stabilize the location of the implantable element, e.g., an adhesive, or closure, e.g., a torque or friction-based closure, e.g., a screw or a pin.
[00186] [00186] The implantable elements included here may be
[00187] [00187] The implantable elements included here can be configured to release a substance, eg, an exogenous substance, eg, a therapeutic agent. In some embodiments, the therapeutic agent is a compound of Formula (I)) or a pharmaceutically acceptable salt thereof. In some embodiments, the therapeutic agent is a biological material. In some embodiments, the therapeutic agent is a cell, cell product, tissue, tissue product, protein, hormone, enzyme, antibody, antibody fragment, antigen, epitope, drug, vaccine or any derivative thereof.
[00188] [00188] The implantable elements here can be configured to change the conformation in response to a signal or movement of the body, eg, an artificial joint, eg, a knee, hip or other artificial joint.
[00189] [00189] Exemplary implantable elements include an endoprosthesis, shunt, dressing, eye device, door, sensor, orthopedic fixation device, implant (e.g., a dental implant, ocular implant, silicone implant, corneal implant, dermal implant , intragastric implant, facial implant, hip implant, bone implant, cochlear implant, penile implant, incontinence control implants), skin covering device, dialysis medium, drug delivery device, artificial or manipulated organ (eg ., a spleen, kidney, liver or heart), drainage device (eg, a bladder drainage device), cell selection system, adhesive (eg, a cement, staple, clip), contraceptive device, intrauterine device, defibrillator, dosimeter, electrode, pump filter (eg, infusion pump), embolization device, closure, fillers, fixator, graft, hearing aid,
[00190] [00190] In some embodiments, an implantable element includes encapsulated or trapped cells or tissues. The cells or tissue can be encapsulated or trapped in a polymer. In some embodiments, an implantable element includes an active cell (eg, an RPE cell), eg, an active cell (eg, an RPE cell) disposed within a polymeric envelope component (eg. alginate).
[00191] [00191] In some embodiments, an implantable element targets or is designed for a certain body system, eg, the nervous system (eg, peripheral nervous system (PNS) or central nervous system (CNS)), vascular system, skeletal system, respiratory system, endocrine system, lymphatic system, reproductive system or gastrointestinal tract. In some modalities, an implantable element is targeted at the CNS. In some embodiments, an implantable element targets or is designed for a certain part of the body, eg blood, eye, brain, skin, lung, stomach, mouth, ear, leg, foot, hand, liver, heart, kidney , bone, pancreas, spleen, large intestine, small intestine, spinal cord, muscle, ovary, uterus, vagina or penis.
[00192] [00192] The implantable elements included here include devices
[00193] [00193] The components or materials used in an implantable element (or the entire implantable element) can be optimized for one or more of biocompatibility (eg, minimizes immune rejection or fibrosis; heat resistance; elasticity; tensile strength ; chemical resistance (eg, resistance to oils, fats, disinfectants, brighteners, processing aids or other chemicals used in the production, use, cleaning, sterilization and disinfection of the device); electrical properties; surface and volumetric conductivity or resistivity, dielectric strength; comparative screening index; mechanical properties; service life, sterilization capacity for long-term durability (eg, able to withstand sterilization processes such as steam, dry heat, ethylene oxide (EtO), electron beam and / or gamma radiation, eg, while maintaining the properties for the intended use of the device), eg, thermal resistance to autoclave / vap conditions or, hydrolytic stability for steam sterilization, chemical resistance to EtO, resistance to high intensity radiation (eg, electron beam, UV and gamma); or crystal structure.
[00194] [00194] An implantable element can be assembled in vivo (eg, injectable substance that forms a structured form in vivo, eg, at body temperature) or ex vivo.
[00195] [00195] An implantable element can have nanodimensions, eg, it can comprise a nanoparticle, eg, nanoparticle made of a polymer described here, eg, PLA. Nanoparticles can be chemically modified nanoparticles, eg, modified to prevent uptake by macrophages and Kupfer cells (eg, a process called opsonization); or to change the circulating half-life of the nanoparticle. Nanoparticles
[00196] [00196] An implantable element can be configured for implantation or implanted or disposed: in a subject's omentum, in a subject's subcutaneous fat, intramuscularly in a subject. An implantable element can be configured for implantation or implanted or disposed on or in; the skin; a mucous surface, a body cavity, the peritoneal cavity (eg, the smaller sac); the CNS, eg, the brain or spinal cord; an organ, eg, the heart, liver, kidney, spleen, lung, lymphatic system, vasculature, the oral cavity, the nasal cavity, the teeth, the gums, the GI tract; bone; hip; fatty tissue; muscle tissue; circulating blood; the eye (e.g., intraocular); breast, vagina; uterus, a joint, eg, the knee or hip joint or the spine. In some embodiments, the implantable element is configured for implantation or implanted or disposed in the peritoneal cavity (eg, the smaller sac).
[00197] [00197] An implantable element may comprise an electrochemical sensor, eg, an electrochemical sensor including a working electrode and a reference electrode. For example, an electrochemical sensor includes a working electrode and a reference electrode that reacts with an analyte to generate a sensor measurement related to an analyte concentration in a fluid to which the eye-mountable device is exposed. The implantable element may comprise a window, e.g., of a transparent polymeric material having a concave surface and a convex surface, a substrate, e.g., at least partially embedded in a transparent polymeric material. An implantable element may also comprise an electronics module including one or more of an antenna; and a controller electrically connected to the electrochemical sense and the antenna,
[00198] [00198] In some embodiments, an implantable element has an average diameter or size that is greater than 1 mm, preferably 1.5 mm or greater. In some embodiments, an implantable element can be as large as 8 mm in diameter or size. For example, an implantable element described here is in a size range of 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm , 1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm, 1.5 mm to 3 mm, 1 , 5 mm to 2 mm, 2 mm to 8 mm, 2 mm to 7 mm, 2 mm to 6 mm, 2 mm to 5 mm, 2 mm to 4 mm, 2 mm to 3 mm, 2.5 mm to 8 mm , 2.5 mm to 7 mm, 2.5 mm to 6 mm, 2.5 mm to 5 mm, 2.5 mm to 4 mm, 2.5 mm to 3 mm, 3 mm to 8 mm, 3 mm to 7 mm, 3 mm to 6 mm, 3 mm to 5 mm, 3 mm to 4 mm, 3.5 mm to 8 mm, 3.5 mm to 7 mm, 3.5 mm to 6 mm, 3.5 mm to 5 mm, 3.5 mm to 4 mm, 4 mm to 8 mm, 4 mm to 7 mm, 4 mm to 6 mm, 4 mm to 5 mm, 4.5 mm to 8 mm, 4.5 mm to 7 mm , 4.5 mm to 6 mm, 4.5 mm to 5 mm, 5 mm to 8 mm, 5 mm to 7 mm, 5 mm to 6 mm, 5.5 mm to 8 mm, 5.5 mm to 7 mm , 5.5 mm to 6 mm, 6 mm to 8 mm, 6 mm to 7 mm, 6.5 mm to 8 mm, 6.5 mm to 7 mm, 7 mm to 8 mm or 7.5 mm to 8 mm . In some embodiments, the implantable element has an average diameter or size between 1 mm to 8 mm. In some embodiments, the implantable element has an average diameter or size between 1 mm to 4 mm. In some embodiments, the implantable element has an average diameter or size between 1 mm to 2 mm.
[00199] [00199] In some embodiments, an implantable element comprises at least one pore or opening, eg, to allow the free flow of materials. In some embodiments, the average pore size of a
[00200] [00200] In some modalities, an implantable element is able to prevent materials above a certain size from passing through a pore or opening. In some embodiments, an implantable element is able to prevent materials larger than 50 kD, 75 kD, 100 kD, 125 kD, 150 kD, 175 kD, 200 kD, 250 kD, 300 kD, 400 kD, 500 kD, 750 kD, 1,000 kD pass.
[00201] [00201] An implantable element (e.g., an implantable element described here) can be provided as a preparation or composition for implantation or administration to a subject. In some modalities, at least 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the implantable elements in a preparation or composition have a characteristic as described here, eg, average pore size.
[00202] [00202] In some embodiments, an implantable element can be used for varying periods of time, ranging from a few minutes to several years. For example, an implantable element can be used for about 1 hour to about 10 years. In some embodiments, an implantable element is used for longer than about 1 hour, 2 hours, 4 hours, 8 hours, 16 hours, 1 day, 48 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1
[00203] [00203] In some embodiments, the implantable element is easily recoverable from a subject, e.g., without causing damage to the subject or without causing significant disruption to the surrounding tissue. In one embodiment, the implantable element can be recovered with minimal or no surgical separation of the implantable element from the surrounding tissue, eg, through minimally invasive inspection, extraction or surgical resection.
[00204] [00204] An implantable element can be configured for limited exposure (e.g., less than 2 days, e.g., less than 2 days, 1 day, 24 hours, 20 hours, 16 hours, 12 hours, 10 hours, 8 hours, 6 hours, 5 hours, 4 hours, 3 hours, 2 hours, 1 hour or less). An implantable element can be configured for prolonged exposure (eg, at least 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months , 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years or more ). An implantable element can be configured for permanent exposure (eg, at least 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years, 3.5 years, 4 years or more).
[00205] [00205] In some embodiments, an implantable element is not an implantable element disclosed in any of WO2012 / 112982,
[00206] [00206] In one embodiment, the implantable element comprises an active cell (eg, an RPE cell) described here. In one embodiment, the implantable element comprises an active cell (eg, an RPE cell), as well as another cell, eg, a recombinant cell or stem cell, which provides a substance, eg, a therapeutic agent described there.
[00207] [00207] In one embodiment, the active cell is a human RPE cell (or a cell derived from it, e.g., an ARPE-19 cell) and the polypeptide is a human polypeptide. In one embodiment, the active cell (e.g. eg RPE cell) provides a substance that alleviates a disease, dysfunction or condition (eg, as described here). Chemical Modification of Implantable Elements
[00208] [00208] The present disclosure features an implantable element comprising an active cell (e.g., an RPE cell), in which the implantable element is chemically modified. Chemical modification can impart an improved property to the implantable element when administered to a subject, e.g., modulation of the immune response in the subject, compared to an unmodified implantable element.
[00209] [00209] In some embodiments, a surface of the implantable element comprising a manipulated active cell (e.g., a manipulated RPE cell) is chemically modified with a compound. In some embodiments, a surface comprises an outer surface or an inner surface of the implantable element. In some embodiments, the surface (e.g., outer surface) of the implantable element comprising a manipulated active cell (e.g., a manipulated RPE cell) is chemically modified with a compound. In some embodiments, the surface (eg, outer surface) is covalently attached to a compound. In some embodiments, the compound comprises at least
[00210] [00210] In some embodiments, the compound is a compound of Formula (I): (I), or a pharmaceutically acceptable salt thereof, wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, –O–, –C (O) O–, –C (O) -, –OC (O) -, –N (RC) -, –N (RC) C (O) -, –C (O) N (RC) -, -N (RC) C (O) (C1-C6 alkylene) -, - N (RC) C (O) (C1-C6 alkenylene) -, –N (RC) N (RD) -, –NCN–, - C (= N (RC) (RD)) O–, –S–, –S (O) x–, –OS (O) x–, –N (RC) S (O) x–, - S (O) xN (RC) -, –P (RF) y–, –Si (ORA) 2 -, –Si (RG) (ORA) -, –B (ORA) - or a metal, wherein each alkyl, alkenyl, alkynyl, alkylene, alkenylene, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is attached to an attachment group (e.g., an attachment group defined here) and is optionally substituted by one or more R1; each of L1 and L3 is independently a bond, alkyl or heteroalkyl, wherein each alkyl and heteroalkyl is optionally substituted by one or more R2; L2 is a link; M is absent, is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R3; P is absent, is cycloalkyl, heterocyclyl or heteroaryl, each of which is optionally substituted by one or more R4; Z is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, - ORA, –C (O) RA, –C (O) ORA, –C (O) N (RC) (RD), –N (RC) C (O ) RA, cycloalkyl, heterocyclyl, aryl or heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is
[00211] [00211] In some embodiments, the compound of Formula (I) is a compound of Formula (I-a):
[00212] [00212] In some embodiments, for Formulas (I) and (Ia), A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, –O–, –C (O) O–, –C (O) -, –OC (O) -, –N (RC) C (O) -, - N (RC) C (O) (C1-C6 alkylene) -, –N (RC) C (O) (C1-C6 alkylene) - or –N (RC) -. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, –O–, –C (O) O–, –C (O) -, - OC (O) - or –N (RC) -. In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, –O–, –C (O) O–, –C (O) -, –OC (O) - or –N (RC) -. In some embodiments, A is alkyl, –O–, –C (O) O–, –C (O) -, –OC (O) or - N (RC) -. In some embodiments, A is –N (RC) C (O) -, –N (RC) C (O) (alkylene-
[00213] [00213] In some embodiments, for Formulas (I) and (I-a), L1 is a bond, alkyl or heteroalkyl. In some embodiments, L1 is a bond or alkyl. In some embodiments, L1 is a link. In some embodiments, L1 is alkyl. In some embodiments, L1 is C1-C6 alkyl. In some modalities, L1 is –CH2–, –CH (CH3) -, –CH2CH2CH2 or –CH2CH2–. In some modalities, L1 is –CH2– or –CH2CH2–.
[00214] [00214] In some embodiments, for Formulas (I) and (I-a), L3 is a bond, alkyl or heteroalkyl. In some modalities, L3 is a link. In some embodiments, L3 is alkyl. In some embodiments, L3 is C1-C6 alkyl. In some modalities, L3 is –CH2–. In some embodiments, L3 is heteroalkyl. In some embodiments, L3 is C1-C6 heteroaryl, optionally substituted by one or more R2 (eg, oxo). In some embodiments, L3 is –C (O) OCH2–, –CH2 (OCH2CH2) 2–, –CH2 (OCH2CH2) 3–, CH2CH2O– or –CH2O–. In some modalities, L3 is –CH2O–.
[00215] [00215] In some modalities, for Formulas (I) and (I-a), M is absent, is alkyl, heteroalkyl, aryl or heteroaryl. In some embodiments, M is heteroalkyl, aryl or heteroaryl. In some modalities, M is absent. In some embodiments, M is alkyl (e.g., C1-C6 alkyl). In some modalities, M is -CH2–. In some embodiments, M is heteroalkyl (eg, C1-C6 heteroalkyl). In some
[00216] [00216] In some modalities, for Formulas (I) and (I-a), P is absent, is heterocyclyl or heteroaryl. In some modalities, P is absent. In some modalities, for Formulas (I) and (I-a), 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 modalities, P is a heteroaryl containing nitrogen, monocyclic. In some modalities, P is a 5-membered heteroaryl. In some embodiments, P is a 5-membered nitrogen containing heteroaryl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl or triazolyl, 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. In some embodiments, P is triazolyl. In some embodiments, P is 1,2,3-triazolyl. In some embodiments, P is.
[00217] [00217] In some modalities, P is heterocyclyl. In some embodiments, P is a 5-membered heterocyclyl or 6-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some
[00218] [00218] In some embodiments, for Formulas (I) and (I-a), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl. In some embodiments, Z is heterocyclyl. In some embodiments, Z is monocyclic or bicyclic heterocyclyl. In some embodiments, Z is a heterocyclyl containing oxygen. In some embodiments, Z is a 4-membered heterocyclyl, 5-membered heterocyclyl or 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, or. In some embodiments, Z is a 4-membered oxygen-containing heterocyclyl. In some modalities, Z is.
[00219] [00219] In some modalities, Z is a heterocyclyl containing bicyclic oxygen. In some embodiments, Z is phthalic anhydridyl. In some embodiments, Z is a sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered sulfur-containing heterocyclyl. In some embodiments, Z is a 6-membered heterocyclyl containing a nitrogen atom and a sulfur atom. In some embodiments, Z is thiomorpholinyl-1,1-dioxidyl. In some modalities, Z is. In some embodiments, Z is a heterocyclyl containing nitrogen. In some embodiments, Z is a 6-membered nitrogen containing heterocyclyl. In
[00220] [00220] In some modalities, Z is a bicyclic heterocyclyl. In some embodiments, Z is a heterocyclyl containing bicyclic nitrogen, optionally substituted by one or more R5. In some embodiments, Z is 2-oxa-7-azospiro [3.5] nonanila. In some modalities, Z is. In some embodiments, Z is 1-oxa-3,8-diazaspiro [4.5] decan-2-one. In O The NH
[00221] [00221] In some modalities, for Formulas (I) and (I-a), Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is monosubstituted phenyl (eg, by 1 R5). In some embodiments, Z is monosubstituted phenyl, where 1 R5 is a group containing nitrogen. In some embodiments, Z is monosubstituted phenyl, where 1 R5 is NH2. In some embodiments, Z is monosubstituted phenyl, where 1 R5 is an oxygen-containing group. In some embodiments, Z is monosubstituted phenyl, where 1 R5 is an oxygen-containing heteroalkyl. In some embodiments, Z is monosubstituted phenyl, where 1 R5 is OCH3. In some embodiments, Z is monosubstituted phenyl, where 1 R5 is in the ortho position. In some embodiments, Z is monosubstituted phenyl, where 1 R5 is in the target position. In some embodiments, Z is monosubstituted phenyl, where 1 R5 is in the para position.
[00222] [00222] In some embodiments, for Formulas (I) and (I-a), Z is alkyl. In some embodiments, Z is C1-C12 alkyl. In some embodiments, Z is C1-C10 alkyl. In some embodiments, Z is C1-C8 alkyl.
[00223] [00223] In some modalities, for Formulas (I) and (I-a), Z is heteroalkyl. In some embodiments, Z is C1-C12 heteroalkyl. In some embodiments, Z is C1-C10 heteroalkyl. In some embodiments, Z is C1-C8 heteroalkyl. In some embodiments, Z is C1-C6 heteroalkyl. In some embodiments, Z is a heteroalkyl containing nitrogen optionally substituted by one or more R5. In some embodiments, Z is a heteroalkyl containing nitrogen and sulfur replaced by 1-5 R5. In some embodiments, Z is N-methyl-2- (methylsulfonyl) ethan-1-aminyl.
[00224] [00224] In some modalities, Z is -ORA or -C (O) ORA. In some embodiments, Z is -ORA (eg, -OH or -OCH3). In some embodiments, Z is -C (O) ORA (eg, –C (O) OH).
[00225] [00225] In some modalities, Z is hydrogen.
[00226] [00226] In some modalities, L2 is a link and P and L3 are independently absent. In some embodiments, L2 is a bond, P is heteroaryl, L3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L3 is heteroalkyl, and Z is alkyl.
[00227] [00227] In some embodiments, the compound of Formula (I) is a compound of Formula (II): (II),
[00228] [00228] In some embodiments, the compound of Formula (II) is a compound of Formula (II-a):
[00229] [00229] In some embodiments, the compound of Formula (II-a) is a compound of Formula (II-b): (II-b), or a pharmaceutically acceptable salt thereof, wherein Ring Z2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; each R3 and R5 is independently alkyl, heteroalkyl, halogen, oxo, –ORA1, –C (O) ORA1 or –C (O) RB1; each RA1 and RB1 is independently hydrogen, alkyl or heteroalkyl; each of p and q is independently 0, 1, 2, 3, 4, 5 or 6; and “” refers to a connection to an attachment group or a polymer described here.
[00230] [00230] In some embodiments, the compound of Formula (II-a) is a compound of Formula (II-c):
[00231] [00231] In some embodiments, the compound of Formula (I) is a compound of Formula (II-d): R2b R2a NNNX Z2 n (R5) p HN m R2c R2d (II-d), or a pharmaceutically acceptable salt thereof , wherein Ring Z2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, it is O or S; each of R2a, R2b, R2c and R2d is independently hydrogen, alkyl or heteroalkyl or each of R2a and R2b or R2c and R2d is taken together to form an oxo group; each R5 is independently alkyl, heteroalkyl, halogen, oxo, –ORA1, –C (O) ORA1 or –C (O) RB1; each RA1 and RB1 is independently hydrogen, alkyl or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4, 5 or 6; and “” refers to a connection to an attachment group or a polymer described here.
[00232] [00232] In some embodiments, the compound of Formula (I) is a compound of Formula (III):
[00233] [00233] In some embodiments, the compound of Formula (III) is a compound of Formula (III-a): (III-a), or a pharmaceutically acceptable salt thereof, wherein L3 is alkyl or heteroalkyl, each of which it is optionally replaced by one or more R2; Z is alkyl or heteroalkyl, each of which is optionally substituted by one or more R5; each of R2a and R2b is independently hydrogen, alkyl or heteroalkyl, or R2a and R2b is taken together to form an oxo group; each R2, R3 and R5 is independently alkyl, heteroalkyl, halogen, oxo, –ORA1, –C (O) ORA1 or –C (O) RB1; each RA1 and RB1 is independently hydrogen, alkyl or heteroalkyl; n is independently 1, 2, 3, 4, 5 or 6; and ““ refers to
[00234] [00234] In some embodiments, the compound of Formula (I) is a compound of Formula (IV): (IV), or a pharmaceutically acceptable salt thereof, wherein Z1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1-5 R5; each of R2a, R2b, R2c and R2d is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl or heteroaryl; or R2a and R2b or R2c and R2d are taken together to form an oxo group; RC is hydrogen, alkyl, alkenyl, where each of alkyl and alkenyl is optionally substituted by 1-6 R6; each of R3, R5 and R6 is independently alkyl, heteroalkyl, halogen, oxo, –ORA1, –C (O) ORA1 or –C (O) RB1; each RA1 and RB1 is independently hydrogen, alkyl or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5 or 6; q is an integer from 0 to 25; and “” refers to a connection to an attachment group or a polymer described here.
[00235] [00235] In some embodiments, the compound of Formula (IV) is a compound of Formula (IV-a): (IV-a), or a pharmaceutically acceptable salt thereof, wherein Ring Z2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; each of R2a, R2b, R2c and R2d is independently hydrogen, alkyl, heteroalkyl, halo; or R2a and R2b or
[00236] [00236] In some embodiments, the compound of Formula (IV-a) is a compound of Formula (IV-b): (IV-b), or a pharmaceutically acceptable salt thereof, where X is C (R ') ( R "), N (R ') or S (O) x; each of R' and R" is independently hydrogen, alkyl, halogen or cycloalkyl; each of R2a, R2b, R2c and R2d is independently hydrogen, alkyl, heteroalkyl, halo; or R2a and R2b or R2c and R2d are taken together to form an oxo group; each of R3 and R5 is independently alkyl, heteroalkyl, halogen, oxo, –ORA1, - C (O) ORA1 or –C (O) RB1; each RA1 and RB1 is independently hydrogen, alkyl or heteroalkyl; one of m and n are each independently 1, 2, 3, 4, 5 or 6; p is 0, 1, 2, 3, 4 or 5; q is an integer from 0 to 25; x is 0, 1 or 2; and “” refers to a connection to an attachment group or a polymer described here.
[00237] [00237] In some embodiments, the compound is a compound of Formula (I). In some embodiments, L2 is a link and P and L3 are independently absent. In some embodiments, L2 is a bond, P is heteroaryl, L3 is a bond, and Z is hydrogen. In some embodiments, P is heteroaryl, L3 is heteroalkyl, and Z is alkyl. In some embodiments, L2 is a link and P and L3 are independently absent. In some
[00238] [00238] In some embodiments, the compound is a compound of Formula (II-b). In some modalities of Formula (II-b), each of R2c and R2d is independently hydrogen, m is 1, q is 0, p is 0, and Z is heterocyclyl (eg, an oxygen-containing heterocyclyl). In some embodiments, the compound of Formula (II-b) is Compound 100.
[00239] [00239] In some embodiments, the compound is a compound of Formula (II-c). In some modalities of Formula (II-c), each of R2c and R2d is independently hydrogen, m is 1, p is 1, q is 0, R5 is –CH3, and Z is heterocyclyl (eg, a heterocyclyl containing nitrogen). In some embodiments, the compound of Formula (II-c) is Compound 113.
[00240] [00240] In some embodiments, the compound is a compound of Formula (II-d). In some embodiments of Formula (II-d), each of R2a, R2b, R2c and R2d is independently hydrogen, m is 1, n is 3, X is O, p is 0, and Z is heterocyclyl (e.g. , an oxygen-containing heterocyclyl). In some embodiments, the compound of Formula (II-d) is Compound 110 or Compound
[00241] [00241] In some embodiments, the compound is a compound of Formula (III-a). In some modalities of Formula (III-a), each of R2a and R2b is independently hydrogen, n is 1, q is 0, L3 is –CH2 (OCH2CH2) 2-, and Z is –OCH3. In some embodiments, the compound of Formula (III-a) is Compound 112.
[00242] [00242] In some embodiments, the compound is a compound of Formula (IV-a). In some modalities of Formula (IV-a), each of R2a, R2b, R2c and R2d is independently hydrogen, each of men is independently 1, p is 0, q is 3, o is 0 or 1, R5, if present, it is –NH2, and Z is aryl or heterocyclyl (eg, a nitrogen-containing heterocyclyl). In
[00243] [00243] In some embodiments, the compound of Formula (I) is not a compound disclosed in WO2012 / 112982, WO2012 / 167223, WO2014 / 153126, WO2016 / 019391, WO 2017/075630, US2012-0213708, US 2016-0030359 or US 2016-0030360.
[00244] [00244] In some embodiments, the compound of Formula (I) comprises a compound shown in the Table of Compound 1 or a pharmaceutically acceptable salt thereof. Compound Table 1: Exemplary Compounds No. of Compound Structure 100 101 102 103 104 105 106
[00245] [00245] In some embodiments, the compound of Formula (I) (e.g., Formulas (Ia), (II), (II-b), (II-c), (II-d), (III) , (III-a), (IV), (IV-a) or (IV-b)), or a pharmaceutically acceptable salt thereof, is selected from:, and N N N
[00246] [00246] In some embodiments, the compound of Formula (I) described here is selected from:,
[00247] [00247] An implantable element can be coated with a compound of Formula (I) or a pharmaceutically acceptable salt thereof or a material comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In one embodiment, the compound of Formula (I) is arranged on a surface, e.g., an internal or external surface, of the implantable element. In some embodiments, the compound of Formula (I) is arranged on a surface, eg, an internal or external surface, of a surrounding component associated with an implantable element. In one embodiment, the compound of Formula (I) is evenly distributed across a surface. In one embodiment, the compound of Formula (I) is distributed unevenly across a surface.
[00248] [00248] In some embodiments, an implantable element (eg, or a surrounding component thereof) is coated (eg, partially or completely covered) with a compound of Formula (I) or a material comprising the Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, an implantable element (eg, or a surrounding component) is coated with a single layer of a compound of Formula (I). In some embodiments, a device is coated with multiple layers of a compound of Formula (I), e.g., at least 2 layers, 3 layers, 4 layers, 5 layers, 10 layers, 20 layers, 50 layers or more.
[00249] [00249] In one embodiment, a first portion of the surface of the
[00250] [00250] In one embodiment, a first portion of the surface of the implantable element comprises a compound of Formula (I) that modulates, e.g., subregulation, an immune response and a second portion of the surface comprises a second compound of Formula (I ), e.g., which over-regulates the immune response, the second portion of the implantable element lacks the compound or has a substantially lower density of the compound.
[00251] [00251] In some embodiments, an implantable element is coated or chemically derivatized in a symmetrical manner with a compound of Formula (I), or a material comprising Formula (I)), or a pharmaceutically acceptable salt thereof. In some embodiments, an implantable element is coated or chemically derivatized in an asymmetrical manner with a compound of Formula (I), or a material comprising Formula (I)), or a pharmaceutically acceptable salt thereof. For example, an exemplary implantable element may be partially coated (eg, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% , 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 99.9% coated) with a compound of Formula (I) or a material comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof.
[00252] [00252] Exemplary implantable elements coated or chemically derivatized with a compound of Formula (I), or a material comprising Formula (I), or a pharmaceutically acceptable salt thereof, can be prepared using any method known in the art, such as through self-assembly (eg through block copolymers, adsorption (eg competitive adsorption), phase separation,
[00253] [00253] In some embodiments, the implantable element comprises a surface exhibiting two or more distinct physical-chemical properties (eg, 3, 4, 5, 6, 7, 8, 9, 10 or more distinct physical-chemical properties ).
[00254] [00254] In some embodiments, the coating or chemical derivatization of the surface of an exemplary implantable element with a compound of Formula (I), a material comprising Formula (I), or a pharmaceutically acceptable salt thereof, is described as the average number of compounds attached by a given area, eg as a density. For example, the coating density or chemical derivatization of an exemplary implantable element can be 0.01, 0.1, 0.5, 1, 5, 10, 15, 20, 50, 75, 100, 200, 400, 500 , 750, 1,000, 2,500 or 5,000 composed of µm square or square mm, eg, on the surface or interior of said implantable element.
[00255] [00255] An implantable element comprising a compound of Formula (I) or a pharmaceutically salt thereof may have a reduced immune response (e.g., an immune response marker) compared to an implantable element that does not comprise a compound of the Formula (I) or a pharmaceutically acceptable salt thereof. An immune response marker is one or more of: cathepsin level or the level of an immune response marker, eg, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL -4, CCL2 or CCL4, as measured, e.g., by ELISA. In some embodiments, an implantable element comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof has an immune response of about 1%, about 5%, about 10%, about 15%, about 20% , about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70% , about 75%, about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% reduced (e.g., a marker
[00256] [00256] An implantable element comprising a compound of Formula (I) or a pharmaceutically salt thereof may have an increased immune response (e.g., an immune response marker) compared to an implantable element that does not comprise a compound of the Formula (I) or a pharmaceutically acceptable salt thereof. An immune response marker is one or more of: cathepsin activity or the level of an immune response marker, eg, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL -4, CCL2 or CCL4, as measured, e.g., by ELISA. In some embodiments, an implantable element comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof has an immune response of about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or about 100% or about 1000% increased (eg a marker of a immune response) compared to an implantable element that does not comprise a compound of Formula (I) or a pharmaceutically acceptable salt thereof. In some embodiments, the increased immune response (eg, a marker of an immune response) is
[00257] [00257] An implantable element may have a smooth surface or may comprise a protuberance, depression, well, crack or orifice or any combination thereof. Said protuberance, depression, well, crack or orifice can be of any size, e.g., from 10 µm to about 1 nm, about 5 µm to about 1 nm, about 2.5 µm to about 1 nm, 1 µm at about 1 nm, 500 nm at about 1 nm or about 100 nm at about 1 nm. The smooth surface or protuberance, depression, well, crack or orifice, or any combination thereof, may be coated or chemically derivatized with a compound of Formula (I), a material comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof .
[00258] [00258] An implantable element can take any suitable shape, such as a sphere, spheroid, ellipsoid, disc, cylinder, torus, cube, stadium, cone, pyramid, triangle, rectangle, square or rod or it can comprise a curved or flat section . Any shaped, curved or flat implantable element can be coated or chemically derivatized with a compound of Formula (I), a material comprising a compound of Formula (I) or a pharmaceutically acceptable salt thereof. Treatment Methods
[00259] [00259] Methods for preventing or treating a disease, dysfunction or condition in a subject by administering or implanting an RPE cell, e.g., encapsulated by a material or device described here, are described here. In some modalities, the methods described here reduce or alleviate directly or indirectly at least one symptom of
[00260] [00260] In some embodiments, the disease, dysfunction or condition affects a system of the body, eg, the nervous system (eg, peripheral nervous system (PNS) or central nervous system (CNS)), vascular system , skeletal system, respiratory system, endocrine system, lymphatic system, reproductive system or gastrointestinal tract. In some embodiments, the disease, dysfunction or condition affects a part of the body, eg, blood, eye, brain, skin, lung, stomach, mouth, ear, leg, foot, hand, liver, heart, kidney, bone , pancreas, spleen, large intestine, small intestine, spinal cord, muscle, ovary, uterus, vagina or penis.
[00261] [00261] In some embodiments, the disease, dysfunction or condition is a neurodegenerative disease, diabetes, a heart disease, an autoimmune disease, a cancer, a liver disease, a lysosomal storage disease, a blood clotting disorder or a dysfunction coagulation, an orthopedic condition, a dysfunction of the metabolism of amino acids.
[00262] [00262] In some modalities, the disease, dysfunction or condition is a neurodegenerative disease. Exemplary neurodegenerative diseases include Alzheimer's disease, Huntington's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and cerebral palsy (CP), dentato-red-pale-Luis atrophy (DRPLA), neuronal intranuclear hyaline inclusion disease (NIHID), dementia with Lewy bodies, Down syndrome, Hallervorden-Spatz disease, prion diseases, dementia with argyrophil grains, cortocobasal degeneration, pugilistic dementia, diffuse neurofibrillary tangles, Gerstmann-Sträussler disease -Scheinker, Jakob-Creutzfeldt disease, Niemann-Pick disease type 3, progressive supranuclear palsy, sclerosing panencephalitis
[00263] [00263] In some embodiments, the disease, dysfunction or condition is an autoimmune disease, eg, scleroderma, multiple sclerosis, lupus or allergies.
[00264] [00264] In some embodiments, the disease is a liver disease, eg, hepatitis B, hepatitis C, cirrhosis, NASH.
[00265] [00265] In some modalities, the disease, dysfunction or condition is cancer. Exemplary cancers include leukemia, lymphoma, melanoma, lung cancer, brain cancer (eg, glioblastoma), sarcoma, pancreatic cancer, kidney cancer, liver cancer, testicular cancer, prostate cancer or uterine cancer.
[00266] [00266] In some modalities, the disease, dysfunction or condition is an orthopedic condition. Exemplary orthopedic conditions include osteoporosis, osteonecrosis, Paget's disease or a fracture.
[00267] [00267] In some modalities, the disease, dysfunction or condition is a disease of lysosomal storage. Exemplary lysosomal storage disorders include Gaucher disease (eg, Type I, Type II, Type III), Tay-Sachs disease, Fabry disease, Farber disease, Hurler syndrome (also known as type I mucopolysaccharidosis ( MPS I)), Hunter syndrome, lysosomal acid lipase deficiency, Niemann-Pick disease, Salla disease, Sanfilippo syndrome (also known as mucopolysaccharidosis type IIIA (MPS3A)), multiple sulfatase deficiency, Maroteaux-Lamy syndrome , metachromatic leukodystrophy, Krabbe disease, Scheie syndrome, Hurler-Scheie syndrome, Sly syndrome, hyaluronidase deficiency, Pompe disease, Danon disease, gangliosidosis or Morquio syndrome.
[00268] [00268] In some embodiments, the disease, dysfunction or condition is a blood clotting disorder or a coagulation disorder.
[00269] [00269] In some embodiments, the disease, dysfunction or condition is a dysfunction of amino acid metabolism, eg, phenylketonuria, tyrosinemia (eg, Type 1 or Type 2), alkaptonuria, homocystinuria, hyperhomocysteinemia, maple syrup urine disease.
[00270] [00270] In some embodiments, the disease, dysfunction or condition is a dysfunction of the fatty acid metabolism, eg, hyperlipidemia, hypercholesterolemia, galactosemia.
[00271] [00271] In some embodiments, the disease, dysfunction or condition is a dysfunction of the purine or pyrimidine metabolism, eg, Lesch-Nyhan syndrome.
[00272] [00272] The present disclosure further comprises methods for identifying a subject having or suspected of having a disease, dysfunction or condition described herein and, after such identification, administering to the subject an implantable element comprising an active cell (e.g., an cell RPE), e.g., optionally encapsulated by a surrounding component and optionally modified with a compound of Formula (I) as described herein or a composition thereof. Pharmaceutical Compositions, Cases and Administration
[00273] [00273] The present disclosure further comprises implantable elements comprising active cells (e.g., RPE cells), as well as pharmaceutical compositions comprising them and their cases.
[00274] [00274] In some embodiments, a pharmaceutical composition comprises active cells (e.g., RPE cells) and a pharmaceutically acceptable excipient. In some embodiments, a pharmaceutical composition comprises manipulated active cells (eg, RPE cells
[00275] [00275] The pharmaceutical compositions described here can be prepared by any method known in the art of pharmacology. In general, such preparatory methods include the steps of placing the active cells (eg, RPE cells or hydrogel capsules encapsulating the RPE cells, ie, “the active ingredient”) in association with a carrier and / or one or more other accessory ingredients and then, if necessary and / or desirable, shaping and / or packaging the product in a desired single or multiple dose unit.
[00276] [00276] Pharmaceutical compositions can be prepared, packaged and / or sold in bulk, as a single unit dose and / or as a plurality of single unit doses. As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject and / or a convenient fraction of such a dosage such as, for example, half or a third of such a dosage.
[00277] [00277] The relative amounts of the active ingredients, the pharmaceutically acceptable excipient and / or any additional ingredients in a pharmaceutical composition of the disclosure will vary, depending on the identity, size and / or condition of the treated subject and additionally depending on the route by which the composition is to be administered. As an example, the composition may comprise between 0.1% and 100% (w / w) of active ingredient.
[00278] [00278] The term "pharmaceutically acceptable excipient" refers to
[00279] [00279] Active cells (eg, RPE cells), implantable elements, and their compositions, can be administered orally, parenterally (including subcutaneous, intramuscular and intradermal), topically, rectally, nasally, intratumorally, intrathecally, buccally, vaginally or through an implanted reservoir. In some embodiments, the compounds or compositions provided are administrable subcutaneously or by implant.
[00280] [00280] In some embodiments, active cells (eg, RPE cells), implantable elements (eg, hydrogel capsule encapsulating
[00281] [00281] In some embodiments, active cells (eg, RPE cells), implantable elements, and their compositions, are administered or implanted in a location other than the central nervous system, eg, the brain, medulla spinal, nerve. In some embodiments, active cells (eg, RPE cells), implantable elements, and their compositions, are administered or implanted in a location other than the eye (eg, retina).
[00282] [00282] The sterile injectable forms of the compositions of this disclosure can be aqueous or oleaginous suspension. These suspensions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, fixed, sterile oils are conventionally employed as a solvent or suspending medium.
[00283] [00283] For ophthalmic use, the pharmaceutically acceptable compositions provided can be formulated as suspensions
[00284] [00284] In order to prolong the effect of the active ingredient, it may be desirable to slow the absorption of the drug from subcutaneous or intramuscular injection.
[00285] [00285] In some embodiments, the active cells (eg, RPE cells) are arranged in a microcarrier (eg, a bead, eg, a polystyrene bead).
[00286] [00286] Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions that are suitable for administration to humans it will be understood by the skilled person that such compositions are generally suitable for administration to animals of all types. The modification of pharmaceutical compositions suitable for administration to humans in order to make the compositions suitable for administration to various animals is well understood, and the skilled veterinary pharmacologist can design and / or perform such modification with common experimentation.
[00287] [00287] Active cells (eg, RPE cells), implantable elements, and their compositions, can be formulated in unit dosage form, eg, single unit dosage form, for ease of administration and uniformity dosage. It will be understood, however, that the total dosage and regimens of use of the compositions of the present invention will be decided by the attending physician within the scope of reliable medical judgment. The specific therapeutically effective dose level for any particular subject or organism will depend on a variety of factors including the disease being treated and the severity of the dysfunction; the activity of the specific active ingredient employed; the specific composition employed; the subject's age, body weight, general health, sex and diet; the time of administration, route of administration and rate of excretion of the specific active ingredient employed; the duration of treatment; drugs used in combination or
[00288] [00288] The exact amount of a composition described here that is required to achieve an effective amount will vary from subject to subject, depending, for example, on a subject's species, age and general condition, severity of side effects or dysfunction, identity of the particular compound (s), method of administration and the like. The desired dosage can be distributed three times a day, twice a day, once a day, every two days, every third day, every week, every two weeks, every three weeks, every four weeks, every three months, every six months, once a year or less frequently. In certain embodiments, the desired dosage can be delivered using multiple administrations (eg, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations). In certain embodiments, the desired dosage of hydrogel capsules encapsulating engineered RPE cells is delivered after removal of all or substantially all of a previous administration of hydrogel capsules.
[00289] [00289] It will be appreciated that the composition, as described herein, can be administered in combination with one or more additional pharmaceutical agents. The compounds or compositions can be administered in combination with additional pharmaceutical agents that improve their bioavailability, reduce and / or modify their metabolism, inhibit their excretion and / or modify their distribution within the body. It will also be appreciated that the therapy employed can achieve a desired effect for the same dysfunction and / or can achieve different effects.
[00290] [00290] The composition can be administered concomitantly with, before or subsequently to one or more additional pharmaceutical agents, which may be useful as, for example, combination therapies. Pharmaceutical agents include therapeutically active agents. The agents
[00291] Exemplary additional pharmaceutical agents include, but are not limited to, antiproliferative agents, anti-cancer agents, anti-diabetic agents, anti-inflammatory agents, immunosuppressive agents and a pain relieving agent. Pharmaceutical agents include small organic molecules such as drug compounds (eg, compounds approved by the US Food and Drug Administration as provided in the Code of Federal Regulations (CFR)), peptides, proteins, carbohydrates, monosaccharides, oligosaccharides, polysaccharides , nucleoproteins, mucoproteins, lipoproteins, polypeptides or synthetic proteins, small molecules bound to proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs, nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides, lipids, hormones, vitamins and cells.
[00292] [00292] Cases (eg pharmaceutical packaging) are also included in the disclosure. Inventive cases can be useful for
[00293] [00293] 1. An implantable element comprising a plurality of manipulated active cells (eg, engineered RPE cells) that produces or releases a therapeutic agent (eg, a nucleic acid (eg, a nucleotide, DNA or RNA), a polypeptide, a lipid, a sugar (e.g., a monosaccharide, disaccharide, oligosaccharide or polysaccharide) or a small molecule), in which: a) the plurality of active cells manipulated (e.g. , manipulated RPE cells) or the implantable element produces or releases the therapeutic agent for at least 5 days, at least 10 days, at least one month or at least 3 months, eg when implanted in a subject or when evaluated by a reference method described herein, e.g., polymerase chain reaction or in situ hybridization to nucleic acids; mass spectroscopy for lipids, sugar and small molecules; microscopy and other imaging techniques for agents modified with a fluorescent or luminescent marker and ELISA or Western blot for polypeptides; b) the plurality of active cells manipulated (eg, cells
[00294] [00294] 2. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell in plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which the plurality of manipulated active cells (eg, engineered RPE cells) produces or releases the polypeptide for at least 5 days, eg, when implanted in a subject or when evaluated by a reference method, eg ELISA or Western blot.
[00295] [00295] 3. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell in plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which the plurality of manipulated active cells (e.g., manipulated RPE cells) produces or releases at least 10 picograms of the polypeptide per day, e.g., produces at least 10 picograms of the polypeptide per day for at least 5 days, eg, when implanted in a subject or when evaluated by a reference method, eg, ELISA or Western blot.
[00296] [00296] 4. An implantable element comprising a plurality of active manipulated cells (eg, manipulated RPE cells),
[00297] [00297] 5. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell in plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which the plurality of manipulated active cells (e.g., manipulated RPE cells) produces or releases the polypeptide for at least 5 days and the amount released per day does not vary by more than 50% (e.g. at least about 40%, about 30%, about 20%, about 10%, about 5% or less), e.g., as assessed by ELISA or Western blot.
[00298] [00298] 6. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell of the plurality an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which, after introducing the element into a subject, sufficient polypeptide is produced or released such that a location at least about 5 cm, about 10 cm, about 25 cm, about 50 cm, about 75 cm, about 100 cm, or about 150 cm away from the element receives an effective concentration (e.g., a therapeutically effective concentration) of the polypeptide (e.g., a therapeutically effective concentration
[00299] [00299] 7. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell of the plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which sufficient polypeptide is produced or released such that, when the element is embedded or implanted in a subject's peritoneal cavity, e.g., a detectable level of the polypeptide, e.g., 10 picograms, it is found in a location at least 5 cm, 10 cm, 25 cm, 50 cm, 75 cm, 100 or 150 cm away from the element.
[00300] [00300] 8. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell of the plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which, after introducing the element into a subject, sufficient polypeptide is produced or released such that about 50% of the polypeptide produced or released (about 60%, about 70%, about 80%, about 90% or about 99% of the therapeutic polypeptide produced or released) enters a subject's circulation (e.g., peripheral circulation).
[00301] [00301] 9. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell in plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which the manipulated active cells (eg, manipulated EPR cell) are capable of phagocytosis, eg, capable of about 99%, about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 60% or about 50% of the phagocytosis level compared to reference unmanipulated active cells (eg, non-RPE cells manipulated), eg as assessed by antibody assay
[00302] [00302] 10. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell in plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which the plurality of manipulated active cells (eg, engineered RPE cells) is capable of autophagy, eg, is capable of about 99%, about 95%, about 90 %, about 85%, about 80%, about 75%, about 70%, about 60%, or about 50% of the level of autophagy compared to reference unmanipulated active cells (eg, unhandled RPE cells), eg as assessed by 5-ethinyl-2'deoxyuridine test, 5-bromo-2'-deoxyuridine (BrdU) assay, cationic amphiphilic tracer (CAT) assay or microscopy (p e.g., fluorescence microscopy (eg, time lapse or spindle formation assessment), LC3 and p62 immunoblot analysis, detection of self formation phagosomes by fluorescence microscopy and monitoring of autophagosome maturation by mRFP-GFP fluorescence microscopy in tandem.
[00303] [00303] 11. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell in plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which the plurality of manipulated active cells (e.g., manipulated RPE cells) is provided having a form factor described here, e.g., as a cluster, spheroid or aggregate of manipulated active cells (eg, manipulated RPE cells).
[00304] [00304] 12. An implantable element comprising a plurality
[00305] [00305] 13. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell in plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which the plurality of manipulated active cells (e.g., engineered RPE cells) is arranged on a non-cellular carrier (e.g., a microtransporter, e.g., a spherule, e.g. a polyester, polystyrene or polymeric bead).
[00306] [00306] 14. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell in plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which the plurality of manipulated active cells (e.g., engineered RPE cells) proliferates or is able to proliferate after encapsulation in the implantable element, e.g., as determined by microscopy.
[00307] [00307] 15. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cell), each cell in plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which the plurality of manipulated active cells (eg, engineered RPE cells) does not proliferate or is not able to proliferate after encapsulation in the implantable element, eg, as determined by
[00308] [00308] 16. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell of the plurality comprising an exogenous nucleic acid that promotes and / or conditions the production of a polypeptide, e.g. ., a therapeutic polypeptide, in which, after introduction, administration or implantation in a subject, sufficient polypeptide is produced or released such that an effective concentration (e.g., a therapeutically effective concentration) of the polypeptide is found in the peripheral bloodstream ( eg a therapeutically effective concentration found in the pancreas, liver, blood or outside the eye).
[00309] [00309] 17. An implantable element comprising a plurality of manipulated active cells (eg, engineered RPE cells) that produces or releases a therapeutic agent (eg, a nucleic acid (eg, a nucleotide, DNA or RNA), a polypeptide, a lipid, a sugar (eg, a monosaccharide, disaccharide, oligosaccharide or polysaccharide) or a small molecule).
[00310] [00310] 18. Any one of modalities 2 to 17, in which the exogenous nucleic acid is an RNA molecule (eg, an mRNA) or a DNA molecule.
[00311] [00311] 19. Any of modalities 1 to 18, in which the polypeptide or therapeutic agent is selected from the group consisting of Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor IX, Factor polypeptides X, Factor XI and Factor XIII.
[00312] [00312] 20. The implantable element of any one of modalities 1 to 19, wherein the polypeptide or therapeutic agent is an insulin polypeptide (e.g., insulin A chain, insulin B chain or proinsulin).
[00313] [00313] 21. The implantable element of any one of modalities 1 to 18, wherein the polypeptide or therapeutic agent is a polypeptide of
[00314] [00314] 22. An implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), each cell in plurality comprising an exogenous nucleic acid encoding a Factor VIII-BDD amino acid sequence (FVIII-BDD) .
[00315] [00315] 23. The implantable element of modality 22, in which the amino acid sequence of FVIII-BDD is selected from the group consisting of: a) SEQ ID NO: 1; b) SEQ ID NO: 3; c) SEQ ID NO: 4; d) SEQ ID NO: 5; e) SEQ ID NO: 6; f) SEQ ID NO: 7; g) SEQ ID NO: 7 with an alanine instead of arginine at position 787 and an alanine instead of arginine at position 790; h) a conservatively substituted variant of the sequence in (a), (b), (c), (d), (f) or (g); and i) a sequence that has 95%, 96%, 97%, 98%, 99% or more of sequence identity with the sequence in (a), (b), (c), (d), (f), (g) or (h);
[00316] [00316] 25. The implantable element of modality 25, wherein the exogenous nucleic acid comprises a coding sequence selected from the group consisting of SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 27.
[00317] [00317] 26. The implantable element of any of the modalities 22 to 25, wherein the exogenous nucleic acid comprises SEQ ID NO: 16 or SEQ ID NO: 27.
[00318] [00318] 27. An implantable element comprising a plurality of active engineered cells (e.g., engineered RPE cells), each cell in the plurality comprising an exogenous nucleic acid encoding a Factor IX (FIX) amino acid sequence.
[00319] [00319] 28. The implantable element of modality 24, wherein the amino acid sequence of FIX is SEQ ID NO: 2, or a conservatively substituted variant thereof, or a sequence that is at least 95%, 96%, 97% , 98%, 99% or more of sequence identity with SEQ ID NO: 2 or the conservatively substituted variant.
[00320] [00320] 28a. The implantable element of modality 24, in which the FIX amino acid sequence is SEQ ID NO: 36, or a conservatively substituted variant thereof, or a sequence that is at least 95%, 96%, 97%, 98%, 99 % or more of sequence identity with SEQ ID NO: 36 or its conservatively substituted variant.
[00321] [00321] 29. The implantable element of either mode 27 or 28, wherein the exogenous nucleic acid comprises a coding sequence that is a) selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 28; or
[00322] [00322] 30. The implantable element of any of the modalities 27 to 29, wherein the exogenous nucleic acid comprises SEQ ID NO: 19 or SEQ ID NO: 28.
[00323] [00323] 31. Manipulated active cell, e.g., an RPE cell, or an implantable element comprising the active cell, wherein the active cell comprises an exogenous nucleic acid comprising a promoter sequence operably linked to a polypeptide coding sequence , wherein the promoter sequence essentially consists of or consists of SEQ ID NO: 23 or has at least 95%, 96%, 97%, 98%, 99% or more of sequence identity with SEQ ID NO: 23.
[00324] [00324] 32. The manipulated active cell or implantable element of modality 30, in which the polypeptide comprises, essentially consists of or consists of an amino acid sequence which is: a) an FVIII-BDD amino acid sequence, e.g. , a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 7 with an alanine instead of arginine in each of positions 787 and 790; b) a FIX amino acid sequence, eg SEQ ID NO: 2 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of sequence identity with SEQ ID NO: 2; c) an amino acid sequence of Interleukin 2 eg SEQ ID NO: 29 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of sequence identity with SEQ ID NO: 29; d) a parathyroid hormone amino acid sequence, eg, SEQ ID NO: 30 or an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more of sequence identity with SEQ ID NO: 30; or
[00325] [00325] 33. The manipulated active cell or implantable element of any of embodiments 31 or 32, wherein the polypeptide comprises SEQ ID NO: 10 and the coding sequence comprises SEQ ID NO: 16 or a sequence having at least 99% sequence identity with SEQ ID NO: 16.
[00326] [00326] 34. The manipulated active cell or implantable element of any of embodiments 30 to 32, wherein the polypeptide comprises, consists essentially of or consists of SEQ ID NO: 2 and the coding sequence comprises, consists essentially of or consists of SEQ ID NO: 19 or a sequence having at least 99% sequence identity with SEQ ID NO: 19.
[00327] [00327] 35. The cell activates implantable element of any of the modalities 30 to 34, wherein the polypeptide additionally comprises SEQ ID NO: 34 or SEQ ID NO: 35.
[00328] [00328] 36. The cell activates an implantable element of any of the modalities 30 to 35, wherein the exogenous nucleic acid comprises a Kozak sequence immediately upstream of the coding sequence.
[00329] [00329] 37. The active cell implantable element of modality 36, in which the Kozak sequence is nucleotides 2094-2099 of SEQ ID NO: 26.
[00330] [00330] 38. The cell activates implantable element of any of the modalities 30 to 37, in which the promoter sequence is SEQ ID NO: 23.
[00331] [00331] 39. A engineered RPE cell (e.g., a engineered ARPE-19 cell) or an implantable element comprising the engineered RPE cell, wherein the engineered RPE cell comprises an exogenous nucleic acid, wherein the exogenous nucleic acid comprises a sequence
[00332] [00332] 40. The manipulated RPE cell or implantable element of modality 39, in which the exogenous nucleic acid comprises SEQ ID NO: 23 operationally linked to the selected coding sequence.
[00333] [00333] 41. The manipulated RPE cell or implantable element of modality 40, in which the exogenous nucleic acid comprises a Kozak sequence immediately upstream of the coding sequence.
[00334] [00334] 42. The manipulated RPE cell or implantable element of any of the modalities 39 to 41, wherein the exogenous nucleic acid comprises SEQ ID NO: 27 or SEQ ID NO: 28.
[00335] [00335] 43. The implantable element or manipulated cell of any of the preceding modalities, which is provided as a treatment for a disease.
[00336] [00336] 44. The implantable element or manipulated cell of modality 43, in which the disease is a blood clotting disease or a disease of lysosomal storage (e.g., a hemophilia (e.g., Hemophilia A or Hemophilia) B), Fabry disease, Gaucher disease, Pompe disease or MPS I).
[00337] [00337] 45. The implantable element or manipulated cell of any of the preceding modalities, which is provided as a prophylactic treatment.
[00338] [00338] 46. The implantable element of any of the preceding modalities, which is formulated for injection in a subject (eg, intraperitoneal, intramuscular or subcutaneous injection) or is formulated for implantation in a subject (eg, in the peritoneal cavity, eg in the smaller sac).
[00339] [00339] 47. The implantable element or manipulated cell of any of the preceding modalities, which is implanted or injected into the smaller sac, omentum or subcutaneous fat of a subject.
[00340] [00340] 48. The implantable element or manipulated cell of any of the preceding modalities, which is administered to a first subject having less than about 50%, 40%, 30%, 25%, 20%, 15%, 10 %, 5%, 2% or 1% of the polypeptide (eg, a blood clotting factor, eg, Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X , Factor XI or Factor XIII) in relation to a second subject (eg, a healthy subject), eg, as determined by a blood test.
[00341] [00341] 49. The implantable element or manipulated cell of any of the preceding modalities, in which the level of a biomarker (eg, a serum biomarker) in a subject is monitored, eg, in order to determine the level of treatment effectiveness.
[00342] [00342] 50. The implantable element of any of the preceding modalities, which comprises a cluster of manipulated active cells (e.g., a cluster of manipulated RPE cells) or a microcarrier (e.g., a spherule or matrix comprising a manipulated active cell (eg, a manipulated RPE cell) or a plurality of manipulated active cells (eg, manipulated RPE cells)).
[00343] [00343] 51. The implantable element of modality 50, wherein the plurality of manipulated active cells (e.g., manipulated RPE cells) or the microcarrier (e.g., a spherule or matrix comprising a plurality of manipulated active cells (e.g., engineered RPE cells)) produces a plurality of polypeptides.
[00344] [00344] 52. The implantable element of any of the preceding modalities, in which the implantable element comprises an enveloping component.
[00345] [00345] 53. The implantable element of modality 52, in which the
[00346] [00346] 54. The implantable element of claim 52, wherein the surrounding component is preformed prior to combining with the manipulated active cell (e.g., manipulated RPE cell) involved, a plurality of manipulated active cells (e.g. eg, manipulated RPE cells) or a microcarrier (e.g., a spherule or matrix) comprising an active cell or active cells.
[00347] [00347] 55. The implantable element of any of the 52-54 modalities, wherein the surrounding component comprises a flexible polymer (eg, PLA, PLG, PEG, CMC or a polysaccharide, eg alginate) .
[00348] [00348] 56. The implantable element of any of the 52-54 modalities, wherein the surrounding component comprises an inflexible polymer or metal housing.
[00349] [00349] 57. The implantable element of any of the preceding modalities, which is chemically modified.
[00350] [00350] 58. The implantable element of any of the modalities 52-57, in which the surrounding component is chemically modified.
[00351] [00351] 59. The implantable element of any of the preceding modalities, in which the implantable element or its surrounding component is modified with a compound of Formula (I): (I), or a salt thereof, where: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, –O–, –C (O) O–, –C (O) -, –OC (O) -, –N (RC) -, -
[00352] [00352] 60. The implantable element of modality 59, wherein the compound of Formula (I) is a compound of Formula (II): (II), or a pharmaceutically acceptable salt thereof, wherein: ring M1 is cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by 1-5 R3; the Z1 ring is cycloalkyl, heterocyclyl, aryl or heteroaryl, optionally substituted by 1-5 R5; each of R2a, R2b, R2c and R2d is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, oxo, cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, it is N (R10) (R11), O or S;
[00353] [00353] 61. The implantable element of modality 60, wherein the compound of Formula (II) is a compound of Formula (II-a): (II-a), or a pharmaceutically acceptable salt thereof, wherein: the ring M2 is aryl or heteroaryl; the Z2 ring is cycloalkyl, heterocyclyl, aryl or heteroaryl; each of R2a, R2b, R2c and R2d is independently
[00354] [00354] 62. The implantable element of modality 60, wherein the compound of Formula (II-a) is a compound of Formula (II-b): (II-b), or a pharmaceutically acceptable salt thereof, wherein: the Z2 ring is cycloalkyl, heterocyclyl, aryl or heteroaryl; each R3 and R5 is independently alkyl, heteroalkyl, halogen, oxo, –ORA1, –C (O) ORA1 or –C (O) RB1; each RA1 and RB1 is independently hydrogen, alkyl or heteroalkyl; each of p and q is independently 0, 1, 2, 3, 4, 5 or 6; and “” refers to a connection to an attachment group or a polymer described here.
[00355] [00355] 63. The implantable element of modality 60, wherein the compound of Formula (II-a) is a compound of Formula (II-c):
[00356] [00356] 64. The implantable element of modality 60, wherein the compound of Formula (II-a) is a compound of Formula (II-d): (II-d), or a pharmaceutically acceptable salt thereof, in which: the Z2 ring is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, it is O or S; each of R2a, R2b, R2c and R2d is independently hydrogen, alkyl or heteroalkyl, or each of R2a and R2b or R2c and R2d is taken together to form an oxo group; each R5 is independently alkyl, heteroalkyl, halogen, oxo, –ORA1, –C (O) ORA1 or –C (O) RB1; each RA1 and RB1 is independently hydrogen, alkyl or
[00357] [00357] 65. The implantable element of modality 59, wherein the compound of Formula (I) is a compound of Formula (III-a): 3 R2b (R) q R2a N N
[00358] [00358] 66. The implantable element of modality 59, wherein the compound of Formula (I) is a compound of Formula (IV-a):
[00359] [00359] 67. The implantable element of any one of embodiments 59 to 66, wherein the compound of Formula (I) is a compound shown in Table of Compounds 1.
[00360] [00360] 68. The implantable element of any of the modalities 59 to 67, in which the compound is selected from:, N N O N S O The N O O
[00361] [00361] 69. The implantable element of any of the modalities 59 to 67, wherein the compound is selected from Compound 110, Compound 112, Compound 113 or Compound 114 from the Compound Table 1.
[00362] [00362] 70. The implantable element of any of the preceding modalities, in which the implantable element is not substantially degraded after implantation in a subject for at least 30 days, 2 months, 3 months, 6 months, 9 months or 12 months.
[00363] [00363] 71. The implantable element of any of the preceding modalities, in which the implantable element is removable from the subject without significant damage to the surrounding tissue, eg, after about 5 days after implantation.
[00364] [00364] 72. A method of treating a subject or providing a product (e.g., a therapeutic product) to a subject, comprising:
[00365] [00365] administering or providing the subject with an implantable element or manipulated active cell of any one of modalities 1 to 69, thereby treating the subject or providing a product (e.g., a therapeutic product) to the subject.
[00366] [00366] 73. Method 72, comprising treatment of the subject.
[00367] [00367] 74. The method of modality 73, comprising supplying a product (e.g., a therapeutic product) to the subject.
[00368] [00368] 75. The method of any of the modalities 72 to 74, in which the subject is a human.
[00369] [00369] 76. The method of any of the modalities 72 to 75, in which the manipulated active cells (eg, manipulated RPE cells) are
[00370] [00370] 77. The method of any of the modalities 72 to 76, wherein the polypeptide is an antibody (e.g., nerve growth antifactor antibody); an enzyme (eg, alpha-galactosidase) or a clotting factor (eg, a blood clotting factor, eg, an activated blood clotting factor).
[00371] [00371] 78. The method of any of the modalities 72 to 77, wherein the plurality of manipulated active cells (e.g., manipulated RPE cells) or the implantable element is provided as a treatment for a disease.
[00372] [00372] 79. Method 78, wherein the disease is a blood clotting disease or a lysosomal storage disease (eg, a hemophilia (eg, Hemophilia A or Hemophilia B), Disease Fabry disease, Gaucher disease, Pompe disease or MPS I).
[00373] [00373] 80. Method 78, in which the disease is diabetes.
[00374] [00374] 81. Method 78, in which the disease is not diabetes.
[00375] [00375] 82. The method of any of the modalities 72 to 77, in which the implantable element is administered to a first subject having less than about 50%, 40%, 30%, 25%, 20%, 15% , 10%, 5%, 2% or 1% of the polypeptide (e.g., a blood clotting factor, e.g., Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI or Factor XIII) in relation to a second subject (eg, a healthy subject), eg, as determined by a blood test.
[00376] [00376] 83. The method of any of the modalities 72 to 82, in which the level of a biomarker (eg, a serum biomarker) in a subject is monitored, eg, in order to determine the level of treatment effectiveness.
[00377] [00377] 84. The method of any of the modalities 72 to 83, in
[00378] [00378] 85. The method of any of the modalities 72 to 83, in which the implantable element is administered to, implanted in or provided to a location at least about 1, 2, 5 or 10 centimeters away from the nervous system central, brain, spine, eye or retina.
[00379] [00379] 86. A method of preparing or manufacturing an implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), comprising: providing a plurality of manipulated active cells (e.g., some engineered RPE cells), eg, engineered active cells described here, and arrangement of the plurality of engineered active cells (eg, engineered RPE cells) in an envelope component, e.g., an envelope component described here , preparing or manufacturing the implantable element.
[00380] [00380] 87. A method of evaluating an implantable element comprising a plurality of manipulated active cells (e.g., manipulated RPE cells), comprising: providing an implantable element comprising a plurality of manipulated active cells (e.g., a manipulated RPE cells) described here; and evaluating a structural or functional parameter of the implantable element or the plurality of manipulated active cells (e.g., the manipulated RPE cells), thereby evaluating an implantable element.
[00381] [00381] 88. The method of modality 87, comprising culturing the plurality of manipulated active cells (e.g., manipulated RPE cells) in
[00382] [00382] 89. The method of modality 87 or 88, comprising evaluation of the plurality of active manipulated cells (eg, manipulated RPE cells), regarding one or more of: viability; the production of a engineered polypeptide; the production of a manipulated RNA; the capture of a nutrient or oxygen; or the production of a waste product.
[00383] [00383] 90. The method of any of the modalities 87 to 89, further comprising: formulation of the implantable element in a drug product if one or more of: viability; production of a engineered polypeptide; production of a manipulated RNA; the capture of a nutrient or oxygen; or the production of a residual product fulfills a predetermined value.
[00384] [00384] 91. The method of any of the modalities 87 to 90, comprising evaluation of a cell parameter related to a form factor, eg, a form factor described here.
[00385] [00385] 92. The method of any of the modalities 87 to 91, in which the evaluation is performed at least 1, 5, 10, 20, 30 or 60 days after disposal of the plurality of manipulated active cells (eg, manipulated RPE cells) in the implantable element.
[00386] [00386] 93. The method of any of the modalities 72-79, in which the evaluation is carried out at least 1, 5, 10, 20, 30 or 60 days after the beginning of the culture of the manipulated active cells (e.g. , manipulated RPE cells).
[00387] [00387] 94. A method of monitoring an implantable element of any of modalities 1 to 70, comprising:
[00388] [00388] 95. The method of modality 94, in which the component is measured in the peripheral circulation, eg in the peripheral blood.
[00389] [00389] 96. The method of any of the modalities 91 to 95, in which the level of the component (eg, polypeptide) is compared with a reference value.
[00390] [00390] 97. The method of any of the modalities 91 to 96, in which, responsive to the level or to the comparison, the subject is classified, eg, as needing or without the need for an additional implantable element or active cells manipulated (eg manipulated RPE cells).
[00391] [00391] 98. The method of any of the modalities 91 to 97, the method comprises (eg, responsive to the level or comparison) recovery of the implantable element or active manipulated cells (eg, manipulated RPE cells) a from the subject.
[00392] [00392] 99. The method of any of the modalities 91 to 98, the level is obtained from about 1 hour to about 30 days after administration (eg, implantation or injection) of an implantable element or manipulated active cells (eg, manipulated RPE cells) or about 1 hour to about 30 days after a previous evaluation.
[00393] [00393] 100. A plurality of active cells (eg, RPE cells) having a pre-selected form factor or form factor disclosed here.
[00394] [00394] 101. The plurality of active cells (eg, RPE cells) of the
[00395] [00395] 102. The plurality of active cells (eg, RPE cells) of the 101 modality, in which the cluster comprises at least about 100, 200, 300, 400 or 500 of active cells (eg, RPE cells).
[00396] [00396] 103. A substrate comprising a plurality of chambers, each chamber of the plurality containing either an active cell (e.g., RPE cell) or an active manipulated cell (e.g., a manipulated RPE cell).
[00397] [00397] 104. The substrate of mode 103, wherein each chamber of the plurality of chambers comprises a plurality of active cells (e.g., RPE cells) or active manipulated cells (e.g., manipulated RPE cells), p , a plurality of manipulated RPE cells having a form factor described here, e.g., a cluster.
[00398] [00398] 105. A microcarrier (e.g., a spherule or matrix), having disposed of a manipulated active cell described here (e.g., an RPE cell, e.g., a manipulated RPE cell) or a grouping of active cells (eg, RPE cells, eg, manipulated RPE cells).
[00399] [00399] 106. The microtransporter of modality 105, wherein the microtransporter comprises a polystyrene bead.
[00400] [00400] 107. A preparation of engineered active cells (e.g., engineered RPE cells), wherein the preparation comprises at least about 10,000; 15,000; 20,000; 25,000; 30,000; 40,000; 50,000; 60,000; or
[00401] [00401] 108. A pharmaceutical composition comprising a plurality of the implantable element or manipulated active cell of any one of modalities 1 to 70. EXAMPLES
[00402] [00402] So that the disclosure described here can be more
[00403] [00403] ARPE-19 cells can be cultured according to any method known in the art, such as according to the following protocol. ARPE-19 cells (from ATCC) in a 75 cm2 culture flask are aspirated to remove the culture medium, and the cell layer is briefly rinsed with 0.05% (w / v) trypsin / EDTA solution at 0.53 mM (“TripsinaEDTA”) to remove all traces of serum that contain a trypsin inhibitor. 2-3 mL of Trypsin / EDTA solution is added to the flask, and the cells were observed under an inverted microscope until the cell layer is dispersed, usually between 5-15 minutes. To avoid clumping, cells are handled with care and flicking or shaking the flask during the dispersion period is discouraged. If the cells do not detach, the flasks are placed at 37 ºC to facilitate dispersion. Once the cells have dispersed, 6-8 ml of complete growth medium is added and the cells are aspirated by gentle pipetting. The cell suspension is transferred to a centrifugal tube and centrifuged at approximately 125 x g for 5-10 to remove Trypsin EDTA. The supernatant is discarded, and the cells are resuspended in fresh growth medium. Appropriate aliquots of cell suspension were added to new culture vessels, which were incubated at 37 oC. The medium was renewed 2-3 times weekly. Example 2A: Preparation of clusters of active cells
[00404] [00404] Spheroid clusters of active cells (eg, RPE cells) were prepared using AggreWell ™ spheroid plants (STEMCELL
[00405] [00405] Alternatively, the ARPE-19 spheroid clusters can be prepared using the following protocol. On Day 1, AggreWell plates were removed from the packaging in a sterile tissue culture chamber. Add 2 mL of Aggrewell Rinse Solution to each well. Centrifuge the plate at 2,000 g for 5 minutes to remove air bubbles. Remove AggreWell Rinse Solution from wells and rinse each well with 2 mL of complete growth medium. Add 2 million ARPE-19 cells in 3.9 μl of the complete growth medium to each well. Centrifuge the plate at 100 g for 3 minutes. Incubate the cells at 37 ° C for 48 hours. On Day 3, the same protocol
[00406] [00406] Unique ARPE-19 cells can be inoculated into commercially available microcarriers (eg, Cultispher® microcarriers, Cytodex® microcarriers, Corning Enhanced Attachment Microcarriers) according to the following protocol.
[00407] [00407] The desired number of ARPE-19 cells (e.g., 20 million cells) and culture medium are added to the microcarriers (optionally coated with collagen) in a conical tube to achieve the desired total volume (e.g. 10 mL). The microcarriers are optionally coated with collagen by combining the desired amount of sterile microcarriers with 0.1 mg / mL rat tail collagen I in phosphate buffered saline (PBS) in a conical tube and then shaking the tube at 200 rpm at RT for at least 2 hours. Collagen-coated microcarriers are washed with PBS three times and then with culture medium twice, allowing the microcarriers to rest for about 5 minutes after each wash before removing the supernatant.
[00408] [00408] The conical tube containing the cells and microcarriers is gently shaken until homogeneous and then placed in a 37 C stationary incubator for about 25 minutes, and these shaking and incubation steps are repeated once. The cells and microcarriers of the conical tube are added to a spinner flask containing the desired amount (eg, 70 mL) of culture medium that is preheated to 37 C, and additional culture medium is added to carry the volume in the flask to the final desired volume (eg, 90 mL). The cells and microcarrier are then incubated at 37 ° C with shaking for about 4 days. A desired volume of the microcarrier / medium composition is transferred to a microcentrifuge tube and the microcarriers washed once in a
[00409] [00409] The procedures below describe methods of preparing exemplary compounds for preparing chemically modified implantable elements. The compounds provided herein can be prepared from readily available starting materials using modifications to the specific synthesis protocols set out below that would be well known to those skilled in the art. It will be appreciated that, where typical or preferred process conditions are given (ie reaction temperatures, times, molar ratios of reagents, solvents, pressures, etc.), other process conditions can also be used unless otherwise affirmed. Optimal reaction conditions may vary with the particular reagents or solvents used, but such conditions can be determined by those skilled in the art by routine optimization procedures.
[00410] [00410] Additionally, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. The choice of a suitable protecting group for a particular functional group 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 references cited there. Huisgen cycleload to give 1,4-substituted triazoles
[00411] [00411] The copper-catalyzed Huisgen [3 + 2] cicloaddition was used to prepare compounds and compositions based on triazole, devices and their materials. The scope and typical protocols were the subject of many
[00412] [00412] In the example shown above, azide is the reactive fraction in the fragment containing connective element A, while alkaline is the reactive component of the pendant group Z. As illustrated below, these functional handles can be exchanged to produce a product of structurally related triazole. The preparation of these alternatives is similar and does not require special considerations.
[00413] [00413] A typical Huisgen cicloaddition procedure starting with an iodide is outlined below. In some cases, iodides are turned into azides during the course of the safety reaction. O I N N O
[00414] [00414] A solution of sodium azide (1.1 eq), sodium ascorbate (0.1 eq), trans-N, N'-dimethylcyclohexane-1,2-diamine (0.25 eq), iodide copper (I) in methanol (1.0 M, limiting reagent) was degassed with bubbling nitrogen and treated with acetylene (1 eq) and aryl iodide (1.2 eq). This mixture was stirred at room temperature for 5 minutes, then heated to 55 ° C for 16 h. The reaction was then cooled to room temperature, filtered through a funnel, and the filter cake washed with methanol. The combined filtrates were concentrated and purified by flash chromatography on silica gel (120 g silica, 0 to 40% gradient (3% aqueous ammonium hydroxide, 22% methanol, remaining dichloromethane) in dichloromethane) to give the material desired target.
[00415] [00415] A typical Huisgen cicloaddition procedure starting with an azide is outlined below.
[00416] [00416] A solution of tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] amine (0.2 eq), triethylamine (0.5 eq), copper (I ) (0.06 eq) in methanol (0.4 M, limiting reagent) was treated with acetylene (1.0 eq) and cooled to 0 ° C. The reaction was allowed to warm to room temperature over 30 minutes, then it was heated to 55 ° C for 16 h. The reaction was cooled to room temperature, concentrated and purified with HPLC (column C18, 0 to 100% gradient (3% aqueous ammonium hydroxide, 22% methanol, remaining dichloromethane) in dichloromethane) to original the desired target material . Huisgen cycleload to provide 1.5-substituted triazoles
[00417] [00417] Huisgen cycloaddition [3 + 2] was also performed with ruthenium catalysts to obtain preferentially 1,5-disubstituted products (eg, as described in Zhang et al., J. Am. Chem. Soc ., 2005, 127, 15998-15999; Boren et al., J. Am. Chem. Soc., 2008, 130, 8923- 8930, each of which is incorporated herein by reference in its entirety).
[00418] [00418] As previously described, the azide and alkaline groups can be exchanged to form similar triazoles as illustrated below.
[00419] [00419] A typical procedure is described as follows: a solution of the alkaline (1 eq) and azide (1 eq) in dioxane (0.8 M) was added dropwise to a solution of pentamethylcyclohydrochloride
[00420] [00420] A mixture of (4-iodophenyl) methanamine (1,843 mg, 3.62 mmol, 1.0 eq), (1S, 2S) -N1, N2-dimethylcyclohexane-1,2-diamine (74 μL, 0.47 mmol, 0.13 eq), Sodium Ascorbate (72 mg, 0.36 mmol, 0.1 eq), Copper Iodide (69 mg, 0.36 mmol, 0.1 eq), Azide sodium (470 mg, 7.24 mmol, 2.0 eq) and 1-methyl-4- (prop-2-in-1-yl) piperazine (2, 0.5 g, 3.62 mmol, 1, 0 eq) in Methanol (9 ml) and water (1 ml) was purged with nitrogen for 5 minutes and heated to 55 ° C overnight. The reaction mixture was cooled to room temperature, concentrated under reduced pressure, and the brownish paste was extracted with dichloromethane. Celite was added to the combined dichloromethane phases 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) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 7.5% to give (4- (4 - ((4-methylpiperazin-1-yl) methyl) -1H-1,2,3-triazol-1-yl) phenyl) methanamine (3, 0.45 g, 43%). LCMS m / z: [M + H] + Calculated for C15H22N6 287.2; Found 287.1. Experimental Procedure for N- (4- (4 - ((4-methylpiperazin-1-yl) methyl) -1H- 1,2,3-triazol-1-yl) benzyl) methacrylamide (4)
[00421] [00421] A solution of (4- (4 - ((4-methylpiperazin-1-yl) methyl) -1H-1,2,3-triazol-1-yl) phenyl) methanamine (3, 1.2 g, 4.19 mmol, 1.0 eq) and triethylamine (0.70 mL, 5.03 mmol, 1.2 eq) in CH2Cl2 (50 mL) was cooled to 0 ° C with an ice bath and methacryloyl chloride ( 0.43 ml, 4.40 mmol, 1.05 eq in 5 ml CH2Cl2) was added. The reaction was stirred for one day until cooling with an ice bath. Ten (10) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (80 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 7.5%. The solvent was removed under reduced pressure and the resulting solid was triturated with diethyl ether, filtered and washed multiple times with diethyl ether to give N- (4- (4 - ((4-methylpiperazin-1-yl) methyl) - 1H-1,2,3-triazol-1-yl) benzyl) methacrylamide (4, 0.41 g, 28% yield) as a white solid. LCMS m / z: [M + H] + Calculated for C19H26N6O 355.2; Found 355.2. Experimental Procedure for (4- (4 - ((2- (2-methoxyethoxy) ethoxy) methyl) -1H- 1,2,3-triazol-1-yl) phenyl) methanamine (6)
[00422] [00422] A mixture of (4-iodophenyl) methanamine (1, 2.95 g, 12.64 mmol, 1.0 eq), (1S, 2S) -N1, N2-dimethylcyclohexane-1,2-diamine (259 μL, 1.64 mmol, 0.13 eq), Sodium Ascorbate (250 mg, 1.26 mmol, 0.1 eq), Copper Iodide (241 mg, 1.26 mmol, 0.1 eq) , Sodium azide (1.64 g, 25.29 mmol, 2.0
[00423] [00423] Experimental Procedure for N- (4- (4 - ((2- (2-methoxyethoxy) ethoxy) methyl) -1H-1,2,3-triazol-1-yl) benzyl) methacrylamide (7)
[00424] [00424] A solution of 4- (4 - ((2- (2-methoxyethoxy) ethoxy) methyl) -1H- 1,2,3-triazol-1-yl) phenyl) methanamine (6, 1.69 g, 5.52 mmol, 1.0 eq) and triethylamine (0.92 mL, 6.62 mmol, 1.2 eq) in CH2Cl2 (50 mL) was cooled to 0 ° C with an ice bath and methacryloyl chloride ( 0.57 mL, 5.79 mmol, 1.05 eq) was added dropwise. The reaction was stirred for 4 h at room temperature. Ten (10) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (80 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 1.25% to give 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 solid
[00425] [00425] A suspension of sodium hydride (27.0 g, 675 mmol, 60% purity) in THF (200 mL) was cooled with an ice bath. Oxetan-3-ol (8.25 g, 337 mmol) was added dropwise and stirred for 30 minutes at 0 ° C. 3-Bromoprop-1-yne (9, 41.2 ml, 371 mmol, 80% purity) was then added dropwise. The mixture was stirred overnight and allowed to warm to room temperature. The mixture was filtered over Celite, washed with THF and concentrated with Celite under reduced pressure. The crude product was purified on silica gel (220 g) and eluted with Hexanes / EtOAc. The concentration of EtOAc in the mobile phase was increased from 0 to 25% to give a yellow oil of (9, 18.25 g, 48%). Experimental Procedure for 3- (4 - ((oxetan-3-yloxy) methyl) -1H-1,2,3-triazol-1-yl) propan-1-amine (11)
[00426] [00426] A mixture of 3- (prop-2-in-1-yloxy) oxetane (9, 7.96 g, 71 mmol, 1.0 eq), 3-azidopropan-1-amine (10, 7.82 g, 78 mmol, 1.1 eq), Tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] -amine (8.29 g, 15.6 mmol, 0.22 eq), Copper Iodide (1.35 g, 7.1 mmol, 0.1 eq) and Triethylamine (2.47 mL, 17.8 mmol, 0.25 eq) 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 concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using
[00427] [00427] A solution of 3- (4 - ((oxetan-3-yloxy) methyl) -1H-1,2,3-triazol-1-yl) propan-1-amine (11, 3.94 g, 18 , 56 mmol, 1.0 eq) and triethylamine (3.1 mL, 22.28 mmol, 1.2 eq) in CH2Cl2 (100 mL) was cooled to 0 ° C with an ice bath and methacryloyl chloride (1 , 99 mL, 20.42 mmol, 1.1 eq) was added dropwise. The reaction was stirred overnight and allowed to warm to room temperature. 20 grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (220 g) using dichloromethane / methanol as the mobile phase. The methanol concentration was gradually increased from 0% to 5% to give N- (3- (4 - ((oxetan-3-yloxy) methyl) -1H-1,2,3-triazol-1-yl) propyl) methacrylamide (12, 3.22 g, 62% yield) as a solid. LCMS m / z: [M + H] + Calculated for C13H20N4O3 281.2; Found 281.0. Experimental Procedure for N- (4- (1H-1,2,3-triazol-1-yl) benzyl) methacrylamide (14)
[00428] [00428] To a solution of (4- (1H-1,2,3-triazol-1-yl) phenyl) methanamine
[00429] [00429] A mixture of (4-iodophenyl) methanamine hydrochloride (5.0 g, 18.55 mmol, 1.0 eq), (1S, 2S) -N1, N2-dimethylcyclohexane-1,2-diamine (0.59 mL, 3.71 mmol, 0.2 eq), Sodium Ascorbate (368 mg, 1.86 mmol, 0.1 eq), Copper Iodide (530 mg, 2.78 mmol, 0.15 eq), Sodium azide (2.41 g, 37.1 mmol, 2.0 eq), Et3N (3.11 mL, 22.26 mmol, 1.2 eq) and 2- (prop-2-in- 1-yloxy) tetrahydro-2H-pyran (2.6 g, 18.55 mmol, 1.0 eq) in Methanol (50 ml) and water (12 ml) was purged with Nitrogen for 5 minutes and heated to 55 ° C overnight. The reaction mixture was cooled to room temperature and filtered through filter paper 413. Celite was added and the solvent was removed under reduced pressure and the residue was purified on silica gel (120 g) using dichloromethane / (methanol containing ammonium hydroxide 12% aqueous (w / w)) as a mobile phase. The concentration of (methanol
[00430] [00430] A solution of (4- (4 - (((tetrahydro-2H-pyran-2-yl) oxy) methyl) - 1H-1,2,3-triazol-1-yl) phenyl) methanamine ( 15, 3.46 g, 12.00 mmol, 1.0 eq) and triethylamine (2.01 mL, 14.40 mmol, 1.2 eq) in CH2Cl2 (40 mL) was cooled to 0 ° C with a bath ice and methacryloyl chloride (1.23 ml, 12.60 mmol, 1.05 eq, diluted in 5 ml CH2Cl2) was added dropwise. The cooling bath was removed and the reaction was stirred for 4 h. 20 grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (80 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 3.75% to give N- (4- (4 - (((tetrahydro-2H-pyran -2-yl) oxy) methyl) -1H-1,2,3-triazol-1-yl) benzyl) methacrylamide (16, 2.74 g, 64% yield) as a white solid. LCMS m / z: [M + H] + Calculated for C19H24N4O3 357.2; Found 357.3. Experimental Procedure for N- (4- (4- (hydroxymethyl) -1H-1,2,3-triazol-1-yl) benzyl) methacrylamide (17)
[00431] [00431] A solution of N- (4- (4- (hydroxymethyl) -1H-1,2,3-triazol-1-yl) benzyl) methacrylamide (16, 1.2 g, 3.37 mmol, 1, 0 eq) was dissolved in Methanol (6 ml) and HCl (1 N, aq., 9 ml) overnight at room temperature. Celite was added and the solvent was removed under reduced pressure. The crude product was purified on silica gel chromatography (24 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 12.5% to give N- (4- (4- (hydroxymethyl) -1H-1,2, 3-triazol-1-yl) benzyl) methacrylamide (17, 0.85 g, 92% yield) as a white solid. LCMS m / z: [M + H] + Calculated for C14H16N4O2 273.1; Found 273.1. Experimental Procedure for (4 - (((tetrahydro-2H-pyran-2-yl) oxy) methyl) benzyl) carbamate (19)
[00432] Benzyl [00432] (4- (Hydroxymethyl) benzyl) carbamate (2.71 g, 10 mmol, 1 eq), 3,4-dihydro-2H-pyran (1.81 mL, 20 mmol, 2 eq) and p-toluenesulfonic acid monohydrate (285 mg, 1.5 mmol, 0.15 eq) in dichloromethane (100 ml) were stirred at room temperature overnight. Celite was added and the solvent was removed under reduced pressure. The crude product was purified on silica gel (24 g) using Hexanes / EtOAc as eluent starting with 100% Hexanes and gradually increasing the concentration of EtOAc to 100% to provide (4 - ((((tetrahydro-2H-pyran -2-yl) oxy) methyl) benzyl) -benzyl carbamate (19, 2.4 g, 68%) as a colorless oil. LCMS m / z: [M + Na] + Calculated for C21H25NO4 378.17 Found 378.17.
[00433] [00433] (4 - (((Tetrahydro-2H-pyran-2-yl) oxy) methyl) benzyl) carbamate (19, 1.5 g, 4.2 mmol, 1 eq), Palladium on carbon (160 mg, 10% by weight) in EtOH were briefly evacuated and then Hydrogen was added via a flask and the mixture was stirred for 1 hour at room temperature. 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) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 25% to give (4 - ((((tetrahydro-2H-pyran-2-yl) oxide) methyl) phenyl) methanamine (20, 890 mg, 95%) as a colorless oil. LCMS m / z: [M + H] + Calculated for C13H19NO2 222.15 Found 222.14. Experimental Procedure for N- (4 - (((tetrahydro-2H-pyran-2-yl) oxy) methyl) benzyl) -methacrylamide (21)
[00434] [00434] A solution of (4 - (((tetrahydro-2H-pyran-2-yl) oxy) methyl) phenyl) methanamine (20, 0.5 g, 2.26 mmol, 1.0 eq) and triethylamine (0.47 mL, 3.39 mmol, 1.5 eq) in CH2Cl2 (10 mL) was briefly evacuated and purged with Nitrogen. Methacryloyl chloride (0.33 mL, 3.39 mmol, 1.5 eq) was added dropwise. The reaction mixture was stirred overnight at room temperature. Ten (10) grams of Celite were
[00435] [00435] A mixture of (4-iodophenyl) methanamine (5.0 g, 21.45 mmol, 1.0 eq), (1S, 2S) -N1, N2-dimethylcyclohexane-1,2-diamine (0 , 44 mL, 2.79 mmol, 0.13 eq), Sodium Ascorbate (425 mg, 2.15 mmol, 0.1 eq), Copper Iodide (409 mg, 2.15 mmol, 0.1 eq) , Sodium azide (2.79 g, 42.91 mmol, 2.0 eq) and 2- (but-3-in-1-yloxy) tetrahydro-2H-pyran (3.36 mL, 21.45 mmol, 1.0 eq) in Methanol (20 ml) and water (5 ml) was purged with Nitrogen for 5 minutes and heated to 55 ° C overnight. The reaction mixture was cooled to room temperature and filtered through filter paper 413. Celite (10 g) was added and the solvent was removed under reduced pressure and the residue was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% aqueous ammonium hydroxide (w / w)) as a mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 5% to give (4- (4- (2 - (((tetrahydro-2H-piran- 2-yl) oxy) ethyl) -1H-1,2,3-triazol-1-yl) phenyl) methanamine (22, 3.15 g, 49%) as a solid LCMS m / z: [M + H ] + Calculated for C16H22N4O2 303.18; Found 303.18.
[00436] [00436] A solution of (4- (4- (2 - ((tetrahydro-2H-pyran-2-yl) oxy) ethyl) - 1H-1,2,3-triazol-1-yl) phenyl) methanamine (22, 3.10 g, 10.25 mmol, 1.0 eq) and triethylamine (1.71 mL, 12.30 mmol, 1.2 eq) in CH2Cl2 (55 mL) was cooled to 0 ° C with an ice bath and methacryloyl chloride (1.05 ml, 12.30 mmol, 1.2 eq, diluted in 5 ml CH2Cl2) was added dropwise. The cooling bath was removed and the reaction was stirred for 4 h. 8 grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (80 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 2.5% to give N- (4- (4- (2 - ((tetrahydro-2H -pyran-2-yl) oxy) ethyl) -1H-1,2,3-triazol-1-yl) benzyl) methacrylamide (23, 2.06 g, 54% yield) as a white solid. LCMS m / z: [M + H] + Calculated for C20H26N4O3 371.2078; Found 371.2085. Experimental Procedure (4- (1- (2 - ((tetrahydro-2H-pyran-2-yl) oxy) ethyl) -1H- 1,2,3-triazol-4-yl) phenyl) methanamine (24)
[00437] [00437] A mixture of (4-ethynylphenyl) methanamine (2.36 g, 18.00 mmol, 1.0 eq), (1S, 2S) -N1, N2-dimethylcyclohexane-1,2-diamine (0 , 56 mL,
[00438] [00438] A solution of (4- (1- (2 - ((tetrahydro-2H-pyran-2-yl) oxy) ethyl) - 1H-1,2,3-triazol-4-yl) phenyl) methanamine (24, 1.5 g, 4.96 mmol, 1.0 eq) and triethylamine (1.04 mL, 7.44 mmol, 1.5 eq) in CH2Cl2 (30 mL) was briefly evacuated and purged with nitrogen . Methacryloyl chloride (0.72 mL, 7.44 mmol, 1.5 eq) was added dropwise. The reaction mixture was stirred for 2 h at room temperature. Ten (10) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (40 g) using Hexanes / EtOAc
[00439] [00439] A hydrochloride mixture of 1- (4-iodophenyl) ethan-1-amine (1.0 g, 4.05 mmol, 1.0 eq), (1S, 2S) -N1, N2-dimethylcyclohexane -1,2-diamine (0.08 mL, 0.53 mmol, 0.13 eq), Sodium Ascorbate (80 mg, 0.40 mmol, 0.1 eq), Copper Iodide (77 mg, 0, 40 mmol, 0.1 eq), sodium azide (526 g, 8.09 mmol, 2.0 eq) and 2- (prop-2-in-1-yloxy) tetrahydro-2H-pyran (0, 57 g, 4.05 mmol, 1.0 eq) in Methanol (9 mL) and water (1 mL) was purged with Nitrogen for 5 minutes and heated to 55 ° C overnight. The reaction mixture was cooled to room temperature and the solvent was removed under reduced pressure. The residue was redissolved in dichloromethane and filtered over a plug of Celite. 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) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 5% to give 1- (4- (4 - (((tetrahydro-2H-pyran-2 -yl) oxy) methyl) -1H-1,2,3-triazol-1-yl) phenyl) ethan-1-amine (26, 0.62 g, 51%) as a yellowish solid. LCMS m / z: [M + H] + Calculated for C16H22N4O2 303.2; Found 303.2.
[00440] [00440] Experimental Procedure for N- (1- (4- (4 - (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-1,2,3-triazol-1-yl ) phenyl) ethyl) methacrylamide (27)
[00441] [00441] A solution of 1- (4- (4 - ((((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-1,2,3-triazol-1-yl) phenyl) ethan-1-amine (26, 0.52 g, 1.7 mmol, 1.0 eq) and triethylamine (0.29 mL, 2.1 mmol, 1.2 eq) in CH2Cl2 (11 mL) was cooled to 0 ° C with an ice bath and methacryloyl chloride (0.18 ml, 1.8 mmol, 1.05 eq, diluted in 11 ml of CH2Cl2) was added dropwise. The cooling bath was removed and the reaction was stirred for 4 h. Five (5) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (40 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 2.5% to give N- (1- (4- (4 - (((tetrahydro- 2H-pyran-2-yl) oxy) methyl) -1H-1,2,3-triazol-1-yl) phenyl) ethyl) methacrylamide (27, 0.49 g, 76% yield) as a white solid. LCMS m / z: [M + H] + Calculated for C20H26N4O3 371.2078; Found 371.2087. Experimental Procedure for (4- (4 - (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-1,2,3-triazol-1-yl) -2- (trifluoromethyl) phenyl ) methanamine (28)
[00442] [00442] A mixture of (4-iodo-2- (trifluoromethyl) phenyl) methanamine (3.0 g, 9.97 mmol, 1.0 eq), (1S, 2S) -N1, N2-dimethylcyclohexane- 1,2-diamine
[00443] [00443] A solution of (4- (4 - ((((tetrahydro-2H-pyran-2-yl) oxy) methyl) - 1H-1,2,3-triazol-1-yl) -2- ( trifluoromethyl) phenyl) methanamine (28, 1.0 g, 2.81 mmol, 1.0 eq) and triethylamine (0.59 mL, 4.21 mmol, 1.5 eq) in CH2Cl2 (25 mL) was briefly evacuated and purged with nitrogen. Methacryloyl chloride (0.41 ml, 4.21 mmol, 1.5 eq) was added dropwise. The reaction mixture was stirred for 6 h at room temperature. Ten (10) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (40 g) using Hexanes / EtOAc as eluent starting with 100% Hexanes and
[00444] [00444] A mixture of 3-azidopropan-1-amine hydrochloride (1.5 g, 14.98 mmol, 1.0 eq), Tris [(1-benzyl-1H-1,2,3-triazole-4 -yl) methyl] -amine (1.99 g, 3.75 mmol, 0.25 eq), Copper Iodide (0.29 g, 1.50 mmol, 0.1 eq) and Triethylamine (0.52 mL , 3.75 mmol, 0.25 eq) 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) tetra -hydro-2H-pyran (2.10 g, 14.98 mmol, 1.0 eq) was added and the reaction mixture was heated to 55 ° C and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, filtered over a plug of Celite and rinsed with methanol (3 x 50 ml). Celite (20 g) was added to the filtrate, 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) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 20% to give 3- (4 - (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-1,2,3-triazol-1-yl) propan-1-amine (30, 2.36 g, 66%). LCMS m / z: [M + H] + Calculated for C11H20N4O2 241.2; Found 241.2. Experimental Procedure for N- (3- (4 - (((tetrahydro-2H-pyran-2-yl) oxy) methyl) -1H-1,2,3-triazol-1-yl) propyl) methacrylamide (31 )
[00445] [00445] A solution of 3- (4 - (((tetrahydro-2H-pyran-2-yl) oxy) methyl) - 1H-1,2,3-triazol-1-yl) propan-1-amine (30, 1.0 g, 4.16 mmol, 1.0 eq) and triethylamine (0.58 mL, 4.16 mmol, 1.0 eq) in CH2Cl2 (20 mL) was briefly evacuated and purged with nitrogen. Methacryloyl chloride (0.40 ml, 4.16 mmol, 1.0 eq) was added dropwise. The reaction mixture was stirred at room temperature overnight. Ten (10) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (40 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 20% to give N- (3- (4 - (((tetrahydro-2H-pyran-2 -yl) oxy) methyl) -1H-1,2,3-triazol-1-yl) propyl) methacrylamide (31, 0.96 g, 75% yield) as a colorless oil. LCMS m / z: [M + H] + Calculated for C15H24N4O3 309.2; Found 309.4. Experimental Procedure for (4- (4 - ((oxetan-3-yloxy) methyl) -1H-1,2,3-triazol-1-yl) phenyl) methanamine (32)
[00446] [00446] A mixture of (4-iodophenyl) methanamine hydrochloride (2.64 g, 9.80 mmol, 1.0 eq), (1S, 2S) -N1, N2-dimethylcyclohexane-1,2-diamine (0.31 mL, 1.96 mmol, 0.2 eq), Sodium ascorbate (198 mg, 0.98 mmol, 0.1 eq),
[00447] [00447] A solution of (4- (4 - ((oxetan-3-yloxy) methyl) -1H-1,2,3-triazol-1-yl) phenyl) methanamine (32, 0.58 g, 2, 23 mmol, 1.0 eq) and triethylamine (0.47 mL, 3.34 mmol, 1.5 eq) in CH2Cl2 (20 mL) was briefly evacuated and purged with Nitrogen. Methacryloyl chloride (0.32 mL, 3.34 mmol, 1.5 eq) was added dropwise. The reaction mixture was stirred for 6 h at room temperature. Ten (10) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by silica gel chromatography (24 g) using Hexanes / EtOAc as eluent starting with 100% Hexanes and gradually increasing the EtOAc concentration to 100% to provide N- (4- (4 - ((oxetan-3 -yloxy) methyl) -1H-
[00448] [00448] A solution of ethyl 1- (2-aminoethyl) -1H-imidazole-4-carboxylate (34, 2.0 g, 10.91 mmol, 1.0 eq) and triethylamine (3.80 mL, 27 , 29 mmol, 2.5 eq) in CH2Cl2 (20 mL) was briefly evacuated and purged with nitrogen. Methacryloyl chloride (1.60 ml, 16.37 mmol, 1.5 eq) was added dropwise. The reaction mixture was stirred for 3 h at room temperature. Fifteen (15) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (40 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 25% to give ethyl 1- (2-methacrylamidoethyl) -1H-imidazole-4-carboxylate (35, 1.28 g, 47% yield) as a colorless solid. LCMS m / z: [M + H] + Calculated for C12H17N3O3 252.1; Found 252.1. Experimental Procedure for N- (4- (1,1- dioxidothiomorpholine) benzyl) methacrylamide (37)
[00449] [00449] To a solution of 4- (4- (aminomethyl) phenyl) thiomorpholine 1,1-dioxide hydrochloride (36, 1.15 g, 4.15 mmol, 1.0 eq) and triethylamine
[00450] [00450] To a mixture of 1-methylsulfonylethylene (4.99 g, 47.03 mmol, 4.13 mL) and Amberlyst-15 ((30% w / w)), N-methylprop-2-in-1- amine (2.6 g, 37.62 mmol) was 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 give: N-methyl-N- (2- (methylsulfonyl) ethyl) prop-2-in-1-amine (38, 6.43 g, 98% ) as an oil. LCMS m / z: [M + H] + Calculated for C7H13NSO2 176.11; Found 176.1. Experimental Procedure for N - ((1- (2- (2- (2- (2-aminoethoxy) ethoxy) ethoxy) ethyl) -1H-1,2,3-triazol-4-yl) methyl) -N-methyl -2- (methylsulfonyl) ethan-1-amine (40)
[00451] [00451] A mixture of N-methyl-N- (2- (methylsulfonyl) ethyl) prop-2-in-1-amine (38, 5.02 g, 28.64 mmol, 1.25 eq), Tris [ (1-benzyl-1H-1,2,3-triazol-4-yl) methyl] -amine (3.04 g, 5.73 mmol, 0.25 eq), Copper Iodide (436 mg, 2.29 mmol, 0.1 eq) and Triethylamine (0.8 mL, 5.7 mmol, 0.25 eq) in Methanol (50 mL) and water (6 mL) was evacuated and purged with Nitrogen (3 times) and cooled with an ice bath. 2- (2- (2- (2-Azidoethoxy) ethoxy) ethoxy) ethan-1-amine (39, 5.02 g, 22.91 mmol, 1.0 eq) was added dropwise, the cooling bath was removed and the mixture was stirred for 5 minutes. The reaction was heated to 55 ºC and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, Celite (20 g) was added and concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 25% to give N - ((1- (2- (2- (2- (2-aminoethoxy)) ethoxy) ethoxy) ethyl) -1H-1,2,3-triazol-4-yl) methyl) -N-methyl-2- (methylsulfonylethane-1-amine (40, 4.98 g, 55%) as an oil LCMS m / z: [M + H] + Calculated for C15H31N5O5S 394.2; Found 394.2. Experimental Procedure N- (2- (2- (2- (2- (4 - ((methyl (2- ( methylsulfonyl) ethyl) amino) methyl) -1H-1,2,3-triazol-1-yl) ethoxy) ethoxy) ethoxy) ethyl) methacrylamide (41)
[00452] [00452] To a solution of N - ((1- (2- (2- (2- (2- (2-aminoethoxy) ethoxy) ethoxy) ethyl) -1H-1,2,3-triazol-4-yl) methyl) -N-methyl-2- (methylsulfonyl) ethan-1-amine (40, 1.0 g, 2.54 mmol, 1.0 eq) and triethylamine (0.43 mL, 3.05 mmol, 1.2 eq ) in CH2Cl2 (15 mL) a solution of methacryloyl chloride (0.30 mL, 3.05 mmol, 1.5 eq) was added dropwise. The reaction mixture was stirred for 5 h at room temperature. Celite was
[00453] [00453] 3-Bromoprop-1-yne (4.4 mL, 39.32 mmol, 1.0 eq) was added to a mixture of 2-oxa-7-aspasospiro [3.5] nonane (8.54 g, 39 , 32 mmol, 1.0 eq), potassium carbonate (17.9 g, 129.7 mmol, 3.3 eq) 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 chromatography on silica gel (220 g) using dichloromethane / methanol as the mobile phase. The methanol concentration was gradually increased from 0% to 5% to give 7- (prop-2-in-1-yl) -2-oxa-7-azospiro [3.5] nonane (42, 4.44 g, 68% ) as an oil. Experimental Procedure for 2- (2- (2- (2- (4 - ((2-oxa-7- azaespiro [3.5] nonan-7-yl) methyl) -1H-1,2,3-triazole-1- il) ethoxy) ethoxy) ethoxy) ethan-1-amine (43)
[00454] [00454] A mixture of 7- (prop-2-in-1-yl) -2-oxa-7-azospiro [3.5] nonane (42, 2.5 g, 15.13 mmol, 1.0 eq), Tris [(1-benzyl-1H-1,2,3-triazol-4-yl) methyl] -amine (1.77 g, 3.33 mmol, 0.22 eq), Copper Iodide (288 g, 1 , 51 mmol, 0.1 eq) and Triethylamine (0.53 ml, 3.8 mmol, 0.25 eq) in Methanol (50 ml) was cooled with an ice bath. 2- (2- (2- (2- Azidoethoxy) ethoxy) ethoxy) ethan-1-amine (39, 3.86 g, 17.70 mmol, 1.17 eq) was added dropwise, the cooling bath was removed and the mixture was stirred for 5 minutes. The reaction was heated to 55 ºC and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, Celite (10 g) was added and concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 10% to give 2- (2- (2- (2- (4 - ((2-oxa -7- azaespiro [3.5] nonan-7-yl) methyl) -1H-1,2,3-triazol-1-yl) ethoxy) ethoxy) ethoxy) ethan-1-amine (43, 4.76 g, 82 %) as an oil. LCMS m / z: [M + H] + Calculated for C18H33N5O4 384.3; Found 384.2. Experimental Procedure for N- (2- (2- (2- (2- (4 - ((2-oxa-7- azospiro [3.5] nonan-7-yl) methyl) -1H-1,2,3-triazole -1- yl) ethoxy) ethoxy) ethoxy) ethyl) methacrylamide (44)
[00455] [00455] A solution of 2- (2- (2- (2- (4 - ((2-oxa-7-azospiro [3.5] nonan- 7-yl) methyl) -1H-1,2,3-triazole -1-yl) ethoxy) ethoxy) ethoxy) ethan-1-amine (43, 2.65 g, 6.91 mmol, 1.0 eq) and triethylamine (1.16 mL, 8.29 mmol, 1.2 eq) in CH2Cl2
[00456] [00456] A mixture of 4- (prop-2-in-1-yl) thiomorpholine 1,1-dioxide (1.14 g, 6.58 mmol, 1.0 eq), Tris [(1-benzyl- 1H-1,2,3-triazol-4-yl) methyl] -amine (768 mg, 1.45 mmol, 0.22 eq), Copper Iodide (125 g, 0.66 mmol, 0.1 eq) and Triethylamine (0.23 ml, 1.65 mmol, 0.25 eq) in Methanol (20 ml) was cooled with an ice bath. 2- (2-Azidoethoxy) ethan-1-amine (1.00 g, 7.70 mmol, 1.17 eq) was added dropwise, the cooling bath was removed and the mixture was stirred for 5 minutes. The reaction was heated to 55 ºC and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, Celite (10 g) was added and concentrated under reduced pressure. The crude product was purified on silica gel (40 g) using dichloromethane / (methanol containing hydroxide
[00457] [00457] A 1,1 - dioxide solution of 4 - ((1- (2- (2-aminoethoxy) ethyl) - 1H-1,2,3-triazol-4-yl) methyl) thiomorpholine (45, 1 , 32 g, 4.35 mmol, 1.0 eq) and triethylamine (0.73 mL, 5.22 mmol, 1.2 eq) in CH2Cl2 (100 mL) was cooled with an ice bath under a nitrogen atmosphere. Methacryloyl chloride (0.47 mL, 4.8 mmol, 1.1 eq) was added dropwise. The cooling bath was removed and the reaction mixture was stirred for 4 h at room temperature. Ten (10) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (120 g) using dichloromethane / (methanol containing 12% aqueous ammonium hydroxide (v / v)) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 1.25% to give N- (2- (2- (4 - ((1,1- dioxidothiomorpholine) ) methyl) -1H-1,2,3-triazol-1-yl) ethoxy) ethyl) -methacrylamide (46, 0.90 g, 56% yield) as a colorless oil. LCMS m / z: [M + H] + Calculated for C15H25N5O4S 372.17; Found 372.15. Experimental Procedure for 4 - ((1- (2- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -1H-1,2,3-triazol-4-yl) methyl) thiomorpholine (47) )
[00458] [00458] A mixture of 4- (prop-2-in-1-yl) thiomorpholine 1,1-dioxide (4.6 g, 26.55 mmol, 1.0 eq), Tris [(1-benzyl- 1H-1,2,3-triazol-4-yl) methyl] -amine (3.1 g, 5.84 mmol, 0.22 eq), Copper Iodide (506 g, 2.66 mmol, 0.1 eq) and Triethylamine (0.93 ml, 6.64 mmol, 0.25 eq) in Methanol (80 ml) was cooled with an ice bath. 2- (2- (2-Azidoethoxy) ethoxy) ethan-1-amine (5.00 g, 28.68 mmol, 1.08 eq) was added dropwise, the cooling bath was removed and the mixture was stirred for 5 minutes. The reaction was heated to 55 ºC and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, Celite was added and concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was gradually increased from 0% to 10% to give 4- ((1- (2- (2- (1,1 ( 2-aminoethoxy) ethoxy) ethyl) -1H-1,2,3-triazol-4-yl) methyl) thiomorpholine (47, 5.26 g, 57%) as a yellowish oil. LCMS m / z: [M + H] + Calculated for C13H25N5O4S 348.1700; Found 348.1700. Experimental Procedure N- (2- (2- (2- (4 - (((1,1-dioxidothiomorpholine) methyl) - 1H-1,2,3-triazol-1-yl) ethoxy) ethoxy) ethyl) methacrylamide (48 )
[00459] [00459] A solution of 1,1 - dioxide of 4 - ((1- (2- (2- (2-aminoethoxy) ethoxy) ethyl) -1H-1,2,3-triazol-4-yl) methyl) thiomorpholine (47, 1.49 g, 4.29 mmol, 1.0 eq) and triethylamine (0.72 mL, 5.15 mmol, 1.2 eq) in CH2Cl2
[00460] [00460] A mixture of 4- (prop-2-in-1-yl) thiomorpholine 1,1-dioxide (5.0 g, 28.86 mmol, 1.0 eq), Tris [(1-benzyl- 1H-1,2,3-triazol-4-yl) methyl] -amine (3.37 g, 6.35 mmol, 0.22 eq), Copper Iodide (550 g, 2.89 mmol, 0.1 eq) and Triethylamine (1.01 ml, 7.22 mmol, 0.25 eq) in Methanol (90 ml) was cooled with an ice bath. 14-Azido-3,6,9,12-tetraoxatetradecan-1-amine (8.86 g, 33.77 mmol, 1.17 eq) was added dropwise, the cooling bath was removed and the mixture was stirred for 5 minutes. The reaction was heated to 55 ºC and stirred overnight under a nitrogen atmosphere. The reaction mixture was cooled to room temperature, Celite (15 g) was added and concentrated under reduced pressure. The crude product was purified on silica gel (220 g) using dichloromethane / (methanol containing 12% (v / v) aqueous ammonium hydroxide) as the mobile phase. The concentration of (methanol containing 12% (v / v) aqueous ammonium hydroxide) was
[00461] [00461] A 4 - ((1- (14-amino-3,6,9,12-tetraoxatetradecyl) -1H-1,2,3-triazol-4-yl) methyl 1,1-dioxide solution) thiomorpholine (49, 1.95 g, 4.79 mmol, 1.0 eq) and triethylamine (0.80 mL, 5.74 mmol, 1.2 eq) in CH2Cl2 (50 mL) was cooled with an ice bath under nitrogen atmosphere. Methacryloyl chloride (0.51 ml, 5.26 mmol, 1.1 eq) was added dropwise. The cooling bath was removed and the reaction mixture was stirred for 4 h at room temperature. Ten (10) grams of Celite were added and the solvent was removed under reduced pressure. The residue was purified by chromatography on silica gel (80 g) using dichloromethane / methanol as the mobile phase. The methanol concentration was gradually increased from 0% to 5% to give N- (14- (4 - ((1,1-dioxidothiomorfolino) methyl) -1H-1,2,3-triazol-1-yl) -3 , 6,9,12- tetraoxatetradecyl) methacrylamide (50, 0.76 g, 32% yield) as a colorless oil. LCMS m / z: [M + H] + Calculated for C21H37N5O7S 504.25; Found 504.20. Example 4: Chemical modification of alginate for cell encapsulation
[00462] [00462] A polymeric material can be chemically modified with compounds of Formula (I) (or its pharmaceutically acceptable salt) prior to the encapsulation of active cells (eg, RPE cells) as described below
[00463] [00463] A polymeric material can be chemically modified with a compound of Formula (I) (or its pharmaceutically acceptable salt) prior to the formation of a device described here (eg, a hydrogel capsule described here) using methods known in the art. technical.
[00464] [00464] For example, in the case of alginate, alginate carboxylic acid is activated for coupling to one or more compounds functionalized with amine to achieve an alginate modified with an afibrotic compound, e.g., a compound of Formula (I ). The alginate polymer is dissolved in water (30 ml / gram of polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 eq) and N-methylmorpholine (1 eq) . To this mixture is added a solution of the compound of interest (e.g., Compound 101 shown in Table 2) in acetonitrile (0.3 M).
[00465] [00465] The amounts of the compound and coupling reagent added depend on the desired concentration of the compound bound to the alginate, e.g., conjugation density. To prepare a solution of CM-LMW-Alg-101-Medium polymer, the dissolved unmodified low molecular weight alginate (approximate MW <75 kDa, ratio G: M ≥ 1.5) is treated with 2-chloro-4 , 6-dimethoxy-1,3,5-triazine (5.1 mmol / g alginate) and N-methylmorpholine (10.2 mmol / g alginate) and Compound 101 (5.4 mmol / g alginate). To prepare a solution of CM-LMW-Alg-101-Very polymer, the dissolved unmodified low molecular weight alginate (approximate MW <75 kDa, ratio G: M ≥ 1.5) is treated with 2-chloro-4 , 6-dimethoxy-1,3,5-triazine (5.1 mmol / g alginate) and N-methylmorpholine (10.2 mmol / g alginate) and Compound 101 (5.4 mmol / g alginate).
[00466] [00466] The reaction is heated to 55 ° C for 16 h, then cooled
[00467] [00467] The conjugation density of a chemically modified alginate is measured by combustion analysis for the percentage of nitrogen. The sample is prepared by dialysis a solution of the chemically modified alginate against water (10,000 MWCO membrane) for 24 hours, replacing the water twice followed by lyophilization to a constant weight.
[00468] [00468] For use in the generation of the hydrogel capsules described in the Examples below, chemically modified alginate polymers were prepared with Compound 101 (shown in Table 1) conjugated to a low molecular weight alginate (approximate MW <75 kDa, ratio G: M ≥ 1.5) at medium (2% to 5% N) or high (5.1% to 8% N) densities, as determined by combustion analysis for the percentage of nitrogen, and are referred to here as CM -LMW-Alg-101-Medium and CM-LMW-Alg-101- High. Unless otherwise specified, the chemically modified alginate in the capsules made in the Examples below is CM-LMW-Alg-101-Medium. Example 5: Formation of Implantable Elements Encapsulated In Situ
[00469] [00469] Clusters of active cells (eg, RPE cells) were encapsulated in alginate to form implantable elements encapsulated in situ configured as hydrogel capsules according to the protocol
[00470] [00470] Before manufacturing the implantable elements encapsulated in situ, the plugs were sterilized by autoclaving, and the alginate solutions were sterilized by filtration through a 0.2 μm filter using aseptic processes. An electrostatic droplet generator was defined as follows: a high voltage power generator 0-100 kV, 20 watt ES series (Gamma ES series, Gamma High-Voltage Research, FL, USA) was connected to the top and bottom of a blunt-tipped needle (SAI Infusion Technologies, IL, USA). This needle was attached to a 5 mL Luer-lock syringe (BD, NJ, USA), which was attached to a vertically oriented syringe pump (Pump 11 Pico Plus, Harvard Apparatus, MA, USA). The syringe pump pumps alginate into a glass plate containing a 20 mM barium crosslinking solution (25 mM HEPES buffer, 20 mM BaCl2 and 0.2 M mannitol). In some experiments, the crosslinking solution also contained 0.01% poloxamer 188. The PicoPlus syringe pump settings were 12.06 mm in diameter and flow rate of about 0.16 mL / min to 0.2 mL / min depending on the target size for the
[00471] [00471] Just before encapsulation, single cultured cells (prepared substantially as described in Example 1), clusters of active cells (prepared substantially as described in Example 2A) or cells in microcarriers (prepared substantially as described in Example 2B) were centrifuged at 1,400 rpm for 1 min 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, MgSO4 × 1.2 mM 7H2O, 135 mM NaCl, pH ,4 7.4, ≈ 290 mOsm). After washing, the cells were centrifuged again and all the supernatant was aspirated. The cell pellet was then resuspended in one of the TMTD alginate solutions: SLG100 (described above) at a range of single cell densities, clusters or microcarriers (eg, number of single cells or clusters or volume of microcarriers per mL of alginate solution). The implantable elements encapsulated in situ were cross-linked using the BaCl2 cross-linking solution, and their sizes were controlled as described above. Immediately after cross-linking, the implantable elements encapsulated in situ (hydrogel capsules) were washed with HEPES buffer (NaCl at 15.428 g, KCl at 0.70 g, MgCl2 · 6H2O at 0.488 g, 50 mL of HEPES buffer solution (1 M ) (Gibco, Life Technologies, California, USA) in 2 liters of deionized water) four times and stored at 4 ° C until use. After formation and before use, implantable elements encapsulated in situ were analyzed by optical microscopy to determine the size and evaluate the quality of the
[00472] [00472] To examine the quality of capsules in a capsule composition, an aliquot containing at least 200 capsules was removed from the composition and transferred to a well plate and the entire aliquot examined by optical microscopy for quality by counting the number of spherical capsules in total. Example 6: Factor VIII-BDD secretion from Implantable Elements Encapsulated In Situ
[00473] [00473] ARPE-19 cells were transfected with a vector encoding human Factor VIII-BDD using standard transfection techniques. The vector also contained a zeocin resistance gene. Two days after transfection, the cell line was cultured as single cells at 37 ° C in complete growth medium supplemented with zeocin, and the cultured cells were then encapsulated as single cells in 1.5 mm alginate implantable elements as outlined in the Example 5.
[00474] [00474] In order to determine the amount of Factor VIII-BDD available, the encapsulated cells (Cap) were centrifuged and the supernatant was collected and analyzed by ELISA (Kit VisuLize FVIII Antigen ELISA, Affinity Biologicals, Inc.) for the presence Factor VIII-human BDD at 4 hours, 24 hours, 48 hours and 72 hours after transfection. These results were compared with non-encapsulated active cells (RPE cells, Culture) and are shown in FIG. 1.
[00475] [00475] The implantable elements were further examined by microscopy to assess the viability of cells as shown in FIGS. 2A-2B. As shown, implantable elements comprising active cells expressing Factor VIII-BDD show high viability over the duration of the experiment. Example 7: Evaluation of Implantable Elements Encapsulated in vivo
[00476] [00476] Encapsulated implantable elements comprising cells
[00477] [00477] Preparation: The mice were prepared for surgery by being placed under anesthesia under a continuous flow of 1-4% isofluorane with oxygen at 0.5 L / min. Preoperatively, all mice received a dose of 0.05-0.1 mg / kg body weight of buprenorphine subcutaneously as a pre-surgical anesthetic, together with 0.5 mL of 0.9% saline solution subcutaneously to prevent dehydration. An epilator with a # 40 cutter blade was used to remove hair to reveal an area of about 2 cm x 2 cm on the ventral midline of the animals' abdomen. The entire shaved area was aseptically prepared with a minimum of 3 cycles of rubbing with povidine (in an external centrifugal direction from the center of the incision site when possible) followed by rinsing with 70% alcohol. A final skin paint with povidine was also applied. The surgical site was draped with sterile disposable paper to prevent the surrounding hair from touching the surgical site, after disinfecting the table top surface with 70% ethanol. Staff wore PPE, lab coat and appropriate surgical gloves
[00478] [00478] Surgical procedure: A sharp surgical blade or scissors was used to cut a 0.5-0.75 mm midline incision through the skin and the white line in the abdomen of the mice in question. The surgeon tried to keep the incision as small as possible with 0.75 cm being the largest possible incision size. A sterile plastic pipette was used to transfer the alginate microcapsules (with or without cells) to the peritoneal cavity. The abdominal muscle was closed by suturing with 5-0 Ethicon black silk or 5.0-6.0 absorbable monofilament thread absorbable by PDS, and the outer skin layer was closed using wound clips. These wound clips were removed 7-10d post-surgery after complete healing was confirmed. Blood and tissue debris was removed from the
[00479] [00479] Intraoperative care: The animals were kept warm using the Deltaphase isothermal pillow. The animal's eyes were hydrated with sterile ophthalmic ointment during the surgery period. Care was taken to avoid moistening the surgical site excessively to avoid hypothermia. The respiratory rate and character were monitored continuously. If vital signs are indicative of extreme pain and distress, the animal was euthanized through cervical relocation.
[00480] [00480] At the desired post-operation time point, the animal was euthanized by asphyxiation with CO2 and the alginate capsules were collected by peritoneal lavage.
[00481] [00481] Exemplary mouse strains used in these experiments include AKXL37 / TyJ; strain deficient in Factor IX B6.129P2-F9tm1Dws / J; a strain deficient in Factor VIII described in Bi, L et al. (1995) Nature 10: 119-121); alpha-galactosidase B6; 129-Glatm1Kul / J spot described in Ohshima, T et al. (1997) Proc Nat'l Sci USA 94: 2540-2544); and the factor IX deficient strain described in Lin, H-F et al. (2017) Blood 90: 3962-3966. Example 8: Comparison of the encapsulation architecture of manipulated active cells
[00482] [00482] A study comparing encapsulation in alginate hydrogel capsules of single manipulated active cells (eg, single RPE cells or derivatives of single RPE cells), clusters of manipulated active cells (eg, clusters of cells Manipulated RPE or groupings of derivatives of RPE cells) and manipulated active cells
[00483] [00483] When the above study was performed using ARPE-19 cells engineered to express an FVIII-BDD protein and encapsulated in 1.5 mm hydrogel capsules as described in Example 5, the FVIII-BDD expression levels and the cell viability were substantially the same regardless of whether the cells had been encapsulated as single cells, clusters of cells or cells attached to a microcarrier (data not shown). Example 9: Comparison of the unmanaged active cell encapsulation architecture
[00484] [00484] The effect of cell architecture on cell packing density, cell viability and quality of capsules was examined using hydrogel alginate capsules (1.5 mm) that encapsulated wild type ARPE-19 cells (ie, not manipulated) in one of the following architectures: single cells, spheroid clusters, cells in Cytodex® 1 microcarriers (Sigma-Aldrich, C0646), cells in Cultispher®-S microcarriers (Sigma-Aldrich, M9043).
[00485] [00485] The hydrogel capsules were formed from an alginate solution (mixture of modified alginate and non-alginate)
[00486] [00486] An aliquot of each of the hydrogel capsule compositions was placed in a well plate and the well plate stored in an incubator at 37 ° C for several hours and then the viability of the encapsulated cells was assessed by staining live / dead (Thermo Fisher Scientific # L3224) followed by visualization of the stained cells using fluorescence microscopy at 4x magnification: viable cells are stained green and dead cells are stained red. The quality of the capsules was determined by examining an aliquot of at least 100 capsules and calculating the percentage of spherical capsules in the aliquot. The number of viable cells per capsule was determined by the CellTiter-Glo® 2.0 Assay (Promega, G9242). The results of these evaluations are shown in Figure 5 (single cells), Figure 6 (spheroids), Figure 7 (Cytodex microcarriers) and Figure 8 (Cultispher microcarriers).
[00487] [00487] As shown in FIG. 5A, spherical capsules containing viable cells were formed with all concentrations of single cell suspensions. However, as the concentration of cells
[00488] [00488] When hydrogel capsules were prepared using suspensions of spheroidal clusters, spherical capsules containing viable cells were formed with all cell concentrations, as shown in FIG. 6A. However, as the concentration of encapsulated cells increased, the overall quality of the capsule preparation was reduced from 97% spherical capsules to 30 M / mL to approximately 93% spherical capsules to 100 M / mL (FIG. 6B) . The number of viable cells per capsule increased with increased cell load in the alginate solution; however, the largest number of viable cells was observed at an intermediate cell concentration of 50 M / mL, which also had> 98% spherical capsules. The quality of the capsules did not correlate directly with the number of cells (FIG. 6C).
[00489] [00489] Spherical capsules containing viable cells were also formed from each of the microcarrier concentrations tested as shown in FIG. 7A and FIG. 8A.
[00490] [00490] However, as shown in FIG. 7B, the overall quality of the capsules in the preparation decreased with increasing concentration of Cytodex microcarriers, ie, the overall quality of the capsule batch was reduced from approximately 98% spherical capsules with the lowest concentration suspension (1: 8) to only 70 % of spherical capsules with the suspension with the highest concentration 1: 0.5 (FIG. 7B). While the number of viable cells per capsule increased with increased microcarrier concentration in the alginate suspension, this corresponded to
[00491] [00491] In contrast, for capsule preparations made from Cultispher microcarrier suspensions, the overall quality of the capsules remained relatively constant as the microcarrier concentration increased, ranging from 91-97% with no clear trend with cell concentration (FIG. 8B). The number of viable cells per capsule increased with increased microcarrier load in the alginate solution (FIG. 8C). Example 10: ARPE-19 cells exhibit contact inhibition in vitro.
[00492] [00492] ARPE-19 cells were plated with 96-well plates at
[00493] [00493] Cells that have been sparsely inoculated (1,000 cells / well) or dense (40,000 cells / well) have many EdU positive cells on day 1 after inoculation; however, by day 7, more cells were positive for EdU and there were more proliferating cells in the wells initially inoculated with 1,000 cells compared to those inoculated with 40,000 cells (data not shown). This demonstrates that
[00494] [00494] PiggyBac transposon expression vectors were created that contained one of several test promoters operationally linked to the Factor IX coding sequence. The ARPE-19 and HS27 cell lines were cultured in 5% CO2 and 37 ° C, transfected with 2.5 µg of each transposon Piggybac DNA expression construct + 0.5 µg of cherry- CAG-HyPBase using lipofectamine method. To generate clusters of stable cells, ARPE-19 cells were selected with puromycin. The cells were maintained and expanded for about 3 weeks and, during this period, fresh medium with selection agent was added every three days. To assess the specific productivity of cells from selected clones, 500,000 cells were inoculated in duplicate in a 6-well plate. After 4 hours, the medium was changed and replaced with fresh medium. After 24 hours, the supernatant medium was collected and the density of viable cells was assessed. The specific productivity of cells (pg / cell / day) was determined by graphing the FIX concentration (determined using an hFIX ELISA) against the number of viable cells.
[00495] [00495] As shown in FIG. 9, ARP-19 cells engineered with different promoters produced different levels of FIX expression. Cells transfected with an expression vector comprising the CAG promoter operably linked to a FIX coding sequence performed better than cells transfected with the same vector
[00496] [00496] RPE cells, e.g., ARPE-19 cells, can be manipulated to express an exogenous polypeptide using the PiggyBac transposon system, which involves co-transfecting RPE cells with two plasmids: (1) a transposon vector containing a transcription unit capable of expressing a polypeptide of interest inserted between inverted terminal repeat (ITR) elements recognized by a PiggyBac transposase and (2) a plasmid that expresses a piggyBac transposase enzyme. The PiggyBac system mediates the transfer of genes through a "cut and paste" mechanism by means of which transposase integrates into the transcription unit and ITRs at TTAA chromosomal sites in RPE cells. Alternatively, RPE cells can be engineered to express a polypeptide of interest from an extrachromosomal vector by transfecting the cells with only the transposon vector.
[00497] [00497] An exemplary transposon vector for manipulating RPE cells is shown in FIG. 10 (SEQ ID NO: 26) and has the vector elements described in the vector table below. Before transfection of RPE cells, the transcription unit to be integrated into chromosomal RPE sites is created by inserting the coding sequence of interest
[00498] [00498] Codon-optimized (CO) sequences encoding the recombinant human FVIII-BDD amino acid sequence shown in FIG. 1 (SEQ ID NO: 1) were generated using a commercially available algorithm. A wild type (e.g., non-optimized) sequence (SEQ ID NO: 8) encoding the same FVIII-BDD polypeptide was used as a control (Native). Each CO and Native sequence was inserted into the vector of
[00499] [00499] To evaluate the effect of using a codon-optimized sequence on other FVIII-BDD variant proteins, the rhFVIII-BDD CO6 sequence (SEQ ID NO: 15) has been modified (by nucleotide substitutions or additions, as appropriate) to generate a codon-optimized sequence encoding the rhScFVIII-BDD 2 variant (rhScFVIII-BDD CO, SEQ ID NO: 16) or a single-stranded inclusion BDD protein variant (inclusion of rhScFVIII-BDD CO; SEQ ID NO: 17). The control coding sequences were the wild type coding sequences (eg, not optimized) encoding the original FVIII-BDD polypeptide variant (SEQ ID NO: 1) (Native), four different single-stranded BDD variants (SEQ ID NOs, 3-6) and the inclusion FVIII variant (SEQ ID NO: 7). Each CO variant and control coding sequence was inserted into the transposon expression vector of FIG. 10, with the insertion site being immediately downstream of the Kozak sequence. ARPE-19 cells were cotransfected with a PiggyBac transposase vector and a transposon vector. The production of FVIII protein (pg / cell / day) by the resulting manipulated cells was evaluated by ELISA. FIG. 12 shows the change in production of single-stranded BDD variants and inclusion FVIII-BDD variants compared to the production of rhFVIII-BDD (SEQ ID NO: 1). Example 13: Codon optimization intensifies the expression of FIX by
[00500] [00500] Codon-optimized (CO) sequences (SEQ ID NOs. 19-21) encoding the recombinant human FIX-Padua variant polypeptide (SEQ ID NO: 2) were generated using a commercially available algorithm. A wild type (e.g., non-optimized) sequence (SEQ ID NO: 18) encoding the same FIX-Padua polypeptide was used as a control (Native). Each CO and Native sequence was inserted into the transposon expression vector of FIG. 10, with the insertion site being immediately downstream of the Kozak sequence. ARPE-19 cells were cotransfected with a PiggyBac transposase vector and a transposon vector. The production of FIX protein (pg / cell / day) by the resulting manipulated cells was evaluated by ELISA. FIG. 13 shows the production of FIX-Padua by cells manipulated with a CO sequence in relation to the production of cells manipulated with the wild type (eg, non-optimized) coding sequence (Native). Example 14: Transfection of RPE cells with multiple FIX transcription units increases FIX expression in engineered RPE cells.
[00501] [00501] RPE cells were engineered to express FIX-Padua (SEQ ID NO: 2) by cotransfecting the cells with a PiggyBac transposase vector and a transposon expression vector (FIG. 10) containing a wild type coding sequence (Native ), the transposon expression vector (FIG. 10) with a codon-optimized sequence (SEQ ID NO: 19) or the same transposon expression vector except with a duplication of the codon-optimized transcription unit, ie, the pCAG promoter, Kozak sequence, SEQ ID NO: 19 and the rBG pA sequence. The production of FIX protein (pg / cell / day) by the resulting engineered cells was evaluated by ELISA and the results are shown in FIG. 14. EQUIVALENTS AND SCOPE
[00502] [00502] This application refers to several issued patents, published patent applications, magazine articles and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the references incorporated and the present report described, the specification should prevail. In addition, any particular form of the present disclosure that resides within the prior art as it is explicitly excluded from any one or more of the claims. As such modalities are considered to be known to one skilled in the art, they can be excluded even if the exclusion is not explicitly presented here. Any particular form of disclosure may be excluded from any claim, for any reason, whether or not it relates to the existence of prior technology.
[00503] [00503] Those skilled in the art will recognize or be able to verify using no more than routine experimentation many equivalents for the specific modalities described here. The scope of the present modalities described here is not intended to be limited by the Description, Figures or Examples above, but instead is as presented in the attached claims. Those skilled in the art will appreciate that various changes and modifications to this description can be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

权利要求:
Claims (36)
[1]
1. manipulated active cell, or an implantable element comprising the manipulated active cell, where the manipulated active cell is characterized by the fact that it comprises a manipulated retinal pigment epithelial (RPE) cell or a manipulated cell derived from an RPE cell, and wherein the engineered active cell comprises an exogenous nucleic acid encoding a polypeptide, wherein the exogenous nucleic acid comprises one or more of the following nucleotide sequences: a) a promoter sequence that consists essentially of or consists of (i) SEQ ID NO: 23 or (ii) a sequence having at least 95%, 96%, 97%, 98%, 99% or more of sequence identity with SEQ ID NO: 23; b) a coding sequence encoding a Factor VIII-BDD polypeptide (FVIII-BDD), wherein the FVIII-BDD polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 7 with an alanine instead of arginine in each of positions 787 and 790; c) a coding sequence encoding a Factor IX (FIX) polypeptide, wherein the FIX polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 2; d) a coding sequence encoding an interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 29; e) a coding sequence encoding a parathyroid hormone (PTH) polypeptide, wherein the PTH polypeptide comprises, essentially consists of or consists of SEQ ID NO: 30 or 31; f) a coding sequence encoding a von Willebrand Factor (vWF) polypeptide, wherein the vWF polypeptide comprises,
2/10 consists essentially of or consists of SEQ ID NO: 32 or 33; g) a coding sequence encoding a conservatively substituted variant of an amino acid sequence in (b), (c), (d), (e) or (f); eh) a coding sequence encoding an amino acid sequence that has at least 90%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the amino acid sequence in (b), (c) , (d), (f) or (g).
[2]
2. Manipulated active cell, or implantable element comprising the manipulated active cell, in which the manipulated active cell, characterized by the fact that it comprises an exogenous nucleic acid encoding a polypeptide, in which the exogenous nucleic acid comprises one or more of the following sequences of nucleotides: a) a promoter sequence that essentially consists of or consists of (i) SEQ ID NO: 23 or (ii) a sequence having at least 95%, 96%, 97%, 98%, 99% or more of sequences with SEQ ID NO: 23; b) a coding sequence encoding a Factor VIII-BDD polypeptide (FVIII-BDD), wherein the FVIII-BDD polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 7 with an alanine instead of arginine in each of positions 787 and 790; c) a coding sequence encoding a Factor IX (FIX) polypeptide, wherein the FIX polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 2; d) a coding sequence encoding an interleukin-2 (IL-2) polypeptide, wherein the IL-2 polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 29;
3/10 e) a coding sequence encoding a parathyroid hormone (PTH) polypeptide, wherein the PTH polypeptide comprises, essentially consists of or consists of SEQ ID NO: 30 or 31; f) a coding sequence encoding a von Willebrand Factor (vWF) polypeptide, wherein the vWF polypeptide comprises, essentially consists of or consists of SEQ ID NO: 32 or 33; g) a coding sequence encoding a conservatively substituted variant of an amino acid sequence in (b), (c), (d), (e) or (f); eh) a coding sequence encoding an amino acid sequence that has at least 90%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the amino acid sequence in (b), (c) , (d), (f) or (g).
[3]
3. Manipulated active cell or implantable element comprising the manipulated active cell according to claim 2, characterized by the fact that the manipulated active cell has one or more of the following characteristics: (a) comprises a retinal pigment epithelial cell (RPE) or a cell derived from it; (b) comprises a cell that was obtained from a less differentiated cell; and (c) comprises a cell that has one or more of the following properties: (i) expresses one or more of the CRALBP, RPE-65, RLBP, BEST1 or αB-crystalline biomarkers; (ii) does not express one or more of the CRALBP, RPE-65, RLBP, BEST1 or αB-crystalline biomarkers; (iii) it is naturally found in the retina and forms a monolayer above the choroidal blood vessels in Bruch's membrane; and
4/10 (iv) is responsible for epithelial transport, light absorption, secretion and / or immune modulation in the retina.
[4]
4. Manipulated active cell or implantable element according to any one of claims 1 to 3, characterized in that the polypeptide is an FVIII-BDD polypeptide or a FIX polypeptide.
[5]
5. Manipulated active cell or implantable element according to any one of claims 1 to 4, characterized in that the polypeptide is an FVIII-BDD polypeptide and the coding sequence comprises, consists essentially of or consists of: a) SEQ ID NO : 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 14, SEQ ID NO: 15 , SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 27; or b) a nucleotide sequence that has at least 95%, 96%, 97%, 98%, 99% or more of sequence identity with any of the coding sequences listed in a).
[6]
6. Manipulated active cell or implantable element according to any one of claims 1 to 5, characterized in that the polypeptide is an FVIII-BDD polypeptide and the coding sequence consists essentially of or consists of SEQ ID NO: 16 or SEQ ID NO: 27.
[7]
7. Manipulated active cell or implantable element according to any one of claims 1 to 4, characterized in that the polypeptide is a FIX polypeptide and the coding sequence comprises, essentially consists of or consists of: a) SEQ ID NO: 18 , SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 28; or b) a nucleotide sequence that has at least 95%, 96%, 97%, 98%, 99% or more of sequence identity with any of the coding sequences listed in a).
[8]
8. Manipulated active cell or implantable element according to
5/10 with any one of claims 1 to 4 or 7, characterized in that the polypeptide is a FIX polypeptide and the coding sequence consists essentially of or consists of: SEQ ID NO: 19 or SEQ ID NO: 28.
[9]
9. Manipulated active cell or implantable element according to any one of claims 1 to 8, characterized by the fact that the manipulated active cell is an ARPE-19 cell.
[10]
Implantable element according to any one of claims 1 to 9, characterized in that it comprises a plurality of the manipulated active cells.
[11]
11. Manipulated active cell, eg, an RPE cell, or an implantable element comprising the manipulated active cell, wherein the manipulated active cell is characterized by the fact that it comprises an exogenous nucleic acid that comprises a promoter sequence operably linked to a coding sequence for a polypeptide, where the promoter sequence consists essentially of or consists of SEQ ID NO: 23 or has at least 95%, 96%, 97%, 98%, 99% or more of sequence identity with SEQ ID NO : 23.
[12]
12. Manipulated active cell or implantable element according to claim 11, characterized in that the polypeptide comprises, essentially consists of or consists of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 or SEQ ID NO: 6.
[13]
13. Manipulated active cell or implantable element according to claim 12, characterized in that the polypeptide comprises SEQ ID NO: 4 and the coding sequence comprises SEQ ID NO: 16 or a sequence having at least 99% sequence identity with SEQ ID NO: 16.
[14]
Active cell manipulated according to claim 11, characterized in that the polypeptide comprises, consists essentially of, or consists of SEQ ID NO: 2.
6/10
[15]
An active cell engineered according to claim 14, characterized in that the polypeptide comprises SEQ ID NO: 2 and the coding sequence comprises SEQ ID NO: 19 or a sequence having at least 99% sequence identity with SEQ ID NO: 19.
[16]
Active cell manipulated according to any one of claims 11 to 15, characterized in that the polypeptide additionally comprises SEQ ID NO: 34 or SEQ ID NO: 35.
[17]
An active cell manipulated according to any one of claims 11 to 16, characterized in that the exogenous nucleic acid comprises a Kozak sequence immediately upstream of the coding sequence.
[18]
18. Active cell manipulated according to claim 17, characterized by the fact that the Kozak sequence is nucleotides 2094-2099 of SEQ ID NO: 26.
[19]
19. The manipulated active cell according to any one of claims 11 to 18, characterized in that the exogenous nucleic acid comprises a polyA signal sequence operably linked to the coding sequence, wherein the polyA signal sequence consists essentially of the or consists of nucleotides 2163-2684 of SEQ ID NO: 26.
[20]
20. Implantable element or active cell manipulated according to any one of the preceding claims, characterized by the fact that the promoter sequence consists of SEQ ID NO: 23.
[21]
21. Implantable element or manipulated active cell according to any one of the preceding claims, characterized by the fact that the manipulated active cell is a human RPE cell.
[22]
22. Implantable element or active cell manipulated according to any one of the preceding claims, characterized by the fact that the exogenous nucleic acid is integrated into a chromosome of the
7/10 active cell manipulated.
[23]
23. Implantable element or active cell manipulated according to any one of claims 1 to 3 or 11, characterized in that the polypeptide is selected from the group consisting of: an antibody (eg, nerve growth antifactor antibody) ; an enzyme (e.g., alpha-galactosidase); and a clotting factor (eg, a blood clotting factor, eg, an activated blood clotting factor).
[24]
24. Implantable element or active cell manipulated according to any one of claims 1 to 3 or 11, characterized in that the polypeptide is an insulin polypeptide (eg, insulin A chain, insulin B chain or pro -insulin).
[25]
25. An implantable element or manipulated cell according to any one of claims 1 to 3 or 11, characterized in that the polypeptide is not an insulin polypeptide (eg, insulin A chain, insulin B chain or pro -insulin).
[26]
26. Implantable element according to any one of claims 1 to 10, 20 to 25, characterized in that the implantable element comprises an envelope component.
[27]
27. Implantable element according to claim 26, characterized in that the surrounding component comprises (i) a flexible polymer (eg, PLA, PLG, PEG, CMC or a polysaccharide, eg, alginate) or (ii) an inflexible polymer or metal housing.
[28]
28. Implantable element according to claim 26 or 27, characterized in that the surrounding component is chemically modified.
[29]
29. Implantable element according to any one of the preceding claims, characterized by the fact that the implantable element or its surrounding component is modified with a compound of Formula (I):
8/10
(I), or a salt thereof, where: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, –O–, –C (O) O–, –C (O) -, - OC (O) -, –N (RC) -, –N (RC) C (O) -, –C (O) N (RC) -, -N (RC) C (O) (C1-C6 alkylene) ) -, - N (RC) C (O) (C1-C6 alkenylene) -, –N (RC) N (RD) -, –NCN–, - C (= N (RC) (RD)) O– , –S–, –S (O) x–, –OS (O) x–, –N (RC) S (O) x–, - S (O) xN (RC) -, –P (RF) y -, –Si (ORA) 2–, –Si (RG) (ORA) -, –B (ORA) - or a metal, where each alkyl, alkenyl, alkynyl, alkylene, alkenylene, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is attached to an attachment group (eg, an attachment group defined here) and is optionally substituted by one or more R1; each of L1 and L3 is independently a bond, alkyl or heteroalkyl, wherein each alkyl and heteroalkyl is optionally substituted by one or more R2; L2 is a link; M is absent, is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, each of which is optionally substituted by one or more R3; P is absent, is cycloalkyl, heterocyclyl or heteroaryl, each of which is optionally substituted by one or more R4; Z is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, - ORA, –C (O) RA, –C (O) ORA, –C (O) N (RC) (RD), –N (RC) C (O ) RA, cycloalkyl, heterocyclyl, aryl or heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R5; each RA, RB, RC, RD, RE, RF and RG is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido,
9/10 cycloalkyl, heterocyclyl, aryl or heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R6; or RC and RD, taken together with the nitrogen atom to which they are attached, form a ring (eg, a 5-7 membered ring), optionally substituted by one or more R6; each R1, R2, R3, R4, R5 and R6 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, –ORA1, - C (O) ORA1, –C (O) RB1, –OC ( O) RB1, –N (RC1) (RD1), –N (RC1) C (O) RB1, - C (O) N (RC1), SRE1, S (O) xRE1, –OS (O) xRE1, - N (RC1) S (O) xRE1, - S (O) xN (RC1) (RD1), –P (RF1) y, cycloalkyl, heterocyclyl, aryl, heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl , heterocyclyl, aryl and heteroaryl is optionally substituted by one or more R7; each RA1, RB1, RC1, RD1, RE1 and RF1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl, where each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl it is optionally replaced by one or more R7; each R7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl or heterocyclyl; x is 1 or 2; and y is 2, 3 or 4.
[30]
30. Implantable element according to claim 29, characterized in that the compound of Formula (I) is a compound shown in the Table of Compounds 1.
[31]
31. Implantable element according to claim 29 or 30, characterized by the fact that the compound is selected from:
10/10
N N O
N S O
The N
O
O
NH and or one of its salt.
[32]
32. Implantable element according to claim 29 or 30, characterized in that the compound is selected from Compound 110, Compound 112, Compound 113 or Compound 114 from the Table of Compounds 1.
[33]
33. Implantable element according to any one of claims 26 to 32, characterized in that the surrounding component is an alginate hydrogel capsule.
[34]
34. Implantable element according to claim 33, characterized by the fact that it comprises at least about 10,000,
15,000 or 20,000 manipulated ARPE-19 cells.
[35]
35. Pharmaceutical composition, characterized by the fact that it comprises a plurality of the implantable element, as defined in any of the preceding claims, in a pharmaceutically acceptable carrier.
[36]
36. An implantable element according to any one of claims 1 to 34, or the pharmaceutical composition according to claim 35, characterized in that it is for use in treating a human subject by a method comprising: administering or providing the subject with the implantable element or composition, thereby treating the subject or providing a product (e.g., a therapeutic product) to the subject.

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RU2020114616A|2021-10-28|

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IL274200D0|2020-06-30|

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JP2020534837A|2020-12-03|

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KR20200057051A|2020-05-25|

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EP3687580A1|2020-08-05|

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CA3077380A1|2019-04-04|

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CN111372612A|2020-07-03|

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法律状态:
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优先权:

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申请号 | 申请日 | 专利标题

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US201762563877P| true| 2017-09-27|2017-09-27|

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US201862652882P| true| 2018-04-04|2018-04-04|

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