Patent Application
20240123040
Mucopolysaccharidosis type VI (VIPS VI) is a rare, autosomal genetic disease characterized by deficient activity of the lysosomal enzyme arylsulfatase B (ARSB) (also known as N-acetylglucosamine 4-sulfatase), which results in lysosome accumulation of the glycosaminoglycans (GAGs) dermatan sulfate (DS) and chondroitin sulfate (CS). Lysosome storage of DS and CS cause a number of problems including bone dysplasia, joint restriction, organomegaly, heart disease, and corneal clouding. MPS VI typically presents in one of two forms: a rapidly advancing form, which leads to severe disease in the majority of patients, and a slowly-progressing form with more attenuated symptoms in a minority of patients.
The most widely used specific treatment for MPS VI disease is enzyme replacement therapy (ERT), which currently is a life-long therapy that requires weekly or twice weekly intravenous infusions with a recombinant version of ARSB. Thus, novel treatment modalities for MPS VI disease are desirable.
Described herein is a mammalian cell (e.g., a human cell) that is engineered to express and secrete a mammalian ARSB protein, as well as compositions, pharmaceutical products, and medical devices comprising the ARSB-secreting cell, and methods of making and using the same. In some embodiments, the mammalian cell is engineered to co-express the mammalian ARSB protein and a mammalian sialytransferase (ST) protein, which co-expression results in a greater amount of ARSB secreted from the cell than the same cell that does not co-express the ST protein. In some embodiments, the compositions, products and devices comprising the ARSB-secreting cell are configured to mitigate the foreign body response when a composition, product or device is administered to, e.g., placed inside, a mammalian subject.
In one aspect, the present disclosure features an isolated polynucleotide (e.g., an expression vector) comprising a first expression cassette which comprises a first promoter sequence and a first polyA signal sequence operably linked to a nucleotide sequence encoding a precursor ARSB protein (e.g., human precursor ARSB). In an embodiment, the promoter sequence is identical to, or substantially identical to, the EF1A promoter sequence shown in
In an embodiment, the isolated polynucleotide further comprises a nucleotide sequence encoding a mammalian sialyltransferase (ST), e.g., a human sialytransferase, e.g., hST3GAL4. In an embodiment, the first expression cassette is multicistronic (e.g., bicistronic) and the ARSB-encoding and the ST-encoding sequences in the first expression cassette flank a sequence encoding a ribosomal codon skipping site (e.g., a 2A peptide sequence as defined herein) or flank an internal ribosome entry site (IRES) sequence (e.g., as defined herein). In an embodiment, the ST-encoding sequence is operably linked to a second promoter sequence and a second polyA signal sequence in a second expression cassette located upstream or downstream of the first expression cassette. In an embodiment, the second promoter sequence is identical to, or substantially identical to, the sequence shown in
In an embodiment, the first expression cassette and any second expression cassette are flanked by a pair of transposon inverted terminal repeat (ITR) sequences. In an embodiment, the isolated polynucleotide comprises SEQ ID NO:18, SEQ ID NO:19 or SEQ ID NO:20.
In another aspect, the present disclosure provides a mammalian cell (e.g., a mouse cell, a human cell, an ARPE-19 cell) engineered to express and secrete a mammalian ARSB protein (e.g., engineered to express a human precursor ARSB protein or a fusion precursor ARSB protein). The engineered cell comprises a first exogenous expression cassette which comprises a first promoter sequence (e.g., SEQ ID NO:16) and a first polyA signal sequence operably linked to a nucleotide sequence encoding a precursor ARSB protein. In some embodiments, the ARSB-secreting cell is also engineered to co-express a sialyltransferase (ST) protein. In an embodiment, the first exogenous expression cassette comprises a nucleotide sequence encoding the ST protein. In another embodiment, the ARSB-secreting cell comprises a second exogenous expression cassette which comprises a second promoter sequence and a second polyA signal sequence operably linked to a nucleotide sequence encoding the ST protein. In an embodiment, the ARSB-secreting cell comprises a transposon which comprises the first expression cassette and any second expression cassette. In an embodiment, the first exogenous expression cassette and any second exogenous expression cassette comprise a transposon component of an extrachromosomal expression vector. In an embodiment, the first exogenous cassette and any second exogenous expression cassette are integrated into at least one location in the genome of the mammalian cell. In an embodiment, the first exogenous expression cassette comprises nucleotide sequence comprises nucleotides 337 to 3,696 of SEQ ID NO:15. In an embodiment, the ST protein co-expressed by an ARSB-secreting cell described herein is human ST3GAL2 or human ST3GAL4. In an embodiment, a mammalian cell co-expressing human ARSB and human ST3GAL4 comprises an exogenous nucleotide sequence selected from the group consisting of nucleotides 337 to 4,824 of SEQ ID NO:18; nucleotides 337 to 5,341 of SEQ ID NO:19; and nucleotides 337 to 5,240 of SEQ ID NO:20. In an embodiment, the mammalian cell is engineered to express ARSB or to co-express ARSB and sialytransferase by transfecting the cell with one of the isolated polynucleotides described herein.
In yet another aspect, the present disclosure provides a device comprising at least one cell-containing compartment which comprises an ARSB-secreting mammalian cell as described herein or a plurality of such cells. In an embodiment, the cell-containing compartment further comprises at least one cell binding-substance (CBS), as defined herein. The device is configured to shield the cell(s) from the recipient's immune system and mitigate the foreign body response (FBR) (as defined herein) to the implanted device. In an embodiment, the device is capable of delivering an ARSB protein for a sustained time period (e.g., one to several months up to one to several years) after implant into a subject.
In the surface of the device comprises a compound or polymer that mitigates the FBR (as defined herein) to the device (e.g., an afibrotic compound or afibrotic polymer). In an embodiment, an afibrotic polymer comprises a biocompatible, zwitterionic polymer, e.g., as described in WO 2017/218507, WO 2018/140834, or Liu et al., Zwitterionically modified alginates mitigate cellular overgrowth for cell encapsulation, Nature Communications (2019)10:5262. In an embodiment, the compound is a compound of Formula (I):
or a pharmaceutically acceptable salt thereof, wherein the variables A, L1, M, L2, P, L3, and Z, as well as related subvariables, are defined herein.
In an embodiment, the device is configured as a two-compartment hydrogel capsule in which an inner compartment comprising the engineered mammalian cell(s) expressing a mammalian ARSB protein, and optionally a mammalian ST, is completely surrounded by a barrier compartment. In some embodiments, the barrier compartment comprises a polymer covalently modified with a compound that mitigates the foreign body response (FBR) to the device.
In an embodiment, a device described herein, or a plurality of the device, is combined with a pharmaceutically acceptable excipient to prepare a device preparation or a composition which may be administered to a subject (e.g., into the intraperitoneal cavity) in need of treatment with the ARSB protein produced by the device. In an embodiment, the subject is a human with MPS VI, the engineered cells are derived from a human cell (e.g., an RPE cell, an ARPE-19 cell) and the device preparation or composition is capable of continuously delivering an effective amount of a human ARSB protein to the subject for a sustained time period, e.g., at least any of 3 months, 6 months, one year, two years or longer.
The present disclosure features mammalian cells (e.g., human RPE cells) engineered to express and secrete a mammalian ARSB protein, and optionally co-express a sialytransferase protein with the ARSB protein, as well as compositions and devices comprising such engineered cells. In some embodiments, the devices comprise a cell-containing compartment which includes a cell binding substance as well as the engineered cells. In some embodiments, the devices are configured to mitigate the FBR when placed inside a subject, e.g., a human subject. In some embodiments, the engineered cells, compositions, and devices are useful for the treatment of MPS VI disease. A variety of engineered ARSB-secreting cells and device configurations are contemplated by the present disclosure. Various embodiments will be described below.
Throughout the detailed description and examples of the disclosure the following abbreviations will be used.
So that the disclosure may be more readily understood, certain technical and scientific terms used herein are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.
As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.
“About” or “approximately” when used herein to modify a numerically defined parameter (e.g., amount of ARSB protein secreted by an engineered cell, a physical description of a device (e.g., hydrogel capsule) such as diameter, sphericity, number of cells encapsulated therein, the number of devices in a preparation), means that the recited numerical value is within an acceptable functional range for the defined parameter as determined by one of ordinary skill in the art, which will depend in part on how the numerical value is measured or determined, e.g., the limitations of the measurement system, including the acceptable error range for that measurement system. For example, “about” can mean a range of 20% above and below the recited numerical value. As a non-limiting example, a hydrogel capsule defined as having a diameter of about 1.5 millimeters (mm) and encapsulating about 5 million (M) cells may have a diameter of 1.2 to 1.8 mm and may encapsulate 4 M to 6 M cells. As another non-limiting example, a preparation of about 100 devices (e.g., hydrogel capsules) includes preparations having 80 to 120 devices. In some embodiments, the term “about” means that the modified parameter may vary by as much as 15%, 10% or 5% above and below the stated numerical value for that parameter.
“Acquire” or “acquiring” as used herein, refer to obtaining possession of a value, e.g., a numerical value, or image, or a physical entity (e.g., a sample), by “directly acquiring” or “indirectly acquiring” the value or physical entity. “Directly acquiring” means performing a process (e.g., performing an analytical method or protocol) to obtain the value or physical entity. “Indirectly acquiring” refers to receiving the value or physical entity from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Directly acquiring a value or physical entity includes performing a process that includes a physical change in a physical substance or the use of a machine or device. Examples of directly acquiring a value include obtaining a sample from a human subject. Directly acquiring a value includes performing a process that uses a machine or device, e.g., using a fluorescence microscope to acquire fluorescence microscopy data.
“Administer,” “administering,” or “administration,” as used herein, refer to implanting, absorbing, ingesting, injecting, placing, or otherwise introducing into a subject, an entity described herein (e.g., a device or a preparation of devices), or providing such an entity to a subject for administration.
“Afibrotic”, as used herein, means a compound or material that mitigates the foreign body response (FBR). For example, the amount of FBR in a biological tissue that is induced by implant into that tissue of a device (e.g., hydrogel capsule) comprising an afibrotic compound (e.g., a hydrogel capsule comprising a polymer covalently modified with a compound listed in Table 4) is lower than the FBR induced by implantation of an afibrotic-null reference device, i.e., a device that lacks any afibrotic compound, but is of substantially the same composition (e.g., same cell type(s)) and structure (e.g., size, shape, no. of compartments). In an embodiment, the degree of the FBR is assessed by the immunological response in the tissue containing the implanted device (e.g., hydrogel capsule), which may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis, using assays known in the art, e.g., as described in WO 2017/075630, or using one or more of the assays/methods described Vegas, A., et al., Nature Biotechnol (supra), (e.g., subcutaneous cathepsin measurement of implanted capsules, Masson's trichrome (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cellular staining and confocal microscopy for macrophages (CD68 or F4/80), myofibroblasts (alpha-muscle actin, SMA) or general cellular deposition, quantification of 79 RNA sequences of known inflammation factors and immune cell markers, or FACS analysis for macrophage and neutrophil cells on retrieved devices (e.g., capsules) after 14 days in the intraperitoneal space of a suitable test subject, e.g., an immunocompetent mouse. In an embodiment, the FBR is assessed by measuring the levels in the tissue containing the implant of one or more biomarkers of immune response, e.g., cathepsin, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4. In some embodiments, the FBR induced by a device of the invention (e.g., a hydrogel capsule comprising an afibrotic compound disposed on its outer surface), is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the FBR induced by an FBR-null reference device, e.g., a device that is substantially identical to the test or claimed device except for lacking the means for mitigating the FBR (e.g., a hydrogel capsule that does not comprise an afibrotic compound but is otherwise substantially identical to the claimed capsule. In some embodiments, the FBR (e.g., level of a biomarker(s)) is measured after about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or longer.
“Arylsulfatase B protein” and “ARSB protein” may be used interchangeably herein and refer to a protein comprising the amino acid sequence of a mature, wild-type mammalian ARSB or any fragment, mutant, variant or derivative thereof that has enzyme activity (e.g., sulfatase activity) that is within 80-120%, 85-115%, 90-110% or 95-105% of the corresponding wild-type mammalian mature ARSB protein, as measured by an ARSB activity assay described herein. ARSB hydrolyses sulfates in the body by metabolizing the sulfate moiety of dermatan sulfate and chondroitin sulfate. The wild-type human ARSB gene encodes a 533 amino acid precursor polypeptide, of which the N-terminal 36 or 38 amino acids constitute a signal peptide. The amino acid sequence for wild-type human precursor ARSB is shown in
“2A peptide sequence”, or “2A linker” as used herein, refers to an amino acid sequence that is identical to or substantially similar to any of the “self-cleaving” or “self-processing” viral-derived peptide sequences that operate by the mechanism of ribosomal codon skipping in which ribosomes skip the synthesis of a peptide bond at the C-terminus of the peptide sequence, leading to cleavage between the peptide and the immediate downstream amino acid sequence. Amino acid and coding sequences for 2A peptides, including P2A, E2A, F2A, and T2A, are described in Kim et ah, PLoS One, 2011, 6(4):e18556. In some embodiments, the 2A peptide sequence is selected from the group consisting of P2A, E2A, F2A, and T2A sequences. In an embodiment, the 2A peptide sequence is at least 90%, 95% or 99% identical to the sequence of P2A, E2A, F2A, or T2A. In an embodiment, the 2A peptide consists essentially of GSGATNFSLLKQAGDVEENPGP (SEQ ID NO:20).
“Cell,” as used herein, refers to an engineered cell or a cell that is not engineered. In an embodiment, a cell is an immortalized cell, or an engineered cell derived from an immortalized cell. In an embodiment, the cell is a live cell, e.g., is viable as measured by any technique described herein or known in the art.
“Cell-binding peptide (CBP)”, as used herein, means a linear or cyclic peptide that comprises an amino acid sequence that is derived from the cell binding domain of a ligand for a cell-adhesion molecule (CAM) (e.g., that mediates cell-matrix junctions or cell-cell junctions). In an embodiment, the CBP is any of the CBPs described in international patent publication WO2020069429. In an embodiment, the CBP is a linear peptide comprising RGD and is less than 6 amino acids in length. In an embodiment, the CBP is a linear peptide that consists essentially of RGD or RGD SP.
“CBP-polymer”, as used herein, means a polymer comprising at least one cell-binding peptide molecule covalently attached to the polymer via a linker. In an embodiment, the polymer in a CBP-polymer is a synthetic or naturally-occurring polysaccharide, e.g., an alginate, e.g., a sodium alginate. In an embodiment, the linker is an amino acid linker (i.e., consists essentially of a single amino acid, or a peptide of several identical or different amino acids), which is joined via a peptide bond to the N-terminus or C-terminus of the CBP. In an embodiment, the CBP-polymer is any of the CBP-alginates defined in WO2020069429.
“Cell-binding substance (CBS)”, as used herein, means any chemical, biological, or other type of substance (e.g., a small organic compound, a peptide, a polypeptide) that is capable of mimicking at least one activity of a ligand for a cell-adhesion molecule (CAM) or other cell-surface molecule that mediates cell-matrix junctions or cell-cell junctions or other receptor-mediated signaling. In an embodiment, when present in a polymer composition encapsulating live cells, the CBS is capable of forming a transient or permanent bond or contact with one or more of the cells. In an embodiment, the CBS facilitates interactions between two or more live cells encapsulated in the polymer composition. In an embodiment, the presence of a CBS in a polymer composition encapsulating a plurality of cells (e.g., live cells) is correlated with one or both of increased cell productivity (e.g., expression of a therapeutic agent) and increased cell viability when the encapsulated cells are implanted into a test subject, e.g., a mouse. In an embodiment, the CBS is physically attached to one or more polymer molecules in the polymer composition. In an embodiment, the CBS is a cell-binding peptide, as defined herein or in WO2020069429.
“Conservatively modified variants” or conservative substitution”, as used herein, refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule, except for having one or more conservative amino acid substitutions in its amino acid sequence. In an embodiment, a conservatively modified variant consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the reference amino acid sequence. A conservative amino acid substitution refers to substitution of an amino acid with an amino acid having similar characteristics (e.g., charge, side-chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) and which has minimal impact on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution tables of functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional features are set forth in Table 2 below.
“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 recited elements or group of elements, and the optional inclusion of other elements, of similar or different nature than the recited elements, that do not materially change the basic or novel properties of the specified molecule, composition, device, or method. As a non-limiting example, An ARSB protein or an ST protein that consists essentially of a recited amino acid sequence may also include one or more amino acids, including substitutions in the recited amino acid sequence, of one or more amino acid residues, which do not materially affect the relevant biological activity of the ARSB protein ST protein, respectively.
“Derived from”, as used herein with respect to a cell or cells, refers to cells obtained from tissue, cell lines, or cells, which optionally are then cultured, passaged, immortalized, differentiated and/or induced, etc. to produce the derived cell(s).
“Device”, as used herein, refers to any implantable object (e.g., a particle, a hydrogel capsule, an implant, a medical device), which contains an engineered cell or cells (e.g., live cells) capable of expressing and secreting an ARSB protein following implant of the device, and has a configuration that supports the viability of the cells by allowing cell nutrients to enter the device.
“Differential volume,” as used herein, refers to a volume of one compartment within a device described herein that excludes the space occupied by another compartment(s). For example, the differential volume of the second (e.g., outer) compartment in a 2-compartment device with inner and outer compartments, refers to a volume within the second compartment that excludes space occupied by the first (inner) compartment.
“Effective amount” as used herein refers to an amount of any of the following: engineered cells secreting a mammalian ARSB protein, a device preparation producing the mammalian ARSB protein, or a component of a device (e.g., amount of a mammalian ST protein co-expressed with ARSB by cells in the device, number of engineered cells in the device, amount of a CBS and/or afibrotic compound in the device) that is sufficient to elicit a desired biological response. In some embodiments, the term “effective amount” refers to the amount of a component of the device (e.g., number of cells in the device, the density of an afibrotic compound disposed on the surface and/or in a barrier compartment of the device, the density of a CBS in the cell-containing compartment.
In an embodiment, the desired biological response is an increase in ARSB levels in a tissue sample removed from a subject treated with (e.g., implanted with) the engineered cells, a device or a device preparation containing such cells. As will be appreciated by those of ordinary skill in this art, the effective amount may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the secreted ARSB, composition or device, the condition being treated, the mode of administration, and the age and health of the subject. An effective amount encompasses therapeutic and prophylactic treatment.
In an embodiment, an effective amount of a ST protein expressed by cells co-expressing ARSB and the ST protein is an amount that increases ARSB secretion by at least any of 10%, 20%, 40% or more as compared to the same cells that do not express the ST protein. In an embodiment, an effective amount of a compound of Formula (I) disposed on or in a device is an amount that reduces the FBR to the implanted device compared to a reference device, e.g., reduces fibrosis or amount of fibrotic tissue on or near the implanted device.
In an embodiment, an effective amount of a CBS disposed with engineered cells in a cell-containing compartment is an amount that enhances the viability of the cells (e.g., number of live cells) compared to a reference device and/or increases the production of ARSB by the cells (e.g., increased ARSB levels in plasma of a subject implanted with the device) compared to a reference device. An effective amount of a device, composition or component (e.g., afibrotic compound, CBS, engineered cells) may be determined by any technique known in the art of described herein.
“Engineered cell,” as used herein, is a mammalian cell (e.g., a human cell, e.g., an RPE cell) having a non-naturally occurring alteration, and typically comprises an exogenous nucleotide sequence (e.g., a vector or an altered chromosomal sequence), encoding a mammalian ARSB protein. In an embodiment, an engineered cell secretes an ARSB protein comprising a human wild-type ARSB amino acid sequence or variant thereof. In an embodiment, the exogenous nucleotide sequence is chromosomal (e.g., the exogenous sequence is disposed in endogenous chromosomal sequence) or is extra chromosomal (e.g., a non-integrated expression vector). In an embodiment, the engineered ARSB-secreting cell comprises an exogenous nucleotide sequence encoding a mammalian sialytransferase (ST) protein. In an embodiment, the exogenous ARSB-encoding nucleotide sequence in an engineered cell comprises a codon optimized coding sequence that achieves higher expression of the ARSB protein than a naturally-occurring ARSB coding sequence. The codon optimized sequence may be generated using a commercially available algorithm, e.g., GeneOptimizer (ThermoFisher Scientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), or Java Codon Adaptation Tool (JCat, www.jcat.de, Grote, A. et al., Nucleic Acids Research, Vol 33, Issue suppl 2, pp. W526-W531 (2005). In an embodiment, an engineered cell (e.g., engineered ARPE-19 cell) is cultured from a population of stably-transfected cells, or from a monoclonal cell line.
“An “exogenous nucleotide sequence,” as used herein, is a nucleotide sequence that does not occur naturally in a subject cell.
An “exogenous polypeptide,” as used herein, is a polypeptide that does not occur naturally in a subject cell, e.g., engineered cell. Reference to an amino acid position of a specific sequence means the position of said amino acid in a reference amino acid sequence, e.g., sequence of a full-length mature (after signal peptide cleavage) wild-type protein (unless otherwise stated), and does not exclude the presence of variations, e.g., deletions, insertions and/or substitutions at other positions in the reference amino acid sequence.
“Expression vector”, as used herein, refers to a recombinant polynucleotide comprising one or more expression constructs encoding one or more proteins to be expressed. Each expression construct contains expression control sequences operatively linked to one or more nucleotide sequences to be expressed. An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. The vector may comprise additional sequence elements used for the expression of and/or the integration of the expression cassette(s) into the genome of a mammalian cell. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide. The expression vectors suitable for use in engineering mammalian cells to express any of the ARSB or ST proteins described herein may also contain a nucleotide sequence encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.
“IRES sequence”, as used herein, refers to an internal ribosomal entry site that allows eukaryotic ribosomes to bind an mRNA transcript and begin translation without binding to a 5′ capped end. A mRNA containing an IRES sequence produces two translation products, one initiating form the 5′ end of the mRNA and the other from an internal translation mechanism mediated by the IRES. The IRES sequence can be a sequence known in the art or a variant thereof, including any viral, chromosomal or artificially designed sequence which initiates cap-independent ribosome binding to mRNA and facilitates the initiation of translation. In one embodiment, the IRES sequence comprises nucleotides 3145-3732 of SEQ ID NO:19. In other embodiment, the IRES sequence is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to nucleotides 3145-3732 of SEQ ID NO:19.
“MPS VI patient” as used herein, refers to an individual who has been diagnosed with or suspected of having MPS VI disease. In an embodiment, an MPS VI patient has a mutated ARSB gene. The patient may be diagnosed using any method known in the art, including one or more of the clinical, biochemical and genetic methods for diagnosing MPS VI described in Vairo et al., The Application of Clinical Genetics Vol. 8, pp. 245-255 (2015).
“Peptide”, as used herein, is a polypeptide of less than 50 amino acids, typically, less than 25 amino acids.
“PolyA” signal, as used herein, refers to any continuous sequence of adenylic acids that terminates transcription of a coding sequence into RNA and directs addition of a polyA tail onto the RNA. The length of a polyA sequence is from 10- to 200 nucleotides and may be controlled variously depending on the allowable size of the backbone of the expression vector. Examples of polyA signals are the rabbit binding globulin (rBG) polyA signal, the SV40 late poly A signal, the SV50 polyA signal, the bovine growth hormone (BGH) poly A signal, the human growth hormone (HGH) polyA signal and synthetic polyA signals known in the art.
“Polymer composition”, as used herein, is a composition (e.g., a solution, mixture) comprising one or more polymers. As a class, “polymers” includes homopolymers, heteropolymers, co-polymers, block polymers, block co-polymers and can be both natural and synthetic. Homopolymers contain one type of building block, or monomer, whereas co-polymers contain more than one type of monomer.
“Polypeptide”, as used herein, is a polymer comprising amino acid residues linked through peptide bonds and having at least two, and in some embodiments, at least 10, 50, 75, 100, 150 or 200 amino acid residues.
“Prevention,” “prevent,” and “preventing”, as used herein, refer to a treatment that comprises administering or applying an ARSB replacement therapy, e.g., administering a composition of devices encapsulating engineered cells (e.g., as described herein), prior to the onset of one or more symptoms of MPS VI disease to preclude the physical manifestation of the symptom(s). In some embodiments, “prevention,” “prevent,” and “preventing” require that signs or symptoms of MPS VI disease have not yet developed or have not yet been observed. In some embodiments, treatment comprises prevention and in other embodiments it does not.
“Promoter sequence”, as used herein refers to a nucleotide sequence that is capable of driving expression in a mammalian cell, e.g., a human cell, e.g., an ARPE-19 cell. In some embodiments, e.g., for driving expression of an ARSB protein described herein, the promoter sequence is from a strong mammalian promoter, e.g., a human promoter sequence. Non-limiting examples of strong promoters for use in ARSB expression cassettes described herein include the EF1A promoter, CAG promoter, PGK (phosphoglycerate kinase) promoter and the ACTB (human beta-actin) promoter. In an embodiment, a promoter sequence useful for driving expression of a ST protein described herein may be from a medium-strength promoter, e.g., the EFS promoter sequence, which is a shortened form of the EF1A promoter sequence.
“RPE cell” as used herein refers to a cell having one or more of the following characteristics: a) it comprises a retinal pigment epithelial cell (RPE) (e.g., cultured using the ARPE-19 cell line (ATCC ® CRL-2302™)) or a cell derived or engineered therefrom, e.g., by stably transfecting cells cultured from the ARPE-19 cell line with an exogenous sequence that encodes an ARSB protein or otherwise engineering such cultured ARPE-19 cells to express an ARSB protein a cell derived from a primary cell culture of RPE cells, a cell isolated directly (without long term culturing, e.g., less than 5 or 10 passages or rounds of cell division since isolation) from naturally occurring RPE cells, e.g., from a human or other mammal, a cell derived from a transformed, an immortalized, or a long term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell culture; b) a cell that has been obtained from a less differentiated cell, e.g., a cell developed, programmed, or reprogramed (e.g., in vitro) into an RPE cell or a cell that is, except for any genetic engineering, substantially similar to one or more of a naturally occurring RPE cell or a cell from a primary or long term culture of RPE cells (e.g., the cell can be derived from an IPS cell); or c) a cell that has one or more of the following properties: i) it expresses one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) it does not express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; iii) it is naturally found in the retina and forms a monolayer above the choroidal blood vessels in the Bruch's membrane; or iv) it is responsible for epithelial transport, light absorption, secretion, and immune modulation in the retina; or v) it has been created synthetically, or modified from a naturally occurring cell, to have the same or substantially the same genetic content, and optionally the same or substantially the same epigenetic content, as an immortalized RPE cell line (e.g., the ARPE-19 cell line (ATCC® CRL-2302™)). In an embodiment, an RPE described herein is engineered, e.g., to have a new property, e.g., the cell is engineered to express and secrete a mammalian ARSB protein. In other embodiments, an RPE cell is not engineered.
“Saline solution” as used herein, means normal saline, i.e., water containing 0.9% NaCl, unless otherwise specified.
“Sequence identity” or “percent identical”, when used herein to refer to two nucleotide sequences or two amino acid sequences, means the two sequences are the same within a specified region, or have the same nucleotides or amino acids at a specified percentage of nucleotide or amino acid positions within the specified when the two sequences are compared and aligned for maximum correspondence over a comparison window or designated region. Sequence identity may be determined using standard techniques known in the art including, but not limited to, any of the algorithms described in US Patent Application Publication No. 2017/02334455 A1. In an 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.
“Sialytransferase protein”, as used herein, refers to any protein with sialyltransferase activity, e.g., capable of transferring N-acetylneuraminic acid (Neu5Ac) from cytidine monophosphate-Neu5Ac to a mammalian ARSB protein (e.g., hARSB) expressed by an engineered cell described herein. In an embodiment, co-expression of a sialytransferase with a mammalian ARSB protein in an engineered mammalian cell described herein (e.g., an engineered ARPE-19 cell) increases secretion of the mammalian ARSB as compared to the same cell that does not express the ST protein. An ST protein may comprise a naturally-occurring amino acid sequence from any prokaryote or eukaryote species or a variant thereof that retains sialytransferase activity. Examples of eukaryotic STs and prokaryotic STs are described in Audrey et al., Glycobiology, Vol. 21, No. 6, pp. 716-726 (2011). Non-limiting examples of sialytransferase proteins that may be expressed by engineered cells described herein include the following human sialyltransferases and their orthologs in other mammalian species:
“Spherical” as used herein, mean a device (e.g., a hydrogel capsule or other particle) having a curved surface that forms a sphere (e.g., a completely round ball) or sphere-like shape, which may have waves and undulations, e.g., on the surface. Spheres and sphere-like objects can be mathematically defined by rotation of circles, ellipses, or a combination around each of the three perpendicular axes, a, b, and c. For a sphere, the three axes are the same length. Generally, a sphere-like shape is an ellipsoid (for its averaged surface) with semi-principal axes within 10%, or 5%, or 2.5% of each other. The diameter of a sphere or sphere-like shape is the average diameter, such as the average of the semi-principal axes.
“Subject” as used herein refers to a human or non-human animal. In an embodiment, the subject is a human (i.e., a male or female) of any age group, e.g., a pediatric human subject (e.g., infant, child, adolescent) or adult human subject (e.g., young adult, middle—aged adult, or senior adult)). In an embodiment, the subject is a non-human animal, for example, a mammal (e.g., a mouse, a dog, a primate (e.g., a cynomolgus monkey or a rhesus monkey). In an embodiment, the subject is a commercially relevant mammal (e.g., cattle, pig, horse, sheep, goat, cat, or dog) or a bird (e.g., a commercially relevant bird such as a chicken, duck, goose, or turkey). In certain embodiments, the animal is a mammal. The animal may be a male or female and at any stage of development. A non-human animal may be a transgenic animal.
“Treatment,” “treat,” and “treating” as used herein refers to one or more of reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of one or more of a symptom, manifestation, or underlying cause, of MPS VI disease. In an embodiment, treating comprises increasing ARSB activity in at least one tissue of a subject in need thereof, e.g., in one or more of liver, kidney and lung. In an embodiment, treating comprises reducing, reversing, alleviating, delaying the onset of, or inhibiting the progress of a symptom or condition associated with MPS VI disease, e.g., reducing urinary GAG levels, reducing levels of dermatan sulfate and optionally chondroitin sulfate (CS) in one or more tissues typically impacted in MPS VI, including liver, kidney and lung. In some embodiments, “treatment,” “treat,” and “treating” require that signs or symptoms associated with MPS VI disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of MPS VI disease, e.g., in preventive treatment. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence. In some embodiments, treatment comprises prevention and in other embodiments it does not.
“Wild-type” (wt) refers to the natural form, including sequence, of a polynucleotide, polypeptide or protein in a species. A wild-type form is distinguished from a mutant form of a polynucleotide, polypeptide or protein arising from genetic mutation(s).
Definitions of specific functional groups and chemical terms are described in more detail below. The chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed., inside cover, and specific functional groups are generally defined as described therein. Additionally, general principles of organic chemistry, as well as specific functional moieties and reactivity, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.
The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.
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 alkyl.
As used herein, “alkyl” refers to a radical of a straight-chain or branched saturated hydrocarbon group having from 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 10 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-C4alkyl”), 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-C6alkyl”). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8) and the like. Each instance of an alkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkyl”) or substituted (a “substituted alkyl”) with one or more substituents, e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
As used herein, “alkenyl” refers to a radical of a straight-chain or branched hydrocarbon group having from 2 to 24 carbon atoms, one or more carbon-carbon double bonds, and no triple bonds (“C2-C24 alkenyl”). 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 (“C2-C6 alkenyl”), 2 to carbon atoms (“C2-C5 alkenyl”), 2 to 4 carbon atoms (“C2-C4 alkenyl”), 2 to 3 carbon atoms (“C2-C3 alkenyl”), or 2 carbon atoms (“C2 alkenyl”). The one or more carbon-carbon double bonds can be internal (such as in 2-butenyl) or terminal (such as in 1-butenyl). Examples of C2-C4 alkenyl groups include ethenyl (C2), 1-propenyl (C3), 2-propenyl (C3), 1-butenyl (C4), 2-butenyl (C4), butadienyl (C4), and the like. Examples of C2-C6 alkenyl groups include the aforementioned C2-4 alkenyl groups as well as pentenyl (C5), pentadienyl (C5), hexenyl (C6), and the like. Each instance of an alkenyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with one or more substituents e.g., for instance from 1 to 5 sub stituents, 1 to 3 substituents, or 1 sub stituent.
As used herein, the term “alkynyl” refers to a radical of a straight-chain or branched hydrocarbon group 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 carbon atoms (“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 instance of an alkynyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted alkynyl”) or substituted (a “substituted alkynyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
As used herein, the term “heteroalkyl,” refers to a non-cyclic stable 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 wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any position of the heteroalkyl group. Exemplary heteroalkyl groups include, but are not limited to: —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 heteroatoms may be consecutive, such as, for example, —CH2—NH—OCH3 and —CH2—O—Si(CH3)3. Where “heteroalkyl” is recited, followed by recitations of specific heteroalkyl groups, such as —CH2O, —NRCRD, or the like, it will be understood that the terms heteroalkyl and —CH2O or —NRCRD are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term “heteroalkyl” should not be interpreted herein as excluding specific heteroalkyl groups, such as —CH2O, —NRCRD, or the like. Each instance of a heteroalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroalkyl”) or substituted (a “substituted heteroalkyl”) with one or more substituents e.g., for instance from 1 to 5 substituents, 1 to 3 substituents, or 1 substituent.
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, alkynylene, or heteroalkylene group may be described as, e.g., a C1-C6-membered alkylene, C2-C6-membered alkenylene, C2-C6-membered alkynylene, or C1-C6-membered heteroalkylene, wherein the term “membered” refers to the non-hydrogen atoms within the moiety. In the case of heteroalkylene groups, heteroatoms can also occupy either or both chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula —C(O)2R′— may represent both —C(O)2R′— and —R′C(O)2—.
As used herein, “aryl” refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 π C electrons shared in a cyclic array) having 6-14 ring carbon atoms and zero heteroatoms provided in the aromatic ring system (“C6-C14 aryl”). In some embodiments, an aryl group has six ring carbon atoms (“Caryl”; e.g., phenyl). In some embodiments, an aryl group has ten ring carbon atoms (“C10 aryl”; e.g., naphthyl such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has fourteen ring carbon atoms (“C14 aryl”; e.g., anthracyl). An aryl group may be described as, e.g., a C6-C10-membered aryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of an aryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted aryl”) or substituted (a “substituted aryl”) with one or more substituents.
As used herein, “heteroaryl” refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π C electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen and sulfur (“5-10 membered heteroaryl”). In heteroaryl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. Heteroaryl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heteroaryl” also includes ring systems wherein the heteroaryl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is either on the aryl or heteroaryl ring, and in such instances, the number of ring members designates the number of ring members in the fused (aryl/heteroaryl) ring system. Bicyclic heteroaryl groups wherein one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like) the point of attachment can be on either ring, i.e., either the ring bearing a heteroatom (e.g., 2-indolyl) or the ring that does not contain a heteroatom (e.g., 5-indolyl). A heteroaryl group may be described as, e.g., a 6-10-membered heteroaryl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety.
In some embodiments, a heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In some embodiments, a heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of a heteroaryl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heteroaryl”) or substituted (a “substituted heteroaryl”) with one or more sub stituents.
Exemplary 5-membered heteroaryl groups containing one heteroatom include, without limitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, without limitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, without limitation, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, without limitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, without limitation, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, without limitation, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, without limitation, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, without limitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.
As used herein, the terms “arylene” and “heteroarylene,” alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.
As used herein, “cycloalkyl” refers to a radical of a non—aromatic cyclic hydrocarbon group having from 3 to 10 ring carbon atoms (“C3-C10 cycloalkyl”) and zero heteroatoms in the non-aromatic ring system. In some embodiments, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-C8cycloalkyl”), 3 to 6 ring carbon atoms (“C3-C6 cycloalkyl”), or 5 to 10 ring carbon atoms (“C5-C10 cycloalkyl”). A cycloalkyl group may be described as, e.g., a C4-C7-membered cycloalkyl, wherein the term “membered” refers to the non-hydrogen ring atoms within the moiety. Exemplary C3-C6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 cycloalkyl groups include, without limitation, the aforementioned C3-C6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (C8), cyclooctenyl (C8), cubanyl (C8), bicyclo[1.1.1]pentanyl (C5), bicyclo[2.2.2]octanyl (C8), bicyclo[2.1.1]hexanyl (C6), bicyclo[3.1.1]heptanyl (C7), and the like. Exemplary C3-C10 cycloalkyl groups include, without limitation, the aforementioned C3-C8 cycloalkyl groups as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), octahydro-1-indenyl (C9), decahydronaphthalenyl (C10), Spiro [4.5] decanyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the cycloalkyl group is either monocyclic (“monocyclic cycloalkyl”) or contain a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic cycloalkyl”) and can be saturated or can be partially unsaturated. “Cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one or more aryl groups wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted cycloalkyl”) or substituted (a “substituted cycloalkyl”) with one or more sub stituents.
“Heterocyclyl” as used herein refers to a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“3-10 membered heterocyclyl”). In heterocyclyl groups that contain one or more nitrogen atoms, the point of attachment can be a carbon or nitrogen atom, as valency permits. A heterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”) or a fused, bridged or spiro ring system such as a bicyclic system (“bicyclic heterocyclyl”), and can be saturated or can be partially unsaturated. Heterocyclyl bicyclic ring systems can include one or more heteroatoms in one or both rings. “Heterocyclyl” also includes ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more cycloalkyl groups wherein the point of attachment is either on the cycloalkyl or heterocyclyl ring, or ring systems wherein the heterocyclyl ring, as defined above, is fused with one or more aryl or heteroaryl groups, wherein the point of attachment is on the heterocyclyl ring, and in such instances, the number of ring members continue to designate the number of ring members in the heterocyclyl ring system. A heterocyclyl group may be described as, e.g., a 3-7-membered heterocyclyl, wherein the term “membered” refers to the non-hydrogen ring atoms, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon, within the moiety. Each instance of heterocyclyl may be independently optionally substituted, i.e., unsubstituted (an “unsubstituted heterocyclyl”) or substituted (a “substituted heterocyclyl”) with one or more substituents. In certain embodiments, the heterocyclyl group is unsubstituted 3-10 membered heterocyclyl. In certain embodiments, the heterocyclyl group is substituted 3-10 membered heterocyclyl.
In some embodiments, a heterocyclyl group is a 5-10 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon (“5-10 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-8 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6 membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In some embodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclyl has one ring heteroatom selected from nitrogen, oxygen, and sulfur.
Exemplary 3- membered heterocyclyl groups containing one heteroatom include, without limitation, azirdinyl, oxiranyl, thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, without limitation, azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, without limitation, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, di hy drothi ophenyl, pyrrolidinyl, dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, without limitation, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, without limitation, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, without limitation, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, without limitation, triazinanyl or thiomorpholinyl-1,1-dioxide. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, without limitation, azepanyl, oxepanyl and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, without limitation, azocanyl, oxecanyl and thiocanyl. Exemplary 5-membered heterocyclyl groups fused to a C6 aryl ring (also referred to herein as a 5,6-bicyclic heterocyclic ring) include, without limitation, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, and the like. Exemplary 6-membered heterocyclyl groups fused to an aryl ring (also referred to herein as a 6,6-bicyclic heterocyclic ring) include, without limitation, tetrahydroquinolinyl, tetrahydroisoquinolinyl, and the like.
“Amino” as used herein refers to the radical —NR70R71, wherein R70 and R71 are each independently hydrogen, C1-C8 alkyl, C3-C10 cycloalkyl, C4-C10 heterocyclyl, C6-C10 aryl, and C5-C10 heteroaryl. In some embodiments, amino refers to NH2.
As used herein, “cyano” refers to the radical —CN.
As used herein, “halo” or “halogen,” independently or as part of another substituent, mean, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.
As used herein, “hydroxy” refers to the radical —OH.
Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., “substituted” or “unsubstituted” alkyl, “substituted” or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl, “substituted” or “unsubstituted” heteroalkyl, “substituted” or “un sub stituted” cycloalkyl, “substituted” or “unsubstituted” heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or “unsubstituted” heteroaryl group). In general, the term “substituted”, whether preceded by the term “optionally” or not, means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent, e.g., a substituent which upon substitution results in a stable compound, e.g., a compound which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, or other reaction. Unless otherwise indicated, a “substituted” group has a substituent at one or more substitutable positions of the group, and when more than one position in any given structure is substituted, the sub stituent is either the same or different at each position. The term “substituted” is contemplated to include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein that result in the formation of a stable compound. The present disclosure contemplates any and all such combinations to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and/or any suitable substituent as described herein which satisfy the valencies of the heteroatoms and results in the formation of a stable moiety.
Two or more substituents may 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 base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring-forming substituents attached to adjacent members of a cyclic base structure 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 base structure create a spirocyclic structure. In yet another embodiment, the ring-forming substituents are attached to non-adjacent members of the base structure.
Compounds of Formula (I) described herein can comprise one or more asymmetric centers, and thus can exist in various isomeric forms, e.g., enantiomers and/or diastereomers. For example, the compounds described herein can be in the form of an individual enantiomer, diastereomer or geometric isomer, or can be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomer. 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 asymmetric syntheses. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The disclosure additionally encompasses compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.
As used herein, a pure enantiomeric compound is substantially free from 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 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight, of the enantiomer. In certain embodiments, the weights are based upon total weight of all enantiomers or stereoisomers of the compound.
Compounds of Formula (I) described herein may also comprise one or more isotopic substitutions. For example, H may be in any isotopic form, including 1H, 2H (D or deuterium), and 3H (T or tritium); C may be in any isotopic form, including 12C, 13C, and 14C; O may be in any isotopic form, including 16O and 18O; and the like.
The term “pharmaceutically acceptable salt” is meant to include salts of the active compounds that are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When compounds of Formula (I) used to prepare devices of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salt, or a similar salt. When compounds used in the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galacturonic acids and the like (see, e.g., Berge et al, Journal of Pharmaceutical Science 66: 1-19 (1977)). Certain specific compounds used in the devices of the present disclosure (e.g., a particle, a hydrogel capsule) contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. These salts may be prepared by methods known to those skilled in the art. Other pharmaceutically acceptable carriers known to those of skill in the art are suitable for use in the present disclosure.
Devices of the present disclosure may contain a compound of Formula (I) in a prodrug form. Prodrugs are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds useful for preparing devices in the present disclosure. Additionally, prodrugs can be converted to useful compounds of Formula (I) by chemical or biochemical methods in an ex vivo environment.
Certain compounds of Formula (I) described herein can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present disclosure. Certain compounds of Formula (I) described herein may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present disclosure and are intended to be within the scope of the present disclosure.
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 herein may be prepared, e.g., in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates and further include both stoichiometric solvates and non-stoichiometric solvates.
The term “hydrate” refers to a compound which is associated with water. Typically, the number of the water molecules contained in a hydrate of a compound is in a definite ratio to the number of the compound molecules in the hydrate. Therefore, a hydrate of a compound may be represented, for example, by the general formula R·x H2O, wherein R is the compound and wherein x is a number greater than 0.
The term “tautomer” as used herein refers to compounds that are interchangeable forms of a compound structure, and that vary in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium through the movement of π electrons and an atom (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomeric forms may be relevant to the attainment of the optimal chemical reactivity and biological activity of a compound of interest.
The symbol “” as used herein refers to a connection to an entity, e.g., a polymer (e.g., hydrogel-forming polymer such as alginate) or surface of an implantable device, e.g., a particle, a hydrogel capsule. The connection represented by “
” may refer to direct attachment to the entity, e.g., a polymer or an implantable element, may refer to linkage to the entity through an attachment group. An “attachment group,” as described herein, refers to a moiety for linkage of a compound of Formula (I) to an entity (e.g., a polymer or an implantable element (e.g., a device) as described herein), and may comprise any attachment chemistry known in the art. A listing of exemplary attachment groups is outlined in Bioconjugate Techniques (3rd ed, Greg T. Hermanson, Waltham, MA: Elsevier, Inc, 2013), which is incorporated herein 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, wherein each of RA, RC, RD, RF, RG, x and y is independently as described herein. In some embodiments, an attachment group comprises an amine, ketone, ester, amide, alkyl. In some embodiments, an attachment group is a cross-linker. In some embodiments, the
attachment group is —C(O)(C1-C1, and R1 is as described herein
. In some embodiments, the attachment group is —C(O)(C1-C6-alkylene)-, wherein alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attachment group is —C(O)C(CH3)2—. In some embodiments, the attachment group is —C(O)(methylene)-, wherein alkylene is substituted with 1-2 alkyl groups (e.g., 1-2 methyl groups). In some embodiments, the attachment group is —C(O)CH(CH3)—. In some embodiments, the attachment group is —C(O)C(CH3)—.
The present disclosure provides an isolated polynucleotide comprising a promoter sequence operably linked to a nucleotide sequence encoding a mammalian precursor ARSB protein or variant thereof, e.g., a precursor ARSB fusion protein.
In an embodiment, the promoter is selected to achieve higher expression of ARSB mRNA in a desired mammalian cell type (e.g., a human cell line) compared to the same ARSB coding sequence operably linked to the promoter in the wild-type mammalian ARSB gene. In an embodiment, the promoter is classified as a strong promoter and achieves higher expression of human ARSB in human cells (e.g., ARPE-19 cells) as compared to at least one, two or three other strong promoters. In an embodiment, the mammalian cells are ARPE-19 cells and the promoter operably linked to a human precursor ARSB coding sequence consists essentially of, or consists of, SEQ ID NO:16 or a nucleotide sequence that is substantially identical to SEQ ID NO:18, e.g., is at least 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:16. In an embodiment, the promoter consists of SEQ ID NO:16.
In an embodiment, the ARSSB precursor protein comprises the mature amino acid sequence from a wild-type human ARSB protein, e.g., 37-533 of SEQ ID NO:1 or a conservatively substituted variant thereof. In an embodiment, the conservatively substituted variant has no more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 conservative substitutions. In an embodiment, the ARSB precursor protein consists of SEQ ID NO: 1.
In an embodiment, the nucleotide sequence encodes a human precursor ARSB fusion protein. The ARSB fusion protein may comprise a signal peptide from a secretory protein other than ARSB operatively linked to an amino acid sequence for mature human ARSB or a conservatively substituted variant thereof. In an embodiment, a conservatively substituted variant of a signal peptide has no more than three, two or one conservative substitutions. In an embodiment, the signal peptide consists of, or consists essentially of, SEQ ID NO:9 or a conservatively substituted variant thereof. In an embodiment, the coding sequence for the signal peptide is SEQ ID NO:10 or a nucleotide sequence that is substantially identical to SEQ ID NO:10, e.g., is at least 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:10.
In an embodiment, the ARSB fusion protein comprises an ARSB wild-type or variant amino acid sequence operatively linked to an amino acid sequence encoding a heterologous polypeptide. The heterologous polypeptide can be any protein or protein domain that confers a longer half-life or other desired property to the fusion protein.
In an embodiment, the isolated polynucleotide comprises a first expression cassette (also referred to as a transcription unit), which further comprises a mammalian Kozak translation sequence immediately upstream of the ATG start codon in the precursor ARSB coding sequence. In an embodiment the Kozak translation sequence is GCCACC. In an embodiment, the expression cassette further comprises a polyA sequence that consists essentially of, or consists of, nucleotides 3175-3696 of SEQ ID NO:15 or a nucleotide sequence that is substantially identical thereto, e.g., is at least 95%, 96%, 97%, 98%, 99% or more identical. In an embodiment, the isolated polynucleotide comprises two, three or more expression cassettes. In an embodiment, the expression cassette(s) are located between a pair of inverted terminal repeats, e.g., a 5′ ITR and a 3′ ITR.
In an embodiment, at least one expression cassette in the polynucleotide comprises a coding sequence for a sialytransferase (ST) protein. In some embodiments, the expression cassette is a second expression cassette, that is different than the first expression cassette. In an embodiment, the ST coding sequence is operably linked to a medium-strength promoter, e.g., nucleotides 3394-3625 of SEQ ID NO:20. In an embodiment the expression cassette comprises nucleotides 3394-5240 of SEQ ID NO:20.
In an embodiment, at least one expression cassette in the polynucleotide is a bicistronic expression cassette which comprises a coding sequence for the mammalian precursor ARSB protein and a coding sequence for the ST protein operably linked to a single promoter sequence and a single polyA signal sequence. The polypeptide includes a cleavage site positioned between the two coding sequences that allows expression of more than one polypeptide from a bicistronic expression cassette. Examples of protein cleavage sites that allow expression of more than one polypeptide are described in Klump et al. Gene Ther.; 8:811 1 (2001), Osborn et al., Molecular Therapy 12:569 (2005), Szymczak and, Vignali, Expert Opin Biol Ther. 5:627 (2005), and Szymczak et al., Nat Biotechnol. 22:589 (2004). In an embodiment, a 2A peptide is positioned between the ARSB and ST coding sequences. In an embodiment, an IRES sequence is positioned between the ARSB ST coding sequences. The ARSB coding sequence may be upstream or downstream of the ST coding sequence. In an embodiment, the ARSB coding sequence is upstream of the ST coding sequence.
In an embodiment, the isolated polynucleotide comprises one of the transposons described in Tables 2A, 2B, 2C and 2D below.
The isolated polynucleotides described above are useful to generate an engineered mammalian cell that expresses and secretes a mammalian ARSB protein and optionally co-express a sialyltransferase protein. The engineered cell may be derived from a variety of different mammalian cell types (e.g., human cells), including adipose cells, epidermal cells, epithelial cells, endothelial cells, fibroblast cells, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, pericytes, keratinocyte cells, subtypes of any of the foregoing and cells derived from any of the foregoing. Exemplary cell types include the cell types recited in WO 2017/075631. In some embodiments, the cells are derived from a cell-line shown in Table 3 below.
In an embodiment, any of the engineered mammalian cells described herein is derived from an RPE cell, e.g., an ARPE-19 cell. In an embodiment, an engineered RPE cell (e.g., an engineered ARPE-19 cell) comprises any of the expression cassettes, transposons and polynucleotides described herein.
Engineered mammalian cells for use in devices, compositions and methods described herein, e.g., as a plurality of engineered cells contained or encapsulated in a hydrogel capsule, may be in various stages of the cell cycle. In some embodiments, at least one engineered cell in the plurality of engineered cells is undergoing cell division. Cell division may be measured using any known method 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 incorporated by reference in its entirety. In an embodiment at least 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, e.g., as determined by 5-ethynyl-2′deoxyuridine (EdU) assay or 5-bromo-2′-deoxyuridine (BrdU) assay. In some embodiments, cell proliferation is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry. In some embodiments, none of the engineered cells in the plurality of engineered cells are undergoing cell division and are quiescent. In an embodiment, less than 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, 5-ethynyl-2′deoxyuridine (EdU) assay, 5-bromo-2′-deoxyuridine (BrdU) assay, microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation), or flow cytometry.
In an embodiment, at least 50%, 60%, 70%, 80%, 90% or more of the engineered cells in the plurality are viable. Cell viability may be measured using any known method 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 may be measured or quantified by an ATP assay, 5-ethynyl-2′deoxyuridine (EdU) assay, 5-bromo-2′-deoxyuridine (BrdU) assay. In some embodiments, cell viability is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry. In an embodiment, at least 80% of the engineered cells in the plurality are viable, e.g., as determined by an ATP assay, a 5-ethynyl-2′deoxyuridine (EdU) assay, a 5-bromo-2′-deoxyuridine (BrdU) assay, microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation), or flow cytometry.
Any of the parameters described herein may be assessed using standard techniques known to one of skill in the art, such as histology, microscopy, and various functional assays.
The activity of ARSB secreted by engineered cells or devices described herein may be measured by any direct or indirect ARSB activity assay known in the art. In an embodiment, ARSB activity may be measured using one or both of the in vitro fluorogenic activity assay and cell-based functional assay described in the Examples below.
An engineered ARSB-secreting cell described herein or a plurality of such cells may be incorporated into an implantable device for use in providing a mammalian ARSB protein to a subject, e.g., to a patient with MPS VI.
An implantable device of the present disclosure comprises at least one barrier that prevents immune cells from contacting cells contained inside the device. At least a portion of the barrier needs to be sufficiently porous to allow proteins (e.g., the ARSB protein) expressed and secreted by the cells to exit the device. A variety of device configurations known in the art are suitable.
The device (e.g., particle) can have any configuration and shape appropriate for supporting the viability and productivity of the contained cells after implant into the intended target location. As non-limiting examples, device shapes may be cylinders, rectangles, disks, ovoids, stellates, or spherical. The device can be comprised of a mesh-like or nested structure. In some embodiments, a device is capable of preventing materials over a certain size from passing through a pore or opening. In some embodiments, a device (e.g., particle) is capable of preventing materials greater 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, or 1,000 kD from passing through.
In an embodiment, the device is a macroencapsulation device. Nonlimiting examples of macrodevices are described in: WO 2019/068059, WO 2019/169089, U.S. Pat. Nos. 9,526,880, 9,724,430 and 8,278,106; European Patent No. EP742818B1, and Sang, S. and Roy, S., Biotechnol. Bioeng. 113 (7): 1381-1402 (2016).
In an embodiment, the device is a macrodevice having one or more cell-containing compartments. A device with two or more cell-containing compartments may be configured to produce two or more proteins, e.g., cells expressing the mammalian ARSB protein and optionally an ST protein would be placed in one compartment and cells expressing a different protein (e.g., a therapeutic protein that can alleviate one or more symptoms of MPS VI) would be placed in a separate compartment. WO 2018/232027 describes a device with multiple cell-containing compartments formed in a micro-fabricated body and covered by a porous membrane.
In an embodiment, the device is configured as a thin, flexible strand as described in U.S. Pat. No. 10,493,107. This strand comprises a substrate, an inner polymeric coating surrounding the substrate and an outer hydrogel coating surrounding the inner polymeric coating. The protein-expressing cells are positioned in the outer coating.
In some embodiments, a device (e.g., particle) has a largest linear dimension (LLD), e.g., mean diameter, or size that is at least about 0.5 millimeter (mm), preferably about 1.0 mm, about 1.5 mm or greater. In some embodiments, a device can be as large as 10 mm in diameter or size.
For example, a device or particle described herein is in a size range of 0.5 mm to 10 mm, 1 mm to 10 mm, 1 mm to 8 mm, 1 mm to 6 mm, 1 mm to 5 mm, 1 mm to 4 mm, 1 mm to 3 mm, 1 mm to 2 mm, 1 mm to 1.5 mm, 1.5 mm to 8 mm, 1.5 mm to 6 mm, 1.5 mm to 5 mm, 1.5 mm to 4 mm, 1.5 mm to 3 mm, 1.5 mm to 2 mm, 2 mm to 8 mm, 2 mm to 7 mm, 2 mm to 6 mm, 2 mm to 5 mm, 2 mm to 4 mm, 2 mm to 3 mm, 2.5 mm to 8 mm, 2.5 mm to 7 mm, 2.5 mm to 6 mm, 2.5 mm to 5 mm, 2.5 mm to 4 mm, 2.5 mm to 3 mm, 3 mm to 8 mm, 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, a device of the disclosure (e.g., particle, capsule) comprises at least one pore or opening, e.g., to allow for the free flow of materials. In some embodiments, the mean pore size of a device is between about 0.1 μm to about 10 μm. For example, the mean pore size may be between 0.1 μm to 10 μm, 0.1 μm to 5 μm, 0.1 μm to 2 μm, 0.15 μm to 10 μm, 0.15 μm to 5 μm, 0.15 μm to 2 μm, 0.2 μm to 10 μm,0.2 μm to 5 μm, 0.25 μm to 10 μm, 0.25 μm to 5 μm, 0.5 μm to 10 μm,0.75 μm to 10 μm, 1μm to 10 μm, 1 μm to 5 μm, 1 μm to 2 μm, 2 μm to 10 μm, 2 μm to 5 μm, or 5 μm to 10 μm. In some embodiments, the mean pore size of a device is between about 0.1 μm to 10 μm. In some embodiments, the mean pore size of a device is between about 0.1 μm to 5 μm. In some embodiments, the mean pore size of a device is between about 0.1 μm to 1 μm.
In some embodiments, the device comprises a semi-permeable, biocompatible membrane surrounding the genetically modified cells that are encapsulated in a polymer composition (e.g., an alginate hydrogel). The membrane pore size is selected to allow oxygen and other molecules important to cell survival and function to move through the semi-permeable membrane while preventing immune cells from traversing through the pores. In an embodiment, the semi-permeable membrane has a molecular weight cutoff of less than 1000 kD or between 50-700 kD, 70-300 kD, or between 70-150 kD, or between 70 and 130 kD.
In an embodiment, the device may contain a cell-containing compartment that is surrounded with a barrier compartment formed from a cell-free biocompatible material, such as the core-shell microcapsules described in Ma, M et al., Adv. Healthc Mater., 2(5):667-672 (2012). Such a barrier compartment could be used with or without the semi-permeable membrane.
Cells in the cell-containing compartment(s) of a device of the disclosure may be encapsulated in a polymer composition. The polymer composition may comprise one or more hydrogel-forming polymers. In addition to the polymer composition in the cell-containing compartment(s), the device (e.g., macrodevice, particle, hydrogel capsule) may comprise or be formed from materials such as metals, metallic alloys, ceramics, polymers, fibers, inert materials, and combinations thereof. A device may be completely made up of one type of material, or may comprise other materials within the cell-containing compartment and any other compartments.
In some embodiments, the device comprises a metal or a metallic alloy. In an embodiment, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all compartments) comprises a metal or a metallic alloy. Exemplary metallic or metallic alloys include comprising titanium and titanium group alloys (e.g., nitinol, nickel titanium alloys, thermo-memory alloy materials), platinum, platinum group alloys, stainless steel, tantalum, palladium, zirconium, niobium, molybdenum, nickel-chrome, chromium molybdenum alloys, or certain cobalt alloys (e.g., cobalt-chromium and cobalt-chromium-nickel alloys, e.g., ELGILOY® and PHYNOX®). For example, a metallic material may be stainless steel grade 316 (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). In metal-containing devices, the amount of metal (e.g., by % weight, actual weight) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
In some embodiments, the device comprises a ceramic. In an embodiment, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all compartments) comprises 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, may also be used. In ceramic-containing devices, the amount of ceramic (e.g., by % weight, actual weight) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
In some embodiments, the device has two hydrogel compartments, in which the inner, cell-containing compartment is completely surrounded by the second, outer (e.g., barrier) compartment. In an embodiment, the inner boundary of the second compartment forms an interface with the outer boundary of the first compartment. In such embodiments, the thickness of the second (outer) compartment means the average distance between the outer boundary of the second compartment and the interface between the two compartments, e.g., the average of the distances measured at each of the thinnest and thickest points visually observed in the outer compartment. In some embodiments (e.g., the device is about 1.5 mm in diameter), the thinnest and thickest distances for the outer compartment are between 25 and 110 micrometers (μm) and between 270 and 480 μm, respectively. In some embodiments, the thickness of the outer compartment is greater than about 10 nanometers (nm), preferably 100 nm or greater and can be as large as 1 millimeter (mm). For example, the thickness (e.g., average distance) of the outer compartment in a hydrogel capsule device described herein may be 10 nm to 1 mm, 100 nm to 1mm, 500 nm to 1 millimeter, 1 micrometer (μm) to 1 mm, 1 μm to 1 mm, 1μm to 500 μm, 1 μm to 250 μm, 1 μm to 1 mm, 5 μm to 500 μm, 5 μm to 250 μm, 10 μm to 1 mm, 10 μm to 500 μm, or 10 μm to 250 μm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is 100 nm to 1 mm, between 1 μm and 1 mm, between 1 μm and 500 μm or between 5 μm and 1 mm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is between about 50 μm and about 100 μm. In some embodiments (e.g., the device is about 1.5 mm in diameter), the thickness of the outer compartment (e.g., average distance) is between about 180 μm and 260 μm or between about 310 μm and 440 μm.
In some embodiments of a two-compartment hydrogel capsule device, the mean pore size of the cell-containing inner compartment and the outer compartment is substantially the same. In some embodiments, the mean pore size of the inner compartment and the second compartment differ by about 1.5%, 2%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some embodiments, the mean pore size of the device (e.g., mean pore size of the first compartment and/or mean pore size of the second compartment) is dependent on a number of factors, such as the material(s) within each compartment and the presence and density of a compound of Formula (I).
In some embodiments, the polymer composition in the cell-containing compartment(s) comprises a polysaccharide or other hydrogel-forming polymer (e.g., alginate, hyaluronate or chondroitin). In some embodiments, the polymer is an alginate, which is a polysaccharide made up of β-D-mannuronic acid (M) and α-L-guluronic acid (G). In some embodiments, the alginate has a low molecular weight (e.g., approximate molecular weight of <75 kD) and G:M ratio ≥1.5, (ii) a medium molecular weight alginate, e.g., has approximate molecular weight of 75-150 kDa and G:M ratio ≥1.5, (iii) a high molecular weight alginate, e.g., has an approximate MW of 150 kDa-250 kDa and G:M ratio ≥1.5, (iv) or a blend of two or more of these alginates. In some embodiments, the cell-containing compartment(s) further comprises at least one cell-binding substance (CBS), e.g., a cell-binding peptide (CBP) or cell-binding polypeptide (CBPP) described in WO2020069429.
In some embodiments, the cell-containing compartment(s) comprises an alginate covalently modified with a linker-cell-binding peptide moiety, e.g., GRGD or GRGDSP. In an embodiment, the cell-binding peptide density in the cell-containing compartment(s) (% nitrogen as determined by combustion analysis, e.g., as described in WO2020198695) to be at least 0.05%, 0.1%, 0.2% or 0.3% but less than 4%, 3%, 2% or 1%. In an embodiment, the total density of the linker-CBP in a cell containing compartment is about 0.1 to about 1.0 micromoles of the CBP per g of CBP-polymer (e.g., a MMW-alginate covalently modified with GRGD or GRGDSP in solution as determined by a quantitative peptide conjugation assay, e.g., an assay described in WO2020198695. In an embodiment, the linker-CBP is GRGDSP and the alginate has a molecular weight of 75 kDa to 150 kDa and a G:M ratio of greater than or equal to 1.5. In an embodiment, the cell-containing compartment also comprises an unmodified alginate with a molecular weight of 75 kDa to 150 kDa and a G:M ratio of greater than or equal to 1.5.
The device may form part of a plurality of substantially the same devices in a preparation (e.g., composition). In some embodiments, the devices (e.g., particles, hydrogel capsules) in the preparation have a mean diameter or size between about 0.5 mm to about 8 mm. In some embodiments, the mean diameter or size of devices in the preparation is between about 0.5 mm to about 4 mm or between about 0.5 mm to about 2 mm. In some embodiments, the devices in the preparation are two-compartment hydrogel capsules and have a mean diameter or size of about 0.7 mm to about 1.3 mm or about 1.2 mm to about 1.8 mm.
In some embodiments, the surface of the device comprises a compound capable of mitigating the FBR upon implant into a subject, an afibrotic compound as described herein below. For devices comprising a barrier compartment surrounding the cell-containing compartment, the afibrotic compound may covalently modify a polymer disposed throughout the barrier compartment and optionally throughout the cell-containing compartment.
In some embodiments, one or more compartments in a device comprises an afibrotic polymer, e.g., an afibrotic compound of Formula (I) covalently attached to a polymer. In an embodiment, some or all the monomers in the afibrotic polymer are modified with the same compound of Formula (I). In some embodiments, some or all the monomers in the afibrotic polymer are modified with different compounds of Formula (I). In some embodiments in which the device is a 2-compartment hydrogel capsule, the afibrotic polymer is present only in the outer, barrier compartment.
One or more compartments in a device may comprise an unmodified polymer that is the same or different than the polymer in any afibrotic polymer that is present in the device. In an embodiment, the first compartment, second compartment or all compartments in the device comprise the unmodified polymer.
Each of the modified and unmodified polymers in the device may be a linear, branched, or cross-linked polymer, or a polymer of selected molecular weight ranges, degree of polymerization, viscosity or melt flow rate. Branched polymers can include one or more of the following types: star polymers, comb polymers, brush polymers, dendronized polymers, ladders, and dendrimers. A polymer may be a thermoresponsive polymer, e.g., gel (e.g., becomes a solid or liquid upon exposure to heat or a certain temperature) or a photocrosslinkable polymer. Exemplary polymers include polystyrene, polyethylene, polypropylene, polyacetylene, poly(vinyl chloride) (PVC), polyolefin copolymers, poly(urethane)s, polyacrylates and polymethacrylates, polyacrylamides and polymethacrylamides, poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), polyesters, polysiloxanes, polydimethylsiloxane (PDMS), polyethers, poly(orthoester), poly(carbonates), poly(hydroxyalkanoate)s, polyfluorocarbons, PEEK®, Teflon® (polytetrafluoroethylene, PTFE), PEEK, silicones, epoxy resins, Kevlar®, Dacron® (a condensation polymer obtained from ethylene glycol and terephthalic acid), polyethylene glycol, nylon, polyalkenes, phenolic resins, natural and synthetic elastomers, adhesives and sealants, polyolefins, polysulfones, polyacrylonitrile, biopolymers such as polysaccharides and natural latex, collagen, cellulosic polymers (e.g., alkyl celluloses, etc.), polyethylene glycol and 2-hydroxyethyl methacrylate (HEMA), polysaccharides, poly(glycolic acid), poly(L-lactic acid) (PLLA), poly(lactic glycolic acid) (PLGA), a polydioxanone (PDA), or racemic poly(lactic acid), polycarbonates, (e.g., polyamides (e.g., nylon)), fluoroplastics, carbon fiber, agarose, alginate, chitosan, and blends or copolymers thereof. In polymer-containing devices, the amount of a polymer (e.g., by % weight of the device, actual weight of the polymer) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
In some embodiments, one or more of the modified and unmodified polymers in the device comprises a polyethylene. Exemplary polyethylenes include ultra-low-density polyethylene
(ULDPE) (e.g., with polymers with densities ranging from 0.890 to 0.905 g/cm3, containing comonomer); very-low-density polyethylene (VLDPE) (e.g., with polymers with densities ranging from 0.905 to 0.915 g/cm3, containing comonomer); linear low-density polyethylene (LLDPE) (e.g., with polymers with densities ranging from 0.915 to 0.935 g/cm3, contains comonomer); low-density polyethylene (LDPE) (e.g., with polymers with densities ranging from about 0.915 to 0.935 g/m3); medium density polyethylene (MDPE) (e.g., with polymers with densities ranging from 0.926 to 0.940 g/cm3, may or may not contain comonomer); high-density polyethylene (HDPE) (e.g., with polymers with densities ranging from 0.940 to 0.970 g/cm3, may or may not contain comonomer) and polyethylene glycol.
In some embodiments, one or more of the modified and unmodified polymers in the device comprises 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.
In some embodiments, one or more of the modified and unmodified polymers in the device comprises a polypropylene. Exemplary polystyrenes include general purpose or crystal (PS or GPPS), high impact (HIPS), and syndiotactic (SPS) polystyrene.
In some embodiments, one or more of the modified and unmodified polymers comprises a comprises a thermoplastic elastomer (TPE). Exemplary TPEs include (i) TPA—polyamide TPE, comprising a block copolymer of alternating hard and soft segments with amide chemical linkages in the hard blocks and ether and/or ester linkages in the soft blocks; (ii) TPC—co-polyester TPE, consisting of a block copolymer of alternating hard segments and soft segments, the chemical linkages in the main chain being ester and/or ether; (iii) TPO—olefinic TPE, consisting of a blend of a polyolefin and a conventional rubber, the rubber phase in the blend having little or no cross-linking; (iv) TPS—styrenic TPE, consisting of at least a triblock copolymer of styrene and a specific diene, where the two end blocks (hard blocks) are polystyrene and the internal block (soft block or blocks) is a polydiene or hydrogenated polydiene; (v) TPU—urethane TPE, consisting of a block copolymer of alternating hard and soft segments with urethane chemical linkages in the hard blocks and ether, ester or carbonate linkages or mixtures of them in the soft blocks; (vi) TPV—thermoplastic rubber vulcanizate consisting of a blend of a thermoplastic material and a conventional rubber in which the rubber has been cross-linked by the process of dynamic vulcanization during the blending and mixing step; and (vii) TPZ—unclassified TPE comprising any composition or structure other than those grouped in TPA, TPC, TPO, TPS, TPU, and TPV.
In some embodiments, the unmodified polymer is an unmodified alginate. In some embodiments, the alginate is a high guluronic acid (G) alginate, and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more guluronic acid (G). In some embodiments, the alginate is a high mannuronic acid (M) alginate, and comprises greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more mannuronic acid (M). In some embodiments, the ratio of M:G is about 1. In some embodiments, the ratio of M:G is less than 1. In some embodiments, the ratio of M:G is greater than 1. In an embodiment, the unmodified alginate has a molecular weight of 150 kDa-250 kDa and a G:M ratio of ≥1.5.
In some embodiments, afibrotic polymer comprises an alginate chemically modified with a Compound of Formula (I). The alginate in the afibrotic polymer may be the same or different than any unmodified alginate that is present in the device. In an embodiment, the density of the Compound of Formula (I) in the afibrotic alginate (e.g., amount of conjugation) is between about 4.0% and about 8.0%, between about 5.0% and about 7.0%, or between about 6.0% and about 7.0% nitrogen (e.g., as determined by combustion analysis for percent nitrogen). In an embodiment, the amount of Compound 101 produces an increase in % N (as compared with the unmodified alginate) of about 0.5% to 2% 2% to 4% N, about 4% to 6% N, about 6% to 8%, or about 8% to 10% N), where % N is determined by combustion analysis and corresponds to the amount of Compound 101 in the modified alginate.
In other embodiments, the density (e.g., concentration) of the Compound of Formula (I) (e.g., Compound 101) in the afibrotic alginate is defined as the % w/w, e.g., % of weight of amine/weight of afibrotic alginate in solution (e.g., saline) as determined by a suitable quantitative amine conjugation assay (e.g. by an assay described in WO2020069429), and in certain embodiments, the density of a Compound of Formula (I) (e.g., Compound 101) is between about 1.0% w/w and about 3.0% w/w, between about 1.3% w/w and about 2.5 w/w or between about 1.5 w/w and 2.2% w/w.
In alginate-containing devices, the amount of modified and unmodified alginates (e.g., by % weight of the device, actual weight of the alginate) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., w/w; less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less.
The alginate in an afibrotic polymer can be chemically modified with a compound of Formula (I) using any suitable method known in the art. For example, the alginate carboxylic acid moiety can be activated for coupling to one or more amine-functionalized compounds to achieve an alginate modified with a compound of Formula (I). The alginate polymer may be dissolved in water (30 mL/gram polymer) and treated with 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 eq) and N-methylmorpholine (1 eq). To this mixture may be added a solution of the compound of Formula (I) in acetonitrile (0.3M). The reaction may be warmed to 55° C. for 16 h, then cooled to room temperature and gently concentrated via rotary evaporation, then the residue may be dissolved, e.g., in water. The mixture may then be filtered, e.g., through a bed of cyano-modified silica gel (Silicycle) and the filter cake washed with water. The resulting solution may then be dialyzed (10,000 MWCO membrane) against water for 24 hours, e.g., replacing the water twice. The resulting solution can be concentrated, e.g., via lyophilization, to afford the desired chemically modified alginate.
In an embodiment, the device comprises at least one cell-containing compartment, and in some embodiments contains two, three, four or more cell-containing compartments. In an embodiment, each cell-containing compartment comprises a plurality of cells (e.g., live cells) and the cells in at least one of the compartments are capable of expressing and secreting a mammalian ARSB protein when the device is implanted into a subject. In some embodiments, the cells in a single cell-containing compartment co-express a human precursor ARSB protein and a human ST protein.
In an embodiment, all the cells in a cell-containing compartment are derived from a single parental cell-type or a mixture of at least two different parental cell types. In an embodiment, all of the cells in a cell-containing compartment are derived from the same parental cell type, but a first plurality of the derived cells are engineered to express the ARSB protein and optionally an ST protein, and a second plurality of the derived cells are engineered to express a different therapeutic protein. In devices with two or more cell-containing compartments, the cells and the protein(s) produced thereby may be the same or different in each cell-containing compartment. In some embodiments, all of the cell-containing compartments are surrounded by a single barrier compartment. In some embodiments, the barrier compartment is substantially cell-free.
In an embodiment, cells to be incorporated into a device described herein, e.g., a hydrogel capsule, are prepared in the form of a cell suspension prior to being encapsulated within the device. The cells in the suspension may take the form of single cells (e.g., from a monolayer cell culture), or provided in another form, e.g., disposed on a microcarrier (e.g., a bead or matrix) or as a three-dimensional aggregate of cells (e.g., a cell cluster or spheroid). The cell suspension can comprise multiple cell clusters (e.g., as spheroids) or microcarriers.
In addition to the ARSB protein secreted by the encapsulated cells, a device (e.g., capsule, particle) may comprise one or more exogenous agents that are not expressed by the cells, and may include, e.g., a nucleic acid (e.g., an RNA or DNA molecule), a protein (e.g., a hormone, an enzyme (e.g., glucose oxidase, kinase, phosphatase, oxygenase, hydrogenase, reductase) antibody, antibody fragment, antigen, or epitope)), an active or inactive fragment of a protein or polypeptide, a small molecule, or drug. In an embodiment, the device is configured to release such an exogenous agent.
In some embodiments, the devices described herein comprise at least one compound of Formula (I):
or a pharmaceutically acceptable salt thereof, wherein:
A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, —O—, —C(O)O—, —C(O)—, —OC(O)—, —N(RC)—, —N(RC)C(O)—, —C(O)N(RC)—, —N(RC)C(O)(1-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)—, —Si(ORA)2—, —Si(RG)(ORA)—, —B(ORA)—, or a metal, each of which is optionally linked to an attachment group (e.g., an attachment group described herein) 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 bond;
M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted by one or more R3;
P is absent, 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, wherein 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, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R6;
or RC and RD, taken together with the nitrogen atom to which they are attached, form a ring (e.g., a 5-7 membered ring), optionally substituted with 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, wherein 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, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted 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.
In some embodiments, the compound of Formula (I) is a compound of Formula (I-a):
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)N(RD)—, N(RC)C(O)(C1-C6-alkylene)-, —N(RC)C(O)(C1-C6-alkenylene)-, —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, each of which is optionally linked to an attachment group (e.g., an attachment group described herein) and 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 bond;
M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted by one or more R3;
P is heteroaryl optionally substituted by one or more R4;
Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which 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, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R6;
or RC and RD, taken together with the nitrogen atom to which they are attached, form a ring (e.g., a 5-7 membered ring), optionally substituted with 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), —(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, wherein 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, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted 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.
In some embodiments, for Formulas (I) and (I-a), 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-alkenylene)-, 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)(C1-C6-alkylene)-, or —N(RC)C(O)(C1-C6-alkenylene)-. In some embodiments, A is —N(RC)—. In some embodiments, A is —N(RC)—, and RC is independently hydrogen or alkyl. In some embodiments, A is —NH—. In some embodiments, A is —N(RC)C(O)(C1-C6-alkylene)-, wherein alkylene is substituted with In some embodiments, A is —N(RC)C(O)(C1-C6-alkylene)-, and R1 is alkyl (e.g., methyl). In some embodiments, A is —NHC(O)C(CH3)2—. In some embodiments, A is —N(RC)C(O)(methylene)-, and le is alkyl (e.g., methyl). In some embodiments, A is —NHC(O)CH(CH3)—. In some embodiments, A is —NHC(O)C(CH3)—.
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 bond. In some embodiments, L1 is alkyl. In some embodiments, L1 is C1-C6 alkyl. In some embodiments, L1 is —CH2—, —CH(CH3)—, —CH2CH2CH2, or —CH2CH2—. In some embodiments, L3 is —CH2CH2—. In some embodiments, L1 is —CH2— or —CH2CH2—.
In some embodiments, for Formulas (I) and (I-a), L3 is a bond, alkyl, or heteroalkyl. In some embodiments, L3 is a bond. In some embodiments, L3 is alkyl. In some embodiments, L3 is C1-C12 alkyl. In some embodiments, L3 is C1-C6 alkyl. In some embodiments, L3 is —CH2—. In some embodiments, L3 is heteroalkyl. In some embodiments, L3 is C1-C12 heteroalkyl, optionally substituted with one or more R2 (e.g., oxo). In some embodiments, L3 is C1-C6 heteroalkyl, optionally substituted with one or more R2 (e.g., oxo). In some embodiments, L3 is —C(O)OCH2—, —CH2(OCH2CH2)2—, —CH2(OCH2CH2)3—, CH2CH2O—, or —CH2O—. In some embodiments, L3 is —CH2O—.
In some embodiments, for Formulas (I) and (I-a), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, for Formulas (I) and (I-a), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, M is heteroalkyl, aryl, or heteroaryl. In some embodiments, M is absent. In some embodiments, M is alkyl (e.g., C1-C6 alkyl). In some embodiments, M is —CH2—. In some embodiments, M is heteroalkyl (e.g., C1-C6 heteroalkyl). In some embodiments, M is (—OCH2CH2—)z, wherein z is an integer selected from 1 to 10. In some embodiments, z is an integer selected from 1 to 5. In some embodiments, M is —(OCH2)2—, (—OCH2CH2—)2, (—OCH2CH2—)3, (—OCH2CH2—)4, or (—OCH2CH25. In some embodiments, M is —OCH2CH2—, (—OCH2CH2—)2, (—OCH2CH2—)3, or (—OCH2CH2—)4. In some embodiments, M is (—OCH2—)3. In some embodiments, M is aryl. In some embodiments, M is phenyl. In some embodiments, M is unsubstituted phenyl. In some embodiments, M is
In some embodiments, M is
In some embodiments, M is phenyl substituted with 1-4 R3 (e.g., 1 R3). In some embodiments, R3 is CF3.
In some embodiments, for Formulas (I) and (I-a), P is absent, heterocyclyl, or heteroaryl. In some embodiments, for Formulas (I) and (I-a), P is absent, heterocyclyl, or heteroaryl. In some embodiments, P is absent. In some embodiments, 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 embodiments, P is a monocyclic, nitrogen-containing heteroaryl. In some embodiments, P is a 5-membered heteroaryl. In some embodiments, P is a 5-membered nitrogen-containing heteroaryl. In some embodiments, P is tetrazolyl, imidazolyl, pyrazolyl, or triazolyl, or pyrrolyl. In some embodiments, P is imidazolyl. In some embodiments, P is 1,2,3-triazolyl. In some embodiments, P is
In some embodiments, P is
In some embodiments, P is
In some embodiments, P is heterocyclyl. In some embodiments, P is heterocyclyl. In some embodiments, P is a 5-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some embodiments, P is
In some embodiments, P is thiomorpholinyl-1,1-dioxidyl. In some embodiments, P is
In some embodiments, for Formulas (I) and (I-a), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. 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, 5-membered heterocyclyl, or 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
In some embodiments, Z is a 4-membered oxygen-containing heterocyclyl.
In some embodiments, Z is
In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments, Z is phthalic 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 embodiments, Z is
In some embodiments, Z is a nitrogen-containing heterocyclyl. In some embodiments, Z is a 6-membered nitrogen-containing heterocyclyl. In some embodiments, Z is
In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic nitrogen-containing heterocyclyl, optionally substituted with one or more R5. In some embodiments, Z is 2-oxa-7-azaspiro[3.5]nonanyl In some embodiments, Z is
In some embodiments, Z is 1-oxa-3,8-diazaspiro[4.5]decan-2-one. In some embodiments, Z is
In some embodiments, 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 (e.g., with 1 R5). In some embodiments, Z is monosubstituted phenyl, wherein the 1 R5 is a nitrogen-containing group. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R5 is NH2. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R5 is an oxygen-containing group. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R5 is an oxygen-containing heteroalkyl. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R5 is OCH3. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R5 is in the ortho position. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R5 is in the meta position. In some embodiments, Z is monosubstituted phenyl, wherein the 1 R5 is in the para position.
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. In some embodiments, Z is C1-C8 alkyl substituted with 1-5 R5. In some embodiments, Z is C1-C8 alkyl substituted with 1 R5. In some embodiments, Z is C1-C8 alkyl substituted with 1 R5, wherein R5 is alkyl, heteroalkyl, halogen, oxo, —ORA1, —C(O)ORA1, —C(O)RB1, —OC(O)RB1, or —N(RC1)(RD1). In some embodiments, Z is C1-C8 alkyl substituted with 1 R5, wherein R5 is —ORA1 or —C(O)ORA1. In some embodiments, Z is C1-C8 alkyl substituted with 1 R5, wherein R5 is —ORA1 or —C(O)OH. In some embodiments, Z is —CH3.
In some embodiments, for Formulas (I) and (I-a), Z is heteroalkyl. In some embodiments, Z is C1-C12 heteroalkyl. I n some embodiments, Z is C1-C10 heteroalkyl. In some embodiments, Z is C1-C8 heteroalkyl. I n some embodiments, Z is C1-C6 heteroalkyl. In some embodiments, Z is a nitrogen-containing heteroalkyl optionally substituted with one or more R5. In some embodiments, Z is a nitrogen and sulfur-containing heteroalkyl substituted with 1-5 R5. In some embodiments, Z is N-methyl-2-(m ethyl sulfonyl)ethan-l-aminyl .
In some embodiments, Z is —ORA or —C(O)ORA. In some embodiments, Z is —ORA (e.g., —OH or —OCH3). In some embodiments, Z is —OCH3. In some embodiments, Z is —C(O)ORA (e.g., —C(O)OH).
In some embodiments, Z is hydrogen.
In some embodiments, L2 is a bond 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, the compound of Formula (I) is a compound of Formula (I-b):
or a pharmaceutically acceptable salt thereof, wherein Ring M1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1-5 R3; Ring Z1 is cycloalkyl, heterocyclyl' aryl or heteroaryl, optionally substituted with 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 each of R2a and R2b or R2c and R2d is taken together to form an oxo group; X is absent, N(R10)(R11), O, or S; RC is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally substituted with 1-6 R6; each R3, R5, and R6 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, —ORA1, —C(O)OR A1, —C(O)RB1, —OC(O)RB1, —N(RC1)(RD1), —N(RC1)C(O)RB1, —C(O)N(RC1), SRE1, cycloalkyl, heterocyclyl, aryl, or heteroaryl; each of R10 and R11 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, —C(O)ORA1, —C(O)RB1, —OC(O)RB1, —C(O)N(RC1), cycloalkyl, heterocyclyl or heteroaryl; each RA1, RB1, RC1, RD1, and RE1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, wherein each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl is optionally substituted with 1-6 R7; each R7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; each m and n is independently 1, 2, 3, 4, 5, or 6; and “” refers to a connection to an attachment group or a polymer described herein. In some embodiments, for each R3 and R5, each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally and independently substituted with halogen, oxo, cyano, cycloalkyl, or heterocyclyl.
In some embodiments, the compound of Formula (I-b) is a compound of Formula (I-b-i):
or a pharmaceutically acceptable salt thereof, wherein Ring M2 is aryl or heteroaryl optionally substituted with one or more R3; Ring Z2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; 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; X is absent, O, or S; each R3 and R5 is independently alkyl, heteroalkyl, halogen, oxo, —ORA1, —C(O)ORA1, or —C(O)RB1, wherein each alkyl and heteroalkyl is optionally substituted with halogen; or two R5 are taken together to form a 5-6 membered ring fused to Ring Z2; each RA1 and RB1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I-b-i) is a compound of Formula (I-b-ii):
or a pharmaceutically acceptable salt thereof, wherein Ring Z2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; each of R2c and R2d is independently hydrogen, alkyl, or heteroalkyl, or R2c and R and taken together to form an oxo group; each R3 and R5 is independently alkyl, heteroalkyl, halogen, oxo, —ORA1, —C(O)ORA1, or —C(O)RB1, wherein each alkyl and heteroalkyl is optionally substituted with halogen; each RA1 and RB1 is independently hydrogen, alkyl, or heteroalkyl; m is 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4, 5, or 6; q is 0, 1, 2, 3, or 4; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (I-c):
or a pharmaceutically acceptable salt thereof, wherein Ring Z2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; each of R2c and R2d is independently hydrogen, alkyl, or heteroalkyl, or R2c and R2d is taken together to form an oxo group; each R3 and R5 is independently alkyl, heteroalkyl, halogen, oxo, —OR, —C(O)ORA1, or —C(O)RB1, wherein each alkyl and heteroalkyl is optionally substituted with halogen; each RA1 and RB1 is independently hydrogen, alkyl, or heteroalkyl; m is 1, 2, 3, 4, 5, or 6; each of p and q is independently 0, 1, 2, 3, 4, 5, or 6; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (I-d):
or a pharmaceutically acceptable salt thereof, wherein Ring Z2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, 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, wherein each alkyl and heteroalkyl is optionally substituted with halogen; each R A1 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 herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (I-e):
or a pharmaceutically acceptable salt thereof, wherein Ring Z2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; X is absent, 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 herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (I-f):
or a pharmaceutically acceptable salt thereof, wherein M is alkyl optionally substituted with one or more R3; Ring P is heteroaryl optionally substituted with one or more R4; L3 is alkyl or heteroalkyl optionally substituted with one or more R2; Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 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, R4, and R5 is independently alkyl, heteroalkyl, halogen, oxo, —ORA1, —C(O)ORA1, or —C(O)RB1; each RA and RB1 is independently hydrogen, alkyl, or heteroalkyl; n is 1, 2, 3, 4, 5, or 6; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (II):
or a pharmaceutically acceptable salt thereof, wherein M is a bond, alkyl or aryl, wherein alkyl and aryl is optionally substituted with one or more R3; L3 is alkyl or heteroalkyl optionally substituted with one or more R2; Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl or —OR5, wherein alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is optionally substituted with one or more R5; RA is hydrogen; 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 a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (II) is a compound of Formula (II-a):
or a pharmaceutically acceptable salt thereof, wherein L3 is alkyl or heteroalkyl, each of which is optionally substituted with one or more R2; Z is hydrogen, alkyl, heteroalkyl or —ORA, heteroalkyl are optionally substituted with 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 heteroalkyl, halogen, oxo, —ORA1, C(O)ORA1, or —C(O)RB1; RA is hydrogen, alkyl, or heteralkyl; each RA1 and RB1 is independently hydrogen, alkyl, or heteroalkyl; n is 1, 2, 3, 4, 5, or 6; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (III):
or a pharmaceutically acceptable salt thereof, wherein Z1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 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, R2c and R2d is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, wherein each of alkyl, alkenyl, alkynyl, or heteroalkyl is optionally substituted with 1-6 R6; each of R3, R5, and R6 is independently alkyl, heteroalkyl, halogen, oxo, —ORA1, or —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 herein.
In some embodiments, the compound of Formula (III) is a compound of Formula (III-a):
or a pharmaceutically acceptable salt thereof, wherein Ring Z2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1-5 R5; 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; m and 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; and “
” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (III-a) is a compound of Formula (III-b):
or a pharmaceutically acceptable salt thereof, wherein Ring Z2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1-5 R5; 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; m is 0, 1, 2, 3, 4, 5, or 6; n is 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, or 4; q is an integer from 0 to 25; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (III-a) is a compound of Formula (III-c):
or a pharmaceutically acceptable salt thereof, wherein X is C(R′)(R″), N(R′), or S(O)x; each of R′ and R″ is independently hydrogen, alkyl, halogen, or cycloalkyl; each of R2a, R2b, R2c, and R2d is independently hydrogen, alkyl, heteroalkyl, or halo; or Ra2 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; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4; q is an integer from 0 to 25; x is 0, 1, or 2; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (III-c) is a compound of Formula (III-d):
or a pharmaceutically acceptable salt thereof, wherein X is C(R′)(R″), N(R′), or S(O)x; each of R′ and R″ is independently hydrogen, alkyl, halogen, or cycloalkyl; each of R2a, R2b, R2c, and R2d is independently hydrogen, alkyl, heteroalkyl, or 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; m and n are each independently 1, 2, 3, 4, 5, or 6; p is 0, 1, 2, 3, 4; q is an integer from 0 to 25; x is 0, 1, or 2; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (III-e):
or a pharmaceutically acceptable salt thereof, wherein Z1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 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 each of R2a and R2b or R2c and R2d is taken together to form an oxo group; RC is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, wherein each of alkyl, alkenyl, alkynyl, or heteroalkyl is optionally substituted with 1-6 R6; each of R3, R5, and R6 is independently alkyl, heteroalkyl, halogen, oxo, —ORA1, —C(O)ORA1, or —C(O)RB1; each R12 is independently deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; 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; w is 0 or 1; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (III-f):
or a pharmaceutically acceptable salt thereof, wherein Ring Z1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1-5 R5; 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; RC is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, wherein each of alkyl, alkenyl, alkynyl, or heteroalkyl is optionally substituted with 1-6 R6; each of R3, R5, and R6 is independently alkyl, heteroalkyl, halogen, oxo, —ORA1, C(O)ORA1, or —C(O)RB1; each R12 is independently deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; each RA1 and RB1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; w is 0 or 1; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (III-g):
or a pharmaceutically acceptable salt thereof, wherein Ring Z1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is optionally substituted with 1-5 R5; RC is hydrogen, alkyl, —N(RC)C(O)RB, —N(RC)C(O)(C1-C6-alkyl), or —N(RC)C(O)(C1-C6-alkenyl), wherein each of alkyl and alkenyl is optionally substituted with 1-6 R6; R2a, R2b, R2c, and R2d is independently hydrogen or alkyl; or R2a and R2b or R2c and R2d are taken together to form an oxo group; each of R3, R5, and R6 is independently alkyl, heteroalkyl, halogen, oxo, —ORA1, —C(O)ORA1, or —C(O)RB1; R12 is hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; 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; x is 0, 1, or 2; and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (III-h):
or a pharmaceutically acceptable salt thereof, wherein RC is hydrogen, alkyl, —N(RC)C(O)RB, —N(RC)C(O)(C1-C6-alkyl), or —N(RC)C(O)(C1-C6-alkenyl), wherein each of alkyl and alkenyl is optionally substituted with 1-6 R6; each of R2a, R2b, R2c, and R2d is independently hydrogen or alkyl; or R2a and R2b or R2c and R2d are taken together to form an oxo group; each of R3, R5, and R6 is independently alkyl, heteroalkyl, halogen, oxo, —ORA1, —C(O)ORA1, or —C(O)RB1; R12 is hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; 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; x is 0, 1, or 2; z is 0, 1, 2, 3, 4, 5, or 6, and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, the compound of Formula (I) is a compound of Formula (III-i):
or a pharmaceutically acceptable salt thereof, wherein X is C(R′)(R″), N(R′), or S(O)x; each of R′ and R″ is independently hydrogen, alkyl, or halogen; RC is hydrogen, alkyl, —N(RC)C(O)RB, —N(RC)C(O)(C1-C6-alkyl), or —N(RC)C(O)(C1-C6-alkenyl), wherein each of alkyl and alkenyl is optionally substituted with 1-6 R6; each of R2a, R2b, R2c, and R2d is independently hydrogen or alkyl; or R2a and R2b or R2c and R2d are taken together to form an oxo group; each of R3, R5, and R6 is independently alkyl, heteroalkyl, halogen, oxo, —ORA1, —C(O)ORA1, or —C(O)RB1; R12 is hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; 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; x is 0, 1, or 2; z is 0, 1, 2, 3, 4, 5, or 6, and “” refers to a connection to an attachment group or a polymer described herein.
In some embodiments, X is S(O)x. In some embodiments, x is 2. In some embodiments, X is S(O)2.
In some embodiments, each of R2a, R2b, R2c, and R2d is independently hydrogen.
In some embodiments, RC is hydrogen, —C(O)(C1-C6-alkyl), or —C(O)(C1-C6-alkenyl). In some embodiments, each of alkyl and alkenyl is substituted with 1 R6 (e.g., —CH3). In some embodiments, RC is hydrogen.
In some embodiments, n is 1. In some embodiments, q is 2, 3, 4, or 5. In some embodiments, q is 3. In some embodiments, m is 1. In some embodiments, p is 0. In some embodiments, R12 is halo (e.g., Cl).
In some embodiments, the compound is a compound of Formula (I). In some embodiments, L2 is a bond and P and L3 are independently absent.
In some embodiments, the compound is a compound of Formula (I-a). In some embodiments of Formula (II-a), 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 bond 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, the compound is a compound of Formula (I-b). In some embodiments, P is absent, L1 is —NHCH2, L2 is a bond, M is aryl (e.g., phenyl), L3 is —CH2O, and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., thiomorpholinyl-1,1-dioxide). In some embodiments, the compound of Formula (I-b) is Compound 116.
In some embodiments of Formula (I-b), P is absent, L1 is —NHCH2, L2 is a bond, M is absent, L3 is a bond, and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (I-b) is Compound 105.
In some embodiments, the compound is a compound of Formula (I-b). In some embodiments of Formula (I-b), each of R2a and R2b is independently hydrogen or CH3, each of R2c and R2d is independently hydrogen, m is 1 or 2, n is 1, X is O, M1 is phenyl optionally substituted with one or more R3, R3 is —CF3, and Z1 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (I-b-i) is Compound 100, Compound 106, Compound 108, Compound 109, or Compound 111.
In some embodiments, the compound is a compound of Formula (I-b-i). In some embodiments of Formula (I-b-i), each of R2a and R2b is independently hydrogen or CH3, each of R2c and R2d is independently hydrogen, m is 1 or 2, n is 1, X is O, p is 0, M2 is phenyl optionally substituted with one or more R3, R3 is —CF3, and Z2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (I-b-i) is Compound 100, Compound 106, Compound 107, Compound 108, Compound 109, or Compound 111.
In some embodiments, the compound is a compound of Formula (I-b-ii). In some embodiments of Formula (I-b-ii), each of R2a, R2b, R2c, and R2d is independently hydrogen, q is 0, p is 0, m is 1, and Z2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl). In some embodiments, the compound of Formula (I-b-ii) is Compound 100.
In some embodiments, the compound is a compound of Formula (I-c). In some embodiments of Formula (I-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 (e.g., a nitrogen-containing heterocyclyl, e.g., piperazinyl). In some embodiments, the compound of Formula (I-c) is Compound 113.
In some embodiments, the compound is a compound of Formula (I-d). In some embodiments of Formula (I-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, e.g., tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (I-d) is Compound 110 or Compound 114.
In some embodiments, the compound is a compound of Formula (I-e). In some embodiments of Formula (I-e), each of R2a, R2b, R2c, and R2d is independently hydrogen, n is 1, m is 2, X is O, and Z2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, e.g., tetrahydropyranyl). In some embodiments, the compound of Formula (I-e) is Compound 107.
In some embodiments, the compound is a compound of Formula (I-f). In some embodiments of Formula (I-f), each of R2a and R2b is independently hydrogen, n is 1, M is —CH2—, P is a nitrogen-containing heteroaryl (e.g., imidazolyl), L3 is —C(O)OCH2—, and Z is CH3. In some embodiments, the compound of Formula (I-f) is Compound 115.
In some embodiments, the compound is a compound of Formula (II-a). In some embodiments of Formula (II-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 (II-a) is Compound 112.
In some embodiments of Formula (II-a), each of R2a and R2b is independently hydrogen, n is 1, L3 is a bond or —CH2, and Z is hydrogen or —OH. In some embodiments, the compound of Formula (II-a) is Compound 103 or Compound 104.
In some embodiments, the compound is a compound of Formula (III). In some embodiments of Formula (III), each of R2a, R2b, R2c, and R2d is independently hydrogen, m is 1, n is 2, q is 3, p is 0, RC is hydrogen, and Z1 is heteroalkyl optionally substituted with R5 (e.g., —N(CH3)(CH2CH2)S(O)2CH3). In some embodiments, the compound of Formula (III) is Compound 120.
In some embodiments, the compound is a compound of Formula (III-b). In some embodiments of Formula (III-b), each of R2a, R2b, R2c, and R2d is independently hydrogen, m is 0, n is 2, q is 3, p is 0, and Z2 is aryl (e.g., phenyl) substituted with 1 R5 (e.g., —NH2). In some embodiments, the compound of Formula (III-b) is Compound 102.
In some embodiments, the compound is a compound of Formula (III-a). In some embodiments of Formula (III-a), each of R2a, R2b, R2c, R2d, and RC is independently hydrogen, m is 1, n is 2, q is 4, p is 0, w is 0, and Z1 is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., thiomorpholinyl-1,1-dioxide). In some embodiments, the compound of Formula (III-a) is Compound 119.
In some embodiments, the compound is a compound of Formula (III-b). In some embodiments of Formula (III-b), each of R2a, R2b, R2c, and R2d is independently hydrogen, m is 1, n is 2, q is 3, p is 0, RC is hydrogen, and Z2 is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., a nitrogen-containing spiro heterocyclyl, e.g., 2-oxa-7-azaspiro[3.5]nonanyl). In some embodiments, the compound of Formula (III-b) is Compound 121.
In some embodiments, the compound is a compound of Formula (III-d). In some embodiments of Formula (III-d), each of R2a, R2b, R2c, and R2d is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O)2. In some embodiments of Formula (III-d), each of R2a and R2b is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O)2. In some embodiments, the compound of Formula (III-d) is Compound 101, Compound 117, Compound 118, or Compound 119.
In some embodiments, the compound is a compound of Formula (III-c). In some embodiments of Formula (III-c), each of R2a, R2b, R2c, R2d, RC and R12 is independently hydrogen, m is 1, n is 2, q is 3, p is 0, z is 1, and R5 is (e.g., —S(O)2(CH3)). In some embodiments, the compound of Formula (III-c) is Compound 123.
In some embodiments, the compound is a compound of Formula (I-b), (I-d), or (I-e). In some embodiments, the compound is a compound of Formula (I-b), (I-d), or (II). In some embodiments, the compound is a compound of Formula (I-b), (I-d), or (I-f). In some embodiments, the compound is a compound of Formula (I-b), (I-d), or (III).
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.
In some embodiments, the compound of Formula (I) comprises a compound shown in Table 4, or a pharmaceutically acceptable salt thereof. In some embodiments, the exterior surface and/or one or more compartments within a device described herein comprises a small molecule compound shown in Table 4, or a pharmaceutically acceptable salt thereof.
Conjugation of any of the compounds in Table 4 to a polymer (e.g., an alginate) may be performed as described in Example 2 of WO 2019/195055 or any other suitable chemical reaction.
In some embodiments, the compound is a compound of Formula (I) (e.g., Formulas (I-a), (I-b), (I-c), (I-d), (I-e), (I-f), (II), (II-a), (III), (III-a), (III-b), (III-c), or (III-d)), or a pharmaceutically acceptable salt thereof and is selected from:
or a pharmaceutically acceptable salt thereof.
In some embodiments, the device described herein comprises the compound of
or a pharmaceutically acceptable salt of either compound.
In some embodiments, a compound of Formula (I) (e.g., Compound 101 in Table 4) is covalently attached to an alginate (e.g., an alginate with approximate MW<75 kDa, G:M ratio ∝1.5) at a conjugation density of at least 2.0% and less than 9.0%, or 3.0% to 8.0%, 4.0-7.0, 5.0 to 7.0, or 6.0 to 7.0 or about 6.8 as determined by combustion analysis for percent nitrogen as described in WO 2020/069429. In an embodiment, the conjugation density of Compound 101 in the modified alginate is determined by quantitative free amine analysis, e.g., as described in WO2020198695, wherein the determined conjugation density is 1.0% w/w to 3.0% w/w, 1.3% w/w to 2.8% w/w, 1.3% w/w to 2.6% w/w, 1.5% w/w to 2.4% w/w, 1.5% w/w to 2.2% w/w, or 1.7% w/w to 2.2% w/w.
A device, device preparation or device composition may be configured for implantation, or is implanted or disposed, into or onto any site or part of the body. In some embodiments, the implantable device or device preparation is configured for implantation into the peritoneal cavity (e.g., the lesser sac, also known as the omental bursa or bursalis omentum). A device, device preparation or device composition may be implanted in the peritoneal cavity (e.g., the omentum, e.g., the lesser sac) or disposed on a surface within the peritoneal cavity (e.g., omentum, e.g., lesser sac) via injection or catheter. Additional considerations for implantation or disposition of a device, device preparation or device composition into the omentum (e.g., the lesser sac) are provided in M. Pellicciaro et al. (2017) CellR4 5(3):e2410.
Engineered ARPE-19 cells for use in manufacturing a device described herein may be generated and cultured using methods known in the art. For example, stably-transfected ARPE-19 cells may be cultured in vitro substantially as described in WO2020198695.
Compounds of Formula (I) and alginates modified with such compounds may be obtained using procedures known in the art, e.g., substantially as those described in WO2020198695.
Alginate solutions for making two-compartment hydrogel capsules may be obtained using procedures known in the art, e.g., substantially as described in WO2020198695.
Two-compartment hydrogel capsules encapsulating engineered mammalian cells described herein may be generated using procedure known in the art, e.g., substantially as described in WO2020198696.
Described herein are methods for preventing or treating MPS VI disease in a subject through administration or implantation of a device containing a plurality of engineered, ARSB-secreting cells described herein. In an embodiment, the device is an implantable device described herein. In some embodiments, the methods described herein directly or indirectly reduce or alleviate at least one symptom of MPS VI disease, or prevent or slow the onset of MPS VI disease. In an embodiment, the method comprises administering (e.g., implanting) an effective amount of a composition of two-compartment alginate hydrogel capsules which comprise in the inner compartment engineered RPE cells and a cell-binding polymer described herein and comprise a Compound of Formula (I), e.g., Compound 101, on the outer capsule surface and optionally within the outer compartment.
1. An isolated polynucleotide which comprises a first expression cassette configured to express a mammalian precursor ARSB protein, wherein the polynucleotide has one or more of the following features:
(a) the first expression cassette comprises a first promoter sequence and a first polyA signal sequence operably linked to a coding sequence for the mammalian precursor ARSB protein;
(b) the first expression cassette is a bicistronic expression cassette and comprises a first promoter sequence and a first polyA signal sequence operably linked to each of (i) a coding sequence for the mammalian precursor ARSB protein and (ii) a coding sequence for a first sialytransferase (ST) protein;
(c) the precursor ARSB protein is an ARSB fusion protein which comprises a heterologous signal peptide operably linked to the N-terminus of a mammalian mature ARSB amino acid sequence;
(d) the polynucleotide comprises a second expression cassette comprising a second promoter and a second polyA signal operably linked to a coding sequence for a second sialytransferase (ST) protein, wherein the first and second promoter sequences may be the same or different, the first and second polyA signal sequences may be the same or different and the first and second ST proteins may be the same or different.
2. The isolated polynucleotide of embodiment 1, which comprises feature (c), optionally wherein the heterologous signal peptide consists essentially of, or consists of, SEQ ID NO:9.
3. The isolated polynucleotide of embodiment 1, which does not have feature (c).
4. The isolated polynucleotide of any one of embodiments 1 to 3, which does not have feature (b).
5. The isolated polynucleotide of any one of embodiments 1 to 4, which has feature (d).
6. The isolated polynucleotide of embodiment 5, which does not have feature (b).
7. The isolated polynucleotide of embodiment 6, wherein the first and second promoter sequences are different.
8. The isolated polynucleotide of embodiment 6, wherein the first and second polyA signal sequences are different.
9. The isolated polynucleotide of any one of embodiments 1 to 8, wherein the first expression cassette and any second expression cassette is flanked by a pair of inverted tandem repeats (ITRs).
10. The isolated polynucleotide of any of embodiments 1 to 9, wherein the first promoter sequence consists essentially of, or consists of, a nucleotide sequence that is identical to, or substantially identical to, SEQ ID NO:16.
11. The isolated polynucleotide of any of embodiments 1 to 10, wherein the first polyA signal sequence consists essentially of a nucleotide sequence that is identical to, or substantially identical to, nucleotides 3175-3696 of SEQ ID NO:15.
12. The isolated polynucleotide of any of embodiments 1 to 10, which has feature (d) and the first polyA signal sequence consists essentially of, or consists of, a nucleotide sequence that is identical to, or substantially identical to, nucleotides 3172-3393 of SEQ ID NO:20.
13. The isolated polynucleotide of any of embodiments 1 to 12, which has feature (d) and the second promoter sequence consists essentially of, or consists of, a nucleotide sequence that is identical to, or substantially identical to, nucleotides 3394-3625 of SEQ ID NO:20.
14. The isolated polynucleotide of any of embodiments 1 to 13, which has feature (d) and the second polyA signal sequence consists essentially of, or consists of, a nucleotide sequence that is identical to, or substantially identical to, nucleotides 4719-5240 of SEQ ID NO:20.
15. The isolated polynucleotide of embodiment 1 or 2, which has feature (b).
16. The isolated polynucleotide of embodiment 15, wherein the ARSB coding sequence is separated from the ST coding sequence by a 2A peptide, optionally wherein the 2A peptide consists essentially of, or consists of, a nucleotide sequence that is identical to, or substantially identical to, nucleotides 3145-3210 of SEQ ID NO:18.
17. The isolated polynucleotide of embodiment 15, wherein the ARSB coding sequence is separated from the ST coding sequence by an IRES sequence, optionally wherein the IRES sequence consists essentially of, or consists of, a nucleotide sequence that is identical to, or substantially identical to, nucleotides 3145-3732 of SEQ ID NO:19.
18. The isolated polynucleotide of any of embodiments 15 to 17, wherein the first promoter sequence consists essentially of, or consists of, a nucleotide sequence that is identical to, or substantially identical to, nucleotides 337-1515 of SEQ ID NO:18.
19. The isolated polynucleotide of any of embodiments 15 to 18, wherein the first poly A sequence consists essentially of, or consists of, a nucleotide sequence that is identical to, or substantially identical to, nucleotides 4308-4824 of SEQ ID NO:18.
20. The isolated polynucleotide of any of embodiments 1 to 19, wherein the ST protein is hST3GAL4.
21. The isolated polynucleotide of any of embodiments 1 to 19, wherein the ST protein is hST3GAL2.
22. The isolated polynucleotide of any of embodiments 1 to 19, wherein the ST protein is hST6GAL2.
23. An isolated polynucleotide which comprises nucleotides 337 to 3696 of SEQ ID NO:15, optionally which further comprises nucleotides 1 to 313 of SEQ ID NO:15 and nucleotides 6110 to 6344 of SEQ ID NO:15.
24. An isolated polynucleotide which comprises nucleotides 337 to 4824 of SEQ ID NO:18, optionally which further comprises nucleotides 1 to 313 of SEQ ID NO:18 and nucleotides 7238 to 7472 of SEQ ID NO:18.
25. An isolated polynucleotide which comprises nucleotides 337 to 5341 of SEQ ID NO:19, optionally which further comprises nucleotides 1 to 313 of SEQ ID NO:19 and nucleotides 7755-7989 of SEQ ID NO:19.
26. An isolated polynucleotide which comprises nucleotides 337 to 5240 of SEQ ID NO:20, optionally which further comprises nucleotides 1 to 313 of SEQ ID NO:20 and nucleotides 7654 to 7888 of SEQ ID NO:20.
27. The isolated polynucleotide of any one of embodiments 1 to 26, which is one strand in an isolated double-stranded DNA molecule.
28. An engineered mammalian cell capable of expressing and secreting a mammalian ARSB protein, wherein the engineered cell comprises an exogenous nucleotide sequence encoding a heterologous signal peptide operably linked to a coding sequence for the mammalian ARSB protein, wherein the heterologous signal peptide consists essentially of, or consists of, SEQ ID NO:9.
29. The engineered mammalian cell of embodiment 28, which further comprises an exogenous nucleotide sequence encoding a sialytransferase (ST) protein.
30. An engineered mammalian cell capable of co-expressing and secreting a mammalian ARSB protein and a sialytransferase (ST) protein, wherein the mammalian cell is transiently or stably transfected with the isolated polynucleotide of any one of embodiments 27.
31. The engineered mammalian cell of embodiment 29 or 30, wherein the ST protein is hST3GAL4.
32. The engineered mammalian cell of embodiment 29 or 30, wherein the ST protein is hST3GAL2.
33. The engineered mammalian cell of embodiment 29 or 30, wherein the ST protein is hST6GAL2.
34. The engineered mammalian cell of any one of embodiments 29 to 33, wherein the cell is derived from an RPE cell, optionally an ARPE-19 cell.
35. The engineered mammalian cell of any one of embodiments 29 to 33, wherein the cell is derived from an induced pluripotent stem cell.
36. A composition comprising a plurality of engineered mammalian cells, wherein each cell in the plurality is an engineered cell as defined by any one of embodiments 29 to 35.
37. The composition of embodiment 36, wherein the plurality of engineered mammalian cells is obtained from a culture of a monoclonal cell line.
38. An implantable device comprising at least one cell-containing compartment and at least one means for mitigating the foreign body response (FBR) when the device is implanted into the subject, wherein the cell-containing compartment comprises the engineered cell of any one of embodiments 29 to 35 or the composition of embodiment 36 or 37.
39. The implantable device of embodiment 38, wherein the cell-containing compartment is surrounded by a barrier compartment comprising an alginate hydrogel and optionally a compound of Formula (I) disposed on the outer surface of the barrier compartment.
40. The implantable device of embodiment 39, wherein the barrier compartment comprises an alginate chemically modified with
or a pharmaceutically acceptable salt thereof.
41. The implantable device of embodiment 40, wherein the barrier compartment comprises an alginate chemically modified with Compound 122 or Compound 123 as shown in Table 4 herein.
42. The implantable device of any one of embodiments 38 to 41 which is a spherical, two-compartment hydrogel capsule of about 0.75 mm to about 2 mm in diameter.
43. A method of treating a human subject for Mucopolysaccharidosis type 6 (MPS VI) disease, comprising administering to the subject the composition of engineered mammalian cells of embodiment 36 or 37 or the implantable device of any one of embodiments 38 to 42.
In order that the disclosure described herein may be more fully understood, the following examples are set forth. The examples described in this application are offered to illustrate the engineered cells, implantable devices, and compositions and methods provided herein and are not to be construed in any way as limiting their scope.
Engineered ARPE-19 cells secreting hARSB were seeded at 400,000 cells/well of a 6-well plate in 2 ml serum-rich medium (DMEM/F12/10% FBS+1 ug/ml puromycin) and were incubated in a temperature-controlled (TC) incubator (37 degrees C., 5% CO2). Twenty to twenty-four hours post-seeding, the conditioned medium was collected and assessed for hARSB activity as described below.
The conditioned medium was diluted 8-fold in Assay Buffer (50 mM sodium acetate, pH 5.6). Fifty microliters (ul) of the diluted conditioned medium were placed in a well of a 96-well black plate. Recombinant human ARSB (rhARSB—R&D Systems catalog #4415-SU) was used as an activity standard. rhARSB was serially diluted in Assay Matrix (composed of 1-part sterile serum-rich medium, 7-parts Assay Buffer) to generate a 7-point standard curve, with the top point set at 75 ng rhARSB in 50 ul Assay Matrix. The standard curve is shown in
Fifty microliters of 5 mM 4-Methylumbelliferyl Sulfate (4-MUS—Sigma Aldrich catalog #M7133-500MG) substrate were combined with 50 ul of diluted conditioned medium or with 50 ul rhARSB standard in the 96-well black plate. The reaction was incubated at ambient temperature in the dark for 1 hour. The reaction was then quenched in 100 ul glycine-carbonate buffer (12.8 grams glycine and 18 grams sodium carbonate dissolved in 200 ml molecular biology-grade water). The assay plate was scanned in a microplate reader for blue fluorescence at excitation and emission wavelengths of 365 nm and 445 nm (top read), respectively, in endpoint mode. The results, which are shown in
MPS VI patient fibroblasts (Coriell GM00538) were seeded at 250,000 cells/well of a 6-well plate in 2 ml serum-rich medium (EMEM/15% FBS) and were incubated in a TC incubator (37 degrees C., 5% CO2). Engineered ARPE-19 cells secreting hARSB were seeded at 400,000 cells/well of a 6-well plate in 2 ml non-selective, serum-rich medium (DMEM/F12/10% FBS—no puromycin) and were incubated in the TC incubator.
Twenty to twenty-four hours post-seeding, the conditioned medium was collected from the engineered cells and was assessed for hARSB activity as described above. The conditioned medium containing active hARSB was diluted to 500 ng hARSB per 1 ml sterile EMEM/15% FBS. Two milliliters of the 500 ng/ml hARSB solution was used to replace the conditioned medium of the MPS VI patient fibroblasts seeded from the day before. The fibroblasts were incubated with the 2 ml solution of 500 ng/ml hARSB for 3 days in the TC incubator. After 3 days, the fibroblasts were rinsed in lx PBS pH 7.4, collected via cell scraping, and lysed in 0.1% Triton-X 100 solution.
Ten microliters of cell lysate were combined with an equal volume of 2× Chondroitinase B reaction mix: 4 ng Chondroitinase B (R&D Systems #6974-GH) in 2× Bacteroides Heparinase Reaction Buffer (New England Biolabs #B0735)/0.1% Triton-X 100 solution. The reaction was incubated in a thermocycler set to 37 degrees C. for 3 days. After 3 days, the entire reaction (20 ul) was combined with 180 ul [83.3: 16.7] [acetonitrile: water] and clarified via centrifugation at 12,000 rpm for 10 minutes at 4 degrees C. One hundred microliters of the sample were analyzed for dermatan sulfate levels via LCMS. The results, which are shown in
Example 3A: Use of heterologous signal peptides. ARPE-19 cells were transfected with four different hARSB expression vectors: one which encoded wild-type precursor hARSB and three that encoded a precursor hARSB fusion protein in which a signal peptide sequence from a heterologous secretory protein was fused to a coding sequence for wild-type human mature ARSB. one of three different secretory proteins. In addition to the native hARSB signal peptide, the signal peptides tested included the consensus MELG-class signal peptide from camelid single domain antibody, the murine Ig kappa (IgK) leader, and the IL-2 signal peptide. As shown in
Example 3B: Co-expression with sialytransferases. ARPE-19 cells stably expressing wild-type human precursor ARSB protein (ARSB-ARPE-19 cells) were transiently transfected with seven commercially available sialytransferase expression constructs (G418 marker). Conditioned culture media were collected from the cells 24 hrs post-transfection and were analyzed for hARSB levels and the results are shown in
hARSB levels were higher in the ARSB-ARPE-19 cells transfected with a sialytransferase expression construct than in the parental ARSB-ARPE-19 cells (Untransfected), with cells transiently transfected with expression vectors for hST3GAL2, hST3GAL4, or hST6GAL2 producing 2.5-fold to 3-fold greater expression/secretion of hARSB when compared to untransfected ARSB-ARPE-19 cells.
These cell lines were then incubated with G418, the selection marker associated with the Sialyltransferase expression constructs, to enrich potentially for cells stably expressing ST as well as hARSB cells. Conditioned culture media were collected from pools of G418-resistant cells and were analyzed for ARSB activity. As shown in
ARPE-19 cells were stably transfected with one of three different co-expression vectors designed to co-express hARSB and one of three sialyltransferases: hST3GAL2, hST3GAL4 or hST6GAL2. Two vectors were bicistronic: with either a P2A linker or an IRES element between the hARSB and ST coding sequences, which were flanked by a single promoter and a single polyA signal sequence, e.g., substantially as shown in
A pool of polyclonal APRE-19 cells stably transfected with a transposon expressing the native hARSB expression construct driven from the EFIA promoter was encapsulated in the inner compartment of two-compartment alginate capsules (spheres) at 50M cells/ml. A dose of 0.5 ml spheres (at 50M/ml) were implanted in the IP space of 5 MPS VI mice (Jackson Lab stock 005598). As a control, a sham surgery was performed on an additional 5 MPS VI mice. At 7 days post-administration, all 10 mice were euthanized, perfused, and tissues were harvested: plasma, liver, spleen, heart, lung, kidney. The tissues were cut in 75-100 mg pieces and placed in 2 ml Lysing Matrix D tubes (MP Biosciences SKU 116913050-CF). Ice-cold lx PBS pH 7.4 supplemented with protease inhibitors (100× Halt™ Protease Inhibitor Cocktail #78438) was added to a final volume of 1.5 ml in the lysing matrix tubes. The tissues were homogenized in PBS in a FastPrep instrument (6 m/second, 40 sec, 3 cycles, 180 sec pause). The tissue homogenates were clarified via centrifugation at 10,000rpm, 4 degrees C., 10 minutes.
Ten microliters of tissue homogenate were combined with an equal volume of 2× Chondroitinase B/AC reaction mix: 4 ng Chondroitinase B (R&D Systems #6974-GH) and 10 ng Chondroitinase AC (R&D Systems #8384-GH) in 2× Reaction Buffer (100 mM Tris-Cl pH 7.5, 100 mM sodium acetate, 10 mM MgCl2, 10 mM CaCl2, 0.1% Triton-X 100). The reaction was incubated in a thermocycler set to 20 degrees C. for 3 days. After 3 days, the entire reaction (20 ul) was combined with 180 ul [83.3: 16.7] [acetonitrile: water] and clarified via centrifugation at 12,000 rpm for 10 minutes at 4 degrees C. One hundred microliters of the sample were analyzed for dermatan/chondroitin sulfate levels via LCMS.
As shown in
This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the disclosure can be excluded from any claim, for any reason, whether or not related to the existence of prior art.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above Description, Figures, or Examples but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.
This application claims priority to U.S. Provisional Application No. 63/146,340, filed Feb. 5, 2021. The disclosure of each of the foregoing applications is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2022/015316 | 2/4/2022 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63146340 | Feb 2021 | US |
