Compositions, Devices, and Methods for Treating CNS Disorders

Abstract

Described herein are BBB transport fusion proteins, polynucleotides encoding the fusion proteins, and mammalian cells genetically modified to express and secrete the fusion proteins, as well as compositions comprising the fusion proteins or genetically modified cells that secrete the fusion proteins, implantable devices and device preparations, and methods of making and using them to treat various CNS diseases and conditions, e.g., any mucopolysaccharidosis type having CNS symptoms.

Description

[Technical field]

[0001] Claiming priority This application claims priority to U.S. Patent Application No. 63 / 304,377, filed January 28, 2022. The entire contents of the aforementioned application are incorporated herein by reference in their entirety. [Background technology]

[0002] Several neurodegenerative and other central nervous system (CNS) disorders are theoretically treatable by proteins such as enzymes, cytokines, and antibodies. However, the promise of using protein-based therapies to treat CNS disorders has been limited by the difficulty of transporting molecules larger than 500 daltons across the blood-brain barrier (BBB), which is formed by brain endothelial cells (BECs) that line blood vessels and connect to each other via tight junctions. Receptor-mediated transcytosis (RMT) is one of a variety of approaches that have been explored to increase the delivery of proteins across the BBB. RMT approaches typically employ intravenous or subcutaneous administration of a fusion protein that links a therapeutic protein to a molecule that binds to one of the endogenous receptors expressed on the surface of BECs. Following receptor binding, the RMT process requires internalization of the receptor-fusion protein complex and exocytosis of the fusion protein at the abluminal side of the cell. Candidate target receptors for RMT include the transferrin receptor, insulin receptor, insulin-like growth factor-1 receptor (IGF1R), low-density lipoprotein (LDL) receptor-related proteins 1 and 2 (LRP-1 and LRP-2), diphtheria toxin receptor, and TMEM30A. Results from preclinical and clinical studies indicate that various factors can affect the amount and / or activity of a therapeutic protein that crosses the BBB, including the plasma half-life of the fusion protein and its binding affinity for the RMT target receptor. Summary of the Invention

[0003] Described herein are BBB transport fusion proteins in which a cargo moiety, e.g., a molecule having an activity useful for treating or diagnosing a central nervous system (CNS) condition or disorder of interest, is placed at the C-terminus to separate it from a domain that binds to human serum albumin (HSA) and the extracellular portion of human IGF1R (hIGF1R). In an embodiment, the IGF1R binding domain is placed between the HSA binding domain and the cargo moiety. In another embodiment, the HSA binding domain is placed between the IGF1R binding domain and the cargo moiety. In an embodiment, a linker moiety is placed between the two binding domains and / or between the hIGF1 binding domain and the cargo moiety. In an embodiment, one or both of the HSA binding domain and the hIGF1R binding domain have a molecular weight of less than about 75 kDa, about 50 kDa, or about 25 kDa. In an embodiment, the amino acid sequence of each of the HSA binding domain and the hIGF1R binding domain is from a single chain Fab (scFab), a single chain Fv (scFv), or a single domain antibody (sdAb). In an embodiment, the cargo moiety (molecule) has a molecular weight of about 1 kD to about 200 kD, or about 2 kD to about 100 kD. In an embodiment, the cargo moiety (molecule) consists essentially of or consists of the amino acid sequence of a polypeptide, e.g., a cytokine, an enzyme, or an antibody. In an embodiment, the linker moiety is a linker peptide that is less than 50 amino acids in length.

[0004] In one aspect, the disclosure features a BBB transport fusion protein including a primary structure defined by formula I: AB-L1-RB-L2-C or formula II: RB-L1-AB-L2-C, where for each formula, AB includes an HSA binding domain, L1 includes a first linker amino acid sequence, RB includes an hIGF1R binding domain, L2 includes a second linker amino acid sequence, and C includes the amino acid sequence of a cargo polypeptide.

[0005] In an embodiment, the fusion protein binds to domain 1 (DI) or domain 2 (DII) of HSA via AB and does not substantially inhibit binding of human FcRn (h-FcRn) to HSA. In an embodiment, the fusion protein binds to HSA via the AB domain with a K of less than about 1 nM to about 100 nM within a pH range of about 5.0 to about 7.4 as determined by surface plasmon resonance at 25° C. D In some embodiments, the fusion protein also binds via AB to at least one mammalian serum albumin ortholog at 25° C. within a pH range of about 5.5 to about 7.4. In embodiments, the albumin ortholog is from a mouse, rat, hamster, rabbit, guinea pig, pig, cat, dog, or non-human primate (e.g., a cynomolgus or rhesus monkey).

[0006] In an embodiment, AB comprises a first, second, and third amino acid sequence corresponding to the three complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of an anti-HSA antibody (e.g., a conventional antibody having two heavy chains and two light chains, scFab, scFv, sdAb). In an embodiment, the CDR1, CDR2, and CDR3 amino acid sequences in AB are GRTFIAYA (SEQ ID NO: 1) or a conservatively substituted variant thereof, ITNFAGGTT (SEQ ID NO: 2) or a conservatively substituted variant thereof, and AADRSAQTMRQVRPVLPY (SEQ ID NO: 3) or a conservatively substituted variant thereof.

[0007] In an embodiment, AB consists essentially of or consists of QVQLVESGGGLVQAGGSLRLSCVASGRTFIAYAMGWFRQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNAKTTVYLQMNSLKPEDTALYYCAADRSAQTMRQVRPVLPYWGQGTQVTVSS (SEQ ID NO: 4) or a conservatively substituted variant thereof. In an embodiment, AB consists essentially of or consists of QVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWFRQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNAKTTVYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPVLPYWGQGTLVTVSS (SEQ ID NO: 5) or a conservatively substituted variant thereof. In embodiments, the AB consists essentially of, or consists of, the amino acid sequence of the heavy chain variable region of an antibody that cross-competes with an sdAb consisting of SEQ ID NO:4 or SEQ ID NO:5 for binding to HSA.

[0008] The fusion protein binds to hIGF1R expressed on the surface of human brain endothelial cells via the RB domain. In some embodiments, the fusion protein does not substantially bind to the human insulin receptor (h-IR). In some embodiments, the fusion protein does not substantially inhibit the binding of insulin, insulin growth factor 1 (IGF1), or insulin growth factor 2 (IGF2) to hIGF1R. In embodiments, the fusion protein binds to an epitope in the hIGF1R extracellular domain comprising FENFLHNSIFVPR (SEQ ID NO: 6) via the RB. In embodiments, the fusion protein binds to hIGF1R via the RB with a K of about 0.5 nM to about 50 nM within a pH range of about 5.0 to about 7.4 as determined by surface plasmon resonance at 25°C. DIn some embodiments, the fusion protein also binds to at least one mammalian IGF1R ortholog via the RB at 25° C. within a pH range of about 5.0 to about 7.4. In embodiments, the IGF1R ortholog is from a mouse, rat, hamster, rabbit, guinea pig, dog, cat, or non-human primate (e.g., a cynomolgus or rhesus monkey). In embodiments, the RB comprises a first, second, and third amino acid sequence corresponding to the three complementarity determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region of an anti-hIGF1R antibody (e.g., a conventional antibody having two heavy chains and two light chains, scFab, scFv, sdAb). In an embodiment, the CDR1, CDR2, and CDR3 amino acid sequences in RB are GRTIDNYA (SEQ ID NO: 7) or a conservatively substituted variant thereof, IDWGDGGX (X is A or T) (SEQ ID NO: 8) or a conservatively substituted variant thereof, and AMARQSRVNLDVARYDY (SEQ ID NO: 9) or a conservatively substituted variant thereof. In an embodiment, the CDR2 sequence in RB is IDWGDGGA (SEQ ID NO: 10).

[0009] In an embodiment, RB consists essentially of or consists of QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPGKDREFVATIDWGDGGARYANSVKGRFTISRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVNLDVARYDYWGQGTQVTVSS (SEQ ID NO: 11) or a conservatively substituted variant thereof. In another embodiment, RB consists essentially of or consists of QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRVNLDVARYDYWGQGTLVTVSS (SEQ ID NO: 12) or a conservatively substituted variant thereof. In embodiments, RB consists essentially of, or consists of, the amino acid sequence of the heavy chain variable region of an antibody that cross-competes with an sdAb consisting of SEQ ID NO:11 or SEQ ID NO:12 for binding to hIGF1R.

[0010] In some embodiments, the cargo moiety in the fusion protein comprises, consists essentially of, or consists of the amino acid sequence of an enzyme deficient in a lysosomal storage disorder (LSD), such as alpha-L-iduronidase (IDUA), iduronate-2-sulfatase (IDS), arylsulfatase B (ARSB), N-sulfoglucosamine sulfohydrolase (SGSH), glucosylceramidase (GBA), alpha-galactosidase A (GLA), and alpha-1,4-glucosidase (GAA). In embodiments, the cargo moiety is not IDS. In other embodiments, the cargo moiety (molecule) comprises, consists essentially of, or consists of the amino acid sequence of an antibody or antigen-binding fragment thereof that binds to a target protein in the brain, such as beta-secretase 1 (BACE1), an immunotherapy target (e.g., programmed death receptor 1 (PD-1)).

[0011] The L1 and L2 amino acid sequences in the BBB transport fusion proteins of the present disclosure can be the same or different. In some embodiments, each of L1 and L2 is 3 to about 35 amino acids in length, 4 to about 30 amino acids in length, or 5 to about 20 amino acids in length. In embodiments, each of L1 and L2 is (GGGGS) n (SEQ ID NO: 13), where n is equal to 3, 4, or 5. In an embodiment, each of L1 and L2 consists essentially of or consists of (GGGGS)4 (SEQ ID NO: 14). In an embodiment, the cargo moiety in the BBB transport fusion protein is an IDUA protein. In an embodiment, the BBB transport IDUA fusion protein comprises, consists essentially of, or consists of the amino acid sequence shown in Figure 6 (SEQ ID NO: 38).

[0012] In another aspect, the disclosure provides a polynucleotide (e.g., an isolated polynucleotide) comprising a nucleotide sequence encoding a BBB transport fusion protein described herein. In an embodiment, the nucleotide sequence is operably linked to a promoter sequence and a polyA signal sequence. In an embodiment, the promoter sequence is identical or substantially identical to one of the promoter sequences in Figures 4A-4C (SEQ ID NO: 32, SEQ ID NO: 33, or SEQ ID NO: 34). In an embodiment, the polyA signal sequence is identical or substantially identical to one of the polyA signal sequences shown in Figure 5 (SEQ ID NO: 35, SEQ ID NO: 36, or SEQ ID NO: 37). In an embodiment, the BBB transport fusion protein consists of the amino acid sequence shown in Figure 6, and the polynucleotide comprises, consists essentially of, or consists of the nucleotide sequence shown in Figures 7-1-7-3.

[0013] In yet another aspect, the disclosure provides mammalian cells (e.g., mouse cells, Chinese Hamster Ovary (CHO) cells, monkey cells, human cells (e.g., RPE cells) that have been genetically modified to express and secrete a BBB transport fusion protein described herein. In embodiments, the mammalian cells are genetically modified by transfection with a polynucleotide (e.g., an expression vector) described herein that encodes a BBB transport fusion protein. In embodiments, the cargo moiety in the fusion protein is an IDUA protein. In embodiments, the mammalian cells are derived from ARPE-19 cells by transfection with an expression vector comprising the nucleotide sequence depicted in Figures 7-1 to 7-3.

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

[0015] In yet another aspect, the disclosure features a device comprising at least one cell-containing compartment comprising a genetically modified mammalian cell or a plurality of such cells as described herein. The device is configured to protect the cell(s) from the recipient's immune system and reduce the foreign body response (FBR) (as defined herein) to the implanted device. In an embodiment, the surface of the device comprises a compound or polymer that reduces the FBR (as defined herein) to the device (e.g., a non-fibrous compound or a non-fibrous polymer). In an embodiment, the non-fibrous polymer comprises a biocompatible, zwitterionic polymer, e.g., as described in WO2017 / 218507, WO2018 / 140834, or Liu et al., Zwitterionically modified alginates mitigate cellular overgrowth for cell encapsulation, Nature Communications (2019) 10:5262. In an embodiment, the compound is a compound of formula (III): [ka] or a pharma- ceutically acceptable salt thereof, wherein the variables A, L 1 , M, L 2 , P., L. 3 , and Z, and associated subvariables, are defined herein. In some embodiments, the compound of formula (III) or a pharma- ceutically acceptable salt thereof (e.g., (III-a), (III-b), (III-bi), (III-b-ii), (III-c), (III-d), (III-e), (III-f), (IV), (IV-a), (V), (Va), (Vb), (Vc), (Vd), (VI-a), (VI-b), (VI-c), (VI-d), or (VI-e)) is a compound described herein, including, for example, one of the compounds shown in Table 6 herein. In some embodiments, the compound of formula (III) is selected from compound 100, compound 101, compound 102, or compound 122 shown in Table 6.

[0016] In one aspect, the device of the present disclosure is a two-compartment hydrogel capsule (e.g., a microcapsule (diameter less than 1 mm) or millicapsule (diameter at least 1 mm)) in which a cell-containing compartment (e.g., an inner compartment) comprising a plurality of live genetically modified cells (and optionally one or more cell-binding substances) as described herein is surrounded by a barrier compartment comprising a non-fibrous polymer (e.g., an outer compartment, e.g., a hydrogel layer). In embodiments, the non-fibrous polymer comprises a non-fibrous compound. In embodiments, the non-fibrous compound is a compound of formula (III).

[0017] In another aspect, the disclosure features a preparation (e.g., composition) that includes a plurality (3, 6, 12, 25, 50, or more) of the cell-containing devices described herein, e.g., hydrogel capsules encapsulating genetically modified ARPE-19 cells. In some embodiments, the preparation is a pharma- ceutically acceptable composition.

[0018] In another aspect, the disclosure features a method of making or manufacturing a device comprising a genetically modified cell as described herein. In some embodiments, the method includes providing a genetically modified cell, or a plurality of such cells, and disposing the cell(s) in a surrounding component, e.g., a cell-containing compartment of a device as described herein. In some embodiments, the surrounding component includes a flexible polymer (e.g., PLA, PLG, PEG, CMC, or a polysaccharide, e.g., alginate). In some embodiments, the surrounding component includes a non-flexible polymer or metal housing. In some embodiments, the surface of the device is chemically modified, e.g., with a compound of formula (III) as described herein.

[0019] In embodiments, the device, or devices described herein, are combined with a pharma- ceutically acceptable excipient to prepare a device preparation or composition that can be administered (e.g., intraperitoneally) to a subject in need of treatment with the BBB transport fusion protein produced by the device. In embodiments, the genetically modified cells are derived from human cells (e.g., RPE cells, ARPE-19 cells), and the device preparation or composition can continuously deliver an effective amount of the BBB transport fusion protein to the subject for a sustained period of time, e.g., at least any of 3 months, 6 months, 1 year, 2 years, or more.

[0020] In another aspect, the disclosure features a method of evaluating a composition, device, or device preparation described herein. In some embodiments, the method includes providing the composition, device, or device preparation and evaluating a functional parameter of the composition, device, or device preparation. In embodiments, the functional parameter is the amount of BBB transport fusion protein produced by cells in the composition, device, or device preparation in vitro (e.g., when placed in a suitable culture medium) and / or in vivo (e.g., after implantation into a subject, e.g., a non-human subject or a human subject).

[0021] In another aspect, the disclosure features a method of treating a subject in need of therapy with a BBB transport fusion protein described herein. In embodiments, the method includes administering to the subject genetically modified cells that express and secrete the fusion protein, or an implantable element (e.g., a device or device preparation) that includes a plurality of such cells. In some embodiments, the administering step includes placing in the subject a pharma- ceutically acceptable preparation that includes a plurality of devices, each capable of producing the BBB transport fusion protein. In some embodiments, the implantable element is administered, placed, or injected into the peritoneal cavity (e.g., the lesser pouch), omentum, or subcutaneous fat of the subject. In embodiments, the method further includes measuring the amount of the BBB transport fusion protein present in a tissue sample removed from the subject, e.g., in plasma separated from a blood sample, or in a tissue sample obtained from the CNS or an organ of interest. In embodiments, the tissue sample is removed from the patient 15, 30, 60, or 120 days after administration, implantation, or placement of the device or device preparation. In some embodiments, the subject is a human. In embodiments, the subject is a human patient diagnosed with a neuropathic MPS disease and the fusion protein comprises a glycosaminoglycan metabolic enzyme that is deficient in the neuropathic MPS disease. In embodiments, the implantable element produces the BBB transport fusion protein in an amount sufficient to reduce one or more symptoms of the neuropathic MPS disease. In embodiments, the treatment results in a reduction in heparan sulfate levels in the brain and, optionally, one or more other organs or tissues outside the CNS, such as the liver, spleen, kidney, lung, and heart. In embodiments, the patient has been diagnosed with Mucopolysaccharidosis Type I (MPSI) and the BBB transport fusion protein comprises a human IDUA protein.

[0022] The details of one or more embodiments of the disclosure are set forth herein. Other features, objects, and advantages of the disclosure will become apparent from the detailed description, drawings, examples, and claims. [Brief description of the drawings]

[0023] [Figure 1A] 1 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in a BBB transport fusion protein described herein: GAA (SEQ ID NO: 15). [Figure 1B] 1 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in the BBB transport fusion proteins described herein. GBA (SEQ ID NO: 16). [Figure 1C] 1 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in a BBB transport fusion protein described herein: GLA (SEQ ID NO: 17). [Figure 1D] 1 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in a BBB transport fusion protein described herein. GNS (SEQ ID NO: 18). [Figure 1E] 1 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in the BBB transport fusion proteins described herein. GUSB (SEQ ID NO: 19). [Figure 1F] 2 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in a BBB transport fusion protein described herein: HGSNAT (SEQ ID NO: 20). [Figure 1G] 2 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in a BBB transport fusion protein described herein. IDS (SEQ ID NO:21). [Figure 1H] 2 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in the BBB transport fusion proteins described herein. IDUA (SEQ ID NO:22). [Figure 1I]1 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in a BBB transport fusion protein described herein: NAGLU (SEQ ID NO:23). [Figure 1J] 1 shows the amino acid sequence of a wild-type, human precursor polypeptide for an exemplary LSD enzyme that may be included as a cargo polypeptide in a BBB transport fusion protein described herein: SGSH (SEQ ID NO:24). [Figure 2A] Exemplary amino acid sequences of precursor forms of human proteins bound by the BBB transport fusion proteins described herein are shown; FIG. 2A (SEQ ID NO: 25) shows the amino acid sequence of the precursor human IGF1R monomer (UniProtKB-P08069), with the signal peptide in italics, the furin cleavage site in lowercase italics, and bold underlined amino acids that are contact points in the putative binding site of certain hIGF1R binding domains described herein. [Figure 2B] FIG. 2B (SEQ ID NO:95) shows the amino acid sequence of precursor human serum albumin, with the signal peptide underlined. [Figure 3A]

[0036] Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) show the amino acid sequences of exemplary BBB transport IDUA fusion proteins of the present disclosure, where the fusion proteins in Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) comprise a parent anti-HSA and one of three different parent anti-IGF1R sdAb sequences, and the fusion proteins in Figures 3D (SEQ ID NO:29), 3E (SEQ ID NO:30), and 3F (SEQ ID NO:31) comprise humanized variants of the anti-HSA and anti-IGF1R sdAb sequences shown in Figures 3A, 3B, and 3C, each of the fusion proteins comprising two linker sequences with 4 or 5 repeats of GGGGS (SEQ ID NO:92). [Figure 3B]

[0036] Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) show the amino acid sequences of exemplary BBB transport IDUA fusion proteins of the present disclosure, where the fusion proteins in Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) comprise a parent anti-HSA and one of three different parent anti-IGF1R sdAb sequences, and the fusion proteins in Figures 3D (SEQ ID NO:29), 3E (SEQ ID NO:30), and 3F (SEQ ID NO:31) comprise humanized variants of the anti-HSA and anti-IGF1R sdAb sequences shown in Figures 3A, 3B, and 3C, each of the fusion proteins comprising two linker sequences with 4 or 5 repeats of GGGGS (SEQ ID NO:92). [Figure 3C]

[0036] Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) show the amino acid sequences of exemplary BBB transport IDUA fusion proteins of the present disclosure, where the fusion proteins in Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) comprise a parent anti-HSA and one of three different parent anti-IGF1R sdAb sequences, and the fusion proteins in Figures 3D (SEQ ID NO:29), 3E (SEQ ID NO:30), and 3F (SEQ ID NO:31) comprise humanized variants of the anti-HSA and anti-IGF1R sdAb sequences shown in Figures 3A, 3B, and 3C, each of the fusion proteins comprising two linker sequences with 4 or 5 repeats of GGGGS (SEQ ID NO:92). [Figure 3D]

[0036] Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) show the amino acid sequences of exemplary BBB transport IDUA fusion proteins of the present disclosure, where the fusion proteins in Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) comprise a parent anti-HSA and one of three different parent anti-IGF1R sdAb sequences, and the fusion proteins in Figures 3D (SEQ ID NO:29), 3E (SEQ ID NO:30), and 3F (SEQ ID NO:31) comprise humanized variants of the anti-HSA and anti-IGF1R sdAb sequences shown in Figures 3A, 3B, and 3C, each of the fusion proteins comprising two linker sequences with 4 or 5 repeats of GGGGS (SEQ ID NO:92). [Figure 3E]

[0036] Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) show the amino acid sequences of exemplary BBB transport IDUA fusion proteins of the present disclosure, where the fusion proteins in Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) comprise a parent anti-HSA and one of three different parent anti-IGF1R sdAb sequences, and the fusion proteins in Figures 3D (SEQ ID NO:29), 3E (SEQ ID NO:30), and 3F (SEQ ID NO:31) comprise humanized variants of the anti-HSA and anti-IGF1R sdAb sequences shown in Figures 3A, 3B, and 3C, each of the fusion proteins comprising two linker sequences with 4 or 5 repeats of GGGGS (SEQ ID NO:92). [Figure 3F]

[0036] Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) show the amino acid sequences of exemplary BBB transport IDUA fusion proteins of the present disclosure, where the fusion proteins in Figures 3A (SEQ ID NO:26), 3B (SEQ ID NO:27), and 3C (SEQ ID NO:28) comprise a parent anti-HSA and one of three different parent anti-IGF1R sdAb sequences, and the fusion proteins in Figures 3D (SEQ ID NO:29), 3E (SEQ ID NO:30), and 3F (SEQ ID NO:31) comprise humanized variants of the anti-HSA and anti-IGF1R sdAb sequences shown in Figures 3A, 3B, and 3C, each of the fusion proteins comprising two linker sequences with 4 or 5 repeats of GGGGS (SEQ ID NO:92). [Figure 4A] An exemplary promoter sequence useful in expression constructs for the BBB transport fusion proteins of the present disclosure is shown: pCAG promoter sequence (FIG. 4A, SEQ ID NO:32). [Figure 4B] Exemplary promoter sequences useful in expression constructs for the BBB transport fusion proteins of the present disclosure are shown: EF1α promoter sequence (FIG. 4B, SEQ ID NO:33). [Figure 4C] An exemplary promoter sequence useful in expression constructs for the BBB transport fusion proteins of the present disclosure is shown: EFS promoter sequence (FIG. 4C, SEQ ID NO:34). [Diagram 5] Exemplary polyA signal sequences useful in expression constructs for the BBB transport fusion proteins of the present disclosure are shown: rBG polyA signal sequence (A, SEQ ID NO:35), SV40 late polyA signal sequence (B, SEQ ID NO:36), BGH polyA signal sequence (C, SEQ ID NO:37). [Figure 6] Shown is the amino acid sequence of an exemplary BBB transport IDUA fusion protein (SEQ ID NO:38), where underline indicates VHH consensus signal peptide, bold indicates anti-HSA sdAb, italics indicates anti-IGF1R sdAb, bold italics indicates wild-type, human mature IDUA amino acid sequence, and dash-dotted underline indicates flexible linker sequence. [Figure 7-1] 6 shows the nucleotide sequence of an exemplary transcription unit useful for expressing the IDUA fusion protein described in FIG. 6 (SEQ ID NO:39), where wavy underline indicates EF1A promoter sequence, straight underline indicates exemplary coding sequence for a VHH consensus signal peptide, bold indicates exemplary coding sequence for an anti-HSA sdAb, italics indicates exemplary coding sequence for an anti-IGF1R sdAb, bold italics indicates exemplary coding sequence for a wild-type, human mature IDUA amino acid sequence, dashed-dotted underline indicates coding sequence for a flexible linker sequence, dotted underline indicates a stop codon, and underlined italics indicates a rBG polyA signal sequence. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 7-3] This is a continuation of Figure 7-2. [Figure 8] FIG. 1 shows IDUA activity in a single IDUA fusion protein containing an exemplary anti-IGF1R sdAb fused to hIDUA via an amino acid linker; A shows in vitro IDUA activity in culture medium of cells expressing one of six different fusion constructs based on sdAb orientation and linker length; B compares in vivo hIDUA activity in liver and plasma samples from MPS-1 mice implanted with encapsulated cells expressing either wild-type hIDUA (light grey bars) or the fusion construct that produced the highest in vitro hIDUA activity in A (IGF1rR-hIDUA, dark grey bars). [Figure 9A]1 is a graph showing in vitro IDUA activity in conditioned medium of cells expressing one of six different dual IDUA fusion proteins containing various orientations of hIDUA, an exemplary anti-IGF1R sdAb (IGF1R5), and an exemplary anti-HSA sdAb (R28). [Figure 9B] FIG. 1 is a graph comparing in vitro IDUA activity in conditioned culture medium of cells expressing an exemplary anti-IGF1R-hIDUA fusion enzyme (IGF1R4-hIDUA, light gray bars) with cells expressing an exemplary anti-HSA-anti-IGF1R-hIDUA fusion enzyme (R28-IGF1R5-hIDUA, dark gray bars). [Figure 10] FIG. 1 is a graph comparing in vivo hIDUA activity in plasma and systemic (non-brain) tissues of MPS-1 mice implanted with encapsulated cells expressing either wild-type hIDUA (light grey bars) or an exemplary anti-HSA-anti-IGF1R-hIDUA fusion enzyme (R28-IGF1R5-hIDUA, dark grey bars). [Figure 11] FIG. 1 is a graph comparing heparan sulfate levels in brain tissue samples from untreated MPS-1 mice (light grey bars) and MPS-1 mice implanted with encapsulated cells expressing an exemplary anti-HSA-anti-IGF1R-hIDUA fusion enzyme (R28-IGF1R5-hIDUA, dark grey bars). [Figure 12] 9A-9C are amino acid sequences of exemplary BBB transport IDS fusion proteins of the present disclosure (SEQ ID NO:93 and SEQ ID NO:94). [Figure 13] 1 is a chart showing a comparison of enzyme activity levels produced by exemplary constructs described herein, as outlined in Example 5. [Figure 14] Graph comparing heparan sulfate levels in liver, spleen, kidney, lung, and heart tissue samples from untreated MPS-1 mice (gray bars) and MPS-1 mice implanted with encapsulated cells expressing an exemplary anti-HSA-anti-IGF1R-hIDUA fusion enzyme (R28-IGF1R5-hIDUA, black bars). [Figure 15]1 is a graph comparing heparan sulfate levels in brain tissue samples from untreated MPS-1 mice (solid black bars) and MPS-1 mice implanted with encapsulated cells expressing an exemplary hIDUA fusion enzyme (gray bars). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The present disclosure features BBB transport fusion proteins comprising HSA- and hIGF1R-binding domains and cargo moieties, mammalian cells (e.g., human RPE cells) genetically modified to express and secrete these fusion proteins, and compositions and devices comprising the genetically modified cells. In some embodiments, the device comprises a cell-containing compartment comprising a cell-binding agent and the genetically modified cells. In some embodiments, the device is configured to reduce FBR when placed inside a subject, e.g., a human subject. In some embodiments, the fusion proteins, genetically modified cells, compositions, and devices are useful for treating CNS conditions or disorders, such as lysosomal storage diseases. Various embodiments are described below.

[0025] Abbreviations and Definitions The following abbreviations are used throughout the detailed description and examples of this disclosure: CNS Central Nervous System CS Chondroitin Sulfate DS Dermatan Sulfate GAA Acid alpha-glucosidase GAG Glycosaminoglycans GBA Beta-Glucosidase GLA Alpha-Galactosidase A GNS N-acetylgalactosamine-6-sulfatase GUSB beta-glucoronidase HS Heparan sulfate HGSNAT Heparan-alpha-glucosaminide N-acetyltransferase protein IDS Iduronate-2-sulfatase protein IDUA Alpha-L-iduronidase protein MPS Mucopolysaccharidoses NAGLU Alpha-N-acetylglucosaminidase SGSH N-sulfoglucosamine sulfohydrolase

[0026] In order that the present disclosure may be more readily understood, certain technical and scientific terms used herein are specifically defined below. Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0027] As used in this specification, including the appended claims, the singular forms of terms such as "a," "an," and "the" include their corresponding plural references unless the context clearly dictates otherwise.

[0028] "About" or "approximately," when used herein to modify a numerically defined parameter (e.g., the amount of fusion protein secreted by engineered cells, the physical description of a device (e.g., hydrogel capsule), e.g., diameter, sphericity, number of cells encapsulated therein, number of devices in a preparation), means that the stated numerical value is within an acceptable functional range for the defined parameter as determined by one of skill in the art, which depends in part on how the measurement system's limits (including the acceptable error range for that measurement system) were measured or determined. For example, "about" can mean a range of 20% above and below the stated numerical value. As a non-limiting example, a hydrogel capsule defined as having a diameter of about 1.5 millimeters (mm) and encapsulating about 5 million (M) cells can have a diameter of 1.2-1.8 mm and encapsulate 4M-6M cells. As another non-limiting example, a preparation of about 100 devices (e.g., hydrogel capsules) includes a preparation having 80-120 devices. In some embodiments, the term "about" means that the modified parameter may vary by as much as 15%, 10% or 5% above or below the numerical value stated for that parameter.

[0029] As used herein, "obtain" or "obtaining" refers to obtaining a value, e.g., a numerical value, or an image, or a physical entity (e.g., a sample), by "directly obtaining" or "indirectly obtaining" the value or physical entity. "Directly obtaining" refers to performing a process (e.g., performing an analytical method or protocol) to obtain a value or physical entity. "Indirectly obtaining" refers to obtaining a value or physical entity from another person or other source (e.g., a third-party laboratory that directly obtained the physical entity or value). Directly obtaining a value or physical entity includes performing a process that involves a physical change of a physical substance or using a machine or device. An example of directly obtaining a value includes obtaining a sample from a human subject. Directly obtaining a value includes performing a process using a machine or device, e.g., using a fluorescent microscope to obtain fluorescent microscopy data.

[0030] "Administer," "administering," or "administration," as used herein, refers to implanting, absorbing, ingesting, injecting, placing, or otherwise introducing into a subject an entity described herein (e.g., a device or preparation of a device), or providing such an entity to a subject for administration.

[0031] "Non-fibrotic" as used herein means a compound or material that mitigates foreign body response (FBR). For example, the amount of FBR in a biological tissue induced by implantation of a device (e.g., a hydrogel capsule) containing a non-fibrotic compound (e.g., a hydrogel capsule containing a polymer covalently modified with a compound listed in Table 6) is lower than the FBR in that tissue induced by implantation of a non-fibrotic null reference capsule lacking any non-fibrotic compound but of substantially the same composition (e.g., same cell type(s)) and structure (e.g., size, shape, number of compartments). In embodiments, the extent of FBR is assessed by immunological responses in the tissue containing the implanted device (e.g., hydrogel capsule), which may include, for example, protein adsorption, macrophages, multinucleated foreign body giant cells, fibroblasts, and angiogenesis, using assays known in the art, for example, as described in WO2017 / 075630, or as described in Vegas, A., et al., Nature 1999, 10, 141-145, 1999, 10, 142-143, 1999, 10, 143-144, 1999, 10, 144-145, 1999, 10, 145-146, 1999, 10, 146-147, 1999, 10, 147-148, 1999, 10, 148-149, 1999, 10, 149 ... Using one or more of the assays / methods described in Biotechnol (ibid.) (e.g., subcutaneous cathepsin measurement of implanted capsules, Masson's trichrome staining (MT), hematoxylin or eosin staining of tissue sections, quantification of collagen density, cell staining and confocal microscopy of macrophages (CD68 or F4 / 80), myofibroblasts (alpha-muscle actin, SMA), or general cellular deposits, quantification of 79 RNA sequences of known inflammatory factors and immune cell markers, or FACS analysis of macrophages and neutrophil cells in devices (e.g., capsules) retrieved after 14 days in the intraperitoneal space of suitable test subjects, e.g., immunocompetent mice). In embodiments, FBR is assessed by measuring the levels of one or more biomarkers of immune response, e.g., cathepsin, TNF-α, IL-13, IL-6, G-CSF, GM-CSF, IL-4, CCL2, or CCL4, in tissues containing the implant.In some embodiments, the FBR induced by a device of the invention (e.g., a hydrogel capsule comprising a non-fibrous compound disposed on its exterior surface) is at least about 80%, about 85%, about 90%, about 95%, about 99%, or about 100% lower than the FBR induced by an FBR null reference device, e.g., a device that is substantially identical to the tested or claimed device except for lacking a means for mitigating the FBR (e.g., a hydrogel capsule that does not comprise a non-fibrous compound but is otherwise substantially identical to the claimed capsule). In some embodiments, the FBR (e.g., the level of biomarker(s)) is measured after about 30 minutes, about 1 hour, about 6 hours, about 12 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 1 week, about 2 weeks, about 1 month, about 2 months, about 3 months, about 6 months, or more.

[0032] "Acid alpha-glucosidase protein," "acid maltase protein," "alpha-1,4-glucosidase protein," and "GAA protein" may be used interchangeably herein and refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GAA gene, or any fragment, mutant, variant, or derivative thereof having GAA enzyme activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature GAA protein as measured by any art-recognized GAA activity assay. The GAA enzyme catalyzes the hydrolysis of alpha(1,4) and alpha(1,6) linkages in glycogen, resulting in free glucose and truncated glycogen polymers. GAA enzyme activity can be measured using any art-recognized assay. The wild-type human GAA gene encodes a precursor pro-polypeptide of 952 amino acids, the N-terminal 27 amino acids of which constitute the signal peptide and amino acids 28-69 of which constitute the pro-peptide (UniProtKB-P10253). In some embodiments, the GAA amino acid sequence in the BBB transport fusion proteins described herein comprises amino acids 70-952 of the human precursor GAA sequence shown in Figure 1A. In embodiments, the human GAA amino acid sequence in the BBB transport fusion protein consists essentially of amino acids 28-952 of the sequence shown in Figure 1A.

[0033] "Alpha-galactosidase A protein", "α-Gal A protein", and "GLA protein" may be used interchangeably herein and refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GLA gene, or any fragment, mutant, variant, or derivative thereof having within 80-120%, 85-115%, 90-110%, or 95-105% of the GLA enzyme activity of the corresponding wild-type mammalian mature GLA protein, as measured by any art-recognized GLA activity assay. The GLA enzyme hydrolyzes terminal alpha-D-galactose residues in glycosphingolipids, particularly globotriaosylceramide (Gb3). GLA enzyme activity can be measured using any art-recognized assay. The wild-type human GLA gene encodes a polypeptide of 429 amino acids, the N-terminal 31 amino acids of which constitute a signal peptide (UniProtKB-P06280). In some embodiments, the human GLA amino acid sequence in a BBB transport fusion protein described herein consists essentially of amino acids 32-429 of the human precursor GLA amino acid sequence shown in FIG. 1C.

[0034] "Alpha-L-iduronidase protein" and "IDUA protein" may be used interchangeably herein and refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) IDUA gene, or any fragment, mutant, variant, or derivative thereof having an IDUA enzymatic activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature IDUA protein as measured by any art-recognized IDUA activity assay (e.g., hydrolysis of the substrate 4-methylumbelliferyl-α-L-iduronide (4MU-iduronide), see Ou, L. et al., Mol Genet Metab. 2014 Feb:111(2):113-115) or any IDUA activity assay described herein. IDUA protein hydrolyzes non-reducing terminal alpha-L-iduronic acid residues in glycosaminoglycans (GAGs) (e.g., dermatan sulfate and heparan sulfate). The wild-type human IDUA gene encodes a precursor protein of 653 amino acids, the N-terminal 26 or 27 amino acids of which constitute a signal peptide (GenBank Accession No. AAA81589.1, GenBank Accession No. AAA51698.1, UniProtKB-P35475). In some embodiments, the mature human IDUA amino acid sequence in the BBB transport fusion proteins described herein comprises amino acids 26, 27, or 28 to amino acid 653 of the precursor human IDUA amino acid sequence shown in FIG. 1H. In embodiments, the mature human IDUA amino acid sequence in the BBB transport fusion protein consists essentially of amino acids 27 to 653 of the amino acid sequence shown in FIG. 1H.

[0035] "Alpha-N-acetyl-glucosaminidase protein", "N-acetyl-alpha-glucosaminidase", and "NAGLU" may be used interchangeably herein to refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) NAGLU gene, or any fragment, mutant, variant, or derivative thereof having an enzymatic activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature NAGLU protein as measured by any art-recognized NAGLU assay. NAGLU catalyzes the hydrolysis of terminal non-reducing N-acetyl-D-glucosamine residues in N-acetyl-alpha-D-glucosaminides. The wild-type human NAGLU gene encodes a precursor polypeptide of 743 amino acids, the N-terminal 23 amino acids of which constitute a signal peptide (UniProtKB-P54802). In some embodiments, the mature human NAGLU amino acid sequence in a BBB transport fusion protein described herein consists essentially of amino acids 24 to 743 of the amino acid sequence shown in FIG. 1I.

[0036] "Beta-glucuronidase protein" and "GUSB protein" may be used interchangeably to refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GUSB gene, or any fragment, mutant, variant, or derivative thereof having within 80-120%, 85-115%, 90-110%, or 95-105% of the enzymatic activity of the corresponding wild-type mammalian mature GUSB protein, as measured by any art-recognized GUSB assay. GUSB catalyzes the hydrolysis of beta-D-glucuronoside to alcohol and D-glucuronate. The wild-type human GUSB gene encodes a precursor polypeptide of 651 amino acids, the N-terminal 22 amino acids of which constitute a signal peptide (UniProtKB PO8236). In some embodiments, the mature human GUSB amino acid sequence in the BBB transport fusion proteins described herein consists essentially of amino acids 23-651 of the sequence shown in FIG. 1E.

[0037] "Beta-glucosidase protein", "acid beta-glucocerebrosidase protein", "glucocerebrosidase protein", "lysosomal acid glucosylceramidase protein", "lysosomal acid GCase protein", and "GBA protein" may be used interchangeably herein to refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GBA gene, or any fragment, mutant, variant, or derivative thereof having within 80-120%, 85-115%, 90-110%, or 95-105% of the enzymatic activity of the corresponding wild-type mammalian mature GBA protein, as measured by any GBA assay known in the art. GBA catalyzes the breakdown of the glycolipid glucosylceramide (GlcCer) to ceramide and glucose within the lysosomal compartment. The wild-type human GBA gene encodes a precursor polypeptide of 536 amino acids, the N-terminal 39 amino acids of which constitute a signal peptide (UniProtKB P04062.3). In some embodiments, the mature human GBA amino acid sequence in a BBB transport fusion protein described herein consists essentially of amino acids 40-536 of the sequence shown in FIG. 1B.

[0038] "Cell" as used herein refers to an engineered cell (e.g., a genetically modified cell) or a non-engineered cell. In embodiments, the cell is an immortalized cell or an engineered cell derived from an immortalized cell. In one embodiment, the cell is a viable cell, e.g., viable as measured by any technique described herein or known in the art.

[0039] "Cell-binding peptide (CBP)" as used herein means a linear or cyclic peptide comprising an amino acid sequence derived from a cell-binding domain of a ligand of a cell adhesion molecule (CAM) (e.g., mediating cell-matrix or cell-cell connections). In an embodiment, the CBP is any of the CBPs described in International Patent Publication No. WO2020 / 069429. In an embodiment, the CBP is a linear peptide comprising RGD (SEQ ID NO: 87) and is less than 10 amino acids in length. In an embodiment, the CBP is a linear peptide consisting essentially of GRGD (SEQ ID NO: 88) or GRGDSP (SEQ ID NO: 89).

[0040] "CBP-polymer" as used herein means a polymer comprising at least one cell-binding peptide molecule covalently bound to the polymer via a linker. In one embodiment, the polymer in the CBP-polymer is a synthetic or naturally occurring polysaccharide, such as an alginate, e.g., sodium alginate. In one embodiment, the linker is an amino acid linker (i.e., consisting essentially of a single amino acid, or a peptide of several identical or different amino acids), which is connected to the N-terminus or C-terminus of CBP via a peptide bond. In an embodiment, the CBP-polymer is any of the CBP-alginates defined in WO2020 / 069429.

[0041] "Cell-binding substance (CBS)" as used herein means any chemical, biological, or other type of substance (e.g., small organic compounds, peptides, polypeptides) capable of mimicking at least one activity of a ligand of a cell-adhesion molecule (CAM) or other cell-surface molecule that mediates cell-matrix or cell-cell connections or other receptor-mediated signaling. In one embodiment, when present in a polymer composition that encapsulates viable cells, the CBS is capable of forming a transient or permanent bond or contact with one or more of the cells. In one embodiment, the CBS facilitates an interaction between two or more viable cells encapsulated in the polymer composition. In one embodiment, the presence of the CBS in a polymer composition that encapsulates a plurality of cells (e.g., viable cells) correlates with one or both of increased cell productivity (e.g., expression of a therapeutic agent) and increased cell viability when the encapsulated cells are implanted into a test subject, e.g., a mouse. In one embodiment, the CBS is physically bound to one or more polymer molecules in the polymer composition. In an embodiment, the CBS is a cell-binding peptide as defined herein or in WO2020 / 069429.

[0042] "Conservatively modified variant" or "conservative substitution" as used herein refers to a variant of a reference peptide or polypeptide that is identical to the reference molecule except for having one or more conservative amino acid substitutions in its amino acid sequence. In one embodiment, a conservatively modified variant consists of an amino acid sequence that is at least 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% identical to the reference amino acid sequence. In some embodiments, a conservatively modified variant of an HSA binding domain sequence and / or an IGF1R binding domain sequence does not include any amino acid substitution within the CDR. A conservative amino acid substitution refers to the replacement of an amino acid with an amino acid that has similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.) and has minimal effect on the biological activity of the resulting substituted peptide or polypeptide. Conservative substitution tables of functionally similar amino acids are well known in the art, and exemplary substitutions grouped by functional characteristics are shown in Table 1 below. [Table 1]

[0043] "Consists essentially of" and variants such as "consist essentially of" or "consisting essentially of" as used throughout this specification and claims indicate the inclusion of any recited element or group of elements, and any inclusion of other elements of similar or different nature to the recited elements that do not substantially alter the basic or novel properties of the identified molecule, composition, device, or method. As a non-limiting example, an HSA binding domain or IGF1R binding domain consisting essentially of a recited amino acid sequence can also include one or more amino acids, including substitutions of one or more amino acid residues in the recited amino acid sequence, that do not substantially affect the relevant biological activity of the HSA binding domain or IGF1R binding domain, respectively.

[0044] "Derived from," as used herein with respect to a cell or cells, refers to cells obtained from a tissue, cell line, or cell, and optionally then cultured, passaged, immortalized, differentiated, and / or induced, etc., to generate the derived cell(s).

[0045] "Device," as used herein, refers to any implantable object (e.g., particle, hydrogel capsule, implant, medical device) that contains an engineered cell or cells (e.g., living cells) that are capable of expressing and secreting a fusion protein after implantation of the device and has a configuration that supports cell survival by allowing cellular nutrients to enter the device.

[0046] "Effective amount," as used herein, refers to any of the following: genetically modified cells secreting a BBB transport fusion protein, a device preparation producing a fusion protein, the number of genetically modified cells in the device, the amount of CBS and / or non-fibrous compound in the device sufficient to elicit a desired biological response. In some embodiments, the term "effective amount" refers to the amount of a component of the device (e.g., the number of cells in the device, the density of non-fibrous compound disposed on the surface and / or in the barrier compartment of the device, the density of CBS in the cell-containing compartment).

[0047] In embodiments, the desired biological response is an increase in the level of cargo molecules (e.g., cargo polypeptides) in tissue samples removed from subjects treated (e.g., implanted) with genetically modified cells, devices containing such cells, or device preparations. As will be understood by those skilled in the art, the effective amount may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the secreted BBB transport fusion protein, composition, or device, the condition being treated, the mode of administration, and the age and health of the subject. Effective amounts include therapeutic and prophylactic treatments.

[0048] In embodiments, an effective amount of a compound of formula (III) disposed on or within the device is an amount that reduces the FBR for the implanted device compared to a reference device, e.g., reduces the amount of fibrosis or fibrous tissue on or near the implanted device.

[0049] In embodiments, an effective amount of CBS placed with the engineered cells in the cell-containing compartment is an amount that enhances the viability of the cells (e.g., number of viable cells) compared to a reference device and / or increases production of the BBB transport fusion protein by the cells (e.g., increased levels of the fusion protein in the plasma of a subject in which the device is implanted) compared to a reference device. Effective amounts of device, composition, or components (e.g., non-fibrous compound, CBS, engineered cells) may be determined by any technique known in the art as described herein.

[0050] An "engineered cell" or "genetically modified cell," as used herein, is a mammalian cell (e.g., a human cell, e.g., an RPE cell, a cell derived from a cell line (e.g., ARPE-19 or other cell line), a stem cell, a cell differentiated from an iPSC) that has a non-naturally occurring mutation and typically comprises an exogenous nucleotide sequence (e.g., a vector or a mutated chromosomal sequence) that encodes a BBB transport fusion protein as described herein. In embodiments, the exogenous nucleotide sequence is chromosomal (e.g., the exogenous sequence is placed in an endogenous chromosomal sequence) or extrachromosomal (e.g., a non-integrated expression vector). In embodiments, the exogenous nucleotide sequence in the genetically modified cell comprises a codon-optimized coding sequence in one, two, or all three of the AB, BBB, or C domains to achieve higher expression of the fusion protein than the naturally occurring coding sequence in each of these domains. Codon-optimized sequences can be generated using commercially available algorithms, such as GeneOptimizer (ThermoFisher Scientific), OptimumGene™ (GenScript, Piscataway, NJ USA), GeneGPS® (ATUM, Newark, CA USA), or 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 embodiments, the cells are also genetically modified to reduce or eliminate expression of one or more proteins naturally expressed by the parent cell. In embodiments, genetically modified cells (e.g., RPE modified cells, modified ARPE-19 cells) are cultured from a population of stably transfected cells or from a monoclonal cell line.

[0051] An "exogenous nucleic acid," as used herein, is a nucleotide sequence that does not naturally occur in a subject cell.

[0052] An "exogenous polypeptide," as used herein, is a polypeptide that does not naturally occur in a subject cell, e.g., an engineered cell. Reference to an amino acid position of a particular sequence refers to the position of that amino acid in the reference amino acid sequence, e.g., the sequence of the full-length mature (after signal peptide cleavage) wild-type protein (unless otherwise stated), and does not exclude the presence of variations, e.g., deletions, insertions and / or substitutions, at other positions in the reference amino acid sequence.

[0053] "Expression vector" as used herein refers to a recombinant polynucleotide comprising one or more expression constructs that encode one or more proteins to be expressed. Each expression construct comprises an expression control sequence operably linked to one or more nucleotide sequences to be expressed. An expression vector comprises sufficient cis-acting elements for expression, and other elements for expression can be supplied by the host cell or in an in vitro expression system. A vector can comprise additional sequence elements used for expression and / or integration of the expression cassette(s) into the genome of a mammalian cell. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate recombinant polynucleotides. Expression vectors suitable for use in engineering mammalian cells to express any of the fusion proteins described herein can also contain a nucleotide sequence encoding a marker for selection of cells containing such a vector. Examples of suitable markers are genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, kanamycin, nourseothricin, or zeocin.

[0054] "Fabry disease", "GLA deficiency", "alpha-galactosidase A deficiency", and "Anderson-Fabry disease" may be used interchangeably herein to refer to an X-linked, LSD caused by insufficient activity of the enzyme alpha-galactosidase A (GLA or GALA), resulting in damaging accumulation of the glycosphingolipid globotriaosylceramide (Gb3) in various tissues and organs. Over 370 different mutations in the human GLA gene have been identified in people with Fabry disease, many of which are unique to a single family. Mutations that eliminate GLA activity lead to the severe classical form of Fabry disease, which typically begins in childhood. Milder, later-onset forms of Fabry disease are correlated with mutations that reduce but do not eliminate GLA activity. A Fabry disease patient refers to an individual who has been diagnosed with or is suspected of having Fabry disease. A Fabry patient may be diagnosed using any method known in the art. In an embodiment, the human Fabry disease patient has insufficient GLA enzyme activity and / or a GLA gene mutation associated with Fabry disease.

[0055] "Gaucher disease" as used herein refers to an autosomal recessive LSD caused by insufficient activity of beta-glucocerebrosidase, leading to intracellular accumulation of the glycolipid glucocerebroside throughout the body. At least five forms of Gaucher disease have been identified, and the forms associated with CNS involvement are named Gaucher disease type 2 and Gaucher disease type 3. The type 2 form, also known as acute neuropathic Gaucher disease, occurs in newborns and infants, and typically results in death within the first three years of life. The type 3 form, also known as chronic neuropathic Gaucher disease, typically occurs during the first decade of life, and is associated with CNS involvement that is slower in onset and progression than type 2 patients. Many mutations in the GBA gene are associated with type 2 and type 3 Gaucher disease, with the L483P substitution being the most common mutation associated with type 3. A Gaucher disease patient refers to an individual who has been diagnosed with or is suspected of having Gaucher disease. In an embodiment, the human Gaucher patient has insufficient GBA enzyme activity and / or a GBA gene mutation associated with Gaucher disease type 3.

[0056] "Heparan-alpha-glucosaminide N-acetyltransferase protein," "heparan acetyl-CoA:alpha-glucosaminide N-acetyltransferase protein," "HGSNAT protein," and "N-acetyltransferase protein" may be used interchangeably herein to refer to a protein comprising a mature amino acid sequence encoded by a wild-type mammalian (e.g., human) gene, or any fragment, mutant, variant, or derivative thereof having HGSNAT enzymatic activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature HGSNAT protein as measured by any art-recognized HGSNAT assay. HGSNAT catalyzes the acetylation of terminal glucosamine residues of intralysosomal heparan or heparan sulfate, converting it into a substrate for hydrolysis by NAGLU. The wild-type human HGSNAT gene encodes a polypeptide of 663 amino acids and contains a predicted signal sequence that is not cleaved upon translocation to the endothelial endoplasmic reticulum (UniProtKB-Q68CP4). In some embodiments, the HGSNAT amino acid sequence in the BBB transport fusion proteins described herein consists essentially of amino acids 1-663 of the human HGSNAT sequence shown in FIG. 1F.

[0057] "High molecular weight alginate" or "HMW-Alg" as used herein means an alginate having an approximate molecular weight of 150 kDa to 250 kDa.

[0058] "Iduronate-2-sulfatase protein," "IDS protein," "I2S protein," and "alpha-L-iduronate sulfate sulfatase protein" may be used interchangeably herein to refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) IDS gene, or any fragment, mutant, variant, or derivative thereof having an IDS enzymatic activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature IDS protein as measured by any art-recognized IDS assay. IDS hydrolyzes the 2-sulfate group of the L-iduronate 2-sulfate units of dermatan sulfate, heparan sulfate, and heparan. The wild-type human IDS gene encodes a 550 amino acid precursor propolypeptide, the N-terminal 25 amino acids of which constitute a signal peptide and the remaining amino acids constitute the pro-polypeptide, which is processed to a mature polypeptide by removal of the pro-peptide at amino acids 26-33, followed by cleavage into two chains formed by amino acids 34-455 and amino acids 456-550. (UniProtKB-P22304). In some embodiments, the IDS amino acid sequence in the BBB transport fusion proteins described herein comprises amino acids 34-550 of the human precursor IDS sequence shown in FIG. 1G. In embodiments, the human IDS amino acid sequence in the BBB transport fusion protein consists essentially of amino acids 26-550 of the sequence shown in FIG. 1G.

[0059] "Low molecular weight alginate" or "LMS-Alg" as used herein means an alginate having a molecular weight of approximately <75 kDa.

[0060] "Lysosomal storage disorder" and "LSD" as used herein refer to an inborn error of metabolism caused by a deficiency of an enzyme or its cofactors that results in insufficient function of lysosomes and accumulation of one or more glycosaminoglycans (GAGs) in various tissues and organs. GAGs, which are long unbranched polysaccharides consisting of repeating disaccharides, include chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS), and hyaluronan. Defective enzymes are responsible for about 70% of LSDs, with the remainder resulting from defects in enzyme activators or associated proteins. Known LSDs include enzyme deficiency LSDs and other LSDs listed in Tables 1 and 2 in Pastores, et al., Neurol Clin. 31(4); 1051-1071 (2013). In an embodiment, the LSD is a mucopolysaccharidosis (MPS) or sphingolipidosis (SP).

[0061] As used herein, "medium molecular weight alginate" or "MMW-Alg" refers to an alginate having a molecular weight of approximately 75 kDa to 150 kDa.

[0062] "Mucopolysaccharidosis" and "MPS" as used herein refer to conditions caused by deficiencies in enzymes involved in glycosaminoglycan metabolism, resulting in the accumulation of glycosaminoglycan fragments in lysosomes, which can lead to bone, soft tissue, and CNS symptoms, especially in neuropathic forms of MPS. Neuronal damage in neuropathic MPS is associated with the storage of undegraded heparan sulfate (HS) and secondary toxic products such as GM2 ganglioside, GM3 ganglioside, inflammatory cytokines, and reactive oxygen species. Neuropathic MPS include MPS-1 Hurler (MPS-1H), MPS-2, MPS-3 (A-D), and MPS-7.

[0063] "Mucopolysaccharidosis type I" and "MPS1" may be used interchangeably herein to refer to an LSD caused by insufficient IDUA enzyme activity and the resulting accumulation of GAGs (mainly DS and HS) in lysosomes in multiple organs and tissues. MPS1 patients refer to individuals (e.g., humans) who have been diagnosed with or are suspected of having MPS1 disease, e.g., severe MPS1 or attenuated MPS1. Patients may be diagnosed using any method known in the art, including clinical, biochemical, and genetic methods for diagnosing MPS1. In embodiments, human MPS1 patients have insufficient IDUA enzyme activity and / or a mutation in the IDUA gene associated with MPS1 disease.

[0064] "Mucopolysaccharidosis type II", "MPS2", and "Hunter syndrome" may be used interchangeably herein to refer to an X-linked LSD caused by insufficient IDS enzyme activity and the resulting accumulation of GAGs (mainly DS and HS) in lysosomes in multiple organs and tissues. MPS2 patients refer to individuals (e.g., humans) who have been diagnosed with or are suspected of having MPS2 disease, e.g., severe early-onset MPS2 (symptoms become apparent before age 2-4) or mild late-onset MPS2. Patients may be diagnosed using any method known in the art, including clinical, biochemical, and genetic methods for diagnosing MPS2. In embodiments, human MPS2 patients have a mutation in the IDS gene associated with insufficient IDS activity and / or MPS2 disease.

[0065] "Mucopolysaccharidosis type IIIA", "MPS3A" and "Sanfilippo syndrome type A" may be used interchangeably herein to refer to an autosomal recessive LSD caused by insufficient SGSH activity and the resulting accumulation of heparan sulfate in the CNS. MPS3A is characterized by severe CNS degeneration. More than 80 different mutations in the SGSH gene have been identified in MPS3A patients. MPS3A patients refer to individuals (e.g., humans) who have been diagnosed with or are suspected of having MPS3A disease. Patients may be diagnosed using any method known in the art, including clinical, biochemical, and genetic methods for diagnosing MPS3A. In embodiments, human MPS3A patients have insufficient SGSH enzyme activity and / or SGSH gene mutations associated with MPS3A disease.

[0066] "Mucopolysaccharidosis type IIIB", "MPS3B" and "Sanfilippo syndrome type B" may be used interchangeably herein to refer to an autosomal recessive LSD caused by insufficient NAGLU enzyme activity and the resulting accumulation of HS in the CNS. Over 100 different mutations in the human NAGLU gene have been associated with the MPS3B phenotype. MPS3B patients refer to individuals (e.g., humans) who have been diagnosed with or are suspected of having MPS3B disease. Patients may be diagnosed using any method known in the art, including clinical, biochemical, and genetic methods for diagnosing MPS3B. In embodiments, human MPS3B patients have insufficient NAGLU enzyme activity and / or NAGLU gene mutations associated with MPS3B disease.

[0067] "Mucopolysaccharidosis type IIIC", "MPS3C" and "Sanfilippo syndrome type C" may be used interchangeably herein to refer to an autosomal recessive LSD caused by insufficient HGSNAT activity and the resulting accumulation of heparan sulfate in the CNS. Onset of MPS3C disease is typically before age 10 and is characterized by progressive CNS degeneration. More than 50 different mutations in the HGSNAT gene have been identified in MPS3C patients. MPS3C patients refer to individuals (e.g., humans) who have been diagnosed with or are suspected of having MPS3C disease. Patients may be diagnosed using any method known in the art, including clinical, biochemical, and genetic methods for diagnosing MPS3C. In embodiments, human MPS3C patients have insufficient HGSNAT enzyme activity and / or HGSNAT gene mutations associated with MPS3C disease.

[0068] "Mucopolysaccharidosis type IIID", "MPS3D", and "Sanfilippo syndrome type D" may be used interchangeably herein to refer to an autosomal recessive LSD caused by insufficient GNS enzyme activity and the resulting accumulation of HS in the CNS. Onset of MPS3D disease is typically between 2 and 6 years of age, and is characterized by severe neurological degeneration in most patients between 6 and 10 years of age. MPS3D patients refer to individuals (e.g., humans) who have been diagnosed with or are suspected of having MPS3D disease. Patients may be diagnosed using any method known in the art, including clinical, biochemical, and genetic methods for diagnosing MPS-3A. In embodiments, human MPS3D patients have insufficient GNS enzyme activity and / or GNS gene mutations associated with MPS3D disease.

[0069] "Mucopolysaccharidosis type VII", "MPS7" and "Sly syndrome" may be used interchangeably herein to refer to an autosomal recessive LSD caused by insufficient GUSB enzyme activity and the resulting accumulation of CS, DS and HS in the lysosomes of multiple tissues. At least 49 different mutations in the GUSB gene have been identified in MPS7 patients. MPS7 patients refer to individuals (e.g., humans) who have been diagnosed with or are suspected of having MPS7 disease. Patients may be diagnosed using any method known in the art, including clinical, biochemical and genetic methods for diagnosing MPS7. In embodiments, human MPS7 patients have insufficient GUSB enzyme activity and / or GUSB gene mutations associated with MPS7 disease.

[0070] "N-acetylgalactosamine-6-sulfatase protein", "glucosamine N-acetyl-6-sulfatase protein", and "GNS protein" may be used interchangeably herein for a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) GNS gene, or any fragment, mutant, variant, or derivative thereof having a GNS enzyme activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature GNS protein as measured by any art-recognized GNS assay. GNS catalyzes the hydrolysis of the 6-sulfate group of the N-acetyl-D-glucosamine 6-sulfate unit of heparan sulfate and keratan sulfate. The wild-type human GNS gene encodes a precursor polypeptide of 552 amino acids, the N-terminal 36 amino acids of which constitute a signal peptide (UniProtKB-P15586). In some embodiments, the mature human GNS amino acid sequence in a BBB transport fusion protein described herein consists essentially of amino acids 37-552 of the human precursor GNS sequence shown in FIG. 1D.

[0071] "N-sulfoglucosamine sulfohydrolase," "SGSH," "sulfamidase," and "heparan-N-sulfatase" may be used interchangeably herein to refer to a protein comprising the mature amino acid sequence encoded by a wild-type mammalian (e.g., human) SGSH gene, or any fragment, mutant, variant, or derivative thereof having SGSH enzymatic activity within 80-120%, 85-115%, 90-110%, or 95-105% of the corresponding wild-type mammalian mature SGSH protein, as measured by any art-recognized SGSH assay. SGSH catalyzes the hydrolysis of N-sulfo-D-glucosamine to D-glucosamine and sulfate. The wild-type human SGSH gene encodes a precursor polypeptide of 502 amino acids, the N-terminal 20 amino acids of which constitute a signal peptide (UniProtKB-P51688). In some embodiments, the mature human SGSH amino acid sequence in a BBB transport fusion protein described herein consists essentially of amino acids 21-502 of the sequence shown in Figure 1J.

[0072] A "peptide," as used herein, is a polypeptide of fewer than 50 amino acids, typically fewer than 25 amino acids.

[0073] "Poly A" signal, as used herein, refers to any contiguous sequence that terminates transcription of a coding sequence into RNA and directs the addition of a poly A tail to the RNA. Examples of poly A signals are the rabbit binding globulin (rBG) poly A signal, the SV40 late poly A signal, the SV50 poly A signal, the bovine growth hormone (BGH) poly A signal, the human growth hormone (HGH) poly A signal, and synthetic poly A signals known in the art.

[0074] A "polymer composition," as used herein, is a composition (e.g., solution, mixture) that includes one or more polymers. As a class, "polymers" include homopolymers, heteropolymers, copolymers, block polymers, and block copolymers, and can be both natural and synthetic. Homopolymers contain one type of building block, or monomer, while copolymers contain multiple types of monomers.

[0075] A "polypeptide," as used herein, is a polymer comprising amino acid residues linked via peptide bonds and having at least 2, and in some embodiments at least 10, 50, 75, 100, 150 or 200 amino acid residues.

[0076] "Pompe disease (PD)", "acid alpha-glucosidase deficiency", and "glycogen storage disease type II" may be used interchangeably herein to refer to an autosomal recessive LSD caused by insufficient GAA enzyme activity and the resulting excess accumulation of lysosomal glycogen primarily in the heart, skeletal muscle, smooth muscle, and nervous system. PD is broadly classified into childhood PD (IPD) and late-onset PD (LOPD). Over 580 mutations in the GAA gene have been identified in PD patients. A PD patient refers to an individual (e.g., a human) who has been diagnosed with or is suspected of having PD. The patient may be diagnosed using any method known in the art, including clinical, biochemical, and genetic methods for diagnosing PD. In an embodiment, a human PD patient has insufficient GAA enzyme activity and / or a GAA gene mutation associated with PD. In an embodiment, the patient is diagnosed with LOPD.

[0077] "Prevention," "prevent," and "preventing," as used herein, refer to treatments that include administering or applying a BBB transport fusion protein described herein, e.g., administering a composition of a device encapsulating modified cells expressing a fusion protein (e.g., as described herein) prior to the onset of one or more symptoms of a CNS condition or disease to prevent the physical manifestation of the symptom(s). In some embodiments, "prevention," "prevent," and "preventing" require that signs or symptoms of the CNS condition / disease have not yet developed or have not yet been observed. In some embodiments, treatment includes prevention, and in other embodiments does not include prevention.

[0078] "Promoter sequence" as used herein refers to a nucleotide sequence that can drive expression in mammalian cells, e.g., human cells, e.g., RPE cells, e.g., ARPE-19 cells. In some embodiments, for example, to drive expression of the BBB transport fusion proteins described herein, the promoter sequence is from a strong mammalian promoter, e.g., a human promoter sequence. Non-limiting examples of strong promoters for use in the expression cassettes described herein include the EF1A promoter, the CAG promoter, the PGK (phosphoglycerate kinase) promoter, and the ACTB (human beta-actin) promoter. In embodiments, the promoter sequence useful for driving expression of the ST proteins described herein can be from a medium-strength promoter, e.g., the EFS promoter sequence, which is a shortened form of the EF1A promoter sequence.

[0079] "RPE cells," as used herein, refer to cells having one or more of the following characteristics: a) retinal pigment epithelial cells (RPE) (e.g., cultured using the ARPE-19 cell line (ATCC® CRL-2302™)), or cells derived or modified therefrom, e.g., by stably transfecting cells cultured from the ARPE-19 cell line with an exogenous sequence encoding a BBB transport fusion protein, cells derived from primary cell cultures of RPE cells, naturally occurring RPE cells, e.g., human or other including cells isolated directly from a mammal (without long-term culture, e.g., less than 5 or 10 passages or rounds of cell division from isolation), transformed, immortalized, or derived from long-term (e.g., more than 5 or 10 passages or rounds of cell division) RPE cell cultures; b) undifferentiated cells, e.g., RPE cells or cells that, except for any genetic manipulation, have been developed, programmed, or otherwise transformed into one or more of RPE cells or cells from naturally occurring RPE cells or primary or long-term cultures of RPE cells (e.g., the cells may be derived from IPS cells). or c) cells obtained from cells that have been programmed or reprogrammed (e.g., in vitro); or c) cells that have one or more of the following characteristics: i) express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; ii) do not express one or more of the biomarkers CRALBP, RPE-65, RLBP, BEST1, or αB-crystallin; iii) are found naturally in the retina and form a monolayer over the choroidal blood vessels in Bruch's membrane; iv) mediate epithelial transport in the retina, light absorption, and or v) have been synthetically produced or have been modified from naturally occurring cells to have the same or substantially the same genetic content, and optionally the same or substantially the same epigenetic content, as an immortalized RPE cell line (e.g., the ARPE-19 cell line (ATCC® CRL-2302™). In embodiments, the RPE cells described herein are genetically modified, e.g., to have new properties, e.g., the cells are modified to express and secrete a fusion protein described herein.In embodiments, the cells are also genetically modified to reduce or eliminate expression of one or more proteins naturally expressed by the parent cell. In other embodiments, the RPE cells are not genetically modified.

[0080] "Sequence identity" or "percent identity," as used herein to refer to two nucleotide sequences or two amino acid sequences, means that the two sequences are the same within a specified region, or have the same nucleotides or amino acids at a specified percentage of nucleotide or amino acid positions within a specified region when the two sequences are compared and aligned for maximum correspondence over a comparison window or designated region. Sequence identity may be determined using standard techniques known in the art, including, but not limited to, any of the algorithms described in U.S. Patent Application Publication No. 2017 / 02334455A1. In one embodiment, the specified percentage of identical nucleotide or amino acid positions is at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0081] As used herein, "spherical" refers to a device with a curved surface (e.g., a hydrogel capsule or other particle) that forms a sphere (e.g., a perfectly round ball) or a sphere-like shape, which may have, for example, waves and undulations on the surface. Spheres and sphere-like objects can be mathematically defined by the rotation of a circle, an ellipse, or a combination around each of three orthogonal axes a, b, and c. In a sphere, the three axes are of equal length. Typically, a sphere-like shape is an ellipsoid (about its average surface) with semi-major axes within 10%, or 5%, or 2.5% of each other. The diameter of a sphere or sphere-like shape is the average diameter, e.g., the average of the semi-major axes.

[0082] "Sphingolipidosis" and "SP" as used herein refer to deficiencies in enzymes involved in sphingolipid metabolism that can cause accumulation of sphingolipids in lysosomes, resulting in symptoms in various organs and tissues, including the visceral and nervous systems. SP diseases include Gaucher disease and Fabry disease.

[0083] "Subject" as used herein refers to a human or a non-human animal. In embodiments, the subject is a human (i.e., male or female) of any age group, e.g., a pediatric human subject (e.g., infant, child, adolescent) or an adult human subject (e.g., young adult, middle-aged adult, or elderly adult). In embodiments, the subject is a non-human animal, e.g., a mammal (e.g., mouse, dog, primate (e.g., cynomolgus or rhesus monkey)). In embodiments, the subject is a commercially relevant mammal (e.g., cow, pig, horse, sheep, goat, cat, or dog) or bird (e.g., commercially relevant bird, e.g., chicken, duck, goose, or turkey). In certain embodiments, the animal is a mammal. The animal can be male or female and at any stage of development. The non-human animal can be a transgenic animal.

[0084] "Treatment", "treat", and "treating" as used herein refer to one or more of reducing, reversing, alleviating, delaying the onset, or inhibiting the progression of one or more of the symptoms, signs, or underlying causes of a CNS condition or disease. In embodiments, treating includes increasing the activity of a therapeutic protein in the CNS. In embodiments, treating includes reducing, reversing, alleviating, delaying the onset, or inhibiting the progression of symptoms associated with a condition or disease. In some embodiments, "treatment", "treat", and "treating" require that a sign or symptom associated with a CNS condition / disease has developed or been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of disease, e.g., in prophylactic 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, e.g., to delay or prevent recurrence. In some embodiments, treatment includes prophylaxis, and in other embodiments, it does not include prophylaxis.

[0085] "Wild type" (wt) refers to the naturally occurring form, including sequence, of a polynucleotide, polypeptide, or protein in a species. The wild type form is distinguished from mutant forms of the polynucleotide, polypeptide, or protein that result from genetic mutation(s).

[0086] Selected Chemical Definitions Definitions of certain functional groups and chemical terms are described in more detail below. Chemical elements are defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 thEd. (inside cover), and specific functional groups are generally defined as described therein. In addition, general principles of organic chemistry, as well as specific functional moieties and reactivities, are described in Thomas Sorrell, Organic Chemistry, University Science Books, Sausalito, 1999; Smith and March, March's Advanced Organic Chemistry, 5th Edition, John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., New York, 1989; and Carruthers, Some Modern Methods of Organic Synthesis, 3rd Edition, Cambridge University Press, Cambridge, 1987.

[0087] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae depicted herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0088] 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 include C1, C2, C3, C4, C5, C6, C1-C6, C1-C5, C1-C4, C1-C3, C1-C2, C2-C6, C2-C5, C2-C4, C2-C3, C3-C6, C3-C5, C3-C4, C4-C6, C4-C5, and C5-C6 alkyl.

[0089] As used herein, "alkyl" refers to the radical of a linear or branched saturated hydrocarbon group having 1 to 24 carbon atoms ("C1-C 24 In some embodiments, an alkyl group is an alkyl group having 1 to 12 carbon atoms ("C1-C 12 alkyl), 1 to 10 carbon atoms ("C1-C12 In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). In some embodiments, an alkyl group has 2 to 6 carbon atoms ("C2-C6 alkyl"). Examples of C1-C6 alkyl groups include methyl (C1), ethyl (C2), n-propyl (C3), isopropyl (C3), n-butyl (C4), tert-butyl (C4), sec-butyl (C4), iso-butyl (C4), n-pentyl (C5), 3-pentanyl (C5), amyl (C5), neopentyl (C5), 3-methyl-2-butanyl (C5), tertiary amyl (C5), and n-hexyl (C6). Additional examples of alkyl groups include n-heptyl (C7), n-octyl (C8), and the like. Each example of an alkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted with one or more substituents; for example, with, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent ("substituted alkyl").

[0090] As used herein, "alkenyl" refers to a radical of a straight-chain or branched hydrocarbon group having 2 to 24 carbon atoms, one or more carbon-carbon double bonds and no triple bonds ("C2-C 24 In some embodiments, an alkenyl group refers to an alkyl group having 2 to 10 carbon atoms ("C-C 10alkenyl"), 2 to 8 carbon atoms ("C2-C8 alkenyl"), 2 to 6 carbon atoms ("C2-C6 alkenyl"), 2 to 5 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 may be internal (e.g., as in 2-butenyl) or terminal (e.g., 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 C2-C6 alkenyl groups discussed above. 2-4 Alkenyl groups include pentenyl (C5), pentadienyl (C5), hexenyl (C6), etc. Each example of an alkenyl group may independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkenyl") or with one or more substituents; for example, substituted with, for example, 1-5 substituents, 1-3 substituents, or 1 substituent (a "substituted alkenyl").

[0091] As used herein, the term "alkynyl" refers to a radical of a linear or branched hydrocarbon group having 2 to 24 carbon atoms and one or more carbon-carbon triple bonds ("C2-C 24 In some embodiments, an alkynyl group refers to an alkynyl group having 2 to 10 carbon atoms ("C-C 10alkynyl"), 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 (e.g., in 2-butynyl) or terminal (e.g., 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 independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkynyl") or substituted with one or more substituents; for example, with, for example, 1 to 5 substituents, 1 to 3 substituents, or 1 substituent (a "substituted alkynyl").

[0092] As used herein, the term "heteroalkyl" refers to an acyclic stable straight or branched chain, or combinations thereof, containing at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein the nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatom may be optionally quaternized. The heteroatom(s) O, N, P, S, and Si may be replaced 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. A "heteroalkyl" is described followed by a particular heteroalkyl group, such as, for example, -CH2O, -NR CR D etc., the term heteroalkyl and -CH2O or -NR C R D It is understood that the terms "heteroalkyl" and "heteroalkyl-alkyl" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are described to provide clarity. Thus, the term "heteroalkyl" refers to specific heteroalkyl groups, such as, for example, -CHO, -NR C R D and the like. Each instance of a heteroalkyl group can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted heteroalkyl") or substituted with one or more substituents; for example, substituted with, for example, 1-5 substituents, 1-3 substituents, or 1 substituent (a "substituted heteroalkyl").

[0093] The terms "alkylene", "alkenylene", "alkynylene", or "heteroalkylene", alone or as part of another substituent, mean a divalent radical derived from an alkyl, alkenyl, alkynyl, or heteroalkyl, respectively, unless otherwise stated. An alkylene, alkenylene, alkynylene, or heteroalkylene group may be described, for example, as a C1-C6 membered alkylene, a C2-C6 membered alkenylene, a C2-C6 membered alkynylene, or a C1-C6 membered heteroalkylene, where the term "membered" refers to a non-hydrogen atom in the moiety. In the case of heteroalkylene groups, heteroatoms may also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(O)2R'- may represent both -C(O)2R'- and -R'C(O)2-.

[0094] As used herein, "aryl" refers to a radical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared by the cyclic array) having 6 to 14 ring carbon atoms and 0 heteroatoms provided in the aromatic ring system (e.g., having 6, 10, or 14 pi electrons shared by the cyclic array). 14 In some embodiments, an aryl group has 6 ring carbon atoms ("C aryl", e.g., phenyl). In some embodiments, an aryl group has 10 ring carbon atoms ("C 10 aryl," e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms ("C 14 Aryl" e.g., anthracyl). Aryl groups are, e.g., C6-C 10 The term "membered" refers to the non-hydrogen ring atoms in the moiety. Aryl groups include phenyl, naphthyl, indenyl, and tetrahydronaphthyl. Each instance of the aryl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted with one or more substituents ("substituted aryl").

[0095] As used herein, "heteroaryl" refers to a radical of a 5-10 membered monocyclic or bicyclic 4n+2 aromatic ring system (e.g., having 6 or 10 pi electrons shared in a cyclic array) having ring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, if valence permits. Heteroaryl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heteroaryl" also includes ring systems in which a heteroaryl ring as defined above is fused with one or more aryl groups with the point of attachment being either on the aryl or heteroaryl ring, and in such instances the number of ring members refers to the number of ring members in the fused (aryl / heteroaryl) ring system. In bicyclic heteroaryl groups where one ring does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, etc.), the point of attachment can be on either ring, i.e., on the ring bearing a heteroatom (e.g., 2-indolyl) or on the ring that does not contain a heteroatom (e.g., 5-indolyl). Heteroaryl groups can be described as, for example, 6- to 10-membered heteroaryl, with the term "member" referring to the non-hydrogen ring atoms in the moiety.

[0096] In some embodiments, the heteroaryl group is a 5-10 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided by the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-10 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-8 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided by the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-8 membered heteroaryl"). In some embodiments, the heteroaryl group is a 5-6 membered aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms provided by the aromatic ring system, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5-6 membered heteroaryl"). In some embodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heteroaryl has one ring heteroatom selected from nitrogen, oxygen, and sulfur. Each instance of a heteroaryl group independently may be optionally substituted, i.e., unsubstituted ("unsubstituted heteroaryl") or substituted with one or more substituents ("substituted heteroaryl").

[0097] Exemplary 5-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyrrolyl, furanyl, and thiophenyl. Exemplary 5-membered heteroaryl groups containing two heteroatoms include, but are not limited to, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, and isothiazolyl. Exemplary 5-membered heteroaryl groups containing three heteroatoms include, but are not limited to, triazolyl, oxadiazolyl, and thiadiazolyl. Exemplary 5-membered heteroaryl groups containing four heteroatoms include, but are not limited to, tetrazolyl. Exemplary 6-membered heteroaryl groups containing one heteroatom include, but are not limited to, pyridazinyl. Exemplary 6-membered heteroaryl groups containing two heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl. Exemplary 6-membered heteroaryl groups containing three or four heteroatoms include, but are not limited to, triazinyl and tetrazinyl, respectively. Exemplary 7-membered heteroaryl groups containing one heteroatom include, but are not limited to, azepinyl, oxepinyl, and thiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, but are not limited to, indolyl, isoindolyl, indazolyl, benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryl groups include, but are not limited to, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Other exemplary heteroaryl groups include heme and heme derivatives.

[0098] The terms "arylene" and "heteroarylene," as used herein, alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively.

[0099] As used herein, "cycloalkyl" refers to a radical of a non-aromatic cyclic hydrocarbon group having 3 to 10 ring carbon atoms and zero heteroatoms in the non-aromatic ring system ("C3-C 10 In some embodiments, a cycloalkyl group refers to a group having 3 to 8 ring carbon atoms ("C3-C8 cycloalkyl"), 3 to 6 ring carbon atoms ("C3-C6 cycloalkyl"), or 5 to 10 ring carbon atoms ("C5-C 10 Cycloalkyl groups may be described, for example, as C4-C7 membered cycloalkyl, where the term "membered" refers to a non-hydrogen ring atom in the moiety. Exemplary C3-C6 cycloalkyl groups include, but are not limited to, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (C5), cyclopentenyl (C5), cyclohexyl (C6), cyclohexenyl (C6), cyclohexadienyl (C6), and the like. Exemplary C3-C8 cycloalkyl groups include, but are not limited to, the C3-C6 cycloalkyl groups previously described 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-C 10 Cycloalkyl groups include, but are not limited to, the C3-C8 cycloalkyl groups described above, as well as cyclononyl (C9), cyclononenyl (C9), cyclodecyl (C 10 ), cyclodecenyl (C 10 ), octahydro-1H-indenyl (C9), decahydronaphthalenyl (C 10 ), spiro[4.5]decanyl (C 10), and the like. As the preceding examples illustrate, in certain embodiments, a cycloalkyl group is monocyclic ("monocyclic cycloalkyl") or contains fused, bridged, or spiro ring systems, e.g., bicyclic systems ("bicyclic cycloalkyl"), and can be saturated or partially unsaturated. "Cycloalkyl" also includes ring systems in which a cycloalkyl ring, as defined above, is fused with one or more aryl groups, with the point of attachment being on the cycloalkyl ring, and in such instances the number of carbons continues to designate the number of carbons in the cycloalkyl ring system. Each instance of a cycloalkyl group may independently be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl") or substituted with one or more substituents ("substituted cycloalkyl").

[0100] "Heterocyclyl," as used herein, is a radical of a 3- to 10-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("3- to 10-membered heterocyclyl"). In heterocyclyl groups containing one or more nitrogen atoms, the point of attachment may be at a carbon or nitrogen atom, where valence permits. Heterocyclyl groups may be either monocyclic ("monocyclic heterocyclyl") or fused, bridged, or spiro ring systems, e.g., bicyclic systems ("bicyclic cycloalkyl"), and may be saturated or partially unsaturated. Heterocyclyl bicyclic ring systems may contain one or more heteroatoms in one or both rings. "Heterocyclyl" also includes ring systems in which a heterocyclyl ring as defined above is fused with one or more cycloalkyl groups with the point of attachment either on the cycloalkyl or heterocyclyl ring, or a heterocyclyl ring as defined above is fused with one or more aryl or heteroaryl groups with the point of attachment on the heterocyclyl ring, and in such instances the number of ring members continues to designate the number of ring members in the heterocyclyl ring system. Heterocyclyl groups may be described, for example, as 3- to 7-membered heterocyclyl, with the term "member" referring to the non-hydrogen ring atoms in the moiety, i.e., carbon, nitrogen, oxygen, sulfur, boron, phosphorus, and silicon. Each instance of heterocyclyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted heterocyclyl") or substituted with one or more substituents ("substituted heterocyclyl"). In certain embodiments, the heterocyclyl group is an unsubstituted 3- to 10-membered heterocyclyl. In certain embodiments, the heterocyclyl group is a substituted 3- to 10-membered heterocyclyl.

[0101] In some embodiments, a heterocyclyl group is a 5- to 10-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, sulfur, boron, phosphorus, and silicon ("5- to 10-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 8-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 8-membered heterocyclyl"). In some embodiments, a heterocyclyl group is a 5- to 6-membered non-aromatic ring system having ring carbon atoms and 1-4 ring heteroatoms, each heteroatom being independently selected from nitrogen, oxygen, and sulfur ("5- to 6-membered heterocyclyl"). In some embodiments, a 5- to 6-membered heterocyclyl has 1-3 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 to 2 ring heteroatoms selected from nitrogen, oxygen, and sulfur. In some embodiments, the 5- to 6-membered heterocyclyl has 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

[0102] Exemplary 3-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azetidinyl, oxetanyl, and thietanyl. Exemplary 5-membered heterocyclyl groups containing one heteroatom include, but are not limited to, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl, and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, dioxolanyl, oxasulfuranyl, disulfuranyl, and oxazolidin-2-one. Exemplary 5-membered heterocyclyl groups containing three heteroatoms include, but are not limited to, triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclyl groups containing one heteroatom include, but are not limited to, piperidinyl, piperazinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, piperazinyl, morpholinyl, dithianyl, dioxanyl. Exemplary 6-membered heterocyclyl groups containing two heteroatoms include, but are not limited to, triazinanyl or thiomorpholinyl-1,1-dioxide. Exemplary 7-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azepanyl, oxepanyl, and thiepanyl. Exemplary 8-membered heterocyclyl groups containing one heteroatom include, but are not limited to, azocanyl, oxecanyl, and thiocanyl. Exemplary 5-membered heterocyclyl groups (also referred to herein as 5,6-bicyclic heterocyclic rings) fused to a C6 aryl ring include, but are not limited to, indolinyl, isoindolinyl, dihydrobenzofuranyl, dihydrobenzothienyl, benzoxazolinonyl, etc. Exemplary 6-membered heterocyclyl groups (also referred to herein as 6,6-bicyclic heterocyclic rings) fused to an aryl ring include, but are not limited to, tetrahydroquinolinyl, tetrahydroisoquinolinyl, etc.

[0103] "Amino" as used herein refers to the radical -NR 70 R 71 (In the formula, R 70 and R 71 are each independently hydrogen, C1-C8 alkyl, C3-C 10 Cycloalkyl, C4-C 10 Heterocyclyl, C6-C 10 Aryl and C5-C 10 In some embodiments, amino refers to NH2.

[0104] As used herein, "cyano" refers to the radical --CN.

[0105] As used herein, "halo" or "halogen," independently or as part of another substituent, means, unless otherwise stated, a fluorine (F), chlorine (Cl), bromine (Br), or iodine (I) atom.

[0106] As used herein, "hydroxy" refers to the radical --OH.

[0107] Alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl groups, as defined herein, are optionally substituted (e.g., "substituted" or "unsubstituted" alkyl, "substituted" or "unsubstituted" alkenyl, "substituted" or "unsubstituted" alkynyl, "substituted" or "unsubstituted" heteroalkyl, "substituted" or "unsubstituted" cycloalkyl, "substituted" or "unsubstituted" heterocyclyl, "substituted" or "unsubstituted" aryl, or "substituted" or "unsubstituted" heteroaryl groups). Typically, the term "substituted," whether preceded by the term "optionally," means that at least one hydrogen present on the group (e.g., a carbon or nitrogen atom) is replaced with an acceptable substituent, e.g., a substituent that provides for a stable compound (e.g., a compound that does not spontaneously undergo transformation by rearrangement, cyclization, elimination, or other reaction). Unless otherwise indicated, a "substituted" group has a substituent at one or more substitutable positions of the group, and when multiple positions in any given structure are substituted, the substituents are the same or different at each position. The term "substituted" is intended to include substitution with all permissible substituents of organic compounds, such as any of the substituents described herein, that result in the formation of a stable compound. The present disclosure contemplates all such combinations to arrive at a stable compound. For purposes of this disclosure, heteroatoms such as nitrogen may have hydrogen substituents and / or any suitable substituents described herein that satisfy the valence of the heteroatom and result in the formation of a stable moiety.

[0108] Two or more substituents may be optionally bonded to form an aryl, heteroaryl, cycloalkyl, or heterocyclyl group. Such so-called ring-forming substituents are typically, but not necessarily, found to be bonded to a cyclic base structure. In one embodiment, the ring-forming substituents are bonded to adjacent members of the base structure. For example, two ring-forming substituents bonded to adjacent members of a cyclic base structure generate a fused ring structure. In another embodiment, the ring-forming substituents are bonded to a single member of the base structure. For example, two ring-forming substituents bonded to a single member of a cyclic base structure generate a spirocyclic structure. In yet another embodiment, the ring-forming substituents are bonded to non-adjacent members of the base structure.

[0109] The compounds of formula (III) and their pharma- ceutically acceptable salts described herein may contain one or more asymmetric centers and therefore may exist in various isomeric forms, such as enantiomers and / or diastereomers. For example, the compounds described herein may be in the form of individual enantiomers, diastereomers or geometric isomers, or may be in the form of a mixture of stereoisomers, including racemic mixtures and mixtures enriched in one or more stereoisomers. Isomers may be isolated from mixtures by methods known to those skilled in the art, including chiral high pressure liquid chromatography (HPLC) and the formation and crystallization of chiral salts; or preferred isomers may be prepared by asymmetric synthesis. See, for example, Jacques et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977); Eliel, Stereochemistry of Carbon Compounds (McGraw-Hill, NY, 1962); and Wilen, Tables of Resolving Agents and Optical Resolutions p. 268 (EL Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, IN 1972). The present disclosure additionally encompasses the compounds described herein as individual isomers substantially free of other isomers, and alternatively, as mixtures of various isomers.

[0110] As used herein, a pure enantiomer compound is substantially free of other enantiomers or stereoisomers of the compound (i.e., in enantiomeric excess). That is, the "S" form of the compound is substantially free of the "R" form of the compound, and is thus in enantiomeric excess of the "R" form. The term "enantiomerically pure" or "pure enantiomer" indicates that the compound contains more than 75% by weight, more than 80% by weight, more than 85% by weight, more than 90% by weight, more than 91% by weight, more than 92% by weight, more than 93% by weight, more than 94% by weight, more than 95% by weight, more than 96% by weight, more than 97% by weight, more than 98% by weight, more than 99% by weight, more than 99.5% by weight, or more than 99.9% by weight of the enantiomer. In certain embodiments, the weight is based on the total weight of all enantiomers or stereoisomers of the compound.

[0111] The compounds of formula (III) described herein may also contain one or more isotopic substitutions. For example, H is: 1 H, 2 H (D or deuterium), and 3 H can be in any isotopic form, including T or tritium; 12 C. 13 C, and 14 It can be in any isotopic form, including C; O is 16 O and 18 It can be any isotopic form including O; and so forth.

[0112] The term "pharmaceutically acceptable salt" is meant to include salts of active compounds prepared using relatively non-toxic acids or bases, depending on the specific substituents found on the compounds described herein. When the compound of formula (III) used to prepare the device of the present disclosure contains a relatively acidic functionality, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, or magnesium salts, or similar salts. When the compound used in the present disclosure contains a relatively basic functionality, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharma- ceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphate, dihydrogenphosphate, sulfuric, monohydrogensulfuric, hydroiodic, or phosphorous acids, and the like, as well as salts derived from organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. Also included are salts of amino acids, e.g., arginates, and the like, and salts of organic acids, such as glucuronic or galacturonic acid (see, e.g., Berge et al, Journal of Pharmaceutical Science 66:1-19 (1977)). Certain specific compounds used in the devices (e.g., particles, hydrogel capsules) of the present disclosure contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. These salts can be prepared by methods known to those of ordinary skill in the art. Other pharma- ceutically acceptable carriers known to those of ordinary skill in the art are suitable for use in the present disclosure.

[0113] The device of the present disclosure may contain the compound of formula (III) in prodrug form. Prodrugs are those compounds that easily undergo chemical changes under physiological conditions to provide compounds useful for preparing devices in the present disclosure. In addition, prodrugs can be converted to useful compounds of formula (III) by chemical or biochemical methods in an ex vivo environment.

[0114] Certain compounds of formula (III) described herein may exist in unsolvated form as well as solvated form, including hydrated form. In general, solvated form is equivalent to unsolvated form and is included within the scope of the present disclosure. Certain compounds of formula (III) described herein may exist in polycrystalline or amorphous form. In general, all physical forms are equivalent for the use contemplated by the present disclosure and are intended to be within the scope of the present disclosure.

[0115] The term "solvate" refers to a form of a compound associated with a solvent, usually by a solvolysis reaction. This physical association may include hydrogen bonding. Conventional solvents include water, methanol, ethanol, acetic acid, DMSO, THF, diethyl ether, and the like. The compounds described herein may be prepared, for example, in crystalline form, and may be solvated. Suitable solvates include pharmaceutically acceptable solvates, further including both stoichiometric and non-stoichiometric solvates.

[0116] The term "hydrate" refers to a compound associated with water. Typically, the number of water molecules contained in a hydrate of a compound is within a defined ratio to the number of compound molecules in the hydrate. Thus, a hydrate of a compound can be represented, for example, by the general formula R·xH2O, where R is a compound and x is a number greater than 0.

[0117] As used herein, the term "tautomer" refers to a compound structure that is an interchangeable form, with changes in the displacement of hydrogen atoms and electrons. Thus, two structures may be in equilibrium between the movement of π electrons and atoms (usually H). For example, enols and ketones are tautomers because they are rapidly interconverted by treatment with either acid or base. Tautomeric forms may be relevant to achieving optimal chemical reactivity and biological activity of a compound of interest.

[0118] Symbols used herein [ka] refers to a connection to an entity, e.g., a polymer (e.g., a hydrogel-forming polymer such as alginate) or to the surface of an implantable device, e.g., a particle, a hydrogel capsule. [ka] The connection represented by may refer to a direct bond with an entity, such as a polymer or an implantable element, or may refer to a linkage with an entity via a linking group. A "linking group" as described herein refers to a moiety for linking a compound of formula (III) with an entity, such as a polymer or an implantable element (e.g., a device) as described herein, and may include any linking chemistry known in the art. A list of exemplary linking groups can be found in Bioconjugate Techniques (3 rd ed. Greg T. Hermanson, Waltham, MA: Elsevier, Inc., 2013), which is incorporated herein by reference in its entirety. In some embodiments, the linking group is an alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -C(O)-, -OC(O)-, -N(R C )-, -N(R C )C(O)-, -C(O)N(R C )-, -N(R C )N(RD )-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F )y -, -Si(OR A) 2-, -Si(R G )(OR A )-, -B(OR A )-, or metal-containing, R A , R C , R D , R F , R G Each of x, x, and y is independently as described herein. In some embodiments, the linking group comprises an amine, a ketone, an ester, an amide, an alkyl. In some embodiments, the linking group is a cross-linker. In some embodiments,

number

number

[0119] Characteristics of BBB transport fusion proteins The BBB transport fusion proteins of the present disclosure comprise an HSA binding domain (AB) and an IGF1R binding domain (RB) located upstream of a cargo moiety (C) (e.g., an amino acid sequence of a therapeutic protein). In some embodiments, a first linker moiety (e.g., a linker peptide) is located between AB and RB, and a second linker moiety (e.g., a linker peptide) is located between RB and the cargo moiety.

[0120] HSA binding domain AB The serum half-life of the BBB transport protein is longer than that of an otherwise identical fusion protein lacking the AB domain. This half-life extension is primarily due to the binding of the fusion protein to HSA via the AB domain. Mature human serum albumin (HSA) is a monomeric protein of 585 amino acids (GenBank accession number AAA98797.1) and contains three homologous domains, DI (amino acids 25-221 in FIG. 2B), DII (amino acids 213-409 in FIG. 2B), and DIII (amino acids 405-609 in FIG. 2B) (Dockal, M., et al., J. Biol. Chem., Vol. 274, No. 41, pp. 29303-29310 (1999)). The long half-life of albumin in blood is driven primarily by two features: (i) the large size (65 kDa) of albumin limits its glomerular filtration, and (ii) albumin binds to FcRn at low pH (pH 6), protecting it from degradation in lysosomes after passive endocytosis in endothelial and epithelial cells by transporting it back from early endosomes to the extracellular environment. In embodiments, the AB domain confers on the BBB transport fusion protein a serum half-life in humans (expressed as t1 / 2-beta) that exceeds any of 6 hours, 12 hours, 24 hours, 72 hours, 1 week, 2 weeks, or up to the half-life of HSA in humans (estimated to be about 19 days).

[0121] In some embodiments, the AB domain is specific for serum albumin, i.e., it does not substantially bind to any non-albumin protein. In some embodiments, the fusion protein does not substantially inhibit the binding of FcRn to HSA, and its binding site is in DIII. In embodiments, less than about 20%, 15%, 10%, 5%, or 1% inhibition of FcRn binding to HSA occurs in the presence of the fusion protein of the present disclosure, which can be determined by any competitive binding assay known in the art, for example, the SPR competitive binding assay described in

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

[0122] In some embodiments, AB (as part of the fusion protein) also binds (e.g., cross-reacts with) serum albumin from any combination of two, three, four, or more of one, two, three, four, or more other mammalian species, such as mouse, rat, guinea pig, hamster, rabbit, cat, dog, pig, sheep, horse, cow, and monkey (e.g., rhesus and / or cynomolgus). In embodiments, the fusion protein binds serum albumin from at least mouse, rat, monkey (rhesus or cynomolgus), and human via the AB domain. In embodiments, the fusion protein binds serum albumin from at least mouse, dog, and human via the AB domain.

[0123] In some embodiments, the AB domain (as part of a fusion protein) binds to HSA, and optionally at least one other mammalian serum albumin, with a desired affinity within a pH range of about 5.0 or about 5.5 up to about 7.4. In embodiments, the desired affinity is a dissociation constant (K) in any of the ranges of about 0.1 nM to about 1,000 nM, about 0.5 nM to about 500 nM, about 1 nM to about 250 nM, about 5 nM to about 50 nM, about 10 nM to about 25 nM, or about 0.5 nM to about 1 nM. D In embodiments, the affinity is determined by surface plasmon resonance (SPR) at 25° C. and a pH range of about 5.5 to about 7.4. In embodiments, the affinity of the fusion protein for serum albumin from mouse, rat, and monkey is similar to the affinity for HSA, e.g., the K D is within 70%-130%, 80%-120%, or 90%-110% of the

[0124] K of anti-HSA antibody and fusion protein D An exemplary SPR assay for measuring affinity in the pH range of 5.0-7.4 is described in US2019 / 0367597A1. The K of anti-HSA antibodies and fusion proteins to HSA and mammalian orthologs is described in US2019 / 0367597A1. D may also be determined substantially as described in WO2019 / 246003 at

[0994] to

[0995] .

[0125] The AB domain of the BBB transport fusion protein of the present disclosure comprises the amino acid sequence of the heavy chain variable region (HCVR) of an anti-HSA antibody. To facilitate expression of the fusion protein by genetically modified cells, in some embodiments, the AB domain has a molecular weight of less than about 75 kDa, about 50 kDa, or about 25 kDa. The AB domain can be derived from any anti-HSA antibody molecule known in the art, including traditional four-chain antibodies, antigen-binding fragments, Fab, Fab', F(ab')2, Fv (double-chain and single-chain (scFv)), minibodies, diabodies, and sdAbs. In embodiments, the AB consists essentially of or consists of the HCVR amino acid sequence of an sdAb.

[0126] In embodiments, the AB domain comprises a set of three CDR amino acid sequences of one of the anti-HSA sdAbs listed in Table I of WO2006 / 122787. In embodiments, the AB consists essentially of, or consists of, the amino acid sequences of one of the anti-HSA sdAbs listed in Table II of WO2006 / 22787 (e.g., Alb-1), or one of the humanized variants of Alb-1 listed in Table III of WO2006 / 22787 (e.g., Alb-8). In embodiments, the AB consists essentially of, or consists of, any of the Alb-23 sequences listed in WO2012 / 175400 (e.g., Alb-23D). In embodiments, the BBB transport fusion protein of the present disclosure cross-competes with any of the anti-HSA sdAbs described in WO2006 / 22787 (e.g., Alb-8) or WO2012 / 175400 (e.g., Alb-23D) for binding to HSA. In embodiments, the BBB transport fusion protein does not include the amino acid sequence of Alb-1. In embodiments, the BBB transport fusion protein does not include the amino acid sequence of Alb-8.

[0127] In another embodiment, the AB domain consists essentially of, or consists of, the amino acid sequence of an albumin binding domain (ABD) set forth in Table 7 of WO2019 / 246003, e.g., LAEAKVLANRELDKYGVSDYYKNLINNAKTVEGVKALIDEILAALP (SEQ ID NO: 40), and has a K of about 1.2 nM for HSA. D In an embodiment, the AB domain comprises the three CDRs of an anti-HSA P367 antibody as set forth in Table 14 of WO2019 / 2460003. In an embodiment, the AB consists essentially of or consists of the amino acid sequence of an anti-HSAP367 antibody or its humanized variant P494, each of which are listed in Table 14 of WO2019 / 2460003. In an embodiment, the BBB transport fusion protein of the present disclosure cross-competes with the P494 sdAb as set forth in WO2019 / 2460003 for binding to HSA.

[0128] In embodiments, the AB domain comprises a set of three heavy chain CDR amino acid sequences of one of the anti-HSA sdAbs listed in Table 5 of WO2021 / 119551. In one embodiment, the AB consists essentially of or consists of the VH amino acid sequence of one of the anti-HSA sdAbs listed in Table 5 of WO2021 / 119551. In embodiments, the BBB transport fusion protein of the disclosure cross-competes for binding to HSA with one or more of the sdAbs listed in Table 5 of WO2021 / 119551.

[0129] In another embodiment, the AB comprises a set of three CDR amino acid sequences found in the T0235002C06 sdAb in Table B of US2019 / 0367597A1. In an embodiment, the AB consists essentially of or consists of the amino acid sequence of T0235002C06 in Table B of US2019 / 0367597A1. In an embodiment, the BBB transport fusion protein of the present disclosure cross-competes with the T0235002C06 sdAb in Table B of US2019 / 0367597A1 for binding to HSA.

[0130] In another embodiment, the AB domain comprises a set of three CDR amino acid sequences found in the T0235005D04 sdAb in Table B of US2019 / 0367596A1. In an embodiment, the AB consists essentially of or consists of the amino acid sequence of T0235005D04 in Table B of US2019 / 0367596A1. In an embodiment, the BBB transport fusion protein of the present disclosure cross-competes with the T0235002D04 sdAb in Table B of US2019 / 0367596A1 for binding to HSA.

[0131] In yet another embodiment, the AB domain comprises a set of three CDR amino acid sequences found in T0235005G01 or T023500043 sdAb as set forth in Table B of US2019 / 0367598A1. In an embodiment, the AB consists essentially of or consists of the amino acid sequence of T0235005G01 or T023500043 as set forth in Table B of US2019 / 0367598A1. In an embodiment, the BBB transport fusion protein of the present disclosure cross-competes with T0235005G01 or T023500043 sdAb as set forth in Table B of US2019 / 0367598A1 for binding to HSA.

[0132] In embodiments, the AB domain comprises a set of three CDR amino acid sequences found in the R28, R11, M75, or M79 sdAbs described in WO2019 / 204925. These CDR sequences are shown in Table 2A below. [Table 2]

[0133] In an embodiment, AB consists essentially of or consists of the amino acid sequence of R28, R11, M75, or M79 sdAb as described in WO2019 / 204925. In an embodiment, AB consists essentially of or consists of the amino acid sequence of one of the humanized variants of R28, R11, M75, or M79 as described in WO2019 / 204925. The amino acid sequences of the parent and humanized variants of R28, R11, M75, and M79 are shown in the sequence listing on pages 46-48 of WO2019 / 204925. In an embodiment, AB consists essentially of or consists of an amino acid sequence selected from the parent and humanized sequences shown in Table 2B herein below. In an embodiment, AB consists essentially of or consists of the parent or humanized amino acid sequence of R28 as shown in Table 2B below. [Table 3]

[0134] IGF1R binding domain RB The RB domain of the BBB transport fusion protein of the present disclosure confers the fusion protein with the ability to cross the BBB via transcytosis mediated by IGF1R, also known as CD221, IGFIR, IGFR, and JTK13. Human IGF1R is synthesized as a monomeric 1367 amino acid pre-proreceptor with a 30 amino acid signal sequence (UniProtKB-P08069). After cleavage of the signal peptide, the proreceptor is glycosylated, dimerized, and transported to the Golgi apparatus where furin cleavage results in alpha and beta subunits that form a disulfide-linked tetramer (beta-alpha-alpha-beta) and are transported to the plasma membrane. The fully mature plasma membrane-bound IGF1R consists of two 130-135 kDa alpha subunits and two 90-95 kDa beta subunits with several alpha-alpha and alpha-beta disulfide bonds. The alpha subunit is entirely extracellular and forms the ligand-binding domain. The beta subunit contains the extracellular, transmembrane, and intracellular domains. Ligand binding induces trans-autophosphorylation and phosphorylation of a wide variety of downstream signaling molecules.

[0135] The ability of the RB domain to confer hIGF1R-mediated transcytosis of the fusion proteins described herein can be evaluated by any method known in the art. For example, in vitro assays can evaluate the internalization of the fusion protein into IGF1-R expressing cells, such as MCF-7 cells or primary human microvascular brain endothelial cells (HMBEC), as described in EP3725806A1

[0317] -

[0315] . A similar assay that can be used when RB cross-reacts with rat IGF1-R uses immortalized rat brain endothelial cells (svARBEC), as described in US10,100,117. Also, known in vitro and in vivo BBB models can be used to evaluate the BBB transport ability of the fusion protein to bind human and rat IGF1R, as described in, for example, Examples 10 and 12 of US10,100,117.

[0136] In some embodiments, the RB domain is specific for IGF1R, i.e., it does not substantially bind to the insulin receptor or any other non-IGF1R protein. The fusion protein containing RB should not induce signaling through IGF1R or IR, nor should it inhibit insulin, IGF-1, or IGF-2-induced signaling through IGF1R or IR. The signaling effect of the BBB transport fusion proteins described herein can be evaluated by analyzing the phosphorylation of IGF1R and IR and / or the receptor-stimulated phosphorylation of the downstream kinase Akt. This evaluation can be performed using any method known in the art, for example, as described in Example 14 of US10,100,117 or in paragraphs

[0325] to

[0327] of EP3725806A1. Any effect of the fusion protein on IGF1 signaling via IGF1R may be assessed using any method known in the art, for example by the MCF-7 cell line proliferation assay described in

[0320] to

[0324] of EP3725806A1.

[0137] The possibility of interference with ligand binding can be reduced by deriving the RB domain from an anti-hIGF1R antibody molecule known not to interfere with ligand binding or by selecting an antibody that does not bind to the alpha subunit. In an embodiment, the fusion protein cross-competes with the IGF1R-5 sdAb described in US10,100,117, the IGF1R-3 sdAb described in US10,106,614, or the IGF1R-5 sdAb described in US10,112,998 for hIGF1R binding via the RB domain. In an embodiment, the fusion protein cross-competes with any of the 996, 1226, and 1564 antibodies described in EP3725806A1 for hIGF1R binding via the RB domain. In an embodiment, the epitope of the RB domain has three binding sites, binding site 1 includes one or more of R650, Y775, P776, F778, E779, S791, and L798, binding site 2 includes one or more of L641, H808, E809, and L813, and binding site 3 includes one or more of V397, W434, D435, Y460, and C488.

[0138] In some embodiments, RB (as part of the fusion protein) also binds to (e.g., cross-reacts with) IGF1R from any combination of two, three, four, or more of one, two, three, four, or more other mammalian species, such as mouse, rat, guinea pig, hamster, rabbit, cat, dog, pig, sheep, horse, cow, and monkey (e.g., rhesus and / or cynomolgus). In embodiments, the fusion protein binds to IGF1R from at least mouse, rat, monkey (rhesus or cynomolgus), and human via the RB domain. In embodiments, the fusion protein binds to IGF1R from at least mouse, dog, and human via the RB domain.

[0139] In some embodiments, the RB domain (as part of a fusion protein) binds to hIGF1R, and optionally at least one other mammalian IGF1R, with a desired affinity within a pH range of about 5.0 or about 5.5 up to about 7.4. In embodiments, the desired affinity is a dissociation constant (K) ranging from (i) about 0.1 nM to about 1,000 nM, (ii) about 0.2 nM to any one of about 500 nM, about 250 nM, about 100 nM, about 50 nM, about 25 nM, or about 10 nM, (iii) about 0.5 nM to any one of about 250 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, or about 5 nM, or (iv) about 1 nM to any one of about 100 nM, about 50 nM, about 25 nM, about 10 nM, or about 5 nM. D In embodiments, the desired affinity is a dissociation constant (K D In embodiments, the affinity is determined by surface plasmon resonance (SPR) at 25° C. and a pH range of about 5.5 to about 7.4. In embodiments, the affinity of the fusion protein for IGF1R from mouse, rat, and monkey is similar to the affinity for hIGF1R, e.g., K D K within 70%~130%, 80%~120%, or 90%~110% of D The K of the BBB-containing fusion protein D An exemplary SPR assay for measuring is described in US 10,100,117.

[0140] The RB domain of the BBB transport fusion protein of the present disclosure comprises the amino acid sequence of the heavy chain variable region (HCVR) of an anti-hIGF1R antibody. To facilitate expression of the fusion protein by genetically modified cells, in some embodiments, the RB domain has a molecular weight of less than about 75 kDa, about 50 kDa, or about 25 kDa. The RB domain can be derived from any anti-IGF1R antibody molecule known in the art, including traditional four-chain antibodies, antigen-binding fragments, Fab, Fab', F(ab')2, Fv (double-chain and single-chain (scFv)), minibodies, diabodies, and sdAbs. In embodiments, the RB consists essentially of or consists of the HCVR amino acid sequence of an sdAb.

[0141] In embodiments, the RB domain comprises a set of three CDR amino acid sequences in an sdAb selected from the group consisting of an IGF1R-5 sdAb described in US 10,100,117, an IGF1R-3 sdAb described in US 10,106,614, and an IGF1R-5 sdAb described in US 10,112,998. These CDR sequences are shown in Table 3A below. [Table 4]

[0142] In an embodiment, RB consists essentially of or consists of the parent amino acid sequence of IGF1R-5, IGF1R-3, or IGF1R-4, or a humanized variant of one of their sdAbs. (i)X1VX2LX3ESGGGLVQX4GGSLRLSCAASGRTIDNYAMAWX5RQAPGKX6X7EX8VX9TIDWGDGGX 10 RYANSVKGRFTISRDNX 11 KX 12 TX 13 YLQMNX 14 LX 15 X 16 EDTAVYX 17 CAMARQSRVNLDVARYDYWGQGTX 18VTVSS, wherein X1 is E or Q, X2 is K or Q, X3 is V or E, X4 is A or P, X5 is V or S, X6 is D or G, X7 is L or R, X5 is F or W, X9 is A or S, 10 is A or T and X 11 is A or S, and X 12 is G or N, and X 13 is M or L, and X 14 is N or R, and X 15 is E or R, and X 16 is P or A, and X 17 is S or Y, and X 18 is Q or L, (ii)X1VX2LX3ESGGGLVQX4GGSLRLSCX5ASEYPSNFYAMSWX6RQAPGKX7X8EX9VX 10 GVSRDGLTTLYADSVKGRFTX 11 SRDNX i2 KNTX 13 X 14 LQMNSX 15 X 16 AEDTAVYYCAIVITGVWNKVDVNSRSYHYWGQGTX 17 VTVSS, wherein X1 is E or Q, X2 is K or Q, X3 is V or E, X4 is A or P, X5 is V or A, X6 is F or V, X7 is E or G, X8 is R or L, X9 is F or W, 10 is A or S, and X 11 is M or I, and X 12 is A or S, and X 13 is V or L, and X 14 is D or Y, and X 15 is V or L, and X 16 is K or R, and X 17 is Q or L, and (iii)X1VX2LX3ESGGGLVQX4GGSLRLSCX5X6SGGTVSPTAMGWX7RQAPGKX8X9EX10 VX 11 HITWSRGTTRX 12 ASSVKX 13 RFTISRDX 14 X 15 KNTX 16 YLQMNSLX 17 X 18 EDTAVYYCAASTFLRILPEESAYTYWGQGTX 19 VTVSS, wherein X1 is E or Q, X2 is K or Q, X3 is V or E, X4 is A or P, X5 is A or E, X6 is V or A, X7 is V or F, X8 is G or E, X3 is L or R, and X 10 is F or W, and X 11 is G or S, and X 12 is V or Y, and X 13 is D or G, and X 14 is N or S, and X 15 is A or S, and X 16 is L or V, and X 17 is K or R, and X 18 is A or S, and X 19 is L or Q.

[0143] In an embodiment, RB consists essentially of or consists of the amino acid sequence of any of IGF1R-5, IGF1R-3, or a humanized variant of IGF1R-5 as described in US10,100,117, US10,106,614, and US10,112,998, respectively. In an embodiment, RB consists essentially of or consists of an amino acid sequence selected from the parent or humanized sequences shown in Table 3B herein below. In an embodiment, RB consists essentially of or consists of the parent or humanized amino acid sequence of IGF1R-5 as shown in Table 3B below. [Table 5]

[0144] Generation of HSA and hIGF1R binding domains The AB and RB domains may be derived from any antibody or antigen-binding fragment thereof having the desired properties as described herein. The antibody or antigen-binding fragment may be known in the art or may be identified by any approach known in the art.

[0145] In some embodiments, one or both of the AB and RB domains are derived from single domain Abs. For example, sdAbs from Camelidae lack light chains and therefore their antigen binding site is V H It consists of one domain, designated H. sdAbs have also been observed in sharks, and NAR Other sdAbs can be engineered based on human Ig heavy and light chain sequences. As used herein, the term "sdAb" refers to VGAs of any origin, such as those derived from human Ig heavy and light chains, through phage display or other techniques. H , V H H, V L , or V NAR These include sdAbs directly isolated from a reservoir, recombinantly produced sdAbs, as well as those sdAbs generated by further modification of such sdAbs by humanization, affinity maturation, stabilization, solubilization, or other methods of antibody engineering. Also encompassed by the present disclosure are homologues, derivatives, or fragments that retain the antigen-binding function and specificity of the parent sdAb.

[0146] V binds to HSA H H sdAb and V binding to hIGF1R H To generate H sdAbs, one of skill in the art can generate phage-displayed V sdAbs from the heavy chain-only antibody repertoire of a llama or other camelid immunized with a desired antigen using any approach known in the art, such as, for example, substantially as described in WO2019 / 204925 and US10,100,117, respectively. HA H library may be generated and screened. In embodiments, the IGF1R antigen used to immunize camelids is a fragment of the precursor hIGF1R, for example comprising amino acids 1-932 of the sequence shown in Figure 1. In embodiments, the antigenic hIGF1R fragment does not include the signal peptide.

[0147] The AB or RB domains derived from the sdAb may comprise the parent framework regions, or alternatively the parent CDRs may be the V domains of other sdAbs. NAR , V H H, V H , or V L The framework regions, or framework regions of other types of antibody fragments or antibody-like molecules (Fv, scFv, Fab) of any source (e.g., human), or proteins of similar size and nature onto which CDRs can be grafted (see, e.g., Nicaise, M. et al, Protein Sci 13:1882-91 2004).

[0148] In some embodiments, the amino acid sequence in one or both of the AB and RB domains is a humanized version (humanized variant) of the parent variable region. Humanization of an antibody or antibody fragment involves replacing amino acids in the sequence with human counterparts, as found in the human consensus sequence, without losing antigen-binding ability or specificity; this approach reduces the immunogenicity of the antibody or fragment thereof when introduced into a human subject. The parent sequence can be humanized using any suitable method known in the art, such as, but not limited to, CDR grafting and veneering.

[0149] In the process of CDR grafting, one or more of the CDRs defined herein are substituted with human variable regions (V H , or V L), to another human antibody (IgA, IgD, IgE, IgG, and IgM), to an antibody fragment framework region (Fv, scFv, Fab), or to a protein of similar size and nature onto which the CDRs can be grafted. CDR grafting is known in the art and is described in at least the following: U.S. Pat. Nos. 6,180,370, 5,693,761, 6,054,297, and European Patent No. 626390.

[0150] Veneering, also referred to as "variable region resurfacing", involves humanizing the solvent exposed positions of an antibody or antibody fragment, thus preserving buried non-human residues that may be important for CDR conformation, while minimizing potential immunological reactions to the solvent exposed regions. Veneering is known in the art and described at least in U.S. Pat. Nos. 5,869,619, 5,766,886, and 5,821,123, and European Patent No. 519596.

[0151] Linker The BBB transport fusion protein may contain, for example, one or more linkers between the AB and RB domains and / or between the RB domain and the cargo moiety (e.g., a therapeutic polypeptide). Each linker should be of sufficient length to allow the linked polypeptides to individually fold into a three-dimensional structure that has the desired functional activity, e.g., binding or therapeutic activity. Also, each linker should not be cleaved by any proteases or other enzymes present in serum.

[0152] In some embodiments, the linker is a peptide linker that includes at least 2, 3, or 4 amino acids and less than about 30 amino acids, such as less than about 25, about 20, about 15, or about 10. Peptide linkers are known in the art and non-limiting examples are described herein.

[0153] The peptide linker may have a naturally occurring or non-naturally occurring sequence. For example, a sequence derived from the hinge region of a heavy chain-only antibody may be used as a linker. See, for example, WO1996 / 34103.

[0154] Suitable linker peptides typically contain G and / or S residues in various forms, and exemplary linkers include GGGG (SEQ ID NO:71), TGGGG (SEQ ID NO:72), GGSSGGSGSSSGSGGSGSSG (SEQ ID NO:73), (GGSS)n (SEQ ID NO:74), (GGGGS)n (SEQ ID NO:13), (SGGGG)n (SEQ ID NO:75), and GGGG(SGGGG)n (SEQ ID NO:76), where "n" in each case is generally a number from 1 to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, but not exceeding a maximum length of about 40 amino acids. Another exemplary peptide linker is SKPTCPPPELLGGPSVFIFPPK (SEQ ID NO:77).

[0155] In an embodiment, the fusion protein comprises two peptide linkers L1 and L2, where L1 is located between AB and RB, and L2 is located between RB and the cargo polypeptide. In an embodiment, each of L1 and L2 is about 15-30 amino acids in length, or about 20-25 amino acids in length. L1 and L2 may have the same or different amino acid sequences. In an embodiment, each of L1 and L2, independently, consists essentially of, or consists of, (GGGGS)4 (SEQ ID NO: 14) or (GGGGS)5 (SEQ ID NO: 78). In an embodiment, each of L1 and L2 is (GGGGS) 4. Consisting essentially of or consisting of.

[0156] Therapeutic and diagnostic cargo The cargo moiety can be any therapeutic or diagnostic molecule that can be bound or linked to the hIGF1R binding domain.

[0157] In embodiments, the therapeutic molecule is a polypeptide, such as a cytokine, an enzyme, a growth factor, or an antibody or an antigen-binding fragment thereof. In embodiments, the polypeptide has an activity useful for treating a neurological disorder in a mammal, such as a human. In embodiments, the neurological disorder is selected from the group consisting of neurological LSD (nLSD), Alzheimer's disease (AD), ataxia (e.g., hereditary ataxias such as Friedreich's ataxia), Huntington's disease, stroke, dementia, muscular dystrophy (MD), multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), cystic fibrosis, Angelman syndrome, Liddle syndrome, Parkinson's disease, Pick's disease, Paget's disease, schizophrenia, depression, encephalitis, prion diseases, cancer, and traumatic brain injury.

[0158] In embodiments, the therapeutic molecule is an enzyme. In embodiments, the enzyme is defective in the LSD, e.g., any of the defective enzymes listed in Table 1 of Edelman, MJ and Maegawa, GHB, Frontiers in Molecular Biosciences, Volume 7, Article 559804 (12 November 2020). In embodiments, the enzyme is IDUA, IDS, SGSH, GLA, or GAA. In embodiments, the LSD is MPS-1 and the enzyme is IDUA. In embodiments, the enzyme is IDS. In embodiments, the enzyme is not IDS.

[0159] In embodiments, the therapeutic molecule is an antibody or antigen-binding fragment thereof that specifically binds to a target protein (e.g., an antigen) in the brain. In embodiments, the target protein is a Tau protein (e.g., cisP-tau), Abeta, BACE1 or its splice isoforms, human epidermal growth factor receptor (2) (HER2), apolipoprotein E4 (ApoE4), alpha-synuclein, CD20, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), interferon receptor 1 (IL1), interferon receptor 2 (IL2), interferon receptor 3 (IL3), interferon receptor 4 (IL4), interferon receptor 5 (IL5), interferon receptor 6 (IL6), interferon receptor 7 (IL7), interferon receptor 8 (IL8), interferon receptor 9 (IL9), interferon receptor 10 (IL9), interferon receptor 11 (IL9), interferon receptor 12 (IL9), interferon receptor 13 (IL9), interferon receptor 14 (IL9), interferon receptor 15 (IL9), interferon receptor 16 (IL9), interferon receptor 17 (IL9), interferon receptor 18 (IL9), interferon receptor 19 ... The tumor necrosis factor (TNF) receptors include, for example, interleukin-gamma, interleukin-1 receptor (IL-1R), interleukin-6 (IL-6), interleukin-6 (IL6R), interleukin-12, interleukin-23, tumor necrosis factor (TNF) receptor (TNFR1), interleukin-1 beta (IL1 beta), caspase 6, interleukin-17 (IL-17), CTLA-4, programmed death receptor 1 (PD-1), programmed death-ligand 1 (PD-L1), programmed death-ligand 2 (PD-L2), TNF-alpha, vascular endothelial growth factor (VEGF), or VEGF receptor (VEGFR).

[0160] In embodiments, the therapeutic molecule is a cytokine or growth factor, e.g., an immunomodulatory cytokine, e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), interferon-beta, nerve growth factor, glial cell line-derived neurotrophic factor (GNDF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF), fibroblast growth factor-2 (FGF2), epidermal growth factor (EGF), or transforming growth factor (TGF)-beta2.

[0161] Genetically modified cells Any of the above BBB transport fusion proteins can be expressed by a mammalian cell(s) genetically modified to express and secrete the fusion protein. The genetically modified cell(s) can be derived from a variety of different mammalian cell types (e.g., human cells), including epithelial cells, endothelial cells, fibroblasts, mesenchymal stem cells, keratinocyte cells, and stem cells, such as embryonic stem cells or induced pluripotent stem cells. Exemplary cell types include those described in WO2017 / 075631. In some embodiments, the cells are derived from the cell lines shown in Table 4. [Table 6]

[0162] In embodiments, any of the genetically modified mammalian cells described herein are derived from RPE cells, e.g., ARPE-19 cells. In embodiments, the genetically modified ARPE-19 cells comprise any of the expression cassettes, transposons, and polynucleotides described herein.

[0163] Cells can be genetically modified to express and secrete the desired BBB transport fusion protein using any of a variety of genetic engineering techniques known in the art. For example, cells can be transfected with an expression vector that includes an exogenous nucleotide sequence(s) encoding the desired fusion protein operably linked to control elements necessary or useful for gene expression, such as promoters, ribosome binding sites, enhancers, polyA signals, etc. In some embodiments, the exogenous nucleotide sequence is part of a transcription unit that is stably integrated into the genome of the parent cell.

[0164] In embodiments, the exogenous sequence comprises a nucleotide sequence encoding a secretory signal sequence for the fusion protein. In embodiments, the signal sequence is from a naturally secreted protein. In embodiments, the signal sequence is MELGLSWVVLAALLQGVQA (SEQ ID NO: 79). In some embodiments, the signal sequence consists essentially of the amino acid sequence shown in Table 5 below. [Table 7]

[0165] The genetically modified mammalian cells for use in the devices, compositions, and methods described herein may be in various stages of the cell cycle, for example, as a plurality of cells in a hydrogel capsule. In some embodiments, at least one cell in the plurality of genetically modified cells is undergoing cell division. Cell division may be measured using any method known in the art, for example, as described in DeFazio A et al (1987) J Histochem Cytochem 35:571-577 and Dolbeare F et al (1983) Proc Natl Acad Sci USA 80:5573-5577, each of which is incorporated by reference in its entirety. In embodiments, at least 1, 2, 3, 4, 5, 10, or 20% of the cells are undergoing cell division, as determined, for example, by 5-ethynyl-2'deoxyuridine (EdU) assay or 5-bromo-2'-deoxyuridine (BrdU) assay. In some embodiments, cell proliferation is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry). In some embodiments, none of the cells in the plurality of genetically modified cells are undergoing cell division and are quiescent. In embodiments, 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).

[0166] In embodiments, at least 50%, 60%, 70%, 80%, 90% or more of the genetically modified cells in the plurality are viable. Cell viability can be measured using any method known in the art, for example, as described in Riss, T. et al (2013) "Cell Viability Assays" in Assay Guidance Manual (Sittapalam, GSet al, eds). For example, cell viability can be measured or quantified by ATP assay, 5-ethynyl-2'deoxyuridine (EdU) assay, 5-bromo-2'deoxyuridine (BrdU) assay. In some embodiments, cell viability is visualized or quantified by microscopy (e.g., fluorescence microscopy (e.g., time-lapse or evaluation of spindle formation) or flow cytometry). In embodiments, at least 80% of the plurality of engineered cells are viable, as determined, for example, 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 assessment of spindle formation), or flow cytometry.

[0167] Any of the parameters described herein may be assessed using standard techniques known to those of skill in the art, such as histology, microscopy, and various functional assays.

[0168] Implantable cell encapsulation devices A genetically modified cell or a plurality of such cells as described herein may be incorporated into an implantable device for use in providing therapeutic or diagnostic cargo to a subject having a CNS disease or condition, e.g., a patient having a LSD, e.g., MPS-I.

[0169] The implantable devices of the present disclosure include at least one barrier that prevents immune cells from contacting cells contained within the device. At least a portion of the barrier must be sufficiently porous to allow proteins (e.g., fusion proteins) expressed and secreted by the cells to exit the device. A variety of device configurations known in the art are suitable.

[0170] The device (e.g., particle) can have any suitable configuration and shape to support the viability and productivity of contained cells after implantation at the intended target site. As non-limiting examples, the device shape can be cylindrical, rectangular, disk-shaped, ovoid, star-shaped, or spherical. The device can be composed of mesh-like or nested structures. In some embodiments, the device can prevent materials over a certain size from passing through the pores or openings. In some embodiments, the device (e.g., particle) can prevent materials over 50 kD, 75 kD, 100 kD, 125 kD, 150 kD, 175 kD, 200 kD, 250 kD, 300 kD, 400 kD, 500 kD, 750 kD, or 1,000 kD from passing.

[0171] In an embodiment, the device is a macroencapsulation device.Non-limiting examples of macrodevices are described in WO2019 / 068059, WO2019 / 169089, U.S. Patent Nos. 9,526,880, 9,724,430, and 8,278,106, European Patent No. 742818B1, and Sang, S. and Roy, S., Biotechnol.Bioeng.113(7):1381-1402(2016).

[0172] In an embodiment, the device is a macro device with one or more cell-containing compartments. A device with two or more cell-containing compartments can be configured to produce two or more proteins, for example, cells expressing a fusion protein are placed in one compartment, and cells expressing a different protein (e.g., a therapeutic protein that can alleviate one or more symptoms of a targeted CNS disease or condition) are placed in another compartment. WO2018 / 232027 describes a device with multiple cell-containing compartments formed in a microassembly and covered by a porous membrane.

[0173] In embodiments, the device is configured as a thin, flexible strand as described in U.S. Patent No. 10,493,107. The strand includes a substrate, an inner polymeric coating surrounding the substrate, and an outer hydrogel coating surrounding the inner polymeric coating. The protein-expressing cells are disposed within the outer coating.

[0174] In some embodiments, the devices (e.g., particles) have a largest linear dimension (LLD), e.g., average diameter, or size, that is at least about 0.5 millimeters (mm), preferably about 1.0 mm, about 1.5 mm, or more. In some embodiments, the devices can be as large as 10 mm in diameter or size. For example, the devices or particles described herein may be 0.5mm to 10mm, 1mm to 10mm, 1mm to 8mm, 1mm to 6mm, 1mm to 5mm, 1mm to 4mm, 1mm to 3mm, 1mm to 2mm, 1mm to 1.5mm, 1.5mm to 8mm, 1.5mm to 6mm, 1.5mm to 5mm, 1.5mm to 4mm, 1.5mm to 3mm, 1.5mm to 2mm, 2mm to 8mm, 2mm to 7mm, 2mm to 6mm, 2mm to 5mm, 2mm to 4mm, 2mm to 3mm, 2.5mm to 8mm, 2.5mm to 7mm, 2.5mm to 6mm, 2.5mm to 5mm, 2.5mm to 4mm, 2.5mm to 3mm, 3mm to 8mm m, in size range of 3mm~7mm, 3mm~6mm, 3mm~5mm, 3mm~4mm, 3.5mm~8mm, 3.5mm~7mm, 3.5mm~6mm, 3.5mm~5mm, 3.5mm~4mm, 4mm~8mm, 4mm~7mm, 4mm~6mm, 4mm~5mm, 4.5mm~8mm, 4.5mm~7mm, 4.5mm~6mm, 4.5mm~5mm, 5mm~8mm, 5mm~7mm, 5mm~6mm, 5.5mm~8mm, 5.5mm~7mm, 5.5mm~6mm, 6mm~8mm, 6mm~7mm, 6.5mm~8mm, 6.5mm~7mm, 7mm~8mm, or 7.5mm~8mm.

[0175] In some embodiments, the devices (e.g., particles, capsules) of the present disclosure include at least one hole or opening, e.g., to allow free flow of material. In some embodiments, the average pore size of the device is about 0.1 μm to about 10 μm. For example, the average pore size may be 0.1 μm to 10 μm, 0.1 μm to 5 μm, 0.1 μm to 2 μm, 0.15 μm to 10 μm, 0.15 μm to 5 μm, 0.15 μm to 2 μm, 0.2 μm to 10 μm, 0.2 μm to 5 μm, 0.25 μm to 10 μm, 0.25 μm to 5 μm, 0.5 μm to 10 μm, 0.75 μm to 10 μm, 1 μm to 10 μm, 1 μm to 5 μm, 1 μm to 2 μm, 2 μm to 10 μm, 2 μm to 5 μm, or 5 μm to 10 μm. In some embodiments, the average pore size of the device is about 0.1 μm to 10 μm. In some embodiments, the average pore size of the device is about 0.1 μm to 5 μm. In some embodiments, the average pore size of the device is between about 0.1 μm and 1 μm.

[0176] In some embodiments, the device includes a semi-permeable biocompatible membrane that surrounds the genetically modified cells that are encapsulated in a polymeric composition (e.g., an alginate hydrogel). The membrane pore size is selected to allow oxygen and other molecules important to cell survival and function to migrate through the semi-permeable membrane while preventing immune cells from traversing through the pores. In embodiments, the semi-permeable membrane has a molecular weight cutoff of less than 1000 kD, or between 50 and 700 kD, between 70 and 300 kD, or between 70 and 150 kD, or between 70 and 130 kD.

[0177] In embodiments, the device may contain a cell-containing compartment surrounded by a barrier compartment formed from a cell-free biocompatible material, such as the core-shell microcapsules described in Ma, M et al., Adv. Healthc Mater., 2(5):667-672 (2012). Such barrier compartments may be used with or without a semi-permeable membrane.

[0178] The cells in the cell-containing compartment(s) of the device of the present disclosure may be encapsulated in a polymer composition. The polymer composition may include 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 include or be formed from materials such as metals, metal alloys, ceramics, polymers, fibers, inert materials, and combinations thereof. The device may be made entirely of one type of material, or may include other materials in the cell-containing compartment and any other compartments.

[0179] In some embodiments, the device comprises a metal or metal alloy. In embodiments, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all of the compartments) comprises a metal or metal alloy. Exemplary metals or metal alloys include titanium and titanium-based alloys (e.g., Nitinol, nickel-titanium alloys, thermal memory alloy materials), platinum, platinum-based alloys, stainless steel, tantalum, palladium, zirconium, niobium, molybdenum, nickel-chromium, chromium-molybdenum alloys, or certain cobalt alloys (e.g., cobalt-chromium and cobalt-chromium-nickel alloys, e.g., ELGILOY® and PHYNOX®). For example, the metal material can be stainless steel grade 316 (SS316L) (composed of Fe, <0.3% C, 16-18.5% Cr, 10-14% Ni, 2-3% Mo, <2% Mn, <1% Si, <0.45% P, and <0.03% S). In metal-containing devices, the amount of metal (e.g., weight %, actual weight) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0180] In some embodiments, the device comprises a ceramic. In embodiments, one or more of the compartments in the device (e.g., the first compartment, the second compartment, or all of the compartments) comprises a ceramic. Exemplary ceramic materials include oxides, carbides, or nitrides of transition elements, such as titanium oxide, hafnium oxide, iridium oxide, chromium oxide, aluminum oxide, and zirconium oxide. Silicon-based materials, such as silica, may also be used. In ceramic-containing devices, the amount of ceramic (e.g., weight %, actual weight) may be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0181] In some embodiments, the device has two hydrogel compartments, an inner cell-containing compartment completely surrounded by a second, outer (e.g., barrier) compartment. In embodiments, the inner boundary of the second compartment forms an interface with the outer boundary of the first compartment. In such embodiments, the thickness of the second (outer) compartment refers to the average distance between the outer boundary of the second compartment and the interface between the two compartments, e.g., the average of the distances measured at each of the thinnest and thickest points visually observed in the outer compartment. In some embodiments (e.g., the device is about 1.5 mm in diameter), the thinnest and thickest distances in the outer compartment are 25-110 micrometers (μm) and 270-480 μm, respectively. In some embodiments, the thickness of the outer compartment is greater than about 10 nanometers (nm), preferably 100 nm or more, and can be as large as 1 millimeter (mm). For example, the thickness (e.g., average distance) of the outer compartment in the hydrogel capsule device described herein can be 10 nm to 1 mm, 100 nm to 1 mm, 500 nm to 1 millimeter, 1 micrometer (μm) to 1 mm, 1 μm to 1 mm, 1 μm to 500 μm, 1 μm to 250 μm, 1 μm to 1 mm, 5 μm to 500 μm, 5 μm to 250 μm, 10 μm to 1 mm, 10 μm to 500 μm, or 10 μm to 250 μm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is 100 nm to 1 mm, 1 μm to 1 mm, 1 μm to 500 μm, or 5 μm to 1 mm. In some embodiments, the thickness (e.g., average distance) of the outer compartment is about 50 μm to about 100 μm. In some embodiments (eg, the device is about 1.5 mm in diameter), the thickness (eg, average distance) of the outer section is about 180 μm to 260 μm or about 310 μm to 440 μm.

[0182] In some embodiments of the two-compartment hydrogel capsule device, the average pore size of the cell-containing inner compartment and the outer compartment are substantially the same. In some embodiments, the average pore size of the inner compartment and the second compartment differs by about 1.5%, 2%, 5%, 7.5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In some embodiments, the average pore size of the device (e.g., the average pore size of the first compartment and / or the average pore size of the second compartment) depends on a number of factors, such as the material(s) in each compartment and the presence and density of the compound of formula (III).

[0183] In some embodiments, the polymer composition in the cell-containing compartment(s) comprises a polysaccharide or other hydrogel-forming polymer (e.g., alginate, hyaluronic acid, or chondroitin). In some embodiments, the polymer is alginate, a polysaccharide composed of β-D-mannuronic acid (M) and α-L-guluronic acid (G). In some embodiments, the alginate has a low molecular weight (e.g., an approximate molecular weight of <75 kDa) and a G:M ratio of ≧1.5, (ii) a medium molecular weight alginate, e.g., an approximate molecular weight of 75-150 kDa and a G:M ratio of ≧1.5, (iii) a high molecular weight alginate, e.g., an approximate MW of 150 kDa-250 kDa and a G:M ratio of ≧1.5, or (iv) a blend of two or more of these alginates. In some embodiments, the cell-containing compartment(s) further comprises at least one cell-binding substance (CBS), such as a cell-binding peptide (CBP) or cell-binding polypeptide (CBPP) described in WO2020 / 069429.

[0184] In some embodiments, the cell-containing compartment(s) comprises alginate covalently modified with a linker-cell-binding peptide moiety, e.g., GRGD or GRGDSP. In embodiments, the cell-binding peptide density (e.g., % nitrogen determined by combustion analysis as described in WO2020 / 198695) in the cell-containing compartment(s) is at least 0.05%, 0.1%, 0.2%, or 0.3%, but less than 4%, 3%, 2%, or 1%. In embodiments, the total density of linker-CBP in the cell-containing compartment is about 0.1 to about 1.0 micromoles of CBP per gram of CBP polymer in solution (e.g., MMW-alginate covalently modified with GRGD (SEQ ID NO: 88) or GRGDSP (SEQ ID NO: 89)) as determined by a quantitative peptide conjugation assay, e.g., the assay described in WO2020 / 198695. In one embodiment, the linker-CBP is GRGDSP and the alginate has a molecular weight of 75 kDa to 150 kDa and a G:M ratio of 1.5 or greater. In an embodiment, the cell-containing compartment also comprises unmodified alginate having a molecular weight of 75 kDa to 150 kDa and a G:M ratio of 1.5 or greater.

[0185] The device may form part of multiple substantially identical devices in the preparation (e.g., composition). In some embodiments, the devices (e.g., particles, hydrogel capsules) in the preparation have an average diameter or size of about 0.5 mm to about 8 mm. In some embodiments, the devices in the preparation have an average diameter or size of about 0.5 mm to about 4 mm or about 0.5 mm to about 2 mm. In some embodiments, the devices in the preparation are two-compartment hydrogel capsules and have an average diameter or size of about 0.7 mm to about 1.3 mm or about 1.2 mm to about 1.8 mm.

[0186] In some embodiments, the surface of the device comprises a compound capable of mitigating the FBR upon implantation in a subject, a non-fibrous compound described below. In devices that include a barrier compartment surrounding a cell-containing compartment, the non-fibrous compound may covalently modify polymers disposed throughout the barrier compartment and optionally throughout the cell-containing compartment.

[0187] In some embodiments, one or more compartments in the device comprise a non-fibrous polymer, e.g., a non-fibrous compound of formula (III) covalently bonded to the polymer. In embodiments, some or all of the monomers in the non-fibrous polymer are modified with the same compound of formula (III). In some embodiments, some or all of the monomers in the non-fibrous polymer are modified with different compounds of formula (III). In some embodiments, where the device is a two-compartment hydrogel capsule, the non-fibrous polymer is present only in the outer barrier compartment.

[0188] One or more compartments within the device may contain an unmodified polymer that is the same or different from the polymer in any non-fibrous polymer present in the device. In embodiments, the first compartment, the second compartment, or all compartments in the device contain an unmodified polymer.

[0189] Each of the modified and unmodified polymers in the device can be linear, branched, or crosslinked polymers, or polymers of a selected molecular weight range, degree of polymerization, viscosity, or melt flow rate. Branched polymers can include one or more of the following types: star polymers, comb polymers, brush polymers, dendrimerized polymers, ladder polymers, and dendrimers. The polymers can be thermoresponsive polymers, such as gels (e.g., that become solid or liquid upon exposure to heat or a selected temperature) or photocrosslinkable polymers. Exemplary polymers include polystyrene, polyethylene, polypropylene, polyacetylene, poly(vinyl chloride) (PVC), polyolefin copolymers, poly(urethanes), polyacrylates and polymethacrylates, polyacrylamides and polymethacrylamides, poly(methyl methacrylate), poly(2-hydroxyethyl methacrylate), polyesters, polysiloxanes, polydimethylsiloxanes (PDMS), polyethers, poly(orthoesters), poly(carbonates), poly(hydroxyalkanoates), polyfluorocarbons, PEEK®, Teflon® (polytetrafluoroethylene, PTFE), PEEK, silicones, epoxy resins, Kevlar®, Dacron® (made from ethylene glycol and terephthalic acid), and the like. Poly(lactic acid), poly(L-lactic acid) (PLLA), poly(lactic-glycolic acid) (PLGA), polydioxanone (PDA), or racemic poly(lactic acid), polycarbonates, (e.g., polyamides (e.g., nylon)), fluoroplastics, carbon fibers, agarose, alginates, chitosan, and blends or copolymers thereof.In a polymer-containing device, the amount of polymer (e.g., % by weight of the device, actual weight of polymer) can be at least 5%, e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0190] In some embodiments, one or more of the modified and unmodified polymers in the device comprises polyethylene. Exemplary polyethylenes include very low density polyethylene (ULDPE) (e.g., 0.890-0.905 g / cm, containing comonomers). 3 with polymers having densities in the range of 0.905 to 0.915 g / cm 3 with polymers having densities in the range of 0.915 to 0.935 g / cm 3 with polymers having densities in the range of about 0.915 to 0.935 g / m 3 with polymers having densities in the range of 0.926 to 0.940 g / cm, with or without comonomers; 3 with polymers having densities in the range of 0.940 to 0.970 g / cm, with or without comonomers; 3 with polymers having densities in the range of 0.1 to 100 MPa, and polyethylene glycol.

[0191] In some embodiments, one or more of the modified and unmodified polymers in the device comprises polypropylene. Exemplary polypropylenes include homopolymers, random copolymers (homophase copolymers), and impact copolymers (heterophase copolymers), as described, for example, in McKeen, Handbook of Polymer Applications in Medicine and Medical Devices, 3-Plastics Used in Medical Devices, (2014): 21-53.

[0192] In some embodiments, one or more of the modified and unmodified polymers in the device comprises polypropylene. Exemplary polystyrenes include general purpose, or crystalline (PS or GPPS), high impact (HIPS), and syndiotactic (SPS) polystyrene.

[0193] In some embodiments, one or more of the modified and unmodified polymers comprises a thermoplastic elastomer (TPE). Exemplary TPEs include: (i) TPA-polyamide TPEs, which comprise block copolymers of alternating hard and soft segments with amide chemical bonds in the hard blocks and ether and / or ester bonds in the soft blocks; (ii) TPC-co-polyester TPEs, which consist of block copolymers of alternating hard and soft segments, where the chemical bonds in the backbone are ester and / or ether; (iii) TPO-olefin TPEs, which consist of blends of polyolefins and conventional rubbers, where the rubber phase in the blend has little or no crosslinking; (iv) at least triblock copolymers of styrene and certain dienes, where the two end blocks (hard blocks) are polystyrene and the inner (v) TPU-urethane TPEs consisting of alternating hard and soft segment block copolymers having urethane chemical bonds in the hard blocks and ether, ester, or carbonate bonds, or mixtures thereof, in the soft blocks; (vi) TPV-thermoplastic rubber vulcanizates consisting of blends of thermoplastic materials and conventional rubbers where the rubber is crosslinked by a process of dynamic vulcanization during the blending and mixing steps; and (vii) TPZ unclassified TPEs which include any composition or structure other than those classified as TPA, TPC, TPO, TPS, TPU, and TPV.

[0194] In some embodiments, the unmodified polymer is an unmodified alginate. In some embodiments, the alginate is a high guluronic acid (G) alginate, comprising about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more guluronic acid (G). In some embodiments, the alginate is a high mannuronic acid (M) alginate, comprising about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more mannuronic acid (M). In some embodiments, the ratio of M:G is about 1. In some embodiments, the ratio of M:G is less than 1. In some embodiments, the ratio of M:G is greater than 1. In embodiments, the unmodified alginate has a molecular weight of 150 kDa to 250 kDa and a G:M ratio of ≧1.5.

[0195] In some embodiments, the non-fibrous polymer comprises an alginate chemically modified with a compound of formula (III). The alginate in the modified non-fibrous polymer may be the same or different from any unmodified alginate present in the device. In embodiments, the density (e.g., amount of conjugation) of the compound of formula (III) in the non-fibrous alginate is about 4.0% to about 8.0%, about 5.0% to about 7.0%, or about 6.0% to about 7.0% nitrogen (e.g., as determined by combustion analysis for nitrogen percentage). In one embodiment, the amount of compound 101 results in an increase in % of N (as compared to unmodified alginate) of about 0.5% to 2%, 2% to 4% N, about 4% to 6% N, about 6% to 8%, or about 8% to 10% N), where % N corresponds to the amount of compound 101 in the modified alginate, as determined by combustion analysis.

[0196] In other embodiments, the density (e.g., concentration) of a compound of formula (III) (e.g., Compound 101) in a non-fibrous alginate is defined as % w / w in a solution (e.g., saline), e.g., % weight of amine / weight of non-fibrous alginate, as determined by a suitable quantitative amine conjugation assay (e.g., by an assay described in WO2020 / 069429), and in certain embodiments, the density of a compound of formula (III) (e.g., Compound 101) is about 1.0% w / w to about 3.0% w / w, about 1.3% w / w to about 2.5% w / w, or about 1.5% w / w to 2.2% w / w.

[0197] In an alginate-containing device, the amount of modified and unmodified alginate (e.g., % by weight of the device, actual weight of alginate) can be at least 5%, e.g., at least 5%, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more, e.g., less than 20%, e.g., less than 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, or less, w / w.

[0198] The alginate in the non-fibrous polymer can be chemically modified with a compound of formula (III) 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 (III). The alginate polymer can be dissolved in water (30 mL / gram polymer) and treated with an activating agent (e.g., 2-chloro-4,6-dimethoxy-1,3,5-triazine (0.5 eq)) and a base (e.g., N-methylmorpholine (1 eq)). To this mixture can be added a solution of the compound of formula (III) (0.3 M) in acetonitrile. The reaction can be warmed to 55° C. for 16 hours, then allowed to cool to room temperature and gently concentrated via rotary evaporation, and the residue can then be dissolved, for example, in water. The mixture can then be filtered, for example, through a bed of cyano-modified silica gel (Silicycle), and the filter cake can be washed with water. The resulting solution can then be dialyzed (10,000 MWCO membrane) against water for 24 hours, for example, with two changes of water. The resulting solution can be concentrated, for example, via lyophilization, to provide the desired chemically modified alginate. The alginate in the non-fibrous polymer can be chemically modified with a compound of formula (III) using any suitable method known in the art, for example, as described in any of WO2021 / 119522, WO2019 / 195055, WO2018 / 067615, WO2017 / 075631, WO2016 / 019391, and WO2012 / 167223.

[0199] In embodiments, the device comprises at least one cell-containing compartment, and in some embodiments, two, three, four, or more cell-containing compartments, hi embodiments, each cell-containing compartment comprises a plurality of cells (e.g., living cells), and the cells in at least one of the compartments are capable of expressing and secreting a BBB transport fusion protein when the device is implanted in a subject.

[0200] In embodiments, all cells in the cell-containing compartment are derived from a single parent cell type or a mixture of at least two different parent cell types. In embodiments, all cells in the cell-containing compartment are derived from the same parent cell type, but a first plurality of derived cells are engineered to express a BBB transport fusion protein and a second plurality of derived cells are engineered to express a different therapeutic protein. In devices with two or more cell-containing compartments, the cells and the protein(s) produced thereby may be the same or different in each cell-containing compartment. In some embodiments, all cell-containing compartments are surrounded by a single barrier compartment. In some embodiments, the barrier compartment is substantially free of cells.

[0201] In embodiments, the cells to be incorporated into the device described herein, for example, into the hydrogel capsule, are prepared in the form of a cell suspension before being encapsulated in the device. The cells in the suspension can be in the form of single cells (e.g., from a monolayer cell culture) or can be provided in another form, for example, arranged on a microcarrier (e.g., a bead or matrix), or as a three-dimensional aggregate of cells (e.g., a cell cluster or spheroid). The cell suspension can include a plurality of cell clusters (e.g., as a spheroid) or microcarriers.

[0202] In addition to the fusion protein secreted by the encapsulated cells, the device (e.g., capsule, particle) may contain one or more exogenous substances not expressed by the cells, such as, for example, a nucleic acid (e.g., an RNA or DNA molecule), a protein (e.g., a hormone, an enzyme (e.g., glucose oxidase, kinase, phosphatase, oxygenase, hydrogenase, reductase) antibody, antibody fragment, antigen, or epitope), an active or inactive fragment of a protein or polypeptide, a small molecule, or a drug. In embodiments, the device is configured to release such exogenous substances.

[0203] Non-fibrotic (e.g., FBR-reducing) compounds In some embodiments, the devices described herein comprise at least one compound of formula (III): [ka] or a pharma- ceutically acceptable salt thereof, wherein: A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-, -N(R C )C(O)-, -C(O)N(R C )-, -N(R C )C(O)(C1-C6-alkylene)-, -N(R C )C(O)(C2-C6-alkenylene)-, -N(R C )N(R D )-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F ) y -, -Si(OR A )2-, -Si(R G )(OR A )-, -B(OR A )-, or a metal, each of which is optionally linked to a linking group (e.g., a linking group described herein), and one or more R 1 is optionally replaced by L 1 and L 3 is independently a bond, an alkyl, or a heteroalkyl, and each alkyl and heteroalkyl is selected from one or more R 2 is optionally replaced by L 2 is a bond, M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from one or more R 3 is optionally replaced by P is absent, cycloalkyl, heterocyclyl, or heteroaryl, each of which is selected from the group consisting of one or more R 4 is optionally replaced by Z is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -OR A , -C(O)R A , -C(O)OR A , -C(O)N(R C )(R D ), -N(R C )C(O)R A , -N(R C )(R D ), cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which may be selected from the group consisting of one or more R 5 is optionally replaced by Each R A , R B , R C , R D , R E , R F , and R G is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of one or more R 6 and is optionally replaced by or R C and R D together with the nitrogen atom to which they are attached, form one or more R 6 forming an optionally substituted ring (e.g., a 5- to 7-membered ring) with Each R 1 , R 2 , R 3 , R4 , R 5 , and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), S.R. E1 , S(O) x R E1 , -OS(O) x R E1 , -N(R C1 )S(O) x R E1 , -S(O) x N(R C1 )(R D1 ), -P(R F1 ) y , cycloalkyl, heterocyclyl, aryl, and heteroaryl, each of which may be selected from the group consisting of one or more R 7 is optionally replaced by Each R A1 , R B1 , R C1 , R D1 , R E1 , and R F1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from the group consisting of one or more R 7 is optionally replaced by Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is 1 or 2; y is 2, 3, or 4.

[0204] In some embodiments, the compound of formula (III) is a compound of formula (III-a): [ka] or a pharma- ceutically acceptable salt thereof, wherein A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )-, -N(R C )C(O)-, -C(O)N(R C )-, -N(R C )N(R D )-, N(R C )C(O)(C1-C6-alkylene)-, -N(R C )C(O)(C1-C6-alkenylene)-, -NCN-, -C(=N(R C )(R D ))O-, -S-, -S(O) x -, -OS(O) x -, -N(R C )S(O) x -, -S(O) x N(R C )-, -P(R F ) y -, -Si(OR A )2-, -Si(R G )(OR A )-, -B(OR A )-, or a metal, each of which is optionally linked to a linking group (e.g., a linking group described herein), and one or more R 1 is optionally replaced by L 1 and L 3 is independently a bond, an alkyl, or a heteroalkyl, and each alkyl and heteroalkyl is selected from one or more R 2 is optionally replaced by L 2 is a bond, M is absent, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from one or more R 3 is optionally replaced by P is one or more R 4 is a heteroaryl optionally substituted by Z is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which may be selected from one or more R 5 is optionally replaced by Each R A , R B , R C , R D , R E , R F , and R G is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halogen, azido, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl is selected from the group consisting of one or more R 6 and is optionally replaced by or R C and R D together with the nitrogen atom to which they are attached, form one or more R 6 forming an optionally substituted ring (e.g., a 5- to 7-membered ring) with Each R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(RC1 )C(O)R B1 , -C(O)N(R C1 ), S.R. E1 , S(O) x R E1 , -OS(O) x R E1 , -N(R C1 )S(O) x R E1 , -S(O) x N(R C1 )(R D1 ), -P(R F1 ) y , cycloalkyl, heterocyclyl, aryl, and heteroaryl, each of which may be selected from the group consisting of one or more R 7 is optionally replaced by Each R A1 , R B1 , R C1 , R D1 , R E1 , and R F1 is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from the group consisting of one or more R 7 is optionally replaced by Each R 7 is independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, oxo, hydroxyl, cycloalkyl, or heterocyclyl; x is 1 or 2; y is 2, 3, or 4.

[0205] In some embodiments, for formula (III) and (III-a), A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, -N(R C )C(O)-, -N(RC )C(O)(C1-C6-alkylene)-, -N(R C )C(O)(C1-C6-alkenylene)-, or -N(R C In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C In some embodiments, A is alkyl, alkenyl, alkynyl, heteroalkyl, -O-, -C(O)O-, -C(O)-, -OC(O)-, or -N(R C In some embodiments, A is alkyl, -O-, -C(O)O-, -C(O)-, -OC(O), or -N(R C In some embodiments, A is -N(R C )C(O)-, -N(R C )C(O)(C1-C6-alkylene)-, or -N(R C )C(O)(C1-C6-alkenylene)-. In some embodiments, A is -N(R C In some embodiments, A is -N(R C )- and R C and R D is independently hydrogen or alkyl. In some embodiments, A is -NH-. In some embodiments, A is -N(R C )C(O)(C1-C6-alkylene)-, where alkylene is R 1 In some embodiments, A is substituted with -N(R C )C(O)(C1-C6-alkylene)-, R 1 is alkyl (e.g., methyl). In some embodiments, A is -NHC(O)C(CH)-. In some embodiments, A is -N(R C )C(O)(methylene)-, and R 1is alkyl (e.g., methyl). In some embodiments, A is -NHC(O)CH(CH3)-. In some embodiments, A is -NHC(O)C(CH3)-.

[0206] In some embodiments, for formula (III) or (III-a), L 1 is a bond, alkyl, or heteroalkyl. In some embodiments, L 1 is a bond or alkyl. In some embodiments, L 1 is a bond. In some embodiments, L 1 is alkyl. In some embodiments, L 1 is C1-C6 alkyl. In some embodiments, L 1 is -CH-, -CH(CH)-, -CHCHCHCH, or -CHCH-. In some embodiments, L 1 is -CH2- or -CH2CH2-.

[0207] In some embodiments, for formula (III) and (III-a), L 3 is a bond, alkyl, or heteroalkyl. In some embodiments, L 3 is a bond. In some embodiments, L 3 is alkyl. In some embodiments, L 3 is C1-C 12 In some embodiments, L 3 is C1-C6 alkyl. In some embodiments, L 3 In some embodiments, L 3 is heteroalkyl. In some embodiments, L 3 is one or more R 2 C1-C optionally substituted with (e.g., oxo) 12 In some embodiments, L is heteroalkyl. 3 is one or more R 2(e.g., oxo). In some embodiments, L 3 is -C(O)OCH-, -CH(OCHCH)-, -CH(OCHCH)-, CHCHO-, or -CHO-. In some embodiments, L 3 is -CH2O-.

[0208] In some embodiments, for formulas (III) and (III-a), M is absent, alkyl, heteroalkyl, aryl, or heteroaryl. In some embodiments, for formulas (III) and (III-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 and z is an integer selected from 1 to 10. In some embodiments, z is an integer selected from 1 to 5. In some embodiments, M is -(OCH2)2-, (-OCH2CH2-)2, (-OCH2CH2-)3, (-OCH2CH2-)4, or (-OCH2CH2-)5. In some embodiments, M is -OCH2CH 2-、 (-OCH2CH 2-)2 , (-OCH2CH2-)3, or (-OCH2CH 2- 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 [ka] In some embodiments, M is [ka] In some embodiments, M is 1 to 4 R 3 (For example, one R 3 In some embodiments, R 3 is CF3.

[0209] In some embodiments, for formulae (III) and (III-a), P is absent, heterocyclyl, or heteroaryl. In some embodiments, for formulae (III) and (III-a), P is absent, heterocyclyl, or heteroaryl. In some embodiments, P is absent. In some embodiments, for formulae (III) and (III-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 [ka] In some embodiments, P is [ka] In some embodiments, P is [ka] It is.

[0210] In some embodiments, P is heterocyclyl. In some embodiments, P is heterocyclyl. In some embodiments, P is 5-membered heterocyclyl. In some embodiments, P is imidazolidinonyl. In some embodiments, P is [ka] In some embodiments, P is thiomorpholinyl-1,1-dioxydyl. In some embodiments, P is [ka] It is.

[0211] In some embodiments, for formula (III) and (III-a), Z is alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl. In some embodiments, for formula (III) or (III-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 6-membered oxygen-containing heterocyclyl. In some embodiments, Z is tetrahydropyranyl. In some embodiments, Z is [ka] In some embodiments, Z is a 4-membered oxygen-containing heterocyclyl. In some embodiments, Z is [ka] It is.

[0212] In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments, Z is a bicyclic oxygen-containing heterocyclyl. In some embodiments, Z is phthalic anhydride. In some embodiments, Z is a sulfur-containing heterocyclyl. [ka] In some embodiments, Z is a 6-membered sulfur-containing heterocyclyl. [ka] In some embodiments, Z is a 6-membered heterocyclyl containing a nitrogen atom and a sulfur atom. In some embodiments, Z is thiomorpholinyl-1,1-dioxydyl. In some embodiments, Z is [ka] In some embodiments, Z is a nitrogen-containing heterocyclyl. In some embodiments, Z is a 6-membered nitrogen-containing heterocyclyl. In some embodiments, Z is [ka] It is.

[0213] In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is a bicyclic heterocyclyl. In some embodiments, Z is selected from one or more R 5 In some embodiments, Z is 2-oxa-7-azaspiro[3.5]nonanyl. In some embodiments, Z is [ka] In some embodiments, Z is 1-oxa-3,8-diazaspiro[4.5]decan-2-one. In some embodiments, Z is [ka] It is.

[0214] In some embodiments, for Formula (III) and (III-a), Z is aryl. In some embodiments, Z is monocyclic aryl. In some embodiments, Z is phenyl. In some embodiments, Z is a monosubstituted phenyl (e.g., one R 5 In some embodiments, Z has one R 5 In some embodiments, Z is a monosubstituted phenyl, wherein R 5 In some embodiments, Z is a monosubstituted phenyl where R 5 In some embodiments, Z is a monosubstituted phenyl, wherein R 5 In some embodiments, Z is an oxygen-containing heteroalkyl, where R 5 In some embodiments, Z is a monosubstituted phenyl where R 5 In some embodiments, Z is a monosubstituted phenyl having one R 5 In some embodiments, Z is a monosubstituted phenyl having one R 5 is a monosubstituted phenyl in the para position.

[0215] In some embodiments, for formula (III) and (III-a), Z is alkyl. In some embodiments, Z is C-C 12 In some embodiments, Z is C-C alkyl. 10 In some embodiments, Z is C1-C8 alkyl. In some embodiments, Z is 1 to 5 R 5In some embodiments, Z is C1-C8 alkyl substituted with one R 5 In some embodiments, Z is C1-C8 alkyl substituted with one R 5 C1-C8 alkyl substituted with R 5 is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , or -N(R C1 )(R D1 In some embodiments, Z is one R 5 C1-C8 alkyl substituted with R 5 -OR A1 OR -C(O)OR A1 In some embodiments, Z is one R 5 C1-C8 alkyl substituted with R 5 -OR A1 Or -C(O)OH. In some embodiments, Z is -CH3.

[0216] In some embodiments, for formula (III) and (III-a), Z is heteroalkyl. In some embodiments, Z is C-C 12 In some embodiments, Z is a C-C 10 In some embodiments, Z is a C1-C8 heteroalkyl. In some embodiments, Z is a C1-C6 heteroalkyl. In some embodiments, Z is selected from one or more R 5 In some embodiments, Z is a nitrogen-containing heteroalkyl optionally substituted with 1 to 5 R 5 In some embodiments, Z is N-methyl-2-(methylsulfonyl)ethane-1-aminyl.

[0217] In some embodiments, Z is -OR A OR -C(O)OR AIn some embodiments, Z is -OR A (e.g., -OH or -OCH3). In some embodiments, Z is -OCH3. In some embodiments, Z is -C(O)OR A (e.g., -C(O)OH).

[0218] In some embodiments, Z is hydrogen.

[0219] In some embodiments, L 2 is a bond, and P and L 3 is independently absent. In some embodiments, L 2 is a bond, P is heteroaryl, and L 3 is a bond and Z is hydrogen. In some embodiments, P is heteroaryl and L 3 is heteroalkyl and Z is alkyl.

[0220] In some embodiments, the compound of formula (III) is a compound of formula (III-b): [ka] or a pharma- ceutically acceptable salt thereof, wherein ring M 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 3 and optionally substituted with ring Z 1 is 1 to 5 R 5 cycloalkyl, heterocyclyl, aryl or heteroaryl optionally substituted with R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R 2a and R 2b Or R 2c and R2d each taken together forms an oxo group, X is absent, N(R 10 )(R 11 ), O, or S, and R C is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 6 R 6 and each R 3 , R 5 , and R 6 are independently alkyl, alkenyl, alkynyl, heteroalkyl, halogen, cyano, azido, oxo, -OR A1 , -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -N(R C1 )(R D1 ), -N(R C1 )C(O)R B1 , -C(O)N(R C1 ), S.R. E1 , cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 10 and R 11 each independently represents hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, -C(O)OR A1 , -C(O)R B1 , -OC(O)R B1 , -C(O)N(R C1 ), cycloalkyl, heterocyclyl, or heteroaryl, and each R A1 , R B1 , R C1 , R D1 , and R E1 are independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, and each of alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is selected from 1 to 6 R 7 and each 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; [ka] refers to a connection to a linking group or polymer as described herein. In some embodiments, each R 3 and R 5 wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl is optionally and independently substituted with halogen, oxo, cyano, cycloalkyl, or heterocyclyl.

[0221] In some embodiments, the compound of formula (III-b) is a compound of formula (III-bi): [ka] or a pharma- ceutically acceptable salt thereof, wherein ring M 2 is one or more R 3 and the ring Z is an aryl or heteroaryl optionally substituted with 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b Or R 2c and R 2d taken together form an oxo group, X is absent, O, or S, and each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1wherein each alkyl and heteroalkyl is optionally substituted with halogen, or two R 5 Together, they form the ring Z 2 A 5- or 6-membered ring is formed by condensing each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; and p is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0222] In some embodiments, the compound of formula (III-bi) is a compound of formula (III-b-ii): [ka] or a pharma- ceutically acceptable salt thereof, 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl; R 2c and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R together form an oxo group, and each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 wherein each alkyl and heteroalkyl is optionally substituted with halogen; and each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; each of p and q is independently 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0223] In some embodiments, the compound of formula (III) is a compound of formula (III-c): [ka] or a pharma- ceutically acceptable salt thereof, 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; R 2c and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2c and R 2d are taken together to form an oxo group, and each R 3 and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR, -C(O)OR, or -C(O)R B1 wherein each alkyl and heteroalkyl is optionally substituted with halogen; and each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m is 1, 2, 3, 4, 5, or 6; and each of p and q is independently 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0224] In some embodiments, the compound of formula (III) is a compound of formula (III-d) [ka] or a pharma- ceutically acceptable salt thereof, 2 is cycloalkyl, heterocyclyl, aryl, or heteroaryl; X is absent, O, or S; R 2a , R2b , R 2c , and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b Or R 2c and R 2d taken together form an oxo group, and each R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 wherein each alkyl and heteroalkyl is optionally substituted with halogen; and each R A1 and R is independently hydrogen, alkyl, or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5, or 6; and p is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0225] In some embodiments, the compound of formula (III) is a compound of formula (III-e) [ka] or a pharma- ceutically acceptable salt thereof, 2 is cycloalkyl, heterocyclyl, aryl or heteroaryl, X is absent, O or S, and R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b Or R 2c and R 2d taken together form an oxo group, and each R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1, -C(O)OR A1 , or -C(O)R B1 And each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; each of m and n is independently 1, 2, 3, 4, 5, or 6; and p is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0226] In some embodiments, the compound of formula (III) is a compound of formula (III-f) [ka] or a pharma- ceutically acceptable salt thereof, wherein M is one or more R 3 and ring P is an alkyl optionally substituted with one or more R 4 and L is heteroaryl optionally substituted with 3 is one or more R 2 and Z is an alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from the group consisting of one or more R 5 Optionally replaced by R 2a and R 2b Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b are taken together to form an oxo group, and each R 2 , R 3 , R 4 , and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 And each R A1 and R B1is independently hydrogen, alkyl, or heteroalkyl; and n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0227] In some embodiments, the compound of formula (III) is a compound of formula (IV): [ka] or a pharma- ceutically acceptable salt thereof, wherein M is a bond, alkyl, or aryl, and the alkyl and aryl are each independently selected from one or more R 3 and optionally substituted with L 3 is one or more R 2 Z is hydrogen, alkyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, or -OR, and the alkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl are each independently selected from one or more R 5 Optionally replaced by R A is hydrogen and R 2a and R 2b Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b are taken together to form an oxo group, and each R 2 , R 3 , and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , or -C(O)R B1 And each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; and n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0228] In some embodiments, the compound of formula (IV) is a compound of formula (IV-a): [ka] or a pharma- ceutically acceptable salt thereof, wherein L 3 is alkyl or heteroalkyl, each of which may be one or more R 2 and Z is hydrogen, alkyl, heteroalkyl, or -OR A and heteroalkyl is one or more R 5 Optionally replaced by R 2a and R 2b Each of is independently hydrogen, alkyl, or heteroalkyl, or R 2a and R 2b are taken together to form an oxo group, and each R 2 , R 3 , and R 5 are independently alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 and R A is hydrogen, and each R A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; and n is independently 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0229] In some embodiments, the compound of formula (III) is a compound of formula (V): [ka] or a pharma- ceutically acceptable salt thereof, 1 is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 Optionally replaced by R 2a , R 2b , R 2c , and R 2d each is independently hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, halo, cyano, nitro, amino, cycloalkyl, heterocyclyl, aryl, or heteroaryl, or R 2a and R 2b Or R 2c and R 2d taken together form an oxo group, R C is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, each of which is selected from 1 to 6 R 6 Optionally replaced by R 3 , R 5 , and R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 And each R 12 is independently deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; and w is 0 or 1; [ka] refers to the connection to a linking group or polymer as described herein.

[0230] In some embodiments, the compound of formula (V) is a compound of formula (Va): [ka] or a pharma- ceutically acceptable salt thereof, 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 Optionally replaced by R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen, alkyl, heteroalkyl, halo, or R 2a and R 2b Or R 2c and R 2d together form an oxo group, and R C is hydrogen, alkyl, alkenyl, alkynyl, or heteroalkyl, each of which is selected from 1 to 6 R 6 Optionally replaced by R 3 , R 5 , and R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 And each R 12 is independently deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; A1 and R B1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; o and p are each independently 0, 1, 2, 3, 4, or 5; q is an integer from 0 to 25; and w is 0 or 1; [ka] refers to the connection to a linking group or polymer as described herein.

[0231] In some embodiments, the compound of formula (V) is a compound of formula (Vb): [ka] or a pharma- ceutically acceptable salt thereof, 1 is cycloalkyl, heterocyclyl, aryl, or heteroaryl, each of which is selected from 1 to 5 R 5 Optionally replaced by R C is hydrogen, alkyl, -N(R C )C(O)R B , -N(R C )C(O)(C1-C6-alkyl), or -N(R C )C(O)(C1-C6-alkenyl), each of the alkyl and alkenyl groups being 1 to 6 R 6 Optionally replaced by R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen or alkyl, or R 2a and R 2b Or R 2c and R 2d together form an oxo group, and R 3 , R 5 , and R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 And each R 12 is independently hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; A1 , R B1 , and R E1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; and x is 0, 1, or 2; [ka] refers to the connection to a linking group or polymer as described herein.

[0232] In some embodiments, the compound of formula (V) is a compound of formula (Vc): [ka] or a pharma- ceutically acceptable salt thereof, wherein R C is hydrogen, alkyl, -N(R C )C(O)R B , -N(R C )C(O)(C1-C6-alkyl), or -N(R C )C(O)(C1-C6-alkenyl), each of the alkyl and alkenyl groups being 1 to 6 R 6 Optionally replaced by R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen or alkyl, or R 2a and R 2b Or R 2c and R 2d together form an oxo group, and R 3 , R 5 , and R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 and R 12 is hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino, and each R A1 , R B1 , and R E1 is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; x is 0, 1, or 2; z is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0233] In some embodiments, the compound of formula (V) is a compound of formula (Vd): [ka] or a pharma- ceutically acceptable salt thereof, wherein X is C(R')(R"), N(R'), or S(O). x each of R′ and R″ is independently hydrogen, alkyl, or halogen; C is hydrogen, alkyl, -N(R C )C(O)R B , -N(R C )C(O)(C1-C6-alkyl), or -N(R C )C(O)(C1-C6-alkenyl), each of the alkyl and alkenyl groups being 1 to 6 R 6 Optionally replaced by R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen or alkyl, or R 2a and R 2b Or R 2c and R 2d together form an oxo group, and R 3 , R 5 , and R 6 each independently is alkyl, heteroalkyl, halogen, oxo, -OR A1 , -C(O)OR A1 , or -C(O)R B1 And each R 12 is independently hydrogen, deuterium, alkyl, heteroalkyl, haloalkyl, halo, cyano, nitro, or amino; A1 , R B1 , and R E1is independently hydrogen, alkyl, or heteroalkyl; m and n are each independently 1, 2, 3, 4, 5, or 6; q is an integer from 0 to 25; x is 0, 1, or 2; z is 0, 1, 2, 3, 4, 5, or 6; [ka] refers to the connection to a linking group or polymer as described herein.

[0234] In some embodiments, X is S(O) x In some embodiments, x is 2. In some embodiments, X is S(O).

[0235] In some embodiments, R 2a , R 2b , R 2c , and R 2d Each of is independently hydrogen.

[0236] In some embodiments, R C is hydrogen, -C(O)(C-C-alkyl), or -C(O)(C-C-alkenyl). In some embodiments, each of alkyl and alkenyl is selected from one R 6 (e.g., -CH3). In some embodiments, R C is hydrogen.

[0237] In some embodiments, n is 1. In some embodiments, q is 2, 3, 4, or 5. In some embodiments, q is 3. In some embodiments, m is 1. In some embodiments, p is 0. In some embodiments, R 12 is halo (e.g., Cl).

[0238] In some embodiments, the compound is a compound of formula (III). 2 is a bond, and P and L 3is, independently, non-existent.

[0239] In some embodiments, the compound is a compound of formula (III-a). In some embodiments of formula (IV-a), L 2 is a bond, P is heteroaryl, and L 3 is a bond and Z is hydrogen. In some embodiments, P is heteroaryl and L 3 is heteroalkyl and Z is alkyl. In some embodiments, L 2 is a bond, and P and L 3 is independently absent. In some embodiments, L 2 is a bond, P is heteroaryl, and L 3 is a bond and Z is hydrogen. In some embodiments, P is heteroaryl and L 3 is heteroalkyl and Z is alkyl.

[0240] In some embodiments, the compound is a compound of formula (III-b). In some embodiments, P is absent and L 1 is -NHCH2, L 2 is a bond, M is aryl (e.g., phenyl), and L 3 is -CH2O and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, such as thiomorpholinyl-1,1-dioxide). In some embodiments, the compound of Formula (Ib) is compound 116.

[0241] In some embodiments of Formula (III-b), P is absent and L 1 is -NHCH2, L 2 is a bond, M is absent, and L 3 is a bond and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl, such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of Formula (III-b) is compound 105.

[0242] In some embodiments, the compound is a compound of formula (III-bi). In some embodiments of formula (III-bi), R 2a and R 2b is independently hydrogen or CH3; 2c and R 2d are each independently hydrogen, m is 1 or 2, n is 1, X is O, p is 0, and M 2 is one or more R 3 phenyl optionally substituted with R 3 is -CF3, Z 2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of formula (III-bi) is Compound 100, Compound 106, Compound 107, Compound 108, Compound 109, or Compound 111.

[0243] In some embodiments, the compound is of formula (III-b-ii). In some embodiments of formula (III-b-ii), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, q is 0, p is 0, m is 1, and Z 2 is heterocyclyl (e.g., an oxygen-containing heterocyclyl, such as tetrahydropyranyl). In some embodiments, the compound of formula (III-b-ii) is compound 100.

[0244] In some embodiments, the compound is of formula (III-c). In some embodiments of formula (III-c), R 2c and R 2d is independently hydrogen, m is 1, p is 1, q is 0, and R 5is -CH3 and Z is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, such as piperazinyl). In some embodiments, the compound of Formula (Ic) is compound 113.

[0245] In some embodiments, the compound is of formula (III-d). In some embodiments of formula (III-d), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 3, X is O, p is 0, and Z is heterocyclyl (e.g., an oxygen-containing heterocyclyl, such as tetrahydropyranyl, tetrahydrofuranyl, oxetanyl, or oxiranyl). In some embodiments, the compound of formula (III-d) is compound 110 or compound 114.

[0246] In some embodiments, the compound is of formula (III-f). In some embodiments of formula (III-f), R 2a and R 2b is independently hydrogen, n is 1, M is -CH-, P is a nitrogen-containing heteroaryl (e.g., imidazolyl), and L 3 is -C(O)OCH2- and Z is CH3. In some embodiments, the compound of Formula (III-f) is compound 115.

[0247] In some embodiments, the compound is a compound of formula (IV-a). In some embodiments of formula (IV-a), R 2a and R 2b is independently hydrogen, n is 1, q is 0, and L 3 is -CH2(OCH2CH2)2 and Z is -OCH3. In some embodiments, the compound of Formula (IV-a) is compound 112.

[0248] In some embodiments of formula (IV-a), R 2a and R 2bis independently hydrogen, n is 1, and L 3 is a bond or -CH2, and Z is hydrogen or -OH 。 In some embodiments, the compound of formula (IV-a) is compound 103 or compound 104.

[0249] In some embodiments, the compound is a compound of formula (V). In some embodiments of formula (V), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 2, q is 3, p is 0, and R C is hydrogen, and Z 1 is R 5 (e.g., -N(CH3)(CH2CH2)S(O)2CH3). In some embodiments, the compound of Formula (V) is compound 120.

[0250] In some embodiments, the compound is a compound of formula (Vb). In some embodiments of formula (Vb), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 0, n is 2, q is 3, p is 0, and Z 2 is one R 5 (e.g., —NH 2 ). In some embodiments, the compound of Formula (III-b) is compound 102.

[0251] In some embodiments, the compound is a compound of formula (Vb). In some embodiments of formula (Vb), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 2, q is 3, p is 0, and R C is hydrogen, and Z 2is heterocyclyl (e.g., a nitrogen-containing heterocyclyl, e.g., a nitrogen-containing spiroheterocyclyl, e.g., 2-oxa-7-azaspiro[3.5]nonanyl). In some embodiments, the compound of Formula (Vb) is compound 121.

[0252] In some embodiments, the compound is of formula (Vd). In some embodiments of formula (Vd), R 2a , R 2b , R 2c , and R 2d is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O). 2a and R 2b is independently hydrogen, m is 1, n is 2, q is 1, 2, 3, or 4, p is 0, and X is S(O). In some embodiments, the compound of Formula (Vd) is Compound 101, Compound 117, Compound 118, or Compound 119.

[0253] In some embodiments, the compound is a compound of formula (III-b), (III-d), or (III-e). In some embodiments, the compound is a compound of formula (III-b), (III-d), or (IV). In some embodiments, the compound is a compound of formula (III-b), (III-d), or (III-f). In some embodiments, the compound is a compound of formula (III-b), (III-d), or (V).

[0254] In some embodiments, the compound of formula (I) is not a compound disclosed in WO2012 / 112982, WO2012 / 167223, WO2014 / 153126, WO2016 / 019391, WO2017 / 075630, US2012 / 0213708, US2016 / 0030359, or US2016 / 0030360.

[0255] In some embodiments, the compound of formula (III) comprises a compound set forth herein below in Table 6, or a pharma- ceutically acceptable salt thereof. In some embodiments, the exterior surface and / or one or more compartments within the devices described herein comprise a small molecule compound set forth in Table 6, or a pharma- ceutically acceptable salt thereof. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8]

[0256] Conjugation of any of the compounds in Table 6 with a polymer (e.g., alginate) can be carried out as described in Example 2 of WO2019 / 195055, or any other suitable chemical reaction.

[0257] In some embodiments, the compound is a compound of formula (III) or a pharma- ceutically acceptable salt thereof: [ka] or a pharma- ceutically acceptable salt thereof.

[0258] In some embodiments, the devices described herein include [ka] or a pharma- ceutically acceptable salt of either compound.

[0259] In some embodiments, the compound of formula (III) (e.g., compound 101 in Table 6) is covalently attached to an alginate (e.g., an alginate having approximately a MW<75 kDa, G:M ratio ≧1.5) with a conjugation density of at least 2.0% and less than 9.0%, or between 3.0% and 8.0%, between 4.0 and 7.0, between 5.0 and 7.0, or between 6.0 and 7.0, or about 6.8, as determined by combustion analysis for nitrogen percentage as described in WO2020 / 069429. In one embodiment, the conjugation density of compound 101 in the modified alginate is determined by quantitative free amine analysis, e.g., as described in WO2020198695, and the determined conjugation density is between 1.0% w / w and 3.0% w / w, between 1.3% w / w and 2.8% w / w, between 1.3% w / w and 2.6% w / w, between 1.5% w / w and 2.4% w / w, between 1.5% w / w and 2.2% w / w, or between 1.7% w / w and 2.2% w / w.

[0260] The device, device preparation, or device composition may be configured for implantation or may be implanted or placed in any part or portion of the body. In some embodiments, the implantable device or device preparation is configured for implantation in the peritoneal cavity (e.g., also known as the omental bursa or bursalis omentum). The device, device preparation, or device composition may be implanted in the peritoneal cavity (e.g., the omentum, e.g., the bursa) or placed on a surface in the peritoneal cavity (e.g., the omentum, e.g., the bursa) via injection or catheter. Additional considerations regarding the implantation or placement of the device, device preparation, or device composition in the omentum (e.g., the bursa) are provided in M. Pellicciaro et al. (2017) CellR45(3):e2410.

[0261] Device fabrication Genetically modified ARPE-19 cells for use in manufacturing the devices described herein can be generated and cultured using methods known in the art, for example, stably transduced ARPE-19 cells can be cultured in vitro substantially as described in WO2020 / 198695.

[0262] Compounds of formula (III) and alginates modified with such compounds can be obtained using procedures known in the art, for example those substantially as described in WO2020 / 198695.

[0263] Alginate solutions for making non-fibrous, two-compartment hydrogel capsules can be obtained using procedures known in the art, for example substantially as described in WO2020 / 198695.

[0264] Two-compartment hydrogel capsules ("shielded capsules") encapsulating genetically modified mammalian cells in the inner compartment and non-fibrous alginate in the outer layer can be produced using procedures known in the art, for example, substantially as described in WO2020 / 198696.

[0265] Treatment method Described herein are methods for preventing or treating a CNS disease or condition (e.g., a lysosomal storage disease, e.g., MPS-1) in a subject by administering or implanting a pharmaceutical composition or genetically modified cells described herein. In an embodiment, the pharmaceutical composition comprises a BBB transport fusion protein and is formulated for intravenous or subcutaneous administration. In another embodiment, the pharmaceutical composition comprises a plurality of cells that have been genetically modified to express a BBB transport fusion protein. In an embodiment, the cells are encapsulated in a hydrogel capsule described herein. In another embodiment, the cells are encapsulated in a macrodevice described herein. In some embodiments, the methods described herein directly or indirectly reduce or alleviate at least one symptom of a CNS disease or condition, or prevent or delay the onset of the disease. In an embodiment, the method comprises administering (e.g., implanting) an effective amount of a composition of a two-compartment alginate hydrogel capsule comprising genetically modified RPE cells and a cell-binding polymer as described herein in the inner compartment, and a compound of formula (III), e.g., compound 101, in the outer capsule surface and, optionally, in the outer compartment.

[0266] Treating MPS-1 The present disclosure provides a method of treating a human patient with MPS-1 by administering to the patient a BBB transport IDUA fusion protein as described herein. In embodiments, the administering step comprises implanting cells engineered to express and secrete the BBB transport IDUA fusion protein, which may be encapsulated in a macrodevice or a two-compartment alginate hydrogel capsule as described herein. In embodiments, the encapsulated cells expressing the BBB transport IDUA fusion protein are implanted in the peritoneal cavity (e.g., also known as the bursa, omental bursa, or bursalis omentum). In embodiments, the BBB transport IDUA fusion protein expressed and secreted by the implanted cells comprises SEQ ID NO:28 or SEQ ID NO:29.

[0267] The therapeutic efficacy of treatment with the BBB transport IDUA fusion protein may be assessed using one or more efficacy measures used in approved experimental MPS1 therapies. Such efficacy measures typically include reduction in urinary glycosaminoglycan (e.g., heparan sulfate) levels, reduction in liver volume, stable forced vital capacity, increase in 6-minute walking distance, improvement in apnea / hypopnea index, increase in shoulder flexion, improvement in Child Health Assessment Questionnaire / Health Assessment Questionnaire disability index. In embodiments, efficacy measures are taken at a desired time point after implantation of cells expressing the GGG transport IDUA fusion protein and compared to baseline levels before implantation. In embodiments, the desired time point is any one or more of 15 days, 30 days, 60 days, 120 days, 1 year, or longer.

[0268] In embodiments, treatment of a subject with a BBB transport IDUA fusion protein described herein results in reduced heparan sulfate levels in the subject's brain, and optionally one or more non-CNS organs and tissues, such as the liver, spleen, kidney, heart, and lungs, hi embodiments, heparan sulfate levels are reduced by at least 10%, 25%, 50%, or more at a desired time point.

[0269] Enumerated exemplary embodiments 1. A fusion protein comprising an N-terminal to C-terminal structure defined by formula I: AB-L1-RB-L2-C or formula II: RB-L1-AB-L2-C, wherein in each of formulas I and II: AB comprises a domain that binds to human serum albumin (HSA); L1, which may be present or absent, comprises a first linker amino acid sequence; RB comprises a domain that binds to the extracellular domain of human IGF1R (hIGF1R); L2 may be present or absent and comprises a second linker amino acid sequence that is the same or different from the first linker amino acid sequence; The fusion protein, wherein C is a cargo moiety.

[0270] 2. The fusion protein of embodiment 1, defined by formula I:AB-L1-RB-L2-C.

[0271] 3. The fusion protein of embodiment 1, defined by formula II: RB-L1-AB-L2-C.

[0272] 4. The fusion protein of any one of the above embodiments, wherein AB has a molecular weight of less than about 75 kDa, about 50 kDa, or about 25 kDa.

[0273] 5. A fusion protein according to any one of the above embodiments, wherein RB has a molecular weight of less than about 75 kDa, about 50 kDa, or about 25 kDa.

[0274] 6. The fusion protein of any one of the preceding embodiments, wherein the cargo moiety has a molecular weight of about 1 kD to about 200 kD, or about 2 kD to about 100 kD.

[0275] 7. The fusion protein of any one of the previous embodiments, wherein the cargo moiety consists essentially of or consists of the amino acid sequence of a polypeptide.

[0276] 8. A fusion protein according to any one of the above embodiments, wherein (i) AB comprises a first, second and third amino acid sequence corresponding to the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of an anti-HSA antibody, or (ii) AB comprises the CDR1, CDR2 and CDR3 sequences of R11 sdAb, R28 sdAb, M75 sdAb or M79 sdAb as shown in Table 2A.

[0277] 9. The fusion protein according to any one of the previous embodiments, wherein AB comprises, consists essentially of, or consists of an amino acid sequence from a single domain antibody (sdAb), optionally wherein said amino acid sequence is selected from the group consisting of the R11, R28, M75, and M79 amino acid sequences disclosed in Table 2B above, the Alb-1 and Alb-8 amino acid sequences described in Tables II and III of WO2006 / 22787, and the Alb-23 amino acid sequence described in WO2012 / 175400.

[0278] 10. The fusion protein of claim 7, wherein the AB CDR1 sequence is GRTFIAYA (SEQ ID NO:1) or a conservatively substituted variant thereof, the AB CDR2 sequence is ITNFAGGTT (SEQ ID NO:2) or a conservatively substituted variant thereof, and the AB CDR3 sequence is AADRSAQTMRQVRPVLPY (SEQ ID NO:3) or a conservatively substituted variant thereof.

[0279] 11. A fusion protein according to any one of the above embodiments, wherein AB comprises, consists essentially of, or consists of an amino acid sequence from an sdAb.

[0280] 12. A fusion protein according to any one of the previous embodiments, wherein AB consists essentially of, or consists of, the parent or humanized sequences shown in Table 2B above.

[0281] 13.AB, 13. The fusion protein of any one of the preceding claims, consisting essentially of or consisting of QVQLVESGGGLVQAGGSLRLSCVASGRTFIAYAMGWFRQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNAKTTVYLQMNSLKPEDTALYYCAADRSAQTMRQVRPVLPYWGQGTQVTVSS (SEQ ID NO: 4), or a conservatively substituted variant thereof.

[0282] 14.AB, 13. The fusion protein of any one of the preceding claims, consisting essentially of or consisting of QVQLVESGGGLVQPGGSLRLSCAASGRTFIAYAMGWFRQAPGKEREFVAAITNFAGGTTYYADSVKGRFTISRDNAKTTVYLQMNSLRAEDTAVYYCAADRSAQTMRQVRPVLPYWGQGTLVTVSS (SEQ ID NO: 5), or a conservatively substituted variant thereof.

[0283] 15. The fusion protein of any one of embodiments 1-10, wherein AB consists essentially of, or consists of, the amino acid sequence of the heavy chain variable region of an antibody that cross-competes with an sdAb consisting of SEQ ID NO:4 or SEQ ID NO:5 for binding to HSA.

[0284] 16. The fusion protein of any one of the above embodiments, wherein the fusion protein binds to domain 1 (DI) or domain 2 (DII) of HSA via AB and does not substantially inhibit binding of human FcRn to HSA.

[0285] 17. The fusion protein has a dissociation constant (K) of less than about 0.1 nM to about 1,000 nM to HSA via the AB domain in a pH range of about 5.0 to about 7.4, as determined by surface plasmon resonance at 25°C. D ) affinity binding to the fusion protein of any one of the above embodiments.

[0286] 18. The fusion protein binds to HSA via the AB domain with a K of about 0.5 nM to about 500 nM, about 1 nM to about 250 nM, about 5 nM to about 50 nM, about 10 nM to about 25 nM, or about 0.5 nM to about 1 nM within a pH range of about 5.5 to about 7.4. D 18. The fusion protein of embodiment 17, wherein the fusion protein binds at

[0287] 19. The fusion protein of any one of the previous embodiments, wherein the fusion protein binds to at least one mammalian serum albumin ortholog via AB at 25°C within a pH range of about 5.5 to about 7.4.

[0288] 20. The fusion protein of any one of the previous embodiments, wherein said fusion protein binds via AB to two or more mammalian serum albumins selected from the group consisting of mouse serum albumin, rat serum albumin, hamster serum albumin, rabbit serum albumin, guinea pig albumin, porcine albumin, feline albumin, canine albumin, and non-human primate serum albumin, and optionally wherein said non-human primate serum albumin is cynomolgus monkey serum albumin or rhesus monkey serum albumin.

[0289] 21. The fusion protein of any one of the previous embodiments, wherein said fusion protein binds to hIGF1R expressed on the surface of human brain endothelial cells via the RB domain.

[0290] 22. The fusion protein of any one of the previous embodiments, wherein the fusion protein does not substantially bind to the human insulin receptor (h-IR).

[0291] 23. The fusion protein of any one of the previous embodiments, wherein said fusion protein does not substantially inhibit the binding of insulin, insulin growth factor 1 (IGF1), or insulin growth factor 2 (IGF2) to hIGF1R.

[0292] 24. The fusion protein of any one of the previous embodiments, wherein said fusion protein binds via RB to an epitope in the hIGF1R extracellular domain comprising FENFLHNSIFVPR (SEQ ID NO: 6).

[0293] 25. The fusion protein binds to hIGF1R via RB with a K of about 0.1 nM to about 1,000 nM in a pH range of about 5.0 to about 7.4 as determined by surface plasmon resonance at 25°C. D 2. The fusion protein of any one of the above embodiments, which binds with affinity.

[0294] 26. The fusion protein binds to hIGF1R via RB with a K of (i) about 0.2 nM to any one of about 500 nM, about 250 nM, about 100 nM, about 50 nM, about 25 nM, or about 10 nM, (ii) about 0.5 nM to any one of about 250 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, or about 5 nM, or (iii) about 1 nM to any one of about 100 nM, about 50 nM, about 25 nM, about 10 nM, or about 5 nM. D 25. The fusion protein of embodiment 24, which binds at

[0295] 27. The fusion protein binds to hIGF1R via RB with a K of 1 nM to 10 nM. D 26. The fusion protein of embodiment 25, which binds at

[0296] 28. A fusion protein according to any one of the above embodiments, wherein the fusion protein binds to at least one mammalian IGF1R ortholog via RB at 25°C and within a pH range of about 5.0 to about 7.4.

[0297] 29. The fusion protein of any one of the above embodiments, wherein the fusion protein binds via RB to two or more mammalian IGF1R proteins selected from the group consisting of mouse IGF1R, rat IGF1R, hamster IGF1R, rabbit IGF1R, guinea pig IGF1R, dog IGF1R, feline IGF1R, and non-human primate IGF1R, and optionally wherein the non-human primate IGF1R is cynomolgus monkey IGF1R or rhesus monkey IGF1R.

[0298] 30. The fusion protein according to any one of the above embodiments, wherein (i) RB comprises a set of first, second and third amino acid sequences corresponding to the three complementarity determining regions CDR1, CDR2 and CDR3 of the heavy chain variable region of an anti-hIGF1R antibody, or (ii) RB comprises a set of first, second and third amino acid sequences selected from the CDR1, CDR2 and CDR3 amino acid sequences of IGF1R-5 sdAb, IGF1R-3 sdAb and IGF1R-4 sdAb shown in Table 3A above.

[0299] 31. The fusion protein of embodiment 30, wherein the RB CDR1 sequence is GRTIDNYA (SEQ ID NO: 7) or a conservatively substituted variant thereof, the RB CDR2 sequence is IDWGDGGX (X is A or T) (SEQ ID NO: 8) or a conservatively substituted variant thereof, and the B3 CDR3 sequence is AMARQSRVNLDVARYDY (SEQ ID NO: 9) or a conservatively substituted variant thereof.

[0300] 32. The fusion protein of embodiment 31, wherein the RB CDR2 sequence is IDWGDGGA (sequence number 10).

[0301] 33. The fusion protein of embodiment 30, wherein RB consists essentially of or consists of the parent or humanized sequences shown in Table 3B above.

[0302] 34. The fusion protein of embodiment 30, wherein RB consists essentially of or consists of QVKLEESGGGLVQAGGSLRLSCAASGRTIDNYAMAWSRQAPGKDREFVATIDWGDGGARYANSVKGRFTISRDNAKGTMYLQMNNLEPEDTAVYSCAMARQSRVNLDVARYDYWGQGTQVTVSS (SEQ ID NO: 11), or a conservatively substituted variant thereof.

[0303] 35. The fusion protein of embodiment 30, wherein RB consists essentially of or consists of QVQLVESGGGLVQPGGSLRLSCAASGRTIDNYAMAWVRQAPGKGLEWVATIDWGDGGTRYANSVKGRFTISRDNSKNTMYLQMNSLRAEDTAVYYCAMARQSRVNLDVARYDYWGQGTLVTVSS (SEQ ID NO: 12), or a conservatively substituted variant thereof.

[0304] 36. A fusion protein according to any one of embodiments 1 to 30, wherein RB consists essentially of, or consists of, the amino acid sequence of the heavy chain variable region of an antibody that (i) cross-competes with an sdAb consisting of SEQ ID NO:11 or SEQ ID NO:12 for binding to hIGF1R, or (ii) cross-competes with any of the 996, 1226 and 1564 antibodies described in EP3725806A1.

[0305] 37. A fusion protein according to any one of the previous embodiments, wherein L1 and L2 are present.

[0306] 38. The fusion protein of embodiment 37, wherein each of L1 and L2 is a linker peptide less than 50 amino acids in length, and optionally each of L1 and L2 is about 15-30 amino acids in length, or about 20-25 amino acids in length.

[0307] 39. The fusion protein of embodiment 38, wherein L1 consists essentially of or consists of (GGGGS)m, where m is 4 or 5.

[0308] 40. The fusion protein according to any one of embodiments 37-39, wherein L2 consists essentially of or consists of (GGGGS)n, where n is 4 or 5.

[0309] 41. The fusion protein of embodiment 39, wherein each of L1 and L2 consists essentially of or consists of GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 14).

[0310] 42. The fusion protein of any one of the previous embodiments, wherein the cargo moiety is a cargo polypeptide selected from the group consisting of an enzyme, a growth factor, a cytokine, or an antibody or antigen-binding fragment thereof.

[0311] 43. The fusion protein of embodiment 42, wherein the cargo polypeptide comprises a mature amino acid sequence encoded by a wild-type human gene.

[0312] 44. The fusion protein of embodiment 42 or 43, wherein the cargo polypeptide is an enzyme.

[0313] 45. The fusion protein according to any one of embodiments 42 to 44, wherein the cargo polypeptide is an acid alpha-glucosidase protein (GAA), an alpha-galactosidase A (GLA) protein, an alpha-L-iduronidase (IDUA) protein, an alpha-N-acetyl-glucosaminidase (NAGLU) protein, a beta-glucoronidase (GUSB) protein, a beta-glucosidase (GBA) protein, an iduronate-2-sulfatase (IDS) protein, a heparan-alpha-glucosaminide N-acetyltransferase (HGSNAT) protein, an N-acetylgalactosamine-6-sulfatase (GNS) protein, or an N-sulfoglucosamine sulfohydrolase (SGSH) protein, and optionally, the cargo polypeptide is not an IDS protein.

[0314] 46. ​​The fusion protein of embodiment 45, wherein the cargo polypeptide is an IDUA protein, and optionally the cargo polypeptide comprises, consists essentially of, or consists of the mature amino acid sequence encoded by the wild-type human IDUA gene.

[0315] 47. The fusion protein of any one of embodiments 42-46, wherein the cargo polypeptide is an IDUA protein and comprises amino acids 26, 27, or 28 to amino acid 653 of the precursor human IDUA amino acid sequence shown in Figure 1H.

[0316] 48. The fusion protein of any one of embodiments 42-47, wherein the cargo polypeptide is an IDUA protein and consists essentially of amino acids 27 to 653 of the amino acid sequence shown in Figure 1H.

[0317] 49. A fusion protein according to any one of embodiments 46 to 48, having an IDUA enzyme activity that is within 80-120% of the corresponding enzyme activity of the wild-type human IDUA protein.

[0318] 50. The fusion protein of any one of embodiments 42 to 45, wherein the cargo polypeptide is an IDS protein, and optionally the cargo polypeptide comprises, consists essentially of, or consists of the amino acid sequence shown in Figure 12A or Figure 12B.

[0319] 51. The fusion protein of embodiment 50, having an IDS enzymatic activity that is within 80-120% of the corresponding enzymatic activity of the wild-type human IDS protein.

[0320] 52. The fusion protein of embodiment 1, comprising, consisting essentially of, or consisting of the amino acid sequence of the hIDUA fusion protein shown in Figure 3A, Figure 3B, Figure 3C, Figure 3D, Figure 3E, or Figure 3F, optionally wherein the fusion protein consists essentially of or consists of the amino acid sequence shown in SEQ ID NO:29, and wherein each of m and n = 4.

[0321] 53. The fusion protein of embodiment 1, comprising formula I: AB-L1-RB-L2-C, wherein AB comprises, consists essentially of, or consists of SEQ ID NO:4 or SEQ ID NO:5; L1 comprises, consists essentially of, or consists of (GGGGS)n, where n is 4 or 5; RB comprises SEQ ID NO:11 or SEQ ID NO:12; L1 comprises, consists essentially of, or consists of (GGGGS)n, where n is 4 or 5; C comprises, consists essentially of, or consists of amino acids 27-653 of SEQ ID NO:22; and L2 comprises, consists essentially of, or consists of (GGGGS)n, where n is 4 or 5.

[0322] 54. The fusion protein of embodiment 1, comprising Formula II: RB-L1-AB-L2-C, wherein AB comprises, consists essentially of, or consists of SEQ ID NO:4 or SEQ ID NO:5; L1 comprises, consists essentially of, or consists of (GGGGS)n, where n is 4 or 5; RB comprises SEQ ID NO:11 or SEQ ID NO:12; L1 comprises, consists essentially of, or consists of (GGGGS)n, where n is 4 or 5; comprises, consists essentially of, or consists of amino acids 27-653 of SEQ ID NO:22; and L2 comprises, consists essentially of, or consists of (GGGGS)n, where n is 4 or 5.

[0323] 55. The fusion protein according to embodiment 53 or 54, wherein AB consists essentially of SEQ ID NO:5 and RB consists essentially of SEQ ID NO:12.

[0324] 56. A polynucleotide comprising a first nucleotide sequence encoding a fusion protein according to any one of the above embodiments.

[0325] 57. The polynucleotide of embodiment 56, wherein the first nucleotide sequence is operably linked to a nucleotide sequence encoding a secretory signal sequence for the fusion protein, and optionally, the secretory signal sequence consists essentially of or consists of one of the amino acid sequences set out in Table 5: (i) MELGLSWVVLAALLQGVQA (SEQ ID NO: 79); or (ii) MELGLSWVVLAALLQGVQA (SEQ ID NO: 79).

[0326] 58. The polynucleotide of embodiment 57, wherein the secretory signal sequence consists essentially of or consists of MELGLSWVVLAALLQGVQA (sequence number 79).

[0327] 59. The polynucleotide according to any one of embodiments 56 to 58, wherein the first nucleotide sequence is operably linked to a promoter sequence and a polyA signal sequence.

[0328] 60. The polynucleotide of embodiment 59, wherein the promoter sequence is the pCAG promoter sequence shown in Figure 4A, the EF1α promoter sequence shown in Figure 4B, or the EFS promoter sequence shown in Figure 4C.

[0329] 61. The polynucleotide of embodiment 60, wherein the promoter sequence is the EF1α promoter sequence shown in Figure 4B.

[0330] 62. The polynucleotide of any one of embodiments 59 to 61, wherein the polyA signal sequence is the rBG polyA signal sequence shown in Figure 5A, the SV40 late polyA signal sequence shown in Figure 5B, or the BGH polyA signal sequence shown in Figure 5C.

[0331] 63. The polynucleotide of embodiment 62, wherein the polyA signal sequence is the rBG polyA signal sequence shown in Figure 5A.

[0332] 64. The polynucleotide according to embodiment 56, comprising the nucleotide sequence shown in Figures 7-1 to 7-3.

[0333] 65. The polynucleotide of any one of embodiments 56 to 64, which is one strand of an isolated double-stranded DNA molecule.

[0334] 66. A genetically modified mammalian cell, transiently or stably transfected with a polynucleotide according to any one of embodiments 56 to 65.

[0335] 67. The genetically modified mammalian cell of embodiment 66, wherein the polynucleotide is inserted at at least one position in the genome of the mammalian cell.

[0336] 68. The genetically modified mammalian cell of embodiment 66 or 67, wherein the cell is derived from a human cell.

[0337] 69. A genetically modified mammalian cell according to embodiment 68, which is an RPE cell, optionally derived from an ARPE-19 cell.

[0338] 70. The genetically modified mammalian cell of embodiment 68, which is derived from an induced pluripotent stem cell (iPSC) or a mesenchymal stem cell.

[0339] 71. A composition comprising a plurality of genetically modified cells, wherein each cell in the plurality is a genetically modified cell defined by any one of embodiments 68 to 70.

[0340] 72. The composition of embodiment 71, wherein the plurality of genetically modified cells is obtained from the culture of a monoclonal cell line.

[0341] 73. An implantable device comprising at least one cell-containing compartment comprising a genetically modified cell according to any one of embodiments 66 to 70 or a composition according to embodiment 71 or 72, and further comprising at least one means for mitigating a foreign body response (FBR) when the device is implanted in a subject.

[0342] 74. The implantable device of embodiment 74=3, wherein the cell-containing compartment comprises a polymer composition, the polymer composition comprising alginate covalently modified with a peptide, the peptide consisting essentially of or consisting of GRGDSP (SEQ ID NO: 89), GGGRGDSP (SEQ ID NO: 90), or GGGRGDSP (SEQ ID NO: 91).

[0343] 75. The implantable device of embodiment 73 or 74, wherein the cell-containing compartment is surrounded by a barrier compartment comprising an alginate hydrogel and, optionally, a compound of formula (III) disposed on the outer surface of the barrier compartment.

[0344] 76. The polymer composition comprises an alginate covalently modified with a peptide, the peptide consisting essentially of or consisting of GRGDSP, and the barrier compartment comprises: [ka] 76. The implantable device according to embodiment 74 or 75, comprising an alginate chemically modified with, or a pharma- ceutically acceptable salt thereof.

[0345] 77. An implantable device described in any one of embodiments 73 to 76, which is a spherical, two-compartment hydrogel capsule having a diameter of about 0.75 mm to about 2 mm.

[0346] 78. A preparation of devices, wherein each device in the preparation is a device according to any one of embodiments 73 to 77.

[0347] 79. A hydrogel capsule comprising: (a) an inner compartment comprising a plurality of genetically modified cells according to any one of embodiments 66 to 70 encapsulated in a first polymer composition, the first polymer composition comprising a hydrogel-forming polymer; (b) a barrier compartment surrounding the inner compartment and comprising a second polymer composition, the second polymer composition comprising an alginate covalently modified with at least one compound of formula (III) or a pharma-ceutically acceptable salt thereof.

[0348] 80. The hydrogel capsule of embodiment 79, wherein the compound of formula (III) is selected from the compounds provided in the table below. [Table 9]

[0349] 81. The selected compound is [ka] 77. The hydrogel capsule of embodiment 75 or 76, wherein

[0350] 82. A hydrogel capsule described in any one of embodiments 79 to 81, wherein the concentration of the genetically modified cells in the inner compartment is at least 40 million cells per ml of the first polymer composition.

[0351] 83. A capsule composition comprising a plurality of hydrogel capsules according to any one of embodiments 79 to 82 in a pharma- ceutically acceptable carrier.

[0352] 84. The capsule composition of embodiment 83, wherein the genetically modified cells in the plurality of hydrogel capsules express and secrete the IDUA fusion protein of embodiment 51.

[0353] 85. A pharmaceutical composition comprising a fusion protein according to any one of embodiments 1 to 55 and a pharma- ceutically acceptable carrier.

[0354] 86. The pharmaceutical composition described in embodiment 81, wherein the fusion protein is an IDUA fusion protein described in embodiment 48.

[0355] 87. A method for preventing or treating a disease or condition in the central nervous system (CNS) of a subject, comprising: (i) administering to said subject a pharmaceutical composition of embodiment 85 or 86; (ii) implanting in said subject a device or device preparation according to any one of embodiments 73 to 78; or (iii) implanting in the subject a capsule composition of embodiment 83 or 84.

[0356] 88. A method of treating a human subject diagnosed with Mucopolysaccharidosis Type 1 (MPS-1) disease, comprising: (a) providing a capsule composition according to embodiment 86; (b) placing the capsule composition within the subject's body.

[0357] 89. The method of embodiment 88, wherein the placing step comprises placing the capsule composition within the intraperitoneal space of the subject.

[0358] 90. The method of embodiment 88, wherein the placing step comprises placing the capsule composition in the abdominal cavity.

[0359] 91. The method of any one of embodiments 87 to 90, wherein the capsule composition produces a BBB transport IDUA fusion protein comprising, consisting essentially of, or consisting of SEQ ID NO:28 or SEQ ID NO:29. EXAMPLES

[0360] In order that the disclosure described herein may be more fully understood, the following examples are presented. The examples described in this application are provided to illustrate the BBB transport fusion proteins, genetically modified cells, implantable devices, and compositions and methods provided herein, and should not be construed in any way as limiting the scope thereof. The MPS-1 mice used in these examples were obtained from Charles River Laboratory (Wilmington, MA) and carry a nonsense mutation at Idua codon W392 (Jackson Laboratory Stock No. 017681).

[0361] Example 1: Effect of fusing an exemplary IGF1R binding domain to hIDUA on in vitro hIDUA activity. DNA expression vectors were engineered to encode six different hIDUA fusion enzymes containing a BBB-penetrating sdAb (IGF1R4) fused to the N-terminus or C-terminus of the hIDUA open reading frame via 3, 4, or 5 repeated variable length G4S linker units. ARPE-19 cells were transfected with these expression vectors, and polyclonal colonies stably expressing the hIDUA fusion enzymes were generated for each of the six different transfections. The expression vectors and transfected cells were named based on the orientation of the coding sequences of the composite modules relative to each other. For example, hIDUA-(G4S)3-IGF1R4 cells express a fusion enzyme in which IGF1R4 is fused to the C-terminus of hIDUA via 3 repeated G4S linkers.

[0362] In vitro IDUA activity of the hIDUA fusion proteins secreted from six polyclonal colonies was assessed by seeding cells from each polyclonal colony at approximately 400,000 cells in 2 ml of fresh medium per well of a 6-well tissue culture plate. After 20-24 hours, conditioned cell culture medium was collected and assayed for IDUA protein concentration using an IDUA activity assay and compared to a known standard (laronidase) essentially as described in Ou, L., et al., (2014). Standardization of α-L-iduronidase enzyme assay with Michaelis-Menten kinetics. Molecular Genetics and Metabolism, 111(2), 113-115. Briefly, 8 ul of cell culture medium was added to 32 ul of 0.4 M sodium formate, pH 3.5 in a black 96-well plate. 20ul of 700um 4-methylumbelliferyl-α-L-iduronide diluted in assay buffer was added to all wells. Plates were incubated at 37°C for 10 minutes. 100ul of stop solution (0.5M NaOH + 0.5M glycine) was added to stop the reaction. α-L-iduronidase catalyzed the cleavage of a non-fluorescent substrate (4MU-iduronide) to a fluorescent product (4-MU). Fluorescence intensity was measured in endpoint mode on a Biotek Cytation 3 with excitation and emission wavelengths of 365nm and 445nm (Topread), respectively. Enzyme activity levels were compared to a standard curve generated with laronidase and the results are shown in Figure 8. The polyclonal culture that produced the highest in vitro IDUA activity was the one expressing the IGF1R4-(G4S)4-hIDUA fusion enzyme.

[0363] Using this fusion expression vector, a new transfection of ARPE-19 cells was performed and a polyclonal pool of higher hIDUA activity was identified. Cells from this pool were encapsulated in the inner compartment of a shield capsule at 50 million cells / ml essentially as described in WO2020 / 198696. Five MPS-1 mice were implanted with a 0.5 ml dose capsule in the IP space of each. Twenty-eight days after administration, the mice were euthanized and the amount of hIDUA activity was assessed in liver and plasma tissue samples. Figure 8B shows the measured hIDUA activity levels compared to typical hIDUA levels observed in a substantially similar experiment using implanted capsules encapsulating ARPE-19 cells genetically modified to express and secrete wild-type hIDUA. This comparison shows that MPS-1 mice implanted with cells producing the IGF1R4-hIDUA fusion did not result in higher liver and plasma hIDUA activity compared to those implanted with cells producing wild-type hIDUA.

[0364] Example 2: Effect of fusing exemplary HSA and IGF1R binding domains to hIDUA on in vitro hIDUA activity. DNA expression vectors were engineered to encode six different hIDUA fusion enzymes containing a BBB-penetrating sdAb (IGF1R5), an anti-HSA sdAb (R28), and different orientations of the hIDUA open reading frame with a (G4S)4 amino acid linker placed between each of the open reading frames. The different orientations evaluated were (i) two fusions in which both sdAbs were fused to the N-terminus of hIDUA (N-terminal fusions), (ii) two fusions in which both sdAbs were fused adjacent to hIDUA (adjacent fusions), and (iii) two fusions in which both sdAbs were fused to the C-terminus of hIDUA (C-terminal fusions). ARPE-19 cells were transfected with these expression vectors and polyclonal colonies stably expressing the hIDUA fusion enzymes were generated for each of the six different transfections. The expression vectors and transfected cells were named based on the orientation of the coding sequences of the composite modules relative to each other. For example, IGF1R5-R28-hIDUA cells express a fusion enzyme in which IGF1R5 is N-terminal to R28, which is itself N-terminal to hIDUA.

[0365] The in vitro hIDUA activity of the hIDUA fusion proteins secreted from the six polyclonal colonies was assessed as described in Example 1, and the results are shown in Figure 9A. The polyclonal pools expressing the N-terminal double fusion enzymes (IGF1R5-R28-hIDUA and R28-IGF1R5-hIDUA) showed higher hIDUA activity levels than cell lines expressing adjacent or C-terminal fusions, a result consistent with the hIDUA activity results obtained in Example 1, which showed higher hIDUA activity produced with a single N-terminal fusion than with a C-terminal fusion. Surprisingly, the orientation of the anti-HSA and anti-IGF1R sdAb in the N-terminal fusion has a profound effect on hIDUA activity, with fusions containing R28 upstream of IGF1R5 yielding approximately 50% higher hIDUA activity than fusions with IGF1R-5 upstream of R28. The difference in activity between each of these six cell lines is statistically significant.

[0366] To assess whether the addition of the HSA binding domain affects the hIDUA activity of the fusion proteins, the top performing double fusion polyclonal pool (R28-(G4S) 4- IGF1R5-(G4S) 4- In vitro hIDUA activity in conditioned medium from cell cultures of IGF1R4-(G4S)4-hIDUA was compared to in vitro hIDUA activity in conditioned medium from cell cultures of the IGF1R4-(G4S)4-hIDUA polyclonal pool used in the MPS-1 mouse experiments described in Example 1. As shown in Figure 9B, there was essentially no difference in the amount of hIDUA activity in the single N-terminus and double N-terminus fusion cultures.

[0367] Example 3: Effect of HSA and IGF1R binding domains on in vivo hIDUA activity. R28-(G4S) 4- IGF1R5-(G4S) 4- Cells from a polyclonal pool secreting hIDUA fusion enzyme were encapsulated in the inner compartment of a shielded capsule at 50 million cells / ml essentially as described in WO2020 / 198696. A 0.5 ml dose capsule was implanted in the IP space of each of six MPS-1 mice (J). Twenty-one days after dosing, the mice were euthanized and the amount of hIDUA activity in plasma and various systemic tissues was assessed.

[0368] 10 shows the measured hIDUA activity levels compared to typical hIDUA levels observed in a substantially similar experiment using implanted shielded capsules encapsulating ARPE-19 cells genetically modified to express and secrete wild-type hIDUA. Mice implanted with cells expressing the R28-IGF1R5-hIDUA fusion enzyme had substantially higher hIDUA activity in tissues (except kidney) and plasma than mice implanted with cells expressing the wild-type hIDUA enzyme.

[0369] Example 4: The effect of implanting MPS-1 mice with shielded capsules producing exemplary BBB transport fusion proteins on brain heparan sulfate levels. Shield capsules encapsulating cells from a polyclonal pool secreting R28-(G4S)4-IGF1R5-(G4S)4-hIDUA fusion enzyme were implanted into the IP space of each of six MPS-1 mice. A group of six untreated MPS-1 mice was observed as a control. On day 21 post-administration, both groups of mice were euthanized and the amount of heparan sulfate in two different brain regions (hippocampus and frontal lobe) was evaluated. Briefly, tissue homogenates of each brain region were obtained and tissue homogenates (10ul) were mixed with an equal volume of heparinase cocktail (a mixture of heparinase I, heparinase II, and heparinase III in heparinase reaction buffer). Individual heparinases and heparinase reaction buffer were from New England Biolabs, catalog numbers P0735S, P0736S, P0737S, and B0735S. The reactions were incubated at 37° C. for 3 days, and the resulting heparan sulfate disaccharides were quantified by LC / MS.

[0370] As shown in FIG. 11, MPS-1 mice implanted with cells secreting the hIDUA BBB transport fusion protein had a statistically significant reduction in heparan sulfate in both brain tissues compared to control mice.

[0371] Example 5: Exemplary dual IDS fusions with different orientations of the HSA and IGF1R binding domains. DNA expression vectors were engineered to encode a BBB-permeable sdAb (IGF1R5), an anti-HSA sdAb (R28), and two different hIDS fusion enzymes containing different orientations of the hIDS open reading frames with a (G4S)4 amino acid linker placed between each of the open reading frames. The different orientations evaluated were R28-(G4S)4-IGF1R5-(G4S)4-hIDS (e.g., Formula I fusion) and IGF1R5-(G4S)4-R28-(G4S)4-hIDS (e.g., Formula II fusion). ARPE-19 cells were transfected with one of the two expression vectors, and polyclonal colonies stably expressing the hIDS fusion enzymes were generated for each of the two transfections.

[0372] In vitro IDS activity of the hIDS fusion proteins secreted from each polyclonal pool was assessed by seeding approximately 400,000 cells from each cell line in 2 ml of fresh medium per well of a 6-well tissue culture plate. After 20-24 hours, conditioned cell culture medium was collected and assayed for IDS protein concentration using a two-step enzymatic IDS activity assay and compared to a known standard (idursulfase). Briefly, 3 ul of cell culture medium was added to 15.6 ul of water and 20 ul of buffer containing 20 mM lead(II) acetate in 0.2 M sodium acetate (pH 5.0) in a black 96-well plate. 1.4 ul of 20 mM substrate (4-methylumbelliferyl-α-L-iduronide 2-sulfate reconstituted in DMSO) was added to all wells. Plates were incubated at 37°C for 10 minutes. To quench the first reaction, 40 ul of 2x McIlvaine's buffer (0.40 M sodium phosphate, 0.20 M citric acid, pH 4.5) supplemented with 5 ug / mL Laronidase was added to each well. The plate was incubated at 37°C for 10 minutes. To stop the second reaction, 100 ul of stop solution was added (0.5 M NaOH + 0.5 M glycine). In the first reaction step, the substrate (4MU-α-iduronide 2-sulfate) is hydrolyzed by iduronate 2-sulfatase (IDS) to generate 4MU-iduronide. In the second reaction step, iduronate 2-sulfatase activity was quenched by the addition of excess phosphate, and α-L-iduronidase catalyzed the cleavage of the non-fluorescent product of the first reaction (4MU-iduronide) to a fluorescent product (4-MU). Fluorescence intensity was measured in endpoint mode on a Biotek Cytation 3 with excitation and emission wavelengths of 365 nm and 445 nm (Topread), respectively. Enzyme activity levels were compared to a standard curve generated with idursulfase, and the results are shown in FIG.The polyclonal pool expressing the R28-(G4S)4-IGF1R5-(G4S)4-hIDS (Formula I) double fusion enzyme exhibited higher hIDS activity levels than the cell line expressing the IGF1R5-(G4S)4-R28-(G4S)4-hIDS (Formula II) double fusion, a result consistent with the hIDUA activity results obtained in Example 3 which showed higher hIDUA activity produced with the Formula I fusion (AB-L1-RB-L2-C) than with the Formula II fusion (RB-L1-AB-L2-C).

[0373] Example 6: Effects of implanting MPS-1 mice with shielded capsules producing exemplary BBB transport fusion proteins on heparan sulfate in systemic tissues. Shield capsules encapsulating cells from a polyclonal pool secreting R28-(G4S)4-IGF1R5-(G4S)4-hIDUA fusion enzyme were implanted into the IP space of each of six MPS-1 mice. A group of six untreated MPS-1 mice was observed as a control. On day 21 post-administration, both groups of mice were euthanized and the amount of heparan sulfate in various organs (liver, spleen, kidney, lung, and heart) was evaluated. Briefly, organ tissue homogenates were obtained and tissue homogenates (10 ul) were mixed with an equal volume of heparinase cocktail (a mixture of heparinase I, heparinase II, and heparinase III in heparinase reaction buffer). Individual heparinases and heparinase reaction buffer were from New England Biolabs, catalog numbers P0735S, P0736S, P0737S, and B0735S. The reactions were incubated at 37° C. for 3 days, and the resulting heparan sulfate disaccharides were quantified by LC / MS.

[0374] As shown in FIG. 14, MPS-1 mice treated with the dual fusion enzyme (black bars) showed a statistically significant decrease in heparan sulfate levels in each of the liver, spleen, kidney, lung, and heart tissue samples compared to untreated mice (gray bars).

[0375] Example 7: Evaluation of the potential immunogenicity of exemplary BBB transport fusion proteins. A commercial vendor (EpiVax, Providence, Rhode Island, USA) performed computational immunogenicity assessments on the following molecules: R28-(G4S)4-IGF1R5-(G4S)4-hIDUA fusion enzyme, (2) R28-H5-(G4S)4-IGF1R5-H2-(G4S)4-hIDUA fusion enzyme, where the R28-H5 component is a humanized R28 variant having the amino acid sequence (SEQ ID NO:5) and the IGF1R5-H2 component is a humanized IGF1R5 variant having the amino acid sequence (SEQ ID NO:12), (3) native human IDUA protein alone (i.e., not fused to any other molecule), and (4) the individual parental and humanized sdAbs present in the two fusion enzymes. Briefly, EpiVax used its proprietary EpiMatrix algorithm to evaluate the individual amino acid sequences containing the molecules in the fusion construct and predicted T cell epitopes. Each molecule was scored for total predicted T cell epitope content and ranked against the EpiMatrix protein immunogenicity scale, which allows molecules to be directly compared to known immunogenic proteins. As shown in the table immediately below, each of the fusion enzymes had a lower predicted immunogenicity score than native hIDUA alone. [Table 10]

[0376] Example 8: In vivo comparison of exemplary humanized and camelid BBB transport fusion proteins. Three groups of MPS-1 mice (six mice per group) were injected with the humanized IDUA fusion protein [R28-H5-(G4S 4- IGF1R5-H2-(G4S) 4- hIDUA or IGF1R5-H2-(G4S) 4-Shield capsules encapsulating cells from a polyclonal pool secreting either the camelid IDUA fusion protein [R28-H5-(G4S)4-hIDUA] or the camelid IDUA fusion protein [R28-(G4S)4-IGF1R5-(G4S)4-hIDUA] were implanted. The IDUA amino acid sequence in each fusion protein was identical. A group of four untreated MPS-1 mice was observed as controls. On day 28 post-treatment, all mouse cohorts were euthanized and the amount of heparan sulfate in the midbrain was evaluated. Briefly, midbrain tissue homogenates were obtained and tissue homogenates (10 ul) were mixed with an equal volume of heparinase cocktail (a mixture of heparinase I, heparinase II, and heparinase III in heparinase reaction buffer). Individual heparinases and heparinase reaction buffers were from New England Biolabs, catalog numbers P0735S, P0736S, P0737S, and B0735S. Reactions were incubated at 37° C. for 3 days, and the resulting heparan sulfate disaccharides were quantified by LC / MS. As shown in FIG. 15, MPS-1 mice treated with any of the three dual fusion enzymes (gray bars) showed a statistically significant reduction in heparan sulfate levels in the midbrain compared to untreated mice (black bars). There was no statistically significant difference in midbrain heparan sulfate reduction between treated mouse cohorts.

[0377] Equivalence and Scope This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification shall control. Also, any particular embodiment of the present disclosure that falls within the prior art may be expressly excluded from any one or more of the claims. Since such embodiments are deemed known to those skilled in the art, they may be excluded even if the exclusion is not expressly set forth herein. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether related to the existence of prior art or not.

[0378] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the present embodiments described herein is not intended to be limited to the above specification, drawings, or examples, but is as set forth in the appended claims. Those skilled in the art will appreciate that various changes and modifications to the specification may be made without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

[Claim 1] A fusion protein comprising an N-terminal to C-terminal structure defined by Formula I: AB-L1-RB-L2-C or Formula II: RB-L1-AB-L2-C, wherein for each of Formulas I and II: AB comprises a domain that binds to human serum albumin (HSA); L1, which may be present or absent, comprises a first linker amino acid sequence; RB comprises a domain that binds to the extracellular domain of human IGF1R (hIGF1R); L2 may be present or absent and comprises a second linker amino acid sequence that is the same as or different from the first linker amino acid sequence; The fusion protein wherein C is a cargo moiety, optionally said cargo moiety comprising, consisting essentially of, or consisting of the amino acid sequence of a polypeptide.

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