Methods and compositions for therapeutic protein delivery

Abstract

Compositions and methods for delivering therapeutic proteins to the central nervous system (CNS) to treat diseases and disorders that impair the CNS, such as the treatment of lysosomal storage diseases, are disclosed. The therapeutic proteins are provided via a therapeutically effective amount of a nucleotide composition encoding the therapeutic protein conjugated to a cell surface receptor-binding protein that crosses the blood-brain barrier (BBB).

Description

[Technical Field]

[0001] Reference to sequence listing submitted as a text file via EFS-WEB The sequence listing set forth in file 10457WO01_ST25.txt is 33.5 kilobytes, was created on January 29, 2019, and is incorporated herein by reference.

[0002] This application is generally directed to compositions and methods for delivering therapeutic proteins to the central nervous system (CNS) to treat diseases and disorders that impair the CNS, such as the treatment of lysosomal storage diseases. This application aims to provide therapeutically effective amounts of nucleotide compositions encoding therapeutic proteins conjugated to one or more delivery domains that cross the blood-brain barrier (BBB). [Background technology]

[0003] Drug delivery approaches have been developed to overcome the blood-brain barrier (BBB), such as nanocarriers, but they have drawbacks. Carriers have shown instability in the blood circulation and undesirable biodistribution profiles (Gelperina S, et al., 2005, Am J Respir Crit Care Med. 172(12):1487-90, incorporated herein by reference in its entirety). Targeting efficiency is also reduced by transport mechanisms at the BBB and whether CNS disease states alter the integrity of the barrier. The appropriate selection of targeting moieties or carriers must take into account neuroinflammatory conditions that affect these transport mechanisms. The delivery of therapeutic proteins via DNA expression in the liver or other tissues provides a convenient approach that eliminates the need for bolus injection of proteins, thus reducing the concern of immunogenicity.Receptor-binding proteins, especially therapeutic proteins conjugated to cell-specific receptors, solve the problem of targeting therapeutic agents to specific tissues.However, there is still a need to provide a method for efficiently delivering therapy to the CNS. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Gelperina S, et al., 2005, Am J Respir Crit Care Med.172(12):1487-90 Summary of the Invention [Means for solving the problem]

[0005] Applicants have discovered that therapeutic proteins, particularly replacement enzymes, when associated with receptor-binding proteins can be effectively delivered to the central nervous system, provided that consistent circulating blood levels are achieved over time. Multidomain therapeutic proteins can be delivered to the liver via gene therapy vectors carrying coding sequences for the therapeutic protein and the binding protein complex.

[0006] In one aspect, the present invention provides a method for delivering a therapeutic protein to the central nervous system (CNS) of a subject, comprising administering to the subject a nucleotide composition encoding a therapeutic protein (tpCSR-BP) conjugated to a cell surface receptor (CSR) binding protein (CSR-BP) via a liver-targeted delivery method sufficient to provide a therapeutically effective amount of tpCSR-BP in the CNS.

[0007] In one embodiment, the CSR-BP is an antibody or antigen-binding fragment thereof that specifically binds to CSR. In another embodiment, the therapeutic protein is a lysosomal enzyme.

[0008] In one embodiment, the enzyme has hydrolase activity, such as a glycosylase, e.g., a glycosidase, such as alpha-glucosidase or alpha-galacosidase A. In one embodiment, the cell surface receptor (CSR) binding protein (CSR-BP) is an antigen-binding protein that binds to an internalizing receptor. In one embodiment, the internalizing receptor is a cell surface molecule that is endocytosed and transported to lysosomes. In a specific embodiment, the internalizing receptor is a CD63 molecule. In one embodiment, the internalizing receptor is an ITGA7 molecule. In a specific embodiment, the CSR-BP is an antibody, antibody fragment, or scFv, such as a single-chain variable fragment (scFv) that binds to CD63 or ITGA7.

[0009] In some embodiments, the multidomain therapeutic proteins described herein comprise one or more delivery domains and an enzymatic domain, wherein the one or more delivery domains bind to the human transferrin receptor (hTfR). In some embodiments, the multidomain therapeutic protein further comprises a second delivery domain that binds to an internalization effector. In some embodiments, the second delivery domain is an internalization effector selected from the group consisting of: (i) CD63, integrin alpha 7 (ITGA7), MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), myelin and lymphocyte protein (MAL), IGF2R, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, and CD36; (ii) CD63, MHC-I, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, and CD36; ATPase, IGF2R, integrin alpha 7 (ITGA7), LRP5, LRP6, LRP8, Kremen-2, LDL receptor, LDL-related protein 1 receptor, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), PRLR, MAL (myelin and lymphocyte protein (MAL)), diphtheria toxin receptor, HBEGF (heparin-binding EGF-like growth factor), glutathione receptor, glutamate receptor, leptin receptor, and folate receptor. (iii) an internalization effector expressed preferentially by bone and / or cartilage, optionally selected from the group consisting of collagen X, integrin alpha 10 (ITGA10), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor isoform C (FGFR3C), hyaluronan and proteoglycan link protein 1 (CRTL1), aggrecan, collagen II, and Kremen-1;(iv) an internalization effector preferentially expressed by monocytes, macrophages, or microglia, optionally selected from the group consisting of scavenger receptor A1-5 (SCARA1-5), SCARB1-3, CD36, MSR1 (macrophage scavenger receptor 1), MRC1 (macrophage mannose receptor 1), VSIG4 (V-set and immunoglobulin domain-containing protein 4), CD68 (macrosialin), and CSF1R (macrophage colony-stimulating factor 1 receptor); (v) an internalization effector preferentially expressed by kidney cells, optionally selected from the group consisting of CDH16 (Cadherin-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (solute carrier family 22 member 13), SLC5A2 (sodium / glucose cotransporter 2), and UMOD (uromodulin). In other specific embodiments, the internalization effector is selected from the group consisting of BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadheri-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAIS; (vi) muscle-specific internalizers, such as ATP1B (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3); (vii) internalization effectors preferentially expressed by liver cells, such as ASGR1 or ASGR2; (vii) BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadherin-15 (viii) an internalization effector preferentially expressed by muscle cells optionally selected from the group consisting of ITGA7, CD9, CD63, ALPL2, MSR1, ASGR1, ASGR2, or PRLR. In some embodiments, the second delivery domain binds to the internalization effector CD63. In some embodiments, at least one of the one or more delivery domains comprises an antigen binding protein. In some embodiments, each of the one or more delivery domains comprises an antigen binding protein, hi some embodiments, at least one of the one or more delivery domains comprises a single chain variable fragment (scFv).In some embodiments, at least one of the one or more delivery domains comprises a half antibody. In some embodiments, the delivery domain that binds hTfR is an scFv, the half antibody binds CD63, and the enzymatic domain is GAA, with GAA conjugated to the carboxy terminus of the half antibody that binds CD63. In some embodiments, each of the one or more delivery domains comprises an scFv. In some embodiments, at least one scFv is fused to an Fc. In some embodiments, the Fc comprises a wild-type human IgG4 isotype or a derivative thereof. In some embodiments, GAA is conjugated to the carboxy terminus of the Fc. In some embodiments, the multidomain therapeutic protein comprises an anti-hTfR scFv and an anti-hCD63 scFv. In some embodiments, both the anti-hTfR scFv and the anti-hCD63 scFv are linked at their carboxy termini to a single GAA enzyme. In some embodiments, the delivery domain is an anti-hTfR scFv, and the enzymatic domain is linked to the carboxy terminus of the VL domain of the scFv. In some embodiments, the multi-domain further comprises a second delivery domain linked to the N-terminus of the VH domain of the anti-hTfR scFv. In some embodiments, the second delivery domain is an anti-hCD63 scFv. In some embodiments, the enzymatic domain comprises the amino acid sequence set forth as SEQ ID NO: 1.

[0010] Also provided is a multidomain therapeutic protein comprising at least two delivery domains and at least one enzymatic domain, wherein each of the two delivery domains is independently selected from the group consisting of an antibody, a half antibody, and an scFv, and wherein at least one or more delivery domains are associated with at least one enzymatic domain, preferably, one or more delivery domains are covalently linked to at least one enzymatic domain. In some embodiments, the multidomain therapeutic protein comprises two or fewer delivery domains. In some embodiments, only one of the delivery domains is associated with at least one enzymatic domain. In some embodiments, each of the at least two delivery domains is covalently linked to an enzymatic domain. In some embodiments, each of the at least two delivery domains is covalently linked to the same enzymatic domain. In some embodiments, each of the at least two delivery domains is covalently linked to a different enzymatic domain. In some embodiments, the multidomain therapeutic protein comprises two or fewer delivery domains, wherein a first delivery domain comprises a half antibody and a second delivery domain comprises an scFv. In some embodiments, the scFv is fused to Fc. In some embodiments, the half antibody is covalently linked at its carboxy terminus to a first enzymatic domain, and / or the scFv is covalently linked at its carboxy terminus to an Fc and, optionally, a second enzymatic domain. In some embodiments, the multidomain therapeutic protein comprises no more than two delivery domains, and the first and second delivery domains each comprise an scFv. In some embodiments, both the first and second scFvs are covalently linked to an enzymatic domain. In some embodiments, the multidomain therapeutic protein comprises, from N-terminus to C-terminus: a first scFv, a second scFv, and an enzymatic domain. In some embodiments, at least one delivery domain binds to a lysosomal transport molecule and at least one delivery domain binds to a transcytosis effector.In some embodiments, the lysosomal transport molecule is selected from the group consisting of CD63, ITGA7, CD9, CD63, CD81, CD82, or CD151, and the transcytosis effector is selected from the group consisting of LDL receptor, IgA receptor, transferrin receptor, neonatal Fc receptor, insulin receptor, CD98, and basigin. In some embodiments, the multidomain therapeutic protein comprises a structure shown in Figure 1C, Figure 1D, Figure 1E, or Figure 1F.

[0011] Also provided herein are polynucleotides encoding the multidomain therapeutic proteins described herein. In some embodiments, the polynucleotides provided herein further comprise a viral nucleic acid sequence and a locus targeting nucleic acid sequence. In some embodiments, the polynucleotide further comprises a viral nucleic acid sequence and a locus targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence. In some embodiments, the polynucleotide further comprises a viral nucleic acid sequence and a locus targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence, wherein the AAV nucleic acid sequence comprises an internal terminal repeat sequence and optionally comprises a tissue-specific regulatory element, such as a liver-specific promoter or a neuron-specific promoter. In some embodiments, the polynucleotide further comprises a viral nucleic acid sequence and a locus targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence comprising an internal terminal repeat sequence comprising SEQ ID NO: 6, SEQ ID NO: 7, or both, and optionally comprises a tissue-specific regulatory element, such as a liver-specific promoter or a neuron-specific promoter. In some embodiments, the polynucleotide further comprises a tissue-specific regulatory element comprising the sequence set forth as SEQ ID NO:8 and / or SEQ ID NO:9.

[0012] In one aspect, the present invention provides a gene therapy vector, such as an AAV vector, containing a nucleic acid sequence encoding a therapeutic protein conjugated to or fused to a CSR-BP, e.g., a polynucleotide described herein. In some embodiments, the gene therapy vector is selected from the group consisting of a viral vector, which may be a natural virus, an engineered virus, or a chimeric virus; a naked polynucleotide comprising a polynucleotide described herein, a polynucleotide complex, optionally a lipid nanoparticle comprising the polynucleotide described in any one of claims 20-25 and a lipid, and any combination thereof. In some embodiments, the gene therapy vector is a viral vector selected from the group consisting of a retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus. In some embodiments, the gene therapy vector is AAV9, Anc80, AAV2 / 8 chimera, and / or AAV pseudotyped for specific tissues, e.g., liver or neuronal tissue.

[0013] In one embodiment, a therapeutic protein, a nucleotide encoding the same, and / or a gene therapy vector comprising the nucleotide encoding the same is used to treat a subject in need of enzyme replacement therapy, e.g., in a method of delivering a therapeutic protein to the central nervous system (CNS), the method comprising administering to the subject a nucleotide composition encoding the multidomain therapeutic protein via a liver-targeted delivery method sufficient to provide a therapeutically effective amount of the multidomain therapeutic protein in the CNS, the multidomain therapeutic protein comprising a delivery domain and an enzymatic domain. In some embodiments, the subject is an animal. In some embodiments, the subject is a human.

[0014] In one aspect, an AAV vector containing a polynucleotide encoding an scFv-hydrolase fusion protein is administered to a human or non-human subject.The polynucleotide is then integrated into a genomic locus in the liver, and the encoded fusion protein is produced.In another embodiment, the polynucleotide is episomally transcribed in the liver, and the encoded fusion protein is produced.In a specific embodiment, the fusion protein is an anti-CD63scFv-GAA fusion protein or an anti-ITGA7scFv-GAA fusion protein, and the human or non-human subject lacks endogenous GAA activity, and GAA activity is effectively restored in the subject.

[0015] In one aspect, the present invention provides a method of treating a subject (human or non-human) having an enzyme deficiency by administering to the patient a gene therapy vector containing a gene encoding a therapeutic protein conjugated to or fused to a CSR-BP.

[0016] Described herein are methods for delivering a therapeutic protein to the central nervous system (CNS) of a subject, comprising administering to the subject a nucleotide composition encoding a multidomain therapeutic protein via a liver-targeted delivery method sufficient to provide a therapeutically effective amount of the multidomain therapeutic protein in the CNS, wherein the multidomain therapeutic protein comprises a delivery domain and an enzymatic domain. In some embodiments, the delivery domain is an antibody or antigen-binding fragment thereof that specifically binds to an internalization effector. In some embodiments, the therapeutic protein is a lysosomal enzyme. In some embodiments, the lysosomal enzyme is GAA. In some embodiments, the nucleotide composition is administered via a viral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the nucleotide composition is administered at a concentration of at least 2 x 10 per kilogram. 12The viral genome (vg / kg) is administered at a dose of 1000 mg / kg. In some embodiments, the internalization effector is expressed on the surface of a cell selected from the group consisting of cells in the CNS, epithelial cells, and cells that cross the blood-brain barrier. In some embodiments, the delivery domain is linked to the internalization effector. In some embodiments, the internalization effector is selected from the group consisting of (i) CD63, integrin alpha 7 (ITGA7), MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), myelin and lymphocyte protein (MAL), IGF2R, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, and CD36; (ii) e.g., CD63, MHC-I, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, and CD36; ATPase, IGF2R, integrin alpha 7 (ITGA7), LRP5, LRP6, LRP8, Kremen-2, LDL receptor, LDL-related protein 1 receptor, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), PRLR, MAL (myelin and lymphocyte protein (MAL)), diphtheria toxin receptor, HBEGF (heparin-binding EGF-like growth factor), glutathione receptor, glutamate receptor, leptin receptor, and folate receptor are expressed in several tissue types, and optionally the subject is diagnosed with Fabry disease, Gaucher disease, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIID, MPS IVB, MPS VI, MPS VII, MPS VIII, MPS VIIIA, MPS VIIIB, MPS VIIIC, MPS VIIID ... IX, Pompe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, Niemann-Pick disease types A, B, and C2, alpha-mannosidosis, neuraminidase deficiency, (iii) exhibit one or more symptoms of a disease selected from the group consisting of sialidosis, aspartylglycosaminuria, mixed saposin deficiency, atypical Gaucher disease, Farber lipogranulomatosis, fucosidosis, and beta-mannosidosis; (iv) exhibit one or more symptoms of a disease selected from the group consisting of: sialidosis, aspartylglycosaminuria, mixed saposin deficiency, atypical Gaucher disease, Farber lipogranulomatosis, fucosidosis, and beta-mannosidosis; (v) exhibit one or more symptoms of a disease selected from the group consisting of: (i) exhibit one or more symptoms of a disease selected from the group consisting of: sialidosis, aspartylglycosaminuria, mixed saposin deficiency, atypical Gaucher disease, Farber lipogranulomatosis, fucosidosis, and beta-mannosidosis; (vi ... (IX), beta-mannosidosis, Gaucher disease, atypical Gaucher disease, mixed saposin deficiency, aspartylglycosaminuria, Farber lipogranulomatosis, sialidosis, neuraminidase deficiency, and alpha-mannosidosis; (iv) a gene encoding, for example, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, CD36, MSR1 (macrophage scavenger receptor 1), MRC1 (macrophage mannosidase receptor 2), or ... receptor 1), VSIG4 (V-set and immunoglobulin domain-containing protein 4), CD68 (macrosialin), and CSF1R (macrophage colony-stimulating factor 1 receptor), which are preferentially expressed by monocytes, macrophages, or microglia, and optionally the subject exhibits one or more symptoms of a disease selected from the group consisting of lysosomal acid lipase deficiency, Gaucher disease, atypical Gaucher disease, mixed saposin deficiency, and Farber lipogranulomatosis;(v) genes preferentially expressed by kidney cells, e.g., CDH16 (Cadherin-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (solute carrier family 22 member 13), SLC5A2 (sodium / glucose cotransporter 2), and UMOD (uromodulin), optionally in a subject exhibiting one or more symptoms of or diagnosed with a disease selected from the group consisting of Fabry disease, Alport syndrome, polycystic kidney disease, and thrombotic thrombocytopenic purpura; (vi) genes preferentially expressed by liver cells, e.g., ASGR1 or ASGR2, optionally in a subject exhibiting one or more symptoms of or diagnosed with lysosomal acid lipase deficiency, Gaucher disease, MPS VI, MPS VII, MPS II, exhibiting one or more symptoms of or being diagnosed with a disease selected from the group consisting of Niemann-Pick disease types A, B, and C2, sialidosis, neuraminidase deficiency, atypical Gaucher disease, mixed saposin deficiency, and Farber lipogranulomatosis; (vii) for example, BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), and cholinergic receptors (nicotinic); alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3), which are preferentially expressed by muscle cells, and optionally the subject exhibits one or more symptoms of or is diagnosed with Pompe disease; (viii) selected from the group consisting of ITGA7, CD9, CD63, ALPL2, MSR1, ASGR1, ASGR2, or PRLR; and / or (ix) CD63. In some embodiments, the delivery domain is a single-chain variable fragment (scFv). In some embodiments, the cell surface receptor (CSR) binding protein (CSR-BP) comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the therapeutic protein comprises a hydrolase. In some embodiments, the therapeutic protein comprises a glycosylase. In some embodiments, the therapeutic protein comprises a glycosidase. In some embodiments, the therapeutic protein comprises an alpha-glucosidase. In some embodiments, the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13, or a fragment thereof. In some embodiments, the therapeutic protein comprises an anti-ABeta or anti-tau antibody. In some embodiments, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11. In some embodiments, the enzymatic domain comprises alpha-glucosidase, and glycogen levels in any CNS tissue of the subject are reduced for at least nine months after treatment. In some embodiments, the subject has Pompe disease. In some embodiments, the administered nucleotide composition provides a multidomain therapeutic protein serum level of at least 1 μg / mL.In some embodiments, the therapeutic protein comprises a glycosidase such as GAA (e.g., SEQ ID NO: 1) or GLA (e.g., UniProtKB No. P06280, aa 32-429, SEQ ID NO: 13), and the patient has Pompe disease or Fabry disease. In some embodiments, the CSR-BP is an antigen binding protein that binds to an internalizing receptor, such as CD63 or ITGA7. In some embodiments, the CSR-BP is an scFv molecule that binds to CD63. In some embodiments, the CSR-BP is an scFv molecule that binds to ITGA7. In some embodiments, the gene therapy vector is an AAV vector comprising a polynucleotide encoding an anti-CD63-GAA fusion therapeutic protein. In some embodiments, the gene therapy vector is an AAV vector comprising a polynucleotide encoding an anti-ITGA7-GAA fusion therapeutic protein.

[0017] In some embodiments, the therapeutic protein comprises a GAA enzyme domain, and elevated serum GAA levels are maintained in the patient's serum for at least 12 weeks after administration of the gene therapy vector. In some embodiments, the therapeutic protein comprises a GAA enzyme, and glycogen levels in the patient's CNS tissue are significantly reduced. In some embodiments, the therapeutic protein comprises a GAA enzyme, and glycogen levels are maintained at wild-type levels 3 months, 6 months, or 9 months after administration of the gene therapy vector. In some embodiments, the therapeutic protein comprises a GAA enzyme, and the patient's muscle strength is restored to wild-type levels after treatment.

[0018] In one aspect, the present invention provides methods for reducing glycogen accumulation in tissues, particularly CNS tissues, of a human or non-human subject by administering a gene therapy vector containing a polynucleotide encoding a therapeutic protein fused to a CSR-BP. In some embodiments, the gene therapy vector is administered at a dose sufficient to provide a threshold serum level of the therapeutic protein fused to a CSR-BP. In some embodiments, the threshold level is at least 1 μg / mL. In some embodiments, the threshold level is at least 2 μg / mL. In some embodiments, the threshold level is at least 3 μg / mL. In some embodiments, the threshold level is at least 4 μg / mL. In some embodiments, the threshold level is at least 5 μg / mL. In some embodiments, the threshold level is at least 6 μg / mL. In some embodiments, the threshold level is at least 7 μg / mL. In some embodiments, the threshold level is at least 8 μg / mL. In some embodiments, the threshold level is at least 9 μg / mL. In some embodiments, the threshold level is at least 10 μg / mL. In some embodiments, the threshold level is at least 11 μg / mL. In some embodiments, the threshold level is at least 12 μg / mL. In some embodiments, the threshold level is at least 13 μg / mL. In some embodiments, the threshold level is at least 14 μg / mL. In some embodiments, the threshold level is at least 15 μg / mL. In one embodiment, the tissue is the cerebellum, spinal cord, or hippocampus. In one embodiment, the human or non-human subject has Pompe disease. In one embodiment, the therapeutic protein comprises an anti-CD63 scFv-GAA fusion protein. In another embodiment, the therapeutic protein comprises an anti-ITGA7 scFv-GAA fusion protein. [Brief explanation of the drawings]

[0019] [Figure 1A]FIG. 1A schematically depicts a multidomain therapeutic protein. Panel A illustrates a multidomain therapeutic protein comprising a bispecific antibody (ii) and a recruited enzyme (i). Panel B illustrates an enzyme-Fc fusion polypeptide (i) associated with an internalization effector-specific half-antibody (ii) to form a multidomain therapeutic protein. Panel C shows a recruited enzyme (hexagon) covalently attached to the C-terminus of the heavy chain of an anti-internalization effector antibody. Panel D shows a recruited enzyme (hexagon) covalently attached to the N-terminus of the heavy chain of an anti-internalization effector antibody. Panel E shows a recruited enzyme (hexagon) covalently attached to the C-terminus of the light chain of an anti-internalization effector antibody. Panel F shows a recruited enzyme (hexagon) covalently attached to the N-terminus of the light chain of an anti-internalization effector antibody. Panel G illustrates a recruiting enzyme (hexagon) covalently linked to the C-terminus of a single-chain variable fragment (scFv) containing a VH region (shaded bar) and a VL region (open bar). Panel H illustrates a recruiting enzyme (hexagon) covalently linked to two scFv domains, with the first scFv (i) serving as the first delivery domain and the second scFv (ii) serving as the second delivery domain. Additional multidomain therapeutic proteins not illustrated in Figure 1A include, but are not limited to, multidomain therapeutic proteins comprising two or more delivery domains and at least one enzyme domain. As non-limiting examples, the antibodies, half antibodies, and scFv domains shown in panels A-H of this figure represent any type of delivery domain, and additional delivery domains or recruiting enzymes can also be associated to create multidomain therapeutic proteins.Non-limiting examples of multidomain therapeutic proteins comprising two or more delivery domains are further shown in Figures 1C, 1D, and 1F, which include a recruiting enzyme (shown, but not limited to, GAA) covalently linked to a first internalization effector-specific half antibody, which is associated with a second internalization effector-specific scFv-Fc fusion, which may or may not be covalently linked to the recruiting enzyme (shown, but not limited to, GAA) to form the multidomain therapeutic protein (Figures 1C and 1D), and the recruiting enzyme (shown, but not limited to, GAA) covalently linked to the C-terminus of each anti-internalization effector-specific half antibody, which functions as the first delivery domain, and the internalization effector-specific scFv-Fc fusion, which functions as the second delivery domain, both anti-internalization effector-specific half antibodies and associated together to form the multidomain therapeutic protein (Figure 1D), and the recruiting enzyme covalently linked to the first scFv, which is linked, for example, via a linker, to the second scFv (Figure 1F).

[0020] [Figure 1B]FIG. 1B provides a non-limiting, exemplary diagram of AAV gene therapy vectors encoding each of the multidomain therapeutic proteins depicted in panel G of FIG. 1A, where the scFv is an anti-human CD63 scFv and the recruitment enzyme is GAA (e.g., anti-hCD63scFv::hGAA; see, e.g., the amino acid sequence set forth as SEQ ID NO: 10). Amino acids 1-117 of SEQ ID NO: 10 provide the amino acid sequence of the heavy chain variable domain (VH) of the H4H12450N antibody, amino acids 118-132 of SEQ ID NO: 10 provide the amino acid linker sequence between the heavy and light chain variable domains of H4H12450N, amino acids 133-240 of SEQ ID NO: 364 provide the amino acid sequence of the light chain variable domain (VL) of the H4H12450N antibody, amino acids 241-245 of SEQ ID NO: 10 provide the amino acid linker sequence between the anti-hCD63scFv and GAA, and amino acids 246-1128 of SEQ ID NO: 10 provide the amino acid sequence of the recruitment enzyme GAA or a biologically active portion thereof. Exemplary 5'ITR and 3'ITR sequences are set forth as SEQ ID NO: 6 and SEQ ID NO: 7, respectively. Panel A of this figure provides an exemplary vector for liver-specific expression, comprising an exemplary liver-specific enhancer (e.g., SerpinA1, described as, but not limited to, SEQ ID NO: 9), an exemplary liver-specific promoter (e.g., TTR, described as, but not limited to, SEQ ID NO: 8), an exemplary signal peptide, a nucleic acid sequence encoding an anti-hCD63scFv::hGAA therapeutic multidomain (SEQ ID NO: 10), and a polyA tail. Panel B of this figure provides an exemplary vector similar to that shown in panel A, with an exemplary ubiquitous promoter in place of the liver-specific enhancer and liver-specific promoter sequence. Panel C of this figure provides an exemplary vector similar to that shown in panel A, with an exemplary neuron-specific promoter in place of the liver-specific enhancer (e.g., SerpinA1) and promoter (e.g., TTR). Panel D of this figure provides an exemplary vector similar to that shown in panel A, with an exemplary neuron-specific promoter in combination with a liver-specific (e.g., SerpinA1) enhancer and promoter (e.g., TTR).

[0021] [Figure 1C] FIG. 1C provides a non-limiting, exemplary diagram of expression vectors encoding each of the multidomain therapeutic proteins, as shown, where the half antibody is an anti-CD63 antibody, the scFv is an anti-human transferrin receptor scFv, and the recruitment enzyme is GAA (e.g., anti-hTfRscFv::hGAA).

[0022] [Figure 1D] FIG. 1D provides a non-limiting, exemplary diagram of expression vectors encoding multidomain therapeutic proteins, each as shown, where the half antibody is an anti-CD63 antibody, the scFv is an anti-human transferrin receptor (TfR) scFv, the Fc domain is human IgG4 Fc, and the recruitment enzyme is GAA (e.g., anti-hTfRscFv::hGAA).

[0023] [Figure 1E] FIG. 1E provides a non-limiting exemplary diagram of expression vectors encoding each of the multidomain therapeutic proteins depicted in panel H of FIG. 1A, where one of the two scFvs is an anti-human CD63 scFv, the other of the two scFvs is an anti-human transferrin receptor (TfR) scFv, and the recruitment enzyme is GAA (e.g., anti-hCD63 scFv::hGAA::anti-TfR scFV).

[0024] [Figure 1F] FIG. 1F provides a non-limiting exemplary diagram of expression vectors encoding multidomain therapeutic proteins, each as shown, where one of the two scFvs is an anti-human CD63 scFv, the other of the two scFvs is an anti-human transferrin receptor (TfR) scFv, and the recruitment enzyme is GAA (e.g., anti-hCD63 scFv::anti-TfR scFV::GAA or anti-TfR scFV::anti-hCD63 scFv::GAA).

[0025] [Figure 1G] FIG. 1G provides a non-limiting, exemplary diagram of expression vectors encoding each of the multidomain therapeutic proteins shown in panel G of FIG. 1A, where the scFv is an anti-human transferrin receptor (TfR) scFv and the recruitment enzyme is GAA (e.g., anti-TfRscFV::GAA).

[0026] [Figure 2] Figure 2 is a bar graph illustrating the amount of stored glycogen in micrograms per milligram of tissue as a function of delivered enzyme. The x-axis illustrates tissues from CD63 hu / hu; GAA - / - mice, from left to right: heart, quadriceps, gastrocnemius, diaphragm, soleus, and extensor digitorum longus (EDL) muscle. The box in lane 1 illustrates the amount of stored glycogen in an untreated mouse Pompe disease model. The box in lane 6 illustrates the amount of stored glycogen in an untreated wild-type mouse model. The box in lane 2 illustrates the amount of stored glycogen in a mouse Pompe disease model treated with AAV-hGAA (an adeno-associated viral vector containing a gene encoding human GAA) at a dose of 10 10 vg. The box in lane 3 illustrates the amount of stored glycogen in a mouse Pompe disease model treated with AAV-hGAA at a dose of 10 11 vg. The boxes in lane 4 and lane 5 show the amount of stored glycogen in a mouse Pompe disease model treated with AAV-anti-hCD63scFv::hGAA (an adeno-associated viral vector containing a gene encoding an anti-human CD63 scFv domain linked to human GAA) at a dose of 10 vg.

[0027] [Figure 3]Figure 3 is a graph illustrating the average glycogen measurements (μg / mg) in skeletal muscle tissue of each mouse 3 months after AAV injection. Each measurement is plotted as a function of GAA exposure (i.e., serum level) per mouse treated with a particular enzyme construct at a particular dose. Black squares represent AAV-hGAA at a dose of 10 vg. Black triangles represent AAV-hGAA at a dose of 10 vg. Black inverted triangles represent AAV-anti-hCD63scFv::hGAA at a dose of 10 vg. Black rectangles represent AAV-anti-hCD63scFv::hGAA at a dose of 10 vg.

[0028] [Figure 4] Figure 4 is a dot plot showing the average myocardial glycogen (µg / mg) measured in cardiac tissue 3 months after AAV injection as a function of GAA exposure (i.e., serum levels) per mouse treated with a particular enzyme construct at a particular dose. Black squares represent AAV-hGAA at a dose of 10 vg. Black triangles represent AAV-hGAA at a dose of 10 vg. Black inverted triangles represent AAV-anti-hCD63scFv::hGAA at a dose of 10 vg. Black rectangles represent AAV-anti-hCD63scFv::hGAA at a dose of 10 vg.

[0029] [Figure 5] Figure 5 is a dot plot illustrating the titer of anti-GAA antibodies 3 months after AAV injection as a function of GAA exposure (i.e., serum levels) per mouse treated with a particular enzyme construct at a particular dose. Open squares represent AAV-hGAA at a 10 vg dose. Open circles represent AAV-hGAA at a 10 vg dose. Open rectangles represent AAV-anti-hCD63scFv::hGAA at a 10 vg dose. Hexagons represent AAV-anti-hCD63scFv::hGAA at a 10 vg dose.

[0030] [Figure 6]Figure 6 is a dot plot illustrating the titer of anti-GAA antibodies 3 months after AAV injection as a function of enzyme construct and dose. Circles represent control mice receiving empty AAV vector. Squares represent AAV-hGAA at a dose of 1010 vg. Triangles represent AAV-hGAA at a dose of 1011 vg. Inverted triangles represent AAV-anti-hCD63scFv::hGAA at a dose of 1010 vg. Rhombuses represent AAV-anti-hCD63scFv::hGAA at a dose of 1011 vg.

[0031] [Figure 7A] Figure 7A is a line graph illustrating serum levels of GAA (arbitrary units "au"; y-axis) as a function of time in weeks after injection of the gene therapy vector. Squares (bottom line) represent AAV-hGAA at a 1010 vg dose. Triangles (second from the top) represent AAV-hGAA at a 1011 vg dose. Inverted triangles (third from the top) represent AAV-anti-hCD63scFv::hGAA at a 1010 vg dose. Rhombuses (top line) represent AAV-anti-hCD63scFv::hGAA at a 1011 vg dose.

[0032] [Figure 7B] Figure 7B is a bar graph illustrating the mRNA ratio (hGAA mRNA to mGADPH mRNA) after administration of AAV constructs in CD63 Humln GAA KO mice (GAA- / -, CD63 hu / hu mice) or GAA+ / +, CD63 hu / hu mice as follows: (1) untreated control, (2) AAV-liver-specific promoter-hGAA (1e10 vg), (3) AAV-liver-specific promoter-hGAA (1e11 vg), (4) AAV-liver-specific promoter-anti-hCD63::hGAA (1e10 vg), (5) AAV-liver-specific promoter-anti-hCD63::hGAA (1e11 vg), or (6) untreated treated control (GAA+ / +, CD63 hu / hu). For all injections of AAV constructs, GAA expression in the liver was detected.

[0033] [Figure 7C]Figure 7C is a dot graph comparing serum GAA levels and RNA expression levels for mice receiving AAV encoding the fusion protein (square) and mice receiving AAV encoding GAA (both constructs were equipped with a liver-specific promoter (LSP) to drive expression).

[0034] [Figure 7D] Figure 7D is a bar graph showing Huh-7 human hepatocytes transiently transfected with liver-specific promoter-driven constructs encoding hGAA, anti-hCD63 scFv::GAA (fusion constructs), or the nonbinding fusion construct scFv::GAA control. Both scFv::GAA fusion constructs resulted in higher protein ratios in the secreted supernatant than hGAA alone 3 days after transfection. Addition of M6P to the supernatant during the experimental period to mitigate CI-MPR-mediated uptake did not affect the ratios. (*=p<0.05, n=3).

[0035] [Figure 7E] Figure 7E is a bar graph showing serum titers of GAA as a function of delivered vector. The X-axis shows serum from CD63-humanized KO mice (GAA- / -; CD63hu / hu) that received plasmids encoding GAA or ScFv-GAA fusions, from left to right: 1) control (no treatment); 2) AAV-LSP-hGAA treatment (1e10 vg / mouse); 3) AAV-LSP-hGAA treatment (1e11 vg / mouse); 4) AAV-LSP-anti-CD63::hGAA treatment (1e10 vg / mouse); and 5) AAV-LSP-anti-CD63::hGAA treatment (1e11 vg / mouse).

[0036] [Figure 8-1]Figure 8 is a set of fluorescence micrographs illustrating lamp1-stained lysosomes in mouse muscle fibers counterstained with DAPI to reveal nuclei. Panels A and A1 illustrate quadriceps muscle cells from untreated wild-type (GAA+ / +) mice stained for lamp1 (Panel A) and nuclei (Panel A1). Panels B and B1 illustrate quadriceps muscle cells from untreated GAA null (GAA- / -) mice stained for lamp1 (Panel B) and nuclei (Panel B1). Panels C and C1 illustrate quadriceps muscle cells from GAA- / - mice treated with an AAV-hGAA construct stained for lamp1 (Panel C) and nuclei (Panel C1). Panels D and D1 depict quadriceps muscle cells from GAA- / - mice treated with the AAV-hCD63scFv::hGAA construct and stained for lamp1 (panel D) and nuclei (panel D1). [Figure 8-2] Same as above.

[0037] [Figure 9] Figure 9 illustrates line graphs showing grip strength and rotarod test performance of mice treated with either AAV-LSP hGAA or AAV-LSP-anti-hCD63::hGAA. Rotarod measurements (A) and forelimb grip strength measurements (B) were obtained at 1-month intervals for 6 months for wild-type GAA mice (inverted triangles), untreated controls (squares), AAV-LSP-hGAA-treated (1e11 vg / mouse) (triangles), or AAV-LSP-anti-hCD63::hGAA-treated (1e11 vg / mouse) (circles). Error bars are + / - standard deviation. N = 8–10 for all groups.

[0038] [Figure 10A]Figures 10A and 10B illustrate the use of other membrane proteins as guides, such as anti-ITGA7 (integrin alpha 7) scFv fusion proteins, to guide GAA. Figure 10A shows the GAA activity (y-axis) of C2C12 mouse myoblasts incubated overnight with anti-mouse CD63-GAA or anti-mouse ITGA7-GAA in the presence or absence of 5 mM M6P. Figure 10B shows CD63-humanized GAA KO mice (GAA- / -; CD63hu / hu) that received plasmids encoding anti-hCD63::GAA in the scFv::GAA format (2) or anti-mouse integrin alpha 7 in the full-length IgG4::GAA format (3) by hydrodynamic delivery (HDD), and then measured tissue glycogen levels 3 weeks after HDD. Untreated control mice, GAA- / -;CD63hu / hu (1), and untreated wild-type GAA control mice, GAA+ / +;CD63hu / hu (4), were also tested for glycogen levels in the same tissues. [Figure 10B] Same as above.

[0039] [Figure 11] Figure 11 shows the serum clearance of full-length anti-CD63 antibody (i.e., full-length IgG4 antibody) fused to GAA in CD63 hu / hu mice compared to wild-type CD63+ / + mice. Plasmids expressing the heavy and light chains of anti-CD63 antibody fused to GAA were injected via the tail vein of each mouse. The serum pK of anti-CD63 (full-length IgG4)::GAA was observed to disappear from the serum within 24 hours.

[0040] [Figure 12A]Figure 12A is a dot plot illustrating serum levels of GAA (arbitrary units, "au"; y-axis) one month after AAV injection as a function of enzyme construct and dose. Squares represent AAV-LSP-Δ8GAA. Triangles represent AAV-anti-hCD63scFv::GAA. Both constructs provided a liver-specific promoter (LSP) to drive expression. Doses are shown as viral genomes (vg) per kilogram (kg) of mouse.

[0041] [Figure 12B] Figure 12B shows dot plots illustrating glycogen levels in micrograms per milligram of tissue (heart, quadriceps, diaphragm, or triceps) as a function of GAA serum levels. Squares represent AAV-LSP-Δ8GAA. Triangles represent AAV-anti-hCD63scFv::GAA. Both constructs provided a liver-specific promoter (LSP) to drive expression.

[0042] [Figure 13] Figure 13 is a bar graph illustrating the amount of stored glycogen in micrograms per milligram of CNS tissue as a function of delivered enzyme / vector (9-month study). The x-axis shows each CNS tissue (spinal cord, cerebellum, or hippocampus) sampled from untreated (lane 1 in each case), or wild-type mice (lane 4 in each case) that received a GAA-encoding plasmid (lane 2 in each case), or an ScFv-GAA fusion (lane 3 in each case), or KO mice (GAA- / -) that received a GAA-encoding vector without any fusion binding domain. An unexpected finding was that mice treated with the fusion protein showed a more robust reduction in glycogen stores in CNS tissue compared to delivery of a GAA-encoding vector without any fusion binding domain.

[0043] [Figure 14A]Figure 14A is a graph showing glycogen stores per mg of spinal cord tissue (µg glycogen / mg tissue; y-axis) 3 months after AAV delivery of GAA with a particular vector construct at a particular dose (vg = viral genome). Glycogen levels are provided in wild-type mice (lane 1; x-axis) compared to KO mice (GAA- / -) that were untreated (lane 2; x-axis) or received a GAA-encoding plasmid (lane 3; x-axis), or were given various doses of anti-CD63 ScFv-GAA fusion (lanes 4-6; x-axis). Increasing doses of ScFv-GAA fusion reduced spinal cord glycogen stores, with a dose of 1 e11 vg providing greater benefit to subjects than an equivalent dose of GAA alone (no fusion).

[0044] [Figure 14B] Figure 14B is a graph showing glycogen reserves per mg of brain tissue (µg glycogen / mg tissue; y-axis) 3 months after AAV delivery of GAA with a particular vector construct at a particular dose (vg = viral genome). Glycogen levels are measured in wild-type mice (lane 1; x-axis) compared to KO mice (GAA- / -) that were untreated (lane 2; x-axis) or received a GAA-encoding plasmid (lane 3; x-axis), or given various doses of anti-CD63 ScFv-GAA fusion (lanes 4-6; x-axis). Increasing doses of ScFv-GAA fusion reduced brain glycogen reserve levels, with a dose of 1 e11 vg providing greater benefit to subjects than an equivalent dose of GAA alone (no fusion). DETAILED DESCRIPTION OF THE INVENTION

[0045] The present invention is not limited to the specific embodiments, compositions, methods, and experimental conditions described, as such embodiments, compositions, methods, and conditions may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention is limited only by the appended claims.

[0046] Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some suitable methods and materials are now described. All publications cited herein are incorporated by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0047] "Blood-brain barrier" refers to the semipermeable membrane barrier that separates blood from the extracellular fluid of the brain and central nervous system. The barrier prevents the passage or selective transport of certain substances into the brain and spinal cord. The blood-brain barrier is formed by brain endothelial cells.

[0048] A "therapeutically effective amount" refers to the amount or dosage of a vector delivered to a subject such that the subject achieves consistent blood levels (serum / plasma levels) of the encoded therapeutic protein. Generally, about 1 x 10 9 ~approx. 1x10 16 Concentrations of genome vectors can be utilized in this manner. The dose for liver delivery is approximately 1 x 10 per kg. 10 ~5x10 13The vector may be an AAV genome. The dosage is adjusted to balance the therapeutic benefit of crossing the blood-brain barrier and achieve the desired effect of the molecule against any side effects, and such dosage may vary depending on the recombinant vector used. The expression level of the transgene can be monitored in the circulation by serum or plasma extraction to determine the frequency of vector administration that achieves a steady state of circulating protein. Those skilled in the art can determine the specific value of the effective amount, for example, by conducting experiments to determine consistent blood levels of the therapeutic protein over consecutive days, weeks, or months after vector delivery. Suitable experiments for testing circulating therapeutic proteins are known in the art, including, but not limited to, Western blot, ELISA, LC-MS, and the like. In one example, a therapeutically effective amount of scFv-GAA fusion protein in the CNS is the amount of viral vector that produces a sufficient amount of scFv-GAA fusion protein to reduce glycogen stores in CNS tissues, such as the spinal cord, cerebellum, or hippocampus.

[0049] CNS disorders Various brain diseases may benefit from the therapeutic protein delivery modes described herein. CNS disorders and disorders with neurological symptoms amenable to protein therapy include, but are not limited to, Alzheimer's disease, brain cancer, Behçet's disease, cerebral lupus, Creutzfeldt-Jakob disease, dementia, epilepsy, encephalitis, Friedreich's ataxia, Guillain-Barré syndrome, Gaucher disease, headache, hydrocephalus, Huntington's disease, increased intracranial pressure, cerebral leukodystrophy, migraine, myasthenia gravis, muscular dystrophy, multiple sclerosis, narcolepsy, neuropathy, Prader-Willi syndrome, Parkinson's disease, Rett syndrome, restless legs syndrome, sleep disorders, subarachnoid hemorrhage, stroke, traumatic brain injury, trigeminal neuralgia, transient ischemic attack, and von Hippel-Lindau disease (angiomatosis).

[0050] Anti-CD63 fusion delivery of therapeutic proteins to the CNS may be particularly beneficial due to its ubiquitous expression, its role as a membrane protein of extracellular vesicles (EVs, e.g., exosomes), and its association with integrins. Other cell surface receptors with properties similar to internalization effectors include ITGA7, CD9, CD63, CD81, CD82, and CD151, and, as discussed throughout this specification, can be tissue- or cell-type-specific to enhance the desired location of uptake.

[0051] Anti-transferrin fusion delivery of therapeutic proteins to the CNS has also been shown to be particularly beneficial. Therefore, the transport and delivery of therapeutic proteins is enhanced by the use of delivery mechanisms such as anti-receptor fusion to specific blood-brain barrier (BBB) ​​targets. Several BBB targets have been shown to be beneficial for CNS uptake (Zuchero, et al., 6 Jan 2016, Neuron, 89(1):70-82; Boado, RJ et al., Mol Pharm, 2014 August 4;11(8):2928-2934; each of which is incorporated herein by reference in its entirety). Other cell surface receptors with BBB uptake properties similar to those of the transferrin receptor include, but are not limited to, the insulin receptor, CD98, and basigin (Bsg).

[0052] In some embodiments, targeted delivery of therapeutic proteins to CNS tissues (e.g., the brain) employs the use of anti-transferrin receptor or anti-insulin receptor, or anti-CD98 or anti-Bsg. Therapeutic proteins may also be fused to internalizing effector antibodies, as described herein throughout. In some embodiments, targeted delivery of therapeutic proteins to CNS and peripheral tissues employs the use of anti-insulin receptor delivery domains, for example, to enhance both brain and peripheral uptake (Boado, RJ et al., 2014, supra; Yu et al., 25 May 2011, Science Transl Med. 3:84ra44, each incorporated herein by reference in its entirety).

[0053] Exemplary anti-transferrin receptor, anti-insulin receptor, anti-CD98, and anti-Bsg antibodies and portions thereof that may be useful as part of the multidomain therapeutic proteins described herein are known in the art (see, e.g., U.S. Patent Nos. 20170174778; 20150196663; 9629801; 20180002433; WO 2016081643; 20180134797; WO 2014189973; 20150110791; 9708406; 20170260 for non-limiting exemplary anti-transferrin receptor antibodies). 292; International Patent Application Publication No. 2016081640; U.S. Patent No. 20180057604; U.S. Patent No. 9611323; International Patent Application Publication No. 2012075037; International Patent Application Publication No. 2018210898, U.S. Patent No. 20180344869, U.S. Patent No. 20180282408, U.S. Patent No. 20170051071, International Patent Application Publication No. 2016207240, International Patent Application Publication No. 2015101588, U.S. Patent No. 20160324984; U.S. Patent No. 20180222993; International Patent Application Publication No. 2017055542; U.S. Patent No. 20180222992; International Patent Application Publication No. 2017055540; Cabezon, I., et al.Mol Pharm.2015 Nov 2;12(11):4137-45;Yu YJ, et al.Sci Transl Med(2014)6:261ra154;Couch, et al.Sci Transl Med.2013 May 1;5(183):183ra57, 1-12;and Yu et al., 2011, supra; for non-limiting exemplary anti-CD98 antibodies, see, e.g., International Patent Application Publication No. 2017214456, International Patent Application Publication No. 2017214458, International Patent Application Publication No. 2017214462; International Patent Application Publication No. 2008017828; International Patent Application Publication No. 2015146132; International Patent Application Publication No. 2016094566; International Patent Application Publication No. 2013078377; International Patent Application Publication No. 2017026497; Hayes GM et al. Int. J.Cancer(2015)137:710-20;and Bixby, et al.American Society Hematology 2015 Meeting, Abstract# 3809 for non-limiting exemplary anti-BSG antibodies, see, e.g., WO 2011112566; U.S. Patent No. 20110223176; U.S. Patent No. 20140079711; WO 2010036460; U.S. Patent No. 8618264; WO 2005092381; WO 2018165619; and WO 2017186182; for non-limiting exemplary anti-insulin receptor antibodies, see, e.g., U.S. Patent No. 8974791; WO 2013081706; U.S. Patent No. 20160152719; U.S. Patent No. 20160208006; U.S. Patent No. 20170114152; Pardridge, W. M. et al. BioDrugs. 2018 Apr;32(2):169-176; Boado RJ et al. Mol. Pharm. (2016) 13:3241-6; Cieniewicz AM, et al. Diabetes (2017) 66:206-217; Bexwada, P. et al. J Pharmacol Exp Ther. 2016 Feb;356(2):466-73; and Bedinger, DH, et al. J Pharmacol Exp Ther. 2015 Apr;353(1):35-43; each of which is incorporated herein by reference in its entirety. Those skilled in the art can readily link these well-known antibodies, or antigen-binding portions thereof (e.g., scFvs derived therefrom, etc.), to the therapeutic proteins described herein to generate and use the multidomain therapeutic proteins described herein.

[0054] Lysosomal storage disorders "Enzyme deficiency diseases" include non-lysosomal storage diseases, such as Krabbe disease (galactosylceramidase), phenylketonuria, galactosemia, maple syrup urine disease, mitochondrial disorders, Friedreich's ataxia, Zellweger syndrome, adrenoleukodystrophy, Wilson's disease, hemochromatosis, ornithine transcarbamylase deficiency, methylmalonic acidemia, propionic acidemia, and lysosomal storage diseases. "Lysosomal storage diseases" include any disorder resulting from defective lysosomal function. Approximately 50 lysosomal storage disorders have been identified, the most well-known of which include Tay-Sachs disease, Gaucher disease, and Niemann-Pick disease. The etiology of these diseases is thought to be the accumulation of incomplete breakdown products in lysosomes, usually due to loss of protein function. Lysosomal storage diseases are caused by loss-of-function or attenuating variants in proteins that normally degrade or regulate the degradation of lysosomal contents. Proteins closely related to lysosomal storage disease include enzymes, receptors, and other transmembrane proteins (e.g., NPC1), post-translational modification proteins (e.g., sulfatase), membrane transport proteins, and non-enzymatic cofactors and other soluble proteins (e.g., GM2 ganglioside activator).Therefore, lysosomal storage disease not only includes the aforementioned disorders caused by defective enzymes themselves, but also includes any disorders caused by any molecular defects.Therefore, as used herein, the term "enzyme" is intended to include the aforementioned other proteins related to lysosomal storage disease.

[0055] The nature of the molecular damage often influences the severity of the disease; in other words, complete loss of function tends to be associated with fetal or neonatal onset and severe symptoms, and partial loss of function is associated with (relatively) milder, later-onset disease. Generally, only a small percentage of activity needs to be restored to correct the metabolic defect in defective cells. Table 1 lists some of the more common lysosomal storage diseases and their associated loss-of-function proteins. Lysosomal storage diseases are generally described in Desnick and Schuchman, 2012.

[0056] Lysosomal storage diseases are a type of rare disease that affect the degradation of various substrates in lysosomes. These substrates, including sphingolipids, mucopolysaccharides, glycoproteins, glycogen, and oligosaccharides, can accumulate in the cells of diseased individuals, leading to cell death. Organs affected by lysosomal storage diseases include the central nervous system (CNS), peripheral nervous system (PNS), lungs, liver, bone, skeletal and cardiac muscles, and reticuloendothelial system.

[0057] Treatment options for lysosomal storage diseases include enzyme replacement therapy (ERT), substrate reduction therapy, pharmacological chaperone-mediated therapy, hematopoietic stem cell transplantation, and gene therapy. An example of substrate reduction therapy is the use of miglustat or eliglustat to treat Gaucher disease type 1. These drugs act by blocking synthase activity, thereby reducing substrate production. Hematopoietic stem cell therapy (HSCT) is used, for example, to ameliorate and delay the negative central nervous system phenotype in patients with some forms of MPS. See R.M. Boustany, “Lysosomal storage diseases—the horizon expands,” 9(10) Nat. Rev. Neurol. 583-98, October 2013, which are incorporated herein by reference in their entireties. Table 1 lists some lysosomal storage diseases and their associated enzymes or other proteins. [Table 1-1] [Table 1-2]

[0058] Two of the most common LSDs are Pompe disease and Fabry disease. Pompe disease, with an estimated incidence of 1 in 10,000, is caused by a defective lysosomal enzyme, alpha-glucosidase (GAA), resulting in defective glycogen processing in lysosomes. Lysosomal glycogen accumulation occurs primarily in skeletal, cardiac, and hepatic tissues. Onset of Pompe disease in infancy, usually before the age of 2 years, leads to cardiac hypertrophy, hypotonia, hepatomegaly, and death from cardiopulmonary failure. Onset of Pompe disease in adulthood can occur in the teens to fifties and typically involves only skeletal muscle. Currently available treatments include Genzyme's MYOZYME® / LUMIZYME® (alglucosidase alpha), a recombinant human alpha-glucosidase produced in CHO cells and administered by intravenous infusion.

[0059] Fabry disease, which has an estimated overall incidence of 1 in 3,000 people, including milder, later-onset cases, is caused by defective lysosomal enzyme alpha-galactosidase A (GLA), resulting in the accumulation of globotriaosylceramide in blood vessels and other tissues and organs. Symptoms associated with Fabry disease include pain due to nerve damage and / or small-vessel occlusion, renal insufficiency and failure, cardiac complications such as hypertension and cardiomyopathy, skin manifestations such as the formation of angiokeratoma, anhidrosis or hyperhidrosis, and eye disorders such as cornea verticillata, radial cataracts, and conjunctival and retinal vascular abnormalities. Currently available treatments include Genzyme's FABRAZYME® (agalsidase beta), a recombinant human alpha-galactosidase A produced in CHO cells and administered by intravenous infusion; Shire's REPLAGAL® (agalsidase alfa), a recombinant human alpha-galactosidase A produced in human fibroblasts and administered by intravenous infusion; and Amicus' GALAFOLD™ (migalastat, 1-deoxygalactonojirimycin), an orally administered small molecule peralone that shifts the folding of abnormal alpha-galactosidase A into a functional conformation.

[0060] Current treatments for lysosomal storage diseases are suboptimal. For example, ERT must generally be administered frequently and at high doses, e.g., up to 40 mg / kg every two weeks. Furthermore, some replacement enzymes are immunologically cross-reactive (CRIM) and stimulate the production of IgG in the subject, which can prevent the enzyme from being delivered to lysosomes via the mannose-6-phosphate (M6P) receptor. IgG can mask the M6P residues of the replacement enzyme, and although the antigen-IgG-antibody complex can be internalized into cellular lysosomes via Fc receptors, the replacement enzyme is preferentially delivered to macrophages.

[0061] Delivery of replacement enzymes to the appropriate affected tissues can also be inefficient (see Table 2, and Desnick & Schuchman, "Enzyme replacement therapy for lysosomal diseases: lessons from 20 years of experience and remaining challenges," 13 Annu. Rev. Genomics Hum. Genet. 307-35, 2012), which is incorporated herein by reference in its entirety. For example, patients receiving long-term enzyme replacement therapy for infantile Pompe may additionally suffer from hypernasality, persistent muscle weakness, ptosis, osteopenia, hearing loss, aspiration risk, dysphagia, cardiac arrhythmias, and swallowing difficulties. The replacement enzyme dose must often be increased over time, up to 40 mg / kg, weekly or biweekly. [Table 2]

[0062] The endogenous mannose-6-phosphate receptor (MPR) mediates the transport of most recombinant enzymes to lysosomes. There are two complementary forms of the MPR: the cation-independent (CI-MPR) and the cation-dependent (CD-MPR). Knockout of either form results in defective transport of lysosomal enzymes. Lysosomal hydrolases are synthesized in the endoplasmic reticulum and translocated to the cis-Golgi network, where they are covalently modified by the addition of a mannose-6-phosphate (M6P) group. The formation of this marker depends on the sequential action of two lysosomal enzymes: UDP-N-acetylglucosamine-l-phosphotransferase (G1cNac-phosphotransferase) and N-acetylglucosamine-l-phosphodiester-α-N-acetyl-glucosaminidase (uncovering enzyme). GlcNAc-phosphotransferase catalyzes the transfer of a GlcNAc-1-phosphate residue from UDP-GlcNAc to the C6 position of a selected mannose residue on the high-mannose oligosaccharide of the hydrolase. A decoating enzyme then removes the terminal GlcNAc, exposing the M6P recognition signal. In the trans-Golgi network, this M6P signal allows lysosomal hydrolases to separate from all other proteins via selective binding to the M6P receptor. The resulting clathrin-coated vesicles bud from the trans-Golgi network and fuse with late endosomes. At the low pH of the late endosome, the hydrolases dissociate from the M6P receptor, and the empty receptor is recycled back to the Golgi apparatus for further rounds of transport.

[0063] With the exception of β-glucocerebrosidase, which is delivered via the mannose receptor, recombinant lysosomal enzymes contain M6P glycosylation and are primarily delivered to lysosomes via the CI-MPR / IGF2R. However, glycosylation / CI-MPR-mediated enzyme replacement delivery does not reach all clinically relevant tissues (Table 2). Improvements in enzyme replacement therapy have been achieved by (i) increasing the surface expression of the CI-MPR using the β2-agonist clenbuterol (Koeberl et al., “Enhanced efficacy of enzyme replacement therapy in Pompe disease through mannose-6-phosphate receptor expression in skeletal muscle,” 103(2) Mol. Genet. Metab. 107-12, 2011, which is incorporated herein by reference in its entirety), (ii) increasing the amount of M6P residues on the enzyme (Zhu et al., “Conjugation of mannose-6-phosphate-containing oligosaccharides to acid alpha-glucosidase improves the clearance of glycogen in Pompe mice,” 279(48) J. Biol. Chem. 50336-41, 2004, which is incorporated herein by reference in its entirety), or (iii) fusing an IGF-II domain to the enzyme (Maga et al., “Glycosylation-independent Our research focuses on improving CI-MPR delivery through “lysosomal targeting of acid alpha-glucosidase enhances muscle glycogen clearance in Pompe mice,” 288(3) J. Biol. Chem. 1428-38, 2013, which is incorporated herein by reference in its entirety.

[0064] Numerous lysosomal storage diseases are inadequately treated by enzyme replacement therapy or gene therapy, primarily due to poor targeting of the replacement enzyme to the relevant tissues or organs, negative immunological reactions in the recipient host, and short serum half-life. There is a need for improved enzyme replacement therapy that enhances and promotes better tissue biodistribution and lysosomal uptake of enzymes, particularly in the brain and spinal cord, without undesirable intrathecal injection. Applicants have developed an improved enzyme replacement therapy that uses CI-MPR-independent binding protein-guided delivery and hepatic expression of enzymes to deliver enzymes to the lysosomes of target diseased tissues, particularly CNS tissues.

[0065] Lysosomal storage diseases can be classified by the type of product that accumulates within the defective lysosomes. Sphingolipid storage diseases are a class of diseases that affect the metabolism of sphingolipids, lipids containing fatty acids linked to aliphatic amino alcohols (reviewed in S. Hakomori, “Glycosphingolipids in Cellular Interaction, Differentiation, and Oncogenesis,” 50 Annual Review of Biochemistry 733–764, July 1981, which is incorporated herein by reference in its entirety). Accumulation products of sphingolipid storage diseases include gangliosides (e.g., Tay-Sachs disease), glycolipids (e.g., Fabry disease), and glucocerebrosides (e.g., Gaucher disease).

[0066] Mucopolysaccharidoses are a group of diseases that affect the metabolism of glycosaminoglycans (GAGS or mucopolysaccharides), which are long, repeating, unbranched chains of disaccharides that help build bone, cartilage, tendons, cornea, skin, and connective tissue (reviewed in J. Muenzer, “Early initiation of enzyme replacement therapy for the mucopolysaccharidoses,” 111(2) Mol. Genet. Metab. 63-72 (Feb. 2014); Sasisekharan et al., “Glycomics approach to structure-function relationships of glycosaminoglycans,” 8(1) Ann. Rev. Biomed. Eng. 181-231 (Dec. 2014), each of which is incorporated herein by reference in its entirety). Storage products of mucopolysaccharidoses include various forms of heparan sulfate, dermatan sulfate, keratin sulfate, chondroitin sulfate, and hyaluronic acid. For example, Morquio syndrome A is caused by a deficiency in the lysosomal enzyme galactose-6-sulfate sulfatase, resulting in the accumulation of keratin sulfate and chondroitin 6-sulfate in lysosomes.

[0067] Glycogen storage diseases (also known as glycogen storage diseases) result from the inability of cells to metabolize (produce or break down) glycogen. Glycogen metabolism is regulated by various enzymes or other proteins, including glucose-6-phosphatase, acid alpha-glucosidase, glycogen debranching enzyme, glycogen branching enzyme, muscle glycogen phosphorylase, liver glycogen phosphorylase, muscle phosphofructokinase, phosphorylase kinase, glucose transporters, aldolase A, beta-enolase, and glycogen synthase. The classic lysosomal storage / glycogen storage disease is Pompe disease, in which glycogen accumulates in lysosomes due to a defect in acid alpha-glucosidase. Symptoms include hepatomegaly, muscle weakness, heart failure, and, in infantile variants, death by age 2 years (see DiMauro and Spiegel, “Progress and problems in muscle glycogenosis,” 30(2) Acta Myol. 96-102 (Oct. 2011), which is incorporated herein by reference in its entirety).

[0068] "Multidomain therapeutic proteins" include (i) a single protein containing two or more functional domains, (ii) a protein containing two or more polypeptide chains, and (iii) a mixture of two or more proteins or two or more polypeptides. The term polypeptide is generally intended to mean a single chain of amino acids linked via peptide bonds. The term protein encompasses not only the term polypeptide but also more complex structures. That is, a single polypeptide is a protein, and a protein can contain one or more polypeptides associated into a higher order structure. For example, hemoglobin is a protein that contains four polypeptides: two alpha globin polypeptides and two beta globin polypeptides. Myoglobin is also a protein, but it contains only a single myoglobin polypeptide.

[0069] Multidomain therapeutic proteins comprise one or more polypeptides and at least two domains that confer two functions. One of these domains is an "enzyme domain" that provides for the replacement of the activity of a defective protein associated with an enzyme deficiency. The other of these domains is a "delivery domain" that provides for the binding of an internalization effector. Thus, a single polypeptide that confers enzyme replacement activity and the ability to bind to an internalization effector (also known as an internalization effector-binding protein) (delivery domain activity) is a multidomain therapeutic protein. Also, a mixture of proteins, where one protein confers the enzymatic function and another protein confers the internalization effector-binding activity, is a multidomain therapeutic protein. Figure 1A illustrates various exemplary multidomain therapeutic proteins. In one example (Figure 1A, panel A), the multidomain therapeutic protein contains an enzyme (represented by a hexagon) and a bispecific antibody (IE-BP) that binds the enzyme (dashed line) and the internalization effector (solid line). Here, one arm of the bispecific antibody noncovalently binds to the enzyme and the other The arm of the enzyme binds non-covalently to an internalization effector, thereby allowing the replacement enzyme to be internalized within a cell or subcellular compartment. In another example (Panel B), the multidomain therapeutic protein comprises a single protein containing two polypeptides, one of which has an enzymatic function and the other has a delivery domain function. Here, the enzyme is fused to an immunoglobulin Fc domain or heavy chain constant region, which associates with the Fc domain of an enzyme half antibody to form a bifunctional multidomain therapeutic protein. The embodiment illustrated in Panel B is similar to that of Panel A, except that the enzyme is covalently attached to one of the half antibodies rather than via an antigen-antibody interaction with the immunoglobulin variable domain of the half antibody.

[0070] In other examples, the multidomain therapeutic protein comprises an enzyme covalently linked (directly or indirectly via a linker) to a delivery domain. In one embodiment, the enzyme is attached to the C-terminus of an immunoglobulin molecule (e.g., a heavy chain or, alternatively, a light chain). In another embodiment, the enzyme is attached to the N-terminus of an immunoglobulin molecule (e.g., a heavy chain or, alternatively, a light chain). In these exemplary cases, the immunoglobulin molecule is the delivery domain. In yet another embodiment, the enzyme is attached to the C-terminus of an scFv molecule that binds to an internalization effector.

[0071] In one embodiment, a multidomain therapeutic protein comprises at least two, and in some embodiments, no more than two, delivery domains, each directed to a different epitope, either on the same antigen or on two different antigens. In one embodiment, the first delivery domain binds to a lysosomal transport molecule or other internalization effector (e.g., CD63) or other similar cell surface receptors, such as ITGA7, CD9, CD63, CD81, CD82, or CD151. In another embodiment, the second delivery domain binds to a transcytosis effector to promote transcytosis of the multidomain therapeutic protein. In one embodiment, the transcytosis effector is, in particular, the LDL receptor, IgA receptor, transferrin receptor, or neonatal Fc receptor (FcRn). In certain embodiments, the transcytosis delivery domain comprises a molecule that binds to the transferrin receptor, such as an anti-transferrin receptor antibody or an anti-transferrin receptor scFv molecule. Tuma and Hubbard, "Transytosis: Crossing Cellular Barriers," Physiological Reviews, 83(3):871-935 (July 1, 2003), is incorporated herein by reference for its discussion of cell surface receptors that mediate transcytosis useful in the practice of the present invention. In one embodiment, the second delivery domain binds to the transferrin receptor or other similar cell surface proteins, such as the insulin receptor, CD98, or basigin (Bsg). Each multidomain therapeutic protein comprising at least two delivery domains also comprises at least one enzymatic domain; for example, each of the at least two delivery domains may or may not be independently associated with an enzymatic domain (e.g., via antigen-antibody interactions, via direct covalent bonds, via indirect covalent bonds) in the methods described herein, and at least one of the at least two delivery domains is associated with an enzymatic domain. Furthermore, each of the at least two delivery domains may independently comprise an antibody, half-antibody, or scFv (e.g., scFv fused to Fc, etc.).

[0072] "Enzyme domain" or "enzyme" refers to any protein involved in the pathogenesis or physiological effects of an enzyme deficiency. Enzymes include actual enzymes, transport proteins, receptors, or other proteins that are defective and believed to have a disease-causing molecular lesion. Enzymes also include any protein that can confer biochemical or physiological activity similar or sufficient to compensate for or bypass the disease's molecular lesion. For example, an "isozyme" may be used as an enzyme. Examples of lysosomal storage disease-associated proteins include those listed as "involved enzymes / proteins" in Table 1 and any known or later discovered proteins or other molecules that bypass the molecular defect of an enzyme deficiency.

[0073] In some embodiments, the enzyme is a hydrolase, including esterases, glycosylases, hydrolases acting on ether bonds, peptidases, linear amidases, diphosphatases, ketone hydrolases, halogenases, phosphoamidases, sulfohydrolases, sulfinases, desulfinases, etc. In some embodiments, the enzyme is a glycosylase, including glycosidases and N-glycosylases. In some embodiments, the enzyme is a glycosidase, including alpha-amylase, beta-amylase, glucan 1,4-alpha-glucosidase, cellulose, endo-1,3(4)-beta-glucanase, inulinase, endo-1,4-beta-xylanase, endo-1,4-b-xylanase, dextranase, chitinase, polygalacturonidase, lysozyme, exo-alpha-sialidase, arachidonic acid ... Examples of enzymes that can be used include alpha-glucosidase, beta-glucosidase, alpha-galactosidase, beta-galactosidase, alpha-mannosidase, beta-mannosidase, beta-fructofuranosidase, alpha,alpha-trehalose, beta-glucuronidase, xylan endo-1,3-beta-xylosidase, amylo-alpha-1,6-glucosidase, hyaluronoglucosaminidase, and hyaluronoglucuronidase.

[0074] In the case of Pompe disease, where the molecular defect is a defect in alpha-glucosidase activity, enzymes include human alpha-glucosidase and "isoenzymes" such as other alpha-glucosidases, engineered recombinant alpha-glucosidases, other glucosidases, recombinant glucosidases, any protein engineered to hydrolyze non-reducing terminal 1-4 linked alpha-glucose residues to release a single alpha-glucose molecule, any EC 3.2.1.20 enzyme, natural or recombinant low pH glycohydrolases for glycogen or starch, and glucosyl hydrolases such as sucrase-isomaltase, maltase-glucoamylase, glucosidase II, and neutral alpha-glucosidase.

[0075] "Internalizing effectors" include proteins, and in some cases receptor proteins, that can be internalized into cells or that are involved in or contribute to retrograde membrane transport. The terms internalization effector, internalizing effector, internalization receptor, and internalizing receptor are used interchangeably herein. In some instances, an internalizing effector is a protein that undergoes transcytosis; i.e., the protein is internalized at one side of the cell and transported to the other side of the cell (e.g., from apical to basal). In many embodiments, the internalizing effector protein is a cell surface-expressed protein or a soluble extracellular protein. However, the present invention also contemplates embodiments in which the internalizing effector protein is expressed within intracellular compartments such as endosomes, the endoplasmic reticulum, the Golgi, lysosomes, etc. For example, proteins involved in retrograde membrane transport (e.g., from early / recycling endosomes to the trans-Golgi network) may function as internalizing effector proteins in various embodiments of the present invention. In either case, the attachment of the delivery domain to an internalizing effector protein results in the entire multidomain therapeutic protein, and any molecules associated therewith (e.g., enzymes), being internalized into the cell. As described below, internalizing effector proteins include proteins that are directly internalized into the cell, as well as proteins that are indirectly internalized into the cell.

[0076] Internalizing effector proteins that are directly internalized into cells include membrane-bound molecules with at least one extracellular domain (e.g., transmembrane proteins, GPI-anchored proteins, etc.) that undergo cellular internalization and are preferably processed via intracellular degradation and / or recycling pathways. Specific, non-limiting examples of internalizing effector proteins that are directly internalized into cells include, for example, CD63, MHC-I (e.g., HLA-B27), Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), MAL (myelin and lymphocyte protein, also known as VIP17), IGF2R, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor (e.g., SCARA1-5, SCARB1-3, CD36), and the like.

[0077] In one embodiment, the internalization effector is expressed in several tissue types and is useful for therapeutics where targeting of both CNS and peripheral cell types is desirable. Internalization effectors useful for delivery to both CNS and peripheral cell types include CD63, MHC-I, vacuolar H+ These include, but are not limited to, ATPase, IGF2R, integrin alpha 7 (ITGA7), LRP5, LRP6, LRP8, Kremen-2, LDL receptor, LDL-related protein 1 receptor, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), PRLR, MAL (myelin and lymphocyte protein (MAL), diphtheria toxin receptor, HBEGF (heparin-binding EGF-like growth factor), glutathione receptor, glutamate receptor, leptin receptor, and folate receptor. In one embodiment, the internalization effector is the prolactin receptor (PRLR). It has been found that PRLR is not only a target for certain therapeutic applications, but also an effective internalization effector protein based on its high rate of internalization and turnover. The potential of PRLR as an internalization effector protein is described, for example, in International Patent Application Publication No. WO 2015 / 026907, where, inter alia, anti-PRLR antibodies have been shown to be effective in It has been shown to be efficiently internalized by PRLR-expressing cells in vitro.

[0078] Targeting internalized effectors expressed by several cell types may be useful in the treatment of diseases such as Fabry disease, Gaucher disease, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIID, MPS IVB, MPS VI, MPS VII, MPS IX, Pompe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, Niemann-Pick disease types A, B, and C2, α-mannosidosis, neuraminidase deficiency, sialidosis, aspartylglycosaminuria, mixed saposin deficiency, variant Gaucher disease, Farber lipogranulomatosis, fucosidosis, and β-mannosidosis, where targeting of both CNS and peripheral cell types is desirable.

[0079] In another embodiment, the internalization effector is expressed in several tissue types. In one example, the internalization effector may preferentially target bone and cartilage. Effectors useful for transport to the CNS and either or both bone and cartilage include, but are not limited to, collagen X, integrin alpha 10 (ITGA10), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor isoform C (FGFR3C), hyaluronan and proteoglycan link protein 1 (CRTL1), aggrecan, collagen II, and Kremen-1. Such effectors are useful in treatments where targeting both the CNS and skeletal and cartilage is desired.

[0080] Targeting internalizing effectors preferentially expressed by bone and cartilage may be useful in the treatment of diseases such as, for example, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIID, MPS IVA, MPS IVB, MPS VI, MPS VII, MPS IX, β-mannosidosis, Gaucher disease, atypical Gaucher disease, mixed saposin deficiency, aspartylglycosaminuria, Farber lipogranulomatosis, sialidosis, neuraminidase deficiency, mucopolysaccharidosis, and α-mannosidosis, where targeting of both the CNS and the skeleton and cartilage is desired.

[0081] In yet another embodiment, the internalization effector is preferentially expressed in specific tissues or cell types, such as macrophages, monocytes, and microglia. Useful effectors for delivery to the CNS and macrophages include, but are not limited to, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, CD36, MSR1 (macrophage scavenger receptor 1), MRC1 (macrophage mannose receptor 1), VSIG4 (V-set and immunoglobulin domain-containing protein 4), CD68 (macrosialin), and CSF1R (macrophage colony-stimulating factor 1 receptor). Such effectors are useful in therapies where targeting both the CNS and macrophages is desired. CNS macrophages are sometimes referred to as microglia.

[0082] Targeting internalized effectors that are preferentially expressed by macrophages (monocytes or microglia) may be useful when targeting both the CNS and macrophages (or microglia) is desired, for example, in the treatment of diseases such as lysosomal acid lipase deficiency, Gaucher disease, atypical Gaucher disease, mixed saposin deficiency, and Farber lipogranulomatosis.

[0083] In certain embodiments, the internalization effector is a kidney-specific internalization effector, such as CDH16 (Cadherin-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (solute carrier family 22 member 13), SLC5A2 (sodium / glucose cotransporter 2), and UMOD (uromodulin).

[0084] Targeting internalizing effectors that are preferentially expressed in the kidney may be useful when targeting of both the CNS and kidney is desired, for example, in the treatment of diseases such as Fabry disease, Alport syndrome, polycystic kidney disease, and thrombotic thrombocytopenic purpura.

[0085] In yet another embodiment, internalization effector is preferentially expressed in specific tissue or cell type, such as liver.The effector that is useful for transporting to CNS and liver includes but is not limited to ASGR1 and ASGR2.This effector is useful in the treatment that is desired to target both CNS and liver.

[0086] Targeting internalized effectors that are preferentially expressed in the liver may be useful when targeting both the CNS and liver is desired, for example, in the treatment of diseases such as lysosomal acid lipase deficiency, Gaucher disease, MPS VI, MPS VII, MPS II, Niemann-Pick disease types A, B, and C2, sialidosis, neuraminidase deficiency, atypical Gaucher disease, mixed saposin deficiency, and Farber lipogranulomatosis.

[0087] In some embodiments, the internalization effector is a muscle-specific internalizer, such as BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3).

[0088] Targeting internalizing effectors that are preferentially expressed in muscle may be useful when targeting of both CNS and muscle tissue is desired, for example, in the treatment of diseases such as Pompe disease.

[0089] In some embodiments, the internalization effector is ITGA7, ITGA10, CD9, CD63, APLP2, MSR1, ASGR1, ASGR2, or PRLR. Antibodies to ITGA7, ITGA10, CD9, CD63, APLP2, MSR1, ASGR1, ASGR2, or PRLR are known in the art (see, e.g., R&D for exemplary, non-limiting anti-IGTA7 antibodies). Systems "Integrin alpha 7: Products"; for exemplary, non-limiting examples of anti-ITGA10 antibodies, see, e.g., U.S. Patent No. 8,048,991, U.S. Patent No. 20120034625, U.S. Patent No. 8,563,255, U.S. Patent No. 20140099716, U.S. Patent No. 20160319023, International Patent Application Publication No. 2018138322, and U.S. Patent No. 10,087,253; for exemplary, non-limiting anti-CD63 antibodies, see, e.g., WO 201102982; WO 2014185908; U.S. Patent No. 20160115229; WO 2017134197; U.S. Patent No. 9,738,717; and de Goeij BE et al. Mol. Cancer Ther. (2016) Nov;15(11):2688-2697; for illustrative, non-limiting examples of anti-APLP2 antibodies, see, e.g., U.S. Pat. No. 543,153A; U.S. Pat. No. 5,441,931A; U.S. Pat. No. 5,677,146A; and U.S. Pat. No. 5,935,854A; for illustrative, non-limiting examples of anti-MSR1 antibodies, see, e.g., R&D Systems product sheet MAB27081; R&D Systems product sheet AF2708; AbCam product sheet ab1515707; Yu X, et al., J.Biol.Chem., 2011;286(21):18795-806; and N Nishijima, et al., Front Immunol, 2017;8(0):379; for illustrative, non-limiting examples of anti-ASGR1 antibodies, see, e.g., International Patent Application Publication No. 2017058944; for non-limiting, exemplary anti-ASGR2 antibodies, see, e.g., www.origene.See Origene Antibodies to ASGR2 available at www.rigene.com / category / antibodies?q=ASGR2&sub_category=Primary+Antibodies&reactivities=Human; for exemplary non-limiting anti-PRLR antibodies, see, e.g., U.S. Patent Nos. 9,649,374; 9,302,015; 2013,017,1147; 10,106,616; WO 2015,026,907; 9,777,063; WO 2011,069,795; 2013,027,2968; 2014,014,1003; WO 2015,026,907; Patent Application Publication No. 2011069799; U.S. Patent No. 20120315276; International Patent Application Publication No. 2012163932; U.S. Patent No. 9241989; International Patent Application Publication No. 2012136519; International Patent Application Publication No. 2011069798; International Patent Application Publication No. 2019011719; U.S. Patent No. 8883979; U.S. Patent No. 20140065158; International Patent Application Publication No. 2015187596; U.S. Patent No. 9353186; International Patent Application Publication No. 2018102304; U.S. Patent No. 20130022606; U.S. Patent No. 9688764; U.S. Patent No. 20130129739; U.S. Patent No. 20160002342; U.S. Patent No. 20150056222; U.S. Patent No. 20150056221; U.S. Patent No. 20170008965; U.S. Patent No. 20150093393; International Patent Application Publication No. 2011069797; U.S. Patent No. 20160251442; U.S. National Patent No. 20180094066; International Patent Application Publication No. 2014036076; U.S. Patent No. 20180185504; U.S. Patent No. 20140271659; International Patent Application Publication No. 2011069794; International Patent Application Publication No. 2014143909; U.S. Patent No. 20160319029; U.S. Patent No. 9545451; U.S. Patent No. 9023357; U.S. Patent No. 20150252116; International Patent Application Publication No. 2011069796 Kelly MP, et al., Mol. Cancer Ther. (2017) Jul; 16 (7): 1299-1311; Otto C. et al.Endocrinology (2015) 156:4365-73 2017-01-20; and Andreev J et al., Mol. Cancer Ther. (2017) Apr;16(4):681-693; each of which is incorporated herein by reference in its entirety.) Those skilled in the art can readily link these well-known antibodies, or antigen-binding portions thereof (e.g., scFvs derived therefrom, etc.), to the therapeutic proteins described herein to generate and use the multidomain therapeutic proteins described herein.

[0090] In embodiments in which an internalization effector (IE) is directly internalized within a cell, the delivery domain can be, for example, an antibody or antigen-binding fragment of an antibody that specifically binds to the IE, or a ligand or portion of a ligand that specifically interacts with the IE. For example, if the IE is Kremen-1 or Kremen-2, the delivery domain can comprise or consist of a Kremen ligand (e.g., DKK1) or a Kremen-binding portion thereof. As another example, if the IE is a receptor molecule such as ASGR1, the delivery domain can comprise or consist of a ligand specific for the receptor (e.g., asialoorosomucoid [ASOR] or beta-GalNAc) or a receptor-binding portion thereof.

[0091] Internalizing effector proteins that are indirectly internalized into cells include proteins and polypeptides that are not themselves internalized, but are internalized into cells after binding or otherwise associating with a second protein or polypeptide that is directly internalized into the cell. Proteins that are indirectly internalized into cells can include, for example, soluble ligands that can bind to receptor molecules expressed on the surface of the internalizing cell. A non-limiting example of a soluble ligand that is internalized (indirectly) into cells through its interaction with a receptor molecule expressed on the surface of the internalizing cell is transferrin. In embodiments where the IE is transferrin (or another indirectly internalized protein), binding of the delivery domain to the IE and the interaction of the IE with the transferrin receptor (or another receptor molecule expressed on the surface of the internalizing cell) results in the internalization of the entire delivery domain and any molecules associated with it (e.g., enzymes) into the cell, along with the internalization of the IE and its binding partner.

[0092] In embodiments in which the IE is indirectly internalized within the cell, the delivery domain can be, for example, an antibody, antigen-binding fragment of an antibody, or scFv that specifically binds to the IE, or a receptor or portion of a receptor that specifically interacts with a soluble effector protein. For example, if the IE is a cytokine, the delivery domain can comprise or consist of the corresponding cytokine receptor or a ligand-binding portion thereof.

[0093] A typical example of IE is CD63, a member of the tetraspanin superfamily of cell surface proteins that span the cell membrane four times. CD63 is expressed in nearly all tissues and is thought to be involved in the formation and stabilization of signaling complexes. CD63 is localized to the plasma membrane, lysosomal membranes, and late endosomal membranes. CD63 is known to associate with integrins and may be involved in epithelial-mesenchymal transition. See H. Maecker et al., "The tetraspanin superfamily: molecular facilitators," 11(6) FASEB J. 428-42, May 1997; and M. Metzelaar et al., "CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells," 266 J. Biol. Chem. 3239-3245, 1991. Each of these references is incorporated herein by reference in its entirety.

[0094] Another typical example of IE is amyloid beta (A4) precursor-like protein 2 ("APLP2"), a ubiquitously expressed member of the APP (amyloid precursor protein) family. APLP2 is a membrane-bound protein known to interact with major histocompatibility complex (MHC) class I molecules (e.g., Kd). It binds to Kd on the cell surface and is internalized along with Kd in a clathrin-dependent manner. See Tuli et al., "Mechanism for amyloid precursor-like protein 2 enhancement of major histocompatibility complex class I molecule degradation," 284 The Journal of Biological Chemistry 34296-34307 (2009); this document is incorporated herein by reference in its entirety.

[0095] Another typical example of IE is the prolactin receptor (PRLR). The prolactin receptor is a member of the type I cytokine receptor family, and upon ligand binding and subsequent dimerization, it activates "tyrosine kinases Jak2, Fyn, and Tec, phosphatase SHP-2, guanine nucleotide exchange factor Vav, and signal transduction inhibitor SOCS" (Clevenger and Kline, "Prolactin receptor signal transduction," 10(10) Lupus 706-18 (2001), Abstract; each of these references is incorporated herein by reference in its entirety). The prolactin receptor can undergo endocytic recycling and be found in the lysosomal compartment. See Genty et al., "Endocytosis and degradation of prolactin and its receptor in Chinese hamster ovary cells stably transfected with prolactin receptor cDNA," 99(2) Mol. Cell Endocrinol. 221-8 (1994); and Ferland et al., "The effect of chloroquine on lysosomal prolactin receptors in rat liver," 115(5) Endocrinology 1842-9 (1984), which are incorporated herein by reference in their entireties.

[0096] As used herein, "immune response" typically refers to a patient's immunological response to a foreign or "non-self" protein. Immunological responses include allergic reactions and the development of antibodies that interfere with the effectiveness of replacement enzymes. Some patients may not produce any non-functional proteins, thus rendering the replacement enzyme a "foreign" protein. For example, repeated injections of recombinant GLA (rGLA) into patients with Fabry disease who are deficient in GLA often cause allergic reactions. In other patients, the production of antibodies against rGLA has been shown to reduce the effectiveness of replacement enzymes in treating the disease. See, for example, Tajima et al. ("Use of a Modified α-N-Acetylgalactosaminidase (NAGA) in the Development of Enzyme Replacement Therapy for Fabry Disease," 85(5) Am. J. Hum. Genet. 569-580 (2009)), which discusses the use of modified NAGA as an "isoenzyme" to replace GLA. This document is incorporated herein by reference in its entirety. Modified NAGA has no immunological cross-reactivity with GLA and was "unreactive with sera from Fabry disease patients who had been repeatedly treated with recombinant GLA" (ibid., abstract).

[0097] "Immunosuppressants" include drugs and / or proteins that result in general immunosuppression and may be used in patients with Pompe disease or Fabry disease to prevent cross-reactive immunological material (CRIM) against replacement enzymes, such as GAA or GLA, respectively. Non-limiting examples of immunosuppressants include methotrexate, mycophenolate mofetil, cyclophosphamide, rapamycin, DNA alkylating agents, anti-CD20 antibodies, anti-BAFF antibodies, anti-CD3 antibodies, anti-CD4 antibodies, and any combination thereof.

[0098] Regulatory elements, for example, promoters specific to tissues such as the liver, enhance the expression of nucleic acid sequences, for example, genes, under the control of such regulatory elements in tissues specific for the regulatory element. For non-limiting examples of liver-specific regulatory elements, for example, liver-specific promoters, see Chuah et al. (2014) Mol. Ther. 22:1605-13. These documents are incorporated herein by reference in their entirety.

[0099] The term "protein" refers to any amino acid polymer having more than about 20 amino acids covalently linked via amide bonds. Proteins contain one or more amino acid polymer chains, commonly known in the art as "polypeptides." Thus, a polypeptide can be a protein, and a protein can contain multiple polypeptides to form a single functional biomolecule. Disulfide bridges (i.e., between cysteine ​​residues, such as those forming cysteine) can be present in some proteins. These covalent bonds can be within a single polypeptide chain or between two individual polypeptide chains. For example, disulfide bridges are essential for the proper structure and function of insulin, immunoglobulins, and protamines. For a recent review of disulfide bond formation, see Oka and Bulleid, "Forming disulfides in the endoplasmic reticulum," 1833(11) Biochim Biophys Acta 2425-9 (2013), which is incorporated herein by reference in its entirety.

[0100] As used herein, "protein" includes biotherapeutic proteins, recombinant proteins used in research or therapy, trap proteins and other Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, scFv fusion proteins, cytokines, chemokines, and peptide hormones. Proteins may be produced using recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia), and mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a recent review discussing biotherapeutic proteins and their production, see Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation," 28 Biotechnol Genet Eng Rev. 147-75 (2012), which is incorporated herein by reference in its entirety.

[0101] The term "antibody," as used herein, includes immunoglobulin molecules comprising four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three domains: CH1, CH2, and CH3. The light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2, and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2, and LCDR3). The term "high affinity" antibodies refers to antibodies whose binding affinity to their target is at least 10 as measured by surface plasmon resonance, e.g., BIACORE™, or solution affinity ELISA. -9 M, at least 10 -10 M, at least 10 -11 M, or at least 10 -12 The term "antibody" refers to an antibody that is M. The term "antibody" can encompass any type of antibody, e.g., monoclonal or polyclonal. Furthermore, the antibody can be of any origin, e.g., mammalian or non-mammalian. In one embodiment, the antibody can be mammalian or avian. In a further embodiment, the antibody can be of human origin and can even be a human monoclonal antibody.

[0102] The term "bispecific antibody" includes antibodies that can selectively bind two or more epitopes. Bispecific antibodies typically contain two different heavy chains, each of which specifically binds a different epitope—either on two different molecules (e.g., antigens) or on the same molecule (e.g., the same antigen). When a bispecific antibody can selectively bind two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope is generally at least one to two, or even three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by bispecific antibodies can be on the same target or on different targets (e.g., on the same protein or on different proteins). Bispecific antibodies can be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleic acid sequences encoding different heavy chain constant regions, and these sequences can be expressed in cells that express immunoglobulin light chains. A typical bispecific antibody has two heavy chains, each having three heavy chain CDRs followed (N- to C-terminus) by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that does not confer antigen-binding specificity but is capable of associating with each heavy chain, or is capable of associating with each heavy chain and binding one or more epitopes bound by the heavy chain antigen-binding region, or is capable of associating with each heavy chain and enabling one or both heavy chains to bind one or both epitopes.

[0103] The phrase "heavy chain" or "immunoglobulin heavy chain" includes immunoglobulin heavy chain constant region sequences from any organism, including a heavy chain variable domain unless otherwise specified. Unless otherwise specified, the heavy chain variable domain includes three heavy chain CDRs and four FR regions. Fragments of heavy chains include CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain (from N-terminus to C-terminus), a CH1 domain, a hinge, a CH2 domain, and a CH3 domain. Functional fragments of heavy chains include those capable of specifically recognizing an antigen (e.g., recognizing an antigen with a KD in the micromolar, nanomolar, or picomolar range), capable of being expressed and secreted from cells, and comprising at least one CDR.

[0104] The term "light chain" includes immunoglobulin light chain constant region sequences derived from any organism, including human kappa light chains and human lambda light chains, unless otherwise specified. A light chain variable (VL) domain typically contains three light chain CDRs and four framework (FR) regions, unless otherwise specified. A full-length light chain generally contains a VL domain, including, from the amino terminus to the carboxyl terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant domain. Light chains that can be used in the present invention include, for example, light chains that selectively bind neither to the first antigen selectively bound by the antigen-binding protein nor to the second antigen. Suitable light chains include those that can be identified by screening the most commonly used light chains in existing antibody libraries (wet libraries or in silico), and do not substantially interfere with the affinity and / or selectivity of the antigen-binding domain of the antigen-binding protein. Suitable light chains include those capable of binding one or both of the epitopes bound by the antigen-binding region of the antigen-binding protein.

[0105] The term "variable domain" includes the amino acid sequence of an immunoglobulin light or heavy chain (modified as desired) which comprises, from N- to C-terminus (unless otherwise specified), the following amino acid regions: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. A "variable domain" comprises an amino acid sequence that can fold into a canonical domain (VH or VL) having a bipartite beta-sheet structure, the beta-sheets being linked by disulfide bonds between residues of the first and second beta-sheets.

[0106] The phrase "complementarity-determining region" or "CDR" includes an amino acid sequence encoded by a nucleic acid sequence of an organism's immunoglobulin gene, which sequence is normally (i.e., in wild-type animals) found between two framework regions in the variable region of the light or heavy chain of an immunoglobulin molecule (e.g., an antibody or T-cell receptor). CDRs can be encoded, for example, by germline sequences or rearranged or unrearranged sequences, e.g., by naive B cells or mature B cells or T cells. Under some circumstances (e.g., with respect to a CDR3), a CDR can be encoded by two or more sequences (e.g., germline sequences) that are not contiguous (e.g., in an unrearranged nucleic acid sequence) but are contiguous in the B-cell nucleic acid sequence, e.g., as a result of splicing or joining of sequences (e.g., VDJ rearrangement to form a heavy chain CDR3).

[0107] The term "antibody fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed by the term "antibody fragment" include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) an F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single antibody arm; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 241:544-546, which is incorporated herein by reference in its entirety); (vi) an isolated CDR; and (vii) an scFv, which consists of the two domains, VL and VH, of an Fv fragment connected by a synthetic linker to form a single protein chain in which the VL and VH domains pair to form a monovalent molecule. Other forms of single-chain antibodies, such as diabodies, are also encompassed under the term "antibody" (see, e.g., Holliger et al. (1993) PNAS USA 90:6444-6448; Poljak et al. (1994) Structure 2:1121-1123, which are incorporated herein by reference in their entireties).

[0108] The phrase "Fc-containing protein" includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that contain at least a functional portion of the CH2 and CH3 regions of an immunoglobulin. "Functional portion" refers to the CH2 and CH3 regions that can bind an Fc receptor (e.g., FcyR or FcRn, i.e., fetal Fc receptor) and / or participate in complement activation. The CH2 and CH3 regions are not functional if they contain deletions, substitutions, and / or insertions, or other modifications that render them incapable of binding any Fc receptor and incapable of complement activation.

[0109] An Fc-containing protein can contain modifications in the immunoglobulin domain, including those that affect one or more effector functions of the binding protein (e.g., modifications that affect FcyR binding, FcRn binding, and therefore half-life and / or CDC activity). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to the EU numbering of immunoglobulin constant regions: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362, 373, 375, 376, 378, 380, 382, ​​383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438, and 439.

[0110] For example, and not intended to be limiting, the binding protein is an Fc-containing protein that exhibits improved serum half-life (compared to the same Fc-containing protein without the described modifications) and has modifications at positions 250 (e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T); or modifications at positions 428 and / or 433 (e.g., L / R / SI / P / Q or K) and / or 434 (e.g., H / F or Y); or modifications at positions 250 and / or 428; or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In another example, modifications can include 428L (e.g., M428L) and 434S (e.g., N434S) modifications; 428L, 2591 (e.g., V259I), and 308F (e.g., V308F) modifications; 433K (e.g., H433K) and 434 (e.g., 434Y) modifications; 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications; 250Q and 428L modifications (e.g., T250Q and M428L); 307 and / or 308 modifications (e.g., 308F or 308P).

[0111] The term "antigen-binding protein," as used herein, refers to a polypeptide or protein (one or more polypeptides complexed as a functional unit) that specifically recognizes an epitope of an antigen, such as a cell-specific antigen and / or a target antigen of the present invention. An antigen-binding protein may be multispecific. The term "multispecific" in reference to an antigen-binding protein means that the protein recognizes different epitopes, either on the same antigen or on different antigens. A multispecific antigen-binding protein of the present invention may be a single multifunctional polypeptide or a multimeric complex of two or more polypeptides associated with each other covalently or noncovalently. The term "antigen-binding protein" includes an antibody or fragment thereof of the present invention, which may be linked to or co-expressed with another functional molecule, e.g., another peptide or protein. For example, an antibody or fragment thereof may be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or otherwise) to one or more other molecular entities, such as proteins or fragments thereof, to produce a bispecific or multispecific antigen-binding molecule having a second binding specificity.

[0112] As used herein, the term "epitope" refers to a portion of an antigen recognized by a multispecific antigen-binding polypeptide. A single antigen (such as an antigen polypeptide) may have more than one epitope. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and are defined as residues that directly contribute to the affinity of the interaction between an antigen-binding polypeptide and an antigen. Epitopes may also be conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three-dimensional structural and / or specific charge characteristics. Epitopes formed from contiguous amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents.

[0113] The term "domain" refers to any part of a protein or polypeptide that has a specific function or structure. Preferably, the domain of the present invention binds to a cell-specific antigen or a target antigen. As used herein, a cell-specific antigen domain or a target antigen-binding domain includes any polypeptide or glycoprotein that is naturally occurring, enzymatically obtainable, synthetic, or genetically modified and specifically binds an antigen.

[0114] The terms "half-body" and "half-antibody" are used interchangeably and refer to half of an antibody that essentially contains one heavy chain and one light chain. Antibody heavy chains can form dimers, and thus the heavy chain of one half-antibody can associate with a heavy chain associated with a different molecule (e.g., another half-antibody) or another Fc-containing polypeptide. Two slightly different Fc domains can "heterodimerize," as in the formation of bispecific antibodies, or other heterodimers, heterotrimers, and heterotetramers. See Vincent and Murini, "Current strategies in antibody engineering: Fc engineering and pH-dependent antigen binding, bispecific antibodies, and antibody drug conjugates," 7 Biotechnol. J. 1444-1450 (2012); and Shimamoto et al., "Peptibodies: A flexible alternative format to antibodies," 4(5) MAbs 586-91 (2012). Each document is incorporated herein by reference in its entirety.

[0115] In one embodiment, the half antibody variable domains specifically recognize an internalization effector, and the Fc domain of the half antibody dimerizes with an Fc fusion protein containing a recruiting enzyme (e.g., a peptibody) (Id., p. 586).

[0116] The term "single-chain variable fragment" or "scFv" includes a single-chain fusion polypeptide containing an immunoglobulin heavy chain variable region (VH) and an immunoglobulin light chain variable region (VL). In some embodiments, the VH and VL are connected by a linker sequence of 10 to 25 amino acids. ScFv polypeptides may also contain other amino acid sequences, such as a CL region or a CH1 region. ScFv molecules may be produced by phage display or by directly subcloning heavy and light chains from hybridomas or B cells. Ahmad et al., Clinical and Developmental Immunology, volume 2012, article ID 98025, is incorporated herein by reference for methods of generating scFv fragments by phage display and antibody domain cloning.

[0117] The terms "alpha-glucosidase" (or "α-glucosidase"), "α-glucosidase activity," "GAA," and "GAA activity" are used interchangeably and refer to any protein that facilitates the hydrolysis of 1,4-alpha bonds in glycogen and starch to glucose. GAA is also known as EC 3.2.1.20, maltase, glucoinvertase, glucoside sucrase, maltase-glucoamylase, α-glucopyranosidase, glucoside invertase, α-D-glucosidase, α-glucoside hydrolase, α-1,4-glucosidase, and α-D-glucoside glucohydrolase, among others. GAA can be found in lysosomes and in the brush border of the small intestine. Patients with Pompe disease lack functional lysosomal α-glucosidase. See S. Chiba, "Molecular mechanism in alpha-glucosidase and glucoamylase," 61(8) Biosci. Biotechnol. 1233-9 (1997); and Hesselink et al., "Lysosomal dysfunction in muscle with special reference to glycogen storage disease type II," 1637(2) Biochim. Biophys. Acta. 164-70 (2003), which are incorporated herein by reference in their entireties.

[0118] The terms "alpha-galactosidase A" (or "α-galactosidase A"), "α-galactosidase A activity," "α-galactosidase," "α-galactosidase activity," "GLA," and "GLA activity" are used interchangeably and refer to any protein that promotes the hydrolysis of terminal α-galactosyl moieties from glycolipids and glycoproteins and hydrolyzes α-D-fucosides. GLA is also known, inter alia, as EC 3.2.1.22, melibiase, α-D-galactosidase, α-galactosidase A, α-galactoside galactohydrolase, and α-D-galactoside galactohydrolase. GLA is a lysosomal enzyme encoded by the X-linked GLA gene. Deficiency of GLA can cause Fabry disease, in which a glycolipid known as globotriaosylceramide (also known as Gb3, GL-3, or ceramide trihexoside) accumulates within blood vessels (i.e., protuberant vasculopathy), resulting in pain and impaired function of the kidneys, heart, skin, and / or cerebrovascular tissue, as well as other tissues and organs. See, e.g., Prabakaran et al., "Mannose 6-phosphate receptor and sortilin-mediated endocytosis of α-galactosidase A in kidney endothelial cells," 7(6) PLoS One e39975 pp. 1-9 (2012), which is incorporated herein by reference in its entirety.

[0119] In one aspect, the present invention provides a method of treating a patient (or subject) suffering from a lysosomal storage disease by administering to the patient a "multidomain therapeutic protein." The multidomain therapeutic protein enters the patient's cells and delivers to the lysosomes an enzyme or enzyme activity (i.e., a "replacement enzyme") that replaces the enzyme (i.e., "endogenous enzyme") or enzyme activity associated with the LSD. In one embodiment, the multidomain therapeutic protein is delivered to the patient via a gene therapy vector containing a polynucleotide encoding the multidomain therapeutic protein.

[0120] LSDs include sphingolipid storage disorders, mucopolysaccharidoses, and glycogen storage disorders. In some embodiments, the LSD is any one or more of Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Niemann-Pick disease type A, Niemann-Pick disease type B, GM1-gangliosidosis, Sandhoff disease, Tay-Sachs disease, GM2-activator deficiency, GM3-gangliosidosis, metachromatic leukodystrophy, sphingolipid activator deficiency, Scheie disease, Hurler-Scheie disease, Hurler disease, Hunter disease, Sanfilippo A, Sanfilippo B, Sanfilippo C, Sanfilippo D, Morquio syndrome A, Morquio syndrome B, Maroteaux-Lamy disease, Sly disease, MPS IX, and Pompe disease. In one particular embodiment, the LSD is Fabry disease. In another embodiment, the LSD is Pompe disease.

[0121] In some embodiments, a multidomain therapeutic protein comprises (a) a recruitment enzyme and (b) a molecular entity (delivery domain) that binds an internalization effector. In some embodiments, the replacement enzyme is any one or more of α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, saposin-C activator, ceramidase, sphingomyelinase, β-hexosaminidase, GM2 activator, GM3 synthase, arylsulfatase, sphingolipid activator, α-iduronidase, iduronidase-2-sulfatase, heparin N-sulfatase, N-acetyl-α-glucosaminidase, α-glucosamide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, and hyaluronidase.

[0122] In some cases, a patient may not make enough of the protein for the replacement enzyme to be recognized as "non-self" by the patient, resulting in an immune response following administration of the replacement enzyme. This is undesirable. Therefore, in some embodiments, the replacement enzyme is designed or produced to avoid inducing an immune response in the subject. One such solution is to use an "isoenzyme" as the replacement enzyme. The isozyme is sufficiently similar to the patient's "self" protein, but has sufficient replacement enzyme activity to alleviate the symptoms of LSD.

[0123] In one particular embodiment where the LSD is Pompe disease and the endogenous enzyme is α-glucosidase (GAA), the isozyme can be any one of acid α-glucosidase, sucrase-isomaltase (SI), maltase-glucoamylase (MGAM), glucosidase II (GANAB), and neutral α-glucosidase (CGNAC). In another particular embodiment where the LSD is Fabry disease and the endogenous enzyme is α-galactosidase A (GLA), the isozyme can be α-N-acetylgalactosaminidase engineered to have GLA activity.

[0124] Provided herein are methods, other than the use of isozymes, for reducing cross-reactive immunological material (CRIM) against a replacement enzyme. As shown in Figures 5 and 6, administration (e.g., via a gene therapy vector) of a multidomain therapeutic protein comprising an internalization effector-binding domain and an enzymatic domain, in combination with administration of a control therapeutic protein (comprising an enzymatic domain that lacks the internalization effector domain), reduces the level of CRIM against the replacement enzyme. Thus, in one embodiment, reducing CRIM against an enzyme in a patient having an enzyme deficiency comprises administering to the patient a multidomain therapeutic protein (or a nucleic acid encoding the same, e.g., a gene therapy vector containing a gene encoding a multidomain therapeutic protein), where the multidomain therapeutic protein comprises a delivery domain (e.g., an internalization effector-binding protein) and an enzymatic domain.

[0125] The multidomain therapeutic protein has an internalization effector binding protein component that allows the replacement enzyme to be taken up into the cell. Thus, in some embodiments, the internalization effector can be CD63, MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL-receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein-2 (APLP2), apelin receptor (APLNR), PRLR (prolactin receptor), MAL (myelin and lymphocyte protein, also known as VIP17), IGF2R, vacuolar H+ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor, SCARA1-5, SCARB1-3, and CD36. In certain embodiments, the internalization effector is a kidney-specific internalizer, such as CDH16 (Cadherin-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (solute carrier family 22 member 13), SLC5A2 (sodium / glucose cotransporter 2), and UMOD (uromodulin). In certain other embodiments, the internalization effector is a muscle-specific internalizer, such as BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3).In some specific embodiments, the internalization effector is ITGA7, CD9, CD63, APLP2, ASGR1, ASGR2, or PRLR.

[0126] In some embodiments, the internalizing effector binding protein comprises an antigen binding protein, such as, for example, a receptor fusion molecule, a trap molecule, a receptor-Fc fusion molecule, an antibody, a Fab fragment, a F(ab')2 fragment, a Fd fragment, a Fv fragment, a single chain Fv (scFv) molecule, a dAb fragment, an isolated complementarity determining region (CDR), a CDR3 peptide, a constrained FR3-CDR3-FR4 peptide, a domain-specific antibody, a single domain antibody, a domain-deleted antibody, a chimeric antibody, a CDR-grafted antibody, a diabody, a triabody, a tetrabody, a minibody, a nanobody, a monovalent nanobody, a bivalent nanobody, a small modular immunopharmaceutical (SMIP), a camelid antibody (a VHH heavy chain homodimeric antibody), and a shark variable IgNAR domain.

[0127] In one embodiment, the molecular entity that binds the internalization effector is an antibody, antibody fragment, or other antigen-binding protein. For example, the molecular entity can be a bispecific antibody, in which one arm binds an internalization effector (e.g., ITGA7, CD9, CD63, PRLR, APLP2, ASGR1, ASGR2) and the other arm binds a replacement enzyme. Here, the multidomain therapeutic protein comprises the bispecific antibody and the replacement enzyme (Figure 1A). In a specific embodiment, the disease to be treated is Fabry disease, and the multidomain therapeutic protein comprises GLA and a bispecific antibody that binds GLA and CD63. In a specific embodiment, the disease to be treated is Fabry disease, and the multidomain therapeutic protein comprises GLA and a bispecific antibody that binds GLA and ITGA7. In another specific embodiment, the disease to be treated is Pompe disease, and the multidomain therapeutic protein comprises GAA and a bispecific antibody that binds GAA and CD63. In another specific embodiment, the disease to be treated is Pompe disease and the multidomain therapeutic protein comprises GAA and a bispecific antibody that binds GAA and ITGA7.

[0128] In another embodiment, the molecular entity that binds the internalizing effector comprises a half antibody and a recruiting enzyme containing an Fc domain (enzyme-Fc fusion polypeptide). In one embodiment, the Fc domain of the enzyme-Fc fusion polypeptide associates with the Fc domain of the internalizing effector-specific half antibody to form a multidomain therapeutic protein (Figure 1B).

[0129] In other embodiments, the recruited enzyme is covalently linked to the internalization effector-binding protein. The enzyme-Fc fusion:half antibody embodiment described in the previous paragraph (see also Figure 1B) belongs to this class because its Fc dimer can be fixed via one or more disulfide bridges. The covalent link between the enzymatically active domain or polypeptide and the internalization-binding domain or polypeptide can be any type of covalent bond, i.e., any bond involving the sharing of electrons. In some cases, the covalent bond is a peptide bond between two amino acids, and thus the recruited enzyme and the internalization effector-binding protein form, in whole or in part, a continuous polypeptide chain, as in a fusion protein. In some cases, the recruited enzyme moiety and the internalization effector-binding protein are directly linked. In other cases, a linker is used to connect the two moieties. See Chen et al., "Fusion protein linkers: property, design and functionality," 65(10) Adv Drug Deliv Rev. 1357-69 (2013).

[0130] The term "linker" or "spacer" typically refers to a short (e.g., 2-25 amino acids) polypeptide that allows for proper folding of one or more linking components of a fusion protein, e.g., the VH associated with the VL of an scFv, or a therapeutic protein (e.g., a recruitment enzyme) linked to a delivery domain (e.g., an anti-internalization effector antibody) of a multidomain therapeutic protein as described herein. The linker provides a flexible junction region for the components of the fusion protein, allowing the two ends of the molecule to move independently and may play an important role in maintaining the proper function of each of the two moieties. Thus, the junction region, in some cases, functions both as a linker that joins the two moieties together and as a spacer that allows each of the two moieties to form a unique biological structure and not interfere with the other moiety. Furthermore, the junction region should create an epitope that is not recognized as foreign by the subject's immune system, or in other words, is not considered immunogenic. The choice of linker can also affect the binding activity of the fusion molecule. (See Huston, et al, 1988, PNAS, 85:16:5879-83; Robinson & Bates, 1998, PNAS 95(11):5929-34; Arai, et al. 2001, PEDS, 14(8):529-32; and Chen, X. et al., 2013, Advanced Drug Delivery Reviews 65:1357-1369.) In one embodiment, the delivery domain is connected to the therapeutic polypeptide or fragment thereof via one or more peptide linkers. In another embodiment, the variable regions of the scFv antibodies are connected to each other or to fragments thereof via one or more peptide linkers.

[0131] The length of the linker chain can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more amino acid residues, but is typically 5 to 25. Examples of linkers include polyglycine linkers such as Gly-Gly, Gly-Gly-Gly (3Gly), 4Gly, 5Gly, 6Gly, 7Gly, 8Gly, or 9Gly. Examples of linkers also include Gly-Ser peptide linkers such as Ser-Gly, Gly-Ser, Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Ser, Ser-Gly-Gly, Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly, Gly-Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly-Gly, Gly-Gly-Gly-Gly-Gly-Gly-Ser, Ser-Gly-Gly-Gly-Gly-Gly-Gly, Gly-Gly-Gly-Gly-Gly-Gly-Gly, (Gly-Gly-Gly-Gly-Ser)n, and (Ser-Gly-Gly-Gly-Gly)n, where n=1-10. (Gly-Gly-Gly-Gly-Ser)n and (Ser-Gly-Gly-Gly-Gly)n are also known as (G4S)n and (S4G)n, respectively.

[0132] In some embodiments, a therapeutic protein, e.g., a recruitment enzyme, is covalently attached to the C-terminus of the heavy chain (see FIG. 1C) or the C-terminus of the light chain (FIG. 1E) of an anti-internalization effector antibody. In some embodiments, a recruitment enzyme is covalently attached to the N-terminus of the heavy chain (see FIG. 1D) or the N-terminus of the light chain (FIG. 1F) of an anti-internalization effector antibody. In some embodiments, an enzyme is linked to the C-terminus of an anti-internalization effector scFv domain (FIG. 1G).

[0133] In some cases, particularly when the therapeutic protein, e.g., the replacement enzyme, is not normally proteolytically processed in the lysosome, a cleavable linker is added to embodiments of multidomain therapeutic proteins comprising an antibody-enzyme fusion. In some embodiments, a cathepsin-cleavable linker is inserted between the antibody and the replacement enzyme to facilitate removal of the antibody in the lysosome, a) to help preserve as much enzyme activity as possible by removing the sterically bulky antibody, and b) to potentially increase the lysosomal half-life of the enzyme.

[0134] In one specific embodiment, the multidomain therapeutic protein is delivered to a patient or cell in a gene therapy vector containing a polynucleotide encoding the multidomain therapeutic protein, hi one embodiment, the multidomain therapeutic protein comprises a delivery domain and an enzymatic domain. In some embodiments, the delivery domain binds to an internalization effector, such as CD63, MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), MAL (myelin and lymphocyte protein (MAL)), IGF2R, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, or CD36. In one embodiment, the delivery domain is a single-chain variable fragment (scFv) that binds CD63 (i.e., anti-CD63 scFv). In another embodiment, the delivery domain is a single-chain variable fragment (scFv) that binds ITGA7 (i.e., anti-ITGA7 scFv).

[0135] In a particular embodiment, the enzymatic domain of the multidomain therapeutic protein comprises a hydrolase. In a particular embodiment, the enzymatic domain comprises a hydrolase that is a glycosylase. In a more particular embodiment, the enzymatic domain comprises a glycosylase that is a glycosidase. In a more particular embodiment, the enzymatic domain is a glycosidase that is an alpha-glucosidase.

[0136] Generally, disclosed herein are compositions and uses comprising polynucleotides, e.g., (m)RNA, DNA, and modified forms thereof, encoding multidomain therapeutic proteins comprising an internalizing effector domain and an enzymatic domain in the treatment of lysosomal storage diseases, e.g., for reducing glycogen and / or increasing immune tolerance to GAA in patients with Pompe disease.

[0137] The term "polynucleotide" includes a polymer of nucleotides (e.g., RNA or DNA) that encodes at least one polypeptide, including fusion polypeptides, e.g., multidomain therapeutic polypeptides comprising an internalizing effector domain and an enzymatic domain. As used herein, polynucleotide encompasses polymers comprising both modified and unmodified nucleotides. A polynucleotide may contain one or more coding and non-coding regions. A polynucleotide can be purified from a natural source, produced using a recombinant expression system, optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, a polynucleotide can include nucleotide analogs, such as analogs with chemically modified bases or sugars, backbone modifications, etc. Unless otherwise indicated, a polynucleotide sequence is presented in a 5' to 3' direction. In some embodiments, polynucleotides are selected from the group consisting of naturally occurring nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, chemically modified bases; biologically modified bases (e.g., methylated bases); intercalating bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0138] In some embodiments, a polynucleotide comprises one or more non-standard nucleotide residues. Non-standard nucleotide residues can include, for example, 5-methyl-cytidine ("5mC"), pseudouridine ("ψU"), and / or 2-thio-uridine ("2sU"). See, for example, U.S. Pat. No. 8,278,036 or International Patent Application Publication No. 2011012316, each of which is incorporated by reference in its entirety for a discussion of such residues and their incorporation into polynucleotides. The presence of non-standard nucleotide residues can render a polynucleotide less stable and / or less immunogenic than a control, i.e., a polynucleotide having the same sequence but containing only standard residues. In yet another embodiment, a polynucleotide may contain one or more non-standard nucleotide residues selected from isocytosine, pseudocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurine cytosine, as well as combinations of these and other nucleobase modifications. Certain embodiments may further include additional modifications to the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., locked nucleic acids (LNAs) with one or more 2'-O-alkyl modifications). In some embodiments, a polynucleotide may be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNAs). In embodiments where the sugar modification is a 2'-O-alkyl modification, such modifications include, but are not limited to, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy modifications. In certain embodiments, any of these modifications may be present in 0% to 100% of the nucleotides, e.g., individually or in combination, in 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the component nucleotides.In some embodiments, a polynucleotide comprises a messenger RNA (mRNA) molecule, which may or may not be modified by well-known methods to increase its stability and / or reduce its immunogenicity, e.g., may or may not contain modified nucleotides. In some embodiments, a polynucleotide comprises a DNA molecule, which may or may not be modified by well-known methods to increase its stability and / or reduce its immunogenicity, e.g., may or may not contain modified nucleotides.

[0139] In some embodiments, the polynucleotide also comprises a "locus targeting nucleic acid sequence." The locus targeting sequence allows for integration of the polynucleotide encoding the multidomain therapeutic protein into the genome of the recipient host cell. In some embodiments, the locus targeting sequence comprises flanking homology arms to enable homologous recombination. In some embodiments, the locus targeting sequence comprises a guide RNA sequence and a type II Cas enzyme (i.e., CRISPR-Cas9 method) to drive integration. In some embodiments, the locus targeting sequence comprises a guide zinc finger nuclease (ZFN) recognition sequence to drive integration. In some embodiments, the locus targeting sequence comprises a transcription activator-like effector nuclease (TALEN) recognition sequence to drive integration. In still other embodiments, the locus targeting sequence comprises a single residue pair nucleotide code used by a BuD-derived nuclease to drive integration.

[0140] In some embodiments, the genomic locus into which the polynucleotide encoding the multidomain therapeutic protein is integrated is a “safe harbor locus.” In one embodiment, a “safe harbor locus” allows for high expression of the multidomain therapeutic protein while not interfering with the expression of essential genes or promoting the expression of oncogenes or other deleterious genes. In one embodiment, the genomic locus is the liver-expressed albumin (Alb) locus, the EESYR locus, the SARS locus, position 188,083,272 on human chromosome 1 or its orthologue in a non-human mammal, position 3,046,320 on human chromosome 10 or its orthologue in a non-human mammal, position 67,328,980 on human chromosome 17 or its orthologue in a non-human mammal, the chromosomal adeno-associated viral site 1 (AAVS1), a naturally occurring integration site for the AAV virus on human chromosome 19 or its orthologue in a non-human mammal, the chemokine receptor 5 (CCR5) gene, a chemokine receptor gene encoding an HIV-1 co-receptor, or the mouse Rosa26 locus or its orthologue in a non-mouse mammal. In one embodiment, the genomic locus is an adeno-associated viral site. In one embodiment, genomic loci for integration are selected according to the method of Papapetrou and Schambach, J. Molecular Therapy, vol. 24(4):678-684, April 2016, which is incorporated herein by reference for stepwise selection of safe harbor genomic loci for use in integration of gene therapy vectors, but see also Barzel et al. Nature, vol. 517:360-364, which is incorporated herein by reference in its entirety, for promoterless gene targeting within the liver-expressed albumin (Alb) locus.

[0141] In some embodiments, the polynucleotide, e.g., DNA, also contains a promoter operably linked to the nucleic acid sequence encoding the multidomain therapeutic protein. In certain embodiments, the promoter is a tissue-specific promoter that drives gene expression in specific tissues. In one embodiment, the tissue-specific promoter is a liver-specific enhancer / promoter derived from Serpinal (e.g., SEQ ID NO: 9) and / or the TTR promoter (SEQ ID NO: 8). In other embodiments, the promoter is a CMV promoter. In other embodiments, the promoter is a ubiquitin C promoter.

[0142] In one embodiment, a "gene therapy vector" encoding a multidomain therapeutic protein is any vector capable of delivering a polynucleotide encoding a multidomain therapeutic protein to a host, such as a patient. In some embodiments, the gene therapy vector targets a specific host cell or organ, e.g., for localized delivery, e.g., tissue-specific delivery. Typically, localized delivery requires the protein (e.g., a multidomain therapeutic protein) encoded by an mRNA to be translated and expressed primarily in and / or by an organ, e.g., the liver, thereby forming a depot, e.g., a liver depot, for production (and secretion) of the protein. In some embodiments, the gene therapy vector delivers a polynucleotide of a multidomain therapeutic protein to the liver of a patient to form a liver depot. See, e.g., Derosa et al., Gene Therapy, vol. 10:699-707, incorporated herein by reference in its entirety. In some embodiments, the gene therapy vector delivers a polynucleotide encoding a multidomain therapeutic protein to muscle tissue in a patient. In some embodiments, the gene therapy vector delivers a polynucleotide encoding a multidomain therapeutic protein to the brain of a patient.

[0143] Any known or future-developed gene therapy delivery vector, whether naturally occurring or engineered, can be used in the practice of the present invention. In some embodiments, the gene therapy vector is a viral vector, e.g., comprising a virus, a viral capsid, a viral genome, etc. In some embodiments, the gene therapy vector is a naked polynucleotide, e.g., an episome. In some embodiments, the gene therapy vector comprises a polynucleotide complex. Exemplary, non-limiting polynucleotide complexes for use as gene therapy vectors include lipoplexes, polymersomes, polypex, dendrimers, inorganic nanoparticles (e.g., polynucleotide-coated gold, silica, iron oxide, calcium phosphate, etc.). In some embodiments, the gene therapy vectors described herein comprise a combination of a viral vector, a naked polynucleotide, and a polynucleotide complex.

[0144] In one embodiment, the gene therapy vector is a virus, including retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus. In one embodiment, the gene therapy vector is an adeno-associated virus (AAV), including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, or engineered or naturally selected variants thereof.

[0145] In one embodiment, the polynucleotide also contains an adeno-associated virus (AAV) nucleic acid sequence. In one embodiment, the gene therapy vector is a chimeric adeno-associated virus containing genetic elements from two or more serotypes. For example, an AAV vector having the rep gene from AAV1 and the cap gene from AAV2 (referred to as AAV1 / 2 or AAV RC1 / 2) may be used as a gene therapy vector to deliver a polynucleotide of a multidomain therapeutic protein to cells or cells of a patient in need thereof.In one embodiment, the gene therapy vector is selected from the group consisting of AAV1 / 2, AAV1 / 3, AAV1 / 4, AAV1 / 5, AAV1 / 6, AAV1 / 7, AAV1 / 8, AAV1 / 9, AAV1 / 10, AAV1 / 11, AAV2 / 1, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2 / 10, AAV2 / 11, AAV3 / 1, AAV3 / 2, AAV3 / 4, AAV3 / 5, AAV3 / 6, AAV3 / 7, AAV3 / 8, AAV3 / 9, AAV3 / 10, AAV3 / 11, AAV3 / 12, AAV3 / 13, AAV3 / 14, AAV3 / 15, AAV3 / 16, AAV3 / 17, AAV3 / 18, AAV3 / 19, AAV3 / 20, AAV3 / 21, AAV3 / 22, AAV3 / 23, AAV3 / 24, AAV3 / 25, AAV3 / 26, AAV3 / 27, AAV3 / 28, AAV3 / 29, AAV3 / 30, AAV3 / 31, AAV3 / 32, AAV3 / 33, AAV3 / 34, AAV3 / 35, AAV3 / 36, AAV3 / 37, AAV3 / 38, AAV3 / 39, AAV3 / 41, AAV3 / 42, AAV3 / 43, AAV3 / 44, AAV3 / 45, AAV3 / 46, AAV3 / 47, AAV3 / 48, AAV3 / 49, AAV3 / 50, AAV3 / 51, AAV3 / 52, AAV3 / 53, AAV3 / 54, AAV3 / 55, AAV3 / 56, AAV AV3 / 9, AAV3 / 10, AAV3 / 10, AAV4 / 1, AAV4 / 2, AAV4 / 3, AAV4 / 5, AAV4 / 6, AAV4 / 7, AAV4 / 8, AAV4 / 9, AAV4 / 10, AAV4 / 11, AAV5 / 1, AAV5 / 2 , AAV5 / 3, AAV5 / 4, AAV5 / 6, AAV5 / 7, AAV5 / 8, AAV5 / 9, AAV5 / 10, AAV5 / 11, AAV6 / 1, AAV6 / 2, AAV6 / 3, AAV6 / 4, AAV6 / 5, AAV6 / 7, AAV6 / 8 , AAV6 / 9, AAV6 / 10, AAV6 / 10, AAV7 / 1, AAV7 / 2, AAV7 / 3, AAV7 / 4, AAV7 / 5, AAV7 / 6, AAV7 / 8, AAV7 / 9, AAV7 / 10, AAV7 / 11, AAV8 / 1, AAV 8 / 2, AAV8 / 3, AAV8 / 4, AAV8 / 5, AAV8 / 6, AAV8 / 7, AAV8 / 9, AAV8 / 10, AAV8 / 11, AAV9 / 1, AAV9 / 2, AAV9 / 3, AAV9 / 4, AAV9 / 5, AAV9 / 6, AAV 9 / 7, AAV9 / 8, AAV9 / 10, AAV9 / 11, AAV10 / 1, AAV10 / 2, AAV10 / 3, AAV10 / 4, AAV10 / 5, AAV10 / 6, AAV10 / 7, AAV10 / 8, AAV10 / 9, AAV10 / 11, AAV11 / 1, AAV11 / 2, AAV11 / 3, AAV11 / 4, AAV11 / 5, AAV11 / 6, AAV11 / 7, AAV11 / 8, AAV11 / 9, AAV11 / 10, chimeric viral vectors or derivatives thereof.Gao et al., "Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy," PNAS 99(18):11854-11859, September 3, 2002, is incorporated herein by reference for its description of AAV vectors and chimeric virus vectors useful as gene therapy vectors, and their construction and use.

[0146] In a more specific embodiment, the gene therapy vector is a chimeric AAV vector having a serotype 2 rep gene sequence and a serotype 8 cap sequence ("AAV2 / 8" or "AAV RC2 / 8").

[0147] In some embodiments, the gene therapy vector is a viral vector that has been pseudotyped (e.g., engineered) to target specific cells, such as hepatocytes. Much of the progress in targeted gene therapy using viral vectors can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modifications of viral vectors, resulting in pseudotyping, expansion, and / or retargeting of the viral vector's natural tropism (reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012:1-15, each of which is incorporated herein by reference in its entirety). Non-genetic approaches typically utilize adapters that recognize both wild-type (unmodified) viral surface proteins and target cells. Soluble pseudoreceptors (wild-type viruses), polymers such as polyethylene glycol, and antibodies or portions thereof have been used as the virus-binding domain of the adapter, while natural peptide or vitamin ligands, as well as antibodies and portions thereof, have been used as the cell-binding domain of the adapter. For example, retargeting of a viral vector to a target cell can be achieved by binding the vector:adapter complex to a protein expressed on the surface of the target cell, such as a cell surface protein.Such approaches have been developed using AAV (Bartlett et al. (1999) Nat. Biotechnol. 74:2777-2785), adenovirus (Hemminki et al. (2001) Cancer Res. 61:6377-81; van Beusechem et al. (2003) Gene Therapy 10:1982-1991; Einfeld, et al. (2001) J. Virol. 75:11284-91; Glasgow et al. (2009) PLOS One 4:e8355), herpesvirus (Nakano et al. (2005) Mol. Ther. 11:617-24), and paramyxovirus (Bian et al. (2005) Cancer Gene Ther. 12:295-303; Bian et al. (2009) PLOS One 4:e8355). al. (2005) Int. J. Oncol. 29:1359-69), and coronaviruses (Haijema et al. (2003) J. Virol. 77:4528-4538; Wurdinger et al. (2005) Gene Therapy 12:1394-1404, each of which is incorporated herein by reference in its entirety).

[0148] A more common approach is recombinant genetic modification of viral capsid proteins, and thus the surface of the viral capsid. In the indirect recombinant approach, the viral capsid is modified with a heterologous "scaffold" that is then linked to an adapter. The adapter binds to the scaffold and to the target cell. (See also Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagen et al. (2002) J. Virol. 76:12900-907; International Patent Application No. 97 / 05266, each of which is incorporated herein by reference in its entirety.) Scaffolds such as (1) Fc-binding molecules (e.g., Fc receptors, protein A, etc.) that bind to the Fc of antibody adapters, (2) (strept)avidin that binds to biotin-labeled adapters, (3) biotin that binds to adapters fused to (strept)avidin, and (4) protein:protein binding pairs that form isometric peptide bonds, such as SpyCatcher that binds to Spy-labeled adapters, have been incorporated into Ad (Pereboeva et al. (2007) Gene Therapy 14:627-637, Park et al. (2008) Biochemical and Biophysical Research Communications 366:769-774, Henning et al. (2002) Human Gene Therapy 13:1427-1439, Banerjee et al. (2011) Bioorganic and Medicinal Chemistry Letters 21:4985-4988), AAV (Gigout et al. (2011) Bioorganic and Medicinal Chemistry Letters 21:4985-4988), and so on have been incorporated into AAV (Gigout et al. (2011) Bioorganic and Medicinal Chemistry Letters 21:4985-4988). al. (2005) Molecular Therapy 11:856-865, Stachler et al. (2008) Molecular Therapy 16:1467-1473), and togaviruses (Quetglas et al. (2010) Virus Research 153:179-196, Ohno et al. (1997) Nature Biotechnology 15:763-767, Klimstra et al. (2005) Virology 338:9-21, each of which is incorporated herein by reference in its entirety).

[0149] In direct recombinant targeting approach, targeting ligand is directly inserted or attached into virus capsid, that is, protein virus capsid is modified to express heterologous ligand.Then, the ligand re-orients, for example, binds to the receptor or marker that is preferentially or only expressed on target cells (Stachler et al. (2006) Gene Ther.13:926-931; White et al. (2004) Circulation 109:513-519, each of which is incorporated herein by reference in its entirety). Direct recombinant approaches include AAV (Park et al., (2007) Frontiers in Bioscience 13:2653-59; Girod et al. (1999) Nature Medicine 5:1052-56; Grifman et al. (2001) Molecular Therapy 3:964-75; Shi et al. (2001) Human Gene Therapy 12:1697-1711; Shi and Bartlett (2003) Molecular Therapy 7:515-525, each of which is incorporated by reference in its entirety), retrovirus (Dalba et al. Current Gene Therapy 5:655-667; Tai and Kasahara (2008) Frontiers in Bioscience 13:3083-3095; Russell and Cosset (1999) Journal of Gene Medicine 10:1052-56; 1:300-311; Erlwein et al. (2002) Virology 302:333-341; Chadwick et al. (1999) Journal of Molecular Biology 285:485-494; Pizzato et al. (2001) Gene Therapy 8:1088-1096), poxvirus (Guse et al. al.(2011)Expert Opinion on Biological Therapy 11:595-608;Galmiche et al.(1997) Journal of General Virology 78:3019-3027; Paul et al. (2007) Viral Immunology 20:664-671), paramyxoviruses (Nakamura and Russell (2004) Expert Opinion on Biological Therapy 4:1685-1692; Hammond et al. (2001) Journal of Virology 75:2087-2096; Galanis (2010) Clinical Pharmacology and Therapeutics 88:620-625; Blechacz and Russell (2008) Current Gene Therapy 8:162-175; Russell and Peng (2009) Current Topics in Microbiology and Immunology 330:213-241), and herpesviruses (Shah and Breakefield (2006) Current Gene Therapy 6:361-370; Campadelli-Fiume et al. (2011) Reviews in Medical Virology 21:213-226, each of which is incorporated herein by reference in its entirety.

[0150] In some embodiments, the gene therapy vectors described herein are pseudotyped into tissues that are particularly suitable for generating regulatory responses, such as tolerance to replacement enzymes. Such tissues include, but are not limited to, mucosal tissues, such as gut-associated lymphoid tissue (GALT), hematopoietic stem cells, and the liver. In some embodiments, the gene therapy vectors or genes encoding the multidomain therapeutic proteins described herein are expressed under the control of promoters specific to those tissues, such as liver-specific promoters.

[0151] In some embodiments, the gene therapy vector described herein comprises a naked polynucleotide. For example, in some embodiments, a polynucleotide encoding a multidomain therapeutic polypeptide may be injected intravenously, for example, intramuscularly, directly into an organ to form a depot. Additional well-known methods for enhancing the delivery of naked polynucleotides include, but are not limited to, electroporation, sonoporation, the use of a gene gun to eject polynucleotide-coated gold particles, magnetic particles, and hydrodynamic delivery.

[0152] In some embodiments, the gene therapy vectors described herein comprise polynucleotide complexes, including, but not limited to, nanoparticles (e.g., polynucleotide self-assembled nanoparticles, polymer-based self-assembled nanoparticles, inorganic nanoparticles, lipid nanoparticles, semiconducting / metallic nanoparticles), gels and hydrogels, polynucleotide complexes with cations and anions, microparticles, and any combination thereof.

[0153] In some embodiments, the polynucleotides disclosed herein can be formulated as self-assembled nanoparticles. As a non-limiting example, the polynucleotides can be used to create nanoparticles that can be used in delivery systems for polynucleotides (see, e.g., International Patent Application Publication No. 2012125987, incorporated herein by reference in its entirety). In some embodiments, the polynucleotide self-assembled nanoparticles can include a polynucleotide core disclosed herein and a polymer shell. The polymer shell can be any of the polymers described herein and known in the art. In additional embodiments, the polymer shell can be used to protect the polynucleotide within the core.

[0154] In some embodiments, these self-assembled nanoparticles can be microsponges formed from long polymeric polynucleotide hairpins that are formed into crystalline "pleated" sheets and then self-assembled into microsponges. These microsponges are densely packed, sponge-like microparticles that can function as efficient carriers and deliver cargo to cells. Microsponges can be 1 μm to 300 nm in diameter. Microsponges can be complexed with other agents known in the art to form larger microsponges. As a non-limiting example, microsponges can be complexed with agents, such as polycationic polyethyleneimine (PEI), to form an outer layer and promote cellular uptake. This complex can form 250 nm diameter particles that can remain stable at high temperatures (150°C) (Grabow and Jaegar, Nature Materials 2012, 11:269-269; incorporated herein by reference in its entirety). Furthermore, these microsponges may provide an extraordinary degree of protection from degradation by ribonucleases. In another embodiment, polymer-based self-assembled nanoparticles, such as, but not limited to, microsponges, may be fully programmable nanoparticles. The geometry, size, and stoichiometry of the nanoparticles can be precisely controlled to create optimal nanoparticles for delivering cargo, such as, but not limited to, polynucleotides.

[0155] In some embodiments, polynucleotides can be formulated into inorganic nanoparticles (see U.S. Pat. No. 8,257,745, which is incorporated herein by reference in its entirety). Inorganic nanoparticles can include, but are not limited to, water-swellable clay-like materials. As a non-limiting example, inorganic nanoparticles can include synthetic smectite clays made from simple silicates (see, e.g., U.S. Pat. Nos. 5,585,108 and 8,257,745, each of which is incorporated herein by reference in its entirety).

[0156] In some embodiments, polynucleotides may be formulated in water-dispersible nanoparticles comprising semiconductive or metallic materials (U.S. Patent Application Publication No. 20120228565, which is incorporated herein by reference in its entirety), or may be formed in magnetic nanoparticles (U.S. Patent Application Publication Nos. 20120265001 and 20120283503, which are incorporated herein by reference in their entirety). Water-dispersible nanoparticles may be hydrophobic or hydrophilic.

[0157] In some embodiments, the polynucleotides disclosed herein can be encapsulated in any hydrogel known in the art that can form a gel when injected into a subject. Hydrogels are networks of hydrophilic polymer chains and can be found as colloidal gels in which water is the dispersion medium. Hydrogels can include highly absorbent (capable of containing more than 99% water) natural or synthetic polymers. Due to their significant water content, hydrogels also possess flexibility very similar to that of natural tissue. The hydrogels described herein can be used to encapsulate biocompatible, biodegradable, and / or porous lipid nanoparticles.

[0158] As a non-limiting example, the hydrogel may be an aptamer-functionalized hydrogel. The aptamer-functionalized hydrogel may be programmed to release one or more polynucleotides using polynucleotide hybridization. (Battig et al., J. Am. Chem. Society. 2012 134:12410-12413; incorporated herein by reference in its entirety.) In some embodiments, the polynucleotides may be encapsulated within lipid nanoparticles, which may then be encapsulated within the hydrogel.

[0159] In some embodiments, polynucleotides may be encapsulated in fibrin gels, fibrin hydrogels, or fibrin glue. In other embodiments, polynucleotides may be formulated into lipid nanoparticles or rapid-clearing lipid nanoparticles before encapsulation in fibrin gels, fibrin hydrogels, or fibrin glue. In yet other embodiments, polynucleotides may be formulated as lipoplexes before encapsulation in fibrin gels, hydrogels, or fibrin glue. Fibrin gels, hydrogels, and glues comprise two components: a fibrinogen solution and a calcium-enriched thrombin solution (see, e.g., Spicer and Mikos, Journal of Controlled Release 2010, 148:49-55; Kidd et al., Journal of Controlled Release 2012, 157:80-85, each of which is incorporated herein by reference in its entirety). The concentrations of the components of the fibrin gel, hydrogel, and / or adhesive can be varied to change the properties of the gel, hydrogel, and / or adhesive, the mesh size of the network, and / or the degradation characteristics, including, but not limited to, changing the release characteristics of the fibrin gel, hydrogel, and / or adhesive. (See, e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148:49-55; Kidd et al., Journal of Controlled Release 2012. 157:80-85; Catelas et al., Tissue Engineering 2008. 14:119-128, each of which is incorporated by reference in its entirety.) This characteristic can be advantageous when used to deliver the polynucleotides disclosed herein.(See, e.g., Kidd et al. Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128, each of which is incorporated by reference in its entirety.)

[0160] In some embodiments, the polynucleotides disclosed herein can include cations or anions. In one embodiment, the formulation includes a metal cation, such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+, and combinations thereof. As a non-limiting example, the formulation can include a polymer and a polynucleotide complexed with a metal cation (see, e.g., U.S. Patent Nos. 6,265,389 and 6,555,525, each of which is incorporated herein by reference in its entirety).

[0161] In some embodiments, polynucleotides may be formulated into nanoparticles and / or microparticles. These nanoparticles and / or microparticles may be formed into any size, shape, and chemistry. As an example, nanoparticles and / or microparticles may be made using PRINT® technology by LIQUIDA TECHNOLOGIES.RTM. (Morrisville, NC) (see International Patent Application Publication No. 2007024323, which is incorporated herein by reference in its entirety).

[0162] In some embodiments, polynucleotides may be formulated in nanojackets and nanoliposomes by Keystone Nano (State College, PA). Nanojackets are made from compounds naturally found in the body, including calcium, phosphate, or compounds that also contain small amounts of silicate. Nanojackets can range in size from 5 to 50 nm and can be used to deliver hydrophilic and hydrophobic compounds, such as, but not limited to, polynucleotides, primary constructs, and / or polynucleotides. Nanoliposomes are made from lipids, including, but not limited to, lipids that occur naturally in the body. Nanoliposomes can range in size from 60 to 80 nm and can be used to deliver hydrophilic and hydrophobic compounds, including, but not limited to, polynucleotides, primary constructs, and / or polynucleotides. In one aspect, the polynucleotides disclosed herein are formulated in nanoliposomes, such as, but not limited to, ceramide nanoliposomes.

[0163] In one embodiment, the multidomain therapeutic protein is an anti-CD63 scFv-GAA fusion protein or an anti-ITGA7 scFv-GAA fusion protein. Administration of the anti-CD63 scFv-GAA fusion protein or the anti-ITGA7 scFv-GAA fusion protein via AAV delivery results in long-term stability of GAA in the serum of patients after administration of a gene therapy vector harboring the multidomain therapeutic protein. In one embodiment, the level of GAA in the serum of a recipient patient is ≥ 1.5-fold to 100-fold, ≥ 1.5-fold to 10-fold, ≥ 2.5-fold, 2.5-fold to 3-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold, 3.5-fold, 3.6-fold, 3.7-fold, 3.8-fold, 3.9-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than the serum level of a patient receiving GAA without an attached delivery domain, 1 month, 3 months, 4 months, 5 months, or 6 months after administration of a gene therapy vector comprising a multidomain therapeutic protein.

[0164] In one embodiment, administration of an anti-CD63 scFv-GAA fusion protein or an anti-ITGA7 scFv-GAA fusion protein via AAV delivery results in a long-term, stable reduction in glycogen storage levels in Pompe disease patients. In one embodiment, glycogen levels in the patient's heart, skeletal muscle, and liver tissues are reduced to wild-type (non-disease) levels. In one embodiment, glycogen levels in the patient's heart, skeletal muscle, and liver tissues are maintained at wild-type levels for 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration of a gene therapy vector harboring a multidomain therapeutic protein.

[0165] In one embodiment, administration of an anti-CD63 scFv-GAA fusion protein or an anti-ITGA7 scFv-GAA fusion protein via AAV delivery results in long-term restoration of muscle strength in Pompe disease patients. In one embodiment, the patient's strength, as measured by grip strength, is restored to normal (i.e., disease-free normal levels) 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months after administration of a gene therapy vector harboring a multidomain therapeutic protein.

[0166] In another aspect, the invention provides a composition comprising an enzymatic activity and an antigen binding protein, where the enzyme is associated with an enzyme deficiency disorder (LSD) and an internalizing effector binding protein. Enzymes (including proteins that are not themselves catalytic) associated with lysosomal storage diseases include, for example, any hydrolase, α-galactosidase, β-galactosidase, α-glucosidase, β-glucosidase, saposin-C activator, ceramidase, sphingomyelinase, β-hexosaminidase, GM2 activator, GM3 synthase, arylsulfatase, sphingolipid activator, α-iduronidase, iduronidase-2-sulfatase, heparin N-sulfatase, N-acetyl-α-glucosaminidase, α-glucosamide N-acetyltransferase, N-acetylglucosamine-6-sulfatase, N-acetylgalactosamine-6-sulfatase, N-acetylgalactosamine-4-sulfatase, β-glucuronidase, and hyaluronidase.

[0167] Internalizing effector binding proteins include, for example, receptor fusion molecules, trap molecules, receptor-Fc fusion molecules, antibodies, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, single-chain Fv (scFv) molecules, dAb fragments, isolated complementarity determining regions (CDRs), CDR3 peptides, constrained FR3-CDR3-FR4 peptides, domain-specific antibodies, single domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies, monovalent nanobodies, bivalent nanobodies, small modular immunopharmaceuticals (SMIPs), camelid antibodies (VHH heavy chain homodimeric antibodies), shark variable IgNAR domains, and other antigen binding proteins.

[0168] Internalization effectors include, for example, CD63, MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), PRLR (prolactin receptor), MAL (myelin and lymphocyte protein, also known as VIP17), IGF2R, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor, SCARA1-5, SCARB1-3, and CD36. In certain embodiments, the internalization effector is a kidney-specific internalizer, such as CDH16 (Cadherin-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (solute carrier family 22 member 13), SLC5A2 (sodium / glucose cotransporter 2), and UMOD (uromodulin). In certain other embodiments, the internalization effector is a muscle-specific internalizer, such as BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3). In some specific embodiments, the internalization effector is ITGA7, CD9, CD63, APLP2, ASGR1, ASGR2, or PRLR.

[0169] In some embodiments, the enzyme is covalently linked to the antigen-binding protein (i.e., electrons are shared between atoms). In one particular embodiment, the internalizing effector-binding protein consists of or contains a half-antibody, and the enzyme is fused to an Fc fusion domain (e.g., at the C-terminus). The Fc domain that is covalently linked to the enzyme then associates with the Fc domain of the antigen-binding protein, such that the association contains one or more disulfide bridges. This particular embodiment is depicted schematically in panel B of Figure 1A.

[0170] In certain other embodiments, the internalizing effector-binding protein (delivery domain) consists of or contains an antibody or antibody fragment, and the enzyme is covalently linked to the antibody or antibody fragment. In certain other embodiments, the delivery domain is an antibody, and the enzyme is covalently linked (either directly via a peptide bond or indirectly via a linker) to the C-terminus of the antibody heavy or light chain (panel C or panel E of Figure 1A, respectively). In certain other embodiments, the delivery domain is an antibody, and the enzyme is covalently linked (either directly via a peptide bond or indirectly via a linker) to the N-terminus of the antibody heavy or light chain (panel D or panel F of Figure 1A, respectively).

[0171] In some embodiments, the enzyme and delivery domain are not covalently linked but are combined in a mixture. The delivery domain and enzyme can associate to form a complex through non-covalent forces. For example, in one particular embodiment, the delivery domain is a bispecific antibody, one arm of which binds an internalization effector and the other arm of which binds an enzyme. This embodiment is depicted schematically in panel A of Figure 1A.

[0172] In some embodiments, the enzyme is GAA or contains GAA activity (e.g., an isozyme with GAA activity), and the internalization effector is ITGA7, CDH15, CD9, CD63, APLP2, ASGR1, ASGR2, or PRLR. In certain embodiments, the enzyme is GAA or contains GAA activity, the internalization domain is CD63, and the delivery domain is a bispecific antibody with specificity for CD63 and GAA. In certain embodiments, the enzyme is GAA or contains GAA activity, the internalization domain is ITGA7, and the delivery domain is a bispecific antibody with specificity for ITGA7 and GAA.

[0173] In some embodiments, the enzyme is GLA or contains GLA activity (e.g., an isozyme with GAA activity), and the internalization effector is ITGA7, CD9, CD63, APLP2, ASGR1, ASGR2, or PRLR. In certain embodiments, the enzyme is GLA or contains GLA activity, the internalization domain is CD63, and the delivery domain is a bispecific antibody with specificity for CD63 and GLA. In certain embodiments, the enzyme is GLA or contains GLA activity, the internalization domain is ITGA7, and the delivery domain is a bispecific antibody with specificity for ITGA7 and GLA. Pharmaceutical Compositions and Their Administration

[0174] Pharmaceutical formulations may additionally contain pharmaceutically acceptable excipients, which, as used herein, include any or all of solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, etc., as appropriate for the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for their preparation. Except insofar as any conventional excipient medium is incompatible with the substance or its derivatives, for example, by producing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of the present invention.

[0175] In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0176] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surfactants and / or emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants, and / or oils. Such excipients may optionally be included in the pharmaceutical composition.

[0177] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, and the like, and / or combinations thereof.

[0178] Exemplary granulating and / or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethylcellulose, cross-linked sodium carboxymethylcellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, and the like, and / or combinations thereof.

[0179] Representative surfactants and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (e.g., stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g., carboxypolymethylene, polyacrylic acid, acrylic acid polymers, and carboxyvinyl polymers), carrageenan, cellulosic derivatives (e.g., sodium carboxymethylcellulose, powdered cellulose, hydroxymethylcellulose, hydroxypropylcellulose, hydrogels, etc.), and the like. hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [SPAN® 40], sorbitan monostearate [SPAN® 60], sorbitan tristearate, [SPAN® 65], glyceryl monooleate, sorbitan monooleate [SPAN® 80]), polyoxyethylene polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®),Polyoxyethylene ethers (e.g., polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC® F68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, and / or combinations thereof.

[0180] Exemplary binders include, but are not limited to, starches (e.g., corn starch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g., acacia, sodium alginate, Chorizoides extract, panwar gum, ghatti gum, isapol skin mucilage, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinylpyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol, and the like, and combinations thereof.

[0181] Exemplary preservatives include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylhydroxyanisole, butylhydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and / or sodium sulfate. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, edetate disodium, edetate dipotassium, edetate, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or edetate trisodium. Exemplary antibacterial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoates, and / or phenylethyl alcohol. Exemplary acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid.Other preservatives include, but are not limited to, tocopherol, tocopheryl acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMABEN® II, NEOLONE™, KATHON™, and / or EUXYL®.

[0182] Exemplary buffering agents include, but are not limited to, citrate buffer solution, acetate buffer solution, phosphate buffer solution, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, sodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, and the like, and / or combinations thereof.

[0183] Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, and the like, and combinations thereof.

[0184] Representative oils include, but are not limited to, almond oil, apricot kernel oil, avocado oil, babassu oil, bergamot oil, black currant oil, borage oil, cade oil, chamomile oil, canola oil, caraway oil, carnauba oil, castor oil, cinnamon oil, cocoa butter oil, palm oil, cod liver oil, coffee oil, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose oil, fish oil, linseed oil, geraniol oil, gourd oil, grape seed oil, hazel oil, hyssop oil, isopropyl myristate, jojoba oil, kukui nut oil, lavandin oil, lavender oil, and lemon oil. , lily of the valley oil, macadamia nut oil, mallow oil, mango seed oil, meadowfoam seed oil, mink oil, nutmeg oil, olive oil, orange oil, orange roughy oil, palm oil, palm kernel oil, peach kernel oil, peanut oil, poppy seed oil, pumpkin seed oil, rapeseed oil, rice bran oil, rosemary oil, safflower oil, sandalwood oil, sasquana oil, savory oil, sea buckthorn oil, sesame oil, shea butter, silicone, soybean oil, sunflower oil, tea tree oil, thistle oil, camellia oil, vetiver oil, walnut oil, and wheat germ oil. Exemplary oils include butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0185] Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents, and the like, can be present in the composition according to the judgment of the formulator.

[0186] delivery

[0187] The present disclosure encompasses delivery of gene therapy vectors (e.g., polynucleotides) by any suitable route taking into account advances in the science of potential drug delivery. Delivery can be naked or formulated.

[0188] Naked Delivery

[0189] The polynucleotides of the present invention can be delivered to cells naked. As used herein, "naked" refers to delivering a polynucleotide without a transfection-facilitating agent. For example, the polynucleotide delivered to a cell may not contain any modifications. Naked polynucleotides can be delivered to cells using routes of administration known in the art and described herein.

[0190] Delivery by formulation

[0191] Polynucleotides can be formulated using the methods described herein.The formulation can contain polynucleotides and can further include, but is not limited to, cell-penetrating agents, pharmaceutically acceptable carriers, delivery agents, biodegradable or biocompatible polymers, solvents, and sustained-release delivery depots.Formulated polynucleotide mRNA can be delivered to cells using administration routes known in the art and described herein.

[0192] Administration

[0193] The polynucleotides of the present invention may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited to, enteral, gastrointestinal, epidural, oral, transdermal, epidural (around the dura), intracerebral (into the cerebrum), intraventricular (into the ventricles of the brain), epicutaneous (applied on top of the skin), intradermal (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous injection (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal (infusion and injection into the peritoneum), intravesical instillation, intravitreal (through the eye), intracavity injection, (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through intact skin for systemic distribution), transmucosal (diffusion through mucous membranes), insufflation (aspiration), sublingual, sublabial, enema, ophthalmic (on the conjunctiva), or ear drops. In certain embodiments, the compositions may be administered in a manner that allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier. Non-limiting routes of administration for the polynucleotides, primary constructs, or mRNA of the invention are described below.

[0194] Parenteral and injectable administration

[0195] Liquid dosage forms for parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and / or elixirs. In addition to the active ingredient, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (e.g., cottonseed oil, underground seed oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, and fatty acid esters of sorbitan, and mixtures thereof. In addition to the inert diluent, oral compositions may contain adjuvants, such as wetting agents, emulsifiers, and suspending agents, sweeteners, flavoring agents, and / or aromatic agents. In certain embodiments for parenteral administration, the composition is mixed with a solubilizing agent such as CREMOPHOR®, alcohol, oil, modified oil, glycol, polysorbate, cyclodextrin, polymer, and / or combinations thereof.

[0196] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, may be formulated according to known techniques using suitable dispersants, wetting agents, and / or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and / or emulsions in non-toxic parenterally acceptable diluents and / or solvents, such as solutions in 1,3-butanediol. Acceptable vehicles and solvents that can be used include water, Ringer's solution, USP, and isotonic sodium chloride solution. Sterile fixed oils are conventionally used as solvents or suspending media. For this purpose, any brand of fixed oil can be used, including synthetic monoglycerides or diglycerides. Fatty acids, such as oleic acid, can be used in the preparation of injectables.

[0197] Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter, and / or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injectable medium before use.

[0198] To prolong the effect of an active ingredient, it is often desirable to slow its absorption from subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends on its rate of dissolution, which in turn may depend on crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form can be achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactic acid-polyglycolic acid. The rate of drug release can be controlled depending on the ratio of drug to polymer and the nature of the particular polymer employed. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by incorporating the drug into liposomes or microemulsions that are compatible with body tissues.

[0199] Depot administration

[0200] As described herein, in some embodiments, the compositions are formulated into a depot for sustained release. Generally, a specific organ or tissue ("target tissue") is targeted for administration.

[0201] In some embodiments of the present invention, the polynucleotide is spatially retained in or near the target tissue. Provided is a method of providing a composition to a target tissue of a mammalian subject by contacting the target tissue (containing one or more target cells) with the composition under conditions such that the composition, particularly the nucleic acid components of the composition, are substantially retained in the target cells, meaning that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 99.99% or more of the composition is retained in the target cells. Advantageously, retention is determined by measuring the amount of nucleic acid present in the composition that enters one or more target cells. For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, or 99.99% or more of the nucleic acid administered to a subject remains intracellular for a period of time following administration. For example, intramuscular injection into a mammalian subject is performed using an aqueous composition containing a polynucleotide and a transfection reagent, and retention of the composition is determined by measuring the amount of ribonucleic acid present in muscle cells.

[0202] Aspects of the present invention are directed to methods of providing a composition to a target tissue of a mammalian subject by contacting the target tissue (containing one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue. The composition contains an effective amount of a polynucleotide such that a polypeptide of interest is produced in at least one target cell. The composition generally contains a cell-penetrating agent and a pharmaceutically acceptable carrier, although "naked" nucleic acid (i.e., nucleic acid free of cell-penetrating agents or other agents) is also contemplated.

[0203] In some situations, the amount of protein produced by cells in a tissue is desirably increased. Preferably, this increase in protein production is spatially restricted to cells in the target tissue. Thus, provided is a method for increasing the production of a protein of interest in the tissue of a mammalian subject. A polynucleotide-containing composition is provided, characterized in that a unit amount of the composition is determined to produce the polypeptide of interest in a substantial percentage of cells contained in a predetermined volume of the target tissue.

[0204] In some embodiments, the composition comprises a plurality of different polynucleotides, wherein one or more of the polynucleotides encode a polypeptide of interest. Optionally, the composition also contains a cell-penetrating agent to aid in intracellular delivery of the composition. A determination is made as to the dose of the composition required to produce the polypeptide of interest in a substantial percentage of cells contained within a given volume of target tissue (generally without inducing significant production of the polypeptide of interest in tissue adjacent to the given volume or distal to the target tissue). Following this determination, the determined dose is introduced directly into the tissue of the mammalian subject.

[0205] In one embodiment, the invention provides a polynucleotide that is delivered in two or more injections or by split-dose injection.

[0206] In one embodiment, the present invention may be held near the target tissue using a small, disposable drug reservoir, patch pump, or osmotic pump. Non-limiting examples of patch pumps include those manufactured and / or sold by BD® (Franklin Lakes, NJ), Insulet, Inc. (Bedford, CA), SteadyMed Therapeutics (San Francisco, CA), Medtronic (Minneapolis, MN) (e.g., MiniMed), Unilife (York, PA), and SpringLeaf Therapeutics (Boston, MA). Non-limiting examples of osmotic pumps include those manufactured by DURECT® (Cupertino, CA) (e.g., DUROS® and ALZET®).

[0207] Dose Administration

[0208] The present invention provides methods comprising administering a gene therapy vector comprising a polynucleotide encoding a multidomain therapeutic polypeptide, and optionally subsequently administering the multidomain therapeutic polypeptide to a subject in need thereof. In some embodiments, the methods comprise administering a gene therapy vector comprising a polynucleotide encoding a multidomain therapeutic polypeptide to a patient in need thereof in a therapeutically effective amount, where the therapeutically effective amount is sufficient to obviate the need for subsequent administration of the multidomain therapeutic polypeptide. Thus, in some embodiments, a method of treating a patient lacking an enzyme in need thereof, e.g., a method of reducing glycogen levels and / or reducing CRIM to GAA in a patient with Pompe disease, comprises administering to the patient a therapeutically effective amount of a gene therapy vector comprising a polynucleotide encoding a replacement enzyme, e.g., an anti-CD63 scFv::GAA fusion protein, e.g., a multidomain therapeutic protein comprising the sequence set forth as SEQ ID NO: 11, where the therapeutically effective amount obviates the need for subsequent administration of a replacement enzyme, e.g., GAA or a derivative thereof, to the patient. In some embodiments, a method of treating a patient lacking an enzyme in need thereof, e.g., a method of reducing glycogen levels and / or reducing CRIM to GAA in a patient with Pompe disease, comprises administering to the patient a therapeutically effective amount of a gene therapy vector comprising a polynucleotide encoding the replacement enzyme, e.g., an anti-CD63 scFv::GAA fusion protein, e.g., a multidomain therapeutic protein comprising the sequence set forth as SEQ ID NO: 11, and further comprises administering a therapeutically effective amount of the replacement enzyme to the patient. The nucleic acid, protein, or complex, or pharmaceutical, imaging, diagnostic, or prophylactic composition thereof, can be administered to a subject using any amount and route of administration effective to prevent, treat, diagnose, or image a disease, disorder, and / or condition (e.g., a disease, disorder, and / or condition associated with deficits in working memory).

[0209] The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.

[0210] The dose of AAV viral vector required to achieve a desired effect or "therapeutic effect" (e.g., a particular serum concentration of a replacement enzyme), e.g., units of vector genome dose per kilogram of body weight (vg / kg), varies based on several factors, including, but not limited to, the route of AAV administration, the expression level required to achieve a therapeutic effect, the particular disease or disorder being treated, and the stability of expression of the multidomain therapeutic protein. One of skill in the art can readily determine a dose range of AAV virions for treating a subject with a particular disease or disorder based on the aforementioned factors, as well as other factors well known in the art. See, e.g., CDER "Guidance for Industry Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers," July 2005, incorporated herein by reference in its entirety. An effective amount of AAV is generally about 10 doses per subject. 9 ~10 16 The volume of solution typically ranges from about 10 μl to about 100 ml, containing 100 genome copies per subject. Other volumes of solution may be used. The volume used typically depends, among other things, on the size of the subject, the dose of AAV, and the route of administration. In some embodiments, about 100 ml of solution per subject is used. 10 ~10 12 A dose of AAV viral genome of 10 or more per subject is appropriate. In some embodiments, the AAV is administered at a dose of 10 or more per subject. 10 , 10 11 , 10 12 , 10 13 , 10 14 , or 10 15 In some embodiments, the AAV is administered at a dose of 10 genome copies per kg. 10 , 10 11 , 10 12, 10 13 , or 10 14 In some embodiments, the dose is at least 2 x 10 viral genomes per kilogram. 12 of viral genome is administered. In some embodiments, the dose provides a threshold multidomain therapeutic protein serum level. In some embodiments, the threshold therapeutic protein level is at least 1 μg / mL. In some embodiments, the dose provides a multidomain therapeutic protein serum level greater than 2 μg / mL. In some embodiments, the dose provides a multidomain therapeutic protein serum level greater than 3 μg / mL. In some embodiments, the dose provides a multidomain therapeutic protein serum level greater than 4 μg / mL. In some embodiments, the dose provides a multidomain therapeutic protein serum level greater than 5 μg / mL. In some embodiments, the dose provides a multidomain therapeutic protein serum level greater than 6 μg / mL. In some embodiments, the dose provides a multidomain therapeutic protein serum level greater than 7 μg / mL. In some embodiments, the dose provides a multidomain therapeutic protein serum level greater than 8 μg / mL. In some embodiments, the dose provides a multidomain therapeutic protein serum level greater than 9 μg / mL. In some embodiments, the dose provides a multidomain therapeutic protein serum level greater than 10 μg / mL. In some embodiments, the dosage provides a multidomain therapeutic protein serum level greater than 11 μg / mL. In some embodiments, the dosage provides a multidomain therapeutic protein serum level greater than 12 μg / mL. In some embodiments, the dosage provides a multidomain therapeutic protein serum level greater than 13 μg / mL. In some embodiments, the dosage provides a multidomain therapeutic protein serum level greater than 14 μg / mL. In some embodiments, the dosage provides a multidomain therapeutic protein serum level greater than 15 μg / mL.

[0211] The compositions of the present invention are typically formulated in unit dosage form for ease of administration and uniform dosage.But it is understood that the total daily use amount of the compositions of the present invention can be determined by attending physician within the scope of sound medical judgment.The specific therapeutically effective, prophylactically effective or suitable imaging dose level for any specific patient will depend on various factors, such as the disorder to be treated and the severity of the disorder; the activity of the specific compound used; the specific compound used; the age, weight, general health, sex and diet of the patient; the administration time, administration route and excretion rate of the specific compound used; the duration of treatment; the drug used in combination with or simultaneously with the specific compound used; and similar factors well known in the medical field.

[0212] Non-limiting and exemplary embodiments are described below. Embodiment 1 A method for delivering a therapeutic protein into the central nervous system (CNS) of a subject, comprising administering to the subject a nucleotide composition encoding a multidomain therapeutic protein via a liver-targeted delivery method sufficient to provide a therapeutically effective amount of the multidomain therapeutic protein to the CNS, wherein the multidomain therapeutic protein comprises a delivery domain and an enzymatic domain. Embodiment 2 2. The method of embodiment 1, wherein the delivery domain is an antibody or antigen-binding fragment thereof that specifically binds to the internalization effector. Embodiment 3 3. The method of embodiment 1 or embodiment 2, wherein the therapeutic protein is a lysosomal enzyme. Embodiment 4 4. The method of embodiment 3, wherein the lysosomal enzyme is GAA. Embodiment 5 The nucleotide composition is administered via a viral vector, and optionally the nucleotide composition is administered in a dose of at least 2 x 10 per kilogram. 12 5. The method of any one of embodiments 1 to 4, wherein the vaccine is administered at a dose of 100 mg / kg of viral genome (vg / kg). Embodiment 6 6. The method of embodiment 5, wherein the viral vector is an AAV vector. Embodiment 7 7. The method of any one of embodiments 1-6, wherein the internalizing effector is expressed on the surface of a cell selected from the group consisting of a cell in the CNS, an epithelial cell, and a cell that crosses the blood-brain barrier. Embodiment 8 The delivery domain is (i) is selected from the group consisting of CD63, integrin alpha 7 (ITGA7), MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), myelin and lymphocyte protein (MAL), IGF2R, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, and CD36; (ii) any of the following proteins expressed in several tissue types: CD63, MHC-I, vacuolar H+ ATPase, IGF2R, integrin alpha 7 (ITGA7), LRP5, LRP6, LRP8, Kremen-2, LDL receptor, LDL-related protein 1 receptor, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), PRLR, MAL (myelin and lymphocyte protein (MAL)), diphtheria toxin receptor, HBEGF (heparin-binding EGF-like growth factor), glutathione receptor, glutamate receptor, leptin receptor, and folate receptor; Optionally, the subject exhibits one or more symptoms of a disease selected from the group consisting of Fabry disease, Gaucher disease, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIID, MPS IVB, MPS VI, MPS VII, MPS IX, Pompe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, Niemann-Pick disease types A, B, and C2, alpha mannosidosis, neuraminidase deficiency, sialidosis, aspartylglycosaminuria, mixed saposin deficiency, variant Gaucher disease, Farber lipogranulomatosis, fucosidosis, and beta mannosidosis; (iii) collagen X, integrin alpha 10 (ITGA10), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor isoform C (FGFR3C), hyaluronan and proteoglycan link protein 1 (CRTL1), aggrecan, collagen II, and Kremen-1, which are preferentially expressed by bone and / or cartilage; Optionally, the subject exhibits one or more symptoms of a disease selected from the group consisting of MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIID, MPS IVA, MPS IVB, MPS VI, MPS VII, MPS IX, beta-mannosidosis, Gaucher disease, atypical Gaucher disease, mixed saposin deficiency, aspartylglycosaminuria, Farber lipogranulomatosis, sialidosis, neuraminidase deficiency, and alpha-mannosidosis; (iv) any of the following proteins, which are preferentially expressed by monocytes, macrophages, or microglia: scavenger receptor A1-5 (SCARA1-5), SCARB1-3, CD36, MSR1 (macrophage scavenger receptor 1), MRC1 (macrophage mannose receptor 1), VSIG4 (V-set and immunoglobulin domain-containing protein 4), CD68 (macrosialin), and CSF1R (macrophage colony-stimulating factor 1 receptor); Optionally, the subject exhibits one or more symptoms of a disease selected from the group consisting of lysosomal acid lipase deficiency, Gaucher disease, atypical Gaucher disease, mixed saposin deficiency, and Farber lipogranulomatosis; (v) a protein preferentially expressed by kidney cells, optionally selected from the group consisting of CDH16 (Cadherin-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (solute carrier family 22 member 13), SLC5A2 (sodium / glucose cotransporter 2), and UMOD (uromodulin); Optionally, the subject exhibits symptoms of or has been diagnosed with one or more of a disease selected from the group consisting of Fabry disease, Alport syndrome, polycystic kidney disease, and thrombotic thrombocytopenic purpura; (vi) optionally, ASGR1 or ASGR2, which is preferentially expressed by hepatocytes; Optionally, the subject exhibits one or more symptoms of or has been diagnosed with a disease selected from the group consisting of lysosomal acid lipase deficiency, Gaucher disease, MPS VI, MPS VII, MPS II, Niemann-Pick disease types A, B, and C2, sialidosis, neuraminidase deficiency, atypical Gaucher disease, mixed saposin deficiency, and Farber lipogranulomatosis; (vii) any of the following proteins preferentially expressed by muscle cells: BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3); Optionally, the subject exhibits one or more symptoms of or has been diagnosed with Pompe disease; (viii) selected from the group consisting of ITGA7, CD9, CD63, ALPL2, MSR1, ASGR1, ASGR2, or PRLR; or (ix) The method of any one of embodiments 1 to 7, wherein the internalization effector is CD63. Embodiment 9 9. The method of any one of embodiments 1 to 8, wherein the delivery domain is a single chain variable fragment (scFv). Embodiment 10 10. The method of any one of embodiments 1 to 9, wherein the cell surface receptor (CSR) binding protein (CSR-BP) comprises the amino acid sequence of SEQ ID NO:2. Embodiment 11 11. The method of any one of embodiments 1-10, wherein the therapeutic protein comprises a hydrolase. Embodiment 12 12. The method of any one of embodiments 1-11, wherein the therapeutic protein comprises a glycosylase. Embodiment 13 13. The method of any one of embodiments 1-12, wherein the therapeutic protein comprises a glycosidase. Embodiment 14 14. The method of any one of embodiments 1-13, wherein the therapeutic protein comprises alpha-glucosidase. Embodiment 15 15. The method of any one of embodiments 1 to 14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13, or a fragment thereof. Embodiment 16 16. The method of any one of embodiments 1-15, wherein the therapeutic protein comprises an anti-ABeta or anti-tau antibody. Embodiment 17 17. The method of any one of embodiments 1 to 16, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11. Embodiment 18 18. The method of any one of embodiments 1-17, wherein the enzyme domain comprises alpha-glucosidase and glycogen levels in any CNS tissue of the subject are reduced for at least nine months after treatment. Embodiment 19 19. The method of any one of embodiments 1-18, wherein the subject has Pompe disease. Embodiment 20 20. The method of any one of embodiments 1-19, wherein the administered nucleotide composition provides a multidomain therapeutic protein serum level of at least 1 μg / mL. Embodiment 21 A multidomain therapeutic protein comprising one or more delivery domains and an enzymatic domain, wherein the one or more delivery domains bind to the human transferrin receptor (hTfR). Embodiment 22 22. The multidomain therapeutic protein of embodiment 21, further comprising a second delivery domain that binds to an internalization effector. Embodiment 23 the second delivery domain comprises: (i) an internalization effector selected from the group consisting of CD63, integrin alpha 7 (ITGA7), MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), myelin and lymphocyte protein (MAL), IGF2R, vacuolar-type H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, and CD36; (ii) an internalization effector expressed in several tissue types, arbitrarily selected from the group consisting of CD63, MHC-I, vacuolar-type H+ ATPase, IGF2R, integrin alpha 7 (ITGA7), LRP5, LRP6, LRP8, Kremen-2, LDL receptor, LDL-related protein 1 receptor, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), PRLR, MAL (myelin and lymphocyte protein (MAL)), diphtheria toxin receptor, HBEGF (heparin-binding EGF-like growth factor), glutathione receptor, glutamate receptor, leptin receptor, and folate receptor; (iii) an internalization effector preferentially expressed by bone and / or cartilage, optionally selected from the group consisting of collagen X, integrin alpha 10 (ITGA10), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor isoform C (FGFR3C), hyaluronan and proteoglycan link protein 1 (CRTL1), aggrecan, collagen II, and Kremen-1; (iv) an internalization effector preferentially expressed by monocytes, macrophages, or microglia, optionally selected from the group consisting of scavenger receptor A1-5 (SCARA1-5), SCARB1-3, CD36, MSR1 (macrophage scavenger receptor 1), MRC1 (macrophage mannose receptor 1), VSIG4 (V-set and immunoglobulin domain-containing protein 4), CD68 (macrosialin), and CSF1R (macrophage colony-stimulating factor 1 receptor); (v) an internalization effector preferentially expressed by kidney cells, optionally selected from the group consisting of CDH16 (Cadherin-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (solute carrier family 22 member 13), SLC5A2 (sodium / glucose cotransporter 2), and UMOD (uromodulin), which in certain other embodiments is a muscle-specific internalizer, such as BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor ( internalization effectors, such as nicotinic (nicotinic) alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3); (vi) optionally an internalization effector preferentially expressed by hepatocytes, such as ASGR1 or ASGR2; (vii) an internalization effector preferentially expressed by muscle cells, optionally selected from the group consisting of BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3); or (viii) The multidomain therapeutic protein of embodiment 22, which binds to an internalization effector protein selected from the group consisting of ITGA7, CD9, CD63, ALPL2, MSR1, ASGR1, ASGR2, or PRLR. Embodiment 24 24. The multidomain therapeutic protein of any one of embodiments 21 to 23, wherein the second delivery domain binds to the internalizing effector CD63. Embodiment 25 25. A multidomain therapeutic protein according to any one of embodiments 21 to 24, wherein at least one of the one or more delivery domains comprises an antigen-binding protein. Embodiment 26 26. The multidomain therapeutic protein of embodiment 25, wherein each of the one or more delivery domains comprises an antigen binding protein. Embodiment 27 27. The multidomain therapeutic protein of any one of embodiments 21 to 26, wherein at least one of the one or more delivery domains comprises a single chain variable fragment (scFv). Embodiment 28 28. The multidomain therapeutic protein of any one of embodiments 21-27, wherein at least one of the one or more delivery domains comprises a half antibody. Embodiment 29 29. The multidomain therapeutic protein of embodiment 28, wherein the delivery domain that binds to hTfR is an scFv, the half antibody binds to CD63, and the enzymatic domain is GAA, wherein GAA is conjugated to the carboxy terminus of the half antibody that binds CD63. Embodiment 30 28. The multidomain therapeutic protein of embodiment 27, wherein each of the one or more delivery domains comprises an scFv. Embodiment 31 31. A multidomain therapeutic protein according to any one of embodiments 27 to 30, wherein at least one scFv is fused to an Fc. Embodiment 32 32. The multidomain therapeutic protein of embodiment 31, wherein the Fc comprises the wild-type human IgG4 isotype, or a derivative thereof. Embodiment 33 33. The multidomain therapeutic protein of any one of embodiments 31-32, wherein GAA is conjugated to the carboxy terminus of Fc. Embodiment 34 31. The multidomain therapeutic protein of embodiment 30, comprising an anti-hTfR scFv, an anti-hCD63 scFv. Embodiment 35 35. The multidomain therapeutic protein of embodiment 34, wherein the anti-hTfR scFv and the anti-hCD63 scFv are both linked at their carboxy termini to a single GAA enzyme. Embodiment 36 28. The multidomain therapeutic protein of any one of embodiments 21 to 27, wherein the delivery domain is an anti-hTfR scFv and the enzymatic domain is linked to the carboxy terminus of the VL domain of the scFv. Embodiment 37 37. The multidomain therapeutic protein of embodiment 36, further comprising a second delivery domain linked to the N-terminus of the VH domain of the anti-hTfR scFv. Embodiment 38 38. The multidomain therapeutic protein of embodiment 37, wherein the second delivery domain is an anti-hCD63 scFV. Embodiment 39 39. The multidomain therapeutic protein according to any one of embodiments 21 to 38, wherein the enzymatic domain comprises the amino acid sequence set forth as SEQ ID NO:1. Embodiment 40 A polynucleotide encoding a multidomain therapeutic protein according to any one of embodiments 21 to 39 or embodiments 50 to 64. Embodiment 41 41. The polynucleotide of embodiment 40, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence. Embodiment 42 42. The polynucleotide of embodiment 40 or embodiment 41, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence. Embodiment 43 43. The polynucleotide of any one of embodiments 40-42, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence, and the AAV nucleic acid sequence comprises an internal terminal repeat sequence and, optionally, a tissue-specific regulatory element, such as a liver-specific promoter or a neuron-specific promoter. EMBODIMENT 44 44. The polynucleotide of any one of embodiments 40-43, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence comprising an internal long terminal repeat sequence comprising SEQ ID NO: 6, SEQ ID NO: 7, or both, and optionally a tissue-specific regulatory element, such as a liver-specific promoter or a neuron-specific promoter. Embodiment 45 45. The polynucleotide of any one of embodiments 40 to 44, further comprising a tissue-specific regulatory element comprising the sequence set forth as SEQ ID NO:8 and / or SEQ ID NO:9. Embodiment 46 A gene therapy vector comprising the polynucleotide of any one of embodiments 40 to 45. Embodiment 47 Gene therapy vectors a viral vector, optionally a naturally occurring virus, an engineered virus, or a chimeric virus; A naked polynucleotide comprising the polynucleotide according to any one of embodiments 20 to 25. A polynucleotide complex, which is optionally a lipid nanoparticle comprising a polynucleotide and a lipid according to any one of embodiments 20 to 25, and 47. The gene therapy vector of embodiment 46, selected from the group consisting of: any combination thereof. Embodiment 48 The gene therapy vector of embodiment 46 or embodiment 47, wherein the gene therapy vector is a viral vector selected from the group consisting of a retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus. Embodiment 49 The gene therapy vector of embodiment 47 or embodiment 48, wherein the gene therapy vector is AAV9, Anc80, AAV2 / 8 chimeric and / or AAV pseudotyped for a specific tissue, such as liver or neuronal tissue. Embodiment 50 A multidomain therapeutic protein comprising at least two delivery domains and at least one enzymatic domain, each of the two delivery domains being independently selected from the group consisting of an antibody, a half antibody, and an scFv, and wherein at least one or more delivery domains are associated with at least one enzymatic domain, preferably wherein the one or more delivery domains are covalently linked to the at least one enzymatic domain. Embodiment 51 51. The multidomain therapeutic protein of embodiment 50, comprising no more than two delivery domains. Embodiment 52 52. The multidomain therapeutic protein of embodiment 50 or embodiment 51, wherein only one of the delivery domains is associated with at least one enzymatic domain. Embodiment 53 53. A multidomain therapeutic protein according to any one of embodiments 50 to 52, wherein each of the at least two delivery domains is covalently linked to the enzymatic domain. EMBODIMENT 54 54. The multidomain therapeutic protein of embodiment 53, wherein each of the at least two delivery domains is covalently linked to the same enzymatic domain. Embodiment 55 54. The multidomain therapeutic protein of embodiment 53, wherein each of the at least two delivery domains is covalently linked to a different enzymatic domain. Embodiment 56 56. A multidomain therapeutic protein according to any one of embodiments 50 to 55, comprising no more than two delivery domains, wherein a first delivery domain comprises a half antibody and a second delivery domain comprises an scFv. Embodiment 57 57. The multidomain therapeutic protein of embodiment 56, wherein the scFv is fused to an Fc. Embodiment 58 58. The multidomain therapeutic protein of embodiment 56 or embodiment 57, wherein the half antibody is covalently linked at its carboxy terminus to a first enzymatic domain and / or the scFv is covalently linked at its carboxy terminus to an Fc and optionally a second enzymatic domain. Embodiment 59 56. A multidomain therapeutic protein according to any one of embodiments 50 to 55, comprising no more than two delivery domains, wherein the first delivery domain and the second delivery domain each comprise an scFv. Embodiment 60 60. The multidomain therapeutic protein of embodiment 59, wherein both the first and second scFvs are covalently linked to the enzymatic domain. Embodiment 61 60. The multidomain therapeutic protein of embodiment 59, comprising from N-terminus to C-terminus: a first scFv, a second scFv, and an enzymatic domain. Embodiment 62 62. The multidomain therapeutic protein of any one of embodiments 50 to 61, wherein at least one delivery domain binds to a lysosomal transport molecule and at least one delivery domain binds to a transcytosis effector. Embodiment 63 63. The multidomain therapeutic protein of embodiment 62, wherein the lysosomal transport molecule is selected from the group consisting of CD63, ITGA7, CD9, CD63, CD81, CD82, or CD151, and the transcytosis effector is selected from the group consisting of LDL receptor, IgA receptor, transferrin receptor, neonatal Fc receptor, insulin receptor, CD98, and basigin. EMBODIMENT 64 64. The multidomain therapeutic protein of any one of embodiments 50-63, comprising the structure shown in Figure 1C, Figure 1D, Figure 1E, or Figure 1F. Embodiment 65 Use of a nucleotide encoding a multidomain therapeutic protein according to any one of embodiments 21 to 39 and 50 to 64, a polynucleotide according to any one of embodiments 40 to 45, or a gene therapy vector according to any one of embodiments 46 to 49 in a method according to any one of embodiments 1 to 20.

[0213] The following examples are provided to further illustrate the method of the present invention. These examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way. [Example]

[0214] Example 1: Construction of anti-hCD63 ScFv::GAA polynucleotide and gene therapy vector AAV2 / 8 viruses encoding expression of human GAA (hGAA; SEQ ID NO: 1; nucleic acid sequence represented by SEQ ID NO: 12) or an anti-human CD63 single-chain variable fragment (ScFv) fused to human GAA at its C-terminus (anti-hCD63 ScFv-hGAA; SEQ ID NO: 10; nucleic acid represented by SEQ ID NO: 11) were generated using a standard triple transfection protocol (see also Gray et al. 2011; "Production of recombinant adeno-associated viral vectors and use in vitro and in vivo administration", Current Protocols in Neuroscience, John Wiley & Sons, New York (1999), pp. 4.17.1-4.17.25, Vol. 1). For production, 1 × 10 7HEK293 cells were seeded onto 15 cm plates and transfected the following day with 8 μg of either (A) a control pAAV vector encoding TTR-driven human GAA containing the liver-specific Serpina1 enhancer (SEQ ID NO: 9) or a test pAAV encoding TTR-driven hCD63 ScFv-hGAA (see Figure 1B) containing the liver-specific Serpina1 enhancer (SEQ ID NO: 9), and (B) a pAAV RC2 / 8-derived vector (Gao, 2002) with 16 μg of pHelper (Agilent, Cat. No. 240074) using PEIpro (Polyplus transfection, New York, NY, Cat. No. 115-100)-mediated transfection at a 1:1 ratio (1 μl PEIpro:1 μg DNA). Seventy-two hours after transfection, cells were harvested and lysed in a buffer consisting of 20 mM Tris-HCl, 1 mM MgCl2, 2.5 mM KCl, and 100 mM NaCl using a standard freeze-thaw method. Benzonase (Sigma, catalog number E1014-25KU) was then added to the sample at a final concentration of 0.5 U / μL, which was then incubated at 37°C for 60 minutes. Virus was then purified using iodixanol gradient ultracentrifugation as described (Zolotukhin et al., 1999, Gene Ther 1999;6:973-985) and subsequently titrated by qPCR.

[0215] AAV samples were treated with DNase I (Thermo Fisher Scientific, catalog no. EN0525) at 37°C for 1 hour and lysed using DNA extract All Reagents (Thermo Fisher Scientific, catalog no. 4403319). Encapsidated viral genomes were quantified using primers directed to the AAV2 ITRs using a QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific). The primer sequences for the AAV2 ITRs were 5'-GGAACCCCTAGTGATGGAGTT-3' (forward ITR; SEQ ID NO: 3) and 5'-CGGCCTCAGTGAGCGA-3' (reverse ITR; SEQ ID NO: 4) (Aurnhammer et al., 2012), derived from the AAV left internal inverted repeat (ITR) sequence (SEQ ID NO: 6) and the AAV right internal inverted repeat (ITR) sequence (SEQ ID NO: 7), respectively. The sequence of the AAV2 ITR probe is 5'-6-FAM-CACTCCCTCTCTGCGCGCTCG-TAMRA-3' (SEQ ID NO: 5) (Aurnhammer C., Haase M., Muether N., et al., 2012, Hum. Gene Ther. Methods 23, 18-28). After a 10-minute activation step at 95°C, a two-step PCR cycle was performed at 95°C for 15 seconds and 60°C for 30 seconds for 40 cycles. TaqMan Universal PCR Master Mix (Thermofisher Scientific, Cat. No. 4304437) was used for qPCR. Absolute titers were determined using DNA plasmid (Agilent, Cat. No. 240074) as a standard.

[0216] Anti-human CD63 antibodies and their fusions include the H5C6 murine anti-human CD63 variable domain (amino acids 1-119 of SEQ ID NO: 10 are the heavy chain variable domain (V H ) and amino acids 135 to 245 of SEQ ID NO: 10 represent the light chain variable domain (V LThe anti-hCD63 ScFv (SEQ ID NO: 2) used here was derived from the H5C6 clone, a murine anti-hCD63 monoclonal IgG1, kappa light chain antibody (H5C6 was deposited at the University of Iowa Developmental Studies Hybridoma Bank by August, JT, and Hildreth, JEK (DSHB Hybridoma Product H5C6; DSHB Catalog No. h5c6, RRID:AB 528158). The ScFv version of the antibody was cloned with the variable domains in heavy-light order, with a glycine-serine linker between them (5'-VH-Gly-Ser-VL-3').

[0217] Example 2: Glycogen content in a mouse Pompe model after AAV To determine the efficacy of AAV-delivered anti-hCD63 ScFv-GAA fusion versus AAV-delivered GAA in a relevant in vivo model of glycogen accumulation, both therapies were delivered to a mouse model of Pompe disease. The mice were homozygous for deletion of the mouse GAA gene and homozygous for expression of human CD63 in place of mouse CD63, on a 75% C57BL / 6, 25% 129SvJ strain background. These mice are referred to herein as CD63HumIn GAA KO mice or CD63hu / hu;GAA - / - It is called a mouse.

[0218] In this experiment, AAV2 / 8 viruses containing either a TTR liver-specific promoter-driven human GAA (AAV-hGAA; described in Example 1) or a TTR liver-specific promoter-driven anti-human CD63 ScFv fused to human GAA at its C-terminus (AAV-anti-hCD63 ScFv-hGAA; described in Example 1) in their genomes were administered to 2-month-old CD63 Humln GAA KO mice via tail vein injection. Both AAV2 / 8 viruses were delivered at one of two doses: 1e10 vg / mouse or 1e11 vg / mouse. As controls, untreated CD63 Humln GAA KO mice and untreated CD63 Humln mice with an intact mouse GAA gene were included in the assay. Mice were housed for 3 months after treatment, during which time blood was collected monthly for serum measurements of GAA levels and anti-GAA antibodies. After 3 months, all mice were sacrificed and individual tissues were collected for glycogen measurement, PAS-H staining, quantification of central nuclei, measurement of lysosomal proliferation, and measurement of LC3b expression. The experimental doses and treatment protocols for the mouse groups are shown in Table 3. [Table 3-1] [Table 3-2]

[0219] The results are also illustrated in Figure 2, which shows that anti-hCD63scFv::GAA, but not GAA alone, reduced glycogen to wild-type levels in skeletal muscle. Treatment with the bidomain anti-hCD63scFv::GAA multidomain therapeutic protein resulted in a much greater reduction in glycogen stores compared to the single-domain GAA replacement enzyme. By plotting quadriceps glycogen levels (Figure 3) or cardiac glycogen levels (Figure 4) over three months for individual mice against total serum expression of GAA or scfv-GAA, we observed that the anti-hCD63scFv::GAA fusion protein removed more glycogen than the GAA enzyme alone, even at similar serum levels (Figures 3 and 4).

[0220] Example 3: Immune response to GAA To measure serum levels of anti-human GAA antibodies, serum from all treatment groups was separated from blood collected during terminal bleeding using serum separator tubes (BD Biosciences, Cat. No. 365967) according to the manufacturer's specifications. Separately, 96-well high protein binding plates (ThermoFisher, Cat. No. 15041) were coated overnight with 20 μg of hGAA (R&D Systems, Cat. No. 8329-GH-025) diluted in PBS. The plates were washed three times with PBS + 0.05% Tween® (PBS-T). The plates were blocked with 0.5% BSA in PBS-T, and a dilution series of mouse serum ranging from 1:300 to 1:5.1e7 was added to the plates overnight. Total anti-mouse IgG (subclasses 1+2a+2b+3) was measured using an HRP-conjugated goat anti-mouse IgG antibody (Jackson ImmunoResearch, catalog number 115-035-164) and the BD Opt EIA substrate kit. The colorimetric reaction was stopped using 1N phosphoric acid. Absorbance was then read at 450 nm on a Spectramax i3 plate reader (Molecular Devices). The dilution curve was fitted to a sigmoidal curve, and titers were calculated from this curve. Titers, expressed as the mean + / - standard deviation of the total IgG titer, are shown in Table 4.

[0221] As shown in Table 4, untreated mice exhibited mean background titers of 1.1E+03. Mice treated with low doses of virus (1e10 vg / mouse) of either AAV-anti-hCD63 ScFv-hGAA or AAV-hGAA exhibited high titers, while mice treated with the high dose (1e11 vg / mouse) had lower titers. Mice treated with 1e11 vg of AAV-anti-hCD63 ScFv-hGAA, which had the highest serum GAA levels, had titers within the range of untreated mice. [Table 4]

[0222] Higher levels of GAA or anti-hCD63scFv::GAA after AAV administration correspond to lower anti-GAA titers. Sera from GAA-null mice treated with high or low titers of AAV-anti-hCD63scFv::GAA or AAV-GAA were evaluated for anti-GAA antibodies over a three-month period after injection. Figure 5 illustrates serum anti-GAA antibody titers versus GAA exposure (i.e., total serum expression of GAA or scfv-GAA over three months) for individual mice. This figure shows a negative correlation between antibody titers to GAA and serum exposure, indicating that mice with high GAA exposure were tolerant to GAA. Similarly, Figure 6 plots anti-GAA antibody titers for various groups infected with AAV encoding GAA or anti-hCD63scFv::GAA proteins, showing that higher doses of the constructs resulted in lower anti-GAA titers.

[0223] Example 4: Serum GAA To measure human GAA serum levels over the course of the experiment, samples were collected monthly via tail bleed. Serum was separated from blood using serum separator tubes (BD Biosciences, catalog no. 365967) according to the manufacturer's specifications. 1 μL of isolated serum was then loaded onto a 4%-20% Novex wedgewell precast gel, run at 220 V for 45 minutes, and transferred to a nitrocellulose membrane for 1 hour at 200 mA using standard protocols. The nitrocellulose membrane was then probed with 12 mL of anti-GAA primary antibody (Abcam, no. ab137068) at a 1:2000 dilution and anti-GAPDH antibody (Abcam, no. AB9484) at a 1:1000 dilution, and incubated overnight at 4°C. After incubation with the primary antibodies, the membrane was washed three times with 1x TBST for 5 minutes per wash. Twelve milliliters of a 1:15,000 dilution of anti-rabbit IgG (LiCor, 926-32211) secondary antibody and anti-mouse IgG (LiCor, 925-68070) secondary antibody (LiCor, Lincoln, NE) were then added to the membrane and incubated for 1 hour at room temperature. After secondary antibody incubation, the membrane was washed twice with 1x TBST for 5 minutes per wash and once with 1x TBS for 5 minutes per wash. The membrane was then imaged and quantified using a LiCor Odyssey instrument (LI-COR Biotechnology). Serum levels of GAA, expressed as mean ± standard deviation (SD) in arbitrary units, are shown in Table 5.

[0224] As shown in Table 5, the high dose tested (10 11 CD63 Humln GAA KO mice treated with AAV-anti-hCD63 ScFv-hGAA or AAV-hGAA at low doses (10 vg / mouse) maintained serum GAA levels over the experimental period, with AAV-anti-hCD63 ScFv-hGAA-treated mice having somewhat higher serum GAA levels than AAV-hGAA-treated mice. 10In mice treated with either AAV-anti-hCD63 ScFv-hGAA or AAV-hGAA (200 mg / mouse), GAA levels decreased over the experimental period, reaching negligible levels in some mice by 12 weeks. [Table 5]

[0225] Expression of GAA or anti-hCD63scfv::GAA was enhanced by a high dose of AAV (10 11 vg / mouse), but was maintained over time in mice receiving lower doses (10 10 The serum levels of GAA were reduced in mice receiving 1000 scFv / mouse (1000 scfv / mouse). Figure 7A depicts a graph plotting serum levels of GAA over time, as probed by Western blot, for various groups infected with AAV encoding GAA or an anti-hCD63scfv fusion with GAA. The fusion protein (scFv::GAA) consistently demonstrated higher levels of serum GAA (e.g., 2.5-3 fold) than the GAA enzyme without the delivery domain (Figure 7A).

[0226] Quantification of expression in the liver, heart, and quadriceps muscle 3 months after injection by real-time PCR is shown in Figure 7B. Liver expression was detected for all AAV constructs, with the highest levels observed for both AAV-hGAA and AAV-anti-hCD63::hGAA (both driven by the liver-specific promoter LSP) at 1e11vg / mouse. We also compared serum GAA levels with GAA RNA expression levels (Figure 7C). The results showed that mice receiving AAV encoding the fusion protein exhibited low GAA RNA expression restricted to the liver at 3 months, although serum GAA levels were high in those particular mice. AAV-LSP-hGAA injection did not result in high serum GAA levels, even though RNA levels were low in the liver. See Figure 7C. These data suggest that AAV encoding the fusion protein (and whose expression is driven by a liver-specific promoter) achieves an improved GAA secretion profile.

[0227] A higher ratio of secreted to intracellular antibody:hGAA versus hGAA alone was also observed in Huh-7 hepatocytes. In one experiment, Huh-7 human hepatocytes were transiently transfected with liver-specific promoter-driven constructs encoding hGAA, an anti-hCD63 scFv:GAA fusion, or a nonbinding scFv:GAA fusion control. Three days after transfection, both scFv:GAA fusion constructs resulted in a higher ratio of protein in the secreted supernatant than hGAA alone (statistically significant p<0.05, n=3). Addition of M6P to the supernatant during the experiment to reduce CI-MPR-mediated uptake did not affect this ratio.

[0228] Example 5: Histological Measurement of Glycogen and Histological Characterization of Muscle Tissue Glycogen Tissue Measurement: To measure the glycogen content of individual tissues, heart, quadriceps, gastrocnemius, diaphragm, soleus, and EDL tissues were excised from mice from all groups immediately after CO2 asphyxiation, then flash-frozen in liquid nitrogen and stored at -80°C. For glycogen measurement, approximately 50 mg of each tissue was lysed in distilled water at a ratio of 1 mg to 25 μL using a benchtop homogenizer with stainless steel beads. Glycogen analysis lysates were heated at 105°C for 15 minutes and centrifuged at 21,000 × g to remove debris. Glycogen measurement was performed using a glycogen assay kit (Sigma-Aldrich, No. MAK016) according to the manufacturer's instructions for the fluorescence assay. Fluorescence of each sample was measured using a fluorescence plate reader (Molecular Devices, Spectramax i3) at an excitation of 535 nm and an emission of 587 nm. The calculated amount of glycogen was calculated using the following formula provided by the manufacturer: The calculated amount of glycogen from each tissue in each treatment group was then averaged and is presented in Table 6 as the mean ± standard deviation (SD).

[0229] As shown in Table 6, loss of Gaa caused a large increase in mean glycogen levels across all tissues measured compared to GAA WT mice. 11 Treatment with vg / mice reduced glycogen to WT or near-WT levels in all tissues tested, in contrast to treatment with AAV-GAA, which only partially reduced glycogen stores. Low doses of both viruses also reduced glycogen, but to a lesser extent than the higher doses. 10 10 vg / mouse dose of AAV-anti-hCD63 ScFv-hGAA was 10 11 vg / mouse dose of AAV-GAA reduced glycogen levels similarly. [Table 6]

[0230] Quadriceps muscle harvest for histopathology and quantification: Quadriceps muscle tissue samples from mice from each group except the low-dose (1e10vg / mouse) treatment group were either snap-frozen in liquid nitrogen immediately after excision and stored at -80°C for quantification of LC3b expression, or placed on blocks containing OCT medium (Tissue-Tek, #4583).

[0231] Tissue samples in OCT medium were sent to Histoserv (Germantown, MD) for sectioning and periodic acid-Schiff (PAS) staining to detect polysaccharides. Additional sections were prepared and returned for staining for central nuclei and lysosomal proliferation.

[0232] PAS staining: PAS-stained sections were imaged using a Leica slide scanner at 20x magnification. The resulting images from representative mice from each treatment group are shown in Figure 8.

[0233] As shown in Figure 8, at 3 months, CD63 HumIn GAA KO mice treated with AAV-anti-hCD63 ScFv-hGAA showed significantly reduced PAS staining compared to both untreated CD63 HumIn GAA KO mice and AAV-hGAA-treated CD63 HumIn GAA KO mice, which both showed high levels of PAS staining. This further indicates that treatment with AAV-anti-hCD63 ScFv-hGAA can reduce polysaccharide accumulation in CD63 HumIn GAA KO mice and can do so in a uniform manner throughout muscle fibers.

[0234] Quantification of central nuclei and lysosomal proliferation: Unstained sections from Histoserv were removed from the freezer and fixed in a staining chamber with 4% paraformaldehyde in PBS for 15 minutes. The fixed slides were then washed twice in PBS for 5 minutes, followed by incubation with blocking buffer (eBiosciences, 00-4953-54) at room temperature for 1 hour. Slides were then stained in a humidified staining chamber with either a rat anti-Lamp-1 antibody (Abcam, No. AB25245) diluted 1:50 in blocking buffer or a rabbit anti-laminin antibody (Sigma, No. L9393) diluted 1:1000 in blocking buffer, or with blocking buffer without antibody, and then transferred to 4°C for overnight incubation. The next day, slides were washed twice in PBS for 5 minutes and then stained with either a goat anti-rabbit IgG (H+L) superclonal secondary antibody conjugated to Alexa Fluor 647 (Lifetech Thermo, No. A27040) or a goat anti-rat IgG (H+L) cross-adsorbed secondary antibody conjugated to Alexa Fluor 555 (Lifetech Thermo, No. A21434) in a staining chamber, followed by incubation at room temperature for 1 hour. The stained slides were then washed twice in PBS for 5 minutes, mounted with Fluoromount-G containing DAPI (Lifetech Thermo, No. 00-4959-52), and imaged using a Zeiss LSM710 instrument (Carl Zeiss Microscopy GmbH). The number of centrally located nuclei was quantified using Halo software (Indica Labs, NM) and is presented in Table 7 as the percentage of fibers showing central nuclei ± standard deviation. Lysosomal proliferation is illustrated in FIG. [Table 7]

[0235] Quantification of LC3b Expression: For quantification of LC3b expression, flash-frozen samples were thawed, homogenized, and then lysed by bead bombardment (MP Biomedical) for 45 seconds in RIPA buffer (150 mM NaCl, 1.0% IGEPAL® CA-630, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM Tris, pH 8.0, Sigma-Aldrich, R0278) at a ratio of 25 μL of RIPA buffer to 1 mg of tissue. Lysates were centrifuged at 21,000 x g to remove insoluble material. Then, 300 µg of lysate in RIPA buffer was loaded onto a 4%-20% Novex wedgewell precast gel, transferred to a nitrocellulose membrane, and analyzed by Western blot using a similar protocol previously described for the analysis of serum GAA levels, substituting a primary antibody recognizing mouse LC3b-I and LC3b-II (Sigma, number L7543) for the primary antibody against GAA. The membranes were then imaged and quantified using a LiCor Odyssey instrument (LI-COR Biotechnology). LC3b-I and LC3b-II levels, expressed as arbitrary units (mean ± standard deviation), are shown in Table 8.

[0236] As shown in Table 8, mice lacking GAA had significantly increased mean LC3b-I and LC3b-II levels compared with CD63 HumLn GAA WT mice. Treatment with AAV-anti-hCD63 ScFv-hGAA reduced mean LC3b-I and LC3b-II levels in CD63 HumLn GAA KO to WT or near-WT levels. CD63 HumLn GAA KO mice treated with AAV-hGAA showed a slight decrease in mean LC3b-I and LC3b-II levels compared with CD63 HumLn GAA KO mice, but this decrease was not as pronounced as with treatment with AAV-anti-hCD63 ScFv-hGAA. [Table 8]

[0237] Example 6: AAV anti-hCD63::GAA treatment produces significant increases in tests of muscle strength and coordination Grip strength and rotarod test performance of mice treated with either AAV-LSP hGAA or AAV-LSP-anti-hCD63::hGAA. Accelerating rotarod measurements (Figure 9A) and forelimb grip strength measurements (Figure 9B) were obtained at 1-month intervals over a 6-month period for wild-type GAA mice, untreated controls, AAV-LSP-hGAA (1e11 vg / mouse), or AAV-LSP-anti-hCD63::hGAA-treated (1e11 vg / mouse). Error bars are + / - standard deviation. N = 8–10 for all groups.

[0238] Example 7: Other membrane proteins as "guides" to target GAA to tissues To guide GAA into tissues to replenish GAA in enzyme-deficient mice, we tested other membrane proteins, such as anti-ITGA7 (integrin alpha 7) fusion proteins. C2C12 mouse myoblasts were incubated overnight with anti-mCD63-GAA or anti-ITGA7-GAA in the presence or absence of 5 mM M6P. For both fusion proteins, active GAA enzyme was detected in myoblast lysates over time (Figure 10A). In a separate experiment, CD63-humanized GAA KO mice (GAA- / -; CD63hu / hu) were given plasmids encoding anti-hCD63::GAA in the scFv::GAA format or anti-integrin alpha 7 in the full-length IgG4::GAA format by hydrodynamic delivery (HDD). The mice were sacrificed 3 weeks after HDD. Tissue glycogen levels were measured in the heart, quadriceps, gastrocnemius, and diaphragm. Untreated control mice with GAA - / - xCD63hu / hu and untreated wild-type GAA control mice GAA+ / +;CD63hu / hu(4) were also tested under the same conditions. Glycogen levels were very low in both the anti-hCD63::GAA-treated and anti-ITGA7::GAA-treated mouse groups, as in wild-type mice. See Figure 10B.

[0239] Example 8: At comparable serum levels, AAV anti-CD63::GAA treatment is more effective than AAV optimized GAA constructs CD63 HumIn GAA KO mice (GAA - / - xCD63 hu / hu Mice were infected with AAV containing a liver-specific enhancer (Serpina1; SEQ ID NO: 9) and a liver-specific promoter (LSP; TTR; SEQ ID NO: 8) driving the expression of an anti-hCD63::GAA multidomain therapeutic (SEQ ID NO: 10). These utilize the signal peptide (SP7) of chymotrypsinogen B2 and contain amino acids 36-952 of human GAA (Δ8GAA), which showed significant improvements in tests of muscle strength and coordination. Three different doses of each virus were administered: 5e11 vg / kg, 2e12 vg / kg, and 4e12 vg / kg. Serum was collected periodically by submandibular bleeding. One month after AAV infection, mice were sacrificed. Cardiac and skeletal muscle tissue samples were collected, flash-frozen in liquid nitrogen, and kept at -80°C for storage. Glycogen in the tissue was measured by homogenizing the tissue by bead bombardment in distilled water. Samples were boiled and centrifuged, and the supernatant was used in a commercially available glycogen assay kit. Serum was quantified using Western blot analysis with an antibody against human GAA, as described in the previous example. For each mouse, glycogen levels in each tissue were plotted against serum levels of the construct at 1 month. A four-parameter curve fit was used to determine the EC50 of the two treatments in each tissue.

[0240] Infection with AAV containing a liver-specific promoter (LSP) encoding either anti-hCD63::GAA or sp7-Δ8GAA resulted in comparable serum GAA levels at each infection dose (Figure 11). However, in each muscle tissue assayed, a 2.2-fold reduction in EC50 was observed when using anti-hCD63::GAA compared to sp7-Δ8GAA, indicating that at comparable serum levels, anti-CD63::GAA removed glycogen more efficiently than the modified GAA expression construct not fused to an antibody (Figure 12).

[0241] Example 9: Glycogen content in the CNS of the mouse Pompe model after AAV treatment with various GAA constructs and doses AAV2 / 8 viruses containing either a TTR liver-specific promoter-driven human GAA (AAV-hGAA; described in Example 1) or a TTR liver-specific promoter-driven anti-human CD63 ScFv fused to human GAA at the C-terminus (AAV-anti-hCD63 ScFv-hGAA; described in Example 1) in their genomes were administered to two-month-old CD63 Humln GAA KO mice via tail vein injection. Both AAV2 / 8 viruses were delivered at 1e11 vg / mouse. As a control, untreated CD63 Humln GAA KO (Gaa - / - ) mice and untreated CD63 HumIn (wild-type) mice with an intact mouse GAA gene were included in the assay. Mice were housed for 9 months after treatment, after which all mice were sacrificed and individual tissues were harvested for glycogen measurements. CNS tissues were dissected on ice and flash-frozen 9 months after AAV transduction. Spinal cord, cerebellar, and hippocampal tissues were homogenized in deionized distilled water using bead bombardment, and glycogen was measured in the supernatant of the tissue lysates using a commercially available fluorescent glycogen assay kit (Figure 13).

[0242] In another similar experiment, knockout mice were treated with AAV constructs encoding GAA (1e11vg) or anti-CD63ScFv-GAA fusion (doses of 1e10vg, 5e10vg, or 1e11vg). The amount of stored glycogen per mg of wild-type mice and CNS tissue (spinal cord: Figure 14A, brain: Figure 14B) was examined 3 months after AAV delivery. Wild-type mice and untreated KO mice (GAA- / -) were used as comparators of stored glycogen levels in CNS tissue for the same period (3 months).

[0243] The ScFv-GAA fusion construct was more effective than GAA alone in reducing glycogen storage levels in diseased mice at a dose of 1e11 vg / mouse (Figures 13-14). A dose of 5e10 vg of the ScFv-GAA fusion provided a reduction in glycogen storage levels equivalent to higher doses of the GAA-only construct (Figures 14A-14B). These reduced glycogen storage levels were shown to be effective by Hordeaux et al. (2017) (e.g., glycogen reduction and improved muscle strength in mice intrathecally injected with AAV GAA). Relative serum levels of the ScFv-GAA fusion protein were detectable at 5e10 vg and above 15 μg / mL in mice receiving 1e11 vg (Table 9). [Table 9]

[0244] Without being bound by any one theory, a detectable serum level, e.g., a serum level of greater than 1 ug / mL of GAA linked to a delivery domain that crosses the blood-brain barrier, is considered therapeutic.

[0245] Example 10: Expression of multidomain therapeutic proteins containing at least two delivery domains CHO cells were transfected with the expression constructs shown in Figure 1C–G, and expression of each construct was confirmed (data not shown). Furthermore, binding of a portion of the multidomain therapeutic proteins encoded by 4W1 and 4M1 (Figure 1F) to CD63 was confirmed by ELISA (data not shown). (Item 1) 1. A method for delivering a therapeutic protein into the central nervous system (CNS) of a subject, comprising administering to the subject a nucleotide composition encoding a multidomain therapeutic protein via a liver-targeted delivery method sufficient to provide a therapeutically effective amount of the multidomain therapeutic protein to the CNS, wherein the multidomain therapeutic protein comprises a delivery domain and an enzymatic domain. (Item 2) 2. The method of claim 1, wherein the delivery domain is an antibody or antigen-binding fragment thereof that specifically binds to an internalization effector. (Item 3) 3. The method of claim 1 or 2, wherein the therapeutic protein is a lysosomal enzyme. (Item 4) 4. The method of claim 3, wherein the lysosomal enzyme is GAA. (Item 5) 5. The method of any one of items 1 to 4, wherein the nucleotide composition is administered via a viral vector. (Item 6) The viral vector is an AAV vector, and optionally the nucleotide composition comprises at least 2 x 10 12 6. The method of item 5, wherein the vaccine is administered at a dose of 100 mg of viral genome (vg / kg). (Item 7) 7. The method of any one of items 1 to 6, wherein the internalization effector is expressed on the surface of a cell selected from the group consisting of a cell in the CNS, an epithelial cell, and a cell that crosses the blood-brain barrier. (Item 8) the delivery domain comprises: (i) is selected from the group consisting of CD63, integrin alpha 7 (ITGA7), MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), myelin and lymphocyte protein (MAL), IGF2R, vacuolar H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, and CD36; (ii) expressed in several tissue types; optionally selected from the group consisting of CD63, MHC-I, vacuolar H+ ATPase, IGF2R, integrin alpha 7 (ITGA7), LRP5, LRP6, LRP8, Kremen-2, LDL receptor, LDL-related protein 1 receptor, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), PRLR, MAL (myelin and lymphocyte protein (MAL), diphtheria toxin receptor, HBEGF (heparin-binding EGF-like growth factor), glutathione receptor, glutamate receptor, leptin receptor, and folate receptor; Optionally, the subject exhibits one or more symptoms of a disease selected from the group consisting of Fabry disease, Gaucher disease, MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIID, MPS IVB, MPS VI, MPS VII, MPS IX, Pompe disease, lysosomal acid lipase deficiency, metachromatic leukodystrophy, Niemann-Pick disease types A, B, and C2, alpha mannosidosis, neuraminidase deficiency, sialidosis, aspartylglycosaminuria, mixed saposin deficiency, variant Gaucher disease, Farber lipogranulomatosis, fucosidosis, and beta mannosidosis; (iii) preferentially expressed by bone and / or cartilage; optionally selected from the group consisting of collagen X, integrin alpha 10 (ITGA10), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor isoform C (FGFR3C), hyaluronan and proteoglycan link protein 1 (CRTL1), aggrecan, collagen II, and Kremen-1; Optionally, the subject exhibits one or more symptoms of a disease selected from the group consisting of MPS I, MPS II, MPS IIIA, MPS IIIB, MPS IIID, MPS IVA, MPS IVB, MPS VI, MPS VII, MPS IX, beta-mannosidosis, Gaucher disease, atypical Gaucher disease, mixed saposin deficiency, aspartylglycosaminuria, Farber lipogranulomatosis, sialidosis, neuraminidase deficiency, and alpha-mannosidosis; (iv) preferentially expressed by monocytes, macrophages, or microglia; optionally selected from the group consisting of scavenger receptor A1-5 (SCARA1-5), SCARB1-3, CD36, MSR1 (macrophage scavenger receptor 1), MRC1 (macrophage mannose receptor 1), VSIG4 (V-set and immunoglobulin domain-containing protein 4), CD68 (macrosialin), and CSF1R (macrophage colony-stimulating factor 1 receptor); Optionally, the subject exhibits one or more symptoms of a disease selected from the group consisting of lysosomal acid lipase deficiency, Gaucher disease, atypical Gaucher disease, mixed saposin deficiency, and Farber lipogranulomatosis; (v) preferentially expressed by kidney cells; arbitrarily selected from the group consisting of CDH16 (Cadherin-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (solute carrier family 22 member 13), SLC5A2 (sodium / glucose cotransporter 2), and UMOD (uromodulin); Optionally, the subject exhibits symptoms of or has been diagnosed with one or more of a disease selected from the group consisting of Fabry disease, Alport syndrome, polycystic kidney disease, and thrombotic thrombocytopenic purpura; (vi) preferentially expressed by hepatocytes; Optionally ASGR1 or ASGR2, Optionally, the subject exhibits one or more symptoms of or has been diagnosed with a disease selected from the group consisting of lysosomal acid lipase deficiency, Gaucher disease, MPS VI, MPS VII, MPS II, Niemann-Pick disease types A, B, and C2, sialidosis, neuraminidase deficiency, atypical Gaucher disease, mixed saposin deficiency, and Farber lipogranulomatosis; (vii) preferentially expressed by muscle cells; optionally selected from the group consisting of BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3); Optionally, the subject exhibits or has been diagnosed with one or more symptoms of Pompe disease; (viii) selected from the group consisting of ITGA7, CD9, CD63, ALPL2, MSR1, ASGR1, ASGR2, or PRLR; or (ix) The method of any one of items 1 to 7, wherein the internalization effector is CD63. (Item 9) 9. The method of any one of items 1 to 8, wherein the delivery domain is a single-chain variable fragment (scFv). (Item 10) 10. The method according to any one of items 1 to 9, wherein the cell surface receptor (CSR) binding protein (CSR-BP) comprises the amino acid sequence of SEQ ID NO: 2. (Item 11) 11. The method of any one of items 1 to 10, wherein the therapeutic protein comprises a hydrolase. (Item 12) 12. The method of any one of items 1 to 11, wherein the therapeutic protein comprises a glycosylase. (Item 13) 13. The method of any one of items 1 to 12, wherein the therapeutic protein comprises a glycosidase. (Item 14) 14. The method of any one of items 1 to 13, wherein the therapeutic protein comprises alpha-glucosidase. (Item 15) 15. The method of any one of items 1 to 14, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13, or a fragment thereof. (Item 16) 16. The method of any one of items 1 to 15, wherein the therapeutic protein comprises an anti-ABeta or anti-tau antibody. (Item 17) 17. The method of any one of items 1 to 16, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11. (Item 18) 18. The method of any one of items 1 to 17, wherein the enzyme domain comprises alpha-glucosidase and glycogen levels in any CNS tissue of the subject are reduced for at least nine months after treatment. (Item 19) 19. The method of any one of items 1 to 18, wherein the subject has Pompe disease. (Item 20) 20. The method of any one of paragraphs 1 to 19, wherein the administered nucleotide composition provides a multidomain therapeutic protein serum level of at least 1 μg / mL. (Item 21) A multidomain therapeutic protein comprising one or more delivery domains and an enzymatic domain, wherein the one or more delivery domains bind to the human transferrin receptor (hTfR). (Item 22) 22. The multidomain therapeutic protein of item 21, further comprising a second delivery domain that binds to an internalization effector. (Item 23) the second delivery domain: (i) an internalization effector selected from the group consisting of CD63, integrin alpha 7 (ITGA7), MHC-I, Kremen-1, Kremen-2, LRP5, LRP6, LRP8, transferrin receptor, LDL receptor, LDL-related protein 1 receptor, ASGR1, ASGR2, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), myelin and lymphocyte protein (MAL), IGF2R, vacuolar-type H+ ATPase, diphtheria toxin receptor, folate receptor, glutamate receptor, glutathione receptor, leptin receptor, scavenger receptor A1-5 (SCARA1-5), SCARB1-3, and CD36; (ii) an internalization effector expressed in several tissue types, arbitrarily selected from the group consisting of CD63, MHC-I, vacuolar-type H+ ATPase, IGF2R, integrin alpha 7 (ITGA7), LRP5, LRP6, LRP8, Kremen-2, LDL receptor, LDL-related protein 1 receptor, amyloid precursor protein-like protein 2 (APLP2), apelin receptor (APLNR), PRLR, MAL (myelin and lymphocyte protein (MAL)), diphtheria toxin receptor, HBEGF (heparin-binding EGF-like growth factor), glutathione receptor, glutamate receptor, leptin receptor, and folate receptor; (iii) an internalization effector preferentially expressed by bone and / or cartilage, optionally selected from the group consisting of collagen X, integrin alpha 10 (ITGA10), fibroblast growth factor receptor 3 (FGFR3), fibroblast growth factor receptor isoform C (FGFR3C), hyaluronan and proteoglycan link protein 1 (CRTL1), aggrecan, collagen II, and Kremen-1; (iv) an internalization effector preferentially expressed by monocytes, macrophages, or microglia, optionally selected from the group consisting of scavenger receptor A1-5 (SCARA1-5), SCARB1-3, CD36, MSR1 (macrophage scavenger receptor 1), MRC1 (macrophage mannose receptor 1), VSIG4 (V-set and immunoglobulin domain-containing protein 4), CD68 (macrosialin), and CSF1R (macrophage colony-stimulating factor 1 receptor); (v) an internalization effector preferentially expressed by kidney cells, optionally selected from the group consisting of CDH16 (Cadherin-16), CLDN16 (Claudn-16), KL (Klotho), PTH1R (parathyroid hormone receptor), SLC22A13 (solute carrier family 22 member 13), SLC5A2 (sodium / glucose cotransporter 2), and UMOD (uromodulin), which in certain other embodiments is a muscle-specific internalizer, such as BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor ( internalization effectors, such as nicotinic (nicotinic) alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3); (vi) optionally an internalization effector preferentially expressed by hepatocytes, such as ASGR1 or ASGR2; (vii) an internalization effector preferentially expressed by muscle cells, optionally selected from the group consisting of BMPR1A (bone morphogenetic protein receptor 1A), m-cadherin, CD9, MuSK (muscle-specific kinase), LGR4 / GPR48 (G protein-coupled receptor 48), cholinergic receptor (nicotinic) alpha 1, CDH15 (Cadherin-15), ITGA7 (integrin alpha 7), CACNG1 (L-type calcium channel subunit gamma 1), CACNAlS (L-type calcium channel subunit alpha 15), CACNG6 (L-type calcium channel subunit gamma 6), SCN1B (sodium channel subunit beta 1), CHRNA1 (ACh receptor subunit alpha), CHRND (ACh receptor subunit delta), LRRC14B (leucine-rich repeat-containing protein 14B), dystroglycan (DAG1), and POPDC3 (Popeye domain-containing protein 3); or (viii) The multidomain therapeutic protein of item 22, which binds to an internalization effector protein selected from the group consisting of ITGA7, CD9, CD63, ALPL2, MSR1, ASGR1, ASGR2, or PRLR. (Item 24) 24. The multidomain therapeutic protein of any one of items 21 to 23, wherein the second delivery domain binds to the internalization effector CD63. (Item 25) 25. The multidomain therapeutic protein of any one of items 21 to 24, wherein at least one of the one or more delivery domains comprises an antigen-binding protein. (Item 26) 26. The multidomain therapeutic protein of claim 25, wherein each of the one or more delivery domains comprises an antigen-binding protein. (Item 27) 27. The multidomain therapeutic protein of any one of items 21 to 26, wherein at least one of the one or more delivery domains comprises a single-chain variable fragment (scFv). (Item 28) 28. The multidomain therapeutic protein of any one of items 21 to 27, wherein at least one of the one or more delivery domains comprises a half antibody. (Item 29) 30. The multidomain therapeutic protein of claim 28, wherein the delivery domain that binds to hTfR is an scFv, the half antibody that binds to CD63, and the enzymatic domain is GAA, wherein GAA is conjugated to the carboxy terminus of the half antibody that binds to CD63. 28. The multidomain therapeutic protein of item 27, wherein each of the one or more delivery domains comprises an scFv. (Item 31) 31. The multidomain therapeutic protein according to any one of items 27 to 30, wherein at least one scFv is fused to an Fc. (Item 32) 32. The multidomain therapeutic protein of item 31, wherein the Fc comprises a wild-type human IgG4 isotype, or a derivative thereof. (Item 33) 33. The multidomain therapeutic protein of any one of items 31 to 32, wherein GAA is conjugated to the carboxy terminus of Fc. (Item 34) 31. The multidomain therapeutic protein according to item 30, comprising an anti-hTfR scFv and an anti-hCD63 scFv. (Item 35) 35. The multidomain therapeutic protein of item 34, wherein the anti-hTfR scFv and anti-hCD63 scFv are both linked at their carboxy termini to a single GAA enzyme. (Item 36) 28. The multidomain therapeutic protein of any one of items 21 to 27, wherein the delivery domain is an anti-hTfR scFv and the enzymatic domain is linked to the carboxy terminus of the VL domain of the scFv. (Item 37) 37. The multidomain therapeutic protein of item 36, further comprising a second delivery domain linked to the N-terminus of the VH domain of the anti-hTfR scFv. (Item 38) 38. The multidomain therapeutic protein of item 37, wherein the second delivery domain is an anti-hCD63 scFV. (Item 39) 39. The multidomain therapeutic protein according to any one of items 21 to 38, wherein the enzymatic domain comprises the amino acid sequence set forth as SEQ ID NO: 1. (Item 40) 1. A multidomain therapeutic protein comprising at least two delivery domains and at least one enzymatic domain, wherein each of the two delivery domains is independently selected from the group consisting of an antibody, a half antibody, and an scFv, and wherein at least one or more of the delivery domains is associated with the at least one enzymatic domain, preferably wherein the one or more delivery domains are covalently linked to the at least one enzymatic domain. (Item 41) 42. The multidomain therapeutic protein of claim 40, comprising no more than two delivery domains. 42. The multidomain therapeutic protein of claim 40 or 41, wherein only one of the delivery domains is associated with the at least one enzymatic domain. (Item 43) 43. The multidomain therapeutic protein of any one of items 40 to 42, wherein each of the at least two delivery domains is covalently linked to an enzymatic domain. (Item 44) 44. The multidomain therapeutic protein of claim 43, wherein each of the at least two delivery domains is covalently linked to the same enzymatic domain. (Item 45) 44. The multidomain therapeutic protein of claim 43, wherein each of the at least two delivery domains is covalently linked to a different enzymatic domain. (Item 46) 46. ​​The multidomain therapeutic protein of any one of items 40 to 45, comprising no more than two delivery domains, wherein the first delivery domain comprises a half antibody and the second delivery domain comprises an scFv. (Item 47) 47. The multidomain therapeutic protein of item 46, wherein the scFv is fused to an Fc. (Item 48) 48. The multidomain therapeutic protein of claim 46 or 47, wherein the half antibody is covalently linked at its carboxy terminus to a first enzymatic domain and / or the scFv is covalently linked at its carboxy terminus to an Fc and optionally a second enzymatic domain. (Item 49) 46. ​​The multidomain therapeutic protein of any one of items 40 to 45, comprising no more than two delivery domains, wherein the first delivery domain and the second delivery domain each comprise an scFv. (Item 50) 50. The multidomain therapeutic protein of item 49, wherein both the first and second scFvs are covalently linked to the enzymatic domain. (Item 51) 50. The multidomain therapeutic protein of item 49, comprising from N-terminus to C-terminus: the first scFv, the second scFv, and the enzymatic domain. (Item 52) 52. The multidomain therapeutic protein of any one of items 40 to 51, wherein at least one delivery domain binds to a lysosomal transport molecule and at least one delivery domain binds to a transcytosis effector. (Item 53) 53. The multidomain therapeutic protein of item 52, wherein the lysosomal transport molecule is selected from the group consisting of CD63, ITGA7, CD9, CD63, CD81, CD82, or CD151, and the transcytosis effector is selected from the group consisting of LDL receptor, IgA receptor, transferrin receptor, neonatal Fc receptor, insulin receptor, CD98, and basigin. (Item 54) 54. The multidomain therapeutic protein according to any one of items 40 to 53, comprising the structure shown in Figure 1C, Figure 1D, Figure 1E, or Figure 1F. (Item 55) 55. A polynucleotide encoding the multidomain therapeutic protein according to any one of items 21 to 54. (Item 56) 56. The polynucleotide of Item 55, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence. (Item 57) 57. The polynucleotide of claim 55 or 56, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence. (Item 58) 58. The polynucleotide of any one of items 55 to 57, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence, and the AAV nucleic acid sequence comprises an internal terminal repeat sequence and, optionally, a tissue-specific regulatory element, such as a liver-specific promoter or a neuron-specific promoter. (Item 59) 59. The polynucleotide of any one of paragraphs 55 to 58, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence comprising an internal long terminal repeat sequence comprising SEQ ID NO: 6, SEQ ID NO: 7, or both, and optionally a tissue-specific regulatory element, such as a liver-specific promoter or a neuron-specific promoter. (Item 60) 60. The polynucleotide of any one of items 55 to 59, further comprising a tissue-specific regulatory element comprising the sequence set forth as SEQ ID NO: 8 and / or SEQ ID NO: 9. (Item 61) A gene therapy vector comprising the polynucleotide according to any one of Items 55 to 60. (Item 62) the gene therapy vector a viral vector, optionally a naturally occurring virus, an engineered virus, or a chimeric virus; A naked polynucleotide comprising the polynucleotide according to any one of Items 20 to 25. A polynucleotide complex, which is optionally a lipid nanoparticle comprising the polynucleotide according to any one of items 20 to 25 and a lipid, and 62. The gene therapy vector of item 61, selected from the group consisting of: a gene therapy vector comprising a nucleotide sequence encoding ... (Item 63) Item 63. The gene therapy vector of item 61 or item 62, wherein the gene therapy vector is a viral vector selected from the group consisting of a retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus. (Item 64) 64. The gene therapy vector of claim 62 or 63, wherein the gene therapy vector is AAV9, Anc80, AAV2 / 8 chimera and / or AAV pseudotyped for a specific tissue, such as liver or neuronal tissue. (Item 65) 6. Use of a nucleotide encoding a multidomain therapeutic protein according to any one of items 21 to 54, a polynucleotide according to any one of items 55 to 60, or a gene therapy vector according to any one of items 61 to 64 in the method according to any one of items 1 to 20. The present invention provides, for example, the following items. (Item A1) 1. A method of delivering a therapeutic protein to the central nervous system (CNS) of a patient, comprising delivering a nucleotide composition encoding a multidomain therapeutic protein to the liver of said patient to form a liver depot for producing and secreting said multidomain therapeutic at consistent serum levels of at least 1 μg / mL for consecutive days, weeks, or months following delivery, thereby providing a therapeutically effective amount of said multidomain therapeutic protein in said CNS; The method, wherein the multidomain therapeutic protein comprises an antibody or antigen-binding portion thereof that binds CD63 or ITGA7 and the enzymatic domain. (Item A2) The method according to item A1, wherein the therapeutic protein is a lysosomal enzyme. (Item A3) The method according to item A2, wherein the lysosomal enzyme is GAA. (Item A4) The method according to any one of items A1 to A3, wherein the nucleotide composition is administered via a viral vector. (Item A5) The viral vector is an AAV vector, and optionally the nucleotide composition comprises at least 2 x 10 12 The method according to item A4, wherein the vaccine is administered at a dose of 100 mg of viral genome (vg / kg). (Item A6) The method of any one of items A1 to A5, wherein the antibody or antigen-binding protein thereof comprises a single-chain variable fragment (scFv). (Item A7) The method according to any one of items A1 to A6, wherein the antibody or antigen-binding protein thereof comprises the amino acid sequence of SEQ ID NO:2. (Item A8) The method according to any one of paragraphs A1 to A7, wherein the therapeutic protein comprises a hydrolase. (Item A9) The method according to any one of paragraphs A1 to A8, wherein the therapeutic protein comprises a glycosylase. (Item A10) The method according to any one of paragraphs A1 to A9, wherein the therapeutic protein comprises a glycosidase. (Item A11) The method of any one of paragraphs A1 to 10, wherein the therapeutic protein comprises alpha-glucosidase. (Item A12) The method according to any one of items A1 to 11, wherein the therapeutic protein comprises the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 13, or a fragment thereof. (Item A13) The method according to any one of items A1 to A12, wherein the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 11. (Item A14) The method of any one of paragraphs A1-13, wherein the enzyme domain comprises alpha-glucosidase and glycogen levels in any CNS tissue of the subject are reduced for at least nine months after treatment. (Item A15) The method according to any one of items A1 to 14, wherein the subject has Pompe disease. (Item A16) The method of any one of items A1 to 15, wherein the serum level is at least 2 μg / mL. (Item A17) The method of any one of paragraphs A1 to 16, wherein the antibody or antigen-binding portion thereof binds to the extracellular domain of CD63 or ITGA7. (Item A18) 1. A multidomain therapeutic protein comprising one or more delivery domains and an enzymatic domain, wherein one of the one or more delivery domains binds to the human transferrin receptor (hTfR) and another of the one or more delivery domains comprises an antibody or antigen binding protein thereof that binds to CD63 or ITGA7. (Item A19) The multidomain therapeutic protein according to item A18, wherein each of the one or more delivery domains comprises an antigen-binding protein. (Item A20) The multidomain therapeutic protein according to item A18 or item A19, wherein at least one of the one or more delivery domains comprises a single-chain variable fragment (scFv). (Item A21) The multidomain therapeutic protein of any one of paragraphs A18 to A20, wherein at least one of the one or more delivery domains comprises a half antibody. (Item A22) The multidomain therapeutic protein of item A21, wherein the delivery domain that binds to hTfR is an scFv, the half antibody binds to CD63, and the enzymatic domain is GAA, wherein GAA is conjugated to the carboxy terminus of the half antibody that binds CD63. (Item A23) The multidomain therapeutic protein according to item A22, wherein each of the one or more delivery domains comprises an scFv. (Item A24) The multidomain therapeutic protein according to any one of paragraphs A20 to A23, wherein at least one scFv is fused to an Fc. (Item A25) The multidomain therapeutic protein according to item A24, wherein the Fc comprises a wild-type human IgG4 isotype, or a derivative thereof. (Item A26) The multidomain therapeutic protein according to any one of paragraphs A24-25, wherein GAA is conjugated to the carboxy terminus of Fc. (Item A27) The multidomain therapeutic protein according to item A23, comprising an anti-hTfR scFv, an anti-hCD63 scFv. (Item A28) The multidomain therapeutic protein according to item A27, wherein the anti-hTfR scFv and anti-hCD63 scFv are both linked at their carboxy termini to a single GAA enzyme. (Item A29) The multidomain therapeutic protein according to any one of items A18 to A28, wherein the delivery domain is an anti-hTfR scFv and the enzymatic domain is linked to the carboxy terminus of the VL domain of the scFv. (Item A30) The multidomain therapeutic protein according to item A29, comprising another delivery domain linked to the N-terminus of the VH domain of said anti-hTfR scFv. (Item A31) The multidomain therapeutic protein according to item A30, wherein the second delivery domain is an anti-hCD63 scFv. (Item A32) The multidomain therapeutic protein according to any one of items A18 to A31, wherein the enzyme domain comprises the amino acid sequence set forth as SEQ ID NO:1. (Item A33) 1. A multidomain therapeutic protein comprising at least two delivery domains and at least one enzymatic domain, wherein each of the two delivery domains is independently selected from the group consisting of an antibody, a half antibody, and an scFv, and wherein at least one or more of the delivery domains is associated with the at least one enzymatic domain, preferably wherein the one or more delivery domains are covalently linked to the at least one enzymatic domain. (Item A34) The multidomain therapeutic protein according to item A33, comprising the structure shown in Figure 1C, Figure 1D, Figure 1E, or Figure 1F. (Item A35) A polynucleotide encoding the multidomain therapeutic protein according to any one of items A18 to A34. (Item A36) The polynucleotide of item A35, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence. (Item A37) The polynucleotide of item A35 or item A36, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence. (Item A38) The polynucleotide of any one of paragraphs A35 to A37, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence, and the AAV nucleic acid sequence comprises an internal terminal repeat sequence and, optionally, a tissue-specific regulatory element, such as a liver-specific promoter or a neuron-specific promoter. (Item A39) The polynucleotide of any one of paragraphs A35 to A38, further comprising a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence comprising an internal long terminal repeat sequence comprising SEQ ID NO: 6, SEQ ID NO: 7, or both, and optionally a tissue-specific regulatory element, such as a liver-specific promoter or a neuron-specific promoter. (Item A40) The polynucleotide according to any one of items A35 to A39, further comprising a tissue-specific regulatory element comprising the sequence set forth as SEQ ID NO: 8 and / or SEQ ID NO: 9. (Item A41) A gene therapy vector comprising the polynucleotide according to any one of items A35 to A40. (Item A42) the gene therapy vector a viral vector, optionally a naturally occurring virus, an engineered virus, or a chimeric virus; A naked polynucleotide comprising the polynucleotide according to any one of items A35 to A40. A polynucleotide complex, which is optionally a lipid nanoparticle comprising a polynucleotide according to any one of paragraphs A34 to A39 and a lipid; and The gene therapy vector according to item A41, selected from the group consisting of: any combination thereof. (Item A43) The gene therapy vector of item A41 or item A42, wherein the gene therapy vector is a viral vector selected from the group consisting of a retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus. (Item A44) The gene therapy vector according to item A42 or item A43, wherein the gene therapy vector is AAV9, Anc80, AAV2 / 8 chimera and / or AAV pseudotyped for a specific tissue, such as liver or neuronal tissue. (Item A45) Use of a nucleotide encoding a multidomain therapeutic protein according to any one of items A18 to A34, a polynucleotide according to any one of items A35 to A40, or a gene therapy vector according to any one of items A41 to A44 in the method according to any one of items A1 to A17.

Claims

1. 1. A composition for delivering a therapeutic protein to the central nervous system (CNS) of a subject in need thereof, comprising a polynucleotide encoding a multidomain therapeutic protein comprising a first delivery domain, a second delivery domain, and an enzymatic domain, the polynucleotide is operably linked to a TTR promoter; the first delivery domain binds to the human transferrin receptor (hTfR) and is an anti-hTfR scFv, and the enzymatic domain is covalently or non-covalently linked to the carboxy terminus of the VL domain of the anti-hTfR scFv; the second delivery domain comprises an antibody or antigen-binding fragment thereof that binds to CD63; the enzyme domain comprises alpha-glucosidase (GAA) or an enzyme containing GAA activity; wherein the composition is administered to the subject via a viral vector and a liver-targeted delivery method sufficient to provide a therapeutically effective amount of the multidomain therapeutic protein to the CNS of the subject, thereby reducing glycogen accumulation in the CNS of the subject.

2. The composition of claim 1 , wherein the second delivery domain comprises a half antibody.

3. The composition of claim 1 , wherein the second delivery domain comprises an scFv.

4. The composition of claim 3 , wherein at least one scFv is fused to an Fc.

5. The composition of claim 4, wherein the Fc comprises a wild-type human IgG4 isotype, or a derivative thereof.

6. The composition of any one of claims 3 to 5, wherein the multidomain therapeutic protein comprises the anti-hTfR scFv and an anti-human CD63 (anti-hCD63) scFv.

7. 7. The composition of claim 6, wherein the anti-hTfR scFv and the anti-hCD63 scFv are both linked at their carboxy termini to a single GAA enzyme.

8. The composition of any one of claims 1 to 7, wherein the second delivery domain is linked to the N-terminus of the VH domain of the anti-hTfR scFv.

9. The composition of any one of claims 1 to 8, wherein the enzyme domain comprises the amino acid sequence set forth as SEQ ID NO:

1.

10. The composition of any one of claims 1 to 9, wherein the multidomain therapeutic protein comprises two delivery domains.

11. The composition of any one of claims 1 to 6 and 8 to 10, wherein only the first delivery domain is covalently or non-covalently bound to the enzyme domain.

12. The composition of any one of claims 1 to 10, wherein each of the first and second delivery domains is covalently linked to the enzyme domain.

13. The composition described in claim 10, wherein the first delivery domain and the second delivery domain each comprise an scFv.

14. The composition of claim 13 , wherein both the first and second scFvs are covalently linked to the enzymatic domain.

15. 14. The composition of claim 13, wherein the multidomain therapeutic protein comprises, from N-terminus to C-terminus: the first scFv, the second scFv, and the enzymatic domain.

16. The composition of any one of claims 1 to 15, wherein the polynucleotide further comprises a viral nucleic acid sequence and a locus-targeting nucleic acid sequence.

17. 17. The composition of claim 16, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence, and the AAV nucleic acid sequence comprises an internal terminal repeat sequence.

18. 16. The composition of any one of claims 1 to 15, wherein the polynucleotide further comprises a viral nucleic acid sequence and a locus-targeting nucleic acid sequence, wherein the viral nucleic acid sequence is an adeno-associated virus (AAV) nucleic acid sequence comprising an internal terminal repeat sequence comprising SEQ ID NO:6, SEQ ID NO:7, or both.

19. The composition of any one of claims 1 to 15, wherein the polynucleotide further comprises a tissue-specific regulatory element comprising the sequence set forth as SEQ ID NO:8 and / or SEQ ID NO:

9.

20. The composition of any one of claims 1 to 19, wherein the viral vector is a naturally occurring virus, an engineered virus, or a chimeric virus.

21. 21. The composition of claim 20, wherein the viral vector is selected from the group consisting of a retrovirus, an adenovirus, a herpes simplex virus, a poxvirus, a vaccinia virus, a lentivirus, and an adeno-associated virus.

22. 22. The composition of claim 20 or 21, wherein the viral vector is AAV9, Anc80, AAV2 / 8 chimera and / or AAV pseudotyped for specific tissues.

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