Variant IGF2 constructs

Abstract

Novel IGF2 peptides, fusion proteins, and nucleotide sequences encoding the novel IGF2 peptides and fusion proteins are provided for the treatment of lysosomal storage disorders.SOLUTION: A nucleic acid construct encoding a fusion protein is provided comprising (a) a nucleic acid sequence encoding a therapeutic protein and (b) a nucleic acid sequence encoding a variant IGF2 (vIGF2) peptide that is at least 95% identical to at least one sequence selected from a plurality of specific sequences. The constructs provided are useful for treating lysosomal storage disorders by both enzyme replacement therapy and gene therapy.SELECTED DRAWING: Figure 48

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 913,677, filed October 10, 2019, and U.S. Provisional Patent Application No. 62 / 929,054, filed October 31, 2019, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Genetic disorders arise through inherited or de novo mutations occurring within gene-coding regions of the genome. In some cases, such genetic disorders are treated by administering a protein that replaces the protein encoded by the mutated gene in an individual with the genetic disorder, or by administering a gene therapy vector encoding such a protein. However, such treatments present challenges because the administered protein or the protein encoded by the gene therapy vector does not always reach the organ, cell, or organelle where the protein is needed. Proteins with improved intracellular targeting (e.g., to lysosomes), and gene therapy vectors encoding same, are desired. Summary of the Invention [Means for solving the problem]

[0003] In certain embodiments, nucleic acid constructs are provided that include: (a) a nucleic acid sequence encoding a therapeutic protein; and (b) a nucleic acid sequence encoding a variant IGF2 (vIGF2) peptide. In some embodiments, the vIGF2 peptide has an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to an IGF2 variant peptide in Table 3. In some embodiments, the vIGF2 peptide comprises an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to an IGF2 variant peptide selected from the group consisting of SEQ ID NOs: 90-123 in Table 3. In some embodiments, the vIGF2 peptide further comprises a linker having a sequence at least 90, 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 181-188. In some embodiments, the vIGF2 peptide has reduced or no affinity for the insulin receptor and IGFR1 compared to a native IGF2 peptide. In some embodiments, the vIGF2 peptide has increased affinity for CI-MPR compared to native IGF2 peptide. In some embodiments, the vIGF2 peptide confers improved expression and / or secretion of the fusion protein compared to native IGF2 peptide. In some embodiments, the vIGF2 peptide can promote uptake of a therapeutic protein into lysosomes within a cell. In some embodiments, the therapeutic protein can replace a defective or deficient protein associated with a genetic disorder in a subject with the genetic disorder. In some embodiments, the genetic disorder is a lysosomal storage disorder.In some embodiments, the genetic disorder is aspartylglucosaminuria, CLN1, CLN2 C-cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase-induced severe combined immunodeficiency (ADA-SCID), chronic granulomatous disease (CGD), and neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is Pompe disease. In some embodiments, the genetic disorder is neuronal ceroid lipofuscinosis.In some embodiments, the therapeutic protein is selected from the group consisting of alpha-galactosidase (A or B), beta-galactosidase, beta-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparan N-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatosulfatase, The therapeutic protein comprises an enzyme selected from the group consisting of N-acetylgalactosamine-6-sulfatase, N-sulfoglucosamine sulfohydrolase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase, alpha-N-acetylneuraminidase (sialidase), ganglioside sialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase, beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase, battenin, protein palmitoylthioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or an enzymatically active fragment thereof. In some embodiments, the therapeutic protein is alpha-glucosidase or an enzymatically active fragment thereof. In some embodiments, the therapeutic protein is protein palmitoyl thioesterase 1 (PPT1). In some embodiments, the therapeutic protein is tripeptidyl peptidase 1 (TPP1). In some embodiments, the therapeutic protein is aspartyl glucosaminidase. In some embodiments, the therapeutic protein is NAGLU (SEQ ID NO: 54).In some embodiments, the Therapeutic protein is the mature peptide of NAGLU, corresponding to amino acids 24-743 of SEQ ID NO:54, remaining after removal of the native signal peptide (SEQ ID NO:180). In some embodiments, the nucleic acid construct further comprises a translation initiation sequence. In some embodiments, the translation initiation sequence comprises a Kozak sequence. In some embodiments, the vIGF2-encoding nucleic acid sequence is 5' to the nucleic acid sequence encoding the Therapeutic protein. In some embodiments, the vIGF2-encoding nucleic acid sequence is 3' to the nucleic acid sequence encoding the Therapeutic protein. In some embodiments, the nucleic acid construct further comprises a linker sequence encoding a linker peptide between the vIGF2 nucleotide sequence and the nucleic acid sequence encoding the Therapeutic protein. In some embodiments, the linker peptide comprises SEQ ID NOs:181-188. In some embodiments, the nucleic acid construct is a viral vector. In some embodiments, the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, a poxviral vector, a vaccinia viral vector, an adenoviral vector, or a herpes viral vector.

[0004] In a further aspect, provided is a pharmaceutical composition comprising a therapeutically effective amount of any one of the nucleic acid constructs provided herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

[0005] In a further aspect, methods of treating a genetic disorder are provided, comprising administering any one of the nucleic acid constructs provided herein or any one of the pharmaceutical compositions provided herein to a subject in need of treatment for the genetic disorder. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is aspartylglucosaminuria, Batten disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Sanfilippo disease type E, Sanfilippo disease type F, Sanfilippo disease type I, Sanfilippo disease type F, Sanfilippo disease type I, Sanfilippo disease type II, Sanfilippo disease type III, Sanfilippo disease type IV, Sanfilippo disease type F, Sanfilippo disease type I, Sanfilippo disease type II, Sanfilippo disease type III, Sanfilippo disease type IV, Sanfilippo disease type I, Sanfilippo disease type III ... In some embodiments, the genetic disorder is selected from the group consisting of Pompe disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase-associated severe combined immunodeficiency (ADA-SCID), chronic granulomatous disease (CGD), and neuronal ceroid lipofuscinosis (Batten disease). In some embodiments, the genetic disorder is Pompe disease. In some embodiments, the genetic disorder is neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is aspartylglucosaminuria. In some embodiments, the administration is intrathecal, intraocular, intravitreal, retinal, intravenous, intramuscular, intraventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneous, or a combination thereof. In some embodiments, administration is performed intrathecally.

[0006] In a further aspect, provided is a pharmaceutical composition comprising any one of the gene therapy vectors provided herein and a pharmaceutically acceptable carrier or excipient for use in treating a genetic disorder. In a further aspect, provided is a pharmaceutical composition comprising any one of the nucleic acid constructs provided herein and a pharmaceutically acceptable carrier or excipient for use in the preparation of a medicament for the treatment of a genetic disorder. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is aspartylglucosaminuria, Batten disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type B, or Sanfilippo disease type C. The genetic disorder is selected from the group consisting of Filippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase-induced severe combined immunodeficiency (ADA-SCID), chronic granulomatous disease (CGD), and neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is Pompe disease. In some embodiments, the genetic disorder is neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is aspartylglucosaminuria. In some embodiments, the composition is formulated for intrathecal, intraocular, intravitreal, retinal, intravenous, intramuscular, intracerebroventricular, intracerebral, intracerebellar, or subcutaneous administration. In some embodiments, the composition is formulated for intrathecal administration.

[0007] In a further aspect, a nucleic acid is provided that encodes a fusion protein having an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 47 to 53. In some embodiments, the nucleic acid is at least 85, 90, 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 60 to 67.

[0008] In a further aspect, there is provided a pharmaceutical composition comprising any one of the above nucleic acids and a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

[0009] In a further aspect, a pharmaceutical composition comprising a fusion protein having an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 47-53 and 60-67, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

[0010] In a further aspect, a gene therapy vector is provided, comprising a nucleic acid encoding an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 47-53 and 60-67; and a nucleic acid encoding an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 106, 109, 111, 119, 120, and 121. In some embodiments, the gene therapy vector is a viral vector. In some embodiments, the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, a poxviral vector, a vaccinia viral vector, an adenoviral vector, or a herpesviral vector, and a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

[0011] In a further aspect, there is provided a method of treating CLN1 / PPT1 disease or CLN2 / TPP1 disease, comprising administering to a subject in need of treatment any one of the nucleic acids described herein, any one of the fusion proteins described herein, any one of the gene therapy vectors described herein, or any one of the pharmaceutical compositions described herein. In some embodiments, administration is performed intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, or a combination thereof.

[0012] In some embodiments, the nucleic acid has a nucleic acid sequence that is at least 85, 90, 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 189-250.

[0013] In a further aspect, there is provided a pharmaceutical composition comprising any one of the nucleic acids herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

[0014] In some embodiments, variant IGF2 (vIGF2) peptides are provided that are at least 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 90-103.

[0015] In some embodiments, the variant IGF2 (vIGF2) peptide is at least 98% identical to at least one sequence selected from SEQ ID NOs: 106, 109, 111, 119, 120, 121. In some embodiments, the vIGF2 peptide is at least 95, 96, 97, 98, or 99% identical to SEQ ID NO: 120 or 121.

[0016] In some embodiments, a fusion protein is provided that includes a variant vIGF2 peptide and a therapeutic protein having an amino acid sequence at least 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NO:4, amino acid residues 21-306 of SEQ ID NO:4, amino acid residues 28-306 of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:46, and SEQ ID NO:54.

[0017] In some embodiments, the fusion protein has an amino acid sequence at least 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 60-67, 47-53, and 54-59. In some embodiments, the fusion protein further comprises a lysosomally cleaving peptide. In some embodiments, the lysosomally cleaving peptide has SEQ ID NO: 188. In some embodiments, the vIGF2 peptide is at the N-terminus of the therapeutic protein. In some embodiments, the vIGF2 peptide is at the C-terminus of the therapeutic protein.

[0018] In some embodiments, the fusion protein comprises a signal sequence, hi some embodiments, the signal sequence has an amino acid sequence at least 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 169-180.

[0019] In some embodiments, the therapeutic protein is PPT1 or an enzymatically active fragment thereof, TPP1 or an enzymatically active fragment thereof, or NAGLU or an enzymatically active fragment thereof.

[0020] In some embodiments, the fusion protein is taken up by target cells more efficiently than the corresponding protein lacking the vIGF2 peptide. In some embodiments, the fusion protein is taken up by cells in the brain. In some embodiments, the fusion protein is taken up by neuronal cells. In some embodiments, the fusion protein is taken up by glial cells.

[0021] Also provided herein is a pharmaceutical composition comprising a fusion protein having a vIGF2 peptide and a therapeutic protein together with a pharmaceutically acceptable carrier or excipient. Also provided herein is a method for treating a lysosomal storage disorder, comprising administering such a pharmaceutical composition to a subject in need of treatment for the lysosomal storage disorder. In some embodiments, the lysosomal storage disorder is selected from the group consisting of CLN1 / PPT1 disease, CLN2 / TPP1 disease, and Sanfilippo type B disease. In some embodiments, the fusion protein or a pharmaceutical composition comprising the fusion protein is administered intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, or a combination thereof.

[0022] In some embodiments, administration of the pharmaceutical composition prevents / reduces or reverses accumulation of autofluorescent material (ASM) in the brain. In some embodiments, administration of the pharmaceutical composition prevents / reduces or reverses elevation of glial fibrillary acidic protein (GFAP) in the brain. In some embodiments, administration of the pharmaceutical composition prevents / reduces or reverses accumulation of autofluorescent material (ASM) in the cortex or thalamus. In some embodiments, administration of the pharmaceutical composition prevents / reduces or reverses elevation of glial fibrillary acidic protein (GFAP) in the cerebral cortex or thalamus.

[0023] Further provided herein are nucleic acids encoding a fusion protein comprising vIGF2 and a therapeutic protein, wherein the nucleic acid is at least 85, 90, 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 189-250.

[0024] In a further aspect, there is provided a pharmaceutical composition comprising any one of the fusion proteins herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

[0025] In a further aspect, a gene therapy vector is provided comprising a nucleic acid encoding an amino acid sequence at least 90% identical to SEQ ID NO: 51. In some embodiments, the gene therapy vector is a viral vector. In some embodiments, the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, a poxviral vector, a vaccinia viral vector, an adenoviral vector, or a herpes viral vector.

[0026] In a further aspect, provided is a pharmaceutical composition comprising any one of the gene therapy vectors provided herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

[0027] In another aspect, a nucleic acid construct is provided that includes: (a) a nucleic acid sequence encoding a therapeutic protein, and (b) a nucleic acid sequence encoding a variant IGF2 (vIGF2) peptide that is at least 95, 96, 97, 98, or 99% identical to at least one sequence selected from SEQ ID NOs: 90-103. In some aspects, the vIGF2 peptide has an amino acid sequence that is at least 95, 96, 97, 98, or 99% identical to an IGF2 variant peptide selected from SEQ ID NOs: 106, 109, 111, 119, 120, and 121. In some embodiments, the vIGF2 peptide comprises an amino acid sequence that is at least 95, 96, 97, 98, or 99% identical to an IGF2 variant peptide selected from the group consisting of SEQ ID NO: 120 and SEQ ID NO: 121.

[0028] In some embodiments, the nucleic acid further comprises a sequence encoding a linker having a sequence at least 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 181-188. In some embodiments, the vIGF2 peptide can increase expression and / or secretion of a therapeutic protein compared to a vIGF2 peptide having the amino acid sequence of SEQ ID NO: 80. In some embodiments, the vIGF2 peptide has increased affinity for CI-MPR compared to a vIGF2 peptide having the amino acid sequence of SEQ ID NO: 80. In some embodiments, the vIGF2 peptide can improve uptake of a therapeutic protein into target cells, such as human brain cells. In some embodiments, the human brain cells are neuronal cells or glial cells.

[0029] In certain aspects, the therapeutic protein can replace a defective or deficient protein associated with a genetic disorder in a subject with the genetic disorder. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is aspartylglucosaminuria, neuronal ceroid lipofuscinosis, CLN1 / PPT1 disease, CLN2 / PPT1 disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sarcopenia, and the like. In some embodiments, the genetic disorder is selected from the group consisting of Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase severe combined immunodeficiency (ADA-SCID), and neuronal ceroid lipofuscinosis. In some embodiments, the genetic disorder is selected from the group consisting of CLN1 / PPT1 disease, CLN2 / PPT1 disease, Pompe disease, and MPS IIIB disease. In some aspects, the genetic disorder is CLN1 / PPT1 disease or CLN2 / PPT1 disease.

[0030] In some embodiments, the therapeutic protein is selected from the group consisting of alpha-galactosidase (A or B), beta-galactosidase, beta-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparan N-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatase, In some embodiments, the therapeutic protein comprises a human enzyme selected from the group consisting of: N-acetylgalactosamine-6-sulfatase, N-sulfoglucosamine sulfohydrolase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase, alpha-N-acetylneuraminidase (sialidase), ganglioside sialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase, beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase, battenin, PPT1, TPP1, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or an enzymatically active fragment thereof. In some embodiments, the therapeutic protein is a human lysosomal enzyme or an enzymatically active fragment thereof. In some embodiments, the human lysosomal enzyme is alpha-glucosidase, PPT1, TPP1, or NAGLU.

[0031] In some embodiments, the nucleic acid construct further comprises a sequence encoding a signal peptide. In some embodiments, the signal peptide is a sequence selected from the group consisting of SEQ ID NOs: 169-180. In some embodiments, the vIGF2-encoding nucleic acid sequence is 5' to the nucleic acid sequence encoding the therapeutic protein. In other embodiments, the vIGF2-encoding nucleic acid sequence is 3' to the nucleic acid sequence encoding the therapeutic protein.

[0032] Also provided herein are gene therapy vectors comprising the nucleic acids described herein. In some embodiments, the gene therapy vector is a viral vector. In some embodiments, the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, a poxvirus vector, a vaccinia virus vector, an adenovirus vector, or a herpesvirus vector.

[0033] In some aspects, the nucleic acid constructs herein are in a plasmid or a bacterial artificial chromosome. In some embodiments, the nucleic acid constructs described herein are in a host cell.

[0034] Further provided are pharmaceutical compositions comprising a therapeutically effective amount of a nucleic acid construct described herein, or a gene therapy vector comprising a nucleic acid construct described herein, together with a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

[0035] Further provided herein are methods of treating a genetic disorder, comprising administering to a subject in need of treatment a nucleic acid construct, gene therapy vector, and / or pharmaceutical composition described herein. In some embodiments, the genetic disorder is a lysosomal storage disorder. In some embodiments, the genetic disorder is aspartylglucosaminuria, neuronal ceroid lipofuscinosis, CLN1 / PPT1 disease, CLN2 / PPT1 disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, or any of the following: In some embodiments, the genetic disorder is selected from the group consisting of: Sanfilippo disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase severe combined immunodeficiency (ADA-SCID), and chronic granulomatous disease (CGD). In some embodiments, the genetic disorder is Batten disease, e.g., CLN1 / PPT1 disease or CLN2 / TPP1 disease. In some embodiments, the genetic disorder is Pompe disease or Sanfilippo disease type B.

[0036] In some embodiments, administration is performed intrathecally, intraocularly, intravitreally, retinal, intravenous, intramuscular, intracerebroventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneously, or a combination thereof.

[0037] In some aspects, administration of the nucleic acid, gene therapy vector, fusion protein, or pharmaceutical composition prevents / reduces or reverses the accumulation of autofluorescent material (ASM) in the brain. In some embodiments, administration of the nucleic acid, gene therapy vector, fusion protein, or pharmaceutical composition prevents / reduces or reverses the elevation of glial fibrillary acidic protein (GFAP) in the brain. In some embodiments, administration of the nucleic acid, gene therapy vector, fusion protein, or pharmaceutical composition prevents / reduces or reverses the accumulation of autofluorescent material (ASM) in the cortex or thalamus. In some embodiments, administration of the nucleic acid, gene therapy vector, fusion protein, or pharmaceutical composition prevents / reduces or reverses the elevation of glial fibrillary acidic protein (GFAP) in the brain cortex or thalamus.

[0038] In some aspects, the nucleic acid encodes a fusion protein having a sequence at least 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 60-67. In some embodiments, the nucleic acid encodes a fusion protein having a sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 47-53.

[0039] In some aspects, the nucleic acid encodes a fusion protein comprising: (a) an amino acid sequence at least 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 106, 109, 111, 119, 120, and 121; and (b) an amino acid sequence at least 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NO: 4, residues 21-306 of SEQ ID NO: 4, residues 28-306 of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 46. In some embodiments, the nucleic acid encodes a vIGF2 that is at least 95, 96, 97, 98, or 99% identical to SEQ ID NOs: 120 and 121. In some embodiments, the nucleic acid encodes a fusion protein comprising: (a) at least one of SEQ ID NOs: 106, 109, 111, 119, 120, or 121; and (b) at least one of SEQ ID NO: 4, residues 21-306 of SEQ ID NO: 4, residues 28-306 of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 46, residues 28-306 of SEQ ID NO: 4, SEQ ID NO: 8, and SEQ ID NO: 46.

[0040] In some embodiments, the nucleic acid further encodes a lysosomal cleaving peptide.

[0041] In some aspects, the fusion protein has a sequence at least 95, 96, 97, 98, or 99% identical to at least one of SEQ ID NOs: 60-67 and SEQ ID NOs: 47-53. In some embodiments, the fusion protein comprises at least one of SEQ ID NOs: 60-67 and SEQ ID NOs: 47-53. In some embodiments, the fusion protein consists of or consists essentially of SEQ ID NOs: 60-67 and SEQ ID NOs: 47-53.

[0042] In a further aspect, a method of treating a lysosomal storage disease is provided, comprising administering to a subject in need of treatment for a lysosomal storage disease a therapeutically effective amount of any one of the nucleic acids herein, any one of the fusion proteins herein, any one of the gene therapy vectors herein, or any one of the pharmaceutical compositions herein. In some embodiments, administration is performed intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, or a combination thereof.

[0043] In a further aspect, there is provided a method of treating Batten disease, including CLN1 / PPT1 disease and CLN2 / TPP1 disease, comprising administering to a subject in need of treatment for Batten disease a therapeutically effective amount of any one of the nucleic acids herein, any one of the fusion proteins herein, any one of the gene therapy vectors herein, or any one of the pharmaceutical compositions herein. In some embodiments, administration is performed intrathecally, intraocularly, intravitreally, retina, intravenously, intramuscularly, intracerebroventricularly, intracerebrally, intracerebellarly, intraventricularly, intraparenchymal, subcutaneously, or a combination thereof.

[0044] In a further aspect, there is provided a pharmaceutical composition comprising any one of the nucleic acids provided herein and a pharmaceutically acceptable carrier or excipient. In some embodiments, the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

[0045] In a further aspect, provided is a pharmaceutical composition comprising any one of the gene therapy vectors provided herein and a pharmaceutically acceptable carrier or excipient.

[0046] In a further aspect, a fusion protein is provided comprising: (a) a lysosomal enzyme and (b) a variant IGF2 (vIGF2) peptide, wherein the vIGF2 peptide comprises an amino acid sequence at least 95, 96, 97, 98, or 99% identical to an IGF2 variant peptide in Table 3. In some embodiments, the vIGF2 peptide comprises an amino acid sequence at least 95, 96, 97, 98, or 99% identical to an IGF2 variant peptide selected from the group consisting of SEQ ID NOs: 69-131. In some embodiments, the vIGF2 peptide comprises an amino acid sequence at least 95, 96, 97, 98, or 99% identical to an IGF2 variant peptide selected from the group consisting of SEQ ID NOs: 90-123. In some embodiments, the vIGF2 is modified to replace residues 31-38 of wild-type IGF2 with four glycine residues (Δ31-38GGGG). In some embodiments, the vIGF2 is further modified by a V43L mutation. In some embodiments, vIGF2 is further modified to replace the serine at position 50 with an acidic residue (aspartic acid or glutamic acid).

[0047] In some embodiments, the vIGF2 peptide further comprises a linker having a sequence at least 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 181-188. In some embodiments, the linker is cleavable. In some embodiments, the vIGF2 peptide has reduced or no affinity for insulin receptor and IGFR1 compared to native IGF2 peptides. In some embodiments, the vIGF2 peptide has increased affinity for CI-MPR compared to native IGF2 peptides. In some embodiments, the vIGF2 peptide can promote the uptake of lysosomal enzymes into lysosomes in cells. In some embodiments, the lysosomal enzymes can replace defective or deficient proteins associated with lysosomal storage disorders. In some embodiments, the lysosomal storage disorder is aspartylglucosaminuria, Batten disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, The lysosomal storage disorder is selected from the group consisting of Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase severe combined immunodeficiency (ADA-SCID), chronic granulomatous disease (CGD), and neuronal ceroid lipofuscinosis. In some embodiments, the lysosomal storage disorder is Pompe disease. In some embodiments, the lysosomal storage disorder is neuronal ceroid lipofuscinosis.In some embodiments, the lysosomal enzyme is selected from the group consisting of alpha-galactosidase (A or B), beta-galactosidase, beta-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparan N-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatosulfatases, and galactosamine-6-sulfatosulfatases. The lysosomal enzyme may comprise an enzyme selected from the group consisting of lysosomal enzymes such as alpha-glucosamine sulfohydrolase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase, alpha-N-acetylneuraminidase (sialidase), ganglioside sialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase, beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase, battenin, protein palmitoyl thioesterase, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or enzymatically active fragments thereof. In some embodiments, the lysosomal enzyme is alpha-glucosidase or an enzymatically active fragment thereof. In some embodiments, the lysosomal enzyme is protein palmitoyl thioesterase. In some embodiments, the lysosomal enzyme is tripeptidyl peptidase 1. In some embodiments, the lysosomal enzyme is aspartyl glucosaminidase.

[0048] Additionally, provided herein is a pharmaceutical composition comprising a therapeutically effective amount of any one of the fusion proteins provided herein and a pharmaceutically acceptable carrier or excipient.

[0049] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0050] The patent application file contains at least one color drawing. Copies of this patent application with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. An understanding of the features and advantages of the present disclosure will be gained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the present disclosure are utilized, and the accompanying drawings, in which: [Brief explanation of the drawings]

[0051] [Figure 1] Figure 1 shows that affinity chromatography using immobilized CI-MPR was used to determine the proportion of GAA that can interact with CI-MPR via phosphorylated oligosaccharides. The first peak represents material that flows through the column and does not contain phosphorylated glycans. The later peak represents material that can bind to the immobilized CI-MPR. This material is eluted with an increasing gradient of M6P. M6P reveals that GAA contains both M6P-containing and M6P-depleted fractions. Because binding to CI-MPR is the essential first step for receptor-mediated endocytosis, only the rhGAA fraction that binds to CI-MPR is capable of effective cellular uptake. [Figure 2] FIG. 2 shows the structure of the CI-MPR, which contains different binding domains for IGF2 and for mono- and bis-phosphorylated oligosaccharides. [Figure 3] Figure 3 shows the sequence and structure of the mature human IGF2 peptide. Site-specific amino acid substitutions are proposed to affect binding to other receptors and serum proteins. [Figure 4] FIG. 4 shows binding of wild-type IGF2 (wtIGF2) peptide to CI-MPR as measured by surface plasmon resonance. [Figure 5] FIG. 5 shows binding of variant IGF2 (vIGF2) peptide to CI-MPR as measured by surface plasmon resonance. [Figure 6] FIG. 6 shows the benefit of adding vIGF2 to alglucosidase alfa to increase binding to the IGF2 / CI-MPR. [Figure 7] FIG. 7 shows the benefit of adding vIGF2 to recombinant human N-acetyl-α-D-glucosaminidase (rhNAGLU) to increase binding to the IGF2 / CI-MPR. [Figure 8] FIG. 8 shows the binding of wild-type human IGF2 to the insulin receptor. [Figure 9] FIG. 9 shows that there is no detectable binding of vIGF2 to the insulin receptor. [Figure 10] FIG. 10 shows the binding of wild-type IGF2 to the insulin-like growth factor 1 receptor. [Figure 11] FIG. 11 shows reduced binding of the vIGF2 peptide to the insulin-like growth factor 1 receptor compared to wild-type IGF2. [Figure 12] Figure 12 shows two examples of gene therapy expression cassettes encoding native and engineered hGAA. Native hGAA is not sufficiently phosphorylated and cannot efficiently bind to the CI-MPR. Engineered hGAA contains added elements for improved CIMPR binding (vIGF2), incorporates a 2GS linker to allow greater interaction of the vIGF2-GAA protein with the CI-MPR, and includes a BiP signal peptide to enhance secretion. [Figure 13] Figure 13 shows a Western blot of PPT1 from cells expressing recombinant human protein palmitoyl thioesterase 1 (PPT1) (PPT1-1), recombinant human PPT1 with the vIGF2 targeting domain (PPT1-2), and recombinant human PPT1 with the vIGF2 targeting domain and BiP signal sequence (PPT1-29). Protein expression can be affected by the IGF variant used. [Figure 14] FIG. 14 shows binding of PPT1 constructs to the CI-MPR. [Figure 15] FIG. 15 shows GAA activity in conditioned medium of CHO cells expressing engineered or native hGAA. [Figure 16] FIG. 16 shows the study design for a 4-week mouse study of gene therapy in GAA knockout mice. [Figure 17] FIG. 17 shows GAA plasma activity in untreated wild-type ("normal") mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 18] FIG. 18 shows GAA levels measured in untreated wild-type ("normal") mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 19] FIG. 19 shows cell surface receptor CI-MPR binding of rhGAA from plasma samples obtained from mice treated as indicated. [Figure 20] Figure 20 shows GAA activity and quad glycogen histopathology scores for the tibialis anterior muscle of untreated wild-type ("normal") mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 21] FIG. 21 shows glycogen PAS in tibialis anterior muscles from untreated wild-type mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 22] FIG. 22 shows hGAA immunohistochemistry of tibialis anterior muscles from untreated wild-type mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 23]Figure 23 shows brain GAA activity, brain glycogen, and spinal cord glycogen histopathology scoring for brains and spinal cords from untreated wild-type ("normal") mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 24] FIG. 24 shows the glycogen PAS of brains from untreated wild-type mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 25] FIG. 25 shows hGAA immunohistochemistry of brainstem and choroid plexus from untreated wild-type ("normal") mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 26] FIG. 26 shows glycogen PAS in spinal cords from untreated wild-type mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 27] FIG. 27 shows hGAA immunohistochemistry of spinal cords from untreated wild-type mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 28] Figure 28 shows GAA activity and glycogen histopathology scoring of quadriceps muscles from untreated wild-type ("normal") mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 29] FIG. 29 shows glycogen luxol / PAS for quadriceps muscles from untreated wild-type mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 30] FIG. 30 shows hGAA immunohistochemistry of quadriceps muscles from untreated wild-type mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 31]FIG. 31 shows GAA activity and histopathology scoring in the triceps muscle for untreated wild-type ("normal") mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 32] FIG. 32 shows glycogen luxol / PAS in triceps muscles from untreated wild-type mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 33] FIG. 33 shows hGAA immunohistochemistry of triceps muscles from untreated wild-type mice or GAA knockout mice treated with gene therapy vector or vehicle as indicated. [Figure 34] FIG. 34 shows engineered and wild-type PPT1 bound to CIMPR. [Figure 35] FIG. 35 shows engineered and wild-type TPP1 bound to CIMPR. [Figure 36] FIG. 36 shows engineered and wild-type AGA bound to CIMPR. [Figure 37] FIG. 37 shows engineered and wild-type GLA bound to CIMPR. [Figure 38] FIG. 38 shows a Western blot of GAA from cells expressing various mutant vIGF2-GAA constructs in conditioned medium. [Figure 39] FIG. 39 shows the secretion of the new IGF2-GAA variants compared to construct vIGF2-GAA in the Western blot of FIG. [Figure 40] FIG. 40 shows CI-MPR binding of various vIGF2-GAA constructs. [Figure 41] FIG. 41 shows the Bmax and Kd values ​​for CIMPR binding of various vIGF2-GAA constructs. [Figure 42] FIG. 42 shows CI-MPR binding of various vIGF2-GAA constructs. [Figure 43] FIG. 43 shows the Bmax and Kd values ​​for CIMPR binding of various vIGF2-GAA constructs. [Figure 44] FIG. 44 shows CI-MPR binding of various vIGF2-GAA constructs. [Figure 45] FIG. 45 shows the Bmax and Kd values ​​for CIMPR binding of various vIGF2-GAA constructs. [Figure 46] FIG. 46 shows the cellular uptake for various vIGF2-GAA constructs. [Figure 47] FIG. 47 shows the cellular uptake for various vIGF2-GAA constructs. [Figure 48] FIG. 48 shows various vIGF2 peptides binding to the CI-MPR or IGF2R. [Figure 49] FIG. 49 shows PPT1 in conditioned medium as quantified by Western blot. [Figure 50] FIG. 50 shows PPT1 in conditioned medium as quantified by Western blot. [Figure 51] FIG. 51 shows PPT1 in conditioned medium, quantified by activity. [Figure 52] FIG. 52 shows the correlation between PPT1 Western blot quantification and activity quantification. [Figure 53] FIG. 53 shows binding of PPT1 constructs to the CI-MPR. [Figure 54] Figure 54 shows structural diagrams of selected PPT1 constructs. [Figure 55] FIG. 55 shows a Western blot of PPT1 secreted into conditioned medium. [Figure 56] FIG. 56 shows the processing of PPT1 in cells by Western blot. [Figure 57] FIG. 57 shows PPT1 in conditioned medium as quantified by Western blot. [Figure 58] FIG. 58 shows the relative PPT1 activity. [Figure 59] Figure 59 shows binding of PPT1 constructs to the CI-MPR. [Figure 60]Figure 60 shows binding of PPT1 constructs to the CI-MPR. [Figure 61] Figure 61 shows the alignment of IGF2-GAA variants (1: vIGF2; 2: vIGF2-17; 3: and 4: IGF2-22). [Figure 62] FIG. 62 shows additional PPT1 constructs. [Figure 63] Figure 63(A) shows the expression of PPT1 constructs normalized to wild-type, untagged PPT1 (construct 100) as measured by Western blot band intensity. The average intensity for four replicate transfections is shown for each sample with standard deviation error bars. (B) shows PPT1 expression / secretion in the medium normalized to wild-type as measured by Western blot band intensity. [Figure 64] Figure 64 shows the uptake of PPT1 constructs into rat cerebral cortical neurons as measured by immunofluorescence. (A) shows the neuronal uptake of purified PPT1-101 and PPT1-104. (B) shows the neuronal uptake of culture medium-derived PPT-1 constructs (unpurified). [Figure 65] Figure 65 shows further NAGLU constructs. [Figure 67] Figure 67(A) shows expression of NAGLU constructs normalized to wild-type, untagged PPT1 (construct 100) as measured by Western blot band intensity. The average intensity for four replicate transfections is shown per sample with standard deviation error bars. (B) shows PPT1 expression / secretion in the medium normalized to wild-type as measured by Western blot band intensity. [Figure 68] Figure 68 shows the expression of TPP1 constructs normalized to wild-type untagged TPP1 as measured by band intensity on a Western blot. [Figure 69] Figure 69 shows CIMPR binding of TPP1 constructs. [Figure 70] Figure 70 shows human CLN1 transgene expression detected by RT-qPCR. [Figure 71] Figures 71-72 show brain autofluorescent material (ASM) accumulation, a correlate of lysosomal dysfunction. [Figure 72] Figures 71-72 show brain autofluorescent material (ASM) accumulation, a correlate of lysosomal dysfunction. [Figure 73] FIG. 73 shows glial fibrillary acidic protein (GFAP), a correlate of astrogliosis and neuroinflammation. DETAILED DESCRIPTION OF THE INVENTION

[0052] Provided herein are novel, engineered IGF2 peptides with enhanced properties, including enhanced expression, secretion, and CIMPR binding. Also provided herein are fusion proteins comprising novel IGF2 peptides and lysosomal enzymes, and nucleic acids encoding the fusion proteins, with enhanced properties, such as increased CIMPR binding and improved expression and secretion. The fusion proteins and nucleic acid constructs provided herein are useful for both enzyme replacement therapy and gene therapy to treat lysosomal storage disorders.

[0053] Gene therapy for single-gene genetic disorders offers a potentially one-time treatment for diseases and disorders with devastating symptoms, some of which appear early in life and can sometimes lead to lifelong disability. Genetic disorders, such as neurological disorders or lysosomal storage disorders, are often treated with enzyme replacement therapy, in which patients are administered a therapeutic protein that is an active form of a protein that is defective or deficient in the disease or disorder state. However, current treatments have challenges, including frequent treatments, the development of immune responses to the therapeutic protein, and difficulty targeting the therapeutic protein to affected tissues, cells, or subcellular compartments. Gene therapy offers advantages, including reduced treatment frequency and long-term efficacy.

[0054] Provided herein are fusion proteins encoded by vectors for administration as enzyme replacement therapy or for use as gene therapy vectors that provide improvements to enzyme replacement therapy or gene therapy, e.g., improving treatment efficacy by providing a more therapeutic protein when needed. Such challenges are addressed herein by improving the expression and cellular uptake, or delivery and intracellular or subcellular targeting of therapeutic proteins. Specific tools or components provided herein include, but are not limited to, signal peptides that increase secretion (e.g., binding immunoglobulin protein (BiP) and Gaussia signal peptides) and peptides that increase endocytosis of therapeutic proteins (e.g., peptides that bind to the CI-MPR with high affinity, enhancing cellular uptake and lysosomal delivery). Such peptides are fused to a therapeutic protein encoded by a gene therapy vector. In some embodiments, the peptide is an IGF2 (insulin-like growth factor 2) peptide or a variant thereof. It is contemplated that the gene therapy vectors provided herein, in some embodiments, comprise a nucleic acid encoding a therapeutic protein fused to a peptide that binds to the CI-MPR with high affinity, optimizing the efficacy of gene therapy.

[0055] A gene therapy construct for enzyme replacement gene therapy was designed. A translation initiation sequence, including but not limited to a Kozak sequence or an IRES sequence, such as the CrPV IRES, located at the 5' end of the construct is followed by a nucleic acid encoding a signal peptide selected from one or more of the GAA signal peptide, a nucleic acid encoding an antitrypsin inhibitor, and a nucleic acid encoding a BiP sequence. This is followed by a nucleic acid encoding a cell-targeting domain, which may be vIGF-2, HIRMab, TfRMab, or other cell-targeting peptide or protein. The gene therapy construct further includes a nucleic acid encoding a linker and a nucleic acid encoding a corrective enzyme or an enzymatically active fragment thereof, wherein the linker links the cell-targeting domain to the corrective enzyme or an enzymatically active fragment thereof. Suitable corrective enzymes include, but are not limited to, alpha-glucosidase (GAA), alpha-galactosidase (GLA), iduronidase (IDUA), iduronate-2-sulfatase (IDS), PPT1, TPP1, NAGLU, or enzymatically active fragments thereof, and other enzymes found to be deficient in the individual.

[0056] Intracellular targeting of therapeutic proteins The N-linked carbohydrates of most lysosomal proteins are modified to contain a special carbohydrate structure called mannose 6-phosphate (M6P). M6P is a biological signal that enables the transport of lysosomal proteins to lysosomes via the membrane-bound M6P receptor. Enzyme replacement therapy for lysosomal storage disorders utilizes the M6P receptor for uptake and delivery of therapeutic proteins to lysosomes. Certain therapeutic agents, such as Cerezyme® and other versions of recombinant human GCase, utilize a mannose receptor that can bind to terminal mannose on protein glycans and deliver them to lysosomes without utilizing the M6P receptor. A problem facing certain enzyme replacement therapeutics is that only low amounts of M6P are present in enzyme therapeutics, requiring higher doses to achieve therapeutic efficacy. This leads to substantially longer infusion times, a higher likelihood of developing an immune response to the therapeutic, and higher drug demands, necessitating increased protein production and increased costs.

[0057] The CI-MPR scavenges M6P-containing lysosomal enzymes from the circulation. The receptor has distinct binding domains for M6P and insulin-like growth factors (domains 1-3 and 7-9, see Figure 2), and is therefore also known as the IGF2 / mannose-6-phosphate receptor or IGF2 / CI-MPR. This receptor can be used to target M6P-, IGF2-, or IGF2 variant-containing enzyme replacement therapeutics. The binding affinity of this receptor for these ligands, including insulin-like growth factors, is shown in Table 1. Notably, IGF2 peptides have a higher binding affinity for the CI-MPR than for mono- or bis-phosphorylated oligosaccharides.

[0058] [Table 1]

[0059] Thus, in some embodiments, it is desirable to design improved variant IGF2 (vIGF2) peptides that form therapeutic fusion proteins with increased stability, CI-MPR binding, cellular uptake, and lysosomal localization in treating diseases such as, for example, lysosomal storage diseases.

[0060] In some embodiments, variant vIGF2 has improved binding to CI-MPR, which is responsible for cellular uptake and delivery of IGF2 to lysosomes for degradation. Some variant IGF2 peptides have reduced affinity for insulin-like growth factor receptor 1 (IGF1R). In some embodiments, IGF2 has reduced or no affinity for integrins. In some embodiments, IGF2 also has reduced or no affinity for at least one insulin-like growth factor binding protein (IGFBP1-6). In some embodiments, IGF2 variants have reduced or no binding to heparin. In some embodiments, IGF2 variants

[0061] The goal in designing vIGF2 peptides may be to improve the biophysical properties of vIGF2 and enhance binding to the CI-MPR / cellular uptake and lysosomal delivery while minimizing other functions. Thus, vIGF2 peptides may (1) improve the stability / solubility of vIGF2; (2) weaken the binding affinity to IR / IGF1R / integrins; and (3) improve the binding affinity to the CI-MPR. In some embodiments, vIGF2 peptides are designed using structure-guided rational design, which identifies critical versus non-critical residues, point mutations, and truncations. In some embodiments, vIGF2 peptides are designed using in silico computational experiments, including alanine scanning mutagenesis (NAMD), which involves systematic mutation studies to determine whether given mutations affect stability and affinity for various binding partners, and / or to improve IGF2 solubility, bioavailability, and / or reduce immunogenicity. In some embodiments, the vIGF2 peptides are designed through directed evolution based on a split GFP assay. In some embodiments, the vIGF2 peptides are designed through directed evolution based on phage display.

[0062] In some embodiments, the vIGF2 peptides are designed using in silico computational experiments, including systematic mutation studies, to determine whether a given mutation affects the stability of the IGF2 peptide. In some embodiments, the stability of the peptides with the mutations is the same or increased compared to wild-type IGF2.

[0063] In some embodiments, vIGF2 peptides are designed to have reduced binding to integrins. In some embodiments, vIGF2 peptides with reduced integrin binding comprise the mutations R24E / R34E, R24E / R37E / R38E, R34E / R37E / R38E, R24E / R37E, R24E / R38E, or R24E / R34E / R37E / R38E. In some embodiments, vIGF2 peptides with mutations at, for example, residues R37, R38, or R40 have reduced binding to integrins and heparin.

[0064] In some embodiments, mutations T16I, T16V, T16L, T16F, T16Y, or T16W increase binding of vIGF2 to the CI-MPR. In some embodiments, mutations T16V or T16Y increase binding of vIGF2 to the CI-MPR. In some embodiments, mutations at D23, e.g., D23K or D23R, increase binding of vIGF2 to the CI-MPR. In some embodiments, mutations at F19, e.g., F19W, increase binding of vIGF2 to the CI-MPR. In some embodiments, mutations at S50, e.g., S50D or S50E, increase binding of vIGF2 to the CI-MPR. In some embodiments, vIGF2 with mutations D23K and S50E has increased binding to the CI-MPR. In some embodiments, vIGF2 with mutations Δ1-4, E6R, Y27L, and K65R has increased binding to the CI-MPR, hi some embodiments, vIGF2 with mutations Δ33-40, D23R, F26E, and S50E has increased binding to the CI-MPR.

[0065] In some embodiments, the vIGF2 peptide is designed to have reduced IGFR1 binding. In some embodiments, the mutations affecting IGF1R binding are on a different face of IGF2 compared to the mutations affecting CI-MPR binding. In some embodiments, F26, Y27, and V43 are important for binding to IGF1R. In some embodiments, vIGF2 peptides with mutations S29N, R34_GS, S39_PQ, R34_GS / S39_PQ, S29N / S39_PQ, or S29N / S39PQ, R43_GS have reduced binding to insulin receptor and IGF1R. In some embodiments, vIGF2 peptides with mutations S39_PQ (insertion of PQ after S39) have reduced binding to insulin receptor and IGF1R. In some embodiments, vIGF2 peptides with mutations at G11, V14, L17, G25, F26, Y27, F28, S29, R30, P31, A32, S33, V35, S36, R37, S39, G41, 142, V43, E44, F48, T53, Y59, C60, or A61 have reduced binding to IGF1R, hi some embodiments, vIGF2 peptides with mutations at G10, L13, V14, L17, F26, Y27, F28, S29, R30, P31, A32, S33, V35, G41, 142, V43, T58, or Y59 have reduced binding to IGF1R. In some embodiments, vIGF2 peptides with mutations V14D / F26A / F28R / V43D have reduced binding to IGF1R, hi some embodiments, vIGF2 peptides with mutations F26S, Y27L, or V43L have reduced binding to IGF1R and / or insulin receptor.

[0066] In some embodiments, the vIGF2 peptide has a deletion within the C domain (e.g., residues 32-41, SRVSRRSR), which reduces the vIGF2 peptide's binding to IGF1R, insulin receptor, heparin, and integrins. In some embodiments, the vIGF2 peptide has mutations Δ1-4, E6R, Δ30-39. In some embodiments, the vIGF2 peptide has mutations Δ1-4, E6R, Δ33-40.

[0067] In some embodiments, the vIGF2 peptide has mutations that decrease its instability index. In some embodiments, IGF2 peptide mutations that increase stability include R38G, R38G / E45W, R38G / E45W / S50G, P31G / R38G / E45W / S50G, or L17N / P31G / R38G / E45W / S50G. In some embodiments, IGF2 peptide mutations that increase stability include R38G, R38G / E45W, R38G / E45W / S50G, P31G / R38G / E45W / S50G, L17N / P31G / R38G / E45W / S50G, L17N / P31G / R38G / E45W / S50G / S66G, L17N / P31G / R38G / E45W / S50G / A64M / S66G, or S5L / L17N / P31G / R38G / E45W / S50G / A64M / S66G.

[0068] In some embodiments, the vIGF2 peptide is mutated to reduce aggregation. In some embodiments, aggregation-prone residues include residues 17-21 (LQFVC), 41-49 (GIVEECCFR), or 53-62 (LALLETYCAT). In some embodiments, the vIGF2 peptide is mutated at F26, Y59, Y27, V14, A1, or L8 to reduce aggregation.

[0069] In some embodiments, the vIGF2 peptide is designed to have reduced binding to IGFBPs. In some embodiments, the vIGF2 peptide has a mutation L8A, V20A, or L56A. In some embodiments, a vIGF2 peptide with a mutation at E6, L8, R24, G25, F26, Y27, or F28 has reduced binding to IGFBP4. In some embodiments, a vIGF2 peptide with a mutation at T7, G10, V14, V43, E44, C47, or F48 has reduced binding to IGFBP4. In some embodiments, a vIGF2 peptide with a mutation at E6 or L8 has reduced binding to IGFBP4. In some embodiments, a vIGF2 peptide with a mutation at E6Q or T7A has reduced binding to human serum binding proteins. In some embodiments, a vIGF2 peptide with a mutation at Q18Y or F19L has reduced binding to human serum binding proteins. In some embodiments, the vIGF2 peptide with the mutations E6Q, T7A, Q18Y, or F19L has reduced binding to human serum binding proteins.

[0070] In some embodiments, the vIGF2 peptide is modified to substitute residues 31-38 with GGGG (vIGF2Δ31-38GGGG), and some of the vIGF2 peptides further contain V43L and S50E or S50D mutations (SEQ ID NOs: 120-121). In some embodiments, vIGF2 peptides at least 95% identical to SEQ ID NOs: 120-121 enhance expression and / or secretion of a therapeutic protein. In some embodiments, the therapeutic protein is PPT1 or TPP1, or an enzymatically active fragment thereof.

[0071] Therapeutic fusion proteins for gene therapy Provided herein are therapeutic fusion proteins generated from gene therapy vectors. In some embodiments, the fusion proteins are secreted by cells transduced with the gene therapy vector encoding the fusion protein. In some embodiments, the transduced cells are located within a tissue or organ (e.g., the liver). Once secreted from the cells, the fusion protein is transported through the patient's vascular system to reach the tissue of interest. In some embodiments, the therapeutic fusion protein is engineered for enhanced secretion. In some embodiments, the fusion protein comprises a signal peptide that enhances secretion levels compared to the corresponding therapeutic protein or fusion protein comprising a therapeutic protein having only the native signal peptide.

[0072] In some embodiments, the provided gene therapy vectors are engineered to improve delivery of therapeutic proteins. For example, in some cases, gene therapy cannot achieve its intended treatment by simply producing sufficient amounts of therapeutic protein in the patient's body if insufficient amounts of therapeutic protein are delivered to cells requiring the therapeutic protein, e.g., due to physical and / or biological barriers that prevent distribution of the therapeutic protein to the required site. Thus, even if gene therapy can fill blood or tissues with saturating concentrations of therapeutic protein, the gene therapy may not be fully therapeutic. In addition, non-productive clearance pathways may remove the majority of the therapeutic protein. Even if the therapeutic protein is delivered from the vasculature to the interstitial space within tissues (e.g., muscle fibers), sufficient therapeutic efficacy is not guaranteed. For effective treatment of lysosomal storage disorders, a therapeutically effective amount of therapeutic protein must be delivered via cellular endocytosis and lysosomal delivery to achieve meaningful efficacy. The present disclosure addresses these problems by providing a gene therapy vector encoding a fusion protein containing a peptide that enables endocytosis of a therapeutic protein into target cells for treatment that results in effective treatment. In some embodiments, the peptide that enables endocytosis is a peptide that binds to the CI-MPR. In some embodiments, the peptide that binds to the CI-MPR is a vIGF2 peptide. Recombinantly expressed GLA is known to be sufficiently phosphorylated to bind to CIMPR. However, surprisingly, GLA expressed in mice is under-phosphorylated and does not bind sufficiently to CIMPR. Therefore, unexpectedly, GLA used in gene therapy requires additional engineering (e.g., an IGF2 tag) to enhance CIMPR binding.

[0073] Provided herein are gene therapy vectors encoding fusion proteins containing a peptide that enables endocytosis of a therapeutic protein into target cells for treatment. In some embodiments, the gene therapy vector encodes a fusion protein containing a therapeutic protein and a peptide that binds to the CI-MPR. When expressed from the gene therapy vector, such a fusion protein targets a therapeutic protein, such as an enzyme-replacement therapeutic, to cells in need thereof, enhances delivery into or cellular uptake by such cells, and targets the therapeutic protein to a subcellular location (e.g., lysosomes). In some embodiments, the peptide is an IGF2 peptide or a variant thereof that can target the therapeutic protein to lysosomes. In some embodiments, the fusion proteins herein further comprise a signal peptide that enhances secretion, such as a BiP signal peptide or a Gaussia signal peptide. In some embodiments, the fusion protein comprises a linker sequence. In some embodiments, the nucleic acid encoding the fusion protein herein comprises an internal ribosome entry sequence.

[0074] Therapeutic fusion proteins for enzyme replacement therapy Provided herein are therapeutic fusion proteins engineered for enzyme replacement therapy. In some embodiments, the provided fusion proteins are engineered to improve delivery of the therapeutic protein. For example, in some cases, if an insufficient amount of the therapeutic fusion protein is delivered to the cells in need of the therapeutic protein, e.g., due to physical and / or biological barriers that prevent the therapeutic protein from being distributed to the required site, the fusion protein may not achieve its intended treatment. Even if the therapeutic protein is transported from the vasculature to the interstitial space within a tissue (e.g., muscle fibers), sufficient therapeutic efficacy is not guaranteed. For effective treatment of lysosomal storage disorders, a therapeutically effective amount of the therapeutic protein must undergo cellular endocytosis and lysosomal delivery to achieve meaningful efficacy. The present disclosure addresses these issues by providing fusion proteins containing peptides that enable endocytosis of the therapeutic protein into target cells for treatment that results in effective treatment. In some embodiments, the peptide that enables endocytosis is a peptide that binds to the CI-MPR. In some embodiments, the peptide that binds to the CI-MPR is a vIGF2 peptide.

[0075] Provided herein are fusion proteins containing peptides that enable endocytosis of a therapeutic protein into target cells for treatment. In some embodiments, the fusion protein contains a peptide that binds to the CI-MPR. Such fusion proteins are used as enzyme replacement therapeutics, increasing the delivery of such proteins into or cellular uptake by cells in need of such proteins and directing the therapeutic protein to a subcellular location (e.g., lysosomes). In some embodiments, the peptide is an IGF2 peptide or a variant thereof that can target the therapeutic protein to lysosomes.

[0076] Therapeutic proteins for enzyme replacement therapy or gene therapy comprising vIGF2 peptides are provided herein. Exemplary proteins are shown in Table 2 below.

[0077] Table 2

[0078] Table 3

[0079] Table 4

[0080] Table 5

[0081] Table 6

[0082] Table 7

[0083] Table 8

[0084] Table 9

[0085] Table 10

[0086] Table 11

[0087] Table 12

[0088] Table 13

[0089] Table 14

[0090] Table 15

[0091] Table 16

[0092] Table 17

[0093] Table 18

[0094] Table 19

[0095] Table 20

[0096] Table 21

[0097] Table 22

[0098] [Table 23]

[0099] [Table 24]

[0100] [Table 25]

[0101] The components of the fusion proteins provided herein are further described below.

[0102] Peptides that bind to CI-MPR (e.g., vIGF2 peptide) Provided herein are peptides that bind to the CI-MPR. Fusion proteins comprising such peptides and a therapeutic protein, when expressed from a gene therapy vector, target the therapeutic protein to cells in need thereof, increase cellular uptake by such cells, and target the therapeutic protein to a subcellular location (e.g., lysosomes). In some embodiments, the peptide is fused to the N-terminus of the therapeutic peptide. In some embodiments, the peptide is fused to the C-terminus of the therapeutic protein. In some embodiments, the peptide is a vIGF2 peptide. Some vIGF2 peptides maintain high affinity binding to the CI-MPR while their affinity for IGF1 receptor, insulin receptor, and IGF-binding proteins (IGFBPs) is reduced or eliminated. Some vIGF2 peptides have increased affinity for binding to the CI-MPR. Thus, some variant IGF2 peptides are substantially more selective and have reduced safety risks compared to wild-type IGF2. vIGF2 peptides herein include those having the amino acid sequences of SEQ ID NOs: 31, 120, and 121. Variant IGF2 peptides further include those having a variant amino acid at position 6, 26, 27, 31-38, 43, 48, 49, 50, 54, 55, or 65 relative to wild-type IGF2 (SEQ ID NO: 68). In some embodiments, the vIGF2 peptide has a sequence with one or more substitutions from the group consisting of E6R, F26S, Y27L, V43L, F48T, R49S, S50E, S50I, A54R, L55R, and K65R. In some embodiments, the vIGF2 peptide has a sequence with an E6R substitution. In some embodiments, the vIGF2 peptide has a sequence with an F26S substitution. In some embodiments, the vIGF2 peptide has a sequence with a Y27L substitution. In some embodiments, the vIGF2 peptide has a sequence with a V43L substitution. In some embodiments, the vIGF2 peptide has a sequence with an F48T substitution. In some embodiments, the vIGF2 peptide has a sequence with an R49S substitution.In some embodiments, the vIGF2 peptide has a sequence with an S50I substitution. In some embodiments, the vIGF2 peptide has a sequence with an S50E substitution. In some embodiments, the vIGF2 peptide has a sequence with an S50E substitution, which has increased binding to CI-MPR. In some embodiments, the vIGF2 peptide has a sequence with an A54R substitution. In some embodiments, the vIGF2 peptide has a sequence with an L55R substitution. In some embodiments, the vIGF2 peptide has a sequence with a K65R substitution. In some embodiments, the vIGF2 peptide has a sequence with E6R, F26S, Y27L, V43L, F48T, R49S, S50I, A54R, and L55R substitutions. In some embodiments, the vIGF2 peptide has an N-terminal deletion. In some embodiments, the vIGF2 peptide has an N-terminal deletion of one amino acid. In some embodiments, the vIGF2 peptide has an N-terminal deletion of two amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of three amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of four amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of four amino acids and substitutions of E6R, Y27L, and K65R. In some embodiments, the vIGF2 peptide has an N-terminal deletion of four amino acids and substitutions of E6R and Y27L. In some embodiments, the vIGF2 peptide has an N-terminal deletion of five amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of six amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of seven amino acids. In some embodiments, the vIGF2 peptide has an N-terminal deletion of seven amino acids and substitutions of Y27L and K65R. In some embodiments, the Bmax for CIMPR binding with SEQ ID NO: 83 is enhanced compared to SEQ ID NO: 80.

[0103] [Table 26]

[0104] Table 27

[0105] Table 28

[0106] Table 29

[0107] Table 30

[0108] Table 31

[0109] Table 32

[0110] Table 33

[0111] Table 34

[0112] Table 35

[0113] Table 36

[0114] [Table 37]

[0115] [Table 38]

[0116] [Table 39]

[0117] Internal ribosome entry sequence Provided herein are gene therapy constructs useful in treating disorders, further comprising an internal ribosome entry sequence (IRES) to increase gene expression by bypassing a translation initiation bottleneck. Suitable internal ribosome entry sequences for optimizing expression for gene therapy include, but are not limited to, cricket paralysis virus (CrPV) IRES, picornavirus IRES, aphthovirus IRES, Kaposi's sarcoma-associated herpesvirus IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, crispavirus IRES, Rhopalococcus aphid virus IRES, Marek's disease virus IRES, and other suitable IRES sequences. In some embodiments, the gene therapy construct comprises a CrPV IRES. In some embodiments, the CrPV IRES is [ka] In some embodiments, the CrPV IRES sequence is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:191.

[0118] signal peptide The gene therapy constructs provided herein, in some embodiments, further comprise a signal peptide that improves secretion of a therapeutic protein from cells transduced with the gene therapy construct. In some embodiments, the signal peptide improves protein processing of the therapeutic protein and promotes translocation of the nascent polypeptide-ribosomal complex to the ER to ensure proper co- and post-translational modifications. In some embodiments, the signal peptide is positioned (i) between the translation initiation sequence and the therapeutic protein, or (ii) downstream of the therapeutic protein. Signal peptides useful in gene therapy constructs include, but are not limited to, binding immunoglobulin protein (BiP) signal peptides from the HSP70 protein family (e.g., HSPA5, heat shock protein family A member 5) and Gaussia signal peptides, as well as variants thereof. These signal peptides have extremely high affinity for signal recognition particles. Exemplary BiP and Gaussia amino acid sequences are shown in Table 5 below. In some embodiments, the signal peptide has an amino acid sequence that is at least 90, 95, 96, 97, 98, or 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 169-180. In some embodiments, the signal peptide differs from a sequence selected from the group consisting of SEQ ID NOs: 169-180 by no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid. In some embodiments, the native signal peptide, referred to interchangeably herein as the "endogenous signal peptide" of the lysosomal protein, is used.

[0119] [Table 40]

[0120] BiP signal peptide-signal recognition particle (SRP) interaction promotes translocation to the ER. This interaction is shown in Figure 20.

[0121] The Gaussia signal peptide is derived from luciferase from Gaussia princeps and directs increased protein synthesis and secretion of a therapeutic protein fused to the signal peptide. In some embodiments, the Gaussia signal peptide has an amino acid sequence at least 90, 95, 96, 97, 98, or 99% identical to SEQ ID NO: 174. In some embodiments, the signal peptide differs from SEQ ID NO: 174 by no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 amino acid.

[0122] Linker In some embodiments, the gene therapy constructs provided herein include a linker between the targeting peptide and the therapeutic protein. Such a linker, in some embodiments, maintains precise spacing to reduce steric clashes between the vIGF2 peptide and the therapeutic protein. In some embodiments, the linker includes repeated glycine residues, repeated glycine-serine residues, and combinations thereof. In some embodiments, the linker consists of 5-20 amino acids, 5-15 amino acids, 5-10 amino acids, 8-12 amino acids, or about 5, 6, 7, 8, 9, 10, 11, 12, or 13 amino acids. Linkers and enzyme replacement therapy constructs suitable for gene therapy herein include, but are not limited to, those shown in Table 6 below.

[0123] [Table 41]

[0124] Translation initiation sequence Gene therapy constructs provided herein include nucleic acids having a translation initiation sequence, e.g., a Kozak sequence, which aids in the initiation of mRNA translation. Kozak sequences contemplated herein have the consensus sequence (gcc)RccATGG, where lowercase letters represent the most common base at that position, bases that vary, and uppercase letters indicate changes to highly conserved bases that vary rarely. R indicates that a purine (adenine or guanine) is always observed at that position. The sequence in parentheses (gcc) is of uncertain significance. In some embodiments, a Kozak sequence comprises the sequence AX1X2ATGA, where each of X1 and X2 is any nucleotide. In some embodiments, X1 comprises A. In some embodiments, X2 comprises G. In some embodiments, a Kozak sequence comprises a nucleic acid sequence at least 85% identical to AAGATGA. In some embodiments, a Kozak sequence differs from the sequence of AAGATGA by one or two nucleotides. In some embodiments, a Kozak sequence provided herein has the sequence AAGATGA. In some embodiments, the Kozak sequence comprises a nucleic acid sequence at least 85% identical to GCAAGATG. In some embodiments, the Kozak sequence differs from the sequence of GCAAGATG by one or two nucleotides. In some embodiments, the Kozak sequence comprises GCAAGATG. In some embodiments, the Kozak sequence comprises a nucleic acid sequence at least 85% identical to CACCATG. In some embodiments, the Kozak sequence differs from the sequence of CACCATG by one or two nucleotides. In some embodiments, the Kozak sequence comprises CACCATG.

[0125] Therapeutic Proteins The gene therapy constructs provided herein comprise a nucleic acid encoding a therapeutic protein for treating a genetic disorder resulting from a genetic defect in an individual that results in an absent or defective protein. The therapeutic protein expressed from the gene therapy construct replaces the absent or defective protein. Thus, the therapeutic protein is selected based on the genetic defect in the individual that requires treatment. In some embodiments, the therapeutic protein is a structural protein. In some embodiments, the therapeutic protein is an enzyme. In some embodiments, the therapeutic protein is a regulatory protein. In some embodiments, the therapeutic protein is a receptor. In some embodiments, the therapeutic protein is a peptide hormone. In some embodiments, the therapeutic protein is a cytokine or chemokine.

[0126] In some embodiments, the gene therapy constructs herein encode an enzyme, e.g., an enzyme that is genetically defective in an individual with a lysosomal storage disorder. In some embodiments, the gene therapy construct encodes a lysosomal enzyme, e.g., a glycosidase, protease, or sulfatase. In some embodiments, the enzymes encoded by the gene therapy constructs provided herein include, but are not limited to, α-D-mannosidase; N-aspartyl-β-glucosaminidase; β-galactosidase; ceramidase; fucosidase; galactocerebrosidase; arylsulfatase A; N-acetylglucosamine-1-phosphotransferase; iduronate sulfatase; N-acetylglucosaminidase; acetyl-CoA:α-glucosaminide acetyltransferase; N-acetylglucosamine 6-sulfatase; β-glucuronidase; hyaluronidase; sialidase; sulfatase; sphingomyelinase; acid β-mannosidase; cathepsin K; 3-hexosidase; Examples of enzymes encoded by the gene therapy constructs provided herein include alpha-glucosidase A, β-hexosaminidase B, α-N-acetylgalactosaminidase, sialin, hexosaminidase A, beta-glucosidase, α-iduronidase, α-galactosidase A, β-glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan α-L-iduronohydrolase, iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, alpha-glucosidase, heparan sulfamidase, gp-91 subunit of NADPH oxidase, adenosine deaminase, cyclin-dependent kinase-like 5, and protein palmitoylthioesterase 1. In some embodiments, the enzyme encoded by the gene therapy constructs provided herein comprises alpha-glucosidase.In some embodiments, the therapeutic protein is associated with a genetic disorder selected from the group consisting of cystic fibrosis, alpha and beta thalassemia, sickle cell anemia, Marfan syndrome, fragile X syndrome, Huntington's disease, hemochromatosis, congenital deafness (asymptomatic), Tay-Sachs, familial hypercholesterolemia, Duchenne muscular dystrophy, Stargardt disease, Usher syndrome, choroideremia, color blindness, X-linked retinoschisis, hemophilia, Wiskott-Aldrich syndrome, X-linked chronic granulomatous disease, aromatic L-amino acid decarboxylase deficiency, recessive dystrophic epidermolysis bullosa, alpha 1-antitrypsin deficiency, Hutchinson-Gilford progeria syndrome (HGPS), Noonan syndrome, and X-linked severe combined immunodeficiency (X-SCID).

[0127] Examples of gene therapy vectors Gene Therapy Vectors and Compositions Provided herein are gene therapy vectors in which a nucleic acid, e.g., DNA, encodes a therapeutic fusion protein, e.g., a vIGF2 fusion, optionally with a signal peptide. The gene therapy vector optionally contains an internal ribosome entry sequence. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its propagation in daughter cells. Lentivirus vectors and adeno-associated virus vectors have an additional advantage over vectors derived from oncoretroviruses, e.g., murine leukemia viruses, in that they can transduce non-proliferating cells, e.g., hepatocytes and neurons. They also have the additional advantage of low immunogenicity.

[0128] Exemplary gene therapy vectors herein encode therapeutic proteins and therapeutic fusion proteins comprising the vIGF2 peptide. Nucleic acids encoding exemplary fusion protein amino acid sequences are shown in Table 7 below.

[0129] [Table 42]

[0130] Table 43

[0131] Table 44

[0132] Table 45

[0133] Table 46

[0134] Table 47

[0135] Table 48

[0136] Table 49

[0137] Table 50

[0138] Table 51

[0139] Table 52

[0140] Table 53

[0141] Table 54

[0142] Table 55

[0143] Table 56

[0144] Table 57

[0145] Table 58

[0146] Table 59

[0147] Table 60

[0148] Table 61

[0149] Table 62

[0150] Table 63

[0151] Table 64

[0152] Table 65

[0153] Table 66

[0154] Table 67

[0155] Table 68

[0156] Table 69

[0157] Table 70

[0158] Table 71

[0159] Table 72

[0160] Table 73

[0161] Table 74

[0162] Table 75

[0163] Table 76

[0164] Table 77

[0165] Table 78

[0166] Table 79

[0167] Table 80

[0168] Table 81

[0169] Table 82

[0170] Table 83

[0171] Table 84

[0172] Table 85

[0173] Table 86

[0174] Table 87

[0175] Table 88

[0176] Table 89

[0177] Table 90

[0178] Table 91

[0179] Table 92

[0180] Table 93

[0181] Table 94

[0182] [Table 95]

[0183] [Table 96]

[0184] [Table 97]

[0185] [Table 98]

[0186] [Table 99]

[0187] In some embodiments, the vector containing the nucleic acid encoding the desired therapeutic fusion protein, e.g., a vIGF2 fusion or a signal peptide fusion, provided herein, optionally with an internal ribosome entry sequence, is an adeno-associated virus vector (A5 / 35).

[0188] In some embodiments, the nucleic acid encoding a therapeutic fusion protein, such as a vIGF2 fusion, optionally has an internal ribosome entry sequence and can be cloned into various types of vectors. For example, in some embodiments, the nucleic acid is cloned into a vector, including, but not limited to, a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0189] Furthermore, in some embodiments, an expression vector encoding a therapeutic fusion protein, such as a vIGF2 fusion or a signal peptide fusion, optionally with an internal ribosome entry sequence, is provided to cells in the form of a viral vector. Viral vector technology is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction enzyme sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0190] Also provided herein are compositions and systems for gene transfer. Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. In some embodiments, a selected gene is inserted into a vector using appropriate techniques and packaged into retroviral particles. The recombinant virus is then isolated and delivered to cells of a subject in vivo or ex vivo. Some retroviral systems are suitable for gene therapy. In some embodiments, adenoviral vectors are used. Some adenoviral vectors are suitable for gene therapy. In some embodiments, adeno-associated viral vectors are used. Some adeno-associated viruses are suitable for gene therapy. In one embodiment, a lentiviral vector is used.

[0191] Gene therapy constructs provided herein include vectors into which a gene of interest has been cloned or which otherwise contain the gene of interest (or gene therapy expression vectors) such that the nucleotide sequence of the vector allows for expression of the gene of interest (either constitutive or otherwise regulated). The vector constructs provided herein include any suitable gene expression vector that can be delivered to a tissue of interest and will achieve expression of the gene of interest in the selected tissue of interest.

[0192] In some embodiments, the vector is an adeno-associated virus (AAV) vector. This is due to the ability of AAV vectors to cross the blood-brain barrier and transduce neuronal tissue. Any serotype of AAV is intended to be used in the methods provided herein. In certain embodiments, the serotype of the viral vector used is selected from the group consisting of AAV1 vector, AAV2 vector, AAV3 vector, AAV4 vector, AAV5 vector, AAV6 vector, AAV7 vector, AAV8 vector, AAV9 vector, AAVrhS vector, AAVrh10 vector, AAVrh33 vector, AAVrh34 vector, AAVrh74 vector, AAV Anc80 vector, AAVPHP.B vector, AAVhu68 vector, AAV-DJ vector, and others suitable for gene therapy.

[0193] AAV vectors are DNA parvoviruses that are nonpathogenic to mammals. Briefly, AAV-based vectors have the rep and cap viral genes, which comprise 96% of the viral genome, removed, leaving two flanking 145-base pair inverted terminal repeats (ITRs) that are used to initiate viral DNA replication, packaging, and integration.

[0194] Further embodiments include the use of other serotype capsids to generate AAV1 vectors, AAV2 vectors, AAV3 vectors, AAV4 vectors, AAV5 vectors, AAV6 vectors, AAV7 vectors, AAV8 vectors, AAV9 vectors, AAVrhS vectors, AAVrh10 vectors, AAVrh33 vectors, AAVrh34 vectors, AAVrh74 vectors, AAV Anc80 vectors, AAVPHP.B vectors, AAV-DJ vectors, and others suitable for gene therapy. In some cases, the AAV viral capsid is AAV2 / 9, AAV9, AAVrhS, AAVrh10, AAV Anc80, or AAVPHP.B.

[0195] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, such promoter elements are located within a region 30 to 110 bp upstream of the start site, although some promoters have been shown to contain functional elements downstream of the start site as well. Frequently, the spacing between promoter elements is flexible, so promoter function is preserved even when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, spacing between promoter elements is often increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to function cooperatively or independently to activate transcription.

[0196] An example of a promoter capable of expressing therapeutic fusion proteins, such as vIGF2 fusion or signal peptide fusion transgenes, optionally with an internal ribosome entry sequence, in mammalian T cells is the EF1a promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl-tRNA to ribosomes. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to be effective in driving expression from transgenes cloned into lentiviral vectors (see, e.g., Milone et al., Mol. Ther. 17(8):1453-1464 (2009)). Another example of a promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any operably linked polynucleotide sequence. However, other constitutive promoter sequences are also occasionally used, including, but not limited to, the chicken β-actin promoter, P546 promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, myosin promoter, elongation factor-1a promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, it is not intended that gene therapy vectors be limited to the use of constitutive promoters. Inducible promoters are also contemplated herein. Inducible promoters provide a molecular switch that can turn on expression of an operably linked polynucleotide sequence when expression is desired and turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline-regulated promoters.

[0197] To assess the expression of therapeutic fusion proteins, such as vIGF fusions or signal peptide fusions, optionally with an internal ribosome entry sequence or portion thereof, the expression vectors to be introduced into cells often contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells desired to be transfected or infected via a viral vector. In other embodiments, the selectable marker is often carried on a separate DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene are sometimes flanked by appropriate regulatory sequences to enable expression in the host cell. Examples of useful selectable markers include antibiotic resistance genes, such as neo.

[0198] Methods and compositions for introducing and expressing genes in cells are suitable for the methods herein. In the context of expression vectors, the vectors are readily introduced into host cells, such as mammalian cells, bacterial cells, yeast cells, or insect cells, by any method in the art. For example, the expression vectors are transferred into host cells by physical, chemical, or biological means.

[0199] Physical methods and compositions for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene gun, and electroporation. Methods for generating cells containing vectors and / or foreign nucleic acids are suitable for the methods herein (see, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0200] Chemical means and compositions for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, nucleic acid-lipid particles, and liposomes. Exemplary colloidal systems used as in vitro and in vivo delivery vehicles include liposomes (e.g., artificial membrane vesicles). Other state-of-the-art methods for targeted delivery of nucleic acids are available, such as delivery of polynucleotides via targeted nanoparticles or other suitable submicron-sized delivery systems.

[0201] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid is associated with a lipid. In some embodiments, the lipid-associated nucleic acid is encapsulated within the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to the liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped within a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained with or complexed with a micelle, or otherwise associated with a lipid. The lipid, lipid / DNA, or lipid / expression vector to which the composition is associated is not limited to any particular structure in solution. For example, in some embodiments, it exists as a bilayer structure, as a micelle, or in a "collapsed" structure. Alternatively, they may simply be dispersed in the solution, possibly forming aggregates with non-uniform size and shape.In some embodiments, lipids are fatty substances, which may be naturally occurring or synthetic lipids.For example, lipids include the naturally occurring lipid droplets in the cytoplasm, and the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0202] Lipids suitable for use are obtained from commercially available sources. For example, in some embodiments, dimyristyl phosphatidylcholine ("DMPC") is obtained from Sigma, St. Louis, Mo.; in some embodiments, dicetyl phosphate ("DCP") is obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is obtained from Calbiochem-Behring in some embodiments; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids are often obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Chloroform or chloroform / methanol stock solutions of lipids are often stored at about -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposome" is a generic term that encompasses a variety of mono- and multi-layered lipid vesicles formed by the formation of closed lipid bilayers or aggregates. Liposomes are often characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Multilamellar liposomes form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures in solution that differ from normal vesicular structures are also encompassed. For example, lipids in some embodiments may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[0203] Regardless of the method used to introduce foreign nucleic acid into host cells or otherwise expose cells to a therapeutic fusion protein, e.g., a vIGF2 fusion or a signal peptide fusion, optionally with an internal ribosome entry sequence, provided herein, it is contemplated that various assays will be performed to confirm the presence of the recombinant DNA sequence within the host cells. Examples of such assays include "molecular biological" assays suitable for the methods herein, such as Southern and Northern blotting, RT-PCR, and PCR; "biochemical" assays, e.g., detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blotting) or by the assays described herein to identify agents within the scope of this specification.

[0204] The present disclosure further provides a vector comprising a nucleic acid molecule encoding a therapeutic fusion protein, e.g., a vIGF2 fusion or a signal peptide fusion, optionally with an internal ribosome entry sequence. In one embodiment, the therapeutic fusion protein vector can be directly transduced into a cell. In one embodiment, the vector is a cloning vector or an expression vector, including, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral vector constructs, and lentiviral vector constructs. In one embodiment, the vector can be used to express a vIGF2 therapeutic fusion protein construct in a mammalian cell. In one embodiment, the mammalian cell is a human cell.

[0205] Uses and methods of treatment Also provided herein are methods of treating genetic disorders using gene therapy, comprising administering to an individual a nucleic acid encoding a therapeutic fusion protein (e.g., a vIGF2 fusion or a signal peptide fusion or a signal peptide-vIGF2 fusion), optionally with an internal ribosome entry sequence, as disclosed herein. Genetic disorders suitable for treatment using the methods herein include disorders in an individual caused by one or more mutations in the genome that result in the lack of expression or the expression of a dysfunctional protein by a mutant gene.

[0206] Further provided herein are pharmaceutical compositions comprising a gene therapy vector, e.g., a gene therapy vector comprising a nucleic acid encoding a therapeutic fusion protein (e.g., a vIGF2 fusion or signal peptide fusion or signal peptide-vIGF2 fusion) disclosed herein, optionally having an internal ribosome entry sequence, and a pharmaceutically acceptable carrier or excipient for use in preparing a medicament for the treatment of a genetic disorder.

[0207] In some embodiments, the genetic disorder suitable for treatment using the methods provided herein is a lysosomal storage disorder. In some embodiments, the lysosomal storage disorder is treated herein using gene therapy to deliver a missing or defective enzyme to a patient. In some embodiments, the methods herein deliver an enzyme fused to vIGF2 or fused to a signal peptide to a patient, delivering the enzyme to cells where it is needed. In some embodiments, the lysosomal storage disorder is selected from the group consisting of aspartylglucosaminuria, Batten disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, and Schindler disease type II. In some embodiments, the lysosomal storage disorder is activator deficiency (GM2-gangliosidosis); GM2-gangliosidosis, AB variant; alpha-mannosidosis (type 2, mild; type 3, neonatal, severe); beta-mannosidosis; aspartylglucosaminuria; lysosomal acid lipase deficiency; cystinosis (late-onset juvenile or adolescent nephropathic; infantile nephropathy); Canarin-Dorfman syndrome; neutral lipid storage disease with myopathy; NLSDM; Danon disease; Fabry disease; Fabry disease type II, Late-onset; Farber disease; Farber lipogranulomatosis; Fucosidosis; Galactosialidosis (combined neuraminidase and β-galactosidase deficiency); Gaucher disease; Type II Gaucher disease; Type III Gaucher disease; Type IIIC Gaucher disease; Gaucher disease due to saposin C deficiency, variant; GM1-gangliosidosis (late-onset infantile / juvenile GM1-gangliosidosis; adult / chronic GM1-gangliosidosis); Globoid cell leukodystrophy, Krabbe disease (late-onset infantile; juvenile; adult onset);Krabbe disease, variant, due to saposin A deficiency; metachromatic leukodystrophy (juvenile; adult); partial cerebroside sulfate deficiency; pseudoarylsulfatase A deficiency; metachromatic leukodystrophy due to saposin B deficiency; mucopolysaccharidosis disorders: MPS I, Hurler syndrome; MPS I, Hurler-Scheie syndrome; MPS I, Scheie syndrome; MPS II, Hunter syndrome; MPS II, Hunter syndrome; Sanfilippo syndrome type A / MPS IIIA; Sanfilippo syndrome type B / MPS IIIB; Sanfilippo syndrome type C / MPS IIIC; Sanfilippo syndrome type D / MPS IIID; Morquio syndrome type A / MPS IVA; Morquio syndrome type B / MPS IVB; MPS IX hyaluronidase deficiency; MPS VI Maroteaux-Lamy syndrome; MPS Sly syndrome VII; mucolipidosis I, sialidosis type II; I-cell disease, LeRoy disease, mucolipidosis II; pseudo-Hurler polydystrophy / mucolipidosis type III; mucolipidosis IIIC / ML III gamma; mucolipidosis type IV; multiple sulfatase deficiency; Niemann-Pick disease (type B; type C1 / chronic neuropathy; type C2; ​​type D / nova) Scotia type); Neuronal ceroid lipofuscinosis: CLN6 disease - atypical late-onset infantile, late-onset variant, early-juvenile type; Batten-Spielmeier-Voigt / juvenile NCL / CLN3 disease; Finnish variant late-onset infantile CLN5; Jansky-Bierschofsky disease / late-onset infantile CLN2 / TPP1 disease; Kufs / adult-onset NCL / CLN4 disease (type B); Northern epilepsy / variant late-onset infantile CLN8; Santavuori-Haltia / infantile CLN1 / PPT disease; Pon Pe's disease (glycogenosis type II); late-onset Pompe disease; pyknodysostosis; Sandhoff disease / GM2 gangliosidosis; Sandhoff disease / GM2 gangliosidosis; Sandhoff disease / GM2 gangliosidosis; Schindler disease (type III / moderate, variable); Kanzaki disease; Salla disease; infantile free sialic acid storage disease (ISSD); progressive spinal muscular atrophy with myoclonic epilepsy (SMAPME); Tay-Sachs disease / GM2 gangliosidosis; juvenile-onset Tay-Sachs disease; late-onset Tay-Sachs disease; Christianson syndrome; Rowe oculocerebrorenal syndrome;Selected from the group consisting of Charcot-Marie-Tooth type 4J, CMT4J; Yunus-Vallon syndrome; bilateral temporo-occipital polymicrogyria (BTOP); X-linked hypercalciuric nephrolithiasis, Dent-1; and Dent disease-2. In some embodiments, the therapeutic protein is associated with a lysosomal storage disorder, and the therapeutic protein is GM2-activator protein; α-mannosidase; MAN2B1; lysosomal β-mannosidase; glycosylasparaginase; lysosomal acid lipase; cystinosin; CTNS; PNPLA2; lysosomal-associated membrane protein-2; α-galactosidase A; GLA; acid ceramidase; α-L-fucosidase; protective protein / cathepsin A; acid β-glucosidase; GBA; PSAP; β-galactosidase-1; GLB1; galactosylceramide β-galactosidase; GALC; PSAP; arylsulfatase A; ARSA; α- Selected from the group consisting of L-iduronidase, iduronate 2-sulfatase, heparan N-sulfatase, N-α-acetylglucosaminidase, heparan acetyl-CoA:α-glucosaminide acetyltransferase, N-acetylglucosamine 6-sulfatase, galactosamine-6-sulfate sulfatase, β-galactosidase, hyaluronidase, arylsulfatase B, β-glucuronidase, neuraminidase, NEU1, the gamma subunit of N-acetylglucosamine-1-phosphotransferase, mucolipin-1, sulfatase-modifying factor-1, acid sphingomyelinase, SMPD1, NPC1, and NPC2;

[0208] In some embodiments, treatment via the methods herein delivers a gene encoding a therapeutic protein to cells in need of the therapeutic protein. In some embodiments, treatment delivers the gene to all somatic cells in an individual. In some embodiments, treatment replaces a defective gene in target cells. In some embodiments, cells engineered ex vivo to express a therapeutic protein are delivered to an individual.

[0209] The gene therapy for disorders disclosed herein provides superior treatment outcomes over conventional treatments, including enzyme replacement therapy, because it does not require long infusion treatments.

[0210] definition As used herein, "ex vivo gene therapy" refers to a method in which patient cells are genetically modified outside of the subject, for example, to express a therapeutic gene. The cells with the new genetic information are then returned to the subject from whom they originated.

[0211] As used herein, "in vivo gene therapy" refers to a method in which a vector carrying a therapeutic gene is directly administered to a subject.

[0212] As used herein, the terms "fusion protein" and "therapeutic fusion protein" are used interchangeably herein and refer to a therapeutic protein linked to at least one additional protein, peptide, or polypeptide. In some instances, a fusion protein is a single protein molecule containing two or more proteins or fragments thereof covalently linked via peptide bonds within each peptide chain without a chemical linker. In some embodiments, the fusion protein includes a therapeutic protein and a signal peptide, or both, that is a peptide that increases endocytosis of the fusion protein. In some embodiments, the peptide that increases endocytosis is a peptide that binds to the CI-MPR.

[0213] As used interchangeably herein, the terms "vector" and "gene therapy vector" refer to a gene therapy delivery vehicle or carrier that delivers a therapeutic gene to a cell. A gene therapy vector is any vector suitable for use in gene therapy, for example, any vector suitable for therapeutic delivery of a nucleic acid polymer (encoding a polypeptide or a variant thereof) into a patient's target cells (e.g., sensory neurons). In some embodiments, a gene therapy vector delivers a nucleic acid encoding a therapeutic protein or a therapeutic fusion protein to a cell, where the therapeutic protein or fusion is expressed and secreted from the cell. The vector may be of any type, for example, a plasmid vector or a minicircle DNA. Typically, the vector is a viral vector. Vectors include both genetically silenced viruses, such as adenoviruses, and non-viral vectors, such as liposomes. Viral vectors may be derived from, for example, adeno-associated viruses (AAVs), retroviruses, lentiviruses, herpes simplex viruses, or adenoviruses. AAV-derived vectors. The vector may comprise the AAV genome or a derivative thereof.

[0214] As used herein, "construct" refers to a nucleic acid molecule or sequence that encodes a therapeutic protein or a fusion protein and, optionally, includes additional sequences, such as a translation initiation sequence or an IRES sequence.

[0215] As used herein, "plasmid" refers to a circular double-stranded unit of DNA that replicates within a cell independently of chromosomal DNA.

[0216] As used herein, a "promoter" refers to a site on DNA to which the enzyme RNA polymerase binds and initiates transcription of the DNA into RNA.

[0217] As used herein, "somatic cell therapy" refers to a method of manipulating gene expression in cells that is corrective to the patient but not inherited by future generations. Somatic cells include all non-germ cells in the human body.

[0218] As used herein, "somatic cells" refers to all somatic cells other than germ cells.

[0219] As used herein, "tropism" refers to the selection of a vector, e.g., a virus, for a particular cell or tissue type. Various factors determine the ability of a vector to infect a particular cell. A virus, for example, must bind to a specific cell surface receptor to enter the cell. A virus typically cannot infect a cell if the cell does not express the required receptor.

[0220] The term "transduction" is used to refer to the in vivo or in vitro administration / delivery of a nucleic acid encoding a therapeutic protein to a target cell via a replication-deficient rAAV of the present disclosure, resulting in expression of a functional polypeptide by the recipient cell. Transduction of a cell with a gene therapy vector, such as the rAAV of the present disclosure, results in sustained expression of the polypeptide or RNA encoded by the rAAV. Thus, the present disclosure provides methods for administering / delivering a gene therapy vector, such as an rAAV, encoding a therapeutic protein to a subject via intrathecal, intraretinal, intraocular, intravitreal, intracerebroventricular, intraparenchymal, or intravenous routes, or any combination thereof. "Intrathecal" delivery refers to delivery into the subarachnoid space of the brain or spinal cord. In some embodiments, intrathecal administration is via intracisternal administration. The present disclosure also provides methods for administering / delivering cells ex vivo transduced with a gene therapy vector, such as an rAAV vector, encoding a therapeutic protein via intrathecal, intraretinal, intraocular, intravitreal, intracerebroventricular, intraparenchymal, or intravenous routes, or any combination thereof.

[0221] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired, as the case may be. A "mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and lab, zoo, sport, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, and monkeys. In some embodiments, the mammal is a human.

[0222] As used herein, the terms "treatment," "treating," and "ameliorating symptoms," etc., refer to administering an agent or performing a procedure to achieve a therapeutic effect, which may include inhibiting, attenuating, lowering, preventing, or altering, as the case may be, statistically significantly or clinically significant, at least one aspect or marker of a disorder. The terms "ameliorate" or "treat" do not state or imply a cure for the underlying condition. As used herein, "treatment" or "ameliorating" (etc.) may include treating a mammal, particularly a human, and includes: (a) preventing a disorder or a symptom of a disorder from occurring in a subject who may be predisposed to the disorder but has not yet been diagnosed as suffering from the disorder (e.g., including disorders that may be associated with or caused by a primary disorder); (b) inhibiting the disorder, i.e., suppressing its onset; (c) palliating the disorder, i.e., causing remission of the disorder; and (d) ameliorating at least one symptom of the disorder. Treating can refer to any indication of success in treating or ameliorating or preventing a disorder, including any objective or subjective parameter, such as decline; remission; reducing symptoms or making the disorder symptoms more tolerable to the patient; slowing the rate of decline or decline; or making the end point of decline less debilitating. Treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Thus, the term "treating" includes administering a compound or agent of the invention to prevent or slow, reduce, suppress or inhibit the progression of symptoms or symptoms associated with a disorder. The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disorder, symptoms of a disorder, or side effects of a disorder in a subject.

[0223] The term "affinity" refers to the strength of binding between a molecule and its binding partner or receptor.

[0224] The phrase "high affinity" as used herein refers to a therapeutic fusion containing a peptide that has, for example, an affinity for the CI-MPR that is about 100-1,000-fold, or 500-1,000-fold higher than the therapeutic protein without the peptide binding to the CI-MPR. In some embodiments, the affinity is at least 100-fold, at least 500-fold, or at least 1,000-fold higher than without the peptide. For example, when a therapeutic protein and a CI-MPR are combined at relatively equal concentrations, a high-affinity peptide will bind to the available CI-MPR, shifting the equilibrium toward a resulting higher concentration of the complex.

[0225] As used herein, "secretion" refers to the release of a protein from a cell, e.g., into the bloodstream, so that it is delivered to a tissue of interest or site of action of the therapeutic protein. When a gene therapy product is secreted into the interstitial space of an organ, secretion may allow for cross-correction of adjacent cells.

[0226] As used herein, "delivery" refers to drug delivery. In some embodiments, the process of delivery refers to the transport of a drug substance (e.g., a therapeutic protein or fusion protein produced from cells transduced with a gene therapy vector) from outside the cell (e.g., blood, tissue, or interstitial space) into the target cells for the drug substance's therapeutic activity.

[0227] As used herein, "engineering" or "protein engineering" refers to the manipulation of the structure of a protein by providing a nucleic acid sequence encoding the protein suitable for resulting in the synthesis of a protein with desired properties or a particular structure.

[0228] A "therapeutically effective amount," as the case may be, means the amount that, when administered to a subject for treating a disorder, is sufficient to effect treatment for that disorder.

[0229] As used herein, the term "about" a numerical value refers to a range extending from less than 10% to more than 10% of the numerical value in question, including the numerical values ​​within the range, e.g., the numerical value itself.

[0230] As used herein, the term "comprising" a claim element refers to that element but does not exclude the inclusion of additional elements. [Example]

[0231] The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the invention in any way. The examples, together with the methods described herein, are presently representative and illustrative of preferred embodiments and are not intended as limitations on the scope of the invention. Modifications and other uses encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

[0232] Example 1: Binding of variant IGF2 peptides to the CI-MPR receptor Surface plasmon resonance (SPR) experiments were performed using Biacore to measure binding of wild-type and variant IGF2 (vIGF2) to the CI-MPR receptor. The wild-type human mature IGF2 peptide (wt IGF2) has the sequence shown in SEQ ID NO: 68. The vIGF2 sequence differs from wt IGF2 in that it lacks residues 1-4 and contains the following mutations: E6R, Y27L, and K65R. The vIGF2 sequence has the amino acid sequence: SRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE (SEQ ID NO: 80). vIGF2 also has an N-terminal linker of the sequence GGGGSGGGG (SEQ ID NO: 181). The combined sequence is Figure 4 shows that, as expected, the wild-type IGF2 peptide binds to the CI-MPR receptor with high affinity (0.2 nM). Figure 5 shows that the variant IGF2 peptide (vIGF2) also binds to the CI-MPR receptor with high affinity (0.5 nM). These data indicate that the vIGF2 peptide has high affinity for the intended CI-MPR receptor for targeting therapeutic agents to lysosomes.

[0233] SPR was used to measure peptide binding to the insulin receptor to assess potential side effects. Insulin binds to the insulin receptor with high affinity (approximately 8 nM; data not shown). Wild-type IGF2 and vIGF2 were tested. Here, vIGF2 has the sequence GGGGSGGGG (SEQ ID NO: 181), an N-terminal linker with the sequence The vIGF2 peptide had the amino acid sequence SRTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE (SEQ ID NO: 80). Figure 8 shows that wild-type IGF2 also binds to the insulin receptor with relatively high affinity (approximately 100 nM). The IGF2 peptide derived from the Biomarin / Zystor IGF2-GAA fusion protein (BMN-701) also binds to the insulin receptor with high affinity and has been shown to cause hypoglycemia in clinical trials. Figure 9 shows that there is no measurable binding of the vIGF2 peptide to the insulin receptor. These data indicate that the vIGF2 peptide confers a superior safety profile compared to wt IGF2 peptide fusions.

[0234] The same SPR binding analysis was used to characterize the vIGF2 peptide interaction with the IGF1 receptor. Figure 10 shows that the wild-type IGF2 peptide binds to the IGF1 receptor with relatively high affinity (approximately 100 nM). Figure 11 shows that there is no measurable binding of the vIGF2 peptide to the IGF1 receptor, indicating an improved safety profile compared to wt IGF2.

[0235] [Table 100]

[0236] Example 2: vIGF2 converts low affinity ligands for the CI-MPR into high affinity ERTs The vIGF2 peptide (SEQ ID NO: 80) with an N-terminal linker (SEQ ID NO: 181) was chemically coupled to alglucosidase alfa, designated here as vIGF2-alglucosidase alfa, to determine whether the vIGF2 peptide could improve its affinity for the CI-MPR. As shown in Figure 6, the binding affinities of alglucosidase alfa and vIGF2-alglucosidase alfa were compared using a CI-MPR plate binding assay in a 96-well plate directly coated with CI-MPR. Unbound enzyme was washed away, and then bound enzyme activity was measured. Various concentrations of both enzyme preparations were used with or without free WT IGF2 peptide. vIGF2 substantially improved the affinity for the CI-MPR. Furthermore, vIGF2-alglucosidase alfa binding was blocked by free WT IGF2, indicating that binding was IGF2-dependent (data not shown). Coupling of the vIGF2 peptide did not attenuate GAA enzyme activity.

[0237] vIGF2 was coupled to recombinant human N-acetyl-α-D-glucosaminidase (rhNAGLU), a lysosomal enzyme lacking M6P, to determine whether the peptide could convert unliganded peptides into high-affinity ligands for the CI-MPR. In this experiment, rhNAGLU and vIGF2-rhNAGLU were compared using a CI-MPR plate binding assay utilizing direct CI-MPR-coated plates. Unbound enzyme was washed away, and then bound enzyme activity was measured. Various concentrations of both enzyme preparations were used with or without free vIGF2 peptide. As shown in Figure 7, vIGF2-rhNAGLU has significantly higher affinity for the CI-MPR than rhNAGLU lacking vIGF2. Furthermore, vIGF2-rhNAGLU binding was blocked by free vIGF2 peptide, indicating that receptor binding was specific to the IGF2 peptide. These results indicate that the vIGF2 peptide can be used to improve drug targeting to lysosomes.

[0238] Example 3: Myoblast uptake of vIGF2-GAA fusion protein A vIGF2-GAA fusion protein (same sequence as in Examples 1-2) was administered and L6 myoblast uptake of the enzyme was measured. Figure 6 shows the superior uptake of vIGF2-rhGAA compared to rhGAA and M6P-GAA. Thus, vIGF2 is effective in targeting GAA to cells.

[0239] Example 4: Constructs for ERT delivered by gene therapy Two different constructs are shown in Figure 12. The top panel contains a construct containing a nucleic acid encoding recombinant human GAA with a Kozak sequence and a native signal peptide encoding "native hGAA" (SEQ ID NO: 189). The middle panel contains a construct encoding "engineered hGAA" (SEQ ID NO: 190), Kozak-BiP-vIGF2-2GS-GAA. This construct is characterized by a Kozak sequence, a nucleic acid encoding the BiP signal peptide, a nucleic acid encoding a vIGF2 peptide having the sequence shown in SEQ ID NO: 80, and a nucleic acid encoding a 2GS linker (SEQ ID NO: 181), followed by a nucleic acid encoding recombinant human GAA (SEQ ID NO: 1) with the N-terminal 60 amino acids removed to prevent premature processing and removal of vIGF2. The amino acid sequence of "engineered hGAA" is shown in SEQ ID NO: 2.

[0240] Example 5: Enhanced secretion of gene therapy constructs The engineered hGAA is more secreted and can interact with cell surface receptors appropriate for cellular uptake and lysosomal targeting. CHO cells expressing engineered or native hGAA, as described in more detail below, were cultured, and conditioned medium was collected for measurement of GAA activity. Figure 15 shows the relative activity of engineered and native hGAA, demonstrating increased activity of the engineered hGAA compared to native hGAA, indicating more efficient secretion of the engineered hGAA.

[0241] Example 6: Analysis of PPT1 in conditioned medium Cloning of PPT1 constructs PPT1 constructs were cloned into the pcDNA3.1 expression vector (ThermoFisher cat# V79020) containing the CMV promoter. Constructs tested included PPT1-1 (WT-PPT1) (SEQ ID NO: 4); PPT1-2 (WT-vIGF2-PPT1) (SEQ ID NO: 5); and PPT1-29 (BiP2aa-vIGF2-PPT1) (SEQ ID NO: 6).

[0242] PPT1 secretion and binding The PPT1 construct was transiently expressed in HEK293T cells for 3 days to allow PPT1 to be secreted into the culture medium. Secreted PPT1 was quantified by Western blotting and assayed for CI-MPR binding using established methods. Secreted PPT1 is shown in Figure 13. CI-MPR binding is shown in Figure 14.

[0243] Example 7: Testing gene therapy vectors in animal models of Pompe disease Pompe gene therapy: Preclinical proof of concept study design Preclinical studies were performed using a high dose in GAA knockout (GAA KO) mice for an initial comparison of the constructs. The constructs are shown in Figure 12. Mice were treated with vehicle or one of two constructs, native-hGAA or engineered-hGAA. Mice were administered 5e11 gc / mouse (approximately 2.5e13 gc / kg). Two-month-old GAA knockout mice were used. Normal (wild-type) mice served as controls. The study design is outlined in Figure 16.

[0244] Pompe Gene Therapy: Plasma Plasma was collected from wild-type (normal) mice or GAA KO mice treated with vehicle or the indicated gene therapy vectors to measure GAA activity and cell surface binding. The data are summarized in Figures 17, 27, and 19. Similar elevated GAA levels were observed in mice treated with the gene therapy vectors (Figures 17, 18). However, more cell-targeted receptor binding was observed with the engineered constructs (Figure 19).

[0245] Pompe Gene Therapy: Quadriceps GAA activity and glycogen storage / cytoplasmic vacuolation were assessed in normal (wild-type) and treated GAA KO mice (Figure 28). GAA activity in the quadriceps was approximately 20-fold higher than in wild-type mice. Glycogen PAS (Figure 29) and immunohistochemistry (Figure 30) were also assessed. Immunohistochemistry demonstrated greater lysosomal targeting of the engineered hGAA compared with wild-type mice. Glycogen depletion was more consistent for the engineered hGAA by PAS staining.

[0246] Pompe Gene Therapy: Triceps GAA activity and glycogen storage / cytoplasmic vacuolation were assessed in normal (wild-type) and treated GAA KO mice (Figure 31). GAA activity was approximately 10-15 times higher than in wild-type mice. Immunohistochemistry and glycogen PAS were also assessed (Figures 32 and 33). Immunohistochemistry demonstrated greater lysosomal targeting of the engineered hGAA compared with wild-type GAA. Glycogen depletion, as measured by PAS staining, was more consistent for the engineered hGAA.

[0247] Pompe gene therapy: tibialis anterior (TA) GAA activity and glycogen storage / cytoplasmic vacuolation were assessed in normal (wild-type) and treated GAA KO mice (Figure 20). GAA activity in the TA was approximately 15-20 times higher than in wild-type mice. Immunohistochemistry and glycogen PAS were also assessed (Figures 21 and 22). Immunohistochemistry demonstrated greater lysosomal targeting of the engineered hGAA compared with wild-type GAA. Glycogen levels were closer to wild-type levels. Glycogen depletion was more consistent for the engineered hGAA by PAS staining.

[0248] Pompe Gene Therapy: Brain and Spinal Cord GAA activity, glycogen content, and glycogen storage / cytoplasmic vacuolization were evaluated in normal (wild-type) and treated GAA KO mice (Figure 23). GAA activity in the brain was approximately 5-fold lower than in wild-type mice. Immunohistochemistry and glycogen PAS were also evaluated (Figures 24, 25, 26, and 27). Immunohistochemistry indicated possible direct transduction of some cells. However, glycogen clearance with the native construct was minimal. Although glycogen levels were close to wild-type levels with the engineered construct, activity was only 20% of that of wild-type. PAS staining in the spinal cord demonstrated little glycogen clearance with the native construct. Glycogen levels close to wild-type with the engineered construct were observed in the ventral horn, which contains motor neurons. Immunohistochemistry demonstrated direct transduction in spinal neurons. The engineered hGAA produced by choroid plexus and neuronal cells was able to lower glycogen by cross-complementation in the spinal cord, whereas little glycogen lowering was observed with naturally occurring hGAA.

[0249] conclusion Overall, the data in this example demonstrate that the tissue uptake and glycogen depletion of the engineered gene therapy construct, including effects in the brain and spinal cord, are dramatically better than wild-type GAA, which is used in conventional treatments.

[0250] Example 8: Animal Study Protocol AAVhu68 vectors were generated and titered by the Penn Vector Core as described (Lock, Alvira et al. 2010, "Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale." Hum Gene Ther 21(10):1259-1271).

[0251] Mus musculus, a Pompe mouse with a Gaa knockout on a C57BL / 6 / 129 background founder, was purchased from Jackson Labs (stock #004154, also known as 6neo mouse).

[0252] Mice received 5 x 10 11 GC (approximately 2.5 × 10 13 Mice received 1000 mg / kg (1000 GC / kg) of AAVhu68.CAG.hGAA (containing either native hGAA (SEQ ID NO: 189) or engineered hGAA (SEQ ID NO: 190)) via the lateral tail vein, were bled for serum isolation on days 7 and 21 post-vector administration, and finally on day 28 post-injection (for plasma isolation) and euthanized by exsanguination. Tissues were rapidly collected, starting with the brain.

[0253] GAA activity Plasma was mixed with 5.6 mM 4-MU-α-glucopyranoside, pH 4.0, and incubated at 37°C for 3 hours. The reaction was stopped with 0.4 M sodium carbonate, pH 11.5. Relative fluorescence units (RFU) were measured using a Victor3 fluorometer with excitation at 355 nm and emission at 460 nm. Activity in nmol / mL / hr was calculated by interpolation from a 4-MU standard curve. Activity in individual tissue samples was further normalized based on the total protein content in the homogenates.

[0254] GAA signature peptides by LC / MS Plasma was precipitated in 100% methanol and centrifuged. The supernatant was discarded. The pellet was spiked with a stable isotope-labeled peptide specific to hGAA as an internal standard, resuspended with trypsin, and incubated at 37°C for 1 hour. Digestion was stopped with 10% formic acid. Tryptic peptides were separated by C-18 reversed-phase chromatography and identified and quantified by ESI-mass spectrometry. Total GAA concentration in plasma was calculated from the signature peptide concentration.

[0255] Cell surface receptor binding assay 96-well plates were coated with receptor, washed, and blocked with BSA. Day 28 plasma from AAV-treated mice was serially diluted to give a series of decreasing concentrations and incubated with the coupled receptor. After incubation, the plate was washed to remove any unbound hGAA, and 4-MU-α-glucopyranoside was added for 1 hour at 37°C. The reaction was stopped with 1.0 M glycine, pH 10.5, and RFUs were read using a Spectramax fluorometer; excitation 370, emission 460. RFUs for each sample were converted to activity (nmol / mL / hr) by interpolation from a 4-MU standard curve. Nonlinear regression was performed using GraphPad Prism.

[0256] histology Tissues were formalin-fixed and paraffin-embedded. Muscle slides were stained with PAS; CNS slides were stained with luxol fast blue / Periodic Acid-Schiff (PaS). A board-certified veterinary pathologist (JH) blindly reviewed the histological slides. Semiquantitative estimation of the total percentage of cells with glycogen accumulation and cytoplasmic vacuolization was performed on scanned slides. A score of 0 to 4 was considered as described in the table below.

[0257] [Table 101]

[0258] Immunohistochemistry (IHC) We studied transgene expression and cellular localization from slides immunostained with anti-human GAA antibody (Sigma HPA029126).

[0259] Example 9: Histology in an Animal Model of Pompe Disease - Tissue Processing - Protocol and Results All tissues were fixed in 10% NBF (neutral buffered formalin). The assays (PAS and IHC) are routinely used in the art.

[0260] PAS staining of quadriceps and triceps muscles (Figures 29 and 32)—Tissues were fixed in 10% NBF and embedded in paraffin. Sections were post-fixed in 1% periodic acid and stained with Schiff's reagent. Sections were then counterstained with hematoxylin. Glycogen appears as magenta aggregates (lysosomal boundaries) or scattered pink (cytosol); nuclei are blue. Based on the images and assuming each is representative of the group, the ranking order for glycogen clearance is: engineered hGAA > native hGAA. The engineered hGAA construct resulted in more staining throughout the image compared to resting. This indicates improved endocytosis of GAA protein mediated by binding of vIGF2 to the CI-MPR.

[0261] PAS staining of spinal cord (Figure 26) - Tissues were fixed in 10% NBF. Post-fixation in 1% periodic acid could be performed before or after paraffin embedding. Sections were stained with Schiff's reagent and counterstained with appropriate methylene blue. Glycogen appears as magenta aggregates (lysosomal boundaries); nerve fibers appear blue. Images focused on glycogen accumulation in the ventral horn of the spinal cord and motor neurons. Engineered hGAA appeared to be the most effective at glycogen depletion among the constructs.

[0262] GAA IHC (Figures 22, 25, 27, 30, and 35) - Tissues were fixed in 10% NBF and embedded in paraffin. Sections were incubated with anti-GAA primary antibody followed by a secondary antibody with an enzyme tag, HRP, that recognizes the primary antibody. An enzymatic reaction was then performed to form a brown precipitate product. Sections were then counterstained with hematoxylin. The constructs showed GAA incorporation into muscle fibers (Figure 31). Engineered hGAA > native hGAA. The BiP-vIGF2 construct had more scattered staining throughout the image compared to rest.

[0263] Compared with other vectors, the engineered hGAA resulted in more GAA IHC signals with a punctum-like appearance inside the muscle fibers, indicating highly effective lysosomal targeting (Figure 22).

[0264] In all, the engineered hGAA consistently demonstrated superior tissue uptake, lysosomal targeting, and glycogen depletion in various tissues among the constructs.

[0265] Example 10: Binding of fusion proteins to CIMPR In this example, a therapeutic enzyme was engineered to target the CI-MPR. The data in this example show that fusion proteins bind better to the CI-MPR when they contain a vIGF2 tag. This was also shown for enzymes known to be highly phosphorylated, such as PPT1.

[0266] Each transgene was cloned into the pIREShyg3 plasmid, and the DNA was transfected into suspension HEK 293K cells using PEI transfection reagent. Cells were grown in FreeStyle293 expression medium. Conditioned medium was harvested from the cells 3-4 days after transfection. The amount of enzyme secreted into the conditioned medium was determined by activity assay or signature peptide assay. These concentrations were used to set up CIMPR binding assays.

[0267] In the binding assay, a plate was first coated with CI-MPR. Then, a sample containing the enzyme of interest was incubated on the plate. The plate was washed so that only the material bound to CI-MPR remained on the plate. The amount of the enzyme of interest bound to the plate was determined by enzyme assay or mass spectrometry. The binding assay was performed over a wide range of concentrations of the enzyme of interest to obtain a binding curve.

[0268] The amount of tagged and untagged enzyme bound to the plate was determined to construct a binding curve. In the case of AGA and TPP1, this was determined by performing an enzyme activity assay. In the other cases, a signature peptide assay was performed to determine the amount of enzyme binding.

[0269] The TPP1 activity assay is described at www.rndsystems.com / products / recombinant-human-tripeptidyl-peptidase-i-tpp1-protein-cf_2237-se#product-details.

[0270] AGA activity assays are described in YaV, et al. Applications of a new fluorometric enzyme assay for the diagnosis of aspartylglucosaminuria. J Inherit Metab Disease 1993 and Banning, et al. Identification of Small Molecule Compounds for Pharmacological Chaperone Therapy of Aspartylglucosaminuria. Sci Rep 2016.

[0271] Figure 34 shows the increased binding of engineered PPT1 compared to wild-type PPT1. Figure 35 shows the increased binding of engineered TPP1 compared to wild-type TPP1. Figure 36 shows the increased binding of engineered AGA compared to wild-type AGA. Figure 37 shows the increased binding of engineered GLA compared to wild-type GLA.

[0272] Example 11: Cloning of PPT1 fusions All PPT1 constructs were assembled into the pcDNA3.1 expression vector using the In-Fusion cloning kit from Takara Bio Inc.

[0273] The linearized pcDNA3.1 vector and each PPT1 gene fragment were recombined via InFusion reaction to obtain the final pcDNA3.1 vector carrying the indicated PPT1 construct.

[0274] Example 12: Cloning vIGF2 mutants All vIGF2 mutants were swapped into the pcDNA3.1-BiP-vIGF2-2GS-GAA expression vector using the In-Fusion cloning kit from Takara Bio Inc.

[0275] Recombination of the ordered vIGF2 fragment with the linearized pcDNA3.1-GAA vector via an InFusion reaction yielded the final pcDNA3.1-BiP-vIGF2 * The -2GS-GAA circular expression vector was obtained.

[0276] Example 13: Characterization of the vIGF2-GAA construct Transient transfection of HEK293T cells with pcDNA3.1-vIGF2-GAA plasmid HEK293T cells were transiently transfected with 1 μg of DNA using Fugene HD transfection reagent. Cultures were incubated at 37°C with 5% CO2 for an additional 2-5 days before harvesting conditioned medium and cell pellets.

[0277] Western blot analysis of vIGF2-GAA in conditioned medium Western blots were performed using standard methods with the Licor Odyssey detection system. The primary antibody used for vIGF2-GAA detection was our in-house rabbit anti-GAA antibody (FL059). The secondary antibody used for GAA was goat anti-rabbit DyLight 800 (ThermoFisher cat#SA5-35571).

[0278] GAA activity assay GAA activity was measured as previously described.

[0279] CI-MPR binding assay CI-MPR binding was measured as previously described.

[0280] Cellular uptake assay The results of generating the 30+IGF2-GAA construct are as follows.

[0281] The vIGF2-GAA constructs that showed secretion / expression levels of 80% or more of the original vIGF2 were vIGF2-4, 5, 10, 11, 14, 16, 17, 31, and 32 (Figures 38 and 39).

[0282] The vIGF2-GAA constructs that showed secretion / expression levels of 50% or more of the original vIGF2 were vIGF2-4, 5, 6, 9-14, 16-23, 25, 27, and 29-34 (Figures 38 and 39).

[0283] All vIGF2-GAA constructs appeared to be correctly processed inside the cells, with the mature GAA peptide fragment of 70 / 76 KDa being observed (FIG. 38).

[0284] vIGF2-17 consistently demonstrated a significantly higher CI-MPR binding Bmax than native vIGF2 (Figures 40, 41, 44, and 45).

[0285] vIGF2-24 bound to the CI-MPR significantly better than native vIGF2 (FIGS. 42 and 43).

[0286] vIGF2-GAA constructs with comparable or better PM25 cell uptake properties relative to native vIGF2 include vIGF2-7, vIGF2-10, vIGF-17, vIGF2-18, vIGF2-20, vIGF2-22, and vIGF2-23 (Figures 46 and 47).

[0287] Example 14: Testing of PPT1 constructs vIGF2 peptides were designed as discussed elsewhere herein. Variants were selected based on increased selective binding to the CI-MPR and improved protein expression. Exemplary peptides and their structures are shown in Figure 48.

[0288] Transient transfection of HEK293T cells with pcDNA3.1-PPT1 plasmid HEK293T cells grown to approximately 80% confluence in 1 mL OptiMEM medium supplemented with 5% FBS in 12-well cultures were transiently transfected with 1 μg of DNA using Fugene HD transfection reagent. Cultures were incubated at 37°C with 5% CO2 for an additional 2–5 days before harvesting the conditioned medium and cell pellets.

[0289] Western blot analysis of PPT1 in conditioned medium Western blots were performed using standard methods with the Licor Odyssey detection system. The primary antibody used for PPT1 detection was a mouse polyclonal antibody from Abcam (catalog cat# ab89022). The secondary antibody used for PPT1 was goat anti-mouse DyLight 800 (ThermoFisher cat# SA5-35521).

[0290] A graph showing the Western blot and band intensity of PPT1 expression is shown in Figure 49. A graph showing PPT1 in conditioned medium as quantified by Western blot is shown in Figure 50.

[0291] PPT1 activity assay The PPT1 activity assay used was essentially that described by Van Diggelen et al. (Mol Genet Metab. 66:240-244, 1999). Briefly, in a typical PPT1 activity assay, 10 μl of conditioned medium containing secreted PPT1 was mixed with 90 μl of reaction buffer containing 75 μM MU-6S-Palm-βGlc (4-methylumbelliferyl-6-thiopalmitate-β-D-glucopyranoside, Cayman Chemical; CAS 229644-17-1), 2 U / mL β-glucosidase (Sigma Chemicals; CAS 9001-22-3; G4511), 20 mM citrate pH 4.0, 5 mM DTT, 0.02% Triton X-100, and 50 mM NaCl in a 96-well black clear-bottom plate (Corning cat#3631). Fluorescence was monitored at 30-second intervals over 1 hour at 25°C using a SpectraMax M2 spectrophotometer, using an excitation wavelength of 330 nm and an emission wavelength of 450 nm. The kinetics of the PPT1 reaction was extracted by fitting the time course fluorescence data with linear regression.

[0292] A graph showing PPT1 in conditioned medium quantified by activity is shown in Figure 51. Activity was found to have a strong correlation with Western blot results. Figure 52 shows the correlation between activity and Western blot quantification.

[0293] PPT1 stability assay Briefly, in a typical stability assay, 180 μL of conditioned medium containing PPT1 was diluted with 20 μL of 10×PBS, pH 7.4, and incubated at 37° C. At different time points, 15 μL aliquots were taken and flash-frozen in dry-ice-cooled ethanol. After the time course experiment was completed, the frozen samples were thawed and PPT1 activity was measured using a PPT1 activity assay.

[0294] CI-MPR binding assay CI-MPR plate binding assays were performed as described above, and the amount of binding was then determined by PPT1 activity assay.

[0295] The binding of PPT1 constructs to CI-MPR in the presence of M6P is in the table below. The binding curves are shown in Figure 53.

[0296] [Table 102]

[0297] Six PPT1 constructs were selected for further analysis. These six constructs are shown in Figure 54. PPT1 secretion into the medium (Figure 55), PPT1 processing within the cells (Figure 56), PPT1 quantification by Western blot (Figure 57), and activity (Figure 58) were determined for these six constructs.

[0298] Example 15 Engineering and testing of additional PPT1 IGF2 fusion constructs Additional PPT1 constructs were designed and cloned as shown in Figure 62. These constructs contain either the endogenous signal sequence with a C6S mutation (SEQ ID NO: 177), optionally with a two-alanine stretch (SEQ ID NO: 178) to improve cleavage, or the modified BiP signal peptide, BiP-2 (SEQ ID NO: 171), a PPT1 sequence comprising amino acid residues 21-306 or 28-306 of wild-type human PPT1 (SEQ ID NO: 4), a GS linker (SEQ ID NOs: 181-187), and variant IGF2-31 or 32 (SEQ ID NO: 120 or 121), separated by the lysosomal cleavage site RPRAVPTQA (SEQ ID NO: 188).

[0299] All PPT1 constructs (Figure 62) were transiently expressed in FreeStyle 293 suspension cells. Briefly, FreeStyle 293 cells were transfected with each PPT1 construct in a pcDNA3.1 backbone using polyethyleneimine (PEI) as the transfection reagent. After 4 days of expression in the FreeStyle 293 expression medium, conditioned medium from each transfection was collected and run on a Western blot using an anti-PPT1 primary antibody. Relative PPT1 levels in the medium were quantified from the band density on these Western blots. Figure 63 shows that several constructs tested were secreted into the medium at levels higher than WT PPT1. The higher PPT1 levels in the conditioned medium reflect both good expression and efficient secretion from the cells. vIGF2-31 (SEQ ID NO: 120) and vIGF2-32 (SEQ ID NO: 32) were designed to improve CIMPR binding and surprisingly, enhanced PPT1 expression and secretion compared to the initial IGF2 variant (SEQ ID NO: 80).

[0300] Neuronal uptake experiments with purified protein constructs PPT1-101 and PPT1-104 demonstrated successful uptake of both proteins, with PPT1-104 being taken up approximately twice as efficiently as PPT1-101 (Figure 64A). For this experiment, rat cerebral cortical neurons were cultured in NeuroCult medium and plated on poly-L-lysine-coated coverslips. Neurons were treated with 5 μg / ml of purified PPT1-101 or PPT1-104 labeled with Alexa Fluor 680 fluorescent dye. After 1 hour of incubation, cells were fixed, permeabilized, and imaged using a Leica SP8 confocal microscope.

[0301] Neuronal uptake experiments with conditioned medium were performed using conditioned medium obtained from FreeStyle 293 cell transfection as described above. The concentration of each PPT1 construct protein in the medium was first determined via Western blot using a standard curve generated using samples of known concentrations of PPT1. Each sample of conditioned medium was concentrated before processing neurons. Rat cerebral cortical neurons were cultured in Primary Neuron Growth Medium and plated on poly-L-lysine-coated coverslips. Neurons were treated with the following concentrations of PPT1 protein in the medium:

[0302] [Table 103]

[0303] After 1 hour of incubation, cells were fixed, permeabilized, and imaged using a Leica SP8 confocal microscope. Uptake by all PPT1 variants was higher than by WT PPT1; PPT1-104 and PPT1-117 showed the highest uptake levels (Figure 64B).

[0304] Example 16 Analysis of NAGLU constructs Mutant fusion proteins containing recombinant human NAGLU protein with an N-terminal vIGF2 tag inserted between the signal peptide and the NAGLU protein were designed as shown in Figure 65. Several variants were prepared, including fusion proteins containing vIGF2 (SEQ ID NO: 80), vIGF2-17 (SEQ ID NO: 106), vIGF2-31 (SEQ ID NO: 120), and vIGF2-32 (SEQ ID NO: 121). Fusion proteins were expressed in HEK293F cells. NAGLU content in lysates and media fractions, as determined by Western blotting with ab214671 (R&D systems), is shown for each fusion protein tested (Figures 66A-66B). Enzyme activity in the conditioned medium for each fusion protein was determined by a 4-MU assay (Figure 66C). Protein amounts in the conditioned medium were not normalized / averaged. Activity data represent the relative secretion of constructs into the conditioned medium, rather than the relative specific activity of equal amounts of protein. As can be seen in Figure 66, the presence of variant IGF2 led to reduced expression and secretion compared to untagged NAGLU. However, CIMPR binding of IGF2-tagged NAGLU was significantly improved compared to untagged NAGLU (Figure 67). Notably, approximately 2.5-fold less IGF2-tagged NAGLU compared to WT was used as input for the binding assay (even more of the tagged compared to WT bound to the immobilized receptor).

[0305] Example 17 Analysis of TPP1 constructs A series of nucleic acid constructs expressing TPP1 fusion proteins linked to IGF2 variants were designed and tested for expression, secretion, and CIMPR binding. The fusion proteins contain a signal peptide (SEQ ID NO:179), variant IGF2 sequences (SEQ ID NOs:80, 106, 111, 133, 119-121), a GS linker (GGGGSGGGGS, SEQ ID NO:186), a lysosomal cleavage site (RPRAVPTQA, SEQ ID NO:188), a TPP1 propeptide (SEQ ID NO:45), and a TPP1 mature peptide (SEQ ID NO:46). Constructs with N- and C-terminal vIGF2 tags were generated and tested. Examples of PPT1 fusion proteins designed and tested are shown in Table 11.

[0306] [Table 104]

[0307] Expression and Secretion For each construct, Freestyle 293 cells (3.7 million cells in 1.5 ml of Freestyle 293 medium) were transfected with 9 μl of 1 mg / ml PEI and 3 μg DNA and grown in 24-well deep-well plates under shaking conditions (37°C, 5% CO2, 80% RH, 250 RPM). Approximately 24 hours after transfection, valproic acid (final concentration 2.2 mM) and an additional 1.5 ml of Freestyle medium were added to the transfection. Cultures were harvested 3 days after transfection and centrifuged to separate cells and conditioned medium. Proteins in the conditioned medium were separated on an SDS-PAGE gel and transferred to a nitrocellulose membrane. Membranes were blocked with 5% milk and probed with anti-TPP1 (abcam EPR16537) and Licor anti-rabbit 800CW (926-32213). The blots were imaged and the bands were quantified on a Licor Odyssey CLX as shown in FIG.

[0308] CIMPR binding CIMPR binding was measured essentially as described in Example 10. The results are shown in Figure 69. rhTPP1 (R&D system #2237-SE-010, expressed in mouse myeloma NS0 cells) and WT TPP1 (SEQ ID NO: 8) were included as controls. As shown in Figure 69, all of the novel TPP1 constructs showed improved binding compared to rhTPP1.

[0309] Example 18 Testing of a Novel PPT1 Variant CLN1 Mouse Model The PPT1-101 (SEQ ID NO: 60) and PPT1-104 (SEQ ID NO: 61) constructs were cloned into CLN1 R151XThis was tested in a mouse model (Miller, 2014, Human Molecular Genetics, 24(1)185-196). Gene therapy constructs containing the coding sequences of PPT1-101 (SEQ ID NO: 228) and PPT1-104 (SEQ ID NO: 235) were prepared. Mice were injected intracerebroventricularly with the viral constructs (or PBS control) at postnatal day 1 (P1) at a dose of 5x1010, 1x1010, or 1x109 vg / animal. Wild-type PPT1 (p546) was included as a control. The transgene was delivered using an AAV9 vector. Outcomes were assessed at 2 months of age.

[0310] Transgene expression Human CLN1 transgene expression was detected by RT-qPCR. As can be seen in Figure 70, brain and spinal cord extracts showed similar gene expression between the various constructs, with higher expression in the cortex.

[0311] Reduction of autofluorescence storage materials Figures 71-72 show the effect of each construct on brain autofluorescent material (ASM) accumulation, a correlate of lysosomal dysfunction. 10 and 1 x 10 10 and 1 × 10 in the thalamus 10 and 1 x 10 9 At this dose, the 101 and 104 constructs lead to a greater reduction in ASM compared to the WT p546 construct.

[0312] Decreased glial fibrillary acidic protein (GFAP) Figure 73 shows the effect of each construct on glial fibrillary acidic protein (GFAP), a correlate of astrogliosis and neuroinflammation. At a dose of 1 x 10 in the cortex, the 10 construct led to a greater reduction in GFAP. At a dose of 1 x 10 in the cortex, the 10 construct led to a greater reduction in GFAP. At a dose of 1 x 10 in the thalamus, the 10 construct led to a greater reduction in GFAP. GFAP-positive cells were morphologically consistent with a reactive astrocyte phenotype.

[0313] Thus, the novel PPT1 101 and PPT1 104 gene therapy constructs showed improved cross-correction compared to wild-type PPT1 in the CLN1 mouse model, leading to greater reductions in both ASM and GFAP in the cortex and thalamus.

[0314] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will readily occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be used. It is intended that the following claims define the scope of the invention, and that methods and structures within the scope of the claims and their equivalents be covered thereby.

Claims

1. (a) a nucleic acid sequence encoding a therapeutic protein, and (b) a nucleic acid sequence encoding a variant IGF2 (vIGF2) peptide that is at least 95% identical to at least one sequence selected from SEQ ID NOs: 90-103. A nucleic acid construct comprising:

2. 2. The nucleic acid construct of claim 1, wherein the vIGF2 peptide has an amino acid sequence that is at least 98% identical to an IGF2 variant peptide selected from SEQ ID NOs: 106, 109, 111, 119, 120, and 121.

3. 2. The nucleic acid construct of claim 1, wherein the vIGF2 peptide comprises an amino acid sequence that is at least 98% identical to an IGF2 variant peptide selected from the group consisting of SEQ ID NO: 120 and SEQ ID NO:

121.

4. The nucleic acid construct according to any one of claims 1 to 3, further comprising a sequence encoding a linker having a sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 181 to 188.

5. The nucleic acid construct of any one of claims 1 to 4, wherein the vIGF2 peptide is capable of increasing the expression and / or secretion of a therapeutic protein compared to a vIGF2 peptide having the amino acid sequence of SEQ ID NO:

80.

6. The nucleic acid construct of any one of claims 1 to 4, wherein the vIGF2 peptide has increased affinity for CI-MPR compared to the vIGF2 peptide having the amino acid sequence of SEQ ID NO:

80.

7. The nucleic acid construct of any one of claims 1 to 4, wherein the vIGF2 peptide is capable of improving the uptake of the therapeutic protein into cells.

8. 8. The nucleic acid construct of any one of claims 1 to 7, wherein the therapeutic protein is capable of replacing a defective or deficient protein associated with a genetic disorder in a subject having the genetic disorder.

9. The nucleic acid construct of claim 8 , wherein the genetic disorder is a lysosomal storage disorder.

10. The genetic disorders include aspartylglucosaminuria, neuronal ceroid lipofuscinosis, CLN1 / PPT1 disease, CLN2 / PPT1 disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Sanfilippo disease type E, Sanfilippo disease type F, Sanfilippo disease type I, Sanfilippo disease type F, Sanfilippo disease type F, Sanfilippo disease type F, Sanfilippo disease type I, Sanfilippo disease type F, Sanfilippo disease type F, Sanfilippo disease type I, Sanfilippo disease type F, Sanfilippo disease type I, Sanfilippo disease type II, Sanfilippo disease type III, Sanfilippo disease type IV, Hurler disease, Hunter disease, Sanfilippo disease type A, Sanfilippo disease type F ...

9. The nucleic acid construct of claim 8, wherein the nucleic acid construct is selected from the group consisting of Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase severe combined immunodeficiency (ADA-SCID), and neuronal ceroid lipofuscinosis.

11. 10. The nucleic acid construct of claim 8 or 9, wherein the genetic disorder is selected from the group consisting of CLN1 / PPT1 disease, CLN2 / PPT1 disease, Pompe disease, and MPS IIIB disease.

12. The nucleic acid construct of claim 11 , wherein the genetic disorder is CLN1 / PPT1 disease or CLN2 / PPT1 disease.

13. The therapeutic protein may be alpha-galactosidase (A or B), beta-galactosidase, beta-hexosaminidase (A or B), galactosylceramidase, arylsulfatase (A or B), beta-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, the lysosomal enzyme acid sphingomyelinase, formylglycine generating enzyme, iduronidase (e.g., alpha-L), acetyl-CoA:alpha-glucosaminide N-acetyltransferase, glycosaminoglycan alpha-L-iduronohydrolase, heparan N-sulfatase, N-acetyl-α-D-glucosaminidase (NAGLU), iduronate-2-sulfatase, galactosamine-6-sulfatesulfatase, N-acetylgalactosamine-6-sulfate sulf ...

13. The nucleic acid construct of any one of claims 1 to 12, comprising a human enzyme selected from the group consisting of galactosamine-6-sulfatase, N-sulfoglucosamine sulfohydrolase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, β-glucuronidase, hyaluronidase, alpha-N-acetylneuraminidase (sialidase), ganglioside sialidase, phosphotransferase, alpha-glucosidase, alpha-D-mannosidase, beta-D-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase, battenin, PPT1, TPP1, and other Batten-related proteins (e.g., ceroid-lipofuscinosis neuronal protein 6), or enzymatically active fragments thereof.

14. 14. The nucleic acid construct of claim 13, wherein the therapeutic protein is alpha-glucosidase, or an enzymatically active fragment thereof.

15. The nucleic acid construct of claim 13 , wherein the therapeutic protein is human PPT1.

16. The nucleic acid construct of claim 13, wherein the therapeutic protein is human TPP1.

17. The nucleic acid construct of claim 13 , wherein the therapeutic protein is human NAGLU.

18. The nucleic acid construct of claim 1 , further comprising a sequence encoding a signal peptide.

19. 19. The nucleic acid construct of claim 18, wherein the signal peptide is one of the sequences selected from the group consisting of SEQ ID NOs: 169 to 180.

20. 20. The nucleic acid construct of any one of claims 1 to 19, wherein the vIGF2-encoding nucleic acid sequence is 5' to the nucleic acid sequence encoding a therapeutic protein.

21. 20. The nucleic acid construct of any one of claims 1 to 19, wherein the vIGF2-encoding nucleic acid sequence is 3' to the nucleic acid sequence encoding a therapeutic protein.

22. A gene therapy vector comprising the nucleic acid construct of any one of claims 1 to 21.

23. The gene therapy vector of claim 22 which is a viral vector.

24. 24. The gene therapy vector of claim 23, wherein the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a retroviral vector, a lentiviral vector, a poxvirus vector, a vaccinia viral vector, an adenovirus vector, or a herpes viral vector.

25. The nucleic acid construct according to any one of claims 1 to 21, wherein the nucleic acid construct is a plasmid.

26. A pharmaceutical composition comprising a therapeutically effective amount of a nucleic acid construct according to any one of claims 1 to 23, or a gene therapy vector according to any one of claims 22 to 24, and a pharmaceutically acceptable carrier or excipient.

27. 26. The pharmaceutical composition of claim 25, wherein the excipient comprises a non-ionic hypo-osmotic compound, a buffer, a polymer, a salt, or a combination thereof.

28. A method for treating a genetic disorder, comprising administering to a subject in need of treatment for the genetic disorder the nucleic acid construct of any one of claims 1 to 23, or the pharmaceutical composition of claim 25 or 26.

29. 28. The method of claim 27, wherein the genetic disorder is a lysosomal storage disorder.

30. The genetic disorders include aspartylglucosaminuria, neuronal ceroid lipofuscinosis, CLN1 / PPT1 disease, CLN2 / PPT1 disease, cystinosis, Fabry disease, Gaucher disease type I, Gaucher disease type II, Gaucher disease type III, Pompe disease, Tay-Sachs disease, Sandhoff disease, metachromatic leukodystrophy, mucolipidosis type I, mucolipidosis type II, mucolipidosis type III, mucolipidosis type IV, Hurler disease, Hunter disease, and Sanfilippo disease type A. , Sanfilippo disease type B, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroteaux-Lamy disease, Sly disease, Niemann-Pick disease type A, Niemann-Pick disease type B, Niemann-Pick disease type C1, Niemann-Pick disease type C2, Schindler disease type I, Schindler disease type II, adenosine deaminase-associated severe combined immunodeficiency (ADA-SCID), and chronic granulomatous disease (CGD).

31. 30. The method of claim 29, wherein the genetic disorder is CLN1 / PPT1 disease.

32. 30. The method of claim 29, wherein the genetic disorder is CLN2 / TPP1 disease.

33. 30. The method of claim 29, wherein the genetic disorder is Sanfilippo disease type B.

34. 33. The method of any one of claims 27 to 32, wherein the administration is performed intrathecally, intraocularly, intravitreally, retinal, intravenous, intramuscular, intracerebroventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneously, or a combination thereof.

35. 32. The method of claim 31, wherein administration of the nucleic acid, gene therapy vector, or pharmaceutical composition prevents / reduces or reverses accumulation of autofluorescent material (ASM) in the brain.

36. 32. The method of claim 31, wherein administration of the nucleic acid, gene therapy vector, or pharmaceutical composition prevents / reduces or reverses elevation of glial fibrillary acidic protein (GFAP) in the brain.

37. 36. The method of claim 35, wherein administration of the nucleic acid, gene therapy vector, or pharmaceutical composition prevents / reduces or reverses accumulation of autofluorescent material (ASM) in the cortex or thalamus.

38. 37. The method of claim 36, wherein administration of the nucleic acid, gene therapy vector, or pharmaceutical composition prevents / reduces or reverses elevation of glial fibrillary acidic protein (GFAP) in the cerebral cortex or thalamus.

39. 39. The method of any one of claims 31, 35 to 38, wherein the nucleic acid encodes a fusion protein having a sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 60 to 67.

40. 33. The method of claim 32, wherein the nucleic acid encodes a fusion protein having a sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 47-53.

41. a. an amino acid sequence at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 106, 109, 111, 119, 120, and 121; and b. an amino acid sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:4, residues 21-306 of SEQ ID NO:4, residues 28-306 of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NOs:54-59, residues 24-743 of SEQ ID NO:54, and SEQ ID NO:46 The nucleic acid according to any one of claims 1 to 13, encoding a fusion protein comprising:

42. 42. The nucleic acid of claim 41, comprising a sequence encoding a fusion protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 120 and 121.

43. 43. The nucleic acid of claim 41 or 42, further comprising a sequence encoding a lysosomal-cleaving peptide.

44. 46. ​​The nucleic acid of any one of claims 43 to 45, wherein the fusion protein has a sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 60 to 67 and SEQ ID NOs: 47 to 53.

45. 45. The nucleic acid of claim 44, wherein the fusion protein has a sequence that is at least 98% identical to a sequence selected from the group consisting of SEQ ID NOs: 60-67 and 47-53.

46. A pharmaceutical composition comprising the nucleic acid of any one of claims 41 to 44 and a pharmaceutically acceptable carrier or excipient.

47. A variant IGF2 (vIGF2) peptide that is at least 98% identical to at least one sequence selected from the group consisting of SEQ ID NOs: 90-103.

48. 48. The variant IGF2 (vIGF2) peptide of claim 47, which is at least 98% identical to at least one sequence selected from SEQ ID NOs: 106, 109, 111, 119, 120, and 121.

49. 49. A fusion protein comprising the variant vIGF2 peptide of claim 47 or 48, further comprising a therapeutic protein having an amino acid sequence at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:4, amino acid residues 21-306 of SEQ ID NO:4, amino acid residues 28-306 of SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:46, SEQ ID NO:54, and amino acid residues 24-743 of SEQ ID NO:

54.

50. 50. The fusion protein of claim 49, having an amino acid sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 60-67, 47-53, and 54-59.

51. 51. The fusion protein of claim 49 or 50, further comprising a lysosomal cleaving peptide.

52. 52. The fusion protein of any one of claims 49 to 51, wherein the vIGF2 peptide is at the N-terminus of the therapeutic protein.

53. 52. The fusion protein of any one of claims 49 to 51, wherein the vIGF2 peptide is at the C-terminus of the therapeutic protein.

54. The fusion protein of any one of claims 49 to 51, wherein the fusion protein comprises a signal sequence.

55. 55. The fusion protein of claim 54, wherein the signal sequence has an amino acid sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NOs: 169-180.

56. 57. The fusion protein of claim 56, wherein the vIGF2 peptide is at least 98% identical to SEQ ID NO: 120 or 121.

57. 58. The fusion protein of claim 57, wherein the therapeutic protein is selected from the group consisting of PPT1 or an enzymatically active fragment thereof, TPP1 or an enzymatically active fragment thereof, and NAGLU or an enzymatically active fragment thereof.

58. 58. The fusion protein of claim 57, wherein the fusion protein is taken up by a target cell more efficiently than a corresponding protein lacking the vIGF2 peptide.

59. A pharmaceutical composition comprising the fusion protein of any one of claims 49 to 58 and a pharmaceutically acceptable carrier or excipient.

60. 60. A method for treating a lysosomal storage disorder, comprising administering the pharmaceutical composition of claim 59 to a subject in need of treatment for a lysosomal storage disorder.

61. 61. The method of claim 60, wherein the lysosomal storage disorder is selected from the group consisting of CLN1 / PPT1 disease, CLN2 / TPP1 disease, and Sanfilippo type B disease.

62. 62. The method of claim 60 or 61, wherein the administration is performed intrathecally, intraocularly, intravitreally, retinal, intravenous, intramuscular, intracerebroventricular, intracerebral, intracerebellar, intraventricular, intraparenchymal, subcutaneously, or a combination thereof.

63. 63. The method of claim 61 or 62, wherein administration of the pharmaceutical composition prevents / reduces or reverses accumulation of autofluorescent material (ASM) in the brain.

64. 63. The method of claim 61 or 62, wherein administration of the pharmaceutical composition prevents / reduces or reverses elevation of glial fibrillary acidic protein (GFAP) in the brain.

65. 64. The method of claim 63, wherein administration of the pharmaceutical composition prevents / reduces or reverses accumulation of autofluorescent material (ASM) in the cortex or thalamus.

66. 65. The method of claim 64, wherein administration of the pharmaceutical composition prevents / reduces or reverses elevation of glial fibrillary acidic protein (GFAP) in the cerebral cortex or thalamus.

67. A nucleic acid encoding a fusion protein comprising vIGF2 and a therapeutic protein, said nucleic acid being at least 85% identical to a sequence selected from the group consisting of SEQ ID NOs: 189-250.

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