Disulfide-stabilized polypeptide compositions and methods of use

Abstract

Provided herein are polypeptides comprising one or more non-natural cysteine ​​residues that form a disulfide bridge between non-natural cysteines within the protein or between non-natural cysteines of two monomers of the protein. Such modified human polypeptides can be used to treat genetic diseases via enzyme replacement therapy and / or gene therapy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 744,069, filed on October 10, 2018, which is hereby incorporated by reference in its entirety. Background Art

[0003] Genetic diseases can be treated by enzyme replacement therapy using recombinant polypeptides or gene therapy using nucleic acids encoding recombinant proteins. For example, Fabry disease can be treated with recombinant α-galactosidase A or small molecule chaperones such as 1-deoxygalactonojirimycin (Migalastat). However, recombinant wild-type polypeptides are generally less stable at neutral pH and rapidly degrade in serum. This greatly limits the half-life of the therapeutic enzyme because it is delivered by intravenous infusion. Summary of the invention

[0004] In certain aspects, a gene therapy vector comprising a nucleic acid construct is provided, the nucleic acid construct comprising: a nucleic acid encoding a stable form of a protein for treating a genetic disorder. In some embodiments, the stable form comprises one or more non-natural cysteine ​​residues that form a disulfide bridge between non-natural cysteines within the protein or between non-natural cysteines of two monomers of the protein. In some embodiments, the protein is selected from the group consisting of α-galactosidase A, β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan α-L-iduronidase, α-L-iduronidase, N-sulfoglucosamine sulfohydrolase (SGSH), N-acetyl-α-glucosaminidase (NAGLU), iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, α-glucosidase, tripeptidyl peptidase 1 (TPP1), palmitoyl protein thioesterase (PPT), neuronal ceroid lipofuscinosis protein (ceroidlipofuscinosis protein neuronal)1, neuronal ceroid lipofuscinosis protein 2, neuronal ceroid lipofuscinosis protein 3, neuronal ceroid lipofuscinosis protein 4, neuronal ceroid lipofuscinosis protein 5, neuronal ceroid lipofuscinosis protein 6, neuronal ceroid lipofuscinosis protein 7, neuronal ceroid lipofuscinosis protein 8, neuronal ceroid lipofuscinosis protein 9, neuronal ceroid lipofuscinosis protein 10, neuronal ceroid lipofuscinosis protein 11, neuronal ceroid lipofuscinosis protein 12, neuronal ceroid lipofuscinosis protein 13, neuronal ceroid lipofuscinosis protein 14, neuronal ceroid lipofuscinosis protein 15, neuronal ceroid lipofuscinosis protein 16 and cyclin-dependent kinase-like 5.In some embodiments, the protein is selected from the group consisting of α-galactosidase A, β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan α-L-iduronidase, α-L-iduronidase, N-sulfoglucosamine sulfohydrolase (SGSH), N-acetyl-α-glucosaminidase (NAGLU), iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, α-glucosidase. enzyme, tripeptidyl peptidase 1 (TPP1), palmitoyl protein thioesterase (PPT), neuronal ceroid lipofuscinosis protein 4, neuronal ceroid lipofuscinosis protein 10 (cathepsin D), neuronal ceroid lipofuscinosis protein 11 (granulin precursor), neuronal ceroid lipofuscinosis protein 13 (cathepsin F), neuronal ceroid lipofuscinosis protein 14 (KCTD7), neuronal ceroid lipofuscinosis protein 15 (TBCK) and cyclin-dependent kinase-like 5. In some embodiments, the stable protein comprises a lysosomal enzyme. In some embodiments, the stable protein comprises a stable α-galactosidase (α-GAL) protein. In some embodiments, the stable α-galactosidase A (α-GAL) protein comprises one or more non-native cysteine ​​residues selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the stable protein comprises a stable palmitoyl protein thioesterase 1 (PPT1). In some embodiments, the stable PPT1 protein comprises non-native cysteine ​​residues A171C and A183C. In some embodiments, the stable protein has a longer half-life at pH 7.4 compared to the corresponding protein without the non-native cysteine. In some embodiments, the stable protein can replace a defective or insufficient protein in the genetic disorder. In some embodiments, the stable protein can alleviate or slow down one or more symptoms associated with the genetic disorder. In some embodiments, the stable protein is more effective in alleviating or slowing down one or more symptoms of the genetic disorder compared to an unstable protein. In some embodiments, the genetic disorder is a neurological disorder. In some embodiments, the genetic disorder is a lysosomal storage disease.In some embodiments, the genetic 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, Maroteau-Lamy disease, Sly disease, 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), infantile, juvenile, and adult forms of neuronal ceroid lipofuscinosis, and CDKL5 deficiency. In some embodiments, the gene therapy vector is a viral vector selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, and a herpes virus vector. In some embodiments, the adeno-associated virus is a serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.74, AAV-B1 and AAV-hu68. In some embodiments, the nucleic acid construct is contained in a viral vector genome. In some embodiments, the viral vector genome comprises a recombinant AAV (rAAV) genome. In some embodiments, the rAAV genome comprises a self-complementary genome. In some embodiments, the rAAV genome comprises a single-stranded genome. In some embodiments, the rAAV genome comprises a first inverted terminal repeat sequence and a second inverted terminal repeat sequence. In some embodiments, the AAV inverted terminal repeat sequence is an AAV2 inverted terminal repeat sequence. In some embodiments, the rAAV genome further comprises an SV40 intron.In some embodiments, the rAAV genome further comprises a polyadenylation sequence. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the nucleic acid sequence is at least 85% identical to one of SEQ ID NOs: 7-12. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the α-GAL protein comprises a sequence at least 85% identical to one of SEQ ID NOs: 1-6. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the nucleic acid sequence comprises a sequence of one of SEQ ID NOs: 8-12. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the α-GAL protein comprises a sequence of one of SEQ ID NOs: 2-6. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the nucleic acid sequence is at least 85% identical to one of SEQ ID NOs: 15-16. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the PPT1 protein comprises a sequence at least 85% identical to one of SEQ ID NOs: 13-14. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the nucleic acid sequence comprises a sequence of SEQ ID NO: 16. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the PPT1 protein comprises a sequence of SEQ ID NO: 14. In some embodiments, the construct further comprises a promoter sequence. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the construct further comprises one or more nucleic acid sequences selected from the group consisting of a Kozak sequence, a CrPV IRES, a nucleic acid sequence encoding a linker, a nucleic acid sequence encoding a signal sequence, and a nucleic acid sequence encoding an IGF2 peptide. In some embodiments, the signal peptide sequence comprises an immunoglobulin binding protein (Bip) signal sequence. In some embodiments, the signal peptide sequence comprises the Bip signal sequence, which comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 29-33. In some embodiments, the construct further comprises an internal ribosome entry sequence (IRES). In some embodiments, the IRES comprises a cricket paralysis virus (CrPV) IRES. In some embodiments, the construct further comprises a nucleic acid sequence encoding a variant IGF2 (vIGF2) peptide. In some embodiments, the vIGF2 peptide comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17-27.In some embodiments, the nucleic acid sequence encoding the vIGF2 peptide is 5' to the nucleic acid sequence encoding the stable form of the protein. In some embodiments, the nucleic acid sequence encoding the vIGF2 peptide is 3' to the nucleic acid sequence encoding the stable form of the protein. In some embodiments, the construct is packaged in a viral capsid.

[0005] In another aspect, a pharmaceutical composition is provided, comprising a gene therapy vector and a pharmaceutically acceptable excipient, carrier or diluent, wherein the gene therapy vector comprises a nucleic acid construct, wherein the nucleic acid construct comprises: a nucleic acid encoding a stable form of a protein for treating a genetic disorder. In some embodiments, the stable form comprises one or more non-natural cysteine ​​residues, wherein the one or more non-natural cysteine ​​residues form a disulfide bridge between non-natural cysteines within the protein or between non-natural cysteines of two monomers of the protein. In some embodiments, the protein is selected from the group consisting of α-galactosidase A, β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan α-L-iduronidase, α-L-iduronidase, N-sulfoglucosamine sulfohydrolase (SGSH), N-acetyl-α-glucosaminidase (NAGLU), iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, α-glucosidase, tripeptidyl peptidase 1 (TPP1), palmitoyl protein thioesterase (PPT), neuronal ceroid lipofuscinosis protein 1, neuronal ceroid lipofuscinosis protein 2, neuronal Neuronal ceroid lipofuscinosis protein 3, neuronal ceroid lipofuscinosis protein 4, neuronal ceroid lipofuscinosis protein 5, neuronal ceroid lipofuscinosis protein 6, neuronal ceroid lipofuscinosis protein 7, neuronal ceroid lipofuscinosis protein 8, neuronal ceroid lipofuscinosis protein 9, neuronal ceroid lipofuscinosis protein 10, neuronal ceroid lipofuscinosis protein 11, neuronal ceroid lipofuscinosis protein 12, neuronal ceroid lipofuscinosis protein 13, neuronal ceroid lipofuscinosis protein 14, neuronal ceroid lipofuscinosis protein 15, neuronal ceroid lipofuscinosis protein 16 and cyclin-dependent kinase-like 5.In some embodiments, the protein is selected from the group consisting of α-galactosidase A, β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan α-L-iduronidase, α-L-iduronidase, N-sulfoglucosamine sulfohydrolase (SGSH), N-acetyl-α-glucosaminidase (NAGLU), iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, α-glucosidase. enzyme, tripeptidyl peptidase 1 (TPP1), palmitoyl protein thioesterase (PPT), neuronal ceroid lipofuscinosis protein 4, neuronal ceroid lipofuscinosis protein 10 (cathepsin D), neuronal ceroid lipofuscinosis protein 11 (granulin precursor), neuronal ceroid lipofuscinosis protein 13 (cathepsin F), neuronal ceroid lipofuscinosis protein 14 (KCTD7), neuronal ceroid lipofuscinosis protein 15 (TBCK) and cyclin-dependent kinase-like 5. In some embodiments, the stable protein comprises a lysosomal enzyme. In some embodiments, the stable protein comprises a stable α-galactosidase (α-GAL) protein. In some embodiments, the stable α-galactosidase A (α-GAL) protein comprises one or more non-native cysteine ​​residues selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the stable protein comprises a stable palmitoyl protein thioesterase 1 (PPT1). In some embodiments, the stable PPT1 protein comprises non-native cysteine ​​residues A171C and A183C. In some embodiments, the stable protein has a longer half-life at pH 7.4 compared to the corresponding protein without the non-native cysteine. In some embodiments, the stable protein can replace a defective or insufficient protein in the genetic disorder. In some embodiments, the stable protein can alleviate or slow down one or more symptoms associated with the genetic disorder. In some embodiments, the stable protein is more effective in alleviating or slowing down one or more symptoms of the genetic disorder compared to an unstable protein. In some embodiments, the genetic disorder is a neurological disorder. In some embodiments, the genetic disorder is a lysosomal storage disease.In some embodiments, the genetic 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, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroto-Ramie disease, Slay 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), infantile, juvenile and adult forms of neuronal ceroid lipofuscinosis, and CDKL5 deficiency. In some embodiments, the gene therapy vector is a viral vector selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, and a herpes virus vector. In some embodiments, the adeno-associated virus is a serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.74, AAV-B1 and AAV-hu68. In some embodiments, the nucleic acid construct is contained in a viral vector genome. In some embodiments, the viral vector genome comprises a recombinant AAV (rAAV) genome. In some embodiments, the rAAV genome comprises a self-complementary genome. In some embodiments, the rAAV genome comprises a single-stranded genome. In some embodiments, the rAAV genome comprises a first inverted terminal repeat sequence and a second inverted terminal repeat sequence. In some embodiments, the AAV inverted terminal repeat sequence is an AAV2 inverted terminal repeat sequence. In some embodiments, the rAAV genome further comprises an SV40 intron. In some embodiments, the rAAV genome further comprises a polyadenylation sequence. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the nucleic acid sequence is at least 85% identical to one of SEQ ID NOs: 7-12. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the α-GAL protein comprises a sequence at least 85% identical to one of SEQ ID NOs: 1-6. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the nucleic acid sequence comprises a sequence of one of SEQ ID NOs: 8-12.In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the α-GAL protein comprises a sequence of one of SEQ ID NOs: 2-6. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the nucleic acid sequence is at least 85% identical to one of SEQ ID NOs: 15-16. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the PPT1 protein comprises a sequence at least 85% identical to one of SEQ ID NOs: 13-14. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the nucleic acid sequence comprises a sequence of SEQ ID NO: 16. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the PPT1 protein comprises a sequence of SEQ ID NO: 14. In some embodiments, the construct further comprises a promoter sequence. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the construct further comprises one or more nucleic acid sequences selected from the group consisting of a Kozak sequence, a CrPV IRES, a nucleic acid sequence encoding a linker, a nucleic acid sequence encoding a signal sequence, and a nucleic acid sequence encoding an IGF2 peptide. In some embodiments, the signal peptide sequence comprises an immunoglobulin binding protein (Bip) signal sequence. In some embodiments, the signal peptide sequence comprises the Bip signal sequence, which comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 29-33. In some embodiments, the construct further comprises an internal ribosome entry sequence (IRES). In some embodiments, the IRES comprises a cricket paralysis virus (CrPV) IRES. In some embodiments, the construct further comprises a nucleic acid sequence encoding a variant IGF2 (vIGF2) peptide. In some embodiments, the vIGF2 peptide comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 17-27. In some embodiments, the nucleic acid sequence encoding the vIGF2 peptide is 5' of the nucleic acid sequence encoding the stable form of the protein. In some embodiments, the nucleic acid sequence encoding the vIGF2 peptide is 3' to the nucleic acid sequence encoding the stabilized form of the protein. In some embodiments, the construct is packaged within a viral capsid.In some embodiments, the excipient is selected from the group consisting of saline, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and the surfactant polyoxyethylene-sorbitan monooleate.

[0006] In a further aspect, a method for treating a genetic disorder in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a gene therapy vector or a pharmaceutical composition thereof, the gene therapy vector comprising a nucleic acid construct comprising: a nucleic acid encoding a stable form of a protein for treating a genetic disorder. In some embodiments, the stable form comprises one or more non-natural cysteine ​​residues, the one or more non-natural cysteine ​​residues forming a disulfide bridge between non-natural cysteines within the protein or between non-natural cysteines of two monomers of the protein. In some embodiments, the protein is selected from the group consisting of α-galactosidase A, β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan α-L-iduronidase, α-L-iduronidase, N-sulfoglucosamine sulfohydrolase (SGSH), N-acetyl-α-glucosaminidase (NAGLU), iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, α-glucosidase, tripeptidyl peptidase 1 (TPP1), palmitoyl protein thioesterase (PPT), neuronal ceroid lipofuscinosis protein 1, neuronal ceroid lipofuscinosis protein 2, neuronal Neuronal ceroid lipofuscinosis protein 3, neuronal ceroid lipofuscinosis protein 4, neuronal ceroid lipofuscinosis protein 5, neuronal ceroid lipofuscinosis protein 6, neuronal ceroid lipofuscinosis protein 7, neuronal ceroid lipofuscinosis protein 8, neuronal ceroid lipofuscinosis protein 9, neuronal ceroid lipofuscinosis protein 10, neuronal ceroid lipofuscinosis protein 11, neuronal ceroid lipofuscinosis protein 12, neuronal ceroid lipofuscinosis protein 13, neuronal ceroid lipofuscinosis protein 14, neuronal ceroid lipofuscinosis protein 15, neuronal ceroid lipofuscinosis protein 16 and cyclin-dependent kinase-like 5.In some embodiments, the protein is selected from the group consisting of α-galactosidase A, β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan α-L-iduronidase, α-L-iduronidase, N-sulfoglucosamine sulfohydrolase (SGSH), N-acetyl-α-glucosaminidase (NAGLU), iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, α-glucosidase. enzyme, tripeptidyl peptidase 1 (TPP1), palmitoyl protein thioesterase (PPT), neuronal ceroid lipofuscinosis protein 4, neuronal ceroid lipofuscinosis protein 10 (cathepsin D), neuronal ceroid lipofuscinosis protein 11 (granulin precursor), neuronal ceroid lipofuscinosis protein 13 (cathepsin F), neuronal ceroid lipofuscinosis protein 14 (KCTD7), neuronal ceroid lipofuscinosis protein 15 (TBCK) and cyclin-dependent kinase-like 5. In some embodiments, the stable protein comprises a lysosomal enzyme. In some embodiments, the stable protein comprises a stable α-galactosidase (α-GAL) protein. In some embodiments, the stable α-galactosidase A (α-GAL) protein comprises one or more non-native cysteine ​​residues D233C and I359C. In some embodiments, the stable protein comprises a stable palmitoyl protein thioesterase 1 (PPT1). In some embodiments, the stable PPT1 protein comprises non-native cysteine ​​residues A171C and A183C. In some embodiments, the stable protein has a longer half-life at pH 7.4 compared to the corresponding protein without the non-native cysteine. In some embodiments, the stable protein can replace a defective or insufficient protein in the genetic disorder. In some embodiments, the stable protein can alleviate or slow down one or more symptoms associated with the genetic disorder. In some embodiments, the stable protein is more effective in alleviating or slowing down one or more symptoms of the genetic disorder compared to an unstable protein. In some embodiments, the genetic disorder is a neurological disorder. In some embodiments, the genetic disorder is a lysosomal storage disease.In some embodiments, the genetic 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, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroto-Ramie disease, Slay 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), infantile, juvenile and adult forms of neuronal ceroid lipofuscinosis, and CDKL5 deficiency. In some embodiments, the gene therapy vector is a viral vector selected from the group consisting of an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, and a herpes virus vector. In some embodiments, the adeno-associated virus is a serotype selected from the group consisting of AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13, AAV rh.74, AAV-B1 and AAV-hu68. In some embodiments, the nucleic acid construct is contained in a viral vector genome. In some embodiments, the viral vector genome comprises a recombinant AAV (rAAV) genome. In some embodiments, in some embodiments, in some embodiments, in some embodiments, in some embodiments, the rAAV genome comprises a self-complementary genome. In some embodiments, the rAAV genome comprises a single-stranded genome. In some embodiments, the rAAV genome comprises a first inverted terminal repeat sequence and a second inverted terminal repeat sequence. In some embodiments, the AAV inverted terminal repeat sequence is an AAV2 inverted terminal repeat sequence. In some embodiments, the rAAV genome further comprises an SV40 intron. In some embodiments, the rAAV genome further comprises a polyadenylation sequence. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the nucleic acid sequence is at least 85% identical to one of SEQ ID NOs: 7-12. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the α-GAL protein comprises a sequence at least 85% identical to one of SEQ ID NOs: 1-6.In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the nucleic acid sequence comprises a sequence of one of SEQ ID NOs: 8-12. In some embodiments, the construct further comprises a nucleic acid sequence encoding an α-GAL protein, wherein the α-GAL protein comprises a sequence of one of SEQ ID NOs: 2-6. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the nucleic acid sequence is at least 85% identical to one of SEQ ID NOs: 15-16. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the PPT1 protein comprises a sequence at least 85% identical to one of SEQ ID NOs: 13-14. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the nucleic acid sequence comprises a sequence of SEQ ID NO: 16. In some embodiments, the construct further comprises a nucleic acid sequence encoding a PPT1 protein, wherein the PPT1 protein comprises a sequence of SEQ ID NO: 14. In some embodiments, the construct further comprises a promoter sequence. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the construct further comprises one or more nucleic acid sequences selected from the group consisting of a Kozak sequence, a CrPV IRES, a nucleic acid sequence encoding a linker, a nucleic acid sequence encoding a signal sequence, and a nucleic acid sequence encoding an IGF2 peptide. In some embodiments, the signal peptide sequence comprises an immunoglobulin binding protein (Bip) signal sequence. In some embodiments, the signal peptide sequence comprises the Bip signal sequence, which comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 29-33. In some embodiments, the construct further comprises an internal ribosome entry sequence (IRES). In some embodiments, the IRES comprises a cricket paralysis virus (CrPV) IRES. In some embodiments, the construct further comprises a nucleic acid sequence encoding a variant IGF2 (vIGF2) peptide. In some embodiments, the vIGF2 peptide comprises an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 17-27. In some embodiments, the nucleic acid sequence encoding the vIGF2 peptide is 5' of the nucleic acid sequence encoding the stable form of the protein. In some embodiments, the nucleic acid sequence encoding the vIGF2 peptide is 3' to the nucleic acid sequence encoding the stable form of the protein. In some embodiments, the construct is packaged in a viral capsid. In some embodiments, the gene therapy vector or the pharmaceutical composition is delivered by intrathecal, intraventricular, intraparenchymal or intravenous injection or a combination thereof.In some embodiments, the gene therapy vector or the pharmaceutical composition reduces or alleviates one or more symptoms of the subject's genetic disorder. In some embodiments, the genetic disorder is a lysosomal storage disease. In some embodiments, the genetic 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, Filippo disease, Sanfilippo disease type C, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroto-Rami 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), neuronal ceroid lipofuscinosis, and CDKL5 deficiency.

[0007] In another aspect, a stable human α-galactosidase A (α-GAL) dimer is provided. In some embodiments, the stable α-GAL dimer comprises one or more non-natural cysteine ​​residues, wherein the one or more non-natural cysteine ​​residues form at least one intermolecular disulfide bond connecting the first subunit and the second subunit of the α-GAL dimer. In some embodiments, the one or more non-natural cysteine ​​residues are selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the one or more non-natural cysteine ​​residues comprise D233C and I359C. In some embodiments, the one or more non-natural cysteine ​​residues comprise M51C and G360C. In some embodiments, the one or more non-natural cysteine ​​residues comprise i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide has a sequence at least 90% identical to one of SEQ ID NOs: 1-6. In some embodiments, the polypeptide is encoded by a nucleic acid at least 85% identical to one of SEQ ID NOs: 7-12. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the polypeptide further comprises a variant IGF2 (vIGF2) peptide.

[0008] In a further aspect, a pharmaceutical composition is provided, which comprises a stable human α-GAL dimer and a pharmaceutically acceptable excipient, carrier or diluent. In some embodiments, the stable α-GAL dimer comprises one or more non-natural cysteine ​​residues, wherein the one or more non-natural cysteine ​​residues form at least one intermolecular disulfide bond connecting the first subunit and the second subunit of the α-GAL dimer. In some embodiments, the one or more non-natural cysteine ​​residues are selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the one or more non-natural cysteine ​​residues comprise D233C and I359C. In some embodiments, the one or more non-natural cysteine ​​residues comprise M51C and G360C. In some embodiments, the one or more non-natural cysteine ​​residues comprise i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide has a sequence at least 90% identical to one of SEQ ID NOs: 1-6. In some embodiments, the polypeptide is encoded by a nucleic acid at least 85% identical to one of SEQ ID NO: 7-12. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the polypeptide further comprises a variant IGF2 (vIGF2) peptide. In some embodiments, the excipient is selected from the group consisting of saline, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N, N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and a surfactant, polyoxyethylene-sorbitan monooleate.

[0009] In another aspect, a method for treating Fabry disease in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a stable human α-GAL dimer or a pharmaceutical composition thereof to a subject in need thereof. In some embodiments, the stable α-GAL dimer comprises one or more non-natural cysteine ​​residues, wherein the one or more non-natural cysteine ​​residues form at least one intermolecular disulfide bond connecting the first subunit and the second subunit of the α-GAL dimer. In some embodiments, the one or more non-natural cysteine ​​residues are selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the one or more non-natural cysteine ​​residues comprise D233C and I359C. In some embodiments, the one or more non-natural cysteine ​​residues comprise M51C and G360C. In some embodiments, the one or more non-natural cysteine ​​residues comprise i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide has a sequence at least 90% identical to one of SEQ ID NOs: 1-6. In some embodiments, the polypeptide is encoded by a nucleic acid at least 85% identical to one of SEQ ID NOs: 7-12. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to the wild-type α-GAL polypeptide. In some embodiments, the polypeptide further comprises a variant IGF2 (vIGF2) peptide. In some embodiments, the excipient is selected from the group consisting of saline, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and a surfactant, polyoxyethylene-sorbitan monooleate. In some embodiments, the stable human α-GAL dimer or the pharmaceutical composition is delivered by intrathecal, intracerebroventricular, intraparenchymal, subcutaneous, intramuscular, ocular, intravenous injection or a combination thereof. In some embodiments, the stable human α-GAL dimer or the pharmaceutical composition reduces or alleviates one or more symptoms of Fabry disease in the subject.

[0010] In another aspect, a stabilized human palmitoyl protein thioesterase 1 (PPT1) molecule is provided. In some embodiments, the stabilized PPT1 molecule comprises one or more non-native cysteine ​​residues, wherein the one or more non-native cysteine ​​residues form at least one intramolecular disulfide bond within the PPT1 molecule.

[0011] In some embodiments, the stabilized PPT1 comprises non-native cysteine ​​residues A171C and A183C. In some embodiments, the polypeptide has a sequence at least 90% identical to one of SEQ ID NOs: 13-14. In some embodiments, the polypeptide is encoded by a nucleic acid at least 85% identical to one of SEQ ID NOs: 15-16. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to the wild-type PPT1 polypeptide. In some embodiments, the polypeptide further comprises a variant IGF2 (vIGF2) peptide.

[0012] In a further aspect, a pharmaceutical composition is provided, comprising a stabilized PPT1 and a pharmaceutically acceptable excipient, carrier or diluent. In some embodiments, the stabilized PPT1 molecule comprises one or more non-natural cysteine ​​residues, wherein the one or more non-natural cysteine ​​residues form at least one intramolecular disulfide bond within the PPT1 molecule. In some embodiments, the stabilized PPT1 comprises non-natural cysteine ​​residues A171C and A183C. In some embodiments, the polypeptide has a sequence at least 90% identical to one of SEQ ID NOs: 13-14. In some embodiments, the polypeptide is encoded by a nucleic acid at least 85% identical to one of SEQ ID NOs: 15-16. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type PPT1 polypeptide. In some embodiments, the polypeptide further comprises a variant IGF2 (vIGF2) peptide. In some embodiments, the excipient is selected from the group consisting of saline, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and the surfactant polyoxyethylene-sorbitan monooleate.

[0013] In another aspect, a method for treating CLN1 disease in a subject is provided, the method comprising administering to the subject a therapeutically effective amount of a stable PPT1 or a pharmaceutical composition thereof to a subject in need thereof. In some embodiments, the stable PPT1 molecule comprises one or more non-natural cysteine ​​residues, wherein the one or more non-natural cysteine ​​residues form at least one intramolecular disulfide bond within the PPT1 molecule. In some embodiments, the stable PPT1 comprises non-natural cysteine ​​residues A171C and A183C. In some embodiments, the polypeptide has a sequence at least 90% identical to one of SEQ ID NOs: 13-14. In some embodiments, the polypeptide is encoded by a nucleic acid at least 85% identical to one of SEQ ID NOs: 15-16. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type PPT1 polypeptide. In some embodiments, the polypeptide further comprises a variant IGF2 (vIGF2) peptide. In some embodiments, the excipient is selected from the group consisting of saline, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether and surfactant polyoxyethylene-sorbitan monooleate. In some embodiments, the modified PPT1 or the pharmaceutical composition is delivered by intrathecal, intracerebroventricular, intraparenchymal, subcutaneous, intramuscular, ocular, intravenous injection or a combination thereof.

[0014] In a further aspect, a modified human α-galactosidase A (α-GAL) polypeptide is provided, comprising a cysteine ​​substitution of an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide comprises a cysteine ​​substitution of D233C and I359C of an α-GAL polypeptide sequence. In some embodiments, the polypeptide comprises a cysteine ​​substitution of M51C and G360C of an α-GAL polypeptide sequence. In some embodiments, the polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide.

[0015] In a further aspect, a nucleic acid molecule is provided, comprising a nucleic acid encoding a modified human α-GAL polypeptide. In some embodiments, the modified human α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide comprises a cysteine ​​substitution of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, the polypeptide comprises a cysteine ​​substitution of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide.

[0016] In a further aspect, a gene therapy vector comprising a nucleic acid molecule is provided, the nucleic acid molecule comprising a nucleic acid encoding a modified human α-GAL polypeptide. In some embodiments, the modified human α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide comprises a cysteine ​​substitution of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, the polypeptide comprises a cysteine ​​substitution of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide.

[0017] In a further aspect, a modified human α-galactosidase A (α-GAL) polypeptide is provided, comprising a cysteine ​​substitution of an α-GAL polypeptide sequence, wherein the cysteine ​​substitution promotes disulfide bond formation between two α-GAL polypeptides to form a homodimer. In some embodiments, the polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide comprises a cysteine ​​substitution of D233C and I359C of an α-GAL polypeptide sequence. In some embodiments, the polypeptide comprises a cysteine ​​substitution of M51C and G360C of an α-GAL polypeptide sequence. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide.

[0018] In a further aspect, a nucleic acid molecule is provided, comprising a nucleic acid encoding a modified human α-GAL polypeptide. In some embodiments, the modified human α-galactosidase A (α-GAL) polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence, wherein the cysteine ​​substitution promotes disulfide bond formation between two α-GAL polypeptides to form a homodimer. In some embodiments, the polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide comprises a cysteine ​​substitution of D233C and I359C of an α-GAL polypeptide sequence. In some embodiments, the polypeptide comprises a cysteine ​​substitution of M51C and G360C of an α-GAL polypeptide sequence. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide.

[0019] In another aspect, a gene therapy vector comprising a nucleic acid encoding a modified human α-GAL polypeptide is provided. In some embodiments, the modified human α-galactosidase A (α-GAL) polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence, wherein the cysteine ​​substitution promotes disulfide bond formation between two α-GAL polypeptides to form a homodimer. In some embodiments, the polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide comprises a cysteine ​​substitution of D233C and I359C of an α-GAL polypeptide sequence. In some embodiments, the polypeptide comprises a cysteine ​​substitution of M51C and G360C of an α-GAL polypeptide sequence. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide.

[0020] In a further aspect, a homodimer comprising two modified human α-GAL polypeptides is provided, wherein each modified human α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, each modified human α-GAL polypeptide comprises a cysteine ​​substitution of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, each modified human α-GAL polypeptide comprises a cysteine ​​substitution of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the homodimer exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL homodimer.

[0021] In another aspect, a homodimer comprising two modified human α-GAL polypeptides is provided, wherein each modified human α-GAL polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence, wherein the cysteine ​​substitution promotes disulfide bond formation between the two α-GAL polypeptides to form a homodimer. In some embodiments, the polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, each modified human α-GAL polypeptide comprises a cysteine ​​substitution of D233C and I359C of an α-GAL polypeptide sequence. In some embodiments, each modified human α-GAL polypeptide comprises a cysteine ​​substitution of M51C and G360C of an α-GAL polypeptide sequence. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the homodimer exhibits increased half-life at pH 7.4 compared to the wild-type α-GAL homodimer.

[0022] In a further aspect, a nucleic acid molecule is provided, comprising a nucleic acid encoding a modified human α-GAL polypeptide, wherein the nucleic acid encodes a cysteine-substituted polypeptide selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C comprising an α-GAL polypeptide sequence. In some embodiments, the nucleic acid encodes a cysteine-substituted polypeptide comprising D233C and I359C. In some embodiments, the nucleic acid encodes a cysteine-substituted polypeptide comprising M51C and G360C. In some embodiments, the polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the nucleic acid is a gene therapy construct. In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid comprises at least a portion of a viral nucleic acid sequence. In some embodiments, the virus is selected from wherein the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus.

[0023] In a further aspect, a nucleic acid molecule is provided, comprising a nucleic acid encoding a modified human α-GAL polypeptide, wherein the nucleic acid encodes a polypeptide comprising a cysteine ​​substitution of an α-GAL polypeptide sequence, wherein the cysteine ​​substitution promotes disulfide bond formation between two α-GAL polypeptides to form a homodimer. In some embodiments, the polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the nucleic acid encodes a polypeptide comprising a cysteine ​​substitution of D233C and I359C. In some embodiments, the nucleic acid encodes a polypeptide comprising a cysteine ​​substitution of M51C and G360C. In some embodiments, the polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the nucleic acid is a gene therapy construct. In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid comprises at least a portion of a viral nucleic acid sequence. In some embodiments, the virus is selected from wherein the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus.

[0024] In another aspect, a nucleic acid construct is provided, comprising at least one promoter and a nucleic acid encoding a modified human α-GAL polypeptide, wherein the modified human α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the nucleic acid encodes a polypeptide comprising cysteine ​​substitutions of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, the nucleic acid encodes a polypeptide comprising cysteine ​​substitutions of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid construct comprises one or more nucleic acids from the group consisting of: CrPV IRES, kozak sequence, nucleic acid encoding a linker, nucleic acid sequence encoding a leader sequence, and nucleic acid encoding an IGF2 peptide. In some embodiments, the nucleic acid construct comprises at least a portion of a viral nucleic acid sequence. In some embodiments, the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus. In some embodiments, the polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the nucleic acid is packaged in a viral capsid protein. In some embodiments, the nucleic acid construct is suitable for gene therapy.

[0025] In a further aspect, a nucleic acid construct is provided, comprising at least one promoter and a nucleic acid encoding a modified human α-GAL polypeptide, wherein the modified human α-GAL polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence, wherein the cysteine ​​substitution promotes disulfide bond formation between two α-GAL polypeptides to form a homodimer. In some embodiments, the polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the nucleic acid encodes a polypeptide comprising a cysteine ​​substitution of D233C and I359C of an α-GAL polypeptide sequence. In some embodiments, the nucleic acid encodes a polypeptide comprising a cysteine ​​substitution of M51C and G360C of an α-GAL polypeptide sequence. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid construct comprises one or more nucleic acids from the group consisting of CrPV IRES, kozak sequences, nucleic acids encoding linkers, nucleic acid sequences encoding leader sequences, and nucleic acids encoding IGF2 peptides. In some embodiments, the nucleic acid construct comprises at least a portion of a viral nucleic acid sequence. In some embodiments, the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus. In some embodiments, the polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the nucleic acid is packaged in a viral capsid protein. In some embodiments, the nucleic acid construct is suitable for gene therapy.

[0026] In a further aspect, a pharmaceutical composition is provided, comprising (a) a modified human α-GAL polypeptide, wherein the modified human α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C; and (b) a pharmaceutically acceptable excipient. In some embodiments, the modified human α-GAL polypeptide comprises a cysteine ​​substitution of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, the modified human α-GAL polypeptide comprises a cysteine ​​substitution of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the modified human α-GAL polypeptide forms homodimers. In some embodiments, the homodimers are stabilized by disulfide bonds. In some embodiments, the modified human α-GAL polypeptide exhibits an increased half-life at pH 7.4 compared to the wild-type α-GAL polypeptide. In some embodiments, the excipient is selected from the group consisting of saline, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether and surfactant polyoxyethylene-sorbitan monooleate. In some embodiments, the composition is suitable for enzyme replacement therapy.

[0027] In another aspect, a method of improving at least one symptom of Fabry disease in a subject in need thereof is provided, the method comprising administering at least one dose of a composition comprising a gene therapy nucleic acid construct comprising at least one promoter and a nucleic acid encoding a modified human α-GAL polypeptide, wherein the modified human α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the nucleic acid encodes a polypeptide comprising cysteine ​​substitutions of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, the nucleic acid encodes a polypeptide comprising cysteine ​​substitutions of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the nucleic acid encodes a polypeptide that forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the nucleic acid encodes a modified human α-GAL polypeptide having an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid comprises at least a portion of a virus. In some embodiments, the virus is selected from wherein the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus. In some embodiments, the nucleic acid is packaged in a viral capsid protein. In some embodiments, the at least one symptom is selected from one or more of pain, skin discoloration, inability to sweat, cloudy eyes, gastrointestinal dysfunction, tinnitus, hearing loss, mitral valve prolapse, heart disease, joint pain, renal failure, and renal dysfunction. In some embodiments, at least one symptom is alleviated by a single administration of the gene therapy nucleic acid construct. In some embodiments, the method further comprises measuring the α-GAL activity in the tissue obtained from the subject after treatment.

[0028] In a further aspect, a method of improving at least one symptom of Fabry disease in a subject in need thereof is provided, the method comprising administering at least one dose of a composition comprising a modified human α-GAL polypeptide, wherein the modified human α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the modified human α-GAL polypeptide comprises a cysteine ​​substitution of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, the modified human α-GAL polypeptide comprises a cysteine ​​substitution of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the modified human α-GAL polypeptide forms homodimers. In some embodiments, the homodimers are stabilized by disulfide bonds. In some embodiments, the modified human α-GAL polypeptide exhibits an increased half-life at pH 7.4 compared to the wild-type α-GAL polypeptide. In some embodiments, the at least one symptom is selected from one or more of pain, skin discoloration, inability to sweat, cloudy eyes, gastrointestinal dysfunction, tinnitus, hearing loss, mitral valve prolapse, heart disease, joint pain, renal failure and renal dysfunction.

[0029] Incorporated by Reference

[0030] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description and accompanying drawings which set forth illustrative embodiments utilizing the principles of the invention, in which:

[0032] Figure 1A and Figure 1B The structure of α-galactosidase A (α-GAL) and proposed sites for amino acid substitutions are shown.

[0033] Figure 2A Modified α-GAL dimer formation is shown.

[0034] Figure 2B The modified α-GAL enzyme activity is shown.

[0035] Figure 3A The stability of α-GAL at pH 4.6 and pH 7.4 over 24 hours is shown.

[0036] Figure 3BThe stability of α-GAL at pH 4.6 and pH 7.4 over 7 days is shown.

[0037] Figure 4A Uptake and enzymatic activity of modified α-GAL by Fabry patient fibroblasts are shown.

[0038] Figure 4B Shown is a decrease in globotriaosylsphingosine (lyso-Gb3) in Fabry patient fibroblasts.

[0039] Figure 5 Shown is the enhanced half-life activity of two α-GAL disulfide dimers in Fabry disease cells.

[0040] Figure 6 Shown is GB3 substrate histology in wild-type mice and GLA knockout mice that did and did not receive treatment with modified α-GAL gene therapy.

[0041] Figure 7 Shown is GLA enzyme activity in wild-type mice and GLA knockout mice that did and did not receive treatment with modified α-GAL gene therapy.

[0042] Figure 8 Shown is GB3 substrate measured in kidney tissue lysates of wild-type mice and GLA knockout mice treated and not treated with modified α-GAL gene therapy.

[0043] Fig. 9 Shown is WinNonlin analysis of palmitoyl protein thioesterase 1 (PPT-1) wild type enzyme activity compared to construct PPT-1 mutant over time. DETAILED DESCRIPTION

[0044] Provided herein are variants of polypeptides for treatment, including constructs with cysteine ​​substitutions for gene therapy, which enable stability due to the formation of disulfide bonds within the molecule or between two subunits in the polypeptide to form a dimer. These disulfide bonds produce recombinases that are more stable at neutral pH (such as the pH of blood). Therefore, a more stable polypeptide with a longer half-life is provided, which can be used to treat diseases caused by mutations, including diseases caused by α-GAL mutations, such as Fabry disease; or diseases caused by PPT-1 mutations, such as CLN1 disease. Polypeptide variants herein (also referred to as "modified polypeptides") include, but are not limited to, variants of α-GAL and PPT-1.

[0045] Modified α-GAL polypeptide

[0046] Provided herein are modified α-GAL polypeptides comprising cysteine ​​substitutions of an α-GAL polypeptide sequence. Contemplated substitutions provided herein include: (i) R49C and G361C; (ii) R49C and G360C; (iii) D233C and I359C; (iv) M51C and G360C; and (v) S276C. In some embodiments, the polypeptide comprises a cysteine ​​substitution of an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the polypeptide comprises a cysteine ​​substitution of D233C and I359C of an α-GAL polypeptide sequence. In some embodiments, the polypeptide comprises a cysteine ​​substitution of M51C and G360C of an α-GAL polypeptide sequence. The modified α-GAL polypeptide can form homodimers that are stabilized by at least one, more preferably two, intermolecular disulfide bonds. The modified α-GAL polypeptides exhibit increased half-life at pH 7.4 compared to wild-type α-GAL polypeptides.

[0047] Wild-type and exemplary modified α-GAL sequences are provided in Table 1

[0048]

[0049]

[0050] Also provided herein are modified α-GAL polypeptides comprising a polypeptide having a sequence containing cysteine ​​residues at positions 51 and 360 and having at least 90% identity with the sequence as set forth in SEQ ID NO: 4. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 51 and 360 and having at least 95% identity with the sequence as set forth in SEQ ID NO: 4. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 51 and 360 and having at least 96% identity with the sequence as set forth in SEQ ID NO: 4. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 51 and 360 and having at least 97% identity with the sequence as set forth in SEQ ID NO: 4. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 51 and 360 and having at least 98% identity with the sequence as set forth in SEQ ID NO: 4. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 51 and 360 and having at least 99% identity to the sequence as set forth in SEQ ID NO: 4. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 51 and 360 and having greater than 99% identity to the sequence as set forth in SEQ ID NO: 4. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence as set forth in SEQ ID NO: 4.

[0051] Also provided herein are modified α-GAL polypeptides comprising a polypeptide having a sequence containing cysteine ​​residues at positions 233 and 359 and having at least 90% identity with the sequence as set forth in SEQ ID NO: 5. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 233 and 359 and having at least 95% identity with the sequence as set forth in SEQ ID NO: 5. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 233 and 359 and having at least 96% identity with the sequence as set forth in SEQ ID NO: 5. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 233 and 359 and having at least 97% identity with the sequence as set forth in SEQ ID NO: 5. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 233 and 359 and having at least 98% identity with the sequence as set forth in SEQ ID NO: 5. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 233 and 359 and having at least 99% identity to the sequence as set forth in SEQ ID NO: 5. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 233 and 359 and having greater than 99% identity to the sequence as set forth in SEQ ID NO: 5. In some embodiments, the modified α-GAL polypeptide comprises a polypeptide having a sequence as set forth in SEQ ID NO: 5.

[0052] Also provided herein are homodimers comprising two modified α-GAL polypeptides, wherein each modified α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence selected from the group consisting of: (i) R49C and G361C; (ii) R49C and G360C; (iii) D233C and I359C; (iv) M51C and G360C; and (v) S276C. In some embodiments, each modified α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, each modified α-GAL polypeptide comprises a cysteine ​​substitution of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, each modified α-GAL polypeptide comprises cysteine ​​substitutions of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the homodimer exhibits an increased half-life at pH 7.4 compared to the wild-type α-GAL homodimer.

[0053] In some embodiments, the modified α-GAL polypeptide has an increased half-life at pH 4.6. In some embodiments, the half-life at pH 4.6 is at least 50% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 150% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 200% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 250% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 300% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 350% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 400% greater than that of a wild-type α-GAL polypeptide.

[0054] In some embodiments, the modified α-GAL dimer has a half-life at pH 4.6 that is increased by at least about 2, 2.5, 3, 3.5, 4, 4.5, or 5 times compared to the half-life of wild-type α-GAL at pH 4.6. More preferably, the modified α-GAL dimer has a half-life at pH 4.6 that is increased by at least about 3, 3.5, or 4 times compared to the half-life of wild-type α-GAL polypeptide at pH 4.6.

[0055] In some embodiments, the modified α-GAL dimer has an intracellular half-life that is increased by at least about 2, 2.5, 3, 3.5, 4, 4.5, or 5 times compared to the intracellular half-life of wild-type human α-GAL. More preferably, the modified α-GAL dimer has an intracellular half-life that is increased by at least about 3, 3.5, or 4, 4.5, or 5 times compared to the intracellular half-life of wild-type α-GAL polypeptide.

[0056] Compared to wild-type human α-GAL, the modified α-GAL dimer has a significantly increased half-life at pH 7.4.

[0057] Nucleic acids encoding modified α-GAL polypeptides

[0058] Also provided herein are nucleic acid molecules comprising nucleic acids encoding modified α-GAL polypeptides. Contemplated nucleic acids include those encoding cysteine-substituted polypeptides comprising α-GAL polypeptide sequences, the substitutions comprising: (i) R49C and G361C; (ii) R49C and G360C; (iii) D233C and I359C; (iv) M51C and G360C; and (v) S276C. In some embodiments, the nucleic acid encodes a cysteine-substituted polypeptide comprising an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the nucleic acid encodes a cysteine-substituted polypeptide comprising D233C and I359C. In some embodiments, the nucleic acid encodes a cysteine-substituted polypeptide comprising M51C and G360C. In some embodiments, the polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the nucleic acid is a gene therapy construct. In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid comprises at least a portion of a viral nucleic acid sequence. In some embodiments, the virus is selected from wherein the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus.

[0059] Also provided herein are nucleic acid constructs comprising at least one promoter and a nucleic acid encoding a modified α-GAL polypeptide. The modified α-GAL polypeptide is expected to comprise a cysteine ​​substitution of the α-GAL polypeptide sequence, the cysteine ​​substitution comprising: (i) R49C and G361C; (ii) R49C and G360C; (iii) D233C and I359C; (iv) M51C and G360C; and (v) S276C. In some embodiments, the nucleic acid encodes a cysteine-substituted polypeptide comprising an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the nucleic acid encodes a cysteine-substituted polypeptide comprising D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, the nucleic acid encodes a cysteine-substituted polypeptide comprising M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid construct comprises at least a portion of a viral nucleic acid sequence. In some embodiments, the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus. In some embodiments, the polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the nucleic acid is packaged in a viral capsid protein. In some embodiments, the nucleic acid construct is suitable for gene therapy.

[0060]

[0061]

[0062]

[0063] In some embodiments, the nucleic acid encoding the modified α-GAL polypeptide herein has an increased half-life compared to the wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 50% greater than the wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 150% greater than the wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 200% greater than the wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 250% greater than the wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 300% greater than the wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 350% greater than the wild-type α-GAL polypeptide.

[0064] Modified PPT-1 polypeptide

[0065] Provided herein are modified PPT-1 polypeptides comprising cysteine ​​substitutions of a PPT-1 polypeptide sequence. Contemplated substitutions provided herein include A171C and A183C. In some embodiments, the polypeptide comprises cysteine ​​substitutions of A171C and A183C of a PPT-1 polypeptide sequence. The modified PPT-1 polypeptide is stabilized by at least one, more preferably two, intramolecular disulfide bonds. The modified PPT-1 polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type PPT-1 polypeptide.

[0066] Wild-type and exemplary modified PPT-1 are provided in Table 3.

[0067]

[0068] Also provided herein are modified PPT-1 polypeptides comprising a polypeptide having a sequence containing cysteine ​​residues at positions 171 and 183 and having at least 90% identity to the sequence as set forth in SEQ ID NO: 14. In some embodiments, the modified PPT-1 polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 171 and 183 and having at least 95% identity to the sequence as set forth in SEQ ID NO: 14. In some embodiments, the modified PPT-1 polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 171 and 183 and having at least 96% identity to the sequence as set forth in SEQ ID NO: 14. In some embodiments, the modified PPT-1 polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 171 and 183 and having at least 97% identity to the sequence as set forth in SEQ ID NO: 14. In some embodiments, the modified PPT-1 polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 171 and 183 and having at least 98% identity to the sequence as set forth in SEQ ID NO: 14. In some embodiments, the modified PPT-1 polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 171 and 183 and having at least 99% identity to the sequence as set forth in SEQ ID NO: 14. In some embodiments, the modified PPT-1 polypeptide comprises a polypeptide having a sequence containing cysteine ​​residues at positions 171 and 183 and having greater than 99% identity to the sequence as set forth in SEQ ID NO: 14. In some embodiments, the modified PPT-1 polypeptide comprises a polypeptide having a sequence as set forth in SEQ ID NO: 14.

[0069] In some embodiments, the modified PPT-1 polypeptide has an increased half-life at pH 4.6. In some embodiments, the half-life at pH 4.6 is at least 50% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 150% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 200% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 250% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 300% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 350% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 400% greater than the wild-type PPT-1 polypeptide.

[0070] In some embodiments, the modified PPT-1 polypeptide has a half-life at pH 4.6 that is increased by at least about 2, 2.5, 3, 3.5, 4, 4.5, or 5 times compared to the half-life of wild-type PPT-1 at pH 4.6. More preferably, the modified PPT-1 polypeptide has a half-life at pH 4.6 that is increased by at least about 3, 3.5, or 4 times compared to the half-life of wild-type PPT-1 polypeptide at pH 4.6.

[0071] In some embodiments, the modified PPT-1 polypeptide has an intracellular half-life that is increased by at least about 2, 2.5, 3, 3.5, 4, 4.5 or 5 times compared to the intracellular half-life of wild-type human α-GAL. More preferably, the modified PPT-1 polypeptide has an intracellular half-life that is increased by at least about 3, 3.5, or 4, 4.5 or 5 times compared to the intracellular half-life of the wild-type PPT-1 polypeptide.

[0072] Compared to wild-type human α-GAL, the modified PPT-1 polypeptide has a significantly increased half-life at pH 7.4.

[0073] Nucleic acid encoding modified PPT-1 polypeptide

[0074] Also provided herein are nucleic acid molecules comprising nucleic acids encoding modified PPT-1 polypeptides. Contemplated nucleic acids include those encoding polypeptides comprising cysteine ​​substitutions of PPT-1 polypeptide sequences, the substitutions comprising: A171C and A183C. In some embodiments, the nucleic acid encodes a polypeptide comprising cysteine ​​substitutions of A171C and A183C. In some embodiments, the polypeptide is stabilized by a disulfide bond. In some embodiments, the polypeptide exhibits an increased half-life at pH 7.4 compared to the wild-type PPT-1 polypeptide. In some embodiments, the nucleic acid is a gene therapy construct. In some embodiments, the nucleic acid further comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid comprises at least a portion of a viral nucleic acid sequence. In some embodiments, the virus is selected from wherein the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus.

[0075]

[0076]

[0077] In some embodiments, the nucleic acid encoding the modified PPT-1 polypeptide herein has an increased half-life compared to a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 50% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 150% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 200% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 250% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 300% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 350% greater than a wild-type PPT-1 polypeptide.

[0078] IGF2 Peptide

[0079] In some cases, the modified polypeptides herein (such as the modified α-GAL or modified PPT-1 polypeptides herein) are fused with insulin-like growth factor 2 (IGF2) peptides for targeting the modified polypeptides to lysosomes where they are needed. Variants of the IGF2 peptide sequence retain high affinity binding to IGF2 / CI-MPR and eliminate binding to IGF1, insulin receptor and IGF binding protein (IGFBP). Compared with conventional IGF2 fusion proteins, the selectivity of variant IGF2 peptides is significantly higher and has a reduced safety risk. The IGF2 peptides herein include those having an amino acid sequence of AYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLA LLETYCATPAKSE (SEQ ID NO: 17). Additional IGF2 peptides have variant amino acid sequences optimized for improved targeting. Variant IGF2 peptides include variant amino acids at positions 26, 27, 43, 48, 49, 50, 54, 55 or 65 of the wild-type IGF2 sequence. These include substitutions at F26, Y27, V43, F48, R49, S50, A54, L55 or K65 of SEQ ID NO: 17. In some embodiments, the IGF2 peptide has a sequence having one or more substitutions from the group consisting of F26S, Y27L, V43L, F48T, R49S, S50I, A54R, L55R and K65R. In some embodiments, the IGF2 peptide has a sequence having a substitution of F26S. In some embodiments, the IGF2 peptide has a sequence having a substitution of Y27L. In some embodiments, the IGF2 peptide has a sequence having a substitution of V43L. In some embodiments, the IGF2 peptide has a sequence having a substitution of F48T. In some embodiments, the IGF2 peptide has a sequence having a substitution of R49S. In some embodiments, the IGF2 peptide has a sequence having a substitution of S50I. In some embodiments, the IGF2 peptide has a sequence having a substitution of A54R. In some embodiments, the IGF2 peptide has a sequence having a substitution of L55R. In some embodiments, the IGF2 peptide has a sequence having a substitution of K65R. In some embodiments, the IGF2 peptide has a sequence having a substitution of F26S, Y27L, V43L, F48T, R49S, S50I, A54R and L55R. In some embodiments, the IGF2 peptide has an N-terminal deletion. In some embodiments, the IGF2 peptide has an N-terminal deletion of one amino acid.In some embodiments, the IGF2 peptide has an N-terminal deletion of two amino acids. In some embodiments, the IGF2 peptide has an N-terminal deletion of three amino acids. In some embodiments, the IGF2 peptide has an N-terminal deletion of three amino acids. In some embodiments, the IGF2 peptide has an N-terminal deletion of four amino acids. In some embodiments, the IGF2 peptide has an N-terminal deletion of five amino acids. In some embodiments, the IGF2 peptide has an N-terminal deletion of six amino acids. In some embodiments, the IGF2 peptide has an N-terminal deletion of seven amino acids. In some embodiments, the IGF2 peptide has an N-terminal deletion of seven amino acids and substitutions of Y27L and K65R.

[0080] Additional substitutions are contemplated to reduce instability while maintaining CI-MPR binding affinity. It is contemplated that these substitutions are combined with any other substitutions described herein. In some embodiments, the IGF2 peptide has a sequence having a substitution of L17N. In some embodiments, the IGF2 peptide has a sequence having a substitution of P31G. In some embodiments, the IGF2 peptide has a sequence having a substitution of R38G. In some embodiments, the IGF2 peptide has a sequence having a substitution of E45W. In some embodiments, the IGF2 peptide has a sequence having a substitution of S50G. In some embodiments, the IGF2 peptide has a sequence having a substitution of R38G and E45W. In some embodiments, the IGF2 peptide has a sequence having a substitution of R38G, E45W, and S50G. In some embodiments, the IGF2 peptide has a sequence having a substitution of P31G, R38G, E45W, and S50G. In some embodiments, the IGF2 peptide has a sequence having substitutions of L17N, P31G, R38G, E45W, and S50G. Exemplary peptide sequences are represented by SEQ ID NOs: 17-27.

[0081]

[0082]

[0083] Internal ribosome entry sequence

[0084] In some embodiments, nucleic acids encoding modified polypeptides herein (such as nucleic acids encoding modified α-GAL and PP-1 polypeptides) further comprise an internal ribosome entry sequence (IRES) for increasing gene expression by bypassing the bottleneck of translation initiation. Suitable internal ribosome entry sequences for optimizing expression for gene therapy include, but are not limited to, cricket paralysis virus (CrPV) IRES, picornavirus IRES, foot-and-mouth disease virus IRES, Kaposi's sarcoma-associated herpesvirus IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, cricket paralysis virus IRES, Rhopalosiphum padi virus IRES, Merek's disease virus IRES, and other suitable IRES sequences. In some embodiments, the gene therapy construct comprises CrPV IRES. In some embodiments, the CrPV IRES has a nucleic acid sequence of CGGUGUCGAAGUAGAAUUUCUAUCUCGACACGCGGCCUUCCAAGCAGUUAGGGAAACCGACUUCUUUGAAGAAGAAAGCUGACUAUGUGAUCUUAUUAAAAUUAGGUUAAAUUUCGAGGUUAAAAAUAGUUUUAAUAUUGCUAUAGUCUUAGAGGUCUUGUAUAUUUAUACUUACCACACAAGAUGGACCGGAGCAGCCCUCCAAUAUCUAGUGUACCCUCGUGCUCGCUCAAACAUUAAGUGGUGUUGUGCGAAAAGAAUCUCACUUCAAGAA (SEQ ID NO: 28).

[0085] signal sequence

[0086] Provided herein are nucleic acid molecules comprising nucleic acids encoding modified polypeptides (such as modified α-GAL polypeptides or modified PPT-1 polypeptides), wherein the nucleic acid molecules further comprise a signal peptide that improves secretion of therapeutic proteins from cells transduced with gene therapy constructs. In some embodiments, the signal peptide improves protein processing of therapeutic proteins, promotes translocation of nascent polypeptide-ribosome complexes to the ER, and ensures correct co-translational and post-translational modifications. In some embodiments, the signal peptide is located at (i) an upstream position of a signal translation initiation sequence, (ii) between a translation initiation sequence and a therapeutic protein, or (iii) a downstream position of a therapeutic protein. Signal peptides useful in gene therapy constructs include, but are not limited to, immunoglobulin binding protein (BiP) signal peptides and variants thereof from the HSP70 protein family (e.g., HSPA5, member 5 of heat shock protein family A). These signal peptides have ultra-high affinity for signal recognition particles. Examples of BiP amino acid sequences are provided in Table 6 below. In some embodiments, the signal peptide has an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID No: 29-33. In some embodiments, the signal peptide differs from a sequence selected from the group consisting of SEQ ID Nos: 29-33 by 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 amino acid.

[0087]

[0088] Kozak sequence

[0089] Provided herein are nucleic acid molecules comprising a nucleic acid encoding a modified polypeptide (e.g., a modified α-GAL polypeptide or a modified PPT-1 polypeptide), wherein the nucleic acid molecule further comprises a nucleic acid having a kozak sequence that facilitates initiation of mRNA translation. The Kozak sequence contemplated herein has a consensus sequence of gccRccAUGG (SEQ ID NO: 34), wherein lowercase letters represent the most common base at the position, and the base changes, and uppercase letters represent highly conserved bases that change only slightly. R indicates that a purine (adenine or guanine) is always observed at the position. The sequence in brackets (gcc) has an uncertain meaning.

[0090] Therapeutic proteins

[0091] The gene therapy construct provided herein comprises a nucleic acid encoding a stable form of a protein for treating a genetic disorder. The therapeutic protein expressed from the gene therapy construct replaces a missing or defective protein. Therefore, the therapeutic protein is selected based on the genetic defect that needs to be treated in an individual. The stable form herein comprises one or more non-natural cysteine ​​residues, and the one or more non-natural cysteine ​​residues form a disulfide bridge between the non-natural cysteines in the protein or between the non-natural cysteines of two monomers of the protein.

[0092] In some embodiments, the gene therapy constructs herein encode enzymes, such as enzymes that are genetically defective in individuals with lysosomal storage diseases. In some embodiments, the enzymes encoded by the gene therapy constructs provided herein include, but are not limited to, α-galactosidase A, β-glucocerebrosidase, glucocerebrosidase, lysosomal acid lipase, glycosaminoglycan α-L-iduronidase, α-L-iduronidase, N-sulfoglucosamine sulfohydrolase (SGSH), N-acetyl-α-glucosaminidase (NAGLU), iduronate-2-sulfatase, N-acetylgalactosamine-6-sulfatase, glycosaminoglycan N-acetylgalactosamine 4-sulfatase, α-glucosidase, tripeptidyl peptidase 1 (TPP1), palmitoyl protein thioesterase, neuronal ceroid lipofuscinosis protein 1, neuronal ceroid lipofuscinosis protein 2, neuronal metaceroid lipofuscinosis protein 3, neuronal ceroid lipofuscinosis protein 4, neuronal ceroid lipofuscinosis protein 5, neuronal ceroid lipofuscinosis protein 6, neuronal ceroid lipofuscinosis protein 7, neuronal ceroid lipofuscinosis protein 8, neuronal ceroid lipofuscinosis protein 9, neuronal ceroid lipofuscinosis protein 10, neuronal ceroid lipofuscinosis protein 11, neuronal ceroid lipofuscinosis protein 12, neuronal ceroid lipofuscinosis protein 13, neuronal ceroid lipofuscinosis protein 14, neuronal ceroid lipofuscinosis protein 15, neuronal ceroid lipofuscinosis protein 16 and cyclin-dependent kinase-like 5.

[0093] Gene therapy vectors and compositions

[0094] Provided herein is a gene therapy vector comprising a nucleic acid construct comprising: a nucleic acid encoding a stable form of a protein for treating a neurological or genetic disorder, the stable form comprising one or more non-natural cysteine ​​residues, the one or more non-natural cysteine ​​residues forming a disulfide bridge between non-natural cysteines within the protein or between non-natural cysteines of two monomers of the protein. In some embodiments, the stable form comprises a modified α-GAL polypeptide or a modified PPT-1 polypeptide.

[0095] In some embodiments, nucleic acids encoding modified polypeptides are cloned into various types of vectors. For example, in some embodiments, the nucleic acids are cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0096] In addition, the expression vector encoding the modified polypeptide is provided to the cell in the form of a viral vector. Viral vector technology is described in, for example, Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual [Molecular Cloning: Laboratory Manual], Vol. 1-4, Cold Spring Harbor Press, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and slow viruses. In general, suitable vectors contain a replication origin, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193) that have a function in at least one organism.

[0097] Also provided herein are compositions and systems for gene transfer. Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. In some embodiments, the selected gene is inserted into a vector and packaged in retroviral particles using suitable techniques. The recombinant virus is then isolated and delivered to the subject's cells in vivo or ex vivo. Many retroviral systems are suitable for gene therapy. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are suitable for gene therapy. In some embodiments, adeno-associated virus vectors are used. Many adeno-associated viruses are suitable for gene therapy. In one embodiment, lentiviral vectors are used.

[0098] The gene therapy constructs provided herein include a vector (or gene therapy expression vector) into which the target gene is cloned, or otherwise includes the target gene in a manner such that the nucleotide sequence of the vector allows the target gene to be expressed (constitutively or otherwise regulated in some way). The vector constructs provided herein include any suitable gene expression vector that can be delivered to a target tissue and will provide expression of the target gene in a selected target tissue.

[0099] In certain embodiments, the vector is an adeno-associated virus (AAV) vector, because the AAV vector has the ability to cross the blood-brain barrier and transduce neuronal tissue. In the method provided herein, it is contemplated that any serotype of AAV is used. The serotype of the viral vector used in certain embodiments is selected from the group consisting of the following: 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 other vectors suitable for gene therapy.

[0100] AAV vectors are small DNA viruses that are non-pathogenic to mammals. Briefly, AAV-based vectors have had the rep and cap viral genes removed, which make up 96% of the viral genome, leaving two 145-base-pair flanking inverted terminal repeats (ITRs) that are used to initiate viral DNA replication, packaging, and integration.

[0101] Further embodiments include using other serotype capsids to produce 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 other vectors suitable for gene therapy. Optionally, the AAV virus capsid is AAV2 / 9, AAV9, AAVrhS, AAVrh10, AAVAnc80 or AAV PHP.B.

[0102] Other promoter elements (e.g., enhancers) regulate the frequency of transcription initiation. Typically, these are located in the region of 30-110bp upstream of the start site, although many promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so when elements are reversed or moved relative to each other, promoter function is retained. In the thymidine kinase (tk) promoter, the spacing between promoter elements is usually increased to 50bp before activity begins to decline. Depending on the promoter, individual elements seem to work collaboratively or independently to activate transcription.

[0103] An example of a promoter capable of expressing a stable protein (such as a modified α-GAL polypeptide or a modified PPT-1 polypeptide transgene) in mammalian T cells is the EF1a promoter. The natural EF1a promoter drives the expression of the α subunit of the elongation factor-1 complex, which is responsible for enzymatic delivery of aminoacyl tRNA to the ribosome. The EF1a promoter has been widely used in mammalian expression plasmids and has been shown to effectively drive the expression of transgenes cloned into lentiviral vectors (see, e.g., Milone et al., Mol. Ther. [Molecular Therapy] 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 that is capable of driving high-level expression of any polynucleotide sequence operably linked thereto. However, other constitutive promoter sequences are sometimes used, including but not limited to the 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 (Epstein-Barr virus) immediate early promoter, Rous sarcoma virus promoter, and human gene promoters (such as but not limited to actin promoter, myosin promoter, elongation factor-1a promoter, hemoglobin promoter and creatine kinase promoter). In addition, gene therapy vectors are not considered to be limited to the use of constitutive promoters. Inducible promoters are also considered here. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to it when such expression is desired, or turn off the 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. In some embodiments, the promoter is an α-GAL promoter.

[0104] In order to assess the expression of modified polypeptides, the expression vector to be introduced into the cell usually contains a selectable marker gene or a reporter gene or both, to facilitate identification and selection of expressing cells from a cell population attempted to be transfected or infected by a viral vector. In other aspects, selectable markers are usually carried on separate DNA fragments and used for co-transfection procedures. Both selectable markers and reporter genes are sometimes flanked by appropriate regulatory sequences to enable them to be expressed in host cells. Available selectable markers include, for example, antibiotic resistance genes, such as neo, etc.

[0105] Methods for introducing genes into cells and expressing them in cells are applicable to the methods herein. In the context of expression vectors, vectors are easily introduced into host cells (e.g., mammalian, bacterial, yeast or insect cells) by any method in the art. For example, expression vectors are transferred to host cells by physical, chemical or biological means.

[0106] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, gene guns, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are applicable to the methods herein (see, e.g., Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Vol. 1-4, Cold Spring Harbor Press, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0107] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods of nucleic acid targeted delivery of the prior art are available, such as delivering polynucleotides with targeted nanoparticles or other suitable submicron-sized delivery systems.

[0108] In the case of utilizing non-viral delivery systems, exemplary delivery vehicles are liposomes. It is expected that nucleic acids are introduced into host cells (in vitro, in vitro or in vivo) using lipid formulations. In another aspect, the nucleic acid is associated with lipids. In certain embodiments, the nucleic acid associated with lipids is encapsulated in the aqueous interior of the liposome, dispersed in the lipid bilayer of the liposome, attached to the liposome via a connecting molecule associated with both liposomes and oligonucleotides, wrapped in the liposome, compounded with the liposome, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in the lipids as a suspension, included or compounded with micelles, or otherwise associated with lipids. The compositions related to lipids, lipid / DNA or lipid / expression vectors are not limited to any specific structure in the solution. For example, in some embodiments, liposomes exist as double-layer structures, as micelles or have a "collapse" structure. Alternatively, liposomes are simply dispersed in the solution, and may form aggregates of uneven size or shape. Lipid is a fatty substance, and in some embodiments, it is a naturally occurring or synthetic lipid. For example, lipids include fat droplets naturally present in the cytoplasm as well as the class of compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0109] Lipids suitable for use are obtained from commercial sources. For example, in some embodiments, dimyristoylphosphatidylcholine ("DMPC") is obtained from Sigma, St. Louis, Missouri; in some embodiments, dicetyl phosphate ("DCP") is obtained from K&K Laboratories (Plainview, New York); in some embodiments, cholesterol ("Choi") is obtained from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids are typically obtained from Avanti Polar Lipids, Inc. (Birmingham, Alabama). Stock solutions of lipids in chloroform or chloroform / methanol are typically stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term covering a variety of monolayer and multilayer lipid vehicles formed by producing closed lipid bilayers or aggregates. Liposomes are generally characterized as having a vesicle structure containing a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they can form spontaneously. The lipid component undergoes self-rearrangement before forming a closed structure, and captures water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology [sugar biology] 5: 505-10). However, compositions having a structure different from a normal vesicle structure in a solution are also contemplated. For example, in some embodiments, lipids take a micellar structure or exist only as an inhomogeneous aggregate of lipid molecules. Lipofectamine (lipofectamine)-nucleic acid complexes are also contemplated.

[0110] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose cells to modified α-GAL polypeptides, in order to confirm the presence of the recombinant DNA sequence in the host cell, it is contemplated that a variety of assays are performed. Such assays include, for example, "molecular biology" assays suitable for use in the methods herein, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, such as by immunological means (ELISA and immunoblotting) or by assays described herein to identify agents falling within the scope of the present invention.

[0111] The disclosure further provides a vector comprising a nucleic acid molecule encoding a modified polypeptide. In one aspect, the therapeutic fusion protein vector can be directly transduced into a cell. In one aspect, the vector is a cloning or expression vector, for example, including but not limited to one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, microcarriers, double minute chromosomes), retroviruses, and lentiviral vector constructs. In one aspect, the vector can express a modified polypeptide construct in a mammalian cell. In one aspect, the mammalian cell is a human cell.

[0112] Pharmaceutical composition

[0113] Provided herein is a pharmaceutical composition comprising a modified polypeptide, i.e., a stable form of a protein for treating a genetic disorder, the stable form comprising one or more non-natural cysteine ​​residues, the one or more non-natural cysteine ​​residues forming a disulfide bridge between the non-natural cysteines within the protein or between the non-natural cysteines of two monomers of the protein; and (ii) a pharmaceutically acceptable excipient. In some embodiments, the modified polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the modified polypeptide exhibits an increased half-life at pH 7.4 compared to the wild-type polypeptide.

[0114] Further provided herein are pharmaceutical compositions comprising (i) a modified α-GAL polypeptide, wherein the modified α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence; and (ii) a pharmaceutically acceptable excipient. Contemplated substitutions include: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the modified α-GAL polypeptide comprises a cysteine ​​substitution of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, the modified α-GAL polypeptide comprises a cysteine ​​substitution of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the modified α-GAL polypeptide forms homodimers. In some embodiments, the homodimers are stabilized by disulfide bonds. In some embodiments, the modified α-GAL polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the composition comprises a molecular chaperone. In some embodiments, the chaperone comprises migalastat.

[0115] Further provided herein are pharmaceutical compositions comprising (i) a modified PPT-1 polypeptide, wherein the modified PPT-1 polypeptide comprises a cysteine ​​substitution of a PPT-1 polypeptide sequence; and (ii) a pharmaceutically acceptable excipient. Contemplated substitutions include A171C and A183C. In some embodiments, the modified PPT-1 polypeptide forms homodimers. In some embodiments, the homodimers are stabilized by disulfide bonds. In some embodiments, the modified PPT-1 polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type PPT-1 polypeptide. In some embodiments, the composition comprises a molecular chaperone.

[0116] Suitable excipients for use in the pharmaceutical compositions herein include, but are not limited to, saline, maleic acid, tartaric acid, lactic acid, citric acid, acetic acid, sodium bicarbonate, sodium phosphate, histidine, glycine, sodium chloride, potassium chloride, calcium chloride, zinc chloride, water, dextrose, N-methylpyrrolidone, dimethyl sulfoxide, N,N-dimethylacetamide, ethanol, propylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, and the surfactant polyoxyethylene-sorbitan monooleate.

[0117] In some embodiments, the pharmaceutical compositions herein comprise a modified α-GAL polypeptide herein having an increased half-life at pH 4.6 compared to a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 50% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 150% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 200% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 250% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 300% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 350% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 4.6 is at least 400% greater than that of a wild-type α-GAL polypeptide.

[0118] In some embodiments, the pharmaceutical compositions herein comprise a modified PPT-1 polypeptide herein having an increased half-life at pH 4.6 compared to a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 50% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 150% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 200% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 250% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 300% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 350% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 4.6 is at least 400% greater than that of a wild-type PPT-1 polypeptide.

[0119] In some embodiments, the pharmaceutical compositions herein comprise a modified α-GAL polypeptide having an increased half-life at pH 7.4. In some embodiments, the half-life at pH 7.4 is at least 50% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 150% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 200% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 250% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 300% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 350% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 400% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 500% greater than that of a wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 600% greater than the wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 700% greater than the wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 800% greater than the wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 900% greater than the wild-type α-GAL polypeptide. In some embodiments, the half-life at pH 7.4 is at least 1000% greater than the wild-type α-GAL polypeptide.

[0120] In some embodiments, the pharmaceutical compositions herein comprise a modified PPT-1 polypeptide having an increased half-life at pH 7.4. In some embodiments, the half-life at pH 7.4 is at least 50% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 150% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 200% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 250% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 300% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 350% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 400% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 500% greater than that of a wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 600% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 700% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 800% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 900% greater than the wild-type PPT-1 polypeptide. In some embodiments, the half-life at pH 7.4 is at least 1000% greater than the wild-type PPT-1 polypeptide.

[0121] Treatment

[0122] Gene therapy approach

[0123] Also provided herein is a method for improving at least one symptom of a genetic disease in a subject in need thereof. Some such methods include administering at least one dose of a composition comprising a gene therapy nucleic acid encoding a stable form of a protein for treating a genetic disorder, the stable form comprising one or more non-natural cysteine ​​residues, the one or more non-natural cysteine ​​residues forming a disulfide bridge between non-natural cysteines within the protein or between non-natural cysteines of two monomers of the protein. In some embodiments, the nucleic acid encodes a polypeptide forming a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the nucleic acid encodes a polypeptide having an increased half-life at pH 7.4 compared to a wild-type polypeptide. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid comprises at least a portion of a virus. In some embodiments, the virus is selected from wherein the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus. In some embodiments, the nucleic acid is packaged in a viral capsid protein. In some embodiments, the at least one symptom is selected from one or more of pain, skin discoloration, inability to sweat, cloudy eyes, gastrointestinal dysfunction, tinnitus, hearing loss, mitral valve prolapse, heart disease, joint pain, renal failure, and renal dysfunction. In some embodiments, at least one symptom is alleviated by a single administration of the gene therapy nucleic acid construct. In some embodiments, the method further comprises measuring activity in a tissue obtained from the subject after treatment.

[0124] In some embodiments, the gene therapy vector or the pharmaceutical composition is administered to the cerebrospinal fluid. In some embodiments, the gene therapy vector or the pharmaceutical composition is delivered by intrathecal, intraventricular, intraparenchymal or intravenous injection or a combination thereof. In some embodiments, the gene therapy vector or the pharmaceutical composition is administered by intrathecal injection. In some embodiments, the gene therapy vector or the pharmaceutical composition is administered via intravenous injection.

[0125] In some embodiments, the genetic disorder is a neurological disorder. In some embodiments, the genetic disorder is a lysosomal storage disease. In some embodiments, the genetic 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, Filippo disease, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroto-Rami disease, Slay 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), infantile, juvenile, and adult forms of neuronal ceroid lipofuscinosis, and CDKL5 deficiency.

[0126] Also provided herein is a method for improving at least one symptom of Fabry disease in a subject in need thereof. Some such methods include administering at least one dose of a composition comprising a gene therapy nucleic acid construct comprising at least one promoter and a nucleic acid encoding a modified α-GAL polypeptide comprising a cysteine ​​substitution of an α-GAL polypeptide sequence. The modified α-GAL polypeptide is expected to comprise a cysteine ​​substitution comprising: (i) R49C and G361C; (ii) R49C and G360C; (iii) D233C and I359C; (iv) M51C and G360C; and (v) S276C. In some embodiments, the nucleic acid encodes a cysteine-substituted polypeptide comprising an α-GAL polypeptide sequence selected from the group consisting of: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the nucleic acid encodes a polypeptide comprising cysteine ​​substitutions of D233C and I359C of an α-GAL polypeptide sequence. In some embodiments, the nucleic acid encodes a polypeptide comprising cysteine ​​substitutions of M51C and G360C of an α-GAL polypeptide sequence. In some embodiments, the nucleic acid encodes a polypeptide that forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the nucleic acid encodes a modified α-GAL polypeptide having an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid comprises at least a portion of a virus. In some embodiments, the virus is selected from wherein the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus. In some embodiments, the nucleic acid is packaged in a viral capsid protein. In some embodiments, the at least one symptom is selected from one or more of pain, skin discoloration, inability to sweat, cloudy eyes, gastrointestinal dysfunction, tinnitus, hearing loss, mitral valve prolapse, heart disease, joint pain, renal failure and renal dysfunction. In some embodiments, at least one symptom is alleviated by a single administration of the gene therapy nucleic acid construct. In some embodiments, the method further comprises measuring α-GAL activity in a tissue obtained from the subject after treatment. In some embodiments, the method further comprises administering a molecular chaperone. In some embodiments, the molecular chaperone comprises migalastat.

[0127] Also provided herein is a method for improving at least one symptom of CLN1 disease in a subject in need thereof. Some such methods include administering at least one dose of a composition comprising a gene therapy nucleic acid construct comprising at least one promoter and a nucleic acid encoding a modified PPT-1 polypeptide comprising a cysteine ​​substitution of a PPT-1 polypeptide sequence. The modified PPT-1 polypeptide is expected to comprise a cysteine ​​substitution comprising A171C and A183C. In some embodiments, the nucleic acid encodes a polypeptide that forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the nucleic acid encodes a modified PPT-1 polypeptide having an increased half-life at pH 7.4 compared to a wild-type PPT-1 polypeptide. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a tissue-specific promoter. In some embodiments, the nucleic acid comprises at least a portion of a virus. In some embodiments, the virus is selected from wherein the virus comprises a retrovirus, an adenovirus, an adeno-associated virus, a lentivirus, or a herpes virus. In some embodiments, the nucleic acid is packaged within a viral capsid protein. In some embodiments, the at least one symptom is selected from one or more of pain, skin discoloration, inability to sweat, cloudy eyes, gastrointestinal dysfunction, tinnitus, hearing loss, mitral valve prolapse, heart disease, joint pain, renal failure and renal dysfunction. In some embodiments, at least one symptom is alleviated by a single administration of the gene therapy nucleic acid construct. In some embodiments, the method further comprises measuring PPT-1 activity in a tissue obtained from the subject after treatment. In some embodiments, the method further comprises administering a molecular chaperone. In some embodiments, the molecular chaperone comprises migalastat.

[0128] In some embodiments, the treatment performed via the methods described herein delivers a gene encoding a therapeutic protein to cells in need of the therapeutic protein. In some embodiments, the treatment delivers the gene to all somatic cells in an individual. In some embodiments, the treatment replaces a defective gene in a targeted cell. In some embodiments, cells treated in vitro to express a therapeutic protein are delivered to an individual.

[0129] In some embodiments, the gene therapy treatment herein comprises administering a nucleic acid encoding a modified α-GAL polypeptide herein having an intracellular half-life increased by at least about 2, 2.5, 3, 3.5, 4, 4.5, or 5 times compared to the half-life of wild-type human α-GAL.

[0130] Enzyme replacement therapy approach

[0131] Also provided is a method for improving at least one symptom of a genetic disease in a subject in need thereof, the method comprising administering at least one dose of a composition comprising a stable form of a protein for treating a genetic disorder, wherein the stable form comprises one or more non-natural cysteine ​​residues, and the one or more non-natural cysteine ​​residues form a disulfide bridge between the non-natural cysteines within the protein or between the non-natural cysteines of two monomers of the protein. In some embodiments, the modified polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the modified polypeptide shows an increased half-life at pH 7.4 compared to the wild-type polypeptide. In some embodiments, the at least one symptom is selected from one or more of mental impairment, epilepsy, loss of speech ability, and loss of motor skills. In some embodiments, the method further comprises administering a molecular chaperone. In some embodiments, the molecular chaperone comprises migalastat.

[0132] In some embodiments, the composition is administered via intrathecal, intracerebroventricular, intraparenchymal, subcutaneous, intramuscular, ocular, intravenous injection, or a combination thereof.

[0133] In some embodiments, the genetic disorder is a neurological disorder. In some embodiments, the genetic disorder is a lysosomal storage disease. In some embodiments, the genetic 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, Filippo disease, Sanfilippo disease type D, Morquio disease type A, Morquio disease type B, Maroto-Rami disease, Slay 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), infantile, juvenile, and adult forms of neuronal ceroid lipofuscinosis, and CDKL5 deficiency.

[0134] Also provided is a method of improving at least one symptom of Fabry disease in a subject in need thereof, the method comprising administering at least one dose of a composition comprising a modified α-GAL polypeptide, wherein the modified α-GAL polypeptide comprises a cysteine ​​substitution of the α-GAL polypeptide sequence. Contemplated cysteine ​​substitutions include: (i) D233C and I359C; and (ii) M51C and G360C. In some embodiments, the modified α-GAL polypeptide comprises a cysteine ​​substitution of D233C and I359C of the α-GAL polypeptide sequence. In some embodiments, the modified α-GAL polypeptide comprises a cysteine ​​substitution of M51C and G360C of the α-GAL polypeptide sequence. In some embodiments, the modified α-GAL polypeptide forms a homodimer. In some embodiments, the homodimer is stabilized by a disulfide bond. In some embodiments, the modified α-GAL polypeptide exhibits an increased half-life at pH 7.4 compared to a wild-type α-GAL polypeptide. In some embodiments, the at least one symptom is selected from one or more of pain, skin discoloration, inability to sweat, cloudy eyes, gastrointestinal dysfunction, tinnitus, hearing loss, mitral valve prolapse, heart disease, joint pain, renal failure, and renal dysfunction. In some embodiments, the method further comprises administering a molecular chaperone. In some embodiments, the molecular chaperone comprises migalastat.

[0135] Also provided is a method of improving at least one symptom of CLN-1 disease in a subject in need thereof, the method comprising administering at least one dose of a composition comprising a modified PPT-1 polypeptide, wherein the PPT-1 polypeptide comprises one or more non-native cysteine ​​residues, the one or more non-native cysteine ​​residues forming a disulfide bridge between non-native cysteines within the protein or between non-native cysteines of two monomers of the protein. Contemplated cysteine ​​substitutions include: A171C and A183C. In some embodiments, the modified PPT-1 polypeptide forms homodimers. In some embodiments, the homodimers are stabilized by disulfide bonds. In some embodiments, the modified PPT-1 polypeptide exhibits an increased half-life at pH 7.4 compared to the wild-type PPT-1 polypeptide. In some embodiments, the at least one symptom is selected from one or more of mental impairment, epilepsy, loss of speech, and loss of motor skills. In some embodiments, the method further comprises administering a molecular chaperone. In some embodiments, the molecular chaperone comprises migalastat.

[0136] In some embodiments, the methods herein include administering a modified polypeptide herein having an increased half-life compared to a wild-type polypeptide. In some embodiments, the half-life is at least 50% greater than a wild-type polypeptide. In some embodiments, the half-life is at least 150% greater than a wild-type polypeptide. In some embodiments, the half-life is at least 200% greater than a wild-type polypeptide. In some embodiments, the half-life is at least 250% greater than a wild-type polypeptide. In some embodiments, the half-life is at least 300% greater than a wild-type polypeptide. In some embodiments, the half-life is at least 350% greater than a wild-type polypeptide.

[0137] In some embodiments, the methods herein include administering a modified α-GAL polypeptide herein having an increased half-life compared to a wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 50% greater than a wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 150% greater than a wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 200% greater than a wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 250% greater than a wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 300% greater than a wild-type α-GAL polypeptide. In some embodiments, the half-life is at least 350% greater than a wild-type α-GAL polypeptide.

[0138] In some embodiments, the methods herein include administering a modified PPT-1 polypeptide herein having an increased half-life compared to a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 50% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 150% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 200% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 250% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 300% greater than a wild-type PPT-1 polypeptide. In some embodiments, the half-life is at least 350% greater than a wild-type PPT-1 polypeptide.

[0139] definition

[0140] As used herein, "stable" with respect to proteins refers to a modified protein (e.g., modified to contain a non-native cysteine ​​residue) that maintains one or more of its biological activities for a longer period of time than a corresponding protein without the modification. In some embodiments, a stable protein maintains biological activity for about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500% longer than a corresponding protein without the modification. In some embodiments, the stable protein maintains biological activity for at least 10% longer, at least 20% longer, at least 30% longer, at least 40% longer, at least 50% longer, at least 60% longer, at least 70% longer, at least 80% longer, at least 90% longer, at least 100% longer, at least 150% longer, at least 200% longer, at least 250% longer, at least 300% longer, at least 350% longer, at least 400% longer, at least 450% longer, or at least 500% longer than the corresponding protein without the modification. In some embodiments, the stable protein has a longer half-life compared to the corresponding protein without the non-natural cysteine. In some embodiments, the stable protein has a longer half-life at pH 4.0 to pH 8.0, or pH 4.0 to 6.0, or pH 6.0 to 8.0 compared to the corresponding protein without the non-natural cysteine. In some embodiments, the stable protein has a longer half-life at pH 4.5 to 5.0 or 7.0 to 7.5 compared to the corresponding protein without the non-natural cysteine. In some embodiments, the stable protein has a longer half-life at pH 7.4 compared to the corresponding protein without the non-natural cysteine. In some embodiments, the stable protein has a longer half-life at pH 4.6 compared to the corresponding protein without the non-natural cysteine.

[0141] As used herein, "ex vivo gene therapy" refers to a method in which a patient's cells are genetically modified outside of a subject, for example to express a therapeutic gene. The cells with the new genetic information are then returned to the subject from which they originated.

[0142] As used herein, "in vivo gene therapy" refers to a method in which a vector carrying one or more therapeutic genes is administered directly to a subject.

[0143] As used herein, "fusion protein" and "therapeutic fusion protein" are used interchangeably herein and refer to a therapeutic protein having at least one additional protein, peptide or polypeptide attached thereto. In some cases, a fusion protein is a single protein molecule containing two or more proteins or fragments thereof, which are covalently linked via peptide bonds within their respective peptide chains without a chemical linker. In some embodiments, the fusion protein comprises a therapeutic protein and a signal peptide, a peptide that increases endocytosis of the fusion protein, or both. In some embodiments, the peptide that increases endocytosis is a peptide that binds CI-MPR.

[0144] As used herein, "plasmid" refers to a circular double-stranded DNA unit that replicates within a cell independently of chromosomal DNA.

[0145] As used herein, "promoter" refers to a site on DNA to which the enzyme RNA polymerase binds and initiates transcription of the DNA into RNA.

[0146] As used herein, "somatic cell therapy" refers to methods of manipulating gene expression in cells that will correct the condition in the patient but will not be inherited by the next generation. Somatic cells include all non-reproductive cells in the human body.

[0147] As used herein, "somatic cells" refers to all cells of the body except germ cells.

[0148] As used herein, "tropism" refers to the preference of a vector (such as a virus) for a certain cell or tissue type. A variety of factors determine the ability of a vector to infect a specific cell. For example, a virus must bind to a specific cell surface receptor to enter a cell. If the virus does not express the necessary receptor, they typically cannot infect the cell.

[0149] As used herein, "vector" or "gene therapy vector" are used interchangeably herein and refer to a gene therapy delivery vehicle or vector that delivers therapeutic genes to cells. A gene therapy vector is any vector suitable for use in gene therapy, for example, any vector suitable for therapeutic delivery of nucleic acid polymers (encoding polypeptides or variants thereof) to patient target cells (e.g., sensory neurons). In some embodiments, the gene therapy vector delivers nucleic acids encoding therapeutic proteins or therapeutic fusion proteins to cells, wherein the therapeutic proteins or fusion proteins are expressed and secreted from the cells. The vector can be of any type, for example, it can be a plasmid vector or a minicircle DNA. Typically, the vector is a viral vector. These include both genetically disabled viruses (such as adenoviruses) and non-viral vectors (such as liposomes). The viral vector can be derived, for example, from an adeno-associated virus (AAV), a retrovirus, a lentivirus, a herpes simplex virus, or an adenovirus. AAV source vector. The vector can include an AAV genome or a derivative thereof.

[0150] As used herein, "construct" refers to a nucleic acid molecule or sequence encoding a therapeutic protein or fusion protein, and optionally comprises additional sequences, such as a translation initiation sequence or an IRES sequence.

[0151] The term "transduction" is used to refer to the administration / delivery of a nucleic acid encoding a therapeutic protein to a target cell in vivo or in vitro via the replication-deficient rAAV disclosed herein, thereby resulting in the expression of a functional polypeptide by the recipient cell. Transduction of cells with a gene therapy vector (such as the rAAV disclosed herein) results in the sustained expression of a polypeptide or RNA encoded by the rAAV. Therefore, the present disclosure provides a method for administering / delivering a gene therapy vector (such as an rAAV encoding a therapeutic protein) to a subject via an intrathecal, intraretinal, intraocular, intravitreal, intraventricular, intraparenchymal or intravenous route or any combination thereof. "Intrathecal" delivery refers to delivery to the space under the arachnoid membrane of the brain or spinal cord. In some embodiments, intrathecal administration is via intracisternal administration.

[0152] The terms "recipient", "individual", "subject", "host" and "patient" are used interchangeably herein and in some cases refer to any mammalian subject, particularly a human, for whom diagnosis, treatment or therapy is desired. "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, livestock and farm animals, and laboratory, zoo, sports or pet animals, such as dogs, horses, cats, dairy cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, etc. In some embodiments, the mammal is a human.

[0153] As used herein, the terms "treatment", "treating", "improvement of symptoms", and the like refer in some cases to the administration of an agent or the performance of a procedure for the purpose of obtaining a therapeutic effect, including inhibiting, attenuating, alleviating, preventing, or altering at least one aspect or marker of a disorder in a statistically significant manner or in a clinically significant manner. The terms "improvement" or "treatment" do not state or imply a cure of the underlying condition. "Treatment" or "improvement" (etc.) as used herein may include treatment of mammals, particularly in humans, and include: (a) preventing a disorder or a symptom of a disorder from occurring in a subject who may be susceptible to the disorder but has not yet been diagnosed with the disorder (e.g., including disorders that may be associated with or caused by a primary disorder); (b) inhibiting the disorder, i.e., curbing its development; (c) alleviating the disorder, i.e., causing the disorder to regress; and (d) improving at least one symptom of the disorder. Treatment may refer to any successful marker of treating or improving or preventing a disorder, including any objective or subjective parameter, such as elimination; alleviation; alleviation of symptoms or making the disorder condition more tolerable to the patient; slowing the rate of degeneration or decline; or making the endpoint of degeneration less debilitating. Treatment or improvement of symptoms is based on one or more objective or subjective parameters; including the results of a doctor's examination. Therefore, the term "treatment" includes the administration of a compound or agent of the present invention to prevent or delay, alleviate or stop or inhibit the development of symptoms or conditions associated with a disorder. The term "therapeutic effect" refers to the alleviation, elimination or prevention of a subject's disorder, a symptom of a disorder, or a side effect of a disorder.

[0154] The term "affinity" refers to the strength of binding between a molecule and its binding partner or receptor.

[0155] As used herein, the phrase "high affinity" refers to, for example, a therapeutic fusion containing such a peptide that binds to CI-MPR, whose affinity for CI-MPR is about 100 to 1,000 times or 500 to 1,000 times greater than the affinity of the therapeutic protein without the peptide. In some embodiments, the affinity is at least 100 times, at least 500 times, or at least 1000 times greater than without the peptide. For example, when a therapeutic protein and CI-MPR are combined at relatively equal concentrations, the high affinity peptide will bind to the available CI-MPR so as to shift the equilibrium toward a high concentration of the resulting complex.

[0156] As used herein, "secretion" refers to the release of a protein from a cell into, for example, the bloodstream to be carried to a target tissue or site of action of the therapeutic protein. When a gene therapy product is secreted into the interstitial space of an organ, secretion can allow cross-correction of neighboring cells.

[0157] As used herein, "delivery" means drug delivery. In some embodiments, the delivery process means transporting a drug substance (e.g., a therapeutic protein or fusion protein produced from a gene therapy vector) from outside a cell (e.g., blood, tissue, or interstitial space) to a target cell for therapeutic activity of the drug substance.

[0158] As used herein, "engineering" or "protein engineering" refers to manipulating the structure of a protein by providing an appropriate nucleic acid sequence that encodes the protein to produce desired properties, or to synthesize a protein with a specific structure.

[0159] In some cases, "therapeutically effective amount" means an amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for the disease.

[0160] As used herein, the term "about" a certain number refers to a range from 10% less than the number to 10% greater than the number and includes the values ​​within the range, like the number itself.

[0161] As used herein, the term “comprising” one or more elements of a claim refers to those elements but does not exclude the inclusion of additional one or more elements.

[0162] Examples

[0163] The following examples are given for the purpose of illustrating various embodiments of the present invention and are not meant to limit the present invention in any way. The present examples together with the methods described herein currently represent preferred embodiments, are exemplary, and are not intended to be limiting of the scope of the present invention. Those skilled in the art will appreciate variations and other uses contained within the spirit of the present invention as defined by the scope of the claims.

[0164] Example 1: Identification of amino acid residues for cysteine ​​substitution of wild-type α-GAL

[0165] The crystal structure of dimerized α-GAL (PDB ID 3HG3) was examined for potential sites for substitution of cysteine ​​residues to create additional disulfide bonds to enhance stability ( Figure 1A ). NAMD with the CHARMM force field was used for analysis. Based on the analysis, the cysteine ​​mutants shown in Table 8 were prepared using standard methods for site-directed mutagenesis.

[0166]

[0167] See also Figure 1B The amino acid sequences are provided in Table 1.

[0168] Example 2: Dimerization and enzyme activity of modified α-GAL

[0169] The formation of disulfide-bonded dimers of modified α-GAL was examined in cell lysates and culture medium ( Figure 2A ). A clone of each α-GAL construct was transiently expressed in 293HEK cells. Cell lysates and culture medium were run on a 4%-12% gradient SDS-PAGE and transferred to nitrocellulose. α-GAL was detected by Western blotting with rabbit monoclonal anti-α-GAL 1:2000 (abcam ab168341).

[0170] Electrophoresis and Western blotting were performed on reduced and non-reduced samples. As shown in Figure 2, the M51C-G360C and D233C-I359C versions of α-GAL readily formed disulfide-bonded α-GAL dimers.

[0171] To prepare the sample, harvest 1x 10^6 cells with transient expression of α-GAL construct. The cells are lysed in 500ul 20mM sodium phosphate buffer pH 6.5, 0.25% TX-100. The cell lysate is centrifuged for 2min at 10,000g, and the supernatant is transferred to a new tube. 40ul cell lysate or culture medium is transferred to a new tube, and 6μl 10X reducing agent (for reducing conditions) is added to 16μl LDS 4X sample buffer. The sample mixture prepared as follows is heated at 95°C for 5 minutes. Electrophoresis is performed using 1x MOPS SDS running buffer.

[0172] To test the enzyme activity, the lysate or culture medium was incubated with 4-methylumbelliferyl-α-D-galactopyranoside (4-MUG) substrate for 1 hour. The enzymatic reaction was then stopped and the α-GAL enzyme activity was measured by excitation at 360 nm and emission at 450 nm. Figure 2B As shown, both the M51C-G360C and D233C-I359C disulfide α-GAL mutants have enzymatic activity. Because the specific activity and amount of α-GAL in each sample were not quantified, Figure 2B A quantitative comparison of the activity between wild-type and mutant versions of α-GAL was not provided.

[0173] Example 3: Stability analysis of modified α-GAL in an acidic environment over time

[0174] To test pH stability over 24 h, transiently expressed mutants and wild-type α-GAL were captured using ConA agarose pull-down according to standard methods. ConA eluates were diluted in pH 4.6 buffer or pH 7.4 buffer. Samples were pre-incubated at pH 4.6 or 7.4 for 0, 0.5, 1, 2, 4, 5, and 24 hours.

[0175] To measure enzyme activity, pH 4.6 buffer was added to each sample and activity was tested on 4-MUG substrate. The reaction mixture was incubated at 37°C for 1 hour. The reaction was terminated by adding 125uL stop buffer (0.4M glycine-NaOH, pH 10.8). Fluorescence was read using a Spectramax plate reader: excitation: 360nm, emission: 450nm. The results are shown in Figure 3A middle.

[0176] For long-term stability testing, transiently expressed modified and wild-type α-GAL were isolated from the culture medium and enriched and purified using ConA agarose beads as described above. The eluted α-GAL was incubated at pH 4.6 or pH 7.4 for time course stability experiments. Time points included 0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 5 hours, 24 hours, 2 days, 5 days, 6 days, and 7 days. Figure 3B It is shown that at pH 4.6, M51C-G360C α-GAL is more stable than the wild-type control over a span of 7 days. Both modified α-GALs are significantly more stable than the wild-type control at pH 7.4 over 7 days.

[0177] Example 4: α-GAL uptake and enzymatic assay in Fabry patient fibroblasts

[0178] Cellular uptake protocol

[0179] In order to carry out the uptake assay, on the first day, 300,000 Fabry patient fibroblasts (R301Q) were seeded into each well of a 6-well plate. On the second day, the culture medium was replaced with 1.8 mL uptake culture medium and incubated for 1 hour at 37°C with 5% CO2. The cells were given 200 uL doses of 250 nM enzymes (Fabrazyme, M51C-G360C, D233C-I359C and WT) prepared in the uptake culture medium in the 6 wells prepared in step 2 for 16-18 hours. On the third day, 300 uL 1 M Tris was added and incubated at room temperature for 30 min. 400 uL 1 M NaH2SO4 was added and mixed. The cell plate was washed twice with 1 ml DPBS. 500 uL water was added to each well and cells were collected from the plate. Matric-green was added and then frozen in a -80°C refrigerator until assayed. The plate was rotated before enzyme and protein assays.

[0180] The protein assay was performed by adding 20uL of cell lysate to 130uL of water. 150uL of BCA working reagent was added and incubated at 37°C for 2 hours. The plate was then read on a Spectramax.

[0181] The enzyme assay was performed by adding 5uL of cell lysate to 15uL of assay buffer, followed by 50uL of 4-MUG substrate. This was incubated at 37°C for 1 hour. 125uL of stop buffer was added and read on the Spectramax.

[0182] As a control, frozen cell lysate was thawed and ultrasonically treated for 5 min at room temperature. 50 μL was transferred to a 13 mL silanized glass tube. 25 μL of IS (concentration 125 ng / mL) was added. 1 mL of methanol was added and the mixture was ultrasonically treated for about 10 min. 500 μL of 1N HCl was added, vortexed, and then ultrasonically treated for about 10 minutes. The mixture was then vibrated at room temperature for about 30 minutes. The sample was centrifuged at room temperature for 10 min at 4,000 rpm. The supernatant was transferred to a pre-conditioned SPE cartridge.

[0183] Solid samples were prepared by adjusting the SPE cartridge with 1ml methanol and 1ml Millipore water. The samples were loaded onto the SPE cartridge. The cartridge was then washed with 2mL 0.1N HCl and 2mL MEOH. The samples were eluted into clean silanized glass with 2mL 5% ammonium hydroxide methanol solution. The samples were evaporated to dryness at 40°C under nitrogen. 25 μL DMSO was added to each extract and vortexed. 125 μL (175 μL was used for running 03) of mobile phase B was added and vortexed. The samples were transferred to a glass vial. 10 μl was injected onto the analytical column.

[0184] Fibroblasts from Fabry disease patients were cultured and seeded in 6-well plates. Cells treated with wild-type α-GAL were used as positive controls for uptake and subsequent enzyme studies. Fibroblasts were incubated with wild-type α-GAL, M51C-G360C α-GAL, or D233C-I359C α-GAL for 16 to 18 hours. The cells were then lysed for α-GAL enzymatic assays as determined by fluorescence output. Figure 4A It was shown that both M51C-G360C and D233C-I359C α-GAL were able to restore α-GAL enzyme activity at least as well as wild-type α-GAL. Figure 4B Globulin (lyso-Gb3) is a biomarker for Fabry disease. Successful treatment of Fabry disease results in a significant reduction in lyso-Gb3 as determined by LC-MS / MS.

[0185] Example 5: FB-14(R301Q) variant homodimer uptake in Fabry patient fibroblasts

[0186] Fabry patient fibroblasts were seeded in 6-well plates for Fabryzin and α-GAL cellular uptake studies. Cells were incubated in uptake medium containing 7 nM Fabryzin, wild-type α-GAL, M51C-G360C α-GAL, or D233C-I359C α-GAL for 16 h at 37C, 5% CO2 incubator. On day 1, cells were washed and further maintained in regular growth medium for another 5 days. Figure 5 Cells were harvested at the indicated time points. Enzyme activity was determined using cell lysates. α-GAL enzyme activity was determined and normalized to nmol / mg protein / h using cell lysate protein concentration. It was determined that after cellular uptake, the half-life of the variant homodimer in the cell was 2-3 times that of the wild type and 3-4 times that of Fabrazyme ( Figure 5 ).

[0187] Example 6: Gene therapy for Fabry disease in a mouse model

[0188] AAV vectors were diluted in sterile PBS. AAV vectors included: AAVhu68.CB7.hGL Anatural.rBG, AAVhu68.CB7.hGLAco.rBG, and AAVhu68.CB7.hGLA-M51C-G360Cco.rBG.

[0189] Vector production

[0190] The reference GLA sequence and variants with methionine to cysteine ​​at position 51 and glycine to cysteine ​​at position 360 were back translated and the nucleotide sequence was codon optimized to generate a cis-plasmid for producing AAV with an expression cassette under the CB7 promoter. In addition, native hGLA (reference sequence) cDNA was sequenced and cloned into the same AAV-cis backbone to compare with the codon-optimized sequence. AAVhu68 vectors were generated and titrated as previously described (Lock, Alvira et al. 2010, "Rapid, simple, and versatile manufacturing of recombinant adeno-associated viral vectors at scale." Hum Gene Ther [Human Gene Therapy] 21 (10): 1259-1271). In brief, HEK293 cells were triple transfected and the culture supernatant was harvested, concentrated and purified with an iodixanol gradient. The purified vector was titered by droplet digital PCR using primers targeting rabbit β-globin polyA sequences as described previously (Lock M, R. Alvira, SJ Chen, and JM Wilson, "Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by droplet digital PCR." Hum Gene Ther Methods 25(2): 115-125 (2014)).

[0191] animal

[0192] Mus musculus (Gla knockout Fabry mice in C57BL / 6 / 129 background founder) were purchased from Jackson Labs (Stock No. 003535 - also known as "α-Gal A KO mice"). Breeding colonies were maintained in a barrier mouse facility AAALAC accredited for gene therapy programs, using heterozygotes to mating heterozygotes to generate null and WT controls within the same litter. Gla knockout mice are a widely used model of Fabry disease.

[0193] The mice appear normal clinically, but they show a gradual accumulation of the GLA substrate globosine (also known as lyso-GB3) in plasma, and a gradual accumulation of globosylceramide (also known as GL3, GB3) in the liver, heart, kidney, skin, small and large intestines, and central nervous system. Small-scale, reproducible phenotypes, and efficient breeding allow for rapid studies, which are optimal for preclinical candidates in in vivo screening.

[0194] The animal housing room was maintained at a temperature range of 64°F-79°F (18°C-26°C) and a humidity range of 30%-70%. Animals were housed with their parents and littermates until weaning, and then housed in standard cages at the Translational Research Laboratories (TRL) GTP animal housing facility, 2 to 5 animals per cage. Cages, water bottles, and bedding substrates were autoclaved in a barrier facility. An automatically controlled 12-hour light / dark cycle was maintained. Each dark period began at 1900 hours (± 30 minutes). Food (Purina, 5053,Irradiated, Rodent Diet 20, 25 lb). All animals had ad libitum access to water via individual water bottles placed in each home cage.

[0195] In vivo studies and histology

[0196] On days 7 and 21 after vehicle administration, 5 × 10 11 GC (about 2.5x10 13 Mice injected with 100 GC / kg) AAVhu68.CB7.hGLA ​​(various hGLA constructs) were bled for serum separation and euthanized by final bleeding (for plasma separation) 28 days after injection. Tissues were collected rapidly, starting with the brain.

[0197] Tissues for histology were formalin fixed and paraffin embedded using standard methods. Spinal cords (in bone) with DRG were fixed in ZF, decalcified in EDTA, and processed according to the standard procedures of GTP Morphology Core. Zinc-formalin was used to obtain good tissue preservation and was used for staining Gb3 stocks by IHC and for morphology (H&E).

[0198] GL3 immunostaining was performed on formalin-fixed paraffin-embedded samples. The sections were dewaxed, blocked with 1% donkey serum in PBS + 0.2% Triton for 15 min, and then incubated with primary antibodies (Amsbio AMS. A2506, anti-Gb3 monoclonal antibody) and biotinylated secondary antibodies diluted in blocking buffer; the signal was detected using an HRP-based colorimetric reaction. The slides were reviewed in a blinded manner by a board-certified veterinary pathologist.

[0199] Fabry- / - mice vehicle PBS controls showed significant accumulation of GL3 (dark staining on IHC stained sections). WT mice and all vehicle-treated mice had near-complete to complete clearance of GL3 stores ( Figure 6 ).

[0200] GLA activity

[0201] The plasma or supernatant of the homogenized tissue was mixed with 6mM 4-MU-α-galactopyranoside pH 4.6, 90mM GalNAc and incubated at 37°C for three hours. The reaction was stopped with 0.4M glycine pH 10.8. Relative fluorescence units (RFUs) were measured using a Victor3 fluorometer, excitation at 355nm and emission at 460nm. The activity in nmol / mL / h was calculated by interpolation from the standard curve of 4-MU. The activity levels in the individual tissue samples were normalized to the total protein content in the homogenate supernatant. Equal volumes were used for plasma samples.

[0202] Fabry- / - mice show a complete lack of α-Gal A activity. Treatment of Fabry mice with the AAVhu68.CB7.hGLA-M51C-G360Cco.rBG GTx vector resulted in >7-fold higher GLA activity in the kidneys than wild-type mice ( Figure 7 ).

[0203] Quantification of globotriaosylceramide (also known as GL3, GB3) by LC-MS / MS

[0204] The GLA substrate GL3 in tissue homogenate was quantified by LC-MS / MS assay. In brief, internal standard was added to the homogenate sample (50 μL), and the sample was processed using C18-based solid phase extraction (SPE). A standard curve of known concentrations of GL3 (8.83 nM to 4.41 μM) was prepared from a stock solution containing twelve ceramide forms. The monitored reactions of all twelve isoforms were summed, and a ratio relative to the internal standard was generated in this assay. The resulting ratio of the study sample was then compared to the prepared curve for GL3 quantification.

[0205] Fabry- / - mice showed >10-fold accumulation of the GLA substrate globotriaosylceramide (GL3). Treatment of Fabry mice with the AAVhu68.CB7.hGLA-M51C-G360Cco.rBG GTx vector resulted in a complete reduction of GL3 in the kidney to wild-type levels ( Figure 8 ).

[0206] Quantification of lyso-GB3 by LC-MS / MS

[0207] The GLA substrate lyso-GB3 in plasma was quantified by LC-MS / MS assay. In brief, a stable C13-labeled internal standard was added to a plasma sample (50 μL), and the sample was processed using a C18 / cation exchange mixed mode solid phase extraction (SPE). A standard curve of known concentrations (0.254 nM to 254 nM) of lyso-GB3 was prepared, and the lyso-GB3 response of the study sample was then compared with the prepared curve for lyso-GB3 quantification.

[0208] GLA signature peptides obtained by LC / MS

[0209] Plasma was precipitated in 100% methanol and centrifuged. The supernatant was discarded. A stable isotope-labeled peptide unique to hGLA was spiked into the precipitate as an internal standard and resuspended with trypsin and incubated at 37°C for two hours. Digestion was stopped with 10% formic acid. Peptides were separated by C-18 reverse phase chromatography and identified and quantified by ESI-mass spectrometry. The total GLA concentration in plasma was calculated from the characteristic peptide concentration.

[0210] Cell surface receptor binding assay

[0211] 96-well plates were coated with receptors, washed, and blocked with BSA. CHO culture conditioned medium or plasma containing equal rhGLA or engineered GLA activity was serially diluted threefold to obtain a series of nine decreasing concentrations and incubated with co-coupled receptors. After incubation, the plates were washed to remove any unbound GLA, and 4-MU-α-pyranogalactoside was added for one hour at 37°C. The reaction was stopped with 1.0 M glycine (pH 10.5), and RFU was read by a Spectramax fluorometer; excitation 370, emission 460. The RFU of each sample was converted to activity in nmol / mL / h by interpolation from a standard curve of 4-MU. Nonlinear regression was performed using GraphPad Prism.

[0212] Example 7: Stable PPT-1 construct

[0213] A stable PPT-1 construct was engineered based on the crystal structure (PDB ID 3GRO). The two cysteines are predicted to form a disulfide bond, which stabilizes the structure and was found to extend the half-life of enzyme function (see data below). This construct was found to express levels in HEG 293 close to those of wild-type PPT-1.

[0214] Improved the stability and half-life of the PPT-1 enzyme

[0215] Intramolecular disulfide bridges were engineered into PPT-1 to stabilize the enzyme as determined by measuring the half-life of the active enzyme. The mutated residues A171C / A183C were chosen because the equivalent residues in the homologous protein PPT-2 form a disulfide bridge. This natural variation in the homologous protein was used to inform the engineering work in PPT-1.

[0216] Stability testing of construct PPT-1

[0217] The construct PPT-1 was transiently expressed in HEK 293T cells and the conditioned medium was harvested five days after transfection. The same procedure was performed for wild-type PPT1. Both sets of conditioned medium were assayed for enzyme activity over the course of 48 or 72 hours. The amount of enzyme activity retained over time was determined in order to compare the double cysteine ​​mutant to the WT. ( Fig. 9 ).

[0218] Half-life was estimated in two ways representing the alpha and beta phases, since activity appears to be eliminated in two phases (see logarithmic plots near the PK table below). The alpha half-life was estimated during the early terminal or distribution phase, and the beta half-life was estimated during the terminal elimination phase. For ATB200 total GAA protein analysis, the alpha phase is usually reported because it is more meaningful to demonstrate the effects of AT2221 on the binding and stabilization of ATB200 during distribution in the blood to tissues.

[0219] The pharmacokinetics of construct PPT-1, including C0 and AUC, are reported. 无穷 is derived from the same elimination rate constant used to estimate the beta half-life.

[0220]

[0221] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art will now appreciate many variations, changes and substitutions. It should be understood that various alternatives to the embodiments described herein may be employed. It is intended that the following claims define the scope of the present invention, and thus cover methods and structures within the scope of these claims and their equivalents. Sequence Listing <110> AMICUS THERAPEUTICS, INC. <120> Disulfide-stabilized polypeptide compositions and methods of use <130> 36003-704.601 <140> PCT / US2019 / 055679 <141> 2019-10-10 <150> 62 / 744,069 <151> 2018-10-10 <160> 34 <170> PatentIn Version 3.5 <210> 1 <211> 429 <212> PRT <213> Homo sapiens <400> 1 Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu 20 25 30 Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile 50 55 60 Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met 85 90 95 Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg 100 105 110 Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly 115 120 125 Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140 Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala 145 150 155 160 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser 165 170 175 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 180 185 190 Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 195 200 205 Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn 210 215 220 His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys 225 230 235 240 Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val 245 250 255 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn 260 265 270 Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala 275 280 285 Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser 290 295 300 Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn 305 310 315 320 Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn 325 330 335 Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 340 345 350 Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala 355 360 365 Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile 370 375 380 Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385 390 395 400 Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln 405 410 415 Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu 420 425 <210> 2 <211> 429 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 2 Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu 20 25 30 Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 Cys Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile 50 55 60 Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met 85 90 95 Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg 100 105 110 Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly 115 120 125 Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140 Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala 145 150 155 160 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser 165 170 175 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 180 185 190 Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 195 200 205 Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn 210 215 220 His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys 225 230 235 240 Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val 245 250 255 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn 260 265 270 Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala 275 280 285 Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser 290 295 300 Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn 305 310 315 320 Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn 325 330 335 Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 340 345 350 Met Ile Asn Arg Gln Glu Ile Gly Cys Pro Arg Ser Tyr Thr Ile Ala 355 360 365 Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile 370 375 380 Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385 390 395 400 Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln 405 410 415 Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu 420 425 <210> 3 <211> 429 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 3 Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu 20 25 30 Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 Cys Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile 50 55 60 Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met 85 90 95 Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg 100 105 110 Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly 115 120 125 Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140 Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala 145 150 155 160 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser 165 170 175 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 180 185 190 Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 195 200 205 Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn 210 215 220 His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys 225 230 235 240 Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val 245 250 255 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn 260 265 270 Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala 275 280 285 Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser 290 295 300 Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn 305 310 315 320 Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn 325 330 335 Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 340 345 350 Met Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser Tyr Thr Ile Ala 355 360 365 Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile 370 375 380 Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385 390 395 400 Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln 405 410 415 Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu 420 425 <210> 4 <211> 429 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 4 Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu 20 25 30 Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 Arg Phe Cys Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile 50 55 60 Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met 85 90 95 Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg 100 105 110 Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly 115 120 125 Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140 Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala 145 150 155 160 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser 165 170 175 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 180 185 190 Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 195 200 205 Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn 210 215 220 His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys 225 230 235 240 Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val 245 250 255 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn 260 265 270 Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala 275 280 285 Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser 290 295 300 Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn 305 310 315 320 Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn 325 330 335 Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 340 345 350 Met Ile Asn Arg Gln Glu Ile Cys Gly Pro Arg Ser Tyr Thr Ile Ala 355 360 365 Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile 370 375 380 Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385 390 395 400 Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln 405 410 415 Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu 420 425 <210> 5 <211> 429 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 5 Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu 20 25 30 Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile 50 55 60 Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met 85 90 95 Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg 100 105 110 Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly 115 120 125 Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140 Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala 145 150 155 160 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser 165 170 175 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 180 185 190 Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 195 200 205 Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn 210 215 220 His Trp Arg Asn Phe Ala Asp Ile Cys Asp Ser Trp Lys Ser Ile Lys 225 230 235 240 Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val 245 250 255 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn 260 265 270 Phe Gly Leu Ser Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala 275 280 285 Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser 290 295 300 Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn 305 310 315 320 Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn 325 330 335 Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 340 345 350 Met Ile Asn Arg Gln Glu Cys Gly Gly Pro Arg Ser Tyr Thr Ile Ala 355 360 365 Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile 370 375 380 Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385 390 395 400 Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln 405 410 415 Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu 420 425 <210> 6 <211> 429 <212> PRT <213> artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 6 Met Gln Leu Arg Asn Pro Glu Leu His Leu Gly Cys Ala Leu Ala Leu 1 5 10 15 Arg Phe Leu Ala Leu Val Ser Trp Asp Ile Pro Gly Ala Arg Ala Leu 20 25 30 Asp Asn Gly Leu Ala Arg Thr Pro Thr Met Gly Trp Leu His Trp Glu 35 40 45 Arg Phe Met Cys Asn Leu Asp Cys Gln Glu Glu Pro Asp Ser Cys Ile 50 55 60 Ser Glu Lys Leu Phe Met Glu Met Ala Glu Leu Met Val Ser Glu Gly 65 70 75 80 Trp Lys Asp Ala Gly Tyr Glu Tyr Leu Cys Ile Asp Asp Cys Trp Met 85 90 95 Ala Pro Gln Arg Asp Ser Glu Gly Arg Leu Gln Ala Asp Pro Gln Arg 100 105 110 Phe Pro His Gly Ile Arg Gln Leu Ala Asn Tyr Val His Ser Lys Gly 115 120 125 Leu Lys Leu Gly Ile Tyr Ala Asp Val Gly Asn Lys Thr Cys Ala Gly 130 135 140 Phe Pro Gly Ser Phe Gly Tyr Tyr Asp Ile Asp Ala Gln Thr Phe Ala 145 150 155 160 Asp Trp Gly Val Asp Leu Leu Lys Phe Asp Gly Cys Tyr Cys Asp Ser 165 170 175 Leu Glu Asn Leu Ala Asp Gly Tyr Lys His Met Ser Leu Ala Leu Asn 180 185 190 Arg Thr Gly Arg Ser Ile Val Tyr Ser Cys Glu Trp Pro Leu Tyr Met 195 200 205 Trp Pro Phe Gln Lys Pro Asn Tyr Thr Glu Ile Arg Gln Tyr Cys Asn 210 215 220 His Trp Arg Asn Phe Ala Asp Ile Asp Asp Ser Trp Lys Ser Ile Lys 225 230 235 240 Ser Ile Leu Asp Trp Thr Ser Phe Asn Gln Glu Arg Ile Val Asp Val 245 250 255 Ala Gly Pro Gly Gly Trp Asn Asp Pro Asp Met Leu Val Ile Gly Asn 260 265 270 Phe Gly Leu Cys Trp Asn Gln Gln Val Thr Gln Met Ala Leu Trp Ala 275 280 285 Ile Met Ala Ala Pro Leu Phe Met Ser Asn Asp Leu Arg His Ile Ser 290 295 300 Pro Gln Ala Lys Ala Leu Leu Gln Asp Lys Asp Val Ile Ala Ile Asn 305 310 315 320 Gln Asp Pro Leu Gly Lys Gln Gly Tyr Gln Leu Arg Gln Gly Asp Asn 325 330 335 Phe Glu Val Trp Glu Arg Pro Leu Ser Gly Leu Ala Trp Ala Val Ala 340 345 350 Met Ile Asn Arg Gln Glu Ile Gly Gly Pro Arg Ser Tyr Thr Ile Ala 355 360 365 Val Ala Ser Leu Gly Lys Gly Val Ala Cys Asn Pro Ala Cys Phe Ile 370 375 380 Thr Gln Leu Leu Pro Val Lys Arg Lys Leu Gly Phe Tyr Glu Trp Thr 385 390 395 400 Ser Arg Leu Arg Ser His Ile Asn Pro Thr Gly Thr Val Leu Leu Gln 405 410 415 Leu Glu Asn Thr Met Gln Met Ser Leu Lys Asp Leu Leu 420 425 <210> 7 <211> 1290 <212> DNA <213> Homo sapiens <400> 7 atgcagctga ggaacccaga actacatctg ggctgcgcgc ttgcgcttcg cttcctggcc 60 ctcgtttcct gggacatccc tggggctaga gcactggaca atggattggc aaggacgcct 120 accatgggct ggctgcactg ggagcgcttc atgtgcaacc ttgactgcca ggaagagcca gattcctgca tcagtgagaa gctcttcatg gagatggcag agctcatggt ctcagaaggc tggaaggatg caggttatga gtacctctgc attgatgact gttggatggc tccccaaaga gattcagaag gcagacttca ggcagaccct cagcgctttc ctcatgggat tcgccagcta 360 gctaattatg ttcacagcaa aggactgaag ctagggattt atgcagatgt tggaataaa acctgcgcag gcttccctgg gagttttgga tactacgaca ttgatgccca gacctttgct 480 gactggggag tagatctgct aaaatttgat ggttgttact gtgacagttt ggaaaatttg gcagatggtt ataagcacat gtccttggcc ctgaatagga ctggcagaag cattgtgtac tcctgtgagt ggcctcttta tatgtggccc tttcaaaagc ccaattatac agaaatccga cagtactgca atcactggcg aaattttgct gacattgatg attcctggaa aagtataag agtatcttgg actggacatc ttttaaccag gagagaattg ttgatgttgc tggaccaggg ggttggaatg acccagatat gttagtgatt ggcaactttg gcctcagctg gaatcagcaa 840 gtaactcaga tggccctctg ggctatcatg gctgctcctt tattcatgtc taatgacctc 900 cgacacatca gccctcaagc caaagctctc cttcaggata aggacgtaat tgccatcaat 960 caggacccct tgggcaagca agggtaccag cttagacagg gagacaactt tgaagtgtgg 1020 gaacgacctc tctcaggctt agcctgggct gtagctatga taaaccggca ggagaattggt 1080 ggacctcgct cttataccat cgcagttgct tccctgggta aaggagtggc ctgtaatcct 1140 gcctgcttca tcacacagct cctccctgtg aaaaggaagc tagggttcta tgaatggact 1200 tcaaggttaa gaagtcacat aaatcccaca ggcactgttt tgcttcagct agaaaataca 1260 atgcagatgt cattaaaaga cttactttaa 1290 <210> 8 <211> 1290 <212> DNA <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic polynucleotide" <400> 8 atgcagctga ggaacccaga actacatctg ggctgcgcgc ttgcgcttcg cttcctggcc 60 ctcgtttcct gggacatccc tggggctaga gcactggaca atggattggc aaggacgcct 120 accatgggct ggctgcactg ggagtgcttc atgtgcaacc ttgactgcca ggaagagcca gattcctgca tcagtgagaa gctcttcatg gagatggcag agctcatggt ctcagaaggc tggaaggatg caggttatga gtacctctgc attgatgact gttggatggc tccccaaaga gattcagaag gcagacttca ggcagaccct cagcgctttc ctcatgggat tcgccagcta 360 gctaattatg ttcacagcaa aggactgaag ctagggattt atgcagatgt tggaataaa acctgcgcag gcttccctgg gagttttgga tactacgaca ttgatgccca gacctttgct 480 gactggggag tagatctgct aaaatttgat ggttgttact gtgacagttt ggaaaatttg gcagatggtt ataagcacat gtccttggcc ctgaatagga ctggcagaag cattgtgtac tcctgtgagt ggcctcttta tatgtggccc tttcaaaagc ccaattatac agaaatccga cagtactgca atcactggcg aaattttgct gacattgatg attcctggaa aagtataag agtatcttgg actggacatc ttttaaccag gagagaattg ttgatgttgc tggaccaggg ggttggaatg acccagatat gttagtgatt ggcaactttg gcctcagctg gaatcagcaa 840 gtaactcaga tggccctctg ggctatcatg gctgctcctt tattcatgtc taatgacctc 900 cgacacatca gccctcaagc caaagctctc cttcaggata aggacgtaat tgccatcaat 960 caggacccct tgggcaagca agggtaccag cttagacagg gagacaactt tgaagtgtgg 1020 gaacgacctc tctcaggctt agcctgggct gtagctatga taaaccggca ggagaattggt 1080 tgccctcgct cttataccat cgcagttgct tccctgggta aaggagtggc ctgtaatcct 1140 gcctgcttca tcacacagct cctccctgtg aaaaggaagc tagggttcta tgaatggact 1200 tcaaggttaa gaagtcacat aaatcccaca ggcactgttt tgcttcagct agaaaataca 1260 atgcagatgt cattaaaaga cttactttaa 1290 <210> 9 <211> 1290 <212> DNA <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic polynucleotide" <400> 9 atgcagctga ggaacccaga actacatctg ggctgcgcgc ttgcgcttcg cttcctggcc 60 ctcgtttcct gggacatccc tggggctaga gcactggaca atggattggc aaggacgcct 120 accatgggct ggctgcactg ggagtgcttc atgtgcaacc ttgactgcca ggaagagcca gattcctgca tcagtgagaa gctcttcatg gagatggcag agctcatggt ctcagaaggc tggaaggatg caggttatga gtacctctgc attgatgact gttggatggc tccccaaaga gattcagaag gcagacttca ggcagaccct cagcgctttc ctcatgggat tcgccagcta 360 gctaattatg ttcacagcaa aggactgaag ctagggattt atgcagatgt tggaataaa acctgcgcag gcttccctgg gagttttgga tactacgaca ttgatgccca gacctttgct 480 gactggggag tagatctgct aaaatttgat ggttgttact gtgacagttt ggaaaatttg gcagatggtt ataagcacat gtccttggcc ctgaatagga ctggcagaag cattgtgtac tcctgtgagt ggcctcttta tatgtggccc tttcaaaagc ccaattatac agaaatccga cagtactgca atcactggcg aaattttgct gacattgatg attcctggaa aagtataag agtatcttgg actggacatc ttttaaccag gagagaattg ttgatgttgc tggaccaggg ggttggaatg acccagatat gttagtgatt ggcaactttg gcctcagctg gaatcagcaa 840 gtaactcaga tggccctctg ggctatcatg gctgctcctt tattcatgtc taatgacctc 900 cgacacatca gccctcaagc caaagctctc cttcaggata aggacgtaat tgccatcaat 960 caggacccct tgggcaagca agggtaccag cttagacagg gagacaactt tgaagtgtgg 1020 gaacgacctc tctcaggctt agcctgggct gtagctatga taaaccggca ggagaatttgt 1080 ggacctcgct cttataccat cgcagttgct tccctgggta aaggagtggc ctgtaatcct 1140 gcctgcttca tcacacagct cctccctgtg aaaaggaagc tagggttcta tgaatggact 1200 tcaaggttaa gaagtcacat aaatcccaca ggcactgttt tgcttcagct agaaaataca 1260 atgcagatgt cattaaaaga cttactttaa 1290 <210> 10 <211> 1290 <212> DNA <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic polynucleotide" <400> 10 atgcagctga ggaacccaga actacatctg ggctgcgcgc ttgcgcttcg cttcctggcc 60 ctcgtttcct gggacatccc tggggctaga gcactggaca atggattggc aaggacgcct 120 accatgggct ggctgcactg ggagcgcttc tgctgcaacc ttgactgcca ggagagcca gattcctgca tcagtgagaa gctcttcatg gagatggcag agctcatggt ctcagaaggc tggaaggatg caggttatga gtacctctgc attgatgact gttggatggc tccccaaaga gattcagaag gcagacttca ggcagaccct cagcgctttc ctcatgggat tcgccagcta 360 gctaattatg ttcacagcaa aggactgaag ctagggattt atgcagatgt tggaataaa acctgcgcag gcttccctgg gagttttgga tactacgaca ttgatgccca gacctttgct 480 gactggggag tagatctgct aaaatttgat ggttgttact gtgacagttt ggaaaatttg gcagatggtt ataagcacat gtccttggcc ctgaatagga ctggcagaag cattgtgtac tcctgtgagt ggcctcttta tatgtggccc tttcaaaagc ccaattatac agaaatccga cagtactgca atcactggcg aaattttgct gacattgatg attcctggaa aagtataag agtatcttgg actggacatc ttttaaccag gagagaattg ttgatgttgc tggaccaggg ggttggaatg acccagatat gttagtgatt ggcaactttg gcctcagctg gaatcagcaa 840 gtaactcaga tggccctctg ggctatcatg gctgctcctt tattcatgtc taatgacctc 900 cgacacatca gccctcaagc caaagctctc cttcaggata aggacgtaat tgccatcaat 960 caggacccct tgggcaagca agggtaccag cttagacagg gagacaactt tgaagtgtgg 1020 gaacgacctc tctcaggctt agcctgggct gtagctatga taaaccggca ggagaatttgt 1080 ggacctcgct cttataccat cgcagttgct tccctgggta aaggagtggc ctgtaatcct 1140 gcctgcttca tcacacagct cctccctgtg aaaaggaagc tagggttcta tgaatggact 1200 tcaaggttaa gaagtcacat aaatcccaca ggcactgttt tgcttcagct agaaaataca 1260 atgcagatgt cattaaaaga cttactttaa 1290 <210> 11 <211> 1290 <212> DNA <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic polynucleotide" <400> 11 atgcagctga ggaacccaga actacatctg ggctgcgcgc ttgcgcttcg cttcctggcc 60 ctcgtttcct gggacatccc tggggctaga gcactggaca atggattggc aaggacgcct 120 accatgggct ggctgcactg ggagcgcttc atgtgcaacc ttgactgcca ggaagagcca gattcctgca tcagtgagaa gctcttcatg gagatggcag agctcatggt ctcagaaggc tggaaggatg caggttatga gtacctctgc attgatgact gttggatggc tccccaaaga gattcagaag gcagacttca ggcagaccct cagcgctttc ctcatgggat tcgccagcta 360 gctaattatg ttcacagcaa aggactgaag ctagggattt atgcagatgt tggaataaa acctgcgcag gcttccctgg gagttttgga tactacgaca ttgatgccca gacctttgct 480 gactggggag tagatctgct aaaatttgat ggttgttact gtgacagttt ggaaaatttg gcagatggtt ataagcacat gtccttggcc ctgaatagga ctggcagaag cattgtgtac tcctgtgagt ggcctcttta tatgtggccc tttcaaaagc ccaattatac agaaatccga cagtactgca atcactggcg aaattttgct gacatttgcg attcctggaa aagtataag agtatcttgg actggacatc ttttaaccag gagagaattg ttgatgttgc tggaccaggg ggttggaatg acccagatat gttagtgatt ggcaactttg gcctcagctg gaatcagcaa 840 gtaactcaga tggccctctg ggctatcatg gctgctcctt tattcatgtc taatgacctc 900 cgacacatca gccctcaagc caaagctctc cttcaggata aggacgtaat tgccatcaat 960 caggacccct tgggcaagca agggtaccag cttagacagg gagacaactt tgaagtgtgg 1020 gaacgacctc tctcaggctt agcctgggct gtagctatga taaaccggca ggagtgcggt 1080 ggacctcgct cttataccat cgcagttgct tccctgggta aaggagtggc ctgtaatcct 1140 gcctgcttca tcacacagct cctccctgtg aaaaggaagc tagggttcta tgaatggact 1200 tcaaggttaa gaagtcacat aaatcccaca ggcactgttt tgcttcagct agaaaataca 1260 atgcagatgt cattaaaaga cttactttaa 1290 <210> 12 <211> 1290 <212> DNA <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic polynucleotide" <400> 12 atgcagctga ggaacccaga actacatctg ggctgcgcgc ttgcgcttcg cttcctggcc 60 ctcgtttcct gggacatccc tggggctaga gcactggaca atggattggc aaggacgcct 120 accatgggct ggctgcactg ggagcgcttc atgtgcaacc ttgactgcca ggaagagcca gattcctgca tcagtgagaa gctcttcatg gagatggcag agctcatggt ctcagaaggc tggaaggatg caggttatga gtacctctgc attgatgact gttggatggc tccccaaaga gattcagaag gcagacttca ggcagaccct cagcgctttc ctcatgggat tcgccagcta 360 gctaattatg ttcacagcaa aggactgaag ctagggattt atgcagatgt tggaataaa acctgcgcag gcttccctgg gagttttgga tactacgaca ttgatgccca gacctttgct 480 gactggggag tagatctgct aaaatttgat ggttgttact gtgacagttt ggaaaatttg gcagatggtt ataagcacat gtccttggcc ctgaatagga ctggcagaag cattgtgtac tcctgtgagt ggcctcttta tatgtggccc tttcaaaagc ccaattatac agaaatccga cagtactgca atcactggcg aaattttgct gacattgatg attcctggaa aagtataag agtatcttgg actggacatc ttttaaccag gagagaattg ttgatgttgc tggaccaggg ggttggaatg acccagatat gttagtgatt ggcaactttg gcctctgctg gaatcagcaa 840 gtaactcaga tggccctctg ggctatcatg gctgctcctt tattcatgtc taatgacctc 900 cgacacatca gccctcaagc caaagctctc cttcaggata aggacgtaat tgccatcaat 960 caggacccct tgggcaagca agggtaccag cttagacagg gagacaactt tgaagtgtgg 1020 gaacgacctc tctcaggctt agcctgggct gtagctatga taaaccggca ggagattggt 1080 ggacctcgct cttataccat cgcagttgct tccctgggta aaggagtggc ctgtaatcct 1140 gcctgcttca tcacacagct cctccctgtg aaaaggaagc tagggttcta tgaatggact 1200 tcaaggttaa gaagtcacat aaatcccaca ggcactgttt tgcttcagct agaaaataca 1260 atgcagatgt cattaaaaga cttactttaa 1290 <210> 13 <211> 306 <212> PRT <213> Homo sapiens <400> 13 Met Ala Ser Pro Gly Cys Leu Trp Leu Leu Ala Val Ala Leu Leu Pro 1 5 10 15 Trp Thr Cys Ala Ser Arg Ala Leu Gln His Leu Asp Pro Pro Ala Pro 20 25 30 Leu Pro Leu Val Ile Trp His Gly Met Gly Asp Ser Cys Cys Asn Pro 35 40 45 Leu Ser Met Gly Ala Ile Lys Lys Met Val Glu Lys Lys Ile Pro Gly 50 55 60 Ile Tyr Val Leu Ser Leu Glu Ile Gly Lys Thr Leu Met Glu Asp Val 65 70 75 80 Glu Asn Ser Phe Phe Leu Asn Val Asn Ser Gln Val Thr Thr Val Cys 85 90 95 Gln Ala Leu Ala Lys Asp Pro Lys Leu Gln Gln Gly Tyr Asn Ala Met 100 105 110 Gly Phe Ser Gln Gly Gly Gln Phe Leu Arg Ala Val Ala Gln Arg Cys 115 120 125 Pro Ser Pro Pro Met Ile Asn Leu Ile Ser Val Gly Gly Gln His Gln 130 135 140 Gly Val Phe Gly Leu Pro Arg Cys Pro Gly Glu Ser Ser His Ile Cys 145 150 155 160 Asp Phe Ile Arg Lys Thr Leu Asn Ala Gly Ala Tyr Ser Lys Val Val 165 170 175 Gln Glu Arg Leu Val Gln Ala Glu Tyr Trp His Asp Pro Ile Lys Glu 180 185 190 Asp Val Tyr Arg Asn His Ser Ile Phe Leu Ala Asp Ile Asn Gln Glu 195 200 205 Arg Gly Ile Asn Glu Ser Tyr Lys Lys Asn Leu Met Ala Leu Lys Lys 210 215 220 Phe Val Met Val Lys Phe Leu Asn Asp Ser Ile Val Asp Pro Val Asp 225 230 235 240 Ser Glu Trp Phe Gly Phe Tyr Arg Ser Gly Gln Ala Lys Glu Thr Ile 245 250 255 Pro Leu Gln Glu Thr Ser Leu Tyr Thr Gln Asp Arg Leu Gly Leu Lys 260 265 270 Glu Met Asp Asn Ala Gly Gln Leu Val Phe Leu Ala Thr Glu Gly Asp 275 280 285 His Leu Gln Leu Ser Glu Glu Trp Phe Tyr Ala His Ile Ile Pro Phe 290 295 300 Leu Gly 305 <210> 14 <211> 306 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 14 Met Ala Ser Pro Gly Cys Leu Trp Leu Leu Ala Val Ala Leu Leu Pro 1 5 10 15 Trp Thr Cys Ala Ser Arg Ala Leu Gln His Leu Asp Pro Pro Ala Pro 20 25 30 Leu Pro Leu Val Ile Trp His Gly Met Gly Asp Ser Cys Cys Asn Pro 35 40 45 Leu Ser Met Gly Ala Ile Lys Lys Met Val Glu Lys Lys Ile Pro Gly 50 55 60 Ile Tyr Val Leu Ser Leu Glu Ile Gly Lys Thr Leu Met Glu Asp Val 65 70 75 80 Glu Asn Ser Phe Phe Leu Asn Val Asn Ser Gln Val Thr Thr Val Cys 85 90 95 Gln Ala Leu Ala Lys Asp Pro Lys Leu Gln Gln Gly Tyr Asn Ala Met 100 105 110 Gly Phe Ser Gln Gly Gly Gln Phe Leu Arg Ala Val Ala Gln Arg Cys 115 120 125 Pro Ser Pro Pro Met Ile Asn Leu Ile Ser Val Gly Gly Gln His Gln 130 135 140 Gly Val Phe Gly Leu Pro Arg Cys Pro Gly Glu Ser Ser His Ile Cys 145 150 155 160 Asp Phe Ile Arg Lys Thr Leu Asn Ala Gly Cys Tyr Ser Lys Val Val 165 170 175 Gln Glu Arg Leu Val Gln Cys Glu Tyr Trp His Asp Pro Ile Lys Glu 180 185 190 Asp Val Tyr Arg Asn His Ser Ile Phe Leu Ala Asp Ile Asn Gln Glu 195 200 205 Arg Gly Ile Asn Glu Ser Tyr Lys Lys Asn Leu Met Ala Leu Lys Lys 210 215 220 Phe Val Met Val Lys Phe Leu Asn Asp Ser Ile Val Asp Pro Val Asp 225 230 235 240 Ser Glu Trp Phe Gly Phe Tyr Arg Ser Gly Gln Ala Lys Glu Thr Ile 245 250 255 Pro Leu Gln Glu Thr Ser Leu Tyr Thr Gln Asp Arg Leu Gly Leu Lys 260 265 270 Glu Met Asp Asn Ala Gly Gln Leu Val Phe Leu Ala Thr Glu Gly Asp 275 280 285 His Leu Gln Leu Ser Glu Glu Trp Phe Tyr Ala His Ile Ile Pro Phe 290 295 300 Leu Gly 305 <210> 15 <211> 2504 <212> DNA <213> Homo sapiens <400> 15 ttattttgat tcaccgcaga gggcggtcta cgagagcgca gagccccact cggccagcgg ggtctggcgg gggacctgtc gcgctgaaag ctccagggta gggccgacgc ccatcaggct gggcatccgt tcgggatgcg caggttgcga tctgcaaccg gcggcgccac gcccaggcgg 180 gcggagcgcg gttcccggag tctcgcgccc gcggtcatgt gacacagcga agatggcgtc 240 gcccggctgc ctgtggctct tggctgtggc tctcctgcca tggacctgcg cttctcgggc 300 gctgcagcat ctggacccgc cggcgccgct gccgttggtg atctggcatg ggatggggaga 360 cagctgttgc aatcccttaa gcatgggtgc tattaaaaaa atggtggaga agaaaatacc 420. tggaattc gtcttatctt tagagattgg gaagaccctg atggaggacg tggagaacag 480 cttcttcttg aatgtcaatt cccaagtaac aacagtgtgt caggcacttg ctaaggatcc taattgcag caaggctaca atgctatggg attctcccag ggaggccat ttctgagggc agtggctcag agatgccctt cacctcccat gatcaatctg atctcggttg ggggacaaca tcaaggtgtt tttggactcc ctcgatgccc aggagagagc tctcacatct gtgacttcat 720 ccgaaaaaca ctgaatgctg gggcgtactc caaagttgtt caggaacgcc tcgtgcaagc 780 cgaatactgg catgacccca taaaggagga tgtgtatcgc aaccacagca tcttcttggc 840 agatataaat caggagcggg gtatcaatga gtcctacaag aaaaacctga tggccctgaa 900 gaagttgtg atggtgaaat tcctcaatga ttccattgtg gaccctgtag attcggagtg 960 gttggattt tacagaagtg gccaagccaa ggaaaccatt cccttacagg agacctccct 1020 gtacacacag gaccgcctgg ggctaaagga aatggacaat gcaggacagc tagtgtttct 1080 ggctacagaa ggggaccatc ttcagttgtc tgaagaatgg tttatgccc acatcatacc 1140 attccttgga tgaaacccgt atagttcaca atagagctca gggagcccct aactcttcca 1200 aaccacatgg gagacagttt ccttcatgcc caagcctgag ctcagatcca gcttgcaact 1260 aatccttcta tcatctaaca tgccctactt ggaaagatct aagatctgaa tcttatcctt 1320 tgccatcttc tgttaccata tggtgttgaa tgcaagttta attaccatgg agattgtttt 1380 acaaactttt gatgtggtca agttcagttt tagaaaaggg agtctgttcc agatcagtgc 1440 cagaactgtg cccaggccca aaggacaa ctaactaag tagtgagata gattctagg 1500 gcaacattt ttccaagtct tgccatattt caagcaaga ggtgcccagg cctgaggtac 1560 tcacataaat gctttgtttt gctggtgat taaccagtgc ttggaaaat cttgcttggc 1620 tatttctgca tcattctta aggctgcctt cctctcag tacgttgcc tctgtgctat 1680 catcttatca tcattatta gataccc actggcctac agtcttgctt ctgcagcacc 1740 cactttgtct cctcaggtag tgatgatta gttgctgtca caaaggagg gaagtagcac 1800 Caattagg Tgcttaag Gaggaatgt Acatcttgta Taacttagg Agcgaagaaa 1860 atgtaggcgc gaagtgaaa agtgaggcag ctagttcttc ctattccatt ctcgaccac 1920 ctgccctttc ttaatatgac tagtgtctt gatgctagag tcacttact ctgttgctgg 1980 ctttagcaga gataggagg aaccatatga aaagatcag gctttctgac ttccatcccc 2040 aaaacacatt taccagcata ctccaactg ttctgatgt gttccatgag aaaggattg 2100 ttgctcaa aagcttggaa atactacac actccctttc tccttctgga gatcaaccca 2160 cattagagtg tctaaggact cctgagaatt cctgttacag taaacaaaac taacgtaatc 2220 taccatttcc tacactattt gagcatggaa atcatagtcc ccactctgtg aaaacttaac 2280 gctttttgga agacatttct gtagcatgtc agtttggaga aatgatgagc tacgccttga 2340 tgaaagaacc gtgttggtgc tgctaagttt agccattatg gtttttcctt tctctctctt 2400 aagccttatt cttcaactaa aagatgagga ttaagagcaa gaagttgggg gggatgtgaa 2460 aataatttta tgaggttgtc taaaataaag agtagtttct tatc 2504 <210> 16 <211> 921 <212> DNA <213> Artificial Sequence <220> <221> Source <223> / Note="Description of artificial sequence: Synthetic polynucleotide" <400> 16 atggcatcac cgggttgcct ctggttgttg gccgttgcgt tgcttccgtg gacatgtgca 60 tcaagagctc ttcaacatct ggatccccca gctcccctgc cgctcgtaat ctggcacggg 120 atgggggatt catgttgtaa cccgttgtca atgggcgcga taaaaaagat ggttgaaaag 180 aagattccag gcatctacgt tctgtccctg gaaatcggta agacactgat ggaagacgtg 240 gagaactcct tctttctcaa cgtcaatagt caggtcacta ccgtctgtca agcattggca 300 aaggacccta aacttcagca ggggtacaat gcgatggggt ttagccaggg cggacagttt 360 cttagagccg tcgcacagcg ctgtccatct cccccgatga ttaaccttat atctgtcggg 420 ggacaacacc agggtgtttt tggtcttcct cgctgtcctg gtgaaagctc ccacatctgt 480 gatttcatac gcaaaacgtt gaacgcagga tgctatagta aagtcgtcca agaacggctt 540 gttcaatgcg agtattggca tgacccaata aaagaagacg tttataggaa tcactctatc 600 ttcttggccg atatcaacca agaacgcgga atcaacgaaa gctacaaaaa gaatcttatg 660 gctctcaaga aatttgttat ggtgaaattc cttaatgact ctatagtaga tcctgtcgat 720 tcagaatggt tcgggttcta caggtctggc caggcgaagg agactattcc cctccaagaa 780 acgtctctct atacacaaga cagactcgga ctgaaagaga tggataatgc gggccagttg 840 gtcttcttgg ctacggaagg cgatcatctc caactctccg aagagtggtt ctatgcccat 900 ataatccccgt tcctgggcta a 921 <210> 17 <211> 67 <212> PRT <213> Homo sapiens <400> 17 Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe 35 40 45 Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> 18 <211> 67 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 18 Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Ser Tyr Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe 35 40 45 Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> 19 <211> 67 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 19 Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Leu Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe 35 40 45 Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> 20 <211> 67 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 20 Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Leu Glu Glu Cys Cys Phe 35 40 45 Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> twenty one <211> 67 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> twenty one Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Thr 35 40 45 Arg Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> twenty two <211> 67 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> twenty two Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe 35 40 45 Ser Ser Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> twenty three <211> 67 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> twenty three Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe 35 40 45 Arg Ile Cys Asp Leu Ala Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> twenty four <211> 67 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> twenty four Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe 35 40 45 Arg Ser Cys Asp Leu Arg Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> 25 <211> 67 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 25 Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Phe Tyr Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Val Glu Glu Cys Cys Phe 35 40 45 Arg Ser Cys Asp Leu Ala Arg Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> 26 <211> 67 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 26 Ala Tyr Arg Pro Ser Glu Thr Leu Cys Gly Gly Glu Leu Val Asp Thr 1 5 10 15 Leu Gln Phe Val Cys Gly Asp Arg Gly Ser Leu Phe Ser Arg Pro Ala 20 25 30 Ser Arg Val Ser Arg Arg Ser Arg Gly Ile Leu Glu Glu Cys Cys Thr 35 40 45 Ser Ile Cys Asp Leu Arg Arg Leu Glu Thr Tyr Cys Ala Thr Pro Ala 50 55 60 Lys Ser Glu 65 <210> 27 <211> 61 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 27 Thr Leu Cys Gly Gly Glu Leu Val Asp Thr Leu Gln Phe Val Cys Gly 1 5 10 15 Asp Arg Gly Phe Leu Phe Ser Arg Pro Ala Ser Arg Val Ser Arg Arg 20 25 30 Ser Arg Gly Ile Val Glu Glu Cys Cys Phe Arg Ser Cys Asp Leu Ala 35 40 45 Leu Leu Glu Thr Tyr Cys Ala Thr Pro Ala Arg Ser Glu 50 55 60 <210> 28 <211> 274 <212> RNA <213> Cricket paralysis virus <400> 28 cggugucgaa guagaauuuc uaucucgaca cgcggccuuc caagcaguua gggaaaccga 60 cuucuuugaa gaagaaagcu gacuauguga ucuuauuaaa auuagguuaa auuucgaggu 120 uaaaaauagu uuuaauauug cuauagucuu agaggucuug uauauuuaua cuuaccacac 180 aagauggacc ggagcagccc uccaauaucu aguguacccu cgugcucgcu caaacauuaa 240 gugguguugu gcgaaaagaa ucucacuuca agaa 274 <210> 29 <211> 18 <212> PRT <213> Homo sapiens <400> 29 Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ser Ala Ala 1 5 10 15 Arg Ala <210> 30 <211> 28 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 30 Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ser Leu Val 1 5 10 15 Ala Ala Met Leu Leu Leu Leu Ser Ala Ala Arg Ala 20 25 <210> 31 <211> 25 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 31 Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Trp Val Ala 1 5 10 15 Leu Leu Leu Leu Ser Ala Ala Arg Ala 20 25 <210> 32 <211> 26 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 32 Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ser Leu Val 1 5 10 15 Ala Leu Leu Leu Leu Ser Ala Ala Arg Ala 20 25 <210> 33 <211> 26 <212> PRT <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic peptide" <400> 33 Met Lys Leu Ser Leu Val Ala Ala Met Leu Leu Leu Leu Ala Leu Val 1 5 10 15 Ala Leu Leu Leu Leu Ser Ala Ala Arg Ala 20 25 <210> 34 <211> 10 <212> RNA <213> Artificial sequence <220> <221> source <223> / Comment="Description of artificial sequence: synthetic oligonucleotide" <400> 34 gccrccaugg 10

Claims

1. A gene therapy vector comprising a nucleic acid construct, the nucleic acid construct comprising: a nucleic acid encoding a stable form of a protein for treating Fabry disease, the stable form comprising one or more non-native cysteine ​​residues, the one or more non-native cysteine ​​residues forming a disulfide bridge between non-native cysteines within the protein or between non-native cysteines of two monomers of the protein; wherein the protein is a stable α-galactosidase (α-GAL) protein; wherein the sequence of the stable α-galactosidase A (α-GAL) protein is SEQ ID NO: 4 or SEQ ID NO:

5.

2. The gene therapy vector of claim 1, wherein the gene therapy vector is a viral vector selected from the group consisting of: Adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors and herpes virus vectors.

3. The gene therapy vector of claim 1, wherein the nucleic acid construct is contained in a viral vector genome.

4. The gene therapy vector of claim 3, wherein the viral vector genome comprises a recombinant AAV (rAAV) genome.

5. The gene therapy vector of claim 4, wherein the rAAV genome comprises a self-complementary genome.

6. The gene therapy vector of claim 4, wherein the rAAV genome comprises a single-stranded genome.

7. The gene therapy vector of claim 4, wherein the rAAV genome comprises a first inverted terminal repeat and a second inverted terminal repeat.

8. The gene therapy vector of claim 7, wherein the AAV inverted terminal repeats are AAV2 inverted terminal repeats.

9. The gene therapy vector of claim 4, wherein the rAAV genome further comprises an SV40 intron.

10. The gene therapy vector of claim 4, wherein the rAAV genome further comprises a polyadenylation sequence.

11. The gene therapy vector of claim 3, wherein the nucleic acid construct further comprises a promoter sequence.

12. The gene therapy vector of claim 11, wherein the promoter is a constitutive promoter.

13. The gene therapy vector of claim 11, wherein the promoter is a tissue-specific promoter.

14. The gene therapy vector of claim 3, wherein the nucleic acid construct further comprises one or more nucleic acid sequences selected from the group consisting of: Kozak sequence, CrPV IRES, a nucleic acid sequence encoding a linker, a nucleic acid sequence encoding a signal sequence, and a nucleic acid sequence encoding an IGF2 peptide.

15. The gene therapy vector of claim 1, wherein the nucleic acid construct is packaged in a viral capsid.

Images