Human alpha-galactosidase variants

Abstract

To provide human alpha-galactosidase variants.SOLUTION: Engineered human alpha-galactosidase polypeptides and compositions thereof are provided. The engineered human alpha-galactosidase polypeptides have been optimized to provide improved thermostability, serum stability, improved cellular uptake, stability under both acidic (pH<4) and basic (pH>7) conditions, reduced immunogenicity, and improved globotriaosylceramide removal from cells. The use of the compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes are described.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 982,949, filed February 28, 2020, which is incorporated by reference in its entirety for all purposes. FIELD OF THE INVENTION

[0002] The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides are optimized to provide improved thermostability, serum stability, reduced immunogenicity, improved cellular uptake, and stability under both acidic (pH<4) and basic (pH>7) conditions, as well as improved clearance of globotriaosylceramide from cells. The present invention also relates to the use of compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes. Reference to a sequence listing, table or computer program

[0003] An official copy of the Sequence Listing is submitted contemporaneously herewith via EFS-Web as an ASCII text file with the filename "CX7-203WO2_ST25.txt," created on February 22, 2021, and measuring 4.43 megabytes in size. The Sequence Listing submitted via EFS-Web is a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Background of the Invention Human alpha-galactosidase ("GLA"; EC 3.2.1.22) is a lysosomal glycoprotein involved in the hydrolysis of terminal alpha-galactosyl moieties from glycolipids and glycoproteins. It acts on many substrates present in various human tissues. Fabry disease (also known as angiokeratoma corporis diffuse, Anderson-Fabry disease, hereditary ectopic lipidosis, alpha-galactosidase A deficiency, GLA deficiency, and ceramide trihexosidase deficiency) is an X-linked inborn error of glycosphingolipid catabolism resulting from deficient or absent activity of alpha-galactosidase A. Patients with Fabry disease accumulate globotriaosylceramide (herein referred to as "Gb3" and "Gb3") and related glycosphingolipids in the plasma and cellular lysosomes of blood vessels, tissues, and organs (see, e.g., Nance et al., Arch. Neurol., 63:453-457

[2006] ). As patients age, the accumulation of these lipids gradually narrows blood vessels, resulting in reduced blood flow and nutrients, particularly to tissues of the skin, kidneys, heart, brain, and nervous system. Thus, Fabry disease is a systemic disorder that manifests as renal failure, cardiac disease, cerebrovascular disease, small-fiber peripheral neuropathy, and skin lesions, as well as other disorders (see, e.g., Schiffmann, Pharm. Ther., 122:65-77

[2009] ). Affected patients exhibit symptoms such as pain in the hands and feet, clusters of small, dark red spots on the skin, decreased ability to sweat, corneal opacity, gastrointestinal problems, tinnitus, and hearing loss. Potentially life-threatening complications include progressive kidney damage, heart attack, and stroke. The disease is estimated to affect 1 in 40,000 to 60,000 men, but it also occurs in women. In fact, heterozygous women with Fabry disease experience serious, life-threatening conditions, including nervous system abnormalities, chronic pain, fatigue, high blood pressure, heart disease, kidney failure, and stroke, and therefore require medical treatment (see, e.g., Want et al., Genet. Med., 13:457-484

[2011] ). Signs of Fabry disease can begin anytime during infancy; symptoms usually begin to appear between the ages of 4 and 8, although some patients exhibit milder, later-onset disease.Treatment is generally supportive and there is no cure for Fabry disease, so there remains a need for safe and effective treatments. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nance et al., Arch. Neurol., 63:453-457

[2006] [Non-patent document 2] Schiffmann, Pharm. Ther., 122:65-77

[2009] [Non-patent document 3] Want et al., Genet. Med., 13:457-484

[2011] Summary of the Invention [Means for solving the problem]

[0006] Summary of the Invention The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides are optimized to provide improved thermostability, serum stability, reduced immunogenicity, improved cellular uptake, and stability under both acidic (pH<4) and basic (pH>7) conditions, as well as improved clearance of globotriaosylceramide from cells. The present invention also relates to the use of compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes.

[0007] The present invention provides recombinant alpha-galactosidase A and / or biologically active recombinant alpha-galactosidase A fragments comprising an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 8. The present invention provides recombinant alpha-galactosidase A and / or biologically active recombinant alpha-galactosidase A fragments comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:8.

[0008] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 44, 44 / 217, 44 / 217 / 316, 44 / 217 / 322, 44 / 217 / 322 / 337, 44 / 247, 44 / 247 / 302, 44 / 247 / 302 302 / 322 / 362 / 373, 302 / 337, 316, 316 / 337, 322, 322 / 337, 362 / 373, and 373, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 44L, 44L / 217F, 44L / 217F / 316L, 44L / 217F / 322M, 44L / 217F / 322M / 337A, 44L / 247N, 44L / 247N / 302Q, 44L / 247N / 302Q / 322M, 44 and 373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of R44L, R44L / R217F, R44L / R217F / D316L, R44L / R217F / I322M, R44L / R217F / I322M / P337A, R44L / D247N, R44L / D247N / K302Q, R44L / D247N / K302Q / I322M, R44L / D247N / I322M, R44L / D247N / P337A, R44L / D247N / Q362K, R44L / K302Q, R44L / P337A, R44L / K373R, R217F / I322M, R217F / K373R, D247N / I322M, D247N / Q362K, K302Q / I322M / Q362K / K373R, K3 and at least one substitution or set of substitutions at one or more positions selected from O2Q / P337A, D316L, D316L / P337A, I322M, I322M / P337A, Q362K / K373R, and K373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.

[0009] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10 / 39 / 44 / 47 / 92 / 166 / 206 / and at least one substitution or set of substitutions at one or more positions selected from 217 / 247 / 261 / 271 / 302 / 316 / 322 / 337 / 362 / 368 / 373 / 392, 44 / 217 / 316, 44 / 217 / 322 / 337, 166 / 362, 217 / 373, and 362 / 373, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10T / 39M / 44L / 47S / 92Y / 166S / 206K / 217F / 24 and at least one substitution or set of substitutions at one or more positions selected from 7N / 261A / 271H / 302Q / 316L / 322M / 337A / 362K / 368W / 373R / 392M, 44L / 217F / 316L, 44L / 217F / 322M / 337A, 166A / 362K, 217F / 373R, and 362K / 373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is and at least one substitution or set of substitutions at one or more positions selected from A / A271H / K302Q / D316L / I322M / P337A / Q362K / A368W / K373R / T392M, R44L / R217F / D316L, R44L / R217F / I322M / P337A, P166A / Q362K, R217F / K373R, and Q362K / K373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.

[0010] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 7, 7 / 48 / 68, 7 / 48 / 68 / 120 / 282 / 299, 7 / 48 / 130 / 282, 7 / 48 / 180, 7 / 68 / 130 / 282 / 365, 7 / 68 / 180, 7 / 88 / 120 / 305 / 365, 7 / 120, 7 / 130, 7 / 282, 7 / 305, 7 / 305 / 365, 7 / 365, 39, 47, 47 / 87 / 95 / 96 / 158 / 162, 47 / 95, 47 / 273, 47 / 343, 48, 48 / 68, 48 / 180 / 282, 48 / 282, 48 / 282 / 305, 67 / 180, 68, 68 / 299 / 300, 71, 87 / 9 1 / 95 / 96 / 158 / 162, 87 / 91 / 95 / 96 / 206 / 343, 87 / 96 / 155 / 273 / 343, 88, 91 / 95, 91 / 95 / 96, 92, 93, 96, 96 / 273, 96 / 312 / 343, 120, 120 / 299 / 305, 151, 158, 158 / 162 / 273, 162, 162 / 273, 162 / 343, 166, 178, 180, 181, 206, 217, 271, 273, 273 / 343, 282, 282 / 3 and at least one substitution or set of substitutions at one or more positions selected from: 65, 293 / 391, 299 / 300, 299 / 300 / 305 / 365, 300, 301, 305, 305 / 365, 314, 333, 336, 337, 343, 345, 363, 365, 370, 389, 393, 394, 396 / 398, 397, and 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:58. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 7L, 7L / 48D / 68E ...7L / 48D / 68E / 120H / 282N / 299R, 7L / 48D / 130E / 282N, 7L / 48D / 180G, 7L / 68E / 130E / 282N / 365V, 7L / 68E / 180G, 7L / 88A / 120H / 305G / 365V , 7L / 120H, 7L / 130E, 7L / 282N, 7L / 305G, 7L / 305G / 365V, 7L / 365V, 39V, 47D, 47D / 87K / 95E / 96L / 158R / 162H, 47D / 95E, 47D / 273P, 47D / 34 3G, 47V, 48D, 48D / 68E, 48D / 180G / 282N, 48D / 282N, 48D / 282N / 305G, 67T / 180G, 68E, 68E / 299R / 300I, 71P, 87K / 91Q / 95E / 96L / 158A / 162 K, 87K / 91Q / 95E / 96L / 206S / 343G, 87K / 96I / 155N / 273P / 343G, 88A, 91Q / 95E, 91Q / 95E / 96L, 92F, 92T, 93I, 96L, 96L / 273P, 96L / 312Q / 34 3G, 120H, 120H / 299R / 305G, 151L, 158A, 158A / 162K / 273G, 158R, 162H / 343D, 162K, 162K / 273P, 162S, 166K, 178G, 178S, 180G, 180L, 18 0T, 180V, 181A, 206K, 206S, 217K, 271R, 273P, 273P / 343G, 282N, 282N / 365V, 293P / 391A, 299R / 300I, 299R / 300I / 305G / 365V, 300I, 301 M, 305G, 305G / 365V, 314A, 333F, 333G, 336V, 337R, 343D, 343G, 345A, 345Q, 363Q, 365A, 365Q, 365V, 370G, 389K, 393V, 394K, 396G / 398T, 397A, 398A, 398P, 398S, and 398V, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:58. In some embodiments, the recombinant alpha-galactosidase A is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%,and R7L / Q68E / F180G, R7L / Q68E / F180G, R7L / Q88A / Y120H / N305G / F365V, R7L / Y120H, R7L / D130E, R7L / D282N, R7L / Q88A / Y120H / N305G / F365V, R7L / Q88A / Y120H, R7L / D130E, R7L / D282N ... , R7L / N305G, R7L / N305G / F365V, R7L / F365V, E39V, T47D, T47D / R87K / S95E / K96L / L158R / R162H, T47D / S95E, T47D / S273P, T47D / K343G, T47V, E48D, E48D / Q68E, E48D / F180G / D282N, E48D / D282N, E48D / D282N / N305G, P67T / F180G, Q68E, Q68E / Q299R / L300I, S71P, R87K / N91Q / S95E / K96L / L158A / R162K, R87K / N91Q / S95E / K96L / A206S / K343G, R87K / K96I / H155N / S273P / K343G, Q88A, N 91Q / S95E, N91Q / S95E / K96L, H92F, H92T, V93I, K96L, K96L / S273P, K96L / P31 2Q / K343G, Y120H, Y120H / Q299R / N305G, D151L, L158A, L158A / R162K / S273G , L158R, R162H / K343D, R162K, R162K / S273P, R162S, P166K, W178G, W178S, F1 80G, F180L, F180T, F180V, Q181A, A206K, A206S, R217K, A271R, S273P, S273 P / K343G, D282N, D282N / F365V, L293P / Q391A, Q299R / L300I, Q299R / L300I / N 305G / F365V, L300I, R301M, N305G, N305G / F365V, S314A, S333F, S333G, I33 6V, P337R, K343D, K343G, V345A, V345Q, L363Q, F365A, F365Q, F365V, S370G,and at least one substitution or set of substitutions at one or more positions selected from T389K, S393V, L394K, D396G / L398T, L397A, L398A, L398P, L398S, and L398V, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 58.

[0011] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 158, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 24 / 202, 39 / 47, 39 / 47 / 217, 39 / 151, 39 / 282 / 337 / 398, 39 / 337 / 343 / 398, 39 / 393 / 398, 47 / 130, 47 / 151, 47 / 343 / 345 / 393, 48, 48 / 68, 48 / 68 / 217 / 333 / 391 / 393, 48 / 68 / 333, 48 / 217, 48 / 333, 48 / 345 / 393, 48 / 393, 59 / 143, 68, 68 / 345, 130, 130 / 158, 130 / 158 / 393, 130 / 345 / 393, 143 / 271, 143 / 333, 143 / 387, 151, 151 / 158 / 217 / 343 / 345 / 393, 151 / 206 / 282 / 337 / 343 / 345 / 398, 151 / 282 / 393, 151 / 345 / 393 / 398, 151 / 393, 158, 158 / 393, 202, 206, 206 / 217, 217, 21 and 393 / 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 158.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 158, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 24S / 202N, 39V / 47D, 39V / 47V / 217K, 39V / 151L, 39V / 282N / 337R / 398A, 39V / 337R / 343G / 398A, 39V / 393V / 398A, 47V / 130E, 47V / 151L, 47V / 343D / 345Q / 393V, 48D, 48D / 68E, 48D / 68E / 217K / 333F / 391 A / 393V, 48D / 68E / 333F, 48D / 217K, 48D / 333F, 48D / 333G, 48D / 345Q / 393V, 48D / 393V, 59A / 143S, 68E, 68E / 345Q, 130E, 130E / 158R, 13 0E / 158R / 393V, 130E / 345Q / 393V, 143S / 271N, 143S / 333N, 143S / 387N, 151L, 151L / 158R / 217K / 343G / 345Q / 393V, 151L / 206S / 282N / 337R / 343D / 345Q / 398A, 151L / 282N / 393V, 151L / 345Q / 393V / 398A, 151L / 393V, 158R, 158R / 393V, 202N, 206S, 206S / 217K, 217K, 217K / 333F, 217K / 333G, 217K / 337R / 345Q / 398A, 271N, 282N / 393V, 333F / 345Q, 333G, 333N, 337R / 343G / 345Q / 398A, 343D, 343D / 345Q / 393V / 398A, 393V, and 393V / 398A, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 158.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of D24S / D202N, E39V / T47D, E39V / T47V / R217K, E39V / D151L, E39V / D282N / P337R / L398A, E39V / P337R / K343 G / L398A, E39V / S393V / L398A, T47V / D130E, T47V / D151L, T47V / K343D / V345 Q / S393V, E48D, E48D / Q68E, E48D / Q68E / R217K / S333F / Q391A / S393V, E48D / Q68E / S333F, E48D / R217K, E48D / S333F, E48D / S333G, E48D / V345Q / S393V, E 48D / S393V, C59A / C143S, Q68E, Q68E / V345Q, D130E, D130E / L158R, D130E / L1 58R / S393V, D130E / V345Q / S393V, C143S / A271N, C143S / S333N, C143S / E387 N, D151L, D151L / L158R / R217K / K343G / V345Q / S393V, D151L / A206S / D282N / P337R / K343D / V345Q / L398A, D151L / D282N / S393V, D151L / V345Q / S393V / L3 98A, D151L / S393V, L158R, L158R / S393V, D202N, A206S, A206S / R217K, R217K , R217K / S333F, R217K / S333G, R217K / P337R / V345Q / L398A, A271N, D282N / S393V, S333F / V345Q, S333G, S333N, P337R / K343G / V345Q / L398A, K343D, K343D / V345Q / S393V / L398A, S393V, and S393V / L398A, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 158.

[0012] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 372, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10, 10 / 39 / 44 / 322, 10 / 39 / 92 / 206 / 217 / 271, 10 / 39 / 92 / 247, 10 / 39 / 92 / 247 / 271 / 316, 10 / 44, 10 / 44 / 47 / 9 2 / 247, 10 / 44 / 47 / 261 / 302 / 322 / 368, 10 / 44 / 92 / 316 / 322, 10 / 44 / 261 / 302 / 316, 10 / 44 / 302 / 337 / 368, 10 / 47 / 217 / 247 / 316 / 392, 10 / 47 / 217 / 322, 10 / 4 7 / 271, 10 / 92, 10 / 92 / 206 / 217 / 247, 10 / 92 / 206 / 247 / 316 / 322 / 392, 10 / 92 / 206 / 247 / 322 / 368, 10 / 92 / 217 / 261 / 302 / 337, 10 / 206 / 217 / 271, 10 / 206 / 247 , 10 / 206 / 261 / 271 / 316, 10 / 261, 10 / 271 / 302, 10 / 302, 10 / 302 / 316, 10 / 302 / 322 / 337, 10 / 316 / 322, 10 / 337 / 392, 10 / 368, 39 / 44 / 92 / 162 / 247 / 302 / 316 / 322, 39 / 44 / 92 / 217 / 322, 39 / 44 / 92 / 247 / 271 / 302, 39 / 47 / 92 / 247 / 302 / 316 / 322, 39 / 47 / 217 / 247 / 368, 39 / 47 / 247, 39 / 92 / 247 / 302 / 316 / 337 / 368, 39 / 92 / 316 / 322, 39 / 247 / 271, 39 / 247 / 271 / 316, 39 / 322, 44 / 47 / 92 / 206 / 217 / 316 / 322, 44 / 47 / 92 / 247 / 261 / 271 / 316 / 337 / 368, 44 / 47 / 206 / 217 / 247 / 271 / 322, 44 / 47 / 247 / 322 / 368, 44 / 47 / 302 / 316 / 322, 44 / 92 / 206 / 247 / 368, 44 / 206 / 337, 44 / 247 / 261 / 302 / 316, 44 / 247 / 261 / 302 / 316 / 322, 47 / 92 / 247 / 271,47 / 217 / 302, 47 / 247, 47 / 247 / 271, 89 / 217 / 247 / 261 / 302 / 316, 92 / 217 / 271, 92 / 247, 92 / 247 / 271 / 322, 92 / 247 / 302 / 322 / 337, 92 / 271 / 337, 92 / 302, 92 / 316, 206 / 217 / 271 / 392, 217 / 247 / 316 / 32 and 368, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 372. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 372, or a functional fragment thereof, and said recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from SEQ ID NO: 372, 247, 247 / 271, 247 / 302, 271, 271 / 302 / 322, 271 / 316 / 322, 302 / 322 / 368, and 368, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 372. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 372, or a functional fragment thereof, and wherein said recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from SEQ ID NO: 372, 247, 247 / 271, 247 / 302, 271, 271 / 302 / 322, 271 / 316 / 322, 302 / 322 / 368, and 368, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 372. P, 10P / 39E / 44R / 322I, 10P / 39E / 92H / 206A / 217R / 271A, 10P / 39E / 92H / 247D, 10P / 39E / 92H / 247D / 271 A / 316D, 10P / 44R, 10P / 44R / 47T / 92H / 247D, 10P / 44R / 47T / 261G / 302K / 322I / 368A, 10P / 44R / 92H / 316D / 322I, 10P / 44R / 261G / 302K / 316D, 10P / 44R / 302K / 337P / 368A, 10P / 47T / 217R / 247D / 316D / 392T, 10P / 47T / 217R / 322I, 10P / 47T / 271A, 10P / 92H, 10P / 92H / 206A / 217R / 247D, 10P / 92H / 206A / 247D / 316D / 32 2I / 392T, 10P / 92H / 206A / 247D / 322I / 368A, 10P / 92H / 217R / 261G / 302K / 337P, 10P / 206A / 217R / 271A, 10P / 206A / 247D, 10P / 206A / 261G / 271A / 316D, 10P / 261G, 10P / 271A / 302K, 10P / 302K, 10P / 302K / 316D,10P / 302K / 322I / 337P, 10P / 316D / 322I, 10P / 337P / 392T, 10P / 368A, 39E / 44R / 92H / 162M / 247D / 302K / 316D / 322I, 39E / 44R / 92H / 217R / 322I, 39E / 44R / 92H / 247D / 271A / 302K, 39E / 47T / 92H / 247D / 302K / 316D / 322I, 39E / 47T / 217R / 247D / 368A, 39E / 47T / 247D, 39E / 92H / 247D / 302K / 316D / 337 P / 368A, 39E / 92H / 316D / 322I, 39E / 247D / 271A, 39E / 247D / 271A / 316D, 3 9E / 322I, 44R / 47T / 92H / 206A / 217R / 316D / 322I, 44R / 47T / 92H / 247D / 26 1G / 271A / 316D / 337P / 368A, 44R / 47T / 206A / 217R / 247D / 271A / 322I, 44R / 47T / 247D / 322I / 368A, 44R / 47T / 302K / 316D / 322I, 44R / 92H / 206A / 247 D / 368A, 44R / 206A / 337P, 44R / 247D / 261G / 302K / 316D, 44R / 247D / 261G / 302K / 316D / 322I, 47T / 92H / 247D / 271A, 47T / 217R / 302K, 47T / 247D, 47T / 247D / 271A, 89I / 217R / 247D / 261G / 302K / 316D, 92H / 217R / 271A, 92H / 2 47D, 92H / 247D / 271A / 322I, 92H / 247D / 302K / 322I / 337P, 92H / 271A / 337

[0023] In some embodiments, the recombinant alpha-galactosidase A has at least one substitution or set of substitutions at one or more positions selected from SEQ ID NO: 372, ...and a polypeptide sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the recombinant alpha-galactosidase A, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of T10P, T10P / M39E / L44R / M322I, T10P / M39E / Y92H / K206A / F217R / H271A, T10P / M39E / Y92H / N247D, T10P / M39E / Y92H / N247D / H271A / L316D, T10P / L44R, T10P / L44R / S4 7T / Y92H / N247D, T10P / L44R / S47T / A261G / Q302K / M322I / W368A, T10P / L44 R / Y92H / L316D / M322I, T10P / L44R / A261G / Q302K / L316D, T10P / L44R / Q302 K / A337P / W368A, T10P / S47T / F217R / N247D / L316D / M392T, T10P / S47T / F21 7R / M322I, T10P / S47T / H271A, T10P / Y92H, T10P / Y92H / K206A / F217R / N247D , T10P / Y92H / K206A / N247D / L316D / M322I / M392T, T10P / Y92H / K206A / N247 D / M322I / W368A, T10P / Y92H / F217R / A261G / Q302K / A337P, T10P / K206A / F2 17R / H271A, T10P / K206A / N247D, T10P / K206A / A261G / H271A / L316D, T10P / A261G, T10P / H271A / Q302K, T10P / Q302K, T10P / Q302K / L316D, T10P / Q302K / M322I / A337P, T10P / L316D / M322I, T10P / A337P / M392T, T10P / W368A, M39E / L44R / Y92H / R162M / N247D / Q302K / L316D / M322I, M39E / L44R / Y92H / F217R / M322I, M39E / L44R / Y92H / N247D / H271A / Q302K, M39E / S47T / Y92H / N247D / Q302K / L316D / M322I, M39E / S47T / F217R / N247D / W368A, M39E / S47T / N247D,M39E / Y92H / N247D / Q302K / L316D / A337P / W368A, M39E / Y92H / L316D / M322I, M39E / N247D / H271A, M39E / N247D / H271A / L316D, M39E / M 322I, L44R / S47T / Y92H / K206A / F217R / L316D / M322I, L44R / S47T / Y92H / N247D / A261G / H271A / L316D / A337P / W368A, L44R / S47T / K206 A / F217R / N247D / H271A / M322I, L44R / S47T / N247D / M322I / W368A, L44R / S47T / Q302K / L316D / M322I, L44R / Y92H / K206A / N247D / W368A , L44R / K206A / A337P, L44R / N247D / A261G / Q302K / L316D, L44R / N247D / A261G / Q302K / L316D / M322I, S47T / Y92H / N247D / H271A, S47T / F217R / Q302K, S47T / N247D, S47T / N247D / H271A, L89I / F217R / N247D / A261G / Q302K / L316D, Y92H / F217R / H271A, Y92H / N247D, Y92H / N 247D / H271A / M322I, Y92H / N247D / Q302K / M322I / A337P, Y92H / H271A / A337P, Y92H / Q302K, Y92H / L316D, K206A / F217R / H271A / M392T, and at least one substitution or set of substitutions at one or more positions selected from F217R / N247D / L316D / M322I / A337P / W368A, N247D, N247D / H271A, N247D / Q302K, H271A, H271A / Q302K / M322I, H271A / L316D / M322I, Q302K / M322I / W368A, and W368A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 372.

[0013] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10 / 36 / 92 / 166 / 247 / 261 / 316 / 392, 10 / 39, 10 / 39 / 44 / 47 / 92 / 206 / 217, 10 / 39 / 44 / 47 / 316, 10 / 39 / 44 / 4 7 / 337, 10 / 39 / 44 / 92 / 166 / 261 / 316 / 322, 10 / 39 / 44 / 92 / 166 / 302 / 322, 10 / 39 / 44 / 92 / 166 / 392, 10 / 39 / 44 / 92 / 217 / 302 / 322, 10 / 39 / 44 / 92 / 302 / 322, 1 0 / 39 / 44 / 166 / 261 / 271 / 316 / 322, 10 / 39 / 44 / 392, 10 / 39 / 47 / 92 / 337, 10 / 39 / 92 / 131 / 166 / 271 / 316 / 322, 10 / 39 / 92 / 166 / 217 / 247 / 271, 10 / 39 / 92 / 217 / 316, 10 / 44 / 47 / 166 / 261 / 271, 10 / 44 / 47 / 166 / 271 / 322 / 368, 10 / 44 / 47 / 217 / 271 / 316 / 322, 10 / 44 / 92, 10 / 44 / 92 / 217 / 247 / 271 / 302 / 316 / 392, 10 / 44 / 166 / 302, 10 / 44 / 206 / 316 / 322, 10 / 47 / 92 / 166 / 271 / 316 / 337, 10 / 47 / 92 / 271 / 302, 10 / 47 / 92 / 316 / 322 / 392, 10 / 47 / 166 / 271, 10 / 47 / 166 / 316, 10 / 92 / 166, 10 / 92 / 166 / 217 / 247 / 261 / 271, 10 / 92 / 166 / 261 / 271 / 392, 10 / 92 / 166 / 261 / 316 / 322 / 337, 10 / 92 / 166 / 337 / 368, 10 / 92 / 302 / 337, 10 / 92 / 316 / 32 2, 10 / 206, 10 / 206 / 247 / 261, 10 / 217 / 322, 10 / 261, 10 / 261 / 337 / 392, 10 / 316 / 392, 10 / 368, 39 / 44 / 47 / 92 / 166 / 206 / 392, 39 / 44 / 47 / 92 / 206 / 247 / 261,39 / 44 / 47 / 92 / 206 / 392, 39 / 44 / 47 / 206 / 337 / 368 / 392, 39 / 44 / 92 / 166 / 247 / 261 / 302 / 337, 39 / 44 / 166 / 271, 39 / 44 / 166 / 271 / 337 / 368 / 392, 39 / 47 / 92 / 316 / 322, 39 / 47 / 92 / 392, 39 / 47 / 166 / 217 / 261 / 392, 39 / 47 / 217 / 247 / 368, 39 / 47 / 247, 39 / 92 / 166 / 217 / 392, 39 / 92 / 261 / 302, 39 / 166 / 217 / 261 / 316 / 368, 39 / 322, 39 / 392, 44 / 47, 44 / 47 / 92 / 217 / 271, 44 / 47 / 92 / 217 / 316 / 322 / 392, 44 / 47 / 92 / 392, 44 / 47 / 166, 44 / 47 / 166 / 271, 44 / 47 / 247 / 271 / 392, 44 / 316 / 322 / 392, 44 / 337, 47 / 166 / 206 / 217 / 247 / 337, 47 / 166 / 217 / 271 / 337, 47 / 206, 47 / 217 / 247 / 261, 47 / 271, 52 / 217 / 302 / 316, 92 / 166 / 206 / 271 / 316, 92 / 166 / 217 / 261 / 271 / 392, 92 / 166 / 217 / 316 / 337 / 392, 92 / 166 / 247, 92 / 166 / 316, 92 / 206 / 322, 92 / 217, 92 / 217 / 271 / 337, 92 / 2 and at least one substitution or set of substitutions at one or more positions selected from 61 / 271, 92 / 271, 166 / 217 / 316 / 322 / 337, 166 / 247 / 271 / 316, 166 / 316 / 322 / 337, 206 / 217, 217 / 392, 247 / 316, 316 / 322 / 368, and 316 / 337 / 392, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10T / 36M / 92Y / 166S / 247N / 261A / 316L / 392M,10T / 39M、10T / 39M / 44L / 47S / 92Y / 206K / 217F、10T / 39M / 44L / 47S / 316L、10T / 39M / 44L / 47S / 337A、10T / 39M / 44L / 92Y / 166S / 261A / 316L / 322M、10T / 39M / 44L / 92Y / 166S / 302Q / 322M、10T / 39M / 44L / 92Y / 166S / 392M、10T / 39M / 44L / 92Y / 217F / 302Q / 322M、10T / 39M / 44L / 92Y / 302Q / 322M、10T / 39M / 44L / 166S / 261A / 271H / 316L / 322M、10T / 39M / 44L / 392M、10T / 39M / 47S / 92Y / 337A、10T / 39M / 92Y / 131G / 166S / 271H / 316L / 322M、10T / 39M / 92Y / 166S / 217F / 247N / 271H、10T / 39M / 92Y / 217F / 316L、10T / 44L / 47S / 166S / 261A / 271H、10T / 44L / 47S / 166S / 271H / 322M / 368W、10T / 44L / 47S / 217F / 271H / 316L / 322M、10T / 44L / 92Y、10T / 44L / 92Y / 217F / 247N / 271H / 302Q / 316L / 392M、10T / 44L / 166S / 302Q、10T / 44L / 206K / 316L / 322M、10T / 47S / 92Y / 166S / 271H / 316L / 337A、10T / 47S / 92Y / 271H / 302Q、10T / 47S / 92Y / 316L / 322M / 392M、10T / 47S / 166S / 271H、10T / 47S / 166S / 316L、10T / 92Y / 166S、10T / 92Y / 166S / 217F / 247N / 261A / 271H、10T / 92Y / 166S / 261A / 271H / 392M、10T / 92Y / 166S / 261A / 316L / 322M / 337A、10T / 92Y / 166S / 337A / 368W、10T / 92Y / 302Q / 337A、10T / 92Y / 316L / 322M、10T / 206K、10T / 206K / 247N / 261A、10T / 217F / 322M、10T / 261A、10T / 261A / 337A / 392M、10T / 316L / 392M、10T / 368W、39M / 44L / 47S / 92Y / 166S / 206K / 392M、39M / 44L / 47S / 92Y / 206K / 247N / 261A, 39M / 44L / 47S / 92Y / 206K / 392M, 39M / 44L / 47S / 206K / 337A / 368W / 392M, 39M / 44L / 92Y / 166S / 247N / 261A / 302Q / 3 37A, 39M / 44L / 166S / 271H, 39M / 44L / 166S / 271H / 337A / 368W / 392M, 39M / 47 S / 92Y / 316L / 322M, 39M / 47S / 92Y / 392M, 39M / 47S / 166S / 217F / 261A / 392M, 3 9M / 47S / 217F / 247N / 368W, 39M / 47S / 247N, 39M / 92Y / 166S / 217F / 392M, 39M / 92Y / 261A / 302Q, 39M / 166S / 217F / 261A / 316L / 368W, 39M / 322M, 39M / 392M, 44L / 47S, 44L / 47S / 92Y / 217F / 271H, 44L / 47S / 92Y / 217F / 316L / 322M / 392M , 44L / 47S / 92Y / 392M, 44L / 47S / 166S, 44L / 47S / 166S / 271H, 44L / 47S / 247N / 271H / 392M, 44L / 316L / 322M / 392M, 44L / 337A, 47S / 166S / 206K / 217F / 247N / 337A, 47S / 166S / 217F / 271H / 337A, 47S / 206K, 47S / 217F / 247N / 261A, 47S / 271H, 52N / 217F / 302Q / 316L, 92Y / 166S / 206K / 271H / 316L, 92Y / 166S / 217 F / 261A / 271H / 392M, 92Y / 166S / 217F / 316L / 337A / 392M, 92Y / 166S / 247N, 92 comprising at least one substitution or set of substitutions at one or more positions selected from Y / 166S / 316L, 92Y / 206K / 322M, 92Y / 217F, 92Y / 217F / 271H / 337A, 92Y / 261A / 271H, 92Y / 271H, 166S / 217F / 316L / 322M / 337A, 166S / 247N / 271H / 316L, 166S / 316L / 322M / 337A, 206K / 217F, 217F / 392M, 247N / 316L, 316L / 322M / 368W, and 316L / 337A / 392M;The amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of P10T / K36M / H92Y / P166S / D247N / G261A / D316L / T392M, P10T / E39M, P10T / E39M / R 44L / T47S / H92Y / A206K / R217F, P10T / E39M / R44L / T47S / D316L, P10T / E39M / R44L / T47S / P337A, P10T / E39M / R44L / H92Y / P166S / G261A / D316L / I322M, P10T / E39M / R44L / H92Y / P166S / K302Q / I322M, P10T / E39M / R44L / H92Y / P166S / T392M, P10T / E39M / R44L / H92Y / R217F / K302Q / I322M, P 10T / E39M / R44L / H92Y / K302Q / I322M, P10T / E39M / R44L / P166S / G261A / A271H / D316L / I322M, P10T / E39M / R44L / T392M, P10T / E39M / T47S / H92 Y / P337A, P10T / E39M / H92Y / W131G / P166S / A271H / D316L / I322M, P10T / E39M / H92Y / P166S / R217F / D247N / A271H, P10T / E39M / H92Y / R217F / D3 16L, P10T / R44L / T47S / P166S / G261A / A271H, P10T / R44L / T47S / P166S / A271H / I322M / A368W, P10T / R44L / T47S / R217F / A271H / D316L / I322M, P10T / R44L / H92Y, P10T / R44L / H92Y / R217F / D247N / A271H / K302Q / D316L / T392M, P10T / R44L / P166S / K302Q, P10T / R44L / A206K / D316L / I322M,P10T / T47S / H92Y / P166S / A271H / D316L / P337A、P10T / T47S / H92Y / A271H / K302Q、P10T / T47S / H92Y / D316L / I322M / T392M、P10T / T47S / P166S / A271H、P10T / T47S / P166S / D316L、P10T / H92Y / P166S、P10T / H92Y / P166S / R217F / D247N / G261A / A271H、P10T、 / H92Y / P166S / G261A / A271H / T392M、P10T / H92Y / P166S / G261A / D316L / I3 22M / P337A、P10T / H92Y / P166S / P337A / A368W、P10T / H92Y / K302Q / P337A、P 10T / H92Y / D316L / I322M、P10T / A206K、P10T / A206K / D247N / G261A、P10T / R 217F / I322M、P10T / G261A、P10T / G261A / P337A / T392M、P10T / D316L / T392M P10T / A368W, E39M / R44L / T47S / H92Y / P166S / A206K / T392M, E39M / R44L / T47S / H92Y / A206K / D247N / G261A, E39M / R44L / T47S / H92Y / A206K / T392M, E3 9M / R44L / T47S / A206K / P337A / A368W / T392M, E39M / R44L / H92Y / P166S / D247N / G261A / K302Q / P337A, E39M / R44L / P166S / A271H, E39M / R44L / P166S / A2 71H / P337A / A368W / T392M, E39M / T47S / H92Y / D316L / I322M, E39M / T47S / H92Y / T392M, E39M / T47S / P166S / R217F / G261A / T392M, E39M / T47S / R217F / D 247N / A368W、E39M / T47S / D247N、E39M / H92Y / P166S / R217F / T392M、E39M / H 92Y / G261A / K302Q、E39M / P166S / R217F / G261A / D316L / A368W、E39M / I322M E39M / T392M, R44L / T47S, R44L / T47S / H92Y / R217F / A271H, R44L / T47S / H92Y / R217F / D316L / I322M / T392M, R44L / T47S / H92Y / T392M, R44L / T47S / P16 6S、R44L / T47S / P166S / A271H、R44L / T47S / D247N / A271H / T392M、R44L / D31 6L / I322M / T392M、R44L / P337A、T47S / P166S / A206K / R217F / D247N / P337A、T47S / P166S / R217F / A271H / P337A, T47S / A206K, T47S / R217F / D247N / G261A, T47S / A271H, D52N / R217F / K302Q / D316L, H92Y / P166S / A206K / A271H / D316L, H92Y / P166S / R217F / G261A / A271H / T392M, H92Y / P166S / R217F / D316L / P337A / T392M, H92Y / P16 6S / D247N, H92Y / P166S / D316L, H92Y / A206K / I322M, H92Y / R217F, H92Y / R217F / A27 and at least one substitution or set of substitutions at one or more positions selected from: 1H / P337A, H92Y / G261A / A271H, H92Y / A271H, P166S / R217F / D316L / I322M / P337A, P166S / D247N / A271H / D316L, P166S / D316L / I322M / P337A, A206K / R217F, R217F / T392M, D247N / D316L, D316L / I322M / A368W, and D316L / P337A / T392M, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374.

[0014] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 704, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 2, 4, 5, 24 / 59, 24 / 143 / 144, 24 / 143 / 202 / 333, 24 / 143 / 202 / 352 / 390 / 391, 24 / 143 / 333 / 352 / 387 / 390 / 391, 24 / 143 / 390 / 391, 24 / 202, 24 / 202 / 271, 24 / 202 / 333 / 352, 24 / 271 / 352, 24 / 352 / 387 / 390 / 391, 24 / 387 / 391, 31, 40, 59, 59 / 143, 59 / 143 / 202, 59 / 143 / 202 / 271 / 333, 59 / 143 / 271, 59 / 143 / 333, 59 / 202, 59 / 202 / 333, 59 / 271 / 387 / 390, 73, 76, 80, 83, 84, 91 / 215 / 361, 122, 123, 143, 143 / 20 2, 143 / 271, 143 / 271 / 352 / 390, 143 / 333, 143 / 333 / 387 / 390, 143 / 387 / 391, 147, 155, 164, 165, 179, 186, 202, 202 / 333, 210, 215 / 218, 218, 218 / 361, 218 / 361 / 398, 218 / 398, 246, 254 / 398, 271, 271 / 333, 271 / 333 / 390 / 391, 271 / 333 / 391, 271 / 352 / 391, 273, 275, 277, 278, 280, 281, 283, 284, 287, In some embodiments, the recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from 300, 303, 304, 325, 331, 332, 333 / 352, 333 / 390 / 391, 333 / 391, 334, 335, 336, 338, 339, 340, 341, 343, 359, 360, 361, 362, 367, 369, 371, 373, 375, 377, 382, ​​382 / 398, 385, 387 / 391, 390, and 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 704.704, or a functional fragment thereof, and the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 704, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 2S, 4L, 5M, 5V, 24S / 59A, 24S / 143S / 144N, 24S / 143S / 202N / 333N, 24S / 143S / 202N / 352N / 390N / 391N, 24S / 143S / 333N / 352N / 387N / 390T / 391N, 24S / 143S / 390T / 391N, 24S / 202N, 24S / 202N / 271N, 24S / 202N / 333N / 352N, 2 4S / 271N / 352N, 24S / 352N / 387N / 390N / 391N, 24S / 387N / 391N, 31F, 31H, 31L, 31T, 31W, 40Q, 59A, 59A / 143S, 59A / 143S / 271N, 59A / 202N, 59T, 59T / 143S / 2 02N, 59T / 143S / 333N, 59T / 202N / 333N, 59V / 143S / 202N / 271N / 333N, 59V / 271 N / 387N / 390T, 73A, 76A, 76F, 76M, 76S, 80T, 83R, 83S, 84G, 84K, 84R, 91S / 21 5S / 361T, 122E, 122N, 122S, 123Q, 123R, 123S, 123T, 143S, 143S / 202N, 143S / 271N, 143S / 271N / 352N / 390N, 143S / 333N, 143S / 333N / 387N / 390T, 143S / 38 7N / 391N, 147L, 147S, 155A, 155D, 155F, 155L, 155R, 155T, 164E, 165I, 179H, 179L, 179R, 179W, 186E, 186F, 186M, 186P, 186R, 186S, 186Y, 202N, 202N / 33 3N, 210I, 215S / 218Y, 218Y, 218Y / 361T, 218Y / 361T / 398F, 218Y / 398F, 246Y, 254T / 398F, 271N, 271N / 333N, 271N / 333N / 390N / 391N, 271N / 333N / 391N, 27 1N / 352N / 391N, 273L, 275A, 275G, 277Q, 277V, 278N, 278R, 278S, 280G, 281I,281M, 283L, 283P, 283T, 283V, 284A, 284E, 284G, 284L, 284M, 284R, 284S, 287R , 300F, 303A, 303C, 303W, 304T, 304V, 304W, 325A, 331M, 332G, 332H, 333N / 352N , 333N / 390N / 391N, 333N / 390S / 391N, 333N / 391N, 334C, 334V, 335A, 335L, 336 F, 336G, 336S, 336T, 338L, 339G, 339N, 339Q, 339V, 340H, 340I, 340K, 340M, 340 and 398F, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:704. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 704, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of D2S, G4L, L5M, L5V, D24S / C59A, D24S / C143S / D144N, D24S / C143S / D202N / G333N, D24S / C143 S / D202N / F352N / M390N / Q391N, D24S / C143S / G333N / F352N / E387N / M390 T / Q391N, D24S / C143S / M390T / Q391N, D24S / D202N, D24S / D202N / A271N, D 24S / D202N / G333N / F352N, D24S / A271N / F352N, D24S / F352N / E387N / M390 N / Q391N, D24S / E387N / Q391N, S31F, S31H, S31L, S31T, S31W, E40Q, C59A,C59A / C143S, C59A / C143S / A271N, C59A / D202N, C59T, C59T / C143S / D202N, C59T / C143S / G333N, C59T / D202N / G333N, C59V / C143S / D202N / A271N / G333N C59V / A271N / E387N / M390T, G73A, Q76A, Q76F, Q76M, Q76S, Q80T, P83R, P83S, H84G, H84K, H84R, N91S / T215S / R361T, D122E, D122N, D122S, I123Q, I123R I123S, I123T, C143S, C143S / D202N, C143S / A271N, C143S / A271N / F352N / M390N, C143S / G333N, C143S / G333N / E387N / M390T, C143S / E387N / Q391N, E147L 、E147S、H155A、H155D、H155F、H155L、H155R、H155T、G164E、R165I、P179H、P 179L、P179R、P179W、T186E、T186F、T186M、T186P、T186R、T186S、T186Y、D202 N, D202N / G333N, S210I, T215S / N218Y, N218Y, N218Y / R361T, N218Y / R361T / L398F, N218Y / L398F, W246Y, A254T / L398F, A271N, A271N / G333N, A271N / G33 3N / M390N / Q391N, A271N / G333N / Q391N, A271N / F352N / Q391N, S273L, Q275A, Q275G, K277Q, K277V, A278N, A278R, A278S, L280G, Q281I, Q281M, K283L, K2 83P、K283T、K283V、D284A、D284E、D284G、D284L、D284M、D284R、D284S、A287 R、L300F、G303A、G303C、G303W、D304T、D304V、D304W、R325A、P331M、R332G、R 332H, G333N / F352N, G333N / M390N / Q391N, G333N / M390S / Q391N, G333N / Q391N, Y334C, Y334V, T335A, T335L, I336F, I336G, I336S, I336T, V338L, A339GA339N, A339Q, A339V, S340H, S340I, S340K, S340M, S340P, S340W, L341F, L341M, K343L, K343R, K343S, K343W, V359F, V359L, V359R, K360H, K360V, R361T, R361V, K362H, E367A, E367D, E367L, E367M, T369D, R and at least one substitution or set of substitutions at one or more positions selected from 371G, R373L, R373S, H375L, H375Q, N377Q, V382I, V382I / L398F, Q385R, E387N / Q391N, M390S, and L398F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 704.

[0015] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from 10, 39, 44, 47, 92, 166, 206, 217, 247, 261, 271, 302, 316, 322, 337, 368, and 392, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 10A, 10C, 10D, 10E, 10F, 10G, 10H, 10HQ, 10I, 10IQ ... 10D, 10E, 10F, 10G, 10H, 10I, 10K, 10L, 10M, 10N, 10Q, 10R, 10S, 10T, 10V, 10W, 10Y, 39A, 39C, 39D, 39F, 39 G, 39H, 39I, 39K, 39L, 39M, 39N, 39P, 39Q, 39R, 39S, 39T, 39V, 39W, 39Y, 44A, 44C, 44D, 44E, 44F, 44G, 44H, 4 4I, 44K, 44L, 44N, 44P, 44Q, 44S, 44T, 44V, 44W, 44Y, 47A, 47C, 47D, 47E, 47F, 47G, 47H, 47I, 47K, 47L, 47M , 47N, 47P, 47Q, 47R, 47S, 47V, 47W, 47Y, 92A, 92C, 92D, 92E, 92F, 92G, 92I, 92K, 92L, 92M, 92N, 92P, 92Q, 9 2R, 92S, 92T, 92V, 92W, 92Y, 166A, 166C, 166D, 166E, 166F, 166G, 166H, 166I, 166K, 166L, 166M, 166N, 166 Q, 166R, 166S, 166T, 166V, 166W, 166Y, 206C, 206D, 206E, 206F, 206G, 206H, 206I, 206K, 206L, 206M, 206N,206P, 206Q, 206R, 206S, 206T, 206V, 206W, 206Y, 217A, 217C, 217D, 217E, 217F, 217G, 217H, 217I, 217K, 217L, 217M, 217N, 217P, 217Q, 217S, 217T, 217 V、217W、217Y、247A、247C、247E、247F、247G、247H、247I、247K、247L、247M 、247N、247P、247Q、247R、247S、247T、247V、247W、247Y、261A、261C、261D、2 61E、261F、261H、261I、261K、261L、261M、261N、261P、261Q、261R、261S、26 1T、261V、261W、261Y、271C、271D、271E、271F、271G、271H、271I、271K、271L 271M, 271N, 271P, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 302A, 302C, 302D, 302E, 302F, 302G, 302H, 302I, 302L, 302M, 302N, 302P, 302Q, 302R, 30 2S, 302T, 302V, 302W, 302Y, 316A, 316C, 316E, 316F, 316G, 316H, 316I, 316K, 316L, 316M, 316N, 316P, 316Q, 316R, 316S, 316T, 316V, 316W, 316Y, 322A 322C, 322D, 322E, 322F, 322G, 322H, 322K, 322L, 322M, 322N, 322P, 322Q, 322R, 322S, 322T, 322V, 322W, 322Y, 337A, 337C, 337D, 337E, 337F, 337G, 337 H, 337I, 337K, 337L, 337M, 337N, 337Q, 337R, 337S, 337T, 337V, 337W, 337Y, 368C, 368D, 368E, 368F, 368G, 368H, 368I, 368K, 368L, 368M, 368N, 368P, 3 68Q, 368R, 368S, 368T, 368V, 368W, 368Y, 392A, 392C, 392D, 392E, 392F, 392G, 392H, 392I, 392K, 392L, 392M, 392N, 392P, 392Q, 392R, 392S, 392V, 392Wand 392Y, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein said recombinant alpha-galactosidase A is selected from the group consisting of P10A, P10C, P10D, P10E, P10F, P10G, P10H, P10I, P10K, P10L, P10M, P10N , P10Q, P10R, P10S, P10T, P10V, P10W, P10Y, E39A, E39C, E39D, E39F, E39G, E39H, E39I, E39K, E39L, E39M, E39N, E39P, E39Q, E39R, E39S, E3 9T, E39V, E39W, E39Y, R44A, R44C, R44D, R44E, R44F, R44G, R44H, R44I, R44K, R44L, R44N, R44P, R44Q, R44S, R44T, R44V, R44W, R44Y, T47A, T 47C, T47D, T47E, T47F, T47G, T47H, T47I, T47K, T47L, T47M, T47N, T47P, T47Q, T47R, T47S, T47V, T47W, T47Y, H92A, H92C, H92D, H92E, H92F , H92G, H92I, H92K, H92L, H92M, H92N, H92P, H92Q, H92R, H92S, H92T, H92V, H92W, H92Y, P166A, P166C, P166D, P166E, P166F, P166G, P166H, P 166I, P166K, P166L, P166M, P166N, P166Q, P166R, P166S, P166T, P166V, P166W, P166Y, A206C, A206D, A206E, A206F, A206G, A206H, A206I, A206K, A206L, A206M, A206N, A206P, A206Q, A206R, A206S, A206T, A206V, A206W, A206Y, R217A, R217C, R217D, R217E, R217F, R217G, R217H,R217I, R217K, R217L, R217M, R217N, R217P, R217Q, R217S, R217T, R217V, R217W, R217Y, D247A, D247C, D247E, D247F, D247G, D247H, D247I, D247K, D247 L、D247M、D247N、D247P、D247Q、D247R、D247S、D247T、D247V、D247W、D247Y、 G261A、G261C、G261D、G261E、G261F、G261H、G261I、G261K、G261L、G261M、G26 1N、G261P、G261Q、G261R、G261S、G261T、G261V、G261W、G261Y、A271C、A271D 、A271E、A271F、A271G、A271H、A271I、A271K、A271L、A271M、A271N、A271P、A 271Q, A271R, A271S, A271T, A271V, A271W, A271Y, K302A, K302C, K302D, K302E, K302F, K302G, K302H, K302I, K302L, K302M, K302N, K302P, K302Q, K302R K302S, K302T, K302V, K302W, K302Y, D316A, D316C, D316E, D316F, D316G, D316H, D316I, D316K, D316L, D316M, D316N, D316P, D316Q, D316R, D316S, D316 T、D316V、D316W、D316Y、I322A、I322C、I322D、I322E、I322F、I322G、I322H、 I322K、I322L、I322M、I322N、I322P、I322Q、I322R、I322S、I322T、I322V、I32 2W, I322Y, P337A, P337C, P337D, P337E, P337F, P337G, P337H, P337I, P337K, P337L, P337M, P337N, P337Q, P337R, P337S, P337T, P337V, P337W, P337Y, A 368C, A368D, A368E, A368F, A368G, A368H, A368I, A368K, A368L, A368M, A368N, A368P, A368Q, A368R, A368S, A368T, A368V, A368W, A368Y, T392A, T392Cand at least one substitution or set of substitutions at one or more positions selected from T392D, T392E, T392F, T392G, T392H, T392I, T392K, T392L, T392M, T392N, T392P, T392Q, T392R, T392S, T392V, T392W, and T392Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374.

[0016] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1022, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is 39 / 44 / 166 / 302, 31 / 47, 31 / 283 / 284, 39, 39 / 44, 39 / 44 / 47, 39 / 44 / 47 / 261 / 283 / 284, 39 / 44 / 283, 39 / 44 / 339, 39 / 47 / 261, 39 / 92, 39 / 206, 39 / 284, 44, 44 / 284 / 302, 84, 84 / 92, 84 / 284 / 302 / 392, 84 / 316, 84 / 368 / 392, 92, 92 / 206 / 217, 92 / 206 / 275, 92 / 206 / 284, 92 / 206 / 302 / 368, 92 / 271, 92 / 271 / 277, 92 / 275 / 284, 92 / 283, 92 / 283 / 392, 92 / 284, 92 / 302, 92 / 316, 92 / 368, 155, 155 / 217, 155 / 368, 166, 166 / 283 / 284, 166 / 302, 206, 206 / 217, and at least one substitution or set of substitutions at one or more positions selected from 206 / 334, 261, 261 / 283, 271, 271 / 368, 275, 283, 283 / 284, 283 / 392, 284, 302, 316, 334, 339, 368, 368 / 392, and 392, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1022. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1022, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of P10G, P10G / T392D, S31T, S31T / E39V / R44V / P166D / K302Y, S31T / T47R, S31T / K283L / D284A, E39L, E39L / H92V, E39L / A206E, E39L / D284S,E39V / R44V, E39V / R44V / T47R, E39V / R44V / T47R / G261S / K283L / D284A, E39V / R44V / K283T, E39V / R44V / A339N, E39V / T47 R / G261S, R44V, R44V / D284E / K302Y, H84K, H84K / H92V, H84K / D284S / K302L / T392A, H84K / D316H, H84K / A368E / T392A, H9 2Q, H92T, H92T / A206E / R217N, H92T / A206E / K302T / A368E, H92T / A271K, H92T / A271K / K277R, H92T / K283P, H92T / K283V / T392W, H92T / D284M, H92T / K302L, H92T / A368E, H92V, H92V / A206E / D284S, H92V / A206Y / Q275A, H92V / Q275A / D284S, H92 V / D284S, H92V / K302L, H92V / D316H, H155F, H155F / R217I, H155F / A368E, P166D, P166D / K283L / D284A, P166D / K302Y, A2 06E, A206E / R217N, A206I, A206Q, A206T / Y334C, A206Y, G261S, G261S / K283L, A271K, A271K / A368E, Q275A, K283L, K283 and at least one substitution or set of substitutions at one or more positions selected from: P / T392W, K283T, K283T / D284E, D284E, D284M, D284S, K302L, K302Y, D316H, Y334C, A339N, A368E, A368E / T392W, T392A, T392D, and T392W, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1022. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1022, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 10G, 10G / 392D, 31T, 31T / 39V / 44V / 166D / 302Y, 31T / 47R ...31T / 283L / 284A, 39L, 39L / 92V, 39L / 206E, 39L / 284S, 39V / 44V, 39V / 44V / 47R, 39V / 44V / 47R / 261S / 283L / 28 4A, 39V / 44V / 283T, 39V / 44V / 339N, 39V / 47R / 261S, 44V, 44V / 284E / 302Y, 84K, 84K / 92V, 84K / 284S / 302L / 39 2A, 84K / 316H, 84K / 368E / 392A, 92Q, 92T, 92T / 206E / 217N, 92T / 206E / 302T / 368E, 92T / 271K, 92T / 271K / 277 R, 92T / 283P, 92T / 283V / 392W, 92T / 284M, 92T / 302L, 92T / 368E, 92V, 92V / 206E / 284S, 92V / 206Y / 275A, 92V / 2 75A / 284S, 92V / 284S, 92V / 302L, 92V / 316H, 155F, 155F / 217I, 155F / 368E, 166D, 166D / 283L / 284A, 166D / 30 2Y, 206E, 206E / 217N, 206I, 206Q, 206T / 334C, 206Y, 261S, 261S / 283L, 271K, 271K / 368E, 275A, 283L, 283P / and at least one substitution or set of substitutions at one or more positions selected from 392W, 283T, 283T / 284E, 284E, 284M, 284S, 302L, 302Y, 316H, 334C, 339N, 368E, 368E / 392W, 392A, 392D, and 392W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1022.

[0017] In some embodiments, the alpha-galactosidase A of the invention comprises at least one mutation at at least one position set forth in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1 and / or 13-1, where these positions are numbered with reference to SEQ ID NO: 2 or another reference sequence as indicated in the tables. In some further embodiments, the recombinant alpha-galactosidase A is derived from human alpha-galactosidase A. In some further embodiments, the recombinant alpha-galactosidase A comprises the polypeptide sequence of SEQ ID NO: 8, 58, 158, 372, 374, 704 and / or 1022.

[0018] In some embodiments, the recombinant alpha-galactosidase A is more thermostable than the alpha-galactosidase A of SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022. In some further embodiments, the recombinant alpha-galactosidase A is more stable at pH 7 than the alpha-galactosidase A of SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022. In still some further embodiments, the recombinant alpha-galactosidase A is more stable at pH 4 than the alpha-galactosidase A of SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022. In still some further embodiments, the recombinant alpha-galactosidase A is more stable at pH 7 and more stable at pH 4 than the alpha-galactosidase A of SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022. In still further embodiments, the recombinant alpha-galactosidase A is more stable to exposure to serum than the alpha-galactosidase A of SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022. In some further embodiments, the recombinant alpha-galactosidase A is more stable to lysosomes than the alpha-galactosidase A of SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022. In yet some further embodiments, the recombinant alpha-galactosidase A is more likely to be taken up by cells than the alpha-galactosidase A of SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022. In some further embodiments, the recombinant alpha galactosidase A depletes more globotriaosylceramide from cells than the alpha galactosidase A of SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022. In yet some further embodiments, the recombinant alpha galactosidase A exhibits improved cellular uptake compared to SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022.In some further embodiments, the recombinant alpha-galactosidase A is less immunogenic than the alpha-galactosidase A of SEQ ID NO: 2, 8, 58, 158, 372, 374, 704, and / or 1022. In some further embodiments, the recombinant alpha-galactosidase A exhibits at least one improved property selected from i) enhanced catalytic activity, ii) increased tolerance to pH 7, iii) increased tolerance to pH 4, iv) increased serum tolerance, v) improved cellular uptake, vi) reduced immunogenicity, or vii) increased depletion of globotriaosylceramide from cells, or any combination of i), ii), iii), iv), v), vi), or vii), compared to a reference sequence. In some embodiments, the reference sequence is SEQ ID NO: 2, 8, 58, 158, 372, 374, 704, and / or 1022. In some further embodiments, the recombinant alpha-galactosidase A is purified.

[0019] The present invention also provides recombinant polynucleotide sequences encoding at least one recombinant alpha-galactosidase A provided herein (e.g., in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and / or 13-1). In some embodiments, the polynucleotide sequence is selected from DNA, RNA, and mRNA. In some embodiments, the recombinant polynucleotide sequence is codon-optimized.

[0020] The present invention also provides expression vectors comprising a recombinant polynucleotide sequence encoding at least one recombinant alpha-galactosidase A provided herein (e.g., Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and / or 13-1). In some embodiments, the recombinant polynucleotide sequence is operably linked to a regulatory sequence. In some further embodiments, the regulatory sequence is a promoter. In some further embodiments, the promoter is a heterologous promoter. In some embodiments, the expression vector further comprises a signal sequence provided herein.

[0021] The present invention also provides a host cell comprising at least one expression vector provided herein. In some embodiments, the host cell comprises an expression vector comprising a recombinant polynucleotide sequence encoding at least one recombinant alpha-galactosidase A provided herein (e.g., Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and / or 13-1). In some embodiments, the host cell is selected from a eukaryote and a prokaryote. In some embodiments, the host cell is a eukaryote. In some further embodiments, the host cell is a mammalian cell.

[0022] The present invention also provides methods for producing an alpha-galactosidase A variant, comprising culturing a host cell provided herein under conditions such that the alpha-galactosidase A encoded by the recombinant polynucleotide is produced. In some embodiments, the method further comprises recovering the alpha-galactosidase A. In some further embodiments, the method further comprises purifying the alpha-galactosidase A. The present invention also provides compositions comprising at least one recombinant alpha-galactosidase A provided herein (e.g., Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and / or 13-1). In some embodiments, the present invention provides pharmaceutical compositions. In some embodiments, the present invention provides pharmaceutical compositions comprising at least one recombinant polynucleotide provided herein. In some further embodiments, the present invention provides pharmaceutical compositions for treating Fabry disease, comprising the enzyme composition provided herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient. In some further embodiments, the pharmaceutical composition is suitable for parenteral injection or infusion into humans.

[0023] The present invention also provides methods for treating and / or preventing symptoms of Fabry disease in a subject, the methods comprising providing a subject with Fabry disease, providing at least one pharmaceutical composition comprising at least one recombinant alpha-galactosidase A provided herein (e.g., Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and / or 13-1), and administering the pharmaceutical composition to the subject. In some embodiments, the symptoms of Fabry disease are improved in the subject. In some further embodiments, a subject to whom a pharmaceutical composition of the present invention has been administered can eat a diet whose fat content is less restricted than the diet required by subjects exhibiting symptoms of Fabry disease. In some embodiments, the subject is an infant or child, while in some alternative embodiments, the subject is an adult or young adult.

[0024] The present invention also provides uses of the compositions provided herein. [Brief explanation of the drawings]

[0025] [Figure 1] FIG. 1 provides a graph showing the relative activity of GLA variants after incubation at temperatures between 30 and 50° C. for 1 hour.

[0026] [Figure 2] FIG. 2 provides a graph showing the relative activity of GLA variants after challenge with human serum at 37° C. for 0 to 24 hours.

[0027] [Figure 3] FIG. 3 provides a graph showing the relative activity of GLA variants after challenge with human lysosomal extract at 37° C. for 0 to 24 hours.

[0028] [Figure 4] FIG. 4 provides a graph showing the cellular uptake of different purified GLA variants, expressed as relative activity compared to wild type, after 4 hours of incubation at 37° C. with cultured Fabry patient fibroblasts.

[0029] [Figure 5] FIG. 5 provides a graph showing the activity of GLA variants compared to SEQ ID NO: 2 in the hearts of Fabry mice at 1, 2 and 4 weeks post-administration.

[0030] [Figure 6] FIG. 6 provides a graph showing residual Gb3 in the hearts of Fabry mice treated with GLA mutants at 1 and 2 weeks post-administration.

[0031] [Figure 7] FIG. 7 provides a graph showing the residual activity of GLA variants after 0-24 hour challenge with human serum.

[0032] [Figure 8] FIG. 8 provides a graph showing the cellular uptake of purified GLA variants in cultured Fabry patient fibroblasts after 4 hours of incubation at 37° C. and a 3 day washout.

[0033] [Figure 9] FIG. 9 provides a graph showing in vivo enzyme activity in the heart of the Fabry mouse model 7 days after the last treatment.

[0034] [Figure 10] FIG. 10 provides a graph showing in vivo enzyme activity in the kidneys of the Fabry mouse model 7 days after the last treatment.

[0035] [Figure 11] FIG. 11 provides a graph showing the degradation of Gb3 in cardiac tissue.

[0036] [Figure 12] FIG. 12 provides a graph showing the degradation of Gb3 in kidney tissue.

[0037] [Figure 13] FIG. 13 provides a graph showing the degradation of lyso-Gb3 in cardiac tissue.

[0038] [Figure 14] FIG. 14 provides a graph showing the degradation of lyso-Gb3 in kidney tissue. DETAILED DESCRIPTION OF THE INVENTION

[0039] Description of the Invention The present invention provides engineered human alpha-galactosidase polypeptides and compositions thereof. The engineered human alpha-galactosidase polypeptides are optimized to provide improved thermostability, serum stability, improved cellular uptake, and stability under both acidic (pH<4) and basic (pH>7) conditions, reduced immunogenicity, and improved clearance of globotriaosylceramide from cells. The present invention also relates to the use of compositions comprising the engineered human alpha-galactosidase polypeptides for therapeutic purposes. In some embodiments, the engineered human alpha-galactosidase polypeptides are optimized for improved cellular uptake while maintaining stability. The present invention also relates to the use of compositions comprising engineered human alpha-galactosidase polypeptides for therapeutic purposes.

[0040] In some cases, enzyme replacement therapy (e.g., FABRAZYME® agalsidase beta, Genzyme) is considered for the treatment of Fabry disease in eligible individuals. Currently used enzyme replacement therapies are recombinantly expressed forms of wild-type human GLA. Intravenously administered GLA is known to circulate, be internalized intracellularly via receptor-mediated endocytosis, primarily via the mannose 6-phosphate receptor (M6PR), and traffic to endosomes / lysosomes in target organs, where it clears accumulated Gb3. These drugs do not completely alleviate patients' symptoms, and neuropathic pain and transient ischemic attacks continue to occur at low rates. Furthermore, GLA uptake by most target organs is poor compared to the highly vascularized, M6PR-rich liver, and the enzyme is unstable at the pH of blood and lysosomes. Therefore, available treatments remain problematic. Furthermore, patients may develop immune responses (IgG and IgE antibodies targeting the administered drug), suffer from severe allergic (anaphylactic) reactions, severe infusion reactions, and even death. The present invention aims to provide a more stable and effective enzyme suitable for the treatment of Fabry disease, with reduced side effects and improved outcomes compared to currently available treatments. Indeed, the present invention aims to provide a recombinant GLA enzyme with increased stability in blood (pH 7.4), which the enzyme experiences upon introduction into the bloodstream. Furthermore, the enzyme has increased stability at lysosomal pH (pH 4.3), where the enzyme is active during treatment. Thus, by using directed evolution of recombinantly expressed human GLA in human HEK293T cells, using high-throughput screening of a diverse enzyme mutant library, novel GLA variants were obtained that maintain stability, improve globotriaosylceramide clearance, and improve cellular uptake. In some embodiments, the GLA variants exhibit reduced immunogenicity. Abbreviations and Definitions

[0041] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, biochemistry, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are well known and commonly used in the art. Such techniques are well known and described in numerous texts and references well known to those skilled in the art. Standard techniques, or variations thereof, are used for chemical synthesis and chemical analysis. All patents, patent applications, articles, and publications mentioned herein, both above and below, are expressly incorporated herein by reference.

[0042] In carrying out the present invention, any suitable method and material similar or equivalent to those described herein can be used, but some methods and materials are described herein.It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described, and these may vary depending on the context in which those skilled in the art use them.Therefore, the terms defined immediately below are more fully explained by referring to this application as a whole.All patents, patent applications, papers, and publications mentioned in this specification, both above and below, are expressly incorporated herein by reference.

[0043] Also, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0044] Numerical ranges are inclusive of the numbers defining the range. Accordingly, every numerical range disclosed herein is intended to include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Also, every maximum (or minimum) numerical limitation disclosed herein is intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0045] The term "about" refers to an acceptable error for a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value's range. In other cases, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0046] Furthermore, the headings provided herein are not intended to limit the various aspects or embodiments of the invention that may be had by reference to this application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to this application as a whole. Nonetheless, to facilitate understanding of the invention, certain terms are defined below.

[0047] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0048] As used herein, the term "comprising" and its cognates are used in an inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0049] As used herein, "EC" numbers refer to the Enzyme Nomenclature of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) Committee on Nomenclature. The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction they catalyze.

[0050] As used herein, "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0051] As used herein, "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0052] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0053] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes. Abbreviations used for genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V). When a three-letter abbreviation is used, the amino acid is represented by the α-carbon (C) unless specifically preceded by "L" or "D" or unless otherwise clear from the context in which the abbreviation is used.α ) can be in either the L- or D-configuration. For example, "Ala" refers to alanine without specifying the configuration about the α-carbon, whereas "D-Ala" and "L-Ala" refer to D-alanine and L-alanine, respectively. When a polypeptide sequence is presented as a series of one-letter or three-letter abbreviations (or combinations thereof), the sequence is presented in the amino (N) to carboxy (C) orientation, in accordance with common convention.

[0054] The abbreviations used for genetically encoded nucleosides are conventional and are as follows: adenosine (A), guanosine (G), cytidine (C), thymidine (T), and uridine (U). Unless specifically indicated, the abbreviated nucleoside may be either a ribonucleoside or a 2'-deoxyribonucleoside. Nucleosides may be identified individually or collectively as either ribonucleosides or 2'-deoxyribonucleosides. When a nucleic acid sequence is presented as a series of single-letter abbreviations, the sequence is presented in the 5' to 3' direction according to common convention, with no phosphates indicated.

[0055] The terms "engineered," "recombinant," "non-naturally occurring," and "mutant," when used with respect to a cell, polynucleotide, or polypeptide, refer to a material that has been modified into a form that does not occur naturally in nature, or a material that is identical to, but produced or derived from synthetic material and / or by manipulation using recombinant techniques, or a material that corresponds to a native or naturally occurring form of, that material.

[0056] As used herein, "UTR" refers to the untranslated region of an mRNA polynucleotide. In some embodiments, the "5' untranslated region" or "5' UTR" is referred to as the "leader sequence" or "leader RNA." This mRNA region is immediately upstream of the start codon. In some embodiments, the "3' untranslated region" or "3' UTR" is the mRNA region immediately downstream of the stop codon. In various organisms (e.g., prokaryotes, eukaryotes, and viruses), both of these regions are important for translation regulation and intracellular transport.

[0057] As used herein, "wild-type," "WT," and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is one that exists in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0058] As used herein, "coding sequence" refers to a portion of a nucleic acid (eg, a gene) that encodes the amino acid sequence of a protein.

[0059] The term "percent (%) sequence identity" is used herein to refer to a comparison between a polynucleotide and a polypeptide and is determined by comparing two optimally aligned sequences through a comparison window, where the portion of the polynucleotide or polypeptide sequence within the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence in the optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue is present in both sequences, or where the nucleic acid base or amino acid residue aligns with a gap to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will appreciate that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by the similarity search method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection as known in the art.Examples of suitable algorithms for determining percent sequence identity and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms described by Altschul et al. (See Altschul et al., J. Mol. Biol., 215:403-410

[1990] and Altschul et al., 1977, Nucleic Acids Res., 3389-3402

[1977] , respectively). Software for performing BLAST analyses is publicly available through the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score T. T is referred to as the neighborhood word score threshold (see Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated for nucleotide sequences using the parameters M (reward score for a pair of matching residues, always greater than 0) and N (penalty score for mismatching residues, always less than 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is stopped when the cumulative alignment score drops by an amount X from its maximum achieved value, when the cumulative score falls below 0 due to the accumulation of one or more negative-scoring residue alignments, or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). For exemplary sequence alignment and determination of percent sequence identity, the BESTFIT or GAP programs from the GCG Wisconsin Software package (Accelrys, Madison WI) can be used using the default parameters provided.

[0060] As used herein, a "reference sequence" refers to a defined sequence used as the basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is a nucleic acid or polypeptide at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, or the full length. Because two polynucleotides or polypeptides can each contain (1) similar sequences (i.e., a portion of the complete sequence) between the two sequences and (2) additional sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" can be based on a primary amino acid sequence, which may have one or more alterations to the primary sequence. A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, where a sequence can be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window can contain 20% or less additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) upon optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, including windows of 30, 40, 50, 100, or more, as desired.

[0061] "Corresponding," "reference to," and "relative to," when used in the context of numbering a given amino acid or polynucleotide sequence, refer to the numbering of residues in a particular reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, residue numbers or residue positions in a given polymer are specified with respect to the reference sequence, not by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of engineered GLA, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, despite the presence of gaps, the numbering of residues in a given amino acid or polynucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0062] As used herein, "amino acid difference" and "residue difference" refer to a difference in an amino acid residue at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in a reference sequence. The position of an amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, a "residue difference at position X44 compared to SEQ ID NO:8" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 44 in SEQ ID NO:8. Thus, if a reference polypeptide of SEQ ID NO:8 has an arginine at position 44, then a "residue difference at position X44 compared to SEQ ID NO:8" refers to an amino acid substitution consisting of any residue other than arginine at the polypeptide position corresponding to position 44 in SEQ ID NO:8. In most cases herein, a specific amino acid residue difference at a position will be designated as "XnY," where "Xn" designates the corresponding position as described above and "Y" is the single-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs from the reference polypeptide). In some cases (e.g., as shown in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and 13-1), the present disclosure also presents specific amino acid differences, designated by the conventional designation "AnB," where A is the single-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and B is the single-letter identifier of the residue substitution in the sequence of the engineered polypeptide. In some cases, the polypeptides of the present disclosure may contain one or more amino acid residue differences relative to the reference sequence, as indicated by a list of the specific positions at which the residue difference exists relative to the reference sequence. In some embodiments, when more than one amino acid can be used at a particular residue position in the polypeptide, the various amino acid residues that can be used are separated by a " / " (e.g., X247D / X247N or X247D / N). In some embodiments, the enzyme variants contain two or more substitutions. These substitutions are separated by a slash for ease of reading (e.g., D24S / D202N). The present application includes engineered polypeptide sequences that contain one or more amino acid differences, including either or both conservative and non-conservative amino acid substitutions.

[0063] As used herein, "mutations" refer to substitutions, insertions, deletions, and other modifications of polypeptide and polynucleotide sequences. It is not intended that the present invention be limited to any particular type of mutation.

[0064] "Conservative amino acid substitution" refers to the replacement of a residue with a different residue having a similar side chain, and thus typically involves replacing an amino acid in a polypeptide with the same or similar amino acid within a defined amino acid class. By way of example and not limitation, an amino acid with an aliphatic side chain may be replaced with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine), an amino acid with a hydroxyl side chain may be replaced with another amino acid with a hydroxyl side chain (e.g., serine and threonine), an amino acid with an aromatic side chain may be replaced with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine), an amino acid with a basic side chain may be replaced with another amino acid with a basic side chain (e.g., lysine and arginine), an amino acid with an acidic side chain may be replaced with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid), and / or a hydrophobic or hydrophilic amino acid may be replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0065] "Non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between defined groups rather than within them, and affect (a) the structure of the peptide backbone in the area of ​​substitution (e.g., proline for glycine), (b) the charge or hydrophobicity, or (c) the bulk of the side chain. By way of example and not limitation, exemplary non-conservative substitutions may be an acidic amino acid substituted with a basic or aliphatic amino acid, an aromatic amino acid substituted with a small amino acid, and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0066] As used herein, "deletion" refers to the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include the removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids, from the reference enzyme, while retaining the enzymatic activity and / or improved properties of the engineered enzyme. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include continuous segments or can be discontinuous.

[0067] As used herein, "insertion" refers to the modification of a polypeptide by adding one or more amino acids from a reference polypeptide. The insertion can be internal to the polypeptide, or at the carboxy or amino terminus. As used herein, insertion includes fusion proteins known in the art. The insertion can be a contiguous segment of amino acids, or can be separated by one or more naturally occurring amino acids of the polypeptide.

[0068] "Functional fragment" and "biologically active fragment" are used interchangeably herein to refer to a polypeptide that has amino- and / or carboxy-terminal deletions and / or internal deletions, but whose remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., a full-length engineered GLA of the present invention), and that retains substantially all of the activity of the full-length polypeptide.

[0069] As used herein, "isolated polypeptide" refers to a polypeptide that is substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. This term encompasses polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., host cells or in vitro synthesis). Recombinant GLA polypeptides may be present intracellularly, in cell culture media, or prepared in various forms, such as lysates or isolated preparations. Thus, in some embodiments, a recombinant GLA polypeptide may be an isolated polypeptide.

[0070] As used herein, "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., more abundant than any other individual macromolecular species in the composition, on a molar or weight basis). A substantially purified composition is generally achieved when the target species constitutes at least about 50% of the macromolecular species present, on a molar or weight percent basis. Generally, a substantially pure GLA composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species present in the composition, on a molar or weight percent basis. In some embodiments, the target species is purified to essential homogeneity, where the composition consists essentially of a single macromolecular species (i.e., contaminating species cannot be detected in the composition by conventional detection methods). Solvent species, small molecules (less than 500 daltons), and elemental ion species are not considered macromolecular species. In some embodiments, an isolated recombinant GLA polypeptide is a substantially pure polypeptide composition.

[0071] As used herein, "improved enzymatic properties" refers to an engineered GLA polypeptide that exhibits improved enzymatic properties compared to a reference GLA polypeptide and / or compared to a wild-type GLA polypeptide or another engineered GLA polypeptide. Improved properties include, but are not limited to, increased gene expression, increased protein production, increased thermal activity, increased thermostability, increased activity at various pH levels, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased substrate resistance and / or product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased resistance to acidic, neutral, or basic pH, increased resistance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity, improved post-translational modifications (e.g., glycosylation), altered temperature profile, increased cellular uptake, increased lysosomal stability, increased ability to deplete Gb3 cells, and increased secretion from GLA-producing cells.

[0072] As used herein, "increased enzymatic activity" and "enhanced catalytic activity" refer to improved properties of an engineered GLA polypeptide, which can be expressed in terms of an increased specific activity (e.g., product produced / time / weight protein) or an increased substrate-to-product conversion rate (e.g., conversion rate of a starting amount of substrate to product in a specified period of time using a specified amount of GLA) compared to a reference GLA enzyme.

[0073] Exemplary methods for determining enzyme activity are presented in the Examples. m , V max or k catAny characteristic associated with enzymatic activity can be affected, including the classical enzymatic characteristics of the enzyme, and the change can result in increased enzymatic activity. The improved enzymatic activity can be about 1.1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold or more than the enzymatic activity of the corresponding wild-type enzyme, or the enzymatic activity over naturally occurring GLA or another engineered GLA from which the GLA polypeptide is derived.

[0074] In some embodiments, the engineered GLA polypeptide has a k of at least 0.1 / sec, at least 0.5 / sec, at least 1.0 / sec, at least 5.0 / sec, or at least 10.0 / sec. cat and in some preferred embodiments, k is greater than 10.0 / sec cat In some embodiments, K mis in the range of about 1 μM to about 5 mM, about 5 μM to about 2 mM, about 10 μM to about 2 mM, or about 10 μM to about 1 mM. In some specific embodiments, the engineered GLA enzyme exhibits improved enzymatic activity after exposure to certain conditions that is 1.5 to 10-fold, 1.5 to 25-fold, 1.5 to 50-fold, 1.5 to 100-fold, or more, than that of a reference GLA enzyme (e.g., wild-type GLA or any other reference GLA, e.g., SEQ ID NO: 8). GLA activity can be measured by any suitable method known in the art (e.g., standard assays such as monitoring changes in the spectrophotometric properties of reactants or products). In some embodiments, the amount of product produced can be measured directly or after o-phthaldialdehyde (OPA) derivatization by high-performance liquid chromatography (HPLC) separation coupled with UV absorbance or fluorescence detection. In some embodiments, the amount of product produced can be measured by monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm) following hydrolysis of the 4-methylumbelliferyl-alpha-D-galactopyranoside (4-MUGal) molecule. Comparisons of enzyme activity are performed using defined preparations of enzymes, defined assays under set conditions, and one or more defined substrates, as described in further detail herein. Generally, when comparing lysates, the number of cells and the amount of protein assayed are measured, and the same expression system and the same host cells are used to minimize variation in the amount of enzyme produced by the host cells and present in the lysates.

[0075] As used herein, the term "improved tolerance to acidic pH" means that the recombinant GLA according to the present invention has increased stability (retains higher activity at about pH 4.8 for a specified period of time (1 hour, up to 24 hours) after exposure to acidic pH) compared to a reference GLA or another enzyme.

[0076] As used herein, the term "improved cellular uptake" means that the recombinant GLA provided herein exhibits increased endocytosis into cells compared to a reference GLA (including wild-type GLA) or another enzyme. In some embodiments, the cells are cultured Fabry patient fibroblasts (wherein greater intracellular activity is retained over a specified period of time after incubation with the cultured cells compared to a reference GLA or another enzyme). In some further embodiments, the recombinant GLA provided herein exhibits greater intracellular activity retained over a specified period of time in cultured cells compared to a reference GLA (including wild-type GLA) or another enzyme. In some further embodiments, the period is about 4 hours, while in some other embodiments, the period is less than 4 hours (e.g., 1, 2, or 3 hours), and in some alternative embodiments, the period is greater than 4 hours (e.g., 5, 6, 7, 8, or more hours).

[0077] As used herein, "physiological pH" refers to the pH range typically found in the blood of a subject (e.g., a human).

[0078] The term "basic pH" (as used, for example, in reference to improved stability to or increased tolerance to basic pH conditions) refers to a pH range of about 7 to 11.

[0079] The term "acidic pH" (as used, for example, in reference to improved stability to or increased tolerance to acidic pH conditions) means a pH range of about 1.5 to 4.5.

[0080] As used herein, "conversion" refers to the enzymatic conversion (or biotransformation) of a substrate to a corresponding product. "Conversion rate" refers to the proportion of a substrate that is converted to a product under specific conditions within a certain period of time. Thus, the "enzyme activity" or "activity" of a GLA polypeptide can be expressed as the "conversion rate" of a substrate to a product in a specific period of time.

[0081] As used herein, "hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Generally, hybridization reactions are performed under lower stringency conditions, followed by washes of varying but higher stringency. The term "moderately stringent hybridization" refers to conditions that allow a target DNA to bind to a complementary nucleic acid having about 60% identity to the target DNA, preferably about 75% identity, about 85% identity, and more than about 90% identity to the target polynucleotide. Exemplary moderately stringent conditions are those corresponding to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "High stringency hybridization" generally refers to a condition that allows a target DNA to bind to a complementary nucleic acid having about 60% identity to the target DNA, preferably about 75% identity, about 85% identity, and more than about 90% identity to the target polynucleotide. Exemplary moderately stringent conditions are those corresponding to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. m High stringency conditions refer to conditions of from about 10°C or less. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65°C (in other words, if a hybrid is not stable in 0.018M NaCl at 65°C, it is not stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization at 42°C under conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization at 65°C in 5x SSC containing 0.1% (w:v) SDS and washing in 0.1x SSC containing 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderate stringency conditions, are described in the references cited above.

[0082] As used herein, "codon-optimized" refers to altering the codons in a polynucleotide encoding a protein so that the encoded protein is more efficiently expressed in a target organism and / or cell. While the genetic code is degenerate in that most amino acids are represented by several codons, known as "synonymous" or "alternative" codons, it is well known that codon usage by a particular organism is non-random and biased toward certain codon triplets. This codon usage bias can be higher for a given gene, for genes of common function or ancestral origin, for highly expressed proteins relative to low copy number proteins, and for aggregated protein-coding regions of an organism's genome. In some embodiments, a polynucleotide encoding a GLA enzyme can be codon-optimized for optimal production from the host organism and / or cell type selected for expression, taking into account GC content, 3' splice sites, transcription termination signals, motifs that may affect RNA stability, and nucleic acid secondary structure, as well as any other factors of interest.

[0083] As used herein, the term "control sequences" refers to all components necessary or advantageous for the expression of the polynucleotides and / or polypeptides of the present application. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, initiation sequence, and transcription terminator. At a minimum, control sequences include a promoter, and transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites to facilitate ligation of the control sequences with the coding region of the nucleic acid sequence encoding the polypeptide.

[0084] As used herein, "operably linked" refers to a configuration in which a control sequence is appropriately positioned (i.e., in a functional relationship) with a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide of interest.

[0085] As used herein, "promoter sequence" refers to a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence comprises a transcriptional control sequence that mediates the expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence, such as a mutant promoter, a truncated promoter, and a hybrid promoter, that exhibits transcriptional activity in a selected host cell, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is either homologous or heterologous to the host cell.

[0086] As used herein, "suitable reaction conditions" refer to the conditions of an enzymatic conversion reaction solution (e.g., ranges of enzyme load, temperature, pH, buffer, cosolvent, etc.) that allow the GLA polypeptide of the present application to convert a substrate into a desired product compound; exemplary "suitable reaction conditions" are provided in the present application and illustrated by the Examples. "Enzyme load" refers to the concentration or amount of a component in a reaction mixture at the start of the reaction. "Substrate" in the context of an enzymatic conversion reaction process refers to a compound or molecule upon which a GLA polypeptide acts. "Product" in the context of an enzymatic conversion process refers to a compound or molecule resulting from the action of a GLA polypeptide on a substrate.

[0087] As used herein, the term "culturing" refers to growing a population of microbial, mammalian, or other suitable cells under any suitable conditions (e.g., using a liquid, gel, or solid medium).

[0088] Recombinant polypeptides can be produced using any suitable method known in the art. A gene encoding a wild-type polypeptide of interest can be cloned into a vector such as a plasmid and expressed in a desired host, such as E. coli, Saccharomyces cerevisiae, or a mammalian cell line (e.g., HEK or CHO cells). Mutants of recombinant polypeptides can be generated by various methods known in the art. Indeed, there are a wide variety of different mutagenesis techniques known to those of skill in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available for performing specific substitutions at defined amino acids (site-directed), specific or random mutations in local regions of a gene (site-directed), or random mutagenesis throughout a gene (e.g., saturation mutagenesis). Many suitable methods for generating enzyme variants are known to those of skill in the art, including, but not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or any other suitable method known in the art. Non-limiting examples of methods used for DNA and protein engineering are set forth in the following patents: U.S. Patent No. 6,117,679; U.S. Patent No. 6,420,175; U.S. Patent No. 6,376,246; U.S. Patent No. 6,586,182; U.S. Patent No. 7,747,391; U.S. Patent No. 7,747,393; U.S. Patent No. 7,783,428; and U.S. Patent No. 8,383,346. After variants are generated, they can be screened for any desired properties (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased thermostability, and / or acidic pH stability, etc.). In some embodiments, "recombinant GLA polypeptides" (also referred to herein as "engineered GLA polypeptides," "mutant GLA enzymes," and "GLA variants") are used.

[0089] As used herein, a "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector operably linked to a suitable control sequence capable of effecting expression of a polypeptide encoded by the DNA sequence in a suitable host. In some embodiments, an "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0090] As used herein, the term "gene therapy vector" refers to a vehicle or carrier suitable for delivering a polynucleotide sequence to a cell. In some embodiments, the vector comprises a gene (e.g., a therapeutic gene) or polynucleotide sequence for delivery to a cell or tissue, including, but not limited to, non-viral vectors such as adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), and liposomes. It is not intended that the present invention be limited to any particular gene therapy vector; any vehicle suitable for a given setting may be used. Gene therapy vectors may be designed to deliver genes to a specific species or host, or may find more general applicability.

[0091] As used herein, the term "expression" includes any step involved in producing a polypeptide, such as, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0092] As used herein, the term "producing" refers to the production of a protein and / or other compound by a cell. The term is intended to encompass any step involved in the production of a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses the secretion of a polypeptide from the cell.

[0093] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is "recombinant" or "heterologous" to another sequence to which it is operably linked if the two sequences are not naturally linked.

[0094] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector containing DNA (e.g., a polynucleotide encoding a GLA variant) provided herein. In some embodiments, a host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0095] The term "analog" refers to a polypeptide having greater than 70% sequence identity to a reference polypeptide, but less than 100% sequence identity (e.g., greater than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity). In some embodiments, "analog" refers to a polypeptide that includes one or more non-natural amino acid residues, such as, but not limited to, homoarginine, ornithine, and norvaline, as well as natural amino acids. In some embodiments, an analog also includes one or more D-amino acid residues and a non-peptide bond between two or more amino acid residues.

[0096] As used herein, the term "therapeutic" refers to a compound having a beneficial or desired medical effect that is administered to a subject exhibiting signs or symptoms of a pathology.

[0097] As used herein, the term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., a human) comprising a pharmaceutically effective amount of an engineered GLA polypeptide encompassed by the present invention and an acceptable carrier.

[0098] As used herein, the term "gene therapy" refers to the delivery of a gene, polydeoxyribonucleotide, or polynucleotide sequence to a cell or tissue using a gene therapy vector to modify the cell or tissue for the treatment of disease prevention. Gene therapy can involve replacing a disease-causing mutant gene with a healthy copy of the gene, or inactivating or "knocking out" an improperly functioning mutant gene. In some embodiments, gene therapy is used to treat a patient's disease.

[0099] As used herein, the term "mRNA therapy" refers to the delivery of mRNA polyribonucleotide sequences to cells or tissues to modify the cells or tissues in order to treat or prevent disease. In some embodiments, the mRNA polynucleotide sequences for delivery to cells or tissues are formulated, for example, but not limited to, in liposomes. In some embodiments, mRNA therapy is used to treat disease in patients.

[0100] As used herein, the term "cell therapy" refers to the delivery of exogenously modified living cells to a patient to provide a defective gene to treat or prevent disease, where the modified cells are then reintroduced into the body.

[0101] As used herein, the term "effective amount" means an amount sufficient to bring about a desired result. One of ordinary skill in the art can determine an effective amount using routine experimentation.

[0102] The terms "isolated" and "purified" are used herein to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that has been removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but rather is intended as a relative definition.

[0103] As used herein, the term "subject" includes mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagomorphs). The term is intended to include both males and females.

[0104] As used herein, the term "patient" means any subject being evaluated for, treated for, or experiencing a disease.

[0105] The term "infant" refers to a child between the ages of 1 month and about 1 year. As used herein, the term "newborn" refers to a child up to 28 days after birth. The term "premature infant" refers to an infant born after the completion of the 20th week of pregnancy but before full term, and generally weighs from about 500 to about 2499 grams at birth. A "very low birth weight infant" is an infant weighing less than 1500 grams at birth.

[0106] As used herein, the term "child" refers to a person who has not reached the legal age for consent to a treatment or research procedure. In some embodiments, the term refers to a person between birth and adolescence.

[0107] As used herein, the term "adult" refers to a person who has reached the legal age of the relevant jurisdiction (e.g., 18 years of age in the United States). In some embodiments, the term refers to any fully grown, mature organism. In some embodiments, the term "young adult" refers to a person who is under the age of 18 but has reached sexual maturity.

[0108] As used herein, "composition" and "formulation" encompass products containing at least one engineered GLA of the present invention intended for any suitable use (e.g., pharmaceutical composition, dietary / nutraceutical supplement, feed, etc.).

[0109] As used herein, the terms "administration" and "administering" a composition means giving a composition of the invention to a subject (e.g., a person affected by Fabry disease).

[0110] As used herein, the term "carrier" when used in reference to a pharmaceutical composition means any of the standard pharmaceutical carriers, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.

[0111] As used herein, the term "pharmaceutically acceptable" means a material that may be administered to a subject without causing undesired biological effects or interacting in a deleterious manner with any of the components it contains, and that possesses the desired biological activity.

[0112] As used herein, the term "excipient" refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than the active pharmaceutical ingredient (API, e.g., an engineered GLA polypeptide of the invention). Excipients are typically included for formulation and / or administration purposes.

[0113] As used herein, the term "therapeutically effective amount" when used in reference to symptoms of a disease / condition refers to an amount and / or concentration of a compound (e.g., an engineered GLA polypeptide) that ameliorates, attenuates, or eliminates one or more symptoms of the disease / condition, or prevents or delays the onset of the symptoms.

[0114] As used herein, the term "therapeutically effective amount" when used with respect to a disease / condition refers to an amount and / or concentration of a composition (e.g., an engineered GLA polypeptide) that ameliorates, attenuates, or eliminates the disease / condition. In some embodiments, the term is used with respect to the amount of a composition that elicits a biological (e.g., medical) response by a tissue, system, or animal subject that is desired by a researcher, physician, veterinarian, or other clinician.

[0115] The terms "treating," "treat," and "treatment" are intended to encompass preventative (e.g., prophylactic) treatment as well as palliative treatment. Engineered GLA expression and activity

[0116] Secretory expression of yeast-codon-optimized mature human GLA was achieved using a synthetic mouse IG signal peptide. Clones were expressed from the pCDNA3.1(+) vector in HEK293T cells. This approach yielded supernatants with measurable activity toward the fluorogenic substrate 4-methylumbelliferyl α-D-galactopyranoside (4-MuGal).

[0117] In some embodiments, a combinatorial library was constructed to generate GLA variants derived from SEQ ID NO:8 to identify mutational diversity with similar stability and improved cellular uptake compared to SEQ ID NO:8. Equal amounts of supernatant were screened under unchallenged conditions (no incubation, pH 4.6) or after 1 hour of incubation in low pH (3.9-4.2), neutral pH (7.0-7.6), or human serum (physiological pH 7.1-8.2) environments. GLA variants with activity due to increased GLA expression or GLA specific activity were identified based on their fold improvement relative to the parent GLA. GLA variants with increased stability were identified by dividing the fold improvement observed under challenged conditions by the fold improvement observed under unchallenged conditions. This approach reduces the bias of selecting variants based on increased expression but no change in specific activity at extreme pH. A composite activity score (product of the fold improvement for all three conditions) and stability (product of the stability scores) were used to rank the mutations in the improved variants for subsequent inclusion in the GLA library. In further embodiments, additional methods and sequences described in the Examples were used. Engineered GLA

[0118] In some embodiments, the engineered GLA exhibiting improved properties has at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% amino acid sequence identity to SEQ ID NO: 2, 8, 48, 158, 372, 374, 704, and / or 1022, and has an amino acid residue difference at at least one or more amino acid positions when compared to SEQ ID NO: 2, 8, 48, 158, 372, 374, 704, and / or 1022. (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 20 or more amino acid positions compared to SEQ ID NOs: 2, 8, 48, 158, 372, 374, 704 and / or 1022, or sequences having at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more amino acid sequence identity to SEQ ID NOs: 2, 8, 48, 158, 372, 374, 704 and / or 1022.) In some embodiments, the residue differences compared to SEQ ID NO: 5 include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions at one or more positions. In some embodiments, the engineered GLA polypeptide is a polypeptide set forth in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and / or 13-1. In some embodiments, the engineered GLA polypeptide comprises SEQ ID NOs: 2, 8, 48, 158, 372, 374, 704, and / or 1022.

[0119] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 44, 44 / 217, 44 / 217 / 316, 44 / 217 / 322, 44 / 217 / 322 / 337, 44 / 247, 44 / 247 / 302, 44 / 247 / 302 302 / 322 / 362 / 373, 302 / 337, 316, 316 / 337, 322, 322 / 337, 362 / 373, and 373, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 44L, 44L / 217F, 44L / 217F / 316L, 44L / 217F / 322M, 44L / 217F / 322M / 337A, 44L / 247N, 44L / 247N / 302Q, 44L / 247N / 302Q / 322M, 44 and 373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of R44L, R44L / R217F, R44L / R217F / D316L, R44L / R217F / I322M, R44L / R217F / I322M / P337A, R44L / D247N, R44L / D247N / K302Q, R44L / D247N / K302Q / I322M, R44L / D247N / I322M, R44L / D247N / P337A, R44L / D247N / Q362K, R44L / K302Q, R44L / P337A, R44L / K373R, R217F / I322M, R217F / K373R, D247N / I322M, D247N / Q362K, K302Q / I322M / Q362K / K373R, K3 and at least one substitution or set of substitutions at one or more positions selected from O2Q / P337A, D316L, D316L / P337A, I322M, I322M / P337A, Q362K / K373R, and K373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.

[0120] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10 / 39 / 44 / 47 / 92 / 166 / 206 / and at least one substitution or set of substitutions at one or more positions selected from 217 / 247 / 261 / 271 / 302 / 316 / 322 / 337 / 362 / 368 / 373 / 392, 44 / 217 / 316, 44 / 217 / 322 / 337, 166 / 362, 217 / 373, and 362 / 373, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10T / 39M / 44L / 47S / 92Y / 166S / 206K / 217F / 24 and at least one substitution or set of substitutions at one or more positions selected from 7N / 261A / 271H / 302Q / 316L / 322M / 337A / 362K / 368W / 373R / 392M, 44L / 217F / 316L, 44L / 217F / 322M / 337A, 166A / 362K, 217F / 373R, and 362K / 373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is and at least one substitution or set of substitutions at one or more positions selected from A / A271H / K302Q / D316L / I322M / P337A / Q362K / A368W / K373R / T392M, R44L / R217F / D316L, R44L / R217F / I322M / P337A, P166A / Q362K, R217F / K373R, and Q362K / K373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.

[0121] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 7, 7 / 48 / 68, 7 / 48 / 68 / 120 / 282 / 299, 7 / 48 / 130 / 282, 7 / 48 / 180, 7 / 68 / 130 / 282 / 365, 7 / 68 / 180, 7 / 88 / 120 / 305 / 365, 7 / 120, 7 / 130, 7 / 282, 7 / 305, 7 / 305 / 365, 7 / 365, 39, 47, 47 / 87 / 95 / 96 / 158 / 162, 47 / 95, 47 / 273, 47 / 343, 48, 48 / 68, 48 / 180 / 282, 48 / 282, 48 / 282 / 305, 67 / 180, 68, 68 / 299 / 300, 71, 87 / 9 1 / 95 / 96 / 158 / 162, 87 / 91 / 95 / 96 / 206 / 343, 87 / 96 / 155 / 273 / 343, 88, 91 / 95, 91 / 95 / 96, 92, 93, 96, 96 / 273, 96 / 312 / 343, 120, 120 / 299 / 305, 151, 158, 158 / 162 / 273, 162, 162 / 273, 162 / 343, 166, 178, 180, 181, 206, 217, 271, 273, 273 / 343, 282, 282 / 3 and at least one substitution or set of substitutions at one or more positions selected from: 65, 293 / 391, 299 / 300, 299 / 300 / 305 / 365, 300, 301, 305, 305 / 365, 314, 333, 336, 337, 343, 345, 363, 365, 370, 389, 393, 394, 396 / 398, 397, and 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:58. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 7L, 7L / 48D / 68E ...7L / 48D / 68E / 120H / 282N / 299R, 7L / 48D / 130E / 282N, 7L / 48D / 180G, 7L / 68E / 130E / 282N / 365V, 7L / 68E / 180G, 7L / 88A / 120H / 305G / 365V , 7L / 120H, 7L / 130E, 7L / 282N, 7L / 305G, 7L / 305G / 365V, 7L / 365V, 39V, 47D, 47D / 87K / 95E / 96L / 158R / 162H, 47D / 95E, 47D / 273P, 47D / 34 3G, 47V, 48D, 48D / 68E, 48D / 180G / 282N, 48D / 282N, 48D / 282N / 305G, 67T / 180G, 68E, 68E / 299R / 300I, 71P, 87K / 91Q / 95E / 96L / 158A / 162 K, 87K / 91Q / 95E / 96L / 206S / 343G, 87K / 96I / 155N / 273P / 343G, 88A, 91Q / 95E, 91Q / 95E / 96L, 92F, 92T, 93I, 96L, 96L / 273P, 96L / 312Q / 34 3G, 120H, 120H / 299R / 305G, 151L, 158A, 158A / 162K / 273G, 158R, 162H / 343D, 162K, 162K / 273P, 162S, 166K, 178G, 178S, 180G, 180L, 18 0T, 180V, 181A, 206K, 206S, 217K, 271R, 273P, 273P / 343G, 282N, 282N / 365V, 293P / 391A, 299R / 300I, 299R / 300I / 305G / 365V, 300I, 301 M, 305G, 305G / 365V, 314A, 333F, 333G, 336V, 337R, 343D, 343G, 345A, 345Q, 363Q, 365A, 365Q, 365V, 370G, 389K, 393V, 394K, 396G / 398T, 397A, 398A, 398P, 398S, and 398V, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:58.

[0122] In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of R7L, R7L / E48D / Q68E, R7L / E48D / Q68E / Y120H / D282N / Q299R, R7L / E48D / D130E / D282N, R7L / E48D / F180G ... 7L / Q68E / D130E / D282N / F365V, R7L / Q68E / F180G, R7L / Q88A / Y120H / N305G / F365V, R7L / Y120H, R7L / D130E, R7L / D282N, R7L / N305G, R7L / N305G / F365V, R 7L / F365V, E39V, T47D, T47D / R87K / S95E / K96L / L158R / R162H, T47D / S95E, T 47D / S273P, T47D / K343G, T47V, E48D, E48D / Q68E, E48D / F180G / D282N, E48D / D282N, E48D / D282N / N305G, P67T / F180G, Q68E, Q68E / Q299R / L300I, S71P, R 87K / N91Q / S95E / K96L / L158A / R162K, R87K / N91Q / S95E / K96L / A206S / K343G, R87K / K96I / H155N / S273P / K343G, Q88A, N91Q / S95E, N91Q / S95E / K96L, H92F , H92T, V93I, K96L, K96L / S273P, K96L / P312Q / K343G, Y120H, Y120H / Q299R / N 305G, D151L, L158A, L158A / R162K / S273G, L158R, R162H / K343D, R162K, R16 2K / S273P, R162S, P166K, W178G, W178S, F180G, F180L, F180T, F180V, Q181A, A206K, A206S, R217K, A271R, S273P, S273P / K343G, D282N, D282N / F365V, L29 3P / Q391A, Q299R / L300I, Q299R / L300I / N305G / F365V, L300I, R301M, N305G,and at least one substitution or set of substitutions at one or more positions selected from N305G / F365V, S314A, S333F, S333G, I336V, P337R, K343D, K343G, V345A, V345Q, L363Q, F365A, F365Q, F365V, S370G, T389K, S393V, L394K, D396G / L398T, L397A, L398A, L398P, L398S, and L398V, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 58.

[0123] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 158, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 24 / 202, 39 / 47, 39 / 47 / 217, 39 / 151, 39 / 282 / 337 / 398, 39 / 337 / 343 / 398, 39 / 393 / 398, 47 / 130, 47 / 151, 47 / 343 / 345 / 393, 48, 48 / 68, 48 / 68 / 217 / 333 / 391 / 393, 48 / 68 / 333, 48 / 217, 48 / 333, 48 / 345 / 393, 48 / 393, 59 / 143, 68, 68 / 345, 130, 130 / 158, 130 / 158 / 393, 130 / 345 / 393, 143 / 271, 143 / 333, 143 / 387, 151, 151 / 158 / 217 / 343 / 345 / 393, 151 / 206 / 282 / 337 / 343 / 345 / 398, 151 / 282 / 393, 151 / 345 / 393 / 398, 151 / 393, 158, 158 / 393, 202, 206, 206 / 217, 217, 21 and 393 / 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 158.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 158, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 24S / 202N, 39V / 47D, 39V / 47V / 217K, 39V / 151L, 39V / 282N / 337R / 398A, 39V / 337R / 343G / 398A, 39V / 393V / 398A, 47V / 130E, 47V / 151L, 47V / 343D / 345Q / 393V, 48D, 48D / 68E, 48D / 68E / 217K / 333F / 391 A / 393V, 48D / 68E / 333F, 48D / 217K, 48D / 333F, 48D / 333G, 48D / 345Q / 393V, 48D / 393V, 59A / 143S, 68E, 68E / 345Q, 130E, 130E / 158R, 13 0E / 158R / 393V, 130E / 345Q / 393V, 143S / 271N, 143S / 333N, 143S / 387N, 151L, 151L / 158R / 217K / 343G / 345Q / 393V, 151L / 206S / 282N / 337R / 343D / 345Q / 398A, 151L / 282N / 393V, 151L / 345Q / 393V / 398A, 151L / 393V, 158R, 158R / 393V, 202N, 206S, 206S / 217K, 217K, 217K / 333F, 217K / 333G, 217K / 337R / 345Q / 398A, 271N, 282N / 393V, 333F / 345Q, 333G, 333N, 337R / 343G / 345Q / 398A, 343D, 343D / 345Q / 393V / 398A, 393V, and 393V / 398A, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 158.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 158, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of D24S / D202N, E39V / T47D, E39V / T47V / R217K, E39V / D151L, E39V / D282N / P337R / L398A, E39V / P337R / K34 3G / L398A, E39V / S393V / L398A, T47V / D130E, T47V / D151L, T47V / K343D / V34 5Q / S393V, E48D, E48D / Q68E, E48D / Q68E / R217K / S333F / Q391A / S393V, E48D / Q68E / S333F, E48D / R217K, E48D / S333F, E48D / S333G, E48D / V345Q / S393V, E48D / S393V, C59A / C143S, Q68E, Q68E / V345Q, D130E, D130E / L158R, D130E / L 158R / S393V, D130E / V345Q / S393V, C143S / A271N, C143S / S333N, C143S / E38 7N, D151L, D151L / L158R / R217K / K343G / V345Q / S393V, D151L / A206S / D282N / P337R / K343D / V345Q / L398A, D151L / D282N / S393V, D151L / V345Q / S393V / L 398A, D151L / S393V, L158R, L158R / S393V, D202N, A206S, A206S / R217K, R217 and S393V / L398A, and wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 158.

[0124] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 372, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10, 10 / 39 / 44 / 322, 10 / 39 / 92 / 206 / 217 / 271, 10 / 39 / 92 / 247, 10 / 39 / 92 / 247 / 271 / 316, 10 / 44, 10 / 44 / 47 / 9 2 / 247, 10 / 44 / 47 / 261 / 302 / 322 / 368, 10 / 44 / 92 / 316 / 322, 10 / 44 / 261 / 302 / 316, 10 / 44 / 302 / 337 / 368, 10 / 47 / 217 / 247 / 316 / 392, 10 / 47 / 217 / 322, 10 / 4 7 / 271, 10 / 92, 10 / 92 / 206 / 217 / 247, 10 / 92 / 206 / 247 / 316 / 322 / 392, 10 / 92 / 206 / 247 / 322 / 368, 10 / 92 / 217 / 261 / 302 / 337, 10 / 206 / 217 / 271, 10 / 206 / 247 , 10 / 206 / 261 / 271 / 316, 10 / 261, 10 / 271 / 302, 10 / 302, 10 / 302 / 316, 10 / 302 / 322 / 337, 10 / 316 / 322, 10 / 337 / 392, 10 / 368, 39 / 44 / 92 / 162 / 247 / 302 / 316 / 322, 39 / 44 / 92 / 217 / 322, 39 / 44 / 92 / 247 / 271 / 302, 39 / 47 / 92 / 247 / 302 / 316 / 322, 39 / 47 / 217 / 247 / 368, 39 / 47 / 247, 39 / 92 / 247 / 302 / 316 / 337 / 368, 39 / 92 / 316 / 322, 39 / 247 / 271, 39 / 247 / 271 / 316, 39 / 322, 44 / 47 / 92 / 206 / 217 / 316 / 322, 44 / 47 / 92 / 247 / 261 / 271 / 316 / 337 / 368, 44 / 47 / 206 / 217 / 247 / 271 / 322, 44 / 47 / 247 / 322 / 368, 44 / 47 / 302 / 316 / 322, 44 / 92 / 206 / 247 / 368, 44 / 206 / 337, 44 / 247 / 261 / 302 / 316, 44 / 247 / 261 / 302 / 316 / 322, 47 / 92 / 247 / 271,47 / 217 / 302, 47 / 247, 47 / 247 / 271, 89 / 217 / 247 / 261 / 302 / 316, 92 / 217 / 271, 92 / 247, 92 / 247 / 271 / 322, 92 / 247 / 302 / 322 / 337, 92 / 271 / 337, 92 / 302, 92 / 316, 206 / 217 / 271 / 392, 217 / 247 / 316 / 32 and 368, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 372. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 372, or a functional fragment thereof, and said recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from SEQ ID NO: 372, 247, 247 / 271, 247 / 302, 271, 271 / 302 / 322, 271 / 316 / 322, 302 / 322 / 368, and 368, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 372. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 372, or a functional fragment thereof, and wherein said recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from SEQ ID NO: 372, 247, 247 / 271, 247 / 302, 271, 271 / 302 / 322, 271 / 316 / 322, 302 / 322 / 368, and 368, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 372. P, 10P / 39E / 44R / 322I, 10P / 39E / 92H / 206A / 217R / 271A, 10P / 39E / 92H / 247D, 10P / 39E / 92H / 247D / 271 A / 316D, 10P / 44R, 10P / 44R / 47T / 92H / 247D, 10P / 44R / 47T / 261G / 302K / 322I / 368A, 10P / 44R / 92H / 316D / 322I, 10P / 44R / 261G / 302K / 316D, 10P / 44R / 302K / 337P / 368A, 10P / 47T / 217R / 247D / 316D / 392T, 10P / 47T / 217R / 322I, 10P / 47T / 271A, 10P / 92H, 10P / 92H / 206A / 217R / 247D, 10P / 92H / 206A / 247D / 316D / 32 2I / 392T, 10P / 92H / 206A / 247D / 322I / 368A, 10P / 92H / 217R / 261G / 302K / 337P, 10P / 206A / 217R / 271A, 10P / 206A / 247D, 10P / 206A / 261G / 271A / 316D, 10P / 261G, 10P / 271A / 302K, 10P / 302K, 10P / 302K / 316D,10P / 302K / 322I / 337P, 10P / 316D / 322I, 10P / 337P / 392T, 10P / 368A, 39E / 44R / 92H / 162M / 247D / 302K / 316D / 322I, 39E / 44R / 92H / 217R / 322I, 39E / 44R / 92H / 247D / 271A / 302K, 39E / 47T / 92H / 247D / 302K / 316D / 322I, 39E / 47T / 217R / 247D / 368A, 39E / 47T / 247D, 39E / 92H / 247D / 302K / 316D / 337 P / 368A, 39E / 92H / 316D / 322I, 39E / 247D / 271A, 39E / 247D / 271A / 316D, 3 9E / 322I, 44R / 47T / 92H / 206A / 217R / 316D / 322I, 44R / 47T / 92H / 247D / 26 1G / 271A / 316D / 337P / 368A, 44R / 47T / 206A / 217R / 247D / 271A / 322I, 44R / 47T / 247D / 322I / 368A, 44R / 47T / 302K / 316D / 322I, 44R / 92H / 206A / 247 D / 368A, 44R / 206A / 337P, 44R / 247D / 261G / 302K / 316D, 44R / 247D / 261G / 302K / 316D / 322I, 47T / 92H / 247D / 271A, 47T / 217R / 302K, 47T / 247D, 47T / 247D / 271A, 89I / 217R / 247D / 261G / 302K / 316D, 92H / 217R / 271A, 92H / 2 47D, 92H / 247D / 271A / 322I, 92H / 247D / 302K / 322I / 337P, 92H / 271A / 337

[0023] In some embodiments, the recombinant alpha-galactosidase A has at least one substitution or set of substitutions at one or more positions selected from SEQ ID NO: 372, ...and a polypeptide sequence having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the recombinant alpha-galactosidase A, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of T10P, T10P / M39E / L44R / M322I, T10P / M39E / Y92H / K206A / F217R / H271A, T10P / M39E / Y92H / N247D, T10P / M39E / Y92H / N247D / H271A / L316D, T10P / L44R, T10P / L44R / S4 7T / Y92H / N247D, T10P / L44R / S47T / A261G / Q302K / M322I / W368A, T10P / L44 R / Y92H / L316D / M322I, T10P / L44R / A261G / Q302K / L316D, T10P / L44R / Q302 K / A337P / W368A, T10P / S47T / F217R / N247D / L316D / M392T, T10P / S47T / F21 7R / M322I, T10P / S47T / H271A, T10P / Y92H, T10P / Y92H / K206A / F217R / N247D , T10P / Y92H / K206A / N247D / L316D / M322I / M392T, T10P / Y92H / K206A / N247 D / M322I / W368A, T10P / Y92H / F217R / A261G / Q302K / A337P, T10P / K206A / F2 17R / H271A, T10P / K206A / N247D, T10P / K206A / A261G / H271A / L316D, T10P / A261G, T10P / H271A / Q302K, T10P / Q302K, T10P / Q302K / L316D, T10P / Q302K / M322I / A337P, T10P / L316D / M322I, T10P / A337P / M392T, T10P / W368A, M39E / L44R / Y92H / R162M / N247D / Q302K / L316D / M322I, M39E / L44R / Y92H / F217R / M322I, M39E / L44R / Y92H / N247D / H271A / Q302K, M39E / S47T / Y92H / N247D / Q302K / L316D / M322I, M39E / S47T / F217R / N247D / W368A, M39E / S47T / N247D,M39E / Y92H / N247D / Q302K / L316D / A337P / W368A, M39E / Y92H / L316D / M322I, M39E / N247D / H271A, M39E / N247D / H271A / L316D, M39E / M 322I, L44R / S47T / Y92H / K206A / F217R / L316D / M322I, L44R / S47T / Y92H / N247D / A261G / H271A / L316D / A337P / W368A, L44R / S47T / K206 A / F217R / N247D / H271A / M322I, L44R / S47T / N247D / M322I / W368A, L44R / S47T / Q302K / L316D / M322I, L44R / Y92H / K206A / N247D / W368A , L44R / K206A / A337P, L44R / N247D / A261G / Q302K / L316D, L44R / N247D / A261G / Q302K / L316D / M322I, S47T / Y92H / N247D / H271A, S47T / F217R / Q302K, S47T / N247D, S47T / N247D / H271A, L89I / F217R / N247D / A261G / Q302K / L316D, Y92H / F217R / H271A, Y92H / N247D, Y92H / N 247D / H271A / M322I, Y92H / N247D / Q302K / M322I / A337P, Y92H / H271A / A337P, Y92H / Q302K, Y92H / L316D, K206A / F217R / H271A / M392T, and at least one substitution or set of substitutions at one or more positions selected from F217R / N247D / L316D / M322I / A337P / W368A, N247D, N247D / H271A, N247D / Q302K, H271A, H271A / Q302K / M322I, H271A / L316D / M322I, Q302K / M322I / W368A, and W368A, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 372.

[0125] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10 / 36 / 92 / 166 / 247 / 261 / 316 / 392, 10 / 39, 10 / 39 / 44 / 47 / 92 / 206 / 217, 10 / 39 / 44 / 47 / 316, 10 / 39 / 44 / 4 7 / 337, 10 / 39 / 44 / 92 / 166 / 261 / 316 / 322, 10 / 39 / 44 / 92 / 166 / 302 / 322, 10 / 39 / 44 / 92 / 166 / 392, 10 / 39 / 44 / 92 / 217 / 302 / 322, 10 / 39 / 44 / 92 / 302 / 322, 1 0 / 39 / 44 / 166 / 261 / 271 / 316 / 322, 10 / 39 / 44 / 392, 10 / 39 / 47 / 92 / 337, 10 / 39 / 92 / 131 / 166 / 271 / 316 / 322, 10 / 39 / 92 / 166 / 217 / 247 / 271, 10 / 39 / 92 / 217 / 316, 10 / 44 / 47 / 166 / 261 / 271, 10 / 44 / 47 / 166 / 271 / 322 / 368, 10 / 44 / 47 / 217 / 271 / 316 / 322, 10 / 44 / 92, 10 / 44 / 92 / 217 / 247 / 271 / 302 / 316 / 392, 10 / 44 / 166 / 302, 10 / 44 / 206 / 316 / 322, 10 / 47 / 92 / 166 / 271 / 316 / 337, 10 / 47 / 92 / 271 / 302, 10 / 47 / 92 / 316 / 322 / 392, 10 / 47 / 166 / 271, 10 / 47 / 166 / 316, 10 / 92 / 166, 10 / 92 / 166 / 217 / 247 / 261 / 271, 10 / 92 / 166 / 261 / 271 / 392, 10 / 92 / 166 / 261 / 316 / 322 / 337, 10 / 92 / 166 / 337 / 368, 10 / 92 / 302 / 337, 10 / 92 / 316 / 32 2, 10 / 206, 10 / 206 / 247 / 261, 10 / 217 / 322, 10 / 261, 10 / 261 / 337 / 392, 10 / 316 / 392, 10 / 368, 39 / 44 / 47 / 92 / 166 / 206 / 392, 39 / 44 / 47 / 92 / 206 / 247 / 261,39 / 44 / 47 / 92 / 206 / 392, 39 / 44 / 47 / 206 / 337 / 368 / 392, 39 / 44 / 92 / 166 / 247 / 261 / 302 / 337, 39 / 44 / 166 / 271, 39 / 44 / 166 / 271 / 337 / 368 / 392, 39 / 47 / 92 / 316 / 322, 39 / 47 / 92 / 392, 39 / 47 / 166 / 217 / 261 / 392, 39 / 47 / 217 / 247 / 368, 39 / 47 / 247, 39 / 92 / 166 / 217 / 392, 39 / 92 / 261 / 302, 39 / 166 / 217 / 261 / 316 / 368, 39 / 322, 39 / 392, 44 / 47, 44 / 47 / 92 / 217 / 271, 44 / 47 / 92 / 217 / 316 / 322 / 392, 44 / 47 / 92 / 392, 44 / 47 / 166, 44 / 47 / 166 / 271, 44 / 47 / 247 / 271 / 392, 44 / 316 / 322 / 392, 44 / 337, 47 / 166 / 206 / 217 / 247 / 337, 47 / 166 / 217 / 271 / 337, 47 / 206, 47 / 217 / 247 / 261, 47 / 271, 52 / 217 / 302 / 316, 92 / 166 / 206 / 271 / 316, 92 / 166 / 217 / 261 / 271 / 392, 92 / 166 / 217 / 316 / 337 / 392, 92 / 166 / 247, 92 / 166 / 316, 92 / 206 / 322, 92 / 217, 92 / 217 / 271 / 337, 92 / 2 and at least one substitution or set of substitutions at one or more positions selected from 61 / 271, 92 / 271, 166 / 217 / 316 / 322 / 337, 166 / 247 / 271 / 316, 166 / 316 / 322 / 337, 206 / 217, 217 / 392, 247 / 316, 316 / 322 / 368, and 316 / 337 / 392, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10T / 36M / 92Y / 166S / 247N / 261A / 316L / 392M,10T / 39M、10T / 39M / 44L / 47S / 92Y / 206K / 217F、10T / 39M / 44L / 47S / 316L、10T / 39M / 44L / 47S / 337A、10T / 39M / 44L / 92Y / 166S / 261A / 316L / 322M、10T / 39M / 44L / 92Y / 166S / 302Q / 322M、10T / 39M / 44L / 92Y / 166S / 392M、10T / 39M / 44L / 92Y / 217F / 302Q / 322M、10T / 39M / 44L / 92Y / 302Q / 322M、10T / 39M / 44L / 166S / 261A / 271H / 316L / 322M、10T / 39M / 44L / 392M、10T / 39M / 47S / 92Y / 337A、10T / 39M / 92Y / 131G / 166S / 271H / 316L / 322M、10T / 39M / 92Y / 166S / 217F / 247N / 271H、10T / 39M / 92Y / 217F / 316L、10T / 44L / 47S / 166S / 261A / 271H、10T / 44L / 47S / 166S / 271H / 322M / 368W、10T / 44L / 47S / 217F / 271H / 316L / 322M、10T / 44L / 92Y、10T / 44L / 92Y / 217F / 247N / 271H / 302Q / 316L / 392M、10T / 44L / 166S / 302Q、10T / 44L / 206K / 316L / 322M、10T / 47S / 92Y / 166S / 271H / 316L / 337A、10T / 47S / 92Y / 271H / 302Q、10T / 47S / 92Y / 316L / 322M / 392M、10T / 47S / 166S / 271H、10T / 47S / 166S / 316L、10T / 92Y / 166S、10T / 92Y / 166S / 217F / 247N / 261A / 271H、10T / 92Y / 166S / 261A / 271H / 392M、10T / 92Y / 166S / 261A / 316L / 322M / 337A、10T / 92Y / 166S / 337A / 368W、10T / 92Y / 302Q / 337A、10T / 92Y / 316L / 322M、10T / 206K、10T / 206K / 247N / 261A、10T / 217F / 322M、10T / 261A、10T / 261A / 337A / 392M、10T / 316L / 392M、10T / 368W、39M / 44L / 47S / 92Y / 166S / 206K / 392M、39M / 44L / 47S / 92Y / 206K / 247N / 261A, 39M / 44L / 47S / 92Y / 206K / 392M, 39M / 44L / 47S / 206K / 337A / 368W / 392M, 39M / 44L / 92Y / 166S / 247N / 261A / 302Q / 3 37A, 39M / 44L / 166S / 271H, 39M / 44L / 166S / 271H / 337A / 368W / 392M, 39M / 47 S / 92Y / 316L / 322M, 39M / 47S / 92Y / 392M, 39M / 47S / 166S / 217F / 261A / 392M, 3 9M / 47S / 217F / 247N / 368W, 39M / 47S / 247N, 39M / 92Y / 166S / 217F / 392M, 39M / 92Y / 261A / 302Q, 39M / 166S / 217F / 261A / 316L / 368W, 39M / 322M, 39M / 392M, 44L / 47S, 44L / 47S / 92Y / 217F / 271H, 44L / 47S / 92Y / 217F / 316L / 322M / 392M , 44L / 47S / 92Y / 392M, 44L / 47S / 166S, 44L / 47S / 166S / 271H, 44L / 47S / 247N / 271H / 392M, 44L / 316L / 322M / 392M, 44L / 337A, 47S / 166S / 206K / 217F / 247N / 337A, 47S / 166S / 217F / 271H / 337A, 47S / 206K, 47S / 217F / 247N / 261A, 47S / 271H, 52N / 217F / 302Q / 316L, 92Y / 166S / 206K / 271H / 316L, 92Y / 166S / 217 F / 261A / 271H / 392M, 92Y / 166S / 217F / 316L / 337A / 392M, 92Y / 166S / 247N, 92 comprising at least one substitution or set of substitutions at one or more positions selected from Y / 166S / 316L, 92Y / 206K / 322M, 92Y / 217F, 92Y / 217F / 271H / 337A, 92Y / 261A / 271H, 92Y / 271H, 166S / 217F / 316L / 322M / 337A, 166S / 247N / 271H / 316L, 166S / 316L / 322M / 337A, 206K / 217F, 217F / 392M, 247N / 316L, 316L / 322M / 368W, and 316L / 337A / 392M;The amino acid positions of the polypeptide sequences are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of P10T / K36M / H92Y / P166S / D247N / G261A / D316L / T392M, P10T / E39M, P10T / E39M / R 44L / T47S / H92Y / A206K / R217F, P10T / E39M / R44L / T47S / D316L, P10T / E39M / R44L / T47S / P337A, P10T / E39M / R44L / H92Y / P166S / G261A / D316L / I322M, P10T / E39M / R44L / H92Y / P166S / K302Q / I322M, P10T / E39M / R44L / H92Y / P166S / T392M, P10T / E39M / R44L / H92Y / R217F / K302Q / I322M, P 10T / E39M / R44L / H92Y / K302Q / I322M, P10T / E39M / R44L / P166S / G261A / A271H / D316L / I322M, P10T / E39M / R44L / T392M, P10T / E39M / T47S / H92 Y / P337A, P10T / E39M / H92Y / W131G / P166S / A271H / D316L / I322M, P10T / E39M / H92Y / P166S / R217F / D247N / A271H, P10T / E39M / H92Y / R217F / D3 16L, P10T / R44L / T47S / P166S / G261A / A271H, P10T / R44L / T47S / P166S / A271H / I322M / A368W, P10T / R44L / T47S / R217F / A271H / D316L / I322M, P10T / R44L / H92Y, P10T / R44L / H92Y / R217F / D247N / A271H / K302Q / D316L / T392M, P10T / R44L / P166S / K302Q, P10T / R44L / A206K / D316L / I322M,P10T / T47S / H92Y / P166S / A271H / D316L / P337A、P10T / T47S / H92Y / A271H / K302Q、P10T / T47S / H92Y / D316L / I322M / T392M、P10T / T47S / P166S / A271H、P10T / T47S / P166S / D316L、P10T / H92Y / P166S、P10T / H92Y / P166S / R217F / D247N / G261A / A271H、P10T、 / H92Y / P166S / G261A / A271H / T392M、P10T / H92Y / P166S / G261A / D316L / I3 22M / P337A、P10T / H92Y / P166S / P337A / A368W、P10T / H92Y / K302Q / P337A、P 10T / H92Y / D316L / I322M、P10T / A206K、P10T / A206K / D247N / G261A、P10T / R 217F / I322M、P10T / G261A、P10T / G261A / P337A / T392M、P10T / D316L / T392M P10T / A368W, E39M / R44L / T47S / H92Y / P166S / A206K / T392M, E39M / R44L / T47S / H92Y / A206K / D247N / G261A, E39M / R44L / T47S / H92Y / A206K / T392M, E3 9M / R44L / T47S / A206K / P337A / A368W / T392M, E39M / R44L / H92Y / P166S / D247N / G261A / K302Q / P337A, E39M / R44L / P166S / A271H, E39M / R44L / P166S / A2 71H / P337A / A368W / T392M, E39M / T47S / H92Y / D316L / I322M, E39M / T47S / H92Y / T392M, E39M / T47S / P166S / R217F / G261A / T392M, E39M / T47S / R217F / D 247N / A368W、E39M / T47S / D247N、E39M / H92Y / P166S / R217F / T392M、E39M / H 92Y / G261A / K302Q、E39M / P166S / R217F / G261A / D316L / A368W、E39M / I322M E39M / T392M, R44L / T47S, R44L / T47S / H92Y / R217F / A271H, R44L / T47S / H92Y / R217F / D316L / I322M / T392M, R44L / T47S / H92Y / T392M, R44L / T47S / P16 6S、R44L / T47S / P166S / A271H、R44L / T47S / D247N / A271H / T392M、R44L / D31 6L / I322M / T392M、R44L / P337A、T47S / P166S / A206K / R217F / D247N / P337A、T47S / P166S / R217F / A271H / P337A, T47S / A206K, T47S / R217F / D247N / G261A, T47S / A271H, D52N / R217F / K302Q / D316L, H92Y / P166S / A206K / A271H / D316L, H92Y / P166S / R217F / G261A / A271H / T392M, H92Y / P166S / R217F / D316L / P337A / T392M, H92Y / P16 6S / D247N, H92Y / P166S / D316L, H92Y / A206K / I322M, H92Y / R217F, H92Y / R217F / A27 and at least one substitution or set of substitutions at one or more positions selected from: 1H / P337A, H92Y / G261A / A271H, H92Y / A271H, P166S / R217F / D316L / I322M / P337A, P166S / D247N / A271H / D316L, P166S / D316L / I322M / P337A, A206K / R217F, R217F / T392M, D247N / D316L, D316L / I322M / A368W, and D316L / P337A / T392M, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374.

[0126] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 704, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 2, 4, 5, 24 / 59, 24 / 143 / 144, 24 / 143 / 202 / 333, 24 / 143 / 202 / 352 / 390 / 391, 24 / 143 / 333 / 352 / 387 / 390 / 391, 24 / 143 / 390 / 391, 24 / 202, 24 / 202 / 271, 24 / 202 / 333 / 352, 24 / 271 / 352, 24 / 352 / 387 / 390 / 391, 24 / 387 / 391, 31, 40, 59, 59 / 143, 59 / 143 / 202, 59 / 143 / 202 / 271 / 333, 59 / 143 / 271, 59 / 143 / 333, 59 / 202, 59 / 202 / 333, 59 / 271 / 387 / 390, 73, 76, 80, 83, 84, 91 / 215 / 361, 122, 123, 143, 143 / 20 2, 143 / 271, 143 / 271 / 352 / 390, 143 / 333, 143 / 333 / 387 / 390, 143 / 387 / 391, 147, 155, 164, 165, 179, 186, 202, 202 / 333, 210, 215 / 218, 218, 218 / 361, 218 / 361 / 398, 218 / 398, 246, 254 / 398, 271, 271 / 333, 271 / 333 / 390 / 391, 271 / 333 / 391, 271 / 352 / 391, 273, 275, 277, 278, 280, 281, 283, 284, 287, In some embodiments, the recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from 300, 303, 304, 325, 331, 332, 333 / 352, 333 / 390 / 391, 333 / 391, 334, 335, 336, 338, 339, 340, 341, 343, 359, 360, 361, 362, 367, 369, 371, 373, 375, 377, 382, ​​382 / 398, 385, 387 / 391, 390, and 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 704.704, or a functional fragment thereof, and the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 704, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 2S, 4L, 5M, 5V, 24S / 59A, 24S / 143S / 144N, 24S / 143S / 202N / 333N, 24S / 143S / 202N / 352N / 390N / 391N, 24S / 143S / 333N / 352N / 387N / 390T / 391N, 24S / 143S / 390T / 391N, 24S / 202N, 24S / 202N / 271N, 24S / 202N / 333N / 352N, 2 4S / 271N / 352N, 24S / 352N / 387N / 390N / 391N, 24S / 387N / 391N, 31F, 31H, 31L, 31T, 31W, 40Q, 59A, 59A / 143S, 59A / 143S / 271N, 59A / 202N, 59T, 59T / 143S / 2 02N, 59T / 143S / 333N, 59T / 202N / 333N, 59V / 143S / 202N / 271N / 333N, 59V / 271 N / 387N / 390T, 73A, 76A, 76F, 76M, 76S, 80T, 83R, 83S, 84G, 84K, 84R, 91S / 21 5S / 361T, 122E, 122N, 122S, 123Q, 123R, 123S, 123T, 143S, 143S / 202N, 143S / 271N, 143S / 271N / 352N / 390N, 143S / 333N, 143S / 333N / 387N / 390T, 143S / 38 7N / 391N, 147L, 147S, 155A, 155D, 155F, 155L, 155R, 155T, 164E, 165I, 179H, 179L, 179R, 179W, 186E, 186F, 186M, 186P, 186R, 186S, 186Y, 202N, 202N / 33 3N, 210I, 215S / 218Y, 218Y, 218Y / 361T, 218Y / 361T / 398F, 218Y / 398F, 246Y, 254T / 398F, 271N, 271N / 333N, 271N / 333N / 390N / 391N, 271N / 333N / 391N, 27 1N / 352N / 391N, 273L, 275A, 275G, 277Q, 277V, 278N, 278R, 278S, 280G, 281I,281M, 283L, 283P, 283T, 283V, 284A, 284E, 284G, 284L, 284M, 284R, 284S, 287R , 300F, 303A, 303C, 303W, 304T, 304V, 304W, 325A, 331M, 332G, 332H, 333N / 352N , 333N / 390N / 391N, 333N / 390S / 391N, 333N / 391N, 334C, 334V, 335A, 335L, 336 F, 336G, 336S, 336T, 338L, 339G, 339N, 339Q, 339V, 340H, 340I, 340K, 340M, 340 and 398F, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:704. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 704, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of D2S, G4L, L5M, L5V, D24S / C59A, D24S / C143S / D144N, D24S / C143S / D202N / G333N, D24S / C143 S / D202N / F352N / M390N / Q391N, D24S / C143S / G333N / F352N / E387N / M390 T / Q391N, D24S / C143S / M390T / Q391N, D24S / D202N, D24S / D202N / A271N, D 24S / D202N / G333N / F352N, D24S / A271N / F352N, D24S / F352N / E387N / M390 N / Q391N, D24S / E387N / Q391N, S31F, S31H, S31L, S31T, S31W, E40Q, C59A,C59A / C143S, C59A / C143S / A271N, C59A / D202N, C59T, C59T / C143S / D202N, C59T / C143S / G333N, C59T / D202N / G333N, C59V / C143S / D202N / A271N / G333N C59V / A271N / E387N / M390T, G73A, Q76A, Q76F, Q76M, Q76S, Q80T, P83R, P83S, H84G, H84K, H84R, N91S / T215S / R361T, D122E, D122N, D122S, I123Q, I123R I123S, I123T, C143S, C143S / D202N, C143S / A271N, C143S / A271N / F352N / M390N, C143S / G333N, C143S / G333N / E387N / M390T, C143S / E387N / Q391N, E147L 、E147S、H155A、H155D、H155F、H155L、H155R、H155T、G164E、R165I、P179H、P 179L、P179R、P179W、T186E、T186F、T186M、T186P、T186R、T186S、T186Y、D202 N, D202N / G333N, S210I, T215S / N218Y, N218Y, N218Y / R361T, N218Y / R361T / L398F, N218Y / L398F, W246Y, A254T / L398F, A271N, A271N / G333N, A271N / G33 3N / M390N / Q391N, A271N / G333N / Q391N, A271N / F352N / Q391N, S273L, Q275A, Q275G, K277Q, K277V, A278N, A278R, A278S, L280G, Q281I, Q281M, K283L, K2 83P、K283T、K283V、D284A、D284E、D284G、D284L、D284M、D284R、D284S、A287 R、L300F、G303A、G303C、G303W、D304T、D304V、D304W、R325A、P331M、R332G、R 332H, G333N / F352N, G333N / M390N / Q391N, G333N / M390S / Q391N, G333N / Q391N, Y334C, Y334V, T335A, T335L, I336F, I336G, I336S, I336T, V338L, A339GA339N, A339Q, A339V, S340H, S340I, S340K, S340M, S340P, S340W, L341F, L341M, K343L, K343R, K343S, K343W, V359F, V359L, V359R, K360H, K360V, R361T, R361V, K362H, E367A, E367D, E367L, E367M, T369D, R and at least one substitution or set of substitutions at one or more positions selected from 371G, R373L, R373S, H375L, H375Q, N377Q, V382I, V382I / L398F, Q385R, E387N / Q391N, M390S, and L398F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 704.

[0127] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from 10, 39, 44, 47, 92, 166, 206, 217, 247, 261, 271, 302, 316, 322, 337, 368, and 392, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 10A, 10C, 10D, 10E, 10F, 10G, 10H, 10HQ, 10I, 10IQ ... 10D, 10E, 10F, 10G, 10H, 10I, 10K, 10L, 10M, 10N, 10Q, 10R, 10S, 10T, 10V, 10W, 10Y, 39A, 39C, 39D, 39F, 39 G, 39H, 39I, 39K, 39L, 39M, 39N, 39P, 39Q, 39R, 39S, 39T, 39V, 39W, 39Y, 44A, 44C, 44D, 44E, 44F, 44G, 44H, 4 4I, 44K, 44L, 44N, 44P, 44Q, 44S, 44T, 44V, 44W, 44Y, 47A, 47C, 47D, 47E, 47F, 47G, 47H, 47I, 47K, 47L, 47M , 47N, 47P, 47Q, 47R, 47S, 47V, 47W, 47Y, 92A, 92C, 92D, 92E, 92F, 92G, 92I, 92K, 92L, 92M, 92N, 92P, 92Q, 9 2R, 92S, 92T, 92V, 92W, 92Y, 166A, 166C, 166D, 166E, 166F, 166G, 166H, 166I, 166K, 166L, 166M, 166N, 166 Q, 166R, 166S, 166T, 166V, 166W, 166Y, 206C, 206D, 206E, 206F, 206G, 206H, 206I, 206K, 206L, 206M, 206N,206P, 206Q, 206R, 206S, 206T, 206V, 206W, 206Y, 217A, 217C, 217D, 217E, 217F, 217G, 217H, 217I, 217K, 217L, 217M, 217N, 217P, 217Q, 217S, 217T, 217 V、217W、217Y、247A、247C、247E、247F、247G、247H、247I、247K、247L、247M 、247N、247P、247Q、247R、247S、247T、247V、247W、247Y、261A、261C、261D、2 61E、261F、261H、261I、261K、261L、261M、261N、261P、261Q、261R、261S、26 1T、261V、261W、261Y、271C、271D、271E、271F、271G、271H、271I、271K、271L 271M, 271N, 271P, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 302A, 302C, 302D, 302E, 302F, 302G, 302H, 302I, 302L, 302M, 302N, 302P, 302Q, 302R, 30 2S, 302T, 302V, 302W, 302Y, 316A, 316C, 316E, 316F, 316G, 316H, 316I, 316K, 316L, 316M, 316N, 316P, 316Q, 316R, 316S, 316T, 316V, 316W, 316Y, 322A 322C, 322D, 322E, 322F, 322G, 322H, 322K, 322L, 322M, 322N, 322P, 322Q, 322R, 322S, 322T, 322V, 322W, 322Y, 337A, 337C, 337D, 337E, 337F, 337G, 337 H, 337I, 337K, 337L, 337M, 337N, 337Q, 337R, 337S, 337T, 337V, 337W, 337Y, 368C, 368D, 368E, 368F, 368G, 368H, 368I, 368K, 368L, 368M, 368N, 368P, 3 68Q, 368R, 368S, 368T, 368V, 368W, 368Y, 392A, 392C, 392D, 392E, 392F, 392G, 392H, 392I, 392K, 392L, 392M, 392N, 392P, 392Q, 392R, 392S, 392V, 392Wand 392Y, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein said recombinant alpha-galactosidase A is selected from the group consisting of P10A, P10C, P10D, P10E, P10F, P10G, P10H, P10I, P10K, P10L, P10M, P10N , P10Q, P10R, P10S, P10T, P10V, P10W, P10Y, E39A, E39C, E39D, E39F, E39G, E39H, E39I, E39K, E39L, E39M, E39N, E39P, E39Q, E39R, E39S, E3 9T, E39V, E39W, E39Y, R44A, R44C, R44D, R44E, R44F, R44G, R44H, R44I, R44K, R44L, R44N, R44P, R44Q, R44S, R44T, R44V, R44W, R44Y, T47A, T 47C, T47D, T47E, T47F, T47G, T47H, T47I, T47K, T47L, T47M, T47N, T47P, T47Q, T47R, T47S, T47V, T47W, T47Y, H92A, H92C, H92D, H92E, H92F , H92G, H92I, H92K, H92L, H92M, H92N, H92P, H92Q, H92R, H92S, H92T, H92V, H92W, H92Y, P166A, P166C, P166D, P166E, P166F, P166G, P166H, P 166I, P166K, P166L, P166M, P166N, P166Q, P166R, P166S, P166T, P166V, P166W, P166Y, A206C, A206D, A206E, A206F, A206G, A206H, A206I, A206K, A206L, A206M, A206N, A206P, A206Q, A206R, A206S, A206T, A206V, A206W, A206Y, R217A, R217C, R217D, R217E, R217F, R217G, R217H,R217I, R217K, R217L, R217M, R217N, R217P, R217Q, R217S, R217T, R217V, R217W, R217Y, D247A, D247C, D247E, D247F, D247G, D247H, D247I, D247K, D247 L、D247M、D247N、D247P、D247Q、D247R、D247S、D247T、D247V、D247W、D247Y、 G261A、G261C、G261D、G261E、G261F、G261H、G261I、G261K、G261L、G261M、G26 1N、G261P、G261Q、G261R、G261S、G261T、G261V、G261W、G261Y、A271C、A271D 、A271E、A271F、A271G、A271H、A271I、A271K、A271L、A271M、A271N、A271P、A 271Q, A271R, A271S, A271T, A271V, A271W, A271Y, K302A, K302C, K302D, K302E, K302F, K302G, K302H, K302I, K302L, K302M, K302N, K302P, K302Q, K302R K302S, K302T, K302V, K302W, K302Y, D316A, D316C, D316E, D316F, D316G, D316H, D316I, D316K, D316L, D316M, D316N, D316P, D316Q, D316R, D316S, D316 T、D316V、D316W、D316Y、I322A、I322C、I322D、I322E、I322F、I322G、I322H、 I322K、I322L、I322M、I322N、I322P、I322Q、I322R、I322S、I322T、I322V、I32 2W, I322Y, P337A, P337C, P337D, P337E, P337F, P337G, P337H, P337I, P337K, P337L, P337M, P337N, P337Q, P337R, P337S, P337T, P337V, P337W, P337Y, A 368C, A368D, A368E, A368F, A368G, A368H, A368I, A368K, A368L, A368M, A368N, A368P, A368Q, A368R, A368S, A368T, A368V, A368W, A368Y, T392A, T392Cand at least one substitution or set of substitutions at one or more positions selected from T392D, T392E, T392F, T392G, T392H, T392I, T392K, T392L, T392M, T392N, T392P, T392Q, T392R, T392S, T392V, T392W, and T392Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374.

[0128] The present invention also provides a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1022, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is 39 / 44 / 166 / 302, 31 / 47, 31 / 283 / 284, 39, 39 / 44, 39 / 44 / 47, 39 / 44 / 47 / 261 / 283 / 284, 39 / 44 / 283, 39 / 44 / 339, 39 / 47 / 261, 39 / 92, 39 / 206, 39 / 284, 44, 44 / 284 / 302, 84, 84 / 92, 84 / 284 / 302 / 392, 84 / 316, 84 / 368 / 392, 92, 92 / 206 / 217, 92 / 206 / 275, 92 / 206 / 284, 92 / 206 / 302 / 368, 92 / 271, 92 / 271 / 277, 92 / 275 / 284, 92 / 283, 92 / 283 / 392, 92 / 284, 92 / 302, 92 / 316, 92 / 368, 155, 155 / 217, 155 / 368, 166, 166 / 283 / 284, 166 / 302, 206, 206 / 217, and at least one substitution or set of substitutions at one or more positions selected from 206 / 334, 261, 261 / 283, 271, 271 / 368, 275, 283, 283 / 284, 283 / 392, 284, 302, 316, 334, 339, 368, 368 / 392, and 392, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1022. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1022, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of P10G, P10G / T392D, S31T, S31T / E39V / R44V / P166D / K302Y, S31T / T47R, S31T / K283L / D284A, E39L, E39L / H92V, E39L / A206E, E39L / D284S,E39V / R44V, E39V / R44V / T47R, E39V / R44V / T47R / G261S / K283L / D284A, E39V / R44V / K283T, E39V / R44V / A339N, E39V / T47 R / G261S, R44V, R44V / D284E / K302Y, H84K, H84K / H92V, H84K / D284S / K302L / T392A, H84K / D316H, H84K / A368E / T392A, H9 2Q, H92T, H92T / A206E / R217N, H92T / A206E / K302T / A368E, H92T / A271K, H92T / A271K / K277R, H92T / K283P, H92T / K283V / T392W, H92T / D284M, H92T / K302L, H92T / A368E, H92V, H92V / A206E / D284S, H92V / A206Y / Q275A, H92V / Q275A / D284S, H92 V / D284S, H92V / K302L, H92V / D316H, H155F, H155F / R217I, H155F / A368E, P166D, P166D / K283L / D284A, P166D / K302Y, A2 06E, A206E / R217N, A206I, A206Q, A206T / Y334C, A206Y, G261S, G261S / K283L, A271K, A271K / A368E, Q275A, K283L, K283 and at least one substitution or set of substitutions at one or more positions selected from: P / T392W, K283T, K283T / D284E, D284E, D284M, D284S, K302L, K302Y, D316H, Y334C, A339N, A368E, A368E / T392W, T392A, T392D, and T392W, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1022. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1022, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 10G, 10G / 392D, 31T, 31T / 39V / 44V / 166D / 302Y, 31T / 47R ...31T / 283L / 284A, 39L, 39L / 92V, 39L / 206E, 39L / 284S, 39V / 44V, 39V / 44V / 47R, 39V / 44V / 47R / 261S / 283L / 28 4A, 39V / 44V / 283T, 39V / 44V / 339N, 39V / 47R / 261S, 44V, 44V / 284E / 302Y, 84K, 84K / 92V, 84K / 284S / 302L / 39 2A, 84K / 316H, 84K / 368E / 392A, 92Q, 92T, 92T / 206E / 217N, 92T / 206E / 302T / 368E, 92T / 271K, 92T / 271K / 277 R, 92T / 283P, 92T / 283V / 392W, 92T / 284M, 92T / 302L, 92T / 368E, 92V, 92V / 206E / 284S, 92V / 206Y / 275A, 92V / 2 75A / 284S, 92V / 284S, 92V / 302L, 92V / 316H, 155F, 155F / 217I, 155F / 368E, 166D, 166D / 283L / 284A, 166D / 30 2Y, 206E, 206E / 217N, 206I, 206Q, 206T / 334C, 206Y, 261S, 261S / 283L, 271K, 271K / 368E, 275A, 283L, 283P / and at least one substitution or set of substitutions at one or more positions selected from 392W, 283T, 283T / 284E, 284E, 284M, 284S, 302L, 302Y, 316H, 334C, 339N, 368E, 368E / 392W, 392A, 392D, and 392W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1022.

[0129] In some embodiments, the recombinant alpha-galactosidase A polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the even-numbered sequences of SEQ ID NOs: 4-1864.

[0130] In some embodiments, engineered GLA polypeptides include functional fragments of engineered GLA polypeptides encompassed by the present invention. Functional fragments have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the activity of the engineered GLA polypeptide (i.e., the parent engineered GLA) from which they are derived. Functional fragments include at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and even 99% of the parent sequence of the engineered GLA. In some embodiments, functional fragments are truncated by fewer than 5, fewer than 10, fewer than 15, fewer than 10, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, and fewer than 50 amino acids. Polynucleotides encoding engineered polypeptides, expression vectors and host cells

[0131] The present invention provides a polynucleotide that encodes the engineered GLA polypeptide described herein.In some embodiments, the polynucleotide is operably linked to one or more heterologous or homologous regulatory sequences that control gene expression, so as to produce a recombinant polynucleotide that can express the polypeptide.The expression construct that comprises the heterologous polynucleotide that encodes the engineered GLA polypeptide can be introduced into suitable host cell to express the corresponding GLA polypeptide.

[0132] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides capable of encoding the subject polypeptides. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for the creation of a vast number of nucleic acids, all of which will encode the engineered GLA polypeptide. Thus, one skilled in the art with knowledge of a particular amino acid sequence can create any number of different nucleic acids by simply altering the sequence of one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present invention specifically contemplates any and all possible variations of polynucleotides that can be made to encode the polypeptides described herein by selecting combinations based on possible codon choices, and all such variations should be considered specifically disclosed for any polypeptide described herein, such as the variants provided in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and / or 13-1.

[0133] In various embodiments, codons are preferably selected to be compatible with the host cell in which the protein is produced. For example, preferred codons used in bacteria are used for expression in bacteria. As a result, a codon-optimized polynucleotide encoding an engineered GLA polypeptide contains preferred codons at more than about 40%, 50%, 60%, 70%, 80%, or 90% of the codon positions in the full-length coding region. In some embodiments, the present invention provides recombinant polynucleotide sequences in which the codons are optimized for expression in human cells or tissues.

[0134] In some embodiments, the present invention provides recombinant polynucleotide sequences having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to SEQ ID NO: 1. In some embodiments, the present invention provides recombinant polynucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1. In some embodiments, the present invention provides recombinant polynucleotide sequences having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to SEQ ID NO: 7. In some embodiments, the present invention provides recombinant polynucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:7. In some embodiments, the recombinant polynucleotide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the odd-numbered sequences of SEQ ID NOs: 3-1863.

[0135] In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 44, 44 / 217, 44 / 217 / 316, 44 / 217 / 322, 44 / 217 / 322 / 337, 44 / 247, 44 / 247 / 302, 44 / and at least one substitution or set of substitutions at one or more positions selected from 247 / 302 / 322, 44 / 247 / 322, 44 / 247 / 337, 44 / 247 / 362, 44 / 302, 44 / 337, 44 / 373, 217 / 322, 217 / 373, 247 / 322, 247 / 362, 302 / 322 / 362 / 373, 302 / 337, 316, 316 / 337, 322, 322 / 337, 362 / 373, and 373, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 44L, 44L / 217F, 44L / 217F / 316L, 44L / 217F / 322M, 44L / 217F / 322M / 337A, 44L / 247N, 44L / 247N / 302Q, 44L / 247N / 302Q / and 373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of R44L, R44L / R217F, R44L / R217F / D316L, R44L / R217F / I322M, R44L / R217F / I322M / P337A, R44L / D247N, R44L / D247N / K302Q, R44L / D247N / K302Q / I322M, R44 L / D247N / I322M, R44L / D247N / P337A, R44L / D247N / Q362K, R44L / K302Q, R44L / P337A, R44L / K373R, R217F / I322M, R217F / K373R, D247N / I322M, D247N / Q362K, K302Q / I322M / Q362K / K373 and at least one substitution or set of substitutions at one or more positions selected from R, K302Q / P337A, D316L, D316L / P337A, I322M, I322M / P337A, Q362K / K373R, and K373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.

[0136] In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10 / 39 / 44 / 47 / 92 / and at least one substitution or set of substitutions at one or more positions selected from 166 / 206 / 217 / 247 / 261 / 271 / 302 / 316 / 322 / 337 / 362 / 368 / 373 / 392, 44 / 217 / 316, 44 / 217 / 322 / 337, 166 / 362, 217 / 373, and 362 / 373, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8. In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10T / 39M / 44L / 47S / 92Y / 166S / 206K / 2 and at least one substitution or set of substitutions at one or more positions selected from 17F / 247N / 261A / 271H / 302Q / 316L / 322M / 337A / 362K / 368W / 373R / 392M, 44L / 217F / 316L, 44L / 217F / 322M / 337A, 166A / 362K, 217F / 373R, and 362K / 373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of P10T / E39M / R44L / T47S / H92Y / P166S / A206K / R217F / D247 and Q362K / K373R, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:8.

[0137] In some embodiments, the present invention provides recombinant polynucleotide sequences having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to SEQ ID NO: 57. In some embodiments, the present invention provides recombinant polynucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 57. In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A, wherein the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58, or a functional fragment thereof; Galactosidase A is 7, 7 / 48 / 68, 7 / 48 / 68 / 120 / 282 / 299, 7 / 48 / 130 / 282, 7 / 48 / 180, 7 / 68 / 130 / 282 / 365, 7 / 68 / 180, 7 / 88 / 120 / 305 / 365, 7 / 120, 7 / 130, 7 / 282, 7 / 305, 7 / 305 / 365, 7 / 365, 39, 47, 47 / 87 / 95 / 96 / 158 / 162, 47 / 95, 47 / 2 73, 47 / 343, 48, 48 / 68, 48 / 180 / 282, 48 / 282, 48 / 282 / 305, 67 / 180, 68, 68 / 299 / 300, 71, 87 / 91 / 95 / 96 / 158 / 162, 87 / 91 / 95 / 96 / 206 / 343, 87 / 96 / 155 / 273 / 343, 88, 91 / 95, 91 / 95 / 96, 92, 93, 96, 96 / 273, 96 / 312 / 343, 120, 120 / 299 / 305, 151, 158, 158 / 162 / 273, 162, 162 / 273, 162 / 343, 166, 178, 180, 181, 206, 217, 271, 273, 273 / 343, 282, 282 / 365, 293 / 391, 299 / 300, 299 / 300 / 305 / 365, 300, 301, 305, 305 / 365, 314, 333, 336, 337, 343, 345, 363, 365,and 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 58. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58, or a functional fragment thereof, wherein said recombinant alpha-galactosidase A is selected from the group consisting of 7L, 7L / 48D / 68E, 7L / 48D / 68E / 120H / 282N / 299R, 7L / 48D / 130E / 282N ... 7L / 48D / 180G, 7L / 68E / 130E / 282N / 365V, 7L / 68E / 180G, 7L / 88A / 120H / 305G / 365V, 7L / 120H, 7L / 130E, 7L / 282N, 7L / 305G, 7L / 305G / 36 5V, 7L / 365V, 39V, 47D, 47D / 87K / 95E / 96L / 158R / 162H, 47D / 95E, 47D / 273P, 47D / 343G, 47V, 48D, 48D / 68E, 48D / 180G / 282N, 48D / 282N, 4 8D / 282N / 305G, 67T / 180G, 68E, 68E / 299R / 300I, 71P, 87K / 91Q / 95E / 96L / 158A / 162K, 87K / 91Q / 95E / 96L / 206S / 343G, 87K / 96I / 155N / 2 73P / 343G, 88A, 91Q / 95E, 91Q / 95E / 96L, 92F, 92T, 93I, 96L, 96L / 273P, 96L / 312Q / 343G, 120H, 120H / 299R / 305G, 151L, 158A, 158A / 162K / 273G, 158R, 162H / 343D, 162K, 162K / 273P, 162S, 166K, 178G, 178S, 180G, 180L, 180T, 180V, 181A, 206K, 206S, 217K, 271R, 273P, 273P / 343G, 282N, 282N / 365V, 293P / 391A, 299R / 300I, 299R / 300I / 305G / 365V, 300I, 301M, 305G, 305G / 365V, 314A, 333F, 333G, 336V, 337R,and at least one substitution or set of substitutions at one or more positions selected from 343D, 343G, 345A, 345Q, 363Q, 365A, 365Q, 365V, 370G, 389K, 393V, 394K, 396G / 398T, 397A, 398A, 398P, 398S, and 398V, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:58. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of R7L, R7L / E48D / Q68E, R7L / E48D / Q68E / Y120H / D282 ... N / Q299R, R7L / E48D / D130E / D282N, R7L / E48D / F180G, R7L / Q68E / D130E / D282N / F365V, R7L / Q68E / F180G, R7L / Q88A / Y120H / N 305G / F365V, R7L / Y120H, R7L / D130E, R7L / D282N, R7L / N305G, R7L / N305G / F365V, R7L / F365V, E39V, T47D, T47D / R87K / S95E / K 96L / L158R / R162H, T47D / S95E, T47D / S273P, T47D / K343G, T47V, E48D, E48D / Q68E, E48D / F180G / D282N, E48D / D282N, E48D / D 282N / N305G, P67T / F180G, Q68E, Q68E / Q299R / L300I, S71P, R87K / N91Q / S95E / K96L / L158A / R162K, R87K / N91Q / S95E / K96L / A2 06S / K343G, R87K / K96I / H155N / S273P / K343G, Q88A, N91Q / S95E, N91Q / S95E / K96L, H92F, H92T, V93I, K96L, K96L / S273P, K96L / P312Q / K343G, Y120H, Y120H / Q299R / N305G, D151L, L158A, L158A / R162K / S273G, L158R, R162H / K343D, R162K, R162K / S273P,R162S, P166K, W178G, W178S, F180G, F180L, F180T, F180V, Q181A, A206K, A206S, R217K, A271R, S273P, S273P / K343G, D282N, D28 2N / F365V, L293P / Q391A, Q299R / L300I, Q299R / L300I / N305G / F365V, L300I, R301M, N305G, N305G / F365V, S314A, S333F, S333G, and at least one substitution or set of substitutions at one or more positions selected from I336V, P337R, K343D, K343G, V345A, V345Q, L363Q, F365A, F365Q, F365V, S370G, T389K, S393V, L394K, D396G / L398T, L397A, L398A, L398P, L398S, and L398V, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 58.

[0138] In some embodiments, the present invention provides recombinant polynucleotide sequences having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to SEQ ID NO: 157. In some embodiments, the present invention provides recombinant polynucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 157.In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A, wherein the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 158, or a functional fragment thereof, and wherein the recombinant alpha-galactosidase A ctosidase A: 24 / 202, 39 / 47, 39 / 47 / 217, 39 / 151, 39 / 282 / 337 / 398, 39 / 337 / 343 / 398, 39 / 393 / 398, 47 / 130, 47 / 151, 47 / 343 / 345 / 393, 48, 48 / 68, 48 / 68 / 217 / 333 / 391 / 393, 48 / 68 / 333, 48 / 217, 48 / 333, 48 / 345 / 393, 48 / 393, 59 / 143 , 68, 68 / 345, 130, 130 / 158, 130 / 158 / 393, 130 / 345 / 393, 143 / 271, 143 / 333, 143 / 387, 151, 151 / 158 / 217 / 343 / 345 / 393, 151 / 206 / 282 / 337 / 343 / 345 / 398, 151 / 282 / 393, 151 / 345 / 393 / 398, 151 / 393, 158, 158 / 393, 202, 206, 206 / 2 and at least one substitution or set of substitutions at one or more positions selected from 17, 217, 217 / 333, 217 / 337 / 345 / 398, 271, 282 / 393, 333, 333 / 345, 337 / 343 / 345 / 398, 343, 343 / 345 / 393 / 398, 393, and 393 / 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 158.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 158, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 24S / 202N, 39V / 47D, 39V / 47V / 217K, 39V / 151L, 39V / 282N / 337R / 398A, 39V / 337R / 343G / 398A, 39V / 393V / 398A, 47V / 130E, 47V / 151L, 47V / 343D / 345Q / 393V, 48D, 48D / 68E, 48D / 68E / 217K / 333F / 391 A / 393V, 48D / 68E / 333F, 48D / 217K, 48D / 333F, 48D / 333G, 48D / 345Q / 393V, 48D / 393V, 59A / 143S, 68E, 68E / 345Q, 130E, 130E / 158R, 13 0E / 158R / 393V, 130E / 345Q / 393V, 143S / 271N, 143S / 333N, 143S / 387N, 151L, 151L / 158R / 217K / 343G / 345Q / 393V, 151L / 206S / 282N / 337R / 343D / 345Q / 398A, 151L / 282N / 393V, 151L / 345Q / 393V / 398A, 151L / 393V, 158R, 158R / 393V, 202N, 206S, 206S / 217K, 217K, 217K / 333F, 217K / 333G, 217K / 337R / 345Q / 398A, 271N, 282N / 393V, 333F / 345Q, 333G, 333N, 337R / 343G / 345Q / 398A, 343D, 343D / 345Q / 393V / 398A, 393V, and 393V / 398A, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 158.In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of D24S / D202N, E39V / T47D, E39V / T47V / R217K, E39V / D151L, E39V / D282N / P337R / L398A, E39V / P337R / K343 G / L398A, E39V / S393V / L398A, T47V / D130E, T47V / D151L, T47V / K343D / V345 Q / S393V, E48D, E48D / Q68E, E48D / Q68E / R217K / S333F / Q391A / S393V, E48D / Q68E / S333F, E48D / R217K, E48D / S333F, E48D / S333G, E48D / V345Q / S393V, E 48D / S393V, C59A / C143S, Q68E, Q68E / V345Q, D130E, D130E / L158R, D130E / L1 58R / S393V, D130E / V345Q / S393V, C143S / A271N, C143S / S333N, C143S / E387 N, D151L, D151L / L158R / R217K / K343G / V345Q / S393V, D151L / A206S / D282N / P337R / K343D / V345Q / L398A, D151L / D282N / S393V, D151L / V345Q / S393V / L3 98A, D151L / S393V, L158R, L158R / S393V, D202N, A206S, A206S / R217K, R217K , R217K / S333F, R217K / S333G, R217K / P337R / V345Q / L398A, A271N, D282N / S393V, S333F / V345Q, S333G, S333N, P337R / K343G / V345Q / L398A, K343D, K343D / V345Q / S393V / L398A, S393V, and S393V / L398A, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 158.

[0139] In some embodiments, the present invention provides recombinant polynucleotide sequences having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to SEQ ID NO: 371. In some embodiments, the present invention provides recombinant polynucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 371. In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A, wherein the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 372, or a functional fragment thereof; Alpha-galactosidase A is 10, 10 / 39 / 44 / 322, 10 / 39 / 92 / 206 / 217 / 271, 10 / 39 / 92 / 247, 10 / 39 / 92 / 247 / 271 / 316, 10 / 44, 10 / 44 / 47 / 92 / 247, 10 / 44 / 47 / 261 / 302 / 322 / 368, 10 / 44 / 92 / 316 / 322, 10 / 44 / 261 / 302 / 316, 10 / 44 / 302 / 33 7 / 368, 10 / 47 / 217 / 247 / 316 / 392, 10 / 47 / 217 / 322, 10 / 47 / 271, 10 / 92, 10 / 92 / 206 / 217 / 247, 10 / 92 / 206 / 247 / 316 / 322 / 392, 10 / 92 / 206 / 247 / 322 / 368, 10 / 92 / 217 / 261 / 302 / 337, 10 / 206 / 217 / 271, 10 / 206 / 247, 10 / 206 / 26 1 / 271 / 316, 10 / 261, 10 / 271 / 302, 10 / 302, 10 / 302 / 316, 10 / 302 / 322 / 337, 10 / 316 / 322, 10 / 337 / 392, 10 / 368, 39 / 44 / 92 / 162 / 247 / 302 / 316 / 322, 39 / 44 / 92 / 217 / 322, 39 / 44 / 92 / 247 / 271 / 302, 39 / 47 / 92 / 247 / 302 / 316 / 322,39 / 47 / 217 / 247 / 368, 39 / 47 / 247, 39 / 92 / 247 / 302 / 316 / 337 / 368, 39 / 92 / 316 / 322, 39 / 247 / 271, 39 / 247 / 271 / 316, 39 / 322, 44 / 47 / 92 / 206 / 217 / 316 / 322, 44 / 47 / 92 / 247 / 261 / 271 / 316 / 337 / 3 68, 44 / 47 / 206 / 217 / 247 / 271 / 322, 44 / 47 / 247 / 322 / 368, 44 / 47 / 302 / 316 / 322, 44 / 92 / 206 / 247 / 368, 44 / 206 / 337, 44 / 247 / 261 / 302 / 316, 44 / 247 / 261 / 302 / 316 / 322, 47 / 92 / 247 / 271, 47 / 217 / 302, 47 / 247, 47 / 247 / 271, 89 / 217 / 247 / 261 / 302 / 316, 92 / 217 / 271, 92 / 247, 92 / 247 / 271 / 322, 92 / 247 / 302 / 322 / 337, 92 / 271 / 337, 92 / 302, 92 / 316, 206 / 217 / 271 / 392, 217 / 247 / 316 / 322 / 3 and 368, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 372. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 372, or a functional fragment thereof, wherein said recombinant alpha-galactosidase A is selected from the group consisting of 10P, 10P / 39E / 44R / 3 22I, 10P / 39E / 92H / 206A / 217R / 271A, 10P / 39E / 92H / 247D, 10P / 39E / 92H / 247D / 271A / 316D, 10P / 44R, 10P / 44R / 47T / 92H / 247D, 10P / 44R / 47T / 261G / 302K / 322I / 368A, 10P / 44R / 92H / 316D / 322I, 10P / 44R / 261G / 302K / 316D,10P / 44R / 302K / 337P / 368A、10P / 47T / 217R / 247D / 316D / 392T、10P / 47T / 217 R / 322I、10P / 47T / 271A、10P / 92H、10P / 92H / 206A / 217R / 247D、10P / 92H / 206A / 247D / 316D / 322I / 392T、10P / 92H / 206A / 247D / 322I / 368A、10P / 92H / 217R / 261G / 302K / 337P、10P / 206A / 217R / 271A、10P / 206A / 247D、10P / 206A / 261G / 2 71A / 316D、10P / 261G、10P / 271A / 302K、10P / 302K、10P / 302K / 316D、10P / 302 K / 322I / 337P、10P / 316D / 322I、10P / 337P / 392T、10P / 368A、39E / 44R / 92H / 16 2M / 247D / 302K / 316D / 322I、39E / 44R / 92H / 217R / 322I、39E / 44R / 92H / 247D / 271A / 302K、39E / 47T / 92H / 247D / 302K / 316D / 322I、39E / 47T / 217R / 247D / 368 A、39E / 47T / 247D、39E / 92H / 247D / 302K / 316D / 337P / 368A、39E / 92H / 316D / 322I、39E / 247D / 271A、39E / 247D / 271A / 316D、39E / 322I、44R / 47T / 92H / 206A / 217R / 316D / 322I, 44R / 47T / 92H / 247D / 261G / 271A / 316D / 337P / 368A, 44R / 47T / 206A / 217R / 247D / 271A / 322I, 44R / 47T / 247D / 322I / 368A, 44R / 47T / 302K / 316D / 322I、44R / 92H / 206A / 247D / 368A、44R / 206A / 337P、44R / 247D / 261G / 302K / 316D、44R / 247D / 261G / 302K / 316D / 322I、47T / 92H / 247D / 271A、47T / 21 7R / 302K, 47T / 247D, 47T / 247D / 271A, 89I / 217R / 247D / 261G / 302K / 316D, 92H / 217R / 271A, 92H / 247D, 92H / 247D / 271A / 322I, 92H / 247D / 302K / 322I / 337Pand at least one substitution or set of substitutions at one or more positions selected from 92H / 271A / 337P, 92H / 302K, 92H / 316D, 206A / 217R / 271A / 392T, 217R / 247D / 316D / 322I / 337P / 368A, 247D, 247D / 271A, 247D / 302K, 271A, 271A / 302K / 322I, 271A / 316D / 322I, 302K / 322I / 368A, and 368A, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 372. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 372, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of T10P, T10P / M39E / L44R / M32 2I, T10P / M39E / Y92H / K206A / F217R / H271A, T10P / M39E / Y92H / N247D, T10P / M39E / Y92H / N247D / H271A / L316D, T10P / L44R, T10P / L44R / S47T / Y92H / N247D, T10P / L44R / S47T / A261G / Q302K / M322I / W368A, T10P / L44R / Y92H / L316D / M3 22I, T10P / L44R / A261G / Q302K / L316D, T10P / L44R / Q302K / A337P / W368A, T10P / S47T / F217R / N247D / L316D / M392T , T10P / S47T / F217R / M322I, T10P / S47T / H271A, T10P / Y92H, T10P / Y92H / K206A / F217R / N247D, T10P / Y92H / K206A / N 247D / L316D / M322I / M392T, T10P / Y92H / K206A / N247D / M322I / W368A, T10P / Y92H / F217R / A261G / Q302K / A337P, T10 P / K206A / F217R / H271A, T10P / K206A / N247D, T10P / K206A / A261G / H271A / L316D, T10P / A261G, T10P / H271A / Q302K,T10P / Q302K, T10P / Q302K / L316D, T10P / Q302K / M322I / A337P, T10P / L316D / M322I, T10P / A337P / M392T, T10P / W368A, M39E / L44R / Y92H / R162M / N247D / Q302K / L316D / M322I、M39E / L44R / Y92H / F217R / M322I、M39E / L44R / Y92H / N2 47D / H271A / Q302K、M39E / S47T / Y92H / N247D / Q302K / L316D / M322I、M39E / S4 7T / F217R / N247D / W368A, M39E / S47T / N247D, M39E / Y92H / N247D / Q302K / L316D / A337P / W368A, M39E / Y92H / L316D / M322I, M39E / N247D / H271A, M39E / N24 7D / H271A / L316D, M39E / M322I, L44R / S47T / Y92H / K206A / F217R / L316D / M322I, L44R / S47T / Y92H / N247D / A261G / H271A / L316D / A337P / W368A, L44R / S47 T / K206A / F217R / N247D / H271A / M322I、L44R / S47T / N247D / M322I / W368A、L4 4R / S47T / Q302K / L316D / M322I、L44R / Y92H / K206A / N247D / W368A、L44R / K20 6A / A337P, L44R / N247D / A261G / Q302K / L316D, L44R / N247D / A261G / Q302K / L316D / M322I, S47T / Y92H / N247D / H271A, S47T / F217R / Q302K, S47T / N247D, S 47T / N247D / H271A, L89I / F217R / N247D / A261G / Q302K / L316D, Y92H / F217R / H271A, Y92H / N247D, Y92H / N247D / H271A / M322I, Y92H / N247D / Q302K / M322I / A337P、Y92H / H271A / A337P、Y92H / Q302K、Y92H / L316D、K206A / F217R / H271A / M392T、F217R / N247D / L316D / M322I / A337P / W368A、N247D、N247D / H271A、and at least one substitution or set of substitutions at one or more positions selected from N247D / Q302K, H271A, H271A / Q302K / M322I, H271A / L316D / M322I, Q302K / M322I / W368A, and W368A, wherein the amino acid positions of said polypeptide sequence are set forth in SEQ ID NO: 372; The numbering is based on the reference.

[0140] In some embodiments, the present invention provides recombinant polynucleotide sequences having at least about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more sequence identity to SEQ ID NO: 373. In some embodiments, the present invention provides recombinant polynucleotide sequences having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 373. In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A, wherein the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof; Recombinant alpha-galactosidase A is 10 / 36 / 92 / 166 / 247 / 261 / 316 / 392, 10 / 39, 10 / 39 / 44 / 47 / 92 / 206 / 217, 10 / 39 / 44 / 47 / 316, 10 / 39 / 44 / 47 / 337, 10 / 39 / 44 / 92 / 166 / 261 / 316 / 322, 10 / 39 / 44 / 92 / 166 / 302 / 322, 10 / 39 / 44 / 92 / 166 / 392, 10 / 39 / 44 / 92 / 217 / 302 / 322, 10 / 39 / 44 / 92 / 302 / 322, 10 / 39 / 44 / 166 / 261 / 271 / 316 / 322, 10 / 39 / 44 / 392, 10 / 39 / 47 / 92 / 337, 10 / 39 / 92 / 131 / 166 / 271 / 316 / 322, 10 / 39 / 92 / 166 / 217 / 247 / 271, 10 / 39 / 92 / 217 / 316, 10 / 44 / 4 7 / 166 / 261 / 271, 10 / 44 / 47 / 166 / 271 / 322 / 368, 10 / 44 / 47 / 217 / 271 / 316 / 322, 10 / 44 / 92, 10 / 44 / 92 / 217 / 247 / 271 / 302 / 316 / 392, 10 / 44 / 166 / 302, 10 / 44 / 206 / 316 / 322, 10 / 47 / 92 / 166 / 271 / 316 / 337, 10 / 47 / 92 / 271 / 302,10 / 47 / 92 / 316 / 322 / 392、10 / 47 / 166 / 271、10 / 47 / 166 / 316、10 / 92 / 166、10 / 92 / 166 / 217 / 247 / 261 / 271、10 / 92 / 166 / 261 / 271 / 392、10 / 92 / 166 / 261 / 316 / 322 / 337、10 / 92 / 166 / 337 / 368、10 / 92 / 302 / 337、10 / 92 / 316 / 322、10 / 206、10 / 206 / 247 / 261、10 / 217 / 322、10 / 261、10 / 261 / 337 / 392、10 / 316 / 392、10 / 368、39 / 44 / 47 / 92 / 166 / 206 / 392、39 / 44 / 47 / 92 / 206 / 247 / 261、39 / 44 / 47 / 92 / 206 / 392、39 / 44 / 47 / 206 / 337 / 368 / 392、39 / 44 / 92 / 166 / 247 / 261 / 302 / 337、39 / 44 / 166 / 271、39 / 44 / 166 / 271 / 337 / 368 / 392、39 / 47 / 92 / 316 / 322、39 / 47 / 92 / 392、39 / 47 / 166 / 217 / 261 / 392、39 / 47 / 217 / 247 / 368、39 / 47 / 247、39 / 92 / 166 / 217 / 392、39 / 92 / 261 / 302、39 / 166 / 217 / 261 / 316 / 368、39 / 322、39 / 392、44 / 47、44 / 47 / 92 / 217 / 271、44 / 47 / 92 / 217 / 316 / 322 / 392、44 / 47 / 92 / 392、44 / 47 / 166、44 / 47 / 166 / 271、44 / 47 / 247 / 271 / 392、44 / 316 / 322 / 392、44 / 337、47 / 166 / 206 / 217 / 247 / 337、47 / 166 / 217 / 271 / 337、47 / 206、47 / 217 / 247 / 261、47 / 271、52 / 217 / 302 / 316、92 / 166 / 206 / 271 / 316、92 / 166 / 217 / 261 / 271 / 392、92 / 166 / 217 / 316 / 337 / 392、92 / 166 / 247、92 / 166 / 316、92 / 206 / 322、92 / 217、92 / 217 / 271 / 337、92 / 261 / 271、92 / 271、166 / 217 / 316 / 322 / 337、166 / 247 / 271 / 316、166 / 316 / 322 / 337、206 / 217、217 / 392、247 / 316、316 / 322 / 368、and 316 / 337 / 392, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein said recombinant alpha-galactosidase A is selected from the group consisting of 10T / 36M / 92Y / 166S / 247N / 261A / 316L / 392M, 10T / 39M, 10T / 39M / 44L / 47S / 92Y / 206K / 217F, 10T / 39M / 44L / 47S / 316L, 10T / 39M / 44L / 47S / 337A, 10T / 39M / 44L / 92Y / 166S / 261A / 316L / 322M, 10T / 39M / 44L / 92Y / 166S / 302Q / 322M, 10T / 39M / 44L / 92Y / 166S / 392M, 10T / 39M / 44L / 92Y / 217F / 302Q / 322M, 10T / 39M / 44L / 92Y / 302Q / 322M, 10T / 39M / 44L / 166S / 26 1A / 271H / 316L / 322M, 10T / 39M / 44L / 392M, 10T / 39M / 47S / 92Y / 337A, 10T / 39M / 92Y / 131G / 166S / 271H / 316L / 322M, 10T / 39M / 92Y / 166S / 21 7F / 247N / 271H, 10T / 39M / 92Y / 217F / 316L, 10T / 44L / 47S / 166S / 261A / 271H, 10T / 44L / 47S / 166S / 271H / 322M / 368W, 10T / 44L / 47S / 217F / 27 1H / 316L / 322M, 10T / 44L / 92Y, 10T / 44L / 92Y / 217F / 247N / 271H / 302Q / 316L / 392M, 10T / 44L / 166S / 302Q, 10T / 44L / 206K / 316L / 322M, 10T / 47S / 92Y / 166S / 271H / 316L / 337A, 10T / 47S / 92Y / 271H / 302Q, 10T / 47S / 92Y / 316L / 322M / 392M, 10T / 47S / 166S / 271H, 10T / 47S / 166S / 316L,10T / 92Y / 166S、10T / 92Y / 166S / 217F / 247N / 261A / 271H、10T / 92Y / 166S / 26 1A / 271H / 392M、10T / 92Y / 166S / 261A / 316L / 322M / 337A、10T / 92Y / 166S / 33 7A / 368W, 10T / 92Y / 302Q / 337A, 10T / 92Y / 316L / 322M, 10T / 206K, 10T / 206K / 247N / 261A, 10T / 217F / 322M, 10T / 261A, 10T / 261A / 337A / 392M, 10T / 316L / 392M, 10T / 368W, 39M / 44L / 47S / 92Y / 166S / 206K / 392M, 39M / 44L / 47S / 92Y / 206K / 247N / 261A, 39M / 44L / 47S / 92Y / 206K / 392M, 39M / 44L / 47S / 206K / 337 A / 368W / 392M、39M / 44L / 92Y / 166S / 247N / 261A / 302Q / 337A、39M / 44L / 166S / 271H、39M / 44L / 166S / 271H / 337A / 368W / 392M、39M / 47S / 92Y / 316L / 322M、3 9M / 47S / 92Y / 392M、39M / 47S / 166S / 217F / 261A / 392M、39M / 47S / 217F / 247N / 368W、39M / 47S / 247N、39M / 92Y / 166S / 217F / 392M、39M / 92Y / 261A / 302Q、3 9M / 166S / 217F / 261A / 316L / 368W、39M / 322M、39M / 392M、44L / 47S、44L / 47S / 92Y / 217F / 271H、44L / 47S / 92Y / 217F / 316L / 322M / 392M、44L / 47S / 92Y / 392 M、44L / 47S / 166S、44L / 47S / 166S / 271H、44L / 47S / 247N / 271H / 392M、44L / 3 16L / 322M / 392M、44L / 337A、47S / 166S / 206K / 217F / 247N / 337A、47S / 166S / 2 17F / 271H / 337A, 47S / 206K, 47S / 217F / 247N / 261A, 47S / 271H, 52N / 217F / 302Q / 316L, 92Y / 166S / 206K / 271H / 316L, 92Y / 166S / 217F / 261A / 271H / 392M92Y / 166S / 217F / 316L / 337A / 392M, 92Y / 166S / 247N, 92Y / 166S / 316L, 92Y / 206K / 322M, 92Y / 217F, 92Y / 217F / 271H / 337A, 92Y / 261A / 271H, 92Y / 271H, 166S / 217F / 316L / 322M / 337A, 166S / 247N / 271H / 316L , 166S / 316L / 322M / 337A, 206K / 217F, 217F / 392M, 247N / 316L, 316L / 322M / 368W, and 316L / 337A / 392M, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, and said recombinant alpha-galactosidase A is T / K36M / H92Y / P166S / D247N / G261A / D316L / T392M, P10T / E39M, P10T / E39M / R44L / T47S / H92Y / A206K / R217F, P10T / E39M / R44L / T47S / D316L, P10T / E39M / R44L / T47S / P337A, P10T / E39M / R44L / H92Y / P166S / G261A / D316L / I322M, P10T / E39M / R44L / H92Y / P166S / K302Q / I322M, P10T / E39M / R44L / H92Y / P166S / T 392M, P10T / E39M / R44L / H92Y / R217F / K302Q / I322M, P10T / E39M / R44L / H92Y / K302Q / I322M, P10T / E39M / R44L / P166S / G261A / A271H / D316L / I322M, P10T / E39M / R44L / T392M, P10T / E39M / T47S / H92Y / P337A, P10T / E39M / H92Y / W131G / P166S / A271H / D316L / I322M, P10T / E39M / H92Y / P166S / R217F / D247N / A271H,P10T / E39M / H92Y / R217F / D316L、P10T / R44L / T47S / P166S / G261A / A271H、P10T / R44L / T47S / P166S / A271H / I322M / A368W、P10T / R44L / T47S / R217F / A271H / D316L / I322M、P10T / R44L / H92Y、P、 10T / R44L / H92Y / R217F / D247N / A271H / K302Q / D316L / T392M, P10T / R44L / P166S / K302Q, P10T / R44L / A206K / D316L / I322M, P10T / T47S / H92Y / P166S / A2 71H / D316L / P337A、P10T / T47S / H92Y / A271H / K302Q、P10T / T47S / H92Y / D316 L / I322M / T392M、P10T / T47S / P166S / A271H、P10T / T47S / P166S / D316L、P10T / H92Y / P166S、P10T / H92Y / P166S / R217F / D247N / G261A / A271H、P10T / H92Y / P166S / G261A / A271H / T392M、P10T / H92Y / P166S / G261A / D316L / I322M / P33 7A, P10T / H92Y / P166S / P337A / A368W, P10T / H92Y / K302Q / P337A, P10T / H92Y / D316L / I322M, P10T / A206K, P10T / A206K / D247N / G261A, P10T / R217F / I322 M、P10T / G261A、P10T / G261A / P337A / T392M、P10T / D316L / T392M、P10T / A36 8W、E39M / R44L / T47S / H92Y / P166S / A206K / T392M、E39M / R44L / T47S / H92Y / A 206K / D247N / G261A, E39M / R44L / T47S / H92Y / A206K / T392M, E39M / R44L / T47S / A206K / P337A / A368W / T392M, E39M / R44L / H92Y / P166S / D247N / G261A / K30 2Q / P337A, E39M / R44L / P166S / A271H, E39M / R44L / P166S / A271H / P337A / A368W / T392M, E39M / T47S / H92Y / D316L / I322M, E39M / T47S / H92Y / T392M, E39M / T47S / P166S / R217F / G261A / T392M、E39M / T47S / R217F / D247N / A368W、E39M / T47S / D247N、E39M / H92Y / P166S / R217F / T392M、E39M / H92Y / G261A / K302Q、E39M / P166S / R217F / G261A / D316L / A368W, E39M / I322M, E39M / T392M, R44L / T47S, R44L / T47S / H92Y / R217F / A271H, R44L / T47S / H92Y / R217F / D316L / I322M / T392M, R44L / T47S / H92Y / T392M, R44L / T47S / P166S, R44L / T47S / P166S / A271H, R44L / T47S / D247N / A271H / T392M, R44L / D316L / I322M / T392M, R44L / P337A, T47S / P166S / A206K / R217F / D247N / P337A, T47S / P166S / R217F / A271H / P337A, T47S / A206K, T47S / R217F / D247N / G261A, T47S / A271H, D52N / R217F / K302Q / D316L, H92Y / P166S / A206K / A271H / D316L, H9 2Y / P166S / R217F / G261A / A271H / T392M, H92Y / P166S / R217F / D316L / P337A / T392M, H92Y / P166S / D247N, H92Y / P166S / D316L, H9 2Y / A206K / I322M, H92Y / R217F, H92Y / R217F / A271H / P337A, H92Y / G261A / A271H, H92Y / A271H, P166S / R217F / D316L / I322M / P33 and at least one substitution or set of substitutions at one or more positions selected from: P166S / D247N / A271H / D316L, P166S / D316L / I322M / P337A, A206K / R217F, R217F / T392M, D247N / D316L, D316L / I322M / A368W, and D316L / P337A / T392M, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374.

[0141] In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 704, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 2, 4, 5, 24 / 59, 24 / 143 / 144, 24 / 143 / 202 / 333, 24 / 143 / 202 / 352 / 390 / 391, 24 / 143 / 333 / 352 / 387 / 390 / 391, 24 / 143 / 390 / 391, 24 / 202, 24 / 202 / 271, 24 / 202 / 333 / 352, 24 / 271 / 352, 24 / 352 / 387 / 390 / 391, 24 / 387 / 391, 31, 40, 59, 59 / 14 3, 59 / 143 / 202, 59 / 143 / 202 / 271 / 333, 59 / 143 / 271, 59 / 143 / 333, 59 / 202, 59 / 202 / 333, 59 / 271 / 387 / 390, 73, 76, 80, 83, 84, 91 / 215 / 361, 122, 123, 14 3, 143 / 202, 143 / 271, 143 / 271 / 352 / 390, 143 / 333, 143 / 333 / 387 / 390, 143 / 387 / 391, 147, 155, 164, 165, 179, 186, 202, 202 / 333, 210, 215 / 218, 218, 2 18 / 361, 218 / 361 / 398, 218 / 398, 246, 254 / 398, 271, 271 / 333, 271 / 333 / 390 / 391, 271 / 333 / 391, 271 / 352 / 391, 273, 275, 277, 278, 280, 281, 283, 284, In some embodiments, the polypeptide comprises at least one substitution or set of substitutions at one or more positions selected from: 287, 300, 303, 304, 325, 331, 332, 333 / 352, 333 / 390 / 391, 333 / 391, 334, 335, 336, 338, 339, 340, 341, 343, 359, 360, 361, 362, 367, 369, 371, 373, 375, 377, 382, ​​382 / 398, 385, 387 / 391, 390, and 398, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 704.The recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 704, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 2S, 4L, 5M, 5V, 24S / 59A, 24S / 143S / 144N, 24S / 143S / 202N / 333N, 24S / 143S / 202N / 352N / 390N / 391N, 24S / 143S / 333N / 391N, 24S / 143S / 333N / 392N, 24S / 143S / 333N / 393N, 24S / 143S / 333N / 394N, 24S / 143S / 333N / 395N, 24S / 143S / 333N / 396N, 24S / 143S / 333N / 397N, 24S / 143S / 333N / 398N, 24S / 143S / 333N / 39 ... 52N / 387N / 390T / 391N, 24S / 143S / 390T / 391N, 24S / 202N, 24S / 202N / 271N, 2 4S / 202N / 333N / 352N, 24S / 271N / 352N, 24S / 352N / 387N / 390N / 391N, 24S / 387 N / 391N, 31F, 31H, 31L, 31T, 31W, 40Q, 59A, 59A / 143S, 59A / 143S / 271N, 59A / 202N, 59T, 59T / 143S / 202N, 59T / 143S / 333N, 59T / 202N / 333N, 59V / 143S / 202 N / 271N / 333N, 59V / 271N / 387N / 390T, 73A, 76A, 76F, 76M, 76S, 80T, 83R, 83S , 84G, 84K, 84R, 91S / 215S / 361T, 122E, 122N, 122S, 123Q, 123R, 123S, 123T, 143S, 143S / 202N, 143S / 271N, 143S / 271N / 352N / 390N, 143S / 333N, 143S / 33 3N / 387N / 390T, 143S / 387N / 391N, 147L, 147S, 155A, 155D, 155F, 155L, 155R, 155T, 164E, 165I, 179H, 179L, 179R, 179W, 186E, 186F, 186M, 186P, 186R, 18 6S, 186Y, 202N, 202N / 333N, 210I, 215S / 218Y, 218Y, 218Y / 361T, 218Y / 361T / 398F, 218Y / 398F, 246Y, 254T / 398F, 271N, 271N / 333N, 271N / 333N / 390N / 39 1N, 271N / 333N / 391N, 271N / 352N / 391N, 273L, 275A, 275G, 277Q, 277V, 278N,278R, 278S, 280G, 281I, 281M, 283L, 283P, 283T, 283V, 284A, 284E, 284G, 284L, 28 4M, 284R, 284S, 287R, 300F, 303A, 303C, 303W, 304T, 304V, 304W, 325A, 331M, 332G , 332H, 333N / 352N, 333N / 390N / 391N, 333N / 390S / 391N, 333N / 391N, 334C, 334V, 3 35A, 335L, 336F, 336G, 336S, 336T, 338L, 339G, 339N, 339Q, 339V, 340H, 340I, 340 and 398F, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO:704. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 704, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of D2S, G4L, L5M, L5V, D24S / C59A, D24S / C143S / D144N, D24S / C143S / D202N / G333N, D 24S / C143S / D202N / F352N / M390N / Q391N, D24S / C143S / G333N / F352N / E 387N / M390T / Q391N, D24S / C143S / M390T / Q391N, D24S / D202N, D24S / D2 02N / A271N, D24S / D202N / G333N / F352N, D24S / A271N / F352N, D24S / F35 2N / E387N / M390N / Q391N, D24S / E387N / Q391N, S31F, S31H, S31L, S31T,S31W, E40Q, C59A, C59A / C143S, C59A / C143S / A271N, C59A / D202N, C59T, C59T / C143S / D202N, C59T / C143S / G333N, C59T / D202N / G333N, C59V / C143S / D20 2N / A271N / G333N, C59V / A271N / E387N / M390T, G73A, Q76A, Q76F, Q76M, Q76S, Q80T, P83R, P83S, H84G, H84K, H84R, N91S / T215S / R361T, D122E, D122N, D12 2S, I123Q, I123R, I123S, I123T, C143S, C143S / D202N, C143S / A271N, C143S / A271N / F352N / M390N, C143S / G333N, C143S / G333N / E387N / M390T, C143S / E 387N / Q391N、E147L、E147S、H155A、H155D、H155F、H155L、H155R、H155T、G16 4E、R165I、P179H、P179L、P179R、P179W、T186E、T186F、T186M、T186P、T186R、 T186S, T186Y, D202N, D202N / G333N, S210I, T215S / N218Y, N218Y, N218Y / R361T, N218Y / R361T / L398F, N218Y / L398F, W246Y, A254T / L398F, A271N, A271 N / G333N, A271N / G333N / M390N / Q391N, A271N / G333N / Q391N, A271N / F352N / Q391N, S273L, Q275A, Q275G, K277Q, K277V, A278N, A278R, A278S, L280G, Q28 1I、Q281M、K283L、K283P、K283T、K283V、D284A、D284E、D284G、D284L、D284M 、D284R、D284S、A287R、L300F、G303A、G303C、G303W、D304T、D304V、D304W、R 325A, P331M, R332G, R332H, G333N / F352N, G333N / M390N / Q391N, G333N / M390S / Q391N, G333N / Q391N, Y334C, Y334V, T335A, T335L, I336F, I336G, I336SI336T, V338L, A339G, A339N, A339Q, A339V, S340H, S340I, S340K, S340M, S340P, S340W, L341F, L341M, K343L , K343R, K343S, K343W, V359F, V359L, V359R, K360H, K360V, R361T, R361V, K362H, E367A, E367D, E367L, E367 M, T369D, R371G, R373L, R373S, H375L, H375Q, N377Q, V382I, V382I / L398F, Q385R, E387N / Q391N, M390S, and L398F, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 704.

[0142] In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374 or a functional fragment thereof, wherein the recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from 10, 39, 44, 47, 92, 166, 206, 217, 247, 261, 271, 302, 316, 322, 337, 368, and 392, and wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of 10A, 10C, 10D, 10E, 10F, 10G, 10H, 10I, 10K, 10L, 10M, 10N, 10Q, 10R, 10S, 10T, 10V, 10W, 10Y, 39A, 39C, 39D, 39F, 39G, 39H, 39I, 39K, 39L, 39M, 39N, 39P, 39Q, 39R, 39S, 39T, 39V, 39W, 39Y, 44A, 44C, 44D, 44E, 44F, 4 4G, 44H, 44I, 44K, 44L, 44N, 44P, 44Q, 44S, 44T, 44V, 44W, 44Y, 47A, 47C, 47D, 47E, 47F, 47G, 47H, 47I, 4 7K, 47L, 47M, 47N, 47P, 47Q, 47R, 47S, 47V, 47W, 47Y, 92A, 92C, 92D, 92E, 92F, 92G, 92I, 92K, 92L, 92M, 92 N, 92P, 92Q, 92R, 92S, 92T, 92V, 92W, 92Y, 166A, 166C, 166D, 166E, 166F, 166G, 166H, 166I, 166K, 166L, 166M, 166N, 166Q, 166R, 166S, 166T, 166V, 166W, 166Y, 206C, 206D, 206E, 206F, 206G, 206H, 206I, 206K,206L, 206M, 206N, 206P, 206Q, 206R, 206S, 206T, 206V, 206W, 206Y, 217A, 217C, 217D, 217E, 217F, 217G, 217H, 217I, 217K, 217L, 217M, 217N, 217P, 217Q 217S, 217T, 217V, 217W, 217Y, 247A, 247C, 247E, 247F, 247G, 247H, 247I, 247K, 247L, 247M, 247N, 247P, 247Q, 247R, 247S, 247T, 247V, 247W, 247Y, 261A 261C、261D、261E、261F、261H、261I、261K、261L、261M、261N、261P、261Q、26 1R、261S、261T、261V、261W、261Y、271C、271D、271E、271F、271G、271H、271I、 271K, 271L, 271M, 271N, 271P, 271Q, 271R, 271S, 271T, 271V, 271W, 271Y, 302A, 302C, 302D, 302E, 302F, 302G, 302H, 302I, 302L, 302M, 302N, 302P, 302Q 302R、302S、302T、302V、302W、302Y、316A、316C、316E、316F、316G、316H、31 6I、316K、316L、316M、316N、316P、316Q、316R、316S、316T、316V、316W、316Y、 322A, 322C, 322D, 322E, 322F, 322G, 322H, 322K, 322L, 322M, 322N, 322P, 322Q, 322R, 322S, 322T, 322V, 322W, 322Y, 337A, 337C, 337D, 337E, 337F, 337G 337H, 337I, 337K, 337L, 337M, 337N, 337Q, 337R, 337S, 337T, 337V, 337W, 337Y, 368C, 368D, 368E, 368F, 368G, 368H, 368I, 368K, 368L, 368M, 368N, 368P 368Q, 368R, 368S, 368T, 368V, 368W, 368Y, 392A, 392C, 392D, 392E, 392F, 392G, 392H, 392I, 392K, 392L, 392M, 392N, 392P, 392Q, 392R, 392S, 392V, 392Wand 392Y, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 374. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 374, or a functional fragment thereof, wherein said recombinant alpha-galactosidase A is selected from the group consisting of P10A, P10C, P10D, P10E, P10F, P10G, P10H, P10I, P10K, P10L, P10M, P10N , P10Q, P10R, P10S, P10T, P10V, P10W, P10Y, E39A, E39C, E39D, E39F, E39G, E39H, E39I, E39K, E39L, E39M, E39N, E39P, E39Q, E39R, E39S, E3 9T, E39V, E39W, E39Y, R44A, R44C, R44D, R44E, R44F, R44G, R44H, R44I, R44K, R44L, R44N, R44P, R44Q, R44S, R44T, R44V, R44W, R44Y, T47A, T 47C, T47D, T47E, T47F, T47G, T47H, T47I, T47K, T47L, T47M, T47N, T47P, T47Q, T47R, T47S, T47V, T47W, T47Y, H92A, H92C, H92D, H92E, H92F , H92G, H92I, H92K, H92L, H92M, H92N, H92P, H92Q, H92R, H92S, H92T, H92V, H92W, H92Y, P166A, P166C, P166D, P166E, P166F, P166G, P166H, P 166I, P166K, P166L, P166M, P166N, P166Q, P166R, P166S, P166T, P166V, P166W, P166Y, A206C, A206D, A206E, A206F, A206G, A206H, A206I, A206K, A206L, A206M, A206N, A206P, A206Q, A206R, A206S, A206T, A206V, A206W, A206Y, R217A, R217C, R217D, R217E, R217F, R217G, R217H,R217I, R217K, R217L, R217M, R217N, R217P, R217Q, R217S, R217T, R217V, R217W, R217Y, D247A, D247C, D247E, D247F, D247G, D247H, D247I, D247K, D247 L、D247M、D247N、D247P、D247Q、D247R、D247S、D247T、D247V、D247W、D247Y、 G261A、G261C、G261D、G261E、G261F、G261H、G261I、G261K、G261L、G261M、G26 1N、G261P、G261Q、G261R、G261S、G261T、G261V、G261W、G261Y、A271C、A271D 、A271E、A271F、A271G、A271H、A271I、A271K、A271L、A271M、A271N、A271P、A 271Q, A271R, A271S, A271T, A271V, A271W, A271Y, K302A, K302C, K302D, K302E, K302F, K302G, K302H, K302I, K302L, K302M, K302N, K302P, K302Q, K302R K302S, K302T, K302V, K302W, K302Y, D316A, D316C, D316E, D316F, D316G, D316H, D316I, D316K, D316L, D316M, D316N, D316P, D316Q, D316R, D316S, D316 T、D316V、D316W、D316Y、I322A、I322C、I322D、I322E、I322F、I322G、I322H、 I322K、I322L、I322M、I322N、I322P、I322Q、I322R、I322S、I322T、I322V、I32 2W, I322Y, P337A, P337C, P337D, P337E, P337F, P337G, P337H, P337I, P337K, P337L, P337M, P337N, P337Q, P337R, P337S, P337T, P337V, P337W, P337Y, A 368C, A368D, A368E, A368F, A368G, A368H, A368I, A368K, A368L, A368M, A368N, A368P, A368Q, A368R, A368S, A368T, A368V, A368W, A368Y, T392A, T392Cand at least one substitution or set of substitutions at one or more positions selected from T392D, T392E, T392F, T392G, T392H, T392I, T392K, T392L, T392M, T392N, T392P, T392Q, T392R, T392S, T392V, T392W, and T392Y, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374.

[0143] In some embodiments, the polynucleotide encodes a recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1022 or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is 0 / 392, 31, 31 / 39 / 44 / 166 / 302, 31 / 47, 31 / 283 / 284, 39, 39 / 44, 39 / 44 / 47, 39 / 44 / 47 / 261 / 283 / 284, 39 / 44 / 283, 39 / 44 / 339, 39 / 47 / 261, 39 / 92, 39 / 206, 39 / 284, 44, 44 / 284 / 302, 84, 84 / 92, 84 / 284 / 302 / 392, 84 / 316, 84 / 368 / 392, 92, 92 / 206 / 217, 92 / 206 / 275, 92 / 206 / 284, 92 / 206 / 302 / 368, 92 / 271, 92 / 271 / 277, 92 / 275 / 284, 92 / 283, 92 / 283 / 392, 92 / 284, 92 / 302, 92 / 316, 92 / 368, 155, 155 / 217, 155 / 368, 166, 166 / 283 / 284, 166 / 302, 206, 206 / and at least one substitution or set of substitutions at one or more positions selected from 217, 206 / 334, 261, 261 / 283, 271, 271 / 368, 275, 283, 283 / 284, 283 / 392, 284, 302, 316, 334, 339, 368, 368 / 392, and 392, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1022. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1022, or a functional fragment thereof, wherein the recombinant alpha-galactosidase A is selected from the group consisting of P10G, P10G / T392D, S31T, S31T / E39V / R44V / P166D / K302Y, S31T / T47R, S31T / K283L / D284A, E39L, E39L / H92V, E39L / A206E,E39L / D284S, E39V / R44V, E39V / R44V / T47R, E39V / R44V / T47R / G261S / K283L / D284A, E39V / R44V / K283T, E39V / R44V / A339 N, E39V / T47R / G261S, R44V, R44V / D284E / K302Y, H84K, H84K / H92V, H84K / D284S / K302L / T392A, H84K / D316H, H84K / A368E / T392A, H92Q, H92T, H92T / A206E / R217N, H92T / A206E / K302T / A368E, H92T / A271K, H92T / A271K / K277R, H92T / K283P, H92T / K283V / T392W, H92T / D284M, H92T / K302L, H92T / A368E, H92V, H92V / A206E / D284S, H92V / A206Y / Q275A, H92V / Q275A / D284 S, H92V / D284S, H92V / K302L, H92V / D316H, H155F, H155F / R217I, H155F / A368E, P166D, P166D / K283L / D284A, P166D / K302 Y, A206E, A206E / R217N, A206I, A206Q, A206T / Y334C, A206Y, G261S, G261S / K283L, A271K, A271K / A368E, Q275A, K283L, K2 and at least one substitution or set of substitutions at one or more positions selected from 83P / T392W, K283T, K283T / D284E, D284E, D284M, D284S, K302L, K302Y, D316H, Y334C, A339N, A368E, A368E / T392W, T392A, T392D, and T392W, wherein the amino acid positions of said polypeptide sequence are numbered with reference to SEQ ID NO: 1022. In some embodiments, the recombinant alpha-galactosidase A comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 1022, or a functional fragment thereof, and the recombinant alpha-galactosidase A is selected from the group consisting of 10G, 10G / 392D, 31T, 31T / 39V / 44V / 166D / 302Y,31T / 47R, 31T / 283L / 284A, 39L, 39L / 92V, 39L / 206E, 39L / 284S, 39V / 44V, 39V / 44V / 47R, 39V / 44V / 47R / 261S / 283L / 284A, 39V / 44V / 283T, 39V / 44V / 339N, 39V / 47R / 261S, 44V, 44V / 284E / 302Y, 84K, 84K / 92V, 84K / 284S / 3 02L / 392A, 84K / 316H, 84K / 368E / 392A, 92Q, 92T, 92T / 206E / 217N, 92T / 206E / 302T / 368E, 92T / 271K, 92T / 271 K / 277R, 92T / 283P, 92T / 283V / 392W, 92T / 284M, 92T / 302L, 92T / 368E, 92V, 92V / 206E / 284S, 92V / 206Y / 275A, 9 2V / 275A / 284S, 92V / 284S, 92V / 302L, 92V / 316H, 155F, 155F / 217I, 155F / 368E, 166D, 166D / 283L / 284A, 166D / 302Y, 206E, 206E / 217N, 206I, 206Q, 206T / 334C, 206Y, 261S, 261S / 283L, 271K, 271K / 368E, 275A, 283L, 283 and at least one substitution or set of substitutions at one or more positions selected from 283T, 283T / 284E, 284E, 284M, 284S, 302L, 302Y, 316H, 334C, 339N, 368E, 368E / 392W, 392A, 392D, and 392W, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1022.

[0144] In some embodiments, as described above, the polynucleotide encodes an engineered polypeptide having GLA activity with the properties disclosed herein, wherein the polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, or 92% similar to the amino acid sequence of a reference sequence (e.g., SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704, and 1022) or any variant disclosed in any of the tables herein. , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity and having one or more residue differences (e.g., at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions) when compared to the amino acid sequence of the reference polypeptide of SEQ ID NO: 8 or any variant disclosed in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and / or 13-1. In some embodiments, the polynucleotide encodes an engineered polypeptide having GLA activity with the properties disclosed herein, wherein the polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more similar to the reference sequence of SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022. and / or 1022, when aligned with SEQ ID NOs: 8, 58, 158, 372, 374, 704, and / or 1022 as appropriate, and have one or more residue differences at residue positions selected from those presented in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1 and / or 13-1 compared to SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704, and / or 1022 as appropriate.

[0145] In some embodiments, the polynucleotide encoding the engineered GLA polypeptide comprises the polynucleotide sequence of SEQ ID NO: 8, 58, 158, 372, 374, 704, and / or 1022. In some embodiments, the polynucleotide is capable of hybridizing to a reference polynucleotide sequence under highly stringent conditions. In some embodiments, the reference sequence is selected from a polynucleotide sequence encoding SEQ ID NO: 1, 7, 57, 157, 275, 371, 373, and / or 1019, or a complement thereof, or any of the variant GLA polypeptides provided herein. In some embodiments, a polynucleotide capable of hybridizing under highly stringent conditions encodes a GLA polypeptide comprising an amino acid sequence having one or more residue differences at a residue position selected from any of the positions set forth in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1 and / or 13-1 when compared to SEQ ID NOs: 2, 8, 58, 158, 372, 374, 704 and / or 1022.

[0146] In some embodiments, an isolated polynucleotide encoding any of the engineered GLA polypeptides provided herein is manipulated in various ways to provide for expression of the polypeptide. In some embodiments, the polynucleotide encoding the polypeptide is provided as an expression vector in which one or more regulatory sequences are present to regulate expression of the polynucleotide and / or polypeptide. Manipulation of the isolated polynucleotide prior to insertion into a vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0147] In some embodiments, the control sequences include, among other sequences, promoters, Kozak sequences, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, DNA-based regulatory elements for gene therapy delivery, and transcription terminators. As is known in the art, appropriate promoters can be selected based on the host cell to be used. Exemplary promoters for filamentous fungal host cells include promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (Aspergillus niger neutral alpha-amylase and Aspergillus Examples of promoters that can be used include promoters from the Saccharomyces cerevisiae gene (e.g., a hybrid of the promoter from the Saccharomyces cerevisiae gene for triosephosphate isomerase), as well as mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, e.g., Romanos et al., Yeast 8:423-488

[1992] ).Exemplary promoters for use in mammalian cells include, but are not limited to, those derived from cytomegalovirus (CMV), the chicken beta-actin promoter fused to the CMV enhancer, from simian vacuolating virus 40 (SV40), from Homo sapiens phosphoglycerate kinase, beta-actin, elongation factor-1a or glyceraldehyde-3-phosphate dehydrogenase, or from chicken beta-actin.

[0148] In some embodiments, the control sequence is a suitable transcription terminator sequence, i.e., a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that is functional in the selected host cell can be used in the present invention. For example, exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (see, for example, Romanos et al., supra). Exemplary terminators for mammalian cells include, but are not limited to, those derived from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), homo sapiens growth hormone (hGH), bovine growth hormone (BGH), and human or rabbit beta globulin.

[0149] In some embodiments, the regulatory sequence is a suitable leader sequence, 5' cap modification, 5' UTR, etc. In some embodiments, these regulatory sequence elements mediate binding to molecules involved in mRNA transport and translation, inhibit 5'-exonucleolytic degradation, and confer resistance to decapping. The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the selected host cell may be used. Exemplary leaders for filamentous fungal host cells are Aspergillus oryzae TAKA amylase and Aspergillus Leaders suitable for yeast host cells include, but are not limited to, those derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP). Suitable leaders for mammalian host cells include, but are not limited to, the 5'-UTR elements present in orthopoxvirus mRNAs.

[0150] In some embodiments, the regulatory sequence includes a 3' untranslated nucleic acid region and a polyadenylation tail nucleic acid sequence, a sequence operably linked to the 3' end of a protein-encoding nucleic acid sequence that mediates binding to proteins involved in mRNA transport and translation and mRNA half-life. Any polyadenylation sequence and 3' UTR that is functional in the selected host cell can be used in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, those derived from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, e.g., Guo and Sherman, Mol. Cell Biol., 15:5983-5990

[1995] ). Useful polyadenylation and 3' UTR sequences for mammalian host cells include, but are not limited to, the 3' UTRs of alpha globin mRNA and beta globin mRNA, which contain several sequence elements that increase mRNA stability and translation.

[0151] In some embodiments, the control sequence is a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the secretory pathway of a cell. The 5' end of the coding sequence of a nucleic acid sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. Any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice may be used for expression of the engineered GLA polypeptides provided herein. Effective signal peptide coding regions for filamentous fungal host cells include those from Aspergillus oryzae. Signal peptide coding regions obtained from the genes for TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucormiehei aspartic proteinase, Humicolainsolens cellulase, and Humicolalanuginosa lipase are included, but are not limited to these. Useful signal peptides for yeast host cells include Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae alpha-factor. Useful signal peptides for mammalian host cells include, but are not limited to, those derived from the gene for immunoglobulin gamma (IgG).

[0152] In some embodiments, the control sequence is a propeptide coding region that encodes an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is sometimes referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

[0153] In another aspect, the present invention also provides recombinant expression vectors comprising a polynucleotide encoding an engineered GLA polypeptide and one or more expression control regions, such as a promoter, terminator, or origin of replication, depending on the type of host into which it will be introduced. In some embodiments, the various nucleic acids and control sequences described above are ligated together to create a recombinant expression vector containing one or more convenient restriction sites that allow for the insertion or substitution of a nucleic acid sequence encoding a variant GLA polypeptide. Alternatively, the polynucleotide sequences of the present invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct containing the polynucleotide sequence into an appropriate vector for expression. In creating an expression vector, the coding sequence is placed in the vector so that the coding sequence is operably linked to appropriate control sequences for expression.

[0154] The recombinant expression vector can be any vector (e.g., a plasmid or virus, including but not limited to, adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), and non-viral vectors such as liposomes) that can be conveniently subjected to recombinant DNA procedures and that can result in expression of the mutant GLA polynucleotide sequence. The choice of vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0155] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome). The vector may include any means for ensuring self-replication. In some alternative embodiments, the vector may be one that, when introduced into a host cell, is integrated into the genome and replicated together with the chromosome into which it has been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, or transposons, may be used that together contain the total DNA to be introduced into the genome of the host cell.

[0156] In some embodiments, expression vectors preferably contain one or more selectable markers that allow for easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and their equivalents. In another aspect, the present invention provides a host cell comprising a polynucleotide encoding at least one engineered GLA polypeptide of the present application, the polynucleotide being operably linked to one or more control sequences for expression of the engineered GLA enzyme in the host cell. Host cells for use in expressing polypeptides encoded by the expression vectors of the present invention are well known in the art and include, but are not limited to, fungal cells, e.g., yeast cells (e.g., Saccharomyces cerevisiae and Pichia pastoris [e.g., ATCC Accession No. 201178]), insect cells (e.g., Drosophila S2 and Spodoptera Sf9 cells), plant cells, animal cells (e.g., CHO, CHO-K1, COS, and BHK), and human cells (e.g., HEK293T, human fibroblast, THP-1, Jurkat, and Bowes melanoma cell lines).

[0157] Thus, in another aspect, the present invention provides a method for producing an engineered GLA polypeptide, comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered GLA polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises isolating and / or purifying the GLA polypeptide described herein.

[0158] Suitable culture medium and growth conditions for the above-mentioned host cells are well known in the art.The polynucleotide for expressing GLA polypeptide can be introduced into cells by various methods known in the art.Techniques include, inter alia, electroporation, biolistic particle bombardment, liposome-mediated transfection, calcium chloride transfection and protoplast fusion.

[0159] The engineered GLA with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding naturally occurring or engineered GLA polypeptide to mutagenesis and / or directed evolution methods known in the art and described herein.Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, for example, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751

[1994] , WO 95 / 22625, WO 97 / 0078, WO 97 / 35966, WO 98 / 27230, WO 00 / 42651, WO 01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include, inter alia, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

[1998] ), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140

[1994] ), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529

[1996] ).

[0160] For example, mutagenesis and directed evolution methods can be readily applied to polynucleotides to generate libraries of variants that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (see, e.g., U.S. Patent Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,277,638, 6,287,861, 6,287,862). No. 6,291,242, No. 6,297,053, No. 6,303,344, No. 6,309,883, No. 6,319,713, No. 6,319,714, No. 6,323,030, No. 6,326,204, No. 6,335, No. 160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,484, No. 6,358,740, No. 6,358,742, No. 6,365,377, No. 6,365,408, No. 6,36 No. 8,861, No. 6,372,497, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. 6,391,552, No. 6,391,640, No. 6,395,547, No. 6,406,855, No. 6 ,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, 6,426,224, 6,436,675, 6,444,468, 6,455,253, No. 6,479,652, No. 6,482,647, No. 6,489,146, No. 6,506,602, No. 6,506,603, No. 6,519,065, No. 6,521,453, No. 6,528,311, No. 6,537,74 No. 6, No. 6,573,098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,613,514, No. 6,653,072, No. 6,716,631, No. 6,946,No. 296, No. 6,961,664, No. 6,995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297 , No. 7,148,054, No. 7,288,375, No. 7,421,347, No. 7,430,477, No. 7,534,564, No. 7 ,620,500, 7,620,502, 7,629,170, 7,702,464, 7,747,391, 7,747 ,393, No. 7,751,986, No. 7,776,598, No. 7,783,428, No. 7,795,030, No. 7,853,410 Nos. 7,868,138, 7,873,499, 7,904,249, 7,957,912, 8,383,346, 8,504,498, 8,849,575, 8,876,066, 8,768,871, 9,593,326, and all related non-U.S. counterparts; Ling et al., Anal. Biochem., 254(2):157-78

[1997] ; Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ; Smith, Ann. Rev. Genet., 19:423-462

[1985] ; Botstein et al., Science, 229:1193-1201

[1985] , Carter, Biochem. J., 237:1-7

[1986] , Kramer et al., Cell, 38:879-887

[1984] , Wells et al., Gene, 34:315-323

[1985] , Minshull et al., Curr. Op. Chem. Biol., 3:284-290

[1999] , Christians et al., Nat. Biotechnol., 17:259-264

[1999] , Crameri et al., Nature, 391:288-291

[1998] , Crameri et al., Nat. Biotechnol., 15:436-438

[1997] , Zhang et al., Proc. Nat. Acad. Sci. USA, 94:4504-4509

[1997] , Crameri et al., Nat. Biotechnol., 14:315-319

[1996] , Stemmer, Nature, 370:389-391

[1994] , St emmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751

[1994] , U.S. Patent Application Publication No. 2008 / 0220990, U.S. Patent Application Publication No. 2009 / 0312196, U.S. Patent Application Publication No. 2014 / 0005057, U.S. Patent Application Publication No. 2014 / 0214391, U.S. Patent Application Publication No. 2014 / 0221216, U.S. Patent Application Publication No. 2015 / 0050658, U.S. Patent Application Publication No. 2015 / 0050659 See U.S. Patent Application Publication No. 0133307, U.S. Patent Application Publication No. 2015 / 0134315, and all related non-U.S. counterparts, WO 95 / 22625, WO 97 / 0078, WO 97 / 35966, WO 98 / 27230, WO 00 / 42651, WO 01 / 75767, and WO 2009 / 152336, all of which are incorporated herein by reference).

[0161] In some embodiments, the enzyme variants obtained after mutagenesis are screened by subjecting the enzyme variants to a defined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatment or other assay conditions. DNA containing a polynucleotide encoding a GLA polypeptide is then isolated from the host cell, sequenced to identify nucleotide sequence changes (if any), and used to express the enzyme in a different or the same host cell. Measurement of enzyme activity from an expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques such as HPLC analysis).

[0162] For engineered polypeptides of known sequence, polynucleotides encoding the enzymes can be prepared by standard solid-phase methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then linked (e.g., by enzymatic or chemical ligation, or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (see, e.g., Beaucage et al., Tetra. Lett., 22:1859-69

[1981] and Matthes et al., EMBO J., 3:801-05

[1984] ), as typically performed in automated synthesis methods. Using the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automated DNA synthesizer), purified, annealed, ligated, and cloned into an appropriate vector.

[0163] Thus, in some embodiments, a method for preparing an engineered GLA polypeptide can include (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequences of any variants set forth in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1, and / or 13-1, and SEQ ID NOs: 8, 58, 158, 372, 374, 704, and / or 1022, and (b) expressing the GLA polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue mutations (e.g., deletions, insertions, and / or substitutions). In some embodiments, the amino acid sequence has 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residue mutations (e.g., deletions, insertions, and / or substitutions), as appropriate. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue mutations (e.g., deletions, insertions, and / or substitutions). In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue mutations (e.g., deletions, insertions, and / or substitutions). In some embodiments, the substitutions may be conservative or non-conservative.

[0164] The expressed engineered GLA polypeptides can be evaluated for any desired improved properties (e.g., activity, selectivity, stability, acid resistance, protease susceptibility, etc.) using any suitable assay known in the art, including, but not limited to, the assays and conditions described herein.

[0165] In some embodiments, any engineered GLA polypeptide expressed in the host cells is recovered from the cells and / or culture medium using one or more of the well-known techniques for protein purification, such as lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others.

[0166] Chromatographic techniques for isolating GLA polypeptides include, among others, reverse-phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, gel electrophoresis, and affinity chromatography. The conditions for purifying a particular enzyme depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, and molecular shape, and will be apparent to those skilled in the art. In some embodiments, affinity techniques can be used to isolate improved mutant GLA enzymes. In some embodiments utilizing affinity chromatography purification, any antibody that specifically binds to a mutant GLA polypeptide is used. In some embodiments utilizing affinity chromatography purification, a protein that binds to the glycan covalently attached to GLA is used. In yet other embodiments utilizing affinity chromatography purification, any small molecule that binds to the GLA active site is used. For antibody production, various host animals, including but not limited to rabbits, mice, and rats, are immunized by injection with a GLA polypeptide (e.g., a GLA mutant) or a fragment thereof. In some embodiments, the GLA polypeptide or fragment is conjugated to a suitable carrier, such as BSA, by a side chain functional group or a linker attached to a side chain functional group.

[0167] In some embodiments, the engineered GLA polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., S. cerevisiae, Daucus carota, Nicotiana tabacum, H. sapiens (e.g., HEK293T), or Cricetulus griseus (e.g., CHO)) comprising a polynucleotide sequence encoding an engineered GLA polypeptide described herein under conditions conducive to production of the engineered GLA polypeptide, and recovering the engineered GLA polypeptide from the cells and / or culture medium.

[0168] In some embodiments, the present invention provides for the production of engineered GLA polypeptides having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a reference sequence (e.g., SEQ ID NOs: 8, 58, 158, 372, 374, 704 and / or 1022) under suitable culture conditions that allow for the production of engineered GLA polypeptides, as appropriate, when aligned with SEQ ID NOs: 8, 58, 158, 372, 374, 704 and / or 1022. , 372, 374, 704 and / or 1022, comprising one or more amino acid residue differences selected from those presented in Tables 2-1, 5-1, 6-1, 7-1, 8-1, 9-1, 11-1, 12-1 and 13-1, and / or combinations thereof, and optionally recovering the engineered GLA polypeptide from the culture and / or cultured cells.

[0169] In some embodiments, once the engineered GLA polypeptides are recovered from the recombinant host cells or cell culture medium, they are further purified by any suitable method known in the art. In some further embodiments, the purified GLA polypeptides are combined with other ingredients and compounds to provide compositions and formulations comprising the engineered GLA polypeptides suitable for different applications and uses (e.g., pharmaceutical compositions). In some further embodiments, the purified or formulated engineered GLA polypeptides are lyophilized. In some embodiments, the engineered GLA polypeptides are produced directly in vivo (i.e., in cells within the body, such as a human or another animal) and are not purified. However, in some alternative embodiments, the engineered GLA polypeptides are produced in vivo (i.e., in cells within the body, such as a human or another animal) and recovered from the body using methods known in the art. In some further embodiments, these harvested engineered GLA polypeptides are purified. In still some further embodiments, these harvested and / or purified engineered polypeptides are introduced into another animal (e.g., a human or another animal) or reintroduced into the body in which the harvested and / or purified engineered GLA polypeptides were originally produced. composition

[0170] The present invention provides a variety of compositions and formats, including, but not limited to, those described below. In some embodiments, the present invention provides engineered GLA polypeptides suitable for use in pharmaceutical and other compositions, e.g., dietary / nutritional supplements.

[0171] These compositions comprising the therapeutically effective amount of engineered GLA according to the present invention are in the form of solid, semi-solid or liquid, depending on the mode of administration.In some embodiments, the compositions comprise other pharmaceutically acceptable components, such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, bulking agents and other components.Details about the techniques for formulation and administration are well known in the art and are described in the literature.In some embodiments, these compositions are directly produced in the human body after being introduced as gene therapy.

[0172] In some embodiments, the engineered GLA polypeptide is formulated for use in a pharmaceutical composition. The present invention utilizes any suitable delivery format for the engineered GLA polypeptide, including, but not limited to, pills, tablets, gel tabs, capsules, lozenges, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, jellies, paints, aerosols, gums, chewables, sticks, solutions, suspensions (including, but not limited to, oily suspensions and oil-in-water emulsions), slurries, syrups, controlled-release formulations, suppositories, and the like. In some embodiments, the engineered GLA polypeptide is provided in a format suitable for injection or infusion (i.e., in an injectable formulation). In some embodiments, the engineered GLA polypeptide polynucleotide sequence is provided in a format suitable for injection. In some embodiments, the engineered GLA polypeptide is provided in a biocompatible matrix, such as a sol-gel, including silica-based (e.g., oxysilane) sol-gels. In some embodiments, the engineered GLA polypeptide is encapsulated. In some alternative embodiments, the engineered GLA polypeptides are encapsulated in nanostructures (e.g., nanotubes, nanocapsules, or microcapsules, microspheres, liposomes, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery formulation and / or delivery means. It is contemplated that the engineered GLA polypeptides be administered by any suitable means known in the art, including, but not limited to, parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, implant, etc.

[0173] In some embodiments, engineered GLA polypeptides are chemically modified by glycosylation, chemical cross-linking reagents, pegylation (i.e., modification with polyethylene glycol [PEG] or activated PEG, etc.), or other compounds (see, e.g., Ikeda, Amino Acids 29:283-287

[2005] ; U.S. Patent Nos. 7,531,341; 7,534,595; 7,560,263; 7,536,533; U.S. Patent Application Publication Nos. 2013 / 0039898; 2012 / 0177722, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery method and / or mechanism.

[0174] In some further embodiments, engineered GLA polypeptides are provided for delivery to cells or tissues via gene therapy, such as viral delivery vectors, including, but not limited to, adenovirus (AV), adeno-associated virus (AAV), lentivirus (LV), or non-viral vectors (e.g., liposomes). In some embodiments, engineered GLA polypeptides are provided for delivery to cells or tissues via mRNA therapy after formulating the polyribonucleotide sequence in an encapsulated delivery vehicle (e.g., liposomes). In some further embodiments, engineered GLA polypeptides are provided for delivery to cells or tissues via cell therapy, in which a polynucleotide sequence encoding the engineered GLA polypeptide is introduced into an exogenous cell, and the cell (or cells) is introduced into a recipient (e.g., a patient exhibiting or at risk of developing Fabry disease).

[0175] In some further embodiments, the engineered GLA polypeptide is provided in the form of a formulation comprising a matrix-stabilized enzyme crystal. In some embodiments, the formulation comprises a crosslinked crystal of the engineered GLA enzyme and a polymer having a reactive moiety that is attached to the enzyme crystal. The present invention also provides an engineered GLA polypeptide in a polymer.

[0176] In some embodiments, compositions comprising engineered GLA polypeptides of the invention comprise one or more commonly used carrier compounds, including, but not limited to, sugars (e.g., lactose, sucrose, mannitol, and / or sorbitol), starches (e.g., corn, wheat, rice, potato, or other vegetable starches), celluloses (e.g., methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose), gums (e.g., acacia, tragacanth, guar, etc.), and / or proteins (e.g., gelatin, collagen, etc.).

[0177] In some embodiments, the present invention provides engineered GLA polypeptides suitable for use in reducing glycolipid concentrations in fluids such as blood and cerebrospinal fluid. The dosage of the engineered GLA polypeptide administered depends on the condition or disease, the subject's general condition, and other factors known to those skilled in the art. In some embodiments, the composition is intended for single or multiple administration. In some embodiments, it is contemplated that the concentration of the engineered GLA polypeptide in a composition administered to a person with Fabry disease will be sufficient to effectively treat and / or ameliorate the disease (e.g., Fabry disease). In some embodiments, the engineered GLA polypeptide is administered in combination with other pharmaceutical and / or dietary compositions. experiment [Example]

[0178] The following examples (including the experiments and results obtained) are provided for illustrative purposes only and are not to be construed as limiting the present invention. In the experimental disclosure that follows, the following abbreviations apply: ppm (parts per million); M (mole); mM (millimole), uM, and μM (micromolar); nM (nanomole); mol (mole); gm and g (grams); mg (milligram); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min(s) (minutes); h(s) and hr(s) (hours); U (units); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); SEM (standard error of the mean); IV (intravenous); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (Coli Genetic Stock) Commonly used laboratory E. coli strains available from the Center for Clinical Chemistry and Scientists [CGSC], New Haven, Connecticut; NHP (non-human primate); HPLC (high pressure liquid chromatography); MWCO (molecular weight cut-off); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); PBS (phosphate buffered saline); DPBS (Dulbecco's phosphate buffered saline); PES (polyethersulfone); CFSE (carboxyfluorescein succinimidyl ester); IPTG (isopropyl β-D-1-thiogalactopyranoside); PMBS (polymyxin B sulfate); NADPH (nicotinamide adenine dinucleotide phosphate); GIDH (glutamate dehydrogenase); FIOPC (fold improvement over positive control); PBMC (peripheral blood mononuclear cells); LB (Luria broth); MeOH (methanol); C max (maximum drug concentration during the dosing interval); RFU (relative fluorescence units); AUC 0-t(area under the curve to last measurable concentration); CL (clearance); Vz (apparent volume of distribution in the terminal phase); TI (test item); Athens Research (Athens Research Technology, Athens, Georgia); ProSpec (ProSpec Tany Technogene, East Brunswick, New Jersey); Sigma-Aldrich (Sigma-Aldrich, St. Louis, Missouri); Ram Scientific (Ram Scientific, Inc., Yonkers, New York); Pall Corp. (Pall Corp., Port Washington, New York); Millipore (Millipore Corp., Billerica, Massachusetts); Difco (Difco Laboratories, BD Diagnostic Systems, Detroit, Michigan); PerkinElmer (PerkinElmer, Waltham, Massachusetts); Molecular Devices (Molecular Devices, LLC, Sunnyvale, California); Kuhner (Adolf Kuhner, AG, Basel, Switzerland; Axygen (Axygen, Inc., Union City, California); Toronto Research Chemicals (Toronto Research Chemicals Inc., Toronto, Ontario, Canada); Cambridge Isotope Laboratories (Cambridge Isotope Laboratories, Inc., Tewksbury, Massachusetts); Applied Biosystems (Applied Biosystems, a division of Life Technologies, Grand Island, NY), Agilent (Agilent Technologies, Inc., Santa Clara, CA); Thermo Scientific (a division of ThermoFisher Scientific, Waltham, MA); Gibco (ThermoFisher Scientific); Pierce (Pierce Biotechnology (now a division of ThermoFisher Scientific), Rockford, IL); ThermoFisher Scientific (Thermo Fisher Scientific, Waltham, MA); Corning (Corning, Inc., Palo Alto, CA); XenoTech (Sekisui XenoTech, LLC, Kansas City, KS); Coriell Institute for Medical Research (Coriell Institute for Medical Research, Camden, NJ); VWR (VWR International, Radnor, PA); Jackson (The Jackson Laboratory, Bar Harbor, ME); Megazyme (Megazyme International, Wicklow, Ireland); Enzo (Enzo Life Sciences, Inc., Farmingdale, NY); GE Healthcare (GE Healthcare Bio-Sciences, Piscataway, NJ); LI-COR (LI-COR Biotechnology, Lincoln, Nebraska); Amicus (Amicus Therapeutics, Cranberry, NJ); Phenomenex (Phenomenex, Inc., Torrance, CA); Optimal (Optimal Biotech Group, Belmont, CA); and Bio-Rad (Bio-Rad Laboratories, Hercules, CA).

[0179] The following polynucleotide and polypeptide sequences are used in the present invention. In some cases (as shown below), the polynucleotide sequence is followed by the encoded polypeptide. Polynucleotide sequence of full-length human GLAc DNA (SEQ ID NO: 1): Polypeptide sequence of full-length human GLA: MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQAD PQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTE IRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 2) Polynucleotide sequence of mature yeast codon-optimized (yCDS) human GLA: TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTC (SEQ ID NO: 3) Polynucleotide sequence of mature human GLA (native hCDS): Polypeptide sequence of mature human GLA: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 5) Polynucleotide sequence of pCK110900i E. coli expression vector: Polynucleotide sequence of pYT-72Bgl secretion yeast expression vector: Polynucleotide sequence of variant number 73yCDS: TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCT GGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTC GGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACTGAGATAAGACAATACTGTAACCATTGGCGTAACTTTGCTGACATAGATGATTCATGGGCTTCAATCAAATCTATCTTGGATTGGACTTCTTTCAACCAGGAAAGAATTGTTGATGTTGCAGGTCCAGGTGGATGGAATGACCCTGATATGCTTGTCATAGGGAACTTTGGGCTATCATGGAATCAACAAGTTACACAAATGGCTTTGTGGGCGATCATGGCCGCACCCCTATTCATGTCTAATGATCTACGTCACATATCACCCCAAGCAAAGGCTTTACTTCAAGATAAGGATGTCATAGCGATCAACCAAGATCCTCTTGGTAAACAAGGTTATCAATTGAGACAAGGTGACAACTTTGAAGTGTGGGAAAGACCATTGTCTGGACTTGCGTGGGCTGTTGCTATGATCAACCGTCAAGAGATCGGAGGGCCAAGATCTTACACTATCGCGGTAGCCTCTTTGGGTAAGGGTGTTGCGTGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATGAGTGGACATCTAGGCTAAGAAGTCACATCAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA(SEQ ID NO: 8) Polynucleotide sequence of variant number 73: Polypeptide sequence of variant no. 73: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 10) Polynucleotide sequence of variant number 218yCDS: TGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGAAAGTTGGGTTTCTATAACTGGACATCTAGGCTAAAAAGTCACATTAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA (SEQ ID NO: 11) Polynucleotide sequence of variant number 218hCDS: Polypeptide sequence of variant number 218: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAIINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYNWTSRLKSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 13) Polynucleotide sequence of variant number 326yCDS: Polypeptide sequence of variant number 326: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFA DIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNWTSRLKSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 15) Polynucleotide sequence of variant number 206yCDS: Polynucleotide sequence of variant number 206hCDS: Polypeptide sequence of variant no. 206: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYNWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 18) Polynucleotide sequence of variant number 205yCDS: TTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTGATGATTGCTGGATGGCTCCACAGCGTGATTCAGAAGGTAGGTTACAAGCTGACCCCCAGAGATTCCCACATGGCATACGTCAGCTTGCAAACTACGTACACAGCAAGGGTCTAAAGTTAGGCATCTACGCTGATGTCGGAAACAAGACATGTGCTGGTTTCCCAGGTTCATTCGGTTACTATGACATAGATGCGCAGACGTTTGCTGATTGGGGTGTTGATTTGTTGAAGTTTGATGGATGCTACTGCGATTCCCTGGAGAACCTAGCCGATGGGTACAAACACATGAGTTTGGCTCTAAACAGGACTGGTAGGAGCATCGTCTATAGTTGTGAATGGCCCTTGTACATGTGGCCGTTTCAGAAGCCAAACTACACT (SEQ ID NO: 19) Polynucleotide sequence of variant number 205hCDS: Polypeptide sequence of variant number 205: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYDWDSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 21) Polynucleotide sequence of variant number 76yCDS: Polynucleotide sequence of variant number 76hCDS: Polypeptide sequence of variant no. 76: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 24) Polynucleotide sequence of Mf alpha signal peptide: ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCT (SEQ ID NO: 25) Polypeptide sequence of Mf alpha signal peptide: MRFPSIFTAVLFAASSALA (SEQ ID NO: 26) Polynucleotide sequence of MMO435: ttaactatatcgtaatacacaggatccaccATGAGATTTCCTTCAATTTTTACTG (SEQ ID NO: 27) Polynucleotide sequence of MMO439: AGTAGGTGTACGGGCTAACCCGTTATCCAAAGCTAATGCGGAGGATGC (SEQ ID NO: 28) Polynucleotide sequence of MMO514: TTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTTTGGATAACGGGTTAGCCCG (SEQ ID NO: 29) Polynucleotide sequence of MMO481: GAGCTAAAAGTACAGTGGGAACAAAGTCGAGGTCGACTTATAACAAATCTTTCAAAGACA (SEQ ID NO: 30) Polynucleotide sequence of synthetic mammalian signal peptide: ATGGAATGGAGCTGGGTCTTTCTCTTCTTCCTGTCAGTAACGACTGGTGTCCACTCC (SEQ ID NO: 31) Polynucleotide sequence of LAKE Fw: CGATCGAAGCTTCGCCACCA (SEQ ID NO: 32) Polynucleotide sequence of Br reverse: CTTGCCAATCCATTGTCCAGGGAGTGGACACCAGTCGTTA (SEQ ID NO: 33) Polynucleotide sequence of Br Fw: TAACGACTGGTGTCCACTCCCTGGACAATGGATTGGCAAG (SEQ ID NO: 34) Polynucleotide sequence of hGLA Rv: CGATCGGCGGCCGCTCAAAGTAAGTCTTTTAATGACA (SEQ ID NO: 35) Polynucleotide sequence of SP-GLA(yCDS): ATGAGATTTCCTTCAATTTTTACTGCAGTTTTATTCGCAGCATCCTCCGCATTAGCTTTGGATAACGGGTTAGCCCGTACACCTACTATGGGTTGGCTTCACTGGGAAAGATTCATGTGTAACTTAGATTGCCAAGAAGAGCCTGACAGCTGTATCTCAGAGAAACTATTCATGGAGATGGCTGAACTAATGGTAAGTGAAGGATGGAAGGATGCTGGTTATGAATACCTATGTATTG Polynucleotide sequence of MFleader-GLA(yCDS): Polypeptide sequence of MFleader: MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYLDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKR (SEQ ID NO: 38) Polynucleotide sequence of variant number 395yCDS: TGCAATCCTGCCTGCTTCATTACACAATTGCTTCCAGTTAAGAGACAATTGGGTTTCTATAACTGGACCTCTAGGCTAAAAAGTCACATTAATCCTACTGGTACGGTATTGTTGCAATTGGAGAACACAATGCAAATGTCTTTGAAAGATTTGTTA (SEQ ID NO: 39) Polypeptide sequence of variant number 395: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNWTSRLKSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 40) Polynucleotide sequence of variant number 402yCDS: Polypeptide sequence of variant number 402: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNWTSRLKSHINPTGTVLLQLENTMQMSLKDLL (SEQ ID NO: 42) Polynucleotide sequence of variant number 625yCDS: Polypeptide sequence of variant number 625: LDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLFMEMAERMVTEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAAPLFMSNDLRAISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNWTSRLKSHINPTGTVLLQLENTMQTSLKDLL (SEQ ID NO: 44) Polynucleotide sequence of variant number 648yCDS: Polypeptide sequence of variant number 648: LDNGLARTPPMGWLHWERFMCNLDCQEEPDSCISEKLFEEMAERMVTEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANHVHSKGLKLGIYADVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRPIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIDDSWASIKSILDWTSRNQERIVDVAGPGGWNDPDMLVIGNFGLSWDQQVTQMALWAIMAGPLFMSNDLRAISPQAKALLQDKDVIAINQDPLGKQGYQLRKGDNFEVWERPLSGDAWAVAIINRQEIGGPRSYTIPVASLGKGVACNPACFITQLLPVKRQLGFYNATSRLKSHINPTGTVLLQLENTMQTSLKDLL (SEQ ID NO: 46) Example 1 Obtaining the GLA gene and constructing an expression vector

[0180] A synthetic gene (SEQ ID NO:1) encoding the wild-type human GLA sequence (SEQ ID NO:2) and derived variants (SEQ ID NO:4, SEQ ID NO:6) were constructed as previously described (see, e.g., U.S. Patent Application Publication No. 2017 / 0360900). For secretory expression and transient transfection in mammalian cells, chimeric GLA expression constructs containing a polynucleotide encoding a synthetic mouse IG signal peptide fused to a synthetic gene encoding a different GLA variant were generated as follows: The oligonucleotides BamHI-pcDNA-GLA-F (SEQ ID NO:63) and XhoI-pcDNA-GLA-R (SEQ ID NO:64) were used to amplify the signal peptide-encoding fragment and the coding sequence for the mature form of the GLA variant. The PCR product was ligated into the BamHI / XhoI-linearized mammalian expression vector pcDNA3.1(+) (Invitrogen) or a vector containing a CMV promoter and BGH-pA (bovine growth hormone polyadenylation) sequence. Directed evolution techniques commonly known to those skilled in the art were used to generate gene variants derived from SEQ ID NO:8 in this plasmid construct (see, e.g., U.S. Pat. No. 8,383,346 and WO 2010 / 144103). Example 2 High-throughput growth and assays High-throughput (HTP) propagation of GLA and GLA mutants

[0181] HEK293T cells were transfected with pcDNA3.1(+) or CMV promoter- and BGH-pA-containing vectors encoding a synthetic mouse IG signal peptide fused to wild-type GLA or GLA mutants using the lipofection method with LIPOFECTAMINE® 3000 Reagent (ThermoFisher Scientific). HEK293T cells were cultured in growth medium (DMEM containing 10% fetal bovine serum [both Corning]). 24 hours before transfection, cells were plated in NUNC® Edge 2.0 96-well plates (ThermoFisher Scientific) at 10°C in growth medium. 5Cells were seeded at a density of 250 μL per well and incubated in an incubator at 37°C and 5% CO2. Cells were incubated at 37°C and 5% CO2 for 24-72 hours to allow expression and secretion of GLA variants. Conditioned medium (50-100 μL) from HEK293T transfections was transferred to Corning 96-well solid black plates (Corning) for activity and / or stability analysis. HTP analysis of the supernatant

[0182] GLA mutant activity was examined by measuring the hydrolysis of 4-methylumbelliferyl α-D-galactopyranoside (MUGal). For the unchallenged assay, 50 μL of HEK293T conditioned medium prepared as described above was mixed with 50 μL of 1 mM MUGal (pH 4.8) in McIlvaine buffer (McIlvaine, J. Biol. Chem., 49:183-186

[1921] ) in a 96-well black, opaque-bottom microtiter plate. The reaction was mixed briefly and incubated at 37°C for 30–180 min before being quenched with 100 μL of 0.5 M sodium carbonate (pH 10.2). Hydrolysis was analyzed using a SPECTRAMAX® M2 microplate reader monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm). The results of this assay are shown in Table 2-1. HTP analysis of acid-pretreated supernatants

[0183] GLA mutants were challenged with acidic buffer to simulate the extreme pH that the mutants may encounter within lysosomes. First, 50 μL of HEK293T conditioned medium and 50 μL of McIlvaine buffer (pH 3.3–4.3) were added to wells of a 96-well round-bottom microtiter plate. The plate was sealed with a PlateLoc® thermal microplate sealer (Agilent) and incubated at 37°C for 1–2 hours. For the pH 4 challenge assay, 50 μL of the acid-pH challenge sample was mixed with 50 μL of 1 mM MUGal in McIlvaine buffer (pH 4.4). The reaction was mixed briefly and incubated at 37°C for 30–180 minutes before being quenched with 100 μL of 0.5 M sodium carbonate (pH 10.2). Hydrolysis was analyzed using a SPECTRAMAX® M2 microplate reader monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm). The results of this assay are shown in Table 2-1. HTP analysis of base-pretreated supernatants

[0184] To simulate the pH the variants would encounter in blood after patient administration, GLA variants were challenged with a basic (neutral) buffer. First, 50 μL of GLA variant HEK293T conditioned medium and 50 μL of McIlvaine buffer (pH 7.0–8.2) were added to wells of a 96-well round-bottom microtiter plate. The plate was sealed and incubated at 37°C for 1–18 hours. For the pH 7 challenge assay, 50 μL of the basic pH challenge sample was mixed with 50 μL of 1 mM MUGal in McIlvaine buffer (pH 4.4). The reaction was mixed briefly and incubated at 37°C for 30–180 minutes before being quenched with 100 μL of 0.5 M sodium carbonate (pH 10.2). Hydrolysis was analyzed using a SPECTRAMAX® M2 microplate reader monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm). The results of this assay are shown in Table 2-1. [Table 2-1] Example 3 Production of GLA mutants Production of GLA in HEK293T cells

[0185] Secretory expression of GLA variants in mammalian cells was performed by transient transfection of HEK293, HEK293T, or Expi293 cells. As described in Example 1, cells were transfected with GLA variants (SEQ ID NOs: 3, 4, 9, 12, 17, 20, 23, and 41) fused to an N-terminal synthetic mammalian signal peptide and subcloned into the mammalian expression vector pLEV113. HEK293 cells were transfected with plasmid DNA and grown in suspension for 4 days using standard techniques known to those skilled in the art. Supernatants were collected and stored at 4°C until analysis. Example 4 Purification of GLA mutants Purification of GLA mutants from mammalian cell supernatants

[0186] WT GLA (SEQ ID NO: 2) was purified from mammalian culture supernatant as described previously (Yasuda et al., Prot. Exp. Pur, 37:499-506

[2004] ). All other GLA mutants were purified as follows: GLA mutants were purified from mammalian culture supernatant essentially as known in the art (Yasuda et al., Prot. Exp. Pur, 37:499-506

[2004] ). Concanavalin A resin (Sigma-Aldrich) was equilibrated with 0.1 M sodium acetate, 0.1 M NaCl, 1 mM MgCl2, CaCl2, and MnCl2 (pH 6.0) (concanavalin A binding buffer). The supernatant was sterile filtered through a 0.2 μm bottle-top filter before loading onto the column. After loading, the column was washed with 10 column volumes of concanavalin A binding buffer, and the bound protein was eluted with concanavalin A binding buffer supplemented with 0.9 M methyl-α-D-mannopyranoside and 0.9 M methyl-α-D-glucopyranoside. The eluted protein was concentrated and buffer exchanged into storage buffer (20 mM sodium phosphate, 150 mM sodium chloride, 185 μM TWEEN®-20 nonionic surfactant, pH 6.0) using an Amicon® Ultra 15 mL filtration unit with a 30 kDa molecular weight cutoff (Millipore) membrane. GLA in the storage buffer was sterile filtered through an ANOTOP® 0.2 μm syringe filter (Whatman) and stored at -80 °C. Purification yielded 2.4–50 μg of purified protein / ml of culture supernatant based on BCA quantification. Protein quantification by BCA protein assay

[0187] Purified GLA was quantified using a bicinchoninic acid (BCA) protein assay (Sigma-Aldrich). In a microtiter plate, 25 μL of protein standard and appropriately diluted purified GLA were mixed with 200 μL of working reagent containing 50 parts BCA Reagent A and 1 part BCA Reagent B. The plate was thoroughly mixed for 30 seconds on a plate shaker and incubated at 37°C for 30 minutes. After the plate was cooled to room temperature, the absorbance of the samples was measured at 562 nm using a plate reader. Example 5 In vitro characterization of GLA mutants Thermostability of GLA mutants expressed in HEK293T cells

[0188] The GLA mutants were exposed to various temperature challenges to assess the overall stability of the enzyme. First, 50 μL of purified HEK293T-expressed GLA and GLA mutants in 1x PBS (pH 6.2) were added to the wells of a 96-well PCR plate (Biorad, HSP-9601). The plate was sealed and incubated for 1 h at 30–50°C using a gradient program on a thermocycler. For the assay, 25 μL of challenged supernatant was mixed with 25 μL of 1 mM MUGal in McIlvaine's buffer (pH 4.4). The reaction was mixed briefly and incubated at 37°C for 60 min before being quenched with 100 μL of 0.5 M sodium carbonate (pH 10.2). Hydrolysis was analyzed using a SPECTRAMAX® M2 microplate reader monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm). The percent residual activity for 1 hour of incubation at temperatures ranging from 30°C to 50°C was calculated by dividing the activity of the challenged sample by the activity of the unchallenged sample, where "unchallenged" is the hydrolysis measured at time 0 and "challenged" is the hydrolysis measured at 1 hour at the specified temperature for each variant. The results of this assay are shown in Table 5-1. Figure 1 provides a graph showing the residual activity of GLA variants after 1 hour of incubation at various temperatures. Serum stability of GLA mutants expressed in HEK293T cells

[0189] To assess the relative stability of the variants in the presence of blood, samples were exposed to serum. First, 100 μL of 7.5 μg / mL purified GLA variants in 1x PBS (pH 6.2) and 90 μL of human serum were added to wells of a COSTAR® 96-well round-bottom plate (Corning). The plate was sealed and incubated at 37°C for 0-24 hours. For the assay, 50 μL of challenged supernatant was mixed with 50 μL of 1 mM MUGal in McIlvaine's buffer (pH 4.4). The reaction was mixed briefly and incubated at 37°C for 90 minutes before being quenched with 100 μL of 0.5 M sodium carbonate (pH 10.2). Hydrolysis was analyzed using a SPECTRAMAX® M2 microplate reader monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm). The percent remaining activity in serum after 24 hours was calculated by dividing the activity of the challenged sample by the activity of the unchallenged sample, where "unchallenged" is the hydrolysis measured at time 0 and "challenged" is the hydrolysis measured at the designated time point for each variant. The results are shown in Table 5.1. Figure 2 provides a graph showing the remaining activity of GLA variants after 0-24 hours of challenge with human serum. Lysosomal stability of GLA mutants expressed in HEK293T cells

[0190] To assess the relative stability of the variants in the presence of lysosomal proteases and other lysosomal components, GLA variants were exposed to human lysosomal lysate (XenoTech, No. H0610.L) according to the manufacturer's instructions with some modifications, as described herein. Briefly, GLA variants were diluted to the appropriate concentration range (0.0625-0.0078125 mM), and 10 μL of the dilution was combined with 10 μL of a 1:20 dilution of human lysosomal lysate in 2x catabolic buffer (XenoTech, No. K5200) in a COSTAR® 96-well round-bottom plate (No. 3798, Corning). The plate was sealed and incubated at 37°C for 0-24 hours. For the assay, 50 μL of challenge supernatant was mixed with 50 μL of 1 mM MUGal in McIlvaine's buffer (pH 4.4). The reactions were mixed briefly and incubated at 37°C for 90 minutes before being quenched with 100 μL of 0.5 M sodium carbonate (pH 10.2). Hydrolysis was analyzed using a SPECTRAMAX® M2 microplate reader monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm). The percent remaining activity in the lysosomal extract after 24 hours was calculated by dividing the activity of the challenged sample by the activity of the unchallenged sample, where "unchallenged" is the hydrolysis measured at time 0 and "challenged" is the hydrolysis measured at 4 and 24 hours as specified for each variant. The results are shown in Table 5.1. Figure 3 provides a graph showing the remaining activity of GLA variants after 0 to 24 hours of challenge with human lysosomal extract. Cellular uptake of purified GLA mutants expressed in HEK293T cells in Fabry fibroblasts.

[0191] The cellular uptake of GLA mutants compared to a reference enzyme (WT GLA [SEQ ID NO: 2]) was examined to assess the overall ability of the mutants to be endocytosed into cultured cells. Fabry fibroblasts (GMO2775, Coriell Institute for Medical Research) were seeded into 12-well culture dishes (VWR, No. 10861-698) containing minimal essential medium (MEM; Gibco No. 11095-080 supplemented with 1% non-essential amino acids (NEAA; Gibco No. 11140-050) and 15% fetal bovine serum (Corning No. 35-016-CV)) and grown to confluence (2–3 days at 37°C, 5% CO). After reaching confluence, the supplemented MEM was removed by sterile vacuum and replaced with 1 mL / well of serum-free MEM + 1% NEAA. Purified enzyme, as described in Example 4, was added to the cells at 10 μg GLA / mL and incubated at 37°C, 5% CO2 for 4 hours. The serum-free medium was aspirated using a sterile vacuum, and the cells were briefly washed with 1 mL of 1x PBS per well, and the PBS was aspirated using a sterile vacuum. The cells were then trypsinized with 200 μL / well of 0.25% trypsin-EDTA (VWR No. 02-0154-0100) and incubated at room temperature for approximately 5 minutes to remove adherent cells from the plate and degrade any remaining extracellular GLA. 500 μL of serum-free MEM was then added to each well, and the samples were transferred to 1.5 mL microcentrifuge tubes. The samples were centrifuged at 8000 RPM for 5 minutes to pellet the cells. The medium was slowly aspirated using a 1000 μL pipette. The cell pellet was resuspended in 500 μL of 1x PBS and repelleted at 8000 RPM for 5 minutes, and the PBS was slowly removed. Then, 100 μL of lysis buffer (0.2% TRITONX-100™ non-ionic surfactant (Sigma No. 93443) diluted in 1x PBS) was added to each sample, followed by sonication for 1-2 minutes and centrifugation at 12,000-14,000 RPM for 10 minutes at 4°C. The supernatant was transferred to a sterile PCR tube for protein and activity assays. For activity assays, 10 μL of cell lysate sample was mixed with 50 μL of 2.5 mM MUGal in McIlvaine's buffer (pH 4.6).The reaction plate was sealed and incubated at 37°C for 60 minutes, after which the reaction was quenched with 140 μL of 0.5 M sodium carbonate (pH 10.2) per well. MUGal hydrolysis was monitored using a SPECTRAMAX® M2 microplate reader monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm). For protein quantification, a BCA assay was performed according to the manufacturer's instructions (Pierce, No. 23225) with the following modifications: 10 μL of cell lysate sample was mixed with 190 μL of BCA working reagent, the plate was sealed, and incubated at 37°C for 60 minutes. Samples were analyzed using a SPECTRAMAX® M2 microplate reader monitoring absorbance (562 nm). Protein concentrations were calculated from a BSA standard curve. Cellular uptake of each GLA variant was calculated by first subtracting the background non-enzyme fluorescence of untreated cells from the enzyme-treated sample, then normalizing to the protein concentration of each well. Cellular uptake FIOPC was calculated by dividing the normalized GLA variant intracellular activity by the corresponding activity of the control (WT). Figure 4 provides a graph of the cellular uptake of purified GLA variants in cultured Fabry patient fibroblasts, expressed as relative activity compared to wild-type (SEQ ID NO: 2), after 4 hours of incubation at 37°C. [Table 5-1] HTP analysis of GLA activity in lysates of Fabry fibroblasts

[0192] The GLA variants produced in the HTP were challenged to ensure they were incorporated into cells and retained activity for 24 to 96 hours. Fabry fibroblasts (GMO2775, Coriell Institute for Medical Research) were seeded and grown to confluence for 24 to 72 hours. After confluence was reached, the medium was removed using an automated BioMek i5 liquid-handling robot. Conditioned medium from HEK293T cells transiently transfected as described above was added to the Fabry fibroblasts, and the cells were incubated with the GLA variants for 2 to 4 hours at 37°C and 5% CO2. The GLA-containing conditioned medium was removed using an automated BioMek i5 liquid-handling robot. The cells were then briefly washed with 150 μL of 1x DPBS / well, and the DPBS was removed using the automated BioMek i5 liquid-handling robot. 200 μL of complete growth medium was then added to each well, and the plate was returned to the incubator for 24 to 72 hours. At the end of the incubation, the complete growth medium was removed using an automated BioMek i5 liquid handling robot. The cells were washed with 150 μL of 1x DPBS / well and then transferred to the automated BioMek i5 liquid handling robot. The DPBS was removed using an i5 liquid-handling robot. Cells were lysed by adding 50 μL of McIlvaine's buffer (pH 4.4) supplemented with 0.2% TRITONX-100™ nonionic surfactant (Sigma No. 93443) and stirring at room temperature for 30 minutes. Activity was assessed by adding 50 μL of 1.5 mM MuGal in McIlvaine's buffer (pH 4.4). Plates were sealed and incubated at 37°C for 360 minutes with stirring at 400 rpm, followed by quenching with 100 μL of 0.5 M sodium carbonate (pH 10.2). Hydrolysis was analyzed using a SPECTRAMAX® M2 microplate reader (excitation wavelength 355 nm, emission wavelength 460 nm) monitoring fluorescence. Cellular uptake FIOPC was calculated by dividing the normalized intracellular activity of the GLA variant by the corresponding activity of the reference sequence. HTP analysis of GLA-induced depletion of globotriaosylceramide in Fabry fibroblasts

[0193] Cells were challenged with the GLA variants produced in the HTP to reduce globotriaosylceramide cellular load. Fabry fibroblasts (GMO2775, Coriell Institute for Medical Research) were seeded and grown to confluence for 24–72 h. After confluence, the medium was removed using an automated BioMek i5 liquid-handling robot. Conditioned medium produced by transiently transfected HEK293T cells as described above was added to the fibroblasts and incubated at 37°C and 5% CO2 for 2–4 h. The GLA-containing conditioned medium was then removed using an automated BioMek i5 liquid-handling robot. The cells were then briefly washed with 150 μL of 1x DPBS / well, and the DPBS was removed using the automated BioMek i5 liquid-handling robot. 200 μL of complete growth medium was then added to each well, and the plate was returned to the incubator for 24–72 h. At the end of the incubation period, the complete growth medium was removed using an automated BioMek i5 liquid-handling robot. Cells were then washed with 150 μL of 1x DPBS per well, and the DPBS was removed using an automated BioMek i5 liquid-handling robot. Globotriaosylceramide was extracted into 200 μL of methanol supplemented with 10 ng / mL N-heptadecanoyl-ceramide trihexoside for 30 minutes at room temperature with gentle agitation. The methanol extract was filtered through a Millipore hydrophobic filter stack into a round-bottom 96-well plate. Cellular globotriaosylceramide was quantified by LC-MS / MS essentially as known in the art (see Provencal et al., Bioanal., 8:1793-1807

[2016] ). Peak integrals for each cell sample were summed, and globotriaosylceramide FIOPC was calculated by dividing the change in globotriaosylceramide levels of the normalized GLA variant by the reference sequence. Example 6 GLA variants derived from SEQ ID NO: 58

[0194] This example describes experiments performed to evaluate the activity of GLA mutants and their Gb3 clearance in Fabry fibroblasts. In this example, SEQ ID NO: 58 was used as the reference sequence (i.e., amino acid differences of the mutants are shown relative to SEQ ID NO: 58, and assay results are reported relative to those obtained with SEQ ID NO: 58). In these experiments, the GLA mutants were tested for MU-Gal activity without preincubation, as described in Example 5. The mutants were also tested for Gb3 depletion in Fabry fibroblasts, as described in Example 5. [Table 6-1-1] [Table 6-1-2] [Table 6-1-3] Example 7 GLA variants derived from SEQ ID NO: 158

[0195] This example describes experiments performed to evaluate the activity of GLA mutants in Fabry fibroblasts, their stability at pH 7.4, and their intracellular activity. In this example, the reference sequence was SEQ ID NO: 158 (i.e., amino acid differences of the mutants are shown relative to SEQ ID NO: 158, and assay results are reported relative to the results of SEQ ID NO: 158). The mutants were tested for GLA MU-Gal activity without preincubation (unchallenged) and after preincubation at pH 7.4, as described in Example 5. The mutants were also tested for MU-Gal activity after lysis of Fabry fibroblasts incubated with the GLA mutants, as described in Example 5. [Table 7-1-1] [Table 7-1-2] Example 8 GLA variants derived from SEQ ID NO: 372

[0196] This example describes experiments performed to evaluate the activity, pH 7.4 stability, Gb3 clearance, and intracellular activity of GLA mutants in Fabry fibroblasts. In this example, the reference sequence was SEQ ID NO: 372 (i.e., amino acid differences of the mutants are shown relative to SEQ ID NO: 372, and assay results are reported relative to the results of SEQ ID NO: 372). These mutants were tested for MU-Gal activity without preincubation (unchallenged) and after preincubation at pH 7.4, as described in Example 5. The mutants were also tested for MU-Gal activity in Fabry fibroblasts incubated with the GLA mutants, as described in Example 5 ("Lysate FIOPC"). The mutants were also tested for Gb3 depletion in Fabry fibroblasts after incubation with the GLA mutants, as described in Example 5 ("Gb3 FIOPC"). [Table 8-1-1] [Table 8-1-2] [Table 8-1-3] Example 9 GLA variants derived from SEQ ID NO: 374

[0197] This example describes experiments performed to examine GLA mutant activity by assaying enzyme activity after serial or independent challenges. These mutants were tested for GLA MU-Gal activity without preincubation and after preincubation at pH 7.4, as described in Example 5. The mutants were also tested for MU-Gal activity after lysis of Fabry fibroblasts incubated with the mutants, as described in Example 5. The mutants were also tested for Gb3 depletion in Fabry fibroblasts after incubation, as described in Example 5. [Table 9-1-1] [Table 9-1-2] [Table 9-1-3] [Table 9-1-4] Example 10 In vivo characterization of GLA mutants

[0198] GLA mutants were characterized in vivo for their activity against accumulated Gb3. Fabry mice (5-month-old female, Jackson, stock no. 3535) with identical genetic backgrounds and age- and sex-matched wild-type mice were used. Mice received a single intravenous injection of the Codexis enzyme mutant (1.0 mg / kg) via the tail vein. At scheduled time points (1 and 2 weeks post-injection), animals were sacrificed using CO2 anesthesia, and disease-related tissues (e.g., heart and kidney) were dissected into two parts (one for enzyme activity assay and the other for Gb3 quantification), frozen on dry ice, and stored at -80°C until analysis. For the enzyme assay, mouse tissues were homogenized in lysis buffer (0.2% TRITONX-100™ non-ionic surfactant (Sigma) diluted in 1x PBS) using a motor-driven TEFLON®-coated pestle of a glass homogenizer. The lysates were homogenized in 20 volumes (w / v) of PBS (No. 93443). The lysates were sonicated and centrifuged at 14,000 rpm for 15 minutes at 4°C, and the supernatant was used for enzyme assays. α-Gal A activity was measured by a standard fluorometric assay using 5 mM 4-methylumbelliferyl-α-D-galactopyranoside at pH 4.4 in the presence of 0.1 M N-acetylgalactosamine (i.e., a specific inhibitor of α-galactosidase B). Protein concentration was measured using a BCA protein assay kit (Pierce, No. 23225). Activity was normalized to protein concentration and expressed as nmol / mg protein / h. Gb3 concentration was measured by mass spectrometry as previously described (see Durant et al., J. Lipid Res., 52:1742-6

[2011] ). Briefly, mouse tissues were homogenized in 20 volumes of ice-cold ultrapure water in a glass homogenizer, and lysates equivalent to 200 μg of total protein were subjected to glycosphingolipid extraction, saponification, and subsequent analysis of Gb3 by mass spectrometry. Gb3 concentrations were expressed as ng / mg protein. Figure 5 provides a graph showing in vivo enzyme activity in the hearts of the Fabry mouse model 1, 2, and 4 weeks after treatment. Figure 6 provides a graph of Gb3 degradation in cardiac tissue of the Fabry mouse model 1 and 2 weeks after treatment compared to untreated animals. Example 11 GLA variants derived from SEQ ID NO: 704

[0199] This example describes experiments performed to evaluate the activity, serum stability, intracellular activity, and Gb3 clearance of GLA mutants in Fabry fibroblasts. In these experiments, GLA mutants were tested for MU-Gal activity in Fabry fibroblast lysates without preincubation and after serum incubation with SEQ ID NO:704 (SEQ ID NO:704 is the amino acid sequence derived from SEQ ID NO:703, which is a mammalian codon-optimized version of the yeast-codon-optimized SEQ ID NO:275), as described in Example 5. The mutants were also tested for Gb3 depletion in Fabry fibroblasts, as described in Example 5. [Table 11-1-1] [Table 11-1-2] [Table 11-1-3] [Table 11-1-4] [Table 11-1-5] [Table 11-1-6] Example 12 GLA variants derived from SEQ ID NO: 374

[0200] This example describes experiments performed to evaluate activity in Fabry fibroblasts, serum stability, intracellular activity, and Gb3 clearance by GLA mutants in Fabry fibroblasts. In these experiments, GLA mutants were tested for MU-Gal activity in Fabry fibroblast lysates without preincubation and after serum incubation, as described in Example 5. The mutants were also tested for Gb3 depletion in Fabry fibroblasts, as described in Example 5. [Table 12-1-1] [Table 12-1-2] [Table 12-1-3] [Table 12-1-4] [Table 12-1-5] [Table 12-1-6] [Table 12-1-7] [Table 12-1-8] [Table 12-1-9] Example 13 GLA variants derived from SEQ ID NO: 1022

[0201] This example describes experiments performed to evaluate activity in Fabry fibroblasts, serum stability, and intracellular activity. In these experiments, GLA mutants were tested for MU-Gal activity in Fabry fibroblast lysates without preincubation and after serum incubation, as described in Example 5. [Table 13-1-1] [Table 13-1-2] [Table 13-1-3] Serum stability of purified GLA variants produced in suspension culture

[0202] To assess the relative stability of the variants in the presence of blood, samples were exposed to serum. First, 100 μL of 7.5 μg / mL purified GLA variants in 1x PBS (pH 6.2) and 90 μL of human serum were added to wells of a COSTAR® 96-well round-bottom plate (Corning). The plate was sealed and incubated at 37°C for 0-24 hours. For the assay, 50 μL of challenged supernatant was mixed with 50 μL of 1 mM MUGal in McIlvaine's buffer (pH 4.4). The reaction was mixed briefly and incubated at 37°C for 90 minutes before being quenched with 100 μL of 0.5 M sodium carbonate (pH 10.2). Hydrolysis was analyzed using an ENVISION® microplate reader (PerkinElmer) monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm). The percent remaining activity in serum after 24 hours was calculated by dividing the activity of the challenged sample by the activity of the unchallenged sample, where "unchallenged" is the hydrolysis measured at time 0 and "challenged" is the hydrolysis measured at the designated time point for each variant. Figure 7 provides a graph showing the remaining activity of GLA variants after 0-24 hours of challenge with human serum. Cellular uptake in Fabry fibroblasts of purified GLA variants produced in suspension culture.

[0203] The cellular uptake of GLA mutants compared to a reference enzyme (WT GLA [SEQ ID NO: 2]) was examined to assess the overall ability of the mutants to be endocytosed by cultured cells. Fabry fibroblasts (GMO2775, Coriell Institute for Medical Research) were seeded into 96-well culture dishes (VWR, No. 10861-698) containing minimal essential medium (MEM; Gibco No. 11095-080 supplemented with 1% non-essential amino acids (NEAA; Gibco No. 11140-050) and 15% fetal bovine serum (Corning No. 35-016-CV)) and grown to confluence (1–3 days at 37°C, 5% CO). After reaching confluence, the supplemented MEM was removed by sterile vacuum and replaced with 1 mL / well of serum-free MEM + 1% NEAA. Purified enzymes described in Example 4 were added to the cells at a dose response of 220 nM to 2 nM and incubated for 4 hours at 37°C, 5% CO2. Cells were washed twice with 100 μL of sterile 1x PBS / well, and the PBS was aspirated by sterile vacuum. Cells were then resuspended in Minimal Essential Medium (MEM; 1% non-essential amino acids (NEAA; Gibco The cells were incubated in PBS containing 100 μL of sterile 1× PBS per well (Gibco No. 11140-050) and 15% fetal bovine serum (Gibco No. 11095-080 [Corning No. 35-016-CV]) supplemented with 15% fetal bovine serum and incubated at 37°C, 5% CO for 3 days. After 3 days, the cells were washed twice with 100 μL of sterile 1× PBS per well, and the PBS was aspirated using a sterile vacuum. Next, 25 μL of lysis buffer (0.2% TRITONX-100™ nonionic surfactant [Sigma No. 93443] diluted in 1x PBS) was added to each sample, followed by 1-2 minutes of sonication and 10 minutes of centrifugation at 12,000-14,000 RPM at 4°C. For activity assays, 25 μL of cell lysate sample was mixed with 25 μL of 2.5 mM MUGal in McIlvaine's buffer (pH 4.6). The reaction plate was sealed and incubated at 37°C for 60 minutes, after which the reaction was quenched with 150 μL of 0.5 M sodium carbonate (pH 10.2) per well. MUGal hydrolysis was examined using an ENVISION® microplate reader (PerkinElmer) monitoring fluorescence (excitation wavelength 355 nm, emission wavelength 460 nm). FIG. 8 provides a graph of the cellular uptake of purified GLA variants in cultured Fabry patient fibroblasts after 4 hours of incubation at 37° C. and a 3 day washout. Example 14 In vivo characterization of GLA mutants

[0204] This example describes experiments conducted using animal models to characterize several GLA variants. An overview of the experimental design for evaluating the pharmacokinetic profile in mice is provided in Table 14-1. The experiment was conducted in a single phase and included 36 male C57B1 / 6 mice (20-25 g). Animals were allowed to acclimate for at least 3 days prior to the experiment. All animals received a single IV injection of 1 mg / kg of either WT GLA (SEQ ID NO: 2) or a GLA variant via the tail vein. Individual doses were calculated based on body weight measured on the day of administration. [Table 14-1]

[0205] At predetermined time points, approximately 150 μL of whole blood was collected into heparinized capillary tubes from six mice per test article (alternating two groups of six for each time point according to Table 14-1), immediately processed for plasma, and stored at −80° C. until assayed. Plasma GLA activity was measured using a standard fluorometric MuGal assay as described in Example 5. Overall, the GLA variants exhibited superior plasma pharmacokinetic profiles compared to SEQ ID NO:2, which had an estimated half-life (t 1 / 2 ) had the fastest clearance. In comparison, the GLA mutant 1 / 2 was approximately 10 times longer. The data are shown in Table 14-2. [Table 14-2]

[0206] In addition to the mouse experiments described above, experiments were conducted to determine the pharmacokinetics of several GLA variants in healthy rats. A simple experimental design is shown in Table 14-3. The first experiment was conducted in three phases and included 21 rats. The animals were allowed to acclimate for at least three days before the experiment. All animals were injected IV with either WT GLA (SEQ ID NO: 2) or a GLA variant via the jugular vein cannula. Individual doses were calculated based on body weight measured on the day of administration. [Table 14-3]

[0207] Blood (approximately 0.25 mL) was collected from each rat at the time points shown in Table 14-3 into EDTA separator tubes, immediately processed for plasma, and stored at -80°C until assayed. Plasma GLA activity was measured using a standard fluorometric MuGal assay as described in Example 5. All GLA variants showed improved PK properties when compared to SEQ ID NO: 2. Data for all PK parameters are shown in Table 14-4. [Table 14-4]

[0208] A brief study design for rat PK study number 2 is shown in Tables 14-5. The study was conducted in one phase and included 18 rats. Animals were acclimated for at least 3 days prior to the study. All animals were injected IV with either WT GLA (SEQ ID NO: 2) or GLA variants via the jugular vein cannula. Individual doses were calculated based on body weight measured on the day of administration. [Table 14-5]

[0209] Blood (approximately 0.25 mL) was collected into EDTA separator tubes at the time points shown in Table 14-5, immediately processed for plasma, and stored at -80°C until assayed. Plasma GLA activity was measured using a standard fluorometric MuGal assay as described in Example 5. All doses of the GLA variants provided improved PK properties when compared to SEQ ID NO: 2. Data for all PK parameters are shown in Table 14-6. [Table 14-6]

[0210] In addition to the mouse and rat experiments described above, experiments were conducted in a primate model. A simplified experimental design is shown in Tables 14-7. The experiment was conducted in one phase and included 12 male cynomolgus monkeys (2-3 kg). The animals were sourced from the testing facility's colony and were considered protein naive. All animals were injected IV with either WT GLA (SEQ ID NO: 2) or the GLA variants. Individual doses were calculated based on body weight measured on the day of administration. [Table 14-7]

[0211] Blood (approximately 0.5 mL) was collected into EDTA separator tubes at the time points shown in Table 14-7, immediately processed for plasma, and stored at -80°C until assayed. Plasma GLA activity was measured using a standard fluorometric MuGal assay as described in Example 5. All doses of the GLA variants provided improved PK properties when compared to SEQ ID NO: 2. Data for all PK parameters are shown in Table 14-8). [Table 14-8]

[0212] A simplified experimental design for the second PK study in monkeys is shown in Tables 14-9. This study was conducted in one phase and included 12 cynomolgus monkeys (3 animals / group). Animals were sourced from the test facility's colony and were considered GLA protein naive. All animals were injected IV with either WT GLA (SEQ ID NO: 2) or GLA variants. Individual doses were calculated based on body weight measured on the day of administration. Blood samples were collected at the following time points: pre-dose (up to 3 hours), 1 minute, 5 minutes, 15 minutes, and 30 minutes after administration, and 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, and 24 hours after administration. [Table 14-9]

[0213] Blood (approximately 1 mL) was collected into EDTA separator tubes at the time points shown in Table 14-7, immediately processed for plasma, and stored at -80°C until assayed. Plasma GLA activity was measured using a standard fluorometric MuGal assay as described in Example 5. All doses of the GLA variants provided improved PK properties when compared to SEQ ID NO: 2. Data for all PK parameters are shown in Table 14-10. [Table 14-10] Efficacy of GLA mutants compared with rhGLA in Fabry knockout mice

[0214] The efficacy of six GLA variants (SEQ ID NOs: 4, 58, 158, 704, 1022, and 1864) compared with WT GLA (SEQ ID NO: 2) was evaluated using Fabry mice (5-month-old female, Jackson, stock no. 3535) and age- and sex-matched wild-type mice with the same genetic background. Mice were administered the enzyme variants (0.1, 0.3, or 1.0 mg / kg via tail vein injection) once weekly for 4 weeks (n = 5 / group). Seven days after the last injection, animals were anesthetized and bled via cardiac puncture into K3 EDTA tubes and then euthanized. Systemic perfusion through the heart with cold saline was performed to remove contaminating blood from the tissues. Disease-related tissues (e.g., heart and kidney) were cut into two sections (one for enzyme activity assay and the other for Gb3 and lyso-Gb3 quantification), frozen on dry ice, and stored at -80°C until analysis. Blood was immediately processed for plasma and stored at -80°C until assayed. Plasma and tissue GLA activity was measured using a standard fluorometric MuGal assay as described in Example 5. Plasma and tissue Gb3 and lysoGb3 levels were assessed using the methods mentioned in Example 10, and tissues were homogenized as described in Example 10. With the exception of some cases of SEQ ID NO: 1864, all GLA variants were more effective than SEQ ID NO: 2 in the Fabry mouse model.

[0215] Figures 9 and 10 provide graphs showing in vivo enzyme activity in the heart and kidney, respectively, of the Fabry mouse model 7 days after the last treatment. In Figure 9, data are presented as mean ± SEM. Results were compared to those of SEQ ID NO:2 using a two-way ANOVA with Dunnett's post-hoc test. "^", p<0.0001, no significant improvement over SEQ ID NO:2 is shown in this figure. In Figure 10, data are presented as mean ± SEM. Results were compared to those of SEQ ID NO:2 using a two-way ANOVA with Dunnett's post-hoc test. "*", p<0.05, "**", p<0.005, and "^", p<0.0001, no significant improvement over SEQ ID NO:2 is shown in this figure. Figures 11 and 12 provide graphs of Gb3 degradation in heart and kidney tissues, respectively. In Figure 11, data are presented as mean ± SEM. Results were compared to those of SEQ ID NO:2 using a two-way ANOVA with Dunnett's post-hoc test. At the 0.1 mg / kg dose, "*", p<0.05 for SEQ ID NOs: 158 and 1022; at the 0.3 mg / kg dose, "^", p<0.0001 for SEQ ID NO: 158, and "**", p<0.005 for SEQ ID NOs: 58, 704, 1022, and 1864. In Figure 12, data are presented as mean ± SEM. Results were compared to those of SEQ ID NO: 2 using a two-way ANOVA with Dunnett's post-hoc test. At the 0.1 mg / kg dose, "^", p<0.0001 for SEQ ID NO: 4, 58, 158, 704, and 1022; at the 0.3 mg / kg dose, "^", p<0.0001 for SEQ ID NO: 158, and "**", p<0.005 for SEQ ID NOs: 4, 58, 704, and 1022. Figures 13 and 14 provide graphs of lyso-Gb3 degradation in heart and kidney tissues, respectively. In Figure 13, data are presented as mean ± SEM. Results were compared to those of SEQ ID NO: 2 using a two-way ANOVA with Dunnett's post-hoc test. In Figure 14, data are presented as mean ± SEM. Results were compared to those of SEQ ID NO: 2 using a two-way ANOVA with Dunnett's post-hoc test.At the 0.1 mg / kg dose, "**", p<0.005 for SEQ ID NO: 4, 58, 158, and "*", p<0.05 for SEQ ID NO: 704.

[0216] Although the invention has been described with reference to specific embodiments, various modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, and equivalents may be substituted thereby to achieve the benefits of the invention without departing from the scope of what is claimed.

[0217] In the United States, all publications and patent documents cited in this disclosure are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not an indication that such document is pertinent prior art, nor does it constitute an admission as to the contents or date thereof. The present invention provides, for example, the following items. (Item 1) Recombinant alpha-galactosidase A and / or biologically active recombinant alpha-galactosidase A fragment comprising an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs: 374, 704, and / or 1022. (Item 2) the recombinant alpha-galactosidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8, or a functional fragment thereof; and the recombinant alpha-galactosidase A is selected from the group consisting of 2, 4, 5, 24 / 59, 24 / 143 / 144, 24 / 143 / 202 / 333, 24 / 143 / 202 / 352 / 390 / 391, 24 / 143 / 333 / 352 / 387 / 390 / 391, 24 / 143 / 390 / 391, 24 / 202, 24 / 202 / 271, 24 / 202 / 333 / 352, 24 / 271 / 352, 24 / 352 / 387 / 390 / 391, 24 / 387 / 391, 31, 40, 59, 59 / 143, 59 / 143 / 202, 59 / 143 / 202 / 271 / 333, 59 / 143 / 271, 59 / 143 / 333, 59 / 202, 59 / 202 / 333, 59 / 271 / 387 / 390, 73, 76, 80, 83, 84, 91 / 215 / 361, 122, 123, 143, 143 / 202, 143 / 271, 143 / 271 / 352 / 390, 143 / 333, 143 / 333 / 387 / 390, 143 / 387 / 391, 147, 155, 164, 165, 179, 186, 202, 202 / 333, 210, 215 / 218, 218, 218 / 361, 218 / 361 / 398, 218 / 398, 246, 254 / 398, 271, 271 / 333, 271 / 333 / 390 / 391, 271 / 333 / 391, 271 / 352 / 391, 273, 275, 277, 278, 280, 281, 283, 284, 287, 300, 303, 304, 325, 331, 2. The recombinant alpha-galactosidase A of item 1, comprising at least one substitution or set of substitutions at one or more positions selected from 332, 333 / 352, 333 / 390 / 391, 333 / 391, 334, 335, 336, 338, 339, 340, 341, 343, 359, 360, 361, 362, 367, 369, 371, 373, 375, 377, 382, ​​382 / 398, 385, 387 / 391, 390, and 398, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 704. (Item 3) 2. The recombinant alpha-galactosidase A of item 1, wherein the recombinant alpha-galactosidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 8, or a functional fragment thereof, and wherein the recombinant alpha-galactosidase A comprises at least one substitution or set of substitutions at one or more positions selected from 10, 39, 44, 47, 92, 166, 206, 217, 247, 261, 271, 302, 316, 322, 337, 368, and 392, and the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 374. (Item 4) the recombinant alpha-galactosidase comprises a polypeptide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 58, or a functional fragment thereof; and the recombinant alpha-galactosidase A is selected from the group consisting of 10, 10 / 392, 31, 31 / 39 / 44 / 166 / 302, 31 / 47, 31 / 283 / 284, 39, 39 / 44, 39 / 44 / 47, 39 / 44 / 47 / 261 / 283 / 284, 39 / 44 / 283, 39 / 44 / 339, 39 / 47 / 261, 39 / 92, 39 / 206, 39 / 284, 44, 44 / 284 / 302, 84, 84 / 92, 84 / 284 / 302 / 392, 84 / 316, 84 / 368 / 392, 92, 92 / 206 / 217, 92 / 206 / 275, 92 / 206 / 284, 92 / 206 / 302 / 368, 92 / 271, 92 / 271 / 277, 92 / 275 / 284, 92 / 283, 92 / 283 / 392, 92 / 284, 92 / 302, 92 / 316, 92 / 368, 155, 155 / 217, 155 / 368, 166, 166 / 283 / 284, 166 / 302, 206, 206 / 217, 206 / 334, 261, 261 / 283, 2. The recombinant alpha-galactosidase A of item 1, comprising at least one substitution or set of substitutions at one or more positions selected from 271, 271 / 368, 275, 283, 283 / 284, 283 / 392, 284, 302, 316, 334, 339, 368, 368 / 392, and 392, wherein the amino acid positions of the polypeptide sequence are numbered with reference to SEQ ID NO: 1022. (Item 5) 2. The recombinant alpha-galactosidase A of item 1, wherein the alpha-galactosidase A comprises at least one mutation at at least one position shown in Tables 11.1, 12.1 and / or 13.1. (Item 6) 6. The recombinant alpha-galactosidase A according to any one of items 1 to 5, which is derived from human alpha-galactosidase A. (Item 7) A recombinant alpha-galactosidase A comprising the polypeptide sequence of SEQ ID NO: 374, 704 and / or 1022. (Item 8) 8. The recombinant alpha-galactosidase A of any one of items 1 to 7, which is more thermostable than the alpha-galactosidase A of SEQ ID NO: 2, 374, 704 and / or 1022. (Item 9) 9. The recombinant alpha-galactosidase A of any one of items 1 to 8, which is more stable at pH 7 than the alpha-galactosidase A of SEQ ID NO: 2, 374, 704 and / or 1022. (Item 10) 10. The recombinant alpha-galactosidase A of any one of items 1 to 9, which is more stable at pH 4 than the alpha-galactosidase A of SEQ ID NO: 2, 374, 704 and / or 1022. (Item 11) 11. The recombinant alpha-galactosidase A of any one of items 1 to 10, which is more stable to exposure to serum than the alpha-galactosidase A of SEQ ID NO: 2, 374, 704 and / or 1022. (Item 12) 12. The recombinant alpha-galactosidase A of any one of items 1 to 11, which is more stable in lysosomes than the alpha-galactosidase A of SEQ ID NO: 2, 374, 704 and / or 1022. (Item 13) 13. The recombinant alpha-galactosidase A according to any one of items 1 to 12, which is more easily taken up by cells than the alpha-galactosidase A of SEQ ID NO: 2, 374, 704 and / or 1022. (Item 14) 14. The recombinant alpha-galactosidase A of any one of items 1 to 13, which depletes more globotriaosylceramide from cells than the alpha-galactosidase A of SEQ ID NOs: 2, 374, 704 and / or 1022. (Item 15) 15. The recombinant alpha-galactosidase A of any one of items 1 to 14, which is purified. (Item 16) 16. The recombinant alpha-galactosidase A of any one of items 1 to 15, exhibiting at least one improved property selected from: i) enhanced catalytic activity, ii) increased tolerance to pH 7, iii) increased tolerance to pH 4, iv) increased serum tolerance, v) increased cellular uptake, vi) increased depletion of globotriaosylceramide from cells, vii) reduced immunogenicity, or any combination of i), ii), iii), iv), v), vi) and / or vii), compared to a reference sequence. (Item 17) 17. The recombinant alpha-galactosidase A of item 16, wherein the reference sequence is selected from SEQ ID NOs: 374, 704 and / or 1022. (Item 18) 18. A composition comprising at least one recombinant alpha-galactosidase A according to any one of items 1 to 17. (Item 19) 19. A recombinant polynucleotide sequence encoding at least one recombinant alpha-galactosidase A according to any one of items 1 to 18. (Item 20) 20. The recombinant polynucleotide sequence according to item 19, wherein the polynucleotide sequence is selected from DNA, RNA and mRNA. (Item 21) 21. The recombinant polynucleotide sequence of item 20, wherein the polynucleotide sequence is codon-optimized. (Item 22) 22. An expression vector comprising the recombinant polynucleotide sequence of items 19, 20 and / or 21. (Item 23) 23. The expression vector of item 22, wherein the recombinant polynucleotide sequence is operably linked to one or more regulatory sequences. (Item 24) 24. The expression vector of item 23, wherein the control sequence is a promoter. (Item 25) 25. The expression vector of item 24, wherein the promoter is a heterologous promoter. (Item 26) A host cell comprising the expression vector according to any one of items 22 to 25. (Item 27) 27. The host cell according to item 26, which is selected from eukaryotes and prokaryotes. (Item 28) 28. The host cell according to items 26 and / or 27, wherein the host cell is a mammalian cell. (Item 29) A method for producing an alpha-galactosidase A variant, comprising culturing the host cell described in any one of items 26 to 28 under conditions in which the alpha-galactosidase A encoded by the recombinant polynucleotide is produced. (Item 30) 30. The method of claim 29, further comprising recovering the alpha-galactosidase A. (Item 31) 31. The method of claim 30, further comprising purifying the alpha-galactosidase A. (Item 32) 19. A pharmaceutical composition for treating Fabry disease, comprising the composition according to item 18. (Item 33) 33. The pharmaceutical composition according to item 32, further comprising a pharmaceutically acceptable carrier and / or excipient. (Item 34) 34. The pharmaceutical composition according to item 32 and / or 33, wherein said composition is suitable for parenteral injection or infusion into humans. (Item 35) 22. A pharmaceutical composition comprising the recombinant polynucleotide according to any one of items 19 to 21. (Item 36) 36. A method of treating and / or preventing symptoms of Fabry disease in a subject, the method comprising providing a subject with Fabry disease and administering to said subject the pharmaceutical composition of any one of items 32 to 35. (Item 37) 37. The method of item 36, wherein the symptoms of Fabry disease are ameliorated. (Item 38) 38. The method of items 36 and / or 37, wherein the subject is able to eat a diet whose fat content is less restricted than the diet required by subjects exhibiting the symptoms of Fabry disease. (Item 39) 39. The method of any one of items 36 to 38, wherein the subject is an infant or a child. (Item 40) 40. The method of any one of items 36 to 39, wherein the subject is an adult or young adult. (Item 41) Use of the composition according to any one of items 18 and 32 to 35.

Claims

1. 1. A recombinant alpha-galactosidase A comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:704, wherein the amino acid sequence comprises at least a substitution selected from T10P, M39E, L44R, S47T, E48D, Y92H, R162K, S166P, K206A, F217R, N247D, A261G, H271A, Q302K, L316D, M322I, S333G, A337P, W368A, and M392T, wherein the mutations are relative to SEQ ID NO:

2.

2. The recombinant alpha-galactosidase A is selected from the group consisting of SEQ ID NOs: 1018, 1020, 1022, 1024, 1026, 1028, 1714, 1716, 1718, 1720, 1722, 1724, 1726, 1728, 1730, 1732, 1734, 1736, 1738, 1740, 1742, 1744, 1746, 1748, 1750, 1752, 1754, 1756, 1758, 1760, 1762, 1764, 1766, 1768, 1770, 1772, 1774, 1776, 1778, 1780, 1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1799, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2 1832, 1834, 1836, 1838, 1840, 1842, 1844, 1846, 1848, 1850, 1852, 1854, 1856, 1858, 1860, 1862, or 1864.

3. The recombinant alpha-galactosidase A of claim 1 or 2, wherein the recombinant alpha-galactosidase A is derived from human alpha-galactosidase A.

4. The recombinant alpha-galactosidase A of any one of claims 1 to 3, wherein the recombinant alpha-galactosidase A is more thermostable than the alpha-galactosidase A of SEQ ID NO:

2.

5. The recombinant alpha-galactosidase A of any one of claims 1 to 4, wherein the recombinant alpha-galactosidase A is more stable at pH 7 than the alpha-galactosidase A of SEQ ID NO:

2.

6. The recombinant alpha-galactosidase A of any one of claims 1 to 5, wherein the recombinant alpha-galactosidase A is more stable at pH 4 than the alpha-galactosidase A of SEQ ID NO:

2.

7. The recombinant alpha-galactosidase A of any one of claims 1 to 6, wherein the recombinant alpha-galactosidase A is more stable to exposure to serum than the alpha-galactosidase A of SEQ ID NO:

2.

8. The recombinant alpha-galactosidase A of any one of claims 1 to 7, wherein the recombinant alpha-galactosidase A is more stable in lysosomes than the alpha-galactosidase A of SEQ ID NO:

2.

9. The recombinant alpha-galactosidase A according to any one of claims 1 to 8, wherein the recombinant alpha-galactosidase A is more easily taken up by cells than the alpha-galactosidase A of SEQ ID NO:

2.

10. The recombinant alpha-galactosidase A of any one of claims 1 to 9, wherein the recombinant alpha-galactosidase A depletes more globotriaosylceramide from cells than the alpha-galactosidase A of SEQ ID NO:

2.

11. The recombinant alpha-galactosidase A of any one of claims 1 to 10, wherein the recombinant alpha-galactosidase A is purified.

12. The recombinant alpha-galactosidase A of any one of claims 1 to 11, wherein the recombinant alpha-galactosidase A exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased resistance to pH 7; iii) increased resistance to pH 4; iv) increased serum tolerance; v) increased cellular uptake; vi) increased depletion of globotriaosylceramide from cells; vii) reduced immunogenicity; or any combination of i), ii), iii), iv), v), vi) and / or vii), compared to SEQ ID NO:

2.

13. A composition comprising at least one recombinant alpha-galactosidase A according to any one of claims 1 to 12.

14. A recombinant polynucleotide encoding at least one recombinant alpha-galactosidase A according to any one of claims 1 to 12.

15. 15. The recombinant polynucleotide of claim 14, wherein the polynucleotide is selected from DNA, RNA, and mRNA.

16. 16. The recombinant polynucleotide of claim 15, wherein the polynucleotide is codon optimized.

17. 17. An expression vector comprising a recombinant polynucleotide according to claim 14, 15 and / or 16.

18. 18. The expression vector of claim 17, wherein the recombinant polynucleotide is operably linked to one or more regulatory sequences.

19. 19. The expression vector of claim 18, wherein the control sequence is a promoter.

20. 20. The expression vector of claim 19, wherein the promoter is a heterologous promoter.

21. A host cell comprising the expression vector according to any one of claims 17 to 20.

22. 22. The host cell of claim 21, wherein the host cell is selected from a eukaryote and a prokaryote.

23. 23. The host cell of claim 21 and / or 22, wherein the host cell is a mammalian cell.

24. A method for producing an alpha-galactosidase A variant, comprising culturing the host cell of any one of claims 21 to 23 under conditions in which the alpha-galactosidase A encoded by the recombinant polynucleotide is produced.

25. 25. The method of claim 24, further comprising recovering the alpha galactosidase A.

26. 26. The method of claim 25, further comprising purifying the alpha galactosidase A.

27. A pharmaceutical composition for treating Fabry disease, comprising the composition of claim 13.

28. 28. The pharmaceutical composition of claim 27, further comprising a pharmaceutically acceptable carrier and / or excipient.

29. 29. The pharmaceutical composition of claim 27 and / or 28, wherein the composition is suitable for parenteral injection or infusion into humans.

30. A pharmaceutical composition comprising the recombinant polynucleotide according to any one of claims 14 to 16.

31. A pharmaceutical composition according to any of claims 27 to 30 for use in the treatment and / or prevention of symptoms of Fabry disease in a subject with Fabry disease.

32. 32. The pharmaceutical composition of claim 31, wherein the symptoms of Fabry disease are ameliorated.

33. 33. The pharmaceutical composition of claim 31 and / or 32, wherein the subject is able to eat a diet whose fat content is less restricted than the diet required by subjects exhibiting the symptoms of Fabry disease.

34. The pharmaceutical composition of any one of claims 31 to 33, wherein the subject is an infant or a child.

35. 35. The pharmaceutical composition of any of claims 31 to 34, wherein the subject is an adult or young adult.

36. Use of a composition as provided in any of claims 13 and 27 to 30.

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