Human α-galactosidase variants

Abstract

To provide engineered human α-galactosidase polypeptides and compositions thereof.SOLUTION: The engineered human α-galactosidase polypeptides are optimized to provide improved stability under both acidic (pH<4.5) and basic (pH>7) conditions. The invention also relates to the use of the compositions comprising the engineered human α-galactosidase polypeptides for therapeutic purposes. The present invention provides recombinant α-galactosidase A and / or biologically active recombinant α-galactosidase A fragment, comprising an amino acid sequence comprising 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:5.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Patent Application No. 62 / 095313, filed December 22, 2014, and U.S. Provisional Patent Application No. 62 / 216452, filed September 10, 2015, both of which are incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention provides engineered human α-galactosidase polypeptides and compositions thereof. The engineered human α-galactosidase polypeptides are optimized to provide improved stability under both acidic (pH < 4.5) and basic (pH > 7) conditions. The present invention also relates to the use of compositions comprising the engineered human α-galactosidase polypeptides for therapeutic purposes.

[0003] References to sequence listings, tables, or computer programs A copy of the Sequence Listing has been submitted concurrently with the specification via EFS-Web as an ASCII text file with the file name "CX7-147WO2_ST25.txt," created on November 30, 2015, and size of 2,545,851 bytes. The Sequence Listing submitted via EFS-Web constitutes a part of the present specification and is incorporated herein by reference in its entirety. [Background technology]

[0004] Background of the Invention Human α-galactosidase ("GLA"; EC 3.2.1.22) is a lysosomal glycoprotein involved in the hydrolysis of terminal α-galactosyl moieties from glycolipids and glycoproteins. It acts on many substrates present in a range of human tissues. Fabry disease (also known as angiokeratoma corporis diffuse, Anderson-Fabry disease, hereditary dystopic lipidosis, α-galactosidase A deficiency, GLA deficiency, and ceramide trihexosidase deficiency) is an X-linked inborn error of glycosphingolipid catabolism resulting from α-galactosidase A deficiency or inactivity. Patients with Fabry disease accumulate globotriosylceramide (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 causes blood vessels to gradually narrow, reducing blood flow and nutrients in tissues, particularly the skin, kidneys, heart, brain, and nervous system. Thus, Fabry disease is a systemic disorder manifesting 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 sweating, corneal opacity, gastrointestinal problems, tinnitus, and hearing loss. Potentially life-threatening complications include progressive kidney damage, heart attack, and stroke. The disease affects an estimated 1 in 40,000 to 60,000 men, but also occurs in women. Indeed, heterozygous women with Fabry disease experience serious life-threatening conditions requiring medical treatment, including neurological abnormalities, chronic pain, fatigue, high blood pressure, heart disease, kidney failure, and stroke (see, e.g., Want et al., Genet. Med., 13:457-484

[2011] ). Symptoms of Fabry disease can begin any time after infancy; symptoms usually begin to appear between the ages of 4 and 8, although some patients exhibit milder, later-onset disease.Generally, treatment is 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., 2006, 63:453-457 [Non-patent document 2] Schiffmann, Pharm. Ther.,

[2009] 122:65~77 [Non-patent document 3] Want et al., Genet. Med.,

[2011] 13:457-484 Summary of the Invention [Means for solving the problem]

[0006] The present invention provides engineered human α-galactosidase polypeptides and compositions thereof. The engineered human α-galactosidase polypeptides are optimized to provide improved stability under both acidic (pH < 4.5) and basic (pH > 7) conditions. The present invention also relates to the use of compositions comprising the engineered human α-galactosidase polypeptides for therapeutic purposes. The present invention provides recombinant α-galactosidase A and / or biologically active recombinant α-galactosidase A fragments comprising an amino acid sequence comprising 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: 5. In some embodiments, the α-galactosidase A comprises at least one mutation at at least one position provided in Tables 2.1, 2.2, 2.4, and / or 2.5, where the position is numbered with reference to SEQ ID NO: 5. In some embodiments, the α-galactosidase A comprises at least one mutation at at least one position provided in Table 2.3, where the position is numbered with reference to SEQ ID NO: 10. In some further embodiments, the recombinant α-galactosidase A is derived from human α-galactosidase A. In some further embodiments, the recombinant alpha-galactosidase A comprises the polypeptide sequence of SEQ ID NO: 15, 13, 10, or 18. In still some further embodiments, the recombinant alpha-galactosidase A is more thermostable than the alpha-galactosidase A of SEQ ID NO: 5. In some further embodiments, the recombinant alpha-galactosidase A is more stable at pH 7.4 than the alpha-galactosidase A of SEQ ID NO: 5, and in further embodiments, the recombinant alpha-galactosidase A is more stable at pH 4.3 than the alpha-galactosidase A of SEQ ID NO: 5. In some embodiments, the recombinant alpha-galactosidase A is more stable at pH 7.4 and pH 4.3 than the alpha-galactosidase A of SEQ ID NO: 5. In still some further embodiments, the recombinant alpha-galactosidase A is deimmunized alpha-galactosidase A. In some embodiments, the recombinant alpha-galactosidase A is a deimmunized alpha-galactosidase A provided in Table 7.1. In still some further embodiments, the recombinant alpha-galactosidase A is purified.In some embodiments, the recombinant α-galactosidase A exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to pH 7.4; iii) increased tolerance to pH 4.3; or iv) reduced immunogenicity; or any combination of i), ii), iii), or iv), relative to a reference sequence. In some embodiments, the reference sequence is SEQ ID NO:5, and in some alternative embodiments, the reference sequence is SEQ ID NO:10.

[0007] The present invention also provides recombinant polynucleotide sequences encoding at least one recombinant alpha-galactosidase A provided herein (e.g., in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and / or 7.1). In some embodiments, the recombinant polynucleotide sequences are codon-optimized.

[0008] The present invention also provides expression vectors comprising a recombinant polynucleotide sequence encoding at least one recombinant alpha-galactosidase A provided herein (e.g., in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and / or 7.1). 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.

[0009] The present invention also provides host cells 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., in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and / or 7.1). In some embodiments, the host cell is eukaryotic.

[0010] 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.

[0011] The present invention also provides compositions comprising at least one recombinant alpha-galactosidase A provided herein (e.g., in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and / or Table 7.1). In some embodiments, the present invention provides pharmaceutical compositions. In some further embodiments, the present invention provides pharmaceutical compositions for treating Fabry disease, the pharmaceutical compositions comprising the enzyme compositions provided herein. In some embodiments, the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier and / or excipient. In some further embodiments, the pharmaceutical compositions are suitable for parenteral injection or infusion into humans.

[0012] The present invention also provides methods for treating and / or preventing symptoms of Fabry disease in a subject, comprising providing a subject with Fabry disease and at least one pharmaceutical composition comprising at least one recombinant alpha-galactosidase A provided herein (e.g., in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and / or 7.1), 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 administered a pharmaceutical composition of the invention can follow a diet whose fat content is less restricted than that required by a subject exhibiting symptoms of Fabry disease. In some embodiments, the subject is an infant or child, and in some alternative embodiments, the subject is an adult or young adult.

[0013] The present invention also provides uses of the compositions provided herein. In an embodiment of the present invention, for example, the following items are provided: (Item 1) Recombinant alpha-galactosidase A and / or a biologically active recombinant alpha-galactosidase A fragment 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:5. (Item 2) 2. The recombinant α-galactosidase A according to item 1, comprising at least one mutation at at least one position provided in Tables 2.1, 2.2, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, and / or 7.1, where the positions are numbered with reference to SEQ ID NO:5. (Item 3) 3. The recombinant alpha-galactosidase A according to item 2, comprising at least one mutation at at least one position provided in Table 2.3, where the positions are numbered with reference to SEQ ID NO: 10. (Item 4) 4. The recombinant α-galactosidase A according to any one of items 1 to 3, which is derived from human α-galactosidase A. (Item 5) A recombinant alpha-galactosidase A comprising the polypeptide sequence of SEQ ID NO: 15, 13, 10, 18, 40, 42, 44, or 46. (Item 6) 6. The recombinant α-galactosidase A according to any one of items 1 to 5, which is more thermostable than the α-galactosidase A of SEQ ID NO: 5. (Item 7) 7. The recombinant α-galactosidase A according to any one of items 1 to 6, which is more stable at pH 7.4 than the α-galactosidase A of SEQ ID NO: 5. (Item 8) 8. The recombinant α-galactosidase A according to item 7, which is more stable at pH 4.3 than the α-galactosidase A of SEQ ID NO: 5. (Item 9) 8. The recombinant alpha-galactosidase A according to item 7, which is more stable to exposure to serum than the alpha-galactosidase A of SEQ ID NO: 5. (Item 10) 10. The recombinant α-galactosidase A according to any one of items 1 to 9, which is a deimmunized α-galactosidase A. (Item 11) The recombinant alpha-galactosidase A according to any of items 1 to 10, which is a deimmunized alpha-galactosidase A provided in Table 7.1. (Item 12) 12. The recombinant α-galactosidase A according to any one of Items 1 to 11, which is purified. (Item 13) 13. The recombinant α-galactosidase A according to any of items 1 to 12, which, compared to a reference sequence, exhibits at least one improved property selected from: i) enhanced catalytic activity; ii) increased tolerance to pH 7.4; iii) increased tolerance to pH 4.3; iv) increased tolerance to serum; or v) reduced immunogenicity; or any combination of i), ii), iii), iv), or v). (Item 14) Item 14. The recombinant alpha-galactosidase A according to Item 13, wherein the reference sequence is SEQ ID NO: 5 or SEQ ID NO: 10. (Item 15) 15. A composition comprising at least one recombinant alpha-galactosidase A according to any one of items 1 to 14. (Item 16) 16. A recombinant polynucleotide sequence encoding at least one recombinant alpha-galactosidase A according to any one of items 1 to 15. (Item 17) 17. The recombinant polynucleotide sequence of item 16, which is codon-optimized. (Item 18) 18. An expression vector comprising the recombinant polynucleotide sequence according to items 16 and / or 17. (Item 19) 19. The expression vector of item 18, wherein the recombinant polynucleotide sequence is operably linked to a regulatory sequence. (Item 20) 20. The expression vector of item 19, wherein the regulatory sequence is a promoter. (Item 21) 21. The expression vector of item 20, wherein the promoter is a heterologous promoter. (Item 22) A host cell comprising the expression vector according to any one of items 18 to 21. (Item 23) 23. The host cell of item 22, which is a eukaryote. (Item 24) 24. A method for producing an alpha-galactosidase A variant, comprising culturing the host cell of item 22 or 23 under conditions in which the alpha-galactosidase A encoded by the recombinant polynucleotide is produced. (Item 25) 25. The method of claim 24, further comprising recovering the alpha-galactosidase A. (Item 26) 26. The method of claim 25, further comprising purifying the alpha-galactosidase A. (Item 27) 16. A pharmaceutical composition for treating Fabry disease, comprising the enzyme composition of item 15. (Item 28) 28. The pharmaceutical composition according to item 27, further comprising a pharmaceutically acceptable carrier and / or excipient. (Item 29) 29. A pharmaceutical composition according to items 27 and / or 28, which is suitable for parenteral injection or infusion into humans. (Item 30) 40. A method for treating and / or preventing symptoms of Fabry disease in a subject, the method comprising the steps of providing a subject with Fabry disease, and providing the subject with the pharmaceutical composition of any of items 27 to 39. (Item 31) 31. The method of item 30, wherein the symptoms of Fabry disease are ameliorated. (Item 32) 32. The method of items 30 and / or 31, wherein the subject is able to consume a diet whose fat content is less restricted than the diet required by subjects exhibiting symptoms of Fabry disease. (Item 33) 33. The method according to any one of items 30 to 32, wherein the subject is an infant or a child. (Item 34) 33. The method according to any of items 30 to 32, wherein the subject is an adult or young adult. (Item 35) Use of the composition according to any one of items 15 and 27 to 29. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1 provides a graph showing the relative activity of different GLA constructs in S. cerevisiae after 2-5 days of culture.

[0015] [Figure 2] FIG. 2 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants after incubation at various pHs.

[0016] [Figure 3] FIG. 3 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants after incubation at various temperatures.

[0017] [Figure 4-1] FIG. 4 provides graphs showing the absolute (panels A and B) and relative (panels C and D) activity of GLA variants after challenge with increasing amounts of serum-containing buffer. [Figure 4-2] FIG. 4 provides graphs showing the absolute (panels A and B) and relative (panels C and D) activity of GLA variants after challenge with increasing amounts of serum-containing buffer.

[0018] [Figure 5] FIG. 5 provides a graph showing the relative activity of GLA variants expressed in HEK293T cells.

[0019] [Figure 6] FIG. 6 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants expressed in HEK293T cells, normalized for activity, and incubated at various pHs.

[0020] [Figure 7] FIG. 7 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants expressed in HEK293T cells, normalized for activity, and incubated at various temperatures.

[0021] [Figure 8] FIG. 8 provides graphs showing remaining GLA variant activity after incubation in acidic (Panel A) or basic (Panel B) solutions.

[0022] [Figure 9] FIG. 9 provides a graph showing restored GLA activity in rat serum after administration of GLA variants. DETAILED DESCRIPTION OF THE INVENTION

[0023] Description of the Invention The present invention provides engineered human α-galactosidase polypeptides and compositions thereof. The engineered human α-galactosidase polypeptides are optimized to provide improved stability under both acidic (pH < 4.5) and basic (pH > 7) conditions. The present invention also relates to the use of compositions comprising the engineered human α-galactosidase polypeptides for therapeutic purposes.

[0024] In some embodiments, the engineered human α-galactosidase polypeptides are optimized to provide improved stability at various levels. The present invention also relates to the use of compositions comprising engineered human α-galactosidase polypeptides for therapeutic purposes.

[0025] For eligible individuals, enzyme replacement therapy (e.g., Fabrazyme® agalsidase beta; Genzyme) is available and will be considered for the treatment of Fabry disease. Currently used enzyme replacement therapy is a recombinantly expressed form of wild-type human GLA. Intravenously administered GLA circulates, is endocytosed, and traffics to the endosomes / lysosomes of target organs, where it is known to reduce Gb3 accumulation. These drugs do not completely relieve patients' symptoms, as neuropathic pain and transient ischemic attacks continue to occur at a low rate. Additionally, GLA is poorly uptaken by most target organs compared to the liver, and the enzyme is unstable at the pH of blood and lysosomes. Therefore, available treatments remain problematic. Additionally, 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 enzyme suitable for the treatment of Fabry disease, with fewer 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), the pH the enzyme encounters upon injection into the bloodstream. Additionally, the enzyme has increased stability at the pH of the lysosome (i.e., where the enzyme is active during treatment) (pH 4.3). Therefore, directed evolution of recombinantly expressed human GLA in Saccharomyces cerevisiae was used to provide novel GLA variants with desired stability characteristics, using high-throughput screening of a diverse enzyme variant library. Additionally, variant enzymes were screened and their amino acid sequences determined to identify novel GLA variants with reduced predicted immunogenicity. By providing GLA variants with increased pH stability and reduced immunogenicity, the present invention provides compositions and methods suitable for use in patients, increasing patient treatment tolerance and providing flexibility in administration and formulation for improved patient outcomes.

[0026] Abbreviations and definitions: Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill 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 those well known and commonly employed in the art. Such techniques are well known and described in numerous texts and references well known to those of ordinary skill in the art. Standard techniques, or modifications thereof, are used for chemical synthesis and chemical analysis. All patents, patent applications, articles, and publications mentioned in this specification (both above and below) are expressly incorporated herein by reference.

[0027] Although any suitable method and material similar or equivalent to those described herein can be used to implement the present invention, 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, because they may vary depending on the circumstances used by those skilled in the art.Therefore, the terms directly defined below will be explained in more detail 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.

[0028] Also, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0029] Numerical ranges are inclusive of the numbers defining the range. Accordingly, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within that broader numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limitation disclosed herein is also intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.

[0030] The term "about" refers to a tolerance for a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some cases, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0031] Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention that may be made by reference to the application as a whole. Accordingly, the terms defined directly below will be more fully described by reference to the application as a whole. Nevertheless, to facilitate understanding of the invention, certain terms are defined below.

[0032] 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.

[0033] As used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0034] "EC" numbers refer to the enzyme nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB). The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction they catalyze.

[0035] "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0036] "NCBI" refers to the National Center for Biological Information and the sequence databases provided therein.

[0037] "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).

[0038] "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. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0039] The terms "engineered," "recombinant," "non-naturally occurring," and "variant," when used with respect to a cell, polynucleotide, or polypeptide, refer to material that corresponds to the native or native form of the material, i.e., material that has been modified in a manner that would not be considered to occur in nature in nature, or material that is identical to the native or native form but that has been produced or derived by synthetic material and / or manipulation using recombinant techniques.

[0040] As used herein, "wild-type" and "naturally occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is one that can be isolated from a natural source and is present in an organism that has not been intentionally modified by human beings.

[0041] "Deimmunized," as used herein, refers to the manipulation of a protein sequence to create a variant that is predicted to be less immunogenic than a wild-type or reference protein. In some embodiments, the predicted deimmunization is complete, in that the variant protein is predicted not to stimulate an immune response in patients to whom the variant protein is administered. This response can be measured by various methods, including, but not limited to, the presence or abundance of anti-drug antibodies, the presence or abundance of neutralizing antibodies, the presence of an anaphylactic response, peptide presentation on major histocompatibility complex-II (MHC-II) proteins, or the prevalence or intensity of cytokine release upon administration of the protein. In some embodiments, the variant protein is less immunogenic than the wild-type or reference protein. In some embodiments, deimmunization involves modifications to subsequences (e.g., epitopes) of a protein that are recognized by human leukocyte antigen (HLA) receptors. In some embodiments, these epitopes are removed by altering their amino acid sequence to generate a deimmunized variant protein in which such subsequences are no longer recognized by HLA receptors. In some other embodiments, these epitopes retain binding affinity to HLA receptors but are not presented. In some embodiments, the deimmunized proteins exhibit lower levels of response in biochemical and cell biological predictors of human immune response, including dendritic cell T cell activation assays or (HLA) peptide binding assays. In some embodiments, these epitopes are removed by altering their amino acid sequence to generate deimmunized variant proteins in which the epitopes are no longer recognized by T cell receptors. In yet other embodiments, the deimmunized proteins induce anergy of their corresponding T cells, activate T regulatory cells, or result in clonal deletion of recognizing B cells.

[0042] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0043] The term "percent (%) sequence identity" is used herein to refer to comparisons between polynucleotides and polypeptides and is determined by comparing two optimally aligned sequences over a comparison window, where the polynucleotide or polypeptide sequence portion in the comparison window may contain additions or deletions (i.e., gaps) when compared to the reference sequence for 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 occurs 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 sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences or where the nucleic acid base or amino acid residue is aligned using gaps 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 sequence identity. Those skilled in the art will recognize that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be achieved using, for example, the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

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

[1970] ), or the homology alignment algorithm of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), 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 percent 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., Nucleic Acids Res., 3389-3402

[1977] , respectively). Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information website. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in a query sequence that match or meet some positive threshold score T when aligned with words of the same length in a database sequence. 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 these word hits. 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 word hits in each direction is halted when the cumulative alignment score falls by an amount X from its maximum achieved value; when the accumulation of one or more negative-scoring residue alignments causes the cumulative score to progress to 0 or less; 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 in the GCG Wisconsin software package (Accelrys, Madison Wis.) can be used using the default parameters provided.

[0044] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence (e.g., a segment of a full-length gene sequence or polypeptide sequence). Generally, a reference sequence is 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 entire length of the nucleic acid or polypeptide. 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, where the reference sequence may have one or more changes in 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 where the portion of the sequence in the comparison window can contain 20% or less additions or deletions (i.e., gaps) when compared to the reference sequence (no additions or deletions) for optimal alignment of the two sequences. The comparison window can be longer than 20 contiguous residues, and optionally includes windows of 30, 40, 50, 100, or longer.

[0045] "Corresponding to," "relating to," or "related to," when used in the context of numbering a given amino acid sequence or polynucleotide sequence, refers to the numbering of residues in a specified reference sequence when the given amino acid sequence or polynucleotide sequence is compared to the reference sequence. In other words, residue numbers or residue positions in a given polymer are designated with respect to the reference sequence rather than by the actual numbered position of the residues in the given amino acid sequence or polynucleotide sequence. For example, a given amino acid sequence (such as the amino acid sequence of an engineered GLA) can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, despite the presence of gaps, residues in the given amino acid sequence or polynucleotide sequence are numbered with respect to the aligned reference sequence.

[0046] An "amino acid difference" or "residue difference" refers to an amino acid residue difference at a position in a polypeptide sequence compared 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 based on the residue difference. For example, a "residue difference at position X93 as compared to SEQ ID NO:2" refers to an amino acid residue difference at a polypeptide position corresponding to position 93 in SEQ ID NO:2. Thus, if a reference polypeptide of SEQ ID NO:2 has a serine at position 93, then a "residue difference at position X93 as compared to SEQ ID NO:2" refers to an amino acid substitution of any residue other than serine at a polypeptide position corresponding to position 93 in SEQ ID NO:2. In most examples herein, a specific amino acid residue difference at a position will be designated as "XnY," where "Xn" designates the corresponding position above and "Y" is a single-letter identifier for the amino acid found in the engineered polypeptide (i.e., the residue that differs from the residue in the reference polypeptide). In some cases (e.g., in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and 6.1), the present disclosure also provides specific amino acid differences, designated by the conventional notation "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 engineered polypeptide sequence. In some examples, the polypeptides of the present disclosure can include one or more amino acid residue differences compared to the reference sequence, which are indicated by a list of the specific positions at which the residue difference occurs when compared to the reference sequence. In some embodiments where more than one amino acid may be used at a particular residue position in the polypeptide, the various amino acid residues that may be used are separated by a " / " (e.g., X307H / X307P or X307H / P). In some embodiments, the enzyme variants include more than one substitution. These substitutions are separated by a slash for ease of reading (e.g., C143A / K206A). 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.

[0047] " Conservative amino acid substitution " refers to the substitution of a residue with a different residue that has a similar side chain, and therefore typically includes the substitution of the amino acid in a polypeptide with the amino acid in the same or similar defined amino acid class.As a non-limiting example, an amino acid with an aliphatic side chain can be substituted with another aliphatic amino acid (for example, alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain can be substituted with another amino acid with a hydroxyl side chain (for example, serine and threonine); an amino acid with an aromatic side chain can be substituted with another amino acid with an aromatic side chain (for example, phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain can be substituted with another amino acid with a basic side chain (for example, lysine and arginine); an amino acid with an acidic side chain can be substituted with another amino acid with an acidic side chain (for example, aspartic acid or glutamic acid); and / or a hydrophobic or hydrophilic amino acid can be substituted with another hydrophobic or hydrophilic amino acid.

[0048] "Non-conservative substitution" refers to the substitution of an amino acid in a polypeptide with an amino acid having a significantly different side chain. Non-conservative substitutions can use amino acids between defined groups rather than within them, and affect (a) the structure of the peptide backbone in the substituted region (e.g., replacing glycine with proline), (b) the charge or hydrophobicity, or (c) the bulkiness of the side chain. By way of non-limiting example, exemplary non-conservative substitutions can 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.

[0049] "Deletion" refers to the modification of a polypeptide by the removal of one or more amino acids from a reference polypeptide. Deletions can remove 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total amino acids, or up to 20% of the total amino acids that make up the reference enzyme, while retaining the enzymatic activity of the engineered enzyme and / or its improved properties. Deletions can be internal and / or terminal to the polypeptide. In various embodiments, deletions can include a contiguous segment or can be discontinuous.

[0050] "Insertion" refers to the modification of a polypeptide by the addition of one or more amino acids to a reference polypeptide. Insertions can be made internally in a polypeptide or at the carboxy or amino terminus. As used herein, insertions include fusion proteins known in the art. Insertions can be contiguous segments of amino acids or can be separated by one or more amino acids in a naturally occurring polypeptide.

[0051] As used interchangeably herein, the terms "functional fragment" and "biologically active fragment" refer to a polypeptide that has amino-terminal and / or carboxy-terminal deletions and / or internal deletions, but in which the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is compared (e.g., the engineered full-length GLA of the present invention), and that retains substantially all of the activity of the full-length polypeptide.

[0052] An "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany the polypeptide. This term includes a polypeptide that has been removed or purified from its naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). Recombinant GLA polypeptides can be present intracellularly, in cell culture medium, or prepared in various forms (such as a lysate or isolated preparation). As such, in some embodiments, a recombinant GLA polypeptide can be an isolated polypeptide.

[0053] "Substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species (i.e., more abundant than any other individual macromolecular species in the composition, on a molar or weight basis). Generally, a substantially purified composition is one in which the target species accounts for at least about 50% of the macromolecular species present in the composition, by molar or weight percentage. 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 the total macromolecular species present in the composition, by molar or weight percentage. In some embodiments, the target species is purified to essential homogeneity (i.e., contaminants in the composition cannot be detected by conventional detection methods), where the composition consists essentially of a single macromolecular species. 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.

[0054] "Improved enzymatic properties" refers to an engineered GLA polypeptide that exhibits improved enzymatic properties compared to a reference GLA polypeptide and / or a wild-type GLA polypeptide or another engineered GLA polypeptide. Improved properties include, but are not limited to, increased protein expression, increased thermoactivity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity or affinity, increased specific activity, increased resistance to substrate or end-product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased tolerance to acidic or basic pH, increased resistance to proteolytic activity (i.e., decreased susceptibility to proteolysis), reduced aggregation, increased solubility, decreased immunogenicity, improved post-translational modifications (e.g., glycosylation), and altered temperature profile.

[0055] "Increased enzyme activity" or "improved catalytic activity" refers to an improved property of an engineered GLA polypeptide, which can be demonstrated by an increase in specific activity (e.g., product produced / time / weight of protein) or an increase in the rate of substrate to product conversion (e.g., the rate of conversion of a starting amount of substrate to product in a specific time using a specific amount of GLA) compared to a reference GLA enzyme. Exemplary methods for determining enzyme activity are provided in the Examples. Any property related to enzyme activity (K, the change of which can increase enzyme activity) can be demonstrated by an increase in specific activity (e.g., product produced / time / weight of protein) or an increase in the rate of substrate to product conversion (e.g., the rate of conversion of a starting amount of substrate to product in a specific time using a specific amount of GLA). Exemplary methods for determining enzyme activity are provided in the Examples. m , V max , or k cat The enzymatic activity can be affected, including classical enzymatic properties such as the enzyme activity of the corresponding wild-type enzyme. The improvement in enzymatic activity can be from about 1.1-fold that of the corresponding wild-type enzyme to 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, or more enzymatic activity over the naturally occurring GLA or another engineered GLA from which the GLA polypeptide is derived.

[0056] In some embodiments, the engineered GLA polypeptides have a k of at least 0.1 / sec, at least 0.5 / sec, at least 1.0 / sec, at least 5.0 / sec, at least 10.0 / sec, and in some preferred embodiments, greater than 10.0 / sec. cat In some embodiments, K m is in the range of about 1 μM to about 5 mM; in the range of about 5 μM to about 2 mM; in the range of about 10 μM to about 2 mM; or in the range of about 10 μM to about 1 mM. In some particular embodiments, the engineered GLA enzyme exhibits improved enzymatic activity in the range of 1.5 to 10-fold, 1.5 to 25-fold, 1.5 to 50-fold, 1.5 to 100-fold, or more, over that of a reference GLA enzyme (e.g., wild-type GLA or any other reference GLA) after exposure to certain conditions. 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 by high-performance liquid chromatography (HPLC) separation coupled with UV absorbance or direct fluorescence detection or subsequent o-phthaldialdehyde (OPA) derivatization. As further described herein, enzyme activity is compared using defined enzyme preparations, defined assays under set conditions, and one or more defined substrates. Generally, when comparing lysates, the same expression system and the same host cells are also used to determine cell number and assay amount of protein to minimize variations in the amount of enzyme produced by the host cells and the amount of enzyme present in the lysates.

[0057] The term "improved tolerance to acidic pH" means that the recombinant GLA of the present invention has increased stability (higher activity is retained for a specified period (1 hour, up to 24 hours) at about pH 4.8 after exposure to acidic pH) compared to a reference GLA or another enzyme.

[0058] "Physiological pH," as used herein, means the pH range generally found in the blood of a subject (eg, a human).

[0059] The term "basic pH" (as used, for example, in reference to improved stability to basic pH conditions or increased resistance to basic pH) refers to a pH range of about 7-11.

[0060] The term "acidic pH" (as used, for example, in reference to improved stability to or increased tolerance to acidic pH conditions) refers to a pH range of about 1.5 to 4.5.

[0061] "Conversion" refers to the enzymatic conversion (or biotransformation) of a substrate to a corresponding product. "Conversion rate" refers to the percentage of a substrate converted to a product within a period of time under specified conditions. Thus, the "enzymatic activity" or "activity" of a GLA polypeptide can be expressed as the "conversion rate" of a substrate to a product over a specified period of time.

[0062] "Hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washing at various but higher stringencies. The term "moderately stringent hybridization" refers to conditions under which a target DNA can bind to a complementary nucleic acid with about 60% identity to the target DNA, preferably about 75% identity, about 85% identity, or more than about 90% identity to the target polynucleotide. Exemplary moderately stringent conditions are conditions equivalent 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 conditions under which the thermal melting temperature T determined under solution conditions for a defined polynucleotide sequence is higher than the thermal melting temperature T mHigh stringency conditions refer to conditions at temperatures from about 10°C or less below 65°C. In some embodiments, high stringency conditions refer to conditions under which only nucleic acid sequences that form stable hybrids at 0.018M NaCl at 65°C can hybridize (i.e., if a hybrid is not stable in 0.018M NaCl at 65°C, it will not be stable under high stringency conditions as intended herein). High stringency conditions can be provided, for example, by hybridization under conditions equivalent to 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at 42°C, followed by a wash in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization in 5x SSC containing 0.1% (w:v) SDS at 65°C and a wash in 0.1x SSC containing 0.1% SDS at 65°C. Other highly stringent and moderately stringent hybridization conditions are described in the references cited above.

[0063] "Codon optimization" refers to the change of the codons of a polynucleotide encoding a protein to those preferentially used in a particular organism so that the encoded protein can be more efficiently expressed in the target organism.Although the genetic code is degenerate in that most amino acids are represented by several codons (called "synonyms" or "synonymous" codons), it is well known that the codon usage frequency of a particular organism is non-random and biased toward certain codon triplets.This codon usage bias may be high for a given gene, a common function or ancestral gene, a highly expressed protein relative to a low copy number protein, and a cohesive protein-coding region 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 selected for expression.

[0064] The term "control sequences" as used herein includes 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 sequence, a polyadenylation sequence, a propeptide sequence, a promoter sequence, a signal peptide sequence, an initiation sequence, and a transcription terminator. At a minimum, a control sequence includes a promoter, a transcriptional stop signal, and a translational stop signal. The control sequences may be provided with linkers to introduce specific restriction sites facilitating ligation of the control sequence within the coding region of the nucleic acid sequence encoding the polypeptide.

[0065] "Operably linked" is defined herein as a configuration in which regulatory sequences are positioned in relation to a polynucleotide of interest such that they direct or control the expression of the polynucleotide and / or polypeptide of interest (i.e., in a functional relationship).

[0066] A "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 transcriptional regulatory sequences that mediate expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in the host cell of choice (including mutant promoters, truncated promoters, and hybrid promoters) and may be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell.

[0067] "Suitable reaction conditions" refer to conditions in an enzymatic conversion reaction solution (e.g., enzyme load, substrate load, temperature, pH, buffer, cosolvent range, 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 in the Examples. "Load" as in "compound load" or "enzyme load" refers to the concentration or amount of a component in the reaction mixture at the start of the reaction. "Substrate" in the context of an enzymatic conversion reaction process refers to a compound or molecule on 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.

[0068] As used herein, the term "culturing" refers to growing a population of microbial cells under any suitable conditions (e.g., using a liquid, gel, or solid medium).

[0069] 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 or S. cerevisiae. Variants of recombinant polypeptides can be produced by various methods known in the art. Indeed, there are a wide variety of different mutagenesis techniques well known to those skilled 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 (region-directed), or random mutagenesis throughout a gene (e.g., saturation mutagenesis). Many suitable methods for generating enzyme variants are known to those skilled 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 in DNA and protein engineering are provided in the following patents: U.S. Patent Nos. 6,117,679; 6,420,175; 6,376,246; 6,586,182; 7,747,391; 7,747,393; 7,783,428; and 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," "variant GLA enzymes," and "GLA variants") are used.

[0070] 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 that is operably linked to a suitable regulatory sequence that can express the polypeptide encoded in 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 termination sequence.

[0071] As used herein, the term "expression" includes any step involved in producing a polypeptide, including, 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.

[0072] As used herein, the term "produce" refers to the production of proteins and / or other compounds 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 the polypeptide from the cell.

[0073] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, termination sequence, etc.) is "heterologous" to another sequence to which it is operably linked if the two sequences are not naturally linked.

[0074] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector containing DNA provided herein (e.g., a polynucleotide encoding a GLA variant). 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.

[0075] The term "analog" refers to a polypeptide having greater than 70% 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%, or 99% sequence identity) with a reference polypeptide. In some embodiments, an analog refers to a polypeptide that includes one or more non-naturally occurring amino acid residues (including, but not limited to, homoarginine, ornithine, and norvaline) and naturally occurring 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.

[0076] The term "therapeutic agent" refers to a compound that is administered to a subject who exhibits signs or symptoms of a medical condition and that has a beneficial or desired medical effect.

[0077] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., a human) that contains a pharmaceutically effective amount of an engineered GLA polypeptide encompassed by the present invention and an acceptable carrier.

[0078] 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 by using routine experimentation.

[0079] The terms "isolated" and "purified" are used 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; rather, it is intended as a relative definition.

[0080] 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 females and males.

[0081] As used herein, the term "patient" means any subject being evaluated for, treated for, or experiencing a disease.

[0082] The term "infant" refers to a child between 1 month and about 1 year of age. As used herein, the term "newborn" refers to a child between birth and 28 days after birth. The term "premature infant" refers to an infant born after 20 full weeks of gestation but before the normal completion of gestation, generally weighing between about 500 and about 2499 grams at birth. A "very low birth weight infant" is an infant weighing less than 1500 grams at birth.

[0083] As used herein, the term "child" refers to a person who has not reached the legal age for consent to treatment or research procedures. In some embodiments, the term refers to a person from birth through adolescence.

[0084] As used herein, the term "adult" refers to a person who has reached the legal age for the relevant jurisdiction (e.g., 18 years of age in the United States). In some embodiments, the term refers to any organism that has reached full growth and maturity. In some embodiments, the term "young adult" refers to a person who is under the age of 18 but has reached sexual maturity.

[0085] As used herein, "composition" and "formulation" encompass products containing at least one engineered GLA of the present invention and intended for any suitable use (e.g., pharmaceutical compositions, dietary / nutritional supplements, feed, etc.).

[0086] The terms "administration" of a composition and "administering" a composition mean providing a composition of the invention to a subject (e.g., one suffering from the effects of Fabry disease).

[0087] The term "carrier," when used in reference to pharmaceutical compositions, refers to any of the standard pharmaceutical carriers, buffers, and excipients (e.g., stabilizers, preservatives, and adjuvants).

[0088] The term "pharmaceutically acceptable" means a material that may be administered to a subject without causing any undesired biological effects and without interacting in a deleterious manner with any of the components it contains, and that possesses the desired biological activity.

[0089] 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.

[0090] 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, reduces, or eliminates one or more symptoms of the disease / condition, or prevents or delays the onset of the symptoms.

[0091] The term "therapeutically effective amount," when used in reference to a disease / condition, refers to an amount and / or concentration of a composition (e.g., an engineered GLA polypeptide) that ameliorates, reduces, or eliminates the disease / condition. In some embodiments, the term is used in reference to an 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.

[0092] The terms "treating," "treat," and "treatment" are intended to encompass preventative (eg, prophylactic) and palliative treatment.

[0093] Engineered GLA Expression and Activity: To drive secretion of yeast-codon-optimized mature human GLA, two strategies for secreted GLA expression were utilized: using the yeast MFα signal peptide (MF-SP) or a longer 83-amino acid leader sequence (MF leader). Clones were expressed from the pYT-72 vector in the S. cerevisiae strain INVSc1. Both approaches provided supernatants with measurable activity toward the fluorogenic substrate 4-methylumbelliferyl α-D-galactopyranoside (4-MuGal). However, the construct with the yeast MFα signal peptide provided threefold higher activity and was used as the starting sequence for directed evolution.

[0094] To identify mutational diversity, a 13-position conserved "homolog" combinatorial library and a 192-position site-saturation mutagenesis library were constructed. Equal volumes of supernatant were screened under non-challenge conditions (no incubation, pH 4.8) or after 1 hour of incubation in low (3.9-4.2) or high (7.1-8.2) pH environments. GLA variants with increased activity due to increased GLA expression or GLA specific activity were identified based on their fold improvement over the parent GLA. GLA variants with increased stability were identified by dividing the fold improvement observed under challenge conditions by the fold improvement observed under non-challenge conditions. This approach reduces the bias against selecting variants based on increased expression without a change in specific activity at extreme pH. A composite activity score (product of fold improvement across all three conditions) and stability (product of stability score) were used to rank the improved variants for inclusion in subsequent GLA libraries.

[0095] Engineered GLA: In some embodiments, an 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 about 100% amino acid sequence identity with SEQ ID NO:5 and amino acid residue differences at one or more amino acid positions compared to SEQ ID NO:5 (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 NO:5, or a sequence 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 NO:5). In some embodiments, the residue differences compared to SEQ ID NO:5 at one or more amino acid positions include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the engineered GLA polypeptide is a polypeptide listed in Table 2.1, 2.2, 2.4, 2.5 or Table 7.1.

[0096] In some embodiments, an 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 about 100% amino acid sequence identity with SEQ ID NO: 10 and amino acid residue differences compared to SEQ ID NO: 10 at one or more amino acid positions (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 NO: 10, or a sequence 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 with SEQ ID NO: 10). In some embodiments, the residue differences compared to SEQ ID NO: 10 at one or more amino acid positions include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions. In some embodiments, the engineered GLA polypeptide is a polypeptide listed in Table 2.3.

[0097] In some embodiments, the engineered GLA exhibiting improved properties has 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%, or at least 99% amino acid sequence identity to SEQ ID NO: 5. In some embodiments, the engineered GLA exhibiting improved properties has 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%, or at least 99% amino acid sequence identity to SEQ ID NO: 10.

[0098] In some embodiments, the engineered GLA polypeptide is selected from SEQ ID NOs: 15, 13, 10, and 18.

[0099] In some embodiments, the engineered GLA polypeptide comprises a functional fragment of the engineered GLA polypeptide encompassed by the present invention. A functional fragment has 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 it is derived. A functional fragment comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or even 99% of the parent sequence of the engineered GLA. In some embodiments, the functional fragment is 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.

[0100] polynucleotides encoding the engineered polypeptides, expression vectors, and host cells; The present invention provides the polynucleotide that encodes the engineered GLA polypeptide described herein.In some embodiments, the polynucleotide is operably linked to one or more heterologous control sequences that regulate gene expression to produce the recombinant polynucleotide that can express polypeptide.The expression construct that comprises the heterologous polynucleotide that encodes engineered GLA polypeptide can be introduced into suitable host cell to express corresponding GLA polypeptide.

[0101] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the corresponding codons for various amino acids describes all polynucleotides capable of encoding a subject polypeptide. Due to the degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, a large number of nucleic acids can be generated, all of which encode the engineered GLA polypeptide. Thus, with knowledge of a particular amino acid sequence, one skilled in the art can generate a number of different nucleic acids by simply modifying the sequence of one or more codons in a manner that does not alter the amino acid sequence of the protein. In this regard, the present invention specifically contemplates each and every possible variation of polynucleotides that can be generated that encode the polypeptides described herein by selecting combinations based on possible codon choices, and all such variations shall be considered specifically disclosed for any polypeptide described herein (including the variants provided in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and 6.1).

[0102] In various embodiments, it is preferred to select codons that are 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.

[0103] In some embodiments, as described above, the polynucleotide encodes an engineered polypeptide having GLA activity with the properties disclosed herein, wherein the polypeptide has an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOs: 5 and / or 10. or any variant disclosed in Tables 2.1, 2.2, 2.3, 2.4, 2.5, or 6.1, and one or more residue differences (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions) when compared to the reference polypeptide of SEQ ID NO: 5, and / or 10, or any variant disclosed in Tables 2.1, 2.2, 2.3, 2.4, 2.5, or 6.1. In some embodiments, the reference sequence is selected from SEQ ID NO: 5 and / or 10. In some embodiments, the polynucleotide encodes an engineered polypeptide having GLA activity with the properties disclosed herein, which polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence of SEQ ID NO: 5 when optimally aligned with the polypeptide of SEQ ID NO: 5 and one or more residue differences compared to SEQ ID NO: 5 at residue positions selected from those provided in Tables 2.1, 2.2, 2.4, 2.5, or 6.1.

[0104] In some embodiments, the polynucleotide encodes an engineered polypeptide having GLA activity with the properties disclosed herein, which polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence of SEQ ID NO: 10 when optimally aligned with the polypeptide of SEQ ID NO: 10 and one or more residue differences compared to SEQ ID NO: 10 at residue positions selected from those provided in Table 2.3.

[0105] In some embodiments, a polynucleotide encoding an engineered GLA polypeptide comprises a polynucleotide sequence selected from the polynucleotide sequences encoding SEQ ID NOs: 10, 13, 15, 18, 21, and 24. In some embodiments, a polynucleotide encoding an engineered GLA polypeptide has at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 93%, 95%, 96%, 97%, 98%, 99% nucleotide residue identity to SEQ ID NOs: 8, 9, 11, 12, 14, 16, 17, 19, 20, 22, and / or 23. In some embodiments, a polynucleotide encoding an engineered GLA polypeptide comprises a polynucleotide sequence selected from the polynucleotide sequences encoding SEQ ID NOs: 8, 9, 11, 12, 14, 16, 17, 19, 20, 22, and 23.

[0106] In some embodiments, a polynucleotide is capable of hybridizing under high stringency conditions to a reference polynucleotide sequence selected from SEQ ID NOs: 8, 9, 11, 12, 14, 16, 17, 19, 20, 22, and 23, or its complement, or to a polynucleotide sequence encoding any of the variant GLA polypeptides provided herein. In some embodiments, a polynucleotide capable of hybridizing under high stringency conditions encodes a GLA polypeptide comprising an amino acid sequence having one or more residue differences compared to SEQ ID NOs: 5 and / or 10 at a residue position selected from any of the positions set forth in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and / or 6.1.

[0107] In some embodiments, an isolated polynucleotide encoding any of the engineered GLA polypeptides provided herein is manipulated in a variety of ways to express 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 control expression of the polynucleotide and / or polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide before insertion into the vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art.

[0108] In some embodiments, regulatory sequences include, inter alia, a promoter, a leader sequence, a polyadenylation sequence, a propeptide sequence, a signal peptide sequence, and a transcription terminator. As is known in the art, an appropriate promoter can be selected based on the host cell to be used. Exemplary promoters for filamentous fungal host cells include promoters from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral α-amylase, Aspergillus niger acid-stable α-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 α-amylase and Aspergillus Examples of promoters that can be used include promoters derived from the Saccharomyces cerevisiae gene (e.g., a hybrid of the promoter derived from the Saccharomyces cerevisiae triosephosphate isomerase gene), mutant promoters, truncated promoters, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the 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 genes. 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 those derived from cytomegalovirus (CMV), simian vacuolating virus 40 (SV40), Homo sapiens phosphorglycerate kinase, beta-actin, elongation factor 1A or glyceraldehyde-3-phosphate dehydrogenase, or Gallus. Examples of suitable actin proteins include, but are not limited to, those derived from β-actin of G. gallus.

[0109] In some embodiments, the regulatory sequence is also an appropriate transcription termination sequence (a sequence recognized by a host cell to terminate transcription). The termination sequence is operably linked to the 3' end of the nucleic acid sequence encoding the polypeptide. Any terminator that functions in the host cell of choice finds use 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 α-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), or Homo sapiens growth hormone.

[0110] In some embodiments, the regulatory sequence is a suitable leader sequence (a nontranslated region of an mRNA important for translation by the host cell). The leader sequence is operably linked to the 5' end of the nucleic acid sequence encoding the polypeptide. Any leader sequence that functions in the host cell of choice can be used. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triosephosphate isomerase. Suitable leaders for yeast host cells include, but are not limited to, those obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae α-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde 3-phosphate dehydrogenase (ADH2 / GAP).

[0111] The regulatory sequence may also be a polyadenylation sequence (a sequence operably linked to the 3' end of a nucleic acid sequence and recognized by a host cell as a signal for adding polyadenosine residues to the transcribed mRNA during transcription). Any polyadenylation sequence that functions in the host cell of choice 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 α-glucosidase. Useful polyadenylation sequences for yeast host cells are also known in the art (see, e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990

[1995] ).

[0112] In some embodiments, the regulatory sequence is a signal peptide coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and that directs the encoded polypeptide into the secretory pathway of a cell. The 5' end of the coding sequence of a nucleic acid sequence can inherently contain a signal peptide coding region that is 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 can 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 is used to express the engineered GLA polypeptides provided herein. Useful signal peptide coding regions for filamentous fungal host cells include those from Aspergillus oryzae Useful signal peptides include, but are not limited to, those derived from the genes for TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those derived from the genes for Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Useful signal peptides for mammalian host cells include, but are not limited to, those derived from the gene for immunoglobulin gamma (IgG).

[0113] In some embodiments, the regulatory sequence is a propeptide coding region that encodes an amino acid sequence positioned at the amino terminus of a polypeptide. The resulting polypeptide is called a "proenzyme" or "propolypeptide" (or, in some cases, a "zymogen"). A propolypeptide can be converted to an active mature polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

[0114] In another aspect, the present invention also provides recombinant expression vectors comprising a polynucleotide encoding an engineered GLA polypeptide and, depending on the type of host into which it will be introduced, one or more expression control regions (such as a promoter and terminator), an origin of replication, etc. In some embodiments, the various nucleic acids and regulatory sequences described above are joined together to produce a recombinant expression vector, which contains one or more convenient restriction sites that allow for the insertion or substitution of a nucleic acid sequence encoding a variant GLA polypeptide at such site. Alternatively, the polynucleotide sequences of the invention are expressed by inserting the polynucleotide sequence or a nucleic acid construct comprising the polynucleotide sequence into a vector suitable for expression. In creating an expression vector, a coding sequence is placed in the vector so that it is operably linked to appropriate regulatory sequences for expression.

[0115] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and that can result in expression of the variant GLA polynucleotide sequence. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0116] In some embodiments, the expression vector is a self-replicating vector (i.e., a vector that exists as an extrachromosomal entity) whose replication is independent of chromosomal replication (e.g., a plasmid, extrachromosomal element, minichromosome, or artificial chromosome). The vector can include any means for ensuring self-replication. In some alternative embodiments, the vector can be a vector that, when introduced into a host cell, integrates into the genome and replicates together with the chromosome into which it has already been integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that together contain all of the DNA to be introduced into the genome of the host cell, or a transposon, can be used.

[0117] In some embodiments, expression vectors preferably contain one or more selectable markers to 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, and the like. 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 equivalents thereof. 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 regulatory sequences for expression of the engineered GLA enzyme in the host cell. Host cells used to express the polypeptides encoded by the expression vectors of the present invention are well known in the art and include fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae cells and Pichia pastoris cells [e.g., ATCC Accession No. 201178]), insect cells (e.g., Drosophila S2 cells and Spodoptera Sf9 cells), plant cells, animal cells (e.g., CHO, COS, and BHK), and human cells (e.g., HEK293T, human fibroblast, THP-1, Jurkat, and Bowes melanoma cell lines).

[0118] Thus, in another aspect, the present invention provides methods 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.

[0119] 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, gene gun particle bombardment, liposome-mediated transfection, calcium chloride transfection and protoplast fusion.

[0120] 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] ; WO95 / 22625; WO97 / 0078; WO97 / 35966; WO98 / 27230; WO00 / 42651; WO01 / 75767 and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include, among others, 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] ).

[0121] For example, mutagenesis and directed evolution can be easily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed.Mutagenesis and directed evolution are well known in the art (see, for example, 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. 6,335,16 No. 0, 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. 6,368 , 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. 6,537,746 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. 6,946,296, 6,961,664, 6,995,017, 7,024,312, 7,058,515, 7,105,297, 7,148,054, No. 7,288,375, No. 7,421,347, No. 7,430,477, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7,629 ,170, 7,702,464, 7,747,391, 7,747,393, 7,751,986, 7,776,598, 7,783,428, 7,795,030, 7,853,410, 7,868,138, 7,873,499, 7,904,249, and 7,957,912, 8, 383,346, 8,504,498, 8,849,575, 8,876,066, 8,768,871 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] ; Stemmer, 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. 201 No. 5 / 0133307, U.S. Patent Application Publication No. 2015 / 0134315 and all relevant non-U.S. counterparts; WO95 / 22625, WO97 / 0078, WO97 / 35966, WO98 / 27230, WO00 / 42651, WO01 / 75767, and WO2009 / 152336 (all of which are incorporated by reference herein).

[0122] In some embodiments, the enzyme variants obtained after mutagenesis treatment 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 comprising 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).

[0123] For engineered polypeptides of known sequence, polynucleotides encoding the enzymes can be prepared by standard solid-phase techniques according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then joined (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, typically implemented in automated synthesis methods (see, e.g., Beaucage et al., Tetra. Lett., 22:1859-69

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

[1984] ). According to the phosphoramidite method, oligonucleotides are synthesized (e.g., in an automated DNA synthesizer), purified, annealed, ligated, and cloned into an appropriate vector.

[0124] Thus, in some embodiments, a method for preparing an engineered GLA polypeptide may include (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of any variant provided in Tables 2.1, 2.2, 2.3, 2.4, 2.5, and / or 6.1 and SEQ ID NOs: 10, 13, 15, 18, 21, and / or 24, 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 10) amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 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 residues. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 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 residues. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 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 residues. In some embodiments, the substitutions may be conservative or non-conservative.

[0125] 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.

[0126] In some embodiments, any of the engineered GLA polypeptides expressed in host cells are recovered from the cells and / or culture medium using any one or more of well-known protein purification techniques, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others.

[0127] 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 variant GLA enzymes. In some embodiments utilizing affinity chromatography purification, any antibody that specifically binds to the variant 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, rats, etc.) are immunized by injection with a GLA polypeptide (e.g., a GLA variant) or a fragment thereof. In some embodiments, the GLA polypeptide or fragment is attached to a suitable carrier, such as BSA, by a side chain functional group or a linker attached to a side chain functional group.

[0128] In some embodiments, an 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.

[0129] In some embodiments, the present invention encompasses a method of producing an engineered GLA polypeptide, comprising culturing a recombinant eukaryotic cell that contains a polynucleotide sequence encoding an engineered GLA polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the reference sequence of SEQ ID NO: 5 and / or 10 when optimally aligned with the amino acid sequence of SEQ ID NO: 5 and / or 10, one or more amino acid residue differences compared to SEQ ID NO: 5 and / or 10 selected from those provided in Tables 2.1, 2.2, 2.4, 2.5, and / or 6.1, and / or combinations thereof, under appropriate culture conditions to allow production of the engineered GLA polypeptide, and optionally recovering the engineered GLA polypeptide from the culture and / or cultured bacterial cells.

[0130] In some embodiments, once the engineered GLA polypeptides are recovered from recombinant host cells or cell culture media, 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 engineered GLA polypeptides suitable for different applications and uses (e.g., pharmaceutical compositions). In some further embodiments, the purified or formulated GLA polypeptides are lyophilized.

[0131] Composition: 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).

[0132] According to the mode of administration, these compositions that contain the therapeutically effective amount of engineered GLA of the present invention are in the form of solid, semi-solid or liquid.In some embodiments, composition comprises other pharmaceutically acceptable components, such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, bulking agents and other components.The details of the techniques for formulation and administration are well known in the art and are described in the literature.

[0133] In some embodiments, the engineered GLA polypeptide is formulated for use in a pharmaceutical composition. Any suitable delivery format for the engineered GLA polypeptide (including, but not limited to, pills, tablets, gel tabs, capsules, lozenges, dragees, powders, softgels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks, solutions, suspensions (including, but not limited to, oil suspensions, oil-in-water emulsions, etc.), slurries, syrups, controlled-release formulations, suppositories, etc.) can be used in the present invention. In some embodiments, the engineered GLA polypeptide is provided in a format suitable for injection or infusion (i.e., an injectable formulation). 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, the present invention is not intended to be limited to any particular delivery formulation and / or delivery means. It is contemplated that the engineered GLA polypeptides will be administered by any suitable means known in the art, including, but not limited to, parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, via implant, etc.

[0134] In some embodiments, the engineered GLA polypeptide is 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, and 7,53,653; U.S. Patent Application Publication No. 2013 / 0039898; U.S. Patent Application Publication No. 2012 / 0177722, etc.). Indeed, the present invention is not intended to be limited to any particular delivery method and / or mechanism.

[0135] In some further embodiments, the engineered GLA polypeptide is provided in a formulation comprising a matrix-stabilized enzyme crystal. In some embodiments, the formulation comprises an engineered crosslinked crystalline GLA enzyme and a polymer having a reactive moiety that is attached to the enzyme crystal. The present invention also provides polymeric engineered GLA polypeptides.

[0136] 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.).

[0137] 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 depends on the condition or disease, the subject's general condition, and other factors known to those of skill in the art. In some embodiments, the composition is for single or multiple administration. In some embodiments, the concentration of the engineered GLA polypeptide in a composition administered to a person with Fabry disease is believed to 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. [Example]

[0138] experiment The following examples, including the experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the invention.

[0139] In the experiments disclosed below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar), uM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min (minutes); h and hr (hours); U (units); MW (molecular weight); rpm (revolutions per minute); °C (degrees Celsius); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); E. coli W3110 (a commonly used research E. coli strain, from the Coli Genetic Stock Center [CGSC], New Haven, CT); HPLC (High Pressure Liquid Chromatography); MWCO (Molecular Weight Cut-Off); SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis); 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); Athens Research (Athens Research Technology, Athens, GA); ProSpec (ProSpec Tany Technogene, East Brunswick, NJ); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Ram Scientific (Ram Scientific, Inc., Yonkers, NY); Pall Corp.(Pall,Corp.,Pt.Washington,NY);Millipore(Millipore,Corp.,Billerica MA);Difco(Difco Laboratories,BD Diagnostic Systems,Detroit,MI);Molecular Devices(Molecular Devices,LLC,Sunnyvale,CA);Kuhner(Adolf Kuhner,AG,Basel,Switzerland);Axygen(Axygen,Inc.,Union City,CA);Toronto Research Chemicals(Toronto Research Chemicals Inc.,Toronto,Ontario,Canada);Cambridge Isotope Laboratories,(Cambridge Isotope Laboratories, Inc.,Tewksbury,MA);Applied Biosystems(Applied Biosystems,part of Life Technologies,Corp.,Grand Island,NY)、Agilent(Agilent Technologies,Inc.,Santa Clara,CA);Thermo Scientific(part of Thermo Fisher Scientific,Waltham,MA);Corning(Corning,Inc.,Palo Alto,CA);Megazyme(Megazyme International,Wicklow,Ireland);Enzo(Enzo Life Sciences,Inc.,Farmingdale,NY);GE Healthcare(GE Healthcare Bio-Sciences,Piscataway,NJ);Pierce(Pierce Biotechnology(now part of Thermo Fisher Scientific),Rockford,IL);LI-COR(LI-COR Biotechnology,Lincoln,NE);Amicus(Amicus Therapeutics,Cranbury,NJ);Phenomenex(Phenomenex,Inc.Optimal (Optimal Biotech Group, Belmont, CA); and Bio-Rad (Bio-Rad Laboratories, Hercules, CA). .

[0140] 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. [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0141] Example 1 Obtaining the GLA gene and constructing an expression vector For optimized gene expression in Saccharomyces cerevisiae, a synthetic gene encoding WT human GLA (SEQ ID NO: 3) was designed, assembled, and subcloned into the E. coli expression vector pCK100900i (SEQ ID NO: 6).

[0142] A chimeric GLA expression construct encoding the 19-amino acid S. cerevisiae MFα signal peptide fused to the mature form of yeast-optimized GLA was generated in a yeast expression vector designed for secretory expression as follows. The fragment encoding the MFα signal peptide (SEQ ID NO:25) was amplified by PCR using oligonucleotides MMO435 (SEQ ID NO:27) and MMO439 (SEQ ID NO:28) derived from S288C genomic DNA, and the fragment encoding synthetic GLA (SEQ ID NO:3) was amplified using primers MMO514 (SEQ ID NO:29) and MMO481 (SEQ ID NO:30). Additional sequence at the 5' ends of these oligonucleotides provides homology for yeast recombination cloning when cotransformed with linearized plasmid pYT-72Bgl (SEQ ID NO:7). In the resulting vector, expression of the fusion protein SP-GLA (SEQ ID NO:36) is driven by the ADH2 promoter. A fusion construct encoding a fusion of the 83-amino acid MFα leader peptide (SEQ ID NO:38) N-terminally fused to GLA (SEQ ID NO:37) was cloned using the same technique. Recombinant cloning and gene expression were carried out in the S. cerevisiae strain INVSc1. A library of gene variants was generated from this plasmid construct using directed evolution techniques commonly known to those skilled in the art (see, e.g., U.S. Patent No. 8,383,346 and WO2010 / 144103).

[0143] A chimeric GLA expression construct encoding a synthetic signal peptide fused to a synthetic gene encoding the mature human GLA coding sequence for secretory expression in transient transfection was generated as follows: A fragment encoding the synthetic signal peptide (SEQ ID NO: 31) was amplified using PCR with oligonucleotides PLEV113Fw (SEQ ID NO: 32) and SPGLARv (SEQ ID NO: 33). A second fragment encoding the native human coding sequence of the mature form of GLA (SEQ ID NO: 4) was amplified using oligonucleotides SPGLAFw (SEQ ID NO: 34) and GLARv (SEQ ID NO: 35). These fragments were recombined using splicing by overlap extension PCR, and the resulting chimeric fragment was ligated into the mammalian expression vector pLEV113 linearized with HindIII / NotI. Specific gene variants were generated from this plasmid construct using directed evolution techniques commonly known to those skilled in the art.

[0144] Example 2 High-throughput proliferation assay High-throughput (HTP) propagation of GLA and GLA variants Yeast (INVSc1) cells transformed with vectors expressing GLA and GLA variants were selected on SD-Ura agar plates using the lithium acetate method. After 72 hours of incubation at 30°C, colonies were plated into wells of an Axygen® 1.1 ml 96-well deep-well plate filled with 200 μl / well of SD-Ura broth (2 g / L SD-Ura, 6.8 g / L amino acid-free yeast nitrogen base [Sigma-Aldrich]), 3.06 g / L sodium dihydrogen phosphate, and 0.804 g / L disodium hydrogen phosphate, pH 6.0, supplemented with 6% glucose. Cells were grown for 20–24 hours in a Kühner shaker (250 rpm, 30°C, and 85% relative humidity). Samples (20 μL) of the overnight culture were transferred to a Corning Costar® 96-well deep-well plate filled with 380 μL of SD-ura broth supplemented with 2% glucose. Plates were incubated in a Kuhner shaker (250 rpm, 30°C, and 85% relative humidity) for 66–84 h. Cells were then pelleted (4000 rpm x 20 min), and the supernatant was isolated and stored at 4°C before analysis.

[0145] HTP analysis of the supernatant GLA variant activity was determined by measuring the hydrolysis of 4-methylumbelliferyl α-D-galactopyranoside (MUGal). For this assay, 5–50 μL of yeast culture supernatant, produced as described above, was mixed with 0–45 μL of McIlvaine buffer (McIlvaine, J. Biol. Chem., 49:183–186

[1921] ), pH 4.8, and 50 μL of 2 mM MUGal in 50 mM citrate, 200 mM KCl, pH 4.6, in a 96-well black opaque-bottom plate. Reactions were mixed briefly and incubated at 37°C for 30–180 min before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm).

[0146] HTP analysis of acid-pretreated supernatants To simulate the extreme pH that variants may encounter in lysosomes, GLA variants were challenged with acidic buffer. First, 50 μL of yeast culture supernatant and 50 μL of McIlvaine buffer (pH 3.3–4.3) were added to wells of a 96-well round-bottom plate. The plate was sealed with a PlateLoc Thermal Microplate Sealer (Agilent) and incubated at 37°C for 1–3 hours. For the assay, 10–50 μL of acidic pH challenge sample was mixed with 0–40 μL of McIlvaine buffer pH 4.8, 25 μL of 1 M citrate buffer pH 4.3, and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reaction was mixed briefly and incubated at 37°C for 30–180 minutes before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm).

[0147] HTP analysis of base-pretreated supernatants To simulate the pH the variants would encounter in the blood after patient administration, GLA variants were challenged with a basic (neutral) buffer. First, 50 μL of yeast culture supernatant and 50 μL of McIlvaine buffer (pH 7.0–8.2) or 200 mM sodium bicarbonate (pH 9.1–9.7) were added to wells of a 96-well round-bottom plate. The plate was sealed and incubated at 37 °C for 1–18 h. For the assay, 10–50 μL of the basic pH challenge sample was mixed with 0–40 μL of McIlvaine buffer pH 4.8, 25 μL of 1 M citrate buffer pH 4.3, and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reaction was mixed briefly and incubated at 37 °C for 30–180 min before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm).

[0148] HTP analysis of supernatants pretreated with bovine serum To simulate the conditions the variants would encounter after infusion into patients, the GLA variants were challenged with bovine serum. First, 20 μL of yeast culture supernatant and 80 μL of bovine serum were added to wells of a 96-well round-bottom plate. The plate was sealed and incubated at 37°C for 1 hour. For the assay, 50 μL of serum challenge sample was mixed with 25 μL of 1 M citrate buffer, pH 4.3, and 25 μL of 4 mM MUGal in McIlvaine buffer, pH 4.8. The reaction was mixed briefly and incubated at 37°C for 180 minutes before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm). [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 [Table 2-23] [Table 2-24] [Table 2-25] [Table 2-26] [Table 2-27] [Table 2-28] [Table 2-29] [Table 2-30] [Table 2-31] [Table 2-32] [Table 2-33]

[0149] Example 3 In vitro characterization of GLA variants GLA production in yeast To produce GLA-containing supernatants, replica HTP cultures of GLA were grown as described in Example 2. Supernatants from the replica cultures (n = 12–36) were combined before further analysis.

[0150] Production of GLA in HEK293T cells Secretory expression of GLA variants in mammalian cells was performed by transient transfection of HEK293 cells. 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 as described in Example 1. HEK293 cells were transfected with plasmid DNA and grown in suspension for 4 days using techniques known to those skilled in the art. The supernatant was collected and stored at 4°C.

[0151] Example 4 Purification of GLA variants Purification of GLA variants from mammalian cell supernatants GLA variants were purified from mammalian culture supernatants essentially as known in the art (see 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 (ConA binding buffer). The supernatant was diluted 1:1 with binding buffer and loaded onto the column. The column was washed with 15 volumes of concanavalin A binding buffer, and the sample was eluted by adding concanavalin A binding buffer containing 0.9 M methyl-α-D-mannopyranoside and 0.9 M methyl-α-D-glucopyranoside. The eluted protein was concentrated and buffer-exchanged three times into ThioGal binding buffer (25 mM citrate-phosphate, 0.1 M NaCl, pH 4.8) using Centricon® Plus-20 filtration units (Millipore) with a 10 kDa molecular weight cutoff. The buffer-exchanged sample was loaded onto immobilized D-galactose resin (Pierce) equilibrated with ThioGal binding buffer. The resin was washed with six volumes of ThioGal binding buffer and eluted with 25 mM citrate-phosphate, 0.1 M NaCl, 0.1 M D-galactose, pH 5.5. The eluted sample was concentrated using Centricon® Plus-20 filtration units with a 10 kDa molecular weight cutoff. Based on Bradford assay, purification yielded 2.4–10 μg of purified protein per ml of culture supernatant.

[0152] SDS-PAGE analysis of GLA variants Samples of yeast culture supernatant, mammalian cell culture supernatant, and purified GLA were analyzed by SDS-PAGE. In the yeast supernatant, GLA levels were too low to be detected by this method. In both the mammalian cell culture supernatant and the purified GLA sample, a band corresponding to the predicted GLA molecular weight of approximately 49 kDa was found.

[0153] Immunoblot analysis of GLA variants Yeast supernatant and mammalian cell culture supernatant samples were analyzed by immunoblotting. Briefly, samples were separated by SDS-PAGE. Proteins were transferred to PVDF membranes using the iBlot dry blot system (Life Technologies). Membranes were blocked with Odyssey blocking buffer (TBS) (LI-COR) for 1 hour at room temperature and probed with rabbit α-GLA IgG (Thermo-Fischer) diluted 1:250 in Odyssey blocking buffer containing 0.2% Tween® 20 for 14 hours at 4°C. Membranes were washed 4 times for 5 minutes with Tris-buffered saline + 0.1% Tween® 20 and probed with IRDye800CW donkey α-rabbit IgG (LI-COR) diluted 1:5000 in Odyssey blocking buffer containing 0.2% Tween® 20 and 0.01% SDS for 1 hour at room temperature. The membrane was washed 4 times for 5 minutes with Tris-buffered saline + 0.1% Tween® 20 and analyzed using an Odyssey Imager (LI-COR). A band corresponding to the predicted GLA molecular weight of approximately 49 kDa was found in both the mammalian cell culture and yeast supernatants. In the S. cerevisiae expression samples, a mutant containing the mutation E367N migrated at a slightly higher molecular weight. This mutation introduces a canonical NXT N-linked glycosylation site (X is any amino acid except P), and the higher molecular weight may be explained by the potential introduction of additional N-linked glycans.

[0154] Example 5 In vitro characterization of GLA variants Optimization of signal peptide for secretory expression of GLA by S. cerevisiae S. cerevisiae transformed with Mf leader-GLA (SEQ ID NO: 7), SP-GLA (SEQ ID NO: 36), or vector control were grown in HTP as described in Example 2. Cultures were grown for 48 to 120 hours before supernatant collection and analysis (n=6) as described in Example 2. Figure 1 provides a graph showing the relative activity of different GLA constructs in S. cerevisiae after 2 to 5 days of culture. As shown in this figure, after 3 days of growth, SP-GLA (SEQ ID NO: 36) produced saturatingly high levels of active enzyme.

[0155] pH stability of GLA variants expressed in S. cerevisiae To assess the overall stability of the enzyme, GLA variants were challenged with different buffers. First, 50 μL of supernatant from GLA variant yeast cultures and 50 μL of McIlvaine buffer (pH 2.86–9.27) or 200 mM sodium carbonate (pH 9.69) were added to wells of a 96-well round-bottom plate (Costar #3798, Corning). The plate was sealed and incubated at 37°C for 1 h. For the assay, 50 μL of challenge supernatant was mixed with 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reaction was mixed briefly and incubated at 37°C for 60–180 min before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm). Figure 2 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants after incubation at various pHs.

[0156] Thermostability of GLA variants expressed in S. cerevisiae To assess the overall stability of the enzyme, GLA variants were challenged at various temperatures in the presence and absence of 1 μM 1-deoxygalactonojirimycin (Migalastat; Toronto Research Chemicals). First, 50 μL of supernatant from GLA variant yeast cultures and 50 μL of McIlvaine buffer (pH 7.65) + / - 2 mM 1-deoxygalactonojirimycin were added to wells of a 96-well PCR plate (Biorad, HSP-9601). The plate was sealed and incubated for 1 h at 30–54 °C using a gradient program on a thermocycler. For the assay, 50 μL of challenge supernatant was mixed with 25 μL of 1 M citrate buffer pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8. The reactions were mixed briefly and incubated at 37°C for 90 minutes before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm). Figure 3 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants after incubation at various temperatures.

[0157] Serum stability of GLA variants expressed in S. cerevisiae To assess the relative stability of the variants in the presence of blood, samples were exposed to serum. First, 20 μL of GLA variant yeast culture supernatant and 0–80 μL of water or 0–80 μL of bovine serum were added to wells of a 96-well round-bottom plate (Costar #3798, Corning). The plate was sealed and incubated at 37°C for 1 hour. For the assay, 50 μL of challenge supernatant was mixed with 25 μL of 1 M citrate buffer, pH 4.3, and 25 μL of 4 mM MUGal in McIlvaine buffer, pH 4.8. The reaction was mixed briefly and incubated at 37°C for 90 minutes before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm). FIG. 4 provides graphs showing the absolute (panels A and B) and relative (panels C and D) activity of GLA variants after challenge with various ratios of serum.

[0158] Relative activity of GLA variants expressed in HEK293T cells Supernatants from GLA variants expressed in HEKT293T cells were serially diluted two-fold with supernatants from non-GLA-expressing yeast cultures. Dilutions (50 μL) were mixed with 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8 and 25 μL of 1 M citrate buffer pH 4.3 in a Corning® 96-well black opaque-bottom plate. The reactions were mixed briefly and incubated at 37°C for 60 minutes before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm). Figure 5 provides a graph showing the relative activity of GLA variants expressed in HEK293T cells. Supernatants from cells transfected with the variant GLA enzyme exhibited significantly higher hydrolase activity compared to the WT enzyme, and the activity per volume was considerably higher than that seen with S. cerevisiae expression.

[0159] pH stability of GLA variants expressed in HEK293T cells To assess overall stability, GLA variants were challenged with different buffers. Supernatants from mammalian cell cultures were normalized to equivalent activity by dilution with supernatants from non-GLA-expressing cultures. Normalized supernatants (50 μL) and 50 μL of McIlvaine buffer (pH 4.06–8.14) were added to wells of a 96-well round-bottom plate (Costar #3798, Corning). The plate was sealed and incubated at 37°C for 3 hours. For the assay, 50 μL of challenge supernatant was mixed with 25 μL of 1 M citrate buffer, pH 4.3, and 25 μL of 4 mM MUGal in McIlvaine buffer, pH 4.8. The reaction was mixed briefly and incubated at 37°C for 3 hours before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm). Figure 6 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants expressed in HEK293T cells, normalized for activity, and incubated at various pHs.

[0160] All enzymes were found to be more stable to pH challenge when compared to WT GLA expressed in S. cerevisiae (compare Figure 2). This difference is likely due to differential glycosylation between the expression hosts. However, the present invention is not limited to any particular mechanism or theory. The mutant enzymes had a broader pH stability profile compared to the WT enzyme expressed in HEK293T.

[0161] Thermostability of GLA variants expressed in HEK293T cells To assess overall stability, GLA variants were challenged at various temperatures in the presence and absence of 1 μM 1-deoxygalactonojirimycin (Migalastat). Supernatants from mammalian cell cultures were normalized to approximately equivalent activity by dilution with supernatants from non-GLA-expressing cultures. Diluted supernatants were added to wells of a 96-well PCR plate (Biorad, HSP-9601). The plate was sealed and incubated for 1 h at 30–54°C using a gradient program on a thermocycler. For the assay, 20 μL of challenge supernatant was mixed with 30 μL of 1 M citrate buffer, pH 4.3, and 50 μL of 4 mM MUGal in McIlvaine buffer, pH 4.8. The reaction was mixed briefly and incubated at 37°C for 90 min before being quenched with 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm). Figure 7 provides graphs showing the absolute (Panel A) and relative (Panel B) activity of GLA variants expressed in HEK293T cells, normalized for activity, and incubated at various temperatures. As shown in this figure, all enzymes were more stable after temperature challenge when compared to WT GLA expressed in S. cerevisiae (compare Figure 2), likely due to differential glycosylation between expression hosts. For GLA variants (SEQ ID NOS: 10 and 13), the T of the enzyme was significantly higher. m The T increased by 2°C and 4°C, respectively. m Although the temperature was increased by 5.5°C, at a final concentration of 0.2 μM in the assay, the activity of the Migalastat-treated sample was reduced by approximately 60%.

[0162] Activity of WT GLA and GLA variants against alternative substrates To confirm that the improved MUGal hydrolysis activity corresponded to a more natural substrate, mammalian cell-expressed GLA variants were assayed using N-dodecanoyl-NBD-ceramide trihexoside (NBD-GB3) as the substrate. HEK293T culture supernatant (10 μL), 100 mM sodium citrate pH 4.8 (80 μL), and NBD-GB3 (0.1 mg / mL) in 10% ethanol (10 μL) were added to a microcentrifuge tube. The sample was mixed by inversion and incubated at 37°C for 1 h. The reaction was quenched by adding 50 μL of methanol, diluted with 100 μL of chloroform, vortexed, and the organic layer was isolated for analysis. A 10 μL aliquot of the organic phase was spotted onto a silica plate and analyzed by thin-layer chromatography (chloroform:methanol:water, 100:42:6) using a 365 nm UV lamp to detect starting materials and products. Significant conversion was observed only in the case of SEQ ID NO: 13, confirming that the variant exhibits improved activity compared to WT-GLA.

[0163] Specific activity of GLA variants GLA variants purified as described in Example 4 were evaluated for their specific activity. 0–0.25 ng of purified enzyme was added to 4 mM MUGal in McIlvaine buffer, pH 4.8 (final pH 4.8). Samples were incubated at 37°C for 60 minutes and quenched by the addition of 100 μL of 1 M sodium carbonate. Hydrolysis was analyzed using a SpectraMax® M2 microplate reader monitoring fluorescence (excitation 355 nm, emission 448 nm) and correlated to absolute amounts of 4-methylumbelliferone by use of a standard curve.

[0164] pH stability of purified GLA variants over time To confirm that the purified GLA variants exhibited the desired pH stability observed after expression in yeast, WT GLA (SEQ ID NO: 5) and SEQ ID NO: 42 were incubated in acidic or basic buffers and analyzed for residual activity. GLA variants (200 ng) were added to McIlvaine buffer pH 4.1 or pH 7.5 and incubated at 37°C for 0-24 hours. Samples (50 μL) were added to a mixture of 25 μL of 1 M citric acid pH 4.3 and 25 μL of 4 mM MUGal in McIlvaine buffer pH 4.8 and incubated at 37°C for 1 hour. Samples were quenched with 100 μL of 1 M sodium carbonate, diluted 1:4 with 1 M sodium carbonate, and analyzed by fluorescence spectroscopy (excitation 355 nm, emission 448 nm). SEQ ID NO:42 was highly stable under both acidic and basic challenge conditions, confirming that the increased stability developed in yeast translated to the protein expressed in mammalian cells (see Figure 8 for a graph of the results).

[0165] Thermostability of purified GLA variants expressed in HEK293T cells To assess overall stability, the thermostability of WT GLA (SEQ ID NO: 5) and SEQ ID NO: 42 was determined. The enzyme, purified as described in Example 4, was diluted in 1× Sypro The GLA was diluted to 20 μg / ml in 1x PBS containing Sypro Orange (Thermo Fischer Scientific) and added to a 96-well PCR plate (Biorad, HSP-9601). The plate was heated from 30°C to 75°C at 0.5°C / min in an RT-PCR machine, and Sypro Orange fluorescence was monitored. Under these conditions, WT GLA melted at 37°C, while SEQ ID NO:42 melted at 55°C.

[0166] Example 6 In vivo characterization of GLA variants Serum pharmacokinetics of purified GLA variants Purified GLA variants produced as described in Example 4 were evaluated for stability in the serum of surviving rats. 1 mg / ml of WT GLA (SEQ ID NO: 5) or SEQ ID NO: 42 was intravenously administered to three naive, jugular-cannulated Sprague-Dawley rats (7-8 weeks old). 200 μL of blood was collected from each rat into EDTA tubes before administration and 5, 15, 30, 60, 120, and 240 minutes after administration. The blood was centrifuged at 4°C and 6000 rpm to generate >80 μL of serum per sample. Samples were frozen and stored on dry ice prior to analysis. For analysis, serum (10 μL) was added to 40 μL of 5 mM MUGal in McIlvaine buffer, pH 4.4, and incubated at 37°C for 1 hour. Samples were quenched with 50 μL of 1 M sodium carbonate, diluted 1:100 in 1 M sodium carbonate, and analyzed by fluorescence spectroscopy (excitation 355 nm, emission 448 nm). Four hours after administration, SEQ ID NO: 42 retained 15.3% of its maximal activity, while WT GLA retained only 0.66% (see FIG. 9).

[0167] Example 7 GLA deimmunization This example describes experiments performed to identify variations that remove predicted T cell epitopes from GLA.

[0168] Identification of deimmunized diversity: To identify mutational diversity that eliminates T cell epitopes, computerized methods were used to identify GLA subsequences that were predicted to efficiently bind to representative HLA receptors.In addition, experimental searches for amino acid mutations were carried out, particularly for mutations that do not affect GLA activity (e.g., in the assay described in Example 2).Then, computerized methods were used to analyze the amino acid sequences of active variants for predicted immunogenicity.

[0169] Computational identification of putative T cell epitopes in WT GLA: Putative T cell epitopes in WT GLA (SEQ ID NO: 5) were identified using art-known immune epitope database (IEDB; immune epitope database and analysis resource website) tools and proprietary statistical analysis tools (see, e.g., iedb.org and Vita et al., Nucl. Acids Res., 38 (Database issue): D854-62

[2010] . Epub 2009 Nov 11). WT GLA was parsed into all possible 15-mer analysis frames (each frame overlapping the last 14 amino acids). Using methods recommended on the IEDB website, the 15-mer analysis frame was evaluated for immunogenicity by scoring the 9-mer core region for predicted binding to eight common class II HLA-DR alleles (DRB1*0101, DRB1*0301, DRB1*0401, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1301, and DRB1*1501), which collectively cover approximately 95% of the human population (see, e.g., Southwood et al., J. Immunol., 160:3363-3373

[1998] ). Statistical analysis tools known in the art were used to identify potential T cell epitope clusters contained within the enzyme (i.e., subregions contained within GLA that are highly likely to be immunogenic). The identified T cell epitope clusters were screened against the IEDB database of known epitopes. These screens identified five putative T cell epitopes in the WT enzyme, hereafter referred to as TCE-I, II, III, IV, and V.

[0170] Deimmunized Library Design: First, the sequences of the active GLA mutants identified in Example 2 are evaluated for the presence of T cell epitopes. Mutations identified as potentially reducing binding to HLA-DR alleles are incorporated into a recombination library. Further libraries are prepared using saturation mutagenesis of all single amino acids within the five T cell epitopes. Hits from these libraries are subjected to further rounds of saturation mutagenesis, HTP screening, and recombination to remove all potential T cell epitopes.

[0171] Deimmunized library construction and screening: The combinatorial and saturation mutagenesis libraries designed as described above were constructed by methods known in the art and tested for activity in the non-challenge assay described in Example 2. Active variants were identified and sequenced. Their activity and mutations relative to WT GLA are provided in the table below.

[0172] Identification of deimmunized diversity: Active variants were analyzed for their level of predicted immunogenicity by assessing binding to eight common class II HLA-DR alleles, as described above. The total immunogenicity score and immunogenic hit count are shown in Table 7.1. The total immunogenicity score (TIS) reflects the overall predicted immunogenicity of the variant (i.e., a higher score indicates a higher level of predicted immunogenicity). The immunogenic "hit count" (IHC) indicates the number of analyzed 15-mer frames that are highly likely to be immunogenic (i.e., a higher score indicates a higher immunogenic potential). Mutations that resulted in a total immunogenicity score and / or immunogenic hit count lower than that of the reference sequence were considered potential "deimmunizing mutations." A collection of the most deimmunizing mutations was recombined to generate several variants that were predicted to be active and significantly less immunogenic than WT GLA. The total immunogenicity score (TIS) and immunogenic hit count (IHC) are provided in the table below. [Table 7-1] Table 7-2 Table 7-3 Table 7-4 Table 7-5 Table 7-6 Table 7-7 Table 7-8 Table 7-9 Table 7-10 Table 7-11 Table 7-12 Table 7-13 Table 7-14 Table 7-15 [Table 7-16] [Table 7-17] [Table 7-18] [Table 7-19] [Table 7-20] [Table 7-21] [Table 7-22] [Table 7-23] [Table 7-24] [Table 7-25]

[0173] Although the invention has been described with reference to specific embodiments, various modifications may be made and equivalents may be substituted to adapt a particular situation, material, composition of matter, process, process step or steps, thereby achieving benefits of the invention without departing from the scope of the claims.

[0174] For all purposes in the United States, each and every publication and patent document cited in this application is incorporated by reference herein as if each such publication or document was specifically and individually indicated to be incorporated 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.

Claims

1. A recombinant alpha-galactosidase A comprising a polypeptide sequence having at least 94% sequence identity to SEQ ID NO: 24, wherein the polypeptide sequence comprises an arginine (R) at position 206 and has increased stability at pH 4.3 compared to an alpha-galactosidase A having a sequence corresponding to SEQ ID NO:

5.

2. The recombinant alpha-galactosidase A of claim 1, wherein the polypeptide sequence of the recombinant alpha-galactosidase A comprises at least 95%, at least 96%, or at least 97% sequence identity to SEQ ID NO:

24.

3. The recombinant alpha-galactosidase A of claim 1, wherein the polypeptide sequence of the recombinant alpha-galactosidase A comprises at least 98%, or at least 99%, sequence identity with SEQ ID NO:

24.

4. The recombinant α-galactosidase A according to any one of claims 1 to 3, which is purified.

5. A composition comprising at least one recombinant alpha-galactosidase A according to any one of claims 1 to 4.

6. A recombinant polynucleotide encoding at least one recombinant alpha-galactosidase A according to any one of claims 1 to 3.

7. The recombinant polynucleotide of claim 6, which is codon-optimized.

8. An expression vector comprising the recombinant polynucleotide of claim 6 or 7.

9. The expression vector of claim 8 , wherein the recombinant polynucleotide is operably linked to a polynucleotide having a regulatory sequence.

10. The expression vector of claim 9, wherein the polynucleotide having the regulatory sequence is a promoter.

11. The expression vector of claim 10 , wherein the promoter is a heterologous promoter.

12. A host cell comprising the expression vector according to any one of claims 8 to 11.

13. The host cell of claim 12, which is a eukaryote.

14. A method for producing an alpha-galactosidase A variant, comprising culturing a host cell described in claim 12 or 13 under conditions in which the alpha-galactosidase A encoded by the recombinant polynucleotide is produced.

15. 15. The method of claim 14, further comprising recovering the alpha-galactosidase A.

16. 16. The method of claim 15, further comprising purifying the alpha-galactosidase A.

17. A pharmaceutical composition for treating Fabry disease, comprising the composition of claim 5.

18. 18. The pharmaceutical composition of claim 17, further comprising a pharmaceutically acceptable carrier and / or excipient.

19. 19. A pharmaceutical composition according to claim 17 or 18, suitable for parenteral injection or infusion into humans.

20. A pharmaceutical composition according to any one of claims 17 to 19 for treating and / or preventing symptoms of Fabry disease in a subject.

21. 21. The pharmaceutical composition of claim 20, which ameliorates the symptoms of Fabry disease.

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