Recombinant alpha-galactosidase a proteins and gene therapy

Abstract

A composition including a gene therapy delivery system and an alpha-galactosidase (GLA) polynucleotide encoding a GLA polypeptide, wherein the GLA polypeptide has at least 95% sequence identity to one of SEQ ID NOS. 1-40, 44-45, 51-52, 57, 61-173 or 212. Also provided are compositions, formulations, fusion proteins, and methods for the treatment of Fabry disease.

Description

RECOMBINANT ALPHA-GALACTOSIDASE A PROTEINS AND GENE THERAPYTECHNICAL FIELD

[0001] The present invention generally relates to the treatment of Fabry disease, particularly novel recombinant polypeptides and gene therapy for the treatment of Fabry disease as well as methods of treating Fabry disease.BACKGROUND

[0002] Many human diseases result from mutations that cause changes in the amino acid sequence of a protein which reduce its stability and may prevent it from folding properly. Proteins generally fold in a specific region of the cell known as the endoplasmic reticulum, or ER. The cell has quality control mechanisms that ensure that proteins are folded into their correct three-dimensional shape before they can move from the ER to the appropriate destination in the cell, a process generally referred to as protein trafficking. Misfolded proteins are often eliminated by the quality control mechanisms after initially being retained in the ER. In certain instances, misfolded proteins can accumulate in the ER before being eliminated. The retention of misfolded proteins in the ER interrupts their proper trafficking, and the resulting reduced biological activity can lead to impaired cellular function and ultimately to disease. In addition, the accumulation of misfolded proteins in the ER may lead to various types of stress on cells, which may also contribute to cellular dysfunction and disease.

[0003] Such mutations can lead to lysosomal storage disorders (LSDs), which are characterized by deficiencies of lysosomal enzymes due to mutations in the genes encoding the lysosomal enzymes. The resultant disease causes the pathologic accumulation of substrates of those enzymes, which include lipids, carbohydrates, and polysaccharides. Although there are many different mutant genotypes associated with each LSD, many of the mutations are missense mutations which can lead to the production of a less stable enzyme. These less stable enzymes are sometimes prematurely degraded by the ER- associated degradation pathway. This results in the enzyme deficiency in the lysosome, and the pathologic accumulation of substrate. Such mutant enzymes are sometimes referred to in the pertinent art as "folding mutants" or "conformational mutants."

[0004] Fabry Disease is a LSD caused by a mutation to the GLA gene, which encodes the enzyme a-galactosidase A (hereinafter “GLA”). GLA is required for glycosphingolipidmetabolism. The mutation causes the substrate globotriaosylceramide (Gb3, GL-3, or ceramide trihexoside) to accumulate in various tissues and organs. Males with Fabry disease are hemizygotes because the disease genes are encoded on the X chromosome. Fabry disease is estimated to affect 1 in 40,000 and 60,000 males, and occurs less frequently in females.

[0005] One approach to treat Fabry disease involves the use of gene therapy. Gene therapy involves replacing or supplementing the defective gene with a nucleic acid sequence that encodes a functional protein. Gene therapies may employ recombinant vectors to deliver nucleic acid sequences that encode the functional protein, or genetically modified human cells that encode the functional protein. Gene therapy is a promising approach, but significant challenges remain. Accordingly, new gene therapies, including polynucleotide sequences encoding more active forms of GLA, are needed to improve upon currently available treatments of Fabry disease.SUMMARY

[0006] Accordingly, various aspects of the invention pertain to new recombinant GLA proteins and gene therapy compositions, which can be used to treat Fabry disease. Other aspects of the invention pertain to methods of producing such recombinant GLA proteins and gene therapy compositions, as well as pharmaceutical compositions, methods of treatment, and uses of such recombinant proteins and gene therapy compositions.DETAILED DESCRIPTION

[0007] Before describing several exemplary embodiments of the invention, it is to be understood that the invention is not limited to the details of construction or process steps set forth in the following description. The invention is capable of other embodiments and of being practiced or being carried out in various ways.

[0008] It has surprisingly been discovered that certain recombinant GLA proteins may have greater activity than wild-type form of human GLA (for example the wild-type GLA given in SEQ ID NO. 211) and greater stability under physiological conditions than wild-type human GLA. Furthermore, it has surprisingly been discovered that the fusion of certain recombinant GLA proteins and one or more of a leader signal polypeptide, a cell-penetrating polypeptide, a promoter, an SV40 intron, or a protease cleavage site may have greater serum activity than wild-type GLA.

[0009] Moreover, it is believed that certain recombinant GLA proteins, as described herein, may have benefits particularly suited for therapeutic administration to patients having from Fabry disease, where the benefits can include improved secretion from host cells during protein production, improved solubility, enhanced ability to cross the blood-brain barrier (BBB), and / or enhanced ability to penetrate target cells.

[0010] Other aspects of the present invention relate to novel cell systems for expressing and secreting recombinant proteins comprising GLA polypeptides (e.g. recombinant GLA variants).

[0011] Other aspects of the present invention relate to gene therapy compositions and methods that utilize a recombinant GLA polynucleotide encoding a recombinant GLA polypeptide as described herein and a gene therapy delivery system.Definitions

[0012] The term “Fabry disease” refers to an X-linked inborn error of glycosphingolipid catabolism due to deficient lysosomal a-galactosidase A activity. This defect causes accumulation of globotriaosylceramide (ceramide trihexoside) and related glycosphingolipids in vascular endothelial lysosomes of the heart, kidneys, skin, and other tissues.

[0013] The term “atypical Fabry disease” refers to patients with primarily cardiac manifestations of the a-Gal A deficiency, namely progressive globotriaosylceramide (GL-3) accumulation in myocardial cells that leads to significant enlargement of the heart, particularly the left ventricle.

[0014] A “Fabry carrier” is a female who has one X chromosome with a defective a- Gal A gene and one X chromosome with the normal gene and in whom X chromosome inactivation of the normal allele is present in one or more cell types. A Fabry carrier is often diagnosed with Fabry disease.

[0015] A “Fabry disease patient” refers to an individual who has been diagnosed with or suspected of having Fabry disease and has a mutated a-Gal A as defined further below. Characteristic markers of Fabry disease can occur in male hemizygotes and female carriers with the same prevalence, although females typically are less severely affected.

[0016] Human a-galactosidase A (a-Gal A) refers to an enzyme encoded by the human GLA gene. The full DNA sequence of a-Gal A, including introns and exons, is available inGenBank Accession No. X14448.1 and shown in SEQ ID NO: 210. The human a-Gal A enzyme consists of 429 amino acids and is available in GenBank Accession Nos. X14448.1 and U78027.1 and shown in SEQ ID NO: 211.

[0017] The term “mutant protein” includes a protein which has a mutation in the gene encoding the protein which results in the inability of the protein to achieve a stable conformation under the conditions normally present in the ER. The failure to achieve a stable conformation results in a substantial amount of the enzyme being degraded, rather than being transported to the lysosome. Such a mutation is sometimes called a “conformational mutant.” Such mutations include, but are not limited to, missense mutations, and in-frame small deletions and insertions.

[0018] As used herein in at least one embodiment, the term “mutant a-Gal A” includes an a-Gal A which has a mutation in the gene encoding a-Gal A which results in the inability of the enzyme to achieve a stable conformation under the conditions normally present in the ER. The failure to achieve a stable conformation results in a substantial amount of the enzyme being degraded, rather than being transported to the lysosome.

[0019] The term “a-Gal A activity” refers to the normal physiological function of a wild-type a-Gal A in a cell. For example, a-Gal A activity includes hydrolysis of GL-3.

[0020] “Deficient a-Gal A activity” refers to a-Gal A activity in cells from a patient which is below the normal range as compared (using the same methods) to the activity in normal individuals not having or suspected of having Fabry or any other disease (especially a blood disease).

[0021] As used herein, the term "gene therapy delivery system" refers to any system that can be used to deliver an exogenous gene of interest to a target cell so that the gene of interest will be expressed or overexpressed in the target cell. In one or more embodiments, the target cell is an in vivo patient cell. In one or more embodiments, the target cell is an ex vivo cell and the cell is then administered to the patient.

[0022] As used herein, the term "carrier" is intended to refer to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Suitable pharmaceutical carriers are known in the art and, in at least one embodiment, are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 18th Edition, or other editions.

[0023] As used herein, the term "enzyme replacement therapy" or "ERT" is intended to refer to the introduction of an exogenous, purified enzyme into an individual having adeficiency in such enzyme. The administered protein can be obtained from natural sources or by recombinant expression. The term also refers to the introduction of a purified enzyme in an individual otherwise requiring or benefiting from administration of a purified enzyme. In at least one embodiment, such an individual suffers from enzyme insufficiency. The introduced enzyme may be a purified, recombinant enzyme produced in vitro, or a protein purified from isolated tissue or fluid, such as, for example, placenta or animal milk, or from plants.

[0024] As used herein, the terms "subject" or "patient" are intended to refer to a human or non-human animal. In at least one embodiment, the subject is a mammal. In at least one embodiment, the subject is a human.

[0025] As used herein, the "therapeutically effective dose" and "effective amount" are intended to refer to an amount of gene therapy composition (e.g. comprising GLA polynucleotides) or recombinant protein (e.g. GLA variants or fusion proteins) which is sufficient to result in a therapeutic response in a subject. A therapeutic response may be any response that a user (for example, a clinician) will recognize as an effective response to the therapy, including any surrogate clinical markers or symptoms described herein and known in the art.GLA Polypeptide Constructs

[0026] The amino acid sequence of a wild-type GLA isoform is provided in SEQ ID NO. 211.

[0027] Various embodiments of the present invention provide novel GLA variants.

[0028] In one or more embodiments, the GLA polypeptide has at least 90%, at least 95%, at least 98%, at least 98.5%, at least 99% or at least 99.5% sequence identity to SEQ ID NOS. 1-40, 44-45, 51 -52, 57, 61 -173 or 212. The GLA polypeptide may contain deletions, substitutions and / or insertions relative to SEQ ID NOS. 1-40, 44-45, 51-52, 57, 61-173 or 212, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more deletions, substitutions and / or insertions to the amino acid sequence described by SEQ ID NOS. 1-40, 44-45, 51-52, 57, 61-173 or 212.

[0029] In one or more embodiments, the GLA polypeptide has at least 90%, at least 95%, at least 98%, at least 98.5%, at least 99% or at least 99.5% sequence identity to SEQ ID NOS. 21-22, 26, 62, 88, 94, 111, 142, 163, 167-168 or 212. The GLA polypeptide may contain deletions, substitutions and / or insertions relative to SEQ ID NOS. 21-22, 26, 62, 88, 94, 111, 142, 163, 167-168 or 212, such as having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or moredeletions, substitutions and / or insertions to the amino acid sequence described by SEQ ID NOS. 21-22, 26, 62, 88, 94, 111, 142, 163, 167-168 or 212.

[0030] In one or more embodiments, the GLA polypeptide comprises one or more affinity-tags. In one or more embodiments, the affinity-tag is located on one or more of the N- terminus or the C-terminus of the GLA polypeptide. Examples of tags that can be added to the fusion proteins include, but are not limited to, epitope tags (e.g. MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, HPC4), glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3xFLAG®, polyhistidine (His), and combinations thereof.

[0031] In one or more embodiments, the GLA polypeptide comprises one or more protease cleavage sites. In some embodiments, the protease cleavage site is located on one or more of the N-terminus or the C-terminus of the GLA polypeptide. Exemplary protease cleavage sites include, but are not limited to, cleavage sites sensitive to thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, and combinations thereof.

[0032] Various alignment algorithms and / or programs may be used to calculate the identity between two sequences, including FASTA, or BLAST which are available as a part of the GCG sequence analysis package (University of Wisconsin, Madison, Wis.), and can be used with, e.g., default setting. For example, polypeptides having at least 90, 95%, 98%, 98.5%, 99% or 99.5% identity to specific polypeptides described herein and preferably exhibiting substantially the same functions, as well as polynucleotide encoding such polypeptides, are contemplated. Unless otherwise indicated a similarity score will be based on use of BLOSUM62. When BLASTP is used, the percent similarity is based on the BLASTP positives score and the percent sequence identity is based on the BLASTP identities score, BLASTP "Identities" shows the number and fraction of total residues in the high scoring sequence pairs which are identical; and BLASTP "Positives" shows the number and fraction of residues for which the alignment scores have positive values and which are similar to each other. Amino acid sequences having these degrees of identity or similarity or any intermediate degree of identity of similarity to the amino acid sequences disclosed herein are contemplated and encompassed by this disclosure. The polynucleotide sequences of similar polypeptides are deduced using the genetic code and may be obtained by conventional means, in particular by reverse translating its amino acid sequence using the genetic code.

[0033] One skilled in the art can readily derive a polynucleotide sequence encoding a particular polypeptide sequence. Such polynucleotide sequence can be codon optimized for expression in the target cell using commercially available products, such as using the OptimumGene™ codon optimization tool (GenScript, Piscataway, New Jersey).Cell-Penetrating Peptides (CPPs)

[0034] A variety of viral and cellular proteins possess basic polypeptide sequences that mediate translocation across cellular membranes. The capacity to translocate across cellular membranes has become an important tool for the delivery of high molecular weight polypeptides across membranes. The phrase "protein transduction domain" (PTD) and "cellpenetrating peptides" (CPPs) are usually used to refer to short peptides (< 30 amino acids) that can traverse the plasma membrane of many, if not all, mammalian cells. After studies to identify the specific properties of the domain that allow them to collectively cross the plasma membrane, researchers have observed that these domains contain a large number of basic amino acid residues such as lysine and arginine. Thus, cell-penetrating peptides fall into two classes: the first consisting of amphipathic helical peptides that contain lysine residues which contribute a positive charge, while the second class includes arginine-rich peptides. These peptides could have therapeutic potential if used in combination with other proteins that are difficult to deliver to intracellular targets. The most frequent experimental uses of PTDs are TAT, Antennapedia (Antp), and other poly-arginine peptides.

[0035] Thus far, TAT has been the best characterized of the PTDs, and has been used to successfully deliver small cargoes, such as short peptides and oligonucleotides, to intercellular targets. HIV-TAT (HIV Transactivator of Transcription) is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1), and many studies have shown that TAT is able to translocate through the plasma membrane and reach the nucleus in order to activate transcription of the viral genome. Studies have also shown that TAT retains its penetration properties when coupled to several different proteins. In an effort to understand which areas of the TAT protein are critical to the translocation property, experiments have been conducted in which different length peptide fragments of TAT are synthesized and their penetration capabilities are assessed. (Lebleu et al. “A Truncated HIV-1 TAT Protein Basic Domain Rapidly Translocates through the Plasma Membrane and Accumulates in the Cell Nucleus.” J. Biol. Chem. 1997, 272:16010-16017). A region of basicamino acids has been identified as the aspect of TAT that retains this penetration property, and experiments in which a TAT protein without this basic amino acid cluster is unable to penetrate the cellular plasma membrane. In some instances, the shorter sequence cellpenetrating peptide has been modified to prevent cleavage during secretion by endoprotease enzymes such as furin. These modifications change the shortened cell-penetrating TAT amino acid sequence from YGRKKRRQRRR to YARKAARQARA, and this short peptide is referred to as TATK.

[0036] The exact mechanism in which TAT is able to translocate across the plasma membrane remains uncertain. Recent work has explored the possibility that a special type of endocytosis is involved with TAT uptake, and a few cell lines have been identified that appear resistant to TAT penetration. The specific cargo to be delivered by TAT may also play a role in the efficacy of delivery. Previous research data have suggested that a TAT fusion protein has better cellular uptake when it is prepared in denaturing conditions, because correctly folded protein cargo likely requires much more energy (delta-G) to cross the plasma membrane due to structural constraints.

[0037] The capacity of the intracellular protein chaperones to refold the TAT cargo likely varies based on the identity and size of the protein cargo to be re-folded. In some instances, TAT-fusion proteins precipitate when placed in an aqueous environment and therefore cannot be prepared in a denatured manner nor remain stable for very long in native conformations. The design of the TAT-fusion protein must also be tailored to the specific cargo to be delivered. If the cargo protein is tightly associated at the N-terminus and the TAT domain is also found at the N-terminus, the TAT translocation domain may be buried in the cargo protein and transduction may be poor.

[0038] Numerous TAT-cargo variants have been successfully delivered into a variety of cell types, including primary culture cells, transformed cells, and cells present in mouse tissue. In culture, the TAT-fusion proteins generally diffuse easily into and out of cells, leading to a very rapid establishment of uniform concentration.

[0039] Many pharmaceutical agents such as enzymes, antibodies, other proteins, or even drug-loaded carrier particles need to be delivered intracellularly to exert their therapeutic action inside the cytoplasm, nucleus, or other specific organelles. Thus, the delivery of these different types of large molecules represents a significant challenge in the development ofbiologies. Current data suggest that TAT is able to cross the plasma membrane through more than one mechanism.

[0040] A TAT transduction domain has also been fused to the enzyme superoxide dismutase (SOD). (Torchilin, “Intracellular delivery of protein and peptide therapeutics.” Protein Therapeutics. 2008. 5(2-3):e95-el03). This fusion protein was used to demonstrate that it could translocate across cell membranes in order to deliver the SOD enzyme to the intracellular environment, and thus here the fusion protein has therapeutic potential in treating enzyme deficiency disorders that lead to higher accumulation of reactive oxygen species and oxidative stress on a host cell.

[0041] TAT fusion proteins have also been shown to transduce across the blood-brain barrier. A TAT domain fused to the neuroprotectant protein Bcl-xL was able to penetrate cells rapidly in culture, and when administered to mice suffering from cerebral ischemia, the fusion protein transduced brain cells within 1-2 hours. After transduction, the cerebral infarct was reduced in size in a dose-dependent manner (Cao, G. et al., “In Vivo Delivery of a Bcl-xL Fusion Protein Containing the TAT Protein Transduction Domain Protects against Ischemic Brain Injury and Neuronal Apoptosis.” J. Neurosci. 22, 5423, 2002.)

[0042] In some embodiments, the GLA variants described herein are operably linked to a CPP such as TAT, modified TAT (TATK), Transportan, Antennapedia or P97. As used herein, TAT can refer to the original TAT peptide having 11 amino acids (designated TATI 1 ) or can refer to a TAT peptide having an additional 16 N-terminal amino acids (designated as TAT28) that are derived from the polylinker of the plasmid used for cloning. Similarly, TATK can refer to a modified version of TAT11 (designated TATKI I) or a modified version of TAT28 (designated TATK28). The TATK28 can be further modified (designated TATKK28) to remove a potential additional weak furin site. The amino acid sequences of the CPPs TAT28, TATK28, TAT11, TATKI I, Transportan, Antennapedia, P97 and TATKK28 are provided in SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36, SEQ ID NO: 37 and SEQ ID NO: 167, respectively.

[0043] In some embodiments, the CPP has at least 90% sequence identity to one of SEQ ID NOS. 176-181. In some embodiments, the CPP has at least 95% sequence identity to one of SEQ ID NOS. 176-181. In some embodiments, the CPP has 100% sequence identity to one of SEQ ID NOS. 176-181. In some embodiments, the CPP has at least 90% sequence identity to one of SEQ ID NOS. 176-181.

[0044] In various embodiments, the CPP can have an N-terminal glycine added. For example, TATK28 and TAT28 would otherwise have an N-terminal aspartate residue, which has a low stability. Adding an N-terminal glycine to the sequence can increase protein stability via the N-end rule. Accordingly, in some embodiments, any of the fusion proteins that have a leader signal polypeptide can have a glycine added at the C-terminal end of the leader signal polypeptide, such that upon cleavage of the leader signal polypeptide, the new N-terminus of the fusion protein will begin with glycine. In an analogous manner, those fusion proteins lacking a leader signal polypeptide can also have a glycine added between the N-terminal methionine and the remainder of the fusion protein. Also in analogous manner, those fusion proteins having a CPP other than TAT28 or TATK28, can also have a glycine added between a leader signal polypeptide and a CPP.

[0045] In one or more embodiments, the CPP is operatively coupled to the N-terminus of the GLA polypeptide. In one or more embodiments, the CPP is operatively coupled to the C- terminus of the GLA polypeptide.

[0046] In one or more embodiments, the CPP comprises one or more affinity- tags. In one or more embodiments, the affinity-tag is located on one or more of the N-terminus or the C-terminus of the CPP. Examples of affinity-tags that can be added to the CPP include, but are not limited to, epitope tags (e.g. MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, HPC4), glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3xFLAG® polyhistidine (His), and combinations thereof.

[0047] In one or more embodiments, the CPP comprises one or more protease cleavage sites. In some embodiments, the protease cleavage site is located on one or more of the N- terminus or the C-terminus of the CPP. Exemplary protease cleavage sites include, but are not limited to, cleavage sites sensitive to thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, and combinations thereof.Fusion Proteins Comprising GLA Variants

[0048] As described above, GLA variants can be used in fusion proteins, such as proteins that also contain a CPP. Other polypeptides can also be incorporated into such fusion proteins, such as leader signal polypeptides to enhance protein secretion or affinity-tags for detecting and / or purifying the fusion proteins, as well as linker polypeptides that can be used to link functional polypeptides.

[0049] Examples of leader signal polypeptides include, but are not limited to, modified fragments of human immunoglobulin heavy chain binding protein (modified BiP), murine IgK chain leader polypeptide (e.g. pSecTag2 from ThermoFisher vectors) or insulin growth factor peptides (IGF2) such as the wild-type IFG2 or variants thereof. Non-limiting examples of such leader signal polypeptides include SEQ ID NOS. 191-209. Examples of modified BiP signal polypeptides include those described in U.S. Patent No. 9,279,007, which is hereby incorporated by reference in its entirety.

[0050] In one or more embodiments, the fusion protein comprises a GLA polypeptide having an N-terminal CPP, optionally with a leader signal polypeptide before the N-terminal CPP. In one or more embodiments, the fusion protein comprises a GLA polypeptide having a C-terminal CPP, optionally with a leader signal polypeptide before the GLA polypeptide. In one or more embodiments, the fusion protein comprises a leader signal peptide and a GLA polypeptide without a CPP.

[0051] Examples of affinity-tags that can be added to the fusion proteins include, but are not limited to, epitope tags (e.g. MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, HPC4), glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3xFLAG®, polyhistidine (His), and combinations thereof.

[0052] Some embodiments of the fusion protein may also include a protease cleavage site. In some embodiments, the protease cleavage site is located on the N-terminus of affinitytag. In some embodiments, the protease cleavage site is located on the C-terminus of affinity - tag. Exemplary protease cleavage sites include, but are not limited to, cleavage sites sensitive to thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV and combination thereof.Methods of Protein Production

[0053] The recombinant protein (e.g. GLA variant or fusion protein) can be expressed in and secreted from host cells using appropriate vectors. For example, mammalian cells (e.g., CHO, HeLa or HEK cells), insect cells (e.g. Sf9 or BTI-Tn-5Bl-4) or bacterial cells (e.g., E. coli or P. haloplanktis TAC 125 cells) can be used. Exemplary plasmids are described in the examples below. Those of skill in the art can select alternative vectors suitable for transforming, transfecting, or transducing cells to produce the GLA variants and fusion proteins described herein.

[0054] After expression and secretion, recombinant protein can be recovered and purified from the surrounding cell culture media using standard techniques. Alternatively, recombinant protein can be isolated and purified directly from cells, rather than the medium.

[0055] In some embodiments, the BTI-Tn-5Bl-4 cells are used to express and purify GLA variant or fusion protein.

[0056] For lysis, the cells expressing the GLA variant or fusion protein may be pelleted and subsequently resuspended into a lysis buffer. The resuspended cells may be then incubated in a cavitation chamber that is charged from about 100 PSI to about 2000 PSI with nitrogen gas. The resuspended cells may be incubated in the charged cavitation chamber for about 5 minutes to about 60 minutes. In some embodiments, the resuspended cells may be incubated in the cavitation chamber charged to 750 PSI with nitrogen gas. In some embodiments, the resuspended cells may be incubated in the charged cavitation chamber for 15 minutes. An effluent from the cavitation chamber after incubation may be then transferred on ice. A detergent may be added in the effluent followed by incubation on ice for about 5 minutes to about 60 minutes. In some embodiments, the detergent is added in the amount of about 0.1% (w / v) to about 5% (w / v). In some embodiments, the detergent is Triton X-100. The effluent with the detergent is then sonicated to lyse the cells. After lysis, soluble fractions and insoluble fractions may be separated. In some embodiments, the soluble fraction and insoluble fraction may be separated by centrifugation. The soluble material may be filtered. In some embodiments, the soluble material may be filtered through 0.45 pm filter.

[0057] For purification of the GLA variants or the fusion protein, the filtered soluble material is then subject to purification. In some embodiments, the GLA variants or the fusion protein is purified by a chromatography technique. In some embodiments, the chromatography technique is an affinity chromatography. In some embodiments, the GLA variant or the fusion protein comprises one or more affinity tags. In some embodiments, the affinity-tag include, but are not limited to, epitope tags (e.g. MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, HPC4), glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3xFLAG®, polyhistidine (His) and combination thereof. In some embodiments, the GLA variant or the fusion protein has a Twin-Strep-tag®. In some embodiments, the GLA variant or the fusion protein with the affinity-tag is purified on a purification resin. In some embodiments of the GLA variant or the fusion protein with a Twin- Strep-tag®, the purification resin is a strep-tactin resin.

[0058] Some embodiments of the GLA variant or the fusion protein may also include one or more protease cleavage sites. In some embodiments, the protease cleavage site is located on the N-terminus of the GLA variant or the fusion protein. In some embodiments, the protease cleavage site is located on the C-terminus of the GLA variant or the fusion protein. In some embodiments, the protease cleavage site is located on N-terminus and C-terminus of the GLA variant or the fusion protein. In some embodiments, the cleavage is performed when the GLA variant or the fusion protein is bound to the purification resin. In some embodiments, the cleavage is performed when the GLA variant or the fusion protein with the Twin-Strep-tag® is bound to the strep-tactin resin.Protein Replacement Therapy

[0059] In one or more embodiments, a subject may be administered with the GLA protein or variants or fusion proteins. In some embodiments, the subjects may be humans, domestic and farm animals, and laboratory, zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys etc. In some embodiments, the subject is a human.

[0060] In one or more embodiments, a cellular uptake of the GLA protein or variants or fusion proteins is determined in cells isolated from the subject. In some embodiments, the cells may be isolated from rats. In some embodiments, the cells may be neuronal cells. In some embodiments, the cells may be embryonic primary cortical neurons. In some embodiments, the embryonic primary cortical neurons may be isolated from rats. In some embodiments, the cells may be cultured and incubated with the GLA protein or variants for a duration of time. The duration of time may be at least 5 minutes, at least 10 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes or at least 60 minutes. In some embodiments the duration of time may be from 5 minutes to 24 hours, 15 minutes to 24 hour, 30 minutes to 24 hour, 1 hour to 24 hour, 4 hour to 24 hour, 8 hour to 24 hour, 12 hour to 24 hour, 5 minutes to 12 hours, 15 minutes to 12 hour, 30 minutes to 12 hour, 1 hour to 12 hour, 2 hour to 12 hour, 4 hour to 12 hour, 6 hour to 12 hour, 8 hour to 12 hour, 10 hour to 12 hour, 5 minutes to 6 hours, 15 minutes to 6 hour, 30 minutes to 6 hour, 1 hour to 6 hour, 1.5 hour to 6 hour, 2 hour to 6 hour, 2.5 hour to 6 hour, 3 hour to 6 hour, 4 hour to 6 hour 5 hour to 6 hour, 5 minutes to 4 hours, 15 minutes to 4 hour, 30 minutes to 4 hour, 1 hour to 4 hour, 1.5 hour to 4 hour, 2 hour to 4 hour, 2.5 hour to 4 hour, 3 hour to 4hour, 5 minutes to 2 hours, 15 minutes to 2 hour, 30 minutes to 2 hour, 1 hour to 2 hour, 1.5 hour to 2 hour, 5 minutes to 1 hours, 15 minutes to 1 hour or 30 minutes to 1 hour.Gene Therapy

[0061] Any of the GLA polypeptides and / or fusion proteins described herein can be utilized in gene therapy via an appropriate polynucleotide (e.g. DNA or RNA) encoding the desired GLA polypeptide and / or fusion protein.

[0062] In various embodiments, gene therapy is provided through the use of a composition comprising a gene therapy delivery system and a GLA polynucleotide. Exemplary gene therapy delivery systems include, but are not limited to, viral vectors, liposomes, lipidnucleic acid nanoparticles, exosomes and gene editing systems. For example, a gene editing system such as Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein 9 (CRISPR-Cas-9), Transcription activator-like effector nucleases (TALEN) or ZNF (Zinc finger proteins) can be used to insert the GLA polynucleotide into the DNA of the host cell.

[0063] Viral vectors include, but are not limited to, adenoviral vectors, adeno- associated viral (AAV) vectors, lenti viral vectors, retroviral vectors, poxviral vectors or herpes simplex viral vectors. Viral vectors typically utilize a viral particle (virion) including an outer protein shell (capsid) and one or more DNA or RNA sequences (viral polynucleotides) encapsulated in the capsid. For example, AAV vectors typically include one or more inverted terminal repeat (ITR) sequences, a replication (Rep) gene sequence, and a capsid (Cap) gene sequence. The ITR, Rep and Cap sequences may be included in the same plasmid (in cis), or may be provided in separate plasmids (in trans). The capsid may be derived from the same serotype as the ITR sequences, or the AAV vector can be a hybrid vector utilizing ITR sequences and capsids derived from different AAV serotypes. Exemplary AAV serotypes include AAV 1, AAV 2, AAV 3, AAV 4, AAV 5, AAV 6, AAV 7, AAV 8, AAV 9, AAV10, AAV11, hybrid serotypes, and synthetic serotypes.

[0064] The viral vectors also may include additional elements for increasing expression and / or stabilizing the vector such as promoters (e.g., hybrid CBA promoter (CBh) and human synapsin 1 promoter (hSynl)), a polyadenylation signals (e.g. Bovine growth hormone polyadenylation signal (bGHpolyA)), stabilizing elements (e.g. Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE)) and / or an SV40 intron. The DNAsequences for CBh and hSynl are provided in SEQ ID NO. 174 and SEQ ID NO. 175, respectively.

[0065] The gene therapy delivery system can be utilized to deliver the GLA polynucleotide to the target cells so that the GLA polypeptide (or fusion protein comprising the same) can be expressed in the target cells. In various embodiments, the GLA polypeptide (e.g. wild-type GLA polypeptides or recombinant variants thereof) (or fusion protein comprising the same) is expressed in the target cell and utilized in the same cell. In other embodiments, the GLA polypeptide (or fusion protein comprising the same) is expressed in a first cell, secreted, and then penetrates into a second cell. In such embodiments, a leader signal polypeptide and / or a cell-penetration may be used to enhance secretion and / or penetration of the GLA polypeptide. Without wishing to be bound by any particular theory, it is believed that secretion and penetration of GLA polypeptide can be used to enhance the effects of gene therapy over conventional gene therapy approaches that only introduce DNA and RNA into the patient, as transduction in gene therapy may only be limited to a certain portion of the patient’s cells e.g. 10% of the target patient cells are successfully transduced with the DNA / RNA). In this way, the successfully transduced cells may be used to express the GLA polypeptide (or fusion protein comprising the same) for both the transduced cells and neighboring cells that were not successfully transduced.Cross-Correction

[0066] Another aspect of the invention can include cross-correction. The genetherapy may not be effective to successfully transfect all defective cells. In one or more embodiments, a genetic defect in non-transfected cells can be corrected by the neighboring successfully transfected cells. For example, the GLA polypeptide or fusion protein may be expressed in a successfully transfected cell, secreted from that cell, and taken up by a neighboring cell that was not successfully transfected. The defect may be cross-corrected by any of the gene therapy methods described herein via an appropriate polynucleotide (e.g. DNA or RNA) encoding the desired GLA polypeptide and / or fusion protein. Any of the GLA polypeptides and / or fusion proteins described herein can be utilized to cross-correct a GLA -related defect.

[0067] In one or more embodiments, a GLA null subject is used for determining the fusion protein induced cross-correction. In some embodiments, the subject is a mouse. In some embodiments, a viral vector may be used to correct the GLA defect. In a particular embodiment, AAV vector was used to correct the GLA defect. In a particular embodiment, theAAV vector comprises a AAV-PHP.B.CBH.BIP-TATK28-GLA.SV40. In a particular embodiment, the viral vector comprising corrective gene is administered in a dose sufficient to correct the genetic defect. In some embodiments, the sufficient dose for correcting genetic defect in mice is in a range of 10 x e2GC / mice to 10 x e15GC / mice. In some embodiments, the sufficient dose for correcting genetic defect in mice may be 10 x e2GC / mice, 10 x e3GC / mice, 10 x e4GC / mice, 10 x e5GC / mice, 10 x e6GC / mice, 10 x e7GC / mice, 10 x esGC / mice, 10 x e9GC / mice, 10 x e10GC / mice, 10 x e11GC / mice, 10 x e12GC / mice, 10 x e13GC / mice, 10 x e14GC / mice or 10 x e15GC / mice. Exemplary routes of administration include, but are not limited to, intrathecal, intravenous, intracisternal, retro-orbital, intraperitoneal, intracerebroventrical or intraparenchymal administration.

[0068] In one or more embodiments, the GLA null mice may be divided into a treatment group and a control group. Each group, the treatment group and the control group, may further be divided into two subgroups based on route of administration. More than one route can be used concurrently, if desired. In one or more embodiments, each subgroup may be administered AAV-PHP.B.CBH.BIP-TATK28-GLA.SV40 dose through either intracerebroventricular (ICV) or retro orbital (RO) route of administration. Each subgroup received AAV-PHP.B.CBH.BIP-TATK28-GLA.SV40 dose in an amount of 10 x e8GC / mice, 10 x e9GC / mice or 10 x e10GC / mice. Three months post-administration, the impact of the vector on behavioral endpoints may be assessed and the mice may be euthanized for transgene expression analysis.

[0069] After euthanizing mice, various section of brain may be taken including but not limited to sagittal section. The sections may be immunostained with DAPI, anti-NeuN antibody, anti-GLA RNA antibody and anti-GLA protein antibody. The sections may be taken from isocortex, striatum, thalamus and hippocampal formation section of brains.

[0070] The immunostained images may be analyzed using Visiopharm software. The immunostained cells may be divided into six groups: (1) DAPI stain to identify cells; (2) NeuN stain to identify neurons; (3) Neurons having GLA mRNA and GLA protein; (4) Neurons having GLA mRNA; (5) Cross-corrected neurons; and (6) Cross-corrected non-neurons. The result of image analysis may be further subject to a statistical analysis for cross-corrected neurons and non-neurons.Formulations, Methods of Treatment and Use

[0071] The gene therapy compositions (e.g. comprising GLA polynucleotides) or the protein replacement therapy compositions (e.g. comprising recombinant proteins including GLA variants or fusion proteins), can be formulated in accordance with the routine procedures as a pharmaceutical composition adapted for administration to human beings. For example, in one or more embodiments, a composition for intravenous administration is a solution in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose / water. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0072] Gene therapy compositions (e.g. comprising GLA polynucleotides) or protein replacement therapy compositions (e.g. comprising recombinant proteins including GLA variants or fusion proteins) (or a composition or medicament containing the gene therapy composition or protein replacement therapy composition) are administered by an appropriate route. In one or more embodiments, the gene therapy composition or protein replacement therapy composition is administered intravenously. In other embodiments, the gene therapy composition or protein replacement therapy composition is administered by direct administration to a target tissue, such as to heart or skeletal muscle (e.g., intramuscular; intra ventricularly), or nervous system (e.g., intrathecal delivery - delivery into the space under the arachnoid membrane of the brain or spinal cord). More than one route can be used concurrently, if desired. Exemplary routes of administration include, but are not limited to, intrathecal, intravenous, intracisternal, intracerebroventrical or intraparenchymal administration.

[0073] The gene therapy composition (e.g. comprising GLA polynucleotides) or protein replacement therapy composition (e.g. comprising recombinant protein including GLA variants or fusion proteins) (or a composition or medicament containing such gene therapy composition or protein replacement therapy) is administered in a therapeutically effectiveamount (e.g., a dosage amount that, when administered at regular intervals, is sufficient to treat the disease, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and / or lessening the severity or frequency of symptoms of the disease). The amount which will be therapeutically effective in the treatment of the disease will depend on the nature and extent of the disease's effects. In addition, in vitro or in vivo assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed will also depend on the route of administration, and the seriousness of the disease, and should be decided according to the judgment of a practitioner and each patient’s circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0074] The therapeutically effective amount of gene therapy composition (e.g. comprising GLA polynucleotides) or protein replacement therapy composition (e.g. comprising recombinant protein including GLA variants or fusion proteins) (or a composition or medicament containing such gene therapy composition or protein replacement therapy) can be administered at regular intervals, depending on the nature and extent of the disease's effects, and / or on an ongoing basis. Administration at a "regular interval," as used herein, indicates that the therapeutically effective amount is administered periodically (as distinguished from a onetime dose). The administration interval for a single individual need not be a fixed interval, but can be varied over time, depending on the needs of the individual.

[0075] The gene therapy composition (e.g. comprising GLA polynucleotides) or protein replacement therapy composition (e.g. comprising recombinant protein including GLA variants or fusion proteins) (or a composition or medicament containing such gene therapy composition or protein replacement therapy composition) may be prepared for later use, such as in a unit dose vial or syringe, or in a bottle or bag for intravenous administration. Kits containing the gene therapy composition (e.g. comprising GLA polynucleotides) or protein replacement therapy composition (e.g. comprising recombinant protein including GLA variants or fusion proteins) (or a composition or medicament containing such gene therapy composition or protein replacement therapy composition), as well as optional excipients or other active ingredients, such as other drugs, may be enclosed in packaging material and accompanied by instructions for reconstitution, dilution or dosing for treating a subject in need of treatment, such as a patient having Fabry disease.EXAMPLES

[0076] The present invention is further illustrated by the following non-limiting examples.

[0077] Galactosidase Alpha (GLA) is an essential lysosomal enzyme responsible for the hydrolysis of glycosphingolipids, which are crucial for cellular functions and integrity. Deficiencies in GLA due to genetic mutations lead to Fabry disease, a rare X-linked lysosomal storage disorder characterized by the accumulation of globotriaosylceramide (Gb3) within various body tissues. This pathological accumulation manifests in a spectrum of symptoms, ranging from pain and angiokeratomas to renal failure, cardiovascular disease, and early mortality. Current treatments, primarily enzyme replacement therapies (ERT) and pharmacological chaperones, aim to supplement or enhance the residual activity of GLA. However, these approaches face limitations due to enzyme instability in circulation at neutral pH and the inability to adequately target all affected tissues (Atta et al. 2020). Thus, protein engineering of GLA presents a promising avenue for developing more effective and durable therapies, potentially overcoming the challenges posed by conventional treatments and significantly improving patient outcomes.

[0078] In some embodiments, polypeptides are provided comprising modified human a-galactosidase polypeptides with enhanced thermostability under neutral pH conditions. In some embodiments, the neutral pH is in a range of from about 7 to about 8. In some embodiments, the neutral pH is about 7.4. Some embodiments provide applications of the compositions containing the modified human a-galactosidase polypeptides in non-human primates (NHPs) for treatment purposes. Some embodiments provide applications of the compositions containing the modified human a-galactosidase polypeptides in humans for treatment purposes. The modified human polypeptides provided in the present disclosure are useful in the treatment of genetic diseases via enzyme replacement therapy and / or gene therapy.

[0079] The instability and consequent inefficacy of therapeutic enzymes such as GLA pose significant challenges to treatment modalities for metabolic disorders. Traditional in silico design methodologies, while valuable, often fall short in addressing the dynamic complexities and nuanced interactions intrinsic to protein structures. To address this gap, our work integrates ProteinMPNN (Dauparas et al., 2022), a cutting-edge machine learning model thatcaptures the intricate molecular dynamics of proteins, facilitating the prediction and rational design of GLA variants with superior stability profiles.

[0080] Additionally, ancestral sequence reconstruction (Zakas et al., 2017) is employed as a complementary strategy, tapping into the evolutionary history of GLA to identify inherently stable and functionally robust predecessors. This method is based on the premise that ancestral proteins, having been subjected to natural selection across diverse environmental conditions, inherently possess qualities of stability and resilience that modern enzymes might lack.

[0081] In tandem with these innovative approaches, traditional protein engineering techniques are also applied, creating a multifaceted and iterative design strategy. This integrative approach not only broadens the scope of potential stabilizing modifications but also provides a robust framework for systematically improving protein therapeutics. We demonstrate that through multiple rounds of engineering, we have enhanced the half-life of GLA at neutral pH from approximately 3 hours to well over 72 hours. Critically, this improved stability has translated to enhanced efficacy in a Gia knockout mouse model.

[0082] GLA is a homodimer, and the protein's stability can be improved through a disulfide linkage at the dimer interface (US20200147241A1). Consequently, we hypothesized that strengthening this interface through rational design could enhance GLA's stability. We applied ProteinMPNN to analyze the entire GLA sequence and specifically targeted regions within 5 A of the symmetric subunit (dimer interface only). Additionally, we used Schrodinger's Prime software to predict the stability and affinity of mutations at the dimer interface. Based on these analyses, particularly focusing on overlapping results from both tools, we generated 20 mutants, each incorporating multiple mutations.

[0083] To streamline the evaluation process, we expressed the constructs in GripTite293 GLA-KnockOut cells and performed T50 assays to determine thermal stability of the active enzyme. Initially, many constructs did not express well; however, several showed enhanced expression and increased thermal stability in the T50 assay (FIG. 2A). For the second batch of 20 constructs, we combined sequences showing improved stability from the first round with additional mutations predicted by Schrodinger Prime and ProteinMPNN. This round yielded more constructs that expressed well and exhibited increased thermal stability (FIG. 3B). Subsequently, we selected several of these mutants for further testing in a neutral pH incubation assay to assess their stability in neutral pH conditions. One mutant,GLAmpnnO26, demonstrated considerably improved stability with a half-life of approximately 20 hours, compared to 3 hours for the wild-type GLA (FIG. 4B).

[0084] In the third and fourth rounds of mutagenesis, we combined mutations from the second round that enhanced thermal stability with new mutations predicted by ProteinMPNN and Schrodinger for improved stability in known immunogenic epitopes of wild-type GLA (Schametzki et al., 2020). This effort resulted in GLAmpnnO62, which exhibited improved activity and a half-life greater than 72 hours, outperforming GLAmpnnO26 with mutations in a known epitope near the active site.

[0085] For the fifth round of mutagenesis, we employed ancestral sequence reconstruction. This technique estimates the amino acid sequences that encoded a protein in a theoretical last common ancestor (LCA) of two clades. Reconstructions of ancestral proteins have often been found to possess higher stability than extant proteins, making them promising candidates for therapeutic biologies (Hendrikse et al., 2020; Thomson et al., 2022; Hartz et al., 2021; Zakas et al., 2017). Several mechanisms to explain this phenomenon have been proposed, including ancestral organisms being exposed to higher temperatures, ancestral organisms having less efficient protein quality control systems, a consensus bias in ancestral reconstructions, and the simple reversal of genetic drift (Trudeau et al., 2016). While the stability of reconstructed enzymes is likely due to a combination of factors, we suspect that the reversal of genetic drift plays a major role, especially in the case of more recent ancestors, such as those within the mammalian lineage.

[0086] Sequences for GLA proteins expressed by human ancestors (e.g. the LCA of all primates, the LCA of primates and rodents, etc.) were estimated using ancestral sequence reconstruction. First, a phylogenetic tree was built from GLA sequences. Then, ancestral residues at each position in the human GLA sequence were inferred using different reconstruction techniques (maximum parsimony, maximum likelihood joint, and maximum likelihood marginal). A selection of these “ancestral” mutations were assessed in the context of the human GLA sequence to determine whether they had beneficial effects on enzyme stability. In total, 107 unique ancestral mutations were found. Of these, 75 (70%) were found by all three methods. Seventy-eight ancestral mutations were assessed in experimental constructs. Ancestral mutations that have been used in late-stage GLA constructs include E74D, K237S, H406R, Q416R, and M421S.

[0087] Using the GLAmpnnO62 template, we introduced single-point mutations based on ancestral analyses, resulting in 25 mutants tested for activity and stability at neutral pH in conditioned media. Two mutants particularly stood out, either exhibiting high expression or exceptional stability with 90% activity at 72 hours. These mutations were further combined to produce Round 5 mutants, among which GLAmpnnl08 and GLAmpnnl l l demonstrated high expression and stability (FIG. 21).

[0088] The seventh round of mutagenesis utilized previously produced data from Amicus Therapeutics to test whether a mutation is amenable to improved activity with the drug Galafold (HEK amenability assay) (Benjamin et al., 2017). Beyond known patient mutations, all amino acid substitutions possible from single point mutations were also tested for activity with and without Galafold. These results included 23 point mutations that improved activity by more than 25% compared to wild-type GLA. However, when these mutations were tested in the GLAmpnnl 11 background, most did not confer benefits to the degree shown in the context of wildtype GLA.

[0089] In the eighth round, we experimented with different combinations of mutations that appeared beneficial beyond the GLAmpnn088 construct. GLAmpnnl42, which combines four of these mutations on top of the GLAmpnn088 background, proved to have the best combination of expression and stability. For these experiments, we increased the pH to 7.44 to further stress the proteins. Subsequent rounds involved individual subtraction of mutations from GLAmpnnl42. In round 9, the removal of the F229S mutation led to significant improvements in stability, while the reversion of the I359R mutation greatly enhanced activity. In round 10, we produced GLAmpnnl68, which combines the F229 and 1359 reversions, resulting in substantial improvements in both activity and stability over GLAmpnnl 42 (FIG. 28).

[0090] In vivo expression in NHP

[0091] For in vivo testing of systemic GLAmpnnl ll expression in Non-Human Primates (NHP), A AV- GLAmpnnl 11 was administered intravenously into cynomolgus macaques (n=3) at a dose of 2.5E+13 GC / kg. Serum was collected at different time points post-injection. The a-Gal A enzyme activities in serum show superphysiological levels compared to the baseline, which were maintained at 300-fold over the baseline through Day 63 post-injection and continued to be elevated at Day 82 (FIGS. 29 and 31). Similarly, the a-Gal A enzyme activity in liver biopsies, taken at Days 30 and 90 post-injection, show a 19-foldincrease in GLA compared to the untreated control (baseline) at Day 30 and continued increase through Day 90 (FIGS. 30 and 32).

[0092] ProteinMPNN

[0093] ProteinMPNN was used with the human alpha-galactosidase crystal structure (PDB:3HG2). Sampling temperatures of 0.1 or 0.2 were used with the vanilla-v_48_002 model and 0.02 backbone noise. The model was set as a homodimer and either the entire protein was not fixed or only residues within 5 A of the dimer partner were not fixed. For each run 5 output sequences were collected.

[0094] In silico residue scanning

[0095] Prime (Schrodinger Release 2022-1; Jacobson et al., 2004; Jacobson et al., 2002) was used to predict the changes in stability and inter-subunit affinity for all possible point mutations along the dimer interface of GLA. The modeling was performed on the 3HG2 structure of GLA protonated for pH 7.4. Mutations predicted to be beneficial for both overall protein stability and dimer affinity were manually reviewed, and the most promising mutations were selected for experimental analysis.

[0096] Ancestral Reconstructions

[0097] The reference human protein sequence for GLA, corresponding to Uniprot entry P06280, was queried in the NCBI databases using PSI-BLAST (Altschul et al., 1997). Three iterative searches were performed, each requesting 500 hits. The final results were filtered to remove precursor proteins, low-quality proteins, shorter isoforms, and predicted proteins for which more confident sequences from the same organism were also in the sequence set. The sequences were then aligned using MAFFT (default settings: BLOSUM62 scoring matrix, gap opening penalty of 1.53, offset value of 0.0, default guide tree, FFT-NS-i alignment method) (Katoh et al., 2019). Sequences with long (>20 residues) insertions or deletions, poorly aligned sequences, and sequences with ambiguous residue calls were removed. The sequence set was then identity -filtered using CD-HIT at a threshold of 0.985 (Fu et al., 2012; Li & Godzik, 2006). The human sequence was re-added to the sequence set (having been removed during identity filtering), and the zebrafish GLA sequence was added for use as an outgroup. Signal peptides were removed from sequences (based on the mature protein starting residue in humans) to prevent spurious signal peptide correspondence from affecting the alignment and tree topology. The sequence set was re-aligned using MAFFT default settings.

[0098] PhyML (version 20120412, Guindon et al., 2010) was used to generate a phylogenetic tree from the multiple sequence alignment (MSA) using a WAG substitution matrix, a discrete gamma distribution of evolutionary rate differences among sites (8 categories, with invariant sites allowed, alpha=0.675), and subtree pruning and regrafting. Sequences that were misplaced in comparison with published phylogenetic trees were deleted.

[0099] The C-termini of the sequences were poorly aligned. To remedy this and improve ancestral sequence inference, the C-terminal ends of the sequences (residues aligned with human residue 389 and later) were aligned together in MAFFT using the G-INS-1 method with an unaligned level of 0.8. The resulting alignment for this region looked slightly better than the alignment generated from whole sequences, so the re-aligned C-termini were substituted into the end of the previous MSA. This C-terminally edited MSA was used for ancestral sequence reconstruction.

[0100] Ancestral residue states were inferred from the phylogenetic tree and MSA using three methods: 1) maximum parsimony as implemented in MEGA version 11.0.13 (Tamura et al., 2021), 2) maximum likelihood marginal reconstruction as implemented in paml version 4.9 (Yang 2007), and 3) maximum likelihood joint reconstruction as implemented in paml version 4.9. For the maximum likelihood reconstructions, an empirical substitution model was used with the WAG substitution matrix, and a single substitution rate was used for all sites.

[0101] The inferred ancestral sequences at each node in the phylogenetic tree from the three inference methods were compared to each other and to the extant human sequence. Mutations relative to the modern human sequence that were found in the inferred ancestral sequences (“ancestral mutations”) were selected for experimental analysis based on their frequency of occurrence in the inferred ancestral sequences, their correspondence with mutations suggested by ProteinMPNN, their location on the protein, and their predicted effects on protein stability (determined using Prime).

[0102] Cloning

[0103] Mutants were cloned into a minimal expression plasmid with EFl -alpha promoter and BGH polyA signal called pSvelte using BspQI cloning (Galloway et al., 2013). Single point mutations were produced using quikchange (Agilent) and multiple mutations were made using eBlocks (Integrated DNA Technologies) and BspQI cloning.

[0104] GLA Knockout Cell Line

[0105] Guide RNA prediction was performed using Benchling’s built-in CRISPR guide prediction tool targeting exon 1 of human GLA (NG_007119.1). The chosen guide RNA 5’-[CGTTTCCTGGGACATCCCTG]-3’ was synthesized by IDT as an Alt-R CRISPR-Cas9 sgRNA. Alt-R S.p. Cas9 Nuclease V3 was purchased from IDT and Invitrogen’s Lipofectamine CRISPRMAX Cas9 Transfection Reagent was purchased from Thermofisher. Primers designed to amplify the gene editing site were synthesized by Azenta (forward primer: 5’-GCGGAAATTTATGCTGTCCGGTCACC-3’, reverse primer: 5’-GCTCAACTGTTCCCGTTGAGACTCTCC-3’, sequencing primer: 5’-AGTTCCCCAAACACACCCAAACACATGG-3’). Wild-type GripTite 293 MSR cells (Invitrogen) maintained according to the manufacturer’s user manual were seeded into a 6- well tissue culture treated plate at 0.3e6 cells per well and incubated at 37C - 5% CO2 overnight. The following day, Cas9-sgRNA RNP complex formation and Cas9 RNP transfection was performed according to Invitrogen’s Lipofectamine CRISPRMAX Cas9 Transfection Reagent protocol and incubated at 37C -5% CO2. Three days after gene editing, cells were subsequently single cell cloned into 96-well tissue culture treated plates by limiting dilution at 0.6 cells / well. Clones were incubated at 37C - 5% CO2 for 12 days until cells achieved ~80%-90% confluency. Clones were subsequently expanded into 6-well tissue culture treated plates and incubated at 37C - 5% CO2 for 5 more days until clones achieved ~80%-90% confluency. Each clone was harvested for cry opreservation and genomic analysis. Genomic DNA from putative GLA knock-out clones was extracted from cell pellets using Qiagen’s DNeasy Blood & Tissue Kit according to manufacturers protocol. PCR amplification of the edit site was performed on genomic DNA from putative GLA knock-out clones and PCR products were gel extracted for sequencing analysis. Amplicons were subsequently sent for Sanger sequencing at Azenta for preliminary screening. Amplicons containing apparent INDELS were sent to Azenta for Next Generation Sequencing using their Amplicon-EZ service for more detailed analysis. NGS analysis identified GripTite GLA knock-out clone #4 to contain complete gene disruption and knock-out. Sequence analysis was validated by anti-GLA western blotting and GLA activity assay.

[0106] Cell Culture and Transfection:

[0107] GripTiteTM 293 MSR GLA-KO cells were seeded in 12-well plates one day prior to transfection (DMEM High Glucose expression media, 10% (V / V) FBS, 0.1 mM MEM Non-essential amino acids, 600 pg / mL Geneticin) and grown overnight. GLA-KO cells weretransfected at a ratio of 3:1 GeneCopoeiaTM Endofectin Max transfection reagent:DNA when the cells reached -70% confluency according to the manufacturer’s protocol (GeneCopoeiaTM) and incubated at 37 oC with 5% CO2. The following day culture medium was replaced, and cells were incubated for 72 hour at 37oC with 5% CO2. At the time of harvest, conditioned media was collected and centrifuged at 8000xg for 10 minutes and supernatant was transferred to a clean, sterile Eppendorf tube. Conditioned media was flash- frozen.

[0108] GLA Activity Assay:

[0109] Frozen conditioned media was thawed on ice and diluted 1:20 in GLA Assay buffer (27 mM Citrate I 46 mM Phosphate Buffer, pH 4.6). 50 pL of 4-methylumbelliferone (4-MU) standard was added to the corresponding wells of each clear, flat-bottom, non-treated 96-well assay plate to generate a 4-MU standard curve (3, 1.5, 0.75, 0.375, 0.1875, 0.0937, 0.0468, 0 nmol). 40 pL of 4-MU-Gal / GalNAc substrate (5 mM 4-methyl-umbelliferyl-alpha- D-galactopyranoside + 98 mM N-Acetyl-D-Galactosamine) in GLA Assay buffer (27 mM Citrate I 46 mM Phosphate Buffer, pH 4.6) was added to each remaining well. 10 pL of diluted conditioned media was added to each sample well and mixed by pipetting (all samples assayed in duplicate), and incubated at 37oC for 1 Hr. After 1 hour, reactions were quenched with 125 pL of Stop buffer (IM Glycine, pH 10.8) and fluorescence was read on SpectraMax M2 (excitation / emission 355 / 460). Raw data were analyzed (Blank-subtracted) using Microsoft Excel, and fluorescence and absorbance counts were converted into nmole / mL / hour in GraphPad Prism.

[0110] T50 Assay:

[0111] Conditioned media was diluted 1: 1 in GibcoTM IX PBS pH 7.4. Samples were incubated at 39.1, 45, 50, 52.5, 56, 61, 65, 71, and 79oC for 10 minutes and cooled to 4oC. Samples were centrifuged briefly to remove condensation from the tubes. Samples were then diluted 1:10 in GLA Assay buffer (27 mM Citrate / 46 mM Phosphate Buffer, pH 4.6) for a final dilution factor of 1:20 and used in the GLA Activity assay as previously described.

[0112] GLA Stability Assay:

[0113] Conditioned media was diluted 1:1 in Mcllvane Buffer (9.15 mM Citrate I 181.7 mM Phosphate Buffer, pH 7.4) and incubated at 37oC. An initial 100 pL aliquot was flash- frozen at time OHr, followed by 100 pL aliquots at 24, 48, and 72Hr. Timepoint samples were thawed on ice and diluted 10-fold in GLA Assay buffer (27 mM Citrate / 46 mM PhosphateBuffer, pH 4.6) to a final dilution factor of 1:20. The GLA Activity assay was performed as previously described.

[0114] NHP AAV administration and sample collection

[0115] On study day 0, cynomolgus macaques received 2.5E+13 genome copies (GC) / kg of AAV-GLAengll l (N = 3) into the saphenous vein. The AAC vector utlized the AAV9 capsid and ubiquitous promoter. Macaques were anesthetized and blood / serum was collected on selected days (at Day 0, 3, 8, 15, 22, 43, 63, 83, 120 and 180) via the femoral vein for analysis and a liver tissue biopsy was taken on Day 30 and Day 90. Necropsy was performed at Day 180 with tissue harvest, biochemical analysis, bioanalytical analysis and histochemical analysis

[0116] GLA Activity Determination in Serum and Liver

[0117] Diluted serum or tissue lysate samples of 20 pL were added to 50 pL of substrate (12mM 4MU-a-D-galactopyranoside + 90 mM N-acetyl-D-galactosamine) in Reaction Buffer (0.1M Citrate Phosphate Buffer, pH 4.6) containing 2% DMSO in Costar 96 well black-wall clear-bottom plate, and the plate was then incubated for 3 hour at 37 °C. Reactions were stopped by addition of 70 pL 0.4 M glycine, pH 10.8. Fluorescence at 460 nm was read with excitation at 355 nm. Raw fluorescence counts were background subtracted (defined by buffers only) and converted to 4-MU concentration by comparing to a 4-MU standard curve ranging from 1 18 nM to 15 pM made using linear regression following log-log transformation. The GLA activity in serum or plasma was calculated and expressed as nanomoles of released 4-MU per mL of sample per hour (nmol / mL / hr). The GLA activity in tissue sample was calculated by normalizing the 4-MU released to the protein concentration and expressed as nanomoles of released 4-MU per milligram protein per hour (nmol / mg protein / hr).

[0118] References:

[0119] Benjamin, E. R. et al. The validation of pharmacogenetics for the identification of Fabry patients to be treated with migalastat. Genet. Med. 19, 430-438 (2017).

[0120] Schametzki, D., Stappers, F., Lenders, M. & Brand, E. Detailed epitope mapping of neutralizing anti-drug antibodies against recombinant a-galactosidase A in patients with Fabry disease. Mol Genet Metab 131, 229-234 (2020).

[0121] Kant, S. & Atta, M. G. Therapeutic advances in Fabry disease: The future awaits. Biomed. Pharmacother. 131 , 1 10779 (2020).

[0122] Galloway, N. R., Toutkoushian, H., N ne, M., Bose, N. & Momany, C. Rapid Cloning For Protein Crystallography Using Type IIS Restriction Enzymes. Crystal Growth & Design 13, 2833-2839 (2013).

[0123] Hendrikse N., Holmberg Larsson A., Svensson Gelius S., Kuprin S., NordlingE., & Syren P-O. (2020). “Exploring the therapeutic potential of modern and ancestral phenylalanine / tyrosine ammonia-lyases as supplementary treatment of hereditary tyrosinemia.” Scientific Reports, 10, 1315.

[0124] Thomson R., Carrera-Pacheco S., & Gillam E. (2022). “Engineering functional thermostable proteins using ancestral sequence reconstruction.” JBC Reviews, 298(10), 102435.

[0125] Hartz P., Strohmaier S., EL-Gayar B., Abdulmughni A., Hutter M., HannemannF., Gillam E., & Bernhardt R. (2021). “Resurrection and characterization of ancestral CYP11A1 enzymes.” The FEBS Journal, 288, 6510-6527.

[0126] Zakas P., Brown H., Knight K., Meeks S., Spencer T., Gaucher E., & DoeringC. (2017). “Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction.” Nature Biotechnology, 35(1), 35-37.

[0127] Trudeau D., Kaltenbach M., & Tawfik D. (2016). “On the potential origins of the high stability of reconstructed ancestral proteins.” Molecular Biology and Evolution, 33(10), 2633-2641.

[0128] Altschul S., Madden T., Schaffer A., Zhang J., Zhang Z., Miller W., & LipmanD. (1997). “Gapped BLAST and PSLBLAST: A new generation of protein database search programs.” Nucleic Acids Research, 25(17), 3389-3402.

[0129] Katoh K., Rozewicki J., & Kazunori Y. (2019). “MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization.” Briefings in Bioinformatics, 20(4), 1160-1166.

[0130] Fu L„ Niu B„ Zhu Z„ Wu S., & Li W. (2012). “CD-HIT: accelerated for clustering the next generation sequencing data.” Bioinformatics, 28(23), 3150-3152.

[0131] Li W. & Godzik A. (2006). “Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences.” Bioinformatics, 22(13), 1658-1659.

[0132] Guindon S., Dufayard J., Lefort V., Anisimova M., Hordijk W., & Gascuel O. (2010). “New algorithms and methods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0.” Systematic Biology, 59(3), 307-321 .

[0133] Tamura K., Stecher G., & Kumar S. (2021). “MEGA11: Molecular Evolutionary Genetic Analysis version 11.” Molecular Biology and Evolution, 38(7), 3022- 3027.

[0134] Yang, Z. (2007). “PAML 4: A program package for phylogenetic analysis by maximum likelihood.” Molecular Biology and Evolution, 24(8), 1586-1591.

[0135] Jacobson M., Pincus D., Rapp C., Day T., Honig B., Shaw D., & Friesner R. (2004). “A hierarchical approach to all-atom protein loop prediction.” Proteins: Structure, Function, and Bioinformatics, 55(2), 351-367.

[0136] Jacobson M., Friesner R., Xiang Z., & Honig B. (2002). “On the role of crystal packing forces in determining protein side chain conformations.” Molecular Biology, 320(3), 597-608.

[0137] The present invention is further illustrated by the following non-limiting examples.Example 1: Protein Engineering Machine Learning Predictions of Affinity and Stability Changes upon Residue Mutations of GLA

[0138] Schrodinger Prime was used to perform in silico saturation mutagenesis of all interface residues. All mutations that were predicted to improve both affinity and stability were selected and manually reviewed.

[0139] Prime (Schrodinger Release 2022-1; Jacobson et al., 2004; Jacobson et al., 2002) was used to predict the changes in stability and inter-subunit affinity for all possible point mutations along the dimer interface of GLA. The modeling was performed on the 3HG2 structure of GLA protonated for pH 7.4. Mutations predicted to be beneficial for both overall protein stability and dimer affinity were manually reviewed, and the most promising mutations were selected for experimental analysis. (Jacobson M., Pincus D., Rapp C., Day T., Honig B., Shaw D., & Friesner R. (2004). “A hierarchical approach to all-atom protein loop prediction.” Proteins: Structure, Function, and Bioinformatics, 55(2), 351-367; and Jacobson M., Friesner R., Xiang Z., & Honig B. (2002). “On the role of crystal packing forces in determining protein side chain conformations.” Molecular Biology, 320(3), 597-608).

[0140] Prime made predictions for 687 mutants across 38 GLA interface residues, 108 of which were predicted to improve both stability and affinity to some degree. Of these potentially beneficial mutations, 96 were tested experimentally. Mutations predicted to be beneficial by the Prime workflow that were used in late-stage GLA constructs include: S62Q, K237R, I359R, and H406R.

[0141] Table 1 shows selected results of the in silico calculations for the mutation of the S62 residue of wild type GLA. AAffinity is the change in Prime predicted binding affinity of the subunits to each other. AStability is the change in Prime predicted energy of the solvated protein. Table 2 shows selected results for mutation of the D233 residue. Table 3 shows results for selected mutations of the 1359 residue.Table 1: Prime results for S26 mutation.Table 2: Prime results for D233 mutation.Moreover, it was found experimentally that D233N D233T, and D233S were stabilizing.Table 3: Prime results for 1359 mutation.Moreover, it was also found experimentally that I359Q and I359K were stabilizing.Example 2: Western Blot Activity Assays, T50 Assays, and Stability Assays of Recombinant GLA

[0142] Activity and T50 assays were performed at pH 4.6 on new GLA mutants in conditioned media from transfected GripTite GLA-KO clone. Activity assays were performed at dilutions of 1:5 and 1:20, and performed using equivalent amounts of conditioned media unless otherwise noted.

[0143] FIG. 1A illustrates Western blot results of several mutants at 1:5 dilution, without normalization. Several of the mutants were stable and showed activity as shown by the presence of Sheep Anti-GLA lines on the Western blot slides at approximately 50 kD.

[0144] It is to be noted that the labels “GLA_mpnnX” correspond to “SEQ ID NO. X.” In other words, GLA_mpnnl has the same sequence as SEQ ID NO. 1, GLA_mpnn2 has the same sequence as SEQ ID NO. 2, and etcetera.

[0145] FIG. 1 B illustrates selected Western blot results at 1:20 dilution. Several constructs showed activity.

[0146] FIG. 2A illustrates a T50 assay showing change in GLA activity as a function of temperature. Several constructs showed activity. FIG. 2B shows residual activity, as percent of the original activity. It can be seen that with increasing temperature, several recombinant constructs have increased activity.

[0147] FIG. 3A shows a GLA 4-MU (4-methylumbelliferone) activity assay of severalGLA constructs at 1:20 dilution. Several recombinant constructs display activity. FIG. 3B shows the results of a T50 assay for several constructs. Several of the constructs have increased GIA activity against GLA substrates. FIG. 3C shows results of a T50 assay showing several constructs with especially beneficial performance. Table 4 provides a summary of several T50 assay results.

[0148] Table 4: Prime results for 1359 mutation.

[0149] GLA-66 corresponds to a (D233C, 1359) recombinant construct.

[0150] FIG. 4A shows measured long-term stability of several recombinant GLA constructs in conditioned media. Particularly, SEQ ID NO. 26 (MPNN26) showed a marked stability increase in conditioned media at 37 °C. FIG. 4B shows durability of different constructs, including SEQ ID NO. 26, in pH 7.4 Mcllvane buffered media.Example 3: Codon Optimization for Durability of Recombinant GLA Constructs

[0151] FIG. 5 shows a schematic illustration of the durability-codon optimization pathway according to some embodiments. FIG. 6 shows GLA-66 (D233C, 1359 recombinant construct) and SEQ ID NO. 26 codon optimizations (i.e. fusion proteins). It was found that IDT, IDTOpt, Gen gave consistently high expression. FIG. 7 shows further results of GLA activity for different fusion proteins.

[0152] pJamaO21 - CB promoter-SV40 intron - MCS-WPRE-PolyA.

[0153] pJamaO23 - CB promoter-SV40 intron - MCS-OPRE-PolyA

[0154] pJamaO25 - EFla promoter(intron) - MCS-WPRE-PolyA

[0155] pJamaO27 - LP1 promoter-MAEL intron- MCS-WPRE-PolyA

[0156] pJamaO28 - LP1 promoter- SV40 intron- MCS-WPRE-PolyA

[0157] pJama033 - CB promoter-SV40 intron - MCS-WPRE- 4xAPC detargeting

[0158] The data was not normalized for dilution amounts. pJama-GLAco66 - 1:20. pJama-wtGLA - 1:40. pJama-Takeda - 1:80. The results in FIG. 7 show that pJamaO23 and pJama025 gave strong expression and minimal decrease in expression with co-expression in M6P plasmid.Example 4: HEPG2 Screening of Recombinant GLA Constructs

[0159] HEPG2 liver cells were seeded in 24-well plates and transfected using X- tremeGENE HP transfection reagent. Conditioned media were harvested 72 hours posttransfection. 4MU-GLA activity assays and Western Blots were performed using the conditioned media, with 5 mM 4MU GLA substrate.

[0160] Results are shown in FIGS. 8A-8B for GLA-66 codon optimized constructs screen in HEPG2, corresponding to two different transfections. It was found that IDT and Genscript had good performance.

[0161] Results are shown in FIGS. 9A-9B for SEQ ID NO. 26 codon optimized constructs screened in HEPG2. The results found that IDT, IDTopt and Gen also had good performance.

[0162] FIG. 10 shows GLA expression in HEPG2 in conditioned media at 1:40 dilution. Good activity was seen.

[0163] FIG. 11 provides a color-coded summary of comparisons between different fusion constructs including the recombinant GLA constructs, and promoters, introns, and / ro codons in AAV9. Example 5: Mouse Short Term Efficacy Study for Fabry Gene Therapy Durability

[0166] Table 6 shows the M6P Combo and treatment groups.

[0167] Table 6: M6P Combo / Treatment Groups.

[0168] Table 6 shows WT and GLA-66 + / - SI S3 co-expression.

[0169] FIG. 12 shows select results of the durability study, showing GLA activity in males and females.

[0170] FIG. 13 shows D28 GLA activity for male mice. It shows that the Takada construct had approximately 20X higher plasma activity than the second best construct GLA- 66-IDT. The constructs can be ranked (1) AAV9-CB-SV40_Takeda, AAV9-CB-SV40 GLA- 66-IDT, AAV9-CB7-GLA-66 (Penn) and AAV9-CB-SV40_GLA-66.

[0171] FIG. 14 shows the D28 GLA activity for all groups (male and female). Takada construct was found to have approximately 18X higher activity than the second best construct GLA-66-IDT. These constructs can also be ranked (1) AAV9-CB-SV40_Takeda, AAV9-CB- SV40 GLA-66-IDT, AAV9-CB7-GLA-66 (Penn) and AAV9-CB-SV40_GLA-66.

[0172] FIG. 15 A shows GLA liver activity in mice. The Takada construct had approximately 7X more liver activity than the second best construct, GLA-66-IDT. FIG. 15B shows GLA heart activity in mice. The Takada construct had approximately 24X more plasma activity than the second best construct. FIG. 15C shows GLA kidney activity in mice. The Takada construct had approximately 14X more kidney activity than the second best construct tested.

[0173] FIG. 15D shows Lyso-Gb3 accumulation in mouse plasma. Several recombinant GLA construct fusion constructs demonstrated good performance. FIG. 15E shows plasma Lyso-GB3 end of life reduction relative to an untreated mutant. The top four constructs were (1) AAV9-CB-SV40 GLA-66-IDT, (2) AAV9-CB-SV40_Takeda, (3) AAV9- CB7-GLA-66 (Penn), and (4) AAV9-CB-SV40-GLA-66. The results showed that GLA plasma activity correlated with lyso-GB3 substrate reduction.

[0174] FIG. 15F shows GL3 accumulation in mouse liver as a percent reduction. FIG. 15G shows GL3 accumulation in mouse heart as a percent reduction.

[0175] FIG. 15H shows ADA IgC titer for several different fusion constructs. A similar titer response for Eng hGLA with different promoters was observed (yellow arrows). The titer response was also lower than wild type with CB-SV40 promoter (green arrow), eng with “IDT” or “IDTopt” and CB-SV40 promoter (purple arrow) and eng with EFla promoter (dark blue arrow). Furthermore, SEQ ID NO. 88 was found to have no titer response at D28 (light purple arrow). APC de-targeting construct (light blue arrow) had a lower titer response compared to original eng construct + / - IDT / ITopt, promoter used, or wild type, as well as alower response than Takeda construct (red arrow). No titer response was observed with AAVhu68 or AAV5 with LP1-MAEL (both wt and eng hGLA, as shown by the blue box).

[0176] FIG. 16 shows alpha-ATBlOl ADA IgG titer at D28-EOL. Note that in PBS in GLA KO, one female mouse was likely an outlier. In wt GLA, the titer was just above baseline without S1S3 present. The presence of S1S3 lowered the titer to just below basline. One male mouse was likely an outlier for the +S1S3 construct (light blue bar - removing the outlier would make titer 0).

[0177] In eng GLA, the titers were already at 0 for D28 without SI S3 present. The titer remained at 0 for +S1S3 engineered construct.Example 6: Ancestral Sequence Reconstruction and Stability Assays of Recombinant GLA constructs

[0178] Ancestral sequence reconstruction is the process of estimating the amino acid sequence that encoded a protein in a theoretical last common ancestor (LCA) of two clades. Reconstructions of ancestral proteins have often been shown to have higher stability than extant proteins, making ancestral reconstructions interesting candidates for therapeutic biologies (Hendrikse et al., 2020; Thomson et al., 2022; Hartz et al., 2021; Zakas et al., 2017). Several mechanisms to explain this phenomenon have been proposed, including ancestral organisms being exposed to higher temperatures, ancestral organisms having less efficient protein quality control systems, a consensus bias in ancestral reconstructions, and the simple reversal of genetic drift (Trudeau et al., 2016). While the stability of reconstructed enzymes is likely due to a combination of factors, we suspect that the reversal of genetic drift plays a major role, especially in the case of more recent ancestors, such as those within the mammalian lineage.

[0179] Sequences for GLA proteins expressed by human ancestors (e.g. the LCA of all primates, the LCA of primates and rodents, etc.) were estimated using ancestral sequence reconstruction. First, a phylogenetic tree was built from GLA sequences. Then, ancestral residues at each position in the human GLA sequence were inferred using different reconstruction techniques (maximum parsimony and maximum likelihood). A selection of these “ancestral” mutations were assessed in the context of the human GLA sequence to determine whether they had beneficial effects on enzyme stability.

[0180] The reference human protein sequence for GLA, corresponding to Uniprot entry P06280, was queried in the NCBI databases using PSI-BLAST (Altschul et aL, 1997). Three iterative searches were performed, each requesting 500 hits. The final results were filtered to remove precursor proteins, low-quality proteins, shorter isoforms, and predicted proteins for which more confident sequences from the same organism were also in the sequence set. The sequences were then aligned using MAFFT (default settings: BLOSUM62 scoring matrix, gap opening penalty of 1.53, offset value of 0.0, default guide tree, FFT-NS-i alignment method) (Katoh et al., 2019). Sequences with long (>20 residues) insertions or deletions, poorly aligned sequences, and sequences with ambiguous residue calls were removed. The sequence set was then identity-filtered using CD-HIT at a threshold of 0.985 (Fu et al., 2012; Li & Godzik, 2006). The human sequence was re-added to the sequence set (having been removed during identity filtering), and the zebrafish GLA sequence was added for use as an outgroup. Signal peptides were removed from sequences (based on the mature protein starting residue in humans) to prevent spurious signal peptide correspondence from affecting the alignment and tree topology. The sequence set was re-aligned using MAFFT default settings.

[0181] A phylogenetic tree was generated from the multiple sequence alignment (MSA) using PhyML (version 20120412, Guindon et al., 2010). Sequences that were misplaced in comparison with published phylogenetic trees were deleted.

[0182] The C-termini of the sequences were poorly aligned. To remedy this and improve ancestral sequence inference, the C-terminal ends of the sequences (residues aligned with human residue 389 and later) were aligned together in MAFFT using the G-INS-1 method with an unaligned level of 0.8. The resulting alignment for this region looked slightly better than the alignment generated from whole sequences, so the re-aligned C-termini were substituted into the end of the previous MSA. This C-terminally edited MSA was used for ancestral sequence reconstruction.

[0183] Ancestral residue states were inferred from the phylogenetic tree and MSA using three methods: 1) maximum parsimony as implemented in MEGA version 11.0.13 (Tamura et al., 2021), 2) maximum likelihood marginal reconstruction as implemented in paml version 4.9 (Yang 2007), and 3) maximum likelihood joint reconstruction as implemented in paml version 4.9. For the maximum likelihood reconstructions, an empirical substitution model was used with the WAG substitution matrix, and a single substitution rate was used for all sites.

[0184] The inferred ancestral sequences at each node in the phylogenetic tree from the three inference methods were compared to each other and to the extant human sequence. Mutations relative to the modern human sequence that were found in the inferred ancestral sequences (“ancestral mutations”) were selected for experimental analysis based on their frequency of occurrence in the inferred ancestral sequences, their correspondence with mutations suggested by ProteinMPNN, their location on the protein, and their predicted effects on protein stability (determined using Prime).

[0185] In total, 107 unique ancestral mutations were found. Of these, 75 (70%) were found by all three methods. Seventy-eight ancestral mutations were assessed in experimental constructs. Ancestral mutations that have been used in late-stage GLA constructs include E74D, K237S, H406R, Q416R, and M421S. (Hendrikse N., Holmberg Larsson A., Svensson Gelius S., Kuprin S., Nordling E., & Syren P-O. (2020). “Exploring the therapeutic potential of modem and ancestral phenylalanine / tyrosine ammonia-lyases as supplementary treatment of hereditary tyrosinemia.” Scientific Reports, 10, 1315; Thomson R., Carrera-Pacheco S., & Gillam E. (2022). “Engineering functional thermostable proteins using ancestral sequence reconstruction.” JBC Reviews, 298(10), 102435. Hartz P., Strohmaier S., EL-Gayar B., Abdulmughni A., Hutter M., Hannemann F., Gillam E., & Bernhardt R. (2021). “Resurrection and characterization of ancestral CYP11A1 enzymes.” The FEBS Journal, 288, 6510-6527; Zakas P., Brown H., Knight K., Meeks S., Spencer T., Gaucher E., & Doering C. (2017). “Enhancing the pharmaceutical properties of protein drugs by ancestral sequence reconstruction.” Nature Biotechnology, 35(1), 35-37. Trudeau D., Kaltenbach M., & Tawfik D. (2016). “On the potential origins of the high stability of reconstructed ancestral proteins.” Molecular Biology and Evolution, 33(10), 2633-2641; Altschul S., Madden T., Schaffer A., Zhang J., Zhang Z„ Miller W„ & Lipman D. (1997). “Gapped BLAST and PSLBLAST: A new generation of protein database search programs.” Nucleic Acids Research, 25(17), 3389- 3402; Katoh K., Rozewicki J., & Kazunori Y. (2019). “MAFFT online service: multiple sequence alignment, interactive sequence choice and visualization.” Briefings in Bioinformatics, 20(4), 1160-1166. Fu L„ Niu B., Zhu Z„ Wu S., & Li W. (2012). “CD-HIT: accelerated for clustering the next generation sequencing data.” Bioinformatics, 28(23), 3150- 3152; Li W. & Godzik A. (2006). “Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences.” Bioinformatics, 22(13), 1658-1659; Guindon S., Dufayard J., Lefort V., Anisimova M., Hordijk W., & Gascuel O. (2010). “New algorithms andmethods to estimate maximum-likelihood phylogenies: Assessing the performance of PhyML 3.0.” Systematic Biology, 59(3), 307-321; Tamura K., Stecher G., & Kumar S. (2021). “MEGA11: Molecular Evolutionary Genetic Analysis version 11.” Molecular Biology and Evolution, 38(7), 3022-3027; Yang, Z. (2007). “PAML 4: A program package for phylogenetic analysis by maximum likelihood.” Molecular Biology and Evolution, 24(8), 1586-1591).

[0186] Sequences were collected using the PSI-BLAST program and aligned using MAFFT, CLUSTAL, and MUSCLE programs. Next, a phylogenetic tree was generated from the alignment. The ancestral sequences were inferred from the phylogenetic tree and alignment using three different methods. In the maximum parsimony method, the ancenstral state that minimizes the number of mutations was chosen. In the maximum likelihood method, the ancestral state that maximizes the likelihood of the sequence data under the selected evolution model was chosen. In the Bayesian inference method, the ancestral states were chosen based on calculating the posterior probability distribution for the phylogenetic tree under the selected evolution model.

[0187] FIG. 16 shows a maximum parsimony inference of ancestral states for alignment position 406 in the GLA MSA.

[0188] Stability of identified genetic reconstructions was tested in assays by the methods described herein. FIGS. 17-26 shows stability data at different timepoints of several GLA mutants according to some embodiments.

[0189] FIGS. 27-28 shows stability testing by measuring GLA activity at pH 7.4 over 72 hours incubation.Example 7: Fabry Gene Therapy Non Human Primate (NHP) Durability Study

[0190] Alpha-galactosidase A (GLA) is a lysosomal enzyme that hydrolyses terminal galactose residues on diverse cellular substrates. Mutations in GLA lead to Fabry disease, an X-linked lysosomal storage disorder characterized by multisystem organ involvement, including kidney and heart pathologies. Approved treatment options for Fabry patients include enzyme replacement therapy (ERT) using systemically infused recombinant human alpha-Gal A (rha-Gal A) and an orally administered molecular chaperone for patients with amenable GLA mutations. Recently, adeno-associated viral (AAV) gene replacement approaches have emerged as a promising therapeutic approach. However, limitations with both ERT and genetherapies include variable uptake into different disease-relevant tissues and short circulating half-life of GLA enzyme at neutral pH. This example presents a newly developed protein engineering platform to generate novel GLA variants with enhanced stability. Through iterative rounds of engineering, constructs demonstrating superior half-lives, enhanced expression, and higher activity in vitro were created. AAV delivery of lead engineered GLA variants in non-human primates resulted in durable long-term expression with a favorable safety profile. The data demonstrate potential solutions to overcome current limitations in developing effective therapies for treating Fabry disease, and the Amicus STABLE engineering platform holds promise for extending its application to a broader range of genetic conditions.

[0191] FIG. 29 shows serum GLA activity durability measured in non-human primates. FIG. 30 shows GLA activity in tissue biopsies of liver and two different muscles (gastrocnemius and tibialis) harvested in-life at day 30.

[0192] FIG. 31 shows serum GLA activity durability measured in non-human primates, extending past day 80 post-injection, showing that AAV9-CB-SV40 vector with second generation engineered GLAengl ll resulted in the highest GLA activity out of all compounds tested. For the compounds that used AAV capsids, the AAV dose was 2.5el3 GC / kg. The dose was the same as that used in the Penn historical samples, also shown in FIG. 31. As labeled in FIG. 31 , “GLAengl l l ” is equivalent to “AAV9-CB-SV40-hGLA l 11 ” shown in FIG. 29. In serum, the AAV9-CB-SV40 vector with second-generation engineered GLAengll l vector resulted in approximately a 250-fold increase in superphysiological GLA activity, compared to an untreated control (labeled as “baseline”). This activity was maintained through day 83 postinjection. In contrast, the hu68-GLA_Penn Tox vector was only about 60-fold over baseline.

[0193] FIG. 32 shows GLA activity in liver biopsies in non-human primates at day 30 and day 90 post-injection. AAV9-CB-S40 vector with second generation engineered GLAengl l l was also found to have the highest GLA activity in the liver biopsies out of all compounds tested. Taken at 30 days post- injection, GLAengl l l showed an approximately 19- fold increase in GLA enzyme activity over the untreated control (labeled as “baseline”). This activity dropped to 16-fold increase (corresponding to minus 14%) at 90 days post-injection. For comparison, the hu68-GLA_Penn Tox vector showed about 14-fold increase over baseline at day 30 post-injection, and only about a 5-fold increase over baseline at day 90 post- injection(corresponding to minus 67%). Thus, AAV9-CB-SV40 had higher activity and higher durability of that activity over 90 days.

[0194] The results in FIGS. 31-32 show that AAV9-CB-SV40 vector with second generation engineered GLAengl l l has high GLA activity and high durability, including at 90 days post-injection.

[0195] FIGS. 33 and 34 provide further data showing the longer-term GLA activity in the NHP treated with AAV gene therapy. The a-Gal A enzyme activities in serum showed superphy siological levels compared to the baseline, which were maintained at 70-fold over the baseline through Day 180 post- injection. Similarly, the a-Gal A enzyme activity in liver biopsies, taken at Day 30 / 90 / 180 post-injection, showed a greater than 2-fold increase in GLA activity compared to the untreated control (baseline).Example 8: In Vitro Characterization of Purified Engineered GLA VariantsExpression and Purification of Engineered GLA

[0196] Engineered GLA constructs were expressed in ExpiCHO-S cells, following manufacturer’s protocol for the transfection of cells. 6.0 x 108 cells were diluted in 100 mL of pre-warmed ExpiCHO Expression Medium and placed into a 500 mL shake flask. In separate tubes 100 ug of engineered GLA plasmid DNA was mixed with 4 mL cold OptiPRO SFM. ExpiFectamine CHO Reagent was mixed with cold OptiPRO SFM. The diluted reagents were then mixed and allowed to incubate at room temperature for 5 minutes before adding slowly to diluted cells. Flasks were placed into shaking incubator overnight at 37°C, 8.0% CO2, 80% relative humidity. On day 1 post transfection, 24 mL of ExpiCHO Feed and 0.6 mL ExpiFectamine CHO Enhancer was added to each flask. Clarified media was harvested on Day 6 post transfection using centrifugation and immediately filtered via 0.2um PES filter. Clarified media was frozen immediately at -80°C until purification.

[0197] Purification of engineered GLA occurred using a two-step purification process. Clarified media was thawed at 4°C the day before purification. Initial capture of GLA was performed through Concavalin A capture. HiTrap Concavalin A 4B (Cytiva), 1 mL, pre- packed column was equilibrated in Equilibration Buffer containing 50 mM sodium phosphate, pH 6.8, 0.1 M NaCl, 1 mM MgC12, 1 mM CaC12, and 1 mM MnC12. Clarified media wasloaded onto the column and washed using Equilibration Buffer. GLA was eluted from column using Elution Buffer (50 mM sodium phosphate pH 6.8, 0.1 M NaCl, 0.9 M methyl-alpha-D- glucopyranoside, 0.9 methyl-alpha-D mannopyranoside). Elution fractions containing GLA were pooled and concentrated to be further purified by size exclusion chromatography. The Superdex 200 Increase 10 300 (Cytiva) column was equilibrated in 50 mM sodium phosphate, pH 6.8, 160 mM NaCl. One milliliter of the concentrated Con A purified GLA eluate was loaded onto the column and eluted with 3 column volumes of the equilibration buffer. Elution fractions were evaluated by SDS-PAGE and Western Blot. Fractions containing GLA were pooled and protein concentration determined by A280.Uncle Thermal Stability

[0198] Uncle was used to characterize GLA thermal stability by measuring the intrinsic fluorescence of tryptophan, tyrosine and phenylalanine exposed as proteins undergo conformational changes from the folded to the unfolded state. Tm (°C) is defined as the temperature at which half the protein has unfolded. Static light scattering (SLS) at wavelengths 266 and 476 nm is monitored concurrently to detect protein aggregation during a thermal ramp from 15 °C to 95 °C using the Uncle instrument (Unchained labs, Pleasanton, CA).

[0199] A working solution of 1 p M of GLA and 100 pM of migalastat were prepared in sodium acetate, pH 5.2 and PBS, pH 7.4 buffer. Total of 9 pL of each sample was loaded into the cuvette of the Unis, ensuring proper sealing to prevent evaporation during the thermal ramp. The temperature range was defined from 15-95 °C, and the ramp rate was 1 °C / minute. Uncle software was used to monitor changes in fluorescence intensity and light scattering as the temperature increases, allowing for the determination of melting temperature (Tm) and aggregation (Tagg). Tm was determined by analyzing the inflection point of the fluorescence intensity curve using Uncle Analysis V6.01. Tagg was detected by using SLS. As protein begins to aggregate, they scatter more light, and this increase in scatter light tracked to determine the Tagg.Enzyme Activity Over Temperature Range

[0200] The T50 assay is a supplemental thermal stability measurement using enzyme activity as a marker of the test protein’s stability. Each of the experimental engineered GLA variants were tested in the T50 assay as follows: 1.5 pg of each enzyme variant was incubated in PBS at a temperature between 39°C - 79°C for 10 minutes in a Bio-Rad Thermocycler. From each of these tubes, duplicate samples containing 1 ng each of the enzyme were transferred to black 96-well Costar plates (Corning Life Sciences, Corning, NY) and assayed for GLA enzyme activity. To each sample, 40 pl of substrate (5 mM 4MU-a-D- galactopyranoside) in Reaction Buffer (0.1 M Citrate Phosphate Buffer, pH 4.6) were added and the plates were incubated at 37°C for 35 minutes. The reactions were stopped by the addition of 125 pl stop buffer (0.4 M glycine, pH 10.8). Fluorescence at 460 nm was read on a SpectraMax M2 (Molecular Devices) instrument with excitation at 355 nm. Raw fluorescence counts were background subtracted (defined by buffers only) and converted to 4-MU concentration by comparing to a 4-MU standard curve ranging from 118 nM to 15 pM made using linear regression following log-log transformation. The GLA activity in each reaction was calculated and expressed as nanomoles of released 4-MU per mL of sample per hour (nmol / mL / hr). These values were analyzed by a dose-response (inhibitor) algorithm using 4PL regression to determine the T50 for each engineered GLA variant. T50 values are reported as degrees Celsius (°C).Stability in PBS pH 7.4

[0201] Fabrazyme, purified WT GLA and engineered GLA proteins were diluted with PBS pH 7.4 to .25 ug / ml or to .33 ug / ml (GLA26, GLA62. GLA111, GLA142). Dilutions were made in triplicate and 264ul of each dilution was loaded in a 96-well plate and incubated at 37°C while shaking at 300 RPM on a Thermomixer C plate shaker (Eppendorf).

[0202] At 0 hr, 1 hr, 2 hr and 4 hr time points, 4MU a-galactosidase activity was measured in triplicate for each sample by mixing lOuL of sample and 40uL of reaction buffer (5mM 4-MU-a-D-Galactopyranoside hydrate, 27mM Citric acid / 46 mM Sodium Phosphate buffer pH 4.6). Reactions were incubated at 37°C for Ihr before addition of 125uL of IM Glycine pH 10.5. Plates were read on a Spectramax M2 (Molecular Devices) at the following parameters: Excitation 370nm, Auto cutoff 455nM, Emission 460 nM. 4MU RFUs were converted to nmol 4MU released / hr by measuring alongside a 4MU standard curve. Data wasplotted as relative activity to 0 hr time-point and the half-life was determined using a one- phase decay model fit with Graphpad Prism.

[0203] Enzyme Kinetics

[0204] For Km and Vmax, GLA activity was measured using constant GLA enzyme concentration and varying substrate 4MU-a-D-galactopyranoside (4MU-Gal) substrate with an 8-point curve. 1.5 ng of enzyme was diluted in GLA assay buffer (0. IM Citrate Phosphate Buffer, pH 4.6) to a total volume of 25 uL / well. Then, 25 uL of appropriately diluted 4MU-Gal substrate at various concentrations was added to each well and incubated for 1 hour at 37°C. The highest substrate concentration used was 15mM. After the incubation, the reaction was stopped with 0.1M Glycine, pH 10.8.

[0205] To measure Specific Activity, GLA activity was assessed using a constant concentration of 4MU-Gal substrate along with an 8-point curve of GLA enzyme concentrations. Specifically, various enzyme concentrations starting from 80ng / mL / well were incubated with 5mM substrate for 1 hour at 37°C. Both the enzyme and substrate were diluted in GLA assay buffer. Following incubation, the reaction was terminated with 0. IM Glycine at pH 10.8.

[0206] Subsequently, the plates were analyzed using the SpectraMax M2 (Molecular Devices) at an emission wavelength of 460 nm with excitation at 370 nm. The raw fluorescence units were background-subtracted and converted to 4-MU concentration via linear regression relative to a 4-MU standard curve. The specific activity of each variant was expressed as mmol 4MU / mg / hr, and the Michaelis-Menten equation model was applied to determine the Km (mM) and Vmax (mmol 4MU / mg / hr).Results

[0207] Table 7 below summarizes the in vitro results from this set of experiments:

[0208] Table 7 In Vitro Characterization of Purified Engineered GLA Variants

[0209] As can be seen from Table 7, each of the tested engineered variants had increased in vitro stability compared to wild-type GLA as measured by Tm, T50 and PBS halflife. Moreover, some engineered GLA variants (167, 168, 171 and 174) had improved enzyme kinetics compared to wild-type GLA as measured by Km, Vmax and / or specific activity.

[0210] Example 9: In Vivo Characterization of Purified Engineered GLA VariantsAAV9-FABRY GENE THERAPY: Mouse Efficacy Study.

[0211] This was a short-term efficacy study in Fabry KO mice. The study details are provided below:

[0212] Animal Model: Fabry model Gia KO (n=5 males / group)

[0213] Age: 12 weeks at AAV dosing

[0214] AAV vector: Capsid: AAV9; Promoter: ubiquitous

[0215] AAV transgenes: 1 wt and 5 GLAeng constructs

[0216] Dose / Route: Tail vein IV: 2ell vg / kg

[0217] Duration: 28-day PoC study

[0218] Weekly blood draws (Day 0, 7, 14, 21) and necropsy at Day 28 with tissue harvest, biochemical analysis, bioanalytical analysis and histochemical analysis.

[0219] Priority readouts: Substrate reduction Iyso-Gb3 / GL3, GLA enzyme activity and anti-GLA titer.

[0220] Table 8 shows the efficacy study treatment groups.

[0221] Table 8: Treatment groups of the efficacy study.

[0222] Tissue Handling and Processing

[0223] Serum and plasma samples were stored in a freezer with temperature set at - 80°C. On the day of analysis, samples were taken out of the -80°C freezer and thawed in a 4°C fridge. Upon thawing, samples were centrifuged at 2,300 x g for 1 min at 4°C as needed to bring the total volume down from the cap and wall of the tubes. Samples were kept on ice, and the total sample volume of each sample was estimated by pipetting and recorded (to understand availability of samples for subsequent analyses and to prioritize key assays if availability is not sufficient for all intended analyses). Then, 5 p L of each sample was pipetted and diluted into245 pL of pre-cooled Fabry Lysis Buffer (0.1M Citrate Phosphate Buffer (CP), pH 4.6) for GLA activity assay, and all remaining volumes were returned to the -80°C freezer immediately. Tissue samples received were stored in a freezer with temperature set at -80°C. For processing, frozen tissues (heart, liver, kidney) were sub-aliquoted on dry ice, then the resulting sub-aliquot thawed in 4°C refrigerator or on ice. The whole piece of thawed tissue was chopped and mixed for homogeneity on ice, and then aliquoted to support various analyses, with weights of aliquots recorded. Unused aliquots and leftover stock samples were returned to -80°C freezer immediately after. On the day of analysis, tissue aliquots werehomogenized in lOx volume of H2O using a Bead homogenizer (Cryolys Evolution, Bertin Corp, Rockville MD), and then centrifuged for 10 minutes at 13,000 x g at 4°C. The GLA enzyme activity and protein concentration of the supernatant was measured, and the rest of supernatant was stored at -80°C.

[0224] Protein Concentration Determination of Tissue Lysates

[0225] A BCA Protein Assay (Pierce, Rockford, IL) was conducted using the tissue lysates, by adding 25 L of lysate to wells in duplicates, as well as adding 2 mg / mL bovine serum albumin to an additional 4 wells then serially diluting to create the protein standard curve which was used to determine total protein concentration in the lysates. BCA working reagent is then added and the plate read at A55O for 1 second on a plate reader.

[0226] GLA Activity Determination of Tissue Lysates or Blood

[0227] Properly diluted tissue lysates or blood samples (serum or plasma) of 20 LIL were added to 50 pL of substrate (12 mM 4MU-a-D-galactopyranoside + 90 mM N-acetyl-D- galactosamine) in Reaction Buffer (0.1 M Citrate Phosphate Buffer, pH 4.6) containing 2% DMSO in Costar 96 well black-wall clear-bottom plate (Corning Life Sciences, Corning, NY), and the plate was then incubated for 3 hours at 37 °C. Reactions were stopped by addition of 70 pL 0.4 M glycine, pH 10.8. Fluorescence at 460 nm was read on a Victor3 plate reader (Perkin Elmer, Waltham, MA) with excitation at 355 nm. Raw fluorescence counts were background subtracted (defined by buffers only) and converted to 4-MU concentration by comparing to a 4-MU standard curve ranging from 118 nM to 15 pM made using linear regression following log-log transformation. The GLA activity in serum or plasma was calculated and expressed as nanomoles of released 4-MU per mL of sample per hour (nmol / mL / hr). The GLA activity in tissue sample was calculated by normalizing the 4-MU released to the protein concentration and expressed as nanomoles of released 4-MU per milligram protein per hour (nmol / mg protein / hr).

[0228] Plasma Lyso-Gb3 Substrate Analysis

[0229] A stock aliquot of 10 mg Iyso-Gb3 was reconstituted to 1 mL in 2:1 CHC13:MeOH for stock solution at a concentration of 10 mg / mL. A 400 pL aliquot of stock solution at 10 mg / mL was diluted to 2 mL in 1:1 MeOH:DMSO for stock A at a concentration of 2 mg / mL. A 20 pL aliquot of stock A at 2 mg / mL was diluted to 2 mL in 1 : 1 MeOH:DMSO for stock B at a concentration of 0.02 mg / mL. Stock B was used to prepare standard calibration samples in 1:1 MeOH:DMSO at ten concentration levels and quality control samples in 1:1MeOH:DMSO at six concentration levels. A 50 pL aliquot of calibration standards in duplicate, quality control standards in triplicate, and plasma samples were transferred into individual wells of a 96-well plate. A 50 qL aliquot of internal standard working solution (75 ng / mL 13C61yso-Gb3 in 1: 1 MeOH:DMSO) was added to each well. Samples were precipitated with the addition of 1-mL MeOH and vortexed for 1 -minute, followed by the addition of 0.5 mL 1 N HC1. Samples for vortexed for 1 minute, sonicated for 10 minutes, and shaken for 45 minutes. The sample plate was then centrifuged for 10 minutes at 4000 rpm. The Oasis MCX (30 pm, 30 mg, Waters) SPE plate was conditioned with 1 mL MeOH and equilibrated with 1 mL Milli-Q water. A 1.5 mL aliquot of the sample supernatant was loaded onto the conditioned SPE plate. Sample wells were washed with 2x1 mL 0.1-N HC1, followed by 2x1 mL MeOH. A 96-well collection plate was placed under the SPE plate and samples were eluted with 1.25 mL 5% NH40H in MeOH and dried under nitrogen at 40 °C. Once dry, samples were reconstituted in 50-pL 1: 1 MeOH:DMSO followed by 125 pL mobile phase B (0.5% formic acid, 5 mM ammonium formate in 95:5 ACN:H2O). Samples were analyzed by injecting 10 pL onto the LC-MS / MS instrument.

[0230] Tissue GL-3 Substrate Analysis

[0231] A stock aliquot of 10 mg GL-3 was reconstituted to 2-mL in 2: 1 CHC13:MeOH for stock solution at a concentration of 5 mg / mL. An 800 pL aliquot of stock solution at 5 mg / mL was diluted to 4 mL in 2: 1 CHC13:MeOH for a working stock solution of 1 mg / mL. The working stock solution at a concentration of 1 mg / mL was used to prepare standard calibration samples in MeOH at eight concentration levels and quality controls sam-ples in MeOH at five concentration levels. A 50 pL aliquot of calibration standards in duplicate, quality control standards in triplicate, and tissue samples were transferred into individual wells of a 96-well plate. A 300 pL aliquot of internal standard working solution (2500 ng / mL lactosylceramide in MeOH) was added to each well. Samples were precipitated with the addition of 300 pL MeOH followed by 120 pL Optima H2O. The sample plate was vor-texed for 1 minute and centrifuged for 10 minutes at 4000 rpm. The Strata C18-E SPE plate (55 pm, 25 mg, Phe-nomenex) was conditioned with 1 mL MeOH and equilibrated with 1 mL 6:1 Me0H:H20. A 700 pL aliquot of the sample supernatant was loaded onto the preconditioned SPE plate and samples were washed with 1 mL 5% MeOH in H2O. A 96-well collection plate was placed under the SPE plate and samples were eluted with 2x500 pL MeOH and driedunder nitrogen at 40 °C. Once dry, samples were reconstituted in 150 pL MeOH. Samples were analyzed by injecting 10 pL onto the LC-MS / MS instrument.

[0232] Results

[0233] FIG. 35 shows plasma GLA activity in Gia KO mice treated with AAV gene therapy. The a-Gal A enzyme activities in serum showed superphysiological levels compared to the WT-PBS and all three engineered variants resulted in superior GLA enzyme activity compared to GLA wildtype control.

[0234] FIGS. 36-38 show tissue (liver, heart and kidney) GLA activity in Gia KO mice treated with AAV gene therapy. The a-Gal A enzyme activities in tissue showed superphysiological levels compared to the WT-PBS and all three engineered variants resulted in superior GLA enzyme activity compared to GLA wildtype control.

[0235] FIGS. 39 and 40 show plasma substrate reduction in Gia KO mice treated with AAV gene therapy. All vectors demonstrated significant reduction of Iyso-Gb3 substrate in serum over time compared to KO-PBS and all engineered variants resulted in superior substrate reduction compared to GLA wildtype control.

[0236] FIGS. 41-43 show tissue (liver, heart and kidney) substrate reduction in Gia KO mice treated with AAV gene therapy. All vectors demonstrated significant reduction of GL3 substrate in liver and heart compared to KO-PBS and all engineered variants resulted in superior substrate reduction compared to GLA wildtype control. In kidney, which is a difficult tissue to target with AAV, only variant GLAengl ll showed a mild reduction of GL-3 substrate.

[0237] Reference throughout this specification to "one embodiment," "certain embodiments," "various embodiments," "one or more embodiments" or "an embodiment" means that a particular feature, structure, material, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of the phrases such as "in one or more embodiments," "in certain embodiments," "in various embodiments," "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, structures, materials, or characteristics may be combined in any suitable manner in one or more embodiments.

[0238] Although the disclosure herein provided a description with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the disclosure. It will be apparent to those skilled in the art that various modifications and variations can be made to the present disclosure without departing from the spirit and scope thereof. Thus, it is intended that the present disclosure include modifications and variations that are within the scope of the appended claims and their equivalents.SEQUENCE LISTING> SEQ ID NO. 1 GLA A (T41I, L45S, H46D, E48V, F50Y, M51Q, C52V, Q57E, E58N, E59Q, D61E, S62T, I64R, S65T, K67Q, F69Y, M70K, E71R, M72Q, V77K, S78E, E79K, W81F, K82L, D83E, E87K, Y88L, C90M, C94G, M96W, Q99S, E103S, R1O5N, L106R, Q111V, H115N, 1117L, R118A, Q119A, N122K, K127L, 1133V, A135T, F145G, I154L, T158Q, D161N, W162D, L166A, F169L, Y184H, H 186R, S2O1H, C202T, M208V, W209L, F211Y, Q212K, C223A, H225S, W226A, N228I, F229C, A230E, D233E, W236F, K237E, 1239 L, K240L, S241A, I242R, L243I, W245F, T246Y, S247A, F248A, E251D, R252D, D255S, G261F, N263I, P265L, L268S, V269I, F273Y, W277R, N278E, Q279E, Q280E, T282L, Q283E, M284A, W287K, M290A, A291K, A292N, F295L, M296L, R3O1S, H302N, P305E, Q306E, A307S, Q312L, K314P, D315E, V316L, I317L, Q321R, K326V, Q327A, Y329R, Q330L, R332W, Q333E, N336G, E338R, V339L, R342K, P343E, L347G, A348R, W349V, A352L, M353V, I354A, R356L, Q357R, E358R, I359R, G361T, P362A, R363Q, S364P, Y365F, T366S, 1367V, A368P, A370R, K374G, V376K, C378F, C382G, F383R, 1384V, V390E, K391R, K393E, F396V, Y397F, E398G, W399Y, T400D, S401D, R402T, R404T, S405T, H406R, 1407V, N408A, T410G, T412A, V413R, L415Y, Q416L, L417V, T420V, M421E, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPIMGWSDWVRYQVN LDCENQPETCRTEQL YKRQAELMKEKGFLEAGYKLLMIDDGWWAPSRDSSGN RQADPVRFPNGLAALAKYVHSLGLKLGVYTDV GN KTCAGGPGSFGYYDLDAQQFAN DGVDALKLDGCYCDSLEN LADGHKRMSLALNRTGRSIVYHTEWPL YVLPYKKPNYTEIRQYANSARICEDIEDSFESLLARIDFYAANQDDIVSVAGPGFWIDLDMSIIGNYGLSREEE VLEAALKAIAKNPLLLSN DLSNISEESKALLLDPELLAINRDPLGVAGRLLWEGDGFRLWEKELSGGRVAVLV ANLRRRGTAQPFSVPVRSLGGGKAFN PAGRVTQLLPERRELGVFGYDDTLTTRVAPGGARLYLVENVEAM SLKDLL> SEQ ID NO. 2 GLA A (T41V, L45S, H46D, E48V, F50Y, M51L, C52N, L54Q, Q57E, E58S, S62T, I64R, S65T, F69Y, M70Y, E71D, M72Q, E74D, E79K, W81F, D83E, Y88L, C90M, C94G, M96Q, Q99S, R100Y, E103N, L106R, Q111V, H115N, R118K, Q119A, N 122D, 1133V, A135T, F145E, Y151R, I154L, T158Q, W162D, L166A, F169V, Y184H, H186K, S201H, C202T, M208V, W209L, F211Y, Q212R, C223A, H225S, W226A, N228L, A230E, D233E, W236R, K237E, I239L, K240L, S241A, I242R, W245F, T246F, S247A, F248A, E251A, R252E, G261F, W262F, N263V, P265L, L268S, V269I, 1270V, F273Y, W277R, N278E, Q279E, Q280E, T282L, Q283E, M284V, A285G,L286F, W287K, M290A, A291K, A292N, F295L, M296L, R301S, H302N, P305E, Q306W, K308R, A309E, Q312L, D313N, N320R, Q321T, K326V, Q327A, Y329R, Q330L, R332W, Q333E, N336G, E338R, V339L, R342Q, L347G, A348Q, W349T, A352L, M353V, I354L, R356L, Q357E, E358R, I359R, S364T, Y365F, T366S, 1367V, A368P, A370S, K374G, V376Q, F383T, 1384V, V390E, K391R, K393A, F396T, Y397F, E398G, W399Y, T400D, S401D, R402T, R404T, S405L, H406R, 1407V, N408P, T410G, T412A, Q416L, E418T, M421D, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPVMGWSDWVRYLN NQDCESEPDTCRTEKL YYDQADLMVSKGFKEAGYELLMIDDGWQAPSYDSNGRRQADPVRFPNGIKALADYVHSKGLKLGVYTDV GN KTCAGEPGSFGRYDLDAQQFADDGVDALKVDGCYCDSLEN LADGHKKMSLALNRTGRSIVYHTEWPL YVLPYRKPNYTEIRQYANSARLFEDIEDSRESLLARLDFFAANQAEIVDVAGPGFFVDLDMSIVGNYGLSREE EVLEVGFKAIAKNPLLLSNDLSN ISEWARELLLN KDVIAIRTDPLGVAGRLLWEGDGFRLWEQPLSGGQTAV LVLN LERRGGPRTFSVPVSSLGGGQACN PACTVTQLLPERRALGTFGYDDTLTLRVPPGGAVLLLLTNTDA MSLKDLL> SEQ ID NO. 3 GLA A(M42A, L45S, H46N, E48V, F50Y, M51G, L54R, C56A, Q57A, E58A, E59N, S62T, C63A, 164V, S65T, K67A, F69Y, M70R, E71R, M72Q, E74D, E79Q, W81F, K82L, D83E, E87T, Y88L, C90M, I91L, M96Q, P98S, Q99S, E103S, R105N, QUIT, R112L, H115N, I117L, R118A, Q119A, N122A, K127L, I133A, A135I, F145G, Y151H, I154Q, D155T, Q157A, T158Q, F159L, D161A, W162A, L166A, F169A, Y184H, H186R, M187L, S201H, C202T, M208V, W209R, P210G, F211Y, Q212R, C223A, H225S, W226A, N228I, F229C, A230E, I232R, D233T, K237E, I239L, K240L, S241A, I242R, W245Y, T246Y, F248A, E251A, R252E, V254R, G261F, W262F, N263V, P265L, L268S, V269I, 1270V, N272R, F273Y, W277R, N278E, Q280E, T282A, Q283E, M284V, L286F, W287N, M290A, A291K, A292N, F295L, M296L, R301S, H302D, P305E, Q306E, K308R, Q312L, K314P, D315E, I317L, N320R, Q321W, K326R, Q327A, Y329R, Q330L, R332W, Q333E, N336G, E338R, V339L, P343E, L347G, A348R, W349V, A352L, M353V, I354L, R356L, Q357V, E358R, I359R, G361T, P362A, S364P, Y365F, T366S, 1367V, A368P, V369L, A370S, K374G, V376R, C378G, N379R, C382A, F383R, 1384V, V390E, K391R, K393E, F396V, Y397F, E398G, W399A, T400G, S401D, R402T, R404T, S405V, H406R, 1407V, N408P, T410G, T412A, Q416L, N419R, T420V, M421E, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTAGWSNWVRYGCN RDAAANPDTAVTEAL YRRQADLMVSQGFLEAGYTLLMLDDCWQASSRDSSGNLQADPTLFPNGLAALAAYVHSLGLKLGAYIDV GN KTCAGGPGSFGHYDQTAAQLAAAGVDALKADGCYCDSLEN LADGHKRLSLALNRTGRSIVYHTEWPL YVRGYRKPNYTEIRQYANSARICEDRTDSWESLLARLDYYSANQAEIRDVAGPGFFVDLDMSIVGRYGLSRE QEVAEVAFNAIAKN PLLLSN DLSDISEEARALLLDPEVLAIRWDPLGRAGRLLWEGDGFRLWERELSGGRV AVLVLNLVRRGTARPFSVPLSSLGGGRAGRPAARVTQLLPERRELGVFGAGDTLTVRVPPGGAVLLLLERVE AMSLKDLL> SEQ ID NO. 4 GLA A(M42A, L45S, H46N, E48V, F50Y, M51G, L54R, C56A, Q57A, E58A, E59N, S62T, C63A, 164V, S65T, K67A, F69Y, M70R, E71R, M72Q, E74D, E79Q, W81F, K82L, D83E, E87T, Y88L, C90M, I91L, M96Q, P98S, Q99S, E103S, R105N, QUIT, R112L, H115N, I117L, R118A, Q119A, N122A, K127L, I133A, A135I, F145G, Y151H, I154Q, D155T, Q157A, T158Q, F159L, D161A, W162A, L166A, F169A, Y184H, H186R, M187L, S201H, C202T, M208V, W209R, P210G, F211Y, Q212R, C223A, H225S, W226A, N228I, F229C, A230E, I232R, D233T, K237E,1239 L, K240L, S241A, I242R, W245Y, T246Y, F248A, E251A, R252E, V254R, G261F, W262F, N263V, P265L, L268S, V269I, 1270V, N272R, F273Y, W277R, N278E, Q280E, T282A, Q283E, M284V, L286F, W287N, M290A, A291K, A292N, F295L, M296L, R301S, H302D, P305E, Q306E, K308R, Q312L, K314P, D315E, I317L, N320R, Q321W, K326R, Q327A, Y329R, Q330L, R332W, Q333E, N336G, E338R, V339L, P343E, L347G, A348R, W349V, A352L, M353V, I354L, R356L, Q357V, E358R, I359R, G361T, P362A, S364P, Y365F, T366S, 1367V, A368P, V369L, A370S, K374G, V376R, C378G, N379R, C382A, F383R, 1384V, V390E, K391R, K393E, F396V, Y397F, E398G, W399A, T400G, S401D, R402T, R404T, S405V, H406R, 1407V, N408P, T410G, T412A, Q416L, N419R, T420V, M421E, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTAGWSNWVRYGCN RDAAANPDTAVTEAL YRRQADLMVSQGFLEAGYTLLMLDDCWQASSRDSSGNLQADPTLFPNGLAALAAYVHSLGLKLGAYIDV GN KTCAGGPGSFGHYDQTAAQLAAAGVDALKADGCYCDSLEN LADGHKRLSLALNRTGRSIVYHTEWPL YVRGYRKPNYTEIRQYANSARICEDRTDSWESLLARLDYYSANQAEIRDVAGPGFFVDLDMSIVGRYGLSRE QEVAEVAFNAIAKN PLLLSN DLSDISEEARALLLDPEVLAIRWDPLGRAGRLLWEGDGFRLWERELSGGRV AVLVLNLVRRGTARPFSVPLSSLGGGRAGRPAARVTQLLPERRELGVFGAGDTLTVRVPPGGAVLLLLERVE AMSLKDLL> SEQ ID NO. 5 GLA A( D233T, F273Y, 1359V, N408P)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADITDSWKSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNYG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQEVGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIPPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 6 GLA A( D233T, F273Y, I359R, N408P)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADITDSWKSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNYG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSH I PPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 7 GLA A( D233T, D234M, F273Y, I359R, N408P)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADITMSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNY GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIPPTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 8 GLA A( D233T, I359R, N408P)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADITDSWKSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSH I PPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 9 GLA A(T41P, F50Y, Q57E, E58T, E59N, D61E, S62N, 164V, S65T, K67E, F69Y, M70K, E71K, M72D, E74D, E79K, W81F, D83A, E87T, Y88I, C90M, M96L, P98S, Q99S, E103N, R105N, L106R, Q111V, H115N, I117L, R118K, Q119A, N122D, 1133V, I154L, Q157K, T158Q, F159W, L166A, A181R, D182K, Y184A, H186E, V199Y, S201H, C202T, M208V, W209L, P210G, F211Y, Q212E, C223A, H225S, W226A, N228L, D233K, W236A, K237E, I239L, K240D, S241A, I242R, W245F, T246Y, S247D, F248A, E251D, R252E, D255S, G261F, W262F, N263V, P265L, L268S, V269I, 1270V, N272R, F273Y, W277R, N278E, Q279E, T282R, Q283E, M284V, A285G, L286A, W287K, M290Y, A291K, A292N, L294Y, M296I, N298T, R301A, H302D, Q306E, K308L, Q312T, D313N, K314P, D315E, I317L, N320R, Q321T, Q327A, Y329R, Q330L, R332W, Q333R, N336G, E338R, V339L, R342Q, P343E, L347G, A348R, W349T, A352L, M353V, R356N, Q357E, E358R, R363K, S364K, Y365F, 1367V, A368P, A370S, K374G, V376K, F383Q, 1384V, V390E, K391R, R392K, K393A, F396I, Y397F, E398G, W399Y, T400D, S401D, R402S, R404T, S405V, H406Y, 1407V, N408P, T410G, T412A, V413R, L415F, Q416L, E418T, T420V, M421D, Q422A, M423H, S424V, L425I, D427R, L429T)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPPMGWLHWERYMCN LDCETNPENCVTEEL YKKDADLMVSKGFKAAGYTILM IDDCWLASSRDSNGNRQADPVRFPNGLKALADYVHSKGLKLGVYADV GN KTCAGFPGSFGYYDLDAKQWADWGVDALKFDGCYCDSLEN LRKGAKEMSLALN RTGRSIYYHTEWPL YVLGYEKPNYTEIRQYANSARLFADIKDSAESLDARLDFYDANQDEIVSVAGPGFFVDLDMSIVGRYGLSRE EQVREVGAKAIYKN PYFISTDLADISPEALALLTNPEVLAIRTDPLGKAGRLLWRGDGFRLWEQELSGGRTA VLVIN NERIGGPKKFTVPVSSLGGGKACN PACQVTQLLPERKALGIFGYDDSLTVYVPPGGARLFLLTNVDA HVIKRLTL> SEQ ID NO. 10 GLA A(T41P, R49Y, F50Y, Q57E, E58N, E59N, D61E, S62L, K67A, F69Y, M70K, E71K, M72Q, E74D, E79Q, W81F, D83E, E87Q, Y88L, C90M, M96L, Q99S, R100F, E103N, R105Y, L106P, Q111V, R112L, H115N, I117L, R118A, Q119A, N 122K, K127L, 1133V, Y151R, I154K, D155F, Q157K, T158W, F159L, W162A, L166A, A181R, D182R, Y184T, H186E, V199I, S201H, C202T, M208V, W209L, F211Y, Q212E, C223A, H225A, W226A, N228L, D233E, W236L, K237E, I239L, K240L, S241A, I242R, L243I, W245F, T246Y, F248A, E251A, R252D, I253W, G261F, N263I, P265L, L268S, V269I, 1270V, N272R, F273Y, W277R, N278E, Q279E, Q280E, T282R, Q283E, M284I, W287K, M290F, A291R, A292N, M296L, N298T, R301S, H302N, P305E, Q306W, A307S, Q312T, D313H, K314P, D315A, I317L, N320R, K326R, Q327A, Y329R, Q330L,R332E, Q333R, N336G, V339L, L347G, A348Q, A352L, M353V, I354T, R356N, Q357I, E358R, G361T, P362K, R363K, S364E, T366S, 1367V, A368P, A370S, K374G, V376Q, A377V, F383R, 1384V, T385R, V390E, K391E, K393E, F396V, Y397F, E398N, W399A, T400S, S401D, R402T, R404T, S405V, H406R, 1407V, N408P, T410G, T412A, L415F, Q416L, E418T, T420V, M421N, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPPMGWLHWEYYMCN LDCENNPELCISEALY KKQADLMVSQGFKEAGYQLLMIDDCWLAPSFDSNGYPQADPVLFPNGLAALAKYVHSLGLKLGVYADVG NKTCAGFPGSFGRYDKFAKWLADAGVDALKFDGCYCDSLEN LRRGTKEMSLALN RTGRSIIYHTEWPLYVL PYEKPNYTEIRQYANAARLFADIEDSLESLLARIDFYSANQADWVDVAGPGFWIDLDMSIVGRYGLSREEE VREIALKAIFRNPLFLSTDLSN ISEWSKALLTHPAVLAIRQDPLGRAGRLLERGDGFELWERPLSGGQWAVL VTN NIRIGTKKEYSVPVSSLGGGQVCN PACRVRQLLPEERELGVFNASDTLTVRVPPGGAVLFLLTNVNAM SLKDLL> SEQ ID NO. 11 GLA A(T41L, F50Y, Q57E, E58N, S62T, 164V, S65T, K67E, F69Y, M70K, E71R, M72D, E74D, E79K, W81F, D83E, Y88L, C90M, M96W, Q99S, R100F, L106R, Q111V, H 115N, I117L, R118K, Q119A, N 122D, 1133V, Y151R, D153E, I154L, Q157K, T158Y, W162C, L166A, L177I, A181I, D182N, Y184S, H186N, M 187I, V199Y, S201H, C202S, M208I, W209E, F211Y, Q212K, C223W, H225S, W226A, N228L, D233K, W236R, K237E, I239L, K240L, S241A, I242R, L243A, W245Y, T246Y, S247A, F248A, E251D, R252D, G261F, N263V, P265L, L268S, V269I, 1270V, N272R, F273Y, W277R, N278E, Q279E, Q280E, T282F, Q283E, M284I, W287K, M290F, A291K, A292N, L294M, F295L, M296L, N298T, R301S, H302N, P305E, Q306E, K308R, Q312L, K314P, I317L, Q327A, Y329R, Q330L, R332W, Q333R, N336G, E338A, V339I, R342K, L347G, A348R, W349V, A352V, M353V, I354A, R356L, Q357I, E358D, S364T, Y365L, I367F, A368P, A370S, K374G, V376K, F383R, 1384V, T385E, Q386E, V390E, K391R, K393E, F396V, Y397F, E398G, W399A, T400D, S401D, R402T, R404T, S405V, H406R, 1407V, N408A, T410G, T412A, V413R, L415F, Q416L, E418T, N419P, T420V, M421E, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPLMGWLHWERYMCN LDCENEPDTCVTEEL YKRDADLMVSKGFKEAGYELLMIDDCWWAPSFDSEGRRQADPVRFPNGLKALADYVHSKGLKLGVYAD VGN KTCAGFPGSFGRYELDAKYFADCGVDALKFDGCYCDSIEN LINGSKN ISLALNRTGRSIYYHSEWPLYIE PYKKPNYTEIRQYWNSARLFADIKDSRESLLARADYYAANQDDIVDVAGPGFWVDLDMSIVGRYGLSREE EVFEIALKAIFKNPMLLSTDLSN ISEEARALLLDPDVLAINQDPLGKAGRLLWRGDGFAIWEKPLSGGRVAV VVANLIDIGGPRTLTFPVSSLGGGKACNPACRVEELLPERRELGVFGADDTLTVRVAPGGARLFLLTPVEAM SLKDLL> SEQ ID NO. 12 GLA A(T41P, F50Y, Q57E, E58N, E59N, D61E, S62T, K67E, F69Y, M70R, E71R, M72D, E79K, W81F, K82L, D83E, E87K, Y88L, C90M, M96L, Q99S, R100F, S102A, E103N, L106P, A108P, D109H, Q111E, R112L, H115D, I117L, R118A, Q119A, N 122E, V124I, K127L, K130L, 1133V, Y151R, I154L, D155W, T158W, F159L, D161E, W162Q, L166G, L177I, A181I, D182N, Y184A, H186Q, V199Y, S201H, C202S, M208I, W209L, F211H, Q212R, C223A, H225S, W226A, N228L, D233E, W236L, K237D, 1239V, K240L, S241A, I242R, L243I, W245F, T246Y, S247D, F248A, E251A, R252D, G261F, W262F, N263V, P265L, L268S, V269I, 1270V, F273Y, W277L, N278E, Q279E, T282R, M284V, L286Y, W287Q, M290A, A291K, A292N, L294F, F295Y, M296L, R301A, H302D, Q306E, A307S, K308L, Q312T, D313N, K314P, I317L, N320R, Q327A,Y329R, Q330L, L331V, R332W, Q333R, N336G, E338R, V339L, R342K, P343E, L347G, A348K, W349V, A352L, M353V, I354T, R356D, Q357I, E358R, G361T, S364K, Y365F, T366S, I367F, A368P, V369A, A370S, L372I, K374G, V376K, A377V, C378F, C382G, F383R, 1384V, V390E, K391R, R392E, K393E, F396V, Y397F, E398G, W399K, T400D, S401D, R402T, L403I, R404T, S405V, H406S, 1407V, N408P, T410Y, T412A, V413R, L415F, Q416L, L417V, T420V, M421E, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPPMGWLHWERYMCN LDCEN N PETCISEELY RRDAELMVSKGFLEAGYKLLM IDDCWLAPSFDANGRPQPHPELFPDGLAALAEYIHSLGLLLGVYADVGN KTCAGFPGSFGRYDLWAQWLAEQGVDGLKFDGCYCDSIEN LINGAKQMSLALNRTGRSIYYHSEWPLYIL PHRKPNYTEIRQYANSARLFADIEDSLDSVLARIDFYDANQADIVDVAGPGFFVDLDMSIVGNYGLSLEEQV RQVAYQAIAKN PFYLSN DLADISPESLALLTNPDVLAIRQDPLGKAGRLVWRGDGFRLWEKELSGGKVAVL VTN DIRIGTPRKFSFPASSIGGGKVFNPAGRVTQLLPEREELGVFGKDDTITVSVPPYGARLFLVENVEAMSL KDLL> SEQ ID NO. 13 GLA A(R38K, T41P, M42R, L45V, E48A, R49Y, F50Y, M51Q, C52N, L54R, C56A, Q57A, E58N, E59D, S62R, C63A, S65T, K67D, F69Y, M70R, E71R, M72H, E74D, E79K, W81F, D83E, E87K, C90M, I91L, C94G, M96Q, Q99E, S102A, E103D, R105N, Q111E, R112L, H115D, I117L, R118A, Q119A, N 122K, 1133V, A135V, D136S, F145D, S148I, Y151H, Y152E, D153K, I154L, D155T, T158W, F159L, W162C, L166M, L167V, F169L, L177T, A181K, D182A, Y184N, K185R, H186A, L189E, R193A, I198M, V199Y, S201H, C202S, W204L, P205A, M208L, W209R, P210N, F211T, Q212E, Y216F, R220A, Y222I, C223A, H225S, W226T, N228V, F229T, A230E, W236A, K237E, I239L, K240D, S241R, I242R, L243I, T246W, S247A, F248A, E251D, R252E, D255S, A257Q, G261F, N263I, P265L, L268S, V269I, 1270V, N272R, F273Y, W277P, N278E, Q279E, T282R, Q283E, M284V, A285G, W287R, M290F, A291K, A292N, L294F, F295M, M296L, N298T, R301A, H302N, Q306E, A309E, Q312L, D313N, K314P, D315E, I317L, N320R, Q321D, L324K, K326V, Q327A, L331R, R332W, Q333A, N336G, E338R, W340F, P343E, G346D, L347G, A348K, W349V, A352L, M353I, I354L, Q357R, E358R, I359R, P362A, S364R, Y365F, T366S, 1367V, A368P, V369L, A370S, K374G, V376K, A377V, C378G, C382A, F383K, 1384V, V390E, R392K, K393E, F396L, Y397F, E398K, W399K, T400D, S401D, R402T, R404T, S405V, H406R, 1407V, T410G, T412A, Q416V, N419K, T420V, M421E, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLAKTPPRGWVHWAYYQN NRDAAN DPDRAITEDL YRRHADLMVSKGFKEAGYKYLMLDDGWQAPERDADGNLQADPELFPDGLAALAKYVHSKGLKLGVYVS VGN KTCAGDPGIFGHEKLTAQWLADCGVDMVKLDGCYCDSTEN LKAGNRAMSEALNATGRSMYYHSEL ALYLRNTEKPNFTEIAQIANSTRVTEDIDDSAESLDRRIDWWAANQDEIVSVQGPGFWIDLDMSIVGRYGL SPEEQVREVGLRAIFKN PFM LSTDLAN ISPEAKELLLN PEVLAIRDDPKGVAGYQRWAGDGFRVFERELSD GKVAVLILNRRRRGGARRFSVPLSSLGGGKVGN PAAKVTQLLPEKKELGLFKKDDTLTVRVN PGGAVLLVLE KVEAMSLKDLL> SEQ ID NO. 14 GLA A(R38K, T41P, M42R, L45D, E48V, F50Y, M51R, C56K, Q57E, E58N, E59D, S62T, C63A, S65T, K67D, F69Y, M70K, E71R, M72A, E74D, L75I, M76L, W81F, D83E, C90V, M96F, Q99E, S102K, R105N, Q111E, R112L, H115N, I117L, R118K, Q119E, N122D, V124I, 1133V, A135V, D136S, F145G, S148I, Y151H, Y152E, D153K, I154L, D155T, T158W, F159L, W162C, L167V, F169V, L177V, A181K, D182A, Y184V, H186A, M 187L, L189E, R193A, I198M,V199L, S201H, C202S, W204A, M208L, W209R, P210G, F211K, Q212E, R220A, Y222W, C223A, H225C, W226A, N228V, F229T, A230E, D233E, W236P, K237E, I239L, K240R, S241R, I242R, T246F, S247E, F248E, E251A, R252D, 1253V, G261F, W262F, N263I, P265L, L268S, V269I, F273Y, W277R, N278E, Q279E, V281I, T282R, Q283E, M284A, L286F, W287R, M290A, A292N, L294Y, M296L, N298T, R301A, H302N, P305E, Q306E, Q312L, K314P, D315E, I317L, N320R, Q321D, K326I, Q327A, Y329R, Q330C, Q333R, D335N, N336D, E338R, V339I, R342K, L347G, A348R, M353V, I354L, R356D, Q357R, E358R, 1359V, P362K, R363K, S364P, Y365F, T366S, 1367V, A368P, A370S, K374G, V376R, C378G, C382A, F383R, 1384V, V390E, K391R, R392K, K393E, F396L, Y397F, E398S, W399R, T400D, S401A, R402T, R404S, S405V, H406R, 1407V, N408A, T410G, T412A, L415F, Q416L, N419R, T420V, M421E, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLAKTPPRGWDHWVRYRCN LDKENDPDTAITEDLY KRAADILVSEGFKEAGYEYLVIDDCWFAPERDKEGN LQADPELFPNGLKELADYIHSKGLKLGVYVSVGN KT CAGGPGIFGHEKLTAQWLADCGVDLVKVDGCYCDSVENLKAGVKALSEALNATGRSMLYHSEAPLYLRGK EKPNYTEIAQWANCARVTEDIEDSPESLRRRLDWFEENQADVVDVAGPGFFIDLDMSIIGNYGLSREEQIR EAAFRAIAAN PYFLSTDLAN ISEEAKALLLDPEVLAIRDDPLGIAGRCLRRGNDFRIWEKPLSGGRWAVAVL N DRRVGGKKPFSVPVSSLGGGRAGNPAARVTQLLPERKELGLFSRDATLSVRVAPGGAVLFLLERVEAMSL KDLL> SEQ ID NO. 15 GLA A( R38K, T41P, M42R, L45D, E48G, R49Y, F50Y, M51R, C52N, C56A, Q57A, E58A, E59D, S62R, C63A, I64R, S65T, K67A, F69Y, M70E, E71R, M72A, E74D, E79K, W81F, D83A, E87R, I91L, C94A, M96L, Q99G, R100F, S102A, E103D, Q107R, Q111E, R112L, H115R, I117L, R118A, Q119A, N 122A, K130E, I133A, A135V, D136S, F145G, S148I, Y151H, Y152E, D153K, I154E, D155T, T158W, F159L, W162C, L167V, F169V, S176N, L177V, A181R, D182A, Y184V, H186A, L189E, R193A, S201G, C202T, W204A, M208E, W209R, P210G, F211T, Q212R, Y216W, R220K, Y222L, C223A, H225Y, W226A, N228I, F229T, A230E, D233E, W236R, K237E, I239L, K240L, S241R, I242R, L243I, T246Y, S247E, F248E, E251D, R252E, G261F, N263V, P265L, L268S, V269M, F273Y, W277R, N278E, Q279E, V281I, Q283E, M284A, A285G, L286Y, W287R, M290A, A291R, A292N, M296L, N298T, R301A, H302D, P305E, Q306E, K308R, Q312L, D313H, K314P, D315E, I317L, Q321D, L324I, K326V, Q327A, Y329R, Q330C, R332W, Q333R, N336D, E338R, P343A, L347G, A348K, W349Y, A352L, M353V, I354L, R356L, Q357R, E358R, I359R, S364R, Y365F, T366S, I367L, A368P, A370S, K374G, V376S, C378G, C382A, F383N, 1384V, V390E, K391R, K393D, F396T, Y397L, E398S, W399S, T400S, S401D, R402T, R404T, S405V, H406R, 1407V, N408A, T410G, T412A, L415F, Q416L, E418T, M421S, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLAKTPPRGWDHWGYYRNN LDAAADPDRARTEAL YERAADLMVSKGFKAAGYRYLCLDDAWLAPGFDADGRLRADPELFPRGLAALAAYVHSKGLELGAYVSVG N KTCAGGPGIFGH EKETAQWLADCGVDLVKVDGCYCDNVEN LRAGVKAMSEALNATGRSIVYGTEAPLY ERGTRKPNWTEIKQLANYARITEDIEDSRESLLRRIDWYEENQDEIVDVAGPGFWVDLDMSM IGNYGLSR EEQITEAGYRAIARNPLFLSTDLADISEEARALLLH PEVLAIN DDPIGVAGRCLWRGDDFRVWERALSGGKY AVLVLNLRRRGGPRRFSLPVSSLGGGSAGN PAANVTQLLPERRDLGTLSSSDTLTVRVAPGGAVLFLLTNTS AMSLKDLL> SEQ ID NO. 16 GLA A(R38L, T41P, L45N, H46N, E48G, R49Y, F50Y, M51R, C52N, C56K, Q57E, E58N, E59D, S62T, C63A, S65T, E66Q, K67D, F69Y, M70K, E71K, M72A, E79K, W81F, D83E,C90M, C94G, M96Q, Q99E, R100F, S102K, E103D, R105N, Q111E, R112L, H115E, I117L, R118K, Q119E, N122K, K127L, 1133V, A135G, D136S, F145K, S148W, Y151H, Y152E, D153K, I154K, D155T, T158W, F159L, W162C, L166A, L167V, F169L, S176N, L177V, A181R, D182K, G183N, Y184A, H186A, L189E, R193A, V199Y, S201H, C202S, P205A, M208E, W209Y, P210N, F211T, Y222I, C223A, H225S, W226F, N228V, F229T, A230E, I232R, D233R, W236R, K237E, I239L, K240L, S241R, I242A, L243M, T246Y, S247A, F248A, E251A, R252D, D255S, A257Q, G261S, N263I, P265L, L268S, V269M, F273H, W277R, N278E, Q283E, M284A, W287R, M290A, A291K, A292N, M296L, N298T, R301A, H302N, P305E, Q306E, K308R, Q312L, D313H, K314P, D315R, I317L, L324V, K326V, Q327A, Q330C, L331R, R332W, Q333E, N336D, E338R, V339I, L347G, A348R, W349V, M353V, I354H, R356L, Q357R, E358D, I359Y, P362E, S364P, Y365F, T366S, 1367V, A368P, K374G, V376R, A377V, C378G, C382A, F383R, 1384V, V390E, K391E, K393D, F396T, Y397L, E398A, W399A, T400G, S401D, R402T, R404S, S405V, H406R, 1407V, N408A, T410W, L415F, Q416L, N419R, T420V, M421A, Q422A)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLALTPPMGWN NWGYYRNNLDKENDPDTAITQDL YKKAAELMVSKGFKEAGYEYLMIDDGWQAPEFDKDGN LQADPELFPEGLKELAKYVHSLGLKLGVYGSVG NKTCAGKPGWFGHEKKTAQWLADCGVDAVKLDGCYCDNVEN LRKNAKAMSEALNATGRSIYYHSEWA LYEYNTQKPNYTEIRQIANSFRVTEDRRDSRESLLRAMDWYAANQADIVSVQGPGSWIDLDMSMIGNHG LSREQQVTEAALRAIAKNPLFLSTDLANISEEARALLLHPRVLAINQDPVGVAGYCRWEGDDFRIWERPLSG GRVAVAVHN LRDYGGERPFSVPVASLGGGRVGN PAARVTQLLPEERDLGTLAAGDTLSVRVAPWGTVLF LLERVAAMSLKDLL> SEQ ID NO. 17 GLA A( D233R, D234L, K237R, I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIRLSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 18 GLA A( D233R, K237R, I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 19 GLA A( I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 20 GLA A( D233R, K237R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. 21 GLA A( K237R, I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 22 GLA A( D233R, K237R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQEIGGPRRYTIAVASLGKGVACN PACFITQLLPVKRKLG FYEWTSRLRSH IN PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. 23 GLA A( S62Q, K237R, I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 24 GLA A( S62Q, D233R, K237R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQEIGGPRRYTIAVASLGKGVACN PACFITQLLPVKRKLG FYEWTSRLRSH IN PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. 25 GLA A( S62Q, D233R, K237R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. 26 GLA A( S62Q, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 27 GLA A( K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 28 GLA A( K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 29 GLA A( K237R, N278R, I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWRQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 30 GLA A( K237R, Q280R, I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQRVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVW ERPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 31 GLA A( K237R, N278R, Q280R, I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWRQRVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 32 GLA A( D233R, K237R, Q280R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFG LSWNQRVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMINRQEIGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTV LLQLENTMQMSLKDLL> SEQ ID NO.33 GLA A( D233R, K237R, N278R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRNFADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWRQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWE RPLSGLAWAVAMINRQEIGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTV LLQLENTMQMSLKDLL> SEQ ID NO.34 GLA A( D233R, K237R, N278R, Q280R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRNFADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWRQRVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWE RPLSGLAWAVAMINRQEIGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTV LLQLENTMQMSLKDLL> SEQ ID NO.35 GLA A( K237R, I359R, S364R, N408P)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRNFADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVW ERPLSGLAWAVAMINRQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIPPTG TVLLQLENTMQMSLKDLL> SEQ ID NO.36 GLA A( D233R, K237R, S364R, N408P)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALNRTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRNFADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWE RPLSGLAWAVAMINRQEIGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIPPTGTV LLQLENTMQMSLKDLL> SEQ ID NO.37 GLA A( D233T, F273Y, I359R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADITDSWKSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNYG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSH I N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 38 GLA A( D233R, K237R, I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LA WAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 39 GLA A( D233C, I359C)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADICDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LA WAVAMIN RQECGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLG FYEWTSRLRSHINPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 40 GLA A( D233C, K237R, I359C, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADICDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAMIN RQECGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 41 Skipped sequence 000> SEQ ID NO. 42 Skipped sequence 000> SEQ ID NO. 43 Skipped sequence000> SEQ ID NO. 44 GLA A( S62Q, K237R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 45 GLA A( S62Q, I359R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 46 Skipped sequence000> SEQ ID NO. 47 Skipped sequence000> SEQ ID NO. 48 Skipped sequence000> SEQ ID NO. 49 Skipped sequence000> SEQ ID NO. 50 Skipped sequence000> SEQ ID NO. 51 GLA A( K237R, I359R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 52 GLA A( K237R, I359R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 53 Skipped sequence000> SEQ ID NO. 54 Skipped sequence000> SEQ ID NO. 55 Skipped sequence000> SEQ ID NO. 56 Skipped sequence000> SEQ ID NO. 57 GLA A( S62Q, M208E, K237R, I359R, S364R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYEWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 58 Skipped sequence000> SEQ ID NO. 59 Skipped sequence000> SEQ ID NO. 60 Skipped sequence 000> SEQ ID NO. 61 GLA A( S62Q, M208E, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYEWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 62 GLA A( S62Q, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 63 GLA A( S62Q, P214G, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKGNYTEIRQYCN HWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 64 GLA A( S62Q, V199I, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIIYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 65 GLA A( S62Q, V199Y, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIYYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 66 GLA A( S62Q, Q212E, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFEKPNYTEIRQYCN HWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 67 GLA A( S62Q, Q212R, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFRKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 68 GLA A( S62Q, F211Y, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPYQKPNYTEIRQYCN HWRNFADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 69 GLA A( S62Q, M208E, K237R, I359R, S364R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YEWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRRYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRINPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 70 GLA A(S62Q, D233R, K237R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIRDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. 71 GLA A( H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 72 GLA A( K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 73 GLA A( S62Q, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 74 GLA A( S62Q, K237R, I359Q, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQEQGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 75 GLA A( S62Q, K237R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 76 GLA A( S62M, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDMCISEKL FMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 77 GLA A( S62Y, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDYCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 78 GLA A( D33N, R38M, L54R, D61Q, 164V, K67R, M70R, E71R, E74D, L89V, Q99K, S102E, E103N, H115N, Q119R, N 122D, F145Y, Q157K, D170A, N 179H, A181D, D182K, H186R, W209R, A230E, D233F, K237Q, 1239V, S247A, F248A, E251D, R252E, N278E, F295L, D313N, Q330R, Q333R, G334E, F337I, G346N, 1354V, R356L, A368S, K374R, V376L, F383L, V390E, K393E, F396L, R402T, S405V, H406R, 1407V, T410H, Q416R, N419E, M421S, S424T, L425S, D427S)MQLRNPELHLGCALALRFLALVSWDIPGARALNNGLAMTPTMGWLHWERFMCN RDCQEEPQSCVSERLFRRMADLMVSEGWKDAGYEYVCIDDCWMAPKRDENGRLQADPQRFPNGIRRLADYVHSKGLKLGIYADVGNKTCAGYPGSFGYYDIDAKTFADWGVDLLKFAGCYCDSLEHLDKGYKRMSLALNRTGRSIVYSCEWPLYMRPFQKPNYTEIRQYCNHWRN FEDIFDSWQSVKSILDWTAANQDEIVDVAGPGGWNDPDMLVIGN F GLSWEQQVTQMALWAIMAAPLLMSN DLRHISPQAKALLQN KDVIAINQDPLG KQGYRLRREDNIEVWE RPLSN LAWAVAMVN LQEIGGPRSYTISVASLGRGLACN PACLITQLLPEKRELGLYEWTSTLRVRVNPHGT VLLRLEETSQMTSKSLL> SEQ ID NO. 79 GLA A(R38M, K67Q, E71Q, E74D, E79D, E103K, H115G, Q119R, N179H, H186Y, D233Y, K237Q, 1239V, F248S, F295L, D313N, Q330R, Q333K, G334E, A368S, K374R, F383L, K393E, R404K, S405T, H406R, Q416R, M421S, M423T, S424T, D427S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLAMTPTMGWLHWERFMCN LDCQEEPDSCISEQL FMQMADLMVSDGWKDAGYEYLCIDDCWMAPQRDSKGRLQADPQRFPGGIRRLANYVHSKGLKLGIYA DVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLEHLADGYKYMSLALNRTGRSIVYSCEWP LYMWPFQKPNYTEIRQYCNHWRN FADIYDSWQSVKSILDWTSSNQERIVDVAGPGGWNDPDMLVIGN FGLSWNQQVTQMALWAIMAAPLLMSNDLRHISPQAKALLQN KDVIAINQDPLGKQGYRLRKEDNFEVW ERPLSGLAWAVAMIN RQEIGGPRSYTISVASLGRGVACNPACLITQLLPVKRELGFYEWTSRLKTRINPTGT VLLRLENTSQTTLKSLL> SEQ ID NO. 80 GLA A( H406R, Q416R, M421S, M423T, S424T, L425S, D427S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGT VLLRLENTSQTTSKSLL> SEQ ID NO. 81 GLA A( R38M)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLAMTPTMGWLHWERFMCN LDCQEEPDSCISEKL FMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYMWPFQKPNYTEIRQYCNHWRN FADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 82 GLA A(L54R, S62Q, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN RDCQEEPDQCISEKL FMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 83 GLA A( S62Q, K67R, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISERLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 84 GLA A( S62Q, K67Q, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEQL FMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 85 GLA A( S62Q, M70R, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF REMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADV GN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLEN LADGYKHMSLALNRTGRSIVYSCEWPLY VWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGL SWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWER PLSGLAWAVAM IN RQERGGPRSYTIAVASLGKGVACN PACFITQLLPVKRKLGFYEWTSRLRSRINPTGTVL LQLENTMQMSLKDLL> SEQ ID NO. 86 GLA A( S62Q, E71R, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MRMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 87 GLA A( S62Q, E71Q, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MQMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 88 GLA A( S62Q, E74D, M208V, K237R, 1359 R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 89 GLA A( S62Q, E79K, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSKGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 90 GLA A( S62Q, R118K, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIKQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 91 GLA A( S62Q, Q119R, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRRLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLEN LADGYKHMSLALNRTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGL SWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWER PLSGLAWAVAM IN RQERGGPRSYTIAVASLGKGVACN PACFITQLLPVKRKLGFYEWTSRLRSRINPTGTVL LQLENTMQMSLKDLL> SEQ ID NO. 92 GLA A( S62Q, H186R, M208V, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKRMSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 93 GLA A( S62Q, M208V, A230G, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FGDIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 94 GLA A( S62Q, M208V, K237Q, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWQSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 95 GLA A( S62Q, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 96 GLA A( S62Q, M208V, K237R, 1239V, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSVKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 97 GLA A( S62Q, M208V, K237R, D313N, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQNKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 98 GLA A( S62Q, M208V, K237R, Q333R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRRG DN FEVWERPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 99 GLA A( S62Q, M208V, K237R, I359R, A368S, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAMIN RQERGGPRSYTISVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. 100 GLA A( S62Q, M208V, K237R, I359R, K374R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQERGGPRSYTIAVASLGRGVACNPACFITQLLPVKRKLGFYEWTSRLRSRINPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 101 GLA A( S62Q, M208V, K237R, I359R, V376L, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGLACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. 102 GLA A( S62Q, M208V, K237R, I359R, H406R, 1407V)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRVN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 103 GLA A( S62Q, M208V, K237R, I359R, H406R, Q416R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLRLENTMQMSLKDLL> SEQ ID NO. 104 GLA A( S62Q, M208V, K237R, I359R, H406R, N419E)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLEETMQMSLKDLL> SEQ ID NO. 105 GLA A( S62Q, M208V, K237R, I359R, H406R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTSQMSLKDLL> SEQ ID NO. 106 GLA A( S62Q, M208V, K237R, I359R, H406R, T410H)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRINPHGT VLLQLENTMQMSLKDLL> SEQ ID NO. 107 GLA A( S62Q, E74D, M208V, K237Q, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWQSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 108 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 109 GLA A( S62Q, E74D, M208V, K237R, D313N, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQNKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 110 GLA A( S62Q, E74D, M208V, K237R, I359R, A368S, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAMIN RQERGGPRSYTISVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. Ill GLA A( S62Q, E74D, M208V, K237R, I359R, H406R, Q416R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLRLENTMQMSLKDLL> SEQ ID NO. 112 GLA A( S62Q, E74D, M208V, K237R, I359R, H406R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTSQMSLKDLL> SEQ ID NO. 113 GLA A( S62Q, M208V, K237Q, D313N, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWQSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQNKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 114 GLA A( S62Q, M208V, K237Q, I359R, A368S, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWQSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRSYTISVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. 115 GLA A( S62Q, M208V, K237Q, I359R, H406R, Q416R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWQSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTMQMSLKDLL> SEQ ID NO. 116 GLA A( S62Q, M208V, K237Q, I359R, H406R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWQSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTSQMSLKDLL> SEQ ID NO. 117 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R, L429P)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLP> SEQ ID NO. 118 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R, L429V)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLV> SEQ ID NO. 119 GLA A( S62Q, E74D, M208V, K237S, I359R, S364A, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQERGGPRAYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRINPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 120 GLA A( S62Q, E74D, M208V, K237S, I359R, S364Y, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQERGGPRYYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRINPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 121 GLA A( S62Q, E74D, M208V, K237S, I359R, S364T, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQERGGPRTYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRINPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 122 GLA A( S62Q, E74D, M208V, F229S, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 123 GLA A( S62Q, E74D, M208V, H225L, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNLWRNFADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGL SWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWER PLSGLAWAVAM IN RQERGGPRSYTIAVASLGKGVACN PACFITQLLPVKRKLGFYEWTSRLRSRINPTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 124 GLA A(: S62Q, E74D, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 125 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 126 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 127 GLA A( S62Q, E74D, M208V, K237S, I359R, H406Y)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSYINPTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 128 GLA A( S62Q, E74D, M208V, N228T, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRTFADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWER PLSGLAWAVAM IN RQERGGPRSYTIAVASLGKGVACN PACFITQLLPVKRKLGFYEWTSRLRSRINPTGTVL LQLENTMQMSLKDLL> SEQ ID NO. 129 GLA A( S62Q, E74D, S126N, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHN KGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 130 GLA A( S62Q, E74D, M208V, K237S, I359R, S405I, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRI Rl N PTGTV LLQLENTMQMSLKDLL> SEQ ID NO. 131 GLA A( S62Q, E74D, M208V, K237S, A307D, 1359 R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQDKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 132 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 133 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 134 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 135 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R, N408S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRISPTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 136 GLA A( S62Q, E74D, P110S, M208V, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADSQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 137 GLA A( S62Q, E74D, K237R, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 138 GLA A( S62Q, K237Q, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWQSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTMQMSLKDLL> SEQ ID NO. 139 GLA A( S62Q, E74D, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 140 GLA A( S62Q, E74D, K237R, I359R, H406R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGNFGLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTG TVLLQLENTSQMSLKDLL> SEQ ID NO. 141 GLA A( S62Q, E74D, F229S, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 142 GLA A( S62Q, E74F, M208V, F229S, K237S, I359R, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAFLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLRLENTSQMSLKDLL> SEQ ID NO. 143 GLA A( S62Q, E74D, M208V, F229S, K237S, I359R, H406R, Q416R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTMQMSLKDLL> SEQ ID NO. 144 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTSQMSLKDLL> SEQ ID NO. 145 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLQLENTSQMSLKDLL> SEQ ID NO. 146 GLA A( S62Q, E74D, M208V, I359R, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTSQMSLKDLL> SEQ ID NO. 147 GLA A( S62Q, E74D, M208V, I359R, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTSQMSLKDLL> SEQ ID NO. 148 GLA A( S62Q, E74D, M208V, K237Q, I359R, H406R, Q416R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWQSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTMQMSLKDLL> SEQ ID NO. 149 GLA A( S62Q, E74D, M208V, F229S, K237Q, I359R, H406R, Q416R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWQSIKSILDWTSFNQERIVDVAGPGGWN DPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTMQMSLKDLL> SEQ ID NO. 150 GLA A( S62Q, E74D, M208V, F229S, K237S, I359R, H406R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTSQMSLKDLL> SEQ ID NO. 151 GLA A( S62Q, E74D, M208V, F229S, K237S, I359R, H406R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTMQMSLKDLL> SEQ ID NO. 152 GLA A( S62Q, E74D, M208V, F229S, K237R, I359R, H406R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRNSADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLQLENTSQMSLKDLL> SEQ ID NO. 153 GLA A(Q2L, S62Q, E74D, M208V, K237R, I359R, H406R, Q416R)MLLRN PELH LGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTMQMSLKDLL> SEQ ID NO. 154 GLA A( R17L, S62Q, E74D, M208V, K237R, I359R, H406R, Q416R)MQLRNPELHLGCALALLFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLRLENTMQMSLKDLL> SEQ ID NO. 155 GLA A( R17L, S62Q, E74D, M208V, K237R, I359R, H406R, Q416R)MQLRNPELHLGCALALLFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLRLENTMQMSLKDLL> SEQ ID NO. 156 GLA A( G11V, S62Q, E74D, M208V, K237R, I359R, H406R, Q416R)MQLRNPELHLVCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCNLDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWRSIKSILDWTSFNQERIVDVAG PGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RP LSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLRLENTMQMSLKDLL> SEQ ID NO. 157 GLA A( R17G, S62Q, E74D, M208V, K237R, I359R, H406R, Q416R)MQLRNPELHLGCALALGFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKL FMEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYA DVGNKTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKHMSLALN RTGRSIVYSCEW PLYVWPFQKPNYTEIRQYCNHWRNFADIDDSWRSIKSILDWTSFNQERIVDVAGPGGWN DPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEV WERPLSGLAWAVAM INRQERGGPRSYTIAVASLGKGVACN PACFITQLLPVKRKLGFYEWTSRLRSRIN PT GTVLLRLENTMQMSLKDLL> SEQ ID NO. 158 GLA A( E74D, M208V, F229S, K237S, I359R, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRINPTGT VLLRLENTSQMSLKDLL> SEQ ID NO. 159 GLA A( S62Q, M208V, F229S, K237S, I359R, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGT VLLRLENTSQMSLKDLL> SEQ ID NO. 160 GLA A( S62Q, E74D, F229S, K237S, I359R, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTSQMSLKDLL> SEQ ID NO. 161 GLA A( S62Q, E74D, M208V, K237S, I359R, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTSQMSLKDLL> SEQ ID NO. 162 GLA A( S62Q, E74D, M208V, F229S, I359R, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSG LAWAVAM I N RQE RGG P RSYTI AVASLG KG VACN PACFITQLLP VKRKLG FYE WTSRLRSRI N PTGTVLLRLENTSQMSLKDLL> SEQ ID NO. 163 GLA A( S62Q, E74D, M208V, F229S, K237S, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FG LSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLRLENTSQMSLKDLL> SEQ ID NO. 164 GLA A( S62Q, E74D, M208V, F229S, K237S, I359R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQERGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHINPTGT VLLRLENTSQMSLKDLL> SEQ ID NO. 165 GLA A( E74D, M208V, F229S, K237S, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLRLENTSQMSLKDLL> SEQ ID NO. 166 GLA A( S62Q, M208V, F229S, K237S, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLRLENTSQMSLKDLL> SEQ ID NO. 167 GLA A( S62Q, E74D, F229S, K237S, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLRLENTSQMSLKDLL> SEQ ID NO. 168 GLA A( S62Q, E74D, M208V, K237S, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLRLENTSQMSLKDLL> SEQ ID NO. 169 GLA A( S62Q, E74D, M208V, F229S, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRNSADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLRLENTSQMSLKDLL> SEQ ID NO. 170 GLA A( S62Q, E74D, M208V, F229S, K237S, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGTV LLRLENTSQMSLKDLL> SEQ ID NO. 171 GLA A( S62Q, E74D, M208V, F229S, K237S, H406R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLQLENTSQMSLKDLL> SEQ ID NO. 172 GLA A( S62Q, E74D, M208V, F229S, K237S, H406R, Q416R)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLFMEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYADVGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPLYVWPFQKPNYTEIRQYCNHWRNSADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLRLENTMQMSLKDLL> SEQ ID NO. 173 GLA A( S62Q, E74D, K237S, H406R, Q416R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNFGLSWNQQVTQMALWAIMAAPLFMSN DLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVWE RPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRIN PTGTV LLRLENTSQMSLKDLL>SEQ ID NO. 174: CBh promoterTTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGC AGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGG GCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGGAGTCGCTGCGTTG CCTTCGCCCCGTGCCCCGCTCCGCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCAAGAGGTAAGGGTTTAAG GGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTGTTTTACAGGCCTGAAATCACTTGGTTTTAG GTTGG>SEQ ID NO. 175: hSynl promoterACTACAAACCGAGTATCTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAG GCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCC CAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCAC TGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCC GCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGT CGTGCCTGAGAGCGCAGCTGTGCTCCTGGGCACCGCGCAGTCCGCCCCCGCGGCTCCTGGCCAGACC ACCCCTAGGACCCCCTGCCCCAAGTCGCAGCCTTCGA>SEQ ID NO. 176 TATK28 cell-penetrating polypeptideDAAQPARRARRTKLAAYARKAARQARA>SEQ ID NO. 177 TATKII cell-penetrating polypeptideYARKAARQARA>SEQ ID NO. 178 Transportan cell-penetrating polypeptide AGYLLGKINLKALAALAKKIL>SEQ ID NO. 179 Antennapedia cell-penetrating polypeptideRQIKIWFQNRRM KWKK>SEQ ID NO. 180 P97 cell-penetrating polypeptideDSSHAFTLDELR>SEQ ID NO. 181 TATKK28 cell-penetrating polypeptideDAAQPARRAARTKLAAYARKAARQARA>SEQ ID NO. 182 DNA sequence for TATKll (human optimized)TACG CCCG G A AG G CCG CCCG G C AG GCCAGAGCC>SEQ ID NO. 182 DNA sequence for TATi<28 (human optimized)GACGCAGCACAGCCCGCAAGAAGAGCAAGAAGAACTAAACTGGCCGCTTACGCAAGGAAGGCAGCA AGACAGGCAAGAGCA>SEQ ID NO. 184 DNA sequence for Antennapedia CPP (human optimized)CGGCAGATCAAGATTTGGTTCCAGAACCGGAGAATGAAGTGGAAGAAG>SEQ ID NO. 185 DNA sequence for Transportan CPP (human optimized)GCCGGCTACCTGCTGGGCAAGATCAACCTGAAGGCCCTGGCCGCCCTGGCCAAGAAGATCCTG>SEQ ID NO. 186 DNA sequence for P97 CPP (human optimized)GACAGCTCCCACGCCTTCACCCTGGATGAGCTGCGG>SEQ ID NO. 187 Exemplary DNA sequence for TATKK28TCTGATGCTGCCCAGCCTGCTAGAAGGGCCGCCAGGACAAAACTGGCCGCCTATGCCAGAAAAGCCG CCAGACAGGCCAGAGCC>SEQ ID NO. 188 Exemplary DNA sequence for TATKK28AGCGACGCCGCTCAACCAGCTCGACGCGCCGCCAGAACCAAGCTGGCCGCCTACGCCCGGAAGGCCG CCAGACAGGCCAGAGCC>SEQ ID NO. 189 Exemplary DNA sequence for TATKK28AGCGACGCCGCCCAGCCCGCCAGAAGAGCCGCCAGAACCAAGCTGGCCGCCTACGCCAGAAAGGCC GCCAGACAGGCCAGAGCC>SEQ ID NO. 190 Exemplary DNA sequence for TATKK28TCTGATGCCGCCCAGCCTGCCAGACGGGCTGCACGGACGAAGCTGGCCGCCTACGCCAGAAAGGCGGCCAGACAGGCCAGAGCC>SEQ ID NO. 191 MBiP leader signal polypeptide MKLSLVAAM LLLLSLVAAM LLLLSAARA>SEQ ID NO.192 MBiP2 leader signal polypeptide MKLSLVAAM LLLLWVALLLLSAARA>SEQ ID NO. 193 MBiP3 leader signal polypeptide MKLSLVAAM LLLLSLVALLLLSAARA>SEQ ID NO. 194 MBiP4 leader signal polypeptide MKLSLVAAM LLLLALVALLLLSAARA>SEQ ID NO. 195 Murine IgK leader signal polypeptide METDTLLLWVLLLWVPGSTG>SEQ ID NO. 196 IGF leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE>SEQ ID NO. 197 IGF F26S leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGSYFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE>SEQ ID NO. 198 IGF Y27L leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPAKSE>SEQ ID NO. 199 IGF V43L leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGILEECCFRSCDLALLETYCATPAKSE>SEQ ID NO. 200 IGF F48T leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCTRSCDLALLETYCATPAKSE>SEQ ID NO. 201 IGF R49S leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFSSCDLALLETYCATPAKSE>SEQ ID NO. 202 IGF S50I leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRICDLALLETYCATPAKSE>SEQ ID NO. 203 IGF A54R leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLRLLETYCATPAKSE>SEQ ID NO. 204 IGF L55R leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGFYFSRPASRVSRRSRGIVEECCFRSCDLARLETYCATPAKSE>SEQ ID NO. 205 IGF F26S, Y27L, V43L, F48T, R49S, S50I, A54R, L55R leader signal polypeptideAYRPSETLCGGELVDTLQFVCGDRGSLFSRPASRVSRRSRGILEECCTSICDLRRLETYCATPAKSE>SEQ ID NO. 206 IGF AI-7, Y27L, K65R leader signal polypeptideTLCGGELVDTLQFVCGDRGFLFSRPASRVSRRSRGIVEECCFRSCDLALLETYCATPARSE>SEQ ID NO. 207 mvBIP leader signal polypeptide MVKLSLVAAM LLLLSLVAAM LLLLSAARA>SEQ ID NO. 208 DNA sequence for mBIP (human optimized)ATGAAGCTGTCCCTGGTGGCCGCTATGCTGCTGCTGCTGTCTCTGGTCGCTGCCATGTTATTACTGCTG TCTGCCGCTAGGGCC>SEQ ID NO. 209 Exemplary DNA sequence for mvBIP ATGAAGCTGTCCCTGGTGGCCGCTATGCTGCTGCTGCTGTCTCTGGTCGCTGCCATGTTATTACTGCTG TCTGCCGCTAGGGCC> SEQ ID NO. 210 (wtGLA nucleotide sequence) atgcagctcagaaacccagagcttcaccttggttgtgctttggctctcagattcctggccctggttagttgggacatacctggagccaga gctcttgataatggcttggctagaactcccactatgggttggctgcactgggagagatttatgtgcaacctggactgccaggaggagcc agatagttgtatttctgaaaaactgttcatggaaatggctgaactcatggtgtctgaaggctggaaagatgctggttatgaatatctttg cattgatgattgctggatggctcctcagagggatagtgaagggagattgcaagcagatccacaaagatttcctcatggcattagacaa ttggctaattatgtgcactctaaaggtttgaaactgggcatttatgcagacgtaggtaacaagacctgtgctggcttccctggatcattt ggatattacgacatagacgctcaaacttttgcagactggggtgtggatttgcttaagtttgatggttgttactgtgactccttggagaact tggctgacggttacaaacacatgtcattggcactgaacagaactggcagaagcattgtttacagctgtgagtggcctctttacatgtgg ccttttcagaaacctaattacacagaaatcaggcaatactgtaatcactggaggaattttgctgacattgatgattcctggaaaagcat caaaagtattctggactggacttcatttaaccaagaaaggattgttgatgtagctggacctggtgggtggaatgaccctgacatgcttg taattgggaactttggcctctcttggaatcaacaagtcacacaaatggccctgtgggctatcatggctgcccctctcttcatgtctaacg accttagacatatatcaccccaagctaaggcacttctgcaagataaggacgtgattgcaatcaaccaggatccattggggaagcagg gctatcagcttagacaaggtgacaattttgaagtgtgggagagacctctgtctggattggcttgggctgtggctatgataaatagacag gagattgggggtccaaggtcttacaccattgctgttgcctcactggggaaaggtgttgcctgtaaccctgcctgtttcattacacagctct tgcctgtgaagaggaaattggggttctacgagtggacctccaggctgagatcacatattaatcccacaggtactgtacttttgcaattg gaaaacaccatgcagatgagcttgaaggacctgctctag> SEQ ID NO. 211 (wtGLA amino acid sequence)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDSCISEKLF MEMAELMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YMWPFQKPNYTEIRQYCNHWRN FADIDDSWKSIKSILDWTSFNQERIVDVAGPGGWNDPDMLVIGNF GLSWNQQVTQMALWAIMAAPLFMSNDLRH ISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDN FEVW ERPLSGLAWAVAMIN RQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSHIN PTGT VLLQLENTMQMSLKDLL> SEQ ID NO. 212 GLA A( S62Q, E74D, M208V, K237S, H406R, M421S)MQLRNPELHLGCALALRFLALVSWDIPGARALDNGLARTPTMGWLHWERFMCN LDCQEEPDQCISEKLF MEMADLMVSEGWKDAGYEYLCIDDCWMAPQRDSEGRLQADPQRFPHGIRQLANYVHSKGLKLGIYAD VGN KTCAGFPGSFGYYDIDAQTFADWGVDLLKFDGCYCDSLENLADGYKH MSLALN RTGRSIVYSCEWPL YVWPFQKPNYTEIRQYCNHWRN FADIDDSWSSIKSILDWTSFNQERIVDVAGPGGWNDPDM LVIGN FGLSWNQQVTQMALWAIMAAPLFMSNDLRHISPQAKALLQDKDVIAINQDPLGKQGYQLRQGDNFEVWERPLSGLAWAVAMINRQEIGGPRSYTIAVASLGKGVACNPACFITQLLPVKRKLGFYEWTSRLRSRINPTGTVLLQLENTSQMSLKDLL

Claims

What is claimed is:

1. A composition comprising: a gene therapy delivery system; and an alpha-galactosidase (GLA) polynucleotide encoding a GLA polypeptide, wherein the GLA polypeptide has at least 95% sequence identity to one of SEQ ID NOS. 1-40, 44-45, 51-52, 57, 61-173 or 212.

2. The composition of claim 1, wherein the GLA polypeptide has at least 95% sequence identity to one of SEQ ID NOS. 21-22, 26, 62, 88, 94, 111, 142, 163, 167-168 or 212.

3. The composition of claim 1 or 2, wherein the sequence identity is at least 98%.

4. The composition of any one of claims 1-3, wherein at least one amino acid residue of the GLA polypeptide is substituted with an arginine residue.

5. The composition of any one of claims 1-4, wherein at least one amino acid residue of the GLA polypeptide is substituted with an arginine residue, a threonine residue, a valine residue, a glutamine residue, or a tyrosine residue.

6. The composition of any one of claims 1-5, wherein at least one lysine residue of the GLA polypeptide is substituted with an arginine residue, or at least one aspartic acid residue of the GLA polypeptide is substituted with an arginine residue or a threonine residue, or at least one phenylalanine residue of the GLA polypeptide is substituted with a tyrosine residue, or at least one serine residue of the GLA polypeptide is substituted with a glutamine residue, or at least one histidine residue of the GLA polypeptide is substituted with an arginine residue.

7. The composition of any one of claims 1-6, wherein the gene therapy delivery system comprises one or more of a viral vector, a liposome, a lipid-nucleic acid nanoparticle, an exosome, or a gene editing system.

8. The composition of claim 7, wherein the gene editing system comprises one or more of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) associated protein 9(CRISPR-Cas-9), Transcription activator-like effector nuclease (TALEN), or ZNF (Zinc finger peptide).

9. The composition of any one of claims 7-8, wherein the gene therapy delivery system comprises a viral vector.

10. The composition of claim 9, wherein the viral vector comprises one or more of an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a poxviral vector or a herpes simplex viral vector.

11. The composition of claim 9 or 10, wherein the viral vector comprises a viral polynucleotide operably linked to the GLA polynucleotide.

12. The composition of any one of claims 9-11, wherein the viral vector comprises at least one inverted terminal repeat (ITR).

13. The composition of any one of claims 9- 12, further comprising one or more of an SV40 intron, a polyadenylation signal, or a stabilizing element.

14. The composition of any one of claims 9-13, further comprising a promoter.

15. The composition of claim 14, wherein the promoter has at least 90% sequence identity to SEQ ID NO. 174 or SEQ ID NO. 175.

16. The composition of any one of claims 1-15, further comprising a polynucleotide encoding a cell-penetrating polypeptide.

17. The composition of claim 16, wherein the cell-penetrating polypeptide has at least 90% sequence identity to one of SEQ ID NOS. 176-181.

18. The composition of claim 16 or 17, wherein the polynucleotide encoding the cellpenetrating peptide has at least 90% sequence identity to one of SEQ ID NOS. 182-190.

19. The composition of any one of claims 1-18, further comprising a polynucleotide encoding a leader signal polypeptide.

20. The composition of claim 19, wherein the leader signal polypeptide has at least 90% sequence identity to one of SEQ ID NOS. 191-207.

21. The composition of claim 19 or 20, wherein the polynucleotide encoding the leader signal polypeptide has at least 90% sequence identity to SEQ ID NO. 208 or SEQ ID NO. 209.

22. A pharmaceutical formulation comprising the composition of any one of claims 1-21 and a pharmaceutically acceptable carrier.

23. A method of treating Fabry disease, the method comprising administering the composition of any one of claims 1-21 or the formulation of claim 22 to a patient in need thereof.

24. The method of claim 23, wherein the composition or the formulation is administered intrathecally, intravenously, intracistnerally, intracerebroventrically or intraparenchymally.

25. A method of treating Fabry disease, the method comprising administering the composition of any one of claims 1-21 or the formulation of claim 22 to an ex vivo cell and administering the ex vivo cell to a patient in need thereof.

26. The method of claim 25, wherein the ex vivo cell is administered intrathecally, intravenously, intracisternally, intracerebroventrically or intraparenchymally.

27. A fusion protein comprising the GLA polypeptide of any one of claims 1-26 and a leader signal polypeptide operatively coupled to the GLA polypeptide.

28. The fusion protein of claim 27, wherein the leader signal polypeptide has at least 90% sequence identity to one of SEQ ID NOS. 191-209.

29. A fusion protein comprising the GLA polypeptide of any one of claims 1-26 and a cellpenetrating polypeptide operatively coupled to the GLA polypeptide.

30. The fusion protein of claim 29, wherein the cell-penetrating polypeptide has at least 90% sequence identity to one of SEQ ID NOS. 182-190.

31. The fusion protein of claim 30, wherein the cell-penetrating polypeptide comprises the sequence of one of SEQ ID NOS. 182-190.

32. The fusion protein of any one of claims 29-31, further comprising a leader signal polypeptide.

33. The fusion protein of claim 32, wherein the leader signal polypeptide has at least 90% sequence identity to one of SEQ ID NOS. 191-209.

34. The fusion protein of claim 32 or 33, wherein the leader signal polypeptide comprises the sequence of one of SEQ ID NOS. 191-209.

35. The fusion protein of any one of claims 27-34, further comprising one or more affinitytags, one or more protease cleavage sites, or a combination thereof.

36. The fusion protein of claim 35, wherein the affinity-tag comprises one or more of MYC, HA, V5, NE, StreplI, Twin-Strep-tag®, glutathione S-transferase (GST), maltose- binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3xFLAG®, polyhistidine (His), HPC4, or a combination thereof.

37. The fusion protein of claim 35 or 36, wherein the protease cleavage site is sensitive to one or more of thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, or a combination thereof.

38. A pharmaceutical formulation comprising: the GLA polypeptide of any one of claims 1-26 or the fusion protein of any one of claims 27- 37; and a pharmaceutically acceptable carrier.

39. A method of treating Fabry disease, the method comprising administering the GLA polypeptide of any one of claims 1-26, or the fusion protein of any one of claims 27-37, or the formulation of claim 38, to a patient in need thereof.

40. The method of claim 39, wherein the GLA polypeptide, the fusion protein or the formulation is administered intrathecally, intravenously, intracisternally, intracerebroventrically or intraparenchymally.

41. A method of producing the GLA polypeptide of any one of claims 1-26 or the fusion protein of any one of claims 27-37, the method comprising: expressing the GLA polypeptide or the fusion protein; and purifying the GLA polypeptide or the fusion protein.

42. The method of claim 41, wherein the GLA polypeptide or the fusion protein is expressed in Chinese hamster ovary (CHO) cells, HeLa cells, human embryonic kidney (HEK) cells, insect cells or Escherichia coli cells.

43. A method of producing a protein comprising a GLA polypeptide, the method comprising: expressing the protein in insect cells; and purifying the protein from the insect cells.

44. The method of claim 43, wherein the insect cells are Sf9 cells or BTI-Tn-5B 1-4 cells.

45. The method of claim 43 or 44, wherein the protein comprises a fusion protein comprising the GLA polypeptide and a cell-penetrating polypeptide operatively coupled to the GLA polypeptide.

46. The method of claim 45, wherein the fusion protein further comprises a leader signal polypeptide.

47. The method of any one of claim 43-46, wherein the fusion protein further comprises one or more affinity-tags, one or more protease cleavage sites, or a combination thereof.

48. The method of claim 47, wherein the affinity-tag comprises one or more of MYC, HA, V5, NE, StrepII, Twin-Strep-tag®, glutathione S-transferase (GST), maltose-binding protein (MBP), calmodulin-binding peptide (CBP), FLAG®, 3xFLAG®, polyhistidine (His), HPC4, or combinations thereof.

49. The method of claim 47 or 48, wherein the protease cleavage site is sensitive to one or more of thrombin, furin, factor Xa, metalloproteases, enterokinases, cathepsin, HRV3C, TEV, or combinations thereof.

50. The method of any one of claims 43-49, wherein the GLA polypeptide has at least 98% sequence identity to one of SEQ ID NOS. 1-40, 44-45, 51-52, 57, 61-173 or 212.

51. The method of any one of claims 43-50, wherein the GLA polypeptide has at least 98% sequence identity to one of SEQ ID NOS. 21-22, 26, 62, 88, 94, 111, 142, 163, 167-168 or 212.

52. An alpha-galactosidase (GLA) polynucleotide encoding a GLA polypeptide, wherein the GLA polypeptide has at least 95% sequence identity to one of SEQ ID NOS. 1-40, 44-45, 51-52, 57, 61-173 or 212.

53. The GLA polynucleotide of claim 52, wherein the GLA polypeptide has at least 95% sequence identity to one of SEQ ID NOS. 21-22, 26, 62, 88, 94, 111, 142, 163, 167-168 or 212.

54. A GLA polypeptide having at least 95% sequence identity to one of SEQ ID NOS. 1- 40, 44-45, 51-52, 57, 61-173 or 212.

55. The GLA polypeptide of claim 54, having at least 95% sequence identity to one of SEQ ID NOS. 21-22, 26, 62, 88, 94, 111, 142, 163, 167-168 or 212.

56. The composition, formulation, fusion protein, or GLA poplypeptide of any one of claims 1-55, wherein an aspartic acid residue at position 233 of the GLA polypeptide is substituted with an arginine residue, or a lysine residue at position 237 of the GLA polypeptide is substituted with an arginine residue, or a serine residue at position 364 of the GLA polypeptide is substituted with an arginine residue.

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