REDUCED IMMUNOGENIC PROTEINS FOR LYSOSOMAL STORAGE DISORDERS

Abstract

Disclosed are methods and compositions for reduced immunogenic proteins used in enzyme replacement therapy for lysosomal storage disorders. More specifically disclosed are genetically engineered variants of N-acetylgalactosamine-6-sulfatase (GALNS), which are less immunogenetic then wild-type GALNS, but maintain enzymatic activity, and may be used to treat Mucopolysaccharidosis IVA (Morquio A disease, MPS IVA).

Claims

1. A modified N-acetylgalactosamine-6-sulfate sulfatase (GALNS) with reduced immunogenic properties and having at least 60 percent GALNS activity.

2. The modified N-acetylgalactosamine-6-sulfate sulfatase (GALNS) of claim 1, selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

3. A modified N-acetylgalactosamine-6-sulfate sulfatase (GALNS) produced by a method comprising: transfecting a cell with a nucleic acid encoding the modified N- acetylgalactosamine-6-sulfate sulfatase (GALNS); and culturing the cell under conditions wherein the cell produces the modified GALNS.

4. The method of claim 3, wherein the cell is a eukaryotic cell.

5. The method of claim 3, wherein the cell is a prokaryotic cell.

6. The method of claim 3, wherein the modified GALNS is secreted from the cell.

7. The method of claim 3 further comprising assaying the modified GALNS for GALNS activity.

8. The method of claim 3 further comprising analyzing the modified GALNS for immunogenicity.

9. The method of claim 3, wherein the modified GALNS has at least 60 percent GALNS activity as compared to wild type GALNS.

10. The method of claim 3, wherein the modified GALNS has at least 80 percent GALNS activity as compared to wild type GALNS.

11. The method of claim 3, wherein the nucleic acid encodes a modified GALNS selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

Description

DESCRIPTION OF THE FIGURES

[0010] The application file contains at least one figure executed in color. Copies of this patent application publication with color photographs will be provided by the Office upon request and payment of the necessary fee.

[0011] FIGS. 1A and 1B Illustrate the locations of immunodominant peptide regions C4, E8, and 110 in 3D structure of GALNS (FIG. 1A) and GALNS immunodominant epitope sequences: C4 (SEQ ID NO:9), E8 (SEQ ID NO:10), I10 (SEQ ID NO:11) (FIG. 1B).

[0012] FIGS. 2A and 2B Example of IEDB and RANKPEP output. Modified sequences showed a decreased immunogenicity when compared to the wild type GALNS in IEDB (FIG. 2A) and RANKPEP (FIG. 2B). Higher percentile rank values from IEDB indicate lower immunogenicity. Lower score values from RANKPEP indicate lower immunogenicity.

[0013] FIG. 3A Molecular docking visualization. The original GALNS Model 1 3D protein structure is depicted in blue. The N-Acetyl-D-Galactosamine (NGA) ligand (orange) binding site is depicted in red with the active site bonds shown in red labeled with their measured distances in angstroms. The calcium (orange asterisk) binding site is depicted in orange with the active site bonds shown in red labeled with their measured distances in angstroms.

[0014] FIG. 3B Molecular docking visualization of modified peptide C4-N6.

[0015] FIG. 3C Molecular docking visualization of modified peptide C4-N6 superimposed onto GALNS.

[0016] FIG. 3D Molecular docking visualization of modified peptide E8-N13.

[0017] FIG. 3E Molecular docking visualization of modified peptide E8-N13 superimposed onto GALNS Model 1.

[0018] FIG. 3F Molecular docking visualization of modified peptide I10-N14.

[0019] FIG. 3G Molecular docking visualization of modified peptide I10-N14 superimposed onto GALNS.

[0020] FIG. 3H Molecular docking visualization of modified peptide GALNS-242.

[0021] FIG. 3I Molecular docking visualization of modified peptide GALNS-242 superimposed onto GALNS.

[0022] FIG. 3J Molecular docking visualization of modified peptide GALNS-201.

[0023] FIG. 3K Molecular docking visualization of modified peptide GALNS-201 superimposed onto GALNS.

[0024] FIG. 3L Molecular docking visualization of modified peptide GALNS-315.

[0025] FIG. 3M Molecular docking visualization of modified peptide GALNS-315 superimposed onto GALNS.

[0026] FIG. 3N Molecular docking visualization of modified peptide GALNS-231.

[0027] FIG. 3O Molecular docking visualization of modified peptide GALNS-231 superimposed onto GALNS.

[0028] FIGS. 4A and 4B illustrate active site molecular docking. Distances in angstroms were measured between the active site residues (79, 310#1, 310#2, 311(O), 311(N), 108(C), 108(O), and 236) and the N-Acetyl-D-Galactosamine substrate (FIG. 4A). Distances in angstroms were measured between the active site residues (79, 39, 40, 288, 289) and the Ca.sup.2+ within the active site (FIG. 4B).

[0029] FIG. 5 illustrates GALNS activity in modified peptides produced in eukaryotic cells, which were co-transfected with Sulfatase-modifying factor 1 (SUMF1).

DETAILED DESCRIPTION OF THE INVENTION

[0030] The Inventors have previously disclosed methods of using bioinformatic tools and in vivo and in vitro immune reactivity assays to identify immunodominant peptides within enzymes administered for enzyme replacement therapy (ERT), including N-acetylgalactosamine-6-sulfatase (GALNS), which is administered for treatment of MPS IVA (see U.S. patent application Ser. No. 13/760,907, incorporated herein by reference in its entirely). The Inventors have shown that immunodominant peptides within GALNS may be used to induce immune tolerance in subject prior to receiving GALNS for ERT. The inventors disclose herein, genetically engineered variants of GALNS, in which these immunodominant peptide regions within GALNS have been modified to present GALNS enzymes with reduced immunogenicity and without an effective reduction in enzyme activity. The inventors believe that this method may also be applied to other enzymes used in ERT, practically those used to treat lysosomal storage disorders (LSDs).

Identification of Immunodominant Peptides and Evaluation of Modifications

[0031] The inventors have identified immunodominant peptides of GALNS using bioinformatics tools in combination with target enzyme deficient animals, and in vitro and in vivo humoral and cellular assays for immune response indicators, as disclosed in U.S. patent application Ser. No. 13/760,907 (published as U.S. Patent Pub. No. 2013/0202633).

[0032] More specifically, the initial identification of potential immunodominant peptides, was done using bioinformatic tools, RANKPEP (Reche et al. (2002) Human Immunology, 63: 701-709.; Reche et al. (2004) Immunogenetics, 56:405-419; Reche and Reinherz (2007) Methods Mol Biol., 409:185-200) and Immune Epitope Data Base (Vita et. al., (2010) Nucleic Acids Res. 2010; 38:D854-62). GALNS immunogenicity and prediction of B-cell epitopes were evaluated by the Immune Epitope Data Base (IEDB) analysis resource. The algorithm is based on the predictions of surface accessibility and flexibility of the molecule, and the presence of -turns and linear epitopes (Zhang et al. (2008) (IEDB-AR). Nucleic Acids Res. 2008: p. W513-8). MHC-II epitopes (H2-IAb) were predicted by IEDB and RANKPEP. Ten peptides were selected by the best scores of IC.sub.50 nM (concentration of peptide that inhibits binding of a standard peptide by 50%) and binding potential, respectively (Kim, et al. (2011) J. Immunol. Methods, 374(1-2): p. 62-9).

[0033] Of 10 peptides identified by bioinformatics techniques, 3 indicated an increased immunodominant response relative to the 7 remaining peptides, and compared to the intact enzyme (see FIG. 1). This selection was done using immunizing enzyme deficient MKC mice (see U.S. patent application Ser. No. 13/760,907, published as U.S. Patent Pub. No. 2013/0202633). The Inventors chose three immunodominant peptide regions, designated C4, E8, and I10, within the GALNS protein as targets for modification to produce GALNS enzymes with reduced immunogenicity.

[0034] The Inventors reasoned that reduced immunogenicity of GALNS may be accomplished by substituting certain amino acid residues within these immunodominant regions. The inventors also reasoned that the tertiary structure of the GALNS protein was an essential consideration when bioengineering GALNS variants, because protein miss-folding is known to be the most common cause of enzyme deficiency in Morquio A disease..sup.2

[0035] GALNS is a homodimeric glycoprotein with 3 domains in each monomer including an N-terminal domain with the active site, a second domain with antiparallel n-strands, and a C-terminal meander..sup.2 Each monomer also contains 3 disulfide bonds, 1 unpaired cysteine, many phosphorylation sites, and 2 N-glycosylation sites at Asn204 and Asn423.sup.2. The GALNS active site contains a calcium ligand bound to 4 residues (D39, D40, D288, N289) and to the catalytic nucleophile DHA79.sup.2. The N acetylgalactosamine (GalNac) substrate binds to the active site at 3 residues (Y108, H236, K310) and the catalytic nucleophile DHA792. GalNac is a subunit of the dimer chondroitin-6-sulfate, which is comprised of GalNac and glucosamine,.sup.2. Keratan sulfate is a dimer comprised of galactosamine and N-acetylglucosamine..sup.2

[0036] When making amino acid substitutions within the immunodominant regions, the Inventors considered the following: (1) amino acid substitutions should be made within the same polarity groups, (2) changes to amino acids with significantly different structures compared to the original sequence should be avoiding, by way of example cysteine was not changed because of its potential to form unique sulfide bonding, and (3) changes to amino acids that would create mutations known to be involved in Morquio A disease were also avoided.

[0037] Working within these 3 immunodominant regions, the Inventors initially created 324 modified sequences, from which 92 modified sequences were initially selected after screening the sequences for predicted reduced immunogenicity using IEDB and RANKPEP analysis. More rigorous selection ultimately lead to 7 selected sequences. FIGS. 2A and 2B show IEDB and RANKPEP analysis respectively, for the final 7 selected sequences. The results from both IEDB and RANKPEP analysis, showed immunogenic variation associated with each amino acid change.

[0038] More specifically, these 7 sequences were selected as follows. From the 92 modified sequences, 21 sequences were further selected after consideration of in silico predictions of phosphorylation sites Table 1A), N-glycosylation sites (Table 1 B), and when the physic chemical properties of the modified sequences were compared to those of the original GALNS sequence (Table 1C).

TABLE-US-00001 TABLE1A PhosphorylationPredictionsGALNSAminoAcidSequence. GPS2.1 NetPhos2.0 NetPhos2.0 Position Code GPS2.1Sequence Score Position Code Sequence Score 56 T YGEPSRETPNLDR 9.526 359 S LTPPSDRAI 0.963 MA SEQID (SEQIDNO:12) NO:24 109 T AHARNAYTPQEIV 9.421 80 S NPLCSPSRA 0.903 GG (SEQID (SEQIDNO:13) NO:25) 181 Y ARPNIPVYRDWEM 9.4 466 S QEALSRITS 0.889 VG (SEQID (SEQIDNO:14) NO:26) 356 T SLALAGLTPPSDRA 9.333 341 S HQLGSIMDL 0.882 I (SEQID (SEQIDNO:15) NO:27) 181 Y ARPNIPVYRDWEM 9.333 264 S EIDDSIGKI 0.834 VG (SEQID (SEQIDNO:14) NO:28) 49 Y GWGDLGVYGEPS 9.333 470 S SRITSVVQQ 0.721 RET (SEQID (SEQIDNO:16) NO:29) 108 Y NAHARNAYTPQEIV 9.25 458 S LSFASAEYQ 0.694 G (SEQID (SEQIDNO:17) NO:30) 356 T SLALAGLTPPSDRA 9.211 135 S AGYVSKIVG 0.63 I (SEQID (SEQIDNO:15) NO:31) 49 Y GWGDLGVYGEPS 9.2 408 S TWTNSWEN 0.592 RET F (SEQIDNO:16) (SEQID NO:32) 249 S SKPFLGTSQRGRY 9.1 56 T PSRETPNLD 0.959 GD (SEQID (SEQIDNO:18) NO:33) 109 T AHARNAYTPQEIV 9 389 T YRGDTLMAA 0.949 GG (SEQID (SEQIDNO:13) NO:34) 240 Y DATHAPVYASKPF 8.667 312 T CGKQTTFEG 0.832 LG (SEQID (SEQIDNO:19) NO:35) 470 S EALSRITSVVQQHQ 8.667 509 T GKCLTPPES 0.767 E (SEQID (SEQIDNO:20) NO:36) 108 Y NAHARNAYTPQEIV 8.667 469 T LSRITSVVQ 0.523 G (SEQID (SEQIDNO:17) NO:37) 80 S YSANPLCSPSRAA 8.667 240 Y HAPVYASKP 0.897 LL (SEQID (SEQIDNO:21) NO:38) 509 T EKLGKCLTPPESIP 8.421 133 Y KKAGYVSKI 0.895 K (SEQID (SEQIDNO:22) NO:39) 49 Y GWGDLGVYGEPS 8.286 98 Y RNGFYTTNA 0.87 RET (SEQID (SEQIDNO:16) NO:40) 80 S YSANPLCSPSRAA 8.25 170 Y HFGPYDNKA 0.736 LL (SEQID (SEQIDNO:21) NO:41) 249 S SKPFLGTSQRGRY 8.111 GD (SEQIDNO:18) 469 T QEALSRITSVVQQH 8.1 Q (SEQIDNO:23) 356 T SLALAGLTPPSDRA 8 I (SEQIDNO:15) 509 T EKLGKCLTPPESIP 8 K (SEQIDNO:22) 469 T QEALSRITSVVQQH 8 Q (SEQIDNO:23)

[0039] Predicted sites of phosphorylation are identified by sequence position (Position) and amino acid identifying code (Code).

TABLE-US-00002 TABLE 1B N-Glycosylation Predictions NetNGlyc 1.0 Potential Jury Sequence Position Sequence Agreement Result N-Glyc GALNS 204 NLTQ 0.7763 (9/9) +++ 423 NVSG 0.5894 (8/9) + GALNS-4 204 NLTQ 0.7762 (9/9) +++ 423 NVSG 0.5894 (8/9) +

TABLE-US-00003 TABLE 1C Physico-Chemical Properties Theoretical Instability Aliphatic Sequence Molec. Wt pl Index (II) Index (AI) GRAVY GALNS 58026.0 6.25 35.37 80.61 0.250 GALNS-4 58062.0 6.25 34.71 83.03 0.224

[0040] Sequences were selected if the modified sequence showed zero or insignificant changes when compared to the original GALNS sequence. By way of example, the original GALNS protein has 2 N-glycosylation sites, which were not changed in the modified proteins. Differences in physico-chemical properties between original GALNS and the modified peptides were insignificant. Several sequences were predicted to have an additional phosphorylation site at T235 and these sequences were also not selected. Sequences with an additional phosphorylation site were also not selected.

[0041] From these 21 sequences, 7 sequences were further selected, after molecular visualization analysis of ligand binding and molecular docking. Table 2 provides a summary of amino acid modifications in the final 7 sequences.

TABLE-US-00004 TABLE 2 Peptide modifications within immunogenic groups of GALNS 7 Selected Sequences Peptide Group Sequence Peptide(s) 1 Peptide C4 C4-N6 E8 E8-N13 I10 I10-N14 2 Peptides C4 + E8 GALNS-242 C4-N7 + E8-N11 C4 + I10 GALNS-201 C4-N6 + I10-N14 E8 + I10 GALNS-315 E8-N14 + I10-N14 3 Peptides C4 + E8 + GALNS-231 C4-N6 + E8-N14 + I10 I10-N14

[0042] The PyMOL Molecular Graphics System, Version 1.7.4 Schrdinger, LLC.sup.14 was used to superimpose the molecular structure of modified proteins onto the wild type GALNS protein. FIGS. 3A-3O show molecular visualization analysis for the final 7 selected sequences. Distances in angstroms were measured between the active site residues and the corresponding ligand as shown in FIG. 4A and calcium binding, as shown in FIG. 4B. A smaller distance indicated that the active site residue and substrate were more likely to bind. Modified sequences were chosen if their measured distances within the active site were very close to or even smaller than the measured distance in the original GALNS sequence. These results are summarized in Table 3a and Table 3b respectively.

Table 3 Distances from Active Site Atom to Substrate in Angstroms

[0043]

TABLE-US-00005 TABLE 3(a) N-Acetyl Galactosamine (NGA) active site. NGA Sequence Peptide(s) 79 310 310 311(O) 311(N) 108(C) 108(O) 236 AVG GALNS Model 1 3.2 4.2 4.8 5.4 3.3 6.6 6.5 4.9 4.9 C4-N6 C4-N6 1.7 3.2 3.7 3.4 5.6 5.9 3.6 4.4 3.9 E8-N13 E8-N13 2.3 3.8 3.5 3.2 5.2 4.0 6.2 4.7 4.1 I10-N14 I10-N14 3.0 4.3 4.4 5.0 3.3 7.3 6.3 4.7 4.8 GALNS-242 C4-N7 + E8-N11 3.9 4.6 5.8 5.4 3.8 2.6 5.6 5.2 4.6 GALNS-201 C4-N6 + I10-N14 5.3 4.5 5.4 3.0 4.9 9.8 9.1 4.3 5.8 GALNS-315 E8-N14 + 3.1 5.5 5.3 5.4 3.4 6.5 4.3 4.8 4.8 I10-N14 GALNS-231 C4-N6 + E8- 3.2 4.6 5.0 3.5 4.8 5.7 5.8 4.6 4.7 N14 + I10-N14

TABLE-US-00006 TABLE 3(b) Calcium active site. Ca.sup.2+ Sequence Peptide(s) 79 39 40 288 289 AVG GALNS Model 1 0.8 3.1 5.4 5.5 6.8 4.3 C4-N6 C4-N6 3.7 2.5 3.3 5.2 7.1 4.4 E8-N13 E8-N13 2.6 2.6 3.0 5.4 7.1 4.1 I10-N14 I10-N14 1.1 2.3 5.8 5.3 6.8 4.3 GALNS-242 C4-N7 + E8-N11 1.2 4.6 5.3 4.8 6.9 4.6 GALNS-201 C4-N6 + I10-N14 2.9 3.7 5.3 5.6 7.0 4.9 GALNS-315 E8-N14 + 1.4 2.4 3.8 5.0 7.0 3.9 I10-N14 GALNS-231 C4-N6 + E8- 2.8 2.5 5.3 5.0 6.5 4.4 N14 + I10-N14

[0044] Corresponding cDNA sequences were then constructed for the selected 7 modified amino acid sequences by in vitro mutagenesis. Corresponding cDNAs were then transcribed using in vitro systems known in the art, by way of example as described in U.S. patent application Ser. No. 13/760,907 (published as U.S. Patent Pub. No. 2013/0202633), incorporated herein by reference in its entirety.

[0045] In addition, post-translational modifications are an important factor affecting enzyme expression and activity in vivo. It is known that GALNS is modified post-translationally by another enzyme, Sulfatase-modifying factor 1 (SUMF1). To confirm that post-translationally modifications are maintained as they occur in vivo, the modified GALNS enzymes were also expressed and secreted from COS-7 cells co-transfected with cDNA for SUMF1. (See Examples)

[0046] Enzyme activity may be assessed through various in vitro techniques, by way of example those described in the examples below, as well as those described in Tomatsu at al., (2007) Mol Genet Metab. May; 91(1):69-78. Modified GALNS with reduced immunogenicity may then be used to treat subjects with Morquio IVA, using methods also know in the art and also as described in U.S. patent application Ser. No. 13/760,907 (published as U.S. Patent Pub. No. 2013/0202633).

[0047] It is expected that any of the modified GALNS enzymes described herein may be used to treat subjects with mucopolysaccharidoses type IVA, including experimental animal subjects and human subjects. Currently, a form of GALNS, branded as VIMIZIM (elosulfase alfa injection, solution, concentrate) (BioMarin Pharmaceutical inc.) is used to treat MPS IVA. The recommended dose is 2 mg per kg given intravenously over a minimum range of 3.5 to 4.5 hours, based on infusion volume, once every week. (See VIMIZIM product insert, and Briefing Document for The Endocrinologic And Metabolic Drugs Advisory Committee (BioMarin Pharmaceutical Inc)). It is expected that these protocols may be followed using equivalent amounts of modified enzyme, adjusted for any differences in activity. The amount of modified enzyme administered may be increased as necessary to compensate for any reduction in enzyme activity as was observed in some of the modified enzymes in vitro.

[0048] It is expected that an effective amount may be from: 0.1 mg to 1.0 mg per kg, mg 1 to 2 mg per kg, 2 mg to 3 mg per kg, 3 mg to 4 mg per kg, 4 mg to 5 mg per kg, 5 mg to 6 mg per kg, 6 mg to 8 mg per kg, 8 mg to 10 mg per kg, 10 mg to 15 mg per kg, 15 mg to 20 mg per kg, 20 mg to 30 mg per kg, 30 mg to 40 mg per kg, 40 mg to 50 mg per kg, or 50 mg to 100 mg per kg, or 100 mg to 200 mg per kg or more. The effective dose may be given, daily, weekly, every two weeks, every four weeks, monthly or combinations thereof.

[0049] Preferred embodiments of the invention are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.

EXAMPLES

Materials and Methods

[0050] Amino acid sequence substitutions. The FASTA format of the original GALNS amino acid sequence and modified GALNS amino acid sequences was utilized for all of the following in silico programs. In silico analyses were performed on new sequences harboring various combinations of amino acid substitutions within the 3 immunodominant peptide regions, C4, E8, and 110, within the wild GALNS sequence.

[0051] Sequences were created by altering one amino acid at a time within individual peptide regions C4, E8, and 110. Individual amino acids were changed based upon their polarity groups: amino acids with positively charged side chains, negatively charged side chains, polar uncharged side chains, and hydrophobic side chains; by way of example, alanine was changed to valine, leucine, isoleucine, and other amino acids with hydrophobic side chains. Additionally, cysteine was not modified because of its unique structure with sulfide bonding. Finally, amino acids were not changed to already known mutations of Morquio A disease. The least immunogenic single and multiple amino acid changes per peptide were selected. These sequences were then combined to create sequences with 2-3 changes within each peptide region, and then combined 1-3 of the mutated C4, E8 and 110 peptide regions until all possible combinations were made to yield a total of 324 mutated sequences.

Immunogenicity Predictions

[0052] Immune Epitope Database.sup.7 (IEDB) and RANKPEP.sup.8 predicted the immunogenicity of the T cell epitope to MHC II binding for the mouse allele H2-IAb of GALNS based upon different collections of previous studies' peptide-binding data sets. IEDB was used via the T Cell Epitope Prediction for Peptide binding to MHC Class II molecules. Here, the FASTA sequences were entered to predict the immunogenicity of Ff2-IAb allele mice by IEDB percentile rank. RANKPEP was used with MHC II and 1-Ab criteria, with the same mutated sequences that were entered in IEDB, to predict the immunogenicity by RANKPEP score. RANKPEP scores>9.0 were determined to be antigenic and were, therefore, excluded from further evaluation. A higher percentile rank indicated less immunogenicity for IEDB.sup.7, and a lower score indicated less immunogenicity for RANKPEP.sup.8.

Physico-Chemical Property Predictions

[0053] Group-based Prediction System version 2.1.sup.9 (GPS 2.1) (also see Xue at al., (2011) Protein Engineering, Design & Selection vol. 24 no. 3 pp. 255-260) and NetPhos 2.0 server.sup.10 (also see Blom et al., (1999) Journal of Molecular Biology: 294(5):1351-1362) determined possible predictions of phosphorylation sites. In GPS 2.1, all phosphorylation kinases were selected for the entered FASTA sequences. In NetPhos 2.0, a single sequence in FASTA format was entered and phosphorylation predictions were selected for tyrosine, serine, threonine, and graphics generation. A higher score according to GPS 2.1 and NetPhos 2.0 indicated that a predicted phosphorylation site was more probable. Criteria for selected sequences included no new or lost phosphorylation sites compared to the original GALNS sequence.

[0054] NetNGlyc 1.0.sup.11 (see Center for Biological Sequence Analysis, Technical University of Denmark) (Gupta et al., (2002) Pacific Symposium on Biocomputing 7:310-322). predicted the N glycosylation sites by simply pasting the FASTA sequences into the server. These N glycosylation predictions were compared to the N-glycosylation predictions of the original GALNS sequence from NetNGlyc 1.0. Sequences were selected if there were no new or lost N-glycosylation sites compared to the original GALNS.

[0055] The FASTA sequences were entered into Expasy ProtParam.sup.12 (see Gasteiger et al., (In) John M. Walker (ed): (2005) The Proteomics Protocols Handbook, Humana Press pp. 571-607) to analyze the physico-chemical properties compared to those of the original GALNS. These properties include molecular weight, theoretical p1, instability index, aliphatic index, and the grand average of hydropathicity (GRAVY). The lowest instability index indicated a more stable protein. The sequences with the most similar physico-chemical properties to the original GALNS sequence were selected.

PyMOL 3D Structural Analysis

[0056] I-TASSER.sup.13 was used to produce the in silico 3D molecular crystallography of protein structure and functions. I-TASSER provided 5 possible models to predict the crystallized structure of the proteins. 1 of 5 models per mutated protein was selected based upon the C-score of the model number. C-score is a confidence score for estimating the quality of predicted models by I-TASSER. C-score is typically in the range 2-5 where a C-score of higher value signifies a model with a high confidence and vice-versa.

[0057] These structures were then visualized via PyMOL vl.leval.sup.14 to analyze the molecular docking and ligand binding of the selected mutated sequences as compared to the structure and binding sites of the original GALNS protein. Mutated sequences were compared to the I-TASSER prediction of GALNS model 1 to maintain consistency within the same protein structure and function prediction server. The two substrates (1) N-acetyl-D-Galactosamine (NGA) and (2) calcium that bind to the active site of GALNS were utilized from the RCSB PDB protein data bank PDB code 4FDJ2 (Berman et al., (2000) Nuclide Acid Research 28: 235-343). Each mutated sequence was superimposed onto GALNS model land the PDB: 4FDJ substrates by aligning the structures on PyMOL. Amino acids within the GALNS active site were determined by selecting residues within 6 angstroms () of the Ca.sup.2+ and NGA. Of those amino acids, PyMOL predicted the polar bonds between amino acids and the substrates based upon, distance between atoms (3.4-4.0 ) and angular cutoffs. These distances were measured between individual atoms of the binding site amino acids and the atoms of the substrate to compare those distances of the mutated proteins and the original GALNS model 1 protein structure. Restriction enzyme predictions.

[0058] Table 4 illustrates wild type GALNS (SEQ ID NO:1), including: the signal peptide at residues 1-26; immunodominant region C4 at residues 163-182; immunodominant region E8 at residues 226-245; and immunodominant region I10 at residues 473-492. Modified GALNS sequences (SEQ ID NO:2-SEQ ID NO:8) are designated according to the modified region(s) followed by the modification. Amino acid residues that have been substituted are indicated by both bold and underlining. The sequences in Table 4 represent polypeptides prior to post-translational modification.

TABLE-US-00007 TABLE4 WildtypeandmodifiedaminoacidsequencesforGALNSwithreduced immunogenicity.Immunodominantpeptideregionsareshaded. Modificationsareboldandunderlined. WildtypeGALNS(SEQIDNO:1) MAAVVAATRWWQLLLVLSAAGMGASGAPQPPNILLLLMDDMGWGDLGVYGEPSR ETPNLDRMAAEGLLFPNFYSANPLCSPSRAALLTGRLPIRNGFYTTNAHARNAYTPQ EIVGGIPDSEQLLPELLKKAGYVSKIVGKWHLGHRPQFHPLKHGFDEWFGSPNCHF GPYDNKARPNIPVYRDWEMVGRYYEEFPINLKTGEANLTQIYLQEALDFIKRQARHH PFFLYWAVDATHAPVYASKPFLGTSQRGRYGDAVREIDDSIGKILELLQDLHVADNT FVFFTSDNGAALISAPEQGGSNGPFLCGKQTTFEGGMREPALAWWPGHVTAGQVS HQLGSIMDLFTTSLALAGLTPPSDRAIDGLNLLPTLLQGRLMDRPIFYYRGDTLMAAT LGQHKAHFWTWTNSWENFRQGIDFCPGQNVSGVTTHNLEDHTKLPLIFHLGRDPG ERFPLSFASAEYQEALSRITSVVQQHQEALVPAQPQLNVCNWAVMNWAPPGCEKL GKCLTPPESIPKKCLWSH C4-N6(SEQIDNO:2) MAAVVAATRWWQLLLVLSAAGMGASGAPQPPNILLLLMDDMGWGDLGVYGEPSR ETPNLDRMAAEGLLFPNFYSANPLCSPSRAALLTGRLPIRNGFYTTNAHARNAYTPQ EIVGGIPDSEQLLPELLKKAGYVSKIVGKWHLGHRPQFHPLKHGFDEWFGSPNCHF GPYDNKIRGQIPVYRDWEMVGRYYEEFPINLKTGEANLTQIYLQEALDFIKRQARHH PFFLYWAVDATHAPVYASKPFLGTSQRGRYGDAVREIDDSIGKILELLQDLHVADNT FVFFTSDNGAALISAPEQGGSNGPFLCGKQTTFEGGMREPALAWWPGHVTAGQVS HQLGSIMDLFTTSLALAGLTPPSDRAIDGLNLLPTLLQGRLMDRPIFYYRGDTLMAAT LGQHKAHFWTWTNSWENFRQGIDFCPGQNVSGVTTHNLEDHTKLPLIFHLGRDPG ERFPLSFASAEYQEALSRITSVVQQHQEALVPAQPQLNVCNWAVMNWAPPGCEKL GKCLTPPESIPKKCLWSH E8-N13(SEQIDNO:3) MAAVVAATRWWQLLLVLSAAGMGASGAPQPPNILLLLMDDMGWGDLGVYGEPSR ETPNLDRMAAEGLLFPNFYSANPLCSPSRAALLTGRLPIRNGFYTTNAHARNAYTPQ EIVGGIPDSEQLLPELLKKAGYVSKIVGKWHLGHRPQFHPLKHGFDEWFGSPNCHF GPYDNKARPNIPVYRDWEMVGRYYEEFPINLKTGEANLTQIYLQEALDFIKRQARHH PFFLIWAVDLTHLPVYASKPFLGTSQRGRYGDAVREIDDSIGKILELLQDLHVADNTF VFFTSDNGAALISAPEQGGSNGPFLCGKQTTFEGGMREPALAWWPGHVTAGQVSH QLGSIMDLFTTSLALAGLTPPSDRAIDGLNLLPTLLQGRLMDRPIFYYRGDTLMAATL GQHKAHFWTWTNSWENFRQGIDFCPGQNVSGVTTHNLEDHTKLPLIFHLGRDPGE RFPLSFASAEYQEALSRITSVVQQHQEALVPAQPQLNVCNWAVMNWAPPGCEKLG KCLTPPESIPKKCLWSH I10-N14(SEQIDNO:4) MAAVVAATRWWQLLLVLSAAGMGASGAPQPPNILLLLMDDMGWGDLGVYGEPSR ETPNLDRMAAEGLLFPNFYSANPLCSPSRAALLTGRLPIRNGFYTTNAHARNAYTPQ EIVGGIPDSEQLLPELLKKAGYVSKIVGKWHLGHRPQFHPLKHGFDEWFGSPNCHF GPYDNKARPNIPVYRDWEMVGRYYEEFPINLKTGEANLTQIYLQEALDFIKRQARHH PFFLYWAVDATHAPVYASKPFLGTSQRGRYGDAVREIDDSIGKILELLQDLHVADNT FVFFTSDNGAALISAPEQGGSNGPFLCGKQTTFEGGMREPALAWWPGHVTAGQVS HQLGSIMDLFTTSLALAGLTPPSDRAIDGLNLLPTLLQGRLMDRPIFYYRGDTLMAAT LGQHKAHFWTWTNSWENFRQGIDFCPGQNVSGVTTHNLEDHTKLPLIFHLGRDPG ERFPLSFASAEYQEALSRITSVVQQHQEALVGIQGQLNVCNWAVMNWAPPGCEKL GKCLTPPESIPKKCLWSH GALNS-201(C4-N6+ I10-N14)(SEQIDNO:5) MAAVVAATRWWQLLLVLSAAGMGASGAPQPPNILLLLMDDMGWGDLGVYGEPSR ETPNLDRMAAEGLLFPNFYSANPLCSPSRAALLTGRLPIRNGFYTTNAHARNAYTPQ EIVGGIPDSEQLLPELLKKAGYVSKIVGKWHLGHRPQFHPLKHGFDEWFGSPNCHF GPYDNKIRGQIPVYRDWEMVGRYYEEFPINLKTGEANLTQIYLQEALDFIKRQARHH PFFLYWAVDATHAPVYASKPFLGTSQRGRYGDAVREIDDSIGKILELLQDLHVADNT FVFFTSDNGAALISAPEQGGSNGPFLCGKQTTFEGGMREPALAWWPGHVTAGQVS HQLGSIMDLFTTSLALAGLTPPSDRAIDGLNLLPTLLQGRLMDRPIFYYRGDTLMAAT LGQHKAHFWTWTNSWENFRQGIDFCPGQNVSGVTTHNLEDHTKLPLIFHLGRDPG ERFPLSFASAEYQEALSRITSVVQQHQEALVGIQGQLNVCNWAVMNWAPPGCEKL GKCLTPPESIPKKCLWSH GALNS-231(C4-N6+ E8-N14+ I10-N14)(SEQIDNO:6) MAAVVAATRWWQLLLVLSAAGMGASGAPQPPNILLLLMDDMGWGDLGVYGEPSR ETPNLDRMAAEGLLFPNFYSANPLCSPSRAALLTGRLPIRNGFYTTNAHARNAYTPQ EIVGGIPDSEQLLPELLKKAGYVSKIVGKWHLGHRPQFHPLKHGFDEWFGSPNCHF GPYDNKIRGQIPVYRDWEMVGRYYEEFPINLKTGEANLTQIYLQEALDFIKRQARHH PFFLIWAVDLTHIPVYASKPFLGTSQRGRYGDAVREIDDSIGKILELLQDLHVADNTFV FFTSDNGAALISAPEQGGSNGPFLCGKQTTFEGGMREPALAWWPGHVTAGQVSH QLGSIMDLFTTSLALAGLTPPSDRAIDGLNLLPTLLQGRLMDRPIFYYRGDTLMAATL GQHKAHFWTWTNSWENFRQGIDFCPGQNVSGVTTHNLEDHTKLPLIFHLGRDPGE RFPLSFASAEYQEALSRITSVVQQHQEALVGIQGQLNVCNWAVMNWAPPGCEKLG KCLTPPESIPKKCLWSH GALNS-242(C4-N7+ E8-N11)(SEQIDNO:7) MAAVVAATRWWQLLLVLSAAGMGASGAPQPPNILLLLMDDMGWGDLGVYGEPSR ETPNLDRMAAEGLLFPNFYSANPLCSPSRAALLTGRLPIRNGFYTTNAHARNAYTPQ EIVGGIPDSEQLLPELLKKAGYVSKIVGKWHLGHRPQFHPLKHGFDEWFGSPNCHF GPYDNKLRGQIPVYRDWEMVGRYYEEFPINLKTGEANLTQIYLQEALDFIKRQARHH PFFLIWLVDATHLPVYASKPFLGTSQRGRYGDAVREIDDSIGKILELLQDLHVADNTF VFFTSDNGAALISAPEQGGSNGPFLCGKQTTFEGGMREPALAWWPGHVTAGQVSH QLGSIMDLFTTSLALAGLTPPSDRAIDGLNLLPTLLQGRLMDRPIFYYRGDTLMAATL GQHKAHFWTWTNSWENFRQGIDFCPGQNVSGVTTHNLEDHTKLPLIFHLGRDPGE RFPLSFASAEYQEALSRITSVVQQHQEALVPAQPQLNVCNWAVMNWAPPGCEKLG KCLTPPESIPKKCLWSH GALNS-315(E8-N14+ I10-N14)(SEQIDNO:8) MAAVVAATRWWQLLLVLSAAGMGASGAPQPPNILLLLMDDMGWGDLGVYGEPSR ETPNLDRMAAEGLLFPNFYSANPLCSPSRAALLTGRLPIRNGFYTTNAHARNAYTPQ EIVGGIPDSEQLLPELLKKAGYVSKIVGKWHLGHRPQFHPLKHGFDEWFGSPNCHF GPYDNKARPNIPVYRDWEMVGRYYEEFPINLKTGEANLTQIYLQEALDFIKRQARHH PFFLIWAVDLTHIPVYASKPFLGTSQRGRYGDAVREIDDSIGKILELLQDLHVADNTFV FFTSDNGAALISAPEQGGSNGPFLCGKQTTFEGGMREPALAWWPGHVTAGQVSH QLGSIMDLFTTSLALAGLTPPSDRAIDGLNLLPTLLQGRLMDRPIFYYRGDTLMAATL GQHKAHFWTWTNSWENFRQGIDFCPGQNVSGVTTHNLEDHTKLPLIFHLGRDPGE RFPLSFASAEYQEALSRITSVVQQHQEALVGIQGQLNVCNWAVMNWAPPGCEKLG KCLTPPESIPKKCLWSH

[0059] The FASTA format of the cDNA sequences were used for the determination of restriction enzyme sites and primer design via in silico programs. Finally, DNASIS MAX v3.0.sup.15 predicted restriction enzyme sites; the FASTA format of the original GALNS cDNA sequence and modified sequences were used for DNASIS MAX v3.0. cDNA sequences were translated to the amino acid sequence from which the restriction enzyme cut map was determined. The GALNS and mutated sequences were compared to find the same restriction enzymes that cut at the same amino acid sequence.

Primer Design

[0060] QuikChange Primer Design.sup.16 was used to computationally determine the primer sequences. Gene synthesis and mutagenesis of 7 selected sequences were ordered through GenScript.

Production of Modified Enzymes and Enzyme Activity

[0061] Corresponding cDNAs were then transcribed using in vitro systems. Seven modified GALNS enzymes were produced using standard cloning techniques and tested in vitro to determine GALNS activity.

[0062] Transfection: HEK-293 cells (2.0-6.010.sup.4 cells) were incubated with 1 g of cDNA and 2 l of Turbofect (ThermoFisher R0531)(cationic polymer in water). The media was changed after 4 hours and incubation continued for 72 hours. Media and cells were then washed 3 times with PBS. Di-deoxycholate (300 l) was added, and the preparation centrifuged. A 30 l aliquot of the pellet was assayed for GALNS activity and protein determination.

Enzyme Activity

[0063] GALNS activity was determined as described in Tomatsu at al., (2007) Mol Genet Metab. May; 91(1):69-78. pCXN, transfected with the plasmid vector alone represents background. Percent activity was calculated relative to wild type GALNS (100 percent).

TABLE-US-00008 TABLE 5 Enzyme activity is expressed as a percentage of wild type GALNS. (n = 3). Sample Percent activity C4-N6 32.004 E8-N13 46.97 I10-N14 40.647 201 64.093 242 84.82 315 81.014 231 90.448 Wild type GALNS 99.995 p.CXN (Plasmid alone) 43.271

[0064] Comparison of GALNS activity in modified GALNS enzymes, relative to wild type GALNS and p.CXN, indicates that GALNS modified with peptides 231, 242, 315, and 201, show acceptable functional activity (see Table 5).

Production of Modified Amino Acid Sequences for GALNS with Reduced Immunogenicity was Also Carried out in COS-7 Cells Co-Transfected with SUMF1.

[0065] To examine the effects amino acid substitutes on post-transcription modifications. Production of modified GALNS in COS-7 cells co-transfected was carried out as follows: COS-7 cells (2.010.sup.5 cells) were incubated with 4 g of cDNA and 8 l of Turbofect (ThermoFisher RO531)(cationic polymer in water). The media was changed after 4 hours and incubation continued for 72 hours. Media and cells were then washed 3 times with PBS. Di-deoxycholate (300 l) was added, and the preparation was centrifuged. Double distilled H.sub.2O (30 l) was added to the pellet and GALNS enzyme activity and protein were assayed as shown in FIG. 5 and Table 6.

TABLE-US-00009 TABLE 6 GALNS activity in modified peptides expressed in COS-7 cells co-transfected with SUMF1. Enzyme Activity Pellet U/mg Percentage C4-N6 + SUMF1 0.576 443.08 E8-N13 + SUMF1 0.737 566.92 I10-N14 + SUMF1 0.738 567.69 201 + SUMF1 0.708 544.62 242 + SUMF1 0.564 433.85 315 + SUMF1 0.732 563.08 231 + SUMF1 0.817 628.46 GALNS 0.130 100.00 GALNS + SUMF1 2.508 1929.23

[0066] As illustrated in FIG. 5 and Table 6, modified GALNS enzymes have enhanced activity when produced in eukaryotic cells co-transfected with SUMF1.

[0067] In each of the following embodiments, and in the claims, the reference to percent GALNS activity is in reference to GALNS, and not in reference to GALNS+SUMF1, as shown in Table 6.

[0068] In one embodiment of the invention, is a modified GALNS enzyme where the amino acid Isoleucine is substituted at position 174, the amino acid Glycine is substituted at position 176, and the amino acid Glutamine is substituted at position 177, by way of non-limiting example, as set forth in SEQ ID NO:2.

[0069] In another embodiment of the invention is the sequence set forth in SEQ ID NO:2 secreted from a eukaryotic cell.

[0070] In another embodiment of the invention is a modified GALNS enzyme where the amino acid Isoleucine is substituted at position 229, the amino acid Leucine is substituted at position 234, and the amino acid Leucine is substituted at position 237, by way of non-limiting example, as set forth in SEQ ID NO:3.

[0071] In another embodiment of the invention is the sequence set forth in SEQ ID NO:3 secreted from a eukaryotic cell.

[0072] In another embodiment of the invention is a modified GALNS enzyme where the amino acid Glycine is substituted at position 481, the amino acid Isoleucine is substituted at position 482, and the amino acid Glycine is substituted at position 484, by way of non-limiting example, as set forth in SEQ ID NO:4.

[0073] In another embodiment of the invention is the sequence set forth in SEQ ID NO:4 secreted from a eukaryotic cell.

[0074] In another embodiment of the invention is a modified GALNS enzyme where the amino acid Isoleucine is substituted at position 174, the amino acid Glycine is substituted at position 176, the amino acid Glutamine is substituted at position 177, the amino acid Glycine is substituted at position 481, the amino acid Isoleucine is substituted at position 482, and the amino acid Glycine is substituted at position 484, by way of non-limiting example, as set forth in SEQ ID NO:5.

[0075] In another embodiment of the invention is the sequence set forth in SEQ ID NO:5 secreted from a eukaryotic cell.

[0076] In another embodiment of the invention, is a modified GALNS enzyme where the amino acid Isoleucine is substituted at position 174, the amino acid Glycine is substituted at position 176, the amino acid Glutamine is substituted at position 177, amino acid Isoleucine is substituted at position 229, the amino acid Leucine is substituted at position 234, the amino acid Isoleucine is substituted at position 237, the amino acid Glycine is substituted at position 481, the amino acid Isoleucine is substituted at position 482, and the amino acid Glycine is substituted at position 484, by way of non-limiting example, as set forth in SEQ ID NO:6.

[0077] In another embodiment of the invention is the sequence set forth in SEQ ID NO:6 secreted from a eukaryotic cell.

[0078] In another embodiment of the invention is a modified GALNS enzyme where the amino acid Leucine is substituted at position 174, the amino acid Glycine is substituted at position 176, the amino acid Glutamine is substituted at position 177, amino acid Isoleucine is substituted at position 229, the amino acid Leucine is substituted at position 234, and the amino acid Leucine is substituted at position 237, by way of non-limiting example, as set forth in SEQ ID NO:7.

[0079] In another embodiment of the invention is the sequence set forth in SEQ ID NO:7 secreted from a eukaryotic cell.

[0080] In yet another embodiment of the invention is a modified GALNS enzyme where amino acid Isoleucine is substituted at position 229, the amino acid Leucine is substituted at position 234, and the amino acid Isoleucine is substituted at position 237, the amino acid Glycine is substituted at position 481, the amino acid Isoleucine is substituted at position 482, and the amino acid Glycine is substituted at position 484, by way of non-limiting example, as set forth in SEQ ID NO:8.

[0081] In another embodiment of the invention is the sequence set forth in SEQ ID NO:8 secreted from a eukaryotic cell.

[0082] Additional embodiments of the invention include each of the above embodiments, secreted from a eukaryote cell, with 60 percent of more GALNS activity.

[0083] In yet another embodiment of the invention is a method of treating a subject with mucopolysaccharidoses type IVA, comprising: administering, intravenously, an effective amount of any of the modified N-acetylgalactosamine-6-sulfate sulfatase (GALNS) described herein, including but not limited to those set forth in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, and secreted from a eukaryote cell.

[0084] In yet another embodiment of the invention is a method of modifying proteins used in enzyme replacement therapy to treat subjects with lysosomal storage disorders to provide modified enzymes reduced immunogenic properties proteins.

[0085] It is recognized that the amino acid substitutions disclosed herein, made in wild type GALNS, are not limited to wild type GALNS, and may be in other variants or mutant forms of GALNS with equal or similar effectiveness.

[0086] Non-limiting examples of enzyme replacement therapy are described in U.S. patent application Ser. No. 13/760,907 (published as U.S. Patent Pub. No. 2013/0202633), incorporated herein by reference in its entirety. However, it is recognized that enzyme replacement therapy may be administered using other methodologies. By way of example, enzyme replacement therapy may be administered using methodology commonly known as gene therapy, wherein an oligonucleotide encoding the target enzyme is administered to the subject in such a manner that the target enzyme is expressed by the subject.

[0087] All publications and patents cited in this specification are hereby incorporated by reference in their entirety. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references.

REFERENCES

[0088] 1. Tomatsu S M A, Qikawa H, Smith M, Barrera L, Chinen Y, Thacker M M, Mackenzie W G, Suzuki V, OrH T. Mucopolysaccharidosis type IVA (Morquio A disease): clinical review and current treatment. Curr Pharm Biotechnol. 2011; 12: 931-945.

[0089] 2. Rivera-Colon Y S E, Kita A Z, Garman S C. The molecular basis of mucopolysaccharidosis IVA, complex with GalNac. J Mol Biol., 2012:736-751.

[0090] 3. Montano A M T S, Gottesman G S, Smith M, Drii T. International Morquio A Registry: clinical manifestation and natural course of Morquio A disease. J Inherit Metab Dis. 2007:30: 165-1 74.

[0091] 4. Tomatsu S M A, Dung V C, Ohashi A, Dikawa H, Oguma T, OrU T, Barrera L, Sly W S. Enhancement of Drug Delivery: Enzyme-replacement Therapy for Murine Morquio A Syndrome. Molecular Therapy. 2010:18: 1094-1102.

[0092] 5. Hendriksz C, Burton, B., Fleming, T., Giugliani, R., Harmatz, P., Hughes, D., Jones, S., Lin, P., Mengel, K., Scarpa, M., Valayannopoulos, V. A multi-national, randomized, double-blind, placebo-controlled study to evaluate the efficacy and safety of BMN 110 treatment for mucopolysaccharidosis IVA (Morquio syndrome type A). Mol Genet Metab. 2013; 108: S17-5102.

[0093] 6. Fireman M, DiMartini, A. F., Armstrong, S. C., Cozza, K. L. Immunosuppressants. Psychosomatics. 2004; 45: 354-360.

[0094] 7. Nielsen M L C, Lund 0. Prediction of MHC class II binding affinity using SMM-align, a novel stabilization matrix alignment method. BMC Bioinformatics. 2007; 8.

[0095] 8. Reche P A GJaRE. Prediction of MHC Class I Binding Peptides Using Profile Motifs. Human Immunology. 2002; 63: 701-709.

[0096] 9. Xue Y R J, Gao X, Jin C, Wen L, Yao X. GPS 2.0, a Tool to Predict Kinase-specific Phosphorylation Sites in Hierarchy. Mol Cell Proteomics. 2008; 7: 1598-1 608.

[0097] 10. Blom N, Gammeltoff, S., Brunak, S. Sequence- and structure-based prediction of eukaryotic protein phosphorylation sites. Journal of Molecular Biology. 1999; 294: 1351-1362.

[0098] 11. Gupta R, Jung, E., and Brunak, S. Prediction of N-glycosylation sites in human proteins, unpublished. 2004.

[0099] 12. Gasteiger E. H C, Gattiker A., Duvaud S., Wilkins M. R., Appel R. D., Bairoch A. Protein Identification and Analysis Tools on the ExPASy Server; (In) John M. Walker (ed): The Proteomics Protocols Handbook, Humana Press. 2005: 57-607.

[0100] 13. Zhang Y. 1-TASSER server for protein 3D structure prediction. BMC Bioinformatics. 2008; 9.

[0101] 14. The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrdinger, LLC.

[0102] 15. DNASIS MAX, Version 3.0. Hitachi Solutions America, Ltd. MiraiBio Group.

[0103] 16. A. Novoradovsky VZ, M. Ghosh, H. Hogrefe, J. A. Sorge and T. Gaasterland. Computational Principles of Primer Design for Site Directed Mutagenesis. Nanotech. 2005; 1: 532-535.