臺灣博碩士論文加值系統

溶酶體貯積症肇因於參與細胞代謝的酵素缺乏，酵素替代療法是一種對多數溶酶體貯積症患者有效的治療方式。然而部分患者在接受酵素注射後所引發的免疫反應會阻礙治療效果並導致嚴重的副作用。我們在此研究一種通用的方式以減少酵素替代療法之免疫原性，即透過改變正常表現之內源性酵素的專一性以補償患者之缺陷酵素。我們試圖鑑別出擁有己醛醣酸鹽水解酵素活性之人類乙型葡萄糖醛酸苷酶，前者的缺乏即為第一型黏多醣症的成因。我們利用可裂解之表面系鏈酵素多用途篩選系統（ECSATSY）來篩選乙型葡萄糖醛酸苷酶文庫，以期分離出對己醛醣酸鹽水解酵素受質MUI具有活性之變異型酵素。相較於野生型乙型葡萄糖醛酸苷酶，我們成功篩選出擁有100至290倍己醛醣酸鹽水解酵素活性之變異型，其酵素專一性亦呈現出7900到24500倍的改變。在治療第一型黏多醣症細胞的體外實驗中，變異型乙型葡萄糖醛酸苷酶顯著地改變其醣胺多醣的累積，改善其細胞型態，並且恢復其溶酶體外觀。這些實驗結果表明了變異型乙型葡萄糖醛酸苷酶對修正第一型黏多醣症細胞表型缺陷的有益效果。總結來說，對於改變正常表現之內源性酵素的專一性以補償另一缺陷酵素的方法，我們的研究提供了原理上的驗證。實驗結果顯示此種策略可能代表一種用以生產具較低免疫原性的治療用酵素之方法。部分溶酶體貯積症患者對於傳統酵素替代療法由於抗體反應而療效不彰，此種策略對這類病患在治療上的應用應該有進一步的研究價值。

Enzyme replacement therapy (ERT) is an effective treatment for many patients with lysosomal storage disorders (LSDs) caused by deficiency in enzymes involved in cell metabolism. However, immune responses that develop against the administered enzyme in some patients can hinder therapeutic efficacy and cause serious side effects. Here we investigated the feasibility of a general approach to decrease ERT immunogenicity by altering the specificity of a normal endogenous enzyme to compensate for a defective enzyme. We sought to identify human beta-glucuronidase variants that display alpha-iduronidase activity, which is defective in mucopolysaccharidosis (MPS) type I patients. A human beta-glucuronidase library was created and screened by the Enzyme Cleavable Surface Tethered All-purpose Screen sYstem (ECSTASY) to isolate variants that exhibited 100 to 290-fold greater activity against the alpha-iduronidase substrate 4-methylumbelliferyl alpha-L-iduronide (MUI) and 7900 to 24500-fold enzymatic specificity shift as compared to wild-type beta-glucuronidase. In vitro treatment of MPS I cells with the beta-glucuronidase variants significantly altered the cellular glycosaminoglycans accumulation, improved cell morphology, and restored lysosome appearance as compared to treatment with wild-type beta-glucuronidase. These results revealed that the beta-glucuronidase variants exhibited beneficial effects toward correction of the phenotype defects of MPS I cells. In conclusion, our study provides a proof of principle that the specificity of a normally expressed endogenous human enzyme can be shifted to compensate for a separate defective enzyme. This strategy may represent a possible approach to produce enzymes that display therapeutic benefits with potentially less immunogenicity. It should be worthy of further investigation for therapeutic applications to LSDs patients, especially who responded poorly to conventional ERT due to antibody responses.

Contents
List of Tables ............................................................................................................................IV
List of Figures ........................................................................................................................... V
List of Appendix......................................................................................................................VII
摘要....................................................................................................................................... VIII
Abstract ..................................................................................................................................... X
Chapter 1 Introduction........................................................................................................... 1
1.1 Lysosomal storage disorders ........................................................................................ 1
1.1.1 Mucopolysaccharidoses (MPS) .......................................................................... 2
1.2 Treatment of lysosomal storage diseases ..................................................................... 4
1.2.1 Enzyme replacement therapy.............................................................................. 4
1.2.2 Substrate reduction therapy ................................................................................ 5
1.2.3 Chaperone therapy.............................................................................................. 5
1.2.4 Gene therapy and hematopoietic stem cell transplantation ................................ 6
1.3 Immune responses to protein therapeutics in LSDs..................................................... 8
1.4 Strategies to deimmunize protein therapeutics ............................................................ 9
1.5 Hypothesis and design ............................................................................................... 10
1.6 References .................................................................................................................. 12
Chapter 2 Creating human beta-glucuronidase variants with enhanced alpha-iduronidase activity…….............................................................................................................................. 23
2.1 Summary .................................................................................................................... 23
2.2 Introduction................................................................................................................ 25
2.3 Materials and methods ............................................................................................... 27
2.3.1 Reagents and antibodies ................................................................................... 27
2.3.2 Cell culture ....................................................................................................... 27
2.3.3 Structure analysis of human beta-glucuronidase and alpha-iduronidase.......... 27
2.3.4 Synthetic DNA library construction ................................................................. 28
2.3.5 Purification of MUI by high-performance liquid chromatography.................. 29
2.3.6 Alpha-iduronidase activity assay with MUI..................................................... 30
2.3.7 Establishment of library cells by retroviral transduction.................................. 30
2.3.8 Flow cytometer analysis and selection of library cells..................................... 30
2.3.9 Surface enzyme release by PI-PLC treatment .................................................. 31
2.3.10 Sandwich enzyme-linked immunosorbent assay (ELISA)............................... 31
2.3.11 Statistical analysis............................................................................................. 32
2.4 Results........................................................................................................................ 33
2.4.1 Purification of commercial 4-methylumbelliferyl alpha-L-iduronide (MUI) .. 33
2.4.2 Hydrolysis of alpha-iduronidase substrate by human beta-glucuronidase ....... 33
2.4.3 Structure analysis of human beta-glucuronidase and alpha-iduronidase.......... 34
2.4.4 Library construction and sequencing................................................................ 34
2.4.5 Optimization of alpha-iduronidase activity assay for beta-glucuronidase library screening....................................................................................................................... 36
2.4.6 Selection of cell model for ECSTASY............................................................. 37
2.4.7 Library screening using ECSTASY.................................................................. 38
2.5 Discussions................................................................................................................. 40
2.6 References .................................................................................................................. 43
Chapter 3 Characterization of human beta-glucuronidase variants displaying enhanced alpha-iduronidase activity ........................................................................................................ 66
3.1 Summary .................................................................................................................... 66
3.2 Introduction................................................................................................................ 68
3.3 Materials and methods ............................................................................................... 70
3.3.1 Reagents and antibodies ................................................................................... 70
3.3.2 Cell culture ....................................................................................................... 70
3.3.3 Production of secreted recombinant human beta-glucuronidase variants ........ 71
3.3.4 Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) ...................................................................................................... 72
3.3.5 Extraction and determination of total GAGs.................................................... 72
3.3.6 Radioactive labelling of cellular GAGs............................................................ 73
3.3.7 Transmission electron microscopy (TEM) ....................................................... 74
3.3.8 Lysosome staining and image acquisition ........................................................ 74
3.3.9 Statistical analysis............................................................................................. 75
3.4 Results........................................................................................................................ 76
3.4.1 Verification of enhanced alpha-iduronidase activity of human beta-glucuronidase variants screened by ECSTASY.................................................... 76
3.4.2 Characterization of selected beta-glucuronidase variants (102H1, 101C7, and 70H1) .......................................................................................................................... 76
3.4.3 Phenotypic effect of beta-glucuronidase variants on cellular GAGs accumulation................................................................................................................. 77
3.4.4 Phenotypic effect of beta-glucuronidase variants on MPS I cell morphology . 79
3.4.5 Phenotypic effect of beta-glucuronidase variants on cellular lysosome appearance .................................................................................................................... 79
3.5 Discussions................................................................................................................. 81
3.6 References .................................................................................................................. 86
Appendix ..........................................................................................................105

1. Settembre, C., et al., A lysosome-to-nucleus signalling mechanism senses and regulates the lysosome via mTOR and TFEB. Embo j, 2012. 31(5): p. 1095-108.
2. Appelqvist, H., et al., The lysosome: from waste bag to potential therapeutic target. J Mol Cell Biol, 2013. 5(4): p. 214-26.
3. Fletcher, J.M., Screening for lysosomal storage disorders--a clinical perspective. J Inherit Metab Dis, 2006. 29(2-3): p. 405-8.
4. Schultz, M.L., et al., Clarifying lysosomal storage diseases. Trends Neurosci, 2011. 34(8): p. 401-10.
5. Neufeld, E.F. and J. Muenzer, The mucopolysaccharidoses, in The Metabolic & Molecular Bases of Inherited Disease, 8th ed, C.R. Scriver, et al., Editors. 2001, McGraw-Hill: New York, NY, USA, . p. 3421-3452.
6. Scott, H.S., et al., Chromosomal localization of the human alpha-L-iduronidase gene (IDUA) to 4p16.3. Am J Hum Genet, 1990. 47(5): p. 802-7.
7. Kakkis, E.D., et al., Enzyme-replacement therapy in mucopolysaccharidosis I. N Engl J Med, 2001. 344(3): p. 182-8.
8. Staretz-Chacham, O., et al., Lysosomal storage disorders in the newborn. Pediatrics, 2009. 123(4): p. 1191-207.
9. Wang, J., et al., Neutralizing antibodies to therapeutic enzymes: considerations for testing, prevention and treatment. Nat Biotechnol, 2008. 26(8): p. 901-8.
10. Pastores, G.M., A.R. Sibille, and G.A. Grabowski, Enzyme therapy in Gaucher disease type 1: dosage efficacy and adverse effects in 33 patients treated for 6 to 24 months. Blood, 1993. 82(2): p. 408-16.
11. Zarate, Y.A. and R.J. Hopkin, Fabry's disease. Lancet, 2008. 372(9647): p. 1427-35.
12. Harmatz, P., et al., Enzyme replacement therapy for mucopolysaccharidosis VI: a phase 3, randomized, double-blind, placebo-controlled, multinational study of recombinant human N-acetylgalactosamine 4-sulfatase (recombinant human arylsulfatase B or rhASB) and follow-on, open-label extension study. J Pediatr, 2006. 148(4): p. 533-539.
13. Kishnani, P.S., et al., Chinese hamster ovary cell-derived recombinant human acid alpha-glucosidase in infantile-onset Pompe disease. J Pediatr, 2006. 149(1): p. 89-97.
14. Amalfitano, A., et al., Recombinant human acid alpha-glucosidase enzyme therapy forinfantile glycogen storage disease type II: results of a phase I/II clinical trial. Genet Med, 2001. 3(2): p. 132-8.
15. Kishnani, P.S., et al., Cross-reactive immunologic material status affects treatment outcomes in Pompe disease infants. Mol Genet Metab, 2009.
16. Linthorst, G.E., et al., Enzyme therapy for Fabry disease: neutralizing antibodies toward agalsidase alpha and beta. Kidney Int, 2004. 66(4): p. 1589-95.
17. Ohashi, T., et al., Reduced alpha-Gal A enzyme activity in Fabry fibroblast cells and Fabry mice tissues induced by serum from antibody positive patients with Fabry disease. Mol Genet Metab, 2008. 94(3): p. 313-8.
18. Sun, B., et al., Enhanced response to enzyme replacement therapy in Pompe disease after the induction of immune tolerance. Am J Hum Genet, 2007. 81(5): p. 1042-9.
19. Ohashi, T., et al., Administration of Anti-CD3 Antibodies Modulates the Immune Response to an Infusion of [alpha]-glucosidase in Mice. Mol Ther, 2012. 20(10): p. 1924-1931.
20. Banugaria, S.G., et al., Persistence of high sustained antibodies to enzyme replacement therapy despite extensive immunomodulatory therapy in an infant with Pompe disease: need for agents to target antibody-secreting plasma cells. Mol Genet Metab, 2012. 105(4): p. 677-80.
21. Patel, T.T., et al., The impact of antibodies in late-onset Pompe disease: a case series and literature review. Mol Genet Metab, 2012. 106(3): p. 301-9.
22. Dickson, P., et al., Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I. J Clin Invest, 2008. 118(8): p. 2868-76.
23. Cox-Brinkman, J., et al., Ultrastructural analysis of dermal fibroblasts in mucopolysaccharidosis type I: Effects of enzyme replacement therapy and hematopoietic cell transplantation. Ultrastruct Pathol, 2010. 34(3): p. 126-32.
24. Schiffmann, R., et al., Randomized, controlled trial of miglustat in Gaucher's disease type 3. Ann Neurol, 2008. 64(5): p. 514-22.
25. Cox, T., et al., Novel oral treatment of Gaucher's disease with N-butyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis. Lancet, 2000. 355(9214): p. 1481-5.
26. Maegawa, G.H., et al., Substrate reduction therapy in juvenile GM2 gangliosidosis. Mol Genet Metab, 2009. 98(1-2): p. 215-24.
27. Elliot-Smith, E., et al., Beneficial effects of substrate reduction therapy in a mouse model of GM1 gangliosidosis. Mol Genet Metab, 2008. 94(2): p. 204-11.
28. Kuter, D.J., et al., Miglustat therapy in type 1 Gaucher disease: clinical and safety outcomes in a multicenter retrospective cohort study. Blood Cells Mol Dis, 2013. 51(2): p. 116-24.
29. Boyd, R.E., et al., Pharmacological chaperones as therapeutics for lysosomal storage diseases. J Med Chem, 2013. 56(7): p. 2705-25.
30. Fan, J.Q., et al., Accelerated transport and maturation of lysosomal alpha-galactosidase A in Fabry lymphoblasts by an enzyme inhibitor. Nat Med, 1999. 5(1): p. 112-5.
31. Khanna, R., et al., The pharmacological chaperone 1-deoxygalactonojirimycin reduces tissue globotriaosylceramide levels in a mouse model of Fabry disease. Mol Ther, 2010. 18(1): p. 23-33.
32. Young-Gqamana, B., et al., Migalastat HCl reduces globotriaosylsphingosine (lyso-Gb3) in Fabry transgenic mice and in the plasma of Fabry patients. PLoS One, 2013. 8(3): p. e57631.
33. Yang, B., et al., Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10. Mol Ther, 2014. 22(7): p. 1299-309.
34. Metcalf, J.A., et al., A self-inactivating gamma-retroviral vector reduces manifestations of mucopolysaccharidosis I in mice. Mol Ther, 2010. 18(2): p. 334-42.
35. Baldo, G., et al., Retroviral-vector-mediated gene therapy to mucopolysaccharidosis I mice improves sensorimotor impairments and other behavioral deficits. J Inherit Metab Dis, 2013. 36(3): p. 499-512.
36. Aronovich, E.L., et al., Systemic correction of storage disease in MPS I NOD/SCID mice using the sleeping beauty transposon system. Mol Ther, 2009. 17(7): p. 1136-44.
37. Shapiro, E.G., et al., Neuropsychological outcomes of several storage diseases with and without bone marrow transplantation. J Inherit Metab Dis, 1995. 18(4): p. 413-29.
38. Visigalli, I., et al., Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model. Blood, 2010. 116(24): p. 5130-9.
39. Wolfe, J.H., et al., Reversal of pathology in murine mucopolysaccharidosis type VII bysomatic cell gene transfer. Nature, 1992. 360(6406): p. 749-53.
40. Medin, J.A., et al., Correction in trans for Fabry disease: expression, secretion and uptake of alpha-galactosidase A in patient-derived cells driven by a high-titer recombinant retroviral vector. Proc Natl Acad Sci U S A, 1996. 93(15): p. 7917-22.
41. Sano, R., et al., Chemokine-induced recruitment of genetically modified bone marrow cells into the CNS of GM1-gangliosidosis mice corrects neuronal pathology. Blood, 2005. 106(7): p. 2259-68.
42. Miranda, S.R., et al., Hematopoietic stem cell gene therapy leads to marked visceral organ improvements and a delayed onset of neurological abnormalities in the acid sphingomyelinase deficient mouse model of Niemann-Pick disease. Gene Ther, 2000. 7(20): p. 1768-76.
43. Shihabuddin, L.S., et al., Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology. J Neurosci, 2004. 24(47): p. 10642-51.
44. Baker, M.P., et al., Immunogenicity of protein therapeutics: The key causes, consequences and challenges. Self Nonself, 2010. 1(4): p. 314-322.
45. Langereis, E.J., et al., Biomarker responses correlate with antibody status in mucopolysaccharidosis type I patients on long-term enzyme replacement therapy. Mol Genet Metab, 2014.
46. Lin, H.Y., et al., Mucopolysaccharidosis I under enzyme replacement therapy with laronidase--a mortality case with autopsy report. J Inherit Metab Dis, 2005. 28(6): p. 1146-8.
47. Ensina, L.F., et al., Laronidase hypersensitivity and desensitization in type I mucopolysaccharidosis: a case report. Pediatr Allergy Immunol, 2014. 25(5): p. 498-9.
48. Parker, A.S., et al., Optimization algorithms for functional deimmunization of therapeutic proteins. BMC Bioinformatics, 2010. 11: p. 180.
49. Cantor, J.R., et al., Therapeutic enzyme deimmunization by combinatorial T-cell epitope removal using neutral drift. Proc Natl Acad Sci U S A, 2011. 108(4): p. 1272-7.
50. Osipovitch, D.C., et al., Design and analysis of immune-evading enzymes for ADEPT therapy. Protein Eng Des Sel, 2012. 25(10): p. 613-23.
51. Choi, Y., K.E. Griswold, and C. Bailey-Kellogg, Structure-based redesign of proteins for minimal T-cell epitope content. J Comput Chem, 2013. 34(10): p. 879-91.
52. Salvat, R.S., et al., Computationally driven deletion of broadly distributed T cell epitopes in a biotherapeutic candidate. Cell Mol Life Sci, 2014. 71(24): p. 4869-80.
53. Peroni, D.G., et al., Effective desensitization to imiglucerase in a patient with type I Gaucher disease. J Pediatr, 2009. 155(6): p. 940-1.
54. Banugaria, S.G., et al., Algorithm for the early diagnosis and treatment of patients with cross reactive immunologic material-negative classic infantile pompe disease: a step towards improving the efficacy of ERT. PLoS One, 2013. 8(6): p. e67052.
55. Durand, P., et al., Structural features of normal and mutant human lysosomal glycoside hydrolases deduced from bioinformatics analysis. Hum Mol Genet, 2000. 9(6): p. 967-77.
56. Islam, M.R., et al., Active site residues of human beta-glucuronidase. Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue. J Biol Chem, 1999. 274(33): p. 23451-5.
57. Nieman, C.E., et al., Family 39 alpha-l-iduronidases and beta-D-xylosidases react through similar glycosyl-enzyme intermediates: identification of the human iduronidase nucleophile. Biochemistry, 2003. 42(26): p. 8054-65.
58. Natowicz, M.R., et al., Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts. Proc Natl Acad Sci U S A, 1979. 76(9): p. 4322-6.
59. Tsukimura, T., et al., Uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts and osteoblasts. Biol Pharm Bull, 2008. 31(9): p. 1691-5.
60. Chen, C.P., et al., ECSTASY, an adjustable membrane-tethered/soluble protein expression system for the directed evolution of mammalian proteins. Protein Eng Des Sel, 2012. 25(7): p. 367-75.
61. Caras, I.W., et al., Signal for attachment of a phospholipid membrane anchor in decay accelerating factor. Science, 1987. 238(4831): p. 1280-3.

1. Chen, C.P., et al., ECSTASY, an adjustable membrane-tethered/soluble protein expression system for the directed evolution of mammalian proteins. Protein Eng Des Sel, 2012. 25(7): p. 367-75.
2. Caras, I.W., et al., Signal for attachment of a phospholipid membrane anchor in decay accelerating factor. Science, 1987. 238(4831): p. 1280-3.
3. Durand, P., et al., Structural features of normal and mutant human lysosomal glycoside hydrolases deduced from bioinformatics analysis. Hum Mol Genet, 2000. 9(6): p. 967-77.
4. Islam, M.R., et al., Active site residues of human beta-glucuronidase. Evidence for Glu(540) as the nucleophile and Glu(451) as the acid-base residue. J Biol Chem, 1999. 274(33): p. 23451-5.
5. Nieman, C.E., et al., Family 39 alpha-l-iduronidases and beta-D-xylosidases react through similar glycosyl-enzyme intermediates: identification of the human iduronidase nucleophile. Biochemistry, 2003. 42(26): p. 8054-65.
6. Natowicz, M.R., et al., Enzymatic identification of mannose 6-phosphate on the recognition marker for receptor-mediated pinocytosis of beta-glucuronidase by human fibroblasts. Proc Natl Acad Sci U S A, 1979. 76(9): p. 4322-6.
7. Tsukimura, T., et al., Uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts and osteoblasts. Biol Pharm Bull, 2008. 31(9): p. 1691-5.
8. Chen, K.C., et al., Membrane-localized activation of glucuronide prodrugs by beta-glucuronidase enzymes. Cancer Gene Ther, 2007. 14(2): p. 187-200.
9. Goding, J.W., Conjugation of antibodies with fluorochromes: modifications to the standard methods. J Immunol Methods, 1976. 13(3-4): p. 215-26.
10. Jain, S., et al., Structure of human beta-glucuronidase reveals candidate lysosomal targeting and active-site motifs. Nat Struct Biol, 1996. 3(4): p. 375-81.
11. Rempel, B.P., L.A. Clarke, and S.G. Withers, A homology model for human alpha-l-iduronidase: insights into human disease. Mol Genet Metab, 2005. 85(1): p. 28-37.
12. Schrodinger, LLC, The PyMOL Molecular Graphics System, Version 1.3r1. 2010.
13. Shih, E.S., R.C. Gan, and M.J. Hwang, OPAAS: a web server for optimal, permuted, and other alternative alignments of protein structures. Nucleic Acids Res, 2006.
34(Web Server issue): p. W95-8.
14. Larkin, M.A., et al., Clustal W and Clustal X version 2.0. Bioinformatics, 2007. 23(21): p. 2947-8.
15. Ye, Y. and A. Godzik, Flexible structure alignment by chaining aligned fragment pairs allowing twists. Bioinformatics, 2003. 19 Suppl 2: p. ii246-55.
16. Barton, G.J., ALSCRIPT: a tool to format multiple sequence alignments. Protein Eng, 1993. 6(1): p. 37-40.
17. Wu, C.H., et al., A simple method for the production of recombinant proteins from mammalian cells. Biotechnol Appl Biochem, 2004. 40(Pt 2): p. 167-72.
18. Matsumura, I. and A.D. Ellington, In vitro evolution of beta-glucuronidase into a beta-galactosidase proceeds through non-specific intermediates. J Mol Biol, 2001. 305(2): p. 331-9.
19. Geddie, M.L. and I. Matsumura, Rapid evolution of beta-glucuronidase specificity by saturation mutagenesis of an active site loop. J Biol Chem, 2004. 279(25): p. 26462-8.
20. Kakkis, E.D., et al., Long-term and high-dose trials of enzyme replacement therapy in the canine model of mucopolysaccharidosis I. Biochem Mol Med, 1996. 58(2): p. 156-67.
21. Dickson, P., et al., Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I. J Clin Invest, 2008. 118(8): p. 2868-76.
22. Hartung, S.D., et al., Correction of metabolic, craniofacial, and neurologic abnormalities in MPS I mice treated at birth with adeno-associated virus vector transducing the human alpha-L-iduronidase gene. Mol Ther, 2004. 9(6): p. 866-75.
23. Hein, L.K., et al., alpha-L-iduronidase premature stop codons and potential read-through in mucopolysaccharidosis type I patients. J Mol Biol, 2004. 338(3): p. 453-62.
24. Chen, K.C., et al., Directed evolution of a lysosomal enzyme with enhanced activity at neutral pH by mammalian cell-surface display. Chem Biol, 2008. 15(12): p. 1277-86.
25. De Duve, C. and R. Wattiaux, Functions of lysosomes. Annu Rev Physiol, 1966. 28: p. 435-92.
26. Freeman, C. and J.J. Hopwood, Human alpha-L-iduronidase. Catalytic properties and an integrated role in the lysosomal degradation of heparan sulphate. Biochem J, 1992.

1. Chen, C.P., et al., ECSTASY, an adjustable membrane-tethered/soluble protein expression system for the directed evolution of mammalian proteins. Protein Eng Des Sel, 2012. 25(7): p. 367-75.
2. Baldo, G., et al., Intraperitoneal implant of recombinant encapsulated cells overexpressing alpha-L-iduronidase partially corrects visceral pathology in mucopolysaccharidosis type I mice. Cytotherapy, 2012. 14(7): p. 860-7.
3. Baldo, G., et al., Retroviral-vector-mediated gene therapy to mucopolysaccharidosis I mice improves sensorimotor impairments and other behavioral deficits. J Inherit Metab Dis, 2013. 36(3): p. 499-512.
4. Boado, R.J., et al., Reversal of lysosomal storage in brain of adult MPS-I mice with intravenous Trojan horse-iduronidase fusion protein. Mol Pharm, 2011. 8(4): p. 1342-50.
5. Cox-Brinkman, J., et al., Ultrastructural analysis of dermal fibroblasts in mucopolysaccharidosis type I: Effects of enzyme replacement therapy and hematopoietic cell transplantation. Ultrastruct Pathol, 2010. 34(3): p. 126-32.
6. Tsukimura, T., et al., Uptake of a recombinant human alpha-L-iduronidase (laronidase) by cultured fibroblasts and osteoblasts. Biol Pharm Bull, 2008. 31(9): p. 1691-5.
7. Wang, D., et al., Engineering a lysosomal enzyme with a derivative of receptor-binding domain of apoE enables delivery across the blood-brain barrier. Proc Natl Acad Sci U S A, 2013. 110(8): p. 2999-3004.
8. Wilkinson, F.L., et al., Neuropathology in mouse models of mucopolysaccharidosis type I, IIIA and IIIB. PLoS One, 2012. 7(4): p. e35787.
9. Unger, E.G., et al., Recombinant alpha-L-iduronidase: characterization of the purified enzyme and correction of mucopolysaccharidosis type I fibroblasts. Biochem J, 1994. 304 ( Pt 1): p. 43-9.
10. Boado, R.J., et al., Genetic engineering of a lysosomal enzyme fusion protein for targeted delivery across the human blood-brain barrier. Biotechnol Bioeng, 2008. 99(2): p. 475-84.
11. Di Natale, P., et al., In vitro gene therapy of mucopolysaccharidosis type I by lentiviral vectors. Eur J Biochem, 2002. 269(11): p. 2764-71.
12. Hartung, S.D., et al., Enzymatic correction and cross-correction of mucopolysaccharidosis type I fibroblasts by adeno-associated virus-mediated transduction of the alpha-L-iduronidase gene. Hum Gene Ther, 1999. 10(13): p. 2163-72.
13. Anson, D.S., J. Bielicki, and J.J. Hopwood, Correction of mucopolysaccharidosis type I fibroblasts by retroviral-mediated transfer of the human alpha-L-iduronidase gene. Hum Gene Ther, 1992. 3(4): p. 371-9.
14. Ou, L., et al., High-dose enzyme replacement therapy in murine Hurler syndrome. Mol Genet Metab, 2013.
15. Di Domenico, C., et al., Gene therapy for a mucopolysaccharidosis type I murine model with lentiviral-IDUA vector. Hum Gene Ther, 2005. 16(1): p. 81-90.
16. Kakkis, E.D., et al., Enzyme-replacement therapy in mucopolysaccharidosis I. N Engl J Med, 2001. 344(3): p. 182-8.
17. Wraith, J.E., et al., Enzyme replacement therapy for mucopolysaccharidosis I: a randomized, double-blinded, placebo-controlled, multinational study of recombinant human alpha-L-iduronidase (laronidase). J Pediatr, 2004. 144(5): p. 581-8.
18. Clarke, L.A., et al., Long-term efficacy and safety of laronidase in the treatment of mucopolysaccharidosis I. Pediatrics, 2009. 123(1): p. 229-40.
19. Giugliani, R., et al., A dose-optimization trial of laronidase (Aldurazyme) in patients with mucopolysaccharidosis I. Mol Genet Metab, 2009. 96(1): p. 13-9.
20. Chen, K.C., et al., Membrane-localized activation of glucuronide prodrugs by beta-glucuronidase enzymes. Cancer Gene Ther, 2007. 14(2): p. 187-200.
21. Wu, C.H., et al., A simple method for the production of recombinant proteins from mammalian cells. Biotechnol Appl Biochem, 2004. 40(Pt 2): p. 167-72.
22. Han, J., et al., Changes in cultured endothelial cell glycosaminoglycans under hyperglycemic conditions and the effect of insulin and heparin. Cardiovasc Diabetol, 2009. 8: p. 46.
23. Mizumoto, S. and K. Sugahara, Glycosaminoglycan chain analysis and characterization (glycosylation/epimerization). Methods Mol Biol, 2012. 836: p. 99-115.
24. Bitter, T. and H.M. Muir, A modified uronic acid carbazole reaction. Anal Biochem, 1962. 4: p. 330-4.
25. Platzer, M., J.H. Ozegowski, and R.H. Neubert, Quantification of hyaluronan in pharmaceutical formulations using high performance capillary electrophoresis and the modified uronic acid carbazole reaction. J Pharm Biomed Anal, 1999. 21(3): p. 491-6.
26. Schneider, C.A., W.S. Rasband, and K.W. Eliceiri, NIH Image to ImageJ: 25 years of image analysis. Nat Methods, 2012. 9(7): p. 671-5.
27. Xu, M., et al., A phenotypic compound screening assay for lysosomal storage diseases. J Biomol Screen, 2014. 19(1): p. 168-75.
28. Di Domenico, C., et al., Limited transgene immune response and long-term expression of human alpha-L-iduronidase in young adult mice with mucopolysaccharidosis type I by liver-directed gene therapy. Hum Gene Ther, 2006. 17(11): p. 1112-21.
29. Herati, R.S., et al., Improved retroviral vector design results in sustained expression after adult gene therapy in mucopolysaccharidosis I mice. J Gene Med, 2008. 10(9): p. 972-82.
30. El-Amouri, S.S., et al., Normalization and improvement of CNS deficits in mice with hurler syndrome after long-term peripheral delivery of BBB-targeted iduronidase. Mol Ther, 2014. 22(12): p. 2028-37.
31. Schrodinger, LLC, The PyMOL Molecular Graphics System, Version 1.3r1. 2010.
32. Linthorst, G.E., et al., Enzyme therapy for Fabry disease: neutralizing antibodies toward agalsidase alpha and beta. Kidney Int, 2004. 66(4): p. 1589-95.
33. Dickson, P., et al., Immune tolerance improves the efficacy of enzyme replacement therapy in canine mucopolysaccharidosis I. J Clin Invest, 2008. 118(8): p. 2868-76.
34. Ohashi, T., et al., Reduced alpha-Gal A enzyme activity in Fabry fibroblast cells and Fabry mice tissues induced by serum from antibody positive patients with Fabry disease. Mol Genet Metab, 2008. 94(3): p. 313-8.
35. Kishnani, P.S., et al., Cross-reactive immunologic material status affects treatment outcomes in Pompe disease infants. Mol Genet Metab, 2009.
36. Patel, T.T., et al., The impact of antibodies in late-onset Pompe disease: a case series and literature review. Mol Genet Metab, 2012. 106(3): p. 301-9.
37. Banugaria, S.G., et al., Algorithm for the early diagnosis and treatment of patients with cross reactive immunologic material-negative classic infantile pompe disease: a step towards improving the efficacy of ERT. PLoS One, 2013. 8(6): p. e67052.
38. Langereis, E.J., et al., Biomarker responses correlate with antibody status in mucopolysaccharidosis type I patients on long-term enzyme replacement therapy. Mol Genet Metab, 2014.
39. Parker, A.S., et al., Optimization algorithms for functional deimmunization of therapeutic proteins. BMC Bioinformatics, 2010. 11: p. 180.
40. Cantor, J.R., et al., Therapeutic enzyme deimmunization by combinatorial T-cell epitope removal using neutral drift. Proc Natl Acad Sci U S A, 2011. 108(4): p. 1272-7.
41. Osipovitch, D.C., et al., Design and analysis of immune-evading enzymes for ADEPT therapy. Protein Eng Des Sel, 2012. 25(10): p. 613-23.
42. Choi, Y., K.E. Griswold, and C. Bailey-Kellogg, Structure-based redesign of proteins for minimal T-cell epitope content. J Comput Chem, 2013. 34(10): p. 879-91.
43. Salvat, R.S., et al., Computationally driven deletion of broadly distributed T cell epitopes in a biotherapeutic candidate. Cell Mol Life Sci, 2014. 71(24): p. 4869-80.
44. Tsujihata, Y., et al., A single amino acid substitution in a self protein is sufficient to trigger autoantibody response. Mol Immunol, 2001. 38(5): p. 375-81.
45. Carreno, B.M., et al., Cancer immunotherapy. A dendritic cell vaccine increases the breadth and diversity of melanoma neoantigen-specific T cells. Science, 2015. 348(6236): p. 803-8.
46. Shi, D., et al., A single mutation in the active site swaps the substrate specificity of N-acetyl-L-ornithine transcarbamylase and N-succinyl-L-ornithine transcarbamylase. Protein Sci, 2007. 16(8): p. 1689-99.
47. Tomasic, I.B., et al., Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases. J Biol Chem, 2010. 285(28): p. 21560-6.

1. Tsujihata, Y., et al., A single amino acid substitution in a self protein is sufficient to trigger autoantibody response. Mol Immunol, 2001. 38(5): p. 375-81.
2. Matsuda, T. and C.L. Cepko, Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci U S A, 2007. 104(3): p. 1027-32.
3. Chen, K.C., et al., Membrane-localized activation of glucuronide prodrugs by beta-glucuronidase enzymes. Cancer Gene Ther, 2007. 14(2): p. 187-200.