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Mapping Immunodominant CD4+ T cell epitopes for the deficient enzyme in Pompe Disease

Permanent Link: http://ufdc.ufl.edu/UFE0043041/00001

Material Information

Title: Mapping Immunodominant CD4+ T cell epitopes for the deficient enzyme in Pompe Disease
Physical Description: 1 online resource (84 p.)
Language: english
Creator: SIVAKUMAR,RAMYA
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: CD4 -- ELISPOT -- EPITOPE -- GAA -- GSD -- MHC -- POMPE
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Pompe disease is a type II glycogen storage disorder caused by an inherited deficiency of the lysosomal enzyme acid alpha glucosidase. Partial or complete deficiency of this enzyme affects the degradation pathway of glycogen inside the lysosomes of the cells.(1, 2) (3)The severity of the disorder is related with its phase of onset and amount of residual enzyme. (4) Since there are no clinically available treatments for this disease, both enzyme replacement and gene replacement therapies have been employed for replacing the dysfunctional enzyme. Myozyme and Lumizyme are the currently approved clinical forms of the GAA protein which have been used in different phases of human trials. And viral vectors such as AAV and Adenovirus have been used for transducing the deficient enzyme in several parts of the body. However the effectiveness of these therapies has been short lived by the formation of antibodies or cross reactive immunologic materials against the administered enzyme. (2) A better understanding of the adaptive immune system is required for preventing such anti-protein inhibitory antibodies and for providing an active functional enzyme to the body. Mapping of the immunodominant CD4+ T cell epitope will present a clear picture of the interactions between the protein and antigen presenting cells and help in identifying the relationship between the cell mediated and humoral immune responses of the body. (5) The peptide sequences IFLGPEPKSVVQ (83), HSRAPSPLYSVE (19), VLQPSPALSWRS (29) were identified as the immunodominant T cell epitopes for the rhGAA protein in 129Sv strain GAA-/- mice using in-vitro techniques such as ELISpot and flow cytometry. Additionally, the message levels and profiles of cytokines expressed by of CD4+ helper T cells were determined using RT-PCR arrays. Thus, a comprehensive study of the T cell mediated immunity for the rare and fatal classical infantile syndrome of the disorder was performed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by RAMYA SIVAKUMAR.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Ogle, William.
Local: Co-adviser: Herzog, Roland.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043041:00001

Permanent Link: http://ufdc.ufl.edu/UFE0043041/00001

Material Information

Title: Mapping Immunodominant CD4+ T cell epitopes for the deficient enzyme in Pompe Disease
Physical Description: 1 online resource (84 p.)
Language: english
Creator: SIVAKUMAR,RAMYA
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2011

Subjects

Subjects / Keywords: CD4 -- ELISPOT -- EPITOPE -- GAA -- GSD -- MHC -- POMPE
Biomedical Engineering -- Dissertations, Academic -- UF
Genre: Biomedical Engineering thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Pompe disease is a type II glycogen storage disorder caused by an inherited deficiency of the lysosomal enzyme acid alpha glucosidase. Partial or complete deficiency of this enzyme affects the degradation pathway of glycogen inside the lysosomes of the cells.(1, 2) (3)The severity of the disorder is related with its phase of onset and amount of residual enzyme. (4) Since there are no clinically available treatments for this disease, both enzyme replacement and gene replacement therapies have been employed for replacing the dysfunctional enzyme. Myozyme and Lumizyme are the currently approved clinical forms of the GAA protein which have been used in different phases of human trials. And viral vectors such as AAV and Adenovirus have been used for transducing the deficient enzyme in several parts of the body. However the effectiveness of these therapies has been short lived by the formation of antibodies or cross reactive immunologic materials against the administered enzyme. (2) A better understanding of the adaptive immune system is required for preventing such anti-protein inhibitory antibodies and for providing an active functional enzyme to the body. Mapping of the immunodominant CD4+ T cell epitope will present a clear picture of the interactions between the protein and antigen presenting cells and help in identifying the relationship between the cell mediated and humoral immune responses of the body. (5) The peptide sequences IFLGPEPKSVVQ (83), HSRAPSPLYSVE (19), VLQPSPALSWRS (29) were identified as the immunodominant T cell epitopes for the rhGAA protein in 129Sv strain GAA-/- mice using in-vitro techniques such as ELISpot and flow cytometry. Additionally, the message levels and profiles of cytokines expressed by of CD4+ helper T cells were determined using RT-PCR arrays. Thus, a comprehensive study of the T cell mediated immunity for the rare and fatal classical infantile syndrome of the disorder was performed.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by RAMYA SIVAKUMAR.
Thesis: Thesis (M.S.)--University of Florida, 2011.
Local: Adviser: Ogle, William.
Local: Co-adviser: Herzog, Roland.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2013-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2011
System ID: UFE0043041:00001


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1 MAPPING IMMUNODOMINANT CD4 + T CELL EPITOPES FOR THE DEFICIENT ENZYME IN POMPE DISEASE By RAMYA SIVAKUMAR A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2011

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2 2011 Ramya Sivakumar

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3 To my family and friends for all their support and e ncouragement

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4 ACKNOWLEDGMENTS I wish to express my deep gratitude to my mentor Professor Roland Herzog, PhD for giving me the opportunity to work on his research program. Without his expert guidance and support this project would not have been possible. I wish to equally thank Dr. Ou C ao for initiating this venture and allowing me to contribute towards its successful completion. I would also like to thank my committee members Dr. William Ogle and Dr. Benjamin Keselowsky for their understanding and co operation towards my project. I wis h to thank Dr. Barry Byrne our project collaborator for rendering his support and for providing us with the protein and GAA / murine models required for this study I am grateful to my Postdoctoral Associate Sushrusha Nayak who has been patiently teaching me all the concepts and techniques associated with epitope mapping She was also kind enough to listen and clarify all my inquisitive questions. Her presence and encouragement throughout the project is invaluable to me. And thanks to Dr. Lee Ann L this study. I am indebted to Dr. Babaq Moghimi, who has been assisting me in all my experiments despite his various research commitments and tight schedules. Additionall y, I wish to take this opportunity to thank Dr. Brad Hoffman, Dr. David Markusic and Irene Zolotukhin and all my other lab members for their valuable inputs and guidance during the early days of my research career. Special thanks are reserved for Mario Coo per and Dr. Debalina Sarkar who have motivated me all along and have made my life in the lab enjoyable.

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5 Finally I wish to express my deepest appreciation to my family for standing by me in all aspects of my academic and personal life. As alwa ys, my friend s will continue to be my pillars of support.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ .. 4 LIST OF TA BLES ................................ ................................ ................................ ............ 8 LIST OF FIGURES ................................ ................................ ................................ .......... 9 LIST OF ABBREV IATIONS ................................ ................................ ........................... 11 ABSTRACT ................................ ................................ ................................ ................... 13 CHAPTER 1 INTRODUCTION ................................ ................................ ................................ .... 15 Overview ................................ ................................ ................................ ................. 15 Biochemistry of the Enzyme ................................ ................................ ................... 15 Pathophysiology: ................................ ................................ .............................. 17 Phenotypes of GSD II ................................ ................................ ....................... 17 Diagnosis: ................................ ................................ ................................ ......... 19 Animal Models for Pompe ................................ ................................ ................ 20 Treatments ................................ ................................ ................................ ....... 20 Enzyme Replacement Therapies: ................................ ................................ .... 21 Gene Therapy: ................................ ................................ ................................ 22 CRIM and GSD II: ................................ ................................ ................................ ... 23 Clinical Trials: ................................ ................................ ................................ ......... 24 Need For Epitope Mapping: ................................ ................................ .................... 25 Epitope: ................................ ................................ ................................ ............ 25 Enigmatic MHC Molecu les: ................................ ................................ .............. 25 MHC Restriction: ................................ ................................ .............................. 26 MHC Haplotype: ................................ ................................ ............................... 28 Study of CD4 T Cell Response? ................................ ................................ ............. 28 Expression of Cytokines ................................ ................................ ................... 28 Anti GAA Formation ................................ ................................ ........................ 29 Epitope Mapping Techniques: ................................ ................................ ................ 30 2 MATERIALS AND METHODS ................................ ................................ ................ 33 Reag ents ................................ ................................ ................................ ................ 33 Equipments and Supplies: ................................ ................................ ...................... 34 Mouse Strain: ................................ ................................ ................................ .......... 34 Construction of the Peptide Library: ................................ ................................ ........ 35 Immunization of Mice: ................................ ................................ ............................. 35 Collection of Blood Samples: ................................ ................................ .................. 35

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7 ELISpot: ................................ ................................ ................................ .................. 36 Coating of Plates: ................................ ................................ ............................. 36 Isolation of Splencoytes: ................................ ................................ .................. 36 Analysis of Blood Samples: ................................ ................................ .............. 37 Cell Counting: ................................ ................................ ................................ ... 37 In vitro Stimulation of Cells with Peptides: ................................ ........................ 38 Addition of Capture Antibody: ................................ ................................ ........... 38 Color Development: ................................ ................................ .......................... 39 Quantification of Spots: ................................ ................................ .................... 39 In Vitro Cytokine Secretion Assay/ Flow Cytometry: ................................ ............... 39 In vitro Stimulation of Cells with Peptides: ................................ ........................ 39 Labeling of Cells: ................................ ................................ .............................. 40 Acquisition and Analysis of Data: ................................ ................................ ..... 4 0 Immunomagnetic Cell Separation: ................................ ................................ ... 40 Intravenous Administration of the Protein ................................ ............................... 42 ELISA to Determine the Antibody Levels Against rhGAA Protein in Mice: ....... 42 Stimulation of Splenocytes with Individual Peptides and Controls for RT PCR: ................................ ................................ ................................ ............. 42 Isolation of B and T cell Lymphocytes: ................................ ............................. 43 Extraction of RNA, Synthesis of cDNA and RT PCR ................................ ........ 43 3 RESULTS ................................ ................................ ................................ ............... 48 Identification of the Dominant Peptides Using ELISPOT: ................................ ....... 48 Dominant Peptide Pools: ................................ ................................ .................. 48 Dominant Peptides: ................................ ................................ .......................... 48 CD4 + Peptides: ................................ ................................ ................................ 49 Quantification of Spots: ................................ ................................ .................... 49 Fluorescence Activated Cell Sorting (FACS): ................................ ......................... 50 Epitope Mapping Algorithms: ................................ ................................ .................. 52 Anti GAA Response ................................ ................................ ............................. 52 4 DISCUSSION ................................ ................................ ................................ ......... 72 APPENDIX A GAA PEPTIDE LIBRARY ................................ ................................ ........................ 75 B T CELL EPITOPE PREDICTION RESULTS ................................ ........................... 78 C SOURCE FIGURE 1 1. ................................ ................................ ......................... 79 LIST OF REFERENCES ................................ ................................ ............................... 80 BIOGRAPHICAL SKETCH ................................ ................................ ............................ 84

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8 LIST OF TABLES Table page 2 1 GAA peptide library using 20 pools of peptides ................................ .................. 44 2 2 GAA peptide library from 32 simple pools of peptides ................................ ........ 45 3 1 Average count of Spot forming units from 20 pools of peptides of Figure 3 2 .... 55 3 2 Average cell c ount from 32 pools of peptides, 8 single peptides and controls (From Figure 3 5) ................................ ................................ ............................... 58 3 3 Immune epitope database T cell epitope prediction tools results .................... 70

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9 LIST OF FIGURES Figure page 1 1 Patient diagnosed with GSD II the tube is connected to aid with his breathing (Use of this photograph has been approved by the patient) ............................... 19 2 1 Isolation of spleen from GAA / 129 Sv mouse immunized with rhGAA ............. 45 2 2 Preparation of Splenocytes ................................ ................................ ................ 46 2 3 In vitro cytokine secretion assay steps ................................ ............................. 46 2 4 Protocol for studying cytokine expression pattern ................................ .............. 47 3 1 Arrangement of 20 peptide pools of 10 peptides /pool (R1 R10 and C1 C10)and controls in IFN R10 and C1 C10 = 20 peptide pools of 10 peptides/pool; DM DMSO, MCK Mock ................................ ........... 53 3 2 IFN (from Figure 3 1) ................................ ................................ ................................ 54 3 3 Average Count for Spot forming cells from 20 pools of peptide and controls (Quantitation of results from Figure 3 2) ................................ ............................. 55 3 4 Arrangement of 32 peptide pools of 3 peptides/ pool and individual peptides tested positive from Figure 3 2 in IFN ................................ ....... 56 3 5 IFN 8 single peptides tested positive from initial 20 pools of peptides (Figure 3 2) containing 10 peptides each ................................ ................................ ............... 57 3 6 Average counts of spot forming cells from 32 pools of peptides and positive and negative controls (Quantitation of results from Figure 3 5) .......................... 59 3 7 Average counts of spot forming cells from single peptides tested positive from 20 pools of peptides and controls (Quantitation of results from Figure 3 5) ................................ ................................ ................................ ........................ 60 3 8 Arrangement of controls and individual peptides tested positive from Figure 3 5 in IFN tes and peripheral blood lymphocytes. ................................ ................................ ................................ ....... 61 3 9 ELISpot with individual peptides that had made up the initial positive pools of 32 p eptide pools containing 3 peptides/pool (from Figure 3 3) ........................... 62

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10 3 10 Average cell count of spot forming units from individual peptides tested positive from the 32 pools of peptides containing 3 peptides/pool and controls (Quantitation of results from Figure 3 9) ................................ ............................. 63 3 11 Arrangement of controls and individual peptides tested positive from Figure 3.5 in IFN ELISpot plate containing CD4 + CD8 + T cells and peripheral blood lymphocytes ................................ ................................ ................................ ........ 64 3 12 IFN ELISpot plate with separated CD4+ and CD8 + T cell populations from rhGAA immunized GAA / 129 Sv mouse ................................ ........................... 65 3 13 Average count of spot forming units/106 cells from CD4+positive T cells of Figure 3.12. ................................ ................................ ................................ ........ 66 3 14 Average count of spot forming units from CD8 + T positive cells of Figure 3 8 .... 67 3 15 IFN + CD4 + T cell frequencies in mice using peptide 83 ................................ ... 67 3 16 IFN + CD4 + T cell frequencies in mice with whole protein rhGAA ..................... 68 3 17 IFN + CD4 + T cell frequencies in GAA / mice with SEB ................................ ... 68 3 18 IFN + CD4 + T cell frequencies in GAA / mice using mock stimulated samples ................................ ................................ ................................ .............. 69 3 19 IFN + CD4 + T cell frequencies in GAA / mice using unstained control ............. 69

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11 LIST OF ABBREVIATION S 129 Sv Inbred strain of mouse AAV Adeno associated virus APC Antigen presenting cells BSA Bovine serum albumin CD4, CD8 Cluster of differentiation 4; 8 cDNA Complementary DNA CFA Complete freunds adjuvant CRIM Cross reactive immunologic material DMEM DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid ELISA Enzyme linked immunosorbent assay ELISpot Enzyme linked Immunosorbent Spot ERT Enzyme replacement therapy FDA Food and Drug Ad ministration GAA GAA / Murine knockout model for GAA gene H 2 Mouse major histocompatibility complex H 2 bc H 2 haplotype of 129 strain mouse HLA Human leukocyte antigen IFN Interferon gamma IgG, IgE Immunoglobulin G; E IL 2 4, 5, 10, 13 Interleukin 2; 4; 5; 10; 13 MHC Major histocompatibility complex

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12 PBMC Peripheral blood mononuclear cell PBS Phosphate buffered saline PMA Phorbol 12 Myristate 13 Acetate rhGAA Recombinant human acid alpha glucosidase RNA Ribonucleic ac id RT PCR Real time polymerase chain reaction SEB Enterotoxin B, Staphylococcal TGF transforming growth factor beta Th1, Th2, Th17 Thymus helper cells 1; 2; 17 TNF Tumor necrosis factor alpha Treg Thymus regulatory cells

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13 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for th e Degree of Master of Science MAPPING IMMUNODOMINANT CD4+ T CELL EPITOPES FOR THE DEFICIENT ENZYME IN POMPE DISEASE By Ramya Sivakumar May 2011 Chair: William Ogle Co chair: Roland Herzog Major: Biomedical Engineering Pompe disease is a ty pe II glycogen storage disorder caused by an in herited deficiency of the lysosomal enzyme acid alpha glucosidase P artial or co mplete deficiency of this enzyme affects the degradation pathway of glycogen inside the lysosomes of the cells (1, 2) (3) The severity of the disorder is related with its phase of onset and amount of residual enzyme (4) Since there are no clinically avai lable treatments for this disease, both e nzyme replacement and gene replacement therapie s have been employed for replacing the dysfunctional enzyme. Myozyme and Lumizyme are the currently approved clinical forms of the GAA protein which have been used in different phases of human trials. And viral vectors such as AAV and Adenovirus have been us ed for transducing the deficien t enzyme in several parts of the body. However the effectiveness of t hese therapies has been short lived by the formation of antibodies or cross reactive immunologic materials against the administered enzyme (2) A better understanding of the adaptive immune system is required for preventing such anti protein inhibitory antibodies and for providing an active functional enzyme to

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14 the body. Mapping of the immunodominant CD4 + T cell epitope will present a clear picture of the interactions between the protein and antigen presenting cells and help in identifying the relationship between the cell mediated and humor al immune responses of the body (5) The peptide sequences IFLGPEPKSVVQ (83), HSRAPSPLYS VE (19), VLQPSPALSWRS (29) were identified as the immunodominant T cell epitopes for the rhGAA protein in 129Sv strain GAA / mice using in vitro techniques such as ELISpot and flow cytometry Additionally, the message levels and profiles of cytokines expressed by of CD4 + helper T cells were determined using RT PCR arrays. Thus, a comprehensive study of the T cell mediat ed im munity for the rare and fatal classical infantile syndrome of the dis order was performed.

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15 CHAPTER 1 INTRODUCTION Overview Pompe dis ease is a debilitating neuromuscular disorder caused by partial or complete deficiency of the lysosomal enzyme acid alpha glucosidase. The autosomal recessive disease which leads to progr essive skeletal muscle weakness has been detected with varying frequencies in several ethnic groups The rare kind of inherited storage disorder has been observed on an average in 1 in 40,000 live births with higher frequency in females compared to males (3, 4, 6) Sir Johannes Cassianus, a Dutch pathologist was the first person to describe the disorder as a progressive generalized muscle weakness associated with severe cardiomegaly in a 7 month pediatric patient in the year 1932. During the same period Putschar reported a parallel observation of a patient (infant) suffering from muscle weakness and card iac dysfunction The actual cause of the disorder was not identified even after 20 years of its initial description by Sir Pompe. F urther developments in the form of discovery of lysosome by C. DeDuve and research of catalytic mechanisms of glycogen by G. T. Cori helped in characterizing the source of the disorder as a lysosomal enzyme deficiency by H.G. Hers in the year 1955. (7) The disorder falls among a class of 49 types of lysosomal storage disorders and is considered as the only glycogen storage disease, which is also a lysosomal storage disorder. Biochemistry of the Enzyme The gene encoding the lysosomal enzyme, acid alpha glucosidase (GAA) is located in the long arm of chromosome 17 in humans and in chromosome 6 of mice.

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16 The 28kb long human GAA gene comprises 20 exons and 19 introns, and contains a 2.7kb long intron between the non translating first exon and coding second exon. The GC rich promoter mimics the characteristics of a housekeeping gene and lacks the TATA and CCAAT motifs, which have been related to the ubiquitous expression of the gene. (8) The 952 a.a long protein encoded by the 3.8kb long cDNA is initially synthesized in the form of a catallytiacally active 110kDa precusor protein. This pre protein undergoes core glycosilation in the ER and obtains mannose 6 phosphate re ceptors from the post ER compartment. Around 70 80% of the pre protein then gets directed to the pre lysososmal or lysosomal organelles by mannose 6 phospahte receptors, where subsequent cleavages of the protein helps in the production of 95 76 and 70 kDA forms of GAA enzyme. These cleavage steps are considered to be essential for activat ion of the enzyme. (1, 8) A small er portio n of the enzyme, (10 20%) which is initially secreted from the cells gets re routed to pre lysosomes of adjacent cells through their mannose 6 phosphate receptors. Both gene replacement and protein replacement therapies have been designed on the basis of this form of the secretory protein. (1) The lysosomal enzyme is soluble in nature and functions optimally at a pH of 4.3. The enz yme is responsible for cleaving 1,6 linkages of glycogen into monosaccharides like glucose and maltose Glucose is taken up by several organs o f the body and is required for maintaining blood sugar levels and as a source of energy during cell turnovers. A notable feature of the GAA enzyme is its sequence similar ity with both the subunits of the intestinal sucrose isomaltase enzyme complex (7, 8)

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17 Pathophysiology: The disorder arises due to genomic mutations of the enzyme At present 287 mutations of the gene have been report ed, among which 67 have been termed to be pathogenic and 197 as non pathogenic GAA is the only available pathway for degradation of lysosomal glycogen, so i n the absence of the enzyme, glycogen starts accumulating in almost all the cells and tissues of the the body. Lysosomal glycogen deposits have also been d etected prior birth in amniotic fluids and in muscles of 18 week old embryos. Glycogen accumulation weakens and enlarges both the cardiac and skeletal muscles of the body. Traces of glycogen have also been observed in satellite cells, liver cells, in motor neurons and glail cells and in nuclei of brain stem and anterior horns. (1, 4, 7) Even though pathophysiology of the disease varies with the expre ssed phenotype, generalized and progressive skeletal muscle weakness has been observed in all the forms of the disorder. As GSDII is a multi system disorder, some of th e organ dsyfunctions including hypertrophic cardiomyop athy, progressive skeletal myopat hy osteopenia, osteoporosis macroglossia, and respiratory insufficiency were observed in patients (7) Phenotypes of GSD II T he GSD II disorder varies accordingly with th e amount of residual enzyme, phase of onset, extent of organ involvement and rate of progression of muscle weakness (4, 7) The infantile phenotype is considered to be the most severe form of the dis order and is usually diagnosed wi thin the first 3 months of life. One of the hallmarks of this form is the almost complete deficiency of the lysosomal enzyme in the cells. The

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18 to ventricular outflow obstructions till feeding problems and organ failures such as cardiomegaly, hypertrophic cardiomyopathy, mild hepatomegaly, severe hypotonia, and macroglos sia. Death usually occurs within a year after birth due to cardio respiratory failures. One in 1, 38,000 babies suffer from this form of fatal disorder which is also commonly referred to as Pompe disease Alternatively the incidence of the disorder varies with the kind of ethnic population e.g frequency of 1 among 100,000 to 1 amo ng 200,000 in outbred Caucasian populations and 1 among 40,000 in African Americans. (3, 4, 6, 7) (9) B oth the juveni le and adult phenotypes are grouped together in the late onset form of the disease Some of the distinguishing features of these forms are the presence of reduced but residual activity of the GAA enzyme and near total absence of cardiac dysfunctions. Patients get dia gnosed within the first decade of their life and start exhibiting symptoms such as mild hepatomegaly, skeletal muscle we aknesses and respiratory insufficiencies One in 57,000 children and adults are affected by this adult form of the disorder (3, 6, 7) (9) A non classical type of the disorder known as the infantile varian t fo rm has been observed in some patients. In this particular phenotype patients do not experience cardiac disorders and usually progress through sympto ms similar to Pompe disease at a slower rate with milder severities. This form is also referred to as the su btype of GSDII. (6, 7)

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19 Figure 1 1. Patient d iagnosed with GSD II the tube is connected to aid with his breathing (Use of this photograph has been approved by the patient ) Diagnosis: The rare condition of this disorder along with its non specific symptoms has made its diagnosi s harder. Nowadays Pompe disease is also being considered as a cause for floppy baby syndromes. (10) Basic diagnosis for the disorder starts with the measurement of GAA activity from either whole blood samples or muscle biopsies and skin fibroblasts. Although detection of GAA from skin fibroblasts is considered to be the gold standard, preference is being given to assays run on dried blood spots for their accuracy and rapid mode of diagnosis (3, 6) (9) Apart from enzyme assays several specific and non specific di agnostic approaches including GAA mutational analysis, e le ctrocardiogram s e chocardiogram s

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20 chest X rays and measurement of creatine kinase leve ls in serum and determining the levels of a specific glucose tetrasaccharide in urine of patie nts are being performed as a part of the diagnosis. Pre natal screening of Pompe disease is also being preferred for detecting the presence of the deficient enzyme at earlier stages. These timely discoveries have led to the presymptomatic diagnosis of 30 d ifferent inborn errors of metabolism. And they could also be used for (10) improving the survival rate of patients via enzyme replacement therapies (6, 7, 11) (9) Animal Models for Pompe Lapl and dog, cats sheep and Brahman and Shorthorn cattle and a particular s train of Japanese quail represent the natur ally occu rring animal models for GSD II. However they could not be used for rese arch studies owing to several constraints including prolonged breeding periods, smaller litter size and evolutionary distance from human models. To overcome these discrepancies five mo use models that more genotypically and phenotypically resembled the human form of the disorder were create d. These models were generated by inducing specific disruptions in GAA gene. Both the phenotypes of GSD II were re created in the knockout murine models by targeting Exon 6, Exon 13 and Exon 14 respectively. Generalized and progressive muscle weakness was observed along with abnormal levels of lysosomal glycogen in the heart, liver and striated muscles of these models Nowadays exon 13 and exon 6 k nockout models have been used extensively for pre clinical studies in gene therapy. (7) Treatments Although there is no available cure for Pompe disease at the moment, both e nzyme replacement and gene replacement therapi e s have been employed for replacing the dysfunctional enzyme These therapies were designed with the aim of

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21 replacing the deficient enzyme with a functional one which would help in clearing the glycogen content from cells and successively improve their card iac and skeletal muscle functions. Enzyme Replacement T herapies: Enzyme replacement therapies for GSD II were introduced in the 1960s using enzyme preparations from Aspergillus niger and human placentas. Due to lack of experience and minimal knowledge of t he disease the trials failed to provide the proposed clinical benefits With experience, the cause of failures was then related to inappropria te choice of enzyme sources and insufficient d osage levels (6) Parallel studies on the mechanisms behind the uptake and functioning of the enzyme helped in unraveling the involvement of cell surface receptors with the uptake of extracellular glycoproteins. As Mannose 6 phosphate cell receptors help with the ingestion of GAA, GAA coupled with the receptor was and still being used as a means of delivering effective and efficient enzymes. Further developments including the cloning of GAA gene helped in opening up more avenues for the large scale production of the recombin ant human enzyme. The recombinant forms of the enzyme were being produced in cell cultures and Chinese hamster ovary cell lines, in milk of transgenic rabbits and even in baculovirus infected insect cells. Currently two types of the recombinant enzymes nam ely Myozyme and Lumizyme were approved by the United States Food and Drug Administration system for application as therapeutic drugs in trials including infantile and late onset disorders Myozyme or aglucosidase alfa was prepared by cloning the human GAA cDNA using a muscle specific promoter in tandem with dihrofolate reductase gene for promoting

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22 methotraxate induced amplifications. This rhGAA was approved in the year 2006 for infantile patients. Lumizyme, which is considered to be a similar version of Myz ozyme was approved by the FDA in 2010 for late onset patients. Both the enzymes have proved to be effective in improving the conditions of the patients and have helped in increasing their survival rates. However a nd require frequent infusions for providing the desired outcome. The enzymes are administered intravenously at a dose of 20 40 mg/kg even in less than a year old patients and are usually tolerated even with their higher infusion rates. Despite these advant facing a major hurdle in the form of anti protein inhibitory antibodies. The efficacy of the therapies have been reduced or completely halted by the formation of IgG and IgE antibodies respectively. Anapyhlactic responses are conside red to be fatal counteractive events and occur rarely. Immune tolerance protocols have been and are being designed for preventing the occurrence of IgG and for providing an efficient enzyme replacement therapy to the patients. Apart from these issues, pati ents have also been suffering without treatments due to the cost factors and paucity of sponsoring health insurance companies. Overall 300 patients worldwide have been receiving therapeutic recombinant human alpha glucosidase enzymes. (6) Gene Therapy: One of the alternatives that hold a promising future for the treatment of Pompe disease is Gene Therapy. Constant supply of the functional enzyme for a prolonged period of time is the main aim of this therapy. Bot h In vivo and In vitro trials have been performed using viral vectors, cell lines and recombinant proteins. GAA enzyme has been transduced into fibroblasts using adenoviral vectors in vitro. Transduction resulted

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23 in an efficient expression of the enzyme an d subsequent secretion of the 11 0kDa precursor protein of GAA. This extracellular GAA was taken up by cells which were able to use it for clearing their lysosomal glycogen. (7) (6) Viral vectors enc oding the cDNA of rhGAA have been used for transducing the enzyme in GAA / mice using muscle and liver specific promoters. Mutant cell lines have also been tested for expression of functional protein in skeletal and cardiac muscles and in the liver upon gene transfer Overall it was identified that transducing and expressing in the muscles was much harder than other organs. Studies done on muscles of murines have always exhibited heterogeneous and discouraging results. Parallel devel opments helped in discovering t he liver as an ideal s ite for expression and functioning of the enzyme. The protein is generally taken up effectively by the cardiac and muscle cells upon its secretion from the liver AAV vector given through th e Intravenous Intramuscular or Intramyocardial routes were found to represent alternative gene therapy approaches (7) Despite encouraging results, possibilities of random integrations of the gene a nd the susceptibility of immune re sponse s against the vector, transgene and its products have decelerated the development of human clinical trials on gene therapy Nowadays f ocus has also been garnered on the use of non viral transfection agents for the treatment of Pompe disease (6) CRIM and GSD II : CRIM is referred to as cross reactive immunogenic material. The enzyme replacement therapies for the infantile and adult form of the disease have been affected by the CRIM status of the patie nts. Patients who are incapable of producing endogenous GAA enzyme due to deleterious mutations in their genome are referred to

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24 as CRIM negative patients, Patients who possess variable amounts of residual enz yme in any form (non functional or partially fun ctional ) are referred to as CRIM positive patients. (2) Rep lacements of the non functional or deficient enzyme by Myzozyme in infantile patients have bee n less effective due to the occurrence of anti e nzyme antibodi es. IgG of high titers against the enzyme were observed much earlier and were more sustained in this group of patients. As a result, they have not been receiv ing the desired outcomes of the treatment such as ventilation free existence, improvement of their cardiac and motor skills and survival rates. (6, 11, 12) Although antibodies have been detected in CRIM positive and adult onset populations, the patients have been benefited wi th improved cardiac and motor development functions. (2, 4) As the CRIM status of the patients has been hampering the goals of ERT, adjunct strategies that can help improve the efficacy of the treatment are currently being tested. These include immunomodulatory and immune suppressive strategies to promote tolerance to the therapeutic GAA protein. Antibody formation often requires activation of B cells by CD4 + T c ells (T helper). Identification of the immunodominant CD4 + T cell epitope will help us understand the mechanism of immune response in this disorder and could successively be used for designing of various tolerance and immune modulating protocols (13, 14) Clinical Trials: Some of the current clinical trials being done on Pompe disease a t University of Florida are

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25 Phase I/II Trial of Diaphragm Delivery of Recombinant Adeno associated Virus Acid Alpha Glucosidase (rAAV1 CMV GAA) Gene Vector in Patients with Pompe Disease Effects of Immunomodulation Therapy on Anti rhGAA Immune Response in Children with Pompe Disease Receiving rhGAA Enzyme Replacement and A Phase I/II Open Label Study of the Safety, Tol erability, Pharmacokinetics, Pharmacodynamic and Preliminary Efficacy of BMN 701 (GILT tagged Recombinant human GAA) in Patients with Late Onset Pompe Disease Need For Epitope Mapping: Epitope : The molecular structure of an antigenic molecule or protein, w hich is recognized by any individual antigen receptor or antibody, is termed as epitope. Immunogens contain several epitopes, and they are capable of binding to various MHC class molecules. Despite the multitude of epitopes for an antigen, T cells respond only against a few of these antigenic determinants in an individual. The power of the immune system to target and regulate the immune responses to a set number of the epitopes is known as immunodominance. (15) The immune system of the body is always going to be focused on these immunodominant epitopes for any individual, so that an initial exposure to these epitopes will help in generating an effective priming response against the antigen and w ill help in conferring protection against successive encounters. E nigmatic MHC M olecules : Major histocompatibility molecules are membrane bound glycoproteins, which are involved with the an tigen presentation to thymocytes and T cells They are highly

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26 poly morphic and polygenic in nature and are encoded by a cluster of genes in a region called the major histocompatibility complex. Major histocompatibility molecules are termed as human leukocyte antigens in humans and as H 2 antigens in mice. There are two ma jor classes of MHC molecules namely the MHC class I and MHC class II molecules. Class I molecules are ex pressed on all nucleated cells and recognized by CD8 positive T cells, while class II molecules are expressed only on antigen presenting cells and reco gnized by CD4 positive T cells The MHC class II gene complex of mice is also known as the I region and is composed of two locis, A and E, which code for the class II molecules. These genes are also referred as Ir (immune response) genes as they determine the level of immune responsiveness of different mouse strains against several antigens. The IA and IE antigens are also jointly referred to as the Ia antigens (5, 6) (5) (16) Class I molecules are known for binding bacterial and viral peptides residues which get degraded and processed within the cytosol of the cell. Class II molecules bind to peptides which get degraded within endocytic vesicles. Once activated nave CD8 T cells get converted into effector T cells or cytotoxic lymphocytes and kill the cells which are infected with f oreign peptides. On the other hand CD4 T cells differentiate into different subsets such as Th1, Th2, Th17 or regulatory T cells. (15) MHC R estriction: A T cell can recognize an antigen only when it is presented as peptide on a specific allelic variant of a MHC molecule and will not recognize the same peptide when it i s presented on another MHC. This unique property of T cells is known as MHC restriction. Specific binding of peptides to MHC molecules occurs due to the interaction of side chains of the peptides with the grooves and pockets of HLA and H 2 molecules.

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27 The polymorphic nature of MHC molecules in the peptide binding groove justifies the preference of different MHC proteins for different sequence motifs an d displays the unique manner in which TCRs recognize MHC allele specific epitopes for restricted recognition of antigens Since there are several genes associated with the synthesis of MHC molecules and since there are several allelic variants of the sa me molecule, an individual is thought to posses 6 different types of MHC class I and class II molecules. So far 2215 HLA class I and 986 HLA class II allelic sequences of MHC molecules ha ve been identified so far in human s (15) Systemic peptide libraries that can bind to almost all possible MHC alleles in an individual have been created. (17) The length of peptides in these libraries plays a decisive role in their binding to MHC class I and MHC class II molecules. Usually to cover the complete immunogenic sequence of a protein, peptides with a length of n 1 sequences (n = length of peptides that can be bound by MHC class I or MHC class II molecules) have be en used for peptide mapping studies. Since the peptide binding groove in MHC class I molecules are closed on either ends, peptide sequences more than 10 amino acids cannot be accommodated in them. So peptide libraries containing 8 10mer sequences of peptid es have been used while mapping the CD8 T cell dominant epitope. (17, 18) (15) The peptide binding groove is open on either end and allows for binding of larger peptides. The normal length of peptides which get processed and presented to the MHC class II molecules vary from from12 25a.a. Peptide libraries consisting of 12 15 mers are generally used for CD4 epitope mapping studies. (18, 19)

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28 MHC H aplotype : The specific pattern of alleles found in a single chromosome is known as an MHC haplotype. The 6 neo /6 neo mice were generated using a mixed strain of 129/C57BL/6 mice. To avoid clinical heterogeneity between the mic e, the mice were backcrossed to the 129Sv strain. (20) The haplotype of 129Sv is referred to as "H 2 bc or I A bc A bc and I A b strains are identical in their classical H 2 and Q regions (21, 22) S tudy of CD4 T Cell R esponse? CD4+ T cells play a vital role in the functioning of both innate and adaptive immune responses. They elicit various functions like promotion of cytotoxic T lymphocytes expansion, maintenance of CD8 + T cells memory, communication with innate immune cells, promo tion of B cell differentiation into plasma cells and their immunoglobulin class switching, assistance to function of memory cells and activation of macrophages through the secretion of effector molecules called cytokines. CD4+ T cells are required for prod ucing a potent primary and memory CD8 + T cell response and for conferring protection against bacterial and viral infections. They can also be used against viral infections by secretion of anti viral cytokines like IFN We are focused on the an tigen presentation and differentiation of CD4 cells. They can branch out to various helper phenotypes like Th1, Th2 and Th17. Th1 cells function by activating the infected macrophages and by aiding B cells for IgG2a secretion and for providing help for CD8 + cells. Although Th2 cells help B cells in the production of antibodies, they are more inclined towards causing a IgG1 or IgE class switch. (15) Expression of Cytokines Cytokines are soluble proteins and cell signaling mole cules, which participate in int r a cellular communications. Th1 cells secrete IFN

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29 cells especially macrophages and other effector cells of the immune system. Th2 cells secrete IL 2, IL 5, IL 1 3, IL 10 cytokines and function by activating B cells and promoting allergic immune responses. While Th17 cells help in recruiting neutrophils to the site of infection and promotion of acute inflammation, Regulatory CD4+ T cells control and level the adapt ive immune responses through the secretion of several inhibitory cytokines like IL 10 and TGF altering the behavior of target cells. (15) Anti GAA F o rmation Both Th1 and Th2 cells are involved in the activation and functioning of B cells like class switching and antibody secretion an d it can be concluded that they are responsible for the co ordination of humoral immune responses. Some of the previous experiments carried out in Pompe mice corrobarate this view rhGAA protein was infused through the Intravenous route at a dose of 20 mg/kg onc e a week for 3 weeks in wt C57BL /6 m ice and CD4 / C57BL6 mice, and their anti GAA IgG reponses were measured. GAA specific IgG levels could be obtained only from the wt C57BL/6 mice and none were found in the CD4 / pompe mice. The level of IgG reponses also differed with the amount of protein that was being ad ministered to the mice The route of immunization is direc tly related to the magnitude and type of immune response elicited by the antigen. Subcutaneous, intramuscular and intravenous are the most widely routes of experimental antigen administratio n. As antigens are taken up by L angerghan cells and expressed effe ctively in the lymph nodes when they are immunized subcutaneously, we chose this method for eliciting GAA specific antibodies and T cells.

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30 The rhGAA was administere d subcutaneously with complete F reunds adjuvant (CFA) for preliminary mapping st udies. CFA is oil in water emulsion consisting of dead mycobaterial components. CFA works by converting the soluble antigen into particulate stimulators of dendritic cells and macrophages. (15) Since the route of immunization and the adjuvant confer a strong inflammatory response, the IFN ELISpot was chosen for studying the effect of antigen presenting cells and for mapping th e immunodominant epitopes against the antigen. Besides these choices, IFN ELISpot was preferred for its high sensitivity and ability to determine the clonal size and function of T cells. (15 ) Epitope Mapping Techniques: Limiting dilution assays, direct lysis in bulk killing, MHC tetramer staining assays, intracellular cytokine staining assays, ELISpot, in vitro cytokine staining are some of the techniques which have been used for exploring th e T cell responses and for mapping the immunogenic peptides of the antigen. The first two methods require radioactively labeled matching MHC cells and are quite cumbersome to perform. Intracellular cytokine staining and MHC tetramer staining procedures wer e aimed to prevent the radioactive labeling of cells and serve as an improved technique, but they were also equally cumbersome and did not eschew the requirement of prior knowledge of T cell epitopes and of MHC usage. (17 19) As ELISpot assays do not require the prior knowledge or presence of MHC specific target cells and are relatively easier to perform and analyze, this technique is currently being preferred over other avail able procedures (17 19) A large collection of

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31 peptides of a protein antigen can be studied by constructing overlapping or non overlapping pools of peptides in the form of a l ibrary. (17) The immunodominant epitope for human GAA in 129Sv strain GAA / mice has been mapped in two successive steps. The splenocytes were tested for their cytokine secreting activities by using peptide matrix po ols and simple pools of peptides. Individual peptides from pools, which had emerged as positive samples in the previous step, were used in the second ELISpot assay for determing the immunodominant epitopes. Alternatively, the types of the responding T cell s were also ascertained by separating them prior to stimulation in the assays. The assay relies on the Cytokine secreting characteristics of antigen experienced T cells. Nave T cells upon prior activation differentiate into effector and memory T cells wh ich can secrete specific cytokines. (16) These cells do not secrete the cytokine in in vitro conditions without prior stimulation with cognate antigens. To prevent the differentiation and expansion of naive T cells i nto their effector counterparts, the assay is limited to 15 hrs of stimulation period. Thus to ascertain the antigen specific response of T cells, the peptides are presented under in vitro conditions to induce cytokine secretion from memory T cells. The im prints of these cytokines are recorded as spot forming cells, which are taken as a direct measure of antigen specific T cell response. (23) CD4 + T cell epitope mapping for the rhGAA protein in 129Sv mice will ser ve as a tool for analyzing both the cell mediated and humoral immune responses of the body. It will also help in studying in detail the peptide and MHC binding motifs and for making predictions on candidate T cell epitopes.

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32 Apart from in vitro assays, bioi nformatic algorithms and in silico tools have been designed for determining MHC class II epitopes. The in silico tools were chosen from the immune epitope database analysis resource (24 30) ( 31 36) We used these tools to compare the results obtained with the epitopes, which were identified using in vitro assays. Thus the CD4 + T cell immunodominant epitopes against the rhGAA protein were identified in GAA / mice belonging to129Sv strain.

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33 CHAP TER 2 MATERIALS AND METHOD S Reagents Peptide Library spanning the mature GAA sequence of 952 Amino Acids was generated in the form of 12 mers with an overlap of 2 amino acids from Anaspec in San Jose CA(purity>70%), Recombinant human acid alpha glucosidase protein was Interferon Gamma Development Module (R&D systems Minneapolis, MN), ELISpot Blue Color Module (R&D systems Minneapolis, MN), Mouse Interferon Gamma Secretion Assa y( Miltenyi Biotec, CA), CD4(L3T4) Microbeads and CD8a (Ly 2) Microbeads for mice were purchased from Miltenyi Biotec in CA, 2 MLC and 5 MLC mouse lymphocyte culture mediums were prepared using DMEM, Sodium Pyruvate, HEPES, Non Essential amino acids,2 B eta Mercaptoethanol, Penicillin/streptomycin and mouse serum, Staphylococcal enterotoxin B (Sigma Aldrich, St.Louis,MO),PMA Phorbol 12 myristate 13 acetate, Ionomycin (Sigma Aldrich, St.Louis,MO),Di methyl sulfoxide, Recombinant Mouse Interferon Gamma(R&D systems Minneapolis, MN), 2A 54 the dominant CD4+ T cell epitope for human FIX, were used as controls for the experiments. Some of the other reagents were Recombinant Mouse Interleukin 2 (Roche Diagnostics, Manheim, Germany),Anti Mouse CD8 eflour450 Anti Mouse CD4 Alexa fluor 700 and 7 AAD( 7 amino actinomycin D), (ebioscience, San Diego, CA) m cell strainer, BD lysing buffer, Trypan blue, ELISA Wash ( Tween 20, 1x PBS and water)

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34 Equipments and Supplies : Multiscreen Filter Plates (Millipore, MA), Haemacytometer, sterile surgical kit, Sonicator, Cellular Technology Limited ELISpot Reader, BD LSR II (BD Biosciences, MD, USA) 1ml, 5ml and 10ml syringes, 25 gage needles, sterile 1.5ml microfuge tubes, Laminar flow cabinet, Sterile pipette tips(10,100,200 and 1000l), Single channel and Multi channel Pipettes, Sterile Pipettes (1ml, 2ml, 5ml, 25ml, 50ml),Centrifuge (Bench Top), tally counter, Cover Slip Phase Contrast Microscope, 37C humidified incubator with 5% CO2 atmosphere, Vortexer, Balance. Mouse Strain: GAA / (GAA knockout) 6 8 weeks old mice were used. They had been created by targeted disruption of exon 6 using a neomycin resistance gene (6neo/6neo). A termination codon and a new EcoRV site had been introduced upstream from the neo gene in exon 6.Even though different types of knockout mice were made (e.g inframe 14 knockout mice exhibited the severe phen otypes of Pompe disease (i.e.) resembling the earlier stage of onset and rapid progression of disease. (20, 37) 6 neo /6 neo mice were generated using a mixed strain of 129/C57BL/6 mice. These mice are ho mozygous in nature and do not synthesize endogenous GAA protein. The strains were maintained by in terc rossing the progeny of the original 129 chimeric male founders and C57BL/6 female. (20, 37) To av oid clinical heterogeneity betwe en the mice, the mice were later backcrossed to the 129SV strain (haplotype H 2 bc ). (20) Center and were housed u nder specific pathogen free conditions in the Animal Facility

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35 at the University of Florida. All the animal procedures were carried out as per IACUC guidelines. Construction of the Peptide Library: Ninety five peptides spanning the mature GAA sequence of 95 2 amino acids were generated as 12 mers which were ov erlapping by 2a.a The peptide stocks were dissolved using Dimethysulfoxide at a concentration of 2 mg/ml. Two dimensional arrays of the peptides were designed to obtain 20 pools of peptides. (17) Each pool was composed of 8 10 peptides, and each peptide was represented in two pools. The final concentration of each peptide in the pool was 4 g/ml. The reconstituted peptides were stored at 70C. Owing to the high concentration of DMSO in the stock, the peptides were re diluted using 5 MLC media and the pools were re designed. Smaller Pools of peptides were constructed this time with each pool consisting of only 3 peptides. Thus 32 pools of peptides were designed a nd the final concentration of each peptide in the pool was 10 g/ ml. Immunization of Mice: rhGAA protein mixture (50 g/mouse ) was prepared by dissolving the protein in 1 ml of Phosphate buffered saline (PBS) and 1 ml of CFA The mixture was sonicated us ing a Branson Sonifier until a creamy white paste was obtained. T his protein (200l) was administered using a 1 ml syringe through the subcutaneous route to 3 mice for every set of experiment. ( Figure 2 3) Collection of Blood Samples: The mice were anaesth etized using 2% isoflurane and hep arinized microhematocrit capillary tubes were used for collecting blood from their retro orbital

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36 plexus. Blood collected from the retro orbital site is considered to be a representative of the venous blood. Th ey were use d for running ELISpots and i n vitro cytokine secretion assays. The results obtained were compared with the results obtained from s plenocytes. Since experiments in humans can be carried out only with blood samples, pilot experiments of this kind were includ ed in the project. ELIS pot: ELISpot kits for Mouse IFN were purchased from R&D systems and used as per Coating of Plates: S terile 96 well multiscreen filter plates were pre wetted using 15 l of 35% ethyl alcohol for a min ute to overcome the hydrophobicity of the membrane.Plates were then washed thrice using 350 l of 1x PBS and coated with anti IFN capture antibody The antibody coated plate was left for overnight incubation at 4C. Isolation of Splencoytes: The mice were sacrificed after 10 12 days of immunization, and their spleens were out aseptically under the laminar hood. The spleens were meshed in 70 m nylon cell strainers using 5 10 ml of 2 MLC media and washed at a speed of 1400 rpm (revolutions per minute) at 4C for 10 min. The supernatant was discarded, and the pellet was resuspended in BD lysing buffer (3 ml/spleen) and left on ice for 3 minutes to lyze the erythrocytes. Afte r incubation, 10 ml of 2 MLC media was added for each spleen and spun at 1400 rpm for 10 min at 4C. The pellet was finally resuspended in 5 MLC media and taken for counting. The final pellet was devoid of erythrocytes and composed of splenocytes.

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37 To check the homogeneity of the spleens, they were harvested separately during the earlier experiments but pooled for the upcoming assays. Analysis of Blood Samples: Blood collected from the 3 mice (750 1000 l/mice) were pooled together and lysed using BD or ACK lysing buffer to eliminate erythrocytes. The cell suspensions were centrifuged and the pellet was resuspended using 5 MLC media and taken for cell counting. Owing to the low cell count obtained by lysing the whole blood, buffy coats were used for the upcom ing experiments. Blood collected from the mice were centrifuged to separate out the buffy coat from plasma and erythrocytes. Buffy coat is made up of leukocytes and platelets and used as an alternative procedure for eliminating erythrocytes from the sample The cells were appropriately diluted to obtain a concentration of 10 6 cells/ml and added in duplicates to the ELISPOT plate. They were treated in the same manner as splenocytes. Since the volume of blood obtained was not enough to run the entire peptide library, only a few peptide pools/peptides and positive controls were tested using blood. Cell Counting: Dilutions of the cells at 1:10, 1:20 were made using trypan blue and 10 15 l of the samples was loaded into the haemocytometer. Since viable cells do not take up trypan blue, this dye was used for differentiating between viable and non viable cells. The cells were viewed under 100 x magnifications under a phase contrast microscope and counted from the four overlying 1mm2 areas of the counting chamber using a tally co unter. The required cell numbers were diluted from the obtained count of live cells.

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38 In vitro Stimulation of Cells with Peptides: The plates were blocked for 2 hours using (200 l/well) ELISPOT blocking buffer (5% sucrose and 1% BSA in PBS) on day 2. Plates were then rinsed and filled with the culture medium until cells were prepared for plating. Splenocytes were resuspended in 5 MLC medium at concentration of 10 6 cells/ml and added in duplicates to the p late (200 l/well). The culture media was also additionally supplemented with recombinant IL 2 (10 u/ml) for promoting T cell growth. In vitro stimulation of cells was induced by adding peptide pools at a concentration of 4 g/ml or 10 g/ml. appropriate c ontrols were included to maintain high performance standards. SEB (1 g/100 l), PMA (0.05 g/ml)/IONOMYCIN (1 g/ml), recombinant IFN g and recombinant human GAA (10 g/ml) were used as positive controls, DMSO as the negative control and 2A 54(10 g/ml) as the irrelevant peptide. The plates were incubated in 5% CO 2 Any Cytokine secreted by the cells due to stimulation during this period will be captured by the membrane bound antibodies. (23, 38) Addition of Capture Antibody: The cells were washed thrice using 350 l of wash buffer and blotted against a paper towel to remove the remaining solution. The required volume of biotinylated detection antibody was calculated and diluted to its working concentration using reagent diluents. 100 l of this antibody was then added to each wel l and incubated overnight at 4C to captu re the secreted cytokines (IFN ).

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39 Color Development: The Ag Ab c omplex was identified by adding appropriate enzyme and substrates conjugates like Streptavidin AP and BCIP /NBT (according to ELISPOT blue development module). Streptavidin AP was preferred for it s linear reaction rate and reduced background staining and BCIP/NBT mixture is the most commonly used substrate for Streptavidin AP. This substrate mixture was chosen for its stable nature. Binding of AP to BCP/NBT induces the formation of intense black blue spots. Since these spots do not fade easily, they can be reanalyzed and stored for a longer period of time. Each colored spot is represen tative of a cell secreting IFN and the spots were enumerated using the Immunospot Analyzer. Quantification of Spots: The colored spots found at the bottom of the wel l were considered to be a quantitative measure of the cells secretion capacity. The quantitative measure was termed as Spot forming cells. The Spots were enumerated using ImmunoSpot Analyzer (Cellular Technology, Shaker Heights, OH). In Vitro Cytokine Sec retion Assay/ Flow Cytometry: Mouse IFN secretion Assay Detection kits were purchased from Mitenyi Biotec and used according to the recommended instructions. In vitro Stimulation of Cells with Peptides: The cells were resuspended in 5 MLC medium and a dded in triplicates to a 96 well round bottom plate (10 6 cells/well). Peptide pools were added at a concentration of 10 g/ml. The media was supplemented with rIL 2 at 10 u/ml for promoting T ce ll proliferation. Positive and n egative controls were added t o standardize the procedure.

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40 SEB (1 g/100l), rhGAA (10 g/ml) were used as the positive controls and DMSO was the negative control. Apart from these con trols unstained and mock stimulated controls were also used for determining the positive cells. The ce lls were incubated along with the peptides in 5% CO 2 Labeling of Cells: Cell surface staining was performed using IFN cells capable of secreting IFN incubated with the catch reagent for 45 minutes under slow rotation at 37c. The cells were then placed on ice and washed using cold buffer. Phycoerythrin conjugated mouse IFN CD4 (Alexa Fluor 700) (1 g/10 6 cells), anti CD8 (eflour 45 0) (1 g/10 6 cells) antibodies were added to the cells and kept on ice for 10 min. The cells were washed and resuspended in cold buffer and taken for the acquiring process. The numbers of apoptotic cells were determined by adding 7 amino actinomycin D just prior to acquisition. Acquisition and Analysis of Data: Data acquisition was carried out in BD LSR II (BD Bioscience) Flow Cytometer and analyzed using Cell Quest and FACS DIVA 6.1. Immunomagnetic Cell Separation: Since we were trying to determine the CD 4+ Immunodominant T cell epitopes, cell separation was carried out to choose the specific subsets of cells. Positive and negative selections are the two commonly used separation techniques. Since ELISpot is a combination of immune and bioassay, the desired cells were depleted or negatively selected from the whole cell population. (38) CD4 (L3T4) Microbeads and CD8a (Ly 2) microbeads were used for this purpose.

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41 As soon as the cells were lysed for erythrocytes and counted, equal volume of cells were divided for separating CD4+ and CD8 + cells from the whole splenocyte population. The cell suspensions were labeled with CD4 and CD8 magnetic beads and passed through cell separation (MS) columns in the presence of a magnetic field. The magnetically labeled cells were retained in the column, while unlabelled cells remained in the flow through. The labeled cells were eluted from the columns using elution buffers. The unlabelled populations from the CD4 + separation were composed of CD8 + cells and antigen presenting cells and were used in the ELISPOT plate for detecting CD8 + epitopes. Conversely, the unlabelled cell populations from CD8 + separation were composed of CD4 + cells with antigen presenting cells and were us e d as samples for detecting CD4 + epitopes. After separation the cells were counted. Duplicates and triplicates of the cell s at a concentration of 10 6 cells/ml and 10 7 cells/ml were used for ELISPOT and in vitro cytokine secretion assays. The separated cells were treated identically to whole splenocytes. To establish the cytokine expression pattern of T cells responding to GAA, RT PCR was performed on cDNA samples extra cted from splenocytes of GAA / mice which were immunized with the whole protein by 3 weekly IV injections at a dosage of 25 mg/kg.

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42 Intravenous Administration of the Protein The GAA / (disruption of exon 6 with neomycin resistance gene) 129 SV mice were im munized with 25 mg/kg of the rhGAA protein through the intravenous route once per week for 3 weeks for cytokine expression assays ELISA to D etermine the Antibody Levels Against rhGAA Protein in M ice: Plasma samples of the immunized mice were obtained at th e end of the intravenous immunization protocol The levels of IgG1, IgG2a and IgG2b antibodies were determined from these samples. P lates were initially coated with 50 l of standards and 50 l of human GAA protein at a concentration of 1 g/ml in coating buffer and left for overnight incubation at 4C The plates were then washed and blocked with the dilution buffer for 1 hr at room temperature. The p lates were again washed and incubated with the plasma samples and secon dary antibodies for 2hrs at 37C r es pectively. OPD substrate was used for detection through calorimetric measurement. Color development is catalyzed by conjugated antibodies. The absorbance s of the samples were read at 450 nm using ELISA reader, and the concentrations of antibodies of the ex perimental samples were determined. Thus ELISA was carried out for testing the immunogenicity of the whole protein administration. Stimulation of Splenocytes with Individual Peptides and Controls for RT PCR: The mice were sacrificied after a period of 3 we eks and their spleens were removed for analysis the functions of antigen specific cells. Splenocytes were isolated by following the same procedure as explained above and were stimulated with the individual peptides for a time period of 48 hours.

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43 Isolation of B and T cell L ymphocytes: The lymphocytes were stained using antibodies against their cell surface proteins, anti CD3 and anti CD4 for T cells and anti CD95 for the B cell population. The cells were sorted through a flow sorter (BD Biosciences, MD, US A ) and the isolated populations were used for extraction and synthesis steps of RT PCR. Extraction of RNA, Synthesis of cDNA and RT PCR RNA was extracted from isolated subpopulation of T cells using the RNeasy kit form QIAGEN according to the instructions by the manufacturer. The extracted RNA was either stored at 80C or used for synthesis of cDNA on the same day. cDNA was synthesized from 5 g whole RNA using the RT 2 First Strand Kit from SABiosciences as instructed by the manufacturer. Two four l of cD NA and random oligo primers were used for the amplification steps of the PCR cycle. The baseline threshold was automatically set by the BIO RAD instrument and 60 80 cycles of amplification were generally preferred for effective amplication of the gene of i nterest. values of the gene of interest from the set of housekeeping genes that were used as controls for the RT PCR assay. Genomic DNA controls were also included to test for efficiency of the first strand of cDNA synthesis.

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44 Table 2 1 GAA peptide library using 20 pools of peptides C1 C2 C3 C4 C 5 C6 C7 C8 C9 c10 R1 1 2 3 4 5 6 7 8 9 10 R2 11 12 13 14 15 16 17 18 19 20 R3 21 22 23 24 25 26 27 28 29 30 R4 31 32 33 34 35 36 37 38 39 40 R5 41 42 43 44 45 46 47 48 49 50 R6 51 52 53 54 55 56 57 58 59 60 R7 61 62 63 64 65 66 67 68 69 70 R8 71 72 73 74 75 76 77 78 79 R9 80 81 82 83 84 85 86 87 88 R10 89 90 91 92 93 94 95

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45 Table 2 2. GAA peptide library from 32 simple pools of peptides Pool 1 1, 2, 3 Pool 12 34, 35, 36 Pool 23 67, 68, 69 Pool 2 4, 5, 6 Pool 13 37, 38, 39 Pool 24 70, 71, 72 Pool 3 7, 8, 9 Pool 14 40, 41, 42 Pool 25 73, 74, 75 Pool 4 10, 11, 12 Pool 15 43, 44, 45 Pool 26 76, 77, 78 Pool 5 13, 14, 15 Pool 16 46, 47, 48 Pool 27 79, 80, 81 Pool 6 16, 17, 18 Pool 17 49, 50, 51 Pool 28 82, 83, 84 Pool 7 19, 20, 21 Pool 18 52, 53, 54 Pool 29 85, 86, 87 Pool 8 22, 23, 24 Pool 19 55, 56, 57 Pool 30 88, 89, 90 Pool 9 25, 26, 27 Pool 20 58, 59, 60 Pool 31 91, 92, 93 Pool 10 28, 29, 30 Pool 21 61, 62, 63 Pool 32 94, 95 Pool 11 31, 32, 33 Pool 22 64, 65, 66 Figure 2 1. Isolation of spleen from GAA / 129 Sv mouse immunized with rhGAA

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46 Figure 2 2. Preparation of Splenocytes Figure 2 3. In vitro cytokine secretion assay steps

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47 Figure 2 4. Protocol for studying cytokine expression pattern

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48 CHAPTER 3 RESULTS Identification of the Dominant P eptides U sing ELISPOT: This ELISpot assay was designed for detecting IFN secreting cells at the single cell level and for determining their frequency. The results were quantified in the form of IFN spot forming units or spot forming cells (SFC) per 10 6 input cells. (23) Usually the threshold for frequency of spot forming units is set at twice the f requency of spots formed with mock stimulated/negative controls. Dominant Peptide Pools: The methodology relies on the potential of the peptides to evoke cytokine response from memory T cells that have been previously exposed to the antigen. (17) This assay is considered to be more sensitive than ELISA and intracellular cytokine staining since they detect T cells based on their functional response to antigens. GAA / mice were immunized with recombinant human acid al pha glucosidase protein through the subcutaneous route, and their spleens were isolated after 10 days of injections. Splenocytes were isolated and cultured using 2 MLC and 5 MLC mediums and plated in duplicates on the membrane backed microplates. The cells were stimulated using the 20 peptide pools {C1 C10 and R1 R10} of the GAA protein to identify those testing positive for the secretion of the cytokine IFN For the initial experim ents, pools containing peptides 54,71,74,80 and 83 (i.e pools R6, R8, R9, C1 and C4) turned out to be positive for secretion of IFN Dominant Peptides: Suboptimal results were obtained in the initial set of experiments, due to the high concentration of DMSO in the stock. Even though positive signals were obtained, from

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49 the sam ples, clearly defined spots could not be detected from the wells. Therefore the peptides were diluted using 5 MLC media and the peptide library was redesigned. New pools with fewer peptides (total of 32 pools comprised of 3 peptides in each) were constructed and tested for the presence of IFN secreting cells. The single peptides which had been identified in the previous experiments were also included in this assay,the homogeneity of the response among the mice was tested by assaying individual po oled spleens in parallel. In this experiment peptides 19,20,21,28,29,30,58,59,60,82,83,84 (i.e pools 7,10,20 and 28) were chosen. Peptide 83 was identified as the dominant one among all the single peptides, which were tested in parallel on the plate. CD4 + Peptides: Once the single peptides were mapped, the following ELISpots were run on separated cells to determine the phenotype of the responding cells S pleno cytes were labeled using CD4 or CD8 magnetic beads and were depleted for CD4 + CD8 + cell popula tion s. Duplicates of the T cell popul ations were added to the ELISpot plates and the frequency of IFN secreting spots was determined. Alternatively, ELISpots were run on peripheral blood mononuclear cells to with results obtained using splenocytes. In eith er case peptide 19, 29 and 83 were found to be dominant among all the other sequences. Quantification of Spots: Quantification of spots was done with the help of CTL ELISpot reader. The reader scans the plate and saves their images in the Tiff or Jpeg for mat. Since all the processes are automated, the machine progresses directly from well to well and uses optical feedback for centering on each of them. Digital encoders are used for identifying

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50 and determining the precise position of each well in the plate. time of scan are recorded with the help of the software. Saved images were analyzed using the Immunospot software 4.0 version. Counting parameters, spot size gates, Minimum and Maximum spot size density, sample spot size, removal of hair and control of backgroun d are some of the features that are set to required levels to aid with the analysis. The whole procedur e was done by applying identical pa rameters to all wells in the plate. The settings were fixed to provide a clear compariso n of the cell count from every well. Despite setting these features, the results from ELISpot were always subjected to quality control to remove counts which might have risen due to artifacts. Image overlays were used to identify the spots which were actua lly counted from each well and for making th e required modifications. All such corrections were recorded and annotated to comply with Good Laboratory P ractice guidelines. The data collected from these counts were used for identifying the CD4 + dominant T ce ll epitope for acid alpha glucosidase protein in GAA / 129Sv mice. Fluorescence Activated Cell Sorting (FACS) : Flow cytometry is used for isolating various cell populati ons based on their phenotypes. The phenotype of cells is usually defined by its cell s urface proteins and they can be detected by using specific monoclonal antibodies which are coupled with fluoresc ent dyes or using flurochromes. In our case lymphocyt es were previously stained with monoclonal antibodies specific to CD4 (Alexa Fluor 700) (1 g/10 6 cells) or CD8 (eflour 450) (1 g/10 6 cells). Dead cells were excluded by adding 10 15 l of 7 amino actinomycin D just prior to acquisition. Fluorescence Activated Cell Sor ter is the machine that was used for

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51 separating cells based on their fluore scence. The mixture of labeled cells was then passed through a nozzle and a single stream of cells was created to cause the laser beam to hit one cell at a time. Laser light gets scattered when each cell passes through it and causes the excitation and emis sion of any fluorescent dye associated with them. The scattered light and emission spectra of cells gets captured by the photomultiplier tubes and provides information about the size and granularity of the cell and successively the expression of their cell surface proteins. Data obtained from the flow cytometer are usually represented in the form of histograms, two dimensional scatter plots or in the form of contour diagrams where the fluorescence of one flurochrome is plotted against the other. Initially the lymphocyte population was gated using a dot plot or contour diagram. Once the required populations were gated, the numbers of live and dead cells in the population were calculated. The required subpopulations of cells were gated from lymphocytes and t heir cell numbers and fluorescence intensity were plotted against each other to obtain the required results. In our experiment the numbers of CD4 + and CD8 + positive cells were determined. F luorescence intensities were compared against each other for all th e tested peptides and results were compared with those of positive and negative controls to determine the actual cell count. Based on the above procedu re and the in vitro assays that had been carried out for th is purpose, peptide 83 was confirmed to be on e of the dominant peptide s for the acid alpha glucosidase protein in GAA / 129Svmice.

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52 Epitope Mapping Algorithms: The peptide sequences were loaded into in silico tools from the immune epitope database analysis resource for comparing the predicted epitope sequences with the e pitopes which were obtained from in vitro T cell assays. Fasta sequences of the whole GAA protein were loaded in all the algorithms for predicting immundominant epitopes specific for the I A bc MHC class II haplotype. The algorit hms were based on neural networks and binding affinities of the peptides with MHC class II molecules. And Peptide 83 was identified a s I A bc res tricted immunodominant CD4 + T cell epitope with the highest score in most of the algorithms. Therefore in silico and experimental results were in excellent agreement. Anti GAA R esponse Plasma samples obtained from all the 3 mice were checked for IgG1, IgG2a and IgG2b levels. And it was found that the amount of IgG1 was higher in a ll the samples followed by minimal amounts of both IgG2a and IgG2b The presence of IgG1 indicates the existence of a Th2 response in the GAA / mice. The results from the RT PCR experiments will help in determining the levels of the various cytokines expressed by these cells.

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53 Fig ure 3 1. Arrangement of 20 peptide pools of 10 pept ides /pool (R1 R10 and C1 C10) and controls in IFN pot plate ; R1 R10 and C1 C10 = 20 pe ptide pools of 10 peptides/pool; DM DMSO, MCK Mock

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54 Fig ure 3 2. IFN of peptide containing 10 peptides/pool (from Fig ure 3 1) A (1 2) H (1 2): Pools R1 R8 A (3 4) B (3 4): Pools R9 R10 C (3 4) H (3 4): Pools C1 C6 A (5 6) D (5 6): Pools C7 C10 E (5 6) GAA; F (5 6) DMSO; G (5 6) SEB ; H (5 6) MOC K CONTROL

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55 Fig ure 3 3. Average Count for Spot forming cells from 20 pools of peptide and controls (Quantitation of results from Fig ure 3 2) Table 3 1. Average count of Spot forming units from 20 pools of peptides of Figure 3 2 1 2 3 4 5 6 A R1 1 R9 429 C7 0 B R2 62 R10 0 C8 1 C R3 1 C1 270 C9 0 D R4 3 C2 6 C10 8 E R5 1 C3 0 GAA 98 F R6 33 C4 252 SEB 40 G R7 0 C5 122 DMSO 0 H R8 0 C6 2

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56 Fig ure 3 4. Arrangement of 32 peptide pools of 3 peptides/ pool and individual peptides tested positive from Fig ure 3 2 in IFN 1 32 (i.e A1 H8) Smaller pools of peptides containing 3 peptides/ pool 51, 54, 71, 74, 80, 81, 83, 84 Single peptides tested positive from earlier IFN ELISpot with 20 peptide pools (Ref Figure 3 2) P/I PMA/Ionomycin DM DMSO, IRP Irrelevant peptide hGA Heat inactivated GAA MCK Mock, rIFN Recombinant IFN

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57 Fig ure 3 5. IFN plate with 32 pools of peptides containing 3 peptides/pool and 8 single peptides tested positive from initial 20 pools of peptides (Fig ure 3 2) containing 10 peptides each A(1 2) H(1 2) Pools 1 8, A(3 4) H(3 4) Pools 9 16, A(5 6) H(5 6) Pools17 24 A (7 8 ) H (7 8) Pools 25 32 A (9 10) H (9 10) Peptides 51, 54,71,74,80,81,83,84 A (11 12) PMA/IONO, B(11 12) SEB, C(11 12) DMSO,D(11 12) GAA,E(11 12) IrrPep, F (11 12) hiGAA, G (11 12) MOCK,H(11 12) rIFN

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58 Table 3 2. Average cell count from 32 pools of pep tides, 8 single peptides and controls (From Figure 3 5) 1 2 3 4 5 6 7 8 9 10 11 12 A POOL1 4.5 POOL9 3 POOL17 2 POOL25 0.5 51 0.5 PMA/IONO B POOL2 3 POOL10 17.5 POOL18 1 POOL26 0 54 0.0 SEB 20.5 C POOL3 0.5 POOL11 0 POOL19 1 POOL27 1 71 1.0 DMSO 0 D POOL4 1.5 POOL12 2 POOL20 13.5 POOL28 37 74 0.5 GAA 98 E POOL5 2 POOL13 3 POOL21 1 POOL29 2.5 80 0.0 IRR PEP 0.5 F POOL6 2 POOL14 3.5 POOL22 0.5 POOL30 1.5 81 0.5 hi GAA 70 G POOL7 10.5 POOL15 1 POOL23 0 POOL31 0.5 83 47.0 MOCK 0.5 H POOL8 1.5 POOL16 2 POOL24 1.5 POOL32 0 84 1 rIFN g

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59 Fig ure 3 6. Average counts of spot forming cells from 32 pools of peptides and positive and negative controls (Quantitation of results from Fig ure 3 5 )

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60 Fig ure 3 7. Average counts of spot forming cells from single peptides tested positive from 20 poo ls of peptides and controls (Quantitation of results from Fig ure 3 5)

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61 Fig ure 3 8. Arrangement of controls and individual peptides tested positive from Fig ure 3 5 in IFN ELISpot plate containing splenocytes and peripheral blood lymphocytes. 19,20,82,21,28,29,30,83,84,58,59,60 Single peptides tested positive from earlier IFN ure 3 5) P/I PMA/Ionomycin ; DM DMSO; IRP Irrelevant peptide ; MCK Mock ; rIFN Recombinant IFN

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62 Fig ure 3 9. ELISpot with individual peptides that had made up the initial positive pools of 32 peptide pools containing 3 peptides/pool (from Fig ure 3 3) A1 D4 Splenocytes with individual GAA peptides 19, 20, 82, 21 28, 29, 30, 83, 84, 58, 59, 60 E3 H4 Mock control; A5 H6 Controls; A7 B10 Peripheral blood lymphocytes with individual GAA peptides 19, 20, 82, 21, 28, 29, 30, 83, 84, 58, 59, 60 C9 C10 Mock control; D9 D10 rhGAA

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63 Fig ure 3 10. Average cell count of spot forming units from individual peptides tested positive from the 32 pools of peptides containing 3 peptides/pool and controls (Quantitation of results from F ig ure 3 9)

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64 Fig ure 3 11 Arrangement of controls and individual peptides teste d positive from Fig ure 3.5 in IFN ELISpot plate containing CD4 + CD8 + T cells and peripheral blood lymphocytes 19,20,82,21,28,29,30,83,84,58,59,60 Single peptides tested positive from earlier IFN Ref er Figure 3 5) P/I PMA/Ionomycin ; DM DMSO; IRP Irrelevant peptide; hGA Heat inactivated GAA;MCK Mock, rIFN Recombinant IFN T+M = Whole splenocytes and mock control; T+G = Whole splencoytes and rhGAA protein T+ 83 = Whole splenocytes and peptide 83; B+G = Peripheral blood lymp hocytes and rhGAA protein; CD8 + GAA & CD8 + 83 = mix of labelled and unlabelled CD8 + T cell populations with rh GAA protein and peptide 83; CD4+ GAA & CD4+ 83 = mix of labelled and unlabelled CD4 + T cell populations with rh GAA protein and peptide 83

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65 Figure 3 12. IFN ELISpot plate with separated CD4+ and CD8 + T cell populations from rhGAA immunized GAA / 129 Sv mouse A (1 2) H (1 2) Peptide Pools 1 8 with 10 peptides/pool A (3 4) H (3 4) Pools 9 16, A (5 6) H (5 6) Pools17 24 A (7 8) H (7 8) Po ols 25 32 A (9 10) H (9 10) Peptides 51, 54,71,74,80,81,83,84 A (11 12) PMA/IONO, B (11 12) SEB, C (11 12) DMSO, D (11 12) GAA, E (11 12) IrrPep, F (11 12) hiGAA, G (11 12) MOCK, H (11 12) rIFN

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66 Fig ure 3 13. Average count of spot forming units/10 6 cells from CD4+ positive T cells of Figure 3.12.

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67 Fig ure 3 14. Average count of spot forming units from CD8 + T positive cells of Figure 3 8 Figure 3 15. IFN + CD4 + T cell frequencies in mice using peptide 83 0.49% 0 50 100 150 200 250 300 350 400 450 Spot forming units/10 6 cells P eptides and C ontrols Average cell count of CD8 + cells AVG CELL COUNT

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68 Figure 3 16. IFN + CD4 + T cell frequencies in mice with whole protein rhGAA Figure 3 17. IFN + CD4 + T cell frequencies in GAA / mice with SEB 0.51 % 0.48%

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69 Figure 3 18. IFN + CD4 + T cell frequencies in GAA / mice using mock stimulated samples Figure 3 19. IFN + CD4 + T cell frequencies in GAA / mice using unstained control 0.22%

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70 Table 3 3 Immune epitope database T cell epitope prediction tools results Lowest score = Highest affinity Allele Position Sequence Consensus Percentile Rank SMM_align Core SMM_align Score SMM_align Percentile Rank NN_align Core NN_align Score NN_align Percentile Rank H2 IAb 1:196 210 ETPHVHSRAPSPLYS 7.74 VHSRAPSPL 479.0 4.96 SRAPSPLYS 837.7 10.52 H2 IAb 1:197 211 TPHVHSRAPSPLYSV 6.12 VHSRAPSPL 406.0 4.19 SRAPSPLYS 584.4 8.05 H2 IAb 1:198 212 PHVHSRAPSPLYSVE 6.55 VHSRAPSPL 455.0 4.7 SRAPSPLYS 617.7 8.41 H2 IAb 1:316 330 NAMDVVLQPSPALSW 2.71 VVLQPSPAL 215.0 2.15 VLQPSPALS 200.8 3.26 H2 IAb 1:317 331 AMDVVLQPSPALSWR 2.45 VVLQPSPAL 214.0 2.14 VLQPSPALS 168.7 2.75 H2 IAb 1:318 332 MDVVLQPSPALSWRS 2.39 VVLQPSPAL 255.0 2.58 VLQPSPALS 137.1 2.2 H2 IAb 1:336 350 ILDVYIFLGPEPKSV 0.56 YIFLGPEPK 62.0 0.39 FLGPEPKSV 55.5 0.73 H2 IAb 1:337 351 LDVYIFLGPEPKSVV 0.45 YIFLGPEPK 59.0 0.36 FLGPEPKSV 44.5 0.53 H2 IAb 1:338 352 DVYIFLGPEPKSVVQ 0.42 YIFLGPEPK 60.0 0.37 FLGPEPKSV 40.8 0.46

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71 Figure 3 20 Anti GAA plasma titer levels from GAA / mice immunized with rhGAA

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72 CHAPTER 4 DISCUSSION Immunodominant epitopes are essential for mounting effective immune responses in the body. In the past decade, more attention has been given towards mapping and characterization of T cell epitopes from protein antigens. CD4 + T cells have been garnering additional importance due to their significant roles in the development and functioning of both the cell mediated and humoral reponses of the adaptive immune system. (19) Although significant improvements have been made in the field of epitope mapping, researchers have been concentrating more towards the humoral immune response of the patients in Pompe (2) As the functioning and differentiation of B cells depends on the help of C D4 + T cells, determining the CD4 + immunodominant epitope against the rhGAA protein in GAA / mice will significantly improve the understanding of the relationship between T and B cell activation and also help in uncovering the peptide and MHC binding motifs f or this disorder. We chose a combination of methods such as ELISpot, in vitro cytokine staining and in silico prediction algorithms, for mapping the immundominant CD4 + epitope against the rhGAA protein in 6 8 weeks old GAA / (GAA knockout) 129Sv mice. The se mice had been created by others through targeted disruption in exon 6 using a neomycin resistance gene (6 neo /6 neo ), (20) and their MHC class II haplotypes were identified as I A bc (21, 22) Consequently, the immunodominant epitopes mapped using the above techniques were specific for this haplotype. Of all the 95 peptides spanning the rhGAA protein that were tested by building simple and matrix pools of pe ptides, peptide 19, 29 and 83 were identified as

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73 candidates for dominant epitopes. Amongst those, peptide 83 emerged as containing the dominant epitopes in all the 3 methods of mapping, clearly showing the prevalence for this portion of the protein during the antigen presentation to CD4 + T cells in the GAA / 129 Sv murine model of severe pompe disease. As the immune responses against the rhGAA protein have in preliminary studies been pivoted on the increased levels of IgG1 antibody, which in other words si gnifies a Th2 type cellular response, (16) we sought to study the different gene expression levels of Th2 and Th1 cytokines in pompe mice that received ERT similar to humans. The results obtained from running an RT P CR on cDNA samples from rhGAA immunized GAA / mice will help in exploring in detail about the T cell mediated responses for this lysosomal storage disorder. Others have reported formation of IgE and fatal anaphylactic reactions in pompe mice during ERT st udies. We hypothesize that the combination of any subclass mapping and the cytokine response from CD4 + T cells to GAA will allow us to propose a mechanism that drives the immune response in ERT. Identifying the helper pathway chosen by CD4 + effector cells will aid in analyzing the different classes of antibodies which can be secreted by these antigen specific cells and a better understanding of the B cell mediated immunity. The epitope can serve as a tool for designing and developing immunomodulatory and im munotherapeutic protocols for this disorder in the future. Use of this epitope will accelerate subsequent me chanistic studies. For example p eptide antigens can be used for stimulation in ICS techniques. These peptides can also be used for inducing toleranc e against the protein and for improving their activities in enzyme replacement therapies.

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74 The results from this mapping can be utilized for tolerance studies similar to the tolerance against FIX (13) and for Induction of tolerance to rhGAA using combinations of the immunosuppressant drug rapamycin (14) While we may not have a prior knowledge of dominant CD4 + T cell epitopes in pompe patients, our approach should be useful to map dominant epitopes in humans with immune responses against the protein. Peptides encoding known epitopes could then be used in immune tolerance protocols. This is of particular import ance for situations where use of protein antigen causes anaphylactic reactions. The future is encouraging for this field with the developments occurring in the areas of enzyme replacement and gene therapy. (4, 9, 11) The inhibitory antibodies which are hindering the effectiveness of these therapies can be effectively countered by using protocols implementing these immunodominant epitopes. Thus the mapping of dominant CD4 T cell epitopes for the deficient enzyme of lysosomal storage disorder in GAA / 129 Sv mice using in vitro T cell assays will help in opening more avenues for research in this field and eventually help in unlocking several ans wers related to T and B cell me diated immunities of the system.

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75 APPENDIX A GAA PEPTIDE LIBRARY

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76

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77

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78 APPENDIX B T CELL EPITOPE PREDI CTION RESULTS NetMHCII 2.2 Server prediction results Technical University of Denmark # Input is in FSA format # Peptide length 15 NetMHCII version 2.2. Strong binder threshold 50.00. Weak binder threshold 500.00. ----------------------------------------------------------------------------Allele pos peptide core 1 log50k(aff) affinity(nM) Bind Level ----------------------------------------------------------------------------------------------H 2 IAb 338 VYIFLGPEPKSVVQQ FLGPEPKSV 0.6606 39.4 SB H 2 IAb 337 DVYIFLGPEPKSVVQ FLGPEPKSV 0.65 73 40.8 SB H 2 IAb 336 LDVYIFLGPEPKSVV FLGPEPKSV 0.6492 44.5 SB H 2 IAb 335 ILDVYIFLGPEPKSV FLGPEPKSV 0.6288 55.5 WB H 2 IAb 475 LIGKVWPGSTAFPDF KVWPGSTAF 0.6185 62.1 WB H 2 IAb 476 IGKVWPGSTAFPDFT KVWPGSTAF 0.6138 65.3 WB H 2 IAb 339 YIFLGPEPKSVVQQY FLGPEPKSV 0.6115 66.9 WB H 2 IAb 781 ALGSLPPPPAAPREP SLPPPPAAP 0.6094 68.5 WB H 2 IAb 474 PLIG KVWPGSTAFPD KVWPGSTAF 0.6084 69.2 WB H 2 IAb 142 SSEMGYTATLTRTTP YTATLTRTT 0.6072 70.1 WB H 2 IAb 780 EALGSLPPPPAAPRE SLPPPPAAP 0.6065 70.7 WB H 2 IAb 143 SEMGYTATLTRTTPT YTATLTRTT 0.6056 71.4 WB H 2 IAb 779 VEALGSLPPPPAAPR SLPPPPAAP 0.6033 73.1 WB H 2 IAb 141 SSSEMGYTATLTRTT YTATLTRTT 0.6025 73.8 WB H 2 IAb 477 GKVWPGSTAFPDFTN KVWPGSTAF 0 .6004 75.5 WB H 2 IAb 778 PVEALGSLPPPPAAP SLPPPPAAP 0.5931 81.7 WB H 2 IAb 144 EMGYTATLTRTTPTF YTATLTRTT 0.5927 82.0 WB

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79 APPENDIX C SOURCE FIGURE 1 1

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80 LIST OF REFERENCES 1. Raben, N., P. Plotz, and B. J. Byrne. 2002. Acid alpha glucosidase deficiency (glycogenosis type II, Pompe disease). Curr. Mol. Med. 2: 145 166. 2. de Vries, J. M., N. A. van der Beek, M. A. Kroos, L. Ozkan, P. A. van Doorn S. M. Richards, C. C. Sung, J. D. Brugma, A. A. Zandbergen, A. T. van der Ploeg, and A. J. Reuser. 2010. High antibody titer in an adult with Pompe disease affects treatment with alglucosidase alfa. Mol. Genet. Metab. 101: 338 345. 3. Tinkle, B. T. and N. Leslie. 1993. Glycogen Storage Disease Type II (Pompe Disease). In GeneReviews, R. A. Pagon, T. D. Bird, C. R. Dolan, and K. Stephens eds. University of Washington, Seattle. All rights reserved, Seattle (WA). 4. ACMG Work Group on Management of Pompe D isease, P. S. Kishnani, R. D. Steiner, D. Bali, K. Berger, B. J. Byrne, L. E. Case, J. F. Crowley, S. Downs, R. R. Howell, R. M. Kravitz, J. Mackey, D. Marsden, A. M. Martins, D. S. Millington, M. Nicolino, G. O'Grady, M. C. Patterson, D. M. Rapoport, A. S lonim, C. T. Spencer, C. J. Tifft, and M. S. Watson. 2006. Pompe disease diagnosis and management guideline. Genet. Med. 8: 267 288. 5. Rosa, D. S., S. P. Ribeiro, and E. Cunha Neto. 2010. CD4+ T cell epitope discovery and rational vaccine design. Arch. I mmunol. Ther. Exp. (Warsz) 58: 121 130. 6. van der Ploeg, A. T. and A. J. Reuser. 2008. Pompe's disease. Lancet 372: 1342 1353. 7. Geel, T. M., P. M. McLaughlin, L. F. de Leij, M. H. Ruiters, and K. E. Niezen Koning. 2007. Pompe disease: current state of treatment modalities and animal models. Mol. Genet. Metab. 92: 299 307. 8. Hoefsloot, L. H., M. Hoogeveen Westerveld, A. J. Reuser, and B. A. Oostra. 1990. Characterization of the human lysosomal alpha glucosidase gene. Biochem. J. 272: 493 497. 9. Byrn e, B. J. e. a. 2011. Pompe disease : Design, methodology, and early findings from Pompe Registry. 10. Howell, R. R., B. Byrne, B. T. Darras, P. Kishnani, M. Nicolino, and A. van der Ploeg. 2006. Diagnostic challenges for Pompe disease: an under recognized cause of floppy baby syndrome. Genet. Med. 8: 289 296. 11. Raben, N., N. Lu, K. Nagaraju, Y. Rivera, A. Lee, B. Yan, B. Byrne, P. J. Meikle, K. Umapathysivam, J. J. Hopwood, and P. H. Plotz. 2001. Conditional tissue specific expression of the acid alpha glucosidase (GAA) gene in the GAA knockout mice: implications for therapy. Hum. Mol. Genet. 10: 2039 2047.

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81 12. Sun, B., S. Li, A. Bird, H. Yi, A. Kemper, B. L. Thurberg, and D. D. Koeberl 2010. Antibody formation and mannose 6 phosphate receptor expression impact the efficacy of muscle specific transgene expression in murine Pompe disease. J. Gene Med. 12: 881 891. 13. Cao, O., E. Armstrong, A. Schlachterman, L. Wang, D. K. Okita, B. Con ti Fine, K. A. High, and R. W. Herzog. 2006. Immune deviation by mucosal antigen administration suppresses gene transfer induced inhibitor formation to factor IX. Blood 108: 480 486. 14. Nayak, S., O. Cao, B. E. Hoffman, M. Cooper, S. Zhou, M. A. Atkinson and R. W. Herzog. 2009. Prophylactic immune tolerance induced by changing the ratio of antigen specific effector to regulatory T cells. J. Thromb. Haemost. 7: 1523 1532. 15. Murphy, K., Travers, P., Walport, M. 2007. Janeway's Immunobiology. 7thed.Garla nd Science, London, UK. 928. 16. Provenzano, M. and G. C. Spagnoli. 2009. T cell epitope mapping by cytokine gene expression assay. Methods Mol. Biol. 514: 107 118. 17. Tobery, T. W. and M. J. Caulfield. 2004. Identification of T cell epitopes using ELIS pot and peptide pool arrays. Methods Mol. Med. 94: 121 132. 18. Anthony, D. D. and P. V. Lehmann. 2003. T cell epitope mapping using the ELISPOT approach. Methods 29: 260 269. 19. Delvig, A. A. and J. H. Robinson. 2001. CD4 T cell epitope mapping. Method s Mol. Med. 66: 349 360. 20. Raben, N., K. Nagaraju, E. Lee, P. Kessler, B. Byrne, L. Lee, M. LaMarca, C. King, J. Ward, B. Sauer, and P. Plotz. 1998. Targeted disruption of the acid alpha glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II. J. Biol. Chem. 273: 19086 19092. 21. Fischer Lindahl, K. 1997. On naming H2 haplotypes: functional significance of MHC class Ib alleles. Immunogenetics 46: 53 62. 22. Ohtsuka, M., H. Ino ko, J. K. Kulski, and S. Yoshimura. 2008. Major histocompatibility complex (Mhc) class Ib gene duplications, organization and expression patterns in mouse strain C57BL/6. BMC Genomics 9: 178. 23. Kalyuzhny, A. E. 2005. Handbook of ELISPOT [electronic reso urce] : methods and protocols. Humana Press, Totowa, New Jersey. 24. Wang P, Sidney J, Dow C, Moth B, Sette A, Peters B. 2008. A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach.

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82 25. Wang P, Sidney J, Kim Y, Sette A, Lund O, Nielsen M, Peters B. 2010. peptide binding predictions for HLA DR, DP and DQ molecules. 26. Zhang, G. L., D. S. Deluca, D. B. Keskin, L. Chitkushev, T. Zlateva, O. Lund, E. L. Reinherz, and V. Brusic. 2010. MULTIPRED2: A comput ational system for large scale identification of peptides predicted to bind to HLA supertypes and alleles. J. Immunol. Methods 27. Bui HH, Sidney J, Peters B, Sathiamurthy M, Sinichi A, Purton KA, Moth BR, Chisari FV, Watkins DI, Sette A. Immunogenetics 304 314. 28. Nielsen M, Lundegaard C, Blicher T, Peters B, Sette A, Justesen S, Buus S, and Lund O. 2008. Quantitative predictions of peptide binding to any HLA DR molecule of known sequence: NetMHCIIpan. 29. Nielsen, M. and O. Lund. 2009. NN align. An artificial neural network based alignment algorithm for MHC class II peptide binding prediction. BMC Bioinformatics 10: 296. 30. Nielsen M, Lundegaard C, Lund O. 2007. Prediction of MHC class II binding affinity using SMM align, a novel stabilization matr ix alignment method. 238. 31. van den Berg, L. E., J. M. de Vries, R. M. Verdijk, A. T. van der Ploeg, A. J. Reuser, and P. A. van Doorn. 2011. A case of adult Pompe disease presenting with severe fatigue and selective involvement of type 1 muscle fibers. Neuromuscul. Disord. 21: 232 234. 32. Luo, F., W. Wu, Q. Geng, F. Li, W. Chen, W. Gan, and J. Xie. 2011. A novel mutation Glu441stop (GAA to TAA) of androgen receptorgene resulting in complete androgen insensitivity syndrome. Zhonghua Yi Xue Yi Chuan Xue Za Zhi 28: 176 179. 33. Zampieri, S., E. Buratti, S. Dominissini, A. L. Montalvo, M. G. Pittis, B. Bembi, and A. Dardis. 2011. Splicing mutations in glycogen storage disease type II: evaluation of the full spectrum of mutations and their relation to pati ents' phenotypes. Eur. J. Hum. Genet. 19: 422 431. 34. Takikita, S., C. Schreiner, R. Baum, T. Xie, E. Ralston, P. H. Plotz, and N. Raben. 2010. Fiber type conversion by PGC 1alpha activates lysosomal and autophagosomal biogenesis in both unaffected and P ompe skeletal muscle. PLoS One 5: e15239. 35. Parenti, G. and G. Andria. 2011. Pompe Disease: from New Views on Pathophysiology to Innovative Therapeutic Strategies. Curr. Pharm. Biotechnol. 36. Fidzianska, A., A. Lugowska, and A. Tylki Szymanska. 2011. Late form of Pompe disease with glycogen storage in peripheral nerves axons. J. Neurol. Sci. 301: 59 62.

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84 BIOGRAPHICAL SKETCH Biotechnology from Anna University,India. She chose to pursue her Masters in the United States of America, and entered the Biomedical Engineering program at the University of Florida during the year 2009. She was working as a Graduate Research Assistant in the Cellular and Molecular therapy division during her course of study. Upon completion of her graduation, she will be joining the PhD program in Biomedical Sciences at UF. Her research interests encompass several areas such as Immunology and genetic and tissue engineering. She intends to continue working in the fields of Immunology in an In dustrial environment in the future. Apart from studies, she loves to travel, read books, and enjoys adventurous sports