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Induction of Immune Tolerance to Factor IX in Gene and Protein Therapy for Hemophilia B

Permanent Link: http://ufdc.ufl.edu/UFE0041421/00001

Material Information

Title: Induction of Immune Tolerance to Factor IX in Gene and Protein Therapy for Hemophilia B
Physical Description: 1 online resource (95 p.)
Language: english
Creator: Nayak, Sushrusha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: aav, aav1, aav2, adaptive, adenoassociated, aicd, antibody, antigen, apc, cd4, cd8, clotting, complement, ctl, dendritic, factor, flt3l, foxp3, gene, gitr, hemophilia, il10, immune, inflamation, inhibitors, innate, interleukin, liver, muscle, neutralizing, pompe, prophylactic, protein, rag, rapamycin, therapy, tolerance, tr1, treg, virus
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Hemophilia B is an X-linked bleeding disorder affecting 1 in 30, 000 male births with varying severity, differing due to the underlying mutations in the Factor IX (F.IX) gene. Treatment of patients with severe hemophilia is complicated by the formation of neutralizing antibodies to the therapeutic Factor IX protein in gene and protein replacement therapies. Prophylactic protocols for the prevention of immune responses are key to successful therapy. Preventive protocols utilizing the immunosuppressant drug rapamycin, cytokines IL-10, IL-2, Fc-GITR-L and antigen specific CD4 epitopes were developed to induce antigen specific tolerance in a test model (DO.11.10tg Rag2-/-) BALB/c mice. The prophylactic protocol utilizing rapamycin, IL-10 cytokine, antigen specific peptide was investigated in detail. The T cell receptor (TCR) transgenic test model showed the induction of CD4+CD25+FoxP3+ regulatory T cells (Treg), a cell type crucial for immune tolerance. Concomitant with Treg induction, depletion of antigen specific effector T cell (Teff) was observed. The Teff cell depletion was caused by activation induced cell death (AICD). Flow cytometric analysis further indicated differential effects of the drug cocktail on Treg vs. Teff. Treg were more resistant to the drug-induced AICD than Teff, hence allowing Treg cells a proliferative advantage. Optimization studies with differing routes of drug cocktail administration showed higher levels of Treg induction and Teff cell depletion in intra-peritoneal, subcutaneous and intravenous drug administration as opposed to oral gavage. Promising results from the (DO.11.10tg Rag2-/-) Balb/c mice warranted an extension to F9 knockout mice (C3H/HeJ-F9-/-), which is a Hemophilia B disease model. Optimal protocols initially identified in the TCR transgenic model were tested for and resulted in prophylactic tolerance induction to F.IX prior to adeno-associated virus serotype1 (AAV1) gene transfer to skeletal muscle in hemophilia B mice. Tolerance was maintained for > 5 months, accompanied by sustained F.IX expression. In addition to prophylactic use, the Rapa/IL-10/specific peptide protocol suppressed a pre-existing immune response initiated by AAV1-hFIX gene transfer in hemophilia B mice and maintained long-term tolerance. The results demonstrate that the prophylactic protocol tested can successfully prevent immune responses to F.IX and may also be applicable to treatment of other inherited systemic protein disorders such as hemophilia A (Factor VIII deficiency) and Pompe disease (a lysosomal storage disorder). The success of the tolerance protocol in eliminating a pre-existing immune response raises the prospect of eventual translation to human therapy for the treatment of patients who have developed an immune response to protein replacement therapy, or correcting unintended immune responses in gene therapy.
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 Sushrusha Nayak.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Herzog, Roland.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041421:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041421/00001

Material Information

Title: Induction of Immune Tolerance to Factor IX in Gene and Protein Therapy for Hemophilia B
Physical Description: 1 online resource (95 p.)
Language: english
Creator: Nayak, Sushrusha
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: aav, aav1, aav2, adaptive, adenoassociated, aicd, antibody, antigen, apc, cd4, cd8, clotting, complement, ctl, dendritic, factor, flt3l, foxp3, gene, gitr, hemophilia, il10, immune, inflamation, inhibitors, innate, interleukin, liver, muscle, neutralizing, pompe, prophylactic, protein, rag, rapamycin, therapy, tolerance, tr1, treg, virus
Immunology and Microbiology (IDP) -- Dissertations, Academic -- UF
Genre: Medical Sciences thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Hemophilia B is an X-linked bleeding disorder affecting 1 in 30, 000 male births with varying severity, differing due to the underlying mutations in the Factor IX (F.IX) gene. Treatment of patients with severe hemophilia is complicated by the formation of neutralizing antibodies to the therapeutic Factor IX protein in gene and protein replacement therapies. Prophylactic protocols for the prevention of immune responses are key to successful therapy. Preventive protocols utilizing the immunosuppressant drug rapamycin, cytokines IL-10, IL-2, Fc-GITR-L and antigen specific CD4 epitopes were developed to induce antigen specific tolerance in a test model (DO.11.10tg Rag2-/-) BALB/c mice. The prophylactic protocol utilizing rapamycin, IL-10 cytokine, antigen specific peptide was investigated in detail. The T cell receptor (TCR) transgenic test model showed the induction of CD4+CD25+FoxP3+ regulatory T cells (Treg), a cell type crucial for immune tolerance. Concomitant with Treg induction, depletion of antigen specific effector T cell (Teff) was observed. The Teff cell depletion was caused by activation induced cell death (AICD). Flow cytometric analysis further indicated differential effects of the drug cocktail on Treg vs. Teff. Treg were more resistant to the drug-induced AICD than Teff, hence allowing Treg cells a proliferative advantage. Optimization studies with differing routes of drug cocktail administration showed higher levels of Treg induction and Teff cell depletion in intra-peritoneal, subcutaneous and intravenous drug administration as opposed to oral gavage. Promising results from the (DO.11.10tg Rag2-/-) Balb/c mice warranted an extension to F9 knockout mice (C3H/HeJ-F9-/-), which is a Hemophilia B disease model. Optimal protocols initially identified in the TCR transgenic model were tested for and resulted in prophylactic tolerance induction to F.IX prior to adeno-associated virus serotype1 (AAV1) gene transfer to skeletal muscle in hemophilia B mice. Tolerance was maintained for > 5 months, accompanied by sustained F.IX expression. In addition to prophylactic use, the Rapa/IL-10/specific peptide protocol suppressed a pre-existing immune response initiated by AAV1-hFIX gene transfer in hemophilia B mice and maintained long-term tolerance. The results demonstrate that the prophylactic protocol tested can successfully prevent immune responses to F.IX and may also be applicable to treatment of other inherited systemic protein disorders such as hemophilia A (Factor VIII deficiency) and Pompe disease (a lysosomal storage disorder). The success of the tolerance protocol in eliminating a pre-existing immune response raises the prospect of eventual translation to human therapy for the treatment of patients who have developed an immune response to protein replacement therapy, or correcting unintended immune responses in gene therapy.
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 Sushrusha Nayak.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Herzog, Roland.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-04-30

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041421:00001


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1 INDUCTION OF IMMUNE TOLERANCE TO FACTOR IX IN GENE AND PROTEIN REPLACEMENT THERAPY FOR HEMOPHILIA B By SUSHRUSHA NAYAK A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FUL FILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

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2 2010 Sushrusha Nayak

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3 To my parents for their unwavering support and encouragement

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4 ACKNOWLEDGMENTS I take th is opportunity to thank my mentor, Dr. Roland W. Herzog for his guidance and extensive support. He has always been available to answer my questions, without judgment, since the day I joined his lab. I appreciate the opportunities he has provided me to work on multiple projects and collaborate with the best researchers in the fields of gene therapy and immunology. What I have gained over my time in this lab and university transcends academic learning and cannot be encompassed in this thesis alone. I would li ke to thank Dr. Ou Cao, who provided me, with the essential training I required to be successful at the bench. I would also like to extend my thanks to my committee members Dr. Mark Atkinson, Dr. Barry Byrne, Dr. Bradley Fletcher and Dr. Laurence Morel who have been instrumental in guiding and directed the theme of my work and bringing this thesis to its successful completion. I thank my lab colleagues for their collaboration and for making arduous tasks easy. I thank my parents, Dr. Rabindranath Nayak and Sukanti Nayak for always believing in me and for their incomprehensible love and support in the things I chose to do, even if they broke from tradition. I thank my brother for his camaraderie; Dr. Arun Srivastava and Dr. Jackie Hobbs for easing my transi tion into a new country and making me feel at home. I thank my friends in the IDP program for all the fun and intellectual conversations and Dr. Aravind Natarajan for all his support.

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5 TABLE OF CONTENTS page ACKNOWLEDGM ENTS ...............................................................................................................4 LIST OF FIGURES .........................................................................................................................7 ABSTRACT .....................................................................................................................................9 CHAPTER 1 INTRODUCTION ..................................................................................................................11 Hemophilia: An Introduction to the Disease ..........................................................................11 Treatment of Hemophilia ........................................................................................................12 Protein Replacement Therapy .........................................................................................12 Gene Therapy for Hemophilia .........................................................................................13 AAVmediated gene transfer ...................................................................................13 Hepatic AAV gene transfer for treatment of hemophilia B .....................................14 Overview of Immune Responses in AAV Mediated Gene Transfer ......................................16 Innate Immune Responses to AdenoAssociated Virus ..................................................16 Adaptive Immune Responses to AAV Vectors and their Transgene Products ...............17 Inhibitors in Hemophilia .........................................................................................................20 Preventing Immune Responses in AAV Mediated Gene Transfer ..................................21 Immune Tolera nce and Regulation of Transgene Product Directed Responses .............22 Potential Roles of Rapamycin, IL 10, IL 2 and Fc GITRL and Flt3L ..........................23 2 MATERIALS AND METHODS ...........................................................................................28 Reagents ..................................................................................................................................28 Mouse Strains .........................................................................................................................28 Admin istration of Drugs .........................................................................................................29 Blood Sample Collection ........................................................................................................30 Analysis of Plasma Samples ...................................................................................................30 Clotting Assays .......................................................................................................................31 Flow Cytometry ......................................................................................................................32 Cytokine Secretion Assay .......................................................................................................33 Adoptive Transfer Assay ........................................................................................................33 In vitro Suppression Assay .....................................................................................................34 Statistical Analysis ..................................................................................................................34 3 ESTABLISHMENT AND MECHANISTIC INVESTIGATION OF NOVEL TOLERANCE PROTOCOLS ................................................................................................37 Introduction .............................................................................................................................37 D epletion Teff Cells and Induction of CD4+CD25+FoxP3+ Treg via Rapamycin/IL 10/Specific peptide Protocol. ..............................................................................................39

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6 Effect of Rapamycin/IL 10/Specific peptide in Lymphoid Organs .......................................40 Activation and Apoptosis of Ova specific CD4+ T Cells. ......................................................41 Induced Treg Suppress Teff and Show Lower Rate of Apoptosis.............................................41 Route Optimization of Rapamycin/IL 10/Specific peptide Protocol .....................................42 Alternative Cytokines for Induction of Treg in the Tolerance Protocol ..................................43 Transient Non specific Changes in Lymphocyte and Neutrophil Counts in Immune Competent Mice ..................................................................................................................43 4 INDUCTION OF TOLERANCE IN HEMOPHILIA B DISEASE MICE RECEIVING GENE THERAPY ..................................................................................................................59 Introduction .............................................................................................................................59 Testing a Short Tolerance Protocol in C57BL/6 Mice ...........................................................60 Prevention of Inhibitor Formation in Intramuscular Gene Therapy for Hemophilia B ..........60 Induction of Functional Treg via the Prophylactic Tolerance Protocol ...................................62 Modified Tolerance Protocol Using an Alternate Route of Tolerance Induction ..................62 Modified Tolerance Protocol Utilizing an Alte rnate Cytokine ..............................................63 Prevention of Inhibitor Formation in Hepatic Gene Therapy for Murine Hemophilia B .......64 Rapamycin/IL 10/Specific Peptide Treatment ................................................................64 Fc GITRL treatment .......................................................................................................65 Inhibition of an Established Anti F.IX Immune Response by the Rapamycin/IL 10/Specif ic Peptide Protocol ...............................................................................................66 Prevention of an Anti F.IX Immune Response in F.IX Protein Therapy ...............................67 5 DISCUSSION .........................................................................................................................79 Creating an Optimal Shift From an Effector to a Regulatory T Cell Population ...................79 Treg are Critical for Many Tolerance Induction Protocols ......................................................81 Implications for Translational Research .................................................................................82 LITERATURE CITATIONS .........................................................................................................86 BIOGRAPHICAL SKETCH .........................................................................................................95

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7 LIST OF FIGURES Figure page 11 Simplified diagram of the intrinsic pathway in the blood clotting cascade. ......................26 12 Overview of immune responses to viral vectors. ...............................................................27 21 Diagram of human factor IX primary amino acid sequence with CD4+ T cell epitopes. .............................................................................................................................35 22 Diagrammatic representation of the effector T cell suppression assay ..............................36 23 Diagrammatic representation of the adoptive transfe r assay .............................................36 31 Flow cytometric analysis of splenocytes from DO.11.10tg Rag/BALB/c mice ............45 32 Average cell percentages from flow cytometric analysis of splenocytes in DO.11.10tg Rag/BALB/c mice.. .....................................................................................................46 33 Cytokine capture in treatment groups ................................................................................47 34 F low cytometric analysis of CD4+ T effector T cell deletion ............................................48 35 Flow cytometric analysis of CD4+ T cell deletion in lymphoid organs. ............................49 36 Flow cytometric analysis of regulatory T cell induction. ..................................................50 37 Measurement of T cell activation in splenocytes. ..............................................................51 38 Measurement of apoptosis and Fas ( CD95) expression in splenocytes .............................52 39 Activation induced cell death (AICD) timeline .................................................................53 310 Differential effects of treatment on Teff and Treg. ...............................................................54 311 Ova specific functional T cell proliferation assay. ............................................................55 312 Opt imization of Rapa/IL 10/specific peptide tolerization protocol utilizing different routes of injection. .............................................................................................................56 313 Optimization of alternate drug combinations. ...................................................................57 314 Effect of tolerance protocol on general immune suppression in immune competent mice over time. ...................................................................................................................58 41 Anti hF.IX formation after IM injection of AAV2 CMVhF.IX in C57BL/6 mice. .........69

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8 42 Prevention of inhibitor formation in hemophilia B mice via rapamycin/IL 10/specific peptide protocol. ................................................................................................................70 43 Treg induced by rapamycin/cytokine/specific peptide protocol are functional ...................71 44 Partial suppression of inhibitor formation in hemophilia B mice via oral administration of rapamyci n and concurrent IP IL 10/specific peptide treatment.. ..........72 45 Partial suppression of inhibitor formation in hemophilia B mice via rapamycin/IL 2/specific peptide protocol .................................................................................................73 46 Prevention of inhibitor formation in hemophilia B mice via rapamycin/IL 10/specific peptide protocol in liver gene transfer. ..............................................................................74 47 Rapamycin/ IL 10/specific peptide protocol evaluation in liver gene transfer to Rag/mice. ...................................................................................................................................75 48 Delay of immune response to hF.IX in hemophilia B mice following administration of Fc GITRL .....................................................................................................................76 49 Elimination of a pre existing/ongoing anti hF.IX immune response.. ..............................77 51 Induction of tolerance to therapeutic proteins by shifting the balance from an effector (CD4+ Teff) to a regulatory response (CD4+ Treg) prior to gene or protein replacement therapy in an antigen specific manner. ..............................................................................85

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9 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy INDUCTION OF IMMUNE TOLERANCE TO THERAPEUTIC PROTEINS IN GENE AND PROTEIN REPLACEMENT THERAPY FOR HEMOPHILIA B By Sushrusha Nayak May 2010 Chair: Roland W. Herzog Major: Medical Sciences Immunology and Microbiology Hemophilia B is an X linked bleeding disorder affecting 1 in 30, 000 male births with varying severity, differing due to the underlying mutations in the Factor IX (F.I X) gene. Treatment of patients with severe hemophilia is complicated by the formation of neutralizing antibodies to the therapeutic Factor IX protein in gene and protein replacement therapies Prophylactic protocols for the prevention of immune responses a re key to successful therapy. Preventive protocols utilizing the immunosuppressant drug rapamycin, cytokines IL 10, IL 2, Fc GITRL and antigen specific CD4 epitopes were developed to induce antigen specific tolerance in a test model (DO.11.10tg Rag2/ ) B ALB/c mice. The prophylactic protocol utilizing rapamycin, IL 10 cytokine, antigen specific peptide was investigated in detail. The T cell receptor (TCR) transgenic test model showed the induction of CD4+CD25+FoxP3+ regulatory T cells (Treg), a cell type crucial for immune tolerance. Concomitant with Treg induction, depletion of antigen specific effector T cell (Teff) was observed. The Teff cell depletion was caused by activation induced cell death (AICD). Flow cytometric analysis further indicated differe ntial effects of the drug cocktail on Treg vs. Teff. Treg were more resistant to the drug induced AICD than Teff, hence allowing Treg cells a proliferative advantage. Optimization studies

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10 with differing routes of drug cocktail administration showed higher levels of Treg induction and Teff cell depletion in intra peritoneal, subcutaneous and intravenous drug administration as opposed to oral gavage. Promising results from the (DO.11.10tg Rag2/ ) Balb/c mice warranted an extension to F9 knockout mice (C3H/H eJF9 / ), which is a Hemophilia B disease model. Optimal protocols initially identified in the TCR transgenic model were tested for and resulted in prophylactic tolerance induction to F.IX prior to adenoassociated virus serotype1 (AAV1) gene transfer to skeletal muscle in hemophilia B mice. Tolerance was maintained for > 5 months, accompanied by sustained F.IX expression. In addition to prophylactic use, the Rapa/IL 10/specific peptide protocol suppressed a pre existing immune response initiated by AAV1hFIX gene transfer in hemophilia B mice and maintained longterm tolerance. The results demonstrate that the prophylactic protocol tested can successfully prevent immune responses to F.IX and may also be applicable to treatment of other inherited systemic protein disorders such as hemophilia A (Factor VIII deficiency) and Pompe disease (a lysosomal storage disorder). The success of the tolerance protocol in eliminating a pre existing immune response raises the prospect of eventual translation to human therapy for the treatment of patients who have developed an immune response to protein replacement therapy, or correcting unintended immune responses in gene therapy.

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11 CHAPTER 1 INTRODUCTION Hemophilia: An Introduction to the Disease Hemophilia is an X linked recessive hemorrhagic disorder resulting from mutations to genes encoding coagulation Factor IX (Hemophilia B) or Factor VIII (F.VIII; Hemophilia A) thereby causing a systemic protein deficiency.1 Males are usually affected and females are typically car riers.2 According to the world federation of hemophilia, an estimated 400,000 people around the world live with hemophilia with only 25% receiving adequate treatment around the globe. It came to the forefront as the royal disease manifested a mong many royal families in Europe. The size of the Factor IX gene is about 34kb, 92% of the gene consists of introns, while there are 8 exons. Factor IX participates in the intrinsic plasmaclotting cascade.3 It occurs as a zymogen and is activated to Factor IXa that further activates Factor X in the presence of Factor VIIIa to further the clotting cascade, which finally results in fibrin clot formation (Fig. 11). Royal disease was recently confirmed via genotype analysis to be hemophili a B caused by mutations that led to altered RNA splicing.4 Mutations may vary from single base substitutions, early/late deletions, missense mutations etc, which affect the severity of the disease and influence the risk of antibody formati on to the missing factor upon treatment with enzyme replacement or gene therapy. Patients with hemophilia exhibit prolonged clotting times (such as activated partial thromboplastin time aPTT) and require factor infusion following trauma or surgery. They m ay also exhibit spontaneous bleeds into joints and internal spaces. Less than 1% of F.VIII or F.IX activity represents severe disease, 25% is moderate and greater than 5% represents mild disease. An increase in the level of circulating clotting factors to above 1% reduces the incidence of bleeding and improves quality of life. There are suggestions that hemophilia A may be the more severe phenotypic form of the two hemophilias. 4 The frequency

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12 of Hemophilia B is about 1 in 30, 000 male bi rths, while the frequency of hemophilia A is about 1 in 5000 male births. Treatment of Hemophilia Protein Replacement Therapy Protein replacement is the choice of treatment for many systemic protein deficiencies such as the hemophilias and glycogen storage diseases (such as Pompe disease and Gaucher disease). Bleeding in hemophilia patients is currently managed by intravenous (IV) infusion of plasma derived or recombinant protein (F.VIII or F.IX) concentrate. Children with severe hemophilia A or hemophilia B can be treated prophylactically with doses of the respective missing factor to prevent haemarthrosis and reduce the regular progression of the disease, which targets joints. Joint damage is a major cause of morbidity of the disease but hemorrhagic stroke may also occur. The treatment usually involves the early start of factor infusion after diagnosis, with an eventual dose escalation. Prophylactic treatment for hemophilia is not commonly practiced; typically, patients are treated with the missing factor in response to a bleeding episode, which does not treat joint damage that has already occurred. In the past, clotting factor from porcine, bovine or human sources raised concerns about viral infection and allergic reactions. The availability of recombinant clotting factors however has reduced this risk. Prophylactic treatment is largely not performed in the United States due to high costs associated with high doses of clotting factor required, and the risk of infection of the catheter needed for intravenous (IV) access. Only 2025% of hemophilia patients are being treated worldwide because of the expense of purified coagulation factors. Maintenance of >1% of factor activity ameliorates disease phenotype by reducing incidences of spontaneous bleeds.5

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13 In protein based therapy for hemophilia, 2030% of hemophilia A and 1.54% of hemophi lia B patients form inhibitors (primarily subjects with gene deletions or premature stop codons).6 These immune responses are de pendent on CD4+ T cells. Targeting F.IX specific CD4+ T cells for tolerance induction is therefore an ideal strategy. It has become clear in recent years that immune regulation plays an important role in many tolerance protocols.7,8 In many ways, hemophilia is an ideal model for the development of gene therapy for genetic disorders. Precise regulation of transgene expression is not required. Although normally expressed by the liver, several cell types are capable of synthesizing biologically active coagulation factor following gene transfer. In addition, there are small and large animal models of the disease (F.VIII and F.IX knockout mice and naturally occurring hemophilia A and B dogs). Gene Therapy for Hemophilia Th e prevalence of the disease (~400,000 individuals) with the low access to treatment in developing countries, combined with the very high cost of treatment begs the need for gene therapy as a treatment modality. Approaches that may be used to ensure consist ent production of F.IX and F.VIII include gene modified cell based therapies and integrating or nonintegrating viral or nonviral vectors. Gene therapy has the potential to provide cost effective, consistent, longterm factor replacement in these individu als.9 AAVmediated gene transfer Adenoassociated virus (AAV) is a non pathogenic, replicationdeficient virus with a 4.7 kb single stranded DNA genome .10 The viral genome is packaged into a viral capsid that is composed of three capsid proteins (VP 1, 2, and 3). In the absence of a helper virus, wildtype AAV undergoes latent infection, characterized by preferential integration into a specific site in human chromosome 19, in vitro however most vector genomes (>95%) persist in episomal forms

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14 in vivo .11,12 AAV replication is dependent on the presence of a helper virus such as adenovirus or herpes virus. In the AAV vector, all viral coding sequences are removed and replaced by an expression cassette for the therapeutic gene. The only remaining vir al sequences are the 145 bp inverted terminal repeats (ITRs), which flank the expression cassette. AAV vectors have a packaging limit of ~5kb. The vector can be produced by triple transfection of HEK 293 cells in a helper virus free system. One plasmid enc odes the vector genome, the second provides AAV wild type rep and cap genes required for replication and packaging of genome, and a third plasmid encodes adenoviral helper functions (E2a, VA, and E4). AAV 2 vectors efficiently transfer genes to non dividing target cells in vivo Sustained high levels of transgene expression have been observed for the life span of a mouse and for >8 years in canine models.1315 Lowtiter Neutalizing Antibody (NAB) to AAV 2 efficiently block hepatic gene transfer.16,17 AAV2 has been shown to be highly i nefficient in transduction of dendritic cells (DC) and other professional antigen presenting cells (APCs) in vivo and in vitro .18,19 This lack of transgene expression in APCs (combined with reduced innate immunity )20 reduces the likelihood of activation of cytotoxic T lymphocytes (CTL) compared to other vector systems. A completed clinical study on treatment of hemophilia B by in vivo viral gene transfer was based on intramuscular (IM) injection of adenoassociated virus serotype 2 (AAV 2) vector to subjects with seve re hemophilia B however pre clinical studies showed an increased risk of immune responses to F.IX in animals with F.IX gene deletions or nonsense mutations. These results make skeletal muscle less attractive as a target of gene transfer, despite the fact l ongterm (albeit sub therapeutic) transgene expression has been demonstrated in the human subjects.21 Hepatic AAV gene transfer for treatment of hemophilia B Hepatocytes are the normal site of F.IX synthesis. Following hepatic gene transfer with AAV2 vector, F.IX levels of 4 to 10% were observed in hemophilia B dogs for > 8 years. This

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15 included animals with a F.IX null mutation, lacking F.IX transcript. Muscle gene transfer however resulted in the development of high titer inhibitors and undetectable F.IX levels .15,22 Expression of a human F.IX transgene at systemic levels of 4 8% of normal was demonstrated in nonhuman primates following infusion of AAV 2 vector into the portal vein.23 Consequently, a Phase I/II clinical trial based on hepatic artery infusion of AAV vector in subjects with severe hemophilia B was performed.16 In contrast to the muscle trial, enrollment was not restricted to subjects with F.IX missense mutation. One patient expressed F.IX at levels >10% of normal for ~1 month after gene transfer, no bleeds occurred during this period of time. However expression of F.IX was gradually lost during weeks 58 following gene transfer. While subjects did not have an immune response to F.IX, activation of AAV capsidspecific cytolytic CD8+ T cells was observed. These CD8+ T cells presumably targeted c ells displaying the specific MHC class I restricted capsid epitope.24 The liver has emerged as a preferable target for gene transfer, when evaluated for immune responses to the transgene product. Dr. Herzogs laboratory has previously demonstrated that hepatic AAV gene transfer can induce immune tolerance to F.IX and other proteins. Immune tolerance results in lack of CD4+ and CD8+ T cell responses and antibody formation even after challenge by administration of F.IX in adjuvant or by secondary viral gene transfer. The advantage of this approach is that the protocol provide s therapy and immune tolerance at the same time. There was evidence for T cell anergy and deletion.25 In addition regulatory CD4+CD25+ T cells are required for tolerance induction and actively suppress antibody formation to F.IX. These regulatory cells are induced by hepatocyte derived transgene expression. Other data demonstrated that the protocol does not provide tolerance in all genetic backgrounds of experimental animals and varies depending upon the type of mutation.26,27

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16 Ad ditionally, the immune response to the AAV capsid in humans is a cause of concern and may limit the success of hepatic gene therapy in human subjects. Therefore, a superior protocol is needed for tolerance induction that can be applicable regardless of gen e or protein based therapy and not bound by the background of the mouse or by the underlying factor mutation. The novel protocol developed in this thesis was tested for efficient immune tolerance induction to F.IX in a strain of hemophilia B mice (C3H/HeJ F9/-) that had previously been refractory to hepatic induced tolerance. Overview of Immune Responses in AAV Mediated Gene Transfer AAV vectors are derived from a non pathogenic replication deficient parvovirus. Numerous serotypes, mostly isolated from hum ans or nonhuman primates, have now been characterized to improve transduction of specific organs and circumvent immune responses. The popularity of AAV as a vector stems from a broad host range, nonpathogenic nature, ability to transduce dividing and nondividing cells, low innate immunity, and low efficiency of transduction of professional APCs such as DCs or macrophages, possibly due to a post entry block.28 Preclinical trials in animal models of human disease have shown long term correction of genetic disease using AAV vectors, and clinical trials have been carried out in a number of diseases.10,29 Nonetheless, immune responses to AAV vectors have been observed in animals as well as in clinical trials and may negate therapy. Innate Immune Responses to Adeno Associated Virus AAV is a weak innate immunogen; microarray studies have shown that AAV does not elicit the robust type I IFN response as is seen for adenoviral vectors.30 Similarly, cytokine and chemokine responses in the transduced tissue are limited and highly transient. AAV triggers Toll like receptor signaling (e.g. TLR 9, which senses DNA).31 AAV has also been shown to interact with complement proteins.32 The complement cascade, an important component of the

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17 innate immune system, leads to opsonization of foreign bodies and lysis of target cells. The three complement pathways include the classical, alternative, and lectin binding pathways, all of which involve C3 convertases. Recent data show that the AAV2 capsid binds to the C3 complement proteins C3, C3b, iC3b and complement regulatory factor H, hence increasing the uptake of AAV into macrophages and enhancing their activation.32 C3 AAV capsid interactions are direct and can occur independently of anti AAV antibodies. However, complement activation by AAV is primarily antibody dependant (classical pathway). Complement dependent activation of macrophages is not restricted to the AAV2 serotype. For example, AAV1 and AAV8 have been found to induce inflammatory gene expression in macrophages. Deficiency of C3 or complement receptor 1/2 result s in the impairment of the humoral response to AAV.32 C3 and CR 1/2 are essential for humoral but not innate immune responses to AAV in vivo Adaptive Immune Responses to AAV Vectors and their Transgene Products Humoral immune responses against AAV capsid or the transgene product can occur following exposure to AAV vectors. Such responses differ depending on the target organ, location within the target o rgan, route of administration, serotype, transgene and expression cassette, and dosing schedule of injection (Fig. 12).28 Humans are natural hosts to AAV. A recent study concerning the prevalence of neutralizing antibody titers (NAB) to the various AAV serotypes spanning humans in 4 continents has shown that the most prevalent NABs are to AAV2 followed by AAV1, while AAV8 and AAV7 have the least prevalent responses.33 Interestingly, the structurally modified AAVrh32.33 serotype was rarely neutralized b y human sera.33 However different studies in mice and rhesus monkeys showed robust T cell responses to AAVrh32.33 capsid and transgene.34,35 IgG1 is the predominant antibody subclass response against AAV capsid antigen in humans.36 Pre existing NAB may not necessarily block in vivo gene transfer to some organs such

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18 as skeletal muscle following intramuscular injection. However injections into blood vessels e.g. portal or peripheral vein injections and direct injection into liver parenchyma resulted in reduced transduction due to the presence of pre existing NAB. Local delivery of the vector outside blood vessels may reduce exposure to NAB.16,17,21 In addition to isolation of novel serotypes, shuffling of capsid sequences between serotypes and molecular evolution techniques are being employed to create AAV particles that are more resistant to neutralization by human sera .37 While longterm expression in skeletal muscle and a lack of inflammatory respons es were observed in a clinical trial in hemophilia B patients using an AAV 2 vector, in a subsequent trial, initial therapeutic expression of the factor IX (F.IX) transgene declined starting 6 weeks after hepatic gene transfer This decline of F.IX express ion in a patient enrolled in the highest dose cohort was accompanied by transient elevations of liver enzyme levels, suggesting destruction of hepatocytes.38 Another subject, who had a similar low titer of pre existing NAB to AAV2, was subsequently treated with a somewhat lower vector dose and showed a lower, but measurable increase in liver enzyme levels, which correlated with emergence of AAV2 capsid specific CD8+ T cells in peripheral blood, indicating T cell mediated immunity.38 Capsid specific CD8+ T cells may have been activated by the infused vector and responded to the vector transduced hepatocytes.39 About 2.5 years after initial vec tor infusion, capsidspecific functional CD8+ T cells were still present and crossreacted with a common epitope of AAV serotypes 1, 6, 7 and 8, suggesting that secondary infusions with different naturally occurring serotypes may not circumvent the T cell response.38 AAV capsid specific CD8+ memory T cells are present in humans at very low freq uency but may become reactivated upon AAV gene transfer. Hepatic AAV2 infusion over a range of doses in mice transgenic for human HLA B*0702 MHC locus failed to elicit capsid specific CD8+ T cell responses.38 It is likely that natural infection with

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19 AAV in the presence of a helper virus causes T cell responses in humans, which would not be the case in animals that are not natural hosts for AAV. Although mice immunized with AAV capsid or adenoviral vectors expressing AAV capsid developed CD8+ T cells against capsid epitopes, these failed to eliminate AAV transduced hepatocytes in several studies.8,4042 This lack of an animal model that reproduces the observations in humans has hampered preclinical studies on immune responses to AAV transduced liver. C onsidering that AAV vectors do not express capsid, input capsid derived from vector particles would have to be efficiently cross presented by the transduced cell to CD8+ T cells via MHC I molecules. AAV capsid is ubiquitinated and degraded by proteosomes, which may occur over a period of time.43 These observations support a model of CTL mediated destruction of transduced cells, but do not explain why capsidspecific CD8+ T cells failed to attack AAV transduced liver in experimental animals. Recently, several strategies have been suggested to avoid CTL responses to AAV capsid antigen. For example, alternate serotypes that do not contain a heparin binding site are processed differently b y dendritic cells and activate CD8+ T cells less efficiently .44 Elimination of surface exposed tyrosine residues on the capsid, enhanced gene transfer to the nucleus and substantially reduced accumulation of ubiquitinated capsids in the cytoplasm.45 AAV capsids with more rapid uncoating/degradation kinetics may also prove advantageous. Mycophenolate mofetil (MMF) and Cyclosporine A blocked T cell responses at least at lower vector dos es .46,47 AAV1 m ediated muscle gene transfer in lipoprotein lipase (LPL) deficiency patients resulted in capsid specific and dose dependant activation of CD4+ and CD8+ T cells. 48 Disorders of the central nervous system (such as P arkinsons disease) are promising targets for AAVbased gene therapies, and despite the fact that the brain is an immune privileged site, immune responses to gene transfer vectors have been observed. AAV vectors may cause

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20 transient innate immune responses at high vector doses in nave mouse brain parenchyma, and additional injections to the opposite hemisphere induced a significantly greater response and reduced transgene expression.49 It has been speculated that the antigen that sparks a brain immune response to rAAV is the capsid protein when injected into the brain striata. Consistent with this hypothesis, delayed re administration of vector, or switching serotype for a second gene transfer to the striata neither r esulted in loss of transgene expression or striatal inflammation, hence overcoming the danger of pre existing immunity. The authors of these studies also concluded that intracellular processing of AAV capsid generates the immunogenic antigen, and that caps id serotypes that are processed more quickly than rAAV2/2 are less immunogenic.50 Inhibitors in Hemophilia Inhibitors in hemophilia B occur in about 9 23% of those with severe disease; up to 50% of patients with F .IX inhibitors have anaphylactic reactions. In protein therapy, 1.54% of hemophilia B and 2030% of hemophilia A patients develop inhibitors to the therapeutic protein. These antibodies generally present during the first two years of treatment after <20 d ays of exposure to product. Hightiter inhibitors (>10 Bethesda units, BU) prevent treatment with clotting factor products. In this case, treatment options include bypass reagents (prothrombin complex concentrates or recombinant activated factor VII, F.VII a). Because of the expense and thrombotic risks associated with prolonged administration, these reagents are only used to provide short term hemostasis. For example, treatment of a single bleeding episode with recombinant F.VIIa may cost >$50,000. Generall y, inhibitor patients are at higher risk for bleeding related morbidity and mortality. Short term treatment with porcine factor products is no longer performed due to potential pathogen contamination. Immune Tolerance Induction (ITI) protocols are based on frequent high dose infusion of F.VIII or F.IX, and often require amounts of products exceeding $1,000,000. These protocols are often less effective for treatment of

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21 inhibitors to F.IX. In addition, some hemophilia B inhibitor patients experience anaphylac tic responses to F.IX. Pre clinical studies in animals have shown that inhibitor formation in gene therapy is dependent on the gene transfer vector, route of administration, vector doses, and other factors. Preventing Immune Responses in AAV Mediated Gene Transfer Immune responses against AAV encoded transgene products vary substantially and are influenced by the target organ, route of delivery and dosing schedule. For example, in Hemophilia B mice with a F.IX gene deletion, intramuscular (IM) vector admini stration caused a local immune response characterized by activation of CD4+ T and B cells to F.IX, which eliminated systemic expression. B cell activation has also been a complication upon over expression of erythropoietin (epo) in skeletal muscle, which i nduced an autoantibody against epo, resulting in autoimmune anemia. Although CD8+ T cell responses occur and are of particular concern in inflamed muscle (which is typical for some forms of muscular dystrophy), these T cells are often not fully functional in healthy muscle.47,51,52 Recent studies found that IM injection of AAV vectors often induces transgene product specific CD8+ T cell that express markers of functional exhaustion and T cell suppression, and ultimat ely undergo programmed cell death in skeletal muscle.53,54 Several immune suppression protocols have been successfully used in different animal models to block humoral and cellular immune responses to transgene products expressed in skeletal muscle.47 This thesis demonstrates the prophylactic use of immunosuppressant drug rapamycin in combination with IL 10 in presence of F.IX dominant CD4+ T cell epitopes induces Treg and long term tolerance to the F.IX transgene product expressed in muscles of hemophilia B mice.55 As opposed to muscle gene transfer, hepatic gene transfer with AAV vectors has been shown to induce immune tolerance to a number of transgene products via induction of Treg and

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22 other mechanisms.8,5658 In addition to direct tolerization of transgene product specific CD4+ T cells by induction of non responsiveness ( anergy) or deletion, B and T cell responses (including CTL responses) are actively suppressed by induction of CD4+CD25+ regulatory T cells (Treg) with an immune suppressive phenotype.8 Antibodymediated depletion studies suggest that Treg are critically required for tolerance to the transgene product following hepatic gene transfer.8,59 IL 10 expression by Treg and Kupffer cells may d irectly suppress immune responses in the liver. Once tolerance is established, the transgene product can safely be expressed in sites that would otherwise predispose to immune responses 60,61 In some cases, one may simply avoid immunity by taking advantage of immune privileged sites. The ocular disease Lebers Congenital Amaurosis ( LCA), caused by an autosomal recessive mutation of RPE65, is being successfully treated in several ongoing clinical trials. Single eye injections of rAAV2 CBSBhRPE65 resulted in an increase of visual sensitivity in several patients.6265 Due to the immune privileged nature of the eye, one should be able to improve treatment by re injection in the pr eviously injected eye or the partner eye, as has been suggested by animal studies in sub retinal vector administration. This route causes a deviant immune response, resulting in a lack of NAB formation. However, intravitreal administration of AAV2 vector i n mice resulted in a humoral response against the capsid that blocked transgene expression on re administration of vector in a partner eye.66 Immune Tolerance and Regulation of Transgene Product Directed Responses Blocking immune responses to the coagulation factor protein is key to succe ssful therapy for hemophilia. Induction of CD4+ T cell tolerance is desirable because inhibitor formation is dependant on CD4 help. To this end, the ability to activate Treg that aid in inducing/maintaining tolerance may be crucial.67 It is known that certain types of Treg can be induced, such as Th3 cells that secrete TGF or Tr1 (regulatory T cells type 1) that secrete large amounts of IL 10.6870

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23 Besides these induced Treg that suppress by cytokine secretion, CD4+CD25+ Treg cells occur naturally (5 10% of CD4+ T cell population).71 These cells are generated during thymic development to maintain tolerance to se lf antigens. CD4+CD25+ Treg constitutively express the IL 2R chain, glucocorticoid induced tumor necrosis factor receptor (GITR)72,73, and CTLA 4 and are potent suppres sors of CD4+CD25T cells. Suppression occurs via a cell contact dependent mechanism in vitro albeit cytokine mediated suppression has been postulated in vivo Expression of transcription factor Forkhead Box P3 (FoxP3) defines this lineage of Treg and rep resents a master control gene for development and function of CD4+CD25+ Treg.74,75 There is now growing evidence in the literature that this type of Treg is not only naturally present but can also be induced upon presentation of exogenous antigen.76,77 Furthermore drugs like rituximab, MMF, dexamethasone and others show some promise in reducing inhibitor responses.78 This thesis project explores a novel strategy toward tolerance to F.IX in gene and protein replacement therapy and seeks to take advantage of immune regulation to facilitate antigen specific tolerance induction using rapamycin/cytokine/specific peptide combinations. Potential Roles of Rapamycin, IL 10, IL 2 and Fc GITRL and Flt3L Rapamycin (sirolimus) is an immun osuppressant used to prevent transplant rejection. It is a macrolide antibiotic produced by Streptomyces hygroscopicus. Rapamycin suppresses T cell signaling in response to cytokines and growth factors through binding to intracellular protein FKBP12 (which in turn inhibits the protein kinase mTOR), but does not interfere with TCR signaling or calcineurin activity. Rapamycin inhibits IL 2 stimulated signal transduction by causing dephosphorylation of p70 S6 kinase and of eIF 4E BP1, which are both regulated by mTOR. These effects lead to a block in cell cycle progression and consequently to inhibition of T cell proliferation.25,79,80 Interestingly, rapamycin does not prevent induction of Treg such as

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24 CD4+CD25+ T cells, nor does it prevent activation induced cell de ath (AICD).25 Published data from Dr. Roncarolos lab demonstrated that in vitro antigen presentation in the presenc e of rapamycin leads to selective expansion of CD4+CD25+ Treg.81 Rapamycin was recently found to ameliorate induced autoimmune encephalomyelitis (EAE) in mice with the elimination of CD4+CD45RB(high) effector T cells and selective expansion of Treg cells bearing the C D4+CD45RB(low)FoxP3+CD25+CD103+ phenotype.82 The cytokine IL 10 has immunomodulatory effects and can be produced by va rious cell types including dendritic cells, macrophages, various subsets of T cells and B cells produce it. IL 10 is known to inhibit MHC class II and co stimulatory molecules B7 1/B7 2 expression on APCs. It also limits the production of pro inflammatory cytokines like IL 1, IL 6, IL 12 and TNF IL 10 can act directly on CD4+ T cells, inhibiting IL 2 production and hence inhibiting proliferation. It can also act via autocrine signaling in DC to prevent chemokine production and prevent its trafficking to lymphnodes.83 Rapamycin combined with interleukin IL 10 applied in a nonspecific fashion prevented Type 1 Diabetes via increasing CD4+CD25+ Treg and T Regulatory Type 1 (Tr1) cell numbers in the absence of chronic immunosuppression in nonobese diabetic (NOD) mice.7 IL 2 is a secreted cytokine produced by several cell types including CD4+ T cells, some other cell types like CD8+ T cells, NK and DCs. IL 2 is known to have autocrine as well as paracrine function. It is important for the regulation of immune responses and proliferation of T and B lymphocytes. The alpha chain of the IL 2 receptor (IL 2R; CD25) is a known marker for FoxP3+ Treg. Treg do not express IL 2 and are thus dependant on exogenous IL 2 produced by other cells. IL 2 plays an important role in controlling the prolif eration and survival of Treg.83 IL 2 and IL 2R knockout mice exhibit severe autoimmunity due to the absence of

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25 CD4+CD25+FoxP3+ Tregs. IL 2 is also known to potentiate activation induced cell death in T cells.84 GlucocorticoidInduced TNF Receptor (GITR) is expressed on normal T lymphocytes and in high levels on Treg cells. The corresponding ligand for GITR (GITR L) is expressed on DCs and other APCs. Initially i t was hypothesized that that the interaction of GITR L on DCs with GITR of Treg resulted in the abrogation of Treg suppression. However, GITRspecific antibodies have been found to stimulate proliferation of Treg cells in the presence of IL 2 but in the absence of a TCR signal. Soluble GITR L has been observed to cause proliferation of Treg cells in the presence of IL 2 and rIL 2 can break the anergic state of Treg.85 Others have shown that GITR L is important in ren dering CD25effector T cells resistant to suppression by Treg. 86 The mechanism of GITR/GITRL control of immune mechanisms is not well understood. Fc GITRL is a chimeric molecule generated by Dr. Gongxian Liao fr om the laboratory of Dr. Cox Terhorst, Beth Israel Deaconess Medical Center, Harvard Medical School. It consists of the Fc region of an antibody, fused with GITR L. The Fc GITRL molecule was observed to induce CD4+FoxP3+ Treg proliferation in vitro and in vivo .87 Fms like tyrosine kinase 3 ligand (Flt3L) is a cytokine required for DC homeostasis in lymphoid tissues. Genetic deletion of Flt3L or treatment with Flt3 tyrosine kinas e inhibitors has been observed to cause a reduction of plasmacytoid dendritic cells (pDCs) and DCs in lymphoid organs. Increased levels of Flt3L cause a massive proliferation of pDCs and conventional DCs (cDCs) in lymphoid and nonlymphoid organs. Repeated injections of Flt3L indirectly lead to an expansion of peripheral naturally occurring Treg.88 Flt3L exists in a soluble or membrane bound form. It is expressed by multiple tissue stromal cells and by activated T ce lls.89

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26 Figure 11. Simplified diagram of the intrinsic pathway in the blood clotting cascade.

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27 Figure 12. Overview of immune responses to viral vectors. Targeting specific organs, engineering viral envelopes, switching serotypes, modifying the transgene cassette, utilizing organ specific promoters or tolerizing dosing schedules increase possibility of immune avoidance or cause immune suppression to the viral vector and transgene product.

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28 CHAPTER 2 MATERIALS AND METHOD S Reagents Peptide A254 ( TEQKRNVIRIIPHHNYNAAI) the dominant CD4+ T cell epitope for human F.IX (hF.IX) in C3H/HeJ mice, peptide 2A 58 (NKYNNHDIALLELDEPLVLNS), an epitope for C57BL/6 mice and chicken ova peptide amino acids 323 to 339 (ISQAVHAAHAEINEAGR) were synthesized by Anaspec ( San Jose, CA) or the ICBR protein core facility at University of Florida (Fig.2 1). 90 Murine IL 10 (Sigma Aldrich, St. Louis, MO), recombinant mouse IL 2 (Roche Diagnostics, Mannheim, Germany), human FLT3L carrier free (Cell signaling technology, MA) rapamycin (LC Laboratories, Woburn, MA) stock solution were made in 0.02% Carboxy methyl cellulose (CMC) and 0.25% Tween80 (Sigma, St. Louis, MO). Rapamycin solution was sonicated before being aliquoted and stored at 20oC. Recombinant coagulation Factor IX (Benefix; Wyeth, Philadelphia PA) was sterile diluted Phosphate buffered saline (PBS). AAVCMVhF.IX vector was as previously published.91 It cont ains the hF.IX cDNA (plus a 1.4kb portion of the F9 gene) expressed from the cytomegalovirus immediate early enhancer/promoter. AAV serotype 1 vector was produced by triple transfection of HEK 293 cells, purified by gradient centrifugation, and titered by quantitative slot blot hybridization. 92 Peptide, interleukin and virus dilutions were made in Dulbeccos Phosphate Buffered Saline (DPBS, Invitrogen, NY) unless otherwise stated. Mouse Strains Male BALB/c mice transgenic for chicken ovalbumin s pecific DO11.10 CD4+ TCR ( OVA peptide, amino acids 323 339) and knock out for Rag2 gene ( recombinase activating gene 2) were used. These DO11.10tg Rag 2/animals do not develop endogenous mature T or B cells.

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29 BALB/c DO11.10tg Rag 2/males were obtai ned from Taconic (Germantown, NY). These animals were used to characterize the mechanism of drug action. In F.IX knockout mice on a C3H/HeJ background (C3H/HeJ F9/-) the promoter and the first three exons of the F9 gene were replaced by neo HPRT, which i s a functional neo gene plus a partially deleted hypoxanthine phosphoribosyl transferase minigene as published. 93 These mice show no transcription of the F9 gene and are completely F.IX antigen deficient. These have <1% clotting activity and are susceptible to internal hemorrhage. Male mice homozygous for Rag1tm1Mom on a C57BL/6 background ( B6.129S7Rag1tm1Mom/J) were from Jackson Laboratories ( Bar Harbor, ME) These animals do not produce mature T cells or B cells and have no CD3+ or T cell receptor (TCR) alpha beta positive cells. The thymus of the mutant mice contains 15 to 130 times fewer cells than heterozygous or wild type siblings. Their thymocytes are CD8-CD4-, and most are IL 2 receptor positive. Male C3H/HeJ and Male C57BL/6 were from Jackson Laborat ories (Bar Harbor, ME). Animals were housed in specific pathogen free conditions and in accordance to local IACUC protocols Administration of Drugs Drug cocktails were administered intraperitoneal (IP) in 200 l of sterile PBS 3 times per week at the foll owing doses. Ovalbumin (ova) peptide (amino acid residues 323339) or hF.IX specific peptide (2A 54) were given at 100 g/dose, rapamycin at 4 mg/kg/dose, and IL 10 at 50 ng/kg/dose, IL 2 at 50ng/kg/dose, FLT3L at 2 g/dose. Experiments involving different routes of injection included IP, Intravenous (IV), and subcutaneous (SQ) on the back of the neck region and oral gavage (oral). Oral gavage of rapamycin was given in 100 l volumes everyday for 1 month or thrice a week, using a special feeding needle with an olive, round tip (Fine Science

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30 Tools). Viral vector diluted in PBS was administered at 1x1011 vector genomes (vg)/mouse in 100 l volume, by intramuscular (IM) injection into quadriceps and tibialis anterior muscles using a Hamilton syringe or by IV admi nistration in the case of AAV8 serotypes. 91 Splenic capsule injections of viral vector in hemophilic mice were done 1 hour after infusion of normal mouse plasma to preclude excessive bleeding and increase survival. Platelet activating factor antagonist (Anti PAF; 50 g/mouse; CV3988, Sigma, USA) was prepared in absolute ethyl alcohol and PBS. Anti histamine Triprolidiene hydrochloride (Sigma) was prepared in PBS; a final dose of 100 g/mouse was injected in conjunction with anti PAF via tail vein injection. The anti histamine combination was used 5 minutes before hemophilic mice were treated with hF.IX whole protein at the 4th tail vein injection in order to avoid hypersensitivity reactions. Blood Sample Collection Blood samples were obtained using heparinized microhematocrit capillary tubes (Fisherbrand, Fisher Scientific, PA) via the retro orbital plexus for ELISA or via tail bleed (after heating the mice for a few minutes under a 125W heating lamp and anesthetizing with isofluorane) into 3.8% sodium citrate buffer for clotting assays or into EDTA tubes; 1.6 mg EDTA/ml blood (Sarstedt, Germany) for whole blood analysis. T he tail wound was then sutured and sealed with silver nitrate. Analysis of Plasma Samples Immunocapture assay for determination of anti hF.IX titers was performed as follows. x: Wyeth, Philadelphia, PA), and mouse plasma samples were applied at a 1:20 dilution. In parallel, wells were coated with 2 fold serial dilutions of purified mouse immunoglobulin IgG (Sigma Aldrich, St Louis, MO) starting at 2000 ng/ml for generation of a standard curve. Anti hF.IX was detected

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31 with rat anti mouse horseradish peroxidase conjugated antibody at 1:2000 dilution (R&D, USA). Antibody levels were measured by optical density (OD) reading at 450 nm following incubation with o phenylene diamine substrate (Sigmafast OPD: Sigma Aldrich) using a microplate reader (Bio Rad, Hercules CA). Levels of hF.IX were determined by an Enzyme linked Immunosorbent Assay (ELISA) as follows. Microtiter plates were coated with 1:850 dilution of mouse monoclonal anti hF.IX (FIX 1 monoclonal anti hF.IX Clone HIX 1; F2645 Sigma). Blocking buffer (5% nonfat milk in wash buffer) was used to block the plate. The standard was diluted from 200 to 3.125 g/ml (TriniCHECK Level 1; Trinity Biotech, Wicklow, Ireland). Samples were diluted to 1:10. Dilute goat anti human FIX HRP (Enzyme Research: GAFIX APHRP 230) 4.5 l in 10.5ml dilution buffer. F.IX levels were measured using O phenylenediamine tablets (Sigmafast OPD from Sigma) with a microplate reader (Bio Rad Laboratories, Her cules, CA) at 450 nm. Clotting Assays Activated partial thromboplastin time (aPTT) was measured with a fibrometer (Fibrosystem; BBL, Cockeysville, MD). Briefly, 50 l of plasma sample was incubated at 370C with 50 l of F.IX deficient plasma (Hematologic T echnologies Inc., Vermont) and 50 l TriniCLOT automated aPTT reagent (Trinity Biotech, Wickow, Ireland) for 3 minutes. Fifty microliter of 25mM CaCl2 is added and the time taken for clotting to occur is measured using the fibrometer (FibrosystemTM, Block Scientific Inc.). Bethesda assay for measuring the titer of inhibitory antibodies was performed as published.91 Brief ly, Sample dilutions are made using Imidazole buffer (MDA, Baltimore, MD) ranging from N to N/8. Control samples at 1:2 and 1:4 dilutions of normal hum an plasma (TriniCHECK Level 1, Trinity Biotech, Ireland) and Imidazole buffer were made. Thirty

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32 microlite r of TriniCHECK was added to all experimental samples and control samples with 60 l to the last dilution of experimental sample. All samples and controls were incubated at 370C for 2 hrs and then transferred to ice. Standards were prepared from 20% to 0% of normal human plasma in Imidazole buffer. Sixty microliter of F.IX deficient plasma was added to all standard tubes. Coagulation times were then determined as aPTT. Residual activity was computed on a standardized chart. One Bethesda unit (BU) represents a residual hF.IX activity of 50%. Flow Cytometry Cells were isolated from the spleen, inguinal, popliteal lymph nodes, thymus and peripheral blood using standard protocols. Briefly, spleen/lymph nodes were isolated and stored in cold PBS and single cell s uspensions were prepared using a 70 m cell strainer (BD Bioscience, MA). Red blood cells are lysed using an ammonium chloride based lysis buffer (Pharmlyse; BD Biosciences, MA), remaining cells were washed with cold PBS or 2 MLC medium (heat inactivated 2 % fetal calf serum, sodim pyruvate, HEPES, penicillin, streptomycin, non essential amino acids and 2mercaptoethanol added to Dulbeccos modification of Eagles medium). Lymph nodes were incubated in collagenase D (Roche) 1 mg/ml for 30 min before straining. Viable cells were enumerated using a hemocytometer in the presence of trypan blue to differentiate between the live and dead cells. Staining for surface and intracellular molecules was carried out according to manufacturers protocols. Antibodies aga inst mouse antigens CD4 (conjugated to APC or FITC), CD69 PE, CD62L APC, CD44 PE, CTLA PE, CD95 PE, CD25 ( PE or FITC), FoxP3 ( Alexa Fluor 647 or FITC) were from eBioscience ( San Diego, CA ). Anti murine GITR cy7 was from BD Biosciences, USA. Isotype controls included Rat IgG1FITC, Rat IgG2b PECy7, Rat IgG2a FITC, Rat IgG2b PE, Rat IgG2a APC, Rat IgG2b PE Cy7, Rat IgG2a Alexa Fluor 647. Other controls were unstained splenocytes and anti Rat Ig

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33 Compensation Particles (BD Biosciences/Pharmingen). Apoptosis was measured with the AnnexinV PE Apoptosis detection kit (BD Biosciences/Pharmingen, USA). Data acquisition was carried out using the BD LSR II and BD FACSCalibur system. Data analysis was done with FACS DIVA 6.1 and Cell Quest software Cytokine Secretion Assay Mouse Cytokine Secretion Assay Detection Kit (PE) (Miltenyi Biotech, Auburn, CA ) was used as per the manufacturers recommendations. Briefly, spleens from DO11.10tg Rag/mice were processed for cell culture using standard protocols. The cell s were resuspended in 5MLC medium at 1 x 107 cells/ml and added to a round bottom 96well culture plate (1 x 106/well in triplicate) for in vitro stimulation with ova peptide (final concentration of 10 g/ml). Three to six hours after peptide stimulatio n, IL 4 and IL 10 expressing CD4+ T cell were identified by flow cytometry using anti murine CD4 FITC, anti murine IL4 PE, and anti murine IL 10APC. Adoptive Transfer Assay Male C3H/HeJ mice were treated with the Rapa/IL 10/2A54 drug protocol for 3 weeks, after which splenocytes were isolated. CD4+CD25+ cells were isolated using magnetic cell sorting regulatory T cell isolation kits (Mylteni Biotech, Auburn, MA). Isolated CD4+CD25+ T cells were enumerated using a hemocytometer and Trypan blue staining. Live CD4+CD25+ T cells (1x106 cells) were injected into recipient mice. Control animals were injected with CD4+CD25or CD4cells. Animals were challenged with F.IX in complete Freunds Adjuvant (cFA; Sigma, St. Louis, MO), which was prepared by mixing 1ml of cFA with 1ml of diluted F.IX antigen in PBS (5 g/100 l). The mixture is sonicated and loaded in syringes. Fifty microliter of the mixture is injected subcutaneously into each of 4 spots on each mouse giving a

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34 total dose of 200 l for each mouse Fig. 23. Plasma is collected 3 weeks after challenge for further analysis. In vitro Suppression Assay Splenic CD4+ cells were isolated using negative selection; CD25+ T cells were isolated from the enriched CD4+ population by positive selection using magnetic cel l sorting (Miltenyi Biotech, Auburn, MA). Cells were resuspended in 2 MLC media and plated in triplicates in flat bottom 96well plates with a gradient of ratios of regulatory T cells, effector T cells and accessory cells. The Celltitre96 Aqueous NonRadio active Cell Proliferation assay (Promega, Madison, WI) was used to assess suppression of effector T cell proliferation in the presence of Treg cells Fig. 2 2. The plate was read at 490 nm using an ELISA reader (Biorad, Hercules CA) Statistical Analysis Re sults are represented in means SD. Assessment of significant differences between means was done by unpaired students t test. P values of <0.05 were considered significant

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35 Figure 21. Diagram of human factor IX primary amino acid sequence with CD4+ T cell epitopes. Boxes indicate peptides that contain dominant CD4+ T cell epitopes in hemophilia B mice on C57BL/6, C3H/HeJ and BALB/c genetic background. Also indicated are domains and protease cleavage sites and numbering of some reference amino acids.90

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36 Figure 22. Diagrammatic representation of the effector T cell suppression assay Figure 23. Diagrammatic representation of the adoptive transfer assay

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37 CHAPTER 3 ESTABLISHMENT AND MECHANISTIC INVESTIGATION OF NOVEL TOLERANCE PROTOCOLS Introduction Therapeutic proteins introduced via protein or gene replacement therapy may be recognized as nonself in patients whose underlying mutations are null mutations. T cells are often critical components of these adaptive immune responses. Antigen presenting cells such as DCs pick up the offending protein that is not recognized as self by the patients immune system. The protein is then processed and presented via MHC Class II on their surface. T lymphocytes confer cell mediated immunity. Antigen specific CD4+ T cells recognize the MHC Class II antigen complex and are signaled to proliferate. CD4+ T cells induce the proliferation of B cells that produce copious amounts of antigen specific antibody. The ant ibody produced is secreted and functions by neutralizing the therapeutic protein. The process of neutralization renders the injected protein or transgene product nonfunctional and hence ineffective. In order to combat this neutralization, higher factor doses at more frequent treatment intervals are required to provide therapy in patients with low titer antibodies. This increase in dosing for patients with anti F.IX or anti F.VIII inhibitors results in increased costs and reduced quality of life. Bypassing reagents such as activated prothrombin complex concentrates and recombinant factor VIIa (rF.VIIa) are used in patients with high titer antibodies. These bypassing reagents however have to be limited in dosing and frequency as they are associated with unwanted thrombosis. rF.VIIa has to be administered every 23 hours, which leads to problems with venous access. Immune tolerance induction therapy (ITI) involves the administration very high concentrations of factor possibly multiple times a day for many mont hs to years. This therapy works for a percentage of F.VIII deficient patients but largely does not work for F.IX deficient

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38 patients. A more effective protocol is therefore desirable to tolerize patients to the therapeutic proteins. The Herzog lab has prev iously demonstrated the importance of CD4+CD25+ Treg in tolerance induction to F.IX. Based on the hypothesis is that a clinically feasible protocol can be designed to reliably induce immune tolerance to coagulation factors; a novel and promising approach t o efficiently induce antigenspecific CD4+ T cell tolerance by transient administration of a cocktail of a specific peptide antigen, an immune suppressive drug, and a cytokine was developed. The function of the drug cocktail was to delete effector CD4+ T c ells that mediate the immune response while simultaneously increasing the number of functional Treg identified as CD4+DC25+FoxP3+. An approach based on TCR transgenic mice to study CD4+ T cell responses to a systemically delivered peptide was adapted. In o rder to identify such a drug or drug/cytokine combination best suited to achieve these results, DO11.10 TCR transgenic BALB/c mice deficient in Rag 2 were used. These animals do not have any endogenous rearranged T cell receptors (TCR) and also lack Treg.94 The DO11.10 TCR (encoded by rearranged V 13 and V 8.2 genes) is specific for a chicken ovalbumin (ova) peptide ISQAVHAAHAEINEAGR (ova amino acids 323339) presented by the MHC class II molecule I Ad to CD4+ T cells, and is expressed in >90% of T cells in the thymus of DO11.10 TCR transgenic mice. Ova specific CD4+ cells can be tracked and counted by FACS analysis using dual antibody stain (anti murine CD4 and DO11.10 clonotype specific KJ1 26 monoclonal antibody). For example conclusive evidence for generation of CD4+CD25+ Treg cells can be obtained in DO11.10 TCR transgenic BALB/c mice deficient in Rag 2 (and thus devoid of re arranged endogenous TCR to other antigens). These mice, as shown by others, are deficient in CD4+CD 25+ Treg (<1% of CD4+ T cells instead of the

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39 normal 510%) due to lack of TCRs to self antigen, and therefore contain almost exclusively CD4+CD25ova specific T cells. The investigation detailed below tested whether this strain could be useful to screen f or antigen/drug combinations that may be optimal for induction of CD4+ T cell tolerance. Depletion Teff Cells and Induction of CD4+CD25+FoxP3+ Treg via Rapamycin/IL 10/Specific peptide Protocol. Using DO11.10tg Rag 2/BALB/c mice, ova peptide (100 g/mo use/dose) was co administered with rapamycin (4 mg/kg/dose) by intraperitoneal (IP) injection. Administration was carried out 3 times per week for a total of 4 weeks (n=4 mice). Spleens were harvested and flow cytometry was utilized to quantify the proport ion of CD4+CD25+FoxP3+ cells. In an identical set of experiments, IL 10 (50 ng/kg/dose) in combination with ova peptide was administered; rapamycin was left out in this group. Finally, a combined protocol was tested, in which mice received ova peptide, rap amycin and IL 10 along with the specific peptide at the above mentioned doses. Control mice received a mixture of rapamycin, IL 10 and an irrelevant peptide. The two groups of mice that received the specific peptide and rapamycin showed a marked reduction (up to 4fold) in ova specific CD4+ T cells when compared to the rapa/IL 10/irr treated controls (Figs. 31 and 32) The effect was greater when IL 10 was included (rapa/IL 10/ova specific peptide group) CD4+CD25+FoxP3+ regulatory T cells were induced in all groups of mice except for the Rapa/IL 10/Irr peptide treated controls. The Treg induction observed was 18fold of CD4+ T cells and up to 148fold of total splenocytes in the Rapa/IL 10/ova specific peptide treated mice and exceeded the Rapa/ova and I L 10/ova mice by a fair margin (Figs. 31, 32). Effects of rapamycin/IL 10 were dependent on TCR signaling and therefore antigenspecific, as can be observed in the differing effects as a result of the presence or absence of the

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40 specific ova peptide. IL 10/ova peptide treated animals showed a Th2 response as was determined by the induction of CD4+IL 10+IL 4+ cells (Fig. 3 3). Upon in vitro stimulation with ova, we were unable to detect IL 10+ or IL 4+ T cells in any experimental group, except for IL 10/ova treated mice, which showed an ova specific CD4+IL 10+IL 4+ response, indicating Th2 activation (Fig.3 3). A substantial decrease in CD4+ T cells was again seen in this assay for rapamycin/IL 10/ova treated mice. No evidence for a Tr1 response was seen in rapamycin/IL 10/ova peptide treated mice, which had the strongest decrease in CD4+ T cells and the most robust induction of CD4+CD25+FoxP3+ cells. These increases in Treg observed on administering the novel rapamycin/IL 10/specific peptide protocol are ~3fold greater than had achieved previously by the Herzog lab with hepatic ova gene transfer in this strain of mice. Virtually all CD4+CD25+ expressed FoxP3. This protocol can serve as a powerful tool to shift the balance from transgene product specific effector to regulatory T cells prior to therapy Effect of Rapamycin/IL 10/Specific peptide in Lymphoid Organs Next, the effect of the most optimal protocol on different lymphoid organs was investigated. Rapamycin/IL 10/ova was administered IP 3times per week for 3 weeks to DO11.10tg Rag 2/mice. Control animals received an irrelevant hF.IX peptide instead of ova. Subsequent flow cytometric analysis showed that CD4+ cell frequencies were markedly reduced in peripheral blood and secondary lymphoid organs (s pleen, inguinal and popliteal lymph nodes) and slightly reduced in the thymus (Fig. 34). Mice receiving an irrelevant peptide showed minor reduction (spleen) or no change (lymph nodes and thymus) in CD4+ T cell numbers compared to nave mice (Fig. 34). M ice treated with rapamycin/IL 10/irrelevant peptide, similar to nave mice, had again no detectable CD4+CD25+FoxP3+ cells (<0.4%) in any of the organs analyzed. In contrast, a substantial induction of Treg to ~20% of CD4+ T cells was observed in peripheral blood and all secondary lymphoid organs in rapamycin/IL 10/ova treated mice (Fig. 3 5). Treg

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41 were also detectable in the thymus, albeit at lower frequency (~0.9%, with one animal achieving 7% of CD4+ T cells compared to 0.1 0.3% in control mice, (Fig. 36 C). Activation and Apoptosis of Ova specific CD4+ T Cells. DO.11.10tg RAG2/ mice were injected IP 3 times per week for 1 week with rapamycin/IL 10/ova. When analyzed 1 day after the last drug administration, splenic CD4+ T cells showed an up regula tion of activation markers like CD25, CD44, and CD69, and the downregulation of marker CD62L (which mediates homing of lymphocytes to sites of infection and is lost after activation occurs) compared to control mice receiving an irrelevant hF.IX peptide in stead of ova, indicating T cell activation due to the drug cocktail administration (Fig. 37). Rapamycin/IL 10/ova treated mice also showed a 2 to 3fold increase in Fas (CD95)+ and in apoptotic (Annexin V+) splenic CD4+ T cells compared to controls (Fig. 38 A B). These cells stained double positive for CD90 and Annexin V (Fig. 38 C). Taken together, the data demonstrate induction of activation induced cell death. As a result of Activation induced cell death, the portion of apoptotic CD4+ T cells increased with increasing doses of drug cocktail administration hence resulting in a decline of CD4+ T cell numbers over time (Fig. 3 9). Induced Treg Suppress Teff and Show Lower Rate of Apoptosis. While antigen presentation in the presence of rapamycin and IL 10 induced apoptosis of CD4+ T cells and consequently a decline in CD4+ T cell numbers, Treg were induced to high frequency; therefore, there had to be a differential effect of the drug cocktail on Teff vs. Treg cells. To test this hypothesis, we measured the frequency of apoptotic cells among Treg and Teff cells 3 weeks after treatment with rapamycin/IL 10/ova (specific peptide) cocktail. In 3 of 4 mice, splenic CD4+CD25-FoxP3Teff cells distinctly contained a higher proportion of Annexin V+ (an apoptosi s marker) cells compared to CD4+CD25+FoxP3+ Treg (Fig. 310). These data indicate a higher resistance of Treg to antigen/rapamycininduced apoptosis. As expected, rapamycin/IL -

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42 10/irrelevant peptide treated control animals showed only a low level of apoptot ic CD4+ cells (which are CD25-FoxP3-). To demonstrate that the Treg cells induced by this novel antigen specific protocol are functional and suppressive, an in vitro suppression assay was performed on splenocytes isolated from DO.11.10tg RAG2/mice aft er 3 weeks of treatment with rapamycin/IL 10/ova. While the tolerized splenocytes failed to proliferate upon stimulation with ova, T cell proliferation was observed in splenocyte cultures depleted for CD4+CD25+ cells (Fig. 3 11). When purified Treg were added back to the depleted cells, proliferation was again suppressed (Fig. 311). Route Optimization of Rapamycin/IL 10/Specific peptide Protocol In order to optimize the rapamycin/IL 10/specific peptide tolerance protocol, various routes of drug cocktail a dministration were tested. The routes tested included intraperitoneal, tail vein, subcutaneous and oral gavage. In the case of oral gavage, only rapamycin was administered orally while IL 10 cytokine and the specific peptide were administered by IP injecti ons due to concerns about their stability in acidic and basic environments of the stomach and intestines. The different routes of administration of the drug combination (3 inj/week for 3 weeks) in Tregdeficient DO11.10tg Rag2/mice showed that Treg induction was more efficient in IP injections (4.7x) compared to nave controls, followed by subcutaneous (3.3x) and tail vein injections (2.8x). Deletion of Teff was more pronounced via IP (2.7x) followed by subcutaneous (2.3x) and tail vein (1.7x) injection s. Oral gavage of rapamycin (3inj/week for 3 weeks) along with IP injections of IL 10/ova specific peptide showed the least deletion (Fig. 312 A) accompanied by the lowest induction of CD4+CD25+FoxP3+ cells (Fig. 3 12 B ), possibly due to lower bioavailability. In summary, the route of administration of drug combination influences the outcome of the treatment modality.

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43 Alternative Cytokines for Induction of Treg in the Tolerance Protocol DO11.10tg Rag 2 /BALB/c mice were administered IP with Flt3L ( 2 g/mouse/dose) co administered with rapamycin ( 4 mg/kg/dose) and specific ova peptide (100 g/mouse/dose) by intraperitoneal (IP) injection. Administration was carried out 3 times per week for a total of 3 weeks. In parallel, Fc GITR L fusion protein (20 0 g/mouse/dose), which has been previously shown to increase levels of Treg, was administered with rapamycin and ova peptide. Alternatively IL 2 cytokine, which is known to be necessary for expansion of Treg was injected (50 ng/kg/dose) along with rapamyc in and specific ova peptide. An additional group of mice was given rapamycin/IL 10/ova peptide for comparison of Treg induction, and a nave control group was also included. Each treatment group included n=4 mice. Spleens were harvested, and flow cytometry was carried out to quantify CD4+ T effector cells and CD4+CD25+FoxP3+ Treg. Rapa/Flt3L/ova administration resulted in a 4.5 fold deletion of CD4+ T cells, and a concomitant 19fold increase in CD4+CD25+FoxP3+ regulatory T cells. Rapa/FcGITR L/ova treatme nt resulted in a 5.5fold reduction in effector T cells with a 18fold increase in CD4+CD25+FoxP3+ T reg. Rapa/IL 2/ova peptide administration resulted in 2fold deletion of CD4+ effector T cells and an 8 fold increase in Treg cells (Fig. 3 13). The combina tion of specific peptide with rapamycin and Flt3L was statistically most significant and therefore shows considerable promise due to synergistic effects of rapamycin and Flt3L on the induction of Treg cells. Transient Non specific Changes in Lymphocyte and Neutrophil Counts in Immune Competent Mice Some general immune suppression is expected by the rapamycin/IL 10 protocol in immune competent mice since Rag+/+ mice have TCRs for multiple antigens and mTOR is also expressed in T and B cells. Wild type C3H/HeJ mice were injected IP 3 times per week with ramapycin/IL -

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44 10 and hF.IX specific A2 54 instead of ova peptide for 4 weeks. Immediately after treatment a 3to 4fold decrease in CD4+, CD8+, and B220+ splenocytes compared to untreated controls was observe d, which was independent of the coadministered peptide (Fig. 314 A C). However, by 5 months after treatment, B and T cell numbers were similar to controls (Fig. 314 D F) Complete blood counts showed that all animals treated with rapamycin/IL 10 (n=6) developed neutrophilia, which was observed up to 4 months after treatment but dissipated 7 months after treatment (this is expected since rapamycin is used for treatment of neutropenia) 95. Peripheral blood analysis included White Blood Cell, neutrophil, lymphocyte, monocyte, eosinophil, basophil, RBC and platelet counts, hemoglobin, mean cell hemoglobin concentration (MCHC), Reticulocyte Count Hematocrit, packed cell volume, nucleated erythrocytes, mean cell or corpuscular volume, mean platelet volume (MPV), and mean cell or corpuscular hemoglobin. Low MCHC and polycythemia was found at random among both treated and untreated, age matched control animals at both time points. Although platelet counts were normal, MPV was low in 40% of C3H/HeJ mice regardless of treatment. All o ther parameters were within the normal range for mice.

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45 A B Figure 31. Flow cytometric analysis of splenocytes from DO.11.10tg Rag/BALB/c mice injected IP 3 times/week for 4 weeks with rapamycin/IL 10/specific peptide (ova), rapamycin/ova, IL 10/ova or control rapamycin/IL 10/irrelavant (2A 54; irr) peptide. A ) Experimental design, B ) Representative examples of CD4+ T cells percentage of total gated lymphocytes as a function of treatment.

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46 Figure 32. Average cell percentages from flow cytometric analysis of splenocytes in DO.11.10tg Rag/BALB/c mice. IP injections 3 times/week for 4 weeks with rapamycin/IL 10/specific peptide (ova), rapamycin/ova, IL 10/ova or control rapamycin/IL 10/irrelavant (2A 54; irr) peptide. A ) Percentage CD4+ T cell deletion; fold decrease compared to control B ) %CD25+FoxP3+ T reg cells gated on CD4+ T cells; fold increase compared to controls C ) CD25+FoxP3+/CD4+ T cells gated on total splenocytes. P<0.0005 ***.

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47 Figure 33. Cytokine capture in treatment groups. Flow cytometric analysis of splenocytes from DO.11.10tg Rag/BALB/c mice injected IP 3 times/week for 4 weeks with A) rapamycin/IL 10/irrelavant (2A 54; irr) peptide in vitro stimulated with ova B ) rapamycin/IL 10/specific peptide (ova) in vi tro stimulated with ova, C ) IL 10/ova stimulated with mock media or D ) IL 10/ova stimulated with ova.

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48 Figure 34. Flow cytometric analysis of CD4+ T effector T cell deletion. in DO.11.10tg Rag/mice were treated for 3 weeks with rapamycin/IL 10/ova or rapmycin/IL 10/A254 Irr peptide or in nave mice. A ) Splenocytes B ) popliteal and inguinal lymph nodes, C ) Thymus and D ) peripheral blood were analyzed for percentage of CD4+ T cell depletion. The figure demonstrates fold decrease of CD4+ T ce lls compared to irrelevant peptide treated controls.

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49 Figure 35. Flow cytometric analysis of CD4+ T cell deletion in lymphoid organs. DO.11.10tg Rag/mice were treated for 3 weeks with rapamycin/IL 10/ova or rapmycin/IL 10/A254 peptide or nave (no treatment). Representative examples of CD4+ cells of total gated lymphocytes depletion in spleen, pooled inguinal and popliteal lymph node cells and thymus.

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50 Figure 36. Flow cytometric analysis of regulatory T cell induction. DO.11.10tg Rag/mice were treated for 3 weeks with rapamycin/IL 10/ova or rapmycin/IL 10/A254 Irr peptide or in nave mice. A ) Splenocytes B ) popliteal and inguinal lymph nodes C ) Thymus and D ) peripheral blood were analyzed for percentage of CD25+FoxP3+ T cells gated on C D4+ cells. The figure demonstrates fold increase of regulatory T cells compared to irrelevant peptide treated controls.

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51 Figure 37. Measurement of T cell activation in splenocytes. DO.11.10tg Rag/mice were treated for 1 week with rapamyc in/IL 10/ova specific peptide or irrelevant peptide (control). A ) Overlay of representative examples of CD62L, CD69, and CD44 expression in CD4+ T cells from a single treated and or nave mouse. Graphs show summaries of results (averages SD, n=4 per group) for percent CD69+ B ), CD44high C ), CD4+ D ) CD62L+, and CD25+ E ) of CD4+ cells. indicates a significant difference with P<0.05.

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52 Figure 38. Measurement of apoptosis and Fas (CD95) expression in splenocytes. DO.11.10tg Rag/mice were treated fo r 1 week with rapamycin/IL 10/ova, rapapamycin/IL 10/A254, or untreated (nave) mice A ) Percent CD95 + of CD4 + T cells. B ) Percent AnnexinV+PIof CD4+ T cells. C ) Percent AnnexinV+PI-CD95+ of CD4+ T cells.

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53 A B Figure 39. Activation induced cell death ( AICD) timeline. A ) Increase in apoptotic cell numbers as a function of time (duration of treatment) with rapamycin/IL 10/ova. B ) Change in ova specific CD4+ T cell frequency as a fun ction of time during treatment with specific or nonspecific protocol, and comparison to untreated nave mice.

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54 Figure 310. Differential effects of treatment on Teff and Treg. Percent apoptotic (AnnexinV+) cells among splenic Teff (CD4+CD25-FoxP3-) an d Treg (CD25+CD25+FoxP3+) from DO.11.10tg Rag/mice after 3 weeks of treatment with rapamycin/IL 10/ova compared to CD4+ T cells from or control rapamycin/IL 10/A254 treated mice. Shown are results for individual mice.

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55 Figure 311. Ova specific fu nctional T cell proliferation assay. Complete splenocyte cultures (total CD4+), CD25+ cell depleted cultures (Teff), and depleted cultures reconstituted with CD4+CD25+ T cells at a ratio of 0.5:1 when compared to CD4+CD25 cell number (Teff : Treg; 1 : 0.5). Splenocytes were from DO11.10tg Rag/mice treated for 3 weeks with rapamycin/IL 10/ova. Results are average SD for quadruplicate cultures.

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56 Figure 312. Optimization of Rapa/IL 10/specific peptide tolerization protocol utilizing different routes of injection. DO.11.10tg Rag/mice (n=4) were treated for 3 weeks with Rapa/IL 10/specific peptide after which the spleens were harvested and flow cytometry was used to determine A ) percentage deletion of CD4+ T effector T cells and B ) CD4+CD25+FoxP3+ T regulatory cells

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57 Figure 313. Optimization of alternate drug combinations. Flow cytometric analysis of splenocytes from DO.11.10tg Rag/mice injected IP for 3 weeks with Ova/Rapa in combination with one of the following drugs: Flt3L, Fc GITRL, IL2 or IL 10. A )Representation of CD25+FoxP3+/CD4+ T cells B ) Percentage CD4+ T cells of total splenocytes C ) Percentage CD25+FoxP3+ cells gated on CD4+ T cells. P<0.0005 ***; P<0.005 **; P<0.05 *.

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58 Figure 314. Effect of tolerance protocol on general immune suppression in immune competent mice over time. Flow cytometric analysis of splenocytes from C3H/HeJ mice injected IP for 3 weeks with Rapa/IL 10/specific peptide protocol. Splenocytes were isolated 1 day after the end of treatment or 5 months after the end of treatment. A,B ) CD8+ cells C,D ) CD4+ cells E,F ) B220+ cells

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59 CHAPTER 4 INDUCTION OF TOLERAN CE IN HEMOPHILIA B DISEASE MICE RECEIVIN G GENE THERAPY Introduction The novel tolerance protocol formulated using Rapamycin and IL 10 (or IL 2 or Fc GITR L) in the presence of specific peptide induced CD4+CD25+FoxP3+ Treg and deleted antigen specific CD4+ effector T cells as observed in the test DO.11.10tg Rag/mice (Chapter 3) In order to extend these findings to a relevant disease model, hemophilia B mice were used to test and optimize the protocol F.IX protein is a vitamin K dependant protein that participates in the coagulation pathway in plasma. It is a serine protease zymogen that converts to activated Factor IXa after vascular injury. The F9 gene DNA sequence is about 1.4 kilo bases in length and can be packaged in an AAV vector.96 Successful gene therapy in the absence of antibody formation in severe hemophilia patients will reduce costs associated with protein replacement therapy and improve the standard of living. Patients with early stop codons and gene deletions develop a neutralizing antibody response against F.IX upon expensive therapeutic protein replacement therapy. The hemophilia B mice used in my experiments are on a C3HeJ background and are knockouts for F.IX (C3H/HeJ F9 / ). They fail to produce hepatic F.IX mRNA and have no circulating F.IX pr otein. These mice exhibit phenotypic characteristics for severe disease such as prolonged bleeds from tail clips, spontaneous internal bleeds and increased mortality (possibly from bleeding due to fight wounds). The Herzog lab was previously unable to completely inhibit the neutralizing antibody response to F.IX by AAV2 gene therapy in C3H/HeJ F9/ (hemophilia B) mice although the protocol was successful using an AAV8 vector.97 Hepatic injection of AAV2 hF.IX containing 1antitrypsin promoter (ApoE/hAAT) after nasal administration of specific peptide suppressed the immune response and long term F.IX expression was

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60 observed. 90 Muscle is an alternative target organ for gene therapy as it can produce biologically active F.IX upon gene transfer. Muscle gene transfer was used in a phase I clinical trial although it resulted in <1% detectable F.IX which was sub therapeutic. F.IX muscle gene transfer in dogs has resulted in longterm therapeutic F.IX levels >4 yrs after gene delivery. AAV1 CMV hF.IX when injected IM has consistently led to high levels of neutralizing antibody production against hF.IX in C3H/HeJ F9 / mice as detected by the Bethesda assay. This muscle gene transfer model was used to test the novel drug cocktail/specific peptide combinations for prevention of the anti hF.IX immune response in the hemophilia B mice. Testing a Short Tolerance Protocol in C57BL/6 Mice Before moving into a therapeutic setting in hemophilia B mice, the Rapa/IL 10/specific peptide tolerance protocol was tested in immune competent C57BL/6 mice to observe the reduction or elimination of an anti F.IX IgG1 response to AAV2CMVF.IX administration in muscle. Control animals that receive d only the gene transfer formed high titer anti hF.IX IgG1 levels (4 36 g/ml plasma in 5/6 mice Fig. 41A) as measured by a sandwich ELISA. An additional group containing experimental mice (n=5) received rapamycin (4mg/kg), IL 10 (50ng/kg) and hF.IX speci fic peptide (CD4+ T cell epitope in C57BL/6 mice; 2A58). The dosing of the mixture was 3 times a week for 6 weeks. The AAV2 CMV h.F.IX vector was administered IM during the third week of treatment. Only 1/5 mice developed an anti F.IX antibody in the toler ized group Fig. 4 1B. Thus, the prophylactic protocol efficiently prevented an antibody response in immune competent mice; these results encouraged the extension of the protocol to a therapeutic hemophilia B model. Prevention of Inhibitor Formation in Intr amuscular Gene Therapy for Hemophilia B In order to test this prophylactic protocol in a therapeutic setting, hF.IX specific peptide (2A 54, containing the dominant CD4+ T cell epitope in C3H/HeJ mice) was co administered

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61 with rapamycin and IL 10 cytokine 3 times per week for 4 weeks to C3H/HeJ hemophilia B mice (n=4 per experimental group). These mice have a targeted F9 gene deletion and show stronger adaptive immune responses to F.IX than other inbred strains 98,99. After the third week of treatment, AAV1 CMVhFIX vector (1x1011 vg/mouse) was injected to intramuscularly (IM) as illustrated in Fig. 4 2A. Control mice received vector only, or rapamycin, IL 10 treatment, except that an irrelevant (ova) peptide was used instead of 2A 54 (n=4 per experimental group). An additional group was given rapamycin with 2A54 specific peptide only. Without immune suppression, mice developed inhibitors of 419 BU within 1 month after gene transfer (Fig. 42B). Mice treated with the hF.IX tolerance protocol (Rapa/IL 10/2A54) had at best very low titer inhibitors (<2 BU, the background of the assay) for the duration of the experiment (5.5 months after gene transfer, Fig.4 2B). Importantly, non specifically immune suppressed mice (Rapa /IL 10/Irrelavent ova peptide) formed low titer inhibitors of 4 5 BU, and therefore showed only partial suppression of the inhibitor response. Systemic hF.IX levels in hF.IX tolerized mice (Rapa/IL 10/2A54) were stable and on average 200250 ng/ml (45% of normal human levels), while both control groups showed substantially lower expression of on average 0 50 ng/ml (Fig. 42C) Clotting times (aPTTs) were partially corrected (~50 sec) in the hF.IX tolerized group compared to an average of 6580 sec in the c ontrol groups, which is identical to untreated mice (Fig. 42D). While hF.IX levels and aPTTs did not improve, inhibitory antibody titers in animals without immune suppression were unchanged for 3.5 months and subsequently declined to lower titers, similar to previously published data (Fig. 4 2B). 91 In the non specifically suppressed group (Rapa/IL10/ova), 3 of 4 animals also showed a decline in Bethesda titers by 6 months, resulting in increased hF.IX expression and improvement of the aPTTs, while one animal still showed 5 BU and lacked systemic expression (Fig. 42D and data not shown). One group treated

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62 with rapamycin and specific peptide without the cytokine IL 10 (Rapa/2A54) showed some reduction of neutralizing antibody levels compared to the untreated controls and lowered clotting times and improved hF.IX expression, although it was not as effective as the treatment group that included IL 10. NonhF.IX specific effects in rapamycin/IL 10 treated immune competent mice included transient reduction in B and T cell frequencies and transient neutropenia. Induction of Functional Treg via the Prophylactic Tolerance Protocol C3H/HeJ F9/mice were treated with rapamycin/IL 10/2A 54 or rapamycin/IL 10/ova for 4 weeks. After tolerization, CD4+CD25+ Treg cells were isolated via MACS. Treg cells ( 1x106) were transferred to C3H/HeJ mice. The recipient mice were given an IM inject ion of AAV1 CMVhF.IX (1x1011 vg/mouse) and later tested for anti F.IX IgG1. The graph (Fig. 4 3) indicates a reduction in levels of anti hF.IX antibodies in the recipients of the CD4+CD25+ Treg cells from tolerized animals when compared to controls. Regul atory T cells induced by rapamycin/cytokine/specific peptide treatment suppress antibody formation against hF.IX as evidenced by this adoptive transfer experiment. Modified Tolerance Protocol Using an Alternate Route of Tolerance Induction Rapamycin is app roved by the FDA for oral administration in patients during transplantation procedures. Therefore, modified version of the novel rapamycin, IL 10, specific peptide protocol was tested for oral tolerance. Rapamycin (4 mg/kg) was orally administered using a gavage needle 3 times/week for 4 weeks while a mixture of IL 10 (50 ng/kg) and specific peptide (hF.IX CD4+ epitope for C3H/HeJ background; 2A54; 100 g/dose/mouse) was injected IP concomitant with the oral gavage of rapamycin. An IM injection of AAV1CMVhF.IX vector (1x1011vg) was given at the third week of tolerization. Control animals received IM gene transfer only. The tolerized group showed a lower anti F.IX IgG1 response averaging <5000 ng/ml over 4 months compared to the gene transfer controls (~600020,000 ng/ml; Fig. 44). A

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63 reduction in neutralizing antibody titer was also observed (~4 5 BU) in treated animals vs. 7 8 BU in controls. There was an increased expression of hF.IX expression in tolerized animals (~90 140 ng/ml) and controls (~10100 ng/ml) over a 4 month period. The marginal reduction of the immune responses may be attributed to the lower bioavailability of the rapamycin via the oral route. Alternatively, the separation of the drug combination in the oral and IP routes may affect their synergistic activity. Modified Tolerance Protocol Utilizing an Alternate Cytokine One group of C3H/HeJ F9 / mice was co administered with rapamycin (4 mg/kg), IL 2 (50 ng/kg) and hF.IX specific peptide for C3H/HeJ background (100 g/dose/mouse). The treatment was given thrice a week for 4 weeks. At the third week of tolerization, AAV1CMVhF.IX vector (1x1011vg) was administered IM. A control group received gene transfer only. The tolerized group had a lower neutralizing antibody response (<= 2.5 BU) compa red to gene therapy only controls (3.213.5 BU). An increase in average hF.IX levels was observed in the tolerized animals (~160 200 ng/ml) over a 4 month period; control animals showed F.IX expression from 50 ng at 1 month and a gradual increase to ~150ng/ml was observed 4 months after gene transfer. This increase in hF.IX levels was observed in other experiments as well and may be attributed to the constant longterm presence of the hF.IX protein expressed by vector transduced cells, eventually leading to some state of hyporesponsiveness. Minimal correction of clotting time was observed in the tolerized animals (60.8 64.5 sec aPTT) over the 4 month period. Controls showed clotting times (aPTTs) of 68.572 sec (Fig. 45). This reduction in clotting times however, was not as effective as the rapamycin/IL 10/specific peptide protocol. In addition to promoting proliferation of Treg, IL 2 also promotes proliferation of Teff. Deletion of

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64 Teff by the dose of rapamycin administered may have been insufficient to c ompletely control the anti F.IX immune response. Prevention of Inhibitor Formation in Hepatic Gene Therapy for Murine Hemophilia B Rapamycin/IL 10/Specific Peptide Treatment Hepatic route of vector administration has previously been shown to be less immunogenic than muscle, possibly resulting from the unique environment of the liver, which promotes an induction of Treg following liver gene transfer. Although AAV2mediated hepatic gene transfer induces robust immune tolerance to hF.IX in several strains of F 9/ mice, an immune response is observed against exogenous hF.IX in F9/ mice of C3H/HeJ background. Expecting to improve tolerance to the hF.IX transgene via AAV2F.IX liver gene transfer, The rapamycin, IL 10, specific peptide IP protocol was combined with liver gene transfer. Rapamycin, IL 10 and the aforementioned specific peptide 2A54 was administered to C3H/HeJ F9 / mice 3 times/week for 4 weeks. At the third week, AAV2 hAAT F.IX was administered to the liver via splenic capsule injection. Control animals received the liver gene transfer only (included in Fig. 48). Contrary to expectations, the treatment group showed a decrease in hF.IX levels (~10 40ng/ml) compared to controls (~50 110 ng/ml) over a 3month period although these results lack signi ficance (average data not shown) Only 1 mouse from the tolerized group had the expected increased F.IX expression accompanied by lowered clotting times (Fig. 46) The anti F.IX IgG1 values and neutralizing antibody response (BU) did not show a significant difference between the control and drug cocktail treated groups, which begged the question whether rapamycin was in some way impeding gene transfer to the liver or had a deleterious effect on F.IX expression in a nonimmune dependant manner ? To test the p ossibility of a nonimmune mediated suppression of liver cell transduction by the rapamycin/IL 10/specific peptide protocol, C57BL/6 Rag/mice (n=5) were treated with

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65 rapamycin, IL 10 and a specific peptide (2A58; hF.IX specific CD4+ T cell epitope for t he C57BL/6 mice). The treatment was done 3 times/week for 4 weeks. At the third week AAV2 hAAT hF.IX (1x1011vg) was administered to the liver via splenic capsule injection. Control animals received the liver gene transfer only. hF.IX levels of 1200 ng/ml w ere observed in the treatment group as well as the control group (Fig 4 7). The result indicates that transduction of hepatocytes is not hampered by the drugs. It can be inferred that the reduction in the hF.IX production in the treated mice was immune function related and may differ depending on the genetic background of the animal albeit the mechanism remains unclear. Fc GITRL treatment In order to test the fusion protein Fc GITRL for its ability to suppress an immune response to AAV2hAAT hF.IX in hem ophilia B mice; Fc GITR injected IP into hemophilia B (C3H/HeJ F9/ ) mice. Seven doses of the fusion protein were 4 days apart over a period of 1 month. After 4 injections, AAV2 hF.IX (1x1011 vg) was administered to the liver via splenic capsule injection. Here, we tested whether the induction of CD4+FoxP3+ Treg cells by in vivo Fc GITRL administration could suppress the response, hence augmenting therapy in this strain of hemophilia B mice. Consistent with the Herzog labs previous findings, C3H/HeJ F9/ mice expressed only low levels of hF.IX following gene transfer and developed inhibitors of 411 BU by 3 months ( Fig. 48). In contrast, administration of Fc GITRL (every 34 days for 4 weeks ) combined with gene transfer (which was performed after the 4th Fc GITRL injection) directed substantially higher levels of hF.IX expression at 1and 2month time points ( Fig. 4 8 ) However, expression decreased to the low levels seen in control mice by 3 months ( Fig.4 8). In agreement with these data, coagulation times were significantly shortened by 2 month, i.e. the peak of hF.IX expression, but then gradually increased (Fig. 48). Antibodies against hF.IX

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66 formed by 5 6 months in 4/5 mice (1 titer inhibitors of 2 3 BU in 2/5 mice by 6 months. Therefore, the effect of Fc GITRL treatment in suppressing anti F.IX responses was transient. Inhibition of an Established Anti F.IX Immune Response by the Rapamycin/IL 10/Specific Peptide Protocol It is of interest for treatment of hemophilia and other systemic protein deficiencies that this novel tolerance protocol can, not only be used prophylactically but also to reverse established immune responses. To address this point, the shortterm rapamycin/IL 10/specific peptide protocol was tested in the case of an ongoing response in hemophilia B mice induced by IM gene transfer of AAV1 CMVhF.IX 1x1011 vg/mouse. Briefly, C3H/HeJ F9/mice were injected with AAV1 CMVhF.IX 1x1011 vg/mouse IM and tested for the presence of high titer anti hF.IX IgG1 that was found to be between 3000 ng/ml to 8000 ng/ml (n=12; Fig. 49). The corresponding neutralizing antibody level was ~7 11 BU (Fig 49), and there was no hF.IX detectable in circulation at this time point. After confirming the presence of high titer anti F.IX antibody, half of the animals (n=6) animals were treated 3 times per week for 4 weeks with rapamycin/IL 10/2A54 specific peptide, at doses as mentioned befor e. Tolerized mice showed a robust drop in neutralizing antibody levels, which remained low for 6 months. By contrast, controls that received gene transfer only showed persistent high levels of neutralizing antibody levels (~7 BU) through the 6month period (Fig. 49). hF.IX levels in the tolerized mice were between 200 and 600 ng/ml during the 6 months as compared to the controls that did not have detectable levels (Fig. 4 9). In agreement with the above observations, clotting times were observed from an average of 80 seconds before tolerization to ~62 seconds after tolerization; clotting times remained low at the 6 month time point. The short term protocol is important since it may be helpful in reversing an existing antiF.IX immune responses in protein re placement

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67 therapy patients who have already developed an immune response (experiments are ongoing). Clearly, future gene therapy trials can benefit from these observations if an immune response is inadvertently induced against the therapeutic protein (or v ector capsid?). The long term beneficial effects of this short protocol are encouraging. An additional experiment in hemophilia B mice wherein animals with an immune response to AAV1 CMV hF.IX (1x1011 vg/mouse) IM injection were administered with a splenic injection of AAV8haat hF.IX (1x1011 vg/mouse) did not result in a significant decrease of the IgG1 response against hF.IX. Prevention of an Anti F.IX Immune Response in F.IX Protein Therapy The rapamycin/IL 10/specific peptide protocol was tested in the context of protein therapy in C3H/HeJ F9/mice Mice were treated with the drug cytokine specific peptide combination 3 times a week for 4 weeks. After the third week of tolerization the animals were challenged with 1 IU hF.IX protein (Benefix) once a we ek for 4 weeks (1 IP to prime the immune system, followed by 3 IV injections). Control animals received IV protein challenge only. Three out of five control animals died shortly after the 4th injection, possibly due to an allergic reaction. The remaining m ice had neutralizing antibody responses of 57 BU (Fig 4 10). There was no mortality among the tolerized mice. Tolerized mice also showed a markedly reduced neutralizing antibody response <2 BU (Fig. 410). An initial experiment in normal C3H/HeJ mice was unsuccessful since the animals did not develop a detectable immune response to IV injections of 1 IU hF.IX via tailvein injections, which was further reflected in C3H/HeJ F9 / Hence the mice from this background were primed with 1 IP injection of 1 IU F.IX followed by three IV injections of hF.IX to observe a strong immune response. Interestingly this protocol resulted in an acute hypersensitivity response accompanied by lower body temperature, piloerection and death in a large percentage of animals.

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68 Ana phylactic responses to therapeutic proteins in patients with inhibitors seriously complicates therapy. IgE mediated hypersensitivity, IgG1 and IgG4 all have been implicated as a cause for the anaphylactic response in hemophilia B patients and the role of c omplement activation has been postulated although the precise mechanism remains unclear. In hemophilia B anaphylaxis is often associated with large/complete gene deletions or early stop codon mutations.100 Combinations of antihistamines and corticosteroids have been utilized during desensitization treatments in patients before ITI.100 Similarly, in order to reduce the mortality rate in the hF.IX injected C3H/HeJ F9/animals, a combination of anti PAF (50 g/mouse) and anti histamine Triprolidiene hydrochloride (100 g/mouse) was administered via tail vein 5 minutes prior to hF.IX protein challenge at the 4th injection. This treatment prolonged the life of the animals and showed an anti hF.IX IgG1 response ( 17600 ng/ml SD 5400; n=16). Mice that did not receive the anti histamine, anti PAF combination resulted in higher immune responses although 2/6 mice died before blood samples could be collected. The single dose of the drug combination resulted in 100% survival after hF.IX challenge as opposed to 40% mortality in control animals. In a related experiment 6 out of the 16 C3H/HeJ F9/mice previously injec ted with hF.IX and showing a high IgG1 response against hF.IX were orally administered with rapamycin 100 g/kg thrice a week combined with IV administration of 0.1 IU hF.IX twice a week. All animals died by the second week of tolerization, due to severe a llergic reactions to hF.IX indicating that the tolerizing protocol was ineffective in adequately suppressing immune responses against hF.IX at the given doses. Other experiments are in progress to test the role of IP administration of rapamycin/IL 10/spec ific CD4+T cell epitope in diminishing an existing immune response to protein therapy.

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69 Figure 41. Anti hF.IX formation after IM injection of AAV2 CMVhF.IX in C57BL/6 mice. The mice were treated IP with Rapamycin/IL 10/B6CD4 specific peptide 3times per week for 4 weeks B ). During the third week of tolerization, AAV2CMVhF.IX 1X1011 vg/mouse was injected IM. Control animals did not receive immun0modualtion A ).

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70 Figure 42. Prevention of inhibitor formation in hemophilia B mice via rapamycin/I L 10/specific peptide protocol. C3H/HeJ F9/mice were treated with rapamycin/IL 10/2A 54 or rapamycin/IL 10/ova for 4 weeks. After the third week of treatment, AAV1 CMVhF.IX (1x1011vg/mouse) was injected IM A ). A third group received IM gene transfer on ly. Shown are the average results SD, (n=4 per group) for inhibitory antibody titers (in BU; A ), hF.IX plasma levels (ng/ml; B ), and clotting times C) ; aPTT in sec, which is 25 35 sec for wildtype mice and >60 sec for untreated hemophilia B mice) as a fu nction of time after vector administration.

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71 Figure 43. Treg induced by rapamycin/cytokine/specific peptide protocol are functional and can be adoptively transferred. C3H/HeJ F9/mice were treated with rapamycin/IL 10/2A 54 or rapamycin/IL 10/ova for 4 weeks. After tolerization, CD4+CD25+ Treg cells were isolated via MACS. 1x106 Treg cells were transferred to C3HeJ mice. The recipient mice were given IM injection of AAV1 CMVhF.IX and later tested for anti F.IX IgG1 responses shown as averages (ng/ml ) SD, (n=4 per group).

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72 Figure 44. Partial suppression of inhibitor formation in hemophilia B mice via oral administration of rapamycin and concurrent IP IL 10/specific peptide treatment. C3H/HeJ F9/mice were treated with rapamycin by oral gav age while IL 10/2A 54 were injected IP 3 times/week for 4 weeks. After the third week of treatment, AAV1 CMVhF.IX (1x1011vg/mouse) was injected IM A ). Control group received IM gene transfer only. The average results SD, (n=6 per group) for anti h.FIX Ig G1 antibody (ng/ml; A ), hF.IX plasma levels (ng/ml; B ), and inhibitory antibody titers (in BU; C ).

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73 Figure 45. Partial suppression of inhibitor formation in hemophilia B mice via rapamycin/IL 2/specific peptide protocol. C3H/HeJ F9/mice were treated with rapamycin/IL 2/2A 54 or rapamycin/IL 2/ova for 4 weeks. After the third week of treatment, AAV1 CMVhF.IX (1x1011vg/mouse) was injected IM A ). Control group received IM gene transfer only. The average results SD, (n=6 per group) for inhibitory anti body titers (in BU; A ), hF.IX plasma levels (ng/ml; B ), and clotting times ( C ; aPTT in sec).

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74 Figure 46. Prevention of inhibitor formation in hemophilia B mice via rapamycin/IL 10/specific peptide protocol in liver gene transfer. C3H/HeJ F9/mice wer e treated with rapamycin/IL 10/2A 54 or rapamycin/IL 10/ova for 4 weeks. After the third week of treatment, AAV2haat hF.IX (1x1011vg/mouse) was injected via spleen capsule A ). A third group received IM gene transfer only. Shown are the average results for individual mice for inhibitory antibody titers (in BU; B ), hF.IX plasma levels (ng/ml; C ), antihF.IX IgG1 (ng/ml; D) and clotting times ( E ; aPTT in sec) as a function of time after vector administration.

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75 Figure 47. Rapamycin/IL 10/specific peptide protocol evaluation in liver gene transfer to Rag/mice. C57BL/6 Rag/mice (n=5) were treated for 4 weeks with Rap/IL 10/hF.IX specific peptide; Control and treatment groups were given liver gene transfer (AAV2 haat hF.IX) 1x1011vg/mouse) via spleen ca psule injection and tested for hF.IX expression by ELISA.

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76 Figure 48. Delay of immune response to hF.IX in hemophilia B mice following administration of Fc GITRL. C3H/HeJ F IX mice (n=5) were treated with 7 doses of FcGITRL IP, AAV2 hAAT hF.IX was injected into the liver via spleen capsule injection 2 weeks into treatment. Controls (n=7) received gene transfer only.

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77 Figure 49. Elimination of a pre existing/ongoing anti hF.IX immune response. C3H/HeJ F9/mice were injected IM with AAV1 C MV hF.IX (1x1011vg/mouse) to generate an immune response. After the proven existence of an immune response, animals were given rapamycin/IL 10/specific peptide for 4 weeks. Controls only received gene transfer A ). Average results SEM, (n=6 per group) for inhibitory antibody titers (in BU; B ), hF.IX plasma levels (ng/ml; C ), anti hF.IX IgG1 antibody (ng/ml; D ) and clotting times (aPTT; E ) as a function of time after vector administration are shown.

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78 Figure 410. Prevention of inhibitor formation in hemophilia B mice receiving protein therapy. C3H/HeJ F9 / mice were treated with rapamycin/IL 10/2A 54 for 4 weeks. A ). After the third week of treatment, weekly administration of Benefix (arrows) was initiated. B ) Inhibitor titers (average SD; n=5) after the 4th protein injection were compared to control mice that had received protein therapy only (results shown for 2 surviving individual mice). C ). Survival of tolerized and control C3H/HeJ F9/ mice after 4 injections of Benefix (n=5 per group)

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79 CHAPTER 5 DISCUSSION The immune system has evolved to fight off infections. In order to achieve an effective and specific response and to create memory, antigen presentation to T lymphocytes results in the generation of effector and memory T cells. CD4+ T cells pr ovide help for activation of B cells, which subsequently form antibodies. However, if the adaptive response is directed against a therapeutic protein, it can become a major complication and source of immunotoxicity in gene and protein replacement therapies for genetic disease. Hemophilic patients with inhibitor formation are at a higher risk for bleedingrelated morbidity and mortality. Patients at high risk for antibody formation in protein replacement therapy for hemophilia and lysosomal storage disease ( LSD) would benefit from prophylactic immune tolerance protocols. Furthermore, safety of gene therapies, which offer the chance of long lasting cures for genetic disease, would increase if such protocols were developed. The possibility of eliminating a pre existing or ongoing immune response must be emphasized, in patients who have previously developed an immune response to protein treatment. Creating an Optimal Shift From an Effector to a Regulatory T Cell Population Experiments presented in this thesis il lustrate the usefulness of the highly defined DO.11.10tg Rag2/mouse model to screen for useful drugs and define their mechanism of action. The drug cocktail protocols proposed were evaluated and are found to be effective in directing a shift from a Tef f to a Treg response upon specific antigen presentation to CD4+ T cells (Fig. 51). Changing the balance of the immune system in favor of regulation is critical for tolerance to the therapeutic protein. Rapamycin, and even more so the combination of rapamycin and IL 10, had a substantial effect by deleting ova specific CD4+ T cells while allowing induction of CD4+CD25+FoxP3+ Treg cells. In the absence of the specific antigen (ova in this

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80 case), the drugs did not change the ova specific T cell population. The presence of the antigen specific CD4+ T cell epitope provides direction to the immune response due to the mechanism of depletion of effector CD4+ T cells. Mechanistically, the data demonstrates activation of CD4+ T cells upon in vivo antigen presentation in the presence of rapamycin/IL 10 and the specific peptide, resulting in apoptotic cell death (AICD). At the same time, we found a greater resistance to antigen/rapamycininduced apoptosis in Treg compared to Teff, which explains why Treg induction and e xpansion is successful in regimens that utilize rapamycin. Rapamycin/FKBP12 complex inhibits IL 2 stimulated signal transduction by preventing phosphorylation of p70 S6 kinase and of eIF 4E BP1, which are both regulated by mammalian target of rapamycin (mT OR), leading to a block in cell cycle progression. A recent study has shown that IL 2 stimulation of Treg, compared to Teff, leads to preferential use of the STAT5 rather than the mTOR pathway, which results in resistance to inhibition of mTOR 101. The Teff cell population regained responsiveness to their specific antigen when Treg were depleted while Treg were able to dampen the antigen specific immune response on in vitro stimulation. This is similar to previously published findings wherein the depletion of CD25+ Treg using anti CD25 antibody resulted in an increased immune response.94 Treg induction by repeated antigen/rapamycin administration was more robust than in hepatic ova gene transfer published previously, which shows the usefulness of the DO.11.10tg Rag2/mouse experimental system for comparison between different treatment approaches. 8 Different routes of adminis tration of the Rapamycin/IL 10 specific peptide protocol resulted in a differential induction of Treg and varying depletion of Teff. Intraperitoneal, intravenous and subcutaneous methods of administration proved to have a significantly better effect than o ral gavage. This difference in results depending on routes may be due to the

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81 bioavailability of the drugs or metabolism in the digestive system or due the splitting of the rapamycin (oral gavage) and IL 10/specific peptide (IP) which would indicate the importance of the presence of the three components of the cocktail in vivo at the same time for adequate antigen presentation, Teff cell deletion and Treg induction. IL 2 cytokine has been previously shown to induce Treg. It is also known to encourage Teff cell proliferation and is produced by T cells for autocrine and paracrine function. The cytokine IL 2 tested in combination with rapamycin and specific peptide showed better Treg cell induction than IL 10 in DO.11.10tg Rag2/mice although its effects were not as significant as the combination of rapamycin and IL 10 in hemophilia B mice; possibly due to the otherwise immune compromised transgenic model system. In addition, IL 2 is known to induce the expansion of Treg and Teff and hence there is a possibili ty of some runaway Teff cell proliferation that slightly dampened the overall antigen specific immune suppression. Flt3L is known to induce Treg and hence is likely to work synergistically with rapamycin. Flt3L in combination with rapamycin and specific peptide resulted in a very significant Teff cell deletion accompanied by a robust Treg cell increase and may be a promising candidate for further testing in hemophilia and other diseases. Fc GITRL also showed promising Treg induction accompanied by antige n specific Teff deletion in combination with rapamycin and specific peptide although its effects tended to have a more variable effect from one mouse to another. Treg are Critical for Many Tolerance Induction Protocols Usefulness of the rapamycin/IL 10/a ntigen cocktail for therapeutic application was demonstrated in the hemophilia B mouse model, where inhibitor formation was effectively blocked using a short (4week) tolerance protocol starting 3 weeks prior to muscledirected gene transfer. Immune tolera nce protocols that utilize immune suppressive drugs will be acceptable for treatment of hemophilia, only if they are limited in time. A recent study by Lillicrap and

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82 colleagues accomplished this goal in hemophilia A mice by short term administration of low doses of a nonFc receptor binding anti CD3 monoclonal antibody. This protocol caused a shift in the Th1/Th2 ratio during the response to F.VIII, and tolerance to F.VIII was dependent on Treg function. 102 Transient blockade of ICOS signaling, a costimulatory pathway during T cell activation, at the time of hepatic gene transfer with plasmid DNA also blocked inhibitor formation to F.VIII and resulted in Treg induction.103 In contrast to these studies, utilizing m ore recently developed and still experimental reagents, our investigation established the usefulness of a drug, rapamycin, which is well established in clinical practice. Therefore, translational research could take advantage of a wealth of experience in humans (including pediatric patients). Furthermore, this protocol resulted in only moderate general immune suppression. Oral administration may also result in Tr1 activation, which was not seen in these experiments or by lab colleagues in the spleen, but wh ich may add another useful subset of regulatory T cells.104 Finally, the importance of Treg fun ction during immune suppression with rapamycin upon F.IX gene transfer is also supported by data in non human primates.46 Implications for Translational Research Prevention of inhibitor formation upon muscle gene t ransfer required co administration of the immune modulatory drugs rapamycin/IL 10 and the hF.IX specific peptide. Without the specific antigen, the drugs were only partially effective in blocking the response to hF.IX expressed in skeletal muscle. These fi ndings indicate that antigenspecific immune tolerance protocols that are initiated prior to the onset of therapy may have more beneficial effects than general immune suppression applied at the time of gene transfer. In this regard, peptide administration is highly effective, because the peptide can be directly loaded onto MHC molecules without a need for antigen processing and because the risk of inducing an adaptive response against the cognate protein antigen in the early phase of the protocol (before tolerance

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83 is established) is substantially reduced.90 However, translation to the clinic would require knowledge of CD4+ epitope for each patient being treated, which although possible is not economically feasible at this point in time. Therefore, it is desirable to develop and optimize the protocol using the entire protein as the antigen for induction of T cell tolerance. However two attempts to devise a protocol with different concentrations of the entire hF.IX protein instead of the CD4+ T cell epitope in hemophilia B mice were not successful. Additional trials in Hemophilia A mice with heat inactivated protein or various low doses of whole protein in I P or IV routes of injection in combination with rapamycin and IL 10 were also not successful, indicating the difficulty of using the whole protein in the tolerization protocol. Nonetheless Dr. Babak Moghimi succeeded in inducing tolerance to F.VIII in Hemophilia A mice using a combination of oral treatments with rapamycin and IV whole F.VIII protein (given at a low dose) in protein replacement therapy (manuscript in preparation). These data show that oral administration of rapamycin and use of protein antigen can be effective but requires significant optimization. The use of rapamycin/IL 10/specific peptide protocol in protein replacement therapy for hemophilia B when delivered via IP administration caused decreased mortality and decreased neutralizing antib ody responses when treatment was started before challenge with hF.IX. Control animals receiving hF.IX challenge only developed severe allergic reactions, accompanied by lower body temperature and piloerection, eventually leading to death; while tolerized a nimals remained healthy. An effort to reduce or eliminate an ongoing immune response in hemophilia B mice brought on by hF.IX administration, by oral administration of rapamycin in combination with IP injections of small doses of whole F.IX protein resulte d in allergic reactions and death of all treated mice within 2 weeks after the beginning of the tolerization protocol. The mice likely

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84 died due to an antibody mediated hypersensitivity response due to their prior exposure to the F.IX protein, which occur red in spite of the presence of rapamycin. Work by others in the laboratory showed that repeated IV delivery of F.IX results in an IgE formation in addition to IgG1. In a different experiment, Hemophilia B mice with an ongoing immune response were given an injection of AAV8 hAAT hF.IX, these mice did not undergo hypersensitivity responses and remained alive. The difference in mortality rate compared with the mice that underwent rapamycin/whole protein tolerization suggests that liver delivered protein antig en did not systemically react with antibody in the way that IV delivered protein did, this aspect requires further study. With regard to rapamycin/IL 10/specific peptide protocol in combination with liver gene therapy, the results were surprising as the ef fect of the treatment was to increase the overall antibody response slightly in treated animals with an actual decrease in hF.IX production compared to the gene transfer controls. This phenomenon was further studied in Rag/ mice to eliminate the possibil ity of reduced transduction of hepatocytes by AAV hF.IX in the presence of rapamycin. Results showed that rapamycin did not block transduction of hepatocytes in the Rag / mice. The strain of mouse used may prove important as the hemophilia B mice were on a C3H/HeJ background but the Rag/ mice used were on a C57BL/6 background.105 This experiment also suggests that the immune system was in some way involved, although the mechanism remains unclear. In conclusion, antigen administration, when combined with a suitable cocktail of immune modulatory drugs, can create a powerful shift from an effector to a regulatory T cell population. This concept can be exploited for the development of prophylactic immune tolerance protocols for treatment of inherited protein deficiencies such as hemophilia

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85 Figure 51. Induction of tolerance to therapeutic proteins by shifting the balance from an effector (CD4+ Teff) to a regulatory response (CD4+ Treg) prior to gene or protein replacement therapy in an antigen specific manner.

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86 LITERATURE CITATIONS 1. Hough C, Lillicrap D. Gene therapy for hemophilia: an imperative to succeed. J Thromb Haemost 2005; 3: 11951205. 2. Giannelli F, Green PM. The molecular basis of hemophilia A and B. In: Lee CA (ed). Clinical haematology: Haemophilia, vol. 9. Bailliere Tindall, 1996, pp 211228. 3. Anson DS, Choo KH, Rees DJ, Giannelli F, Gould K, Huddleston JA et al The gene structure of human anti haemophilic factor IX. EMBO J 1984; 3: 10531060. 4. Makris M. Is VIII worse than IX? Blood 2009; 114: 750751. 5. Carcao MD, Aledort L. Prophylactic factor replacement in hemophilia. Blood Rev 2004; 18: 101 113. 6. Ljung R, Petrini P, Tengborn L, Sjorin E. Haemophilia B mutations in Sweden: a populationbased study of mutational heterogeneity. Br J Haematol 2001; 113: 8186. 7. Battaglia M, Stabilini A, Draghici E, Migliavacca B, Gregori S, Bonifacio E et al Induction of Tolerance in Type 1 Diabetes via Both CD4+CD25+ T Regulatory Cells and T Regulatory Type 1 Cells. Diabetes 2006; 55: 15711580. 8. Cao O Dobrzynski E, Wang L, Nayak S, Mingle B, Terhorst C et al Induction and role of regulatory CD4+CD25+ T cells in tolerance to the transgene product following hepatic in vivo gene transfer. Blood 2007; 110: 1132 1140. 9. Viiala NO, Larsen SR, Rasko JE. G ene therapy for hemophilia: clinical trials and technical tribulations. Semin Thromb Hemost 2009; 35: 8192. 10. Warrington KH, Jr., Herzog RW. Treatment of human disease by adenoassociated viral gene transfer. Hum Genet 2006; 119: 571603. 11. Nakai H, Yant SR, Storm TA, Fuess S, Meuse L, Kay MA. Extrachromosomal recombinant adenoassociated virus vector genomes are primarily responsible for stable liver transduction in vivo. J Virol 2001; 75: 69696976. 12. Schnepp BC, Reed K, Klemanski DL, Pacak CA, Johnson PR. Genetic fate of recombinant adenoassociated virus vector genomes in muscle. J Virol 2003; 77: 34953504. 13. Herzog RW, High KA. AAVmediated gene transfer of factor IX for treatment of hemophilia B by gene therapy. Thr Haemost 1999b; 82: 540 546. 14. Herzog RW, Hagstrom JN, Kung Z H, Tai SJ, Wilson JM, Fisher KJ et al Stable gene transfer and expression of human blood coagulation factor IX after intramuscular injection of recombinant adeno associated virus. Proc Natl Acad Sci USA 1997; 94: 58045809.

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87 15. Niemeyer GP, Herzog RW, Mount J, Arruda VR, Tillson DM, Hathcock J et al Longterm correction of inhibitor prone hemophilia B dogs treated with liver directed AAV2 mediated factor IX gene therapy. Blood 2009; 113: 797 806. 16. Manno CS, A rruda VR, Pierce GF, Glader B, Ragni M, Rasko J et al Successful transduction of liver in hemophilia by AAV Factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342347. 17. Scallan CD, Jiang H, Liu T, PatarroyoWhite S, Somme r JM, Zhou S et al Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice. Blood 2006; 107: 18101817. 18. Jooss K, Yang Y, Fisher KJ, Wilson JM. Transduction of dendritic cells by DNA viral vectors d irects the immune response to transgene products in muscle fibers. J Virol 1998; 72: 42124223. 19. Sarukhan A, Camugli S, Gjata B, von Boehmer H, Danos O, Jooss K. Successful interference with cellular immune responses to immunogenic proteins encoded by recombinant viral vectors. Journal of Virology 2001; 75: 269277. 20. Zaiss AK, Liu Q, Bowen GP, Wong NC, Bartlett JS, Muruve DA. Differential activation of innate immune responses by adenovirus and adenoassociated virus vectors. J Virol 2002; 76: 45804590. 21. Manno CS, Chew AJ, Hutchinson S, P.J. L, Herzog RW, V.R. A et al A phase I safety study in patients with severe hemophilia B using adeno associated viral (AAV) vector to deliver the gene for human factor IX to skeletal muscle. Blood 2003; 101: 29632972. 22. Mount JD, Herzog RW, Tillson DM, Goodman SA, Robinson N, McCleland ML et al Sustained phenotypic correction of hemophilia B dogs with a factor IX null mutation by liver directed gene therapy. Blood 2002; 99: 26702676. 23. Nathwani AC, Dav idoff AM, Hanawa H, Hu Y, Hoffer FA, Nikanorov A et al Sustained highlevel expression of human factor IX (hFIX) after livertargeted delivery of recombinant adenoassociated virus encoding the hFIX gene in rhesus macaques. Blood 2002; 100: 16621669. 24. Maus MV, Mingozzi F, Sabatino DE, Hui D, Ragni MV, High KA. T cell responses to AAV vector capsid in normal donors and subjects who have undergone liver directed AAV mediated gene transfer. Mol Ther 2006; 13: S282. 25. Wells AD, Li XC, Li Y, Walsh MC, Z heng XX, Wu Z et al Requirement for T cell apoptosis in the induction of peripheral transplantation tolerance. Nat Med 1999; 5: 13031307. 26. Mingozzi F, Schuttrumpf J, Arruda VR, Liu Y, Liu YL, High KA et al Improved hepatic gene transfer by using an adenoassociated virus serotype 5 vector. J Virol 2002; 76: 1049710502.

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95 BIOGRAPHICAL SKETCH Sushrusha Nayak was born in 1981 in Manhattan, New York, USA to Dr. Rabindranath Nayak and Mrs. Sukanti Nayak. She grew up in Bangalore, India within the ramparts of the Indian Institute of Science. The association with a major academic institution in India afforded her the opportunity to test her interest in fields like ethology and biomedical science with Dr. Gadagkar, Dr. Ramananda Rao, Dr. Lakshmi Sita and Dr. R Nayak. She completed her undergraduate degree in Microbiology, Zoology and Chemistry in 2003 and received her Master of Science in biotechnology in 2005 from Bangalore University. During her masters program she was drawn towards the study of immunology, gene t herapy and ribosomal RNA based research. She proceeded to join the Interdisciplinary Program in Biomedical Science at the University of Florida in 2005 with ample support from her parents. She trained in the laborato ry of Dr. Alfred Lewin, Dr. William W. Hauswirth, Dr. Susan L. Semple Rowland and Dr. Roland Herzog during rotations, before joining the laboratory of Dr. Roland Herzog in 2006. The Herzog lab focused on gene therapy and immunological studies in hemophilia She received the Medical Guild research incentive award in 2007 and research competition award in 2009. She published her first two first author papers in 2009, including a research and a review article. She undertook an MS degree in management at the Wa rrington Business School, University of Florida, concurrent with her Ph.D. work in 2008, which gave her an insight into the working of business organizations in the USA. Sushrusha plans to continue developing her career in translational medicine, gene the rapy and immunology with an eye open for collaborations with the industry. She hopes to keep her professional and personal life moving in a positive direction with reasonable harmony.