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Alpha 1 Antitrypsin Gene Therapy and Its Immunoregulatory Function for Preventing Type 1 Diabetes in Non-Obese Diabetic Mouse

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Alpha 1 Antitrypsin Gene Therapy and Its Immunoregulatory Function for Preventing Type 1 Diabetes in Non-Obese Diabetic Mouse
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TANG, MEI ( Author, Primary )
Copyright Date:
2008

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Antibodies ( jstor )
Cells ( jstor )
Diabetes complications ( jstor )
Diseases ( jstor )
Dosage ( jstor )
Gene therapy ( jstor )
Insulin ( jstor )
Protein isoforms ( jstor )
Type 1 diabetes mellitus ( jstor )
Type 2 diabetes mellitus ( jstor )

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University of Florida
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University of Florida
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Copyright Mei Tang. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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12/31/2011

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ALPHA 1 ANTITRYPSIN GENE THERAPY AND ITS IMMUNOREGULATORY FUNCTION FOR PREVENTING TYPE 1 DIABETES IN NON-OBESE DIABETIC MOUSE By MEI TANG A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by MEI TANG

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This dissertation is dedicated to my parents, my husband and my daughter.

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ACKNOWLEDGMENTS I would like to express my deep appreciation and grateful thanks to Dr. Sihong Song for his guidance and support during my Ph.D. study. The things I learned from him and his lab in the past four years will be appreciated and cherished my entire lifetime. I would also like to thank Dr. Mark Atkinson for his broad knowledge of diabetes, intelligent guidance and generous support. It has really been a wonderful experience to learn immunology and diabetes in his laboratory. Without their consistent assistance, I could never have finished my Ph.D. I would also like to thank the members of my supervisory committee, Dr. Jeffery Hughes and Dr. Sean Sullivan, for their support and valuable advice. I would like to thank the exchange students Tomas, Ena, Holger for their assistance and friendship. I would like to thank all the people in Dr. Song’s lab, Hong Li, Christian Grimstein, Dr. Bin Zhang, Dr. Young-Kook Choi and Dr. Yuanqing Lu for sharing everyday fun and their technical support. I am deeply grateful for the guidance of Clive Wasserfall and Todd Brusko in Dr. Mark Atkinson’s lab. Their technical support has been invaluable for me to complete my projects. I would like to thank Ke Ren and Dr. Edwin M. Meyer for their generous help with my project and their friendship. I would like to thank the secretaries in the Department of Pharmaceutics, Mr. James Ketcham, Mrs. Patricia Khan, and Mrs. Andrea Tucker, for their support. iv

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I would like to thank all my friends, especially Yaning Wang, Ke Ren, and Yan Gong, I appreciate their friendship and I enjoyed spending time with them. Lastly, I would thank my parents and my grandparents, my parents in-law, my brother and his wife, my husband and my little daughter. Without their love and support, I could never have finished this dissertation. v

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES.............................................................................................................x ABSTRACT.....................................................................................................................xiii CHAPTER 1 INTRODUCTION........................................................................................................1 Type 1 Diabetes............................................................................................................1 Classification of Diabetes......................................................................................2 Causes of Type 1 Diabetes....................................................................................4 Immune Characteristics of Type 1 Diabetes.................................................................7 NOD Mice as a Model for Type 1 Diabetes...............................................................12 Alpha 1 Antitrpsin (AAT) and Its Immunoregulatory Properties..............................14 Elafin...........................................................................................................................15 Therapeutic Strategies for Type 1 Diabetes................................................................16 Insulin Replacement Therapy..............................................................................16 Pancreas/Islet Cell Transplantation.....................................................................17 Islet Encapsulation...............................................................................................19 Implantable Insulin Pumps..................................................................................20 Gene Therapy......................................................................................................23 Recombinant Adeo-Associated Viral Vectors.....................................................24 Prevention Strategies..................................................................................................26 Aims of This Study.....................................................................................................28 2 MATERIALS AND METHODS...............................................................................31 Animals.......................................................................................................................31 ELISA Analysis of Protein Expression......................................................................31 Large Scale Preparation of Plasmid DNA for Packaging it into rAAV Vectors........33 Packaging of rAAV Serotype 1 Virus........................................................................34 Western Blot Analysis of Protein Expression............................................................35 Histopathology and Immunohistochemistry...............................................................36 Isolation of Splenocytes..............................................................................................37 vi

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Immune Profiling of Flow Cytometry........................................................................38 3 MOLECULAR CLONING OF RECOMBINANT AAV VECTORS.......................40 Construction of rAAV Vectors Expressing Mouse Alpha 1 Antitrypsin...................40 Construction of rAAV Vector Expressing Mutant Human Alpha 1 Antitrypsin.......47 4 EFFECTS OF TIMING AND DOSE OF RAAV1-CB-HAAT THERAPY ON PREVENTING TYPE 1 DIABETES IN NOD MICE...............................................49 Introduction.................................................................................................................49 Materials and Methods...............................................................................................50 Mice.....................................................................................................................50 Blood Glucose Analysis......................................................................................50 Flow Cytometry...................................................................................................53 Results.........................................................................................................................54 Effects of Dose and Time on hAAT Gene Therapy for Type 1 Diabetes Prevention........................................................................................................55 AAV1 Mediate Transgene Expression was Dose and Time Dependent.............56 Immune Response to hAAT................................................................................57 Effect of rAAV1-CB-hAAT Therapy on Insulitis...............................................58 Influence of AAV1-hAAT on the Frequency of Regulatory T Cells..................60 Discussion...................................................................................................................62 5 IDENTIFICATION OF THE FUNCTIONAL DOMAIN(S) OF ALPHA 1 ANTITRYPSIN FOR THE PREVENTION OF TYPE 1 DIABETES......................65 Introduction.................................................................................................................65 Methods......................................................................................................................66 Animal.................................................................................................................66 Vector Construction.............................................................................................66 Cellular Transfection and Transduction..............................................................66 Detection of hAAT and hNE Complex...............................................................67 Anti-elastase Activity Assay...............................................................................67 Construction of pCB-MouseAAT.......................................................................67 Generate Isoform Specific Antibodies................................................................68 Detection of Transgene Expression.....................................................................68 Results.........................................................................................................................69 Construction and Expression of a Human AAT Mutant without Reaction Center...............................................................................................................70 Interaction with Neutrophil Elastase...................................................................71 Humoral Immune Response to Human AAT and Hd(6)AAT.............................73 Construction and Injection of rAAV1 Vectors Expressing Mouse AAT Isoforms...........................................................................................................73 Immunohistochemistry........................................................................................77 Survival Curves of In Vivo Treated NOD mice...................................................77 Effect of rAAV1-CB-hd(6)AAT on the Insulitis Lesion....................................80 vii

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Splenocyte Proliferation Assay...........................................................................81 Discussion...................................................................................................................81 6 EFFECT OF ALPHA 1 ANTITRYPSIN ON NATURAL KILLER (NK) CELL KILLING ACTIVITY................................................................................................84 Introduction.................................................................................................................84 Materials and Methods...............................................................................................85 Results.........................................................................................................................86 Discussion...................................................................................................................86 7 CONCLUSIONS........................................................................................................90 LIST OF REFERENCES...................................................................................................92 BIOGRAPHICAL SKETCH.............................................................................................99 viii

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TABLE Table page 5-1 Study design for prevention of T1D in NOD mice (n=10/group)............................69 ix

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LIST OF FIGURES Figure page 3-1 Sequences of the DNA probes to identify 5 different isoforms of mouse AAT......43 3-2 Dot blot screening of isoform specific clones..........................................................44 3-3 Alignment of amino acid sequences of mouse AAT isoform 1 from the gene bank (PI-1) with the sequence of one of the clone of pCR2.1-TOPO-mAAT1 (Mouse AAT 23)......................................................................................................45 3-4 Alignment of amino acid sequences of mouse AAT isoform 2 (GB PI-2, gi|191844|gb|AAC28865.1|) from the gene bank with the sequence of one of the colonies of pCR2.1-TOPO-mAAT2 (mAAT27).....................................................46 3-5 Cloning of rAAV1-CB-mouseAAT.........................................................................47 3-6 Construction of rAAV-d(6)AAT vector...................................................................48 4-1 Injection scheme of rAAV1-CB-hAAT therapy in NOD mice to prevent type 1 diabetes.....................................................................................................................52 4-2 The rAAV1-CB-hAAT (A), rAAV1-CB-elafin (B) constructs................................55 4-3 Alpha 1 antitrypsin prevents type 1 diabetes in a dose dependent manner..............56 4-4 Effect of dose and timing of vector injection on transgene expression in NOD mice..........................................................................................................................57 4-5 Influence of dose and timing on local production of hAAT....................................58 4-6 AAV1-hAAT administration results in the production of anti-hAAT immune responses..................................................................................................................59 4-7 AAT reduces insulitis progression...........................................................................60 4-8 Pancreatic immunostaining for B220 and CD3 lymphocytes..................................61 4-9 Influence of AAV-AAT on T regulatory cells.........................................................62 4-10 Frequency of CD4+CD25+ cells in diabetic and non-diabetic NOD mice. (n=4/group)...............................................................................................................63 x

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5-1 The sequences of the peptides used to generate antibody which can be isoform specific and the common antibody can recognize all the isoforms of mouse AAT..........................................................................................................................68 5-2 293 cells were transfected with rAAV-CB-hAAT or rAAV-CB-hd(6)AAT plasmid, The concentration of hAAT or d(6)AAT in the serum free medium was measured by ELISA.................................................................................................70 5-3 Western Blot of human AAT and D(6) AAT from transfected medium with neutrophil elastase....................................................................................................71 5-4 Comparison of anti-elastase activity of human AAT and d(6)AAT........................72 5-5 Cohorts of female NOD mice were IM injected at 4 weeks of age with rAAV-CB-hAAT or rAAV-CB-hd(6)AAT (5x10 11 particles). Transgene product (wild type hAAT(B) and hd(6)AAT (A) were detected by ELISA...................................72 5-6 Antibody against human AAT in the NOD mice serum..........................................73 5-7 Rabbit anti-mouse AAT serum. ELISA was used to detect the anti-serum titer.....74 5-8 Mouse AAT expression in the serum compared with UF11.Rabit anti-mouse AAT1 serum was use as the antibody to detect AAT in the serum by ELISA........77 5-9 Immunostaining for mAAT1 in rAAV1-mAAT1 vector injected muscle using rabbit anti-mouse AAT (common region-KLH) antiserum.....................................78 5-10 Kaplan Meier survival curve for different treatment groups. Comparison of different groups administered with rAAV1-hAAT, rAAV1-mouseAAT, rAAV1-d(6)AAT ,rAAV1-GFP and saline...........................................................................79 5-11 Kaplan Meier survival analysis for different treatment group after CY injection. d(6)AAT injected mice 3 weeks after CY injection still completely abolished diabetes in NOD mice..............................................................................................79 5-12 Mutant hAAT, d(6)AAT attenuate insulitis lesion. A shows the percentage of islets at each stage in 3 different groups. B shows the number of islets at each stage in 3 different groups........................................................................................81 5-13 Comparison of splenocytes proliferation in d(6)AAT or PBS treated groups. ConA was used as the stimuli..................................................................................82 6-1 Experimental design for cytotoxicity assay.............................................................87 6-2 hAAT inhibits NK cell mediated cell killing. The effector cells were pretreated with hAAT for 1hr, before mixed with the effector cells were added. Note that the inhibition of hAAT on cell killing is dose dependent........................................87 xi

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6-3 Incubate human AAT together with effector cells and target cells for 3 hours. The target cells were protected from being killed by the NK cells..........................88 6-4 Pre-incubate the effectors with hAAT for 1 hour, then the cells were washed for 3 times with PBS. The target cells were added and co-incubated for 3 hours. There were no significant differences between treatments......................................88 xii

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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 ALPHA 1 ANTITRYPSIN GENE THERAPY AND ITS IMMUNOREGULATORY FUNCTION FOR PREVENTING TYPE 1 DIABETES IN NON-OBESE DIABETIC MICE By Mei Tang December 2006 Chair: Sihong Song Major: Pharmaceutical Sciences Type 1 diabetes is an autoimmune disease resulting in the destruction of pancreatic insulin producing cells that leads to insulin deficiency and hyperglycemia. Although the exact cause of this disease is not well understood, increasing evidence has shown that immuno-suppressive and anti-inflammatory agents can prevent type 1 diabetes. Alpha 1 antitrypsin (AAT) is a major serine proteinase inhibitor (serpin); we have recently shown that an early (4wks) intramuscular injection of recombinant adeno-associated virus (rAAV) vector expressing hAAT prevented type 1 diabetes in NOD mice. However, the mechanisms by which hAAT suppresses the autoimmunity and protects islets are largely unknown. The goals of this project are to understand the protective and immune regulatory function of hAAT in type 1 diabetes and to optimize AAV mediated hAAT gene delivery to a safe and efficient therapy in type 1 diabetes prevention. xiii

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In order to test the effect of the time and dose of hAAT gene therapy on the prevention of this disease, female NOD mice were IM injected at three different ages (4, 8 or 12wks of age) with rAAV1-CB-hAAT at three different doses (4x10 11 , 4x10 10 or 4x10 9 particles/mouse). We monitored blood glucose weekly until 32 weeks of age. The transgene expression was dose dependent. Prevention of type 1 diabetes was also dose dependent (30%, 100%, 80% incidence of diabetes respectively in high, medium and low dose rAAV1-hAAT treated group). The well-known function of hAAT is inhibition of a class of proteinases by the suicide interaction between its reaction center and the enzymes. However, it is unknown whether the reaction center is critical for the prevention of type 1 diabetes. We constructed a rAAV1 vector encoding a mutant human AAT (hAAT without the reaction center, 6 amino acid deletion, hd(6)-AAT), and tested it for the prevention of type 1 diabetes in NOD mice. In addition, we also tested rAAV1 vectors expressing mouse AAT for the prevention of type 1 diabetes in NOD mice. The rAAV1-d(6)AAT injection completely abolished type 1 diabetes development while saline or rAAV1-GFP injection did not. NK cell can kill the target cells through the perforin and granzyme (serine proteases) passway. Our in vitro experiments showed that the major serine protease inhibitor AAT can inhibit NK cell mediated cell killing. In conclusion, 1) rAAV1 mediated AAT gene therapy prevented type 1 diabetes in NOD mice in a dose dependent manner; 2) AAT without reaction center efficiently prevented type 1 diabetes; and 3) AAT blocked the NK cell mediated cell killing in vitro. xiv

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CHAPTER 1 INTRODUCTION This chapter discusses type 1 diabetes (T1D), the animal model for this disease, serum proteinase inhibitor alpha 1 antitrypsin and its potential role in treatment of type 1 diabetes. Type 1 Diabetes Type 1 diabetes mellitus affects about 1 in 300 people in North America and Europe. Epidemiological studies indicate that the incidence, and thus prevalence, of type 1 diabetes is rising worldwide. One out of every five healthcare dollars in the US is used to treat diabetes mellitus and its complications. The disease severely affects the quality of life of the patient and necessitates a dramatic change of habits, especially involving diet and social activity (Eisenbarth, G.S., and K. Lafferty, 1996). Diabetes mellitus is a disorder of glucose homeostasis manifested by uncontrolled hyperglycemia, caused either by an absence of the glucoregulatory hormone insulin, or by an insensitivity to the effects of insulin by tissues that normally take up glucose. Insulin is a dimeric protein produced by cells within the islets of Langerhans in the pancreas and is responsible for inducing cellular uptake and storage of glucose from the blood stream. In response to rising blood glucose levels, secretary granules within cells release preformed insulin. The insulin then binds to and stimulates glucose transporters located on the surfaces of muscle and fat cells. Upon insulin activation, these transporters facilitate the movement of glucose across their respective cell membranes. Therefore, any disruption in this metabolic process, either by inefficiency or a deficiency in insulin, will 1

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2 disrupt the necessary homeostasis of blood glucose levels, leading to the common symptoms of diabetes mellitus (Scobie, I.N.2002). In contrast to type 2 diabetes mellitus, which results from insulin resistance and relative insulin deficiency, type 1 diabetes mellitus is primary insulinopenic diabetes as defined in the 1997 American Diabetes Association classification scheme. Type 1 diabetes results from chronic autoimmune -cell destruction (Rossini, 2004). Approximately 10% of all cases of diabetes in westernized countries are type 1. Patients usually present with the classic, short-term clinical symptoms of polyuria, polydipsia and weight loss. With lack of attention to these symptoms, diabetic ketoacidosis may ensue. A majority of the long-term complications of T1D fall into four groups: cardiovascular disease, nephropathy, retinopathy and neuropathy. According to the latest statistics by the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), coronary heart disease is the leading cause of the diabetes-related deaths, accounting for approximately one-half of the deaths in T1D. Each year, diabetes represents the leading cause of end-stage renal failure, new asses of blindness each year, and lower limb amputations in the United States. Also, persons with diabetes are more susceptible to bacterial and viral infections, strokes, high blood pressure and periodontal disease. Given the number of complications associated with diabetes, it is not surprising that people afflicted with T1D have a 33% reduced life expectancy (Costantino, 1999). Classification of Diabetes While a formal classification system of diabetes was not established until 1979, distinction between various degrees of the disease has been evident as early as the nineteenth century. Prior to the isolation of insulin in the 1920’s, patients suffering from

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3 diabetes were diagnosed as having either of two types, based solely on the age of the onset of symptoms. Patients becoming symptomatic during childhood were classified as having juvenile diabetes. This type of diabetes was considered more severe and led to almost certain death within one year of diagnosis. In contrast, patients afflicted with diabetes later in life were diagnosed as having adult-onset diabetes. This form of the disease was usually not immediately life threatening and could be treated with a combination of diet, exercise and hypoglycemic agents (National Diabetes Data Group, 1979). After insulin becomes the standard treatment for type 1 diabetes, further classification of diabetes mellitus was established base on the varying requirements for insulin. As a result, diabetic patients were described as having either an insulin-dependent (IDDM) or non-insulin dependent diabetic mellitus (NIDDM). Although the basis for classification became more pathologic, it still appeared that the timing of onset was vital in the severity of disease (National Diabetes Data Group, 1979). Recently, the classification of diabetes mellitus has been further refined, favoring a system based on the underlying pathogenesis of the disease rather than on age of onset or insulin requirement. The new system, as established by the American Diabetes Association (ADA), the NIDDK, and the centers for Disease Control and Prevention (CDC), urges the use of Type 1 diabetes (T1D) in place of IDDM and type 2 diabetes (T2D) in place of NIDDM. (Expert Committee on the Diagnosis and Classification of Diabetes Mellitus, 2001). Two other types of diabetes were introduced that were formerly not considered. Type 3 diabetes was termed to classify diabetes resulting from all other factors not

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4 involving the other types of diabetes, while type 4 diabetes was named exclusively for gestational diabetes (Tilil,H., 1998) Causes of Type 1 Diabetes This disease is genetically determined and is triggered by environmental factors. The genetics of the disease primarily are located on the region of chromosome six known as the human leukocyte antigen (HLA). HLA antigens are surface glycoproteins that are responsible for presenting antigens to the immune system via the MHC complexes. The MHC class I locus codes for HLA types A, B or C that function to present antigens to the T cell receptor (TCR) of CD8+ lymphocytes. The MHC class II locus also codes for three subclasses known as HLA-DR, DQ and DP and function to present antigens to the TCR of CD4+ lymphocytes. A number of other loci have also demonstrated suggestive association with the disease. Among the MHCII molecules, it has been found that individuals who have certain HLA-DR or –DQ have an increased risk of developing T1D. Statistically, 95% of individuals with T1D have HLA-DR3 or DR4 or both. Specifically, it has been shown that those with HLA-DQB1*0302 locus have the highest risk of developing T1D (Bowman,M.A. and M.A.Atkinson, 1996). Despite the strong correlation between T1D and HLA type, correlation of disease is imperfect as 50% of the non-diabetic populations are also positive for these genotypes (Baquerizo,H., 1989). Additionally, the loci DQB1*602, located within the same locus as the diabetogenic loci, has been shown to have a potential protective phenomenon (Gaillat-Zucman,S.,1996). HLA genotype is not the only genetic determinant related to T1D development. Several other genetic loci have been implemented as potential factors involved in T1D development. Each new discovery is given a name of IDDM with a number preceding it signifying the order in which it was discovered. HLA, it being the first genetic locus

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5 related to diabetes development, was therefore named IDDM1. The regions outside of the IDDM1 locus have been found not to be as important immunologically as the HLA region, but enough polymorphisms in certain genes could lead to the development of autoimmunity. IDDM2, for instance, is a locus located on the 5’ region of the insulin gene that has a series of variable number of tandem repeats (VNTR). The 5’ VNTR has been shown to fold into a conformation that can be recognized as a foreign antigen by the immune system resulting in an immune response to insulin. The ever-increasing list of IDDM loci has reached 20, and is continually being added to as a result in T1D humans and animal models. Overall, the exactness of a genetic predisposition to develop T1D has not been completely convincing. This data further proves that genetics alone can not be used as an independent marker for diabetes development. However, the genetic correlation among diabetics suggests that genetic screening can be a useful tool in helping to predict those at risk for disease. Additionally, based upon what is known of the loci involved in T1D, further understanding of the predisposing genetic factors may help explain how T1D develops (Lyons,P.A. and L.s.Wicker., 1999, Froguel.P.,1997). The environmental factors include diet and viral infections. The autoimmune destruction of the cell has been linked to dietary of nutritional factors. Epidemiological studies show that the incidence of T1D is significantly different in geographic and temporal areas for those with the genetic predisposition for diabetes. Obviously, due to cultural and environmental differences, the intake of certain foods and vitamins vary greatly; the importance of quantity of food, what type of food and when that food is eaten are all considered variables in determining the risk of T1D development. High interest, Swedish scientists have shown a link between the duration of breast-feeding and

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6 increased risk of T1D. This study shows that the early introduction of cow’s milk proteins to infants less than 3 months of age have higher risks of developing T1D diabetes in contrast to those who breast-feed longer than three months (Van Beresteijn, E.C., 1996). The between cow’s milk proteins and the autoimmune effect triggering T1D is unknown; however, Elliot et al performed studies showing that bovine serum albumin (BSA) has a similar amino acid homology with certain MHC II HLA-DQ and –DR haplotypes, and that antibodies formed against BSA can cross react with certain cell antigens. Thus, the possible introduction of certain bovine proteins at ages younger than three months can trigger autoimmune diabetes through possible cross reactivity. Consumption of nitrosamines, nitrates and/or nitries may also lead to the development of T1D. Nitrosamine is toxic to -cells in a dose dependent manner. Thus, as mentioned before, free radicals from -cells can result in cross reactivity and initiation of the autoimmune process (Virtanen, S.M., 1994). Vial-induced diabetes has been suggested on several accounts, but there has only been empirical evidence of the viral-induced mechanisms triggering autoimmune diabetes. The increased incidences of T1D of individuals who are afflicted with the viruses rubella, cytomegallavirus, herpes, mumps and enteroviruses prior to disease development have helped substantially this claim. The most prevalent and widely studied scenario is that of the enterovirus family of coxsackieviruses (CSV), specifically that of the Class B viruses (Froguel,P.,1997). Several therories have been put forth trying to explain the mechanism by which CSV initiates autoimmunity. One heavily favored theory, is the possibility of molecular mimicry between the P2-C protein of CBV serotype 4 and glutamic acid decarboxylase (GAD). The P2-C protein codes for a viral

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7 protein used for replication that coincidentally has seven amino acid homology with the GAD self-antigen. This suggests that when the immune system is primed against the P2_C antigen after infection that there is a possible cross reactivity of the self-antigen GAD. In fact, there have been reports showing that peripheral blood mononuclear cells from some T1D will mount an immune response against the P2-C peptide. Moreover, studies have linked specific CSV serotype infect ability to HLA-DR and HLA-DQ genotypes suggesting a predisposed higher susceptibility to infection. Another possible theory in regard to the initiation of autoimmunity by viral toxicity is the hit and run theory. This theory suggests that the autoimmune process is initiated or exacerbated by chronic or acute viral infection of the pancreas as the result of the lytic cycle that some viruses possess or by necrosis and phagocytosis of the infected cell. Although viral infections are often transient, it is believed that the lesion resulting from viral infection can result in the initial release and/or presentation of lesion resulting from viral infection can result in the initial release and/or presentation of the autoreactive cellular contents of cells. This scenario, as mentioned previously, would only affect those who have a genetic predisposition for developing diabetes as those not having a genetic predisposition only suffer from minor cell death due to the viral toxicity. One obvious avenue that should be considered to prevent infection by known diabetogenic viruses is the use of immunization. Immunizing those at high-risk for T1D could mean the difference from disease onset and disease-free (Falke,D.,1988). Immune Characteristics of Type 1 Diabetes Autoimmunity is characterized by the immune-mediated destruction of self-tissue as a result of a failure to discriminate self-antigens from foreign antigens. Self-tolerance is established in the thymus where T cells go through an educational process known as

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8 positive and negative selection. Normally, autoreactive cells are clonally deleted or rendered nonreactive when introduced to self-antigens. In the case of type 1 diabetes, studies of animal models have suggested that negative selection process does not take place for certain cell antigens. The autoimmune response against cell antigens in T1D includes both cellular and humoral responses. The primary autoimmune destruction of cells is thought by most to occur via a T cell mediated response. T cells are grouped into two primary categories: CD8+ and CD4+. CD8+ cells, termed cytotoxic T lymphocytes, are responsible for cell mediated immunity, and possess the ability to kill any cell expressing a certain peptide: major histocompability complex (MHC) class I combination. A key function of CD8+ effector cells is to kill cells that are infected by virus and secrete anti-viral cytokines. CD4+ cells are specialized lymphocytes that activate antigen presenting cells (APC); including B cells and marophages containing specific peptide: MHC class II complexes. CD4+ cells are responsible for humoral immunity, activating APCs, and can be further classified depending upon their cytokine secretion profile. Based on their pattern of cytokine production and their functional responses T lymphocytes are subdivided into those that participate in cell-mediated immune responses such as inflammation, delayed type hypersensitive reactions, and macrophage activation (i.e., Th1 subset), from those releasing cytokines that induce B lymphocytes to secrete antibodies (i.e., Th2 subset). Since the original definition of the Th1/Th2 clones, several additional cytokines have become associated with each subset; such that Th1 cells are defined by their production of interferon gamma (IFN) and tumor necrosis factor alpha (TNF), while Th2 cells produce interleukin (IL) such as IL-4, IL-6, IL-10, and IL-13.

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9 T regulatory cells (Treg) with the CD4+ and CD25+ phenotype are known suppressor cells, and have been implicated in diabetes prevention. This CD4+ T lymphocytes subset constitutes 5-10% of peripheral CD4+ T lymphocytes and is capable of inhibiting the responses of CD4+CD25and CD8+ T lymphocytes in vitro and in vivo (Marie, J.C., 2005). CD4+CD25+FoxP3+ regulatory T cells play a major role in the maintenance of immune tolerance to self and in the control of autoimmunity (Sakaguchi, S, 2001). Treg inhibit autoimmunity in a number of experimental models, including T1D. They have been shown to prevent onset of autoimmunity by being involved in regulation of effector T lymphocyte homeostasis. In terms of effector function, it appears that naturally occurring Treg in the immune system produce IL-10 and IL-4 to mediate their suppressive activity of autoimmune cells. In addition to this, Treg are able to interfere with APC-effector T lymphocyte engagement and weaken the T cell activation, leading to antigen specific anergy. CD4+/CD25+ suppressor cells express the CTLA-4 molecule, known for its anergy inducing capabilities through the B7.2 and .2 receptors, are 5-6 fold less prevalent in NOD mice in comparison to other mouse models. Alternatively, recent data suggests that the ability of CD4+CD25+ Treg ability to suppress in NOD mice is time dependent within the autoimmune response and to suppress CD4+CD25T effectors is time dependent. Therefore it has been proposed that it will be necessary for a true therapy for the prevention of T1Dto control T lymphocyte responses by reducing the T effector response and to increase the Treg response. Experiments with polyclonal T lymphocyte deactivators such as anti-CD3 antibody (Walker, M.R., 2003) and low dose of nominal antigen (Apostolou, I., 2004) have been shown to induce the development of FoxP3+ CD4+CD25+ T lymphocytes in normal and NOD mice and reduce the incidence

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10 of T1D. Further more, diabetes prevalence in NOD mice is greatly reduced when additional CD4+/CD25+ cells are introduced. Thus, the lack of regulation due to T-cell deficiencies and/or irregularities can provide possible explanations as to the development of immunoresponses against cell-specific antigens. Autoreactive-lymphocyte activation is initiated by adaptive immune response mechanisms (Rohane, P.W., 1995). Unlike that of the innate immune system, adaptive immunity can produce a sustained immune response against antigens that are recognized as foreign. Naive or unstimulated lymphocytes are not reactive until they encounter their specified antigen via the T cell receptor and receive the necessary co-stimulatory factors. Once a lymphocyte receives the necessary stimulatory signals, it undergoes clonal expansion proliferating into both effector and memory cells reactive against a certain antigen. In the case of T1D, it is believed that cell-antigen stimulation is a result of cell destruction resulting from environmental stresses that leads to the development of anti-islet effector cells. Subsequent priming and expansion of autoreactive cells occurs during insulitis when primarily CD8+, and to a lesser extent, CD4+, macrophages, Natural Killer and B cells are found clustered in and around the islet. However, once the effector populationis created, this population no longer needs secondary signals in order to maintain their function. In T1D, CD8+ effector cells are primed against cell antigens, and maintain their cytotoxicity without the need for secondary activation. This is evident as CD8+ effector splenocytes from diabetic NOD mice can transfer disease at a rapid rate when injected into NOD.scid mice (a genetically identical mouse strain that cannot produce lymphocytes).

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11 –cell specific cytotoxic effector cell populations alone do not fully explain the immune process involved in T1D. Studies have shown that T1D requires a prolonged immune reaction against cells, and possible constant activation of cytotoxic cells in order to result in sufficient cell death causing T1D (Szelachowska, M., 1998). The insulin-producing cells are a part of the pancreatic endocrine tissue known as the islet of Langerhans. In addition to cells, islets also contain glucagons-producing cells, somatostatin-producing cells, autoimmunity in T1D targets specifically antigens. Indeed, most patients with T1D possess evidence of cell specific reactions by the presence of autoantibodies and autoreactive T cells against cell antigens. Many lines of evidence indicate that antigen-presenting cells (APC), especially dendritic cells (DC) are pathologically active in orchestrating the process of insulitis(Bottino,. 2003). APC within islets respond to micro-environment triggers including -cell death and impaired -cell apoptosis, and initiate the insulitis process by migrating out of the islet and into the peripheral pancreatic lymph nodes. APC trigger the activation and proliferation of -cell reactive T cells. The activated T cells infiltrate and destroy islet cells resulting in type 1 or insulin dependent diabetes (Hui, 2004). Homeostatic control of -cell mass in normal and related pathological conditions is based on the balance of proliferation, differentiation, and apoptosis of these cells. Stimulation or induction of -cell apoptosis leads to the development of type 1 diabetes. It has been shown that both direct cytotoxic T cells mediated and indirect cytokine-dependent (i.e., IL-1, TNF-, INF-) mechanisms are responsible for -cell apoptosis. The cytotoxic mechanism involves the release of cytotoxic granule contents (perforin and granzymes) into the intercellular space between CTL and the target cells. The target cells

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12 can uptake granzymes efficiently and rapidly by receptor-mediated endocytosis. Granzymes can also enter the target cells through cell surface pores formed by perforin. Granzymes play a critical role in triggering apoptotic cell death through the mitochondrial pathway or by the activation of cellular caspases. Therefore, it is possible that serine proteinase inhibitor AAT can inhibit granzymes, and thus block CTL mediated -cell killing and protect islet cells. A great body of evidence indicates that cytokines, secreted by islet-infiltrating cells (T cells, B cells, NK cells and macrophages), play an important role in the pathogenesis of type 1 diabetes. Several studies have shown that IL-1 along or in combination with TNF-, INFleads to islet cell dysfinction and death (Constant, S.L., 1997). The signal transduction by these cytokines involves interaction with specific receptors, activation of MAPK pathways, mobilization of transcription factors and upregulation or down regulation of down stream gene transcription. The mechanisms by which cytokines destroy cells are complex and under active investigation. It is possible that (the) cytokine induced cell death also involves caspase activation. NOD Mice as a Model for Type 1 Diabetes The NOD mouse is a well-accepted spontaneous model used to investigate both disease pathology and intervention strategies to prevent human type 1 diabetes (Atkinson and Maclaren 1994; Bach 1994; Delovitch and Singh 1997). NOD mice were serendipitously discovered in Japan when a group of scientists crossbred two strains of mice for during a cataracts study. 80 percent of the female offspring, compared to 10 percent of the males, manifested symptoms parallel to that of human T1D. Not only do these mice possess the clinical manifestation of diabetes, they also have a genetic and

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13 autoimmune process similar to that of T1D in human. Due to the extreme inbreeding of the NOD mouse, many scientists consider experiments using NOD mice to be one case study. Be that as it may, these mice possess similar genetic predisposition for diabetes development and autoimmune processes that are present in human T1D development. Since the similarities of the NOD and human T1D are numerous, this model may help to elucidate the beginning at approximately five weeks of age, a mononuclear cell infiltrates of the pancreatic ducts and venues initiates with eventual progression to the pancreatic islets (i.e., insulitis). Whereas these early insulitis stages appear non-destructive, intra-islet invasion occurs at 12-16 weeks of age with this latter infiltrate associated with selective destruction of the insulin-secreting -cells. The cellular infiltrate is heterogeneous, with a predominance of T cells followed by various percentages of macrophages, dendritic cells, and B-lymphocytes. Multiple lines of evidence suggest that both CD4+ T-helper and CD8+ T cells play a role in the disorder (Like, Biron et al. 1986; Bendelac, Carnaud et al. 1987; Sibley and Sutherland 1987; Wang, Hao et al. 1987; Haskins, Portas et al. 1988; Miller, Appel et al. 1988) . Although multiple factors are thought to contribute to this disease, an imbalance of the immune regulatory pathways appears to play an important role in the disease development. Therefore, immune regulatory approaches hold great potential for the prevention of this disease. Recent studies have shown that increases of CD4+CD25+ regulatory T cells, as well as blockade of blockade of CD8+ T cell receptor (NKG2D), can prevent autoimmune diabetes in the NOD mouse model of type 1 diabetes.

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14 Alpha 1 Antitrpsin (AAT) and Its Immunoregulatory Properties AAT is the major serine protease inhibitor (serpin) of human plasma with a molecular weight of about 52kDa. Human Serum concentration of AAT is about 1-2mg/ml, and it has a half-life of about 5-6 days. AAT has an extraordinarily broad range of enzyme inhibitory activity, it can inhibit neutrophil elastase, pancreatic elastase and proteinase 3 with high efficiency, and cathepsin G, thrombin, trysin and chymotrypsin with lower efficiency (Ray, Desmet et al. 1977; Boskovic and Twining 1998; Johansson, Malm et al. 2001). These enzymes are directly responsible for the majority of the tissue destruction in inflammatory response due to their effects on substances such as elastin, cartilage and structural collagen, basement membrane, fibrin, and fibronectin. Additionally, they generate chemotactic factor of IgG, and mediators such as complement, kininogen, and angiotensinogen and activate other cells such as lymphocytes and monocytes. It is known that the mean AAT concentration and function were lower in the non-smoking young insulin-dependent diabetic subjects. It would therefore appear very likely that in type 1 diabetes, with reduced concentration and impaired function of AAT, the decreased inhibition of these enzymes would result in increased islet injury and indirect injury through activation of other cells and mediator systems. This tissue destruction, through the production of increased amounts of altered self antigens, may perhaps also provide extra impetus for the development of autoimmune diseases. Abnormalities of lymphocyte function in AAT deficient patient may well be of relevance in determining its association with immune disorders. AAT deficient subjects exhibit marked serum-mediated enhancement of lymphocyte responsiveness to PHA especially at suboptimal dose (Ranes, J., 2005). AAT appears to have a similar but

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15 somewhat less marked effect on Con A responses but no effect on pokeweed mitogen (PWM)-induced proliferation (Ranes, J., 2005). AAT is able to inhibit the cytotoxic reactions of lymphocytes including antibody-dependent cell-mediated cytotoxicity, T-cell-mediated cytotoxicity, and natural killer activity. The latter possibly is due to interaction with an elastase-like enzyme on the cell surface. It is still unknown if the inhibition of these cytotoxic functions is due to an effect on the activation of the cytotoxic cell or due to interaction with substance mediating target cell destruction. AAT may prevent diabetes because the inhibitor prevents inflammatory cytokine production, prolongs islet allograft survival in mice, inhibits a variety of immune responses, including mixed lymphocyte reaction, mitogen-stimulated lymphocyte proliferation, T cell mediated cytotoxicity (Ranes, J., 2005). Elafin Elafin (neutrophil elastase inhibitor, a 6-kDa peptide) was originally isolated from the scales of patients with psoriasis(Wiedow, Schroder et al. 1990) and in lung secretions(Sallenave and Ryle 1991; Tremblay, Sallenave et al. 1996), but it is also present at mucosal sites in many tissues. It is present in sputum, in tracheal biopsies and bronchoalveolar lavage from both normal subjects and the patients with lung inflammation. Elafin is synthesized by Clara cells and type II cells in lung. Elafin levels were highly correlated with lung inflammatory cell numbers, especially lymphocytes(Tremblay, Sallenave et al. 1996). It has recently been observed that macrophages also express elafin. The sequence of the gene showed that it is approximately 2.3 kb long, and is composed of three exons and two introns. The 5’ regulatory sequences contain activator protein-1 and nuclear factor-B sites. A positive

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16 regulatory cis-element present in the region between and bp is responsible for the upregulation of the elafin gene in normal breast epithelial cells. The peptide is composed of 117 amino acid residues including a hydrophobic signal peptide of 22 residues. Elafin can be divided into two domains, the carboxy-terminal domain containing the antiproteinase active site and the amino-terminal domain containing characteristic VKGQ sequences. These sequences allow the elafin molecule to glue itself into polymers and bind other interstitial molecules through transglutamination. This feature could make elafin maximally effective as a tissue-bound inhibitor. Elafin has also been suggested to have a locally protective role against neutrophilic damage, presumably because of its small size and negative charge. Elafin has been shown to be more specific in its spectrum. It inhibits pancreatic elastase, neutrophil elastase and proteinase-3. Therapeutic Strategies for Type 1 Diabetes At present, insulin replacement therapy is the first line treatment for T1D. Other experimental efforts to reverse disease include: pancreas/islet cell transplantation, islet encapsulation, implantable insulin pumps, Immunosuppressant therapy, gene therapy. Each has advantages and disadvantages which will be discussed in the following section. Insulin Replacement Therapy Insulin is the standard treatment for type 1 diabetes. Dr.Gred Sanger won a Nobel Prize in 1953for using insulin in the first-ever experiment where the amino acid sequence of a molecule was elucidated. The success of in vitro production of insulin opened up a new field of science where recombinant DNA is used to produce proteins for research and pharmacological purposes. Since the use of insulin as a treatment for diabetes mellitus, the acute-mortality of the disease has dramatically reduced, particularly in type 1 diabetes. However, the extensive list of chronic complications associated with the

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17 disease combine not only to enhance morbidity, but also to cause accelerated and premature mortality, still manifest even with insulin therapy. Chronic complications of diabetes result from the loss of glucose homeostasis and usually develop 10 to 15 years following the onset of symptoms. The primary function of insulin is to regulate the body’s level of glucose in the blood. Insulin is a highly conserved 6 kd protein synthesized as a preprohormone known as preproinsulin. Upon translocation of the preprohormone into the endoplasmic reticulum (ER), the signal region is cleaved off to produce proinsulin. Proinsulin is then further reduced to C-peptide and the active form of insulin comprising alpha and beta subunits linked by disulfide bonds. C-peptide and insulin are then packaged into secretary granules within the golgi and are stored in the cell’s cytoplasm. The secretary granules consist primarily of C-peptide and active insulin; however, there is a small percentage of inactive proinsulin present. Insulin granules are released into the blood stream by unknown mechanisms but empirical data suggest that serum glucose levels regulate this process. One theory explaining this phenomenon suggests that elevated intracellular glucose levels leads to an increased intercellular ATP/ADP ratio resulting in the opening of K+ channels and increasing calcium influx. Increased intracellular calcium concentration then leads to the degranulation of the cells and exocytose of insulin and C-peptide. Pancreas/Islet Cell Transplantation Transplantation of pancreas or purified islet cells have shown the potential to restore islet cells the insulin secretary responses to metabolic needs, correct glucose homeostasis, and thus limit the incidence and severity of the degenerative complications that are commonly seen in diabetic patients on insulin treatment. Over the past 20 years,

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18 the surgical procedures for whole-pancreas transplantation have remarkably improved. This technique can now be performed with one-year success rates that are close to those for kidneys alone or for other solid organs. Pancreas graft survival after one year is 82%, 74%, and 76% when the organ is implanted after the kidney, simultaneously with the kidney, or alone, respectively. The majority of one year-surviving grafts maintains functioning cells for a decade. Pancreas graft survival is almost invariably associated with normalized glucose levels and states of insulin independence, which also improve the quality of life for the recipients. However, these benefits do not occur without risks. First, the surgical procedure is still technically cumbersome and associated with a high morbidity. Moreover, the need for chronic immune suppression carries the same infectious and tumorigenic risks as other organ transplantations. The decision for pancreas transplantation is therefore usually delayed until late in the course of diabetes when renal failure raises the need for a kidney graft. At this later stage, simultaneous implantation of a pancreatic organ will not bring a major benefit in terms of prevention of secondary complications, as these are already advanced to an irreversible level. Islet transplants have long since been proposed as a safer alternative to whole-organ transplants. Studies in rodents have demonstrated that islet grafts can be implanted in different sites using simple techniques. In several models they correct diabetes, rapidly and for extended periods. For many years, none of these promising features could be reproduced in T1D patients. The reasons were probably multiple, ranging from technical difficulties in preparing viable and metabolically adequate grafts to biologic obstacles of inflammatory and immune nature. Over the years, Progess has been made in the isolation of human islet tissue and its use in auto-and allo-transplantations. Human islet grafts were

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19 shown to correct T1D in patients who had received a donor kidney prior to, or simultaneously with, the islet graft. The gain in safety is, however, associated with a less favorable donor-to-recipient ratio. In the recently successful series of islet transplantations by the Edmonton group (i.e., showing 90% restoration of euglycemia at one year), metabolic correction required islet preparations from two to four pancreas that were procured under optimized conditions. The shortage in donor organs, and in particular those that are procured under the better conditions, is currently a major limitation to the clinical future of islet cell transplantation. This combined with the same immune problems of rejection that plague whole pancreas transplantation, make the future use of islet transplantation difficult to conceptualize as a mass answer to a major therapeutic need. Islet Encapsulation A bioartificial pancreas has the potential as a promising approach to preventing or reversing complications associated with this disease. Bioartificial pancreatic constructs are based on encapsulation of islet cells with a semipermeable membrane so that cells can be protected from the host’s immune system. Encapsulation of islet cells eliminates the requirement of immunosuppressive drugs, and offers a possible solution to the shortage of donors as it may allow the use of animal islets or insulin-producing cells engineered from stem cells. During the past 2 decades, several major approaches for immunoprotection of islets have been studied. The microencapsulation approach is quite promising because of its improved diffusion capacity, and technical ease of transplantation. It has the potential for providing an effective long-term treatment or cure of Type 1 diabetes.

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20 The successful development of a bioartificial pancreas involves several considerations. Two major obstacles in islet transplantation are the limited supply of islet cells and the use of immunosuppressive drugs to prevent transplant rejection. It is hoped that the use of encapsulated islets as a form of bioartificial pancreas would overcome these obstacles. Three potential sources of islet cell tissue, including human or allogeneic cells, porcine or xenogenic cells, and engineered cells, are currently under investigation.( Kizilel, S 2005) In summary, a potential cure for Type 1 diabetes could be the use of bioartificial pancreatic constructs based on insulin-secreting cells (allogeneic or xenogeneic islet cells) that are immunoisolated with the microencapsulation technique. Additionally, enhanced survival of the graft might be supported with a novel approach to induce neovascularization, which could make this technology a clinical reality in the near future. Implantable Insulin Pumps Over a lifetime, the Diabetes Control and Complications Trial (DCCT)-defined intensive therapy using Continuous Subcutaneous Insulin Infusion (CSII) reduces complications, improves quality of life, and can be expected to increase length of life. From a health service perspective, intensive therapy is shown by the DCCT to be 'well within the range of cost-effectiveness seen to represent good value.' These figures do not include any cost savings accrued by delaying the progression of complications by an overall 60%, as was found by the DCCT intensive management group. These savings are potentially enormous, without considering improved quality of life for those who are not blind, on kidney support, or who have not suffered limb amputation. Cardiovascular and psychological treatment savings were also not included two significant potential savings (DCCT Research Group, November 1996).

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21 In a recent American Diabetes Association abstract by Dr. Hirsch, a group of 107 CSII patients studied yielded the following results: (1) A reduction in HbAlc from 7.6% to 7.1%; (2) A reduction in severe hypoglycaemia from 73.2 to 19.2 episodes/100 pt. years. Dr. Hirsch concludes that in the near future as more data is available regarding the advantages of CSII therapy, Endocrinologists may need to re-think their position on prescribing CSII. Crawford, Sinha, Odell et al. (2000), investigated the effect of CSII on plasma glucose to identify factors associated with improved glycaemic control in Type I diabetics. 9 adults aged between 30 and 58 were followed-up after changing from MDI to CSII. relative to changes in weight; insulin requirement and HbAlc. Crawford et al. reported results of a daily insulin requirement reduction from an average 45.2 units to 37.1 units; HbAlc average from 8.4% to 7.7%; and an insulin-to-weight ratio from 0.66 u/kg to 0.53 u/kg. The bolus-to-basal ratio on MDI was 1:2, on CSII this was 1:1 at follow-up. The shift in ratios is associated with better glycaemic control. Therefore, Crawford concludes CSII therapy in patients with Type 1 diabetes improves glycaemic control and lowers the total basal insulin dose, without affecting the patient's weight. (Renard 2004; Catargi B. 2004; Hovorka R. 2006). Stem/Progenitor Cells The considerable genetic manipulations that are required to convert non-cells into efficient glucose-sensing, insulin-secreting cells have led other investigators into considering means of expanding adult or neonatal -cells or of harnessing the developmental potential of islet precursor cells and embryonal stem cells. However, despite the culture conditions and manipulations, commitment to -cells and insulin

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22 production has not always been consistent. Much excitement has also surrounded observations that adult stem cells from bone marrow or from other tissues could 'transdifferentiate' into a number of other lineage-different cell types. Such stem cells have been described and sometimes physically isolated in the nervous system, pancreas, epidermis, mesenchyme, liver, bone, muscle and endothelium. Hematopoietic stem cells, in some studies, were proven able to yield endothelial, brain, muscle, liver and mesenchymal cells. In some studies, hematopoietic cells could also be generated from neuronal or muscle stem cells. A number of issues, however, have tempered the enthusiasm with which these observations were initially greeted. The contamination of hematopoietic stem cells with mesenchymal precursors or the programming by growth factors in culture, and more recently, the phenomenon of fusion of stem cells with tissue cells are perhaps the most important variables to better test. Recent developments, however, strengthen the belief that mesenchymal cells in bone marrow may be a multipotent source of cells. This characteristic can be exploited; however, there are no data on whether such cells can be differentiated along the islet and -cell lineage. Clearly, the ability to manipulate blood-borne progenitors into the -cell lineage should provide a significant breakthrough for surrogate -cell technology as insulin replacement.( Hovorka R. 2006; Melton DA. 2006) Despite the current controversy and the serious ethical issues raised by cloning technology, it is likely that therapeutic cloning, under strict and defined conditions, will find its place in stem cell therapies. In this regard, one possible means of propagating cells or progenitors while avoiding the complications involved with the immune response could entail the removal of DNA or nucleus from somatic cells of a patient, its transfer

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23 into an enucleated embryonal stem cell, and its expansion into an appropriate -cell lineage. While this remains highly speculative at present, the rapid pace of basic work in this area, despite restrictions, will likely yield insight into such manipulations (Lakey JR 2006). Immortalization of islet cells with a -cell phenotype has been attempted and successfully achieved. Insulin production, however, seems to be linked to terminal differentiation of the cell, an event normally reached with growth arrest. This problem has so far limited the utility of cell immortalization. Also, this approach carries with it the possibility of oncogenic transformation. Although still controversial, there are data indicating that mature human cells can be induced to replicate under the effects of hepatocyte growth factor (HGF). The limitation of this approach, however, rests on the loss of differentiation of the induced -cell along with a substantial decrease in insulin production. Conditional replication of non-human cells has been achieved by placing the SV-40 T antigen under the control of an inducible promoter. In these studies, cells were able to replicate and to maintain differentiated function under inducible conditions. No data exist on whether such an approach is feasible in human cells ( Pileggi A 2004; Trucco M. 2006). Gene Therapy Introducing new genes into cells has the potential to correct defects in several major diseases, including cystic fibrosis, cancer, and cardiovascular disease. But cells do not easily take up foreign genes. One of the biggest challenges to using gene therapy for treating or curing diseases is finding a way to deliver genes safely into the cells where they're needed (Goudy KS 2006).

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24 Viruses are naturally evolved vehicles which efficiently transfer their genes into host cells. This ability made them desirable for engineering virus vector systems for the delivery of therapeutic genes. The viral vectors recently in laboratory and clinical use are based on RNA and DNA viruses processing very different genomic structures and host ranges. Particular viruses have been selected as gene delivery vehicles because of their capacities to carry foreign genes and their ability to efficiently deliver these genes associated with efficient gene expression. These are the major reasons why viral vectors derived from retroviruses; adenovirus, adeno-associated virus, herpesvirus and poxvirus are employed in more than 70% of clinical gene therapy trials worldwide. Among these vector systems, adeno-associated virus vectors represent the most prominent delivery system, since these vectors have high gene transfer efficiency and mediate high expression of therapeutic genes with modest immune response (Giannoukakis N 2005). Recombinant Adeo-Associated Viral Vectors Adeno-associated virus (AAV) is a single-stranded DNA parvovirus with a 4.7 kb genome and a particle diameter of approximately 20nm. The AAV genome is flanked by two identical inverted terminal repeat (ITR) sequences (Lusby, 1980)These ITRs provide all the cis-acting sequences required for replication, packaging and integration (Samulski,1989). There are two large open reading frames (Srivastava, Lusby et al. 1983). The open reading frame in the right half of the genome (cap) encodes 3 overlapping coat proteins (VP1, VP2, VP3 ). The open reading frame in the left half (rep gene) encodes 4 regulatory proteins with overlapping sequences which are known as Rep proteins (Rep 78, Rep68, Rep52 and Rep40), because frame shift mutations at most locations within the open reading frame inhibit viral DNA replication (Hermonat, 1984). The Rep proteins are multi-functional DNA binding proteins. The functions of the Rep

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25 proteins in viral DNA replication include helicase activity and a site-specific, strand-specific endonuclease (nicking) activity (Ni, Zhou et al. 1994). AAV infects a broad spectrum of vertebrates from birds to humans, although in nature specific types are species specific (Berns and Giraud 1996). In humans AAV can infect a large variety of cells derived form different tissues. The infection of AAV is ubiquitous within the population with about 90% of adults being seropositive. In spite of its omnipresence, AAV has never been associated with any human disease. In this sense, rAAV is the safest of the currently used gene therapy vectors. Because of its propensity to establish latency and because it has not been implicated as a pathogen, AAV has been of considerable interest as a potential vector for human gene therapy (Flotte and Ferkol 1997). In general, rAAV vectors are produced by deleting the viral coding sequences and substituting the transgene of interest under control of a non-AAV promoter between the two AAV ITRs. When the rep and cap proteins are expressed in Ad-infected cells, rAAV genomes can be efficiently packaged. Unlike adenovirus vectors, rAAV vectors are remarkably nonimunogenic with little host response (Jooss, Yang et al. 1998; Song, Morgan et al. 1998). In addition to all above unique features, rAAV vectors have mediated long-term transgene expression in a wide variety of tissues, including muscle(Kessler, Podsakoff et al. 1996; Xiao, Li et al. 1996; Clark, Sferra et al. 1997; Snyder, Spratt et al. 1997; Song, Morgan et al. 1998), lung(Flotte, Afione et al. 1993), liver(Snyder, Miao et al. 1997; Xiao, Berta et al. 1998; Song, Embury et al. 2001; Xu, Daly et al. 2001), brain (Kaplitt, Leone et al. 1994) and eyes(Flannery, Zolotukhin et al. 1997). Thus rAAV vectors appear to have significant advantages over other commonly used viral vectors

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26 Prevention Strategies In order to prevent the disorder, one must be able to identify first with a sufficient degree of confidence individuals who are at very high risk for developing type I diabetes. While inheritance of susceptibility alleles at loci linked to andor associated with the disorder is an important risk factor, it alone cannot guarantee that the individual will in fact become diabetic. This is the main reason for the ongoing debates on prevention based on genetic screening. While outright prevention based only on genetic screening may not be yet acceptable, other strategies that fall inside the realm of 'prevention' can be acceptable. There is data indicating that newly onset diabetics still possess adequate -cell mass to sustain normoglycemia if the autoimmune inflammation can be promptly controlled. The time between diagnosis and elimination of -cell mass adequate to sustain normoglycemia has been termed the 'honeymoon' period. One can exploit immunoregulatory networks to promote hyporesponsiveness of autoaggressive immune cells in this period as a viable means of improving or restoring normoglycemia. Supporting this approach are the studies where treatment of newly onset diabetic NOD mice with an anti-CD3 antibody restored normoglycemia in a substantial portion of mice for a sustained period of time. Very recently, human trials using the same approach also seem quite promising. Although clinical diabetes onset has most often been associated with -cell death, it is possible that the low levels of insulin production are because of the effects of cytokines that modulate their production. If this is the case, this process can be reversed. Some data strongly suggest that suppression of the activity of the insulitic cells by the induction of immune hyporesponsiveness in clinically diabetic individuals may

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27 promote either -cell neogenesis andor rescue of the cytokine-suppressed cells in the insulitic environment. Inherent in this philosophy is the ability to promote TIDM-specific autoantigen tolerance or TIDM-specific autoantigen immune hyporesponsiveness. To acheieve this, one can target genes andor cells to the thymus, or one can manipulate the peripheral immune effectors using cells alone or gene-engineered cells.The evidence suggesting that a preventive approach manipulating the thymic environment of antigen presentation is possible was initially obtained by generating transgenic NOD mice with different H2 (major histocompatibility complex) genes. Mice carrying H2 transgenes conferring resistance did not develop diabetes. Additionally, diabetes in the NOD mouse was also prevented by thymic inoculation of soluble islet antigens in the form of cellular lysates or by expression of putative -cell autoantigens in the thymus. Could this approach be clinically applicable? Recent data on plasticity of bone marrow stem cells seem to imply that culture conditions could be defined in which bone marrow progenitors could be propagated towards 'thymic' APC. These cells could be engineered using a number of viral or nonviral vector methods (gene vectors to be described in a later section) to present autoantigen. These cells could then be injected into the host where they could eventually populate the recipient thymus. To obviate the problems associated with graft versus host disease in an allogeneic context, one could envisage the use of hematopoietic stem cells propagated from peripheral blood precursors of the recipient. Preliminary evidence seems to suggest that the newly generated insulin-generating cells may not have the same phenotypic makeup of normal cells and because of this characteristic; they may be able to escape the recurrence of pre-existing autoimmunity.

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28 A number of studies have shown that allogeneic bone marrow transplantation into NOD or BB rats with the aim of inducing a state of chimerism can also prevent diabetes and facilitate alloand xenograft islet transplantation (Balamurugan, A., 2006). While the mechanisms are believed to involve central and peripheral chimerism, the applicability of this approach in humans is impeded by the use of very high radiation conditioning of the recipient. The need for complete or partial myeloablative treatment and of allogeneic donors could be obviated by genetically engineering peripheral blood-derived autologous hematopoietic stem cells with transgenes promoting the induction and activity of immunoregulatory networks. Independently of the means utilized to abrogate autoimmunity, a state in which the diabetic patient is free of autoreactive T cells and their assault on pancreatic cells is optimal to allow or promote the rescue or regeneration of enough insulin-secreting cells in the endogenous pancreas. This may allow physiologic euglycemia. Alter-native measures to control the glycemia during the possibly long recovery period must also be implemented. Aims of This Study Type 1 diabetes is an autoimmune disease resulting in the destruction of pancreatic insulin producing cells that leads to insulin deficiency and hyperglycemia. The destruction of islet cells involves the dysfunction of both CD4 + and CD8 + T cells leading to a complex inflammation. Although the exact cause of this disease is not well understood, increasing evidence has shown that immuno-suppressive and anti-inflammatory agents can prevent type 1 diabetes. Alpha 1 antitrypsin (AAT) is a major proteinase inhibitor (serpin), it has been recently shown that an early (4wks) intramuscular injection of recombinant adeno-associated virus (rAAV) vector expressing

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29 hAAT prevented type 1 diabetes in NOD mice. Unlike tightly regulated hormones, growth factors and cytokines, hAAT has a large range of concentration in the circulation (0.8-4 mg/ml). Over expression of this protein by gene therapy resulted in no identifiable side-effects in animal models and thus far, in humans subjected to the therapy. These results suggest a promising potential for the clinical application of this approach in the prevention of type 1 diabetes. However, the mechanisms by which hAAT suppresses the autoimmunity and protects islets are largely unknown. The goals of this project are to understand the protective and immune regulatory function of hAAT in type 1 diabetes and optimize AAV mediated hAAT gene delivery to a safe and efficient therapy in type 1 diabetes prevention. The specific aims of this project are to test the following hypotheses: Specific Aim 1: Evaluation of the time and dose of rAAV-CB-hAAT gene therapy on preventing type 1 diabetes in NOD mice. Although the pathology of type 1 diabetes is not fully understood, it is clear that level of the autoimmunity is increased as the disease develops. We hypothesize that the time of the treatment and the level of the transgene expression (hAAT) will be critical for (the) optimal prevention. To test this hypothesis, Female NOD mice of three different ages (4wks, 8wks and 12wks-old) will be given an i.m. injection of rAAV-CB-hAAT at three different doses (4x10 11 , 4x10 10 and 4x10 9 particles/mouse). We will monitor blood glucose weekly until 32 weeks of age. We will detect transgene expression at different time points and measure antibody against hAAT in the serum. We will also compare the insulitis in different groups. Specific Aim 2: Identification of the functional domain(s) of AAT for prevention of type 1 diabetes. The wellknown function of hAAT is inhibition of a class of proteinases by the suicide interaction between its reaction center and the enzymes. However, it is

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30 unknown whether the reaction center is critical for the prevention of type 1 diabetes. We hypothesize that the reaction center plays an important role in this protective effect, and that a change in the amino acid in the P1 position will change the function of AAT. To test this hypothesis, we will construct a rAAV1 vector encoding a mutant human AAT (hAAT without the reaction center, 6 amino acid deletion, hd(6)-AAT), and test it for the prevention of type 1 diabetes in NOD mice. In addition, we will also test rAAV1 vectors expressing mouse AAT isoforms for the prevention of type 1 diabetes in NOD mice. The 5 reaction centers of the 5 mouse AAT isoforms are very diverse and can interact with different classes of proteinase, while the rest of the amino acid sequences are highly conserved. These variants of AAT will be used to test the importance of the inhibitory effects and humoral immunity to human AAT on prevention of type 1 diabetes in NOD mice. Specific Aim 3: Effect of alpha 1 antitrypsin on NK cell killing activity. Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) play an important role in the cell destruction. We will evaluate the ability of AAT on inhibition of NK cells on target cell killing.

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CHAPTER 2 MATERIALS AND METHODS General methods used in the experiments are described in great detail in this chapter to enable researchers to use the experiments presented. Animals Female NOD (NOD/ltj) mice were purchased at 4 weeks of age from the Jackson Laboratory (Bar Harbor, Maine). All mice were housed in specific pathogen-free facilities at the University of Florida. The Institution Animal Care and Use Committees at the University of Florida approved all animal manipulations. Mice were monitored weekly for hypergylcemia until they became diabetic, as defined by two consecutive (>24 hours apart) non-fasting blood glucose levels >240 mg/dl. ELISA Analysis of Protein Expression Human alpha 1 antitripsin expression in the serum was routinely done in our laboratory. Briefly, Microtiter plates (Immoulon 4, Dynex Technologies, Chantilly, VA, USA) are coated with 100l of first antibody goat anti-hAAT (1:200 diluted Sigma Immunochemical, St. Louis, MI, USA) in Voller’s buffer overnight at 4 o C. Duplicated standard curves (hAAT, Athens Research & Technology, Inc., Athens, GA) and serially diluted unknown samples are incubated in the plate at 37 o C for 1 hour. After blocking with 3% bovine serum albumin (BSA), a second antibody, rabbit anti-hAAT (1:1000 diluted, Roche Molecular Biochemicals, Indianapolis, IN) is reacted with the captured antigen at 37 o C for 1 hour. A third antibody, goat anti-rabbit IgG conjugated with peroxidase (1:800 diluted, Roche Molecular Biochemicals, Indianapolis, IN, USA) is 31

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32 incubated at 37 o C for 1 hour. . The plate is washed with PBS-Tween 20 between reactions for 3 times. After reaction with the substrate (o-phenylenediamine, Sigma Immunochemical, St. Lous, MI, USA) plates were read at 490nm on a MRX microplate reader (Dynex Technologies, Chantilly, VA, USA). It is notable that no or very little cross-reaction to hAAT has been observed using this ELISA. Serum Elafin was detected by a kit (Human pre-Elafin/SKALP ELISA test kit, cell sciences, Canton, MA, USA). Make standard series of elafin by diluting the reconstituted standard in dilution buffer (protein stabilized phosphate buffered saline, preservative: 2-chloroacetamide) in polypropylene tubes in order to achieve a standard range from 156 to 10,000 pg/ml. Use 8 polypropylene tubes, number them 1-8. Tube 8 set aside with 500 l dilution buffer, it will be used as control value. Add 158 l dilution buffer to tube 1 (the amount of dilution buffer, that must be put into tube 1 is given on the batch control), Fill tube 2-7 with 225 l dilution buffer. Transfer 25 l standards to tube nr. 1 and dilute 1:2 further by mixing well and pipetting 225 l over to tube 2 and again 225 l to the next tube and so on until tube nr. 7. Use tubes 1-8 as the standards in the assay. Dilute samples with dilution buffer. Serum or plasma samples should be diluted at least 10 times. Transfer 100 l in duplicate from each standard, each unknown sample and the controls to the assigned wells, following the scheme on previously prepared data collection sheet. Cover the tray with adhesive cover and incubate 2 hours at room temperature (18-25 o C). Aspirate all wells using a multichannel pipette, wash 3 times with 200 l diluted wash buffer (PBS-Tween20), and empty the tray to remove the remaining wash buffer. Add 100 l of diluted tracer (biotin labeled antibody to human elafin/SKALP in protein stabilized buffer, preservative: 2-chloroacetamide) to each well, cover the tray with the

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33 adhesive cover and incubate for 1 hour at room temperature (18-25 o C). Prepare the substrate tetramethylbenzidine (TMB) solution just prior to the end of the incubation period with the conjugate. Aspirate all wells, and wash 3 times with 200 ul diluted wash buffer. Finally add 100 ul of TMB solution to each well and read the plate at 490nm on a MRX microplate reader (Dynex Technologies, Chantilly, VA, USA). Large Scale Preparation of Plasmid DNA for Packaging it into rAAV Vectors A loopfull of glycerol stock bacterial containing plasmid DNA was inoculated into 1000ml of Luria-Bertani (LB) Broth with ampicillian (100g/mL) and incubated at 37 C overnight shaking at 200rpm. After about 16-24 hours, Pellet cells at 4000rpm , 30 min, at 4C in (4) 250 ml bottles. The liquid was poured off and the cells were resuspended in 21 ml of TEG (50mM Glucose, 25mM Tris, 10mM EDTA, pH=8.0, 100ml) per bottle until no clumps are visible. Then add 42 ml fresh base solution (0.2M NaCl, 1%SDS, 100ml) to each bottle, mix by inverting tube 6 times, Incubate on ice for 10 min, do not vortex. Then add 31 ml of acid solution (3.0M Kac pH=5.5, 200ml) to each bottle, mix by inverting the tube 6 times, incubate on ice for 5 min, do not vortex. Then spin the bottles at 9000 rpm for 30 min at 4C. Then filter the supernatant though tissue and split it into 2 bottles (250ml). Then 2 volumes of ethanol was added to each bottle, place the bottle at -70C for 1 hour or -20C overnight. The bottles were centrifuged at 9000 rpm for 45min at 4C. The supernatant was discarded and 2 or 3 pellets were combined in 10 ml Tris-EDTA (TE) (10mM Tris/0.1mM EDTA buffer, pH=7.4), and then transfered it to an oakridge tube. 5 ml of 7.5 M Ammonium acetate was then added and the tube was incubated on ice for 30min. After centrifuged at 12,000-rpm for30 min, the supernatant was transferred to a new oakridge tube and 2 volume of ethanol was added to each tube. After a 10 minutes incubation at room temperature the tubes were centrifuged at 12,000

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34 rpm at 4Cfor 30 min. The supernatant was discarded and the pellet was allowed to air-dry and resuspend in 3.69ml of TE per centrifuge tube. 20ul of RNAase was added to each tube, then the tubes were incubated at room temperature for 5 min. For each tube, 5.247ml of CsTFA and 10ul of Ethidium bromide were added. Then the mix was transferred to the ultracentrifuge tubes (Beckman, Optiseal TM 361623) and centrifuged in a 75.1 Ti rotor at 60,000 rpm at 20C for 18hours. The next day, the lower plasmid band was removed with needle and a gauge syringe and placed into a 1.5 ml eppendorf tube and precipitated with 0.7 volume isopropanol. After 5 minutes centrifugation in micro centrifuge at 14,000 rpm, the pellet was allowed to air-dry and resuspended in 1 ml TE buffer. To the tube containing the dissolved DNA, 500ul of phenol-chloroform was added, vortexed and centrifuged for 5 minutes at 4k rpm. The top aqueous layer was transferred to a new tube. This was repeated once more with phenol/chloroform and once with chloroform. Then the DNA was precipitated by adding 0.1 volume of 3M NaAc (PH=5.2) and 2 volume of ethanol to each tube. DNA was pellet by centrifugation 10 minutes at 14,000 rpm. Let the pellet Air dry and resuspended in TE (Ph 8.0).Then the OD of the final solution of DNA was measured and the concentration of DNA was calculated. After digested the DNA with corresponding enzyme at 37C for 2 hours, the DNA was analyzed by gel electrophoresis. Packaging of rAAV Serotype 1 Virus A cell factory (Nalge Nunc International; Rochester, NY) of 70% 80% confluent HEK 293 cells was cotransfected with the rAAV vector and the helper plasmid pXYZ1 using CaPO 4 transfection. Forty-eight hours later the cells were harvested and lysed by resuspending the cells in lysis buffer ( 20nM Tris, 150mM Nacl, pH=8), freezing and thawing three times, followed by Benzonase digestion (25Units/ml). Following digestion,

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35 the lysate centrifuged at 3,500 times gravity for 20 minutes and the supernatant loaded onto the iodixanol step gradient. Following Optiprep iodixanol (Optiprep Co; Oslo, Norway) gradient centrifugation, the vector-containing fraction was collected by side puncture of the centrifugation tube and diluted 1:1 using a low salt (20mM Tris, 15mM NaCl, pH=8.5) buffer. The diluted Iodixanol fractions were loaded onto 5nL high Trap Q HP column (Amersham) at a flow rate of 5mL/minute. The column was washed at a flow rate of 5mL/minute from the column using the elution buffer (20nM Tris and 350mM NaCl, pH=8.5) and collecting the peak. Buffer exchange and concentration of the final stock was accomplished using a Centrifugal Spin Concentrator, Apollo 20mL High-performed three times and resulted in a final formulation of the vector in lactated Ringer’s solution. Western Blot Analysis of Protein Expression Protein to be run on for the Western Blot was denatured at 100C for 5 minutes, then 100ng (nanograms) of protein was loaded onto a precast 10% Tris/glycine polyacrylamide gel (invitrogen; Carlsbad, CA) along with a broad range protein marker. The gels were run in an electrophoresis apparatus (BioRad; Hercules, CA) for 2 hours at 110 volts in 1X SDS running buffer (1.5M TrisHCL / 0.4% Sodium Dodecyl Sulfate (SDS), pH=8.8). The gels were then transferred onto a nitrocellulose membrane and placed in between two pieces of filter paper and 4 sponges. It was then placed a transfer box and placed back into the electrophoresis apparatus, The transfer was run at 25 volts over night in transfer buffer (200mL methanol, 1.45g Tris, 7.2g Glycine, H 2 O to 1 liter). The nitrocellulose paper was removed and washed with blocking solution ( 1X phosphate buffered saline (PBS), 0.1% Tween20, 5% nonfat dry milk) on a shaker at 75rpm for 1 hour. The buffer was pored off and the membrane was washed 3 times with

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36 PBS/Tween20 for 10 minutes on a plate shaker at room temperature. After the final wash, the primary antibody, diluted in 10mL of blocking buffer, was applied to the membrane. After one hour incubation, the membrane was washed 3 times again with PBS/Tween20. After the third wash, 1l of secondary horse radis peroxidase (HRP) antibody diluted in 10mL of blocking buffer was applied to the membrane at incubated room temperature on a shaker for 30 minutes. The secondary antibody was then poured off and washed 3 times with PBS/Tween20. The membrane was then placed on a piece of saran wrap and blotted dry with filter paper. The ECL developing solution (Amersham; Uppsala, Sweden) was applied (2mL solution A and 50L solution B) and incubated in the dark at room temperature for 5 minutes. The membrane was then blotted with another piece of filter paper to dry and the membrane was wrapped in saran wrap. The membrane was then brought to the dark room and exposed to develop using x-ray film. Histopathology and Immunohistochemistry Pancreas, muscle (injection site and non-injected muscle), salivary gland, spleen, kidney, liver, heart, lung, ovary, and jejunum from all study mice were fixed in 10% neutral buffered formalin or 2% paraformaldehyde-periodate-lysine-(PLP) buffer, embedded in paraffin, and sectioned at 4 microns. All sections were stained with hematoxylin and eosin for histological assessment. Insulitis was scored in a blind fashion by one observer as previously reported (Goudy et al., 2001; Song et al., 2004). Briefly, the degree of lymphocytic infiltration in each islet was scored according to the following scale: 0: none, 1: peri-islet infiltrates, 2: <50% intra-islet infiltrates, 3: >50% intra-islet infiltrates. The severity of inflammation in the perimysium and muscle fibers at the injection site was also scored in a blinded fashion according to the following scale: 0=normal, 1=mild focal, 2=mild multifocal, 3=moderate multifocal, 4=severe multifocal.

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37 The injection site was evaluated for hAAT transgene expression as previously reported (Song et al., 2004). Briefly, paraffin sections were deparaffinized and rehydrated with buffer. Antigen retrieval was performed using trypsin digestion for 5 min at 37C. Following a 10% serum block, sections were incubated with rabbit anti-human AAT antibodies (1:200, Research Diagnostics Institute) for one hour. Antibody binding was visualized following incubation with a biotinylated secondary antibody, avidin-biotin complex with alkaline phosphatase (Rabbit AP kit, Vector) with vector blue chromogen containing levamisole (5g/ml). Sections were counterstained with nuclear fast red (DAKO). Positive controls consisted of normal human liver and liver from a patient with AAT deficiency while a normal mouse liver served as a negative control sample. Negative controls were also performed on injected muscles using normal rabbit IgG for the primary antibody. Images were photographed using a Zeiss Axioskop Plus microscope and Axiocam camera and compiled with Adobe Photoshop. Isolation of Splenocytes Spleen was removed from the sacrificed mouse, and put into a 50mL plastic tube with 5mL of Hank’s balanced salt solution (Media Tech). Put sterile, 70 m cell strainer on top of a new, sterile, 50mL plastic tube , make sure to handle the strainer only by the handle (strainer = 70m Nylon BD Falcon). Then the spleen was placed in the cell strainer and a 5ml syringe with 27-G needle was used to pierce spleen and inject with 1-2ml of HANK’s. During the injection the spleen should become lighter in color and the outer membrane should rupture. Then the plunger of the syringe was used to macerate the spleen on the filter until all that remains is the white connective tissue. Then a sterile transfer pipette was used to wash the strainer, repeated wash for 3 times until the volume

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38 of the tube was 30ml with HANK’s (using the graduated lines on the side of the tube). Centrifuge the tube at 10g for 8 minutes (1200rpm). Then the supernatant was pulled off, taking care not to disturb the pellet and the pellet was resuspended in the small remaining volume by flicking the bottom of the tube. Then, 2ml of cold ammonium chloride was added to the tube and incubated on ice for 2 minutes. Then HANK’s was added to the tube until volume reach 20ml, centrifuged at 10g for 8 minutes. Then after repeating this washing steps two times the pellet was resuspended and 5ml of RPMI was added into the tube with sterile pipette. Then the cells were counted by adding 10l of cell cuture to 90l trypan blue solution (0.4%) (Signa Cell Culture), 10l of trypan blue mixture was then added to hemocytometer and the light colored cees were counted int he triple-lined grid under microscope. (200 cells =20 x10 6 cells/ml). Immune Profiling of Flow Cytometry For each sample, 1X10 6 cells were aliqoted into 5mL Falcon tubes (Becton Dickinson Inc; Palo Alt, Ca) per test. In addition, 1X10 6 cells were added for each test’s isotype control. For Calibration purpose, 1 tube for each individual florescent-tagged antibody used, and 1 tube of cells with no antibodies was prepared containing 1X10 6 cells. The cells were then centrifuged at 500g for 10 minutes at 4C. The media was aspirated out and the cells were resuspended in 1mL Flow Cytometry Staining Buffer. (1X PBS, 0.1M bovine serum albumin (BSA), and 0.09M Na Azide) followed by lightly vortexing the tube. The cells were centrifuged again and resuspended in 100L flow buffer. To each tube, 10L of normal rat serum was added and the tubes were incubated for 15 minutes at 4C in the dark. The fluorescently labeled antibody mixtures were then added to the appropriate tubes. The cells were then vortexed lightly and incubated with the antibodies at 4C for 30 minutes in the dark. After 30 minutes, 2mL flow buffer was

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39 added to each tube and then centrifuged for 10 minutes. The buffer was removed and the cells were washed once more with an additional 2mL flow buffer. The cells were then resuspended in 400L flow buffer and made ready for reading on the flow cytometer. Flow cytometric analysis of prepared cells was carried out using the FACScan Flow Cytometer (Becton Dickinson Inc). First, unlabeled cells were placed into the cytometer and the forward scater/side scatter setting was made. This was followed by the Fluorescein Isothiocyanate (channel-1), Phycoerythrin (channel-2), peridinin Chlorophyll-a Protein (channel 3), and Allophycocyanin (channel-4) markers. Alternatively, ten minutes before the cells were read by the flow cytometer, 5L of 7Amino-Actinomycin D (7-AAD) was then added to a tube of unlabeled cells and calibrated in channel 3. Once the calibration settings were established, the settings were strored and 50100,000 cells wee counted from each sample. The files were saved to the computer and analyzed using FCS Express Flow Cytometer analysis program. (De Novo Software; Thornhill, Canada).

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CHAPTER 3 MOLECULAR CLONING OF RECOMBINANT AAV VECTORS Construction of rAAV Vectors Expressing Mouse Alpha 1 Antitrypsin Alpha 1 antitrypsin is predominantly produced in liver; thus, RNA was extracted from the liver of C57BL/6 mouse using RNeasy Mini Kit (Qiagen, Valencia, CA). The cDNA of AAT was obtained by reverse transcriptase polymerase chain reaction (RT PCR) from the purified liver RNA. The first strand (RT mix) synthesis was carried out in the nuclease-free tube, first add 1g total RNA and 1l oligo dT (dioxythymidine) (500g/ml), mix well, then put it on a heat blocker at 65 C for 10 min, next add the following components: 2l 10X RT buffer, 1l PCR dNTP (dioxyucleotidetriphospate) mix (10mM each), 0.5l mulv. RT enzyme and 10.5l H 2 O, then incubate in the water bath at 37 C for 1 hour, after the RT reaction, the mixture was inactivated at 65 C for 10 min, then put it on ice and ready for following PCR. The cDNA of AAT was amplified by PCR high fidelity platinum pfx DNA polymerase (Invitrogen, Carlsbad, CA) using specific primers. The following components were added to the nuclease free PCR tubes: 5l 10X pfx amplification Buffer, 1.5l dNTP mix (10mM each) 1l MgSO 4 (50mM), 1.5l forward primer, 1.5l backward primer, 1l RT mix, 1l pfx DNA polymerase and 37.5l dH 2 O. The tubes was capped and placed into the RoboCycler Gradient 96 thermocycler (Stratagene, La Jolla, CA). After 3 minutes 95C denaturation step, 35 cycles at 94C for 30 seconds, 55C for 30 seconds, and 72C for 1 minute was performed. A final extension at 72C for 10 minutes finished the reaction. 40

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41 The PCR products were separated by 1% agarose gel with a 1 kilo base pair (KB) ladder at 100 volts for 1.5 hours. The 1.3KB fragment for the AAT was removed with sterile scalpel and placed into a 1.5ml centrifuge tubes. The DNA was purified from the agarose gel using the QIAquick Gel Extraction Kit (Qiagen; Hilden, Germany). Next, the RT-PCR product was moved into a sequencing vector. It was ligated into the pCR2.1-TOPO vector (Invitrogen Corp) using the following procedure. To a sterile microcentrifuge tube, 4L RT-PCR product, 1L sterile H 2 O, and 1L pCR2.1-TOPO vector added. The ligation reactions were mixed gently, incubated for 5 minutes at room temperature, and then placed on ice. To transform the DNA into bacterial cells, 2L of the ligation reaction was added into a vial of competent cells (One Shot Chemically Competent TOP10). After a 30minute incubation on ice, the cells were heat-shocked for 30 seconds in a 42C water bath. The tube was then immediately transferred on ice. Then, 250L of room temperature Luria-Bertani (LB) broth was added to the cells and incubated in a shaker (200rpm) at 37C. After one hour, 50L of the transformation was spread on a pre-warmed LB agar plate containing 50g/ml (amp)icillin and 40mg/ml 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside in demethylformamide and incubated overnight at 37C. The next day, 10 white colonies were picked from each construct using sterile toothpicks and placed into 15mL conical tubes containing 3mL LB Broth (100g/ml amp). The tubes were then placed in a shaking 37C incubator (200rpm) overnight. The next morning, the colonies were screened using a QIAprep Minipre (Qiagen Corp).

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42 A restriction enzyme digestion was then performed on the DNA to screen for clones that have the PCR product inserts. Plasmid DNA (1g) from each clone was digested by restriction enzymes EcoR1 and Not1 (New England Biolabs Inc; Beverly, MA). at 37C for two hours. DNA fragments were separated by 1% agarose gel. Clones with the band at 1.3 Kb fragment were considered as a insert containing clone. In order to screen clones containing each isoform of mouse AAT cDNA, Southern dot blot analysis were performed using isoform specific probes (Figure 3-1). Briefly, denatured plasmid DNA from each insert containing clone ( boil the DNA plasmid for 5 minutes ) in a 100 l sample volume was loaded on a nitrocellulose membrane in 6xSSC (diluted from 20xSSC, 20xSSC contains 3M sodium chloride and 0.3M sodium citrate) using Bio-Dot apparatus. The membrane was rinsed with 2xSSC, air-dryed, and baked for 2 hours at 80C before hybridization. The isoform specific probes (PI 1-5, Figure 3-1) were labeled using DIG oligonucleotide 3’end labeling kit (Roche, USA). Briefly, 100 pmol oligonucleotide was diluted with sterile double distilled water to a final volume of 10L, and mixed with the following reagents on ice: 4L of 5 x reaction buffer (1 M postassium cacodylate, 0.125 M Tris-HCL, 1.25 mg/ml bovine serum albumine, pH=6.6), 4L CoCl 2 – solution (25mM), 1L DIG-ddUTP solution (25 L 1mM Digoxigenin-11-ddUTP, in double distilled water), 1L Terminal transferase (25L terminal transferase, in 60mM K-phosphate pH=7.2, 150 mM KCl, 1mM 2-mercaptoehanol, 0.5% Triton X-100, 50% glycerol). The reaction was incubated at 37C for 15 minutes and placed on ice. Finally the reaction was stopped by adding 2L 02 M EDTA (0.2 M ethylenediamino-tetaadetic acid, pH=8.0).

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43 PI-1 CAGTTACAGTCTTACAAATGGTTCCTATGTCTATGCCCCCTATCCTGCGC PI-2 CAGCTACAGTCTTTGAAGCCGTTCCTATGTCTATGCCCCCTATCCTGCGC PI-3 CAGCTACAGTCTTACTAGCCGTTCCTTATTCTATGCCCCCTATCGTGCGC PI-4 CAGCTACAGTCTTACAAGTCGCTACTTATTCTATGCCCCCTATCGTGCGC PI-5 CAGCTACAGTCTTACAAGGCGGTTTTTTGTCTATGCCCCCTATCTTGCAC Figure 3-1: Sequences of the DNA probes to identify 5 different isoforms of mouse AAT which is also the reaction center loop sequence of 5 isoforms The membrane was hybridized with the Dig labeled probe at 42C over night using DIG easy Hyb solution (Roche, USA). Briefly, DIG Easy Hyb (approximately 20 ml/100cm 2 ) was pre-heated to hybridization temperature (42C in this case). Then, the membrane was incubated for 15-30 minutes with gentle agitation.After the DIG-labeled DNA probes (5-25ng/ml hybridization solution) were denatured by boiling for 5 minutes and rapidly cooling on ice-water, the denatured DNA probe was added to pre-heated DIG easy Hyb (at 3.5ml/100 cm 2 membrane) and mixed well. Then the prehybridization solution was poured off and the probe/DIG Easy Hyb mixture was immediately added to the membrane. The membrane was incubated with gentle agitation overnight at 42C. The hybrydization was detected by DIG luminescent detection kit (Roche, USA). Briefly, after hybridization and stringency washes, the membrane was rinsed for 1-5 minutes in Washing Buffer (0.1 M maleic acid, 0.15 M NaCl; PH 7.5, 0.3 %( v/v) Tween 20), and incubated for 30 minutes in 100 ml blocking solution (Prepare a 1 x working solution by diluting the 10 x blocking solution 1:10 in freshly prepare Maleic acid buffer) followed by a 30 minute incubation in 20 ml antibody solution (anti-digoxigenin-AP, 1:10,000 (75mU/ml) diluted in blocking solution).After 2 x 15-minutes washing in 20 ml washing buffer, the membrane was equilibrated 5 minutes in 20 ml detection buffer (0.1 M Tris-HCl, 0.1 M naCl, PH 9.5). After incubation with 2 ml diluted DSPD (dilute CSPD 1:100 in detection buffer) at room temperature for 5 minutes, excess liquid was squeezed out and the development folder was sealed. The damp membrane was incubated for 5-15

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44 minutes at 37C to enhance the luminescent reaction. Finally, the membrane was exposed to X-ray film for 5-30 minutes at 15-25C. Since luminescence continues for at least 24 hours and signal intensity remains almost constant during the first hours, multiple exposures were performed to achieve the desired signal strength. Figure 3-2 is a representative dot blot image showing the isoforms-specific positive clones. Figure 3-2: Dot blot screening of isoform specific clones. After TA cloning, the colonies were screened by isoforms specific probes (PI-1 to PI-5) Separately. The positive DNA plasmid for each isoform was sequenced (DNA sequencing core, ICBR, University of Florida) using an ABI Prism 377XL sequencing system (Applied Biosytems Inc; Foster City, CA). The clones with the correct sequence (Figure 3-3 and Figure 3-4) were stored as pCR2.1-TOPO-mAAT1, pCR2.1-TOPO-mAAT2, pCR2.1-TOPO-mAAT3, pCR2.1-TOPO-mAAT4, pCR2.1-TOPO-mAAT5 in 50% glycerol in -80C freezer. In order to generate recombinant AAV vector constructs, 5g pCR2.1-TOPO-mAAT1 or pCR2.1-TOPO-mAAT2 was digested with restriction enzymes EcoR1 and Not1. The 1.3 kb fragments from the pCR2.1-TOPO-mAAT1 or pCR2.1-TOPO-mAAT2 were isolated using Qiaquick Gel extraction Kit and inserted into rAAV-CB-AAT

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45 construct to replace hAAT cDNA using the Rapid DNA ligation Kit (Roche; Indianapolis, In.) as described in Figure 3-5. Figure 3-3: Alignment of amino acid sequences of mouse AAT isoform 1 from the gene bank (PI-1) with the sequence of one of the clone of pCR2.1-TOPO-mAAT1 (Mouse AAT 23). Briefly, a mixture of the insert and backbone (3:1 molar ratio) was diluted in 1 x concentration DNA dilution buffer to a final volume of 10l and mixed with 10l T4 DNA ligation buffer, 1l T4 DNA ligase. The reaction was incubated at room temperature for five minutes. The ligated DNA was added to 100l of SURE 2 cells (Stratagene Inc; La Jolla, Ca) in a pre-chilled 15 ml polypropylene round-bottom tubes. The cells were incubated on ice for 30 minutes. After a heat-pulse in a 42C water bath for 30 seconds, the cells were incubated for an additional 2 minutes on ice. After mixed with 0.9 ml of preheated (42C) LB broth, the cells were incubated at 37C for 1 hour

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46 with shaking at 225-250 rpm, plated on LB/amp agar plates and incubated at 37C overnight. Plasmid DNA from each colony was purified by Qiapre miniprep and digested with SamI. to confirm the sequence. The clones, pAAV1-CB-mAAT1 or pAAV1-CB-mAAT2 with fragment sizes about 1.3 kb was selected for future use. mAAT27 MTPSISWGLLLLAGLCCLVPSFLAEDVQETDTSQKDQSPASHEIATNLGD 50 GB PI-2 ----------LAGLCCMVPSFLAEDVQETDTSQKDQSPASHEIATNLGD 39 ******:******************************** mAAT27 FAISLYRELVHQSNTSNIFFSPVSIATAFAMLSLGSKGDTHTQILEGLQF 100 GB PI-2 FAISLYRELVHQSNTSNIFFSPVSIATAFAMLSLGSKGDTHTQILEGLQF 89 ************************************************** mAAT27 NLTQTSEADIHKSFQHLLQTLNRPDSELQLSTGNGLFVNNDLKLVEKFLE 150 GB PI-2 NLTQTSEADIHKSFQHLLQTLNRPDSELQLSTGNGLFVNNDLKLVEKFLE 139 ************************************************** mAAT27 EAKNHYQAEVFSVNFAESEEAKKVINDFVEKGTQGKIVEAVKELDQDTVF 200 GB PI-2 EAKNHYQAEVFSVNFAESEEAKKVINDFVEKGTQGKIVEAVKELDQDTVF 189 ************************************************** mAAT27 ALANYILFKGKWKKPFDPENTEEAEFHVDKSTTVKVPMMMLSGMLDVHHC 250 GB PI-2 ALANYILFKGKWKKPFDPENTEEAEFHVDKSTTVKVPMMMLSGMLDVHHC 239 ************************************************** mAAT27 SILSSWVLLMDYAGNASAVFLLPEDGKMQHLEQTLNKELISKILLNRRRR 300 GB PI-2 SILSSWVLLMDYAGNASAVFLLPEDGKMQHLEQTLNKELISKILLNRRRR 289 ************************************************** mAAT27 LVQIHIPRLSISGDYNLKTLMSPLGITRIFNNGADLSGITEENAPLKLSK 350 GB PI-2 LVQIHIPRLSISGDYNLKTLMSPLGITRIFNNGADLSGITEENAPLKLSK 339 ************************************************** mAAT27 AVHKAVLTIDETGTEAAAATVFEAVPMSMPPILRFDHPFLFIIFEEHTQS 400 AVHKAVLTIDETGTEAAAATVFEAVPMSMPPILRFDHPFLFIIFEEHTQS 389 ************************************************** mAAT27 PIFVGKVVDPTHK 413 GB PI-2 PIFVGKVVDPTHK 402 ************* Figure 3-4: Alignment of amino acid sequences of mouse AAT isoform 2 (GB PI-2, gi|191844|gb|AAC28865.1|) from the gene bank with the sequence of one of the colonies of pCR2.1-TOPO-mAAT2 (mAAT27). One amino acid sequence difference was found.

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47 B A Figure 3-5: Cloning of rAAV1-CB-mouseAAT.A: strategy to replace the human AAT gene in rAAV1-CB-hAAT with mouse AAT. B: Gel electrophoresis showed that the pAAV1-CB-mouse AAT was digested by EcoR1 and Not1, the top band is the vector bone, and the lower band is the mouse AAT gene. Construction of rAAV Vector Expressing Mutant Human Alpha 1 Antitrypsin In order to generate the vector that expressing a mutant hAAT without reaction center, plasmid rAAV-CB-hAAT was used as parental template. Three PCR reactions were performed using AAT 4 specific primers and high fidelity DNA polimerase to deleted the 6 amino acid of the reaction center. The sequences of the primers are: primer 1: 5’-GCTGGTTATTGTGCTGTCTC-3’ primer 2: 5’-TGACCTCGGGGGCCTCTAAAAACATGG-3’ primer 3: 5’-TTTAGAGGCCCCGAGGTCAAGTTCAA-3’ primer 4: 5’-TCGCTATTACGCCAGGCTGC-3’. As described in Figure 3-6, 5’-end of primer 2 and 3’-end of primer 3 are complementary. Therefore, products of PCR1 and PCR2 can be annealed and amplified in PCR3 by primer 1 and 4. The product of PCR3 was cloned into the pCR2.1-TOPO vector (Invitrogen Corp). Plasmids were digested by the EcoR1 and Not1. The plasmids

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48 with 1.3 kb fragment were made glycerol stock, and 3 of them were sequenced (DNA sequencing Core, ICBR, University of Florida). The plasmid with the correct sequence was then digested with EcoR1 and Not1. The mutant AAT cDNA fragment (1.3 kb) was purified using Qiaquick Gel extraction Kit, and inserted into the rAAV1-CB-hAAT backbone to replace hAAT cDNA between EcoRI and NotI sites thus created rAAV-CB-d(6)AAT vector construct.This plasmid was used to package rAAV1-d(6)AAT vector for in vitro and in vivo studies in Chapter 5. A B C Figure 3-6: Construction of rAAV-d(6)AAT vector: A. Gel electrophoresis showed the corresponding 3 PCR products. B. PCR strategies for generation of d(6)AAT cDNA. C. Structure and sequences of rAAV1-CB-d(6)AAT.

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CHAPTER 4 EFFECTS OF TIMING AND DOSE OF RAAV1-CB-HAAT THERAPY ON PREVENTING TYPE 1 DIABETES IN NOD MICE This chapter discusses the background, methods and results of the experiments for the prevention of type 1 diabetes in NOD mice using different doses of rAAV1 vectors encoding alpha 1 antitrypsin injected at different time points. Introduction Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the selective destruction of pancreatic -cells that leads to insulin deficiency and hyperglycemia. Although the exact cause of this disease is unknown, many reports have shown that anti-inflammatory and immunoregulatory strategies hold great potential for preventing T1D. Alpha 1 antitrypsin (AAT) is the major serine proteinase inhibitor (sepin), normal human plasma concentration of AAT is 0.8-2.4 mg/ml. It inhibits neutrophil elastase and proteinase 3 with high efficiency. Of particular importance are the findings that recombinant adenoassociated virus (rAAV) mediated AAT gene therapy modulated cellular immunity and efficiently prevented type 1 diabetes in NOD mice. rAAV vectors become increasingly recognized as having some super priority over other viral gene delivery system with regard to safety, modest immune response, sustained action without known pathology. Multiple papers have described the paradox of using the same gene therapy for the prevention of type 1 diabetes. For example, there are different reports on the effect of 49

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50 GAD65 on type 1 diabetes development. Those different results might due to different doses of GAD used and resulted in different level of transgene expression or different time points of intervention. In this study we sought to evaluate the effectiveness of the dose and timing of AAT gene therapy on the prevention of type 1 diabetes in NOD mice. Knowledge gained from this project will form the foundation for future studies and will enable us to improve the efficiency and efficacy of the vector system. Materials and Methods Mice Female non-obese diabetic (NOD)/Ltj mice were obtained at 4 -8 wk of age from The Jackson Laboratory (Bar harbor, ME). All mice were housed in the special pathogen free (SPF) animal care facility at University of Florida. The animal use protocols were approved by University of Florida Institutional Animal Care and Use Committee (IACUC) and Animal Care Services (ACS). Blood Glucose Analysis For monitoring blood glucose, mice in all treatment groups were bled by tail artery once per week using the Medisense Optium meter (Abbott Laboratories, Alameda, CA). If two consecutive (>24 hours apart) non-fasting blood glucose levels are greater that 240 mg/dl, the mouse will be considered as diabetic. rAAV Vector Construction, Production and Administration The rAAV-CB-AAT vector construct was produced as previously described (Song et al., 2001). Briefly, this vector contains human AAT cDNA driven by CMV enhancer and chicken actin promoter. Human elafin cDNA with FLAG and poly A sequences was amplified from a plasmid (Elafin in pBS-SK, a kind gift from Dr. M. Rabinovitch) by PCR (Zaidi et al., 1999). EcoR I and Not I sites were designed in the 5’

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51 and 3’ primers, respectively. The EcoRI-NotI fragment from the PCR product was inserted into the rAAV-CB-AAT plasmid to replace the AAT cDNA (EcoRI-NotI), thus creating the rAAV-CB-elafin vector construct. Both vectors containing AAV2 ITR were packaged into AAV serotype 1 capsids by co-transfection of vector plasmid and helper plasmid (XYZ1) into 293 cells. rAAV1 vectors were purified by iodixanol gradient centrifugation followed by anion exchange chromatography. The physical particle titers of vector preparations were assessed by quantitative dot blot analysis. All vector preparations lacked any detectable wild type AAV by either physical particle or infectious unit measurement. Figure 4-1 shows the experimental design, briefly; rAAV1-CB-hAAT and control were diluted into 100l lactated saline and injected intramuscularly into the caudal muscle of the pelvic limb at 3 time points (4 weeks, 8 weeks and 12 weeks of age) , in the 4-wk-old injection group, we used 3 different doses of rAAV1-CB-hAAT(figure 4-1). ELISA for Detection of hAAT, Elafin and the Antibodies Against Human AAT The detection of hAAT in mouse serum was performed as previously described in chapter two, we used purified hAAT as an antigen standard (Athens Research & Technology, Inc., Athens, GA). Detection of human elafin in mouse serum was performed using an ELISA kit, the Hbt human pre-elafin/SKALP kit (Hycult Biotechnology, The Netherlands). Antibodies against hAAT were detected by ELISA as described previously in chapter 2. The antibodies HRP-rabbit anti-mouse IgG 2a and HRP-rabbit anti-mouse IgG 2b were purchased from Zymed Laboratories (South San Francisco, CA). The antibodies HRP-goat anti-mouse IgG1 and HRP-goat anti-mouse

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52 IgG3 were purchased from Roche (Indianapolis, IN). HRPgoat anti-mouse IgM was purchased from KPL (Gaithersburg, MD). Figure 4-1: Injection scheme of rAAV1-CB-hAAT therapy in NOD mice to prevent type 1 diabetes. The arrow indicates the time of intervention (4-, 8-, and 12-wk-old female NOD mice; n=10/group per time point). Zymed Laboratories (South San Francisco, CA). The antibodies HRP-goat anti-mouse IgG1 and HRP-goat anti-mouse IgG3 were purchased from Roche (Indianapolis, IN). HRPgoat anti-mouse IgM was purchased from KPL (Gaithersburg, MD). Histopathology and Immunohistochemistry Tissue blocks of pancreas, muscle (injection site and non-injected muscle), salivary gland, spleen, kidney, liver, heart, lung, ovary and jejunum from all sacrificed mice were harvested, placed into cassettes, fixed in 10% formalin or 2% paraformaldehyde-periodate-lysine-(PLP) buffer, processed and sectioned (UF Pathology Core facility). All sections were stained with hematoxylin and eosin for general pathological examinations. Insulitis was scored on H&E stained pancreas in a blind fashion by one observer as previously reported (Goudy et al., 2001; Song et al., 2004). In

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53 brief, the degree of lymphocytic infiltration in each islet was scored according to the following scale: 0: none, 1: peri-islet infiltrates, 2: <50% intra-islet infiltrates, 3: >50% intra-islet infiltrates.4: complete insulitis. The pancreas was stained for B220 and CD3 for analysis of the lymphocyte profiled, and was stained for F80 for neutraphils for mice sacrificed at the 32 weeks of age. The injection site was evaluated for hAAT transgene expression as previously reported (Song et al., 2004). Briefly, paraffin sections were deparaffinized and rehydrated with buffer. Antigen retrieval was performed using trypsin digestion for 5 min. at 37C. Following a 10% serum block, sections were incubated with rabbit anti-human AAT antibodies (1:200, Research Diagnostics Institute) for one hour. Antibody binding was visualized following incubation with a biotinylated secondary antibody, avidin-biotin complex with alkaline phosphatase (Rabbit AP kit, Vector) with vector blue chromogen containing levamisole (5g/ml). Sections were counterstained with nuclear fast red (DAKO). Positive controls consisted of normal human liver and liver from a patient with AAT deficiency while a normal mouse liver served as a negative control sample. Negative controls were also performed on injected muscles using normal rabbit IgG for the primary antibody. Images were photographed using a Zeiss Axioskop Plus microscope and Axiocam camera and compiled with Adobe Photoshop. Flow Cytometry Splenocytes from 32 week old sacrificed mice were harvested and immunostained with anti-CD4-Allophycocyanin (APC), anti-CD3-Fluorescein Isothiocyanate (FITC), anti-CD25-PE (eBioscience Inc.). The cells were counted by a flow cytometer, FACSCaliber (Becton Dickinson) to determine the percentage of T lymphocytes in the

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54 different treatment groups. All data were analyzed on FCS express software (De Novo Software, Thornhill, Ontario, Canada). Results Previous study has shown that systemic treatment of nonobese diabetic mice with high dose rAAV1-CB-hAAT at early time point (4 wk of age) effectively prevented type 1 diabetes. Although the pathology of type 1 diabetes is not fully understood, it is clear that the autoimmunity increases as the disease develops. We hypothesize that the treatment time of gene therapy and the level of the transgene expression will be critical for the optimal prevention. To test this hypothesis, we injected high dose rAAV1-CB-hAAT at 3 different time points (4-, 8-, and 12-wk-old female NOD mice; N=10/group), at the 4wk-old time point, we used different doses of rAAV1-CB-hAAT (high dose: 4 x 10 11 , Medium dose: 4 x 10 10 and Low dose: 4 x 10 9 particles/mouse). With respect to time, it was interesting to determine the effects of this treatment would have on mice treated at later time points in the effector of prediabetes. The recombinant rAAV1-CB-hAAT (Figure 4-2) was diluted to proper doses with saline (100l/mouse) and intramuscularly (IM) injected into the skeletal muscle of the hind limb. At each time point, there are 2 control groups; mice were injected with saline or 4 x 10 11 particles rAAV1-CB-elafin (control vector, figure 4-2). Treated mice were bled by tail artery for analysis of blood glucose levels weekly until 32 weeks of age. After two consecutive blood glucose readings above 240mg/dL over two days, the mouse was classified as diabetic and euthanized.

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55 Figure 4-2: The rAAV1-CB-hAAT (A), rAAV1-CB-elafin (B) constructs. AAV serotype 1 vector cassette map, where ITR is the rAAV inverted terminal repeat, CMV-beta actin promoter is the CMV enhancer and chickenactin promoter with a hybrid chicken-rabbit -globin intron. An is the SV40 poly (A) signal. Effects of Dose and Time on hAAT Gene Therapy for Type 1 Diabetes Prevention In the 4-wk-old injected group, mice receiving the high dose rAAV1-CB-hAAT vector (4 x 10 11 particles/mouse) effectively abrogated the development of diabetes (7 of 10 diabetic free; 30% incidence at 32 weeks of age; p<0.01 vs. saline or rAAV1-CB-elafin); while the medium dose (4 x 10 10 particles/mouse) and low dose (4 x 10 9 particles/mouse) treatment failed to prevent the diabetes development (90% and 80% incidence of diabetes, respectively) and was similar to the results of control vector or saline injected group (90% incidence of diabetes in both groups, figure 4-3A). Surprisingly, injection of 4x10 11 particles of rAAV1-CB-AAT vector (high dose) at 8 or 12 weeks of age also prevented type 1 diabetes (60% diabetes free in both groups, p > 0.05 vs. 4-wk-old high dose AAT group, figure 4-3B, 4-3C). Results from these experiments confirm our previous observations that AAT gene therapy efficiently prevents type 1 diabetes. These results also showed that AAT protective effect is dose

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56 dependent and that hAAT gene therapy significantly delayed diabetes development even at later time point in the prediabetes phase. Figure 4-3: Alpha 1 antitrypsin prevents type 1 diabetes in a dose dependent manner Kaplan Meier survival analyses of the percentage of mice diabetic free until 32 weeks of age (n=10/group). A, NOD mice were injected at 4 weeks of age. B, NOD mice were injected at 8 weeks of age, C, NOD mice were injected at 12 weeks of age. The arrow indicates the time of injection. All mice were treated with rAAV1-CB-hAAT at the above-mentioned dose, the control vector rAAV1-CB-elafin, or saline. All the animals were tested for blood glucose levels once per week. AAV1 Mediate Transgene Expression was Dose and Time Dependent To asses the transgene expression, serum levels of hAAT were detected by human AAT specific ELISA. As shown in Figure 4-4A, transgene expression was sustained until 32 weeks of age. Serum hAAT levels in the high dose group were higher than the medium dose group, while the hAAT levels in the low dose group were undetectable.

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57 These results indicate that the transgene expression levels from rAAV1-CB-hAAT were dose dependent.., Serum hAAT levels in the high dose12-wk-old injected group was significantly lower than the high dose 4-wk-old injected group suggesting that early injection resulted in higher transgene expression. Figure 4-4B shows that serum levels of human elafin in the rAAV1-CB-elafin injected group (vector control) were around 100ng/ml. Figure 4-4: Effect of dose and timing of vector injection on transgene expression in NOD mice. A) Comparison of serum hAAT production in NOD mice injected at different doses and different time. B) Elafin production in the serum for mice injected at 3 different time points. In addition, to evaluate the hAAT gene expression in the vector injected muscle, immunostaining for hAAT was performed in all treatment groups. Results from these studies also showed that transgene expression levels at the injection sites was dose dependent (Figure 4-5). Immune Response to hAAT Antibodies against hAAT were detected in all rAAV1-hAAT treated NOD mice. Interestingly, antibody levels are also time dependent. As shown in figure 4-6, anti-hAAT in high dose 4-wk-old injected group was significantly lower, than high dose 8-wk-old or

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58 Figure 4-5: Influence of dose and timing on local production of hAAT. Immunostaining of h AAT of skeletal muscle at the injection site confirmed that transgene expression is dose and time dependent. (W4-H) week 4 injection high dose group (4x10 11 particles/mouse), (W4-M) Week 4 injection medium dose group (4 x10 10 particles/mouse), (W4-L) Week 4 injection low dose group (4 x10 9 particles/mouse), (W8-H) Week 8 injection high dose group (4x10 11 particles/mouse), (W12-H) Week 12 injection high dose group (4x10 11 particles/mouse), (Non-injected muscle) Week 4 injection saline group. 12-wk-old injected group (p<0.03). Similar to human type 1 diabetes, autoimmunity in NOD mice increases as the age increases, thus, at early time point, immune response to human AAT was lower and results in higher serum levels of hAAT. Effect of rAAV1-CB-hAAT Therapy on Insulitis Infiltration of the endocrine pancreas by autoreactive mononuclear cells is termed insulitis, which is a key feature in the pathogenesis of type 1 diabetes in NOD mice. To exam the anti-inflammatory effects of rAAV1-CB-hAAT on the progression of insulitis,

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59 Figure 4-6: AAV1-hAAT administration results in the production of anti-hAAT immune responses. A) Comparison of anti-AAT level versus weeks of age in different treatment groups. B) Comparison of anti-AAT levels at different treatment group at 14 weeks of age. nondiabetic mice were sacrificed at 32 weeks of age from 4, 8 and 12 weeks injected groups. Organs were harvested, fixed in periodate-lysine-paraformaldehyde buffer, and processed in the UF molecular pathology core. General pathological analysis was performed on pancreas, kidney, liver, leg muscle, lung, ovary, jejunum, salivary gland, spleen and lymph nodes. No abnormality was observed in any of the animals sacrificed at the onset of T1D or at 32 weeks of age. Histological examination of pancreatic islets was performed and insulitis score was determine as described in the materials and methods. The effect of rAAV1-CB-hAAT therapy on the preservation of islets (stage 0 and stage 1) was dose and time dependent. Animals treated with high dose rAAV-CBhAAT at 4 weeks of age had more preserved islets (Figure 4-7). General insulitis levels of medium dose or low dose group was similar to that in saline or rAAV1-elafin controls treated groups. There was no significant difference (p>0.39) in insulitis between three 8-wk-old groups or three 12-wk-old groups.

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60 Figure 4-7: AAT reduces insulitis progression. The upper pictures are the representative pictures of H&E stained pancreatic sections (40x) obtained from NOD mice used in theses experiments, displayed to show the scoring categories of insulitis. Stage 0, normal islet, devoid of lymphocytes; stage 1, peri-insulitis only; stage 2, insulitis involving <50% of the islet in cross-section; stage3, insulitis involving>50% of the islet; stage 4, complete infiltrate of the islet. The lower pictures show the percentage of each stage of insulitis in different groups. Immunostaining assay showed that rAAV1-CB-hAAT therapy reduced B220 and CD3 positive cell infiltration of pancreatic islets (Figure 4-8). Influence of AAV1-hAAT on the Frequency of Regulatory T Cells Given the introductory importance of T regulatory (Treg) cells, we performed the flow cytometric analysis of splenocytes to examine the effect of rAAV-AAT gene therapy on the frequency of Treg cells. Splenocytes were harvested from 32 week old sacrificed NOD mice from 4-wk-old high dose, medium dose and saline injected groups and was immunostained with anti-CD4, anti-CD25, and anti-CD3. Frequency of CD4+CD25+ cells in all groupe (Fig 4-9).

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61 A B C Figure 4-8: Pancreatic immunostaining for B220 and CD3 lymphocytes. A) Example of infiltrate scoring of islets. B) B220 scoring of different experimental rAAV treated groups. C) CD3 scoring of different experimental rAAV treated groups.

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62 Figure 4-9: Influence of AAV-AAT on T regulatory cells. Splenocytes were isolated and suspended to a concentration of 1x10 6 /ml. Splenocytes were stained with 1g of anti-CD4-APC, anti-CD25-PE, and anti-CD3PITC per million cells and were incubated in dark for 30 minutes. A) Representative graph of CD3 positive cells from one animal in the 4-wk-old 4x10 11 particles/mouse AAV-AAT treated group. B) Representative graph of CD4+CD25+ cells (upper right region) from one animal in the 4-wk-old 4x10 11 particles/mouse AAV-AAT treated group. C) For these analysis, quadrants were established based upon the isotype controls scatters from each animal. CD3+ positive cells were further gated as CD4+CD25+ and CD4+CD25cells. CD4+CD25+ cell populations were determined as percentage of total CD4+ cells. Data shows that Tr cells are capable of preventing/resolving autoimmunity. One question is: Does the presence or absence of diabetes influence the frequency of regulatory T cells? To address this issue, we performed flow cytometric analysis of CD4+CD25+ cells in 20-wk-old nondiabetic and diabetic mice. There was no significant difference between these two groups (p=0.45). Discussion Our previous study has shown that rAAV-CB-AAT gene therapy leaded to long term systemic transgene expression and effectively prevented type 1 diabetes in NOD mice. This study further our previous study in demonstrating that AAT gene therapy

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63 Figure 4-10: Frequency of CD4+CD25+ cells in diabetic and non-diabetic NOD mice. (n=4/group). prevented T1D in NOD mice in a dose and time dependent manner. High dose AAT gene therapy resulted in preventing T1D in NOD mice while medium dose or low dose could not. It also showed that treatment in younger age resulted in better prevention than in older age. Regulatory cells have been implicated in inducing tolerance and regulating diabetes development in NOD mice when cotransferred with diabetogenic T lymphocytes. The regulatory properties of the CD4+CD25+ cells are thought to be conferred in a cell contact-dependent and/or -independent fashion. In our study, no significant difference was observed of the frequency of CD4+CD25+ cells between AAT gene therapy treated mice or saline controlled mice. So, there must be other mechanisms involved for AAT to prevent type 1 diabetes in NOD mice. hAAT is well known as a serine proteinase inhibitor (serpin) in the circulation, and can inhibit a broad range of proteinases including neutrophil elastase and proteinase-3 (Lomas and Parfrey, 2004). It has been reported that treatment with AAT can reduce apoptosis and affect the activities of cellular enzymes and transcription factors (Daemen

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64 et al., 2000; Churg et al., 2001). AAT also inhibits human immunodeficiency virus (HIV) production in infected cells (Shapiro et al., 2001). Antibodies against hAAT were detected in the mouse serum. These observations are consistent with observations that autoimmune mouse models are more sensitive to vectors and their transgene products (Zhang et al., 2004). Lu et al., 2006). Importantly, sustained serum hAAT levels demonstrated that the immune responses did not block efficient transgene expression. It is important to consider the safety and possible side effects of gene therapy for the treatment of type 1 diabetes. Interestingly, to date, in our studies of hundreds of animals (including C57BL/6, BALB/c, and NOD mice; rats; and baboons), no clinically relevant side effects have been observed. Indeed, the University of Florida happens to be a site testing rAAV-human-AAT in a phase 1 dose escalation trial and among the first 12 patients, no adverse events have been reported. In addition, clinical studies of humans involving infusion of purified human AAT protein did not result in any adverse side effects. AAT is a major serum protein, with normal levels ranging from 1 to 1.5 mg/ml in humans. Under inflammation conditions this level can increase 3to 4-fold. Hence, we believe that this large range will allow AAT gene therapy to be safe. Results from the present study suggest the promise of hAAT as a means to prevent type 1 diabetes. In summary, these data clearly demonstrate that AAT gene therapy efficiently prevented type 1 diabetes development in NOD mice in a dose dependent fashion and suggest a potential clinical application of this approach for type 1 diabetes prevention.

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CHAPTER 5 IDENTIFICATION OF THE FUNCTIONAL DOMAIN(S) OF ALPHA 1 ANTITRYPSIN FOR THE PREVENTION OF TYPE 1 DIABETES The following chapter will discuss the background, methods and results for the primary prevention experiments using rAAV vectors expressing mouse AAT and human AAT without the reaction center loop. Introduction AAT is the serine proteinase inhibitor (serpin), which can interact with several proteinases through its reaction center. This inhibitory function plays an important role in protecting tissues from damage (Daemem,M.A., 2000). We have demonstrated that hAAT can prevent type 1 diabetes (Lu, Y., 2006). However, it is not clear which domain is critical. The reactive-center loop (RCL) domain of AAT determines target protease specificity, we hypothesize that the RCL plays an important role in this protective effect, and change the amino acid in the P1 position will change the function of AAT. To test the functions of the reaction center in the prevention of type 1 diabetes, we have generated a rAAV vector (rAAV-CB-hd(6)AAT) that expresses a mutant hAAT without the reaction center and tested for the prevention of T1D in NOD mice.. Unlike humans that have a single copy of AAT gene, the mouse has at least 5 isoforms of AAT. All the isoforms are highly conserved except the reaction center. The reaction centers interact with and inhibit different classes of proteinase. We have generated 6 polyclonal antibodies recognizing mouse AAT isoforms, one is the common .antibody which can react with all 5 mouse AAT genes, and the other 5 antibodies are 65

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66 isoform specific.To test the effect of mouse AAT on prevention of type 1 diabetes, we have cloned and sequenced mouse AAT isoform 1 and 2. rAAV1-CB-mouseAAT1 has been packaged and injected into female NOD mice (n=10) at 4 weeks of age to test its function. AAT is the serine proteinase inhibitor (serpin), which can interact with several proteinases through its reaction center. This inhibitory function plays an important role in protencting tussues from damage. Through the mutant human AAT (d(6)hAAT), we can know if the reaction center is critical or not for the prevention of T1D.The mouse AAT study helps further understand which domain is important in preventing T1D. Methods Animal Female non-obese diabetic (NOD)/Ltj mice were obtained at 4 -8 wk of age from The Jackson Laboratory (Bar harbor, ME). All mice were housed in the special pathogen free (SPF) animal care facility at University of Florida. The animal use protocols were approved by University of Florida Institutional Animal Care and Use Committee (IACUC) and Animal Care Services (ACS). Vector Construction Plasmid CB-hd(6)AAT was generated from pCB-AT by relacing hAAT cDNA (at 5’EcoR and 3’ Not I sites) with the d(6) cDNA fragment generated by the PCR. The detailed cloning procedure was shown in chapter 3. Cellular Transfection and Transduction C2C12 murine myoblasts were grown in 35-mm wells (4 10 5 cells/well) and transfected with 5 ug of each plasmid DNA by using lipofectamine 2000 (invitrogen). Secretion of hAAT into the medium was assessed 2 days after transfection by using an

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67 antigen-capture ELISA assay with standards which have been previously described in specific aim 1. A plasmid CB-UF5 (GFP) was used as control. For transduction experiments, cells were transduced with multiplicities of infection ranging from 4 10 5 to 4 10 6 particles per cell.Viral packaging, production, administration and detection of transgene expression uses the same method described in chapter 2. Detection of hAAT and hNE Complex Purified hAAT (Athens Research Technology, Athens,GA, USA) or medium containing wild type hAAT or d(6)AAT was mixed with human neutrophil elastase (hNE) and incubated at 37 C for 15 min. Proteins were separated by 10% sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Intact hAAT, hAAT/hNE complex and cleaved hAAT were detected by Western blot analysis using rabbit anti-hAAT antibodies. Anti-elastase Activity Assay A commercial EnzCHek Elastase assay kit (Molecular Probes, Inc., Eugene, OR, USA) was used. In this assay, elastase from pig pancreas serves as enzyme. Soluble bovine neck ligament elastin was labeled with BODIP FL dye and served as a substrate. The non-fluorescent (quenched) substrate can be digested by elastase and yield highly fluorescent fragments. We used mouse serum mixed with hAAT as a control. Mouse serum from the AAT gene therapy treated group with known concentration of hAAT was tested. Construction of pCB-MouseAAT In striking contrast to the human genomes, wherein AAT is represented by a single gene, five isoforms (PI-1 to PI-5) are found in the mouse. We extracted RNA from C57/BL6 mouse, and use RT-PCR to amplify the mouse AAT gene, TA cloning, then use

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68 dot blot to screen each plasmid by 5 gene specific probe, and finally verify them by sequence analysis. The detailed cloning procedures are described in chapter 3. The P1 position of PI is mersinine which is the same as the human AAT, so we will first test PI-1 gene therapy in NOD mice. Generate Isoform Specific Antibodies Based on the reaction center sequences of mouse AAT and the common region of all 5 isoforms, each isoform specific peptide and the common peptide were synthesized, (sequences are shown in figure 5-1), conjugated to KLH, then injected them to rabbits. After two immunizations, rabbits were bled to collect the polyclonal antibody containing serum. The antibody title was measured by ELISA. Common: CEDVQETDTSQKDQS PI-1: CVTVLQMVPMSMP PI-2: CTVFEAVPMSMP PI-3: CTVLLAVPYSMP PI-4: CTVLQVATYSMP PI-5: CTVLQGGFLSMP Figure 5-1: The sequences of the peptides used to generate antibody which can be isoform specific and the common antibody can recognize all the isoforms of mouse AAT. Detection of Transgene Expression Mouse AAT in the serum was detected by ELISA. Microtiter plate (Immoulon 4, Dynex Technologies, Chantilly, VA) was coated with mouse serum, we used rabbit anti-mouse AAT as the first antibody, and goat anti-rabbit IgG conjugated with peroxidase as the second antibody. Vector packaging, production and administration were described in chapter 4. rAAV1-CB-hAAT, rAAV1-CB-mouseAAT1, rAAV1-CB-d(6)AAT , control vector and saline are intramuscularly injected to female NOD mice at 4 weeks of age.(table 5-1).

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69 Table 5-1. Study design for prevention of T1D in NOD mice (n=10/group). Vector Dose (particles/mouse) N= rAAV1-CB-hAAT 6x10 11 10 rAAV1-CB-d(6)AAT 6X10 11 10 rAAV1-CB-mouseAAT1 1X10 11 10 rAAV1-CB-GFP(UF11) 1X10 11 10 Saline 10 Results This section will discuss results from the primary prevention experiments using rAAV vectors expressing the mouse AAT and human AAT without the reaction center. Four week old female NOD mice were injected intramuscularly with rAAV1-CB-hAAT, rAAV1-CB mAAT1, rAAV1-CBd(6)hAAT, rAAV1-CB-hElafin or saline (n=10).All mice were bled by tail artery for blood glucose levels. After two consecutive blood glucose reading above 240mg/dl over two days the mouse was classified as diabetic and sacrificed. Interestingly, 40 weeks after vector administration, all animals in rAAV1 d(6) human AAT group remain diabetes free. In order to test the strength of the protective effect of d(6) human AAT against auto immunity. We injected these animals with cyclosphamide(CY). CY is commonly used for accelerate type 1 diabetes in NOD mice. Single IP injection of 200mg/kg of cyclosphamide will result in diabetes in 2-3 weeks. The experiment was performed as described in table 5-1. All mice were IP injected with 200mg/kg of CY or saline (100ul), and blood glucose level was monitored daily.

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70 Construction and Expression of a Human AAT Mutant without Reaction Center AAT is the serine proteinase inhibitor (serpin), which can interact with several proteinases through its reaction center. To test the functions of the reaction center in the prevention of type 1 diabetes, we have generated a recombinant AAV vector (rAAV-CB-d(6)hAAT) that expresses a mutant human AAT without the reaction center . We sequenced the coding sequences and confirmed that the cDNA was mutated as designed. Transfection of 293 cells by the vector plasmid showed that the mutant hAAT in the medium can be detected by ELISA, and the concentration is comparable with the transfected wild type human AAT in the medium (Figure 5-2). This vector was packaged into rAAV1 vector and has been injected intramuscularly into a cohort of 4-week-old NOD female mice. As shown in figure 5-5, rAAV1-CB-hd(6)AAT gives high level of transgene expression. Figure 5-2: 293 cells were transfected with rAAV-CB-hAAT or rAAV-CB-hd(6)AAT plasmid, The concentration of hAAT or d(6)AAT in the serum free medium was measured by ELISA

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71 Interaction with Neutrophil Elastase One of the major functions of human AAT is binding to the neutrophil elastase through its reaction center and inhibiting its activity. In order to test if the hd(6)AAT can bind to neutrophil elastase and inhibit its activity, we transfected 293 cells by rAAV1-hAAT plasmid or rAAV1-hd(6)AAT plasmid. Human AAT and hd(6)AAT concentration in the medium was measured by ELISA. Western blot shows that human AAT was cleaved by neutrophil elastase and formed a complex with it while hd(6)AAT did not form the complex (Figure 5-3). This data confirms that the reaction center loop has been deleted in the mutant human. In order to test the ability of hd(6)AAT to inhibit the neutrophil elastase, we performed anti-elastase activity assay using EnzChek Elastase Assay Kit (Molecular Probes, Inc., Eugene, OR). Figure 5-3: Western Blot of human AAT and D(6) AAT from transfected medium with neutrophil elastase. Wild type hAAT formed complex with NE while d(6)AAT did not.

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72 Elastase activity assay shows that medium containing human AAT has significantly higher elastase inhibitory ability than the medium containing hd(6)AAT (figure 5-4). This data indicates that mutant AAT without the reaction center has significantly lower ability to inhibit neutrophil elastase. Figure 5-4: Comparison of anti-elastase activity of human AAT and d(6)AAT. The clinical grade AAT has the highest inhibition activity of NE, The medium containing hd(6)AAT has significantly lower anti-NE activity than the medium containing wild type human AAT.(p=0.02) A B Figure 5-5: Cohorts of female NOD mice were IM injected at 4 weeks of age with rAAV-CB-hAAT or rAAV-CB-hd(6)AAT (5x10 11 particles). Transgene product (wild type hAAT(B) and hd(6)AAT (A) were detected by ELISA by antibodies against wild type AAT.

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73 Humoral Immune Response to Human AAT and Hd(6)AAT In order to know the humoral response to human AAT and Hd(6)AAT, we measured the antibody against human AAT in the NOD mouse serum at 2 weeks, 5 weeks, 7 weeks post vector injection and at the end of the experiment. We used the same ELISA protocol to assess the anti-hAAT and anti-hd(6)AAT, and the assay was carried out on the same plate. As shown in Figure 5-6A, the antibody against human increases after vector administration, and decreases a lot at the end of the experiment; while in Figure 5-6B, we can see the antibody against h(6)AAT was sustained until 45 weeks of age, and the antibody level was almost doubled as compared with that of human AAT. This might indicate that deletion of the reaction center of human AAT, make the protein more immunogenic. A B Figure 5-6: Antibody against human AAT in the NOD mice serum. Left plot shows anti-AAT in human AAT treated group. Right plot shows the anti-AAT level in d(6)AAT treated group. Construction and Injection of rAAV1 Vectors Expressing Mouse AAT Isoforms Unlike humans that have a single copy of AAT gene, the B57BL/6 mouse has at least 5 isoforms of AAT. All the isoforms are highly conserved except the reaction center. The reaction centers interact with and inhibit different classes of proteinasse. We have generated 6 polycolonal antibodies recognizing mouse AAT isoforms, one is the

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74 common antibody which can react with all 5 mouse AAT genes, and the other 5 antibodies are isoform specific. The titer of each of the antibodies is shown in figure 57 (A, B, C, D, E, and F). Dilution 1 10 100 1000 10000 0 0.5 1 1.5 2 UFl, Common CEDVQE; Rbt 2632 6/2/04 y=(A-D)/(1+(x/C)^B)+D: A B C D R^2 2632 (2632: Concentration vs MeanValue) 0.97 0.995 641.685 0.018 0.936 A Dilution 10 100 1000 10000 100000 1e6 1e7 0 0.5 1 1.5 2 U of Fl, PI-1 CVTVLQ; Rbt 2633 6/2/04 y=(A-D)/(1+(x/C)^B)+D: A B C D R^2 2633 (2633: Concentration vs MeanValue) 2.053 0.579 36964.87 0.282 0.954 B Figure 5-7: Rabbit anti-mouse AAT serum. ELISA was used to detect the anti-serum titer. A) Rabbit anti-mouse AAT common antibody to recognize all 5 isoforms. B) Rabbit anti mouse AAT isoform1. C) Rabbit anti mouse AAT isoform2. D) Rabbit anti mouse AAT isoforms 3. E) Rabbit anti mouse AAT isoforms 4. F) Rabbit anti mouse AAT isoform5.

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75 Dilution 10 100 1000 10000 100000 1e6 1e7 0 0.5 1 1.5 2 2.5 U of Florida, PI2 CTVFEA; Rbt 2634 6/2/04 y=(A-D)/(1+(x/C)^B)+D: A B C D R^2 2634 (2634: Concentration vs MeanValue) 2.167 0.496 1.01e5 0.584 0.85 C Dilution (1:X) 10 100 1000 10000 100000 1e6 1e7 0 0.5 1 1.5 U of Florida, PI3 CVTLLA; Rbt 2635 6/2/04 y=(A-D)/(1+(x/C)^B)+D:A B C D R^2 2635 (2635: Concentration vs MeanValue)1.8860.64733811.490.1620.932 D Figure 5-7. Continued

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76 E Dilutions (1:X) 10 100 1000 10000 100000 1e6 1e7 0 0.5 1 1.5 U of Florida, PI4 CTVLQA; Rbt 2636 6/2/04 y=(A-D)/(1+(x/C)^B)+D:A B C D R^2 2636 (2636: Concentration vs MeanValue)1.4550.7356192.2780.0040.956 Dilutions (1:X) 10 100 1000 10000 100000 1e6 1e7 0 0.5 1 1.5 2 2.5 U of Florida, PI5 CTVLQG; Rbt 2637 6/2/04 y=(A-D)/(1+(x/C)^B)+D:A B C D R^2 2637 (2637: Concentration vs MeanValue)2.2420.4941.62e50.7130.849 F Figure 5-7. Continued To test the effect of mouse AAT on prevention of type 1 diabetes, we have cloned and sequenced mouse AAT isoform 1 and 2. rAAV1-CB-mouseAAT1 was packaged and

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77 injected into female NOD mice (n=10) at 4 weeks of age. As shown in figure 5-8, the vector mediated high level and sustained transgene expression (mouse AAT isoform 1). 05001000150020002500W6W9W11Weeks of ageRelative Units UF11 mAAT Figure 5-8: Mouse AAT expression in the serum compared with UF11.Rabit anti-mouse AAT1 serum was use as the antibody to detect AAT in the serum by ELISA. Immunohistochemistry Organs including heart, lungs, liver, gonads, skeletal muscle, intestine, kidney, and spleen from all sacrificed mice were harvested for immunohistological analysis. Organs were put into cassettes, stored in 10% formalin, and sent to the Pathology Core facility at University of Florida for H&E staining for general pathology. Apart from a localized myositis at the site of injection, no unusual pathology was observed in any of the mice sacrificed at onset of Type 1 diabetes Survival Curves of In Vivo Treated NOD mice The incidence of T1D through 41 weeks post-injection in NOD/Ltj female mice in different rAAV vectors or saline treated groups are shown in Figure 5-10.

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78 Figure 5-9: Immunostaining for mAAT1 in rAAV1-mAAT1 vector injected muscle using rabbit anti-mouse AAT (common region-KLH) antiserum. NOD mice treated with 1x 10 11 particles of rAAV1-CB-mouseAAT did not prevent diabetes development (70% diabetic) as compared to saline (70% diabetic) or rAAV1-CB-GFP (70% diabetic) treated mice. However, NOD mice treated with 6x 10 11 particles of rAAV1-CB-d(6)hAAT completely abolished diabetes onset by 45 weeks of age (100% diabetic free) as compared to saline or rAAV1-GFP treated mice. The incidence of T1D through 3 weeks after cyclosphamide or saline injection in 45-wk-old rAAV1-d(6)hAAT treated or saline treated NOD mice was shown in Figure 5-11. 45-wk-old rAAV1-d(6)hAAT treated NOD mice received cyclosphamide remained diabetes free (five of five, 0% incidence at 3 weeks post cyclosphamide injection, p<0.05 vs saline controls). No animal in the 45-wk-old rAAV1-d(6)hAAT treated NOD mice plus saline injection group developed diabetes (0% incidence),while 60% of the animals in the 45-wk-old saline treated group developed diabetes 3 weeks after cyclosphamide injection.

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79 Figure 5-10: Kaplan Meier survival curve for different treatment groups. Comparison of different groups administered with rAAV1-hAAT, rAAV1-mouseAAT, rAAV1-d(6)AAT ,rAAV1-GFP and saline. Figure 5-11: Kaplan Meier survival analysis for different treatment group after CY injection. d(6)AAT injected mice 3 weeks after CY injection still completely abolished diabetes in NOD mice.

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80 Effect of rAAV1-CB-hd(6)AAT on the Insulitis Lesion Insulitis represents the inflammation of the islet, which is an important feature in the development of type 1 diabetes in NOD mice. To examine the anti-inflammatory effects of rAAV1-CB-hd(6)AAT has on the insulitis lesion, we monitored the insulitis in all the nondiabetic mice sacrificed 2 weeks after cyclosphamide or saline injection. Insulitis scoring was carried out in a blinded fashion on the H&E stained pancreas by the UF Pathology Core facility. The over all number of islets in each stage was counted and averaged per group (Figure 5-12A), the quantity of islets varied from 9-16/mouse, the rAAV1-CB-hd(6)AAT treated group without cyclosphamide injection has the highest number of islets, comparing the two groups both injected with CY, the rAAV1-CB-hd(6)AAT group has significant higher number of islets than the saline or UF11 injected group. The hd(6)AAT treated group without CY injection has highest number of islets in stage 0, the hd(6)AAT treated group with CY injection has more islets in stage 1 and 2 than the control with CY injection group. We also converted these data into percentages of the stage of islets compared with the overall number of islets (Figure 5-12B). The rAAV1-hd(6)AAT treated group without CY injection has 56% of islets in stage 0 versus mice treated with rAAV1-hd(6)AAT plus CY injection with 25% or control group plus CY injection with 24% stage 0 islets. These data suggests that cyclosphamide induces a rapid infux of inflammatory cells into the pancreatic islets. There was no significant differences in general insulitis of rAAV1-hd(6)AAT treated plus CY injection group over saline or rAAV1-GFP treated plus CY injection group.

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81 . B A Figure 5-12: Mutant hAAT, d(6)AAT attenuate insulitis lesion. A shows the percentage of islets at each stage in 3 different groups. B shows the number of islets at each stage in 3 different groups Splenocyte Proliferation Assay Two weeks after cyclosphamide or saline injection, Splenocytes from all three groups were isolated and stimulated with 1g/ml con A, After 72 hours, H 3 was added to each well and incubated for additional 16 hours. The H 3 thymidine incorporation was then measured using a -scintillation counter. The proliferative ability of the splenocytes was calculated as counts per minute (cpm) based on H 3 thymidine incorporation. NOD mice treated with rAAV1-hd(6)AAT plus CY injection significantly reduces the proliferation ability of splenocytes compared with the mice treated with saline or GFP Plus CY injection (p<0.05) (Figure 5-13). Discussion The mouse has at least five isoforms of AAT, each encoded by a separate gene (Borriello and Krauter, 1991; Goodwin et al., 1997; Barbour et al., 2002). Although these proteins share high homology for most of their sequences, their reaction center regions are

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82 different. Sequence analysis of AAT isoforms indicates that they may inhibit different proteinases (Forsyth et al., 2003). Only isoforms 1 and 2 have methionine at the P1 CPM Figure 5-13: Comparison of splenocytes proliferation in d(6)AAT or PBS treated groups. ConA was used as the stimuli, without stimuli, proliferation was very low in both groups; while in conA treated splenocytes, the d(6)AAT group has significantly lower proliferation than the PBS group. position, similar to human AAT, and therefore it may inhibit neutrophil elastase activity. To date, the relationship between serum level and the degree of functional activity for each isoform of murine AAT remains largely unknown. We have developed a polyclonal antibody that recognizes a common region of murine AAT. Using this antibody, we are able to detect the mouse AAT both at the injection site and in the serum. Mouse AAT isoform 1 did not prevent type 1 diabetes in NOD mice. We will try other isoforms of mouse AAT later to further understand the effect of mouse AAT on T1D in NOD mice and also further understand the which domain or amino acid sequence is critical for prevention of T1D in NOD mice.

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83 Surprisingly, d(6)AAT prevented T1D more efficiently, which indicate that the reaction center loop is not required for the prevention of diabetes. More interestingly, the mice treated with rAAV1-d(6)AAT also resistant to cyclosphamide induced diabetes. 1-Antitrypsin (AAT), a major endogenous inhibitor of serine proteases, plays an important role in minimizing proteolytic injury to host tissue at sites of infection and inflammation. There is now increasing evidence that AAT undergoes post-translational modifications to yield by-products with novel biological activity. One such molecule, the C-terminal fragment of AAT, corresponding to residues 359 (C-36 peptide) has been reported to stimulate significant pro-inflammatory activity in monocytes and neutrophils in vitro (Subramaniyam, D., 2006).. Thus one possible explanation for the better efficacy of d(6) AAT versus wild type AAT on the prevention of type 1 diabetes is that deletion of the reaction center makes d(6)AAT unable to generate the C-terminal pro-inflammatory peptide .

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CHAPTER 6 EFFECT OF ALPHA 1 ANTITRYPSIN ON NATURAL KILLER (NK) CELL KILLING ACTIVITY Introduction Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) play an important role in the cell destruction. Studies have shown that cell destruction is mediated by T lymphocytes, in particular autoreactive CD8 + CTL. CTL and NK cells induce apoptosis of target cells through either the granule exocytosis pathway or the Fas/FasL pathway. The granule contains perforin and granzymes (serine proteases). It has been reported that inhibition of granzyme B along is sufficient to block apoptosis even by whole CTL (which can kill either by perforin or Fas-based mechanisms). Both direct cytotoxic (T-cell and natural killer cells mediated) and indirect cytokine-dependent (i.e. IL-1, TNF-, INF-) mechanisms are responsible for -cell apoptosis. CTL and NK induce apoptosis of target cells primarily through the exocytosis of granules containing perforin and granzymes, or by the interaction of Fas ligand (FasL) with the Fas on the target cells. Interestingly, granzymes are serine proteinases, thus the major serine proteinase inhibitor, alpha 1 antitrypsin could possibly block the granzymes, then protect CTL or NK cell mediated -cell apoptosis and provide a potential therapy to circumvent the development of type 1 diabetes. In this chapter, we evaluated the ability of AAT on inhibiting the grazymes in the NK cells and CTL granules and thus block the NK cells and CTL mediate target cell killing. 84

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85 Materials and Methods The CTL Activity was measured by the Calcein Release Assay. Calcein-AM (Molecular Probes, USA) is a nonfluorogenic substrate that readily enters cells. Cytoplasmic esterase activity of viable cells cleaves calcein-AM to the intensely fluorescent calcein. K562 target cells were resuspended in cRPMI-2.5 at 2 10 6 cells/ml and labeled with 10 M calcein-AM for 30 min at 37C in room air. The cells were washed 4 times with culture medium and adjusted to a concentration of 5 10 4 cells/ml. Rhesus PBMC were washed twice with and resuspended in cRPMI-2.5. Cytotoxicity assays were carried out in round-bottom microculture plates (Costar, Cambridge, Mass.). Effector cells (NK-92CI) were prepared in triplicate by serial dilutions of the stock preparation to give effector-to-target cell ratios of 25:1, 10:1, 5:1, and 2.5:1. Target cells (K562) were added, and the plates were incubated for 4 h at 37C in an atmosphere of 95% air-5% CO 2 . Target cells were also incubated in medium alone and with 2% Triton (Sigma) for estimations of spontaneous and maximum release. Aliquots of 110 l of supernatant were removed from each well and transferred to 96-well flat-bottom microtiter plates (Microfluor-Black; Dynex Technologies, Chantilly, Va.) for reading calcein fluorescence in each well using an automated fluorescence measurement system (HTS 7000 Bio Assay reader; Perkin-Elmer, Branchburg, N.J.) with an excitation filter setting of 482/20 nm and an emission filter setting of 530/25 nm. The value of the background fluorescence was subtracted from the values of the maximum, spontaneous, and experimental fluorescence, and the percentage of killing for each condition was calculated as (experimental fluorescence spontaneous fluorescence) 100/(maximum fluorescence spontaneous fluorescence).

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86 Results Studies in both humans and NOD mice have shown that cell destruction is mediated by T lymphocyte and NK cells. NK-92CI is an interleukin-2 (IL-2) independent Natural Killer Cell line; it kills K562 cells in chromium release assays. To test the effect of hAAT on protecting K562 cells from being killed by NK-92CI cells, we pre-incubated clinical grade human AAT at 2 different concentrations (0.5 or 1.0 mg/ml) with the effectors (NK 92-CI) for 1 hours, then performed cytotoxity assay at 3 different E/T ratios (2.5:1, 5:1, 10:1). As shown in figure 6-2, treatment of human AAT dramatically inhibit NK cell mediated cell killing. This result is consistent with our in vivo observation that AAT gene therapy reduced insulitis and demonstrated hAAT could protect against cell death. Figure 6-3 shows the without pre-incubate the effectors with the hAAT for 1 one hour, AAT still protected the target cells from being killed by the NK cells. Figure 6-4 shows that if only pretreat the effectors with hAAT, the target cells will be killed by NK cells. These results demonstrated that hAAT can protect the target cells against the killing of effector cells. Discussion Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) play an important role in the cell destruction. These experiments demonstrated that hAAT can efficiently protect the target cells K562 against the killing of NK cell (NK92 CI). The mechanism behind this protection remains to be further investigated. The development of diabetes in the NOD mouse is preceded by a prolonged period of islet inflammation and the switch to hyperglycemia occurs only after a sufficient number of -cells have been destroyed, and insufficient insulin is secreted to maintain

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87 Figure 6-1: Experimental design for cytotoxicity assay. Figure 6-2: hAAT inhibits NK cell mediated cell killing. The effector cells were pretreated with hAAT for 1hr, before mixed with the effector cells were added. Note that the inhibition of hAAT on cell killing is dose dependent.

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88 Figure 6-3: Incubate human AAT together with effector cells and target cells for 3 hours. The target cells were protected from being killed by the NK cells. Figure 6-4: Pre-incubate the effectors with hAAT for 1 hour, then the cells were washed for 3 times with PBS. The target cells were added and co-incubated for 3 hours. There were no significant differences between treatments.

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89 normal blood glucose levels. CTL or NK cells induce apoptosis of target cells primarily through the exocytosis of granules containing perforin and granzyme B, or by the interaction of Fas ligand (FasL) with Fas on the target cell surface (Kagi, D.,1994). One possible mechanism of AAT inhibiting NK cell mediated killing is that AAT can block the serum proteinase-granzyme B, and thus prevent cell apoptosis by inhibiting the perforin-granzyme B passway.

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CHAPTER 7 CONCLUSIONS Type 1 diabetes is an autoimmune disorder resulting in the selective destruction of the insulin-producing pancreatic cells. Typically, this disease occurs in juveniles as a result of a chronic state of insulitis leading to overt diabetes. Nearly all patients diagnosed with Type 1 diabetes will rely on insulin injections for the rest of their lives in order to maintain normal glucose levels. Genetic predispotion, environmental factors, and immunophenotype have all been associated in the development of this disease. Epidemiological studies have shown that immunopheotypes biased toward Th2 responses have been associated with disease prevention/protection. Specifically, IL-4 and IL-10 are associated with preventing disease onset. Using a rAAV gene therapy approach in the mouse model for type 1 diabetes, we demonstrated that human AAT prevented T1D in a dose and time dependent fashion. Additionally, human AAT treatment reduced the insulitis scores in comparison with controls. But hAAT treatment did no change the frequency of CD4+CD25+ cells as compared with the controls. The wellknown function of hAAT is inhibition of a class of proteinases by the suicide interaction between its reaction center and the enzymes. However, it is unknown whether the reaction center is critical for the prevention of type 1 diabetes. Our studies clearly showed that hAAT without the reaction center prevented T1D more efficiently indicating that the reaction center is not critical for the prevention of diabetes. 90

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91 Mouse AAT has 5 different isoforms. The 5 reaction centers of the 5 mouse AAT isoforms are very diverse and can interact with different classes of proteinase, while the rest of the amino acid sequences are highly conserved. In our study, the onset of T1D in the rAAV1-CB-mouseAAT isoform treated NOD mice has no significant difference compared with the controls. Other isoforms of mouse AAT should be tested to identify the functional domains of AAT for prevention of T1D. Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) play an important role in the cell destruction. We demonstrated that AAT played an important role on blocking the NK cell mediate target cell killing. Collectively, AAT has multiple functions in protecting islet cells and preventing type 1 diabetes. Results from these studies imply a therapy for prevention or reversal of type 1 diabetes in humans.

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BIOGRAPHICAL SKETCH Mei Tang was born on March 24 th 1974, in Chengdu, Sichuan, P.R. China. She got her M.D. in West China University of Medical Sciences. She did her residency in internal medicine and pediatrics for one year at the regional hospital of Sichuan province. Then she became a faculty member in the Department of Immunology and did her practice in internal medicine with a focus on clinical immunology. In August, 2002, she entered the Ph.D. program in the Department of Pharmaceutics at the University of Florida. 99