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1 ALPHA-1 ANTITRYPSIN PROTEIN AND GENE THERAPIES FOR THE PREVENTION OF RHEUMATOID ARTHRITIS IN MOUSE MODELS By CHRISTIAN ALEXANDER GRIMSTEIN 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 2008
2 2008 Christian Alexander Grimstein
3 To my parents, Herbert and Ursula Grimstein
4 ACKNOWLEDGEMENTS I would like to express my si ncere thanks to m y mentor Dr. Sihong Song for giving me the opportunity to conduct my doctoral research in his laboratory and for creating an inspiring working environment. I highly appreciate his gu idance, experience and dedication which helped me pursuing my goals while working on this project. Without the help and valuable suggestions I received from my lab mates the research presented here could not have been conducted. Therefore I would like to thank Dr.Yangquin Lu, Dr. Bin Zhang, Hong Li, Matthias Fueth and espe cially Dr. Young-Kook Choi for their great support and assistance. I also appreciate the suggestions, valuable advices and support of my committee members Dr. Hartmut Derendorf, Dr. Jeffery Hughes and Dr. Minoru Satoh. The completion of this dissertation would not be possible without their timely feedback and advice. I would like to acknowledge Dr. Martha Cam pbell-Thompson and the Pathology Core at University of Florida for the assistance in hi stological evaluation, im munohistochemistry and H&E staining of tissue and joints. I would like to extend my thanks to C live Wasserfall and Mark Atkinson in the Department of Pathology, Immunology and Laboratory Medicine for their support in performing the cell proliferation assay. My personal thanks go to my mother and my father for their love, support, guidance and encouragement throughout my life. Finally, I woul d like to express my sincerely gratefulness to my girlfriend (Manuela) for her s upport, friendship, kindness and love.
5 TABLE OF CONTENTS page ACKNOWLEDGEMENTS .............................................................................................................4LIST OF FIGURES .........................................................................................................................7ABSTRACT ...................................................................................................................... ...............9CHAPTER 1 INTRODUCTION AND BACKGROUND ...........................................................................11Rheumatoid Arthritis .......................................................................................................... ....11Pathogenesis and Treatment of Rheumatoid Arthritis ..................................................... 11Animal Models for Rheumatoid Arthritis ............................................................................... 14Collagen Induced Arthritis (CIA) ....................................................................................15Pristane Induced Arthritis (PIA) ......................................................................................16Gene therapy ...........................................................................................................................17Gene Therapy for Rheumatoid Arthritis ..........................................................................17Adeno-Associated Virus (AAV) Mediated Gene Delivery ............................................. 19Alpha-1 Antitrypsin (AAT) ....................................................................................................20Doxycycline ............................................................................................................................21Study Overview ......................................................................................................................212 MATERIALS AND METHODS ...........................................................................................24Recombinant Adeno Associated Vi rus (rAAV) Vector Production ....................................... 24Human AAT Protein, Doxycycline and rAAV Vector Administration .................................24Animals ....................................................................................................................... ............25ELISA for the Detection of hAAT and Antibodies ................................................................26Cell Proliferation Assay ..........................................................................................................28Histological Assessment ....................................................................................................... ..28Immunohistochemistry .......................................................................................................... .29Cell Culture .............................................................................................................................29Interleukin-6 Assay .................................................................................................................30Statistical Analysis .......................................................................................................... ........303 ALPHA-1 ANTITRYPSIN PROTEIN AND GENE THERAPIES IN COLLAGEN INDUCE D ARTHRITIS MODEL ......................................................................................... 33Introduction .................................................................................................................. ...........33Experimental Design ........................................................................................................... ...34Protein Therapy Study .....................................................................................................34Gene Therapy Study ........................................................................................................35Results .....................................................................................................................................35Arthritis Development in DBA/1 Mice is Suppressed by hAAT Protein Therapy ......... 35
6 Inhibition of T-Cell Prolifera tion by hAAT Protein Therapy ......................................... 36Human AAT Protein Therapy Reduces th e Levels of Anti-bCII and Anti-mCII Autoantibodies ............................................................................................................. 36Human AAT Gene Therapy Suppres ses Arthritis Development .................................... 37Levels of Anti-CII Autoantibodies are Redu ced in rAAV8-cb-hAAT Injected Mice ....37Discussion .................................................................................................................... ...........374 COMBINATION THERAPY USING DOXYCYCLINE AND AAV8 -TET-ON VECTOR EXPRESSING AAT ..............................................................................................51Introduction .................................................................................................................. ...........51Experimental Design ........................................................................................................... ...52Results .....................................................................................................................................53Administration of AAV8-t et-on-hAAT and Doxycycline Resulted in Sustained Expression of hAAT in DBA/1 Mice .......................................................................... 53Gene Delivery Using rAAV8-tet-on-hAAT in Combination with Doxycycline Suppressed Arthritis Development in CIA Mice ......................................................... 53Histopathological Changes in Mouse Joints are Improved by hAAT and Doxycycline Combination Therapy .............................................................................54Human AAT and Doxycycline Suppress IL6 Expression from NIH/3T3 Cells ........... 54Discussion .................................................................................................................... ...........555 ADENO-ASSOCIATED VIRUS MEDIATED HUMAN AAT GENE THERAP Y IN PRISTANE INDUCED ARTHRITIS MODEL ..................................................................... 67Introduction .................................................................................................................. ...........67Experimental Design ........................................................................................................... ...68Results .....................................................................................................................................68Serum Levels of hAAT and anti-hAAT in PIA Mouse Model Following rAAV8cb-hAAT Mediated Gene Therapy ..............................................................................68Pristane Injection Results in Mild Arthritis Development in Treatment Groups ............ 69Lupus Nephritis Development Tends to be Suppressed by hAAT and Doxycycline Monotherapy ................................................................................................................ 69Effect of Monoand Combination Therapy on Development of Autoantibodies ........... 70Human AAT or Doxycycline Treatment Does Not Change Type of Antibody Response ...................................................................................................................... 70Discussion .................................................................................................................... ...........716 SUMMARY AND PERSPECTIVES .....................................................................................81Summary ....................................................................................................................... ..........81Perspectives .................................................................................................................. ..........84BIOGRAPHICAL SKETCH .......................................................................................................100
7 LIST OF FIGURES Figure page 2-1Vector construct of rAAV-CB-hAAT. ..............................................................................312-2Vector construct of rAAV-tet-on-hAAT. ..........................................................................323-1Experimental design of hAAT protei n therapy in CIA mouse model. .............................. 413-2Severity grades of arthritis development in mouse paws. .................................................. 423-3Serum level of hAAT and anti-hAAT antibodies in CIA mouse model. ........................... 433-4Anti arthritic effect of h AAT in CIA mouse model. .......................................................... 443-5Protein therapy using hAAT reduces splenocyte proliferation. .........................................453-6Anti-collagen II (CII) antibody levels after hAAT treatment. ........................................... 463-7Experimental design for AAV8-CB mediated hAAT gene delivery in CIA mouse model..................................................................................................................................473-8Stable and prolonged expression of hAAT af ter rAAV8-CB-hAAT vector injection. ..... 483-9Human AAT gene therapy delays diseas e progression in CI A mouse model. ..................493-10 Effect of hAAT gene ther apy on autoantibody production. ..............................................504-1Experimental design for combination thera py of rAAV8 tet-on mediated hAAT gene therapy and doxycycline in CIA mouse model. ................................................................. 604-2Human AAT serum and anti-hAAT level in DBA/1 mice received rAAV8-tet-on hAAT and doxycycline. .....................................................................................................614-3Anti-arthritic effect of combinati on therapy in CIA mouse model. ................................... 624-4Tissue protective effect of combinat ion therapy in CIA mouse model. ............................ 634-5Histopathological evaluation of arthritis development. .....................................................644-6Effect of hAAT and doxycycline (DOX) on LPS-induced IL-6 release from mouse embryonic fibroblast cells (NIH/3T3).. ............................................................................. 654-7Effect of hAAT and doxycycline on TNFstimulated IL-6 release from mouse embryonic fibroblast cells (NIH/3T3). .............................................................................. 665-1Experimental design of pristane indu ced autoimmunity in DBA/1 mice. .........................75
8 5-2Human AAT and anti-hAAT antibody levels after rAAV8 mediated hAAT gene delivery in pristane induced mouse model......................................................................... 765-3Evaluation of arthritis development on pristane induced arthritis model. ......................... 775-4Pristane injection caused renal diseas e in pristane injected DBA/1 mice. ........................ 775-5Autoantibody production in pristane treat ed DBA/1 mice after receiving different treatments.. .................................................................................................................. .......785-6Production of lupus autoantibodies after pristane injection in DBA/1 mice. ....................795-7Expression of antibody subclasses in pristane treated DBA/1 mice. ................................. 80
9 Abstract of Dissertation Pres ented 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 PROTEIN AND GENE THERAPIES FOR THE PREVENTION OF RHEUMATOID ARTHRITIS IN MOUSE MODELS By Christian Alexander Grimstein December 2008 Chair: Sihong Song Major: Pharmaceutical Sciences Rheumatoid arthritis (RA) is an autoi mmune disease characterized by immune cell infiltration, synovial hyperplasia, and progressive destruction of bone and cartilage, affecting about 1% of the population in the United States. Recent studies have suggested that RA results from autoimmune self destruction and imbalance between inflammatory and anti-inflammatory network. Human alpha 1 antitrypsin (hAAT) is a multi-functional protein with anti-inflammatory and protective properties. We ha ve previously shown that treatme nt of hAAT prevents type 1 diabetes development and protects islet cells fr om apoptosis in mouse models. However, the effect of hAAT on RA has not been tested. The goal of this study is to determine the pr otective effect of hAAT protein therapy and gene therapy on the development of RA and auto antibody formation in collagen-induced arthritis (CIA) and pristane induced ar thritis (PIA) mouse model. First, hAAT was delivered either intrap eritoneally as a human protein or with a recombinant adeno-associated virus vector expressing hAAT (rAAV8-CB-hAAT) in CIA model. Using these different delivery ap proaches, we examined the feasibility of hAAT as a therapeutic
10 agent for RA. We were able to show that bo th approaches successfully suppressed collagen II autoantibodies and delayed arthritis onset in DBA/1 mice. We intended to further improve our therap eutic approach and de livered rAAV8-tet-onhAAT vector expressing hAAT in combination with doxycycline. Interestingly the combination therapy was more effective in suppressing arthri tis development than monotherapy of hAAT or doxycycline alone. In addition, we tested the feas ibility of hAAT therapy in pr istane induced arthritis mouse model. Although the effect of hAAT on arthritis scores was not signi ficant due to th e variation of the model, hAAT and doxycycline combination therapy was able to suppress lupus related autoantibody anti-Su. These results suggest that hAAT has therapeu tic potential for the prevention of disease progression and the treatment of RA. The therapeutic effect of hAAT appear s to be at least in part through the control of autoantibody development and can be further improved by combination with doxycycline.
11 CHAPTER 1 INTRODUCTION AND BACKGROUND Rheumatoid Arthritis Rheum atoid arthritis (RA) is a systemic auto immune disease, and affects about 0.5 to 1% of the population in the industrialized world (1). As the most fre quent of all inflammatory joint diseases, it is characterized by chronic joint inflammation and synovial hyperplasia leading to bone and joint destruction. Insufficiently treated, RA leads to disability resulting in loss of working capacity, early retirement as well as pr emortal death and theref ore has not only a great impact on quality of life for the individual but also on health car e and socioeconomic systems (2, 3). Pathogenesis and Treatment of Rheumatoid Arthritis Although the exact causes or the initiating stim uli remain uncl ear, extensive research over the last decades have shown that multiple geneti c as well as environmental factors interact and trigger the onset of RA (4, 5). Multiple cell type s, associated with the immune system, enter the synovium via activated endotheli al cells which express vari ous adhesion molecules. Among those cells are dendritic cells, neutrophils, macrophages, fibroblasts, mast cells, natural killer (NK) cells, NKT-cells, T-cells as well as plasma cells. Especially dendritic cells express pattern recognition molecules such as Toll-like receptors, which can bind foreign antigens and self structures, become subsequently activated and act on cells of the adap tive immune system. One of the key players in arthritis pathogenesis are T-cells. They r ecognize antigens which are associated with arthritis and are presente d by professional antigen presenting cells (APCs) such as dendritic cells, macrophage s or activated B-cells. The antig enic peptides are presented by class II MHC molecules, located on the surface of APCs. It has been shown that 80% of RA patients carry a so called shared epitope of the HLA-DRB1*04 cl uster indicating a relationship
12 between disease susceptibility and certain antige nic peptides (6). These peptide-MHC complexes lead to stimulation and expansion of auto antigen specific T-cells in joints or lymph nodes, or both. Besides T-cell receptor e ngagement with peptide-MHC complexes, full T-cell activation requires a co-stimulatory signal which is medi ated through CD80 and CD86, receptors located on the APC surface and the T-cell receptor CD28 (7). To avoid over stimulation, T-cells also e xpress cytotoxic T-lymphocyte antigen 4 (CTLA4) which binds with higher affinity than CD28 to APC receptors CD80/CD86 and conveys inhibitory signals. Abatacept is a recombinan t protein of CTLA-4 and blocks CD80 or CD86 mediated co-stimulation and activation of T-cells and is therefore used therapeutically in rheumatoid arthritis. T-cells in the synovium usually belong to Th1 subset and their up regulation is associated with the expression of various cytokines su ch as interleukin 2 (IL-2) and interferon (INF). Once stimulated, these T-cell are able to induce macrophages, B-cel ls, fibroblasts, and osteoclasts (8, 9). B-lymphocytes express vari ous surface receptors such as their antigen receptors, immunoglobulins, and differentiation antigens, such as CD20 and CD22. They are able to differentiate into plasma cells which secrete antibodies, including autoantibodies to IgG (rheumatoid factor), citrullinated peptides such as vementin, fibrinoge n, cyclic citrullinated peptide or collagen II (10). These autoantibodies are able to form immune complexes that stimulate proinflammatory cytokine production vi a complement and Fc receptor activation (11). It has been shown in different studies that presen ce of autoantibodies is associated with severe rheumatoid arthritis (12, 13). Sin ce B-cells also can serve as APCs, they are also able to activate T-cells and contribute to perpet uation of the autoimmune respons e (14, 15). The survival and activation of B-cells, including expression of di fferent immunoglobulin isotypes, are mediated
13 and maintained by the interaction with T-ce lls and co-stimulation. Rituximab, a monoclonal antibody against CD20 is another recently licensed agent that targets and depletes B cells. In addition to immune cell infiltration into the inflamed synovium we also observe neovascularisation and a large increase in fibroblasts like synoviocytes, which are highly activated and produce cytokines and inflammatory mediators such as pros taglandins and matrix metalloproteinases (MMP) (16, 17). Fibroblasts like s ynoviocytes secrete matrix metalloproteinases (MMPs) direct ly into the synovial fluid a nd also invade the surrounding tissues, leading to destruction of cartilage and bone (18, 19). Many cytokines are secreted by activated cell populations in the synovium (20). Proinflammatory cytokines, such as TNF, IL-1 or IL-6 secreted by activated macrophages and fibroblasts have shown to be important contributors for disease pathogenesis. These cytokines can therefore be therapeutic targets. TNFinhibitors such as Infliximab, Adalimumab and Etanercept, IL-1 receptor antagonist Anakinra, or humanized IL-6 receptor Tocilizumab have been approved and tested in recent years for the treatment of rheumatoid arthritis in humans (21). It is interesting to note that when mo notherapies fail, the therapeutic effect of these biologics can be further enhanc ed by combination therapies with methotrexate, which inhibits lymphocyte proliferation and is a cornerstone drug in RA treatment. (3, 22-25). The use of biologics and disease modifyi ng drugs (DMARD) such as methotrexate, leflonomide, sulfasalazin and hydroxychloroquin e in monotherapy as well as in combination therapy have shown improved treatment success in rheumatoid arthritis patients (26). However, despite promising effects regarding effective suppression of disease development, current therapies are accompanied by side effects and lack of efficiency in some patients. Increased risk of liver damage, infections, malignancies (anti-cy tokine therapy) and te ratogenesis have been
14 reported after treatment in RA patients (27-29). Th ree quarters of patients treated with biological agents do not reach American College of Rheu matology 70% improvement score (21). Therefore there is still need to optimize current therapie s as well as developing new drugs and treatment strategies. Animal Models for Rheumatoid Arthritis There are a num ber of adequate animal m odels for rheumatoid arthritis which are commonly used in arthritis res earch. Their heterogeneity in di sease stimulating factors and presentation of disease progression resemble the heterogeneity of clinical symptoms among patients. They are used to simulate pathogene sis and progression of the human disease and may predict efficiency of therapeutic agents in hum ans. Most models are easy to perform, show reasonable duration of active disease state and reproducibility of data. In many studies arthritis models of rats or mice are used and arthritis development is induced in different ways. Streptococcal cell wall (SCW) induced arthritis is the best characterized arthritis animal m odel in rats. A single injection of peptidoglycan polysaccharide leads to chronic, severe a nd erosive arthritis development within days. The model is characterized by a high incidence of 95% and females are more effected then male, similar to what is observed in humans (30). Interestingly SCW induced arthritis in mice displays a short active state of disease of less then 7 days and absence of the chronic phase (31). The SCW induced arthritis model as well as collagen induced arthritis, which is de scribed below, develop arthritis due to administration of foreign an tigens following an antibody dependent immune response targeting the joint tissue. It also ha s been shown that inje ction of anti-collagen antibodies cocktail or immune complexes carryin g joint unrelated proteins are able to induce severe and chronic arthritis in susceptible mouse strains. In travenous injection of heatinactivated polyclonal rabbit anti -lysozyme serum, followed by an injection with poly-lysine-
15 coupled lysozyme leads to immune complex form ation and subsequent in flammatory arthritis (32). In contrast to the described models above, some mouse strains deve lop arthritis without prior administration of external antigens, adjuvant or antibodies. Due to their genetic background K/BxN or NZB/NZW mice develop inflammatory and progressive arthritis spontaneously. K/BxN mice are characterized by a mutation in a T-cell receptor and therefore develop antibodies to ubiquitous glucose-6-phosphate isomerase. Formation of immune complexes including these antibodies is responsible for arthritis development in these mice at the age of 3 weeks (33). NZB/NZW mice also spontaneously develop inflammatory arthritis which is characterized by generation of IgG and IgM rheumatoid factor, similar to human disease. As in pristane induced arthritis mouse model which is re viewed in more detail below, these mice also develop lupus related autoantibodies and are th erefore used as a model for systemic lupus erythematosus(34). Other genetic models include gene modifications in TNFgene or knockout of IL-1 receptor antagonist leading to inflammatory arth ritis and emphasizing the importance of these two cytokines in dis ease pathogenesis(34). For our studies, we employ collagen induced arthritis mouse model which is commonly used, well described and displays reproducibility, high incidence and convenient induction. The pristane model we used displays slightly differe nt disease conditions beca use it is different in terms of time of disease onset a nd duration of dis ease progression. Collagen Induced Arthritis (CIA) Collagen induced arthritis (CIA) has been pr oven to be an excellent animal m odel for rheumatoid arthritis since it shar es several characteristics with the human disease. Among those are genetic, pathological and im munological features. CIA has be en developed in a number of
16 species including mice, rats and monkeys. Although initially developed in rats, CIA is now the most widely used mouse model for studying autoimmune arthritis pathogenesis. After immunization with type II collagen these animal s develop progressive and inflammatory arthritis within days as well as high serum levels of autoreactive antibodies against collagen II. These auto-antibodies are involved in triggering inflammatory pathways via the activation of the complement cascade (35). The susceptibility is clear ly associated with the expression of specific class II MHC genes and it has been shown that bo th T and B cells responses are required to establish the pathogenesis. Pristane Induced Arthritis (PIA) Pristan e (2,6,10,14-tetramethylpentadecane) is a mineral oil which induces a chronic inflammatory arthritis in mice after intraperitoneal injection (36). Like ot her models of arthritis such as adjuvant-induced arthritis, collageninduced arthritis, and streptococcal cell wall induced arthritis, PIA resembles the joint inflammation and destruction seen in human RA. The main histological features are synovial hyperplasi a, inflammatory cell in filtrate, bone erosions, cartilage erosions, and pannus-like formation. PIA demonstrates a prolonged, delayed clinical time course of joint inflammation which can star t between 60 days and 18 0 days after pristane administration (36, 37). The condition is normally permanent, thus providing extended periods of disease for experimental investigation. Like CIA, pristane induced arthritis is an inflammatory joint disease accompanied by hypergammaglobulinemia with a profile of antibodi es against HSP and collagen II as well as the presence of rheumatoid factor. In addition to that, susceptible mice also have been shown to develop lupus specific autoantibodies against nucl ear proteins (anti-Su, anti-RNP, anti-Sm, antiDNA, anti-P).
17 The precise mechanism underlying PIA remains unclear, but it has been shown that pristane facilitates autoimmune responses via immune activation in response to antigens found on prevalent microbes in the environment (38, 39) Therefore, housing mice in a pathogen-free environment precludes development of PIA, wher eas returning such mice to normal environment reestablishes their susceptibility. Gene therapy In recent years, gene thera py has becom e a promising appro ach to mediate expression of therapeutic proteins in target tissues. Viral vect ors such as lentivirus, retrovirus, adenovirus and adeno-associated virus or non-vira l vectors such as liposomes or plasmid DNA are used for gene delivery (40, 41). Since the firs t clinical study using gene tran sfer was conducted by Rosenberg and his colleagues in 1990, the field has rapidly grown moving from preclinical studies to clinical studies for many diseases. Due to cons tant improvements of ge ne therapy vectors in terms of efficiency and safety, we are now able to ensure long term gene expression resulting in high levels of therapeutic proteins in serum and various tissues. Several clinical gene therapy tr ials have been conducted or are currently in progress. They target diseases such as cystic fibrosis, al pha-1 antitrypsin defici ency, muscular dystrophy, Parkinson disease, cancer, hemophilia, Alzheimers disease and severe combined immunodeficiency (42, 43). Gene Therapy for Rheumatoid Arthritis Gene therapy is a prom ising therapeutic approach for RA treatment since it overcomes some drawbacks of currently applie d protein therapies. It resemble s an effective, inexpensive and continual delivery system in which regular inje ctions of protein is not necessary. The most prominent viral vectors used in arthritis therapy include retrovir uses, lentiviruses, adenoviruses, herpes simplex viruses as well as adeno-associated viruses (44).
18 Several studies conducted in animal models showed that gene therapy delivering IL-1 receptor antagonist (IL-1RA), IL-18 binding protein, or TNFreceptor variants effectively reduced collagen induced arthritis developmen t in mouse model (45-50 ). Besides targeting cytokines, other studies using ge ne therapy showed that deliveri ng transgenes which target the degradation of extr acellular matrix has signifi cant effect on suppression of arthritis development in rodents. Delivering tissue i nhibitor of matrix metalloprotei nases such as TIMP1, TIMP3 and TIMP4 or ribozymes targeting MMP-1 and cathepsin L mRNA have shown promising results using adenoviruses, retroviruses or electroporation (51-54). In addition anti-angiogenic gene therapy employing adenovirus or adeno associated virus to deliver proteins with anti-angiogenic properties such as angiostatin, human kallistatin TSP-1 or soluble sFlt -1 showed promising potential to reduce arthritis development in mice and rats (55-57). Targeting apoptosis using adenovirus to deliver Fas-ligand or TRIAL or delivering anti-infla mmatory cytokines such as IL4 and IL-10 also showed that ther e is indeed a wide range of poten tial target molecules which are currently used for anti-arthrit ic gene therapy (58-65). Less than 10 gene therapy clini cal trials have been initiated for the treatment of rheumatoid arthritis to date (66). For most of them results ha ve not been published yet. In one clinical trial, an IL-1RA transgene has been ex vivo transduced by an retrovirus and a dose escalation study in 9 volunteers confirmed f easibility and safety as well as ev idence supporting symptomatic relief (67, 68). Another study, sponsored by ISIS pharm aceuticals, was designed to deliver antisense oligonucleotides, reducing the production of TN F by blocking mRNA. The study aimed to assess safety and efficacy in a randomized double blind, placebo control design. No results have been published yet, but a press release on the compan ies website suggests pos itive disease response in patients receiving the highest dose.
19 The most recent human clinical arthritis trial performed by Targ eted Genetics, in which i.a. delivery of a TNFreceptor was mediated by adeno-associ ated virus serotype 2 (rAAV2) also revealed promising results. The phase 1/ 2 study showed improvement of joint symptoms, function and pain. Although one pa tient died after receiving a s econd injection of AAV2 and the FDA put the trial temporarily on hold, it was c onfirmed that AAV gene therapy was not related to the incidence and a severe histoplasmosis infection most likely caused the patient death (69). Using gene delivery vehicles to specifically target cells and mediators involved in disease development reveal a promising pot ential for effective arthritis th erapy. Further research has to be conducted to elucidate safety and long term effi ciency of this novel treatment approaches. Adeno-Associated Virus (AAV) Mediated Gene Delivery Adeno-associated viruses (AAV) are sm all, non enveloped singlestranded parvoviruses that depend on a viral helper, such as adenovirus or herpes simplex virus, to facilitate productive infection and replication. In th e absence of a helper virus, AAV establishes a latent infection within the cell, resulting in long-term expression in vivo, either by site-specific integration into the host genome or by persisting in episomal fo rms. AAV has never been associated with any human disease and recombinant AAV vectors have never led to significant inflammatory responses or toxicity in animal models and human c linical trials (70). In this sense, rAAV is the safest of the currently used gene therapy vector s. Because of its propensity to establish latency and because it has not been implicated as a pathogen, AAV has been of consid erable interest as a potential vector for human gene therapy (71) In addition rAAV has mediated longterm transgene expression in a wide va riety of tissues, including muscle (72-76), lung (77), liver (76, 78-80), brain (81) and eye (82). Therefore rAAV vectors appear to have significa nt advantages over other commonly used viral vectors.
20 So far, 12 primate rAAV serotypes have been isolated and cloned. Serotype 2 (AAV2) is the best-characterized and has been predominantly used in gene th erapy studies. Apparailly et al. recently demonstrated that rAAV5 is the preferred serotype for local gene delivery to joints in RA since it efficiently transduces synovial cells (83). rAAV 8 has been shown to be able to very successfully transduce murine liver (84) and muscle cells (85) in addition to a wide range of other tissues. The different tr opism of AAV serotypes provide s opportunities to optimize the transduction efficiency in different target cells. It is therefore the vector of choice when systemic gene delivery is desired. Alpha-1 Antitrypsin (AAT) Hum an alpha-1 antitrypsin (hAAT) is a 52 kD a serum glycoprotein, synthesized primarily in the liver. It is also expre ssed in other types of cells in cluding neutrophils, monocytes, macrophages, alveolar macrophages, intestinal ep ithelial cells, carcinoma cells and the cornea (86-89). The normal serum level of hAAT is 12 mg/ml. During inflammation, hAAT level, as an acute phase reactant, can increase 3-4 folds. This makes it an ideal candidate protein as potential anti-inflammatory agent since it could be used in a wide therapeutic range without toxic side effects. Increasing eviden ce indicates that hAAT is imm unoregulatory, anti-inflammatory and may be used for the treatmen t of RA. It inhibits neutrophil elastase and proteinase 3 with high efficiency, as well as cat hepsin G, thrombin, trypsin and chymotrypsin with lower efficiency (90). Most of thes e proteases target receptor protei ns, involved in proinflammatory cytokine expression and cell signalin g (91). It also has been re ported that neutrophil elastase inhibitors reduce incidence as well as severity of collagen-induced arthritis (CIA) in both rats and mice (92). Human AAT is able to completely eliminate the acute inflammatory infiltration and connective tissue breakdown in the lung in a cigarette smoke induced emphysema mouse model (93). It also inhibits LPS stimulated release of TNFand IL-1 and enhances the
21 production of anti-inflammatory cytokine IL10 (94-96). Human AAT si gnificantly protects against the lethality induced by TNFor endotoxin in mice (97). It can also induce expression of IL1-Ra in human PBMCs (98) and reduces ischemia-induced apoptosis and inflammation (99). Doxycycline Doxycycline, commonly used as an anti-m icrobi al agent, has also shown to hold tissue protective properties. It is able to inhibit bon e and cartilage breakdown in an animal model of osteoarthritis, and it also inhibits matrix meta lloproteinases specifically the collagenases MMP8 and MMP13 (100, 101). Both collagenases are upregul ated in RA patients and contribute to disease development of rheumatoid arthriti s (102-104). Doxycycline also increases RNA degradation of nitric oxide synthase which usually increases ca rtilage breakdown and stimulates MMP expression in synovial cells (105). Fu rthermore, doxycyclin e also possesses antiinflammatory effects. It induces FAS/FAS-Liga nd mediated apoptosis in T-lymphocytes (106), and inhibits staphylococcal exotoxin stimulated T-cell prolif eration and proinflammatory cytokine/chemokine producti on in human peripheral blood mononuclear cells (107). Recently it has been shown, that combinati on therapy using doxycycline and methotrexate showed a significant American College of Rheumatology 50% improvement (ACR50) response in early seropositve rheumatoid arthritis patients when compared to methotrexate monotherapy (108). This study revealed promising potential for the use of doxycycline in combination therapies with other antiarthritic drugs. Study Overview The ultim ate goal of the research presented in th is dissertation is to test the feasibility of AAT as a therapy for rheumatoid arthritis. In recent years new developments have been made concerning RA pathogenesis and it has been shown, that reduction of inflammation and cartilage
22 destruction can be effectively achieved through targeting proinf lammatory cytokines such as TNFand IL1. AntiTNFand anti-IL1cytokine therapy is now widely used for RA treatment and we believe that AAT with its anti-inflammatory and immune modulatory proper ties is effective in suppressing arthritis development by reducing joint inflammation. It may also avoid various side effects which are commonly seen in standard RA therapy. Chapter 2 describes the experi mental procedures used. Chap ter 3 covers studies which were performed to test the feasibility of AAT to suppress arthritis deve lopment. We initially performed a protein therapy study to determine whether regular injection of commercially available hAAT protein (prolastin) is able to reduce arthritis developm ent in collagen induced arthritis mouse model. In addition we performed AAV mediated hAAT gene therapy in CIA model. Delivering hAAT in two different ways by employing protein as well as gene therapy intended to reveal the general potential effect of hAAT on RA development. Furthermore we also aimed to display a possible mechanis m describing how AAT suppresses arthritis development. Therefore we determined levels of common RA biomarkers such as antibodies to collagen II in mouse serum and also tested hAAT effect on T-cell proliferation. In Chapter 4 we specify the development of a combination therapy in CIA mouse model using hAAT and doxycyline. This approach has reference to common RA treatment practice since most therapies employed today are combina tion therapies of commonly used anti-arthritic drugs. Drug combination showed improved treat ment effects in many RA patients. The experimental approach described in Chap ter 5 will further reveal the potential of hAAT on suppressing arthritis development and reducing autoantibodies. By using pristane induced arthritis mouse model we further widen th e experimental setup in which anti-arthritic
23 properties of hAAT are tested. In addition we also intend to test the feasibility of the combination therapy using hAAT and doxycyline in this model and its potential to reduce autoantibodies. Chapter 7 summarizes major findings and emphasizes future directions of the projects described.
24 CHAPTER 2 MATERIALS AND METHODS Recombinant Adeno Associated Virus (rAAV) Vector Production The rAAV-CB-hAAT v ector construct was pr oduced and packaged as previously described (82). Briefly, this vector carries hAAT cDNA driven by the cytomegalovirus (CMV) enhancer and chicken -actin promoter and contains AAV2 inverted terminal repeats (ITRs) (Figure 2-1). The rAAV-tet-on-h AAT vector contains a bidirectional promoter with a tetracycline response elements (TRE) flanked by two mini-CMV promoters controlling hAAT and reverse transcriptional transactivator (rtTA) genes. In the presence of doxycycline, rtTA will bind to the TRE and activate hAAT and rtTA ge nes. The rtTA gene serves as a positive regulatory system for hAAT gene transcription (Figure 2-2). The respective plasmids were packaged into AAV serotype 8 capsid by cotransf ection of vector plasmid and helper plasmid (XYZ8) into 293 cells. rAAV8-CB-hAAT and r AAV8-tet-on-hAAT vectors were purified by iodixanol gradient centrifugation followed by anion-exchange chromatography. The physical particle titers of vector preparations were assessed by dot blot analysis. Human AAT Protein, Doxycycline and rAAV Vector Administration For hAAT protein therapy studies, D BA/1 mice were intraperitoneally (IP) injected with 0.5 mg of hAAT in 100 l saline (Prolastin, Bayer Cor p., Elkhard, IN). The control group received saline injection. The injections were performed twice per week, starting from 6 days before the first bovine type II collagen (bCII) immunization. For hAAT gene therapy studies, DBA/1 mice were IP injected with rAAV8-CB-hAAT vector (2x1011 particles/mouse) or sa line two weeks before th e first bCII immunization. The combination therapy study consisted of f our treatment groups, receiving a single IP injection containing rAAV8-tet-on-hAAT vector (1x1011 particles/mouse) or saline four weeks
25 before the first CII immunization. Some gr oups also received doxycycline during the study. Doxycycline was administered with food (ad li bitum, 200mg/kg food). The groups represent hAAT and doxycycline combination therapy, doxycycline monothera py, hAAT monotherapy and saline control group. For studies using pristane indu ced arthritis model, cohorts of DBA/1 mice were IP injected with rAAV8-cb-hAAT (2x1011 particles/mouse) or saline. Some of the vector or saline injected mice also received doxycycline-co ntaining food (ad libitum, 200mg /kg food). Therefore, there are 4 groups of mice: hAAT plus doxycyclin e, hAAT, doxycycline and saline group. Three weeks after vector inject ion, all animals received a single pris tane injection to induce arthritis and lupus development. Animals Housing : D BA/1 mice were purchased from Harlan Sprague Dawley, Inc. (Indianapolis, IN), housed in a specific pathogen-free room and handled as approved by the University of Florida Institutional Animal Care and Use Committee. Collagen induced arthritis (CIA) mouse model: For induction of arthritis, bovine type II collagen (bCII, Chondrex LLC, Redmond, WA ) was dissolved in 0.05N acetic acid at a concentration of 2mg/ml by stirri ng overnight at 4C and emulsifi ed with an equal volume of Complete Freunds Adjuvant (CFA, Chondrex LLC, Redmond, WA) or incomplete Freunds Adjuvant (IFA, Difco, Detroit, MI). Six week-old male DBA/ 1 mice were first immunized by subcutaneous injection at the base of th e tail with 0.1ml of emulsion containing 100 g of bovine type II collagen. Three weeks after priming (day 21), the mice were boost ed with 0.1 ml of bovine collagen II (100 g) emulsified in equal volume of incomplete Freunds Adjuvant (IFA, Difco, Detroit, MI).
26 Pristane-induced arthritis mouse model: For pristane induced arthritis study, twelve week-old female DBA/1 mice received a single intraperitoneal injecti on of 0.5ml pristane (Aldrich Chemical Co., St Louis, MO) to induce arthritis and lupus development. Evaluation of arthritis and lupus development: For assessment of arthritis, all mice were regularly monitored by the same person bli nded to the treatment group and evaluated for incidence of arthritis and clinical score. An arthritis score system ranging from stage 0 4 was used (Figure 3-2): 0: no swelling or redness; 1: detectable arthri tis with erythema; 2: significant swelling and redness; 3: severe sw elling and redness from joint to digit; 4: joint stiffness or deformity with ankylosis (109). The clinical score was expressed as the average cumulative value of all four paws with a maximum score per animal of 16. Severe arthritis was defined as arthritis score >3 for the purpose of comparing data between groups. Serum was collected every 2 to 4 weeks for determination of hAAT and an tibodies in serum. At 34 weeks after pristane injection, proteinurea was determ ined in urine using albustix (B ayer, Elkhart, IN), where 0 = absent, 1+ 30 (mild), 2+ = 100 (moderate), 3+ = 300(severe), and 4+ = 2000 mg/dl (very severe). All animals were sacrificed at the end of th e respective study. ELISA for the Detection of hAAT and Antibodies Detection of hAAT and anti-hAAT antibodies in mouse serum was perform ed by standard ELISA as previously described (110). Purified hAAT was used as standard for detection of hAAT protein in serum (Athens Research & Technology, Athens, GA). Hi gh titer antibody sera were used as standard for determinat ion of respective an tibody levels. Anti-type II collagen antibodies in mouse seru m were also detected by a standard ELISA. Briefly, microtiter plates (Immulon 4, Dynex Technologies, Chantilly, VA) were coated with bCII or mCII (0.5 g/well, Chondrex LLC, Redmond, WA) in Vollers buffer overnight at 4C. After blocking with 3% bovine serum albumin, d iluted serum samples were added and incubated
27 at room temperature for 2 h. HRP-conjugated go at anti-mouse IgG antibodies (1:1,000 dilution, Sigma, St. Louis MO), goat anti-mouse IgG1 antibodies (1:1,500 dilution, Roche, Indianapolis, IN) or goat anti-mouse IgG2 a antibodies (1:1,500 dilution, Roche, Indianapolis, IN) were incubated for 1 h at RT. The plates were wash ed with PBS-Tween 20 be tween reactions. After adding the substrate (o-phenylened iamine, Sigma, St Louis, MO) plates were read at 490 nm on an MRX microplate reader (D ynex Technologies, Chantilly, VA) Optical densities were converted into units based on a standard curve ge nerated with high titer sera from DBA/1 mice immunized with bCII. Anti-chromatin antibodies were detected in mouse serum (at a 1:500 dilution) using an ELISA with chicken erythrocyte chromatin, as desc ribed previously (111). In brief, microtiter plates (Immulon 4, Dynex Technologies, Chantilly, VA) were coated with 5 g/ml chicken erythrocyte chromatin in borate buffered saline ov ernight at 4C. Sera were tested at a 1:500 dilution, followed by alkaline phosphatase c onjugated goat antimouse IgG (1:1,000 dilution, SouthernBiotech, Birmingham, AL) and p-nitroph enyl phosphate substrate (Sigma, St Louis, MO). Antigen-capture ELISAs were performed to detect anti-nRNP/Sm, anti-ribosomal P and anti-Su in the mouse serum (at a 1:500 dilution) as described previ ously (111, 112). Briefly, microtiter plates (Immulon 4, Dynex Technologi es, Chantilly, VA) were coated with 1 g P peptide or 50 l of cell lysate from K592 cells (for antiSu and anti-nRNP/Sm) overnight at 4C. Sera were tested at 1:500 dilution followed by alkaline phosphatase conjugated goat anti-mouse IgG (1:1,000 dilution, SouthernBiotech, Birm ingham, AL)) and p-nitrophenyl phosphate substrate (Sigma, St Louis, MO).
28 Levels of anti-dsDNA and antisingle-str anded DNA (anti-ssDNA) antibodies were determined in the sera using S1 nucleasetreated calf thymus DNA as antigen (heat denatured for ssDNA) (111). In brief, microtiter plates (Imm ulon 4, Dynex Technologies, Chantilly, VA) were coated with 3 g/ml DNA in Pierce Reacti-Bind DNA co ating solution (Pierce Biotechnology Inc. Rockford, IL) overnight at 4C. Sera were tested at a 1:500 dilution, followed by alkaline phosphatase conjugated goat antimouse IgG (1: 1,000 dilution, SouthernBiotech, Birmingham, AL) and p-nitrophenyl phosphate substrate (Sigma, St Louis, MO). Total immunoglobulin levels were measured by ELISA as described previously (113). Briefly, 96-well microtiter plat es (Immulon 4, Dynex Technologies Chantilly, VA) were coated at 4C overnight with 50 l/well of goat anti-mouse kappa/lambda light chain antibodies (9:1 ratio, Southern Biotechnology, Birmingham, AL). Murine sera were tested at 1:200,000 dilution followed by a 1:1000 dilution of alkaline phosph atase-labeled goat anti-mouse antibodies specific for IgG1, IgG2a, IgG2b, IgG3 or Ig M (Southern Biotechnology Birmingham, AL) and p-nitrophenyl phosphate substrat e (Sigma, St Louis, MO). Cell Proliferation Assay To test the effect of hAAT protein adm inistration on splenocyte proliferation, spleens from treatment and control group were ha rvested. Splenocytes were isolated and cultured in triplicates (4 x 105 cells/well, in 96-well plate) in serum free X-VIVO medium (Cambrex, Walkersville, MD) in the presence or absence of ConA (0.5 g/ml, Sigma, St.Louis, MO). After culturing for 3 days, 1 Ci/well of [3H] TdR was added. Cells were cultured for additional 18h and [3H] TdR uptake was measured using a scintillation counter. Histological Assessment For the ana lysis of acute arthritis in CIA m odel, mice were anesthetized and sacrificed by cervical dislocation. The two hind limbs of mice in treatment or control group were removed.
29 Specimens were fixed in formalin and decalcifi ed in RDO solution (Apex, Aurora, IL) for 10-20 min depending on tissue size and then checked manually for pliability. Joint tissues were cut into 4 m thick sections and stained with hematoxylin and eosin according to standard methods. Histological evaluation was performed by tw o independent and blinded pathologists. Infiltration of immune cells, hyperplasia, pannus formation and bone deformation was determined for each paw using an evaluation sc ale ranging from 0-4 according to severity of histopathological changes (0: normal, 1: mild, 2: moderate, 3: seve re, 4: very severe). The score was then averaged for each treatment group. Immunohistochemistry In order to detect transgene (hAAT) expr ession fro m rAAV8-cb-hAAT vector, saline and vector injected animals were s acrificed. From each animal, liver was harvested, fixed in 10% neutral-buffered formalin and submitted for rout ine paraffin processing. Formalin-fixed paraffinembedded tissue sections (4 m) were sequentially deparaffinized, rehydrated, and blocked for endogenous peroxidase activity. Follo wing antigen retrieval in Targ et Retrieval solution (DakoCytomation, Carpinteria, CA), s ections were blocked with serum and incubated with either rabbit anti-hAAT (1:100; Research Dia gnostic Institute, Flanders, NJ ) or normal rabbit serum as a negative control. Antibody binding was detected using the Mach2 Polymer (Biocare, Concord, CA) and DAB+ (Biocare, Concord, CA). Slides we re counterstained using hematoxylin (Vector Labs, Burlingame, CA) and mounted. Digital im ages were captured using a Zeiss Axioskop equipped with an Axiocam camera. Camera expos ure settings were constant for all images. Cell Culture Mouse em bryo fibroblasts cells (NIH/3T3) were purchased from American Type Culture Collection (Rockville, MD) and maintained in DMEM/10% Bovine Calf Serum (BCS) culture
30 medium (DMEM containing 10% heat-inactiv ated BCS, 100 units/ml of penicillin, 100 g/ml streptomycin) at 37C in a 5% CO2 incubator. For measuring IL-6 release, cells were seeded at 1 105/ml in 12 well plates. After 48 h at 37C, the culture medium was replaced with 0.5 ml of fresh, DMEM/10% BCS culture medium and used for experimental studies. Cells were incubated with hAAT (Pro lastin), doxycycline or both agents in combination for 6h and then stimulated with LPS (1 g/ml) or TNF(10ng/ml). 16-20 hours after stimulation, IL-6 secretion into the culture me dium was determined by ELISA. Interleukin-6 Assay Murine IL-6 in culture m edium of NIH/3T 3 cells was determined by standard ELISA according to manufacture instructions (eBioscience, Inc., San Diego, Ca.). The level of quantification was 4 500 pg/ml. Statistical Analysis Analysis of the data was perform ed using Gr aphPad Prism 4.0 (GraphPad Software). Each data set was tested for variance and normality. Ba sed on the results, the appropriate parametric or nonparametric tests were applied. P < 0.05 was considered statistical ly significant.
31 Figure 2-1. Vector construct of rAAV-CB-hAAT. The construct is flanked by two AAV inverted terminal repeats (ITR) and consists of a hAAT gene under the control of a CBpromotor followed by polya denylation site (An).
32 Figure 2-2. Vector construct of rAAV-tet-on-hAAT. Bidirecti onal promoter that contains tetracycline response elements (TRE) fla nked by two mini-CMV promoters controls hAAT and rtTA genes. In the presence of doxycycline (Dox), rt TA will bind to the TRE and activate hAAT and rtTA genes. The rtTA gene serves as a positive regulatory system for hAAT gene tran scription. An = polyadenylation site
33 CHAPTER 3 ALPHA-1 ANTITRYPSIN PROTEIN AND GENE THE RAPIES IN COLLAGEN INDUCED ARTHRITIS MODEL Introduction Rheum atoid arthritis (RA) is a chronic infl ammatory disease affecting about 1% of the population in the United States. Recent studies have suggested that RA results from autoimmune self destruction and imbalance between infl ammatory and anti-inflammatory network and extensive research over the last decades revealed new treatment strategies. These mainly include biologics targeting key players of synovial inflammation such as TNFand IL-1 which have shown promising results in a number of patients. However, this new generation of cytokine targeting drugs does have serious si de effects such as increased risk of infections, concerns about malignancies and other adverse out comes. Therefore development of safe and effective treatment approaches is desirable. Our previous studies in NOD mouse model showed that human alpha-1 antitrypsin (hAAT) gene therapy decreased levels of insulin auto an tibodies, attenuated cellular autoimmunity and prevented autoimmune diabetes implying that hAAT may be used for other autoimmune diseases (110). In the present study we intended to elucidate th e feasibility of hAAT for the treatment of RA We hypothesize that hAAT as a mu lti-functional protein with antiinflammatory, immunoregulatory an d tissue protective properties di splays therapeutic potential for RA. To test our hypothesis we developed hAAT protein as well as adeno-associated virus mediated gene therapy in collagen induced arthritis, a commonly used, well established mouse model for RA. The first study described in this chapter em ployed hAAT protein therapy as a convenient approach to determine the potential of hAAT in arthritis therapy. Human AAT protein was
34 delivered intraperitoneally into DBA/1 mice and arthritis development was evaluated according to paw swelling and joint deformity. AAT protein therapy in humans is both expensive and inconvenie nt due to the necessity of repeating injections, therefore we also evaluated an alternative approach using rAAV serotype 8 to deliver the hAAT gene. Since antibodies against type II collagen ar e essential for disease development in CIA mice, and it has been shown that inhibition of their development is acco mpanied with a reduction of disease severity, we also tested the effect of hAAT therapy on autoantibody development in CIA mouse model (114, 115). Experimental Design Protein Therapy Study At the age o f 8 weeks, arthritis was induced in DBA/1 mice by two bCII injections as described in Material & Methods. One group of mice (n=9) received hAAT protein injections twice/week starting six days befo re the first bCII immunization, another group (n=7), served as the control group and received saline injections (Fig.3-1). Arthritis evaluation was star ted after the second collagen injection on day 21 and was performed by a blinded investigator three time s per week. The evaluation was based on a well established grading scale for arth ritis development as described in Materials & Methods. Serum samples were taken every other week to determin e hAAT protein level as well as anti-hAAT and anti-CII antibody levels by ELISA. On day 70 after immunization, mice were sacr ificed and splenocytes were isolated to perform T-cell proliferation assay.
35 Gene Therapy Study DBA/1 m ice (n=10) were IP injected with rAAV8-CB-hAAT vector (2x1011 particles/mouse, Figure 2-1). The control group (n= 10) received a single saline injection at the same time (Figure 3-7). Two weeks later, ar thritis was induced by tw o bCII injections as described in Materi als and Methods. As for protein therapy, arthritis evaluation started after the second bCII immunization and was performed three times a week by a blinded i nvestigator using the 0 to 4 scoring scale for arthritis development. Every other week, seru m samples were collected to measure hAAT protein as well as anti-hAAT and anti-CII anti body levels by ELISA. Mice were sacrificed on day 56 after the first immunization. Results Arthritis Development in DBA/1 Mice is Suppressed by hAAT Protein Therapy In order to investigate the effect of hAAT on developm ent of arthritis, we first examined the feasibility of hAAT protein therapy in CIA mouse model. Ad ministration of hAAT resulted in sustained high levels of hAAT in mouse serum (Figure 3-3A). Although anti-hAATantibodies were detected (Figur e 3-3B) serum levels of hAAT did not decrease over time. A few days after the second immunization wi th bCII (day 21), mice in control group developed arthritis in multiple joints, which was manifested by redness, severe joint swelling and joint stiffness as well as ankylos is as the disease progressed. The severity of arthritis as measured by the arthritic score rapidly increased in control group until a maximum score of 15 1 was reached at day 63. Interestingly, the deve lopment of arthritis was suppressed in hAAT treated group as indicated by a significantly re duced clinical score (F igure 3-4A) and lower incidence of severe arthritis (score >3) (p = 0.0025, logrank test, Figure 3-4B). Moreover, mice in hAAT treated group had significantly delayed ons et of arthritis compared with control group.
36 The clinical signs of severe arthritis started on day 47.3 8.7 in hAAT treated group compared to day 36.0 5.8 in control group (p = 0.01 by stude nts t-test). In addition, the numbers of arthritic paws were also signi ficantly reduced in hAAT treated group (p< 0.05 by Fishers exact test, Figure 3-4C). These results showed that treatment with hAAT protein led to a delayed arthritis onset and amelioration of dis ease progression in CIA mouse model. Inhibition of T-Cell Proliferati on by hAAT Protein Therapy Since CIA is a T-cell-mediated autoimmune dis ease, the effect of hAAT on T-cell function was examined by a T-cell proliferation assa y. As shown in Figure 3-5, ConA induced proliferation of splenocytes from hAAT protein treated group was significantly lower than that of control group (p<0.05). Human AAT Protein Therapy Reduces the Levels of Anti-bCII and Anti-mCII Autoantibodies It has been shown that high levels of serum anti-collagen II autoantibodies are pathognomonic and associated with the development of arthritis (115, 116). To test the effect of hAAT on autoantibody production, we evaluated the le vels of anti-CII autoan tibodies in total Ig, and IgG1 and IgG2a subclass at early (day 35) and late (day 49) stage of the disease. As shown in Figure 3-6A, hAAT treatment did not result in a significant change of total autoantibody levels against bCII (total anti-bCII-Ig). However, hAAT treatment significantly reduced the pathognomonic IgG2a (anti-bCII-IgG2a) levels at day 35 (Figure 3-6B), and increased IgG1 (anti-bCII-IgG1) levels at day 49 (Figure 3-6C), consistent w ith a shift from a Th1 to Th2 response. Interestingly, total Ig autoantibodies against endogenous mouse co llagen II (total antimCII-Ig) were significantly lowe r in hAAT protein treated group than that in control group (p<0.05) (Figure 3-6D).
37 Human AAT Gene Therapy Suppresses Arthritis Development To further confirm our observation that hAAT is effective in suppressi ng arthritis, we used recombinant adeno-associated virus vector (rAAV) to delive r the hAAT gene. A single IP injection of rAAV8-CB-hAAT vect or resulted in sustained levels of hAAT in the circulation (Figure 3-8A). Interestingly, we did not observe the development of antibodies to hAAT after AAV8 mediated gene delivery (Figure 3-8B, comp are vs. Figure 3-3B in mice with hAAT protein therapy). Similar to th e results from hAAT protein th erapy, rAAV-mediated hAAT gene therapy significantly reduced arthritis developm ent (Figure 3-9A) and delayed the onset of the disease in CIA mouse model as indicated by a reduced severe ar thritis incidence in treatment groups compared to control (p=0.028 by logrank test, Figure 3-9B). We also observed fewer arthritic paws in mice receivi ng rAAV8-CB-hAAT compared to c ontrol (p< 0.05, Figure 3-9C). Levels of Anti-CII Autoantibodies are Re duced in rAAV8 -cb-hAAT Injected Mice As shown in Figure 3-10, rAAV8-mediated hAAT gene therapy resulted in a significant suppression of anti-CII autoantibody production. The le vels of total Ig anti-bCII (Figure 3-10A) and IgG2a anti-bCII (Figure 3-10B) were signi ficantly reduced in hAAT gene therapy group. Although IgG1 anti-bCII levels (F igure 3-10C) were also lower in hAAT gene therapy group, the ratio of IgG2a anti-bCII to Ig G1 anti-bCII (Figure 3-10D) was significantly decreased in hAAT gene therapy group, suggesting a shift from Th1 to Th2 skewed response. Importantly, hAAT gene therapy also reduced levels of autoantibodi es against mCII and the ra tio of IgG2a anti-mCII to IgG1 anti-mCII (Figure 3-10E-H). Discussion RA is a complex system ic autoimmune disease with unknown etiology. Although recently developed biologicals that target TNF-alpha have provided dramatic improvement in controlling disease activity in many patients, continued searches for more efficient and safer treatments are
38 still needed. In the present st udy we showed that hAAT, admini stered as protein or through rAAV8 mediated gene therapy, reduced anti -CII autoantibodies and significantly delayed arthritis development in a mouse model. To date, this is the first report on beneficial effects of hAAT in tr eatment of arthritis. Although the exact mechanisms underlying the th erapeutic effect rema in to be further investigated, several possible mechanisms may be involved. One is through the inhibition of proinflammatory cytokine producti on. It is well known that various proinflammatory cytokines, including TNFand IL1, play major roles in the pathogen esis of RA (117, 118). Strategies targeting these cytokines have proven to be effective in treatment of RA (21). Previous work done by Janciauskiene and her colleagues clearly demonstrated that hAAT inhibited LPSinduced TNF, IL-6 and IL-1 production by human monocytes (94, 95). In addition, hAAT completely suppressed macrophage inflammatory protein-2 (MIP-2)/monocyte chemotactic protein-1 (MCP-1) gene expression in lung (1 19). hAAT also enhanced anti-inflammatory cytokine IL-10 production from monocytes (94). It is possible that the eff ects of hAAT on cytokine produc tion contribute to shift the immune response from Th1 to Th2 pathway an d inhibit autoantibody pr oduction. In previous studies we showed that hAAT reduced anti-i nsulin autoantibodies (IAA) and attenuated cellmediated autoimmunity (110, 120). Consistent w ith these results, this study shows that hAAT reduces T-cell proliferation and the levels of anti-CII autoantibodies. In addition, hAAT reduces the IgG2a/ IgG1 ratios of anti-CII autoantibodies (mCII and bCII), consis tent with a shift of immune response from Th1 to Th2 pathway. As a consequence of interfering with the cytokine/chemokine network, hAAT may also inhibit polymorphonuclear leukocyte (PMN) invasion in to the joint. Churg et al. demonstrated
39 that hAAT inhibited silica-induced PMN influx into the lung and partially suppressed nuclear transcription factor B (NFB) translocation and incr eased inhibitor of NFB (IB) levels in a mouse model of acute PMN me diated inflammation (119). Another possible mechanism of hAAT suppr essing arthritis development is through inhibition of proteinases to pr otect tissue injury and joint destruction. Human AAT is well known as a serine proteinase inhibitor (serpin). It inhibits trypsin, proteinase 3, neutrophil elastase, and cathepsin G, which are all involv ed in tissue injury. Human AAT can also reduce ischemia-induced apoptosis, inflammation, and acute phase response in the kidney (99). We have recently shown that hAAT directly inhibits ca spase 3 activity and protects islet cells from cytokine and chemically-induced apoptosis (121, 122). In the protein therapy studies, we used Prolastin, which is clinical grade of hAAT purified from human plasma. Repeated IP injection of hAAT induced strong humoral immune response against hAAT in DBA/1 mice (Figure 3-3B). It is possible that non-spec ific inflammation caused by repeated IP injection is res ponsible for inhibition of arthritis. In order to rule out this possibility, we performed rAAV8 mediated h AAT gene therapy. AAV serotype 8 vector is unique for this purpose because it can mediate long term and high levels of transgene expression in the liver and muscle, but is not able to tran sduce dendritic cells and has low immunogenicity (123, 124). Indeed, after a single injection of rAAV-CB-hAAT vector, sustained high levels of hAAT were detected in the circulation, while no detectable levels of anti-hAAT antibodies were present (Figure 3-8C) in contrast to mice that received h AAT protein therapy (Figure 3-3B). Importantly, we have observed similar protectiv e effects and reduction of autoantibodies by both methods. These results strongly s upport our hypothesis that hAAT is able to reduce inflammation in autoimmune diseases, such as RA and type 1 diabetes.
40 In summary, our results from protein and gene therapy showed that hAAT is effective in suppressing arthritis in a mouse model of CIA. They indicate that hAAT has immunoregulatory and immunomodulatory effects and has great potential as a new treat ment for RA. Future studies will focus on improvement of the therapeutic e ffect by optimizing the dose and timing of hAAT delivery, and by combination therapy with other anti-arthritic drugs.
41 Figure 3-1. Experimental design of hAAT protein therapy in CIA mouse model.
42 Severity 0 Severity 1 Severity 2 Severity 3 Severity 4 Figure 3-2. Severity grades of arthritis development in mouse paws. Arthritis development was evaluated based on a well established m acroscopic scoring system. Solid arrows indicate swollen digits, the dotted arrow indicates early stages of necrosis.
43 Figure 3-3. Serum level of hAAT and anti-hAAT antibodies in CIA mouse model. Human AAT (Prolastin) was intraperitoneally injected in DBA/1 mice, starting 6 days before CII immunization ( ). A, Serum hAAT protein levels in DBA/1 mice were measured by ELISA (at a 1:2000 dilution) (mean SD). B, Serum anti-hAAT antibody levels in DBA/1 mice were measured by ELISA on day 49 after bCII immunization (at a 1:8000 dilution) (relative units). Each dot repr esents antibody levels of an individual mouse.
44 Figure 3-4. Anti arthritic effect of hAAT in CIA mouse model. A, Arthritis score. For each paw, 0 is normal and 4 is most severe arthritis. For each animal the maximum score is 16. Each line represents the scores from hAAT treated group (open triangles) or control group (open circles, mean SD) indi cates p<0.05, ** indicates p<0.01, by MannWhitney U test. B, Incidence of severe arthritis. Mice were considered to be severe arthritic if arthritic scor e/mouse >3 (P=0.0025 by logrank test). Dotted line, saline injected control group; Solid line, hAAT tr eated group. C, Number of arthritic paws. Paws were considered to be arthritic when arthritis scores greater than 1. The total numbers of the arthritic paws in percent (arthritis score>1) in hAAT treated group (open triangles) and control group (open circles) were plotted over time (mean SD). indicates p<0.05, ** indicates p< 0.01 by Fishers exact test.
45 Figure 3-5. Protein therapy using hAAT reduces splenocyte proliferation. Splenocytes (4 x 105 cells/well, in 96-well plate) from hAAT treated group (black bar) and control group (open bar) were stimulated with ConA (0.5 g/ml). Each bar (n =3) represents the average of 3H-thymidine incorporation (count per minute, CPM) (mean SD). *p<0.05 by student t-test.
46 Figure 3-6. Anti-collagen II (CII) antibody levels after hAAT tr eatment. Anti-CII antibodies at day 35 and day 49 were tested by ELISA. E ach dot represents the antibody levels (relative units) of an individual mouse. Th e horizontal line indicat es median. A, Levels of total Ig antibodies against bCII (total anti-bCII-Ig). B, Levels of IgG2a against bCII (anti-bCII -IgG2a). C, Levels of IgG1 ag ainst bCII (anti-bCII-IgG1). D, Levels of total Ig antibodies against mCII (total anti-mCII-Ig). p<0.05 by MannWhitney U test.
47 Figure 3-7. Experimental design for AAV8-CB me diated hAAT gene delivery in CIA mouse model.
48 Figure 3-8. Stable and prolonge d expression of hAAT after r AAV8-CB-hAAT vector injection. DBA/1 mice were intraperitoneally inj ected with rAAV8-CB-hAAT vector (2x1011 particles/mouse, n=10) or saline (n=10) at two weeks before immunization. Control group received saline. A, Serum hAAT protei n in vector injected group was detected by ELISA (mean SD). indicates the injection time. B, Immunostaining for hAAT. left representative liver section from hAAT gene therapy treated mice; right, representative liver section from saline injected mice. Brown stained cells express hAAT. Magnification: 200x. C, Anti-hAAT antibody levels. Serum anti-hAAT antibodies in vector injected group were undetectable by ELISA (at 1:50 dilution, day 56 after immunization, relativ e units). Each dot repres ents antibody level of an individual mouse.
49 Figure 3-9. Human AAT gene therapy delays disease progression in CIA mouse model. A, Arthritis score. Each line represents th e average scores from hAAT treated group (open triangles) or control group (open ci rcles) (mean SD). Arrow indicates the second CII immunization. indicates p< 0.05, ** indicates p<0.01 by Mann-Whitney U test. B, Incidence of severe arthritis. Mice were considered to be severe arthritic if arthritic score/mouse > 3, (p=0.028 by logrank te st). C, Number of arthritic paws in percent. Paws were considered to be arthri tic when arthritis scores greater than 1. The total numbers of the arthriti c paws in hAAT treated group (open triangles) and control group (open circles) were pl otted over time (mean SD). indicates p<0.05 by Fishers exact test.
50 Figure 3-10.Effect of hAAT gene therapy on autoantibody production. Anti-CII antibodies at day 28, 42 and 56 were detected by ELISA. Black bars represent the average levels (n=10, meanSD, relative units) of antibodi es in hAAT gene therapy treated group. Open bars represent the average levels (n =10, mean SD, relative units) of antibodies in saline injected group. A, Total antibodies against bovine CII (total anti-CII-Ig). B, Levels of IgG2A against bovine CII (anti-bC II-IgG2A). C, Levels of IgG1 against bovine CII (anti-bCII-IgG1). D, The ratio of anti-bCII-IgG2A to anti-bCII-IgG1 (antibCII-IgG2A/IgG1 ratio), E, Total antibodies against mouse CII (total anti-mCII-Ig). F, Levels of IgG2A against mouse CII (anti-mCII-IgG2A). G, Levels of IgG1 against mouse CII (anti-mCII-IgG1). H, The rati o of anti-mCII-IgG2A to anti-mCII-IgG1 (anti-mCII-IgG2A/IgG1). *indicates p<0.05, **indicates p<0.01, ***indicates p<0.001 by Mann-Whitney U test.
51 CHAPTER 4 COMBINATION THERAPY USING DOXYC YCLINE AND AAV8 -TET-ON VECTOR EXPRESSING AAT Introduction As described in Chapter 1, rheum atoid arthritis is known as a complex disease with various factors being involved in diseas e development. Various cell types such as T-cells, B-cells, macrophages as well as fibroblasts participate in disease pathogenesis and they have the ability to secrete mediators which activate the complex network of a proinflamma tory immune response (117). It has been shown that currently applied monotherapies of disease modifying drugs (DMARDS) are ineffectiv e in some patients and serious si de effects can occur. Increased susceptibility to infections as well as malignancies are mainly caused by a suppressed immune system due to anti-TNFor anti IL-1 therapy (125). In order to effectively treat rheumatoid arthritis with its complex pathogenesis, an advanced treatment strategy is necessary and it has been shown that combination therapy of antia rthritic drugs are highl y effective (29). The combination therapy of methothrexate and TNFor IL-1 inhibitors has proven to be a promising alternative to esta blished monotherapies because of improved treatment efficiency (21, 108, 126). It seems reasonable that targetin g different mediators important for disease development increases treatment efficiency as well as may lower therapeutic doses and consequently side effects (29). Another study re cently revealed that dr ugs, different from DMARDs, also have been shown to be highly effective in reducing arthritis development when combined with methotrexate. Combination ther apy using doxycycline and methotrexate showed a significant American College of Rheumatol ogy 50% improvement (ACR50) response in early seropositive rheumatoid arthritis patients when compared to me thotrexate monotherapy (108). In our study, we propose to test the th erapeutic effect of the combination of doxycycline and hAAT.
52 Because RA is characterized by recurrent peri ods of joint inflammation, it is desirable to limit therapeutic gene expression to the acute phases of inflammation. Constitutive over expression of transgenes can lead to detrimental effects in disease conditions. In order to control hAAT gene expression in our study, we deliv ered an AAV vector containing a doxycycline dependent tet-on gene expressi on system. This system enables us to control hAAT gene expression with doxycycline, which in a ddition has therapeu tic properties. Experimental Design Cohorts of DBA/1 m ice received either a si ngle IP injection of rAAV8-tet-on-hAAT vector or saline. In addition mi ce in vector injected (n=9) or saline treated group (n=11) also received doxycycline ad libitum administered with food ( 200mg/kg food). A control group (n=11) received a saline injec tion. Arthritis was induced 4 weeks after vector injection by two bovine collagen II injections as descri bed in Materials & Methods (Fig.4-1). Evaluation of arthritis was perf ormed three times per week by a blinded investigator based on a well established arthritis scoring scale as described in Materials and Methods. Every other week, serum samples were collected to determine hAAT serum levels. On day 56 after immunization mice were s acrificed. Liver was harvested for hAAT immunostaining and joints were collected and prepared for H&E staining to determine and evaluate histopathol ogical changes. To further evaluate the mechanism involved in hAAT and doxycycline mediated arthritis suppression, we conducted in vitro studies using mouse embryonic fibrobla st cell line NIH/3T3. Simulating IL-6 secretion from activated synovial fibroblasts, a process involved in arthritis development, the cells were stimulated with LPS or TNF. In the presence of hAAT and/or doxycycline, IL-6 secretion into cultur e medium was evaluated by ELISA.
53 Results Administration of AAV8-tet-on-hAAT and Doxyc ycline Resulted in Sustained Expression of hAAT in DBA/1 Mice Our previous studies showed that AAV m ediat ed hAAT gene delivery suppress arthritis in CIA model. To further improve the treatment eff ect we tested the feas ibility of AAV mediated hAAT gene therapy in combination with doxycyclin e. We delivered an AAV vector, carrying the hAAT gene under control of the doxycycline inducible tet-on promot er (Figure 2-2). Four weeks after vector administration and starting doxycycline diet (200mg/ kg food, ad libitum), a sustained level of hAAT protein was detected in mouse seru m (Figure 4-2A). Consistent with our previous results, rAAV8 mediated gene de livery resulted in undetectable or weak immune response to the transgene product (hAAT). As seen in Figure 4-2B only 25% of vector injected mice developed low titer anti-hAAT antibodies. Gene Delivery Using rAAV8-tet-on-hAAT in Combination w ith Doxycycline Suppressed Arthritis Development in CIA Mice As shown in Figure 4-3A, combination of doxycycline and hAAT results in a sustained suppression of arthritis development in mice (p< 0.05 by Mann-Whitney). When evaluating incidence of severe arthritis (clinical scor e/mice >3), we observed, that mice receiving combination therapy developed significantly less severe arthritis compared to control group. Only combination therapy was able to signifi cantly reduce incidence of severe arthritis development (p< 0.05 by logrank test, Figure 4-3B), whereas monotherapy using doxycycline alone did not result in a significant reduction. Evaluation of arthritic paws revealed similar results. Only combination therapy was able to reduce the number of arthriti s paws significantly compared to control and monotherapy (p=0.0011 by Fishers exact test adjusted for multiple comparison, Figure 4-3C). These results
54 showed that adding doxycycline to hAAT therapy significantly improved the treatment in reducing paw swelling and arthritis development. Histopathological Changes in Mouse Joints are Improve d by hAAT and Doxycycline Combinatio n Therapy To further determine the effect of the combination therapy, we evaluated joint sections from treatment and control groups. Figure 4-4 show s representative joint tissue sections from mice receiving combination therapy (Figure 4-4A-C ) or saline injections (control group, Figure 4-4D-F). Combination therapy results in less infl ammation and infiltration of immune cells into the joint space. Hyperplasia, pannus formation a nd cartilage erosion are significantly reduced in treatment group receiving doxycycline a nd hAAT compared to control group. Joint tissue sections from all mice in respectiv e groups were prepared and evaluated by two independent and blinded pathologists. The eval uation was based on grading the infiltration of immune cells into the joints, hyperplasia and pan nus formation of synovial tissue as well as bone degradation. As presented in Fi gure 4-5, all histopathological crit eria were significantly lowered in mice receiving combination therapy compared to mice receiving monotherapy or control (p< 0.05 by students t-test). These re sults confirm the macroscopic observations described in Figure 4-4 and further underline that the combination therapy effect ively suppresses immune cell infiltration, invasive, joint damagi ng tissue proliferation as well as cartilage and bone erosion. Human AAT and Doxycycline Suppress IL6 Expression from NIH/3T3 Cells To evaluate the m echanism of the anti-arth ritic effect we obser ved after combination therapy we tested the effect of hAAT and doxycycline on disease associated IL-6 secretion from fibroblasts. Following incubation with different concentr ations of hAAT, ranging from 0.1mg/ml to 2.0 mg/ml, LPS induced IL-6 release was signi ficantly reduced in a dose dependent manner
55 (Figure 4-6A). Interestingly, we did not observe this effect after incubation with hAAT and TNFinduced IL-6 release (Figure 4-7A), indicating that the hAAT inhibitory effect may be more specific for the LPS signaling pathway. After incubating cells with differe nt doses of doxycycline, LPS or TNFinduced IL-6 expression was significantly re duced in the presence of 100g/ml doxycycline (Figure 4-6B and Figure 4-7B). Simulating the combination therapy applied in the presented study, we were also able to show that IL-6 concentration in cultu re medium was significantly reduced when cells were stimulated with LPS and incubated us ing combination of hAAT (1.0mg/ml) and doxycycline (10g/ml and 100g/ml) compared of hA AT alone (Figure 4-6C). The presence of hAAT (1mg/ml) and doxycycline (100g/ml) also resulted in significant reduction of TNFinduced IL-6 secretion into culture medium compar ed to hAAT alone (Figure 4-7C). This effect is probably due to the presence of doxycycline, since hAAT alone did not suppress TNFinduced IL-6 expression (Figure 4-7D). As summarized in Figure 4-6D, hAAT or doxycycline alone reduced LPS induced IL-6 secretion from fibroblast cells. This effect was further signi ficantly enhanced by using a combination of these agents. Discussion Finding an effective treatm ent for rheumatoid ar thritis is very challe nging and until today it is not possible to cure the dis ease. One reason is certainly that it is still unknow n what exactly triggers the disease onset. Since RA is driven by interaction of various immune cells, including T-cells, B-cells and macrophages, communica ting through a network of cytokine and chemokines, it is challenging to determine what kind of intervention is effective in order to restore joint homeogenesis without causing unwanted side effects. Current treatments targeting cytokines such as TNFand IL-1 are promising approaches a nd have shown to be highly
56 effective in clinical settings ( 29). However, since we are now ab le to examine long term effects of the new treatment strategies, it became obvious that they also have their drawbacks. AntiTNFtherapy failed to be effective in some pa tients accompanying with occurrence of side effects such as increased risk of infection, concerns about malignancies and other adverse outcomes (127-131). Several studies have shown, that combination therapies, using drugs which target different pathways of disease pathogenesis are able to improve RA outcome signi ficantly and therefore indicate to be a promising alternative treatment option (126). This strategy may also result in reduction of therapeutic dose accompanied with re duction of unwanted side effects. In our study we tested a combination therapy using h AAT and doxycycline. Both agents employ antiinflammatory and immunomodulatory effects and we were able to show th at they are effective when used as monotherapy. According to our hypot hesis their treatment effect will be improved when used in combination. Doxycycline as a pot ent MMP inhibitor is ab le to reduce protease activity in the join t and also effectively suppresses proinf lammatory cytokine release in vivo and in vitro (100, 101, 107). Similar, hAAT has been shown to reduce TNF, IL-1 and IL-8 mRNA and protein expression in LPS stimulated m acrophages (94, 132). Our study revealed that combination therapy using hAAT and doxycycline effectively suppresses arthritis development and is more effective than respective monotherapies. Since RA is known as a chronic disease with recurrent active phases, characterized by acute inflammation, it is desirable to limit drug exposure to the active di sease state. Employing gene therapy for this kind of disease characteris tics has advantages because vector systems with controllable gene expression are available. Their introduction into RA therapy may also reduce side effects and minimize drug exposur e during non acute disease states.
57 In accordance to the success of combination therapy in RA patients our study aimed to develop a unique combination therapy with a doxycycline dependent gene expression system. The uniqueness is, that doxycycline not only co ntrols gene expression but also employs therapeutic potential. It not only targets the ongoing inflammato ry autoimmune response during arthritis development but also turns on anti-i nflammatory hAAT gene expression from the delivered AAV vector containing the tetracy cline dependant gene expression system. In order to further investigate the mechan ism which leads to the suppression of CIA development in our study, we performed in vitr o studies to test the ability of hAAT and doxycycline to reduce cytokine rele ase from fibroblasts. Fibroblasts like synovial cells are key sources for proinflammatory cytokines, prosta glandins, metalloproteina ses as well as other mediators of inflammation in the synovial cavity and are considered to be a major mediator of pathologic RA join t processes (133). It has been shown that fibroblasts are a main source of IL-6 in RA patients and increased IL-6 activity correlates with elev ated serum levels of acute phas e reactants and other signs of inflammation such as fever and anemia (134). IL-6 has also been implicated as a major factor for the generation of auto antibodies (including rheumatoid factor). It also activates T-cells by inducing IL-2 production and IL-2 receptor expression and may act synergistically with IL-2 in driving the differentiation of T-ce lls into cytotoxic Tcells (135, 136). Playing an important role in establishing the RA synovial fibroblast (SF) phenotype, it increases SF proliferation in conjunction with its soluble re ceptor in the synoviu m of RA patients (137). Arthritis development was significantly suppressed in IL-6 deficient CIA mice indicat ing its major role in arthritis development(138). Targeting IL-6 is also effective in CIA mouse model after administration of a rat-anti-murine IL-6R monocl onal antibody earl y after arthritis induction as
58 shown by Takagi et al (139). Anti -IL-6 strategy is already successfu lly introduced in treatment of human RA. Tocilizumab, a humanized murine antibody to IL-6R which binds membrane associated as well as soluble IL-6R and theref ore inhibits IL-6 from binding, shows promising potential for RA treatment in humans (29). In the present study, we showed that both, h AAT and doxycycline are ab le to suppress LPS induced IL-6 release from fibroblasts in vitro, indicating a possible mechanism of our therapy for inhibition of arthritis development. Using a co mbination of both agents further suppressed the IL-6 production. These observations are in accordance with the results we received from the animal studies in which combination therapy us ing hAAT and doxycycline was more efficient in suppressing arthritis development compared to monotherapies. Our results also showed that hAAT alone was not able to suppress TNFinduced IL-6 production in NIH/3T3 cells. This indicates that the suppressive effect of hAAT on LPS induced IL-6 production may be more specific for the L PS induced cell signaling pathway. Consistent with our observations, Nita et al. have shown that hAAT was able to decrease CD14 and Tolllike receptor 4 (TLR4) expressions in human mo nocytes in vitro (132). CD14 and TLR4 are important cell surface proteins of the LPS induced signaling pathway. Interestingly, we also reveale d, that in contrast to hAAT, doxycycline was able to suppress not only LPS induced IL-6 expression but also TNFinduced IL-6 expression, indicating a different mechanism of action compared to hAAT It has been shown by Cazalis and al. that pretreatment of macrophages with 10 M ( = 5g/ml) doxycycline prior to LPS stimulation resulted in a marked decrease in the phosphorylation of extrace llular signal-regulated kinase (ERK1/2) (140). ERK is involved in both TNFand LPS mediated responses and therefore might be the target for doxycycline inhibition in our experiment (141, 142). Doxycycline also
59 downregulates proteinkinase C (PKC) pathways, as shown in a study of its effects on granuloma formation (143). TNF(141, 144-146) as well as LPS (147) pathways reportedly signal through PKC family members in different cell types, resulting in expressi on of proinflammatory cytokines. We therefore suggest that the significant suppression of IL-6 release into the cell medium observed after treatment with the drug combination after LPS treatment might be due to inhibition of hAAT on CD14/TLR4 expression, as well as inhibition of ERK phosphorylation and downregulation of PKC pathways by doxycycline. Further studies have to be conducted to confirm this hypothesis and also its tran slation to in vivo settings. In conclusion, our results show a new and pr omising approach to apply gene therapy for treatment of rheumatoid arthri tis. Combining doxycycline and hAAT while being able to control gene expression of hAAT is a unique approach to target two different pathways involved in disease pathogenesis. The ability of hAAT and doxycycline to i nhibit IL-6 expression from LPS stimulated fibroblasts also reveal s a new property of this protease inhibitor. Future studies will focus on optimizing the treatment dose as well as using tissue specific AAV-vectors homing for disease affected tissues. In addition, deliv ering hAAT under self-limiting, inflammationresponsive promoter control is another promis ing approach for future arthritis treatment.
60 Figure 4-1. Experimental design for combination therapy of rAAV8 tet-on mediated hAAT gene therapy and doxycycline in CIA mouse model.
61 Figure 4-2. Human AAT serum and anti-hAAT level in DBA/1 mice received rAAV8-tet-on hAAT and doxycycline. A, shown is hAAT level in mouse serum as determined by standard ELISA (meanSD). rAAV8-tet-onhAAT was injected intraperitoneally 28 days before CII immunization. B, anti-hAAT antibody level was determined by standard ELISA (at dilution 1:50). Each dot represents anti-hAAT level (measured as optical density (OD) at 490n m) of an individual mouse. Horizontal line represents median.
62 Figure 4-3. Anti-arthritic effect of combination th erapy in CIA mouse model. A, Arthritis score. For each paw, 0 is normal and 4 is most severe arthritis. For each animal the maximum score is 16. Each line represents the scores from hAAT + doxycycline treated group (open triangle s), doxycycline treated group (clo sed triangles) or control group (open circles) (mean SD). i ndicates p<0.05 by Mann-Whitney U test. B, Incidence of severe arthritis. Mice were cons idered to be severe arthritic if arthritic score/mouse >3. Dotted line (short dots) re presents saline injected control group; dotted line (long dots) represents doxycyclin e treated group; solid line represents hAAT + doxycycline treated group. *indicate s p<0.05 by log-rank test. C, Number of arthritic paws. Paws were considered to be arthritic when arthritis scores greater than 1. The total numbers of the arthritic paws in percent (arthritis score>1) in hAAT + doxycycline treated group (open triang les), doxycycline treated group (closed triangles) and control group (open circles) were plot ted over time. ** indicates p<0.01, by Fishers exact test ad justed for multiple comparison.
63 A D 100x B E 200x C F 400x Figure 4-4. Tissue protective eff ect of combination therapy in CIA mouse model. Mice were sacrificed on day 56 after CII immunization, hind limbs were harvested and processed for histological assessment. A-C, representative joint section from mice receiving combination therapy. D-F, representativ e joint section from mice in control group(saline injection). Magnification: A, D: 100x; B,E: 200x; C,F: 400x. S= synovium, C= cartilage, P= pannus. indicates cartilage erosion.
64 Figure 4-5. Histopathological eval uation of arthritis development. Mice in combination therapy group (black bars), doxycyclin e monotherapy group (striped bars) or control group (empty bars) were evaluated according to histopathological changes by two blinded pathologists. Each hind paw was evaluate d based on a scale ranging from 0-4. (0: normal, 1: mild, 2: moderate, 3: severe, 4: very severe). The score was then averaged for each treatment group (meanSD). *p=0.05, **p=0.01 by students t-test.
65 Figure 4-6. Effect of hAAT and doxycycline (DOX) on LPS-induced IL-6 release from mouse embryonic fibroblast cells (NIH/3T3). Cells were incubated with hAAT and/or doxycycline for 6h and stimulated with LPS (1 g/ml) for 20h. IL-6 release into the culture medium was determined by standard ELISA. A, IL-6 level in cell medium after incubation with indicated concentrations of hAAT. B, IL-6 level in cell medium after incubation with indicated concentra tions of doxycycline. C, Incubation with hAAT (1mg/ml) and indicated concentrations of Doxycycline. D, Comparison of IL-6 level in cell medium after incubation with hAAT or doxycycline alone and in combination. *p<0.05, **p<0.01, ***p<0.001 to contro l group or as indicated using students t-test.
66 Figure 4-7. Effect of hAAT and doxycycline on TNFstimulated IL-6 release from mouse embryonic fibroblast cells (NIH/3T3). Cells were incubated with hAAT and/or doxycycline for 6h and stimulated with TNF(10ng/ml) for 20h. IL-6 release into cell medium was determined by standard ELI SA. A, IL-6 level in cell medium after incubation with indicated con centrations of hAAT. B, IL-6 level in cell medium after incubation with indicated concentrations of doxycycline. C, Incubation with hAAT (1mg/ml) and different concentrations of doxycycline. D, Comparison of IL-6 level in cell medium after incubation with hAAT or doxycycline alone and in combination. *p<0.05, **p<0.01, ***p<0.001 to control group or as indicated using students t-test.
67 CHAPTER 5 ADENO-ASSOCIATED VIRUS MEDIATED HUMAN AAT GENE THERAP Y IN PRISTANE INDUCED ARTHRITIS MODEL Introduction As we have reviewed in the previous chapte rs, rh eumatoid arthritis is a very complex disease with multiple dysfunctional components of immunity in the pathogenesis. It has been reported that different cyt okines such as IL-1, TNF, IL-6 and RANKL (receptor activator of nuclear factor nfB ligand) play important roles in disease pathogenesis indicating the complexity of the cytokine network involved (117). Observed heterogeneity of disease manifestations, clinical course and different treatment responses in RA patients further indicate the diversity of the disease and suggest that possible treatment approaches have to be examined very carefully. Due to this complexity and dive rsity it has been suggested that therapeutic strategies should be evaluated in different animal models. Several animal models with different propertie s have been used for RA studies. Collagen induced arthritis and adjuvant i nduced arthritis show a high incide nce as well as reliable onset and a robust and easy measurable poly-arthritic paw inflammation. MRL/lpr mice spontaneously develop mild arthritis with variable onset and incidence. For our studies we decided to use pristane induced arthritis because arthritis can be induced by a single i.p. injection of pristane and a prolonged and delayed clinical timecourse of joint inflammation can be observed. An extended and permanent period of active disease state enables us to better investigate the treatment effect. In addition to the development of arthritis, pristane injected DBA/ 1 mice also develop lupus like autoimmunity characte rized by autoantibody production ag ainst nuclear proteins.
68 Using this model for our studies elucidates the potential of hAAT and its combination therapy with doxycycline to suppress autoantibody production and will further reveal the therapeutic applicability of hAAT therapy in autoimmune diseases. Experimental Design Fe male DBA/1 mice received either a single i.p. injection of recombinant AAV8-cb-hAAT vector (n=20) or saline (n=20). The mice were randomly assigned to four groups (n=10 each). In addition to the AAV8-CB-hAAT vector, one gro up resembling the combination therapy also received a doxycycline containing diet. Other groups received rAAV8-CB-hAAT alone (hAAT monotherapy), doxycycline alone (doxycycline monotherapy) or a single saline injection (control group) (Fig.5-1). Two weeks after vector injection, mice were i.p. in jected with a single dose of 0.5 ml pristane oil to induce arthri tis and lupus like autoimmunity. Every four weeks, serum samples were collect ed to determine hAAT levels and anti-hAAT as well as lupus related auto-an tibodies. 12 weeks after pristane injection evaluation of arthritis development was started using the commonly accepte d arthritis grading scale as described in Materials & Methods. At 34 weeks after pristane injection mice we re sacrificed and proteinurea was determined in urine to test for Lupus Nephritis development. Results Serum Levels of hAAT and anti-hAAT in PIA Mouse Model Following rAAV8-cb-hAAT Mediated Gene Therapy Based on our prelim inary data, we decided to use rAAV8 mediated hAAT gene therapy to test the feasibility of hAAT therapy in PIA model. As shown in Figure 5-2A, 6 weeks after vector injection (or 4 weeks after pristane inje ction), serum level of hAAT protein serum level was about 40 g/ml in both groups which received hAAT gene therapy. Doxycycline, as expected did not have an effect on hAAT gene expres sion. We also evaluated the immune response to
69 hAAT by measuring anti-hAAT antibodies. As illustrated in Figur e 5-2B, only few mice developed a minor immune res ponse against the transgene. Pristane Injection Results in Mild Arth ritis Development in Treatment Groups We evaluated the effect of hAAT monotherapy and doxycylin e monotherapy as well as their combination therapy in the PIA mouse model. 12 weeks after pristane injection, mice started to develop arthritis characterized by joint redness and swelling. Arthritis development was then evaluated once a week until the experiment was terminated on week 34. As shown in Figure 5-3A, all groups showed slow and sustained arthritis development and reached the maximum average score between 4.5 and 7.5 at about 26 weeks after immunization. With a maximum possible score of 16, this indicates on ly mild arthritis development in all groups. Interestingly, we were not able to observe any differences based on clinical score between treatment groups. Similarly, when evaluating arthritis in cidence in mice with score >1 as shown in Figure 5-3B we also were not able to observe any significant di fference between groups. These results indicate that further optimizati on of the combination is required to get the therapeutic effect for ar thritis in the model. Lupus Nephritis Development Tends to be Suppressed by hAAT and Doxycycline Monothera py Besides arthritis development, pristane injection results in occurrence of lupus nephritis in mice which is characterized by proteinurea. We examined the effect of hAAT gene therapy on the induction of lupus nephritis. As shown in Figure 5-4, >1 prot einurea was detected in 7 of 10 mice from control group. Although not statistically significant, only 3 of 10 mice in hAAT monotherapy (p= 0.179 by Fishers exact test compared to cont rol) and 2 of 10 in doxycycline monotherapy (p= 0.06 by Fishers exact test compared to control) indicated at least a trend of a suppressive effect on proteinu rea of hAAT as well as doxycycline monotherapy. However,
70 combination therapy using hAAT and doxycycline did not show such convincing results. 4 of 9 mice developed >1 proteinurea in this group (p=0.36 by Fishers exact test co mpared to control). Effect of Monoand Combination Therapy on Development of Autoantibodies The develop ment of lupus like autoimmunity in PIA model is characterized by production of autoantibodies against nuclear proteins such as anti-RNP, anti-rP, anti-Su, anti-chromatin, anti-ssDNA and anti-dsDNA. To examine the effect of hAAT gene thera py and its combination with doxycycline on autoantibody development we de termined antibody levels in mouse serum 3 month and 8.5 month after pristane injection by ELISA. As shown in Figure 5-5 A,C, 12 weeks after pristane injection, only low antibody levels against chromatin and DNA were detectable. Figure 5-5B, D, E, illustrate, that although respec tive autoantibody levels were increased at 34 weeks compared to levels at 12 weeks, we di d not determine significant differences between treatment groups. Interestingly, Figure 5-6A shows, that comb ination therapy resulted in suppression of autoantibodies against Su protein at 12 weeks (p= 0.0185 by Mann-Whitney). In addition we were also able to observe a trend of anti-Su suppression at 34 weeks (Figure 5-6B, p=0.144 by Fishers Exact test) indicati ng a suppressive effect of h AAT and doxycycline combination therapy on development of certain autoantibodies in PIA model. Similar to anti-Su, autoantibody levels of anti -rP and anti-RNP increased over time (Figure 5-6 C-F). However, we were not able to determ ine any significant differences in anti-RNP and anti-rP autoantibody development between treatment groups and control. Human AAT or Doxycycline Treatment Does No t Change Type of Antibody Response To further characterize the antibody response that was elicited in our model and the ability of hAAT gene therapy or doxycycline treatment to modify this response, we determined the serum levels of different antibody classes. Depending on the kind of immune cells, involved in
71 the immune response, we will obs erve a characteristic pattern of antibodies in serum. Usually, pristane injection results in high titer IgG2a antibodies in mi ce resulting from a Th1 skewed response. We determined levels of IgG1, IgG2a, IgG 2b, IgG3 and IgM by ELISA. As seen in Figure 5-7, hAAT or doxycycline treatment did not cha nge serum levels of these antibody classes. Discussion Anim al models for rheumatoid arthritis are widely used to test the potential of anti-arthritic drugs for suppression of disease development because they resemble pathogenic and inflammation patterns similar to those seen in humans. Although animal models help us to understand the complex immunological and pathologi cal patterns important for the disease, none of them exactly represents human RA. This sugge sts using different animal models to test for therapeutic efficiency. In addition, differences and diversity in seve rity, clinical sy mptoms of RA as well as response to treatment in patients al so recommend examination of new anti-arthritis drugs in different animal models in order to cover a broad range of pathological manifestations. The animal models we used in our studies, co llagen induced arthritis and pristane induced arthritis, simulate human diseas e in different ways. The auto-antigen collagen II which induces CIA, elicits an autoimmune response which is ch aracterized by a relativel y short period of acute joint inflammation (max 6-7 weeks) followed by joint erosion and deformation. This model is commonly used when therapeutic effects on disease onset and early stages of disease progression are investigated. In contrast, pr istane induced arthritis is char acterized by a delayed, slow and chronic inflammation of the joint. The inflam mation lasts 3-6 month and results in cartilage erosion and joint deformation after 5-6 month, similar to human RA which also progresses slowly. Using these two animal models therefore enable us to determine the effect of AAT on arthritis development from different perspec tives. One being the acute inflammation in the
72 beginning of disease progression, th e other is focusing on the long te rm effects while the disease progresses. Our data using CIA model showed that both, pr otein as well as gene therapy using hAAT are effective. For PIA model we preferred the gene therapy approach since a single vector injection as it is performed for gene therapy is much more convenient than regular injections of protein. Especially when applie d in PIA model in which diseas e progresses slowly over several month. We decided to use AAV serotype 8 to deliv er the hAAT gene due to its constant gene expression and its promising e ffects on suppressing arthritis a nd autoantibody development as observed in the previous studies Interestingly, the average hAAT level in PIA model was lower compared to the serum level in CIA model when in jecting the same vector. This might be due to an increased antiviral type 1 interferon expr ession, commonly seen in response to pristane injection, and resulting in decrease of effective vector concentration. The immune response against hAAT seems to be slightly stronger than it has been seen in previous studies using AAV8 as a gene delivery vector. Three out of 20 mice developed elevated levels of hAAT antibodies (Figure 5-2). This might be due to an unspecific inflammatory response caused by the administration of pristane. In a sustained inflammatory state as a result of pristane injection, dendritic ce lls may more likely uptake the hAAT protein, which is then processed and presented to T-cells, eliciti ng the humoral response against hAAT. Although we were able to detect elevated se rum levels of hAAT, we did not observe an anti-arthritic effect in any of the applied ther apies in PIA model. Neither hAAT nor doxycycline monotherapy nor the combination therapy was eff ective in this model. A possible reason could be that the hAAT level was not high enough for a sustained suppression of arthritis development over time. As seen in Chapter 3, hAAT effectiv ely suppresses disease on set and development in
73 the early phase of arthritis but showed less therapeutic effect in inhibiting disease progression. Since PIA progresses slowly with major presenta tion of the chronic, disease progressing state, the hAAT effect on disease onset in this m odel might not be powerful enough to suppress arthritis progression in the long run. Another reason for the absence of a therapeutic effect could be that our therapies were effective to a certain grade, but since the severity of arthritis development in the control group was rela tively low, the effects observed were undistinguishable. Our mice were hold in SPF ro oms and PIA progression may be inhibited due to lack of various factors such as ligands fo r patternrecognition recep tors, which are commonly abundant in normal environment and necessary for PIA development. In addition to arthritis development, pristane injection also leads to development of lupus like autoimmunity characterized by elevated se rum levels of autoantibodies against nuclear proteins in susceptible mice. As we learned from the previous studies, hAAT is able to inhibit autoantibody production in CIA mice. We ther efore determined if hAAT also inhibits autoantibody production against nuclear proteins in PIA mous e model. Interestingly, the combination therapy showed the most prom ising results and revealed a reduction of autoantibodies against Su-protein. Unfortunately, we were not able to determine such an effect for any of the other lupus re lated autoantibodies anti-RNP, anti-ssDNA, anti-dsDNA, antiChromatin and anti-rP. The long term inhibitory effect of hAAT on autoantibody production might be limited and a suppressive effect can just be observed shortly after disease onset as seen in the previous studies described in chapter 3. This hypothesis is underlined by the ability of combination therapy to significantly suppress anti-Su development after 12 weeks and a less significant reduction after 34 weeks (Figure 5-6).
74 Further studies have to be conducted to eliminat e uncertainties in the effect of hAAT in the PIA model. We need to further optimize disease onset and increase incidence and severity of arthritis development in this animal model. We are optimistic to observe a better hAAT treatment effect if we are able to increase overall disease severity in the control group. This will most likely also lead to more severe lupus development accompanied with higher levels of autoantibodies and increased incidence of proteinur ea. In this case a hAAT effect may also more likely be detected.
75 Figure 5-1. Experimental design of pristane induced autoimmunity in DBA/1 mice. Mice developed arthritis as well as lupus like autoimmunity se veral weeks after receiving a single i.p. injection of 0.5ml pristane.
76 Figure 5-2. Human AAT and anti-hAAT antibody levels after rAAV8 mediated hAAT gene delivery in pristane induced mouse mode l. A, hAAT serum level after rAAV8-cbhAAT gene delivery was determined by sta ndard ELISA. Shown are levels of hAAT gene therapy alone (open triangles) and in combination with doxycycline (closed circles) (meanSD). B, Serum antibody levels against hAAT were determined on week 34 after pristane injection. Shown ar e serum anti-hAAT levels of individual mice. Horizontal line represents median.
77 Figure 5-3. Evaluation of arthritis development on pristane induced arthritis model. A, Arthritis score. For each paw, 0 is normal and 4 is most severe arthritis. For each animal the maximum score is 16. Each line represents the scores from hAAT treated group (closed diamonds), doxycycline treated gr oup (open triangles), hAAT+doxycycline treated group (open circles) or control group (closed triangels) (mean SD). B, Incidence of arthritis. Mice were considered to be arthritic if arthritic score/mouse >1. Dotted line (short dots), saline injected control group; dotted line (long dots) doxycycline treated group; dotted line (shor t&long dots), doxycycline treated group; solid line, hAAT+doxycycline treated group. Figure 5-4. Pristane injection ca used renal disease in pristane injected DBA/1 mice. Presence of proteinurea defined as 1 on dipstick analysis was id entified in all groups, with highest presence in control group. Shown ar e protein levels de fined according to dipstick analysis of individual mice. Horizontal line represents median.
78 Figure 5-5. Autoantibody production in pristane treated DBA/1 mice afte r receiving different treatments. Serum levels of lupus autoantibodies were determined by standard ELISA. A,B, Serum levels of anti-chromatin antibodies were determined using ELISA for detection of reactivity with ch icken erythrocyte chromatin at indicated timepoints. C,D, Sera from treatment or control groups were tested for presence of anti-double-stranded antibodies (anti-ds-DNA) at indicated time points. E, Antibodies against single stranded DNA (anti-ssDNA) were detected 34 weeks after pristane injection. Horizontal lines i ndicate median for each group.
79 Figure 5-6. Production of lupus auto antibodies after prista ne injection in DBA/1 mice. A,B, antiSu antibodies; C,D, anti-nuclear RNP anti bodies, E,F, anti-ribosomal P antibodies were measured by ELISA at indicated time points in saline control groups and the indicated treatment groups. Horizontal line represents the median in each group. *p<0.05 determined by Mann-Whitney U test.
80 Figure 5-7. Expression of antibody subclasses in pristane treated DBA/1 mice. A, Ig2a antibodies (the predominant isotype in pristane treated mice), B, IgG2b antibodies, C, IgG3 antibodies, D, IgG1 antibodies, E, IgM antibod ies, F, ratio of IgG2a/IgG1 antibodies. Total antibody subclass levels were determined in sera 34 weeks after pristane injection. Each dot represents level of an individual mouse as determined by standard ELISA. Horizontal line represents median.
81 CHAPTER 6 SUMMARY AND PERSPECTIVES Summary Therapeutic approaches for the treatment of rheumatoid a rthritis changed dramatically within the last two decades. We now know that pr oinflammatory cytokines play a pivotal role for disease onset and progression. A dvances in the field of imm unology, molecular biology as well as a better understanding of dis ease pathogenesis resulted in the development of drugs with antiinflammatory and immunomodulatory pr operties such as blockade of TNFand IL-1 as well as inhibition of T-cells and B-cells. Introducti on of these biologics significantly changed the standard treatment regimen for RA. Although methot rexate is still a corn erstone in rheumatoid arthritis, todays standard therapy uses the ne w biologics or combinations with methotrexate. Their introduction showed significant improveme nt in many patients including those which showed no response to the applied standard therapies with methotrexate (29). Unfortunately, increasing evid ence suggests that these new bi ologics are not as promising as anticipated. Although most studies showed grea t efficiency, some long term studies revealed their inefficiency in selected patients and occurrence of severe side effects (21). As mentioned before, it has been shown that anti-TNFtherapy increased the risk of infections as well as malignancies (27-29). In addition prescribing these drugs for treatment relieves the symptoms and prevents some patients from further joint damage, but they do not fight the actual cause and therefore have to be taken life long, which cr eates a financial burde n for many patients. Therefore, there is still the need to deve lop new strategies and drugs for effective treatment. The studies described here are a new and promising approach which may lead to future therapies applicable for rheumatoid arthritis. Human AAT, which is the therapeutic protein we used in our study, has anti-inflammatory properties and its serum level increases 3-4
82 fold in response to inflammation. It theref ore may be a major contributor for suppressing inflammation in the body. Due to its high endogenous concentration, side effects may be limited when applied in high therapeutic concentrations in clinical settings. Our studies and those from other groups also indicate that hAAT has anti -inflammatory properties by not targeting a single cytokine but many with medium efficiency ( 95). This might be advantageous since total blockade of a specific cytokine can cause severe side effects as seen in commonly applied anticytokine therapy for RA. Current therapies are accompanied with hi gh costs and the requirement of regular injections, reducing patients compliance. For trea tment, the protein has to be administrated in high doses to ensure effective concentrations in target tissues, therefore increasing the risk of unwanted side effects. Our approach of using gene therapy should demonstrat e that it is possible to circumvent these regular inje ctions of high protein doses and still ensuring therapeutic protein serum levels. We used the AAV8 vector to deliver the hAAT gene since it has been shown that AAV8 is able to infect various tissues, including muscle and liver, ensuring systemic expression of the delivered protein. This is desirable in a model for rheumatoid arthritis in which multiple joints ubiquitous in the body are affected. Local injection in e ach single joint would be time consuming and less efficient especially in sma ller joints. AAV8 also has a low immunogenicity compared to other AAV serotypes or other vira l vectors. Especially when compared with different vectors such as ade novirus or retrovirus the low immunogenicity results in higher and more effective gene expression. After receiving promising results that hAAT is effective in reducing arthritis development by using protein as well as gene therapy, we in tended to further improve and optimize the gene delivery approach. In the initial studies we us ed a CB-promoter resulting in constant hAAT
83 expression in the infected cells; even in non-acu te phases of the diseas e in which hAAT might not be needed. As mentioned before, it is desira ble to control gene expression in order to limit high therapeutic protein levels to active disease stages. For this purpose we administered AAV8 vector which delivered hAAT gene under contro l of a tetracycline dependent promoter. Using this expression system not just enables us to control hAAT gene expres sion but also requires doxycycline administration which has an additional therapeutic effect. In this unique approach, doxycycline is used as a therapeu tic agent and to regulate hAAT expression. We were able to show that administration of both hAAT and doxyc ycline improves the treatment effect compared to monotherapies. As mentioned before AAV8 mediated ge ne delivery is characterized by low immunogenicity of the vector. We observed that antibody development against the transgene is almost absent and a high sustained serum level of the therapeutic protein hAAT indicates that an immune response against the vector also seems to be very low. This is certainly an interesting and unexpected observation since previous studie s showed that other AAV serotypes such as AAV2 do elicit an immune response against the transgene or the vector (70, 71, 148). In addition we also observed that autoantibodies against coll agen II in CIA model are initially reduced after hAAT protein therapy and this effect is even more enhanced and more sustained after gene delivery of hAAT. This observation might rev eal the great potential of AAV8-hAAT mediated gene delivery for autoimmune diseases where au toantibodies play a pivo tal role. We therefore transferred the gene therapeutic approach to a different arthritis animal model to test the feasibility of hAAT therapy and its effect on autoantibody development. We used pristane induced arthritis mouse model which is characterized by development of inflammatory arthritis accompanied with development of lupus specific autoantibodies. Although we were not able to
84 observe an anti-arthritic effect in this model we saw a trend that hAAT in combination with doxycycline is able to suppress lupus autoantibod y anti-Su. This observation encourages us to believe that AAV8-hAAT maybe a promising ag ent to suppress autoan tibody development in autoimmune diseases. Perspectives The studies presented in this dissertation describe alternative approaches to current therapies for rheumatoid arthritis. The observatio n that promising treatments with new biologics which evolved in the last two decades have shown to cause side effects and also turned out to be inefficient in some patients, emphasizes the n eeds to further improve and develop new therapy approaches in arthritic diseases. Due to the fact that rheumatoid arthritis is a complex disease where several pathways have to be regulated in order to achieve a sufficient therapeutic effect, monotherapies are often not as successful as combination therapies. The introduction of doxycycline is therefore reasonable si nce it adds therapeutic potential to the thera py by targeting metalloproteinases in the synovium. The anti-arthritic potential of doxycycline monotherapy in humans seems to be questionable as seen in a study recently published (149). Interestingly, it is able to improve the therapeutic effect when it is combined with methotrexate (108). It would therefore be interesting to see if doxycycline is also able to improve the therapy effect when it is combined with other commonly used drugs in arthritis such as the new biologicals targeting TNFor IL-1 Our approach to combine doxycycline w ith hAAT also revealed an improved therapeutic effect. It ha s still to be elucidated if blocking of MMPs is the only therapeutic effect of doxycycline. Our studies suggest that doxycycline in addition is able to reduce proinflammatory cytokine releas e such as IL-6 from fibroblas ts. As described previously, antibiotics administered in RA patients might al so eliminate potential environmental triggers
85 involved in disease onset suggest ing an antimicrobial effect being responsible for disease suppression (150). When examining current anti-cyt okine therapies, the concern of potential side effects in response to a complete blocking of a singl e cytokine arises. The complexity of cytokine/chemokine network and its integrity in the immune syst em suggest that it is quite reasonable to expect unwanted c onsequences. It is therefore reas onable to develop an approach where relevant cytokines are affected but not as mu ch as in current therapies. Further research is required to test the efficiency of arthritis suppression by targeting multiple cytokines in a moderate manner. Our approach, using hAAT might be a first step in this direction since it has been shown that hAAT has moderate i mmunomodulatory effects by down regulating proinflammatory IL-1 TNF, IL-8 and up regulating anti-inf lammatory IL-10 (94). If this mechanism is indeed responsible for the effects we have seen in the presen ted studies has still to be elucidated. Further studies also have to reveal to what extent hAAT regulates the affected cytokines. A critical aspect when delivering proteins is to ensure that they are available at effective concentrations at the side of ac tion. Due to their structure and prope rties, proteins might elicit an immune response which results in their eliminat ion. In the studies described, we showed that hAAT serum level is elevated after protein and gene therapy. Although they vary using different delivery methods and animal models, they seem to be in therapeutic range since we observed an anti-arthritic effect. Neverthele ss it is definitely desirable to determine whether further increase of hAAT serum level would also improve the therapeutic effect. Different options to increase local hAAT con centrations are available. Concerning protein therapy we just need to inject higher concentrations of hAAT pr otein e.g. 1mg/ml or 2mg/ml or
86 even higher doses and determine actual serum level as well as testing for therapeutic effects. The increase of protein concentration using a gene therapy approach would be more challenging. We have to optimize gene delivery by either using a higher vector dose, a different AAV serotype, perform local vector administration or perform ex vivo gene delivery approaches. Increasing the vector dose may be the easiest to perform but it is also a very expensive alternative. In addition we need to determine whether this results in high protein levels w ithout causing noteworthy immune responses. Using a different serotype is also challenging because efficient infect ion of target tissues has to be ensured and most so far known AAV vectors do elicit some immune responses which limits transgene delivery. Local vector administra tion would ensure high ve ctor concentration at the target tissue but as mentioned before since RA is a systemic disease and multiple joints are affected and it is challenging to inject the vector in every single affected joints, including the very small ones. A certainly interesting approach would be ex vivo gene transf er to cells which are major players in RA pathogenesis and therefore target affected joints. Such cells include macrophages, T-cells and B-cells. They would home to the sides of inflammation and the transgene is expressed locally where it is needed. To circumvent the obstacles seen in protei n therapy such as being time consuming and very costly, gene therapy might be the delivery method of choice for future RA therapy. Despite all the drawbacks seen in the field in the last decade, including malignancies and failed therapeutic efficiency, researcher s in the field are still convinced that it has a promising future. Continuous vector improvement and understandi ng of its biology are certainly necessary to
87 achieve the proposed goals and en suring that gene therapy resembles a promising future therapy providing a save, economic, efficient and convenient alternative of drug delivery.
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100 BIOGRAPHICAL SKETCH Christian Alexander Grim stein was born in Georgsmarienhuette, Germany in 1978. After graduating from high school in 1998 he performed civilian service at a ho spital in Osnabrueck, Germany in the Department of Laboratory Medi cine. He started pharm acy school at Saarland University in 1999 and received his bachelors degree in pharmaceutical sciences in 2003. He became Registered Pharmacist in Germany in 2004. Af ter that he started the graduate program in the Department of Pharmaceutics at University of Florida, Gainesville in January 2005. He worked under supervision of Dr. Sihong Song on prot ein and gene therapy of alpha-1 antitrypsin for experimental rheumatoid arthritis.