ADULT STEM CELL-BASED GENE THERAPY FOR ALPHA 1-ANTITRYPSIN DEFICIENCY

By HONG LI

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009 1

© 2009 Hong Li

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To my parents, Guoji Li and Baozhu Zheng, and my sister, Xia Li

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ACKNOWLEDGMENTS I would like to express my deepest gratitude to my mentor, Dr. Sihong Song, for giving me the opportunity to pursue my Ph.D. degree in his laboratory. Dr. Song was always there to help me, listen to me and offer advice. He showed me different ways to approach research problems and guided me development as a scientist. His enthusiasm for science continues to inspire me to overcome the difficulties during my research. I would also like to thank the members of my dissertation committee: Dr. Guenther Hochhaus, Dr. Jeffrey Hughes, and Dr. Bryon Petersen; for their valuable suggestion, and advice. Furthermore, this project would not have been possible without the kind help of Dr. Young-Kook Choi and Dr. Bin Zhang, who both taught me molecular cloning techniques; Dr. Yuanqin Lu, who assisted with my animal studies; Dr. Rafal Witek, who taught me how to do liver transplantations; and Marda Jorgensen, for teaching me Y- FISH. I would also like to acknowledge Dr. Martha Campbell-Thompson and the staff of the Pathology Core; especially, Amy Wright and Dontao Fu, for their assistance with immunohistochemistry assays. Also many thanks go out to all the members of Dr.Song’s lab, in particular, Dr.Christian Grimstein and Matthias Fueth, who supported me in all my endeavors. Last, but not the least, I would like to thank my parents, Guoji Li and Baozhu Zheng for their unconditional support and guidance in my life. Without their tremendous support, I would not have been able to realize this dream. I also extend a special thanks to my sister, Xia Li. She encouraged me to study abroad and face the challenge. She is always by my side, listens patiently to my frustrations, and helps me through the tough times. I also thank my 3-yr-old niece, Minghui Zhang, for her to bring happiness to my life.

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TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................................... 4 LIST OF FIGURES .............................................................................................................................. 8 ABSTRACT ........................................................................................................................................ 10 CHAPTER 1

LITERATURE REVIEW ........................................................................................................... 12 Introduction ................................................................................................................................. 12 Alpha 1-Antitrypsin Deficiency ................................................................................................. 12 AAT Biology........................................................................................................................ 13 AAT Deficiency Pathogenesis ............................................................................................ 14 Current Therapy for AAT Deficiency ................................................................................ 16 Recombinant AAV Vector ......................................................................................................... 19 AAV Genome ...................................................................................................................... 19 AAV Entry ........................................................................................................................... 20 AAV Serotype ...................................................................................................................... 22 Self-Complementary AAV.................................................................................................. 24 AAV Integration .................................................................................................................. 26 Recombinant AAV Vector and Its Application for Stem Cell Transduction .................. 28 Lentiviral Vector ......................................................................................................................... 30 Adult Stem Cells ......................................................................................................................... 33 Hepatic Oval Cell ........................................................................................................................ 33 Induction and Isolation of Oval Cell .................................................................................. 33 The Origin of Oval Cells ..................................................................................................... 35 Signal Pathway of Oval Cell Activation ............................................................................ 36 Bone Marrow Cells ..................................................................................................................... 37 Hematopoietic Stem Cells (HSCs) ..................................................................................... 37 Isolation and phenotype of HSCs ................................................................................ 37 HSCs in stem cell-based gene therapy ........................................................................ 38 Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) ....................................... 39 Isolation and in vitro characteristics of BM-MSCs.................................................... 39 Differentiation capacity and immunosupression of BM-MSCs ................................ 40 Transdifferentiation and Cell Fusion .................................................................................. 41 Adipose Tissue-Derived Mesenchymal Stem Cells (AT-MSCs) ............................................. 42 AT-MSCs vs BM-MSCs ..................................................................................................... 42 Isolation and Characterization of AT-MSCs ..................................................................... 43 Proliferation and Differentiation Capacity of AT-MSCs .................................................. 44 Liver Anatomy ............................................................................................................................ 46

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MATERIALS AND METHODS ............................................................................................... 48

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Hepatic Oval Cell Induction and Isolation from Mouse Liver ................................................. 48 Bone Marrow Isolation ............................................................................................................... 48 AT-MSCs Isolation and Culture ................................................................................................ 49 Recombinant AAV Vector Construction and Production ........................................................ 49 Lentiviral Vector Construction and Production ........................................................................ 50 Animal.......................................................................................................................................... 51 In vitro Transduction ................................................................................................................... 51 In vivo Injection of Vectors into Mouse Liver and Muscle ...................................................... 51 Monocrotaline Treatment ........................................................................................................... 52 Adipogenic and Osteogenic Differentiation of AT-MSCs ....................................................... 52 Adipogenesis ........................................................................................................................ 52 Osteogenesis......................................................................................................................... 52 Liver Directed Transplantation of Adult Stem Cells ................................................................ 53 Immunohistochemistry for Human AAT, GFP and Mouse Albumin...................................... 53 Immunofluorescent Staining of AT-MSCs................................................................................ 54 Y-chromosome Fluorescence in situ Hybridization.................................................................. 55 Human AAT Specific ELISA ..................................................................................................... 55 3

HEPATIC OVAL CELL-BASED LIVER GENE DELIVERY .............................................. 58 Introduction ................................................................................................................................. 58 Animal Experimental Design ..................................................................................................... 58 Results .......................................................................................................................................... 59 Ex vivo Transduction Efficiency on Oval Cells by rAAV Vector .................................... 59 Lentiviral Vector Construction ........................................................................................... 59 Ex vivo Transduction and Transplantation of Oval Cells .................................................. 60 Discussion .................................................................................................................................... 61

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EX VIVO TRANSDUCTION AND TRANSPLANTATION OF BONE MARROW CELLS FOR LIVER GENE DELIVERY OF ALPHA 1-ANTITRYPSIN ............................ 70 Summary ...................................................................................................................................... 70 Introduction ................................................................................................................................. 70 Animal Experimental Design ..................................................................................................... 72 Results .......................................................................................................................................... 73 Bone Marrow Cells Transduction ....................................................................................... 73 Liver Transplantation of ex vivo Transduced Bone Marrow Cells ................................... 73 Bone Marrow Cell Transplantation Resulted in Sustained Levels of hAAT in Recipient Circulation ....................................................................................................... 74 Discussion .................................................................................................................................... 75

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ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELL-BASED LIVER GENE DELIVERY ..................................................................................................................... 86 Summary ...................................................................................................................................... 86 Introduction ................................................................................................................................. 87 Experimental Design ................................................................................................................... 89

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In vivo Transduction by ssAAV and dsAAV Vectors ....................................................... 89 Ex vivo Transduction and Transplantation of AT-MSCs .................................................. 90 Results .......................................................................................................................................... 90 Isolation and Characterization of AT-MSCs ..................................................................... 90 Optimization of rAAV vecotors ......................................................................................... 91 Liver Transplantation of ex vivo Transduced AT-MSCs .................................................. 92 Discussion .................................................................................................................................... 93 6

SUMMARY AND FUTURE DIRECTION ............................................................................ 105 Summary .................................................................................................................................... 105 Future Direction ........................................................................................................................ 107

LIST OF REFERENCES ................................................................................................................. 109 BIOGRAPHICAL SKETCH ........................................................................................................... 130

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LIST OF FIGURES page

Figure 2-1

rAAV vectors construct. ........................................................................................................ 57

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Experimental outline of oval cell study.. .............................................................................. 64

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Flow cytometric quantification of green fluorescent oval cells after transduction of rAAV-CB-GFP vectors.......................................................................................................... 65

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Construct of Lenti-CB-hAAT vectors.. ................................................................................ 66

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Ex vivo transduction of oval cells.......................................................................................... 67

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Detection of expression of hAAT in recipient liver after transplantation of ex vivo transduced oval cells by immunostaining. ............................................................................ 68

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hAAT expressed from engrafted oval cells.. ........................................................................ 69

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Experimental Outline of BM cell study.. .............................................................................. 78

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Ex vivo transduction of BM cells. ......................................................................................... 79

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Detection of expression of human alpha1-antitrypsin (hAAT) in recipient liver after transplantation of viral vector infected BM cells by immunostaining................................ 80

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Detection of transgene expression from the engrafted donor BM cells by fluorescence double immunostaining for human alpha 1-antitypsin (hAAT) and green fluorescent protein (GFP). ........................................................................................... 81

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Detection of donor cells in recipient liver after BM cell transplantation by fluorescence in situ hybridizations (FISH) for Y-chromosome. ......................................... 82

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Detection of coexpression of human alpha 1-antitypsin (hAAT) and mouse albumin by immunostaining.. ............................................................................................................... 83

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Multi-ogran homing of transplanted BM cells.. ................................................................... 84

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Detection of expression of human alpha1-antitrypsin (hAAT) in the recipient serum.. ... 85

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Experimental outline of AT-MSC study............................................................................... 96

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Characterization of AT-MSCs............................................................................................... 97

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In vivo muscle or liver transduction by ssAAV and dsAAV vectors.. ............................... 98

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Ex vivo AT-MSCs transduction efficiency of rAAV vectors.. ............................................ 99

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Detection of expression of human alpha 1-antitrypsin (hAAT) in recipient liver after transplantation of ssAAV1-CB-hAAT infected AT-MSCs by immunostaining.. ........... 100

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Detection of donor cells in recipient liver after AT-MSCs transplantation by fluorescence in situ hybridizations for Y-chromosme. ...................................................... 101

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Detection of coexpression of human alpha 1-antitypsin (hAAT) and mouse albumin by immunostaining.. ............................................................................................................. 102

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Multi-ogran homing of transplanted AT-MSCs.. ............................................................... 103

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Detection of expression of human alpha 1-antitrypsin (hAAT) in the serum.. ................ 104

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy ADULT STEM CELL-BASED GENE THERAPY FOR ALPHA 1-ANTITRYPSIN DEFICIENCY By Hong Li August 2009 Chair: Sihong Song Major: Pharmaceutical Sciences Alpha 1-antitrypsin (AAT) deficiency is a genetic defect caused mostly by a single base substitution in the AAT gene, and leads to hepatocyte dysfunction or lung destruction. Protein replacement therapy is the only available treatment for AAT deficiency associated lung disease and requires weekly repeated intravenous infusion of human AAT (hAAT) protein. For AAT deficiency associated liver disease, no effective therapy is available except liver organ transplantation which is limited by the shortage of donor organ. Recent studies showed that adult stem cell gene therapy which replaces the patients’ disease-causing gene with the healthy counterparts in their own stem cells holds great potential for the treatment of genetic diseases. To test the feasibility of adult stem cell-mediated liver gene therapy for treatment of AAT deficiency, we performed a series of experiments using three types of adult stem cells including liver progenitor cells (oval cells), bone marrow (BM) cells and adipose tissue-derived mesenchymal stem cells (AT-MSCs) for ex vivo transduction and transplantation. Using oval cells, we confirmed the feasibility of recombinant adeno-associated virus (rAAV) vector mediated ex vivo transduction and transplantation. Considering isolation of oval cell for autologous transplantation is not clinically applicable, we have test the use of BM cells. We showed that both lentiviral vector and rAAV vectors can transduce BM cells. Transplantation of 10

transduced BM cells showed that BM cells transdifferentiated into hepatocytes and mediated transgene (hAAT) expression in the liver. Importantly, sustained serum levels of hAAT were detected in the recipient mice. Similarly, we have employed AT-MSCs since they can be obtained easily (or less invasively) in large quantities. Results from this study demonstrated that AT-MSCs were transduced efficiently by rAAV serotype 1 vector. After transplantation, these cells engrafted into recipient liver, transdifferentiated into hepatocytes, contributed to liver regeneration, and served as platform for transgene expression. Sustained serum levels of hAAT in the recipients implied a potential application for future treatment for AAT deficiency. AT-MSC-based gene therapy presents a novel approach for the treatment of human genetic diseases, such as AAT deficiency. Future studies will focus on achieving therapeutic levels of transgene expression, and gene correction in AT-MSCs for AAT deficiency associated liver disease.

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CHAPTER 1 LITERATURE REVIEW Introduction Alpha 1-antitrypsin (AAT) deficiency is an autosomal recessive condition caused mostly by a single gene mutation in the AAT coding sequence. This mutation results in abnormal aggregation of AAT protein in hepatocytes and consequent absence of AAT protein in the systemic circulation, which leads to hepatocyte dysfunction or lung destruction. Adult stem cell gene therapy provides a promising treatment for long-term gene correction by taking advantage of self-renewal and multiple differentiation potential of adult stem cells. Adult autologous stem cell therapy also avoids the ethical quandaries presented by embryonic stem cells and the complications from a triggered immune response. To outline the potential of a therapy for AAT deficiency by using adult stem cells, this chapter reviews the biology, pathogenesis, and current treatment for AAT deficiency. Current research on recombinant adeno-associated virus (rAAV) vector, lentiviral vector and adult stem cells (including hepatic oval cells, bone marrow cells and adipose tissue-derived mesenchymal stem cells) are also reviewed to provide theoretical support for the adult stem cell-based gene therapy. Alpha 1-Antitrypsin Deficiency AAT deficiency is a genetic disorder resulting from mutations of AAT gene. The mutation results in a reduction of serum levels of alpha 1-antitrypsin and consequently an increased risk of developing early onset pulmonary emphysema and severe forms of liver disease, including cirrhosis, neonatal hepatitis and hepatocellular carcinoma.1, 2 An estimated 80,000 to 100,000 Americans are living with severe AAT deficiency, but fewer than 10 percent have been diagnosed.3

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AAT Biology AAT, a serine protease inhibitor, is mainly synthesized by hepatocytes and secreted into the circulatory system with a serum concentration ranging from 20 to 53 µM. It is the second abundant plasma protein next to albumin and has a serum half-life of 4 to 5 days.2 The AAT molecule is a 52kD glycoprotein with 394-residue and three asparagines-linked carbohydrate side chains.4-6 The active inhibitory site of the AAT molecule is positioned on a protrusion from the surface of its globular molecule.7 The primary function of AAT is to protect delicate tissue such as lung alveoli, of which major component is elastin, against the excessive proteolytic damage of neutrophil elastase (NE) by combining the AAT inhibitory site (Met358 - Ser359) with the active proteolytic site (catalytic triad Ser 173 -His41 -Asp88) of the protease in a mousetrap mechanism. NE is an enzyme produced by neutrophils in response to inflammation and functions to digest damaged tissue and bacteria. Once AAT binds NE, the AAT-NE complex remains intact and rarely comes apart.8 As a result, AAT inhibit the activity of NE. The AAT gene locus, designated by their protease inhibitor (PI), is located on chromosomal segment 14q32.1 and composed of four coding exons II-V and three untranslated exons in the 5’ region IA, IB, and IC.9 The reactive serine protease inhibitory site of AAT, Met 358

is located in exon V.7 The length of hAAT mRNA depends on the site of synthesis. In

hepatocytes, the major site of synthesis, AAT mRNA is 1.6 kb in length. AAT is also transcribed in the minor extrahepatocyte sites such as mononuclear phagocytes and neutrophils with mRNA of 1.8 and 2.0 kb in length.9, 10 Over 100 AAT variants have been discovered and named according to their migration rate in a pH 4-5 isoelectric focusing gel, such as fast (F), medium (M), slow (S), and very slow (Z). For clinical classification purposes, AAT variants may be divided into at least four different categories: normal variants, with normal AAT serum levels and without an association with lung or liver disease, (i.e. M variant); deficient variants, with 13

reduced but detectable AAT serum concentration and associated with lung and/or liver disease, (i.e. Z and S variants); dysfunctional variants, associated with altered function, (i.e. AAT Pittsburgh, a thrombin inhibitor instead of a neutrophil elastase inhibitor); and null variants, with no serum AAT and an increased risk of lung disease.11, 12 AAT Deficiency Pathogenesis AAT deficiency was discovered in 1963 by C.-B. Laurell and S.Eriksson in Malmö, Sweden13 when they described that three of five individuals lacking an alpha 1-globulin band in electrophoretic analysis of serum had significant pulmonary disease. It was known that about 90% of the alpha 1-globulin band was a single protein capable of inhibiting the proteolytic action of trypsin; hence, the term alpha 1-antitrypsin deficiency was used to define this disease state.13 After decades of AAT deficiency research, “alpha 1-antitrypsin” was recognized as a misnomer since NE is the major target of AAT. Therefore, although it is correct that “alpha 1-antitrypsin” inhibits trypsin, it also inhibits a number of serine proteases (proteolytic enzymes with serine at the active site). Despite campaigns to change the name to alpha 1-antiprotease, alpha 1antitrypsin is still commonly used to refer to this molecule. AAT deficiency is an autosomal recessive disorder that requires the inheritance of two defective AAT alleles from each parent to substantially increase the risk of disease. Among over 100 AAT variants, the Z variant and S variant are the two most common mutated alleles. The Z allele contains a G-to-A mutation within exon V which results in a change from a negatively charged glutamate (GAG) at position 342 to a positively charged lysine (AAG) within the protein. This amino acid substitution causes abnormal folding and the accumulation of AAT within the endoplasmic reticulum. The Z mutation accounts for >95% of all AAT deficiency alleles.14 Individuals with two homozygous Z alleles have an 85% deficit in plasma AAT concentrations and a lower association rate constant than that of the normal M allele.13 The S 14

variant is an A-to-T mutation within exon III, which results in a change from glutamate (GAG) at position 264 to valine (GTG). This resulting protein has an increased susceptibility to intracellular degradation.15 Individuals with two S alleles have a 40% decrease in plasma AAT concentrations.13 Consequently, AAT plasma concentrations in both ZZ homozygotes and SZ heterozygotes are insufficient to ensure the lifetime protection of tissues from the proteolytic damage of NE and cause clinical deficiency of AAT. Compound heterozygotes for Z allele and S allele are also at risk if the AAT serum levels fall below the protective threshold of 11µM.16, 17 AAT deficiency is more prevalent in Caucasians, although it has been identified in virtually every population, culture and ethnic group.18, 19 The allelic frequency of Z variant is of 0.01-0.02 in North American Caucasians and 0.02-0.03 in Northern Europeans.20 The two major clinical manifestations of AAT deficiency are lung disease, emphysema, and liver disease (e.g. neonatal jaundice and cirrhosis). Emphysema develops when the elastin fibers in the lung parenchyma which normally support the structure of the alveoli are destroyed by NE. It is now known that the destruction of elastin in the lung is the result of a disturbance of the physiologic balance between anti-protease and protease within the lung, which explains why AAT deficiency will increase the probability of developing emphysema. There are two major features that differentiate AAT-deficiency associated emphysema from its non-AAT deficient counterpart. First, the age of onset is much earlier in AAT-deficient patients, for whom symptom will present by age 35 to 50 years.21, 22 In contrast, non-AAT deficient individuals typically don’t develop the symptoms until their 60s or 70s. Second, the disease location in the lung is more prevalent in the lower part of the lung for AAT deficient individuals, whereas in the non-AAT related emphysema, the disease affects the upper lung region. Cigarette smoking significantly increases the risk and fastens the onset of AAT-deficiency associated emphysema by 10-

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15years.23 Oxidants, particularly oxidants in cigarette smoke can easily oxidize the Met358 , the active inhibitory site of AAT, and thus render AAT impotent as an inhibitor of NE.24 AAT deficiency accounts for about 2% of the cases of emphysema seen by clinicians in the US.20 The pathogenesis of AAT-deficiency-associated liver disease is not well understood as that of the lung disease. The mechanism by which PI*Z mutant AAT protein accumulates within the endoplasmic reticulum of hepatocytes is likely due to the polymerization of AAT protein, a result of a change in the three-dimensional structure secondary to the Glu342 →Lys substituation.7 Aggregated mutant AAT protein is targeted for proteasome-mediated degradation. Inefficient degradation of mutant AAT protein may predispose patients to liver disease.23 The clinical presentation of AAT-deficiency-related liver disease is quite different from that of AATdeficiency-related lung disease. Only approximately 10-15% of AAT-deficient individuals with two homozygous Z alleles will develop the liver disease23, while lung disease will occur in any AAT-deficient individual with a serum AAT level of < 11µM. The liver disease presents in infancy or early childhood causing progressive cirrhosis and often leading to liver failure, whereas the lung disease presents in the early adulthood. Furthermore, the lung and liver disease rarely coexist in the same individual.25 There is no available therapy for AAT deficiency-related liver disease other than liver transplantation. Nevertheless, liver transplantation has a number of important drawbacks, including shortage of donors, lifelong immunosuppressive therapy and a significant mortality rate. Current Therapy for AAT Deficiency The prevention of lung disease in AAT deficiency is a relatively straightforward concept, since the serum levels at 11µM or 800µg/ml and above is a clear indicator of having restored anti-neutrophil elastase defense.23 Protein replacement therapy is the only FDA approved treatment for individuals with emphysema due to AAT deficiency. The regimen of once-weekly 16

infusion of human plasma-derived AAT at a dose of 60 mg/kg effectively sustains AAT serum level above the protective threshold level of 11 µM and directly augments anti-NE protection in the lung of individual with AAT deficiency.1 AAT protein replacement therapy appears to be safe and well tolerated. However, there are several limitations for this therapy. One is the potential for transmitting infectious agents in the donor’s plasma, from which AAT protein is made. Fortunately, there has been no reported cases of blood born diseases (HIV or hepatitis) linked to receiving this therapy. Most common reported side effects include headaches, fever, urticaria, and fatigue, while serious side effects such as anaphylaxis and precipitation of heart failure are rare.26 Another is the limited production capacity for AAT protein. Thousands of doses of human AAT protein replacement have been administered, but still many patients are out of this treatment. Alternative sources have been sought to address this shortage. Alternative sources have been sought to address the shortage of AAT protein. A yeastderived recombinant AAT (rAAT) and a transgenic sheep/goat-derived AAT product have been generated and tested in clinical trials for safety and efficacy.27-31 Unlike human AAT protein, these rAAT products derived from yeast and transgenic animals have different glycosylation. For example, yeast-derived rAAT lacks carbohydrate side chains 29, 32 and thus has a high renal clearance and a much shorter plasma half-life than natural human AAT protein, which makes the intravenous administration impractical. As an alternative, inhalation administration of aerosolized rAAT to the lower respiratory tract of AAT-deficiency individuals has been evaluated.33 Studies showed that aerosolize rAAT can be deposited on the alveolar epithelium and can move from the epithelial surface into the lung interstitium, the critical site requiring protection from NE degradation.33 Once or twice daily aerosol administration of 200mg rAAT should result in sustained levels of anti-NE protection to the lower respiratory tract.33 Inhaled

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route of administration also reduce the dose of AAT protein in replacement therapy. The protective threshold level in the lung is 1.2µM, 10% of the AAT plasma concentration34 , and only 2% of the intravenously infused AAT protein reaches the lung.35 For a 70kg individual, the amount of AAT protein administrated by weekly intravenous infusion of a dose of 60mg/kg will reduce from 4,200mg/week to 1,400mg/week for inhaled therapy, a reduction by two-thirds in the amount.33 Unfortunately, this approach needs more clinical proof and long-term efficacy data before it can be employed in the general population. Gene therapy for lung disease in AAT deficiency is a direct extension of commonly used protein replacement strategies. Besides avoiding the major concerns associated with protein replacement (i.e. pathogen transmission), gene therapy has the capacity to produce a stable plasma level of AAT over a prolonged period of time from a single administration. Gene therapy also prevents the wide-range of fluctuations in AAT levels that is seen in protein replacement therapy. Most importantly, gene therapy can provide a potential treatment for AAT deficiency associated liver disease by implementing the strategy of delivering shRNA to correct or block the mutant AAT gene in hepatocytes. Although the costliness of gene therapy often comes into question, protein replacement therapy is also very expensive. The mean annual cost of protein therapy was from $ 30,000 to $40,000, based on a dosage of 60mg/kg BW and the 1999 average wholesale price for Prolastin®, the first marketed human AAT product, of $0.21/mg.36 Several gene therapy approaches for AAT gene delivery have been evaluated in vivo in animal model including the non-viral delivery systems (e.g. cationic liposome, naked DNA injection, and gene-particle bombardment delivery), and the viral delivery systems (e.g. adenoviral, retroviral and Adeno-associated viral vector). A variety of ectopic sites (e.g. bronchial epithelium, peritoneal surface, liver, skin and skeletal muscle) are capable of secreting

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biologically active AAT into serum.23 A phase I clinical trial evolving the intramuscular injection of recombinant adeno-associated virus serotype 2 alpha 1-antitrypsin (rAAV2-AAT) vector in 12 AAT-deficiency adults has been done. Four dose cohorts ranging from 2.1×1012 vector genome (VG) to 6.9×1013 VG have been tested. No vector-related severe side effects have been observed, and especially no evidence showed that the germ line cells were infected by rAAV vectors. The data support the general safety of this approach up to the highest dose of 6.9×1013 VG per patient. But no therapeutic effect was detected either. A serum level of the transgene product was detectable transiently in only one subject and the level was approximately 125-fold below the lower end of the therapeutic range.23 Another phase I clinical trial using a rAAV serotypes 1 (rAAV1) vector for muscle delivery of AAT gene is ongoing since rAAV1 has demonstrated an efficiency advantage of hundred-fold over rAAV2 in mouse muscle.37 Recombinant AAV Vector AAV Genome Adeno-associated virus (AAV) is a non-pathogenic DNA parvovirus with a linear singlestranded genome of 4.7 kb and a non-enveloped capsid of approximately 22 nm in diameter.38 The AAV genome consists of two open reading frames (ORFs), which comprise the rep and cap genes, and two identical 145-bp inverted terminal repeats (ITRs) that flank either end of AAV genome.39 The rep gene encodes four non-structural regulatory proteins (Rep78, Rep68, Rep52, and Rep40) by utilizing two promoters (p5 and p19) and alternative splicing. The Rep78 and Rep68 proteins are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases.40 During AAV DNA replication, Rep78 and Rep68 bind to 22-bp Rep-binding element (RBE), tandem repeats of the tetramer GAGC, and produce a sitespecific, single-stranded nick into the terminal resolution site (trs) located in the viral ITRs.41 Rep78 and Rep68 proteins also bind RBE homologous at AAV p5 promoter and the proviral 19

integration locus on human chromosome 19 to regulate viral transcription and proviral integration.42 The Rep52 and Rep40 proteins play roles in virus assembly by generating and accumulating single-stranded viral genomes from double-stranded replicative intermediates and driving ssAAV genome translocation into the preformed capsid.43, 44 The cap gene encodes three structural viral capsid proteins (VP1, VP2 and VP3) from a single promoter (p40) through a combination of alternative splicing and alternative start codons.45 These three capsid proteins assemble into a near-spherical protein shell of a total 60 copies of VP1,VP2, and VP3 at a molar ratio of 1:1:10.46 The ITR is the only cis-acting elements required for viral replication, packaging and integration.47-49 The first 125bp of ITR constitute a palindrome and fold on itself to form Tshaped hairpin structure and the other 20 bases, called D sequence, remained unpaired.50 AAV Entry AAV entry in target cell is not well understood yet. Current mechanism for AAV vector to achieve transgene expression comprises receptor binding, internalization/endocytosis, trafficking to the nuclear, uncoating, and conversion of single-stranded (ss) AAV genome to doublestranded (ds) molecule. AAV2 gains entry into target cell by binding to primary attachment receptor heparin sulphate proteoglycans (HSPG).51 Fibroblast growth factor receptor-1 (FGFR1)52 , αVβ5 integrin heterodimers,53 and hepatocyte growth factor receptor (HGFR)54 serve as co-receptors to facilitate the internalization. AAV2 internalizes rapidly by clathrin-mediated endocytosis from clathrin-coated pits (half-time 50% homology, which are first fragmented and then reassembled based on partial homology, resulting in libraries of chimeric genes.84 A single AAV type2/tpye8/type9 chimera, AAV-DJ, was generated by using this technology. AAV-DJ show limited distribution to the liver (and a few other tissues), superb liver performance, and the ability to evade preexisting human immunity.84 Self-Complementary AAV To improve AAV vector as a gene delivery tool, virus genome modification is as important as virus capsid engineering in order to achieve desired transduction efficiency. Transgene expression mediated by rAAV vector is not observed until 1-2 weeks post infection and reaches to the plateau at week 4-6. The delayed transgene expression is thought to be hindered by the 24

conversion of ssAAV genome to dsAAV genome either through host-cell DNA polymerasemediated second-strand synthesis or intermolecular hybridization between plus and minus DNA strand, the rate-limiting step. Even though recent studies show that AAV uncoating efficacy determines the ability of conversion from ssDNA to dsDNA.64, 85 Regardless of the mechanism, the step can be bypassed in scAAV vector which is generated by mutating one of the AAV ITRs to force the generation of dimeric over monomeric replicative forms, e.g. deleting the TRS sequence from one ITR to inhibit Rep protein-mediated site-specific nicking. After uncoating, scAAV genome folds back into dsDNA through intramolecular base pairing due to selfcomplementary nature with a covalently closed ITR at one end and two open-ended ITRs at the other. The folded molecules are ready for serving as template for transcription and result in faster onset of transgene expression and higher transduction efficiency.86, 87 In vitro studies of 293, Hela, and COS-7 cells show dsAAV2-CMV-GFP vector yielded 10-20% green fluorescent cells one day after infection at a dose of 500 v.g./cell compared to less than 1% green fluorescent cells of ssAAV2-CMV-GFP under the same condition.87 In vivo studies show 25-50% hepatocytes displayed transgene expression 6 week after tail vein injection of dsAAV2 vector at 5×105 MOI, but less than 5 % hepatocytes were permissive to conventional ssAAV2 vector for long-term transgene expression.86 The important trade-off for this efficiency is the loss of half the packaging capacity of the AAV vector with a total packaging capacity of 4.7kb, while small coding sequence and RNA-based therapy (shRNA and microRNA, ribozymes) can be accommodated, e.g. efficient delivery of siRNA into multidrug-resistant human breast cancer cells to suppress MDR1 gene expression resulting in a substantial reversion of the drug-resistant phenotype.88 What’s more, recent observation of larger genome, up to 8.9kb, being packaged into an AAV5 capsid greatly expands the therapeutic potential of these vectors.89

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AAV Integration After entry into the host cell nucleus, AAV can follow either lytic or latent life-cycle depending on the presence or absence, respectively, of the helper virus such as adenovirus (Ad) or herpesvirus (HSV). When AAV2 infects a human cell alone, its gene expression is autorepressed and establishes latency by integrating virus genome into human chromosome 19q13.3qter designated AAVS1 with a frequency of more than 70%.90, 91 AAVS1 locus (8.2kb) is near several muscle-specific genes, p85,TNNT1 and TNNI3.90 The AAVS1 region itself is an upstream part of a recently described gene, MBS85, whose product has been shown to be involved in actin organization.92 Tissue culture experiment shows that AAVS1 is a safe integration site.46 AAV site-specific integration is directed by both cellular DNA sequence and viral components. A 33bp minimum AAVS1 sequence containing a RBE-like and a TRS-like sequence separated by an eight-nucleotide spacer is necessary and sufficient to Rep-protein dependent site-specific integration.93, 94 Many RBEs have been identified in human genome but AAVS1 is the only site that has RBE and TRS in close proximity to each other.46 The AAV components required for integration include the ITRs (in cis), Rep78/68 (in trans), and a 138bp cis element termed the integration efficiency element (IEF), located within the P5 promoter.95 It is proposed that Rep protein bind to REBs situated in both AAV genome and AAVS1 site to form a Rep protein/AAV-DNA complex. Within this complex, AAV genome and AAVS1 site are tethered to each other. Site-specific endonuclease activities of Rep78/68 introduce a strandspecific nick at the TRS in AAVS1 and initiate a non-homologous recombination, resulting in integration of the AAV genome.96 Once AAV is integrated, it will remain stable within the infected cells for prolonged periods of time, up to 100 passages.23

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Recombinant AAV vector lacks Rep protein so that the integration is inefficient and random, not targeted to chromosome 19. The majority of rAAV genome persists in the transduced cell as extrachromosomal, not integrated, genomes which are primarily responsible for stable rAAV-mediated gene expression.97 AAV2 vectors integrate to host genome at a low frequency of 1% in liver and no integration was undetectable in muscle (60% transgene integration specifically at AAVS1. More importantly, the cloned cell lines with the AAVS1 site-specific integrated GFP were healthy and stably expressed GFP for 35 passages. More excitingly, Smith et al (2008) has

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shown robust and persistent transgene expression in human embryonic stem cells can be achieved with AAVS1-targeted integration.102 A Rep-expression plasmid and an ITR flanked EGFP reporter gene were co-transfected into hESCs using either electroporation or Lipofectamine 2000. 4.16% of hESC clones achieved AAVS1-targeted integration. The AAVS1targeted hESCs retained their phenotypes and differentiated into all three primary germ layers. EGFP expression from AAVS1-targeted clones showed significantly reduced variegated expression and reduced tendency to undergo silencing when compared to randomly targeted controls. In addition, transgene expression from AAVS1 locus was shown to be stable during hESCs differentiation with more than 90% of cell expressing EGFP after 15 days of differentiation. An insulator in AAVS1 locus was proposed to be one of the possible explanations why the resistance to transgene silencing observed at the AAVS1 site. In addition, the authors showed AAV ITR also impart insulation on the expression cassette in hESCs, resulting in less variegated EGFP expression. Recombinant AAV Vector and Its Application for Stem Cell Transduction The first infectious clone of AAV2 was constructed in 1982 by deleting viral rep and cap genes and inserting a transgene expression cassette between the two ITRs with Rep and Cap genes provided in trans.103 rAAV2 has been tested in preclinical studies for a variety of diseases such as cystic fibrosis, hemophilia, alpha-1 antitrypsin deficiency, Parkinson’s disease, muscular dystrophy, and arthritis.104 Data on safe, broad tropism and long-term expression make rAAV vector rapidly gain popularity in gene therapy application. Since 1995 AAV2-based vector was first administrated to a human subject for treating cystic fibrosis,105 over 40 clinical trials have been approved involving 14 diseases and 4 serotype rAAV vectors so far.106 These studies indicate that in vivo gene transfer is feasible and relatively safe, but also suggest that the transduction efficiency of AAV2 vectors fall short of requirement for adequate and organ28

specific transgene expression. As a result, ongoing research efforts are focused on developing new rAAV vector by modifying both AAV genome and capsid protein as described above. Another direction of future therapy is the combination of rAAV-mediated gene delivery with stem cell therapy. The capability of self-renewal and differentiation of stem cells makes them to be the promising target of virus vector for long-term gene correction. Although AAV vectors can transduce a broad range of tissues and cells, the transduction efficiency of rAAV vector in stem cell remain further improvement. Only few researches have been shown the successful transduction of HSCs by rAAV vectors. Santat et al. (2005) showed efficient rAAV2 transduction of primitive human cord blood HSCs (CD34+ CD38-) capable of serial engraftment in NOD/SCID mice.107 rAAV2-transduced HSCs differentiated into all expected cell lineages including myeloid cells and B lymphocytes after transplantation. Furthermore, transduced CD34+ stem/progenitor cells were continuously detected throughout the analysis in primary and secondary recipients. All of these indicate that rAAV2-transduced HSCs maintain their multipotential differentiation, long-term persistence and self-renewal capacity. Paz et al. (2007) further demonstrated that rAAV2 vector preferentially targets quiescent subpopulation of human CD34+ HSCs, the CD34+CD38- cell population.108 In addition, CD34+CD38- and CD34+G0 cells, the more quiescent cells, were found to possess higher levels of chromosomally integrated forms of rAAV than did CD34+ G1/S/G2 /M cells, the rapidly dividing cells. One of the obstacles that limit high-efficiency rAAV2-mediated transduction of human HSCs is sub-optimal levels of expression of the cell surface receptor and co-receptor for AAV2. By applying an approach of random 7-residue peptide library insertion into the AAV2 capsid sequence developed by Muller et al.(2003), Sellner et al. (2008) obtained a highly efficient hematopoietic progenitor-targeted

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rAAV2 vector resulting in up to 8 fold increase on transduction efficiency of primary human CD34+ peripheral blood progenitor cells compared to standard rAAV2 vectors.109 Unlike HSCs, rAAV can transduce mesenchymal stem cells (MSCs) efficiently. Stender et al. (2007) showed human MSCs could indeed be transiently transduced in vitro by rAAV2 vector with efficiency of up to 65%. The transgene express reached peak at 4 days post transduction and declined rapidly toward 0% after day 8. Transient process is ideal in the case that temporary transgene expression is beneficial without risking potential adverse effects of long-term transgene expression, such as the healing process. Importantly, transduced MSCs retained multipotential activity comparable to untransduced controls demonstrated by retaining the capability of osteogenesis, adipogenesis, and chondrogenesis.110 Ex vivo gene therapy for osteoporosis in mouse model has shown that bone marrow-derived MSCs can be modulated to function as continuous source of progeny osteoblasts after transduction by rAAV vector encoding bone morphogenic protein (BMP-2), known to induce osteoblast differentiation.111 Although studies have demonstrated that supplementation of BMP-2 either by a purified protein or through direct gene transfer into muscle can result in osteoinduction, these approaches appear limited because of the half-life of protein and lack of target cell specificity of gene transfer approcaches.111 However, rAAV transduction on MSCs seems to be species-specific. Rat MSCs has been found to be refractory to transduction by AAV serotype 1-6, in contrast to rabbit MSCs tested at the same time.112 Lentiviral Vector Lentivirus is a member of retrovirus, a RNA virus family, of which particle consists of 2 identical single-stranded RNA molecules.113 A unifying feature of these viruses is that replication involves the process of conversion of the viral RNA genome into double-stranded DNA, hence the designation “retro” (backward) virus.114 Lentivirus is unique among retrovirus 30

because of its ability to infect nondividing and terminally differentiated cells such as HSCs and neurons, something that other retroviruses cannot do.115 The ability to integrate into the host genome of nondividing cells makes lentivirus particularly attractive for permanent genetic modification in the treatment of chronic diseases and genetic defects. Lentiviral vectors are derived from different species such as human immunodeficiency virus (HIV-1and2)116-118, simian immunodeficiency virus (SIV)119, feline immunodeficiency virus (FIV)120-122 , equine infectious anemia virus (EIAV)123, 124, caprine arthritis encephalitis virus (CAEV)125, and bovine immunodeficiency virus (BIV)126, 127. Among those, the HIV-1 based lentiviral vectors are prototypical and predominantly used in current studies116. HIV-1 genome contains three structural genes: gag, pol and env. The gag gene encodes three protein subunits: matrix (MA), essential for virion assembly and infection of nondividing cells; capsid (CA), which forms the hydrophobic core of the virion and is essential for virion assembly and maturation; nucleocapsid (NC), which coats viral RNA stochiometrically; and several additional polypeptides of small size and unknown function, such as p1, p2 and p6. The pol gene encodes three enzymes required for viral replication: protease (PRO), reverse transcriptase (RT) and integrase (IN). The env gene is essential for viral binding and entry into the host cells. It encodes the precursor glycoprotein, gp160, which is cleaved into a surface moiety, gp120 (SU), and a transmembrane moiety, gp41 (TM). SU is required for binding to cellular receptors and TM is responsible for the fusion with cellular membrane. Different from simple retroviruses, HIV genome encodes two additional regulatory genes, tat and rev, and four accessory genes, vif, vpu, vpr, and nef, all of which are involved in the viral pathogenesis.114 HIV-1 genome is flanked by long terminal repeats (LTRs) on either end. The LTR consists of repeat region (R), unique 5’ and unique 3’ sequence at U3-R-U5 manner. Two viral integrase

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attachment sites (att) are located in the 5’ (U3) and 3’ (U5) termini of LTRs, important for integration into host chromosome. The LTRs control viral transcriptional initiation and termination.128 HIV-1 based lentiviral vectors are generated by co-tranfecting 3 plasmids into human cells, including transducing vector (TV), help plasmid (HP), and vesicular stomatitis virus G (VSV-G) envelope expression plasmid. TV contains the transgene expression cassette and cis-acting sequences (modified lentiviral LTRs) for efficient transduction. HP comprises gag and pol gene in order to supply reverse transcriptase and integration function in trans. HIV-1 lentiviral vector is pseudotyped with VSV-G envelope protein to improve the tropism of HIV-1 vector.128 Promising results with lentivirial vector have been achieved in animal model for HIV-1 infection129,β-thalassaemia130 , sickle cell disease131, Parkinson’s disease132, muscular dystrophy 133

, etc. A phase I open-label nonrandomized clinical trial for HIV infection has been completed.

Autologous CD4+ T cells from HIV-infected subjects were transduced ex vivo with HIV-1-based lentiviral vector, named VRX496, which contains a 937-base antisense gene against the HIV envelope. VRX496 directly interferes with wild-type HIV (wt-HIV) expression via anti-env antisense expression in vector transduced CD4+ T cells that become infected with wt-HIV and thus decrease productive HIV replication from CD4+ T cells. Five subjects with chronic HIV infection who had failed to respond to at least two antiviral regimens were enrolled. A single IV infusion of gene-modified autologous CD4+ T cells was well tolerated in all patients. Viral loads were stable, and one subject exhibited a sustained decrease in viral load. CD4+ T cell counts remained steady or increased in four subjects, and sustained gene transfer was observed. There is no evidence for insertional mutagenesis after 21-36 months of observation. Immune function improved in four subjects134, 135 . Lentiviral vectors appear promising for gene transfer to human.

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Adult Stem Cells An adult stem cell is defined as an undifferentiated cell found in a tissue or organ, can renew itself, and differentiate to yield the major specialized cell types of the tissue or organ. The primary roles of adult stem cells are to maintain and repair the tissues where they are found. Regenerative medicine devotes to rebuilding damaged organs from stem cells including cloned cells, embryonic or fetal stem cells, or adult stem cells. Of all the different stem cell types, only adult stem cells might provide more medical solutions because of avoiding the ethical and legal problems of cloning and embryonic-stem-cell approaches. Recently studies have suggested that adult stem cells are plastic, meaning that they can differentiate not only into their original source tissue, but also into cells of unrelated tissue. Hepatic Oval Cells Hepatic oval cells are bipotential progenitors that can differentiate into two types of epithelial cells within the liver, hepatocytes and bile ductular cells, when severe hepatic injury can not be corrected by replication of mature hepatocytes.136 Oval cells can be isolated from animal model in large quantity and form colony and proliferate in in vitro culture supplemented with growth facts such as stem cell factor (SCF), Flt-3 ligand, and interleukin-3 (IL-3).136 Furthermore, oval cells can differentiate into insulin-producing pancreatic cells137, and neural cells under certain condition138. Induction and Isolation of Oval Cell Oval cells were first described by Kinosita et al. who observed small ovoid cells in the livers of rats exposed to the carcinogenic azo dye ‘Butter Yellow’.139 Later on, E. Farber termed these cells ‘oval cells’ because of their characteristic morphology, ovoid nucleus, small size (compared to hepatocytes), and large nucleus to cytoplasm ratio.140 Oval cells didn’t invoke attention until Thorgeirsson et al. showed that oval cells can differentiate into hepatocytes in the

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1980s.141, 142 Under normal conditions, oval cells are quiescent and reside in the Canals of Hering (also called cholangioles, terminal ducts, or ductules).143, 144 When severe liver damage occurs and the proliferation of hepatocytes is blocked by exposure to hepatotoxins or carcinogens, oval cells are activated, and proliferate in the periportal region of liver. As the liver damage progresses, they infiltrate into the parenchyma along the bile canaliculi between the hepatic cord.139 In rat models, the common protocol of oval cell activation employs a two-step induction with 2-acetylaminofluorene (2-AAF) and either LD50 dose of carbon tetrachloride (CCl4) or a two-thirds partial hepatectomy (PHx).145 2-AAF, a carcinogen, hinders hepatocytes proliferation and PHx or CCl4 cause physical/chemical liver damage to create a regenerative stimulus. However, mouse oval cell compartment doesn’t response to this two-step induction protocol used in the rat model. Instead, mouse was placed on a diet containing the chemical 3, 5diethoxycarbonyl-1, 4-dihydrpcollodine (DDC) at 0.1% concentration.146 0.1% DDC diet cause chronic liver injury and induces very consistent and massive oval cell accumulation after 4 to 6 weeks.147 In contrast to the rat oval cell regimen, DDC does not completely block hepatocytes proliferation, but rather induces a chronic regenerative state in the liver.148 The DDC-induced murine oval cells were indeed capable of liver repopulation and could rescue a metabolic liver disease.148 Morphologically, mouse oval cells share many similarities to rat oval cells, small in size (approximately 10µm) and a large nucleus to cytoplasm ratio; they radiate from the periportal region forming primitive ductular structures with a poorly defined lumen. 149 To isolate oval cells from the liver, gradient density centrifugation is applied to separate nonparenchymal compartment (NPC) fraction containing oval cells from hepatocytes after twostep collagenase perfusion of the liver.150 To further enrich oval cells from NPC, oval cell

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surface markers are utilized. Oval cells share molecular marker with adult hepatocyte (albumin, cytokeratin 8 and 18), fetal hepatocytes (AFP), bile duct cell (cytokeratin 7 and 19, γ-glutamyltranspeptidase (GGT), OV-6 for rat and A6 for mouse).145, 151-155 Oval cell also express hematopoietic stem cell marker (Thy-1, CD34, Flt-3 and c-kit).152, 156, 157 In rat, by using Thy-1 antibody and flow cytometric method, 95% to 97% of pure Thy-1+ oval cells can be isolated, which also expressed the traditional oval cell markers of AFP, CK19, GGT,OC.2 and OV6152 , but were negative for desmin, a marker for Ito cells . In mice, by using Sca-1 antibody in conjugation with magnetic activated cell sorting (MACS), more than 90% of oval cells (A6 +and AFP +) can be enriched.149 So far, none of these protocols to induce oval cells from murine liver would fulfill the requirements for clinical application to isolate human oval cells since they involve administration of carcinogens. Liver progenitor cell activation has been observed in the chronic liver diseases, such as hepatic cirrhosis due to hepatitis B158 , and is also seen in the hepatocellular carcinoma and cholangiocarcinoma development.159 The Origin of Oval Cells The origin of oval cells has been discussed for decades but still remains controversial. Traditionally, oval cells have been believed to originate in the liver within the Canals of Hering, the junction between the hepatocytes canalicular system and the terminal bile ducts.139 But this can not rule out the possibility that oval cell might be derived from other cells of either intrahepatic origin or extrahepatic origin. Actually, most studies have implied the existence of an undifferentiated oval cell precursor that proliferates and gives rise to oval cells.144, 160, 161 Extrahepatic origin of oval cell precursor is supported by the observation that classic hematopoietic markers, including Thy-1, c-kit, and CD34, are also expressed by oval cells. Additionally, sex-mismatched BM transplantation in lethally irradiated DPPIV¯ rat treated with 2-AAF/CCl4 has firstly demonstrated cells in the bone marrow were capable of repopulating the 35

injured liver.145 Bone marrow transplantation of c-kit+, Thylo , Lin- , and Sca l+ (KTLS) hematopoietic stem cells rescued the FAH-/- mouse, an animal model of tyrosinemia type I, and restored the biochemical function of its liver.162 However, Wang et al.(2003) and Menthena et al.(2004) argued that oval cells originated from endogenous liver progenitors but not arise through transdifferentiation from BM cells, because the oval cells isolated from the BM transplanted mice/rats lacked the genetic markers of the original bone marrow donor.148, 163 Furthermore, the cell fusion of BM progenitors with resident hepatocytes might be the explanation to the observation of BM-originated hepatocytes. This discrepancy might be explained by the timing effect of exposure to hepatotoxic chemicals including monocrotalin (MCT) and retrorsine, which plays an antimitotic activity on hepatocytes and bone marrow cells.164 Of course, more data are needed to clarify this controversy. Signal Pathway of Oval Cell Activation The interaction between stromal derived factor-1 alpha (SDF-1α) and its receptor CXCR4, a mediator of hematopoiesis165, has been proposed to be the possible mechanism by which oval cells are activated and participate in liver regeneration.166 When oval cells are involved in the regenerative process, it was found that oval cells expressed the stromal derived factor-1 alpha (SDF-1α) receptor, CXCR4, while SDF-1α protein expression was up-regulated by hepatocytes in the injured liver. Oval cells migrate along a SDF-1α chemotactic gradient to the injured liver parenchyma. However, under non-oval cells-aided regeneration, SDF 1α expression was not detected. SDF-1α/CXCR4 interaction possibly recruits a second wave of bone marrow cells to the injuried liver as a percentage of oval cells are of hematopoietic origin.166 Other molecular pathways involve the mitogenic cytokines for oval cell, i.e., tumor necrosis factor (TNF) and interleukin-6 (IL-6). The cytokine TWEAK (TNF-like weak inducer of apoptosis) selectively stimulates oval cell proliferation in mouse liver through its receptor Fn14 with no detectable 36

mitogenic effect on hepatocytes.167 Three primary growth factors, Hepatocyte growth factor (HGF), epidermal growth factor (EGF) and transforming growth factor-α (TGF-α) , are highly upregulated in liver regeneration via the stem cell compartment. HGF acts as a strong promoter of differentiation toward the hepatic lineage.168 Both oval cells and HSCs express the HGF receptor c-Met.169 Transforming growth factor-beta1 (TGF-β1) and tumor necrosis factor-alpha (TNF-α) are also being upregulated to suppress the differentiation of HSCs into megkaryocytes and down the myeloid lineage.170, 171 These cellular factors, plus yet undetermined factors, control the process of homing, engrafting and differentitiating into a hepatic lineage. Bone Marrow Cells BM is the reservoir of stem cells including two major populations, hematopoietic stem cell (HSC) and mesenchymal stem cell (MSC). BM stem cells are one of the first stem cells to be used successfully in humans for treating blood disease (e.g. leukemia) and BM transplantation gave rise to the 1990 Nobel Prize in Medicine. Hematopoietic Stem Cells (HSCs) Isolation and phenotype of HSCs HSCs are the best-studied and well-characterized population of stem cells mainly found in the bone marrow. HSCs can reconstitute the whole hematopoietic system because of their capability of giving rise to all the blood cell types including myeloid and lymphoid lineages and their ability to replenish themselves by self-renewal. The hematopoietic tissue contains cells with long-term and short-term regeneration capacities and mulitpotent or lineage-committed progenitors. In human bone marrow, 1% of cells are CD34+ progenitor cells which are lineage committed progenitor cells, including lymphoid, myeloid and erythroid progenitor cells. 0.1-1% of total CD34+ cells are thought to represent pluripotent progenitors capable of self-renewal and differentiation along any of the hematopoietic lineage.172 Strategies for preparing highly enriched

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HSCs may consist of combination of lineage depletion followed by positive selection of Linsubset that expresses specific hematopoietic markers. In lineage depletion step, cells that express lineage markers (e.g. CD3 for T cells, B220 for B cells and some NK cells, Ly6g/Gr-1 for granulocytes, CD11b/Mac-1 for monocyte /marcophages and TER119 for erythroid cells173) are removed. Subsequent Lin- population is subject to positive selection for c-Kit+, Thylo , and Sca l+, so called KTLS cells, a virtually pure population of multilineage HSCs. Thirty of KTLS cells are sufficient to save 50% of lethally irradiated mice.173 HSCs in stem cell-based gene therapy To date, approximately 40% of the more than 450 gene therapy clinical trials conducted in the US have been cell-based.174 Of these, about 30% have used human stem cells, specifically HSCs, as the means for delivering transgene into patients.175 Clinical trials have used genetically modified HSCs to correct severe genetic immunodeficiency diseases, such as X-SCID, ADASCID and CGD.176-178 These clinical trails proved the concept of stem cell-based gene therapy but also pointed out potential barriers to the development of the treatments using HSCs. First, gene transfer into HSCs did not lead to efficient transduction rates of these cells. Second, over time, the transgene get turned off, known as gene silencing, due to cellular mechanisms that alter the structure of the area of chromosome where the therapeutic gene has been inserted.179-183 Third, HSCs can not be expanded ex vivo. In recent years, scientists have overcome some of these limitations. To improve the transduction efficiency of HSCs, virus vectors that can transduce non-dividing cells (e.g. HSCs) have been explored, such as lentiviral vector and AAV. Another approach has been to stimulate HSCs to divide without differentiating by using cytokines, i.e., flt3-ligand and stem cell factor. The inability to expand HSCs ex vivo hinders the current application of HSCs in both cell and cell-based gene therapy. This is especially true in cases where the number of available stem cells 38

is limiting. One group of researchers showed the possibility of 40-fold expansion of mouse HSCs by overexpressing the homeobox B4 (HOXB4) gene by retroviral gene tranafer.184 Beside the blood lineages, HSCs also show capability of differentiating into brain, muscle, and liver cells.162, 185, 186 These observations implies even broader applications of HSCs in both cell and stem cell-based gene therapy. Bone Marrow-Derived Mesenchymal Stem Cells (BM-MSCs) MSCs are a heterogeneous population of plastic-adherent, spindle-shaped and fibroblastlike cells which can be extensively expanded in vitro and differentiate into mesenchymal lineage such as bone (osteoblasts), cartilage (chondrocytes), adipose (adipocytes). When cultured at lowdensity, MSCs are able to form fibroblastic colonies, termed colony-forming unit-fibroblasts (CFU-F). As another distinct stem cell population which resides in bone marrow, MSCs are a population of stem cell responsible for the maintenance of non-hematopoietic bone marrow elements which promote HSC proliferation and differentiation. The history of MSCs can be traced back 130 years ago when the German pathologist Cohnheim first suggested the presence of non-hematopoietic stem cells in his study of wound repair.187 It was not until the mid-1970s that Friedenstein and his colleagues first successfully isolated fibroblast-like cells from bone marrow.188 Isolation and in vitro characteristics of BM-MSCs To date, Friedenstein’s procedure of MSCs preferential attachment to tissue culture plastic is still considered as the standard approach to isolate MSCs. Marrow aspirate, from the tibias and femurs of the rodent experimental animals or the iliac crest of human, is applied to density gradient centrifugation to isolate the BM mononuclear cells (BM-MNCs). BM-MNCs are plated on the tissue culture plastic in medium with 10%FBS. MSCs representing approximatly1 in 10,000 BM-MNCs attach and grow as fibroblastic cells that develop into visible symmetric 39

colonies at about 5 to 7 days after initial plating189, 190. HSCs and non-adherent cells are washed way over time in culture by changing the medium. Other protocols have been investigated to enrich more homogenous population of MSCs. The approach of negative selection for MSCs lacking the expression of endothelial marker (e.g., CD31) and HSC maker (e.g., CD34,CD45,and CD 14) is more widely used. 191 The in vitro cultured MSCs are both morphologically heterogeneous, ranging from narrow spindle shaped cells to large polygonal cells, and phenotypically heterogeneous due to culture medium, plating density, and species from which MSCs are isolated. In general agreement, MSCs do not express either hematopoietic markers CD 34, CD45, CD14, and CD11 or endothelial markers CD31, but they do express stromal-associated marker CD105 (SH2), CD73 (SH3/4), CD44, CD90, CD71 and Stro-1 as well as the adhesion molecules CD106 (VCAM-1), CD166 (ALCAM), ICAM-1 and CD29.190 Unfortunately, no single unique marker is specific to MSCs. Multilineage potential is another important criterion for identifying the putative MSC population. Though not immortal, MSCs can expand in vitro many-fold and still retain the multilineage potential. By culturing MSCs under conditions that are favorable for adipogenic, chondrogenic or osteogenic differentiation for 1 to 3 weeks, MSCs were highly differentiated without evidence of the other lineages.189 In contrast, cultured primary fibroblasts, the mature mesenchymal cells, don’t undergo any such differentiation under same induction condition. Differentiation capacity and immunosupression of BM-MSCs In addition to differentiation into its native derivatives, the mesenchymal tissue such as bone, cartilage, adipose, tendon, and muscle, MSCs have the potential to differentiation into other cell types such as hepatic, renal, cardiac, and neural cells 192-198. Both Toma et al. (2002) and Barbsh et al. (2003) demonstrated that MSCs can migrate and engraft in infracted myocardium and appeared to differentiate into cardiomyocytes after either site-directed or 40

systemic delivery of MSCs. 193, 194 The two-step protocol with the use of hepatocytes growth factor and oncostatin M has been developed to in vitro effectively induce hepatic induction of MSCs. Differentiated MSCs gained cuboidal morphology of hepatocytes and expressed liver specific marker gene in a time-dependent manner. Differentiated cells further demonstrated in vitro functional characteristic of liver cells including albumin production, glycogen storage, urea secretion, uptake of low-density lipoprotein and cytochrome P450 activity.198 Hepaticpreconditioned human MSCs functionally integrated into hepatectomized mouse liver administered by intrasplenic injection.195 An important advantage of using MSCs is that in some situation they don’t need to be matched, since the immune phenotype of MSCs (widely described as MHC I+, MHC II-,CD40,CD80-, and CD86-) is considered as non-immunogenic; therefore transplantation into an allogeneic host may not induce immunoresponse.190 This provides the possibility to use MSCs as an “off-the-shelf” product. Furthermore, MSCs exhibit immunomodulatory and anti-proliferative effects on T cells. When MSCs are present in mixed lymphocyte culture (MLT), T-cell proliferation is suppressed in a dose-dependent fashion.199-201 A phase II clinical trial has been conducted by using ex vivo expanded MSCs to treat patients with steroid-resistant, severe, acute graft-versus-host disease (GVHD) after hematopoietic-stem-cell transplantation. For 55 patients, 30 patients lost all the symptoms of acute GVHD and 9 patients showed improvement.202 Transdifferentiation and Cell Fusion Transdifferentiation, the conversion of adult stem cells from one lineage to another, has been proposed to explain the observation that transplanted bone marrow stem cells can turn into unexpected lineages such as myocytes, endothelial cells, hepatocytes, neurons and many others. 145, 203-205

If this concept, transdifferentiation, is true, it would remove the need to collect the stem

cells from embryo and thus avoiding many of the political and ethical barriers to stem cell 41

therapy. In order to identify the molecules that are responsible for reprogramming the adult stem cells to acquire these new lineages, Terada et al. (2002) and Ying et al. (2002) set out to show that embryonic stem (ES) cells can induce the BM stem cells or neural stem cells to transdifferentiate into embryonic-like plurioptent stem cells by culturing them with embryonic stem cells in vitro, respectively. Instead, they found that ES cells and BM cells fused with each other to create tetraploid cells which carried markers for both the ES cells and the adult stem cells206, 207. It therefore started a new debate on whether cell fusion, rather than transdifferentiation, is responsible for adult stem cell plasticity. However, cell fusion can not explain all the cases of transdifferentiation. Adult stem cells can adopt new fates in vitro when ES cells are not present, suggesting the enviromental influences can switch cell lineage. Zhang et al. (2004) also demonstrated that cell fusion and transdifferentiation may account for the transformation of peripheral blood CD34+ cells into cardiomyocytes in vivo.208 In view of the greater aims of gene therapy, as long as the resulting cells are healthy and functional, either transdifferentiation or cell fusion can be used as a means for stem cell gene therapy in which adult stem cells serve as vehicle. Nonetheless, more studies are needed for the future of stem cell-based regenerative medicine. Adipose Tissue-Derived Mesenchymal Stem Cells (AT-MSCs) AT-MSCs vs. BM-MSCs MSCs have traditionally been isolated from bone marrow aspirates, although recent studies have reported that MSCs can be isolated from other tissues such as cord blood, peripheral blood, fetal liver, and adipose tissu0065.209-212 Among these tissues, adipose tissue presents a promising stem cell resource of repeatable access, replenishment, easy isolation and minimal patient discomfort. There is little to no difference between BM derived-MSCs and AT-MSCs regarding the morphology, immune phenotype, yield of adherent stromal cells, growth kinetics, cell 42

senescence, multilineage differentiation capacity or transduction efficiency.197, 213Compared with bone-marrow-derived MSCs, adipose tissue-derived MSCs do have an equal potential to differentiation into mesenchymal lineage such as adipocytes, osteocytes, and chondrocytes, etc.214 More over, the colony frequency is higher in adipose tissue than in bone marrow e.g. the number of CFU-F was 557±673 for adipose tissue vs. 83±61 for bone marrow at an initial plating density of 1×106 cells per cm2 , as well as the maintenance of proliferating ability in culture.197, 215, 216 Harvesting of BM is also a highly invasive procedure and the number, differentiation potential, and maximum life span of MSCs from BM decline with increasing age 217-219

. Although the attachment and proliferation capacity are more pronounced in AT-MSCs

derived from younger donors compared with older donors, the differentiation capacity is maintained with aging.220 Taking all of these issues into account, adipose tissue might be a more attractive alternative to BM in isolating MSCs. Isolation and Characterization of AT-MSCs Adipose tissue, like bone marrow, is derived from embryonic mesoderm and contains a heterogeneous stromal cell population including a putative stem cell population.210 Approximately 400,000 liposuction surgeries are performed in the US each year and these surgeries yield anywhere from 100ml to >3L of lipoaspirate tissue.221 Recent researchers have found the liposuction waste are an alternative source of adult stem cells that are believed to contribute to repair and healing as regenerative medicine for tissue engineering. At least 5 different types of adipose tissue exist: bone marrow adipose tissue, brown adipose tissue, mammary adipose tissue, mechanical adipose tissue, and white adipose tissue. Each adipose tissue type serves a distinct biological function.214 Of great interest to regenerative medicine is the stem cells-derived from white adipose tissue, which can differentiate along multiple pathways in vitro.222 43

Rodbell and Jones presented the first in vitro isolation method for mature adipocytes and progenitor cells from the rat fat pad in the 1960s.221 Tissue was minced into small fragments, digested with collagenase Type I at 370C, and fractionated by differential centrifugation. The supernatant contained the mature adipocytes which floated due to their high lipid content. The pellet contains the stromal vascular fraction (SVF) which consisted of circulation blood cells, fibroblasts, pericytes, endothelial cells, adipose progenitors and putative stem cells. SVF was plated in the tissue culture plastic to enrich the plastic adherent population. The mean number of nucleated SVF cells was determined at 308,849 cells per ml of lipoaspirate tissue and the initial nucleated SVF cells contained colony-forming unit fibroblasts at a frequency of 1:32.222 Besides AT-MSCs, a variety of names have been used to described the fibroblast-like, plastic adherent, multilineage potential cell population isolated from collagenase digests of adipose tissue, such as adipose-derived stem/stromal cells (ASCs), adipose-derived adult stem (ADAS) cells, adipose mesenchymal stem cells (ADMSCs), and processed lipoaspirate (PLA) cells, etc. The surface immunophenotype of human AT-MSCs resembles that of human BM-derived MSCs, with > 90% -identity based on direct comparison221, that is negative to endothelial marker ( CD144) or HSC maker ( CD34, CD45, CD 14) but positive to typical MSC marker (CD29,CD44,CD73, CD90,and CD105).197 Discrepancy has been observed in published reports due to the lack of consistency between laboratories with respect to the isolation and cell passage. The immunophenotype of AT-MSCs progressively change with passage such that classic stromal cell markers presents only on 0.8%-54% of the initial SVF, and on up to 98% of AT-MSCs at late passage.222 Proliferation and Differentiation Capacity of AT-MSCs Following isolation, human AT-MSCs remain inactive with an initial lag time of 5-7 days, and then enter a proliferative phase, reaching confluence within 48hrs.210 Human AT-MSCs 44

display a cell doubling time of 2 to 4 days depending on culture medium and passage number222, 223

or 60hrs in average under standard culture condition (i.e., 10%FBS)210 . In vitro culture, MSCs

demonstrate a limited life span and finally undergo replicative arrest or senescence demonstrated by loss of proliferation and altered morphology.197 Izadpanah et al. (2006) showed that human AT-MSCs can be expanded routinely beyond 180-210 population doublings223, greater than the upper limit of most somatic cells (80 population doublings)224. With prolonged passage for > 4 months, human AT-MSCs have been observed to undergo malignant transformation characterized by abnormal karyotype and tumor formation when implanted into immunodeficient mice, which might be due to the overexpression of oncogene c-myc. 225 Not only can AT-MSCs differentiate into mesodermal lineages e.g., adipogenic, osteogenic, chondrogenic, and myogenic cells but also into ecto- and endodermal lineages as well (e.g., neurons, endocrine pancreatic cells, hepatocytes, endothelial cells, and cardiomyocytes). 202, 226-231 Evidence that AT-MSCs can be applied as a source of hepatocytes is the observation that the human CD105+ fraction of AT-MSCs reveal several liver-specific markers and functions, such as albumin production, low density lipoprotein uptake, and ammonia detoxification, after treatment with a hepatic induction growth factor cocktail (HIFC).232 More importantly, CD105+ AT-MSC-derived hepatocyte-like cells can be transplanted and incorporated into the host liver parenchyma. In addition, AT-MSCs support complete differentiation of hematopoietic progenitor into myeloid and B lymphoid cells.233 This suggests that the addition of AT-MSCs infusion may improve and accelerate hematopoietic stem-cell engraftment in recipients who have undergone BM ablation. The molecular mechanism of the lineage-specific differentiation into cells and tissues of mesodermal origin is well documented in the review paper by Schaffler and Buchler (2007). Yet, the molecular event

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behind the “cross-differentiation” is far from clear and more research is needed to decode this puzzle for future AT-MSC-related cell therapy and tissue engineering. For clinical purposes, adipose tissue derived stem cells might appear superior to bone marrow cells in view of their availability in large quantities at a low risk to patients. Liver Anatomy Liver is the largest gland in the body consisting of several separate lobes and accounting for 2% of the body weight in the human and 5% in the mouse.234 It is the only organ with two separate afferent blood supplies. Hepatic artery provides oxygenated blood and the portal vein supplied the liver with venous blood rich in nutrients and hormones from the intestines and pancreas. Roughly 75% of the blood entering the liver is venous blood. Arterial and venous blood mix as they enter the sinusoids, distensible vascular channels lined with discontinuous and fenestrated endothelial cells and bounded by hepatocytes, in the liver. When blood flows through the sinusoid, oxygen, carbon dioxide, nutrients, proteins and wastes are exchanged between blood and hepatocytes, and finally blood empty into the central vein. Hepatocytes are arranged in cords, cellular arrays of one-cell-wide, radiating from the central vein with their basal surfaces facing and surrounding the sinusoids and the apical faces of adjoining hepatocytes form canaliculi. Bile secreted by the hepatocytes is collected in the canaliculi and flow parallel to the sinusoid, but in the opposite direction to the blood flows. At the end of the bile canaliculi, bile drains into bile ducts that lie in very close proximity to the terminal branches of the portal vein and hepatic artery. Collectively, these three structures are called the portal triad. The main cell type of the liver is parenchymal cells including hepatocytes and bile duct epithelia. Non-parenchymal cells of the liver include kuffer cells (marcophages in the hepatic sinusoid), stellate cells (located under the sinusoid), vascular endothelial cells, and pit cells (natural killer cells). Among these, hepatocytes are responsible for the majority of liver function, 46

and constitute ~60% of the liver cell population and 90% of liver mass. An adult mouse liver contains about 5×107 hepatocytes, and an adult human liver contains about 8×1010 hepatocytes.235 Hepatocytes are the largest and polygonal-shaped epithelial cells (30-40µm) with diploid or tetraploid nuclei and a large proportion of adult hepatocytes are binucleated.236 Zone1,-2 and -3 hepatocytes are distinguished based on the basis of their relative position within the lobule. Zone-1 hepatocytes surround the portal triad, zone-3 hepatocytes are around the central vein, and zone-2 hepatocytes are the inter-zonal hepatocytes. Hepatocytes at different zone are heterogeneous in the size and metabolic /biosynthetic function, having smaller and usually single-nucleated hepatocytes in zone 1 and predominantly bi-nucleated hepatocytes in zone-3. Liver performs a variety of biochemical functions including the metabolism of amino acids, lipids, and carbohydrates, the detoxification of xenobiotics, the synthesis of serum proteins and secretion of bile. Additionally, liver has an astounding capacity to regenerate after injury. Animals (including humans) can survive surgical removal of up to 75% of the liver mass.237 The residual lobes enlarge to make up for the removed mass, although the resected lobes never grow back. The original number of cell is restored within 1 week and the original tissue mass with 2 to 3 weeks.236, 238 Parenchymal regeneration after the surgical loss of liver tissue principally originates from the proliferation of the remaining mature hepatocytes rather than from liver stem/progenitor cells. Typically 10-12 hrs after partial hepatectomy (PHx), hepatocytes in the remaining liver initiate the DNA synthesis which peaks at 24-48 hrs depending on the species237. Hepatocyte proliferation starts in the periportal area and proceeds to the pericentral area237. Following the hepatocytes, the other hepatic cell types enter into DNA synthesis 24 hours later.

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CHAPTER 2 MATERIALS AND METHODS Hepatic Oval Cell Induction and Isolation from Mouse Liver Adult male C57BL/6 mice (8 week old) were fed with a diet containing 0.1% DDC (BioServe, Frenchtown, NJ) for 4 -6 weeks. This diet has been approved to be very effective for inducing and enriching murine oval cells in liver.146 A two-step collagenase perfusion was applied for hepatocyte and nonparenchymal (NPC) isolation.150 Low –speed centrifugation (500 g) separated NPC fraction containing oval cells from hepatocytes. The oval cells were further enriched by magnetic activated cell sorting using Sca-I antibody conjugated magnetic beads (Miltenyi Biotec, Germany). Bone Marrow Isolation BM cells were isolated from the femurs and tibias of C57BL/6 male mice. The bone was sterilized by immersion in 70% ethanol, and the skin and muscles were removed. BM was exposed by cutting the ends of the bones, and extruded by inserting a 20-gauge needle attached to a 3ml syringe and forcing 1-2 ml of DMEM (Mediatech, Inc., Manassas, VA) containing 2% FBS (HyClone laboratories, Inc., Logan, Utah) through the bone shaft. To make a single cell suspension, BM was triturated by gently aspirating several times using the same needle and syringe and passed through a 70µm nylon mesh strainer (Becton Dickinson Labware, Franklin Lakes, NJ). Cells were treated with red blood cell (RBC) lysing buffer for 2 minutes at room temperature to deplete RBC. After that cells were cultured for 1h at 37℃ to remove the macrophages which will attach to the bottom of the cell culture dish. Those remaining suspension cells contained mesenchymal and hematopoietic stem cells, and blood progenitor cells and were ready for virus vector transduction. Transduction was performed in a 1ml reaction

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volume of DMEM supplemented with 10% FBS for 2 h at 37℃ and 5% CO 2. Cells were the washed three times with PBS and resuspended in 100ul saline for transplantation or cultured in murine myeloid long-term culture medium (MyeloCultTM M5300, StemCell Technologies Inc., Vancouver, BC) for in vitro transduction efficiency study. AT-MSCs Isolation and Culture Mouse AT-MSCs were isolated from peritoneal adipose tissue excised from the abdominal region of 6 to 8-week-old male C57BL/6 mice. Adipose tissue was enzymatically digested with 0.075% collagenase (type I; Sigma-Aldrich, St. Louis, MO) in PBS for 1hr at 37°C with gentle agitation. The collagenase was inactivated with an equal volume of DMEM (Mediatech, Inc., Manassas, VA) supplemented with 10% fetal bovine serum (FBS, HyClone laboratories, Inc., Logan, Utah), and the infranatant was centrifuged at 1,000 rpm for 5 min at room temperature. The cell pellet was resuspended in 160mM NH 4Cl (StemCell Technologies Inc, Vancouver,BC) and incubated at room temperature for 2 min to eliminate contaminating red blood cells and filtered through a 100-µm nylon mesh strainer (Becton Dickinson Labware, Franklin Lakes, NJ) to remove debris. The resulting AT-MSC-containing cell pellet is collected by centrifugation as described above, and resuspended in a DMEM/10%FBS medium at 1-2×106 cells/100 mm plastic tissue culture dishes. Non-adherent cell population was poured off after 12-16hr culture. Adherent cells were washed with PBS and cultured in DMEM with 10% FBS for expansion. The cells with 70-80% confluence were harvested with 0.25% trypsin-EDTA and reseeded at 1.0×105 cells/60mm dish. Recombinant AAV Vector Construction and Production Recombinant single-stranded AAV vectors used in this study has been described previously.239, 240 Briefly, plasmid ssAAV-CB-hAAT contained full length AAV2 ITRs, hAAT

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cDNA flanked by two ITRs and driven by CMV enhancer/chicken-β-Actin promoter, intron and ploy(A) sequence (Figure 2-1A). dsAAV vector construction was described previously.87 In brief, dsAAV vector plasmid was made from the ssAAV-CB-hAAT plasmid by deleting Dsequence and terminal resolution site (trs) of 5’-ITR. To make the vector plasmid smaller than 2.5kb for dsAAV packaging, the Neor cassette was deleted and the CB promoter was replaced by duck hepatitis B virus (DHBV) promoter (Figure 2-1B) or CMV promoter (Figure 2-1C). Plasmid ssAAV-CB-hAAT, dsAAV-DHBV-hAAT, and dsAAV-CMV-hAAT were packaged into AAV serotype 1 and 8 capsids, respectively, as described previously.241 In brief, , vector plasmid was co-transfected with helper plasmid(s) which contain genes from adenovirus and corresponding serotype AAV cap and rep genes into 293 cells. Cells were harvested and disrupted by freeze-thaw lysis to release virions. The rAAV vectors were purified by iodixanol gradient centrifugation followed by heparin affinity or anion exchange chromatography. The physical particle titers of vector preparations are routinely assessed by quantitative dot blot analysis. Lentiviral Vector Construction and Production In order to generate a lentiviral vector expressing hAAT (Lenti-CB-hAAT), hAAT cDNA fragment and CB promoter was released from plasmid AAV-CB-hAAT and inserted into pTYFlinker vector which was derived from an LTR-modified recombinant HIV-1 plasmid. This lentiviral vector was packaged by as previously described. Briefly, pHP-helper (packaging helper construct), pHEF-VSV-G (envelop expression construct) and pLenti-CB-hAAT (transducing construct) were cotransfected into 293T cells using superfect reagent (Qiagen Inc.). The lentiviral vector was harvested at 48hr after transfection by collecting the cell culture medium and was concentrated by centrifuging at 2500g under 4℃ for 20 minutes x 2times using the

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Amicon Ultra-15 centrifugal filter device (Millipore). The virus titer was estimated at 1×109 viral particles/ml.242 Animal C57BL/6 mice were purchased from Jackson Laboratory, housed in a specific pathogenfree room. All animal work was conducted under the protocols approved by the University of Florida Institutional Animal Care and Use Committee. In vitro Transduction Cells were seeded in 24-well plates (Costar, Corning Inc.) with 1×104cells/well and infected with virus vectors at different multiplicities of infection (MOI) in triplicate. Oval cells were cultured in Iscove’s Modified Dulbecco’s medium (StemCell Technologies,Inc.,Vancouver, BC) with 10% FBS, 1000 I.U./ml penicillin and streptomycin, 100ng/ml IL-6, 100ng/ml Flt-3, 100ng/ml SCF, and 20ng/ml GM-CSF. All these growth factors were purchased from StemCell Technologies (Vancouver BC). BM cells and AT-MSCs were culture in DMEM medium (Mediatech, Inc., Manassas, VA) with 10% FBS and 1000 I.U. /ml penicillin and streptomycin. The accumulative hAAT secretion in the culture medium was collected and measured by hAAT specific ELISA. In vivo Injection of Vectors into Mouse Liver and Muscle For muscle injection, 8-week-old female C57BL/6 mice were anesthetized by isoflurane inhalation, and aliquots of vectors were injected percutaneously into the quadriceps femoris muscles of both hind limbs.243 For portal vein injection, mice were anesthetized by isoflurane inhalation and a ventral midline abdominal incision was made into the peritoneal cavity to expose the portal vein.239 Vectors were injected into the portal vein using a 30-gauge needle. Hemostasis was achieved by application of a small piece of cotton directly onto the injection site. The volume of vector was 100ul and the total amount of virus injected per mouse is 2×1010

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particles. For monitoring the hAAT expression, serum samples were collected via tail vein and subjected to hAAT specific ELISA. Monocrotaline Treatment Monocrotaline (MCT) was purchased from Sigma Aldrich (St.Louis, MO). Solution was prepared as previously described.244 Briefly, 500mg MCT was dissolved in 2ml acidified PBS (pH 3.0) using 2N HCl by gentle stirring. After complete dissolution, solution was adjusted to pH7.0 with 5N NaOH and additional PBS was added to achieve total volume of 10ml and final concentration of 50mg/ml. The standard MCT treatment consisted of two intraperitoneal injection of MCT at 50mg/kg BW with a 2-week interval. After the final injection, mice were housed for two more weeks before further studies were conducted. Adipogenic and Osteogenic Differentiation of AT-MSCs Adipogenesis At passage 3, AT-MSCs were seeded in 6-well plate and grown to 100% confluence for differentiation. Adipogenic differentiation was induced by culturing cells in the adipogenic medium for 2 weeks with medium changes twice weekly. Adipogenic medium consists of DMEM supplemented with 10% FBS, 0.5mM 3-isobutyl-1-methylxanthine, 1µM dexamethasone, 200µM indomethacin, and 10 µg/ml bovine insulin (all Sigma). Adipogensis was assessed by staining for intracellular lipid droplets with Oil Red O stain (Sigma). Osteogenesis Cultured cells at passage 3 were seeded in 6-well plate and grown to 100% confluence for differentiation. Osteogenic differentiation was induced by culturing cells in the osteogenic medium for 2 weeks with medium changes twice weekly. Osteogenic medium consists of DMEM supplemented with 10% FBS, 0.1µM dexamethasone, 10mM β-glycerophosphate, and

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50 µM ascorbate-2-phosphate (all Sigma). Osteogenesis was assessed by staining for calcium depositions with Alizarin Red S (pH 4) stain (Sigma). Liver Directed Transplantation of Adult Stem Cells Adult female C57BL/6 mice received MCT (50mg/kg BW) at a two-week interval by i.p. injection for inhibiting the endogenous liver cell proliferation.245 Two weeks after second injection, the mice were partially hepatectomized to remove 70% liver (large median and left lateral lobes of the liver) under general anesthesia.246 In the meantime, adult stem cells suspended in 100ul saline were transplanted into the remaining liver immediately after PHx by portal vein injection as previously described.239 To transplant adult stem cells by intrasplenic injection, cells were injected into the inferior tip of the spleen of mice right after PHx using a 30gauge needle. To aid in the coagulation process, splenic injection site was ligated using sterile absorbable surgical suture (Ethicon, INC., Somerville, NJ). Post-surgery, mice were placed back in specific pathogen-free room. Blood sample was collected from the tail vein each week. At the end of experiment (8-14 weeks post surgery), samples of liver, lung, kidney, ovary, pancreas, brain, heart, intestine, and bone were collected and subjected to OCT embedding or Paraffin embedding. Immunohistochemistry for Human AAT, GFP and Mouse Albumin Organ tissues were fixed in 10% neutral buffered formalin (NBF) and embedded in paraffin. For hAAT and GFP immunostaining, tissue sections (5μm) were de-paraffinized, rehydrated, and blocked for endogenous peroxidase with 3% hydrogen peroxide in methanol for 10 minutes. To detect hAAT expression, tissue sections were incubated with primary antibody, rabbit anti-human AAT (1:800, RDI/Fitzgeral Industries, Concord, MA, USA), for overnight at 4°C. Staining was detected using ABC-Rb-HRP and DAB kits (Vector laboratories, Burlingame, CA). Antigen retrieval was performed in Digest-All™ (trypsin) (Zymed® Laboratories, 53

Carlsbad, CA, USA) for 5 minutes at 37°C, followed by incubation in Trilogy (Cell Marque Corp., Rocklin, CA, USA) for 25 minutes at 95°C. Immunostaining of GFP was performed using rabbit anti-GFP antibody (1: 10,000, AbCam Ltd, Cambridge, MA). Staining was detected using ABC-Rb-HRP and DAB kits (Vector laboratories, Burlingame, CA). Antigen retrieval was performed in Trilogy (Cell Marque Corp., Rocklin, CA, USA) for 25 minutes at 95°C. To detect albumin expression, antigen retrieval was performed using citrate retrieval for 30 minutes in a steamer. The tissues were incubated with goat anti-mouse albumin (1:5,000, Abcam, Ltd., Cambridge, MA) overnight at 4°C, followed by incubation with biotinylated horse anti-goat (1:200, Vector Laboratories) for 30 minutes. Staining was developed by Vectastain ABCAlkaline Phosphatase kit (Vector Laboratories) Vulcan Fast Red (VFR) chromagen (Biocare Medical, Concord, CA, USA). Immunofluorescence double staining for human AAT and GFP was performed as previously described with minor modification.245 Co-localization of hAAT and GFP were detected by staining sequentially with anti-GFP (1:500, AbCam Ltd, Cambridge, MA) and anti-hAAT (1:100, RDI/Fitzgeral Industries, Concord, MA, USA). The FITC (1:1000) - or rhodamine (1:1000)-conjugated secondary antibody were applied. Immunofluorescent Staining of AT-MSCs The cells were plated onto glass chamber slides for a day, and then fixed for 15 min in 4% paraformaldehyde in 100mM sodium phosphate buffer (pH 7.0). The cells were washed for 10 min in 100mM glycine in PBS (PBS/glycine) and blocked for 1 h in immunofluorescent blocking buffer (IBB) containing 5% bovine serum albumin (BSA), 10% FBS, PBS, 0.1% Triton X-100. The cells were subsequently incubated for 1 h in IBB containing the following anti-mouse monoclonal antibodies: CD31, CD34, CD44, CD45, CD90, CD105, and CD133 (1:100, eBioscience, San Diego, CA). The cells were washed extensively with PBS/glycine and

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incubated for 1 h in IBB containing a fluoroisothiocyanate (FITC)-conjugated secondary antibody. The cells were washed with PBS/glycine and mounted with glass coverslips with DAPI (Vector, Burlingame, CA). Y-chromosome Fluorescence in situ Hybridization 5μm sections of paraffin embedded liver tissue samples were used for detecting Ychromosome. Liver sections were de-paraffinized 2×5 min in fresh xylene and rehydrated 2×2 min in 100% ethanol, 2min in 95% ethanol, 1 min in 70% ethanol and 1 min in water. Deparaffinized liver sections were first treated with 0.2N HCl for 30 min at RT and retrieved in 1M NaSCN for 30 min at 850C. Sections were then digested in 4mg/ml Pepsin (Sigma) diluted in 0.9%NaCl (pH2.0) for 11-15 min at 370C. Digested sections were equilibrated 1 min in 2×SSC and then dehydrated through graded alcohols. Sections were incubated with FITC-conjugated Ychromosome probes (Cambio,UK) and performed denaturation at 650C for 10 min followed by hybridizatation at 370C overnight using Hybrite (Vysis,IL). After hybridization, sections were washed using 50% Formamide/2×SSC, 2×SSC, and 4×SSC+0.1% Igepal (NP-40) at 460C for 7 min, respectively. For detection, air dry slide at RT in dark and mount glass coverslips with DAPI (Vector, Burlingame, CA). Human AAT Specific ELISA Microtiter plates were coated with 100ul of goat anti-hAAT (1:200, Sigma Immunochemical, St.Louis, MI, USA) in voller’s buffer overnight at 4oC. Blocking buffer, 3% bovine serum albumin (BSA, Sigma, St.Louis,MI,USA), was added to saturate the remaining sites for protein binding on the microtiter plate and incubate 1 hour at 37oC. After blocking, duplicated standard curves (Sigma Immunochemical, St.Louis,MI,USA) and diluted sample serum or cell culture medium were loaded and incubated 1hr at 37oC. A second antibody, rabbit anti-hAAT (1:1000, Roche Molecular Biochemicals, Indianapolis,IN, USA),was added and 55

reacted with captured hAAT at 37oC for 1 hour. A third antibody, goat anti-rabbit IgG conjugated with peroxidase (1:80, Roche Molecular Biochemicals, Indianapolis,IN, USA) was incubated at 37oC for 1 hour. The plate was washed with PBS-Tween 20 three times between reactions. After reacting with the substrate (o-phenylenediamine, Sigma Immunochemical, St.Louis, MI, USA), Microtiter plate was read at 490nm on a MRX microplate reader (Dynex Technologies, Chantilly, VA, USA).

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A

ITR

CB promoter

hAAT

pA

ITR

B

Delt-ITR DHBV promoter

hAAT

pA

ITR

pA

ITR

C

Delt-ITR CMV promoter

hAAT

Figure 2-1. rAAV vectors constructs. (A) rAAV-CB-hAAT. Plasmid rAAV-CB-hAAT contained full length AAV2 ITRs, hAAT cDNA driven by CMV enhancer/chicken-β-Actin promoter, intron and ploy(A) sequence. (B) rAAV-DHBV-hAAT. D-sequence and terminal resolution site (trs) of 5’-ITR is deleted to make self-complementary dsAAV vector. 3’-ITR remains intact. hAAT is driven by DHBV promoter. (C) rAAV-CMVhAAT. D-sequence and terminal resolution site (trs) of 5’-ITR was deleted to make self-complementary dsAAV vector. 3’-ITR remains intact. hAAT is driven by CMV promoter.

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CHAPTER 3 HEPATIC OVAL CELL-BASED LIVER GENE DELIVERY Introduction Our previous study has shown that hepatic oval cells can be transduced by rAAV vectors and transplanted into the recipient liver.245 Transgene (hAAT) was expressed in the engrafted donor cells and transgene product (hAAT protein) was secreted into the circulation of recipient. The transgene expression was also sustained throughout the experiment, 14 weeks post transplantation. Results from previous studies formed solid base for future investigations of adult stem cell transplantation studies in a model for adult. However, the level of the transgene expression remain improvement.247 The goal of this study is to further evaluate the potential of viral vector-mediated adult stem cell-based therapy. Since transgene expression is affected by transduction efficiency of viral vector, transplantation efficiency, and engraftment capability of adult stem cells, we have tested the possibility of enhancing oval cell transduction efficiency by optimizing rAAV vectors and employing a lentiviral vector. Animal Experimental Design Freshly isolated liver oval cells (2×106 cells) from male C57BL/6 mice were infected with rAAV1-CB-hAAT at 1×104 vg/cell or Lenti-CB-hAAT at 5vg/cell, respectively, for 2 hr, washed with PBS three times, and transplanted into partially hepatectomized female C57BL/6 mouse liver (n=5) by intrasplenic injection. Donor mice were treated with a diet containing 0.1% diethyl 1, 4-dihydro-2, 4, 6-trimethyl-3, 5-pyridine-dicarboxylate (DDC) for 4 weeks to stimulate oval cell proliferation. Recipient mice were injected with monocrotaline (MCT) 50mg/kg BW twice at a 2-week interval to inhibit the endogenous liver cell proliferation. Two weeks after the second injection, recipient mice were partially hepatectomized to remove 70% of the liver to create liver injury and thus enhance the environment for the proliferation of transplanted oval cells (Figure 3-

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1). A species-specific hAAT ELISA was performed to measure serum hAAT protein level from the transgene expression. At the end of experiment, liver tissues were collected for immunostaining for hAAT. Results Ex vivo Transduction Efficiency on Oval Cells by rAAV Vectors To optimize the transduction efficiency of rAAV vectors on oval cells, oval cells were infected with 4 different serotypes of rAAV vectors derived from human or nonhuman primates or bovine rAAV vector using GFP as a reporter gene, respectively. An identical genome, AAVCB-GFP, were packaged into each of four different AAV serotype capsids (serotype 1, 7, 8, 9) and bovine AAV capsid. rAAV-CB-GFP vector transduced oval cells were subjected to flow cytometry analysis to quantify the GFP positive oval cells. As seen in Figure 3-2, rAAV1-CBGFP vector yielded 2.17% green fluorescent cells 7 day after infection at a dose of 104vg/cell. rAAV7-CB-GFP and rAAV8-CB-GFP vectors yielded 2.11% and 1.63% green fluorescent cells, respectively, under the same condition. rAAV9 and bovine rAAV vector gave less than 1% green fluorescent cells. Consistent with our previous observation, rAAV1 vector showed the highest transduction efficiency than other 4 rAAV vectors, although the transduction efficiency was low. Lentiviral Vector Construction In order to generate a lentiviral vector expressing hAAT, a lentiviral virus vector plasmid, pTYF-linker derived from HIV-1 was used as a parental plasmid (Figure 3-3A). Human AAT expression cassette including hAAT cDNA driven by CB promoter (Figure 3-3B) was inserted into pTYF-linker plasmid. Briefly, the DNA fragment of hAAT cDNA and the CB promoter was released from pCB-hAAT plasmid by enzyme digestion with BglII and BstEII. Two fragments (2639bp and 600bp) were obtained. Since the Ploy A sequences can not be included in RNA viral vector we have use PCR amplification to remove the poly A sequences at 3’-end of hAAT

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cDNA. We have inserted an enzyme digestion site SpeI into the primer before the polyA site for cloning purpose. PCR was applied to amplify the fragment between BstEII (2802) and BstEII (3426) of pCB-hAAT. The amplified fragment was cloned into TA clone and released by enzyme digestion with BstEII and SpeI. The BglII-BstEII (2639 bp) fragment and the PCR amplified BstEII-SpeI fragment were cloned into pTYF-linker between BamHI and SpeI to yield the Lenti-CB-hAAT construct (Figure 3-3C). Restriction enzyme digestion showed this construct is correct (Figure 3-3D). This construct was packaged into lentiviral particles by Dr. Chang’s laboratory. To evaluate the transduction efficiency of Lenti-CB-hAAT vector on oval cells, oval cells were infected with Lenti-CB-hAAT vector at 5 MOI (multiplicity of infection). To compare the transduction efficiency between lentiviral vector and rAAV vector, oval cells were also infected with rAAV1-CB-hAAT and rAAV8-CB-hAAT vector at 104 MOI, respectively. Supernatant media were assayed for transgene product (hAAT protein) using species-specific hAAT ELISA. As shown in Figure 3-4, Lenti-CB-hAAT vector can transduce oval cells and mediate higher transgene expression than rAAV vectors, 100-fold higher than rAAV1 and rAAV8, while the transgene expression levels from rAAV 1 and 8 vectors are comparable. Ex vivo Transduction and Transplantation of Oval Cells Based on above results, we chose rAAV1-CB-hAAT and Lenti-CB-hAAT vectors for transplantation studies. In this study, each recipient mouse received rAAV1-CB-hAAT or LentiCB-hAAT vector transduced oval cells (2×106 cells/mouse). Ten weeks post transplantation, recipients were sacrificed and the liver sections were subjected to hAAT immunostaining. Results demonstrated that transgene hAAT was expressed in the recipient liver cells of both groups (Figure 3-5). However, lentiviral vector resulted in fewer hAAT positive cells and much lower transgene expression than rAAV1 vectors. Blood samples were collected every week for evaluating the hAAT serum level. As Figure 3-6A showed, one out of five mice transplanted 60

with rAAV1-CB-hAAT vector transduced oval cells exhibited transient elevation (2,500ng/ml) of serum hAAT at week 2 post transplantation. Other mice in both Lenti-CB-hAAT vector and rAAV1-CB-hAAT vector groups didn’t show any significant increase in serum hAAT level compared to saline group in which mice were transplanted with untransduced oval cells. Immune responses to the transgene product (hAAT protein) were examined. Serum anti-hAAT IgG level increased as the serum hAAT concentration increased (Figure 3-6B). Discussion Results from these studies confirmed rAAV vector can be used for genetic modification of oval cells. Several rAAV vectors including serotype 1, 7, 8, 9 and bovine AAV were tested for transduction efficiency on oval cells using flow cytometry for quantification. Although the transduction efficiencies for all vectors were low, rAAV1 is the best among the vectors tested. Using GFP as report gene, flow cytometry demonstrated less than 3% oval cells were green cells one week after infection. The transduction efficiency estimated by GFP positive cells might be underestimated for several possible reasons. First, rAAV vector is single stranded DNA vector and the transgene expression requires the conversion of ssDNA to dsDNA which depends on the host cell DNA replication machinery. Host cell stage may also have effect on the transgene expression. Some cells demonstrated early transgene expression while others were on the late stage. Usually it takes 4-6 weeks for rAAV transgene expression to reach maximum in vivo. However, in vitro propagation of stem/progenitor cells will dilute out the rAAV vector. Therefore, the evaluation of oval cell transduction was relative and helpful for selection of better vector. This approach can not be suitable for absolute quantification of transduction, which may require quantification of vector DNA in the cells. Nevertheless, our results consists previous observation and showed rAAV1 is the best one that mediated the highest transgene expression on

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oval cells among different AAV serotypes (serotype 1-5 and 7-9), and bovine AAV. Hence rAAV1-CB-hAAT vectors were used for in vivo transplantation study. We observed transgene expression in the recipient liver after transplanting rAAV1-CBhAAT vector transduced oval cells. We also noticed the large variation among the animal on transgene expression. In contrast to previous study, current study only observed transient transgene product (hAAT protein) in the serum. One possible explanation is that 5 times lower MOI was applied in this study. In addition, ELISA demonstrated a significant increase in serum anti-hAAT IgG level, as we observed in the study of rAAV1- mediated hAAT gene deliver to skeletal muscle.76 No antibody response was seen when there is a lack of detectable circulating hAAT. These results suggest that anti-hAAT antibody might target and neutralize the transgene product (hAAT), and thus limited the concentration of circulating hAAT. For future studies, SCID mice may be used as recipient to rule out this problem. In order to enhance the transgene expression, lentiviral vector was constructed and used for hAAT gene delivery into the oval cells. We showed that Lenti-CB-hAAT vector infected oval cells efficiently and mediated efficient transgene expression in cell culture condition. However, after transplantation of transduced cells, hAAT expression in the recipient liver was very low or nearly undetectable. There are two possible explanations. Studies conducted by Brown and colleagues showed that in vivo administration of lentiviral vector to mice triggered a type I interferon response.248 This innate immune response in turn triggered an adaptive response against transgene product and also promoted vector clearance. In addition, lentiviral vector integrates into the host genome randomly. It has been demonstrated that transgene expression is subjected to negative influence of chromosomal sequence flanking the integration sites and often leads to transcriptional silencing.131, 249, 250 The interaction between the cis-acting elements of

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provirus and trans-factors of stem cells results in epigenetic modifications including DNA methylation and chromatin structure modulation, which may contributes to the lentiviral transgene silencing.249, 251, 252 Transcriptional silencing is most pronounced in stem cell. The trans-factors scan for foreign DNA and establish silencing in stem cells and maintain silencing in their progeny.252 Strategies for overcoming these limitations are under the development including addition of chromatin insulator element to protect the transgene from negative position effect or deletion/mutation of the retrovirus silencer elements.251, 253-255 Although our studies have clearly demonstrate the feasibility of stem cell mediated hAAT gene delivery using rAAV vector and oval cells, isolation of oval cell for autologous transplantation in human is not practical. Therefore, next studies will focus on finding new stem cell resources for liver directed hAAT gene delivery.

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Figure 3-1. Experimental outline of oval cell study. The female recipients (C57BL/6) were IP injected twice (2 weeks interval) with 50mg/kg of MCT and received partial hepatectomy (PHx) before transplantation. Liver oval cells were isolated from male GFP transgenic C57BL/6 mice treated with a diet containing 0.1% DDC for 4 weeks. Magnetic cell sorting (MACS) system was applied to isolate Sca l+ cell population from the nonparenchymal cell compartment from 2-step liver perfusion. The newly purified Sca l+ oval cells were infected with rAAV1-CB-hAAT vector and Lenti-CBhAAT vector for 2 hours, respectively, washed and transplanted into the recipient liver by intrasplenic injection. Serum hAAT levels were monitor by ELISA. Liver tissues were subjected to hAAT specific immunostaining.

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Figure 3-2. Flow cytometric quantification of green fluorescent oval cells after transduction of rAAV-CB-GFP vectors.

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Figure 3-3. Construct of Lenti-CB-hAAT vectors. (A) Lentiviral vector backbone. pTYF-Linker is derived from an LTR-modified recombinant HIV-1 plasmid. (B) hAAT expression cassette. hAAT is driven by CB promoter. (C) Restriction map of Lenti-CB-hAAT plasmid. (D) Gel electrophoresis results. H: HindIII; SB: SpeI & BamHI; S: SpeI; B: BamHI.

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Figure 3-4. Ex vivo transduction of oval cells. Mouse oval cells were grown in the 24-well plate (1×104 cells/well; n=3) and were infected with the Lenti-CB-hAAT vector at 5 MOI, rAAV1-CB-hAAT and rAAV8-CB-hAAT vectors at 1×104 MOI, respectively. The accumulative hAAT in the culture medium was measured by ELISA. Open triangle, lentiviral vector; Closed circle, rAAV1 vector; Open square, rAAV8 vector.

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Figure 3-5. Detection of expression of hAAT in recipient liver after transplantation of ex vivo transduced oval cells by immunostaining. (A) Liver section from C57BL/6 mouse transplanted with ssAAV1-CB-hAAT infected oval cells stained for hAAT (Brown). (B) Enlarged image of A. (C) Liver section from C57BL/6 mouse transplanted with Lenti-CB-hAAT infected oval cells. (D) Larger image of C. (E) Liver section from normal human serves as positive control. (F) Liver section from an untransplanted C57BL/6 mouse serves as negative control.

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A

B

Figure 3-6. hAAT expressed from engrafted oval cells. (A)1-2×106 fresh isolated oval cells were infected with Lenti-CB-hAAT at 5moi, rAAV1-CB-hAAT at 104moi or saline for 2hr, respectively, and transplanted into hepatectomized mouse liver ( n=5 each group) by intrasplenic injection. Serum samples were collected very week for detection of hAAT. (B) Serum anti-hAAT IgG levels were estimated by ELISA. Open circle: Lenti-CB-hAAT; Open square: rAAV1-CB-hAAT; Cross, Saline; Open triangle: one mouse in rAAV1-CB-hAAT group with transient increase in serum hAAT level at week 2 post transplantation. Dash is lower limit of quantification (LLOQ).

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CHAPTER 4 EX VIVO TRANSDUCTION AND TRANSPLANTATION OF BONE MARROW CELLS FOR LIVER GENE DELIVERY OF ALPHA 1-ANTITRYPSIN Summary Adult stem cell-based gene therapy holds several unique advantages including avoidance of germline or other unwanted cell transduction. We have previously showed that liver progenitor (oval) cells can be used as a platform for liver gene delivery of human alpha 1antitrypsin (hAAT). However, this cell source can not be used in humans for autologous transplantation. In the present study, we tested the feasibility of bone marrow (BM) cell-based liver gene delivery of hAAT. In vitro studies showed that bone marrow cells can be transduced by lentiviral vector (Lenti-CB-hAAT) and recombinant adeno-associated viral vectors (rAAV1CB-hAAT, and rAAV8-CB-hAAT). Transplantation studies showed that transplanted bone marrow cells homed into liver, differentiated into hepatocytes and expressed hAAT in the liver. Importantly, we showed that transplantation of rAAV8-CB-hAAT vector transduced bone marrow cells resulted in long-term and sustained levels of hAAT in the systemic circulation of recipient mice. These results demonstrated that rAAV vector mediated, bone marrow cell-based liver gene therapy is feasible for the treatment of AAT deficiency and implies a novel therapy for the treatment of liver diseases. Introduction Alpha 1-antitrypsin deficiency (AATD) is a genetic defect caused mostly by a single base substitution in the alpha 1-antitrypsin (AAT) gene which encodes a 52kDa glycoprotein.256 This mutation results in accumulation of polymerization of mutant AAT protein in the hepatocytes where AAT is mainly synthesized and secreted into the circulation, and consequently leads to a reduced level of AAT in the serum.257 As a serine protease inhibitor, the primary function of AAT is to protect delicate tissue such as pulmonary interstitial elastin against the excessive

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proteolytic damage of neutrophil elastase (NE). Deficiency of AAT in the serum could cause alveolar elastin exposure to NE and lead to an increasing risk of developing early onset pulmonary emphysema by ages 35 to 50 years if the AAT serum level is less than 11µM (approximately 800ug/ml).247 Aggregated mutant AAT in the endoplasmic reticulum of hepatocytes would result in the liver disease such as neonatal jaundice and hepatic cirrhosis which take places in one portion of patients homozygous for PI*Z mutation.247 For AAT deficiency-associated lung disease, restoring anti-NE protection in the lung has been achieved by boosting serum AAT level via weekly intravenous infusion of human plasma AAT.258 Strategies of overexpression of wild-type AAT gene to correct the deficiency of AAT by gene transfer to muscle are being investigated in the phase I clinical trial using recombinant adeno-associated virus vector (rAAV) serotype 1 & 2.106 For AAT deficiency- associated liver disease, no effective therapy is available except liver organ transplantation which is limited by the shortage of donor organ and immune rejection. Adult stem cells offer a platform for ex vivo genetic manipulation followed by autologous transplantation, which will overcome many limitations, including immune rejection of allogeneic cells and ethical issue of embryonic stem cells. Non-specific targeting, such as germline transduction, is one of the major concerns of conventional gene therapy, direct infusion of gene into a patient. Using stem cell as a mean for delivering gene into patients could minimize the unwanted cell transduction. More importantly, stem cell, a regenerative medicine, can self-renew and continue replenishing the aged or damaged tissue cells, and thus stem cell-based gene therapy may reduce or eliminate the need for repeated administration of the gene therapy. Adult stem cell gene therapy which replaces the patients’ disease-causing gene with the healthy

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counterparts in their own stem cells will offer a hope for those who are running out of treatment options and are tired of life-long medication. Bone marrow is the reservoir of stem cells including two major populations, hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). BM stem cell is one of the first stem cells to be used successfully in the clinical for treating blood disease (e.g. leukemia). In addition, BM has been proposed to be an extrahepatic origin of liver progenitor cells.145, 162, 164 Sexmismatched BM transplantation in lethally irradiated DPPIV¯ rat treated with 2-AAF/CCl4 has firstly demonstrated cells in the bone marrow are capable of repopulating the injured liver.145 Both cell fusion with host hepatocytes and hepatic transdifferentiation of BM cells have been proposed as the underlying principle.206, 207 AAV vector, a nonpathogenic vector, is able to transduce a broad range of tissues and cells and mediate long-term transgene expression. However, the transduction efficacy of rAAV vector in HSCs is not conclusive. Whereas some researches showed that HSCs can be successfully transduced by rAAV vector and the rAAV-transduced HSCs maintain their multipotential differentiation, long-term persistence and self-renewal capacity, others have reported the HSCs are impervious to rAAV vector.107, 108 Unlike HSCs, MSCs can be efficiently transduced by rAAV vector. rAAV vector have been applied for gene targeting in MSCs.110 In the present study, we tested the feasibility of BM cell mediated liver gene delivery of hAAT in mouse model. Animal Experimental Design The recipients, 4-week old female C57BL/6, were IP injected twice (2 weeks interval) with MCT at 50mg/kg BW and received PHx before transplantation. BM cells were isolated from the femurs and tibias of 6 to 8-week-old male C57BL/6 GFP transgenic mice. The newly isolated BM cells were infected with Lenti-CB-hAAT vector at 1×102 MOI, rAAV1-CB-hAAT and 72

rAAV8-CB-hAAT vector at 1×104 MOI, respectively, for 2 hours. After transduction, BM cells were washed 3 times with PBS and then resuspended in saline solution at an approximate concentration of 5×106 cells/100ul. Transduced BM cells were transplanted into the recipient liver (5×106 cells/mouse) by intrasplenic injection (n=4) or portal vein injection (n=5). Serum samples were collected every week post transplantation. Serum hAAT levels were monitored by ELISA. Recipient mice were sacrificed at 8 or 14 weeks post transplantation, and liver tissues were harvested for immunostaining to evaluate the engraftment efficiency and transgene expression (Figure 4-1). Results Bone Marrow Cells Transduction In order to test the transduction efficiency of bone marrow cells, total bone marrow cells were isolated and infected with Lenti-CB-hAAT, rAAV1-CB-hAAT and rAAV8-CB-hAAT vectors. As shown in Figure 4-2, all vectors can transduce BM cells, but with different efficiency. Lenti-CB-hAAT infection resulted in the highest levels of hAAT in culture medium. Although hAAT levels in rAAV vector infected cells are much lower than that in lentiviral vector infected cells, they are clearly detectable. Relatively, rAAV1-CB-hAAT mediated higher levels of hAAT than rAAV8-CB-AAT. These results suggest that both lentiviral vector and rAAV vectors can be useful in bone marrow cell transplantation studies. Liver Transplantation of ex vivo Transduced Bone Marrow Cells Next, we tested the feasibility of bone marrow cell transplantation for liver gene delivery of alpha 1-antitrypsin. As described in Figure 4-1, male GFP transgenic mice were used as donor animals and female C57BL/6 mice were used as recipients. The recipients were treated with monocrotaline (MCT) to inhibit endogenous hepatocyte proliferation. The recipients also received partial hepatectomy (PHx) before transplantation to create liver injury, thus enhancing

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the environment for the proliferation and differentiation of transplanted bone marrow cells. In this experiment, freshly isolated total bone marrow cells (donor cells) were infected with LentiCB-hAAT (MOI=100), rAAV1-CB-hAAT (MOI=1x104) and rAAV8-CB-hAAT (MOI=1x104) vectors, respectively. After thorough washes, 5x106 cells were transplanted into recipient liver through portal vein injection. As shown in Figure 4-3, rAAV8-CB-hAAT vector mediated more hAAT positive cells in the recipient liver, while Lenti-CB-hAAT and rAAV1 vector mediated few hAAT positive cells. To confirm that the hAAT positive cells were derived from donors, we had performed co-immunostaining experiments. As shown in Figure 4-4, hAAT positive cells from rAAV8-CB-hAAT vector treatment group were also GFP positive. Bone Marrow Cell Transplantation Resulted in Sustained Levels of hAAT in Recipient Circulation In order to further enhance transgene (hAAT) expression, we have performed an additional experiment with a modified procedure.245 In this experiment, we transplanted 2x107 rAAV8-CBhAAT infected bone marrow cells (from male C57BL/6) into the recipients through intrasplenic injection. As expected, Y-chromosome positive cells were detected in the recipient liver demonstrating that donor cell could migrate and integrate into recipient liver (Figure 4-5). Immunostaining studies showed that most of hAAT positive donor cells were also positive for mouse albumin in the liver indicating that majority of donor bone marrow cells in liver transdifferentiated into hepatocytes (Figure 4-6, black arrow). To test the possibility that some bone marrow cells could home to other organs and express transgene, we had performed Y-FISH and immunostaining in spleen, bone and lung. Both Y-FISH and AAT-immunostaining showed some donor cells retained in spleen (Figure 4-7A and 4-7B). AAT-immunostaining also showed some AAT-positive cells in lung and bone marrow indicating that intrasplenic injection of bone marrow cells also resulted in multi-organ homing of these cells (Figure 4-7C and 4-7D, black

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arrow). Importantly, long-term and sustained serum levels of hAAT were obtained in this experiment (Figure 4-8). These results imply that transplantation of rAAV8-transduced bone marrow cells represents a novel therapy for AAT deficiency. Discussion Adult stem cells offer a platform for ex vivo genetic manipulation followed by autologous transplantation. Adult stem cell-mediated gene delivery may overcome many limitations, including immune rejection of allogeneic cells and ethical issue of embryonic stem cells, the shortage of donor organs, and non-specific targeting (such as germline transduction). In addition, adult stem cells-mediated gene therapy can serve as a regenerative medicine to replace diseased cells with patient’s stem cells carrying healthy gene(s). We have previously showed that liver stem (or progenitor) cell mediated liver gene delivery of hAAT was feasible in mouse model.245 However, liver stem cells can not be used in humans for autologous transplantation. Considering clinical practice, we investigated the possibility of transplanting genetically modified BM cells. In the present study, we showed that rAAV8-CB-hAAT vector transduced BM cells differentiated into hepatocytes and mediated sustained serum levels of hAAT in mouse model. These results imply a novel therapy the treatment of alpha 1-antitrypsin deficiency in humans. In the present study, we showed that Lenti-CB-hAAT vector transduced BM cells efficiently in vitro. Interestingly, transplantation of these transduced cells resulted in undetectable levels of hAAT in the circulation, although some hAAT positive cells were detected in the recipient mouse liver. These results were consistent with the previous oval cell studies. The possible mechanisms discussed in Chapter 3 may apply here as well. BM cells can be transduced by both rAAV1 and rAAV8 vectors in vitro. However, much higher levels of hAAT were detected in liver and serum from recipients received rAAV8-CBhAAT infected BM cells than that from recipients received rAAV1-CB-hAAT infected BM 75

cells. It is possible that the transdifferentiation of BM cells into hepatocytes provide favorable cellular environment for the intracellular process of rAAV8 vector including cytoplasmic trafficking, uncoating, and nuclear entry. Recent studies have shown that mutations of capsid proteins can affect rAAV2 vector trafficking and enhance transgene expression, and might support above hypothesis.59 Future studies will investigate the effect of stem cell transdifferentiation on rAAV vector intracellular processing. BM cells contain HSCs and MSCs, and both have been shown to possess hepaticdifferentiation potential.162, 195, 198 Furthermore, considering the possible contribution of cell to cell interaction to stem cell proliferation and differentiation, the present study employed total bone marrow cells. After intrasplenic injection, donor cells were found not only in the liver, but in spleen, lung, and bone marrow. It was expected that some donor bone marrow cells were trapped in the injection site, spleen, while some cells home back to the bone. The BM migrating or homing to lung might due to the pulmonary toxicity induced by MCT, a pyrollizidine alkaloid (PA) plant toxin. MCT is bioactivated by cytochromes P450 in hepatocytes to its active compound monocrotaline pyrrole (MCTP) that produces both hepatic and pulmonary toxicity.259 The results of the present study suggested that BM cell-based gene therapy approach is a promising therapy for genetic diseases. However, further improvement on transgene expression in the donor cells is required to achieve therapeutical application. This issue could be addressed mainly from two aspects, transduction efficiency by viral vectors and the transplantation/engraftment efficiency of adult stem cells. Rapid advances in gene delivery vector provide future gene therapy with lots of options. Self-complementary AAV vectors circumvent rate-limiting second-strand synthesis in single-stranded AAV vector genome and thus facilitate robust and highly efficient transduction.87 Site-specific integration AAV vector can

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establish long-term and persistent gene expression. Integration to AAVS1 locus could resist to transgene silencing, a major obstacle of integration viral vector such as retroviral vector.102 The efficiency of differentiation of BM-derived MSCs can be improved by modifying the culture conditions such as adding growth factors or cytokines, or by delivering an expression cassette to regulate hepatic differentiation.195, 198, 260 Pre-conditioned MSCs demonstrated higher liver engraftment potential. Here we showed that BM cells can be transduced by rAAV8 vector and that transplantation of these cells resulted in hepatic differentiation and transgene expression in the liver and detectable levels of transgene product (hAAT protein) in the serum. Detailed studies to elucidate the mechanism underlying interaction between viral vectors and adult stem cell, stem cell regulation on expression of foreign gene, migration and homing of stem cell will enhance the use of BM cell-based gene therapy for the treatment of AAT deficiency.

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Figure 4-1. Experimental outline of BM cells study. The recipients (female C57BL/6) were IP injected twice (2 weeks interval) with 50mg/kg of monocrotaline (MCT) and received partial hepatectomy (PHx) to remove 70% of liver mass before transplantation. BM cells were isolated from the femurs and tibias of male C57BL/6 mice. The newly purified BM cells were infected with Lenti-CB-hAAT, rAAV1-CB-hAAT, rAAV8CB-hAAT vector, respectively, for 2 hours, washed, and transplanted into the recipient liver by portal vein injection or intrasplenic injection. Serum hAAT levels were monitor by human AAT specific ELISA. Liver repopulation was measured by immunostaining.

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Figure 4-2. Ex vivo transduction of BM cells. Mouse Bone marrow cells were seeded in 24-well (1×104 cells/well; n=3) and were infected with the Lenti-CB-hAAT vector at 100 moi, rAAV1-CB-hAAT, rAAV8-CB-hAAT at 104 moi, and PBS, respectively. The accumulative hAAT in the culture medium was measured by ELISA. Circle, LentiCB-hAAT; Triangle, rAAV1-CB-hAAT; Square, rAAV8-CB-hAAT; Dash, lower limit of quantification (LLOQ). hAAT level of PBS group ( negative control) was below LLOQ.

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Figure 4-3. Detection of expression of human alpha1-antitrypsin (hAAT) in recipient liver after transplantation of viral vector infected BM cells by immunostaining. (A) Liver section from C57BL/6 mouse transplanted with Lenti-CB-hAAT infected BM cells (Brown). (B) Image A view at larger magnification. (C) Liver section from C57BL/6 mouse transplanted with rAA1-CB-hAAT infected BM cells. (D) Image C view at larger magnification. (E) Liver section from C57BL/6 mouse transplanted with rAAV8-CB-hAAT infected BM cells. (F) Image E view at larger magnification. (G) Human liver section served as positive control. (H) Liver section from untransplanted C57BL/6 mouse served as negative control.

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Figure 4-4. Detection of transgene expression from the engrafted donor BM cells by fluorescence double immunostaining for human alpha 1-antitypsin (hAAT) and green fluorescent protein (GFP). (A) Liver section from C57BL/6 mouse transplanted with rAAV8-CBhAAT infected BM cells showing hAAT expression (red). (B) Liver section same as in A stained for GFP (green). (C) Merge image of A and B. Representative slides were viewed at ×100 magnification.

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Figure 4-5. Detection of donor cells in recipient liver after BM cell transplantation by fluorescence in situ hybridizations (FISH) for Y-chromosome. (A, B, C) Female mice treated with MCT/PHx and BMTx from male mouse. x, X chromosome; Y, Y chromosome.

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Figure 4-6. Detection of coexpression of human alpha 1-antitypsin (hAAT) and mouse albumin by immunostaining. (A, C) Liver section from C57BL/6 mouse transplanted with rAAV8-CB-hAAT infected BM cells stained for hAAT (brown). (B,D) Liver section adjacent to the section in A and C, respectively, stained for albumin (red). Black arrow point to both AAT positive and albumin positive cells. Asterisks: location indicator. Representative slides were viewed at ×20 magnification.

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Figure 4-7. Multi-organ homing of transplanted BM cells. Tissue sections were from female C57BL/6 mouse at 8 weeks after transplantation with rAAV8-CB-GFP vector infected male BM cells. (A) Spleen section subjected to FISH for detecting Ychromosome. (B) Spleen section stained for GFP (brown). (C) Bone section stained for GFP. (D) Lung section stained for GFP. Black arrowheads indicate the observed GFP staining.

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Figure 4-8. Detection of expression of human alpha1-antitrypsin (hAAT) in the recipient serum. BM cells from C57BL/6 mice were infected with ssAAV8-CB-hAAT vector at 1×104 particles/cells for 2 h and transplanted into liver of partially hepatectomized C57BL/6 recipient (2 x107 cells/mouse; n=4) by intrasplenic injection. The transgene expression was monitored by measuring the serum level of hAAT. Square is the serum from the treatment group; Dash is lower limit of quantification (LLOQ). The serum level of hAAT from untransplanted C57BL/6 mouse (negative control) was below the LLOQ.

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CHAPTER 5 ADIPOSE TISSUE-DERIVED MESENCHYMAL STEM CELL-BASED LIVER GENE DELIVERY Summary Adipose tissue represents an accessible, abundant, and replenishable source of adult stem cells for potential application in regenerative medicine. Adipose tissue-derived mesenchymal stem cells (AT-MSCs) resemble bone marrow-derived mesenchymal stem cells (BM-MSCs) regarding morphology, immune phenotype and multiple differentiation capability, while possess the advantage of less invasive procurement and obtainability in large quantity. In light of recent observation of hepatic differentiation of AT-MSCs, our study investigated the feasibility of ATMSC-based liver gene delivery to correct a genetic disease, alpha 1-antitryspin deficiency (AATD). In vitro study showed AT-MSCs can be efficiently transduced by recombinant adenoassociated viral vector serotype 1(rAAV1-CB-hAAT). After transplanting to MCT/PHx injured liver, ex vivo transduced AT-MSCs displayed sustained transgene expression and secreted transgene product, human alpha 1-antitrypisn (hAAT) into circulating system resulting in a serum hAAT level of 100-200ng/ml. Immunostaining for hAAT on recipient liver section revealed that about 5-10% recipient liver was repopulated from approximately 1.6 ×106 ex vivo genetically modified AT-MSCs, 8 weeks post transplantation. More importantly, AT-MSCderived hepatocyte-like cells demonstrated liver-specific marker, albumin. In conclusion, results from this study demonstrated that AT-MSCs can be transduced by rAAV vectors, engrafted into recipient liver, contributed to liver regeneration, and served as platform for transgene expression. AT-MSC-based gene therapy presents a novel approach for the treatment of human genetic diseases, such as AAT deficiency.

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Introduction Alpha 1-antitrypsin (AAT) deficiency is a genetic disorder resulting from a single gene mutation on AAT coding gene, which results in a reduction of serum levels of AAT and accumulation of AAT protein in hepatocytes. Consequently this mutation causes an increased risk of developing early onset pulmonary emphysema and severe forms of liver disease, including neonatal jaundice, cirrhosis, and hepatitis.1, 2 Protein replacement therapy consisting of weekly repeated intravenous infusion of human AAT (hAAT) is the only available treatment for AAT deficiency-associated lung disease so far, while this therapy is expensive, not a cure, and temporary effect. For AAT deficiency-associated liver disease, no effective therapy is available except liver organ transplantation which is hampered by the shortage of donor organ and immune rejection. To circumvent this dilemma, we propose to replace dysfunctional hepatocytes with ex vivo genetically modified adult stem cells which carry the correct AAT gene. An urgent need for an adequate supply of hepatocytes for liver repopulation drives researchers to investigate in generating hepatocytes from extrahepatic adult stem cells, such as MSCs. MSCs are a heterogeneous population of plastic-adherent, spindle-shaped and fibroblastlike cells which can be extensively expanded in vitro whilst retaining their multi-lineage differentiation potential such as osteogenesis, chondrogenesis, adipogenesis.189, 190 In addition to differentiation into its native derivatives, mesenchymal tissues, MSCs also have the potential to differentiate into hepatocytes in vitro and in vivo.195, 198 MSCs have been traditionally isolated from bone marrow aspirates with a yield of approximately 1 MSC per 105 BM nucleated cells.190 To be clinical usefulness, such low cell number necessitate ex vivo expansion to obtain clinically significant cell numbers, which is time consuming and risk of cell contamination. Furthermore, differentiation potential and maximum life span of MSCs from BM decline with increasing age. 217-219

Fortunately, MSCs can also be isolated from adipose tissue.221, 261 There is little to no 87

difference between BM-MSCs and AT-MSCs regarding the morphology, immune phenotype, yield of adherent stromal cells, growth kinetics, cell senescence, multilineage differentiation capacity or transduction efficiency.197, 213 More importantly, the frequency of colony-forming unit-fibroblasts (CFU-F) in adipose tissue is hundred-fold higher than that of bone marrow.197 From practical standpoint, adipose tissue may represent an idea autologous stem cell source of repeatable access, replenishment, easy isolation, and minimal patient discomfort. Adeno-associated virus (AAV) is a linear single-stranded DNA parvovirus with a genome of 4.7 kb and a non-enveloped capsid of approximately 22 nm in diameter.38 To date, total 12 AAV serotypes and over 100 AAV variants have been discovered from human/nonhuman primate tissues. AAV serotypes display distinct and broad cell and tissue affinities such as muscle, liver, lung, and central nervous system.262 Not any human diseases have been shown to be associated with AAV. The lack of pathogenicity, low risk of insertional mutagenesis, many available serotypes, and broad tissue tropisms have make rAAV vector rapidly gain popularity in gene therapy application. Since 1995 AAV2-based vector was first administrated to a human subject for treating cystic fibrosis,263 over 40 clinical trials have now been approved involving 14 diseases so far.106 These studies indicate that in vivo gene transfer is feasible and relatively safe, but also suggest that the transduction efficiency of AAV2 vectors fall short of requirement for adequate and organ-specific transgene expression. As a result, ongoing research efforts are focused on developing new AAV vector by modifying both AAV genome and capsid protein. For example, self-complementary double-stranded AAV (dsAAV) vectors generated by mutating one of the AAV ITRs exhibit faster onset of gene expression and higher transduction efficiency than single-stranded AAV (ssAAV) vectors in muscle, liver and brain.86, 87 The underlying mechanism could be dsAAV vectors bypass the rate-limiting step that host cell mediates

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synthesis of dsDNA from the ssDNA.86 Other efforts have focused on engineering capsid protein such as transcapsidation, adsorption modification, mosaic capsid, and chimeric capsid.65 For instance, AAV2 ITR has been cross-packaged into AAV1 capsid and tested in clinical trial for muscle–directed gene therapy for AATD because rAAV1 vectors have shown hundred-fold more potency for murine muscle transduction than rAAV2 vectors.37, 76 Furthermore, rAAV vectors have been demonstrated to be capable of transducing MSCs efficiently and the transduced MSCs retained multipotential activities.110 The combination of rAAV-mediated gene delivery with stem cell therapy is the future direction. The capability of self-renewal and differentiation of stem cells make them to be the promising target of virus vector for long-term gene correction. By putting viral vectors in the stem cell, we can limit the undesired side effect resulting from the nonspecific targeting by systemic delivery of rAAV vectors. Here, we demonstrated that MSCs from mouse peritoneal adipose tissue can be genetically modified by rAAV vector and transplanted into liver parenchyma. Engrafted AT-MSCs was able to mediate long-term transgene expression. Experimental Design In vivo Transduction by ssAAV and dsAAV Vectors Female C57BL/6 mice (8-week old) were injected with ssAAV and dsAAV vectors to compare the transduction efficiency. Four rAAV vectors were selected, ssAAV1-CB-hAAT, dsAAV1-CMV-hAAT, ssAAV8-CB-hAAT, and dsAAV8-DHBV-hAAT. Two groups of mice (n=5, each) received rAAV1 (ssAAV and dsAAV) by percutaneous injection into the quadriceps femoris muscles of both hind limbs with 2×1010 particles per mouse in 50ul saline, respectively. The other two groups of mice (n=5, each) received rAAV8 (ssAAV and dsAAV) by portal vein injection with 2×1010 particles per mouse in 50ul saline. Serum samples were taken from tail vein

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every week post injection and subjected to hAAT specific ELISA to evaluate the transgene, hAAT, expression. Ex vivo Transduction and Transplantation of AT-MSCs The recipients, 4-week old female C57BL/6, were IP injected twice (2 weeks interval) with MCT at 50mg/kg BW and received PHx before transplantation. AT-MSCs were isolated from the peritoneal adipose tissue of 6 to 8-week-old male C57BL/6 mice. The newly isolated ATMSCs were infected with ssAAV1-CB-hAAT vector at 5×104 MOI for 2 hours. After transduction, AT-MSCs were washed 3 times with PBS and then resuspended in saline solution at an approximate concentration of 1.6×106 cells/100ul. Transduced AT-MSCs were transplanted into the recipient liver (1.6×106 cells/mouse, n=5) by intrasplenic injection. Serum samples were collected every week post transplantation. Serum hAAT levels were monitored by ELISA. 8 weeks post transplantation, liver tissue of recipient mice were harvested for immunostaining (Figure 5-1). Results Isolation and Characterization of AT-MSCs Mouse AT-MSCs were isolated from peritoneal adipose tissue of male C57BL/6 mice as described in Chapter 2. These cells were characterized by immunofluorescence and multiple differentiation potential upon exposure to adipogenic and osteogenic induction medium. Immunofluorescence staining revealed that the cells isolated from the mouse peritoneal adipose tissue expressed stromal-associated marker CD44, CD90 and CD105 but didn’t express either hematopoietic markers CD34 and CD45 or endothelial marker CD31. The expression of CD133 was low (Figure 5-2A). The cell surface phenotypes were consistent with those reported in the literature for adipose tissue derived stem cells.222 Multiple differentiation potential of AT-MSCs was demonstrated by induced differentiation into adipocytes and osteocytes. Two weeks after

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exposure to adipogenic induction medium, intracellular lipid droplets were observed within the adipogenic-differentiated AT-MSCs using Oil Red O staining. Osteogenic differentiation resulted in extracellular calcium phosphate precipitates as revealed by Alizarin Red S staining (Figure 5-2B). Optimization of rAAV Vecotors AAV is a single stranded DNA virus. After infection, the viral DNA undergoes second stranded DNA synthesis using host cellular enzymes. Therefore, transgene expression from conventional single stranded rAAV vector depends on the second-stranded DNA synthesis. Recently double-stranded AAV vectors (dsAAV), or self-complementary AAV vectors(scAAV) have been developed by deleting the D-sequence (the packaging sequence) and the adjacent terminal resolution site (trs) of one of the ITRs. It has been shown that dsAAV can avoid secondstranded synthesis in the host cells thus mediated quicker and higher levels of transgene expression. In order to achieve optimal levels of hAAT expression in AT-MSCs, we have generated two dsAAV vectors, dsAAV-DHBV-hAAT and dsAAV-CMV-hAAT. Due to the limitation of the packaging capacity of dsAAV (2.4 kb, half of the full packaging capacity of ssAAV vector of 4.7 kb), two smaller promoters were used. Duck hepatitis B virus (DHBV) promoter has been shown as an active liver specific promoter.264 CMV promoter was also used since it is active in most of the stem cells and muscle cells. The dsAAV-CMV-hAAT vector plasmid was packaged into rAAV1 vector for muscle gene delivery. As shown in Figure 5-3A, dsAAV1-CMV-hAAT mediated detectable levels of hAAT expression. However, the levels were lower than that from matched dose of ssAAV1-CB-hAAT vector. Similarly, the dsAAV-DHBVhAAT vector plasmid was packaged into rAAV8 vector for liver gene delivery. As shown in Figure 5-3B, dsAAV8-DHBV-hAAT mediated sustained levels of hAAT, but the levels were much lower than that from ssAAV8-CB-hAAT vector. These results suggested that advantage of 91

dsAAV vector in muscle and liver was limited and did not overcome the advantage of CBpromoter. In order to select the most efficient rAAV vectors for AT-MSCs, matched dose of all above vectors were used to infect AT-MSCs. As shown in Figure 5-4A, ssAAV1-CB-hAAT vector mediated the highest transgene expression than the other three rAAV vectors, dsAAV1CMV-hAAT, ssAAV8-CB-hAAT, and dsAAV8-DHBV-hAAT, as indicated by more than 10fold increase in hAAT serum level at day 9 post transduction. It was interesting that rAAV1 mediated more than 25-fold higher levels of hAAT than rAAV8 in AT-MSCs. Furthermore, double infection of AT-MSCs using ssAAV1-CB-hAAT vector at 12hr interval could further increase the transgene expression (Figure 5-4B). Together, above results clearly demonstrated the ssAAV1-CB-hAAT vector was the best vector among those tested and displayed two advantages in vector transduction and transcription of transgene. Therefore, we decided to use ssAAV1-CB-hAAT vector for the following studies. Liver Transplantation of ex vivo Transduced AT-MSCs We hypothesis that ex vivo transduced AT-MSCs with rAAV1-CB-hAAT could serve as a platform for liver expression of hAAT after autologous transplantation. To test this hypothesis, AT-MSCs (1.6 ×106) from male mice were infected with ssAAV1-CB-hAAT (MOI=5x104) and were transplanted into the liver of MCT-treated and partial-hepatectomized female C57BL/6 recipients (Figure 5-1). MCT, a pyrrolizidine alkaloid, is metabolized in the liver to its active derivatives within a few hours or days but able to induce persistent inhibition effect on recipient hepatocyte proliferation and thus provide subsequently transplanted cells with a proliferative advantage over endogenous hepatocytes. Partial hepatectomy was applied to create a liver damage model for enhancing the engraftment and proliferation of transplanted AT-MSCs. Recipient organs were harvested 8 week post transplantation and subjected to hAAT 92

immunostaining. Immunostaining for hAAT revealed that 5-10% of total hepatocytes displayed hAAT transgene expression (Figure 5-5). Those hAAT positive hepatocytes indicated that rAAV- transduced AT-MSCs were able to migrate into liver from the injection site of spleen, engraft into the recipient liver parenchyma, contribute to liver repopulation, and give rise to transgene expression. More importantly, those AAT positive cells were morphologically similar to hepatocyte. Y-chromosome fluorescent in situ hybridization (Y-FISH) further confirmed the presence of male donor cells in the female recipient liver (Figure 5-6). To test the hypothesis that AT-MSCs can transdifferentiate into hepatocytes after liver transplantation, serial sections of recipient liver tissue were subjected to mouse albumin and human AAT immunostaining, respectively. As shown in Figure 5-7, most of hAAT positive cells were also positive to mouse albumin. These results indicated that adipose tissue-derived MSCs were able to differentiate into functional hepatocytes with capability of producing albumin. Using GFP as reporter gene, donor cells were also detected in spleen, lung and bone marrow after intrasplenic injection by using immunostaining for GFP (Figure 5-8). In order to quantify transgene product generated and secreted from engrafted rAAVtransduced AT-MSCs, serum hAAT levels were measured serially for 8 weeks by hAAT specific ELISA. All animals showed sustained transgene expression throughout the study with an average serum hAAT concentration between 100ng/ml and 200ng/ml (Figure 5-9). These results demonstrated that AT-MSCs can be used as platform for liver-directed hAAT gene delivery. Discussion AT-MSCs represent an excellent cell source for regenerative medicine. However, the use of AT-MSCs for liver regeneration and gene delivery remain elusive. In this study, we isolated and characterized mouse AT-MSCs. We showed that these AT-MSCs can be efficiently transduced by ssAAV1-CB-hAAT vector. Transplantation of rAAV transduced AT-MSCs 93

resulted in hepatic differentiation and sustained long-term transgene expression in the liver and the serum of the recipients. Results from this study demonstrated that it is feasible to use ATMSCs as a cell vehicle for liver gene delivery and implied a novel therapy for the treatment of liver diseases. AAT is a major serum protective protein. It is generally accepted that patients with serum AAT levels below 11µM or 800µg/ml may develop emphysema. Therefore, the serum level of hAAT obtained from AT-MSCs transplantation in this study remain further improvement to be therapeutic (approximately 500-800ug/ml)12 . Several possible approaches may be employed to enhance hAAT expression levels. Banas and his colleagues demonstrated that CD105+ fraction of AT-MSCs exhibited high hepatic differentiation ability.232 Therefore, enrichment of stem cell population by isolating CD105+ AT-MSCs may increase the total number of hepatocytes derived from donor AT-MSCs and thus enhance hAAT levels in the recipient serum. Similarly, other cell markers may also be used to enrich stem cell population, such as p75 neurotrophin receptor (p75NTR), which has also been used to isolate and collect putative multipotent stem cell from mouse adipose tissue-derived stromal vascular fraction culture cells (ADSVF cells).265 Secondly, hepatic-differentiation potential of AT-MSCs can be further improved by in vitro preconditioning toward hepatocyte-like cells such as incubating MSCs with specific growth factors e.g., hepatocyte growth factor (HGF), epidermal growth factor (EGF) or fibroblast growth factor (FGF).226, 232 In addition, improvement of transduction efficiency of AT-MSC by further optimize rAAV vectors may also enhance hAAT levels in the receipt serum. In the present study, we showed rAAV1 was more efficient than rAAV8. Other serotypes of AAV vectors and recently developed mutant AAV vectors might mediate higher transduction efficiency in AT-MSCs. In contrast to previous study 87 , our study showed dsAAV vectors didn’t

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demonstrate superior transduction efficiency to ssAAV vector. This inconsistency to the welldocumented characteristics of dsAAV might reflect the inferior promoter activity of DHBV and CMV, compared to the CB promoter. Previous studies have shown a 10-100 fold higher promoter activity of CB promoter than that of CMV promoter depending on the vector dose.239, 266

This superior activity of CB promoter, noted in our study, even defeated the advantage of

dsAAV genome of the faster and stronger transgene expression. Importantly, the promoter remained active after host cell differentiated into hepatocytes. Finally, site-specific integration system of AAV vector may be employed. Considering the dilution of episomal rAAV vector during cell division, site-specific integration AAV vector system may not only enhance but also ensure a long-term transgene expression. Importantly, AAVS1 site has been reported to be a safe integration site.46 A bipartite rAAV vector has been designed to fulfill this concept.101 In addition to the feature of easy isolation in large quantity from adipose tissue and expendable in vitro, AT-MSCs, like bone-marrow derived MSCs, also exhibit immunomodulatory and anti-proliferative effects on T cells.267, 268 Therefore, it is possible to transplant allogeneic AT-MSCs from normal individual to AAT deficient patients without severe immune response. This strategy can be tested using our recently-developed hAAT transgenic mouse as donor. Transplantation of AT-MSCs from hAAT transgenic mice to genetically mismatched recipients will not only avoid the ex vivo transduction, but provide better understanding of hepatic differentiation of AT-MSCs. In summary, results from our study using rAAV vector expressing hAAT gene consisted and further extended the previous observations232 thus paved a path to both basic and clinical studies.

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Figure 5-1. Experimental outline of AT-MSCs study. The recipients (female C57BL/6) were IP injected twice (2 weeks interval) with 50mg/kg BW of monocrotaline (MCT) and received partial hepatectomy (PHx) to remove 70% of liver mass before transplantation. AT-MSCs were isolated from the femurs and tibias of male C57BL/6 mice. The newly purified BM cells were infected with rAAV1-CB- rAAV-CB-hAAT vector for 2 hours, washed three times with PBS, and transplanted into the recipient liver by intrasplenic injection. Serum hAAT levels were monitor by human AAT specific ELISA. Liver repopulation was measured by immunostaining.

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A.

B.

Figure 5-2. Characterization of AT-MSCs. (A) Expression of cell surface markers in AT-MSCs by immunofluorescence staining. (B) Multiple differentiation potential of AT-MSCs. Left, undifferentiated AT-MSCs; Middle, AT-MSCs were cultured for 2 weeks in an adipogenic induction medium and stained with Oil Red O for lipid droplets; Right, AT-MSCs were cultured for 2 weeks in an osteogenic induction medium and stained with Alizarin Red S (pH 4) for calcium phosphates.

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B

Figure 5-3. In vivo muscle or liver transduction by ssAAV and dsAAV vectors. C57BL/6 female mice were injection 2×1010 particle rAAV vectors by intramuscular injection or portal vein injection to liver. A) In vivo muscle transduction by rAAV vectors. Solid square, ssAAV1-CB-hAAT vector; Open square, dsAAV1-CMV-hAAT vector; Solid triangle, saline group serve as negative control. B) In vivo liver transduction by rAAV vectors. Solid square, ssAAV8-CB-hAAT vector; Open square, dsAAV8-DHBVhAAT vector; Solid triangle, saline group serve as negative control.

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Figure 5-4. Ex vivo AT-MSCs transduction efficiency of rAAV vectors. (A) Optimization for AT-MSCs transduction efficiency of four rAAV vectors. Mouse AT-MSCs (passage=3) were seeded in 24-well (5×104cells/well, n=3) and infected with rAAVhAAT vector at 1x104 particles/cell. The accumulative hAAT in the culture medium was measured by hAAT ELISA. Solid triangle, ssAAV1-CB-hAAT vector; Open circle, dsAAV1-CMV-hAAT vector; Open square, ssAAV-CB-hAAT vector; Cross, dsAAV-DHBV-hAAT vector; Dash, lower limit of quantification (LLOQ). hAAT level of PBS group ( negative control) was below LLOQ. (B) Double transduction of AT-MSCs by ssAAV1-CB-hAAT vector. Mouse AT-MSCs (passage=1) were seeded in 24-well (1×104cells/well, n=3) and infected with ssAAV1-CB-hAAT vector at 5x104 particles/cell. The accumulative hAAT in the culture medium was measured by hAAT ELISA. Triangle, one infection; Circle, two infections at 12 hr interval; Dash, lower limit of quantification (LLOQ). hAAT level of PBS group ( negative control) was below LLOQ.

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Figure 5-5. Detection of expression of human alpha 1-antitrypsin (hAAT) in recipient liver after transplantation of ssAAV1-CB-hAAT infected AT-MSCs by immunostaining. (A, C, D) Liver section from C57BL/6 mouse transplanted with ssAAV1-CB-hAAT infected AT-MSCs stained for hAAT (Brown). (B) Liver section from the same animal as in A, C, and D stained by anti-rabbit immunoglobulin G serving as negative control. (E) Liver section from normal human serves as positive control. (F) Liver section from an untransplanted C57BL/6 mouse serves as negative control.

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Figure 5-6. Detection of donor cells in recipient liver after AT-MSCs transplantation by fluorescence in situ hybridizations for Y-chromosome. (A) Male liver served as positive control. (B, C) Female mice treated with MCT/PHx and transplanted with AT-MSCs from male mouse. Y, Y chromosome.

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Figure 5-7. Detection of coexpression of human alpha 1-antitypsin (hAAT) and mouse albumin by immunostaining.(A) Liver section from mouse transplanted with ssAAV1-CBhAAT vector infected AT-MSCs stained for hAAT (brown). (B) Liver section adjacent to the section in A, stained for mouse albumin (red). (C) Human liver section staining for hAAT served as positive control. (D) Normal mouse liver section staining for mouse albumin served as positive control.

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Figure 5-8. Multi-organ homing of transplanted AT-MSCs. Tissue sections were from female C57BL/6 mouse at 8 weeks after transplantation with rAAV8-CB-GFP vector infected male AT-MSCs. (A) Spleen section stained for GFP (brown). (C) Lung section stained for GFP. (D) Bone section stained for GFP. Black arrowheads indicate the observed GFP staining. Images were viewed at ×20 magnification.

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Figure 5-9. Detection of expression of human alpha 1-antitrypsin (hAAT) in the serum. ATMSCs from C57BL/6 mice were infected with ssAAV1-CB-hAAT vector at 5x104 particles/cells for 2 h and transplanted into liver of partially hepatectomized C57BL/6 recipient (1-2 x106 cells/mouse; n=3). The transgene expression was monitored by measuring the serum level of hAAT. Square is the serum from the treatment group. Dash is lower limit of quantification (LLOQ). The serum level of hAAT from untransplanted C57BL/6 mouse (negative control) was below the LLOQ.

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CHAPTER 6 SUMMARY AND FUTURE DIRECTION Summary Protein replacement therapy has been only optional treatment for AAT deficiency for more than 20 years. Several new ideas have come up, implemented and tested for sake of the advances in the field of molecular biology, disease pathogenesis, and biotechnology, etc. In the last 10 years, strides have been made in treating a secreted protein disorder such as AAT deficiency with the use of rAAV gene therapy and bring it to clinical trials. At the meantime, stem cell has gone from basic research to clinical application as regenerative medicine to increase our standard of lives. For instance, bone marrow transplantation extends the lives of people suffering from leukemia, lymphoma and other inherited blood disorders. A combination of gene therapy and stem cell will broad their application than using either single therapy alone and will address some intrinsic problem of gene therapy or stem cell therapy such as nonspecific targeting of viral vector and differentiation potential of stem cells. In 1990, the first gene therapy trial was a cellbased gene therapy. Patients’ T-cells were isolated and transduced with MoMLV-ADA vector ex vivo, followed by returning to patients. Patients benefited and suffered no harmful effect from this therapy, however, since T-cells have a limited life-span, patients need to receive periodic infusion of their genetically –modified T cells. From this point, researches see the prospect of using stem cell to develop a permanent cure, the stem cell-based gene therapy. Our studies tested the feasibility of using adult stem cell-based gene therapy approach to treat one of the common secreted protein disorders, AAT deficiency. We investigated two types of viral vectors rAAV vector and lentiviral vector. rAAV vector is the safest viral vector among all of the viral vectors, which leads to over 40 clinical trials involving 14 diseases and 4 serotype rAAV vectors so far. Lentiviral vector is known by its high transduction efficiency and long-

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term transgene expression by integrating viral genome into host chromosome. Both viral vectors represent unique property in the gene therapy. To answer the question that whether genetically modified adult stem cells can be utilized to correct the genetic defect, we first transduced liver progenitor cells (oval cells) with rAAV and lentiviral vector expressing hAAT and transplanted these cells into the mouse liver. Results from these studies showed that oval cells can be transduced by both rAAV and lentiviral vectors. Transgene (hAAT) expression can be detected in the recipient liver and transgene produce could be secreted into the circulation to boost serum hAAT level. Results from oval cell study were promising and indicated it is feasible to use adult stem cell for liver gene delivery. However, isolation of oval cells is not clinically applicable, although oval cells can be isolated from animal in large quantity. In order to avoid this problem, in the second set of studies, we transduced and transplanted BM cells into mouse livers. BM cells have been proved to be capable of converting into hepatocytes and contributing to liver regeneration. More importantly, BM cells can be used for autologous transplantation to eliminate rejection problem from allograft transplantation. These results showed that BM cells can be transduced by rAAV and lentiviral vectors and engrafted into liver resulting in transgene expression and transgene product in the serum. rAAV8 vector demonstrated superior transduction efficiency than rAAV1 vector and lentiviral vector. To obtain enough cell number from BM for clinical application is challenging. Adipose tissue represents an ideal source of autologous stem cells, AT-MSCs. Liposuction results in minimal patient discomfort and adipose tissue can be obtained in large volume for yielding enough cells for clinical practice. Hence, we performed the third set of studies using AT-MSCs. Results demonstrated that AT-MSCs can be efficiently transduced by rAAV 1 vector. Ex vivo

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transduced and transplanted AT-MSCs can mediate transgene expression in the liver and result in sustained transgene product, hAAT, in the circulation. Importantly, engrafted AT-MSCs presented hepatocyte cell function e.g. albumin production. In conclusion, this study showed adult stem cells can serve as carrier for gene delivery. Adult stem cell engrafted into target organ and played as a platform for transgene expression after transplantation. Adult stem cell-based gene therapy presents a novel approach for treatment of human genetic disease. In this study, we have develop two stem cell (BM and AT-MSC) based gene therapies for the treatment of AAT deficiency. Future Direction Achieving therapeutical serum level of transgene product (hAAT) is the final goal. Efficiency of adult stem cell-based gene therapy is determined by three main factors, transduction efficiency, engraftment efficiency, and transgene expression. Optimization in any of these three factors will definitely contribute to achieve our goal. For increasing transduction efficiency of viral vectors, engineering viral capsid and genome have been carried out. Tissue and cell specific targeting and site-specific integration are the two attractive properties that future viral vectors want to pursuit. Specific targeting to a special cell type will decrease the possibility of site effect resulting from nonspecific binding to unwanted cells. At the mean time, it will enhance the viral vectors concentration in the targeted cells and leads to increase transgene expression. Site-specific integration provides a long-term treatment and reduces the risk of tumorigenesis resulting from randomly integrating into promoter region of oncogene. Clarifying and understanding of stem cell homing will in turn aid application of stem cell transplantation. But stem cell homing and engraftment are a complex and multistep process. These evolve various adhesion receptors and ligands that mediate cell-to-matrix and cell-to-cell interaction, including selectins, integrins, and Ig family and numbers of others undefined factors. 107

Several signaling pathways (e.g.SDF1-α/CXCR4 signaling) have been put forward, but more researches are needed to solve this puzzle. Tumor formation is a big issue for stem cell therapy. Detail examination of the tumorigenesis of stem cell is required to make stem cell an effective and safe therapy.

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BIOGRAPHICAL SKETCH Hong Li was born in Fuzhou, Fujian Province, P. R. China in 1979. After graduation from high school at Fuzhou No. 8 Middle School in 1997, she started her college education at China Pharmaceutical University in Nanjing, P. R. China. She was awarded her bachelor’s degree in biotechnical pharmaceutics in 2001 and master’s degree in microbiology and biochemical pharmacy in 2004. She then joined the Department of Pharmaceutics at University of Florida and began her doctoral study under the guidance of Dr. Sihong Song in August 2004. Her research focused on adult stem cell-based gene therapy for alpha 1-antitrypsin deficiency. At the meantime, she obtained her master’s degree in statistics in 2008 from University of Florida, Department of Statistics.

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